{"paper_id":"8152ef77-863b-4056-8f6c-1613a70683d7","body_text":"Abstract\n!\nAim: The etiology of endometriosis remains un-\nknown, but increasing evidence suggests that im-\nmune regulation may be important. Our study\naimed to evaluate peripheral blood lymphocyte\nsubpopulations during the menstrual cycle in\nwomen with peritoneal and ovarian endometrio-\nsis relative to healthy women.\nMethods: In this study, 65 women with endome-\ntriosis (37 in the follicular phase and 28 in the lu-\nteal phase of the menstrual cycle) and 61 healthy\nwomen (33 in the follicular phase and 28 in the\nluteal phase) were enrolled. Flow cytometric\nanalysis measured peripheral blood lymphocyte\nsubpopulations. The serum levels of cortisol were\nalso determined.\nResults: In healthy controls, we detected an in-\ncreased concentration of cytotoxic (CD8 +) T cells\nand activated (HLA ‑DR) T cells in the luteal phase\ncompared with the follicular phase of the men-\nstrual cycle (p = 0.020 and p = 0.045), whereas no\nsuch fluctuation was detected in endometriosis.\nHowever, a marked increase in regulatory T-cell\nconcentration in the luteal phase was detected\nonly in endometriosis patients (p = 0.005). Wom-\nen with endometriosis had higher levels of serum\ncortisol (p = 0.022), which correlated with the\nconcentration of regulatory T cells (p = 0.048).\nConclusions: Women with endometriosis do not\nexhibit fluctuations in the concentrations of cyto-\ntoxic and activated peripheral blood lymphocytes\nduring the menstrual cycle. The marked fluctua-\ntion of regulatory T cells detected in endometrio-\nsis could be attributed to altered immune re-\nsponse.\nZusammenfassung\n!\nEinleitung: Endometriose ist ein häufiges, jedoch\nkomplexes gynäkologisches Syndrom unbekann-\nter Pathogenese, von dem Frauen im gebärfähigen\nAlter befallen sind. Die Ätiologie der Endometri-\nose bleibt unbekannt, aber immer häufigere Be-\nweise zeigen, dass das Immunsystem dabei eine\ngroße Rolle spielt. Unsere Studie zielte darauf ab,\ndie peripheren Blutlymphozytensubpopulationen\nwährend des Menstruationszyklus bei Frauen mit\nPeritoneal- und Ovarialendometriose im Ver-\ngleich zu gesunden Frauen zu bewerten.\nMethoden: Die Studie umfasste 65 Frauen mit\nEndometriose (37 in der Follikelphase und 28 in\nder Lutealphase des Menstruationszyklus) und\n61 gesunde Frauen (33 in der Follikelphase und\n28 in der Lutealphase des Menstruationszyklus).\nDie Lymphozytensubpopulationen wurden mit-\ntels Durchflusszytometrie festgestellt. Der Serum-\nspiegel von Cortisol wurde ebenfalls bestimmt.\nErgebnisse: Bei gesunden Probanden fanden wir\nim peripheren Blut eine erhöhte Konzentration\nvon zytotoxischen (CD8\n+) T-Zellen und aktivier-\nten (HLA ‑DR) T-Zellen in der Lutealphase gegen-\nüber der Follikelphase des Menstruationszyklus\n(p = 0,020 und p = 0,045), während keine solche\nSchwankung in der Endometriose festgestellt\nwurde. Ein deutlicher Anstieg der regulatorischen\nT-Zellen-Konzentration in der Lutealphase wurde\nnur bei Frauen mit Endometriose ermittelt. Frau-\nen mit Endometriose hatten einen höheren Se-\nrumspiegel von Cortisol (p = 0,022), der mit der\nKonzentration von regulatorischen T-Zellen\n(p = 0,048) korreliert.\nSchlussfolgerung: Frauen mit Endometriose zei-\ngen keine Konzentrationsschwankungen von zy-\ntotoxischen und aktivierten Lymphozyten im pe-\nripheren Blut während des Menstruationszyklus.\nIn der Endometriose nachgewiesene Schwankun-\ngen von regulatorischen T-Zellen können auf ver-\nänderte Immunantwort zurückgeführt werden.\nCytotoxic T-Cells in Peripheral Blood\nin Women with Endometriosis\nZytotoxische T-Zellen im peripheren Blut bei Frauen mit Endometriose\nAuthors N. Slabe 1, H. Meden-Vrtovec 1, I. Verdenik 1, R. Kosir-Pogacnik 1,A .I h a n2\nAffiliations 1 Obstetrics and Gynecology, University Clinical Centre Ljubljana, Ljubljana, Slovenia\n2 Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia\nKey words\nl\" endometriosis\nl\" gynecology\nl\" infertility\nl\" regulatory T cells\nl\" cell immunity\nSchlüsselwörter\nl\" Endometriose\nl\" Gynäkologie\nl\" Infertilität\nl\" regulatorische T‑Zellen\nl\" Zell‑Immunität\nreceived 16. 4. 2013\nrevised 3. 6. 2013\naccepted 17. 6. 2013\nBibliography\nDOI http://dx.doi.org/\n10.1055/s-0033-1350702\nGeburtsh Frauenheilk 2013; 73:\n1042–1048 © Georg Thieme\nVerlag KG Stuttgart · New York ·\nISSN 0016‑5751\nCorrespondence\nDr. Nina Slabe\nUniversity Clinical Centre\nLjubljana\nObstetrics and Gynecology\nSlajmerjeva 3\n1000 Ljubljana\nSlovenia\nnina.slabe@t-2.net\n1042\nSlabe N et al. Cytotoxic T-Cells in … Geburtsh Frauenheilk 2013; 73: 1042 –1048\nGebFra Science\n\n\nIntroduction\n!\nEndometriosis is a common gynecological condition that affects\n5–15 % of women of reproductive age. It is characterized by the\ngrowth and expansion of endometrial-like glands and stroma\noutside the uterine cavity, most often in the peritoneal cavity\nand ovaries [1]. The disease process typically involves the surface\nof the ovaries and pelvic peritoneum. It is a risk factor for specific\ninvasive subtypes of ovarian cancer. However, the steps in malig-\nnant transformation of ectopic endometrium still need to be\nunderstood [2]. Endometriosis represents an important cause of\nmorbidity and affects quality of life in affected women.\nThe etiology of endometriosis remains poorly understood, but\ncertain features suggest that immune regulation may be impor-\ntant. Characteristics frequently found in women with endome-\ntriosis are multiorgan involvement, familial occurrence, a genetic\nbasis, environmental factors, responsiveness to hormonal ther-\napy, previous tissue damage, polyclonal activation of B lympho-\ncytes, immunological abnormalities in the function of T and B\nlymphocytes, and other associated autoimmune diseases [3 –9].\nRisk factors often mentioned for endometriosis are menstrual\ncycle length, duration of menstrual bleeding, number of preg-\nnancies and miscarriages, body mass index and smoking status\n[10].\nIt is not known how the immune system recognizes ectopic en-\ndometrial lesions. It would be necessary for the immune cells\nthat are responsible for the destruction of ectopic endometrial le-\nsions – natural killer cells, cytotoxic T cells, and macrophages – to\nbe well regulated. This would allow the immune system to de-\nstroy only ectopic endometrial cells, and not endometrium with-\nin the uterine cavity. The main regulators of this process are reg-\nulatory T cells (Treg) [11–17].\nThe endometrium in the uterine cavity is infiltrated by a number\nof different immune cells, whose role is to maintain immunolog-\nical homeostasis within the uterus. Berbic et al. [18, 19] showed\nthat Treg populations are altered in the endometrium of women\nwith endometriosis during the luteal phase of the menstrual\ncycle. The dramatic decline in Treg cells in the endometrium dur-\ning the late follicular phase of the menstrual cycle – typical for\nhealthy women of childbearing age – is absent in endometriosis.\nThe concentration of Treg cells in the uterus in endometriosis re-\nmains elevated throughout the menstrual cycle. This can be at-\ntributed to the increased quantity of endometrial antigens and\nuterine exposure to estrogen, which stimulate proliferation of\nTreg cells in endometriosis [20].\nThough we know how Treg cells fluctuate in the endometrium,\nthere is yet no data addressing Treg concentration changes in\nwomen with endometriosis in the peripheral blood (PB). Our\nstudy aimed to evaluate peripheral blood lymphocyte subpopu-\nlations during the menstrual cycle in women with peritoneal\nand ovarian endometriosis relative to healthy women in order\nto find possible connection between endometriosis and altered\nimmune response.\nMaterials and Methods\n!\nThis study was approved by the National Medical Ethics Commit-\ntee in the Ministry of Health, Republic of Slovenia.\nSubjects\nWe determine the required sample size according to power anal-\nysis [21]. The assumption was that the difference in concentra-\ntion of Treg cells between luteal and follicular phase in endome-\ntriosis patients would be 20 % with common standard deviation,\nafter appropriate normalization. That called for 28 patients in\neach group. To account for possible difficulties in recruiting pa-\ntients in different phases of menstrual cycle, we decided to enroll\n65 endometriosis patients altogether. A similar number of\nhealthy women were enrolled in a control group.\nThe study group comprised 65 women with endometriosis ad-\nmitted to the hospital for laparoscopic operation. Diagnosis was\nconfirmed by a histological sample taken by laparoscopy. The in-\nclusion criterion was peritoneal and ovarian endometriosis. We\nexcluded women with deep rectovaginal endometriosis because\nof the currently favored etiological hypothesis of coelomic meta-\nplasia and the concept of tissue injury and repair [1, 3, 22, 23]. In\nthe control group, we enrolled 61 healthy women at reproduc-\ntive age who had been admitted to the hospital for laparoscopic\nsterilization (tubal electrocoagulation).\nThe exclusion criteria for both groups were the presence of pelvic\ninflammatory disease, polycystic ovary syndrome, gynecological\ncarcinoma, ovarian cysts of a different etiology than endometrio-\nsis, and the use of hormonal treatment in the last 3 months. We\nexcluded 12 women with rectovaginal endometriosis, 3 women\nwhere endometriosis was not histologically confirmed and 2\nwomen with dermoid ovarian cyst. All patients were previously\ninformed of the purpose of the research, participated voluntarily,\nand provided signed, written consent.\nAll patients completed a self-administered questionnaire regard-\ning their menstrual status, and reproductive, personal, and fami-\nlial history. Additionally, the history of previous immune dis-\neases, such as Hashimoto thyroiditis, asthma, systemic lupus ery-\nthematosus, Sjögrenʼs syndrome, rheumatoid arthritis, and vitili-\ngo was collected from documentation. Women were asked about\nthe clinical features most common in endometriosis, dysmenor-\nrhea, dyspareunia and irregular menstrual cycle.\nPeripheral blood samples were collected from January 2009 to\nDecember 2010 in the Department of Obstetrics and Gynecology\nat University Medical Centre Ljubljana from 126 women of repro-\nductive age, both with and without endometriosis. Seventy sam-\nples were collected in the follicular phase of the menstrual cycle\n(37 and 33 from women with and without endometriosis, respec-\ntively) and 56 samples were collected in the luteal phase of the\nmenstrual cycle (28 and 28 from women with and without endo-\nmetriosis, respectively). All samples were taken at late morning\nhours. Evaluation of the endometrium, ovaries, and phase of\nmenstrual cycle was determined by ultrasound examination\n(Samsung Medison Europe B. V., Samsung Medison Sonoace X6,\nHoofddorp, Netherlands).\nLymphocyte subpopulations analysis\nThe types of immune abnormality have been identified with im-\nmunological investigations at the cellular level: subtypization of\nlymphocytes, cytotoxic and Treg cells, and natural killer (NK)\ncells.\nFor the analysis, 2 mL of peripheral venous blood were collected\nin a vacuum tube that contained an anticoagulant (ethylenedia-\nminetetraacetate [EDTA]). With flow cytometry (Becton Dickin-\nson FACS), the concentration of lymphocyte subpopulations was\ndetermined: B-lymphocytes (CD19\n+), T-lymphocytes (CD3 +), T-\nhelper cells (CD4 +), cytotoxic T-lymphocytes (CD8 +), NK cells\n1043\nSlabe N et al. Cytotoxic T-Cells in … Geburtsh Frauenheilk 2013; 73: 1042 –1048\nOriginal Article\n\n\n(CD56+/CD16+), and regulatory T lymphocytes (CD3 +/CD25++). All\nmonoclonal antibodies were purchased from Becton and Dickin-\nson, USA. Differential leukocyte counts were determined using\nhematology analyzers (Coulter GEN ‑S).\nCortisol levels\nCortisol was quantified in the serum of all subjects using chemi-\nluminescence on an automatic analyzer (Immulite 2000 XPi, Sie-\nmens), using commercially available kits (Cortisol Immulite\n2000). The reference range for cortisol in serum in the morning\nranges from 138–690 nmol/L.\nStatistical analysis\nStatistical analysis was performed using IBM SPSS software (ver-\nsion 19.0, USA). The distribution of the data was checked for nor-\nmality using a normal probability plot. Abnormally distributed\nparameters were normalized or nonparametric tests were ap-\nplied. Dichotomous variables between groups were compared\nwith the chi-square test. For continuous variables, differences\nwere determined by 2-tailed unpaired t-test or Mann-Whitney\ntest. To analyze the effect of menstrual cycle phase and taking in-\nto account other influential variables, analysis of variance was\nused. The phases and subpopulations were analyzed using de-\nscriptive statistics. Pearsonʼs correlation was used to analyze cor-\nrelations between the levels of cortisol and lymphocyte subpopu-\nlations. The characteristics of cases and controls are presented as\nmeans and standard deviations or counts and percentages. The\ndifferences between groups were considered to be statistically\nsignificant for p < 0.05.\nResults\n!\nThe differences between groups are shown in l\" Table 1.\nEnrolled women were 20–46 years old; the mean age in endome-\ntriosis patients (study group) was 32.86 ± 6.09 years and in the\ncontrol group, 40.49 ± 3.96 years (p < 0.001). By law, Slovenian\nwomen can opt for sterilization at age 35 year or older. The im-\npact of age on lymphocyte subpopulations is shown in l\n\" Table 2.\nLymphocyte subpopulations in the follicular\nand luteal phase of the menstrual cycle\nin women with and without endometriosis\nPB lymphocyte subpopulations are different in the follicular and\nluteal phases of the menstrual cycle. In healthy subjects, we de-\ntected a statistically significant increase in the concentration of\ncytotoxic (CD8\n+) T cells and activated (HLA ‑DR) T cells in the lu-\nteal phase compared with the follicular phase of the menstrual\ncycle (p = 0.020 and p = 0.045), whereas no such fluctuation of cy-\ntotoxic and activated T cells was detected in endometriosis. How-\never, a significant increase of Treg cells in the luteal phase relative\nto the follicular phase was detected in endometriosis patients\n(p = 0.005). The CD4\n+/CD8+ ratio was higher in control subjects\nin the follicular phase (p = 0.008). There is an increase in the con-\ncentration of CD8\n+ cells during the luteal phase in control sub-\njects (p = 0.020) and no differences in the concentration of CD4 +\ncells were detected among groups or phases. We did not detect\nluteal- or follicular-phase fluctuation in any other PB lymphocyte\nsubpopulations (B cells, NK cells, CD4\n+ cells; l\" Fig. 1 a and b,\nFig. 2 a and b).\nThe PB concentration of Treg cells in the control group did not\nfluctuate during the menstrual cycle, whereas in women with\nendometriosis, we detected a significant increase in the luteal\nphase relative to the follicular phase (p = 0.005; l\n\" Fig. 3). The in-\ncrease of Treg cells in study group relative to controls was statis-\ntically significant in luteal phase of menstrual cycle (p = 0.030;\nl\n\" Fig. 2 b).\nSerum cortisol levels and peripheral blood lymphocyte\nsubpopulations\nWomen with endometriosis had higher serum cortisol levels\n(417 ± 202 nmol/L) compared with control subjects\n(341 ± 165 nmol/L; p = 0.022). Higher serum cortisol correlated\nwith changes in Treg cells in women with endometriosis\n(p = 0.048; l\n\" Table 3).\nDiscussion\n!\nThe difference in age between study group and controls was sta-\ntistically significant. By law, Slovenian women can opt for sterili-\nzation at age 35 year or older. All women enrolled in our study\nwere at reproductive age. The analysis of variance showed there\nis no impact of age on lymphocyte subpopulations. Concentration\nand function of lymphocyte subpopulations are altered signifi-\ncantly with aging and contribute to the aging-related decline of\nimmune responses and lead to the higher risk of immune-medi-\nated diseases in aged individuals [24 –26].\nTable 1 Differences between women with endometriosis (study group) and\nwithout endometriosis (control group). Data are presented as means ± stan-\ndard deviation or counts and percentages.\nStudy group Control group p-value*\nn=6 5 n=6 1\nAge (years) 32.86 ± 6.09 40.49 ± 3.96 < 0.001\nFollicular phase 37 (56.9 %) 33 (54.1 %) 0.750\nLuteal phase 28 (43.1 %) 28 (45.9 %) 0.750\nDysmenorrhea 45 (69.2 %) 4 (6.6 %) < 0.001\nDyspareunia 23 (35.4 %) 2 (3.3 %) < 0.001\nIrregular menstrual\ncycle\n22 (33.8 %) 7 (11.5 %) 0.003\nAssociated immune\ndisease\n27 (41.5 %) 23 (37.7 %) 0.660\nCortisol (nmol/L) 417 ± 202 341 ± 165 0.022\n* p-value was judged by Mann-Whitney or unpaired t-test\nTable 2 Impact of age on lymphocyte subpopulations. Lymphocyte sub-\npopulations: T lymphocytes (CD3 +); B lymphocytes (CD19 +); T helper cells\n(CD4+); cytotoxic T lymphocytes (CD8 +); natural killer cells (NK); regulatory T\nlymphocytes (CD3+/CD25++); activated T cells (HLA ‑DR).\nLymphocyte subpopulations Impact of age\np-value*\nCD3+ 0.474\nCD19+ 0.308\nCD4+ 0.265\nCD8+ 0.715\nCD4/8 0.072\nNK 0.060\nCD3\n+/CD25++ 0.448\nHLA‑DR 0.327\n* p-value was judged by analysis of variance (ANOVA)\n1044\nSlabe N et al. Cytotoxic T-Cells in … Geburtsh Frauenheilk 2013; 73: 1042 –1048\nGebFra Science\n\n\nLymphocyte concentration\n(× 10 cells/L)\n9\nLymphocyte concentration\n(× 10 cells/L)\n9\nLymphocyte subpopulations\nLymphocyte subpopulations\na\nb\nCD 3\nCD 3\nCD 19\nCD 19\nCD 4\nCD 4\nCD 8\nCD 8\nCD 4/8\nCD 4/8\nNK\nNK\nTreg\nTreg\np = 0.005\np = 0.045\np = 0.020\nHLA-DR\nHLA-DR\n3.0\n2.5\n2.0\n1.5\n1.0\n0.5\n0\n3.0\n2.5\n2.0\n1.5\n1.0\n0.5\n0\nFollicular phase\nLuteal phase\nFollicular phase\nLuteal phase\nFig. 1 a and b Peripheral blood lymphocyte sub-\npopulations in follicular and luteal phase of men-\nstrual cycle in women with endometriosis ( a)a n d\nwithout endometriosis ( b). Data are presented as\nmean ± SD and p-value as analysed by unpaired\nt-test (* p < 0.05). Lymphocyte subpopulations:\nT lymphocytes (CD3\n+); B lymphocytes (CD19 +);\nT helper cells (CD4 +); cytotoxic T lymphocytes\n(CD8+); natural killer cells (NK); regulatory T lym-\nphocytes (CD3+/CD25++); activated T cells\n(HLA‑DR).\nLymphocyte concentration\n(× 10 cells/L)\n9\nLymphocyte concentration\n(× 10 cells/L)\n9\nLymphocyte subpopulations\nLymphocyte subpopulations\na\nb\nCD 3\nCD 3\nCD 19\nCD 19\nCD 4\nCD 4\nCD 8\nCD 8\nCD 4/8\nCD 4/8\nNK\nNK\nTreg\nTreg\np = 0.008\np = 0.010p = 0.030\nHLA-DR\nHLA-DR\n3.0\n2.5\n2.0\n1.5\n1.0\n0.5\n0\n3.0\n2.5\n2.0\n1.5\n1.0\n0.5\n0\nWomen with\nendometriosis\nWomen without\nendometriosis\nWomen with\nendometriosis\nWomen without\nendometriosis\nFig. 2 a and b Peripheral blood lymphocyte sub-\npopulations in women with endometriosis and\nwithout endometriosis in follicular ( a) and luteal\nphase of menstrual cycle ( b). Data are presented\nas mean ± SD and p-value as analysed by unpaired\nt-test (* p < 0.05). Lymphocyte subpopulations:\nT lymphocytes (CD3\n+); B lymphocytes (CD19 +);\nT helper cells (CD4 +); cytotoxic T lymphocytes\n(CD8+); natural killer cells (NK); regulatory T lym-\nphocytes (CD3+/CD25++); activated T cells\n(HLA‑DR).\n1045\nSlabe N et al. Cytotoxic T-Cells in … Geburtsh Frauenheilk 2013; 73: 1042 –1048\nOriginal Article\n\n\nIn patients with endometriosis, lymphocyte-mediated cytotoxic-\nity plays a major role in the clearance of ectopic endometrial\ncells. Treg cells are important for modulating immune response\nand function to maintain immunological homeostasis, which in-\nhibit the development of immune diseases [27, 28].\nLymphocyte subpopulations and regulatory T cells in the perito-\nneal fluid of women with endometriosis have already been ana-\nlyzed such as those published by Konincx et al. [29], Bedaiwy et\nal. [30], Mier-Cabrera et al. [31], and Rusdi et al. [32]. Rusdi et al.\n[32] showed that the percentage and the total number of regula-\ntory T cells in peritoneal fluid did not significantly differ between\npatients with endometriosis and those without endometriosis.\nThe study included 12 patients without endometriosis and 21\nwith endometriosis. The patients who had endometriosis were\nclassified as having minimal-mild- or moderate-severe-stage en-\ndometriosis according to the criteria of the American Fertility So-\nciety (AFS). The endometriosis stage did not differ in terms of reg-\nulatory T cells in peritoneal fluid. According to that we concluded\nthat examination of regulatory T cell expression in peritoneal flu-\nid is not informative. The study performed by Olkowska-Trucha-\nnowicz et al. [33] on regulatory T cells in peripheral blood and in\nthe peritoneal fluid of patients with endometriosis had a study\ndesign similar to that of our study; however, that study included\nonly subjects with ovarian endometriosis. They showed the per-\ncentage of Treg cells was significantly increased in the peritoneal\nfluid of women with endometriosis. The proportion of Treg cells\nwas significantly decreased in peripheral blood. Podgaec et al.\npublished [34] that the peritoneal fluid of women with endome-\ntriosis had a higher percentage of Treg cells in peritoneal fluid\ncompared to women without the disease.\nIn this study, we report significant differences between endome-\ntriosis patients and health control subjects with respect to men-\nstrual cycle fluctuations of PB lymphocyte concentrations.\nChanges in the concentration of CD8\n+ cells in control subjects\nare reflected by significant changes in the CD4 +/CD8+ ratio in the\nfollicular phase between groups. These results are not in accord-\nance with Lee ʼs study [35] that detected similar changes in the\nCD4\n+/CD8+ ratio in healthy women between phases because of\nthe decrease of CD4 + cells. In his study, the CD8 + cell concentra-\ntion remained constant between phases.\nIn patients with endometriosis, lymphocyte-mediated cytotoxic-\nity plays a major role in the clearance of ectopic endometrial cells\n[36]. A constant number of CD8\n+ cells during the menstrual cycle\nin the PB in endometriosis patients may reflect absent regulation\nof CD8 + cell activity. Because most HLA ‑DR T cells are cytotoxic\nCD8+ cells, the lack of cycle fluctuation in activated T cells may al-\nso reflect absent regulation of cell cytotoxicity in endometriosis\npatients. In our study, we detected an increased concentration\nof HLA ‑DR T cells in the control group during the luteal phase.\nThis fluctuation was absent in endometriosis patients. In Mier-\nCabreraʼs study [31], however, no changes in the concentration\nof HLA ‑DR T cells were found in women with or without endo-\nmetriosis.\nBecause we analyzed PB lymphocyte subpopulations, we are\naware that fluctuations in lymphocyte subpopulations primarily\nreflect a redistribution of cytotoxic cells between the blood, en-\ndometrium, and lymph nodes in different phases of the menstru-\nal cycle. In our study, we did not detect any fluctuation of Treg\ncells during the menstrual cycle in healthy women in PB. Our re-\nsults are consistent with Prieto and Rosenstein [20] who also did\nnot detect any changes in Treg number in PB during the menstru-\nal cycle in healthy, nonpregnant women of childbearing age.\nIn the endometrium of healthy women of reproductive age, the\nTreg tissue concentration linearly increases from the early to late\nfollicular phase and decreases between the late follicular and\nearly luteal phase of the menstrual cycle [18, 19]. Similar Treg dy-\nnamics were described by Arruvito et al. [11] in PB, although the\nredistribution of lymphocyte subpopulations may not follow the\nsame pattern in PB and tissues. In our study, the concentration of\nTreg cells in endometriosis patients failed to decline during the\nluteal phase, which may be responsible for the decreased ability\nof newly recruited leukocytes to initiate effective immune re-\nsponses against viable endometrial segments. Berbic et al. [18,\n19] observed an increase in the endometrial concentration of\nTreg cells during the entire luteal phase of the menstrual cycle\nin endometriosis patients compared with healthy controls.\nIn our study, we showed that the PB concentration of Treg cells in\nthe follicular phase was similar in women with and without en-\ndometriosis, but in the luteal phase, an increase in the concentra-\ntion of Treg cells in women with endometriosis, compared with\nhealthy control subjects, was detected. Budiu et al. [37] demon-\nstrated an increased presence of Treg cells in endometrial lesions.\nHowever, in the PB of women with endometriosis, no increase in\nTable 3 Cortisol and correlations with peripheral blood (PB) lymphocyte sub-\npopulations. Lymphocyte subpopulations: T lymphocytes (CD3 +); B lympho-\ncytes (CD19 +); T helper cells (CD4 +); cytotoxic T lymphocytes (CD8 +); natural\nkiller cells (NK); regulatory T lymphocytes (CD3 +/CD25++); activated T cells\n(HLA‑DR).\nLymphocyte\nsubpopulations\nCortisol correlation\ncoefficient\np-value*\nCD3+ − 0.126 0.159\nCD19+ − 0.072 0.423\nCD4+ − 0.125 0.164\nCD8+ − 0.055 0.538\nCD4/8 − 0.054 0.549\nNK 0.051 0.574\nCD3+/CD25+ − 0.176 0.048\nHLA‑DR − 0.118 0.189\n* p-value was judged by Mann-Whitney test\nTreg concentration (× 10 cells/L)\n9\nWomen with\nendometriosis\nWomen without\nendometriosis\np = 0.005\n0.16\n0.14\n0.12\n0.10\n0.08\n0.06\n0.04\n0.02\n0\nFollicular phase\nLuteal phase\nFig. 3 Regulatory T lymphocytes (Treg) during the menstrual cycle\nin women with and without endometriosis in peripheral blood. Data are\npresented as mean ± SD and p-value as analysed by unpaired t-test\n(* p < 0.05).\n1046\nSlabe N et al. Cytotoxic T-Cells in … Geburtsh Frauenheilk 2013; 73: 1042 –1048\nGebFra Science\n\n\nTreg cell concentration above normal was detected. Basta et al.\n[17] did not observe any fluctuation in the percentage of Treg\ncells over the course of menstrual cycle in endometriosis tissue\nsamples; additionally, the percentage of Treg cells in endometrio-\nsis patients was not statistically higher compared with healthy\nwomen who underwent curettage as an additional procedure\nduring uterine cervix biopsy for a diagnosis of cervical intraepi-\nthelial neoplasia. The absence of menstrual cycle fluctuation of\nTreg tissue concentration can be linked to an immunoregulatory\ndefect associated with the development of endometriosis.\nLimaʼs [38] study and our study demonstrated that the serum\ncortisol concentrations were elevated in endometriosis patients\ncompared with fertile women without endometriosis. The higher\nlevels of serum cortisol in endometriosis may be associated with\npersistent physical or emotional stress, which may contribute to\nthe development of the disease. An increased cortisol level inhib-\nits PB lymphocyte activity and could favor the development of\nendometriosis in predisposed women. The negative correlation\nbetween concentration of Treg cells and cortisol level was shown\nalso in our study. Higher levels of cortisol are responsible for an\nenhanced migration of Treg cells from PB into the tissues and re-\nticuloendothelial system.\nIn studies assessing the impact of stress on cortisol levels, those\nlevels were fluctuating within physiological range. Physiological\nrange for serum cortisol was determined according to the endo-\ncrinological diseases, wheather the hypotalamic-pituitary-adre-\nnal axis is over- or insufficiently active. The pathogenesis of en-\ndometriosis remains unknown but in the last years considerable\nevidence has pointed to immune changes that may contribute to\ndevelopment of disease. Several lines of evidence indicate stress-\nrelated hormones as immunosuppressive agents [39–41]. We be-\nlive that further studies should be done in order to clarify the role\nof cortisol in development of endometriosis and to verify if the\nincrease in serum cortisol concentration is one of the risk factors\nor just a consequence of the complex disease.\nIn some studies, the association of endometriosis with immune\ndiseases, such as Hashimoto thyroiditis, systemic lupus ery-\nthematosus, Sjögrenʼs syndrome, rheumatoid arthritis, and asth-\nma, were analyzed [42, 43]. Because the etiology of immune dis-\nease is closely related to Treg cell functions, a positive correlation\nbetween immunity and endometriosis would be expected [44,\n45]. However, recent studies did not confirm that relationship,\nbecause they discovered that the frequency of immune diseases\nin women with endometriosis is equal to that in healthy subjects\n[46, 47]. This is consistent with our findings ( l\n\" Table 1).\nOur present study is limited to patients with ovarian and perito-\nneal endometriosis. The sample size was small and further inves-\ntigations are needed to reveal whether changes are similar in\nlarger groups.\nConclusions for Practice\n!\nWe conclude that women with endometriosis do not exhibit fluc-\ntuations in the concentration of cytotoxic and activated PB lym-\nphocytes during the menstrual cycle. However, a marked in-\ncrease in regulatory T cell concentration in the luteal phase was\ndetected only in endometriosis patients. 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