Molecular Heterogeneity Within Normozoospermia: A Pilot Pooled Small RNA Sequencing Study

Normozoospermia, defined by semen parameters above the WHO lower reference limits, is conventionally treated as a single category. Increasing evidence suggests that this classification may encompass molecular heterogeneity. This pilot-pooled sequencing study investigated whether men occupying distinct phenotypes within the normozoospermic range exhibit different sperm miRNA profiles. Thirty normozoospermic men were stratified into three investigator-defined groups based on sperm concentration and normal morphology: an intermediate concentration/morphology group (NORMAV), a higher-concentration group (>80 × 106/mL; NORMCOUNT), and a higher-morphology group (>9% normal forms; NORMMOR). Sperm small RNA sequencing was performed using two biological replicate pools per group, followed by differential expression (q-value < 0.01, |log2FC| > 1) and functional analyses restricted to strongly validated miRNA–target interactions. Pool-level differences in miRNA abundance were identified across all pairwise comparisons, with miR-34c-3p, miR-891b, miR-145-5p, and miR-1-3p among the recurrently dysregulated miRNAs. Functional enrichment of database-curated miRNA targets suggested convergence on proliferation, apoptosis, development, and intracellular signaling, including the PI3K–Akt, MAPK, and FoxO pathways. 568 genes were shared across all comparisons, with VEGFA, IGF1R, and PTEN among the most recurrent targets. These exploratory findings indicate pool-level sperm miRNA variation among the investigated normozoospermic groups and identify candidate miRNAs for subsequent individual-level validation. Larger studies using individual-sample sequencing and orthogonal validation are required to determine whether these differences are consistently observed across participants.