Expression of Progesterone and Androgen Receptors in the Breast of Premenopausal Women, Considering Menstrual Phase.

BACKGROUND: Progesterone and androgens are important for normal development and tumorigenesis of the breast. PATIENTS AND METHODS: Breast tissue samples from 49 premenopausal women were obtained. The progesterone receptors (PRA, PRB, PGRMC1 and PGRMC2) and the androgen receptor (AR) were determined in malignant and benign breast tumors and control tissues. RESULTS: The PRB and AR mRNA levels were highest in tumors. PGRMC1 and PGRMC2 mRNA levels were higher in malignant tumors compared to their paired normal tissues. PRA protein showed most immunostaining in benign tumors. PRB immunostaining varied according to menstrual phase. AR immunostaining was highest in the glands of malignant tumors. CONCLUSION: Progesterone and androgen receptors are differently regulated in tumors compared to normal breast tissues. A malignant breast tumor could appear PR-negative if collected in the luteal phase, but positive in the follicular phase. This finding may have clinical implications.

Expression of cyclooxygenase-1 and cyclooxygenase-2, syndecan-1 and connective tissue growth factor in benign and malignant breast tissue from premenopausal women.

Stromal factors have been identified as important for tumorigenesis and metastases of breast cancer. From 49 premenopausal women, samples were collected from benign or malignant tumors and the seemingly normal tissue adjacent to the tumor. The factors studied, with real-time polymerase chain reaction (PCR) and immunohistochemistry, were cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), syndecan-1 (S-1) and connective tissue growth factor (CTGF). COX-1 and S-1 mRNA levels were higher in the malignant tumors than in normal and benign tissues. The COX-2 mRNA level was lower in the malignant tumor than in the normal tissue, while CTGF mRNA did not differ between the groups. COX-1 immunostaining was higher in stroma from malignant tumors than in benign tissues, whereas COX-2 immunostaining was higher in the malignant tissue. Glandular S-1 immunostaining was lower in malignant tumors compared to benign and normal tissues, and the opposite was found in stroma. Conclusively, mRNA levels of COX-1 and COX-2 were oppositely regulated, with COX-1 being increased in the malignant tumor while COX-2 was decreased. S-1 protein localization switched from glandular to stromal cells in malignant tissues. Thus, these markers are, in premenopausal women, localized and regulated differently in normal/benign breast tissue as compared to the malignant tumor.

Expression of Estrogen Receptors in Relation to Hormone Levels and the Nottingham Prognostic Index.

BACKGROUND: Estrogen hormones have a large impact on both normal development and tumorigenesis of the breast. MATERIALS AND METHODS: Breast tissue samples from 49 women undergoing surgery were included. The estrogen receptors (ERα and ERß), ERα36 and G-coupled estrogen receptor-1 (GPER) were determined in benign and malignant breast tissue. RESULTS: The ERα36 and ERα mRNA levels were highest in malignant tumors. Stromal ERß immunostaining in benign tumors was higher than in the paired normal tissue. GPER expression was lowest in benign tumors. In the malignant tumors, the Nottingham Prognostic Index (NPI) correlated positively with stromal GPER and the serum testosterone level. The serum insulin-like growth factor-1 (IGF-1) level correlated negatively with GPER mRNA and glandular ERα. CONCLUSION: The expression of ERα36 is stronger in malignant breast tissue. The strong positive correlation between NPI and GPER in malignant breast stroma indicates an important role for GPER in breast cancer prognosis.

IVF outcome in women with endometriosis in relation to tumour necrosis factor and anti-Müllerian hormone.

This study reports on anti-Müllerian hormone (AMH) in serum and follicular fluid (FF) in relation to inflammatory parameters in women with and without endometriosis undergoing IVF. Serum and FF samples were obtained from 72 women, with (n = 34) and without (n = 38) endometriosis, undergoing IVF. The concentrations of AMH, FSH, tumour necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and several interleukins were analysed. Women with endometriosis had significantly lower AMH in serum and FF (serum: 6.38 versus 12.8 pM; P < 0.01, FF: 14.0 versus 19.6 pM; P < 0.05). TNF was increased in FF (40.0 versus 30.8 pg/ml, P < 0.05) from women with endometriosis and significantly higher concentrations of IL-15 and GM-CSF were detected in FF (both P < 0.05). During IVF, women with endometriosis responded well to FSH but had lower fertilization rates. Women with endometriosis have elevated concentrations of several cytokines in FF. They respond adequately to exogenous FSH but may have impaired oocyte quality, reflected in lower fertilization rates, presumably resulting from an inflammatory process in the ovaries. Further studies are needed to elucidate the role of AMH in predicting ovarian reserve in women with endometriosis.

Expression of the androgen receptor and syndecan-1 in breast tissue during different hormonal treatments in cynomolgus monkeys.

OBJECTIVES: To analyze the expression of the androgen receptor(AR) and syndecan-1 in breast tissue during long-term hormonal treatment in cynomolgus monkeys. METHODS: Sixty oophorectomized macaques were randomized to receive either tibolone, conjugated equine estrogens (CEE), CEE + medroxyprogesterone acetate (MPA) or no hormonal treatment. Breast tissue was collected at necropsy after 2 years and stained for AR and syndecan-1. RESULTS: Apparent differences were seen between treatment groups as compared to untreated animals. AR expression was markedly increased by tibolone and suppressed by combined CEE/MPA. Both treatments increased syndecan-1 in stromal tissue, whereas CEE alone had no significant effect. CONCLUSIONS: We found alternative regimens for hormonal therapy to differ in their influence on two markers of importance for the development of breast cancer. The results may be relevant for the ongoing clinical discussion on the long-term safety of different hormonal treatments.

Peak bone mineral density in Vietnamese women.

SUMMARY: This cross-sectional study showed that peak bone mineral density in Vietnamese women is comparable to that in Caucasian women; however, the prevalence of osteoporosis in post-menopausal Vietnamese women was slightly higher than in Caucasian women. The age of achieving peak bone mass in Vietnamese women was between 26 and 30 years. INTRODUCTION: While peak bone mass and its determinants have been well-documented in Caucasian populations, little has been studied in Asian populations. The present study was designed to estimate the peak bone mineral density (BMD), age of its attainment, and to examine the prevalence of osteoporosis in Vietnamese women aged 50+. METHODS: The study was designed as a cross-sectional study with 328 women aged between 10 and 65 years (average age: 41) who were randomly selected from two districts around Hanoi city according to a stratified sampling scheme. BMD at the lumbar spine, femoral neck and total hip was measured by a DXA instrument (GE Lunar Prodigy, WI, USA). BMD was modeled as a cubic function of age, from which peak BMD and age at peak BMD were estimated. Bootstrap method was utilized to estimate the 95% confidence interval of peak BMD and age at peak BMD. From the peak BMD, T-score was calculated for each woman, and using the World Health Organization criteria, any woman with femoral neck BMD T-score

Effects of tibolone and conventional HRT on the expression of estrogen and progesterone receptors in the breast.

OBJECTIVE: There is evidence that long-term hormone replacement therapy (HRT) is associated with an increased breast cancer risk. The aim of this study was to assess the effects of tibolone on estrogen and progesterone receptors in comparison to the effects of conventional HRT in the breast of surgically postmenopausal macaques. METHOD: Sixty macaques were bilaterally ovariectomized 3 months before hormonal treatment was initiated. The animals were randomized into four treatment groups, including tibolone (TIB), conjugated equine estrogens (CEE), conjugated equine estrogens+medroxyprogesterone acetate (CEE+MPA) and control animals (C). After 2 years treatment, breast tissues were collected, fixed and paraffin embedded. Immunohistochemistry assays with monoclonal antibodies for estrogen receptors (ERalpha and ERbeta) and progesterone receptors (PRA and PRB) were performed. RESULTS: The expression of ERalpha was markedly decreased in the CEE+MPA group as compared to C and TIB groups. The TIB group was not different from the C and CEE groups. No significant differences were found for ERbeta immunostaining. The expression of PRA was strongly increased in the TIB group as compared to the C and CEE+MPA groups. Immunostaining of PRB was increased in the CEE and TIB treated animals as compared to both C and CEE+MPA groups. CONCLUSIONS: Tibolone increased the expression of both PRA and PRB, without affecting ERalpha and ERbeta expression in the macaque breast. These findings indicate that the effects of tibolone in breast tissue could be mediated via differential regulation of PRA and PRB isoforms and therefore distinct from those observed with conventional HRT.

Safety of tibolone in the treatment of vasomotor symptoms in breast cancer patients--design and baseline data 'LIBERATE' trial.

Many patients with a history of breast cancer (BC) will suffer from vasomotor symptoms, which can be induced or exacerbated by treatment with tamoxifen or aromatase inhibitors. The LIBERATE trial was designed as a randomized, double-blind, multicenter trial to demonstrate that tibolone 2.5mg/day (Livial) is non-inferior to placebo regarding BC recurrence in women with vasomotor symptoms surgically treated for primary BC within the last 5 years. Secondary objectives are effects on vasomotor symptoms as well as overall survival, bone mineral density and health-related quality of life. Mean age at randomization was 52.6 years, and the mean time since surgery was 2.1 years. The mean daily number of hot flushes and sweating episodes was 7.3 and 6.1, respectively. For the primary tumor, Stage IIA or higher was reported for >70% of the patients. In subjects whose receptor status was known, 78.2% of the tumors were estrogen receptors positive. At randomization, tamoxifen was given to 66.2% of all patients and aromatase inhibitors to 7%. Chemotherapy was reported by 5% at randomization. The adjuvant tamoxifen use in LIBERATE allows a comparison with the Stockholm trial (showing no risk of BC recurrence associated with hormone therapy), which was stopped prematurely subsequent to HABITS. The LIBERATE trial is the largest, ongoing, well-controlled study for treatment of vasomotor symptoms in BC patients.

Expression of syndecan-1 in paired samples of normal and malignant breast tissue from postmenopausal women.

BACKGROUND: The mammary stroma is important for modulating epithelial breast cell response to sex steroid hormones. Proteoglycans, such as syndecan-1, promote the integration of cellular signals. MATERIALS AND METHODS: The immunohistochemical expression of syndecan-1 and of the androgen receptor (AR) was analyzed in paired samples of cancer and adjacent normal tissue from postmenopausal women. RESULTS: Normal and cancer tissue showed dramatic differences in the expression of syndecan-1. In malignant breast stroma, mean values were more than 10-fold higher than in normal tissue (p<0.001). There was also a marked redistribution from the epithelium to the stroma. The expression of AR was on average 2-fold higher in cancerous than in normal tissue (p<0.01). CONCLUSION: Breast cancer patients have very different prognoses. Syndecan-1 and the AR may be new molecular markers relevant to clinical outcome. The redistribution from the epithelium and the dramatic increase of syndecan-1 in cancerous stroma may be related to the natural history of the disease.

Neutral effect of ultra-low-dose continuous combined estradiol and norethisterone acetate on mammographic breast density.

OBJECTIVE: To compare the effects of two different ultra-low doses of continuous combined hormone therapy and placebo on mammographic breast density in postmenopausal women. METHODS: A subpopulation of 255 postmenopausal women from the CHOICE trial were randomly assigned to 0.5 mg 17beta-estradiol (E2) + 0.25 mg norethisterone acetate (NETA), 0.5 mg E2 + 0.1 mg NETA, or placebo. Women using hormone replacement therapy (HRT) up to 2 months prior to the study were excluded; 154 women fulfilled the inclusion criteria. Mammograms were performed at baseline and after 6 months. Breast density was evaluated by visual classification scales and a computer-assisted digitized technique. RESULTS: No significant differences were detected between the active treatment groups and the placebo group in the digitized quantification. The mean baseline values for density around 20% were unchanged after 6 months. Also, visual classifications showed no increase in breast density in any study group. CONCLUSION: In contrast to currently available bleed-free regimens, the new ultra-low-dose combination of 0.5 mg E2 and 0.1 mg NETA seems to have very little or even a neutral effect on the breast. Both digitized quantification and visual assessment of breast density were unchanged after 6 months. Larger prospective studies should be performed to confirm this new finding.

Testosterone addition during menopausal hormone therapy: effects on mammographic breast density.

OBJECTIVE: To study the effect on mammographic breast density of testosterone addition during combined estrogen/progestogen therapy in postmenopausal women. METHODS: A prospective, randomized, double-blind, placebo-controlled trial. A total of 99 women were given 2 mg 17beta-estradiol and 1 mg norethisterone acetate in combination with either a testosterone patch (300 mug/24 h) or a placebo patch. Mammographic breast density at baseline and after 6 months was assessed by visual classification scales and by digitized quantification. A standardized questionnaire was used to quantify subjective breast symptoms. RESULTS: Visual classifications showed an increase in mammographic density in 18-30% of the women, with no significant differences between the treatment groups. The mean increase of the area of dense breast during treatment according to digitized assessment was 7.4% in the placebo group and 5.4% in the testosterone group. Breast symptoms showed a positive association with the increase in density (r(s) = 0.34; p < 0.01). Symptoms were most pronounced at 2 months of treatment. Density, both at baseline (r(s) = -0.35; p < 0.01) and change during treatment (r(s) = -0.28; p < 0.01) showed a negative association with free testosterone levels. CONCLUSION: The addition of testosterone does not appear to influence mammographic breast density in women concurrently treated with a common oral estrogen/progestogen regimen for a period of 6 months.

Expression of Syndecan-1 in histologically normal breast tissue from postmenopausal women with breast cancer according to mammographic density.

OBJECTIVE: To analyze the expression of Syndecan-1 in dense and non-dense human breast tissue. METHODS: Specimens of histologically normal tissue were obtained from postmenopausal women undergoing surgery for breast cancer. Each tissue block was subject to radiological examination and pair-wise samples of dense and non-dense tissue were collected. Semi-quantitative assessment of immunohistochemical staining intensity for Syndecan-1 and estrogen receptor subtypes was performed. RESULTS: The expression of Syndecan-1 in all tissue compartments was significantly higher in dense than in non-dense specimens. The strongest staining was recorded in stromal tissue. There was a strong correlation between epithelial estrogen receptor alpha and stromal cell Syndecan-1 expression in dense tissue (rs = 0.7; p = 0.02). This association was absent in non-dense tissue. CONCLUSION: An increase of Syndecan-1 in all tissue compartments and a redistribution from epithelium to stroma may be a characteristic feature for dense breast tissue.

Are estrogen receptor content in breast cancer and effects of tamoxifen on sex hormone-binding globulin markers for individual estrogen sensitivity?

Individual women differ with respect to their sensitivity to estrogen and serum levels of sex hormone-binding globulin (SHBG) may reflect the individual response. We found a significant correlation between estrogen receptor (ER) concentrations in breast cancer tissue and SHBG levels during tamoxifen treatment. Estrogen sensitivity may be a general characteristic common to various organs and different between individual women.

Expression of sex steroid receptor subtypes in normal and malignant breast tissue - a pilot study in postmenopausal women.

Female sex steroids are implied in breast cancer development. The estrogen (ER) and progesterone (PR) receptor subtypes may have different roles to modulate the cellular response. Paired samples of cancer and adjacent normal tissue were collected from postmenopausal women at surgery for ductal breast cancer. The expression of ERa, ERss, PRA and PRB was quantified by immunostaining and digitized image analysis. We found ERss to be significantly reduced in breast cancer tissue (35% vs 50%; p?=?0.001) and there was also a decrease of the ERss/ERa ratio. Among women using hormones at the time of diagnosis tumor tissue showed higher values for both PRB and PRA, as compared to women without such treatment. The results extend previous animal data to be valid also in women. There is evidence that loss of ERss expression may relate to estrogen dependent tumor progression. Increased PR expression could possibly relate to breast cancer risk during combined estrogen/progestogen treatment.

Tamoxifen and megestrol acetate for postmenopausal breast cancer: diverging effects on liver proteins, androgens, and glucocorticoids.

AIM: To compare the effects of tamoxifen and megestrol acetate on liver proteins, androgens, and glucocorticoids during adjuvant treatment for postmenopausal breast cancer. METHODS: A subgroup of women within a large prospective multicenter trial were followed with blood sampling every 3 mo during 2 yr. Women were randomized to receive either continuous tamoxifen 40 mg/d or repeated sequential treatment with tamoxifen and megestrol acetate (MA) 160 mg/d. RESULTS: We found profound and distinct differences between the two regimens. Tamoxifen increased steroid-binding proteins (SHBG and CBG) and suppressed circulating androgens and IGF-I. In contrast, the metabolic effects of tamoxifen were clearly antagonized by MA. There was a rise in IGF-I and marked suppression of steroid-binding proteins. Levels of free testosterone were reduced by 70%. MA also caused apparent adrenal suppression. CONCLUSION: The different effects on anabolic/catabolic balance and adrenal function may relate to certain clinical effects during treatment.

The effects of tibolone and oestrogen-based HT on breast cell proliferation and mammographic density.

Tibolone is a tissue-selective compound used for the treatment of climacteric symptoms and the prevention of osteoporosis in post-menopausal women. In this review some in vitro data and clinical studies indicating that the effects of tibolone on breast tissue are different from those seen with oestrogen-based hormone therapy (HT) are briefly discussed. From a clinical perspective, an increase in mammographic density and breast cell proliferation should be regarded as an unwanted side-effect of HT. Efforts should therefore be made to define treatment regimens for post-menopausal women that have minimal effects on the breast but still maintain the many advantages of HT. Data suggest that tibolone may be such an alternative.

A comparative study of breast cell proliferation during hormone replacement therapy: effects of tibolon and continuous combined estrogen-progestogen treatment.

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.

Serum lipids in oophorectomized women during estrogen and testosterone replacement therapy.

OBJECTIVE: To evaluate the effects of giving testosterone undecanoate (TU) in addition to estrogen replacement on serum lipids in oophorectomized women. METHOD: Women with surgically induced menopause (n = 50) were randomly assigned to oral treatment with 2 mg of estradiol valerate in combination with 40 mg of TU or placebo for 24 weeks. The study was double-blind with cross-over to the other regimen for further 24 weeks of treatment. Forty-four women completed the study. Their serum concentrations of total, high density lipoprotein (HDL)- and low density lipoprotein (LDL)-cholesterol, triglycerides, lipoprotein-(a) (Lp-(a)), total testosterone, estradiol and sex hormone-binding globulin (SHBG) were analyzed at baseline and after 24 weeks of each treatment. RESULTS: Serum levels of total testosterone increased markedly from a baseline mean of 0.8-4.9 nmol/l during testosterone addition. The levels of free testosterone significantly increased during the combined treatment and fell when given estrogen alone. Total and LDL-cholesterol levels were significantly reduced by both treatments as also were those of Lp-(a) although the difference was not significant. We found a 13% reduction in HDL-cholesterol levels when testosterone was added, but no change with estrogen alone. Triglyceride levels were increased by estrogen treatment, but not affected by the combination of estrogen plus testosterone. CONCLUSIONS: These findings suggest that 40 mg of TU can be given in addition to estrogen replacement with only little side-effects on the pattern of circulating lipids. Although supraphysiological concentrations of testosterone were induced a significant reduction in total and LDL-cholesterol levels occurred.

Clinical efficacy and safety of combined estradiol valerate and dienogest: a new no-bleed treatment.

A combination of 2 mg estradiol valerate with 2 mg dienogest (E2V/DNG) (Climodien, Schering AG, Berlin, Gemany) is the first continuous combined postmenopausal hormone replacement therapy (HRT) preparation to contain a progestogen with substantial antiandrogenic activity. A study of its clinical efficacy and safety in a comparative study versus a combination of 2 mg estradiol with 1 mg norethisterone acetate (E2/NETA) has shown both preparations to be highly effective in achieving a rapid response in women with postmenopausal symptoms, in terms of hot flushes and the Kupperman index. Biopsy and ultrasound studies have demonstrated that E2V/DNG quickly and effectively achieved endometrial atrophy in the vast majority of subjects, suggesting a protective role in endometrial proliferation. Data on PP-14 (glycodelin) levels may indicate that E2V/DNG is even more effective than E2/NETA in maintaining endometrial atrophy. No-bleed rates with E2V/DNG at 1, 6 and 12 months were at least as favorable as those with other standard HRT products, with evidence that the no-bleed state is attained more quickly with E2V/DNG. The proportions of women with the no-bleed state in a large-scale study (n = 1501) at 1, 6 and 12 months were 71.8%, 76.6% and 86.4%, respectively. Women with irregular bleeding before treatment responded to E2V/DNG in a manner similar to those without bleeding; this concordance was especially marked after five cycles of treatment. In the comparative study, the mean number of days of bleeding over 12 cycles was significantly lower for E2V/DNG than for E2/NETA. Overall, the profiles of adverse events recorded in clinical use were similar in the two preparations, whilst the safety profile of E2V/DNG in the large-scale study was similar to that of other HRT preparations and gave no cause for clinical concern. The 2 mg E2V/2 mg DNG preparation was associated with a favorable lipid profile, whilst a similar combination (2 mg E2V/3 mg DNG) showed no impact on carbohydrate metabolism or hemostasis, compared to placebo. In summary, 2 mg E2V/2 mg DNG is a novel continuous combined HRT preparation that is effective in treating postmenopausal symptoms rapidly, and has a highly favorable bleeding profile. Studies of the safety of 2 mg E2V/2 mg DNG in clinical use have uncovered no factors likely to be disadvantagous in comparison with other HRT products in widespread use.

Effects of long-term HRT and tamoxifen on the expression of progesterone receptors A and B in breast tissue from surgically postmenopausal cynomolgus macaques.

Estrogen is a well-known mitogen in breast epithelium but the role of progesterone is complex and incompletely understood. In contrast to what is seen in the endometrium, combined estrogen/progestogen treatment for postmenopausal replacement (HRT) may carry a risk for breast cancer beyond that of estrogen alone. The ratio of the two progesterone receptor (PR) isoforms, PRA/PRB may define the response to progesterone in reproductive tissues. In a primate model for long-term HRT, surgically, postmenopausal cynomolgus macaques were treated for 35 months with conjugated equine estrogens (CEE), medroxyprogesterone acetate (MPA), CEE + MPA and tamoxifen (n = 5 in all groups). The immunohistochemical expression of PRA, PRB and the androgen receptor (AR) in breast tissue was quantified by image analysis. Over all, the total PR immunostaining in glandular epithelium was more abundant during CEE (mean 12%) and tamoxifen ( 1%) treatment as compared to CEE/MPA (5%), MPA (4%) and untreated controls (6%). Differences in PRB expression were observed between treatment groups (p < 0.05). In the CEE group levels of PRA were unchanged while there was a decline in the CEE/MPA group. The mean PRA/PRB ratio in the CEE group was 2.7 and in the CEE/MPA group 0.2. Treatment with tamoxifen had effects similar to those of estrogen. There was in all groups a weak positive nuclear AR immunostaining. This is the first in vivo study on the effects on long-term hormonal treatment on the expression of PR isoforms in normal primate breast tissue. The results suggest that hormonal treatments have a different influence on the PRA/PRB balance in the breast.