Abstract

Adenomyosis (AM) is traditionally explained by biochemical dysregulation at the endometrial–myometrial interface, leading to passive displacement of endometrial stromal cells (ESCs). However, the uterus is a highly mechanosensitive organ. Aberrant myometrial hypercontractility in AM may expose ESCs to chronic mechanical stimulation. Whether such mechanical cues can modulate ESCs’ behavior under in vitro conditions remains incompletely understood.This study hypothesizes that cyclic mechanical strain–independent of biochemical cues–can induce a pro-migratory and pro-invasive ESC phenotype in vitro. Immortalized human endometrial stromal cells (hESCs) were subjected to cyclic uniaxial stretching (12% strain, 0.2 Hz, 48 hours) to model sustained mechanical loading. We evaluated cellular morphology, migration and invasion (wound healing and Transwell assays), cytoskeletal remodeling (F-actin), extracellular matrix (ECM) related proteins, transcriptomic changes (RNA-sequencing) and MAPK pathway activation. The functional involvement of p38 MAPK was tested using the selective inhibitor Adezmapimod.The results indicated that cyclic stretch induced marked cellular elongation without increasing apoptosis. RNA-sequencing revealed 580 differentially expressed genes enriched in migration and ECM remodeling. Functionally, stretched hESCs exhibited enhanced migration and invasion capacity in vitro, accompanied by elevated F-actin, fibronectin and CTGF. Mechanistically, mechanical strain triggered selective activation of the p38 MAPK pathway, without affecting ERK or JNK. Pharmacological inhibition of p38 attenuated stretch-associated cytoskeletal remodeling, ECM-related protein expression, and cell motility. Together, these findings demonstrate that cyclic mechanical stretch, as an underexplored non-biochemical factor, can promote a motile and invasive stromal cell phenotype in vitro via a p38 MAPK/F-actin-associated mechanism. While these results do not establish causality for A