Table3_A case report of a family with developmental arrest of human prokaryotic stage zygote.XLSX

To study the genetic variation leading to the arrest phenotype of pronuclear (PN) zygotes. We recruited a family characterized by recurrent PN arrest during in vitro fertilization (IVF) and intracytoplasmic sperm injection cycles (ICSI) and performed whole-exome sequencing for 2 individuals. The transcriptome profiles of PN-arrest zygotes were assessed by single-cell RNA sequencing analysis. The variants were then validated by PCR amplification and Sanger sequencing in the affected individuals and other family members. A family characterized by recurrent PN arrest during IVF and ICSI cycles were enrolled after giving written informed consent. Peripheral blood samples were taken for DNA extraction. Three PN-arrest zygotes from patient III-3 were used for single-cell RNA-seq as described. This phenotype was reproduced after multiple cycles of egg retrieval and after trying different fertilization methods and multiple ovulation regimens. The mutant genes of whole exon sequencing were screened and verified. The missense variant c. C1630T (p.R544W) in RGS12 was responsible for a phenotype characterized by paternal transmission. RGS12 controls Ca2+ oscillation, which is required for oocyte activation after fertilization. Single-cell transcriptome profiling of PN-arrest zygotes revealed defective established translation, RNA processing and cell cycle, which explained the failure of complete oocyte activation. Furthermore, we identified proximal genes involved in Ca2+ oscillation–cytostatic factor–anaphase-promoting complex (Ca2+ oscillation–CSF–APC) signaling, including upregulated CaMKII, ORAI1, CDC20, and CDH1 and downregulated EMI1 and BUB3. The findings indicate abnormal spontaneous Ca2+ oscillations leading to oocytes with prolonged low CSF level and high APC level, which resulted in defective nuclear envelope breakdown and DNA replication. We have identified an RGS12 variant as the potential cause of female infertility characterized by arrest at the PN stage during multiple IVF and ICSI.

本研究旨在探究导致原核（pronuclear, PN）受精卵阻滞表型的遗传变异。我们招募了一个在体外受精（in vitro fertilization, IVF）及卵胞浆内单精子注射（intracytoplasmic sperm injection, ICSI）周期中反复出现原核阻滞的家系，并对2名个体开展全外显子测序（whole-exome sequencing）。通过单细胞RNA测序（single-cell RNA sequencing）分析，我们对原核阻滞受精卵的转录组特征进行了评估。随后，通过PCR扩增（PCR amplification）及桑格测序（Sanger sequencing），对受累个体及其他家系成员的变异位点进行了验证。
本研究在获取书面知情同意后，纳入了另一个在IVF及ICSI周期中反复出现原核阻滞的家系。采集外周血样本以提取DNA。取自患者III-3的3枚原核阻滞受精卵被用于后续单细胞RNA测序，具体操作如前文所述。该表型在多次取卵周期、尝试不同受精方式及多种促排卵方案后均得到重现。我们对全外显子测序发现的突变基因进行了筛选与验证。
RGS12基因中的错义变异c.C1630T（p.R544W）为父系遗传相关表型的致病因素。RGS12可调控钙振荡（Ca2+ oscillation），而钙振荡是受精后卵母细胞激活所必需的过程。对原核阻滞受精卵的单细胞转录组分析显示，其翻译起始、RNA加工及细胞周期均存在缺陷，这解释了卵母细胞完全激活失败的原因。此外，我们还鉴定出参与钙振荡-细胞静止因子（cytostatic factor, CSF）-后期促进复合物（anaphase-promoting complex, APC）信号通路的近端调控基因，包括上调表达的CaMKII、ORAI1、CDC20及CDH1，以及下调表达的EMI1和BUB3。
本研究结果表明，异常的自发性钙振荡会导致卵母细胞内细胞静止因子水平持续偏低、后期促进复合物水平偏高，进而引发核膜破裂及DNA复制缺陷。我们已确认RGS12基因变异是导致多次IVF及ICSI周期中出现原核阶段阻滞的女性不孕症的潜在病因。