P-103 Three-dimensional oviductal organoid model to study spermatozoa-oviduct interaction in humans

Abstract Study question Is it possible to establish an in vitro model that recapitulating human oviduct investigate sperm-oviduct interaction Summary answer Three-dimensional (3D) oviductal organoid was established from either primary tissues or immortalized OE-E6/67 cell line, based on our protocol. What is known already The sperm reservoir formed maintain the fertilizing capacity of spermatozoa until ovulation by binding epithelial lining oviduct, which also significant select with normal chromatin structure and superior fertilization ability. regulation mechanism formation still unknown, mainly due ethical issue technical difficulty obtaining enough oviduct. We previously demonstrated fucosyltransferase-5 (sFUT5), a carbohydrate-binding protein spermatozoa, responsible for spermatozoa-oviduct humans. design, size, duration Human line HPV 16 E6/E7 open reading frame retroviral expression system employed co-culture model. Fresh samples (human spermatozoa,N = 30; 1-2 cm tissue,N 15) were collected hospital signed consent. Fluorescent pre-labelled co-cultured organoids 2 hours. At least three replicates conducted each assay. Participants/materials, setting, methods Morphological characterization markers expressions determined immunohistochemistry confocal microscopy analysis. studied system. Sperm organoid-bound assessed flow cytometry-based TUNEL Affinity chromatography followed nano-liquid chromatography–mass spectrometry used identify sFUT5-binding proteins ovidutal cells (OECs) their OECs validated immunostaining/flow cytometry. Main results role chance exhibited hollow surrounded single layer OECs. Immunohistochemistry analysis revealed high marker E-cadherin (basolateral membrane), secretory pair-box 8 (PAX8) proliferative Ki67. Expression ciliated marker, acetylated tubulin (AcTUB) can be detected. modification culture environment successfully reversed OEC polarity as immunostaining using antibodies against ZO-1 (apical marker) marker), making accessible during co-culture. These apical-out have significantly higher capacities than basal-out counterparts. Furthermore, possessed more intact DNA unbound spermatozoa. indicate relevance organoid-spermatozoa tool assess By immuno-affinity mass analysis, adhesion molecule 4 (CADM4) then identified potential sFUT5-interacting This result further supported cytometry immunofluorescent staining. 3-D demonstrates profound investigation physiology. Limitations, reasons caution Our 3D may not fully represent entire cellular complexity tubular architecture epithelium. Further morphology integrity transcriptome will enhance understanding reproduction. Wider implications findings give clue future study whether applied clinically functional selection fertilization. tubal ectopic pregnency. Trial registration number applicable