Supplemental Material for: SEMA3C Promotes the Progression of Endometriosis by Inducing Ferroptosis and Enhancing the Viability and Migration of Endometrial Stromal Cells

Objectives: To examine SEMA3C expression in endometriosis (EMs) and its relationship with ferroptosis in human endometrial stromal cells (HESCs).Design: Experimental study using patient tissue samples and in vitro gain- and loss-of-function assays.Participants/Materials: Endometrial tissues from 30 patients with EMs and 30 controls, including paired ectopic and eutopic tissues from 10 patients, and primary HESCs.Setting: Affiliated Women and Children's Hospital of Ningbo University and associated laboratory facilities.Methods: SEMA3C expression was assessed by immunohistochemistry, Western blotting, and quantitative real-time PCR. Primary HESCs were subjected to SEMA3C overexpression or siRNA-mediated knockdown. Cell viability, migration, iron content, lipid peroxidation, antioxidant activity, inflammatory cytokines, and ferroptosis regulators were analyzed. Ferrostatin-1 was used for rescue experiments.Results: SEMA3C was upregulated in ectopic tissues versus controls (P < 0.001) and correlated with disease severity (stage III–IV vs. I–II, P < 0.01). Ectopic lesions showed higher SEMA3C mRNA than matched eutopic endometrium (P < 0.001). Ectopic tissues and SEMA3C-overexpressing HESCs showed increased ferroptosis-related oxidative and inflammatory changes. SEMA3C overexpression enhanced HESC viability and migration, increased ACSL4, and decreased Nrf2, GPX4, and SLC7A11 expression (P < 0.001). SEMA3C knockdown or Ferrostatin-1 attenuated these alterations and suppressed HESC viability and migration (P < 0.01).Limitations: The findings are based on clinical tissues and in vitro HESC experiments; in vivo validation and prospective confirmation are required.Conclusions: SEMA3C promotes HESC viability and migration through ferroptosis-related oxidative and inflammatory mechanisms, identifying the SEMA3C-ferroptosis axis as a potential pathogenic pathway in EMs.