{"paper_id":"eb678587-19c6-4877-8443-4cd7958e5900","body_text":"BioMed Central\nPage 1 of 9\n(page number not for citation purposes)\nReproductive Biology and \nEndocrinology\nOpen AccessResearch\nComparison of RCAS1 and metallothionein expression and the \npresence and activity of immune cells in human ovarian and \nabdominal wall endometriomas\nLukasz Wicherek*1, Magdalena Dutsch-Wicherek2, Krystyna Galazka3, \nTomasz Banas2, Tadeusz Popiela4, Agata Lazar1 and Beata Kleinrok-\nPodsiadlo4\nAddress: 1Department of Gynecology and Infertility of the Jagiellonian University, 23 Kopernik Str, 31-501 Krakow, Poland, 2Department of \nPathomorphology of the Jagiellonian University, 17 Grzegorzecka Str, 31-531 Krakow, Poland, 3ENT Department of the Jagiellonian University, \n2 Sniadeckich Str, 31-531 Krakow, Poland and 4Department of the General Surgery of the Jagiellonian University, 40 Kopernik Str, 31-501 Krakow, \nPoland\nEmail: Lukasz Wicherek* - mowicher@cyf-kr.edu.pl; Magdalena Dutsch-Wicherek - mowicher@cyf-kr.edu.pl; Krystyna Galazka - bengot@wp.pl; \nTomasz Banas - tbanas@mp.pl; Tadeusz Popiela - msjpopie@cyf-kr.edu.pl; Agata Lazar - bengot@wp.pl; Beata Kleinrok-\nPodsiadlo - msjpopie@cyf-kr.edu.pl\n* Corresponding author    \nAbstract\nBackground: The coexistence of endometrial and i mmune cells during decidualization is\npreserved by the ability of endo metrial cells to regulate the cyto toxic immune activity and their\ncapability to be resistant to immune-mediated apoptosis. These phenomena enable the survival of\nendometrial ectopic cells. RCAS1 is responsible for regulation of cytotoxic activity. Metallothionein\nexpression seems to protect endometrial cells against apoptosis. The aim of the present study was\nto evaluate RCAS1 and metallot hionein expression in human ov arian and scar endometriomas in\nrelation to the presence of immune cells and their activity.\nMethods: Metallothionein, RCAS1, CD25, CD69, CD 56, CD16, CD68 antigen expression was\nassessed by immunohistochemistry in ovarian and scar endometriomas tissue samples which were\nobtained from 33 patients. The secretory endometrium was used as a control group (15 patients).\nResults: The lowest metallothionein expression wa s revealed in ovarian endometriomas in\ncomparison to scar endometriomas and to the control group. RCAS1 expression was at the highest\nlevel in the secretory endometrium and it wa s at comparable levels in ovarian and scar\nendometriomas. Similarl y, the number of CD56-positive ce lls was lower in scar and ovarian\nendometriomas than in the secretory endometr ium. The highest number of macrophages was\nfound in ovarian endometriomas. RCAS1-positive  macrophages were observed only in ovarian\nendometriomas. CD25 and CD69 antigen expres sion was higher in scar and ovarian\nendometriomas than in the control group.\nConclusion: The expression of RCAS1 and metallothion ein by endometrial cells may favor the\npersistence of these cell s in ectopic localization both in sc ar following cesarean section and in\novarian endometriosis.\nPublished: 14 August 2006\nReproductive Biology and Endocrinology 2006, 4:41 doi:10.1186/1477-7827-4-41\nReceived: 17 June 2006\nAccepted: 14 August 2006\nThis article is available from: http://www.rbej.com/content/4/1/41\n© 2006 Wicherek et al; licensee BioMed Central Ltd.\nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), \nwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nReproductive Biology and Endocrinology 2006, 4:41 http://www.rbej.com/content/4/1/41\nPage 2 of 9\n(page number not for citation purposes)\nBackground\nThe ovary is the most common location of the ectopic\nendometrium occurrence in the pelvic genital organs.\nEndometriosis is also found outside the genital tract. The\ncesarean scar was the most common site of extragenital\nendometriosis [1]. It was suggested in the 1950s that\nendometrioma occurring in a cesarean scar might result\nfrom specific endometrial changes depending on the preg-\nnancy development [2]. Ovarian endometriosis, however,\nwas thought to be associated with retrograde menstrua-\ntion [3]. The aforementioned two hypotheses in combina-\ntion with the current opinion indicating that the\nendometrial tissue acquires a secondary gestagen resist-\nance [4] indicates the natural ability of endometrial cells\nto coexist with adjacent activated immune cytotoxic cells\nwithin the decidua. This phenomenon is secondary to the\nparticipation of endometrial cells in reproduction and\nenables the creation of maternal immune tolerance\nagainst fetal antigens.\nThe cells of the ectopic endometrium preserve an ability\nof eutopic endometrial cells to regulate the cytotoxic\nimmune activity, for example, by the expression of many\nimmunomodulating factors (IL-1, IL-6, IL-8 and others)\n[5] and by their resistance to immune-mediated apopto-\nsis. A lower sensitivity to immune-induced apoptosis was\nnoticed in endometrial cells in ovarian cysts [6]. Both fea-\ntures seem to be crucial for endometrial cell survival in\nectopic localization.\nRCAS1, which until now has been thought to be responsi-\nble for tumor escape from immune surveillance in various\nhuman cancer cells [7-9] seems to be an important factor\nfor cytotoxic activity regulation in the endometrium [10].\nThe expression of RCAS1 has been detected in the bone\nmarrow, the Waldeyer's ring, the placenta, the\nendometrium, and the tubal mucosa thus indicating its\nrole in immune cells regulation [11-13].\nMetallothionein (MT) is a cysteine-rich, low molecular\nweight cytoplasmic protein. Its expression is related with\nboth processes concerning cell proliferation and cell death\n[14]. MT expression correlated in cancer tissue with\nreduced apoptosis in carcinoma cells [15]. Its expression\nwas observed in the endometrium and it was significantly\nhigher in the secretory than in the proliferative phase with\na peak during the implantation window [10,16,17]. MT\nexpression was also demonstrated in endometrial cancer\nand ovarian endometriosis [16]. MT expression seems to\nprotect endometrial cells against apoptosis enabling them\nto acquire resistance to immune-mediated apoptosis [10].\nThe absence of pelvic endometriosis is typical in patients\nwith abdominal wall endometrioma [18]. The reported\nincidence of cesarean scar endometriosis ranges between\n0.03 and 0.047 per cent and is reported until 2 years after\nthe surgical procedure [19]. The pelvic ectopic\nendometrium might undergo decidualization during\npregnancy even in ovarian endometriosis, where the initi-\nation of implantation starts in the early proliferative\nphase. On the contrary, scar endometriosis may form\nchocolate cysts although it starts from the implantation of\na decidual cell. The formation of the ectopic decidua in\novarian endometrioma is a well documented phenome-\nnon. Deciduosis is usually an asymptomatic phenome-\nnon and continues undetected throughout pregnancy\n[20]. The ectopic endometrium preserves the ability to\nundergo reversible decidualization, which is a phenome-\nnon typifying normal eutopic endometrium.\nThe aim of the present study was to evaluate RCAS1 and\nmetallothionein expression in ovarian and scar endome-\ntriomas in relation to immune cell presence and their\nactivity.\nMaterials and methods\nSubjects\nForty-eight patients were included in our study. The mate-\nrial was collected during routine surgical procedure in the\nDepartment of Gynecology and Infertility of the Jagiello-\nnian University in Krakow, Poland between January and\nNovember 2005. No patient included in our study\nreceived any hormonal treatment. Patients' consent was\nobtained in all cases. The approval of the research pro-\ngram by the Jagiellonian University Ethical Committee\nwas obtained prior to the study (KBET/89/B/2005). The\ntissue samples were obtained during routine surgical pro-\ncedures, were immediately fixed in 10% buffered formal-\ndehyde solution and sent to the Pathomorphology\nDepartment of the Jagiellonian University. Two experi-\nenced pathomorphologists (K.G. and A.L) independently\nevaluated the routinely stained (hematoxylin and eosin)\nslides prepared from paraffin-embedded tissue material,\nand selected the material adequate for further analysis.\nParaffin blocks were cut and used for immunohistochem-\nistry.\nOvarian endometriomas\nEctopic human endometrium tissues were obtained from\n19 non-menopausal infertile women, aged 25–37 years\nfrom ovarian lesions during laparoscopic cyst enuclea-\ntion. Before surgery the patients complained of dysmenor-\nrhoea (12 cases), dyspareunia (14 cases) and chronic\npelvic pain (10 cases).\nAbdominal wall endometriomas\nScar endometriomas were diagnosed in 15 patients aged\n25–35 years in the incisional scar after cesarean elective\ndelivery with the presence of subcutaneous nodules infil-\ntrating the fascia (10 cases) and muscle (5 cases). The sur-\n\nReproductive Biology and Endocrinology 2006, 4:41 http://www.rbej.com/content/4/1/41\nPage 3 of 9\n(page number not for citation purposes)\ngical procedure was performed less than two years after\ncesarean section. The patients complained of a cyclic local\npain and tenderness at the time of menstruation. Patients\nafter cesarean section with multiple pregnancies or exist-\ning pregnancy complications such as preterm deliveries,\nhypertension, diabetes mellitus, and cases of fetal demise\nwere excluded from this study.\nControl group\nEutopic human endometrium tissues were obtained from\n14 non-menopausal fertile women, aged 29–41 years.\nThese patients underwent hysterectomy because of a\nbenign gynecological indication (leiomyomas). Tissue\nsamples were classified according to the menstrual cycle\nphases. We included into the study only the tissue sam-\nples from the mid secretory cycle phase. The control sam-\nples from uterine corpus included the entire thickness of\nthe endometrium (basal and superficial part, composed\nof stromal cells and glandular epithelial cells).\nImmunohistochemistry\nImmunohistochemical analysis was performed in the\nPathology Department of the Jagiellonian University.\nFive-micrometer sections from each case were stained to\nvisualize expression of RCAS1, MT and CD16-, CD25-,\nCD69-, CD56-positive cells (mainly lymphocytes) as well\nas CD68+ cells, or macrophages. In all cases immunohis-\ntochemistry was performed applying Envision method\nusing Dako Autostainer. The following antibodies were\napplied: mouse monoclonal antibody Anti-RCAS1 (Med-\nical and Biological Laboratories, Naka-ku Nagoya, Japan\nin DAKO Antibody Diluent with Background Reducing\nComponents-DAKO, Denmark, dilution 1:1000), mono-\nclonal mouse antibody ImmunOTM (MP Biomedicals,\nInc., clone 1A12 in dilution 1:1000), CD56 (NCAM; NCL-\nCD56-504, Novocastra) in dilution 1:100, CD69 (NCL-\nCD69, Novocastra) in dilution 1:25, CD25 (Interleukin-2\nReceptor, NCL-CD25-305, Novocastra) in dilution 1:25,\nCD16 (NCL-CD16, Novocastra) in dilution 1:40, CD68\n(Klone PG-M, Dako) in dilution 1:50, according the man-\nufacturer's instructions. Visualization of reaction products\nwas performed using AEC (3-amino-9-ethyl-carbazole) as\na chromogen (AEC Substrate Chromogen ready-to-use,\nDAKO, Denmark) for 10 minutes at room temperature.\nSections were counterstained with hematoxylin and\nmounted in glycergel. As a positive control a tonsil speci-\nmen was taken for RCAS1 and a breast cancer specimen\nfor metallothionein. All stainings were performed with\nthe same procedure but with the omission of the primary\nantibody as a negative control. RCAS1 expression was\nevaluated in entire slides in endometriosis area, in glan-\ndular epithelium (superficial and of the glands) and the\nstromal cells (fibroblasts), considering the per cent of pos-\nitive cells and the intensity of the colour reaction. The\ndegree of metallothionein positivity was quantified as the\npercentage of MT-positive cells in the endometriosis\nlesion. The staining in epithelial and stromal cells of\nendometriosis was evaluated. The scales used for estima-\ntion of both marker staining are shown in Table 1.\nThe immune cells were calculated in an entire specimen,\nin the region of endometriosis and an average cell number\nper 1 hpf (high power field, objective magnification ×40)\nwas calculated. Variable scales were used to evaluate sem-\niquantitatively an amount of the cells, depending on their\ngeneral number in the specimen, summarized in Table 2.\nThe evaluation of immunohistochemical reactions was\nperformed independently by two histopathologists (K.G.\nand A.L.).\nStatistical analysis\nThe distribution of variables in the study groups of\nwomen checked with the use of the Shapiro-Wilk test\nshowed that all of them were different from normal.\nTherefore, nonparametric testing was employed. Statisti-\ncal significance between the groups was determined by\nthe Kruskal-Wallis analysis of variance (ANOVA) test. The\nMann-Whitney U test was then used as applicable. The\nSpearman rank test was used to evaluate interclass corre-\nlation coefficients. All calculations were carried out with\nthe use of STATISTICA software v. 6 (StatSoft, USA, 2001).\nTable 1: The scale used for evaluation of metallothionein and RCAS1 expression.\nAntigen Immunoreactivity\n0+ 1 + 2 + 3\nRCAS1 No reactivity Weak (when observed any, also \ngranular in paranuclear region) \ncytoplasmic staining pattern in up \nto 10% of positive cells\nMarked cytoplasmic (sometimes \ntogether with membranous) \nstaining in 11–30% of the cells\nHigh expression – more than \n30% of positive cells\nMetallothionein Lack of any positivity W eak staining in less than 5% of \nthe cells\nModerate – various staining \nintensity but in <50% of the cells,\nStrong – staining of more than \n50% of the cells.\n\nReproductive Biology and Endocrinology 2006, 4:41 http://www.rbej.com/content/4/1/41\nPage 4 of 9\n(page number not for citation purposes)\nResults\nRCAS1 immunoreactivity was visible in the endometrial\nepithelium without positive reaction in the proper stro-\nmal cells.\nRCAS1 immunopositivity was revealed in 57% of ovarian\nendometriosis tissue samples, in 60% of scar endometri-\noma tissue samples and in all examined secretory\nendometrial tissue samples (Figure 1.) The differences in\nthe distribution of MT immunoreactivity were found\nbetween stromal cells and glandular cells in the tissue\nsamples derived from the scar and ovarian endometriosis.\nNo such alterations in MT distribution were observed\nbetween samples in the control group (Figure 2.) Metal-\nlothionein immunoreactivity was observed in glandular\nepithelium of 21% ovarian endometriosis tissue samples,\n94% scar endometriomas and in all secretory\nendometrium tissue samples. Metallothionein immunop-\nositivity was shown in stromal cells of 69% ovarian\nendometriosis tissue samples and in all stromal cells of\nscar endometriomas. No MT immunoreactivity was\nobserved in stroma of secretory endometrium. Immune\ncell presence (CD68, CD56 and CD16) and activity\n(CD69, CD25) were assessed within stroma.\nThe statistical analysis of examined factors in ovarian\nendometriosis, scar endometriomas and secretory\nendometrium was performed by the Kruskal-Wallis anal-\nysis of variance (ANOVA) test and the results are pre-\nsented in Table 3.\nAnalysis of RCAS1 immunoreactivity\nStatistically significant lower RCAS1 expression was\nobserved in ovarian endometriosis than in the control\ngroup (p = 0.002). Similarly, statistically significant lower\nRCAS1 expression was identified in scar endometriosis in\ncomparison to the control group (p = 0.002). RCAS1\nexpression was on comparable levels in scar and ovarian\nendometriosis (Table 4.) The RCAS1 positive macro-\nphages were found in ovarian endometriomas, but they\nwere not detected either in the eutopic endometrium or in\nscar endometriomas (Figure 3.)\nRCAS1 expression in: scar endometriomas (A,B), ovarian endometriomas (C,D) and secretory endometrium (E,F)Figure 1\nRCAS1 expression in: scar endometriomas (A,B), ovarian \nendometriomas (C,D) and secretory endometrium (E,F). A – \nWeak positive glandular reaction in scar endometrioma (hor-\nizontal arrows). Obj. magn. ×40; B-Very weak immunoreac-\ntivity in glandular epithelium in scar endometrioma \n(horizontal arrow). Obj. magn. ×40; C – Weak immunoreac-\ntivity in glandular epithelium of ovarian endometrioma (hori-\nzontal arrow). Obj. magn. ×60; D – Strong immunoreactivity \nin glandular epithelium of ovarian endometrioma (horizontal \narrows). Obj. magn. ×60; E – Strong immunoreactivity in \nglandular epithelium of the secretory endometrium, also of \nits basal part (horizontal arrows). Obj. magn. ×10; F – Strong \nimmunoreactivity in glandular epithelium of secretory \nendometrium (horizontal arrow). Obj. magn. ×40.\nTable 2: The scale used for evaluation of CD25, CD56, CD68, CD69, and CD16 antigens expression.\nAntigen Immun oreactivity\n0 1 +2 +3 +4 +\nCD25 Lack of positive cells Single positive cells 1–5 positive cells/1 hpf More than 5 positive cells/\n1 hpf\n-\nCD56 Lack of positive cells Single positive cells 1–5 positive cells/1 hpf More than 5 positive cells/\n1 hpf\n-\nCD69 Lack of positive cells Single positive cells 1–5 positive cells/1 hpf More than 5 positive cells/\n1 hpf\n-\nCD68 Lack of positive cells 1–5 positive  cells/1 hpf 6–10 cells/1 hpf 11–20 positive cells/1 hpf More than 20 positive \ncells/1 hpf\nCD16 Lack of positive cells 1–5 positive cells per 1 hpf 6–10 cells/1 hpf 11–20 positi ve cells/1 hpf More than 20 positive \ncells/1 hpf\n\nReproductive Biology and Endocrinology 2006, 4:41 http://www.rbej.com/content/4/1/41\nPage 5 of 9\n(page number not for citation purposes)\nAnalysis of MT immunoreactivity\nA statistically significant lower MT immunoreactivity in\nthe glandular epithelium was identified in the ovarian\nendometriosis in comparison to the scar endometriosis (p\n< 0.001) and was also statistically significantly lower than\nin the control group (p < 0.001). No differences in MT\nexpression in the glandular epithelium were observed\nbetween scar endometriosis and the control group (Table\n5.) MT immunoreactivity was statistically significantly\nhigher in the stroma of scar endometriosis when com-\npared to ovarian endometriosis (p < 0.001).\nThe difference in MT expression between the ovarian glan-\ndular epithelium and ovarian stroma was observed and\nwas statistically significant (p = 0.01). However, no differ-\nences of MT expression in scar endometriosis between the\nglandular epithelium and the stroma were found.\nAnalysis of immune cells presence (Table 6)\nThe number of macrophages was significantly higher in\novarian endometriosis in comparison to the control\ngroup, similarly it was observed to be statistically signifi-\ncantly higher in scar endometriosis than in the control\ngroup (p = 0.03).\nCD69 antigen expression was on comparable levels in\novarian and scar endometriosis, while in ovarian endome-\ntriosis it was significantly higher than in the control\ngroup. Similarly, CD69 antigen expression was signifi-\ncantly higher in scar endometriosis than in the control\ngroup. CD25 antigen expression was statistically signifi-\ncantly higher in scar endometriosis than in the control\ngroup (p = 0.02). No differences in CD25 expression were\nidentified between ovarian endometriosis and the control\ngroup. Statistically significantly lower CD25 expression\nwas detected in ovarian than in scar endometriosis (p =\n0.005). The number of CD56-positive cells was identified\nin ovarian and scar endometriosis on comparable levels,\nbut it was in both cases statistically significantly lower\nthan in the control group (p < 0.001). No differences in\nCD16 antigen expression were observed between the\nstudied tissue samples and the control group.\nDiscussion\nRCAS1 immunoreactivity was more prominent in the\nsecretory endometrium than in scar and ovarian endome-\ntriomas. Stronger Metallothionein expression was deter-\nmined in scar endometriomas and in secretory\nendometrium than in ovarian endometriosis. The altera-\ntions in RCAS1 and Metallothionein expression are\naccompanied by changes in CD25, CD56 and CD69 anti-\ngens expression on immune cells.\nA decrease of CD25 antigen expression with a concomi-\ntant increase of CD69 expression has been observed in the\nendometrium according to the menstrual cycle changes\n[21]. Chao et al. concluded that there might exist a factor\nor factors capable of selective suppression of activated\ncytotoxic cells. RCAS1 could be considered as a factor to\nplay that role. Its expression increases significantly during\nthe secretory cycle phase. In our previous report a signifi-\ncantly lower RCAS1 expression was noticed in ovarian\nendometriosis in comparison to endometrial carcinoma\n[10]. RCAS1 expression was growing in the eutopic\nendometrium simultaneously with the growth of the\nnumber of CD56-positive cells and CD69 antigen expres-\nsion. In the present study a correlation between CD56 and\nCD69 expression was observed in ovarian endometrio-\nmas (R = 0.53, p = 0.018) as well as in scar endometrio-\nmas (R = 0.52, p = 0.04). A decrease of the number of\nMT expression in: scar endometriomas (A,B), ovarian endometriomas (C,D) and secretory endmetrium (E,F)Figure 2\nMT expression in: scar endometriomas (A,B), ovarian \nendometriomas (C,D) and secretory endmetrium (E,F). A – \nModerate positive stromal reaction in scar endometrioma \ntissue (vertical arrows). Obj. magn. ×40; B-Strong immunore-\nactivity in glandular epithelium of scar endometrioma (hori-\nzontal arrow) Obj. magn. ×10; C – Moderate \nimmunoreacitvity in stromal cells of ovarian endometrioma \n(vertical arrows). Obj. magn. ×40; D – Very weak immunore-\nactivity in glandular epithelium of ovarian endometrioma \n(vertical arrows). Obj. magn. ×40; E – Strong immunoreactiv-\nity in glandular epithelium of secretory endometrium (hori-\nzontal arrows) and negative stromal cells (x). Obj. magn. ×40. \nF – Strong MT immunoreactivity in glandular epithelium of \nthe secretory endometrium (horizontal arrow). Obj. magn. \n×60.\n\n\nReproductive Biology and Endocrinology 2006, 4:41 http://www.rbej.com/content/4/1/41\nPage 6 of 9\n(page number not for citation purposes)\nCD56-positive cells in scar and ovarian endometrioses\nseems to remain in agreement with described NK cell dys-\nfunction, which is considered to contribute to the patho-\ngenesis of endometriosis [22]. Diminished NK cell activity\nhas been observed in the peritoneal fluid of women with\nendometriosis [23]. The main task of scientists is focused\non the question whether this dysfunction is primary or\nsecondary to the development of ovarian endometriosis.\nThe expression of the killer inhibitory receptors (KIRs; for\nexample CD158a) on NK cells associated with the\ndecrease of the NK cell activity might indicate that the NK\ncell changes are the primary event [22]. On the contrary, a\nhigher expression of class I human lymphocyte antigens\non endometrial cells surviving in ectopic localization [24]\nsuggests the secondary resistance to natural killer-medi-\nated cytolysis [25]. The RCAS1 expression observed in this\nstudy in both scar and ovarian endometriomas points to\nthe possibility of inhibition of cytotoxic immune\nresponse, which might be secondary to the physiological\nability of endometrial cells to regulate immune cell activ-\nity. The decreased RCAS1 expression in scar and ovarian\nendometriomas in comparison to the eutopic secretory\nendometrium might result from the lower number of NK\ncells. NK dysfunction in endometriosis was found to be\nrelated to the diminished macrophages ability to present\nantigens [26].\nMacrophages seem to play a central role in the immunob-\niology of endometriosis [27]. The recent evidence suggests\nthat ovarian macrophages migration is a result of RANTES\nsecretion into the environment of endometriomas [28],\nand their numerous products (IL-1beta, TNF-alfa, Il-6, Il-\nDestroyed epithelial layer of an endometrial ovarian cystFigure 3\nDestroyed epithelial layer of an endometrial ovarian cyst. \nThe covering of it composed mainly of macrophages, with \nstrong RCAS1 expression (B, C, D). RCAS1-positive macro-\nphages present also loosely in the cyst fluid (A). Obj magn.: \nA, C, D ×40; B ×20.\nTable 3: The results of statistical analysis of metallothionein (MT), RCAS1, CD68, CD56, CD16, CD25 and CD69 expression in ovarian \nendometriosis, scar endometriomas and secretory endometrium performed by Kruskal-Wallis analysis of variance (ANOVA) test. (± \nSE – standard error).\nAntigens Ovarian endometriosis Sca r endometriosis Control group p\nRCAS1 0.684 (± 0.149) 0.666 (± 0.168) 1.583 (± 0.173) 0.0026\nMT – glandular epithelium 0.211 (± 0.159) 1.800 (± 0.179) 2.167 (± 0.185) <0.001\nMT – stroma 1.000 (± 0.162) 2.200 (± 0.182) 0.000 (± 0.189) <0.001\nCD56 0.579 (± 0.135) 0.400 (± 0.152) 2.000 (± 0.186) <0.001\nCD16 1.789 (± 0.240) 1.600 (± 0.270) 2.300 (± 0.331) 0.292\nCD68 2.579 (± 0.227) 2.000 (± 0.256) 2.800 (± 0.313) 0.024\nCD25 0.421 (± 0.239) 1.667 (± 0.268) 0.300 (± 0.329) 0.009\nCD69 0.895 (± 0.259) 1.200 (± 0.292) 0.300 (± 0.357) 0.167\nTable 4: The immunhistochemical analysis of RCAS1 expression in glandular epithelium\nGroups Part of endometriu m RCAS1 immunoreactivity*\n0+ 1 + 2 + 3\nOvarian endometriosis (n = 19) Gland ular epithelium 42 (8) 47 (9) 11 (2) -\nScar endometriosis (n = 15) Glandular epithelium 40 (6) 53 (8) 7 (1) -\nControl group (n = 14) Glandula r epithelium - 57 (8) 35 (5) 8 (1)\n*percentage of cases; (n-total number of cases)\n\nReproductive Biology and Endocrinology 2006, 4:41 http://www.rbej.com/content/4/1/41\nPage 7 of 9\n(page number not for citation purposes)\n8, MMP-9, VEGF and others) may be involved in the onset\nand development of endometriosis [27]. In our present\nstudy the highest number of macrophages was identified\nin ovarian endometriosis when compared to scar\nendometriosis and to the control group. Interestingly,\nonly ovarian macrophages were RCAS1 positive (Fig. 3).\nThe presence of RCAS1-positive macrophages in ovarian\nendometriosis was already reported in our previous pre-\nliminary study [30]. Until now RCAS1-positive macro-\nphages were observed in the bone marrow, peripheral\nblood of patients with Hodgkin lymphoma, nasal polyps\nand in immune mediated liver disease [11,31,32]. Per-\nhaps the alteration of macrophage-NK cells and macro-\nphage-cytotoxic T lymphocyte interactions might help the\ncells survive in ectopic localization. In scar endometrio-\nmas no RCAS1-positive macrophages were observed, but\nthis is the situation when the primary lesion develops dur-\ning the immune tolerance against fetal antigens in preg-\nnancy maintenance and this protection seems to facilitate\nthe development of the endometriomas foci. Our results\nseem to confirm the clinical observations of scar endome-\ntriomas. Although the implantation of decidual cells\nmight be induced iatrogenically by mechanical transplan-\ntation, endometriomas do not develop in all scars follow-\ning cesarean sections and occur more frequently following\nelective cesarean sections performed during the mainte-\nnance of immune tolerance during pregnancy [33-35].\nImplanting endometrial cells within the cesarean scar pos-\nsesses both the ability to regulate the activity of immune\nresponse and a high resistance to immune mediated\napoptosis similar to eutopic endometrial cells during the\nsecretory cycle phase [6,29]. The recruitment of lym-\nphocytes to decidua takes place during the whole course\nof pregnancy [36], pregnancy termination is related with\nthe termination of their activity inhibition [37], decidual\ncells are extremely exposed to immune mediated apopto-\nsis. The resistance to immune mediated apoptosis of the\nendometrial cells can be related to MT expression [38-41].\nThe increase of MT endometrial immunoreactivity during\nthe implantation window seems to be secondary to the\ngrowing cytotoxic activity. A higher level of MT expression\nhas been demonstrated in the eutopic endometrium\nsimultaneously with the increasing number and activity of\ncytotoxic cells [10]. Endometriosis cells are characterized\nby the inability to transmit a \"death\" signal [42]. In the\npresent study MT expression in ovarian endometriosis was\nat the lowest level when compared to the eutopic\nendometrium and scar endometriosis. However, it was at\nthe comparable level in scar endometriosis and the con-\nTable 6: Density of CD56+, CD16+, CD68+, CD25+ and CD69+ cells in the endometrial stroma.\nTissues samples CD – antigens The number of lymphocytes\n0 + 1+ 2+ 3+ 4\nOvarian endometriosis (n = 19) CD56 47 (9) 47 (9) 6 (1) - -\nCD16 6 (1) 41 (8) 26 (5) 21 (4) 6 (1)\nCD68 - 22 (4) 26 (5) 26 (5) 26 (5)\nCD25 78 (15) 22 (4) - - -\nCD69 47 (9) 26 (5) 15 (3) 12 (2) -\nScar endometriosis (n = 15) CD56 66 (10) 27 (4) 7 (1) - -\nCD16 19 (3) 27 (4) 27 (4) 27 (4) -\nCD68 11 (2) 11 (2) 37 (5) 41 (6) -\nCD25 41 (6) - 11 (2) 48 (7) -\nCD69 48 (7) 19 (3) 7 (1) 19 (3) 7 (1)\nControl group (n = 10) CD56 - 10 (1) 80 (8) 10 (1) -\nCD16 - 20 (2) 40 (4) 30 (3) 10 (1)\nCD68 - 20 (2) 80 (8) - -\nCD25 70 (7) 30 (3) - - -\nCD69 80 (8) 10 (1) 10 (1) - -\n*percentage of cases; (n-total number of cases)\nTable 5: The immunhistochemical analysis of metallothionein expression in the glandular epithelium and stroma.\nGroups Part of endometrium Meta llothionein immunoreactivity*\n0+ 1 + 2 + 3\nOvarian endometriosis (n = 19) Glan dular epithelium 78 (15) 22 (4) - -\nStroma 31 (6) 38 (7) 31 (6) -\nScar endometriosis (n = 15) Glandular epithelium 7 (1) 27 (4) 46 (7) 20 (3)\nStroma - 27 (4) 27 (4) 46 (7)\nControl group (n = 14) Glandular epithelium - 28 (4) 44 (6) 28 (4)\nStroma 100(14) - - -\n*percentage of cases; (n-total number of cases)\n\nReproductive Biology and Endocrinology 2006, 4:41 http://www.rbej.com/content/4/1/41\nPage 8 of 9\n(page number not for citation purposes)\ntrol group. High MT expression in scar endometriomas\nwas accompanied by the growing number of CD25- and\nCD69-positive immune cells, while low MT expression in\novarian endometriomas accompanied by a lower number\nof CD25- and CD69-positive immune cells coexisted with\nthe presence of RCAS1-positive macrophages. Thus, mac-\nrophages probably play an additional role in the NK cells\nactivity restrictions. Moreover, differences in MT expres-\nsion between epithelial cells and stroma were identified,\nwhat seems to confirm the reports on differences between\napoptosis levels in the glandular epithelium and stroma\nin ovarian endometrioid cyst [43,44].\nThe expression of RCAS1 and metallothionein by\nendometrial cells is important for the coexistence of acti-\nvated immune cells together with endometrial cells dur-\ning decidualization in secretory cycle phase. This\nexpression may favor the persistence of endometrial cells\nin ectopic localization both in scar following cesarean sec-\ntion and in ovarian endometriosis.\nAbbreviations\nReceptor associated cancer antigen presenting on SiSo\ncells (RCAS1); metallothionein (MT); class I human lym-\nphocyte antigens (HLA-I); natural killer cells (NK); tumor\nnecrosis factor (TNF); cytotoxic T lymphocytes (CTLs).\nAcknowledgements\nThis work was financially supported by the KBN Grant number: \nN40601231/0201.\nReferences\n1. Douglas C, Rotimi O: Extragenital endome triosis – a clinico-\npathological review of a Glas gow hospital experience with\ncase illustrations.  J Obstet Gynaecol 2004, 24:804-808.\n2. Scott RB, Telinde RW: Clinical external endometriosis: proba-\nble viability of menstrually shed fragments of endometrium.\nObstet Gynecol 1954, 4:502-510.\n3. Sampson JA: Endometrial carcinoma of  the ovary, arising in\nendometrial tissue of that organ.  Arch Surg 1925, 10:1-72.\n4. 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