{"paper_id":"37096727-1a2e-4277-9e21-f0dfda2f912e","body_text":"Endometriosis, characterized by presence of endometrial\ntissue outside of uterine cavity, is a chronic inflammatory\ndisorder that involves 6-10% of reproductive age\nwomen ( 1 ,  2 ).\nDespite numerous investigations regarding the pathogenesis\nof endometriosis, the definite aetiology remains\nundetermined. Many factors such as genetic predisposition,\nhormonal imbalance, environmental factors and, especially,\nimmune system disturbances are potential aetiologic\nfactors ( 3 - 5 ).\nSystemic and local changes in immune responses that include\nimpaired CD4+ T cells have been frequently reported\nas contributing factors in endometriosis pathogenesis ( 6 - 8 ).\nCD4+ T cells exert their potential role in endometriosis through cytokines that are involved in implantation and\nproliferation of ectopic endometrial cells, inflammation\nand angiogenesis ( 7 ,  9 - 12 ). Aberrations in CD4+ T cell\npopulations have been assessed in several endometriosis\nstudies ( 11 - 16 ); however, only one or two of these CD4+\nT cells were investigated in each of these studies. To date,\nthe trend in changes in all four CD4+ T subsets in early\nand advanced stages of endometriosis and between peripheral\nblood (PB) and peritoneal fluid (PF) in each stage\nof this disease have not been assessed in a single study.\nHence, the current study was designed and implemented\nto answer the following questions:\n1. Are there deviations in the numbers of T helper (Th)1,\nTh2, Th17 and regulatory T (Treg) cells in peripheral\nblood between stages I, II and III, IV of endometriosis\nand a control group?\n2. Are there deviations in the numbers of Th1, Th2,\nTh17 and Treg cells in PF between stages I, II and III,\nIV of endometriosis and a control group?\n3. Are there any changes in the number of Th1, Th2,\nTh17 and Treg cells between blood and PF in endometriosis\nstages I, II and III, IV and control group?\n\nThis case-control study enrolled 20 women with endometriosis\nconfirmed by observation of endometriotic lesions\nduring laparoscopy and pathological confirmation\nof disease in biopsies that were taken from endometriotic\nfoci as the case group and a control group comprised\nof 10 women with no evidence of endometriosis during\nlaparoscopy. All participants were 24 to 40 years of age.\nThe presence of endometriosis was confirmed by a gynaecologist\nduring laparoscopy. According to the revised\nAmerican Society for Reproductive Medicine classification\nof endometriosis ( 17 ), we divided the endometriosis\ngroup into two subgroups - 10 women with early stage\nendometriosis (stage I, II) and 10 women with advanced\nstage of this disease (stage III, IV). All control women\nunderwent laparoscopy for other diseases (dermoid or\nfollicular cysts) and endometriosis was not detected in\nany of these women. Women who had a history of autoimmunity,\ninflammatory disorders (including allergies)\nor other gynaecological diseases (e.g., polycystic ovary\nsyndrome) and those who received hormonal treatment\nduring three months before taking samples were excluded\nfrom the study.\nThe study was approved by the Ethics Committee at Tehran\nUniversity of Medical Sciences (TUMS), Tehran, Iran\n(Ethics code: IR.TUMS.MEDICINE.REC.1395.1073).\nThe samples were taken from women who referred to\nYas and Arash hospitals, both of which are TUMS affiliated\nwomen’s hospitals. All women signed an informed\nconsent form for study participation before entering the\nstudy. A total of 5 mL of peripheral blood (PB) was collected\nfrom the antecubital vein of each patient before\nthey underwent general anaesthesia. PF was aspirated by\nthe surgeon after insertion of the second trocar at the beginning\nof the laparoscopic procedure. The volume of PF\nvaried from 2 to 8 mL in different cases. In each group,\nthe samples were classified as follicular or luteal phase\nbased on the date of the patient’s last menopausal period,\nwhich was reported by each patient at the time of sampling\nand confirmed by pathologic reports in cases where\nsamples of endometriotic lesions were obtained for pathological\ninvestigations.\nPB and PF were collected in heparinized tubes and\ntransferred in sterile, cold conditions to the laboratory\nwhere they were diluted with phosphate-buffered saline\n(PBS) at a 1:1 ratio. The diluted PB or PF were layered\non Ficoll-Hypaque (Inno-train, Germany) and centrifuged\n(1000 g, 20 minutes). The cells in the interphase layer\nwere collected and transferred into new tubes and washed\ncompletely with PBS. After discarding the supernatant,\nthe precipitated mononuclear cells were suspended in culture\nmedium, and the number and viability of these cells\nwere determined by vital staining.\nPB or PF mononuclear cells were divided into two portions.\nOne part was used for detection of Treg cells and\nthe other for stimulation and recognition of Th1, Th2 and\nTh17 cells.\nTreg cells were considered to be CD4+CD25+CD127-\nFOXP3+ cells. For determination and evaluation of\nthe Treg cells, we stained the mononuclear cells with\nFITC-labelled anti-CD4, PE/Cy7-labelled anti-CD25,\nand APC-labelled anti-CD127 antibodies (Biolegend,\nCA, USA); after cell fixation and permeabilisation, the\ncells were stained with PE-conjugated anti-FOXP3 antibody.\nFor detection of Th1, Th2 and Th17 cells in PB or PF, the mononuclear cells were cultured\nin Roswell Park Memorial Institute medium (RPMI) 1640 (Biosera, France) supplemented with\n10% FBS (Gibco, UK), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, UK) in\n24-well cell culture plates and stimulated with phorbol myristate acetate (PMA, Sigma, St.\nLouise, MO, USA) at 50 ng/mL and ionomycin (Sigma-Aldrich, St. Louise, MO, USA) at 1 μg/mL\nconcentrations and incubated at 37°C and 5% CO 2 . After an hour, 10 μg/mL\nBrefeldin A (eBioscience, San Diego, CA, USA) was added. The cells were harvested after a\n5-hour incubation period by using PMA, Ionomycin and Brefeldin A, followed by staining\nwith FITC-conjugated anti-CD4 antibody. After fixation and permeabilisation, intracellular\ncytokines were stained with PE-Cy7-conjugated anti-interferon gamma (IFNγ), APC-conjugated\nanti-interleukin 4 (IL-4) and PE-conjugated anti-IL-17 antibodies. CD4+IFNγ+, CD4+IL-4+\nand CD4+IL-17+ cells were considered to be Th1, Th2 and Th17 cells, respectively.\nIsotype-matched fluoro- phore-conjugated antibodies were used as the controls.\nThe stained cells (10 5  cells) were investigated by BD FACSCalibur instrument\n(Becton Dickinson, CA).\nThe lymphocytes were gated according to forward and side scatters, which were\nrepresentative of the cell size and granularity. In the group of stimulated cells, we\nconsidered Th1 cells to be CD4+IFNγ+, Th2 were CD4+IL-4+ and Th17 were CD4+IL-17+. For\nTreg cell discrimination, first the CD4+CD127- cells were gated from the lymphocytes.\nThen, from these gated cells, CD25+FOXP3+ were specified. The percentage of the\nCD4+CD25+FOXP3+CD127- cells from the lymphocyte population was defined as the frequency of\nthe Treg cells. These percentages were calculated using FlowJo software (Version 7.6.1).\nThe gating procedure was similar for both PB and PF ( Fig . 1 ); however, the percentage of\nthe lymphocytes was different in PB and PF.\nBecause of the non-normal distribution of the samples, we used the Kruskal-Wallis test to compare the frequency of the Th1, Th2, Th17 and Treg cells in PB and PF samples between the three groups. The Wilcoxon test was used to compare the percentage of each T cell population in each group between PB and PF. P values <0.05 were considered to be statistically significant. SPSS version 19 and GraphPad prism Version.6 software was used for data analysis and for drawing the plots.\nGating strategy for the detection of regulatory T cells (Treg) (upper row) and T\nhelper (Th)1, Th2 and Th17 cells (lower row) in peripheral blood (PB). The sample is\nfrom a patient in stage III, IV endometriosis (follicular phase).\nCD4+CD25+FOXP3+CD127- were defined as Treg cells. CD4+IFNγ+ were considered to be Th1\ncells, CD4+IL-4+ were Th2 cells and CD4+IL-17+ were considered to be the Th17\ncells.\n\nAfter considering the inclusion and exclusion criteria, 10 samples were selected in each group. We had six follicular and four luteal phase samples in each of the control and advanced endometriosis (stage III, IV) groups; however, in the early stages of endometriosis (stage I, II), we collected seven follicular and three luteal phase samples. The ages (median, minimum-maximum) of women in the three groups were similar: control (33.5,  27 -40 years), endometriosis stage I, II ( 32 ,  24 -38 years) and endometriosis stage III, IV ( 32 ,  25 -38 years) (P value: 0.875). The age medians were similar between groups when compared according to menstrual phase of sampling.\nOur results indicated similar numbers of Th1, Th2 and Th17 in blood samples of the three groups (P values: 0.78, 0.298, and 0.228,  Fig . 2 ). The Kruskal-Wallis test results showed different numbers of Treg cells between the three groups (P=0.042) with mean ranks of 13.10 (control), 12.20 (stage I, II), and 21.20 (stage III, IV); however, a pairwise comparison between each pair of groups indicated that none of the P values were statistically different and the adjusted P values were 0.067 (stage I, II vs. stage III, IV of endometriosis), 0.119 (control vs. stage III, IV of endometriosis) and 1 (control vs. stage I, II of endometriosis). Our results showed no significant differences in the four blood CD4+ T cell subsets when they were compared based on menstrual phases.\nThe numbers of Th1, Th2 and Treg cells were not\ndifferent in the PF of the three groups; however, there\nwas only an increased number of Th2 cells in the PF of\nendometriosis stage I, II (mean rank=10) compared to\nstage III, IV (mean rank=3.5) in the luteal phase (P=0.035).\nThere were increased numbers of Th17 cells in the PF of\nadvanced endometriosis (stage III, IV) cases compared to\ncontrols (adjusted P=0.003, mean rank of 22.4 in advanced\nendometriosis vs. 9.30 in the control). This trend was\nseen in both the follicular (P=0.059) and luteal [P=0.045,\nmean ranks were 8.25 (advanced endometriosis) and 2.75\n(control)] phases. The frequency of Th17 cells did not\ndiffer between the other groups (Figes . 2 ,  3 ).\nFrequency of T helper (Th)1, Th2, Th17 and regulatory T (Treg) cells compared in\nperipheral blood (PB) (upper row) and peritoneal fluid (PF) (lower row) amongst the\ncontrol, early stages of endometriosis (stage I, II) and advanced endometriosis (stage\nIII, IV) groups. Each box plot represents 25-75% quartiles with median.\nFrequency of T helper (Th)1, Th2, Th17 and regulatory T (Treg) cells in peritoneal\nfluid (PF) in the control, early endometriosis (stage I, II) and advanced\nendometriosis (stage III, IV) groups in the follicular (upper row) and luteal (lower\nrow) phases. Each box plot represents 25-75% quartiles with the median. *; P\nvalue<0.05.\nFrequency of T helper (Th)1, Th2, Th17 and regulatory T (Treg) cells were compared in\nperitoneal fluid (PF) with peripheral blood (PB) in the control (upper row), early\nendometriosis (stage I, II) (middle row) and advanced endometriosis (stage III, IV)\n(lower row) groups in the follicular phase. Each box plot represents 25-75% quartiles\nwith median. *; P value<0.05.\nThe control group had increased numbers of Treg and Th2 cells in the PF compared to PB (both, P=0.046). This up-regulation was only detected in the follicular phase when compared with the luteal phase ( Fig . 4 ).\nIn the early stages (I, II) of endometriosis, we observed increased numbers of Th2, Th17 and Treg cells in the PF compared to PB (P values were 0.005, 0.047 and 0.013, respectively). This observation was only noted in the follicular phase (P-values were 0.018, 0.028 and 0.018, respectively). Th17 cells were the only CD4+ T cell subsets that increased in the PF of stage III, IV endometriosis (P=0.013,  Fig . 4 ). This change was also observed in the follicular phase (P=0.046). We did not observe any change in quantity of these T cell subsets between the PB and PF in the luteal phase in any of the groups.\n\nEndometriosis is a complex disorder with variable immune disturbances ( 18 ,  19 ); however, the role of CD4+ T lymphocytes deviation in different stages of disease is not completely elucidated.\nIn this study, we compared the frequencies of Th1, Th2, Th17 and Treg cells between early and advanced stages of endometriosis, and in women without endometriosis, as a control group. Because the population of immune cells varies according to hormonal changes in the menstrual cycle ( 20 ), we also compared these cells in the follicular and luteal phases. We found no systemic deviation in frequency of CD4+ T subsets in different stages of endometriosis; however, Th17 cells were increased in the PF in patients with advanced endometriosis compared to controls.\nFirst, we compared the numbers of four CD4+ T subsets in peripheral blood between the three groups. Our results did not show any significant differences in frequency of blood Th1, Th2, Th17 and Treg cells between the different groups. The systemic deviations of CD4+ T cell numbers between three groups were not reflected in our study. We found one study by Takamura et al. ( 18 ) that compared all four CD4+ T subsets concurrently in blood from patients with stage III, IV endometriosis (10 samples; 6 follicular\nand 4 luteal phases) with 10 normal individuals (4 follicular\nand 6 luteal phases). Similar to our results, they indicated\nno systemic changes in Th2, Th17 and Treg cells between\nthe endometriosis and control subjects ( 18 ). However,\nthese researchers confirmed an increased number of Th1\ncells in the blood of patients with endometriosis ( 18 ). This\nfinding differed from our results.\nRegarding Th1 and Th2 cells, Szyllo et al. reported diminished IFN-γ levels and augmented\nIL-4 production by stimulated blood T cells of endometriosis patients; however, the authors\ndid not divide the T cells into CD8 or CD4 cells ( 21 ). Also, the results of another study\nconfirmed increased systemic CD4+IL-4+ and CD4+IL-10+ cells in patients with endometriosis\ncompared to a control group ( 10 ). Our findings about circulatory Th2 frequencies were not\nconsistent with these studies ( 10 ,  21 ).\nIn terms of blood Th17 cells, three independent studies\ndemonstrated no significant differences in PB Th17 cell\nnumbers between endometriosis and control groups ( 14 ,\n 18 ,  22 ), which supported our findings.\nThere are contradictory results concerning the frequency\nof Treg cells in blood samples between endometriosis and\nhealthy women ( 11 ,  12 ,  18 ,  22 ). Although Olkowska-\nTruchanowicz et al. have reported decreased numbers of\nblood Treg cells in endometriosis ( 12 ), other investigators\n( 11 ,  18 ,  22 ) reported that blood Treg cells remained\nunchanged between endometriosis and controls, which\nsupported our results. Olkowska-Truchanowicz et\nal. compared the number of Treg cells in 17 blood\nsamples obtained during the follicular phase from\nwomen with advanced stages (III, IV) of endometriosis\nto 15 samples from the follicular phase in control\nwomen without endometriotic foci. They introduced\nCD4+CD25highFoxP3+ cells as the Treg cell population\n( 12 ). On the other hand, Gogacz et al. investigated the\nnumbers of Tregs in two groups with endometriosis (15\nsamples in early stages and 7 samples with advanced\nendometriosis) and 20 women with unexplained infertility\nwithout any evidence of endometriosis. These researchers\nintroduced CD4+CD25+FoxP3+ cells as Tregs and\nreported similar numbers of Treg cells between mild and\nadvanced endometriosis ( 11 ). Khan et al. evaluated the\nfrequency of Tregs amongst three groups – early stage\nendometriosis (n=15), advanced stage endometriosis\n(n=24) and control women (n=21), who were in different\nmenstrual phases. Like other researchers, this group\nintroduced CD4+CD25+FoxP3+ as Tregs and reported\nsimilar numbers of blood Tregs in endometriosis and\ncontrol samples ( 22 ).\nNext, we compared the number of different CD4+ T\nsubsets locally in the PF. Th17 cells were the only CD4+\nT cells increased in the PF of stage III, IV endometriosis\nrelative to the control group. This trend was observed\nin both the follicular and luteal phases. We could not\nfind any article that compared the number of all four\nCD4+ T subsets in the PF between endometriosis and\ncontrol groups; however, Takamura et al. compared all\nfour subsets in endometriotic and normal endometrial\ntissues, and reported increased Th17 cells in ectopic\ntissue compared to eutopic endometrium ( 18 ). Gogacz et\nal. compared the numbers of CD3+CD4+IL-17+ T cells\nbetween 22 infertile women with endometriosis (mild\nendometriosis n=15; severe endometriosis n=7) and 20\ninfertile women without any evidence of endometriosis.\nAll samples were taken during the follicular phase. These\nresearchers reported that the percentage of Th17 cells in\nthe PF was higher in the higher stages of endometriosis\n(III, IV) compared to the lower stages (I, II) in the\nfollicular phase; however, they did not observe any\nsignificant difference in the number of Th17 cells in the\nPF between the endometriosis and healthy groups. They\nconcluded that a correlation existed between the frequency\nof Th17 in PF and severity of endometriosis ( 14 ). Chang\net al. reported increased numbers of Th17 cells in the PF\nin endometriosis (stage I, II and stage III, IV) compared\nto control women. In comparison with other studies, the\nsample size of their study was more similar to our study\nand the results that pertained to the number of Th17 cells\nbetween stage III, IV and controls were concordant with\nour results ( 23 ). Our results suggested predominant Th17\nresponses in stage III, IV of endometriosis relative to the\ncontrol group.\nTh17 cells play prominent roles in induction of\ninflammation and development of endometriosis. IL17\nstimulates secretion of IL-8 by endometrial stromal cells\n(ESCs), expression of cyclooxygenase 2 and proliferation\nof ectopic endometrial cells. IL-8 could induce the\nadhesion of stromal cells to fibronectin. In this way, IL-17\ncould increase endometriotic lesions ( 24 - 26 ).\nAlthough some studies demonstrated increased levels\nof CD4+CD25+FoxP3+ cells in the PF ( 12 ,  22 ,  27 ) and\nectopic tissue ( 28 ) of endometriosis subjects, we did not\ndetect this increase in our study. On the other hand, some\nresearchers reported unvarying numbers of Tregs in PF\n( 11 ) or ectopic lesions ( 18 ,  29 ) in endometriosis, which\nsupported our results.\nWe did not detect any significant difference in\nfrequency of Th2 cells in the PF from different stages of\nendometriosis and controls; however, our results indicated\nhigher numbers of Th2 cells in the PF exclusively in the\nluteal phase in the early stages of endometriosis compared\nto advanced stages. There are studies that illustrated\ndeviation to Th2 immune responses, directly through\ninvestigating the expression of Th2 related cytokines or\ntranscription factors ( 10 ,  30 ) or, indirectly ( 18 ) through\ndeviation of Th1/Th2 proportions in endometriosis.\nIn the third step, we compared the frequency of\ndifferent CD4+ T cells between the PF and PB in the\ndifferent groups. We performed this comparison based\non the menstrual cycle. We observed a fluctuation in\nCD4+ numbers between PB and PF only in the follicular\nphase. This finding could be related to the dominancy\nof oestrogen in the follicular phase and of progesterone in the luteal phase ( 31 ). Endometriosis is characterized by a dependency on oestrogen ( 32 ) and resistance to progesterone ( 33 ), which may guide us to observed alterations in follicular phase.\nOur findings indicated that Th2 and Treg cells increased in the PF of the control group relative to blood. High doses of oestrogen could deviate immune responses from Th1 to Th2 ( 34 ). On the other hand, oestrogen levels increase from the pre-ovulatory to the end of the menstrual phase in the PF compared to blood ( 35 ,  36 ). Thus, in the last days of the follicular phase, which was compatible with the time of sampling, high peritoneal doses of oestrogen might cause shift of immune responses to Th2 ( 34 ). This could confirm the up-regulation of Th2 cells in the PF in control samples from the follicular phase. On the other hand, oestrogen could bind to its receptor on the surface of CD4+CD25-T cells and convert them to CD4+CD25+T cells that express FOXP3 and IL-10 ( 37 ). Increased oestrogen levels in PF in the pre-ovulatory phase could lead to upraised levels of Treg cells in PF compared to blood in the control samples. Compatible with our results, Khan et al. reported a modest increase in Treg cells in PF of normal subjects compared to PB ( 22 ).\nThe number of Th17 cells, as well as Th2 and Treg cells, in the early stages of\nendometriosis (stage I, II) were elevated in the PF compared to blood. The increased levels\nof Th17 cells in the PF compared to blood in stage (I, II) endometriosis could be explained\nby two assumptions. The first is related to production of C-C chemokine ligand 20 (CCL20) by\nectopic endometrial cells. CCL20 is stimulated by inflammatory cytokines such as tumour\nnecrosis factor-α (TNF-α) and IL-1β in the local microenvironment and it can attach to C-C\nchemokine receptor type 6 on Th17 cells, which leads to recruitment of these cells to the PF\n( 19 ,  38 ). The second assumption is related to increased differentiation of naive T cells to\nTh17 at the local site of endometriotic tissue, which is rich in transforming growth factor\nbeta (TGF-β), IL-23 and IL-6 ( 9 ,  39 ,  40 ).\nWith progression of endometriosis to stage III or IV, we found that only Th17 cells were increased in PF compared to PB. Similar to our findings, Gogacz et al. reported that there were elevated levels of Th17 cells in the PF of stage III, IV endometriosis, which was proportional to blood ( 14 ); however, other researchers reported no changes in the numbers of Th17 cells between the PF and PB in endometriosis subjects ( 22 ). It seems that Th17 cells could be involved in endometriosis development by production of inflammatory cytokines and angiogenic factors. Chang et al. confirmed that IL-27 from macrophages or ESCs could stimulate production of IL-10 by Th17 cells, which induces progression of endometriosis ( 23 ). In the higher stages of endometriosis, Th17 cells cause cyclooxygenase 2 induction, inflammation and cell adhesion. In this way, these cells could promote the development of endometriosis.\nWe did not locate any article that compared all four subsets of CD4+ T cells in both PB and PF between early and advanced stages of endometriosis and controls. However, our study had some limitations such as the small sample size. In addition, we did not use anti-CD3 antibody for more specific determination of Th and Treg cells.\nThis study provided a comprehensive view of systemic and local changes in Th1, Th2, Th17 and Treg cells in early and advanced stages of endometriosis. Identification and application of local factors that affect Th17 cells can provide novel approaches for future treatments of advanced endometriosis.\n\nIn this study, apparent changes in systemic CD4+ T cells were not found between different stages of endometriosis and the controls; however, our results indicated a predominance of Th17 cells in the local microenvironment of the PF. Th17 superiority could be related to disease progression because we observed a higher number of Th17 cells in the more advanced stages of endometriosis. However, future studies should be conducted to evaluate this relationship at the cellular and molecular levels.","source_license":"CC0","license_restricted":false}