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Primary ovarian insufficiency (POI) is defined as the loss of ovarian function before 40 years of age. It clinically manifests as amenorrhea, infertility, and signs of estrogen insufficiency. POI is frequently induced by chemotherapy. Gonadotoxic chemotherapy reagents damage granulosa cells, which are essential for follicular function and development. Our recently published studies demonstrated that intraovarian transplantation of human mesenchymal stem cells (hMSCs) can restore fertility in a chemotherapy-induced POI mouse model. However, the regenerative mechanism underlying the hMSC effect in POI mice is not fully understood. Here, we report that the hMSC secretome increased the proliferation of human granulosa cells (HGrC1). We showed by FACS analysis that treatment of HGrC1 cells with hMSC-conditioned media (hMSC CM) stimulates cellular proliferation. We also demonstrated that the expression of steroidogenic enzymes involved in the production of estrogen, CYP19A1 and StAR, are significantly elevated in hMSC CM-treated HGrC1 cells. Our data suggest that hMSC CM stimulates granulosa cell proliferation and function, which may explain the therapeutic effect of hMSCs in our chemotherapy-induced POI animal model. Our findings indicate that the hMSC secretome may be a novel treatment approach for restoring granulosa cell and ovarian function in patients with POI.

Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3′,5′-cyclic adenosine 5′-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals.

Context The role of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) on aromatase regulation is poorly understood in humans. Objective Determine GDF9 and BMP15 effects on FSH stimulation of estradiol production in primary human cumulus granulosa cells (GCs). We hypothesized that the combination of GDF9 and BMP15 potentiates FSH-induced aromatase expression. Design Primary human cumulus GCs in culture. Setting University infertility center. Patients or Other Participants GCs of 60 women undergoing in vitro fertilization were collected. Interventions Cells were treated with GDF9 and/or BMP15 (GB) in the presence or absence of FSH, dibutyryl cAMP, or SMAD inhibitors. Main Outcome Measures Promoter activity, mRNA, protein, and estradiol levels were quantified. Results FSH and GB treatment increased CYP19A1 promoter activity, mRNA, and protein levels as well as estradiol when compared with cells treated with FSH only. GB treatment potentiated cAMP stimulation of aromatase and IGF2 stimulation by FSH. GB effects were inhibited by SMAD3 inhibitors and IGF1 receptor inhibitors. GB, but not FSH, stimulates SMAD3 phosphorylation. Conclusion The combination of GDF9 and BMP15 potently stimulates the effect of FSH and cAMP on CYP19a1 promoter activity and mRNA/protein levels. These effects translate into an increase in estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway and involves, at least in part, the effect of the IGF system.

Context Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown. Objective To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs. Design Primary human cumulus GCs in culture. Setting University infertility center. Patients or Other Participants GCs of women undergoing in vitro fertilization. Intervention(s) Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic homolog (SMAD) inhibitors. Main Outcome Measure(s) Quantification of mRNA, protein, promoter activity, and DNA methylation. Results FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P < 0.0001) in a concentration-dependent manner showing a maximal effect at 5 ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P < 0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P < 0.009). Conclusions For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.

The objective of this study was to evaluate basal anti-Müllerian hormone as a marker for ovarian responsiveness to fertility treatment.Frozen basal menstrual cycle day 3 serum samples were evaluated retrospectively for anti-Müllerian hormone, inhibin B, and follicle-stimulating hormone levels in 123 in vitro fertilization cycles (93 patients) and compared with in vitro fertilization records.Anti-Müllerian hormone values correlated the best with the number of retrieved oocytes (r = 0.539; P.001) relative to age (r = -0.323; P.01), follicle-stimulating hormone (r = -0.317; P.01), inhibin B (P.05), luteinizing hormone (P.05), and estradiol (r = -0.190; P.05). Receiver operating characteristic curve analysis demonstrated that, for the prediction of4 oocytes retrieved, anti-Müllerian hormone had the largest area under the curve (AUC = 0.81; P = .0001) relative to age (r = 0.74; P = .005), follicle-stimulating hormone (0.71; P = .02), inhibin B (0.66; P = .03), and estradiol (0.54; P.05). Similarly, for the prediction ofor=15 retrieved oocytes, anti-Müllerian hormone had the largest area under the curve (0.80; P = .0001) relative to age (0.63; P = .02), follicle-stimulating hormone (0.64; P = .005), inhibin B (r = 0.57; P.05), and estradiol (0.58; P.05).Anti-Müllerian hormone correlates better than age, follicle-stimulating hormone, luteinizing hormone, inhibin B, and estradiol with the number of retrieved oocytes. Receiver operating characteristic curves estimated that anti-Müllerian hormone accurately predicts ovarian responsiveness to controlled ovarian stimulation with high sensitivity and specificity.