应用冷冻环玻璃化冷冻小鼠囊胚的效果观察

The vitrification of mouse blastocysts at different stages using cryoloop technique

目的比较冷冻环玻璃化冷冻小鼠各阶段囊胚的存活率及继续发育能力。方法应用冷冻环以15%二甲基亚砜(DMSO)+15%乙二醇(EG)为主要组成的玻璃化冷冻液,冷冻小鼠早期囊胚112个、囊胚120个、扩张囊胚105个,以同期未冷冻囊胚(100个)作对照;并于冷冻前人工皱缩小鼠囊胚30个,以同期未皱缩的囊胚(50个)为对照。比较冷冻复苏后胚胎存活率及继续发育能力。结果小鼠早期囊胚、囊胚、扩张囊胚各组的存活率分别为88.4%、69.2%和55.2%,囊胚孵出率分别为82.8%、65.1%和60.3%。早期囊胚组复苏后的存活率和囊胚孵出率均显著高于囊胚组和扩张囊胚组(P<0.05)。冷冻前人工皱缩扩张期囊胚,冷冻复苏后存活率达93.8%,孵出率78.1%,与未皱缩组比较差异有显著性(P<0.05)。结论玻璃化冷冻囊胚的效果与囊胚的发育阶段有关;冷冻前人工皱缩囊腔体积能明显提高扩张囊胚玻璃化冷冻的效率。

Objective： To compare the survival rate and development capacity of mouse early blastocysts, blasto cysts and expanded blastocysts cryopreserved by vitrification using cryoloop technique. Methods： Cryoloops technique was used to vitrify 112 early blastocysts, 120 blastocysts, 105 expanded blastocysts, and 30 expanded blastocysts artificially shrunk by mechanical pipetting with a fine handdrawn glass pipette slightly smaller in diameter than that of the blastocysts. The main vitrification medium was composed of 15% dimethlsulphoxide （DMSO）, 15% ethylene glycol （EG）. The survival rates and hatching rates of the frozen thawed blastocysts were observed. Results： The survival rates of early blastocysts, blastocysts and expanded blastocysts were 88. 4%, 69.2%respectively, and the hatching rates were 82.8%, 65.1% and 60.3% respectively. Both the survival rates and the hatching rates in early blastocysts group were higher than those in blasto cysts and expanded blastocysts groups （P〈0.05）. The survival rate of blastocyst group was higher than that of the expanded blastocyst group （P〈0.05）. The survival rate and hatching rate of the shrunk blastocysts were higher than those without shrinking （P〈0.05）. Conclusion： The efficiency of the blastocyst vitrification method was dependent on the stage of blastocysts. The artificial shrinkage prior to vitrification could improve the survival rate and hatching rate of the vitrified expanded blastocyst.