Ortega Sandoval, K. I.; Dave, R. M.; Gonyea, C. R.; Mitchum, K.; Aristimuno Millan, A.; Suryakumar, S.; Frolova, A. I.; Raghavan, S. A.

Forceful and coordinated contractions of the uterine myometrium are essential for successful labor, delivery, and postpartum uterine involution. Failure of the uterus to generate or sustain contractile force (uterine atony) after delivery results in postpartum hemorrhage, a leading cause of maternal mortality globally. Paradoxically, uterine atony is exacerbated by prolonged oxytocin exposure used to induce or augment labor through a process of contractile desensitization. Despite its prevalent use in obstetrics, the direct impact of oxytocin desensitization on myometrial contractile force generation remains poorly defined. Current model systems are inadequate to address this gap: ex vivo myometrial tissue strips are limited by tissue availability, donor variability, and lack of genetic tractability, while existing in vitro models provide only indirect readouts of contractility without direct force quantification. Here, we introduce engineered myometrial microtissues (EMMIs), a platform enabling the direct, isometric measurement of contractile force in response to physiological agonists like oxytocin. By embedding and molding immortalized human myometrial smooth muscle cells within a collagen hydrogel, we induced significant structural and molecular maturation over six days. Upon maturation, EMMIs were characterized by circumferential cellular alignment, sustained expression of smoothelin, upregulation of connexin-43, and a transcriptomic shift toward a contractile phenotype. Mature EMMIs generated calcium-sensitive, dose-dependent contractions to oxytocin and potassium chloride. Genetic deletion of the oxytocin receptor abolished oxytocin-induced contractility, establishing receptor specificity. Finally, we utilized EMMIs to recapitulate clinical oxytocin desensitization, providing a direct link between prolonged oxytocin exposure and diminished contractile output. Together, these findings establish engineered myometrial microtissues (EMMIs) as a genetically manipulable, and reproducible system for investigating myometrial contractile physiology to improve obstetric outcomes.