O-253 Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males

Abstract

Abstract Study question Is there any novel variants and mutant hotspot in DNAH1 gene related to multiple morphological abnormalities of sperm flagella (MMAF) and male infertility in humans? Summary answer We identified eighteen variants of DNAH1 gene in eleven MMAF-affected Chinese men, including fourteen novel variants, four previously reported variants, and a candidate mutant hotspot. What is known already As a specific type of asthenoteratozoospermia, MMAF is characterized by composite flagellar defects including absent, short, coiled, angulation, and irregular-caliber, and usually presents ultrastructural abnormalities of axonemal and peri-axonemal structures. Genetic defects are the major cause of MMAF and biallelic variants of DNAH1 have been found to account for approximately 30% of studied human MMAF cohorts. Males with DNAH1 variants usually present with primary infertility owing to abnormal sperm morphology and motility. Intracytoplasmic sperm injection (ICSI) is the main strategy applied for patients with DNAH1 variants to conceive. Study design, size, duration A total of thirty-five patients with MMAF phenotype were recruited from the center for reproductive medicine from August 2019 to June 2022. A next-generation sequencing (NGS) panel of 22 MMAF-related genes was applied for genetic testing and the hereditary pattern was confirmed with Sanger sequencing. Sperm morphological analysis, immunostaining assays, and assisted reproductive therapy were performed in 2021 and 2022. Participants/materials, setting, methods The DNAH1 variants were identified by NGS and confirmed by Sanger sequencing. Pedigree analysis and in silico analysis further confirmed the pathogenicity or likely pathogenicity of these variants. Papanicolaou-staining, scanning and transmission electronic microscopy, and immunostaining were used to characterize the morphology and ultrastructure of spermatozoa. ICSI was applied for the assisted reproductive therapy of DNAH1-mutant patients and the outcomes of fertilization, embryo cleavage, and delivery were analyzed. Main results and the role of chance We totally identified 18 different DNAH1 variants (NM_015512.5) in 11 of 35 MMAF-affected males, including 9 missense variants (c.7690G>A, p.A2564T; c.10970C>G, p.T3657R; c.5584G>A, p.G1862R; c.6887T>C, p.L2296P; c.12122C>T, p.T4041I; c.1832T>C, p.L611P; c.2738C>A, p.A913D; c.5795G>A, p.R1932Q; c.7066C>T, p.R2356W) and 9 loss-of-function variants (c.11726_11727delCT, p.P3909Rfs*33; c.12172C>T, p.Q4058*; c.5104C>T, p.R1702*; c.12210del, p.V4071Cfs*54; c.8533del, p.D2845Mfs*2; c.12178_12190del, p.E4060Pfs*61; c.2301-1G>T; c.4552C>T, p.Q1518*; c.12118_12119delCA, p.Q4040Dfs*33). Moreover, 77.8% (14/18) of the identified variants were novel. The results of Papanicolaou-staining, scanning electronic microscopy, and immunofluorescence demonstrated the typical characteristics of MMAF, consisting of absent, short, coiled, angulation, and irregular-caliber flagella, of the spermatozoa affected by DNAH1 variants. Transmission electronic microscopy further revealed the compound changes of flagella ultrastructure, including loss of central pair and disorganization of microtubule doublets and outer dense fibers. Six of eleven underwent assisted reproductive therapy with ICSI in our clinic. The rates of fertilization, usable embryos and clinical pregnancy for patients with DNAH1 variants following ICSI were 81.5%, 52.8% and 66.7%, respectively. To date, three couples have given birth to a total of five healthy children. Limitations, reasons for caution Because of distance and inconvenience, five of the eleven DNAH1-affected men didn’t provide additional semen samples for functional analysis. In addition, as the absence of specific antibody of DNAH1, immunostaining for DNAH1 was not performed in this study. Wider implications of the findings This study significantly expands the variant spectrum of DNAH1 gene related to MMAF and male infertility in humans. The systematic description of assisted reproduction therapies will provide clinicians with new insights into molecular diagnosis and genetic counseling. Trial registration number not applicable