Study On Vitrification And It's Damage Of In-vitro Matured Oocytes

PartⅠThe role of cytochalasin B in the vitrification of in vitro matured oocytesObjective: To study the role of Cytochalasin B(CCB) in the vitrification of in-vitro matured oocytes, and observe the clinical outcome of in-vitro matured oocytes after vitrification and thawing.Methods: 395 immature oocytes were collected from stimulated ovarians of intracytoplasmic sperm injection (ICSI) cycle (include GV stage and MⅠstage). All the immature oocytes were in vitro matured(IVM) for 24h～48h, 269 oocytes were matured(with the extrusion of second polarbody), and randomized into three groups to vitrification. Group A: 66 oocytes pretreated with CCB for 20min before vitrification; Group B: 68 oocytes pretreated with CCB for30 min. Group C: 63oocytes pretreated with CCB for 40 min Group D: 72 oocytes under vitrification without CCB pretreated. Group E: 30 oocytes in vivo matured under vitrifiation without CCB pretreated as the controlled group. All the oocytes were thawed after three weeks and then fertilized by ICSI. Compared the revival rate, fertilized rate, cleavage rate and blastocyst rate.Results: After vitrification, the revival rate, fertilized rate, cleavage rate and blastocyst rate of IVM oocytes were significantly lower than those of in vivo matured ooctyes(P<0.05). There were no significant difference among the four groups in revival rate,fertilized rate and cleavage rate (P>0.05). There were no blastocysts in group A , group B and groupC , only 1 blastocyst in group D.Conclusion: The revival rate, fertilized rate, cleavage rate and blastocyst rate of IVM oocytes after vitrification were declined, the potential of the oocytes development was reduced significantly. There were no positive effects of CCB in the vitrification of IVM ooctyes. Pretreated with CCB cannot improve the revival rate﹑fertilized rate﹑cleavage rate and the development potential of the IVM oocyte after vitrification.PartⅡSpindle and chromosome configuration of human in vivo matured oocytes after vitrification : effect of Cytochalasin B pretreatedObjective: To investigate the effect of Cytochalasin B pretreated on the spindle and chromosome configuration of human oocytes after vitrification.Methods: 71 in vivo matured oocytes were collected from stimulated ovarians of in vitro fertilization - embryo transfer (IVF-ET)cycles and intracytoplasmic sperm injection (ICSI) cycles, and randomized into four groups for vitrification. Group A: 17 oocytes pretreated with CCB for 20mins before vitrification; Group B: 20 oocytes pretreated with CCB for 30 mins. Group C: 17 oocytes pretreated with CCB for 40 mins. Group D: 17 oocytes under vitrifiation without CCB. All oocytes were thawed after three weeks and then immunostained for tubulin and chromatin and at last visualized by confocal laser scanning microscopy.Results: No statistical difference was found in survival rate among group A,B,C,D(64.7%,50.0%,64.7% and 70.6%),(P>0.05). There was no significant difference in the frequencies of normal spindle and chromosome in group A,B,C,D(27.3%,30.0%,45.5% and 33.3%),(P>0.05).Conclusion: Spindle and chromosome configuration of human MⅡoocytes were damaged in vitrification, which can not improved by CCB pre-treatment before vitrification.PartⅢSpindle and chromosome configuration of human in vivo matured oocytes after vitrification : effect of Cytochalasin B pretreatedObjective: To investigate the effect of Cytochalasin B pretreated on the spindle and chromosome configuration of human in vitro matured oocytes after vitrification. Methods: Immature oocytes were collected from stimulated ovarians of intracytoplasmic sperm injection (ICSI) cycles (include GV stage and MⅠstage),and then they were in vitro matured(IVM) for 24h～48h,170 oocytes were matured(with the extrusion of first polarbody), and randomized into five groups to vitrification. Group A: 37 oocytes pretreated with CCB for 20mins before vitrification; Group B: 38 oocytes pretreated with CCB for 30 mins. Group C: 33 oocytes pretreated with CCB for 40 mins. Group D: 36 oocytes under vitrifiation without CCB. Group E: 26 ooctyes whitout vitrification and CCB pretreated as the controlled group. All oocytes were thawed after three weeks and then immunostained for tubulin and chromatin and at last visualized by confocal laser scanning microscopy.Results: No statistical differences was found in survival rate among group A,B,C,D(80.39%,64.86%,55.36% and 78.79%),(P>0.05). There were also significantly differences in the frequencies of normal spindle and chromosome in group A,B,C,D(21.05%,26.30%,27.78% and 23.81%),(P>0.05), all groups'frequencies of normal spindle and chromosome were significantly lower than group E (61.54%),(P<0.05).Conclusion: Spindle and chromosome configuration of human in vitro matured oocytes were damaged in vitrification, no effect of CCB pre-treatment before vitrification.