Project Summary/Abstract This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 21-100. One in five opioid-naïve patients undergoing mastectomy is a persistent opioid user one year after surgery, highlighting the burden of opioid use in women with breast cancer. Preclinical studies have shown that opioids adversely impact the gut microbiome through bacterial translocation and the release of inflammatory cytokines, which may influence secondary effects such as gastrointestinal disorders and even the progression of certain cancers. The gut microbiome is also the site of steroid hormone metabolism. Specifically, bacteria of the distal gut microbiome "reactivate” conjugated estrogens excreted into bile, allowing for re-entry into circulation. The microbiome component responsible for liberating these biologically active estrogens is termed the “estrobolome.” Increased estrogen metabolite concentration appears to be strongly associated with gut microbial diversity, and a low ratio of estrogen metabolites to serum estradiol and estrone is associated with an increased risk of breast cancer. Therefore, the estrobolome may influence the systemic concentration of sex steroids and oncologic outcome. This proposal seeks to characterize the impact of perioperative opioids on the estrobolome and its metabolites in a diverse cohort of women with breast cancer by comparing perioperative changes in the gut microbiome between two groups of patients undergoing breast cancer surgery: those that receive standard opioid-based pain management and those that receive a validated, opioid-sparing multimodal analgesia regimen. Stool and blood samples will be collected before and one week after surgery to determine whether perioperative opioid administration is associated with adverse changes in the gut microbiome. Specific microbial strains associated with opioid-induced changes in the estrobolome will be stratified by antibiotic receipt (none, low, high) to account for the potential influence of perioperative antibiotics. Aside from an additional impetus for other surgeons to adopt similar opioid-sparing perioperative protocols, characterizing the impact of opioids on the gut estrobolome in women with estrogen-sensitive breast cancer provides a novel and innovative method to inform the development of future targets of intervention.