Anti-Müllerian hormone is decreased in women with superficial peritoneal endometriosis and associated with an elevated inflammatory profile

22 Study question: How are the levels of anti -Müllerian hormone and inflammatory cytokines 23 influenced by superficial peritoneal endometriosis (SPE)? 24 Summary answer: Fertility metrics (Endometriosis Fertility Index (EFI), and serum anti-Müllerian 25 hormone (AMH) levels) are reduced in women with SPE. Simultaneously, inflammatory markers 26 are elevated in the circulation and local pelvic peritoneal microenvironment , with distinct 27 changes in each compartment. 28 What is known already: Between 25-40% of women with endometriosis experience infertility , 29 though the mechanisms behind this are poorly understood. Ovarian endometriosis is known to 30 decrease AMH levels and contribute to infertility, but little is known about SPE-associated 31 infertility, and how the peritoneal microenvironment might play a role in infertility for women with 32 SPE. 33 Study design, size, duration: Venous blood samples from women with suspected endometriosis 34 were collected prior to diagnostic laparoscopy (n=105). Pelvic peritoneal fluid was also collected 35 from a subset of the women (n= 38). The Endometriosis Fertility Index (EFI) was calculated after 36 surgery, and levels of AMH and inflammatory cytokines measured by ELISA or multiplex Luminex. 37 Participants/materials, setting, methods: Based on their surgical findings, women were 38 classified as no endometriosis observed (no endo; n=39), superficial peritoneal lesions only 39 (SPE; n=43), or SPE with an ovarian endometrioma (SPE+OE; n=23). Women were further grouped 40 by their use of hormone treatments to manage their endometriosis symptoms (no endo : no 41 hormones n=14, hormones n=25; SPE : no hormones n=20, hormones n=23; SPE+O E: no 42 hormones n=17, hormones n=6). Data are described as either mean ± standard deviation, or 43 median [interquartile range]. 44 Main results and the role of chance: SPE+OE women were older (3 1.73±6.31) than SPE 45 (27.77±6.14; p=0.04) and control women (27.65±5.81; p=0.02). Both SPE and SPE+OE groups had 46 lower EFI scores compared to women with no endometriosis (no endo 9.41±0.50; SPE 8.63±1.11 47 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint p=0.04, SPE+OE 6.95±1.60 p<0.0001). Serum AMH levels were lower for SPE alone (p=0.009) and 48 SPE+OE women (0.73ng/mL [0.32, 1.19], p=0.002) compared to women with no endometriosis 49 (1.15ng/mL [0.75, 1.94] ) when accounting for age. When also accounting for hormone use , 50 women with SPE+OE had lower AMH levels compared to women with no endometriosis (p=0.02), 51 while women with SPE alone did not (p=0.069) . Moreover, women with SPE not using hormones 52 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-a 53 compared to women with no endometriosis (4.28pg/mL, [3.37, 5.88] vs 1.99pg/mL, [1.49, 3.43], 54 p=0.03), while pelvic peritoneal fluid levels of IL -23 were elevated in women with SPE not using 55 hormones (212.4pg/mL, [184.0, 244.5] vs 121.3, [46.37, 147.60], p=004). These di@erences were 56 not significant in women using hormones. 57 Limitations, reasons for caution: Due to the limited sample size of women not using hormones, 58 we were unable to determine if serum IL-17 or TNF-a, or pelvic peritoneal IL-23 levels negatively 59 correlated with AMH levels. 60 Wider implications of the findings: Women with SPE, with or without OE, have lower AMH levels 61 - indicative of reduced ovarian reserve - compared to women without endometriosis. Among 62 those with SPE, diminished AMH was associated with increased serum levels of IL-17 and TNF-α 63 and elevated IL-23 in the pelvic peritoneal fluid, suggesting compartment-specific inflammatory 64 profiles. Notably, changes to circulating inflammatory cytokines were di@erent when use of 65 hormonal therapy was taken into account , highlighting such treatments may modulate 66 inflammation linked to endometriosis. Taken together, our data support the need for further 67 investigation into inflammation as a potential mechanism underlying infertility in women with 68 SPE in the absence of OE. 69 Study funding/competing interest(s): Deanery of Clinical Sciences Funding Challenge, 70 University of Edinburgh awarded to MJG. 71 Trial registration number: University of Edinburgh Lothian Ethics Committee REC 20/LO/1298. 72 73 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Key words (up to 10) 74 Endometriosis, infertility, AMH, ovarian reserve, superficial peritoneal endometriosis, 75 inflammation, cytokines 76 77 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint

78 Endometriosis is a chronic, hormone-dependent neuroinflammatory disease where 79 endometrial-like tissue grows outside the uterus. It a@ects about 10% of reproductive age women 80 worldwide, and currently, there is no cure (As-Sanie, Mackenzie et al. 2025). The four subtypes of 81 endometriosis - superficial peritoneal (about 80% of cases), deep, ovarian (endometriomas), and 82 extrapelvic - can occur alone or together. Superficial peritoneal endometriosis (SPE) is typically 83 located on the surface of abdominal or pelvic organs and the pelvic wall . Deep endometriosis 84 invades pelvic tissues or organs, like the bowel or bladder. Ovarian endometriomas (OE) are cysts 85 lined by endometrial tissue within the ovary. Extrapelvic endometriosis involves lesions outside 86 the pelvis and can a@ect many organs, including the diaphragm, thoracic organs, or even the 87 brain. 88 A frequent symptom for up to half of women with endometriosis is infertility. Alone, infertility has 89 detrimental impacts on mental health and quality of life for those wanting to conceive, which is 90 further increased by endometriosis (Mori, Zaia et al. 2024) . Clinically, both endometriosis and 91 infertility su@er delays with diagnosis and a lack of e@ective, accessible, treatment options. 92 In international guidelines for endometriosis, it is recommended women with SPE consider 93 surgery to remove SPE lesions based on evidence that this may improve spontaneous pregnancy 94 rates within the first 12 months post-surgery (NICE 2017, Becker, Bokor et al. 2022). In many UK 95 centres, the waitlist for a diagnostic laparoscopy for endometriosis is two or more years 96 (Endometriosis UK 2024, Royal College of Obstetricians and Gynaecologists 2024) . For these 97 women, assisted reproductive technologies (e.g. IVF) provide one potential solution , although 98 this is also subject to access to NHS provision (in the UK), and is costly. A 2023 study found 3.2 99 million women of reproductive age in England had no or limited access to a fertility clinic in their 100 area of residence , while the highest household income areas had the best access to fertility 101 clinics (Jones, Peri-Rotem et al. 2023) . Importantly, there is a group of women who are only 102 diagnosed with endometriosis when they attend fertility clinics when seeking to become 103 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint pregnant and the majority of these are cases of SPE (Van Gestel, Bafort et al. 2024) . If these 104 women are of advanced maternal age, it is unlikely that they will want to wait several years for a 105 laparoscopy to remove the endometriosis in the hope it may e nable them to conceive 106 spontaneously. To provide better and more equitable options for these women , we must better 107 our understanding of the mechanisms behind how SPE contributes to infertility. 108 The Endometriosis Fertility Index (EFI) predicts post-surgery pregnancy success for women with 109 endometriosis based on the surgeon’s assessment of damage to Fallopian tubes, fimbria and 110 ovaries, endometriosis staging score, patient age, and pregnancy history (Adamson and Pasta 111 2010). Since its inception, it has been validated in a variety of settings and shown to accurately 112 predict non -IVF pregnancy success for women with endometriosis post -surgery (Tomassetti, 113 Geysenbergh et al. 2013, Garavaglia, Pagliardini et al. 2015) . It has also recently been shown it 114 can be accurately completed without surgical intervention (Tomassetti, Bafort et al. 2021) . 115 However, to date, i ts utility has been limited to testing on combined datasets containing all 116 subtypes of endometriosis. 117 In clinics o@ering assisted reproducti ve technologies , circulating levels of anti -Müllerian 118 hormone (AMH) are typically measured to predict the capacity of the ovary to respond to hormone 119 stimulation and production of viable oocytes. Notably, AMH is an indirect marker of the quiescent 120 pool of primordial follicles that make up the ovarian reserve and is produced by the proliferating 121 granulosa cells surrounding an oocyte during development, prior to ovulation. AMH has 122 previously been measured in cohorts of endometriosis patients, most commonly those with 123 ovarian endometrioma. In these cases, AMH often decreases after surgical removal of an 124 endometrioma via cystectomy (Wang, Liu et al. 2020, Muraoka, Osuka et al. 2021, Sarbazi, Akbari 125 et al. 2021, Fakehi, Davari Tanha et al. 2022, Mansouri, Safinataj et al. 2022, Shi, An et al. 2022, 126 Tang and Li 2022, Crestani, Merlot et al. 2023). Some reports suggest AMH levels increase again 127 during follow-up periods, however they never return to pre-surgery levels (Kostrzewa, Wilczyński 128 et al. 2019, Sadullayev and Medvediev 2022). To date, only one study has reported AMH levels in 129 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint a cohort of women with SPE , demonstrating no change in AMH compared to age -matched, 130 population controls (n=62 in each group) (Lessans, Gilan et al. 2023) . In another study , 40 131 expectantly managed women (no surgical intervention to treat their ovarian endometriosis) were 132 reported to have AMH levels which declined faster than control women without endometriosis 133 (Kasapoglu, Ata et al. 2018) providing the strongest evidence to date that there is a mechanism 134 by which endometriosis drives infertility. In the current study, serum AMH was measured in a 135 cohort of women with SPE , and levels were compared to both women without endometriosis 136 (confirmed absence of lesions by laparoscopy), and a group of women also with an 137 endometrioma (SPE+OE), to investigate how AMH may be altered by SPE. Additionally, we 138 measured systemic and peritoneal inflammation as potential mechanisms driving 139 endometriosis-associated infertility and associated changes to AMH levels. 140 141

142 Ethical approval 143 Participants were recruited in South-East Scotland between October 2015 and July 2023 from the 144 Royal Infirmary of Edinburgh (NHS Lothian, REC 20/LO/1298) and gave informed consent to be 145 involved in the study and for biospecimens to be collected during surgery. 146 147 Participants samples 148 Participants were scheduled to undergo diagnostic laparoscopy for suspected endometriosis, or 149 other gynaecological procedures (‘no endo’ group only). A venous blood sample was taken prior 150 to surgery, and where possible, a peritoneal fluid sample collected during surgery. 151 Biospecimens were processed in accordance with WERF EPH ect protocols (Rahmioglu, 152 Fassbender et al. 2014) . Serum was collected from whole blood samples via centrifugation at 153 2500g for 10 minutes at 4°C and aliquots stored at -80°C. Peritoneal fluid was similarly processed 154 by centrifugation at 900g for 5 minutes at 4°C and aliquots stored at -80°C. 155 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint 156 Clinical information 157 Participants were categorised by an endometriosis surgeon according to surgical findings; no 158 endometriosis (no lesions observed ; no endo ), superficial peritoneal endometriosis (only 159 superficial lesions present ; SPE ), or superficial peritoneal endometriosis with an ovarian 160 endometrioma (SPE+OE) (Table 1). Exclusion criteria included participants currently pregnant or 161 breastfeeding, known reproductive malignanc ies, and previous history of endometriosis, 162 specifically endometrioma. Current use of hormonal treatment s to manage endometriosis 163 symptoms was used to stratify samples. Hormone treatments included the combined oral 164 contraceptive pill (COCP), progesterone only pill (POP), Depo-ProveraTM, levonorgestrel 165 intrauterine system (LNG-IUS), NexplanonTM implant, norethisterone, or a contraceptive patch . 166 Their prevalence of use in this cohort is summarised in Table 1. The Endometriosis Fertility Index 167 (EFI) was calculated for each participant as previously described (Adamson and Pasta 2010). 168 169 AMH ELISA 170 Serum samples were diluted 1:10 and assayed in duplicate using the human picoAMH ELISA (AL-171 124-r, Ansh Labs, United States) according to manufacturer’s directions. Absorbance was read 172 using Clariostar plate reader (BMG Labtech) at 450nm. 173 174 Luminex 175 Circulating inflammatory cytokines were quantified using custom 13 -plex Discovery Luminex 176 (Bio-Techne, United States) to detect CD163, IL-1b, IL-8/CXCL8, IL-23, MIF , TGF-α, b-NGF , IL-1α, 177 IL-6, IL-17A, IL-33, NRG1, and TNF -α according to manufacturer’s instructions. Serum samples 178 were diluted 1:2 , and peritoneal fluid diluted 1:10. All samples were assayed in duplicate. 179 Luminex detection was completed using a Luminex xMAP INTELLIFLEX analyser using LX200 low 180 sensitivity setting. 181 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint 182 PGE2 ELISA 183 For measurement of p rostaglandin E2 levels serum and peritoneal fluid were diluted 1:2 and 184 assayed in duplicate according to manufacturer’s instructions ( 500141, Cayman Chemical , 185 United States). Absorbance was read using Clariostar plate reader (BMG Labtech) at 420nm. 186 187 CD14 ELISA 188 Serum and peritoneal fluid samples were diluted 1:2 and assayed in duplicated according to 189 manufacturer’s instruction ( A75809, antibodies.com, United Kingdom). Absorbance was read 190 using Clariostar plate reader (BMG Labtech) at 450nm. 191 192 CCL18 ELISA 193 Serum and peritoneal fluid samples were diluted 1:100 and assayed in duplicate according to 194 manufacturer’s instruction (DCL180B, R&D Systems, United States). Absorbance was read using 195 Clariostar plate reader (BMG Labtech) at 450nm. 196 197 Statistical analysis 198 Analyses were completed in GraphPad Prism 10. ELISA results were interpolated from standard 199 curve generated from each assay’s unique standards and corresponding blank corrected 200 absorbance values. Luminex analyses were completed using Quantist software (BioTechne , 201 United States ). Data were assessed for normality using Shapiro -Wilk normality test and the 202 appropriate statistical test chosen accordingly. Linear regression modelling was performed using 203 RStudio (Version 2024.12.1+563) using lm() function within the ‘stats’ package including a two-204 way interaction term to assess the relationship between endometriosis subtypes and hormone 205 use, with age as a covariate . Comparisons of two groups used Welch’s t -test for normal 206 distribution, or Mann Whitney U test for non -normally distributed data. Comparison of three 207 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint groups utilised one -way ANOVA with Tukey’s post-hoc test for normal distribution, or Kruskal -208 Wallis with Dunn’s post-hoc test for non-normal distribution. 209 210

211 Women with superficial endometriosis have lower EFI and serum AMH levels compared to 212 women without endometriosis 213 Demographic information including age and BMI of participants and their use of hormone 214 treatments are summarised in Tables 1 and 2. There was no di@erence in age between the no 215 endometriosis and SPE groups, but the SPE+OE women were older than both no endometriosis 216 and SPE women (Table 2, Figure 1A). Body mass index (BMI) was comparable between all groups 217 (Table 2, Figure 1B). Most women had no history of being diagnosed with infertility and 43-60% of 218 women in each group had been pregnant in the past (Table 2). Women with SPE had lower EFI 219 scores than women with no endometriosis (Table 3, Figure 1C), while SPE+OE women had lower 220 EFI scores compared to both women with no endometriosis, and those with SPE alone (Figure 221 1C). Serum AMH levels for SPE+OE were lower than no endometriosis women (Table 3), however 222 linear regression modelling was performed to allow AMH levels between groups to be compared 223 while accounting for confounding variables like age and use of hormone treatments (Table 4). 224 Both SPE and SPE+O E groups had significantly lower AMH levels compared to the no 225 endometriosis control when controlling for participant age (p =0.009 and p=0.002, respectively; 226 Table 4). When accounting for both age and hormone use, SPE+OE women had significantly lower 227 AMH levels (p=0.02), whil e women with SPE alone did not (p=0.069) , suggesting hormone use 228 may contribute to di@erences observed in AMH. 229 230 Elevated inflammatory cytokines observed in circulation of women with SPE 231 We next sought to understand potential driving mechanisms of this change in AMH with SPE and 232 turned to inflammation as a hallmark of endometriosis. Inflammatory cytokines and proteins 233 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint were quantified using multiplex Luminex and ELISA’s on the same serum samples used to 234 measure AMH and are summarised in Table 5. When cohorts were separated by use of hormones 235 for the majority of cytokines in serum were not significantly changed between SPE and no 236 endometriosis groups (Table 5). The two exceptions were IL-17 (Figure 2A) and TNF-a (Figure 2B) 237 both of which were significantly increased in women with SPE not using hormones (n=9) 238 compared to controls (n=6); notably these di@erences were not significant is women were using 239 hormone therapies. 240 241 Inflammation in the local peritoneal microenvironment diMers from the circulation 242 In a subset of women (n=15), pelvic peritoneal fluid samples from those not using hormones were 243 used to assess inflammation levels in the local pelvic peritoneal microenvironment. 244 Concentrations of peritoneal fluid inflammatory markers are summarised in Table 6. Whilst most 245 factors were detected , variation in levels meant most did not reach statistical significance , 246 including those previously shown to be elevated in serum. Interestingly, one exception was IL-23 247 that was significantly elevated in women with SPE not using hormones compared to those 248 without endometriosis (Figure 2C). 249 250

251 The mechanisms underlying endometriosis -associated infertility are poorly understood and 252 rarely characterised by subtype of endometriosis . Here, we demonstrate for the first time that 253 serum AMH is decreased in women who only have lesions identified as the SPE subtype, 254 compared to women confirmed to have no endometriosis lesions detected during surgery . 255 Interestingly, this finding was associated with elevated pro -inflammatory cytokine levels in the 256 same biospecimens, suggesting inflammation is one mechanism contributing to endometriosis-257 associated delays in conception in women with SPE. 258 259 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Notably, in our cohort, we found the EFI was lower for women with SPE or SPE+OE compared to 260 no endometriosis controls, suggesting a lower probability of future pregnancy success for women 261 with either subtype of endometriosis even though some had already achieved a pregnancy. Since 262 its inception, the EFI has been validated extensively and can be reliably and routinely calculated 263 to predict pregnancy success for non -ART conceptions (Adamson and Pasta 2010, Tomassetti, 264 Geysenbergh et al. 2013, Vesali, Razavi et al. 2020). However, until our study, it had not been used 265 specifically to evaluate future fertility in women with endometriosis by making a direct 266 comparison to a similar cohort of women without endometriosis. The fact the EFI was reduced in 267 our cohort of women with SPE is supported by a recent population study from Finland 268 demonstrating women with endometriosis have a lower overall fertility rate compared to women 269 without endometriosis (Tuominen, Saavalainen et al. 2025). Notably, in their data was a historical 270 population-based cohort study (1998 -2012) with data from 18,320 fertile -aged women with a 271 surgical diagnosis of endometriosis; 5786 of whom had a diagnosis of SPE alone. They reported 272 lower fertility rates in the endometriosis group compared to a reference group . A lthough, 273 consistent with our data, 68% did achieve a pregnancy during follow up. While registry studies 274 provide large datasets, they are not able to provide insight on whether their lower fertility rate is 275 due to a n underlying biological reason or less sexual activity due to debilitating symptoms 276 associated with endometriosis. Our study starts to bridge this gap by providing evidence of lower 277 EFI and serum AMH in the same women with SPE alone, or SPE with O E compared to no 278 endometriosis controls. 279 280 In this study, we focused on SPE as this is the most common subtype of endometriosis and one 281 that receives less attention in fertility studies than ovarian disease. We also focus on the impact 282 to ovarian function and fertility, rather than the endometrium as this has already been the subject 283 of several previous studies (reviewed (Gri@iths, Horne et al. 2024). A strength of our cohort is the 284 precise phenotyping of the participants and their disease subtypes . P revious studies have 285 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint assessed fertility in groups of women with endometriosis, irrespective of the subtype (reviewed 286 (Gri@iths, Horne et al. 2024). Our cohort was stratified into groups based on their surgical findings 287 and medical history to confirm the absence of any previous diagnosis of an endometrioma that 288 could confound the AMH results. Moreover, the control ‘no endo’ population has advantages over 289 previously reported studies as women in this group were confirmed as having no observable 290 endometriosis lesions after undergoing an identical diagnostic laparoscopy to those confirmed 291 to have endometriosis lesions . In contrast, p revious studies investigating endometriosis -292 associated infertility have either utilised age -matched controls where the presence of 293 asymptomatic and undiagnosed endometriosis has not been ruled out surgically, or women 294 attending fertility clinics (Lessans, Gilan et al. 2023) . In both cases there are likely to be other 295 factors contributing to infertility, and as such neither is an ideal control (‘fertile’) group to 296 compare to the fertility in a group of women with endometriosis. This concern is also based on 297 reports describing that almost half of women presenting to fertility clinics with unexplained 298 infertility are subsequently found to have endometriosis , most of which have SPE (Van Gestel, 299 Bafort et al. 2024). 300 301 Interestingly, changes in AMH levels were influenced by hormone use and a significant reduction 302 in AMH levels for women with SPE was not seen when hormone use was taken into account in 303 linear regression modelling. The ability of hormone treatments to alter AMH has been reported 304 previously (Bernardi, Weiss et al. 2021, Hariton, Shirazi et al. 2021, Nelson, Ewing et al. 2023) , 305 however, the influence of hormone use on AMH levels for women who also have endometriosis 306 has never been investigated to the best of our knowledge. It is possible the lack of change here is 307 also driven by a smaller sample size once part icipants are stratified by their use of hormones. 308 Therefore, studies in a larger sample size are warranted to confirm and validate these findings. 309 310 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint To investigate a potential mechanism by which the pelvic microenvironment may contribute to 311 depleted AMH, the associated inflammatory environment in the peritoneal fluid recovered at the 312 time of surgery from the same women was characterised. From the panel of cytokines 313 investigated here, which were selected based on previous works and evidence in the literature , 314 only IL-17 and TNF-a, and IL-23 were found to be elevated with SPE in the serum and peritoneal 315 fluid, respectively. These results align with previous works demonstrating a dysregulated IL-17/IL-316 23 axis, and elevated TNF -a levels with endometriosis (Harada, Yoshioka et al. 1997, Sisnett, 317 Zutautas et al. 2024) . For example, Sisnett et al. (2024) showed elevated IL -23 in the plasma of 318 patients with endometriosis (n=13) compared to healthy, fertile controls (n=19) (Sisnett, Zutautas 319 et al. 2024) . Elevated levels of TNF-a (Harada, Yoshioka et al. 1997) and IL-17 (Zhang, Xu et al. 320 2005) in the peritoneal fluid of women with endometriosis has been previously reported . A 321 strength of our study over previous work is our subgroup analysis focussed on SPE or SPE+OMA, 322 rather than a general endometriosis versus control comparison. Interestingly, Zhang et al. (2005) 323 also reported an even greater increase in IL -17 levels for patients with endometriosis and a 324 diagnosis of infertility compared to endometriosis alone (Zhang, Xu et al. 2005) . Due to limited 325 sample size, we were unable to complete the same analyses. Taken together with our study 326 showing IL-17 is elevated in the same biospecimens where AMH and EFI are reduced, it would be 327 interesting for future studies to attempt to disentangle th e relationship between IL -17 and 328 endometriosis pathogenesis and endometriosis-associated infertility. 329 330 Interestingly, di@erences in circulating IL-17 and TNF-a were unique to the participants not using 331 hormone treatments . Interestingly, this appeared to be due to a similar elevation in these 332 cytokines in the no endometriosis grou p using hormone treatments. While enhanced 333 inflammatory responses to acute stressors have been reported previously in women using 334 hormone treatments (Larsen, Cox et al. 2020, Mengelkoch, Gassen et al. 2024), to the best of our 335 knowledge this has not been assessed in women with a more chronic inflammatory profile, as 336 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint with endometriosis. Moreover, it is unclear what the consequences may be on levels of 337 proinflammatory cytokines and subsequently AMH levels for women with SPE or SPE+OE if they 338 stop taking hormone treatments to attempt to conceive. These are all relevant avenues for future 339 work characterising how inflammation contributes to endometriosis-associated infertility. 340 341 Surprisingly, in our study no di@erences were observed in well-established factors known to play 342 a role in endometriosis-associated symptoms including PGE2 (Rakhila, Carli et al. 2013) and IL-343 8 (Jørgensen, Hill et al. 2017) . This may be due to di@erences in the biospecimens investigated 344 with the Rakhila (2013) study focused on levels in ectopic lesion tissue. The study from Jørgensen 345 and colleagues (2017) measured levels of peritoneal fluid and reported significantly higher levels 346 of IL-8 in 56 endometriosis patients compared to 38 without endometriosis attending a fertility 347 clinic in biospecimens recovered during the luteal/secretory phase. In our study, we had only 8 348 samples from women not on hormones and the increase detected compared with controls was 349 not statistically significant. 350 IL-33 has been studied in endometriosis previously and shown to be elevated with deep 351 endometriosis (Santulli, Borghese et al. 2012, Mbarik, Kaabachi et al. 2015, Miller, Monsanto et 352 al. 2017). This may explain why it is unchanged in our study, as patients with deep endometriosis 353 were not investigated. Mbarik et al. (2015) divided their cohort by the revised American Society 354 for Reproductive Medicine staging and found stage I-II endometriosis (closest equivalent to the 355 SPE group in the current study) had similar circulating IL-33 levels compared to control but 356 elevated levels for the stage III-IV group in both the serum and peritoneal fluid (Mbarik, Kaabachi 357 et al. 2015). A limitation to our study is also one of its aforementioned strengths. While our control 358 group are confirmed to have no endometriosis lesions visualised at the time of laparoscopy, they 359 all experience pelvic pain justifying their investigation for suspected endometriosis. It is possible 360 alterations in the levels of these cytokines may be driven by inflammatory processes similarly 361 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint contributing to pelvic pain which may explain the lack of di@erences observed be tween groups 362 here, particularly for the cytokines previously extensively published in relation to endometriosis. 363 It is also possible these inflammatory peritoneal processes present for those with chronic pelvic 364 pain contribute to infertility. Serum AMH levels in our ‘no endo’ control group were lower than 365 those reported in previous literature. The mean value for our ‘no endometriosis’ control group was 366 1.15ng/mL (compared to 0.89ng/mL in the SPE group). The previous study by Lessans et al. (2023) 367 reported a mean AMH value of 3.0ng/mL for their age -matched control group (2.8ng/mL for 368 peritoneal endometriosis), and a study combining all subtypes of endometriosis reported mean 369 AMH of 2.30ng/mL for healthy controls (1.99ng/mL for endometriosis) (Lessans, Gilan et al. 2023, 370 Ramezani Tehrani, Mousavi et al. 2025) . Together, these data may suggest a mechanism where 371 the pelvic microenvironment (including pelvic inflammation) , irrespective of presence of 372 endometriosis lesions contributes to diminished AMH and subfertility. Women with other pelvic 373 inflammatory conditions such as C rohn’s disease or inflammatory bowel disease are known to 374 impact fertility if they have a flare up of their condition during attempts to conceive and during 375 pregnancy itself (Nguyen, Seow et al. 2016, Mahadevan, Robinson et al. 2019, Rosiou and 376 Selinger 2023, Torres, Chaparro et al. 2023) . While this requires further investigation in the 377 settings of pelvic pain and endometriosis, the notion of an inflammatory peritoneum 378 compromising fertility is plausible. 379 380

381 Here, for the first time, we demonstrate EFI scores and AMH levels are reduced for women with 382 SPE compared to women without endometriosis. These changes are accompanied by greater 383 levels of IL -17 and TNF -a in the serum, and IL -23 in the peritoneal fluid. Interestingly, these 384 di@erences were only observed in women not using hormone treatments. Our findings provide 385 valuable information for clinicians to utilise when counselling women with endometriosis about 386 their future fertility, with or without an endometrioma present. 387 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint 388

389 The authors would like to thank the EXPPECT Edinburgh clinical team for study recruitment and 390 sample collection, and specifically Dr Lucy Whitaker for clinical input. Also, thanks to the 391 expertise from the University of Edinburgh SURF Biomolecular & Assay Core, specifically Dr 392 Kirsten Wilson and Linda Ferguson for Luminex technical assistance. 393 394 Author’s roles 395 Study design: MJG, DAG, PTKS, AWH. Experimental procedures, statistical analysis: MJG, MB, FC. 396

interpretation: MJG, DAG, PTKS, CED, AWH. Manuscript writing: MJG, DAG, PTKS, AWH. 397 Manuscript editing: MJG, MB, FC, DAG, CED, PTKS, AWH. 398 399 Funding 400 M.J.G secured an internal University of Edinburgh Deanery of Clinical Sciences Funding 401 Challenge grant to fund part of this work. 402 A.W.H receives grants from the National Institute for Health and Care Research Health 403 Technology Assessment, Chief Scientist O@ice, Wellbeing of Women, Roche Diagnostics, and 404 European Union. 405 406 Conflict of interest 407 P.T. K . S .’s i n s t i t u t i o n ( U n i v e r s i t y o f E d i n b u r g h ) r e c e i v e s p a y m e n t f o r c o n s u l t a n c y f o r G e s y n t a , 408 Rathlin, Roche and Gideon Richter. 409 A.W.H.’s institution (University of Edinburgh) receives personal fees from Rathlin, Theramex and 410 Gedeon Richter. 411 412 413 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint

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No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Table 1. Participant hormone treatment use. No endo % (n) SPE % (n) SPE+OMA % (n) COCP 10.25 (4) 11.63 (5) 4.34 (1) POP 20.50 (8) 16.27 (7) 4.34 (1) LNGIUS 12.82 (5) 18.60 (8) 13.04 (3) Depo-Provera 10.25 (4) 0.00 (0) 4.34 (1) Other 10.25 (4) 6.97 (3) 0.00 (0) No hormones 35.89 (14) 46.51 (20) 73.91 (17) No endo – no endometriosis lesions visualised at laparoscopy, SPE – superficial peritoneal endometriosis, SPE+OMA – superficial peritoneal and ovarian endometriosis, COCP – combined oral contraceptive pill, POP – progesterone only pill, LNGIUS – levonorgestrel intrauterine system, other includes norethisterone, patch, Nexplanon implant. Table 2. Participant demographic information. No endo SPE SPE+OMA Sample size (n) Whole cohort 39 43 23 No hormones 14 20 17 Hormones 25 23 6 Age Mean±SD 27.65 ± 5.81 27.77 ± 6.14 31.73 ± 6.31*^ BMI Mean±SD 25.14 ± 5.29 25.68 ± 5.44 27.43 ± 4.92 History of infertility (n) Yes 4 2 8 No 32 36 11 Information unavailable 3 5 4 History of pregnancy (n) Yes 16 26 10 No 19 14 10 Information unavailable 4 3 3 No endo – no endometriosis lesions visualised at laparoscopy , SPE – superficial peritoneal endometriosis, SPE+OMA – superficial peritoneal and ovarian endometriosis, BMI – body mass index, SD – standard deviation. *p<0.05 vs no endo, **p<0.01 vs no endo, ***p<0.001 vs no endo, ^p<0.05 vs SPE. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Table 3. Endometriosis fertility index and levels of c irculating AMH . Mean±SD or median [IQR]. No endo SPE SPE+OMA EFI Mean±SD 9.41 ± 0.50 (29) 8.63 ± 1.11 (27) 6.95 ± 1.60 (24) AMH (ng/mL) Median [IQR] (n) 1.15 [0.75, 1.94] (36) 0.89 [0.53, 1.52] (41) 0.73 [0.32, 1.19] (29) Mean ± SD (n) 1.29 ± 0.80 (36) 1.02 ± 0.66 (41) 0.77 ± 0.52 (29) No endo – no endometriosis lesions visualised at laparoscopy, SPE – superficial peritoneal endometriosis, SPE+OMA – superficial peritoneal and ovarian endometriosis , EFI – endometriosis fertility index, AMH – anti-Mullerian hormone, SD – standard deviation, IQR – interquartile range. Table 4. Linear regression model of AMH. Relationship between AMH and SPE/SPE+OMA vs control, with age or age and hormone use taken into consideration. *p<0.05, **p<0.01. Predictor Estimate Standard error t-value p-value Significance SPE+age -0.64 0.24 -2.68 0.009 ** SPE+OMA+age -0.77 0.25 -3.12 0.002 ** SPE+age+hormones 0.57 0.31 1.83 0.069 ns SPE+OMA+age+hormones 0.96 0.43 2.21 0.02 * All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Table 5. Cytokine levels in the circulation of women with and without superficial endometriosis. Cytokine Hormone treatment No endo (n=17) SPE (n=22) bNGF (pg/mL) No hormones 2.67 [2.25, 3.92] 3.87 [3.18, 4.50] Hormones 2.96 [2.82, 4.42] 3.70 [2.47, 4.16] CCL18 (ng/mL) No hormones 588.1 [324.1, 773.9] 641.6 [390.0, 770.3] Hormones 486.9 [367.6, 557.1] 363.8 [307.0, 570.5] CD14 (ng/mL) No hormones 4.07 [3.44, 5.69] 4.14 [3.10, 5.36] Hormones 3.19 [2.68, 3.45] 3.60 [2.96, 4.96] CD163 (ng/mL) No hormones 804.8 [196.7, 942.4] 358.3 [305.4, 963.3] Hormones 447.8 [208.3, 566.4] 613.1 [313.9, 808.6] IL-1a (pg/mL) No hormones 0.0 [0.0, 0.03] 0.0 [0.0, 0.18] Hormones 0.0 [0.0, 0.0] 0.0 [0.0, 0.12] IL-1b (pg/mL) No hormones Undetected Undetected Hormones Undetected Undetected IL-6 (pg/mL) No hormones 0.49 [0.07, 1.73] 0.54 [0.07, 4.16] Hormones 0.56 [0.13, 1.06] 0.22 [0.09, 1.66] IL-8 (pg/mL) No hormones 8.97 [4.60, 29.59] 7.34 [3.53, 13.51] Hormones 5.00 [3.55, 6.77] 6.30 [1.68, 12.74] IL-17 (pg/mL) No hormones 3.84 [3.54, 4.19] 4.45 [4.26, 4.88]* Hormones 4.55 [4.15, 4.66] 4.19 [4.07, 4.59] IL-23 (pg/mL) No hormones Undetected 0.0 [0.0, 15.63] Hormones 0.0 [0.0, 32.8] 0.0 [0.0, 17.7] IL-33 (pg/mL) No hormones 0.67 [0.22, 7.85] 1.23 [0.52, 1.73] Hormones 0.93 [0.06, 9.40] 0.89 [0.18, 25.14] MIF (pg/mL) No hormones 662.9 [0.0, 1561] 0.0 [0.0, 3713] Hormones 1029 [341.5, 1341] 506.8 [138.9, 1355] NRG1 (pg/mL) No hormones 0.0 [0.0, 141.8] Undetected Hormones 1.58 [0.0, 22.61] 0.0 [0.0, 22.77] PGE2 (ng/mL) No hormones 337.3 [187.0, 654.0] 215.1 [74.46, 403.4] Hormones 893.4 [469.6, 2203] 378.1 [178.6, 707.8] TGF-a (pg/mL) No hormones 7.79 [3.48, 17.93] 9.58 [6.42, 16.11] Hormones 9.99 [8.39, 18.49] 13.88 [9.91, 16.83] TNF-a (pg/mL) No hormones 1.99 [1.49, 3.43] 4.28 [3.37, 5.88]* Hormones 2.83 [2.03, 3.73] 2.95 [2.50, 4.35] No endo – no endometriosis lesions visualised at laparoscopy, SPE – superficial peritoneal endometriosis, IQR – interquartile range. Median [IQR], *p<0.05 vs no endo. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Table 6. C ytokine levels in the peritoneal fluid from women with and without superficial endometriosis. Cytokine Hormone treatment No endo (n=6) SPE (n=8) bNGF (pg/mL) No hormones 5.07 [3.47, 7.38] 3.91 [2.12, 8.06] CCL18 (ng/mL) No hormones 355.9 [232.2, 621.4] 372.9 [269.7, 683.9] CD163 (ng/mL) No hormones 720.0 [505.1, 772.3] 701.4 [647.5, 919.9] IL-1a (pg/mL) No hormones 4.08 [1.77, 29.07] 5.98 [4.72, 6.49] IL-1b (pg/mL) No hormones 5.52 [2.97, 16.89] 3.86 [1.69, 7.81] IL-6 (pg/mL) No hormones 156.2 [16.64, 968.4] 36.06 [15.92, 86.30] IL-8 (pg/mL) No hormones 11.03 [7.64, 117.5] 16.54 [7.24, 25.47] IL-17 (pg/mL) No hormones 10.98 [3.77, 27.04] 6.92 [4.73, 9.81] IL-23 (pg/mL) No hormones 121.3 [46.37, 147.6] 212.4 [184.0, 244.5]* IL-33 (pg/mL) No hormones 0.21 [0.00, 1.71] 0.00 [0.00, 0.93] MIF (pg/mL) No hormones 11904 [3995, 23856] 14089 [6821, 18922] NRG1 (pg/mL) No hormones 0.00 [0.00, 12.39] 0.00 [0.00, 0.00] PGE2 (ng/mL) No hormones 141.1 [97.84, 495.2] 119.9 [83.87, 324.4] TGF-a (pg/mL) No hormones 2.17 [0.14, 9.56] 1.70 [1.00, 2.46] TNF-a (pg/mL) No hormones 3.16 [0.74, 6.43] 1.58 [1.08, 3.28] No endo – no endometriosis lesions visualised at laparoscopy, SPE – superficial peritoneal endometriosis IQR – interquartile range. Median [IQR], *p<0.05 vs no endo. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Figure 1. Women with SPE have impaired fertility. A) SPE+OE women are older than no endo controls and those with SPE only (n=26-36), with BMI similar across groups (B ; n=22 -43). Endometriosis Fertility Index (EFI) is reduced for both SPE alone and SPE+OMA groups (C ; n=24-29). Data are mean±SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Shapiro-Wilk test for normality, one -way ANOVA with Tukey’s post -hoc test (A) or Kruska l-Wallis with Dunn’s post-hoc test (B, C,). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint Figure 2. Women with SPE have elevated inflammatory cytokines, seen only in women not using hormone treatments. Levels of IL17 (A) and TNFa (B) in the circulation, and IL23 (C) in the peritoneal fluid are elevated in women with SPE not using hormones, compared to no endo. Data are mean±SD, n=6-13/group, *p<0.05, **p<0.01. Shapiro-Wilk test for normality, one-way ANOVA with Tukey’s post-hoc test (A, B), or Welch’s t-test (C) for significance. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.25.25334349doi: medRxiv preprint