吴成亮 苏占营 辜敏 巢时斌

[摘要] 目的 探讨在卵子冷冻液中添加合适浓度的褪黑素能否改善卵子存活率和受精能力。方法 回顾性分析2017年3月至2020年10月在本中心进行卵胞浆内单精子注射（ICSI）时临床废弃未受精卵母细胞（判断标准：受精评估时无原核和第二极体出现），根据实验目的不同，分为两个阶段。第一阶段探寻最佳的褪黑素添加浓度，经过第一阶段实验分析发现添加10-9 M浓度组卵子存活率最高（88.64% vs. 74.12%，P<0.05）;第二阶段实验是用玻璃化冷冻及解冻方法冷冻及解冻卵子后进行卵胞浆内单精子注射（ICSI），观察及分析10-9 M褪黑素添加组172枚（51.04%）和对照组（未添加褪黑素）165枚（48.96%）卵子受精能力。 结果 在冷冻液添加褪黑素后卵子冷冻复苏存活率明显改善，褪黑素浓度10-9 M为效果最佳（88.64% vs. 84.93% vs. 84.21% vs. 74.12%，P<0.05），添加10-9 M褪黑素组比未添加褪黑素组卵子正常受精率明显升高，差异有统计学意义（66.86% vs. 51.52%，P<0.01）。结论 褪黑素可以显著改善卵子冷冻复苏效果。

[关键词] 褪黑素;卵子冷冻;存活率;受精

[中图分类号] R321          [文献标识码] A          [文章编号] 1673-9701（2021）34-0028-03

[Abstract] Objective To investigate whether adding a suitable concentration of melatonin to the oocyte freezing solution can improve the oocyte survival rate and fertilization ability. Methods A retrospective analysis was conducted on the unfertilized oocytes during intracytoplasmic sperm injection（ICSI） in our center from March 2017 to October 2020 （judgment criteria： no pronucleus and second polar body appear during fertilization assessment）. According to the different purposes of the experiment， it was divided into two stages. In the first stage， the optimal melatonin concentration was explored. After the first stage of experimental analysis， it was found that the egg survival rate of the 10-9 M concentration group was the highest （88.64% vs. 74.12%， P<0.05）. The second stage of the experiment was to freeze and thaw the eggs by vitrification and thawing， and then perform intracytoplasmic sperm injection（ICSI）. The fertilization abilities of 172 （51.04%） eggs in the 10-9 M melatonin group （with 10-9 M melatonin added） and 165 （48.96%） eggs in the control group （without melatonin added） were observed and analyzed. Results After melatonin was added to the freezing solution， the oocyte survival rate during freezing and resuscitation was significantly improved， and the melatonin concentration of 10-9 M was the optimal concentration（88.64% vs. 84.93% vs. 84.21% vs. 74.12%， P<0.05）. The normal fertilization rate of oocyte in the 10-9 M melatonin group was significantly higher than that in the control group， with the difference statistically significant （66.86% vs. 51.52%， P<0.01）. Conclusion Melatonin can significantly improve the effect of oocyte freezing and resuscitation.

[Key words] Melatonin; Oocyte freezing; Survival rate; Fertilization

人類辅助生殖技术经过40多年的发展，已经取得了巨大的进步。在生育力保存领域也取得了较好的效果，尤其是在胚胎冷冻方面，玻璃化冷冻技术的成熟应用，使胚胎冷冻复苏存活率提升到95%以上[1-2]。但是卵子冷冻技术的应用目前还不是十分的成熟，原因在于卵子的超微结构不同于胚胎，不论是传统的慢速程序化冷冻技术还是玻璃化冷冻技术，都对冷冻卵子的超微结构、卵膜的渗透性、线粒体功能等方面有不可逆的损伤[3-4]。因此导致冷冻复苏后卵子的存活率低，发育潜能差[5]。

本研究拟验证在卵子冷冻液中添加合适浓度褪黑素改善卵子冷冻复苏效果。此外，卵子冷冻技术的进步缓慢另一个重要原因是因为人类卵子稀缺和珍贵性及必须符合伦理监督原则，临床无法提供足量的样本进行深入研究。本研究采用进行单精子卵胞浆内注射技术后16～18 h受精观察时未受精卵子（未见原核形成和第二极体排出），这些卵子无任何临床使用价值，临床的处理原则是直接丢弃，但是这部分卵子能够保持一定的受精能力，具有一定的科研价值。……