Unraveling the Molecular Mechanisms of Total Fertilization Failure: Insights into Transcriptional Dysregulation

Background Fertilization involves interactions between sperm and metaphase II (MII) oocyte, initiating a cascade of events including oocyte activation, calcium release, resumption of meiosis, and zygote formation. Total fertilization failure (TFF), characterized by disruption of any of these processes, occurs in 1-3% of intracytoplasmic sperm injection (ICSI) cycles. The genetic basis of TFF remains largely unexplored. Methods We recruited a familial case of TFF. We assessed sperm parameters, binding ability, and fertilization outcomes using molecular assays and confocal microscopy. The whole genome sequencing and comprehensive genetic analyses were performed on the patients and family members to identify potential loss-of-function variants in genes involved in oogenesis and fertilization. Additionally, single-cell RNA sequencing of the patient's MII oocytes was conducted to assess transcriptomic profile. Two ICSI cycles were performed, one of which included assisted oocyte activation (AOA) using a calcium ionophore. Integrated bioinformatic analysis using RNA-seq and ATAC-seq was used to map genome-wide regulatory landscape in normal oocytes and early embryos. An enhancer-mediated gene regulatory network (eGRN) was constructed to assess the impact of mutations on transcription factor binding and gene regulation across the different GO BP and Reactome pathways. Results Despite normal sperm parameters in the husband and the absence of genetic variants related to oocyte activation in both partners, RNA-seq revealed aberrant expression of genes involved in meiosis, zona pellucida biogenesis, and oocyte activation. RNA-seq and ATAC-seq data identified 22 key transcription factors whose binding is significantly affected by the mutational landscape of the patient's oocytes and that may disrupt downstream pathways involved in oogenesis, fertilization, and early embryonic development. ZNF121, TFAP2D, VEZF1 and YY1 were the top 4 hub TFs discovered in GO terms network. Additionally, TFAP2D, YY1, ZFP28 and ZNF121 were the most connected TFs to different Reactome pathways. Conclusions Our findings suggest that transcriptional dysregulations play a central role in total fertilization failure (TFF), by impacting downstream gene expression. Although, assisted oocyte activation with calcium ionophore helped successfully achieve fertilization, leading to the development of an 8-cell embryo. The insights from our study provide valuable opportunities for targeted interventions and potential therapeutic strategies in reproductive medicine. Overall design: low-input RNA sequencing (RNAseq) at the single-oocyte resolution level of inseminated MII oocytes of an infertile patient.

背景 受精过程涉及精子与第二次减数分裂中期卵母细胞（metaphase II, MII）的相互作用，可触发一系列级联反应，包括卵母细胞激活、钙离子释放、减数分裂恢复以及合子形成。完全受精失败（total fertilization failure, TFF）以上述任一过程紊乱为特征，在卵胞浆内单精子注射（intracytoplasmic sperm injection, ICSI）周期中的发生率为1%~3%，但其遗传基础目前仍未得到充分阐明。
方法 本研究纳入1例完全受精失败的家系病例。通过分子实验与共聚焦显微镜成像评估患者的精子参数、结合能力以及受精结局。对患者及其家庭成员进行全基因组测序与综合遗传分析，以筛选参与卵子发生与受精过程的基因中潜在的功能丧失变异。此外，对患者的MII期卵母细胞开展单细胞RNA测序以分析其转录组特征。本研究共完成2次ICSI周期，其中1次采用钙离子载体实施辅助卵母细胞激活（assisted oocyte activation, AOA）。通过整合RNA测序与转座酶可及性测序（ATAC-seq）的生物信息学分析，绘制正常卵母细胞与早期胚胎的全基因组调控图谱。构建增强子介导的基因调控网络（enhancer-mediated gene regulatory network, eGRN），以评估突变对不同基因本体生物学过程（GO BP）及Reactome通路中转录因子结合与基因调控的影响。
结果 尽管男方精子参数正常，且夫妻双方均未携带与卵母细胞激活相关的遗传变异，但RNA测序结果显示，参与减数分裂、透明带生物发生以及卵母细胞激活的基因存在异常表达。RNA测序与ATAC-seq数据共筛选出22个关键转录因子，其结合活性受患者卵母细胞突变谱的显著影响，可能破坏参与卵子发生、受精以及早期胚胎发育的下游通路。在基因本体术语网络中，ZNF121、TFAP2D、VEZF1与YY1为排名前4的枢纽转录因子。此外，TFAP2D、YY1、ZFP28与ZNF121是与不同Reactome通路关联最紧密的转录因子。
结论 本研究结果表明，转录失调通过影响下游基因表达，在完全受精失败中发挥核心作用。尽管采用钙离子载体实施辅助卵母细胞激活成功实现了受精，并培育出8细胞胚胎，但本研究的发现为生殖医学领域的靶向干预与潜在治疗策略提供了宝贵的研究思路。
整体实验设计：对1例不育患者的受精后MII期卵母细胞开展单细胞分辨率的低起始量RNA测序（low-input RNA sequencing）。