Single-cell analysis of testicular bacterial microbiome changes during aging and effect on reproductive capacity in mice.

The testis supports spermatogenesis through a tightly regulated microenvironment, and the bacterial microbiome (BM) may influence host cells through immune and metabolic pathways, thereby impacting reproductive capacity. Here, we applied invasion-adhesion-directed expression sequencing (INVADE-seq), a single-cell RNA sequencing approach that simultaneously captures host and bacterial transcripts, to examine how bacterial signals shape testicular cell states. We detected a sparse but widespread bacterial presence across multiple cell types, with somatic and early germ cells outside the blood-testis barrier (BTB) showing relatively higher bacterial abundance. Bacterial load increased with age, coinciding with transcriptional signatures of reduced BTB function. At the cellular level, bacterial-positive Leydig cells exhibited activation of steroidogenic genes, whereas macrophages upregulated pathways related to autophagy and immune modulation. These findings not only deepen our understanding of testicular microbiome biology but also hold promise for the discovery of novel diagnostic biomarkers and therapeutic targets for BM-related and age-associated male subfertility.