Deep immune profiling of endometrial and peripheral blood cells in endometriosis.

study questionHow is endometrial and systemic immunity modulated throughout the menstrual cycle and are there changes in women with endometriosis? SUMMARY ANSWER: Endometriosis is associated with reduced endometrial early natural killer (NK) cells and increased mucosal-associated invariant T (MAIT)-like CD8+ T cells, with cyclical variation. WHAT IS KNOWN ALREADY: The endometrial mucosa contains innate and adaptive immune cells that fluctuate across the menstrual cycle. Immune dysregulation is found in endometriosis, however few studies have broadly assessed endometrial immune single-cell proteome phenotypes. STUDY DESIGN, SIZE, DURATION: This observational cross-sectional immune phenotyping study included 40 participants (28 with surgically confirmed endometriosis and 12 controls). PARTICIPANTS/MATERIALS, SETTING,

methodsEndometrial and peripheral blood samples were analysed by spectral flow cytometry using a 36-parameter immune phenotyping panel and a 13-parameter MAIT-specific panel. Totals of 1 950 292 circulating and 1 023 215 endometrial immune cells were profiled. Full-thickness uterine biopsies (n = 3) underwent multiplex immunohistochemical imaging to assess spatial organization across the menstrual cycle. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with controls, patients with endometriosis exhibited decreased endometrial early NK cells (P.adj = 0.006, log2FC = -1.369) and increased MAIT-like CD8+ T cells (CD161+CD8+CD3+) (P.adj = 0.033, log2FC = 1.415). The MAIT cells (CD161+Va7.2+CD3+) peaked during ovulation and the implantation window (P.adj < 0.05). Peripheral immunity also showed cyclical variation with increased early NK cells (P.adj = 0.001, log2FC = 1.052) and decreased effector CD4 T (P.adj = 0.002/log2FC = -2.010) and effector CD8 T cells (P.adj = 0.002, log2FC = -1.180) in the endometriosis group. LIMITATIONS, REASONS FOR CAUTION: The cytometric panel design was biased towards acquired immunity, and the endometriosis patient sample size prevented subtype analysis. WIDER IMPLICATIONS OF THE

findingsMAIT cell dysregulation represents a novel feature of endometriosis, potentially contributing to subfertility and providing new avenues for therapeutic development. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Nuffield Department of Women's and Reproductive Health, University of Oxford, and also supported from the University of Oxford Medical Sciences HIDI Internal Fund Award (0010398), Academy of Medical Science Award (SBF007\100078) and, British Society for Immunology Career Enhancing Grant. C.M.B. has a consultancy role with ObsEva, Theramex, Roche Diagnostics, Sumitovant, Gedeon Richter, Gesyntha, and they have Research Grants from Bayer, Gesyntha, and Serac Life Services. K.Z. is a Board member (non-remunerated) of the World Endometriosis Research Foundation. She has consultancy status with Roche Diagnostics and Gedeon Richter. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TRIAL REGISTRATION NUMBER: N/A.