Project Summary The endometrium is a complex mucosal barrier that lines the uterine muscle and undergoes a remarkable, hormonally-driven scarless healing process to regenerate the ~1 cm thick functionalis layer, which, absent embryo implantation, is shed each month from the permanent stem cell-containing basalis layer. This process is a source of tragic illness for an estimated 200 million girls and women worldwide who suffer debilitating pain and infertility from chronic diseases in which the endometrium grows ectopically, in the myometrium (adenomyosis) or outside the uterus, invading deep into abdominal organs and migrating throughout the body (endometriosis). Ectopic lesions undergo cyclic hormonally-induced changes that cause local bleeding and inflammation, leading to progressive invasion and fibrosis and growth of lesions from small (~0.1mm) epithelial acinar structures with associated stroma, to large (~ cm) fibrotic lesions. Animal models do not capture the spectrum of behaviors of the human condition. Therefore, we propose to build a microphysiological system (MPS) model of early-stage lesions. In the first phase of the project, we integrate 3 independent MPS platform technologies to solve outstanding technical problems in modeling metabolically-active tissues where microvasculature and inflammation (extravasation of circulating immune cells to form tissue-resident cells) are crucially involved, incorporating a previously-developed tissue engineered static model of endometrium and endometrial lesions. After validating the platform performance and basic MPS function, we then compare the behavior of lesions with different properties. A major emphasis of this work is characterizing how reproducible the outcomes are within a single donor, and the variation among donors. A second major emphasis is gaining quantitative insights into inflammatory cell-cell communication networks in MPS systems. We use the platform for 3 Aims: AIM 1 - Define the range of phenotypic responses and molecular signatures for lesions as a function of donor status and hormonal cycle status, determining factors that influence the reproducibility for repeated experiments with the same donor, and those between donors AIM 2 – Evaluate how lesions recruit circulating monocytes immune cells in a hormone cycle-dependent manner, and characterize the evolution of recruited monocyte phenotype in tissues as a function of donor state, in terms of cytokine signatures. AIM 3 – Evaluate of lesion responses to established and experimental therapies as a function of lesion progression state and donor cell hormonal response status.