输精管梗阻通过附睾精子miRNA簇影响胚胎发育的机制研究

梗阻性无精子症一般用单精子卵胞浆内注射（ICSI）生育子代，目前优胚率和妊娠率偏低；用睾丸精子还是附睾精子也不一致，缺乏依据和机制的支持。近期研究表明附睾是精子miRNA变化的重要场所，有些miRNAs在附睾头部丢失在尾部重新获得（失而复得），生理情况下对胚胎发育起必要作用。我们在输精管梗阻（VDO）小鼠中发现，其附睾尾部精子一些miRNAs显著下降，大部分生理情况下属于“失而复得”模式，且集中于三个miRNA簇；同时，用VDO附睾尾部精子行ICSI，出生率显著降低。我们推测VDO致附睾微环境变化，影响附睾尾部精子miRNAs的正常获得，而不利于胚胎发育和/或着床。本课题进一步研究VDO致miRNA簇下降的机制，明确VDO附睾尾部精子影响胚胎发育异常的主要阶段，筛选出主要有效miRNA簇，并揭示其致胚胎发育异常的主要通路。旨在为梗阻性无精子症精子的选择，和提高其成功率提供依据和新思路。

Infertility due to obstructive azoospermia (OA) may be treated effectively by retrieval of sperm from the epididymis or testis, followed by in vitro fertilization with intracytoplasmic sperm injection (ICSI). However, the pregnancy rate remains relatively low with low rate of good quality embryo and occasional occurrence of early miscarriage. And whether to use testicular or epididymal sperm for ICSI is a controversial issue which lacks sufficient clinical proof and underlying mechanism to verify it. Recent studies showed the small RNA payload of mammalian sperms underwent dramatic change during post-testicular maturation in the epididymis. Some miRNAs lost in the caput of epididymis but regained in the cauda played an essential role in embryonic development under normal physiological conditions. We had generated the mice model of vas deferens obstruction (VDO) and found some miRNAs dropped distinctly in the cauda epididymis of VDO mice, those miRNAs were mainly distributed among 3 miRNA clusters and generally in accordance with the lost and regained pattern in epididymis. Meanwhile, our results demonstrated that the sperm retrieved from the cauda of VDO used for ICSI resulted in significantly lower rate of pregnancy and live birth of mice. Presumably, we deduced that VDO can cause irregular micro-environment of epididymis and lead to abnormal miRNA regaining process of the cauda sperm, which then have adverse effect on embryonic implantation and development. We aimed to explore the potential mechanism of decreased level of the 3 miRNA clusters caused by VDO model and identify the major phase of embryonic development influenced by the sperm obtained from cauda epididymis of VDO. Furthermore, we will screen out the causal miRNA cluster and uncover the molecular pathway of abnormal embryonic development. Results of this project can provide novel experimental evidence and mechanism to support sperm chosen, and shed lights on developing treatment and improving the pregnancy rate of ICSI in OA patients.