Development of Apical-Out Human Fallopian Tube Organoids for Modeling Sperm-Epithelium Interactions

Abstract Elevated sperm DNA fragmentation index (DFI) is closely associated with adverse pregnancy outcomes, although the underlying mechanisms remain unclear. The fallopian tube is a key site for sperm selection and functional regulation, yet whether it differentially responds to sperm with varying DFI levels remains unknown. Reliable in vitro models for investigating sperm-fallopian tube interactions are currently lacking. Therefore, the development of reliable in vitro models to dissect the process of sperm selection by the fallopian tube will provide a new perspective for elucidating the mechanisms high DFI associated infertility. Primary epithelial cells isolated from the human fallopian tube isthmus were used to establish fallopian tube organoids (FTOs), and a suspension culture strategy was applied to reverse epithelial polarity, producing an apical-out configuration. Comprehensive histological, immunofluorescence, ultrastructural, and gene expression analyses confirmed that epithelial differentiation and barrier integrity were maintained following polarity reversal. A co-culture system was subsequently developed to enable direct sperm-epithelium contact. Apical-out FTOs supported sperm adhesion and improved sperm viability by reducing apoptosis and intracellular reactive oxygen species levels. Transcriptomic profiling revealed that normal sperm primarily induced pathways related to immune modulation and extracellular matrix remodeling, whereas high-DFI sperm induced inflammatory responses. Collectively, this study introduces and validates a novel apical-out human FTOs model that overcomes structural limitations of conventional systems. This platform enables relevant investigation of sperm-epithelium interactions and reveals that the tubal epithelium differentially responds to sperm DNA integrity, offering new insight into female tract potential implications in male factor infertility.