A Novel Homozygous Mutation in PMFBP1 Associated with Acephalic Spermatozoa Defects.

Background/Objectives: Acephalic spermatozoa syndrome (ASS) is a rare subtype of male infertility characterized by headless sperm due to defective head-tail coupling. Genetic factors are recognized as the primary etiology of ASS; however, known pathogenic mutations only explain a subset of ASS cases. Further investigations are required to elucidate the underlying genetic pathogenesis of ASS and the implications of such genetic defects for assisted reproductive technology (ART) outcomes. This study aimed to identify a novel PMFBP1 mutation in an ASS patient; investigate the effects of the identified mutation on sperm ultrastructure and PMFBP1 protein expression/stability, and assess ART outcomes using the patient's sperm. Methods: One 34-year-old infertile male with ASS was enrolled. Genetic screening was performed via whole-exome sequencing (WES), followed by Sanger sequencing for mutation validation. Sperm morphological characteristics were evaluated using Diff-Quik staining (for general morphology), transmission electron microscopy (TEM, for ultrastructural analysis), and peanut agglutinin (PNA) staining. Protein expression and stability were analyzed by Western blot and cycloheximide (CHX)/MG132 assays. ART outcomes were compared between the in vitro fertilization (IVF) cycles using the patient's sperm and those using donor sperm. Results: In IVF cycles, donor sperm achieved normal fertilization (characterized by two pronuclei [2PN] formation), whereas the patient's sperm failed to form 2PN and leading to embryo fragmentation. Genetic sequencing identified a novel nonsense mutation in PMFBP1 (c.2641C>T), which introduced a premature stop codon and resulted in a premature protein product (p.Arg881Ter). Morphologically, this mutation led to complete sperm head-tail detachment, and abnormalities in acrosome structure and sperm head-neck junction. The absence of PMFBP1 protein in the patient's spermatozoa was observed. The in vitro assays showed the c.2641C>T mutation induced expression of the truncated PMFBP1 protein and significantly altered PMFBP1 protein stability. Conclusions: The PMFBP1 c.2641C>T mutation impairs sperm head-tail adhesion, thereby contributing to the pathogenesis of ASS. This study expands the clinical mutational spectrum of PMFBP1-associated male infertility and provides valuable insights for the genetic diagnosis of ASS patients. Additionally, these findings may lay a foundation for the choice of therapeutic strategies targeting PMFBP1-related ASS.