BASIC STUDIES FOR IMPROVEMENT OF IN VITRO FERTILIZATION AND CRYOPRESERVATION

比较了两种冷冻方法(常规程序冷冻机慢速冷冻和液氮直接玻璃化冷冻)对2-细胞胚胎发育和囊胚发育的影响，并将新鲜的和冷冻保存的2-细胞胚胎体外发育为囊胚。玻璃化冷冻胚胎的囊胚率显著低于新鲜冷冻胚胎和慢速冷冻胚胎。尽管每个冷冻过程的囊胚形态正常，囊胚中的细胞数量和体外滋养层细胞铺展情况都没有显著差异，但它们的功能活性存在显著差异。首先，玻璃化冷冻和解冻的2-细胞胚胎的葡萄糖掺入活性(摄取[(3)H]2-脱氧葡萄糖(2-DG))显著低于新鲜或慢速冷冻和解冻的2-细胞胚胎。体外培养的新鲜2-细胞胚胎、慢速冷冻或玻璃化冷冻2-细胞胚胎的2-DG摄取量分别为105+/-75、43.0+/-28.3和22.O+/-11.4fmol/h。玻璃化冷冻2-细胞胚胎体外培养的囊胚率(10.2%)显著低于新鲜2-细胞胚胎(30.8%)和慢速冷冻2-细胞胚胎(22.1%)。由于这些数据表明，冷冻保存可能会对胚胎的功能发育产生不可告人的后果，而且玻璃化冷冻可能比缓慢控制率冷冻产生更有害的影响，因此在临床计划中常规使用玻璃化冷冻之前，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.