Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare disease that causes primary infertility. However, the genetic causes for approximately half of MMAF cases are unknown. Whole exome sequencing analysis of the 27 patients with MMAF identified several CFAP44 mutations (3 homozygous: c.2935_2944del: p.D979*, c.T1769A: p.L590Q, c.2005_2006del: p.M669Vfs*13; and putative compound heterozygous: c.G3262A: p.G1088S and c.C1718A: p.P573H.) and CFAP43 acceptor splice-site deletion (c.3661-2A>-) mutations in 5 and 1 patients, respectively. Real-time quantitative polymerase chain reaction assays also demonstrated that CFAP44 expression was very weak in patient (P)1 and P3, and CFAP43 expression was lower in P6 than in the control. Immunofluorescence analysis of CFAP43 showed lower CFAP43 protein expression levels in P6 than in the normal control. This study demonstrated that biallelic mutations in CFAP44 and CFAP43 cause MMAF. These results provide researchers with a new insight to understand the genetic etiology of MMAF and to identify new loci for genetic counselling of MMAF.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.