Identification Of A New Candidate Gene DNHD1 For Asthenoteratozoospermia With Male Infertility

Objective:Asthenoteratozoospermia,defined as decreased sperm motility and abnormal sperm morphology,accounts for approximately19%of male infertility cases.It has been widely recognized that genetic factors are likely involved in a large proportion of asthenoteratozoospermia with sperm flagellum defects.Briefly,a series of cilia and flagella associated protein（CFAP）genes,dynein axonemal heavy chain（DNAH）family member genes,and other genes such as USP26,WDR19,AKAP3 and DZIP1 have been reported to cause asthenoteratozoospermia.Identifying candidate genes of asthenoteratozoospermia and their specific clinical phenotypes will promote the understanding of the etiology of asthenoteratozoospermia and assist the development of related treatment strategies.Methods:In this study,whole-exome sequencing（WES）was performed on 314 unrelated infertile men with asthenoteratozoospermia from CITIC-Xiangya Reproductive and Genetics Hospital to screen potential candidate pathogenic variants,and Sanger sequencing,bioinformatics analysis was performed to assess the pathogenicity of the identified mutations.Further through in vitro functional experiments,we used CRISPR/Cas9 to construct mouse model to clarify the pathogenicity of the mutations.Follow-up and record the outcome of ICSI assisted pregnancy treatment with the pathogenic mutation and provide fertility treatment reference for such patients.Results:In this study,we found DNHD1 biallelic mutations of four patients with asthenoteratozoospermia from four different families,all of which were consistent with family co-segregation.Common light microscopy and scanning electron microscopy showed that there were various morphological abnormalities of sperm flagella.Transmission electron microscopy showed that some patients had central pair of microtubules（CP）deletion and mitochondrial sheath（MS）abnormalities.The results of sperm immunofluorescence staining showed that the expression of DNHD1,CP component SPAG6 and sperm flagella assembly-related protein SPEF2 were absent,and MS component TOMM20 was expressed missharpen,unevenly distributed and swelling of the flagella mid-piece.The Dnhd1^-/-male mice showed complete sterility,decreased sperm concentration and motility,and presented various morphological abnormalities of sperm flagella as the patients.In addition,four patients with biallelic mutations in DNHD1 were treated with ICSI,and the wives of two patients had successful pregnancy.Conclusions:In conclusion,we identified a gene causing male infertility due to asthenoteratozoospermia.The genetic and experimental evidence from DNHD1-associated men and Dnhd1^-/-male mice strongly suggests that DNHD1 plays a vital role in sperm flagellogenesis.DNHD1deficiency causes abnormalities in both axonemes and peri-axonemes,leading to asthenoteratozoospermia.Our study provides insights into the understanding of asthenoteratozoospermia and the counseling of individuals with asthenoteratozoospermia.