人类成熟卵母细胞慢速程序化冷冻复苏后培养时间对纺锤体和染色体的影响

目的 研究慢速程序化冷冻人类成熟卵母细胞复苏后培养时间对纺锤体和染色体的影响.方法 慢速程序化冷冻人成熟卵母细胞102枚,选择复苏后外观存活良好的卵母细胞共64枚,随机分为A组20枚、B组22枚、C组22枚,分别培养不同时间(1、3、5 h)后进行固定、荧光染色;另取新鲜成熟卵母细胞18枚固定、染色作为对照组,采用激光共聚焦显微技术,观察并分析纺锤体和染色体的形态学变化.结果 (1)A、B、C 3组正常纺锤体比例分别为10%(2/20)、46%(10/22)、41%(9/22),均明显低于对照组(83%,15/18),差异均有统计学意义(P＜0.05);A组分别和B组、C组比较,差异也有统计学意义(P＜0.05).纺锤体缺失率A组为45%(9/20),明显高于对照组(6%,1/18),差异有统计学意义(P＜0.01);A组分别和B组(14%,3/20)、C组(14%,3/20)比较,差异也有统计学意义(P＜0.05);B、C组之间比较,差异无统计学意义(P＞0.05).(2)A组正常染色体比例为30%(6/20),与B组(68%,15/22)、C组(64%,14/22)和对照组(78%,14/18)比较均明显降低,差异均有统计学意义(P＜0.05);B、C、对照组之间两两比较,差异均无统计学意义(P＞0.05).结论 慢速程序化冷冻复苏后培养3～5 h可以促进部分卵母细胞纺锤体和染色体形态的恢复.

Effect of culture time on the spindle and chromosome configurations of human thawed oocytes with slow-cooHng and raid-thawing protocol

0bjectlve To examine the metaphase II spindle and chromosome configurations of human oocytes cultured for different times after thawing.Methods Using slow.cooling and raid—thawing protocol combined with 0.3 mol/L sucrose and 1.5 mol/L 1,2-propanedio 1(1,2-PROH)to cryoprotect human mature oocytes(n=102),the 64 survival oocytes without abnormal zona pellucida and cytoskeletal were randomly assigned to three groups after thawing:group A:culture 1 hour(n=20),group B:culture 3 hour(n=22),group C:culture 5 hours(n=22),the flesh oocytes served as control group(n=18).Immunocytochefifical staining and fluorescence microscopy were used to assess the morphology of the metaphase II spindle and chromosome.Results (1)The normal spindle rates of groups A,B and C were 10％(2/20),46％(10/22)and 41％(9/22)respectively,significantly decreased compared with control group(83％,15/18;P<0.05).The rates of absent spindle in group A(45％,9/20)was significantly higher than control group(6％,1/18;P<0.01).Also,the rates of absent spindle in group A was higher than groups B(14％,3/20)and C(14％,3/20;P<0.05).However,no significant differences were observed in groups B and C(P>0.05).(2)A significant increase in abnormal chromosome rate was observed in group A(30％,6/20)compared to groups B(68％,15/22),C(64％,14/22)and control group(78％,14/18;尸<0.05).No differences in chromosome morphology were observed in groups B,C and control group(P>0.05).Conclusions The cryoproteetant protocol leads to a deleterious effect on the organization of the meiotic spindle and chromosome at MI stage.The 3—5 hours post—thawing incubation could permit restoration of the meiotic spindles and chromosome.