精子DNA 氧化应激损伤定位及对早期胚胎发育的影响机制研究

Oxidative stress in sperm preparation and sperm freezing caused sperm DNA damage in assisted reproductive technology(ART) and finally resulted in poor clinical pregnancy outcomes because of the failure of egg-sperm fusion and poor development of embryos. The underlying etiology of sperm oxidative stress DNA damage on egg-sperm fusion and embryo development is still unclear. Our pre-experimental results indicated that sperm DNA damage always localized in some specific nucleosome regions where gene promoter and the genes related with embryo development are highly expressed. So we propose a hypothesis: sperm DNA damage has negative effects on development regulators in the nucleosome regions and subsequently has detrimental impacts on the expression of genes involved in embryo development, ultimately resulted in poor quality embryos and poor clinical pregnancy outcome. In order to uncover the mechanism involved in this hypothesis,oxidative stress DNA damage sperm models of mice and human will be established, the effects of sperm DNA damage on embryo development will be analyzed in details by immunofluorescence, RT-PCR and SNP-Chips. In all, our research in this project will provide the theoretical basis for improving the clinical pregnancy outcome when sperm DNA damages occurs.

在辅助生殖技术中，精液处理和精子冷冻等过程中的氧化应激反应可能会造成精子DNA损伤，导致精卵结合障碍和胚胎发育异常而影响临床妊娠结局。目前精子DNA氧化应激损伤对精卵结合和胚胎发育影响机制依然知之甚少。我们的预实验结果提示小鼠和人的精子DNA损伤发生在特定的核小体区域，既往多项研究证实此区域内富含基因启动子和调节胚胎发育的基因位点，为此我们提出假说，精子DNA氧化应激损伤造成核小体区域内的调控基因受累，从而影响后续发育相关基因的表达，最终导致胚胎质量欠佳和临床妊娠结局低下。为了验证这一假说，我们将通过建立小鼠和人精子DNA氧化应激损伤模型，采用免疫荧光、实时PCR、基因芯片等技术手段探讨精子DNA损伤对胚胎发育的影响，为改善精子DNA损伤的临床妊娠结局提供理论基础和指导。

不育男性中多种因素引发的氧化应激损伤比较常见，易造成精子核DNA受损，最终导致临床助孕结局欠佳，具体机制不详。本研究提出精子核DNA氧化应激损伤累及区域内的胚胎发育相关基因，最终导致早期胚胎发育受阻的假说。本课题收集男性不育患者行精子核完整性检测后的废弃精液标本，根据精子DNA碎片化指数（DFI）分组，采用免疫荧光技术进行氧化应激损伤共定位，通过实时定量PCR技术检测并计算胚胎发育相关基因的损伤率；回顾性分析接受常规体外受精-胚胎移植技术（IVF-ET）不育患者临床资料，比较不同DFI组的胚胎质量指标，比较time-lapse延时摄影培养系统中的胚胎动力学指标，研究精子氧化应激损伤对早期胚胎发育的影响，进一步阐明相关作用机制，为临床治疗男性不育和改善助孕结局提供理论支持和指导。同时，研究褪黑素对卵子氧化应急损伤及后续胚胎质量的影响及作用机制，为精子氧化应激损伤及促进胚胎发育的干预提供理论和策略支持。研究发现人精子DNA损伤易发生在富含组蛋白的核小体区域和精子基质处，累及胚胎发育相关基因FSHB，并且随着精子DNA损伤程度的加重而基因损伤率增加；同时，精子DNA损伤也影响早期胚胎发育，随着DFI的增加，可移植胚胎率与优质胚胎率下降，早期胚胎的发育速度变缓。此外，发现褪黑素可以明显减轻卵子氧化应激损伤和改善胚胎发育，为进一步干预处理精子DNA损伤奠定基础。总之，探讨精子DNA损伤对早期胚胎发育的影响，进一步阐明详细作用机制，为ART临床改善男性不育的助孕结局提供理论支持和指导。