Asthenozoospermic spermatozoa manifest mitochondrial dysfunction and DKK3 deficiency is a pathogenic factor

Abstract Asthenozoospermia (AS), a prevalent contributor to male infertility, remains incompletely characterized at the metabolic level. This study aimed to define the energy metabolic profile of AS spermatozoa and identify key proteins associated with observed dysregulations. Sperm samples from 19 AS patients and 21 normozoospermic (NS) controls were analyzed. Aliquots from each sample were used to quantify energy-metabolic parameters (OXPHOS/glycolytic ATP production rates, electron transport chain complex activities, reactive oxygen species ROS levels, mitochondrial membrane potential MMP) and perform proteomic sequencing. Differentially expressed proteins (DEPs) underwent functional enrichment analysis. Spearman correlation linked DEPs to energy-metabolic parameters to identify pathogenesis-associated candidates. AS spermatozoa demonstrated significantly reduced OXPHOS-derived ATP production, impaired ETC complex activities, decreased MMP, and elevated ROS levels compared to NS controls. Proteomic analysis identified 205 DEPs, with aerobic respiration as the top enriched pathway. Correlation analysis revealed significant associations between OXPHOS ATP/MMP parameters and six DEPs (DKK3, KAD9, TMCO1, DRC10, NDUAD, DCAF8). DKK3 exhibited pronounced expression in human reproductive tissues and localized to the sperm mitochondrial sheath. Dkk3 knockout mice displayed phenotypes mirroring clinical AS, including reduced motility and mitochondrial dysfunction. Collectively, our integrated human and murine data demonstrate significant OXPHOS impairment with elevated ROS levels in asthenozoospermic sperm, phenotypes recapitulated in Dkk3-KO models. These findings support DKK3 deficiency as a contributor to asthenozoospermia pathogenesis through mitochondrial bioenergetic dysregulation. Further interventional studies remain essential to define mechanistic links between DKK3 loss and compromised sperm mitochondrial function and motility.