Longitudinal evaluation of primary uterine leiomyoma cultures reveals changes in molecular identity and drug responsiveness

Uterine leiomyomas (ULs) are common benign tumors that can adversely affect female fertility and reproductive health, particularly when their size or location results in distortion of the uterine cavity. Despite their clinical importance, primary two-dimensional (2D) cultures used to study UL biology and therapeutic responses remain poorly characterized with respect to their phenotypic stability during serial passaging. Herein, we systematically evaluated how experimental parameters and serial 2D passaging influence the molecular identity and drug responsiveness of patient-derived UL cells. Morphological, transcriptomic, and functional analyses revealed a rapid culture-induced loss of native tissue identity. Targeted gene profiling demonstrated that estrogen and progesterone receptor mRNA levels dropped precipitously by passage 2, followed by a progressive decline in the smooth muscle marker ACTA2 . Although the manually formulated culture medium transiently enhanced selected molecular and early hormone-responsive characteristics at early passages, it failed to prevent long-term phenotypic drift. Furthermore, drug responses to hormone receptor modulators were highly context-dependent, significantly altered by prolonged culture and media composition. These results demonstrate that primary UL cells undergo rapid transcriptional reprogramming, leading to unstable pharmacological behavior. Consequently, careful selection and reporting of passage number and culture conditions are important for improving the reproducibility and interpretability of in vitro pharmacological evaluations.