Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1-7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6-7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6-7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH.The prediction of an individual patient's response to FSH is essential to optimize treatment outcome for assisted reproductive techniques and to minimize the associated risk of ovarian hyperstimulation syndrome. Previous clinical studies have demonstrated differences in the ovarian response depending on a single nucleotide polymorphism (SNP) at amino acid position 680 of the FSH receptor (FSHR). This study was designed to evaluate in vitro the underlying cellular mechanisms. It included two patient groups homozygous for the two variants of the FSHR SNP 680 (NN or SS) undergoing ovarian stimulation for assisted reproductive techniques. Granulosa cells (GC) as the target of FSH action in the ovary and main source of female sex steroids were isolated during oocyte retrieval and cultured for 1-7 days. Basal concentrations of oestradiol and progesterone were measured after short-term culture and oestradiol, but not progesterone, was significantly higher in the NN compared to the SS variant. As GC were irresponsive to FSH stimulation, the culture period was extended and re-appearance of FSHR expression was demonstrated by immunofluorescence microscopy with increasing days in culture. Upon stimulation with FSH, GC at day 6-7 of culture displayed a dose-dependent increase of cyclic adenosine monophosphate (cAMP), oestradiol and progesterone, but no statistically significant difference in the kinetic values between both FSHR variants. In conclusion, we have established a suitable system to study the effects of FSH ex vivo in primary long-term GC cultures. To fully explain the clinically observed differences in the FSHR variant-mediated response to FSH, factors downstream of P production or external to GC might be involved.