Dongdong Tang1,2,3, Yanwei Sha4,5, Yang Gao1,2,3, Jingjing Zhang1, Huiru Cheng1,2,3, Junqiang Zhang1,2,3, Xiaoqing Ni1,2,3, Chao Wang1,2,3, Chuan Xu1,2,3, Hao Geng1,2,3, Xiaojin He6,7,8, Yunxia Cao9,10,11. 1. Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei, 230022, Anhui, China. 2. NHC Key Laboratory of study on abnormal gametes and reproductive tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China. 3. Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China. 4. Department of Andrology, United Diagnostic and Research Center for Clinical Genetics, School of Public Health & Women and Children's Hospital, Xiamen University, Xiamen, 361005, Fujian, China. 5. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, 361102, China. 6. Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei, 230022, Anhui, China. hxj0117@126.com. 7. NHC Key Laboratory of study on abnormal gametes and reproductive tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China. hxj0117@126.com. 8. Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China. hxj0117@126.com. 9. Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei, 230022, Anhui, China. caoyunxia6@126.com. 10. NHC Key Laboratory of study on abnormal gametes and reproductive tract (Anhui Medical University), No 81 Meishan Road, Hefei, 230032, Anhui, China. caoyunxia6@126.com. 11. Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No 81 Meishan Road, Hefei, 230032, Anhui, China. caoyunxia6@126.com.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.
BACKGROUND: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. METHODS: 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. RESULTS: 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. CONCLUSIONS: We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.