Functional evidence for two distinct mechanisms of action of progesterone and selective progesterone receptor modulator on uterine leiomyomas.

OBJECTIVE: To study the specific mechanisms through which progesterone and selective progesterone receptor modulators impact the growth, synthesis, and accumulation of the extracellular matrix in uterine leiomyomas. DESIGN: Laboratory study. SETTING: Academic Research Institutions. PATIENTS (S): This study involved reproductive-age women diagnosed with infertility associated uterine leiomyomas who underwent myomectomy either after selective progesterone receptor modulator ulipristal acetate (UA) treatment or without any pharmacological pretreatment. Control samples included healthy myometrium tissue (n = 100). Specimens were obtained from the Department of Reproduction and Gynecological Endocrinology and Biobank, Medical University of Bialystok, Poland. INTERVENTIONS: Daily (5 mg/d) UA treated for 2 months (n = 100) and untreated (n = 150) patients with uterine leiomyomas or normal healthy myometrium (n = 100) tissue samples immediately after surgery were collected for transcriptional analysis and assessments. MAIN OUTCOME MEASURES: Progesterone-induced activation of the signaling pathways related to uterine leiomyomas extracellular matrix synthesis, deposition, and growth, as well as the expression profile of progesterone receptors in uterine leiomyomas, were assessed. RESULTS: The results indicated that progesterone activated the transforming growth factor-ß and SMAD3 signaling pathways and promoted proliferation, growth, and extracellular matrix remodeling in uterine leiomyomas by up-regulating SMAD3, transforming growth factor-ß (TGF-ß) receptor type 1 and II, Ras homolog A, vascular endothelial growth factor, or increasing the fibrosis-related gene collagen, type I, ɑ-1, and procollagen, type I, ɑ-1 production. In contrast, UA had inhibitory effects on these processes. The study also showed that both nuclear and membrane progesterone receptors play distinct roles in uterine leiomyoma pathobiology. CONCLUSIONS: We showed that both nuclear and membrane progesterone receptors were relevant in the treatment of uterine leiomyomas, especially when combined with selective progesterone receptor modulators. Novel therapeutic approaches combining selective progesterone receptor modulators with or without direct and indirect extracellular matrix targeting through selected specifically TGF-ß and SMAD3 (SMAD3, TGF-ß receptor types 1 and II, Ras homolog A, vascular endothelial growth factor, collagen, type I, ɑ-1) signaling pathways could therefore be a treatment option for uterine leiomyomas.

Serum expression levels of selected microRNAs and their association with glucose metabolism in young women with polycystic ovary syndrome.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is associated with metabolic disturbances, such as insulin resistance and prediabetes, and the risk for their occurrence is especially increased in hyperandrogenic (HA) phenotypes of PCOS. Circulating microRNAs (miRNAs) may be involved in PCOS pathogenesis and regulation of metabolic processes. OBJECTIVES: The aim of the study was to assess expression levels of selected circulating miRNAs in women with PCOS and to investigate the relationship of these miRNAs with glucose metabolism. PATIENTS AND METHODS: The study included 95 patients with HA­PCOS and 76 healthy women similar to the study group in age and body mass index. Measurements of sex hormone concentrations, oral glucose tolerance test (OGTT), and transvaginal ultrasonography were performed. Serum levels of selected miRNAs (miR­27a, miR­34a, miR­106b, miR­193b, miR­181a, miR­181b, and miR­320) were assessed with real­time polymerase chain reaction, and their association with PCOS and glucose metabolism parameters was studied. RESULTS: Serum levels of all studied miRNAs, except for miR­34a, differed between the patients with HA­PCOS and healthy women (all P <0.05). In HA­PCOS, miR­27a and miR­320 levels correlated with fasting glucose (R = 0.33; P = 0.001 and R = -0.35; P <0.001, respectively) and insulin concentrations (R = 0.26; P = 0.01 and R = -0.23; P = 0.03, respectively). Additionally, the level of miR­27a correlated with mean glucose concentration during OGTT (R = 0.26; P = 0.01). No such correlations were observed in the healthy women. In linear regression analyses, both miR­27a and miR­320 were associated with fasting glucose concentrations after adjustment for potentially confounding factors in the HA­PCOS group only. CONCLUSIONS: The expression profile of circulating miRNAs is altered in patients with HA­PCOS. Circulating miR­27a and miR­320 could serve as potential biomarkers of glucose metabolism disturbances in PCOS.

New Insights on the Progesterone (P4) and PGRMC1/NENF Complex Interactions in Colorectal Cancer Progression.

The literature data regarding the risk of colorectal cancer (CRC) in the context of hormone therapy (HT), including both estrogen-progestogen combinations and estrogen alone, are inconclusive. The precise relationship underlying the action of progesterone (P4) and progesterone receptors in CRC has yet to be determined. We characterized the expression profiles of both nuclear and membrane progesterone receptors and their potential cofactors in CRC tissues. Additionally, we analyzed the P4 and NENF treatment effects on the cell proliferation and invasion of DLD-1 and HT-29 colorectal cancer cells. We observed a weak expression of the nuclear P4 receptor (PGR), but an abundant expression of the P4 receptor membrane component 1 (PGRMC1) and neuron-derived neurotrophic factor (NENF) in the CRC tissues. P4 treatment stimulated the proliferation of the DLD-1 and HT-29 CRC cells. The co-treatment of P4 and NENF significantly increased the invasiveness of the DLD-1 and HT-29 cells. A functional analysis revealed that these effects were dependent on PGRMC1. AN immunocytochemical analysis demonstrated a cytoplasmic co-localization of PGRMC1 and NENF in the CRC cells. Moreover, the concentration of serum NENF was significantly higher in CRC patients, and P4 treatment significantly increased the release of NENF in the DLD-1 cells. P4 or NENF treatment also significantly increased the IL-8 release in the DLD-1 cells. Our data may provide novel insights into the action of P4 and PGRMC1/NENF in CRC progression, where NENF may act as a potential PGRMC1 co-activator in non-classical P4 signaling. Furthermore, NENF, as a secreted protein, potentially could serve as a promising circulating biomarker candidate for distinguishing between colorectal cancer patients and healthy individuals, although large-scale extensive studies are needed to establish this.

Molecular insights underlying the adverse effects of bisphenol A on gonadal somatic cells' steroidogenic activity.

Bisphenol A (BPA) exposure may impair gonadal steroidogenesis, although the underlying mechanism is not well known. Hereby, we assessed BPA action on human primary granulosa (hGC) and mouse Leydig cells (BLTK-1) proliferation, cytotoxicity, hormone secretion, and steroidogenic enzyme/receptor gene profile. hGC and BLTK-1 cells were stimulated with increasing concentrations of BPA (10-12 M to 10-4 M for cell proliferation assay, 10-8 M to 10-4 M for LDH-cytotoxicity assay, and 10-9 M to 10-5 M for hormone secretion and genes expression analysis). BPA at low concentrations (pM - nM) did not affect cell proliferation in either cell type, although was toxic at higher (µM) concentrations. BPA stimulation at low nM concentrations decreased the production of estradiol (E2) and testosterone (T) in BLTK-1, E2, and progesterone in hGCs. BPA down-regulated Star, Cyp11a1, and Hsd17b3, but up-regulated Cyp19a1, Esr1, Esr2, and Gpr30 expression in BLTK-1 cells. In hGC, BPA down-regulated STAR, CYP19A1, PGRMC1, and PAQR7 but up-regulated ESR2 expression. Estrogen receptor degrader fulvestrant (FULV) attenuated BPA inhibition of hormone production in both cell lines. FULV also blocked the BPA-induced Gpr30 up-regulation in BLTK-1 cells, whereas in hGC, failed to reverse the down-regulation of PGRMC1, STAR, and CYP19A1. Our findings provide novel mechanistic insights into environmentally-relevant doses of BPA action through both nuclear estrogen receptor-dependent and independent mechanisms affecting cultured granulosa and Leydig cell steroidogenesis.

Functional Implications of Estrogen and Progesterone Receptors Expression in Adenomyosis, Potential Targets for Endocrinological Therapy.

Adenomyosis is a common gynaecological disease associated with the presence of endometrial lesions in the uterine myometrium. Estrogens have been proven to be the crucial hormones driving the growth of adenomyosis. Little is known about the distinct mechanisms of progesterone action in adenomyosis. Hence, in this study, we decided to characterize the expression of all nuclear and membrane estrogen and progesterone receptors. Additionally, as a functional investigation, we monitored prolactin production and cell proliferation after estradiol and progesterone treatments. We confirmed the presence of all nuclear and membrane estrogen and progesterone receptors in adenomyotic lesions at gene and protein levels. The expression of membrane progesterone receptors α and ß (mPRα, mPRß) as well as estrogen receptor ß (ERß) was upregulated in adenomyosis compared to normal myometrium. Estradiol significantly increased adenomyotic cell proliferation. Progesterone and cAMP upregulated prolactin secretion in adenomyosis in the same pattern as in the normal endometrium. In the present study, we showed the functional link between estradiol action and adenomyotic cell proliferation, as well as progesterone and prolactin production. Our findings provide novel insights into the sex steroid receptor expression pattern and potential regulated pathways in adenomyosis, suggesting that all receptors play an important role in adenomyosis pathophysiology.

The Association between Bisphenol A, Steroid Hormones, and Selected MicroRNAs Levels in Seminal Plasma of Men with Infertility.

Bisphenol A (BPA), the most common endocrine-disrupting chemical, has been associated with male reproductive dysfunctions. Recently, it has been shown that BPA may also affect miRNAs expression. Herein, we aimed to evaluate the association of BPA levels with steroid hormone concentration and circulating miRNAs levels to investigate the potential direct effect of BPA on homeostasis in the testis environment. The level of BPA in the seminal plasma of azoospermic men was significantly higher compared to the healthy control. The concentrations of estradiol (E2) and androstenedione (A) were significantly decreased in the seminal plasma of azoospermic men compared to the normospermic men. The levels of miR-let-7a, miR-let-7b, and miR-let-7c were significantly up-regulated, and the level of miR-518f was significantly down-regulated in the seminal plasma of the azoospermic men compared to the healthy control. The level of BPA correlated negatively with sperm concentration and normal semen morphology. A significant positive correlation was found between BPA levels and miR-let-7a and miR-let-7c levels, whereas BPA negatively correlated with miR-518f levels. Our results suggest that BPA may negatively affect sperm quality. Moreover, BPA correlated with the miR-let-7a, miR-let-7c, and miR-518f levels in seminal plasma, which suggests that BPA may act directly in seminal plasma, affecting the testicular environment.

Mifepristone Treatment Promotes Testicular Leydig Cell Tumor Progression in Transgenic Mice.

The selective progesterone receptor modulator mifepristone (MF) may act as a potent antiproliferative agent in different steroid-dependent cancers due to its strong antagonistic effect on the nuclear progesterone receptor (PGR). Hereby, we analyzed the effects of MF treatment on Leydig cell tumor (LCT) progression in a transgenic mouse model (inhibin-α promoter-driven SV40 T-antigen), as well as on LCT (BLTK-1 and mLTC-1) cell proliferation. MF significantly stimulated the proliferation of LCT in vitro. Similarly, a 1-mo MF or P4 treatment stimulated LCT tumor growth in vivo. Traceable/absent classical Pgr or nonclassical membrane PRs α, ß, γ and Pgrmc2, but abundant membrane Pgrmc1 expression, was found in LCTs. MF did not activate glucocorticoid or androgen receptors in LCTs. Functional analysis showed that PGRMC1 is required for MF and P4 to stimulate the proliferation and invasiveness of LCTs. Accordingly, MF and P4 induced PGRMC1 translocation into the nucleus and thereby stimulated the release of TGFß1 in LCT cells. MF and P4 treatments upregulated Tgfbr1, Tgfbr2, and Alk1 expression and stimulated TGFß1 release in LCT cells. Our findings provide novel mechanistic insights into the action of MF as a membrane PR agonist that promotes LCT growth through PGRMC1 and the alternative TGFß1 signaling pathway.

Oxytocin antagonism reverses the effects of high oestrogen levels and oxytocin on decidualization and cyclooxygenase activity in endometrial tissues.

RESEARCH QUESTION: What is the in-vitro effect of oxytocin receptor (OTR) antagonism on parameters of receptivity in human endometrial explants and endometrial stromal cell lines cultured in oestradiol-rich conditions mimicking ovarian stimulation? DESIGN: Experimental in-vitro study on endometrial tissue explants collected by aspiration biopsy from 30 women undergoing fertility treatment and cultured endometrial tHESC cell line. The study examined the effects of high oestradiol, oxytocin and OTR antagonist on parameters of decidualization (cell viability and prolactin secretion) as well as cyclooxygenase-1/2 (COX-1/2) activity and prostaglandin F2α (PGF2α) secretion. Changes in expression of OXTR and COX-2 genes were examined using quantitative polymerase chain reaction (qPCR). RESULTS: In experiments on cultured endometrial cell line, high oestradiol and oxytocin similarly limited the viability of cells. In cultured endometrial explants both also decreased the secretion of prolactin (a marker of decidualization) and augmented endometrial COX-2 activity and formation of PGF2α. Oxytocin antagonist atosiban was confirmed to reverse the above effects, both in the endometrial line and endometrial explants. Addition of atosiban to cultures acted analogously in experiments employing both oxytocin and high oestradiol. CONCLUSIONS: Oxytocin antagonist reversed the effects of high oestradiol and oxytocin on parameters related to endometrial receptivity in conditions mimicking ovarian stimulation. This might point to a novel, endometrium-related mechanism to support embryo implantation achieved by the application of oxytocin antagonist prior to embryo transfer.

Molecular mechanisms underlying mifepristone's agonistic action on ovarian cancer progression.

BACKGROUND: Recent clinical trials on ovarian cancer with mifepristone (MF) have failed, despite in vitro findings on its strong progesterone (P4) antagonist function. METHODS: Ovarian cancer human and murine cell lines, cultured high-grade human primary epithelial ovarian cancer (HG-hOEC) cells and their explants; as well as in vivo transgenic mice possessing ovarian cancer were used to assess the molecular mechanism underlying mifepristone (MF) agonistic actions in ovarian cancer progression. FINDINGS: Herein, we show that ovarian cancer cells express traceable/no nuclear P4 receptor (PGR), but abundantly P4 receptor membrane component 1 (PGRMC1). MF significantly stimulated ovarian cancer cell migration, proliferation and growth in vivo, and the translocation of PGRMC1 into the nucleus of cancer cells; the effects inhibited by PGRMC1 inhibitor. The beneficial antitumor effect of high-doses MF could not be achieved in human cancer tissue, and the low tissue concentrations achieved with the therapeutic doses only promoted the growth of ovarian cancers. INTERPRETATION: Our results indicate that treatment of ovarian cancer with MF and P4 may induce similar adverse agonistic effects in the absence of classical nuclear PGRs in ovarian cancer. The blockage of PGRMC1 activity may provide a novel treatment strategy for ovarian cancer. FUND: This work was supported by grants from the National Science Centre, Poland (2013/09/N/NZ5/01831 to DP-T; 2012/05/B/NZ5/01867 to MC), Academy of Finland (254366 to NAR), Moikoinen Cancer Research Foundation (to NAR) and EU PARP Cluster grant (UDA-POIG.05.01.00-005/12-00/NCREMFP to SW).

Functional Expression of FSH Receptor in Endometriotic Lesions.

CONTEXT: FSH receptor (FSHR), besides being expressed in gonads, is also expressed in some extragonadal tissues at low levels. OBJECTIVE: We examined the functional expression of FSHR in different types of endometriotic lesions. DESIGN: Extensive studies were carried out to detect functional FSHR expression and FSH-stimulated estrogen production in ovarian endometriomas and recto-vaginal endometriotic nodules (RVEN). Normal endometrium, ovary, and myometrium tissues from nonpregnant cycling women served as controls. SETTINGS: This laboratory-based study was carried out on tissue specimens from patients with endometriosis and healthy donors. RESULTS: Endometriotic lesions and normal secretory-phase endometrium showed FSHR expression at both mRNA and protein level. RVEN and ovarian endometrioma demonstrated up-regulated CYP19A1, dependent on the activation of CYP19A1 proximal promoter II. Estrogen receptor-ß (ESR2) expression was significantly increased in RVEN vs normal endometrium. Recombinant human FSH stimulation of RVEN explants significantly increased estradiol production and CYP19A1 and ESR2 expression. FSHR was up-regulated in recombinant human FSH-stimulated endometrial and decidualized stromal cells with increased CYP19A1 expression. CONCLUSIONS: We described a novel functional FSHR expression, where FSH-stimulated CYP19A1 expression and estrogen production in RVEN are demonstrated. This locally FSH-induced estrogen production may contribute to the pathology, development, progression, and severity of RVEN.