Human myometrial cell fate under chronic oxidative stress for leiomyomagenesis

Uterine Leiomyoma are the most common benign tumors in women of reproductive age. Studies show that both leiomyoma and adjacent myometrium exist in a state of high oxidative stress, characterized by reactive oxygen species (ROS) production and impaired antioxidant defenses including defects of manganese superoxide dismutase (MnSOD). Oxidative stress has been linked to leiomyoma initiation by promoting oxidative DNA damage leading to mutations in MED12 and leiomyoma growth. We hypothesize that chronic oxidative stress in human myometrial cells induces cellular remodeling and genomic alterations that promote adaptation and contribute to early events in leiomyoma development. To test this hypothesis, we generated stable human myometrial cell lines (Myo-hTERT) of wild type, MnSOD 68K and mutant MnSOD 68Q that impairs MnSOD antioxidant activity thereby imposing sustained oxidative stress. Experiments were performed both in vitro using three-dimensional spheroid cultures and in vivo using xenograft models in immunocompromised mice, with some studies extending up to 10 months to model chronic oxidative stress. Cells were exposed to the oxidative inducer paraquat (PQ) and analyzed using cellular and molecular approaches including spatial transcriptomics and MED12 mutational analysis. Cells expressing MnSOD 68Q exhibited increased intracellular ROS and oxidative DNA damage, as indicated by accumulation of 8-hydroxy-deoxyguanosine (8-OHdG). Transcriptomic analysis revealed activation of pathways associated with oxidative stress responses including collagen/extracellular matrix, cellular senescence and IGF/AKT signaling. Xenografts of primary myometrial cells subjected to prolonged oxidative stress demonstrated similar molecular responses and showed an increased frequency of MED12 mutations. These changes were accompanied by modest increases in cell proliferation with minimal induction of cell death. Together, these findings provide mechanistic evidence that sustained oxidative stress can drive molecular and genomic changes in myometrial cells and support a causal role for chronic redox imbalance in the initiation of uterine leiomyomas.