Changes in seminal plasma microecological dynamics and the mechanistic impact of core metabolite hexadecanamide in asthenozoospermia patients

Asthenozoospermia (AZS) is a prevalent contributor to male infertility, characterized by a substantial decline in sperm motility. In recent years, large-scale studies have explored the interplay between the male reproductive system's microecology and its implications for reproductive health. Nevertheless, the direct association between seminal microecology and male infertility pathogenesis remains inconclusive. This study used 16S rDNA sequencing and multi-omics analysis to conduct a comprehensive investigation of the seminal microbial community and metabolites in AZS patients. Patients were categorized into four distinct groups: Normal, mild AZS (AZS-I), moderate AZS (AZS-II), and severe AZS (AZS-III). Microbiome differential abundance analysis revealed significant differences in microbial composition and metabolite profiles within the seminal plasma of these groups. Subsequently, patients were classified into a control group (Normal and AZS-I) and an AZS group (AZS-II and AZS-III). Correlation and cross-reference analyses identified distinct microbial genera and metabolites. Notably, the AZS group exhibited a reduced abundance of bacterial genera such as Pseudomonas, Serratia, and Methylobacterium-Methylorubrum in seminal plasma, positively correlating with core differential metabolite (hexadecanamide). Conversely, the AZS group displayed an increased abundance of bacterial genera such as Uruburuella, Vibrio, and Pseudoalteromonas, with a negative correlation with core differential metabolite (hexadecanamide). In vitro and in vivo experiments validated that hexadecanamide significantly enhanced sperm motility. Using predictive metabolite-targeting gene analysis and single-cell transcriptome sequencing, we profiled the gene expression of candidate target genes PAOX and CA2. Protein immunoblotting techniques validated the upregulation protein levels of PAOX and CA2 in sperm samples after hexadecanamide treatment, enhancing sperm motility. In conclusion, this study uncovered a significant correlation between six microbial genera in seminal plasma and the content of the metabolite hexadecanamide, which is related to AZS. Hexadecanamide notably enhances sperm motility, suggesting its potential integration into clinical strategies for managing AZS, providing a foundational framework for diagnostic and therapeutic advancements. In the onset of asthenozoospermia (AZS), the presence of six genera of bacteria (Pseudomonas, Serratia, Methylobacterium-Methylorubrum, Uruburuella, Vibrio, and Pseudoalteromonas) in the seminal plasma potentially induces dynamic changes that ultimately diminish the synthesis of hexadecanamide. This reduction in hexadecanamide content in seminal plasma contributes to a subsequent decline in sperm motility. This reduced motility can be attributed to the downregulation of the levels of two key proteins, PAOX and CA2, within sperm cells.Highlightsimage This study substantiates the potential clinical diagnostic utility of microbial composition and specific metabolites within seminal plasma for the asthenozoospermia (AZS) identification.Divergent microbial compositions and metabolite profiles within seminal plasma are discernible across varying degrees of AZS severity.Validation via in vitro and in vivo experiments corroborates hexadecanamide as a crucial metabolite influencing sperm motility in AZS, coupled with the dysregulation of target proteins (PAOX and CA2).