Single-nucleus Transcriptome Profiling of Human Ovary Reveals Role of Distinct Immune Cells in Classic Galactosemia Leading to Premature Ovarian Insufficiency

Eunice Kennedy Shriver National Institute of Child Health and Human Development, Division of Pediatric and Adolescent Gynecology, Bethesda, Maryland, USA

Abstract Text:

Classic galactosemia (CG) is an autosomal recessive disorder and an inborn error of galactose metabolism caused by mutations in the GALT gene encoding the enzyme galactose-1-phosphate uridylyltransferase. Early symptoms of this disease in infants include failure to thrive, liver damage, kidney failure, jaundice, E. coli sepsis, and neonatal death. Premature ovarian insufficiency (POI) is a later complication that affects 80 - 90% of women with CG due to a significant decline in ovarian reserve (primordial follicle pool). The immune system plays a critical role in ovarian physiology by regulating the ovarian follicle pool and in the development of pathological conditions, including POI. However, the molecular mechanism involving ovarian immune cells in triggering early onset of POI in CG patients has not been explored. In this study, we performed single-nucleus RNA sequencing (snRNA-seq) on ovary tissue biopsies from pre-pubertal and pubertal girls [5-10 years of age range] diagnosed with and without CG [3 patients per group] to investigate dynamic changes in gene expression and altered signaling pathways in major immune cell populations. Functional enrichment analysis was performed using Qiagen IPA platform to identify significantly enriched canonical signaling pathways. To study cell–cell communication changes among different cell types, we used the R package “CellChat” analysis. We successfully constructed a single-nucleus transcriptomic atlas of human ovary which revealed a diverse ovarian microenvironment with eight major ovarian cell types: oocytes, granulosa cells, endothelial cells, epithelial cells, smooth muscle cells, immune cells and stromal cells. Sub-clustering analysis identified distinct subsets of immune cells which include macrophages, T cells, B cells, NK cells and dendritic cells. We found increased number of immune cells specifically macrophages and CD8+ cytotoxic T cells in CG group compared to controls. Our snRNA-seq analysis on all the immune cells revealed activation of Interferon, T cell receptor, antigen presentation, macrophage activation, phagosome formation and Th1 signaling pathways in CG group. The signaling by IL-12 in macrophages which plays a crucial role in promoting inflammation via IFN-γ was significantly enriched in CG ovary and may induce stromal tissue damage & phagocytic cell-death of follicles. We also found a marked increase in expression of pro-inflammatory cytokines like TNF-a, IFN-γ and CCL3/4/5 chemokines in CG ovaries. Functional enrichment analysis on differentially expressed genes in macrophages showed several genes involved in phagosome formation, IL-12, IL-13, interferon, macrophage activation, Th1/Th2 activation, ferroptosis and pyroptosis pathways were significantly enriched in CG group. Furthermore, CellChat analysis reveal increased ligand-receptor pair interactions among immune cells in CG compared to control groups. Our findings provide molecular insights into the robust cellular immune response elicited by pro-inflammatory signaling pathways adversely effecting primordial follicle growth and survival that can explain the etiology of POI in CG patients. This study has implications in the development of future therapeutic interventions to preserve ovarian function and promote reproductive health.