Mechanism study of phthalate exposure promoting endometriosis: Based on the ferroptosis perspective

Phthalates (PAEs), typical endocrine-disrupting chemicals (EDCs) that accumulate in the human body to induce reproductive toxicity, have epidemiological links to endometriosis (EM), but the underlying micro-mechanisms remain unclear. Ferroptosis is a key pathological driver of EM, yet research connecting PAE exposure, ferroptosis, and EM remains insufficient. This study integrated bulk RNA-seq, scRNA-seq, and spatial transcriptomics data, combined with network toxicology, multi-omics analysis, molecular docking, and clinical validation, to systematically investigate the mechanism by which PAEs exposure promotes EM from the perspective of ferroptosis. First, multiple machine learning approaches identified ARF6 as a key ferroptosis-related gene (FRG) under PAEs exposure. ARF6 plays a potential role in disrupting the immune microenvironment of EM, thereby creating preconditions for promoting epithelial-mesenchymal transition (EMT). Specifically, this study found that ARF6 expression was significantly positively correlated with the stemness of Epithelial (Lumenal + Ciliated) and the abundance of Mesenchymal (Fibroblasts). Accordingly, this study constructed an EMT model of "high expression of ARF6, promoting Lumenal + Ciliated → Fibros" in EM. Subsequently, the scTenifoldKnk algorithm was employed to perform virtual knockout of ARF6 in Fibros cells to investigate the disturbance effects on its downstream ferroptosis genes. Results revealed that disturbed genes were significantly enriched in responses to oxidative stress, revealing ARF6's role in mediating ferroptosis mechanisms during EMT, and finally proposed the "PAEs - Ferroptosis - EMT - Endometriosis" axis. In summary, these findings not only identify novel therapeutic target for EM based on the "PAEs-EM" axis but also provide an environmental exposure prevention perspective for EM.