ABSTRACT/SUMMARY Endometriosis is an inflammatory gynecological disease of reproductive-age women. The two major symptoms are chronic pelvic pain and infertility, which profoundly affect reproductive health and life quality. The pro- inflammatory microenvironment of the endometriotic lesions, endometrium, and the peritoneal cavity are the hallmarks of endometriosis, which leads to chronic pelvic pain and infertility. Emerging evidence suggests that pelvic pain in endometriosis is a combination of visceral inflammatory and neuropathic pain. The long-term goals are to understand the molecular and cellular pathogenesis of endometriosis and identify non-steroidal therapy for endometriosis. Withania somnifera, an ancient medicinal plant, has been used to treat inflammatory disorders, neurological diseases/disorders, neuropathic and inflammatory pain, and cancers. A wide range of therapeutic effects of Withania somnifera is exerted through its active steroidal lactone Withaferin-A. However, no studies have investigated the role of Withania somnifera/Withaferin-A in endometriosis. The proposed timely needed hypothesis-driven novel preclinical research is focused on the therapeutic effects of Withaferin-A in endometriosis. Overarching Hypothesis: Treatment of Withaferin-A suppresses the growth and survival of endometriotic lesions, ameliorates pelvic pain, and restores the endometrial microenvironment to support fertility. Objectives: Determine the therapeutic effects of Withaferin-A on (i) growth and survival of the endometriotic lesions, (ii) pro-inflammatory state of the peritoneal cavity, endometriotic lesions, and pain-brain areas, (iii) pro- inflammatory and hormonal microenvironment of the endometrium, (iv) epithelial and stromal cell-specific gene signatures in the endometriotic lesions and endometrium, (v) ascending and descending pain pathway and central sensitization and the underlying molecular and cellular mechanisms, and (vi) pelvic pain threshold and spontaneous pain. Specific Aim-1 will determine the therapeutic effects of Withaferin-A on endometriotic lesions and endometrium. Specific Aim-2 will determine the therapeutic effects of Withaferin-A on central pain mechanisms in endometriosis. The experimental approaches include the preclinical allograft mouse model of endometriosis, RNA-Seq, electrophysiology of brain neurons, ascending and descending pain pathways, pain behavior, in vivo bioimaging, fluorescence microscopy, and flow cytometry. Innovation and Impact: The new information obtained should fill the gap in the knowledge on therapeutic effects of Withaferin-A in (i) central mechanisms of pelvic pain, gene expression signatures in the pain-brain areas, and their association with chronic pelvic pain in endometriosis; (ii) epithelial and stromal cell-specific gene expression signatures in the endometriotic lesions and their association with growth and survival of endometriotic lesions; and (iii) epithelial and stromal cell-specific gene expression signatures in the endometrium and their association with infertility. (iv) The novel results will establish a molecular therapeutic and preclinical basis to use Withaferin-A as a non- steroidal and non-opioid therapy to treat pelvic pain and infertility in women with endometriosis.

PROJECT NARRATIVE Endometriosis is an inflammatory gynecologic disease of reproductive-age women and is characterized by two primary symptoms chronic pelvic pain and infertility. Withania somnifera (active component Withaferin-A), an ancient medicinal plant, has been used to treat inflammatory disorders, neurological diseases or disorders, neuropathic and inflammatory pain, and cancers; and thus, it provides a therapeutic opportunity to treat endometriosis in women. Successful completion of the proposed research is expected to expand the understanding of the therapeutic effects of Withania somnifera/Withaferin-A and establish a novel preclinical and molecular therapeutic basis to use Withaferin-A as a non-steroidal and non-opioid therapy for the treatment of pelvic pain and infertility in women with endometriosis.