辅助激活受精对含DNA损伤精子遗传风险的影响及其机制研究

Severe weak sperm is often accompanied by high DNA damage. Assisted oocyte activation (AOA) can help severe weak sperm to fertilization and then carry the DNA damage to the zygote. If the AOA can improve or disturbe or be independent of the regulation of DNA damage repair in zygote, the AOA will give different impact on the genetic risk in the offspring, which is very anxious. However, no research related this impact and its mechanism can be found. This is mainly because it is hard to have effective simulation of animal models for study. Our previous studies have created a new thchnique to simulate the human intracytoplasmic sperm injection in mice and a new technique to reduce the ability of sperm to active oocyte in control. And our previous study has also found that AOA can improve the development of embryo from the sperm with weak oocyte activation ability, and restore the expression of its large number of genes, including the related genes of DNA damage repair, to normal expression. This project, in further, intends to control and detect the DNA damage of sperm and the degree of oocyte activation ability quantitatively, to establish mice models of different periods of development from AOA fertilization, monitoring the relations between the AOA factors and the ability and related moleculars of DNA damage repair in embryo, and analyze the genetic risk in different developmental periods to verify these relations. Thus, we will definite clearly the impact of AOA on genetic risk in different period of development, and its role on DNA damage repair in early embryo and its molecular pathways. These will find out an important link and its mechanism associated the impact on genetic risk, and will provide the scientific basis for promoting optimization of the human AOA technology.

人严重弱精常伴有高DNA损伤，辅助激活（AOA）能帮助本不能受精的严重弱精受精而将DNA损伤带入受精卵。若AOA能改善或干扰或无关受精卵DNA损伤修复的调控，则AOA将给子代遗传风险带来不同影响，令人堪忧。然而，有关该影响及其机制还是研究空白。难以进行动物模型模拟是该研究的主要障碍。我们前期研究创建了能模拟人的小鼠显微授精新技术和可控降低精子激活力技术，并发现AOA能改善弱激活力精子受精的早期发育，恢复其大量基因包括DNA损伤修复相关基因的正常表达。本项目拟进一步通过精子DNA损伤和AOA激活力的量化控制与检测，建立各期发育模型，监测胚胎中DNA损伤修复能力及相关分子变化与AOA的关系，并检测发育各期遗传风险以验证这些关系，从而明确AOA对发育各期遗传风险有何影响，及其对早期胚胎DNA损伤修复的作用与分子途径。这将发现影响遗传安全的一个重要环节与机制，并为人AOA技术优化提供科学依据。

在临床辅助生殖治疗中，辅助激活（AOA）是改善弱激活力精子受精结局的重要手段。然而，在人类弱激活精子中，往往携带有较多的DNA损伤。因此，辅助激活是否会导致精子携带的DNA损伤引入受精卵，并给后代的健康带来风险，令人堪忧。故我们的研究项主要着眼于探究辅助激活是否会对受精过程DNA损伤及其修复产生影响，从而对其分子机制进行研究，并寻找可能的优化途径。项目立项以来我们进行了一系列的研究探索：（1）借助非Piezo依赖的小鼠ICSI操作技术建立高效的人类精子激活力评估手段；（2）成功构建了模拟人类激活力减弱并伴有DNA损伤的小鼠精子模型；（3）基于不同激活力强度，对辅助激活可能对DNA损伤及其修复的影响进行分类；（4）发现适度激活可能有利于改善卵子能量代谢，从而产生更多的ATP，促进DNA损伤的修复；（5）发现较高强度的激活可能急剧改变卵子钙震荡的模式，而导致能量代谢的失衡，使ROS水平增加，从而加剧DNA损伤，但这种损伤可以通过施加抗氧化剂进行挽救；（6）同时我们对临床数据进行了回溯分析，发现在辅助激活导致优胚率下降的分组中，其受精率在辅助激活和未激活亚组之间并没有显著性差异，进一步佐证了激活力过强可能导致胚胎发育质量下降；（7）除此以外，我们还针对临床激活力减弱的病例进行了基因筛查，发现了6种未经报道的PLCZ1突变，并对采用正常PLCZ1 cRNA补充（实验研究）和辅助激活（临床实践）手段对改善其受精结局进行了探究和实践，并最终获得3名健康活产婴儿。综上，本项目探究并揭示了辅助激活对受精和胚胎发育过程中的利弊影响及其机制，并为辅助生殖的AOA临床应用提示出：当受精的激活力不足时适当辅助激活有利于受精与发育，而当本来受精的激活力正常时，辅助激活反而存在损害DNA的风险，不利于胚胎发育。总之，辅助激活技术要以评估是否存在受精弱激活力为依据，本项目建立的人精子激活力评估技术和辅助激活抗氧化方法也将为解决这一临床问题提供可行的新技术途径。