Apoptosis and pAKT levels in cumulus cells of patients undergoing in vitro fertilization program with specific polymorphisms of gonadotropins and their receptors: a case-control study.

Study question Is there a difference in oocyte competence among patients with different gonadotrophin polymorphisms, after ovarian stimulation with r-FSH? Summary Answer Higher DNA Fragmentation Index and cleaved caspase-3 related to lower level of pAKT has been observed in patients with specific gonadotrophin polymorphism (FSHR and LHB). What is known already • In our experience, the DFI, the percentage of cleaved caspase-3 and the pAKT on cumulus cells can be used as molecular markers of oocyte competence; • The polymorphic variant of LHB is characterized by an extra glycosylation signal into the β subunit. This molecular variation influences the pharmacokinetic properties of v-betaLH, showing an elevated bioactivity in vitro, but a significantly shorter half-life in circulation if compared with the wild type LH. These patients show sub-optimal ovarian response after pharmacological ovarian stimulation with r-FSH; We know that FSH receptor (FSHR) variants, Thr307/Asn680 and Ala307/Ser680, are involved in the response to ovarian stimulation. Study design, size, duration The aim of the study was to determine the apoptosis levels (in terms of percentage of DNA fragmentation and percentage of cleaved caspase-3) and the pAKT levels as potential markers of oocyte competence in the cumulus cells of individual patients with specific FSHR and LHB polymorphisms. It was a retrospective study. It was included 36 patients. The duration of the study was 12 months. Participants/materials, setting, methods Patients were normo-responder patients with a normal basic level of FSH (<12 IU/mL), body mass index <28, aged < 38 years old, with a minimum of 3 MII oocytes collected. The patients were treated with a GnRH agonist and r-FSH. Cumulus cells were collected after treatment with a hyaluronidase solution. SNPs of FSHR and LHB gene will be amplified by PCR in different tubes, using different primers. Main results and the role of chance In this study population we found the following phenotypes: - for FSHR: A/T – S/N n=18; A/A S/S n=6; T/T N/N n=12 - for LHB: W/W I/I n=23; W/R I/T n=13 A higher level of apoptosis in terms of DFI and amount of active protein caspase-3 (p <0.05 vs. all other combinations) has been observed in patients with phenotype A / TS / N associated with W / RI / T (double heterozygous), correlating with an inverse proportion of survival factor pAKT. The analysis of clinical data between the double heterozygous and other combinations showed that there are no statistically significant differences in terms of total number of oocytes, oocytes in Metaphase I, zygotes, high quality embryos. No statistical difference was found also in the pregnancy rate (25% in double heterozygous compared to 30% in other combinations). Limitations, reasons for caution The main limitations could be the low number of patients with polymorphic variants among the cohort of patients included in the study. Wider implications of the findings We expect that patients with double polymorphism for LHB and FSHR will produce oocytes with a limited competence after ovarian stimulation with r-FSH. So, it will be possible to personalize the ovarian stimulation according to polymorphic condition of the individual patient. Study funding/competing interest(s) Founded by a research grant from Merck Serono S.p.A, Italy Trial registration number The trial is an observational study and no registration is needed.