Loss-of-function variant in TEX44 cause male infertility with asthenoteratozoospermia in humans and mice

Background Asthenoteratozoospermia, characterized by impaired sperm motility and abnormal morphology, is a major cause of male infertility. However, its genetic basis remains largely unclear in many idiopathic cases. Testis-expressed protein 44 (TEX44) is critical for murine sperm flagellar development, but its role in human asthenoteratozoospermia is remains poorly defined. Methods Whole-exome sequencing (WES) was performed on 535 unrelated infertile men to identify TEX44 variants, followed by Sanger sequencing validation. SWISS-MODEL and IUPred3 were used for TEX44 protein structure and disordered region prediction. A Tex44-knockout (Tex44) mouse model was established via CRISPR/Cas9. Sperm quality was assessed by computer-assisted sperm analysis (CASA). Fertility assays (in vivo fertilization, IVF, ICSI) and ultrastructural observations (TEM, SEM) were conducted to evaluate phenotypic defects. Transcriptome sequencing was used to explore underlying mechanisms. Clinical ICSI outcomes of patients with TEX44 variants were analyzed. Results Eight distinct TEX44 variants (seven missense: c.31G > C, c.236A > G, c.541C > T, c.736T > C, c.781T > C, c.794C > T, c.1156C > T; one frameshift deletion: c.429_432del) were identified in seven patients. Functional prediction and 3D modeling confirmed the pathogenicity of these variants, which correlated with patients' asthenoteratozoospermia phenotypes. Tex44 mice recapitulated the human sperm defects, showing drastically reduced motility and fertilization rates. TEM revealed core ultrastructural abnormalities: disruption of the axonemal 9 + 2 microtubule structure (specifically loss of the 7th peripheral doublet microtubule) and defective mitochondrial sheath assembly.Transcriptome analysis showed downregulated flagellar movement-related genes and upregulated mitochondria-associated genes in Tex44 testes. Notably, ICSI effectively rescued fertility in Tex44 mice and achieved favorable pregnancy outcomes in variant-carrying patients. Conclusion TEX44 variants are novel genetic causes of asthenoteratozoospermia, acting by impairing sperm axoneme integrity and mitochondrial sheath assembly. ICSI is a promising therapeutic strategy for affected individuals, highlighting the translational value of TEX44 as a diagnostic marker and therapeutic target. Genetic mutations in TEX44 further expand the genetic landscape underlying male infertility.