Insights into the Behavior of Triple-Negative MDA-MB-231 Breast Carcinoma Cells Following the Treatment with 17ß-Ethinylestradiol and Levonorgestrel.

Oral contraceptives (OCs) are widely used due to their efficiency in preventing unplanned pregnancies and treating several human illnesses. Despite their medical value, the toxicity of OCs remains a public concern. Previous studies indicate the carcinogenic potential of synthetic sex hormones and their link to the development and progression of hormone-dependent malignancies such as breast cancer. However, little is known about their influence on the evolution of triple-negative breast carcinoma (TNBC), a malignancy defined by the absence of estrogen, progesterone, and HER2 receptors. This study reveals that the active ingredients of modern OCs, 17ß-Ethinylestradiol, Levonorgestrel, and their combination induce differential effects in MDA-MB-231 TNBC cells. The most relevant behavioral changes occurred after the 24 h treatment with 17ß-Ethinylestradiol, summarized as follows: (i) decreased cell viability (64.32% at 10 µM); (ii) cell roundness and loss of confluence; (iii) apoptotic aspect of cell nuclei (fragmentation, membrane blebbing); and (iv) inhibited cell migration, suggesting a potential anticancer effect. Conversely, Levonorgestrel was generally associated with a proliferative activity. The association of the two OCs exerted similar effects as 17ß-Ethinylestradiol but was less effective. Further studies are necessary to elucidate the hormones' cytotoxic mechanism of action on TNBC cells.

Comparative effects of progesterone and the synthetic progestin norethindrone on neuroprotection in a model of spontaneous motoneuron degeneration.

The Wobbler mouse has been proposed as an experimental model of the sporadic form of amyotrophic lateral sclerosis (ALS). The administration of natural progesterone (PROG) to Wobbler mice attenuates neuropathology, inhibits oxidative stress, enhances the expression of genes involved in motoneuron function, increases survival and restores axonal transport. However, current pharmacological treatments for ALS patients are still partially effective. This encouraged us to investigate if the synthetic progestin norethindrone (NOR), showing higher potency than PROG and used for birth control and hormone therapy might also afford neuroprotection. Two-month-old Wobbler mice (wr/wr) were left untreated or received either a 20 mg pellet of PROG or a 1 mg pellet of NOR for 18 days. Untreated control NFR/NFR mice (background strain for Wobbler) were also employed. Wobblers showed typical clinical and spinal cord abnormalities, while these abnormalities were normalized with PROG treatment. Surprisingly, we found that NOR did not increase immunoreactivity and gene expression for choline-acetyltransferase, drastically decreased GFAP + astrogliosis, favored proinflammatory mediators, promoted the inflammatory phenotype of IBA1+ microglia, increased the receptor for advanced glycation end products (RAGE) mRNA and protein expression and the activity of nitric oxide synthase (NOS)/NADPH diaphorase in the cervical spinal cord. Additionally, NOR treatment produced atrophy of the thymus. The combined negative effects of NOR on clinical assessments (forelimb atrophy and rotarod performance) suggest a detrimental effect on muscle trophism and motor function. These findings reinforce the evidence that the type of progestin used for contraception, endometriosis or replacement therapy, may condition the outcome of preclinical and clinical studies targeting neurodegenerative diseases.

Rapid masculinization and effects on the liver of female western mosquitofish (Gambusia affinis) by norethindrone.

Natural and synthetic progestins in receiving streams can disrupt the normal endocrine systems of fish. Norethindrone (NET) is a widely used synthetic progestin that often appears in wastewater effluents. For this research, adult female western mosquitofish (Gambusia affinis) were exposed to NET at three concentrations. The effects of NET on the following biological factors were evaluated: the histology of the ovaries and livers, the anal fin morphology, and transcription of genes related to steroidogenesis signaling pathways in the livers. After 42 d exposure to NET at 33.0 ng L-1 and 347.5 ng L-1, rapid masculinization, an increase in the number of atretic and postovulatory follicles in the ovary, enhanced vascularization, degenerated hepatocytes and irregular nuclei in the livers were observed. Exposure to NET did not affect the expression of the androgenic and estrogenic receptor genes and Cyp19a except for a significant up-regulation of Erα. However, the expression of Vtg A, Vtg B, and Vtg C were markedly inhibited in the females exposed to three concentrations of NET. Compared to the control female, exposure to NET at 33.0 ng L-1 and 347.5 ng L-1 caused a 4.4- and 5.8-fold increase in the expression of Hsd17ß3 in the livers, respectively. The results demonstrate that NET can cause rapid masculinization of female G. affinis, hepatopathological alterations and inhibited expressions of Vtg A, Vtg B, and Vtg C. The results imply that G. affinis populations might be threatened in NET-contaminated environment.

Reproductive effects of synthetic progestin norgestrel in zebrafish (Danio rerio).

The aim of this study was to assess the adverse effects of synthetic progestin norgestrel (NGT) on the reproduction of zebrafish by measuring the egg production, histology and transcriptional expression profiles along the hypothalamic-pituitary-gonadal (HPG) axis in adult zebrafish. After a pre-exposure period of 7 days, adult zebrafish were exposed to 6, 29 and 69 ng L-1 NGT for 21 days. The results showed that exposure to 69 ng L-1 NGT led to a significant up-regulation of follicle stimulating hormone, beta polypeptide (fshb), luteinizing hormone, beta polypeptide (lhb), progesterone receptor (pgr), estrogen receptor 1 (esr1) and androgen receptor (ar) genes in the brains, as well as significant up-regulation of hydroxysteroid 20-beta dehydrogenase (hsd20b) and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2) genes and down-regulation of 11-beta-hydroxylase (cyp11b) gene in the ovaries of females. In the testes of males, an overall down-regulation of steroidogenic acute regulatory protein (star), cytochrome P450-mediated side-chain cleavage enzyme (cyp11a1), cyp11b, hsd20b, hydroxysteroid 17-beta dehydrogenase type 3 (hsd17b3), hsd11b2 and ar genes were observed following exposure to different treatments of NGT. These transcriptional alterations imply that NGT could exhibit the potent progestogenic and androgenic activities in zebrafish. Egg production as well as histology in the ovaries and testes was not affected by NGT. Taken together, the overall results demonstrated that NGT could significantly affect transcriptional expression levels of genes related to HPG axis in zebrafish, and whether that change translates to additional physiological effects is needed further research.

Improvement of Low Sexual Desire Due to Antiandrogenic Combined Oral Contraceptives After Switching to an Oral Contraceptive Containing 17ß-Estradiol.

OBJECTIVES: To investigate the effects of a combined oral contraceptive (COC) containing 17ß-estradiol (E2) 1.5 mg and nomegestrol acetate 2.5 mg (NOMAC/E2) on the sexual health of women affected by low sexual desire due to COCs containing ethinylestradiol. MATERIALS AND METHODS: Eighty-three women (age range 19-32) participated in the study. Sex hormone-binding globulin (SHBG), total testosterone (TT), and free androgen index (FAI) were measured. The Female Sexual Function Index (FSFI) and the Female Sexual Distress Scale (FSDS) questionnaires were used to assess sexual function and distress, respectively. Hormonal levels were measured and questionnaires were administered before the women switched COC NOMAC/E2 usage (baseline) and at the 3-month (first) and 6-month (second) follow-ups. RESULTS: SHBG reduction (p < 0.001), TT (p < 0.05), and FAI increases (p < 0.001) were observed during the first and second follow-ups with respect to baseline values. Sexual desire increased from baseline to the first and second follow-ups (p < 0.001). At baseline, the total FSFI score was 22 ± 1.5 and the FSDS score was 16.6 ± 1.3, both indicating sexual dysfunction with sexual distress. At the first follow-up, the total FSFI score and the FSDS score increased toward sexual health values, being 28.3 ± 1.6 and 12.1 ± 1.5, respectively (p < 0.001). At the second follow-up, the FSFI score had risen to 30.6 ± 1.3 (p < 0.001) and the FSDS score had dropped to 8.3 ± 1.4 (p < 0.001). CONCLUSIONS: COCs containing E2 are an innovation that could help women to not suffer from low sexual desire during hypoandrogenic COC usage.

The synthetic progestin norgestrel acts to increase LIF levels in the rd10 mouse model of retinitis pigmentosa.

PURPOSE: Retinal degenerative conditions affect thousands of people worldwide. Retinitis pigmentosa (RP) is among the most common, but it is currently incurable. It is characterized by the progressive death of photoreceptor cells, eventually leading to blindness. Neurotrophic factors play an important role in such retinopathies, and much research has been performed on their use as treatments. Our group previously demonstrated the ability of the synthetic progestin norgestrel to rescue photoreceptors from cell death, the mechanism of which is believed to include upregulation of the neurotrophic factor basic fibroblast growth factor (bFGF). The objective of the present study was to investigate whether the protection provided by norgestrel is likely to be mediated by other neurotrophins. METHODS: The 661W photoreceptor cells and retinal explants from P30 to P40 wild-type (wt) C57BL/6 mice were treated with norgestrel over time. Homozygous rd10/rd10 mice that mimic the human form of RP were fed either a control or a norgestrel-containing diet. Changes in neurotrophic factor expression in response to norgestrel were detected with real-time PCR, western blotting, or immunofluorescence staining. Using specific siRNA, leukemia inhibitory factor (Lif) expression was knocked down in 661W photoreceptor cells that were stressed by serum starvation. Cells were treated with norgestrel followed by measurement of cell viability with (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. RESULTS: LIF, a potent neuroprotective cytokine, was found to be upregulated in response to norgestrel in vitro and in vivo. Upregulation of LIF in degenerating rd10 retinas coincided with preservation of the photoreceptor layer. We also found LIF was necessary for the norgestrel-mediated rescue of stressed photoreceptor cells from cell death in vitro. CONCLUSIONS: LIF was upregulated in response to norgestrel in all models studied and is necessary for the protective effects of norgestrel in vitro. The increase in LIF expression in rd10 mice undergoing retinal degeneration was concurrent with rescue of the photoreceptor cell layer. These results highlight the ability of norgestrel to induce prosurvival molecules in the compromised retina, underlining norgestrel's potential as a viable drug for treatment of RP.

Clotting factor changes during the first cycle of oral contraceptive use.

OBJECTIVES: The risk of venous thromboembolism (VTE) is highest during the initial months of oral contraceptive (OC) use. We sought to evaluate the extent of hemostatic variable changes during the initial OC cycle and if such changes are related to systemic ethinyl estradiol (EE2) exposure. STUDY DESIGN: Participants provided multiple blood samples during a 21-day OC cycle (30mcg EE2; 150mcg levonorgestrel) and after a single dose following a washout period. Analytes included D-dimer, factor VIII activity, protein C total antigen and the hepatic proteins corticosteroid-binding globulin (CBG) and sex-hormone-binding globulin (SHBG). EE2 pharmacokinetic analyses related to the 24h after the first OC tablet (OC1) and at steady state (OC21). RESULTS: Seventeen women completed the study. D-dimer more than doubled by OC6 (p=.013) and remained elevated at OC21 (p=.012). D-dimer levels within women varied widely from day to day. Factor VIII increased 27% by OC2 (p<.001) but declined to a 9% increase by OC21. Protein C increased only 6%. EE2 steady-state area-under-the-curve ranged from 488 to 1103pg∙h/mL; higher levels were not correlated with greater increases in clotting variables. CBG and SHBG increased significantly but were not significantly correlated with levels of EE2 or with the hemostatic variables. CONCLUSIONS: D-dimer increases during the first OC cycle were at least as great as increases seen with longer OC use. These results provide support for the increased VTE risk during initial OC use. The extreme variability in D-dimer levels may be an important component of this risk. IMPLICATIONS: This study showed that increases in D-dimer are clearly evident in the first cycle of OC use and may be larger than are seen after a longer duration of use and thus provide biological support for the increased VTE risk during initial OC use found in epidemiological studies.

Unique effects on hepatic function, lipid metabolism, bone and growth endocrine parameters of estetrol in combined oral contraceptives.

OBJECTIVES: Estetrol (E4) is a natural estrogen produced by the human fetal liver. In combination with drospirenone (DRSP) or levonorgestrel (LNG), E4 blocks ovulation and has less effect on haemostatic biomarkers in comparison with ethinylestradiol (EE) combined with DRSP. This study evaluates the impact of several doses of E4/DRSP and E4/LNG on safety parameters such as liver function, lipid metabolism, bone markers and growth endocrine parameters. METHODS: This was a dose-finding, single-centre, controlled study performed in healthy women aged 18 to 35 years with a documented pretreatment ovulatory cycle. Participants received 5 mg or 10 mg E4/3 mg DRSP; 5 mg, 10 mg or 20 mg E4/150 µg LNG; or 20 µg EE/3 mg DRSP as a comparator for three consecutive cycles in a 24/4-day regimen. Changes from baseline to end of treatment in liver parameters, lipid metabolism, bone markers and growth endocrinology were evaluated. RESULTS: A total of 109 women were included in the study. Carrier proteins were minimally affected in the E4/DRSP and E4/LNG groups, in comparison with the EE/DRSP group, where a significant increase in sex hormone-binding globulin was observed. Similarly, minor effects on lipoproteins were observed in the E4 groups, and the effects on triglycerides elicited by the E4 groups were significantly lower than those in the EE/DRSP group. No imbalances in bone markers were observed in any groups. No alterations in insulin-like growth factor were observed in the E4 groups. CONCLUSIONS: E4-containing combinations have a limited effect on liver function, lipid metabolism, and bone and growth endocrine parameters.

A randomised study comparing the effect on ovarian activity of a progestogen-only pill (POP) containing desogestrel and a new POP containing drospirenone in a 24/4 regimen.

OBJECTIVES: Progestogen-only pills (POPs) are safer with respect to cardiovascular risks than contraceptives containing estrogens. Despite the increased contraceptive efficacy of a desogestrel-only pill compared with a traditional POP, POPs are still not widely used due to an unpredictable bleeding pattern. A new POP containing 4 mg drospirenone has been developed with a 24/4 intake regimen which may improve the bleeding pattern. The objectives of this study were to investigate ovulation inhibition with the new drospirenone-only pill in comparison with the desogestrel-only pill and, in addition, to assess the effects on cervical mucus permeability and bleeding. METHODS: Sixty-four healthy volunteers with proven ovulatory cycles were randomised and treated with either the drospirenone-only or the desogestrel-only pill during two 28-day cycles. Follicular diameter, endometrial thickness, and serum estradiol (E2) and progesterone concentrations were measured and Hoogland scores were determined. Additionally, cervical mucus scores, bleeding and return of ovulation were assessed. RESULTS: Both treatments effectively inhibited ovulation. Follicular diameter, E2 levels and Hoogland scores were equal, demonstrating efficient ovarian suppression. One subject in each group had a Hoogland score of 6, but the criteria for normal luteal activity were not fulfilled. In both groups, ovulation did not occur before day 9 of the post-treatment cycle. Cervical mucus permeability was suppressed in both groups. The median number of bleeding and spotting days was lower in the drospirenone group. CONCLUSIONS: The new drospirenone-only pill inhibited ovulation as effectively as the desogestrel-only pill despite the 4-day hormone-free interval.

Molecular action of norgestimate: new developments.

BACKGROUND: Acne occurs because the sebaceous glands are overstimulated by high levels of androgens or are hypersensitive to normal levels of testosterone. In women with mild or moderate acne, the association of norgestimate (NG), and ethinyl estradiol (EE) is an effective treatment. This is related to the effect of oral contraceptives on androgen production and transport and the antiandrogenic properties of NG itself. DESIGN: The present work was undertaken to find out whether NG and its derivative, 17-deacetylnorgestimate(dNG), present steroid activities other than antiandrogen activities, using human progesterone receptor(PR), estrogen receptor α(ERα) and ß(ERß), glucocorticoid receptor(GR) and mineralocorticoid receptor(MR)-responsive cell lines. RESULTS: We confirmed that NG and its metabolite were progestogen partial agonists (EC50 of 13 and 11.1 nM) and ERα selective agonists (EC50 of 30.4 and 43.4 nM), as well as full antagonists of low affinity for GR (IC50 of 325 and 255 nM) and moderate affinity for MR (IC50 of 81.2 and 83.7). CONCLUSION: We demonstrated that NG and dNG have full progestogen and weak estrogenic (through ERα) properties, which could explain in part the efficacy of NG in association with EE for the treatment of moderate acne in women. Moreover, their antagonist MR activity might have a favorable impact on cardiovascular risk, atherosclerosis and lipid profiles.

Ovulatory effects of three oral contraceptive regimens: a randomized, open-label, descriptive trial.

OBJECTIVE: This study describes ovarian activity suppression of a 21/7-active low-dose combined oral contraceptive (COC) regimen that included only ethinyl estradiol (EE) during the traditional hormone-free interval (HFI) and two commercially available 28-day regimens, a 24/4 and a 21/7 regimen. STUDY DESIGN: The randomized, open-label, parallel-group descriptive study was conducted at two US sites. Healthy, reproductive-aged women (n=146) were randomized to one of three groups for three consecutive 28-day cycles, as follows: treatment 1 (n=39 completed): 21/7-active COC [21 days of 150 mcg desogestrel (DSG)/20 mcg EE, followed by 7 days of 10 mcg EE (DSG/EE+7 days EE)], treatment 2 (n=39 completed): 24 days of 3mg drospirenone (DRSP)/20 mcg EE, followed by 4 placebo (PBO)-pill days (DRSP/EE+4 days PBO) and treatment 3 (n=42 completed): 21 days of 100 mcg levonorgestrel (LNG)/20 mcg EE, followed by 7 PBO-pill days (LNG/EE+7 days PBO). The primary outcome was ovarian activity suppression assessed by transvaginal ultrasound and serum hormone concentrations and classified using the Hoogland and Skouby (H/S) method. RESULTS: Ovarian activity rate (H/S grade 4 or 5) was low for all three treatments: 0% [95% confidence interval (CI) 0-2.8] for DSG/EE+7 days EE, 1% (95% CI 0.2-5.2) for DRSP/EE+4days PBO and 1% (95% CI 0-3.9) for LNG/EE+7 days PBO. All three treatments showed similar suppression of serum progesterone, 17ß-estradiol, follicle-stimulating hormone and luteinizing hormone levels. CONCLUSIONS: The 21/7-active low-dose COC regimen (DSG/EE+7 days EE) showed ovarian activity suppression that was similar to the 24/4 (DRSP/EE+4 days PBO) and 21/7 (LNG/EE+7days PBO) regimens. IMPLICATIONS: The 21/7-active low-dose COC regimen (DSG/EE+7 days EE) that included only EE during the traditional HFI showed suppression of ovarian follicular activity that was similar to the 24/4 (DRSP/EE+4days PBO) and the 21/7 (LNG/EE+7 days PBO) comparator regimens.

Depot medroxyprogesterone acetate in combination with a twice-daily lopinavir-ritonavir-based regimen in HIV-infected women showed effective contraception and a lack of clinically significant interactions, with good safety and tolerability: results of the ACTG 5283 study.

We conducted an open-label, steady-state pharmacokinetic (PK) study of drug-drug interactions between depot medroxyprogesterone acetate (DMPA) and twice-daily lopinavir (LPV) plus low-dose ritonavir (RTV) (LPV/r) among 24 HIV-infected women and compared the results to those for HIV-infected women receiving DMPA while on no antiretroviral therapy or on nucleosides only (n = 14 subjects from the control arm of AIDS Clinical Trials Group [ACTG] study 5093). The objectives of the study were to address the effect of LPV/r on DMPA and to address the effect of DMPA on LPV/r therapy. PK parameters were estimated using noncompartmental analysis with between-group comparisons of medroxyprogesterone acetate (MPA) PKs and within-subject comparisons of LPV and RTV PKs before and 4 weeks after DMPA dosing. Plasma progesterone concentrations were measured every 2 weeks after DMPA dosing through week 12. Although the MPA area under the concentration-time curve and maximum concentration of drug in plasma were statistically significantly increased in the study women on LPV/r compared to those in the historical controls, these increases were not considered clinically significant. There were no changes in LPV or RTV exposure after DMPA. DMPA was well tolerated, and suppression of ovulation was maintained. (This study has been registered at ClinicalTrials.gov under registration no. NCT01296152.).

A comparative study of the androgenic properties of progesterone and the progestins, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A).

The importance of investigating the molecular mechanism of action of medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A), two clinically important progestins used in hormone therapy (HT), has been highlighted by clinical evidence showing that MPA and norethisterone (NET) increase the risk of the development of breast cancer in HRT users, and that MPA may increase susceptibility to- and transmission of HIV-1. The aim of this study was to compare the molecular mechanisms of action of MPA, NET-A and progesterone (Prog) via the androgen receptor (AR) in a cell line model that can minimize confounding factors such as the presence of other steroid receptors. This study is the first to determine accurate apparent Ki values for Prog, MPA and NET-A toward the human AR in COS-1 cells. The results reveal that these ligands have a similar binding affinity for the AR to that of the natural androgen 5α-dihydrotestosterone (DHT) (Ki's for DHT, Prog, MPA and NET-A are 29.4, 36.6, 19.4 and 21.9 nM, respectively). Moreover, in both transactivation and transrepression transcriptional assays we demonstrate that, unlike Prog, MPA and NET-A are efficacious AR agonists, with activities comparable to DHT. One of the most novel findings of our study is that NET-A, like DHT, induces the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C-interaction) of the AR independent of promoter-context, while MPA does not induce the N/C interaction on a classical ARE and does so only weakly on an AR-selective ARE. This suggests that MPA and NET-A may exert differential promoter-specific actions via the AR in vivo. Consistent with this, molecular modeling suggests that MPA and NET-A induce subtle differences in the structure of the AR ligand binding domain. Taken together, the results from this study suggest that unlike Prog, both MPA and NET-A used in hormonal therapy are likely to compete with DHT and exert significant and promoter-specific off-target transcriptional effects via the AR, possibly contributing to some of the observed side-effects with the clinical use of MPA and NET-A.

Mifepristone: a potential clinical agent based on its anti-progesterone and anti-glucocorticoid properties.

Nowadays, unwanted pregnancy is a major globe tragedy for millions of women, associated with significant direct and indirect costs, no matter for individuals or society. The progesterone receptor antagonist steroid, mifepristone has been widely and effectively using throughout the world for medical abortion, but to a lesser extent for emergency contraception. In this review, we hope to explore the role of mifepristone as a contraceptive, particularly for emergency contraception. Studies of mifepristone have also been expanding to the fields of endometriosis and uterine fibroids. Furthermore, this initially considered reproductive medicine has been investigated in some psychotic diseases and various disorders of hypercortisolism, because of its glucocorticoid receptor antagonism. Mifepristone was approved suitable for patients with hyperglycemia secondary to Cushing's syndrome by the United States Food and Drug Administration (FDA) in 2012. The aim of this article is to review published reports on the anti-progesterone and anti-glucocorticoid properties of mifepristone as a clinical agent. There is a new insight into systematically describing and evaluating the potential efficiency of mifepristone administrated in the field of endocrine and neuroendocrine, not only in obstetrics and gynecology.

Overexpression of progesterone receptor membrane component 1: possible mechanism for increased breast cancer risk with norethisterone in hormone therapy.

OBJECTIVE: Clinical trials have demonstrated an increased risk of breast cancer during estrogen/norethisterone (NET) therapy. With this in mind, the effects of estrogen/NET combination on the proliferation of breast cancer cells overexpressing the progesterone receptor membrane component 1 (PGRMC1) were examined. The same combination was used for the first time in a mouse xenograft model to determine its effects on tumor development. METHODS: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12 cells) or empty vector control (pcDNA-3HA). NET, medroxyprogesterone acetate (MPA), and progesterone were tested alone and sequentially and continuously combined with estradiol (E2). Six-week-old nude mice were inoculated with E2 pellets 24 hours before the injection of tumor cells into both flanks (n = 5-6 mice per group). After 8 days, animals were inoculated with a NET pellet or with placebo pellets, and tumor volumes were recorded twice a week. RESULTS: NET alone significantly increased the proliferation of WT-12 cells, MPA was effective only at the two highest concentrations, and progesterone had no effect. The twofold to threefold E2-induced increase (10 M) was not significantly influenced by the addition of the various progestogens. In contrast, 10 M E2 had no effect; however, addition of MPA and NET triggered a significant proliferative response. In vivo, a sequential combination of NET and E2 also significantly increased the tumor growth of WT-12 cells; empty vector cells did not respond to NET. CONCLUSIONS: We have demonstrated for the first time that an E2/NET combination increases the proliferation of PGRMC1-overexpressing breast cancer cells, both in vivo and in vitro. Our results suggest that undetected tumor cells overexpressing PGRMC1 may be more likely to develop into frank tumor cells in women undergoing E2/NET hormone therapy.

Effects of short-term estradiol and norethindrone acetate treatment on the breasts of normal postmenopausal women.

OBJECTIVE: The aim of this study was to evaluate among postmenopausal women the effects of a 3-month treatment with estradiol (E2) alone or in combination with norethindrone acetate (NA) on expression of hormone receptors and proliferation in the breast as well as on lipids and climacteric symptoms. METHODS: Sixty healthy postmenopausal women were computer-randomized into two groups, with one group receiving 1 mg of E2 and the other group receiving 1 mg of E2 and 0.5 mg of NA daily for 12 weeks. Before and after treatment, middle-needle biopsies were obtained for histology and investigation of the expression levels of estrogen receptors (ERs; ER-α and ER-ß), progesterone receptors (PRs; PR-A and PR-B), androgen receptor (AR), the proliferation marker Ki67, and collagen. Climacteric symptoms were recorded, and serum was collected to measure lipoprotein levels. RESULTS: Fifty-six women finished the 12-week study. Proliferating cells (Ki67-positive) were very rare in all but a few of the untreated women. There were proliferating cells in both E2- and E2/NA-treated groups; however, these were not widespread and limited to nests of cells that amounted to 2% of the total epithelial cells. Some of these nests were positive for human epithelial growth factor receptor 2. Treatments caused no marked changes in the expression of ER-α, ER-ß, or AR. However, both treatments resulted in an increase in PR-A and PR-B expressions. The presence of collagen was clearly associated with a mammographic diagnosis of dense breasts, but neither hormone treatment affected breast density. Both E2 and E2/NA treatments were effective in relieving hot flashes and sweating without adverse effects on blood pressure, weight, and liver, kidney, and thyroid functions. A decrease in cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol was induced by E2/NA but not by E2. CONCLUSIONS: This short-term prospective study shows that E2 and estrogen-progestogen treatment can up-regulate PRs but do not significantly affect ERs, AR, proliferation, or breast density.

Investigating the anti-mineralocorticoid properties of synthetic progestins used in hormone therapy.

A more detailed understanding of the affinities and efficacies for transcriptional regulation by the synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A) via the mineralocorticoid receptor (MR) is required, to better understand their relative risk profiles. Both MPA and NET-A bind to the MR, although with about 100-fold lower affinities than that of Prog. MPA and NET-A exhibit no agonist activity, but NET-A, unlike MPA, has similar antagonistic efficacy to Prog on the endogenous mineralocorticoid/glucocorticoid response element (MRE/GRE)-containing genes, α-glycolytic protein or orosomucoid-1 (Orm-1) and plasminogen activator inhibitor-1 (PAI-1). This study is the first to show that NET-A, but not MPA, can dissociate between transrepression and transactivation via the MR. Given the relatively low affinity and potency of MPA and NET-A for the MR, our results suggest that these progestins are unlikeley to exert significant effects via the MR at doses used in hormonal therapy. However, considering their relative free concentrations compared to endogenous hormones, the possibility that NET-A may exhibit significant MR antagonist activity, with some possible cardiovascular protective benefits, should not be excluded.

Functional role of progestin and the progesterone receptor in the suppression of spermatogenesis in rodents.

Synthetic progestins such as levonorgestrel (LNG) are used in combination with testosterone (T) in male contraceptive clinical trials to suppress gonadotropins secretion, but whether progestins have additional direct effects on the testis are not known. This study aimed to examine the effect of a potent progestin, (LNG), alone or in combination with testosterone (T) on spermatogenesis in adult rats, and to evaluate the functional role of the progesterone receptors (PRs) in the testis. In comparison with a low dose of LNG treatment in adult rats for 4 weeks, T and T + LNG treatment decreased testicular sperm count to 64.1 and 40.2% of control levels respectively. LNG induced germ cell apoptosis at stages I-IV and XII-XIV; T increased apoptosis at stages VII-VIII; LNG + T treatment induced greater germ cell apoptosis at a wider range of seminiferous epithelial stages. RT-PCR and Western Blots showed that PR was present in testes and up-regulated during suppression of spermatogenesis induced by testicular hormonal deprivation. PR knockout (PRKO) mice had larger testes, greater sperm production, increased numbers of Sertoli and Leydig cells. Suppression of gonadotropin and intratesticular T by GnRH-antagonist treatment induced PR promoter driven LacZ expression in Leydig cells of PRKO mice. This suggests that GnRH-antagonist treatment while inducing germ cell apoptosis also up-regulates PR. We conclude that (i) LNG + T induced greater suppression of spermatogenesis through increase in germ cell apoptosis involving a wider range of seminiferous epithelial stages than either treatment alone, (ii) up-regulation of PR was associated with inhibition of spermatogenesis, (iii) PR knockout mice showed increased sperm production suggesting that testicular PR activated events play a physiological and pharmacological inhibitory role in the testis. These data support the hypothesis that in addition to its known suppressive effects on gonadotropins, progestins may have direct inhibitory actions on the testis.