Objectives To determine the effects of continuous combined hormone therapy, tibolone, black cohosh, and placebo on digitized mammographic breast density in postmenopausal women. Study design A prospective, double-blind, placebo-controlled study of 154 postmenopausal women randomized to estradiol 2 mg/norethisterone acetate 1 mg (E2/NETA), tibolone 2.5 mg or placebo and a prospective, open, uncontrolled drug safety study, of which 65 postmenopausal women were treated with black cohosh. Mammograms, at baseline and after six months of treatment, were previously classified according to visual quantification scales. Main outcome measures Reanalysis of assessable mammograms by digitized quantification of breast density. Results Treatment groups were comparable at baseline. During treatment, both E2/NETA and tibolone significantly increased breast density (mean increase 14.3%, p < 0.001 and 2.3%, p < 0.001, respectively), while black cohosh and placebo did not. Twenty-four out of the 43 women on E2/NETA had an increase in density exceeding 10% and 6 women had an increase of 30% or more. In the tibolone group, only one woman had an increase in density of more than 10%. The difference in increase in breast density between E2/NETA on the one hand and tibolone, black cohosh and placebo on the other was highly significant (p < 0.0001). Conclusions Digitized mammographic breast density is a highly sensitive method confirming significant increase in density by standard E2/NETA treatment and to a lesser extent by tibolone, whereas black cohosh does not influence mammographic breast density during six months treatment. Digitized assessment also yields data on individual variation and small increases left undetectable by visual classification.

BACKGROUND Progestins as well as estrogens have a role in breast cell proliferation and the development of breast cancer. Here, the effect of mifepristone on cell proliferation in human breast tissue in vivo was studied in premenopausal women. METHODS A group of 30 women, scheduled for surgical treatment of leiomyomas, were randomized to either 50 mg mifepristone or placebo every other day, for 3 months. Fine needle aspiration biopsies were obtained at baseline and after 3 months. Immunocytochemical analysis of Ki-67 was performed to reflect breast epithelial cell proliferation. Samples from 14 women were included in the final analyses. RESULTS The Ki-67 index was significantly reduced after mifepristone treatment compared with baseline (P = 0.012). Furthermore, less individual variation in the Ki-67 index was seen in the mifepristone group. Treatment with mifepristone did not affect cortisol levels, whereas an increase in serum testosterone was noted. Breast symptoms like soreness and swelling were reduced, whereas the incidence of flushes increased. CONCLUSIONS The ability of mifepristone to block breast epithelial cell proliferation in premenopausal women may prove beneficial when used for contraceptive purposes or for other gynaecological indications. Future studies should address a possible antiproliferative effect in the post-menopausal breast tissue during hormone replacement therapy. Our results implicate a possible protective effect of mifepristone on the breast epithelium. ClinicalTrials.gov NCT00579475.

Objective: To perform a pilot study of the effects on the breast by low-dose intrauterine progestogen combined with estrogen.Design: A prospective pilot study.Setting: University hospital.Patient(s): Twenty postmenopausal women without any previous breast disorder.Intervention (s): Women were treated with a low-dose intrauterine system releasing 20 mu g/24 hours of levonorgestrel in continuous combination with 2 mg of oral E-2 valerate. The effects on mammographic breast density, breast cell proliferation, and hormonal levels were followed for 18 months.Main Outcome Measure(s): Change in mammographic breast density and breast cell proliferation. Correlations with levels of hormones, growth factors, and binding proteins.Result(s): Three women showed an apparent increase in density. For the remaining 17 women the changes were only a few percent. Digitized assessment of density showed strong correlations with Visual classification scales (r(s) = 0.96-0.97). There was no increase in proliferation as expressed by the percentage of MIB-1-positive breast cells in fine-needle aspiration biopsies. Increase in breast density displayed a positive correlation with patients age (r(s) = 0.52) and an inverse relationship with levels of E-2 (r(s) = -0.50) and free T (r(s) = -0.50).Conclusion(s): Low-dose intrauterine administration progestogen may develop into an attractive alternative for hormonal therapy in postmenopausal women as endometrial protection may be achieved at very low systemic levels.

OBJECTIVE:To use the fine-needle aspiration (FNA) biopsy technique to compare the effects of tibolone, conventional hormone replacement therapy (HRT) and placebo on breast cell proliferation in postmenopausal women.METHODS:A total of 91 women were randomized to receive either estradiol 2 mg plus norethisterone acetate 1 mg (E2/NETA), tibolone 2.5 mg or placebo for 6 months in a prospective double-blind trial. Breast cell proliferation was assessed using the Ki-67/MIB-1 monoclonal antibody.RESULTS:From the 83 women who completed the study, a total of 166 FNA biopsies were obtained, and 118 of these aspirates (71%) were evaluable for MIB-1 content. Women with assessable biopsies were younger, had a lower body mass index, and had higher levels of sex hormone binding globulin and insulin-like growth factor-I than women in whom the cell yield was insufficient. During treatment with E2/NETA, there was an increase in proliferation (percentage of MIB-1) from a mean value of 2.2 to 6.4% after 6 months (p < 0.01). No significant changes were recorded during treatment with tibolone or placebo. There was a negative association between proliferation and serum levels of total (r(s) = -0.29, p < 0.05) and free (rs = -0.31, p < 0.03) testosterone.CONCLUSIONS:Tibolone seems to have little influence on breast cell proliferation.

OBJECTIVE:To study the effect on mammographic breast density of two different continuous combined regimens for hormone therapy.DESIGN:Randomized clinical study.SETTING:University hospital.PATIENT(S):Postmenopausal women without any previous history of breast disorder.INTERVENTION(S):The women received either estradiol valerate/dienogest or estradiol/norethisterone acetate. Mammograms and venous blood samples were obtained at baseline and after 6 months.MAIN OUTCOME MEASURE(S):Change in mammographic breast density. Correlations with levels of hormones, growth factors, and binding proteins.RESULT(S):An increase in mammographic density was recorded in approximately 50% of the women, and there were no differences between treatments. Increased density showed a positive correlation with estradiol, estrone, and sex hormone-binding globulin and showed a negative association to free T. Among hormonal factors, levels of free T were the most important for predicting increased density.CONCLUSION(S):Continuous combined hormone therapy with different progestogens has a marked impact on the breast.

The basis of breast cancer risk associated with hormonal therapies may lie in the regulation of cell proliferation. In a prospective, double-blind, randomized study postmenopausal women were given continuous combined hormone replacement therapy (HRT) either as estradiol valerate 2 mg/dienogest 2 mg, (E2V/DNG) or estradiol 2 mg/noretisterone acetate 1 mg (E2/NETA) for 6 months. Fine needle aspiration (FNA) biopsies were used for immunocytochemical analysis of breast cell proliferation before and during treatment. From 45 women completing the study 135 biopsies were obtained. In the total material there was a more than 4-fold increase in proliferation between baseline and 3 months (p < 0.001). The mean percentage of MIB-1 positive breast cells increased from 2.2 to 9.1%. In some individual women values were as high as 25%. No further increase was recorded at 6 months. While numerical values were somewhat lower in the E2V/DNG group, there were no significant differences between treatments. There was a positive correlation between breast cell proliferation (MIB-1%) and circulating levels of both estradiol (rs = 0.54, p < 0.01) and estrone (rs = 0.53, p < 0.01) after 3 and 6 months of treatment. No correlations with other endogenous hormones, proteins or with the two exogenous progestogens dienogest and norethisterone were observed. Increased breast cell proliferation should probably be regarded as an unwanted side-effect during HRT. Means to identify those women with the most pronounced proliferative response should be developed. The FNA biopsy technique may be a useful tool to monitor and evaluate the proliferative response to HRT in the normal breasts of postmenopausal women.

OBJECTIVE:Our purpose was to compare the effects of tibolone, continuous combined hormone replacement therapy, and placebo on mammographic breast density. STUDY DESIGN:A prospective, randomized, double-blind placebo-controlled study was performed. A total of 166 postmenopausal women were equally randomized to receive tibolone 2.5 mg, estradiol 2 mg/norethisterone acetate 1 mg (E(2)/NETA), or placebo. Mammograms were performed at baseline and after 6 months of treatment. Mammographic density was quantified according to the Wolfe classification and by the percentage area of the breast that had a dense pattern. RESULTS:An increase in mammographic density was much more common among women receiving continuous combined hormone replacement therapy (46%-50%) than among those receiving tibolone (2%-6%) and placebo (0%) treatment. The difference between E(2)/NETA and placebo was highly significant (P <.001). Treatment with tibolone did not differ from that with placebo. The relative risk of an increase in breast density for E(2)/NETA versus tibolone was found to be 8.3 (95% CI 2.7-25.0). CONCLUSION:An increase in mammographic density should be regarded as an unwanted side effect of hormone replacement therapy. In contrast to estrogen/progestogen treatment, tibolone seems to exert little stimulation of breast tissue.

Objective: The aim of this study was to evaluate the impact of different hormone replacement therapy (HRT) regimens on mammographic breast density. Study design: Mammographic density was recorded in women participating in a population-based screening program. At first mammogram, all women were non-users of HRT, and thereafter reported continuous use of the same HRT regimen. The study population comprised 158 women: a total of 52 women were using continuous combined HRT (conjugated equine estrogen 0.625 mg plus medroxyprogesterone acetate 5 mg); 51 women were using low-dose oral estrogen alone (estriol 2 mg daily); and 55 women were using unopposed transdermal estrogen given as a patch (estradiol 50 μg/24 h). Films were coded and analyzed for mammographic density by an independent radiologist blinded to treatments. Mammographic density was classified according to Wolfe. Results: An increase in mammographic density was much more common among women taking continuous combined HRT (40%) than for those using oral low-dose estrogen (6%) and transdermal (2%) treatment. The increase in density was already apparent at the first visit after starting HRT. During long-term follow-up, there was very little change in mammographic status. Conclusion: HRT regimens were shown to have different effects on the normal breast. There is an urgent need to clarify the biological nature and significance of a change in mammographic density during treatment and, in particular, its relation to symptoms and breast cancer risk.

Using the ovariectomized macaque model of postmenopausal women's health, we investigated the effects of long-term treatments (5 weeks-3 years) with estradiol, conjugated equine estrogens (CEE), esterified estrogens, progestins such as medroxyprogesterone acetate (MPA) and nomegestrol acetate, CEE + MPA, tamoxifen, soybean phytoestrogens (SPEs), a variety of putative selective estrogen receptor modulators (SERMs), and androgens. Agents tested were selected on the basis of beneficial effects on arteries and/or bone. Doses were scaled on a caloric or serum-concentration basis to approximate human clinical doses. We evaluated endometrial and mammary gland histopathology and morphometry and used immunohistochemistry to evaluate cell proliferation and expression of estrogen receptor alpha and progesterone receptor (PR). Both estradiol and CEE induced endometrial hyperplasia. MPA antagonized epithelial proliferation induced by CEE in endometrium and induced pseudodecidual stromal hyperplasia in some animals. Tamoxifen induced endometrial polyps, cystic hyperplasia, stromal fibrosis, and PR expression but not Ki-67 expression. SPEs were not estrogenic at dietary doses and antagonized estrogen-induced proliferation in the endometrium and breast. Nandrolone induced mucometra and an adenomyosis-like change. The potential SERM 17 alpha dihydroequilenin did not have uterotrophic or mammotrophic effects. In general, experimental findings in macaques have been predictive of outcomes in human clinical trials of the same agents.

Objective: The aim was to evaluate fine-needle-aspiration (FNA) cytology as a method of following breast epithelial proliferation in postmenopausal women during hormone replacement therapy (HRT). Methods: Twelve healthy postmenopausal women were recruited and randomized to two different types of sequential HRT during 4 months of treatment. The women were administered continuous estradiol 50 μg/24 h with the addition of progestogen sequentially in the form of either vaginal progesterone gel 8 mg every 2nd day or medroxyprogesterone acetate 5 mg/day orally during 12 days per month for the complete treatment period. Fine-needle-aspiration biopsies were performed twice during the estrogen phases and twice during the estrogen plus progestogen phases of treatment. Breast epithelial proliferation was analyzed in these samples by immunocytochemistry to measure the content of the nuclear antigen Ki-67/MIB-1, which is expressed in proliferating cells. Results: From the 12 women, a total number of 47 FNA biopsies were taken. Thirty-eight of these aspirates, 19 from each of the estrogen and the estrogen plus progestogen phases, were evaluable for MIB-1 content (81%). There was a nonsignificant increase in levels of proliferation during the combined estrogen-progestogen phase (2.1%) compared with the estrogen-only phase (1.4%). These values were similar to those previously observed during the menstrual cycle in young fertile women. Conclusions: We conclude that the FNA biopsy technique is feasible for studying proliferation not only in young, normally cycling women but also in the postmenopausal breast.

The mechanisms behind increased breast tissue proliferation and a possibly increased breast cancer risk in women using hormonal contraception (HC) and hormonal replacement therapy (HRT) are incompletely understood. We analyzed breast tissue from 20 premenopausal and seven postmenopausal women undergoing reduction mammoplasties for estrogen receptor (ER) and progesterone receptor (PR) content as well as mRNA levels for ER, PR and insulin-like growth factor-1 (IGF-1). The receptor values were correlated to IGF-1 mRNA concentrations and levels of steroid and peptide hormones and SHBG. In women using HC, we found significantly lower ER values (p = 0.02) but non-significantly lower ER mRNA levels compared to those in naturally cycling women. PR and PR mRNA were no different. Women on HC displayed a higher breast tissue proliferation (p = 0.05) expressed as Ki-67, MIB-1 positivity, which was correlated with IGF-1 mRNA (r(s) = 0.82, p = 0.04). Since the concentration of sex steroid receptors in breast tissue is comparatively low and steroid receptors are down-regulated during hormonal treatment, mechanisms other than direct sex steroid receptor action are likely to be present. Our results suggest a role for IGF-1 in the proliferative response of breast tissue during exogenous hormonal treatment.

Estrogens are important for both normal cell growth and malignant proliferation in the mammary gland as well as in the endometrium. Tamoxifen is a non‐steroidal anti‐estrogen widely used in breast cancer treatment. In recent years reports have been made of an increased risk of endometrial carcinoma during tamoxifen treatment. We used surgically menopausal cynomolgus macaques to study proliferation and p53 expression during hormonal replacement therapy (HRT) and tamoxifen treatment. Animals were treated continuously for 35 months with either conjugated equine estrogens (CEE; n = 20); medroxyprogesterone acetate (MPA; n =17); the combination of CEE + MPA (n = 13) or tamoxifen (n = 17) for 35 months. We found an increased expression of p53 in normal breast and endometrial tissue linked to CEE but not tamoxifen treatment. In the breast alveoli there was an association between proliferation measured by morphometry and p53 expression in all groups. However, in the endometrium CEE induced significantly more p53 positivity than tamoxifen, 9/20 vs. 3/17 in glands and 9/19 vs. 0/17 in stroma, respectively. If indeed long‐term treatment with tamoxifen as in the present study could inactivate the tumor‐suppressive function of p53, endometrial cells might thereby become more susceptible to genetic lesions associated with carcinogenesis.

Objective: Our purpose was to investigate the effects of various hormone replacement regimens on mammographic breast density. Study Design: Mammographic density was recorded in women participating in a population-based screening program. All women were nonusers of hormone replacement therapy at first mammogram and thereafter reported continuous use of the same treatment: estrogen alone (n = 50) or estrogen in cyclic (n = 75) or continuous (n = 50) combination with progestogen. Mammographic density was quantified according to the Wolfe classification. Results: An increase in mammographic density was much more common among women receiving continuous combination hormone replacement therapy (52%) than among those receiving cyclic (13%) and estrogen-only (18%) treatment. The increase in density was apparent already at first visit after the start of hormone replacement therapy. There was little change in mammographic status during long-term follow-up. Conclusion: Regimens of hormone replacement therapy were shown to have different effects on the normal breast. There is an urgent need to clarify the biologic nature and significance of a change in mammographic density during treatment and, in particular, its relation to symptoms and breast cancer risk. (Am J Obstet Gynecol 1999;181:348-52.)

Numerous women are treated with a combination of oestrogen and progestogen for contraception and hormone replacement therapy worldwide. A possible increased risk of cancer in target organs has been discussed vividly for many years. While oestrogens are clearly mitogenic for breast epithelial cells, there has been considerable uncertainty about the effects of progestogens. This article reviews current knowledge on this field, including our own data. Oestrogen receptors are down-regulated during the luteal phase, while progesterone receptors remain at a high level throughout the menstrual cycle. According to most studies, in vivo proliferation of normal breast epithelial cells is higher during the luteal phase in the vast majority of women. Normal breast tissue can convert oestrone sulphate to oestradiol. A negative correlation between the levels of circulating oestradiol and the enzyme converting oestrone into oestradiol suggests a local regulatory mechanism of tissue oestradiol formation. Serum progesterone levels correlate positively with sulphatase activity while 19-norsteroid progestogens may be inhibitory. We found that long-term continuous combined hormonal treatment with conjugated equine oestrogens and medroxyprogesterone acetate induced a proliferative response in the breasts of surgically postmenopausal macaques. The effect of combined treatment was more pronounced than that of oestrogen treatment alone. Both endogenous progesterone and exogenous progestogens increase proliferation of breast epithelial cells. Exogenous progestogens down-regulate both oestrogen and progesterone receptors. Oestrogen and progestogens may have both direct and indirect stimulating effects on proliferation. The finding of a positive correlation between insulin-like growth factor I messenger RNA and proliferation found in hormonally treated women with low receptor levels suggests the possibility of nonreceptor-mediated effects of sex steroids on proliferation, which needs to be investigated further.

Our purpose was to assess 17β-hydroxysteroid dehydrogenase (17HSD) type 1 protein expression in normal breast tissue during the menstrual cycle and hormonal contraception. We analyzed 17HSD type 1 protein expression by immunohistochemistry during the regular menstrual cycle (n = 12) and hormonal contraception (n = 7) in women undergoing reduction mammoplasty. 17HSD type 1 protein was detected in normal breast epithelial cells throughout the menstrual cycle and in all women using hormonal contraception. Mean 17HSD type 1 staining intensity was higher in alveolar epithelial cells in women using hormonal contraception (2.14) than in untreated women (1.25; P < 0.04). For ducts, this difference approached significance (2.29 vs. 1.41; P = 0.06). There was a negative correlation between serum estradiol (E2) levels and 17HSD type 1 protein expression for both alveolar (rs = −0.68; P = 0.004) and ductal (rs = −0.75; P = 0.002) breast epithelial cells. Enhanced 17HSD type 1 protein expression might increase the con...

OBJECTIVE:To define the relative proliferative response and hormone receptors status in ten sites in the mammary gland of surgically postmenopausal cynomolgus macaques given hormone replacement therapy.METHODS:Surgical postmenopausal cynomolgus macaques were given either no treatment (n = 4), conjugated equine estrogens (CEE, n = 4), or combined therapy with CEE and medroxyprogesterone acetate (n = 4). The drugs were administered in the diet, at doses equivalent on a caloric basis to 0.625 mg/woman/day for CEE and 2.5 mg/woman/day for medroxyprogesterone acetate. Immunostaining of mammary sections was done for estrogen receptor, progesterone receptor, and the proliferation marker Ki-67 MIB-1 (MIB). Comparisons were made between central and peripheral gland, by quadrant, left versus right, and with respect to distance from the nipple within each quadrant.RESULT:There were no significant differences in hormone receptor or MIB expression within different sites within the gland.CONCLUSIONS:In the surgically postmenopausal, hormone-treated macaque, regional differences in estrogen and progesterone receptors and MIB staining are not apparent. The assumption of homogeneity throughout the gland makes aspiration cytology and multiple biopsy studies feasible in this species.

The breast is a target organ for reproductive hormones but basic knowledge on hormonal effects is very poor. Available data indicate that the breast is regulated in a specific manner which is distinct from the endometrium and other target organs. It seems clear that the breast undergoes cyclic changes during the menstrual cycle and that in vivo there is a direct stimulatory action of progestogens on the breast. In surgically postmenopausal female macaques continuous combined estrogen/progestogen therapy was found to induce greater proliferation than estrogen alone.

OBJECTIVE: Our purpose was to define the proliferative response and receptor status in the mammary glands of surgically postmenopausal macaques given hormone replacement therapy, equivalent for monkeys to that given women. STUDY DESIGN: Surgically postmenopausal adult female cynomolgus macaques (Macaca fascicularis) were given either no treatment (n = 26), conjugated equine estrogens (n = 22), or combined therapy with conjugated equine estrogens and medroxyprogesterone acetate (n = 21). Drugs were administered in the diet, at doses equivalent on a caloric basis to 0.625 mg per woman per day for conjugated equine estrogens and 2.5 mg per woman per day for medroxyprogesterone acetate, for 30 months. Mammary gland proliferation was assessed subjectively and by morphometric and stereologic means. Estrogen receptor and progesterone receptor content and proliferation were studied by immunohistochemistry. RESULTS: In this model combined therapy with conjugated equine estrogens and medroxyprogesterone acetate induced greater proliferation than did conjugated equine estrogens alone. The percentage of estrogen receptor - positive cells was decreased in the conjugated equine estrogens plus medroxyprogesterone acetate group. The percentage of progesterone receptor - positive cells was increased by treatment with conjugated equine estrogens alone. CONCLUSION: These results indicate a proliferative response of mammary gland epithelium to therapy with conjugated equine estrogens plus medroxyprogesterone acetate in postmenopausal macaques. The clinical implication of this finding may be a greater risk for development of breast neoplasms in women receiving combined hormone replacement therapy. (AM J OBSTET GYNECOL 1996;174:93-100.)

The breast is a target organ for reproductive hormones but basic knowledge on hormonal effects is very poor. Available data indicate that the breast is regulated in a specific manner which is distinct from the endometrium and other target organs. It seems clear that the breast undergoes cyclic changes during the menstrual cycle and that in vivo there is a direct stimulatory action of progestogens on the breast. In surgically postmenopausal female macaques continuous combined estrogen/progestogen therapy was found to induce greater proliferation than estrogen alone.