Phthalate Metabolite, Mono(2‐Ethyl‐5‐Hydroxyhexyl) Phthalate ( <scp>MEHHP</scp> ), Promotes Uterine‐Fibroid–Associated Phenotypes in Myometrial Stem Cell‐Derived <scp>3D</scp> Organoids

This study investigates how phthalate exposure contributes to uterine fibroid (UF) development by studying the effects of the Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), a metabolite of Di(2-ethylhexyl) phthalate, on myometrial stem cells (MMSCs). MMSCs from normal (MYON) and at-risk (MYOF) uterine tissues were cultured in 3D organoids and treated with 1.6 μM MEHHP for 48 h. Functional assays investigated cell viability, apoptosis, and mitochondrial activity, whereas RT-PCR, immunohistochemistry (IHC), and RNA sequencing evaluated markers of proliferation, apoptosis, extracellular matrix (ECM), and oxidative stress (OS). Cytokines and growth factors secretion were analyzed using a multiplex ELISA. Results showed that MEHHP exposure significantly increased cell viability and inhibited apoptosis in MYOF compared to MYON organoids. Proliferation markers (PCNA, Ki67), anti-apoptotic markers (BCL2/BAX ratio), and ECM markers (fibronectin and COL1A1) were significantly upregulated, whereas pro-apoptotic markers (Caspase-3) were downregulated in MYOF organoids. MEHHP-treated MYOF organoids exhibited elevated secretion of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-8) and growth factors (e.g., PDGF, VEGF, TGFβ1), indicative of impaired tissue repair and fibrosis. RNA sequencing identified increased OS in MYOF organoids, validated by differential expression of genes such as CA9 and GPX3. Mitochondrial analysis revealed enhanced oxidative phosphorylation (OXPHOS) and elevated oxygen consumption rates, implicating mitochondrial dysfunction as a driver of cytokine release and UF pathogenesis. In conclusion, MEHHP was shown to promote the transformation of MYOF organoids into a UF phenotype by driving proliferation, inhibiting apoptosis, and inducing cytokine-mediated inflammation via mitochondrial dysfunction. These findings related to MYOF-specific effects, as compared to MYON, emphasize that these differences are statistically significant and relevant to UF risk. It can shed insight on how phthalates exposures may impact UF pathogenesis and provide a basis for exploring targeted therapeutic strategies.