Human Reproduction, Vol.25, No.11 pp.

2851–2858, 2010
Advanced Access publication on September 2, 2010 doi:10.1093/humrep/deq241

ORIGINAL ARTICLE Reproductive biology

Effect of dienogest administration on

angiogenesis and hemodynamics in a
rat endometrial autograft model
Hiroko Katayama 1, Tomihiro Katayama 1,*, Kazuhiko Uematsu 1,
Mie Hiratsuka 2, Masaki Kiyomura 1, Yutaka Shimizu 3, Atsuro Sugita 4,
and Masaharu Ito 1
1
Department of Obstetrics & Gynecology, Graduate School of Medicine, Ehime University, Shitsukawa Toon, Ehime 791-0295, Japan 2Fukui
Women’s Clinic, Hoshinooka, Matsuyama, Ehime 790-0922, Japan 3Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd,

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Gotemba 412-8524, Japan 4Pathology Division, Ehime University Hospital, Shitsukawa Toon, Ehime 791-0295, Japan

*Correspondence address: E-mail: tkatayam@m.ehime-u.ac.jp

Submitted on March 31, 2010; resubmitted on August 5, 2010; accepted on August 10, 2010

background: We aimed to establish an endometrial autograft model in rats that would allow for repetitive in vivo analyses of angio-
genesis. Dienogest (DNG) is an orally active progestin used for the treatment of endometriosis. We investigated whether DNG would affect
angiogenesis of the ectopic endometrium in our model.
methods: Mechanically isolated endometrial fragments were transplanted into dorsal skinfold chambers in rats. We analyzed the effect of
DNG on angiogenesis of the ectopic endometrium on Days 0, 2, 4, 7, 10 and 14 after transplantation using intravital fluorescence
microscopy.
results: The DNG-administered group showed significant suppression of angiogenesis of endometrial autografts, as indicated by the
reduced size of the microvascular network and decreased microvessel density compared with those of control animals. The newly
formed microvessels of the DNG-administered group showed consistently elevated diameters and centerline red blood cell velocity was
decreased. Immunohistochemistry revealed a significant reduction in the level of perivascular a-smooth muscle actin within endometrial
grafts of the DNG-administered group.
conclusions: DNG inhibited angiogenesis of the ectopic endometrium, with confirmed structural changes in the microvessels.
Key words: dienogest / endometriosis / angiogenesis / dorsal skinfold chamber / intravital fluorescence microscopy

fragments pass backward along the Fallopian tubes and are implanted
Introduction inside the peritoneal cavity (Sampson, 1927). According to this theory,
Endometriosis is a common disease affecting about 1 in 10 women of angiogenesis is a major prerequisite for the initiation and progression
reproductive age. It is characterized by the growth of endometrium at of the disease (Healy et al., 1998).
ectopic sites, such as the abdominal cavity and the ovary. Patients with Laschke et al. (2005) established an endometrial autograft model
endometriosis often suffer from dysmenorrhea, dyspareunia, dysuria that allowed for repetitive in vivo analyses of angiogenesis in hamsters.
and chronic abdominal or pelvic pain as well as infertility, resulting in Furthermore, they found that treatment with the selective
a severely limited quality of life (Strathy et al., 1982; The Practice Com- cyclooxygenase-2 inhibitor NS398 (Laschke et al., 2007) and rapamy-
mittee of the American Society for Reproductive Medicine, 2004). cin (Laschke et al., 2006a) induced the regression of ectopic endome-
Although endometriosis is one of the most intensively investigated trium by inhibiting vascularization and cell proliferation.
diseases in gynecology, many questions about its etiology and patho- Dienogest (DNG) is an orally active progestin used for the treat-
genesis are still unanswered. Today, Sampson’s implantation theory ment of endometriosis. We investigated whether DNG might affect
is the most widely accepted explanation of this common gynecological angiogenesis of ectopic endometrium in an animal model using confo-
disease. This postulates that, during menstruation, endometrial cal laser scanning microscopy.

& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
2852 Katayama et al.

unit (CSU-X1; Yokogawa Electric Corporation, Tokyo, Japan) and an elec-

Materials and Methods tron multiplier charge-coupled device (MC285SPD-LOBO; Texas Instru-
ments, Inc., Dallas, TX, USA). We observed the 488 nm excitation
Animals wave of the Krypton/Argon laser (643-YB-A01; CVI Melles Griot
Female Wistar rats (7 – 8 weeks) were purchased from CLEA Japan Corp., Albuquerque, NM, USA). The microscopic images were recorded
(Tokyo). The animals were housed (one/cage) at the animal experimental in TIFF format.
center of Ehime University, Graduate School of Medicine under controlled
conditions (238C, 55% humidity, 12 h light/dark cycle) with free access to
tap water and a diet of standard pellets (CLEA). Rats (8– 10 weeks) with a Microcirculatory analysis
body weight of 180– 220 g were used for the study. All experimental pro- We analyzed the images and determined the size of the transplanted
cedures were conducted in accordance with the Guide for Animal Exper- endometrial fragments (mm2), the microvessel density as determined by
imentation of Ehime University, and were approved by the animal ethical the length of red blood cell (RBC)-perfused microvessels per graft area
committee of Ehime University (approval number: 05-HE-32-2). The (in cm/cm2), the diameters of the microvessels (in mm) and the centerline
number of animals used during the experiment was kept to a minimum. RBC velocity (VRBC; in mm/s).

Preparation of the dorsal skinfold chamber Experimental protocol

The dorsal skinfold chamber preparation (Medium Dorsal KIT; APJ DNG (Mochida Pharmaceutical Co. Ltd, Tokyo, Japan; orally administered
Trading Co., Inc., Ventura, CA, USA) contains one layer of striated at 1 mg/kg body weight/day) was suspended in 0.5% carboxymethylcellu-

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muscle, subcutaneous tissue and skin, and allows for intravital microscopic lose solution. The experimental protocol consisted of studies on four
observation of the microcirculation in conscious animals for up to 3 weeks. groups. The first group of five rats was treated with DNG from Day 0
The chamber technique and its implantation have been described in detail when we transplanted the endometrial fragments (DNG from the Day 0
(Menger et al., 2002). Briefly, with rats under sodium pentobarbital group). The second group of five rats received DNG from Day 4 after
anesthesia (50 mg/kg body weight i.m.), two symmetrical titanium transplantation, at which time we were able to confirm angiogenesis
frames were implanted in the extended dorsal skinfold of the rats, so (DNG from the Day 4 group). The third group, as a control group (n ¼
that they sandwiched the double layer of skin. One layer of skin was 5), was treated with vehicle suspended in 0.5% carboxymethylcellulose
then completely removed in a circular area of 15 mm in diameter and solution (control group). Finally, the fourth group consisted of five bilater-
the remaining layers, consisting of striated skin muscle, subcutaneous ally ovariectomized rats treated with vehicle suspended in 0.5% carboxy-
tissue and skin were covered with a removable coverslip that was incor- methylcellulose solution (ovariectomized group).
porated into one of the titanium frames. After the preparation, the The macroscopic appearances of the skinfold chamber preparations and
animals were allowed to recover from anesthesia and surgery for at the implanted grafts were documented daily. Intravital fluorescence
least 48 h. microscopy of the microcirculation was performed on Days 0 (day of
transplantation), 2, 4, 7, 10 and 14 after transplantation of endometrial
Isolation and transplantation of endometrial fragments. Microvessel density was measured [the length of microvessels
fragments per graft area (cm/cm2)], and microvascular diameters and microhemody-
For isolating endometrial fragments, rats equipped with a dorsal skinfold namic parameters were determined by analyzing 10 microvessels per
chamber were anesthetized with pentobarbital sodium (50 mg/kg body region of interest. At the end of the in vivo experiments (i.e. Day 14
weight i.m.). After laparotomy, one uterine horn was removed aseptically after transplantation of endometrial fragments), the animals were eutha-
and placed in a 50-mm diameter Falcon plastic Petri dish filled with 378C nized with an overdose of pentobarbital and the dorsal skinfold
warm Dulbecco’s modified Eagle’s medium (with 10% fetal calf serum and chamber preparations were processed for histological and immunohisto-
0.1 mg/ml gentamicin) and the fluorescent dye bisbenzimide H33342 chemical studies.
(200 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) for staining endometrial
tissue. The isolated uterus horn was opened longitudinally and the endo- Histology and immunohistochemistry
metrium was dissected from the uterine muscle under a stereomicro- Specimens resected from the rats at Day 14 after transplantation were for-
scope. Then, the endometrium was transferred into 378C warm malin fixed, embedded in paraffin wax and 4-mm-thick sections were cut.
bisbenzimide H33342-free Dulbecco’s modified Eagle’s medium and For light microscopy, sections were stained with hematoxylin and eosin.
microdissected into endometrial fragments of similar size (0.5 mm2) For immunohistochemical detection of pericytes and endothelial cells of
(Laschke et al., 2005). newly formed blood vessels within the endometrial grafts, anti-a-smooth
For autologous transplantation of endometrial fragments, the cover glass muscle actin (a-SMA) (1A4, prediluted, mouse monoclonal; Nichirei,
of the dorsal skinfold chamber was removed and two or three fragments Tokyo, Japan) and anti-CD31 (sc-1506-R, 1:250, rabbit polyclonal; Santa
were placed on the striated muscle within each chamber (Laschke et al., Cruz Biochemicals, Santa Cruz, CA, USA) were used as primary anti-
2006b). Before transplantation, all animals were confirmed to be in bodies. Paraffin was removed from all sections with a series of xylene
pro-estrus by microscopic evaluation of vaginal smears. baths, and rehydration was performed with a series of graded alcohol sol-
utions. To retrieve antigens, the sections were heated in sodium citrate
Intravital fluorescence microscopy buffer (pH 6.0) for 40 min at 958C. After blocking the endogenous peroxi-
For in vivo microscopy, the animals were immobilized on a plastic stage and dase activity with 3% hydrogen peroxidase for 10 min, sections were incu-
the dorsal skinfold preparation was attached to the microscopic stage. bated with primary antibody for 1 h at room temperature. Saline was used
After i.v. injection of 0.15 ml of 5% fluorescein isothiocyanate instead of primary antibody as a negative control. Histofine Simple Stain
(FITC)-labeled dextran 150000 (Sigma-Aldrich) (contrast enhancement Max-PO (M or R; Nichirei) was used according to the manufacturer’s
by intravascular staining of blood plasma), intravital fluorescence instructions. Tissue samples were washed in phosphate-buffered saline
microscopy was performed using a fixed stage microscope (upright micro- and 3,3′ -diaminobenzidine (Nichirei) was used as a chromogen; Mayer’s
scope model FN1; Nikon, Tokyo, Japan) equipped with a confocal scanner hematoxylin was the counterstain.
Dienogest and anti-angiogenesis in endometriosis 2853

Quantification of pericytes and endothelial DNG from the Day 4 group (0.39 + 0.04 mm2), in the control group
cells of newly formed blood vessels within the (0.41 + 0.03 mm2) and in the ovariectomized group (0.41 + 0.05 mm2).
The development of new blood vessels could be observed in all
endometrial grafts experimental groups. Angiogenesis had commenced at Day 2 after
a-SMA can be used to identify pericytes in the endometrium (Rogers transplantation and was characterized by capillary sprouts growing
et al., 2000; Hull et al., 2003), but it also labels vascular smooth muscle
into the endometrial grafts. Over the following days, in the control
cells in large luminal blood vessels; these were excluded from the pericyte
group, these sprouts interconnected with each other and finally
count (Sugino et al., 2005). Thirty small luminal vessels showing crosscuts
(perpendicular to longitudinal axis) on each histological section were formed new microvascular networks with a glomerulus-like structure
selected, and a-SMA-positive cells were counted. The numbers of (Fig. 1A and D). In the control group, the whole area of the endo-
CD31 positive cells were also counted; CD31 is highly specific to endo- metrial grafts was almost completely vascularized by Day 7 (92%,
thelial cells in the endometrium (Jondet et al., 2006). 302 cm/cm2) after transplantation. However, the ovariectomized
group showed a significant restriction of angiogenesis when compared
Statistics with the control; the vascularized area of the graft from Day 7 to the
Differences in results between groups were examined by one-way ANOVA end of the 14-day observation period was reduced to 59 –73% (120–
with Fisher’s multiple comparison test; P , 0.05 was considered significant. 204 cm/cm2; Fig. 1B and D). Endometrial grafts of the DNG from the
Day 0 group exhibited a significantly lower density of microvessels
Results (157–231 cm/cm2; 69 –85%) at Days 7 and 14 when compared

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with the control (Fig. 1C). In the DNG from the Day 4 group,
After isolation and transplantation, the initial size of the endometrial grafts blood vessels increased until DNG administration began, but after
was similar in the DNG from the Day 0 group (0.41 + 0.04 mm2), in the

Figure 1 (A– C) Intravital fluorescence microscopy of the newly formed microvascular network of endometrial grafts at Day 14 after autologous
transplantation into the dorsal skinfold chamber of Wistar rats: the control group (A), the ovariectomized group (B) and the DNG from the Day 0
group (C). The graft in the control group exhibited a dense network of newly formed blood vessels with a glomerulus-like angioarchitecture (A).
However, substantial parts of the endometrial graft in the ovariectomized group still lacked vascularization (asterisks) (B). The graft in the DNG
from the Day 0 group showed a smaller vascularized area and a lower microvessel density, but larger microvessel diameter (C). The excitation
and emission wavelengths employed for 5% FITC-labeled dextran 150000 were 480 and 520 nm, respectively. Scale bars ¼ 150 mm. (D) Microvessel
density (in cm/cm2) of endometrial grafts after autologous transplantation into the dorsal skinfold chambers of Wistar rats: the control group (open
triangles; n ¼ 11 fragments), the ovariectomized group (closed triangles; n ¼ 11), the DNG from the Day 4 group (open circles; n ¼ 12 fragments) and
the DNG from the Day 0 group (closed circles; n ¼ 11 fragments), as assessed by intravital fluorescence microscopy and computer-assisted image
analysis. Values are the mean + SD. *P , 0.05 versus the control group at corresponding time points.
2854 Katayama et al.

DNG administration microvessel density did not show any further sig-
nificant increase, and the microscopic images were similar to those of
the DNG from the Day 0 group (Fig. 1D).
In the control group, the size of the endometrial grafts on Day 14
after transplantation showed only a minor reduction to 86% of the
size measured immediately after transplantation. In contrast, the
ovariectomized group showed a significant decrease of the graft
size at Day 14 – 66% of that measured at Day 0, and endometrial
grafts in the DNG from the Day 0 group were significantly reduced
to 78% of the size measured immediately after transplantation.
Graft size in the DNG from the Day 4 group showed a reduction
to 82% of the size measured immediately after transplantation
(Fig. 2).
In endometrial grafts of both the control group and the ovari-
ectomized group, sequential analysis of the diameters of the newly
Figure 2 Size of endometrial grafts (as percentages of the initial formed microvessels revealed a significant decrease from 19 to
size) at Day 14 after autologous transplantation into dorsal skinfold 20 mm on Day 2 to 10.5 –11.7 mm on Day 14 after transplantation

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chambers of Wistar rats: the control group (n ¼ 11 fragments); the (Fig. 3A, B and D). In contrast, the capillaries of the DNG from the
ovariectomized group (n ¼ 11 fragments); the DNG from the Day Day 0 group presented with consistently elevated diameters of
0 group (n ¼ 11 fragments) and the DNG from the Day 4 group
16– 20.9 mm throughout the observation period. In the DNG from
(n ¼ 12 fragments). Sizes were assessed by intravital fluorescence
the Day 4 group, vessel diameters did not decrease after DNG admin-
microscopy and computer-assisted image analysis. Values are the
mean + SD. *P , 0.05 versus the control group. istration and on Day 14 the microscopic images were similar to those
of the DNG from the Day 0 group (Fig. 3C and D).

Figure 3 (A – C) Intravital fluorescence microscopy of endometrial grafts at Day 14 after autologous transplantation into the dorsal skinfold chamber of
Wistar rats: the control group (A), the ovariectomized group (B) and the DNG from the Day 0 group (C). Note that DNG treatment suppressed vessel
maturation as indicated by large vessel calibers (C). Blue-light epi-illumination with contrast enhancement for 5% FITC-labeled dextran 150000. Scale
bars ¼ 100 mm. (D) Microvessel diameters (mm) of endometrial grafts after autologous transplantation into the dorsal skinfold chambers of Wistar
rats: the control group (open triangles; n ¼ 11 fragments), the ovariectomized group (closed triangles; n ¼ 11), the DNG from the Day 4 group (open
circles; n ¼ 12 fragments) and the DNG from the Day 0 group (closed circles; n ¼ 11 fragments). Diameters were assessed by intravital fluorescence
microscopy and computer-assisted image analysis. Values are the mean + SD. *P , 0.05 versus the control group at corresponding time points.
Dienogest and anti-angiogenesis in endometriosis 2855

Centerline VRBC increased progressively in endometrial grafts of epithelial cells were not identified in the ovariectomized group
the control group throughout the observation period, up to (Fig. 5B). Immunohistochemistry experiments revealed significant
189 mm/s. Endometrial fragments in the ovariectomized group pre- decreases in the number of a-SMA-positive pericytes in the DNG
sented with significantly lower VRBC values of 126 mm/s at Day 7 from the Day 0 group and the ovariectomized group when compared
after transplantation, and a subsequent increase in the VRBC values with the control (Fig. 6A– C and G). There were no differences in
at Day 10 (179 mm/s). In contrast, endometrial fragments in the CD31 immunostaining for vascular endothelium in the control
DNG from the Day 0 group presented with significantly lower group, the ovariectomized group, and the DNG from the Day 0
VRBC values of 97–113 mm/s at Days 7 and 14. The VRBC did group (Fig. 6D–F and H). The microscopic images of the DNG
not increase after DNG administration in the DNG from the Day 4 from the Day 4 group (data not shown) were similar to those of
group (Fig. 4). the DNG from the Day 0 group.
Histology of the dorsal skinfold preparations in a Wistar rat from
the control group (Fig. 5A) and the DNG from the Day 0 group
(Fig. 5C) at Day 14 after transplantation revealed typical endometriotic Discussion
lesions consisting of cyst-like dilated endometrial glands. Glandular
Current guidelines for the treatment of endometriosis recommend
either surgical or hormonal therapies that suppress ovarian function
to reduce the serum estradiol concentration and thus shrink the endo-

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metriotic lesions (Olive and Pritts, 2001). According to the European
Society of Human Reproduction and Embryology guidelines,
gonadotrophin-releasing hormone (GnRH) agonists, danazol, gestri-
none, medroxyprogesterone acetate (MPA) and combined oral con-
traceptives are equally effective but their side-effects and cost
profiles differ (Kennedy et al., 2005). GnRH agonists are associated
with symptoms of estrogen deprivation (including hot flushes, vaginal
dryness, headache, decreased libido) and bone mineral loss that
limits treatment to 6 months in the absence of add-back therapy
which adds further cost to the treatment (Winkel and Scialli, 2001;
Mounsey et al., 2006). Danazol is characterized by adverse changes
in lipid metabolism and androgenic adverse effects, including weight
gain, edema, acne, hirsutism and oily skin, which lead to low compli-
Figure 4 Centerline RBC velocity (VRBC, mm/s) of endometrial ance with therapy (Winkel and Scialli, 2001; Selak et al., 2007). Proges-
grafts after autologous transplantation into the dorsal skinfold tins offer long-term efficacy in treating endometriosis, but a number of
chambers of Wistar rats: the control group (open triangles; n ¼ 11 these agents are associated with weight gain and androgenic effects
fragments), the ovariectomized group (closed triangles; n ¼ 11 frag-
when administered at the high doses required for efficacy (Mahutte
ments), the DNG from theDay 4 group (open circles; n ¼ 12 frag-
and Arici, 2003; Vercellini et al., 2003). Long-term use of depot
ments) and the DNG from the Day 0 group (closed circles; n ¼ 11
fragments). VRBC values were assessed by intravital fluorescence
MPA is shown to impact adversely on bone mineral density, while
microscopy and computer-assisted image analysis. Values are the the delay in resumption of ovulation that may follow discontinuation
mean + SD. *P , 0.05 versus the control group at corresponding of this therapy is a contraindication to use in women wishing to con-
time points. ceive in the near future (Vercellini et al., 2003; Crosignani et al., 2006;
Schlaff et al., 2006). Combined oral contraceptives, although widely

Figure 5 Histological appearance of endometrial grafts at Day 14 after transplantation. Endometrial grafts were planted onto striated muscle within
dorsal skinfold chambers fixed on Wistar rats: the control group (A), the ovariectomized group (B) and the DNG from the Day 0 group (C). In the
control group (A) and DNG from the Day 0 group (C), the grafts were characterized by cyst-like dilated endometrial glands with an intact glandular
epithelium surrounded by a vascularized endometrial stroma. In the ovariectomized group (B), endometrial glands were not detected. Stain: HE. Scale
bars ¼ 300 mm.
2856 Katayama et al.

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Figure 6 (A – F) Immunohistochemical detection of a-SMA (A– C) and CD31 (D –F) in endometriotic lesions of Wistar rats: the control group
(A and D), the ovariectomized group (B and E) and the DNG from the Day 0 group (C and F) at Day 14 after transplantation of endometrial fragments
into the dorsal skinfold chamber. (G) The a-SMA-positive cell count/vessel. The number of a-SMA-positive pericytes decreased significantly in the
DNG from the Day 0 group and the ovariectomized group compared with the control. (H) The CD31 positive cell count/vessel. CD31 is a vascular
endothelial marker. There were no significant differences between the numbers of CD31 positive cells in the control group, the ovariectomized group
and the DNG from the Day 0 group. Scale bars ¼ 10 mm.

used to manage symptoms of endometriosis, are not approved for this et al. (2006) indicated that progestins induce microvascular changes,
indication in most countries and lack a solid body of supportive clinical consisting of a reduction in the number and a concomitant dilatation
trial evidence (Davis et al., 2007). Therefore, there is a need for new of the microvessels in endometrium and endometriotic lesions. In
therapeutic drugs that are well tolerated and have efficacy against accordance with these data, DNG exposure caused a reduction in
endometriosis through long-term therapeutic use. the number of vessels and vascular dilatation in the endometrial
DNG, a 19-nortestosterone derivative lacking androgenicity, is a grafts in our study.
safe progestin that is highly selective for progesterone receptors Perivascular a-SMA is significantly reduced around the
(Foster and Wilde, 1998). Because DNG exhibits antiovulatory activity progestin-exposed eutopic endometrial microvasculature (Rogers
(Moore, 1999), antiproliferative action on endometrial cells (Okada et al., 2000). In our experiments, the level of perivascular a-SMA
et al., 2001) and inhibits cytokine production by endometriotic cells within endometrial grafts of the DNG-administered group was signifi-
(Horie et al., 2005), it is expected to have a therapeutic effect cantly reduced as compared with the control. This was consistent with
against endometriosis (Strowitzki et al., 2010). findings that endometrial perivascular pericytes decrease in number
In this study, the administration of DNG to two groups of animals following the administration of progestins.
was found to affect angiogenesis of endometrial grafts. The first group Vascular endothelial growth factor (VEGF) is associated with angio-
began DNG treatment from Day 0, when we transplanted the endo- genesis, and is important for the establishment and maintenance of
metrial fragments. This allowed us to analyze the effects of DNG while ectopic endometrial lesions (Hull et al., 2003). Elevated levels of angio-
angiogenesis was occurring. The second group received DNG from genic growth factors, particularly VEGF, have been demonstrated in
Day 4 after transplantation. This allowed us to observe the effects the peritoneal fluid, and eutopic and ectopic endometrial tissues
of DNG once angiogenesis had occurred. Our results indicated that from patients with endometriosis (Laschke and Menger, 2007).
DNG affected the vasculature even after the onset of angiogenesis. In our study, the ovariectomized group showed delayed graft vascu-
Previous reports have stated that the eutopic endometrial micro- larization with a significantly reduced vascularized area and microvessel
vascular appearance is altered to superficial vascular dilatation and density compared with the control. Estrogen has been shown to
neovascular formation by progestin exposure (Hickey and Fraser, promote the expression of VEGF and matrix metalloproteinases
2000). It seems that administration of DNG was equally effective (MMPs) in association with angiogenesis (Cullinan-Bove and Koos,
against the ectopic endometrium in our study. In addition, Jondet 1993; Huang et al., 1998; Osteen et al., 2002). Therefore, the
Dienogest and anti-angiogenesis in endometriosis 2857

altered vascularization of the ovariectomized group was probably a B inactivation in endometriotic stromal cells. Fertil Steril 2005;
response to a lack of ovarian estrogen. It is known that progestins 83:1530– 1535.
have antiestrognic effects (Levy et al., 1980; Lessey et al., 1988). Huang JC, Liu DY, Dawood MY. The expression of vascular endothelial
Therefore, in this study, DNG may have inhibited angiogenesis via growth factor isoforms in cultured human endometrial stromal cells and
its regulation by 17beta-oestradiol. Mol Hum Reprod 1998;4:603 – 607.
anti-estrogenic effects. In addition, it is reported that DNG has
Hull ML, Charnock-Jones DS, Chan CL, Bruner-Tran KL, Osteen KG,
direct effects on endometrial cells, such as reductions in cytokine pro-
Tom BD, Fan TP, Smith SK. Antiangiogenic agents are effective
duction and cell proliferation (Horie et al., 2005; Fu et al., 2008). Thus,
inhibitors of endometriosis. J Clin Endocrinol Metab 2003;88:2889– 2899.
in addition to its anti-estrogenic effects, DNG may also directly inhibit Jondet M, Vacher-Lavenu MC, Chapron C. Image analysis measurements
angiogenesis in ectopic endometrial lesions. of the microvascularisation in endometrium, superficial and deep
To elucidate the role of angiogenic factors, Laschke et al. (2006b) endometriotic tissues. Angiogenesis 2006;9:177– 182.
investigated whether blocking the actions of VEGF, fibroblast growth Kennedy S, Bergqvist A, Chapron C, D’Hooghe T, Dunselman G, Greb R,
factor (FGF) and platelet-derived growth factor (PDGF) affected angio- Hummelshoj L, Prentice A, Saridogan E. ESHRE guideline for the diagnosis
genesis of ectopic endometrium in an in vivo model similar to ours. and treatment of endometriosis. Hum Reprod 2005;20:2698–2704.
Combined inhibition of VEGF, FGF and PDGF significantly suppressed Laschke MW, Menger MD. In vitro and in vivo approaches to study
angiogenesis of endometrial grafts, as indicated by a reduced size of angiogenesis in the pathophysiology and therapy of endometriosis.
Hum Reprod Update 2007;13:331 – 342.
the microvascular network and decreased microvessel density. Neo-
Laschke MW, Elitzsch A, Vollmar B, Menger MD. In vivo analysis of
vascularity in the combined inhibitor-treated endometrial fragments
angiogenesis in endometriosis-like lesions by intravital fluorescence

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showed consistently elevated vessel diameters and significantly lower
microscopy. Fertil Steril 2005;84(Suppl. 2):1199 – 1209.
VRBC values. Furthermore, immunohistochemistry revealed a signifi- Laschke MW, Elitzsch A, Scheuer C, Holstein JH, Vollmar B, Menger MD.
cant reduction in the level of pericytes, as indicated by a decrease in Rapamycin induces regression of endometriotic lesions by inhibiting
perivascular desmin, within endometrial grafts. These results are con- neovascularization and cell proliferation. Br J Pharmacol 2006a;
sistent with those of our study. Progestins have been shown to sup- 149:137– 144.
press angiogenic factors including MMPs, VEGF-A, cysteine-rich Laschke MW, Elitzsch A, Vollmar B, Vajkoczy P, Menger MD. Combined
angiogenic inducer (CYR61) and basic FGF in grafted endometrial inhibition of vascular endothelial growth factor (VEGF), fibroblast
tissue (Monckedieck et al., 2009). We found here that DNG adminis- growth factor and platelet-derived growth factor, but not inhibition of
tration regulates angiogenesis in ectopic endometrial lesions, and we VEGF alone, effectively suppresses angiogenesis and vessel maturation
in endometriotic lesions. Hum Reprod 2006b;21:262 – 268.
suggest that this pharmacological action of DNG might contribute to
Laschke MW, Elitzsch A, Scheuer C, Vollmar B, Menger MD. Selective
an effective treatment for endometriosis.
cyclo-oxygenase-2 inhibition induces regression of autologous
endometrial grafts by down-regulation of vascular endothelial growth
factor-mediated angiogenesis and stimulation of caspase-3-dependent
Funding apoptosis. Fertil Steril 2007;87:163– 171.
This study was supported by grant No. 19591933 from the Ministry of Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS Jr.
Immunohistochemical analysis of human uterine estrogen and
Education, Science, Sports and Culture of Japan.
progesterone receptors throughout the menstrual cycle. J Clin
Endocrinol Metab 1988;67:334 – 340.
Levy C, Robel P, Gautray JP, De Brux J, Verma U, Descomps B, Baulieu EE.
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