M2 macrophages undergoing ferroptosis regulate uterine smooth muscle contraction to facilitate labor onset

Labor onset is a complex process influenced by multiple factors. Although the mechanisms initiating labor remain incompletely understood, accumulating evidence suggests that maternal immune responses serve as critical drivers of this process. Ferroptosis, an iron-dependent form of programmed cell death, has emerged as a potential modulator of the immune microenvironment. But its role in labor onset remains largely unexplored. This study examined ferroptosis occurrence and how it affects cell functions during labor onset. We analyzed multi-cohort transcriptome datasets to identify immune cell infiltration using CIBERSORT and ssGSEA. Meanwhile, with via LASSO regression, we identified four core immune cells including M2 macrophages in labor onset. Differential analysis revealed 49 ferroptosis-related differential genes (FRDGs) in this process. Ferroptosis and iron ion homeostatic pathways were enriched by KEGG and GO analysis. Our ROC model using the key FRDGs for diagnosing labor onset performed excellently. We further identified a significant negative correlation between M2 macrophages and the FRDGs HMOX1 by Spearman. Finally, we performed in vitro and in vivo experiments to confirm the ferroptosis involvement in myometrium and to affect the specific functions of human uterine smooth cells (HUSMCs) and THP1. Several key FRDGs are significantly differentially expressed at both mRNA and protein levels in the labor myometrium. In addition, elevated Fe²⁺, decreased T-GSH, and increased MDA reflected higher levels of ferroptosis during labor. In vitro, we detected a shift towards a pro-inflammatory phenotype induced by ferroptosis in M2 macrophages using flow cytometry and PCR arrays, which enhanced cytokine secretion and promoted uterine smooth muscle contraction. Conversely, inducing ferroptosis in HUSMCs suppressed cytoskeletal remodeling and gap junction protein expression, impairing contraction. The study aims to provide a new theoretical basis for the mechanism of labor onset and a potential therapeutic target for abnormal timing of delivery.