# paper.pdf ## Metadata - PDFFormatVersion=1.6 - Language=en - IsLinearized=true - IsAcroFormPresent=false - IsXFAPresent=false - IsCollectionPresent=false - IsSignaturesPresent=false - Author=Ioannis Simitsidellis; Rebecca J. Ainslie; Angela E. Taylor; Lorna C. Gilligan; Fozia Shaheen; Samira Blanke; Craig Anderson; Dharani Hapangama; Wiebke Arlt; Andrew W. Horne; Philippa T.K. Saunders; Douglas A. Gibson - CreationDate=D:20260707065018+05'30' - Creator=Servigistics Arbortext Advanced Print Publisher 11.1.4546/W-x64 - Custom.EPSprocessor=PStill version 1.84.42 - Keywords=androgen; endometriosis; 11-oxygenated androgens; adrenal - ModDate=D:20260707065024+05'30' - Producer=PDFlib+PDI 9.0.7p3 (C++/Win64) - Subject=DOI: 10.1093/ejendo/lvag109; European Journal of Endocrinology, 195, 1, 2026-07-07.; Abstract: Objective: Endometriosis is a chronic, hormone-dependent condition affecting an estimated 190 million women worldwide. Our understanding of hormonal contributions to endometriosis pathophysiology is incomplete, hindering the identification of diagnostic biomarkers and novel therapeutic targets. Although the role of estrogens is well established, research on androgens in endometriosis is limited and the contribution of adrenal-derived 11-oxygenated androgens remains largely unknown. Methods: We performed steroid androgen profiling to measure androgen concentrations in serum from healthy controls and women with laparoscopically confirmed endometriosis. We found that women with endometriosis had a distinct hormone signature characterized by systemic differences in adrenal androgen concentrations and 11-ketotestosterone excess.Using metabolomic data, we generated statistical models that showed robust discrimination between healthy controls and women with endometriosis (AUC = 0.99; positive predictive power = 96.84%, negative predictive power = 92.86%) consistent with an endometriosis-specific signature. Data were partitioned into train and validation groups to assess diagnostic potential and a refined model identified >95% of endometriosis patients in a blinded sample set. Conclusions: Collectively, these data reframe endometriosis as an androgen-dependent disorder and highlight 11-oxygenated androgens as potential diagnostic biomarkers and future therapeutic targets. - Title=Steroid hormone profiling reveals altered adrenal androgen production in endometriosis - dc:publisher=Oxford University Press - dc:title=Steroid hormone profiling reveals altered adrenal androgen production in endometriosis - dc:creator=Ioannis Simitsidellis,Rebecca J. Ainslie,Angela E. Taylor,Lorna C. Gilligan,Fozia Shaheen,Samira Blanke,Craig Anderson,Dharani Hapangama,Wiebke Arlt,Andrew W. Horne,Philippa T.K. Saunders,Douglas A. Gibson - dc:description=DOI: 10.1093/ejendo/lvag109; European Journal of Endocrinology, 195, 1, 2026-07-07.; Abstract: Objective: Endometriosis is a chronic, hormone-dependent condition affecting an estimated 190 million women worldwide. Our understanding of hormonal contributions to endometriosis pathophysiology is incomplete, hindering the identification of diagnostic biomarkers and novel therapeutic targets. Although the role of estrogens is well established, research on androgens in endometriosis is limited and the contribution of adrenal-derived 11-oxygenated androgens remains largely unknown. Methods: We performed steroid androgen profiling to measure androgen concentrations in serum from healthy controls and women with laparoscopically confirmed endometriosis. We found that women with endometriosis had a distinct hormone signature characterized by systemic differences in adrenal androgen concentrations and 11-ketotestosterone excess.Using metabolomic data, we generated statistical models that showed robust discrimination between healthy controls and women with endometriosis (AUC = 0.99; positive predictive power = 96.84%, negative predictive power = 92.86%) consistent with an endometriosis-specific signature. Data were partitioned into train and validation groups to assess diagnostic potential and a refined model identified >95% of endometriosis patients in a blinded sample set. Conclusions: Collectively, these data reframe endometriosis as an androgen-dependent disorder and highlight 11-oxygenated androgens as potential diagnostic biomarkers and future therapeutic targets. - dc:subject=androgen,endometriosis,11-oxygenated androgens,adrenal - dc:rights=© The Author(s) 2026. Published by Oxford University Press on behalf of European Society of Endocrinology. ## Contents ### Page 1 # Steroid hormone profiling reveals altered adrenal androgen production in endometriosis Ioannis Simitsidellis, Rebecca J. Ainslie, Angela E. Taylor, Lorna C. Gilligan, Fozia Shaheen, Samira Blanke, Craig Anderson, Dharani Hapangama, Wiebke Arlt,,, Andrew W. Horne, Philippa T.K. Saunders and Douglas A. Gibson Centre for Reproductive Health, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh EH16 4UU, United Kingdom Metabolism and Systems Science, School of Medicine and Health, University of Birmingham, Birmingham B15 2TT, United Kingdom School of Mathematics and Statistics, University of Glasgow, 132 University Pl, Glasgow G12 8TA, United Kingdom Department of Women’s & Children’s Health, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, United Kingdom Institute of Clinical Sciences, Imperial College London, London W12 0HS, United Kingdom MRC Laboratory of Medical Sciences, London W12 0HS, United Kingdom Corresponding author: Centre for Reproductive Health, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh BioQuarter, 4-5 Little France Crescent, Edinburgh EH16 4UU, United Kingdom. Email: I.S. and R.J.A. contributed equally to the work. Abstract Objective Endometriosis is a chronic, hormone-dependent condition affecting an estimated 190 million women worldwide. Our understanding of hormonal contributions to endometriosis pathophysiology is incomplete, hindering the identification of diagnostic biomarkers and novel therapeutic targets. Although the role of estrogens is well established, research on androgens in endometriosis is limited and the contribution of adrenal-derived 11-oxygenated androgens remains largely unknown. Methods We performed steroid androgen profiling to measure androgen concentrations in serum from healthy controls and women with laparoscopically confirmed endometriosis. We found that women with endometriosis had a distinct hormone signature characterized by systemic differences in adrenal androgen concentrations and 11-ketotestosterone excess. Using metabolomic data, we generated statistical models that showed robust discrimination between healthy controls and women with endometriosis (AUC = 0.99; positive predictive power = 96.84%, negative predictive power = 92.86%) consistent with an endometriosis-specific signature. Data were partitioned into train and validation groups to assess diagnostic potential and a refined model identified >95% of endometriosis patients in a blinded sample set.Conclusions Collectively, these data reframe endometriosis as an androgen-dependent disorder and highlight 11-oxygenated androgens as potential diagnostic biomarkers and future therapeutic targets.Keywords androgen, endometriosis, 11-oxygenated androgens, adrenalSignificanceEndometriosis occurs when cells similar to those lining the womb are found elsewhere in the body. These cells can grow and change in response to hormones, causing pain and inflammation. While estrogens are known to affect endometriosis, the role of other hormones, such as androgens, isn’t as well understood. In this study, we measured androgens in blood and found that women with endometriosis have a distinct hormone pattern, including higher levels of an androgen called 11-ketotestosterone. We used this information to create a statistical model that could accurately identify women with endometriosis based on androgen concentrations. These findings could help with earlier diagnosis and the development of new treatments for endometriosis.Received: December 1, 2025. Revised: May 19, 2026. Accepted: June 1, 2026 © The Author(s) 2026. Published by Oxford University Press on behalf of European Society of Endocrinology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-com-mercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.European Journal of Endocrinology, 2026, 195, 22–34https://doi.org/10.1093/ejendo/lvag109Published: 7 July 2026Original Research Conclusions Collectively, these data reframe endometriosis as an androgen-dependent disorder and highlight 11-oxygenated androgens as potential diagnostic biomarkers and future therapeutic targets. Keywords androgen, endometriosis, 11-oxygenated androgens, adrenal Significance Endometriosis occurs when cells similar to those lining the womb are found elsewhere in the body. These cells can grow and change in response to hormones, causing pain and inflammation. While estrogens are known to affect endometriosis, the role of other hormones, such as androgens, isn’t as well understood. In this study, we measured androgens in blood and found that women with endometriosis have a distinct hormone pattern, including higher levels of an androgen called 11-ketotestosterone. We used this information to create a statistical model that could accurately identify women with endometriosis based on androgen concentrations. These findings could help with earlier diagnosis and the development of new treatments for endometriosis. Received: December 1, 2025. Revised: May 19, 2026. Accepted: June 1, 2026 © The Author(s) 2026. Published by Oxford University Press on behalf of European Society of Endocrinology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (), which permits non-com-mercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. European Journal of Endocrinology, 2026, 195, 22–34Published: 7 July 2026Original Research ### Page 2 ## Introduction Endometriosis is a chronic, hormone-dependent condition that affects an estimated 190 million women worldwide. It is charac-terized by the presence of “endometrial-like” tissue (referred to as “lesions”) outside the uterus, most commonly within the pel-vic peritoneal cavity. Women experience life-altering symptoms including chronic pelvic pain and infertility. In addition to im-pacts on quality of life, endometriosis also has serious financial consequences limiting health, well-being, and life potential. Our understanding of hormonal contributions to endometriosis pathophysiology is incomplete, which limits our capacity to iden-tify disease-relevant biomarkers and therapeutic targets. Endometriosis is primarily an estrogen-dependent condition, with research focused on the role of local hormones within endometriotic lesions. These studies demonstrate that local estrogen production is a key driver of disease by promoting inflam-mation and tissue growth within lesions. In contrast, systemic hormonal influences, particularly those of androgens, are less well understood. Androgens play established roles in regulating endometrial func-tion as hormone precursors as well as direct mediators. Androgen precursors, such as dehydroepiandrosterone (DHEA) and andro-stenedione (A4), are locally activated in the endometrium during the establishment of pregnancy where they promote tissue remodeling. Active androgens, such as testosterone (T) and dihydrotestosterone (DHT), regulate endometrial proliferation, tissue remodeling, angiogenesis, and inflammation, which are processes associated with establishment and growth of endomet-riosis lesions. Concentrations of T are increased in endometriosis lesions, and this is associated with changes in androgen-regulated genes indicating the potential for direct androgen action within lesions. Androgens can also have indirect roles through conversion to estrogens and activation of pro-disease pathways. Thus, changes in systemic androgen bioavailability have the potential to directly and indirectly alter disease processes in endometriosis. However, profiling of androgens in endometriosis has been limited, both in range of analytes and in sample size, making it difficult to determine their role in endometriosis pathophysiology. 11-Oxygenated androgens have historically been overlooked in endometriosis, but they are increasingly recognized as import-ant components of the androgen pool, particularly in women.11-Oxygenated androgens are a group of adrenal-derived ste-roids that include both inactive precursors and potent andro-gens. They are characterized by the presence of an oxygen atom at carbon 11 and are among the most abundant androgens produced by the adrenal gland. 11-Ketotestosterone (11KT), the most potent metabolite, circulates at concentrations equivalent to or greater than T in women and has similar andro-genic activity, consistent with a potential role in regulating androgen-dependent processes. Changes in 11-oxygenated an-drogens have been described in polycystic ovarian syndrome (PCOS), but whether they are also altered in endometriosis is yet to be determined. Given the potential importance of androgen signaling to endometriosis pathophysiology, charac-terizing the abundance of 11-oxygenated androgens is crucial in order to understand if they are an important feature of the condition. In this study, we performed comprehensive androgen profiling to measure and compare androgen concentrations in serum from healthy controls and women with laparoscopically con-firmed endometriosis. We aimed to identify systemic differences that could be used to characterize a distinct hormone signature in endometriosis. Serum samples from women with laparoscop-ically confirmed endometriosis ( = 159) and healthy controls (n =57) were profiled using a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay to simultaneously measure clas-sic and 11-oxygenated androgens. We combined androgen profil-ing with statistical modeling to characterize hormonal differences, hypothesizing that women with endometriosis ex-hibit a distinct androgen profile and that adrenal-derived 11-oxygenated androgens are key to this unique signature. ## Results ### Endometriosis is characterized by altered concentrations of androgenic hormones Androgens and their precursors were quantified using LC-MS/MS in serum samples from healthy controls (HC; n = 57) and women with endometriosis (ENDO; n = 159). The concentrations of “clas-sic” androgens, DHEA, A4, T, and DHT, and adrenal-derived 11-oxygenated androgens, 11OHA4, 11OHT, 11KA4, and 11KT, were determined ( and). Concentrations of DHEA (; P < .0001), A4 (;P < .0001), and T (; P < .0001) were significantly higher in serum samples from women with endometriosis compared to HC ( “All’). Concentrations of DHT were not significantly dif-ferent between groups (). The 11-oxygenated androgen precursor metabolites, 11OHA4 (; P < .05) and 11KA4 (; P < .0001), were decreased, and the active 11-oxygenated androgens, 11OHT (; P < .0001) and 11KT (; P < .0001), were increased in women with endo-metriosis ( “All’). Consistent with previous reports,11-oxygenated androgens did not differ by menstrual cycle phase (). Neither classic nor 11-oxygenated androgen concen-trations correlated with age, although 11OHT trended to positive correlation (r = 0.1267, P = .0636). Only 11KA4 negatively corre-lated with body mass index (BMI; r = −0.2029, P = .0037;). ### Altered adrenal androgen production is detected in all stages of endometriosis To assess whether androgen concentrations were associated with disease severity in endometriosis, data were stratified ac-cording to the revised American Society for Reproductive Medicine (rASRM) staging. Broadly, there were no significant dif-ferences in steroid concentrations between the rASRM stages for any of the analytes assessed. Analytes that were increased in the unstratified group (ENDO/“All”) were also increased in each rASRM stage compared to HC (;). Notably, the median concentrations of DHEA (P < .0001), A4 (P < .0001), and T (P < .0001) were highest in Stage I endometriosis and sig-nificantly higher than healthy controls (;). Similarly, concentrations of 11OHT (P < .001) and 11KT (P <.0001) were highest in Stage I endometriosis and significantly higher than healthy controls (;). ### Page 3 ![For image description, please refer to the figure legend and surrounding text.]() ### Page 4 Concentrations of T and 11KT were significantly elevated in women with Stage I, II, III, and IV endometriosis compared to healthy controls, whereas 11KA4 was significantly reduced in women with all stages of endometriosis compared to healthy controls (). The greatest difference in androgen con-centrations compared to healthy controls was in patients classi-fied as Stage I. Median concentrations of T, 11KA4, and 11KT were modestly, inversely decreased with stage, but overall, there was no distinct steroid signature associated with a given rASRM stage. ### Endometriosis is characterized by altered adrenal androgen metabolism that is biased toward production of 11-ketotestosterone The correlation among different serum androgen analytes was assessed in healthy controls and women with endometriosis us-ing Pearson correlation (,). In healthy controls, closely related metabolites exhibited a significant positive correlation. This included classic androgens A4 and T (r = 0.8, P = 1.59E-12) and T and DHT (r = 0.5, P = 6.37E-06), as well as 11-oxygenated androgens 11OHA4 and 11KA4 (r = 0.6,P = 8.2E-07) and 11OHT and 11KT (r = 0.9, P = 5.53E-20). In wom-en with endometriosis, additional metabolites that were not cor-related in healthy controls were found to be positively correlated. This included a significant positive correlation be-tween classic and 11-oxygenated androgens, as well as a greater correlation amongst all 11-oxygenated androgen metabolites. This was characterized by DHEA, A4, T, and DHT each being sig-nificantly positively correlated with all 11-oxygenated androgen metabolites (,). Additionally, there was a significant positive correlation between 11KT and 11OHA4 (r = 0.6, P = 1.82E-19), 11KT and 11KA4 (r = 0.7, P = 7.68E-22), 11OHT and 11OHA4 (r = 0.7, P = 2.14E-24), and 11OHT and 11KA4 (r = 0.6, P = 3.94E-17) in women with endometriosis that was absent in healthy controls (,). To investigate if these changes were indicative of altered metab-olism in women with endometriosis, we estimated the activities of enzymes required for 11-oxygenated androgen production by cal-culating product to substrate ratios (; 11-β-hydroxylase (CYP11B1), 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) and Aldo-keto reductase family 1 member C3 (AKR1C3)). ### Page 5 ![For image description, please refer to the figure legend and surrounding text.]() ### Page 6 CYP11B1 and HSD11B2 enzymes mediate interconversion and acti-vation of 11-oxygenated androgens. The ratios of 11OHA4/A4 and 11OHT/T (mediated by CYP11B1) were significantly decreased in women with endometriosis (), consistent with de-creased bioavailability of the precursors 11OHA4 and 11OHT. The ratio of 11KA4/11OHA4 (mediated by HSD11B2) was significantly decreased in women with endometriosis (, P < .0001), consistent with decreased bioavailability of the precursor 11KA4. In contrast, the ratio of 11KT/11OHT was modestly increased between healthy controls and women with endometriosis (, P < .0001), suggesting that metabolism favors the pro-duction of 11KT. Ratios for the reciprocal inactivation of 11KA4 and 11KT, mediated by HSD11B1, were also calculated. The ratio of 11OHA4/11KA4 was increased (, P < .0002) and 11OHT/11KT controls were decreased (, P < .0002) in women with endometriosis compared to healthy controls. The ra-tio of T/A4 did not differ between groups (). AKR1C3 is a multifunctional enzyme that plays a pivotal role in all pathways to androgen production including that of T, 11OHT and 11KT. The ratio of T/A4 (mediated by AKR1C3) did not differ between groups (), but other AKR1C3 activities deter-mined by the ratios of 11OHT/11OHA4 and 11KT/11KA4 were sig-nificantly higher in women with endometriosis (;P < .0001), suggestive of greater production of 11OHT and 11KT in women with endometriosis. Taken together, these data are consistent with a shift in metabolism towards production of 11KT (). ### Women with endometriosis have a disease-specific hormone signature characterized by altered androgen concentrations Receiver operating characteristic (ROC) curve analysis of se-rum androgen concentrations was performed to identify if dif-ferences in androgen metabolites or ratios were robust enough to discriminate between healthy controls and women with endometriosis (). The androgen analytes T, 11KA4, and 11KT exhibited best sensitivity and specificity with area under the ROC curve (AUC) values of 0.815 (P <.001), 0.822 (P < .001), and 0.815 (P < .001), respectively (). ROC curve analysis of calculated enzyme ratios was also per-formed as these data have reduced inter-sample variability and provide a better indicator of the proposed altered metabolism found in women with endometriosis. Enzyme ratios associated with metabolism of 11-oxygenated androgens were assessed (). The ratios 11OHA4/A4, 11OHT/11OHA4, and 11KT/ 11KA4 had best sensitivity and specificity with AUC values of 0.857 (P < .001), 0.817 (P < .001), and AUC of 0.968 (P < .001), re-spectively (). The ratio of 11KT/11KA4 provided ro-bust discrimination between healthy controls and women with endometriosis with a sensitivity of 92.36% and specificity of 90.91% and a likelihood ratio of 10.16 (). These data suggest that differences in androgen metabolism in endo-metriosis represent a consistent, disease-specific hormone signature. ### Development of a predictive model using supervised machine learning confirms specificity of androgen signature to endometriosis diagnosis To complement and extend these findings, androgen data were used to train a predictive model using multiple logistic regression. Androgen concentrations, DHEA, A4, T, DHT, 11OHA4, 11OHT, and 11KT, were used as dependent varia-bles, and diagnosed endometriosis (as a binary) was the out-come variable. BMI was also included in the model to account for any independent effects of this variable (). ROC curve analysis demonstrated excellent discrimination be-tween healthy controls and women with endometriosis with AUC of 0.99 (; P < .0001, CI 0.9878-1.000), providing further improvement on discrimination informed by single an-alytes or ratios (). The predicted probability for outcome was calculated for each sample using a classifica-tion cut-off of 0.5. The negative predictive power of the mul-tiple logistic regression model was 92.86% and the positive predictive power was 96.84% () consistent with androgen concentrations providing a robust and accurate model for predicting diagnostic outcome in endometriosis. ### Validation of a refined statistical model highlights potential for androgen concentrations to robustly predict diagnostic outcome in a blinded cohort To assess how a predictive model could perform when blinded to outcome, the whole data set was partitioned into training and validation groups. The training set contained dependent and outcome variables for an arbitrary subset of healthy con-trols (n = 21) and women with endometriosis (n = 21), and the validation set contained dependent variables but was blinded to outcome variable (n = 138). Comparative modeling was used initially to identify the most robust analytes to include in a refined predictive model (not shown). Multiple logistic re-gression was used to fit a predictive model using BMI, 11OHA4, 11KA4, and 11KT as dependent variables and diag-nosed endometriosis as the outcome variable (). ROC curve analysis of the simplified model demonstrated ex-cellent discrimination between healthy controls and women with endometriosis with an AUC of 0.97 (; P < .0001, CI 0.9124-1.000). The estimated negative predictive power of the model was 94.74% and the positive predictive power was 95.24% (). The actual performance of the model was assessed using the remaining data with outcome blinded (validation set) and in-cluded separate performance estimates according to stratifica-tion by rASRM stage (). The predicted outcome was calculated for each sample, using a classification cut-off of 0.5. In the validation dataset, the sensitivity of the refined model was 95.65% with accurate prediction observed for all disease ### Page 7 ![For image description, please refer to the figure legend and surrounding text.]() ### Page 8 stage stratifications (), including grouped minimal/mild ENDO (Stage I + II; 94% sensitivity), moderate/severe ENDO (Stage III + IV; 96.55% sensitivity), or by individual rASRM stage (I-IV; range 91.2%-100% sensitivity). Consistent with univariate analysis, 11KT had a large odds ratio estimate, indicating the im-portance of this variable in determining diagnostic outcome. These data confirm the utility of using androgen concentrations to predict diagnostic outcome, with a refined model accurately identifying >95% of women with endometriosis in a blinded cohort. ## Discussion We conducted androgen profiling in women with endometriosis and discovered a disease-specific hormone signature character-ized by altered adrenal androgen production. When used alone or in combination with multiple logistic regression, androgen concentrations effectively differentiated between healthy con-trols and women with endometriosis, establishing altered adrenal androgens as a key feature of endometriosis pathophysi-ology. When data were partitioned into training and validation ![For image description, please refer to the figure legend and surrounding text.]() ### Page 9 cohorts, a refined statistical model demonstrated over 95% sen-sitivity in a blinded sample set, highlighting the potential for an-drogen concentrations to be used for diagnostic purposes. These findings represent a significant breakthrough in the search for disease-relevant blood biomarkers, positioning 11-oxygenated androgens at the core of future diagnostic approaches for endometriosis. We found that 11-oxygenated androgens are increased in endometriosis but whether they are key drivers of disease is yet to be determined. 11KT can activate androgen-dependent processes with a similar potency to T and in our study was more abundant than T in women with endometriosis. Androgens can regulate processes associated with the formation of endometriosis lesions and the androgen receptor has been identified as an endometriosis-associated transcription factor.We propose that androgen excess, described herein, may drive endometriosis pathophysiology through direct regulation of androgen-dependent processes (via T and 11KT) and by acting as obligate substrates for aromatase (via A4 and T) to fuel estro-genic processes in lesions. Changes in androgen metabolism may also have systemic effects that increase the risk of lesion forma-tion over the life course, including changes in innate and adap-tive immune cell function and inflammation, which has been reported in other contexts of androgen excess such as PCOS.This may explain how retrograde menstruation can lead to lesion formation in some women but not others. While our findings demonstrate a positive correlation between circulating androgens and endometriosis, a recent study utilizing genetic methods to examine the causal relationship between an-drogens and risk of endometriosis showed an inverse relationship between the two. Specifically, Gjorgoska et al. performed two- sample Mendelian randomization analysis to investigate the causal effects of the androgen precursor, DHEAS, T, and the andro-gen metabolite, androsterone sulfate, on different sub- phenotypes of endometriosis and showed that genetically pre-dicted DHEAS and T levels are causally associated with decreased risk of endometriosis, while genetically predicted total T levels are inversely associated with risk of peritoneal endometriosis. The po-tential discrepancy between the current study and that reported by Gjorgoska et al. likely reflects differences in the biological ex-posure captured by each approach. While mass spectrometry pro-vides direct measurement of hormones at point of sampling, Mendelian randomization estimates the effect of lifelong genetic-ally predicted hormone exposure and risk of endometriosis. Further work integrating observational and genetic approaches will be essential to delineate causality and determine how this may change across the life course and natural history of disease. If 11-oxygenated androgens are driving pathophysiology then targeting their synthesis or action could be an effective therapeutic approach. Notably, the concentrations of 11KT are not diminished by oral contraceptives or gonadotropin-releasing hormone analog- mediated ovarian suppression. Although widely used, a recent Cochrane review suggested the overall evidence for gonadotropin- releasing hormone analogs reducing pain in endometriosis was low to very low quality. Thus, if 11KT drives endometriosis pathogen-esis, this may explain why gonadotropin-releasing hormone ana-logs are ineffective in some women. Previously, the synthetic androgen danazol was found to reduce endometriosis-associated pain, but its use was stopped due to unacceptable masculinizing side effects. The mechanism of action of danazol in endometriosis has not been fully characterized, but in other contexts, it has been shown to inhibit adrenal androgen syn-thesis via suppression of androgen metabolizing enzyme activity.We have found that enhanced adrenal androgen synthesis was a key feature of endometriosis; as characterized by altered activities of the metabolizing enzymes HSD11B2, HSD11B1, and AKR1C3 and the enhanced biosynthesis of 11KT. Thus, the efficacy of danazol in endometriosis may be due to its action on androgen metabolism, which reframes our perception of the pharmacodynamics of this drug. Although danazol was poorly tolerated, our results support revisiting danazol (eg, administered by a local delivery system) or a more selective derivative as a therapeutic approach in endomet-riosis. The findings in the current study provide a platform for fur-ther research into the role of adrenal hormones in endometriosis pathophysiology, with the potential to develop new therapeutic approaches that can transform endometriosis care. Proposed blood biomarkers for endometriosis include the glyco-protein CA-125, inflammatory cytokines (eg, IL-6, IL-8, TNF-α, and MCP-1 [CCL2]), and the pro-angiogenic factor VEGF., However, these markers lack specificity and have not been validated in larger studies. Emerging results from a proteomic screen of serum sam-ples from a large cohort of 805 patients showed promise using a combination of 10 serum protein biomarkers with strong predictive accuracy for the diagnosis of endometriosis, particularly those with severe endometriosis (Stage IV). Similarly, an undisclosed panel of salivary miRNAs has been reported to detect endometriosis with high sensitivity but whether any of these markers vary across different populations, endometriosis subtypes or menstrual cycle stage is still unknown., Finding biomarkers for endometriosis is challenging because they must accurately reflect endometriosis pathophysiology, be consistent across subtypes and not vary with menstrual cycle stage or with hormone treatment. 11-Oxygenated androgens show significant promise in this regard because they meet many of these criteria. However, larger and more representative cohorts will be needed to understand if this hormone signature is generalizable across different demographics and endometriosis subtypes. Our study primarily included participants of European ethnicity, and future research should ensure broader representation to reduce bias. Comparisons to other endocrine disorders will also be important to understand distinct features of endometriosis and enable differential diagnosis from other conditions. Notably, the androgen profile in endometriosis, as described in our study, is different from that reported for PCOS. In PCOS, median concentrations of 11OHA4, and 11KA4, as reported by O’Reilly et al., are 31.7 nmol/L and 13.4 nmol/L, respectively, and are increased relative to healthy controls. In contrast, these analytes were decreased compared to healthy controls in endometriosis in our study with lower me-dian concentrations of 11OHA4 and 11KA4 in endometriosis; 5.67 and 1.64 nmol/L, respectively. An integrated analysis of andro-gen profiles from both disorders is warranted as this could pro-vide resolution on their distinct hormone features with the potential to support differential diagnoses. In the context of endometrial cancer, Gjorgoska et al. evaluated the diagnostic and prognostic utility of preoperative serum steroid profiles, in-cluding both classic and 11-oxygenated androgens, alongside es-tablished biomarkers. They found that steroid measurements alone had modest diagnostic and prognostic performance, but their incorporation into multivariable models enhanced overall ### Page 10 diagnostic accuracy. A similar integrative approach as outlined in the present study may also prove valuable in the context of endo-metriosis diagnosis. One of the limitations of this study is the inference of enzym-atic activities from ratios of products/substrates instead of meas-uring enzymatic activities directly. This caveat needs to be considered for data interpretation, especially because the prod-ucts of the reactions can be metabolized further. Measuring en-zymatic activities directly is challenging in the current paradigm since the cells contributing to the altered androgen metabolism are not known, necessitating further investigation. Potential sources of altered androgen metabolism in endometri-osis could be the lesions themselves, as endometrial cells have been shown to have the capacity to express the full complement of the steroidogenic genes involved in androgen and estrogen synthesis. Adipose tissue also impacts the bioavailability of 11-oxygenated androgens through AKR1C3-dependent activa-tion of 11KA4 to 11KT. General indicators of adiposity such as BMI may therefore influence the interpretation of 11-oxygenated androgen concentrations in different cohorts. The BMI of the endometriosis cohort was greater than healthy controls in the current study, but neither cohort was in the obese range. To limit bias in interpretation of direct comparisons between cohorts, we included BMI in our statistical models to account for any inde-pendent effects of this variable. Notably, 11-oxygenated andro-gens robustly distinguished between healthy controls and women with endometriosis when BMI was included in predictive models, consistent with a distinct association between endomet-riosis and 11-oxygenated androgen concentrations. In summary, we found that women with endometriosis had a distinct hormone signature characterized by systemic differen-ces in adrenal androgen concentrations. Together, these findings redefine endometriosis as an androgen-dependent disorder and highlight the potential for 11-oxygenated androgens to be used as diagnostic biomarkers and therapeutic targets. ## Materials and methods ### Study design Serum was collected from women with laparoscopically con-firmed endometriosis (ENDO) and healthy controls (HC). Samples were analyzed using LC–MS/MS to comprehensively measure serum androgen hormones in endometriosis. Steroid measurements were compared between groups and used to gen-erate statistical models to predict diagnostic outcome. Research was conducted in compliance with the Declaration of Helsinki. ### Study approvals Written informed consent was obtained prior to study participa-tion from healthy volunteers identified through advertisement. Ethical approval was granted by the Science, Technology, Engineering and Mathematics Ethical Review Committees of the University of Birmingham, UK (ERN_17-0494, ERN_17-0494B). Written informed consent was obtained from all endometriosis study participants prior to surgery. Ethical approval was granted by the Lothian Research Ethics Committee (LREC 11/AL/0376), South Central-Hampshire A research ethics committee (IRAS:237815 REC reference 19/SC/0449) and Wales REC 6 A re-search ethics committee (IRAS 268806; REC ref: 19/WA/0271). Methods were carried out in accordance with the local Tissue Governance guidelines and international EPHect guidelines (). ### Patient cohorts #### Healthy controls Inclusion criteria for healthy controls were pre-menopausal women aged 18 years or above. A standardized questionnaire was used to record demographic data including age and BMI; the use of hormonal contraceptives and menopausal status were recorded. Healthy control data were selected from a broad-er cohort of participants as detailed in Schiffer et al. to match the age range of the patient cohort in the current study. #### Endometriosis Eligible participants were women with chronic pelvic pain (aged 18-50 years) of >3 months duration who were undergoing diagnos-tic laparoscopy for suspected endometriosis in NHS Lothian. Pelvic pain was defined as pain located within the true pelvis (between and below the anterior iliac crests). Participants at the Liverpool Women’s Hospital were those undergoing diagnostic laparoscopy for suspected endometriosis who were recruited from endometri-osis or general gynecology clinics. Diagnostic outcome, age, BMI, menstrual cycle stage, and hormone status were obtained and re-corded along with other key clinical data. Diagnosis of endometri-osis was confirmed macroscopically at laparoscopy. Endometriosis was subsequently classified according to the revised scoring sys-tem of the American Society for Reproductive Medicine (rASRM) which ranges from Stage I to Stage IV based on the location and extent of lesions, as well as adhesions, visualized at surgery. #### Menstrual cycle phase Menstrual cycle phase was determined based on the day of the start of the last menstrual bleed (defined as day 1) and samples categorized as either proliferative (days 1-14) or secretory phase (days 14 or greater) based on the day of sample collection relative to the day of last menstrual bleed. In the endometriosis cohort, the menstrual cycle phase was further corroborated by histologic-al staging of endometrial tissue by an expert pathologist. #### Exclusion criteria Exclusion criteria for both cohorts were any acute or chronic dis-ease affecting steroid biosynthesis or metabolism (including PCOS) and the intake of any medication known to interfere with steroid biosynthesis or metabolism. Participants using hor-monal contraceptives (combined oral contraceptives, contracep-tive depot injection, or implant) were excluded. #### Serum steroid analysis All venous blood samples were collected in the morning and, in the case of women with suspected endometriosis, prior to anes-thesia on the morning of surgery. All blood samples were col-lected in serum-separating tubes. After centrifugation, aspirated serum was aliquoted and stored at −80 °C. ENDO ### Page 11 (n = 159) and HC (n = 57) serum samples were profiled using a sensitive LC–MS/MS assay to simultaneously measure androgen hormones/metabolites. Serum steroids were quantified using a previously published and validated approach for multi-steroid profiling using LC– MS/MS. Briefly, 200 μL of serum was mixed with stable isotope- labeled internal standards, and following protein precipitation with 50 μL acetonitrile, the samples were extracted by liquid– liquid extraction with 1 mL methyl tert-butyl ether (MTBE). The MTBE organic phase was removed, dried, and reconstituted in 50/50 methanol/water. Steroids were chromatographically sepa-rated using a Phenomenex Luna Omega C18 column (1.6 μm, 100Å, 2.1 mm × 50 mm) and a water (0.1% formic acid)– methanol gradient. Ammonium fluoride was introduced by post-column infusion to aid ionization. Steroids were quantified relative to a calibration series ranging from 0.02 to 250 ng/mL with inclusion of a blank calibration point. Analysis was per-formed on a Waters Xevo TQ-XS mass spectrometer using elec-trospray ionization in positive ion mode. #### Analyte key Serum analytes were grouped as follows: classic androgens, DHEA, A4, T, and DHT; 11-oxygenated androgens, 11β-hydroxy androstenedione (11OHA4), 11β-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), and 11-ketotestosterone (11KT). ### Statistical analysis Statistical analysis was performed using GraphPad Prism 10. Undetected steroid concentrations or data below the lower limit of quantification (LLOQ) were replaced by 0.5×LLOQ for statistical purposes. Data were summarized using mean ±SD, presented for HC (N = 57) and ENDO (N = 159) or as median± interquartile range in tables, as indicated. T test was used to compare the difference in the means of the two groups. Two-way ANOVA was used to determine the significance be-tween treatments in grouped data. Non-parametric testing was utilized where sample sizes were insufficient to confirm the normality of data distribution. The Mann–Whitney test was used to assess variance between two groups, and the Kruskal–Wallis test was used to assess differences between multiple groups. Criterion for significance was P < 0.05. Correlation between analytes was assessed using the Pearson matrix. Discrimination between cohorts using metab-olite concentrations or ratios was assessed by ROC curve ana-lysis. Predictive modeling for multiple analytes was performed using multiple logistic regression. ## Acknowledgments We thank members of the EXPPECT Edinburgh group, Frances Collins and Ann Doust, for support with participant recruitment and biosample collection. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) li-cence to any Author Accepted Manuscript version arising from this submission. ## Authors’ contributions Ioannis Simitsidellis (Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review & editing [equal]), Rebecca J Ainslie (Formal analysis, Methodology, Writing—original draft, Writing—review & editing [equal]), Angela Taylor (Data curation, Formal analysis, Methodology, Writing—original draft, Writing—review & editing [equal]), Lorna C. Gilligan (Data curation, Formal analysis, Methodology [equal]), Fozia Shaheen (Data curation, Formal analysis, Methodology [equal]), Samira Blanke (Writing—original draft, Writing—review & editing [equal]), Craig Anderson (Data curation, Formal ana-lysis [equal]), Dharani K. Hapangama (Methodology, Resources [equal]), Wiebke Arlt (Formal analysis, Funding acquisition, Writing—review & editing [equal]), Andrew W Horne (Resources, Writing—original draft, Writing—review & editing [equal]), Phillipa Saunders (Funding acquisition, Writing—original draft, Writing—review & editing [equal]), and Douglas A Gibson (Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing—original draft, Writing—review & editing [lead], Data curation, Investigation, Methodology, Resources [equal]) ## Supplementary material Supplementary material is available at online. ## Conflicts of interest A.W.H. reports receiving grants from the UK Research and Innovation, UK National Institute for Health and Care Research, Scotland’s Chief Scientist’s Office, Wellbeing of Women, and Roche Diagnostics; consultancy fees from Roche Diagnostics, Gesynta, and Thramex; lecture fees from Gedeon Richter; serving as the President of the World Endometriosis Society, Co-editor in chief of Reproduction and Fertility, Trustee and Medical Adviser to Endometriosis UK, and Specialty Adviser to the Scottish Government’s Chief Medical Officer for Obstetrics and Gynaecology. P.T.K.S.’s institution (University of Edinburgh) re-ceives payment for consultancy from Gesynta and Heranova. D.A.G., P.T.K.S., and A.W.H. are listed as co-inventors on UK Patent Application (No. 2217921·2). ## Funding This work was supported by the Wellcome Trust (Fellowship 220656/Z/20/Z to D.A.G., Investigator Award 209492/Z/17/Z to W.A.), Medical Research Council (Grant MRC/IAA/002 to D.A.G., program grant MR/N024524/1 to P.T.K.S., and program grant MC_UP_1605/15 to W.A.), and the Institute for Regeneration and Repair Innovators award (funded by WT ITPA to D.A.G.). Research conducted by the Edinburgh EXPPECT group that con-tributed to the collection of biospecimens has been supported by grants from the MRC, Wellcome Trust, NIHR, CSO, and Wellbeing of Women. ### Page 12 ## Data availability All data are available in the main text or the. ## References Horne AW, Missmer SA. Pathophysiology, diagnosis, and management of endometriosis. BMJ. 2022;379:e070750. 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