冻融胚胎移植对子代神经发育相关印记基因甲基化修饰的影响及其与认知行为的相关性研究

Imprinted genes play an important role in the development of the nervous system. Frozen-thawed embryo transfer technology has been widely used in the field of assisted reproduction in recent years, but this occurs during a critical window of gene imprinting removal, establishment and maintenance. It is not clear whether it will alter the methylation of imprinted genes,especially those related to neurodevelopment, and affect cognitive behavior in offspring. Our previous study found there were individuals hospitalized for neurological diseases in offsprings born after frozen-thawed embryo transfer. Therefore, based on our establised“follow-up cohort of assisted reproductive technology”，this prospective study is carried out in offspring born after frozen-thawed embryo transfer and fresh embryo transfer. We will evaluate cognitive behaviors with the Gesell developmental index scale in infants at 12 and 24 months.The developmental level of cognitive behaviors in offsprings born after frozen-thawed embryo transfer and fresh embryo transfer will be assessed to analyze the potential risk of frozen-thawed embryo transfer for neuraldevelopment of offsprings from the population level. We will detect the methylation status of neurodevelopmen-related imprinted genes to analyse the effect of frozen-thawed embryo transfer on the methylation modification of imprinted genes related with neuraldevelopment. Furthermore, we will explore the gene expression of DNA methylation-related enzymes to reveal the molecular mechnism. In addition, the regulation mechanism of epigenetic modify-related enzymes on imprinted genes will be explored by adding inhibitors of epigenetic modifiy-related enzyme into the culture medium to construct the frozen-thawed embryo transplantation model in rats. This study aims to explore effect of frozen-thawed embryo transfer on DNA methylation of imprinted genes associated with neurodevelopment in offspring born after assisted reproductive technology and analyze its associations with cognitive behavior, providing scientific basis for the safety assessment of frozen-thawed embryo transfer.

印记基因对神经系统发育有重要作用。冻融胚胎移植术在辅助生殖领域应用广泛，但该操作发生在印记基因去除、建立和维持的关键窗口期，是否会影响子代神经发育相关印记基因甲基化修饰而改变认知行为目前尚不明确。我们前期研究发现冻融胚胎移植出生子代中有患神经系统疾病个体。为此，本项目依托已建立的“辅助生殖技术后随访队列”对冻融胚胎和新鲜胚胎移植子代开展前瞻性研究。采用Gesell发育量表在婴幼儿12、24个月测量其认知行为；对比冻融胚胎和新鲜胚胎移植子代认知行为发展水平，评估冻融胚胎移植子代神经发育异常风险。同时检测脐血中神经发育相关印记基因甲基化水平，分析冻融胚胎移植对神经发育印记基因甲基化修饰的影响；并检测表观遗传修饰相关酶的表达，探索可能作用机制。进一步通过在培养液中添加表观遗传修饰酶抑制剂构建冻融胚胎移植大鼠模型探究表观遗传修饰相关酶对印记基因的调控机制，为冻融胚胎移植安全性评估提供参考依据。

研究背景：冻融胚胎移植技术作为辅助生殖技术中不可或缺的组成部分，其安全性是关注的重点。近年来很多研究认为ART操作可能会增加神经发育性疾病的风险，但关于冻融胚胎移植术后出生子代神经发育的流行病学研究并不多见。.主要研究内容：本研究从接受辅助生殖治疗开始建立前瞻性出生队列，并追踪随访孕妇直至子代出生后24个月。在婴幼儿12、24个月时采用国际上通用的Gesell儿童发育指数量表进行认知行为发展水平测评，定量分析冻融胚胎移植对婴幼儿认知行为发展水平的影响。开展动物实验，构建小鼠冻融胚胎移植模型，进行全转录组基因测序，分析冻融胚胎技术对早期胚胎基因表达的影响，探究其可能的分子作用机制。.重要结果：我们尚没有发现胚胎冻存时间长短与子代出生健康结局存在有统计学意义的关联；未发现冻融胚胎移植会增加子代12/24月龄的神经行为发育异常的风险。构建小鼠冻融胚胎移植模型，通过全转录组基因测序找到了受早期胚胎冻融技术影响的神经性疾病相关通路和基因，这提示我们还需对子代进行长期健康随访。.关键数据：多因素线性回归模型分析，冻融胚胎移植并未增加子代12/24月龄的大动作、精细动作、适应性、语言等各能区神经发育异常风险。冻融胚胎组小鼠胚胎有642个基因上调，1266个基因表达下调，同时，确定了上调和下调转录本的前10个Gene Ontology (GO)条目和前3个Kyoto Encyclopedia of Genes and Genomes (KEGG) 通路。在差异通路分析中，我们重点关注了和神经相关的差异信号通路，表达上调基因参与的信号通路：帕金森病（Parkinson disease）调控通路，阿尔茨海默病（Alzheimer disease）调控通路，神经退行性多种疾病（Pathways of neurodegeneration - multiple diseases）调控通路，并且分析出参与以上3种信号通路的是COX4I2基因。表达下调基因参与的信号通路：长期的抑郁（Long-term depression）相关通路，调控此信号通路的差异基因为：GRID2和GUCY1B3基因。动物实验结果提示仍需长期关注冻融胚胎移植子代的神经发育。.科学意义：本研究以丰富科学的数据探讨了冻融胚胎移植对子代神经行为发育的影响，为冻融胚胎移植技术的安全性评价提供了客观的科学依据。