{"paper_id":"d9012951-058f-49fa-9a7b-68f00e30b224","body_text":"1\nScientific  RepoRts  | 6:26117 | DOI: 10.1038/srep26117\nwww.nature.com/scientificreports\nLimited value of pro-inflammatory \noxylipins and cytokines as \ncirculating biomarkers in \nendometriosis – a targeted ‘omics \nstudy\nYie Hou Lee1,*, Liang Cui2,*, Jinling Fang3, Bernard Su Min Chern4, Heng Hao Tan5 & \nJerry K. Y. Chan1,5,6\nEndometriosis is a common, complex gynecologic disorder characterized by the presence of \nendometrial-like tissues at extrauterine sites. Elevation in protein and lipid mediators of inflammation \nincluding oxylipins and cytokines within the peritoneum characterize the inflamed pelvic region \nand may contribute to the survival and growth of displaced endometrial tissues. The presence of a \nclinically silent but molecularly detectable systemic inflammation in endometriosis has been proposed. \nThus, we examined serum oxylipin and immunomodulatory protein levels in 103 women undergoing \nlaparoscopy to evaluate systematically any involvement in systemic pathophysiological inflammation \nin endometriosis. Oxylipin levels were similar between women with and without endometriosis. \nStratification by menstrual phase or severity did not offer any difference. Women with ovarian \nendometriosis had significantly lower 12-HETE relative to peritoneal endometriosis (−50.7%). Serum \noxylipin levels were not associated with pre-operative pain symptoms. Changes to immunomodulatory \nproteins were minimal, with IL-12(p70), IL-13 and VEGF significantly lower in mild endometriotic \nwomen compared to non-endometriotic women (−39%, −54% and −76% respectively). Verification \nusing C-reactive protein as a non-specific marker of inflammation further showed similar levels between \ngroups. The implications of our work suggest pro-inflammatory mediators in the classes studied may \nhave potentially limited value as circulating biomarkers for endometriosis, suggesting of potentially \ntenuous systemic inflammation in endometriosis.\nEndometriosis is a complex gynecologic disorder characterized by the presence of endometrial-like tissues at sites \noutside of the uterine cavity, affecting 2–10% of women, and half of women with subfertility. Pain and infertility \nare two prominent symptoms most commonly associated with the endometriosis and have been attributed to \nchronic inflammatory state of the pelvic peritoneal area with altered immunological and inflammatory milieu in \nthe microenvironment\n1. This can be deduced by two main features found in the peritoneal environment–i) the \nincrease in immune cells and ii) the elevation of pro-inflammatory immunomodulatory proteins (cytokines and \nchemokines) and lipid mediators such as prostaglandins in the peritoneum and peritoneal fluids of women with \nendometriosis\n2–7. There are several reports of increased circulating cytokines such as IL-6 and TNFα  in women \nwith endometriosis2–5 but discrepancies continue to pervade the literature in terms of reproducibility of the find-\nings4,8,9. This has led to the questioning of whether endometriosis is accompanied by a clinically silent systemic \n1KK Research Centre, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, 229899, Singapore. 2Singapore-\nMIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602, Singapore. 3Saw \nSwee Hock School of Public Health, National University of Singapore, 12 Science Drive 2, #10-01, 117549, Singapore. \n4Division of Obstetrics & Gynaecology KK Women’s and Children’s Hospital, Singapore, 100 Bukit Timah Road, \n229899, Singapore. 5Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore, 100 \nBukit Timah Road, 229899, Singapore. 6Cancer and Stem Cell Biology, Duke-NUS Medical School, 8 College Road, \n169857, Singapore.  *These authors contributed equally to this work. Correspondence and requests for materials \nshould be addressed to Y.H.L. (email: lee.yie.hou@kkh.com.sg)\nreceived: 10 February 2016\nAccepted: 27 April 2016\nPublished: 19 May 2016\nOPEN\n\nwww.nature.com/scientificreports/\n2\nScientific  RepoRts  | 6:26117 | DOI: 10.1038/srep26117\ninflammation10 and resulted in searches for circulating inflammatory markers which could potentially predict \nendometriosis.\nInflammation is biochemically modulated by oxylipins cooperating with cytokines and chemokines 11. \nOxylipins, collectively, includes bioactive, oxidized lipid mediators synthesized from free omega-6 polyunsatu-\nrated fatty acids (n-6 PUFA) including arachidonic acid (AA), linoleic acid (LA), and dihomo-gamma-linolenic \nacid (DGLA), or omega-3 polyunsaturated fatty acid (n -3 PUFA) including eicosapentenoic acid (EPA), doco-\nsahexanoic acid (DHA), and alpha-linolenic acid (ALA). Upon liberation from membrane bound phospholipids \nby activation of phospholipase A 2 and subsequent oxidation by cyclooxygenase (COX), lipoxygenase (LOX), \nand cytochrome P450 epoxygenase (CYP450) systems, oxylipins such as prostaglandins (PG), leukotrienes (LT), \nthromboxanes (TBX) and hydroxyeicosatetraenoic acids (HETEs) are generated. In general, the n-6 PUFAs AA \nand LA are the precursors of pro-inflammatory lipid mediators while EPA and DHA derived lipid mediators \nresolve inflammation\n12. Oxylipins are known to exhibit paracrine, autocrine and increasingly endocrine effects, \nacting on both local and distant targets by secretion into the circulation13.\nConsidering the pivotal physiological and pathophysiological roles of oxylipins and immunomodulatory pro-\nteins in inflammation, we used them as assessors of systemic inflammation in endometriosis. We previously \nemployed a targeted lipidomics approach using a liquid chromatography-tandem mass spectrometry (LC-MS/\nMS) to analyze serum sphingolipids in endometriosis\n14 and herein, we employed a combined targeted ‘omics \napproach comprising LC-MS/MS and multiplex immunoassay to assess the levels of serum pro-inflammatory \noxylipins, cytokines and chemokines. We report limited systemic inflammation in women with endometriosis \nand the results was corroborated with the non-specific inflammation marker, C-reactive protein\n15. Our extensive \nprofiling here from well-defined cases and controls suggests that systemic pro-inflammatory mediators are likely \nto have limited diagnostic value in endometriosis. Our results inform prospective researchers in the search for \nbiomarker discovery of endometriosis diagnosis to refrain from pro-inflammatory mediators in the circulation.\nMethods\nPatients and sample collection. The study population comprising of 120 patients presenting with sub-fer-\ntility in addition to a general gynaecological case-mix was recruited in KK Women’s and Children’s Hospital, \nSingapore. Exclusion criteria include patients who are menstruating, anovulatory, post-menopausal, on hormonal \ntherapy for at least three months before laparoscopy or anti-inflammatory medication a day before laparoscopy, \nand other potentially confounding diseases such as diabetes, adenomyosis or any other chronic inflammatory \ndiseases (rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis, etc). The following patients were \nexcluded: seven menstruating patients, two with undeterminable menstrual phase, two with irregular menstrual \nphase, two anovulatory patients, and one patient with undetermined endometriosis severity scoring. Women \nprovided written informed consent for collection of samples and were carried out in accordance with stipulated \nguidelines and regulations under Centralised Institutional Research Board approval (CIRB 2010-167-D).\nA diagnostic laparoscopy was performed with careful inspection of the uterus, fallopian tubes, ovaries, pouch \nof Douglas and the pelvic peritoneum. Presence of endometriosis is scored according to the revised American \nFertility Society (rAFS) classification of endometriosis\n16. For the purpose of this study, we grouped rAFS Stages \nI and II as “mild” (EM +  Mild; n =  19) and rAFS Stages III and IV as “severe” (EM +  Sev; n =  38). 57 patients were \ndiagnosed as having endometriosis (EM+  ), and 46 women who did not have endometriosis or have benign \ngynecological presentations such as uterine fibroids and benign ovarian cysts were taken as the control group \n(EM−  ). Further details on patient characteristics can be found in Supplemental Table 1. Peritoneal or ovarian \nendometriosis (endometrioma) were determined based on endometriosis entity grouping by Chapron et al.\n17,18. \nThere were no patients with deep infiltrating endometriosis. We estimated the sample size based on a conservative \nlower limit of the disease prevalence at 20% in a population and an anticipated Area Under Curve (AUC) of any \ncandidate biomarkers of 0.8, at 90% power with Type I error (false positives) fixed at 5% and Type II error (false \nnegatives) fixed at 5%. To this, 14 Stage I/II endometriotic subjects, 14 endometriotic Stage III/IV subjects and 56 \nnon-endometriosis women subjects was required to power the study. A value of AUC 0.5 is of no diagnostic value \nand 1 representing 100% sensitivity and specificity. While slightly underpowered in terms of non-endometriosis \nsubjects, in this exploratory study the sample size serves as a useful guide for future studies.\nMenstrual cycle phase was determined according to cycle history of the patients. Blood was collected in BD \nVacutainer\n®  SST II and serum prepared by spinning the tubes at 1,200× g for 10 min and the top yellowish layer \ntransferred to a clean 15 mL tube. Subsequently, the tube was spun at 3,600×  g for 10 min. The supernatant was \ncarefully removed and transferred into 1 mL aliquots and stored at − 80 °C until use.\nMass spectrometry analysis.  The LC-MS/MS analysis followed a published report with some modifi -\ncations19. Deuterium-labeled and non-deuterium-labeled oxylipins standards were obtained from Cayman \nChemicals (MI, USA). Oxylipins were extracted from 50 μ L serum by methanol–based protein precipitation and \ndeuterated standards were added as internal standards (ISTD).\nBriefly, Reversed-phase Liquid Chromatography (RPLC)-MS analysis was performed with Agilent 1290 Ultra \nPressure Liquid Chromatography (UPLC, Waldbronn, Germany) coupled to an electrospray ionization with iFun-\nnel Technology on a triple quadrupole mass spectrometer (6490 QQQ, Agilent Technologies). Chromatographic \nseparation was achieved using HT Zorbax SB-C18 column (2.1 ×  100 mm, 1.8 μ m; Agilent Technologies, CA, \nUSA) with a flow rate of 0.40 mL/min at 40 °C. The initial condition was set at 15% B, a 11 min linear gradient to \n60% B was applied, followed by a 17 min gradient to 100% B which was held for 5 min, then returned to starting \nconditions over 0.1 min., while using Solvents A, 0.1% aqueous acetic acid, and B, 50:50 v/v acetonitrile/ isopro-\npanol. The auto-sampler was cooled at 4 °C and 10 μ L of the extract was injected. Electrospray ionization was per-\nformed in negative mode with the following source parameters: drying gas (N2) temperature 200 °C with a flow of \n14 L/min, nebulizer gas pressure 30 psi, sheath gas temperature 400 °C with a flow of 11 L/min, capillary voltage \n\nwww.nature.com/scientificreports/\n3\nScientific  RepoRts  | 6:26117 | DOI: 10.1038/srep26117\n3,000 V and nozzle voltage 800 V . Oxylipins were quantified in Multiple Reaction Monitoring (MRM) mode and \ntheir nomenclature as defined in Supplemental Tables 2 and 3. Data acquisition and processing were performed \nusing MassHunter software (Agilent Technologies, CA, US).\nRecoveries were evaluated by spiking defined amounts of deuterated ISTDs into aliquots of unprocessed \nserum and calculated by comparing peak areas from serum against mean peak areas of three equal amounts of \nunprocessed compounds in pure solvent. The recoveries generally ranged from 55.0% to 65.2%. For intra-batch \nand inter-batch precision and accuracy, the relative standard deviation (RSD) values ranged from 2.5% to 18.9% \nand 1.5% to 15.9%, respectively. Because chromatography separated oxylipin classes according to different \nretention time groups, we used the closest eluting internal standard (based on structural similarity) for relative \nquantitation estimates. Oxylipins were quantified by normalizing to their corresponding ISTDs as described in \nSupplemental Table 2. Equation 1 was used for peak area normalizations for oxylipin\ni of samplej and ISTDk:\n=×oxylipin oxylipin /ISTDc oncentration ISTD (1)ij normalized ij raw k,,\nMultiplexed immunoassay.  The Luminex xMAP multiplexing technology and the Bio-Plex ®  platform \n(Bio-Rad Laboratories, CA, USA) were used as previously employed20. The method uses 5.5 μ m polystyrene beads \nlabelled with two fluorescent dyes in different ratios, which assigns them to specific antibodies and thus allows \nthe simultaneous measurement of 27 immunomodulatory proteins (cytokine, chemokine and growth factor) in \n25 μ L of serum. Serum was diluted four times prior to analysis. Data analysis of experimental data was carried out \nusing five-parameter logistic regression modeling on the Bio-Plex system (Bio-Rad). Calibrations and validations \nwere performed prior to analysis and on a monthly basis respectively. Measured immuno-modulators and assay \nparameters are reported in Supplemental Table 4.\nC-reactive protein analysis. Serum CRP levels were determined via Architect C8000 (Abbott Diagnostics, \nIllinois, USA) according to manufacturer’s protocol.\nStatistics. Data were first analyzed using D'Agostino-Pearson and Shapiro-Wilk normality tests to evaluate if \nthey followed Gaussian distribution or not. Subsequently, the appropriate tests were used to test for statistical sig-\nnificance–Mann Whitney and Kruskal-Wallis test for non-Gaussian distributed data and Student’s t-test and 1-way \nANOV A for Gaussian distributed data. Changes were deemed significant when p <  0.05 and % fold change > 50%.\nResults\nData from a total of 103 women who underwent diagnostic laparoscopy were used to assess changes of \nendometriosis-associated systemic inflammation. Of the 103 subjects, 46 women did not have endometriosis \n(designated “EM− ”), 19 women with rAFS Stage I/II (designated “EM+ \nMild”) and 38 with Stages III/IV (desig-\nnated “EM+ Sev”) (Supplemental Table 1). The mean age was 34.6 ±  7.2 years (mean ±  SD) with no statistical dif-\nference between groups. Chinese women form the majority (67.3%), followed by Malays (14.4%), Indians (7.7%) \nand women of other Southeast Asian heritage (10.6%). Women with proliferative or secretory phase were not \nsignificantly different between EM−  and EM+ . There was significant difference in the menstrual cycle comparing \nEM− , EM+ \nMild and EM+  Sev where numbers of EM+  Mild women at proliferative phase were lower (p  =  0.036). \nThere was a significant difference of the endometriosis types between EM+  Mild and EM+  Sev (peritoneal versus \novarian endometriosis; p <  0.0001).\nOur targeted LC-MS/MS method allowed the quantification of 50 oxylipins (validated with 50 external stand-\nards) and 5 internal standards (Supplemental Tables 2 and 3) which afforded compound identity and quantifica-\ntion reliability and accuracy, and also a targeted analysis of pro-inflammatory LA and AA-derived n-6 oxylipins. \nAmong these 50 oxylipins, 20 were readily detectable in sera, and the four most abundant oxylipins were AA, \nLA, 9-HODE and 13-HODE in decreasing order (mean ±  standard deviation: 68.5 ±  15.9 nM, 19.3 ±  4.2 nM, \n3.3 ±  2.4 nM, 3.1 ±  2.5 nM respectively). The mean concentration levels of the remaining detectable oxylipins were \n< 1 nM. No significant difference in serum oxylipins between EM−   and EM+ women was found. Stratification \naccording to rAFS stages (I and II versus III and IV) or pre-operative pain symptoms did not result in significant \ndifferences relative to EM− . Stratifying by endometriosis type (ovarian/peritoneal), women with predominant \nendometriomas had significantly lower serum 12-HETE relative to EM−   (− 50.7%; p =  0.03) (Table 1). When \nmatched for menstrual phase (proliferative versus  secretory), EM−   women had significantly higher 8-HETE \n(54.7%; p =  0.04), 11-HETE (61.6%, p =  0.02), 15-HETE (57.65, p =  0.03) and 5-oxoETE (52.4%; p =  0.04) in the \nproliferative phase compared to the secretory phase (Table 2). While 14,15-DHET was statistically decreased in \nEM+ \nSev (p =  0.03), it was only 30.4% lower in the proliferative and was deemed insignificant (Table 2).\nAmong the 27 serum immunomodulatory proteins analyzed, 21 were detected (Table 3). The four most abun-\ndant immunomodulatory proteins were PGDF-bb, IP-10, IFNγ  and IL-1rα  in decreasing order (mean ±  standard \ndeviation: 7548.8 ±  4873.2 pg/mL, 1086.0 ±  501.3 pg/mL, 147.3 ±  89.2 pg/mL, 130.0 ±  70.0 pg/mL). Of the 27 fac-\ntors, IL-12(p70) and IL-13 were significantly decreased by 32% and 47% between EM−  and EM+  (p =  0.03 and \n0.02 respectively; Table 2, Fig. 1A). In our study, IL-1rα  , IL-6 and TNFα  results were statistically insignificant \nbetween EM+  to EM−  (p =  0.90, 0.31, 0.65 respectively), EM+  Mild to EM−   (p =  0.24; p =  0.42) and EM+  Sev to \nEM−  (p =  0.83; p =  0.77). Interestingly, IL-12(p70), IL-13 and VEGF were significantly lower by 39%, 54% and \n76% respectively in EM+ Mild compared to EM−  (Fig. 1B; Table 3).\nAdditionally, we used serum CRP as a non-specific marker of systemic inflammation. No difference in circu-\nlating CRP levels in EM− , EM+ Mild and EM+ Sev patients was found (p =  0.32; Fig. 1C), with median values con-\nsistent with that of healthy volunteers15. CRP levels were independent of the menstrual phases (pproliferative =  0.53 \nand psecretory =  0.35).\n\nwww.nature.com/scientificreports/\n4\nScientific  RepoRts  | 6:26117 | DOI: 10.1038/srep26117\nDiscussion\nEndometriosis is commonly associated with inflammation of the pelvic area and peritoneum. This hallmark has \nled to searches of inflammatory markers in the circulation which could potentially predict the presence of endo-\nmetriosis, and the possibility of a clinically silent systemic inflammatory state in women with endometriosis\n10. \nOur results, which covered three classes of molecules associated with systemic inflammation, namely oxylipins, \nimmunomodulatory proteins and CRP , were largely similar with minimal differences at a level which precludes \nAnalyte\nEM− Average \nconc. (nM)\nEM + Average \nconc. (nM)\nEM− vs EM+ \n(p-value)\nEM− vs EM+ Mild \n(p-value)\nEM− vs EM+ Sev \n(p-value)\nEM− vs EM+ ovar \n(p-value)\nEM− vs EM+ perit \n(p-value)\n5,6-DHET 0.075 0.079 0.82 0.16 0.47 0.66 0.33\n8,9-DHET 0.006 0.006 0.82 0.53 0.55 0.73 0.94\n11,12-DHET 0.053 0.050 0.60 0.06 0.81 0.81 0.19\n14,15-DHET 0.063 0.061 0.76 0.13 0.70 0.97 0.14\n12,13-DIHOME 0.693 0.632 0.58 0.67 0.65 0.44 0.86\n9,10-DIHOME 0.559 0.412 0.22 0.40 0.30 0.17 0.78\n5-HETE 0.061 0.060 0.91 0.95 0.85 0.83 0.77\n8-HETE 0.016 0.018 0.65 0.42 0.93 0.71 0.32\n11-HETE 0.227 0.201 0.66 1.00 0.54 0.66 0.91\n12-HETE 0.219 0.108 0.06 0.19 0.13 0.03 0.49\n13-HODE 3.041 3.211 0.74 0.90 0.59 0.45 0.86\n15-HETE 0.066 0.065 0.95 0.74 0.73 0.95 0.68\n20-HETE 0.004 0.004 0.47 0.08 0.94 0.91 0.20\n5-HEPE 0.016 0.016 0.90 0.58 0.67 0.61 0.50\n9-HODE 3.193 3.401 0.66 0.57 0.37 0.46 0.76\n9,10-EODE 0.499 0.442 0.46 0.85 0.34 0.51 0.74\n12,13-EODE 0.559 0.520 0.62 0.94 0.42 0.65 0.57\n5-OxoETE 0.016 0.019 0.55 0.22 0.99 0.85 0.15\nAA 69.094 67.976 0.72 0.62 0.85 0.71 0.49\nLA 19.025 19.528 0.55 0.48 0.22 0.35 0.57\nTable 1.  Summary of serum oxylipins in women with endometriosis (EM+) and without (EM−). Bold, \nsignificantly changed oxylipin (% change >  50%, p <  0.05).\nAnalyte\n% change of \nEM− (Prol)/EM− (Secr)\nEM− Prol vs \nSecr (p-value)\n% change of  \nEM+ Mild (Prol)/EM+ Mild (Secr)\nEM+ Mild Prol vs \nSecr (p-value)\n% change of  \nEM+ Sev (Prol)/EM+ Sev (Secr)\nEM+ Sev Prol vs \nSecr (p-value)\n9,10-DiHOME − 106.8 0.13 61.3 0.18 57.6 0.19\n12,13-DiHOME − 70.3 0.07 57.3 0.12 45.4 0.13\n5,6-DHET 8.1 0.55 − 7.1 0.59 3.0 0.99\n8,9-DHET − 3.5 0.81 − 43.3 0.99 − 6.5 0.55\n11,12-DHET − 12.8 0.45 − 34.8 0.68 − 27.3 0.07\n14,15-DHET − 23.0 0.19 − 46.6 0.27 − 30.4 0.03\n5-HETE 34.6 0.15 − 0.8 0.87 35.3 0.38\n8-HETE 54.7 0.04 61.0 0.36 115.6 0.11\n11-HETE 61.6 0.02 58.9 0.34 75.3 0.20\n12-HETE 43.5 0.23 35.9 0.55 − 43.1 0.30\n13-HODE 30.4 0.14 43.7 0.42 49.9 0.20\n15-HETE 57.6 0.03 62.2 0.30 80.6 0.18\n20-HETE − 20.2 0.31 − 8.5 0.79 − 25.5 0.14\n5-HEPE 18.6 0.41 − 19.3 0.38 17.6 0.73\n9-HODE 27.2 0.12 21.4 0.80 39.1 0.26\n9,10-EODE 17.8 0.53 36.8 0.57 − 15.6 0.46\n12,13-EODE 3.4 0.94 40.1 0.49 − 12.8 0.49\n5-OxoETE 52.4 0.04 51.4 0.47 48.3 0.41\nAA 5.4 0.25 − 13.9 0.71 1.5 0.98\nLA 2.8 0.59 − 1.0 0.88 3.5 0.77\nTable 2.  Summary of serum oxylipins in women with and without endometriosis at different menstrual \nphases. EM− , women without endometriosis; EM+ Mild, women with rAFS I or II endometriosis; EM+ Sev, \nwomen with rAFS III or IV endometriosis. Prol, proliferative phase; Secr, secretory phase. Bold, significantly \nchanged oxylipin (% change >  50%, p <  0.05).\n\nwww.nature.com/scientificreports/\n5\nScientific  RepoRts  | 6:26117 | DOI: 10.1038/srep26117\ntheir use as diagnostic biomarkers for endometriosis. This may explain why there has been no unequivocal con-\nsensus of the circulating cytokine levels in endometriosis4,8,9.\nLimited changes to systemic pro-inflammatory immunomodulatory proteins and oxylipins are consistent with \nreports of peripheral blood immune cell activation or cytokines in women with or without endometriosis21,22. We \ndid not find alterations in cytokines such as IL-1Rα , IL-6, TNFα  as reported by other groups2,23,24. Similar EM+ \noxylipin levels in the proliferative or secretory phase is congruent with others7,25. Additionally, comparing pain \nsymptomatic and asymptomatic groups, did not yield any significant differences between the two groups. This \ncan be plausibly reasoned by the increased expression of neurotrophic factors and nerve fibres in endometriotic \nlesions, eutopic endometrium and the peritoneum, and consequently the frequent association of pain with the \npelvic and uterine regions, rather than throughout the body\n26. Among the significantly different factors, with the \nexception of IL-13, are involved in a variety of physiologic and pathophysiologic events, and not just inflamma-\ntion, such as growth factor-like (IL-12(p70), VEGF and 12-HETE). IL-12(p70) mediates anti-angiogenic effects\n27, \nwhile 12-HETE and VEGF are a potent angiogenic factors plausibly involved in the maintenance of endometriotic \nlesions\n28,29. The significant differences in immunomodulatory proteins in EM+ Mild compared to EM−  are consist-\nent with other reports5. This suggests that incremental changes in immunomodulatory proteins are likely to take \nplace in the early phase of endometriosis development but was subverted by other unknown mechanisms later \nin the disease. Given the difference in our results with some reported\n2 and not others21,30,31 such conflicting data \nmay be attributed to (i) the heterogeneity of the disease and/or (ii) the use of different controls in different studies: \nwomen without endometriosis but may present other benign gynecological disorders, healthy women or a com-\nbination. Alternatively, there remains a possibility that larger study cohorts may result in statistically significant \nfindings and this study needs further verification. In addition, we did not find significant differences in serum \nCRP , consistent with a recent study\n32. Our results differed from another study which reported CRP levels to be \nsignificantly different in rAFS Stage III/IV endometriosis women in the first three days of the menstrual cycle33. \nPossible reasons for discrepancies include the report’s relatively smaller sample size and the temporally broader \ntiming of sampling in our study cohort. One would note with interest that a combination of 5 proteins, permu-\ntating between plasma annexin V , VEGF , CA-125, glycodelin or sICAM-1 could predict endometriosis\n34–none of \nwhich are of pro-inflammatory nature.\nTargeted ‘omics is a rapidly emerging bioanalytical field enabling the quantitative analysis of a large number \nof analytes associated with diseases 35,36. We have previously demonstrated the elevated levels of serum sphin-\ngolipids in women with endometriosis, suggesting a different pathophysiological mechanism of these bioactive \nlipids to that of oxylipins in endometriosis\n14. The imbalance of n-3 and n-6 PUFAs may lead to inflammation12 \nand suggests that targeted profiling of n-3 PUFAs may further clarify if the role of inflammatory resolving oxylip-\nins in endometriosis. Similarly, global ‘omics technologies including metabolomics and proteomics may fur -\nther test the hypothesis of endometriosis as a systemic inflammatory disease through the potential identification \nof pro-inflammatory circulating metabolites or proteins. Indeed, innovation global LC-MS/MS proteomics of \nthe serum may unravel disease-specific biomarkers\n37,38. Interestingly, while serum 1H-NMR and LC-MS/MS \nNo. Analyte\nEM− Average \nConcentration (pg/mL)\nEM+ Mild Average \nConcentration (pg/mL)\nEM+ Sev Average \nConcentration (pg/mL)\n% change \nEM+ Mild to EM−\n% change \nEM+ Sev to EM−\n1 IL-1β 1.66 1.52 1.76 8.8 − 6.0\n2 IL-1rα 128.29 118.58 138.18 7.6 − 7.7\n3 IL-4 3.51 3.33 3.90 5.3 − 11.0\n4 IL-6 7.54 8.12 10.87 − 7.7 − 44.2\n5 IL-7 14.70 11.60 15.13 21.1 − 2.9\n6 IL-8 14.60 12.28 14.10 15.8 3.4\n7 IL-9 15.79 8.51 16.36 46.1 − 3.6\n8 IL-10 12.60 7.63 9.94 39.5 21.1\n9 IL-12 (p70) 28.66 11.74 23.33 59.0 18.6\n10 IL-13 7.12 1.61 4.47 77.4 37.2\n11 IL-17 17.17 2.15 9.08 87.5 47.2\n12 Eotaxin 59.97 63.23 52.41 − 5.4 12.6\n13 FGF basic 27.38 13.23 26.87 51.7 1.9\n14 G-CSF 32.28 27.82 33.85 13.8 − 4.9\n15 IFN-γ 144.70 131.87 158.86 8.9 − 9.8\n16 IP-10 1186.68 826.18 1101.85 30.4 7.1\n17 MCP-1 17.90 20.21 22.55 − 12.9 − 26.0\n18 PDGF-bb 8491.17 5277.85 7611.71 37.8 10.4\n19 MIP-1β 107.28 76.52 87.59 28.7 18.4\n20 TNF-α 25.25 23.03 30.69 8.8 − 21.5\n21 VEGF 104.87 25.43 96.96 75.7 7.5\nTable 3.  Summary of serum immunomodulatory proteins in women with endometriosis (EM+) and \nwithout (EM−). EM− , women without endometriosis; EM+ Mild, women with rAFS I or II endometriosis; \nEM +  Sev, women with rAFS III or IV endometriosis.\n\nwww.nature.com/scientificreports/\n6\nScientific  RepoRts  | 6:26117 | DOI: 10.1038/srep26117\nmetabolomics of endometriosis patients revealed differential serum metabolites between endometriosis patients \nand those without, these metabolites were not considered of pro-inflammatory status39,40.\nThis study is the first to provide extensive profiles of pro-inflammatory protein and lipid mediators in the \ncirculation of women with endometriosis and our results reflected a limited systemic inflammation in endome-\ntriosis. The implications of our work include the (i) pro-inflammatory mediators in the classes studied may have \nlimited value as biomarkers for endometriosis, and (ii) further ‘omics work in identifying other related markers \nmay be warranted to definitively test the hypothesis that there is systemic inflammation in endometriosis.\nReferences\n1. Herington, J. L., Bruner-Tran, K. L., Lucas, J. A. & Osteen, K. G. Immune interactions in endometriosis. Expert Rev. Clin. Immunol. \n7, 611–626 (2011).\n2. Bedaiwy, M. A et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum. \nReprod. 17, 426–31 (2002).\n3. Seeber, B. et al. Panel of markers can accurately predict endometriosis in a subset of patients. Fertil. Steril. 89, 1073–81 (2008).\n4. Gupta, S. et al. Serum and peritoneal abnormalities in endometriosis  58, 527–551 (2006).\n5. Pizzo, A. et al. Behaviour of cytokine levels in serum and peritoneal fluid of women with endometriosis. 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Methods 7, 6125–6132 (2015).\nAcknowledgements\nWe thank Dr. Clement Goh and Johnson Setoh from KKH Clinical Chemistry laboratory for running the CRP \nanalysis. This study is funded by SingHealth Foundation (SHF/FG560P/2014) and National Medical Research \nFoundation (NMRC/BNIG/2033/2015).\nAuthor Contributions\nThe study was designed by L.Y .H. performed analysis of cytokines. C.L. and F .J.L. performed oxylipin analyses. \nL.Y .H. and J.C.K.Y . interpreted the data and wrote the manuscript. B.C., T.H.H. and J.C.K.Y . phenotyped the \npatients and obtained samples.\nAdditional Information\nSupplementary information accompanies this paper at http://www.nature.com/srep\nCompeting financial interests:  Chan J. K.Y . received salary support from the National Medical Research \nCouncil, Singapore (NMRC/CSA/043/2012). \nHow to cite this article: Lee, Y . H. et al. Limited value of pro-inflammatory oxylipins and cytokines as \ncirculating biomarkers in endometriosis – a targeted ‘omics study. Sci. Rep. 6, 26117; doi: 10.1038/srep26117 \n(2016).\nThis work is licensed under a Creative Commons Attribution 4.0 International License. The images \nor other third party material in this article are included in the article’s Creative Commons license, \nunless indicated otherwise in the credit line; if the material is not included under the Creative Commons \nlicense, users will need to obtain permission from the license holder to reproduce the material. To view a copy \nof this license, visit http://creativecommons.org/licenses/by/4.0/","source_license":"CC0","license_restricted":false}