Oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n = 154) and from perimenopausal and postmenopausal women (n = 28). Serum concentrations of BMP15 and GDF9 were analyzed in women relative to age, anti-Müllerian hormone, number of oocytes retrieved, and polycystic ovary syndrome (PCOS) after superovulation for in vitro fertilization. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/mL, respectively), and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64-fold and 15-fold, respectively, between women, but they did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15 concentration, was associated with oocyte number retrieved in patients without PCOS (P = 0.018). GDF9 and BMP15 associations with oocyte number differed significantly (P < 0.05) with PCOS status. GDF9 concentrations were lower in poor responders (women with fewer than four oocytes retrieved or with cancelled cycles; P = 0.020). Serum BMP15, but not GDF9, was lower in women >55 years of age, compared with women of reproductive age (P < 0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.
The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally and it is hypothesised that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer, termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate, and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine if cumulin regulates granulosa cell inhibin and activin production, and if this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from IVF patients were cultured ± FSH with various forms of recombinant cumulin (native and cysteine mutants, and with/without the pro-domains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature-and pro-forms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin ± FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced
Bone morphogenetic protein-15 (BMP15) and growth differentiation factor-9 (GDF9) are oocyte-secreted factors critical for folliculogenesis, oocyte developmental competence and fertility. BMP15 and GDF9 are produced only by gametes and despite known associations with reproductive pathologies (1), concentrations in serum have not previously been reported. We have developed novel immunoassays that allow quantitative measurement of BMP15 and GDF9 in serum and have applied these to samples collected from women undergoing IVF to investigate the possibility that these proteins may be useful biomarkers of female reproductive function. The BMP15 and GDF9 immunoassays were developed in-house and validated for sensitivity (24 and 26 pg/ml, respectively), specificity (<0.01% and <0.03%, respectively) and reproducibility (inter-assay CV <10%). BMP15 and GDF9 were not detectable in a significant number of samples (33% for BMP15; 71% for GDF9) and these were assigned the sensitivity value for subsequent analyses. The effect of superovulation of patients undergoing IVF treatment on serum BMP15 and GDF9 was assessed using samples collected immediately before and on multiple days during FSH stimulation in antagonist treatment cycles (56 bloods from 14 women). BMP15 and GDF9 varied 64-and 15-fold respectively between women, but, within an individual were unchanged throughout superovulation, and were independent of FSH dose (P>0.05). Further analyses including an additional 141 women treated for infertility demonstrated no difference in BMP15 or GDF9 between GnRH antagonist (n=69) and agonist (n=17) stimulation cycles, or between these stimulated cycles and unstimulated cycles (n=41). Serum GDF9 positively correlated with the number of oocytes retrieved from non-PCO/PCOS women after superovulation (r=0.439, P=0.05, n=27), but not in PCO/PCOS patients. Serum GDF9, but not BMP15, was significantly lower in poor responders (women with <4 oocytes retrieved or cycle cancellation due to insufficient follicles on ultrasound (2)), compared with normal responders (27.2±0.8 vs 50.6±7.0 pg/ml; P<0.05). Serum BMP15 and GDF9 did not correlate with age, however BMP15 positively correlated with day 2 baseline FSH (r=0.305, P<0.05, n=52). This is the first report of quantitative measurement of BMP15 and GDF9 levels in human serum, correlating with reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine. References: (1) Persani L et al., HRU 2014; 20(6):869-883. (2) Ferraretti et al., Hum Reprod 2011;26:1616-24. Funding : National Health and Medical Research Council, Australia.
Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are TGF-β proteins that regulate key processes throughout folliculogenesis and are determinants of mammalian fecundity (1). They are uniquely produced predominantly by the oocyte and have potential clinical application as markers of oocyte quality and quantity (2). However, no studies have been conducted to assess whether serum concentrations alter across the different phases of the menstrual cycle, and thus if assessment should be confined to specific cycle stages. The aim of this study was to measure serum concentrations of these proteins during the menstrual cycle in women at different stages of reproductive life. Serum was collected every 1-3 days throughout the menstrual cycle from 41 healthy ovulatory women from three cohorts: menses to late luteal phase (21-29 years of age; n=16; University of Otago) and across one interovulatory interval (18-35 years of age; n=10; and 45-50 years of age; n=15; University of Saskatchewan), with simultaneous ultrasound scans confirming ovulation. Serum concentrations of GDF9, BMP15, estradiol, FSH, LH, progesterone, inhibin A and B and AMH were measured. GDF9 and BMP15 were detectable in 54% and 73% of women and varied 236- and 52-fold between women, respectively. To detect changes, mean concentrations and variances across the cycle were statistically modelled using a generalized additive model of location, shape and scale (GAMLSS). Across the menstrual cycle, there were minimal changes in serum GDF9 or BMP15 within a woman for all cohorts, with no significant differences detected in modelled mean concentrations. However, modelled variances were highest in the luteal phases of all women for BMP15 immediately following ovulation, regardless of age, suggesting a possible underlying cyclic pattern. These results suggest that serum BMP15 and GDF9 are not overtly affected by menstrual cycle dynamics but may be more stable in the follicular phase. Larger studies with more frequent sampling should establish if BMP15 and presumably GDF9 demonstrate clinically relevant cyclic variation. References: (1) Gilchrist RB et al., HRU 2008; 14:159-77. (2) Riepsamen AH et al., Endocrinol 2019; 160:2298-313.
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