Characterization Of Expression, Mutation And Function Of GDF-9 In Women With Diminished Ovarian Reserve

Ovarian reserve refers to the number and quantity of follicle in relation to women's reproductive potential that remains in the ovary at any given time. Diminished ovarian reserve is a condition where the ovaries presented reduced ability to produce quality oocytes that are essential for conception. Diminished ovarian reserve will develop into premature ovarian failure (POF), and it was regarded as the most threatening condition against women's reproductive health. Most researchers can only rely on biomarkers that predict and evaluate ovarian status, as the amount of data available to reveal the precise molecular mechanisms of diminished ovarian reserve are minimal.Chronological age is generally considered as a primary factor in ovarian function. However, the ovarian aging in accordance to human physiological age is highly variable. This ovarian characteristic indicated that the intra-ovarian factors are more likely to be the determinants. The abnormal follicular development and follicular atresia resulted from granulosa cells apoptosis dedicated diminished ovarian reserve. Thus, identification of the genes involved in this process is critical for revealing the pathogenesis of diminished ovarian reserve. There are accumulating evidences that oocyte-secreted factors play an important role in regulating the function of surrounding somatic cells during folliculogenesis. Growth differentiation factor-9 (GDF-9) is the first reported oocyte-secreted factor that belongs to TGF-βsuperfamily. Previous studies confirmed that GDF-9 was involved in several aspects of ovarian development, including promoted follicle progress and survive, stimulated granulosa cells proliferation and inhibit apoptosis, controlled ovulation, enhanced oocyte potential and regulated hormone in ovary. Due to its critical involvement in follicle development, GDF-9 has been proposed as a potential candidate gene for POF and PCOS, and several mutations has been reported. In addition, mutations were also found in mothers of dizygotic twins. All these results suggest that GDF-9 gene might play a role in the ovarian function. There is no published diminished ovarian reserve.In this study, we aim to explore the implications of GDF-9 in diminished ovarian reserve by analyzing the relationship between GDF-9 SNPs and diminished ovarian reserve, characterizing the functional significance of mutation, and identifying the GDF-9 protein level in diminished ovarian reserve and its role in granulosa cell apoptosis.Part I Expression of the GDF-9 in women with diminished ovarian and its regulation in granulosa cell apoptosisObjective:To detect the GDF-9 level in follicular fluid (FF) and p53 mRNA level in granulosa cells in women with diminished ovarian reserve, and investigated its role in granulosa cell apoptosis.Methods:68 ovarian poor responders and 97 normal responders were examined. Western Blot was applied to analysis the GDF-9 level in FF, Real-time PCR was performed to detect the p53 mRNA expression in granulosa cells. Colleted the human granulose cells from women underwent IVF and cultured them. After the primary cultured granulosa cells were treated with various concentration of human recombined GDF-9 for 24h, the expression of p53 mRNA level was examined by real-time, the protein expression was examined by Western Blot.. Results:Western Blot results showed that the GDF-9 level of poor responders was significantly lower than normal responders. Real-time analysis showed that p53 mRNA expression was significantly up-regulated in granulosa cells of poor responders. p53 mRNA and protein expression in primary cultured granulosa cells was decreased in a dose-dependent manner after treated with GDF-9.Conclusion:Decreased GDF-9 level up-regulated the p53 mRNA expression in granulosa cells which may induce granulosa cells apoptosis, and resulted in diminished ovarian reserve.PartⅡMolecular screening of GDF-9 gene in women with diminished ovarian reserveObjective:To investigate the association of growth differentiation factor-9 (GDF-9) polymorphisms and diminished ovarian reserve, and to explore the mechanism of diminished ovarian reserve related poor IVF outcome..Methods:We sequenced GDF-9 gene in 106 women with diminished ovarian reserve, with 123 women with normal ovarian reserve as control. Analyze allele and genotype frequencies of polymorphisms in both group, and association with IVF outcomes, include ovarian response, good quality embryos and pregnancy rate.Results:The GA/AA genotype and A allele frequencies of G546A was significantly higher (P<0.05) in women with diminished ovarian reserve compared with women with normal ovarian reserve. Within diminished ovarian reserve group, carrier of GDF9 A allele more often had poor ovarian response (64.7% vs.23.6%, P<0.001), lower fertilization rate, good quality embryos and pregnancy rate than non-carriers (P<0.001). In contrast, there are no differences in those parameters in women with normal ovarian reserve. C447T polymorphism was unrelated to ovarian reserve and ovarian stimulation outcome in either women with diminished ovarian reserve or women with normal ovarian reserve. Conclusions:Genetic variants of GDF-9 G546A polymorphism is linked with diminished ovarian reserve and may account for poor IVF outcome in women with diminished ovarian reserve.PartⅢExpression and functional analysis of GDF-9 mutation causing dinminshed ovarian reserveObjectives:To report the genetic screening in a group of young women with diminished ovarian reserve and examine the functional outcomes of the observed mutations.Methods:DNA sequencing of GDF-9 gene were performed in all patients. Mutant protein secretion was analyzed using the media from the HEK293T cell transfencted with expression vectors containing mutated and wild-type human GDF-9 cDNA. Mutant protein activity was performed on human primary granulosa cells.Results:Sequencing analysis revealed two mutations (p.D57Y and p.R164C). GDF-9 p.R164C was absent from control population. Functional analysis indicated that this mutation reduced mature protein secretion, impaired ability to stimulate human gramulosa cells proliferation and activate Smad2 phosphoralation. In contrast, GDF-9 p.D57Y was found both diminished ovarian reserve and control women (6 vs.2). We did not observe any deleterious effects of this mutation on protein function.Conclusion:This first report suggests that in young women with diminished ovarian reserve, a GDF-9 mutation should be sought. A GDF-9 p.R146C mutation leading to a defective functional protein production may be a new mechanism of early onset of ovarian failure.