9 Endometriosis

Tasuku Harada
Editor
Endometriosis
Pathogenesis
and Treatment
123
Endometriosis
Tasuku Harada
Editor
Endometriosis
Pathogenesis and Treatment
Editor
Tasuku Harada
Department of Obstetrics
and Gynecology
Tottori University
Faculty of Medicine
Yonago, Tottori, Japan
ISBN 978-4-431-54420-3 ISBN 978-4-431-54421-0 (eBook)

DOI 10.1007/978-4-431-54421-0
Springer Tokyo Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014942646
Springer Japan 2014

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Preface
Endometriosis remains an enigmatic disease despite a long history of research into

its basic science and clinical aspects. Sampson published his pioneer article in 1927
theorizing that retrograde menstruation may cause peritoneal endometriosis. Since
that time, researchers throughout the world have wrestled with the mysteries of the
disease. In this book, we bring you a compendium of the present understanding of
the pathogenesis of endometriosis as well as how this information may be applied in
current therapies. As editor, I invited an international group of distinguished
scientists to contribute chapters and was happy that all graciously agreed to do
so. In particular, many Asian scientists made significant contributions.
Our hope is that this book will provide useful information and fresh knowledge
to all physicians and scientists interested in endometriosis, and that it will stimulate
further research and lead to more efficacious treatment modalities.
Yonago, Tottori, Japan Tasuku Harada
v
Contents
Part I Introduction
1 Endometriosis: A Mysterious Disease . . . . . . . . . . . . . . . . . . . . . . . 3

Tasuku Harada
Part II Basic Science
2 Pathological Aspect and Pathogenesis of Endometriosis . . . . . . . . . 9

Ritsuo Honda and Hidetaka Katabuchi
3 Visible and Invisible (Occult) Endometriosis . . . . . . . . . . . . . . . . . 19
Khaleque Newaz Khan
4 Role of Stem Cells in the Pathogenesis of Endometriosis . . . . . . . . 33
Tetsuo Maruyama
5 Role of NK Cells in Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . 49
Nagamasa Maeda
6 Macrophages in Pathophysiology of Endometriosis . . . . . . . . . . . . 61
S.F. Ahmad, N. Michaud, H. Rakhila, and A. Akoum
7 Inflammation and Cytokines in Endometriosis . . . . . . . . . . . . . . . . 87
Tomio Iwabe and Tasuku Harada
8 Epigenetics in Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Masao Izawa, Fuminori Taniguchi, and Tasuku Harada
9 Roles of Prostaglandin E2 in Endometriosis . . . . . . . . . . . . . . . . . . 125
Kuei-Yang Hsiao, Meng-Hsing Wu, and Shaw-Jenq Tsai
10 Sex Steroids and Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Jo Kitawaki
vii
viii Contents
11 Aromatase Expression in Endometriosis and Its Significance . . . . . 155

Hiroshi Ishikawa and Makio Shozu
12 Apoptosis in Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Apostolos Kaponis, Georgia Kollia, Fuminori Taniguchi,
Tasuku Harada, and George Decavalas
13 Role of Nerve Fibres in Endometriosis . . . . . . . . . . . . . . . . . . . . . . 191
Natsuko Tokushige
14 Endometriosis in Experimental Models . . . . . . . . . . . . . . . . . . . . . 213
Fuminori Taniguchi and Tasuku Harada
15 Malignant Transformation of Endometriosis:
Underlying Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Masaki Mandai, Ken Yamaguchi, Noriomi Matsumura,
and Ikuo Konishi
16 Potential New Drugs for Endometriosis:
Experimental Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Kaei Nasu, Yukie Kawano, Masakazu Nishida,
Akitoshi Tsuno, Akitoshi Yuge, Wakana Abe,
Kentaro Kai, Mamiko Okamoto, and Hisasshi Narahara
17 Altered Biological Characteristics of Eutopic
and Ectopic Endometrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Cassia G. T. Silveira, Admir Agic, Geraldine O. Canny,
and Daniela Hornung
Part III Clinical Science
18 Prevention of Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

Ebru H. Biberoglu and Kutay O. Biberoglu
19 MR Imaging of Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Shinya Fujii
20 Biomarkers of Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Amelie Fassbender, Dorien O, Bart De Moor,
Etienne Waelkens, Christel Meuleman, Carla Tomassetti,
Karen Peeraer, and Thomas DHooghe
21 Classification of Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Hans-Rudolf Tinneberg, Frank Tuttlies, Eniko Berkes,
Gabriele Anja Krombach, Andreas Hackethal,
Lutz Konrad, and Frank Oehmke
Contents ix
22 The Association of Endometriosis with Ovarian Cancer:

A Critical Review of Epidemiological Data . . . . . . . . . . . . . . . . . . . 363
Sun-Wei Guo
23 Surgical Management of Endometriosis . . . . . . . . . . . . . . . . . . . . . 385
Imari Deura and Tasuku Harada
24 Systematic Laparoscopic Surgery for Complete Obliteration
of the Cul-de-sac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Yoshiaki Ota, Masaaki Andou, Keiko Ebisawa, Kazuko Fujiwara,
Tomonori Hada, Hiroyuki Kanao, and Ikuko Ota
25 Prevention of Recurrence After Surgery . . . . . . . . . . . . . . . . . . . . . 411
Yutaka Osuga, Yuri Takemura, Masashi Takamura,
and Kaori Koga
26 Ovarian Reserve in Patients with Endometriosis . . . . . . . . . . . . . . 419
Michio Kitajima and Hideaki Masuzaki
27 Infertility Treatment of Endometriosis Patients . . . . . . . . . . . . . . . 431
Kaori Koga, Osamu Yoshino, Yasushi Hirota, Tetsuya Hirata,
Miyuki Harada, and Yutaka Osuga
28 Pregnancy Complications Associated with Endometriosis . . . . . . . . 445
Ivo Brosens and Giuseppe Benagiano
29 Malignant Transformation of Endometriosis . . . . . . . . . . . . . . . . . 457
Hiroshi Kobayashi
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Part I
Introduction
Chapter 1
Endometriosis: A Mysterious Disease
Tasuku Harada
Abstract Endometriosis is an enigmatic disease, despite a long history of research

into its basic science and clinical aspects. Sampson published his pioneer article in
1927, theorizing that retrograde menstruation may cause peritoneal endometriosis.
Since that time, researchers throughout the world have wrestled with the mysteries
of the disease. In this book, we bring you a compendium of the present understand-
ing of the pathogenesis of endometriosis as well as how this information may be
applied in current therapies. As editor, I invited an international group of distin-
guished scientists to contribute a chapter and was happy that all graciously agreed
to do so. Our hope is that this book will provide useful information and fresh
knowledge to all doctors and scientists interested in endometriosis, and that it will
stimulate further research, and lead to more efficacious treatment modalities.
Keywords Autotransplant Epigenetics Eutopic endometrium Implantation

theory Pathogenesis
1.1 Introduction
Endometriosis is an enigmatic disease, despite a long history of research into its

basic science and clinical aspects. Sampson published his pioneer article in 1927,
theorizing that retrograde menstruation may cause peritoneal endometriosis
[1]. Since that time, researchers throughout the world have wrestled with the
mysteries of the disease. In this book, we bring you a compendium of the present
understanding of the pathogenesis of endometriosis as well as how this information
may be applied in current therapies. As editor, I invited an international group of
T. Harada, M.D., Ph.D., D.M. Sci. (*)

Department of Obstetrics and Gynecology, Faculty of Medicine,
Tottori University, 36-1 Nishi-cho, Yonago 683-8504, Japan
e-mail: tasuku@med.tottori-u.ac.jp
T. Harada (ed.), Endometriosis: Pathogenesis and Treatment, 3

DOI 10.1007/978-4-431-54421-0_1, Springer Japan 2014
4 T. Harada
distinguished scientists to contribute a chapter and was happy that all graciously
agreed to do so. Our hope is that this book will provide useful information and fresh
knowledge to all doctors and scientists interested in endometriosis, and that it will
stimulate further research, and lead to more efficacious treatment modalities.
The prevalence of endometriosis in women varies widely: 0.711 % in
populations presenting for health care, 222 % when undergoing surgical steriliza-
tion, 1747 % among infertile women, and 274 % in women with chronic pelvic
pain [2]. An estimated 2.6 million Japanese women have this disease, extrapolating
a prevalence rate of 10 % among reproductive age women. An increase in the
incidence of endometriosis is worrisome because the birth rate in Japanese has
decreased and age of primiparous is reportedly over 30 years old. The number of
newborn babies is decreasing in Japan, while the population over 65 years old is
increasing. In such circumstances, infertility due to endometriosis of women is a
serious socioeconomical problem in Japan.
The assumption that the incidence of endometriosis is increasing is based on the
combination of the implantation theory of retrograde menstruation and the data of
well-known studies on epidemiology. Several well-designed epidemiological stud-
ies reported that shorter menstrual cycles, longer duration of flow, heavier men-
strual flow, and low parity are risk factors for endometriosis [3], indicating an
increased number of menstruation and retrograde reflux may increase risk of the
disease.
The classic theory on the pathogenesis of endometriosis includes implantation
theory, coelomic metaplasia theory, and embryonic Mullerian rests. A recent study
found that extrauterine stem cells originating from bone marrow may differentiate
into endometriotic tissues [4]. Although many researchers argue that modern
technologies of molecular medicine are unveiling the unsolved issues regarding
the pathogenesis of endometriosis, Sampsons implantation theory has never been
discarded.
Redwine raised a question about an intriguing point in Sampsons theory. His
paper entitled Was Sampson wrong? proposed that endometriotic tissues may not
be autotransplanted tissue. After reviewing more than 200 papers on autotransplants
and comparisons of endometriosis and endometrium, Redwine pointed out that a
majority of studies found multiple and significant alterations between ectopic
endometrium (endometriosis) and original endometrium. He suggested that endo-
metriosis is not a simple autotransplant, indicating that endometriosis is not a
displacement (implantation) of normal endometrium. Redwine concluded that
Sampson may be wrong [5].
We investigated the expression of inflammatory cytokines and prostaglandins in
eutopic endometrium and endometriotic tissues [68]. Recently, we obtained inter-
esting data regarding differential expression of inflammatory genes in eutopic
endometrium and endometriosis. We obtained endometriotic tissue from ovarian
chocolate cysts at the time of laparoscopic surgery, collected the two types of
eutopic endometrial tissues, and classified them as follows: (1) the disease-free
(F-Em: patients with benign ovarian tumor) and (2) endometriosis with ovarian
chocolate cysts (C-Em). Gene expression of interleukin-6 (IL-6), IL-8, and
1 Endometriosis: A Mysterious Disease 5
100 b 10.0
80 8.0 b 80
COX-2 mRNA
b
IL-6 mRNA
IL-8 mRNA
60 6.0 60
40 4.0 40
a a
20 2.0 20
a a a a
0 0 0
F-Em C-Em C F-Em C-Em C F-Em C-Em C
Fig. 1.1 Quantitative analysis of IL-6, IL-8, and COX-2 gene expression in human endometrial
and endometriotic tissues. The mRNA levels were evaluated by real-time RT-PCR. The abbrevi-
ations of the three types of tissues are as follows: (1) eutopic endometrial tissues of disease-free,
F-Em (n 15); (2) eutopic endometrium with ovarian chocolate cyst, C-Em (n 15); and (3) the
ovarian chocolate cyst, C (n 15). The mRNA level of F-Em (proliferative phase) set arbitrarily at
1.0. Data are the mean SE of three independent experiments. Bars that do not share a letter are
significantly different (P < 0.05)
cyclooxygenase 2 in endometriotic tissue is enhanced compared with that in both

types of eutopic endometrial tissues (Fig. 1.1). With regard to IL-6 and IL-8 mRNA
expression, eutopic endometrium in women with endometriosis exhibited greater
expression than the disease-free endometrium.
These observations suggest that the biological character of endometriosis tissue
regarding inflammatory mediators is quite distinguishable from the eutopic endo-
metrium of disease-free women. Therefore, this implies that endometriosis tissue is
not derived from eutopic endometrium as Redwine described. It may also not be an
autotransplant disease. On the other hand, we can speculate (1) that eutopic
endometrium had already been altered within the uterine cavity before its implan-
tation to the peritoneum or (2) that endometriotic tissues may change their biolog-
ical character during separation from the uterus, escape from immune clearance,
attachment, invasion, establishment of local neurovascularity, and continued
growth. In the latter case, however, altered gene expression in the eutopic endo-
metrium of patients with endometriosis cannot be explained. Why modest alter-
ations occur in eutopic endometrium of patients with endometriosis is observed.
Taylor and colleagues published interesting papers, reporting significant
changes in multiple markers of endometrial receptivity in the eutopic endometrium
after induction of endometriosis in a mouse model [9]. They showed reduction in
the Hoxa 10 gene in the eutopic endometrium of the endometriosis model mouse, a
similar finding to that observed in women with endometriosis. They also found
hypermethylation of the Hoxa 10 gene in the eutopic endometrium of mouse
endometriosis model. These data suggest that the presence of endometriotic tissue
may induce changes in eutopic endometrial gene expression. The alteration of gene
expression is regulated through epigenetic transcriptional repression. In Taylors
subsequent study, cells from endometriotic lesions when induced in the mouse
model migrated to the eutopic endometrium [10]. Together with these observations,
experimentally induced ectopic endometrium may influence eutopic endometrium
through direct movement of cells and influence via epigenetic changes.
6 T. Harada
Experimental studies in autotransplant animal models used eutopic endometrium

in the non-menstrual phase. The transplanted ectopic endometrial tissues gained
distinct biological character compared with the eutopic endometrium [11, 12]. These
distinct characteristics are observed in human endometriotic tissues, suggesting that
biological character of eutopic endometrium can be altered in humans also.
Although conclusive data are still not available, we know much more about the
details of the nature of transplanted endometrium than our great senior doctors, like
Sampson. Endometriosis is still a mysterious disease. The accumulation of research
data slowly unveils its character and its application in endometriosis patient care is
urgently awaited.
References
1. Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial

tissue into the peritoneal cavity. Am J Obstet Gynecol. 1927;14:42269.
2. Peterson CM, Johnstone EB, Hammoud AO, Stanford JB, Varner MW, Kennedy A, Chen Z,
Sun L, Fujimoto VY, Hediger ML, Buck Louis BM. Risk factors associated with endometri-
osis: importance of study population for characterizing disease in the ENDO study. Am J
Obstet Gynecol. 2013;208:451.
3. Eskenazi B, Warner ML. Epidemiology of endometriosis. Obstet Gynecol Clin North
Am. 1997;24:23558.
4. Sasson IE, Taylor HS. Stem cells and the pathogenesis of endometriosis. Ann N Y Acad Sci.
2008;1127:10615.
5. Redwine DB. Was Sampson wrong? Fertil Steril. 2002;78:68693.
6. Harada T, Iwabe T, Terakawa N. Role of cytokines in endometriosis. Fertil Steril.
2001;76:110.
7. Iba Y, Harada T, Horie S, Deura I, Iwabe T, Terakawa N. Lipopolysaccharide-promoted
proliferation of endometriotic stromal cells via induction of tumor necrosis factor alpha and
interleukin-8 expression. Fertil Steril. 2004;82:103642.
8. Takenaka Y, Taniguchi F, Miyakoda H, Takai E, Terakawa N, Harada T. Lipopolysaccharide
promoted proliferation and invasion of endometriotic stromal cell via induction of
cyclooxygehase-2 expression. Fertil Steril. 2010;93:3257.
9. Lee B, Du H, Taylor HS. Experimental murine endometriosis induces DNA methylation and
altered gene expression in eutopic endometrium. Biol Reprod. 2009;80:7985.
10. Santamaria X, Massasa EE, Taylor HS. Migration of cells from experimental endometriosis to
the uterine endometrium. Endocrinology. 2012;153:556674.
11. Sharpe KL, Vernon MW. Polypeptides synthesized and released by rat ectopic uterine
implants differ from those of the uterus in culture. Biol Reprod. 1993;48:133440.
12. Machado DE, Berardo PT, Palmero CY, Nasciutti LE. Higher expression of vascular endo-
thelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) and metalloproteinase-9
(MMP-9) in a rat model of peritoneal endometriosis is similar to cancer disease. J Exp Clin
Cancer Res. 2010;29:4.
Part II
Basic Science
Chapter 2
Pathological Aspect and Pathogenesis
of Endometriosis
Ritsuo Honda and Hidetaka Katabuchi
Abstract Historically, endometriosis was first described in Egyptian scrolls in the

sixteenth century BC. The first scientific description of endometriosis was published
by Carl Freiherr von Rokitansky in 1860. At this time, the concept of endometriosis
as a disease entity was not established and it was assumed to be an enigmatic
disease with an unknown pathology. Although extensive basic and clinical research
has been carried from the last century until present, the pathogenesis of endome-
triosis is still controversial. Major theories on the pathogenesis of endometriosis are
(1) metastases of endometrial tissues to an ectopic location (transplantation theory),
(2) metaplastic development of endometrial tissue on the ectopic site (metaplastic
theory), and (3) changes of the embryonic duct remnant epithelium into endome-
trial epithelium (mullerian remnants theory). A comprehensive understanding of
the histopathogenesis of endometriosis is essential to the novel clinical approaches
for the enigmatic disease.
Keywords Endometriosis Pathogenesis Pathophysiology
2.1 Introduction
Endometriosis is a common gynecological disease of unknown etiology which

affects an estimated 1045 % in infertile females. Endometriosis is defined as the
presence of endometrial tissue, consisting of both glandular epithelium and stroma,
in ectopic locations. Clinically, it can be associated with many distressing and
debilitating symptoms, such as pelvic pain, severe dysmenorrhea, dyspareunia, and
infertility, or it may be asymptomatic and incidentally discovered at laparoscopic
surgery.
R. Honda, M.D., Ph.D. (*) H. Katabuchi

Department of Obstetrics and Gynecology, Faculty of life Sciences, Kumamoto University,
1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
e-mail: ritz@kuamoto-u.ac.jp

10 R. Honda and H. Katabuchi
In 1860, the first histological description of endometriosis was given by von

Rokitansky [1]. In 1896, Cullen suggested that adenomyoma, as he called it, resem-
bled the histological features of normal endometrial epithelium and stroma [2].
The term endometriosis was first described by Sampson in 1925 [3] and his
studies became a pioneering figure of research on the pathogenesis of endometriosis
[35]. Endometriosis is believed a benign disease, but it has been confirmed that it
may lead to recurrence and metastasis and be secondary ovarian cancer from
chocolate cysts. Because of the different locations of the disease, its pathogenesis
also has been attributed either to transplantation of viable endometrial cell fragments,
to coelomic metaplasia, or to embryonic remnants, and several contributing factors
are also involved, including retrograde menstruation, disturbances of hormonal
conditions, or familial and genetic factors.
2.2 Sites of Endometriosis
Endometriosis is defined as a pathological disease caused by the presence and

proliferation of ectopic endometrial tissue in sites other than the endometrial cavity.
Endometriosis often develops in the ovary, pouch of Douglas, and uterosacral
ligaments and sometimes in the fallopian tubes, uterine cervix, vagina, colon and
rectum, vermiform appendix, urinary bladder and tract, and others, that is, mostly in
the peritoneum of the pelvic cavity and associated deep tissue [6].
Endometriosis is also occasionally observed in remote organs such as lymph
nodes, pudendum, umbilicus, and lung [6]. Cases of endometriosis can occur in
tissues beyond the pelvis and have diverse symptoms that depend on the site of
development, including pain and/or bleeding, associated with the menstrual cycle.
Rarely, endometriosis has been reported in males. However, they were all older
men suffering from prostate cancer, who were treated with high doses of estrogens.
In these cases, a hypertrophic change of the mullerian remnants was observed in the
prostate [7].
2.3 The Theory of Secondary Mullerian System
The term secondary mullerian system was first described by Lauchlan in 1972 [8].
He noted the propensity of the peritoneum which covers the surface of the ovary
and the peritoneal cavity to mullerian differentiation. The surface mesothelial and
the submesothelial stroma of peritoneum exhibit a full spectrum of mullerian
differentiation from benign to malignant. Many lesions of secondary mullerian
system are described, which are benign, such as endosalpingiosis, of the low
grade of malignancy, serous and mucinous and malignant, described as
extraovarian peritoneal serous and mucinous carcinomas, and endometriosis is
considered as the main lesson of the secondary mullerian system [9]. It is proposed
2 Pathological Aspect and Pathogenesis of Endometriosis 11
that the development of peritoneal mullerian lesions may be secondary to the

proximity of pelvic peritoneum to tubal fimbria and the exposure of the peritoneal
surfaces to external agents, such as talc and asbestos, that stimulate the peritoneal
mullerian differentiation [10].
2.4 Microscopic Findings of Endometriosis
The focus of endometriosis is basically formed of three components: the endome-

trial glandular epithelium, an endometrium-specific stroma, and a stroma with
fibrosis that forms the lesion in regions of chronic inflammation associated with
endometriosis. Findings of fibrosis are associated with infiltration of inflammatory
cells such as macrophages, mast cells, monocytes, eosinophil granulocytes, and
basophil granulocytes; proliferation of fibroblasts; and smooth muscle metaplasia,
angiogenesis, and innervation. Similar to the original endometrium, the ectopic
glandular and stromal cells also express estrogen and progesterone receptors. In
response to the expression of the sex steroid receptors, stromal cells show a
decidual reaction in the pregnant women.
The cell morphology of glandular epithelium may change to that of the epithe-
lium of mullerian ducts; epithelial cells of the fallopian tubes [11], the glandular
epithelium of the endocervical canal (Fig. 2.1b), with apocrine-like cells (Fig. 2.1c)
and intestinal epithelium (Fig. 2.1d). These changes usually may occur in response
to inflammation and others, and it enables epithelial cells to change to their
surrounding circumstances to better adapt to their environment [12].
2.5 Theories on the Pathogenesis of Endometriosis
Because of the different locations, possible organs, appearances, and hormone

responsiveness, many theories on the pathogenesis of endometriosis have been
proposed over about one century. However, no single theory is sufficient to explain
the development of this enigmatic disease.
2.5.1 Transplantation
The theory of transplantation implies that the endometrium is replaced from the
uterus to another location inside the body. Many different ways of dissemination of
endometrial tissue are involved in this concept. Iatrogenic, lymphogenic, and
hematogenic spread account for uncommon, extraperitoneal lesion of endometri-
osis [13, 14]. The most easily understood, scientifically supported, and widely
accepted mechanism for the histogenesis of endometriosis is that, at menstruation,
some effluent flows retrograde through the lumen of fallopian tubes into the
Fig. 2.1 Metaplasia of epithelial cells involved in endometriosis. (a) Metaplasia of tubal epithe-
lial cells. (b) Metaplasia of endocervical canal glandular cells. (c) Apocrine metaplasia. (d)
Intestinal metaplasia. Hematoxylineosin stain, (ad) 200 (Reprinted from Okamura and
Katabuchi [12] with permission of Int Rev Cytol.)
peritoneal cavity (Fig. 2.2). Indeed, the high frequency of this phenomenon is
supported by the finding of menstrual blood in the peritoneal fluid of up to 90 %
of women with patent fallopian tubes undergoing laparoscopy during the
perimenstrual period [16]. Although retrograde menstruation explains the physical
displacement of endometrial fragments into the peritoneal cavity, additional steps
are necessary for the development of endometriotic implants. Escape from the
immune clearance system, during the courses of attachment to the ovarian surface
epithelium and peritoneal mesothelium, invasion of the epithelium, establishment
of local neurovascularity, and continued growth and survival are necessary if
endometriosis is to develop from retrograde passage of the endometrium.
2.5.2 Coelomic Metaplasia
The theory of coelomic metaplasia suggests that the mesothelium of the peritoneum
including ovarian surface epithelium (OSE) can be transformed into endometrium
by metaplasia. The mullerian ducts, which constitute the primordial uterus, are
generated through intrusion of the coelomic epithelium in the antenatal stage.
For this reason, it has been proposed secondary mullerian system; the organs derived
Fig. 2.2 Histogenesis of endometriosis. (a) Exfoliated endometrial tissue (left arrow) floating in
the tubal cavity. (b) Endometrial tissue (left arrow) adhering to the peritoneum. Hematoxylin
eosin stain, (a) 40, (b) 100 (Reprinted from Katabuchi [15] with permission of J Japan Societ
Endo.)
from mullerian duct are generated from the same origin as that of the peritoneal
mesothelium and OSE [8, 17]. The peritoneal mesothelium and OSE may undergo
metaplasia into the endometrial epithelium and stroma, thereby inducing endome-
triosis. We rarely come across the histological evidence for this metaplastic progres-
sion in the ovarian surface as shown in Fig. 2.3 [17]. Moreover, in the observation
of morphological changes in a collagen-embedded culture system of the human
OSE [18], the OSE and the coculture of OSE with endometrial stromal cells
(ES) showed a luminal structure with estradiol (E2) supplementary (Fig. 2.4ac),
and, in the OSE/ES coculture, the nuclear position was deviated toward the basal
side and the appearance of cilia was observed [19]. The OSE/ES coculture was
positive for epithelial membrane antigen (EMA) and cytokeratin, indicating differ-
entiation into glandular cells, while the epithelium of the OSE culture was negative
for EMA. These findings suggest that the OSE can differentiate into glandular cells
and that E2 and endometrial stromal cells are involved in this process. Thus, flow of
endometrial stromal cells in the menstrual blood back through the fallopian tube may
be an important factor in the development of endometriosis [1820].
2.5.3 M
ullerian Ducts Remnants
In the antenatal phase, the coelomic epithelium gives rise to the mullerian ducts,
which constitute the fallopian tubes and the uterus and the upper portion of the
vagina. During the course of differentiation and migration of the mullerian ducts
and fetal organogenesis, some primordial cells might spread in the posterior pelvic
floor. This might explain the findings that endometriosis is frequently found in the
pouch of Douglas, uterosacral ligaments, and rectovaginal septum and even the
presence of endometriosis among young women with MayerRokitanskyKuster
Hauser syndrome.
Fig. 2.3 Progression of a cyst enclosed in the ovary to endometriosis. Single layer of the surface
epithelium in the cortex of the ovary, which formed the cyst, moved to the glandular epithelium of
the endometrium (right arrow). Hematoxylin-eosin stain, 150 (Reprinted from Okamura and
Katabuchi [17] with permission of Ital J Anat Embryol.)
Fig. 2.4 Three-dimensional collagen gel-embedded cultures of the ovarian surface epithelium
(OSE). (a) In the OSE culture, a cavity formed after addition of estradiol 17 (10 ng/mL). (b, c) In
a coculture of OSE with the endometrial stoma, an epithelium-stoma structure formed, the surface
epithelium formed a cavity with a threedimensional structure, the position of the nucleus was
biased toward the basal side, and cilia appeared. Hematoxylineosin stain, (a) 200, (b) 400;
Uranyl acetate-lead citrate stain, (c) 17,000 (Reprinted from Ohtake et al. [19] with permission
of Fertil Steril.)
2.6 The Factors Affecting the Development

of Endometriosis
2.6.1 Disturbances of Hormonal Conditions
Hormonal alterations may influence the ability of endometrial cells to proliferate,

attach to the mesothelium, and/or evade immune-mediated clearance
[11]. Sex steroids including estradiol (E2) are found intracellularly in patients
with endometriosis. Aromatase is specifically expressed in endometrial tissue and
converts androstenedione to estrone (E1), which in turn is converted to E2 by
17-hydroxysteroid dehydrogenase (17-HSD) type 1. 17-HSD type 2, which
converts E2 to E1 for regulation of E2 activity, is found in the normal endometrium,
but seems not to be expressed in the tissue of patients with endometriosis, with a
consequent increase in the local concentration of E2 [21]. In addition to estrogen
dependence, there is increasing evidence to support a profile of P resistance in the
pathophysiology of endometriosis [22]. Endometriotic lesions exhibit an overall
reduction in P receptor expression relative to eutopic endometrium and absence of
P receptor-B [23]. Additionally, endometrial expression profiling has documented
dysregulation of P-responsive genes in the luteal phase [24]. An incomplete tran-
sition of endometrium from the proliferative to a secretory phase has significant
molecular implantation of refluxed endometrial cells.
2.6.2 Familial and Genetic Factors
The upregulation of the antiapoptotic gene BCL-2 in eutopic and ectopic endome-
trium from women with endometriosis is reported, and a genetic alteration of
endometrial cells influencing their tendency to implant may be hereditary [25].
Linkage analysis has elucidated candidate genes with biological plausibility. The
largest of these involved over 1,000 families more than two affected sib pairs and
established significance for a susceptibility locus in the regions of chromosome
10q26 and 7p15 [26, 27].
2.6.3 Inflammation
From the studies of macrophages in the female reproductive organ since the
1980s [28, 29], the conceptualization of endometriosis as a pelvic inflammatory
condition is established. In patients with endometriosis, the peritoneal fluid is
remarkable for an increased number of activated macrophages and important
differences in the cytokine/chemokine profile. Macrophages produce and secrete
diverse physiologically active substances including cytokines, coagulation factors,

fibrinolytic factors, components of complement, plasma proteins, lipids, and
enzymes. A proteomics approach identified a unique protein structurally similar
to haptoglobin in the peritoneal fluid in patients with endometriosis [30]. This
protein was subsequently found to bind to macrophages, reduce their phagocytic
activity, and increase their production of IL-6. Other cytokines or chemokines
found to be increased in the peritoneal fluid of patients with endometriosis include
macrophage migration inhibitory factor, TNF-, IL-1, IL-6, and IL-8, regulated on
activation normal T expressed and secreted, and monocyte.
2.7 Conclusions
The pathogenesis of endometriosis has been studied among major theories; how-
ever, no single theory is sufficient to explain the development of the disease. It has
been suggested that peritoneal endometriosis, chocolate cysts of the ovary, and
adenomyotic nodules of the rectovaginal septum or deeply infiltrating endometri-
osis are three different disease entities, each with a different pathogenesis. This
concept leads that diverse pathological conditions underlie endometriosis and leads
to the proposal of endometriosis as a series of syndromes that develop through
different mechanisms depending on the host tissue or organ. Recently, a combina-
tion of the metastatic and metaplastic theories has been favored to explain that
endometriosis represents a polygenetic disorder, with alterations in multiple bio-
logical pathways leading to a metaplastic process under the irritating effect of
endometrial tissue shed during retrograde menstrual flow. Thus, endometriosis
has a long medical and historical background and is appropriately referred to as
an enigmatic disease. Clinical treatment in line with individual pathology is
required because of the diverse symptoms and findings. Further assessment of the
pathology of endometriosis may open the way for development of new drugs for
this disease.
Co-workers in our Department Okamura H, Matsuura K, Ohba T, Tashiro H, Fukumatsu Y,

Nakamura M, Ohtake H, Motohara K, Miyamura S.
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14. Victory R, Diamond MP, Johns DA. Villars node: a case report and systematic review of
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A but not B is expressed in endometriosis. J Clin Endocrinol Metab. 2000;85:2897902.
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LC. Gene expression analysis of endometrium reveals progesterone resistance and candidate
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K. Activated macrophages in the peritoneal fluid of women with endometriosis: examination
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Chapter 3
Visible and Invisible (Occult) Endometriosis
Khaleque Newaz Khan
Abstract Endometriosis is a chronic disease characterized by endometrial tissue

located outside of the uterine cavity and is associated with chronic pelvic pain and
infertility. However, an in-depth understanding of the pathophysiology of endome-
triosis is still elusive. Once generated within pelvis due to retrograde entry of
menstrual debris, peritoneal endometriotic lesions time-dependently change their
color appearance resulting from certain biochemical change within lesions. A
variable pattern of endometriotic lesions within pelvis can be detected by laparos-
copy as visible peritoneal endometriosis. It is generally believed that besides
ovarian steroid hormones, the growth of endometriosis can be regulated by innate
immune system in pelvic microenvironment by their interaction with endometrial
cells and immune cells. We conducted a series of studies in perspectives of pelvic
inflammation that is triggered primarily by bacterial endotoxin (lipopolysaccharide,
LPS) and is mediated by Toll-like receptor 4 (TLR4) and showed their involvement
in the growth regulation of visible peritoneal endometriosis. Even with the careful
eye of an expert surgeon, we may sometimes miss to detect peritoneal lesion within
peritoneal cavity or deep into peritoneum. In such a case, random collection of
normal peritoneum may carry the possibility to identify some hidden endometriotic
lesions by microscopy and these lesions can be named as invisible endometriosis.
Here, we discuss the color appearance of peritoneal lesions, role of innate immune
system in visible endometriosis, and finally our recent findings on invisible micro-
scopic endometriosis and their biological and clinical significance.
Keywords Visible endometriosis Bacterial endotoxin Innate immunity TLR4

Invisible endometriosis
K.N. Khan, M.D., Ph.D. (*)

Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Nagasaki
University, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan
e-mail: nemokhan@nagasaki-u.ac.jp

20 K.N. Khan
3.1 Visible Endometriosis: Introduction
Endometriosis, the presence of functional endometrium outside of the uterine

cavity, is a common disease, causing abdominal pain, dysmenorrhea, dyspareunia,
and infertility in about 10 % of the female population [1]. Besides metaplastic
transformation of endometrial and peritoneal mesothelial cells, the transplantation,
implantation, and growth of exfoliated menstrual debris on the peritoneal and
ovarian surfaces are the widely accepted mechanisms of endometriosis [2, 3].
With the elapse of many decades and publication of abundant number of literatures,
exact physiopathology or pathogenesis of endometriosis is still debatable. The
potential role of ovarian steroid hormones in the regeneration of endometrium
after menstruation and the growth of endometriosis has been demonstrated [4].
However, as a nonself lesion in pelvic environment, the growth or persistence of
visible endometriosis can also be regulated by innate immune system. Therefore,
we cannot rule out the involvement of an immuno-endocrine crosstalk in the pelvis
of women with visible endometriosis.
3.1.1 Pathogenesis and Natural Course

of Peritoneal Endometriosis
3.1.1.1 Common Concepts in the Pathogenesis of Endometriosis
In addition to transplantation and implantation theory of Sampson [2] and coelomic

metaplasia theory of Meyer [5], immuno-surveillance of the refluxed endometrial
cells is another attractive theory for the development of pelvic endometriosis. The
immune tolerance or immune defect theory could be responsible for a deficiency in
the rejection of the autologous cells derived from the eutopic endometrium in the
peritoneal cavity after menstrual reflux. This rejection in the clearance of endome-
trial cells could be contributed by a dysfunctional immune response in the pelvic
cavity [6].
According to the retrograde menstruation theory, endometrial fragments flow back
through the patent fallopian tubes, reach the peritoneal cavity, attach on the pelvic
mesothelium, invade the peritoneum, and develop into endometriotic lesions [2].
Limited information still exists regarding early endometrial-peritoneal attachment
and invasion in the development of endometriosis.
3.1.1.2 Role of Cell Adhesion Molecules in Endometriosis
After overcoming a phase of immune tolerance, a key step in the development of

early endometriosis is the ability of endometrial cells to adhere to mesothelium and
invade the extracellular matrix. These effects are contributed by a number of
3 Visible and Invisible (Occult) Endometriosis 21
intercellular adhesion molecules (ICAMs) and subcellular matrix degrading

metalloproteinases (MMPs) [7]. The expressions of these ICAMs such as integrins
and E-cadherin and MMPs are already detected in cells derived from menstrual
effluent, endometrium, peritoneal fluid, peritoneum, and endometriosis [8].
3.1.1.3 Role of Heme Metabolism in Endometriosis
A potential implication of hemoglobin in the pathogenesis of peritoneal endome-

triosis has been recently reported [9]. A simple hypothesis is that hemoglobin, being
released into the peritoneal cavity after red blood cell lysis, may activate cell
adhesion molecules and induce cytokine production, cell proliferation, and the
process of neovascularization. Degradation of hemoglobin yields biologically
active molecules, heme, and its products of oxidative cleavage by heme oxygenases
such as iron, carbon monoxide, biliverdin, and bilirubin. Accumulation of heme in
the peritoneal cavity might have a number of deleterious effects including induction
of oxidative stress, stimulation of cell adhesion, and cytokine production by
macrophages (M). All these biological events are finally involved in the genera-
tion of visible peritoneal lesions.
3.1.1.4 Color Changes of Visible Peritoneal Lesions
The lesions of early endometriosis are either transparent or translucent because they
still lack formation of vasculatures around them. We named these early lesions as
nonopaque lesions [10] because these lesions contain either of watery, serous, or
mucinous secretion and there is no collection of blood in the stroma by histology.
Once cellular attachment and invasion of endometrial cells are established, the
subsequent growth or maintenance of endometriotic lesions is maintained by
promotion of mitogenesis and angiogenesis with the continuation of menstrual
cycle. The growth-promoting effect of endometriosis is contributed by an orches-
trated action of estrogen and other inflammatory or proinflammatory mediators.
Over proliferation of microvessels in the growing endometriotic lesions causes
oozing of blood in the stroma and appears as blood-filled opaque red lesions by
laparoscope [10]. With the progression of time, there is deoxygenation process from
hemoglobin to methemoglobin or hemosiderin leading to color changes of these
opaque red lesions to black lesion or related lesions. In this stage, collection of
blood in the stroma disappears. Black lesion again changes to white lesion due to
collection of bilirubin or biliverdin and accumulation of fibrous tissue. In this stage,
gland gradually becomes smaller and stroma sometimes disappears due to deposi-
tion of fibrous tissue. Finally old lesions disappear and there is new focus of
endometriosis due to continuation of menstrual reflux. These sequential events
indicate that once exfoliated, the endometrium enters into the pelvic cavity and
becomes attached to the mesothelial layer, and then a process of angiogenesis,
22 K.N. Khan
Fig. 3.1 Shows the diagrammatic representation of the natural course of visible peritoneal
endometriosis in pelvic cavity. After initial attachment of refluxed endometrial cells with perito-
neal cells producing early endometriotic lesions, the consequent events of mitosis, angiogenesis,
metabolic degradation of heme, and appearances of fibrosis result in the generation of different
morphological appearances of peritoneal endometriosis as shown in this figure
heme metabolism, and fibrosis ensue to maintain the natural course of endometri-
osis (Fig. 3.1). A panel of nonopaque lesion, blood-filled opaque lesion, blue berry
spots, and their corresponding histological pictures is shown in Fig. 3.2.
3.1.2 Role of M in Pelvic Inflammation and Growth

of Visible Endometriosis
3.1.2.1 Infiltration of M in Eutopic and Ectopic Endometria
As a cell component of innate immune system, peritoneal fluid (PF) and eutopic/
ectopic endometria derived from women with endometriosis have been shown to
contain higher numbers of activated M than in control women [11, 12]. This
results in the secretion of higher concentrations of growth factors and cytokines in
the PF as produced by the stimulated M in these patients [11]. Red peritoneal
lesions and their adjacent peritoneum had the greatest M infiltration, compared
with black/white lesions or chocolate cyst walls. These results indicate that early
endometriosis with red peritoneal lesions induces a higher inflammatory response
Fig. 3.2 Shows the laparoscopic and corresponding histologic appearance of nonopaque trans-
parent/translucent lesion (A, B), blood-filled opaque red lesion (C, D), and blue berry spots (E, F)
in pelvic cavity of women with peritoneal endometriosis. It can be noted here that nonopaque
endometriotic lesions such as vesicular bleb (A) lack oozing of blood in stroma (B) and opaque red
lesions such as blood bleb with ecchymosis (C) are accompanied by oozing of blood in the stroma
of these lesions (D). In contrast, black lesions such as blue berry spots (E) are manifested by color
change and disappearance of blood from the stroma of these lesions (F)
in the pelvic cavity than advanced endometriosis [12]. The inflammatory reactions
in the eutopic and ectopic endometria suggest that the growth of endometriosis does
not depend on the fibrotic extension of disease; rather, it depends on the tissue
activity of endometriosis. We presume that extension of disease could be related to
pelvic pain, but higher tissue activity of endometriosis associated with abundant
recruitment and infiltration of M could be related to infertility.
3.1.2.2 Role of Endotoxin or Lipopolysaccharide (LPS)

in M-Mediated Pelvic Inflammation
We examined the ability of peritoneal M to synthesize different macromolecules

in basal conditions and after treatment with lipopolysaccharide (LPS), a bacterial
endotoxin derived from the cell wall extract of Gram-negative bacteria. We spec-
ulated that LPS could be a primary inflammatory mediator of M stimulation in
pelvic microenvironment. We found that activated M synthesize and secrete
variable amount of different secondary inflammatory mediators such as IL-1,
IL-6, IL-10, TNF-, and other growth factors in response to LPS [13, 14].
24 K.N. Khan
3.1.2.3 TLR4-Mediated Cytokine Production and Growth

of Endometrial Cells
Bacterial endotoxin (LPS) is recognized by Toll-like receptor 4 (TLR4), a pattern

recognition receptor, and transmits NF-B-mediated cellular signals in association
with other accessory molecules [15]. We confirmed gene and protein expression of
TLR4 in M, gland cells, and stromal cells derived from the eutopic and ectopic
endometria of women with or without endometriosis [16]. In an attempt to examine
that the stimulating effect of LPS in the production of cytokines and growth factors
is mediated by TLR4, we pretreated peritoneal M and glandular epithelial cells/
stromal cells derived from eutopic/ectopic endometria with antibody against TLR4
and then again treated them with LPS. We found that blocking of TLR4 was able to
significantly suppress M-mediated production of HGF/VEGF/IL-6/TNF- as well
as growth of endometriotic cells [16]. These results indicate that LPS-mediated
inflammatory reaction and growth of endometriotic cells are mediated by TLR4.
3.1.3 Source of Bacterial Endotoxin in Endometriosis
3.1.3.1 Endotoxin Levels in Body Fluids
We measured endotoxin levels in the menstrual fluid (MF) and PF of women with
and without endometriosis. We found that the concentration of bacterial endotoxin
is two- to fourfold higher in the MF when compared with that in PF. Endotoxin
level in MF/PF was also significantly higher in women with endometriosis than in
control women [16]. We found the highest endotoxin level during the menstrual
phase and persistence of a small amount of endotoxin in the pelvis either in the
proliferative phase or in the secretory phase of the menstrual cycle [16]. This
indicates that MF of women with endometriosis is highly enriched with bacterial
endotoxin followed by the presence of a modest amount in the PF.
3.1.3.2 Source of Endotoxin in Intrauterine and Pelvic Environment
There is a possibility that the lower genital tract of women with or without
endometriosis is contaminated with a number of normal bacterial florae including
Escherichia coli (E. coli). Therefore, we speculated that there might be an ascend-
ing migration of E. coli from the vaginal lumen up into the uterine cavity that causes
contamination of menstrual blood and resulting in the subsequent release of endo-
toxin into menstrual blood and back to the peritoneal fluid. After bacteria culture
analysis, we found a significantly higher colony formation (CFU/ml) of E. coli in
the menstrual blood of women with endometriosis than that in control women [16].
The CFU of E. coli was also significantly higher in women containing red perito-
neal lesions than in women having only chocolate cyst.
Based on these findings of our serial experiments, we proposed a new bacterial
contamination hypothesis that may be involved in the growth regulation of visible
peritoneal endometriosis via LPS/TLR4 cascade [16]. This E. coli contamination of
menstrual blood is responsible for higher endotoxin levels in the MF and PF of
women with endometriosis. In search of a mechanistic basis of bacterial contam-
ination of menstrual blood, we found that higher concentrations of PGE2 in MF and
PF of women with endometriosis were involved in E. coli growth by its direct
bacterial proliferation effect or indirect immunosuppressive effect [17].
3.1.4 Inflammation, Stress Reaction, and TLR4

in Endometriosis
In addition to pelvic inflammation, a wide variety of stressful stimuli, such as heat

shock, ultraviolet radiation, viral or bacterial infection, internal physical stress (cell
growth, differentiation, invasion), chemical stress (ligand/receptor interaction),
oxidative stress, neurogenic stress, pain sensation, and pelvic inflammation, may
induce a variable degree tissue stress reaction in pelvis and release stress-induced
proteins, such as heat shock proteins (HSPs) [17, 18]. As a danger signal, the effect
of HSPs has been reported to be mediated by TLR4 either alone or in combination
with LPS.
We demonstrated a variable amount of soluble HSP70 in MF, PF, and in
different peritoneal lesions [18, 19]. These body fluid and tissue concentrations of
HSP70 were significantly higher in women with endometriosis than in control
women. In an in vitro cell culture system, we found that HSP70 was able to induce
TLR4-mediated proinflammatory response and growth of endometriotic cells, and a
combined effect was observed between HSP70 and LPS in further promoting pelvic
inflammation and growth of endometriotic cells [19]. Although polymyxin B, a
potent LPS antagonist, or anti-HSP70 antibody, was unable to suppress combined
LPS+HSP70-mediated growth of endometriosis, blocking of TLR4 alone signifi-
cantly suppressed LPS+HSP70-mediated inflammation and growth of endometri-
osis [19]. Recently it has been demonstrated that in addition to the effects of
endogenous danger signals via TLRs, tissue oxidative stress itself may promote
NF-B-mediated or TLR4-mediated growth of endometriosis [20]. In fact, LPS
itself has the capacity to produce ROS by M [15]. These findings are consistent
with the understanding that LPS, endogenous danger signals, and oxidative stress
may promote the onset and progression of visible endometriosis after activation of
TLR4 and/or NF-B signaling.
26 K.N. Khan
3.1.5 Inflammation, Ovarian Steroid, and TLR4

in Endometriosis
Basically endometriosis is an estrogen-dependent disease and induces an inflam-

matory reaction in pelvic environment. Irrespective of the phases of the menstrual
cycle, a small amount of estrogen is available in the PF of women with and without
endometriosis [21]. Therefore, it is important to know the combined effect of
estrogen and inflammation in the growth of endometriosis. Based on published
reports, gland cells/stromal cells of eutopic/ectopic endometria and M retain ER
and PR [18, 21]. Recently, we reported that M-mediated production of
HGF/VEGF/IL-6/TNF- in response to ovarian steroid was further enhanced after
treatment with LPS [22]. An additive effect was observed between E2 and LPS on
cytokine production and growth of eutopic/ectopic endometrial stromal cells when
compared with their individual treatment. This combined effect of E2+LPS on
pelvic inflammation and cell growth was markedly abrogated after pretreatment
of cells with anti-TLR4 antibody and ICI, an ER antagonist [21]. Our findings
suggest that a TLR4/ER-mediated immuno-endocrine crosstalk in pelvis may be
involved in the growth or progression of endometriosis.
3.2 Invisible (Occult) Endometriosis: Introduction
The detection and visible diagnosis of peritoneal endometriosis is usually performed

by laparoscopy, a gold standard modality, and is microscopically confirmed by
histopathology. Even with the careful eyes of expert surgeons, there is obvious
chance to miss or overlook hidden lesions in visually normal peritoneum. Therefore,
immense interest could arise to randomly collect visually normal peritoneum
from different anatomical location in pelvis and to investigate the nature of these
visually undetectable lesions of endometriosis. The concept of microscopic endo-
metriosis in visually normal peritoneum was first reported by Murphy in 1986 [23]
and subsequently confirmed with an incidence rate of 613 % [24].
We histologically examined all biopsy specimens derived from visually normal
peritoneum of women with and without endometriosis to detect the possible
occurrence of invisible (occult) microscopic endometriosis (IME). The designation
of visually normal peritoneum was based on modified criteria of Redwine [25].
The question still remains, if hidden endometriosis lesions could be detected in
normal peritoneum, are these invisible lesions really inactive as proposed in a
previous report [26], or do they truly retain some biological activity? If tissue
activity of IME is there, this could be a clinically important issue. To address this
question, we recently investigated the expression patterns of some tissue activity
markers including ovarian steroid receptors and cell proliferation marker in histo-
logically confirmed IME lesions.
Fig. 3.3 Microscopically detected three patterns of invisible (occult) endometriosis in visually
normal peritoneum. Pattern I shows presence of typical gland/stroma; pattern II shows reactive
hyperplastic change of endometrioid epithelial cells with surrounding stroma; and pattern III
shows single-layered epithelium-lined cystic lesions with surrounding stromal cells (all in HE
stain, upper column). The identification of glandular epithelial cells, stromal cells, and peritoneal
mesothelial cells was confirmed by the immunoreaction to Ber-EP4, CD10, and calretinin,
respectively, and are shown against each HE-stained slide. Flat mesothelial cells derived from
normal peritoneum and mesothelioma cells as a positive control (inset) immunoreactive to
calretinin are shown at the right panel. The immunoreactions to nonimmune mouse IgG as a
negative control are shown on the extreme right panel. HE stain, hematoxylin and eosin stain.
Magnification of slides (200)
3.2.1 Pattern of IME in Normal Peritoneum
After careful observation and analysis, we collected 227 visually normal peritoneal
samples from 151 women with visible endometriosis and 78 samples from
62 women without any visible peritoneal lesions (control). We detected three
patterns of IME: (I) presence of typical gland/stroma, (II) reactive hyperplastic
change of endometrioid-like epithelium with surrounding stroma, and (III) single-
layered mesothelium- or epithelium-lined cystic lesions with surrounding rim of
stromal cells. All these IME lesions were confirmed by their immunoreactivity to
Ber-EP4 (marker of gland epithelium), CD10 (marker of stroma), and nonreactivity
to calretinin (marker of mesothelial cells) (Fig. 3.3).
We could detect variable patterns of IME in the normal peritoneum derived from
23 women with endometriosis (biopsy samples, n 27) and 4 control women
(biopsy samples, n 4) without visible endometriosis. The detection rate of IME
was as follows: for endometriosis, 15.2 % (23/151) and 11.8 % (27/227) and for
28 K.N. Khan
control women, 6.4 % (4/62) and 5.1 % (4/78) by the number of patients and
number of collected samples, respectively. A higher tendency in the incidence of
IME was found in women with visible endometriosis than in control women
( p 0.06 by patient number and p 0.07 by sample number) [27].
A predominance of IME occurrence was observed in pouch of Douglas and
uterovesical space than in other anatomical sites in pelvis. A dominant presence of
r-ASRM stages III endometriosis and red/black lesions and complaints of dys-
menorrhea were observed in women with visible endometriosis harboring IME in
their peritoneum [27].
3.2.2 ER/PR/Ki-67 Expression in IME Lesions
A variable ER and PR immunoexpressions were observed in all IME lesions

detected in women with visible endometriosis and control women. The
immunoreaction of PR as measured by quantitative-histogram (Q-H) score
appeared to be higher in all patterns of IME lesions comparing to ER expression.
A stronger immunoreaction of Ki-67 (cell proliferation marker) was found in
pattern I/II IME lesions than in pattern III IME lesions detected in women with
visible endometriosis. A weak Ki-67 expression was found in pattern I/III IME
lesions diagnosed in control women. Ki-67 index (mean percentage of Ki-67-
positive nuclei among total cells) was significantly higher in pattern I/II IME
lesions than in pattern III IME lesions found in women with visible
endometriosis [27].
We are against the argument by Donnez et al. [26] that IME lesions are quiescent
and they are nonactive or inactive and that these lesions are clinically irrelevant.
Our findings indicate that a proportion of occult lesions are indeed biologically
active and a substantial amount of estrogen and different inflammatory mediators in
pelvis might be involved in the growth promotion of IME lesions even if it is
undetectable by naked eyes. With the influence of both systemic and local estrogen,
these IME lesions, even if it is minute in size, may time-dependently increase in
size to be recognized by histology. This could be responsible for the subsequent
recurrence/occurrence of endometriotic lesions or persistence/recurrence of pain
symptoms even after successful excision or ablation of visible peritoneal lesions by
laparoscopy. Further studies in human or in primates are necessary to establish this
possibility. From the logical point of view, it is difficult to trace these growing
lesions on the peritoneal surface by repeated surgical procedures in human.
3.2.3 Origin of IME Lesions
The most alarming questions may arise now, how can we decide the origin of IME
lesions? Or is Sampsons theory enough to explain IME lesions? There is no
Fig. 3.4 Some hypothetical proposals that may be linked to the possible origin of invisible
(occult) microscopic endometriosis (IME) lesions. After initial attachment of menstrual debris
to the peritoneum, a sequence of epithelial-mesenchymal transition (EMT) or mesenchymal-
epithelial transition (MET) may be involved in the generation of IME. Hematogenous or
lymphogenous dissemination of shedding endometrial cells during menstruation could be another
mechanism. In addition to possible origin from progenitor cells/stem cells, cellular change of flat
mesothelium to cuboidal or columnar cells (metaplasia theory) or activated cells derived from
residing coelomic epithelium within peritoneum (mulleriosis, induction theory) in response to
pelvic inflammation could be linked to the origin of IME. Cells or tissues derived from mullerian
rests in association with peritoneal pockets can be another explanation for the development of IME
definite answer at this moment. But we argue that we can link each and every
theory supporting the origin of visible endometriosis [28] to the pathogenesis of
IME lesions. If Sampsons theory does not directly support the origin of IME
lesions, it can be indirectly explained by lymphatic or hematogenous spread of
menstrual debris and subsequent localization deep into peritoneum. We cannot
exclude the possibility of genetic factor or metaplastic transformation of peritoneal
mesothelial cells (metaplasia theory) in response to estrogen, inflammation, or
environmental factors. Despite possible origin of IME as a result of epithelial-
mesenchymal transition/mesenchymal-epithelial transition or from stem cells
[29, 30], activation of cells derived from coelomic epithelium (mulleriosis, induc-
tion theory) within peritoneum may be another possible mechanism to explain the
origin of IME [31]. Some hypothetical proposals that might be linked to the
possible origin of IME lesions are shown in Fig. 3.4.
3.3 Summary and Perspective
We proposed for the first time a new concept bacterial contamination hypothesis in
endometriosis. Our results suggest that a substantial amount of endotoxin in perito-
neal fluid due to reflux of menstrual blood is involved in pelvic inflammation and
30 K.N. Khan
may promote TLR4-mediated growth of endometriosis. When there is persistence

or recurrence of pain even after successful surgical ablation of visible endometriosis
in pelvis, we should keep in mind about its possible link with invisible or occult
endometriosis, although emerging evidence supporting this phenomenon is still
unknown. In order to avoid confusion in the use of the term invisible or non-
visible microscopic endometriosis among laparoscopists, we can also use the term
occult microscopic endometriosis (OME) instead of IME to indicate any hidden
lesion in visually normal peritoneum (27). Since the growth regulation of endome-
triosis is difficult to explain uniformly by a single factor, we believe that a mutual
orchestration among inflammatory mediator, tissue stress reaction, and estrogen may
be involved in the growth, maintenance, or progression of endometriosis once it is
generated in the female pelvis. Our ongoing study targeting to find the evidence of
a subclinical vaginal infection in women with endometriosis may hold new thera-
peutic potential in addition to conventional estrogen suppressing agent.
Acknowledgement I gratefully thank Dr. Michio Kitajima of Nagasaki University Hospital and
Dr. Akira Fujishita of Saiseikai Nagasaki Hospital for their kind assistance in sample collection;
Prof. Masahiro Nakashima of Atomic Bomb Disease Institute, Nagasaki for his experimental
advice; and Prof. Hideaki Masuzaki of Nagasaki University Hospital for reading/advice.
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Chapter 4
Role of Stem Cells in the Pathogenesis
of Endometriosis
Tetsuo Maruyama
Abstract Endometriosis can be defined as a benign estrogen-dependent disorder in

which endometrium-like tissues reside outside of the uterine cavity. Studies now
indicate that multiple genetic and epigenetic changes (reminiscent of neoplastic
processes) are involved in the pathophysiology of endometriosis. Given the molec-
ular similarities between endometriosis and cancer, it is reasonable to apply the
cancer stem cell model concept to the pathogenesis of endometriosis. In this
article, I review and discuss the stem cell model for endometriosis in which
endometriosis originates from endometrial stem/progenitor cells within eutopic and
ectopic sites.
Keywords Cancer Endometriosis Endometrium Epithelialmesenchymal

transition Stem cells
4.1 Introduction
Endometriosis can be defined as the presence of endometrium-like tissues outside

of the uterine cavity and is frequently associated with dysmenorrhea and
dyspareunia [1, 2]. Endometriosis is commonly found associated with the ovaries,
pelvic peritoneum, uterine ligaments, and the rectovaginal septum. More rarely,
endometriotic sites can include pelvic lymph nodes, the cervix, the intestine, the
bladder, and the vagina, and fallopian tubes. More distant sites can include the
lungs, skin, kidneys, brain, and spinal column [13]. Microscopically, glandular
components surrounded by endometrium-like stroma are observed [4]. Smooth
muscle cell components (perhaps via smooth muscle metaplasia) can be associated
T. Maruyama (*)
Department of Obstetrics and Gynecology, School of Medicine, Keio University,
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
e-mail: tetsuo@a5.keio.jp

34 T. Maruyama
with endometriotic lesions, particularly deeply infiltrating endometriosis [5]. The

lesions are often accompanied by angiogenesis and innervation [6, 7]. A number of
causes of endometriosis have been proposed. These include retrograde menstrua-
tion, iatrogenic direct implantation, coelomic metaplasia, lymphatic and vascular
metastasis, embryonic rest, and mesenchymal cell differentiation (induction).
Importantly, none of the theories can completely account for all types of
endometriotic lesions. Thus, the pathophysiology might be complex, involving
several mechanisms.
Current analyses of endometriosis indicate that multiple genetic, epigenetic,
environmental, immunological, and/or endocrine processes are involved [8]. Thus
far, research has failed to identify one or more specific susceptibility genes.
Endometriosis is in fact a benign disorder. However, the underlying molecular
mechanisms appear similar to those of cancer [8]. Indeed, evidence indicates that
both endometriosis and tumors are monoclonal in origin [912]. Furthermore, both
endometriotic cells and cancer cells are invasive [13]. To support this interpreta-
tion, we point out the enhanced susceptibility to ovarian clear-cell and
endometrioid cancers in patients with endometriosis [8]. Given this background,
it is reasonable to examine whether the pathophysiology of endometriosis can be
explained, at least in part, by the cancer stem cell (CSC) hypothesis. CSCs have the
abilities to self-renew and to give rise to differentiated tumor cells. They are also
responsible for the overall organization of tumors [14, 15]. Here, I review and
discuss an emerging concept and hypothesis, i.e., the endometriosis stem cell
theory in which endometriosis arises from stem cell(s) originating from bone
marrows or eutopic and/or ectopic endometrial stem/progenitor cells [1618].
4.2 Cancer Stem Cell Theory
Before discussing the stem cell theory for the pathogenesis of endometriosis, I will
briefly introduce the current paradigm regarding tissue stem cells (adult stem cells),
CSCs, and their roles in cancer formation and metastasis.
4.2.1 Tissue Stem Cells
Tissue-specific stem cells (also termed somatic stem cells or adult stem cells) are
found in a quiescent, undifferentiated state throughout the body [19]. They self-
renew through symmetric and/or asymmetric cell divisions. Growth is modulated
by physiological signals originating from the microenvironment or stem cell
niche. Asymmetric divisions generate lineage-committed cells that differentiate
and thereby maintain the tissue of origin. The tissue-specific stem cells and the
4 Role of Stem Cells in the Pathogenesis of Endometriosis 35
Stem cell
Undifferentiated
Self-renewal
Quiescent
Multidifferentiation Progenitor cell
Self-tissue reconstitution
Transit amplifying (TA)

cells
Terminally differentiated
cells
Fig. 4.1 Hierarchy of adult stem cell differentiation. Within its microenvironmental niche, adult
stem cells generally remain quiescent. Under the proper conditions, the cells can be stimulated to
undergo asymmetric divisions to renew themselves and produce daughter/progenitor cells. The
latter cell further divides to produce transit-amplifying (TA) cells. The TA cells promptly
proliferate and differentiate into a variety of mature, functional cells
microenvironmental niche work together to keep a balance between the need for
cell replacement and the necessity of retaining a pool of primitive cells. In this
fashion, the structural and functional requirements of organs and tissues can be met
[19]. The hierarchy of tissue stem cells is illustrated in Fig. 4.1.
4.2.2 CSCs and Primary Tumor Formation
The classical or stochastic model (clonal evolution model) posits that any normal
(stem) cell, upon acquiring genetic and/or epigenetic modification(s) giving it
selective growth advantage, gives rise to a neoplastic clone of homogenous neo-
plastic cells [14, 15]. Upon the acquisition of additional genetic and/or epigenetic
change(s), it expands as a tumor. In this model, all cells within a tumor have equal
tumorigenic potentials [14, 15].
More recently, a hierarchical or CSC model has been developed. This model is
based upon the hypothesis that tumors consist of a heterogeneous population of
cells, only a small proportion of which are CSCs, also termed tumor-initiating cells
[14, 15, 20]. As depicted in Fig. 4.2, a small, self-renewing population of CSCs is
responsible for tumor initiation and growth maintenance. Thus, CSCs have been
operationally defined by their ability to generate tumors, and tumor-initiating cells
are thought to reflect the operational definition of CSCs [20]. The model states that
CSCs could originate from tissue stem cells or even differentiated cells that
acquired stem cell-like properties (including self-renewal) as a result of genetic
and/or epigenetic modifications [14, 15]. In the CSC model, completely mature
cells can fully dedifferentiate to become CSCs. In addition to genetic/epigenetic
36 T. Maruyama
Primary tumor formation
Differentiation
Angiogenesis
Colonization
Primary tumor
EMT
Migration/Invasion
Intravasation
Systemic dissemination
Secondary tumor Extravasation
formation
Metastasis
De-differentiation
Dormancy
Colonization
Angiogenesis
MET
Normal stem cell Normal non-stem cell Cancer stem cell (CSC)
Non-CSC progeny Genetic and epigenetic Niche

modifications
Fig. 4.2 Cancer stem cell (CSC) model for tumor formation and metastasis. Within tumors, a
small population of self-renewing CSCs is responsible for initiating tumor growth and maintaining
its presence. CSCs might be derived from tissue stem cells or more differentiated cells that gained
stem cell properties (including self-renewal) upon genetic and/or epigenetic modifications. Even
completely differentiated cells can develop into CSCs through dedifferentiation. The tumor
microenvironment (niche) maintains stem cell properties. CSCs that migrate away from the
primary site are capable of invasion, intravasation, systemic dissemination, and extravasation.
This behavior might be due to the EMT. Upon reaching distant tissues, the cells can reverse the
epithelial state by undergoing the MET. This promotes growth and the initiation of angiogenesis.
Alternatively, EMT programs per se might lead to the generation of CSCs
modifications, the tumor microenvironment (the CSC niche) is required for the
maintenance of stem cell properties. It likely includes many components, such as
stromal cells, blood vessels, extracellular matrix (ECM), growth factors, and
cytokines in a hypoxic environment. Exposure of non-CSCs to niche factors
might result in their acquisition of stem cell properties [14, 15].
4.2.3 Cell Metastasis and Tumor Formation
The metastatic cascade includes tumor cell migration away from the site of the
primary tumor. It is now believed by many researchers that this process requires the
epithelialmesenchymal transition (EMT) [20, 21]. The EMT is a biological process
in which epithelial cells lose their cell polarity and cellcell adhesive properties. In
exchange, the cells gain migratory and invasive properties and undergo multiple
biochemical changes, thereby exhibiting a mesenchymal cell phenotype [22].
The EMT is observed in many processes, including mesoderm and neural tube
formation as well as wound healing, tissue regeneration, and organ fibrosis [22].
Our own data indicated that endometrial epithelial cells undergo EMT during
embryo implantation [23].
CSCs are likely involved in the formation of metastases [20, 21], as they exhibit
invasion, intravasation, systemic dissemination, and extravasation. It is believed that
such processes are enhanced by the EMT conversion [20, 21]. Subsequently, CSCs
that metastasize to distant tissues show colonization and reversion to an epithelial
state through reversal of the EMT, i.e., the mesenchymalepithelial transition
(MET), giving rise to metastatic lesions accompanied by angiogenesis [20].
Alternatively, the activation of EMT programs has been implicated in the generation
of CSCs [20, 21]. Furthermore, in addition to CSCs, non-CSC progeny cancer cells
might become CSCs through dedifferentiation at the metastatic sites and thereby
generate metastatic cancer lesions [20, 21] (Fig. 4.3).
For reference, Fig. 4.2 provides a representative CSC model. However, recent
studies have revealed complexities, such as plasticity of stem cell properties and
clonal diversity of CSCs in certain tumor types requiring revision of the original
CSC model. As a result, more complex models such as the dynamic stemness model
and the combinatory CSC and stochastic models have recently emerged [15].
4.3 Endometriosis Stem Cell Theory
Based on the principles of the cancer stem cell model illustrated in Fig. 4.2, I here
propose a stem cell model for the pathogenesis of endometriosis as depicted in
Fig. 4.3. This model incorporates the implantation theory, which is the most widely
accepted version among the many theories in this field. Note, however, that the
coelomic metaplasia theory and embryonic rest theory are also compatible with the
second part of this model in which some types of mesothelial cells and/or embry-
onic cell rests of mullerian origin behave as endometriosis-initiating cells
(endometriotic stem cells) and thereby give rise to endometriotic lesions at ectopic
site(s) [18].
38 T. Maruyama
Eutopic endometrium
Menstruation
EMT
Transtubal dissemination
De-differentiation
Hematogenous and lymphatic
dissemination
Intravasation
Extravasation
I. II. III.
Ectopic implantation
Attachment
Migration/Invasion
MET/Differentiation
Colonization
Angiogenesis
Establishment of
endometriotic lesions
Niche, esp. at ectopic site(s) Progeny of endometrial stem cells

Endometrial stem cells
differentiated endometrial cells
Endometriosis-initiating cells Progeny of endometriosis-initiating

endometriotic stem cells Genetic and epigenetic modifications
cells (endometriotic stem cells)
Fig. 4.3 Three proposed stem cell models for the pathogenesis of endometriosis extrapolated
from the CSC model. Model I. Endometrial stem cells (EmSCs) are released from eutopic
endometrium most likely upon menstruation. EmSCs are transported to ectopic sites via retrograde
menstruation, lymphatic and vascular dissemination, direct migration, and invasion or some
combination. The EmSCs remain at the ectopic site through attachment, implantation, and/or
extravasation followed by invasion. EmSCs initiate self-renewal and/or asymmetric division to
generate progenitor cells, transit-amplifying cells, and more differentiated endometrial cells.
Because EmSCs and their progeny require a blood supply, angiogenesis is initiated, supporting
the expanding colony/endometriotic lesions. Model II. EmSCs undergo genetic and/or epigenetic
modifications at the eutopic or ectopic sites and thereafter behave as endometriosis-initiating cells
(EmoICs), giving rise to endometriotic lesions as shown in model I. Model III. EmSCs undergo
more extensive genetic and/or epigenetic modifications than in model II. Alternatively, terminally
differentiated endometrial cells undergo multiple and profound genetic and epigenetic changes
and thereby dedifferentiate into EmSC-like cells (not depicted in the schema). In either case, the
EmoICs are capable of generating a variety of endometrial cell components in a fashion similar to
model I
4.3.1 A Stem Cell Model for the Development

of Endometriosis
A stem cell model for endometriosis development is presented below in three

variations, i.e., models I, II, and III (Fig. 4.3). In model I, endometrial stem cells
are released from eutopic endometrium upon menstruation. The cells could move to
ectopic sites via many routes, including lymphatic and vascular dissemination,
direct migration and invasion, retrograde menstruation, or some combination
thereof. The endometrial stem cells settle ectopically in new sites by attachment,
implantation, extravasation, and, finally, invasion. Upon reaching the ectopic site,
the endometrial stem cells might undergo self-renewal or divide asymmetrically. In
this way, they can self-renew as well as produce progenitor cells, transit-amplifying
cells, and more differentiated endometrial cells (Fig. 4.3, model I). This process is
based on the behavior of general tissue stem cells (Fig. 4.1). Endometrial stem cells
and their proliferating daughter cells require a blood supply to flourish. Therefore,
they induce angiogenesis, permitting them to undergo clonal growth. In this
fashion, endometriotic lesions are formed.
In model II, endometrial stem cells are subject to genetic (or epigenetic) mod-
ifications within the eutopic or ectopic sites. The modifications permit them to act
as endometriosis stem cells, also termed endometriosis-initiating cells, produc-
ing endometriotic lesions. Notably, in this model, they retain the proliferative and
differentiative potential of endometrial stem cell-like cells, allowing them to
generate components of the endometrium, including glandular, stromal, endothe-
lial, and smooth muscle cells. Like CSCs and tumor-initiating cells as mentioned
previously [20], I here operationally define endometriosis stem cells by their stem
cell-like properties and ability to generate endometriosis with high efficiency, and
endometriosis-initiating cells, therefore, reflect the operational definition of
endometriosis stem cells. Thus, endometriosis-initiating cells is used herein
as a synonym of endometriosis stem cells.
In model III, endometrial stem cells undergo additional genetic and/or epige-
netic modifications and thereafter behave as endometriosis-initiating cells, pro-
ducing endometriotic lesions. Alternatively, terminally differentiated endometrial
cells could undergo multiple profound genetic and epigenetic modifications
resulting in dedifferentiation into endometrial stem cell-like cells capable of gen-
erating a variety of endometrial cell populations, including glandular, stromal,
endothelial, and smooth muscle cells. However, it seems likely that such profound
modifications would destroy the cells capability for multipotential differentiation.
Hence, this pathway might result in carcinogenesis. Thus, model III might explain
endometriosis-originated cancers such as clear-cell carcinoma and endometrioid
cancer. In fact, Pten and K-ras double mutations in the mouse give rise to
endometrioid adenocarcinoma of the ovary, whereas single mutation of K-ras
results in pelvic endometriosis [24].
It is important to note that the processes displayed in models IIII are likely
modulated by the microenvironments in which the cells associate themselves.
Microenvironmental influences could include menstrual efflux, peritoneal fluids,
inflammation, and the ECM of the peritoneum, all of which might regulate molec-
ular/cellular events. Thus, invasion, intravasation, extravasation, colonization,
angiogenesis, EMT, and MET could all be affected by the microenvironment. It
should be emphasized that EMT and MET could play critical roles in the passage of
endometrial stem cells from eutopic sites to ectopic sites and the resultant gener-
ation of endometriotic lesions.
40 T. Maruyama
4.3.2 Evidence Supporting the Endometriosis

Stem Cell Model
The stem cell theory for the pathogenesis of endometriosis is not merely a hypothesis.
Rather, a number of studies have emerged that support this model [18, 16]. The
supportive evidence is summarized below.
4.3.2.1 EMT and MET Appear to Be Involved in the Pathogenesis

of Endometriosis
The metastatic cascades initiated by CSCs and non-CSC cancer cells likely involve
the EMT and the MET [20, 21]. In fact, generation of CSCs probably involves the
activation of EMT programs [20, 21]. Thus, it is intriguing that EMT- and
MET-like processes are also involved in the pathogenesis of endometriosis [25].
For example, menstrual effluent can induce the EMT in mesothelial cells [26].
Consider also side population (SP) cells, a population with a high efflux ability of
Hoechst 33342 dye defined by flow cytometric techniques [27]. SP cells constitute
an undifferentiated population that resides in a number of tissues [28]. SP cells have
been isolated from an endometrial cancer cell line, and they represent likely
candidates for endometrial CSCs. These cells possess a high capacity to develop
into mesenchymal cell lineages, a reflection of EMT activity [29]. These data are
consistent with the endometriosis stem cell model. In other words, menstruation
might induce EMT pathways in endometrial stem cells and their progeny. In turn,
this might induce the cells to migrate away from the eutopic endometrium. Once
implanted in an ectopic microenvironment, invasion and establishment of
endometriotic lesions could occur through MET at the ectopic site.
4.3.2.2 Expression of Stem Cell Markers in Endometriotic Lesions
In models II and III, Fig. 4.3, primitive endometriosis-initiating cells are thought to
express several stem cell markers. Thus, clonal expansion generates endometriotic
lesions and the endometriosis-initiating cells retain their original stem cell markers.
Experiments have shown that cells present in or adjacent to endometriotic lesions
express several stem cell markers including OCT4/POU5F1 and ABCG2 [3033].
OCT4/POU5F1 is expressed by embryonic stem cells, germ cells, and some types
of adult stem cells. This protein plays a crucial role in maintaining stem cell
pluripotency [34]. Another stem cell marker is adenosine triphosphate-binding
cassette transporter G2 (ABCG2). ABCG2 is highly expressed in a variety of
stem cells. It is responsible for removing exogenously added fluorescent dye,
Hoechst 33342, and produces the SP phenotype characteristic of stem cells [35].
Thus, the expression of these stem cell markers in endometriotic lesions supports
the presence of stem cells and provides indirect evidence for the stem cell model.
4.3.2.3 Tumor Clonality
Most neoplasms are monoclonal in origin. Thus, it is particularly intriguing that

endometriotic lesions can also be clonal in nature. The clonality of an endometriotic
lesion provides clues to its developmental history. For example, several studies have
demonstrated that ovarian endometriomas are in fact monoclonal in origin [911].
These molecular findings support the single-cell derivation of endometriomas.
However, the developmental history of endometriotic lesions can be more complex.
For example, whereas peritoneal endometriotic lesions are polyclonal [12, 36],
individual glands of endometriotic lesions are monoclonal [12]. Thus, either single
or multiple precursors might give rise to a single peritoneal endometriotic lesion,
while the glands arise individually from single stem/progenitor cells [12].
4.3.2.4 Endometriotic Lesions Contain Self-Renewing Mesenchymal

Stem Cells
The endometriosis stem cell model predicts that endometriotic lesions should
contain a stem cell-like cell population. Chan et al. reported that ovarian
endometriomas contain a subset of cells displaying a number of somatic stem cell
properties. They include colony-forming activity, self-renewal capacity, and
multipotency [37].
4.3.3 The Origin of Stem Cells
Several theories have been proposed to explain both the origin and pathogenesis of
endometriosis. These hypotheses include retrograde menstruation (implantation),
coelomic metaplasia, and the embryo rest theories [2, 18, 16, 3840]. Retrograde
menstruation (implantation) is the most widely accepted because it is a reasonable
explanation for various types of endometriosis, including peritoneal endometriosis
[38, 40] and even prepubertal endometriosis when neonatal bleeding is taken into
account as a possible cause [41, 42]. In the implantation theory, endometriosis stem
cells originate from stem cells that are present in eutopic endometrium and reach
ectopic sites via many possible routes as discussed above.
Sasson and Taylor [16] and others proposed that the pathogenesis of endome-
triosis could be due to endometriosis stem cells that originate from the bone marrow
of humans [43, 44], data supported by work in mice [45, 46]. These theories are
plausible because a variety of stem/progenitor cells reside in the bone marrow.
In this regard, the presence of mesenchymal stem cells is of particular interest. In
support of this theory, a murine model of endometriosis was used to demonstrate
that bone marrow-derived cells participate in the genesis of epithelial and stromal
cells when endometrium was ectopically transplanted into the peritoneum [45].
42 T. Maruyama
Theories that propose different stem cell origins do not necessarily contradict
one another. It is quite likely that endometrial SP cells include endometrial stem
cells [4751]. Our laboratory showed that endometrial SP cells possess phenotypic
properties that are similar to endothelial progenitor cell (EPC)-like cells [49]. Given
that EPCs originate from bone marrow [52, 53], it is entirely possible that endo-
metrial SP cells have a similar origin. The fact that bone marrow-derived cells are
incorporated into human endometrium at a low level [43, 44] suggests that resident
endometrial stem/progenitor cells such as endometrial SP cells are more likely
responsible for the cyclic renewal and regeneration of endometrium and also
possibly for the establishment of endometriosis than circulating bone marrow-
derived cells [54].
4.3.4 Endometrial Stem Cells and Their Relevance

to the Pathogenesis of Endometriosis
The best current theory for the pathogenesis of endometriosis posits that stem/
progenitor cells in the eutopic endometrium reach ectopic sites through retrograde
menstruation or systemic dissemination. At that point, they give rise to
endometriotic lesions. Integral to this theory is the presence of EmSCs. Below,
the identity and function of EmSCs are discussed.
4.3.4.1 Endometrial Stem Cells
Endometrial stem/progenitor cells and related cells have been isolated and
characterized by a number of laboratories [18, 17, 55]. Some of the precursors
show plasticity, i.e., the capacity to differentiate into a variety of endometrial
tissue types. For example, endometrial SP cells can differentiate into endothelial,
glandular, smooth muscle, and stromal cells, both in vitro and in vivo [4751].
Endometrial SP cells have been observed in the functional layer of the human
endometrium [49]. Hence, they might contribute to renewal of the endometrium [19].
Intriguingly, when endometrial SP cells are transplanted under the mouse kidney
capsule, they migrate into the kidney parenchyma and initiate the formation of blood
vessels [49]. Endometrial SP cells can be found in the vascular walls of endometrial
small vessels in functional and basal layers, and they have functional properties like
EPCs [49]. Endometrial SP cells might initially trigger neovascularization followed by
propagation and differentiation into various cellular components of the human
endometrium [49].
In addition to endometrial SP cells, endometrial mesenchymal stem cells and
endometrial epithelial progenitor cells have been identified and isolated. This was
achieved through the use of cell surface markers such as CD146, CD140b/
PDGFR-b, EPCAM, and W5C5 [5659]. These cell populations are capable of
self-renewal and multilineage differentiation, at least in in vitro experiments,
except for W5C5-positve cells whose stem cell-like properties are verified in vivo
as well as in vitro [59].
Laboratories isolating endometrial SP cells are not in complete agreement with
regard to the cells properties [4750]. While the endometrial SP cells share some
properties, they appear different in regard to their expression of surface markers,
clonal efficiency, culture requirements, and location within the normal endome-
trium. Thus, it is not clear whether there are multiple types of stem cells in the
human endometrium or whether these differences are the result of subtle variations
in laboratory techniques. In any event, it is an open question whether the endome-
trium contains populations of precursor cells differing in phenotype and function. If
this is indeed the case, it is important to determine their hierarchical relationship.
To better define the nature of endometrial stem cells, our laboratory established a
novel in vivo endometrial stem cell assay [51]. This approach has a number of
advantages, the most important of which is that we could follow multipotential
differentiation through use of cell tracking in combination with in vivo model of
human endometrial regeneration [51, 60]. We found that the efficiency with which
endometrial SP cells reconstituted the endometrium increased when unfractionated
endometrial cells were included as support cells. These data clearly showed the
importance of the microenvironment in supporting primitive undifferentiated cells
[51]. When SP and non-SP cells were labeled with a fluorescent marker by lentiviral
labeling, we found that endometrial SP cells had a greater capacity to differentiate
into vascular, glandular, and stromal structures in vivo compared with non-SP
endometrial cells [51]. These experiments verified that endometrial precursors
possess a range of differentiation potentials. This newly developed in vivo endo-
metrial stem cell assay [51] should prove useful for the identification and analysis
of human endometrial stem/progenitor cells.
4.3.4.2 Properties of Endometriosis-Initiating Cells
Based upon the pathogenesis of endometriosis, we can readily predict that

endometriosis-initiating cells should possess three sets of properties. In the first
case, recall that the functional layer sheds during menstruation and is partially
refluxed into the peritoneal cavity through the fallopian tubes. The retrograde
menstruation (implantation) theory requires large numbers of endometriosis-
initiating cells in the functional layer. In agreement, our data have shown that
endometrial SP cells are present in both the basalis and functional layers [49]. How-
ever, it has been postulated that stem cells present solely within the basal layer
which are not shed during menstruation will then give rise to the new functional
layer [61, 62]. There is evidence that fragments of the shed endometrial basalis
are found more frequently in the menstrual blood of women with endometriosis
than in that of healthy control subjects [63]. Importantly, the expression of stem
cell-related markers such as SSEA-1 by endometriotic cells and basal layer
44 T. Maruyama
cells is similar [64]. The data of the two studies suggest that endometriosis may
originate from the basal layer, which, however, does not exclude a possibility that
endometrial stem cells such as endometrial SP cells present in the functional layer
may also contribute to the cyclic renewal and regeneration of eutopic endometrium
and also the establishment of endometriosis.
Second, attachment, migration, and angiogenesis are essential for the implanta-
tion and survival of endometriosis-initiating cells at ectopic site(s). These cells,
therefore, should have migratory potential and angiogenic capability. Third, to give
rise to endometriotic lesion(s) containing glandular structures, endometriosis-
initiating cells should demonstrate multi-differentiation potentials to produce a
variety of endometrial cell components.
In summary, several groups have shown that endometrial SP cells satisfy most of
the predicted properties of endometriosis-initiating cells. That is, endometrial SP
cells are present in both the functional and basal layers and have migratory,
angiogenic, and stem cell-like properties [49, 50]. These findings strengthen the
stem cell theory of endometriosis and support the retrograde menstruation theory.
4.3.5 The Strength and Weakness of the Stem Cell Model
The stem cell theory can account for the weakness of the implantation theory. It can
also address the unique characteristics and behavior of endometriosis.
The retrograde menstruation theory has a number of weaknesses. Specifically, it
has been extremely difficult to detect the initial pathological steps, i.e., the attach-
ment of endometrial tissue to the peritoneum and its secondary proliferation and
invasion [6567]. A modified version of the stem cell theory explains this short-
coming. This theory posits that endometriosis arises from EmSCs and/or progenitor
cells in the implanted endometrial fragments. In other words, EmSCs and/or
progenitor cells are both necessary and sufficient for the establishment of endome-
triosis and can be initiated by a single or very few cells. In this scenario, EmSCs
and/or progenitor cells (and not endometrial tissues) implant and give rise to
endometriotic lesions. As such, it would be almost impossible to detect the initial
attachment and proliferation events of these cells. Thus, endometriotic lesions
would only become microscopically detectable after completion of initial events.
Note that non-stem/progenitor cells in the endometrium (the bulk of endometrial
cells) will not give rise to persistent endometriosis even when a large numbers are
present ectopically.
We emphasize that endometrial SP cells, the most likely candidate for endome-
trial stem cells, constitute only ~2 % of the endometrial cell population [49].
Consequently, the chances are low that endometrial stem/progenitor cells will
find an ectopic supportive microenvironment and initiate an endometriotic lesion.
This might explain the apparent discrepancy between the incidence of endometri-
osis and the frequent occurrence of retrograde menstruation.
The stem cell theory of endometriosis is weakened by the fact that the charac-
teristics of endometrial and endometriosis stem cells are not universally accepted.
Without such consensus, the theory necessarily remains an attractive hypothesis.
4.4 Conclusions
The stem cell theory for the pathogenesis of endometriosis seems to account for
many aspects of the pathophysiology of this disease. The theory provides testable
hypotheses and suggests new approaches to the development of novel diagnostic
tests and treatments. The theory, however, largely depends on determining
unequivocally the existence, identity, and function of EmSCs. Such evidence is
growing. However, a consensus has not yet been established. Once EmSC
populations are defined, significant progress in understanding the pathophysiology
of the disease and new clinical approaches can be anticipated.
Acknowledgements I thank Hirotaka Masuda, Masanori Ono, Kaoru Miyazaki, Takashi

Kajitani, Hiroshi Uchida, and the other members of my research group for their generous
assistance and discussion and Hideyuki Okano and Yumi Matsuzaki for their collaboration with
the endometrial and endometriotic stem cell project. We acknowledge the secretarial assistance of
Rika Shibata. This work was partly supported by Grant-in-Aid from the Japan Society for the
Promotion of Science (to T.M and Y.Y.), Grant-in-Aid from Keio University Sakaguchi-Memorial
Medical Science Fund (to T.M.), and Grant-in-Aid from the Japan Medical Association (to T.M.).
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Chapter 5
Role of NK Cells in Endometriosis
Nagamasa Maeda
Abstract Impaired natural killer (NK) cell activity in women with endometriosis
is thought to promote implantation and the progressive growth of endometrial tissue
in accordance with Sampsons hypothesis. However, the mechanisms responsible
for decreased NK cell activity and the antigens recognized by NK cells in these
women are not clear.
Decreased NK cell activity in the peripheral blood (PB) and peritoneal fluid
(PF) of women with endometriosis was first reported by Oosterlynck et al. and
subsequent investigators have identified the depression of NK cell function in
women with this disorder. Decreased NK cell activity in women with endometriosis
is thought to allow the implantation of endometrial tissue in the manner of a graft,
but the mechanisms underlying the decline of NK cell activity remain uncertain.
We focused on the expression of HLA-G, a ligand of NK cell receptors, and its
changes in eutopic endometrium during the menstrual cycle. HLA-G expression
was only identified in eutopic endometrium during the menstrual phase, but not
during the proliferative or secretory phases. HLA-G-expressing cells were also
detected in peritoneal fluid during the menstrual phase.
Retrograde menstruation may allow HLA-G-expressing endometrial tissue to
enter the peritoneal cavity, where it should be scavenged by the immune surveil-
lance system. Because peritoneal NK cells play an important role in this system,
impairment of their cytotoxicity via HLA-G could allow the survival and implan-
tation of peritoneal endometrial cells.
In this article, we discuss the pathogenesis of endometriosis from the perspective
of intraperitoneal interactions between NK cell receptors and their ligands (anti-
gens) that enter the peritoneal cavity on cells shed from eutopic endometrium via
retrograde menstruation.
N. Maeda, M.D. (*)

Department of Obstetrics and Gynecology, Kochi Medical School,
Kohasu, Oko-cho, Nankoku-city, Kochi 783-8505, Japan
e-mail: maedan@kochi-u.ac.jp

50 N. Maeda
Keywords Endometriosis Endometrium HLA-G KIR Natural killer cell

NK receptor Retrograde menstruation Stress
5.1 Natural Killer (NK) Cells
NK cells are cytotoxic lymphocytes that constitute a major component of the innate
immune system. Because these cells can attack target cells without requiring
antigen sensitization, they are called natural killer cells [1]. NK cells participate
in host defenses against infection [2], have antitumor activity [3], and are involved
in graft rejection [4]. NK cells can also have an adverse influence on pregnancy [5].
These cells usually express the surface markers CD16 (FcRIII) [6] and CD56
(neural cell adhesion molecule: NCAM) [7] in humans. In addition to killing target
cells, NK cells secrete various cytokines, such as the antiviral cytokine interferon-
(IFN-) [8, 9] and the inflammatory and antitumor cytokine tumor necrosis factor-
(TNF-) [10].
NK cell activity can be assessed by measuring cytotoxicity for NK-sensitive
K562 cells (a chronic myelogenous leukemia cell line) [11, 12]. NK cells do not
require activation in order to kill target cells that lack expression of major histo-
compatibility complex (MHC) class I antigens. Karre et al. [13, 14] have proposed
the missing self hypothesis, which states that NK cells act against target cells that
do not express MHC determinants characteristic of self, while toxicity is
inhibited when cells express these determinants.
This missing self hypothesis has been supported by the identification of killer
cell immunoglobulin-like receptors (KIRs) on NK cells that recognize autologous
MHC class I antigens [15, 16] and inhibit NK cytotoxicity against target cells
bearing these determinants.
5.1.1 Activation of NK Cells
Because of their strong cytotoxicity and the potential for autoreactivity, the activity
of NK cells is strictly regulated by several factors. Cytokines play a crucial role in
the regulation of NK cells. Cytokines involved in the activation of these cells
include interleukin (IL)-2 [8], IL-12 [17], IL-15 [18], IL-18 [19], IL-21 [20],
IFN- [8], and granulocyte-macrophage colony-stimulating factor (GM-CSF) [21].
NK cells, as well as macrophages and several other cell types, express the
Fc receptor (FcRIII: CD16/Leu-11 antigen), an activating receptor that binds
the Fc portion of antibodies and allows NK cells to lyse target cells through
antibody-dependent cellular cytotoxicity (ADCC) [22, 23]. Thus, NK cells are
5 Role of NK Cells in Endometriosis 51
fundamentally different from T cells in their lineage and mechanism of nonself

recognition.
In addition to the Fc receptor, NK cells express various activating and inhibitory
receptors, which maintain the balance of positive and negative signals that controls
their cytolytic activity. The KIR family of receptors recognizes and binds
both classical and nonclassical MHC class I molecules. Although receptors of
this family can have activating or inhibitory cytoplasmic domains, most of these
receptors mediate the inhibition of NK cell cytotoxicity. In addition,
the nonclassical MHC I molecule HLA-E is recognized by the lectin-like
CD94/NKG2 receptor family, which includes both activating and inhibitory recep-
tors [2426]. Thus, NK cells can simultaneously express both activating and
inhibitory receptors.
5.1.2 NK Cells and Endometriosis
In 1991 and 1992, Oosterlynck et al. reported that the NK cell activity against
autologous endometrium in PB and PF was reduced in women with endometriosis
[27, 28]. Impaired NK cell activity in women with this condition is thought to allow
the implantation of endometrial tissue as a graft according to Sampsons
hypothesis [29].
The finding of decreased NK cell activity in women with endometriosis and the
positive correlation of NK cell activity in both the PB and PF with the severity of
this disease has led to consensus about its pathogenesis [3033].
After removal of endometriotic lesions, decreased NK cell activity and the
impaired cytotoxicity of autologous and heterologous lymphocytes against the
endometrium are unchanged, and cytotoxicity is still significantly decreased com-
pared with that in women who do not have endometriosis. These findings suggest a
primary deficiency of NK cell activity in women with endometriosis, which could
explain its frequent relapse after treatment [34].
Survival of endometrial cells in the peritoneal cavity of women with endome-
triosis [35] is mainly due to decreased NK cell activity, but is also related to
resistance of these cells to NK cytotoxicity [3638].
The missing self hypothesis has been supported by the identification of KIRs
on NK cells that recognize self-determinants among MHC class I antigens
andinhibit NK cell cytotoxicity against target cells bearing these determinants. In
women with endometriosis, expression of inhibitory killer immunoglobulin-like
receptors on NK cells from PB and PF is significantly upregulated compared with
the level of expression in women without endometriosis [3941], indicating a
decrease of NK cell activity and cytotoxicity for endometriotic cells.
52 N. Maeda
5.2 NK Cell Receptors
5.2.1 Killer-Cell Immunoglobulin-Like Receptors (KIRs)
The KIR genes are polymorphic and highly homologous genes that are located in a
cluster on chromosome 19q13.4 [25]. The KIR proteins are classified by the number
of immunoglobulin (Ig)-like extracellular domain receptors (2 or 3) and by whether
they have a long or short cytoplasmic tail. KIR proteins with a long cytoplasmic tail
transduce inhibitory signals upon ligand binding via an immunoreceptor tyrosine-
based inhibitory motif (ITIM) after binding tyrosine phosphatase SHP1/SHP2,
while KIR proteins with a short cytoplasmic tail that lacks the ITIM associate
with the tyrosine kinase-binding protein ZAP-70/Syk instead transduce activating
signals via an immunoreceptor tyrosine-based activation motif (ITAM) [4244].
Most KIRs are inhibitory, indicating that recognition of MHC antigens by these
receptors suppresses the cytotoxic activity of NK cells, while only a limited number
of KIRs have the ability to activate these cells. The ligands for several KIRs are
subsets of both classical HLA-Ia antigens (HLA-A, HLA-B, HLA-C) and also
nonclassical HLA-Ib antigens (HLA-G) [45, 46].
5.2.1.1 KIR2DL1 and Endometriosis
In women with endometriosis, the decrease of NK cell activity may be related to the
inhibitory KIRs expressed by NK cells.
The percentage of cells expressing KIR2DL1 among NK cells in PF and PB was
reported to be significantly higher in women with endometriosis than in controls,
suggesting that this receptor is probably related to suppression of NK cell activity
in endometriosis [39]. The elevated percentage of KIR2DL1+ NK cells in PB
from women with endometriosis was not reduced by laparoscopic surgery or by
gonadotropin-releasing hormone agonist treatment. Thus, KIR2DL1 overexpression
may be a primary event that represents a risk factor for both the development of
endometriosis and its recurrence after treatment [40].
5.2.1.2 KIR2DL4 and Endometriosis
KIR2DL4-expressing NK cells have been identified in both the PB and PF [47].

Expression of HLA-G (a ligand for KIR2DL4), ILT-2, and ILT-4 by eutopic
endometrium was only detected during the menstrual phase and was not found in
either proliferative or secretory endometrium [47]. Because HLA-G-expressing
cells were detected in PF during the menstrual phase, retrograde menstruation
may allow eutopic endometrial cells bearing HLA-G to enter the peritoneal cavity
and react locally with KIR2DL4.
Due to the innate immunological activity of KIR2DL4 targeting HLA-G,

KIR2DL4-expressing NK cells in the PF may be cytotoxic for HLA-G-expressing
endometrial cells that enter the peritoneal cavity through retrograde menstruation.
Mutant (9A)/wild-type (10A) polymorphism would modulate the response of
peritoneal NK cells to membrane-bound HLA-G on endometrial cells, and inhib-
itory ILT-2 and ILT-4 receptors may also influence the local immune response
together with KIR2DL4. Thus, impaired cytotoxicity for endometrial cells could
allow these cells to survive and result in ectopic implantation that produces
peritoneal endometriosis.
5.2.2 CD94/NKG2A
CD94/NKG2A is a member of the C-type lectin receptor family composed of a

CD94 chain covalently bound with a member of the NKG2 family. The ligand of
CD94/NKG2A is HLA-E, a nonclassical HLA-Ib molecule. After binding of CD94
with NKG2A that has the ITIM, the resulting CD94/NKG2A heterodimer forms an
inhibitory receptor, whereas association of CD94 with an NKG2 family member
lacking the ITIM (NKG2C) may not form an inhibitory receptor.
5.2.2.1 CD94/NKG2A and Endometriosis
Despite its interesting inhibitory function, little has been reported concerning
CD94/NKG2A and endometriosis. Women with stage III and stage IV endometri-
osis were found to have a significantly higher percentage of CD94/NKG2A+
peritoneal NK cells than control women [48], and HLA-E (the CD94/NKG2A
ligand) has been identified in endometriotic tissues [48]. Because target cells
expressing HLA-E show resistance to NK cell-mediated cytotoxicity in a CD94/
NKG2A-dependent manner, the increased expression of CD94/NKG2A by perito-
neal NK cells may mediate the resistance of endometriotic tissues to NK cell
cytotoxicity and thus contribute to the progression of endometriosis [48].
5.2.3 Immunoglobulin-Like Transcript (ILT)
The immunoglobulin-like transcript (ILT) gene family has up to 11 members in

humans. The extracellular part of each ILT molecule includes at least two and
usually four immunoglobulin domains. ILT-2 through ILT-5 are all inhibitory
members of this family with different numbers of cytoplasmic ITIM domains.
ILT1, ILT7, and ILT8 have a short cytoplasmic tail and a charged amino acid
residue in the transmembrane domain, and these family members deliver an acti-
vating signal through the cytoplasmic ITAM of the associated common g chain of
Fc receptor (FcRg).
54 N. Maeda
5.2.3.1 Immunoglobulin-Like Transcript (ILT) and Endometriosis
ILT-2 and ILT-4 bind to HLA-G with a three- to fourfold higher affinity than that
for classical MHC class Ia [49], suggesting that ILT/HLA-G recognition may play a
dominant role in regulating the activation of NK cells, T cells, and monocytes.
Although ILT is thus thought to have a role in the pathogenesis of endometriosis,
surprisingly, there has been no report concerning its interaction with this disease.
5.3 NK Receptor Ligands on Endometrial Cells
5.3.1 HLA Class Ia
In 1990, expression of HLA class I molecules was identified on human endometrial

and endocervical epithelial cells [50]. This suggests that KIRs expressed on NK
cells can respond to their HLA class I ligands on eutopic endometrium [3941]. It
was also reported that progesterone induces HLA class I mRNA expression by
cultured cells from secretory endometrium [51].
In women with endometriosis, HLA class I expression by both glandular cells
and stromal cells is significantly elevated compared with that in controls [52].
Furthermore, women with endometriosis show significantly higher expression of
HLA class I molecules by eutopic endometrial cells compared with controls [52].
Our investigations have suggested that HLA-C-expressing endometrial
cells may enter the peritoneal cavity via retrograde menstruation and react with
peritoneal NK cells expressing KIR2DL1 [3941]. Impaired KIR2DL1 expression
by peritoneal NK cells could allow endometrial cells to survive in the peritoneal
cavity and undergo ectopic implantation, favoring the onset and progression
of endometriosis.
5.3.2 HLA Class Ib
5.3.2.1 HLA-E
HLA-E is one of a family of molecules known as HLA class Ib and it has a very
specialized role in the recognition of other cells by NK cells. HLA-E is very highly
conserved and presents a small repertoire of peptides of various origins [53]. NK
cells recognize the HLA-E/peptide complex via the heterodimeric inhibitory recep-
tor CD94/NKG2A or the activating receptor CD94/NKG2C. When CD94/NKG2A
is stimulated, it has an inhibitory effect on NK cell cytotoxic activity and prevents
target cell lysis [54].
Expression of HLA-E, the ligand of CD94/NKG2A, has been detected in

endometriotic tissues. Target cells bearing HLA-E show resistance to NK cell
cytotoxicity in a CD94/NKG2A-dependent manner. Therefore, increased expres-
sion of CD94/NKG2A by peritoneal NK cells may mediate the resistance of
endometriotic tissue to NK cell cytotoxicity, thus contributing to the progression
of endometriosis [48].
5.3.2.2 HLA-G
5.3.2.2.1 Receptors for HLA-G
HLA-G appears to be mainly recognized by ILT-2 and ILT-4 receptors, which are
expressed by T and B lymphocytes, as well as NK cells and monocytes, and inhibit
the activating signals received by these cells. In addition to ILT, HLA-G can also
react with KIR2DL4 expressed on NK cells and T cells. Although the expression of
ILT2, ILT3, ILT4, and KIR2DL4 is known to be upregulated by HLA-G in antigen-
presenting cells, NK cells, and T cells [55], whether the HLA-G/KIR2DL4 inter-
action leads to activation or inhibition of NK cells remains a complicated and
controversial issue.
5.3.2.2.2 HLA-G and Endometriosis
Hornung et al. reported that HLA-G was not expressed by eutopic endometrium or
endometriotic tissue [56] and HLA-G protein was not detectable in peritoneal fluid
from endometriosis patients or controls. Moreover, ectopic and normal endometrial
tissues and stromal cells did not express HLA-G during the proliferative phase.
Accordingly, they concluded that immune cell inhibition in endometriosis is medi-
ated by factors other than HLA-G.
In another study, HLA-G expression was identified in the glandular epithelium
of peritoneal endometriotic implants, but not in eutopic endometrium [57]. The
authors concluded that differential expression of HLA-G suggests peritoneal
inflammation or cellular stress may upregulate mechanisms promoting the survival
of ectopic endometrium.
According to our recent findings, KIR2DL4-expressing NK cells can be identi-
fied in both the PB and PF. Interestingly, we found that HLA-G (a ligand of
KIR2DL4) was only expressed by eutopic endometrium during the menstrual
phase and not during the proliferative or secretory phases [47]. In addition, we
detected HLA-G-expressing cells in PF during the menstrual phase [47]. This
suggests that cells bearing HLA-G may enter the peritoneal cavity through retro-
grade menstruation, allowing the antigen to react locally with KIR2DL4.
In 2000, Ibrahim et al. reported that stress, including heat shock and arsenite
treatment, induces an increase in the expression of various HLA-G alternative
56 N. Maeda
transcripts without affecting other MHC class I transcripts (HLA-A, HLA-B,

HLA-E, and HLA-F) [58].
A heat shock element (HSE) that binds to heat shock factor 1 (HSF1) trimers
under stress conditions has been identified within the HLA-G promoter. When
stresses such as heat shock, bacterial infection, and poisons (including arsenite)
affect cells, cytoplasmic heat shock factor (HSF)-1, which is usually regulated by
HSF-2 and HSF-4, undergoes translocation into the nucleus where it forms trimers
and binds with the HSE located in the promoter region of the heat shock protein
(Hsp) and HLA-G genes (37), resulting in the induction of hsp70 and HLA-G [58].
We detected HLA-G expression by eutopic endometrial cells during menstrua-
tion [47], which presumably reflects the stressful intrauterine environment at
this time.
In the normal menstrual cycle, a decrease of the serum progesterone level
induces uterine smooth muscle contraction and spiral artery spasm, resulting in
menstruation [59]. Transient bacterial invasion and inflammation may also affect
the eutopic endometrium during menstruation, and increased expression of Hsp70
and HLA-G by the endometrium may result from such physiological stress.
Hsp70- and HLA-G-expressing endometrial cells that enter the peritoneal cavity
via retrograde menstruation may react with immunocompetent cells such as NK
cells in the PF and be eliminated. However, an impaired NK cell response to these
antigens could lead to endometrial cell survival in the PF and implantation with
subsequent development of endometriosis (Fig. 5.1).
5.4 Conclusions
We demonstrated that HLA-G was expressed in the eutopic endometrium during

menstruation, suggesting the existence of physiological stress during the menstrual
phase. HLA-G-expressing endometrial cells may enter the peritoneal cavity via
retrograde menstruation and react with immunocompetent cells, particularly NK
cells. Retrograde menstruation involves the backflow of menstrual discharge from
the uterine cavity through the fallopian tubes into the peritoneal cavity. It is a
physiological phenomenon that is observed in women with patent tubes.
There is probably a physiological system that removes displaced tissues or
debris from the peritoneal cavity and peritoneal NK cells presumably play an
important role in this system. Expression of HLA-G by endometrial cells during
the menstrual phase and entry of such cells into the peritoneal cavity may induce a
local immune response from immunocompetent cells including KIR2DL4-
expressing NK cells, while impaired NK cell activity could allow the survival
and ectopic implantation of endometrial cells.
Impairment of the physiological immune surveillance system (particularly NK
cell function) may favor endometrial cell implantation and thus trigger the onset of
endometriosis. Further investigation of the molecular mechanisms leading to
Physiological stress during menstruation

progesteron withdraw, smooth muscle contraction, spasm of spiral artery,
ischemia-reperfusion, oxidative stress, infection
Endometrial cell
HSF-1
Nucleus
Promotor Hsp gene
HSE
Chromosome 6 Hsp gene HLA-G gene
HSE HSE
Hsp70 HLA-G Enter into peritoneal cavity via

retrograde menstruation
NK cell
Cell lysis
KIR2DL4 (10A/9A)
Ectopic implantation? Survival HLA-G
Endometrial cell ILT-2

NK cell
Fig. 5.1 In the usual menstrual cycle, physiological stresses such as progesterone withdraw,
smooth muscle contraction, spasm of spiral artery, ischemia-reperfusion, oxidative stress, and
transient bacterial infection may occur in the uterus. Increased expression of Hsp70 and HLA-G
in the endometrium during menstruation may result from such stress pathway. Hsp70- and
HLA-G-expressing endometrial cells in the retrograde menstruation may react with immunocom-
petent cells such as NK cells in PF and finally disappear from peritoneal cavity or survive and
develop to endometriosis. HSF heat shock factor, HSE heat shock element, HSP heat shock
protein, KIR killer immunoglobulin-like receptor, ILT immunoglobulin-like transcript
expression of antigens during menstruation and the role of immunocompetent

responder cells in the PF may contribute to better understanding of the pathogenesis
of endometriosis.
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Chapter 6
Macrophages in Pathophysiology
of Endometriosis
S.F. Ahmad*, N. Michaud*, H. Rakhila*, and A. Akoum
Abstract The mechanisms that sustain endometrial tissues at ectopic sites in

patients with endometriosis are poorly understood. It is well established now that
endometriosis is associated with changes in population and functions of various
leukocytes, including macrophages. Macrophages are the most abundant cells
found in the peritoneal fluid and are the consistent feature of endometriotic lesion.
They infiltrate endometriotic lesions where they undergo alternative activation as a
consequence of signals generated within the invaded tissue. However, instead of
clearing endometrial cells from the peritoneal cavity and restoring local homeosta-
sis, macrophages appear to enhance their survival and proliferation by secreting
growth, remodelling and inflammatory factors which could contribute to the devel-
opment of endometriosis as well as to the disease-associated chronic pelvic inflam-
mation and symptoms. Thus, unveiling the molecular mechanisms that underlie
macrophage dysfunctions is a critical area of research, which would lead to the
development of novel medical treatments for endometriosis. In this chapter, we
described how macrophages can play a critical role in the pathophysiology of
endometriosis not only via their weakened phagocytic functions but also via other
major mechanisms revealed to date.
Keywords Cytokines Endometriosis In vivo model Infertility Macrophages

Pain Prostaglandins
*Author contributed equally with all other contributors.

S.F. Ahmad N. Michaud H. Rakhila A. Akoum (*)
Faculte de Medicine, Endocrinologie de la Reproduction, CHU de Quebec,
HSFA, Universite Laval, Quebec, QC, Canada G1L 3L5
e-mail: ali.akoum@crsfa.ulaval.ca

62 S.F. Ahmad et al.
Abbreviations
CD Cluster of differentiation
Cox-2 Cyclooxygenase-2
DCs Dendritic cells
E2 Oestrogen
FGF Fibroblast growth factor
ICAM-l Intercellular adhesion molecule-l
IL Interleukin
IFN- Interferon gamma
ISO-1 ((S,R) 3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic methyl
ester
LFA-1 Leukocyte function antigen-l
LPS Lipopolysaccharide
MAPK Mitogen-activated protein kinase
MCP-1 Monocyte chemotactic protein-1
MIF Macrophage migration inhibitory factor
MMPs Matrix metalloproteinases
NF-B Nuclear factor kappa B
NGF Nerve growth factor
NK Natural killer
PGE2 Prostaglandin-E2
PGF2 Prostaglandin-F2
PGs Prostaglandins
PlGF Placental growth factor
RANTES Regulated on activation, normal T cell expressed and secreted
StAR Steroidogenic acute regulatory protein
TGF Tumour growth factor
TIMPs Tissue inhibitors of MMPs
TNF- Tumour necrosis factor-alpha
uNK Uterine natural killer
VEGF Vascular endothelial growth factor
6.1 Introduction
Endometriosis is associated with changes in the components of the immune system

as the peritoneal fluid of women with endometriosis contains increased numbers of
immune cells. Dysfunction of the immune system may play an important role in the
development of endometriosis since in normal women, menstrual tissue is refluxed
into the peritoneal cavity, but it is cleared by immune cells such as macrophages,
uterine natural killer (uNK) cells and lymphocytes. However, in women with
endometriosis, these subsets of immune cells have been found to be altered in the
6 Macrophages in Pathophysiology of Endometriosis 63
Fig. 6.1 Macrophage in endometriosis: proposed model of positive feedback loops
peritoneal fluid [1]. Macrophages are the most abundant nucleated immune cells
found in the peritoneal fluid [2], and their role in endometriosis has been studied for
more than three decades. Haney et al. first reported an increase in the number of
peritoneal macrophages in women with endometriosis [3]. Several studies con-
firmed this observation and further reported an increased activation of these cells in
women with endometriosis [47]. However, activated peritoneal macrophages in
women with endometriosis seem to have a reduced capacity to eliminate misplaced
endometrial cells. Paradoxically, they rather seem to facilitate endometrial cell
survival, invasiveness and growth by secreting growth, angiogenic and tissue
remodelling factors and thereby contribute to the development of endometriosis.
Interestingly, it was reported that even peripheral blood monocytes enhance eutopic
endometrial cell proliferation in women with endometriosis but suppress endome-
trial cell proliferation in healthy women [8]. Puzzling at first, this phenomenon is
being progressively elucidated. It is thus likely that a combination of defects either
in macrophages or in the inherent capability of endometrial cells themselves to
resist immune suppression and apoptosis in the peritoneal environment is involved.
We also believe that activating positive feedback loops and a mutual crosstalk
occur within the pelvic cavity of women with endometriosis (where the disease is
frequently found) between local immune cells, particularly macrophages, and
misplaced or implanted endometriotic cells, which lead to the establishment of a
chronic inflammatory process and a local environment that favours ectopic tissue
growth (Fig. 6.1).
This chapter provides an overview of the current state of knowledge on macro-
phages in endometriosis, the functional impairment of these key immune cells and
their possible role in the pathophysiology of this disease.
6.2 Physiology of Macrophages
Macrophages are mononuclear phagocytic cells derived from blood monocytes [9].
Monocytes originate from haematopoietic stem cells present in bone marrow,
which undergo differentiation. They represent immune effector cells, equipped
with chemokine receptors and pathogen recognition receptors that mediate migra-
tion from blood to tissues during infection. Extravasation of monocytes from blood
to tissue initiates maturation processes leading to mature macrophages or dendritic
cells (DCs) [10]. After migration to peripheral tissue, activated monocytes differ-
entiate to inflammatory DCs and macrophages, which are determined by the
inflammatory milieu, and pathogen-associated pattern recognition receptors present
on the surface of these immune cells. The developmental origin and the function of
tissue macrophage subsets, such as microglia (macrophages in the central nervous
system), dermal macrophages and splenic marginal zone and metallophilic macro-
phages, remain insufficiently understood [11]. Resident macrophages in lymphoid
and non-lymphoid tissues are the phagocytic cells involved in steady-state tissue
homeostasis, and they undergo local activation in response to various inflammatory
and immune stimuli. These macrophages are classified as being elicited, as in the
antigen-non-specific response to a foreign body or a sterile inflammatory agent or as
being classically activated or alternatively activated by an antigen-specific
immune response. It is difficult to distinguish originally resident macrophages
from more recently recruited, elicited or activated macrophages, because cells
adapt to a particular microenvironment [12].
6.2.1 Types and Functions
Macrophages are the professional phagocytes expressing numerous membrane

adhesion molecules including scavenger receptors such as the cluster of differenti-
ation 36 (CD-36) receptors ensuring targets fixation and endocytosis [9, 13]. Based
on membrane-expressed molecules and secretion, macrophages are commonly
classified as M1 or M2 phenotype. M1-macrophages are the pro-inflammatory
macrophages expressing and secreting cytokines and antimicrobial molecules
such as interleukin-1 (IL-1). M2-macrophages are the anti-inflammatory macro-
phages secreting a majority of growth factors and anti-inflammatory cytokines
[14]. It is important to mention that macrophages are dynamic and flexible
cells that can change from a phenotype to another depending on their microenvi-
ronments [15]. It is a matter of fact that interferon- (IFN-) and lipopolysaccharide
(LPS) generate M1-macrophages, whereas IL-4 and IL-13 activate
M2-macrophages [16]. Furthermore, a pallet of phenotype exists between
the common M1-macrophages and M2-macrophages activation such as decidual
macrophages present in endometrium, which are hybrid M1M2 phenotype
[10, 17, 18]. Scientists finally conclude that the subtle composition of macrophage
environment determines their expression profile and thus their role [10].
6.2.2 Macrophages Along the Menstrual Cycle
Macrophages are ubiquitously present as resident cells monitoring tissue environ-

ment and ready to react to any discrete change [15]. Actually, oestrogen (E2) has
been discovered as a regulator of M2-phenotype and is responsible for decidual
macrophages efficiency especially in pregnancy [19]. Indeed, resident macro-
phages in endometrium are differentially expressed along the menstrual cycle.
During proliferative phase, macrophages represent only 12 % of all cells and
express tumour growth factor (TGF), IL-10, metalloproteinases (MMPs) and
tissue inhibitors of MMPs (TIMPs) contributing to endometrial regeneration,
enhancing extracellular matrix turnover and fibroblasts recruitment and prolifera-
tion [20, 21]. When progesterone level rises, macrophages represent 35 % of cells
probably due to resident macrophages proliferation. They express numerous
chemoattractant molecules such as macrophage chemotactic protein (MCP)-1,
regulated on activation, normal T cell expressed and secreted (RANTES) and
IL-8 to allow immune cells infiltration into endometrium where they will prepare
for the eventual feto-implantation. Finally, progesterone withdrawal, marking
menstrual phase, is characterised by an increase in number of macrophages due
to chemoattracted monocyte extravasation [20]. These M1-like macrophages
secrete MMP-9, tumour necrosis factor-alpha (TNF)- and many other
pro-inflammatory cytokines and proteolytic enzymes, leading to tissue breakdown
and menstruation [21].
In the peritoneal fluid, macrophages are the most abundant cell type. Normally,
the peritoneal fluid contains 0.520 106 leukocytes/ml, of which 85 % are mac-
rophages [2]. In the absence of pathogens, peritoneal fluid macrophages are present
at low level and contribute to tissue homeostasis [22]. Their concentration appears
to fluctuate during the menstrual cycle and is maximal during the menses [23]. This
is most likely related to the menstrual reflux phenomenon, which is common in
almost 90 % of reproductive age women during menstruation. Briefly, a small part
of endometrial debris travels through fallopian tubes to the peritoneal compartment
instead of being evacuated in vaginal secretions [24, 25], where they are normally
phagocytised by peritoneal macrophages [26].
Concisely, macrophages are the phagocytes implicated in an intimate crosstalk
between endometrial tissue and the local immune system. Any perturbation in their
close environment or intrinsic modifications could trigger profound imbalance in

these interactions.
6.3 Pathophysiology of Macrophages in Endometriosis:

A Weakened Immune System May Lead
to Endometriosis
The discovery that macrophages play a major role in the pathophysiology of

endometriosis dates back to 1981 when Dmowski and his group first reported the
existence of a deficient cellular immunity in women with endometriosis and a
number of functional changes in several immunologic components of the peritoneal
fluid [1]. In the same year 1981, other investigators revealed that the number of
peritoneal macrophages is increased in infertile women with endometriosis com-
pared with normal women or women with other causes of infertility [3], and an
increased phagocytosis of spermatozoa by peritoneal macrophages in patients with
endometriosis was then shown [27]. Later on, it was found that endometriosis is
associated with an increased influx of macrophages that undergo further maturation
and activation and release active substances such as prostaglandins (PGs), which
can have adverse effects on the fertility [5, 28]. Data from other studies confirmed
these findings and further suggested that peritoneal macrophages in women with
endometriosis are in an advanced level of differentiation, which may interfere with
gametes and pre-implantation embryos and contribute to endometriosis-associated
subfertility [4]. By the end of 1980s, it was well established that concentration,
activation and abnormal maturation of peritoneal macrophages may facilitate the
onset of endometriosis. A wide array of factors produced by activated macrophages
appear to be involved in endometrial cell survival, adhesion, invasion, proliferation
and the formation of endometriotic lesions. A key role for macrophages in
decreased immunologic surveillance, recognition and destruction of misplaced
endometrial cells and possible facilitation of ectopic endometrial tissue growth
seems well plausible.
Many aspects of macrophage involvement in endometriosis-related symptoms
have also drawn a considerable interest, but the mechanisms by which activated
macrophages cause infertility, menstrual disorders and pelvic pain in women with
endometriosis remain unclear.
6.3.1 Suppression of Phagocytosis and Apoptosis

by Macrophages
Braun et al. have observed a reduction in the number of macrophages in the eutopic
endometrium of women with endometriosis [8]. Their results were surprising
because an increase in macrophages number in the peritoneal cavity of these

women was well documented [6, 29]. Such a phenomenon would reflect altered
chemokine gradients that favour macrophage mobilisation to the ectopic environ-
ment due to the presence of cyclical, inflammatory stimuli within that environment.
Intrinsic resistance to apoptosis, in conjunction with a physiologic disturbance in
macrophage trafficking in the eutopic endometrial environment, would be expected
to favour the survival of endometrial cells and lead to their establishment in the
ectopic sites. Another study has shown a decrease in the capacity of macrophage-
mediated cytolysis of misplaced endometrial cells in the peritoneal locations and an
increased resistance of these cells to apoptosis [30].
Taken together, the evidences available to date suggest that defective cytolytic
function in macrophages within the peritoneal cavity coupled with the inherent
resistance of the endometrial cells to programmed cell death may be fundamental to
the pathophysiology of endometriosis.
It is relevant to mention that in women with endometriosis, the ectopic endo-
metrial tissue also seems to escape immune surveillance in the peritoneal cavity. On
one hand, several studies have shown that peritoneal macrophages phagocytise and
degrade spermatozoids and that peritoneal macrophages isolated from these women
have a greater phagocytic ability than those from fertile or infertile women without
endometriosis [27, 31]. These results suggest that, if peritoneal macrophages from
women with endometriosis enter the reproductive tract via the oviducts, they might
adversely influence fertilisation by phagocytising sperm cells [32]. On the other
hand, other studies have shown a decreased phagocytic activity against endometrial
cells [33, 34]. Thus, instead of eliminating endometrial debris following retrograde
menstruation, peritoneal macrophages of patients with endometriosis would favour
the development of endometriotic lesions because of the absence of adequate
phagocytosis and the large amounts of cytokines and other growth factors released
into the peritoneal cavity [35]. It has been suggested that endometrial cells of
women with endometriosis contribute to their own survival and escape of immune
suppression by secreting various molecules that are capable of interfering with the
process of recognition of immune cells [34]. An example of such a phenomenon is
the expression of the soluble form of intercellular adhesion molecule (ICAM)-l.
ICAM-1 is a co-receptor that interacts with the integrin leukocyte function antigen
(LFA)-l present on the surface of leukocytes in order to participate in cell recog-
nition. It has been demonstrated that eutopic endometrial stromal cells of women
with endometriosis express a higher concentration of the ICAM-1 soluble form than
those of healthy women. In addition, the expression of this form is even higher in
ectopic endometrium. Thus, it is possible that the soluble form of ICAM-1 com-
petes with its cellular form to bind to LFA-1, which will disrupt recognition by
leukocytes target cells [36].
In most circumstances, macrophages secrete MMP-9 to destroy the extracellular
matrix to disperse endometrial tissues into small pieces [37]. In addition, CD-36
receptor is highly expressed on the cell membrane of macrophages to facilitate
phagocytosis of these small fragments of endometrial debris. However, in the
presence of high concentration of prostaglandin E2 (PGE2), the expression of
MMP-9 and CD-36 is suppressed [38, 39]. This significantly inhibits the phagocytic
ability of macrophages and may favour abnormal endometrial tissue growth into the
ectopic host sites.
It is worth mentioning here that an apparent inconsistency seems to emerge
considering the ability of macrophages to phagocytise sperm cells and their inabil-
ity to eliminate ectopic endometrial cells. While it is possible that such a difference
is at least partly due to inherent properties of endometrial cells of women with
endometriosis, the underlying mechanisms remain to be clarified, given their likely
impact on the pathogenesis of the disease and the symptoms it causes.
6.3.2 Macrophage Secretions in Endometriosis
Elevated concentrations of cytokines, growth and pro-inflammatory and angiogenic

factors have been found in the peritoneal fluid of women with endometriosis. These
different factors can originate from a wide variety of sources including
endometriotic lesions themselves, macrophages, T cells and follicular fluid after
ovulation [40]. IL-1, IL-8, macrophage migration inhibitory factor (MIF), MCP-1,
PGE2 and prostaglandin F2 (PGF2) are among the main cytokines and eicosa-
noids that are present at high levels in the peritoneal fluid of women with endome-
triosis [34, 4044] and known to be overproduced by activated macrophages. Our
previous studies have for the first time detected the existence of endometrial
dysfunctions in patients with endometriosis. Thus, endometrial cells are more
sensitive to activation by IL-1, a cytokine that is mainly released by activated
monocytes/macrophages and found at high concentrations in the peritoneal fluid of
patients with endometriosis [45]. IL-1, also known as catabolin, can enhance the
development of endometriosis by causing the release of factors for blood vessel
growth. Other data suggest that IL-1 can also be involved in the up-regulation of
MIF expression by ectopic endometrial implants [46].
IL-8, known as an -chemokine that shows chemotactic activity and acts as
a potent angiogenic agent [47], is believed to play a significant role in endometrial
physiology and endometriosis. Mainly produced by peripheral macrophages as
well [48], IL-8 was detected at higher concentration in the peritoneal fluid of
women with endometriosis. In addition to its chemotactic and activating properties
for granulocytes, IL-8 was recently found to stimulate proliferation of endometrial
cells [49].
MCP-1 is a P-chemokine having a potent chemotactic activity for monocytes,
macrophages and lymphocytes [50] and produced by different cell types. In endo-
metriosis, however, MCP-1 was found to be over-expressed in eutopic and ectopic
endometrial cells as well as in peritoneal fluid macrophages of women with
endometriosis [51, 52], and its peritoneal concentrations appeared to correlate
with the stage of the disease [53, 54]. This factor may play a role in the development
of endometriotic lesions not only by recruiting and stimulating peritoneal macro-
phages but also by directly stimulating endometrial cell proliferation [54] and
angiogenesis [55, 56]. MCP-1 has potent direct angiogenic effects on endothelial
cells [57] and may act indirectly via its ability to stimulate macrophage recruitment
and activation and the release of angiogenic factors [52, 58, 59].
RANTES, one of the members of CC chemokine family that mediate monocyte
chemotactic activity, was found to be elevated in peritoneal fluid of women with
endometriosis [60]. RANTES peritoneal fluid levels also correlate with advanced
endometriosis stage, suggesting that it might contribute to the progression of this
disease [60]. Interestingly, our and other studies showed that macrophage-/mono-
cyte-derived pro-inflammatory cytokines such as IL-1 trigger endometriotic cells
to produce this monocyte chemoattractant chemokine [61, 62] and point thereby to
feedforward amplification loops underlying chronic pelvic inflammation in women
with endometriosis.
MIF, originally identified as a protein factor secreted by T cells, inhibits the
migration of macrophages in vitro (hence its name: macrophage migration inhibitory
factor) [63, 64]. MIF is actually expressed by most immune cells such as monocytes,
macrophages or B cells. It has been shown that MIF plays an important role in the
cell-mediated immune response by promoting the Th1 response by the production of
IL-12 by macrophages [65, 66]. MIF is able to inhibit the immunosuppressive effects
of glucocorticoids on the production of pro-inflammatory cytokines such as IL-1 in
activated macrophages [6669]. To illustrate this property, it has been described that
MIF induces the expression of cytoplasmic phospholipase A2 which is an important
component of the pro-inflammatory cascade and usually blocked by the action of
glucocorticoids [70]. Our studies showed that MIF is overproduced by activated
peritoneal macrophages in women with endometriosis [52] and further found abun-
dant levels of this factor in peritoneal fluid [43], peripheral blood [44] and eutopic
endometrial tissue of endometriosis patients [71]. Furthermore, MIF levels varied
according to endometriosis stage and were particularly elevated in women suffering
from endometriosis but also complaining from pelvic pain and/or infertility. This,
together with the increased peritoneal fluid levels of MIF, was corroborated by other
investigators [72, 73]. Our subsequent studies showed that MIF expression is also
elevated in the early stage and highly vascularised endometriotic lesions [43] and
appeared to act at multiple coordinated levels in the PG biosynthesis cascade,
thereby inducing cyclooxygenase-2 (Cox-2) expression in these cells and stimulat-
ing PGE2 [74]. From a cellular mechanism point of view, our work suggests the
involvement of the transcription factor nuclear factor kappa B (NF-B) in MIF gene
activation in ectopic endometrial cells in response to IL-1 [75]. In addition, our
group revealed for the first time the presence of a positive feedback loop by which E2
acts directly on ectopic endometrial cells to up-regulate the expression of MIF,
which, in turn, displays the capability of inducing the expression of aromatase, the
key and rate-limiting enzyme for E2 synthesis [76]. Our studies also revealed that
MIF exerts a potent indirect angiogenic effect by interacting with ectopic endome-
trial cells and inducing the secretion of major angiogenic factors via CD-44 and
CD-74 and mitogen-activated protein kinase (MAPK) signalling pathways [77] and
provide evidence for a possible new mechanism underlying endometriosis develop-
ment and pathophysiology. Angiogenesis or the formation of new blood vessels is
essential for the development and the maintenance of endometriotic lesions. Vascu-
lar endothelial growth factor (VEGF), one of the major angiogenic factors endowed
with the capability of stimulating mitogenesis, migration and differentiation of
endothelial cells, is strongly expressed in endometriotic tissue as well as in perito-
neal macrophages [78, 79]. Peritoneal-activated macrophages are the major source
of VEGF in endometriosis and that this expression is regulated directly by ovarian
steroids [80]. Ovarian steroids regulate the production of this growth factor through
peritoneal macrophages. E2 acts on various macrophage signalling pathways,
influencing in particular those related to sustain the recruitment of inflammatory
cells and the remodelling of inflamed tissues, such as MAPK, phosphatidylinositide-
3-kinase/protein kinase B and NF-B. As a consequence, a deregulated response to
steroids might influence the survival of ectopic endometrial cells and promote the
vascularisation of the lesions [8185]. It is quite established that hypoxia induces the
expression of VEGF [81, 86, 87]. The effects of hypoxia are mainly mediated by
hypoxia-inducible factor-1 (HIF-1) protein complex, which is composed of two
subunits, HIF-l, the inducible unit, and HIF-1, the constitutive unit [88]. In
endometriotic tissue, abnormally high levels of complex HIF-l were found [89],
which makes clear that hypoxia is involved in VEGF production in endometriotic
lesions and presumably by peritoneal macrophages.
It is of note that peritoneal macrophages strongly express Cox-2 [90], one of the
rate-limiting enzyme for PGs secretion. As described in several reports [26, 84, 91,
92], PGE2 and PGF2 themselves induce the over-expression of Cox-2 in macro-
phages and endometriotic stromal cells, leading to an elevated concentration of
their own levels in the peritoneal fluid. On one hand, high levels of PGs act on
macrophages to suppress their phagocytic ability by down-regulating MMP-9 and
CD-36 [37, 39]. On the other hand, PGs stimulate the steroidogenic capacity of
endometriotic stromal cells by up-regulating steroidogenic acute regulatory protein
(StAR) and aromatase [93], which induces aberrant biosynthesis of E2. E2 further
stimulates the production of mitogens such as fibroblast growth factor-9 (FGF-9),
which induces endometriotic cell proliferation [94, 95]. PGE2 can also induce
FGF-9 expression by PGE2 receptor 3-dependent transcriptional up-regulation
[96, 97]. Other main functions of PGs include the induction of the expression of
angiogenic factors such as VEGF and FGF-2 to induce angiogenesis [98, 99]. These
effects may play an important role in the survival and proliferation of endometriotic
cells. Furthermore, elevated PGs concentrations in endometriosis women is
suspected to be involved in pelvic pain and infertility, though more studies will
be required to further elucidate the underlying mechanisms [90, 100, 101].
Obviously, macrophages have a rather heterogeneous array of characteristics
and dramatically modify their environment not only via the production of cytokines
and other pro-inflammatory factors but also via reactive oxygen species (ROS)
[102104]. Production of ROS is known to increase after activation of immune
cells, especially macrophages [105] and their production was reported to be
increased in serum and peritoneal fluid of patients with endometriosis. In addition,
markers of oxidative stress have been found elevated [102104]. ROS role, as a
second messenger of cellular proliferation, has been described. McCubrey
et al. found that normal cell proliferation correlated with production of endogenous
ROS through the activation of growth-related signalling pathways [106]. Endome-
triosis is considered a benign disease but shares some features with cancer, such as
propensity to invasion, unrestrained growth, neo-angiogenesis, and distant spread-
ing [19, 107]. The known correlation between ROS and proliferation of cancer
cells, along with the increased production of ROS in response to chronic inflam-
mation in endometriosis, thus suggests a possible role for ROS in the regulation of
endometriotic cell proliferation.
6.4 Deleterious Effects Induced by Macrophages

in Endometriosis
The available literature argues in favour of a significant role for macrophages in the
growth and development of endometriotic lesions, the generation of pain through
interaction with nerve fibres and infertility via the impediment of spermatozoids
and/or oocyte functions and endometrial receptivity. It is obvious that little evi-
dence is available as to the existence of a direct cause and effect relationship
between endometriosis-associated macrophage dysfunctions and pain or infertility.
However, chronic pelvic inflammation in women with endometriosis and many
inflammatory and embryotoxic factors involved, abnormally expressed and known
for being secreted by activated endometrial and peritoneal macrophages let, in an
indirect way, believe in such a role.
6.4.1 Macrophages in Pain Related to Endometriosis
The pathophysiology of pelvic pain associated with endometriosis and macro-

phages is unclear, especially that there is no correlation between the existence of
pain and the extent of damage. Main inflammatory factors, such as PGs (e.g. PGE2
and PGF2) or cytokines (e.g. MIF), released by macrophages can also irritate
tissues, stimulate nerve fibres and cause severe, painful reaction even when very
small areas are involved. The eutopic endometrium of women with endometriosis
has higher rates of MIF than the eutopic endometrium of normal women; this is
correlated with the degree of advancement of the disease. In addition, this increase
of the MIF expression is even more obvious in patients with pelvic pain [71]. It is
interesting to note that MIF concentration varies during the menstrual cycle. This
variation of expression suggests that MIF would be dependent of ovarian hormones
[108]. Recent studies have also shown that MIF stimulates Cox-2 expression and
PGE2 production in the ectopic endometrial cells [74] leading to chronic
inflammation.
The density of nerve fibres in peritoneal endometriotic lesions is much higher

than in normal peritoneum. Many inflammatory substances can potentially stimu-
late the nerve endings of these fibres. Recent studies have demonstrated that VEGF
can also act as potent neurotrophic factor in addition to its effects on vessels
[109, 110]. But the most important growth factor that plays a role in facilitating
development, growth and repair of nerve fibre is the nerve growth factor (NGF).
An elevated concentration of PGE2 is thought to stimulate production of NGF [111]
which is released from macrophages, as well as endometriotic lesions, and induces
smooth muscle metaplasia and innervations. Subsequently, the contraction of
smooth muscle cells and hyperalgia of sensory nerve cells, derived from innerva-
tions in interstitial lesions, induce endometriotic pain [112].
PGE2 and PGF2 play an important role in inflammation by increasing the
vascular permeability, cause of oedema, by generating a fever or by regulating
blood flow and are also involved in the sensation of pain [113, 114]. Over their
nociceptive properties, PGs seem to be involved in pelvic pain often present in
women with endometriosis. Cyclooxygenase inhibitors, such as nonsteroidal anti-
inflammatory drugs, are frequently used in the treatment of mild endometriosis and
can significantly reduce pain in these patients [90]. The inhibition of certain
macrophage activities may have a benefit in the future relief of endometriosis-
related pain [22].
In conclusion, several relevant studies provide a plausible link between perito-
neal inflammation, increased macrophage number and the development of new
nerve fibres throughout the peritoneal cavity, which may be associated with the
generation of the pelvic pain sensations in women with endometriosis.
6.4.2 Macrophages in Infertility Related to Endometriosis
It is now well known that macrophages are located into decidua (endometrium of
the pregnant uterus) during the implantation window and implicated in the physi-
ological processes of implantation, establishment and maintenance of pregnancy
and labour control. Indeed, decidual macrophages (2025 %) and uNK cells are
predominant decidual leukocytes [115, 116]. While other uterine leukocytes are
diminished during pregnancy, macrophages proportion remains unchanged. During
implantation, macrophages cooperate with developing embryo to insure trophoblast
endovascular invasion and anchorage. At this site, only M2-macrophages are found
infiltrated into endometrial basalis where they will secrete MMP-7 and MMP-9
degrading extracellular matrix to facilitate trophoblast invasion into myometrium
[117]. Moreover, M2-macrophages are able to produce VEGF and placental growth
factor (PlGF) implicated in the profound remodelling of uterine vasculature during
embryonic growth. Furthermore, those phagocytes participate in maternal cell
apoptosis and trophoblast renewal. In fact, maternal decidua cell apoptosis insures
trophoblast invasion to remodelling and development of embryo. It is important to
notice that phagocytosis of apoptotic bodies are vital to avoid secondary necrosis
and so a harmful inflammatory response generating tissue damage [118, 119]. Even
more, macrophages are fundamental actors of maternal immune tolerance in preg-
nancy. Indeed, these immune cells are not only able to secrete molecules such as
IL-10, known to inhibit T cell activation, but are also to diminish expression of
co-stimulatory molecules (CD-80 and CD-86) and indoleamine 2,3-dioxygenase
[116]. They create a microenvironment capable of containing immune response to
hemi-allogeneic fetal cells even during infection [120]. Interestingly, macrophages
secrete IL-15, an uNK cell chemoattractant, and down-regulate uNK cell cytotoxic
capacity [121]. uNK cells are crucial for implantation knowing that they are
responsible for trophoblast chemoattraction and invasion by secreting IL-8 and
IFN-inducible protein-10 that binds to trophoblasts membrane receptors [122].
Intriguingly, in endometriosis, the number of M2-macrophages is significantly
increased in decidua and peritoneal fluid. As explained in the previous section,
macrophages are differentially activated in endometriosis overproducing a myriad
of pro-inflammatory factors and key processes are crucial for implantation. Decid-
ual macrophages in endometriosis have been shown to secrete elevated concentra-
tions of angiogenic factors (VEGF, MCP-1, IL-8) and to mediate the development
of an anarchic vasculature similar to unstructured tumour vasculature [123,
124]. Such an abnormal angiogenic process might disfavours the functional
crosstalk between maternal decidua and trophoblasts, which is essential for embryo
survival [125]. Acute and controlled inflammation is necessary for pregnancy, but
any chronic inflammation leads to spontaneous abortion [126]. As described pre-
viously, women with endometriosis are characterised by an exaggerated inflamma-
tion that may contribute to infertility in endometriosis [127]. First, disruption of the
phagocytic function in endometriosis may promote accumulation of apoptotic
bodies and secondary necrosis responsible for inflammation. Second, the cocktail
of inflammatory molecules secreted in endometrium generates an acute inflamma-
tion, which is deleterious for trophoblastic implantation. In fact, some relate
miscarriage to TNF- increase and normal pregnancy with high concentrations of
IL-10 [128]. Chronic inflammation may inhibit trophoblast invasion and immune
tolerance [129]. Macrophage phagocytic function is also an issue since sperm
phagocytosis has been described in endometriosis patient. Moreover, an activation
of specific sperm engulfment surely prevents or disturbs oocyte fertilisation [32]. It
seems that endometrial cells express or secrete molecules able to impair phagocy-
tosis in macrophages, but further investigations could provide interesting insights
on this phenomenon. In the meantime, endometriosis is also characterised by an
oxidant environment inauspicious for pregnancy [130]. Both endometriotic cells
and macrophages are responsible for ROS high concentration in peritoneal fluid of
women with endometriosis. More precisely, endometriotic lesions activate induc-
ible nitrogen oxide synthase (iNOS) in macrophages leading to an augmentation of
NO production [131]. Moreover, the antioxidant machinery (secreting ascorbic
acid, GPx, thiol) is down-regulated creating an imbalance in the redox status of
peritoneal environment [132134]. Interestingly, ROS are known to be related to
age-related fertility decline, in vitro fertilisation failure and cigarette smoke
subfertility [135137]. In endometriosis, such redox status may affect sperm via-
bility in peritoneal microenvironment, oocyte and embryo quality [130].
Since macrophages are crucial cells in implantation and pregnancy, it is not
surprising that their dysfunctions may be a significant possible cause of infertility in
endometriosis.
6.4.3 Endometriosis, Macrophages and Cancer
In recent years, a possible link between endometriosis and certain types of cancer,
such as ovarian cancer, has been suggested. It is well established that endometriosis
shares a number of features with cancer such as abnormal cell proliferation and
invasion, the development of new blood vessels [138, 139], the decrease in the
number of cells undergoing apoptosis [140] and its stem cell-like activity
[141]. Macrophages are important inflammatory mediators and have been impli-
cated in endometriosis through their role in chronic inflammation. As discussed
previously in this chapter, macrophages produce a wide range of cytokines which
can also induce the progression of tumorigenesis [78]. IL-1 is a potent mediator of
inflammation produced by macrophages and it promotes tumorigenesis by stimu-
lating the production of other cytokines and growth factors. Among these, VEGF
has been detected in endometriosis-associated ovarian carcinoma [142]. In addi-
tion, it has been proposed that VEGF acts as a growth factor for tumours regardless
of its role in angiogenesis as there was no correlation observed between VEGF
expression and micro-vessel density in ovarian tumours [139].
Taken together, it seems that macrophages might play a role in the development
of cancerous transformation at the site of endometriosis by secreting important
mediators which are responsible for tumour progression and development. How-
ever, further studies are required to ascertain any possible link between endome-
triosis and cancer.
6.5 Animal Models to Study the Role of Macrophages

in Endometriosis
In order to study endometriosis in vivo, an animal model of the disease is required

where endometriosis could be induced experimentally by implanting the human
endometrial tissue intra-peritoneally. Since endometriosis occurs spontaneously
only in humans and some non-human primates, animal models of induced endo-
metriosis have been developed and are of high value for the evaluation of patho-
physiological mechanisms underlying the development of this disease. There are
two main groups of animal models for endometriosis: rodents and non-human
primates. This section of the chapter will summarise the study of macrophages in
the animal models for endometriosis.
6.5.1 Rodents
Mice and rats are the two established experimental animal models among
the rodents. Since rodents do not shed their endometrial tissue and therefore
do not develop endometriosis spontaneously, endometriosis can be induced by
transplanting endometrial tissue to ectopic sites. These models are classified into
two types, homologous and heterologous models. Homologous models have been
employed utilising the surgical transplantation of endometrium of the same or
syngeneic animals in immunocompetent animals, whereas in heterologous models,
human endometrial fragments are transferred either intra-peritoneally or subcuta-
neously to immunodeficient mice.
Nude and Knockout Mice. As mentioned, animal models represent a useful tool
to study in vivo early steps of this disease. The latter approach relies on the transfer
of fragments of endometrial tissue harvested from syngeneic donor mice and
recapitulates important aspects of the disease [143]. The first experimentally
induced endometriosis in mice was reported in 1984 [144], where normal and
ectopic human endometrial tissues were successfully transplanted into the perito-
neal cavity of athymic nude mice [144]. This study demonstrated that endometrial
tissue keeps intact structure and shows the presence of glands and stroma with an
infiltration of macrophages. Since then, several groups have used fluorescent
markers such as green fluorescent protein [16] or bioluminescent markers such as
luciferase expression system to generate endometriosis mouse models [145]. Recent
studies on Tie-2 knockout mice demonstrated that alternatively activated macro-
phages can infiltrate endometriotic lesions and promote angiogenesis
[146, 147]. These data indicate that the recruited macrophages have more than
one effect. Also, it has been shown that knockdown of annexin A2 inhibited the
phagocytic function of macrophages, whereas treatment with annexin A2 recom-
binant protein enhanced phagocytosis [148]. In addition, we have successfully used
the athymic nude mice model to study the role of MIF in the development of
endometriosis [149]. As described previously in this chapter, MIF has been a
regulator of immune system that promotes the pro-inflammatory functions of
immune cells, and its role in angiogenesis, tumorigenesis and autoimmune diseases
is well established [74, 150154]. We have reported previously that expression of
MIF is increased in eutopic endometrial tissue of women with endometriosis, which
is related to the stage of endometriosis [71]. Furthermore, our research in agreement
with others has shown a significant elevation in circulating and local peritoneal
levels of MIF [43, 44]. Taking the advantage of athymic nude mice model of
experimentally induced endometriosis, we have developed a treatment model of
endometriosis. In this study, we have used the in vivo model of experimentally
induced endometriosis and challenge MIF in order to discover a possible target for
the treatment of endometriosis. We used (S,R) 3-(4-hydroxyphenyl)-4,5-dihydro-
5-isoxazole acetic methyl ester (ISO-1), a specific antagonist of MIF [155] where
human endometrial tissue was allowed to implant and grow prior to any treatment.
Thus our group is the first to report that macrophage migration inhibitory factor can
be a suitable target for the treatment of endometriosis as there is no specific targeted
treatment available at present [149].
Rat. Among the rodents, rat is another choice as an experimental model for the
endometriosis for the researchers. Matsubavashi et al. reported the first study where
rats with auto-transplanted endometrium showed the same immunologic changes as
humans with endometriosis [156]. In this study the effect of ectopic endometrial
lesions on the changes of leukocyte subpopulation in the rat model of endometriosis
was reported. This was supported by another independent research where it was
reported that the stromal tissue of uterus-attached peritoneum showed proliferation
and infiltration of macrophages in rat endometriosis models [157]. Furthermore, it
was discovered that an increase in the number of activated macrophages in the
endometriotic lesions has a positive correlation with VEGF [158]. Recently, lipo-
somal bisphosphonate [159] and a selective Cox-2 inhibitor [158] have been used as
therapeutic drugs targeting macrophages in a rat experimental endometriosis
model. These findings have suggested that macrophage depletion effectively
inhibits the initiation and growth of endometriotic implants in a rat endometriosis
model, and further studies are required to confirm these findings in order to use this
approach as a treatment for endometriosis.
6.5.2 Non-human Primates
In the recent past, researchers have started using rhesus macaque (aka rhesus
monkey) as an animal model for endometriosis since spontaneous development of
the disease requires menstrual shedding. Endometriosis occurs naturally only in
some non-human primate species, making development of lesions more compara-
ble to the establishment of disease in humans. Compared with rodents, the
non-human primate model of endometriosis is advantageous due to a close reca-
pitulation of human disease and physiology [160]. In a recent study, it has been
shown that the activation status of macrophages in endometriosis in the rhesus
monkey is more oriented towards the M2 phenotype, in exactly the same way as
humans [161].
Concisely, non-human primates have been extensively used for the investigation
of endometriosis, but the very high cost of animal handling limits their use. For this
reason, the establishment of rodent models for endometriosis via the intra-
peritoneal transplantation of pieces of endometrial tissue has been greatly exploited
in the recent years.
6.6 Future Research and Perspectives
Altogether, it is evident that impaired functions of macrophages enable

endometriotic tissue proliferation and their secretory products are implicated in
the pathophysiology of endometriosis and the major symptoms of the disease
such as pain and infertility. Presently, there is an urgent need for new approaches
to the medical treatment of endometriosis. Since it is obvious that macrophages are
closely associated with endometriosis, future treatment strategies may be based on
immunological approaches. Currently, treatments are mostly focused on cytokines
and growth factors secreted by macrophages which showed abnormal concentra-
tions in the peritoneal fluid and peripheral blood of endometriosis patients. To
regulate angiogenesis, the effect of anti-VEGF antibody has been investigated with
an endometriosis mouse model. Bevacizumab, first used for cancer chemotherapy,
has been shown to decrease VEGF levels in serum and prevent lesion
vascularisation and thus trigger endometriotic cell apoptosis [162]. The most
promising molecule is metformin, an anti-inflammatory and aromatase inhibitor
known as a biguanide insulin sensitiser. Metformin is able to decrease IL-8, IL-6
and VEGF concentration and improve endometriosis symptoms in women with
endometriosis [163]. In the same field, statins seem to prevent MCP-1 expression
and to reduce the number and size of endometriotic implants in mice [164]. How-
ever, there is no treatment available targeting specifically and directly macro-
phages. An increased understanding of the immune aspects in endometriosis
would be beneficial to set up such novel treatment strategies. Inhibiting MIF
might be a promising strategy for future therapies targeting endometriosis, and
recent in vivo data from our lab using ISO-1, a potent MIF inhibitor, support this
hypothesis [149]. Other approaches to MIF inhibition may include anti-MIF or anti-
MIF receptor antibodies. Moreover, macrophages can express and secrete various
molecules depending on their activation status. Clearly, in endometriosis, perito-
neal macrophages show a different secretory and membrane profile. It would be
interesting to target macrophage activation and polarisation in order to control the
disease. Actually, some strategies already focus on a modulation of macrophage
polarisation. For example, in ovarian cancer administration of INF- induces
macrophage phenotype modifications promoting tumoricidal activity [165,
166]. Similar treatment strategies applied to endometriosis could open new prom-
ising avenues for the management of this disease.
In summary, a growing body of evidence indicates that a combination of
endometrial and immune cell dysfunctions plays a major role in the pathogenesis
of endometriosis and its major symptoms. Macrophages appear to be critically
involved via a wide spectrum of secretory products and functional changes that for
the most part remain to be elucidated. A better understanding of the role of
macrophages and its intriguing and particular interaction with ectopic endometrial
cells is crucial for the development of new medical treatments for this serious
disease.
During menstruation, endometrial cells reach the peritoneal cavity as a conse-

quence of retrograde menstruation. These cells resist apoptosis and immune sup-
pression, activate the recruitment of immune cells, particularly macrophages, which
have a reduced phagocytic ability, and contribute to angiogenesis, pain innervation,
abnormal steroid production and ectopic endometrial cell growth. The reciprocal
interactions of misplaced endometrial cells and macrophages create deleterious and
activating feedback loops that trigger chronic inflammation and other processes
involved in endometriosis pathophysiology.
Acknowledgement This study is supported by CIHR grants MOP 93716, 120769 and 123259 to
Pr. Ali Akoum, Chercheur National, FRQ-S.
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Chapter 7
Inflammation and Cytokines
in Endometriosis
Tomio Iwabe and Tasuku Harada
Abstract Endometriosis, a common disease among women of reproductive age, is

characterized by the presence and growth of endometrial tissue (glands and stroma)
outside the uterus. Dysmenorrhea and infertility common in endometriosis compro-
mise the quality of life of reproductive age women. Despite a long history of clinical
experience and experimental research, endometriosis remains an enigma, and its
pathogenesis is still controversial. The peritoneal fluid (PF) of women with endome-
triosis contains an increased number of activated macrophages that secrete a variety
of local products, such as growth factors and cytokines. In this chapter, we review the
current understanding of the role of cytokines in the pathogenesis of endometriosis.
Keywords Angiogenesis Cytokine Peritoneal fluid Retrograde menstruation

Transcriptional factor
7.1 Introduction
Endometriosis was first acknowledged more than about 100 years ago, and the usual
definitions of this disease are based on histology and related to glands, stroma,
hemosiderin, and fibromuscular metaplasia [1]. Endometriosis, which occurs in
10 % of reproductive age women, is characterized by the growth of endometrial-
like tissue outside the uterus. Dysmenorrhea and infertility, which are common
symptoms of endometriosis, compromise the quality of life. This disease is thought
to be estrogen dependent. However, the pathogenesis of endometriosis is poorly
T. Iwabe (*)
Sanin Rosai Hospital, Yonago, Japan
e-mail: iwabe@saninh.rofuku.go.jp
T. Harada
Tottori University, Yonago, Japan

88 T. Iwabe and T. Harada
understood despite decades of experimental and clinical study. Endometriosis is

associated with infertility even among affected women who ovulate and have
anatomically patent fallopian tubes. The exact mechanism by which endometriosis
interferes with fertility is not known, but data suggest that an aberrant immunolog-
ical mechanism is involved in its pathophysiology. An important general concept is
that of endometriosis as a local pelvic inflammatory process with altered function of
immune-related cells in the peritoneal environment. Supporting this concept are
recent studies suggesting that the peritoneal fluid (PF) of women with endometri-
osis contains an increased number of activated macrophages that secrete a variety
of local products, such as growth factors and cytokines. Therefore, secreted
cytokines in chronic pelvic inflammation is a key factor in the pathogenesis
of endometriosis [2].
7.2 Peritoneal Environment
7.2.1 Retrograde Menstruation
Retrograde menstruation, in which fragments of endometrium are refluxed through

the fallopian tubes into the peritoneal cavity, occurs only in women and nonhuman
primates and in a few exceptional species, such as the elephant and bat. Spontaneous
endometriosis, which is clinically confirmed endometriosis in nature, has been
reported only in humans and some primates, the rhesus macaque (Macaca mulatta),
the Japanese macaque (Macaca fuscata), the pig-tailed macaque (Macaca
nemestrina), and the Kenya baboon (Papio doguera) [3]. The reproductive organs
of women are anatomically similar to that of female baboons; therefore these
nonhuman primates are thought to be an adequate animal experimental model.
Patients with endometriosis almost always have patent tubes; thus blood and endo-
metrial fragments are able to pass out of the uterus by way of the tubes (Fig. 7.1).
Cyclic and retrograde menstruation are common phenomena in both humans and
these animals. Several studies suggest that retrograde menstruation strongly relates
to endometriosis. Shorter cycles and heavier and longer menstrual flow often occur
in women with endometriosis [4]. From the above observation, retrograde menstru-
ation is one of the most important factors in the pathophysiology of endometriosis.
The most widely accepted hypothesis for endometriosis is Sampsons theory of
retrograde menstruation [5]. Retrograde menstruation occurs in 7690 % of inves-
tigated women, and endometrial cells have been observed in the peritoneal fluid of
5979 % of women during menstruation or the early follicular phase; however,
endometriosis was diagnosed in only 10 % of this population [6, 7]. Endometriosis
was hypothesized to be caused by decreased clearance of peritoneal fluid in
endometrial cells due to reduced natural killer (NK) activity and/or decreased
macrophage activity [8]. However, the development of endometriosis cannot be
explained by this phenomenon. Recently, Khan et al. reported that the menstrual
7 Inflammation and Cytokines in Endometriosis 89
Fig. 7.1 Laparoscopic findings in pelvic cavity during menstrual period. (a) Retrograde menstru-
ation from fimbria. (b) Cul-de-sac
blood of women with endometriosis is more frequently contaminated with

Escherichia coli than that of the controls and corresponds to higher levels of
endotoxin in the menstrual fluid. Consequently, endotoxin levels are high in the
PF due to reflux of menstrual blood into the pelvis. The colony formation of E. coli
and endotoxin levels in the menstrual fluid was markedly higher in women
with red lesion in r-ASRM stage III endometriosis patients than in women with
endometrioma of stage IIIIV endometriosis [9]. We also reported LPS
(endotoxin)-promoted proliferation and invasion of endometriotic stromal cells
via upregulation of cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) [10].
These findings, including that of bacterial contamination, would be a suitable area
for future research into the pathophysiology of endometriosis.
7.2.2 Peritoneal Fluid
Peritoneal fluid (PF) containing immune-related cells is often seen in the vesico-
uterine cavity or the pouch of Douglas during gynecologic surgery. PF bathes the
pelvic cavity, uterus, fallopian tubes, and ovaries and may be a major player
controlling the peritoneal microenvironment that influences the development and
progression of endometriosis and endometriosis-associated infertility.
The peritoneal cavity is normally empty except for a thin film of fluid that keeps
surfaces moist. Peritoneal fluid arises primarily from two sources: plasma transu-
date and ovarian exudate. Other sources of peritoneal fluid are tubal fluid, retro-
grade menstruation, and immune cell secretions. Peritoneal fluid depends on
follicular activity, corpus luteum vascularity, and hormonal production. The vol-
ume of PF within the peritoneal cavity varies during the menstrual cycle, reaching a
peak of 20 mL at the time of ovulation [11].
Changes in fluid volume and the presence of various cells, hormones, and other
compounds during normal menstrual cycles and in pathologic conditions have been
described. Syrop and Halme analyzed PF volume in 426 patients and found that
women with endometriosis had a greater PF volume than fertile controls, patients
with adhesive disease, or those with unexplained infertility [12]. The PF volume in
women with unexplained infertility was also higher than in controls. Therefore,
increased PF volume may be commonly associated not only with endometriosis but
also with long-lasting, unexplained infertility.
PF contains various free-floating cells, including macrophages, mesothelial
cells, lymphocytes, eosinophils, and mast cells. Normally, PF contains leukocytes
in concentrations of 0.52.0 106 mL1, of which approximately 85 % are mac-
rophages. Halme et al. postulated that peritoneal macrophage activation might be a
central contributor to the pathogenesis of endometriosis [13]. Activated macro-
phages in the peritoneal cavity of women with endometriosis are potent producers
of cytokines. Thus, PF contains a rich cocktail of cytokines. The cytokines are
multifunctional proteins whose biological properties suggest a key role in hemato-
poiesis, immunity, infectious disease, tumorigenesis, homeostasis, tissue repair,
and cellular development and growth.
7.3 Cytokines
7.3.1 Cytokines in Peritoneal Fluid
Cytokines, a large family of more than 100 low molecular weight proteins that
function as growth and differentiation factors and immune cell modulators, also
play a major role in the regulation of immune and inflammatory responses. Immune
cell activation results in a burst and cascade of inflammatory cytokines. These
cytokines have pleiotropic and redundant activities culminating in the recruitment
of numerous cell types to the site of inflammation.
The development of enzyme-linked immunosorbent assay has made it possible
to measure a number of cytokines in the PF of women with endometriosis. These
include interleukin-1 (IL-1) [14], IL-4 [15], IL-5 [16], IL-6 [1721], IL-8 [2224],
IL-10 [20, 25, 26], IL-12 [27, 28], IL-13 [29], IL-17 [30], IL-23 [31], IL-33 [32],
interferon (INF) [19], tumor necrosis factor (TNF) [14, 21], RANTES [33],
monocyte chemotactic protein-1 (MCP-1) [3436], macrophage colony-stimulating
factor (MCSF) [37], transforming growth factor (TGF) [38], and vascular
endothelial growth factor (VEGF) [39, 40]. A number of studies report that the
level of many cytokines is increased in the PF of women with endometriosis.
Cytokines may regulate the actions of leukocytes in the PF or could act directly
on the ectopic endometrium, where they may play various roles in the pathogenesis
and pathophysiology of endometriosis.
7.3.2 Source of Cytokines
Increased levels of cytokines in the PF of women with endometriosis may reflect

increased synthesis of cytokines by the peritoneal macrophages, lymphocytes,
ectopic endometrial implants, and/or mesothelial cells of the peritoneum, all of
which are capable of cytokine production [41, 42]. The main source of cytokines is
thought to be the macrophages, which originate in bone marrow, circulate as
monocytes, and then migrate to various body cavities.
7.3.2.1 Macrophages
Macrophages are main regulators of the innate response to injured, infected, and
neoplastic tissues. The peritoneal macrophages (PMs) are the major resident cells in
the peritoneal cavity. They kill cells, such as retrograde endometrial tissues, and
their presence is commonly associated with an inflammatory process. Most studies
revealed increased cell numbers and activity of PMs in cases of endometriosis,
although some studies did not [43, 44]. The increased number of PMs in women
with endometriosis may indicate that the presence of endometrial tissue in the
peritoneal cavity represents a foreign entity and needs to be removed. Activated
PMs might synthesize and secrete different cytokines into the PF including various
cytokines and growth factors.
7.3.2.2 T-Lymphocytes
T-lymphocytes are also implicated in the pathogenesis of endometriosis. T-helper

cells can be classified into two subsets: type 1 (Th1) and type 2 (Th2). Th1 cells
produce IL-2, IL-12, and INF, which are potent inducers of cell-mediated immu-
nity. Th2 cells produce mainly IL-4, IL-5, IL-10, and IL-13, which are involved in
the suppression of cell-mediated immunity. Hsu et al. investigated the expression of
Th1 (IL-2 and INF) and Th2 (IL-4 and IL-10) cytokines in the peripheral blood
monocytes and PF from patients with endometriosis [15]. They found that cytokine
secretion by Th1 and Th2 cells is altered in women with endometriosis. The shift in
the balance of Th1/Th2 toward the Th2 arm may contribute to the derangement of
an immunologic defense mechanism in endometriosis. Interleukin-10 is well
known to be an anti-inflammatory cytokine in regulating inflammatory responses.
We also observed that IL-10 expression levels in endometriotic stromal cells were
lower than in endometrial stromal cells [26].
7.3.2.3 Others
Recent studies suggest that endometriotic implants also produce cytokines [45, 46].
We demonstrated that endometriotic cells constitutively express IL-6 mRNA and
produce IL-6 protein and that adding TNF stimulated IL-6 gene and protein
expression in a dose-dependent manner [47]. When we compared IL-6 production
by macrophages and endometriotic stromal cells in patients with endometriosis,
similar levels of IL-6 were found to be produced in stromal cells derived from an
endometrioma and by macrophages under basal- and TNF-stimulated conditions.
Numerous leukocytes are harbored in both stromal and intraepithelial parts of
normal eutopic endometrium. In women with endometriosis, the member of lym-
phocytes is increased both in eutopic and ectopic endometria. We postulated that
immune cells, such as leukocytes or lymphocytes, and endometriotic tissue
interacted via the paracrine mechanism on the source of the cytokines. Therefore,
they may contribute to cytokine production in PF and be involved in cellular growth
and inflammatory reaction. The findings suggest that endometriotic tissue may be
another important source of this cytokine.
7.3.3 Role of Cytokines
The pathogenesis of endometriosis is still a matter of debate, despite extensive

research efforts since Sampsons landmark article in 1927 [5]. Sampsons theory of
retrograde menstruation, which describes endometrial cells that may attach,
implant, and grow, seems plausible because peritoneal lesions are most frequently
found in the ovaries and the posterior cul-de-sac where regurgitated menstrual
material pools.
One study demonstrated that endometrium can attach to the mesothelial surface
of the peritoneum in vitro [48]. The authors described that in all cases of adhesion to
intact mesothelium, the endometrium was attached via stromal cells. Another
theory, the metaplasia theory, is also attractive since it can explain some rare
cases of endometriosis, such as those with the absence of menstruation
(RokitanskyKusterHauser syndrome) [49]. A recent in vitro study supports the
metaplasia theory [50]. Unfortunately, neither theory can explain all cases of
endometriosis.
7.3.3.1 Implantation of Endometrium
Recent studies demonstrated increased IL-6 production by endometriotic cells in

both basal- and cytokine-stimulated conditions compared with their normal coun-
terpart [45]. Tseng et al. examined eutopic endometrium from patients with endo-
metriosis and found an increased basal- and IL-1-stimulated production of IL-6
compared with patients without endometriosis [46]. This is an important aspect for
recent investigation because it suggests that endometrial cells of women who
develop endometriosis may function differently from those who do not.
In order to implant and grow, endometrial cells must establish cellcell or cell
extracellular matrix (ECM) interactions with the peritoneal lining. In these circum-
stances, cell adhesion molecules are of great importance [51]. A recent report
clearly showed that endometrial stromal cells are the critical cells in endometrial
attachment to the mesothelial surface of the peritoneum and that endometrial
epithelial cells fail to attach to the mesothelium [48]. Most of these interactions
between endometrial cells and ECM are mediated by the integrin family of cell
surface receptors, which are capable of transducing intracellular signals. It has also
been suggested that cellularadhesion itself stimulates chemokine expression [52].
Garcia-Velasco and Arici showed that increasing the dose of IL-8 stimulates
endometrial stromal cells ability to adhere to an ECM protein, fibronectin [53].
They also showed that the adhesion of endometrial stromal cells to different ECM
proteins induces variable levels of IL-8 gene expression and protein secretion and
that this event is integrin-mediated [54]. IL-8 may be relevant for the attachment of
endometrial implants in the pathogenesis of endometriosis.
According to Sampsons theory of retrograde menstruation, deficient cellular
immunity, in particular impaired natural killer (NK) cell function, is one of the
etiological factors that could contribute to the survival and implantation of refluxed
endometrial cells. Several investigators showed a decrease in NK cell activity in the
PF of women with endometriosis compared with women without endometriosis
[8, 55, 56]. This observation suggests that the clearing mechanism of retrograde
menstruated endometrial cells may be impaired in women with endometriosis
because of a defect in the local immune defense system. Oosterlynck et al. found
increased TGF activity in the PF of women with endometriosis [37]. Transforming
growth factor may be a cytokine that inhibits NK activity in the PF of women with
endometriosis.
Intercellular adhesion molecule (ICAM)-1-mediated cellcell adhesion is essen-
tial for various immunological functions, including NK cell-mediated cytotoxicity
against endometrium. Recently, Somigliana et al. reported that soluble intercellular
adhesion molecules (sICAM)-1 were constitutively shed from the surface of endo-
metrial stromal cells obtained from patients with endometriosis into the culture
medium [57]. The enhanced release of sICAM may allow the endometrial stromal
cells of patients with endometriosis to escape immunosurveillance and, therefore, to
implant in ectopic sites. More interestingly, sICAM-1 production from the macro-
phages of patients with endometriosis was upregulated by INF and IL-6 [58].
Interleukin-12 acts on T and NK cells, inducing cytokine production (primarily
INF), enhancing NK cell cytotoxic activity, and favoring the generation of
T-helper 1 response [59, 60]. Concentrations of IL-12 in the PF are low regardless
of the presence or absence of endometriosis, but they are detectable [26]. The
administration of IL-12 was recently shown to significantly prevent ectopic endo-
metrial implantation in a murine model of endometriosis [61]. A direct growth
inhibitory effect on endometrial cells seems unlikely since endometrial cells do not
express receptors for IL-12. A potential explanation for these results is that IL-12
may enhance the growth and augment the cytolytic activity of both NK and T cells.
These data support the idea that manipulation of cytokine activity in PF is a novel
management approach to controlling the establishment of endometriosis.
7.3.3.2 Angiogenesis
Angiogenesis seldom occurs in adult organs with normal tissues under physiologic
conditions. The endometrium represents an exception: the tightly regulated fluctu-
ation of ovarian steroids, estrogen and progesterone concentrations, cyclically
triggers the remodeling of the organ vasculature, with angiogenesis and
lymphangiogenesis. Angiogenesis, which is the process of generating new capillary
blood vessels, occurs in a variety of normal and pathologic processes. It consists of
the following steps: dissolution of the basement membrane by the protease derived
from vascular endothelial cells, migration and proliferation of the endothelial cells,
and formation of the capillary tube [62]. Each step is regulated by various angio-
genic factors. Neovascularization is likely to be required for the implant to grow
beyond 23 mm during tumor growth [63]. An angiogenic mechanism could be
involved in the pathogenesis of endometriosis. We can postulate that further
outgrowth of these ectopic endometrial implants will depend on new capillary
growth according to several studies that indicate that tumors are angiogenesis
dependent [64].
Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor of
3046 KDa, which is active as a disulfide-linked homodimer and is a potent
mitogen, morphogen, and chemoattractant for endothelial cells. The angiogenic
activity of PF, as well as levels of VEGF in PF, is elevated in women with
endometriosis [38, 65]. McLaren et al. demonstrated that PF macrophages are the
principal source of the angiogenic growth factor, VEGF, and that the anti-VEGF
antibody abolished the enhanced endothelial cell proliferation induced by a condi-
tioned medium from macrophages isolated from the peritoneal cavity of women
with endometriosis [39]. McLarens study suggests that activated macrophages are
a major source of VEGF in endometriosis and that estradiol and progesterone
directly regulate this expression. Since endometriosis is characterized by pro-
nounced vascularization within and surrounding the ectopic tissue, elevated levels
of the potent angiogenic growth factor, VEGF, in the PF and the presence of VEGF-
positive macrophages within the ectopic tissue are clinically important in this
disease. VEGF-induced angiogenesis may therefore be a critical aspect of the
pathophysiology of endometriosis.
IL-8, which is a chemoattractant for neutrophils and an angiogenic agent, induces
the proliferation of human melanoma and glioma cells [66, 67]. Arici et al. reported
that IL-8 is produced in the human endometrium in vivo, mainly in glandular cells,
and that IL-8 induces proliferation of endometrial stromal cell as a potential auto-
crine growth factor [68, 69]. We demonstrated that IL-8 exerts its growth-promoting
actions in endometriotic as well as in normal endometrial cells [24, 70].
TNF, a secretory product of activated macrophages and a potent inducer of new

blood vessel growth, also stimulates proliferation of endometriotic stromal cells.
These angiogenic cytokines may play a role in the angiogenesis of endometriosis.
Hypoxia is the stimulus that triggers vessel remodeling in injured and
regenerating tissues as well as in tumors. It elicits an adaptive response, which is
largely mediated by the hypoxia-inducible transcription factor-1 alpha (HIF1),
under hypoxic conditions. HIF1 translocates to the nucleus where it enhances and
accelerates the transcription of genes with appropriate response elements, including
angiopoietin 2 (Ang-2), CXCL12, and VEGF [7173].
In experimental models of endometriosis, early phases of lesion establishment
are characterized by a transient hypoxia, which results in the upregulation of
HIF1, with downstream expression of VEGF [74, 75]. Limited ischemia of the
endometrium in the early and middle secretory phase occurs: the event is apparently
associated with the upregulated expression of VEGF in the late secretory phase of
the menstrual cycle in endometriotic women [76]. Interestingly, endometrial frag-
ments from women in which a transient ischemia had been induced by repeated
clamping/ unclamping of the uterine artery transplanted onto the chick embryo
chorioallantoic membrane demonstrated higher VEGF expression and better sur-
vival: this mechanism could facilitate implantation and establishment of the endo-
metrium at ectopic sites [77]. Moreover, response to ischemia is likely to play a role
in established lesions of endometriotic patients: the relative expression of HIF1
and VEGF differs at various sites within endometriotic lesions, possibly accounting
for some of their heterogeneous histological characteristics [78].
7.3.3.3 Progression and Infiltration
Surrey and Halme demonstrated a direct stimulatory effect of the cell-free fraction
of PF samples derived from patients with endometriosis on the proliferation of
normal uterine endometrial cells in a short-term culture, indicating that factors in
the PF are involved in the progression of endometriosis [79]. Several cytokines,
such as IL-8 and TNF, have growth-promoting effects on endometrial and
endometriotic cells [24, 69, 70]. These findings suggest that elevated PF levels of
cytokines promote the progression and spread of endometriotic implants in the
peritoneal cavity.
We revealed that PF levels of IL-8 significantly enhanced the proliferation of
stromal cells derived from ovarian endometriomas [24]. Expression of IL-8 recep-
tor type A (CXCR3A) mRNA was detected in endometriotic stromal cells. These
results suggest that IL-8 may promote the progression of endometriosis [70]. We
tested the hypothesis that TNF elevated in PF of patients with endometriosis may
contribute to the progression of endometriosis by inducing the production of IL-8.
Gene and protein expression of IL-8 in the stromal cells of endometriotic tissues are
upregulated by TNF, and TNF also stimulated the proliferation of the
endometriotic stromal cells. This stimulatory effect of TNF was abolished by
adding either anti-TNF antibody or anti-IL-8 antibody. Therefore, the action of
TNF on stromal cells may occur by mediating the proliferative effects of IL-8. The
expression of type I and type II receptors for TNF was observed in endometriotic
stromal cells. This evidence suggests that TNF action mediated by IL-8 may not
only be an initiating factor that facilitates adhesion of endometrial cells to the
peritoneum, but may also contribute to the development and progression of
endometriosis.
We found that the extent of superficial red endometriotic lesions was related to
increased levels of IL-6, IL-8, and TNF in the PF [21]. Red lesions, such as red
flame-like lesions, gland-like lesions, and red vesicles, were classified as active
lesions of endometriosis because angiogenesis is more pronounced in red lesions
than in black or white lesions and because early red lesions invade the ECM. Thus,
cytokines may play a role in the early stage of endometriosis.
Hepatocyte growth factor (HGF) was originally characterized as a potent mito-
gen for adult hepatocytes. HGF is known as a mesenchymal (stromal)-derived
pleiotropic growth factor that elicits mitogenic and morphogenic activities on
various types of epithelial cells, usually as a paracrine factor [80, 81]. In normal
uterine endometrium, stromal-derived HGF promotes proliferation, migration, and
lumen formation of endometrial epithelial cells [82]. Overexpression of Met, the
receptor for HGF, was observed in several malignant tumors, such as uterine
endometrium and ovary [83]. We also showed that the peritoneum and
endometriotic stromal cells may be major sources of HGF in peritoneal fluid.
Endometrial and endometriotic stromal cells expressed the Met receptor, which
was activated by endogenous and exogenous HGF [84]. HGF enhanced stromal cell
proliferation and invasion. We also demonstrated that the HGF-stimulated stromal
cell invasion was due in part to the induction of urokinase-type plasminogen
activator, a member of the extracellular proteolysis system. HGF increased in PF
and produced by endometrial stromal cells may induce critical changes in mor-
phology of mesothelial cells and then enhance the endometrial cell attachment and
invasion.
7.3.3.4 Infertility
Pelvic endometriosis is frequently associated with infertility even if affected

women ovulate and have functional, patent tubes. The exact mechanism by which
endometriosis interferes with fertility is not fully understood. A recent study
suggests that cytokines are related to infertility in women with endometriosis [2].
Since the ovaries and fallopian tubes are bathed in PF, substances present in PF
have the potential to impact greatly on the reproductive function by affecting tubal
motility, ovum pickup, or ovulation. It is speculated that a substance or substances
from endometriotic tissues enter the PF, interfering with the reproductive process.
Interleukin-6, a pleiotropic cytokine produced by a variety of cell types, plays
a pivotal role as a mediator of numerous physiologic and pathogenic processes.
Fig. 7.2 Pathophysiology of endometriosis-associated infertility
It has also been suggested that IL-6 has important functions in reproductive
physiology, including the regulation of ovarian steroid production, folliculogenesis,
and early events related to implantation [85]. We demonstrated that the addition of
human recombinant IL-6 to culture medium suppressed the rate of blastocyst
formation of mouse embryos, suggesting that increased IL-6 in the PF of endome-
triosis patients may contribute to infertility by adversely affecting embryonic
development [86]. Recently, Banerjee J et al. showed that IL-6 caused the deteri-
oration in morphology of the microtubule and chromosomal alignment in
metaphase-II mouse oocytes [87]. IL-6, generated in the process of oxidative stress,
directly affects the quality of the oocyte and may contribute to infertility.
We used a steroidogenic human granulosa-like tumor cell line, KGN cells, as a
model for granulosa cells collected during the follicular phase [88]. We demon-
strate that IL-6 may reduce aromatase activity and E2 production via the MAPK
signal pathway in human granulosa cells [89]. The results may support the notion
that IL-6 is related to impaired estrogen biosynthesis in patients with endometriosis.
Half of the cause of infertility is the male factor. The key predictors of fertili-
zation capability are sperm count and motility. We showed that IL-6 and sIL-6R
significantly reduced the percentage of motile and rapidly moving sperm [90]. The
inhibition of sperm motility by IL-6 may be involved in the infertility of at least
some patients with endometriosis who have highly elevated levels of IL-6 in PF. PF
diffusing into the tubal and endometrial environment may affect the sperm and their
interaction with the oocyte and embryo development. Many authors have demon-
strated that the PF of patients with endometriosis has detrimental effects on several
steps of the reproductive process.
These findings suggest that endometriotic implants, which can produce various
cytokines, may contribute to reduced fecundity in patients with endometriosis. The
role of PF and cytokines in the pathophysiology of endometriosis-associated infer-
tility is summarized in Fig. 7.2. However, data on cytokines and their role in
infertility are still incomplete, and future investigation that can reveal the critical
roles of cytokines is still needed.
Fig. 7.3 Signal

transduction of MAPK
pathway
7.3.4 Signal Transduction in Endometriotic Cells
Signal transduction is defined as the response of a cell to the application of an

external stimulus. Numerous factors, including cytokines, hormonal factors,
genetic predisposition, environmental toxins, and immunological dysfunction,
may contribute to the aberrant progression of endometriotic tissue.
7.3.4.1 MAPK Pathway
Many studies have demonstrated that MAPK is involved directly in regulating the
pathogenesis of endometriosis [9193]. MAPK pathways seem to play a pivotal role
as intracellular and extracellular signal transducers in endometriotic cells. In the
MAPK pathway, the activation of p38, c-jun N-terminal kinase (JNK), and ERK1/2
is important for inflammatory cytokine secretion in endometriotic stromal cells. The
most extensively studied mitogen-activated protein kinase (MAPK) pathway is the
extracellular signal-regulated kinase (ERK) pathway in which the MAPKKK is Raf,
the MAPKK is MEK, and the MAPK is ERK. The activation of the MAPK pathway
induced by TNF through Ras, Raf, MEK, and ERK also affected the activation
of AP-1 (Fig. 7.3). We showed that TNF induced the activation of the signal
molecule ERK1/2 of the MAPK cascade in endometriotic cells [94].
Recent studies have shown that p38 mitogen-activated protein kinase (p38
MAPK), an intracellular signal-transducing molecule, plays an important role in
the regulation of a variety of inflammatory responses, including expression of
proinflammatory cytokines, leukocyte adhesion, and chemotaxis. A number of
studies indicated that the p38 MAPK pathway might play an important role in the
development and progression of endometriosis [94]. Increased p38 MAPK activa-
tion in eutopic and ectopic endometrium indicated that p38 MAPK might be one of
the main factors regulating the inflammatory process in endometriosis [9597].
Yoshino et al. revealed that FR 167653, a p38 mitogen-activated protein kinase
inhibitor, inhibits the development of endometriosis, possibly by suppressing peri-
toneal inflammatory status [98]. Zhou et al. also demonstrated that SB203580,
a p38 mitogen-activated protein kinase inhibitor, may suppress the development
Fig. 7.4 Signal transduction of NF-B pathway
of EM by inhibiting the expression of proinflammatory cytokines and proteolytic

factors. p38 MAPK might play a key role in the progression of endometriosis [99].
These findings suggest that estrogen might to some extent exert its effects on the
endometrium through the p38 MAPK pathway.
Many studies have demonstrated that MAPK is involved directly in regulating the
pathogenesis of endometriosis. MAPK pathways seem to play a pivotal role as
intracellular and extracellular signal transducers in the pathogenesis of endometriosis.
7.3.4.2 NF-kappaB Pathway
Nuclear factor-kappaB (NF-B) is a family of transcription factors modulating

hundreds of genes involved in inflammation, cell proliferation, apoptosis, invasion,
angiogenesis, and other cell processes [100]. Therefore, accurate monitoring of
NF-B activation in target cells is crucial to investigating its signal transduction.
NF-B is composed of homo- and heterodimers of five members of the Rel family
including p50, p52, p65, RelB, and c-Rel. NF-B dimers are sequestered in the
cytosol of unstimulated cells via noncovalent interactions with a class of inhibitor
proteins, called IkBs. NF-B is normally bound to IB in cytosol; this binding
prevents its movement into the nucleus. Proinflammatory stimuli activate the IB
kinase (IKK) complex and NF-B essential modulator. Activated IKK phosphory-
lates IB, resulting in its polyubiquitination and degradation. The degradation of
IB exposes the nuclear localization signal of NF-B, resulting in the translocation
of the p50/p65 NF-B dimer to the nucleus in which it can be bound to NF-B
recognition elements in the promoter of proinflammatory cytokines such as TNF,
IL-6, and IL-8 (Fig. 7.4). We demonstrated that NF-B activation has been involved
in the induction of IL-8 in endometriotic tissues [101].
Fig. 7.5 Mechanism of

signal transduction by the
TAK1 cascade
Multiple publications have strongly suggested participation of the NF-B path-

way in endometriosis pathophysiology. In vitro studies have shown positive regu-
lation of growth factors and proinflammatory and antiapoptotic proteins mediated by
NF-B activation in human endometrial and endometriotic cells [102, 103]. In vivo
research in animal models treated with NF-B inhibitors has revealed reduction of
endometriosis development by diminishing inflammation and cell proliferation and
inducing apoptosis of endometriotic cells [104, 105]. Constitutive activation of
NF-B was shown to be increased in red endometriotic lesions relative to black
endometriotic lesions in women, and iron-mediated NF-B activation in pelvic
macrophages and endometriotic cells has been proposed as a possible mechanism
contributing to endometriosis establishment and maintenance [106]. Thus, NF-B
pathways seem to play a pivotal role in the pathophysiology of endometriosis.
TAK1, a serine/threonine kinase, is an essential intracellular signaling compo-
nent in inflammatory signaling pathways [107]. TAK1 has proven to be a crucial
factor in regulating inflammatory responses by controlling production and function
of various other cytokines. An accumulation of evidence suggests that TAK1 plays
an important role as a second messenger in the activation of NF-B, p38, and JNK.
The activation of TAK1, which works on the TNF-inducible phosphorylation of
both the NF-B and MAPK pathways, may be indispensable for inflammatory
response and progression of endometriosis. TAK1 has been widely accepted as a
regulator of the rapid activation of JNK/p38 MAPKs and IB kinase signaling
pathways in response to cellular stimuli (Fig. 7.5).
We showed that TNF and its downstream TAK1, which are key mediators for
NF-B and MAPK pathways, may be involved in the pathogenesis of
endometriosis [108].
7.4 Conclusion
Pathogenesis of endometriosis is very complex, so various factors and directions of

studies were needed. Cytokines, which are produced by many cell types in PF, play
a diverse role in constructing the peritoneal environment that induces the
development and progression of endometriosis. Intense basic research into the

specific role of these cells and soluble factors may improve our understanding of
endometriosis and result in novel therapeutic modalities for endometriosis.
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Chapter 8
Epigenetics in Endometriosis
Masao Izawa, Fuminori Taniguchi, and Tasuku Harada
Abstract There is accumulating evidence supporting the concept that endometriosis

is a disease associated with an epigenetic disorder. Epigenetics is one of the most
expanding fields in the current biomedical research. The word epigenetics refers to
the study of mitotically and/or meiotically heritable changes in gene expression that
occur without changes in the DNA sequence. The disruption of such changes
(epigenetic aberration or disorder) underlies a wide variety of pathologies. Epige-
netic regulation includes DNA methylation and histone modifications and is respon-
sible for a number of gene transcriptions associated with chromatin modifications
that distinguish the states of diseases. As an introduction, we summarize our findings
of epigenetic disorder in endometriotic cells and then overview recent studies
focused on DNA methylation in endometriosis. We describe our recent challenge
and advanced studies from other laboratories using genome-wide (GW) analysis.
Finally, we refer to environmental factors as a potential background of epigenetic
disorder in endometriosis.
Keywords Aberrant DNA methylation Aberrant histone modification Aberrant

transcription Epigenetic disorder
8.1 Why Epigenetics in Endometriosis?
Epigenetics is one of the most promising and expanding fields in the current
biomedical research. The word epigenetics refers to the study of mitotically
and/or meiotically heritable changes in gene expression that occur without changes
in the DNA sequence [1]. The disruption of such changes (epigenetic aberration or
M. Izawa, Ph.D. (*) F. Taniguchi T. Harada

Department of Obstetrics and Gynecology, Tottori University Faculty of Medicine,
36-1 Nishi-cho, Yonago 683-8504, Japan
e-mail: 1mizawa@med.tottori-u.a.jp

108 M. Izawa et al.
disorder) underlies a wide variety of pathologies including cancer [2, 3]. Epigenetic
regulation includes DNA methylation and histone modifications [4, 5] and is
responsible for a number of gene transcriptions associated with chromatin modifi-
cations that distinguish the various cell types and the states of diseases. Cancer and
many other diseases show aberrant epigenetic regulation [6]. In terms of DNA
methylation, cancer cells show genome-wide (GW) hypomethylation and
site-specific hypermethylation of promoter CpG islands [2]. This may lead to
transcriptional silencing and aberrant transcription from incorrect transcription
start sites [4]. In addition, a recent study comparing colorectal cancer tissue with
its normal counterpart suggests changes at the CpG island shores [7]. In normal
cells, CpG islands and CpG island shores are under the control of physiological
methylation, allowing normal gene transcription.
There is accumulating evidence supporting the concept that endometriosis is an
epigenetic disease [8]. The concept of aberrant DNA methylation in endometriosis
is expanding [9]. Here we describe how the field of epigenetics is reshaping the
current thinking about endometriosis. Firstly, we present our study shedding light
on aberrant aromatase expression in endometriosis from the viewpoint of epigenetic
disorder and then summarize recent advances in endometriosis research using the
epigenetic approach. We subsequently describe the advanced technologies of GW
methylation analysis and GW association study (GWAS) in endometriosis research.
Finally, we refer to environmental factors as a potential background of epigenetic
disorder in endometriosis.
8.2 Estrogen Environment in Endometriosis
Endometriotic tissue growth depends on ovarian steroids; thus, medical treatments

aim to reduce ovarian steroidogenesis. Continuous exposure to GnRH agonist
results in desensitization or downregulation of GnRH receptors leading to reduced
serum gonadotropin levels and reduced ovarian hormone production. Treating
endometriosis with GnRH agonist reduces both the number of observable
endometriotic implants and the frequency and severity of associated pain [10, 11].
Likewise, inhibition of estrogen production by progestins or aromatase inhibitors
reduces endometriotic lesions and clinical symptoms [12]. Three major sites for
estrogen production are recognized in women with endometriosis: (a) de novo
synthesis in the ovary, (b) an intrinsic system that depends on aromatase, which
converts circulating androstenedione to estradiol (intracrine) in the skin and adi-
pose tissue, and (c) a de novo system and an intracrine system in endometriotic
tissues [13, 14]. Among these sites, local estrogen production, which depends on
aberrantly expressed aromatase in endometriotic implants, plays an important role
in the pathophysiology of endometriosis [1517]. Local estrogen production by
these implants may contribute to the progression of endometriosis even under the
hypoestrogenic environment produced by GnRH agonist exposure [17].
8 Epigenetics in Endometriosis 109
8.2.1 Aromatase Upregulation in Endometriosis
Aromatase, an enzyme that catalyzes the conversion of androgens to estrogens, is a

key molecule for estrogen production. Aromatase is encoded by a single-copy gene
CYP19 on chromosome 15q21. CYP19 expression is regulated in a tissue-specific
manner, in which alternative usage of multiple promoters with each unique cis-
acting element has been known [18]. Therefore, identifying the promoter usage
becomes the first step in understanding the molecular background of aromatase
expression in specific tissues. Bulun et al. previously reported that promoter II is the
most potent promoter functioning in endometriotic cells from endometrioma [17].
In addition to promoter II, we recently demonstrated that two proximal promoters,
I.3 and I.6, are used additionally in endometriotic cells [14]. At the same time, we
observed that aromatase transcription in endometrial cells was at a marginal
level depending on the same 3 promoters as those of endometriotic cells [14].
From these observations, we hypothesized that the upregulation of aromatase gene
in endometriotic cells may be an epigenetic disorder, since hypermethylation
of promoter region in tumor suppressor gene associated with gene silencing has
been known.
8.2.2 DNA Demethylation and Aromatase Upregulation

in Endometriotic Cells
An epigenetic disorder may lead to the aromatase upregulation in endometriotic

cells. We challenged this hypothesis: after treating endometrial cells with
5-aza-deoxycytidine (5-aza-dC, competitive inhibitor for DNA methyltransferase)
for 96 h, aromatase transcription was markedly upregulated in the cells (Fig. 8.1).
This is the first demonstration that epigenetic modification enhances aromatase
mRNA expression [14]. The enhanced aromatase mRNA expression was dependent
on the same promoters as those in endometriotic cells (Fig. 8.2). When the effect of
trichostatin A (TSA), instead of 5-aza-dC, on aromatase mRNA expression
was examined, we observed little effect, suggesting that one of the major factors
that suppresses aromatase mRNA expression in endometrial cells is the methylation
of aromatase gene and/or its trans-acting factor gene. Alternatively, the observation
suggests that a disorder of a putative methylation-dependent suppression mecha-
nism may lead to the upregulation of aromatase mRNA expression in endometriotic
cells.
110 M. Izawa et al.
Fig. 8.1 Aromatase mRNA

induction in response to
5-aza-dC in endometrial
cells. (a) RT-PCR: lane
1, untreated control, lanes
2 and 3, treated with
5-aza-dC, and lanes 4 and
5, treated with TSA. (b)
Semiquantitative analysis
Fig. 8.2 Promoter usage of aromatase mRNA expression. Left: 5-aza-dC-treated endometrial
cells. Right: endometriotic cells
8.2.3 Hypomethylated CpG Island Within the Aromatase

Gene in Endometriotic Cells
We searched for the unmethylated CpG locus within the aromatase gene in
endometriotic cells [19]. We predicted a CpG island at approximately 20 kb
upstream from the end of exon II (Fig. 8.3). In endometriotic cells, the CpG
sequence was hypomethylated, while in endometrial cells, the upstream half was
hypermethylated and recognized by the methyl-CpG binding proteins, MBD1 and
MeCP2 [19]. The downstream half was hypomethylated in both endometrial and
endometriotic cells. Because the CpG sequence is located at the promoter-distal
region, we speculate that the sequence may act as a cis-acting element under the
control of methylation.
Fig. 8.3 A hypomethylated CpG island at 20 kb upstream from the end of exon II in endometriotic
cells
8.3 Nuclear Receptor Genes Under Epigenetic Aberration

in Endometriosis
Estrogen receptor (ER) plays pivotal roles in the pathogenesis and progression of
endometriosis. Earlier studies have focused on the expression of ER as well as
ER in the eutopic endometrium and in endometriotic lesions. Simultaneous
expression of ER and ER indicates that the estrogen action might be transmitted
in a cooperative manner [20, 21]. In contrast to the high ER/ER expression ratio
in the eutopic endometrium, a lower expression of ER and a markedly higher
expression of ER in ovarian endometriomas have been reported [22, 23]. The
higher ER expression in endometriotic tissue may depend on the hypomethylation
of the ER-promoter region [24]. The increased ER expression in endometriotic
tissues may suppress ER expression [25].
8.3.1 ER Gene: Hypomethylation and Upregulation
That the development and progression of endometriosis depends on estrogen is well

known [26, 27]. ER and ER function as transcription factors and are believed to
play key roles in tissue growth of endometrium and endometrioma [22, 23]. Previ-
ous studies demonstrated markedly higher levels of ER and lower levels of ER in
endometriotic tissues and endometriotic stromal cells [2123]. Differences in the
ER/ER ratio between endometriotic and endometrial stromal cells are suggested
to have important functional implications [24]. Recently, Xue et al. [24] demon-
strated that the ER promoter is hypomethylated in endometriotic cells.
Hypomethylation caused the higher expression of ER gene in endometriotic
cells, while hypermethylation silenced the expression in endometrial cells. Treat-
ment with a demethylating agent significantly increased ER mRNA expression in
endometrial cells. They proposed that enhanced ER transcription in endometriotic
cells takes over the ER promoter activity, thus favoring the suppression of ER
levels [25].
112 M. Izawa et al.
8.3.2 Progesterone Receptor (PR) Gene: Hypermethylation

and Downregulation
Progesterone induces differentiation of endometrial stromal cells to decidualized

cells and glandular epithelial cells to the secretory phenotype. Representative
molecular markers of progesterone action include increased production of epithelial
glycodelin and stromal prolactin in the endometrium [28, 29]. Progesterone resis-
tance is known as a feature in some endometriosis and can be attributed to the low
level of PR in endometriotic tissue [3032]. PR-A and PR-B forms are expressed in
the stromal and epithelial components of the endometrium [32]. In the endome-
trium, expressions of PR-A and PR-B are progressively upregulated during the
proliferative phase to their highest level immediately before ovulation, and dimin-
ish thereafter, suggesting that estradiol stimulates PR expression [25]. PR level may
be related to the responsiveness to progesterone in patients [33, 34]. Wu et al. [35]
have shown that the promoter region of PR-B is hypermethylated in endometriosis,
which may lead to the PR-B downregulation. They recently showed that prolonged
stimulation of TNF- induced partial methylation in the promoter region of PR-B
associated with decreased expression of PR-B in an immortalized epithelial-like
endometriotic cell line [36]. This seems to provide evidence that phenotypic
changes in endometriosis, such as chronic inflammation associated with increased
production of proinflammatory cytokines, may cause epigenetic aberrations leading
to changes in gene expression [37].
8.4 Other Genes Under Epigenetic Aberration

in Endometriosis
8.4.1 Steroidogenic Factor-1 (SF-1) Gene: Hypomethylation

and Upregulation
SF-1 is a transcriptional factor essential for the activation of multiple steroidogenic

genes for estrogen biosynthesis, such as the genes for steroid acute regulatory
(StAR) and aromatase [3840]. SF-1 is usually undetectable in eutopic endometrial
cells [41]. It has been demonstrated that SF-1 mRNA and protein levels in
endometriotic cells were significantly higher than those in eutopic endometrial
cells [40, 41]. Xue et al. identified a classical CpG island at the promoter region
of the SF-1 gene and showed that the SF-1 promoter has increased methylation in
eutopic endometrial cells [41]. In endometrial cells, the silencer-type transcription
factor MBD2 is recruited to the methylated SF-1 promoter and prevents its inter-
action with transcriptional activators, resulting in silencing of the SF-1 gene. On the
other hand, the SF-1 promoter is hypomethylated in endometriotic cells. Steroido-
genic factor-2 (SF-2), a transcription factor highly expressed in endometriotic
tissues, binds to the unmethylated SF-1 promoter and activates its transcription in
endometriotic cells [42]. The SF-1 expression is under epigenetic control that
permits the binding of activator complexes to the SF-1 promoter [41, 42]. SF-1
expression in endometriosis may enhance aromatase expression. Treatment with a
demethylating agent has been shown to increase SF-1 mRNA levels in eutopic
endometrial cells [26].
8.4.2 E-Cadherin Gene: Hypermethylation

and Downregulation
Downregulation of E-cadherin, a known metastasis-suppressor protein in epithelial

tumor cells [43], has been shown in endometriotic cells [44]. In two immortalized
endometriotic cell lines, the E-cadherin gene was found to be hypermethylated at
the promoter region, and treatment with a histone deacetylase inhibitor TSA
induced expression [45]. Interestingly, the increased promoter methylation of the
E-cadherin gene during aging has been demonstrated [46].
8.4.3 HOXA10 Gene: Hypermethylation

and Downregulation
HOXA10 has been expressed in the endometrium, and its expression is under the
control of estrogen and progesterone [4648]. The roles in endometrial develop-
ment during the menstrual cycle and in establishing uterine receptivity have been
suggested [47, 48]. In women with endometriosis, HOXA10 expression is signif-
icantly decreased in the eutopic endometrium during the secretory phase, indicating
functional defects in uterine receptivity [47, 49]. The promoter region of HOXA10
gene was found to be hypermethylated in the eutopic endometrium from women
with endometriosis [50]. As promoter hypermethylation has been suggested as an
epigenetic marker of gene silencing, the promoter hypermethylation may be related
to the HOXA10 downregulation in the eutopic endometrium of women with
endometriosis [47].
8.5 Genome-Wide (GW) Profiling of DNA Methylation

and GW Association Study (GWAS) in Endometriosis
Methylation of DNA provides a layer of epigenetic controls that has important

implications for diseases including endometriosis. There has been a revolution in
DNA methylation analysis technology. Analyses can now be performed on a
114 M. Izawa et al.
Fig. 8.4 Differentially methylated CpGs in endometriotic cells (a) CpGs, which show more than
10-fold difference, were extracted (b) the classification
genome-scale and entire methylomes can be characterized at single-base-pair

resolution [51]. In endometriosis, a number of aberrant gene expressions have
been demonstrated [52, 53]. These aberrations may be related to aberrant DNA
methylations. However, it is unknown whether global alterations in DNA methyl-
ation patterns occur in endometriosis and to what extent they are involved in its
pathogenesis. A whole-genome scanning of CpG methylation status in more than
25,000 promoters has been conducted previously [54]. The results showed highly
similar methylation profiles between endometrium and endometriotic lesions.
Recently, a new generation of genome-wide DNA methylation BeadChip array
including 485,577 CpGs (Infinium HumanMethylation450 BeadChip Array) has
been developed [55, 56]. The array covers more than 99 % of human genes and
96 % of CpG islands. Using the array, we recently identified 1,811 CpGs (0.38 %)
differentially methylated not only in the promoter region but also in promoter-distal
CpGs (Fig. 8.4) [57]. Among them, 954 CpGs (52.7 %) were hypermethylated,
while 857 CpGs (47.3 %) were hypomethylated in endometriotic cells. The results
indicate that the overall methylation profile in endometriotic cells was highly
similar to that in endometrial cells. The observation supports the retrograde men-
struation theory by Sampson [58] for the pathogenesis of endometriosis. It is
important to note that the CpGs hypomethylated or hypermethylated in
endometriotic cells were demonstrated not always in promoter CpG islands.
These observations show a facet of epigenetic disorder in endometriosis.
Through a GWAS and a replication study using a total of 1,907 Japanese
individuals with endometriosis and 5,292 controls, a significant association of
endometriosis with rs10965235 (P 5.57 10(12), odds ratio 1.44), which is
located in CDKN2BAS on chromosome 9p21, encoding the cyclin-dependent
kinase inhibitor 2B antisense RNA was identified [59]. The findings suggest that
these regions are susceptible loci for endometriosis. A GWAS was performed using
two case-control cohorts genotyped with the Affymetrix Mapping 500K Array or
Genome-Wide Human SNP Array 6.0 in Japanese women with endometriosis [60].
A GWAS in 3,194 individuals with surgically confirmed endometriosis and 7,060
controls from Australia and the UK was conducted [61]. The strongest association
signal was reported on 7p15.2 (rs12700667) for endometriosis. rs12700667 is

located in an intergenic region upstream of the plausible candidate genes
NFE2L3 and HOXA10.
8.6 Epigenetic Aberrations and Environmental Factors

in Endometriosis
The known epigenetic modifications are DNA methylation and histone modifica-
tions, including methylation, acetylation, ubiquitylation, and phosphorylation [62].
The functional and biological significance of the epigenetic alterations accumulate
over a life span. The earliest studies found a pattern of low global DNA methylation
levels in aged mammalian tissues [63]. A recent monozygotic (MZ) twin study
showed a line of evidence that epigenetic variants accumulate during aging inde-
pendently of the genetic sequence [64]. The study tested the epigenetic contribution
to twin discordance and elucidated the effect of environmental characteristics on
gene function. The results revealed that epigenetic difference between siblings is
associated with phenotypic discordance, which might be attributed to an unshared
environment.
8.6.1 Effect of Environmental Factors on Epigenome
The association between environmental factors and phenotypic discordance within

MZ twins has been noticed [64]. However, little is known about the molecular basis
by which environmental factors influence gene functions [65]. Typical examples
are the abnormal intrauterine environment associated with epigenetic
downregulation of genes [66, 67] and the maternal diet associated with the DNA
methylation profile of offspring [6870]. Gene function and chromatin structure can
be modulated by environmental factors [71, 72]. In response to a methyl-deficient
diet, a significant decrease in repressive dimethyl-H3K9 associated with the
upregulation of targeted gene was demonstrated in mice [73]. Environmental fac-
tors, including endocrine disrupting chemicals, may affect the epigenome leading to
the onset of endometriosis in utero. There might be epigenetic changes during
ontogenic development [74, 75].
8.6.2 DNA Methylation, Aging, and Endometriosis
The great fidelity with which DNA methylation patterns in mammals are inherited
after each cell division is ensured by the DNA methyltransferases (DNMTs).
116 M. Izawa et al.
However, the aging cell undergoes a DNA methylation drift. Early studies showed
that global DNA methylation decreases during aging in many tissue types [63]. The
loss of global DNA methylation during aging is probably mainly the result of the
passive demethylation of DNA as a consequence of a progressive loss of DNMT
activity [76]. Several specific regions of the genomic DNA become
hypermethylated during aging [77]. Methylation of promoter CpG islands in
nontumorigenic tissues has been reported for several genes, including ER [77].
Interestingly, genes with increased promoter methylation during aging include the
E-cadherin gene [46], which is downregulated and hypermethylated in
endometriotic cells [65]. Aberrant methylation of CpG islands in 50 promoters has
been suggested to be associated with transcriptional silencing or upregulation in
endometriosis [24, 33, 36, 50]. Recent GW methylation studies suggest that tissue-
and cell-type-specific methylation is present only in a small percentage of CpG
islands in 50 promoters, while a far greater proportion of CpG island methylation is
across gene bodies [78, 79]. In addition, functionally different types of DNA
modification, methylation, and hydroxymethylation have been identified [78,
80]. Therefore, methylation of CpG islands in 50 promoters alone may not be a
major player in the aberrant gene expression.
8.6.3 Histone Modification, Aging, and Endometriosis
Histone modifications have a defined profile during aging. For example, the
trimethylation of H4-K20, which is enriched in differentiated cells [81], increases
with age [82, 83] and decreases in cancer cells [8387]. A decrease in the histone
trimethylation has been observed in the liver after the long-term treatment with
the hepatocarcinogen tamoxifen [88]. The loss of trimethylated H4-K20 in cancer
can be caused by the loss of expression of the H4-K20-specific methyltransferase
Suv4-20h [74]. Although approximately 100 histone methyltransferases and
demethylases have been identified in human genome, only a subset of histone
methyltransferase inhibitor is in clinical trials for cancer treatment [89].
Acetylation levels of histones are controlled by a balance between histone
acetyltransferases and histone deacetylases (HDACs). Histone acetyltransferases
transfer acetyl groups from acetyl-CoA to lysine residues on the amino-terminal
region of histones and activate gene transcription. Conversely, HDACs restore the
positive charge on lysine residues by removing the acetyl groups and prevent
transcription. HDACs comprise large multiprotein complexes that target promoter
sites through their interaction with sequence-specific transcription sites. HDAC
inhibitors (HDACIs) can inhibit cell proliferation, induce cell differentiation and
cell cycle arrest, and stimulate apoptosis of various cell types [90]. Hyperacetylation
of histones H3 and H4 is often associated with activated transcription, and
hypoacetylation of histones H3 and H4 correlates with transcriptional silencing
or repression [91]. Kawano et al. [92] recently demonstrated that treatment with
HDACIs induced the accumulation of acetylated histones associated with some
cell cycle-related gene expressions in endometriotic cells. The advanced human

epigenome projects [51, 93] may provide further insight into understanding epige-
netic aberrations in endometriosis.
8.7 Conclusion
Epigenetics is one of the most promising and expanding fields in the current
biomedical research of diseases including endometriosis. Most important is its
translational application. One of the immediate questions to be clarified in endo-
metriosis is which genome is under epigenetic aberration. Here we focused mostly
on DNA methylation and reviewed current epigenetics studies in endometriosis.
Epigenetics is currently expanding from DNA methylation to histone modifica-
tions (Fig. 8.5) [94]. The development of high-throughput technologies including
Fig. 8.5 Organization and composition of epigenome. (a) A beads-on-a-string chromatin fiber
includes nucleosome cores connected by linker DNA and linker histone H1. Me: methyl group. (b)
The distribution of DNA methylation and a small subset of histone markings, linker histones, and
core histone variants represent a different regulation at active promoters, gene bodies, and
enhancers (top) as compared to silenced and repressed chromatin (bottom)
118 M. Izawa et al.
GW analysis associated with next-generation sequencer is accelerating the study of

epigenetic aberration in endometriosis. Using the epigenetic concept as a tool, new
diagnostic marker or therapy may be developed to overcome serious problems in
patients with endometriosis.
Acknowledgment This work was supported in part by Grants-in-Aid for Scientific Research
(C) from Japan Society for the Promotion of Science (Nos. 22591824 and 18591800 to M.I.).
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Chapter 9
Roles of Prostaglandin E2 in Endometriosis
Kuei-Yang Hsiao, Meng-Hsing Wu, and Shaw-Jenq Tsai
Abstract Endometriosis is one of the most common gynecological diseases,

affecting approximately 10 % of women in reproductive age. It is characterized
as the presence of endometrial-like glands and stroma outside the uterus, commonly
on the pelvic peritoneum and ovaries. The major symptoms of endometriosis
include pelvic pain, dysmenorrhea, dyspareunia, and infertility. During the past
decade, intensive investigations on molecular mechanisms responsible for the
pathological processes of endometriosis have been conducted. Although many
factors have been reported to be involved in these processes, prostaglandin E2
(PGE2) no doubt represents as one of the most critical regulators of all. Accumu-
lating data demonstrate that PGE2 controls many critical functions, such as ste-
roidogenesis, angiogenesis, proliferation, and immune suppression that contribute
to the pathogenesis of endometriosis. Herein, we will summarize the most up-to-
date information regarding the functional roles of PGE2 in the development and
maintenance of endometriosis.
Keywords Angiogenesis Hypoxia Macrophage Phagocytosis Proliferation

Prostaglandin Steroidogenesis Stromal cells
K.-Y. Hsiao S.-J. Tsai, Ph.D. (*)

Department of Physiology, College of Medicine, National Cheng Kung University,
1 University Road, Tainan 70101, Taiwan
e-mail: seantsai@mail.ncku.edu.tw
M.-H. Wu
Department of Obstetrics and Gynecology, College of Medicine,
National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan

126 K.-Y. Hsiao et al.
9.1 Introduction
Although the etiology of endometriosis remains unclear, there are several hypoth-
eses proposed including retrograde menstruation theory, embryonic rest theory,
coelomic metaplasia theory, and new combined theory [15]. Among these,
Sampsons retrograde menstruation theory is the most accepted one in which it
states that the origin of endometriosis is caused by implanted cast-off endometrial
tissues, the retrograde menstruation [3, 4]. Several clinical observations, which
described that women with cervical or vaginal obstruction have higher risk of
developing endometriosis, are in line with this hypothesis [68]. In addition, an
animal model of baboon showed that the ligation of cervices increases the incidence
of endometriosis [9]. A more convincing evidence is that endometriosis is observed
exclusively in species that menstruate [10]. Nevertheless, retrograde menstruation
theory is insufficient to explain why only 510 % of women with reflux menstru-
ation developed endometriosis when 90 % of women in reproductive age have
retrograde menstruation [11]. Furthermore, before the infiltration of vasculature,
the retrograded tissues have to escape from the surveillance of immune system, gain
the capacity of steroidogenesis (because endometrial cells are highly dependent on
estrogen), and establish a self-support system to maintain proliferation to survive in
the ectopic sites. Therefore, it is clear that other local or even epigenetic factors
must exist to contribute to the pathological processes of this disease. It is obvious
that we cannot discuss all the factors involved in endometriosis pathogenesis;
therefore, in this review chapter, we will primarily focus on the functional roles
of prostaglandin E2 (PGE2) and some other factors that are involved in the regula-
tion of PGE2 biosynthesis.
9.2 Biosynthesis of PGE2
Prostaglandins, a group of biologically active long-chain fatty acids derived from

arachidonic acid, are short-lived eicosanoids that are produced locally in response
to numerous stimuli. PGs regulate numerous physiological and pathological pro-
cesses including but not limited to inflammation, reproduction, respiration, angio-
genesis, coagulation, photo-sensing, sleep and awakeness, stem cell generation, and
cancer progression [1216]. In mammals, every cell can synthesize one or more
kinds of PGs. The synthesis of PGs involves multiple enzymes and sequential
processes. First, it begins with the cleavage of arachidonic acid from diacylglycerol
and phospholipids by phospholipase C and A2 (PLC and PLA2), respectively
(Fig. 9.1). Then prostaglandin H synthase-1 and synthase-2 (also known as cyclo-
oxygenase-1/cyclooxygenase-2, COX-1/COX-2) convert arachidonic acid to com-
mon prostaglandin precursor PGH2. This enzymatic reaction is the rate-limiting
step in the biosynthesis of the 2-series of PGs (PGD2, PGE2, PGF2, PGI2, PGJ2,
and thromboxane A2). Third, a group of PG synthases, such as microsomal PGE
9 Roles of Prostaglandin E2 in Endometriosis 127
Fig. 9.1 Biosynthesis of prostaglandin E2. Membrane-bound phospholipids, such as

diacylglycerol and phospholipids, are cleaved by phospholipase C and A2 (PLC, PLA2) to yield
arachidonic acid, the common precursor of all PGs. Prostaglandin G/H synthase (cyclooxygenase,
COX-1/COX-2) then converts arachidonic acid into PGH2, which is further metabolized by
microsomal PGE synthase (mPGES) to produce PGE2. PGE transporter (PGT) and multidrug
resistance-associated protein 4 (MRP4) are responsible for the uptake and secretion of PGE2,
respectively
synthase (mPGES) and PGF synthases, act on PGH2 to produce PGE2 and PGF2,
respectively [1719]. These enzymes coordinate with preferred partners in specific
tissue to produce PGs. For example, in the process of PGE2 synthesis, cytosolic
PGES (cPGES) and COX-1 account for constitutive production of PGE2 [19, 20],
while the coupling of mPGES-1 and COX-2 is responsible for inflammation-
induced PGE2 production [17]. As is the rule for locally acting lipid mediators,
PGE2 is not stored but rapidly metabolized once synthesized. The major enzymes
responsible for the rapid (within minutes) inactivation of PGE2 are the cytosolic
enzymes 15-ketoprostaglandin 13-reductase and 15-hydroxyprostaglandin dehy-
drogenase (15-PGDH).
9.3 Source of PGE2 in Endometriosis
Early in the mid-1980s, it was discovered that concentrations of PGE2 are elevated
in the peritoneal fluid collected from women with endometriosis compared to that
derived from otherwise healthy women [21]. Later, it became clear that the majority
of PGE2 comes from two cell typesperitoneal macrophages and endometriotic

stromal cells [2225]. Macrophages are the major immune cells recruited to the
sites where endometriotic tissues reside within first few hours and it has been found
that women with endometriotic lesion have more macrophages [26, 27], especially
the activated macrophages [24]. COX-2 was overexpressed in peritoneal macro-
phages from women with endometriosis while COX-1 was expressed at very low
level and elevated COX-1 is only seen in the severe stage of endometriosis. It
should be noted that COX-2 levels are undetectable in monocytes (precursor of
macrophages) isolated from peripheral blood regardless of their stages [24], indi-
cating that local modulators in the peritoneal cavity promote COX-2
overexpression. Proinflammatory cytokines are commonly elevated in the perito-
neal fluid from women with endometriosis [28] and are potentially responsible for
COX-2 induction. Indeed, PGE2, tumor necrosis factor- (TNF-), and interlukin-
1 (IL-1) have been demonstrated to promote the expression of COX-2 in perito-
neal macrophages [24]. This provides evidence to demonstrate that overproduction
of PGE2 by peritoneal macrophages in women with endometriosis is likely due to
the activation of COX-2 by a group of proinflammatory cytokines in the peritoneal
fluid.
In parallel to macrophages, ectopic endometriotic stromal cells also produce
high levels of PGE2 [22, 23, 25]. Phospholipase A2 was found to increase in lesion
tissues [29, 30], and mPGES was elevated in both epithelial and stromal cells from
women with endometriosis [29, 31]. These elevated PLA2 and mPGES work
together with the upregulated COX-2 to increase PGE2 production.
Aberrant expression of COX-2 in endometriotic stromal cells has been reported
by several groups [22, 23, 25]. However, one intriguing phenomenon is that
although the ectopic endometriotic tissue consists of the same genetic backgrounds
as the eutopic endometrium, it possesses distinct biochemical nature compared to
its eutopic counterpart. It has been shown that ectopic stromal cells are at least
100 times more sensitive to IL-1 treatment than eutopic endometrial stromal cells
in terms of COX-2 expression. The distinct sensitivity is due to increased transcrip-
tional activity of COX-2 promoter in ectopic but not eutopic endometrial stromal
cells [25]. Because endometriosis is a chronic inflammatory disease and many
proinflammatory cytokines, including IL-1, are elevated in the peritoneal fluid,
increased sensitivity enables ectopic endometriotic stromal cells to respond to the
level of proinflammatory cytokines that eutopic endometrial stromal cells normally
do not respond. This phenomenon may explain why COX-2 and, to some extent, its
downstream target genes are consistently overexpressed in ectopic endometriotic
tissues.
The underlying molecular mechanism responsible for the increased sensitivity of
ectopic stromal cells to stimuli is an area of interest to be investigated because it
may provide important information for designing new treatment regimens. Recent
studies reveal that predisposition to hypoxic stress may account for the distinct
responses of ectopic endometriotic cells. The retrograded tissues encountered
hypoxic stress before the formation of new blood vessels in the ectopic site.
Typically, cells sense the hypoxia via an oxygen-dependent hydroxylation on
hypoxia-inducible factors (HIFs). There are two kinds of HIF members: the and
subunits, both of which are constitutively transcribed and translated but undergo
differential posttranslational modifications. Under normoxic condition, the
subunit undergoes hydroxylation at two proline residues (Pro402 and Pro564),
which ultimately results in 26S proteasome-mediated degradation of HIF-
protein [32]. In response to hypoxia, the subunit accumulates due to the lack of
oxygen-induced hydroxylation and degradation. In contrast, the subunit does not
respond to oxygen-dependent degradation and is constitutively expressed. Thus, the
level of subunit determines the gene expression profile of a cell. In endometriotic
stromal cells, levels of HIF-1 mRNA and protein were elevated compared to the
eutopic endometrial stromal cells [33]. Elevation of HIF-1 protein or treatment
with hypoxia causes the downregulation of dual-specificity phosphatase-2
(DUSP2), a downstream inactivator of mitogen-activated protein kinase (MAPK)
signaling [3436]. Downregulation of DUSP2 results in a prolonged activation of
ERK and p38 MAPK in ectopic endometriotic stromal cells, which explains the
increased sensitivity of COX-2 promoter to IL-1 stimulation because it is medi-
ated by ERK- and p38 MAPK-dependent signaling pathway [25].
In addition to altered COX-2 regulation, PGE2 transporter/carrier may also play
important roles on the PGE2 production. Up to date, only few transporters/carriers
of PGE2 have been found and are capable of facilitating the uptake or clearance of
PGE2 to regulate pericellular PG levels. The co-expression of prostaglandin trans-
porter, preferentially transporting PGE2, and 15-PGDH, metabolizing PGE2 resides
in cytoplasm, indeed supporting the idea that uptake of PGE2 is essential for its
metabolism [37]. Multidrug resistance-associated protein 4 (MRP4) is one of the
few proteins with high specificity to export PGE1 and PGE2 [38]. Interestingly,
endometriotic tissue expressed lower 15-PGDH but higher MRP4 mRNA compared
to eutopic ones and correlated to higher PGE2 production/release [29, 39].
9.4 Actions of PGE2
9.4.1 Control of Steroidogenesis by PGE2
The development and maintenance of endometriosis are highly dependent on

estrogen. This notion was supported by several lines of evidences. First, symptoms
of endometriosis usually appear after menarche and regress in menopausal or
ovariectomized women [40]. Second, a study using the monkey animal model
also demonstrated that only the group of animals that received capsules with
estrogen or progesterone developed endometriosis [41]. During the menstrual
cycle, the level of estrogen reaches its maximum before ovulation and maintains
at a certain level in the luteal phase. However, after luteolysis and before the
production of estrogen by follicles of the next cycle, the estrogen level drops to a
minimal level. This raises the question: what is the source of estrogen supporting
Fig. 9.2 Prostaglandin E2 promotes cell proliferation by two independent EP receptor-mediated

pathways. PGE2 binds to EP2 receptor and activates adenylyl cyclase (AC) to generate cyclic
AMP (cAMP), which then activates protein kinase A (PKA). Activated PKA translocates to the
nucleus to phosphorylate cAMP response element-binding protein (CREB), which binds to StAR
(and aromatase) gene promoter. Phosphorylated CREB recruits CREB-binding protein (CBP), a
histone acetyl transferase (HAT), to initiate chromatin remodeling and promote StAR and aroma-
tase gene transcription. Upregulation of StAR and aromatase leads to biogenesis of 17- estradiol
(E2) that stimulates fibroblast growth factor-9 (FGF9) production in an autocrine manner. On the
other hand, PGE2 binds to EP3 receptor to activate the protein kinase C (PKC)-Raf-MEK-ERK
signaling pathway that directly results in upregulation of FGF9 transcription. Overexpressed FGF9
then stimulates endometriotic cell proliferation via autocrine and/or paracrine regulations
the survival of endometriotic tissue when the ovarian estrogen is not available? The
answers to this question were revealed by multiple studies showing that
endometriotic stromal cells actually are capable of producing estrogen.
Endometriotic stromal cells not only express proteins/enzymes required for de
novo synthesis of estrogen but even express at higher levels compared to its
endometrial counterpart. The pro-steroidogenic proteins, including steroidogenic
acute regulatory protein (StAR), P450 side chain cleavage enzyme,
3-hydroxysteroid dehydrogenase, 17-hydroxylase 17, 20 lyase, aromatase,
and 17-hydroxysteroid dehydrogenase type I, are either aberrantly expressed or
elevated in ectopic stromal cells [4244], whereas the anti-steroidogenic
protein, 17-hydroxysteroid dehydrogenase type II, is suppressed. Among these
proteins, StAR and aromatase control two of the most important steps for
estrogen production. StAR governs the first step in which hydrophobic cholesterol
is carried through the double-membraned mitochondria where the P450 side chain
cleavage enzyme resides and aromatase converts androstenedione to estrone.
Expression of StAR in endometriotic stromal cells is induced by PGE2 (Fig. 9.2).
This stimulation is unique to endometriotic stromal cells, but not found in eutopic
endometrial stromal cells or epithelial cells [44]. PGE2-induced StAR expression is

mediated via EP2 receptor [45]. Treatment with PKA inhibitor attenuates PGE2-
induced StAR expression, indicating that the EP2 signaling is mediated via typical
Gs and PKA/cAMP pathway. In line with this observation, treatment with cell-
permeable cAMP also stimulates StAR expression in endometriotic stromal cells.
The PKA/cAMP pathway further leads the phosphorylation of cAMP response
element-binding protein (CREB), which binds to a CCAAT/enhancer-binding
protein (C/EBP) response element in StAR promoter [46]. Phosphorylated CREB
recruits CREB-binding protein (CBP), a histone acetyltransferase, causing histone
H3 acetylation around StAR promoter and facilitating local nucleosome
decondensation, which allows the assembly of transcription complexes [45]. The
phosphorylated CREB (15 min), histone H3 acetylation (60 min), and newly
transcribed nascent RNA (2 h) were nicely coordinated as the fact that the peak
of each molecular event takes place sequentially.
Along with the aberrant expression of StAR in endometriotic stromal cells,
expression of aromatase is also regulated by PGE2 via the EP2 receptor-mediated
signaling pathway [47, 48]. Aberrant expression of steroidogenic factor-1 (SF-1) in
endometriotic stromal cells alters its sensitivity to PGE2. It has been found that
competition between SF-1 and COUP-TFII on the same DNA-binding site in
aromatase promoter may contribute to the change of PGE2 sensitivity in
endometriotic stromal cells [49], whereas in endometrial cells, due to the lack of
SF-1, COUP-TFII occupies the aromatase promoter, rendering its transcriptional
activity. Since the expression of StAR and aromatase is regulated in a similar and
parallel aspect, this mechanism enables PGE2 to induce de novo estrogen produc-
tion from the readily available precursor, cholesterol, without depending on the
transport of intermediate metabolites from other organs.
9.4.2 Induction of Peptide Growth Factors by PGE2
Although it is clear that endometriosis is an estrogen-dependent disease, estrogen

per se is not a mitogen. The mitogenic effect of estrogen usually is mediated by one
or more peptide growth factors. Several well-known peptide growth factors, such as
insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and fibroblast
growth factor-2 (FGF2), have been shown to exert such estrogen-induced growth
effect in other cell types [5053]. However, evidences that link the expression
patterns of these growth factors with the pathogenesis of endometriosis are either
inconsistent or even controversial. For example, levels of IGF-1 in peritoneal fluid
of women with endometriosis were higher [54], not different [55], or even
lower [56] as compared with that in peritoneal fluid of women without endometri-
osis. The levels of EGF and EGF receptor are not different between ectopic
endometriotic lesions and eutopic endometrial tissues [57, 58]. Concentrations of
FGF-2 in peritoneal fluid and immunoreactive FGF-2 in pelvic endometriotic cells
are not different from those of normal or eutopic counterparts [57, 59, 60].
In contrast to the aforementioned peptide growth factors, FGF9 seems to be a

promising candidate that carries estrogens mitogenic effect in endometriotic cells.
First, expression of FGF9 is induced by estrogen in normal endometrial stromal
cells [61]. Second, FGF-9 is consistently expressed by ectopic endometriotic tissue
with greater amounts in early stage compared to that in late stage [62], which
correlates with the concentrations of estrogen in the peritoneal fluid of women with
endometriosis [44]. Third, expression of FGF9s high-affinity receptors, including
FGFR2IIIb, FGFR2IIIc, FGFR3IIIb, and FGFR3IIIc, is detected in ectopic
endometriotic stromal cells [61]. Fourth, FGF9 dose-dependently induces endome-
trial stromal cell proliferation [61, 63]. Taking these four lines of evidence together
with the notion that PGE2 is able to induce estrogen production, it strongly suggests
that FGF9 is an estromedin that transmits PGE2s action in promoting the prolifer-
ation of endometriotic cells. Indeed, treatment of endometrial stromal cells with
PGE2 dose-dependently induces FGF9 expression, and PGE2-preteated conditioned
medium is able to induce stromal cell proliferation, an effect that can be blocked by
the addition of anti-FGF9 antibody [64]. Interestingly, it was shown that blocking
estrogen signaling by estrogen receptor antagonist, ICI182,780, only partially
inhibits PGE2s action [64]. This observation leads to a new discovery that PGE2
also directly induces FGF9 expression independent of estrogenic effect [64].
How can PGE2 regulate the same gene expression through two different path-
ways? To understand this, it is necessary to review the signaling transduction
pathways of PGE2. PGE2 regulates various physiological and pathological pro-
cesses by binding to its receptors on the plasma membrane. In mammals, there are
four distinct subtypes of PGE2 receptor, namely EP1, EP2, EP3, and EP4, which are
encoded by different genes [65]. All EP receptors are G-protein-coupled receptors.
EP2 and EP4 couple to Gs and activate adenylyl cyclase and protein kinase A
signaling pathway. Compared to EP2 and EP4, EP1 and EP3 have more compli-
cated pathways. Both EP1 and EP3 have different isoforms generated via alterna-
tive splicing varied in their C-terminal cytoplasmic domain, which largely accounts
for interaction with G proteins. EP1 has been reported to couple to Gq or Gi/o
proteins and promotes the increase of intracellular Ca2+ level and/or inhibition of
PKA. EP3 couples to Gs, Gq, or Gi proteins and activates the PKA/PKC/MAPK
pathway, immobilization of intracellular Ca2+, or inactivation of PKA [65]. There-
fore, different signaling pathway(s) can be regulated by PGE2 dependent on which
specific EP was bound [16]. Three EP receptors, EP2, EP3, and EP4, are expressed
in human endometrial and endometriotic stromal cells [45].
The effect of PGE2 on the induction of StAR and aromatase is mediated through
binding to the EP2 receptor [45, 47]. In contrast, the induction of FGF9 by PGE2 is
mediated by EP3 receptor and its downstream signaling pathway (Fig. 9.2). Treat-
ment with PGE2 or selective EP3 agonist, sulprostone, activates PKC, which leads
to phosphorylation of ERK. Phosphorylated ERK translocates to the nucleus and
activates transcription factor ELK-1, which binds to two response elements residing
in the promoter of human FGF9 gene [64]. This direct effect of PGE2 represents the
quick response, which occurs between 8 and 12 h after treatment. On the other
hand, the PGE2-estrogen-FGF9 axis represents a delayed response, which occurs
between 24 and 48 h. Taken together, via two different types of receptors, PGE2 is
able to induce the critical survival and proliferating factor, FGF9, to ensure the
progression of endometriosis.
9.4.3 Suppression of Phagocytosis by PGE2
One of the most intriguing questions in the pathogenesis of endometriosis is why

the immune system fails to clear the retrogradely transported tissues. Normally,
apoptotic tissues such as the shed endometrium will be destroyed and engulfed by
immune cells. However, in the case of endometriosis, these tissues obviously have
some sort of immune privilege that prevent them from being phagocytosed. During
the development of endometriosis, the retrograded endometrial tissues induce local
inflammation that recruits immune cells, mainly the macrophage, to the peritoneum
[66, 67]. Macrophages represent the first line of defense system that either directly
phagocytose these aberrantly present cells or activates other immune cells (such as
dendritic cells, natural killer cells, and lymphocytes) to launch the antiproliferation
responses [68]. However, peritoneal macrophages isolated from patients with
endometriosis have greater ability in producing inflammatory agents and poorer
capability in phagocytosis [69, 70]. This phenomenon has puzzled researchers for
more than three decades. Recently, through a series of investigations, the mecha-
nism of immune deficiency in endometriotic macrophage becomes more and more
clear.
The phagocytosis process begins with secreting proteases by activated macro-
phages. Matrix metalloproteinases (MMPs) are a group of proteases that participate
in extracellular matrix degradation [71]. Macrophages can secrete MMP-2,
MMP-7, MMP-9, and MMP-12 to degrade elastin and have been implicated to
play an important role in the pathogenesis of emphysema and aortic aneurysm
[7275]. In addition, MMP-9 can facilitate the destruction of the type IV collagen-
containing basement membrane which separates the epithelial and stromal com-
partment [76]. This feature makes MMP-9 becoming the prime candidate secreted
by macrophages to destroy the retrogradely transported tissues in the peritoneal
cavity.
Another important molecule that also contributes to macrophages phagocytic
function is annexin A2. Annexin A2 has diverse biological functions depending on
its cellular localization. When annexin A2 is expressed in membrane-bound form, it
promotes the ability of macrophages in remodeling extracellular matrix. Annexin
A2 acts as a fibrinolytic receptor that activates plasmin by facilitating the interac-
tion between tissue plasminogen activator and plasminogen [77]. Plasmin serves as
a physiological activator which, in turn, converts pro-MMP-9 to active MMP-9.
Thus, the activation of membrane annexin A2 will lead to MMP-9 activation. In
addition, annexin A2 can be externalized or secreted. Soluble annexin A2 protein
activates human monocyte-derived macrophages through toll-like receptor
4 resulting in enhancing phagocytosis [78]. Furthermore, it has been reported that
Fig. 9.3 Prostaglandin E2 inhibits phagocytosis. Under low-PGE2 condition, macrophages are
recruited to the peritoneal cavity due to retrogradely transported endometrial tissue-induced
inflammation. Recruited macrophages express high levels of annexin A2 (membrane form and
soluble form), MMP9, and CD36 to facilitate phagocytosis. Therefore, the retrograded tissues will
be engulfed and removed. However, under high concentrations of PGE2 (indicated by red lines),
the expression of annexin A2, MMP9, and CD36 is all suppressed. Thus, macrophages lose their
phagocytic ability and retrogradely transported endometrial tissues are able to implant and grow in
the peritoneum
membrane externalization of annexin A2 in macrophages mediates apoptotic cell

clearance [79].
The third line of phagocytic activity involves expression of scavenger receptors
on the macrophages to enhance the uptake and degradation of cell debris [80,
81]. Scavenger receptors are a family of structurally diverse receptors having
broad ligand specificity that includes low-density lipoprotein, phosphatidylserine,
polyanion, and apoptotic cells [8284]. The known scavenger receptors that par-
ticipate in phagocytosis of apoptotic cells by macrophages include class A scaven-
ger receptors (SR-AI, SR-AII, SR-AIII) [83] and class B scavenger receptors
(SR-BI, SR-BII, and SR-BIII) [85, 86]. Reduced expression of one of these
scavenger receptors may result in loss of phagocytic ability.
By using peritoneal macrophages isolated from individuals with or without
endometriosis, we have demonstrated that levels of MMP-9, annexin A2, and
SR-BIII (better known as CD36) are all reduced in endometriotic macrophages
[8789] (Fig. 9.3). The decrease in MMP-9, annexin A2, and CD36 is due to
exposure to soluble factors in the peritoneal fluid of individuals with endometriosis.
Treatment of normal macrophages with peritoneal fluids collected from individuals
with endometriosis recaps the phenomenon seen in endometriotic macrophages. In
contrast, normal macrophages treated with peritoneal fluids collected from normal
individuals are not affected. Interestingly, not only the mRNA and protein levels of
MMP-9 are reduced, the MMP-9 enzymatic activity is also inhibited [89]. This
markedly impairs macrophages ability to digest the basement membrane of retro-
gradely transported tissues and ultimately contributes to reduced macrophages
phagocytic ability. Since CD36 is one of the first macrophage receptors to be
implicated in the recognition of aged or apoptotic cells [90, 91] and annexin A2
participates in both MMP-9 activation and phagocytosis, reduced expression of
CD36 and annexin A2 severely impairs the phagocytic ability of macrophages.
Indeed, when CD36 or annexin A2 is knocked down from the normal macrophages,
the phagocytic ability is reduced. In contrast, when peritoneal macrophages isolated
from individuals with endometriosis are transfected with exogenous CD36 or
annexin A2, the phagocytic ability is restored [88, 92].
The next question to ask is which factor in the endometriosis peritoneal fluid
exerts such inhibitory effect. Because endometriosis is a chronic inflammatory
disease and many proinflammatory cytokines are elevated in the peritoneal fluid
of endometriosis [24, 9396], it is likely that reduced phagocytic ability of perito-
neal macrophage is mediated by one or some of these proinflammatory cytokines.
Through a series of screens, we identified that PGE2 is the primary factor to inhibit
the expression of MMP-9, annexin A2, and CD36 in peritoneal macrophages [88,
89, 92]. PGE2, via binding to the EP2 and EP4 receptors, activates PKA signaling
pathway to suppress the expression of MMP-9, annexin A2, and CD36. As
expected, treatment of macrophages with PGE2 inhibits phagocytic ability, which
can be reversed by adding EP2 receptor antagonist [92]. The in vitro results of PGE2
action in suppressing phagocytosis are further supported by in vivo autologous
transplanted mouse model. In this model, uterine endometria from donor mice are
peeled off, minced into small pieces, and injected into peritoneal cavity of recipient
mice. Recipient mice are treated with or without PGE2 or selective COX-1/COX-2
inhibitors to blunt the biosynthesis of PGE2 for 4 weeks. Mice treated with PGE2
have more endometriotic tissue-like lesions, lower levels of CD36 and annexin A2
in peritoneal macrophages, and reduced phagocytic ability of peritoneal macro-
phages. In contrast, mice that received selective COX inhibitors develop fewer
lesions, express more CD36 and annexin A2 by the peritoneal macrophages, and
exert greater phagocytic ability of peritoneal macrophages [88, 92]. Taken alto-
gether, these data reveal that PGE2 utilizes the same receptor-mediated signaling
pathway to target three different molecules involved in phagocytosis. Such a
safeguarding system to efficiently inhibit macrophages phagocytic ability by
controlling three target proteins simultaneously is likely due to the evolutional
advantage.
9.4.4 Induction of Angiogenesis by PGE2
Establishment of an effective blood supply is a prerequisite for the survival of

retrogradely transported endometrial tissues to develop as endometriotic lesions.
Newly formed vessels play an indispensible role in the development and
progression of endometriosis by providing nutrients, growth factors, and oxygen

[9799]. The newly formed blood vessel can be easily observed on day 4 after the
transplantation of mouse endometrium to the peritoneal cavity [27]. Several
pro-angiogenic factors that may be involved in blood vessel formation during the
development of endometriosis had been reported. Among those, vascular endothe-
lial growth factor (VEGF) and cysteine-rich angiogenic inducer 61 (CYR61) are the
most well studied. Concentrations of VEGF in the peritoneal fluid of individuals
with endometriosis are greater compared with controls [100]. This VEGF may
come from both endometriotic lesions and infiltrated neutrophils and macrophages
[27, 97]. Estrogen and COX-2 have been shown to play critical roles in angiogen-
esis in various tumor models. Both can stimulate VEGF expression and induce
endothelial cell proliferation [101103]. In endometriosis, estrogen has been shown
to enhance VEGF production in neutrophils and macrophages [27] while the
expression of COX-2 and VEGF is highly correlated in endometriotic lesions
[104]. In vitro study reveals that treatment of endometrial epithelial cells with
celecoxib, a selective COX-2 inhibitor, inhibits VEGF expression in comparison
to that treated with vehicle [105]. In vivo studies also demonstrate inhibitory effects
of selective COX-2 inhibitors in the growth of endometriotic lesion and the
development of microvascular networks. Treating rats with rofecoxib induced a
decrease in the endometriotic lesion size accompanied by a decrease in peritoneal
fluid VEGF levels [106]. Ozawa et al. demonstrate that another selective COX-2
inhibitor, NS398, decreases the size of implants in a xenograft model that implants
human ovarian endometrioma to peritonea of SCID mice [107]. Laschke
et al. report that the expression of proliferating cell nuclear antigen and VEGF as
well as the microvessel density within the endometrial grafts is decreased in
NS398-treated golden hamster [108]. Machado et al. also report that parecoxib,
also a selective COX-2 inhibitor, reduced lesion size, microvessel density, the
number of macrophages, and the expression of VEGF in a rat model of peritoneal
CYR61, a member of the CCN family of growth regulators, is a pro-angiogenic
factor that mediates several distinct functions in cell proliferation, adhesion, migra-
tion, differentiation, apoptosis, and extracellular matrix production. Cyr61-
deficient mice suffer embryonic death due to vascular defects [110]. Expression
of CYR61 mRNA is rapidly induced in an immediate early fashion by a spectrum of
stimuli such as growth factors, cytokines, and estrogens [111]. The expression of
CYR61 in endometrium is elevated in the proliferative phase and menstrual efflu-
ents [112, 113]. Aberrant expression of CYR61 has been found in the ectopic
lesions of endometriotic women and baboon [35, 114, 115]. The expression of
CYR61 is upregulated by PGE2 and hypoxia in human endometrial stromal cells
and colon cancer cells, respectively [34, 112]. While PGE2-induced CYR61 is a
direct effect, hypoxia-mediated CYR61 may be mediated via downregulation of
DUSP2. It has been shown that reduced expression of DUSP2 by hypoxia causes a
prolonged phosphorylation of ERK and p38 MAPK, which ultimately leads to
upregulation of COX-2 [35, 36]. Therefore, hypoxia-induced CYR61
overexpression is likely also mediated by PGE2.
Taking together all currently available data, it is clear that COX-2-derived PGE2
does play a key role in establishing an effective blood supply system either directly
or indirectly during the development of endometriosis. Targeting COX-2-derived
PGE2 signaling pathway to blunt new blood vessel formation may be a plausible
approach to prevent the development of endometriosis.
9.5 Feed-Forward Loop of PGE2
The consistent production of self-supporting factors is a sophisticated mechanism

that keeps endometriotic cells alive despite of the cyclic rises and falls of estrogen
and is also the main reason why there is no effective therapeutic regimen for
endometriosis. The central piece of this feed-forward self-supporting system is
PGE2. There are at least three feed-forward loops to maintain PGE2 at the high
concentration in the endometriotic tissues and surrounding local environment
(Fig. 9.4). The first positive loop involves estrogen and COX-2. COX-2-derived
PGE2 stimulates StAR and aromatase expression in ectopic stromal cells, which
leads to aberrant production of estrogen. Autonomous production of estrogen by
ectopic tissues induces several known peptide growth factors such as VEGF and
FGFs that serve as autocrine (for endometriotic cell) and paracrine (for endothelial
cell) factors to stimulate cell proliferation and angiogenesis. On the other hand,
estrogen can induce COX-2 expression and thus PGE2 production to form the feed-
forward auto-amplification loop [116].
The second positive loop requires the cooperation between peritoneal macro-
phages and endometriotic stromal cells. Peritoneal macrophages secrete
proinflammatory cytokines such as IL-1 and PGE2 to induce COX-2 expression
in ectopic endometriotic stromal cells and peritoneal macrophages. As a result,
more PGE2 is produced. The elevated PGE2 not only induces more COX-2 expres-
sion by macrophages but also inhibits phagocytosis. Attenuated phagocytic ability
of macrophages enables ectopic endometriotic tissues to grow and produce more
PGE2 upon stimulation by proinflammatory cytokines or estrogen.
The third positive loop, to some extent, may be the most important one to initiate
the whole pathological process of endometriosis. It starts with the increase of
hypoxic stress due to lack of blood supply to the endometrium right before the
onset of menstruation. Increased hypoxic stress causes the accumulation of HIF-1,
which then translocates to the nucleus and dimerizes with HIF-1 to regulate gene
expression. Hypoxia-mediated downregulation of DUSP2 causes a prolonged acti-
vation of ERK and p38 MAPK, which results in increased sensitivity of COX-2
gene to proinflammatory cytokine stimulation. As a result, the endometriotic
lesions are more vulnerable to exogenous stimulation and produce more PGE2.
Moreover, the activation of ERK enhances nuclear accumulation of HIF-1 [117],
which further suppresses DUSP2 and thus enhances ERK activation. This may
explain why HIF-1 protein [33] and phosphorylated ERK [118] are elevated in
ectopic endometriotic lesions. Because ERK and p38 MAPK signaling controls
Fig. 9.4 Positive feed-forward loops of PGE2 actions in endometriosis. Regulation of PGE2
biosynthesis in ectopic endometriotic stromal cells is regulated by predisposition to hypoxic stress,
which inhibits expression of dual-specificity phosphatase-2 (DUSP2) and leads to a prolonged
activation of ERK. Phosphorylated ERK stabilizes HIF-1 and, at the same time, enhances
proinflammatory cytokine-induced COX-2 expression and PGE2 production. These
proinflammatory cytokines also induce COX-2 expression and PGE2 production by peritoneal
macrophages. Elevated concentration of PGE2 stimulates steroidogenesis and angiogenesis to
promote cell proliferation and inhibits phagocytosis to prevent ectopic endometriotic tissues
from destroyed by macrophages. The effects of PGE2 are augmented by three feed-forward
positive regulatory loops: (1) hypoxia-HIF-1-ERK loop, (2) COX-2-PGE2-estrogen loop, and
(3) COX-2-PGE2 proinflammatory cytokine loop. See text for details
many endometrial functions such as IL-1 and macrophage migration inhibitory

factor-induced COX-2 expression [25, 119, 120], macrophage migration inhibitory
factor-stimulated production of angiogenic factors [121], basal endometrial cells
survival [122], and estrogen-stimulated cell proliferation [118], downregulation of
DUSP2 in ectopic endometriotic cells points out the importance of hypoxia and/or
HIF-1 in the pathogenesis of endometriosis.
As we can see in Fig. 9.4, these three positive feed-forward loops are not
separated but rather interlinked to form a very complicated auto-amplification
gene regulatory network. It involves multiple genes and environmental factors.
Thus, how to disrupt these tightly regulated, self-supporting feed-forward loops
represents a great challenge in treating endometriosis.
9.6 Targeting PGE2 Signaling Pathway as a Potential

Therapeutic Approach
As reviewed above, PGE2 exerts multiple pathological functions to regulate the

development of endometriosis and minimize or eradicate those effects triggered by
PGE2 and represents a plausible approach to prevent or cure endometriosis.
Inhibiting the production of PGE2 by ectopic endometriotic stromal cells and by
peritoneal macrophages might be an option. However, given the short half-life of
COX inhibitors and the unfavorable side effects caused by long-term use of
NSAIDs, suppressing PGE2 production to cure endometriosis may not be an ideal
choice. An alternative approach is to block the downstream signaling pathways to
terminate PGE2s action. For example, it has been shown recently that blocking
EP2/EP4 may prevent PGE2 to transactivate EGF receptor and induce apoptosis in
SV40-immortalized endometriotic stromal and epithelial cells. Similar approach
may be applicable to block EP3 receptor on endometriotic stromal cells to inhibit
the production of FGF-9 or to target EP2 on macrophages to prevent suppression of
phagocytic ability by PGE2. In addition, inhibition of multiple enzymes to disrupt
the positive feedback loops of PGE2 overproduction may be an alternative. Indeed,
dienogest, a synthetic progestin that inhibits COX-2 and aromatase activities, was
found to be effective in ameliorating endometriotic symptoms [123125]. In light
of this recent finding, developing effective small molecules to terminate PGE2-
mediated signaling is more likely a promising therapeutic strategy to treat
endometriosis.
Acknowledgment This work was supported by grants from National Science Council of Taiwan
(NSC-101-2320-B-006-030-MY3 to SJT and NSC-101-2314-B-006-043-MY2 to MHW). KYH
wrote the first draft and proof-read the final manuscript. MHW and SJT edited and wrote the final
draft. SJT designed and supervised the project. All authors read and approved the final draft.
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Chapter 10
Sex Steroids and Endometriosis
Jo Kitawaki
Abstract Endometriosis develops mostly in women of reproductive age and

regresses after menopause, suggesting that the growth of lesions is estrogen depen-
dent. Estrogen metabolism differs considerably in women with a normal endome-
trium compared to those with estrogen-dependent uterine diseases, including
endometriosis, adenomyosis, or fibromas. Altered expression patterns of estrogen
receptor (ER)-/ER-, progesterone receptor (PR)-A/PR-B, and 17-
-hydroxysteroid dehydrogenase type 1 (HSD17B1)/type 2 (HSD17B2) in
endometriotic tissue may upregulate aromatase and increase local estrogenic activ-
ity. Polymorphisms in ESR1, ESR2, PR, HSD17B1, 17-hydroxylase (CYP17A1),
and aromatase (CYP19) genes have been investigated putative associations with
endometriosis susceptibility.
Keywords Endometriosis Endometrium Estrogen Polymorphism Steroid

receptors
10.1 Introduction
Endometriosis, a common gynecological disorder in women of reproductive age, is

characterized by the presence of endometrium-like lesions outside the uterine
cavity. The main symptoms are pelvic pain, including dysmenorrhea, chronic
pelvic pain and deep dyspareunia, and infertility [1]. Endometriosis develops in
approximately 10 % of women of reproductive age and regresses after menopause
or ovariectomy. The occurrence of endometriosis before menarche has not been
reported. The suppression of estrogen levels using a gonadotropin-releasing
J. Kitawaki (*)
Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine,
Graduate School of Medical Science, Kyoto 602-8566, Japan
e-mail: kitawaki@koto.kpu-m.ac.jp

148 J. Kitawaki
hormone agonist causes regression of the lesions. The recovery of estrogen levels
after discontinuing therapy causes a relapse. Endometriosis may relapse in post-
menopausal women who have been treated with estrogen-replacement therapy,
suggesting that endometriosis progresses and regresses in an estrogen-dependent
fashion.
10.2 Mechanisms of Estrogen-Dependent Growth
Breast cancer, endometrial cancer, endometriosis, adenomyosis, and fibromas are

diseases that progress in an estrogen-dependent fashion. These lesions commonly
contain ER, PR, and androgen receptors. Interestingly, all of the estrogen-
dependent diseases of the breast and uterus contain not only ER but also aromatase,
an enzyme that catalyzes the conversion of androgens to estrogens. Together with the
circulating estrogen, local estrogen production increases the estrogen concentration
and stimulates the growth of tissue mediated by the ER. Immunohistochemical studies
have shown that ER and PR are localized to the nucleus of both glandular and stromal
cells [29], whereas aromatase is exclusively localized to the cytoplasm of epithelial
glandular cells [9].
Expression patterns of steroid-related molecules, including steroid receptors and
enzymes involved in estrogen metabolism, differ between endometriotic tissues and
eutopic endometria. Electron microscopic analysis has shown that one-third of
endometriotic implants are out of phase with the menstrual cycle [10], and a light
microscopic study showed that only 13 % of endometriotic implants were synchro-
nous with the corresponding eutopic endometria [11].
10.2.1 Steroid Receptors
ER- levels are higher and ER- levels are lower in women with endometriosis than
in those with a normal endometrium. Higher ER- levels are caused by
hypomethylation of a CpG island at the promoter region of this gene. In
endometriotic cells, the excess expression of ER- is responsible for the estrogen-
dependent responses, and this expression inhibits ER- action [12].
The single PR gene is translated into one of two isoforms (PR-A or PR-B) by
activating the corresponding promoters. PR-A is a truncated form of PR-B that
lacks the N-terminal 164 amino acids; PR-A acts as a repressor of PR-B function. In
endometriotic tissue, PR-B is undetectable [13] due to hypermethylation of the PR
gene and PR-A is markedly reduced [14]. Furthermore, excess ER- occupies the
estrogen responsive element at PR promoter region and blocks transcription of PR
that is normally activated by ER- [15].
10 Sex Steroids and Endometriosis 149
10.2.2 Estrogen Metabolism
10.2.2.1 Aromatase
Steroidogenic factor 1 (SF1), an orphan nuclear receptor, is expressed at a 12,000

times higher concentration in women with endometriosis than in those with a
normal endometrium. SF1 is involved in the prostaglandin E2 (PGE2)-stimulated
increase in aromatase expression [15]. In endometrial cells, a CpG island at the SF1
promoter is highly methylated and binds to a silencer-type transcription factor,
whereas in endometriotic cells, SF1 promoter is unmethylated and binds to a
coactivator.
A positive feedback cycle indicates that aromatase and cyclooxygenase-2
(COX-2), a PGE2 synthase, are responsible for the continuous local formation of
estrogen and PGE2 in endometriotic stromal cells [16]. PGE2 is a major mediator of
endometriosis-associated pain. Aromatase expression might be induced by PGE2 in
endometriotic cells. Aromatase produces estradiol, which consequently promotes
PGE2 production by inducing COX-2 expression [17]. Danazol and dienogest, a
fourth-generation progestin, which is used for relieving endometriosis-associated
pelvic pain, downregulate expression of aromatase and COX-2 in endometriotic
cells [18].
In the normal endometrium of women, aromatase is not expressed, but it is
expressed in the endometrium of women with estrogen-dependent uterine diseases
such as endometriosis, adenomyosis, or fibromas [9, 19, 20]. In endometriotic cells,
the increased expression of SF1 activates aromatase transcription [21]. Immunohis-
tochemical detection of aromatase P450 protein in endometrial biopsies can be used
as a screening test for endometriosis [22]. However, detection of aromatase mRNA
is less specific for screening of endometriosis [23].
10.2.2.2 17-Hydroxysteroid Dehydrogenase Type 1 and Type 2
In endometriotic tissues, HSD17B1, an enzyme that converts estrone to the more

potent estradiol, predominates, whereas expression of HSD17B2, an enzyme
responsible for weakening the activity of estradiol and converting it into less active
estrone, is undetectable [24] or at a lower level than HSD17B1 [20, 25]. This is
partially responsible for raising the local estrogen activity level.
In contrast in the endometrium, an oxidative reaction that weakens the activity of
estradiol to estrone by HSD17B2 predominates and the expression of HSD17B1 is
undetectable [26] or extremely low [25]. During the proliferative phase, the expres-
sion level and activity of HSD17B2 are comparable in endometria of women with
estrogen-dependent diseases and women without disease. However, during the
secretory phase, while the expression and activity of HSD17B2 increase four- to
sixfold in diseased endometria, there is no cyclical change in normal endometrium
[20, 27]. Although it is not possible to distinguish histologically between normal
150 J. Kitawaki
and diseased endometria, estrogen metabolism is different between the two states.
Furthermore, in endometriotic tissues, progesterone does not stimulate HSD17B2
because there is no PR-B [13], whereas endometrium of women with endometriosis
acquires ability to stimulate HSD17B2 by progesterone.
10.3 Polymorphisms in Genes Involved

in Steroid Metabolism
Both environmental and genetic factors have been implicated in the pathogenesis of
endometriosis. Family and twin studies have indicated that there is a two- to
ninefold increase in the risk of developing endometriosis in first-degree relatives
of women with endometriosis, suggesting it is a genetic disorder with polygenic or
multifactorial inheritance [2830]. Studies have revealed associations between the
polymorphisms of many genes, including the genes involved in steroid metabolism
and endometriosis susceptibility.
10.3.1 ESR1
Polymorphisms in the gene coding for ER- (ESR1) have been investigated in
European and Asian women. Single-nucleotide polymorphisms in intron 1 of
the ESR1 gene have been assessed in PvuII (398 T/C) and XbaI (351A/G)
restriction fragment length polymorphisms. Several studies found statistically sig-
nificant differences between the cases and controls in the distribution of PvuII
alleles [3134], but other studies found no association with PvuII polymorphisms
[3538]. One study found an association with an XbaI polymorphism [34], and
three studies found an association with the TA dinucleotide repeat polymorphism
[31, 36, 39, 40].
10.3.2 ESR2
One study in Japanese women showed association with the gene coding for
ER- (ESR2) AluI polymorphism [35], but other studies found no association
[33, 41]. One study showed that shorter CA dinucleotide repeats in the ESR2
gene were linked to endometriosis in women without infertility [40].
10.3.3 PR
Several studies in Austria, Italy, and Brazil showed association between the 306-bp
insertion polymorphism in intron G of the PR gene (PROGINS) and the risk of
endometriosis risk [4244].
10.3.4 HSD17B1
Several studies showed that, in an A/G polymorphism of the HSD17B1 gene, the A
allele was found to be associated with a significantly increased risk of endometriosis
[40, 45].
10.3.5 CYP17
One study showed a possible association of endometriosis in Taiwanese women

with a MspA1 (T/C) polymorphism in the CYP17A1 gene [39], whereas other
studies found no association between any CYP17A1 gene polymorphisms and
endometriosis risk [4648].
10.3.6 CYP19
In intron 4 of the CYP19 gene, there is a TTTA repeat microsatellite polymorphism,

and 50-bp upstream of the TTTA polymorphism, a 3-base pair (CTT) insertion/
deletion polymorphism. A study in Japanese women showed an association between
this 3-base pair insertion/deletion polymorphism and endometriosis risk [46].
The TTTA repeat polymorphism was found to increase endometriosis risk in
Greek women [49]. A study in Italian women showed a significant association of
AA and CC genotypes in Val80 and C1558T polymorphisms with endometriosis
risk [50]. However, two other studies showed no association between CYP19 gene
polymorphisms and endometriosis susceptibility [40, 45].
10.4 Conclusions
Altered expression patterns of a range of receptors and enzymes involved in steroid

metabolism (ER-/ER-, PR-A/PR-B, and HSD17B1/HSD17B2) in endometriotic
tissue may upregulate aromatase and increase local estrogenic activity, thus
152 J. Kitawaki
stimulating the growth of lesions. Substantial differences in estrogen metabolism

exist between normal endometrium and the endometrium in women with uterine
diseases, including endometriosis, adenomyosis, or fibromas. Polymorphisms in
ESR1, ESR2, PR, HSD17B1, CYP17A1, and CYP19 genes have all been investi-
gated with varying success. Further investigations to distinguish pathologic from
normal endometria and genetic polymorphism associations will contribute to a
better understanding of endometriosis and help develop novel therapeutic strategies
for this disease.
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Chapter 11
Aromatase Expression in Endometriosis
and Its Significance
Hiroshi Ishikawa and Makio Shozu
Abstract Endometriosis is a chronic inflammatory disease frequently observed

in the ovary, pelvic peritoneum, and rectovaginal septum. The growth and
progression of an endometriotic lesion depends on a sex steroid, estrogen.
Aromatase, a key enzyme in estrogen biosynthesis, is highly expressed in the
endometriotic tissue, resulting in in situ production of estrogen that, in addition to
endocrine estrogen from the ovary, may contribute to the etiology and progression
of endometriosis. The aberrant expression of aromatase together with the elevated
expression of 17-hydroxysteroid dehydrogenase type 1 and the absence of
17-hydroxysteroid dehydrogenase type 2 observed in the endometriotic tissue
would contribute to an increase in the tissue concentration of estrogen. Aromatase
expression is regulated at multiple levels, from the transcription of CYP19A1 and
epigenetic codes to posttranslational modification and degradation of the protein.
Among the multiple promoters of CYP19A1, the most proximal promoter PII
is the most active in endometriosis and is regulated by cAMP, prostaglandin E2,
steroidogenic factor-1, and possibly the end product estrogen. Hypomethylation
of CpG islands on CYP19A1 observed in the endometriotic tissue may contribute
to the upregulation of aromatase expression.
Similar to spontaneous menopause, inhibition of in situ estrogen biosynthesis may
regress endometriosis. The use of aromatase inhibitors (AIs), which selectively inhibit
aromatase activity in human tissues, is a possible treatment for inhibiting local estrogen
biosynthesis in endometriosis. AIs have been used as monotherapy or in combination
therapies with progestins, oral contraceptive pills, and gonadotropin-releasing
hormone agonists to reduce endometriosis-related pain in premenopausal women.
Keywords Aromatase Aromatase inhibitor Estrogen Letrozole
H. Ishikawa, M.D., Ph.D. M. Shozu, M.D., Ph.D. (*)

Department of Reproductive Medicine, Graduate School of Medicine, Chiba University,
1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
e-mail: shozu@faculty.chiba-u.jp

156 H. Ishikawa and M. Shozu
11.1 Introduction
Endometriosis is defined as the proliferation of the endometrial gland-like epithelium

and surrounding stroma outside the uterus. It is frequently observed in the pelvic
peritoneum, ovary, and rectovaginal septum. In total, 610 % of women of reproduc-
tive age are suffering from endometriosis-related pain, i.e., menorrhagia, dyspareunia,
and chronic pelvic pain. Moreover, endometriosis may cause infertility. Although
considerably rare, ovarian endometrioma can transform and give rise to ovarian cancer
during long-term management of the disease.
Endometriosis becomes symptomatic in the reproductive age and is self-limited
after spontaneous menopause. Similar to spontaneous menopause, long-term inhi-
bition of ovulation using progestins, oral contraceptive pills (OCPs), and
gonadotropin-releasing hormone (GnRH) agonists alleviates the symptoms. On
the other hand, estrogen use in postmenopausal women induces symptomatic
regrowth of endometrial lesions. These findings suggest that the progression of
endometriosis is closely associated with estrogen as well as ovulation.
The ovary synthesizes estrogens de novo from cholesterol and supplies most of
the circulating estrogens in women of reproductive age. In addition to the ovary, a
small amount of estrogens can be synthesized in situ at peripheral fat, bones, the
skin, the brain, and vessels, which are unable to synthesize estrogen from choles-
terol de novo but are able to convert circulating androgens to estrogens [1, 2].
There are 2 rate-limiting steps in estrogen biosynthesis. The first step is the
mobilization of cholesterol across the mitochondrial membrane into the mitochon-
dria. This step is mediated by the steroidogenic acute regulatory protein (StAR)
expressed in the mitochondria. The second step is the enzymatic reaction
converting androgens to estrogens by aromatase. Aromatase is a member of the
cytochrome P450 superfamily and is expressed in the endoplasmic reticulum.
Aromatase is a unique enzyme that can catalyze aromatization. The rate of estrogen
synthesis depends on these 2 steps in the ovary, where estrogen is synthesized de
novo. On the other hand, the synthesis depends only on the second step in peripheral
organs. In these peripheral organs, expression levels of aromatase determine the
production rate of estrogen as long as circulating androgen is supplied [3].
Estrogen plays essential roles in reproduction, bone development, and epiphy-
seal closure in puberty, bone mineral metabolism, and possibly cognitive function.
In addition to the physiological roles, recent studies have focused on the patholog-
ical roles and revealed that the overproduction of estrogen in situ causes diseases.
Breast cancer tissues express a high level of aromatase, and the resulting in situ
estrogen promotes cancer cell growth in postmenopausal women. Endometriosis is
another example in which the aberrant expression of aromatase is involved in
disease pathogenesis. The inhibition of in situ estrogen synthesis using an aroma-
tase inhibitor (AI) is therefore a potential choice of treatment, similar to that seen in
the case of breast cancer.
Here, we review the aromatase expression in endometriotic tissues and the
mechanisms underlying the aberrant expression. Following this, we discuss the
11 Aromatase Expression in Endometriosis and Its Significance 157
pathophysiological significance of in situ estrogen in endometriotic tissues and of

aromatase as a molecular target treatment for refractory endometriosis.
11.2 Aromatase Genes and Their Expression

in Endometriotic Tissues
11.2.1 Genomic Structure of CYP19A1
Aromatase is a unique enzyme responsible for the conversion of androgens to

estrogens in humans. Aromatase is encoded by CYP19A1 on chromosome 15q22,
which spans approximately 132 kb and is composed of at least 9 alternative first
exons and their 9 downstream coding exons (exons 210). Each first exon contains
a unique 50 -untranslated sequence and a unique upstream promoter sequence. Each
promoter possesses multiple transcriptional regulatory element-binding sites,
which enable tissue-specific and promoter-specific regulation of aromatase
expression.
The transcription of aromatase begins from 1 of the 9 first exons, is extended
to exon 10, and ends at 1 of the 2 poly A signals located at the end of the
30 -untranslated region of exon 10. The resulting primary transcripts give rise to
mRNA by splicing all introns. Because there are 9 different first exons, at least
9 different mRNAs are formed; however, all of these encode the same aromatase
protein because the protein coding sequence is located in the shared exon 2 and its
downstream.
11.2.2 Regulation of Aromatase Expression
11.2.2.1 Expression of Aromatase and In Situ Estrogen
Aromatase is highly expressed in ovarian granulosa cells of Graafian follicles and in

placental cytotrophoblasts. Aromatase is also expressed in Sertoli cells, neurons,
adipose stromal cells, vascular smooth muscle cells, skin fibroblasts, bone osteo-
blasts and osteoclasts, hepatocytes, and uterine myometrium. More recently, aro-
matase expression has been reported in the stomach, lung, colon, and macrophages,
albeit at a low level [47]. Almost all estrogen receptor (ER)-positive cells express
aromatase, although the expression levels vary.
In addition to normal tissues, aromatase is expressed in some pathological
conditions such as breast cancer, endometriosis, uterine fibroids, and endometrial
cancer. Estrogens synthesized in situ within these peripheral organs can bind to ERs
of their own cells or those of neighboring cells and stimulate proliferation, thereby
playing a role in disease pathogenesis. The biological actions of this in situ estrogen
are more potent than those expected from a comparable amount of endocrine
estrogen, possibly because in situ estrogen directly acts on neighboring cells with
neither dilution into the circulating volume nor binding to proteins that interfere
with the hormonal action.
11.2.2.2 Tissue-Specific Regulation of Aromatase
As described above, aromatase expression is regulated in a tissue-specific manner

in multiple tissues by the alternate use of multiple promoters. For example, aroma-
tase transcription is driven in the ovary by the most proximal promoter PII, which is
regulated by cAMP downstream of a gonadotropin, follicle-stimulating hormone
(FSH). The transcription in the brain is driven by promoter 1f. A further upstream
promoter, I.4, drives the transcription in adipose tissues. In these promoters,
specific steroid hormones are essential for expression: testosterone and glucocorti-
coid for promoters 1f and I.4, respectively. Local factors such as growth factors and
cytokines also play regulatory roles in these promoters. For the placenta, promoter
I.1 is a predominant promoter, while all other promoters are transcribed at very low
levels. Promoter I.1 is quite different from other promoters in terms of its structure
and function: it does not have a nuclear half site for binding of the NR5A (SF1) and
permits constitutive expression, while others strictly downregulate basal
expression.
The promoter-specific regulation is realized by relatively short upstream seg-
ments of promoters (less than approximately 500 bp), where tissue-specific
enhancers and regulatory cis-elements possibly exist. Thus, a part of genomic
DNA containing these short sequences mimics the physiological regulation by
factors such as FSH, steroid hormones, cytokines, and prostaglandins. Using
transgenic mice, it has been shown that these short sequences are capable of
tissue-specific expression in the ovary and placenta [8]. However, the absolute
level of expression of these transgenes (promoter constructs) is generally lower than
that observed in vivo, suggesting that undiscovered enhancers exist outside the
promoter region. Another explanation has recently been reported for the low level
of in vitro expression compared with that of in vivo expression. Estrogen as a
product of aromatase binds to the TSPYL5 promoter and induces the expression of
TSPYL5, which in turn is integrated into the transcriptional machinery of aroma-
tase and efficiently enhances its expression [9]. This explains the reason why
expression levels of aromatase demonstrated by promoter constructs that lack
estrogen synthesizing activity are generally low.
11.2.2.3 Promoter Switching
Physiological expression of aromatase in human tissues, except for the placenta, is

basically downregulated, and upregulation occurs only when appropriate stimuli
exist. In comparison, it has been known that high levels of aromatase are
constitutively expressed in pathological conditions of the breast. For example,

aromatase is highly expressed in adipose tissues surrounding breast tumors in
postmenopausal women [10]. Similarly, aberrant expression of aromatase has
been confirmed in other pathological conditions such as endometriosis, uterine
fibroids, endometrial cancer, and lung cancer [3, 1113].
Promoter switching is a possible event underlying the aberrant expression of
aromatase in pathological conditions. This was initially described in breast adipose
tissues of women with or without breast cancer [14]. The normal breast adipose
tissue of cancer-free women expresses low levels of aromatase by the predominant
use of promoter I.4, whereas the adipose tissue surrounding breast tumors expresses
high levels of aromatase by the predominant use of promoters PII and I.3. A
possible explanation for promoter switching is that cancer-derived or cancer-
prone local factors drive aromatase expression. Consistent with this, local factors
drive aromatase expression. A promoter construct composed of tandem sequences
of mini-promoters of CYP19A1 mimics promoter switching in the breast [15].
11.2.3 CYP19A1 Polymorphisms and Risk of Endometriosis
Endometriosis is 67 times more frequent among first-degree relatives and is

presumed to have a multifactorial inheritance. Several studies have reported poly-
morphisms of CYP19A1 in endometriosis, including dysequilibrium for both breast
cancer and endometrial cancer. As shown in Table 11.1, the polymorphisms of
CYP19A1 have been associated with the risk of endometriosis; however, the results
are controversial [1624].
Recent genome-wide association studies (GWAS) identified genetic variants in
multiple loci that have been associated with endometriosis. These single-nucleotide
polymorphisms (SNPs) have never been identified by traditional candidate gene
strategies for specific diseases. GWAS-identified endometriosis-associated SNPs
have ethnic differences [25]. No SNPs on chromosome 15q22, where CYP19A1
maps, have been identified by GWAS.
11.2.4 Epigenetic Regulation of Aromatase
An epigenetic change is defined as heritable changes in gene expression that do not

represent changes in the DNA sequence. DNA methylation and histone modifica-
tion are the most often explored epigenetic mechanisms. Transcriptional regulation
by noncoding RNAs, particularly micro RNAs (miRNAs), is another distinct
epigenetic mechanism.
160
Table 11.1 Association between CYP19A1 gene polymorphisms and the risk of endometriosis
Staging of cases
Nucleotide Range of age Range of age (number of
polymorphisms Location Author Cases (mean SD) Control (mean SD) subjects)a Results
rs 10046 30 untranslated Szczepanska 115 2039 197 1939 I (59), II (56) No associationb
C1558T region of et al.
Exon 10 Lamp et al. 150 1845 199 3050 I (53), II (39), III No association
(32.1 6.1) (39.8 5.3) (36), IV (22)
Vietri et al. 104 2245 86 1848 Undescribed Genotype CC was
(36.8 6.7) (37.8 5.1) overpresented in
cases (48.1 %
vs. 30.2 %)
Hur et al. 224 Undescribed 188 Undescribed III to IV (224) No association
Huber et al. 32 (52.3 5.4) 790 (34.6 7.0) I (0), II (21), III (10), No association
IV (1)
rs700519 Exon7 Wang et al. 300 (34.3 6.9) 337 (52.2 4.2) I (7), II (52), III No association
C > T, (165), IV (76)
Arg264Cys Tsuchiya et al. 79 2045 59 2045 I (21), II (10), III No association
(23), IV (25)
Huber et al. 32 (52.3 5.4) 790 (34.6 7.0) I (0), II (21), III (10), No association
IV (1)
rs2236722 Exon2 Wang et al. 300 (34.3 6.9) 337 (52.2 4.2) I (7), II (52), III No association
T > C, (165), IV (76)
Trp39Arg Hur et al. 224 Undescribed 188 Undescribed III to IV (224) No association
240 A > G, At codon80 Vietri et al. 104 2245 86 1848 Undescribed Genotype AA was
Val80 in exon3 (36.8 6.7) (37.8 5.1) overpresented in
cases (50.0 %
vs. 32.6 %)
Hur et al. 224 Undescribed 188 Undescribed III to IV (224) No association
H. Ishikawa and M. Shozu
11
3 bp insertion/ 50 bp upstream Lamp et al. 150 1845 199 3050 I (53), II (39), III No association
deletion from the (32.1 6.1) (39.8 5.3) (36), IV (22)
(TTTA)n Kado et al. 140 2448 177 (63.8 6.1) I to II (32), III to IV Del/Del was fre-
tract in (36.3 8.1) (108) quently
intron4 observed in
cases
rs1004982 Intron Trabert et al. 256 1849 567 Matched to Undescribed Increased risk of
cases endometriosis
rs18700479 Intron Increased risk of
endometriosis
rs936307 Intron Increased risk of
endometriosis
(TTTA) repeat Intron4 Lamp et al. 150 1845 199 3050 I (53), II (39), III No significant asso-
number (32.1 6.1) (39.8 5.3) (36), IV (22) ciation between
[TTTA] repeat
<7 and [TTTA]
repeat 813
Hur et al. 224 Undescribed 188 Undescribed III to IV (224) No significant asso-
ciation between
[TTTA] repeat
<7 and [TTTA]
Aromatase Expression in Endometriosis and Its Significance
repeat 813
Kado et al. 140 2448 177 (63.8 6.1) I to II (32), III to IV No significant asso-
(36.3 8.1) (108) ciation between
[TTTA] repeat
<7 and [TTTA]
repeat 813
Arvanitis et al. 275 2137 346 2653 Undescribed [TTTA] 10 allele
(27.2 3.2) (34.5 7.4) increased risk by
4.99 times for
endometriosis
a
The staging was assessed by the revised American Society for Reproductive Medicine score
161
b
No statistically significant association was observed among different genotypes
11.2.4.1 DNA Methylation of CYP19A1
Changes in the methylation status of CpG islands of CYP19A1 have been reported
in endometriosis [26]. A CpG island located approximately 70 kb downstream from
exon 1.1 of CYP19A1 is hypomethylated in endometriosis (stromal cells obtained
from ovarian chocolate cysts) but hypermethylated in the eutopic endometrium
(stromal cells obtained from disease-free women). The methyl-CpG-binding pro-
teins MBD1 and MeCP2, which contain an amino-terminal methyl-CpG-binding
domain and a carboxy-terminal transcriptional repression domain, bind to the
hypermethylated region of the CpG island in eutopic tissues, which in turn may
suppress aromatase expression in the eutopic endometrium [27].
The treatment of endometrial stromal cells with 5-aza-deoxycytidine
(an irreversible inhibitor of DNA methyltransferase1, which is essential for
maintaining the methylation status of genomic DNA) induces aromatase expression
in endometrial stromal cells obtained from disease-free women. The demethylation of
the CpG islands in CYP19A1 may be relevant to the upregulation of aromatase [28],
although other explanations are possible because 5-aza-deoxycytidine alters the
expression level of a broad spectrum of genes that may indirectly or directly affect
aromatase expression.
11.2.4.2 Histone Modification
Histone modifications affect the chromatin structure and the subsequent interaction of
transcription factors with their response elements in the promoters. The acetylation of
histone H3 and histone H4 activates transcription by loosening the chromatin structure
and allowing the recruitment of transcription factors to their response elements.
In contrast, trimethylation of lysine at sites 9 and 27 on histone H3 inactivates
transcription by causing the chromatin to become more condensed. Histone modifi-
cations affect Cyp19a1 mRNA expression in rat granulosa cells [29]. We found
that the acetylation of H3 and H4 at the promoter I.4 region occurs during the
induction of aromatase expression by dexamethasone in breast cancer cell lines.
No histone modification has been reported for aromatase regulation in endometriosis
till date.
11.2.4.3 Regulation of Aromatase by miRNA
miRNAs are short noncoding RNAs that act by targeting partially complementary
sequences within mRNAs. They consist of 1925 nucleotides and commonly exist
in the 30 -untranslated region of the target genes. Traditionally, miRNAs negatively
regulate the transcription of their target genes. A single miRNA may target many
genes, and each of them may in turn be regulated by different miRNAs [30].
The transcriptional repression of aromatase mRNA by miRNAs has been

reported in mammalian ovarian tissues (miR-503 and miR-378 directly inhibit
aromatase, miR-224 and miR-383 indirectly inhibit aromatase), trophoblast differ-
entiation (miR-19b and miR-106a downregulate aromatase), and endometrial can-
cer (miR98 represses aromatase) [3133]. There has been no report on miRNA
regulation of aromatase mRNA in endometriosis.
11.2.5 Posttranslational Regulation of Aromatase
In addition to the mRNA level, aromatase activity is regulated at the protein level. It
has been reported that phosphorylation and dephosphorylation of amino acids in the
aromatase protein alter the enzymatic activity [34, 35].
Recently we found a novel mechanism underlying posttranslational regulation
by autophagy [36]. Insulin-like growth factor-1 enhances aromatase activity over
50 % as early as 1 h in THP-1 myeloid leukemia cells through the inhibition of
autophagy. A part of the newly synthesized aromatase protein is continuously
transported to the lysosome and is degraded when aromatase protein synthesis is
increased.
Another mechanism underlying posttranslational regulation has been proposed.
A series of transfection experiments revealed that mRNA stability and protein
translation efficiency vary among the 50 -noncoding sequence of exon 1 of aroma-
tase [37]. The 50 -noncoding sequence of exons I.3 and I.4 contains the cis-acting
elements responsible for the modulation of aromatase levels.
No study has reported the posttranslational regulation of aromatase in endome-
triosis where aromatase is highly expressed and translated.
11.3 Aromatase Expression in Endometriosis
11.3.1 Aberrant Biosynthesis of Estrogen in Endometriosis
There is unequivocal clinical evidence that the development and progression of

endometriosis depends on ovarian estrogen. In addition to endocrine estrogen,
numerous studies have shed light on the role of estrogen synthesized by
endometriotic cells in situ. Although there have been no reports on direct measure-
ments of the concentration of estrogen in the endometriotic tissues, comprehensive
accumulated data have indicated the overproduction of estrogen in situ and its role
in the pathogenesis in endometriotic tissues.
The endometriotic tissue expresses higher levels of aromatase than the eutopic
endometrium, and it efficiently converts androstenedione to estrogen [3842].
Moreover, endometriotic tissues do not inactivate estradiol by conversion to estrone
because of the reduced expression of 17-hydroxysteroid dehydrogenase (17-HSD)

type 2, whereas the eutopic endometrial epithelium does [43, 44]. Thus, endometriosis
has more available estrogen, which possibly stimulates disease progression.
Interestingly, the eutopic endometrium of women with endometriosis overexpresses
aromatase before actual implantation to the abdominal cavity. This supports the notion
that the overexpression of aromatase in the endometrium is a pathogenic factor that
causes ectopic implantation and growth of endometrial tissues in regurgitating men-
strual blood [45]. This remains to be determined in future.
The diagnostic use of the aberrant expression of aromatase has been examined.
A Japanese group collected endometrial biopsy specimens from patients and
immunostained the specimens for aromatase. Receiver operating characteristic
curve analysis revealed a cut value of 20 H-scores with 91 % sensitivity, 100 %
specificity, 100 % positive predictive value, and 72 % negative predictive value to
distinguish the eutopic endometrium from women with or without
endometriosis [46].
In addition to a high aromatase activity and reduced expression of 17-HSD type
2, the expression of 17-HSD type 1 is higher in the endometriotic tissues than in
the eutopic endometrium. 17-HSD type 1 converts estrone, a primary estrogen
synthesized from androstenedione, to estradiol, an active form of estrogen, thereby
enhancing the action of estrogen as a product. Compared with the eutopic endo-
metrium, the aberrant expression of aromatase, the elevated expression of 17-HSD
type 1, and the reduced expression of 17-HSD type 2 in the endometriotic tissue
collectively give rise to elevated local levels of estradiol [47]. Another study
reported that compared with the normal endometrium, aromatase, 17-HSD types
1 and 7 (but not type2), sulfatase, and ER were statistically significantly
upregulated, while ER was significantly downregulated in ovarian endometrioma.
There were no significant differences in 17-HSD type 2, sulfotransferase, and
progesterone receptors A and B gene expression [48]. Aromatase and 17-HSD
type1 mRNA levels were extremely low in the normal human endometrium, while
mRNAs for 17-HSD types 2 and 4, expressed sequence tags, and sequence-tagged
sites were readily detectable [49].
Endometriotic tissues express a complete set of steroidogenic enzymes to syn-
thesize estrogen: cholesterol side-chain cleavage enzyme (P450scc), 3-
-hydroxysteroid dehydrogenase 2 (HSD3B2), 17-hydroxylase/17,20-lyase
(P450c17), and aromatase. The rate-limiting steps in estrogen synthesis are choles-
terol mobilization into the side-chain cleavage enzyme (the initial step) and aro-
matization (the last step). Both these steps are enhanced in endometriotic tissues:
StAR, which facilitates the entry of cytosolic cholesterol into the mitochondria, is
overexpressed in endometriotic tissues compared with the normal endometrium. A
master transcription regulator for steroidogenesis, steroidogenic factor 1 (SF1 or
Ad4BP), also called NR5A1, is expressed at a higher level in endometriotic tissues
than in endometrial tissues [39]. This explains the overexpression of a series of, if
not all, steroidogenic enzymes in endometriotic tissues.
Prostaglandin E2 (PGE2) is another gateway regulator of the aberrant expression
of steroidogenic enzymes in endometriotic tissues. Inflammatory endometriotic
tissues express a high level of cyclooxygenase-2 (COX-2) and synthesize substan-

tial amounts of PGE2, which in turn induces a series of steroidogenic enzymes,
resulting in the production of progesterone, estrone, and estradiol. Estrogen in turn
enhances the expression of COX-2. Thus, PGE2 and estrogen form a different cycle
along with their synthesizing enzymes [50, 51]. Increased PGE2 and estrogens
synergistically stimulate the progression of endometriosis [39].
Compared with the normal and eutopic endometrium, the expression of ER and
ER is increased in endometriotic tissues [52]. Predominant expression of ER
mRNA compared with that of ER mRNA has been reported [53]. In addition, the
downregulation of ER and upregulation of ER in ovarian endometrioma com-
pared with those in the normal endometrium have been reported [54]. A role of ER
in the regulation of ER in endometriotic stromal cells has been elucidated. ER
knockdown significantly increased ER mRNA and protein levels in endometriotic
stromal cells, whereas ER overexpression decreased ER mRNA and protein
levels. High levels of ER suppress ER expression and the response to estradiol
in both endometrial and endometriotic stromal cells via binding to classic and
nonclassic DNA motifs in alternatively used ER promoters [55]. Estrogen is
believed to have a strong effect on endometriotic tissues through binding of ER
and ER.
11.3.2 Regulation of Aromatase In Situ in Endometriosis
The overexpression of aromatase mRNA has been confirmed in endometriotic

tissues and endometriotic stromal cells isolated and cultured from ovarian
endometrioma. The level of the transcript correlates with the 17-estradiol-produc-
ing activity in vitro in endometriotic stromal cells [3, 39, 41]. The aromatase
activity of endometriotic stromal cells is stimulated by the peritoneal fluid, tumor
necrotizing factor-, and interleukin-6 [56].
Macrophage migration inhibitory factor (MIF), a major pro-inflammatory and
growth-promoting factor expressed in active endometriotic lesions, enhances the
aromatase activity of endometriotic cells by posttranscriptional mRNA stabilization
of aromatase. The resulting aromatase increases in situ estrogen, which in turn
upregulates MIF expression. Accordingly, MIF and aromatase produce a positive
feedback loop to enhance their mutual expression and promote endometriosis.
Whether miRNA-mediated regulation is involved in this reciprocal enhancement
between MIF and aromatase remains unclear. The inhibition of endogenous MIF
may be a therapeutic candidate for endometriosis [57].
As described earlier, COX-2 is another regulator of aromatase expression for
reciprocal enhancement in endometriotic tissues. Compared with the eutopic endo-
metrium, COX-2 is overexpressed in endometriotic tissues of healthy women,
enhancing PGE2 production in situ. PGE2 induces aromatase, and the resulting
estrogen in turn stimulates COX-2 expression. This may prove to be another
mechanism underlying the transcriptional enhancement of aromatase [39].
SF1 is a master transcription factor of steroidogenic enzymes, as explained

earlier. SF1 induces the transcription of StAR, an essential protein in the initiation
of steroidogenesis via cholesterol translocation into the mitochondria. SF1 binds to
nuclear half sites of promoter PII of CYP19A1 and induces aromatase expression in
endometriotic tissues. SF1 efficiently induces the transcription from promoter PII in
cooperation with the transcriptional cAMP response element-binding protein [42].
In comparison, the chicken ovalbumin upstream promoter transcription factor
(COUP-TF) alternatively binds to the nuclear half sites of promoter PII in the
eutopic endometrium. This may explain why aromatase is constitutively suppressed
in the eutopic endometrium, particularly in healthy women.
Why does the transcription factor switch from COUP-TF to SF1 in
endometriotic tissues? A CpG island located upstream of SF1 promoter is highly
methylated in the normal eutopic endometrium but unmethylated in endometriotic
tissues. Thus, cancelation of silencing by genomic methylation seems to be a major
mechanism underlying the reactivation of SF1 in endometriotic tissues, resulting in
an overexpression of aromatase [58].
Many factors that may contribute to the induction and subsequent maintenance
of aromatase expression in endometriosis have been reported. Some of these
interact with each other and reciprocally increase expression. The contribution of
each gene to the pathogenesis of endometriosis remains to be determined.
11.4 Aromatase Inhibitor (AIs)
11.4.1 AIs
AIs were originally developed for the treatment of breast cancer. By the 1990s, it
had been revealed that breast cancer tissues synthesize estrogen in situ, which
promotes breast cancer cell growth. After the molecular cloning of aromatase in
the late 1990s, the aberrant expression of aromatase was identified as a cause of the
overproduction of estrogen in situ. AIs were then developed as a new endocrine
treatment of breast cancer. Initial trials showed that AIs reduced the level of
estrogen in situ and suppressed the progression of cancer in postmenopausal
women [59, 60]. Subsequent large-scale studies confirmed the therapeutic advan-
tages over tamoxifen and reported the reduction of cancer development in the
contralateral breast, suggesting a cancer preventive effect [61]. The role of in situ
estrogen in pathology was first exemplified in breast cancer, and this established
aromatase as a molecular target for endocrine therapy.
AIs are now widely used for the treatment of ER-positive breast cancer in
postmenopausal women. The use of AIs shows a survival benefit compared with
that of other endocrine therapies in women with advanced breast cancer [62].
Table 11.2 Three Aromatase inhibitor

generations of aromatase
inhibitors Generation Nonsteroidal Steroidal
First Aminoglutethimide
Second Fadrozole Formestane
Third Letrozole anastrozole Exemestane
11.4.2 Pharmacology
The functional aromatase enzyme complex is composed of 2 polypeptides: aroma-

tase cytochrome P450 and NADPH-cytochrome P450 reductase. The former is a
product of CYP19A1, a single gene on chromosome 15q22, which binds to andro-
gens and hydroxylates them twice at the C19 position located between the A and B
rings, resulting in aromatization of the A ring. The latter is a flavoprotein, ubiqui-
tously distributed in most cells. NADPH-cytochrome P450 reductase confers an
electron from NADP to aromatase for the enzymatic reaction.
AIs are classified into three generations according to the history of development
(Table 11.2). The first-generation inhibitor aminoglutethimide is not selective to
aromatase and causes a medical adrenalectomy, leading to lethargy, skin rashes,
and nausea. The second-generation inhibitors include fadrozole and formestane,
which are more selective than aminoglutethimide. A risk of glucocorticoid sup-
pression is reduced but still exists. The third-generation AIs include letrozole,
anastrozole, and exemestane (6-methylenandrost-1, 4-diene-3, 17-dione). These
are more selective and potent; thus, they are excellent for use in clinical practice
[63, 64]. The former two are nonsteroidal, while the latter is steroidal. Both types
bind to a substrate-binding site of aromatase as a false substrate. The next step in the
binding differs between the 2 types of compounds. A steroidal compound forms an
unbreakable complex with the aromatase protein, and the enzymatic activity of the
aromatase is thus permanently blocked once the binding occurs. This may lead to
accelerated degradation of the aromatase protein [65]. Thus, enzymatic activity
does not recover even if all unattached inhibitors are removed, and the enzymatic
activity can only be restored by new enzyme synthesis [66]. In contrast, nonsteroi-
dal compounds can dissociate from aromatase, resulting in recovery of the enzy-
matic activity. The difference in inhibitory mechanisms between steroidal and
nonsteroidal AIs is expected to provide rationale for alternate treatments for
patients with breast cancer who are resistant or refractory to other types of AIs [67].
The third-generation AIs can decrease the local concentration of estradiol by 97
99 %. The therapeutic dose of AIs for breast cancer significantly reduces circulating
estrogen levels in postmenopausal women. In comparison, the inhibition of estra-
diol synthesis in premenopausal women is not sufficient, with only a 2030 %
reduction in circulating estrogen levels [68, 69]. This less effective suppression in
premenopausal women is because of androgen availability for nonsteroidal inhib-
itors: androgen as a substrate is present at micromolar concentrations in the ovary
(follicle), while circulating androgen at nanomolar concentrations is the only source
of androgen for extraglandular (peripheral) estrogen synthesis. Competitive binding

is insufficient in androgen-rich follicles, resulting in insufficient inhibition.
Another mechanism underlying the insufficient suppression observed in
premenopausal women is the compensatory increase in FSH. The suppression of
estrogen synthesis causes an increase in FSH secretion, which stimulates follicular
development and aromatase expression and eventually restores estradiol secretion
to some extent [70].
11.4.3 Side Effects of AIs
AIs cause undesired health issues related to the presence or absence of estrogen
suppression. Bone loss and fracture are the most important issues, particularly in
postmenopausal women who use AIs for more than 6 months. Concomitant use of
bisphosphonate is recommended for women who have additional risk factors for
fracture [71]. Compared with the use of tamoxifen, long-term use of AIs increases
the odds of cardiovascular disease [72]. Other side effects associated with the use of
AIs are hot flushes, headache, joint stiffness or pain, leg cramps, arthralgia, nausea,
and diarrhea. All of them are generally mild and are therefore tolerable.
It has been reported that AIs + norethindrone acetate or other OCs prescribed for
the treatment of endometriosis cause bone mineral loss, as seen in the case of breast
cancer. The change is significant but not symptomatic. This is partly because of the
shorter duration of therapy compared with that for patients with breast cancer and
partly because of the bone protective action of concomitantly used sex steroids [64].
11.4.4 AIs for the Treatment of Endometriosis
In most cases, the progression of endometriotic lesions and endometriosis-related

symptoms can be controlled by the use of various types of medications, surgery, or
a combination of both. Thus, trials of AIs are limited to patients with severe pain,
including deep pareunia and chronic pain, who are refractory to established treat-
ment options. The use of AIs in endometriosis was initially reported for postmen-
opausal women with refractory endometriosis of the rectovaginal space [73]. The
therapeutic effect is now tested in premenopausal women suffering from refractory
alleviation of endometriosis-related pain.
As described earlier, the application of AIs to premenopausal women leads to an
elevation in FSH levels and subsequent follicular development, which increases
estradiol, thereby potentially nullifying the therapeutic effect. Thus, additional
agents to control ovarian activation are required. To this end, progesterone or
progestins, OCs, and GnRH agonists have been tested. All of these are used in
the conventional therapy for endometriosis.
11.4.4.1 AI Monotherapy
A few reports have provided evidence of the efficacy of AI monotherapy for the
treatment of endometriosis-related pain in premenopausal women. In these studies,
vaginally administered anastrozole (0.25 mg/day) combined with oral elemental
calcium (1.2 g/day) and cholecalciferol (800 IU/day) for 6 months improved
rectovaginal endometriosis-related dysmenorrhea [74].
A prospective randomized clinical trial has been conducted to compare the effect
of short-term letrozole and a GnRH agonist (triptorelin) versus case control on the
pregnancy rate and recurrence of endometriosis after laparoscopic surgery [75]. The
overall pregnancy and recurrence rate of the letrozole group (2.5 mg/day for
2 months) were similar to those of the triptorelin group. The pregnancy and
recurrence rate among the 3 groups were as follows: 23.4 and 6.4 % in the letrozole
group, 27.5 and 5 % in the triptorelin group, and 28.1 and 5.3 % in the control
group, respectively.
Another case report has revealed the efficacy and side effects of AIs in the
treatment of premenopausal patients with endometriosis-related chronic pelvic pain
refractory to conventional treatment. Four premenopausal patients were treated with
either anastrozole (1 mg/day) combined with alendronate (10 mg/day) or letrozole
(2.5 mg/day) combined with calcium (1.5 g/day) and vitamin D (800 U/day) for
6 months, and marked improvement of pelvic pain was observed in all the patients
without significant hormonal change and bone mineral loss [76]. The most common
side effect was irregular bleeding with anastrozole and joint pains with letrozole.
Thus, AI monotherapy for endometriosis-related pain is tolerable for a short
duration, if the prevention of bone mineral loss is adequate.
11.4.4.2 Combination of AIs with Progesterone or Progestins
A prospective noncomparative study on the efficacy of letrozole + norethindrone

acetate for endometriosis-related chronic pelvic pain has been conducted [77].
This study included 10 women who were refractory to conventional medication
for endometriosis-related pain. The diagnosis of endometriosis was confirmed by
laparoscopic biopsy. The combined use of letrozole (2.5 mg/day), norethindrone
acetate (2.5 mg/day), calcium citrate (1,250 mg/day), and vitamin D (800 IU/day)
for 6 months relieved the pain in 9 of 10 women. The authors concluded that
letrozole is a candidate for the medical management of refractory endometriosis-
related pain.
Two refractory cases with severe pain were treated with the cyclic administra-
tion of anastrozole combined with progesterone. One treatment course consisted
of 21 days of treatment with anastrozole (1 mg/day), progesterone (200 mg/day),
and calcitriol (1,25-dihydroxyvitamin D3; 0.5 g/day), followed by 7 days
without treatment. In addition, rofecoxib (a selective COX-2 inhibitor; 12.5
50 mg/day) was continuously administered. A six-course treatment resulted in
a rapid, progressive reduction in pain, and the remission lasted over 24 months
after treatment [78].
Another large-scale, prospective, open-label, nonrandomized trial including
82 women with pain caused by rectovaginal endometriosis has been conducted.
Patients received either norethisterone acetate (2.5 mg/day) alone or a combination
of letrozole (2.5 mg/day), norethisterone acetate (2.5 mg/day), elemental calcium
(1,000 mg/day), and vitamin D3 (880 IU/day) for 6 months. Both regimens were
similarly effective for dysmenorrhea; however, the efficacy differed between the
2 regimens with respect to chronic pelvic pain and deep dyspareunia. The reduction
rate of the intensity of the pain scale in the combination therapy was 1.41.5-fold
larger than that in therapy with norethisterone acetate alone [79]. A carryover effect
was not observed in both regimens in terms of pain relief. Side effects, including
joint pain (n 5) and myalgia (n 5), hair loss (n 1), and decreased libido
(n 1), were only reported for the combination therapy (n 41). However, with-
drawal due to side effects was not statistically different between the combination
group (n 4) and the norethisterone acetate group (n 3).
Overall, the use of AIs enhances the therapeutic efficacy of progesterone/progestins
with regard to pelvic pain and deep dyspareunia that are refractory to established
treatments. However, the improvement in the pain relapses soon after the comple-
tion of treatment, and the side effects, albeit minor, are increased. For extended
treatment, great caution is required regarding side effects related to long-term use
of AIs, such as bone loss and arthritis, as reported in case of long-term
AI monotherapy for breast cancer.
11.4.4.3 Combination of AIs with OCPs
The efficacy of AIs for refractory endometriosis has been also examined in
combination with OCPs. A phase 2 prospective open-label trial was conducted on
15 premenopausal women with documented refractory endometriosis and chronic
pelvic pain [80]. The use of anastrozole (1 mg/day) and 1 tablet of ethinyl estradiol
(20 g/day)/levonorgestrel (0.1 mg/day) daily for 6 months significantly relieved the
pain. Side effects were mild, and no adverse effects on major organs were observed.
The efficacy of AIs + OCPs for premenopausal patients with ovarian
endometriomas and chronic pelvic pain, who were previously treated with surgery
and medication with an unsatisfactory result, has been reported. Five women received
letrozole (2.5 mg/day), desogestrel (0.15 mg/day), ethinyl estradiol (0.03 mg/day),
calcium (1,200 mg/day), and vitamin D (800 IU/day) daily for 6 months. Ovarian
endometriomas disappeared and the pain was relieved in all 5 women. Loss of bone
mineral density was not observed [81]. This result is excellent; however, interpretation
is limited because of the small number of patients and the fact that the study was
nonrandomized and did not include controls. In addition, the primary target of the
treatment was ovarian endometrioma and not rectovaginal endometriosis.
Further randomized trials are necessary to elucidate the benefits of a
combination of AIs with OCPs.
11.4.4.4 Combination of AIs with GnRH Agonists
One prospective randomized trial examined whether the addition of anastrozole to

goserelin is superior to goserelin alone in terms of adjuvant therapy after conser-
vative surgery [82]. Forty women with stage IV (the revised American Society for
Reproductive Medicine score > 40) severe endometriosis received either goserelin
(3.6 mg/4 weeks) alone or goserelin + anastrozole (1 mg/day). Compared with
goserelin alone, 6 months of treatment with goserelin + anastrozole significantly
increased the pain-free interval (1.7 months versus >2.4 months) and decreased
symptom recurrence rates until 2 years (35 % versus 7.5 %). The postmenopausal
quality of life and bone mineral density at 2 years after medical therapy remained
unaffected.
These studies were designed to evaluate the efficacy of AIs combined with
progestins or GnRH agonists and actually suggested that the addition of AIs is
beneficial for pain relief but may cause unfavorable side effects. Therefore, which
is better for combination with AIs in terms of risk and benefit: progestins or GnRH
agonists? A randomized prospective study was designed to compare the efficacy
and tolerability of AIs combined with either progestin or a GnRH agonist [83].
Women with rectovaginal endometriosis were treated with letrozole (2.5 mg/day)
for 6 months and were randomized to also receive either oral norethisterone acetate
(2.5 mg/day) or triptorelin (11.25 mg every 3 months). The reduction in the
intensity of nonmenstrual pelvic pain and deep dyspareunia did not differ between
the 2 medications, whereas the interruption of treatment and bone mineral
loss were more frequent and severe and patient satisfaction was therefore lower
in the triptorelin group. AIs possibly enhance the efficacy of conventional medi-
cines for endometriosis in premenopausal women; however, they may cause
intolerable side effects.
11.4.5 AIs for the Treatment of Infertility in Women

with Endometriosis
In 2001, a new application of letrozole for ovulation induction in clomiphene citrate

(CC)-resistant anovulatory women was reported [84]. The administration of
letrozole in the early follicular phase would release the pituitary/hypothalamic
axis from estrogenic negative feedback and increase FSH, somewhat similar to
the mechanisms of CC.
The clinical features of AIs as ovulation-inducing medicines differ from those of
CC. CC causes thinning of the endometrium during the therapeutic cycle and
decreases the cervical mucus, whereas AIs do not. This negative effect on the
endometrium explains the reason why the pregnancy rate of CC-treated women is
somewhat lower than that expected from the ovulation rate. AIs do not have the
negative effects on the endometrium. A reason for the difference is the half-life of
the medicine: CC, particularly an isomer of CC existing in the medicine, lasts

longer in the body and exerts an antiestrogenic action for a longer period than
letrozole: the half-lives are 57 days and 45 h for CC and letrozole, respectively.
In addition, CC increases circulating estrogen, which binds to ER and accelerates
its degradation. Thus, it takes time to recover estrogen responsibility through de
novo synthesis of ER after CC is eliminated from the blood. In comparison, AIs do
not induce ER degradation and are rapidly eliminated from the body after
the cessation of administration. Thus, the endometrium maintains its sensitivity
to estrogen and quickly recovers to the level of untreated cycles after the cessation
of medication, whereas estrogen levels are decreased to half to one-third of the
levels observed in natural cycles. This supports the absence of any direct
antiestrogenic effects of letrozole on the endometrium [85].
Despite the lack of a negative effect on the endometrium, meta-analysis does
not support that AIs achieve higher pregnancy rates than CC for women with
polycystic ovary syndrome. Meta-analyses of 6 randomized controlled trials
comparing letrozole with CC demonstrated that letrozole improved the ovulation
rate per patient, without a statistically significant difference in the ovulation rate
per cycle or pregnancy, live birth, multiple pregnancy, or miscarriage rates [86].
Treatment with letrozole for unexplained infertility is almost equally effective to
that with CC; however, it may have some advantages in terms of low serum
estradiol levels, the pregnancy rate per cycle, and the abortion rate [70, 87, 88].
There is a report on the efficacy of AIs for infertility in women with endometri-
osis: the pregnancy rate with letrozole was the same as that with CC alone in an
intrauterine insemination program for women with minimal to mild endometriosis
who did not achieve pregnancy after 612 months following laparoscopic
treatment [89].
The use of AIs for ovulation induction is an off-label use and the safety has
not been established. A concern about teratogenicity was reported in an abstract
in 2005; however, there have been no additional studies published since then. In
contrast, a large multicenter retrospective study showed that compared with CC,
AIs do not increase congenital cardiac anomalies [90].
11.4.6 AIs for the Treatment of Endometriosis

in Postmenopausal Women
Endometriosis spontaneously resolves after menopause; however, endometriosis

may progress and cause symptoms, albeit rare. Such patients have been treated with
surgery, including hysterectomy, at earlier ages and experience recurrent
endometriosis-related pain. AIs have been reported to be successful in the treatment
of postmenopausal women with endometriosis [91, 92]. As expected, no additional
medication is required to prevent FSH increases during AIs use.
11.5 Conclusions
Endometriotic tissues overexpress aromatase and synthesize estrogen in situ, which

may play roles in pathogenesis and progression by enhancing auto-implantation,
proliferation, and angiogenesis. Researchers propose several mechanisms underly-
ing this overexpression, namely switching of the transcriptional regulators from
COUP-TF to SF1; overexpression of COX-2, MIF, and cytokines; and changes in
the methylation status of CpG islands in CYP19A1.
Clinical trials of AIs have been conducted and have revealed that AIs reduce
endometriosis-related pain, particularly in recurrent cases. The effectiveness sup-
ports the notion that locally synthesized estrogen plays a role in the progression of
endometriosis, unlike anticipated. Short-term use of AIs (less than 6 months) either
as monotherapy or in combination with other medications for ovulation inhibition
reduced endometriosis-related pain without significant severe side effects. Future
studies will be required to confirm these conclusions.
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Chapter 12
Apoptosis in Endometriosis
Apostolos Kaponis, Georgia Kollia, Fuminori Taniguchi, Tasuku Harada,

and George Decavalas
Abstract Endometriosis is an inflammatory, estrogen-dependent disease charac-

terized by the growth of endometrial tissues outside the uterus. The eutopic
endometrium from women with endometriosis has some fundamental differences
compared with the normal endometrium of women without endometriosis. The
differences could contribute to the survival of regurgitating endometrial cells into
the peritoneal cavity and the development of endometriosis. One mechanism that
gained a lot of interests is the finding that apoptosis appeared in eutopic and ectopic
endometrium of patients with endometriosis. A common characteristic of
endometriotic cells is their ability to evade the apoptotic machinery. Endometriosis
could result from increased cellular proliferation or decreased apoptosis in response
to appropriate stimuli. This chapter focused on the physiological role of apoptosis
in normal endometrium and the alterations in regulation of apoptosis in eutopic and
ectopic endometrium from women with endometriosis. Finally, the role of apopto-
sis in the treatment of endometriosis is reviewed to link the basic research findings
into clinical applications.
Keywords Apoptosis Bcl-2 Endometriosis Fas/FasL Medical treatment
A. Kaponis, M.D., Ph.D. (*) G. Kollia G. Decavalas

Department of Obstetrics and Gynecology, Patra University School of Medicine,
Rio, 26441, Peloponnisos, Patra, Greece
e-mail: kaponisapostolos@hotmail.com
F. Taniguchi T. Harada
Department of Obstetrics and Gynecology, Tottori University Faculty of Medicine,
Yonago, Japan

180 A. Kaponis et al.
12.1 Introduction
Endometriosis is a common and enigmatic disease characterized by the presence of

endometrium-like glandular tissue and stroma outside the uterus. The pathophysi-
ology of the disease remains a mystery. Nearly all women of reproductive age
exhibit some degree of reflux of endometrial debris [1]. Menstrual effluents,
retrogradely shed into the peritoneal cavity, were observed to contain viable
endometrial cells [2]. These mechanisms are necessary but insufficient to explain
why only some patients develop the disease. The fact that the eutopic endometrium
of women with endometriosis shares changes with ectopic tissue and that these
changes are not found in the eutopic endometrium of disease-free women has
advanced the view that the primary defect in endometriosis is to be found in the
eutopic endometrium [3]. Cells and tissue elements, derived from such an altered
eutopic endometrium and shed into the peritoneal cavity, have been proposed to
have a higher potential for implantation and growth on peritoneal surfaces and
development into endometriosis [4]. Many differences are observed between
eutopic endometrium of disease-free women and ectopic tissue of a patient with
endometriosis. These differences can be explained as the direct influence of the
different environment of peritoneal fluid (PF) [5]. One of the endometrial alter-
ations appearing in eutopic and ectopic endometrium from women with endome-
triosis refers to the regulation of apoptosis. In particular, decreased susceptibility of
endometrial tissue to apoptosis may contribute to the pathogenesis of
endometriosis.
12.2 Apoptosis in Pathophysiology of Endometriosis
Endometrial cells from women with and without endometriosis have fundamental
differences. Endometrial cells from women with endometriosis has enhanced
proliferation and increased ability to implant and survive in ectopic locations.
Impaired sensitivity of endometrial tissue to apoptosis contributes to abnormal
implantation and growth of endometrium at ectopic sites. The inability of endome-
trial cells to transmit a death signal or their ability to avoid cell death is associated
with increased expression of antiapoptotic factors (e.g., Bcl-2) and decreased
expression of proapoptotic factors (e.g., BAX) [6]. It is unclear whether the
abnormal apoptosis in the eutopic endometrium from patients with endometriosis
is primary in origin or secondary after establishment of pelvic endometriosis
process. This could be attributed to the fact that at the time of clinical presentation
and diagnosis most women have already established disease and therefore, it is very
difficult to investigate the early developmental stages of the endometriosis.
Reflux of endometrial fragments during menstruation into the peritoneal cavity
is a common phenomenon. Under normal conditions, cells that do not adhere to
their extracellular matrix enter apoptosis as they receive different signals from their
12 Apoptosis in Endometriosis 181
adhesion receptors [7]. In endometriosis, these cells have the ability to adhere to
mesothelial cells of peritoneum, to proliferate, and to produce neoangiogenesis
resulting in the development of active endometriosis. The effect of MMPs on
apoptotic factors and their regulation by steroid hormones may provide a link
between endometrial turnover and the invasive process necessary for the develop-
ment of endometriosis. Immunoglobulin-like cell adhesion molecules (nectins and
Necls) involved in apoptosis and cell proliferation have also stronger expression in
eutopic and ectopic endometrium of women with endometriosis [8]. High levels of
VEGF and IL-1 have been found in the PF of patients with endometriosis. VEGF
and IL-1 reduce apoptosis and decrease Bax expression in endometrial epithelial
cells from patients with endometriosis. VEGF and IL-1 may protect endometriotic
cells from undergoing apoptosis favoring the establishment and progression of
endometriotic lesions by promoting the formation of new blood vessels and by
protecting endometriotic cells from undergoing cell death [9].
Intrinsic abnormalities in transplanted eutopic endometrium are contributed to
the pathogenesis of endometriosis. Abnormal signaling pathways in the eutopic
endometrium of women with endometriosis have been recently reported. Two
different groups demonstrated increased activity of the protein kinase A and B
pathways regulating the function of many cellular proteins involved in apoptosis
and proliferation [10, 11]. It was suggested that increased Akt phosphorylation may
be related to the altered apoptosis/proliferation harmony in endometriosis. Another
pathway whose activation confers a resistant to apoptosis phenotype in
endometriotic cell is the NF- [12]. In vivo, NF-B inhibition in early-stage
endometriotic lesions induced in nude mice was found to decrease the proliferation
of endometriotic cells and stimulate their apoptosis [13].
cDNA microarray analysis has provided an interesting insight for altered gene
expression profiles in patients with endometriosis. Arimoto et al. found
97 upregulated and 337 downregulated genes in women with endometriosis [14].
Genes related to apoptosis (GADD34, GADD45A, GADD45B, PIG11) and the
tumor suppressor TP53 gene were downregulated in endometriotic tissues. These
findings are inconsistent with the decreased spontaneous apoptosis observed in
eutopic endometrium from women with endometriosis.
Survivin is a member of the inhibitors of apoptosis family (IAP). IAP proteins
directly inhibit the terminal effector caspases 3 and 7 and thus protect cells from
apoptosis. Endometriotic cells express more survivin genes than normal endome-
trial cells without endometriosis [15]. Survivin plays a critical role in susceptibility
of endometriotic stromal cells to apoptosis and survivin inhibitors may be effective
as treatment for endometriosis [16]. Increased survivin expression was present in
eutopic and ectopic epithelial cells, but only ectopic epithelial cells lost the cyclic
variation of survivin expression during menstrual cycles.
Recently, aberrant miRNA expression has been shown to play an important role
in the pathogenesis of endometriosis as a part of epigenetic mechanisms. A global
miRNA microarray technique used to evaluate the expression of miRNAs in
endometriotic cyst stromal cells [17]. In normal endometrial stromal cells
miR-196b targets c-myc and Bcl-2 expression, inhibits proliferation, and induces
apoptosis. In contrast, in endometriotic cells, the expression of miR-196b was

repressed by DNA hypermethylation of the miR-196b gene and this repression
may be involved in the development of proliferative and antiapoptotic characteris-
tics of endometriosis [17].
Steroid hormones are able to modulate the apoptotic machinery in endometriotic
cells. Estradiol has proinflammatory and antiapoptotic effects in endometrial cells,
and these effects appear to exacerbate in women with endometriosis. In these
women, physiological estradiol concentrations are able to induce an enhanced
inflammatory response mediated by local chemokine production and to reinforce
mechanisms of cell survival mediated by extracellular signal-regulated kinases and
Bcl-2 [18]. On the other hand, the main effect of progesterone is to inhibit
interleukin-8 (IL-8) and other chemokines in stromal cells from both eutopic and
ectopic endometrium. Progesterone is effective to induce apoptosis through the
inhibition of Bcl-2 and NF- [18].
12.3 Apoptosis in the Normal Endometrium
Apoptosis helps to maintain cellular homeostasis during the menstrual cycle by elim-
inating senescent cells from the functional layer of the uterine endometrium [19].
In normal endometrium, apoptotic cells were identified in the glandular epithelium
of late secretory and menstruating endometrium due to progesterone withdrawal,
while very little apoptosis was detected during the proliferative phase or at the begin-
ning of the secretory phase [20]. The pattern of apoptosis negatively correlates to serum
estradiol concentrations in the proliferative phase [20]. Considering the cyclical nature
of apoptosis in normal endometrium, it seems likely that estrogen and progesterone
can regulate the signals that result in apoptosis in this tissue.
Bcl-2 has been considered to inhibit apoptosis in the endometrium during the
proliferative phase. Bcl-2 cyclically expressed in endometrial glandular and stromal
cells peaks during the late proliferative phase, while it decreases dramatically in the
early and mid-secretory phase to reappear in the late secretory phase. In contrast,
myometrial smooth muscle cells showed consistent Bcl-2 immunoreactivity
throughout the menstrual cycle [21]. Higher expression of Bcl-2 was observed in
the basal layer, whereas death receptor Fas and caspase-3 were higher in the
functional layer of the endometrium [22]. These results fit well with the functional
biology of endometrium. Since the basal layer remains relatively constant through-
out the menstrual cycle, apoptosis is less common in this layer. The functional layer
that undergoes cyclical growth, differentiation, and shedding appears with
increased level of apoptosis.
Susceptibility of any given cells to a potential apoptotic stimulus may be
determined by the ratio of pro- and antiapoptotic Bcl-2 family members presented
in the cell at that time [23]. Bax and Bak are Bcl-2 family members promoting cell
death susceptibility, possibly by countering the effect of Bcl-2 on cellular survival
through heterodimer interaction. BAX and BAK were upregulated in the glandular
epithelial cells during the secretory phase of the normal menstrual cycle [24, 25].
These data imply the existence of a dynamic interplay among many members of the
Bcl-2 family in triggering apoptosis.
The Fas ligand (FasL) belongs to the tumor necrosis factor superfamily. The
Fas/FasL interaction is essential in inducing apoptosis. Fas expression seems to be
unchanged in the different phases of the menstrual cycle [26]. FasL exhibits peak
expression during the secretory and menstrual phases [27]. Taken together, Bcl-2 is
expressed in human endometrial glandular cells during the proliferative phase but
not during the late secretory phase. In contrast, both Fas and FasL are expressed
throughout the cycle in weak or moderate amount, except relatively higher expres-
sion of FasL in the late secretory phase. It is generally accepted that Bcl-2 blocks
apoptosis via the mitochondrial pathway and not the death receptor pathway
induced by the Fas/FasL system. Therefore, in the normal human endometrium,
caspase-8 is initially activated by the Fas/FasL signal, resulting in the caspase
cascade. Activated caspase-8 can switch on both the death receptor pathway and
the mitochondrial pathway via Bid degradation [28]. It is possible that both the
mitochondrial and the death receptor pathways are involved in apoptosis of human
endometrial cells.
12.4 Apoptosis in Endometriosis
The percentage of apoptosis in sloughed endometrial cells is greatly reduced among

women with endometriosis implying that the number of surviving cells that enter
the peritoneal cavity is greater in women who develop endometriosis. The apoptosis
indices in the eutopic endometrium of women with endometriosis were lower
compared to women without endometriosis [29]. This difference caused primarily
by a significant decrease in apoptosis during the late secretory/menstrual and early
proliferative phases in women with endometriosis. The cyclic variability of apo-
ptosis may be lost in these women.
12.4.1 Bcl-2 Family in Endometriosis
The expression of Bcl-2 in endometrial glandular cells has a cyclic pattern in

eutopic endometrium in patients with endometriosis, but that cyclic changes were
not apparent in peritoneal and ovarian endometriotic tissues [21]. Jones et al. did
not detect apoptosis in stromal cells from peritoneal endometriotic tissues [30]. In
accordance with these findings, Bcl-2 is expressed to a greater extent in stromal
cells from ectopic tissues. This overexpression may be directly correlated to the
increase in the number of estrogen receptors expressed by ectopic stroma [31].
ncreased expression of Bcl-2 protein was found in proliferative eutopic
endometrium from women with endometriosis when compared with normal endo-
metrium from healthy women [32]. BAX expression was absent in proliferative
endometrium, whereas there was an increase in its expression in secretory endo-
metrium from women with endometriosis and healthy women. The Bcl-xL/Bcl-xS
ratio (antiapoptotic/proapoptotic) was substantially higher in eutopic endometrium
from women with endometriosis compared to endometria from women without
endometriosis [33]. Altered expression of Bcl-2 family members in eutopic endo-
metrium of women with endometriosis resulted to a decreased number of apoptotic
cells and consequently to their abnormal survival in the ectopic locations.
Increased prostaglandin E2 (PGE2) signaling was observed in ectopic
endometriotic tissues compared with eutopic endometrium tissues during the men-
strual cycle [34]. The ability of endometriotic cells to circumvent apoptotic signals
can be the result of increased PGE2 signaling, which is associated with abundant
expression of the antiapoptotic Bcl-2 and Bcl-XL proteins, low expression of
proapoptotic Bax protein, phosphorylation/inactivation of proapoptotic Bad protein,
and activation of multiple cell survival signaling pathways (ERK1/2, Akt, nuclear
factor-, -catenin) [35].
12.4.2 Fas/FasL System in Endometriosis
Few studies have been published on the expression of Fas in endometriotic tissues.
Harada et al. found that Fas is expressed randomly in both eutopic and ectopic
glandular endometrial cells [36]. Fas expression was constant in both tissues
throughout the menstrual cycle. Abundant expression of Fas antigen was found in
NKcells of PF of women with early stages of endometriosis [37]. The activated PF
NK-cells can be intensively eliminated via the Fas/FasL apoptosis, thus providing
conditions for the survival of ectopic endometrium cells and the development of the
disease at the initial stages of endometriosis.
In contrast with Fas, many studies indicated that higher expression of FasL by
endometriotic cells contributes to their survival and the development of endome-
triosis. The levels of soluble/active FasL are higher in serum and PF in women with
moderate to severe endometriosis than in women with early-stage disease or in
disease-free women [38]. Higher levels of soluble FasL in the PF of women with
endometriosis may contribute to increased apoptosis of Fas-bearing immune cells
in the peritoneal cavity, leading to their decreased scavenger activity [31]. This may
result in prolonged survival of endometrial cells into the peritoneal cavity.
The sources of the elevated levels of soluble FasL in the peritoneal cavity were
endometriotic lesions and PF leukocytes. Endometrial glandular and stromal cells
presented with increased FasL expression. Peritoneal macrophages in endometri-
osis might stimulate a Fas-mediated apoptosis of immune cells [38]. FasL expres-
sion in the endometriotic cells may protect them from attack by T lymphocytes.
Consequently, ectopic endometrium cells escaping from immune surveillance in
the peritoneal cavity may contribute to the maintenance of the disease. It is,
therefore, possible that many endometriotic cells not only become resistant to
Fas-mediated apoptosis, but additionally they have acquired the ability to utilize
this pathway to their advantage by launching a Fas counterattack against the
hosts immune system.
Upregulation of FasL expression by endometriotic cells could be induced after
the adhesion of these cells to the extracellular matrix proteins laminin, fibronectin,
and collagen IV [39]. MMPs have been implicated in the conversion of FasL to
active/soluble forms, suggesting that these molecules can activate or release factors
involved in the apoptotic process [40]. FasL expression that occurs when endome-
trial stromal cells attach to the extracellular matrix may be one of the critical events
in the development of endometriosis.
Interleukin-8 (IL-8), a chemokine for neutrophils and a potent angiogenic agent,
is elevated in the PF of women with endometriosis [41]. IL-8 promotes proliferation
of stromal cells derived from endometriotic tissues [42], suggesting that it may
facilitate growth of endometriotic implants. Selam et al. demonstrated a
concentration-dependent increase in IL-8-induced FasL expression in endometrial
stromal cells. Elevated IL-8 levels in PF, via stimulation of FasL, induce apoptosis
in activated T lymphocytes and contribute to an immune-privileged environment
around the endometriosis implants supporting their survival [43]. On the other
hand, IL-8 exerts a chemotactic activity primarily on neutrophils and inhibits
their apoptosis even in the presence of Fas engagement. IL-8 is one of the neutro-
phil survival factors in the PF of endometriosis patients. IL-8 exerts the
antiapoptotic effects by activating the PTEN/Akt pathway and mediating the
expression of survivin and Bcl-2 [44]. IL-2 also enhanced survival and invasiveness
of endometrial stromal cells in an autocrine manner by activating Akt and MAPK/
Erk1/2 signal pathway [45]. The impaired clearance of cells responsible for innate
immunity in the PF of patients with endometriosis may be associated with the
development of the disease.
12.4.3 Apoptosis and Treatment of Endometriosis
Endometriosis is an estrogen-dependent disease. Current therapeutic alternatives

consist of various hormone treatments aimed at decreasing circulating estrogen to
postmenopausal levels. Incubation with GnRH agonists increased the apoptotic rate
in eutopic and ectopic endometrium cells from women with endometriosis [46].
Treatment with GnRH-a affects the expression of a diverse range of genes, includ-
ing those that encode apoptotic factors. GnRH-a significantly induced apoptosis, as
was shown from the increased expression of activated caspase-3, in eutopic endo-
metrium cells and lesions from women with endometriosis [47]. GnRH-a inhibit
cell proliferation and increase the apoptotic rate in eutopic endometrium cell
cultures, an effect that appears to be mediated by an increase in the expression of
the proapoptotic proteins Bax and FasL and a decrease in the expression of the
antiapoptotic protein Bcl-2[48]. GnRH-a treatment attenuated IL-8 expression
by reducing TNF--induced NF-B activation [49]. Inhibitors of NF-B activity

(BAY 11-7085, apigenin, pyrrolidine dithiocarbamate) were used to examine the
potential application for the treatment of endometriosis. It has been shown that
NF- inhibitors significantly inhibited the cell proliferation and induced apoptosis
[5052].
Mutants of estrogen receptor genes delivered to endometriotic cells via an
adenovirus vector decreased cell proliferation, induced apoptosis, and decreased
cytokine production, suggesting that adenovirus-mediated gene therapy may rep-
resent a potential therapy for endometriosis in the future [53]. Aromatase
overexpression is observed in endometriotic tissues. Aromatase inhibitors, letrozole
and anastrozole, produced significant and positive effects on apoptosis of epithelial
endometrial cells from patients with endometriosis [54]. Combined oral contra-
ceptives (OC) can be administered to women with endometriosis in order to reduce
pain symptoms and to prevent progression or recurrence of the disease [55]. OC can
enhance programmed cell death (decreased Bcl-2/BAX expression ratio) in the
eutopic endometrium of women with endometriosis [56]. Progestogens exert inhib-
itory effects on endometrial proliferation and enhance apoptosis in the endome-
trium [27]. Levonorgestrel increased Fas expression and enhanced apoptotic index
in eutopic and ectopic endometrium of patients with endometriosis [57].
Several new compounds have been investigated as new treatment modalities
for endometriosis in in vitro or in animal models (bufalin, beta-
hydroxyisovalerylshikonin, raloxifene, fasudil). These compounds act as
apoptosis-induced agents in endometriotic cells [5861]. Histone deacetylase inhib-
itors have been shown also to induce apoptosis in endometriotic cells [62]. Selective
inhibition of cyclooxygenase-2 prevented survival, migration, and invasion of
human endometriotic epithelial and stromal cells, which was due to decreased
PGE2 production [63]. Modulation of apoptotic factors may result in the effective
treatment of endometriosis.
Apoptosis has an important role in the development of endometriosis. Manipu-
lation of cell death processes could be used to treat endometriosis. However, it is
important to remember that no biochemical pathway stands on its own. Apoptosis
represents the final execution step that defines the fate of a cell. However, the
decision for the survival or the death of a cell has been taken earlier through various
and complicated gene regulations. Advances in molecular biology and genetics will
help us to understand these issues and may yield prevention and treatment modal-
ities for endometriosis in the near future.
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Chapter 13
Role of Nerve Fibres in Endometriosis
Natsuko Tokushige
Abstract Endometriosis is an oestrogen-dependent inflammatory disease.

Endometriosis is often associated with pain symptoms such as dysmenorrhoea,
dyspareunia, dyschezia, dysuria and low back pain. Although increased immune
cells in peritoneal fluid, adhesions, retrograde menstruation and prostaglandins are
considered to be causes of pain symptoms in endometriosis, the underlying mech-
anisms by which pain is generated still remain unknown. Recently numerous
studies have focused on nerve fibres and neurotrophins in eutopic endometrium
and endometriotic lesions from women with endometriosis as well as in animal
models as a source of pain generation. Nerve fibres in eutopic and ectopic endo-
metrium may be activated and/or sensitised by many inflammatory mediators to
cause pain and tenderness. Neurotrophins are known to regulate the survival,
development and function of nerve fibres. However, many other molecules may
act as a neurotrophic factor in endometriosis. Increased numbers of nerve fibres,
increased amount of neurotrophins and different types of nerve fibres in endome-
triosis may explain why women with endometriosis experience pain.
Keywords Endometriosis Nerve fibres Pain
13.1 Different Types of Nerve Fibres
Nerve fibres in the body arise from cell bodies in dorsal root ganglia (DRG) and
nerve fibres in the head arise from cell bodies in trigeminal ganglion. Nerve fibres
are categorised into six groups, namely A, A, A, A, B and C fibres. Cell bodies
with the largest diameters give rise to myelinated A, A and A fibres, and cell
bodies with small and medium diameter give rise to thinly myelinated A and B and
N. Tokushige (*)
Department of Obstetrics and Gynaecology, University of Tottori, Tottori, Japan
e-mail: natsuko_tokushige31@yahoo.co.jp

192 N. Tokushige
unmyelinated C fibres. A, A and A fibres are large (515 m in diameter) with

myelin sheaths around them and they conduct at a rapid rate (20100 m/s). A, A
and A fibres carry the sensation of light pressure to deep muscle and soft touch to
skin and do not produce pain. Pain impulses originate at nociceptors, which are
sensory neurons that respond to potentially damaging stimuli. When receptors
receive pain stimuli, the stimuli are transmitted to the central nervous system
(CNS) by either A fibres or C fibres via DRG [1]. A fibres are smaller fibres
with myelin sheaths and they conduct at a slower rate (1025 m/s) than A or A
fibres due to their size (15 m in diameter). C fibres are small unmyelinated fibres
(0.31 m in diameter) and they conduct at a rate of 0.52 m/s. A fibres mediate
rapid, acute and sharp pain (first pain), and C fibres mediate dull and diffuse pain
(second pain) to the CNS [2].
13.2 Nociceptors and Pain Transmission
There are two types of nociceptors, namely high threshold mechanoreceptors and
polymodal nociceptors. High threshold mechanoreceptors respond to mechanical
damage such as cutting, crushing or pinching, and polymodal nociceptors respond
to all kinds of damaging stimuli, such as irritating chemicals released from injured
tissue. Information from high threshold mechanoreceptors are rapidly transmitted
to the brain mainly by A fibres, and the location of pain can be recognised.
However, information from polymodal nociceptors is transmitted to the brain
slowly and the location of pain is usually unrecognised.
Nociceptors can be further divided into peptidergic which contains peptides,
such as substance P (SP) and calcitonin gene-related peptide (CGRP), and
non-peptidergic which binds isolectin B4 (IB4) [3]. A and C fibres express
many of the molecules that have been implicated in the pain activation and
sensitisation. These molecules include numerous ion channels and receptors for
chemical mediators in their sensory endings. Most nociceptors express only part of
the receptors, but prolonged inflammation can lead to an up-regulation of receptors
for excitatory compounds. It is likely that the expression of some receptors for
chemical mediators may be increased in endometriosis.
The pain from the periphery enters the spinal cord via DRG and it is passed
to the nociceptive second-order neurons in the dorsal horn of the grey matter.
Second-order neurons are divided into two groups, namely nociceptive-specific
(NS) neurons and wide dynamic range (WDR) neurons [4]. Neurotransmitters such
as glutamic acid and SP are released from the nerve terminals and they can activate
DL--amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA) and
N-methyl-D-aspartic acid (NMDA) receptors on the second-order neurons. The pain
goes to the thalamus and then enters the cerebral cortex or cerebral limbic system.
Not all neurotransmitters activate AMPA or NMDA, and some neurotransmitters
such as gamma-aminobutyric acid (GABA) and serotonin suppress pain sensation.
Neurotransmitters can actually control pain transmission in the brain and spinal cord.
13 Role of Nerve Fibres in Endometriosis 193
13.3 Pain-Producing Substances
The sensitisation and activation of nociceptors after inflammation result from the
release of a variety of chemicals by damaged cells and tissues in the vicinity of the
injury. These substances include bradykinin, histamine, prostaglandins (PGs),
serotonin and nerve growth factor (NGF). They sensitise (lower the threshold) or
activate the terminals of the nociceptor by interacting with cell-surface receptors
expressed by these neurons. Primary sensory neurons have been shown to contain
bioactive peptides that can cause local inflammation. These peptides include SP,
CGRP, neuropeptide Y and vasoactive intestinal polypeptide (VIP). Inflammatory
mediators activate the non-selective cation channel transient receptor potential
vanilloid 1 (TRPV1) which leads to an influx of calcium. Tryptase released from
degranulated mast cells cleaves PAR-2 at the plasma membrane of sensory nerve
endings. Increased calcium causes depolarisation of the nerve and activated PAR-2
stimulates the release of bioactive peptides including SP and CGRP from sensory
nerve endings.
SP and CGRP can contribute to the inflammatory response by causing vasodila-
tion, plasma extravasation (leakage of proteins and fluid from postcapillary venules)
and cellular infiltration by interacting with endothelial cells, arterioles, mast cells,
neutrophils and immune cells [5]. SP acts on mast cells in the vicinity of sensory
nerve endings to evoke degranulation and the release of histamine, which further
induces a release of SP and NGF. SP also acts on platelets to release serotonin,
providing a positive feedback [6]. CGRP inhibits SP degradation by a specific
endopeptidase (SPE) [7] and enhances SP release, amplifying the effects [6].
VIP induces vasodilation and histamine release from mast cells. Histamine and
serotonin levels rise in the extracellular space, secondarily sensitising nearby
nociceptors. This leads to a gradual spread of hyperalgesia and/or tenderness.
13.4 Research on Nerve Fibres in the Uterus
Research on nerve fibres in the human uterus began about 1680 [8]. Kilian [9] was
one of the first researchers to investigate nerve fibres in the human endometrium [9].
Frankenhauser [10] reported unmyelinated nerve fibres to the smooth muscle of the
myometrium with branches to the stroma and the lining epithelium of the human
endometrium [10]. Patenko [11], Kostlin [12] and Clivio [13] also demonstrated a
fine plexus of unmyelinated nerve fibres in the submucosa from which fine fibrils
extended to the epithelium of the human endometrium. Von Gawronsky [14]
showed large nerve bundles extending parallel to the endometrial-myometrial junc-
tion, with fine branches into the human endometrial stroma [14]. Labhardt [15] and
Mabuchi [16] were unable to demonstrate nerve fibres in the human endometrium;
however, Dahl [17] described numerous fine nonmodulated nerve fibres in the
human endometrium, ending in small treelike branches in the stroma and as straight
194 N. Tokushige
fibres between the cells of the epithelium. Stohr [18] reported nerve fibres in the
mucosa of the human uterus, even fine fibres to the epithelium. Davis [19] did not
find nerve fibres beneath the mucosa of the body of the human uterus. Brown and
Hirsch [20] reported nerve fibres in the basal layer of the endometrium in the
infantile uterus but were unable to demonstrate the mode of termination in those
tissues. These investigators used the silver reduction methods to stain nerve fibres in
the human uterus. There is a discrepancy between these studies and it is considered
that the silver staining method did not clearly differentiate the nerves and reveal the
nerve fibres in the uterus compared with immunohistochemistry.
State and Hirsch [21] used Goldners modification of Massons trichrome stain
to demonstrate nerve fibres in human uteri with no obvious pathologic changes.
Some nerve fibres were present in the lower third of the basal layer of the
endometrium that have branches terminating in the stroma, in the basolar arterioles
and at the origin of the spiral arterioles. No nerve fibres were detected beyond
the basal layer of the endometrium. They also demonstrated some nerve fibres in the
myometrium and at the endometrial-myometrial junction. In this study, the nerve
fibres in the endometrium were found to be unmyelinated. Koppen [22], Stohr [23],
Krantz [24] and Witt [25] reported that branches of nerve fibres accompanying
arteries had been clearly revealed only in the basal layer of the endometrium and
there were no nerve fibres in the outer two-thirds of the endometrium by Goldners
modification of Massons trichrome stain.
Lerner et al. [26] reported abnormal innervation in the myometrium from
women with chronic pelvic pain and dysmenorrhoea. There was a marked prolif-
eration of unmyelinated nerve bundles in the myometrium, but nerve fibres in the
endometrium were not mentioned in this study. Quinn and Kirk [27] investigated
uterine innervation in normal and some clinical conditions such as adenomyosis
and chronic pelvic pain. They performed immunohistostaining using an antibody
against protein gene product (PGP9.5). They demonstrated nerve bundles at the
endometrial-myometrial interface and throughout the myometrium in nulliparous
and parous subjects with no histologic abnormality. There was nerve fibre prolif-
eration throughout the myometrium with small-diameter nerve fibres eccentric
courses throughout the myometrial stoma in some subjects with chronic pelvic pain.
Some researchers have identified types of nerve fibres in the human uterus. SP- and
CGRP-immunoreactive nerve fibres were present in the human myometrium [28].
Heinrich et al. [29] demonstrated neuropeptide (NPY), SP, neurotensin (NT)
and VIP-immunoreactive nerve fibres in the basal layer of the endometrium and
myometrium in nonpregnant women. Lynch et al. [30] and Helm et al. [31] also
detected VIP-immunoreactive nerve fibres in the endometrium and myometrium in
women with no pathologic abnormality.
Nerve fibres in the endometrium in animals have also been investigated by some
researchers. A number of tyrosine hydroxylase (TH), dopamine -hydroxylase
(DH), NPY and VIP-immunoreactive nerve bundles and fibres were present in
equine endometrium [32], and CGRP-immunoreactive nerve fibres were in equine
and rat endometrium [32, 33], secretoneurin (SN)-immunoreactive nerve fibres

were in rat endometrium [34], VIP-immunoreactive nerve fibres were in rat
and porcine endometrium [35, 36], adrenergic and acetylcholine (ACh)-
immunoreactive nerve fibres were in sheep endometrium [37], and AChE- and
NPY-immunoreactive nerve fibres were in rat endometrium [38].
TH-immunoreactive nerve fibres are considered to regulate uterine contractility,
uterine blood flow and endometrial secretion as those nerve fibres are often
associated with blood vessels, endometrial glands and myometrial smooth muscle.
SP and CGRP are co-expressed in a subpopulation of nerve fibres (sensory A and
C). SP induces contraction in the human uterus [28] and controls blood flow [39]. In
contrast, CGRP is a potent vasodilator [40] and inhibitor of spontaneous contractile
activity in the human uterus [28]. Also CGRP inhibits uterine contractility caused
by SP in rat uterus [41], regulates sensory transmission and glandular secretion [42,
43] and has a proliferative effect on human endothelial cells [43]. NPY is known to
co-exist with noradrenaline and considered to regulate vascular tone and exert
inhibitory effect on myometrial contractility [28]. VIP is a potent vasodilator of
the uterine artery and involves in smooth muscle relaxation, blood flow increase
and secretion [36]. ACh is associated with myometrial and vascular smooth muscle
[44] and evokes contraction of the myometrium [45]. VIP co-exists with ACh [46]
and promotes relaxation of the myometrium through inhibition of the excitatory
action of ACh [47]. CGRP also inhibits ACh-stimulated uterine contraction and this
is dose dependent [33]. SN is contained in sensory C fibres and parasympathetic
neurons [34] and co-exists with SP and CGRP [48]. SN may regulate vascular
control and smooth muscle contraction and play a role in the process of neurogenic
inflammation that involves the activation of sensory C fibres, which release SP,
neurokinin A, CGRP and nitric oxide from their peripheral terminals to increase
vascular permeability, protein extravasation, tissue oedema, vasodilation and acti-
vation and recruitment of inflammatory immune cells. TH and DH are present in
adrenergic nerve fibres and they are associated with vascular and nonvascular
smooth muscle and participate in the regulation of myometrial contractions and
blood flow [49]. It would be interesting to see how these nerve fibres are correlated
with variation in pain symptoms and response to hormone treatment.
13.5 Nerve Fibres and Neurotrophins in the Uterus from

Women with Endometriosis
Quinn and Kirk [27] reported that there was widespread nerve fibre proliferation
(small-diameter nerve fibres) in the uterine isthmus suggesting nerve fibre damage
in the uterine isthmus in women complaining of chronic pelvic pain. Few of these
women had endometriosis, but they did report widespread nerve fibre proliferation
in the uterus and cervix in one woman with advanced endometriosis and with
painful periods and intercourse. These nerve fibres were prominently seen around
196 N. Tokushige
arteries and veins throughout the uterine isthmus. Quinn and Armstrong [50]
reported widespread nerve fibre proliferation with extensive perivascular nerve
fibre proliferation around both arteries and veins in the myometrium in an endo-
metriosis patient with dysmenorrhoea and dyspareunia. In another study, increased
numbers of nerve fibres were seen in the myometrium from women with advanced
endometriosis compared with women without endometriosis [51]. These studies
demonstrated the proliferation of nerve fibres; however, types of nerve fibres in the
endometrium and myometrium from women with endometriosis were not investi-
gated in these studies. It has been demonstrated that no nerve fibres were detected in
the functional layer of the endometrium from women without endometriosis;
however, there were several small nerve fibres present in the functional layer of
the endometrium from women with endometriosis (the mean density of nerve
fibres: 13.4 mm2) [52]. There were more nerve fibres in the basal layer of the
endometrium and myometrium from women with endometriosis than women
without endometriosis. Several thick nerve trunks were seen in the basal layer of
the endometrium or at the endometrial-myometrial interface in women with endo-
metriosis, but these nerve fibres were not seen in women without endometriosis.
Many small nerve fibres were also present throughout the basal layer of the
endometrium, but only a few nerve fibres were seen in women without endometri-
osis. Many more nerve fibres and nerve trunks were seen in the myometrium in
women with endometriosis than in women without endometriosis. In women with
endometriosis, nerve fibres in the functional layer of the endometrium were sensory
C fibres; sensory C, sensory A and adrenergic fibres in the basal layer; and
sensory C, sensory A, adrenergic and cholinergic fibres in the myometrium [53].
These results indicate that abnormal innervation in the uterus may be associated
with pain generation in women with endometriosis who suffer from chronic
pelvic pain.
Progestogens and combined oral contraceptives are often used to treat women
with endometriosis-associated pain. Progestogens and combined oral contracep-
tives significantly reduced the nerve fibre density in the functional and the basal
layers of the endometrium and myometrium from women with hormonally treated
endometriosis compared with that from women with untreated endometriosis [54].
Hormonal treatments may reduce pain symptoms by decreasing nerve fibres in
the endometrium and myometrium in women with endometriosis.
13.6 Conscious Pain Mapping
Several groups have studied conscious pain mapping since it has been assumed that
endometriotic lesions are the source of localised pain and tenderness, and conscious
pain mapping may help to identify potential areas causing pelvic pain. Palter and
Olive [55] first described conscious pain mapping to identify a focal source of pain
and generalised visceral hypersensitivity in a majority of women with chronic

pelvic pain (CPP). They found that deep infiltrating sclerotic endometriosis of the
rectum, active peritoneal endometriosis and an adhesion of small bowel were
apparent sources of cause of pain. Demco [56] found that most women localised
or mapped their pain to their endometriotic lesions and the surrounding peritoneum.
In this study, pain extended beyond the lesions to normal-looking peritoneum.
Almeida and Val-Gallas [57] found 48 positive findings in 50 women with con-
scious pain mapping and Howard et al. [58] also reported that conscious pain
mapping successfully identified tender lesion in 70 % of women. It seems that
most endometriotic lesions themselves appear to be capable of generating pain
stimuli, and conscious pain mapping can be useful in detecting a source of pain.
However, not all endometriotic lesions produce pain and it is likely that there may
be other sources of pain generation in women with endometriosis.
13.7 Nerve Fibres in Endometriotic Lesions
Tamburro et al. [59] demonstrated the expression of transforming growth factor 1

(TGF1) in nerve fibres in endometriotic lesions from women with dysmenorrhoea
associated with endometriosis. There was a significant correlation between the
dysmenorrhoea and maximal intensity of staining of TGF1 and a relationship
between the colour of the lesions and maximal intensity of staining of TGF1.
Greater maximal intensity of staining of TGF1 was seen in red, deep lesions than
in black and normal peritoneum. TGF1 is increased in the peritoneal fluid of
women with endometriosis [60] and can increase cyclooxygenase-2 (COX-2)
activity to produce more PGs [59]. Since there were no morphologic differences
in the nerve fibres between endometriotic peritoneal lesions and normal perito-
neum, substances that are present in the nerve fibres appear to be associated with
causing pelvic pain in women with endometriosis. Tulandi et al. [61] demonstrated
nerve fibres in endometriotic lesions in women with endometriosis by using an
antibody against neurofilament (NF). They reported that the distance between
endometrial glands and nerve fibres in endometriotic lesions from women with
pain was closer than in women with no pain. Quinn and Kirk [62] reported
widespread nerve fibre proliferation (small-diameter nerve fibres) in endometriotic
lesions from a woman with pelvic pain. Berkley et al. [63] demonstrated nerve
fibres in endometriotic lesions in a rat model and these nerve fibres were protein
gene product 9.5 (PGP9.5), CGRP, SP and vesicular monoamine transporter
(VMAT) immunoreactive. SP and CGRP are present in myelinated and unmyelin-
ated sensory nerve fibres (A fibres and C fibres), and VMAT is present in
sympathetic fibres.
In peritoneal endometriotic lesion from endometriosis patients who presented
pain symptoms, nerve fibres were innervated by sensory C, sensory A, cholinergic
and adrenergic fibres, and the mean density of nerve fibres was 16 mm2. Pro-
gestogens and combined oral contraceptives significantly reduced nerve fibre
198 N. Tokushige
density in peritoneal endometriotic lesions from hormone-treated women with

endometriosis (the mean density of nerve fibres: 10.6 mm2) compared with perito-
neal endometriotic lesions from untreated women with endometriosis (the mean
density of nerve fibres: 16.3 mm2) [65]. Nerve fibres expressing growth-associated
protein 43, which is a marker of neuronal development and sprouting in peritoneal
endometriotic lesion as well as SP, have also been reported by another group [66].
The same group has shown the relationship between the severity of pain symptoms
and the density of nerve fibres in peritoneal endometriotic lesions [67]. There were
more nerve fibres stained with PGP 9.5 and NF in endometriosis patients whose
pain score was at least 3 or more than those whose pain score was 2 or less. This
study has shown that there is a correlation between the density of nerve fibres and
pain severity in peritoneal endometriotic lesions in women with endometriosis.
Alvarez et al. have developed peritoneal endometriotic lesions in a rat model by
implanting with autologous uterus in the gastrocnemius muscle [68]. Those lesions
showed peptidergic nerve fibres (CGRP-positive), non-peptidergic nerve fibres
(IB4-positive) as well as GAP43-positive nerve fibres. Since no studies have
demonstrated IB4-positive nerve fibres in endometriotic lesions from women with
endometriosis, it would be interesting to investigate the presence of IB4-positive
nerve fibres and to find out the roles of these nerve fibres in endometriosis.
Deep infiltrating endometriosis (DIE) is often associated with severe pain and is
defined as endometriotic lesions penetrating into the retroperitoneal space or the
wall of the pelvic organs for a distance of 5 mm or more [69]. Patients with deep
pelvic infiltrating endometriosis usually have much stronger pain than those with
other types of endometriosis [6971]. Anaf et al. [70] have demonstrated that
endometriosis patients with DIE had significantly higher preoperative pain scores
than patients with peritoneal or ovarian endometriosis. In their study, mast cells
located <25 m from nerve fibres were significantly more abundant in DIE than in
peritoneal and ovarian endometriosis. Wang et al. have reported that there were
more nerve fibres in DIE (the mean density of nerve fibres: 68 mm2) than in
peritoneal endometriosis (the mean density of nerve fibres: 16 mm2) [72]. Those
nerve fibres were sensory C, sensory A, cholinergic and adrenergic fibres. When
compared with the locations of DIE, more nerve fibres were detected in DIE in the
sigmoid colon, appendix and rectum (the mean density of nerve fibres: 172 mm2)
than in the uterosacral ligament, cul-de-sac or peritoneal sidewall (the mean density
of nerve fibres: 68 mm2).
There are many substances secreted from endometriotic lesions including
PGs [73], tumour necrosis factor- (TNF-) [74] and NGF [75]. These substances
could sensitise and/or activate sensory and sympathetic nerve fibres that are present
in endometriotic lesions. TRPV1, a key molecule in nociception, has been identified
in the human cervix uteri in the nonpregnant state [76]. The levels of TRPV1 can be
increased in painful inflammatory diseases, and TRPV1 can trigger the release of
SP and CGRP from peripheral nerve terminals during inflammation [77]. It would
be interesting to see whether TRPVI may be present in ectopic endometrium in
women with endometriosis.
Most women with endometriosis present some kind of pain symptoms such as
dysmenorrhoea or dyspareunia. However, a few endometriosis patients do not show
any pain symptoms. When peritoneal endometriotic lesions from women with
endometriosis who did not have any pain symptoms were stained with PGP 9.5,
some nerve fibres were still detected. Therefore, not only the presence of nerve
fibres but also types of nerve fibres or molecules in endometriotic lesions may be
associated with pain generation in women with endometriosis. There may be some
molecules which can desensitise nerve fibres in those who do not experience pain
symptoms. Further studies will be required as to nerve fibres and substances
secreted from endometriotic lesions from endometriosis patients who do not have
any pain symptoms.
13.8 Neurotrophins (NGF, BDNF, NT-3, NT-4/5)

in Eutopic and Ectopic Endometrium
NGF belongs to the neurotrophin family, together with brain-derived neurotrophic

factor (BDNF) [78], neurotrophins-3 (NT-3) [79] and neurotrophins-4/5
(NT-4/5) [80]. NGF exerts its effects by binding to a tyrosine kinase A receptor
(TrkA), and BDNF and NT-4/5 bind TrkB, and NT-3 binds TrkC instead [81]. p75,
a low-affinity glycoprotein, also binds all neurotrophins [82].
NGF was strongly expressed near endometriotic glands in peritoneal and ovarian
endometriosis [64, 75], and neurotrophin-3 (NT-3) expression was also observed in
peritoneal endometriosis [66]. In ovarian endometriosis, NGF, BDNF, NT-3, NT-4/5
and NTRK2 mRNA expressions were detected by quantitative reverse transcriptase-
polymerase chain reaction (RT-PCR) [83]. Browne et al. have demonstrated that
NT-4/5 and BDNF mRNAs concentrations in eutopic endometrium detected on the
antibody microarrays and reverse transcriptase-polymerase chain reaction were
much higher in endometriosis patients than in women without endometriosis [84].
However, another study has reported that there was no difference in amounts of NGF,
BDNF, NT-3, TrkA and NGFRp75 in eutopic endometrium between women with and
without endometriosis using immunofluorescence staining, Western blot and a neu-
ronal growth assay [85]. The discrepancy may be due to different kinds of pain
symptoms, severity of pain or the density of nerve fibres among those patients.
Much more detailed studies will be needed to determine the expression of these
neurotrophins.
NGF has several effects through a variety of mechanisms. NGF stimulates the
development, growth and survival of neurons, particularly of sensory neurons [86].
NGF increases synthesis and release of neuropeptides such as SP and CGRP from
sensory nerve endings [87] and stimulates degranulation of mast cells. Recent
evidence has implicated NGF as a key mediator of inflammation and pain. NGF
is up-regulated in nerve fibres associated with the inflamed area during inflamma-
tion. NGF can increase the sensitivity and excitability of peripheral neurons to
200 N. Tokushige
cause pain by directly or indirectly sensitising sensory nerve fibres [88]. NGF in
turn activates mast cells and neutrophils which can release additional inflammatory
mediators such as histamine, serotonin and bradykinin to cause hypersensitivity
[89]. NGF has also been reported to promote angiogenesis [90]. NGF induces
proliferation of endothelial cells [91] and of vascular smooth muscle cells [92]
and stimulates the production of vascular endothelial growth factor (VEGF)
[93]. NGF is expressed in vascular endothelial cells [94] and a variety of cell
types such as T and B lymphocytes [95], mast cells [96], eosinophils [97], mono-
cytes/macrophages [98], neutrophils and basophils [99]. Increased angiogenesis in
eutopic endometrium in women with endometriosis demonstrated by several stud-
ies [100, 101] and increased numbers of immune cells may lead to further produc-
tion of NGF, promoting further nerve fibre outgrowth and pain sensation.
13.9 Effects of Oestrogen on Nerve Fibres
It is well established that oestrogen has multiple effects on the female reproductive
system and peripheral nervous system including pain sensitivity and neural regu-
lation of vascular function. Endometriosis is oestrogen dependent for continued
growth and proliferation, and it usually becomes less active with menopause as the
oestrogen level decreases. However, oestrogen replacement therapy can reactivate
the disease. Oophorectomy significantly decreased NGF and BDNF levels while
oestrogen treatment increased these levels [102, 103], and oestrogen also
up-regulated NGF and BDNF expressions in the endometrium [104106]. Several
studies have shown that neuroprotective effects of oestrogen were blocked by ER
antagonists [107109]. Ovariectomy caused a significant decrease in NGF protein
content in the uterus, and short-term treatment of ovariectomised mice with
oestrogen and/or progesterone increased uterine NGF mRNA and restored NGF
protein to concentrations similar to intact control mice [110].
In animals, stronger immunostaining of NGF and TrkA was observed in luminal
epithelial cells and glandular cells in the estrous period and early pregnancy as
compared to the non-breeding period in the uterus of the wild ground squirrels [111].
p75 was immunolocalised only in luminal epithelial and glandular cells during the
estrous period, early pregnancy and non-breeding period. The mean mRNA levels of
NGF and TrkA and p75 were significantly higher in the estrous period and early
pregnancy as compared to the non-breeding period.
Strong immunostaining of NGF and its receptors NTRK1 and TNFRSF1B was
observed in uteri of golden hamsters on the day of proestrus as compared to the
other stages of the estrous cycle [112]. There was a positive correlation between
uterine NGF expression and plasma concentrations of estradiol-17beta, and
estradiol-17beta stimulated expression of NGF and its two receptors in the uterus.
Neutral endopeptidase (NEP) is the enzyme responsible for degradation of SP and
oestrogen treatment of ovariectomised rats resulted in a four-fold decrease in uterine
NEP relative to control ovariectomised rats, resulting in increased SP levels [113].
Angiotensin-converting enzyme (ACE) is an enzyme responsible for degradation of

bradykinin and treatment of postmenopausal women with oestrogen caused an
increase in bradykinin and a decrease in ACE [114]. Steroidal hormones may induce
further production of neuropeptides and neurotransmitters by altering enzyme expres-
sions to sensitise sensory nerve terminals to cause prolonged pain sensation.
Oestrogen receptor-alpha (ER-alpha) and ER-beta are expressed in sensory
neurons of the dorsal root ganglia (DRG) [115], so oestrogen could directly act
on sensory nerve fibres to enhance axon outgrowth. Receptors for other steroidal
hormones such as progesterone may be expressed in nerve fibres and they may also
have direct effects on nerve fibres.
13.10 Prostaglandins and Neurotrophins
The correlation between prostaglandins and endometriosis-associated pain is well

established. PGE2 is present in sensory and sympathetic nerve fibres and can
sensitise and activate sensory nerve fibre terminals to induce pain [116]. Prosta-
glandins may act neuroprotectively to induce the production and release of
neurotrophins to promote the survival and outgrowth of nerve fibres. Toyomoto
et al. [117] reported that PGE2 induced and stimulated the secretion and synthesis of
NGF and BDNF, and Kanda et al. [118] also demonstrated that PGE2 enhanced the
production of neurotrophin-4 (NT-4) via EP3 receptor.
COX-2 is an enzyme which catalyses the synthesis of prostaglandins from
arachidonic acid and COX-2 may have a key function with respect to inflammation
and pain. Previous studies have shown that COX-2 is expressed in neurons and
COX-2 expression in eutopic endometrium in women with endometriosis was
higher than in those without endometriosis [119]. Also, increased COX-2 expres-
sion in endometriotic lesions of different anatomical sites (ovarian, peritoneal and
deep infiltrating endometriosis) has been reported [120122], resulting in further
modification of the production of prostaglandins in different sites.
13.11 Molecules That May Be Associated with Nerve Fibre

Growth in Endometriosis
Several molecules are present in eutopic endometrium and endometriotic lesions in

women with endometriosis and some of them may have effects on nerve fibre
outgrowth. Insulin-like growth factor I (IGF-I) was present in eutopic endometrium
and endometriotic lesions in women with endometriosis [123]. IGF-1 induced sensory
nerve fibre growth and potentiated the NGF-induced neuritogenesis [124, 125].
Oestrogen also regulated the expression and synthesis of IGF-I and its receptor
[126, 127].
202 N. Tokushige
Bcl-2 was present in eutopic endometrium and endometriotic lesions in women

with endometriosis [128]. Bcl-2 is expressed in neurons and promoted axonal
growth rates in sensory nerve fibres [129, 130]. Oestrogen up-regulated the expres-
sion of Bcl-2 [131], and oestrogen treatment increased the number of Bcl-2
immunoreactive nerve fibres [132]. Both oestrogen and progesterone can increase
the expression of Bcl-2 [133, 134] and Bcl-2 has been shown to inhibit neuronal
death [133]. Therefore oestrogen and progesterone may directly affect cell survival
or prevent neuronal cell death in neurons by Bcl-2-induced inhibition of cell death
and axonal growth, resulting in increased nerve fibre densities.
Hepatocyte growth factor (HGF) was up-regulated in eutopic endometrium in
women with endometriosis [135]. HGF alone had no outgrowth-promoting activity,
but it co-operated with NGF in enhancing axonal growth of sensory nerves and also
enhanced the neurotrophic activities of NGF [136]. Oestrogen also increased the
production of HGF by peritoneal macrophages in women with endometriosis [137].
Significantly increased expression of heat shock protein 27 (HSP 27) was
reported in eutopic endometrium in women with endometriosis [138]. HSP27 was
expressed by sensory nerve fibres [139, 140] and significantly increased survival for
rat sensory and sympathetic nerve fibres after axotomy or NGF withdrawal [141].
HSP expression was stimulated by oestrogen in the endometrium [142].
Fibroblast growth factor 9 (FGF-9) was present in endometriotic lesions in
women with endometriosis [143]. FGF-9 enhanced survival of cholinergic nerve
fibres [144] and administration of 17-estradiol induced FGF-9 expression in
endometriotic lesions [143].
These molecules may be associated with increased nerve fibre densities, spe-
cially sensory and autonomic neurons in women with endometriosis to induce pain,
and steroidal hormones may promote the synthesis of these molecules in women
with endometriosis
13.12 Angiogenic Molecules
Novella-Maestre et al. [145] have demonstrated that antiangiogenic treatment with

cabergoline significantly reduced the number of immature blood vessels, nerve
fibres, mast cells and macrophages in endometriotic lesions in a mouse model of
endometriosis. Vascular endothelial growth factor (VEGF) is a secreted vascular
mitogen that is specific for endothelial cells and plays an important regulatory role
in vascular growth during development [146]. VEGF is increased in DIE [147] and
peritoneal fluid [148] in women with endometriosis compared with women without.
It is accumulating evidence that VEGF acts as a neurotrophic and neuroprotective
factor [149]. VEGF application to cultured peripheral adult ganglia caused signif-
icant neuritic outgrowth [150] and peripheral nerve regeneration in vivo has been
shown to be enhanced by VEGF application [151]. There are some proangiogenic
factors, such as IL-8 [152], hepatocyte growth factor (HGF) [153], erythropoietin
[154], angiogenin [155], macrophage migration inhibitory factor [156], neutrophil-
activating factor [157] and TNF- [158], and these proangiogenic factors may also
have neurotrophic effects and be involved in nerve fibre growth in endometriosis.
13.13 Conclusions
Increased numbers of nerve fibres in eutopic endometrium (both the functional

layer and the basal layer), myometrium and endometriotic lesions may contribute in
some way to the mediation of pain in women with endometriosis. It was always
been assured that the pain of endometriosis is generated in the endometriotic
lesions, but the findings in some studies allow the possibility that some of the
pain may actually arise within the endometrium.
Endometriosis is an inflammatory condition and many chemical and inflamma-
tory mediators such as bradykinin, prostaglandins as well as neurotrophins can be
released from both eutopic endometrium and endometriotic lesions. These sub-
stances can directly activate and/or sensitise sensory nerve endings by interacting
with cell-surface receptors and trigger the release of pain mediators from other cells
and afferent nerve fibres. Those pain mediators sensitise nerve endings, resulting in
an increased response to painful stimuli. Prostaglandins and other arachidonic acid
derivatives can increase the sensitivity of nerve endings to bradykinin or other pain-
producing substances, leading to a secondary sensitisation of nearby sensory nerve
fibres.
There may be a correlation between pain severity and nerve fibre density;
however, it seems that some nerve fibres in eutopic and ectopic endometrium are
not associated with pain generation. Further research will be needed to better
understand the roles of nerve fibres and molecules secreted from eutopic and
ectopic endometrium from women with endometriosis.
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Chapter 14
Endometriosis in Experimental Models
Fuminori Taniguchi and Tasuku Harada
Abstract To investigate the factors involved in the initiation and progression of

endometriosis, an animal model of this disease has been employed. Animal models
are often used to investigate factors affecting the initiation and progression of
endometriosis. This chapter describes these models and focuses on the murine
model of endometriosis as a potential tool to evaluate therapies. The traditional
explanation for the existence of endometrium in ectopic locations is based on the
common occurrence of retrograde menstruation. This implantation hypothesis is
widely accepted as the etiology of endometriosis. Animal models are crucial for
elucidating the mechanisms underlying endometriosis. Several animal models of
endometriosis have been used in the past, most of which consist of transplanting
endometrium into the peritoneal cavity. Because primates spontaneously develop
endometriosis, primate models most closely resemble the disease in women; how-
ever, rodent models are more cost effective and readily available. Nevertheless,
since rodents do not menstruate, rodent models used for the research in endome-
triosis have certain limitations. We addressed the question whether a murine
endometriosis model is suitable for evaluating drugs employed in human endome-
triosis. We concluded that the murine endometriosis model may be a valuable and
reliable tool for evaluating new therapeutic approaches in human endometriosis.
Keywords Cystic lesion Experimental model Mouse endometriosis-like lesion

Parthenolide
The authors have no conflicts to disclose relative to this work.

F. Taniguchi, M.D., Ph.D. (*) T. Harada, M.D., Ph.D.
Department of Obstetrics and Gynecology, Faculty of Medicine, Tottori University,
36-1 Nishi-cho, Yonago 683-8504, Japan
e-mail: tani4327@med.tottori-u.ac.jp

214 F. Taniguchi and T. Harada
14.1 Introduction
Endometriosis is commonly believed to occur via retrograde menstruation, also

known as the Sampson hypothesis [1], in which viable endometrial tissue flows
retrograde through the fallopian tube and into the peritoneal cavity where it can
attach to and invade tissues and organs within the cavity. At least 90 % of women
experience retrograde menstruation, but endometriosis occurs in only 1014 % of
reproductive age women, suggesting that additional elements impact its etiology [2].
Since endometriosis impairs the quality of life of severely affected women,
improved research and new treatment paradigms are needed.
Current medical therapies for endometriosis aim to decrease ovarian estrogen
production and/or counteract estrogen effects with the use of GnRH agonists,
progestins (including oral contraceptives), and androgens, but undesirable side
effects limit their long-term use [2]. Likewise, the scope of surgical treatment is
also limited by a high recurrence rate, which may eventually lead to extreme
measures, such as removal of the uterus and ovaries. There is therefore a need for
focused mechanistic research that can be translated into expanded therapeutic
capability for this widely prevalent disease.
Animal models, which are important for elucidating the mechanisms underlying
endometriosis and are used in the early stages of drug testing, usually rely on
non-menstruating rodents with induced endometriosis-like lesions. One of the
major limitations in endometriosis research is the paucity of robust animal disease
models. Ideally, a disease model should mimic human disease and allow scientific
investigation into the effects of both intrinsic (e.g., genes) and extrinsic (e.g.,
environment) factors on disease progression.
Several animal models of endometriosis have been established, most consisting
of transplantation of endometrium into the peritoneal cavity, which is by far the
most common site of disease. Besides rodents, primates, such as monkeys that
spontaneously develop endometriosis or that have been transplanted intraperitone-
ally with endometrium, can be used to study drug candidates. Indeed, because
endometriosis occurs spontaneously in monkeys, the only nonhuman animal
model that has cyclic menstrual periods, such an animal model is undoubtedly the
most reliable one [37]. However, high cost and difficulties maintaining these
animals have led most researchers to concentrate their studies on smaller mamma-
lian models, such as rats [812] and rabbits [13, 14]. Nevertheless, the murine
model was considered as a possible candidate for this purpose, and these studies
were restricted to nude [15, 16] or SCID mice [17], which were implanted with
human endometrium (xenotransplantation). Since mice are much smaller than
rabbits or rats, microsurgical procedures of tissue transplantation become even
more complicated.
The murine model may provide advantages in terms of new therapeutic
approaches. In recent decades, many knockout or transgenic mice have been
generated. The availability of an endometriosis model in mice is crucial because
it can be used to investigate some aspects of endometriosis.
14 Endometriosis in Experimental Models 215
14.2 Murine Endometriosis Model
Mice are the most common animal models capable of investigating the pathophys-
iology of endometriosis; however, they do not spontaneously develop endometri-
osis. To induce endometriosis in mice, endometrial tissue must be transplanted into
the peritoneal cavity using several methods [1821], which can be classified into
two basic types, homologous and heterologous. Both models produce comparable
phenotypes, which are then morphometrically evaluated.
In homologous or autologous models, normal endometrial tissue is transplanted
into the peritoneal cavity of immunocompetent recipients and starts to grow in an
estrogen-dependent manner. In almost all models, uterine endometrial fragments
from a donor mouse are directly introduced via injection into the peritoneal cavity
of an immunocompetent syngeneic recipient without suturing the implants. In
heterologous models, human endometriotic lesions are transplanted into the peri-
toneal cavity of immunodeficient mice [22, 23]. Human endometriotic implants
were sutured to the peritoneum of immunocompromised mice [24, 25]. This xeno-
transplantation model using the nude mouse is also used, but is limited by the lack
of a normal immune system. Despite the advantage of being based on human
endometrial tissues, the number of endometriotic lesions in the heterologous
model that will develop varies from one animal to another. In both models, with
or without suturing the endometrial implant, the drugs influence on growth of
endometrial or endometriotic transplants is evaluated.
These established endometriosis models in mice are under discussion. Indeed,
mice do lack a menstrual cycle and do not develop spontaneous endometriosis. It
should be established whether transplanting normal endometrium into the perito-
neal cavity of a non-menstruating species reflects all pathophysiological aspects of
human endometriosis. Although the murine endometriosis model is not exactly the
same as human endometriosis, their endometriotic lesions develop in some ways
the same as human endometriotic lesions.
The murine model of endometriosis is a versatile one used to study how the
immune system, hormones, and environmental factors affect endometriosis and
how endometriosis affects fertility and pain. A novel study design also enables the
evaluation of molecular mechanisms that are critical for disease initiation [26, 27].
In Table 14.1, the recent representatives of mouse endometriosis models are shown.
Additional and ideal models of endometriosis are needed. In the next section, we
demonstrate the results in a murine endometriosis model that expands the capability
of conducting both mechanistic and translational research.
Table 14.1 Representatives of mouse endometriosis models

Characteristics of
Author (year) Donor Host Drug models References
Takai et al. (2013) M M Parthenolide Large cyst [28]
formation
Sokalska et al. (2013) H M Simvastatin [29]
Daftary et al. (2013) M M Adhesion scoring [30]
system
Rudzitis-Auth M M Resveratrol [31]
et al. (2013)
Burns et al. (2012) M M Estrogen receptor [27]
KO
Novella-Maestre H NM Cabergoline Assessment nerve [32]
et al. (2012) fibers
Han et al. (2012) M M SRC-1 KO [33]
Mariani et al. (2012) M M Vitamin D receptor [34]
agonist
Wieser et al. (2012) M M Retinoic acid GFP mice [35]
Cakmak et al. (2012) M M Statins [36]
Leconte et al. (2011) M M Temsirolimus Deep infiltrating [37]
implants
Olivares et al. (2011) M M Celecoxib, [38]
Rosiglitazone
Kulak Jr et al. (2011) M M Bazedoxifene [39]
Lu et al. (2010) M M Trichostatin A Autotransplant [40]
model
Styer et al. (2008) M M Leptin receptor [26]
antagonist
Hirata et al. (2005) M M GFP mice [21]
M mouse, H human, NM nude mouse, KO knockout mouse, GFP green fluorescent protein
14.3 Our Animal Model of Endometriosis Using

Homologous Mice
14.3.1 Care and Treatment
According to the implantation theory, we established the readily available murine

model to evaluate the development of endometriosis-like lesions [27, 28]. Female
mice (6 weeks of age, BALB/c) were purchased. Before initiating the experiments,
animals were allowed to acclimate to the following conditions for 7 days. Mice
were in a controlled temperature range (7274 F) on a 12-h light, 12-h dark cycle.
Mice were given food and water ad libitum. Recipient mice were ovariectomized
through two 0.5-cm dorsolateral skin incisions and were then divided into two
treatment groups, estradiol valerate (0.5 g/mouse week) in corn oil or only
corn oil vehicle. Mice were dosed subcutaneously once per week for 2 weeks
before inducing experimental endometriosis. Donor mice were primed 41 h before

removing the uterus with pregnant mare serum gonadotropin (10 IU intraperitone-
ally). The donor uterus was removed en bloc after euthanasia, cleaned of excess
tissue, and washed thrice in sterile PBS. The uterus was slit with a linear incision
longitudinally and minced (1.5 mm). Recipient mice were anesthetized using
isoflurane/oxygen and given buprenorphine (0.1 mg/kg) for pain management.
A 0.5-cm right dorsolateral incision was made; minced donor tissue (1:2 donor
uterus to host ratio) in 500 L PBS was injected into the peritoneal cavity of the
recipient and gently massaged to disperse the tissue. An equivalent amount
(~100 mg) of minced tissue was transferred into all recipients. Mice were treated
for 4 additional weeks with estradiol valerate or the vehicle. After 4 weeks, mice
were euthanized with CO2, the peritoneal cavity was opened, and endometriosis-
like lesions were removed. To assess the effects of drug candidates on ectopic
uterine tissue, ectopic lesions were photographed to document in situ
endometriosis-like lesions. Endometriosis-like lesions were visualized, dissected,
measured, and weighed. Endometriosis-like lesions were removed and either fixed
in 10 % formalin or snap-frozen on dry ice and stored at 80 C until use.
14.4 Effects of Parthenolide on the Endometriosis-Like

Lesions in a Murine Model
We undertook a study of feverfew as a potential therapy for endometriosis that

illustrated the effectiveness of the murine model. The medical herb feverfew has
long been used as a folk medicine for treating fevers, migraine, rheumatoid arthritis,
and dysmenorrhea. Parthenolide is considered the primary bioactive compound in
feverfew having antitumor and anti-inflammatory properties [41]. Parthenolide has
produced anti-tumorigenesis effects against human acute myeloid leukemia and
solid tumors, such as breast and pancreatic cancer [4244]. We therefore used an
experimental mouse model to evaluate the effect of parthenolide as therapy for
endometriosis [28].
A murine model was established by injecting tissue suspension intraperitoneally.
Endometriosis-like lesions had grown in the abdominal cavity of all mice. The
deposits appeared as cystic lesions bulging under the serosal coat. Most of the
lesions were observed around the abdominal incision, the intestinal membrane, and
the renal capsule (Fig. 14.1a). The size of lesions ranged from approximately 2 to
8 mm in diameter (Fig. 14.1b). Histologic sections stained with hematoxylin and
eosin were endometriotic in character. The monolayer epithelial cell lining of the
cyst was revealed by HE staining (Fig. 14.1c, d) [28]. We confirmed that
cytokeratin (a marker of epithelial cells) and vimentin (a marker of stromal cells)
in the mouse endometriosis-like lesions were positive, whereas calretinin (that of
mesothelial cells) was negative, indicating that these cystic lesions originated from
the injected endometrial tissues, not the peritoneal cells.
Fig. 14.1 Cystic lesion in the murine endometriosis model. (a) An endometriosis-like lesion
developed in the murine abdominal cavity. (b) Representative of a large excised lesion. (c) and (d)
HE staining of lesion
In view of the above considerations, we proposed a hypothesis that parthenolide

may have an inhibitory effect on the development of endometriosis. After
parthenolide treatment for 4 weeks, the total number of lesions (5.8 vs. 3.9/mouse)
was significantly reduced, and the average weight (65.6 vs. 29.6 mg/mouse) and the
surface area (50.3 vs. 25.7 mm2/mouse) of lesions were decreased by approximately
50 % of controls. To evaluate the proliferative activity of lesions, the ratio of
Ki67-stained cells was calculated. In endometrial glands epithelia, the percentage
of Ki67-positive cells decreased after parthenolide treatment (17.8 vs. 8.5 %).
14.5 Discussion
Convenient and reliable endometriosis animal models are needed to accelerate

emerging therapeutic alternatives. According to the Sampson implantation the-
ory, we established the syngeneic immunocompetent mice model. In this model,
we and the other investigators provided crucial evidence of both the development of
endometriosis-like lesion growth and donor tissue responsiveness. Recently, Pelch
et al. described a detailed method of a mouse surgically induced endometriosis
model by autotransplantation of uterine tissue [45].
The rodent endometriosis models using mice or rats are widely used in research,
but may have limitations and may not mimic all aspects of human pathophysiology.
For example, in homologous rodent models, healthy uterus is cut into fragments
and transplanted into the peritoneum, whereas it has been suggested that the eutopic
endometrium of women suffering from endometriosis may already be abnormal [2].
Nude mice lacking an intact immune system are employed in the heterologous
model, which cannot mimic the inflammatory response normally seen in human
endometriotic lesions [23]. Although heterologous rodent endometriosis models are
responsive to drugs and manipulations that induce a hypoestrogenic state, such as
ovariectomy, GnRH agonists, aromatase inhibitors, danazol, and selective estrogen
receptor modulators, it may be difficult to analyze novel target families in which,
for example, the murine ligand does not bind to the receptor of the human
transplant.
Whereas cell-based in vitro experiments provide a framework for testing molec-
ular mechanisms, eventually confirming their role in disease causality in vivo can
only be accomplished by a suitable animal model. For a disease as diverse as
endometriosis, a single animal model would unlikely be sufficient to represent the
entire diversity in etiology, pathogenesis, and pathology. Each model has design-
related strengths and limitations. For example, a recently described disease model
consists of introducing endometrial tissue via direct injection into the peritoneal
cavity of immunocompetent mice without suturing [21, 27, 28]. In mice, the
injected tissue forms cyst-like endometriosis lesions; however, the injection method
does not seem to work in rats because the tissue fails to attach to and invade the
peritoneal cavity [9]. Since all endometriosis lesions in each model are attached to
the peritoneum and/or mesenterium with or without suturing, evaluating attachment
or invasion of lesions would be difficult. Furthermore, to quantify the injected
endometrial fragments, this model may be insufficient to evaluate precisely the
endometriotic lesions.
The rodent model is used extensively to study the etiology, pathology, and risk
factors of endometriosis [21, 22, 27, 30, 33, 35, 46, 47] as well as to explore novel
therapeutics [28, 29, 31, 32, 34, 3640, 4851]. In conclusion, this murine model of
endometriosis provides an important tool to evaluate a therapeutic approach to the
disease. This model will help to better understand disease evolution in the living
animal and permit faster and more accurate characterization of a drugs effect on
experimental endometriosis.
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apoptosis of pancreatic cancer cells in vitro. J Exp Clin Cancer Res. 2010;29:108.
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parthenolide in combination with docetaxel reduces metastasis and improves survival in a
xenograft model of breast cancer. Mol Cancer Ther. 2005;4:100412.
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promotion of endometriotic lesion growth by TCDD in adult female rats and mice. Toxicol
Sci. 1999;52:459.
47. Sharpe-Timms KL, Piva M, Ricke EA, Surewicz K, Zhang YL, et al. Endometriotic lesions
synthesize and secrete a haptoglobin-like protein. Biol Reprod. 1998;58:98894.
48. Yavuz E, Oktem M, Esinler I, Toru SA, Zeyneloglu HB. Genistein causes regression of
endometriotic implants in the rat model. Fertil Steril. 2007;88:112934.
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involvement in endometriosis. Pain. 2010;151:70310.
50. Efstathiou JA, Sampson DA, Levine Z, Rohan RM, Zurakowski D, et al. Nonsteroidal
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51. Becker CM, Sampson DA, Short SM, Javaherian K, Folkman J, et al. Short synthetic
endostatin peptides inhibit endothelial migration in vitro and endometriosis in a mouse
model. Fertil Steril. 2006;85:717.
Chapter 15
Malignant Transformation of Endometriosis:
Underlying Mechanisms
Masaki Mandai, Ken Yamaguchi, Noriomi Matsumura, and Ikuo Konishi
Abstract Although it is well known that ovarian cancer, especially clear cell and
endometrioid carcinoma, sometimes develops from endometriotic cyst, the precise
mechanism of carcinogenesis is not clarified yet. Recently, several molecules,
including HNF-1, AKT/PI3K/Met, and ARID1A, have been shown to be involved
in this carcinogenic process. Some of them are included in the OCCC signature
genes which we identified as a gene group specifically expressed in clear cell
carcinoma among ovarian cancers. OCCC signature genes contain many stress-
related genes and were induced by treatment with the fluid of endometriotic cysts.
The fluid of endometriotic cysts contained significantly high concentration of free
iron and oxidative stress-related products. These findings suggest that microenvi-
ronment within the endometriotic cyst may play an important role in the malignant
transformation of endometriosis and development of clear cell carcinoma, a rare
histotype among ovarian cancers.
Keywords Carcinogenesis Clear cell carcinoma Iron Microenvironment

Ovarian cancer
M. Mandai (*)
Department of Obstetrics and Gynecology, Faculty of Medicine, Kinki University,
377-2 Ono-Higashi, Osaka-Sayama, Osaka 589-5811, Japan
e-mail: mandai@med.kindai.ac.jp
K. Yamaguchi N. Matsumura I. Konishi
Department of Gynecology and Obstetrics, Graduate School of Medicine,
Kyoto University, Kyoto, Japan

224 M. Mandai et al.
15.1 Introduction
Endometriosis affects approximately 510 % of women of reproductive age and is

estimated to be increasing due to late marriage and low birth rates in some countries
including Japan. Although the causes of endometriosis have been extensively
investigated, as shown in detail in this book, we do not have definite answers
yet. Endometriosis is clinically associated with three major disorders, namely,
endometriosis-associated pain, endometriosis-associated infertility, and
endometriosis-associated ovarian cancer (EAOC). The former two are disorders
that occur during reproductive age, but EAOC frequently occurs after menopause
when endometriosis itself regresses. This fact indicates two issues: Clinically, it is
very important to follow the patient with endometriosis even after menopause, and
if there is a sign of malignant transformation, prompt surgery should be considered.
From the basic science perspective, it is possible that the menopausal status may
somehow contribute to the occurrence of EAOC.
EAOC primarily consists of endometrioid and clear cell subtypes, both of which
are relatively rare histotypes among ovarian cancers (Fig. 15.1). However, there is
no clear explanation why these particular histologies are associated with endome-
triosis. As described in detail in another section of this book, the frequency of
malignant transformation of endometriotic cysts is apparently higher than that of
other benign epithelial ovarian cysts, including serous and mucinous cystadenoma.
However, again, the reason is unclear. In this chapter, by reviewing possible
mechanisms responsible for the malignant transformation of endometriosis, we
will discuss the characteristic carcinogenesis of endometriosis.
32 y.o. G0P0 1 year 2 years 3 years
Endometriotic cyst Solid part CDDP/CPT-11 Normal Pregnancy/

of 3cm was found appeared x3course ovarian child birth
by US, MRI within the cyst function
SO/omentectomy
with post-operative
pathology of clear
cell carcinoma
Fig. 15.1 Typical case of malignant transformation of endometriotic cyst

15 Malignant Transformation of Endometriosis: Underlying Mechanisms 225
15.2 Endometriosis as a Precursor of Ovarian Cancer
15.2.1 Molecular Evidence
Endometriosis is currently classified as a tumorlike lesion according to the WHO

classification, although it is not fully described. However, a variety of genetic
analyses have demonstrated that endometriosis, especially the endometriotic cyst
of the ovary, is a monoclonal lesion. In the late 1990s, X chromosome-linked
polymorphism analysis revealed that an endometriotic cyst consisted of monoclo-
nal epithelium [13]. Later, fluorescence in situ hybridization (FISH) analysis
demonstrated the possibility that specific chromosomal loss or gain plays a role in
the development and/or progression of endometriosis [4]. Similarly, another FISH
study indicated that perturbations of chromosome 17 and the p53 locus occur
frequently in severe, late-stage endometriosis [5]. In an analysis of DNA from
40 cases of endometriosis, 11 cases (28 %) demonstrated LOH at one or more loci,
although no mutations were detected in the p53 or K-ras genes [6]. Most other
similar analyses [13, 7, 8] revealed the monoclonal nature of endometriotic
epithelium, except for one paper [9] in which the interpretation of the data may
be inadequate. These findings clearly demonstrated that a majority of the
endometriotic cysts are monoclonal and neoplastic. Moreover, genetic events
may accumulate in endometriotic epithelia in parallel with the development of
endometriosis.
15.2.2 Pathological View
Given the fact that endometriosis is a neoplastic disorder, it may have precancerous
potential. Most endometrioses show a benign character, but malignant transformation
may accompany some morphologic and genetic alterations. In pathology, we some-
times encounter the so-called atypical endometriosis, a putative intermediate between
benign endometriosis and endometriosis-associated ovarian cancer (EAOC).
Sampson first described ovarian cancer in endometriosis and defined a criterion
for EAOC [10]. In a review of 194 cases of ovarian endometriosis, Czernobilsky
and Morris found severe cytological atypia in 3.6 % of the cases [11]. Moreover,
in their study, 2.0 % of the cases showed adenomatous, hyperplasia-like lesions.
LaGrenade and Silverberg presented four cases of ovarian carcinomas contiguous
with atypical glandular epithelial changes in endometriosis [12]. Subsequently,
Fukunaga et al. reported that as many as 61 % of the cases of endometriosis coexisting
with ovarian cancer had atypical endometriosis foci, while only 1.7 % of the cases
of endometriosis without ovarian cancer exhibited atypical lesions [13]. Ogawa
et al. reported that atypical endometriosis was found in 78 % of the cases with
EAOC [14]. In their investigations, the transition from typical to atypical endometri-
osis was detected in 22 of 37 cases, and the transition from atypical endometriosis to
carcinoma was found in 23 cases. These pathological observations suggest that benign
endometriosis develops into ovarian cancer in some cases via atypical endometriosis,
a premalignant stage.
15.2.3 Links Between the Pathology and Molecular Findings
Several studies have demonstrated that atypical endometriosis, which is a putative

pathological transition between benign endometriosis and EAOC, has actually
shown an intermediate nature by molecular analyses. Sainz de la Cuesta
et al. evaluated the immunohistochemical expression of p53 in normal endome-
trium, endometriosis, atypical endometriosis, and ovarian cancer associated with
endometriosis. They found that 82.4 % of cancers associated with endometriosis
and all the atypical endometriosis samples showed P53 overexpression, whereas
only 11.8 % of the endometriosis samples, and none of the endometrium samples,
showed P53 overexpression [15]. In the report by Obata et al., frequent LOH was
observed on chromosome 6q (60.0 %) and chromosome 10q (40.0 %) in ovarian
atypical endometriosis [16]. However, not all the endometriosis cases that are
coincident with ovarian cancer have atypical endometriosis lesions, and the bio-
logical significance of atypical endometriosis is still unclear.
15.3 Molecular Events Associated with Malignant

Transformation of Endometriosis
15.3.1 Loss of Heterozygosity (LOH)
There are several ways of analyzing LOH. LOH is usually used to estimate the
locus of a tumor suppressor gene associated with a corresponding event, the
development and malignant transformation of endometriosis in this case. Using
DNA from 40 cases of endometriosis, Jiang et al. analyzed candidate ovarian tumor
suppressor loci on chromosome arms 6q, 9p, 11q, 17p, l7q, and 22q [17]. LOH was
detected on chromosomes 9p (18 %), 1lq (18 %), and 22q (15 %), and, in total, 11 of
40 (28 %) cases demonstrated LOH at one or more of these loci. The same
investigators subsequently examined 14 cases of endometriosis synchronous with
ovarian cancer for LOH on 12 chromosome arms and for X chromosome inactiva-
tion. In all four of the cases in which the carcinoma had arisen within the endome-
triosis and in five of the seven cases in which the carcinoma was adjacent to the
endometriosis, common genetic lesions were detected to be consistent with a
common lineage [17]. Prowse et al. analyzed LOH in 10 EAOCs with coexisting
endometriosis using 82 microsatellite markers and found that, of 63 LOH events
detected in the carcinoma samples, 22 were also detected in the corresponding
endometriosis samples [18]. Goumenou et al. reported that LOH in p16, GALT,
and p53, as well as APOA2, a region frequently lost in ovarian cancer, occurred in
endometriosis, even in stage II of the disease [19]. Sato et al. examined 20 ovarian
endometrioid carcinomas, 24 clear cell carcinomas, and 34 solitary endometrial
cysts of the ovary for LOH at 10q23.3 [20]. In five endometrioid carcinomas
synchronous with endometriosis, three cases displayed LOH events common to
both the carcinoma and the endometriosis. In seven clear cell carcinomas that are
synchronous with endometriosis, three displayed LOH events common to both
the carcinoma and the endometriosis. No LOH events were found in solitary
endometriosis. These findings indicate that attenuation of tumor suppressor genes
is associated with the malignant transformation of endometriosis.
15.3.2 Mutation and Altered Expression of Oncogenes

and Tumor Suppressor Genes
Mutations and altered expression of several genes have been implicated in the
malignant transformation of endometriosis.
15.3.2.1 Augmented Expression of HNF-1
Hepatocyte nuclear factor-1 is a transcription factor that is expressed specifically

in clear cell carcinoma. Kato et al. examined expression of HNF-1 in 30 clear cell
tumors (26 malignant, three borderline, and one benign) and in 40 endometriotic
cysts [21]. All of the 30 clear cell tumors expressed HNF-1. In 9 of 12 cases with
the endometriotic epithelium, expression of HNF-1 was detected in the
endometriotic epithelium as well as in the clear cell tumor. Furthermore, 16 of
40 (40 %) endometriotic cysts without neoplasms also expressed HNF-1. They
concluded that early differentiation into the clear cell lineage takes place in ovarian
endometriosis, not only in atypical endometriosis but also in endometriosis with
degenerative and regenerative changes.
15.3.2.2 The AKT/PI3K/Met Pathway
An array-based comparative genomic hybridization (CGH) analysis by Yamashita

et al. revealed Met gene amplification in 4/13 ovarian clear cell carcinomas and 2/8
cell lines [22]. Amplification of the AKT2 gene was also observed in 5/21 samples. In
73 ovarian clear cell cases, 37.0 % demonstrated Met gene amplification (>4 copies),
and 8.2 % had AKT2 amplification, suggesting that the Met signaling pathway plays
an important role in clear cell carcinogenesis.
According to Yamamoto et al., exons 9 and 20 of the PIK3CA gene were analyzed
in 23 clear cell carcinomas with synchronous putative precursor lesions [23].
Somatic mutations of the PIK3CA gene were detected in 10/23 (43 %) carcinomas
and in the coexisting endometriotic epithelium adjacent to the carcinoma in 9/10
(90 %) cases. Moreover, in six of the nine lesions, the mutation was identified even in
the endometrioses lacking cytological atypia. The authors suggested that mutations
of the PIK3CA gene are early events in tumorigenesis, most likely initiating the
malignant transformation of endometriosis.
15.3.2.3 ARID1A Mutations
ARID1A encodes BAF250a, one of the components of the SW1/SNF chromatin

remodeling complex. In 2010, two groups identified mutations of the ARID1A gene
in EAOC and in ovarian clear cell carcinoma. Genome-wide mutational analysis
exhibited ARID1A mutations in almost one half of the clear cell carcinomas, one
third of the endometrioid carcinomas, and none of the serous carcinomas of the
ovary [24]. BAF250a protein expression was also lost in these cases. In some cases,
loss of BAF1a expression was also found in atypical endometriosis adjacent to
the cancer lesions. In another study, ARID1A mutations were detected in 57 %
of the cases as well as PI3CA mutations in 40 % of the cases [25]. Although
ARID1A is thought to play a role as a tumor suppressor gene [26], the precise
effects of mutations in this gene are not fully understood.
Clinically, loss of ARID1A expression was significantly correlated with
advanced FIGO stage, high CA125 levels, and with shorter progression-free sur-
vival of patients with clear cell carcinomas treated with platinum-based chemo-
therapy [27]. Although loss of ARID1A protein expression is thought to be an early
event in the development of ovarian clear cell adenocarcinoma [28], its biological
effect on the malignant transformation of endometriosis should be further clarified.
15.4 Environmental Factors That Affect Malignant

Transformation of Endometriosis
15.4.1 Microenvironments in Endometriotic Cysts
15.4.1.1 Oxidative Stress and Carcinogenesis
In our bodies, live cells are continuously exposed to oxidative stresses, which
are produced internally or externally. Internal stress consists of mitochondrial
respiratory stress, cytochrome P450 metabolism, and inflammatory responses.
External stresses are generated by various agents, including chemicals, xenobiotics,
irradiation, and metal ions, including Fe ions. One of the well-known mechanisms
Free iron concentration LPO (oxidative stress marker)
120 80
100
60
80
60 40
40
20
20
0 0
endometriotic other epithelial endometriotic other epithelial
cyst cyst cyst cyst
Fig. 15.2 Differences in Fe concentration/oxidative stress between endometriotic cysts and other
benign epithelial cysts
by which metal ions produce intracellular ROS is called the Fenton reaction [29].
During this reaction, highly toxic dOH and anoxidized metal ions are generated
from H2O2. Metal-induced ROS causes DNA damage, including single- or double-
strand breaks, base modifications, deoxyribose modifications, and DNA cross-
linking, which ultimately contributes to carcinogenesis.
In normal conditions, ROS overproduction is avoided by endogenous antioxi-
dants, including superoxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPx). If the balance between the cellular antioxidant defense and
ROS generation is impaired, excessive ROS can cause oxidative stress. Prolonged
and excessive oxidative stress mediates a variety of chronic and degenerative
diseases, including cancers, inflammation, aging, and neuronal disorders [29].
15.4.1.2 Oxidative Stress in Endometriotic Cysts
The content of endometriotic cysts consists of old blood containing a high concen-
tration of iron derived from hemoglobin. We compared the concentrations of free
iron in endometriotic cysts with those in other ovarian cysts, including serous and
mucinous cystadenoma, and found that the concentrations were significantly higher
in endometriotic cysts [30] (Fig. 15.2). Likewise, lactose dehydrogenase (LDH)
(a marker of tissue damage), potential antioxidant (PAI) (an antioxidant marker),
lipid peroxidase (LPO) (Fig. 15.2) (a marker of oxidative stress), and 8-hydroxy-2-
deoxyguanosine (a marker of DNA damage) were all significantly elevated in
endometriotic cysts compared with other ovarian cysts. These data strongly suggest
that the epithelial cells of the endometriotic cyst are constantly exposed to exces-
sive oxidative stress and are subjected to cellular and DNA damage (Fig. 15.3).
To elucidate the mutagenic property of the fluid in chocolate cysts, we
conducted an experiment in vitro, which demonstrated that the fluid in chocolate
Fig. 15.3 Intrinsic and

extrinsic factors affecting Estrogen
malignant transformation of
endometriotic cyst
DNA injury/mutation
Oxidative stress
Old blood
containing Fe
cysts is more mutagenic than that in other cysts [30]. There are several reports
suggesting a link between a stressful microenvironment and cancer development.
(1) Chromosomal aberrations are more frequent in ovarian endometriotic cysts than
in extra-gonadal endometriosis [31]. (2) Malignant transformation of endometriotic
cysts increases with the duration of endometriosis [32].
15.4.2 Clear Cell Carcinoma and the Stress Response
If the microenvironment in endometriotic cysts affects cancer development, is it

also associated with the phenotype of the ovarian cancer that arises as a result of
malignant transformation? To assess this possibility, we began by identifying the
gene signature that distinguishes clear cell carcinoma from other types of ovarian
cancer using microarray datasets [33]. The signature composed of 437 genes was
designated as the ovarian clear cell carcinoma (OCCC) signature. By using a
categorical analysis, we demonstrated that the OCCC signature contains genes in
three major categories, that is, stress response, sugar metabolism, and coagulation.
Stress-related genes were estimated to play a central role, suggesting that the stress
response is the fundamental feature of clear cell carcinoma. Actually, the OCCC
signature was shown to involve a signal network consisting of many stress-related
genes, including HIF1-, IL-6, and SOD2. Moreover, when ovarian surface epi-
thelial cells were treated with the contents of endometriotic cysts, the OCCC
signature was increased in a time-dependent manner. The constitutive expression
of the OCCC signature in clear cell carcinoma may reflect gene induction in
response to the microenvironment in endometriotic cysts.
These data raise a novel and important concept that the specific microenviron-
ment induces unique gene expression and leads to a cancer of a special phenotype.
The local microenvironment, including ROS or inflammation, is likely not only to
be implicated in cancer development [34], but it is also likely to be related to the
occurrence of a specific cancer phenotype.
15.5 The Carcinogenic Scheme of Endometriotic Cysts

and Future Applications
15.5.1 The Natural History of Endometriosis

and Malignant Transformation
As mentioned earlier, endometriosis itself is largely a monoclonal, neoplastic

disorder and has a potential for malignant transformation. Because endometriotic
epithelium is a mullerian-type epithelium, it may be subject to mullerian organ
carcinogenesis, leading to the development of various types of cancers, including
serous carcinoma, mucinous carcinoma, and borderline tumors such as mucinous
mullerian borderline tumors [35]. However, these types of cancer are relatively
rare. A major type of cancer that develops from endometriosis is endometrioid
carcinoma. The carcinogenic process of development of endometrioid carcinoma
from endometriosis may resemble that in the endometrium under the influence of
(unopposed) estrogen. In this case, progestin may play a prophylactic role, although
no definite data are available yet.
The third mechanism, which is associated with the occurrence of clear cell
carcinomas, is strongly influenced by the unique microenvironment within the
chocolate cyst [35]. Continuous exposure to oxidative stress, partly caused by
iron, results in the expression of stress-responsive genes, the OCCC signature.
Constitutive expression of the OCCC signature genes is closely associated with
the stress-resistant and slow-growing phenotype of malignancy, that is, clear cell
carcinoma [36] (Fig. 15.4). Regarded as the prevention of clear cell carcinoma,
surgical treatment to improve the microenvironment may be the best option.
ovarian surface epithelium? peritoneal mesothelium? endometriotic implant?
genetic alteration
endometriosis = neoplastic lesion
(unopposed) oxidative stress

estrogen inflammation
hormone dependent stress induced Mullerian type

carcinogenesis carcinogenesis carcinogenesis
endometrioid clear cell serous carcinoma,

carcinoma carcinoma mucinous carcinoma,
MMBT, etc.
Fig. 15.4 Hypothetical mechanism of malignant transformation of endometriotic cyst

Thus, clarification of the precise natural course of malignant transformation of

endometriotic cysts should lead to the proper management of endometriosis in
terms of malignant transformation.
15.5.2 Future Treatment Strategy of EAOC, Especially

Ovarian Clear Cell Carcinoma
Recent advances in understanding the molecular and biological features of clear

cell carcinoma enable us to consider several new treatment strategies. Frequent
activation of the AKT/PI3K/Met pathway in EAOC suggests that mTOR inhibitors
may be effective in the treatment of these cancers. Mabuchi et al. showed that the
mTOR inhibitor RAD001 is effective in the treatment of clear cell carcinoma of
the ovary [37]. We have shown that sorafenib, a multikinase inhibitor, showed
therapeutic effects in the RMG-2 clear cell cancer cell line, which is resistant to
cisplatin [38]. In the future, the exploration of more precise mechanisms of malig-
nant transformation of endometriosis may lead not only to various molecular
targeted therapies but also to therapies targeting neovascularization or cancer
metabolism [36].
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31. Korner M, Burckhardt E, Mazzucchelli L. Higher frequency of chromosomal aberrations in
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endometrioid adenocarcinoma. Mod Pathol. 2006;19(12):161523.
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33. Yamaguchi K, Mandai M, Oura T, Matsumura N, Hamanishi J, Baba T, Matsui S, Murphy SK,
Konishi I. Identification of an ovarian clear cell carcinoma gene signature that reflects inherent
disease biology and the carcinogenic processes. Oncogene. 2010;29(12):174152.
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36. Mandai M, Matsumura N, Baba T, Yamaguchi K, Hamanishi J, Konishi I. Ovarian clear cell
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Chapter 16
Potential New Drugs for Endometriosis:
Experimental Evidence
Kaei Nasu, Yukie Kawano, Masakazu Nishida, Akitoshi Tsuno,

Akitoshi Yuge, Wakana Abe, Kentaro Kai, Mamiko Okamoto,
and Hisasshi Narahara
Abstract Endometriosis, a disease affecting 310 % of women of reproductive

age, is characterized by the ectopic growth of endometrial tissue. Recent basic
studies have revealed that the dysregulation of apoptosis, fibrosis, and epigenetic
factors plays important roles in the pathogenesis of this enigmatic disease.
Contraceptive steroids, progestogens, agonists of gonadotropin-releasing hor-
mone, androgens, and nonsteroidal anti-inflammatory agents have been used to
treat endometriosis. Endometriosis treatments designed to lower circulating estra-
diol concentrations can be used only for a limited time due to unacceptable side
effects. The development of medical treatments based on novel strategies to prevent
or treat endometriosis has thus become a research priority.
Regarding the development of novel medical treatments for endometriosis,
many researchers have been evaluating new drugs including molecular-targeting
agents and herbal medicine as well as the newly developed hormonal agents. This
chapter is a review of the findings from recent basic research on the pathogenesis of
endometriosis and the evaluations of novel medical treatments for this disease,
especially focusing on the inhibitors of nuclear factor-B, the mevalonate-Rho/
ROCK pathway, and histone deacetylase. These agents are now considered prom-
ising agents for the treatment and prevention of endometriosis.
Keywords Endometriosis Histone deacetylase inhibitor Mevalonate-Rho/

ROCK pathway Nuclear factor-B
K. Nasu, M.D., Ph.D. (*) Y. Kawano M. Nishida A. Tsuno A. Yuge W. Abe

K. Kai M. Okamoto H. Narahara
Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University,
Idaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan
e-mail: nasu@oita-u.ac.jp

236 K. Nasu et al.
16.1 Introduction
Endometriosisthe benign, estrogen-dependent, tumorlike disease characterized

by chronic pelvic pain, dysmenorrhea, dyspareunia, and/or subfertilityis due to
the uncontrolled ectopic growth of proliferative endometrial tissue. Women of
reproductive age are most commonly affected by endometriosis, which usually
occurs in the peritoneum, ovaries, and rectovaginal septum [1]. The symptoms of
endometriosis may markedly reduce a womans quality of life.
Several surgical and medical strategies have been used to treat endometriosis.
Contraceptive steroids, progestogens, and agonists of gonadotropin-releasing hormone,
androgens, and nonsteroidal anti-inflammatory agents have been attempted [2].
Endometriosis treatments designed to lower circulating estradiol concentrations can
be used only for a limited time due to unacceptable side effects. The current medical
treatments inhibit the growth of endometriotic implants by suppressing ovarian steroids
and inducing a hypoestrogenic state; they have been demonstrated to be effective in
relieving endometriosis-associated pain [2]. However, high recurrence rates, up to
45 %, after the completion of medical treatments remain a significant problem [3].
An international consensus group proposed the development of nonhormonal medical
treatments to prevent or treat endometriosis as a research priority [4]. In addition, many
research groups have been evaluating new drugs for endometriosis such as molecular-
targeting agents and herbal medicine, as well as the newly developed hormonal agents.
In this chapter, we review the recent basic studies on the development of novel
medical treatments for endometriosis based on distinct strategies. We focus on the
recent publications about the nuclear factor (NF)-B inhibitors, mevalonate-Ras
homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK) inhib-
itors, and histone deacetylase (HDAC) inhibitors (HDACIs) as potential candidates
for the next era in endometriosis treatment and prevention.
16.2 Potential New Drugs
16.2.1 NF-B Inhibitors
Apoptosis plays a critical role in maintaining tissue homeostasis, and its function is
to eliminate excess cells and dysfunctional cells. Apoptosis can be initiated by
extracellular or intracellular death signals, and it results from a series of related
morphologic and biochemical processes. Morphologically, apoptotic cells present
with condensed chromatin, multiple membrane-bound organelles (apoptotic bod-
ies), and shrunken appearance. Biochemically, apoptosis is characterized by mono-
meric or multimeric 180-base pair nucleosomal fragments resulting from the
cleavage of double-stranded nuclear DNA [5]. Apoptosis is controlled by the
expression of a number of regulatory genes, including c-myc, p53, Fas, NF-B,
and members of the B-cell lymphoma/leukemia (Bcl)-2 family [611].
16 Potential New Drugs for Endometriosis: Experimental Evidence 237
Estradiol
TNF- Lipopolysaccharide
Receptors

NF-
MAPKK NIK Akt NF-B
inhibitors
IKK
Phosphorylated
NF-B
inhibitors
NF-
Autologous activation ECM
stimulation remodeling
Inflammation Cell cycle
Cytokine Proliferation
production
Apoptosis
Progression of endometriosis
Fig. 16.1 Involvement of the NF-B-mediated pathway in the pathogenesis of endometriosis.

ECM extracellular matrix, IB inhibitor B, IKK IB kinase, NIK NF-B-inducing kinase,
MAPKK MAPK kinase, TNF tumor necrosis factor
Histologically, endometriotic tissues and normal proliferative endometrium are

similar. However, endometriosis is increasingly being recognized as a condition in
which endometriotic cells at the ectopic sites exhibit abnormal proliferative and
apoptotic regulation in response to appropriate stimuli [1222]. It has been dem-
onstrated that the degree of apoptosis in endometriotic lesions is less than that in the
endometrium of the same patients and that of healthy women [1315, 2325]. The
resistance of endometriotic cells to apoptosis is suspected to be either intrinsic or
brought about by environmental factors. Aberrantly expressed proliferation-related
and apoptosis-related molecules in endometriosis include the decreased expressions
of homeobox (HOX) A10 [26] and caspase-1 [27] and the enhanced expressions of
c-myc [28], cellular inhibitor of apoptosis protein (cIAP) 1 [29], X chromosome-
linked IAP [29], B-cell lymphoma/leukemia (Bcl)-2 [19], Bcl-XL [19], and NF-B
[30, 31] in endometriotic cells.
The pleiotropic transcription factor NF-B has been identified as a critical
component of several signal transduction pathways [29]. Figure 16.1 shows the
238 K. Nasu et al.
proposed functions of NF-B in the pathogenesis of endometriosis. One important

function of NF-B is its ability to protect cells from apoptosis by activating
antiapoptotic genes [9, 10]. Wieser et al. [32] demonstrated the constitutive acti-
vation of NF-B in endometriotic cells. It is suggested that NF-B has a significant
role in the proliferation of endometriotic lesions [30, 31]. The activation of NF-B
by lipopolysaccharide (LPS) induces the proliferation of endometriotic stromal
cells [33].
A number of substances have been presented in the endometriosis literature as
NF-B inhibitors. The reported selective inhibitors of NF-B include an IB
protease inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) [33], tha-
lidomide [34], BAY 117085 [35], pyrrolidine dithiocarbamate (PDTC) [3638],
costunolide [39], and parthenolide [40]. The urinary preparation human chorionic
gonadotropin A (hCG-A) [41], gonadotropin-releasing hormone (GnRH) agonists
[30], progesterone [42], and danazol [42] have been demonstrated to inhibit NF-B
activity in endometriosis. NF-B inhibitors have been shown to significantly block
the proliferation of endometriotic stromal cells [33, 35, 40] and they induce
apoptosis and the G0/G1 phase cell-cycle arrest of endometriotic stromal cells
[35, 38, 39]. Additionally, after endometriotic stromal cells were treated with
NF-B inhibitors, the downregulation of the expression of antiapoptotic factors
(Bcl-2 and Bcl-XL), inflammatory cytokines (interleukin-6 [IL-6], IL-8, monocyte
chemoattractant protein-1 [MCP-1], and granulocyte-macrophage colony-stimulat-
ing factor [GM-CSF]), inflammatory mediators (COX-2 and PGE2), extracellular
matrix remodeling mediators (MMP-2, MMP-3, MMP-7, MMP-9), CD44, and
vascular endothelial growth factor [VEGF] with simultaneous activation of
caspase-3, caspase-8, and caspase-9 was observed [30, 3441].
The suppression of NF-B activity by proteasome inhibitors also suppresses the
proliferation of endometriotic cells in vitro [31]. The NF-B inhibitors BAY 11
7085 and SN-50 significantly reduced the development of endometriotic lesions in
a nude mice model [43]. Takai et al. [40] also demonstrated that parthenolide
reduced the growth of endometriotic lesions in a murine model. The antioxidant
PDTC reduced the growth and vascularity of experimental peritoneal endometriotic
lesions in a rat model [44].
16.2.2 Mevalonate-Rho/ROCK Inhibitors
During the development and progression of endometriotic lesions, excess fibrosis

may lead to scarring, chronic pain, and the alterations of tissue function that are
characteristic of this disease [4548]. It has been suggested that type I collagen is a
major contributor to endometriosis-associated fibrosis [46, 49], whereas -smooth
muscle actin (SMA)-positive fibroblastic cells were frequently detected in the
fibrotic areas associated with endometriosis of the peritoneum, ovary, rectovaginal
septum, and uterosacral ligaments [45, 48, 50]. An immunohistochemical analysis
led Anaf et al. [50] to suggest that endometriotic stromal cells can differentiate to
-SMA-positive myofibroblasts.
To establish a model of fibrosis formation in endometriosis, we used a 3D
collagen culture system with human endometriotic stromal cells [51, 52]. We
cultured the cells in floating collagen lattices to reorganize the collagen fibers and
make them compact, resulting in contraction of the collagen gels. This culture
system provided a model for mechanically relaxed tissue with low tensile strength,
comparable to the early stages of an endometriotic lesion. We found that
endometriotic stromal cells cultured in floating 3D gels have an enhanced contrac-
tile profile compared to normal endometrial stromal cells [51]. This suggested that
endometriotic stromal cells may acquire fibrogenetic ability or fail to avoid
fibrogenesis during the pathogenesis of endometriosis.
Members of the Rho family of small guanosine triphosphatase (GTPase) are
known to regulate cell shape, motility, cell-substratum adhesion, and cell contrac-
tion through the reorganization of actin cytoskeletons [5365]. The active form of
Rho is GTP-bound [54, 62], and many polypeptides have been reported as targets of
activated Rho, including ROCK-I/p160ROCK and Rho-kinase/ROCK-II [66].
ROCKs phosphorylate the myosin light chain (MLC) [67] and the myosin-binding
subunit of myosin phosphatase [68], and they inhibit myosin II regulatory light
chain phosphatase activity [68] in cultured fibroblasts. Rho and ROCKs have been
implicated in myosin II-dependent force generation [69]. Thus, the activation of
ROCKs by Rho can promote the assembly of focal adhesions, actin stress fiber
formation, and contraction of non-muscle cells [62, 68], in which RhoA regulates
-SMA expression [70].
Based on these observations in fibroblasts, we have investigated the signaling
pathway underlying endometriotic stromal cell-mediated contractility by using 3D
cultures. Our data indicated that human endometriotic stromal cells undergo
myofibroblastic differentiation and show increased expression of RhoA, ROCK-I,
and ROCK-II proteins, resulting in enhanced contractility [51]. Thus, we evaluated
several inhibitors of mevalonate-Rho/ROCK pathways as candidate drugs for the
treatment and prevention of endometriosis-associated fibrosis [51, 52, 7173].
16.2.3 Statins
Statins are potent inhibitors of cholesterol biosynthesis that are widely used to
reduce serum cholesterol levels in hyperlipidemic patients [74, 75]. Statins are
divided into three categories: natural statins (i.e., lovastatin and pravastatin),
semisynthetic statins (i.e., simvastatin), and synthetic statins (i.e., atorvastatin,
fluvastatin, and cerivastatin) [75, 76]. These three subtypes of statin exhibit mark-
edly different hydrophobicities, with simvastatin as the most hydrophobic and
pravastatin as the most hydrophilic.
Statins competitively inhibit 3-hydroxy-3-methylglutarylcoenzyme A
(HMG-CoA) reductase to block the conversion of HMG-CoA to L-mevalonate, a
240 K. Nasu et al.
rate-limiting step in cholesterol synthesis [77]. By inhibiting the initial step of the
cholesterol synthesis pathway, statins reduce the synthesis of several important
lipid intermediate compounds including isoprenoids such as farnesyl pyrophos-
phate (FPP), a precursor of cholesterol, and geranylgeranyl pyrophosphate (GGPP),
which is synthesized from FPP [78]. Both FPP and GGPP are important isoprenoid
intermediates and serve as lipid attachments for a variety of intercellular proteins to
the plasma membrane, including Rho proteins, resulting in their activation [59, 79].
By inhibiting the activation of Rho proteins, statins also regulate both the
Rho/ROCK pathways and the mevalonate pathway [80, 81].
Simvastatin has been demonstrated to inhibit the proliferation of endometriotic
stromal cells as well as the collagen gel contraction mediated by these cells [71].
Attenuation of the endometriotic stromal cell attachment to collagen fibers is
involved in this mechanism. Lovastatin also inhibited cell proliferation and angio-
genesis in endometriosis [82], whereas simvastatin and mevastatin inhibited the
MCP-1 production by endometriotic cells [83]. Atorvastatin also exhibited
antiproliferative and anti-inflammatory effects in endometriotic cells [84]. Oktem
et al. [85] demonstrated that atorvastatin induced the regression of endometriotic
implants in a rat model. Similarly, Bruner-Tran et al. [86] demonstrated that
simvastatin induced the regression of endometriotic implants in a nude mouse
model.
16.2.4 ROCK Inhibitors
Several selective ROCK inhibitors including Y-27632 and fasudil hydrochloride

are now available for basic and clinical studies [58, 63, 87, 88]. We demonstrated
that Y-27632, a pyridine derivative that acts as a specific inhibitor of ROCK-I and
ROCK-II [58, 63], inhibits endometriotic stromal cell-mediated contractility [51].
Interestingly, Y-27632 was found to exert a greater effect on the contractility
of endometriotic stromal cells than on the contractility of normal eutopic
endometrial stromal cells, whereas fasudil hydrochloride also inhibited
endometriotic stromal cell-mediated contractility, myofibroblastic differentiation,
and cell proliferation [72].
16.2.5 Heparin
Heparin is an analog of heparan sulfate, a unique class of macromolecules that is

widely expressed on the cell surface and in the extracellular matrix [89]. It is a
commonly used anticoagulant drug [90]. Heparin was shown to inhibit the gel
contraction mediated by dermal fibroblasts [91, 92]. We demonstrated that heparin
attenuates the contractility of endometriotic stromal cells by suppressing the cells
attachment to collagen fibers, the inhibition of myofibroblastic differentiation, and
the suppression of the Rho/ROCK-mediated pathway [73]. However, the precise

target molecule of heparins action on endometriotic stromal cells has not yet been
elucidated.
16.2.6 HDACIs
Epigenetics refers to the stable inheritance of phenotypes of cells and organisms

without changes in DNA sequence or DNA content [93]. The epigenetic pheno-
types are conferred via nuclear processes such as DNA methylation and chromatin
modifications (e.g., acetylation, biotinylation, isomerization, methylation, phos-
phorylation, ribosylation, sumoylation, and ubiquitination of histones) and underlie
the regulation of all genome functions, including gene expression, DNA replication,
and genome stability [9496]. Epigenetic phenotypes are also conferred via
microRNA and double-stranded noncoding RNA, which are interconnected and
may work together to establish and/or maintain specific gene activity states in
normal cells [93, 96, 97]. Epigenetic processes are known to be involved in
development, health, disease, and aging and are responsible for phenomena such
as X-chromosome inactivation and genomic imprinting [98, 99]. Of these epige-
netic regulatory mechanisms, histone acetylation is the most studied.
The acetylation levels of histone are controlled by a balance between histone
acetyltransferases and histone deacetylases (HDACs). Histone acetyltransferases
transfer acetyl groups from acetyl-CoA to lysine residues on the aminoterminal
region of histones and activate genetic transcription. Conversely, HDACs restore
the positive charge on lysine residues (by removing the acetyl groups) and prevent
transcription. HDACs are large multiprotein complexes that target promoter sites
through their interaction with sequence-specific transcription. They play an impor-
tant role in the regulation of gene transcription through the remodeling of chromatin
structure and dynamic changes in the nucleosomal packaging of DNA [100].
The hyperacetylation of histones H3 and H4 is often associated with activated
transcription, and the hypoacetylation of histones H3 and H4 correlates with
transcriptional silencing or repression [101], whereas Choi et al. [102] suggested
that the substrates of HDACs are not restricted to histones but include transcrip-
tional regulators, such as p53, E2F-1, Mad-1, BCL-6, and ETO. In this regard,
global gene expression analyses have shown that HDACIs affect the expression
levels of 220 % of genes in the genome, of which about half are upregulated and
half downregulated [103].
Accumulating evidence indicates that several epigenetic aberrations are
involved in the pathogenesis of endometriosis [104110]. We observed that the
levels of acetylated histones H3 and H4 were significantly lower in unstimulated
endometriotic stromal cells compared to normal endometrial stromal cells,
suggesting that aberrant histone modifications are present in endometriosis [108].
This initial finding encouraged us to evaluate the efficacy of HDACIs for the
treatment of endometriosis. Our subsequent experiments demonstrated that
242 K. Nasu et al.
HDACIs significantly inhibited the proliferation of endometriotic stromal cells, and

they also induced significant levels of cell-cycle arrest at the G0/G1 or G2/M phases
and significant apoptosis of these cells [108].
Interestingly, HDACIs showed marginal to weak effects on normal endometrial
stromal cells compared to endometriotic stromal cells. Moreover, HDACI treat-
ment significantly inhibited HDAC activity and resulted in the accumulation of
acetylated histones H3 and H4 in total cellular chromatin and in the promoter
regions of the p16INK4a, p21Waf1/Cip1, p27Kip1, and cell-cycle checkpoint kinase
2 (chk2) genes in these cells. A Western blot analysis revealed the increased protein
levels of p16INK4a, p21Waf1/Cip1, p27Kip1, and chk2, the suppression of Bcl-2 and
Bcl-XL protein levels, and the activation of caspase-3 and caspase-9 in
endometriotic stromal cells after treatment with HDACIs [108]. Recently, we
found that the expression of CCAAT/enhancer-binding protein (C/EBP)-, a
tumor suppressor gene, and death receptor 6, a TNF receptor superfamily gene,
was epigenetically suppressed by histone deacetylation (our unpublished data).
Histone deacetylation appears to be a potent regulator of gene expression in
endometriosis, which raises the prospect of using HDACIs as therapeutic tools in
endometriosis [108]. Several classes of HDACIs have been identified, including
(a) organic hydroxamic acids [e.g., trichostatin A and suberoylanilide
bishydroxamine (SAHA)], (b) short-chain fatty acids [e.g., butyrates and valproic
acid (VPA)], (c) benzamides (e.g., MS-275), (d) cyclic tetrapeptides (e.g.,
trapoxin), and (e) sulfonamide anilides [108]. The molecular events that mediate
the biological effects of HDACIs are incompletely understood. Inhibition of HDAC
by HDACIs increases histone acetylation and maintains chromatin structure in a
more open conformation, resulting in the reactivation of transcriptionally silenced
pathways or the suppression of aberrantly expressed genes through the recruitment
of repressors [111, 112]. HDACIs can reactivate genes silenced by promoter
hypermethylation as well as the demethylation agents [113]. HDACIs also cause
mitotic defects through non-transcriptional mechanisms [114].
We demonstrated that HDACIs, including VPA, SAHA, and apicidin, can inhibit
the proliferation, induce cell differentiation and cell-cycle arrest, and stimulate the
apoptosis of endometriotic cells [108]. Guo and his colleagues have demonstrated
that HDACIs, such as VPA and trichostatin A, can suppress the proliferation, induce
cell-cycle arrest, inhibit IL-1-induced cyclooxygenase-2 expression and NF-B
activation, upregulate peroxisome proliferator-activated receptor , p21Waf1/Cip1 and
PR-B expression, attenuate invasiveness, and reactivate the silenced E-cadherin
gene expression of endometriotic cells [115120]. Romidepsin, an HDACI, was
shown to specifically reduce HDAC enzymatic activity, resulting in inhibited cell
proliferation, cell-cycle arrest, increased apoptosis, and reduced expression of
VEGF mRNA and protein in endometriotic cells [121, 122]. Treatment with
trichostatin A and VPA has been shown to significantly reduce the growth of
endometriotic lesions in a murine model [123, 124]. Histone deacetylation appears
to be a potent regulator of gene expression in endometriosis, which raises the
prospect of using HDACIs as therapeutic tools in endometriosis [108, 117].
VPA was used in a pilot study of three patients with endometriosis and
adenomyosis who had moderate to severe dysmenorrhea [125]. A VPA dose of
1,000 mg/day was used for 3 months. Complete relief of pain in all cases and an
average reduction of one-third in uterine size were reported. The disappearance or
reduction of palpable tender nodules in the cul-de-sac was also reported.
16.3 Conclusions
Basic research on endometriosis offers new opportunities to understand the patho-

genesis of this enigmatic disease and to provide novel medical treatments other than
hormonal therapy. As shown in this chapter, NF-B inhibitors and mevalonate-Rho/
ROCK inhibitors as well as HDACIs seem to be promising therapeutic strategies
for the treatment of endometriosis.
Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science (no. 13237327 to K. Nasu, no. 25861500 to
Y. Kawano, and no. 23592407 to H. Narahara).
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Chapter 17
Altered Biological Characteristics of Eutopic
and Ectopic Endometrium
Cassia G.T. Silveira, Admir Agic, Geraldine O. Canny,

and Daniela Hornung
Abstract As it has been presented thus far, the pathogenesis of endometriosis is

complex and multifactorial. The intrinsic endometrial abnormalities thought to be
associated with endometriosis include abnormal gene expression, altered endome-
trial responses to hormones such as progesterone, impaired immunological
response, increased nerve density, and oxidative stress. Also interesting is the fact
that such biological alterations have also been observed in the eutopic endometrium
of patients with endometriosis, which strongly indicates their critical role in the
pathophysiology of the disease. Indeed, it has been suggested that the evaluation of
eutopic endometrium is an important line of investigation which may help to
achieve a fuller understanding of endometriosis pathogenesis. Hence, we present
herein a literature review and a comprehensive evaluation of the involvement of the
eutopic endometrium in endometriosis. The biological characteristics of both
eutopic and ectopic endometrial tissues as well as their clinical correlations with
the disease are highlighted, with the primary objective of understanding the role of
the eutopic endometrium in this enigmatic gynecological disorder.
Keywords Apoptosis Endometrium Estrogen Signaling Inflammation Inva-

sion L1CAM YB-1
C.G.T. Silveira
Department of Gynecology and Obstetrics, University of Lubeck, Lubeck, Germany
A. Agic
Department of Gynecology and Obstetrics, Diakonissenkrankenhaus Karlsruhe Ruppurr,
Diakonissenstr. 28, 76199 Karlsruhe, Germany
G.O. Canny
Geneva Foundation for Medical Education and Research, Versoix, Switzerland
D. Hornung (*)
Department of Gynecology and Obstetrics, University of Lubeck, Lubeck, Germany
Department of Gynecology and Obstetrics, Diakonissenkrankenhaus Karlsruhe Ruppurr,
Diakonissenstr. 28, 76199 Karlsruhe, Germany
e-mail: D.Hornung@gmx.de

252 C.G.T. Silveira et al.
17.1 Introduction
17.1.1 The Biology of Eutopic and Ectopic Endometria
The endometrium is a highly specialized tissue composed of a surface epithelium

and associated glands surrounded by a cell-rich connective tissue stroma containing
a rich supply of blood vessels. Within the endometrium, two distinct layers can be
clearly distinguished: the first layer is the transient superficial functionalis, which
comprises the upper-two-thirds of the endometrium and which includes loose
stroma and differentiating glands; it is constantly regulated by sequential changes
in circulating sex steroid hormones through the menstrual cycle and is shed during
menstruation; the second layer is the basalis (germinal layer), which comprises
undifferentiated glands embedded in stroma and which is responsible for producing
a new functionalis in the subsequent cycle; it is relatively insensitive to hormonal
changes persisting from cycle to cycle [14].
Remarkably, the human endometrium is a dynamic tissue that undergoes cycles
of regeneration (proliferative phase), differentiation (secretory phase), and shed-
ding (menstrual phase) over the reproductive life of women. These successive
events of cellular proliferation and differentiation are finely orchestrated by sequen-
tial changes in circulating sex steroid hormones across the menstrual cycle, which is
also accompanied by significant variations in histological and gene expression
profiles [5] and by immune mediators [6].
Prior to ovulation, increasing levels of ovarian estrogens produced in response to
follicle-stimulating hormone promotes the reepithelialization of the luminal edge
and growth of stromal and glandular components, preparing the tissue for the
subsequent secretory transformation of the glands triggered by the post-ovulation
production of progesterone. In addition to glandular cell differentiation, progester-
one also mediates growth and coiling of spiral arteries, influx of distinct immune
cells, and pre-decidualization of stromal compartments. Subsequently, in the
absence of implantation and upon steroid hormone withdrawal, the stromal com-
partment signals the activation of matrix-degrading enzymes to mediate tissue
desquamation and endometrial bleeding [7].
The actions of estrogen and progesterone are mediated by specific intracellular
and membrane bound receptor proteins in stromal and epithelial endometrial
cells [8]. In particular, estrogen-related effects on endometrial cell proliferation
and survival are predominantly mediated via estrogen receptor (ER1) [9], while
its isoform (ER2), expressed at low levels in the endometrium, promotes epithe-
lial differentiation through negative regulation of ER1-mediated responses [10]. In
the secretory phase, the antiproliferative effect of progesterone on the endometrial
epithelium is mediated via progesterone receptor A (PR-A) [11].
During the menstrual cycle, locally produced growth factors also participate in
endometrial regeneration exerting mitogenic effects of estrogen and differentiating
effects of progesterone through autocrine and/or paracrine interactions between
epithelial and stromal cells. For instance, the synthesis and secretion of
17 Altered Biological Characteristics of Eutopic and Ectopic Endometrium 253
transforming growth factor (TGF), epidermal growth factor (EGF) and EGF
receptors, as well as insulin-like growth factor-1 (IGF-1) in response to estrogen
were shown and associated with endometrial cell proliferation and decidualization.
Interestingly, these growth factors are also believed to modulate the effects of
estrogen or progesterone or each other by altering growth factor receptor or binding
protein expression [1214].
Indeed, the architecture and function of the endometrium are maintained by the
dynamic relationship between epithelial and stromal cells and the complex micro-
environment that includes the extracellular matrix (ECM), diffusible growth fac-
tors, cytokines, and other paracrine messengers, as well as a variety of other cell
types such as endothelial cells, lymphocytes, and macrophages [15, 16]. Thus, it has
become evident that the abnormal interactions between these cellular components
and their microenvironments may disrupt tissue homeostasis and precede the
development of several endometrial pathologies, including endometriosis. So far,
numerous studies have pointed out that the onset and progression of endometriosis
are potentially supported by the interruption of this well-balanced cellular
equilibrium [15, 1721] potentially resulting from genetic [22] and epigenetic
changes [23].
Similar to intrauterine endometrium, ectopic endometrial tissue consists of
endometrial-like epithelial and stromal cells although cases of only stromal endo-
metriosis have also been reported [24]. In ectopic loci, such as the peritoneum and
ovary, both endometriotic epithelial and stromal cells are functional and respond
actively to steroid hormones during the menstrual cycle. On the other hand,
endometriotic tissue displays a distinct production of cytokines and prostaglandins,
diverse estrogen biosynthesis and metabolic pathways, as well as an altered
response to progesterone, e.g., progesterone resistance [25], that seem to favor
ectopic endometrial growth and maintenance.
In addition to altered steroid content and metabolism, ectopic endometrium also
retains other peculiar characteristics that contribute to disease development.
Although peritoneal, ovarian, and rectovaginal endometriotic lesions are consid-
ered distinct entities with a different pathogenesis [2628], a general consensus
exists regarding the critical role of the cross talk between endometriotic cells, host
peritoneum and infiltrating leukocytes, endothelial cells, and fibroblasts. This all
appears to support the development of the disease [16].
17.1.2 Eutopic Endometrium in Endometriosis:

Villain or Victim?
Although a number of studies have been focused on elucidating the etiology of

endometriosis, a unifying theory regarding its origin has remained elusive. Instead,
several theories have emerged to account for the disparate observations regarding
the pathogenesis, and these can generally be categorized as those suggesting that
implants originate from uterine endometrium and those proposing that implants
arise from tissues or cells other than the uterus. Intrinsic to these theories are
inciting factors (i.e., immune, endocrine, and environmental factors) and genetic
susceptibilities whose roles are beginning to be delineated, although they are still
insufficient to fully explain the cause and development of disease. For instance,
acquired genomic alterations and several physiological changes were shown to
represent a potential source for a conferred proliferation and survival advantage to
endometriotic implants [29].
In this context, the potential involvement of eutopic endometrium seems to be
particularly critical in the pathogenesis of endometriosis, evinced by Sampsons
retrograde menstruation theory in 1921 [30], the most widely accepted hypothesis,
which nonetheless does not fully explain the etiology. In accordance with this
theory, it is presumed that the establishment of endometrial cells from refluxed
menses in ectopic sites is supported by the following characteristics: (a) the endo-
metrial debris, including both epithelial and stromal cells, must exist in the reflux
menses; (b) these two cellular components must be viable and able to evade
immune attack within the pelvic cavity; (c) both epithelial and stromal cells must
have the potential ability to attach and implant into the pelvic mesothelium; and
(d) ultimately, once implanted at an ectopic location, endometriotic cells must be
able to survive and develop a neovascular system [31]. Indeed, the link between
altered eutopic endometrium and the risk for developing endometriosis may possi-
bly justify the morbidity of the disease in only 1015 % of the women with
menstrual reflux [32, 33] and at least partially explain the high rate of recurrence
after medical and/or surgical treatment [34].
In fact, important functional and biochemical differences between eutopic endo-
metrium of women with and without endometriosis have been reported in the
literature [35, 36]. In particular, a large number of differentially expressed gene
transcripts, miRNAs, and proteins have been detected and they appear to mostly
encode molecular signals mediating cross talk between epithelial and stromal cells,
cell proliferation, differentiation and survival, as well as endometrial receptivity
[3743]. For instance, aberrant production of cytokines, growth and angiogenic
factors, different steroid receptor expression and signaling, and abnormal expres-
sion of specific cancer-related genes presented in eutopic endometrial cells are
thought to predispose to growth and survival of endometrial foci outside the uterus
and to produce disease-related symptoms such as reduced fertility and implantation
failures.
The role of the eutopic endometrium as a major and active contributor for the
development of endometriotic lesions has also been supported by the accumulat-
ing evidence implying the involvement of stem cells. Stem cells are rare undiffer-
entiated cells characterized by high proliferative, self-renewal, and differentiation
potential that are present in virtually all adult tissues and organs [44]. The
presence of aberrant stem cells has been associated with the pathogenesis of
several tumors and proliferative disorders in female reproductive organs [45].
As thoroughly addressed in Chap. 4, endometrium-derived stem/progenitor cells
residing in the basalis and/or functionalis have been suggested to reach the
peritoneal cavity via menstrual reflux or lymphovascular dissemination and

establish endometriotic implants [46, 47].
Bearing in mind that the eutopic endometrium of women with endometriosis shows
a wide variety of anomalies both in the tissue architecture and biochemistry and
this tissue being a potential source of endometrial stem cells, it is plausible that the
primary defect in endometriosis may be the eutopic endometrium [4850].
Nevertheless, it is becoming increasingly apparent that interplay between eutopic
endometrium and endometriotic lesions is more complex than abnormal eutopic
endometrium resulting in establishment of endometriotic lesions [51]. In addition
to critical interactions between shed endometrial fragments and peritoneum
(i.e., peritoneal mesothelium and environment) during lesion establishment [16, 52],
it is likely that the biology of eutopic endometrium is altered in response to the
presence of endometriotic lesions as it has currently been demonstrated in in vivo
models of endometriosis [51].
So far, the abnormalities described in endometriotic endometrium and suggested
to predispose to endometriosis were in fact identified when the disease was already
present, thus making it difficult to define whether these endometrial changes are a
cause or an effect of endometriosis. Alternatively, by using mouse and non-primate
models with no previous history of disease, it was possible to evaluate the endo-
metrium before and after induction of endometriosis and to obtain strong evidence
that the presence of endometriotic lesions directly influences the endometrial
environment.
In particular, data obtained from baboon models of experimentally induced
endometriosis indicate that a complex series of changes in endometrial gene
and protein expression occurs at different time points during disease progression
[53, 54], potentially as a result of epigenetic modifications triggered in the presence
of ectopic lesions [55]. Similar to anomalies in eutopic endometrium of women with
endometriosis, collective findings in this model show a local increase in proliferation
and angiogenesis and a significant alteration in steroid hormone receptor distribution
and signaling with an evident progesterone resistance [53, 54, 5660]. Moreover,
it has recently been demonstrated in a mouse model of endometriosis that cells from
endometriotic lesions can migrate to and preferentially populate the endometrial
basal layer, thus further supporting the bidirectional interaction between eutopic
and ectopic endometrial tissue [61].
In women with endometriosis, the effects of ectopic lesions on eutopic endome-
trium remain uncertain, and due to a range of reasons (mainly ethics-related issues),
this relationship is extremely difficult to study. However, the interaction between
lesions and eutopic endometrium is likely to occur and contribute to both endome-
triosis establishment and progression. From animal model data, it is speculated that
the interconnecting roles of the vascular, lymphatic, and nervous systems in both
the uterine and peritoneal environments with concurrent participation of eutopic
endometrium, ovarian steroids, and inflammatory mediators may favor the devel-
opment of endometriosis and its associated symptoms. A hypothesized pathway has
been suggested [51].
17.2 Eutopic and Ectopic Endometria: Similarities

and Dissimilarities
As addressed above, eutopic endometrium of endometriosis patients retains several

abnormalities that in fact resemble the biological characteristics found in ectopic
endometrial tissues such as augmented proliferation, aromatase activity, unusual
cytokine expression, decreased apoptosis, as well as altered local hormone metab-
olism [62, 63].
Although the morphological appearance of endometriotic implants may reflect
that of eutopic endometrium, these similarities are often only limited. Indeed, a
variety of investigations have also revealed important differences between eutopic
and ectopic endometria involving cell structure, gene expression, and responsive-
ness of various proteins [16, 6368].
Morphologically, both endometriotic glandular and stromal cells retain ultra-
structural features that are recognizably different from those of eutopic endome-
trium of patients with endometriosis. In contrast to eutopic tissue, endometriotic
tissue was shown to encompass a wide range of morphological development from
poorly to highly differentiated glands [69]. Interestingly, such variations were
detected not only from gland to gland but also within the same gland. Moreover,
while complete proliferative development has been detected in some endometriosis
patients, full secretory transformation seems to frequently be absent in ectopic
implants [69]. Additionally, the comparative observations of Yu et al. [70] of
eutopic and ectopic endometrial cells cultured in vitro described an enlarged
nucleus and manifold chromatin in endometriotic glandular cells, whereas
endometriotic stromal cells appeared to be smaller and characterized by many
tiny villi and protuberances on the plasma membrane.
Apart from morphological features, the dissimilarities between endometriotic
lesions and eutopic endometrium are even more evident at the molecular level.
With the advent of microarray-based technologies, several investigators were able
to explore the distinct pattern of gene expression in eutopic and ectopic endome-
trium. Large-scale transcriptional profiling analyses using microarrays are widely
employed for analyzing the expression of thousands of genes simultaneously in a
single experiment supported by the availability of the complete nucleotide
sequence of the human genome. Microarray studies provide extremely important
information enabling a better understanding of the etiology and pathophysiology of
a variety of diseases and the development of new diagnostic and therapeutic
strategies [71, 72].
To date, a considerable number of studies comparing the global expression
profiles of paired eutopic endometrium and endometriosis specimens have been
published although the data available in the public domain is still restricted and
occasionally divergent. In fact, it is important to note that some discrepancies may
be a result of the potential impact of one or more factors such as demographic
characteristics, site of endometriosis, fertility history, severity of stages, and phases
of the menstrual cycle that were not fully considered in most studies despite their
well-known influences on gene expression in eutopic and ectopic tissues [7375].
In spite of these limitations, significant and interesting findings have been
provided by microarray-based analyses of eutopic and ectopic endometrium
which have been confirmed by subsequent investigations using other conventional
and sophisticated methodologies. Collectively, these published data highlight dif-
ferences in the expression of gene-encoding proteins involved in multiple biolog-
ical process and hypothesized to be responsible for the establishment of ectopic
endometrial implants, including cell adhesion, extracellular matrix remodeling,
migration, proliferation, apoptosis, immune system regulation, inflammatory
pathways, sex hormone-related signaling and neuroangiogenesis [16, 6468, 75]
that will be thoroughly addressed in the following sections.
Also noteworthy are the observations on the differential expression pattern of
microRNAs (miRNAs) in ectopic versus eutopic endometrium [76, 77]. miRNAs
are small noncoding RNA molecules that have a critical role in posttranscriptional
regulation of gene expression by repression of target mRNA translation. A rela-
tively recent microarray-based investigation in paired eutopic and ectopic endome-
tria has identified more than 80 differentially expressed miRNAs. Interestingly,
most of them are predicted to regulate a large fraction of protein-coding genes
associated with genetic (i.e., cancer) and immunological disorders and involved in
cellular growth and proliferation, apoptosis, cellular movement, cell-mediated
immune response, cell-to-cell signaling and interaction, as well as gene expression
regulation [78].
Evidence for molecular abnormalities in eutopic and ectopic endometria has also
been revealed through proteomic approaches. In particular, large-scale proteomic
studies including mass spectrometry and protein microarray-based technologies
have enabled the simultaneous evaluation of several hundred proteins in eutopic
and ectopic endometrium as well as biological fluids and provided valuable infor-
mation regarding their expression pattern, functions, localization, posttranslational
modifications, and interactions [79]. Investigations focusing on the comparative
proteomic analysis of paired eutopic and ectopic endometrial tissues are still limited
although they are currently gaining more attention. For instance, a recent study
combining two-dimensional electrophoresis and mass spectrometry demonstrated a
remarkable difference in the protein repertoire of endometriotic lesions compared
with its uterine counterpart [80]. Interestingly, significant differences comprised
proteins primarily involved in cellular spreading and attachment, cell homeostasis
and survival, mRNA processing and transport, immune response, and protein
trafficking [80].
In addition to differences between eutopic and ectopic endometrium, increasing
evidence in the literature has also suggested that the biological characteristics of
endometriotic cells might differ between different forms of endometriosis [28, 81].
These findings may possibly account for the high heterogeneity observed in endo-
metriosis pathogenesis and disease-related symptoms and imply that a single
universal treatment is perhaps not effective for all forms of endometriosis.
17.3 Endometrial Cell Proliferation, Survival,

and Invasion
Endometriosis could result from increased cellular proliferation or decreased

apoptosis in response to appropriate stimuli. Eutopic endometrium from women
with endometriosis has several differences compared with normal endometrium of
women without endometriosis. These differences may contribute to the survival of
retrograded endometrial cells into the peritoneal cavity and thus to the development
of endometriosis.
Nearly all women of reproductive age exhibit some degree of endometrial debris
reflux [82]. Menstrual effluent retrogradely shed into the peritoneal cavity was
observed to contain viable endometrial cells [8387]. Summarizing previous
knowledge of the disease, Vinatier et al. [88] presented two theories to explain
why only some patients develop the disease. The first theory is based on disorders of
the ectopic endometrial tissue whereby it resists normal peritoneal cleaning. The
second suggests that the disease is secondary to abnormalities of cellular and
humoral immunity, which induces excessive receptivity of the peritoneal mesothe-
lium, hyperactivated macrophages, and NK cell abnormalities. Mediators in the
peritoneal environment may alter a genetically predisposed endometrium which
then exhibits increased invasion. It is also possible that an excess of refluxed
endometrium or altered endometrium has the potential to contribute to the devel-
opment of a proinflammatory environment favorable to disease establishment [88].
17.3.1 Apoptosis in Endometriosis
Accumulating evidence suggests that endometrial cells from women with and
without endometriosis exhibit fundamental differences in their apoptotic capacity.
Endometrial cells from women with endometriosis have enhanced proliferation and
an increased ability to implant and survive in ectopic locations. Impaired sensitivity
of endometrial tissue to spontaneous apoptosis contributes to the abnormal implan-
tation and growth of endometrium at ectopic sites.
The inability of endometrial cells to transmit a death signal or their ability to
avoid cell death is associated with increased expression of anti-apoptotic factors
(Bcl-2) and decreased expression of pre-apoptotic factors (BAX) [89]. However, it
remains unclear whether abnormal apoptosis in the eutopic endometrium from
patients with endometriosis is a primary or a secondary process after establishment
of the pelvic endometriosis process. This could be attributed to the fact that at the
time of clinical presentation and diagnosis, most women already have established
disease and, therefore, it is difficult to investigate the early developmental stages of
endometriosis.
Reflux of endometrial fragments during menstruation into the peritoneal cavity
is a common phenomenon. Under normal conditions, cells which do not adhere to
their extracellular matrix enter apoptosis as they receive different signals from their
adhesion receptors [90]. However, in women with endometriosis, these cells have
the ability to adhere to mesothelial cells of the peritoneum, proliferate, and induce
neoangiogenesis resulting in the development of active endometriotic implants. The
effect of MMPs on apoptotic factors and their regulation by steroid hormones may
provide a link between endometrial turnover and the invasive process necessary for
the development of disease.
Recent studies from Gonzalez-Ramos et al. [91, 92] showed constitutive NF-B
activation in peritoneal endometriosis. They reported that the NF-B pathway is
implicated in the development of endometriotic lesions in vivo and that NF-B
inhibition reduces intracellular adhesion molecule-1 expression and cell prolifera-
tion, but increases apoptosis of endometriotic lesions, diminishing the initial devel-
opment of endometriosis in an animal model. This and other observations support
the notion that endometriosis is characterized by persistent inflammation and
proliferation.
Murk et al. [93] described extracellular signal-regulated kinase (ERK1/2) activ-
ity in human endometrium. ERK1/2 plays key intracellular roles in activating
cellular survival and differentiation processes. The authors found that ERK1/2-
mediated steroid inhibition in endometrial stromal cells reduced proliferation and
increased apoptosis. They suggested that abnormally high levels of ERK1/2 activity
may be involved in endometriosis, possibly by stimulating endometrial cell
survival.
Some studies have suggested that genetic factors are likely to influence
individual susceptibility to endometriosis. Genetic alterations in somatic chromo-
somes [94] and DNA deletions that inactivate some tumor suppressor genes
(e.g. PTEN) are likely to be involved in the initiation, persistence, and progression
of endometriosis [95, 96].
cDNA microarray analysis provided an interesting insight into altered gene
expression profiles in endometriosis patients. Using this method, Arimoto
et al. [37] found 97 upregulated and 337 downregulated genes in women with
endometriosis. Genes related to apoptosis (GADD34, GADD45A, GADD45B,
PIG11) and the tumor suppressor TP53 gene were downregulated in endometriotic
tissues. On the other hand, Eyster et al. [66] found that only a few genes in apoptosis
resistance pathways were differentially expressed in endometriosis cells compared
to normal endometrial cells. They suggested that the presence of ligands to activate
the apoptotic pathway or the apoptosis resistance pathways may be more important
than whether specific members of the pathway are over- or underexpressed.
Braun et al. [97] demonstrated that the transcript abundance ratio of anti-
apoptotic to pro-apoptotic isoforms of the Bcl-X gene favors survival in eutopic
and ectopic endometrial cells from women with endometriosis, but not control
endometrial cells. This was found throughout the menstrual cycle for ectopic
endometrial cells. Eutopic but not ectopic endometrial cells also expressed
increased transcript abundance of the anti-apoptotic DAD-1 gene in endometriosis.
Eutopic and ectopic endometrial cells from women with endometriosis expressed
decreased transcript abundance of p53 and caspase-1 compared to endometrial cells
from women without endometriosis. Dysregulation of pro- and anti-apoptotic

regulatory gene expression characterized eutopic and ectopic endometrial cells
from women with endometriosis, consistent with apoptotic resistance and enhanced
survival of endometrial cells in endometriosis. This data should provide useful
information for finding candidate genes whose products might regulate the apopto-
tic machinery in endometriosis and, additionally, could serve as molecular targets
for diagnosis or treatment of endometriosis.
Othman et al. [98] reported that mutants of estrogen receptor genes delivered to
endometriotic cells via an adenovirus vector (Ad-DNER) decreased cell prolifera-
tion, induced apoptosis, and decreased cytokine production. The authors suggested
that adenovirus-mediated gene therapy may represent a potential therapeutic option
for endometriosis in the future.
17.3.2 Invasion in Endometriosis
Endometriosis is a benign and frequently progressive disease with a high preva-

lence in the female population of reproductive age. Nevertheless, endometriosis
shows behavior similar to malignant tumors. Deep invasion in different tissues such
as the peritoneum, ovary, and intestines is presumably occurs as a result of
migrating epithelial and stromal cells of modified endometrium adhering to distant
tissues [99]. Endometriotic cells are capable of attaching to the peritoneal
mesothelium, breaking the peritoneal lining, and destroying ECM, thereby invading
surrounding tissue.
Endometriosis is associated with an increased risk for several types of malignant
diseases [100, 101]. Especially the endometrioid subtype of ovarian cancer has a
high probability for developing on the basis of existing endometriosis [102105].
Like malignancy, endometriosis displays features of atypia, adherence, invasion,
and metastases. The risk of direct malignant transformation of ovarian endometri-
osis has been estimated as 0.71.6 % over an average of 8 years.
Among several investigated potential invasion markers, two proteins, YB-1 and
L1CAM, seem to have very important roles in the progression of endometriosis.
17.3.2.1 Cold Shock Domain Family Member YB-1 Expression

in Endometrium and Endometriosis
The Y-box binding protein YB-1, an evolutionarily conserved 48 kDa protein,

belongs to the superfamily of cold shock domain proteins with pleiotropic biolog-
ical functions. Eukaryotic YB proteins are involved in the regulation of DNA
transcription and repair, in translational control of protein synthesis, as well as in
cellular responses to a wide variety of stressors [106108].
The YB-1 protein is highly expressed in a number of malignant diseases, and it

seems to promote tumor growth and multidrug resistance [109] through the induc-
tion of specific growth factors [110113].
Based on multiple biological functions of YB-1 and its close association with
tumorigenesis, YB-1 expression in human endometrium, ovarian endometriosis,
and peritoneal macrophages was investigated [114]. YB-1 gene and protein expres-
sion was significantly higher in ovarian lesions, eutopic endometrium, and perito-
neal macrophages of patients with endometriosis in comparison to the control
group. Interestingly, the strongest YB-1 expression was observed in the epithelial
compartment of endometrial tissues. In the 12Z endometriotic epithelial cell
line, YB-1 knockdown resulted in significant cell growth inhibitory effects
including reduced cell proliferation and increased spontaneous and TNF-induced
apoptosis rate. Significantly higher RANTES (regulated upon activation, normal
T cell expressed and secreted chemokine) expression and decreased cell
invasion in vitro were also associated with YB-1 inactivation. Elevated YB-1
expression seems to have an impact on the development and progression of
17.3.2.2 L1 Cell Adhesion Molecule (L1CAM) as a Pathogenic Factor

in Endometriosis
L1 cell adhesion molecule (L1CAM, CD171) is a type I transmembrane glycopro-

tein (200220 kDa) which belongs to the immunoglobulin superfamily [115].
Initial studies showed that L1CAM plays an important role in the development of
the nervous system [115117] and have implicated L1CAM also in the ontogeny
of human tumors including melanomas, neural tumors, renal carcinomas, colon
carcinoma, and endometrial and ovarian carcinomas [118121]. The expression of
L1CAM in carcinomas augments dissemination of tumor cells by enabling cell
migration and invasion [122124].
Given the role of L1 in endometrial and ovarian carcinomas, L1CAM was also
investigated as a pathogenetic factor in endometriosis [125].
L1CAM was found present in endometriotic tissue of women with endometri-
osis and increased at gene and protein levels using short-term cultures of endo-
metriosis lesions. A significantly higher expression of L1CAM Significantly
higher L1CAM expression in the epithelial cell fraction in rAFS stages III and
IV (AFS 1997) of endometriosis was found. The nerve growth-promoting activity
of the conditioned medium of endometrial epithelial cell cultures from women
with endometriosis was studied using a chicken ganglion assay. The conditioned
medium of endometrial epithelial cell cultures from patients with endometriosis
stimulated strong neurite growth which was blocked by co-incubation with anti-
L1-mAb in a dose-dependent fashion [125].
17.4 Estrogen-Related Signaling and Progesterone

Resistance in Endometrium and Endometriotic
Lesions
It is well known that endometriosis is a hormone-responsive disease and that

disease progression is inhibited by an antiestrogenic environment. In postmeno-
pausal women undergoing in vitro fertilization with donor eggs, the exogenous
administration of only estradiol (E2) and progesterone is sufficient to prepare the
endometrium for implantation in the absence of ovarian function. This observation
underscores the essential roles of these steroids in uterine physiology. Indeed, both
E2 and progesterone are master regulators of endometrial tissue [25]. Each hor-
mone is estimated to regulate expression of hundreds of genes during various
phases of the menstrual cycle [126]. Endometriotic and eutopic endometrial tissues
respond to E2 and progesterone with apparently similar histological changes, and
both tissues contain immunoreactive ERs and PRs. The eutopic endometrium
predictably becomes atrophic in response to prolonged progestin therapy or oral
contraceptives that contain progestins. Treatment with these agents, however, does
not predictably suppress endometriotic tissue growth. Endometriotic tissue in
ectopic locations, such as the peritoneum or ovary, is fundamentally different
from eutopic endometrium within the uterus in terms of production of cytokine
and prostaglandin production, estrogen biosynthesis and metabolism, and clinical
response to progestins [127129].
17.4.1 Estrogen Action in the Endometrium

and Endometriosis
Both circumstantial and laboratory evidence strongly supports the notion that
estrogen is an extremely strong mitogen for endometriotic tissue. The biologically
active estrogen 17 beta estradiol (E2), which is secreted by the ovary or locally
produced by in endometriotic tissue, acts as a classical steroid hormone to regulate
growth of endometrial or endometriotic tissue. E2 enters cells and binds to the ER
in estrogen-responsive cells. ER subtypes and are proteins with high affinity for
E2 and are encoded by separate genes. The classical human ER was cloned in
1986, and a second estrogen receptor, ER, was cloned from rat prostate and human
testis in 1996 [130132]. Although both ER and ER are present in the endome-
trium, ER seems to be the primary mediator of estrogenic action in this tissue
[133]. Studies using ER-deficient mice have revealed the central role of this
receptor in reproductive function, at all levels of the hypothalamicpituitary
gonadal axis [134]. Despite its sensitivity to estrogen, endometriosis appears to
have an altered steroid hormone receptor profile compared with that of its normal
tissue counterpart, the eutopic endometrium. For example, several investigators
reported markedly higher levels of ER and lower levels of ER in human
endometriotic tissues and primary stromal cells compared with eutopic endometrial
tissues and cells [56, 135, 136]. Moreover, the levels of both isoforms of PR,
particularly PR-B, are significantly lower in endometriosis compared with eutopic
endometrium [137, 138].
The E2-receptor complex acts as a transcription factor that becomes associated
with the promoters of E2-responsive genes via direct DNA binding or binding to
other docking transcription factors at basal promoter regions [139]. This interaction
brings about ER-specific initiation of gene transcription, which promotes the
synthesis of specific mRNAs and proteins [139]. PR is one of many
E2-responsive genes, and E2 acts in eutopic endometrial tissues and stromal cells
to promote endometrial responsiveness to progesterone [140]. In contrast, PR
mRNA and protein levels are not elevated in biopsied endometriotic tissues
exposed to high E2 levels during late proliferative phase or in endometriotic cells
treated with E2, indicating that E2 induction of PR expression in endometriosis is
markedly blunted [138].
17.4.2 Progesterone Resistance in Eutopic Endometrium

and Endometriotic Lesions
E2 is the best-defined mitogen for growth and inflammation processes in ectopic

endometriotic tissue that commonly resides on pelvic organs. Progesterone and
progestins may relieve pain by limiting growth and inflammation in endometriosis,
but some patients with pelvic pain do not respond to treatment with progestins.
Moreover, progesterone-induced molecular changes in eutopic endometrial tissue
of women with endometriosis are either blunted or undetectable. These in vivo
observations are indicative of resistance to progesterone action in endometriosis.
The molecular basis of progesterone resistance in endometriosis may be related to
an overall reduction in levels of progesterone receptors (PRs) and the lack of the PR
isoform progesterone receptor B (PR-B). In normal endometrium, progesterone acts
on stromal cells to induce secretion of paracrine factor(s). These unknown factor(s)
act on neighboring epithelial cells to induce the expression of the enzyme
17-hydroxysteroid dehydrogenase type 2 (17-HSD-2), which metabolizes E2 to
the less active estrone (E1). In endometriotic tissue, progesterone does not induce
epithelial 17-HSD-2 expression due to a stromal cell defect. The inability of
endometriotic stromal cells to produce progesterone-induced paracrine factors that
stimulate 17-HSD-2 may be due to the lack of PR-B and very low levels of
progesterone receptor A (PR-A) observed in vivo in endometriotic tissue. The end
result is deficient E2 metabolism in endometriosis giving rise to high local concen-
trations of this mitogen [137].
Other mechanisms proposed to explain progesterone resistance are altered
expression or function of chaperone proteins like FKBP52 [141] and
co-regulators such as HIC-5/ARA55 [142].
PR is a target gene of ER in several cell types including breast cancer epithelial

cells. ER mediates E2-induced PR expression. It has been reported that two
distinct E2-regulated promoters generate transcripts encoding two functionally
different human PR isoforms, PR-A and PR-B [143]. Previous studies have dem-
onstrated that maximal PR mRNA and protein levels are reached after human breast
cancer cells have been exposed to E2 for 3 days [144146]. Two major transcrip-
tional start sites have been identified. The upstream transcription start site gives rise
to a full-length mRNA species that encodes the PR-B protein. Another mRNA
species with a further downstream transcription initiation site gives rise to the
truncated PR-A form. Despite the fact that both proposed PR promoter sequences
are E2 responsive, neither contains a classical palindromic ERE sequence [147].
Several nonclassic regulatory elements (e.g., AP1, Sp1) in the human PR promoter
have been reported. These sites have been shown to bind ER and on one occasion
ER [140, 147155]. Bulun et al. speculate that a critical level of ER may be
necessary for E2-dependent induction of PR in endometrial stromal cells.
The occupancy of the PR promoter regions with varying ratios of ER to ER
may be critical in determining the effect of E2 on PR expression. A strikingly
lower ER-to-ER ratio in endometriotic stromal cells may be responsible for a
shift from E2 stimulation to E2 inhibition of PR expression in endometriotic
stromal cells [25].
17.5 The Inflammatory Response in Eutopic

and Ectopic Endometria
The immune status has been suggested to play an important role in both initiation and
progression of endometriosis. T and B lymphocytes and natural killer cells seem to
play essential roles in determining if endometrial and endometriotic cells accept or
reject survival, implantation, and proliferation [156, 157]. Several studies have shown
a reduced activity of cytotoxic T cells and NK cells, cytokine secretion by helper T
cells, and autoantibody production by B lymphocytes in women with endometriosis
[156158]. As alluded to in a previous section, NF-B transcriptional activity modu-
lates inflammatory key cell processes contributing to the initiation and progression of
endometriosis [159]. It has further been shown that immuneendocrine interactions are
likely to be involved in the pathogenesis of endometriosis.
Endometriosis is associated with an increased inflammatory activity, as seen
by elevated serum levels of inflammatory markers such as CA-125 [160] and
C-reactive protein [161]. Changes in peritoneal fluid inflammatory markers of
peritoneal fluid have also been observed in women with endometriosis [162]. The
generalized inflammatory activity may lead to more generalized clinical effects
where some women with endometriosis suffer from fever and a general feeling of
sickness, especially AT times when they experience more pain.
17.6 Neurogenesis in Endometrial and Endometriotic

Tissue
Recent evidence indicates that ectopic endometriotic implants recruit their own
unique neural and vascular supplies through neuroangiogenesis. It is believed that
these nascent nerve fibers in endometriotic implants influence dorsal root neurons
within the central nervous system, increasing pain perception in patients [163].
Several studies have suggested that endometrial biopsies with the detection of
nerve fibers provide a reliability of diagnosing endometriosis [164, 165]. Newer
studies however were not able to demonstrate any differences in the amount of
nerve fibers or neuronal markers in endometrium of women with endometriosis
compared to women without endometriosis. The neuronal markers, PGP9.5,
NGFp75, and VR1, are expressed in the endometrium at levels that do not differ
between women with and without endometriosis [166].
Endometrial functional layer nerve fibers were identified in 22 % of biopsies
overall including 19 % of cases with histologically confirmed peritoneal endome-
triosis and 29 % of cases without endometriosis. There was no correlation between
the presence of functional layer nerve fibers and the presenting symptoms, endo-
metrial histology, or current hormonal therapy. Endometrial functional layer nerve
fibers assessment performed using standard immunohistochemical techniques on
routine biopsy specimens proved neither sensitive nor specific for the diagnosis of
endometriosis [167]. In conclusion, much rigorous research to better understanding
this complex pathology and to find specific and sensitive biomarkers remains
to be done.
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Part III
Clinical Science
Chapter 18
Prevention of Endometriosis
Ebru H. Biberoglu and Kutay O. Biberoglu
Abstract There is an association between the presence of endometriosis and

common autoimmune and atopic diseases and some of the cancers. There are also
concerns about the risk for birth defects in the children of women with endometri-
osis. Prevention of endometriosis may also reduce the risk for these other health
problems and their sequelae. To be able to prevent or delay the development of
endometriosis and hopefully other associated comorbidities, risk factors for endo-
metriosis should be defined. Exposure to environmental chemicals recently has
been proposed to contribute to several gynecologic pathologies including endome-
triosis, especially when exposures occur during critical periods of development.
Although potential role in the pathogenesis of endometriosis has not been
established, exposure to certain endocrine-disrupting chemicals is shown to be
higher in women affected by endometriosis compared to women without the
disease. Although there is extensive scientific and clinical data applicable to
endometriosis when it is regarded as a systemic inflammatory, endocrine, and
immunological disease, the prevention of endometriosis per se, have not been
addressed fully in the medical literature.
Keywords Chemicals Endometriosis Environment Life style Nutrition

Prevention
E.H. Biberoglu (*)

Dr. Zekai Tahir Burak Women Health Care, Education and Research Hospital Ankara, Turkey
e-mail: ebru2513@gmail.com
K.O. Biberoglu
Gazi University Medical School, Professor of Obstetrics and Gynecology, Gynecological
Endocrinology and Infertility Unit Besevler, Ankara, Turkey
e-mail: kobiber@gmail.com

278 E.H. Biberoglu and K.O. Biberoglu
18.1 Risk Factors
Although science has not yet addressed directly, there are extensive scientific and
clinical resources applicable to the prevention of endometriosis, especially when
regarded as the systemic inflammatory, autoimmune and endocrine disease. Fur-
ther, dioxin and endocrine-disrupting environmental toxicants that modify the
inflammatory process have been strongly associated with endometriosis [1].
Endometriosis shares pathophysiological characteristics such as immune
response alterations, increased inflammation, elevated levels of tissue-remodeling
components, altered apoptosis, increased local and/or systemic levels of cytokines.
Growth factors including fibroblast growth factor (FGF), vascular endothelial
growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth
factor (EGF), transforming growth factor-h (IGF-h) and granulocyte/macrophage-
colony-stimulating factor (GM-CSF) and inflammatory mediators like IL-1, TNF-a,
IL-2, IL-6, IL-8, IL-10, IL-11, MCP-1, and interferon-g (IFN-g) produced by
peritoneal leukocytes are all elevated in the peritoneal fluid of women with
endometriosis.
There is an association between the presence of endometriosis and common
autoimmune and atopic diseases, such as systemic lupus erythematosus, Sjogrens
syndrome, rheumatoid arthritis, Crohns disease, psoriasis. chronic fatigue immune
dysfunction syndrome, multiple sclerosis, hypothyroidism and fibromyalgia.
Research has suggested that endometriosis may increase the risk of ovarian and
breast cancer, non Hodgkins lymphoma, melanoma, thyroid, kidney and brain
tumors. There are also concerns about the risk for birth defects in the children of
women with endometriosis. Therefore, prevention of the disease processes involv-
ing endometriosis may also reduce the risk for these other health problems and their
sequelae [13].
To be able to prevent or delay the development of endometriosis and hopefully
other associated comorbidities, known risk factors for endometriosis should be
defined at the first place. The plethora of risk factors for endometriosis may reflect
varying methodologies such as study populations, definitions utilized for risk
factors, and diagnostic accuracy. Infertility by itself is a significant risk factor for
endometriosis. An infertility history increases the odds of an endometriosis diag-
nosis in both the operative (OR, 2.43; 95 % CI, 1.573.76) and population (OR,
7.91; 95 % CI, 1.6937.2) cohorts [4]. Increasing age, alcohol use, early menarche,
family history of endometriosis, infertility, intercourse during menses, low body
weight, prolonged menstrual flow, and short cycle interval are known risk factors
[58]. Endometriosis has been negatively associated with exercise and smoking
[9]. Recently, red hair [10], blue or green eyes, and freckles have been reported to
increase the odds of diagnosis [11]. It is possible that there may not be a classic set
of risk factors generic to all women with endometriosis. Rather, risk factors may
need tailoring to the subgroups of women by their behavioral and clinical
characteristics.
18 Prevention of Endometriosis 279
18.1.1 Family History
First degree relatives of a woman with endometriosis carry 410 times higher
risk of also having endometriosis when compared to the general population.
Candidate genes such as ESR1, COMT, IL6, IL10, CYP17A1, CYP19A1,
CYP1A1, MMP1, and MMP9 studied in genomic DNA showed no association
with endometriosis [12].
In more than 1,000 families with two or more members with surgically documented
endometriosis from Australia and the UK, significant linkage to 10q26 and 20p13
was demonstrated. However, no causative gene was identified [13]. It is likely that
endometriosis is a common polygenic/multifactorial disease caused by an interaction
between genes as well as the environment [14, 15].
18.1.2 Menstrual Cycle Characteristics
Early age at menarche (11 years old) might increase a womans exposure to
menstruation during her reproductive lifetime and consequently increase the risk of
endometriosis. The data, however, do not present strong evidence for the clinical
utility of a history of early menarche in the evaluation of endometriosis [16]. The
lowest risk was seen in those whose age at menarche was 15 years [17].
Increased exposure to menstruation in terms of short cycle length, long duration
of flow, and low parity have frequently been identified as possible risk factors. The
use of tampons does not seem to confer a risk for endometriosis.
Dysmenorrhea is likely to be a precursor to disease development, and shorter
cycles may possibly suggest increased risk [18, 19].
In contrast to past studies, data of a recent study found no relationship between
endometriosis and menstrual cycle history, including age at menarche, average
cycle length, and number of menstrual cycles in the past 12 months. However,
>80 % of the women in all groups in this study had a history of oral contraceptive
use. This contraceptive use may have altered both recent menstrual cycle patterns or
possibly the presence or absence of endometriosis [4].
Therefore, the potential role of menstrual cycle characteristics in the actual
development of endometriosis remains an open question. At best they may be
used to guide diagnostic and therapeutic strategies if other symptoms point to
endometriosis as a possible diagnosis.
18.1.3 Lean Body Mass
Several studies have found that a lower body mass index (BMI) during adolescence
and early adulthood is a risk factor for endometriosis. Taller women tend to have
higher follicular-phase estradiol levels and thus may have an increased risk
of endometriosis. This evidence was supported by a retrospective study that found

that women with endometriosis have lower BMI and are less frequently obese than
control subjects [20]. Indeed, for every unit increase in BMI, 1214 % decrease in
the likelihood of having endometriosis was claimed [21]. In a recent study, BMI
(OR, 0.95; 95 % CI, 0.930.98) was found to decrease the odds of diagnosis of
endometriosis [4]. The 20 years follow-up within the Nurses Health Study II
prospective cohort revealed that BMI at age 18 and current BMI were each
significantly inversely associated with endometriosis (P < 0.0001). Both associa-
tions were stronger among infertile women. Obese infertile women with current
BMIs of 3539.9 kg/m2 and 40 kg/m2 had a 55 % (95 % CI 0.300.67) and a
62 % (95 % CI 0.230.62) lower risk of endometriosis, respectively, compared
with the low-normal BMI referent (18.522.4 kg/m2). Rates of endometriosis
were nearly threefold higher in women with waist-to-hip ratios,0.60 (RR 2.78,
95 % CI 1.385.60) compared with those with waist-to-hip ratios between 0.70
and 0.79 [22].
18.1.4 DES Exposure
The incidence rates of diagnosis of laparoscopically confirmed endometriosis was

found to be 80 % greater among women exposed to diethylstilbestrol (DES)
(RR 1.8, CI 1.22.8) [23]. Exposure to DES in utero has been associated with
cervical stenosis, uterine smooth muscle abnormalities, and altered estrogen recep-
tor expression in both mice and women [24]. In addition, it was reported that
exposed women with vaginal epithelial changes had 50 % more autoimmune
disease than exposed women without vaginal epithelial changes [25]. Therefore
the relation between DES exposure and endometriosis may result from a combined
effect of increased retrograde menstruation, immune dysfunction, and exogenous
estrogen exposure.
18.1.5 Environmental Exposures
Endocrine-disrupting chemicals (EDC) directly through induction of gene expres-

sion or indirectly impair female reproduction by interfering with the production,
release, transportation, metabolism, action, or elimination of natural hormones.
Also the neuroendocrine (monitoring the environment and sending signals to the
endocrine system) and the epigenetic (altering transcriptional capabilities without
changing DNA sequence) routes could have been involved in the pathogenesis. In
epigenetic disruption, the chemicals modify histones altering the DNAnuclear
protein interactions or promote DNA methylation. Most importantly, the resultant
chromatin modifications can be passed on to future generations and increase the
likelihood of a disease state later in life across several generations [26, 27].
Although potential role in the pathogenesis of endometriosis has not been

established, exposure to certain EDCs is shown to be higher in women affected
by endometriosis compared to women without the disease [2831].
A recent study demonstrated that infertile patients affected by endometriosis had
higher percentage of serum sample with bisphenol A (BPA), perfluorooctane
sulfonate (PFOS), perfluorooctanoic acid (PFOA), di-(2-ethylhexyl) phthalate
(DEHP) and mono-ethylhexyl phthalate (MEHP) levels compared with infertile
patients without endometriosis. The study group of infertile patients had also a
significantly higher expression of several nuclear receptors that represent potential
EDC targets, namely, estrogen receptor alpha (ERa) and beta (Erb), androgen
receptor (AR), pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR), and
peroxisome proliferator-activated receptor gamma (PPARg) [32].
While some evidence from laboratory animal studies suggests that endometri-
osis can be promoted by many organochlorines, a class of xenobiotic chemicals
including the dioxin TCDD, the pesticides methoxychlor and dichlorodiphenyltri-
chloroethane (DDT), and many polychlorinated biphenyls (PCBs) with dioxin-like
effects, some others fail to find any significant relationship between the two. The
hypothesis that EDC exposure during embryogenesis increases susceptibility for
endometriosis, but subsequent adult hormone, immune, and/or EDC irregularities
are required for disease onset was supported by the finding of larger implanted
endometriotic lesions when exposure of the fetus to the dioxin 2,3,7,8-tetrachlor-
odibenzo-p-dioxin (TCDD) on gestational day 8 was combined with the adult
exposure in the mouse model [33, 34].
It is possible that fetal exposure to TCDD promotes adult endometriosis through
altered P action, because PR expression is reduced in the uterus of adult mice that
were exposed to TCDD in utero [35]. It is also possible that TCDD which is an
immunosuppressant promotes endometriosis through altered immune function
enabling establishment and growth of peritoneal endometriosis under the influence
of E2, proangiogenic, proliferative, and antiapoptotic factors [36]. Alternatively,
TCDD could activate specific signaling pathways causing overexpression of K-ras
thereby promoting peritoneal endometriosis [37].
Endometriotic lesions have increased expression of aromatase and 17-HSD
type 1 and decreased expression of 17-HSD types 2 and 4, resulting in an increase
in production of E2 [38]. If this expression pattern is established during fetal
development via epigenetic mechanisms, then endometriosis could manifest during
adulthood after estrogenic exposures [39].
Studies have found increased risk of endometriosis-associated infertility among
workers exposed to formaldehyde, video display terminals, chemical dusts, or
organic solvents, and among workers in agricultural industries and occupations,
in particular farmworkers. Having ever worked as a flight attendant, service station
attendant, or health worker, particularly as a nurse or health aide, is associated with
increased risk of endometriosis (flight attendant: OR 9.80, 95 % CI 1.0889.02;
service station attendant: OR 5.77, 95 % CI 1.0332.43; health worker: OR 1.49,
95 % CI 1.032.15) [40].
18.1.6 Cigarette Smoking, Alcohol, and Caffeine
Active cigarette smoking in adulthood or adolescence, with the known

antiestrogenic effect of inhaled tobacco smoke, has generally been associated
with decreased endometriosis risk in previous research [9]. Whereas, a deleterious
effect on endometriosis risk through exposure to polycyclic aromatic hydrocarbons
[41] or dioxins [42] in tobacco smoke may dominate. A negative effect of passive
smoking during childhood on endometriosis risk has also been suggested but further
research is needed to confirm this relationship [43]. In a recent study where
smoking habits in endometriosis patients are studied, no correlation between
smoking habits and the risk of any form of endometriosis (superficial peritoneal
endometriosis, ovarian endometriomas, and deep infiltrating endometriosis) and
with the revised American Fertility Society stages or scores have been demon-
strated [44]. Alcohol and caffeine consumption have also been shown to increase
the risk of endometriosis. It is known that alcohol increases estrogen levels in the
body and may disrupt the immune response during the menstrual cycle. The effect
of caffeine is likely related to its influence on the immune system as well [45].
18.1.7 History of Allergic, Inflammatory,

and Autoimmune Diseases
Alterations in inflammatory response and in both cellular/humoral immunity ends

up with overproduction of prostaglandins, metalloproteinases, cytokines, and
chemokines, thus favoring an optimal environment for the survival and prolifera-
tion of endometriotic implants [46]. This might explain why women with endome-
triosis report symptoms of not only pain but also of nervousness, tension, anxiety,
headaches, depression, fatigue, insomnia, indigestion, bloating, recurrent vaginitis,
recurrent cystitis, autoimmune diseases, asthma, and allergies [47, 48].
Women with endometriosis have a higher prevalence of allergies on medica-
tions, allergic rhinitis, asthma, and family history of allergic diseases compared to
control subjects. In epidemiological studies, 48 % of women with endometriosis
were reported to be allergic to at least one medication. Of these, 85 % had
complaints of sinus and 14 % had suffered from asthma. Additionally, 80 % had
a parent with allergic disease [49]. In another study, 61 % of the women with
endometriosis reported allergies (compared with 18 % of the general female
population), and 12 % had asthma (compared with 5 % of the general female
population) [50]. In a recent study, a 4.6-fold increased frequency of allergic
women with endometriosis compared with the control group was reported and the
most prevalent allergen was found to be penicillin in this group [51].
Evidence available to date indicates that immune and inflammatory factors,
whether they are released by immune or peritoneal, endometrial, and endometriotic
cells, may play a critical role in the ectopic survival, implantation, and growth of
endometrial tissue. Higher incidence of autoimmune diseases, abnormalities in

T- and B-cell function, increased polyclonal B cell activity, high B-cell and
T-cell counts, reduced natural killer (NK) cell activity, and the familial inheritance
pattern and its recurring nature support an autoimmune aspect of endometriosis
[47]. A recent genome-wide study suggests that endometriosis exhibits a gene
expression signature in terms of increased presence and activation of plasma cells
and macrophages and upregulation of complement system [52]. A macrophage
product B lymphocyte stimulator (BLyS) which is a member of TNF superfamily is
found to be elevated in the serum of women with in rheumatoid arthritis, systemic
lupus erythematosus, and Sjogrens syndrome [52] and with endometriosis in
association with BLyS-817CT polymorphism [53].
Recent studies support the contributing role of inflammation in endometriosis-
related pain. The proinflammatory peptides facilitate endometriotic cell survival by
stimulating cell proliferation and inhibiting apoptosis of endometriotic cells. The
main cell processes that NF-B regulates, contributing to endometriosis develop-
ment, are inflammation, cell proliferation, and inhibition of apoptosis. Iron overload
in the pelvic cavity of endometriosis patients is very probably an important facil-
itator or inductor of chronic NF-B activation, enhancing the NF-B-mediated
inflammatory reaction and endometriotic cell survival and growth [54].
From the clinical perspective, patients with endometriosis have been shown to
have a higher prevalence of several generalized autoimmune diseases, including
systemic lupus, erythematosus, rheumatoid arthritis, and Sjogrens syndrome, and
also of irritable bowel syndrome, painful bladder syndrome, migraine head ache,
and fibromyalgia [5557].
18.1.8 Fibromyalgia, Chronic Fatigue Syndrome, Irritable

Bowel Syndrome, Painful Bladder Syndrome,
and Migraine
In recent years, epidemiologic studies have identified an association between

endometriosis and some other pain syndromes (such as fibromyalgia, chronic
fatigue syndrome, interstitial cystitis, and irritable bowel syndrome) as well as
various autoimmune and atopic conditions as already been discussed [50]. A recent
study described a high prevalence of comorbid chronic pain syndromes (56 %) and
mood disorders (48 %) in adolescents and young women with endometriosis [58].
Nevertheless, irritable bowel syndrome, painful bladder syndrome, and chronic head-
ache were detected in 25 % versus 65 % [59]; in 16 % versus 65 % [60] and 19 %
versus 38 % [61] in adolescent/young women compared to adult endometriosis
patients, respectively. Fibromyalgia and chronic fatigue syndrome prevalences were
found in 7 % and 4 % of young girls versus 6 % and 5 % of adult endometriosis
women, respectively [50]. Women with endometriosis have a 30 % increased
risk of migraines. There is also an increased prevalence of endometriosis in women
with migraine. The subgroup of migraineurs with endometriosis is more likely to have
other comorbid conditions affecting mood and pain [56]. Angiogenic cytokines are
hypothesized to play a critical role in the pathogenesis of endometriosis and migraine,
possibly by stimulating matrix metalloproteinases (MMPs) [62]. There may also be a
neuro-immuno-endocrine link between endometriosis and migraine, fibromyalgia,
irritable bowel syndrome, chronic fatigue syndrome, interstitial cystitis (painful
bladder), and mood disorder through increased mast cell activation [63]. Mast cell
activation without allergic degranulation has been documented to occur in response to
stress and lead to painful sterile inflammatory states [64]. An increased prevalence of
hypothyroidism, fibromyalgia and chronic fatigue syndrome, and autoimmune inflam-
matory diseases in women with endometriosis compared with the general female
population was previously reported. The coexistence of all these conditions suggests
an underlying role for the immune system in fibromyalgia and chronic fatigue
syndrome [50].
18.1.9 Anatomical Obstruction of the Uterus and Surgical

Scar Endometriosis
Sampsons theory of retrograde menstruation and implantation is supported by

evidence that obstructive Mullerian anomalies that enhance retrograde menstrua-
tion such as a narrowed or completely blocked cervix, a malformed or absent
cervix, absence of the vagina, or a completely blocking hymen have been associ-
ated with endometriosis in adolescent women, and repair of these anomalies has
been associated with resolution of endometriosis [6568].
Endometriosis has been reported at or near the site of surgical scars at the
perineum, abdominal wall, even at the laparoscopic trocar port site, likely due to
the mechanical transplantation of endometrial tissue during previous episiotomy,
cesarean section, hysterectomy, hysteroscopy, tubal ligation, vulvar surgery, and
accidental trauma [6973].
Timing of abdominal surgery was suggested to play role in the development of
the disease. In women with endometriosis, the recurrence rate was higher in those
who had surgery near the end of the menstrual cycle (days 2228) than in those who
had surgery earlier in their cycle [74].
18.1.10 Contraception
If nonsteroidal anti-inflammatory drugs fail in alleviating dysmenorrhea, oral

contraceptive pills (OCP) are commonly offered to young women. The symptoms
of dysmenorrhea usually disappear with the suppression of ovulation but recur once
pill taking is discontinued [75]; therefore long-term use is recommended [76].
The risk of endometriosis appears to rise with greater lifetime number of

ovulatory cycles. Besides suppressing ovulation, OCPs increase the low apoptotic
activity of the endometrium of women with endometriosis [77]; progestins in OCPs
prevent implantation of regurgitated endometrium, inhibit angiogenesis and also
expression of matrix metalloproteinases, and reduce the inflammation of the
endometriotic implants and the consequent immune response [78].
The findings of the reports studying the link between OCP and endometriosis are
contradictory. Decreased [17, 79, 80] or increased [81, 82] risk of endometriosis
among OCP users have been published. On the other hand, some of the studies
failed to find any association between the two [83, 84].
A cross-sectional study on a large series of patients revealed that past use of OCP
if particularly given for treating severe dysmenorrhea was associated with all stages
of endometriosis, especially deep infiltrative endometriosis (DIE) whereas no
association was shown with endometriosis and current OCP use [85]. Meta-analysis
are in agreement with decreased endometriosis prevalence in current but increased
prevalence in past OCP users. Data from cohort studies (excluding casecontrol
studies) demonstrate a protective effect of current OC use (relative reduction, 43 %;
95 % CI, 2060 %), whereas previous use seems to increase the risk by 60 % (95 %
CI from 40 to 82 %) [86]. The pill may reduce the risk of endometriosis by
suppressing ovulation.
On the other hand, it was experimentally demonstrated that the regurgitated
endometrial tissue into the peritoneum of castrated female monkeys survived only
if estradiol was supplemented [87]. Therefore, it is quite possible that the estrogen
in the pill may act as a rescue factor for regurgitated endometrial glands that would
otherwise be resorped during hypo-estrogenic menstrual milieu.
The observed link might not be a causal one. It is well known that hormonal
therapies are effective on pain symptoms [88], and women who receive OCP
especially following failed NSAIDs for dysmenorrhea may already have developed
endometriosis, but is still undiagnosed [89]. Since OCP use reduces pelvic pain
symptoms, current users tend not to be investigated for endometriosis. On the other
hand, women with endometriosis-induced dysmenorrhea might have been selec-
tively excluded from the never OC users category, with a consequent increased
risk for past users as a group.
In the end, it appears unlikely that OCs influence the risk of endometriosis to any
great extent, because a consistent doseresponse effect for lifetime duration of use
has not been observed. Furthermore, also the pattern of risk with time since last use
does not support a causal relationship [86].
If retrograde menstruation is involved in the etiology of endometriosis, exposure
to nonhormonal intrauterine devices (IUD), by increasing the menstrual flow, may
be a risk factor for endometriosis. The results of the studies are contradictory.
Several studies have suggested that IUD use does not influence the development of
endometriosis [81, 9092]. Others have reported an increased prevalence of endo-
metriosis among former IUD users [9, 93, 94]. Even a weak protection has been
observed in a subgroup for subjects who stopped using IUD more than 10 years
ago [90].
There is no question that levonorgestrel-containing intrauterine systems (LNG

IUS) which induce endometrial atrophy and decidual transformation of the stroma
downregulate endometrial cell proliferation, increase apoptotic activity, which also
has anti-inflammatory and immunomodulatory effects, and play a definite role in
the treatment of endometriosis-associated symptoms [95, 96]. The LNG IUS also
reduce the risk of recurrence of dysmenorrhea after conservative surgery for
endometriosis [97].
Tubal ligation has been linked to development of endometriosis. Overall, no
relationship between tubal ligation and prevalence of endometriosis was found in a
group of women. On the other hand, tubal ligation was significantly associated with
severity of disease [P 0.036, crude OR (95 % CI) 0.17 (0.020.85), adjusted
OR (95 % CI) 0.21 (0.041.08)]. In subgroups, moderatesevere endometriosis
was found in 8.7 % and 36.4 % among patients with and without sterilization,
respectively [98]. In another group of 3384 multiparous women who underwent
tubal sterilization, endometriosis was detected in 126 patients (3.7 %), which was
not different from the control group [92].
18.2 Boosting the Immune System and Reducing

the Inflammation Stress
Activation of the hypothalamopituitaryadrenal (HPA) and sympatheticadrenal

medullary axes in the presence of stress lead to abnormal corticotropin-releasing
factor (CRF) secretion pattern, overexpression of glucocorticoid receptors, and
chronic overreaction of the bodys stress system [99]. The release of cortisol also
affects the immune system, in addition to its effects on the brain. Acute stressors are
associated with an upregulation of the immune system, while prolonged increase in
cortisol levels has been shown to depress immunologic function [100].
In women with chronic diseases such as gastrointestinal (GI) disorders causing
chronic pelvic pain, chronic fatigue syndrome, dysmenorrhea, and mood disorders
(e.g., anxiety, depression, posttraumatic stress syndrome), besides elevated levels of
inflammatory cytokines, findings reflecting abnormal HPA responses and decreased
cortisol levels have been reported. Chronic stress and hypocortisolism have been
hypothesized to cause a deregulation in the neuroendocrineimmune axis leading to
diseases including endometriosis. In fact, women with endometriosis have lower
salivary and follicular fluid cortisol levels [101], but higher serum cortisol levels are
detected in infertile women with advanced endometriosis [102]. It might be that
other neuroendocrine factors, like CRF levels, could be more accurate and informa-
tive markers of HPA axis deregulation than systemic cortisol levels.
It is hypothesized that stressful life events can impact the immunological health
of an individual. It is also known that endometriosis is associated with increased
secretion of cytokines and impaired cell-mediated immunity, modulating the
growth of ectopic endometrial implants [103]. The fibrosis and inflammation,

particularly the mast cells in the infiltrate surrounding ectopic endometrial
tissue, suggest that a hypersensitivity reaction, specifically, is strongly related to
endometriosis [104, 105]. Increased activated and degranulating mast cells and its
histological relationship with nerves in deeply infiltrating endometriosis lesions
may be contributing to intense and typical deep pelvic pain [106, 107]
The increased frequency of having shorter cycle length which is a known risk
factor for endometriosis in women with stressful jobs compared with those who did
not consider their jobs stressful and twofold elevated dysmenorrhea prevalence in
women reporting high levels of stress in the preceding menstrual cycle are indirect
evidences supporting the link between stress and endometriosis [108, 109].
Although it is unknown whether stress is a causal or exacerbating factor in the
development of endometriosis, establishment of psychological, behavioral, and
stress-reduction interventions as part of multidisciplinary preventive management
should be taken into consideration.
18.2.1 Vaccines
The use of bacillus CalmetteGuerin (BCG) and granulocytemacrophage colony-

stimulating factor (GM-CSF), among others, as boosters for the immune system in
oncology cases, stimulated researchers to study their possible role in the prevention
of endometriosis. In animal studies, systemic prophylaxis with BCG caused an
inhibitory effect on endometrial transplantation [110].
The effects of mycobacteria in altering the ability of peripheral blood mononu-
clear cells (PBMCs) and natural killer (NK) cells to kill endometrial stromal cells
have been assessed in in vitro model and endometrial stromal cell susceptibility to
killer cells has been demonstrated [111].
Research on possible immunomodulatory role of pentoxifylline did not show
any impact on future fertility in infertile women with asymptomatic minimal and
mild endometriosis [112].
After vaccination with RESAN which is a complex of molecules extracted from
xenogeneic tissues containing glycoproteins, peptides, and carbohydrate fragments
of more than 40 different common tumor antigens, a reduction in endometriosis
induction from 69.6 to 4.3 % was obtained in the rat model [113].
18.2.2 Retinoic Acid
Cytokines, chemokines, proteases, and angiogenic factors in the peritoneal cavity

which are derived mainly from activated peritoneal macrophages promote the
development and progression of endometriosis [114]. Defects in macrophage
activation may lead to chronic immune activation with accompanying reduction
in immune response contributing to the growth of endometriotic lesions [115].

Retinoic acid (RA) has been shown to modulate inflammation in autoimmune
disease by enhancing regulatory T-cell (Treg) suppression of proinflammatory
cells [116, 117]. In model systems involving activated monocytes/macrophages,
RA decreases proinflammatory cytokines while increasing anti-inflammatory pro-
teins such as interleukin-10 [118] also decrease the peritoneal fluid levels of
interleukin-6 (IL-6) and macrophage chemotactic factor-1(MCP-1), which have
been implicated in its pathogenesis of endometriosis [119]. These in vivo findings
emphasize the potential use of retinoids to prevent and to treat women with
18.2.3 Melatonin as an Antioxidant
Proper regulation of matrix metalloproteinases (MMPs) is essential for physiolog-

ical functioning of the endometrium, for invasion characteristics, and for
remodeling of the extracellular matrix. Derangement of MMP regulation is critical
in the development of endometriosis. Both MMP-2 and MMP-9 are activated by
reactive oxygen species (ROS), and their expressions seem to be regulated by
oxidant stress [121]. In the animal model, antioxidant enzymes like superoxide
dismutase and catalase prevent intraperitoneal adhesions of endometriotic tissues in
the peritoneal cavity [122].
Melatonin and its metabolites as antioxidants protect cellular components,
stimulate secretion of progesterone, and have oncostatic, antiproliferative, and
antiestrogenic effects [123, 124].
The role of melatonin in prevention and regression of endometriotic lesions
is through upregulation of proMMP-9 and antiestrogenic activities. Also a new
diagnostic marker, MMP-9/TIMP-1 (tissue inhibitors of matrix metalloproteinases)
expression ratio in judging disease progression and severity have been demon-
strated in animal model [125].
18.2.4 Anti-inflammatory Modulators
Endometriotic cells respond to TNF- with increased secretion of MCP-1, which is

a factor found to be elevated in peritoneal fluid of patients with endometriosis [126].
Experimental data emerging from treatments with anti-inflammatory modulators
such as cyclooxygenase 2 inhibitors [127], peroxisome proliferator-activated recep-
tor- agonist [128], and TNF- inhibitors like TNF-binding protein (TBP)-1,
TNF-soluble high-affinity receptor complex, infliximab, and etanercept
[129, 130] are promising and suggest potential for targeting the immune system
to treat patients with endometriosis.
Macrophage migration inhibitory factor (MIF), an important regulator of the

host immune system that promotes the proinflammatory functions of immune cells,
plays a role in angiogenesis, tumorigenesis, as well as in many inflammatory and
autoimmune diseases. Circulating and local peritoneal levels and expression of MIF
which is a product of activated peritoneal macrophages are found to be elevated in
the presence of early, vascularized, and most active endometriotic lesions. In
in vivo model of endometriosis, ISO-1 [(S,R) 3-(4-hydroxyphenyl)-4,5-dihydro-
5-isoxazoleacetic methyl ester)], a highly specific inhibitor of MIF, has been shown
to lead to regression of ectopic endometrial implants; downregulation of angio-
genic, tissue remodeling, and survival factors such as integrins v and 3, VEGF,
IL8 and COX2; and the expressions of MMP2 and MMP9 and Bcl2 [131].
Several previous studies showed the benefit of targeting MIF and also of managing
inflammatory diseases such as asthma, sepsis, and viral infection [132, 133].
18.2.5 25-Hydroxyvitamin D and Selective Vitamin D

Receptor Agonist (VDR) as Immunoregulatory
and Anti-inflammatory Agents
Endometriosis risk may be influenced by dietary vitamin D intake and plasma

25-hydroxyvitamin D concentration through immunomodulatory effects [134]. In a
large prospective study, a significantly lower rate of laparoscopically confirmed
endometriosis among women with greater predicted plasma 25(OH)D levels and
among women with a higher intake of dairy foods has been demonstrated. Calcium,
vitamin D, and magnesium intakes from foods were also inversely related to endo-
metriosis [135]. Several other studies reported contradictory results [136138].
Moreover, data in humans suggest that high magnesium intake may be associated
with lower levels of inflammatory markers, including interleukin-6 and tumor necrosis
factor alpha receptor 2 [139]. Magnesium has also been shown to relax smooth
muscles [140] and as a result may influence endometriosis through its effect on
retrograde menstruation.
Several immunomodulatory effects could be mediated by the capacity of VDR
agonists to inhibit the NF-B [141, 142]. In addition, the inhibition of leucocyte
infiltration into inflammatory sites by treatment with VDR agonists is associated
with their capacity to inhibit chemokine production by cells in the target organ via
the inhibition of NF-B activation which results in interference with the growth of
experimentally induced endometriosic lesions [142, 143].
It has been shown that the VDR agonist, elocalcitol, inhibits lesion development
in a mouse model of endometriosis [144].
By administering elocalcitol during the perimenstrual and menstrual phase of
the cycle, inhibition of inflammation, endometrial cell adhesion, and lesion orga-
nization could be exerted with the maximal efficacy; thereby prevention of
the development of endometriotic implants could be feasible in subjects at high
risk of disease recurrence.
18.2.6 Simvastatin
Statins reduce the rate of endometrial stromal growth and angiogenesis, interfere
with the development and attachment of endometriotic implants, and also protect
the subject from the development of endometriosis by virtue of their anti-
inflammatory and antioxidant properties [145]. Considering its safety and minimal
side effects, use of statins in treatment of endometriosis holds promise [146].
18.2.7 Pentoxifylline
A methylxanthine acting as a phosphodiesterase inhibitor, an anti-inflammatory

agent, and also an immunomodulator which has been used for many years to
modify blood viscosity and improve tissue oxygen delivery in the management of
defective microcirculation reduces the production and action of cytokines such as
TNF- and interleukin-1 and thereby inhibits the inflammatory activation
of polymorphonuclear neutrophils and also inhibits phagocytosis and the gener-
ation of toxic oxygen species and proteolytic enzymes by macrophages
and granulocytes. Experimentally it has been shown that pentoxifylline can
modulate rat endometriotic implant growth and production of implant-specific
proteins [147]. Therefore, immunomodulation of peritoneal inflammatory cell
hyperactivation with pentoxifylline may represent a new modality to specifically
manage the pathophysiology of endometriosis. On the other hand, up until now,
there still appears to be little evidence to support using pentoxifylline in the
management of endometriosis [112, 148].
18.2.8 Sorafenib: An Antiangiogenic and Tyrosine

Kinase Inhibitor
Various epigenetic aberrations have been described in endometriosis [149].

Mesenchymal stem cells (MSC), located in the microenvironment of the ectopic
endometriotic lesion, may be modulated epigenetically and lead to the survival of
the MSC cells with enhanced migratory, proliferative, and angiogenic properties.
Since endometrial MSC do not express ER [150], the current use of antiestrogenic
medications is likely to spare MSC and target only ER-positive cells which explains
why symptom relief is just temporary and eradication of the disease is not possible.
Researchers just recently observed that sorafenib treatment inhibited the
increased phosphorylation of ezrin which plays a major role in the regulation of
cell morphology, migration, and attachment in ectopic MSC, and consequently
limited the increased migration of ectopic MSC. Targeting the stem cell population
may be relevant in achieving the complete eradication of endometriotic
implants [151].
18.2.9 Curcumin as an Antioxidant, Anti-inflammatory,

and Antiproliferative Agent
Curcumin is a naturally occurring polyphenolic yellow-/turmeric-colored com-

pound derived from the rhizome of Curcuma longa which is widely used as a
spice and coloring agent in several foods such as curry, mustard, and potato chips
and also in cosmetics and drugs. The anti-inflammatory effects are mediated
through interference with multiple key signaling molecules, including nuclear
factor-kappaB (NF-B). The increased MMP-9 activity and expression of tumor
necrosis factor-alpha (TNF-) in endometriotic tissues can be reversed by admin-
istration of curcumin in experimental models. Moreover, lipid peroxidation and
protein oxidation in endometriotic tissues are prevented by curcumin [152]. Another
study documents the Curcumins effect on regulation of matrix metalloproteinase
(MMP-2) activity by tissue inhibitor of MMP (TIMP-2) during the early phase of
endometriosis development [153]. It has also been demonstrated that curcumin can
effectively suppress ICAM-1 and VCAM-1 gene and protein expression, as well as
secretion of IL-6, IL-8, and MCP-1, by inhibiting the activation of NF-B induced
by TNF- in human ectopic endometriotic stromal cells [154]. All these findings
provide a novel rationale for the potential of curcumin in the prevention and
treatment of endometriotic disease in humans.
18.2.10 Green Tea as a Potent Antiangiogenesis Agent
Endometriosis is an angiogenesis-dependent disorder. Since endometriotic lesions

require new vessel formation to deliver the nutrient supply, dense vascularization is
a typical pathological feature of endometriosis. Antiangiogenesis is one of the most
well-characterized biological properties of green tea. The polyphenols, especially
epigallocatechin-3-gallate (EGCG) in the leaves of the tea plant Camellia sinensis,
have potent antioxidative, antimitotic, and antiangiogenic properties [155, 156].
In mice experimental endometriosis model, pro-EGCG, inhibits the develop-
ment, growth, and angiogenesis of the implants [157, 158]. EGCG selectively
suppresses vascular endothelial growth factor C (VEGFC) and tyrosine kinase
receptor VEGF receptor 2 (VEGFR2) expressions in experimental endometriosis
in vivo and endothelial cells in vitro [158].
Antiangiogenesis for the management of endometriosis has the potential advan-

tage of lower recurrence rates and less endocrine side effects compared to conven-
tional surgical and hormonal therapies.
18.2.11 Resveratrol: A Phytochemical Compound
Resveratrol (trans-3,5,40 -trihydroxystilbene) is a phytochemical compound of

grapes, red wine, nuts, and different berries, affecting multiple cellular processes,
including proliferation, apoptosis, and oxygen radical formation [159], and also
suppressing the development of new blood vessels. Moreover, resveratrol dose-
dependently suppresses the development of new blood vessels [160]. The most
significant concentrations of resveratrol are found in the skin of grapes and there-
fore in red wines but not white wines. Resveratrol has been suggested as a
promising therapeutic agent for the treatment of cancer [161] as well as several
inflammatory, metabolic [162], and cardiovascular diseases [163]. A group of
researchers just recently have shown in mice that resveratrol inhibits the establish-
ment of endometriotic lesions by decreasing proliferative activity and by
upregulating apoptotic cell death inside the lesions [164]. Another group have
also demonstrated that resveratrol treatment suppresses the development of new
microvessels and inhibits the proliferation of both stromal and glandular endome-
trial cells in peritoneal and mesenteric lesions [165]. Better understanding of the
basic mechanisms of action of EGCG and resveratrol, as well as their bioavailabil-
ity, is needed to determine the potential usefulness of these natural compounds as
endometriosis-preventive agents [166].
18.2.12 Chinese Herbal Medicine: Puerarin

as a Phytoestrogen
Puerarin, the main isoflavone glycoside derived from the Chinese medicinal herb
Radix puerariae, exhibits antiestrogenic activity by suppressing P450arom and
interferes with the invasion of endometrial stromal cells (ESC) and angiogenesis
of ectopic tissues, in a model of endometriosis. This might be a good option for
avoiding the relapse of endometriosis after the initial surgical and/or medical
therapy, since it can be used for long periods without severe side effects, unlike
the classical antiestrogenic medical treatment modalities [167].
According to several clinical studies in the medical literature, treatment with
Chinese herbal medicine (CHM) involving formulae of between 10 and 20 separate
herbal ingredients selected from a materia medica of several hundred commonly
herbs that are administered as pills, enemas, and intramuscular injections prevents
the recurrence of endometriosis after a conservative operation with fewer adverse
reactions when compared with conventional western medicine. Better quality

randomized controlled trials are needed to investigate a possible role for CHM in
the prevention and management of endometriosis [168, 169].
18.3 Life Style
18.3.1 Pregnancy and Vaginal Parturition
It has been established that there is a higher prevalence of endometriosis in infertile

women (48 %) than in fertile women (5 %) [170], and infertile women are 68 times
more likely to have endometriosis than fertile women [171]. Gravidity (OR, 0.49;
95 % CI, 0.320.75) and parity (OR, 0.42; 95 % CI, 0.280.64) decrease the
odds of diagnosis of endometriosis [4]. It is possible that pregnancy may indeed
suppress the growth and inflammation of endometriotic lesions due to elevated
progesterone levels.
Among parous women, parity and lifetime duration of lactation are associated
with decreased risk. Among parous women, there is a linear decrease in risk with the
number of liveborn children (rate ratio of 0.5 comparing >3 with 2 children; 95 % CI
0.40.7) and lifetime duration of lactation if time since last birth is less than 5 years
(rate ratio of 0.2 comparing >23 months with never; 95 % CI 0.10.4) [17].
The recurrence rate of endometriosis has been found to be significantly lower in
women who had vaginal parturition than in nulliparous women and those who
delivered by cesarean section. Enlargement of the internal cervical ostium has
been inversely related to the recurrence of endometriosis, confirming the role of
retrograde bleeding in the occurrence and recurrence of the disease. Regardless of
the presence of endometriosis, a relief in dysmenorrhea has been observed only in
women who deliver vaginally [172].
18.3.2 Diet
Over the past decade, many studies have provided evidence that higher intakes of
fruit and vegetables, rich in antioxidants, among other micronutrients, improve the
function of the immune system and fight free radical damage [173]. Manipulation
of dietary polyunsaturated fatty acid (PUFA) composition demonstrably affects the
proinflammatory activities of many cell types involved in the immune response,
inflammatory reactions, and cytokine network and on the synthesis and biological
activity of prostaglandins and cytokines such as IL-1, IL-2, IL-6, TNF, and inter-
feron [174, 175]. In the presence of high n-6:n-3 PUFA ratios of dietary intake,
biosynthesis of their metabolites steadies a prominent production of 2-series
prostaglandins (PGE2, PGF2a), thromboxane A2, and 4-series leukotrienes, in
contrast to high n-3:n-6 PUFA ratios. Moreover, a diet based on vitamin B,

vegetables, fibers, and antioxidants decreases estrogenic state-related body fat
excess implicated in the estrogen-dependent growth of endometriotic tissue
[174]. Dietary supplementation induces enzymes of estradiol metabolism, and
the subsequent defective formation and metabolism of steroid hormones are respon-
sible for the promotion and development of endometriosis [176]. Although not well
characterized, some observational studies have shown that plant-based and high in
fiber diets decrease concentrations of bioavailable estrogen by increasing its excre-
tion and thus lower endometriosis risk [177, 178]. Additionally, high-fat diets have
been associated with increased estradiol levels in premenopausal women [177,
179], suggesting that diets low in fat and high in fiber may modify endometriosis
risk by altering steroid hormone metabolism. The published reports on this issue are
somewhat contradictory [34, 173]. A recent large cohort study using 12 years of
prospectively collected data have failed to show any association between total
dietary fat intake and endometriosis risk, but a decreased risk with increased
long-chain n-3 fatty acid consumption and an increased risk with trans-fat intake
have been demonstrated [180]. A recently published population-based casecontrol
study [181] suggests a possible inverse risk of disease with dietary fat and dairy
consumption and an increased risk of endometriosis with -carotene and higher
servings of fruit, but these findings have not been confirmed elsewhere and require
further evaluation in a prospective investigation. Unfortunately, there are only few
well-designed, randomized, controlled trials to evaluate the efficacy and safety of
complementary dietary therapy to manage endometriosis. From the accumulated
data, one can conclude that the effect of dietary fat on the risk and incidence of
endometriosis, if any, is marginal and is not clinically relevant. There is no
adequate scientific support to the suggestion that fish oil consumption is beneficial
for the prevention of endometriosis. Keeping in mind that the diagnosis of endo-
metriosis can be made by laparoscopy especially in women with pain and that
women with a high fatty acid intake are less likely to undergo a laparoscopy since a
high fatty acid intake can reduce menstrual pain, the association between the risk of
undergoing a laparoscopy and fatty acid intake will therefore probably be as
significant as the association between endometriosis and fatty acid intake. The
impact of diet on endometriosis risk is urgently needed to be further studied before
development of population-based strategies to prevent endometriosis can be
suggested.
18.3.3 Physical Activity
Physical activity has been hypothesized to be protective since endometriosis is an

estrogen-dependent disease, and physical activity may increase levels of sex
hormone-binding globulin (SHBG), which would reduce estrogens. Regular exer-
cise has been associated with a 4080 % reduction in risk for endometriosis in
several casecontrol studies. Four casecontrol studies have found inverse
associations between physical activity and the risk of endometriosis, with relative
risks ranging from 0.2 to 0.6 [9, 34, 182]. 70 % decreased risk of developing an
endometrioma with recent, frequent, and regular high intensity physical activity, as
characterized by 3 times/week, 30 min/episode, 10 month/year for 2 years.
Another study found a 40 % lower risk for women who reported regular exercise
for 37 h/week and an 80 % risk reduction for those who exercised more than 7 h/
week when compared with nonexercisers [183]. On the contrary to the others, in the
Nurses Health Study II, activity reported 6 years prior to diagnosis and inactivity
have not been found to be associated with endometriosis [184].
Although adult physical activity has been mostly associated with lower endo-
metriosis risk [9, 34, 182, 183, 185], little is known about the influence of childhood
or adolescent physical activity on endometriosis.
One of the studies reported a 27 % increased risk of endometrioma for any
physical activity at 1221 years of age [182], and the other one, the Nurses Health
Study II, also found a positive linear relationship between strenuous physical
activity at 1213 years of age and endometriosis risk [186], suggesting that the
early adolescent period is a critical window of exposure for the implantation of
endometriosis lesions, which physical activity might promote at that age.
18.4 The Timing of Exposure to Environmental Factors
Exposure to environmental chemicals recently has been proposed to contribute to

several gynecologic pathologies including endometriosis, especially when expo-
sures occur during critical periods of development. There are limited data on the
prevalence of conditions that affect womens reproductive health. Hormone-related
diseases such as endometriosis and uterine fibroids, pubertal developmental abnor-
malities, and polycystic ovary syndrome are more common, although few data on
population-based trends are available. The toxic chemicals altering reproductive
health in females have been demonstrated by the consequences of diethylstilbestrol
(DES) use by pregnant women. Other synthetic chemicals which are called
endocrine-disrupting compounds (EDCs) used in commerce are known to mimic
hormones and have been shown to contribute to disease onset [187].
The impact of the environment on reproductive physiology can be a direct
inducer of gene expression, acting directly as hormones or disrupting the metabo-
lism or synthesis of endogenous hormones, or through a neuroendocrine route,
whereby the nervous system monitors the environment sending signals to the
endocrine system, and an epigenetic route could have been chosen, whereby
altering transcriptional capabilities without changing DNA sequence [188].
The data collected from the Nurses Health Study have revealed that DES
daughters have an 80 % increased risk (relative risk [RR] 1.8, [CI] 1.22.8) of the
development of endometriosis [23]. At the same time, in mice model, exposure
to the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gestational day 8
increases the size of implanted endometriotic lesions when combined with an
adult exposure [33]. Based on these evidences, one can hypothesize that during
embryogenesis, EDC exposure has an organizational effect that increases suscep-
tibility for endometriosis, but subsequent adult hormone, immune, and/or EDC
irregularities are required for disease onset.
Although estrogen is necessary for the progression of endometriosis, other
factors also influence this progression. The most toxic dioxin, TCDD, induces
endometriosis, not in ovariectomized mice but with an intact ovary [34]. Also, in
women with peritoneal endometriosis, immune dysfunction by TCDD has been
blamed since the immune system fails to prevent implantation of endometrial
debris, despite high levels of activated macrophages and inflammatory cytokines
in the peritoneal environment [189], suggesting that the progression of endometri-
osis is dependent on both hormonal and immune environments.
Increasing experimental evidence suggests an influence of environmental organ-
ochlorines, a class of xenobiotic chemicals on endometriosis development, includ-
ing dioxin or dioxin-like compounds [190, 191] which are known to disrupt
endocrine and immune functions [192].
Human endometrium is a known site for estrogen, and many environmental
chemicals have been detected there [193] or may induce inflammation and the
chronic stimulation of proinflammatory cytokines. At the same time, they have
been associated with immunologic changes, downregulating natural killer cells or
interleukin-1 and interleukin-12 [194].
Recently, organochlorine pesticides have also been shown to increase endome-
triosis risk in a laparoscopic cohort of US women [195] and other studies have
reported increased risks with higher concentration of phthalates [196, 197],
polychlorinated dibenzodioxins and polychlorinated dibenzofurans, and
polychlorinated biphenyls [198, 199].
Experiments on rodents suggest that both adult and in utero exposure to dioxin
can promote endometriosis during adulthood. Increased endometriotic lesion size
was observed in mice exposed to TCDD during both perinatal and adult life
stages [33].
Since in utero and lactational exposure to TCDD reduces circulating estradiol
in vivo [200] and decreases ovarian estradiol production in cultures [201], and also
causes degradation of ER- [202], it is possible that fetal exposure to TCDD
promotes adult endometriosis through altered P action, because PR expression is
reduced in the uterus of adult mice that were exposed to TCDD in utero [35] and P
insensitivity is characteristic of women with endometriosis [189, 203]. TCDD
which is an immunosuppressant [36] might promote endometriosis by altering the
immune function, thereby enabling establishment and growth of peritoneal endo-
metriosis under the influence of E2, proangiogenic, proliferative, and antiapoptotic
factors. Alternatively, TCDD could activate the expression of K-ras in the ovarian
surface resulting in peritoneal endometriosis [37]. Endometriotic lesions have
increased expression of aromatase and 17-HSD type 1 and decreased expression
of 17-HSD types 2 and 4, resulting in an increase in production of estradiol [39].
If this expression pattern is established during fetal development via
epigenetic mechanisms, then endometriosis could manifest during adulthood after
estrogenic exposures. In ectopic endometrial tissue, ER- is upregulated and acts as

the mediator of endometrial proliferation [204, 205]. Therefore, adult exposures to
high doses of ER- agonists are hypothesized to promote ectopic endometrium
growth after retrograde menstruation.
18.4.1 The Developmental Origins of Adult Disease

Hypothesis
According to Barker hypothesis, adverse influences early in development, particu-

larly during intrauterine life, can result in permanent changes in physiology and
metabolism resulting in increased disease risk in adulthood, which is speculated to
occur largely through epigenetic mechanisms [206]. As a typical example, low birth
weight (LBW) is associated with a list of chronic diseases ranging from coronary
artery disease (CAD), type II diabetes mellitus (T2DM), cancer, and osteoporosis to
various psychiatric illnesses [207]. As stated by Dr. Ian Donald, The first 38 weeks
of human life spent in the allegedly protected environment of the amniotic sac are
medically more eventful and more fraught with danger than the next 38 years in the
lifespan of most human individuals [208].
Regarding endometriosis, development of permanent increased estrogen sensi-
tivity due to imprinting the regulatory gene HOXA10, in offspring exposed to
bisphenol A (BPA) during pregnancy, has been reported [209].
Unfortunately, placenta instead of being a protective barrier for the fetus allows
many toxic chemicals to pass to the fetus. BPA, for example, in animal model,
passes to the fetus reaching to higher than maternal blood levels in less than 30 min
after exposure [210, 211]. The embryo or fetus also cannot or partially detoxify the
chemicals since the key enzyme, cytochrome P450 activity, is lacking or not fully
developed in the fetus, even in young children [212].
Breastfed infants exposed to EDCs have much higher blood levels than formula-
fed infants [213]. It has also been emphasized that breastfed infants receives about
50 times the daily PCB intake of adults and up to 18 % higher than those of formula-
fed infants [214].
It is unknown whether breastfeeding can counteract or not, the detrimental
effects of prenatal toxicant exposure. Given that toxic chemicals are being removed
from the mothers stores during lactation and that the longer the lactation, the more
toxins removed, it might be expected that breastfeeding reduces breast cancer and
endometriosis risk. In fact, among the benefits of breastfeeding to the mother are
reduced risk of breast cancer and reduced risk of recurrence of endometriosis in
women who have had children, not longer than 5 years ago [215, 216].
In addition to the recommendations to choose breastfeeding or not, mother to be
should be as healthy as possible even before conception, lose weight if necessary,
eat organically, be supplemented with antioxidants and other detoxifiers, and avoid
all toxic exposures possible while breastfeeding [217, 218]. By losing weight in the
postpartum period, whether there will be any remobilization or not of adipose tissue
resulting in increased circulating levels of previously stored EDCs, thereby increas-
ing the levels in breast milk, is unknown [219].
Parents should be advised to avoid plastic bottles and not to store foods including
breast milk or formula for baby in plastics to avoid bisphenol A. There are also
toxins including dioxin, xylene, ethylbenzene, and styrene in disposable diapers
made of bleached paper and plastic [220]. Infants whose skin are exposed to lotion,
powder, and shampoo reveal increased urinary concentrations of phthalates [221].
Since food is the primary exposure to EDCs, eating organic instead of geneti-
cally modified foods is very critical [222]. Another way to reduce exposure is to eat
more vegetables, grains, fruits, and less animal products [223]. One might skip meat
completely and go vegetarian. On the other hand, soy, corn, potatoes, squash,
canola oil, cottonseed oil, papaya, and tomatoes are among the most commonly
genetically engineered foods. Consuming lignin-containing vegetables like cab-
bage, cauliflower, broccoli, and Brussels sprouts, which helps in the removal of
excess estrogen, is another way to reduce exposure [224]. In brief, contaminated
fish, meats, dairy, eggs, processed oils and fast foods, fried foods, and refined
processed foods should better be reduced or even avoided.
Avoiding pesticides and herbicides from other, non-food, sources is also important.
Pesticides have been linked to some immune abnormalities seen in endometriosis,
infections, asthma, and allergies. PVC, a source of phthalates, is prevalent worldwide
in building materials, plumbing, shoes, rain gear, shower curtains, flooring, and toys.
Dental sealants, used to protect teeth from decay-causing bacteria, typically contain
bisphenol A [225]. Children who have been exposed to pesticides are 37 times
more likely to develop non-Hodgkins lymphoma than children who have not been
exposed to pesticides and this risk was similar for pesticide exposure to the
mother during pregnancy and direct exposure after birth [226]. Since women with
endometriosis have 40 % higher risk for developing hematopoietic malignancies,
mainly non-Hodgkin lymphoma, this may be a problem which requires extra
caution [227].
Menarche is correlated to percent body fat with about 17 % body fat required for
menarche and 22 % body fat reported to be required to maintain or restore
menstruation [228]. Since fat cells produce estrogen, heavier girls usually begin
sexual development and periods earlier [229]. Fat cells also make cytokines,
therefore keeping down fat should also avoid inflammation [230]. The fat con-
sumed as a child may be even more an important risk than the fat consumed as an
adult. Exercise is another way to keep body fat low and achieve the goal of delaying
puberty and menarche [231].
The Nurses Health Study II (NHS II), in a well-characterized cohort, has
reported that low birth weight, multiple gestation, and DES are associated with a
diagnosis of endometriosis [23]. Another study demonstrated lower odds of the
diagnosis with in utero exposure to cigarette smoking [232]. Women eventually
diagnosed with endometriosis were leaner from childhood through diagnosis
relative to women without endometriosis [21]. This finding was subsequently
corroborated in the large Nurses Health III Cohort Study [233]. Despite some
indirect evidence suggestive of an early origin for endometriosis, some recent
studies failed to demonstrate an association between in utero exposures and
increased odds of an endometriosis diagnosis [234, 235].
18.5 Conclusion
In conclusion, medical literature have not yet addressed the prevention of endome-
triosis. However, there is extensive scientific and clinical data applicable to pre-
vention of endometriosis when it is regarded as a systemic inflammatory,
endocrine, and immunological disease. We hope this review will stimulate further
basic and clinical research on this very critical health problem of women.
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Chapter 19
MR Imaging of Endometriosis
Shinya Fujii
Abstract MR imaging has played a significant role as a noninvasive method,

although laparoscopy is the gold standard for diagnosis. We describe MR imaging
findings about endometriotic cyst, endometriotic implant, adhesion, deep endome-
triosis, decidualized endometriotic cyst, and malignant neoplasms arising from
endometriosis.
Keywords Endometriosis MRI Ovary
19.1 Endometriotic Cyst
The signal intensity of endometriotic cysts is hyperintense on T1-weighted imaging

similar to that of fat. T2-weighted imaging finding of endometriotic cyst is
hypointense (shading) in most cases (Fig. 19.1), but can be hyperintense. Besides,
multiplicity of the cysts is another important finding. MR imaging for the diagnosis of
endometriotic cysts has a sensitivity of 9092 %, a specificity of 9198 %, and an
accuracy of 9196 % [14]. Therefore, MR imaging is very important and useful for
the diagnosis of endometriotic cysts. This characteristic signal intensity is considered
to reflect aged blood and its viscosity [1]. The signal intensity of endometriotic cysts
on T2-weighted imaging has a significant relationship with the iron concentration,
although there is no significant relationship with the signal intensity on T1-weighted
imaging [5]. Fat-suppressed T1-weighted imaging should always be performed for
not only the differentiation from mature cystic teratoma showing hyperintensity on
T1-weighted imaging but also the detection of small lesions such as endometriotic
implants. Another characteristic finding is thick wall with hypointensity on both T1-
and T2-weighted imaging. This finding reflects the fibrous nature of the cyst wall with
S. Fujii (*)
Division of Radiology, Department of Pathophysiological and Therapeutic Science,
Faculty of Medicine, Tottori University, Yonago, Japan
e-mail: sfujii@med.tottori-u.ac.jp

312 S. Fujii
Fig. 19.1 A 39-year-old woman with bilateral ovarian endometriotic cysts. Axial T1-weighted
MR image (a) shows bilateral hyperintense ovarian masses (arrows). T2-weighted image shows
shading with hyperintensity in both lesions (arrows) (b). On fat-suppressed T1-weighted image,
these masses remain hyperintense (c)
Fig. 19.2 A 32-year-old woman with left ovarian endometriotic cyst. Axial T2-weighted images
demonstrate left ovarian hypointense cystic mass (arrow). The shading sign is more prominent at
3 T (a) than at 1.5 T (b)
hemosiderin-laden macrophages [6]. Susceptibility weighted imaging can detect this

hemosiderin deposit [7]. Meanwhile, 3.0 T MR imaging is more useful for the
diagnosis of endometriotic cysts than that of 1.5 T, because 3.0 T MR imaging can
demonstrate shading sign on T2-weighted images [8] (Fig. 19.2).
19 MR Imaging of Endometriosis 313
19.2 Endometriotic Implants
Fat-suppressed T1-weighted imaging is mandatory sequence for the detection of

endometriotic implants [2, 3, 911]. The sensitivity in detecting peritoneal
implants is significantly higher with fat-suppressed T1-weighted imaging
(61 %) than with conventional T1-weighted imaging (27 %) due to better
visualization of hemorrhagic implants with signal suppression of surrounding
fat tissue [9]. Additionally, fat-suppressed T1-weighted imaging can detect
endometriotic implants that are not detected during surgery because of overlying
adhesions [12, 13]. However, the sensitivity is not so high because even
fat-suppressed T1-weighted images cannot detect tiny peritoneal lesions and
the lesions which do not contain enough methemoglobin [12]; some small
endometriotic implants can show hypointensity on T1-weighted images and
hyperintensity on T2-weighted images [6].
19.3 Adhesion, Posterior Cul-de-sac Obliteration
Adhesion is diagnosed when there is obliteration of fat planes with a lack of a clear
interface between adjacent organs, spiculated hypointensity stranding between the
organs, and angulation and distortion of adjacent bowel loops [14]. These findings
are usually subtle, and we should detect the findings carefully.
The following findings are related to posterior cul-de-sac obliteration:
retroflexed uterus, elevated posterior vaginal fornix, intestinal tethering and/or a
tethered appearance of the rectum in the direction of the uterus, faint strands
between the uterus and intestine, and fibrotic plaque and/or nodule covering the
serosal surface of the uterus [14] (Fig. 19.3). Particularly, the following findings are
proposed as the major criteria for diagnosing cul-de-sac obliteration because of
their good specificity: intestinal tethering and/or a tethered appearance of the
rectum in the direction of the uterus, strands between the uterus and intestine, and
a fibrotic plaque covering the serosal surface of the uterus. Additionally, the most
accurate combination of two findings is intestinal tethering in the direction of the
uterus and fibrotic plaque in the uterine serosal surface, and the next highest
combination is retroflexed uterus and intestinal tethering in the direction of the
uterus [14].
19.4 Deep Pelvic Endometriosis
Deep infiltrating endometriosis is defined as the presence of endometriotic implants,

fibrosis, and muscular hyperplasia penetrating >5 mm into the peritoneum.
Associated symptoms include dysmenorrhea, dyspareunia, noncyclical pelvic
314 S. Fujii
Fig. 19.3 A 34-year-old woman with bilateral ovarian endometriotic cysts and posterior
cul-de-sac obliteration. The uterus is retroflexed on sagittal T2-weighted image (a). An irregular
hypointense area at posterior serosal surface of uterus is found (curved arrows). The posterior
vaginal fornix (arrow) is elevated toward this area. Axial T2-weighted image (b) shows faint
strands between uterus and rectum (arrowhead), tethered appearance of the rectum in the direction
of the uterus (bold arrow)
pain, dysuria, and lower gastrointestinal symptoms. The lesion can involve the
posterior cul-de-sac, uterosacral ligaments, rectovaginal septum, ureters, bowel,
and bladder. Endometriotic implants usually elicit an intense desmoplastic response
in the surrounding tissues, leading to the formation of adhesions, fibrotic bands, and
plaques. The histologic finding of deep pelvic endometriosis is mainly characterized
by fibromuscular hyperplasia surrounding foci of endometriosis. Previous report has
demonstrated the high accuracy in the prediction of deep pelvic endometriosis
with a sensitivity of 90.3 %, specificity of 91 %, and accuracy of 90.8 % [15].
Additionally, 3.0 T MR imaging has high accuracy in the diagnosis and staging of
deep endometriosis [16].
Deep endometriosis shows typically hypointensity with punctate hyperintensity
on T1-weighted imaging, hypointensity on T2-weighted imaging, and contrast
enhancement, which findings correspond to fibrous tissue. Punctate foci of
hyperintensity reflect hemorrhage surrounded by solid fibrous tissue. Additionally,
tiny hyperintensities within the lesion, which represent endometrial glands, can be
shown on T2-weighted imaging.
Irregular fibrotic thickening and nodularity with regular or stellate margins along
the course of the uterosacral ligament suggests deep pelvic endometriosis
(Fig. 19.4) [15]. MR imaging for the diagnosis of uterosacral ligament endometri-
osis has a sensitivity of 69.290 % and a specificity of 7694.3 % [17]. Thin-section
oblique axial T2-weighted imaging can improve the depiction of uterosacral liga-
ment endometriosis [18]. However, 3D T2-weighted imaging in combination with a
multi-planar reconstruction technique has no significant different accuracy from
that of conventional 2D axial T2-weighted imaging [17].
Fig. 19.4 A 29-year-old woman with deep pelvic endometriosis of bilateral uterosacral ligaments.
Axial T2-weighted image (a) demonstrates hypointense fibrotic plaque (arrow) with punctate
hyperintense foci on fat-suppressed T1-weighted image (arrow) (b) and irregular thickening of the
right uterosacral ligament (arrowheads)
Endometriosis of rectovaginal septum is mainly classified to massive lesion of

the deepest portion of the pouch of Douglas and retroperitoneal deep endometriotic
lesion originating from the metaplasia of mullerian remnants located in the
rectovaginal septum. Laparoscopic diagnosis is difficult because of complete oblit-
eration of the cul-de-sac. The lesion demonstrates ill-defined hypointense mass
between posterior vaginal fornix and rectum and can cause rectal stenosis and
hydronephrosis.
The rectum and distal sigmoid colon account for most of the bowel endome-
triosis. The endometriotic implants are usually serosal, but can eventually erode
through the subserosal layers and cause marked thickening and fibrosis of the
muscularis propria. Inflammatory response to cyclic hemorrhage can lead to
adhesions, bowel stricture, and gastrointestinal obstruction. Bowel endometriosis
shows mushroom or fan-shaped configuration, demonstrating isointensity com-
pared to muscle and slightly hyperintense at the luminal side of the bowel wall on
T2-weighted imaging [19, 20] (Fig. 19.5). The lesions sometimes demonstrate
hemorrhagic foci that are hyperintense on fat-suppressed T1-weighted imaging.
MR imaging has a sensitivity of 100 % and specificity of 75 % for predicting
muscular infiltration [20]. Diffusion-weighted imaging depicts the lesion as
hypointensity and is useful for the differentiation from colorectal cancer
[21]. Additionally, contrast-enhanced imaging can allow easier recognition of
colorectal endometriosis [22].
Involvement of bladder is seen in less than 1 % of patients with endometriosis.
Bladder endometriosis is mostly found on the posterior wall or in the dome of
the bladder. Bladder endometriosis can be demonstrated as localized or diffuse
bladder wall thickening showing hypointensity on T1- and T2-weighted imaging.
316 S. Fujii
Fig. 19.5 A 30-year-old woman with deep pelvic endometriosis infiltrating the rectosigmoid.
Rectosigmoid involvement shows hypointense fan-shaped configuration on sagittal T2-weighted
image (a) and contrast enhancement on enhanced T1-weighted image (arrow). An irregular
hypointensity area at posterior serosal surface of uterus is also found (arrowhead)
Hyperintense foci on fat-suppressed T1-weighted and T2-weighted imaging

indicative of hemorrhage and endometrial glands may be seen within the lesion
[23, 24].
Parametrial involvement can also occur, representing a severe form of endome-
triosis, and is important for surgical planning for deep infiltrating endometriosis
because a parametrectomy can be required for the complete removal of deep
infiltrating endometriosis. Recent report demonstrates the MR findings indicating
parametrial involvement as follows: the presence of a hypointense area in the
paracervical or paravaginal region on T2-weighted imaging or pelvic wall involve-
ment and ureteral dilatation [25].
19.5 Prediction of Hormone Therapy Response
MR imaging can predict the hormone therapy response of endometriotic cyst.

Response of the therapy is not good for endometriotic cysts with shading on
T2-weighted imaging, while endometriotic cysts without shading and multiplicity
indicate a good response to hormone therapy [26] (Fig. 19.6). The signal intensity
of responding endometriotic cysts decreases after the therapy on T2-weighted
imaging.
Fig. 19.6 A 34-year-old woman with bilateral ovarian endometriotic cysts. Bilateral
endometriotic cysts (arrows) show hyperintensity with subtle shading on axial T2-weighted
images obtained before hormone therapy (a). The size and signal intensity of the cysts (arrows)
decreased after hormone therapy (b)
19.6 Decidualized Endometrioma
Decidual changes of the ectopic endometrial stroma during pregnancy are well
known. Decidual changes of endometrial tissue in endometriomas during preg-
nancy may manifest as mural nodules and mimic malignant transformation. The
mural nodules indicating decidualization are demonstrated as linear, small nodular,
broad-based nodular, or polypoid structures, which show isointensity with the
nomotopic decidualized endometrium on T1- and T2-weighted images, balanced
fast field echo images, and diffusion-weighted images [2729]. Additionally, the
apparent diffusion coefficient of decidualized mural nodules is significantly higher
than that of ovarian cancers [29]. These MR imaging characteristics can help to
differentiate decidualized endometriotic cyst from malignant transformation.
19.7 Malignant Tumor Arising in Endometriotic Cyst
Malignant transformation is a rare complication of endometriosis and is estimated

to occur in 0.60.8 % of women with ovarian endometriosis. Approximately 75 %
of malignancies arising in endometriosis are found in ovarian endometriosis, and
25 % are found in extraovarian endometriosis [30]. Malignancies arising in ovarian
endometriosis are composed of endometrioid carcinomas in about 70 %, clear cell
carcinoma in 13.5 %, and sarcoma in 11.6 %, while malignancies arising in
extraovarian endometriosis are composed of endometrioid carcinomas in 65 %,
sarcoma in 25 %, and clear cell carcinoma in 4.5 % [28].
The following findings suggesting endometriotic cysts with malignant transfor-
mation are endometrioma with increasing size, larger size than those of
318 S. Fujii
Fig. 19.7 A 45-year-old woman with endometrioid adenocarcinoma associated with

endometriotic cyst. Left endometriotic cyst shows hyperintensity on axial T1-weighted image
(a) and absence of shading on T2-weighted image (b). The solid component (arrows) demonstrates
contrast enhancement on contrast-enhanced T1-weighted image (c) and hyperintensity on
diffusion-weighted image (d)
contralateral endometriotic cysts, loss of shading on T2-weighted images, and

enhancing mural nodules on contrast-enhanced images [31] (Fig. 19.7). The
enhancement of mural nodules is sometimes difficult to evaluate on conventional
contrast-enhanced T1-weighted images. Dynamic subtraction imaging can improve
the detection of enhancing mural nodule against intrinsically hyperintense back-
ground signal of endometriotic cyst [31] and is useful for the differentiation of the
tumors from intracystic blood clots. Meanwhile diffusion-weighted images can
provide a little information about its differentiation because some clots can show
hyperintensity [32]. Additionally, we should recognize that the small enhancing
nodules have the possibility of benign conditions such as granulomatous tissue and
benign endometriotic tissue [33].
Mullerian mucinous borderline tumor (MMBT) should be recognized as malig-
nant tumor arising from endometriosis because of coexistence with ovarian or
pelvic endometriosis in about 30 %. MMBTs are unilocular or paucilocular in
80 % of cases, bilateral in 7.740 %, and show prominent hyperintensity on
T2-weighted images reflecting intraluminal mucinous material and stromal
edema [34].
Malignant tumors arising in extraovarian endometriosis occur in various

anatomic locations, such as rectovaginal sites, colorectal sites, and bladder, which
seem to reflect the distribution of extraovarian endometriotic implants. Malignant
tumors arising from extraovarian endometriosis typically manifest as solid lesions
showing intermediate signal intensity on T1- and T2-weighted images, enhanced
by contrast material, and hyperintensity on diffusion-weighted images [35].
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Chapter 20
Biomarkers of Endometriosis
Amelie Fassbender*, Dorien O*, Bart De Moor, Etienne Waelkens,

Christel Meuleman, Carla Tomassetti, Karen Peeraer,
and Thomas DHooghe
Abstract Endometriosis is a benign gynecological disease defined by the ectopic

presence of endometrium and associated with pelvic pain and infertility. The etiology
and pathogenesis remain unclear. The gold standard of diagnosing endometriosis
is laparoscopy followed by histological confirmation, associated with an 8-year
delay in the diagnosis of endometriosis. A clinically reliable test for endometriosis
can be expected to allow early diagnosis and treatment, with profound impact on
the reduction of health care and individual costs. A noninvasive diagnostic test
could be developed for serum or plasma, urine, and endometrial or menstrual fluid.
*Amelie Fassbender and Dorien O are joint first authors

A. Fassbender D. O (*)
Department of Development and Regeneration, Organ systems,
KU Leuven, Leuven, Belgium
Department of Obstetrics and Gynaecology, Leuven University Fertility Centre,
University Hospital Leuven, UZ Gasthuisberg, Leuven, Belgium
B. De Moor
Department of Electrical Engineering (ESAT-SCD), KU Leuven,
Leuven, Belgium
E. Waelkens
Department of Molecular Cell Biology, Campus Gasthuisberg, Leuven, Belgium
C. Meuleman C. Tomassetti K. Peeraer
T. DHooghe (*)
Department of Development and Regeneration, Organ systems,
KU Leuven, Leuven, Belgium
Division of Reproductive Biology, Institute of Primate Research, Karen, Nairobi, Kenya
e-mail: thomas.dhooghe@uzleuven.be

322 A. Fassbender et al.
A semi-invasive test could be developed for peritoneal fluid and eutopic endometrium.
The development of such a test from initial biomarker discovery to be a clinically
approved biomarker assay is a long, difficult, and uncertain process and includes four
different phases. A review of the existing literature is provided in this chapter. Overall,
most endometriosis biomarker studies remain at the level of phase I, with only a few
currently in phase II of biomarker development. There is a need for a well-designed
multinational study including academic and industrial partners for phase II and
phase III trials.
Keywords Diagnostic test Endometriosis Ultrasound negative
20.1 Introduction
Endometriosis is one of the most common gynecological disorders, affecting

approximately 10 % of women in the reproductive age group and up to 50 % of all
infertile women [1, 2]. This estrogen-dependent disease is characterized by the
presence of endometrial-like tissue at ectopic sites like the pelvic peritoneum, the
ovaries, the rectovaginal septum, and in some cases even the pericardium, the pleura,
and the brain [1]. Infertility is frequently associated with endometriosis as it is
estimated that up to 50 % of endometriosis patients are subfertile (any form of
reduced fertility with prolonged time of unwanted non-conception) [3]. Another
important symptom that accompanies endometriosis is pain. Endometriosis-
associated pain typically occurs as chronic pelvic pain in 90 % of women suffering
from painful menstruations (dysmenorrhea), in 42 % of women suffering from pain
during intercourse (dyspareunia), and in 39 % of women suffering from
non-menstrual pain [4, 5]. The degree of pain and the severity of endometriosis
are poorly related and the exact mechanism of endometriosis-associated pain has yet
to be determined [6]. Pain might be caused by pressure of large nodules on visceral
organs, pelvic adhesions, tissue damage by infiltration of lesions, or an inflammatory
response in the peritoneal cavity triggered by cyclical bleeding of lesions
[4, 7]. Recent evidence suggests that endometriosis-associated nerve fibers might
be a pain-causing feature. Indeed, in endometriotic lesions, ingrowth of blood
vessels along with nerve fibers has been observed [8, 9].
The diagnosis of endometriosis is currently made by means of laparoscopy to
identify endometrial-like tissue at ectopic places [1]. The severity of the disease is
determined according to the revised American Fertility Society classification sys-
tem. The classification of the disease stage (stages I to IV or minimal to severe) is
based on acquired information during laparoscopic surgery on the morphology and
depth of the implants, the presence, place, and type of lesion, and the presence,
place, and type of adhesion [10]. Although laparoscopy is considered the golden
standard, it can fail to detect very small lesions and lesions might not be noticed
because of their location [11]. Furthermore, an average delay of 8 years precedes an
accurate diagnosis. Five to six of these years can be attributed to a delay in the
search of medical help and the remainder to obtaining the correct diagnosis [12].
20 Biomarkers of Endometriosis 323
The etiology and pathogenesis of endometriosis is still controversial and is

almost certainly multifactorial [13]. Despite several established theories such as
retrograde menstruation, abnormal immune system, coelomic metaplasia, genetic
and epigenetic factors, and the stem cell theory, researchers still have not found the
possible mechanism and cause of endometriosis.
Treatment options are directed against endometriosis itself, against
endometriosis-associated pain, or against endometriosis-related infertility. Surgery
aims to remove all visible endometriotic lesions [14]. However, as it does not
manage the underlying mechanisms of the disease, recurrences of endometriotic
lesions are common. Currently, different medical treatments are used in the clinic
as well [15]. These treatments focus on altering the hormonal environment, hereby
creating a suboptimal milieu for the growth and maintenance of the lesions. Still,
these medications have many side effects and their efficiency is not ideal [14]. It has
been indicated that, in order to improve diagnostic tools and treatment strategies,
the underlying mechanism of endometriosis needs to be clarified [16]. In recent
years, new more targeted therapies are being developed, e.g., aromatase inhibitors,
angiogenesis inhibitors, and immunomodulating agents [17].
A noninvasive diagnostic test could be developed for serum or plasma, urine,
and endometrial or menstrual fluid that can be recovered from the posterior vaginal
fornix and from the cervix during speculum examination. A semi-invasive test
could be developed in peritoneal fluid, obtained after transvaginal ultrasound-
guided aspiration, or in endometrial, obtained after transcervical endometrial
biopsy. Whatever method is used, the most important goal of the test is that no
women with endometriosis or other significant pelvic pathology are missed who
might benefit from surgery [18]. To achieve this, a test with a high sensitivity is
needed, which is the probability of a test of being positive when endometriosis is
present. At present, such a test does not exist [19, 20].
Researchers and clinicians need to realize that a diagnostic test may do more
harm than good, e.g., by subjecting patients to unnecessary or even potentially
harmful procedures [21] since the benefits of treating women with asymptomatic
endometriosis are unclear [19]. Therefore, we do not recommend the development
or use of a blood test for screening purpose in asymptomatic women. However, up
to 45 % of subfertile women with a regular cycle whose partner has normal sperm
quality, with or without pelvic pain, and with normal clinical examination and a
normal pelvic ultrasound may have endometriosis [20, 22].
Most gynecologists are not sure if endometriosis is present if a woman has, for
example, subfertility, a regular cycle, and a partner with a normal sperm examina-
tion and if they have been unsuccessful in trying to conceive for more than 1 year
without moderate severe cyclic pelvic pain. In addition, if a woman has chronic
pelvic pain (requiring at least cyclic or chronic use of pain killers), combined with a
normal clinical examination and a normal pelvic ultrasound, many gynecologists are
in doubt about the value of a diagnostic laparoscopy. From a clinical perspective, it is
unlikely that these women will have moderatesevere endometriosis, but they may
have extensive peritoneal endometriosis with or without adhesions associated
with subfertility and possibly mild pain [18]. For this population, a noninvasive or
semi-invasive diagnostic test would be useful to discriminate between women

without endometriosis who need to avoid having unnecessary surgery and
those with endometriosis, most likely minimalmild disease, who are known to
benefit from surgical therapy for both subfertility and pain and from controlled
ovarian stimulation in combination with intrauterine insemination for subfertility
[15, 18, 20, 23]. In summary, a noninvasive test for endometriosis would be useful
for women with pelvic pain and/or subfertility with normal ultrasound. This would
include nearly all cases of minimalmild endometriosis, some cases of moderate
severe endometriosis without clearly visible ovarian endometrioma, and cases with
pelvic adhesions and/or other pelvic pathology, who might benefit from surgery to
improve pelvic pain and/or subfertility [18, 20].
20.2 Noninvasive Test
The last few years, tremendous work has been published regarding biomarkers.
In 2009 and 2013, researchers proposed that the development of reliable noninva-
sive test of endometriosis is one of the top research priorities in endometriosis
[16, 24]. A clinically reliable test for endometriosis can be expected to have a
profound impact on the reduction of health care and individual costs by:
1. Reducing time to diagnosis and the time wasted to see numerous health-care
professionals
2. Subsequently reducing the time before individualized specialist care is invoked
3. Subsequently reducing expensive hit-and-miss treatments
4. Subsequently reducing expensive fertility treatments if the disease is under
control before fertility is impaired [25]
Highly relevant markers known to be involved in the pathogenesis of endometriosis
have been studied such as glycoproteins, inflammatory and noninflammatory
cytokines, adhesions, and angiogenic and growth factors [20]. At present, neither a
single biomarker nor a panel of biomarkers measurable in peripheral blood has been
validated as a noninvasive test for endometriosis [19]. The measurement of serum
CA-125 levels has no value as a diagnostic tool compared to laparoscopy [15].
Although previous studies have shown that various tumor markers, cytokines, and
angiogenic and growth factors show altered levels in peripheral blood (plasma or
serum) of women with endometriosis when compared to controls [19, 26], so far none
of them, alone or in combination, have been validated as a noninvasive test for
endometriosis [19]. Furthermore, at present there is no consensus on the value of
inflammatory factors as biomarkers for endometriosis [20]. Comparable serum IL-6
[27, 28], IL-8 [27, 29], TNF-alpha, and IL-1 [27, 28, 30] levels were previously
reported in women with and without endometriosis. However, other investigators
reported elevated peripheral levels of IL-6 [30, 31], IL-8 [32, 33], TNF-alpha [31, 34],
and IFN-gamma [30] in endometriosis patients compared with controls [20].
So far, studies evaluating panels of biomarkers [3538] have been limited with
respect to the number of biomarkers analyzed, the statistics used, and the lack of
validation in an independent test set of patients [20]. One study proposed two panels
of four biomarkers (annexin V, VEGF, CA-125, and glycodelin or sICAM-1)
measured in plasma samples obtained during menstruation allowed the detection
of ultrasound-negative endometriosis with high sensitivity (82 %) and acceptable
specificity (6375 %) in an independent test data set [39]. In the same study, three
biomarkers (VEGF, annexin V, and CA-125) present in plasma obtained during
menstruation allowed the diagnosis of endometriosis (minimalsevere endometri-
osis, both with and without ultrasound evidence) with 8594 % sensitivity and 62
75 % specificity in an independent test data set [39]. These results are promising but
need to be validated in a prospective study. Surprisingly, inflammatory molecules
did not emerge as biomarkers in this study [39].
Recently, a diagnostic model including patient-reported clinical data derived
from a validated questionnaire predicted any-stage endometriosis poorly, but stages
III and IV accurately, with menstrual dyschezia and a history of benign ovarian
cysts as the strongest predictive factors [40]. More research is needed to add clinical
factors to diagnostic models based on plasma or endometrial analysis.
The development of a noninvasive diagnostic test, from initial biomarker dis-
covery to a clinically approved biomarker assay, is a long, difficult, and uncertain
process [41] and occurs in four different phases as described below:
Phase IPreclinical discovery phase. This phase consists of exploratory preclinical
studies aiming to identify potential biomarkers. In endometriosis research, the
state of the art in this field has recently been reviewed by May et al. [19].
Phase IIPreclinical assay development and validation of a clinically useful
noninvasive diagnostic test in the preclinical setting, as has been done in the
context of endometriosis in our most recent paper [39].
Phase IIIProspective clinical validation and determination of clinical utility. This
phase establishes the diagnostic accuracy and predictive value in the target
population, but this phase has not yet been reached in endometriosis biomarker
research so far.
Phase IVCommercialization: product development by industry, which has not
yet been done successfully for noninvasive endometriosis biomarkers.
Overall, most endometriosis biomarker studies remain at the level of phase I [19]
and only a few have made it to phase II studies. There is a need for well-designed
phase II and phase III trials to make progress in this field.
20.2.1 Nerve Fibers
The most promising efforts in developing a semi-invasive diagnostic test reported

an increased nerve fiber density in the functional layer of eutopic endometrium of
women with endometriosis, uniting the concept of an alternation of eutopic

endometrium with the presence of nerve fibers provoking pelvic pain [42].
The first study to investigate the presence of nerve fibers in eutopic endometrium
as a potential endometriosis biomarker reported a higher density of small nerve
fibers in the functional layer of women with endometriosis compared with women
without endometriosis [43]. Uterine curettage (endometriosis n 25 and control
n 47) and hysterectomy samples (endometriosis n 10 and control n 35) were
immunostained with antibodies against protein gene product 9.5 (PGP 9.5) as
pan-neural marker for both unmyelinated and myelinated nerve fibers and
neurofilament (NF) for myelinated nerve fibers. The most outspoken difference
could be found in the functional layer of the endometrium where PGP 9.5-positive
nerve fibers were observed in all endometriosis cases (on average for all cycle
phases 11 5 mm2 for hysterectomy specimens and 10 5 mm2 for curettage
specimens), but never in control patients (P < 0.001). The difference was the most
obvious in the secretory phase of the cycle, but was also significant in the menstrual
and proliferative phases [43].
To determine the type of small nerve fibers in the endometrium, the same
research group published an additional study [44] in which uterine blocks were
obtained after hysterectomy from women with endometriosis (n 10) and controls
(n 35). Positive staining of the nerve fibers in the functional layer for SP
(1.0 0.2 mm2), CGRP (1.7 0.4 mm2), VIP (8.5 2.3 mm2), and NPY
(9.6 2.8 mm2) and the absence of staining with TH or VAChT antibodies
indicated the presence of different types of sensory C nerve fibers in women with
endometriosis. This finding might be relevant to the mechanism of pain generation
in women with endometriosis.
While the previous studies [43, 44] focused on full endometrial curettage and
full uterine blocks after hysterectomy, clinical relevance of an endometrial bio-
marker is only valid if an endometrial biopsy can generate the same sensitivity and
specificity [45]. Endometrial curettage requires general anesthesia and the need for
hospitalization, while endometrial biopsy sampling can be done in an outpatient
setting [46]. A pilot study including 20 endometriosis patients and 17 controls
compared the diagnostic value of an endometrial biopsy and a full curettage [45].
Much care was given to the correct sampling of the endometrial biopsy, using a
cannula to take a tissue column representing full thickness of the endometrium.
Despite the wide range in nerve fiber densities observed in the functional layer of
the endometrium, previous findings of increased nerve fiber density in endometrium
obtained in hysterectomy specimens were confirmed in endometrium obtained by
endometrial sampling (26.7 55.9 mm2) or by curettage (20.4 33.1 mm2),
suggesting equal diagnostic capacity of the two collection methods. Sensitivity,
specificity, positive predictive value (PPV), and negative predictive value (NPV)
were all 100 %.
To further prove the diagnostic relevance of the detection of nerve fibers in an
endometrial biopsy, a double-blind study [47] was conducted using endometrial
biopsies from 99 consecutive patients who received a laparoscopy for pelvic pain
and/or infertility, including 64 cases with endometriosis and 35 controls without
endometriosis. Immunohistochemical staining with PGP 9.5 showed a mean nerve

fiber density of 2.7 3.4 nerve fibers per mm2 in the functional layer of endome-
trium from nearly all endometriosis patients and an absence of nerve fibers in most
control patients (except in six cases), resulting in a statistically significant differ-
ence between the two groups (P < 0.001). The specificity and sensitivity of the test
were 83 and 98 %. PPV and NPV were 91 and 96 %, respectively.
The accuracy of making an endometrial-based diagnosis of endometriosis was
also investigated by two other independent research groups [48, 49]. In a paper from
Bokor et al. [49], endometrial samples from only the secretory phase were included,
because the initial Tokushige pilot study had reported a higher nerve fiber density in
this phase [43]. Only patients with minimal (n 10) or mild (n 10) endometriosis
were recruited, as they have the greatest need of a semi-invasive diagnostic test [49].
Another 20 patients with a normal pelvis were included as controls. Prevalence
of pain symptoms was comparable in both groups. PGP 9.5, NF, SP, VIP, NPY,
and CGRP expression was detected by immunohistochemistry and showed a
14-fold higher density of small nerve fibers in the functional layer of women
with endometriosis (1.96 2.73 mm2) compared with the control group
(0.14 0.46 mm2) (P < 0.0001). Multivariate analysis showed that the combined
assessment of VIP, PGP 9.5, and SP had the greatest diagnostic accuracy to predict
endometriosis with a sensitivity of 95 %, a specificity of 100 %, a PPV of 100 %, and
an NPV of 95 %. In contrast to the Bokor study (84), results were not controlled for
cycle phase or disease severity in another study [48]. In spite of this limitation, nerve
fibers positive for PGP 9.5 or NF were present in all 12 endometriosis cases (mean
13.1 3.3 mm2) and only in 3 of 15 patients without endometriosis (mean
2.2 4.7 mm2) resulting in a significant difference between the two groups,
although no exact p-value was mentioned [48]. Sensitivity and specificity were not
mentioned, but can be calculated based on the published data to be 100 and 80 %,
respectively. No significant differences in the pain characteristics of both groups
were reported.
An alternative study showed the added value of detection of endometrial PGP
9.5-stained nerve fibers to IL-6 measurement in serum for diagnosis of minimal/
mild endometriosis [50] in endometriosis patients (n 35) compared with controls
(n 40). Specificity and sensitivity of the test for predicting minimal/mild endo-
metriosis regarding the presence of nerve fibers were 92 and 80 %, respectively
(PPV and NPV were 81 and 91 %). These values raised to 100 and 92.5 % (PPV and
NPV 92.7 and 100 %) when IL-6 was added. The exact nerve fiber densities in
endometriosis patients were not mentioned.
A meta-analysis of several studies [43, 4749] included 131 women with
endometriosis and 152 controls [51]. Mean nerve fiber density per mm2 was
significantly higher in endometriosis patients (5.67 12.08) versus controls
(0.78 3.39, P < 0.005) [51]. This meta-analysis demonstrated that PGP 9.5
staining of eutopic endometrium can be used as a semi-invasive test and could
reduce in the delay of diagnosis; however, further research is necessary [51].
Despite the promising data shown in the previously discussed papers, nerve fiber
density in eutopic endometrium is not yet ready to be used in clinical practice.
The abovementioned data were not confirmed by Newman and coworkers, com-
paring 20 patients with endometriosis (minimalmild n 20, moderate n 4, and
severe n 4) to 25 controls [52]. No significant difference was observed between
the disease and control group in PGP 9.5-positive nerve fibers, visualized by
immunohistochemistry [52]. Using western blotting as a quantitative method to
detect the neural markers, all three markers were expressed in endometrium from
both endometriosis cases and controls (PGP 9.5: P 0.0991, VR I: P 0.0621,
NGFp75: P 0.2586) [52].
In another study, no statistical difference was observed in endometrial nerve
fiber density, determined by PGP 9.5 staining, between women with (n 47) and
without endometriosis (n 21) [53]. This study, however, was marked by several
methodological drawbacks. Firstly, threshold for endometrial biopsy quality was
low (one low-power field of well-oriented endometrial mucosa sample perceived to
be sufficient to being classified as satisfactory). Secondly, the exact endometrial
nerve fiber density was not mentioned, since data were classified as positive or
negative depending on the presence or absence of nerve fibers [53]. This study
highlighted that methodological factors such as the method of endometrial biopsy
sampling and image analysis might influence the consistency of results for this
semi-invasive diagnostic test [53]. Taking into account the uneven distribution of
the nerve fibers in a biopsy, reliable results may require inspection of more than one
section per biopsy [45]. Furthermore, the whole surface of an endometrial section
should ideally be examined [49] as opposed to counting endometrial nerve fibers in
randomly chosen fields [45]. Additionally, it is crucial that background staining
during immunohistochemistry is reduced as much as possible, which can be
challenging. In a research context, results should be analyzed by an experienced
pathologist. While it is essential to refine methodology, it is equally important to
select and phenotype the study population. All cases in the endometriosis group
should have laparoscopically confirmed endometriosis, preferably with histological
confirmation of the presence of endometrial glands and stroma in the lesions [49].
In some of the studies, this criterion was not met, as only in a part of the cases
histological confirmation of endometriosis was available [45, 47]. Further,
according to the modified QUADAS (Quality Assessment of Diagnostic Accuracy
Studies) criteria [19], detailed information should be available regarding the demo-
graphics such as disease stage and cycle phase [49]. Study patients ideally have not
received hormonal medication 3 months prior to surgery, because systemic expo-
sure to hormonal medication might reduce the presence of nerve fibers in eutopic
endometrium [43]. However, this medication-free period was as short as 1 month in
one study [50] and could have been even shorter in another study stating that
patients were not on medication only at the time of laparoscopy [45]. Additionally,
one study included three patient groups (one on current hormonal therapy (n 11),
one without hormonal therapy (n 44), and a group with unknown status (n 13))
and found an overall poor sensitivity for the test that was independent of the
treatment status [53]. This lack of significance might have been due to the
low sample size in each group. The control group should be equally well charac-
terized and should have absence of endometriosis confirmed by laparoscopy
(QUADAS criteria [19]). Included controls were only described in detail in a

number of studies [43, 44]. They consisted of women undergoing tubal sterilization,
assessment prior to tubal reanastomosis or investigation of infertility [43] and
women with uterine fibroids, uterine prolapse, abdominal adhesions, and abnormal
menstrual bleeding [44]. Two studies included only controls with a normal pelvis
[49, 50]. Pain characteristics should be comparable in case and control groups,
although this did not always seem to be the case. In the double-blind study by
Al-Jefout, patients with pain symptoms were present in 87.5 % of the endometriosis
cases, but only in 45.7 % of controls [47]. This could influence the results as
previous studies have postulated a link between pain and nerve fiber ingrowth in
ovarian or deep infiltrating endometriosis [54, 55]. Interestingly, the association
between pain and the presence of nerve fibers in endometrium exists in women with
other gynecological disorders such as adenomyosis or uterine fibroids, suggesting
that the presence of nerve fibers is linked to the diagnosis of pelvic pain rather than
to the occurrence of endometriosis [20, 56].
Implemented statistics were comparable in most studies with the use of univar-
iate analysis such as the MannWhitney U-test as a nonparametric test to compare
two groups [43, 44, 47] and the KruskalWallis chi-square test for the comparison
of multiple groups [47]. The KolmogorovSmirnov test [48] was used by one group
to determine normality, before performing a student t-test or related nonparametric
test to evaluate nerve fibers [48]. The use of the KolmogorovSmirnov test is
discouraged, because of its poor performance in assessing normality [57]. Other
groups mentioned using the student t-test, but did not state whether they had
verified a normal data distribution first [50, 53]. Advanced multivariate statistical
analysis was only performed in one study by Bokor and coworkers, using multi-
variate logistic regression and leave-one-out cross-validation (LOO-CV) analysis
with least-squares support vector machines (LS-SVM) modeling [49].
In conclusion, it can be stated that the determination of nerve fiber density in the
endometrium as a diagnostic test for endometriosis is a promising prospect. How-
ever, the techniques and study populations should be randomized and further
validation with larger patient groups should be performed before this test can be
used in the clinic. Up to now, none of the studies evaluating nerve fibers in
endometrial biopsies have reached phase III of biomarker development. To make
progress in this field, there is a need for well-designed phase II and III studies.
Validation should be carried out in a patient population experiencing pain/infertility
with a 30 % prevalence of endometriosis.
20.2.2 MicroRNAs and Endometriosis
Since endometriosis is a multifactorial and polygenic disease, aberrant expression

of microRNA (miRNA) has been proposed as a potential pathogenic mechanism
[20, 58]. Essentially, miRNAs are short (~22 nucleotides) single-stranded noncod-
ing RNAs with the capacity to regulate gene expression at a posttranscriptional level
through translational repression or messenger RNA (mRNA) degradation [59].

One miRNA may regulate the expression of several hundreds of mRNAs [60].
Diagnostic use of miRNA signatures has been proposed for various diseases,
such as cancer, cardiovascular diseases, rheumatic diseases, and neurological
disorders [61]. In cancer, miRNAs perform better than mRNAs to classify poorly
differentiated tumors [62].
20.2.2.1 Endometrium
Between eutopic endometrium and endometriotic lesions, differential expression of

a number of miRNAs has been shown and some of their mRNA targets, as predicted
by in silico algorithms, have previously been identified as dysregulated in endo-
metriosis [6366]. A number of cellular events involved in the development of
endometriotic lesions has been linked to miRNA dysfunction, such as hypoxic
injury, inflammation, tissue repair, disrupted cell cycling, extracellular matrix
remodeling, angiogenesis, cellular movement, and DNA methylation [6568].
More importantly, differences in miRNA expression between eutopic endome-
trium of endometriosis patients and controls have been found, providing a potential
role for miRNAs as biomarkers or therapeutic tools in endometriosis [20]. In a
study conducted by Toloubeydokhti et al., miR-17-5p, miR-23a, miR-23b, and
miR-542-3p were upregulated in eutopic endometrium (n 5) of patients with
endometriosis, compared with controls (n 5) [69]. These miRNAs have previ-
ously been linked to cancer [70]. The predicted downstream targets were known to
be involved in endometriosis (steroidogenic acute regulatory protein (StAR), aro-
matase, and COX-2) and were confirmed to be upregulated, using RT-PCR [69].
However, an independent research group found reduced expression of miR-23a and
miR-23b in eutopic endometrium of endometriosis cases compared with disease-
free controls [71]. This reduction in miRNA expression correlated with a 2.26-fold
increase (P 0.041) in steroidogenic factor 1 (SF-1) expression [71]. In another
study, endometrial miR-9 and miR-34 were significantly reduced in patients with
moderate/severe endometriosis (n 4) compared with controls with uterine
leiomyomata (n 3) [72]. Their targets were predicted to enhance the proliferative
capacity of the endometrium [72]. In another study, an increased miR-21 expres-
sion throughout the menstrual cycle was reported to allow distinction between
severe and mild endometriosis (controls: n 12, mild endometriosis: n 19, severe
endometriosis: n 44) and associated with a downregulation of tumor suppressor
genes such as PTEN [73]. In another study, it was found that miR135a/b expression
was significantly upregulated in eutopic endometrium of endometriosis patients
(n 32) compared with controls (n 50) in the proliferative phase (miR135a and
miR135b) and the secretory phase (miR135b) [74]. This upregulation of miR135a/b
was marked by a downregulation of HOX10 mRNA and protein (a regulator of
endometrial receptivity) which was reversible by the addition of miR-135a/b
inhibitors [74]. In yet another paper, the downregulation of miR-126 was observed
in ectopic endometrium (n 16) and in eutopic endometrium (n 31) from women
with endometriosis (n 31) when compared with controls (n 27) [75].

This downregulation of miRNA was inversely correlated with the expression of
Crk, an oncogene [75].
20.2.2.2 Peripheral Blood
Recently, several studies of miRNA in peripheral blood have been performed in the
context of endometriosis [7678]. One study, investigating circulating miRNAs in
plasma, showed a significant downregulation of miR-17-5p ( p 0.011), miR-20a
( p 0.0020), and miR-22 ( p 0.0002) in women with endometriosis (n 23)
compared with women without endometriosis (n 23) [78]. Receiver operating
characteristic (ROC) curve analysis permitted the calculation of the area under the
curve (AUC) (0.74, 0.79, and 0.85 for miR-17-5p, miR-20a, and miR-22, respec-
tively) after which a cutoff value could be set to determine sensitivity (70.0, 60.0,
90.0 % for miR-17-5p, miR-20a, and miR-22, respectively) and specificity (70.0,
90.0, 80.0 % for miR-17-5p, miR-20a, and miR-22, respectively) to differentiate
between women with and without endometriosis [78]. Combined assessment of the
three miRNAs resulted in an AUC of 0.90 [78]. Additionally, another study showed
that discrimination was possible between plasma of healthy controls (n 20) and
endometriosis patients (n 33) with 88 % sensitivity and 60 % specificity
(AUC 0.90), based on the assessment of miR-16, miR-191, and miR-195 which
were all highly expressed in endometriosis [77]. Endometriosis and endometriosis-
associated ovarian cancer (EAOC) (n 14) could be distinguished from each other
through a combination of miR-21, miR-362-5p, and miR-1274a with 57 % sensi-
tivity and 91 % specificity and an AUC of 0.92. Distinction of endometriosis and
serous ovarian cancer (SOC) (n 21) was possible with 90 % sensitivity and 73 %
specificity (AUC 0.88), based on the assessment of miR-362-5p, miR-628-3p, and
miR-1915 [77]. A trend of elevated plasma miRNA expression compared with
healthy controls was found in endometriosis cases and even more so in EAOC
cases. This suggests that endometriosis might be a precursor stage of EAOC and the
possibility for miRNA signatures to act as disease progression markers [77].
In serum of patients with endometriosis (n 60) and controls (n 25) the
combination of miR-199a, miR-122, miR-145*, and miR-542-3p could predict
endometriosis with 93.22 % sensitivity and 96.00 % specificity (AUC 0.994) [76].
Currently, there is no agreement on which circulating miRNA can be used for
data normalization. One group selected U6 as a normalization control because of its
use as internal control in other studies [76]. However, other investigators preferred
miR-16 as endogenous control because it is more stable and less variable in
circulation than other miRNAs [78]. Yet another research group used miR-132
for data normalization, as they found this miRNA to be homogeneously expressed
across all samples [77].
It is also important to distinguish the presence of miRNAs in either plasma
or serum. Although higher miRNA concentrations were observed in plasma than
in serum in one study [79], this was not confirmed in another study [80].
This discrepancy might have been due to pre-analytical variability concerning
blood tube type or differences in sample-processing protocols [79]. Conversely,
other studies have shown an increased concentration of miRNAs in serum samples
when compared to plasma samples of the same patient, although they conclude that
plasma might be the sample of choice because miRNAs that are released during
the coagulation process in serum samples may interfere with the true miRNA
profile [81].
Interestingly, miRNAs are exceptionally stable in plasma, serum, and tissue
samples, making them excellent candidates for biomarker research [80]. Possibly
miRNA is protected from RNases in blood by being packaged in vesicles or bound
to RNA-binding proteins [82].
Despite the potential advantages of a miRNA-based blood test for endometri-
osis, some possible pitfalls should be taken into consideration. It should be noted
that apart from their best known function as transcriptional repressor, miRNAs can
act as translational activators [83]. Therefore the relationship between miRNA
and target mRNA is not necessarily an inverse one [72]. Additionally, in most
miRNA studies, specific mRNA targets are often not experimentally verified, but
only predicted by various computational algorithms [65, 72]. Beside the influence
of the choice of mathematical algorithm, one mRNA can be the target of multiple
miRNAs that might not all be altered, rendering validation in an in vitro
setup essential to determine the effect of the dysregulation of the miRNA of interest
[65, 72].
Since miRNA patterns change under the influence of reproductive hormones and
are thus altered in different phases of the menstrual cycle [84], an adequate control
group should be chosen, preferably without any other illness as this could also
influence the miRNA profile [78].
The discrepancies observed in different miRNA results can be explained by
differences in patient selection, microarray protocol, and choice of housekeep-
ing genes for data normalization. Therefore a standardized methodological
approach needs to be determined and controls and patients need to be selected
according to the QUADAS criteria. The rationale for implementing next-
generation sequencing is that a larger amount of miRNAs can be examined,
without being dependent on the availability of probes [66]. Exceptionally
important is the role of advanced statistical methodology in studies where
many variables are compared. Therefore, all data should be controlled for
multiple testing [85].
In conclusion, miRNAs are interesting subjects in the further development of
biomarker research, although more extensive research in a larger population and
validation in independent test sets should be conducted with full awareness of
methodological issues and the complexity of miRNA mechanisms.
20.3 Conclusions
Despite its gradual progress, the biomarker research field still faces the challenge of
successfully developing a clinically approved biomarker assay for endometriosis [20].
Up to now, no semi- or noninvasive diagnostic test exists for endometriosis [19].
Most studies so far have included limited numbers of patients, limited assessment
of different cycle phases and endometriosis stages, limited number of biomarkers
analyzed, limited statistical analysis (mostly univariate statistical analysis only),
and the lack of validation in an independent test set of patients [20].
In the past, collaborations between research centers have been limited and
standard operating procedures (SOPs) are different among centers [20]. Most
biomarker studies remain at the level of phase I, the preclinical discovery phase,
as reviewed by May et al. [19, 42]. Only a few biomarkers make it to phase II, the
preclinical assay development and validation [39, 47, 86].
To develop a diagnostic test for endometriosis, semi-invasive techniques utiliz-
ing endometrial biopsies have been explored [87]. An increased amount of small
unmyelinated nerve fibers in the functional layer of eutopic endometrium of
endometriosis patients has been reported [45, 49, 88]. However, prospective vali-
dation of this method in a blinded fashion is needed in a larger patient population,
using a standardized biopsy sampling technique and standard immunohistochemi-
cal and statistical methods. Validation should be carried out in a patient population
experiencing pain/infertility with a 30 % prevalence of endometriosis.
At present, neither a single biomarker nor a panel of biomarkers measurable in
peripheral blood has been validated as a noninvasive test for endometriosis [19],
although panels with reasonable specificity and sensitivity have recently been
published [39]. Panels of biomarkers that have been proposed as diagnostic tests
in phase I and II trials should be validated in prospective phase III studies [20].
In this phase, diagnostic accuracy of the proposed panel needs to be confirmed in
an independent test population with infertility/pain scheduled for surgery [20].
The predicted outcome should be compared with the actual presence of endome-
triosis. Future studies should also focus on the large and clinically relevant
population of endometriosis patients using hormonal medication, which is under-
represented in published biomarker discovery studies.
In order to improve the specificity and sensitivity of previously proposed
diagnostic models, several choices are possible: using advanced protein technology
to discover new and unknown biomarkers, combining a blood test with a semi-
invasive test, or adding clinical factors to a model.
Advanced protein technologies such as antibody-based large-scale protein
arrays, allowing concurrent detection of up to 1,000 proteins, might be required
to improve the diagnostic power of a noninvasive blood test for endometriosis.
Antibody-based microarrays have already been applied in biomarker screening for
cancer research [89]. Combined assessment of a blood sample and an endometrial

biopsy is another option to increase the power of a diagnostic test. A study has been
published, combining a noninvasive test measuring IL-6 serum levels with a semi-
invasive test for the presence of nerve fibers [50]. Despite the high sensitivity and
specificity obtained by this method, it might not be suitable for clinical use due to
the need for two interventions (blood and endometrial biopsy sampling). Addition-
ally, the diagnostic accuracy of a test might be enhanced by the incorporation of
clinical factors into a non- or semi-invasive test. A diagnostic method published by
Nnoaham et al. predicted any-stage endometriosis poorly, but stages III and IV
accurately [40]. The most predictive factors for the presence of endometriosis were
menstrual dyschezia and a history of benign ovarian cysts [40].
While genomics, transcriptomics, and proteomics have already been covered
extensively in endometriosis research, metabolomics is an underdeveloped field
with only one study published up to date [90]. More extensive research in this topic
should clarify whether the alteration of the metabolomic profile in the blood of
women with endometriosis could serve as a diagnostic test for endometriosis.
Another field of interest could be stem cell markers and endometriosis. Stem
progenitor cells may serve as early markers and also detect recurrence [91]. The
endometrium contains endometrial/stem progenitor cells that have been reported to
be present in the peritoneal cavity in women that have retrograde menstruation [91].
Positive immunostaining for stem cell markers such as CD9, CD34, c-Kit, Oct-4,
and Musashi-1 was detected in isolated epithelial and stromal cells in eutopic and
ectopic endometrium [92]. The expression of stemness-related markers suggested
that endometriosis arises as a clonal proliferation with the putative involvement of
stem cells (2). The limitation of this study was the small sample size [20].
In conclusion, biomarker research will have to proceed from the discovery phase to
the validation phase, focusing on successfully conducting phase III and IV trials [20].
To achieve phase III biomarker validation in both semi- and noninvasive testing,
independent patient populations should be investigated, preferably with a realistic
prevalence of endometriosis (30 % in women with infertility and 3050 % in women
with pelvic pain) [20]. According to one of the QUADAS criteria, patient character-
ization regarding cycle phase and endometriosis stage should be clearly stated and the
control group should be standardized [19, 20]. Patients should have laparoscopically
confirmed presence or absence (for controls) of endometriosis [19]. The different
stages and clinical classifications of endometriosis should be taken into account in
further research, treating peritoneal, ovarian, and rectovaginal septum endometriosis
as different entities [93, 94]. Because of the different types (superficial, deep, cyst)
and location of endometriosis, it is possible that a different subset of biomarkers may
be required for the diagnosis of different stages of endometriosis [16], i.e., women
with peritoneal endometriosis may have different markers when compared
to those with rectovaginal endometriosis [19, 20]. This requires more genomic
analysis of the different lesion types and different locations.
Statistical approaches should be multivariate, instead of the univariate
approaches used in many studies now, and the involvement of biostatisticians is
crucial to extract a diagnostic test from raw data [20]. Standardized protocols for
sample collection, processing, and storage along with reporting uniform clinical
information are crucial to compare results between studies and to enable a multi-
center biobanking approach [16, 95]. Collaborations are essential to obtain the large
sample sizes that are required for the statistical power of validation studies [16].
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Chapter 21
Classification of Endometriosis
Hans-Rudolf Tinneberg, Frank Tuttlies, Eniko Berkes,

Gabriele Anja Krombach, Andreas Hackethal, Lutz Konrad,
and Frank Oehmke
Abstract As endometriosis is a chronic disease which debilitates women of

reproductive age and has a negative impact on work force and quality of life in
general, a disease classification would help to estimate the extent of impact on
various aspects.
The most common classification has been established by the American Fertility
Society and finally revised as American Society of Reproductive Medicine Classi-
fication. It is based on a laparoscopic evaluation and scoring and stages from I to IV
can be applied, reflecting the extent of disease.
Since this rASRM classification does not clearly reflect a prognosis for infertility
patients, the endometriosis fertility index (EFI) was developed, which is eased on
the rASRM classification, but in addition includes predicted ovarian and tubal
function and historical parameters.
Since these two scoring algorithms do not consider deep infiltrating endometri-
osis, a classification, the Enzian classification was developed according to the TNM
classification for cancer. It describes the extent of disease within three different
pelvic compartments classified into three sizes.
The development of MRI led to an accurate preoperative mapping of respective
endometriotic lesions and enabled an MRI-based radiological classification system
H.-R. Tinneberg (*) E. Berkes L. Konrad F. Oehmke

Direktor der Frauenkilnik, Department of Obstetrics and Gynaecology,
University of Giessen and Marburg, Klinikstrae 33, 35385 Gieen, Germany
e-mail: Hans-Rudolf.Tinneberg@gyn.med.uni-giessen.de
F. Tuttlies
Department of Obstetrics and Gynaecology, Public Hospital Villach, Villach, Austria
G.A. Krombach
Department of Radiology, University of Giessen, Gieen, Germany
A. Hackethal
Queensland Centre of Gynaecological Cancer, Royal Brisbane and Womens Hospital,
Brisbane, Australia

342 H.-R. Tinneberg et al.
(MARIE) that reflects both the localisation of endometriosis and concomitant

reproductive function.
Up to date no classification exists that concludes all aspects of the disease, the
pathophysiology, localisation, progression, pain and infertility, treatment, progno-
sis and recurrence.
Keywords Adhesions Classification Deep infiltrating endometriosis Pain

Sterility
21.1 Introduction
Since the first descriptions of endometriosis by Lockyer et al. in 1918 [1] and
Sampson et al. in 1921 [2] it became necessary to classify the disease with the aim
to include histological differentiation as well as differentiation according to loca-
tion and severity of the disease. Ideally, a classification has a common language that
specifies the diagnosis, thereby allowing a standardisation of disease assessment.
Furthermore, it enables research, as well as the clinical community to compare
findings.
The early classification systems were based on the anatomical localisation of the
disease and its similarity to malignancy. The classification systems before 1973
described mainly the anatomical distribution of endometriosis and did not correlate
with the clinical outcome; thus they have not received widespread clinical accep-
tance. The Acosta classification [3] was the first one where a direct relationship
could be established with different stages of the disease and clinical pregnancy
rates. As a further development, the staging system of Kistner [4] tried to reflect
the natural progression of the disease with moving from early peritoneal implants
to ovarian and fallopian tube-ovarian involvement and dissemination within the
pelvis. One of the most precise and detailed classification systems was the Buttram
classification [5], which, however, has not received widespread acceptance.
Table 21.1 gives an overview of the most common classification systems, which
have been made since 1918.
An ideal classification would require that a consensus be reached over empiri-
cally as well as scientifically based data that are comprehensive for all cases. Terms
would need to be defined unambiguously, and anatomic lesions, extent of disease,
severity of pain, impact on fertility and organ function within the pelvis as well as
social impact would need a simple translation into a verbal description. Founding
variables would need to be recognised and the risk of complications should be
indicated. Finally the ideal classification would also guide the treatment and
estimate risk for recurrence.
To date, we are far from having an ideal endometriosis classification.
In this chapter we are introducing and analysing the currently used classification
systems.
21 Classification of Endometriosis 343
Table 21.1 The most important historical classification systems of endometriosis

Classification
Year system Characteristics
1918 Lockyer [1] First descriptions of the disease
1921 Sampson [2]
1949 Wicks and Grade I: The wall of the cavity is lined by large bloated phagocytic
Larson [6] cells containing blood pigment and cellular debris, most abundant
on the inner side of the wall
Grade II: The epithelium remains and the individual epithelial cells
appear atrophic. The stroma is partially or completely replaced
by bloated phagocytic cells
Grade III: Epithelium and stroma are both present. Neither the
epithelium nor the stroma appears to be materially influenced
by the cyclic hormonal situation of the ovary
Grade IV: The lesion contains endometrium resembling that seen
at some stage of the menstrual cycle as found in the uterus. Glands
are always present and are supported by an abundant endometrial
stroma
1951 Huffman [7] Stage I
a. Limited to uterosacral ligaments and/or
b. Limited to ovaries and/or
c. Superficial peritoneal implants
Stage II
a. Extensive involvement of one ovary, with lesser involvement of
second ovary and/or
b. Superficial implants both ovaries and/or
c. Superficial bowel implants and/or
d. Infiltrating lesions of uterus and uterosacral ligaments
Stage III
a. Extensively infiltrating both ovaries and/or
b. Bilateral ovarian endometriotic cysts and/or
c. Deeply invading rectovaginal lesions and/or
d. Infiltrating nonobstructing bowel implants
Stage IV
a. Vesical invasion and/or
b. Intestinal invasion, obstructive and/or
c. Ureteral involvement
1954 Sturgies Stage I: Early development
and Call [8] Stage II: Active development
Stage III: Endometrial inactivity (postmenopause)
1962 Riva [9] Staging categories are defined according to the cumulative count
of pelvic structures involved and surrounding adhesions. The first
scale which tries to define who might benefit from the therapy
1966 Beecham [10] Stage I: Scattered, small (12 mm) spots anywhere in the pelvis at
laparotomy
Stage II: Uterosacral ligaments, broad ligaments, cervix and ovaries
are, collectively or individually, fixed, tender, nodular and slightly
enlarged
(continued)
Table 21.1 (continued)

Classification
Stage III: The same as stage II, with ovaries at least twice normal size;
uterosacral ligaments, rectum and adnexa are confluent and the
cul-de-sac is obliterated
Stage IV: Massive involvement, internal pelvic viscera cannot be
clearly distinguished by palpation
Stages IIIV may be used to describe either the palpable finding at the
physical examination or the palpable-visual findings at operation
1973 Acosta [3] Mild
1. Scattered, fresh lesions (i.e. implants not associated with scarring or
retraction of the peritoneum) in the anterior or posterior cul-de-sac
or pelvic peritoneum
2. Rare surface implant on ovary, with no endometrioma, without
surface scarring and retraction, and without periovarian adhesions
3. No peritubular adhesions
Moderate
1. Endometriosis involving one or both ovaries, with several surface
lesions, with scarring and retraction, or small endometriomas
2. Minimal periovarian adhesions associated with ovarian lesions
described
3. Minimal peritubular adhesions associated with ovarian lesions
described
4. Superficial implants in the anterior/posterior cul-de-sac with scar-
ring and retraction. Some adhesions, but not sigmoid invasion
Severe
1. Endometriosis involving one or both ovaries with endometrioma
>2 2cm (usually both)
2. One or both ovaries bound down by adhesions associated with
endometriosis, with or without tubal adhesions to ovaries
3. One or both tubes bound down or obstructed by endometriosis;
associated adhesions or lesions
4. Obliteration of the cul-de-sac from adhesions or lesions associated
with endometriosis
5. Thickening of the uterosacral ligaments and cul-de-sac lesions from
invasive endometriosis with obliteration of the cul-de-sac
6. Significant bowel or urinary tract involvement
1977 Kistner [4] Stage I: Areas of endometriosis are present on the posterior pelvic
peritoneum (cul-de-sac, uterosacral ligaments) or on the surface of
the broad ligaments but do not exceed 5 mm in diameter. Avas-
cular adhesions may involve the tubes, but the fimbriae are free.
The ovaries may show a few avascular adhesions, but there is no
ovarian fixation. The surfaces of the bowel and the appendix are
normal
Stage IIA: Areas of endometriosis are present on the posterior pelvic
peritoneum (cul-de-sac, uterosacral ligaments) and the broad
ligaments but do not exceed 5 mm in diameter. Avascular adhe-
sions may involve the tubes, but the fimbriae are free. Ovarian
involvement has been subclassified as follows: IIA-1, endometrial
cyst or surface is 5 cm or less; IIA-2, endometrial cyst or surface is
(continued)

Classification
over 5 cm; IIA-3, ruptured endometrioma, the bowel and the
appendix are normal
Stage IIB: The posterior leaf of the broad ligament is covered by
adherent ovarian tissue. The tubes present adhesions not remov-
able by endoscopic procedures. The fimbriae are free. The ovaries
are fixed to the broad ligament and show areas of endometriosis
over 5 mm in diameter. The cul-de-sac presents multiple implants,
but there is no adherent bowel nor is the uterus in fixed position.
The bowel and the appendix are normal
Stage III: The posterior leaf of the broad ligament may be covered by
adherent tube or ovary. The tubal fimbriae are covered by adhe-
sions. The ovaries are adherent to the broad ligament, and tube
may or may not show surface endometriosis or endometriomas.
The cul-de-sac shows multiple areas of endometriosis, but there is
no evidence of adherent bowel or uterine fixation. The bowel and
the appendix are normal
Stage IV: Endometriosis involves the bladder serosa, and the uterus is
in fixed, third-degree retroversion. The cul-de-sac is covered by
adherent bowel or is obliterated by the fixed uterus. The bowel is
adherent to the cul-de-sac, uterosacral ligaments or uterine corpus.
The appendix may be involved by the endometriotic process
1974 Mitchell and Similar staging system to that used in gynaecological malignancies
Farber [11] with stage V for malignant transformation
1979 Buttram [5] Stage I (Peritoneum)
A. No peritoneal involvement
B. Scattered superficial surface endometrial implants on the pelvic
peritoneum (anterior or posterior cul-de-sac, uterosacral ligaments
or the broad ligaments), which do not exceed 5 mm in diameter.
Neither tubal nor ovarian involvement
C. Same as for B, but invasive endometriosis or plaques or endome-
trial implants >5 mm in diameter. Fine, filmy adhesion may be
present that may be lysed without great danger of resultant
adhesions
Stage II (Ovarian): 1, right; 2, left; 3, bilateral
A. No ovarian involvement
B. Superficial surface endometrial implants of ovary of <5 mm in
diameter, which can be removed by scraping or fulguration with-
out great danger of resultant adhesions. Fine, filmy adhesions may
be present and lysed without great danger of resultant adhesions
C. Invasive endometriosis (plaques or endometrioma) >5 mm but
<2 cm that requires surgical removal. Fine, filmy adhesion may be
present, which may be lysed without great danger of resultant
adhesions
D. Invasive endometriosis >2 cm that requires surgical removal or a
ruptured endometrioma of any size. Fine, filmy adhesion may be
adhesions
E. B, C or D with sufficient dense adhesions to fix ovary to adjacent
tissue (usually posterior leaf of broad ligament)
(continued)

Classification
Stage III (Tubal): 1, right; 2, left; 3, bilateral
A. No tubal involvement
B. Superficial endometrial implants on tube that do not exceed 5 mm
in diameter and can be removed by craping or fulguration without
great danger of resultant adhesions. Fine, filmy adhesions may be
adhesions
C. Invasive endometriosis (plaques or endometrioma) >5 mm but
<2 cm that requires surgical removal. Fine, filmy adhesions may
be present, which may be lysed without great danger of resultant
adhesions
D. Tube involved with adhesions that distort tubal anatomy and/or
limit tubal movement. Fimbriae are free and tube is patent. C may
be present
E. Fimbriae are covered by adhesions or distal end of tube is
occluded. B, C or D may be present
Stage IV (Cul-de-sac)
A. Neither B nor C is present
B. Invasive endometriosis of bladder or colon
C. Posterior cul-de-sac obliterated and/or uterus fixed and retroverted.
Bowel or adnexa may be adherent to cul-de-sac area. B is usually
present
21.2 Classification of Superficial Endometriosis
21.2.1 The rASRM Classification
None of the classifications before 1978 have been widely accepted in the clinical
practice, which motivated the American Fertility Society (AFS) to form a panel and
introduce a new classification that has been published in 1979 [12]. The first
revision was published in 1985 [13] and appeared in its final version in 1996
when the society had changed its name into American Society of Reproductive
Medicine [14].
Even though it is called a classification, it rather is a scoresheet, where the
peritoneum, the ovaries, the tubes and the cul-de-sac are listed (Fig. 21.1).
The size and depth of lesion corresponds to points, which by analogy are also
assigned for adhesions on the ovaries and fallopian tubes as well as points for partial
or complete obliteration of the cul-de-sac. A schematic drawing is provided where
the localisation of lesions and adhesions can be drafted. The summing up of all
points yields in a score, which then allows classifying the endometriosis into four
grades of severity: stage I (minimal endometriosis: 15 points), stage II (mild
endometriosis: 615 points), stage III (moderate endometriosis: 1640 points) and
Fig. 21.1 The revised American Society for Reproductive Medicine classification of endometri-
osis. With permission from Elsevier (Fertility and Sterility, Licence number: 3184950406638)
Fig. 21.1 (continued)
stage IV (severe endometriosis >40 points). In order to exemplify the different

stages of the disease, examples are given which show the mode of scoring and the
summing up of points.
The rASRM classification is currently the best known and the most widely used
system for clinical and scientific applications throughout the world. Especially with
examples given, it is easy to use and the four stages of severity can easily be
understood by health professionals as well as by patients.
Unfortunately, this staging system involves a major potential of observer errors

and has to be regarded as an arbitrary scoring system. Its reproducibility is limited
and it fails to consider the different morphological lesion types (e.g. black or dark
bluish lesions, red spots, white opacification, red-flame-like lesions, yellowish
patches), as it has been described by Mettler et al. [15]. With respect to correlation
to clinical symptoms Vercellini [16] reported a poor correlation between the extent
of the disease and pelvic pain and Fujishita [17] a poor correlation between the
extent of the disease and infertility. In particular the rASRM classification does not
take into account the involvement of reputable structures which in essence means
that deep infiltrating endometriosis (DIE) as the most impacting endometriosis is
not represented.
Despite the disadvantages of the rASRM classification for classifying endome-
triosis, it is still widely used as the most popular endometriosis scoring system. It is
easy to apply and publications can compare stages of severity.
21.2.2 The Endometriosis Fertility Index
The most widely used rASRM and Enzian scores (see below) describe properly the
anatomical distribution of the respective superficial and deep infiltrating
endometriotic lesions and concomitant adhesions, but are not eligible to provide
information about the clinical outcome, the pain reduction and the reproductive
performance after surgery.
In 2010 Adamson and Pasta analysing the clinical characteristics and reproduc-
tive results after surgical intervention of 569 infertile endometriosis patients in the
USA in a prospective study, proposed a new staging system, the endometriosis
fertility index (EFI) [18]. EFI predicts pregnancy rates in patients with surgically
scored endometriosis who attempt non-ART conception. EFI could be regarded as a
specifically further developed form of the rASRM classification, focusing on the
reproductive outcome, and is not intended to assess the pain symptoms or predict
the pain-reducing effect of the surgery.
The EFI score is based on three surgical factors and on one history factor
that are presented in Fig. 21.2.
In the first step the least function (LF) score at conclusion of surgery is defined.
LF reflects the predicted function of the fallopian tubes, the fimbriae and the
ovaries, each scored from 0 to 4, depending on absent or nonfunctional state, severe
dysfunction, moderate dysfunction, mild dysfunction and normal state. Table 21.2
represents the description of least function terms.
The better the function the higher the score. Scores are added and give the least
function score. The LF score is completed with the categorised and valued rASRM
lesion score as well as the rASRM total score.
The historical factors contribute to age, duration of infertility and history of prior
pregnancies. The lower the age, the lower the duration of infertility, and the higher
the number of previous pregnancies, the higher the historical score (Fig. 21.2).
Fig. 21.2 Endometriosis fertility index (EFI). With permission from Elsevier (Fertility and
Sterility, licence number: 3184941294427)
The total historical as well as the total surgical scores sum up to a score ranging
between 0 and 10, with 0 representing the poorest and 10 the best prognosis. A
coloured graph shows the respective estimated percentage of pregnancy likelihood
depending on time after surgery and EFI score.
Table 21.2 Least function terms after Adamson and Pasta [18]
Structure Dysfunction Description
Tube Mild Slight injury to serosa of the fallopian tube
Moderate Moderate injury to serosa or muscularis of the fallopian tube; moderate
limitation in mobility
Severe Fallopian tube fibrosis or mild/moderate salpingitis isthmica nodosa;
severe limitation in mobility
Nonfunctional Complete tubal obstruction, extensive fibrosis or salpingitis isthmica
nodosa
Fimbria Mild Slight injury to fimbria with minimal scarring
Moderate Moderate injury to fimbria, with moderate scarring, moderate loss of
fimbrial architecture and minimal intrafimbrial fibrosis
Severe Severe injury to fimbria, with severe scarring, severe loss of fimbrial
architecture and moderate intrafimbrial fibrosis
Nonfunctional Severe injury to fimbria, with extensive scarring, complete loss of
fimbrial architecture, complete tubal occlusion or hydrosalpinx
Ovary Mild Normal or almost normal ovarian size; minimal or mild injury to
ovarian serosa
Moderate Ovarian size reduced by one-third or more; moderate injury to ovarian
surface
Severe Ovarian size reduced by two-thirds or more; severe injury to ovarian
surface
Nonfunctional Ovary absent or completely encased in adhesions
Besides introducing the new EFI score, Adamson has prospectively validated
and proven the effectiveness of the new scoring system analysing the predicted and
actual reproductive results of 222 North American surgically treated endometriosis
patients [18]. Three further external studies, performed in China, France and
Belgium, have validated as well the clinical usefulness of the EFI score. In the
study of Wei et al., 350 infertile patients were studied retrospectively. Within
3 years after surgery the cumulative pregnancy rates with EFI scores 8, 9 and
10 were 62.5, 69.8 and 81.1 % and with EFI scores 5, 6 and 7, 49.8, 43.9 and 41.6 %
respectively, in accordance with the estimated pregnancy rates [19]. A French study
of Yacoub et al. investigated in a retrospective study 132 infertile endometriosis
patients and found that EFI showed a significant association between the severity of
endometriosis, infertility and postoperative cumulative birth rates. However, the
rASRM score fall short to predict pregnancies. The authors suggested that the EFI
should be the main component in the choice of the postoperative ART
management [20].
A further study performed in a Belgian population with 233 infertile endome-
triosis patients has also validated the effectiveness of the EFI and found the LF
score the most important contributor to the total EFI score among all the other
variables [21]. The authors concluded that the EFI classification system is a useful
tool in counselling infertile endometriosis patients about their reproductive chances
after surgery.
21.3 Classification of Deep Infiltrating Endometriosis
Several approaches have been published in the literature to classify deep infiltrat-
ing endometriosis (http://www.endometriose-sef.de) [2224]. The intention of
Chapron et al. was to propose a classification based on where the DIE lesions are
located. The deep infiltrating lesions have been divided into two major compart-
ments, anterior (DIE of the bladder) and posterior compartment (DIE of the
sacrouterine ligaments, the vagina and intestines) and a subsequent operative proce-
dure has been defined [22]. Another descriptive classification system of Koninckx
tried to reflect all the possible manifestation and severity of endometriosis, classifying
the so-called subtle, typical, cystic, deep and adenomyotic lesions [23]. In the
meantime the group of German-speaking gynaecologist has developed a classifica-
tion system, called Enzian, with the intention to describe deep infiltrating lesions in
those compartments where the appropriate surgical removal can be performed.
21.3.1 The Enzian Classification
In 2005 Tuttlies et al. [24] published the Enzian classification, which has been revised
in 2010 and 2011. The latest version was published in 2012 at the homepage of the
SEF (Stiftung fur Endometriose-Forschung) (http://www.endometriose-sef.de).
The Enzian classification is exclusively devoted to describe deep infiltrating
endometriosis and is supposed to be used additionally with the rASRM classifica-
tion. Enzian is not an acronym or abbreviation for endometriosis issues but refers to
a beautiful blue-coloured flower and also to the name of a hotel in the Alpes, where
a group of Austrian and German experts since 2002 annually meet under the
patronage of the SEF in order to discuss endometriosis-related problems.
The development of this classification followed in the early versions the master
model of the TNM classification for cervical cancer inspired by the fact that deep
infiltrating endometriosis shows significant characteristics of a malignant tumour,
like crossing organ boundaries and likely infiltrating adjacent structures like blad-
der, ureter or intestines.
When comparing the rASRM classification, which has been established over
decades, with the Enzian classification, the list of common sites demonstrates only
little overlapping. The Enzian classification was designed to describe exclusively
deep infiltrating endometriosis, which means that it is limited to a special, but
challenging, clinically relevant situation. The Enzian classification is based on
different topographic areas following a surgical way of separation of involved
anatomic structures.
There are two main subclasses introduced to describe the clinical presentation of
deep infiltrating endometriosis. On the one hand there is a group of three topo-
graphic relevant compartments in the posterior pelvis and on the other hand there
Fig. 21.3 Example for surgical route for successful separation of compartments A and
C. Segmental rectum resection of two transmural infiltrative endometriotic lesions of 4 and
2 cm, respectively, Enzian C3 FI Sigma
is a group of different typical endometriotic infiltrations in distant organ sites.

The three pelvic compartments of the Enzian classification describe the topographic
anatomy of the pelvis. Successful and secure performance of surgical procedures is
based on the initial separation and demonstration of the relevant anatomic struc-
tures which are involved (Fig. 21.3); in other words, the anatomical compartments
for the Enzian classification follow the surgical procedure lining up the structures
that are equally involved in typical presentation of typical DIE. Radical surgery for
the management of DIE depends on initial reconstruction of the anatomy.
Fig. 21.4 The Enzian compartments A, B and C
The three posterior pelvic compartments were entitled A, B and C (Fig. 21.4)
and embrace the pelvic manifestation of deep infiltrating endometriosis including
the rectovaginal space, the vagina, the rectum and also the sacrouterine ligament
with the pelvic sidewall.
The second group with the capital letter F (far) was designed to add important
information about the location of infiltrative endometriosis, which is not directly
involved to the pelvic site or distant from the cardinal posterior compartments of the
pelvis as described above. Only important and typical presentations of DIE are
listed, such as infiltration of the bladder, intrinsic ureter endometriosis,
adenomyosis as infiltration of the uterus and distant bowel infiltration.
Compartment A (signed with red colour) includes the rectovaginal space from
the pouch of Douglas along a longitudinal direction downwards to the vagina.
Compartment B (signed with green colour) follows a horizontal line divided in a
left and a right part starting from the sacrouterine ligament; further lateral obstruc-
tions like the external ureter compression as well as the involvement of the cardinal
ligament up to the pelvic sidewall are included. Involvement of the splanchnic
nerves may also be an important issue in extended pelvic sidewall infiltration.
Compartment C (signed with blue colour) describes the dimension of rectal
Fig. 21.5 The Enzian classification system
infiltration with respect to histological confirmation of muscular layer infiltration as

an indispensable prerequisite.
Multiple bowel infiltrations are summarised under the description Enzian FI
(far intestine) with a topographic information, for example, FI Sigma or FI
terminal ileum.
The three pelvic compartments A, B and C are subjected to a consistent metric
level system; 1 indicates that the lesions are less than 1 cm, 2 indicates between
1 and 3 cm and 3 over 3 cm (Fig. 21.5). The metric system classifies the dimension
of the infiltration in each compartment.
The presence of adenomyosis is classified as FA (far adenomyosis), the
transmural infiltration of the bladder wall as FB (far bladder) and the very rare
presentation of intrinsic ureter endometriosis as FU (far ureter). The latter has to be

distinguished from the more common extrinsic obstruction of the ureter, which
would appear in the B3 group of the Enzian classification.
The Enzian classification of DIE is expressed as a multiple string code of the
involved compartments starting with the initial Enzian letters. The involved
compartments A, B and C are added with the appropriate metric level 1, 2 or
3, followed by the F subgroup. The complexity of the disease can be easily
expressed by using an abstract code summarising the involved anatomical struc-
tures. The description of the more complex disease will result in an accordingly
longer string.
An example of string may look like the following: Enzian: A2, B3, C3, FA, FB,
FI Sigma, which means deep infiltrating endometriosis of the vagina 13 cm, ureter
compression on one side by extrinsic endometriosis with dilatation of the urinating
system above the obstruction, bowel infiltration of the rectum more than 3 cm,
additional adenomyosis, infiltration of the bladder wall and bowel infiltration in the
sigma.
With reference to the rASRM score peritoneal superficial endometriosis or the
involvement of the fallopian tube-ovarian unit had been excluded from the Enzian
scoring system and fertility aspects should be scored using the endometriosis
fertility index (EFI). It is the distinguished purpose of the Enzian classification to
describe the topographic manifestation of DIE and the size and extent of organ
destruction; it is an easy-to-use system following an empiric pathway essential for a
successful radical surgery.
21.4 Magnetic Resonance Imaging of Endometriosis

(MARIE): Classification
Of uppermost importance for the imaging of patients suffering from deep infiltrat-
ing endometriosis is the detection and description of all manifestations of endome-
triosis in order to provide a reliable roadmap for surgical and conservative therapy.
MRI has been established for diagnostic and pretherapeutic imaging of patients
suspected to suffer from endometriosis [25]. Inherent to the method in contrast to
ultrasound, clinical examination or laparoscopy, which offers only a limited field of
view, spaces of the pelvis are equally accessible. For complete diagnostic evalua-
tion of all pelvic spaces including the rectum, retrocervical space and vaginal
fornices, careful preparation of the patient is indispensable [26]. For most scanning
protocols 50 mL aqueous gel (ultrasound gel) is administered intravaginally (for
distension of the vaginal cavity, assessment of the retrocervical area and vaginal
fornices) and 150200 mL water is administered into the rectum to obtain disten-
sion and increase contrast between bowel wall and lumen. Scopolamine-N-butyl
bromide is intravenously injected immediately prior to MRI in order to reduce
bowel movements and contractions of the uterus [27]. The bladder should be filled
moderately, rendering the evaluation of the bladder wall possible. A reliable

possibility to obtain moderate filling of the bladder is to ask patients not to empty
the bladder one hour prior to scanning.
T2-weighted images in axial, sagittal and coronal orientation obtained with high
resolution are crucial for the delineation of endometriotic lesions. T1-weighted 3D
sequences with fat suppression are obtained prior and after intravenous injection of
a gadolinium chelate contrast medium. On T1-weighted images obtained prior to
the administration of contrast medium methaemoglobin appears with high signal
intensity (bright). Methaemoglobin will be present in endometriosis 3 days up to
4 weeks after bleeding. Thereafter it is degraded into hemosiderin, which has low
signal intensity on T1-weighted MR images. Fibrous components of
endometriomas appear with low signal intensity on T2-weighted images and may
enhance after the injection of contrast medium, depending on the size of the
extracellular space in the lesions. Inflammatory lesions are also dark on
T2-weighted images but strongly enhance after the injection of contrast medium
on T1-weighted sequences. Endometriomas which do not contain predominantly
blood or degraded blood components appear dark on T2-weighted sequences [25].
The involvement of the different anatomical structures in the pelvis has an
unequal impact regarding fertility, physiologic function and required operative
technique. Consequently a classification system for MRI should take these differ-
ences into account, and weighting of endometriotic lesions in different locations
should be different regarding fertility and physiologic function (Table 21.3) [28].
In order to completely assess all present lesions, the compartments of the pelvis
must be assessed in a systematic order. A structured report of a comprehensive MR
scan for the detection of endometriosis should start with the description of site of
lesions in the respective compartments, size of the lesion and structures involved. In
the anterior compartment the bladder, vesicouterine pouch and vesicovaginal
septum can be affected. The urethra is only rarely involved. Endometriosis of the
anterior compartment usually does not cause infertility. Thus weighting regarding
infertility is low (Table 21.3). On the other hand, the involvement of the urethra or
bladder requires technically demanding operative strategies, so that weighting
factor for structure is relatively high. The middle compartment contains the vagina,
uterus, ovaries and uterine ligaments. The middle compartment is the most common
site of endometriomas and the involvement of its structures often leads to infertility.
This is mirrored in high scores regarding fertility for the respective anatomical
structures of the middle compartment.
The rectovaginal pouch, uterosacral ligaments, posterior vaginal fornix and
rectum are located in the posterior compartment. Most endometriotic lesions are
located in the cul-de-sac [27]. The rectosigmoid is the most commonly affected part
of the bowel. The involvement of the bowel requires technical demanding operation
strategies and has a high impact on function, while fertility is rarely affected.
Adhesions on the other hand might overlap the compartments and potentially
have a high impact on fertility, depending on the structures involved.
For performing the MARIE classification, the three compartments are system-
atically assessed for endometriosis, lesion location is described and the size of
Table 21.3 Scoring system for MARIE classification

1 12 cm >2 cm
Anterior compartment
Bladder 2s 3s 4s
Vesicouterine pouch 0.5 s 1s 1.5 s
Vesicovaginal septum 1s 1.5 s 2s
Urethra 3s 4s 5s
Middle compartment
Ovary (unilateral) 1f 2f 3f
Ovaries bilateral, second side (value must be added to 3f 4f 5f
that of the first side)
Fallopian tube (unilateral) 4f 4.5 f 5f
Fallopian tubes, bilateral, second side (value must be 7f 8f 9f
added to that of first side)
Uterus (surface) 0.5 f 1f 1.5 f
Vagina 0.5 s 1s 1.5 s
Posterior compartment
Rectovaginal septum 0.5 s 1s 1.5 s
Cul-de-sac 1s 2s 3s
Cul-de-sac and rectum 2s 3s 4s
Rectum/sigmoid 3s 4s 5s
Uterosacral ligament unilateral 1/0.5 s/f 2/1 s/f 3/1.5 s/f
Uterosacral ligament bilateral 1.5/1 s/f 2.5/1.5 s/f 3.5/2 s/f
Other
Adhesions between bowel and ovaries/fallopian tubes 3/4 s/f 4/5 s/f 5/6 s/f
Adhesions between both ovaries 1/4 s/f 1.5/5 s/f 2/6 s/f
Adhesions between bowel and uterus 2/2 s/f 2.5/2.5 s/f 3/3 s/f
Adhesions without involvement of uterus, ovaries and 2.5 s 3s 3.5 s
fallopian tubes
Lesions outside the compartment of the pelvis but in 2s 3s 4s
pelvic region, except subcutaneous
Subcutaneous lesions, fascia not penetrated 0.5 s 1s 1.5 s
Sciatic nerve 3s 4s 5s
Points Class Class
fertility structures
MARIE classification
0.52 MARIE 1 f MARIE 1 s
2.56.5 MARIE 2 f MARIE 2 s
>7 MARIE 3 f MARIE 3 s
lesions is measured. Points are added separately for f (fertility) and s (structures)
according to Table 21.3 (Fig. 21.6 represents an example of MRI classification of
a 28-year-old patient with deep infiltrating endometriosis). In cases of bilateral
involvement (i.e. ovaries or fallopian tubes), first the more severely affected side is
rated followed by the side with the smaller lesion. According to Table 21.3 MARIE
classification for f and e values is assigned following the scheme MARIE f s.
Fig. 21.6 Sagittal and axial

T2-weighted images of a
28-year-old patient with
deep infiltrating
endometriosis. Lesions of
both ovaries (right ovary
diameter 3 cm, score 3f, left
ovary 2 cm, score 4f),
adhesion between right
ovary and rectum with a
diameter of 1.5 cm (score
4 s, 5f) lesion of the spatium
rectovaginale (diameter
2 cm, score 1 s) and cul-de-
sac (diameter 4 cm, score
3 s) are present (scores
summing up to 12 f 8 s),
resulting in MARIE 3 f 3 s
21.5 Discussion
Even though many classifications as well as scoring systems have been proposed
since the first mentioning of endometriosis as a disease entity, no widespread
agreement on a classification for endometriosis is obtained. This review describes
four examples in more detail. The rASRM classification differentiates endometri-
osis in minimal, mild, moderate and severe stages and provides a score that includes
superficial endometriotic implants as well as adhesions. The assignment of points
according to the clinical situation was not developed on the basis of empirical data,
but based on theoretical background and estimations. In case of a superficial
non-infiltrating endometriosis, which is only manifested on the peritoneal surface,
this scoring system including a graph makes a lot of sense; however, any sub- and
retroperitoneal deep infiltrating manifestation is not considered with the rASRM
classification. The most commonly used classification system to describe deep
infiltrating endometriosis is the Enzian string code, which is used additionally to the
rASRM. In addition to the rASRM and Enzian classification Hackethal et al. [29]
showed that even in stage 1 endometriosis (rASRM classification) 25 % of those
patients were suffering from deep infiltrating endometriosis. Even though this is in
accordance with the initial aim of the rASRM classification omitting DIE can lead to
a marked misjudgement of the impact of the disease and necessary treatment [30].
Since the EFI is a specific further development of the rASRM classification, DIE
with no peritoneal, ovarian or tubal infiltration would not have been reflected in the
EFI score; however it could be responsible for infertility. This would apply to DIE of
the rectovaginal space as well as adenomyosis. However, only limited data are
available on the impact of such manifestations of DIE on fertility. Therefore, the
EFI will likely not be suitable to fully reflect the impact of different locations and
manifestations of endometriosis on fertility. It is probably because of this reason that
EFI gives major importance on historical factors as it is very well known that the
duration of infertility, age of a patient and prior history of pregnancies are extremely
strong predictors.
Haas et al. [31] compared the rASRM classification with the Enzian classifica-
tion. They clearly concluded that the Enzian classification is a clear supplement to
the rASRM classification with regard to the description of the manifestations of
DIE. They found an overlapping of description, especially in peritoneal disease of
the pouch of Douglas or cul-de-sac, which could be repetitive in the Enzian
classification as well as in rASRM classification.
The development of a radiological classification system is extremely useful in
the preparation of the surgical procedure and counselling patients preoperatively
about the required surgical steps. It has to be kept in mind that endometriosis
surgery is almost never easy and straightforward. A presurgical adequate classifi-
cation of disease can potentially improve patients outcome by the organisation of
multidisciplinary surgical teams or referral of patients to specialised surgical
endometriosis centres.
Unfortunately there is no ideal classification of endometriosis at the moment that
would be able to reflect all the aspects of endometriosis, the pathogenesis, anatom-
ical distribution, clinical manifestation, progression and recurrence. The way to
define the perfect classification system is long and lots of basic research as well as
well-conducted clinical trials in a large multicentre set-up are needed to better
understand the clinical nature of the disease and develop a classification system,
which encompass all these aspects.
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be validated. Gynecol Surg. 2011;8:16.
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Chapter 22
The Association of Endometriosis
with Ovarian Cancer: A Critical Review
of Epidemiological Data
Sun-Wei Guo
Abstract Although endometriosis is well recognized as a benign gynecologic

condition, its association with ovarian cancer has frequently been reported. Many
review papers on this topic have been published, yet there seems to be no consensus
as to whether or not endometriosis is a precursor of ovarian cancer and whether or
not any actions should be taken based on our current knowledge on the
endometriosis-ovarian cancer association. In this chapter, I shall critically review
epidemiological and clinical prevalence data for and against the link, point out the
challenges in proving the causal link, and, in the end, sketch some ways to solve this
problem. I shall provide a tutorial review of epidemiological studies, measures of
effect size, and confounding and point out the connection between the prevalence
data and the relative risk estimate. Funnel plots are used to examine the asymmetry
of risk estimates, in which the risk estimates such as OR are plotted on a logarithmic
scale against the inverse of their corresponding measures of precision. Areas in
need of further research are also outlined.
Keywords Association Case-control studies Endometriosis Epidemiology

Ovarian cancer
22.1 Introduction
Although endometriosis is well recognized as a benign gynecologic condition, its

association with ovarian cancer has often been reported and mentioned in literature.
A PubMed search with the keyword, endometriosis, yielded a total of 19,621
S.-W. Guo, M.Med., Ph.D. (*)

Shanghai OB/GYN Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011, China
Department of Biochemistry, Shanghai College of Medicine, Fudan University,
Shanghai, China
e-mail: hoxa10@gmail.com

364 S.-W. Guo
papers. Using the combination of endometriosis and ovarian cancer, it turned

up 2,544 papers or 13.0 % of the total literature on endometriosis. In contrast, 3,722
(19.0 %), 3,542 (18.0 %), and 515 (2.6 %) papers were on endometriosis and
infertility, endometriosis and pain, and endometriosis and inflammation, respec-
tively (accessed June 18, 2013). Many review papers on this topic have been
published, yet there is no consensus as to whether or not endometriosis is a
precursor of ovarian cancer [1]. While many scientists sit on the fences, some
investigators think that the ovarian endometriomas could be viewed as a neoplastic
process, considering the malignant transformation of endometriosis as rather
obvious [2]. Other investigators are more cautious, arguing that, among the nine
criteria of causality, proposed by Hill [3], many are still unfulfilled [4].
However, a Lancet Oncology paper, published early last year, seemed to tip the
balance of this debate towards the conclusion that endometriosis is a precursor of
ovarian cancer. From a pooled analysis with primary data from 13 case-control
studies, Pearce et al. reported that women with self-reported endometriosis were
associated with a significantly increased risk of clear cell (odds ratio (OR) 3.05),
low-grade serous (OR 2.11), and endometrioid invasive (OR 2.04), but not
high-grade serous invasive ovarian cancer [5]. The authors of that paper concluded
that we will develop a risk stratification model that combines genetic and epide-
miological risk to better stratify women into high-risk, intermediate-risk, and
low-risk categories, allowing better individualization of prevention and early
detection approaches such as risk-reduction surgery and screening. The lead
author, Dr. Celeste Leigh Pearce, was even more optimistic, stating, in a journal
news release, that This breakthrough could lead to better identification of women
at increased risk of ovarian cancer and could provide a basis for increased cancer
surveillance of the relevant population, allowing better individualization of preven-
tion and early detection approaches such as risk-reduction surgery and screening
(http://health.usnews.com/health-news/news/articles/2012/02/22/endometriosis-could-
raise-risk-of-3-ovarian-cancers, accessed January 15, 2013). In other words, endome-
triosis is indeed a precursor of ovarian cancer and, as such, certain measures should be
taken accordingly.
Ovarian cancer is by far the most lethal malignancy of the female reproductive
system. Over 90 % of ovarian cancers arise from the surface epithelium [6]. Despite
advances in radical surgery and chemotherapy, the overall survival has changed
very little in the last 30 years [7]. With the advent of molecular biology, a great deal
of efforts have been focused on early detection, yet this attempt often brings with
roller-coaster experience and consequently it has so far not resulted in any tangible
survival benefit to the patients. Faced with such an abject failure, it is important to
identify the precursor(s) of ovarian cancer, even for some specific histotypes.
With this in perspective, it is perhaps understandable as why there have been so
much attention on the endometriosis-ovarian cancer link. However, how solid is the
evidence for this link? Would the evidence, gathered so far, warrant any actionable
measures such as that suggested by Dr. Pearce? These are simple yet weighty issues
that are worth careful examination.
22 The Association of Endometriosis with Ovarian Cancer: A Critical Review. . . 365
In this chapter, I shall critically review the evidence in support of the link, point
out the challenges in proving the causal link, and, in the end, sketch some ways to
solve this problem. Due to space limitation, I shall restrict my attention to epide-
miological and clinical data. While there is a growing body of literature
documenting shared molecular aberrations between endometriosis and ovarian
cancer, suffice to say that, due to the nature of these studies, many such aberrations
are indicative of association, only suggestive for a causal link, simply because the
temporality of the link is difficult to prove.
22.2 Methods
A systematic and comprehensive search of PubMed was performed for all studies
published up to June 18, 2013, using the following combination of search terms of
endometriosis, ovarian cancer, epidemiology, and association. The studies
had to report epigenetic aberrations in endometriosis. The search was limited to
publications written in English.
For each retrieved case-control studies, the OR and its 95 % confidence interval
(CI) and the standard error (SE) of the log OR were extracted. The choice of log OR
was simply due to the fact that, in contrast to the OR, its SE is unaffected by the
magnitude of the log OR. For cohort studies, the standardized incidence ratio (SIR),
rate ratio (RR), or hazard ratio (HR) and their SEs were extracted.
Funnel plots were used to examine asymmetry, in which the risk estimates such
as OR were plotted on a logarithmic scale against the inverse of their corresponding
standard errors, a measure of precision [8]. If bias is absent, small studies will have
ORs that are widely scattered but symmetric about the OR estimates provided by
larger, more precise studies. In this case, the plot would resemble an inverted funnel
with the tip pointing roughly towards the true log OR. If publication bias is present,
the plot will be asymmetric because some negative studies are not published
All computations were made with R statistics software system version 3.0.9 [9].
The statistical routine rmeta was used.
22.3 The Role of the Funding Source
The sponsor of the study had no role in study design, data collection, data analysis,
data interpretation, or writing of this report.
366 S.-W. Guo
22.4 Evidence from Clinical Series
22.4.1 Early Criteria
As early as 1925, Sampson proposed histopathological criteria for inferring that the
malignancy arose from endometriosis, or the causal relationship between endometri-
osis and malignancy: (1) clear evidence of endometriosis close to the tumor (prox-
imity); (2) the carcinoma must be seen to arise in endometriosis and not to be
invading it from other sources (arising from endometriosis); and (3) presence of
tissue resembling endometrial stroma surrounding characteristic glands (endometrial
stroma plus glands) [10]. Scott later added one more criterion: the demonstration of a
histology-proven transition from benign endometriosis to cancer (transition) [11].
Clearly, these criteria are all based on histological evidence, which, in turn, are
based on tissue samples taken from patients. While the proximity and endome-
trial stroma plus glands criteria may be relatively easy to establish, the inference of
arising from endometriosis and transition has, by necessity, to be based on a
single snapshot, in contrast to serial observations, of the histological images or
morphologic features during a presumably long period of tumorigenesis and, as
such, can be challenging to establish. It is no wonder that these criteria, considered
to be stringent, are rarely fulfilled [1, 12].
The morphologic data can tell us something, but only to a certain extent. For
example, ovarian cancer was once regarded as a single disease since by morphology
the tumor seemingly originated from ovary but now a dualistic model of carcino-
genesis of ovarian cancer based on distinctive clinicopathologic and molecular
genetic features seems to have replaced the older view [7, 13]. Also, just by
morphology or histological data, it would be very difficult to find that there are
4 main subtypes of breast cancer caused by different subsets of genetic and
epigenetic abnormalities [14]. In addition, since the choice of tissue sections entails
certain degree of selection, it may be susceptible to attribution error, especially
when the pathologists are inexperienced.
22.4.2 Prevalence Data: An Intimate Connection

with the Odds Ratio
About a dozen reports on the prevalence of endometriosis in women with ovarian

cancer have been published. If we denote the prevalence of endometriosis (E) as P
(E), the prevalence of women with both endometriosis and ovarian cancer (O)
(often called endometriosis-associated ovarian cancer or EAOC) as P(E, O), and
the proportion of E among women with O as the conditional probability P(E|O),
then we can denote the relative
risk of having O in women with E vs. women
without E (E) as RR P OE =P OE , that is, the relative risk of having O when a
woman has E vs. woman who does not. This is the quantity that most, if not all,
epidemiological studies attempt to estimate. Here, the age dependency is often

ignored for ease of exposition.
Since both P(O|E) and P OE are small (because the lifetime risk of developing
O is about 1 % or 0.01
and even lower
for some specific histotypes of ovarian
cancers), 1 P OE 1 P OE 1,

P O E

P O E 1P OE
RR OR
P O E P O E

1P OE
That is, when ovarian cancer is rare (indeed it is), the OR obtained from case-
control studies is merely an approximation to RR.
By Bayes theorem and after some simple algebra, it is easy to see that RR
P(E|O)[1P(E)]/(P(E)/[1P(E|O)]). That is, the true RR depends only on the
prevalence of endometriosis (which we have a fairly good idea) and the prevalence
of endometriosis among women with ovarian cancer (which can be estimated from
data, at least in theory). Note that this RR does not depend on the prevalence of O.
Hence we can calculate RR even for some specific histotypes of ovarian cancer, such
as clear cell or endometrioid ovarian cancers. It is noted that RR can be expressed in
a more revealing form, as RR rO/r, where rO P(E|O)/[1P(E|O)], the odds
of having endometriosis given that the woman has O, and r P(E)/[1P(E)],
the odds of having endometriosis in the general population.
22.4.3 Prevalence Data from Published Studies
The prevalence data extracted from 13 studies reporting the prevalence of endo-
metriosis in women with ovarian cancers, along with the estimated RR using either
the 10 % prevalence for endometriosis or the 5 % for ovarian endometriomas, are
listed in Table 22.1. It can be seen that there are enormous variations in the reported
prevalence, and, consequently, the RR estimates also vary greatly from study to
study. It should be noted that the reported prevalence of endometriosis in women
with ovarian cancer is likely to be an underestimate of P(E|O), since some
endometriotic lesions may not have been found by the surgeon who performed
the operation, or pathologists who performed histologic examination may not have
found any lesion which might still exist. On the other hand, P(E|O) is prone to be
overestimated if one is single-mindedly trying to find all cases of ovarian cancer-
associated endometriosis while dismissing or discarding cases with ovarian cancer
but no endometriosis, consciously or otherwise. Anyhow, it can be seen from
Table 22.1 that some RR estimates based on the reported prevalence still
deviate greatly from the OR estimates reported from case-control studies, espe-
cially for clear cell and endometrioid histotypes. In fact, all, except one, OR
368
Table 22.1 Prevalence of endometriosis in women with ovarian cancers and the estimated relative risk of developing ovarian cancer in women with
endometriosis vs. women without
Overall Clear cell and endometrioid
Year of RR RR RR RR
ID Author publication Prevalence (%) (P 10 %) (P 5 %) Prevalence (%) (P 10 %) (P 5 %) Remark
A71 Aure et al. [15] 1971 4.2 (35/831) 0.39 0.83 12.5 (34/271) 1.29 2.71
K72 Kurman et al. [16] 1972 NR 10.2 (5/49) 1.02 2.16
R79 Russell [17] 1979 11.3 (46/407) 1.15 2.42 34.3 (36/105) 4.70 9.92
B89 Brescia et al. [18] 1989 NR 18.4 (14/76) 2.03 4.28
V93 Vercellini et al. [19] 1993 11.1 (52/466) 1.12 2.37 25.0 (38/152) 3.00 6.33
C96 De La Cuesta et al. 1996 NR 40.0 (16/40) 6.00 12.67
J97 Jimbo et al. [20] 1997 14.5 (25/172) 1.53 3.22 35.6 (16/45) 4.96 10.50
F97 Fukunaga et al. [21] 1997 24.1 (54/224) 2.86 6.03 49.4 (40/81) 8.79 18.55
O00 Ogawa et al. [22] 2000 29.1 (37/127) 3.69 7.80 66.0 (33/50) 17.47 36.88
V00 Vercellini et al. [23] 2000 43.5 (91/209) 6.93 14.63 60.7 (54/89) 13.90 29.35 Only unilateral
cancer and
left-sided lesions
were considered
O03 Oral et al. [24] 2003 7.7 (14/182) 0.75 1.59 26.3 (5/19) 3.21 6.79
D11 Dzatic-Smiljkovic 2011 11.0 (23/210) 1.11 2.35 33.3 (19/57) 4.49 9.49
et al. [25]
K12 Kondi-Pafiti et al. [26] 2012 5.9 (1/17) 0.56 1.19 0.0 (0/16) 0.00 0.00
S.-W. Guo
Funnel plot of prevalence-based log RR
0.25
K12
O03
0.20
1/sqrt(sample size)
C96
0.15
J97
K72 O00
D11
B89
F97
0.10
V00
R79
V93
A71
-4 -2 0 2 4
log RR
Fig. 22.1 Funnel plot for the 13 log-transformed prevalence-based RRs, using data extracted from
Table 22.1 and assuming a 5 % prevalence of endometriosis in the general population. The dashed
line represents RR 1. The alphabet-numeric combinations are the IDs shown in Table 22.1, and
each ID represents one study. Note that for study K12, a Bayes estimate for binomial distribution
assuming a non-informative prior (uniform distribution) was used, since otherwise the data would
yield a RR of 0
estimates are pretty large. It is unclear as to whether some seemingly high preva-
lence estimates are genuine or a result of ascertainment bias, population idiosyn-
crasy, or chance events.
Based on all published prevalence estimates listed in Table 22.1, one can get a
pooled estimate of prevalence for all histotypes of ovarian cancer weighted by the
sample size, which is 12.9 %, yielding an RR estimate of 1.33 if a prevalence of
10 % is assumed, or 2.81 if a prevalence of 5 % is assumed. For clear cell and
endometrioid ovarian cancers, the pooled estimate of prevalence is 28.0 % and
the corresponding RR estimates are 3.51 and 7.40, respectively, depending on the
endometriosis prevalence.
Note that, since RR rO/r, where rO P(E|O)/[1P(E|O)] and r P(E)/[1
P(E)], r is a constant when P(E) is assumed to be a fixed number (say, 5 %); hence
we can calculate the standard error of log RR, which is equivalent to the standard
error of log rO, or log[P(E|O)]log[1P(E|O)]. By delta method, it is easy to
see that the standard error of log RR is inversely proportional to the squared root of
the sample size n. Hence, we can plot the log RR listed in Table 22.1 against the
squared root of the sample size n reported in the study (Fig. 22.1).
It is interesting to see from Fig. 22.1 that when the estimated log RRs were
plotted against the squared root of the sample size of the study, the seemingly large
RRs seen in Table 22.1, when all placed in the funnel plot in Fig. 22.1, seem to
gravitate to a point close to 1, but perhaps slightly greater than 1.
370 S.-W. Guo
22.5 Epidemiological Evidence
22.5.1 Cohort Studies
In a typical cohort study, two cohorts or two groups of people, one with and one
without a particular attribute (in our case, women with or without endometriosis),
are identified and followed up longitudinally. The incidence of a particular event
quite often, disease (ovarian cancer, in our case)in the two groups is evaluated
and compared. In this way, whether having the particular attribute (exposure)
would increase or decrease the incidence can be investigated.
Due to constraint in time and resources, cohort studies are seldom conducted
concurrently or truly prospectively. Instead, many cohort studies are conducted
retrospectively. In the latter case, the cohorts are identified and assembled in the
past based on archived records. In this case, the occurrence of the event of interest is
often retrieved from the records as well. While retrospective cohort studies require
much less resources and time, their major disadvantage is their exclusive reliance
on available information, sometimes the subjects own memory. Consequently, the
quality of exposure or disease data can be compromised (e.g., recall bias).
In epidemiology, the outcome is frequently a disease. One common outcome
measure in cohort studies is incidence, or the risk of developing certain disease
within a specified period of time given no occurrence prior to that time period. It
can be expressed either in cumulative incidence (incidence proportion) or incidence
rate with a denominator (called incidence density rate or person-time incidence
rate). In analytical epidemiology, one measure that is used very frequently is the
standardized incidence ratio (SIR), which is a ratio or percentage quantifying the
increase or decrease in incidence of a study cohort with reference to the general
population.
While the SIR provides a succinct measure of the change from the incidence
from the reference population or cohort, it becomes a bit cumbersome when there
are some confounding factors that need to be controlled for, especially in retro-
spective cohort studies. In these circumstances, the rate ratio (RR, also called ratio
of incidence densities) or hazard rate (HR) would be more convenient, and their use
would render the use of some sophisticated statistical models such as the Cox
regression model possible, facilitating elaborate statistical analysis. The RR gives
the ratio of the event rate in the cohort of interest (say, women with endometriosis)
vs. that in the reference group after adjustment for other known confounders. Both
SIR and RR provide a measure of causality (or, rather, association, especially for
retrospective cohort studies) between exposures and outcomes.
So far, seven cohort studies, with varying qualities, on endometriosis-ovarian
cancer link can be identified (Table 22.2). Some of them were population-based and
prospective studies, and others were hospital-based or retrospective cohort studies.
These studies yielded either SIR or RR estimates, along with 95 % confidence
intervals (CIs) (Table 22.2).
Table 22.2 Published cohort studies reporting ovarian cancer risk in women with endometriosis
Length
Year of #
of Cohort follow- of
ID Author publication size up cases SIR or RR S.E. 95 % CI
B97 Brinton 1997 20,686 11.4 29 1.92 0.38 1.32.8
et al. [27]
O02 Olson 2002 1,392 13 13 0.78* 0.74 0.25
et al. [28] 2.44
B04 Brinton 2004 1,919 18.8 13 1.26* 0.29 0.62.6
et al. [29]
B05 Brinton 2005 2,491 NA 50 1.69* 0.09 1.27
et al. [30] 2.25
M06 Melin 2006 66,187 12.7 122 1.43 0.133 1.19
et al. [31] 1.71
M07 Melin 2007 63,630 13.4 134 1.37 0.12 1.14
et al. [32] 1.62
K07 Kobayashi 2007 6,398 12.8 46 8.95 2.85 4.12
et al. [33] 15.3
A10 Aris [34] 2010 2,854 10 333 1.6* 0.10 1.12
2.09
S12N Stewart 2012 3.11* (for 0.52 1.13
et al. [35] nulli- 8.57
parous
women)
S12P 1.52* (for 0.76 0.43
parous 6.75
women)
SIR standard incidence ratio, RR rate ratio, SE standard error, CI confidence interval
*SIR or RR column are RR estimates
From these SIRs and their CIs, their standard errors (SEs) can be easily calcu-
lated. Since all the RRs were calculated based on logistic or Poisson regression
models after adjusting for possible confounding factors, the SE of the
log-transformed RR can be calculated based on RRs and their CIs.
In evaluating risk estimatesbe it SIR, RR, or OR if from case-control studies
from various studies, funnel plots are often employed [36]. In funnel plots, risk
estimates, such as RRs or log RRs, are placed on the horizontal axis against some
measure of study size or precision, such as their standard errors, on the vertical axis.
The funnel plot is so named because of its shape: in the absence of selection biases
(such as publication bias and bias in inclusion criteria), true heterogeneity (i.e., size
of effect differs according to study size), and data irregularities (such as poor
methodological design of small studies, inadequate analysis, and fraud), studies
of large or small sample sizes should be more or less symmetrically scattered
around the true log RR. Hence the plot should have the shape of a funnel with
wide opening on the top (due to sampling variability), with the tip of the funnel
pointing to the bottom and centering on the true log RR [36]. The choice of log OR
instead of OR is due to the fact that the standard error of RR is related with the odds
372 S.-W. Guo
Fig. 22.2 Funnel plot for Funnel plot for log SIRs/RRs
the 9 log-transformed SIRs/
K07
RRs, using data extracted
from Table 22.2. The
2.5
dashed line represents
SIR 1 or RR 1.
The alphabet-numeric
2.0
combinations are the IDs
Standard error
shown in Table 22.2,
and each ID represents
1.5
one study
1.0
O02
0.5
S12P
B97
B04
M06
M07A10 S12N
B05
0.0
-2 -1 0 1 2
log SIR/RR estimate
ratio, while the standard error of log OR is purely a function of sample sizes in
different exposure-disease status combinations. The use of log OR also renders ORs
that are greater than 1 or less than 1 symmetric about 1 (0 on the log scale).
When plotting the 9 log-transformed SIRs/RRs against their SEs in a funnel plot
(Fig. 22.2), two features can be noted. First, the plot looks like an asymmetric
funnel, with its tip gravitating towards somewhere near log SIR or log RR 0, i.e.,
SIR 1 or RR 1. Since there is no indication of bias in inclusion criteria or
heterogeneity, this suggests that there may be a publication bias towards favoring
positive studies and higher estimates of odds ratios may well be a chance variation.
In addition, the plot seems to suggest that the true SIR or RR is near 1.
Second, the SIR estimate, K07 in the plot, provided by Kobayashi et al. [33] is
situated at the rim of the funnel, suggesting that while it gave a larger SIR estimate,
it is not a precise estimate.
The paper by Aris [34] gave P(E) 0.107 and P(E|O) 0.14, yielding
RR 1.36 as discussed above. This is very close to the RR estimate of 1.6 reported
by the paper.
22.5.2 An Overview on Case-Control Studies
Case-control studies are an alternative to cohort studies for investigating the

association between exposure (in our case, having endometriosis) and disease
(ovarian cancer). The basic questions for such studies are the degree of association
between risk for disease and the factor(s) under investigation, the extent to which
the observed association may result from bias, confounding, and/or chance, and the
extent to which they may be described as causal [37]. A case-control study
compares cases (in our case, women with ovarian cancer) and controls (women
without ovarian cancer, say) with respect to their exposure (or lack thereof) or
levels of exposure to a suspecting risk factor (in our case, having endometriosis).
When the risk factor at hand is a dichotomous variable, such as having endometri-
osis or not, the outcome measure is typically the odds of exposure in cases as
compared with that in controls, or OR. When the occurrence of disease is rare, such
as ovarian cancer, the OR estimated from case-control studies becomes an accept-
able approximation to the relative risk. Case-control studies can be a powerful tool
in the investigation of exposure-disease relationship when both the disease and the
exposure are rare. A prime example is the uncovering of the relationship between in
utero exposure to diethylstilbestrol (DES) and vaginal adenocarcinomas in the
daughters [38]. That study was based on just eight cases, each with four matched
controls. Seven out of eight cases had been exposed to DES in utero, but in contrast
none of the 32 controls had.
As with SIR or RR used in cohort studies, the OR or relative risk (RR) used in
case-control studies is the measure of association between disease and exposure.
However, the association could be causal but also could be merely a correlation. For
women with endometriosis (E), or with ovarian cancer (O), the association between
E and O could be due to a variety of scenarios. Figure 22.3 shows several scenarios in
which E and O can be found to be associated. Scenario a is the case where factor
X has a causal relationship with both E and O. E and O are associated simply because
of the presence of the common risk factor X, which may or may not be measured in a
study. It should be noted that E and O share at least one common risk factor, that is,
the incessant ovulation/menstruation. Incessant ovulation or unopposed estrogen
exposure is a known major risk factor for ovarian cancer [39]. Similarly, incessant
menstruation is a known major and consistently identified risk factor for endome-
triosis [40]. Figure 22.4 shows two numerical examples, perhaps somewhat extreme
but nonetheless not unusual cases. In example a, failure to control for the
confounding factor gives rise to spurious results. It is interesting to point out that,
while the OR for the O-E association in each stratum of factor X is 1, the OR for the
association with pooled levels of the factor is 1.35 > 1. Of course, the failure to
control for confounders can also go to the other direction, in which the pooled OR
can be smaller than ORs in each stratum (example b).
In Fig. 22.3, scenario b shows the case in which both factors X and E represent
the same underlying cause for O, such as the case when X and E represent different
aspects of the same factor. Scenario c is the case where E leads to X, which, in turn,
has a causal relationship with O. In the case of E-O association, it is possible that the
diagnosis of endometriosis may result in the use of danazol, an androgenic agent,
which could increase the risk of O in light of the androgen hypothesis of ovarian
cancer [41, 42]. In other words, it could be the exposure to an androgenic agent,
once a popular therapeutic for endometriosis, that increases the risk of ovarian
cancer, not the endometriosis itself. Scenario d is the case in which E-O has a causal
relationship.
374 S.-W. Guo
Fig. 22.3 Diagrams showing 4 different scenarios in which E and O can be found associated
Fig. 22.4 Two hypothetical examples showing that failure to control for confounding can lead to
spurious results. (a) Artificially inflated OR; (b) underestimated OR
It should be noted that factor X in scenario a is considered a confounding factor.

Confounding is the distortion of a disease/exposure association brought about by
the association of other factors with both disease (O) and exposure (E) [37].
The magnitude of OR is a measure of the strength of association, or the effect
size. In general, when an OR is large, say greater than 10, the association is likely to
be genuine. For example, the relative risk of having cervical cancer in women with
HPV positivity vs. negativity is about 1000. Depending on the number of daily
cigarettes consumed, the OR for smoking-lung cancer association ranges from 7 to
about 27. The OR for the association between the DES exposure and vaginal cancer
is about 40. In contract, for an association with an OR < 2, it is likely that the OR
estimate could be a result of confounding or bias and needs to be scrutinized

rigorously even though the association could also be genuine [43].
22.5.3 Case-Control Studies
Eleven case-control studies can be identified (Table 22.3). As expected, these

studies vary in the types of ovarian cancer and the selection of controls (women
with endometriosis or endometriomas, or infertility). Note that the last study listed
in Table 22.3 is somewhat different from the rest, since both cases and controls had
endometriosis, but the risk (protective) factor of interest was whether or not the
subject had all visible endometriotic lesions removed [53]. Regardless, the forest
plot revealed that the pooled (raw) OR estimate is 1.54 (95 % CI 1.431.66,
excluding the last study; Fig. 22.5), suggesting that overall, the OR value is
moderate. In addition, there is little heterogeneity ( p 0.47 for heterogeneity test).
As with the SIR/RR estimates, the funnel plot of the log ORs from the ten studies
indicates that the plot also looks like an asymmetric funnel, with its tip pointing
towards somewhere near log OR 0, i.e., OR 1 (Fig. 22.6). Since there is no
indication of bias in inclusion criteria or heterogeneity, this suggests that there may
be a publication bias towards favoring positive studies and higher estimates of odds
ratios may well be a chance variation. In addition, the plot seems to suggest that the
true OR is quite moderate.
The study by Melin et al. [53] is of particular interest since, unlike other case-
control studies, it examined the effect of surgical treatment on the endometriosis-
ovarian cancer association. By linkage to the National Swedish Cancer Register, it
identified all women diagnosed with epithelial ovarian cancer at least 1 year after
the endometriosis diagnosis (cases). Two controls per case with no ovarian cancer
before the date of cancer diagnosis of the case were randomly selected from the
study base and matched for the year of birth. It found an OR of 0.30 (95 %
CI 0.120.74) for women who received a complete removal of all visible endo-
metriosis. That is, for a woman with endometriosis, her risk of developing ovarian
cancer could be cut by 70 % if she had all visible endometriotic lesions removed. It
is worth noting that so far no other case-control studies have taken surgical
completeness into consideration, since the study by Melin et al. strongly suggests
this can be a protective factor.
22.5.4 Other Considerations
The control for shared risk (and/or protective) factors between E and O appears to
be a big challenge in sorting out the relationship between E and O association.
Besides the scenario depicted in Fig. 22.4a, it is known that E and O share
some other common risk/protective factors, for example, the use of oral
376
Table 22.3 Published case-control studies reporting ovarian cancer risk in women with endometriosis
Year of # of # of # case # controls Adjusted
ID Author publication cases controls w/ endo w/ endo OR (95%CI) Remark
N00 Ness et al. [44] 2000 764 1,364 66 85 1.7 (1.22.4)
N02 Ness et al. [45] 2002 3,678 5,268 51 39 1.73 (1.102.71) Originally considered infertility as a
possible risk factor
but also looked at patients with
infertility due to endometriosis
M04 Modugno et al. [46] 2004 2,089 2,943 177 184 1.32 (1.061.65) This study also included some data
from Ness et al. [44]
B04 Borgfeld et al. [47] 2004 27,050 81,254 81 181 1.34 (1.031.75)
M08 Merritt et al. [48] 2008 1,555 1,500 124 87 1.31 (0.971.78)
N08 Nagle et al. [49] 2008 87 1,450 13 87 3.0 (1.55.9) For endometrioid and clear cell
ovarian cancers
R08 Rossing et al. [50] 2008 585 1,293 64 94 1.6 (1.12.3) For invasive ovarian cancer
W09 Wu et al. [51] 2009 604 679 51 37 1.66 (1.012.75)
P12 Pearce et al. [5] 2012 7,911 13,226 738 818 1.46 (1.311.63) Invasive clear cell and endometrioid
ovarian cancers
M13 Merritt et al. [52] 2013 358 2,100 51 165 1.92 (1.362.71) Low-grade serous, endometrioid/
mixed, mucinous and clear cell
E13 Melin et al. [53] 2013 197 402 52 201 0.30 (0.120.74) All cases and controls had endome-
triosis. The factor of
interest was whether or not the
subject had a radical extirpation
of all visible endometriosis
OR odds ratio, SE standard error, CI confidence interval, # numbers, endo endometriosis, w/ with
S.-W. Guo
Ness(2000)
Ness(2002)
Study Reference Modugno(2004)
Borgfeld(2004)
Merritt(2008)
Nagle(2008)
Rossing(2008)
Wu(2009)
Pearce(2012)
Merritt(2013)
Summary
1.26 1.58 2.00 2.51 3.16 3.98 5.01

Odds Ratio
Fig. 22.5 Forest plot summarizing the results from 10 case-control studies using data from
Table 22.3
Fig. 22.6 Funnel plot for Funnel plot for log odds ratios
the log ORs, using data
0.4
extracted from Table 22.3.

The dashed line represents
OR 1. The alphabet- N08
numeric combinations are

0.3
the IDs shown in

Table 22.3, and each ID
Standard error
W09
represents one study N02
0.2
R08
N00M13
M08
B04
M04
0.1
P12
0.0
-1.0 -0.5 0.0 0.5 1.0

Log(OR)
378 S.-W. Guo
contraceptives (OC) and age at menarche. These factors are very likely to be
causally associated with both E and O, effectively making them confounding
factors when assessing the E-O association in case-control studies. However,
while some studies did control for OC use, few, if any, controlled for the number
of ovulations/menstrual cycles.
While the mean age at onset of ovarian cancer is about 56 years [4], the onset of
endometriosis occurs mostly and typically during womens reproductive age. This
has been taken as a support for temporality requirement in the Hills 9 criteria of
causality [4]. Indeed, the reported mean age of EAOC cases is often significantly
younger than ovarian cancer patients without endometriosis but older than women
with endometriosis alone [34].
However, the case-control studies published so far have not demonstrated a
clear, graded temporal relationship between endometriosis and ovarian cancer.
Most epithelial tumors take a latent period of at least 15 years to develop [4]. If
endometriosis is a precursor of certain types of ovarian cancer, then it should take a
certain latent period, likely to be shorter than 15 years, for ovarian cancer to
develop. Consequently, one would see that after excluding some cases with
endometriosis, say, 3 years of interval between the diagnosis of endometriosis
and of ovarian cancer, the OR would go up since this would effectively remove
many noisy cases which would dilute the association signal. Unfortunately, we
actually see the opposite from the study by Pearce et al. [5]. Figure 22.7 is a
graphical rendition of its sensitivity analysis (Table 4 in [5]). One can see that once
the cases who had at least 3, 5, or 10 years of interval between the diagnosis of
endometriosis and of ovarian cancer were removed, the OR estimate goes down
considerably.
22.6 Should Any Action Be Taken?
Given the somewhat consistent but rather moderate increase in OR, some investi-
gators believe that ovarian cancer originates from endometriosis, at least for clear
cell carcinoma and endometrioid adenocarcinoma [54]; hence, screening, labora-
tory, and imaging evaluation should be recommended for early detection of
malignant disorders in women with endometriosis [55]. Some even show that
patients with EAOC actually had a more favorable prognosis [5658]. However,
other studies do not find such evidence [59, 60].
Due to the low incidence of ovarian cancer and the rather moderate increase in
risk, extreme caution needs to be exercised when conveying the message to
the public and also in the context of screening. For clear cell ovarian cancer, the
prevalence is reported to be 13 per 100,000 women (Surveillance Epidemiology
and End Results: http://seer.cancer.gov/statfacts/html/ovary.html, accessed
January 17, 2013). Assuming, perhaps too optimistically, that a screening test exists
that is 99 % sensitive and 99 % specific. Even with this rosy scenario, the
corresponding positive predictive value is a disappointing 3.7 %. In other words,
3.0
C
C: Clear-cell
E: Endometrioid
C L: Low-grade serous
2.5
C
OR estimates
C
L
E
2.0
L L
L
E
E
1.5
None <=3 yrs <=5 yrs <=10 yrs
Exclusions
Fig. 22.7 A graphical rendition of the sensitivity analysis for the association of endometriosis and
risk of invasive ovarian cancer based on timing (time interval) of diagnosis between the two
diseases, as reported by Pearce et al. [5] (their Table 4). When patients with the time interval less
than or equal to 3 years, 5 years, and 10 years are excluded, the decrease in the OR estimate is seen
out of 100 women who have tested positive, fully 96 would have a false positive
result and be likely to be subjected to invasive procedures. Therefore, given the low
incidence and also the moderate increase in OR, it is perhaps premature to talk
about screening.
22.7 Conclusion
From the funnel plots for the SIR/RRs reported from cohort studies and the ORs
from case-control studies, it seems that there may be a publication bias towards
favoring positive studies. In addition, the plots seem to suggest that the true effect
size is very moderate. Yet the vast discrepancy between RRs estimated from
prevalence of endometriosis in women with ovarian cancer and ORs reported
from published case-control studies is puzzling. Since the prevalence is likely an
underestimate, the true RR is likely to be higher, which would highlight the
discrepancy even more. It is unclear as to what factors contributed to the discrep-
ancy. Have all epidemiological studies published so far underestimated the effect
size due to failure to control for some, yet to be identified, confounders or certain
biases of unknown sources? Or have many studies reporting the prevalence of
endometriosis in ovarian cancer somehow overreported, perhaps unwittingly,
380 S.-W. Guo
because of ascertainment or selection bias and population idiosyncrasy or have

simply fallen into the trap of attribution error? There is no answer as of now, and to
address these questions would warrant more studies.
While the presence of ovarian endometriomas may generate a pro-inflammatory
microenvironment that may be conducive to the development of ovarian cancer, it
is noted that most, if not all, diseases, especially those associated with pain, have
signs of inflammation. Even obesity has signs of inflammation. What is unclear is
how the pro-inflammatory milieu in endometriosis per se leads to ovarian cancer. It
is also unclear as to whether the peritoneal or vagino-rectal deep infiltrating
endometriosis would also increase the risk of ovarian cancer more than that of
other gynecological cancers. Moreover, the failure in providing or adjustment for
information on treatment in many published epidemiological studies raises the
question as to whether a surgery or drug treatment can actually reduce the risk of
ovarian cancer. It also raises the question as to whether the use of danazol, an
androgenic agent and once a popular therapeutics, could increase the risk of ovarian
cancer. Finally, due to the nature of case-control studies, it cannot rule out that both
endometriosis and ovarian cancer (especially clear cell or endometrioid type) may
simply share some common risk and/or protective factorssuch as the incessant
menstruation and OC use, or yet to be identifiedso that an elevated OR is still an
association, but the relationship is by no means causal.
The finding reported by Melin et al. [53] that the risk of developing ovarian
cancer in women with endometriosis could be cut by 70 % if they had all visible
endometriotic lesions removed is particularly interesting. So far almost all other
case-control studies published failed to control the effect of surgical treatment on
the endometriosis-ovarian cancer association, even though the diagnosis of endo-
metriosis is usually established by laparoscopic visualization of lesions, which is
almost always followed by surgical removal of the lesions. Of course, some sub-
types of endometriotic lesions, such as deep infiltrating endometriosis, can be
challenging to remove completely. But does this mean that those women who had
a complete removal of all their visible lesions are those who had less severe
endometriosis? How does this surgical completeness or radicality interact with
the extensiveness or severity of endometriosis and impact the risk of developing
ovarian cancer? There are no data to answer these questions.
While younger age at diagnosis of endometriosis compared to that of ovarian
cancer is often taken as a proof of temporality in the causal link, many epidemio-
logical studies have not clearly demonstrated a temporal relationship between
endometriosis and ovarian cancer. Since it is now well documented that, similar
to cancer, endometriotic lesions are monoclonal in origin [61], one way to prove the
temporal relationship, perhaps once for all, and to provide a convincing proof that
some histotypes of ovarian cancer originate from endometriosis is to reconstruct a
phylogenetic trees delineating the relationship between endometriotic lesions and
ovarian cancer based on molecular clock. A proof-of-concept study demonstrating
the utility of the molecular clock in reconstructing the geological relationship
among pieces of endometrial fragments demonstrates that the phylogenetic
approach is feasible using todays genetic technology [62].
In summary, while published clinical and epidemiological studies strongly

implicated the risk, though moderate, of developing certain histotypes of ovarian
cancer in women with endometriosis, many stones are still left unturned. Future
studies need to determine whether the association is causal with a clear temporal
relationship or merely association due to exposure to shared risk factors. Given the
moderate association, it is perhaps premature to institute any actionable measures
as of now. Future studies also need to resolve an apparent discrepancy in estimated
effect size between the clinical data and epidemiological data and to further
delineate the molecular pathways linking endometriosis and ovarian cancer. Care
should be taken to avoid making just-so stories.
Acknowledgment I would like to thank Professor Paolo Vercellini for stimulating the discussion
when preparing for this chapter. This research was supported in part by grant 81270676 from the
National Science Foundation of China and financial support from Fudan University and the
Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases.
Conflict of Interest Statement. None declared.
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Chapter 23
Surgical Management of Endometriosis
Imari Deura and Tasuku Harada
Abstract Endometriosis, a common disease affecting about 10 % of women

of reproductive age, causes pelvic pain and infertility. Pelvic endometriosis is
histologically categorized into peritoneal superficial endometriosis, ovarian
endometrioma, and deep infiltrating endometriosis (DIE). Surgical treatment for
endometriosis aims to relieve symptoms and preserve fertility, in most cases by
restoring anatomy, by lysing adhesions, and by removing endometriotic lesions.
Laparoscopic surgery, which is the standard surgical procedure for endometriosis,
reduces pelvic pain and improves fertility by means of excision and ablation of
endometriotic lesions. Managing endometriomas in women who wish to conceive is
controversial because two main risks may occur after conservative surgery: recur-
rence of the disease and significant reduction in ovarian reserve. Surgical treatment
for endometriosis should be tailored to the individual according to clinical presen-
tation and personal wishes. In this chapter, we describe laparoscopic conservative
surgery for pelvic endometriosis, particularly for ovarian endometrioma.
Keywords Cystectomy Infertility Laparoscopic surgery Ovarian

endometrioma Ovarian reserve
23.1 Surgical Approach for Endometriosis
Laparoscopy compared with laparotomy is considered the gold standard surgical

treatment for endometriosis, especially when endometriomas are present. The two
approaches do not differ in terms of pain relief, fertility outcome, and risk of
recurrence [14]. One randomized controlled trial (RCT) revealed that laparoscopic
I. Deura (*) T. Harada

Department of Obstetrics and Gynecology, Tottori University
School of Medicine, Yonago, Japan
e-mail: imari@med.tottori-u.ac.jp

386 I. Deura and T. Harada
surgery for endometriomas was associated with less pain after surgery, shorter
hospital stay, and faster recovery compared with laparotomy and that the
complication rate and operative time were similar in the two approaches [5].
A meta-analysis of 27 RCTs comparing the outcome of laparoscopic surgery and
laparotomy for benign gynecologic pathologies also showed that the two
approaches exposed patients equally to complications [6]. These results suggest
that laparoscopic surgery, minimum invasive technique, is feasible and safe as well
as laparotomy and that it should be used as a first-line choice in conservative
surgery for endometriosis.
23.2 Surgical Treatment

for Endometriosis-Associated Pain
Endometriosis is present in approximately 70 % of patients with pelvic pain [7].

Endometriotic lesions and adhesions are clearly associated with pain symptoms,
which include dysmenorrhea, non-menstrual chronic pelvic pain, dyspareunia, and
dyschezia. These symptoms significantly affect the quality of life of women of
reproductive age. Lesions and adhesions are included in several scoring systems,
for example, the commonly used revised American Fertility Society (rAFS) clas-
sification, which describes the severity of endometriosis, but not the severity of
pain. The relationship between the stage or lesion type of endometriosis and
severity of pelvic pain has been studied, but the results are inconsistent [8]. Severe
pelvic pain is associated with DIE; however, the depth of endometriosis does not
affect the stage of rAFS classification. This may be one reason that this scoring
system failed to show severity of pain.
Endometriosis can be treated either medically or surgically. Surgery allows
visual diagnosis and is usually used when medical treatment fails or produces
unacceptable side effects or when conception is desired. The aim of surgical
treatment is to restore anatomy by dividing adhesions and removing visible
endometriotic lesions, thus relieving pain. Endometriosis often develops in
women of reproductive age; therefore, laparoscopic surgery commonly needs to
preserve fertility.
The RCT reported by Sutton et al. showed that laparoscopic laser ablation
resulted in statistically significant pain relief at 6 months after surgery in women
with minimal to moderate endometriosis compared with diagnostic laparoscopy [9].
A follow-up study of Suttons RCT demonstrated that pain relief after laparoscopic
laser ablation continued at 1 year in 90 % of those who initially responded [10].
Abbott et al. performed a randomized, placebo-controlled trial of patients with
minimal to severe endometriosis, comparing laparoscopic excision with placebo,
and found that 80 % of the excision group had reduced pain and improved quality of
23 Surgical Management of Endometriosis 387
life by 6 months compared with only 32 % in the placebo group. Surprisingly,

diagnostic laparoscopy was associated with a 20 % placebo response rate [11].
A meta-analysis consisting of five RCTs also demonstrated an advantage in using
laparoscopic surgery for pelvic pain associated with endometriosis compared with
diagnostic laparoscopy alone [12]. The two RCTs reported that 2038 % of patients
had no improvement in symptoms after operative laparoscopy [9, 11], implying that
surgical treatment alone for endometriosis-associated pain has limitations.
Which of the laparoscopic surgical modalities is most effective for pain relief in
endometriosis is inconclusive. Healey et al. showed no difference in pain relief
between laparoscopic ablation and excision at 12 months after surgery for patients
with minimal to severe endometriosis [13]. A similar result was reported in another
study of minimal to mild endometriosis [14].
Several surgical procedures to interrupt pelvic nerve pathways have been
performed to reduce pelvic pain caused by endometriosis, such as uterine nerve
ablation, presacral neurectomy, uterosacral ligament resection, and so on. However,
there is insufficient evidence of pain relief to recommend their use [15, 16].

for Endometriosis-Associated Infertility
Endometriosis is present in 2068 % of subfertile women [17]. Severe endometri-

osis may impair fertility due to anatomical distortion caused by adhesions. How-
ever, endometriosis is associated with infertility even in the early stages without
adhesions. The exact mechanism by which endometriosis interferes with fertility is
not fully understood, and therefore the strategy for treatment is controversial.
Medical treatment that suppresses ovarian function has no effect to improve fertility
in women with endometriosis and should not be offered to patients wishing to
conceive [18]. Adamson et al. demonstrated that surgical treatment was superior to
medical treatment regarding the pregnancy rate of infertile women with minimal
and mild endometriosis [19]. Whether surgical treatment or artificial reproductive
technique should be performed first has been debated.
Endometriosis is treated surgically by lysing adhesions and excising or ablating
endometriotic lesions.
A meta-analysis consisting of two RCTs demonstrated that laparoscopic surgery,
including excision and ablation with adhesiolysis, improves fertility in patients with
minimal and mild endometriosis compared with diagnostic laparoscopy alone [20].
There seems to be a negative correlation between the stage of endometriosis and the
spontaneous cumulative pregnancy rate after surgical excision of endometriosis.
No evidence recommends choosing laparoscopic surgery first for patients with
moderate or severe endometriosis complaining of infertility alone [21].
23.4 Ovarian Endometrioma
23.4.1 Surgery for Endometrioma
Ovarian endometrioma, also known as chocolate cyst, is a form of endometriosis

located on the ovaries. Between 17 and 44 % of patients with endometriosis have
endometriomas [2224]. Endometriosis by itself does not always cause
endometriosis-related symptoms. No consensus has been reached on the definitive
intervention for endometrioma, particularly in infertile women.
Surgical treatment is usually recommended for large symptomatic
endometrioma, and the indication for surgery also depends on the risks of rupture,
infection, torsion of the ovary, and malignant formation. Laparoscopic surgery is
the gold standard for endometrioma [5], but which is the best modality of conser-
vative surgery remains controversial. Drainage of endometriomas alone is not
recommended because of the high recurrence rate [25]. Two main modalities are
used: ablation of the cyst wall and cyst excision, also called cystectomy. Laparo-
scopic cystectomy has been considered a first-line choice of surgical treatment. A
meta-analysis demonstrated that laparoscopic cystectomy for endometriomas larger
than 3 cm in diameter increased spontaneous conception in subfertile women with
less recurrence of pain symptoms and fewer endometriomas compared with abla-
tion alone. Ovarian response to gonadotrophin stimulation after surgery was similar
between the two modalities despite concerns over the more damaging effect of
cystectomy on ovarian reserve [26].
23.4.2 ART and Endometrioma
Endometrioma may interfere with the outcome of artificial reproductive technology

(ART). ART cycles of women with endometriomas have several problems, includ-
ing difficulty monitoring ovarian response by ultrasound, poor ovarian response to
controlled ovarian stimulation (COH), and infection after oocyte pick up [2729].
Gupta et al. showed decreased ovarian response to COH with in vitro fertilization
(IVF) cycles of patients with endometriomas compared with controls [30], which
implies a decline of ovarian reserve due to endometrioma itself.
Endometriomas larger than 3 cm are generally treated surgically before ART,
but surgery may actually decrease the success of ART due to damaged ovarian
reserve. Demirol et al. evaluated the outcome of intracytoplasmic sperm injection
(ICSI) in patients with prior cystectomy for endometrioma between 3 and 6 cm in
diameter compared with ICSI alone. Cystectomy resulted in longer COH, higher
FSH requirement, and lower mature oocyte number, but fertilization, pregnancy,
and implantation rates did not differ [31]. Two meta-analyses concluded that
surgical treatment of endometriomas prior to ART did not improve reproductive
outcome [32, 33].
Lumen
Epithelium of endometrioma
Fibrotic layer
Cortex
Medulla
200mM
Fig. 23.1 Normal ovarian tissue adjacent to the cyst wall of endometrioma. The tissue specimen
of the stripped cyst wall of endometrioma contains normal ovarian tissue. Arrows ovarian follicle
23.4.3 Ovarian Reserve and Endometrioma
The pathogenesis of endometrioma is controversial. Endometrioma is generally

believed to result initially from a deposit of endometrium that passed through the
fallopian tube causing adherence of the ovary to the pelvic peritoneum and pro-
gressive vagination of the ovary [3436]. Considering that endometrioma is a
pseudocyst, cyst excision may involve removing some ovarian tissue [37]. One
concern over performing cystectomy for endometrioma is that it may damage
ovarian reserve when normal ovarian cortex is removed and thermal coagulation
is used for hemostasis, resulting in the loss of follicles (Fig. 23.1).
Ovarian reserve is defined as the total ovarian follicle pool including primordial
and growing follicles [38]. Serum anti-Mullerian hormone (AMH) is the most
useful, reliable, and sensitive marker of ovarian reserve compared with other
known serum markers [39]. AMH is stable throughout the menstrual cycle and
sensitive to decline in ovarian reserve with aging and is not affected by the use of
hormones [40]. Serum AMH correlates to antral follicle count (AFC) measured by
ultrasound, which is also a reliable marker of ovarian reserve [41].
A meta-analysis of eight prospective cohort studies to investigate the effect of
cystectomy for endometrioma on ovarian reserve as determined by serum AMH level
found that serum concentration of AMH significantly decreased after surgery [42].
Multivariate analyses showed that the risk factors associated with reduction of serum
AMH after cystectomy were as follows: bilateral endometriomas, presurgical serum
AMH level, and the presence of normal ovarian tissue in the enucleated cyst
[4345]. Var et al. compared postsurgical ovarian reserve determined by AFC
between laparoscopic cystectomy and ablation for bilateral endometriomas.
AFC was significantly decreased in cystectomized ovaries compared with ablated
ovaries [46]. One surgical modality used as an alternative to cystectomy, a three-step
procedure proposed by Donnez et al., involves drainage of the cyst during
laparoscopy, GnRH agonist treatments, and then laser vaporization of the remains
during a second laparoscopy [47]. Ovarian reserve, determined by AMH and AFC,
was less diminished after the three-step procedure for endometrioma compared with
cystectomy [48, 49]. Donnez et al. also proposed a combined technique consisting of
excision of a large part of the endometrioma and laser vaporization of the remaining
1020 % of the cyst wall close to the hilus. AFC after the combined technique was
similar to that of women without endometriosis or contralateral normal ovaries
[50]. Use of electrosurgical coagulation to achieve hemostasis after stripping the
endometrioma may amplify damage to ovarian reserve. Why ovarian reserve declines
after cystectomy is not precisely understood, but it may relate to the methods used for
hemostasis, including suturing the ovaries and bipolar coagulation [51, 52]. Details of
the studies evaluating ovarian reserve before and after cystectomy are shown in
Table 23.1 [5357].
We know that laparoscopic cystectomy for endometrioma has a negative impact
on ovarian reserve, but we should take into account that the presence of
endometrioma per se is also associated with a decrease in ovarian reserve [58].
23.4.4 Recurrence of Endometrioma
The recurrence of endometrioma after conservative surgery is a serious problem.

Postoperative recurrence rates vary between 6 and 78 % after 25 years, depending
on the surgical modality and the length of postoperative time [5966]. Details of the
studies evaluating the recurrence rates of endometriomas after surgery are shown in
Table 23.2. Long-term follow-up is necessary to assess endometrioma recurrence
after surgery. The risk factors for recurrent endometrioma are often varied, and the
rAFS staging system is not predictive of recurrence [67, 68].
Cochrane study comparing the recurrence of endometriomas and pain symptoms
after surgery showed that cystectomy is more advantageous than ablation [26].
Carmona et al. found an earlier recurrence of endometriomas at 5 years of follow-up
after ablation compared with cystectomy [69].
Repeated surgery for recurrent endometriomas is not recommended. The proba-
bility of conception after secondary surgery is almost half that after a primary surgery.
The repetitive damage to the ovaries should be avoided to preserve the already reduced
reproductive potential.
Curing endometriosis by conservative surgery alone is difficult, and studies have
shown that postoperative medical treatment can only delay the recurrence of
endometrioma [70]. However, a recent report showed that treating with long-term
oral contraceptives decreases the recurrence of endometrioma.
The patients should be informed of the risk of postoperative recurrence, and
long-term adjuvant treatment to suppress ovulation suggested until pregnancy is
desired.
23
Table 23.1 Ovarian reserve before and after cystectomy
Sample Follow-up Outcome

Study Design size (month) Variable Preoperative Postoperative P value
Tsolakidis 2009 [49] RCT 10 10 AMH 3.9 1.3 2.9 0.6 0.002
Pados 2010 [48] RCT 10 12 AFC 2.0 1.3 2.4 0.8 NS
Biacchiardi 2011 [53] Prospective cohort 43 9 AMH 3.0 0.4 1.3 0.3 <0.001
Surgical Management of Endometriosis
Celik 2012 [45] Prospective cohort 65 6 AMH 1.8 1.7 0.7 0.8 <0.001
Ercan 2010 [54] Prospective cohort 64 1 AMH 1.6 1.1 1.4 1.2 NS
Ercan 2011 [55] Prospective cohort 36 3 AMH 2.0 0.4 1.95 0.6 NS
Kitajima 2011 [44] Prospective cohort 19 3 AMH 4.3 3.0 3.0 2.5 NA
Hirokawa 2011 [45] Prospective cohort 38 1 AMH 3.9 2.5 2.1 1.6 <0.001
Hwu 2011 [56] Prospective cohort 31 3 AMH 3.9 0.4 2.01 0.2 <0.01
Lee 2010 [57] Prospective cohort 13 3 AMH 4.7 2.5 3.3 2.1 <0.05
Uncu 2013 [58] Prospective cohort 30 6 AMH 2.8 2.2 1.8 1.3 0.02
Var 2011 [46] RCT 48 6 AFC 5.6 1.1 3.67 1.3 0.001
Note: Values are mean SD. AMH levels are reported in nanograms per milliliter. P < 0.05 was statistically significant
RCT randomized controlled trial, AMH anti-Mullerian hormone, AFC antral follicle counts, NS not statistically significant, NA not available
391
Table 23.2 Recurrence rate of endometrioma after cystectomy or ablation

Recurrence rate
Sample Follow- Cystectomy Ablation P
Study Design size up (year) (%) (%) value
Alborzi 2004 [59] RCT 100 2 17.3 31.3 0.16
Beretta 1998 [60] RCT 64 2 6.2 18.8 NS
Busacca 1999 [61] Retrospective 366 4 11.7 NA
follow-up
Carmona 2011 [69] RCT 74 5 22.2 36.8 0.2
Fedele 2006 [62] Descriptive 305 5 18.9 NA
Hart 2011 [26] Meta-analysis 164 2 13.1 26.3 <0.05
Hemmings 1998 [63] Retrospective 103 3 8.0 12.0 NS
Kikuchi 2006 [64] Retrospective 315 3 27.0 NA
Koga 2006 [65] Retrospective 224 2 30.4 NA
Saleh 1999 [66] Retrospective 231 4 25.0 78.0 0.0003
Note: P < 0.05 was statistically significant. RCT randomized clinical trial, NS not statistically
significant, NA not available
23.5 Laparoscopic Techniques for Endometriosis
Laparoscopic conservative surgery for endometriosis aims to restore anatomy by

means of adhesiolysis and by destructing endometriotic lesions. Advanced techni-
cal skills and systematic surgical procedures are required to prevent damage to
adjacent organs, particularly in cases with an obliterated cul-de-sac.
Peritoneal endometriosis can be destructed by excision or ablation, the latter
being commonly used. Symptom relief after surgery does not differ between
excision and ablation [71]. Bipolar coagulation and vaporization with CO2 laser
or plasma energy are often used as ablation techniques. Laser vaporization pene-
trates tissue at a shallow depth, which is useful to avoid deep thermal tissue damage.
Delicate manipulation of forceps is needed to dissect pelvic adhesions. There are
two types of adhesions in endometriosis, filmy adhesions and dense adhesions with
fibrotic tissue. Filmy adhesions can be dissected bluntly, while dense adhesions
require sharp dissection. A contralateral traction should be applied with the proper
instruments (e.g., a uterine manipulator). Dissection techniques are used to lyse
adhesions surrounding the ovaries, remove the ovarian cysts, and open the obliter-
ated cul-de-sac.
To free adhesions surrounding the ovaries, contralateral traction should be
applied (Fig. 23.2a). Fibrotic tissue can be dissected sharply using monopolar
diathermy with pure cutting current. Sharp and blunt dissections are repeated
alternatively until the whole length of the utero-ovarian ligament is visible. The
cyst is almost always ruptured during this procedure.
The procedures for ovarian cystectomy are followed. The content of the cyst is
aspirated. The ovarian incision obtained by cyst rupture is enlarged (Fig. 23.2b).
The cleavage plane between the cyst wall and the ovarian tissue should be ade-
quately identified (Fig. 23.2c). The cyst wall is bluntly stripped from ovarian tissue
Fig. 23.2 Laparoscopic techniques for endometriosis. (a) The adhesion surrounding the left
ovary is freed. (b) The ovarian incision obtained by cyst rupture is enlarged. (c) The cleavage
plane between the cyst wall and normal ovarian tissue is adequately identified. (d) The cyst wall
is bluntly stripped from normal ovarian tissue. (e) Laser vaporization is applied to the cyst
wall close to the left ovarian hilus. (f) The spaces outside the right uterosacral ligament are
opened and the right ureter is separated from the right uterosacral ligaments. Rt. USL the right
uterosacral ligament. (g) The space inside the right uterosacral ligament is opened and the rectum
is separated from the right uterosacral ligaments. The right uterosacral ligament. (h) Sharp
dissection using the scissors is applied between the uterus and the rectum. (i) The obliterated
cul-de-sac is opened
with atraumatic forceps and scissors applying a contralateral traction (Fig. 23.2d).
The boundary between the cyst wall and the ovarian tissue should be exposed
constantly. After removal of the cyst wall, hemostasis is achieved with the pinpoint
bipolar coagulation of bleeding sites on the ovary. The combined technique is
sometimes used for patients with bilateral or multiple endometriomas [50]
(Fig. 23.2e). The vasopressin injection technique may be useful to decrease bleed-
ing after stripping the endometrioma [72], but an inadequate cleavage plane
resulting from hydrodissection may cause the removal of normal ovarian tissue.
The procedures to open an obliterated cul-de-sac are followed. First, the bilateral
ureters are identified. The spaces outside the bilateral uterosacral ligament are
opened and the ureters are separated from the uterosacral ligaments (Fig. 23.2f).
After separation of the ureters, the spaces inside the uterosacral ligaments are
opened and the rectum is separated from the uterosacral ligaments (Fig. 23.2g).
The adhesion between the rectum and the uterine cervix can be dissected bluntly
with a contralateral traction when the adhesion is not dense. Sharp dissection is
needed to dissect dense adhesions with fibrotic tissue. Monopolar diathermy with
pure cutting current can dissect dense adhesions surrounding the rectum. Sharp
dissection using the scissors should be used to avoid thermal injury of the rectum
(Fig. 23.2h). After these procedures to open the obliterated cul-de-sac, it is possible
to remove the DIE lesions of the uterosacral ligaments and the rectovaginal septum
(Fig. 23.2i).
23.6 Conclusions
Surgical candidates might include the following patients with endometriosis: those
with severe pain symptoms, large ovarian endometriomas, and infertility caused by
minimal and mild endometriosis.
Surgery for endometriosis has an important role in relieving pain symptoms and
improving fertility by lysing adhesions and removing lesions. However, the optimal
procedure is as yet undetermined because of several controversial issues: the
recurrence of disease and ovarian reserve decline after conservative surgery.
Complete cure of endometriosis is not currently possible by surgery alone, partic-
ularly the conservative procedure. Surgical treatment of endometriosis should be
tailored to the individual according to clinical presentation and personal wishes,
and combined treatment with medical treatment or ART after surgery is needed.
We cannot deal with all patients with endometriosis in the same way.
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Chapter 24
Systematic Laparoscopic Surgery
for Complete Obliteration of the Cul-de-sac
Yoshiaki Ota, Masaaki Andou, Keiko Ebisawa, Kazuko Fujiwara,

Tomonori Hada, Hiroyuki Kanao, and Ikuko Ota
Abstract Endometriosis has been reported as a major cause of pelvic pain. Most
notably, deep infiltrating endometriosis is a very active disease that occurs in 20 %
of women with endometriosis. We have been actively dissecting deep infiltrating
diseased areas within the sacral ligaments around the uterus in order to improve
dysmenorrhea and chronic pelvic pain caused by deep infiltrating endometriosis.
Laparoscopic surgery is an ideal option to treat deep infiltrating endometriosis
involving complete cul-de-sac obliteration due to its minimal invasiveness and
ability to achieve an appropriate depth of surgical field. It is important to prevent
pain recurrence by providing systematic surgery and removing the deep infiltrating
endometriosis safely and widely. To reduce recurrence, it is ideal to provide
postoperative education to maintain the effect of surgery.
Keywords Complete cul-de-sac obliteration Deep infiltrating endometriosis

(DIE) Laparoscopic surgery
24.1 Introduction
Endometriosis occurs in 610 % of women of reproductive age [1]. There are three
histological classifications: peritoneal endometriosis, endometrioma, and deep
infiltrating endometriosis [2]. Treatment is largely divided into either surgical or
pharmacological intervention.
Y. Ota (*) M. Andou K. Ebisawa K. Fujiwara T. Hada H. Kanao

Department of Obstetrics and Gynecology, Kurashiki Medical Center, Kurashiki, Japan
e-mail: yoshimon@cj8.so-net.ne.jp
I. Ota
Department of Obstetrics and Gynecology, Kurashiki Heisei Hospital,
Kurashiki, Japan

400 Y. Ota et al.
In pharmacological intervention, recently, it has become a trend to use either

low-dose estrogenprogestin (LEP) or dienogest but neither option is practical for
women who are preparing to conceive.
On the other hand, the surgical option is expected to provide early pain relief.
Endometriosis has been reported as a major cause of pelvic pain. Most notably,
deep infiltrating endometriosis is a very active disease that occurs in 20 % of
women with endometriosis and is strongly associated with pelvic pain [3, 4].
Furthermore, deep infiltrating endometriosis is not only a cause of infertility, but
also may lead to functional impairments of retroperitoneal organs by causing
compartment pressure and constriction.
We have been actively dissecting deep infiltrating diseased areas within the
sacral ligaments around the uterus in order to improve dysmenorrhea and chronic
pelvic pain caused by deep infiltrating endometriosis.
It is important to prevent possible residual diseases and ureteral and rectal injury
when considering cases specifically involving complete cul-de-sac obliteration.
Laparoscopic surgery is an ideal option to treat deep infiltrating endometriosis
involving complete cul-de-sac obliteration due to its minimal invasiveness and
ability to achieve an appropriate depth of surgical field.
In this article, focusing on deep infiltrating endometriosis with complete
cul-de-sac obliteration, we report our surgical methods and results.
24.2 Subjects
Between January 2008 and July 2012, we performed 622 laparoscopic surgical
resections of endometriosis in patients of childbearing age. Among them,
those with and without deep infiltrating endometriosis (DIE) numbered
369 and 253, respectively. Therefore, 60 % of cases had DIE (Fig. 24.1).
We divided our cohort into four groups, as follows: 372 (age: 33.91 5.75)
cases without postoperative pharmacological treatment, 123 (age: 31.60 5.70)
cases with postoperative low-dose estrogenprogestins (LEP), 35 (age:
30.94 5.97) cases with postoperative treatment with 1 mg of dienogest,
and 92 (age: 33.32 6.23) cases of postoperative treatment with 2 mg of
dienogest (Table 24.1).
41%
59%
Fig. 24.1 Sixty percent of

patients with DIE: 187
cases had deep infiltrating
endometriosis patients without DIE: 185
24 Systematic Laparoscopic Surgery for Complete Obliteration of the Cul-de-sac 401
Table 24.1 Between January 2008 and July 2012, we performed 622 laparoscopic surgical
resections of endometriosis in patients of childbearing age
Dienogest Dienogest
No postoperative COC treatment treatment
pharmacotherapy treatment 1 mg/day 2 mg/day
(n 372) (n 123) (n 35) (n 92) P-value
Age (years) 33.91 5.75 31.60 5.70 30.94 5.97 33.32 6.23 0.01
Presence of deep infiltrating endometriosis
185 48 8 12 0.01
+ 187 75 27 80
Beecham classification
Stage I 16 1 1 1 0.08
Stage II 209 65 14 26
Stage III 60 21 7 17
Stage IV 87 36 13 48
Laterality of endometriotic cysts
31 5 2 10 0.075
+
Monolateral 196 76 17 40
Bilateral 145 42 15 43
24.3 Surgical Methods: Excision of Cul-de-sac

Obliteration and DIE Method
Firstly, in the case of complete cul-de-sac obliteration, deep infiltrating endometri-

osis is distributed around the uterosacral ligament.
Deep infiltrating endometriosis is attached to the rectum and urinary tubes.
Therefore, when removing the deep infiltrating endometriosis, it is important to
separate the rectum and urinary tube from the diseased area in order to prevent
tissue damage and avoid leaving residual diseases.
Therefore, we have been dissecting interstitial spaces between deep infiltrating
endometriosis and the urinary tube which center around the uterosacral ligament.
This interstitial space is Okabayashis pararectal space, and we have termed the
outside of the uterosacral ligament the lateral pararectal space, LPRS.
Furthermore, we refer to the interstitial space between the rectum and lateral
pararectal space as the median pararectal space, MPRS (Fig. 24.2).
24.3.1 Method to Separate Ureter
In most cases, deep infiltrating endometriosis is distributed around the uterosacral

ligament and cervix uteri, and almost all cases involve adhesion of the lower
urinary tube to the outer side of the uterosacral ligament.
402 Y. Ota et al.
Fig. 24.2 Anatomy of cul-de-sac obliteration. The lateral pararectal space is consistent with the
outside of the uterosacral ligament. The medial pararectal space is consistent with the interstitial
space between the rectum and lateral pararectal space
Especially, adhesion occurs between the uterosacral ligament and either the urinary
tube or rectum, and the urinary tube may be unpredictably shifted; therefore, identi-
fying and separating the urinary tube first may facilitate a safe operation.
In cases of cul-de-sac obliteration, we identify the urinary tube where it crosses
the common iliac artery and trace it down as far as possible.
We locate and unfold the posterior side of the broad ligament of the uterus, and
identify the urinary tube.
Next, we open the LPRS which consists of the internal aspect of the urinary tube
and extraluminal space of the uterosacral ligament.
This leads to separation of the urinary tube from the deep infiltrating endome-
triosis centered around the uterosacral ligament (Fig. 24.3).
24.3.2 Method to Obliterate Complete Cul-de-sac Adhesion
In the case of complete cul-de-sac adhesion, the strongest adhesion is at the

posterior uterine cervix and anterior aspect of the rectum.
When the direction of the rectum is not clear, it is possible to cause rectal
damage while dissecting between the posterior uterine cervix and anterior aspect
of the rectum.
Therefore, we have been approaching from the lateral rectal side.
We dissect the lateral rectal fossa from the rectum and then deeply obliterate the
space outside the lateral rectal fossa.
Fig. 24.3 LPRS lateral pararectal space. Developing on the right side of the LPRS. The ureter was
separated from the sacral ligament
This space is the MPRS described earlier. Then, we proceed to dissect the MPRS
further anterior toward the posterior part of the vagina.
This leads to complete separation of the deep infiltrating endometriosis centered
around the uterosacral ligament and rectum.
Furthermore, it has become possible to laterally identify part of the anterior
aspect of the rectum.
In this way, dissecting from the lateral side leads to gradual identification of the
rectal outline.
Approaching the right and left lateral sides leads to identification of the remaining
strongest point of adhesion between the posterior uterus and anterior rectum.
At this step, since we have clearly outlined the anterior aspect of the rectum, it is
safe to proceed with dissecting the center part of the rectum.
Dissection of the remaining center part of the rectum leads to separation of the
cul-de-sac obliteration (Fig. 24.4).
24.3.3 Method of Deep Infiltrating Endometriosis Excision
Firstly, disseminate the LPRS to separate the ureter and deep infiltrating endome-
triosis centered around the uterosacral ligament. Then, by disseminating the MPRS
to separate the rectum, deep infiltrating endometriosis will be isolated, attaching to
the posterior aspect of the uterus in the shape of an upside-down U.
This isolated deep infiltrating endometriosis will be removed from the posterior
side of the uterus without injuring the deep uterine vein.
Since the urinary tube and rectum have been isolated, it is possible to further
separate the diseased area systematically (Fig. 24.5).
404 Y. Ota et al.
Fig. 24.4 MPRS medial pararectal space. Developing on the right side of the MPRS. The rectum
was separated from the side of the sacral ligament
Fig. 24.5 Isolation of DIE in the shape of a horseshoe. The LPRS and MPRS should be developed
to isolate DIE from the lateral pararectal space. It is shaped like a horseshoe
24.3.4 Checking Damage After Excision

of Cul-de-sac Obliteration
As described earlier, it is important to check for rectal damage after disseminating

the MPRS and removing the cul-de-sac obliteration.
Firstly, we perform digital rectal examination by extending the dissected area,
and confirm that damage is not present laparoscopically.
Then, we perform a leak test. The leak test is performed by pumping 50100 mL
of air into the rectum using a large soft catheter (Figs. 24.6 and 24.7).
Fig. 24.6 Air leak test. To check for rectal injury, the air leak test is important. Nelatons catheter
was inserted into the anus and 50 mL of air was pumped into the rectum using a syringe
Fig. 24.7 Air leak test. Checking air leak from the rectum using a laparoscope from the inter-
abdominal side. Sometimes, pin hole rectal injury occurred in the pararectal area. If rectal injury is
overlooked, it will cause serious postoperative complications
406 Y. Ota et al.
24.4 Surgical Outcome
Three hundred and seventy-two subjects who did not receive pharmacological
treatment after surgery were divided into two groups: 187 cases with surgical
resection of deep infiltrating endometriosis, and 185 cases without deep infiltrating
endometriosis. We defined recurrence of chocolate cyst of 2 cm or larger by either
ultrasound or MRI, and recurrence of pelvic pain as a VAS score equal to or greater
than the score assessed prior to the operation. The cumulative risk of recurrence was
calculated using the KaplanMeier method, and the log-rank test was performed.
The cumulative risk of recurrence in the group with deep infiltrating endome-
triosis resection was 6 % at 3 years postoperatively, while that of those who did not
require resection was 10 %. No significant difference was observed between these
two groups (Fig. 24.8).
Since these results were compared among groups without postoperative phar-
macological intervention, they indicate adequate resection of deep infiltrating
endometriosis and pain control. Therefore, these results show that our operating
method of systematic resection of deep infiltrating endometriosis is effective.
On the other hand, recurrence of chocolate cyst at 4 years postoperatively was
30 % in both groups (Fig. 24.9).
10%
6%
1.0
No DIE (n = 185)
Recurrece-free subjects (%)
0.8 Needed DIE excision

(n = 187)
0.6
0.4
0.2
Log-rank ^2 = 0.039
NS
0.0 P = 0.844
0 10 20 30 40 50 60
Time after surgery (months)
Fig. 24.8 The effect on pain of systematic DIE and endometrioma excision. Comparison of the
recurrence rate of pain in the presence/absence of deep infiltrating endometriosis (372 women
receiving no medical therapy after surgery). The recurrence rate of pain was 10 % after surgery at
4 years. There was no the significant difference between the No DIE group and Needed DIE
excision group for 4 years. Systematic DIE excision mostly controlled pain recurrence for 4 years
1.0
30%
Recurrece-free subjects (%) 30%
0.8
No DIE (n = 185)
0.6 Needed DIE excision
(n = 187)
0.4
0.2
0.0
0 10 20 30 40 50 60
Time after surgery (months)
Fig. 24.9 The effect on endometrioma recurrence of systematic DIE and endometrioma excision.
Comparison of recurrence rate of endometrioma by the presence/absence of deep endometriosis
(372 women who received no medical therapy after surgery). The recurrence rate of endometrioma
was 30 % at 3 years. There was no significant difference between the No DIE group and Needed
DIE excision group for 4 years. One reason is that surgery is often incomplete to maintain the
ovarian reserve. Endometrioma will recur in 30 % after surgery within 4 years
24.5 Discussion
Treatment for endometriosis is largely divided into two types: surgical intervention
and pharmacological treatment.
Recently, it has become a trend to use pharmacological treatment with either
low-dose estrogenprogestin (LEP) or dienogest, but neither option is practical for
women who are preparing to conceive.
On the other hand, surgery provides rapid pain relief, but, among surgical
methods, the biggest setback of fertility preserving surgery has been the high
recurrence rate. It has been reported that the recurrence rate at 2 to 5 years
postoperatively of the chocolate cyst was 12 to 30 % [510], while the recurrence
rate of pain was 1049 % [5, 8, 11, 12].
Also, it has been clearly reported that deep infiltrating endometriosis has
an association with strong pelvic pain, and in those with disease around the
uterosacral ligament, the odds ratio of chronic pelvic pain was 2.1, while the odds
ratio of dyspareunia was 2.0 [2]. Therefore, we have completely removed deep
infiltrating endometriosis and optimized surgery to prevent ureteral and
rectal injury.
Specifically, in cases with complete cul-de-sac obliteration, we performed LPRS
and MPRS to systemically separate the urinary tube and rectum from deep infil-
trating endometriosis.
408 Y. Ota et al.
With this method, the 4-year postoperative pain recurrence rate was similar in
both groups, that is, almost 15 % for both patients with or without deep infiltrating
endometriosis.
These results are from the study of cases without postoperative pharmacological
intervention and, therefore, our systematic resection was considered effective for
pain control at 4 years postoperatively.
On the other hand, the cumulative risk of recurrence of ovarian chocolate cyst
was 30 % at 4 years postoperatively. Recurrence of pain was considered to be the
recurrence of deep infiltrating endometriosis, and the recurrence rate of ovarian
chocolate cyst was 3 to 5 times higher than that of deep infiltrating endometriosis.
The ovarian reserve needs to be considered in cases of ovarian chocolate cyst
surgery; however, it is possible to enucleate large fields in the case of deep
infiltrating endometriosis. The recurrence rate may be associated with differences
in both operative backgrounds. Therefore, it is important to prevent pain recurrence
by providing systematic surgery and removing the deep infiltrating endometriosis
safely and widely. On the other hand, in cases of ovarian chocolate cyst, we provide
surgery while considering the ovarian reserve. In terms of recurrence, as Vercellini
and others reported that recurrence can be reduced to 37 % at 3 years postopera-
tively by providing combined oral contraceptives (COCs), we think that it is
optimal to provide combined oral contraceptives (COCs) postoperatively [13].
24.6 Conclusion
In cases of deep infiltrating endometriosis with complete cul-de-sac obliteration, it

is ideal to provide systematic enucleation to avoid rectal and urinary tube damage
with the LPRS and MPRS in order to prevent pain recurrence.
References

2. Chapron C. Ovarian endometrioma: severe pelvic pain is associated with deeply infiltrating
endometriosis. Hum Reprod. 2012;27(3):70211.
3. Vercellini P. Endometriosis: what a pain it is. Semin Reprod Endocrinol. 1997;15(3):25161.
4. Chapron C. Operative management of deep endometriosis infiltrating the uterosacral liga-
ments. J Am Assoc Gynecol Laparosc. 1999;6(1):317.
5. Busaca M. Recurrence of ovarian endometrioma after laparoscopic excision. Am J Obstet
Gynecol. 1999;180:51923.
6. Saleh A. Reoperation after laparoscopic treatment of ovarian endometriomas by excision and
by fenestration. Fertil Steril. 1999;72:3224.
7. Ghezzi F. Recurrence of endometriosis and anatomical location of the primary lesion. Fertil
Steril. 2001;75:13640.
8. Jones KD. Recurrence of chocolate cysts after laparoscopic ablation. J Am Assoc Gynecol
Laparosc. 2002;9:31520.
9. Vercellini P. Coagulation or excision of ovarian endometriomas? Am J Obstet Gynecol.

2003;188:60610.
10. Koga K. Recurrence of ovarian endometrioma after laparoscopic excision. Hum Reprod.
2006;21:21714.
11. Vercellini P. Association between endometriosis stage, lesion type, patient characteristics and
severity of pelvic pain symptoms: a multivariate analysis of over 1000 patients. Hum Reprod.
2007;22:26671.
12. Liu X. Patterns of and risk factors for recurrence in women with ovarian endometriomas.
Obstet Gynecol. 2007;109(6):141120.
13. Vercellini P. Postoperative oral contraceptive exposure and risk of endometrioma recurrence.
Am J Obstet Gynecol. 2008;198:504. e1504e5.
Chapter 25
Prevention of Recurrence After Surgery
Yutaka Osuga, Yuri Takemura, Masashi Takamura, and Kaori Koga
Abstract Recurrence of ovarian endometrioma after excision imposes profound

encumbrance on the suffered women, with increasing pain, subfertility/infertility,
and a risk of ovarian cancer. Postoperative recurrence is relatively common, the rate
ranging from 11 to 32 % in 15 years. To date, a number of risk factors for the
recurrence have been reported, i.e., younger age, larger cysts, high rASRM scores,
previous medical treatment, previous surgery for endometriosis, and short-term
postoperative medical treatment. In contrast, postoperative pregnancy reduces the
risk. Several biomarkers are suggested to predict the recurrence but not yet vali-
dated in clinical practice. Postoperative OC use for a long period has been demon-
strated to reduce the recurrence remarkably. Postoperative GnRH analogue use is
also effective for the reduction of the recurrence. There still remain some issues on
the duration and the drugs most suitable for the prevention.
Keywords Endometrioma Oral contraceptive Recurrence Surgery
25.1 Introduction
Endometriosis is a disease defined as the presence of endometrium-like lesions

outside the uterus. Although the exact etiology has not yet been determined, it is
widely accepted that endometrial cells in the retrograde menstruation attach,
invade, and grow in the peritoneum to cause the disease. It occurs in women of
reproductive age and deteriorates the quality of life of the affected women by
inducing infertility/subfertility and intractable pain. Treatment of the disease is
mainly conducted with drugs and/or surgery. A mainstream of medical treatment is
Y. Osuga (*) Y. Takemura M. Takamura K. Koga

Department of Obstetrics and Gynecology, School of Medicine, the University of Tokyo,
Japan, 7-3-1 Hongo Bunkyo, Tokyo 113-8655, Japan
e-mail: yutakaos-tky@umin.ac.jp

412 Y. Osuga et al.
hormonal treatment. However, hormonal drugs cannot be used for those who want
to conceive since the drugs inhibit ovulation. In addition, some pain symptoms are
occasionally resistant to medical treatment. Likewise, hormonal drugs are often not
effective to eradicate developed lesions, such as endometrioma. On the other hand,
surgical treatment is appropriate for removing developed lesions and often effective
to mitigate drug-resistant pain. Widespread use of laparoscopic surgery makes it
less invasive to treat endometriosis by surgery.
Surgical treatment for endometriosis, while having relatively effective outcome
in the short term, has a problem in the long period after surgery. Hysterectomy with
bilateral oophorectomy is radical enough to cure endometriosis, but the procedure is
not acceptable for patients who want to maintain fertility. For these patients,
excision of endometriosis lesions (conservative surgery) is a method of choice.
This treatment often entails recurrence both in symptoms and in histology.
Recently, the concern is gaining traction because more and more women need
conservative surgery due to increasing tendency to childbearing in late
reproductive ages.
This problem, however, is not so new. An incipient report that examined the
recurrence after conservative surgery, while performed by open surgery in those
days, indicated several noticeable findings. The authors followed 423 patients
treated with conservative surgery and found 13.5 and 40.3 % cumulative recurrence
rates in 3 and 5 years, respectively [1]. They found that severity of disease was not
predictive of recurrence and pregnancy delayed recurrence.
There are a number of reports also in the era of laparoscopic surgery. When we
read these papers, we notice the complexity of the problems regarding recur-
rence. Some papers examine recurrence of pain, while others examine recurrence
of endometriosis lesions detected by laparoscopy or imaging. Some papers study
recurrence of endometriosis in general while others study recurrence of a specific
type of endometriosis, e.g., peritoneal lesion, ovarian endometrioma, and deep
endometriosis at the cul-de-sac. The operation methods are either not uniform,
including excision of the cyst (cystectomy) and coagulation or vaporization using
electric devises or lasers. In this chapter, we focus on the recurrence of
endometrioma, which can be easily detected by transvaginal ultrasound, after
laparoscopic excision.
25.2 Recurrence of Endometrioma After

Laparoscopic Excision
25.2.1 Recurrence Rate
Postoperative recurrence rate for endometrioma ranges from 11 to 32 % in

15 years [211]. The variation partly comes from the difference of the definition of
recurrence; some define it by the presence of ovarian cysts greater than 3 cm [5, 10]
25 Prevention of Recurrence After Surgery 413
while others define it by the size at least 2 cm [6, 8]. Instinctively, the time after
surgery is related to the recurrence rate. However, unless patients are regularly
followed up, as often with the case of asymptomatic patients, it is difficult to
accurately quantify the time of recurrence. It is thus not obvious in which time
period the patient is most susceptible to recurrence, or whether there is a specific
period of time after which a patient is less likely to develop recurrence. According
to Evers et al. the recurrence rate during the first 5 years gradually increases [12].
This might imply that endometriosis will eventually reappear in all patients who
underwent complete removal of the lesions. In accordance with this notion, a
longitudinal study by Liu et al. showed that the recurrence rates continued to
increase with time, 7.8, 17.7, and 32.3 % at 1, 2, and 3 years, respectively [10].
In contrast, Kikuchi et al. drew a cumulative recurrence rate curve and demon-
strated that the cumulative rate reaches a plateau at around 48 months after
surgery [6]. Further studies with longer follow-up are needed to settle the issue.
Whether the recurrence of endometrioma occurs by de novo or the relapse of
residual endometrioma is poorly understood as well as the origin of endometrioma
per se. To address the issue, the recurrence rate was studied with analysis of the side
in which endometrioma recurred, supposing that recurrence in the contralateral
ovary should be de novo. Exacoustos et al. analyzed 62 patients with recurrent
endometrioma and found that 12 patients had recurrence on the counter-lateral
untreated ovary [13]. Kikuchi et al. also analyzed 26 cases with recurrence after
hemilateral surgery and found that 11 cases had recurrence on the counter-lateral
untreated ovary [6]. These findings suggest that the recurrence of endometrioma is
not totally dependent on the presence of the residual endometrioma but in part on de
novo occurrence. Of course, it cannot be denied that a lesion on the contralateral
ovary undetected by the initial laparoscopy may have progressed after surgery.
Although the difference between de novo and relapse is critical for optimal man-
agement of this disorder, it is impossible at the moment to discriminate clearly the
origin by just looking at and following up the patents and the lesions. Further
ingenious studies seem to be necessary to resolve the issue. Collectively, there is no
doubt that the recurrence of endometrioma after laparoscopic excision is a common
and serious problem, although the actual recurrence rate varies among studies.
25.2.2 Risk Factor and Biomarker of Recurrence
Since endometriosis has characteristics of a chronic disease, it would be useful if

doctors are able to estimate the probability of recurrence before surgery. To this
end, a number of studies have been conducted analyzing various factors which
might associate with recurrence. We retrospectively studied a total of 224 patients
who had undergone laparoscopic cystectomy of endometrioma [8]. The patients had
a minimum of 2 years of postoperative follow-up after laparoscopic cystectomy.
Recurrence was defined as the presence of endometrioma more than 2 cm in size,
detected by ultrasonography within 2 years of surgery. We assumed that
414 Y. Osuga et al.
Table 25.1 Univariate and logistic regression analysis of factors related to the recurrence of
ovarian endometrioma
Univariate
analysis Logistic regression analysis
Odds ratio
Factors P values P values (95 % confidence interval)
Age (years) NS
Infertility NS
Pain NS
Presence of uterine myoma NS
Presence of adenomyosis NS
Previous medical treatment <0.05 <0.01 2.324 (1.2324.383)
of endometriosis
Previous surgery of ovarian NS
endometrioma
Multiple cysts NS
Largest cyst diameter (cm) <0.05 <0.05 1.182 (1.0041.391)
Bilateral involvement NS
Coexistence of deep endometriosis NS NS 0.456 (0.1981.052)
Revised ASRM score NS NS 1.010 (1.0001.021)
Postoperative medical treatment NS
Postoperative pregnancy <0.05 <0.05 0.292 (0.0280.317)
ASRM American Society for Reproductive Medicine [8]
14 variables (age, presence of infertility, pain, uterine myoma, adenomyosis,

previous medical treatment of endometriosis, previous surgery for ovarian endo-
metriosis, single or multiple cysts, the size of the largest cyst at laparoscopy,
unilateral or bilateral involvement, coexistence of deep endometriosis, revised
American Society for Reproductive Medicine (ASRM) score, postoperative med-
ical treatment, and postoperative pregnancy) might associate with the recurrence
and evaluated these variables to assess their independent effects on the recurrence
using logistic regression analysis. As a result, the overall rate of recurrence was
30.4 % (68/224). Significant factors that were independently associated with higher
recurrence were previous medical treatment of endometriosis [odds ratio (OR)
2.324, 95 % confidence interval (95 % CI) 1.2324.383, P 0.0092) and larger
diameter of the largest cyst (OR 1.182, 95 % CI 1.0041.391, P 0.0442). As
shown in Table 25.1, postoperative pregnancy was associated with lower recur-
rence (OR 0.292, 95 % CI 0.0280.317, P 0.0181). It seems intuitively
reasonable that the larger cyst associates with the higher recurrence rate. In
contrast, it appears to be difficult to construe the association between previous
medical treatment and the higher recurrence risk. The finding may be explained by
two possible mechanisms. The first is that the medication may mask endometriotic
lesions and allow them to escape from removal at operations. Because more than
half of the women who were categorized into previous medical treatment group had
continued their medication until the time of operation, it may be possible that the
medication might yield latent lesions that remain and recur after the operation. The
other possible reason for a negative impact of medical treatment on endometrioma

recurrence is that hormonal-suppressive therapy may alter some genomic charac-
teristics of endometriotic lesions. As for malignant transformation of endometri-
osis, it is proposed that hormonal ablative treatments may cause negative selection,
suppress the normal, eukaryotic cells more than aneuploid cells bearing chromo-
somal aberrations, and increase the rate of dyskaryotic cells in the endometriotic
implants [14]. We conjecture that negative selection may also contribute to the
recurrence of disease, making the lesion more active, progressive, and liable to
recurrence. The association of postoperative pregnancy and lower rate of recur-
rence is an interesting finding but needs careful interpretation. A possible scenario
is that subsequent pregnancy may have a protective effect on endometrioma
recurrence. An absence of retrograde menstruation and formation of corpora
lutea [15], both of which are known to cause endometriosis, and persistent exposure
to progesterone are thought to contribute to the protective effect of pregnancy.
Conversely, those who tended not to recur had more probability to be pregnant
given that endometriosis causes infertility/subfertility.
Several risk factors have been reported by others. In general, the more advanced
the endometriosis, the more the disease recurs. Larger cysts [8] and high rASRM
scores [6, 7, 10, 16, 17] are reported to be risk factors for recurrence. Factors that
may correlate with the severity of endometriosis such as previous medical treatment
[8, 10], previous surgery for endometriosis [3, 17], and short-term postoperative
medical treatment [9] are also identified as risk factors. Younger age at surgery is
reported as a risk factor by two studies [6, 10]. A younger age at surgery would
correlate with a younger age at onset and possibly a disease type that is more
aggressive and prone to recurrence than the endometrioma associated with an older
age of onset.
A few researches are trying to find out biomarkers for recurrence of ovarian
endometriomas. Increased expression of progesterone receptor isoform B, nuclear
factor kappa-B, SLIT/ROBO1, and cyclooxygenase-2 are shown to be associated
with higher risk of recurrence [1820]. However, whether they are practically
useful in predicting the recurrence awaits further studies.
25.2.3 Postoperative Medical Managements

to Prevent Recurrence
Until recently, postoperative medical treatment that prevents recurrence has not
been developed successfully. In the face of the fact that postoperative pregnancy
decreases the risk of recurrence, as described above, we came up with the idea if
postoperative oral contraceptives might decrease recurrence. We thus conducted a
before and after study on postoperative OC treatment. In 2005, our clinic introduced
the OC recommendation, that is, at the time of the operation, we provided each
patient with information about OC and the known possible benefits and risks and let
416 Y. Osuga et al.
Eligible Patients
n = 87 (20)
( ) the number
of recurrence
Initial OC take
n = 48 ( 3 )
OC entire period Discontinuous Never OC

n = 34 ( 1 ) n = 14 ( 2 ) n = 39 (17)
2.9 % 14.3 % 43.6 %
Fig. 25.1 Recurrence after laparoscopic excision of endometrioma. Flowchart of the patients who
underwent laparoscopic excision of endometrioma after May 2005, for the retrospective cohort
study. A total of 87 patients were followed up for 24 months. Of the 87 patients, 48 started to take
OCs, but 39 did not. Of the 48 patients who had started OC, 34 continued OC for the entire study
period (24 months), whereas 14 discontinued
the patient decide whether or not to take OC. Women who chose to take OC were
given a cyclic (21 days pills/7 days no pill), monophasic OC containing ethinyl
estradiol (0.035 mg) and norethisterone (1.0 mg) (Ortho-M 21, Mochida, Tokyo,
Japan), in the first menstrual cycle after laparoscopy. We then conducted a histor-
ical study to compare the 2-year recurrence rate before and after the introduction of
the OC recommendation. The overall recurrence rate in patients who underwent
laparoscopy after the introduction of the OC recommendation was significantly
lower than that in patients who received laparoscopy before the introduction (18.6
versus 33.1 %, relative risk 0.56, 95 % CI 0.320.97, P < 0.05) [21]. Figure 25.1
depicts the recurrence rate in those who used OC which was significantly lower than
others (non-OC users plus those who quit OC) (2.9 versus 35.8 %, relative risk 0.08,
95 % CI 0.010.48, P < 0.001) [21]. This study indicated that postoperative OC use
reduces the risk of ovarian endometrioma after laparoscopic excision.
In addition to our study, there have been several studies that evaluate the role of
postoperative OC on the recurrence of endometrioma. In contrast to the classical
report showing OC had no effect on disease recurrence when used for up to
6 months [22], all studies that tested postoperative OC for 2 years or more
demonstrated protective effect of OC on recurrence [11, 21, 23]. The different
outcomes between short-term and long-term studies indicate that the duration of
treatment with OC affects recurrence. Indeed, Vercellini et al. compared cumula-
tive recurrence according to the duration of postoperative OC use and found that
women who used OC for less than 12 months were at higher risk of recurrence than
women using OC for 12 months or more [11].
GnRH analogue also has an effect to mitigate recurrence. Jee et al. analyzed the
influence of postoperative GnRH analogue according to the duration of the treat-
ment and found that a 6-month treatment had a beneficial impact compared with a
3- and 4-month treatment and expectant management, although the differences did
not reach statistical significance [24]. In another report, patients who received 3
6 months of GnRH analogue therapy alone and patients who received OC after
GnRH analogue were compared. As a result, recurrent endometrioma after
60 months was significantly lower in OC plus GnRH analogue group than in
GnRH analogue alone group (6.1 versus 43.3 %) [25]. It seems that GnRH analogue
treatment longer than 6 months reduces the recurrence, and additional OC treatment
may maintain the effect. However, the benefit of GnRH analogue should be
weighed against the risk of adverse effects associated with estrogen deprivation.
25.3 Summary
Postoperative recurrence of endometrioma is a common event and prevention of the

recurrence is needed to manage women with endometriosis successfully for a long
period. Thanks to recent extensive studies, it has been established that long-term
administration of OC after surgery is safe and tolerable and recommended for those
who do not want to conceive immediately after the surgery. However, there still
remain some issues regarding what kind of drugs are most suitable and how long the
drug should be used. Further studies are warranted to improve strategies to prevent
the recurrence.
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14. Blumenfeld Z. Hormonal suppressive therapy for endometriosis may not improve patient
health. Fertil Steril. 2004;81(3):48792.
15. Vercellini P, Somigliana E, Vigano P, Abbiati A, Barbara G, Fedele L. Blood On The Tracks
from corpora lutea to endometriomas. BJOG. 2009;116(3):36671.
16. Busacca M, Bianchi S, Agnoli B, Candiani M, Calia C, De Marinis S, Vignali M. Follow-up of
laparoscopic treatment of stage III-IV endometriosis. J Am Assoc Gynecol Laparosc. 1999;6
(1):558.
17. Porpora MG, Pallante D, Ferro A, Crisafi B, Bellati F, Benedetti Panici P. Pain and ovarian
endometrioma recurrence after laparoscopic treatment of endometriosis: a long-term prospec-
tive study. Fertil Steril. 2010;93(3):71621.
18. Shen F, Liu X, Geng JG, Guo SW. Increased immunoreactivity to SLIT/ROBO1 in ovarian
endometriomas: a likely constituent biomarker for recurrence. Am J Pathol. 2009;175
(2):47988.
19. Shen F, Wang Y, Lu Y, Yuan L, Liu X, Guo S-W. Immunoreactivity of progesterone receptor
isoform B and nuclear factor kappa-B as biomarkers for recurrence of ovarian endometriomas.
Am J Obstet Gynecol. 2008;199(5):486.e1486.e10.
20. Yuan L, Shen F, Lu Y, Liu X, Guo S-W. Cyclooxygenase-2 overexpression in ovarian
endometriomas is associated with higher risk of recurrence. Fertil Steril. 2009;91
(4, Suppl):13036.
21. Takamura M, Koga K, Osuga Y, Takemura Y, Hamasaki K, Hirota Y, Yoshino O, Taketani
Y. Post-operative oral contraceptive use reduces the risk of ovarian endometrioma recurrence
after laparoscopic excision. Hum Reprod. 2009;24(12):30428.
22. Muzii L, Marana R, Caruana P, Catalano GF, Margutti F, Panici PB. Postoperative adminis-
tration of monophasic combined oral contraceptives after laparoscopic treatment of ovarian
endometriomas: A prospective, randomized trial. Am J Obstet Gynecol. 2000;183(3):58892.
23. Seracchioli R, Mabrouk M, Frasca C, Manuzzi L, Montanari G, Keramyda A, Venturoli
S. Long-term cyclic and continuous oral contraceptive therapy and endometrioma recurrence:
a randomized controlled trial. Fertil Steril. 2010;93(1):526.
24. Jee BC, Lee JY, Suh CS, Kim SH, Choi YM, Moon SY. Impact of GnRH agonist treatment on
recurrence of ovarian endometriomas after conservative laparoscopic surgery. Fertil Steril.
2009;91(1):405.
25. Lee D-Y, Bae D-S, Yoon B-K, Choi D. Post-operative cyclic oral contraceptive use after
gonadotrophin-releasing hormone agonist treatment effectively prevents endometrioma recur-
rence. Hum Reprod. 2010;25(12):30504.
Chapter 26
Ovarian Reserve in Patients
with Endometriosis
Michio Kitajima and Hideaki Masuzaki
Abstract The number of follicles present in human ovaries is finite. Ovarian

reserve is currently defined as the number and quality of the follicles left in the
ovary at any given time. As several novel tests for ovarian reserve, such as anti-
Mullerian hormone (AMH) and antral follicle count (AFC), are widely introduced
into gynecological practice recently, the knowledge on the physiology of follicular
development and mechanism of maintenance of ovarian reserve are rapidly accu-
mulating. Diminished ovarian reserve is a major concern in women with
endometriosis-associated infertility. Cystectomy for endometriomas could nega-
tively impact on postoperative ovarian reserve. Some women who had surgery for
endometriomas suffer from poor ovarian response, which is directly linked to
treatment results in infertility care. In addition, endometriomas themselves may
be a cause of diminished ovarian reserve. Chronic inflammation in the local pelvic
environment may affect the status of the dormant follicle in the ovarian cortex.
Determination of ovarian reserve may serve as an important role in the management
of reproductive health issues in women with endometriosis.
Keywords Anti-Mullerian hormone (AMH) Antral follicle count (AFC)

Endometrioma Endometriosis Ovarian reserve
M. Kitajima (*) H. Masuzaki

Department of Obstetrics and Gynecology, Graduate School
of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
e-mail: m-kita@nagasaki-u.ac.jp

420 M. Kitajima and H. Masuzaki
26.1 Ovarian Reserve Testing in Women

with Endometriosis
26.1.1 Physiological Basis of Ovarian Reserve Testing
The human ovary contains limited numbers of primordial follicles. These dormant
follicles are activated and also demised incessantly from fetus to the age of
menopause. Loss of nongrowing follicles in womens ovary is age dependent.
Age-related declining curve of ovarian reserve resembles decline of womens
fecundability along with age [1]. However, there are substantial variations in the
decline of reproductive capacity with age [2]. These variations may be defined in
part by several confounding variables. Indeed, Wallace et al. [3] showed that the
estimated number of nongrowing follicle present in the ovaries gets wider variation
after the age of 25 years, which indicates that factors other than age (e.g., smoking,
BMI, parity, stress, systemic disease, etc.) become more important in determining
the rate at which nongrowing follicles are lost through atresia.
Ovarian reserve is currently defined as the number and quality of the follicles left
in the ovary at any given time [4]. The number of remaining primordial follicles in
the ovary may define the number of follicular pool to be selected [3]. The size of
follicular pool is closely related to selectable follicular cohort in stimulation cycles.
Thus, women with decreased ovarian reserve will not be able to produce sufficient
multiple follicular growth in IVF treatment, which is a major determinant of
treatment success [5]. Response to gonadotropin may represent the size of the
cohort of FSH-sensitive follicles in the ovaries and is directly related to the
magnitude of ovarian reserve.
However, since not all the women with infertility receive ovulation induction and
as individual ovarian reserve may show wide variation, tests of ovarian reserve to
identify each reproductive potential may be valuable in particular clinical situations.
In ART settings, testing ovarian reserve before initiation of treatment cycle may be
useful in tailoring stimulation protocol. Ovarian reserve testing may also be useful to
predict womens age at menopause. On the other hand, the markers of ovarian
reserve can be used to evaluate surgical damage to normal ovarian tissue when
one attempts to perform fertility sparing surgery for several indications including
endometriosis. These tests may also be used to evaluate the effects of pharmaceu-
tical chemicals, anticancer drugs, and hormonal agents on ovarian reserve.
26.1.2 The Varieties of Tests for Ovarian

Reserve Determination
There are several clinical tests to estimate ovarian reserve [4]. However, it is still
unclear that these tests can measure quality and quantity of remaining primordial
follicles in ovaries precisely [4, 6]. Counting all the follicles present in serial
26 Ovarian Reserve in Patients with Endometriosis 421
sections of whole ovary by histological analysis after oophorectomy may be a direct

way to evaluate remaining primordial follicles in ovaries, but it cannot be applica-
ble to reproductive aged women. Since follicles in the ovarian cortex may not
distribute homogenously, a biopsied small sample of the ovarian cortex may not
always represent remaining primordial follicles [7, 8]. Considering its invasiveness
and in the context of ovarian reserve testing, ovarian biopsy may not serve as a
useful test to estimate ovarian reserve [9].
Classically, serum FSH and estradiol (E2) levels in the early follicular phase (i.e.,
cycle day 24) had long been utilized as markers for ovarian reserve. Inhibin-B is
also reported as a serum marker for ovarian reserve. Stimulation test using clomi-
phene citrate, gonadotropin, or GnRH agonist had been reported. Alternatively,
ultrasonographic determination of antral follicle count (AFC) in the early follicular
phase and ovarian volume may serve as surrogate markers for ovarian reserve
instead of blood sampling [10, 11]. Although various markers and tests of ovarian
reserve had been reported, the clinical value of testing for basal FSH and inhibin-B
value is limited [4, 12]. Ultrasonographic markers, such as AFC and ovarian volume,
correlate well with age-related decline of ovarian reserve. However, it is difficult to
assess the exact number of antral follicles and ovarian volume of the cystic ovary,
such as endometrioma, before cystectomy [13].
26.1.3 AMH: A Novel Marker for Ovarian Reserve
Recently, serum anti-Mullerian hormone (AMH) levels had got wide popularity to
predict ovarian responsiveness in ART settings. AMH is a dimeric glycoprotein,
belongs to the transforming growth factor- family, and is produced solely by the
granulosa cells of the recruited follicles until they become sensitive to FSH [14,
15]. The serum level of AMH declines with age, is menstrual cycle independent,
and is unaffected by gonadotropin or GnRH agonist administration [16]. In
addition, it is very sensitive to changes in ovarian reserve with advancing age
and correlates well with antral follicle count [17]. Therefore, measurements of
serum AMH may be superior to other markers of ovarian reserve, given its
reliability and convenience, to indicate the number of growing follicles and
estimate ovarian follicular reserve. However, although compromised response
to controlled ovarian stimulation can be diagnosed, decreased AMH may not
always be an absolute determinant of womens fecundability. For example,
women with undetectable serum AMH value occasionally become pregnant
[18]. As these limitations may be due to threshold of the present assay system,
development of more sensitive detection method may bring about further under-
standing of the relationship between ovarian reserve testing by AMH and ovarian
functions.
26.1.4 Role of Ovarian Reserve Tests in Women

with Endometriosis
Infertility is the main concern of women with endometriosis. The cause of infertility
in endometriosis is multifactorial and pathogenesis of endometriosis-associated
infertility remains uncertain [19]. The ovary is one of the frequent anatomical
locations in which endometriosis may develop. Although it is still controversial,
diminished ovarian reserve after surgical intervention to ovarian endometriosis had
been a large clinical concern in infertility care [20]. In addition, ovarian function
may be distorted by the disease itself [21]. On the other hand, some reports argued
that endometriosis is a significant confounding variable that affects the age of
menopause [22, 23]. In this view, endometriosis and its associated events through
womens reproductive life span, such as chronic inflammation, infertility, and
repeated pelvic surgery, may bring about considerable effects on ovarian functions.
Diminished ovarian reserve may be a key element of endometriosis-associated
infertility and possible confounders in health issue in women with endometriosis
at late reproductive stage. Therefore, accurate estimation of ovarian reserve in
women with endometriosis may serve an important clinical role.
26.2 Surgery for Endometriosis and Ovarian Reserve
26.2.1 Influence of Cystectomy for Endometrioma

on Ovarian Reserve
26.2.1.1 Ovarian Response After Cystectomy for Endometrioma
Laparoscopic cystectomy for endometrioma is common and seems to be feasible

in terms of postoperative fecundability and recurrence rate compared with that of
fenestration and coagulation of the cyst wall [24, 25]. However, the safety of this
technique with respect to residual ovarian damage has been questioned [20]. On
the other hand, there had been conflicting reports on ovarian response to ovulation
induction in infertile women with previous surgical excision of endometrioma
[2629]. Under gonadotropin stimulation, the damage by surgery may be masked.
Even skilled hands show excellent results, cystectomy for endometrioma may
cause unavoidable risk of surgical injury to residual normal ovarian tissue
[30]. This may cause loss of ovarian follicles in women who were operated on
for endometrioma [31, 32]. According to a recent report, absence of follicular
growth was observed in 13 % of operated ovaries, although this event never
occurred in the contralateral gonad [33].
26.2.1.2 Effects of Cystectomy for Endometrioma

on Serum AMH Levels
We previously demonstrated that serum AMH levels significantly decreased in

women with endometrioma compared to that of women with other benign ovarian
cyst after unilateral laparoscopic cystectomy [34]. Interestingly, postsurgical
changes in serum AMH levels were significantly more prominent in women with
normal ovarian tissue found in dissected cyst wall. In addition, multivariate
analysis with several confounding variables revealed that type of the cyst
(endometrioma) and presence of normal ovarian tissue in dissected specimen
were significantly associated with postsurgical decline in serum AMH levels.
However, when we analyzed the model with these two variables together, the
statistical significance for the type of the cyst was lost. In other words, proper
surgery avoiding damage to surrounding normal ovarian tissue may not result in
overt loss of ovarian reserve compared to similar surgery applied to other normal
benign ovarian cyst. Meta-analysis and systematic review of literatures evaluating
the impact of surgery for endometriomas on ovarian reserve as determined by
serum AMH showed a statistically significant decrease in serum AMH concentra-
tion after ovarian cystectomy [35, 36]. Studies utilizing several tests of ovarian
reserve other than AMH, such as AFC and inhibin-B, also showed negative impact
of cystectomy for endometriomas on ovarian reserve [3739].
26.2.1.3 Time Course Change of Marker for Ovarian Reserve After

Cystectomy for Endometrioma
Recently, several authors reported time course change in serum AMH value
postsurgery up to 1 year. Sugita et al. [40] analyzed the pattern of sequential
changes in the serum AMH levels within 1 year after cystectomy for
endometriomas. Although serum AMH levels decreased in almost all cases imme-
diately (at 1 month) after surgery, they found that 51 % of patients showed partial
recovery of AMH levels at 1 year after surgery, and in these women, the number of
follicles removed by surgery was significantly more compared to that of women
who showed persistent decrease in AMH value at 1 year after surgery. Celik
et al. [41] also reported serial change in postsurgical AMH levels and its associa-
tions with other clinicopathological factors. They found that serum AMH decreased
significantly at the sixth month (61 %) postoperatively. The FSH level increased
significantly at the sixth week but returned to normal at the sixth month. The AFC
increased significantly at the sixth week and at the sixth month. These results may
indicate that the acute decrease in the serum AMH levels caused by surgical
removal of the ovarian cortex may result in alterations of selectable follicular
cohort, which may affect the values of several ovarian reserve markers after ovarian
cystectomy.
26.2.2 Influence of Other Surgical Techniques

for Endometrioma on Ovarian Reserve
Surgical technique other than cystectomy, such as laser ablations or electrocoa-

gulations of the cyst wall, may show different effects on ovarian reserve after
surgery. Even the decreases in AFC and ovarian volume were found for both
coagulation and cystectomy, but the decrease was statistically significantly more
frequent in cystectomized ovaries than in coagulated ovaries. Also, in the in vitro
fertilization cycles, the ovarian response to ovulation induction was statistically
significantly reduced in cystectomized ovaries as compared with coagulated
ovaries [42]. When compared with plasma energy ablation, cystectomy is respon-
sible for a statistically significant decrease in ovarian volume and a statistically
significant reduction in AFC. This data should be taken into account in therapeutic
decision-making concerning women attempting pregnancy, especially where there
are other risk factors for postoperative ovarian failure [43]. Three-stage laparo-
scopic management with CO2 laser and GnRH analogs showed favorable results
compared to cystectomy in terms of postsurgical serum AMH levels and
echographic parameters for ovarian reserve [44, 45]. Combined technique with
CO2 laser vaporization and stripping of the cyst wall with postsurgical GnRHa
therapy did not compromise postsurgical ovarian volume and AFC [46]. These
results may indicate that selection of surgical technique may improve postoperative
reduction of ovarian reserve. The overall efficacy of each surgical technique for
endometriomas may be judged by postoperative fecundability including mainte-
nance of ovarian reserve as well as recurrence rate and symptom improvement.
Some reports argued associations between recurrence and postsurgical ovarian
reserve as they found higher ovarian responsiveness in gonads that developed
recurrent endometriomas [47].
26.3 Ovarian Reserve in Women with Endometriosis Did

Not Undergo Ovarian Surgery
26.3.1 The Effects of Unoperated Endometrioma

on Ovarian Reserve
The effects of endometriosis on ovarian reserve are largely contributed by surgical

intervention to ovarian endometriosis. However, even women with endometriosis
before surgical interventions or women with endometriosis without ovarian
involvement may show diminished ovarian reserve. Endometriomas themselves
could be linked to reduced ovarian reserve, and damage to normal ovarian tissue
may precede surgery. In IVF patients with unilateral ovarian lesion, the presence
of ovarian endometriomas is associated with a reduced responsiveness to
gonadotropins comparing to those of contralateral intact gonads. This deleterious

effect was more evident in women with larger and multiple lesion, and in those who
were more responsive to ovarian hyperstimulation [48]. However, another study
showed that the presence of an endometrioma does not markedly affect respon-
siveness to hyperstimulation [49]. By the determination of serum AMH levels, the
presence of ovarian endometriomas is associated with a significant reduction in
ovarian reserve. In addition, bilateral endometrioma exerts a more profound neg-
ative impact on ovarian reserve than unilateral endometrioma, regardless of either
conservative or surgical intervention [50]. On the other hand, the rate of spontane-
ous ovulations significantly decreased in the ovaries with endometriomas [51]. As
ovulation induction may compensate decreased ovarian function in ovaries with
endometriomas, the presence of endometriomas per se may deteriorate ovarian
reserve.
We recently demonstrated decreased follicular density, associated fibrosis, and
deterioration of normal structure of ovarian cortex in ovaries that are affected by
endometriomas [21]. By serial histological analysis, microscopic endometriotic
foci on the surface of ovarian cortex were found and they showed extensive
hemorrhage conjoined with infiltration of inflammatory cells and fibrosis [21]. In
addition, the size of primordial follicles is significantly smaller and increased
recruitment and atresia of early follicles were evidenced in the cortex of affected
ovaries. These results may support endometriomas themselves that are the cause of
diminished ovarian reserve in women with endometriosis. Deterioration of ovarian
reserve may precede surgery and destructive surgery may exacerbate their repro-
ductive potential further.
26.3.2 The Effects of Non-ovarian Endometriosis

on Ovarian Reserve
Endometriosis that develops in other anatomical sites besides the ovary, such as
pelvic peritoneal implants and rectovaginal nodules, is a common feature of
endometriosis, and their pathogenesis may be different from ovarian
endometriomas [52]. Presence of deep infiltrating endometriosis in addition to
ovarian endometriomas negatively affects ovarian reserve in terms of antral follicle
count and number of oocytes retrieved [53]. In infertile patients with minimal/mild
endometriosis, although serum FSH did not show any difference compared to
control women without endometriosis, decreased serum AMH levels were signif-
icantly lower [54]. However, other investigators reported that peritoneal endome-
triosis and ovarian endometriomas per se do not result in lower AMH levels. AMH
levels are decreased in women with previous endometrioma surgery independently
of the presence of current endometriomas [55]. Several confounding variables
affecting ovarian reserve such as severity of intrapelvic inflammation and adhesion
should be taken into account in women with and without ovarian involvement.
26.4 Summary and Perspective
Endometriosis can be regarded as a chronic disease that requires long-term medical

or surgical management through womens reproductive life span. Cystectomy for
endometrioma may cause significant detrimental effects on ovarian reserve. In
addition, inflammation provoked by endometriosis may affect the homeostasis of
dormant follicles in the ovarian cortex. Development and selection of surgical
methods and search for suitable medical treatment to minimize the damage to
normal ovarian tissue in women with endometriosis are urgently expected [56].
Further research may be warranted to determine the mechanism of diminished
ovarian reserve in women with endometriosis.
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ovarian endometriomas is associated with a reduced responsiveness to gonadotropins. Fertil
Steril. 2006;86:1926.
49. Benaglia L, Pasin R, Somigliana E, Vercellini P, Ragni G, Fedele L. Unoperated ovarian
endometriomas and responsiveness to hyperstimulation. Hum Reprod. 2011;26:135661.
50. Hwu YM, Wu FS, Li SH, Sun FJ, Lin MH, Lee RK. The impact of endometrioma and
laparoscopic cystectomy on serum anti-Mullerian hormone levels. Reprod Biol Endocrinol.
2011;9:80.
51. Benaglia L, Somigliana E, Vercellini P, Abbiati A, Ragni G, Fedele L. Endometriotic ovarian
cysts negatively affect the rate of spontaneous ovulation. Hum Reprod. 2009;24:21836.
nodules of the rectovaginal septum are three different entities. Fertil Steril. 1997;68:58596.
53. Papaleo E, Ottolina J, Vigano P, Brigante C, Marsiglio E, De Michele F, Candiani M. Deep

pelvic endometriosis negatively affects ovarian reserve and the number of oocytes retrieved for
in vitro fertilization. Acta Obstet Gynecol Scand. 2011;90:87884.
54. Lemos NA, Arbo E, Scalco R, Weiler E, Rosa V, Cunha-Filho JS. Decreased anti-Mullerian
hormone and altered ovarian follicular cohort in infertile patients with mild/minimal endome-
triosis. Fertil Steril. 2008;89:10648.
55. Streuli I, de Ziegler D, Gayet V, Santulli P, Bijaoui G, de Mouzon J, Chapron C. In women
with endometriosis anti-Mullerian hormone levels are decreased only in those with previous
endometrioma surgery. Hum Reprod. 2012;27:3294303.
56. Jadoul P, Kitajima M, Donnez O, Squifflet J, Donnez J. Surgical treatment of ovarian
endometriomas: state of the art? Fertil Steril. 2012;98:55663.
Chapter 27
Infertility Treatment of Endometriosis
Patients
Kaori Koga, Osamu Yoshino, Yasushi Hirota, Tetsuya Hirata,

Miyuki Harada, and Yutaka Osuga
Abstract Endometriosis is common in infertile patients, but infertility treatment

of endometriosis patients is controversial and varied. This article summarizes
evidence concerning infertility treatment of endometriosis patients. Endometriosis
impairs fertility by causing both anatomical and biochemical distortion in female
reproductive system. Medical treatment of endometriosis does not improve fertility,
whereas there is some evidence that surgery for mild endometriosis does. There is
conflicting evidence regarding removal of endometriomas due to the potential impact
on ovarian reserve. Assisted reproductive technology (ART) improves pregnancy
rates, although the pregnancy rates are lower than for women without endometriosis.
Some women with infertility and endometriosis may benefit from a combination of
medical treatment, ART, and surgery. The decision about whether to undergo
laparoscopy or superovulation (SO) with intrauterine insemination (IUI) or pursue
ART will depend on a variety of factors such as the patients age and symptoms, other
infertility factors, risk of surgery, the presence of endometrioma, and ovarian reserve.
Keywords Assisted reproductive technology (ART) Endometriosis Infertility

Laparoscopy
27.1 Introduction
Management of infertility with endometriosis raises a number of complex clinical

questions. This article will review the current literature regarding endometriosis-
associated infertility including its pathophysiology and treatment.
K. Koga (*) Y. Hirota T. Hirata M. Harada Y. Osuga

Obstetrics and Gynecology, The University of Tokyo, Tokyo, Japan
e-mail: kawotan-tky@umin.ac.jp
O. Yoshino
Obstetrics and Gynecology, University of Toyama, Toyama, Japan

432 K. Koga et al.
27.1.1 Epidemiology of Endometriosis and Infertility
The fecundity rate in normal couples is in the range of 1520 % per month and
decreases with age of the female partner [1]. In contrast, in women with untreated
endometriosis, monthly fecundability is 210 % [2]. Early studies suggested that
2550 % of infertile women have endometriosis and that 3050 % of women with
endometriosis are infertile [3]. Other reports have confirmed that infertile women
are 6 to 8 times more likely to have endometriosis than fertile women [4].
27.1.2 Mechanisms by Which Endometriosis Adversely

Impacts Fertility
Endometriosis impacts fertility in both mechanical and biochemical manners.

Mechanically, pelvic adhesions accompanied with endometriosis cause anatomical
distortions and these impair oocyte release from the ovary or inhibit ovum pickup or
transport by fallopian tubes. Adhesions may also cause abnormal myometrial
contractions and impair fertilization and embryo transport [5]. Biochemically,
endometriosis is known to alter peritoneal immune environment, endocrine status,
and oocyte/embryo quality. Many studies demonstrated that peritoneal fluid even in
mild endometriosis contains increased concentrations of prostaglandins, proteases,
and inflammatory cytokines such as IL-1, IL-6, and TNF [6] and these alterations
may have adverse effects on oocyte, sperm, embryo, and fallopian tube functions. It
has also been proposed that women with endometriosis may have endocrine and
ovulatory disorders, including luteinized unruptured follicle syndrome, luteal phase
dysfunctions, abnormal follicular growth, and premature as well as multiple
luteinizing hormone (LH) surges [7]. In addition, abnormalities of oocyte and
embryo quality have been described in women with endometriosis. In oocyte
donation cycles, when donor oocytes from women with endometriosis are trans-
ferred into women without endometriosis, implantation rates are lower, suggesting
that the embryo quality is reduced in women with endometriosis [8]. At the same
time, newer research identifies alternations in gene expression and genetic defects
in the endometrium of women with endometriosis [9, 10]. Taken together, it seems
to be multifactorial, involving mechanical and biochemical mechanisms by which
endometriosis causes infertility, and this indicates that controlling of this disease
may improve fertility.
27.2 Expectant Management
Women with mid-moderate endometriosis are able to conceive without any medical
or surgical intervention, although the fecundability is significantly lower compared
with women without endometriosis. Multiple studies evaluating patients with
27 Infertility Treatment of Endometriosis Patients 433
endometriosis who undergo expectant management report their fecundity rate to be

around 2.403.0 per 100 person-months [11, 12]. Therefore, the option of expectant
management is not unreasonable for patients with mid-moderate disease, especially
for young patients. In contrast, in women with more severe disease, pregnancy rates
are much lower [13], and expectant management in those with severe disease is
only delaying the start of effective treatment.
27.3 Medical Treatment
Medications used for controlling endometriosis are hormonal medications includ-

ing combined oral contraceptives (OC), progestins, danazol, and gonadotropin-
releasing hormone agonist (GnRHa). Although these medications may help reduce
pain, they have shown no benefit in the management of endometriosis-associated
infertility. A large meta-analysis of 23 trials including more than 3,000 women
demonstrated that there was no difference in pregnancy or live birth rates with
preceding ovulation suppression with OC, progestins, or danazol in subfertile
women with endometriosis (odds ratio (OR) 1.02, 95 % confidence interval
(CI), 0.701.52, P 0.082) [14]. Not only was there no benefit from ovulation
suppression, but it also delayed the patient from conceiving while taking the
suppressive agents. Therefore, medical therapy should be discouraged in patients
with endometriosis who wish to conceive [13].
Surgery for endometriosis can be both diagnostic and therapeutic. Surgical treat-
ment of endometriosis aims to remove macroscopic endometriosis and restore
normal pelvic anatomy, as well as normal pelvic immunological and hormonal
environment. Surgery, however, may not be able to completely reverse the chronic
inflammatory state or repair severe anatomical distortion and might even negatively
affect fertility by for instance reducing ovarian function. It is therefore important to
weigh up the benefits and the harm when measuring the effect of surgery.
27.4.1 Mild Endometriosis
The Canadian Collaborative Group on Endometriosis conducted a RCT with

341 women to determine whether laparoscopic surgery enhanced fecundity in
infertile women with minimal or mild endometriosis. They found that either
resection or ablation of minimal and mild endometriosis significantly enhanced
fecundity in infertile women compared with diagnostic laparoscopy alone
434 K. Koga et al.
(cumulative probability of pregnancy 30.7 % and 17.7 %, P 0.006) [11]. However,

another RCT of 101 women with minimal to mild endometriosis demonstrated no
difference in live birth rates between women who underwent laparoscopic
treatment of endometriosis by ablation or resection compared with diagnostic
laparoscopy alone (19.6 % and 22.2 %, OR 0.75, 95 % CI, 0.301.85) [15].
Subsequently, a Cochrane review concluded that in infertile patients with early-
stage endometriosis, surgical treatment, compared with diagnostic laparoscopy
alone, had significant benefits in clinical pregnancy and in ongoing pregnancy
after 20 weeks (OR 1.66, 95 % CI, 1.092.51 and OR 1.94, 95 % CI, 1.09
2.51, respectively) [16, 17].
These studies suggest that there may be a role for laparoscopic surgery of mild-
to-moderate disease to improve fertility. However, it should be kept in mind that
overall pregnancy rate in these studies remains still very low. The overall absolute
difference is 8.6 % in favor of laparoscopic surgery [18], and this means that the
number of women who needed to undergo laparoscopic surgery for one additional
clinical pregnancy is approximately 12. Thus, although there is objective evidence
that surgery is better than no treatment, surgery may not always be the best
treatment to improve fertility.
27.4.2 Severe Endometriosis
There are few RCT studying the effects of surgery on fecundity in advanced-staged
diseases, and thus, there is insufficient evidence to recommended surgery for the
treatment of infertility in severe disease. Our previous study analyzed pregnancy
outcome in 186 infertile women for a follow-up period of 18 months after laparos-
copy and found that the pregnancy rate for women with minimal/mild endometri-
osis appeared to be the highest, followed by the non-endometriosis group and
the moderate/severe endometriosis group (45.1, 33.8, and 27.6 %, respectively)
[19, 20]. Accordingly, the benefit of laparoscopic surgery in increasing fecundity
seems high in mild endometriosis but limited in severe endometriosis, and alterna-
tive therapies should be considered for those with severe endometriosis.
27.4.3 Ovarian Endometrioma
A 2008 Cochrane review examined the current literature regarding laparoscopic

ablation versus excision of endometriomas and found that the excision of the cyst
was associated with a subsequent increased spontaneous pregnancy rate in women
who had documented prior subfertility (OR 5.21, 95 % CI, 2.0413.29) [21]. This
review also identified an RCT which demonstrated an increased ovarian follicular
response to gonadotropin in those who underwent excisional surgery compared
with ablative surgery [21]. These and other observational studies suggest that, in
women with stage III/IV endometriosis who have no other identifiable infertility
factors, surgery may increase fertility [22]; however, one may be aware of a
possible adverse consequence, the loss of viable ovarian cortex [23]. After the
first infertility operation, additional surgery for recurrent endometriosis has only
rarely increased fecundity, and these patients may be better treated by assisted
reproductive technology (ART) [24].
27.4.4 Deep Endometriosis
Vercellini et al. conducted a nonrandomized study of 105 women comparing

surgery with expectant management. They found no difference in a 12-month
probability of conception (20.5 % in surgical group and 34.7 % in expectant
P 0.12) [25]. A large prospective cohort study of 500 women treated with
laparoscopic rectal shaving of endometriotic lesions found that 57 % of women
wishing to conceive had conceived naturally in a mean follow-up of 3.1 years [26].
A nonrandomized study by Stepinewska et al. looked at the effect of removing
bowel endometriosis and found that women who had a colorectal segmental
resection for bowel endometriosis had a higher monthly fecundity rate (MFR)
than women with bowel disease left unexcised (MFR 2.3 % in resection of bowel
disease and 0.84 % in bowel disease left P 0.03) [27]. However, the complication
rate in the bowel resection was considerably high. Therefore, surgery should be
conducted only when the benefit overweighs the complication risk. For instance,
deep endometriosis is always accompanied with severe pain requiring ovulation
suppression, but when a patient wishes to conceive, she should discontinue hor-
monal treatments. Therefore, surgery for deep endometriosis should be primarily
aimed for reducing pain and in turn allowing patients to conceive and should not be
performed only for the aim of improving fertility.
27.5 Superovulation (SO) and Intrauterine

Insemination (IUI)
In a crossover RCT among patients with unexplained infertility or surgically

corrected endometriosis, the pregnancy rate per cycle was significantly higher
with four cycles of clomiphene citrate/IUI than with four cycles of timed inter-
course (9.5 % vs. 3.3 %, respectively) [24]. An RCT among 49 women with stage
I/II endometriosis and infertility compared three cycles of gonadotropin/IUI with
6 months of expectant management and found that the pregnancy rate per cycle
was 0.15 % in the gonadotropin/IUI group and 0.045 % in the untreated group
(P < 0.05) [28]. Another study reported increased fecundity with gonadotropin
therapy compared to no treatment (7.3 % vs. 2.8 %, respectively) in women with
436 K. Koga et al.
infertility and minimal/mild endometriosis [29, 30]. A much larger study by

Tummon et al. randomized 103 patients (311 cycles) and found that the cumulative
live birth rates was fivefold higher following SO/IUI (OR 5.6, 95 % CI,
1.817.4) [30]. Nevertheless, there is strong evidence from a large number of
observational studies that the outcomes following SO/IUI in women with endome-
triosis are more unfavorable compared with women with other etiologies. The
largest of these studies analyzed 14,968 cycles in 3,371 couples and found that
women with endometriosis had a 30 % lower chance of achieving pregnancy than
women without endometriosis (adjusted OR 0.71, 95 % CI, 0.540.92) [31]. Col-
lectively, there is evidence to support SO/IUI in women with endometriosis,
especially with mild, surgically corrected endometriosis; however, the benefit is
still lower than in infertile women without endometriosis. It is also important to
mention that SO with clomiphene or gonadotropin may potentially enhance the
progress of endometriosis and thus in turn may negatively impact on the long-term
fecundity.
27.6 Assisted Reproductive Technology (ART)
In vitro fertilization (IVF) is currently the most effective treatment of

endometriosis-associated infertility, although the presence of endometriosis
seems to adversely affect IVF results.
27.6.1 ART and Endometriosis
Barnhart et al. performed a meta-analysis of 22 observational studies and concluded

that women with endometriosis have poorer IVF outcomes than women with tubal
infertility (OR 0.56, 95 % CI, 0.440.70) [32]. In addition, women with more
severe disease had worse outcomes than women with minimal/mild disease
(OR 0.60, 95 % CI, 0.420.87) [32]. This same study also showed that there
were significant decreases in fertilization and implantation rates and in the number
of oocyte retrieved in patients with endometriosis [32]. While endometriosis may
affect IVF results, IVF maximizes cycle fecundability for those with endometriosis,
especially in those with distortion of pelvic anatomy due to moderate or severe
disease. In one RCT, a subgroup of 21 women with endometriosis and infertility
had IVF (n 15) or expectant management (n 6). None of the women in the
expectant management group became pregnant compared to five of the 15 women
who received IVF (33 %) [33]. These findings suggest that woman with endome-
triosis, especially with severe condition, should be encouraged to undergo ART,
although at the same time she should be informed that the success rate of ART is
lower than that in women with other infertility factors.
Table 27.1 Pros and cons of expectant and surgical management of endometriomas prior to ART
Expectant Surgery
Pros
Avoid surgery Exclusive malignancy
Lower FSH doses Relieve pain
Increased E2 Reduce the risk of cyst complications
(e.g., rupture)
Increased follicles Facilitate access to oocyte retrieval
Cons
Pain Risk of damage of normal ovarian tissue
No histological diagnosis Reduced number of oocytes collected
Risk of pelvic infection following oocyte retrieval Risks of surgical complication
27.6.2 Surgical Treatment Prior to ART
27.6.2.1 Ovarian Endometrioma
The benefit of surgical treatment of endometriomas prior to IVF is still contro-

versial. There are no randomized trials comparing laparoscopic excision to
expectant management before IVF/intracytoplasmic sperm injection (ICSI)
cycles. One systematic review found that surgery (aspiration or cystectomy)
versus expectant management showed no evidence of a benefit for clinical
pregnancy with either technique [34]. Another meta-analysis of five nonrand-
omized trials found that excision of an endometrioma is no better than no
treatment prior to IVF [35]. These findings suggest that surgery for endom-
etrioma prior to scheduled IVF/ICSI does not improve the result of ART.
However, there are possible benefits of surgical treatment for endometrioma
prior to ART, including prevention of possible ruptured endometrioma, facilitation
of oocyte retrieval, detection of occult malignancy [36], avoidance of contamina-
tion of follicular fluid with endometrioma content, and prevention of progression of
endometriosis and these benefits should be taken into account when surgery is
considered. On the contrary, clinicians should be also aware of the disadvantages of
surgery including surgical trauma, surgical complications, economic costs, and,
most importantly, the potential of decreasing ovarian response [37, 38]. There is
evidence that ovarian surgery reduces the number of oocytes retrieved, reduces
peak estradiol levels, and increases total FSH requirement [38, 39]. A prospective
study also demonstrated that ovarian surgery led to complete ovarian failure in the
operated ovary in 13 % of the cases [40]. Therefore, a removal of an endometrioma
is not warranted for infertility alone, and clinicians balance the benefits and the risk
when considering surgery prior to ART. The pros and cons of the management of
endometriomas prior to ART are summarized in Table 27.1.
438 K. Koga et al.
27.6.2.2 Deep Endometriosis
A nonrandomized prospective cohort study of 179 women with deep infiltrating

endometriosis and infertility examined the impact of surgery prior to IVF versus
IVF alone. The pregnancy rate in the surgery group following IVF was 41 %, and
the no surgery group was 24 % (P 0.004) suggesting a benefit to removing deep
disease prior to IVF [41]. However, surgery for deep endometriosis is a major
surgery with inherent morbidity and thus, decision to perform surgery must be made
on an individual basis with extensive counseling.
27.6.3 Medical Treatment Prior to ART
Multiple studies have shown that prolonged GnRHa treatment before IVF may
improve fertility rates in advanced endometriosis [4244]. A Cochrane review
summarized the findings of these three RCTs, which collectively comprised
165 women with infertility and severe endometriosis. The pretreatment with
GnRHa significantly increased the clinical pregnancy and the live birth rates
compared with no pretreatment (OR 4.28, 95 % CI, 2.009.15 and OR 9.19,
95 % CI, 1.0878.22, respectively). Proposed mechanisms are via increased
retrieved oocytes, higher implantation rates, and reduced preclinical abortions
[45, 46]. Similar to GnRHa, the use of OC has been shown to improve outcomes.
De Ziegler et al. conducted a nonrandomized comparison and suggested that ART
outcomes following 68 weeks of OC in women with endometriosis are comparable
with the outcomes of age-matched controls without endometriosis [47]. Regarding
patients with endometriomas, the effectiveness of GnRHa and OC is rather contro-
versial. A Cochran review in 2010 concluded that administration of GnRHa does
not significantly affect the clinical pregnancy rate when given before ART in
patients with endometriomas, despite the pretreatment improved ovarian response
and increased the number of mature oocytes aspirated. In contrast, the study by
De Ziegler et al. showed improvement using pre-ART continuous OC therapy for
68 weeks even in those with endometriomas. Collectively, it seems that medical
treatment for endometriosis seems to benefit on ART outcomes; however, one
should also be aware that the medical treatment delays the commencement of
ART, and this may also impact the outcome of ART especially in patients with
advanced age.
27.7 Conclusions
Given the abovementioned non-RCT and RCT evidences, the Japanese Society of
Obstetrics and Gynecology (JSOG) published a guideline and recommendations for
the management of women with endometriosis (Fig. 27.1) (JSOG) [48].
Fig. 27.1 Algorism for management of infertile patents with endometriosis published by the
Japanese Society of Obstetrics and Gynecology (JSOG)
For infertile women with suspected endometriosis, laparoscopy is

recommended. If laparoscopy is performed, ablation or excision of visible endo-
metriosis should be considered. Expectant management after laparoscopy is an
option for younger women.
For infertile women with stage I/II endometriosis, factors such as the womens
age, duration of infertility, and other infertility factors must be taken into consid-
eration. When laparoscopy is undergone, ablation or excision of visible endome-
triosis should be performed. Expectant management or timed intercourse is an
option for women in younger age (age < 30) and shorter infertility period
(<3 years) and no other infertility factor. If a woman does not conceive after
36 months of expectant management, SO with clomiphene or gonadotropin with
IUI should be considered and if this also fails, step-up to ART is recommended. For
women with advanced age (age > 30) or with longer infertility duration (>3 years)
or with other infertility factor, physicians should not manage them expectantly, but
consider starting SO with IUI immediately. For women with severe tubal adhesion,
immediate ART is recommended.
For infertile women with stage III/IV endometriosis, adhesiolysis and removal
of peritoneal lesion should be done at laparoscopy. If the endometrioma is large
(>34 cm), removal should be performed to confirm the diagnosis histologically.
Following the laparoscopy, the fecundity rate without assisted treatment is very
440
Table 27.2 International guidelines on surgical treatment of endometriosis-associated infertility

Recommendation
Clinical condition ESHRE 2005 [49] ASRM 2012 [18] RCOG 2006 [50] JSOG 2010 [48]
Minimal/mild endometriosis Limited benefit: surgery Small benefit: surgery Demonstrated benefit: Small benefit: surgery recommended
(stage III disease) recommended recommended surgery recommended
Moderate/severe endometriosis Possible but unproven benefit: Possible benefit: sur- Possible benefit: recom- Possible benefit: surgery
(stage IIIIV disease) surgery recommended gery recommended mendation uncertain recommended though still
controversial
Postoperative adjuvant No benefit: not recommended No benefit: not No benefit: not No recommendation
treatment recommended recommended
Surgery before IVF Recommended if Doubtful benefit: no Recommended if Recommended if endometrioma
endometrioma 4 cm recommendation endometrioma 4 cm >3 4 cm
Recurrent endometriosis No recommendation Second-line surgery No recommendation Second-line surgery not
not recommended recommended
K. Koga et al.
low, thus discouraging expectant management. ART should be considered if 68

cycles of SO and IUI failed in younger (age < 37) women or immediately after
laparoscopy in women with advanced age (age > 38). Table 27.2 summarizes the
international guidelines on surgical treatment of endometriosis-associated infertil-
ity [18, 48, 49, 50].
In any stage and condition of endometriosis, female age is the most important
factor in designing therapy. After age 35, there is a significant decrease in fecundity.
In addition, fecundity may be decreased due to the additive adverse effects of
endometriosis progression as age increased. Consequently, in the older infertile
women with endometriosis, a more aggressive therapeutic plan especially with
ART may be reasonable. The patient with endometriosis should be informed that
she may have a decreased success rate after ART compared to a woman undergoing
ART for another indication, and the pregnancy rate will further decrease as her age
advances.
In conclusion, the association between infertility and endometriosis is complex,
and treatment alternatives for infertility with endometriosis have both risks and
benefits. Complete and detailed information on these risks and benefits must be
provided to infertile patients to allow unbiased choices among possible options.
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50. The investigation and management of endometriosis. Guideline No. 24. London: RCOG; 2006.
Chapter 28
Pregnancy Complications Associated
with Endometriosis
Ivo Brosens and Giuseppe Benagiano
Abstract An association between endometriosis and infertility has been confirmed

by a large number of studies, although mechanisms are still debated. The availabil-
ity of in vitro fertilization represented a major step forward in achieving pregnancy
in women with endometriosis, although in both peritoneal and ovarian disease,
there is an adverse effect on ovulation rates, markers of ovarian reserve, and
response to ovarian stimulation. Results have improved using intracytoplasmic
sperm injection.
When achieved, pregnancy may be complicated by spontaneous
hemoperitoneum, a rare but potentially fatal event. Data on incidence of preeclamp-
sia, small for gestational age (SGA), and preterm birth are not univocal. A large
cohort study found an increased risk of preterm birth; another observed no evidence
for an association between endometriosis and risk of pregnancy hypertension, or
preeclampsia; a third found increased rates of both preterm birth and SGA. In
pregnancies complicated by the presence of ovarian endometrioma no differences
have been observed in late pregnancy and neonatal outcomes.
Endometriosis patients seem to be at an increased risk of placenta previa and
postpartum hemorrhage. An association has been found also when pregnancy is
achieved through in vitro fertilization, suggesting that events around the time of
implantation may be responsible.
With regard to pathophysiology, the hypothesis has been proposed that defective
spiral artery remodeling may be the cause of major obstetrical syndromes in
endometriosis and adenomyosis and that endometrial and myometrial junctional
zone (MJZ) abnormalities represent a risk factor for the vascular development of
the placental bed.
I. Brosens, M.D. (*)

Leuven Institute for Fertility and Embryology, Catholic University Leuven, Leuven, Belgium
e-mail: ivo.brosens@med.kuleuven.be
G. Benagiano, M.D.
Department of Gynecology, Obstetrics and Urology, Sapienza University, Rome, Italy

446 I. Brosens and G. Benagiano
Available evidence supports the idea of a reversibility of MJZ changes by

appropriate medication inducing a prolonged hypoestrogenic period.
Keywords Adenomyosis Endometriosis Obstetrical complications Spontaneous

hemoperitoneum
28.1 Introduction
A clinical association between endometriosis and infertility has been suspected for
more than 60 years [1, 2] and confirmed by a large number of studies, although
mechanisms are still debated [3, 4]. Indeed, a study conducted some time ago
reported that between 30 and 50 % of women with endometriosis are infertile and
the prevalence of endometriosis in women with infertility may be between 25 and
50 % [5]. Recently, an opinion by the American Society for Reproductive Medicine
[5] confirmed such an association stating that endometriosis typically present with
pelvic pain, infertility, or an adnexal mass, and may require surgery.
Although decreased fertility in women with endometriosis is today well
established, causes seem to be multifactorial, involving mechanical, molecular,
genetic, and environmental ones [46].
In the event of severe disease causes are usually evident, since pelvic anatomy
becomes distorted and, when this happens, mechanical factors, such as pelvic
adhesions, may directly impair fertility disrupting oocyte release or pickup, altering
sperm motility, disorganizing myometrial contractions, and impairing fertilization
and tubal embryo transport [7].
In 2010, de Ziegler et al. [4] summarized possible mechanisms through which
endometriosis may impair fertility at all stages. These include:
Changes in the composition and characteristics of peritoneal fluid capable of
affecting fertilization and associated with pelvic inflammation. As a result,
sperm motility may be impaired and sperm capacitation inhibited; in addition,
oocytesperm interactions may also be hindered, sperm binding to the zona
pellucida decreased, and the acrosome reaction and spermoocyte fusion
impaired.
A direct effect of endometriosis on oocyte and embryo quality has been pro-
posed, although there is no agreement on this point.
In the presence of an ovarian endometrioma, the age-dependent decline in the
number of ovarian follicles can occur earlier in life.
The eutopic endometrium is altered in women with endometriosis and may
become less receptive because of a local production of estradiol and of proges-
terone resistance.
In conclusion, endocrine and paracrine pathways in human endometriotic
cells that are modulated by estrogens and progestogens, including chemotaxis
and apoptosis, are perturbed in women with endometriosis, contributing to
28 Pregnancy Complications Associated with Endometriosis 447
inflammatory responses. These phenomena promote adhesion formation and

infertility [8].
The availability of in vitro fertilization (IVF) followed by embryo transfer
(ET) has been considered as a major step forward in achieving pregnancy in
women suffering from endometriosis. Unfortunately, it was soon found that in
both peritoneal and ovarian diseases, there is an adverse effect on ovulation rates,
markers of ovarian reserve, and response to ovarian stimulation [9]. In particular,
the presence of an endometrioma can reduce ovarian reserve and decrease the
number of oocytes retrieved [10, 11]; this effect has been attributed to endometri-
osis itself [10], although these findings are controversial [1214].
In a recent Chinese cohort study, following surgery for endometrioma, the bFSH
level was higher and the numbers of oocytes retrieved were lower [15]. It seems
therefore that, on the one hand, cystectomy carries away ovarian follicles and, on
the other, endometriosis itself seems to have a detrimental effect on ovarian
follicles.
The latter hypothesis is substantiated by studies involving patients with perito-
neal endometriosis: back in 2001, Hock et al. found that women with stage III/IV
(rAFS) have a reduced ovarian reserve compared to women with stage I/II [16].
This observation is consistent with progressive loss of ovarian reserve in women
with increasing stages of endometriosis, independent of age. As pointed out by
Hauzman et al. [17], in patients with endometriosis, endometrial receptivity is also
compromised; this is evidenced in oocyte donation cycles, where lower implanta-
tion rates can be observed in subjects without endometriosis if they receive an
oocyte from a patient with endometriosis. Interestingly, oocytes donated by healthy
subjects provide the patient with endometriosis similar chances to achieve preg-
nancy to women without the disease.
The solution proposed for women with endometriosis who wish to achieve
pregnancy through IVF-ET is the use of intracytoplasmic sperm injection (ICSI).
Indeed, a recent Norwegian study [18] found that, using ICSI and leaving aside
patients with endometrioma, infertile women with various stages of endometriosis
have the same success rates as patients with tubal factor. On the basis of their results
these authors have urged the European Society of Human Reproduction and
Embryology (ESHRE) to modify its recommendations.
Notwithstanding the abovementioned problems, a large number of women with
endometriosis-associated infertility can today conceive following surgery and/or
IVF-ET. This makes mandatory an evaluation of pregnancy and its outcome in
women with endometriosis, given that recent epidemiological studies are drawing
the attention to changes in the uterine environment in association with this condi-
tion. The cellular and molecular changes in the endometrium in association with
endometriosis and adenomyosis have been reviewed by Benagiano et al. [19].
Changes in the inner myometrium, the so-called myometrial junction zone (JZ),
have been observed since it became possible to diagnose adenomyosis by magnetic
resonance (MR) imaging. These structural modifications were first described by
Bazot et al. [20, 21] and Kunz et al. [22, 23]. However, endometriosis and
adenomyosis are at present diagnosed, respectively, by laparoscopy and imaging
techniques (MR and ultrasonography) and the two are not both routinely
performed as complementary examinations when endometriosis or adenomyosis
is diagnosed [24]. At any rate, in reviewing obstetrical complications in association
with endometriosis, the presence of alteration in the myometrial JZ is of critical
importance. Therefore, this review will also include studies on pregnancy compli-
cations in women with adenomyosis. After reviewing the obstetrical complications
the underlying mechanisms in the endometrium and myometrial JZ will be
discussed.
28.2 Spontaneous Hemoperitoneum in Pregnancy

and Postpartum
28.2.1 Rare, but Dramatic
Pregnancy may have a beneficial effect on endometriotic implants, but carries an

increased risk of spontaneous hemoperitoneum in pregnancy (SHiP), fortunately a
rare event, but an important cause of maternal and fetal death [25]. A review of case
reports suggests a major role of pelvic endometriosis in the pathogenesis of
SHiP [26]. Even cases where no endometriosis is noted at the time of intervention
may be caused by endometriosis, since peritoneal endometriotic implants undergo
decidual changes in the first trimester of pregnancy, characterized by loss of
pigmentation and fibrosis, which renders visual diagnosis more difficult [27]. If
the lesion is not biopsied at the time of an emergency laparotomy, the diagnosis of
endometriosis as a cause for SHiP will often be missed.
Massive spontaneous hemoperitoneum associated with mild endometriosis
has also been described in the postpartum period and at the time of menstruation
[2830]. In addition, rupture of the sigmoid or appendix during late pregnancy in
nulliparous women has been related to decidualization of endometriotic implants
[3133].
28.2.2 Pathogenesis
Invasiveness of severe endometriosis has been suggested as a reason for SHiP, but
there is no apparent correlation between SHiP and stage of endometriosis. An
alternative explanation is that SHiP results from involution of decidualized ectopic
endometrium during pregnancy. In the differentiation of mesenchymal cells,
decidualization represents the point of no return; after which the cellular integrity
becomes inextricably dependent upon sustained progesterone signaling [34]. Falling
progesterone levels not only reverse the decidual phenotype, but also induce the
expression of a gene network that encodes for chemokines, proinflammatory
cytokines, matrix metalloproteinases, and apoptotic factors, leading to influx of

inflammatory cells, proteolytic breakdown of the extracellular matrix, cell death,
and bleeding. Interestingly, emerging evidence suggests that endometriosis is
associated with progesterone resistance, characterized by suboptimal expression
of target genes [35]. Therefore, it is tempting to speculate that functional pro-
gesterone withdrawal triggers involution of the decidual phenotype of the ectopic
endometrium surrounding distended parametrial veins, leading to peritoneal bleed-
ing of unpredictable severity.
28.3 Obstetrical Complications Associated

with Endometriosis
28.3.1 Preeclampsia, Small for Gestational Age (SGA),

and Preterm Birth
In 2003, a matched case-control study of 137 pregnant patients with endometriosis,

including IVF patients, found no difference in pregnancy outcome [36]. A few
years later, a retrospective case-control study of infertile women with laparoscopy-
confirmed endometriosis from the University of Ghent found a significantly lower
incidence of preeclampsia and pregnancy-induced hypertension in women with
endometriosis than in a control group of male-factor infertility (0.8 % versus 7.5 %,
95 % CI: 1.733.3) [37]. These studies set the stage for several other investigations
evaluating the risk of obstetrical complications in women with endometriosis
(Table 28.1).
A nationwide Swedish study including 1,442,675 singleton births between 1992
and 2006 reported in 13.090 singleton births among 8,922 women diagnosed with
endometriosis, an increased risk of preterm birth irrespective of assisted reproduc-
tion technology (ART), antepartal bleeding/placental complications, preeclampsia,
and Caesarean section. There was no association between endometriosis and SGA
or stillbirth [38]. On the other hand, a population-based, longitudinal Australian
Table 28.1 Obstetrical complications of pregnancy after ART in women with endometriosis
In comparison with
Nature of complication Naturally conceived ART without endometriosis
Preterm birth Increased (38, 40)a
Preeclampsia Increased (38)
Antepartum hemorrhage Increased (38)
Caesarean section Increased (38)
Placenta previa Increased (50) Increased (50)
SGA birth No increase (38)
Stillbirth No increase (38)
a
Increased only if endometrioma
study found in 3,239 women with endometriosis aged between 15 and 45 years no
evidence for an association between endometriosis and subsequent risk of either
pregnancy hypertension or preeclampsia, even after adjusting for age and gesta-
tional age [39]. A retrospective cohort study by Fernando et al. [40] found in
95 singletons ART babies from patients with ovarian endometriomas increased
rates of preterm birth and SGA in comparison with community birth records and
with other forms of endometriosis.
The association between endometriosis and preeclampsia remains controversial.
Unfortunately, the epidemiological studies were not controlled for changes in the
myometrial JZ that plays a critical role in the pathogenesis of pregnancy compli-
cations such as preeclampsia or SGA and to a lesser extent in preterm birth and
preterm premature rupture of the membranes [4143]. It should be noted that a
case-control study of preterm delivery in patients with adenomyosis by Juang
et al. [44] found an increased risk of both spontaneous preterm delivery and preterm
premature rupture of the membranes. This finding underlines the interest to evaluate
potential changes in the myometrial JZ in studies on obstetrical complications in
women with endometriosis.
28.3.2 Presence of Ovarian Endometrioma

During Pregnancy
Pregnancy complicated by ovarian endometrioma is a rare event [45]. Benaglia

et al. [46] analyzed data from patients achieving singleton clinical pregnancies
through IVF comparing the pregnancy outcome between 78 pregnant women with
endometriomas at the time of IVF and 156 patients who achieved pregnancy
through IVF without endometriomas. No differences were observed in late preg-
nancy and neonatal outcomes between the two groups. In particular, the rate of
preterm birth and SGA was similar. However, the study included all hemorrhagic
cysts from 1 cm persisting for 2 months. Garcia-Velasco and Somigliana [47]
recommended proceeding directly to IVF to reduce time to pregnancy, to avoid
potential surgical complications, and to limit patient costs. On the other hand, no
attention is paid to the progressive vascular sclerosis in the endometrioma bed as
demonstrated by the color Doppler sonographic studies of Qiu et al. [48] indicating
that the delayed diagnosis and surgery are the main factors of follicular loss.
28.3.3 Placenta Previa and Postpartum Hemorrhage
ART has been suspected for some reason to increase the risk of obstetrical hemor-
rhages including placenta previa, a life-threatening complication of pregnancy. The
retrospective cohort study by Healy et al. [49] compared the prevalence of
antepartum hemorrhage, placenta previa, placental abruption, and primary

postpartum hemorrhage in women with singleton births between 1991 and 2004.
Endometriosis patients had more placenta previa (1.7; 1.22.4) and postpartum
hemorrhage (1.3; 1.11.6) than those without endometriosis. The exploratory
analysis of factors in the IVF/ICSI group, showing associations with fresh embryo
transfers in stimulated cycles, endometriosis, and hormone treatments, suggested
that events around the time of implantation may be responsible and that suboptimal
endometrial function is the critical mechanism. Takemura et al. [50] confirmed by
logistic regression analysis in a group of consecutive 318 pregnancies conceived by
ART that the risk of placenta previa in relation of ten variables (maternal age,
gravidity, parity, male or female fetus, previous abortion, previous Caesarean
delivery, endometriosis, ovulatory disorder, tubal disease, and male infertility) is
related to endometriosis (odds ratio 15.1; 95 % CI 7.6500.0) and tubal disease
(odds ratio 4.4; 95 % CI 1.126.3). According to the study of Sazonova
et al. [51] blastocyst transfer increases the risk of placenta previa after IVF in
singleton pregnancies.
In a recent retrospective study, Vercellini et al. [52] assessed pregnancy outcome
in 419 women who achieved a first spontaneous singleton pregnancy after surgery
for different types of endometriosis. The study found an incidence of placenta
previa of 7.6 % in 150 women with rectovaginal lesions; 2.1 % in 69 with ovarian
endometriomas plus peritoneal implants; and 2.4 % in 100 women with peritoneal
implants only. No case of placenta previa was observed in 100 women with ovarian
endometriomas only.
28.3.4 Pathophysiology of the Myometrial Junction Zone

in Endometriosis
Endometriosis, a chronic inflammatory disorder, disrupts coordinated progesterone

responses throughout the reproductive tract, including in the endometrium. This
phenomenon is increasingly referred to as progesterone resistance. Emerging
evidence suggests that progesterone resistance in endometriosis is not just a con-
sequence of perturbed progesterone signal transduction caused by chronic inflam-
mation, but is associated with epigenetic chromatin changes that determine the
intrinsic responsiveness of endometrial cells to differentiation cues [53]. Petraglia
et al. [54] speculated that an exaggerated inflammatory reaction or the lack of a
response can activate the inflammatory process in placental membranes and
myometrium. An overlap of molecules and mechanisms may explain the evidence
that preterm birth is a common outcome in pregnant patients with endometriosis. A
correlation with preterm birth has been suggested for both endometriosis [38, 40]
and adenomyosis [44].
In the human, the process of tissue remodeling in preparation of deep placenta-
tion starts in the secretory phase of the menstrual cycle. Successful pregnancy
requires full transformation of the spiral arteries in the placental bed artery from
their origin in the myometrial junction zone [55]. Recently Brosens et al. [56]
discussed the hypothesis of defective spiral artery remodeling as a cause of major
obstetrical syndromes in endometriosis and adenomyosis. The process is first
characterized by an influx of specialized uterine natural killer cells and
decidualization of the endometrial stroma and its vasculature; then, after implanta-
tion, the interstitial and endovascular trophoblast invasion begins. The final process
results in transformation of the spiral arteries into large uteroplacental arteries in the
endometrium and myometrial JZ. Kim et al. [42, 43] suggested that in the absence
of an adequate decidual effect endovascular trophoblast cell invasion is arrested at
the level of the endometrialmyometrial junction and failed to progress into the
myometrial spiral arteries. This could explain the vascular resistance in preterm
premature rupture of the membranes and preterm birth. Defective endovascular
trophoblast invasion can also be secondary to absence of natural killer cells in the
thickened myometrial JZ. It is generally accepted that natural killer cells, which are
present around spiral arteries in the basal decidua, but not deeper in the
myometrium, play a role in determining the depth of interstitial and endovascular
trophoblast [57, 58].
The question then arises whether the endometrial and myometrial JZ abnormal-
ities associated with endometriosis at the time of implantation represent a risk
factor for the vascular development of the placental bed. Unfortunately, no studies
have yet been performed on biopsies of the placental bed in women with endome-
triosis to investigate the pattern and extent of deep placentation decidualization.
Despite the lack of histopathological investigations, clinical studies have reported
an association between endometriosis and disorders such as preterm delivery that
are associated with defective deep placentation.
28.4 Pituitary Downregulation and Pregnancy Outcome

in Endometriosis and Adenomyosis
The vital question is whether the uterine microenvironment in endometriosis and

adenomyosis can be improved by medical therapy and pregnancy rates in infertile
patients enhanced.
Recently, Maubon et al. [59] investigated in a retrospective study the impact of
the JZ structure on the likelihood of pregnancy after IVF-ET treatment and found
that a pelvic MR scan showing a thickened uterine JZ represents a negative
predictive factor for embryo implantation after IVF-ET.
Nakagawa et al. [60] suggested that treatment with a superagonist gonadotropin-
releasing hormone analog (GnRHa) prior to IVF-ET could improve the implanta-
tion rate following IVF in infertile patients with endometriosis. The results of this
study were confirmed in a retrospective study of 74 infertile patients with surgically
proven endometriosis and adenomyosis who were treated with IVF-ET [61]. All
patients were pretreated with long-term GnRHa prior to IVF-ET. The contemporary
presence of adenomyosis had apparently no adverse effects on IVF-ET outcomes in
women with endometriosis when pretreated with long-term pituitary
downregulation. In a small case series Tremellen and Russell [62] described four
women, who previously had undergone multiple unsuccessful IVF cycles because
of failure of implantation of good quality embryos who had a coexisting uterine
adenomyosis. The inactivation of adenomyosis by an ultra-long pituitary
downregulation regime promptly resulted in successful pregnancy for all four
women. Given that the majority of fertility clinics are now moving towards the
more patient-friendly antagonist protocol, where patients are not placed in a
hypoestrogen state before commencing ovarian stimulation, the question of
whether adenomyosis has an impact on IVF success rates in GnRHa antagonist-
stimulated IVF treatment needed to be examined. In a recent retrospective cohort
study of 748 patients who underwent a screening transvaginal ultrasound to identify
possible pelvic pathology before commencing their IVF treatment, Talluri and
Tremellen [63] identified 213 patients who were eligible to be included in the
adenomyosis study as they had no obvious underlying uterine or embryonic factors
that could have interfered with successful implantation. The adenomyosis group
had a viable clinical pregnancy rate of 23.6 compared with 44.6 in the
non-adenomyosis group (P 0. 017). This is the first study to clearly describe an
implantation problem in a relatively large cohort exclusively undergoing GnRHa
antagonist cycles in women with ultrasound-diagnosed adenomyosis. Thus, avail-
able evidence supports the idea of a reversibility of myometrial JZ changes by
appropriate medication inducing a prolonged hypoestrogenic period. More studies
will be required to evaluate the beneficial effect of pituitary downregulation during
the cycle of conception on deep placentation in women with adenomyosis and the
potential of this treatment for the prevention of major obstetrical complications
associated with defective deep placentation.
28.5 Conclusions
Subtle lesions and symptoms of endometriosis often disappear during pregnancy

and postpartum. The shedding of decidualized tissue may cause bleeding and the
differential diagnosis of spontaneous hemoperitoneum should be taken into account
in pregnancy and postpartum, since both maternal and fetal outcomes can be
dramatic.
The available data on the association between endometriosis and obstetrical
complications are still controversial for several reasons. In the first place, studies
are frequently based on laparoscopy for the diagnosis of endometriosis and fail to
take into account myometrial JZ thickening or adenomyosis as a potential risk of
obstetrical complications. Secondly, the diagnosis of the endometrioma is still
based on laparoscopy and ultrasound, but fails to take into account the degree of
vascular sclerosis and follicular loss, as demonstrated by the color Doppler flow
sonographic studies of Qiu et al. [48]. Finally, the potential effect of prolonged
hypoestrogenic treatment may modify risk factors at the time of conception. It is
clear that, together with studies reporting an increased risk for preterm birth in
women with endometriosis, physicians must be aware that close antenatal follow-
up and early diagnosis of vascular complications are crucial.
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45. Rossman F, DAblaing 3rd G, Marrs RP. Pregnancy complicated by ruptured endometrioma.
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JA. Pregnancy outcome in women with endometriomas achieving pregnancy through IVF.
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47. Garcia-Velasco JA, Somigliana E. Management of endometriomas in women requiring IVF: to
touch or not to touch. Hum Reprod. 2009;24:496501.
48. Qiu JJ, Liu M-H, Zhang Z-X, Chen L-P, Yang Q-C. Transvaginal color Doppler sonography
predicts ovarian interstitial fibrosis and microvascular injury in women with ovarian
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Y. Increased risk of placenta previa is associated with endometriosis and tubal factor infertility
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outcome in women with peritoneal, ovarian and rectovaginal endometriosis: A retrospective
cohort study. Br J Obstet Gynaecol. 2012;119:153843.
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54. Petraglia F, Arcuri F, de Ziegler D, Chapron C. Inflammation: a link between endometriosis
and preterm birth. Fertil Steril. 2012;98:3640.
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preeclampsia. Obstet Gynecol Annu. 1972;1:17791.
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cause of major obstetrical syndromes in endometriosis and adenomyosis. Placenta.
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HLA class I molecules by decidual NK cell receptors e a review. Placenta. 2000;21(Suppl1):
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a remodelling partnership. Hum Reprod Update. 2012;18:45871.
59. Maubon A, Faury A, Kapella M, Pouquet M, Piver P. Uterine junctional zone at magnetic
resonance imaging: a predictor of in vitro fertilization implantation failure. J Obstet Gynaecol
Res. 2010;36:6118.
60. Nakagawa K, Yamano S, Nakasaka H, Komatsu J, Hinokio K, Aono T. Effectiveness of
pre-treatment with gonadotropin-releasing hormone agonist to the patients with endometriosis
in in vitro fertilization and embryo transfer. Jpn J Fertil Steril. 2000;45:16.
61. Mijatovic V, Florijn E, Halim N, Schats R, Hompes P. Adenomyosis has no adverse effects on
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regulation before IVF/ICSI. Eur J Obstet Gynecol Reprod Biol. 2010;151:625.
62. Tremellen K, Russell P. Adenomyosis is a potential cause of recurrent implantation failure
during IVF treatment. Aust N Z J Obstet Gynaecol. 2011;51:2803.
63. Thalluri V, Tremellen KP. Ultrasound diagnosed adenomyosis has a negative impact on success-
ful implantation following GnRH antagonist IVF treatment. Hum Reprod. 2012;27:348792.
Chapter 29
Malignant Transformation of Endometriosis
Hiroshi Kobayashi
Abstract The association between endometriosis and epithelial ovarian cancer has
been supported by years of epidemiologic research. Approximately 1.0 % of women
with endometriosis may undergo malignant transformation. The malignant transfor-
mation is believed to progress in a stepwise fashion through an intermediary
endometriotic lesion, atypical endometriosis. The greatest risk is associated with
epithelial ovarian cancer of endometrioid and clear cell histology. Endometriosis
and ovarian cancer may share a common pathogenic mechanism: hyperestrogenism,
excess oxidative stress, and inflammation derived from repeated hemorrhage and
iron, contributing to ovarian tumorigenesis. The iron-induced signals can contribute
to carcinogenesis via three processes: step 1, by increasing oxidative stress, which
facilitates the accumulation of somatic mutations, contributing to endometriosis-
associated ovarian cancer initiation; step 2, by creating an estrogen-dependent micro-
environment, supporting endometrioid adenocarcinoma progression; and step 3, by
surviving stressful periods, thereby contributing to clear cell carcinoma progression.
In conclusion, some endometriosis lesions may predispose to ovarian cancer, but
future studies are needed to know the exact mechanisms of endometriosis-associated
ovarian cancer.
Keywords Endometriosis Inflammation Iron Ovarian cancer Oxidative

stress
H. Kobayashi, M.D., Ph.D. (*)

Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho,
Kashihara 634-8522, Nara, Japan
e-mail: hirokoba@naramed-u.ac.jp

458 H. Kobayashi
29.1 Introduction
Endometriosis is a benign hormone-dependent condition and a common gyneco-

logic disorder, with an estimated frequency of approximately 10 % among women
of reproductive age. This disease often results in a serious clinical problem because
of its potential for adverse sequelae, including severe dysmenorrhea, chronic pelvic
pain, infertility, and possibly developing ovarian cancer [1]. The exact etiology and
pathogenesis of endometriosis have yet to be elucidated. It may mainly cause
retrograde menstruation, implantation of menstrual tissue, and peritoneal metapla-
sia in a woman with an improper immune response and a genetic predisposition to
developing endometriotic lesions. Thus, the pathogenesis of endometriosis is mul-
tifactorial, including the role of genetics, hormonal factors, environmental factors,
and immune system.
The pathogenesis of endometriosis has been an area of active investigation,
including retrograde menstruation theory, coelomic metaplasia theory, embryonic
rest theory, lymphovascular metastasis theory, stem cell theory, and others [2]. Sev-
eral studies exploring the differential expression of genes between autologous
eutopic and ectopic endometrium from patients with endometriosis or between
eutopic endometrium from women with or without endometriosis may provide a
better understanding of the pathogenesis and pathophysiology of the disorder. The
genome-wide profiling and pathway-based enrichment analysis revealed that genes
related to cell cycle, growth factors, signal transduction, transcription factors,
hormones, cytokines, chemokines and (pro)inflammation, proteases, cell adhesion
and motility, stress response and detoxification, immune response, and metabolism
were affected in the pathogenesis process of endometriosis [316].
Furthermore, it is generally assumed that histologically benign endometriotic
lesions may be caused by the genetic defects that are permissive for malignant
transformation. Studies have documented loss of heterozygosity and mutations of
tumor suppressor genes in endometriosis (see Sect. 29.3). This disorder exhibits
high genetic instability that is involved in the cell phenotype changes that take place
during cancer progression [17]. Endometriosis shows a monoclonal pattern in
origin, suggesting that individual glands of the lesion are derived from single
precursor cells. Also, the genome-wide transcriptome of endometriosis does resem-
ble that found in ovarian cancer.
29.2 Epidemiologic Research
Epithelial ovarian cancers have been classified into four major histologic subtypes:
serous (60 %), endometrioid (1020 %), clear cell (<10 %), and mucinous
(<5 %). Serous, endometrioid, clear cell, and mucinous ovarian tumors histologi-
cally resemble the phenotypes of the fallopian tube, proliferative endometrium,
gestational endometrium, and endocervix/gastrointestinal tract, respectively.
29 Malignant Transformation of Endometriosis 459
Comparing the profile of epithelial ovarian cancer between Japanese and Cauca-
sians, clear cell carcinomas (27.6 vs. <10 %) are more common in Japan, possibly
with fewer serous adenocarcinomas (40.7 vs. 60 %). One possibility is that the
Japanese may exhibit a lower proportion of serous adenocarcinoma compared to the
United States population. This may reflect a proportional change.
The investigators have focused on latest knowledge of the genetic and environ-
mental factors affecting the development of epithelial ovarian cancer and outline
future challenges in its pathogenesis research [18]. The time trend analyses of
incidence between 1973 and 2005 in the United States exhibited a decline by
27 % in epithelial ovarian cancer incidence [19, 20]. The incidence trend of ovarian
cancer in the United States is similar to the trends observed in most of the European
countries. In contrast, an increase in epithelial ovarian cancer rates has been
reported in Japan. It is generally accepted that oral contraceptive (OC) use reduces
the risk of ovarian cancer and endometrial cancer. Although the exact reasons for
the higher ovarian cancer incidence rates in Japan are unknown, the trends may be
due to changes in risk factors, such as diet and environmental factors and the low
prevalence of OC use (23 %) in Japan. Although some part of the pathogenesis has
been unveiled, the complete events of genetic and epigenetic changes associated
with epithelial ovarian cancer remain to be identified.
The association between endometriosis and malignant transformation has often
been described in the medical literature. A literature search of MEDLINE (online
PubMed database) was conducted for published articles from 1966 to October 2010
using the keywords endometriosis combined with malignant transformation [21].
The search revealed an increase in reports describing endometriosis-associated
malignant transformation. Overall, more than 400 articles were included following
a process of independent review of each article and six were graded as good
quality [2227]. Numerous epidemiologic studies have shown an association
between endometriosis and ovarian cancer [28, 29]. Epidemiologic studies have
shown an increased risk of epithelial ovarian cancer, especially endometrioid and
clear cell histologies, among women with endometriosis. Brinton et al. examined
the records of 20,686 women hospitalized with endometriosis between 1969 and
1983 [22]. Standardized incidence ratios (SIRs) of cancers were calculated to
compare the cancer incidence of the study cohort with that of the general popula-
tion. After adjustment for age, period, and comorbidities, the hazards ratio was 1.9
for the endometriosis group compared with the control group, indicating that this
cohort had an increased overall risk of ovarian cancer. They also found further
increases in ovarian cancer risk among women with long-standing histories of
ovarian endometrioma (SIR, 4.2) [22]. The same group reported that patients
with endometriosis had the risk (4.19-fold) compared with the general population
if they presented with primary infertility [24].
There is one unique epidemiologic study in Japan, supporting the hypothesis
that ovarian endometrioma increases the subsequent risk of developing ovarian
cancer [30]. A cohort of 6,398 women with a clinically documented ovarian
endometrioma enrolled between 1985 and 1995 in the prefecture-wide Shizuoka
Cohort Study on Endometriosis and Ovarian Cancer Programme has prospectively
460 H. Kobayashi
analyzed, with follow-up through 2002. During follow-up of up to 17 years of the

ovarian endometrioma cohort, 46 incident ovarian cancers were identified, yielding
that the ovarian cancer risk was elevated significantly among women with ovarian
endometrioma (SIR, 8.95). The elevated risk of developing ovarian cancer was
mainly restricted to women with ovarian endometrioma diagnosed after age
40 (advancing age). Tumor size 9 cm or greater in diameter was an independent
predictive factor of patients with development of ovarian cancer [31]. This analysis
was restricted to those with a clinically diagnosed ovarian endometrioma. There-
fore, it has been proposed that ovarian endometrioma has been identified as a
possible risk factor for ovarian cancer.
Surveillance of endometriosis might result in a number of newly diagnosed cases
of ovarian cancer [20]. It is generally accepted that the incidence of malignant
transformations ranges between 0.7 and 1.0 % in women with endometriosis in
Japan. Although this finding is consistent with the results of six studies that support
a positive association between endometriosis and risk of ovarian cancer [2227],
Kobayashis group found an SIR of 8.95, compared with other studies reporting SIR
less than 5.0. These data provide novel and exciting possibilities. A number of
factors contribute to the results including ethnic disparities and differences in
genetic predisposition. A significantly increased risk may be found for Japanese.
Ovarian endometrioma may have higher cancer risk than pelvic endometriosis.
Ovarian cancer in Japan is a growing concern because long-term ovarian cancer
trends in incidence show rising rates.
29.3 Ovarian Cancer Susceptibility Genes
Although the etiology and the ovarian carcinogenesis still need clarification, the link
between ovarian carcinogenesis and (epi)genetic mutations is well established [18].
The investigators have utilized genome-wide gene expression analysis and associ-
ation studies to identify a specific gene signature distinguishing ovarian cancer from
controls and which served as a molecular signature for complicated histologies.
Recent high-throughput whole genome or targeted sequencing studies have also
identified numerous somatic mutations across the whole exome in a variety of
neoplasms.
High-grade serous ovarian carcinomas develop rapidly without a definite
precursor lesion. Multiple genetic and epigenetic changes are involved in the
molecular pathogenesis of serous adenocarcinoma, for example, high-grade serous
carcinomas are characterized by the tumor suppressor gene TP53 mutations. They
also have germline or somatic loss-of-function mutations in BRCA1 or BRCA2 or
promoter methylation of BRCA1. Mucinous adenocarcinoma most probably arises
via an adenoma-borderline tumor-carcinoma sequence. KRAS mutation (up to
75 %) and lack of TP53 mutations are common in mucinous tumors. Mutations
of Wnt/CTNNB1 (beta-catenin) are common in endometrioid adenocarcinoma.
Loss-of-function mutations of PIK3CA (phosphatidylinositol-4,5-bisphosphate
3-kinase, catalytic subunit alpha)/PTEN (phosphatase and tensin homolog) are
common in low-grade endometrioid carcinoma [18]. In contrast, high-grade

endometrioid carcinomas harbored TP53 mutations and lacked CTNNB1,
PIK3CA, or PTEN mutations [18]. Mutations of PIK3CA are observed most
frequently in clear cell carcinoma. Recent studies implicated a tumor suppressor
gene ARID1A (AT-rich interactive domain 1A (SWI-like)) as frequently disrupted
in endometriosis-associated ovarian cancer and clear cell and endometrioid adeno-
carcinomas. ARID1A plays a role in chromatin remodeling, which leads to cell
cycle arrest and cell death in the event of DNA damage. The loss-of-function
mutations of ARID1A may result in susceptibility to carcinogenesis through a
defect in the repair or replication of damaged DNA. All atypical endometriosis
and 86 % of non-atypical endometriosis lost ARID1A expression. ARIDA expres-
sion was retained in areas of endometriosis from sites distant from the malignant
lesion [32]. These data suggest that loss of ARID1A expression occurs as a very
early event in the endometriosis-atypical endometriosis sequence [33]. In
endometriosis-associated ovarian cancer, clear cell carcinomas harbor frequent
mutations of ARID1A and PIK3CA genes and moderate mutations of PPP2R1A
(protein phosphatase 2, regulatory subunit A, alpha) and KRAS [32, 33], while
endometrioid cancer harbors mutations of PTEN, CTNNB1, and KRAS [34, 35].
The investigators failed to identify one potential driver mutation in endometriosis-
associated ovarian cancer samples. The development of cancer takes place in a
multi-step process during which cells acquire a series of mutations, including
ARID1A, PPP2R1A, PIK3CA, PTEN, KRAS, or Wnt/CTNNB1. These mutations
may be significant factor for endometriosis-associated ovarian carcinogenesis, but
various questions have yet to be answered. This model postulates the existence of
additional mutations that establish carcinogenesis after acquisition of these muta-
tions. Future studies will represent the remarkable genetic research achievements in
the pathogenesis of endometriosis-associated ovarian cancer.
29.4 Pathogenesis of Endometriosis-Associated

Ovarian Cancer
Epidemiologic studies account for the fact that endometriosis has been associated
with an increased risk of epithelial ovarian cancer. Genome-wide studies demon-
strate that endometriosis shares several genetic characteristics with ovarian cancer.
The same pathophysiology (immune alterations, excess oxidative stress and inflam-
mation, estrogen excess, and steroid hormone interaction) orchestrates the progres-
sion of endometriosis and its transformation to ovarian cancer. These facts show
that some endometriosis has been shown to undergo malignant transformation.
Ovarian cancer precursor lesions are known to be atypical endometriosis, which
was identified in ~50 % of these histologic subtypes. Many investigators agree that
the potential of invasive epithelial malignancies arises in atypical endometriosis
[3638]. Malignant transformation of endometriosis is not a single entity; rather it
462 H. Kobayashi
is a term defining a group of histologically distinct tumors. Each histologic

subtype is clinically and genetically unique. The malignant transformation of
endometriosis is classified into three groups: (i) epithelial ovarian cancers
(endometrioid adenocarcinoma and clear cell carcinoma), (ii) other Mullerian-
type tumors, including Mullerian-type mucinous borderline tumor and serous
borderline tumor, and (iii) sarcomas such as adenosarcoma and endometrial stromal
sarcoma in the female pelvic cavity [21]. Epithelial ovarian cancer is a popular
tumor of malignant transformation [39].
Genome-wide studies have facilitated the genetic basis of pathogenesis and
pathophysiology of endometriosis and endometriosis-associated ovarian cancer
owing to the advent of new network-based analysis methods. Several investigators
identified the endometriosis susceptibility genes and pathways that may be potential
pathophysiology of endometriosis progression [316]. The endometriosis suscep-
tibility genes were grouped into pathways or networks based on functional anno-
tation. Interestingly, current molecular studies have sought to link endometriosis
with endometriosis-associated ovarian cancer through pathways or networks
related to inflammation, oxidative stress, and hyperestrogenism [40].
29.5 Inflammation and Immunity
Previous epidemiologic observations suggest that a number of factors that suppress

ovulation or menstruation, including gravidity, breast feeding, oral contraception,
and gynecologic surgery including hysterectomy and tubal ligation, reduced the
risk of ovarian cancer. Endometriosis, perineal talc use, and asbestos exposure
increase the risk, while aspirin or NSAIDs use decreases the risk. Ovulation,
endometriosis, and talc use may be associated with inflammatory responses of the
ovarian surface epithelium and pelvic peritoneum, contributing to ovarian tissue
remodeling, proliferation, and tumorigenesis. These risk factors support that per-
sistent pelvic inflammation may play a role in ovarian cancer risk [41]. In general,
chronic inflammation has been implicated in a variety of cancers, including gastric
cancer (Helicobacter pylori infection), colorectal cancer (ulcerative colitis and
Crohns disease), lung cancer (tobacco smoking-associated chronic inflammation),
malignant mesothelioma and lung cancer (asbestos exposure), hepatocellular car-
cinoma (hepatitis B and C infection), ovarian cancer (a long history of endometri-
osis), and others.
Recent advances in innate immunity illuminate the molecular mechanism under-
lying inflammation-induced carcinogenesis. Innate immunity is made possible by a
network of pattern-recognition receptors (PRRs), which include the toll-like recep-
tors (TLRs), Nod-like receptors (NLRs), RIG-like receptors (RLRs), and cytosolic
DNA receptors. TLRs mediate interactions between environmental stimuli and
innate immunity and trigger inflammatory signals. TLRs are involved in not only
the host defense against microbial infections but also stimulation of tumor cell
growth and carcinogenesis. Overexpression of PRRs in endometriosis stimulates
chronic inflammation pathways, accelerates endometriosis proliferation, and sub-

sequently causes carcinogenesis. Yamada et al. discussed the role of innate immu-
nity in the pathogenesis of endometriosis-associated ovarian cancer, with respect to
endogenous ligands, their PRRs, and their signaling pathways [42].
29.6 Oxidative Stress
Increased generation of reactive oxygen species (ROS) is implicated in the patho-

genesis of a variety of human diseases, which include cancer, atherosclerosis,
diabetes, neurodegenerative diseases, cardiovascular disease, and aging. Repetitive
hemorrhage and the accumulation of heme and iron within endometriotic cysts and
peritoneal cavity play a role in the development of ovarian cancer through the
formation of ROS under a Fenton reaction [43, 44]. Excessive iron increases cancer
risk by free radical-induced chromosomal instability. Persistent oxidative stress
induced by endometriosis-dependent hemorrhage might be associated with
carcinogenesis.
Recent studies have noted a set of genes that distinguished clear cell carcinoma
from non-clear cell carcinoma and confirmed specific expression of a transcription
factor, hepatocyte nuclear factor-1beta (HNF-1beta), in clear cell carcinoma, and
genetic alteration may be involved in oxidative stress [45]. HNF-1beta is signifi-
cantly upregulated in ovarian clear cell carcinoma and rarely expressed in non-clear
cell carcinoma specimens [46]. Of the clear cell carcinoma susceptibility
genes, 87 % are redox-related genes, including anti-oxidative and detoxification
enzymes [45]. Forty-one percent of the genes upregulated in clear cell carcinoma
samples are downstream targets of HNF-1beta [47]. HNF-1beta is thought to play a
role in anti-apoptosis, detoxification, survival, cell cycle regulation, and glycogen
synthesis. Sixty percent of the endometriosis cases also exhibited the
overexpression of HNF-1beta [48]. Endometriosis has evolved adaptive mecha-
nisms to cope with oxidative stress. Excess hemorrhage and iron can induce high
levels of oxidative stress that may have deleterious effects on endometriotic cell
growth. Endometriotic cells exhibit a higher production of HNF-1beta to detoxify
ROS and survive under oxidative stress conditions. HNF-1beta also plays key roles
in triggering DNA damage response without causing cell death and regulating
timely cell cycle arrest. HNF-1beta upregulation is sufficient to accumulate the
iron-induced genomic instability, which may be enhanced and accumulated with
increasing cell passage. Genomic instability might be increased even further upon
exposure to iron, ultimately resulting in carcinogenesis. Endometriotic cells have
developed efficient ways to cope with oxidative stress and seemingly survive
stressful periods by launching a minimal set of protection mechanisms and by
temporarily bringing several key genes such as HNF-1beta. These data allow us
to speculate that excess oxidative stress might be implicated in the development of
endometriosis-associated clear cell carcinoma.
464 H. Kobayashi
29.7 Estrogen
Endometriosis and endometrioid adenocarcinoma share a common hormonal mech-

anism. Hyperestrogenism is a common finding with development of estrogen-
dependent lesions and is a significant risk factor for the development of cancer
from endometriosis [49]. Endometrioid adenocarcinoma of the ovary and endome-
trium develops in the setting of excess endogenous and exogenous estrogen expo-
sure. Estrogen drives cell proliferation and activates the PI3K and MAPK
proliferative pathways, which are frequently dysregulated in cancer. Estrogen is
supposed to participate in the early stages of endometrial tumorigenesis, through
the accumulation of random genetic errors and increased telomerase activity.
Genomic and nongenomic estrogen receptor (ER) signaling pathways play a role
in the onset and development of tumors arising from or outside the reproductive
system. Endometrioid adenocarcinoma of the ovary is predominantly positive for
ER; however, the molecular link between estrogen and endometrial carcinogenesis
remains poorly understood.
In contrast, clear cell carcinoma specifically exhibits negative ER expression and
estrogen independency. The iron-mediated ROS oxidatively modifies genomic
DNA and, subsequently, ER depletion may be observed, possibly through DNA
methylation of the promoter region, histone deacetylation, heme and iron binding,
chromatin remodeling, and ubiquitin ligase activity [50, 51]. ER is thought to be
inactivated mainly through aberrant DNA methylation [52]. Loss of estrogen
function may be a turning point in clear cell carcinoma progression and aggres-
siveness. Endometriosis-associated ovarian cancer has a dual pathway in carcino-
genesis, estrogen-dependent ovarian carcinogenesis with an endometrioid
morphology, and estrogen-independent, oxidative stress-dependent carcinogenesis
with the clear cell morphology [53].
29.8 Conclusion
The endometriosis contains abundant iron due to repeated episodes of hemorrhage.

Iron is a mutagenic and carcinogenic compound and causes oxidative stress due to
generation of ROS. The iron-induced ROS signaling cascades can contribute to
carcinogenesis via three major processes: step 1, by increasing oxidative stress,
which facilitates the accumulation of somatic mutations and promotes DNA muta-
genesis, histone modification, chromatin remodeling, and gene products activation/
inactivation, thus contributing to endometriosis-associated ovarian cancer initia-
tion; step 2, by creating a microenvironment that supports sustained growth,
angiogenesis, migration, and invasion of cancer cells via estrogen-dependent mech-
anisms, thus supporting tumor progression of endometrioid adenocarcinoma; and
step 3, by surviving stressful periods via temporarily HNF-1beta overexpression,
thereby contributing to clear cell carcinoma progression (Fig. 29.1). The high
The carcinogenic pathways

Excess oxidative stress
and inflammation
Mutations of
HNF-1beta-positive
ARID1A
ER-negative Clear cell PIK3CA
Iron-mediated oxidative stress Ov. Ca. ZNF217
and inflammation PPP2R1A
Step 1
Endometriosis Atypical Endo.
Endometrioid
KRAS, BRAF, ERBB2, CTNNB1, PTEN
PIK3CA, ARID1A, and PPPR1A mutations HNF-1beta-negative Ov. Ca. Mutations of
ER-positive ARID1A
CTNNB1
hyperestrogenism PTEN
PIK3CA
Genetic mutation PPP2R1A
Fig. 29.1 The carcinogenic pathways. Endometriosis may contribute to carcinogenesis via three
processes: step 1, by increasing oxidative stress, which facilitates the accumulation of somatic
mutations, contributing to endometriosis-associated ovarian cancer initiation; step 2, by creating
an estrogen-dependent microenvironment, supporting endometrioid adenocarcinoma progression;
and step 3, by surviving stressful periods via temporarily HNF-1beta overexpression, thereby
contributing to clear cell carcinoma progression
incidence of malignant transformation in high-risk women with endometriosis

(advancing age and tumor size 9 cm or greater in diameter) further supports
intensive targeted surveillance.
Acknowledgments Grant support: Supported by Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, and Culture of Japan to the Department of Obstetrics and
Gynecology, Nara Medical University (H. Kobayashi).
Conflict of Interest The authors declare no conflict of interest.
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Index
A Anti-apoptotic factors, 180

Ablation, 388 Antibody-based microarrays, 333
ACE. See Angiotensin-converting Anti-Mullerian hormone (AMH), 421, 423
enzyme (ACE) Antral follicles count (AFC), 421, 423, 424
ACh. See Adrenergic and acetylcholine Apoptosis, 67, 236, 258
(ACh) ARID1A. See AT-rich interactive
Acosta classification, 342 domain 1A (ARID1A)
Activator protein 1 (AP-1), 98 Aromatase, 97, 109, 130, 132, 148, 149,
Ad4BP/Steroidogenic factor-1 (SF-1), 164 156, 163, 186, 330
Adenomyosis, 355 Aromatase inhibitors (AIs), 156, 167
Adhesion, 313 Artificial reproductive technology
Adrenergic and acetylcholine (ACh), 195 (ART), 388
Adrenergic fibres, 196 Assisted reproductive technology
AFC. See Antral follicles count (AFC) (ART), 435
AFS. See American Fertility Society (AFS) Asthma, 282
Aging, 116 Atopic diseases, 278
AIs. See Aromatase inhibitors (AIs) Atorvastatin, 240
AKT2, 227 AT-rich interactive domain
Alcohol, 282 1A (ARID1A), 228, 463
Allergic rhinitis, 282 Atypical endometriosis, 225
American Fertility Society (AFS), 346 Autophagy, 163
American Society of Reproductive Autotransplants, 4
Medicine, 346 5-Aza-deoxycytidine, 109, 162
AMH. See Anti-mullerian
hormone (AMH)
Anastrozole, 169, 170, 171 B
Androgen(s), 156 Baboon models, 88, 255
hypothesis, 373 Bacillus Calmette-Guerin (BCG), 287
receptors, 148 Bacterial contamination hypothesis, 29
Androstenedione, 163 Bak, 182
Angiogenesis, 94, 136 Barker hypothesis, 297
Angiotensin-converting enzyme (ACE), 201 Basalis, 252
Animal models, 214 BAX, 181, 182, 258
Annexin A2, 133 BAY 11-7085, 238
Annexin V, 325 Bayes theorem, 367

DOI 10.1007/978-4-431-54421-0, Springer Japan 2014
470 Index
B-cell lymphoma 2 (Bcl-2), 15, 182, Chemokine, 67

183, 202, 237, 258 Chicken ovalbumin upstream promoter
B-cell lymphoma-extra large transcription factor (COUP-TF), 166
(Bcl-XL), 237, 259 Chinese herbal medicine, 292293
BCG. See Bacillus Calmette-Guerin (BCG) Cholinergic fibres, 196
Bcl-2. See B-cell lymphoma 2 (Bcl-2) Chromatin, 108
Bcl-xL/Bcl-xS, 184 Chronic fatigue syndrome, 283
BDNF. See Brain-derived neurotrophic Chronic pelvic pain (CPP), 197, 322, 386
factor (BDNF) Cigarette, 282
Ber-EP4, 27 Class A scavenger receptors, 134
Beta-hydroxyisovalerylshikonin, 186 Class B scavenger receptors, 134
Biomarkers, 324, 415 Clear cell carcinoma, 230, 461
Birth defects, 278 Clear cell ovarian cancer, 378
Bisphenol A (BPA), 281, 297 Cluster of differentiation 10 (CD10), 27
Bladder endometriosis, 315 Cluster of differentiation 16 (CD16), 50
B lymphocyte stimulator (BLyS), 283 Cluster of differentiation 36 (CD36), 64,
Body mass index (BMI), 279 67, 134, 135
Bone loss, 168 Cluster of differentiation 56 (CD56), 50
Bowel endometriosis, 315, 435 Cluster of differentiation 94 (CD94), 53
Bowel infiltrations, 355 c-myc, 237
BPA. See Bisphenol A (BPA) Coelomic epithelium, 13
Bradykinin, 193 Coelomic metaplasia, 12
Brain-derived neurotrophic COH. See Controlled ovarian
factor (BDNF), 199 stimulation (COH)
Breast cancer, 159 Cohort study, 370
Breast cancer 1 (BRCA1), 462 CO2 laser. See Carbon dioxide
Breast cancer 2 (BRCA2), 462 laser (CO2 laser)
Breastfeeding, 297 Compartment A, 354
Bufalin, 186 Compartment B, 354
Buttram classification, 342 Compartment C, 354
Butyrates, 242 Confounding factor, 374
Conscious pain mapping, 196
Controlled ovarian stimulation
C (COH), 388
CA-125, 324, 325 Costunolide, 238
Caffeine, 282 COX-2. See Cyclooxygenase-2 (COX-2)
Calcitonin gene-related peptide Cox regression model, 370
(CGRP), 192, 193, 194, 195, 197 CPP. See Chronic pelvic pain (CPP)
Calretinin, 27, 217 CREB-binding protein (CBP), 131
cAMP response element-binding protein Cul-de-sac obliteration, 400
(CREB), 132 retroflexed uterus, 313
Carbon dioxide laser (CO2 laser), 424 Cullen, 10
Carcinogenesis, 224 Cumulative incidence, 370
Case-control studies, 372 Curcumin, 291
Caspase-1, 237 Cyclin-dependent kinase inhibitor 2B, 114
Caspase-3, 185 Cyclooxygenase-1 (COX-1), 126
Catalase (CAT), 229 Cyclooxygenase-2 (COX-2), 5, 89, 126, 137,
CBP. See CREB-binding protein (CBP) 149, 165, 197, 201, 238, 330
cDNA microarray, 181, 259 inhibitors, 136
Cells, 62 Cyclooxygenase (COX) inhibitors, 139
CGRP. See Calcitonin gene-related CYP19, 151
peptide (CGRP) CYP17A1, 151
Chaperone proteins, 263 CYP19A1, 157
Index 471
CYR61. See Cysteine-rich angiogenic EFI. See Endometriosis fertility index (EFI)
inducer 61 (CYR61) EGF. See Epidermal growth factor (EGF)
Cystectomy, 388, 412 Endocrine-disrupting, 278
of endometrioma, 413 Endocrine-disrupting chemicals
Cysteine-rich angiogenic inducer (EDCs), 280
61 (CYR61), 136 Endocrine-disrupting compounds
Cytokeratin, 217 (EDCs), 297
Cytokines, 50, 68, 88, 90, 278, 432 Endometrial biopsy, 326
Endometrial cancer, 159
Endometrial curettage, 326
D Endometrial-myometrial junction, 452
DAD-1. See Defender against cell Endometriomas, 385, 388, 412417,
death 1(DAD-1) 437, 447, 462
Danazol, 238, 433 Endometriosis, 180
DDT. See Dichlorodiphenyltrichloroethane Endometriosis-associated ovarian cancer
(DDT) (EAOC), 224
Death signal, 180 Endometriosis fertility index (EFI), 349
Decidual changes, 317 Endometriotic cells, 92
Decidualization, 448 Endometriotic cysts, 311, 317319
Deep endometriosis, 435 Endometriotic stromal cells, 128
Deep infiltrating endometriosis (DIE), 198, Endometrium, 252
313, 352, 353, 386, 400, 438 Endotoxin, 88, 89
Deep placentation, 452 Environmental factors, 115
Defender against cell death 1(DAD-1), 259 Enzian, 352
Delta method, 369 Enzian classification, 352
Dendritic cells, 64 Epidermal growth factor (EGF), 131, 253
Density of nerve fibres, 197 receptors, 253
Dental sealants, 298 Epigenetic aberrations, 365
DES. See Diethylstilbestrol (DES) Epigenetic change, 5, 159
Diagnostic laparoscopy, 434 Epigenetics, 107
Diagnostic test, 323 Epigenetics refers, 241
Dichlorodiphenyltrichloroethane (DDT), 281 EP2 receptor, 131
DIE. See Deep infiltrating EP receptors, 132
endometriosis (DIE) Escherichia coli (E. coli), 24
Diethylstilbestrol (DES), 280, 295, 298 Estradiol (E2), 182, 262, 421
Dioxin, 281 Estrogen, 137, 156, 163, 231, 466
DL--amino-3-hydroxy-5-methylisoxazole-4- production, 108
propionic acid receptor (AMPA), 192 Estrogen receptor 1 (ESR1), 150
DNA methylation, 108, 466 Estrogen receptor 2 (ESR2), 150
DNA methyltransferases (DNMTs), 115 Estrogen receptor (ER), 28, 111,
Dorsal root ganglia (DRG), 191 148, 157, 466
Douglas, 10 Estrogen receptor alpha (ER), 111,
Drainage, 388 148, 150, 165, 201, 252, 262
Dual-specificity phosphatase-2 Estrogen receptor beta (ER), 111, 148, 150,
(DUSP2), 129, 137 165, 201, 252, 262
Dyschezia, 386 Estromedin, 132
Dysmenorrhea, 279, 322, 386 Etiology, 126
Dyspareunia, 322, 386 Eutopic endometrium, 55, 92, 128, 180
Excision, 388, 434
Expectant management, 433
E Exposure-disease relationship, 373
E-cadherin, 113 Extracellular signal-regulated
Ectopic endometrium, 4 kinase (ERK1/2), 259
472 Index
F Hemoglobin, 21
Family history, 279 Hemosiderin, 21
Far adenomyosis (FA), 355 Heparin, 240
Far bladder (FB), 355 Hepatocyte growth factor (HGF), 96, 202
Farnesyl pyrophosphate (FPP), 240 Hepatocyte nuclear factor-1 (HNF-1beta),
Far ureter (FU), 356 227, 465
Fas, 184 HIFs. See Hypoxia-inducible factors (HIFs)
Fas ligand (FasL), 183 Hills 9 criteria, 378
Fasudil, 186 Histamine, 193
Fat cells, 298 Histone acetyltransferases, 116
Fat-suppressed T1-weighted imaging, 313 Histone deacetylases (HDACs), 116, 241
Fc receptor, 50 Histone deacetylases inhibitors
Fibroblast growth factor-2 (FGF2), 131 (HDACIs), 116
Fibroblast growth factor-9 (FGF9), 132, 202 Histone H3, 162
Fibromuscular hyperplasia, 314 Histone H4, 162
Fibromyalgia, 283 Histone modifications, 116, 162
Fibrotic plaque, 313 Histopathological criteria, 366
Follicle-stimulating hormone (FSH), 421 Historical score, 349
Follicular pool, 420 HOX10, 330
Food, 298 HOXA10, 5, 113, 115, 237
Functionalis, 252 HSPs. See Heat shock proteins (HSPs)
Funnel plots, 365, 371 Human heat shock protein 70 (HSP70), 25
Human leukocyte antigen (HLA), 54
Human leukocyte antigen G
G (HLA-G), 55, 56
GABA. See Gamma-aminobutyric acid 3-Hydroxy-3-methylglutaryl coenzyme A
(GABA) (HMG-CoA), 239
Gamma-aminobutyric acid (GABA), 192 17-Hydroxysteroid dehydrogenase
Genome-wide (GW), 108 (17-HSD), 15
Genome-wide association study 17--Hydroxysteroid dehydrogenase type
(GWAS), 108 1 (HSD17B1), 149, 164
Genome-wide DNA methylation, 114 17--Hydroxysteroid dehydrogenase type
Geranylgeranyl pyrophosphate (GGPP), 240 2 (HSD17B2), 149, 164, 263
Glutathione peroxidase (GPx), 229 Hypermethylation, 5, 108
Gonadotropin-releasing hormone Hypomethylation, 108
(GnRH), 238 Hypoxia, 95
analogs, 424 Hypoxia-inducible factors (HIFs), 129
Gonadotropin-releasing hormone agonist HIF-, 129
(GnRHa), 185, 433, 438, 453 HIF-1, 129, 137, 230
Gravidity, 293 Hypoxia-inducible transcription
Green tea, 291292 factor-1alpha (HIF1), 95
Growth factors, 88, 278 Hypoxic stress, 128
Guanosine triphosphate (GTPase), 239
I
H IAP. See Inhibitors of apoptosis family (IAP)
Haptoglobin, 16 ICAMs. See Intercellular adhesion molecules
Hazard rate (HR), 370 (ICAMs)
Heat shock element (HSE), 56 IGF-1. See Insulin-like growth factor-1 (IGF-1)
Heat shock factor 1 (HSF1), 56 IME. See Invisible microscopic
Heat shock protein 27 (HSP 27), 202 endometriosis (IME)
Heat shock proteins (HSPs), 25 Immune cells, 64
Heme, 21 Immune clearance, 5
Index 473
Immune surveillance, 67 K
Immunoglobulin-like transcript (ILT), 53 Ki-67, 28, 218
Implantation, 20 Killer-cell immunoglobulin-like
Implantation theory, 4, 218 receptors (KIRs), 52
Incessant menstruation, 373 KIR2DL1, 52
Incidence, 370 KIR2DL4, 52, 55
Incidence rate, 370 Kistner, 342
Inclusion criteria, 371 Knockout, 214
Infertility, 96, 278, 387, 422, 431, 446 KRAS, 225, 462
Inhibin-B, 421, 423
Inhibitors of apoptosis family (IAP), 237
Inhibitors of NF- (NF-) activity, 186 L
Inhibitory killer cell immunoglobulin-like Laparoscopic ablation, 434
receptors (KIRs), 52 Laparoscopic cystectomy, 422
Insulin-like growth factor-1 (IGF-1), Laparoscopy, 322, 385
131, 201, 253 Lateral pararectal space (LPRS), 401, 403
Intercellular adhesion molecule-1 Lauchlan, 10
(ICAM-1), 67, 93 L1 cell adhesion molecule
Intercellular adhesion molecules (L1CAM), 261
(ICAMs), 21 Leak test, 404
Interferon (INF), 50, 90 Letrozole, 169, 170, 171
Interleukin-1 (IL-1), 90 Lipopolysaccharide (LPS), 23
Interleukin-2 (IL-2), 185 Logistic regression, 371
Interleukin-2 (ILT-2), 55 Loss of heterozygosity (LOH), 226227
Interleukin-4 (IL-4), 90 Lovastatin, 240
Interleukin-4 (ILT-4), 55 Low birth weight (LBW), 297
Interleukin-5 (IL-5), 90 Low-dose estrogen-progestin (LEP), 407
Interleukin-6 (IL-6), 4, 90, 230, 288 Luteinized unruptured follicle
Interleukin-8 (IL-8), 4, 90, 93, 95, syndrome, 432
182, 185
Interleukin-10 (IL-10), 9092
Interleukin-12 (IL-12), 90, 93 M
Interleukin-13 (IL-13), 90 Macrophage chemotactic factor-1
Interleukin-17 (IL-17), 90 (MCP-1), 288
Interleukin-23 (IL-23), 90 Macrophage chemotactic protein
Interleukin-33(IL-33), 90 (MCP)-1, 65
Interleukin-1 (IL-1), 68, 128, 137 Macrophage colony stimulating factor
Interstitial cystitis, 283 (MCSF), 90
Intestinal tethering, 313 Macrophage migration inhibitory
Intrauterine devices (IUD), 285 factor (MIF), 165, 289
Intrauterine insemination (IUI), 435436 Macrophages, 62, 90, 91
Invisible (occult) endometriosis, 2629 Magnetic resonance imaging (MRI), 356
Invisible microscopic endometriosis Major histocompatibility complex
(IME), 26 (MHC), 50
In vitro fertilization (IVF), 388, 436 Malignant transformation, 317, 364, 463
Irritable bowel syndrome, 283 MAPK. See Mitogen-activated protein
Ischemia, 95 kinase (MAPK)
IUD. See Intrauterine devices (IUD) Mass spectrometry, 257
IVF. See In vitro fertilization (IVF) Matrix metalloproteinases
(MMPs), 133, 259
MBD1, 110, 162
J MCP-1. See Macrophage chemotactic
Junction zone (JZ), 447, 450452 factor-1 (MCP-1)
474 Index
MeCP2, 110, 162 N

Median pararectal space (MPRS), 401, 403 Natural killer (NK)
Medical treatment, 387, 414 cells, 50, 56, 62, 88, 93, 184, 283, 452
Melatonin, 288 cytotoxicity, 51
Menarche, 279, 298 Nerve fiber density, 327
Menopause, 224 Nerve fibers, 191, 193195, 325
Menstrual cycle, 182 Nerve growth factor (NGF), 193, 198, 199
Menstrual fluid (MF), 24 Neurofilament (NF), 197, 326
Mesothelial cells, 91 Neurokinin A, 195
Met, 96, 227 Neuropeptide (NPY), 193, 194, 195
Metalloproteinase-9 (MMP-9), 67 Neurotensin (NT), 194
Metalloproteinases (MMPs), 21 Neurotrophin-4 (NT-4), 201
Metaplasia theory, 4, 92 Neurotrophins, 199200
Methemoglobin, 21 Neurotrophins-3 (NT-3), 199
Mice, 215 Neurotrophins-4/5 (NT-4/5), 199
Microarray, 256 Neutral endopeptidase (NEP), 200201
Microglia, 64 NFE2L3, 115
MicroRNAs (miRNAs), 162, 181, 257, 329 NGF. See Nerve growth factor (NGF)
Microscopic endometriosis, 26 NGFRp75, 199
Microscopic findings, 11 NKG2, 53
MIF. See Macrophage migration inhibitory N-methyl-D-aspartic acid (NMDA), 192
factor (MIF) Nociceptive-specific (NS), 192
Migraines, 283 Nociceptors, 192
Missing self, 50 Nod-like receptors (NLRs), 464
Mitochondrial pathway, 183 Noninvasive diagnostic test, 323
Mitogen-activated protein kinase Noninvasive test, 324332
(MAPK), 98, 129 Nonopaque lesions, 21
M, 22, 26 Nonsteroidal antiinflammatory drugs
M1-macrophages, 64 (NSAIDs), 139
M2-macrophages, 64 NR5A (SF1), 158
MMBT. See Mullerian mucinous N-tosyl-L-phenylalanine chloromethyl
borderline tumor (MMBT) ketone (TPCK), 238
MMPs. See Matrix metalloproteinases NTRK1, 200
(MMPs) Nuclear factor-kappa B (NF-), 24, 99,
Monoclonal, 225 181, 237, 259, 264, 283
Monocyte chemotactic protein-1 inhibitors, 236238
(MCP-1), 90 Nude, 214
MPRS. See Median pararectal
space (MPRS)
MRI-based radiological classification O
system (MARIE), 357 Obese, 279
MRP4. See Multidrug resistance-associated Obstetrical complications, 448
protein 4 (MRP4) OCP. See Oral contraceptive pills (OCP)
mTOR, 232 Odds ratio (OR), 366367, 373
Mullerian ducts, 12, 13 Oral contraceptive pills (OCP), 284
Mullerian mucinous borderline tumor Oral contraceptives (OC), 186,
(MMBT), 318 433, 438, 461
Mullerian remnants, 315 OSE. See Ovarian surface
Mullerian rests, 4 epithelium (OSE)
Multidrug resistance-associated protein Ovarian cancer, 363, 364, 460
4 (MRP4), 129 Ovarian failure, 437
Mutations, 227 Ovarian reserve, 420, 422, 425, 447
Index 475
Ovarian surface epithelium (OSE), 12, 464 Primates, 214

Ovulation, 96 Primordial follicles, 420
Ovulation induction, 171 Pro-apoptotic factors, 180
Ovum pick-up, 96 Progesterone, 112, 182
Oxidative stress, 228229 Progesterone receptor
A (PR-A), 112, 148, 252
Progesterone receptor B (PR-B), 112, 148
P Progesterone receptors (PRs), 28, 148, 263
p53, 225, 226, 259 Progestins, 411
Pain recurrence rate, 408 Promoter switching, 159
Paracrine factor, 96 Prospective studies, 370
Parametrial involvement, 316 Prostaglandin E2 (PGE2), 25, 89, 126,
Pararectal space, 401 127, 137, 149, 164, 184, 201, 238
Parity, 293 receptor, 132
Parthenolide, 217, 238 Prostaglandin E2 receptor (EP2), 132
Pathogenesis, 1114, 20, 180, 323, 460 Prostaglandin E3 receptor (EP3), 132
Pattern-recognition receptors (PRRs), 464 Prostaglandins (PGs), 66, 193, 198, 432
PCB. See Polychlorinated biphenyls (PCBs) Prostate cancer, 10
PDTC. See Pyrrolidine dithiocarbamate Protein gene product 9.5 (PGP 9.5), 194,
(PDTC) 197, 326, 327
Pelvic inflammation, 88 Protein microarray, 257
Pentoxifylline, 290 PTEN. See Phosphatase and tensin
Peripheral blood, 331 homolog (PTEN)
Peritoneal fluid (PF), 63, 65, 88, 89, Publication bias, 371, 375, 379
278, 446 Puerarin, 292
volume, 90 PUFA. See Polyunsaturated fatty
Peritoneal macrophages (PMs), 91, acid (PUFA)
128, 184 Pyrrolidine dithiocarbamate (PDTC), 238
Phagocytes, 6568
Phagocytosis, 133
Phosphatase and tensin homolog Q
(PTEN), 259, 330, 462 Quality Assessment of Diagnostic Accuracy
Phosphatidylinositol-4,5-bisphosphate Studies (QUADAS), 328
3-kinase, catalytic subunit alpha criteria, 332
(PIK3CA), 228, 462
Phospholipase A2 (PLA2), 128
Physical activity, 294 R
Placenta previa, 450 Raloxifene, 186
Poisson regression, 371 Randomized controlled trial
Polychlorinated biphenyls (PCBs), 281 (RCT), 385, 386
Polymorphisms, 150 rASRM classification, 346349
Polyunsaturated fatty acid (PUFA), 293 Rate ratio (RR), 370
Postmenopausal women, 172 Reactive oxygen species (ROS), 465, 466
Postoperative medical treatment, 415 Recall bias, 370
Postpartum hemorrhage, 451 Rectovaginal septum, 315
Preeclampsia, 450 Recurrence, 236
Pregnancy, 412 Recurrence of chocolate cyst, 406
Pregnancy-induced hypertension, 450 Recurrence rates, 412413
Preterm birth, 450, 451 Red lesion, 89
Prevalence, 4 Regulated upon activation, normal T cell
data, 367 expressed and secreted chemokine
of endometriosis, 366 (RANTES), 90, 261
Prevention, 278 Relative risk (RR), 367, 373
476 Index
Resveratrol, 292 Statistics, 329

Retinoic acid (RA), 288 Stem cell markers, 334
Retrograde menstruation, 12, 20, 56, 8893, Stem cells, 4, 254
126, 214, 411 Steroidogenic acute regulatory
Retrospective cohort studies, 370 protein (StAR), 112, 130, 132,
Rho, 239 156, 330
Rho-associated coiled-coil-forming protein Steroidogenic factor-1 (SF-1), 112113,
kinase (ROCK), 239 131, 149, 164, 166
inhibitors, 240 Steroidogenic factor-2 (SF-2), 112
RIG-like receptors (RLRs), 464 Suberoylanilide bishydroxamine
Risk factor, 373 (SAHA), 242
Rodents, 214 Substance P (SP), 192, 193, 194, 195, 197
Rofecoxib, 169 Superoxide dismutase (SOD), 228
Rokitansky, 10 Surgical treatment, 434
Rupture, 388 Survivin, 181
S T
SAHA. See Suberoylanilide TAK1, 100
bishydroxamine (SAHA) 2,3,7,8-Tetrachlorodibenzo-p-dioxin
Sample size, 369 (TCDD), 281, 295
Sampson, 10, 20, 214 TGF. See Transforming growth
Sampsons retrograde menstruation, 254 factor (TGF)
Sampsons theory, 28 Thalidomide, 238
SCID mice, 214 T-helper cells, 91
Secondary Mullerian system, 10 T-helper cells 1(Th1), 91
Secretoneurin (SN), 195 T-helper cells 2(Th2), 91
Selection biases, 371 Tissue inhibitors of MMPs (TIMPs), 65
Sensory C, 326 TLR4. See Toll-like receptor 4 (TLR4)
Serotonin, 192, 193 3.0 T MR imaging, 312
Serous ovarian carcinomas, 462 TNFRSF1B, 200
SF-1. See Steroidogenic factor-1 (SF-1) Toll-like receptor 4 (TLR4), 24, 25
Siagnostic test, 333 Toll-like receptors (TLRs), 464
Signal-regulated kinase (ERK), 98 TP53, 462
Simvastatin, 240 TPCK. See N-tosyl-L-phenylalanine
Single nucleotide polymorphisms chloromethyl ketone (TPCK)
(SNPs), 159 Transforming growth factor 1
SIR. See Standardized incidence (TGF1), 197
ratios (SIRs) Transforming growth factor
Small for gestational age (TGF), 90, 253
(SGA), 449450 Transgenic mice, 214
Small nerve fibres, 196 Transient receptor potential vanilloid
Smooth muscle actin (SMA), 238 1 (TRPV1), 193, 198
SN-50, 238 Translational activators, 332
SOD2, 230 Translational repression, 330
Soluble FasL, 184 Transplantation, 11, 214
Sorafenib, 290 Tricostatin A (TSA), 109, 242
Sperm, 97 Tubal motility, 96
Spiral artery remodeling, 452 Tumor necrosis factor- (TNF-), 50, 90,
Spontaneous hemoperitoneum, 448 95, 128, 198
Standardized incidence ratios Tumour growth factor, 65
(SIRs), 370, 461 Tyrosine kinase A receptor
Statins, 239, 290 (TrkA), 199, 200
Index 477
U VIP. See Vasoactive interstinal

Ureter tube, 400403, 407 polypeptide (VIP)
Urinary tube, 401, 402 Visible endometriosis, 2026
Uterosacral ligament, 314 Vitamin D, 289
VMAT. See Vesicular monoamine
transporter (VMAT)
V VPA. See Valproic acid (VPA)
Vaginal parturition, 293
Valproic acid (VPA), 242, 243
Vascular endothelial growth factor (VEGF), W
90, 94, 136, 181, 200, 202, 238, 325 WDR. See Wide dynamic
Vasoactive interstinal polypeptide range (WDR)
(VIP), 193, 194, 195 Wide dynamic range (WDR), 192
VEGF. See Vascular endothelial growth Wnt/CTNNB1, 462
factor (VEGF)
Vesicular monoamine transporter
(VMAT), 197 Y
Vimentin, 217 YB-1, 260

9 Endometriosis

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What is the book about?

What is the book about?

What is the editor's background?

What is the editor's background?

Tasuku Harada

ISBN 978-4-431-54420-3 ISBN 978-4-431-54421-0 (eBook)

Springer Japan 2014

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Endometriosis remains an enigmatic disease despite a long history of research into

Yonago, Tottori, Japan Tasuku Harada

1 Endometriosis: A Mysterious Disease . . . . . . . . . . . . . . . . . . . . . . . 3

Part II Basic Science

2 Pathological Aspect and Pathogenesis of Endometriosis . . . . . . . . . 9

11 Aromatase Expression in Endometriosis and Its Significance . . . . . 155

Part III Clinical Science

18 Prevention of Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

22 The Association of Endometriosis with Ovarian Cancer:

Abstract Endometriosis is an enigmatic disease, despite a long history of research

Keywords Autotransplant Epigenetics Eutopic endometrium Implantation

Endometriosis is an enigmatic disease, despite a long history of research into its

T. Harada, M.D., Ph.D., D.M. Sci. (*)

T. Harada (ed.), Endometriosis: Pathogenesis and Treatment, 3

cyclooxygenase 2 in endometriotic tissue is enhanced compared with that in both

Experimental studies in autotransplant animal models used eutopic endometrium

1. Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial

Ritsuo Honda and Hidetaka Katabuchi

Abstract Historically, endometriosis was first described in Egyptian scrolls in the

Keywords Endometriosis Pathogenesis Pathophysiology

Endometriosis is a common gynecological disease of unknown etiology which

R. Honda, M.D., Ph.D. (*) H. Katabuchi

T. Harada (ed.), Endometriosis: Pathogenesis and Treatment, 9

In 1860, the first histological description of endometriosis was given by von

2.2 Sites of Endometriosis

Endometriosis is defined as a pathological disease caused by the presence and

2.3 The Theory of Secondary Mullerian System

that the development of peritoneal mullerian lesions may be secondary to the

2.4 Microscopic Findings of Endometriosis

The focus of endometriosis is basically formed of three components: the endome-

2.5 Theories on the Pathogenesis of Endometriosis

Because of the different locations, possible organs, appearances, and hormone

2.5.2 Coelomic Metaplasia

2.6 The Factors Affecting the Development

2.6.1 Disturbances of Hormonal Conditions

Hormonal alterations may influence the ability of endometrial cells to proliferate,

2.6.2 Familial and Genetic Factors

diverse physiologically active substances including cytokines, coagulation factors,

Co-workers in our Department Okamura H, Matsuura K, Ohba T, Tashiro H, Fukumatsu Y,

1. von Rokitansky C. Ueber Uterusdrusen-Neubilding in Uterus- und Ovarial Sarcomen. Ztschr

5. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue

28. Fukumatsu Y, Katabuchi H, Miyamura S, Matsuura K, Okamura H, Naito M, Takahashi

Khaleque Newaz Khan

Abstract Endometriosis is a chronic disease characterized by endometrial tissue

Keywords Visible endometriosis Bacterial endotoxin Innate immunity TLR4

K.N. Khan, M.D., Ph.D. (*)

T. Harada (ed.), Endometriosis: Pathogenesis and Treatment, 19

3.1 Visible Endometriosis: Introduction

Endometriosis, the presence of functional endometrium outside of the uterine

3.1.1 Pathogenesis and Natural Course