Spatial transcriptomics in the human adult ovary: insights into key signalling pathways during follicular atresia.

study questionAre key signalling pathways WNT, TGFβ/BMP, NOTCH, and HH involved in follicular atresia in the human adult ovary? SUMMARY ANSWER: In this study, we used spatial transcriptomics to investigate the progression of follicular atresia, focusing on genes of interest associated with steroidogenesis and key signalling pathways WNT, TGFβ/BMP, NOTCH, and HH. WHAT IS KNOWN ALREADY: While extensive research has focused on the mechanisms driving follicular growth, much less is known about the process of follicular atresia, despite its relevance for ovarian aging and reproductive longevity. Follicular atresia is characterized by complex molecular and cellular changes, that lead to the degeneration of granulosa and theca cells. STUDY DESIGN, SIZE, DURATION: Spatial transcriptomics was conducted on 16 regions of human ovarian tissue from different donors (N = 6) containing 21 small antral follicles (diameter 0.5-4 mm) healthy and at different stages of atresia. PARTICIPANTS/MATERIALS, SETTING,

methodsWe selected 80 genes to facilitate cell type identification in the ovary and to investigate the key signalling pathways WNT, TGFβ/BMP, NOTCH, and HH. Haematoxylin and eosin staining was used to manually select different follicle types for spatial transcriptomics. The Molecular Cartography platform (Resolve BioSciences) multiplexing single-molecule fluorescence in situ hybridization on cryo-sections was used for spatial transcriptomics. The cell segmentation masks were obtained from Resolve BioSciences and transcripts for each gene were assigned to individual cells based on the segmentation mask. Downstream visualization and quantification were performed using AnnData and Python. MAIN RESULTS AND THE ROLE OF CHANCE: By comparing the molecular signature of cell types present in healthy small antral follicles to those observed during the progression of atresia, we revealed a profound cellular and molecular shift. Key signalling pathways exhibited a general downregulation in granulosa cells, whereas expression in internal theca cells increased transiently at the onset of atresia, in line with ongoing cellular degeneration and follicular remodelling. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was conducted on ovarian tissue from transmasculine donors. We cannot exclude that testosterone therapy impacts follicular dynamics. WIDER IMPLICATIONS OF THE

findingsOur study provides novel insights into the spatial and molecular mechanisms of follicular atresia, contributing to a deeper understanding of the ovarian biology in humans. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Fonds National de la Recherche Scientifique de Belgique (T.0004.20 to H.V. and C.A.A.), the Novo Nordisk Foundation (NNF21CC0073729, reNEW to F.W., H.C., and S.M.C.S.L.), the Dutch Organization for Health Research and Development (ZonMW PSIDER-2021-10250022120001 to S.M.C.S.L.), and the China Scholarship Council (CSC 202008450034 to F.W., CSC 202008320362 to H.C.). The authors have no conflicts of interest to declare.