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 4559 Downloaded from https://academic.oup.com/jcem/article/84/12/4559/2864741 by guest on 19 June 2022 LORNE J. HOFSETH, AHMED M. RAAFAT, JANET R. OSUCH, DOROTHY R. PATHAK, CAROL A. SLOMSKI, AND SANDRA Z. HASLAM 4560 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 Downloaded from https://academic.oup.com/jcem/article/84/12/4559/2864741 by guest on 19 June 2022 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. Downloaded from https://academic.oup.com/jcem/article/84/12/4559/2864741 by guest on 19 June 2022 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. 4561 4562 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 Downloaded from https://academic.oup.com/jcem/article/84/12/4559/2864741 by guest on 19 June 2022 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. Downloaded from https://academic.oup.com/jcem/article/84/12/4559/2864741 by guest on 19 June 2022 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 Downloaded from https://academic.oup.com/jcem/article/84/12/4559/2864741 by guest on 19 June 2022 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. References 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. increases proliferation and decreases oestrogen receptor content of epithelial cells in the normal human breast. Int J Cancer. 48:206 –210. Potten CS, Watson RJ, Williams GT, et al. 1988 The effect of age and menstrual cycle upon proliferative activity of the normal human breast. Br J Cancer. 58:163–170. Masters JRW, Drife JO, Scarisbrick JJ. 1977 Cyclic variation of DNA synthesis in human breast epithelium. J Natl Cancer Inst. 58:1263–1265. Going JJ, Anderson TJ, Battersby S, Macintyre CCA. 1988 Proliferative and secretory activity in human breast during natural and artificial menstrual cycles. Am J Pathol. 130:193–204. Johansson CM, Anderson TJ, Bergstrom R, Lindgren A, Persson IR. 1998 Epithelial proliferation in the normal human breast in relation to endogenous hormones and oral contraceptive use. The Breast. 7:162–167. Haslam SZ. 1988 Progesterone effects on deoxyribonucleic acid synthesis in normal mouse mammary glands. Endocrinology. 122:464 – 470. Wang S, Counterman LJ, Haslam SZ. 1990 Progesterone action in normal mouse mammary gland. Endocrinology. 127:2183–2189. Cline JM, Soderqvist G, von Schoultz E, Skoog L, vonSchoultz B. 1996 Effects of hormone replacement therapy on the mammary gland of surgically postmenopausal cynomolgus macaques. Am J Obstet Gynecol. 174:93–100. Cline JM, Soderqvist G, vonSchoultz E, Skoog L, vonSchoultz B. 1998 Effects of conjugated estrogens, medroxyprogesterone acetate, and tamoxifen on the mammary glands of macaques. Br Cancer Res Treat. 48:221–229. Laidlaw IJ, Clarke RB, Howell A, Owen AWMC, Potten CS, Anderson E. 1995 The proliferation of normal human breast tissue implanted into athymic nude mice is stimulated by estrogen but not progesterone. Endocrinology. 136:164 –171. McManus MJ, Welsch CW. 1984 The effect of estrogen, progesterone, thyroxine and human placental lactogen on DNA synthesis of human breast ductal epithelium maintained in athymic nude mice. Cancer. 54:1920 –1927. Pike MC. 1996 Experiments on proliferation of normal human breast tissue in nude mice do not show that progesterone does not stimulate breast cells. Endocrinology. 137:1505. Sadi MV, Barrack ER. 1991 Determination of growth fraction in advanced prostate cancer by Ki-67 immunostaining and its relationship to the time to tumor progression after hormonal therapy. Cancer. 67:3065–3071. Yu CCW, Filipe MI. 1993 Update on proliferation-associated antibodies applicable to formalin-fixed paraffin-embedded tissue and their clinical applications. Histochem J. 25:843– 853. Rosen PP. 1997 Chapter 1: Anatomy. Rosen PP, ed. Rosen’s Breast Pathology. Philadelphia: Lippincott-Raven Publishers; 1–15. Horwitz KB. 1992 The molecular biology of RU486. Is there a role for antiprogestins in the treatment of breast cancer? Endocr Rev. 13:146 –163. Mockus MB, Horwitz KB. 1982 Progesterone receptors in human breast cancer. Stoichiometric translocation and nuclear receptor processing. J Biol Chem. 258:4778 – 4783. Shyamala G, Haslam SZ. 1980 Estrogen and progesterone receptors in normal mammary gland during different functional states. In: Bresciani F, ed. Perspectives in Steroid Receptor Research. New York: Raven Press:193–216. Bravo R, Macdonald-Bravo H. 1987 Existence of two populations of cyclinproliferaing cell nuclear antigen during the cell cycle. Association with DNA replication sites. J Cell Biol. 105:1549 –1554. Shivji MKK, Kenny MK, Wood RD. 1992 Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 69:367–374. Hargreaves DF, Knox F, Swindell R, Potten CS, Bundred NJ. 1998 Epithelial proliferation and hormone receptor status in the normal postmenopausal breast and the effects of hormone replacement therapy. Br J Cancer. 78:945–949. Prestin-Martin S, Pike MC, Ross RK, Jones PA, Henderson BE. 1990 Increased cell division as a cause of human cancer. Cancer Res. 50:7415–7421. Pike MC, Spicer DV, Dahmoush L, Press MF. 1993 Estrogens, progestogens, normal breast cell proliferation, and breast cancer risk. Epidemiol Rev. 15:17–35. Foidart JM, Colin C, Denoo X, et al. 1998 Estradiol and progesterone regulate the proliferation of human breast epithelial cells. Fertil Steril. 69:963–969. Downloaded from https://academic.oup.com/jcem/article/84/12/4559/2864741 by guest on 19 June 2022 1. Hutchinson TA, Polansky SM, Feinstein AR. 1979 Postmenopausal estrogens protect against fractures of the hip and distal radius. A case-control study. Lancet. 2:705–709. 2. Weiss NS, Ure CL, Ballard JH, Williams AR, Daling JR. 1980 Decreased risk of fractures of the hip and lower forearm with postmenopausal use of estrogens. N Engl J Med. 303:1195–1198. 3. Ross RK, Paganini-Hill A, Mack TM, Arthur M, Henderson BE. 1981 Menopausal estrogen therapy and protection from death from ischemic heart disease. Lancet. 1:858 – 860. 4. Stampfer MJ, Willett WC, Colditz GA, Rosner B, Speizer FE, Hennekens CH. 1985 A prospective study of postmenopausal estrogen therapy and coronary heart disease. N Engl J Med. 313:1044 –1049. 5. Tang MX, Jacobs D, Stern Y, et al. 1996 Effect of oestrogen during menopause on risk and age at onset of Alzheimer’s disease. Lancet. 348:429 – 432. 6. Sillero-Arenas M, Delgado-Rodriguez M, Rodigues-Canteras R, BuenoCavanillas A, Galvez-Vargas R. 1992 Menopausal hormone replacement therapy and breast cancer: a meta-analysis. Obstet Gynecol. 79:286 –294. 7. Beral V, Bull D, Doll R, Key T, Peto R, Reeves G, and the Collaborative Group on Hormonal Factors in Breast Cancer. 1997 Breast cancer and hormone replacement therapy: collaborative reanalysis of data from 51 epidemiological studies of 52,705 women with breast cancer and 108,411 women without breast cancer from 54 epidemiological studies. Lancet. 350:1047–1059. 8. Colditz GA, Hankinson SE, Hunter DJ, et al. 1995 The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. N Engl J Med. 332:1589 –1593. 9. Bergkvist L, Adami H-O, Persson I, Hoover R, Schairer C. 1989 The risk of breast cancer after estrogen and estrogen-progestogen replacement. N Eng J Med. 321:293–297. 10. Magnusson C, Baron JA, Correia N, Bergstrom R, Adami H-O, Persson I. 1999 Breast-cancer risk following long-term oestrogen- and oestrogen-progestinreplacement therapy. Int J Cancer. 81:339 –344. 11. Persson I, Adami HO, Bergkvist L, et al. 1989 Risk of endometrial cancer after treatment with oestrogens alone or in conjunction with progestogens: results of a prospective study. Br Med J. 298:147–151. 12. Hulka BS, Chambless LE, Kaufman DG, Fowler Jr WC, Geensberg BG. 1982 Protection against endometrial carcinoma by combination product oral contraceptives. J Am Med Assoc. 247:475– 477. 13. Gambrell Jr R, Massey F, Castaneda T, Ugenas A, Ricci C, Wright J. 1980 Use of the progestogen challenge test to reduce the risk of endometrial cancer. Obstet Gynecol. 70:732–738. 14. SoderqvistG, Isaksson E, von Schoultz B, Carlstrom K, Tani E, Skoog L. 1997 Proliferation of breast epithelial cells in healthy women during the menstrual cycle. Am J Obstet Gynecol. 176:123–128. 15. Olsson H, Jernstrom H, Alm P, et al. 1996 Proliferation of the breast epithelium in relation to menstrual cycle phase, hormonal use, and reproductive factors. Breast Cancer Res Treat. 40:187–196. 16. Meyer JS. 1977 Cell proliferation in normal human breast ducts, fibroadenomas, and other ductal hyperplasias measured by nuclear labeling with tritiated thymidine. Effects of menstrual phase, age, and oral contraceptive hormones. Hum Pathol. 8:67– 81. 17. Anderson TJ, Battersby S, King RJB, McPherson K, Going JJ. 1989 Oral contraceptive use influences resting breast proliferation. Hum Pathol. 20:1139 –1144. 18. Williams G, Anderson E, Howell A, et al. 1991 Oral contraceptive (OCP) use 19. 4565