Selection of the best three oocytes for intracytoplasmicsperm injection (ICSI) using apoptotic analysis of cumulus cells

Introduction: We studied the apoptosis rate of the cumulus cells of individualcumulus-oocyte complex (COC), to verify a relationship with clinical outcomes,in terms of pregnancy and implantation rates. Usually oocytes areselected using morphological criteria. We tried to verify if cumulus cell apoptoticrate could be used as a molecular criteria in selecting oocytes with higherimplantation potentiality.Material and Methods: The study design consisted in two different trials: inthe first we investigated apoptosis rate in cumulus cells of the three selectedoocytes, according to Italian law, to be fertilized by intracytoplasmic sperminjection (ICSI). In a second trial, average apoptosis rate of the cumulus cellscoming from the three selected oocytes to be fertilized by ICSI and thepooled remaining oocytes were compared, when more than 5 COCs were aspirated.In a first trial we included 22 consecutive couples undergoing ICSIcycles, 20 in a second one, for a total of 42 patients. We selected the threeoocytes for (ICSI) on the basis of the morphological appearance of thecumulus, according to Veek’s criteria. The cumulus cells of each COC was submittedto apoptotic assays. The patients were classified, on the basis of pregnancysuccess, in A Group (pregnant patients) and B Group (patients withnegative bhCG).Results: Both trials showed that apoptosis in the cumulus cells was remarkablylower in the A Group if compared with B Group. The apoptosis rate in theselected COCs was similar to pooled COCs, confirming that apoptosis rate incumulus cells is characteristic for each patient. Out of 22 patients involvedin the first trial, 8 were pregnant (36.3% A Group) and 14 were not pregnant(B Group). In the second trial 4 of a total of 20 patients were pregnant (20%).In the first trial a total of 58 metaphase II oocytes and 56 in the second trialwere studied. In the second trial 38 oocytes where pooled to compare apoptosisrate with the three selected oocytes pools. In the first trial the incidence of DNAfragmentation, evaluated by TUNEL assay, of the cumulus cells from individualtreated oocytes, was lower in A Group than in B Group (6.7% rangingbetween 2.2–13.3 vs 13.19% ranging between 6.2–34.9 respectively, p ,0.05). To confirm if DNA fragmentation was related to apoptosis process, weperformed caspase-3 immunoassay in the same cells. Data showed a lowercapase-3 activity in cumulus cells of pregnant than in those of non pregnantpatients (5.2% ranging between 1.2–8.6 vs 11.8% ranging between 5.6–14.8, p , 0.05). It is noteworthy to underline that pregnant patients usuallyexhibited, at least, one COC with a DNA fragmentation rate (TUNEL) lessthan 10% and caspase-3 activity rate less than 7%.Four (A Group) of 20 patients involved in the second trial were pregnant but2 aborted at 8–9 weeks. The low number of pregnant patients didn’t allow us tohave a powerful statistical analysis of apoptotic rate in cumulus cells, but itseems evident that an higher apoptotic rate in cumulus cells is associated tothe pregnancy failure (B Group) and in aborted patients of A Group, rangingfrom 10 to 60.3%.Conclusion: The data seem to demonstrate that apoptosis may be a marker forthe selection of the three best oocytes to be submitted to ICSI treatment. Allpregnant patients showed a lower apoptosis rate in cumulus cells if comparedwith patients with pregnancy failure.