Objective  To systematically analyze the mutation spectrum of the androglobin gene (ADGB) in infertile males and investigate the association between its genotypes and phenotypes related to male infertility, thereby elucidating the critical role of ADGB in male reproductive function.

Methods  This study recruited 781 Chinese males diagnosed with primary infertility. Whole exome sequencing (WES) combined with Sanger sequencing was used to screen for and identify potential pathogenic mutations in the ADGB gene among infertile males. Bioinformatics analysis of ADGB evolutionary conservation was conducted using MEGA11 software. Western blotting was performed to detect ADGB expression in patients' sperm. Semen analysis and modified Papanicolaou staining were used to evaluate sperm motility and morphological characteristics, while transmission electron microscopy (TEM) was used to observe abnormalities in sperm ultrastructure. Additionally, real-time fluorescence quantitative PCR was used to detect Adgb mRNA expression levels in various mouse tissues and testicular developmental stages. Immunofluorescence staining was used to analyze the localization and expression distribution of ADGB in human and mouse germ cells across different developmental stages. Finally, proteins associated with ADGB were screened using STRING data and validated by co-immunoprecipitation.

Results  Among 781 infertile men, 148 ADGB gene mutation sites were identified. According to the American College of Medical Genetics and Genomics (ACMG) Variant Classification Guidelines, 45 sites were classified as benign mutations, one site as a potentially benign mutation, 96 sites as variants of uncertain significance, and 6 sites as potentially pathogenic mutations. Two potentially pathogenic mutations (c.4859G > A and c.4981A > G) were identified in a compound heterozygous state in a patient with idiopathic oligoasthenoteratozoospermia (OAT). Conservation analysis suggested that these mutations may disrupt functionally conserved domains of the ADGB gene across species, indicating potential pathogenicity. Western blot analysis revealed absent ADGB protein expression in the patient's sperm. Morphological analysis showed marked head abnormalities (microcephaly, conical heads) and tail defects (short tails, acutely angled curved tails, coiled tails, and tailless sperm) in the patient's sperm. TEM observations showed spermatozoa with acrosome loss, disorganized mitochondrial sheath helical structures, and disrupted or partially absent “9 + 2” microtubule structures in the axoneme. Additionally, ADGB was found to be highly expressed after puberty in both human and mouse testes and was localized to the acrosome and flagella of spermatogenic cells. Co-immunoprecipitation experiments confirmed TTC29 and CFAP47 as interacting proteins of ADGB. The patient carrying the pathogenic ADGB mutation achieved a successful pregnancy through intracytoplasmic sperm injection (ICSI) treatment.

Conclusion This study expands the mutation and phenotypic spectrum of ADGB in male infertility, confirming its biological significance in maintaining human fertility.