卵激活强度相关的胚胎碎片形成机制及临床策略研究

Fragmentation during embryonic cleavage is quite common in human in-vitro fertilization embryos, but the underlying mechanism is unclear. Our previous clinical observation and the model research through new established technique to artificially decrease mice sperm activation ability, suggest that activation intensity of oocyte (AIO) correlates with embryo fragmentation. On this basis, this project will further clarify the corelation between AIO and fragmentation and provide clinical strategy. To construct divers AIO embryo models, sperms with different artificially decreased activation ability will be used in intracytoplasmic sperm injection, and combine with different intensity of assisted oocyte activation. Comparing these models, we will first analyze the correlation between Ca2+ message and submicron change of fragmentation. At the same time, the gene expression pattern and cytoskeleton correlated gene expression of AIO models will be detected to determine the molecular mechanism of embryo fragmentation caused by abnormal AIO. Further,we will construct adult mice models to detect the effects of AIO on embryonic late development. Also, we will use the new technique that we have set up to examine the activation ability of human sperms, so to validate the correlation of AIO and embryo fragmentation in clinic. Then we will detect expression patterns of the associated genes as above in the denoted human embryos, so to validate the mechanism of fragmentation found in mouse embryonic model in human embryo. We will also evaluate the assisted oocyte activation techniques following intracytoplasmic sperm injection based on clinical samples. Our research will not only describe the AIO correlated mechanism of embryo fragmentation, but also provide novel theory for evaluation of human embryo quality and strategies for application of assisted oocyte activation technique following intracytoplasmic sperm injection.

卵裂中胚胎碎片形成是人类体外受精常见现象，但其形成机制尚不清楚。申请者通过临床观察及建立的模拟小鼠精子激活力减弱技术，发现卵激活强度（AIO）不足与胚胎卵裂碎片形成相关。本项目将在此基础上进一步阐明AIO与胚胎碎片形成的关系及临床对策。拟采用激活力减弱小鼠精子进行显微授精，联合辅助激活法，构建不同AIO胚胎模型，观测碎片化的亚微结构改变与钙信号的联系，比较基因表达谱及骨架蛋白动力学调控相关基因的表达，以分析AIO异常导致胚胎碎片形成的分子机制。且建立出生小鼠模型，进一步探究AIO对胚胎后期发育影响。同时通过人精子激活力检测的新技术，在临床上验证AIO与胚胎碎片的关系，通过检测人类捐赠废胚的相关分子，在人类胚胎验证上述发现的碎片形成机制，并综合临床显微授精辅助激活的资料分析辅助激活的利弊。本项目将不仅阐明AIO相关的胚胎碎片形成机制，而且为人胚质量分析提供新理论，对辅助激活应用提供新策略。

卵裂中胚胎碎片形成是人类体外受精常见现象，其形成机制尚不清楚。本课题拟阐明卵激活强度（AIO）与胚胎碎片形成的关系及临床对策。项目实现了激活力减弱程度量化可控的小鼠精子模型，进行显微授精，联合辅助激活法，构建不同AIO胚胎模型。研究发现，激活力降低导致胚胎碎片增多，发育潜能降低，早期胚胎发育和基因表达谱的改变，而辅助激活有利于恢复其正常；进一步研究发现，在低激活力组细胞凋亡显著增加，而对低激活力加以辅助激活则恢复正常。检测发现众多与细胞凋亡、生存以及细胞分裂相关的基因表达在低激活力组发生了显著变化。这三个细胞过程的改变影响早期胚胎正常发育，且最重要的诱因常常是DNA损伤，而后者有报道可能是胚胎碎片形成的一个重要原因之一。本项目深入研究发现，卵激活强度与DNA损伤修复相关基因表达改变有关，而在对精子DNA损伤的修复中，弱激活力组显著低于正常对照组和弱激活力加辅助激活组。研究还测试了与DNA损伤有关的抗氧化培养，发现在胚胎早期发育过程中，强化线粒体介导的抗氧化反应能提高胚胎发育质量、减少碎片形成。然而，激活力强弱还与卵子的胞质成熟状态有关。因此，本项目结合临床促排卵策略，研究发现高孕激素下促排卵在可人为控制延长卵泡发育时间的同时，有利于包括减少胚胎碎片在内的胚胎质量的提高。总之，本项目证实了弱激活力是胚胎碎片形成的重要因素，其机理是激活力强度影响受精后一系列重要基因正常表达，包括细胞凋亡、生存以及细胞分裂相关的基因表达，尤其是DNA损伤修复相关表达；针对弱激活力精子的辅助激活、抗DNA损伤的抗氧化培养及控制延长卵泡发育成熟时间的临床策略等有利于降低胚胎碎片的形成。这些研究为指导临床优化治疗策略，提高胚胎发育质量提供了理论依据和技术途径。