{"paper_id":"bac278f2-d470-4a0e-9046-a3234e71b996","body_text":"1 \n \nSteroid Metabolome Profiling Identifies a Unique Androgen Hormone Signature \nAssociated with Endometriosis \nIoannis Simitsidellis1‡, Rebecca Ainslie1‡, Angela Taylor2, Lorna C. Gilligan2, Fozia Shaheen2, \nSamira Blanke1, Craig Anderson3, Dharani Hapangama4, Wiebke Arlt2,5,6, Andrew W Horne1, \nPhilippa T.K. Saunders1 and Douglas A. Gibson1* 5 \n1Centre for Reproductive Health , Institute for Regeneration and Repair , University of \nEdinburgh, Edinburgh BioQuarter, 4-5 Little France Cresc ent, Edinburgh EH16 4 UU, UK . \n2Metabolism and Systems Science, School of Medicine and Health, University of Birmingham, \nBirmingham, B15 2TT, UK. 3School of Mathematics and Statistics, University of Glasgow, 132 \nUniversity Pl, Glasgow G12 8TA, UK. 4Department of Women’s & Children’s Health, Institute 10 \nof Life Course and Medical Sciences, University of Liverpool , UK; 5Institute of Clinical \nSciences, Imperial College  London, London W12 0HS, UK.  6MRC Laboratory of Medical \nSciences, London, W12 0HS, UK.  \n‡These authors contributed equally to the work \n*Corresponding Author  15 \nDouglas Gibson, Principal Investigator & Sir Henry Dale Fellow \nInstitute for Regeneration and Repair  \nThe University of Edinburgh  \nEdinburgh BioQuarter  \n4-5 Little France Drive  20 \nEdinburgh  \nEH16 4UU  \nE-mail: D.A.Gibson@ed.ac.uk   \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n2 \n \nAbstract \nEndometriosis is a chronic, hormone -dependent condition that affects 190  million women 25 \nworldwide. There are no validated biomarkers for endometriosis and this delays diagnosis and \ntreatment. \nWe performed serum steroid metabolome profiling in healthy controls (n=57) and women with \nlaparoscopically-confirmed endometriosis (n=159) using liquid chromatography-tandem mass \nspectrometry. Women with endometriosis had a distinct steroid signature characterised by 30 \nincreased concentrations of classic and 11 -oxygenated androgens, and altered metabolism \nassociated with 11-ketotestosterone production. \nMetabolomic data were used to generate a supervised machine learning model to predict \ndiagnostic outcome. ROC curve analysis demonstrated robust discrimination between healthy \ncontrols and endometriosis patients (AUC=0.99) with 96.84% positive-, and 92.86% negative-35 \npredictive power. Data were partitioned into train and validation groups, and a refined model \nidentified >95% of endometriosis patients in a blinded sample set.  \nThese data reframe endometriosis as an androgen-dominant condition and present a unique \nopportunity to develop novel diagnostic approaches using 11 -oxygenated androgens as \nbiomarkers.   40 \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n3 \n \nIntroduction \nEndometriosis is a chronic, hormone-dependent condition that affects an estimated 190 million \nwomen worldwide 1. It is characterised by the presence of ‘endometrial-like’ tissue (referred to \nas ‘lesions’) outside the uterus, most commonly within the pelvic peritoneal cavity. Women \nexperience life-altering symptoms including chronic pelvic pain and infertility. In addition to 45 \nimpacts on  quality of life, endometriosis also has serious financial consequences  limiting \nhealth, wellbeing and life potential 2.  \nDiagnosis is a key clinical challenge in endometriosis, which prolongs suffering and delays \ntreatment. Internationally, overall time to diagnosis ranges from 5 to 12 years3, with a median \ndelay of 8 years reported in the UK4. Establishing a correct diagnosis of endometriosis is often 50 \nproblematic because the presenting symptoms are associated with other conditions and this \nmakes clinical recognition of endometriosis challenging 5,6. Thus, the d iagnosis of \nendometriosis usually involves a combination of clinical evaluation and imaging techniques 7. \nHowever, the gold standard for diagnosing endometriosis, as recommended by international \nguidelines, is laparoscopic visualisation of the lesions  performed by specialised 55 \ngynaecologists 8-10. Endometriosis is broadly categorised into three subtypes : superficial \nperitoneal, ovarian and deep. Nevertheless, it is most commonly described according to the \nrevised scoring system of the American Society for Reproductive Medicine (rASRM) which \nranges from Stage I to Stage IV based on the location and extent of lesions, as well as \nadhesions, visualized at surgery 11. Although widely adopted, this surgical classification does 60 \nnot correlate with symptom severity 12. Additionally, diagnostic laparoscopy is invasive, costly, \nand carries significant risks, including injury to pelvic organs  13. Non-invasive diagnostic \napproaches for endometriosis could provide earlier definitive diagnosis and eliminate surgery-\nassociated complications. However, unlike other similarly common diseases, there are no \nknown biomarkers for endometriosis. 65 \nResearch focused on identifying endometriosis biomarkers has been hampered by low -\npowered studies, inter-study variability and by the identification of markers with low predictive \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n4 \n \nvalue 14. Until now, there has been no single  blood or urinary biomarker approach that has \nbeen validated 15. Recent attention has focussed on screening salivary miRNAs 16 and the \nserum proteome as potential diagnostic approach es but independent validation studies are 70 \nyet to be reported  17,18. A common limitation of endometriosis biomarker studies is that they \nseek to screen out differential targets without consideration of the underlying biology, limiting \ntheir utility for further development as therapeutic targets and prognostic markers.  \nEndometriosis is a hormone -dependent disorder  but utilising endocrine differences for \ndiagnostic purposes is challenging. Estrogens are considered the main disease driver due to 75 \ntheir established roles in promoting inflammation, angiogenesis and proliferation of  \nendometriosis lesions 19. Although estrogen concentrations are increased within lesions there \nare no reported differences in  circulating estrogens in women with endometriosis  20,21. \nFurthermore, estrogen concentrations are highly variable across the menstrual cycle, limiting \ntheir potential utility as diagnostic biomarkers. In contrast , androgens, which can be derived 80 \nfrom both the ovary and the adrenal,  have limited cyclical variation  in women. C lassic \nandrogens, such as testosterone (T) and dihydrotestosterone (DHT), regulate various cellular \nprocesses implicated in endometriosis, inc luding proliferation, tissue remodeling, and \ninflammation 22. Testosterone concentrations are increased in endometriosis lesions and this \nis associated with changes in androgen-regulated genes 23.  85 \nRecent studies suggest that adrenal -derived 11 -oxygenated androgens  are importan t \ncomponents of the androgen pool, particularly in women 24, but little is known about the \npotential contribution of 11-oxygenated androgens to endometriosis pathophysiology. 11KT \ncirculates at concentrations equivalent to or greater than T in women and has similar  \nandrogenic activity , consistent with a potential role in regulating androgen -dependent 90 \nprocesses 25. 11-oxygenated androgens are now recognised for the ir roles in endocrine \ndisorders such as polycystic ovarian syndrome (PCOS) 26 but whether they also play a role in \nendometriosis is yet to be determined.  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n5 \n \nGiven the potential importance of androgens to endometriosis pathophysiology and the limited \ndata on profiling of androgen metabolites, we sought to define the androgen metabolome in 95 \nendometriosis and investigate its potential utility as a diagnostic approach. Serum samples \nfrom women with laparoscopically-confirmed endometriosis (n= 159) and healthy controls \n(n=61) were profiled using a liquid chromatography–tandem mass spectrometry (LC-MS/MS) \nassay to simultaneously measure classic and 11 -oxygenated androgens.  Women with \nendometriosis had a distinct steroid signature characterised by increased concentrations of 100 \nclassic and 11 -oxygenated androgens and altered metabolism associated with 11 KT \nproduction. In predictive statistical models,  11KT and related metabolites demonstrated \nsignificant potential as diagnostic biomarkers and could robustly distinguish between healthy \ncontrols and women with endometriosis. These data reframe endometriosis as an androgen-\ndependent disorder and highlight the potential for 11-oxygenated androgens to be used as 105 \ndiagnostic biomarkers.  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n6 \n \nResults \nEndometriosis is associated with an altered androgen hormone signature \nAndrogens and their precursors were quantified using LC -MS/MS in serum samples from \nhealthy controls (HC; n= 57) and women with endometriosis ( ENDO; n= 159). The 110 \nconcentrations of ‘classic’ androgens; DHEA, A4, T and DHT and 11-oxygenated androgens; \n11OHA4, 11OHT, 11KA4 and 11KT were determined (Figure 1 and Table 1).  \nDHEA (Figure 1A; p<0.0001), A4 (Figure 1B; p<0.0001)  and T (Figure 1C; p<0.0001) were \nsignificantly higher in serum samples from women with endometriosis compared to healthy \ncontrols (Table 1 ‘All’). Concentrations of DHT were not significantly different between groups 115 \n(Figure 1D). The 11-oxygenated androgen precursor metabolites , 11OHA4 (Figure 1 E; \np<0.05) and 11KA4 (Figure 1 F; P<0.0001), were decreased, and the active 11-oxygenated \nandrogens, 11OHT (Figure 1G; p<0.0001) and 11KT (Figure 1H; p<0.0001), were increased \nin women with endometriosis (Table 1 ‘All’) . Consistent with previous reports 27 ,11-\noxygenated androgens did not differ by menstrual cycle phase ( Supplementary Table 1). 120 \nNeither classic nor 11 -oxygenated androgen concentrations correlated with age, although \n11OHT trended to positive correlation (r=0.1267, p=0.0636). Only 11KA4 negatively correlated \nwith BMI (r=-0.2029, p=0.0037; Supplementary Table 2). \nClassic and 11-oxygenated androgens do not differ by rASRM stage \nTo assess whether androgen concentrations were associated with disease severity in 125 \nendometriosis, data were stratified according to rASRM stage. Broadly, there were no \nsignificant differences in steroid concentrations between rASRM stages for any of the analytes \nassessed. Analytes that were increased in the unstratified group (ENDO /‘All’) were also \nincreased in each rASRM stage compared to healthy controls (HC) (Figure 1 I-P; Table 1). \nNotably, median concentrations of DHEA (p<0.0001), A4 (p<0.0001) and T (p<0.0001) were 130 \nhighest in Stage I endometriosis and significantly higher than healthy controls (Figure 1 I-P; \nTable 1). Similarly, concentrations of 11OHT (p<0.001) and 11KT (p<0.0001) were highest in \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n7 \n \nStage I endometriosis and significantly higher than healthy controls (Figure 1I-P; Table 1). \nConcentrations of T and 11KT were significantly elevated in women with Stage I, II, III and IV \nendometriosis compared to healthy controls, whereas 11KA4 was significantly reduced in 135 \nwomen with all stages of endometriosis compared to healthy controls  (Figure 1I-P). The \ngreatest difference in androgen concentrations compared to healthy controls was in patients \nclassified as Stage I. Median concentrations of T, 11KA4 and 11KT were modestly, inversely \ndecreased with stage but overall, there was no distinct steroid signature associated with a \ngiven rASRM stage. 140 \nAndrogen metabolism is  altered in endometriosis and  biased towards production of 11 -\nketotestosterone \nThe correlation between serum androgen analytes was assessed in healthy contro ls and \nwomen with endometriosis using Pearson correlation (Figure 2A and B, Supplementary Table \n3). In healthy controls, closely related metabolites exhibited a significant positive correlation. 145 \nThis included  classic androgens ; A4 and T  (r=0.8, p=1.59E-12) and T and DHT ( r=0.5, \np=6.37E-06), as well as  11-oxygenated androgens; 11OHA4 and 11KA4 ( r=0.6, p=8.2E-07) \nand 11OHT and 11KT (r=0.9, p=5.53E-20). In women with endometriosis , additional \nmetabolites that were not correlated in healthy controls were found to be positively correlated. \nThis included a significant positive correlation between classic and 11-oxygenated androgens, 150 \nas well as a greater correlation amongst all 11-oxygenated androgen metabolites. This was \ncharacterised by DHEA, A4, T and DHT each being significantly positively correlated with all \n11-oxygenated androgen metabolites (Figure 2A and B, Supplementary Table 3). Additionally, \nthere was a significant positive correlation between 11KT and 11OHA4 (r=0.6, p=1.82E-19), \n11KT and 11KA4 (r=0.7, p=7.68E-22), 11OHT and 11OHA4 (r=0.7, p=2.14E-24), and 11OHT 155 \nand 11KA4 ( r=0.6, p=3.94E-17) in women with endometriosis that was absent in healthy \ncontrols (Figure 2A and B, Supplementary Table 3). \nTo investigate  if these changes  were indicative of altered metabolism  in women with \nendometriosis, we estimated the activities of enzymes required for 11-oxygenated androgen \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n8 \n \nproduction by calculating substrate to product ratios (Figure 2; 11-β-hydroxylase (CYP11B1), 160 \n11β-hydroxysteroid dehydrogenase type 2  (HSD11B2) and Aldo-keto reductase family 1 \nmember C3 (AKR1C3)). CYP11B1 and HSD11B2 enzymes mediate interconversion and \nactivation of 11-oxygenated androgens. The ratios of 11OHA4/A4 and 11OHT/T (mediated by \nCYP11B1) were significantly decreased in women with endometriosis  (Figure 2C and D ), \nconsistent with decreased bioavailability of the precursors 11OHA4 and 11OHT. The ratio of 165 \n11KA4/11OHA4 ( mediated by  HSD11B2) was significantly decreased in women with \nendometriosis (Figure 2E, p<0.0001), consistent with decreased bioavailability of the \nprecursor 11KA4. In contrast, the ratio of 11KT/11OHT was modestly increased between \nhealthy controls and women with endometriosis (Figure 2F, p<0.0001) suggesting metabolism \nfavouring production of 11KT . Ratios for the reciprocal inactivation of 11KA4 and 11KT , 170 \nmediated by HSD11B128, were also calculated. The ratio of 11OHA4/11KA4 was increased \n(Supplementary figure 1A, p<0.0002) and 11OHT/11KT controls was decreased \n(Supplementary figure 1B, p<0.0002) in women with endometriosis compared to healthy  \ncontrols. \nAKR1C3 is a multifunction al enzyme that plays a pivotal role in all pathways to androgen 175 \nproduction including that of T, 11OHT and 11KT. The ratio of T/A4 (mediated by AKR1C3) did \nnot differ between groups (Supplementary figure 1C), but other AKR1C3 activities determined \nby the ratios of 11OHT/11OHA4 and 11KT/11KA4 were significantly higher in women with \nendometriosis (Figure 2G and H; p<0.0001), suggestive of greater production of 11OHT and \n11KT in women with endometriosis. Taken together, these data are consistent with a shift in 180 \nmetabolism towards production of 11KT (Figure 2I). \nAndrogen concentrations can be used to discriminate between healthy controls and women \nwith endometriosis \nReceiver operating characteristic (ROC) curve analysis of serum androgen concentrations \nwas performed to identify if specific androgen metabolites or ratios could be used to 185 \ndiscriminate between healthy controls and women with endometriosis (Supplementary Table \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n9 \n \n4). The androgen analytes T, 11KA4 and 11KT exhibited best sensitivity and specificity with \narea under the receiver operating characteristic curve (AUC) values of 0.815 (p<0.001), 0.822 \n(p<0.001), and 0.815 (p<0.001) respectively (Figure 3A-C).  \nROC curve analysis of calculated enzyme ratios was also performed as these data have 190 \nreduced inter -sample variability and provide a better indicator of the  proposed altered \nmetabolism found in women with endometriosis. Enzyme ratios associated with metabolism \nof 11 -oxygenated androgens  were assessed ( Supplementary Table 4). The ratios \n11OHA4/A4, 11OHT/11OHA4 and 11KT/11KA4 had best sensitivity and specificity with AUC \nvalues of 0.857 (p<0.001), 0.817 (p<0.001), and AUC of 0.968 (p<0.001) respectively (Figure 195 \n3D-F). The ratio of 11KT/11KA4 provided robust discrimination between healthy controls and \nwomen with endometriosis  with a sensitivity of 92 .36% and specificity of 9 0.91% and a \nlikelihood ratio of 10.16 (Figure 3G).  These data suggest that  changes in  androgen \nmetabolism could be used as predictors of endometriosis diagnostic outcome.  \nDevelopment of a predictive model using supervised machine learning 200 \nTo complement and extend these findings, androgen metabolome data were used to train a \npredictive model using multiple logistic regression. Androgen concentrations; DHEA, A4, T, \nDHT, 11OHA4, 11OHT  and 11KT, were used as dependent variables and diagnosed \nendometriosis (as a binary) was the outcome variable. BMI was also included in the model to \naccount for any independent effects of th is variable (Figure 3H). ROC curve analysis 205 \ndemonstrated excellent di scrimination between healthy controls  and women with \nendometriosis with AUC of 0.99 (Figure 3I; p<0.0001, CI 0.9878 to 1.000)  providing further \nimprovement on discrimination informed by single analytes or ratios (Figure 3A -G). The \npredicted probability for outcome was calculated for each sample using a classification cut-off \nof 0.5. The negative predictive power of the multiple logistic regression model was 9 2.86% 210 \nand positive predictive power 96.84% (Figure 3H and J) consistent with a robust and accurate \nmodel for predicting diagnostic outcome in endometriosis. \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n10 \n \nTrain and validation using a refined statistical model has robust positive predictive power for \ndiagnostic outcome \nTo assess how a predictive model could perform when blinded to outcome, the whole data set 215 \nwas partitioned into train and validation groups. The training set contained dependent and \noutcome variables for an arbitrary subset of healthy controls  (n=21) and women with \nendometriosis (n=21) and the remaining validation set contained dependent variables but was \nblinded to outcome variable (n=138). Comparative modelling was used initially to identify the \nmost robust analytes to include in a refined predictive model (not shown). Multiple logistic 220 \nregression was used to fit a predictive model using BMI, 11OHA4, 11KA4 and 11KT as \ndependent variables and diagnosed endometriosis as the outcome variable (Figure 4A, B). \nROC curve analysis of the simplified model demonstrated excellent discrimination between \nhealthy controls and women with endometriosis with AUC of 0. 97 (Figure 4C; p<0.0001, CI \n0.9124 to 1.000). The estimated negative predictive power of the model was 94.74% with a 225 \npositive predictive power of 95.24% (Figure 4A and D).  \nThe actual performance of the model was assessed using the remaining data with outcome \nblinded (validation set) and included separate performance estimates according to \nstratification by rASRM stage (Figure 4E). The predicted outcome was calculated for each \nsample, using a classification cut -off of 0.5 . In the validation dataset, the sensitivity of the 230 \nrefined model was 95.65% with accurate prediction observed for all disease stage \nstratifications (Figure 4E); including grouped minimal/mild ENDO (Stage I + II; 94% sensitivity), \nmoderate/severe ENDO (Stage III + IV; 96.55% sensitivity) or by individual rASRM stage (I -\nIV; range 91.2-100% sensitivity). Consistent with univariate analysis, 11KT had a large odds \nratio estimate, indicating the importance of this variable in determining diagnostic outcome. 235 \nThese data confirm the utility of using androgen concentrations to predict diagnostic outcome, \nwith a refined model accurately identifying >95% of women with endometriosis in a blinded \ncohort. \n \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n11 \n \n 240 \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n12 \n \nDiscussion \nIn summary, we sought to define the androgen metabolome in endometriosis and investigate \nits potential utility as a diagnostic approach. We discovered a disease -specific hormone \nsignature that identifies endometriosis with high specificity and sensitivity. This is 245 \ncharacterised by increased concentrations of classic and 11-oxygenated androgens in serum, \nreframing endometriosis as an androgen -dependent disorder. Elevated androgens were \nassociated with changes in activities of androgen synthesis enzymes, including AKR1 C3, \nindicative of altered metabolism that results in increased production of 11KT in women with \nendometriosis. When used alone or in combination with multiple logistic regression, androgen 250 \nmeasurements provided a  strong predictive performance for endometri osis using both \ncomprehensive and targeted analyte statistical models. When data were partitioned into train \nand validation cohorts, a refined model using only 11OHA4, 11KA4 and 11KT in combination \nwith BMI was sufficient to correctly identify >95% of women  with endometriosis in a blinded \nsample set. These data provide a significant breakthrough in the search for endometriosis 255 \nblood biomarkers and place 11-oxygenated androgens at the cornerstone of a new diagnostic \napproach for endometriosis.  \nSeveral blood biomarkers for endometriosis have been proposed , such as the glycoprotein \nCA-125, inflammatory cytokines (e.g. IL-6, IL-8, TNF-α and MCP -1 [CCL2]), as well as the \npro-angiogenic factor VEGF. Although these markers have been found to be elevated in 260 \nwomen with endometriosis, they lack specificity and have not been validated in larger studies. \nA recent multi -centre study that assessed 54 blood -based biomarkers in samples from 919 \nwomen with endometriosis found CA-125 offered best discrimination between endometriosis \ncases and pathology -free symptomatic controls. However, the AUC for CA -125 in this \ncomparison was 0.645 and thus had limited accuracy as a diagnostic biomarker 29. Critically, 265 \nCA-125 is elevated in other conditions including ovarian and endometrial cancers, further \nlimiting its utility as a diagnostic biomarker in endometriosis 30,31. The use of CA -125 as a \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n13 \n \ndiagnostic or screening tool is  not recommended within the European Society of Human \nReproduction and Embryology endometriosis guidelines 32. \nEmerging results from a proteomic screen of serum samples from a large cohort of 805 270 \npatients showed promise using a combination of ten serum protein biomarkers with strong \npredictive accuracy for the diagnosis of endometriosis, particularly those with severe \nendometriosis (Stage IV) 18. Further studies are needed to establish if these candidate markers \nare specific to endometriosis and if their expression is affected by age, menstrual cycle stage \nor hormone treatment. Similarly, an undisclosed panel of 109 salivary miRNAs were reported 275 \nto detect endometriosis with high sensitivity (96.2%) and specificity (95.1%) in an interim \nvalidation study, but whether these markers are variable across populations and \nendometriosis subtypes is not yet known 33. \nAn ideal biomarker for endometriosis would need to overcome the limitations of current \napproaches and should have high sensit ivity and specificity, reflect endometriosis 280 \npathophysiology, be consistent across subtypes and not vary by menstrual cycle stage or with \nhormone treatment. Based on our results, 11-oxygenated androgens represent a significant \nimprovement on other endometriosis biomarkers as they fulfil all of these key criteria. To the \nbest of our knowledge, our study is the first to profile 11 -oxygenated androgens in \nendometriosis. However, larger and more representative cohorts will be needed to understand 285 \nif this profiling is generalisable across different demographics and endometriosis subtypes . \nThe participants in our study were mostly of European ethnicity and future studies should \nensure broader representation and inclusion to limit bias in this factor. Compari sons to other \nendocrine disorders will  also be important to understand distinct features of endometriosis. \nAlthough there are major differences between  the androgen analyte concentrations in 290 \nendometriosis in our study and those previously published for PCOS, such as increased rather \nthan decreased concentrations of 11OHA4 and 11KA4 in PCOS, an integrated analysis of \nboth disorders would provide  the greatest resolution on their distinct hormone profiles. We \nhave profiled samples from all stages of endometrio sis, but our study cohort was more \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n14 \n \nrepresentative of patients with minimal/mild disease ( Stage I and II; 62.9% of cohort) rather 295 \nthan moderate/severe disease ( Stage II and IV; 37.1% of cohort), and predominantly \npresented with pain rather than infertility symptoms. We did not detect a distinct hormone \nprofile associated with endometriosis subtypes but it is possible that naturally occurring groups \nwithin the data could be identified using cluster analysis approaches in larger datasets. This \ncould add further resolution to existing staging and subtyping methods used in endometriosis. 300 \nThis is important because current disease staging approaches do not correlate with severity \nof symptoms 12 which in part compounds the diagnostic challenges associated with \nendometriosis.  \nWe found no correlation between an drogen concentrations and pain scores in women with \nendometriosis but the majority of patients in our cohort presented with pelvic pain which may 305 \nbias interpretation. Androgens play a complex role in pain perception in women with \nendometriosis that is yet to be fully defined. An inverse correlation between measures of \nchronic pain and androgen levels has been reported in women with dysmenorrhea but this \nstudy did not directly assess women with endometriosis 34.  Notably, in our study we measured \nmultiple androgen analytes in women with laparoscopically -confirmed endometriosis and 310 \ncorrelated these with pain scores based on the pain domain of the EHP -30 questionnaire: a \nreliable and validated questionnaire for assessing the health-related quality of life in women \nwith endometriosis  35. This direct comparison found no significant correlation between \nandrogens and pain in women with endometriosis. \nAlthough androgens did not correlate with endometriosis symptoms or subtypes, we believe 315 \nthat they may have a direct role in endometriosis pathogenesis. Androgens can regulate \nprocesses associated with formation of endometriosis lesions and the androgen receptor has \nbeen identified as an endometriosis -associated transcription factor 36. 11KT has similar \npotency to T in activating androgen -dependent processes 25 and was detected at higher \nconcentrations than T in women with endometriosis in the current study. We propose that 320 \nandrogen excess in endometriosis drives pathophysiology through direct regulation of \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n15 \n \nandrogen-dependent processes (via T and 11KT) in endome trial and endometriotic tissues \nand by acting as obligate substrates for aromatase in lesions (via A4 and T) to fuel estrogenic \nprocesses. Changes to androgen metabolism may also have systemic effects that increase \nrisk of lesion formation over the life course, including changes to innate and adaptive immune 325 \ncell function and inflammation as reported in other contexts of androgen excess such as PCOS \n37. This may explain how retrograde menstruation can lead to lesion formation in some women \nbut not others. \nConcentrations of 11KT are not dimin ished by oral contraceptives or gonadotropin-releasing \nhormone analogue-mediated ovarian suppression 27. If 11KT is driving endometriosis 330 \npathogenesis, this may explain why these widely used medical treatments for endometriosis-\nassociated pain are ineffective in some women 27. Previously, the synthetic androgen danazol \nwas found to be effective in the medical management of endometriosis-associated pain 38 but \nits use was stopped due to unacceptable masculinizing side-effects. The mechanism of action \nof danazol in endometriosis has not been fully characterised but it has been shown to inhibit 335 \nadrenal androgen synthesis via suppression of androgen metabolising enzyme activity in other \ncontexts 39. We have found that adrenal androgen production  was a key feature of \nendometriosis; as characteri sed by altered activities of HSD 11B2, HSD11B1 and AKR1C3 \nand the enhanced biosynthesis of 11KT. Thus, the efficacy of danazol in endometriosis may \nbe due to its action on androgen metabolism,  which reframes our perception of the 340 \npharmacodynamics of this drug. Although danazol was poorly tolerated, these results support \nrevisiting danazol (e.g. administered by a local delivery system) or a more selective derivative \nas a therapeutic approach in endometriosis.  \n11KT is now considered the most biologically relevant androgen in women  24 and we have \nidentified that endometriosis is associated with 11KT excess. These data highli ght the 345 \nimportance of androgens in endometriosis, both as a fundamental feature of the \npathophysiology and as a potential disease driver. When combined with predictive modelling \nandrogen metabolite concentrations can be used to robustly distinguish between  healthy \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n16 \n \ncontrols and women with endometriosis. Collectively, these data signify a paradigm shift in our \nunderstanding of endometriosis and represent a unique opportunity to develop rapid, non -350 \nsurgical approaches for endometriosis diagnosis and management. \nAcknowledgements \nThis work was supported by the Wellcome Trust (Fellowship 220656/Z/20/Z to DAG , \nInvestigator Award 209492/Z/17/Z to WA), Medical Research Council (Grant MRC/IAA/002 to \nDAG, program grant MR/N024524/1 to PTKS, and program grant MC_UP_1605/ 15 to WA ) 355 \nand the Institute for Regeneration and Repair Innovators award (funded by WT ITPA to DAG). \nResearch conducted by the Edinburgh EXPPECT group  that contributed to collection of \nbiospecimens has been supported by grants from the MRC, Wellcome Trust, NIHR, CSO, and \nWellbeing of Women. We thank members of the EXPPECT Edinburgh group, Frances Collins \nand Ann Doust, for support with participant recruitment and biosample collection.  For the 360 \npurpose of open access, the author has applied a Creative Commons  Attribution (CC BY) \nlicence to any Author Accepted Manuscript version arising from this submission.   \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n17 \n \nMaterials and Methods \nStudy approval \nWritten informed consent was obtained prior to study participation from healthy volunteers 365 \nidentified through advertisement. Ethical approval was granted by the Science, Technology, \nEngineering and Mathematics Ethical Review Committees of the University of  Birmingham, \nUK (ERN_17-0494, ERN_17-0494B).  \nWritten informed consent was obtained from all endometriosis study participants prior to \nsurgery. Ethical approval was granted by the Lothian Research Ethics Committee (LREC 370 \n11/AL/0376), South Central-Hampshire A research ethics committee  (IRAS:237815 REC \nreference 19/SC/0449) and Wales REC 6 A research ethics committee  (IRAS 268806; REC \nref: 19/WA/0271). Methods were carried out in accordance with Local Tissue Governance \nguidelines and international EPHect guidelines (https://endometriosisfoundation.org/ephect/).  \nPatient cohorts 375 \nInclusion criteria for healthy controls  (HC) were pre-menopausal women aged 18 years or \nabove. A standardized questionnaire was used to record demographic data including age and \nBMI; the use of hormonal contraceptives and menopausal status were recorded. Healthy \ncontrol data were selected from a broader cohort of participants as detailed in Schiffer et al . \nto match age range of patient cohort in the current study 27.  380 \nEligible participants were women with chronic pelvic pain (aged 18 –50 years) of >3 months \nduration who were undergoing diagnostic laparoscopy for suspected endometriosis in NHS \nLothian. Pelvic pain was defined as pain located within the true pelvis (between and below the \nanterior iliac crests). Participants at the Liverpool Women’s Hospital were those undergoing \ndiagnostic laparoscopy for suspected endometriosis  that were recruited from endometriosis 385 \nor general gynaecology clinics. Diagnostic outcome, age, BMI, menstrual cycle stage and \nhormone status were obtained and recorded along with other key clinical data.  Diagnosis of \nendometriosis was confirmed macroscopically at laparoscopy ( ENDO). Endometriosis was \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n18 \n \nsubsequently classified according to the revis ed scoring system of the American Society for \nReproductive Medicine (rASRM) which ranges from Stage I to Stage IV based on the location 390 \nand extent of lesions, as well as adhesions, visualized at surgery 11. \nExclusion criteria were any acute or chronic disease affecting steroid biosynthesis or \nmetabolism (including polycystic ovarian syndrome; PCOS) and the intake of any medication \nknown to interfere with steroid biosynthesis or metabolism.  Participants using hormonal \ncontraceptives (combined oral contraceptives, contraceptive depot injection, or implant) were 395 \nexcluded. \nSerum steroid analysis \nAll venous blood samples were collected in the morning and, in the case of women with \nsuspected endometriosis, prior to anaesthesia on the morning of surgery. All blood samples \nwere collected in serum-separating tubes. After centrifugation, aspirated serum was aliquoted 400 \nand stored at -80oC. Serum samples from women with endometriosis (n= 159) and healthy \ncontrols (n= 57) were profiled using a sensitive liquid chromatography tandem -mass \nspectrometry (LC-MS/MS) assay to simultaneously measure 21 steroid \nhormones/metabolites. \nSerum steroids were quantified using a previously published and validated approach for multi-405 \nsteroid profiling using liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay \n40. Briefly, 200 μL of serum was mixed with stable isotope -labelled internal standards and \nfollowing protein precipitation with 50 μL acetonitrile samples were extracted by liquid –liquid \nextraction with 1 mL methyl tert-butyl ether (MTBE). The MTBE organic phase was removed, \ndried and reconstituted in 50/50 methanol/water. Steroids were chromatographically 410 \nseparated using a Phenomenex Luna Omega C18 column (1.6 µm, 100Å, 2.1 mm × 50 mm) \nand a water (0.1% formic acid) –methanol gradient. Ammonium fluoride was introduced by \npost-column infusion to aid ionisation. Steroids were quantified relative to a calibration series \nranging from 0.02 to 250 ng/mL with inclusion of a blank calibration point. Analysis was \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n19 \n \nperformed on a Waters Xevo TQ -XS mass spectrometer using electrosp ray ionization in 415 \npositive ion mode.  \nAnalyte key \nSerum analytes were grouped as follows . Classic Androgens ; dehydroepiandrosterone \n(DHEA), androstenedione (A4), testosterone (T) and dihydrostestosterone (DHT).  11-\noxygenated androgens ; 11β-hydroxyandrostenedione (11OHA4), 11β -hydroxytestosterone 420 \n(11OHT), 11-ketoandrostenedione (11KA4) and 11-ketotestosterone (11KT).  \nStatistical analysis \nStatistical analysis was performed using GraphPad Prism 10. Undetected steroid \nconcentrations or data below the LLOQ were replaced by 0.5×LLOQ for statistical purposes . \nData were summarized using mean ± SD, presented for healthy controls (N=57) and women 425 \nwith endometriosis (N=159) or as median ± interquartile range in tables, as indicated . T test \nwas used to compare the difference in the means of the two groups. Two -way ANOVA was \nused to determine the significance between treatments in grouped data. Non -parametric \ntesting was utili sed where sample sizes were insufficient to confirm normality of data \ndistribution; Mann–Whitney test to assess variance between two groups or Kruskal–Wallis test 430 \nwas used to assess differences between multiple groups . Criterion for significance was P < \n0.05. Correlation between analytes was assessed using Pearson matrix. Discrimination \nbetween cohorts using metabolite concentrations or ratios was assessed by ROC curve \nanalysis. Predictive modelling for multiple analytes was performed using multiple logisti c \nregression. 435 \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n20 \n \nReferences \n1. Horne, A.W. & Missmer, S.A. Pathophysiology, diagnosis, and management of \nendometriosis. Bmj 379, e070750 (2022). \n2. Berkley, K.J., Rapkin, A.J. & Papka, R.E. The pains of endometriosis. Science 308, 1587-1589 440 \n(2005). \n3. De Corte, P., Klinghardt, M., von Stockum, S. & Heinemann, K. Time to Diagnose \nEndometriosis: Current Status, Challenges and Regional Characteristics—A Systematic \nLiterature Review. BJOG: An International Journal of Obstetrics & Gynaecology 132, 118-130 \n(2025). 445 \n4. 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Journal of Obstetrics and Gynaecology Canada 46(2024). \n11. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. \nFertil Steril 67, 817-821 (1997). 460 \n12. Vercellini, P., et al. Association between endometriosis stage, lesion type, patient \ncharacteristics and severity of pelvic pain symptoms: a multivariate analysis of over 1000 \npatients. Human Reproduction 22, 266-271 (2007). \n13. Roy, S.N. The risks of laparoscopic surgery: I. Gynecological Surgery 3, 315-319 (2006). \n14. Brulport, A., et al. An integrated multi-tissue approach for endometriosis candidate 465 \nbiomarkers: a systematic review. Reprod Biol Endocrinol 22, 21 (2024). \n15. Nisenblat, V., et al. Blood biomarkers for the non‐invasive diagnosis of endometriosis. \nCochrane Database of Systematic Reviews (2016). \n16. Bendifallah, S., et al. MicroRNome analysis generates a blood-based signature for \nendometriosis. Sci Rep 12, 4051 (2022). 470 \n17. Scheck, S.M., et al. Non-invasive tests for endometriosis are here; how reliable are they, and \nwhat should we do with the results? Aust N Z J Obstet Gynaecol 64, 168-170 (2024). \n18. Schoeman, E.M., et al. Identification of plasma protein biomarkers for endometriosis and the \ndevelopment of statistical models for disease diagnosis. Hum Reprod (2024). \n19. Chantalat, E., et al. Estrogen Receptors and Endometriosis. in International Journal of 475 \nMolecular Sciences, Vol. 21 (2020). \n20. Huhtinen, K., et al. Endometrial and endometriotic concentrations of estrone and estradiol \nare determined by local metabolism rather than circulating levels. J Clin Endocrinol Metab \n97, 4228-4235 (2012). \n21. Mercorio, A., Giampaolino, P., Romano, A., Dällenbach, P. & Pluchino, N. Is intracrinology of 480 \nendometriosis relevant in clinical practice? A systematic review on estrogen metabolism. \nFront Endocrinol (Lausanne) 13, 950866 (2022). \n22. Simitsidellis, I., Saunders, P.T.K. & Gibson, D.A. Androgens and endometrium: new insights \nand new targets. Molecular and Cellular Endocrinology 465, 48-60 (2018). \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n21 \n \n23. Huhtinen, K., et al. Intra-Tissue Steroid Profiling Indicates Differential Progesterone and 485 \nTestosterone Metabolism in the Endometrium and Endometriosis Lesions. The Journal of \nClinical Endocrinology & Metabolism 99, E2188–E2197 (2014). \n24. Storbeck, K.-H. & O’Reilly, M.W. The clinical and biochemical significance of 11-oxygenated \nandrogens in human health and disease. European Journal of Endocrinology 188, R98-R109 \n(2023). 490 \n25. 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Multicenter evaluation of blood-based biomarkers for the detection of \nendometriosis and adenomyosis: A prospective non-interventional study. Int J Gynaecol \nObstet 164, 305-314 (2024). 505 \n30. Duk, J.M., Aalders, J.G., Fleuren, G.J. & de Bruijn, H.W. CA 125: a useful marker in \nendometrial carcinoma. Am J Obstet Gynecol 155, 1097-1102 (1986). \n31. Charkhchi, P., et al. CA125 and Ovarian Cancer: A Comprehensive Review. Cancers (Basel) \n12(2020). \n32. Kuznetsov, L., Dworzynski, K., Davies, M. & Overton, C. Diagnosis and management of 510 \nendometriosis: summary of NICE guidance. BMJ 358, j3935 (2017). \n33. Bendifallah, S., et al. Validation of a Salivary miRNA Signature of Endometriosis — Interim \nData. NEJM Evidence 2, EVIDoa2200282 (2023). \n34. Evans, S.F., et al. The Relationship Between Androgens and Days per Month of Period Pain, \nPelvic Pain, Headache, and TLR4 Responsiveness of Peripheral Blood Mononuclear Cells in 515 \nYoung Women with Dysmenorrhoea. J Pain Res 14, 585-599 (2021). \n35. Jones, G.L., et al. A systematic review to determine use of the Endometriosis Health Profiles \nto measure quality of life outcomes in women with endometriosis. Hum Reprod Update 30, \n186-214 (2024). \n36. Yang, H., Kang, K., Cheng, C., Mamillapalli, R. & Taylor, H.S. Integrative Analysis Reveals 520 \nRegulatory Programs in Endometriosis. Reprod Sci 22, 1060–1072 (2015). \n37. Shabbir, S., et al. The interplay between androgens and the immune response in polycystic \novary syndrome. Journal of Translational Medicine 21, 259 (2023). \n38. Selak, V., Farquhar, C., Prentice, A. & Singla, A. Danazol for pelvic pain associated with \nendometriosis. Cochrane Database Syst Rev, Cd000068 (2007). 525 \n39. Stillman, R.J., Fencl, M.D., Schiff, I., Barbieri, R.L. & Tulchinsky, D. Inhibition of Adrenal \nSteroidogenesis by Danazol in Vivo**Presented at the Thirty-Sixth Annual Meeting of The \nAmerican Fertility Society, March 18 to 22, 1980, Houston, Tex. Fertil Steril 33, 401-406 \n(1980). \n40. Schiffer, L., et al. Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of 530 \nammonium fluoride. Journal of Chromatography B 1209, 123413 (2022). \n \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n22 \n \nResults – figures and tables \nTable 1 535 \n \nTable 1. Serum steroid concentrations of classic and 11-oxygenated androgens in healthy controls \nand women with endometriosis. Age (years) median (min-max), BMI (kg/m2) median (min-max), \nserum steroid concentrations (median and 25th-75th centile range; nmol L–1). Values below the \nlower limit of quantification are indicated as < lower limit of quantification. Steroid concentrations 540 \nbelow the LLOQ were replaced by 0.5×LLOQ for statistical purposes. P value vs healthy control; * \np<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.  a vs Stage I, b vs Stage II. \n  \n \n CONTROLS ENDOMETRIOSIS \n All All Stage I Stage II Stage III Stage IV \nn 57 159 66 34 26 33 \nAge 30 (22-48) 32.00 (19-50) 28.00 (19-47) 36.00 a (22-46) 37.00 a (22-50) 34.00 a (23-47) \nBMI 22.04 (17.3-37.3) 25.00 \n(18.0-62.0) *** \n25.00** \n(18.0 – 62.0) \n25.25** \n(18.0 – 37.5) \n25.00* \n(19.2 – 35.0) \n25.65 \n(18.0 – 39.5) \nCycle stage \npro/sec/ND \n19/22/16 \n33.3%/38.6%/28% \n75/67/17 \n47.2%/42.1%/10.7% \n32/30/4 \n48.5%/45.5%/6.1% \n16/14/4 \n47.0%/41.2%/11.2% \n17/9/0 \n65.4%/34.6%/0% \n10/15/8 \n30.3%/45.5%/24.2% \nDHEA 9.6 \n(6.02-14.63) \n20.80 \n(12.23-32.48) **** \n25.40**** \n(16.35-39.45) \n18.50*** \n(11.70-33.55) \n14.65a \n(10.00-22.40) \n19.40** \n(10.20-30.60) \nA4 2.83 \n(1.76-4.01) \n4.72 \n(3.37-6.59) **** \n5.55**** \n(3.71-7.51) \n4.52*** \n(3.25-7.41) \n4.56 \n(2.88-5.51) \n4.61* \n(2.76-5.83) \nT 0.63 \n(0.50-0.93) \n1.15 \n(0.86-1.49) **** \n1.26**** \n(0.97-1.59) \n1.25**** \n(1.00-1.80) \n1.07*** \n(0.80-1.44) \n1.03* b \n(0.69-1.33) \nDHT 0.17 \n(0.17-0.50) \n0.40 \n(0.17-0.60) \n0.48* \n(0.17-0.70) \n0.44 \n(0.17-0.59) \n0.26 \n(0.17-0.47) \n0.36 \n(0.17-0.57) \n11OHA4 7.26 \n(5.35-10.12) \n5.67 \n(3.80-8.21) ** \n6.27 \n(4.49-8.54) \n6.34 \n(4.11-8.34) \n5.38* \n(3.08-7.10) \n5.18 \n(3.78-7.35) \n11KA4 3.18 \n(2.28-3.75) \n1.64 \n(1.11-2.39) **** \n1.88**** \n(1.24-2.72) \n1.63**** \n(1.07-2.32) \n1.53**** \n(1.08-2.05) \n1.42**** \n(0.97-2.28) \n11OHT 0.36 \n(0.17-0.52) \n0.51 \n(0.17-0.72) **** \n0.57*** \n(0.36-0.73) \n \n0.53* \n(0.31-0.68) \n0.43 \n(0.17-0.67) \n0.44 \n(0.25-0.71) \n11KT 0.69 \n(0.44-0.97) \n1.3 \n(0.99-1.80) **** \n1.49**** \n(1.12-2.19) \n1.25**** \n(0.96-1.80) \n1.18*** \n(0.87-1.72) \n1.15** \n(0.72-1.71) \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n23 \n \n \nFigure 1. Serum concentrations of classic androgens and 11-oxygenated androgens in healthy 545 \ncontrols (HC) or women with endometriosis (ENDO). Serum concentrations of DHEA, A4, T, and \n11OHA4, 11KA4, 11OHT, and 11KT were by LC-MS/MS. DHEA, A4, T, were significantly elevated in \nENDO compared to HC (A-C). Concentrations of DHT did not differ between groups (D). 11-\noxygenated androgen precursors, 11OHA4 (E) and 11KA4 (F), were decreased in ENDO, while potent \n11-oxygenated androgens, 11OHT (G) and 11KT (H) were significantly elevated in ENDO. ENDO 550 \npatients were stratified according to rASRM stage (I-P). DHEA and A4 were significantly elevated in \nStage I, II and IV endometriosis (I, J). T was elevated in all endometriosis stages (K), and DHT was \nsignificantly elevated in Stage I endometriosis (L). Concentrations of 11OHA4 did not significantly \ndiffer between groups (M) while 11KA4 was decreased in all stages (N). In contrast, 11OHT was \nsignificantly elevated in Stage I endometriosis (O) while 11KT was elevated in all endometriosis 555 \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n24 \n \nstages (P). Median serum steroid concentrations (25th-75th centile range; nmol L–1) for each stage \n(I). Error bars represent the standard deviation of the mean. Dotted lines denote LLOQ for each \nanalyte. Undetected steroids or those detected below LLOQ were replaced by 0.5×LLOQ for statistical \npurposes.  Statistical comparison Mann Whitney U test or Kruskal-Wallis test with multiple \ncomparisons. *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001, ns – non significant. 560 \n \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n25 \n \n \nFigure 2. Endometriosis is associated with altered androgen metabolism characterised by increased \nproduction of 11KT. A Pearson correlation matrix of key analytes in HC demonstrates strong 565 \ncorrelation between related steroids within relevant subgroupings including A4 and T, 11OHA4 and \n11KA4 as well as 11KT and 11OHT. B In ENDO patients there was a strong correlation between both \nclassic and 11-oxygenated androgen metabolites indicative of altered metabolism. Enzyme activity \nbased on substrate to product ratios (product/substrate) were calculated based on measured serum \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n26 \n \nconcentrations in healthy control women (HC) and patients diagnosed with endometriosis (ENDO). 570 \nThe ratios required for sequential production of 11KT were assessed including CYP11B1 (C, D), \nHSD11B2 (E, F) and AKR1C3 (G, H). The ratios of 11OHA4/A4 (C), 11OHT/T (D) and 11KA4/OHA4 (E) \nwere significantly decreased in ENDO compared to HC. In contrast, the ratios of 11KT/11OHT (F), \n11OHT/11OHA4 (G), 11KT/11KA4 (H) were significantly increased in ENDO compared to HC. \nCollectively these metabolism changes are associate with increased production of 11KT (I). Statistical 575 \ncomparison Mann Whitney U test. *p<0.05, ****p<0.0001.  \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n27 \n \n \nFigure 3. Assessing the utility of androgen concentrations for discrimination between HC and ENDO. \nROC curve analysis of serum analyte concentrations (A-C) and ratios (D-F) was assessed (G). Robust 580 \ndiscrimination between HC and ENDO was observed for single analytes; T (AUC 0.8152; p<0.001), \n11KA4 (AUC 0.8145; p<0.001) and 11KT (AUC 0.8219; p<0.001), and  for each ratio; 11OHA4/A4 (AUC \n0.8556; p<0.001), 11OHT/11OHA4 (AUC 0.817; p<0.001), and 11KT/11KA4 (AUC 0.9676; p<0.001). \nSensitibity, specificity and likelihood ratio were summarised and the ratio of 11KT/11KA4 provided \nthe most robust discrimination between HC and ENDO (G). H Multiple logistic regression modelling 585 \nwas used to fit a predictive model using androgen concentrations, Age and BMI as dependent \nvariables and diagnosed endometriosis as the outcome variable. I ROC curve analysis of model \ndemonstrated excellent discrimination between HC and ENDO with AUC of 0.99 (p<0.0001, CI 0.9794 \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n28 \n \nto 1.000). J The predictive power of the model was calculated based on observed vs predicted \noutcomes. The negative predictive power of the model was 92.86% and the positive predictive power 590 \nwas 96.84%. Statistical comparison Mann Whitney U test. ****p<0.0001, ns – non significant. \n \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint \n\n29 \n \n \nFigure 4. Actual performance of simplified predictive model for diagnostic status using androgen 595 \nconcentrations. The whole data set was partitioned into train and validation groups. The training set \ncontained dependent and outcome variables for HC (n=21) and ENDO (n=21) and the validation set \ncontained dependent variables but was blinded to outcome variable (n=138). Multiple logistic \nregression was used to fit a refined predictive model using BMI, 11OHA4, 11KA4 and 11KT as \ndependent variables and diagnosed endometriosis as the outcome variable (A, B). Samples with 600 \nmissing data were excluded as part of the regression analysis. C ROC curve analysis of the model \ndemonstrated excellent discrimination between HC and ENDO with AUC of 0.965 (p<0.0001, CI \n0.9124 to 1.000). The estimated negative predictive power of the model was 94.74% with a positive \npredictive power of 95.24%. (A, D). The actual performance of the model was assessed using the \nremaining data with outcome blinded (validation set). E The sensitivity for ENDO was 95.65% with 605 \naccurate prediction observed in all disease stage stratifications; including grouped Minimal/Mild \n(Stage I + II), Moderate/Severe (Stage III + IV) or by individual rASRM stage (I-IV).  \n \n \n . CC-BY-NC 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 25, 2025. ; https://doi.org/10.1101/2025.05.24.25328281doi: medRxiv preprint","source_license":"CC0","license_restricted":false}