冷冻环冷冻促排周期未成熟卵母细胞效果的研究

Cryopreservation by Cryoloop Damages Human Immature Oocytes

目的:本文通过对冷冻环冷冻促排周期未成熟卵母细胞后成活率和体外成熟(IVM)能力的比较,及卵母细胞免疫荧光染色后纺锤体和染色体形态的分析,探索未成熟卵母细胞冷冻的方法。方法:获得的GV期卵母细胞随机分为3组:①对照组:GV期卵母细胞体外成熟为MⅡ期(n=68);②GV期冷冻组:卵母细胞在GV期冷冻后体外成熟(n=111);③MⅡ期冷冻组:卵母细胞IVM为MⅡ期予以冷冻(n=69)。获得的MⅡ期卵母细胞予以免疫荧光染色,激光扫描共聚焦显微镜(LSCM)观察纺锤体和染色体的形态。结果:GV期冷冻组卵母细胞存活率(89.11%)显著性高于MⅡ期冷冻组(77.05%),而GV期冷冻组卵母细胞的体外成熟率(77.78%)和对照组(86.74%)没有明显的差异(P>0.05)。GV期冷冻组成熟后卵母细胞正常形态的纺锤体(14.1%)和染色体(12.5%)都显著低于对照组(29.1%,32.7%,P<0.05),MⅡ期冷冻组正常形态的纺锤体(11.4%)显著性低于对照组(P<0.05),染色体(15.9%)也低于对照组,但是无显著性差异(P>0.05)。结论:玻璃化冷冻对GV期和MⅡ期卵母细胞纺锤体和染色体都会带来一定的损伤。IVM技术和玻璃化冷冻技术还需要进一步提高。

Objective： To examine the influence of cryoloop on the spindle and chromosome configurations of human oocytes cryopreserved in the germinal vesicle （GV） and metaphase Ⅱ （MⅡ ） stages, as well as on the survival rate and potential for in vitro maturation （IVM）. Methods： GV oocytes were randomly assigned into a control group （ matured in vitro into the M Ⅱ stage） , a GV cryopr- eserved group （ cryopreserved in the GV stage and then matured in vitro） , and an M Ⅱ cryopreserved group （ matured in vitro and cryo- preserved in the M Ⅱ stage）. After cryopreservation and IVM, immunostaining of the tubulin and chromatin was performed followed by visualization using laser scanning confocal microscopy （LSCM）. Rusults ： A significantly higher survival rate was observed in the GV cryopreserved group than in the M Ⅱ, but the maturation rate showed no significant difference between the GV cryopreserved group and the control （ P 〉 0.05 ）. Compared with the control group, there was a statistically significant decrease in the rates of normal meiotic spindles and chromosomes in the GV cryopreserved group （ P 〈 0.05 ）. A significantly lower rate of normal spindles was noted in the M Ⅱ cryopreserved group than in the control, but no statistical difference was shown in the rate of normal meiotic chromosomes between the two groups （ P 〉 0.05 ）. Conclusion ： Cryopreservation by cryoloop has a damaging effect on the spindle and chromosome of human oocytes in the GV and M Ⅱ stages.