Integrative Single-Cell Analysis Reveals Iron Overload-Induced Senescence and Metabolic Reprogramming in Ovarian Endometriosis-Associated Infertility

Endometriosis, particularly ovarian endometriosis (OE), significantly impacts fertility by often leading to decreased oocyte quality and impaired ovarian function. Iron overload has been identified as a critical factor driving disease progression in endometriosis lesions. This study aims to investigate the effects of iron overload on follicular function in OE-associated infertility (OEI). Using single-cell sequencing technologies, we first identified oocyte dysfunction in OEI patients and established a single-cell atlas of iron overload in follicular fluid prior to ovulation in OEI patients. This atlas revealed dynamic changes in iron metabolism and identified iron-induced senescence phenotypes. We also examined the differentiation trajectories of granulosa cells and the relationship between macrophage M1/M2 phenotypes and iron metabolism. Additionally, we utilized Stereo-seq (spatiotemporal enhanced resolution omics-sequencing) to construct a spatial transcriptomic map of iron-overloaded mouse ovaries, uncovering various enriched senescence phenotypes. Finally, we extended our findings by exploring the dynamics of iron metabolism imbalance in aging human ovaries. Overall, these findings enhance our understanding of iron overload-related cellular interactions and molecular characteristics in ovarian pathology, providing new therapeutic targets for improving oocyte quality in OEI patients. Overall design: Bulk RNA-sequencing was performed on granulosa cells from infertile patients' follicular fluid. Follicular fluid was collected separately from patients with EMS-related infertility and male factor infertility, as described previously. Cells were pelleted by centrifugation at 4°C, 2000 rpm for 10 minutes, and resuspended in 10 ml PBS. The cell suspension was carefully layered over lymphocyte separation medium (1:1 volume) along the tube wall and centrifuged at 4°C, 2000 rpm for 20 minutes. The cloudy white layer was gently aspirated into a 15 ml centrifuge tube. After addition of red blood cell lysis buffer and gentle agitation to lyse red blood cells, the suspension was centrifuged at 4°C, 2000 rpm for 10 minutes. The resulting cell pellet was washed twice with PBS and RNA was extracted from granulosa cells using TRIzol reagent (Invitrogen) for subsequent Bulk RNA-seq.