Depot Pure GnRH Antagonist for Long-term Treatment of Ovarian Hyperthecosis Monitored by Multi-Steroid LCMS Profiling

Ovarian hyperthecosis (OHT), severe hyperandrogenism after menopause in the absence of ovarian or adrenal tumors, is usually treated by surgical excision. We report a 58-year-old woman presenting with severe hyperandrogenism (serum testosterone 15.7-31.0 nmol/L, normal female <1.8 nmol/L) with menopausal gonadotropins and virilization but no adrenal or ovarian lesions. Multi-steroid profiling by liquid chromatography-mass spectrometry (LCMS) of adrenal and ovarian vein samples identified strong gradients in left ovarian vein (10-30-fold vs peripheral blood in 17OHP4, 17 OHP5, A4, T, DHEA) but right ovarian vein could not be cannulated with the same findings in a second ovarian vein cannulation. OHT diagnosis was confirmed by an injection of a depot pure GnRH antagonist (80 mg Degarelix, Ferring) producing a rapid (< 24hr) and complete suppression of ovarian steroidogenesis as well as serum LH and FSH lasting at least 8 weeks with reduction in virilization but injection site reaction and flushing and vaginal spotting, ameliorated by an estradiol patch. Serum testosterone remained suppressed at 313 days after the first dose despite recovery of menopausal gonadotropins by day 278 days. This illustrates use of multi-steroid LCMS profiling for confirmation of the OHT diagnosis by ovarian and adrenal vein sampling and monitoring of treatment by peripheral blood sampling. Injection of a depot pure GnRH antagonist produced rapid and long-term complete suppression of ovarian steroidogenesis maintained over 10 months. Hence a depot pure GnRH antagonist can not only rapidly confirm the OHT diagnosis but also induce long-term remission of severe hyperandrogenism without surgery.

Zika virus infection in the ovary induces continuously elevated progesterone level and compromises conception in interferon α/β receptor-deficient mice

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impacts of ZIKV infection on nonpregnant female reproductive health are not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/β receptor-deficient ( Ifnar1 -/- ) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle, and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months post infection, the damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE ZIKV, a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barre syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on genital tract of non-pregnant female. This study investigated the effects of ZIKV on the ovary in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function, and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.

PSXII-23 Sirtuins expression and activity in bovine granulosa cells are regulated by hormones that influence ovarian steroidogenesis

Sirtuins (SIRTs) are a family of seven NAD+-dependent histone deacetylases that regulate several biological reactions. How SIRTs regulate ovarian steroidogenesis in cattle remains to be fully unveiled. We hypothesize that SIRTs expression and activity are regulated by hormones that influence steroidogenesis in bovine granulosa cells (GC). Bovine ovaries were collected at an USDA-inspected commercial slaughterhouse and GC were isolated from small antral follicles (1–5 mm on surface diameter). Cells were treated with hormones that regulate ovarian folliculogenesis: FSH, IGF1, fibroblast growth factor (FGF) 2, FGF9, and their combinations. Cells were cultured for 24h for total RNA isolation (n = 6 pools) with miRNeasy microkit (Qiagen) or for 48h for isolation of nuclear and cytoplasmic extracts (n = 3 pools) with EpiQuik Nuclear Extraction Kit (Epigentek) according to the manufacturers’ instructions. Relative mRNA abundance was quantified via qPCR and expressed as 2-ΔΔCt using the relative comparative threshold cycle (Ct) whereas SIRTs activity in nuclear (SIRTs 1, 6, and 7) and cytoplasmic (SIRTs 2, 4, and 5) extracts was analyzed with the Epigenase Universal SIRT Activity/Inhibition assay kit (Epigentek) following the manufacturer’s instructions. Data were analyzed via ANOVA with GLM procedures of SAS for Windows. In terms of mRNA relative abundance, FSH+IGF1+IGF9 increased mRNA relative expression of SIRTs 2 to 7 in comparison to negative control and of SIRTs 2, 3, 4, 6, and 7 in comparison to FSH+IGF1; FSH+IGF1+IGF2 increased mRNA relative abundance of SIRTs 2 and 6 in comparison to FSH+IGF1; FGF2 alone increased SIRT1 in comparison to negative control (P < 0.05). In term of SIRTs activity, FGF2 alone increased nuclear SIRTs activity in comparison to FSH, IGF1, FSH+IGF1, and FGF9 alone; FSH+IGF1+IGF2 increased cytoplasmic SIRTs activity in comparison to all treatments (P < 0.05). Taken together, our data demonstrate that SIRTs expression and activity in bovine GC are regulated by hormones that influence steroidogenesis.

Urinary bisphenol A in women with polycystic ovary syndrome – a possible suppressive effect on steroidogenesis?

Objectives There is a growing evidence indicating an impact of endocrine distrupting chemicals such as bisphenol A (BPA) on human reproduction. Its higher levels in serum or urine have been documented in women with polycystic ovary syndrome (PCOS), however the relationship to ovarian steroidogenesis remains unclear. Aim of the study was to compare urinary BPA (U-BPA) concentrations among PCOS women and control group. Second aim was to assess the relationship of U-BPA to ovarian steroidogenesis in the group with PCOS. Methods Eighty six Caucasian women (age 28.5 ± 5.1 years) diagnosed with PCOS and 32 controls of age 24.9 ± 4.4 years were included in the study. Fasting blood samples were analyzed for biochemical parameters and steroid hormones. U-BPA was measured in the morning urine sample using high pressure liquid chromatography. Results PCOS women had significantly higher U-BPA as compared with control group (p=0.0001). Those with high levels of U-BPA (U-BPA ≥2.14 ug/g creatinine) demonstrated higher serum insulin (p=0.029) and HOMA IR (p=0.037), lower serum estrone (p=0.05), estradiol (p=0.0126), FSH (p=0.0056), and FAI (p=0.0088), as compared with low-BPA group (U- BPA <2.14 ug/g creatinine). In PCOS women, U-BPA positively correlated with age (p=0.0026; R2=0.17), negatively with estradiol (p=0.0001, R2=0.5), testosterone (p=0.0078, R2=0.15), free-testosterone (p=0.0094, R2=0.12) and FAI (p=0.0003, R2=0.32), respectively. Conclusions PCOS women have significantly higher U-BPA concentrations than healthy controls. U-BPA positively correlates with age and negatively with ovarian steroid hormones suggesting a possible suppressive effect of bisphenol A on ovarian steroidogenesis.