纳秒脉冲电场激活ICSI未受精卵母细胞及其定量调控胞浆内钙振荡的机制研究

钙振荡是卵母细胞激活和胚胎发育的必要条件。常用的人工卵母细胞激活方法因无法模拟钙振荡，在用于治疗反复卵胞浆内单精子注射（ICSI）受精失败或低下患者时，无法获得与生理状态下相似的激活效率和胚胎发育潜能。前期研究首次发现纳秒脉冲电场（nsPEFs）能激活小鼠卵母细胞，并可定量调控钙振荡，提示nsPEFs可通过定量调控钙振荡的方式激活人卵母细胞。本课题以ICSI未受精卵母细胞为研究对象，利用不同电学剂量的nsPEFs刺激人卵母细胞，观察激活率、囊胚形成率及其诱导的钙振荡模式；通过设计拮抗实验，研究nsPEFs诱导卵胞浆内钙振荡的生物学机制；通过构建nsPEFs调控人卵母细胞钙振荡动力学模型，研究可控激活人卵母细胞的实现路径；通过对卵母细胞亚细胞结构和激活胚胎的多重组学分析，评价nsPEFs作为高效、可控的人工激活方法的安全性。本课题可望为反复ICSI受精失败和低下病人提供全新的治疗方法。

Calcium (Ca2+) oscillations is a prerequisite for oocyte activation and subsequent embryonic development. Intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA) could not achieve perfect activation efficiency and embryonic development because the current AOA methods cannot mimic the natural pattern of calcium oscillations after sperm penetration. Our previous study firstly demonstrated that nanosecond pulsed electric field (nsPEFs) technology could activate mouse oocytes by quantitively regulating Ca2+ oscillation. The results indicate that nsPEFs may activate human oocytes by inducing Ca2+oscillation. In this study, nsPEFs technology is innovatively introduced to activate unfertilized oocytes after ICSI. Different absorption dose of nsPEFs are used to stimulate unfertilized oocytes after ICSI. The activation rate, blastocyst rate and the calcium oscillation patterns are observed correspondingly, and then the biophysical correlation among them are analyzed. Furthermore, the antergic experiments are designed to explore the underlying mechanisms of nsPEFs quantitively regulate calcium oscillation. A nsPEFs-mediated Ca2+-channel kinetic model is constructed to regulate the absorption dose to mimic Ca2+ oscillation for activating human oocytes. Finally, in order to verify the safety of nsPEFs as a novel AOA methods for its potential application in clinic, the effect of nsPEFs on the subcellular structure of unfertilized oocyte after ICSI is evaluated , while the genetic and epigenetic characteristics and transcriptome information of activated embryos will be detected by single-cell triple omics sequencing. This study may provide a new and feasible treatment for infertility couples with repeated total fertilization failure or low fertilization following ICSI, which has important scientific significance and social value.