1 Department of Pathomorphology, Jagiellonian University, Kraków, Poland2 Department of Gynecology and Oncology, Lukaszczyk Oncological Center in Bydgoszcz and Chair of Gynecology, Oncologyand Gynecological Nursing, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, JagiellonianUniversity, Kraków, Poland3 Department of Gynecology and Oncology, Jagiellonian University, Kraków, Poland4 Department of Anesthesiology, Lukaszczyk Oncological Center in Bydgoszcz, Poland

Archives of Perinatal Medicine 19(3), 166-172, 2013 ORIGINAL PAPER

The immunohistochemical analysis of RCAS1 in the endometrium during menstrual bleeding

KRYSTYNA GAŁĄZKA1, MARCIN OPŁAWSKI2, MARIOLA MAŚLIŃSKA2, PAWEŁ MACH3,ADAM KWIATKOWSKI4, PAOLA FRANCZAK2, MARTA DRZEWIECKA2, ALEKSANDRA MAZUR2,

KARINA LEKS2, ŁUKASZ WICHEREK2

Abstract

Introduction: The presence of the suppressive microenvironment of the endometrium is crucial for ensuring theproper course of reproductive processes. This phenomenon is related to the intensity of the expression of pro-teins such as RCAS1 that are able to suppress the immune cytotoxic cells infiltrating the endometrium. The aimof our study has been to gather information on the immunoreactivity level of RCAS1 within endometrial cellsduring menstrual bleeding. Methods: We analyzed the immunoreactivity levels of RCAS1 in the endometrial cellsduring both the late secretory cycle phases and menstrual bleeding. We also analyzed the immunoreactivity ofsuch antigens as CD69, CD25, and CD3 within the immune cells infiltrating the endometrium. Results: RCAS1immunoreactivity levels in the endometrium were significantly higher during menstrual bleeding than during thelate secretory cycle phases. During menstruation, the immunoreactivity level of CD69 decreases while the im-munoreactivity level of CD25 increases in comparison to those levels found in the endometria of women in thelate secretory cycle phases. Conclusion: The intensity of the suppressive profile of the endometrium related toRCAS1 expression changes significantly during menstruation as compared to the late secretory cycle phases.

Key words: RCAS1, endometrium, menstruation, CD25+, CD69+

Introduction

The implantation of the human embryo is associatedwith the growth of the suppressive microenvironment ofthe endometrium. This microenvironment is generatedmainly by the suppressive activity of both fetal and endo-metrial cells. Many processes in the endometrium thatsupport implantation start earlier, for example, duringthe late proliferative or early secretory cycle phases[1-6]. If successful implantation does not occur, all ofthese processes cease after the secretory phase whichis followed by menstruation. One of the molecular me-chanisms enabling the proper course of reproductiveprocesses is the generation of the suppressive profile ofthe endometrial microenvironment. The main role ofthese processes is related to the expression on the endo-metrial cell membrane proteins involved in the regula-tion of immune cell activity. Proteins such as: RCAS1,HLA-G, or B7H4, are effectively able to suppress im-mune cell activity, and the profile of the expression of

these proteins fluctuates according to the particularmenstrual cycle phase [7-14]. For instance, it is wellknown that during the implantation window – that is,during the mid-secretory cycle phases the suppressiveprofile dominates in the endometrium. During this pe-riod the infiltration of mononuclear immune cells to theendometrium increases greatly and decidual NK cellsdominate in the immune cell population [15-16]. Whileisolated from the human endometrium, these cells areable to stimulate the apoptosis of trophoblast cells, butwithin the actual microenvironment of the endometriumthey are selectively suppressed. Thus they limit thetrophoblast infiltration within decidua, but support pla-cental growth by regulating the neovascularization of thematernal-fetal interface [17]. The expression of CD69antigens was observed on these cells and is linked withdNK cells activity. During the secretory cycle phase,however, other markers of activity, such as CD25, noti-

The immunohistochemical analysis of RCAS1 in the endometrium during menstrual bleeding 167

ceably decrease [7, 8]. This phenomenon is directly con-nected with the intensity of the suppressive profile ofthe endometrium. Little is known, however, about theprofile of the endometrium during menstruation. Wehave chosen RCAS1 for our analysis because it is animportant factor in the homeostasis of the endometrium.RCAS1 is a protein that may stimulate the suppressionand apoptosis of both T lymphocytes and NK cells[18, 19]. The presence of these proteins has been ob-served in the endometrium and decidua as well as withinthe tumor microenvironment and various types of neo-plasms. For example, RCAS1 has been identified withinthe microenvironment of different types of adenocar-cinoma and squamous cell carcinoma [19, 20]. Spe-cifically, Sonoda et al. have demonstrated that the ex-pression of RCAS1 within cervical cancer cells is relatedto the presence of lymph node metastases and to cancernest growth [20]. Moreover, the degree of RCAS1 ex-pression within the endometrium changes according tothe particular menstrual cycle phase and is higher in thesecretory than in the proliferative cycle phases. Fluctua-tions in RCAS1 concentration levels within the peri-pheral blood of the women have been observed, and thehighest concentration level has been found during men-struation [21]. No such studies, however, have beenperformed for analyzing the immunoreactivity levels ofRCAS1 within the endometrium during menstruation.For our study, we therefore decided to measure the im-munoreactivity levels of RCAS1 in the endometrium atthe start of menstruation. In order to more effectivelyanalyze the changes in the levels of RCAS1 we chose twodifferent antigens, CD25 and CD69, and measured theimmunoreactivity levels of both of these within the mo-nonuclear cells infiltrating the endometrium during men-struation.

Materials and methods

PatientsThe tissue samples evaluated in our study were

obtained from 19 patients on whom a diagnostic biopsyof the endometrium was performed following a diagnosisby ultrasound examination of endometrial hypertrophy.The ultrasound diagnosis was performed on those pa-tients who had been suffering from inappropriate men-strual bleeding. No hormonal treatment was adminis-tered prior to the diagnosis. The patients included in thestudy averaged 41 ± 3 years of age. All of them had beenpregnant prior to the age of 30. In the study we includedonly those patients in whom upon histopathologicalverification, no endometrial hypertrophy was found and

in whom no additional pathological disturbance in theendometrium was observed. Based on histopathologi-cal verification, we chose for our analysis those tissuesamples taken from the endometria of patients in thelate secretory and menstrual phases.

The tissue samples were collected during the sur-gical procedures performed in the Department of Gyne-cology and Oncology of the Lukaszczyk Oncological Cen-ter in Bydgoszcz, Poland between November, 2009 andDecember, 2010. The immunohistochemical stainingwas performed in the Department of Pathomorphologyof the Jagiellonian University, Kraków, Poland. Prior tothe present study we obtained the approval of the Nico-laus Copernicus University Ethical Committee for ourresearch program (KB/54/2009), Bydgoszcz, Poland.

ImmunohistochemistryImmunohistochemical analysis was performed in the

Pathomorphology Department of the Jagiellonian Uni-versity, manually, with application of the Ultravision LP-Value Detection System (Thermo Scientific, Lab VisionCorporation, Fremont, CA, USA). For visualization ofreaction products AEC (3-amino-9-ethyl-carbazole) asa chromogen (AEC substrate Chromogen ready to use,DAKO, Denmark) was used for 10 min at room tempera-ture.

Sections were counterstained with Meyer’s hemato-xylin and mounted in glycergel.

As a positive control for RCAS1 a ductal breast can-cer specimen was used. The staining was performed onbreast cancer slides with the same procedures but omit-ting the primary antibodies as a negative control.

Four-micrometer slides cut from curettage materialroutinely fixed in 10% solution of formaldehyde and em-bedded in paraffin were stained to visualize the expres-sion of RCAS1 secretion in the endometrium of secretoryand menstrual cycle phases. For RCAS1 immunostaining,the slides were treated with the mouse monoclonalantibody anti-RCAS1 (Medical and Biological Laborato-ries, Naka-ku Nagoya, Japan in DAKO Antibody Diluentwith Background Reducing Components – DAKO, Den-mark, dilution 1 :1000) in a moist chamber overnight.

Further, the following antibodies were also used:CD69 (NCL-CD69; Novocastra) in dilution 1:25, CD25(interleukin-2 receptor, NCL-CD25-305; Novocastra) indilution 1 : 25, CD3 (NCL-CD3p, rabbit polyclonal anti-body; Novocastra) in dilution 1:100, according to themanufacturer’s instructions.

A semi-quantitative interpretation of immunohisto-chemical results was carried out by an experienced

K. Gałązka, M. Opławski, M. Maślińska et al.168

pathologist (G.K). A percentage of RCAS1-positive endo-metrial glandular cells was evaluated at least in 5 hpfs(high power field – objective magnification × 40, NikonEclipse 50i Microscope). Depending on percentage ofpositive cells the reaction was classified as: 0 – lack ofimmunopositive cells; +1 – 1-10% of the glandular cellwith positivity for RCAS1 , +2 – 11-30%, +3 – more than30% of the positive cells. The various types of lympho-cytes in the endometrium were also evaluated. The num-ber of immune cells in an entire specimen was countedand an average cell number per 1 hpf calculated. Thefollowing scale was used to evaluate the number of CD3-positive lymphocytes semi-quantitatively: 0 – lack ofpositive cells or only single positive cells in the entirespecimen; +1 – 2 to 5 positive cells '1 hpf; +2 – 6 to 10positive cells '1hpf, +3 – more than10 positive cells' 1hpf. Because of the scarcity of CD25- and CD69-positivelymphocytes, the other three-pointed scale was appliedto evaluate their number: 0 – lack of positive cells, +1 –presence of single cells, up to two per 1 hpf, +2 – morethan two positive lymphocytes per 1 hpf.

Statistical analysis The distribution of variables in the study groups of

women checked with the use of the Shapiro-Wilk testshowed that each of the women was different from nor-mal. The statistical significance between the groups wasdetermined by the Kruskal-Wallis test, one-way analysisof variance by ranks. The Mann-Whitney U test was thenused as applicable. All statistical analyses were carriedout with the Statistica 8.0 software program. A p value< 0.05 was considered indicative of statistical significance.

Results

RCAS1 immunoreactivityRCAS1-immunopositive endometrial cells were found

in all of the tissue samples from menstrual endometria(Fig. 1 a, b) and in 88% of the samples from late sec-retory endometria. Moreover, when a direct comparisonwas made of the samples taken from women in the latesecretory cycle phases with those taken from womenduring the menstrual period, it revealed a statisticallysignificantly increase in the RCAS1 immunoreactivitylevel during menstrual period (Fig. 2).

CD25, CD69, and CD3 immunoreactivityCD25 immunopositive mononuclear cells were

found in 55% of the tissue samples derived from men-strual endometria and in 40% of the samples from latesecretory endometria.

CD69-positive immune cells were found in 33% ofthe tissue samples taken from women during theirmenstrual period, and in 66% of the tissue samples takenfrom those in the late secretory cycle phases. Fur-thermore, we found CD3-positive immune cells in all thetissue samples derived from menstruating women, andin 50% of the samples taken from women in the latesecretory cycle phases. Moreover, when a direct com-parison was made between the samples from the womenin the late secretory cycle phase and those from thewomen having their menstrual period, it revealeda significant increase in the CD25-positive immune cellinfiltration. Likewise, we observed a slight increase inthe CD3-positive cell infiltration to the endometrium inthe menstruating women.

Fig. 1a. Moderate immunoexpression in RCAS1 in menstrual endometrium (ca. 30% of glandular cells with weak positivity). Obj. magn. × 20

The immunohistochemical analysis of RCAS1 in the endometrium during menstrual bleeding 169

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Fig. 1b. Strong RCAS1 expression in the breakdown part (on the right part of the photo) of menstrual endometrium, weaker (on the left) in parabasal glands of endometrium. Obj. magn. × 20

Fig. 2. RCAS1 immunoreactivity level differences corre-lated with the menstrual cycle phases (LS – late secretory,

M – menstrual period)

Fig. 3. CD25 immunoreactivity level differences corre-lated with the menstrual cycle phases (LS – late secretory,

M – menstrual period)

Fig. 4. CD69 immunoreactivity level differences corre-lated with the menstrual cycle phases (LS – late secretory,

M – menstrual period)

By contrast, the decrease in the infiltration of CD69-positive cells correlated with hormonal fluctuations oc-curring between the late secretory cycle phases andmenstrual period.

Discussion

We found statistically significant increase in the im-munoreactivity levels of RCAS1 in the endometriaduring menstruation in comparison to the levels ob-served in the tissue samples of women during the latesecretory cycle phases. To our knowledge, this is thefirst investigation to focus specifically on the immu-noreactivity levels of RCAS1 within the endometriumduring menstruation. For our study, we performedRCAS1 immunoreactivity analysis on the endometrium

K. Gałązka, M. Opławski, M. Maślińska et al.170

in correlation with the menstrual cycle phases. We ana-lyzed the tissue samples from the endometria of womenin the proliferative and secretory cycle phases (that is,the early-, mid- and late-proliferative and secretory pha-ses) in detail [22, 23]. Moreover, RCAS1 soluble formconcentration levels in the blood serum were assessedin the women during all the menstrual cycle phases,including the menstrual period. We found in our pre-vious study that the RCAS1 soluble form concentrationlevel in the blood was the highest in women duringmenstruation [21]. Likewise this study showed that theRCAS1 immunoreactivity level was significantly higherduring menstruation than in the late secretory cyclephases. RCAS1 helps to inhibit the maturation and apop-tosis of the cytotoxic immune cells such as thoselymphocytes and T, B, or NK cells [18] that infiltrate theendometrium. An increase in RCAS1 expression is thusgenerally linked with the strong suppression of thecytotoxic mononuclear cells infiltrating the tumor micro-environment [18-20, 24-26]. Similarly, in the endometrialmicroenvironment, an increase in the degree of RCAS1expression was observed in comparison to the prolife-rative and secretory cycle phases and was associatedwith an increase in the selective suppression of the cy-totoxic immune cells infiltrating the endometrium [22].During the subsequent menstrual cycle phases, not onlydoes the level of immune cell activity change, but sodoes the type of immune cell dominating the infiltration.For example, in the secretory cycle phase, dNK cells arepre-dominant in the endometrium, but during menstrua-tion, neutrophils and macrophages dominate [15-16, 27].Moreover, RCAS1 has been observed on the surface ofmacrophages in the tumor microenvironment in diffe-rent types of cancerous tumors (such as brain, ovarian,and salivary gland tumors) and in metastatic tumors[12, 28-31]; additionally, it has been found in the organsof patients with different types of chronic inflammatorydiseases, such as those of the liver [32] and nasal muco-sa [33], and it has furthermore been observed in womenwith ovarian endometriosis [30]. Thus far no suchmacrophages have been found in the normal endome-trium of women during the proliferative and secretorymenstrual cycle phases [30]. In our study, however, wedid observe RCAS1-positive macrophages in menstrualendometria. While RCAS1 macrophages are generallyinvolved in regulating erythropoiesis [19], they are alsocrucial for the development of the suppressive micro-environment surrounding the tumor cells [27] and forthe suppressive profile of the microenvironment of theendometrium. In our study we observed a significant

decrease in CD69 antigens levels accompanied by anincrease in CD25 antigen levels when we compared thelevels found in the tissue samples derived from womenduring the menstrual period with those of women in thelate secretory cycle phases. CD69 markers representimmune cell activity, and the level seems be linked withRCAS1 level, as a result of the role of RCAS1 plays inthe suppression of the immune response [24-26]. Theincrease in RCAS1 is followed by a decrease in CD69antigen expression as has been observed in deciduaduring placental abruption [9]. However, given that sup-pressive lymphocyte T (Treg) cells are also CD3 andCD25+, CD25 cells are not the only markers of immunecell activity [34]. Probably the increasing infiltration ofCD3+ cells in conjunction with the increase in CD25+immune cells is connected with an infiltration of Tregcells to the endometrium. Treg cells participate in ge-nerating the suppressive profile of the endometrium andare also a primary factor in creating the suppressiveprofile in different organs and tumors [1, 2, 5, 34-36]. Inour study we found CD3-positive cells in all the endo-metrial tissue samples derived from women during men-struation. Further detailed study is needed, however, inorder to elucidate the precise role CD3 positive cellsplay in the homeostasis of human endometrium. In sum,the suppressive profile of the endometrium serves tomaintain this organ’s homeostasis even as it is beingmassively infiltrated by active immune cells, and RCAS1is a major participant in the regulation of this suppres-sive phenomenon.

Conclusion

It appears that the intensity of the suppressive pro-file of the endometrial microenvironment may be relatedto RCAS1 expression within endometrial cells. More-over, the intensity of this phenomenon fluctuates duringmenstruation in response to changes in the immune cellinfiltration to the endometrium.

Acknowledgments We would like to thank Dr. Z. Pawlowicz for generating theconditions advantageous for our research. We would also liketo thank Christine Maisto for her assistance with our manu-script.

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J Łukasz WicherekDepartment of Gynecology and OncologyLukaszczyk Oncological Center in Bydgoszcz85-796 Bydgoszcz, Romanowskiej 2, Polande-mail: mowicher@cyf-kr.edu.pl