{"paper_id":"e8fbc2c8-48fc-4f3c-815f-f2a92c041b6b","body_text":"R E S E A R C H Open Access\nCD4+/CD8+ mucosa-associated invariant T\ncells foster the development of\nendometriosis: a pilot study\nCaihua Li 1,2†, Zhimin Lu 1,2†, Kaihuan Bi 1,2, Kangxia Wang 1, Yuping Xu 1, Peipei Guo 1, Ya Chen 1, Ping Zhou 1,2,\nZhaolian Wei 1,2, Huanhuan Jiang 1* and Yunxia Cao 1,2*\nAbstract\nBackground: Immune dysregulation is one of the mechanisms to promote endometriosis (EMS). Various T cell\nsubpopulations have been reported to play different roles in the development of EMS. The mucosa-associated\ninvariant T cell (MAIT) is an important T cell subset in the pathogenesis of various autoimmune diseases. Evidence\nhas indicated that there are three functionally distinct MAIT subsets: CD4+, CD8+ and CD4/CD8 −/− (double\nnegative, DN) MAIT cells. Till now, the associations between endometriosis and MAIT have not been studied. Our\nresearch investigates different MAIT subpopulations in peripheral blood (PB) and peritoneal fluid (PF) from EMS\npatients.\nMethods: Thirty-two EMS patients and eighteen controls were included. PB and PF were collected. Tests of\ncytokines in plasma and PF were performed by ELISA kit. Characterisations of MAIT were done by flow cytometry.\nMAIT cells have been defined as CD3 + CD161 + V α7.2+ cells. Based on CD4 and CD8 expression, they were divided\ninto CD8 +MAIT, CD4 +MAIT and DN MAIT.\nResults: Enrichments of MAIT cells, especially CD4 and CD8 MAIT subsets were found. Moreover, CD8 MAIT cells\nhad a high activation in the EMS group. EMS patients produced higher level of IL-8/12/17 as compared to these\nfrom controls. On the contrary, control patients exhibited an impressive upregulation of DN MAIT cells, however,\nthese DN MAIT cells from controls showed a higher expression of PD-1. Lastly, we performed the relevance analysis,\nand discovered that the accumulation of PB MAIT cells positively correlated with an elevated level of serum CA125\nproduction in EMS group.\nConclusion: These results suggest that different MAIT subsets play distinct roles in the progression of\nendometriosis.\nKeywords: Endometriosis, Mucosa-associated invariant T (MAIT) cells, IL-17, Peritoneal fluid\nIntroduction\nEndometriosis (EMS) is a chronic disease which is charac-\nterised by the presence of endometrial cells outside the\nuterus [1, 2]. It affects up to 10% of women of reproduct-\nive age, who suffer symptoms, such as dysmenorrhea,\nchronic pelvic pain, pelvic inflammatory reactions as well\nas infertility [ 3, 4]. The pathogenesis of endometriosis has\nbeen studied for decades, however, a clear answer is still\nmissing.\nMany reports have indicated that the process of endo-\nmetriosis is related to a dysregulation of the host immune\nand some researchers even consider EMS to be an auto-\nimmune disorder [5–12]. On one hand, some studies have\nbeen focused on downregulation of anti-endometrial im-\nplants cells, such as NK cells, CD4 +/CD8+ T cells, B cells\nand so on [ 5–8], on the other hand, some studies demon-\nstrated that increased immunosuppressive cells could pro-\nmote the progression of endometriosis, such as Tregs\n(regulatory T cells), TH2 (T helper) cells and even MDSCs\n© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\n* Correspondence: jhh198673@163.com; caoyunxia6@126.com\n†Caihua Li and Zhimin Lu are joint first authors\n1Reproductive Medicine Center, Department of Obstetrics and Gynecology,\nthe First Affiliated Hospital of Anhui Medical University, Wanshui Road Nr.120,\n230000 Hefei, People ’s Republic of China\nFull list of author information is available at the end of the article\nLi et al. Reproductive Biology and Endocrinology           (2019) 17:78 \nhttps://doi.org/10.1186/s12958-019-0524-5\n\n(myeloid derived suppressor cells), which have been sug-\ngested recently to promote the implantation of endomet-\nrial tissue [ 13, 14]. All these evidences pointed to the fact\nthat the impaired immune response exists in\nendometriosis.\nMucosal-associated invariant T (MAIT) cells are non-\nclassical T lymphocytes characterised by a semi-\ninvariant T cell receptor (TCR) which has been evolu-\ntionarily conserved [ 15–18]. This TCR consists of a re-\nstricted α chain (V α7.2-Jα33 in humans and V α19-Jα33\nin mice) and one of the several β chains [ 16, 17]. It has\nbeen known that MAIT cells take part in both innate\nand adaptive immune response and can be found in per-\nipheral blood and other tissues [ 18, 19]. However, till\nnow there is no study about the relationship between\nMAIT cells and EMS, even though other kinds of T cells\nhave been fully studied, such as TH17, TH1, TH2 and\nso on [ 10, 12, 20, 21]. In humans, MAIT cells have been\ndefined as CD3 +CD161+Vα7.2+ cells. Based on CD4 and\nCD8 expression, MAIT cells can be divided into\nCD8+MAIT cells and CD4/CD8 −/− (double negative,\nDN) MAIT cells which are the major population of\nMAIT cells, and CD4 +MAIT cells which are only up to\n2–11% of MAIT cells in human blood [ 18, 19]. MAIT\ncells have the ability to recognize microbial-derived vita-\nmin B metabolites presented by the major histocompati-\nbility complex class I (MHC I) - related protein 1 (MR1)\nand allow them to detect various strains of bacteria and\nyeasts in vitro and in vivo [ 22, 23]. Recently, multiple ev-\nidences showed that human MAIT cells with high ex-\npression of IL-18 α could be activated by the pro-\ninflammatory cytokines IL-12 and IL-18 in a TCR inde-\npendent manner [ 24–26]. Once activated, MAIT cells\nshow cytotoxic properties, and secrete pro-inflammatory\ncytokines such as IL-17 and IFN- γ [23, 27]. It has been\nreported that both blood and peritoneal fluid from endo-\nmetriosis patients are rich in IL12 and IL18 [ 28]. There-\nfore, we have the hypothesis that MAIT cells can be\nactivated in endometriosis patients.\nNext to the role of MAIT cells in mediating anti-\nmicrobial defenses, they have been implicated in the de-\nvelopment of autoimmune and immunological diseases\n(multiple sclerosis, inflammatory bowel disease and in-\nflammatory arthritis) [ 26, 29–31]. This suggests that\nMAIT cells may play a role in the manifestation of in-\nflammatory responses in the absence of infection. Due\nto the functions of MAIT cells, many studies about dif-\nferent types of cancer suggested that MAIT cells have a\ngreat ability to destroy cancer cells [ 32–34]. However,\nsome recent researches revealed that MAIT cells showed\na potential to promote the progression of tumor by en-\nhancing an immunosuppressive microenvironment\nwhich is induced by the cytokines produced by MAIT\ncells [ 35, 36]. For instance, it has been indicated that\nMAIT cells have the ability to facilitate the recruitment\nof MDSCs [ 37]. Since endometriosis has been reported\nto be a chronic inflammatory disease with malignant ac-\ntivities, what kind of role will MAIT cells play in endo-\nmetriosis? Will they promote or prevent the\ndevelopment of endometriosis? Our study aims to assess\nthe immune disorder of endometriosis by analyzing\nMAIT cell subpopulations and cytokine levels in the PF\nand PB from patients with endometriosis and controls.\nThe present study reveals the relation between MAIT\ncells and endometriosis, thus finds out dysregulation of\nMAIT cells might be related to the immune disorder of\nendometriosis.\nMethods\nPatient selection\nThe study group comprised 32 patients with a diagnosis\nof endometriosis. They had their laparoscopy at the De-\npartment of Gynecology, the First Affiliated Hospital of\nAnhui Medical University from January 2018 to Febru-\nary 2019. Eighteen women with benign ovarian cyst (ser-\nous or dermoid) or uterine leiomyoma who underwent\nlaparoscopy were recruited as control group. The selec-\ntion of our control group is according to previous stud-\nies [ 9, 11, 38]. Table 1 displays the age, serum CA125\nand menstrual days from both cases and control groups.\nThe same medical team performed the surgeries for all\npatients. All patients had normal menstrual cycle, and\nthe samples were taken when patients were at 5 –14 days\nof their menstruation, and the antral follicular diameter\nfrom all patients were ≤ 10 mm which was measured via\nvaginal ultrasound before surgery. Peripheral blood (PB)\nwas obtained shortly before the surgery. Peritoneal fluid\n(PF) was collected during laparoscopy. And peritoneal\nfluid samples with contamination of blood were dis-\ncarded. Therefore, we got 29 PF samples from EMS pa-\ntients and 10 samples from controls. The stage of\nendometriosis was scored according to the proposed re-\nvised American Society for Reproductive Medicine\n(rASRM) classification (Revised American Society for\nReproductive Medicine classification of endometriosis,\n1996) [ 39]. Table 1 shows the percentage of patients\nwith different staged endometriosis: 7 - stage I (21.9%),\n10 - stage II (31.2%), 8 - stage III (25.0%) and 7 - stage\nIV (21.9%) (Table 1).\na) Age at the sample collecting; b) CA125 before sur-\ngery; NS Not statistically significant\nSample preparation\nHeparinised PB samples from all patients were taken in a\nsterile condition and centrifugated with the density gradi-\nent centrifugation by Biocoll (Biochrom, Berlin, Germany).\nThe procedure was conducted according to the manufac-\nturer’s instructions. Peripheral blood mononuclear cells\nLi et al. Reproductive Biology and Endocrinology           (2019) 17:78 Page 2 of 9\n\n(PBMCs) and plasma were collected. Clear PF samples\nwere centrifuged with a speed of 2000 rpm for 10 min.\nPF supernatant and plasma was stored at − 80 °C. The\nPBMC and cell pellets from PF were counted using a\nNeubauer counting chamber and adjusted to 10 7 cells.\nAll cell samples were cryopreserved in the medium\ncontaining X-VIVO supplemented with 30% human\nserum and 10% DMSO at − 80 °C. All samples were\nstored until needed for analysis avoiding freeze-thaw\ncycles.\nFlow cytometry analysis\nThe staining procedure was the same as described be-\nfore. In short, after thawing, samples were treated with\nFcR Blocking Reagent (Miltenyi Biotech, Germany) and\nstained accordingly with human anti-bodies (mAbs).\nThey were anti-CD8-PerCP-Cy5.5, anti-CD3-APC-Cy7\n(SP34–2), anti-CD4-FITC, anti-V α 7.2-PE, anti-CD161-\nAPC, anti-CD38-PE-Cy7, anti-CD279-PE-Cy7 and all of\nthem were from BD Biosciences (Heidelberg, Germany).\nThen samples were washed. Acquisition was carried out\nby six-color flow cytometry using FACSVerse ™ flow cy-\ntometry (BD Biosciences) with FACSuite software (BD\nBiosciences). Analyses of the data were made by FlowJo\nsoftware (Tree Star, Ashland, OR, USA).\nCytokines measurements in plasma and PF from patients\nby ELISA\nIL-8, 12, 18, 17, MMP-9, INF- γ from the peritoneal fluid\nand plasma were analysed by ELISA kit (Multisciences\nBiotech, Hangzhou, China). The procedure was per-\nformed as the manufacturer indicated. Briefly, a polystyr-\nene microplate of 96 well pre-coated with monoclonal\nantibody specific for each cytokine was used for each\ntest. After final staining and washing, the optical density\nwas determined.\nStatistical analysis\nGraphPad Prism software (GraphPad Software, San Diego,\nUSA) was used for statistical analysis. A one-way ANOVA\ntest and an unpaired Student ’s t test were performed\nrespectively for multiple groups ’ results or two groups ’\nresults. Spearman analysis was performed for correlation\ntest. Correlation coefficient is presented as r.A p value\nless than 0.05 was considered statistical difference.\nResults\nPresence of MAIT cells in PB and PF from patients\nTo clarify the role of MAIT cells in the pathogenesis of\nendometriosis, we first tried to examine their existence\nin the PB (Fig. 1a) and PF (Fig. 1b) from endometriosis\npatients and controls. We characterised MAIT cells as\nCD3+CD161+Vα 7.2+ cells, and divided them into\nCD8−CD4−, CD8 +CD4− and CD8 −CD4+ three subpopu-\nlations. Figure 1 shows the gating way of MAIT cells.\nEnrichment of the cytokines IL-8/12/17 in EMS patients\nTo clarify if MAIT cells would be functional in the pro-\ngression of EMS, we analysed IL-8, 12, 18, 17, MMP-9,\nINF-γ in plasma and PF samples from all patients using\nthe ELISA kit. PF samples from EMS patients displayed\na significant higher production of IL-8/12/17 (Fig. 2a, b,\nc) as compared to those from controls (11.30 ± 2.46 vs\n21.50 ± 3.04, P = 0.0447; 4.23 ± 0.59 vs 8.70 ± 1.38, P =\n0.0485; 4.50 ± 0.61 vs 12.56 ± 2.86, P = 0.0431) (Add-\nitional file 1: Table S1). When we divided all endometri-\nosis patients into two groups: early staged group (stages\nI and II) and late staged group (stages III and IV), late\nstaged EMS patients were found to produce increased\nlevels of PF IL-8/12/17 (Fig. 2d, e, f) as compared to those\nfrom controls (11.30 ± 2.46 vs 26.37 ± 4.86, P = 0.0288),\nand the early staged EMS group showed higher secretion\nof PF IL-12 and IL-17 (Fig. 2e, f) (4.23 ± 0.59 vs 8.15 ±\n1.50, P = 0.0220; 4.23 ± 0.59 vs 9.42 ± 2.62, P = 0.0356;\nTable 1 Characteristics of EMS and control patients\nBaseline All stage EMS\nEarly stage\nLate stage CG P value\nSubjects (n) 3 21 71 51 8\nEMS stage, n (%)\nI 7 (21.9%) 7\nII 10 (31.2%) 10\nIII 8 (25.0%) 8\nIV 7 (21.9%) 7\nUterine leiomyoma 10 (55.6%)\nOvarian benign cysts 8 (44.4%)\nAge (years)a 32.6 ± 1.10 33.8 ± 1.53 31.3 ± 1.58 33.3 ± 1.23 NS\nMenstrual days 27.6 ± 0.63 27.4 ± 0.88 27.9 ± 0.92 27.8 ± 0.76 NS\nCA125b 83.0 ± 10.2 72.4 ± 14.9 95.1 ± 13.7 9.48 ± 0.86 < 0.0001\nLi et al. Reproductive Biology and Endocrinology           (2019) 17:78 Page 3 of 9\n\n4.50 ± 0.61 vs 11.19 ± 3.10, P = 0.0406; 4.50 ± 0.61 vs\n14.20 ± 5.24, P = 0.0499). We could not find any differ-\nences for cytokines in blood (Additional file 1: Table S1).\nEnhancement of different MAIT cells in EMS patients and\ncontrols\nTo elucidate the involvement of MAIT cells in endomet-\nriosis development, we first analysed them in PB from\nall patients. Our results showed that EMS patients dis-\nplayed a remarkably higher level of PB MAIT cells as\ncompared to these from controls (Fig. 3a) (3.10 ± 0.43 vs\n5.70 ± 0.52, P = 0.0013). Next we tried to find any differ-\nence for the three MAIT subpopulations: CD8 −CD4−,\nCD8+CD4− and CD8 −CD4+MAIT cells. We could not\nfind any difference for these three subpopulations in PB\nfrom different groups. Subsequently, we wanted to dis-\ncover the MAIT cells ’ patterns in the microenvironment\nof endometriosis. We characterised MAIT cells in the\nPF from all patients. The frequency of PF MAIT cells\nwas detected to be increased in EMS patients as com-\npared to controls (Fig. 3b) (5.82 ± 0.79 vs 10.56 ± 1.08,\nP = 0.0175). Out of the three subpopulations, CD4 and\nCD8 MAIT cells subsets rose significantly in EMS\npatients (Fig. 3c, d) (0.30 ± 0.05 vs 0.67 ± 0.09, P =\n0.0295; 2.77 ± 0.42 vs 6.52 ± 1.05, P = 0.0454), Contrary\nto these results, EMS patients, especially the early stage\ngroup showed a dramatic deletion of DN MAIT cells\n(Fig. 3e, f) (3.39 ± 0.56 vs 1.73 ± 0.22, P = 0.0018; 3.39 ±\n0.56 vs 1.43 ± 0.28, P = 0.0023). We also found that there\nwere significant differences for the proportion of these\nthree MAIT subpopulations in PB and PF from patients\nwith EMS and controls (Table 2).\nFunctional activation of MAIT cells from EMS patients and\nrapid consumption of MAIT cells in controls\nAt the end, we tried to find the activities of MAIT cells.\nCD38 was used to be a marker of MAIT cells activation\nand PD-1 was used as a marker of MAIT cells dysfunc-\ntion (Fig. 4a, b) [ 22]. The expression of CD38 was ele-\nvated in the CD8 + MAIT cells in PF from EMS group\n(Fig. 4c) (0.36 ± 0.09 vs 2.98 ± 0.53, P = 0.0071), espe-\ncially the early stage group as compared to these from\ncontrols (Fig. 4d) (0.36 ± 0.09 vs 3.00 ± 0.76, P = 0.0282),\nwhereas, the control group showed DN MAIT cells with\nan increased expression of PD-1 in PF as compared to\nthose from EMS patients (Fig. 4e) (1.74 ± 0.46 vs\nFig. 1 Cytometric characterisation of MAIT cells. Cells were from one EMS patient and one control. a and b exhibit PB sample and PF sample\nrespectively. Live cells were gated (CD3 +CD161+), and then V α7.2+ cells were gated. The last gating step was based on CD4 and CD8, and three\nsubsets were identified: CD8 MAIT cells, DN MAIT cells and CD4 MAIT cells. Data is shown as pseudocolor\nLi et al. Reproductive Biology and Endocrinology           (2019) 17:78 Page 4 of 9\n\nFig. 3 Different distribution of MAIT subsets in EMS and control patients. a and b show a significant increase of PB and PF MAIT cells (CD3 +\nCD161+ TCRV α 7.2+) from EMS group as compared with the control group. c and d display higher levels of PF CD4 and CD8 MAIT cells\nsubpopulations from EMS patients as compared to those from controls. e and f exhibit a remarkable downregulation of PF DN MAIT cells in EMS\ngroup. Scatter plot represents the frequencies of MAIT cells in EMS group ( n = 32 in PB and n = 29 in PF) and control group ( n = 18 in PB and\nn = 10 in PF). * indicates P < 0.05 and ** indicates P < 0.01\nFig. 2 Comparation of cytokines in PF between EMS group and CG by ELISA. a–f Graphical representations display levels of IL-8/12/17 in PF from\nstudy and control groups. Concentrations are shown as the mean ± SEM. * indicates P < 0.05\nLi et al. Reproductive Biology and Endocrinology           (2019) 17:78 Page 5 of 9\n\n0.63 ± 0.10, P = 0.0012), both early and late stage\ngroups (Fig. 4f) (1.74 ± 0.46 vs 0.67 ± 0.15, P = 0.0123;\n1.74 ± 0.46 vs 0,58 ± 0.14, P = 0.0089).\nThe correlation between different MAIT cells and related\ncytokines and CA125 in EMS patients\nThe serum CA125 level from EMS groups was remark-\nably elevated compared to the control group (Table 1).\nA positive association between the PB MAIT cells and\nCA125 from patients with endometriosis was discovered\n(r = 0.39 and P < 0.05, Fig. 5a), whilst DN MAIT cells\nwere found to be positively related to CD8 MAIT cells\nin PF from endometriosis ( r = 0.62 and P < 0.01, Fig. 5b).\nWe could not find more correlation between other\nparameters.\nDiscussion\nEndometriosis has been extensively studied for de-\ncades, however the clear mechanisms for its patho-\ngenesis are still poorly understood, due to the\ncomplexity of its initiation and process. One of the\nmost known hypotheses is th e retrograde menstru-\nation theory of Sampson [ 40]. His theory indicated\nthat endometrial cells (epithelium and/or glands)\nflowed through the fallopian tubes to the pelvis, and\nthen implanted on other organs in the pelvis or even\noutside pelvis [ 40]. The haze is why only 10% of\nwomen have endometriosis, while retrograde\nmenstrual flow happens to a high number of women\n[9, 10]. One explanation could be the microenviron-\nment, especially the immunological and inflammatory\nfactors.\nAn area of great interest in endometriosis is soaring in\nimmunological activity and its function in development\nof this condition [ 6, 9, 10, 12]. The endometriosis-\nassociated immunological reactions were well reported\nin previous studies indicating the abnormalities in the\nfrequency and function of T cells and their associated\nTable 2 Frequencies of MAIT subpopulations in PB and PF\nCD8 MAIT DN MAIT CD4 MAIT P value\nPB (%) CG 2.45 ± 0.34 1.42 ± 0.19 1.03 ± 0.09 0.0002 a, b\nEMS 2.50 ± 0.33 1.70 ± 0.24 1.29 ± 0.13 0.003 b\nPF (%) CG 2.77 ± 0.42 3.39 ± 0.56 0.30 ± 0.05 < 0.0001 b, c\nEMS 6.52 ± 1.05 1.73 ± 0.22 0.67 ± 0.09 < 0.0001 a, b\nPercentages presented as mean ± SEM\naGroup A significantly different from Group B,\nbGroup A significantly different from Group C,\ncGroup B significantly different from Group C\nThe comparison between endometriosis patients and controls are displayed in\nFig. 3\nFig. 4 Activation and exhaustion of MAIT cells in peritoneal microenvironment from different groups. CD38 and PD-1 were used as a marker for\nactivation of MAIT cells and exhaustion of MAIT cells respectively ( a and b). All EMS patients and early stage group have higher expression of\nCD38 on CD8 MAIT subset ( c and d). The control group shows a significantly higher level of PD-1 expression on DN MAIT cells as compared to\nthose from EMS patients ( e and f). * indicates P < 0.05 and ** indicates P < 0.01\nLi et al. Reproductive Biology and Endocrinology           (2019) 17:78 Page 6 of 9\n\ncytokines [9, 10, 20]. Our study indicates that alterations\nin circulating and local MAIT cells may be one mechan-\nism which causes immunological disorder in\nendometriosis.\nMAIT cells are abundant in the host immune system\n[17, 41]. As the name implies, they reside in the mucosa,\nbut they are also found in other organs or tissues such\nas the peripheral blood, lymphoid tissues, lung and liver\n[23, 30]. We are the first study to analyse them in the\nperitoneal fluid. Firstly, we found that MAIT cells also\nexist in the peritoneal fluid. They are CD8 +CD4−MAIT\ncells, CD8 −CD4−MAIT cells, and CD8 −CD4+MAIT cells.\nIn accordance with other studies, our results showed\nthat for endometriosis patients in both PB and PF the\nCD8 MAIT cells were the major subpopulation and\nCD4 MAIT cells were the minority, with DN MAIT cells\nin between (Fig. 1 and Table 2). As IL-17 producing\ncells, MAIT cells have been reported to have a similar\nfunction of TH17 cells [ 23, 30]. Multiple studies have\ndiscovered that TH17 cells are enriched in EMS patients\nand play a critical role in the progression of EMS [ 10,\n20, 42]. Therefore, we assumed that augment of MAIT\ncells could also contribute to the pathogenesis of EMS,\nand our results showed that EMS patients had increased\nfrequencies of MAIT cells in PB and PF. Furthermore,\nwe found that PF IL-17 was higher in EMS patients as\ncompared to controls, which was identical to other re-\nports [ 20].\nMAIT cells and their ability of IL-17 production were\npreviously studied in many autoimmune diseases and\nimmunological disorder, such as multiple sclerosis, in-\nflammatory arthritis, Type 1 diabetes, primary Sjogren ’s\nsyndrome and so on [ 26, 27, 30]. It was well studied that\nIL-17 was increased and took part in the pathological\nprocess in the above-mentioned diseases [ 30, 31]. Mean-\nwhile, some researches also suggested that MAIT cells\nwere equipped to launch TH17-skewed immune reac-\ntions [ 30]. Taken together, MAIT, TH17 and IL-17\ncould initiate a pro-inflammatory condition and even\ninduce an immunosuppressive microenvironment. Inter-\nestingly, Rudak and his colleagues proposed that MAIT\ncells may promote the tumor development in tumori-\ngenesis due to their potency of inducing MDSCs [ 37].\nHowever, the relation between these cells needs further\ninvestigation. Moreover, our findings revealed that PF\nsamples from EMS patients had elevated levels of IL-8\nand IL-12 (Fig. 2). These results give further evidences\nthat MAIT cells can be activated and function in endo-\nmetriosis. Unfortunately, we could not find any associ-\nation between measured cytokines and the frequencies\nof MAIT cells. Although, we discovered that in periph-\neral blood there was a positive relation between frequen-\ncies of MAIT cells and the level of serum CA125.\nAnother study also found that TH17 cells positively cor-\nrelated with serum CA125 [ 20]. This result might give a\nfurther hint that increased levels of MAIT cells are asso-\nciated with the severity of endometriosis.\nAs mentioned before, there are three subpopulations\nof MAIT cells, and they were reported to have different\nfunctions and participate in various diseases [ 43–45].\nThe present study showed an interesting outcome. In\nour study, we found increased PF CD4 and CD8 MAIT\ncells in EMS group and highly activated CD8 MAIT cells\nin EMS group and early EMS group. In contrast, there\nwas an elevation of DN MAIT cells within control group\nas compared to EMS patients, both early and late stage\ngroups. One recent study by Dias et al. indicated that\nDN MAIT cells might derive from CD8 MAIT subset,\nbut with different functions. In their study, they pointed\nout that DN MAIT cells had less cytolytic effect and\nwere more prone to apoptosis [ 44]. Similar to their\nstudy, there was a positive relation between CD8 MAIT\ncells and DN MAIT cells. We also found that DN MAIT\ncells had a high expression of PD-1 in control group.\nMeanwhile, these two subsets of MAIT cells play oppos-\nite roles in endometriosis. One explanation would be\nthat DN MAIT cells function as guardians in the im-\nmune system, and unlike the two other subsets, they\nFig. 5 Correlation between PB MAIT cells and CA125, and association between DN MAIT cells and CD8 MAIT cells in PF from patients with\nendometriosis. a shows the correlation between PB MAIT cells and CA125 ( r = 0.39 and P < 0.05). b exhibits the correlation between PF DN MAIT\ncells and PF CD8 MAIT cells ( r = 0.62 and P < 0.01)\nLi et al. Reproductive Biology and Endocrinology           (2019) 17:78 Page 7 of 9\n\nexhaust quickly without producing overloaded pro-\ninflammatory cytokines. However, further researches\nabout these mechanisms are needed.\nConclusion\nOur study revealed the role of MAIT cells in endometri-\nosis and different profiles of their three subpopulations\n(i.e. CD8 MAIT, CD4 MAIT and DN MAIT cells). The\noutcomes of our research have identified that the dis-\norder of MAIT cells might contribute to the immune\ndysregulation of endometriosis patients. CD4 and CD8\nMAIT cells could be drivers in the development of in\nendometriosis, whereas DN MAIT cells might be protec-\ntors for the host. Therefore, manipulation of these cells\nmight open new therapeutic strategies in the future.\nSupplementary information\nSupplementary information accompanies this paper at https://doi.org/10.\n1186/s12958-019-0524-5.\nAdditional file 1: Table S1. Cytokine levels in PB and PF.\nAbbreviations\nCG: Control group; DN: Double negative; EMS: Endometriosis; IFN: Interferon;\nIL: Interleukin; mAbs: Human anti-bodies; MAIT cells: Mucosa-associated\ninvariant T cells; MDSCs: Myeloid derived suppressor cells; MHC: Major\nhistocompatibility complex class; MMP: Matrix metalloprotein; MR: MHC I-\nrelated protein; NK: Natural killer cell; PB: Peripheral blood; PBMCs: Peripheral\nblood mononuclear cells; PD: Programmed cell death protein; PF: Peritoneal\nfluid; rASRM: revised American Society for Reproductive Medicine; TCR: T cell\nreceptor; TH: T helper; Tregs: Regulatory T cells\nAcknowledgments\nThe authors acknowledge Nicky Werner, an English teacher in Anhui Medical\nUniversity, for proof reading the article. We would like to thank the doctors\nand nurses who performed the surgeries for our patients.\nAuthors’ contributions\nHJ and YC participated in study design and manuscript drafting; CL and ZL\ncollected the samples and performed the main experiments. KB, KW, PG and\nYX helped to collect the clinical data and flow cytometry experiments; YC,\nPZ and ZW helped with the sample preparations. All authors read and\napproved the final manuscript.\nFunding\nThis work was supported by the National Natural Science funds of China for\nYoung Scholar [grant number 81701421] and the Province Natural Science\nfunds of Anhui [grant number 1808085QH273].\nAvailability of data and materials\nThe data supporting the conclusions of this article are available from the\ncorresponding author on reasonable request.\nEthics approval and consent to participate\nThis study was approved by the Ethics Review Board of the First Affiliated\nHospital of Anhui Medical University, China (No. 20170026). Written informed\nconsent was signed by all patients.\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthor details\n1Reproductive Medicine Center, Department of Obstetrics and Gynecology,\nthe First Affiliated Hospital of Anhui Medical University, Wanshui Road Nr.120,\n230000 Hefei, People ’s Republic of China. 2Anhui Province Key Laboratory of\nReproductive Health and Genetics, Hefei, People ’s Republic of China.\nReceived: 12 June 2019 Accepted: 23 September 2019\nReferences\n1. Vercellini P, Viganò P, Somigliana E, Fedele L. Endometriosis: pathogenesis\nand treatment. Nat Rev Endocrinol. 2014;10:261 –75.\n2. Peiris AN, Chaljub E, Medlock D. Endometriosis. JAMA. 2018;320:2608.\n3. Leeners B, Damaso F, Ochsenbein-Kölble N, Farquhar C. The effect of\npregnancy on endometriosis-facts or fiction? Hum Reprod Update. 2018;24:\n290–9.\n4. Giudice LC. Endometriosis. N Engl J Med. 2010;362:2389 –98.\n5. 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