VeriXiv supplementary materials: Nanobodies targeting the sperm-egg fusion synapse block human fertilization

Modern contraceptives are highly effective, yet systemic side effects contribute to discontinuation of hormonal methods, highlighting the need for safe, reversible, and accessible non-hormonal alternatives. Here, we describe a first-in-class contraceptive strategy targeting sperm-egg fusion, a specific and essential step in human fertilization. The interaction between IZUMO1 on sperm and its oocyte partner JUNO is essential for gamete fusion. We identified seven anti-JUNO nanobodies with nanomolar affinities that inhibited human in vitro fertilization by up to 99% inhibition, even at sperm concentrations more than two-orders of magnitude above physiological levels. Structural modeling revealed that nanobody epitopes overlap the IZUMO1-binding interface on JUNO, consistent with direct competitive inhibition. Selected nanobodies exhibited high target specificity, favorable solubility profiles, and strong stability and serum integrity. These findings establish proof-of-concept for pharmacological disruption of the sperm-egg fusion machinery and validate IZUMO1-JUNO as a tractable target for a new class of non-hormonal contraceptives.