Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei, 230022, Anhui, China.
NHC Key Laboratory of study on abnormal gametes and reproductive tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China.
Reprod Biol Endocrinol. 2021 Feb 20;19(1):27. doi: 10.1186/s12958-021-00709-0.
Asthenozoospermia is one of the most common causes of male infertility, and its genetic etiology is poorly understood. DNAH9 is a core component of outer dynein arms in cilia and flagellum. It was reported that variants of DNAH9 (OMIM: 603330) might cause primary ciliary dyskinesia (PCD). However, variants in DNAH9 lead to nonsyndromic severe asthenozoospermia have yet to be reported.
Whole exome sequencing (WES) was performed for two individuals with nonsyndromic severe asthenozoospermia from two non-consanguineous families, and Sanger sequencing was performed to verify the identified variants and parental origins. Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition). Transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail. Quantitative realtime-PCR and immunofluorescence staining were performed to detect the expression of DNAH9-mRNA and location of DNAH9-protein. Furthermore, assisted reproductive procedures were applied.
By WES and Sanger sequencing, compound heterozygous DNAH9 (NM_001372.4) variants were identified in the two individuals with nonsyndromic severe asthenozoospermia (F1 II-1: c.302dupT, p.Leu101fs*47 / c.6956A > G, p.Asp2319Gly; F2 II-1: c.6294 T > A, p.Phe2098Leu / c.10571 T > A, p.Leu3524Gln). Progressive rates less than 1% with normal sperm morphology rates and normal vitality rates were found in both of the two subjects. No respiratory phenotypes, situs inversus or other malformations were found by detailed medical history, physical examination and lung CT scans etc. Moreover, the expression of DNAH9-mRNA was significantly decreased in sperm from F1 II-1. And expression of DNAH9 is lower in sperm tail by immunofluorescence staining in F1 II-1 compared with normal control. Notably, by intracytoplasmic sperm injection (ICSI), F1 II-1 and his partner successfully achieved clinical pregnancy.
We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.
弱精症是男性不育症最常见的原因之一，但其遗传病因尚不清楚。DNAH9 是鞭毛和纤毛中外辐动蛋白的核心组成部分。据报道，DNAH9（OMIM：603330）的变体可能导致原发性纤毛运动障碍（PCD）。然而，导致非综合征性严重弱精症的 DNAH9 变体尚未报道。
对来自两个非近亲家庭的两名非综合征性严重弱精症患者进行全外显子组测序（WES），并进行 Sanger 测序以验证鉴定的变体和亲本来源。根据世界卫生组织 2010 年（第 5 版）指南进行精子常规分析、精子活力率和精子形态分析。使用透射电子显微镜（TEM、TECNAI-10、80kV、Philips、Holland）观察精子尾部的超微结构。进行实时定量 PCR 和免疫荧光染色以检测 DNAH9-mRNA 的表达和 DNAH9-蛋白的位置。此外，还应用了辅助生殖程序。
通过 WES 和 Sanger 测序，在两名非综合征性严重弱精症患者中发现 DNAH9（NM_001372.4）复合杂合变体（F1 II-1：c.302dupT，p.Leu101fs*47/c.6956A>G，p.Asp2319Gly；F2 II-1：c.6294>T/A，p.Phe2098Leu/c.10571>T/A，p.Leu3524Gln）。在这两个人中，精子的运动率小于 1%，形态正常，活力正常。通过详细的病史、体检和肺部 CT 扫描等未发现呼吸表型、 situs inversus 或其他畸形。此外，F1 II-1 中的 DNAH9-mRNA 表达显著降低。与正常对照相比，F1 II-1 中 DNAH9 的免疫荧光染色显示精子尾部的表达较低。值得注意的是，通过胞浆内精子注射（ICSI），F1 II-1 和他的伴侣成功实现了临床妊娠。
我们将 DNAH9 鉴定为非综合征性严重弱精症的新型致病基因，ICSI 可有助于这些患者获得良好的妊娠结局。
