DataSheet_1_Novel HYDIN variants associated with male infertility in two Chinese families.docx

IntroductionInfertility is a major disease affecting human life and health, among which male factors account for about half. Asthenoteratozoospermia accounts for the majority of male infertility. High-throughput sequencing techniques have identified numerous variants in genes responsible for asthenoteratozoospermia; however, its etiology still needs to be studied.
MethodIn this study, we performed whole-exome sequencing on samples from 375 patients with asthenoteratozoospermia and identified two HYDIN compound heterozygous variants, a primary ciliary dyskinesia (PCD)-associated gene, in two unrelated subjects. H&E staining, SEM were employed to analyze the varies on sperm of patients, further, TEM was employed to determine the ultrastructure defects. And westernblot and immunostaining were chose to evaluate the variation of structural protein. ICSI was applied to assist the mutational patient to achieve offspring.
ResultWe identified two HYDIN compound heterozygous variants. Patient AY078 had novel compound heterozygous splice variants (c.5969-2A>G, c.6316+1G>A), altering the consensus splice acceptor site of HYDIN. He was diagnosed with male infertility and PCD, presenting with decreased sperm progressive motility and morphological abnormalities, and bronchial dilatation in the inferior lobe. Compared to the fertile control, HYDIN levels, acrosome and centrosome markers (ACTL7A, ACROSIN, PLCζ1, and Centrin1), and flagella components (TOMM20, SEPT4, SPEF2, SPAG6, and RSPHs) were significantly reduced in HYDIN-deficient patients. Using intracytoplasmic sperm injection (ICSI), the patient successfully achieved clinical pregnancy. AY079 had deleterious compound heterozygous missense variants, c.9507C>G (p. Asn3169Lys) and c.14081G>A (p. Arg4694His), presenting with infertility; however, semen samples and PCD examination were unavailable.
DiscussionOur findings provide the first evidence that the loss of HYDIN function causes asthenoteratozoospermia presenting with various defects in the flagella structure and the disassembly of the acrosome and neck. Additionally, ICSI could rescue this failure of insemination caused by immobile and malformed sperm induced by HYDIN deficiency.

引言
不育症是影响人类生命健康的重大疾病，其中男性因素占比约半数。弱畸精子症（asthenoteratozoospermia）是男性不育的主要病因。高通量测序技术已发现众多与弱畸精子症相关的基因变异，但该病的病因学仍有待深入研究。
方法
本研究对375例弱畸精子症患者的样本实施全外显子组测序（whole-exome sequencing），在两名无关患者中鉴定出原发性纤毛运动障碍（primary ciliary dyskinesia, PCD）相关基因HYDIN的复合杂合变异。采用苏木精-伊红（H&E）染色、扫描电子显微镜（SEM）分析患者精子的形态异常，进一步通过透射电子显微镜（TEM）观察其超微结构缺陷。此外通过蛋白质印迹（westernblot）与免疫染色实验评估结构蛋白的表达变化。采用卵胞浆内单精子注射（intracytoplasmic sperm injection, ICSI）辅助携带该变异的患者获得子代。
结果
本研究成功鉴定出HYDIN基因的两种复合杂合变异。患者AY078携带新型复合杂合剪接变异（c.5969-2A>G、c.6316+1G>A），该变异破坏了HYDIN基因的保守剪接受体位点。该患者被诊断为男性不育合并原发性纤毛运动障碍，表现为精子前向运动能力下降、形态异常，以及下叶支气管扩张。与可育对照组相比，HYDIN蛋白缺陷患者的HYDIN表达水平、顶体与中心体标志物（ACTL7A、ACROSIN、PLCζ1及Centrin1）以及鞭毛组分（TOMM20、SEPT4、SPEF2、SPAG6及RSPHs）的表达均显著降低。通过卵胞浆内单精子注射（ICSI）辅助生殖，该患者成功实现临床妊娠。患者AY079携带有害复合杂合错义变异c.9507C>G（p.Asn3169Lys）与c.14081G>A（p.Arg4694His），表现为男性不育，但未能获取其精液样本与原发性纤毛运动障碍相关检查结果。
讨论
本研究首次证实HYDIN功能缺失可引发弱畸精子症，患者表现为鞭毛结构多种缺陷以及顶体与颈部解离。此外，卵胞浆内单精子注射（ICSI）可挽救因HYDIN缺陷导致的精子活动力缺失与形态异常所引发的受精失败。