PROJECT SUMMARY Endometriosis, which causes pain in the pelvis and lower abdomen, afflicts 1 in 10 women between 15 and 49 years of age in the United States. Nearly half of these women experience chronic pelvic pain, and many find that available treatments (hormone therapy and surgery) have negative side effects and do not prevent recurrences. A well-accepted theory is that endometriosis occurs when endometrial tissue enters the peritoneal cavity via retrograde menstruation and implants onto pelvic organs and peritoneal surfaces. However, whereas up to 90% of women experience retrograde menstruation, only 10% of women develop endometriosis, suggesting that unknown factors contribute to development of endometriosis. Thus, identifying such causal factors is essential to develop new tools to diagnose and treat this painful disease. This proposal will test the central hypothesis that whereas some gut bacteria promote endometriosis by inducing macrophage-mediated inflammation, others protect against endometriosis by fermenting fiber to produce short chain fatty acids (SCFAs). This idea is built on several key pieces of preliminary and published data. First, in a syngeneic injection model of endometriosis, microbiota-depleted mice developed significantly smaller endometriotic lesions and had less peritoneal inflammation than control mice. However, lesion size was restored in mice orally gavaged with feces from mice with endometriosis. Second, the peritoneal fluid of mice with endometriosis contained less of the SCFAs acetate, propionate, and butyrate than peritoneal fluid from mice without endometriosis. Third, butyrate inhibited both in vivo endometriotic lesion growth in mice and in vitro growth of human cells derived from endometriotic lesions. Finally, recent reports indicate that women with endometriosis have different gut bacteria compositions than women without endometriosis. The work proposed here will build on these strong preliminary data and test the hypothesis by pursuing the following specific aims: (Aim 1) Determine the mechanism by which gut bacteria promote endometriosis; (Aim 2) Determine the mechanism by which SCFAs affect endometriosis; (Aim 3) Identify human gut bacteria associated with endometriosis, and determine the effect of gut bacteria on human endometriosis growth in mice. At the level of basic science, this project will identify gut bacteria and inflammatory profiles that confer sensitivity to developing endometriosis and identify mechanisms by which SCFAs protect against endometriosis. Of translational significance, this work will identify bacterial candidates that promote or protect against endometriosis in reproductive-age women. Together, this work will help advance one of the Aspirational Goals stated in the NICHD 2020 Strategic Plan: to "accelerate efforts to definitively diagnose, prevent, and treat endometriosis".