Ghandy Nasibeh, Karimpur Malekshah Abbas Ali
Department of Anatomy, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Molecular and Cell Biology Research Center, Department of Anatomy, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Int J Fertil Steril. 2017 Jan-Mar;10(4):357-362. doi: 10.22074/ijfs.2016.5086. Epub 2016 Nov 1.
Vitrification has been shown as one of the most effective methods of cryopreservation for mammalian embryos. However, there is no consensus which stage of embryonic development is the most appropriate for vitrification with subsequent maximal development after thawing. This study was carried out to explore and compare the effect(s) of vitrification on mouse 2-cell, 4-cell, 8-cell, morula and blastocyst stage embryos and subsequent blast formation and hatching after thawing.
In this experimental study, 2-cell embryos were obtained from the oviducts of super ovulated female NMRI mice. Some embryos were randomly selected and vitrified through a two-step media protocol and cryotop. Other embryos were cultured to assess their development. During the ensuing days, some of these cultured embryos were vitrified at 4-cell, 8-cell, morula and blastocyst stages. After 10 to 14 days, the embryos were thawed to assess their survival and also cultured to determine the rate of blastocyst formation and hatching. The results were analyzed using one-way ANOVA and Tukey's post-hoc tests.
There was no significant difference in the survival rates of vitrified embryos at 2-cell, 4-cell, 8-cell, morula and blastocyst stages after thawing (P>0.05). The blastocyst formation rate of vitrified 8-cell embryos was significantly higher than that of 2-cell embryos (P<0.05). The hatching rate of vitrified 4-cell, 8-cell and blastocysts were significantly higher than that of 2-cell embryos (P<0.05).
Vitrification is suitable for cryopreservation of all stages of mouse embryonic development. However, the best tolerance for vitrification was observed at 4and 8-cell stages of development. Accordingly, the development of vitrified embryos to blastocysts, following thawing, was most efficacious for 4 and 8-cell embryos. Compared to mouse 2-cell embryos, embryos vitrified as blastocysts had the highest rate of hatching.
玻璃化冷冻已被证明是哺乳动物胚胎最有效的冷冻保存方法之一。然而，对于胚胎发育的哪个阶段最适合玻璃化冷冻并在解冻后实现最大程度的发育，目前尚无定论。本研究旨在探索并比较玻璃化冷冻对小鼠二细胞、四细胞、八细胞、桑葚胚和囊胚阶段胚胎的影响，以及解冻后的囊胚形成和孵化情况。
在本实验研究中，从超排后的雌性NMRI小鼠输卵管中获取二细胞胚胎。随机选取部分胚胎，通过两步介质方案和冷冻环进行玻璃化冷冻。其他胚胎用于培养以评估其发育情况。在随后的几天里，将部分培养的胚胎在四细胞、八细胞、桑葚胚和囊胚阶段进行玻璃化冷冻。10至14天后，将胚胎解冻以评估其存活率，并进一步培养以确定囊胚形成率和孵化率。结果采用单因素方差分析和Tukey事后检验进行分析。
解冻后，二细胞、四细胞、八细胞、桑葚胚和囊胚阶段玻璃化冷冻胚胎的存活率无显著差异（P>0.05）。玻璃化冷冻的八细胞胚胎的囊胚形成率显著高于二细胞胚胎（P<0.05）。玻璃化冷冻的四细胞、八细胞胚胎和囊胚的孵化率显著高于二细胞胚胎（P<0.05）。
玻璃化冷冻适用于小鼠胚胎发育各阶段的冷冻保存。然而，在发育的四细胞和八细胞阶段观察到对玻璃化冷冻的耐受性最佳。因此，解冻后玻璃化冷冻胚胎发育至囊胚的效率在四细胞和八细胞胚胎中最高。与小鼠二细胞胚胎相比，玻璃化冷冻的囊胚孵化率最高。
