RELATIONSHIP BETWEEN DNA FRAGMENTATION INDEX AND pAKT IN CUMULUS CELLS: NEW MARKERS OF OOCYTE COMPETENCE

The specific LH and FSH polymorphisms could influence the growth of follicles and oocytes.Some studies have shown that certain single nucleotide polymorphisms of FSHR are associated with changes in the ovarian activity, having functional implications in human reproduction. Carriers of polymorphic variant of betaLH show sub-optimal ovarian response to the standard long GnRH-agonist down-regulation protocol, when stimulated with recombinant FSH. No studies have been designed relating the polymorphic variants of FSHR and LHB with the oocyte competence. In previous studies, we demonstrated the correlation between the apoptosis rate and the expression level of some survival pathways molecules, as pAKT, in cumulus cells, as potential markers of oocyte competence. The aim of this study is to investigate the apoptosis levels of cumulus cells pool collected from the cumulus-oocyte complex of individual patients with specific FSHR and LHB polymorphisms. Cumulus cells, obtained from 36 selected patients, were used for in situ immunocytochemistry by cleaved caspase-3, pAKT and by TUNEL assays. Genomic DNA was extracted from whole blood samples. SNPs of FSHR and LHB gene was amplified by PCR using different primers. 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 Cumulus cells of patients with phenotype A/T–S/N associated with W/R–I/T (double heterozygous) showed an higher level of apoptosis in terms of DNA Fragmentation Index (DFI) and percentage of active protein caspase-3 P<0.05 vs all other combinations, and an inverse proportion of the pAKT levels. However no statistical difference was found in clinical data. In conclusion, these patients with double heterozygous will produce oocytes with a limited competence after ovarian stimulation with rFSH. So, it will be possible to personalize the ovarian stimulation according to polymorphic condition of the individual patient.