种属和发育期及不同实验条件对卵裂期胚胎冷冻复苏结局的影响

背景：人卵裂期胚胎冷冻复苏的研究中,不同的实验条件及实验动物模型是否与人类胚胎具有相同的敏感性从而反映出实验方案的优劣值得探讨。目的：观察胚胎种属和发育期及不同冷冻条件对卵裂期胚胎冷冻复苏结局的影响。方法：将人卵裂期胚胎作为对照组,将KM小鼠胚分为2细胞,4细胞,8细胞组。各组胚胎随机采用以下实验方案：①冷冻操作环境温度18~20 ℃、24~26 ℃和37 ℃。②慢速程序化方案、自制straw叶片玻璃化方案和CPS玻璃化方案。③与玻璃化液接触时间< 40 s、40~60 s和60~90 s。各组胚胎比较不同实验条件下胚胎复苏率和培养24 h发育率。结果与结论：①对照组24~26 ℃冷冻操作环境温度复苏率高于37 ℃冷冻操作环境(P < 0.05)。②对照组和4细胞鼠胚组采用自制straw叶片玻璃化方案复苏率高于慢速程序化方案(P < 0.05),培养24 h胚胎发育率差异无显著性意义(P > 0.05)。③各组胚胎与冷冻保护剂接触不同时间胚胎复苏率差异无显著性意义(P > 0.05)。表明卵裂期胚胎玻璃化冷冻复苏效果优于慢速程序化,24~26 ℃操作环境、减少冷冻保护剂剂量和缩短接触时间可改善玻璃化冷冻复苏结局;相同冷冻条件下,胚胎种属和发育阶段对冷冻复苏结局有影响,4细胞鼠胚更适合作为研究人类卵裂期胚胎冷冻复苏的实验模型。

Effects of species,development periods and different experimental conditions on the outcomes of embryo freezing and thawing in cleavage stage

BACKGROUND: In the studies of frozen-thawed human cleavage embryos, whether the experimental conditions and experimental animal model have the same sensitivity with human to reflect the experiment quality is worth exploring. OBJECTIVE:To investigate the effects of embryo species, development periods and different frozen conditions on the outcomes of embryo freezing and thawing in cleavage stage.METHODS:Human embryos in cleavage stage served as control group, KM mouse embryos were randomly divided into 2-cell-stage group, 4-cell-stage group and 8-cell-stage group. Experiment scheme: Operating environment temperature were 18-20 ℃, 24-26 ℃ and 37 ℃. Slow programmed cryopreservation, self-made Straw-leaf vitrification cryopreservation and close pulled straw vitrification cryopreservation were used in this study. Contact durations of vitrified solution were less than 40 seconds, 40-60 seconds and 60-90 seconds. The embryo survival rates and developmental rates after cultivating for 24 hours were compared among groups.RESULTS AND CONCLUSION:The survival rate of control group under the environment temperature of 24-26 ℃ was higher than that of 37 ℃ temperature (P < 0.05). The survival rate of control group and 4-cell-stage group by self-made Straw-leaf vitrification cryopreservation was higher than that of the two groups by slow programmed cryopreservation (P < 0.05). The embryo developmental rates after cultivating for 24 hours showed no significant difference (P > 0.05). There was no significant difference in embryo survival rates among groups with different contact durations of vitrified solution (P > 0.05). These findings suggest that the cleavage embryo resuscitation effect of vitrification is better than that of slow recovery process. Under the temperature of 24-26 ℃, the outcomes of embryo freezing and thawing can be improved by reducing cryoprotectant dose and contact duration. Different embryo species and developmental stages may produce different outcomes under the same conditions. Compared with other stages of embryo development, 4-cell mouse embryo is a more suitable experimental model for the freezing and thawing studies of human cleavage stage embryos.