Association of rs6166 polymorphism with FSH receptor transcript variants and steroid production in human granulosa cell cultures

Abstract

Follicle stimulating hormone receptor (FSHR) genetic variation at position 2039 A > G (rs6166, p.Asn680Ser) was repetitively shown to correspond to the measures of ovarian sensitivity and the outcomes of gonadotropin stimulation. However, to date, there has been no study revealing the mechanisms behind the associations observed. The aim of the present research was to investigate the relationship between rs6166 and mRNA expression of FSHR-dependent genes such as LHCGR, CYP19A1, and FSHR itself with particular reference to the FSHR transcript variants (deletions of exon 2, 6, and 9 and insertion of a novel exon between exons 8 and 9) in the cell culture model. Steroid production and its dependency on FSHR genotype were also assessed. A total of 22 normoovulatory patients undergoing IVF treatment were recruited. Granulosa cells were obtained by ovary puncture and cultured for 7 days to regain responsiveness to FSH in a gonadotropin-free medium. Stimulation was carried out for 24 hours in a serum-depleted environment using 0.5 UI/l rhFSH. Gene expression was assessed by real-time PCR and genotype was determined by allele-specific PCR. The distribution of p.Asn680Ser genotypes was as follows: 10 homozygotes Asn/Asn, 8 heterozygotes Asn/Ser, and 4 homozygotes Ser/Ser. Expression of total FSHR in all samples studied increased by a mean factor of 1.9 (95% CI: 0.39-11.53, p < 0.001) upon stimulation. All of the analyzed FSHR transcript variants were detectable in non-stimulated and stimulated cells. The only distinct transcript that followed up-regulation was deletion of exon 2. Homozygotes Asn/Asn tended to have higher rhFSH-induced expression of FSHR as compared to the carriers of Ser/Ser genotype. Relative expression of LHCGR and CYP19A1 although up-regulated showed no significant difference with respect to the FSHR genotype. Variable modulation of FSHR expression by its own ligand is likely to explain different clinical behavior of patients with FSHR genetic variants. The putative contribution of rs6166 requires further investigation.

Ala307Thr and Asn680Ser Polymorphisms ofFSHRGene in Human Reproduction Outcomes

“…Allelic variants of FSHR determine different FSHR sensitivity's [20,24,26]. Because of that we expected to find that FSHR 's variants influence in the reproduction outcomes, in our results we observed that the number of MII oocytes and embryos are higher in Ala307Ala women.…”

“…Perez Mayorga et al [20] was the first group that observed differences in Asn680Ser genotypes with dose of rFSH in COH. Other groups observed that the FSHR variants influence the basal FSH and estradiol levels with conflicting results [23,24,25,26,27], for this reason, the FSHR gene and others gene variations may play a role in modulating receptor sensitivity and intracellular second messenger cascades to exogenous hCG and other gonadotrophins [24,28], thus the FSHR SNPs may cause changes in the phosphorylation and glycosylation molecular mechanisms [29,30]. …”

Mutations and polymorphisms in FSH receptor: functional implications in human reproduction

“…The level of FSHR expression at transcript and protein levels was significantly reduced in women with AA genotype when compared with GG genotype Desai et al (2011) R242 S S Desai and others stimulation did not differ significantly between the three genotypes, suggesting that the probable contribution of polymorphism at position 680 needs further investigation in long-term GC culture system (Nordhoff et al 2011, Zalewski et al 2013. The level of FSHR expression also has an impact on the FSH action.…”

MECHANISMS IN ENDOCRINOLOGY: Genetics of FSH action: a 2014-and-beyond view

“…hGLC homozygous for each FSHR haplotype responded identically in terms of cAMP, progesterone and estradiol (E 2 ) production, measured at saturation (31). Another experiment suggested that hGLC homozygous for Asn at amino acid position 680 only 'tended' toward higher FSHinduced FSHR expression, whereas the expression of other well-known FSHR-dependent genes, such as LHCGR and CYP19A1, was not affected (32). We recently have analyzed the effects of FSHR exon 10 SNPs in vitro more in depth, looking at the kinetics of response and at different signal transduction pathways.…”