Study question: How are the levels of anti-Mullerian hormone and inflammatory cytokines influenced by superficial peritoneal endometriosis (SPE)? Summary answer: Fertility metrics (Endometriosis Fertility Index (EFI), and serum anti-Mullerian hormone (AMH) levels) are reduced in women with SPE. Simultaneously, inflammatory markers are elevated in the circulation and local pelvic peritoneal microenvironment, with distinct changes in each compartment. What is known already: Between 25-40% of women with endometriosis experience infertility, though the mechanisms behind this are poorly understood. Ovarian endometriosis is known to decrease AMH levels and contribute to infertility, but little is known about SPE-associated infertility, and how the peritoneal microenvironment might play a role in infertility for women with SPE. Study design, size, duration: Venous blood samples from women with suspected endometriosis were collected prior to diagnostic laparoscopy (n=105). Pelvic peritoneal fluid was also collected from a subset of the women (n=38). The Endometriosis Fertility Index (EFI) was calculated after surgery, and levels of AMH and inflammatory cytokines measured by ELISA or multiplex Luminex. Participants/materials, setting, methods: Based on their surgical findings, women were classified as no endometriosis observed (no endo; n=39), superficial peritoneal lesions only (SPE; n=43), or SPE with an ovarian endometrioma (SPE+OE; n=23). Women were further grouped by their use of hormone treatments to manage their endometriosis symptoms (no endo: no hormones n=14, hormones n=25; SPE: no hormones n=20, hormones n=23; SPE+OE: no hormones n=17, hormones n=6). Data are described as either mean ±standard deviation, or median [interquartile range]. Main results and the role of chance: SPE+OE women were older (31.73± 6.31) than SPE (27.77± 6.14; p=0.04) and control women (27.65± ;5.81; p=0.02). Both SPE and SPE+OE groups had lower EFI scores compared to women with no endometriosis (no endo 9.41± 0.50; SPE 8.63±1.11 p=0.04, SPE+OE 6.95±1.60 p<0.0001). Serum AMH levels were lower for SPE alone (p=0.009) and SPE+OE women (0.73ng/mL [0.32, 1.19], p=0.002) compared to women with no endometriosis (1.15ng/mL [0.75, 1.94]) when accounting for age. When also accounting for hormone use, women with SPE+OE had lower AMH levels compared to women with no endometriosis (p=0.02), while women with SPE alone did not (p=0.069). Moreover, women with SPE not using hormones had elevated serum IL-17 (4.45pg/mL [4.26, 4.88] vs 3.84pg/mL [3.54, 4.19], p=0.02) and TNF-α ; compared to women with no endometriosis (4.28pg/mL, [3.37, 5.88] vs 1.99pg/mL, [1.49, 3.43], p=0.03), while pelvic peritoneal fluid levels of IL-23 were elevated in women with SPE not using hormones (212.4pg/mL, [184.0, 244.5] vs 121.3, [46.37, 147.60], p=004). These differences were not significant in women using hormones. Limitations, reasons for caution: Due to the limited sample size of women not using hormones, we were unable to determine if serum IL-17 or TNF-α ;, or pelvic peritoneal IL-23 levels negatively correlated with AMH levels. Wider implications of the findings: Women with SPE, with or without OE, have lower AMH levels - indicative of reduced ovarian reserve - compared to women without endometriosis. Among those with SPE, diminished AMH was associated with increased serum levels of IL-17 and TNF-α and elevated IL-23 in the pelvic peritoneal fluid, suggesting compartment-specific inflammatory profiles. Notably, changes to circulating inflammatory cytokines were different when use of hormonal therapy was taken into account, highlighting such treatments may modulate inflammation linked to endometriosis. Taken together, our data support the need for further investigation into inflammation as a potential mechanism underlying infertility in women with SPE in the absence of OE.

The authors have declared no competing interest.

M.J.G secured an internal University of Edinburgh Deanery of Clinical Sciences Funding Challenge grant to fund part of this work. A.W.H receives grants from the National Institute for Health and Care Research Health Technology Assessment, Chief Scientist Office, Wellbeing of Women, Roche Diagnostics, and European Union.

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