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Objective: To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing in vitro fertilization., Design: Basic science research with a preclinical allosteric FSHR agonist., Setting: University hospital., Patient(s): Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS., Intervention(s): Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle., Main Outcome Measure(s): Estradiol (E 2 ) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells., Result(s): TOP5300 consistently stimulated E 2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS., Conclusion(s): TOP5300 appears to stimulate E 2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E 2 production., Competing Interests: Declaration of interests J.Z.G. reports funding from NIH R01 HD032067 and Baylor College of Medicine Department of Obstetrics and Gynecology for the submitted work. D.M. has nothing to disclose. H.Y. is the Chief executive officer and reports stock options from CanWell Pharma. F.S.S. has nothing to disclose. H.L.J. has nothing to disclose. W.E.G. has nothing to disclose. M.M.M. has nothing to disclose. S.P. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects., Design: Case-control., Setting: University research setting., Patient(s): Premenopausal women., Intervention(s): Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions., Main Outcome Measure(s): Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) β., Result(s): Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERβ-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERβ based on small interfering RNA knockdown and chromatin immunoprecipitation of ERβ followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor., Conclusion(s): ERβ leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To define the expression and function of DNA methyltransferases (DNMTs) in response to decidualizing stimuli in endometriotic cells compared with healthy endometrial stroma., Design: Basic science., Setting: University research center., Patient(s): Premenopausal women with or without endometriosis., Intervention(s): Primary cultures of stromal cells from healthy endometrium (E-IUM) or endometriomas (E-OSIS) were subjected to in vitro decidualization (IVD) using 1 μM medroxyprogesterone acetate, 35 nM 17β-estradiol, and 0.05 mM 8-Br-cAMP., Main Outcome Measure(s): Expression of DNMT1, DNMT3A, and DNMT3B in E-IUM and E-OSIS were assessed by quantitative real-time polymerase chain reaction and immunoblotting. Recruitment of DNMT3B to the promoters of steroidogenic factor 1 (SF-1) and estrogen receptor α (ESR1) was examined by chromatin immunoprecipitation., Result(s): IVD treatment reduced DNMT3B messenger RNA (74%) and protein levels (81%) only in E-IUM; DNMT1 and DNMT3A were unchanged in both cell types. Significantly more DNMT3B bound to the SF-1 promoter in E-IUM compared with E-OSIS, and IVD treatment reduced binding in E-IUM to levels similar to those in E-OSIS. Enrichment of DNMT3B across 3 ESR1 promoters was reduced in E-IUM after IVD, although the more-distal promoter showed increased DNMT3B enrichment in E-OSIS after IVD., Conclusion(s): The inability to downregulate DNMT3B expression in E-OSIS may contribute to an aberrant epigenetic fingerprint that misdirects gene expression in endometriosis and contributes to its altered response to steroid hormones., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To define altered gene expression networks in endometriosis., Design: Experiments using endometriotic tissues and primary cells., Setting: Division of Reproductive Biology Research, Northwestern University., Patient(s): Premenopausal women., Intervention(s): Matched samples of eutopic endometrium and ovarian endometriosis (n = 8 patients) were analyzed by microarray and verified in a separate set of tissues (n = 6 patients). Experiments to define signaling pathways were performed in primary endometriotic stromal cells (n = 12 patients)., Main Outcomes Measure(s): Using a genome-wide in vivo approach, we identified 1,366 differentially expressed genes and a new gene network favoring increased glucocorticoid levels and action in endometriosis., Result(s): Transcript and protein levels of 11β-hydroxysteroid dehydrogenase (HSD11B1), which produces cortisol, the biologically active glucocorticoid, were strikingly higher, whereas messenger RNA (mRNA) levels of the cortisol-degrading HSD11B2 enzyme were significantly lower in endometriotic tissue. Glucocorticoid receptor mRNA and protein levels were significantly higher in endometriosis. The inflammatory cytokine tumor necrosis factor robustly induced mRNA and protein levels of HSD11B1 and glucocorticoid receptor but suppressed HSD11B2 mRNA in primary endometriotic stromal cells, suggesting that tumor necrosis factor stimulates cortisol production and action. We also uncovered a subset of genes critical for prostaglandin synthesis and degradation, which favor high eicosanoid levels and activity in endometriosis., Conclusion(s): The proinflammatory milieu of the endometriotic lesion stimulates cortisol synthesis and action in endometriotic lesions., (Published by Elsevier Inc.)