BASIC STUDIES FOR IMPROVEMENT OF IN VITRO FERTILIZATION AND CRYOPRESERVATION

比较了两种低温保存方法（常规可编程冷冻机慢速冷冻和直接注入液氮玻璃化冷冻）对2细胞胚胎及其囊胚发育的影响，将新鲜或低温保存的2细胞小鼠胚胎体外发育为囊胚。玻璃化冷冻胚胎发育成囊胚的比例明显低于新鲜和慢速冷冻胚胎。尽管每种低温保存方法的囊胚形态正常，囊胚细胞数量和体外滋养细胞扩散均无显著差异，但其功能活力存在显著差异。首先，与新鲜或慢速冷冻和解冻的2细胞胚胎相比，玻璃化和解冻的2细胞胚胎中[(3)H]2-脱氧葡萄糖（2- dg）摄取方面的葡萄糖掺入活性显著降低。其次，新鲜2细胞胚胎和慢速冷冻或玻璃化2细胞胚胎体外培养的囊胚对2-DG的摄取分别为105 +/- 75,43.0 +/- 28.3和22。O +/- 11.4 fmol/胚/h。第三，玻璃化2-细胞胚胎体外培养囊胚的着床率（10.2%）明显低于新鲜2-细胞胚胎（30.8%）或缓慢控制率冷冻2-细胞胚胎（22.1%）。由于这些数据表明冷冻保存可能对胚胎的功能发育有潜在的影响，并且玻璃化可能比缓慢的控制速率冷冻产生更有害的影响，因此在玻璃化常规应用于临床计划之前，应更多地关注其安全性。

Effects of two cryopreservation procedures (conventional slow controlled-rate freezing using a programmable freezer and vitrification by direct plunging into liquid nitrogen) were compared on 2-cell embryos and their subsequent development to blastocysts, fresh or cryopreserved 2-cell mouse embryos were developed into blastocysts in vitro. The percentage of vitrified embryos which developed into blastocysts was significantly lower than that of fresh and slow controlled-rate frozen embryos. Although blastocysts from each cryopreservation procedure appeared morphologically normal and neither number of cells in the blastocysts nor in-vitro trophoblast spreading differed significantly, there were significant differences in their functional viability. First, the glucose incorporation activity in terms of [(3)H]2-deoxyglucose (2-DG) uptake in vitrified and thawed 2- cell embryos significantly decreased compared with fresh or slow controlled-rate frozen and thawed 2-cell embryos. Second, 2-DG uptake by blastocysts developed in vitro from fresh 2-cell embryos and from slow controlled-rate frozen or vitrified 2-cell embryos was 105 +/- 75, 43.0 +/- 28.3 and 22.O +/- 11.4 fmol/embryo/h respectively. Third, the implantation rate of blastocysts developed in vitro from vitrified 2-cel1 embryos (10.2%) was significantly lower than that from fresh 2-cell embryos (30.8%) or slow controlled-rate frozen 2-cell embryos (22.1%). Since these data suggest that cryopreservation may have ulterior consequences on the functional development of embryos and that vitrification may exert a more harmful effect than slow controlled-rate freezing, more attention should be paid to its safety before vitrification is used routinely in a clinical programme.