An Association And Functional Study Of Thr307Ala And Asn680Ser Gene Polymorphisms With Ovarian Response

Objective:We aimed to determine if FSHR gene polymorphisms were associated with therapeutic efficacy of FSH in Chinese infertility patients, and to investigate whether FSHR gene polymorphisms affect the expression and function of the FSHR.Methods:450Chinese women were recruited in an assisted reproductive technology program from October2011to March2012. FSHR polymorphisms at the positions307and680were examined by PCR-RFLP. Serum FSH and estradiol level, FSH amount, ovarian response and pregnancy rate were recorded during treatment. The haplotype analysis for FSHR was performed using PHASE. Linkage disequilibrium between SNPs was estimated using Haploview. ANCOVA, Chi-square analysis and logistic regression analysis were used to analyze the data. Furthermore, the two candidated SNPs were recreated by site-directed mutagenesis and tested for their effect on FSHR expression and function in stable transfected HEK293cell lines. Real-time PCR and western blot were used to determine the mRNA and protein expression. At last, the effect of mutations on FSHR-mediate cAMP pathway was determined using ELISA (enzyme linked immunosorbent assay) assay.Results:1. The basal FSH levels were higher in AA [7.38±2.07vs6.34±1.75,6.63±1.94, P<0.05,95%CI (6.75,8.01)] and SS [7.51±2.01vs6.31±1.75,6.66±1.96, P<0.05,95%CI (6.88,8.15)] compared to other genotypes respectively; the days for ovulation induction was slightly longer in AA and SS.2. Women with AA and SS have higher rates of poor response compared to carriers of other genotypes (P<0.05). There was no significant association between the occurrence of OHSS and the genotypes at either position307or680; 3. Furthermore, there is a nearly complete linkage between these two polymorphisms in Chinese women (D’=0.95, r2=0.84).4. The FSHR mRNA and protein expression levels were not affected by the Thr307Ala and Ser680Asn polymorphisms. None of these mutations had any effect on cAMP levels in HEK293cell line.Conclusion:In Chinese women receiving ART, the subjects with AA and SS genotypes require higher amounts of FSH for ovarian stimulation. SS genotypes are associated with an increased risk of poor response. The Thr307Ala and Ser680Asn polymorphisms did not affect FSHR expression, and independent of FSHR-mediate cAMP pathway. Twelve figures,5tables,68references were included in this paper.