Relationship between endometriosis diagnosis, staging, and typology and inflammatory cytokines

Among the 395 Utah operative cohort women in the ENDO study with stored biospecimens, 166 (42.0%) participants were diagnosed with endometriosis. Of the 349 women with adequate samples for the cytokine panel, 10% had elevated IL-6, 7% had elevated IL-8, and 13% had elevated TNF-α; while 26% had at least one elevated marker. The average age was 32.5 years (SD = 7.0), and most participants were non-Hispanic white (n = 310, 78.5%), with comparable distributions for age and race/ethnicity across both groups. Participants with endometriosis were more likely to have incomes above 180% of the federal poverty level (83.4% vs. 70.6%) and have a normal BMI (47.0% vs. 33.6%) ( Table 1 ). Participants with endometriosis also had a lower frequency of cotinine <10 ng/mL (13.3% vs. 20.0%) compared with their counterparts. Menstrual phases were similarly distributed between the groups. Table 1 Population characteristics by endometriosis status among ENDO Study participants in the Utah operative cohort that had a baseline blood draw (n = 395 in the operative cohort). Population characteristics Overall N = 395 Endometriosis Yes n = 166 (42.0%) No n = 229 (58.0%) Age at visit (y), Mean (SD) 32.5 ± 7.0 31.7 ± 6.8 33.1 ± 7.1 Race/ethnicity  Hispanic 48 (12.2) 20 (12.1) 28 (12.2)  Non-Hispanic white 310 (78.5) 130 (78.3) 180 (78.6)  Non-Hispanic black 4 (1.0) 1 (0.6) 3 (1.3)  South Asian 17 (4.3) 8 (4.8) 9 (3.9)  Multiracial 9 (2.3) 2 (1.2) 7 (3.1)  Other 7 (1.8) 5 (3.0) 2 (0.9) Income  Below poverty line 49 (12.5) 17 (10.4) 32 (14.0)  ≤180% of poverty line 45 (11.5) 10 (6.1) 35 (15.4)  >180% of poverty line 297 (76.0) 136 (83.4) 161 (70.6) Marital status  Married 283 (72.2) 123 (74.6) 160 (70.5)  Cohabiting/domestic partners 19 (4.9) 8 (4.9) 11 (4.9)  Single 57 (14.5) 24 (14.6) 33 (14.5)  Divorced, separated or widowed 33 (8.4) 10 (6.1) 23 (10.1) BMI (kg/m 2 ), Mean (SD) BMI (kg/m 2 ), Category 28.2 ± 8.2 26.4 ± 7.2 29.5 ± 8.7  Underweight (<18.5) 18 (4.6) 10 (6.0) 8 (3.5)  Normal weight (18.5–24.99) 155 (39.2) 78 (47.0) 77 (33.6)  Overweight (25–29.99) 88 (22.3) 37 (22.3) 51 (22.3)  Obese (≥ 30.0) 134 (33.9) 41 (24.7) 93 (40.6) Serum cotinine (ng/mL)  ≥10 ng/mL 67 (17.0) 22 (13.3) 45 (19.7)  ≥5 ng/mL 68 (17.2) 22 (13.3) 46 (20.1) Caffeinated beverages/day  None 77 (19.5) 36 (21.7) 41 (17.9)  Low (1–2) 175 (44.3) 78 (47.0) 97 (42.4)  Moderate (3–6) 86 (21.8) 31 (18.9) 55 (24.0)  High (>6) 57 (14.4) 21 (12.7) 36 (15.7) Current consumer of alcohol 150 (38.1) 58 (34.9) 92 (40.4) Menstrual cycle phase  Follicular 170 (52.0) 75 (52.5) 95 (51.6)  Luteal 157 (48.0) 68 (47.6) 89 (48.4) Primary reason for surgery  Tubal ligation 39 (9.9) 8 (4.8) 31 (13.5)  Pelvic pain 189 (47.9) 108 (65.1) 81 (35.4)  Pelvic mass 60 (15.2) 20 (12.1) 40 (17.5)  Infertility 31 (7.9) 6 (3.6) 25 (10.9)  Fibroids 21 (5.3) 5 (3.0) 16 (7.0)  Menstrual irregularities 19 (11.5) 19 (11.5) 36 (15.7) Prior diagnosis of PCOS 28 (7.1) 14 (9.4) 14 (6.1) Prior diagnosis of hirsutism 16 (4.1) 7 (4.2) 9 (3.9) Prior use of oral hormonal contraception  Ever 339 (85.8) 146 (88.0) 193 (84.3)  Prior 2 years 69 (23.2) 35 (27.1) 34 (20.2) Prior diagnosis of diabetes 16 (4.1) 7 (4.2) 9 (3.9) Prior diagnosis of high blood pressure 29 (7.3) 12 (7.2) 17 (7.4) Prior diagnosis of cardiovascular disease 9 (2.3) 1 (0.6) 8 (3.5) Physical activity  Low 61 (17.6) 23 (15.8) 38 (19.0)  Moderate 123 (35.6) 57 (39.0) 66 (33.0)  High 162 (46.8) 66 (45.2) 96 (48.0) Note: n (%) unless otherwise noted. Missing n = 1 age at visit; n = 0 race/ethnicity; n = 4 income, n = 3 marital status, n = 0 serum cotinine; n = 1 alcohol, n = 68 cycle phase, n = 0 oral hormonal contraceptive, n = 0 prior diagnosis of diabetes, high blood pressure, or cardiovascular disease, and n = 49 physical activity. BMI = body mass index; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; n = total sample size; N = total population size; PCOS = polycystic ovary syndrome; SD = standard deviation. Population characteristics by endometriosis status among ENDO Study participants in the Utah operative cohort that had a baseline blood draw (n = 395 in the operative cohort). Note: n (%) unless otherwise noted. Missing n = 1 age at visit; n = 0 race/ethnicity; n = 4 income, n = 3 marital status, n = 0 serum cotinine; n = 1 alcohol, n = 68 cycle phase, n = 0 oral hormonal contraceptive, n = 0 prior diagnosis of diabetes, high blood pressure, or cardiovascular disease, and n = 49 physical activity. BMI = body mass index; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; n = total sample size; N = total population size; PCOS = polycystic ovary syndrome; SD = standard deviation. Participants with elevation in at least one inflammation biomarker (IL6, IL8, or TNF-α), compared with none, also had comparable age and race/ethnicity, but were slightly more likely to be below federal poverty line (16.9% vs. 10.6%), obese (37.8% vs. 32.8%), a current alcohol (39.3% vs. 36.7%) or caffeine consumer (83.3% vs. 78.0%), and less likely to have high physical activity (43.2% vs. 48.9%). ( Supplemental Table 1 , available online). Participants with elevated inflammatory biomarkers also had a higher frequency of cotinine <10 ng/mL (17.8% vs. 15.4%), were less likely to have high physical activity (43.2% vs 48.9%), and more likely to be in their luteal phase at the time of biospecimen collection compared with their counterparts. Participants with endometriosis had similar levels of serum IL-6 (10% vs. 10%), higher levels of TNF- α (16% vs. 12%), and lower levels of IL-8 (5% vs. 8%) compared with those without endometriosis. Overall, there was no association observed between endometriosis and elevated interleukins (IL-6 PR 1.17, 95% CI 0.59, 2.30; IL-8 PR 0.60, 95% CI 0.20, 1.84, and TNF- α PR 1.30, 95% CI 0.68, 2.51) after adjusting for age at baseline, race/ethnicity, marital status, BMI, serum cotinine, and use of oral hormonal contraception within the past 2 years ( Table 2 ). Continuous assessment of inflammatory biomarkers also revealed a null association. Our assessment, whereby we compared women with endometriosis with women without endometriosis or other gynecologic pathology as the reference group, showed similar null findings ( Supplemental Table 2 ). Table 2 Endometriosis diagnosis and unadjusted and adjusted prevalence of elevated inflammatory markers in ENDO operative cohort (n = 395) a . Categorical Endometriosis n = 166 (42.0%) No endometriosis n = 229 (58.0%) n (%) Unadjusted PR (95% CI) Adjusted PR (95% CI) b n (%) Unadjusted PR (95% CI) Adjusted PR (95% CI) b IL-6, pg/mL, ≥2 15 (10) 1.00 (0.54, 1.88) 1.17 (0.59, 2.30) 21 (10) 1.0 (ref) 1.0 (ref) IL-8, pg/mL, ≥3 7 (5) 0.62 (0.26, 1.46) 0.60 (0.20, 1.84) 16 (8) 1.0 (ref) 1.0 (ref) TNF-α, pg/mL, ≥7.5 23 (16) 1.29 (0.77, 2.19) 1.30 (0.68, 2.51) 25 (12) 1.0 (ref) 1.0 (ref) Continuous Unadjusted % difference (95% CI) Adjusted % difference (95% CI) b Unadjusted % difference (95% CI) Adjusted % difference (95% CI) b IL-6, pg/mL 0.39 (−3.94, 4.72) 1.31 (−3.97, 6.60) 0.0 (ref) 0.0 (ref) IL-8, pg/mL −4.27 (−8.13, −0.41) −4.61 (−9.46, 0.24) 0.0 (ref) 0.0 (ref) TNF-α, pg/mL 7.05 (−5.95, 20.05) 2.12 (−12.32, 16.56) 0.0 (ref) 0.0 (ref) Note :. a Among the 395 Utah operative cohort women in the ENDO study with stored biospecimens, 349 women had adequate samples for the cytokine panel. Addressed potential missing data bias in our multiple imputation models. b Adjusted for age (continuous), BMI (continuous), race/ethnicity (non-Hispanic white vs. other), serum cotinine (continuous), and use of oral hormonal contraceptives within the past 2 years (yes/no). BMI = body mass index; CI = confidence interval; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; IL-6 = interleukin 6; IL-8 = interleukin 8; n = total sample size; PR = prevalence ratio; TNF-α = tumor necrosis factor alpha. Endometriosis diagnosis and unadjusted and adjusted prevalence of elevated inflammatory markers in ENDO operative cohort (n = 395) a . Note :. Among the 395 Utah operative cohort women in the ENDO study with stored biospecimens, 349 women had adequate samples for the cytokine panel. Addressed potential missing data bias in our multiple imputation models. Adjusted for age (continuous), BMI (continuous), race/ethnicity (non-Hispanic white vs. other), serum cotinine (continuous), and use of oral hormonal contraceptives within the past 2 years (yes/no). BMI = body mass index; CI = confidence interval; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; IL-6 = interleukin 6; IL-8 = interleukin 8; n = total sample size; PR = prevalence ratio; TNF-α = tumor necrosis factor alpha. When stratified by stage ( Table 3 ), participants with moderate to severe endometriosis had higher levels of serum IL-6 (12% vs. 10%) and TNF-α (16% vs. 12%), but lower levels of IL-8 (5% vs. 8%). After adjustment, we observed no associations between endometriosis staging and inflammatory markers, whether assessed categorically or continuously, or in our sensitivity analyses comparing women with endometriosis with women without endometriosis or other gynecologic pathology ( Supplemental Table 3 ). Table 3 Endometriosis staging and prevalence of elevated inflammatory markers in ENDO operative cohort (n = 395) a . Categorical Minimal to mild 124 (31.4%) Moderate to severe 42 (10.6%) No endometriosis n = 229 (58.0%) n (%); Adjusted PR (95% CI) b IL-6, pg/mL, ≥2 13 (12); 1.41 (0.69, 2.87) 2 (5); 0.60 (0.14, 2.52) 21 (10); 1.0 (ref) IL-8, pg/mL, ≥3 5 (5); 0.51 (0.13, 1.98) 2 (5); 0.80 (0.18, 3.58) 16 (8); 1.0 (ref) TNF-α, pg/mL, ≥7.5 17 (16); 1.16 (0.55, 2.42) 6 (16);1.69 (0.71,4.03) 25 (12); 1.0 (ref) IL-6, pg/mL 2.71 (−2.63, 8.06) −3.16 (−6.54, 0.20) 0.0 (ref) IL-8, pg/mL −4.05 (−8.48, 0.37) −5.76 (−9.59, −2.30) 0.0 (ref) TNF-α, pg/mL 7.09 (−7.59, 21.79) 2.47 (−19.35, 24.29) 0.0 (ref) Note :. a Among the 395 Utah operative cohort women in the ENDO study with stored biospecimens, 349 women had adequate samples for the cytokine panel. Addressed potential missing data bias in our multiple imputation models. b Adjusted for age (continuous), BMI (continuous), race/ethnicity (non-Hispanic white vs. other), serum cotinine (continuous), and use of oral hormonal contraceptives within the past 2 years (yes/no). BMI = body mass index; CI = confidence interval; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; IL-6 = interleukin 6; IL-8 = interleukin 8; n = total sample size; PR = prevalence ratio; TNF-α = tumor necrosis factor alpha. Endometriosis staging and prevalence of elevated inflammatory markers in ENDO operative cohort (n = 395) a . Note :. Among the 395 Utah operative cohort women in the ENDO study with stored biospecimens, 349 women had adequate samples for the cytokine panel. Addressed potential missing data bias in our multiple imputation models. Adjusted for age (continuous), BMI (continuous), race/ethnicity (non-Hispanic white vs. other), serum cotinine (continuous), and use of oral hormonal contraceptives within the past 2 years (yes/no). BMI = body mass index; CI = confidence interval; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; IL-6 = interleukin 6; IL-8 = interleukin 8; n = total sample size; PR = prevalence ratio; TNF-α = tumor necrosis factor alpha. Patterns by endometriosis typology were consistent with null results by diagnosis and staging for IL-6 and IL8, whether the comparison group was women with no endometriosis ( Table 4 ) or women without endometriosis or other gynecologic pathology ( Supplemental Table 4 ). Women with OE plus DE, compared with those without endometriosis, did have suggestively higher prevalence of elevated TNF- α ≥ 7.5 (27% vs. 14%), with an adjusted prevalence ratio (aPR) of 3.06 (95% CI: 1.15, 8.16). However, results by typology should be interpreted with caution because of low precision reflected in the wide confidence intervals. Table 4 Endometriosis typology and prevalence of elevated inflammatory markers in ENDO operative cohort (n = 395) a Categorical Superficial n = 104 (26.3%) Deep infiltrating n = 26 (6.6%) Endometrioma n = 19 (4.8%) Endometrioma and deep infiltrating n = 17 (4.3%) No endometriosis n = 229 (58.0%) n (%); Adjusted PR (95% CI) b IL-6, pg/mL, ≥2 11 (12); 1.40 (0.68, 2.88) 1 (4); NA 2 (12); 1.29 (0.31, 5.38) 1 (7); 0.93 (0.12, 6.71) 21 (10); 1.0 (ref) IL-8, pg/mL, ≥3 4 (4); 0.43 (0.09, 1.97) 1 (4); 0.75 (0.09, 6.13) 1 (6); 0.79 (0.09, 6.80) 1 (7); 1.03 (0.15, 6.91) 16 (8); 1.0 (ref) TNF-α, pg/mL, ≥7.5 15 (17); 1.13 (0.52, 2.45) 1 (4); 0.50 (0.07, 3.74) 3 (18); 1.75 (0.56, 5.43) 4 (27); 3.06 (1.15, 8.16) 48 (14); 1.0 (ref) IL-6, pg/mL 2.97 (−4.14, 10.07) −3.13 (−6.64, 0.37) 0.24 (−9.67, 10.15) −1.17 (−7.56, 3.26) 0.0 (ref) IL-8, pg/mL −4.99 (−9.49, −0.50) 0.77 (−14.27, 15.81) −7.00 (−11.70, −2.31) −6.78 (−11.74, −1.82) 0.0 (ref) TNF-α, pg/mL −0.92 (−17.21, 15.36) −11.71 (−33.06, 9.63) 16.71 (−18.55, 51.98) 21.14 (−20.98, 63.27) 0.0 (ref) Note :. a Among the 395 Utah operative cohort women in the ENDO study with stored biospecimens, 349 women had adequate samples for the cytokine panel. Addressed potential missing data bias in our multiple imputation models. b Adjusted for age (continuous), BMI (continuous), race/ethnicity (non-Hispanic white vs. other), serum cotinine (continuous), and use of oral hormonal contraceptives within the past 2 years (yes/no). BMI = body mass index; CI = confidence interval; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; IL-6 = interleukin 6; IL-8 = interleukin 8; n = total sample size; PR = prevalence ratio; TNF-α = tumor necrosis factor alpha. Endometriosis typology and prevalence of elevated inflammatory markers in ENDO operative cohort (n = 395) a Note :. Among the 395 Utah operative cohort women in the ENDO study with stored biospecimens, 349 women had adequate samples for the cytokine panel. Addressed potential missing data bias in our multiple imputation models. Adjusted for age (continuous), BMI (continuous), race/ethnicity (non-Hispanic white vs. other), serum cotinine (continuous), and use of oral hormonal contraceptives within the past 2 years (yes/no). BMI = body mass index; CI = confidence interval; ENDO = Endometriosis, Natural History, Diagnosis, and Outcomes; IL-6 = interleukin 6; IL-8 = interleukin 8; n = total sample size; PR = prevalence ratio; TNF-α = tumor necrosis factor alpha. Results were consistent after additional covariate adjustment ( Supplemental Table 5 ). Although we did not observe effect modification by menstrual cycle phase or a prior diagnosis of PCOS, we did observe modification by primary reason for surgery (Wald P <.001 for IL-6, IL-8, and TNF- α ). Again, precision was low, especially for more severe disease (OE and/or DE); however, results indicated that among women coming in for tubal ligation, IL-6 and TNF- α concentrations were lower among women diagnosed with, vs. without, incident endometriosis ( Supplemental Table 6 ). Finally, our multiple imputation models indicated little missing data bias because complete case analysis had similar estimates to our imputed models.

This analysis utilizes data from the Endometriosis, Natural History, Diagnosis, and Outcomes (ENDO) study, from the Utah site ( 34 ). The ENDO study (2007–2009) aimed to explore the extent and impact of endometriosis based on diagnostic methods and choice of comparison group; and examine the association between endocrine-disrupting chemicals and the development of gynecologic conditions, such as endometriosis ( 34 ). Eligible women were currently menstruating, aged 18–44 years, without a prior history of laparoscopically confirmed endometriosis to ensure identification of incident disease. Further, eligibility criteria included ability to communicate in English or Spanish; had not been breastfeeding for ≥6 months; had no injectable hormonal treatment within the past 2 years; and no cancer history save for nonmelanoma skin cancer. The ENDO study operative cohort consisted of individuals scheduled to undergo gynecologic laparoscopy or laparotomy, irrespective of clinical indication, at one of five participating hospitals in Utah. Surgical indications included pelvic pain (44%), pelvic mass (16%), irregular menses (13%), fibroids (10%), tubal ligation (10%), and infertility (7%). The University of Utah Institutional Review Board (IRB) approved the study (IRB #00021614), and all participants provided written informed consent before enrollment. Standardized data collection involved a baseline personal interview, biospecimen collection, and anthropometric assessment performed before or on the day of surgery (median = 4 days; interquartile range [IQR] = 2, 10 days). Participants provided self-reported information on sociodemographics (age, race/ethnicity, income, marital status), health history (hormonal medications, prior diagnoses of polycystic ovary syndrome [PCOS], and cardiometabolic disease), and lifestyle factors (caffeine and alcohol intake, physical activity). Height and weight were measured using portable stadiometers and electronic scales from which body mass index (BMI) was calculated ( 35 ). Confirmation of tobacco use was determined using serum cotinine levels (>5 and >10 ng/mL, with >5 ng/mL considered the cutoff for current smokers) ( 36 ). To capture menstrual cycle phase (follicular vs. luteal) at the time of biospecimen collection, women reported the date of their last menstrual cycle. Physical activity was assessed using the International Physical Activity Questionnaire-Short Form (IPAQ-SF) ( 37 ) and categorized into low, moderate, and high physical activity. The exposure of interest among the operative cohort was surgically visualized endometriosis diagnosis (yes/no), along with endometriosis stage and typology. For endometriosis, all surgeons participating in the ENDO study were trained in the diagnosis and staging of endometriosis. The surgeons completed a standardized operative report immediately after surgery to capture the degree of endometriosis and gynecologic and pelvic pathology, including endometriosis, uterine fibroids, pelvic adhesions, benign ovarian cysts, neoplasms, and congenital müllerian anomalies. Endometriosis staging was categorized using the revised American Society for Reproductive Medicine (rASRM) disease stage ( 38 ). The rASRM classification uses a weighted point score to categorize endometriosis staging: stage I, minimal (scores 1–5); stage II, mild (scores 6–15); stage III, moderate (scores 16–40); and stage IV, severe (scores >40). Our prior research showed substantial agreement between gynecologic surgeons on endometriosis diagnosis using the rASRM criteria ( 39 ). Typology was determined using the rASRM standardized form if there was information on both the location and size of endometriosis lesions (n = 180 [95%] out of the 190 women with an endometriosis diagnosis) ( 28 ). Women with superficial lesions on the ovary or peritoneum were considered to have SE. Deep lesions (>5 mm invasion), noted in the peritoneum or obliteration of the posterior cul-de-sac, were considered to be DE. Deep lesions of any size noted in the ovary were considered to be OE. Women who had deep ovarian and peritoneal lesions were considered to have OE and DE. Women without information on lesion location, size, and depth (n = 10) were assumed to have SE. All blood serum samples were stored at −80°C from the time of collection and processing until 2024, when they were shipped on dry ice to Utah for analysis. Cytokine biomarkers IL-6, IL-8, and TNF- α were assayed via quantitative multiplex bead using banked (−80° Fahrenheit) serum specimens by ARUP Laboratories (Salt Lake City, UT) that had not previously been thawed. Upon arrival, samples were thawed, aliquoted, and processed within 24 hours. Elevated serum cytokine concentrations were defined continuously and by standard reference cut points: IL-6 ≥2 pg/mL, IL-8 ≥3 pg/mL, and TNF-α ≥7.5 pg/mL (ARUP Laboratories, Salt Lake City, UT: Cytokine Panel 13, Serum, https://ltd.aruplab.com/Tests/Pub/0051394 ). Mean ± standard deviation (SD) or median (IQR), where appropriate, were reported for continuous population characteristics (age at baseline, BMI, serum cotinine) and frequency (N) and relative frequency (%) were reported for categorical characteristics (race/ethnicity [Hispanic, non-Hispanic white, non-Hispanic black, South Asian, multiracial, other], income [180% of poverty line], marital status [married; cohabiting/domestic partner; single; divorced, separated, or widowed], BMI [underweight, normal weight, overweight, obese], serum cotinine [≥5 ng/mL] ( 36 ) caffeinated beverages/day over last year [none, 1–2, 3–6, >6], current alcohol consumer [yes/no], menstrual cycle phase [follicular, luteal], report of ever having used oral hormonal contraception [yes/no] or used oral hormonal contraception within the past 2 years [yes/no], and prior diagnosis of PCOS/hirsutism [yes/no], diabetes [yes/no], high blood pressure [yes/no], or CVD [yes/no]). Generalized linear models with a Poisson family, log link, and robust standard errors were utilized to calculate prevalence ratios (PR) and 95% confidence intervals (CI) for associations between inflammatory markers (above and below reference value) and the prevalence of endometriosis diagnosis, staging, and typology. Our primary models adjusted for baseline age (continuous), race, and ethnicity (non-Hispanic White vs. other), BMI (continuous, kg/m 2 ), serum cotinine (continuous, ng/mL), and report of oral hormonal contraception use within the past 2 years (yes/no). We also generated models assessing adjusted association between endometriosis diagnosis, staging, and typology and continuous measures of log-transformed inflammatory biomarkers via multivariable generalized linear models with a gamma family and log link. The percentage difference in inflammatory markers (exp(β)-1)×100 and the 95% CI were reported for each model. Finally, we evaluated whether the association between endometriosis and inflammatory biomarkers differed by menstrual cycle phase and primary reason for surgery. To assess effect modification on the multiplicative scale, we included an interaction term between endometriosis status and each potential modifier. After fitting each model, we conducted a joint Wald test of the interaction terms to determine whether inclusion of the interaction significantly improved model fit. A P value <.05 from the joint test was considered evidence of effect modification, and the report stratified results accordingly. We conducted several sensitivity analyses. Although our primary models included all women without incident endometriosis (n = 229) as our reference category, we ran sensitivity analyses whereby our reference category was all women without incident endometriosis and no other gynecologic pathology (i.e., no uterine fibroids, pelvic adhesions, benign ovarian cysts, neoplasms, or congenital müllerian anomalies). A sensitivity analysis was also done adjusting for other important confounding factors, including income, marital status, caffeine, alcohol, physical activity, and prior diagnosis of PCOS/hirsutism, diabetes, high blood pressure, and CVD. Finally, we addressed missing data for both predictors and outcomes using multiple imputation by chained equations (MICE) under the assumption of missing at random. Thirty imputations were generated, and all variables in the analysis, including inflammatory biomarker outcomes (IL-6, IL-8, TNF-α), endometriosis status, and covariates, were included in the imputation model to preserve associations among variables.

In summary, this study found no clear correlation between endometriosis diagnosis, staging, typology, and inflammatory markers IL-6, IL-8, and TNF- α . These findings underscore the complexity of endometriosis-associated inflammation and suggest that circulating cytokines alone are insufficient as biomarkers. Future research should prioritize longitudinal designs, larger and more diverse populations, and refined phenotyping alongside multimodal biomarker panels to elucidate immune contributions to disease pathogenesis and improve diagnostic accuracy.

This study examined associations between incident endometriosis diagnosis, staging, and typology, and cytokine concentrations, particularly focusing on IL-6, IL-8, and TNF- α . We observed no clear associations between endometriosis and elevated serum IL-6, IL-8, and TNF- α after multivariable adjustment. Although some subgroups showed suggestive trends, such as women with OE plus DE having elevated TNF- α concentrations, confidence intervals were consistently wide and encompassed the null. These findings emphasize the complexity of inflammatory profiles in endometriosis and suggest that variation by disease severity and phenotype may exist but was not detectable in this sample. Interleukin 6, IL-8, and TNF- α have well-established roles in reproductive and inflammatory processes relevant to endometriosis pathophysiology. The IL-6 is critical for normal endometrial function and implantation, influencing corpus luteum activity and reproductive success ( 40 ). The IL-8 promotes angiogenesis, cellular invasion, and migration, processes that facilitate ectopic endometrial tissue establishment and maintenance ( 19 , 41 , 42 ). The TNF- α acts as a potent macrophage activator and mediates inflammatory cascades implicated in ovarian dysfunction ( 43 ). Elevated concentrations of these cytokines have been consistently observed in peritoneal fluid from endometriosis patients, reflecting a localized inflammatory microenvironment ( 44 , 45 ). However, systemic levels may not accurately mirror this localized immune response, complicating their utility as circulating biomarkers. Prior research has established cytokines as central mediators of the inflammatory processes underlying endometriosis ( 18 , 21 , 23 ). Elevated concentrations of IL-6 and TNF- α have been consistently reported in peritoneal fluid, suggesting local inflammatory activity proximal to lesions ( 32 , 44 , 45 , 46 , 47 , 48 ). Several studies have also noted that IL-6 levels tend to be higher in more advanced stages of the disease ( 32 , 46 , 47 , 48 ). However, findings from systemic measurements of these cytokines have been inconsistent. Although some studies demonstrate significant elevations in serum IL-6 and TNF- α among affected individuals ( 32 , 46 ), others, including large cohort investigations and meta-analyses, have failed to detect significant differences ( 25 , 49 ). The equivocal nature of IL-8 associations further reflects this complexity, with reports of variable elevation depending on disease stage and study design ( 22 , 24 ). Our results align with those demonstrating no significant systemic cytokine elevation, reinforcing the notion that circulating cytokines may lack sensitivity and specificity as diagnostic biomarkers. The absence of statistically significant associations warrants careful interpretation. Endometriosis is a heterogeneous disease, characterized by variable lesion phenotypes and clinical presentations that likely contribute to inconsistent inflammatory signatures. Peritoneal fluid cytokine profiles show that each lesion subtype is associated with a unique inflammatory environment ( 50 , 51 ). Additionally, cytokine levels exhibit temporal variability and may be influenced by acute physiological or environmental factors, thereby limiting the utility of single serum measurements as biomarkers of chronic disease. Additionally, the ‟no endometriosis” reference group was defined based on surgical evaluation but may encompass individuals with subclinical or microscopic disease undetectable by laparoscopy, as well as those with other gynecologic inflammatory conditions. Such misclassification bias would attenuate effect estimates toward the null, thereby obscuring potential true associations. However, in sensitivity analyses where the comparison group was restricted to those without other gynecologic conditions, the findings did not appreciably change. This highlights a fundamental challenge in biomarker research for endometriosis and underscores the necessity for improved methods to define control populations with greater specificity. This study advances the field by incorporating typology into analyses. Although associations between cytokine concentrations and lesion subtypes did not reach statistical significance, observed trends toward elevated TNF- α in ovarian and combined deep lesions, support the hypothesis that distinct lesion phenotypes may exhibit differential inflammatory profiles. These findings warrant further investigation in larger cohorts with adequate power to elucidate subtype-specific immune mechanisms. This study had several strengths, including being one of the few studies that directly analyzed elevated cytokine concentrations with endometriosis at the time of incident diagnosis, with the novel addition of analyzing disease typology alongside diagnosis and staging. The ENDO study is considered generalizable to other women undergoing laparoscopy because of few exclusion criteria. However, the study was made up primarily of white, urban, non-Hispanic women of higher socioeconomic status. Other limitations include the cross-sectional study design, which limits the assessment of cytokine levels over time or in relationship with disease progression. Furthermore, although cytokines were properly stored at −80 °C at Fisher BioServices Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Repository, with no risk of freeze/thaw minimizing degradation, prior research has shown variability in stability for long-period storage ( 52 ). We would expect any resulting measurement error to be nondifferential with respect to endometriosis diagnosis. Given the well-documented diagnostic delay in endometriosis ( 53 , 54 ), it remains unclear when the disease truly begins. Without longitudinal data, it is difficult to determine whether cytokine elevations contribute to disease onset or are a reflection of an established inflammatory environment. Future prospective studies are needed to track cytokine levels in individuals at risk of endometriosis to better establish temporal and causal relationships. Additionally, the small sample sizes limited our precision in determining relationships between cytokine levels and endometriosis. Finally, while adjusting for our a priori determined sociodemographic, health, and lifestyle confounding factors did not appreciably alter our inflammatory marker estimates, we cannot rule out that there may be residual confounding factors we did not account for.

M.S. has nothing to disclose. J.T.R. has nothing to disclose. E.A. has nothing to disclose. L.V.F. has nothing to disclose. A.Z.P. has nothing to disclose. C.M.P. has nothing to disclose. K.M.R. has nothing to disclose. R.H. has nothing to disclose. M.P. has nothing to disclose. B.H.B. has nothing to disclose. S.T.S. has nothing to disclose. M.W.V. has nothing to disclose. K.C.S. has nothing to disclose.