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.