Male infertility is often linked to genetic mutations that impair sperm motility and morphology. Multiple morphological abnormalities of the sperm flagella (MMAF) are recognized as a significant cause of male infertility and are characterized by a spectrum of structural defects. However, the underlying etiology of MMAF remains largely unknown. In this study, we recruited a consanguineous family with a history of primary male infertility affecting two brothers diagnosed with MMAF, without obvious signs of primary ciliary dyskinesia. Whole-exome sequencing and Sanger sequencing were used to identify potentially pathogenic variants, and the effects of the identified mutation were evaluated in vitro and in silico. Papanicolaou staining, scanning electron microscopy, and transmission electron microscopy were performed to analyze sperm morphology. The localization and expression of outer dense fiber 2 (ODF2) in spermatozoa were examined by immunofluorescence and Western blot. We identified a novel homozygous missense mutation, c.539G>A (p.R180K), in ODF2 in the two infertile brothers. Sanger sequencing confirmed autosomal recessive inheritance. The patients exhibited asthenoteratozoospermia, with abnormal flagellar morphology and reduced motility. Further ultrastructural analysis revealed axonemal disorganization consistent with MMAF. Although the identified ODF2 mutation did not affect the overall protein expression, the mutant ODF2 protein was absent from the sperm flagella. Cell transfection experiments demonstrated the aberrant localization of the mutated protein, and bioinformatics analyses predicted the disruption of a conserved residue within a coiled-coil domain of ODF2. This study underscores the pivotal role of ODF2 in sperm flagellar development and broadens the mutational spectrum associated with human MMAF.

Keywords: male infertility; multiple morphological abnormalities of the sperm flagella; ODF2; whole-exome sequencing