EVALUATION OF OOCYTE QUALITY IN GRANULOSA AND CUMULUS CELLS OF PATIENTS UNDERGOING PMA

Abstract

Background. We investigate the apoptosis rate of individual granulosa cell–oocyte and cumulus cell–oocyte (COC), associated with the levels of molecules playing a critical role in the regulation of cell death or survival. These molecular analyses have been done to verify the difference of competence between oocytes producing embryos able to reach the blastocyst stage compared with embryos arrested during the in vitro culture. Methods. From each single follicle: granulosa cells were processed for Western blotting analyses, using the following antibodies: pAKT, ERK 1/2, pERK 1/2; cumulus cells were used for in situ immunofluorescence with the same antibodies. DNA fragmentation rate was measured by TUNEL assay. Results. We have involved 58 patients and recovered 255 MII oocytes, of which 197 were fertilized and the derived embryos had the following evolution: 117 transferred, 57 vitrified and 23 arrested; 58 oocytes failed the fertilization or were in GV or MI stages. In the cumulus cells: we found a significant inverse correlation between oocytes resulting in transferred and arrested embryos in the ratio pAKT/TUNEL; nuclear localization of pERK1/2 showed a significant inverse correlation pERK1/2/TUNEL and a significant direct correlation with the intracellular accumulation of pERK1/2/pAKT. In granulosa cells: oocytes able to produce blastocysts, ERK1/2 /TUNEL ratio was higher than in cells of arrested embryos. Conclusions. Cumulus and granulosa cells showed different levels of expression of the investigated molecules. We found that in the cumulus cells of the oocytes able to produce blastocysts, the pAKT/TUNEL ratio is higher than in cumulus cells of arrested embryos, indicating that pAKT is involved in survival pathways. Moreover, pERK1/2 has an anti-apoptotic effect, when translocated into the nucleus. In granulosa cells: ERK1/2 indicates that it is involved in survival pathways. Briefly, we demonstrated that DNA fragmentation rate related to specific molecular levels could be considered a molecular marker of oocyte competence, for the evaluation of a prognostic pattern of blastocyst formation.