Abstract
OBJECTIVE: The aim of this study was to compare vitrified and slowly frozen oocytes in terms of their post-warm/thaw morphology, meiotic spindle configuration and DNA integrity in order to assess possible effects on subcellular organization after cryopreservation. DESIGN: Prospective randomized study. MATERIALS AND METHODS: A total of 88 donor's oocytes (23.2±2.7) included in the study were set in to: Group 1 (N=36) where oocytes were slowly cooled using 1.5 M 1,2-propendiol and 0.2M sucrose; Group 2, including 36 vitrified using 15% ethylene glycol and 15% dimethyl sulfoxide according to Cryotop protocol; and Control Group (N=16 fresh oocytes). After thawing/warming, changes in cellular volume were estimated by calculating the mean average oocyte volume (MAV). Cytoplasmic and zona pellucida (ZP) appearance were also evaluated. Spindle assessment was performed before and two to three hours after cryopreservation by the use of Oosight™ Imaging System. DNA integrity was assessed by the TUNEL-based DNA fragmentation assay in both cryopreserved and fresh oocytes. Statistical analysis was carried out by t-test and χ2 test. RESULTS: Our preliminary results show: Survival rate was higher for vitrified oocytes (55.6% vs. 88.9%; p<0.05). There were no differences regarding the MAV for group 1 or group 2 oocytes as compared with the values observed prior to cryopreservation and two hours after thawing. During dilution, group 1 did not show a significant change in MAV. However group 2 lost 13.8% of their MAV in first step after warmed vs. initial volume (p<0.05). No significant changes were observed in cytoplasmic appearance, neither in ZP thickness.The proportion of oocytes showing meiotic spindles prior to cryopreservation was 52% and 65.2% and 47% for group 1, 2 and controls respectively (ns) and 75% and 90.3% after thawing and warming respectively (p<0.05). In 88.9%, 100% and 85.7% the spindle appeared aligned in a 0–30° with the polar body (PB) (ns). After thawing 66.6% (ns) and warming 32.2% (p<0.05) of the oocytes showed that spindle alignment. No DNA fragmentation was observed in either slow cooled or vitrified oocytes. CONCLUSIONS: Vitrification is more effective than slow cooling, as shown by higher survival rate and spindle assessment. However due to the higher misalignment between the spindle and the PB in vitrified oocytes, the spindle assessment during ICSI should be recommended.The DNA integrity of cryopreserved oocytes is not altered after the procedure, although further studies are needed to confirm this data.
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