0021-972X/99/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 1999 by The Endocrine Society
Vol. 84, No. 12
Printed in U.S.A.
Hormone Replacement Therapy with Estrogen or
Estrogen plus Medroxyprogesterone Acetate Is
Associated with Increased Epithelial Proliferation
in the Normal Postmenopausal Breast*
Departments of Physiology (L.J.H., A.M.R., S.Z.H.), Surgery (J.R.O., C.A.S.), and Epidemiology
(D.R.P.), Michigan State University, East Lansing, Michigan 48824
ABSTRACT
The relative effects of postmenopausal hormone replacement
therapy (HRT) with estrogen alone vs. estrogen1progestin on
breast cell proliferation and on breast cancer risk are controversial.
A cross-sectional observational study was carried out to examine
the proliferative effects of HRT with estrogen or estrogen plus the
progestin, medroxyprogesterone acetate, in breast tissue of postmenopausal women. Benign breast biopsies from 86 postmenopausal women were analyzed with antiproliferating cell nuclear
antigen (anti-PCNA) and Ki67 antibodies to measure relative levels of cell proliferation. Epithelial density and estrogen and progesterone receptor status were also determined. The women were
categorized either as users of: 1) estrogen (E) alone; 2) estrogen1
medroxyprogesterone acetate (E1P); or 3) no HRT. Compared with
no HRT, the breast epithelium of women who had received either
E1P or E alone had significantly higher PCNA proliferation indices, and treatment with E1P had a significantly higher index
(PCNA and Ki67) than treatment with E alone. Breast epithelial
density was significantly greater in postmenopausal women
treated with E and E1P, compared with no HRT. Thus, the present
study shows that postmenopausal HRT with E1P was associated
with greater breast epithelial cell proliferation and breast epithelial cell density than E alone or no HRT. Furthermore, with E1P,
breast proliferation was localized to the terminal duct-lobular unit
of the breast, which is the site of development of most breast
cancers. Further studies are needed to assess the possible association between the mitogenic activity of progestins and breast cancer risk. (J Clin Endocrinol Metab 84: 4559 – 4565, 1999)
P
a greater extent than estrogen alone (23, 24). Similarly, in
adult, ovariectomized cynomolgus macaques, estrogen1
progestin induces greater breast epithelial cell proliferation
than estrogen alone (25, 26). In contrast, in human/mouse
xenograft studies, estrogen1progesterone increased proliferation but no more than estrogen alone (27, 28). The doses
of estrogen and progesterone used in these xenograft studies,
however, have been questioned because of the absence of sex
steroid binding globulin in the serum of mice vs. its presence
in humans (29).
To investigate the effects of HRT more directly, normal
breast tissue from postmenopausal women who had taken
estrogen, estrogen1progestin, or no HRT was analyzed for
cell proliferation, epithelial cell density, and steroid receptor
levels. We found that treatment with estrogen1progestin or
estrogen alone was associated with significantly higher mitogenic activity in the terminal ductal lobular unit (TDLU) of
the breast and greater breast epithelial density than women
not receiving HRT.
OSTMENOPAUSAL hormone replacement therapy
(HRT) with estrogen alone or estrogen1progestin
alleviates menopausal symptoms and reduces the risk of
osteoporosis, coronary artery disease, and possibly Alzheimer’s disease (1–5). However, HRT may increase breast
cancer risk (6 –10). Because of the prevalence of HRT, understanding the effects of estrogen vs. estrogen1progestin
on the postmenopausal breast is essential. This, in turn,
can help us understand how these hormones may play a
role in the genesis of cancer.
Whether estrogen, progestin, or both enhance proliferation of normal breast epithelial tissue is controversial. Progestins decrease endometrial cell proliferation in the uterus
(11–13), but their effect on the breast is less clear. In premenopausal women, proliferation of breast epithelial tissue
is greatest during the luteal phase of the menstrual cycle,
when progesterone levels are maximal (14 –22). In animal
studies, progestins increase mammary gland epithelial cell
proliferation. In the mouse, progesterone, when combined
with estrogen, is mitogenic in the adult mammary gland to
Received July 6, 1999. Revision received August 16, 1999. Accepted
August 26, 1999.
Address correspondence and requests for reprints to: Sandra Z.
Haslam, Ph.D., Department of Physiology, 108 Giltner Hall, Michigan
State University, East Lansing, Michigan 48824. E-mail: shaslam@
pilot.msu.edu.
* This work was supported by National Institute of Aging Grant
R01-AG13059 (to S.Z.H.).
Materials and Methods
Subjects
A cross-sectional, observational study was carried out to study breast
tissue from cycling, premenopausal (n 5 56) and postmenopausal (n 5
86) women undergoing surgical breast biopsy at Lansing, Michigan area
hospitals. Biopsies were carried out to diagnose suspicious palpable
lesions upon physical exam or suspicious mammographic densities.
Profiles of the study populations of premenopausal and postmenopausal women are summarized in Table 1. Postmenopausal women
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LORNE J. HOFSETH, AHMED M. RAAFAT, JANET R. OSUCH,
DOROTHY R. PATHAK, CAROL A. SLOMSKI, AND SANDRA Z. HASLAM
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HOFSETH ET AL.
JCE & M • 1999
Vol 84 • No 12
TABLE 1. Characteristics of premenopausal and postmenopausal subjects
Characteristic
Number of subjects
Mean age (yr) (range)
Body mass index (kg/m2)
Time on HRT (yr) (range)
Postmenopausal
Luteal
No HRT
E
E1P
n 5 28
42.2 6 7.0a
(22–50)
27.8 6 5.6
N/Ab
n 5 28
41.6 6 7.8
(24 –52)
24.6 6 6.9
N/A
n 5 29
65.2 6 9.2
(45– 87)
30.1 6 8.3
N/A
n 5 32
60.8 6 10.3
(41– 82)
28.6 6 6.3
14.2 6 10.5
(0.25–39)
n 5 25
59.5 6 7.8
(47– 87)
28.1 6 7.7
6.7 64.1c
(2–20)
N/A
N/A
N/A
N/A
86.2
13.8
37.5
62.5d
88.0
12.0
14.3
78.6
7.1
10.8
82.1
7.1
24.1
70.0
5.9
12.5
78.1
9.4
16.0
68.0
16.0
85.7e
14.3
82.1f
17.9
31.0
69.0
46.9
53.1
72.0g
28.0
14.3
85.7
2.8 (1– 6)
2.1 (1– 4)
26.9 (19 – 40)
35.7
64.3
2.5 (1– 6)
1.8 (0 – 4)
25.3 (17–33)
10.3
89.7
3.8 (1–9)
2.9 (0 – 6)
22.8 (18 – 47)
15.6
84.4
3.3 (1– 6)
3.0 (1– 6)
22.4 (16 –33)
0.0
100.0
3.1 (2–7)
3.0 (2– 6)
22.0 (17–29)
a
Means 6 SD.
N/A, Not applicable.
c
Significant difference from other postmenopausal groups (P 5 0.0001).
d
Significant difference from other postmenopausal groups (P 5 0.001).
e
Current users, n 5 3.
f
Current users, n 5 3.
g
Significant difference from other postmenopausal groups (P 5 0.009).
b
were defined as those who had experienced 12 consecutive months of
amenorrhea, had a bilateral ovariectomy at least 1 yr before biopsy, or
were 55 yr of age or older. Subjects were placed into one of three
categories: 1) no HRT, defined as not having taken hormones for 1 yr
before surgery; 2) estrogen alone; or 3) estrogen1progestin. HRT subjects were defined as those taking hormones for at least 3 months continuously up to the day of surgery. Premenopausal subjects were divided into two categories depending on the phase of the menstrual cycle:
1) follicular, days 1–14; or 2) luteal, days 15–28.
All hormones were taken on a continuous, daily basis. Subjects had
taken E in one of three forms: conjugated equine estrogens (CEEs; dose,
0.3–2.5 mg; n 5 27); micronized estradiol (dose, 0.5–1 mg; n 5 4); or
ethinyl estradiol (dose, 0.05 mg; n 5 1). The progestin taken was medroxyprogesterone acetate (MPA; dose, 2.5–5 mg; n 5 25), which was
taken in combination with CEE (n 5 19) or micronized estradiol (n 5 6).
Herein, all types of estrogens are referred to as E, and MPA is referred
to as P.
Tissue samples
The protocol for tissue collection was approved by the University
Committee on Research Involving Human Subjects and Institutional
Research Review Boards of the participating hospitals; written informed consent was obtained from each patient. Biopsies collected for
study were kept on ice, then snap-frozen or fixed in 3.7% buffered
formalin within 2 h of surgery for paraffin embedding. Only benign
biopsies were used in the study; those diagnosed with atypical hyperplasia or cancer were excluded. Adjacent serial sections (5 mm)
were mounted onto 3-aminopropyl-triethoxysilane (Sigma, St. Louis,
MO)-coated cover slips, and assayed in parallel, by immunohistochemistry, for proliferating cell nuclear antigen (PCNA, clone PC-10;
DAKO Corp., Carpinteria, CA) and Ki67 (MIB-1, Immunotech, Marseille, France). Estrogen receptor (ER) and progesterone receptor (PR;
Zymed Laboratories, Inc., San Francisco, CA) were analyzed independently on nonserial sections. The number of tissue samples assayed for proliferation indices (PCNA and Ki67), epithelial density,
or receptor status varied because, in some cases, there was not enough
tissue for all assays, or some tissue sections only contained lobules
or ducts, and not both.
Immunohistochemistry
Adjacent sections were incubated in PCNA antibody (1:100 dilution),
Ki67 antibody (1:50 dilution), or normal serum (negative control). Signals were amplified with a biotinylated rabbit antimouse IgG (1:200
dilution, DAKO Corp.) followed by horseradish peroxidase-conjugated
avidin-biotin complex (Vector Laboratories, Inc., Burlingame, CA); the
chromogen was diaminobenzidine tetrahydrochloride (Pierce Chemical
Co. Biotec Co., Rockford, IL). Ki67 antibody immunoreactivity required
antigen retrieval, which was achieved by boiling the sections for 10 min
in 10 mm citrate buffer (pH 6.0) before antibody treatment. ER and PR
were detected as described above for Ki67, including antigen retrieval,
using anti-ER (1:2 dilution) or anti-PR (1:1 dilution) antibodies according
to the manufacturer’s recommendations. Cells with clearly identifiable
nuclear staining were considered to be positive for ER or PR; no cytoplasmic staining was observed.
Quantification of immunohistochemical staining
Using a double-blind approach, PCNA, Ki67, ER, and PR were
quantified in specific breast structures: inter- and intralobular ducts
(ducts) or terminal ductules and lobules (terminal duct-lobular unit,
TDLU). Only normal areas of tissues were scored. Areas with fibrocystic changes, hyperplasia, microcalcifications, and metaplasias
were not analyzed. Frozen tissue sections were analyzed for PCNA,
ER, or PR only when paraffin sections were not available; quantitative
comparison of results, obtained with frozen and paraffin sections,
revealed no significant differences. Quantification was facilitated by
use of a computer-interfaced morphometric digitizing system (Bioquant II, R and M Biometrics, Nashville, TN), as described previously
(23). The mean unit area of an epithelial cell was determined by
measuring cell areas for over 10,000 cells (mean 6 sd, 4.2 6 0.25 unit
area/cell). No significant difference in cell sizes was observed for the
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Menopausal status (%)
Natural
Surgically induced
Family history of breast cancer (1° relative) (%)
Yes
No
Unknown
Oral contraceptives, ever (%)
Yes
No
Reproductive history
Nulliparous (%)
Parous (%)
Mean no. pregnancies (range)
Mean no. deliveries (range)
Age at 1st delivery (yr) (range)
Premenopausal
Follicular
HRT AND THE NORMAL BREAST
various study groups. For each section, 1000 – 4000 cells were scored.
It was determined, according to the method of Sadi and Barrack (30),
that a minimum of 1000 cells was required to obtain a reliable estimate
of the Ki67 proliferation index. All tissue samples were analyzed
within 3 yr of collection. Interindividual and intraindividual differences in scoring were monitored for each assay and were calculated
to be within 5%.
Epithelial density
Statistical analysis
Results are expressed as mean 6 sem (except for Table 1, where
values are expressed as mean 6 sd). Differences between no-HRT,
E-alone, and E1P groups were examined using one-way ANOVA and
analysis of covariance, adjusting for age. Multiple comparisons between groups were carried out using the Newman-Keuls test and the
pairwise comparisons of least-square means, respectively. Differences between premenopausal follicular and luteal groups and between ducts and TDLU were examined using the Student’s t test. The
above analyses were repeated using nonparametric equivalents, the
Kruskal-Wallis test, and the Mann-Whitney test; and the conclusions
remained the same. Multiple regression was used to assess the effect
of length of HRT use (up to 20 yr) on cell proliferation. All statistical
analyses were carried out using the SAS program, with a 5 0.05 as
the statistical significance level.
adjusted by including age as one of the variables in our
regression models. This was done for both pre- and postmenopausal women. The results were the same when the
means were age adjusted.
The PCNA index (Fig. 1A) was significantly higher in both
the E-alone and E1P HRT groups than in the no-HRT group.
For the E-alone group, the PCNA index was the same for the
TDLU and ducts. In contrast, in the E1P group, the PCNA
index was significantly higher in the TDLU than in ducts. The
PCNA index in the TDLU of the E1P group was significantly
greater than in the TDLU of E-alone group. Results with Ki67
(Fig. 1B) were comparable to the PCNA results, except that
the difference between the E-alone vs. no-HRT groups did
not reach statistical significance.
For premenopausal women, in agreement with past studies (14 –22), our results with both anti-PCNA and Ki67 antibodies (Fig. 1) showed that epithelial cell proliferation in the
TDLU was greater in the luteal phase of the menstrual cycle
as compared with the follicular phase.
Regression analysis was carried out to assess the influence
of length of time of HRT use (E and E1P groups) or length
of time postmenopause (no-HRT group) on cell proliferation
Results
Population profiles
Table 1 compares the three postmenopausal and the two
premenopausal groups analyzed in this study. Some differences were noted among the postmenopausal groups. For the
E-alone HRT group, 62.5% had experienced surgical menopause, and their mean time on HRT was twice as long as that
of women taking E1P (14.2 vs. 6.7yr). As expected, the majority of women receiving no HRT or E1P experienced natural menopause. There was a higher percentage of women
in the E1P group (72%) who had taken oral contraceptives,
as compared with the no-HRT (31.0%), and E-alone (46.9%)
groups.
Effect of HRT on cell proliferation
Anti-PCNA and Ki67 antibodies detect two different
proteins present in cycling cells and have both been widely
used to measure relative levels of proliferative activity in
human tissues (31). Both antibodies were used herein to
obtain two measures of proliferation that could be compared to each other and to published results. Although the
percentage of epithelial cells detected by the anti-PCNA
antibody was significantly greater than that detected with
the anti-Ki67 antibody, relative differences between the
postmenopausal groups were comparable, regardless of
the antibody used. Because breast cell proliferation has
been shown to be negatively related to age in premenopausal women (19), all proliferation data were age
FIG. 1. Epithelial proliferation indices in normal breast tissue of
postmenopausal women and cycling premenopausal women. The
number under each bar represents the number of individuals for
whom ducts or TDLU could be analyzed. *, P 5 0.002– 0.0001 that the
percentages of PCNA- (A) and Ki67- (B) positive cells in the TDLU of
the E1P group were significantly greater than in TDLU of no-HRT
or E-alone groups; †, P 5 0.007– 0.002 that the percentages of PCNApositive cells in the TDLU or ducts of the E group or ducts of the E1P
group were significantly greater than in the TDLU or ducts of the
no-HRT group; ‡, P , 0.05 that the percentages of PCNA- and Ki67positive cells in the TDLU of the luteal-phase group were significantly
greater than in the TDLU of the follicular-phase group; §, P , 0.05
that the percentages of PCNA- and Ki67-positive cells were greater
in TDLU than in the ducts of the same group.
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The same sections assessed for proliferation and steroid receptors
were analyzed for epithelial density, using a video camera (Sony, CCD
color camera) attached to a light microscope (Nikon Eclipse E400; Mager
Scientific Inc., Dexter, MI). After capture of the image, using an NIH
ImagePC program (Scion Corporation, Frederick, MD), the total area of
the tissue and of the epithelium was quantified by digital tracing at 2003
magnification. Only the area occupied by epithelial cells was measured;
area occupied by lumina or stromal components was excluded. The area
of the epithelium was then expressed as a percentage of the total area
of the section.
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HOFSETH ET AL.
JCE & M • 1999
Vol 84 • No 12
the acini had larger lumina (Fig. 4C). This latter morphology
is similar to that observed during the luteal phase in cycling
women (32).
FIG. 2. Effects of length of time on HRT on proliferation in TDLU of
postmenopausal women. Labeling indices determined by PCNA (A)
and Ki67 (B) in TDLU were regressed on years of HRT or years
postmenopause. Each straight line represents the best-fit line for each
group. P 5 0.03 that an increase in Ki67 labeling index was observed
with each yr of E1P treatment.
(Fig. 2). For the PCNA index, an increase in slope was observed with each yr of HRT use for both E and E1P; however
this did not reach statistical significance. The Ki67 index
showed no significant increase in proliferation with time on
E alone. However a significant increase (P 5 0.03) was observed with time on E1P.
Effects of HRT on breast epithelial density and morphology
The number of epithelial cells present in breast tissue at
any given time is determined by proliferative cell addition
and cell loss caused by cell death. Because anti-PCNA and
Ki67 antibodies provide relative measures of proliferative
activity, it was of interest to determine the net effects of HRT
on breast tissue. The epithelial density was significantly
higher in women receiving E alone or E1P, compared with
the no-HRT group (Fig. 3). Epithelial density of the E1P
group was also significantly higher than the E-alone group.
Because large variations in tissue section size could skew
results of epithelial density calculations, the mean area
(mm2) per tissue section for each group was determined and
found to be similar (mean 6 sem: no-HRT 5 41.0 6 4.0, E 5
35.2 6 4.4, E1P 5 36.0 6 3.2).
Histological analysis of breast tissues from the three
groups also revealed differences in the appearance of the
epithelium (Fig. 4). In the no-HRT and E-treated groups,
lobules were small, compact, and similar in appearance.
There were more lobules per unit area in the E-treated group
than in the no-HRT group (Fig. 4, A and B). In contrast, in the
E1P group, lobules were much larger and less compact, and
Steroid receptor status
The induction of epithelial progesterone receptors (PRs)
by estrogen is believed to be the basis for the synergistic effect
of estrogen1progestin on epithelial cell proliferation in the
rodent (23, 24) and primate mammary gland (25, 26). The
current model of progestin-induced proliferation in the
mammary gland, based on animal studies, is that estrogen
increases PR levels in mammary epithelial cells, and the
subsequent binding of progestins to the PR leads to a proliferative response. Progestin binding to PR also results in the
down-regulation of PR levels (33). Therefore, we analyzed
the effect of HRT on PR content in postmenopausal human
breast tissue. Cells were identified as PR positive, based upon
the presence of nuclear staining; no cytoplasmic staining was
observed. The percentage of PR-positive cells was about
3-fold higher in ducts and TDLU of the E-alone group, as
compared with the no-HRT group (Fig. 5A). PR-positive cells
were found to be equally distributed in ducts and TDLU. No
PR-positive cells were observed in the mammary stroma. The
higher number of PR-positive cells in the E-alone HRT group
is consistent with the effect of E to up-regulate PR, and it
indicates that the postmenopausal breast is responsive to this
action of E. Treatment with E1P resulted in lower PR levels
than in the E-alone group, consistent with PR down-regulation by P as a consequence of P action (25, 34, 35).
ER-positive cells were identified by presence of nuclear
staining. The percentage of ER-positive cells in ducts and
TDLU ranged from 4.0 6 1.4 to 10.0 6 2.3 (mean 6 sem). No
significant differences were observed among the three postmenopausal groups (Fig. 5B).
Discussion
Our results demonstrate, for the first time, that: 1) combination HRT with E1P is associated with higher levels of
epithelial proliferation in the TDLU of the postmenopausal
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FIG. 3 Effects of HRT on breast epithelial density in postmenopausal
women. The number under each bar represents the number of individuals for whom epithelial density was determined. *, P 5 0.001–
0.01 that the percentages of epithelial area in the E-alone or E1P
groups were significantly greater than that of the no-HRT group. †,
P 5 0.02 that the percentage of epithelial area in the E1P group was
significantly greater than that of the E-alone group.
HRT AND THE NORMAL BREAST
4563
FIG. 4. Photomicrographs of breast tissue from postmenopausal
women; no HRT (A), E alone (B), E1P (C). TDLUs and ducts are
indicated by arrows. Magnification, 340.
breast, based on PCNA and Ki67 indices; and 2) E alone and
E1P HRT are positively associated with greater epithelial
density. In addition, the morphology of the breast epithelium
in the E1P group is consistent with the morphology seen in
mammary tissue during the luteal phase in cycling women.
The amount of epithelial proliferation was also similar to that
of luteal-phase premenopausal women. These results also
indicate that the mitogenic activity associated with progestins is localized to the TDLU of the human breast.
E-alone HRT was associated with a smaller effect than
E1P on proliferation index and epithelial density. Although
the proliferation indices for the E-alone group, obtained with
PCNA and Ki67, were both higher than the no-HRT group,
this difference was statistically significant only for the PCNA
index. However, epithelial density, another indicator of proliferative activity in the breast, was also significantly greater
in the E-alone group. It is likely that the low percentage of
Ki67-positive cells, combined with variability among samples, contributed to the fact that statistical significance was
not achieved with the Ki67 antibody in the E-alone HRT
group. It has been reported in the literature that the differences between the raw values obtained with PCNA vs. Ki67
antibodies could be attributable to the following reasons.
First, PCNA has a longer half-life than Ki67 and, therefore,
may be detectable in cells that have recently left the cell cycle
(36). Second, PCNA has been shown to be detectable in cells
undergoing DNA repair (37). However, both methods were
used in this paper because both PCNA and Ki67 have been
used extensively in the literature as proliferation markers.
The observed difference with anti-PCNA and Ki67 antibodies and measurement of epithelial density underscores the
importance of using multiple methods to assess cell
proliferation.
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FIG. 5. Effects of HRT on PR (A) and ER (B) in breast tissue of
postmenopausal women. The number under each bar represents the
number of individuals for whom ducts or TDLU could be analyzed. *,
P 5 0.002– 0.04 that the percentage of PR -positive cells in the ducts
of the E-alone group was significantly greater than in the ducts of the
no-HRT or E1P groups. †, P 5 0.0007 and 0.04, respectively, that the
percentages of PR-positive cells in the TDLU of the E-alone and E1P
groups were significantly greater than in the TDLU of the no-HRT
group.
4564
HOFSETH ET AL.
proliferation associated with E1P was localized to the TDLU,
which is the site of origin of most breast cancers.
With regard to the progestins used, in our study, all subjects received MPA, which is a synthetic progestin structurally related to progesterone. In the Hargreaves et al. study,
89.7% of the subjects received norethisterone, levonorgestrel,
and norgestrel, which are synthetic progestins structurally
related to testosterone; 7.7% received tibolone and 2.6%
(1 subject) MPA (D.F. Hargreaves, personal communication).
Further direct comparative study will be required to determine whether there are differences in the mitogenic activities
of various progestins. We have previously shown that
estradiol1MPA was highly effective in inducing mammary
epithelial cell proliferation in the mouse mammary gland
(24). Similarly, in surgically postmenopausal cynomolgus
macaques, treatment with CEE1MPA caused a significant
increase in breast epithelial cell proliferation, above that obtained with CEE alone (25, 26). Furthermore, the histological
appearance of lobules in the no-HRT, CEE-alone, and
CEE1MPA-treated monkey glands were strikingly similar to
human breast tissues obtained from similar postmenopausal
HRT groups herein (26). Thus, the greater proliferation observed in postmenopausal women treated with E1MPA
herein is consistent with the mitogenic effect of E1MPA in
mouse and monkey mammary glands.
Others have reported that progesterone decreases the proliferative effect of estrogens in the postmenopausal breast. In
a prospective study, postmenopausal women were withdrawn from HRT for 12 weeks, then received 14 daily topical
treatments with hydroalcoholic gels containing placebo, estradiol, progesterone or estradiol1progesterone applied directly to their breasts (41). Estradiol alone increased the proliferation index (PCNA) of breast epithelium 100-fold,
progesterone alone increased proliferation 15-fold and combination treatment with estradiol1progesterone only increased proliferation 13-fold. The results of that study (41)
are possibly due to the experimental protocol (pretreatment
hormone withdrawal, the use of progesterone or the route
and length of hormone administration), compared to the
standard protocols of HRT treatment experienced by postmenopausal women in our study.
In summary, the present study provides compelling evidence that postmenopausal HRT with E1P was positively
associated with higher levels of epithelial cell proliferation in
the TDLU of the breast. There was also a positive association
between higher levels of proliferation and increasing length
of time on HRT. Both E alone and E1P HRT were associated
with greater epithelial density in the postmenopausal breast.
The possibility is raised that there may be potential differences in the biological activity of various progestins in breast
tissue. Further investigation is needed to assess the possible
association between the mitogenic effects of progestins in the
breast and breast cancer risk.
Acknowledgments
The authors gratefully acknowledge the expert technical assistance
provided by Ms. Jessica M. Bennett, Ms. Bridget M. Mahon, and Ms. Lisa
M. Coram and the cooperation and contributions of Drs. Laura L. Morris,
Keith Appelgren, L. Rao Kareti, Edward Lanigan, Rafael De Los Santos,
Lee Colony, Hugh J. Lindsey, James Harkema, and John Kisala in the
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There were notable differences in the characteristics of the
postmenopausal groups (Table 1). Significantly more women
receiving no HRT or E1P had undergone natural menopause
vs. women in the E-alone group, who were more likely to
have experienced surgical menopause and whose time on
HRT was twice as long as that of women taking E1P (14.2
vs. 6.7 yr). Because E alone is usually prescribed for women
lacking a uterus, early surgically induced-menopause is the
likely explanation for the longer time on HRT for the E-alone
group. Analysis of the influence of length of time of HRT
showed a trend of greater proliferation, with increasing
length of time on E1P. This suggests that there may be a
cumulative effect of E1P on proliferation. However, because
the maximum length of time on E1P was 20 yr (vs. 39 yr on
E) and the numbers of subjects at each individual time point
was low, further studies will be required to resolve this
question.
There was a significantly higher prevalence of past oral
contraceptive use in the E1P group (72%), as compared with
the no-HRT and E-alone groups (31% and 46.9%, respectively). We questioned whether this could have influenced our
results. We therefore analyzed proliferation indices for
women who had ever taken contraceptives vs. those who had
never taken contraceptives, in each treatment group, and
found no significant differences (data not shown). This is not
surprising, because contraceptives were taken during their
cycling years, and there may not be any residual effects of the
hormones after menopause. Previous studies of the effects of
current use of oral contraceptives on breast proliferation in
premenopausal women have shown either no effect (19, 21)
or only a slight effect (17, 18, 22). Furthermore, studies analyzing past use of oral contraceptives have not found any
influence on proliferation in the premenopausal breast
(15, 17).
Recently, Hargreaves et al. (38), using the Ki67 antibody as
a proliferation marker, retrospectively examined epithelial
cell proliferation indices in postmenopausal breast tissue of
women who had taken estrogen alone or estrogen1
progestin, in England. They found no association between
either estrogen or estrogen1progestin HRT and epithelial
proliferation, results different from those observed in the
present study. Possible explanations for this difference are:
1) the length of time on HRT; and 2) the progestins used. In
our study women had taken estrogen1progestin for up to 20
yr; whereas, in the Hargreaves et al. study, women took
estrogen1progestin for a maximum of 5.5 yr. In this regard,
our analyses of proliferation indices, as a function of time on
HRT (Fig. 2), demonstrates that there is a trend of greater
proliferation associated with longer time on estrogen1
progestin HRT. Interestingly, the epidemiological data indicate that breast cancer risk also increases with length of time
on estrogen or estrogen1progestin HRT (7–10). A recent
epidemiological study has also shown a positive association
with breast cancer risk for estrogen1progestin HRT, which
was especially pronounced with continuous combined regimens (10). HRT is hypothesized to play a role in increasing
uterine and breast cancer risk through enhanced cell proliferation and potential accumulation of DNA damage (39, 40).
In relation to the association of HRT with increased risk of
breast cancer, it is of interest to note that, in our study,
JCE & M • 1999
Vol 84 • No 12
HRT AND THE NORMAL BREAST
Department of Surgery, Dr. Jianping He in the Department of Epidemiology, and Dr. Terry L. Woodward in the Department of Physiology
at Michigan State University. We also would like to thank Lansing area
hospital pathologists, Drs. W. De Mendoca-Calaca, R.N. Horowitz,
Ernesto B. Quiachon, Ralph R. Edminster, M. Costa-Fox, N.C. Caliman,
Adonica L. Walker, Stanley B. Katlein, Mary A. Flynn, and Alan G.
Kaplan.
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