Plasma metabolites associated with endometriosis in adolescents and young adults

STUDY QUESTION What are the plasma metabolomics profiles associated with endometriosis in adolescents and young adults? SUMMARY ANSWER Our findings show dysregulation of plasma metabolomic profiles in adolescents and young adults with endometriosis, revealing systemic elevation of fatty acyls and ceramides in endometriosis cases compared to controls. WHAT IS KNOWN ALREADY Endometriosis is a gynecologic disease often presenting with severe pelvic pain impacting around 200 million reproductive-aged women worldwide. However, little is known about the pathophysiology and molecular features of endometriosis diagnosed during adolescence and young adulthood. STUDY DESIGN, SIZE, DURATION We conducted a cross-sectional analysis including 190 laparoscopically confirmed endometriosis cases and 120 controls who participated in The Women’s Health Study: From Adolescence to Adulthood, which enrolled participants from 2012 to 2018. Control participants were females without a diagnosis of endometriosis enrolled from the same clinics as the cases or recruited from the general population. Among the cases, 81 had blood samples collected before and after surgery. PARTICIPANTS/MATERIALS, SETTING, METHODS Plasma metabolites were measured in blood collected at enrollment using liquid chromatography–tandem mass spectrometry, and a total of 430 known metabolites were evaluated in our analysis. We used linear regression adjusting for age at blood draw, BMI, hormone use, and fasting status at blood draw. Metabolite set enrichment analysis (MSEA) was used to identify metabolite classes. Number of effective tests (NEF) and false discovery rate (FDR) were used for multiple testing correction. MAIN RESULTS AND THE ROLE OF CHANCE The median age was 17 years for endometriosis cases and 22 years for controls. The majority of endometriosis cases had rASRM stage I or II (>95%). We identified 63 plasma metabolites associated with endometriosis (NEF < 0.05). Endometriosis cases had higher levels of plasma metabolites associated with proinflammatory response [e.g. eicosatrienoic acid (β = 0.61, 95% CI = 0.37, 0.86)], increased oxidative stress response [e.g. xanthine (β = 0.64, 95% CI = 0.39, 0.88)], and downregulation of metabolites related to apoptosis [glycocholic acid (β = −0.80, 95% CI = −1.04, −0.56)]. MSEA revealed increased fatty acyls (FDR = 2.3e−4) and ceramides (FDR = 6.0e−3) and decreased steroids and steroid derivatives (FDR = 1.3e−4) in endometriosis cases compared to controls. When we examined the changes in plasma metabolite profiles before and after surgery among endometriosis cases, 55 endometriosis-associated metabolites significantly changed from before to after surgery. MSEA revealed steroids and steroid derivatives (FDR = 8.1e−4) significantly increased after surgery, while fatty acyls (FDR = 1.2e−4) significantly decreased after surgery. Ceramides did not change from pre- to post-surgery and were elevated in post-surgical blood compared to controls (FDR = 3.9e−3). LIMITATIONS, REASONS FOR CAUTION Our study population mainly consists of self-reported non-Hispanic, white individuals and endometriosis cases with superficial peritoneal lesions only, so the generalizability may be limited. Furthermore, despite our large study population of adolescents and young adults with endometriosis, sample size was limited to conduct detailed stratified analyses of plasma metabolomic profiles, especially by post-surgical pelvic pain outcomes. WIDER IMPLICATIONS OF THE FINDINGS Our study includes the utilization of state-of-the-art metabolomics technology with high reproducibility to comprehensively investigate the metabolites that were associated with endometriosis diagnosed in adolescents and young adults. Our results suggest a positive impact of endometriosis-related surgery for some, but not all, on systemic metabolic dysregulation in young patients with endometriosis. These results warrant further investigation on whether and how persistent systemic changes despite treatment may lead to long-term chronic disease risk among those diagnosed with endometriosis. STUDY FUNDING/COMPETING INTEREST(S) Financial support for establishment of and data collection within the A2A cohort was provided by the J. Willard and Alice S. Marriott Foundation, and support for assay costs was in part provided by the Peery family. This project was funded by Eunice Kennedy Shriver National Institute of Child Health and Human Development R21HD107266. S.A.M., A.L.S., and K.L.T. were supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development R01HD094842. S.A.M. received grant funding from AbbVie, National Institutes of Health, Department of Defense, and Marriott Family Foundation; received honoraria from WERF, Huilun Shanghai, and University of Kansas Medical Center; travel support from SRI, ESHRE, FWGBD, University of Michigan, MIT, ASRM, LIDEA Registry, Taiwan Endometriosis Society, SEUD, Japan Endometriosis Society, NASEM, Endometriosis Foundation of America, Gedeon Richter Symposium at ESHRE; Board member receiving financial remuneration from AbbVie, Roche, LIDEA Registry, Editor of Frontiers in Reproductive Health, Roundtable participation for Abbott; Board member without financial remuneration from NextGen Jane and Statistical Advisory Board member of Human Reproduction; leadership role in Society for Women’s Health Research, World Endometriosis Society, World Endometriosis Research Foundation, ASRM, ESHRE. N.S. and K.L.T. receive grant funding from Aspira Women’s Health unrelated to this project. The remaining authors have no disclosures relevant to this manuscript. TRIAL REGISTRATION NUMBER N/A.

endometriosis, adolescence, biomarkers, metabolomics, inflammation

Endometriosis is characterized by the presence of endometrial-like tissue outside the uterus that responds to hormonal cues and is estimated to affect about 10% of reproductive-aged women, corresponding to an estimated 200 million women and girls worldwide (Adamson et al., 2010; Zondervan et al., 2018; Zondervan et al., 2020). Although menstrual pain during adolescence is a frequent early symptom of endometriosis, the requirement of surgical visualization is a barrier to timely diagnosis, leading to an average 7-year delay from symptom onset to diagnosis (Nnoaham et al., 2011). Consequently, women with endometriosis suffer prolonged pain, decreased quality of life, and increased risk of comorbidities, resulting in substantial social and economic burden (Nnoaham et al., 2011; Youngster et al., 2013; Weintraub et al., 2014; Kvaskoff et al., 2015). Being able to diagnose endometriosis earlier in the life course, during adolescence and young adulthood, may lead to earlier intervention and improved clinical outcomes. In fact, two-thirds of those who were diagnosed with endometriosis in adulthood reported their symptom onset in adolescence, suggesting endometriosis development may be starting from adolescence (Nnoaham et al., 2019). However, little is known about the pathophysiology and molecular features of endometriosis diagnosed during adolescence. Adolescent endometriosis typically presents with superficial peritoneal lesions only (DiVasta et al., 2018), which is different from adult-diagnosed endometriosis often presenting with superficial peritoneal lesions and deep fibrotic lesions (Shah and Missmer, 2011; DiVasta et al., 2018; Shafrir et al., 2018). Thus, capturing the molecular profiles in adolescents and young adults may yield novel biological pathways for interventions for endometriosis. Metabolites are the downstream products of cellular activities regulated by the genome and modified by environmental factors (Gerszten and Wang, 2008). Thus, metabolomic profiles integrate genetic and environmental interactions and potentially capture cellular responses to past exposures, furthering our understanding of disease pathophysiology. While there have been studies reporting metabolites associated with endometriosis (Dutta et al., 2012; Jana et al., 2013; Lee et al., 2016; Dutta et al., 2018; Li et al., 2018), prior studies focused on endometriosis diagnosed in adulthood, and many studies have been limited by small sample sizes and used a control group undergoing surgery for other benign gynecologic indications that may have similar associated underlying metabolic pathways (Shafrir et al., 2018; Gallagher et al., 2019; Missmer, 2019). In this study, we aimed to identify novel blood metabolomics profiles associated with endometriosis diagnosed in adolescents and young adults compared to population-based controls.

Study population The Women’s Health Study: From Adolescence to Adulthood (A2A) is an ongoing observational longitudinal cohort study, with 1549 participants (787 laparoscopically confirmed endometriosis cases and 762 control participants) enrolled from 2012 to 2018, as described in detail previously (Shafrir et al., 2022; Sasamoto et al., 2023). In brief, endometriosis cases were enrolled from two tertiary medical centers, Boston Children’s Hospital (BCH) and Brigham and Women’s Hospital (BWH). Control participants were females without a diagnosis of endometriosis, representing the underlying general population that gave rise to the endometriosis cases. The controls were identified from the surrounding communities served by the two tertiary medical centers through local clinics, local advertisements, online postings, and/or word of mouth. The participants completed an extensive baseline questionnaire and annual follow-up questionnaires, in which the baseline questionnaire is an expanded version of the standard clinical questionnaire of the World Endometriosis Research Foundation Endometriosis Phoneme and Biobanking Harmonization Project (WERF EPHect) (Vitonis et al., 2014). Surgical characteristics for endometriosis were captured using the WERF EPHect standard surgical form (Becker et al., 2014). Also described in detail previously (Shafrir et al., 2022), a subset of endometriosis cases answered a postoperative questionnaire assessed 5 weeks to 6 months after their endometriosis-related surgery (Supplementary Fig. S1A). Blood samples were collected for participants who consented to a blood draw at baseline and 5 weeks to 6 months after surgery, following a standard protocol (Rahmioglu et al., 2014) with one deviation to the protocol (i.e. blood were centrifuged at 1790 g for 10 min). When blood samples were collected, participants also completed a biospecimen form including information such as fasting status and hormonal medication use (Rahmioglu et al., 2014). Ethical approval This study was approved by the BCH Institutional Review Board on behalf of both BCH and BWH. All participants provided written informed consent, with participant assent and parental consent provided for girls <18 years. Study variables We collected the following covariates at baseline: age at blood draw (years), BMI (kg/m2), self-reported race (White, Black, Asian, Mixed race, and other race), self-reported ethnicity (Hispanic and non-Hispanic), smoking status (never, former, and current), fasting status at blood draw (no, yes), hormone use at blood draw (no, yes), and analgesic use at blood draw (no, yes). Among participants who were not using hormones at blood draw, menstrual cycle phase was determined using information of the participants’ last menstrual cycle date and their following menstrual cycle date (follicular phase, luteal phase, missing/unknown cycle phase). For participants 20 years or older, BMI was categorized based on World Health Organization criteria: underweight (BMI <18.5 kg/m2), normal (BMI 18.5–24.9 kg/m2), overweight (BMI 25–29.9 kg/m2), or obese (BMI ≥30 kg/m2) (WHO, 1995). For women younger than 20 years, the age- and gender-specific BMI Z-score is categorized as underweight (Z-score ≤ −2), normal (−2 < Z-score 2) (Barlow, 2007). Additional information was collected for endometriosis cases, including age at endometriosis diagnosis (years), age at symptom onset (years), revised American Society of Reproductive Medicine (rASRM) stage (I, II, III, IV), and endometriosis macrophenotype (superficial only, endometrioma, deep infiltrating, and both endometrioma and deep infiltrating) from an expanded WERF EPHect surgeon-completed surgical form (Becker et al., 2014). Detailed pelvic pain information was collected using the baseline questionnaire and postoperative questionnaire that was assessed in a subset of endometriosis cases (Shafrir et al., 2022; Sasamoto et al., 2023). At baseline, participants were asked to report the severity of both their usual menstrual pain and acyclic pain in the past 3 months using an 11-point numeric rating scale (NRS) (Ferreira-Valente et al., 2011; Birnie et al., 2019). On the postoperative questionnaire, they reported their pain during the last month using the same NRS. Change in pelvic pain from pre- to post-surgery was assessed by subtracting the higher severity rating for menstrual pain and acyclic pain at baseline from the postoperative pain severity rating. Change in severity of pain from pre- to postop was categorized into two groups as pain improved (severity score decreased by >2 points) or pain persisted or worsened (severity score decreased by 2 points or less or increased). Plasma metabolomics measurement All plasma metabolites were measured at the Broad Institute of the Massachusetts Institute of Technology and Harvard University (Cambridge, MA) in 2019 and 2023 using four complimentary liquid chromatography–tandem mass spectroscopy (LC-MS) methods, as previously described (Mascanfroni et al., 2015; Paynter et al., 2018; Sasamoto et al., 2022). The two plasma metabolomics datasets were processed using TraceFinder (Thermo Fisher Scientific). Briefly, metabolomic data were measured using complimentary LC-MS methods designed to measure polar metabolites and lipids as well as free fatty acids on four platforms, including polar and non-polar lipids (C8-pos), metabolites (C18-neg), cationic polar metabolites (HILIC-pos), and anionic polar metabolites (HILIC-neg). The metabolomics platforms measure metabolites with different features of ionization, polarity, and hydrophilicity. Hydrophilic interaction liquid chromatography (HILIC) analyses of water-soluble metabolites in the positive ionization mode (HILIC-pos) were conducted using an LC-MS system comprised of a Shimadzu Nexera X2 U-HPLC (Shimadzu Corp., Marlborough, MA) coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). HILIC analyses of water-soluble metabolites in the negative ionization mode (HILIC-neg) were conducted by using the Nexera X2-Q Exactive system for HRAM profiling and a UPLC (Waters) coupled to a QTRAP 5500 (SCIEX) for targeted profiling. Plasma lipids (C8-pos) were profiled using a Shimadzu Nexera X2 U-HPLC (Shimadzu Corp., Marlborough, MA). Metabolites of intermediate polarity (C18-neg), including free fatty acids and bile acids, were profiled using a Nexera X2 U-HPLC (Shimadzu Corp., Marlborough, MA) coupled to a Q Exactive (Thermo Fisher Scientific, Waltham, MA). Raw data from Orbitrap mass spectrometers were processed using TraceFinder 3.3 software (Thermo Fisher Scientific, Waltham, MA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK), and targeted data from the QTRAP 5500 system were processed using MultiQuant (version 2.1; SCIEX, Framingham, MA). For each method, metabolite identities were confirmed using authentic reference standards or reference samples. Among 692 known metabolites measured, we removed 65 duplicated metabolites that were measured in multiple platforms, 127 metabolites with coefficient of variation ≥25% in blinded quality control samples, and 2 metabolites with cross-sample missingness ≥20%. Additionally, because plasma samples from endometriosis cases and controls were measured in different batches, metabolite reproducibility was calculated as an intraclass correlation (ICC) or Spearman correlation ≥0.75 comparing 30 drift samples that were measured in both batches and removed 68 metabolites with low reproducibility (Tworoger et al., 2004). As a result, 430 metabolites were included in the analysis. Statistical analysis Among the 787 laparoscopically confirmed endometriosis cases enrolled in the study, 297 provided a blood sample collected within 90 days of surgery and completed a baseline questionnaire up to 90 days before or after surgery. Among these, we excluded cases who had no plasma (n = 3), had their baseline blood collected after surgery (n = 6), did not have superficial peritoneal lesions observed at surgery (n = 6), and those with missing data (n = 11). Due to other future analyses utilizing these samples, we further excluded those who did not complete the follow-up questionnaire 1 year after baseline (n = 81). Among the 762 controls, we excluded participants who did not provide a baseline blood sample (n = 193), did not complete the baseline questionnaire (n = 47), and those with missing data on the baseline questionnaire (n = 49). Among the 473 remaining, we included 73 who had previously been selected for a prior study (Sasamoto et al., 2022) and randomly selected an additional 47 for inclusion. The final sample included 190 laparoscopically confirmed endometriosis cases and 120 controls (Supplementary Fig. S1B). Both endometriosis cases and controls had plasma metabolites measured using blood collected at baseline. Among the endometriosis cases, 81 had both baseline (pre-surgical) and post-surgical blood metabolites measured, in which post-surgical blood were drawn 5 weeks to 6 months after the surgery (median 16.2 weeks). For metabolite values with missingness <20%, we assigned 50% of the minimum metabolite value for that metabolite, as we assumed these missingness were due to the metabolite values being below the detection limits (Sasamoto et al., 2022). We then transformed the continuous metabolite values into probit scores to reduce the impact of skewed distributions of metabolite values and to scale metabolite values on the same range. To identify individual plasma metabolites associated with endometriosis, we used linear regression adjusting for age at blood draw, BMI, fasting status, and hormone use at blood draw to calculate mean metabolite level differences and 95% CIs between endometriosis cases and controls. To examine whether menstrual cycle phase influenced the identified blood metabolite levels, we calculated the geometric means adjusted for age, BMI, and fasting status of the identified metabolites overall and by menstrual cycle phase among controls not on hormonal medication at blood draw. We also conducted a stratified analysis by hormone use at blood draw. We conducted a sensitivity analysis restricting to 65 controls with no self-reported acyclic or cyclic pelvic pain, as the self-reported pelvic pain may imply undiagnosed endometriosis. We further conducted stratified analyses on potential effect modifiers, including age at blood draw (<20-year-olds vs ≥20-year-olds), BMI (underweight/normal weight vs overweight/obese), and time between symptom onset and diagnosis (<3 years vs ≥3 years). Cochran’s Q test was used to test for heterogeneity by effect modifiers (Cochran, 1950). To examine changes in plasma metabolites from before to after endometriosis-related surgery, we conducted pairwise comparisons of pre- and post-surgical blood metabolites among 81 endometriosis cases using conditional logistic regression adjusting for fasting status and stratified by individual cases. We also compared metabolite levels in post-surgical blood and controls using linear regression adjusting for age at blood draw, BMI, fasting status, and hormone use at blood draw. We further conducted secondary analysis stratified by the timing of post-surgical blood collection (i.e., 5–8 weeks, 9–16 weeks, 17–24 weeks, 25+ weeks) and change in pelvic pain from pre- to post-surgery (i.e., improved, persistent/worsening pain). Metabolite set enrichment analysis (MSEA) (Xia and Wishart, 2016) was used to identify metabolite classes that were enriched. Number of effective tests (NEF) and false discovery rate (FDR) were used for multiple testing corrections in individual metabolite analysis and MSEA, respectively. The analysis was conducted in R version 4.4.0.

The baseline characteristics of 190 endometriosis cases and 120 controls are summarized in Table 1. The median age at baseline was 19 years overall; endometriosis cases had slightly younger age at baseline, with median age of 17 years compared to controls with median age of 22 years. The majority of the participants were White (90.6%) and normal weight (66.5%) with similar proportions between endometriosis cases and controls. Compared to controls, endometriosis cases had higher proportions of those on hormone use at blood draw (92% vs 66%) and higher proportions with cyclic (93% vs 30%) or acyclic (64% vs 14%) pelvic pain, while similar proportions of analgesic use at blood draw (27% vs 26%). In terms of endometriosis characteristics, the dominant clinical characteristics were rASRM stage I (83%) and superficial peritoneal lesions only (99%). Table 1. | Endometriosis cases | Controls | | |---|---|---| | (N = 190) | (N = 120) | | | Age at blood draw, years | || | Median [25th, 75th quartiles] | 17.0 [16.0, 20.8] | 22.0 [19.0, 24.0] | | Race, n (%) | || | White | 172 (90.5%) | 98 (81.7%) | | Black | 4 (2.1%) | 6 (5.0%) | | More than one race | 9 (4.7%) | 3 (2.5%) | | Other/unknown | 5 (2.6%) | 13 (10.8%) | | Ethnicity, n (%) | || | Non-Hispanic | 178 (93.7%) | 111 (92.5%) | | Hispanic | 12 (6.3%) | 9 (7.5%) | | BMI, n (%) 1 | || | Underweight | 0 (0%) | 1 (0.8%) | | Normal | 123 (64.7%) | 83 (69.2%) | | Overweight | 51 (26.8%) | 20 (16.7%) | | Obese | 16 (8.4%) | 16 (13.3%) | | Fasting status at blood draw, n (%) | || | No | 127 (74.3%) | 106 (93.0%) | | Yes | 44 (25.7%) | 8 (7.0%) | | Hormone use at blood draw, n (%) | || | No | 16 (8.4%) | 40 (34.2%) | | Yes | 174 (91.6%) | 77 (65.8%) | | Acyclic pelvic pain, n (%) 2 | || | None | 67 (36.0%) | 103 (85.8%) | | Mild | 5 (2.7%) | 6 (5.0%) | | Moderate | 19 (10.2%) | 7 (5.8%) | | Severe | 95 (51.1%) | 4 (3.3%) | | Cyclic pelvic pain, n (%) 3 | || | None/mild | 8 (6.3%) | 75 (70.1%) | | Moderate | 41 (32.0%) | 26 (24.3%) | | Severe | 79 (61.7%) | 6 (5.6%) | | Age at endometriosis diagnosis, years | || | Median [25th, 75th quartiles] | 16.0 [15.0, 18.0] | NA | | rASRM stage, n (%) | || | Stage I | 156 (82.5%) | NA | | Stage II | 28 (14.8%) | NA | | Stage III | 0 (0%) | NA | | Stage IV | 5 (2.6%) | NA | | Endometriosis macrophenotype, n (%) | || | Superficial peritoneal lesions only | 188 (98.9%) | NA | | Endometrioma | 0 (0%) | NA | | Deep infiltrating | 2 (1.1%) | NA | rASRM, revised American Society of Reproductive Medicine. Number of missing characteristics: race (n = 12 controls), fasting status (n = 19 cases, n = 6 controls), hormone use at blood draw (n = 3 controls), acyclic pelvic pain (n = 4 cases), cyclic pelvic pain (n = 62 cases, n = 13 controls), rASRM stage (n = 1 case). The majority (72/75) of subjects with missing cyclic pelvic pain information because they were on hormone therapy so that they did not experience menstrual cycles. BMI is calculated as weight (kilograms) divided by the square of height (meters); For women 20 years or older, BMI was categorized based on World Health Organization criteria: underweight (BMI 30 kg/m2). For women younger than 20 years, the age- and gender-specific BMI Z-score is categorized as underweight (Z-score ≤ −2), normal (−2 < Z-score 2). Severity of general pelvic pain in the last three months among those who reported acyclic pelvic pain in the last 3 months. Usual severity of pelvic pain with periods among those who reported cycling in the last 3 months. A total of 63 individual metabolites were associated with endometriosis (NEF <0.05) (Fig. 1 and Supplementary Table S1). Compared to controls, endometriosis cases showed higher plasma levels of individual metabolites suggesting activation of proinflammatory response [i.e., eicosatrienoic acid (β = 0.61, 95% CI = 0.37, 0.86), oleic acid (β = 0.60, 95% CI = 0.35, 0.84), palmitoleic acid (β = 0.50, 95% CI = 0.26, 0.74), and alpha-linolenic acid (β = 0.46, 95% CI = 0.21, 0.70)], and increase in oxidative stress and generation of reactive oxygen species (ROS) [i.e., xanthine (β = 0.64, 95% CI = 0.39, 0.88), palmitic acid (β = 0.45, 95% CI = 0.22, 0.69), and ceramide 18:1; O2/18:1 (β = 1.10, 95% CI = 0.87, 1.34)]. Conversely, endometriosis cases had lower plasma levels of individual metabolites compared to controls, which indicated reduction of anti-inflammatory response [i.e., tryptophan (β = −0.47, 95% CI = −0.74, −0.20), alpha-tocopherol (β = −0.45, 95% CI = −0.70, −0.19), and phenylalanine (β = −0.57, 95% CI = −0.84, −0.31)], decrease of aerobic glycolysis [i.e. aconitate (β = −0.77, 95% CI = −0.93, −0.60) and citrate/isocitrate (β = −0.74, 95% CI = −0.91, −0.57)], and decrease of neuro-biochemical alterations [i.e. serotonin (β = −0.53, 95% CI = −0.80, −0.27)]. Additionally, endometriosis cases had statistically significantly lower bile acid metabolite levels [i.e. taurochenodeoxycholic acid (TCDCA) (β = −0.77, 95% CI = −1.0, −0.53), glycocholic acid (GCA) (β = −0.80, 95% CI = −1.04, −0.56), and tauroursodeoxycholic acid (TUDCA) (β = −0.52, 95% CI = −0.76, −0.28)] compared to controls. MSEA revealed enrichment of several metabolite classes associated with endometriosis (Fig. 2; Supplemental Table S2). Fatty acyls (normalized enrichment score (NES) = 2.3, FDR = 2.3e−4) and ceramides (NES = 1.9, FDR = 6.0e−3) were positively associated with endometriosis, while steroids and steroid derivatives (NES = −2.1, FDR = 1.3e−4) were negatively associated with endometriosis. Similar results were observed in a sensitivity analysis where we restricted controls to those who did not self-report pelvic pain (Supplementary Fig. S2; Supplementary Table S3). We observed similar direction of association for the 63 metabolites when stratified by hormone use at blood draw and also confirmed that these 63 metabolites were not associated with pelvic pain among controls. We also investigated whether menstrual cycle phase influenced the 63 individual metabolite levels by examining geometric means of these metabolites overall and by menstrual cycle phase among controls not on hormonal medication at blood collection and generally observed similar results, although the sample size was limited (n = 28 controls with menstrual cycle phase information). Furthermore, stratified analyses showed no significant heterogeneity by age at blood draw, BMI, and time between age at symptom onset and age at endometriosis diagnosis (Supplementary Fig. S3; Supplemental Table S4). However, while not statistically significant, stratified analysis by time between age at symptom onset and age at endometriosis diagnosis suggested a stronger magnitude of association for fatty acyls and steroids and steroid derivatives among endometriosis cases who experienced a longer diagnostic delay (≥3 years) compared to shorter diagnostic delay (<3 years). We then examined the changes in plasma metabolite profiles before and after surgery by comparing post-surgical and pre-surgical blood metabolite levels in 81 paired blood samples. We identified 55 individual metabolites that statistically significantly changed from before to after endometriosis-related surgery (NEF <0.05; Supplementary Table S5), in which 28 were associated with endometriosis at baseline compared to controls. MSEA presented multiple plasma metabolite classes that changed from pre- to post-surgery. Compared to levels in pre-surgical blood, metabolite levels of likely saturated triglycerides (number of double bounds ≤3) (NES = 2.1, FDR = 2.0e−4), steroids and steroid derivatives (NES = 2.0, FDR = 8.1e−4) significantly increased after surgery, while fatty acyls (NES = −2.5, FDR = 1.2e−6) and cholesteryl esters (NES = −1.9, FDR = 1.6e−2) significantly decreased after surgery (Fig. 3). Interestingly, while fatty acyls and steroids and steroid derivatives were elevated in endometriosis cases compared to controls at baseline, and their levels significantly declined after surgery, there were no statistically significant changes in ceramides when comparing pre- to post-surgery despite ceramides being elevated in endometriosis cases compared to controls at baseline. To further verify this observation, we examined the differences in plasma metabolites between post-surgical blood and controls (Supplementary Fig. S4). Compared to controls, ceramides (NES = 1.8, FDR = 3.9e−3) were still significantly higher after surgery, while fatty acyls (NES = −1.8, FDR = 3.1e−2) were significantly lower after surgery, and there were no statistically significant differences in levels of steroids and steroid derivatives. Furthermore, to examine if the change in plasma metabolites from pre- to post-surgery differed by the post-surgical pelvic pain outcomes, we stratified by change in pelvic pain severity between pre- and post-surgery (Fig. 3). To examine if the change in plasma metabolites from pre- to post-surgery differed by the timing of the post-surgical blood collection, we stratified by the timing of post-surgical blood collection (Supplementary Fig. S5). In general, we observed similar direction of associations in each of the post-surgical time periods compared to the overall differences in metabolite profiles between pre- and post-surgery (left panel). Interestingly, the largest difference in plasma metabolite profiles between pre- and post-surgical blood was observed at 8–24 weeks after surgery. No individual metabolites showed statistically significant change from pre- to post-surgery in 35 endometriosis cases with improved pelvic pain after surgery, while 36 individual metabolites showed significant change from pre- to post-surgery in 46 endometriosis cases with persistent or worsening pelvic pain after surgery. Similar enrichment patterns of changes in plasma metabolite classes from pre- to post-surgery were observed in the stratified analysis as in the overall analysis, including steroids and steroid derivatives, likely saturated triglycerides (number of double bounds ≤3), and fatty acyls. Interestingly, there was a significant increase in lysophosphatidylcholine (LPC) (NES = 1.6, FDR = 4.0e−2) from pre- to post-surgery among endometriosis cases with persistent or worsening pelvic pain after surgery but not among those with improved pelvic pain.

We identified 63 plasma metabolites that were significantly dysregulated in adolescents and young adults with endometriosis compared to those without, with higher levels of fatty acyls and ceramides and lower levels of steroids and steroid derivatives being significantly associated with endometriosis compared to controls. Furthermore, our data suggest that endometriosis-related surgery may reshape some of the blood metabolomics landscape so the plasma metabolomic profiles for several classes become more similar to those without, although ceramides remained elevated despite surgical treatment. We identified a systematic proinflammatory environment in participants with endometriosis compared to those without, which is consistent with previous research on endometriosis among older adults (Zondervan et al., 2018). Our results showed elevation of proinflammatory eicosatrienoic acid in endometriosis cases compared to controls, which is known to induce inflammatory immune response through a lipid signaling complex and leads to redness, pain, swelling, and sensation of heat at the site of inflammation (Lone and Taskén, 2013). We also observed lower levels of anti-inflammatory metabolites of tryptophan, indolelactic acid, phenylalanine, medium-chain fatty acid, hydrocinnamic acid, caffeine, and trigonelline in endometriosis cases compared to controls. Tryptophan and its metabolite indolelactic acid (Kim et al., 2022; Zhou et al., 2022) have been associated with antioxidant activity, immune regulation, and inflammation reduction (Suzuki et al., 2013; Huang et al., 2021; Laursen et al., 2021). One study reported phenylalanine inhibits the production of IL1-β in proinflammatory macrophages (Zhang et al., 2023). Furthermore, hydrocinnamic acid and medium-chain fatty acids (i.e. caprylic acid and capric acid) are related to suppression of the NF-kB signaling pathway and reduction of proinflammatory adipokine expression (Sprong et al., 2001; Huang et al., 2014; Alam et al., 2016; Roopashree et al., 2021). Last, caffeine and its bioactive marker trigonelline can inhibit phosphodiesterase, which would lead to higher cyclic adenosine monophosphate levels suppressing inflammatory pathways (Eichwald et al., 2023). Our results were in line with prior studies reporting reduction of aerobic glycolysis and increase in oxidative stress in endometriosis diagnosed in adulthood compared to those without (Fong et al., 2011; Dutta et al., 2012). We observed reduction of plasma metabolites involved in the tricarboxylic acid cycle (TCA), indicating reduction of aerobic glycolysis in the endometriosis cases compared to controls, in which hexose, aconitate, and citrate/isocitrate are direct substrates of TCA. Alterations of metabolic signature in endometriosis are also supported on the endometriotic lesion level (Marečková et al., 2024; Sarsenova et al., 2024). A study comparing transcriptomics profiles of paired ectopic and eutopic endometrium at a single-cell level reported over- and under-expression of genes associated with glycolysis metabolism. Our results captured metabolite signals related to the downregulation of glycolysis metabolism, which could be explained by the observed downregulation of glycolysis activators, such as PFKFB3 and PFKFB4. Interestingly, we did not observe elevation of plasma lactate levels, an indicator of increased anaerobic glycolysis that has also been previously associated with endometriosis diagnosed in adulthood (Dutta et al., 2012; Dutta et al., 2018). One possible explanation is that the elevation of anaerobic glycolysis may be more likely to occur systemically from within deep lesions due to a poorly oxygenated environment (Dutta et al., 2012), and since our study population consisted primarily of young patients with superficial peritoneal lesions only, systemic elevation of lactose levels may not have been observed. In addition, previous studies revealed oxidative stress being related to endometriosis with elevated advanced oxidation protein products and reduced levels of antioxidants such as catalase and glutathione (Jana et al., 2013). Our study concurred with these previous findings, although there was no significant difference in urine 8-hydroxy-2′-deoxyguanosine (8-OHdG) between endometriosis cases and controls in the A2A cohort (Shafrir et al., 2024). It is possible this discrepancy may be due to 8-OHdG measuring different aspects of oxidative stress pathways, as 8-OHdG is a biomarker of DNA oxidative stress and does not reflect lipid or protein oxidation. Interestingly, genes related to oxidative metabolism were reported upregulated in ectopic endometrium (Sarsenova et al., 2024). In particular, upregulated genes of ACADL, CPT1A, and CYP2U1 indicate the dysregulation of fatty acid-associated metabolism may provide biological explanation for the upregulated plasma fatty acid levels observed in our endometriosis patients compared to controls. We observed lower levels of methionine sulfoxide and alpha-tocopherol in individuals with endometriosis. These metabolites are both antioxidants, and therefore lower levels of methionine sulfoxide and alpha-tocopherol suggest an increase in oxidative stress in endometriosis compared to controls (Yao et al., 1994; Lee and Gladyshev, 2011). On the contrary, we observed higher levels of plasma xanthine, palmitic acid, and ceramides in endometriosis cases compared to controls, suggesting elevation in oxidative stress. Oxidative stress converts sphingomyelins to ceramides, leading to higher ceramide levels and lower sphinganine levels observed in endometriosis patients (Kitatani et al., 2008; Corre et al., 2010). Similarly, oxidative stress dysregulates methionine sulfoxide biosynthesis, resulting in lower methionine sulfoxide levels in endometriosis cases (Lee and Gladyshev, 2011). We identified nine bile acids that had lower levels in endometriosis cases compared to controls. The association of bile acids and endometriosis has not been well studied, but the potential mechanism has been hypothesized that bile acids, as indicators of liver function, are associated with liver-metabolized estrogen and gut microbiota function, which further contribute to endometriosis development (Liu et al., 2018; Li et al., 2022). Bile acids are also associated with inflammation and pro-apoptotic activities (Chen et al., 2019; Režen et al., 2022). Among endometriosis-associated bile acids identified in our study, glycochenodeoxycholic acid (GCDCA) triggers hepatocyte apoptosis via activating S1PR2 (Karimian et al., 2013), which is highly expressed in macrophages involved in multiple immunological pathways (Hughes et al., 2008; Grigorova et al., 2009). Furthermore, tauroursodeoxycholic acid (TUDCA) enhances CPT-11- and DLC1-induced apoptosis (Lim et al., 2010; Chung et al., 2011). Conversely, taurodeoxycholic acid (TDCA) is anti-apoptotic via upregulating NOX5-S expression (Hong et al., 2010; Liu et al., 2018) and is associated with oxidative stress through TGR5 (Hong et al., 2010). Thus, our results observing lower levels of these bile acid metabolites in endometriosis suggest reduction in apoptosis, which is consistent with prior literature reporting alterations in regulation of apoptosis in eutopic and ectopic endometrium of older adult women with endometriosis (Harada et al., 2004). This is also supported by our previous work reporting downregulation of apoptosis pathway in endometriosis cases compared to controls, evident through plasma proteomics markers (Sasamoto et al., 2022). We observed decreases in plasma serotonin levels in endometriosis cases compared to controls. Serotonin is involved in the central and peripheral regulation of the nociceptive signal and alteration (Millan, 1994; Millan, 1995; Rosenthal et al., 1998), where the association of low levels of serotonin and depression has been widely reported (Coppen, 1967; Paredes et al., 2019). The reduction of serotonin observed in endometriosis cases provides a potential metabolomics explanation of high proportion of depression among endometriosis patients reported in the previous study (Estes et al., 2021; Hu et al., 2023). Interestingly, we observed elevations of several fatty acids in endometriosis cases compared to controls, including monounsaturated fatty acids [i.e. oleic acid, palmitoleic acid, 10Z-heptadecenoic acid and nonadeca-10Z-enoic acid], polyunsaturated fatty acid [i.e. alpha-linolenic acid, 4,7,10,13,16-docosapentaenoic acid, gamma-linolenic acid (GLA), linoleic acid, myristoleic acid, and arachidonic acid], and odd-chain saturated fatty acid (OCFA) [i.e. pentadecylic acid]. Higher circulating free fatty acid levels can impact metabolism and induce insulin resistance. One study reported higher serum levels of oleic acid, linoleic acids, eicosatrienoic acid, and arachidonic acids were observed in patients with nonalcoholic fatty liver disease compared to healthy controls (Gambino et al., 2016). In the prospective cohort study of the Nurse’s Health Study II, greater intake of trans-saturated fat was associated with increased endometriosis risk (RRquintile 5vs1 = 1.48, 95% CI = 1.17, 1.88) (Missmer et al., 2010). Palmitoleic acid has been associated with increased risk of dyslipidemia and insulin resistance (Warensjö et al., 2006; Paillard et al., 2008; Mozaffarian et al., 2010). Our results observing elevated levels of these plasma fatty acids in adolescents and young adults with endometriosis may suggest early metabolism dysregulation in endometriosis. Furthermore, recent publications provided evidence of fatty acid metabolism dysregulation in endometriotic lesions. A single-cell RNAseq study comparing ectopic to eutopic endometriotic lesions observed a variety of genes that were associated with fatty acid biosynthetic metabolism that were up- or downregulated in multiple cell types, such as OLAH and ACSBG1 in perivascular cells and ASCL5 in endothelial cells (Sarsenova et al., 2024). Taken together, the elevated fatty acid metabolomic levels observed in our study may be due to the abnormal cellular metabolism reflected by the dysregulation of biosynthetic-related genes under the inflammatory environment. Our results comparing plasma metabolomics profiles before and after surgery among endometriosis cases implied that endometriosis-related surgery removing endometriotic lesions may have a positive effect—at least in the short term—on the endometriosis-associated systemic metabolic dysregulation, with reduction of metabolites associated with inflammation and oxidative stress and increase of bile acid metabolites. Furthermore, when stratified by post-surgical pelvic pain outcome, LPC was enriched in endometriosis cases suffering from persistent or worsening pelvic pain after surgery. LPC has been related to chronic pain, with 18:0 LPC being involved in the development of persistent pain by activating the transient receptor potential ion channels via modifying the lateral pressure in the cytoplasmic membrane (Friston et al., 2023). Our study investigating the plasma metabolomics in association with endometriosis in adolescents and young adults exhibited plasma metabolomic dysregulation in this young population with elevation in circulating fatty acyls and ceramides. Furthermore, exploration of change in plasma metabolites before and after endometriosis-related surgery provided novel biological insight into the effect of surgical treatment on endometriosis-associated systemic metabolic dysregulation. Interestingly, while two of the metabolite classes that were significantly dysregulated in endometriosis compared to controls (i.e. fatty acyls and steroids and steroid derivatives) showed significant change from pre- to post-surgery among endometriosis cases, ceramides did not show significant change after surgery, suggesting that ceramides may be persistently elevated in those diagnosed with endometriosis despite surgical treatment. This is interesting because epidemiological data support that those with endometriosis are at greater risk of chronic diseases (Missmer, 2009; Kvaskoff et al., 2015), such as cardiovascular disease (Mu et al., 2016; Mu et al., 2017), autoimmune conditions (Harris et al., 2016; Shigesi et al., 2019), and ovarian cancer (Pearce et al., 2012; Kvaskoff et al., 2021), and ceramides have been associated with increased risk of these chronic disease outcomes (Ichi et al., 2006; De Mello et al., 2009; Haus et al., 2009; Spijkers et al., 2011; Wang et al., 2017; Zeleznik et al., 2020). While further research is necessary, our study suggests that there may be potential persistent systemic changes in those diagnosed with endometriosis that may link the association between endometriosis and development of chronic diseases decades later. Strengths of this study include the utilization of state-of-the-art metabolomics technology with high reproducibility to comprehensively investigate the metabolites that were associated with endometriosis. Secondly, our unique study population consists of adolescents and young adults who represent a timeframe close to disease onset. Third, we were able to examine change in plasma metabolite levels using paired blood samples collected at both pre- and post-surgery from endometriosis cases. However, our study population mainly consists of self-reported non-Hispanic, white individuals and endometriosis cases with superficial peritoneal lesions only, so the generalizability may be limited. Furthermore, despite our study having the largest study population addressing plasma metabolomic profiles in adolescents and young adults with endometriosis, sample size was limited to conduct detailed stratified analyses, especially by post-surgical pelvic pain outcomes. It is possible that menstrual cycle phase at blood draw may influence blood metabolite levels (Urbano-Márquez et al., 1991). The majority of our study participants were on hormonal medication at blood collection, and therefore we were not able to account for menstrual cycle phase in our analyses. However, when we calculated the geometric means of 63 identified endometriosis-associated metabolites by menstrual cycle phase among controls, the metabolite values were generally similar compared to all controls, suggesting minimal impact of menstrual cycle phase on these blood metabolite values.

This study presents the dysregulation of plasma metabolomic profiles in adolescents and young adults with endometriosis, revealing increase in fatty acyls and ceramides in endometriosis cases compared to controls. Furthermore, our results suggest a positive impact of endometriosis-related surgery for some but not all systemic metabolic dysregulation observed in young patients with endometriosis, which warrants further investigation of whether and how persistent systemic changes despite treatment may lead to long-term chronic disease risk among those diagnosed with endometriosis. Supplementary Material

The authors would like to thank all the participants of the Women’s Health Study: From Adolescence to Adulthood for their valuable contributions and the staff of the Boston Center for Endometriosis. Contributor Information Nan Lin, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA. Oana A Zeleznik, Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA. Allison F Vitonis, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; Boston Center for Endometriosis, Boston Children’s Hospital and Brigham and Women’s Hospital, Boston, MA, USA. Ashley Laliberte, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; Boston Center for Endometriosis, Boston Children’s Hospital and Brigham and Women’s Hospital, Boston, MA, USA. Amy L Shafrir, Boston Center for Endometriosis, Boston Children’s Hospital and Brigham and Women’s Hospital, Boston, MA, USA; Department of Health Sciences and Nutrition, School of Nursing and Health Sciences, Merrimack College, North Andover, MA, USA; Division of Adolescent and Young Adult Medicine, Department of Pediatrics, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA. Julian Avila-Pacheco, Broad Institute of MIT and Harvard, Boston, MA, USA. Clary Clish, Broad Institute of MIT and Harvard, Boston, MA, USA. Kathryn L Terry, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; Boston Center for Endometriosis, Boston Children’s Hospital and Brigham and Women’s Hospital, Boston, MA, USA; Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Stacey A Missmer, Boston Center for Endometriosis, Boston Children’s Hospital and Brigham and Women’s Hospital, Boston, MA, USA; Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA; Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids, MI, USA. Naoko Sasamoto, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; Boston Center for Endometriosis, Boston Children’s Hospital and Brigham and Women’s Hospital, Boston, MA, USA. Data availability Data are not publicly available due to information that could compromise research participants’ privacy and consent. However, experienced scientists who would like to inquire regarding use of data from this study to address specific hypotheses or replicate the analyses in this study may submit an application and research proposal. Data requests must be reviewed and approved by the BWH Institutional Review Board (https://www.brighamandwomens.org/research/research-administration). All inquiries should be directed to the A2A cohort leadership committee ([email protected]). Data sharing will require a fully executed Data Usage Agreement. Authors’ roles N.L.: writing—review and editing, writing—original draft, and formal analysis; O.A.Z.: writing—review and editing; A.F.V.: writing—review and editing and data curation; A.L.: writing—review and editing; A.L.S.: writing—review and editing and funding acquisition; J.A.-P.: writing—review and editing; C.C.: writing—review and editing; K.L.T.: writing—review and editing and funding acquisition; S.A.M.: writing—review an editing and funding acquisition; N.S.: writing—review and editing, validation, supervision, and funding acquisition. Funding Financial support for establishment of and data collection within the A2A cohort was provided by the J. Willard and Alice S. Marriott Foundation, and support for assay costs was in part provided by the Peery family. This project was funded by Eunice Kennedy Shriver National Institute of Child Health and Human Development R21HD107266. S.A.M., A.L.S., and K.L.T. were supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development R01HD094842. Conflict of interest S.A.M. received grant funding from AbbVie, National Institutes of Health, Department of Defense, and Marriott Family Foundation; received honoraria from WERF, Huilun Shanghai, and University of Kansas Medical Center; travel support from SRI, ESHRE, FWGBD, University of Michigan, MIT, ASRM, LIDEA Registry, Taiwan Endometriosis Society, SEUD, Japan Endometriosis Society, NASEM, Endometriosis Foundation of America, Gedeon Richter Symposium at ESHRE; Board member receiving financial remuneration from AbbVie, Roche, LIDEA Registry, Editor of Frontiers in Reproductive Health, Roundtable participation for Abbott; Board member without financial remuneration from NextGen Jane and Statistical Advisory Board member of Human Reproduction; leadership role in Society for Women’s Health Research, World Endometriosis Society, World Endometriosis Research Foundation, ASRM, ESHRE. N.S. and K.L.T. receive grant funding from Aspira Women’s Health unrelated to this project. The remaining authors have no disclosures relevant to this manuscript.

- Adamson GD, Kennedy S, Hummelshoj L. Creating solutions in endometriosis: global collaboration through the World Endometriosis Research Foundation. J Endometr 2010;2:3–6. [Google Scholar] - Alam MA, Subhan N, Hossain H, Hossain M, Reza HM, Rahman MM, Ullah MO. Hydroxycinnamic acid derivatives: a potential class of natural compounds for the management of lipid metabolism and obesity. Nutr Metab 2016;13:27–13. [DOI] [PMC free article] [PubMed] [Google Scholar] - Barlow SE; Expert Committee. Expert committee recommendations regarding the prevention, assessment, and treatment of child and adolescent overweight and obesity: summary report. Pediatrics 2007;120 Suppl 4:S164–S192. [DOI] [PubMed] [Google Scholar] - Becker CM, Laufer MR, Stratton P, Hummelshoj L, Missmer SA, Zondervan KT, Adamson GD; WERF EPHect Working Group. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project: I. Surgical phenotype data collection in endometriosis research. Fertil Steril 2014;102:1213–1222. [DOI] [PMC free article] [PubMed] [Google Scholar] - Birnie KA, Hundert AS, Lalloo C, Nguyen C, Stinson JN. Recommendations for selection of self-report pain intensity measures in children and adolescents: a systematic review and quality assessment of measurement properties. Pain 2019;160:5–18. [DOI] [PubMed] [Google Scholar] - Chen ML, Takeda K, Sundrud MS. Emerging roles of bile acids in mucosal immunity and inflammation. Mucosal Immunol 2019;12:851–861. [DOI] [PubMed] [Google Scholar] - Chung GE, Yoon J-H, Lee J-H, Kim HY, Myung SJ, Yu SJ, Lee S-H, Lee S-M, Kim YJ, Lee H-S. Ursodeoxycholic acid-induced inhibition of DLC1 protein degradation leads to suppression of hepatocellular carcinoma cell growth. Oncol Rep 2011;25:1739–1746. [DOI] [PubMed] [Google Scholar] - Cochran WG. The comparison of percentages in matched samples. Biometrika 1950;37:256–266. [PubMed] [Google Scholar] - Coppen A. The biochemistry of affective disorders. Br J Psychiatry 1967;113:1237–1264. [DOI] [PubMed] [Google Scholar] - Corre I, Niaudet C, Paris F. Plasma membrane signaling induced by ionizing radiation. Mutat Res 2010;704:61–67. [DOI] [PubMed] [Google Scholar] - De Mello V, Lankinen M, Schwab U, Kolehmainen M, Lehto S, Seppänen-Laakso T, Orešič M, Pulkkinen L, Uusitupa M, Erkkilä A. Link between plasma ceramides, inflammation and insulin resistance: association with serum IL-6 concentration in patients with coronary heart disease. Diabetologia 2009;52:2612–2615. [DOI] [PubMed] [Google Scholar] - DiVasta AD, Vitonis AF, Laufer MR, Missmer SA. Spectrum of symptoms in women diagnosed with endometriosis during adolescence vs adulthood. Am J Obstet Gynecol 2018;218:324. e321–324. e311. [DOI] [PubMed] [Google Scholar] - Dutta M, Joshi M, Srivastava S, Lodh I, Chakravarty B, Chaudhury K. A metabonomics approach as a means for identification of potential biomarkers for early diagnosis of endometriosis. Mol Biosyst 2012;8:3281–3287. [DOI] [PubMed] [Google Scholar] - Dutta M, Singh B, Joshi M, Das D, Subramani E, Maan M, Jana SK, Sharma U, Das S, Dasgupta S et al. Metabolomics reveals perturbations in endometrium and serum of minimal and mild endometriosis. Sci Rep 2018;8:6466. [DOI] [PMC free article] [PubMed] [Google Scholar] - Eichwald T, Solano AF, Souza J, de Miranda TB, Carvalho LB, Dos Santos Sanna PL, da Silva RAF, Latini A. Anti-inflammatory effect of caffeine on muscle under lipopolysaccharide-induced inflammation. Antioxidants (Basel) 2023;12:1–15. [DOI] [PMC free article] [PubMed] [Google Scholar] - Estes SJ, Huisingh CE, Chiuve SE, Petruski-Ivleva N, Missmer SA. Depression, anxiety, and self-directed violence in women with endometriosis: a retrospective matched-cohort study. Am J Epidemiol 2021;190:843–852. [DOI] [PMC free article] [PubMed] [Google Scholar] - Ferreira-Valente MA, Pais-Ribeiro JL, Jensen MP. Validity of four pain intensity rating scales. Pain 2011;152:2399–2404. [DOI] [PubMed] [Google Scholar] - Fong MY, McDunn J, Kakar SS. Identification of metabolites in the normal ovary and their transformation in primary and metastatic ovarian cancer. PLoS One 2011;6:e19963. [DOI] [PMC free article] [PubMed] [Google Scholar] - Friston DA, Cuddihy J, Souza Luiz J, Truong AH, Ho L, Basra M, Santha P, Oszlacs O, de Sousa Valente J, Marczylo T, Junttila S. Elevated 18:0 lysophosphatidylcholine contributes to the development of pain in tissue injury. Pain 2023, 164:e103.e115. [DOI] [PMC free article] [PubMed] [Google Scholar] - Gallagher CS, Mäkinen N, Harris HR, Rahmioglu N, Uimari O, Cook JP, Shigesi N, Ferreira T, Velez-Edwards DR, Edwards TL, et al. ; 23andMe Research Team. Genome-wide association and epidemiological analyses reveal common genetic origins between uterine leiomyomata and endometriosis. Nat Commun 2019;10:4857. [DOI] [PMC free article] [PubMed] [Google Scholar] - Gambino R, Bugianesi E, Rosso C, Mezzabotta L, Pinach S, Alemanno N, Saba F, Cassader M. Different serum free fatty acid profiles in NAFLD subjects and healthy controls after oral fat load. Int J Mol Sci 2016;17:479. [DOI] [PMC free article] [PubMed] [Google Scholar] - Gerszten RE, Wang TJ. The search for new cardiovascular biomarkers. Nature 2008;451:949–952. [DOI] [PubMed] [Google Scholar] - Grigorova IL, Schwab SR, Phan TG, Pham TH, Okada T, Cyster JG. Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells. Nat Immunol 2009;10:58–65. [DOI] [PMC free article] [PubMed] [Google Scholar] - Harada T, Kaponis A, Iwabe T, Taniguchi F, Makrydimas G, Sofikitis N, Paschopoulos M, Paraskevaidis E, Terakawa N. Apoptosis in human endometrium and endometriosis. Hum Reprod Update 2004;10:29–38. [DOI] [PubMed] [Google Scholar] - Harris HR, Costenbader KH, Mu F, Kvaskoff M, Malspeis S, Karlson EW, Missmer SA. Endometriosis and the risks of systemic lupus erythematosus and rheumatoid arthritis in the Nurses’ Health Study II. Ann Rheum Dis 2016;75:1279–1284. [DOI] [PMC free article] [PubMed] [Google Scholar] - Haus JM, Kashyap SR, Kasumov T, Zhang R, Kelly KR, DeFronzo RA, Kirwan JP. Plasma ceramides are elevated in obese subjects with type 2 diabetes and correlate with the severity of insulin resistance. Diabetes 2009;58:337–343. [DOI] [PMC free article] [PubMed] [Google Scholar] - Hong J, Behar J, Wands J, Resnick M, Wang LJ, DeLellis RA, Lambeth D, Souza RF, Spechler SJ, Cao W. Role of a novel bile acid receptor TGR5 in the development of oesophageal adenocarcinoma. Gut 2010;59:170–180. [DOI] [PMC free article] [PubMed] [Google Scholar] - Hu P-W, Zhang X-L, Yan X-T, Qi C, Jiang G-J. Association between depression and endometriosis using data from NHANES 2005-2006. Sci Rep 2023;13:18708–18708. [DOI] [PMC free article] [PubMed] [Google Scholar] - Huang W-C, Tsai T-H, Chuang L-T, Li Y-Y, Zouboulis CC, Tsai P-J. Anti-bacterial and anti-inflammatory properties of capric acid against Propionibacterium acnes: a comparative study with lauric acid. J Dermatol Sci 2014;73:232–240. [DOI] [PubMed] [Google Scholar] - Huang W, Cho KY, Meng D, Walker WA. The impact of indole-3-lactic acid on immature intestinal innate immunity and development: a transcriptomic analysis. Sci Rep 2021;11:8088. [DOI] [PMC free article] [PubMed] [Google Scholar] - Hughes JE, Srinivasan S, Lynch KR, Proia RL, Ferdek P, Hedrick CC. Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages. Circ Res 2008;102:950–958. [DOI] [PMC free article] [PubMed] [Google Scholar] - Ichi I, Nakahara K, Miyashita Y, Hidaka A, Kutsukake S, Inoue K, Maruyama T, Miwa Y, Harada-Shiba M, Tsushima M, et al. ; Kisei Cohort Study Group. Association of ceramides in human plasma with risk factors of atherosclerosis. Lipids 2006;41:859–863. [DOI] [PubMed] [Google Scholar] - Jana SK, Dutta M, Joshi M, Srivastava S, Chakravarty B, Chaudhury K. 1H NMR based targeted metabolite profiling for understanding the complex relationship connecting oxidative stress with endometriosis. Biomed Res Int 2013;2013:329058. [DOI] [PMC free article] [PubMed] [Google Scholar] - Karimian G, Buist-Homan M, Schmidt M, Tietge UJF, de Boer JF, Klappe K, Kok JW, Combettes L, Tordjmann T, Faber KN et al. Sphingosine kinase-1 inhibition protects primary rat hepatocytes against bile salt-induced apoptosis. Biochim Biophys Acta 2013;1832:1922–1929. [DOI] [PubMed] [Google Scholar] - Kim K, Kim H, Sung GY. Effects of indole-3-lactic acid, a metabolite of tryptophan, on IL-4 and IL-13-induced human skin-equivalent atopic dermatitis models. Int J Mol Sci 2022;23:13520. [DOI] [PMC free article] [PubMed] [Google Scholar] - Kitatani K, Idkowiak-Baldys J, Hannun YA. The sphingolipid salvage pathway in ceramide metabolism and signaling. Cell Signal 2008;20:1010–1018. [DOI] [PMC free article] [PubMed] [Google Scholar] - Kvaskoff M, Mahamat-Saleh Y, Farland LV, Shigesi N, Terry KL, Harris HR, Roman H, Becker CM, As-Sanie S, Zondervan KT et al. Endometriosis and cancer: a systematic review and meta-analysis. Hum Reprod Update 2021;27:393–420. [DOI] [PubMed] [Google Scholar] - Kvaskoff M, Mu F, Terry KL, Harris HR, Poole EM, Farland L, Missmer SA. Endometriosis: a high-risk population for major chronic diseases? Hum Reprod Update 2015;21:500–516. [DOI] [PMC free article] [PubMed] [Google Scholar] - Laursen MF, Sakanaka M, von Burg N, Mörbe U, Andersen D, Moll JM, Pekmez CT, Rivollier A, Michaelsen KF, Mølgaard C et al. Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut. Nat Microbiol 2021;6:1367–1382. [DOI] [PMC free article] [PubMed] [Google Scholar] - Lee BC, Gladyshev VN. The biological significance of methionine sulfoxide stereochemistry. Free Radic Biol Med 2011;50:221–227. [DOI] [PMC free article] [PubMed] [Google Scholar] - Lee YH, Cui L, Fang J, Chern BSM, Tan HH, Chan JK. Limited value of pro-inflammatory oxylipins and cytokines as circulating biomarkers in endometriosis—a targeted ‘omics study. Sci Rep 2016;6:26117. [DOI] [PMC free article] [PubMed] [Google Scholar] - Li J, Guan L, Zhang H, Gao Y, Sun J, Gong X, Li D, Chen P, Liang X, Huang M et al. Endometrium metabolomic profiling reveals potential biomarkers for diagnosis of endometriosis at minimal-mild stages. Reprod Biol Endocrinol 2018;16:1–10. [DOI] [PMC free article] [PubMed] [Google Scholar] - Li Y, Wang K, Ding J, Sun S, Ni Z, Yu C. Influence of the gut microbiota on endometriosis: potential role of chenodeoxycholic acid and its derivatives. Front Pharmacol 2022;13:954684. [DOI] [PMC free article] [PubMed] [Google Scholar] - Lim S-C, Choi JE, Kang HS, Han SI. Ursodeoxycholic acid switches oxaliplatin‐induced necrosis to apoptosis by inhibiting reactive oxygen species production and activating p53‐caspase 8 pathway in HepG2 hepatocellular carcinoma. Int J Cancer 2010;126:1582–1595. [DOI] [PubMed] [Google Scholar] - Liu R, Li X, Hylemon PB, Zhou H. Conjugated bile acids promote invasive growth of esophageal adenocarcinoma cells and cancer stem cell expansion via sphingosine 1-phosphate receptor 2-mediated Yes-associated protein activation. Am J Pathol 2018;188:2042–2058. [DOI] [PMC free article] [PubMed] [Google Scholar] - Liu X, Xue R, Yang C, Gu J, Chen S, Zhang S. Cholestasis-induced bile acid elevates estrogen level via farnesoid X receptor-mediated suppression of the estrogen sulfotransferase SULT1E1. J Biol Chem 2018;293:12759–12769. [DOI] [PMC free article] [PubMed] [Google Scholar] - Lone AM, Taskén K. Proinflammatory and immunoregulatory roles of eicosanoids in T cells. Front Immunol 2013;4:130. [DOI] [PMC free article] [PubMed] [Google Scholar] - Marečková M, Garcia-Alonso L, Moullet M, Lorenzi V, Petryszak R, Sancho-Serra C, Oszlanczi A, Icoresi Mazzeo C, Wong FCK, Kelava I et al. An integrated single-cell reference atlas of the human endometrium. Nat Genet 2024;56:1925–1937. [DOI] [PMC free article] [PubMed] [Google Scholar] - Mascanfroni ID, Takenaka MC, Yeste A, Patel B, Wu Y, Kenison JE, Siddiqui S, Basso AS, Otterbein LE, Pardoll DM et al. Metabolic control of type 1 regulatory T cell differentiation by AHR and HIF1-α. Nat Med 2015;21:638–646. [DOI] [PMC free article] [PubMed] [Google Scholar] - Millan MJ. Serotonin and pain: evidence that activation of 5-HT1A receptors does not elicit antinociception against noxious thermal, mechanical and chemical stimuli in mice. Pain 1994;58:45–61. [DOI] [PubMed] [Google Scholar] - Millan MJ. Serotonin (5-HT) and pain: a reappraisal of its role in the light of receptor multiplicity. Semin Neurosci 1995;7:Elsevier, 409–419. [Google Scholar] - Missmer SA. Commentary: Endometriosis—epidemiologic considerations for a potentially ‘high-risk’ population. Int J Epidemiol 2009;38:1154–1155. [DOI] [PubMed] [Google Scholar] - Missmer SA. Why so null? Methodologic necessities to advance endometriosis discovery. Paediatr Perinat Epidemiol 2019;33:26–27. [DOI] [PubMed] [Google Scholar] - Missmer SA, Chavarro JE, Malspeis S, Bertone-Johnson ER, Hornstein MD, Spiegelman D, Barbieri RL, Willett WC, Hankinson SE. A prospective study of dietary fat consumption and endometriosis risk. Hum Reprod 2010;25:1528–1535. [DOI] [PMC free article] [PubMed] [Google Scholar] - Mozaffarian D, Cao H, King IB, Lemaitre RN, Song X, Siscovick DS, Hotamisligil GS. Circulating palmitoleic acid and risk of metabolic abnormalities and new-onset diabetes. Am J Clin Nutr 2010;92:1350–1358. [DOI] [PMC free article] [PubMed] [Google Scholar] - Mu F, Rich-Edwards J, Rimm EB, Spiegelman D, Forman JP, Missmer SA. Association between endometriosis and hypercholesterolemia or hypertension. Hypertension 2017;70:59–65. [DOI] [PMC free article] [PubMed] [Google Scholar] - Mu F, Rich-Edwards J, Rimm EB, Spiegelman D, Missmer SA. Endometriosis and risk of coronary heart disease. Circ Cardiovasc Qual Outcomes 2016;9:257–264. [DOI] [PMC free article] [PubMed] [Google Scholar] - Nnoaham KE, Hummelshoj L, Webster P, d'Hooghe T, de Cicco Nardone F, de Cicco Nardone C, Jenkinson C, Kennedy SH, Zondervan KT, World Endometriosis Research Foundation Global Study of Women's Health consortium. Impact of endometriosis on quality of life and work productivity: a multicenter study across ten countries. Fertil Steril 2011;96:366–373.e368. [DOI] [PMC free article] [PubMed] [Google Scholar] - Nnoaham KE, Hummelshoj L, Webster P, d'Hooghe T, de Cicco Nardone F, de Cicco Nardone C, Jenkinson C, Kennedy SH, Zondervan KT, World Endometriosis Research Foundation Global Study of Women's Health consortium. Reprint of: Impact of endometriosis on quality of life and work productivity: a multicenter study across ten countries. Fertil Steril 2019;112:e137–e152. [DOI] [PubMed] [Google Scholar] - Paillard F, Catheline D, Duff FL, Bouriel M, Deugnier Y, Pouchard M, Daubert JC, Legrand P. Plasma palmitoleic acid, a product of stearoyl-CoA desaturase activity, is an independent marker of triglyceridemia and abdominal adiposity. Nutr Metab Cardiovasc Dis 2008;18:436–440. [DOI] [PubMed] [Google Scholar] - Paredes S, Cantillo S, Candido KD, Knezevic NN. An association of serotonin with pain disorders and its modulation by estrogens. Int J Mol Sci 2019;20:5729. [DOI] [PMC free article] [PubMed] [Google Scholar] - Paynter NP, Balasubramanian R, Giulianini F, Wang DD, Tinker LF, Gopal S, Deik AA, Bullock K, Pierce KA, Scott J et al. Metabolic predictors of incident coronary heart disease in women. Circulation 2018;137:841–853. [DOI] [PMC free article] [PubMed] [Google Scholar] - Pearce CL, Templeman C, Rossing MA, Lee A, Near AM, Webb PM, Nagle CM, Doherty JA, Cushing-Haugen KL, Wicklund KG, et al. ; Ovarian Cancer Association Consortium. Association between endometriosis and risk of histological subtypes of ovarian cancer: a pooled analysis of case-control studies. Lancet Oncol 2012;13:385–394. [DOI] [PMC free article] [PubMed] [Google Scholar] - Rahmioglu N, Fassbender A, Vitonis AF, Tworoger SS, Hummelshoj L, D'Hooghe TM, Adamson GD, Giudice LC, Becker CM, Zondervan KT, et al. ; WERF EPHect Working Group. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: III. Fluid biospecimen collection, processing, and storage in endometriosis research. Fertil Steril 2014;102:1233–1243. [DOI] [PMC free article] [PubMed] [Google Scholar] - Režen T, Rozman D, Kovács T, Kovács P, Sipos A, Bai P, Mikó E. The role of bile acids in carcinogenesis. Cell Mol Life Sci 2022;79:243. [DOI] [PMC free article] [PubMed] [Google Scholar] - Roopashree P, Shetty SS, Kumari NS. Effect of medium chain fatty acid in human health and disease. J Funct Foods 2021;87:104724. [Google Scholar] - Rosenthal NE, Mazzanti CM, Barnett RL, Hardin TA, Turner EH, Lam GK, Ozaki N, Goldman D. Role of serotonin transporter promoter repeat length polymorphism (5-HTTLPR) in seasonality and seasonal affective disorder. Mol Psychiatry 1998;3:175–177. [DOI] [PubMed] [Google Scholar] - Sarsenova M, Lawarde A, Pathare ADS, Saare M, Modhukur V, Soplepmann P, Terasmaa A, Käämbre T, Gemzell-Danielsson K, Lalitkumar PGL et al. Endometriotic lesions exhibit distinct metabolic signature compared to paired eutopic endometrium at the single-cell level. Commun Biol 2024;7:1026. [DOI] [PMC free article] [PubMed] [Google Scholar] - Sasamoto N, Ngo L, Vitonis AF, Dillon ST, Missmer SA, Libermann TA, Terry KL. Circulating proteomic profiles associated with endometriosis in adolescents and young adults. Hum Reprod 2022;37:2042–2053. [DOI] [PMC free article] [PubMed] [Google Scholar] - Sasamoto N, Shafrir AL, Wallace BM, Vitonis AF, Fraer CJ, Sadler Gallagher J, DePari M, Ghiasi M, Laufer MR, Sieberg CB et al. Trends in pelvic pain symptoms over 2 years of follow-up among adolescents and young adults with and without endometriosis. Pain 2023;164:613–624. [DOI] [PMC free article] [PubMed] [Google Scholar] - Sasamoto N, Zeleznik OA, Vitonis AF, Missmer SA, Laufer MR, Avila-Pacheco J, Clish CB, Terry KL. Presurgical blood metabolites and risk of postsurgical pelvic pain in young patients with endometriosis. Fertil Steril 2022;117:1235–1245. [DOI] [PMC free article] [PubMed] [Google Scholar] - Shafrir AL, Farland LV, Shah DK, Harris HR, Kvaskoff M, Zondervan K, Missmer SA. Risk for and consequences of endometriosis: a critical epidemiologic review. Best Pract Res Clin Obstet Gynaecol 2018;51:1–15. [DOI] [PubMed] [Google Scholar] - Shafrir AL, Vitonis AF, Wallace B, DiVasta AD, Sadler Gallagher J, Sasamoto N, Laufer MR, Terry KL, Missmer SA. Cohort profile: the Endometriosis pain QUality aftEr Surgical Treatment (EndoQUEST) Study. PLoS One 2022;17:e0269858. [DOI] [PMC free article] [PubMed] [Google Scholar] - Shafrir AL, Wallace B, Laliberte A, Vitonis AF, Sieberg CB, Terry KL, Missmer SA. Pelvic pain symptoms and endometriosis characteristics in relation to oxidative stress among adolescents and adults with and without surgically-confirmed endometriosis. F1000Res 2024;13:34. [DOI] [PMC free article] [PubMed] [Google Scholar] - Shah DK, Missmer SA. Scientific investigation of endometriosis among adolescents. J Pediatr Adolesc Gynecol 2011;24:S18–S19. [DOI] [PubMed] [Google Scholar] - Shigesi N, Kvaskoff M, Kirtley S, Feng Q, Fang H, Knight JC, Missmer SA, Rahmioglu N, Zondervan KT, Becker CM. The association between endometriosis and autoimmune diseases: a systematic review and meta-analysis. Hum Reprod Update 2019;25:486–503. [DOI] [PMC free article] [PubMed] [Google Scholar] - Spijkers LJA, van den Akker RFP, Janssen BJA, Debets JJ, De Mey JGR, Stroes ESG, van den Born B-JH, Wijesinghe DS, Chalfant CE, MacAleese L et al. Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide. PLoS One 2011;6:e21817. [DOI] [PMC free article] [PubMed] [Google Scholar] - Sprong RC, Hulstein MF, Van der Meer R. Bactericidal activities of milk lipids. Antimicrob Agents Chemother 2001;45:1298–1301. [DOI] [PMC free article] [PubMed] [Google Scholar] - Suzuki Y, Kosaka M, Shindo K, Kawasumi T, Kimoto-Nira H, Suzuki C. Identification of antioxidants produced by Lactobacillus plantarum. Biosci Biotechnol Biochem 2013;77:1299–1302. [DOI] [PubMed] [Google Scholar] - Tworoger SS, Yasui Y, Chang L, Stanczyk FZ, McTiernan A. Specimen allocation in longitudinal biomarker studies: controlling subject-specific effects by design. Cancer Epidemiol Biomarkers Prev 2004;13:1257–1260. [PubMed] [Google Scholar] - Urbano-Márquez A, Casademont J, Grau JM. Polymyositis/dermatomyositis: the current position. Ann Rheum Dis 1991;50:191–195. [DOI] [PMC free article] [PubMed] [Google Scholar] - Vitonis AF, Vincent K, Rahmioglu N, Fassbender A, Buck Louis GM, Hummelshoj L, Giudice LC, Stratton P, Adamson GD, Becker CM, et al. ; WERF EPHect Working Group. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: II. Clinical and covariate phenotype data collection in endometriosis research. Fertil Steril 2014;102:1223–1232. [DOI] [PMC free article] [PubMed] [Google Scholar] - Wang DD, Toledo E, Hruby A, Rosner BA, Willett WC, Sun Q, Razquin C, Zheng Y, Ruiz-Canela M, Guasch-Ferré M et al. Plasma ceramides, Mediterranean diet, and incident cardiovascular disease in the PREDIMED trial (Prevención con Dieta Mediterránea). Circulation 2017;135:2028–2040. [DOI] [PMC free article] [PubMed] [Google Scholar] - Warensjö E, Ohrvall M, Vessby B. Fatty acid composition and estimated desaturase activities are associated with obesity and lifestyle variables in men and women. Nutr Metab Cardiovasc Dis 2006;16:128–136. [DOI] [PubMed] [Google Scholar] - WHO. Physical status: the use and interpretation of anthropometry. Report of a WHO Expert Committee. World Health Organ Tech Rep Ser 1995;854:1–452. [PubMed] [Google Scholar] - Weintraub A, Soriano D, Seidman D, Goldenberg M, Eisenberg V. Think endometriosis: delay in diagnosis or delay in referral to adequate treatment. J Fertil 2014;2:2–7. [Google Scholar] - Xia J, Wishart DS. Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis. Curr Protoc Bioinformatics 2016;55:14.10.1–14.10. 91. [DOI] [PubMed] [Google Scholar] - Yao T, Degli Esposti S, Huang L, Arnon R, Spangenberger A, Zern M. Inhibition of carbon tetrachloride-induced liver injury by liposomes containing vitamin E. Am J Physiol 1994;267:G476–G484. [DOI] [PubMed] [Google Scholar] - Youngster M, Laufer MR, Divasta AD. Endometriosis for the primary care physician. Curr Opin Pediatr 2013;25:454–462. [DOI] [PubMed] [Google Scholar] - Zeleznik OA, Clish CB, Kraft P, Avila-Pacheco J, Eliassen AH, Tworoger SS. Circulating lysophosphatidylcholines, phosphatidylcholines, ceramides, and sphingomyelins and ovarian cancer risk: a 23-year prospective study. J Natl Cancer Inst 2020;112:628–636. [DOI] [PMC free article] [PubMed] [Google Scholar] - Zhang Q, Chen S, Guo Y, He F, Fu J, Ren W. Phenylalanine diminishes M1 macrophage inflammation. Sci China Life Sci 2023;66:2862–2876. [DOI] [PubMed] [Google Scholar] - Zhou Q, Xie Z, Wu D, Liu L, Shi Y, Li P, Gu Q. The effect of indole-3-lactic acid from Lactiplantibacillus plantarum ZJ316 on human intestinal microbiota in vitro. Foods 2022;11:3302. [DOI] [PMC free article] [PubMed] [Google Scholar] - Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Viganò P. Endometriosis. Nat Rev Dis Primers 2018;4:9. [DOI] [PubMed] [Google Scholar] - Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Viganò P. Endometriosis. Nat Rev Dis Primers 2018;4:9. [DOI] [PubMed] [Google Scholar] - Zondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med 2020;382:1244–1256. [DOI] [PubMed] [Google Scholar] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials Data Availability Statement Data are not publicly available due to information that could compromise research participants’ privacy and consent. However, experienced scientists who would like to inquire regarding use of data from this study to address specific hypotheses or replicate the analyses in this study may submit an application and research proposal. Data requests must be reviewed and approved by the BWH Institutional Review Board (https://www.brighamandwomens.org/research/research-administration). All inquiries should be directed to the A2A cohort leadership committee ([email protected]). Data sharing will require a fully executed Data Usage Agreement.