Functional heterogeneity of sperm binding to the ZP2 N terminus across normospermic and infertile phenotypes

Human gamete recognition is mediated by the N-terminal domain of the zona pellucida protein ZP2. Conventional semen analysis often fails to identify functional subpopulations within heterogeneous ejaculates, particularly in men with defective spermiogenesis. This study sought to evaluate if a recombinant ZP2^39-154 peptide can serve as a high-stringency selection tool to identify and enrich for morphologically superior and binding competent sperm.

Sperm samples from 10 normospermic and 12 infertile participants (oligozoospermia, asthenozoospermia, and teratozoospermia) were incubated with agarose beads conjugated to recombinant ZP2^39-154. Binding efficiency was quantified using confocal microscopy. Morphometric analysis, including nuclear area and flagellum length, was performed on bound sperm and compared to sperm bound to native human zonae pellucidae.

Normospermic individuals exhibited nearly four-fold inter-individual heterogeneity in ZP2^39-154 binding efficiency (2.88 to 9.04 sperm/bead), which did not correlate with standard concentration, motility, or morphology metrics. In the infertile cohort, ZP2^39-154 beads identified a reservoir of binding-competent sperm even in severe cases of oligoasthenoteratozoospermia. While the global functional reservoir was reduced in defective spermiogenesis, the top-tier binding events frequently overlapped with the normospermic range. Furthermore, the ZP2^39-154 significantly enriched for morphologically normal sperm, increasing the proportion from a 1-5% baseline in raw semen to as high as 90% on the beads. Notably, 70-94% of bound sperm in the infertile group remained structurally atypical, suggesting molecular binding competence can be preserved despite gross morphological defects.

Recombinant ZP2^39-154 beads provide a standardized, high-throughput functional proxy for the human zona pellucida. This assay transcends the diagnostic limitations of light microscopy by identifying physiologically competent gametes, offering a robust tool for targeted sperm recovery in assisted reproduction.