The profile of steroid hormones in human fetal and adult ovaries.

BACKGROUND: Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. METHODS: We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. RESULTS: Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17ß-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. CONCLUSIONS: Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

Identification of biomarkers and outcomes of endocrine disruption in human ovarian cortex using In Vitro Models.

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women. However, there is a lack of information regarding mechanisms and effects in humans. Our study aims to identify mechanisms of endocrine disruption using two EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ)1. Human ovarian cortical tissue obtained from Caesarean section patients was exposed to 10-9 M - 10-5 M KTZ and 10-10 M - 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied via histology analysis and liquid-chromatography-mass spectrometry-based steroid quantification. RNA-sequencing was performed on COV434, KGN, and primary ovarian cells that were exposed for 24 h. Significantly lower unilaminar follicle densities were observed in DES 10-10 M group, whereas low KTZ exposure reduced secondary follicle density. KTZ 10-5 M reduced levels of pregnenolone and progesterone. RNA-sequencing revealed that 445 and 233 differentially expressed genes (false discovery rate < 0.1) altogether in DES and KTZ exposed groups. Gene set variation analysis showed that both chemicals modulated pathways that are important for folliculogenesis and steroidogenesis. We selected stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) for further validation. Up-regulation of both genes in response to KTZ was confirmed by qPCR and in situ RNA hybridization. Further validation with immunofluorescence focused on the expression of SCD in growing follicles in exposed ovarian tissue. In conclusion, SCD may serve as a potential novel human-relevant biomarker of EDC exposure and effects on ovaries.