温度、冻存液体积和操作时间对小鼠卵子Cryotop玻璃化冻融复苏率的影响

Effect of the temperature,volume and treatment duration of CPA microdrop on mouse oocytes vitrification

目的 探讨Cryotop法中冷冻操作环境温度、加载覆盖的保护液微滴体积以及投入液氮前暴露于高浓度冷冻保护剂（CPA）的时间对小鼠MⅡ期卵子玻璃化冻融效果的影响。方法 对6周龄KM系小鼠促排卵、脱颗粒细胞以获得形态良好的MⅡ期卵子,用Cryotop法对其进行玻璃化冻融,卵母细胞随机分组后实验方案如下：根据冷冻环境温度的不同,设置37℃操作组和22℃操作组;根据薄膜上包被的保护液微滴体积的不同,设置〈0.1μL组、0.1~0.5μL组和〉0.5~1.0μL组;根据与CPA的接触时间的不同,将冷冻卵子分为30~40 s暴露组、〉40~90 s暴露组和〉90~180 s暴露组。随后对各组卵母细胞的复苏率进行统计比较。结果 22℃操作组小鼠卵子的复苏率为81.4%,显著高于37℃操作组的50.7%,差异有高度统计学意义（P〈0.01）。选择22℃的操作环境,〈0.1μL组、0.1~0.5μL组、〉0.5~1.0μL组复苏率（85.7%、90.5%、83.2%）比较,差异无统计学意义（P〉0.05）。〉90~180 s CPA暴露组的卵子复苏率为74.7%,低于30~40 s暴露组（83.3%）和〉40~90 s暴露组（78.9%）,差异无统计学意义（P〉0.05）。结论 使用Cryotop法冻融卵母细胞时,不必刻意追求〈0.1μL的保护液加载体积。卵子在高浓度CPA中的暴露时间不宜过长,且控制在30~90 s内对其存活率无明显影响。在常温（22℃）环境下进行操作可改善复苏结局。

Objective To evaluate the effects of the temperature, volume and treatment duration of loading CPA solu- tion on the survival rate of oocytes vitrification. Methods Oocytes were obtained from the female KM mice aged six weeks after superovulation. Matured oocytes at M II stage were frozen with Cryotop vitrification method at the environ- mental temperature of 37℃ or 22℃, microdrop volume of 〈0.1, 0.1-0.5, 〉0.5-1.0 μL, and time of oocyte treated in CPA solution of 30-40, 〉40-90, 〉90-180 s. The survival rates of warmed oocytes in the different treatments were compared. Results The oocyte survival rate at 22℃ of environmental temperature was 81.4% which was significantly higher than that at 37℃ （50.7%） （P 〈 0.01）. At the temperature of 22℃, the volume changes of loading mierodrops of CPA solution did not influence the survival rate significantly （85.7%, 90.5% and 83.2% in 〈0.1, 0.1-0.5 and 〉0.5-1.0 txL, respec- tively （P 〉 0.05）. The survival rate seemed to be decreased with the increased treatment duration of oocyte in CPA solu- tion （74.7% in 〉90-180 s, 78.9% in 〉40-90 s and 83.3% in 30-40 s）, but the differences did not reach to statistical significance （P〉 0.05）. Conclusion It would not be necessary to minimize the CPA solution drop＇s volume to less than 0.1 μL. Treatment duration of oocyte in CPA should not be too long, and inside 30-90 s has no significant effect on its survival. Controlling environmental temperature under 22℃ can improve the survival rate of oocyte vitrification.