The frequency of CD25+CD4+ and FOXP3+ regulatory T cells in ectopic endometrium and ectopic decidua

During the normal menstrual cycle the endometrium within the uterus is widely infiltrated by immune cells [1‐3]. The specific activities of these immune cells are crucial for the proper course of such reproductive processes as menstruation and implantation. The suppressive profile of the endometrium is what enables the respective activities of these infiltrating immune cells. This suppressive phenomenon of the endometrium is a result both of the ability of endometrial cells to suppress immune cells and of the infiltration of the endometrium by such immune regulatory cells as regulatory T (Treg) cells or suppressive macrophages (B7H4) [4‐6]. In some cases, however, the endometrium, which constitutes an active form of immunoregulatory tissue, is located outside the uterus. In other cases, as in ectopic pregnancy (e.g., within the Fallopian tube), the trophoblast cells may be located outside the uterus, even though the infiltrating trophoblast cells within the tubal wall are linked to the decidualization of epithelial and stromal cells. In cases of ovarian endometriosis and of ectopic pregnancy, mononuclear immune cells are recruited to the microenvironment of ectopic lesions; after this Treg cells infiltrate the tissue [7, 8].

Treg cells are crucial for the tolerance and prevention of autoimmunity and have been described as pivotal both for the survival of allogeneic organ grafts and for the evasion of immune surveillance by fetal cells within the uterus [4‐13]. Moreover, it has been observed that the number of Treg cells in the peripheral blood not only increases in women during pregnancy but also that their level of infiltration within the endometrium and decidua becomes elevated [11]. While Tilburgs et al. have shown that fetus-specific Treg cells are recruited to the decidua from the periphery [14], Arruvito et al. have demonstrated that the changes that take place in the Treg cell population in the peripheral blood over the different menstrual cycle phases are crucial for the proper course of reproductive processes. More specifically, the authors of the latter study found that the deregulation of the frequency of Treg cells in women with recurrent spontaneous abortion (RSA) may contribute to reproductive failure [15]. Recently, Schumacher et al. have shown that Treg cell attraction to decidua is associated with the high HCG concentration that occurs during intrauterine pregnancy development [16]. By contrast, the Treg cell concentration within the decidua derived from women with ectopic pregnancy or who suffer from spontaneous abortion is lower because the concentration level of beta HCG is lower in such conditions than it is in normal pregnancy [16]. Moreover, the infiltration level of Treg cells within decidua is decreased in patients suffering from spontaneous abortion compared with the level of infiltration found in the decidua of those undergoing induced abortions [9], and spontaneous abortion is strongly associated with an increase in the activity of the maternal immune system [17‐19]. Furthermore, it has been shown that the rupture of the tubal wall is a result not only of trophoblast cell infiltration, but more importantly of the accumulation of immune cytotoxic cells within the ectopic ovum microenvironment [6]. In contrast to the situation with ectopic decidua in the Fallopian tube, local suppression of immune cytotoxic cells has been observed within ectopic endometrium (such as ovarian endometrioma) during the development of endometriosis [20]. Recently, Berbic et al. have demonstrated the presence of Treg cells within both the ectopic and eutopic endometrium of women suffering from peritoneal endometriosis. They showed that the FOXP3 expression within the eutopic endometrium derived from women with peritoneal endometriosis differs from that observed in healthy women [21]. An alteration in Treg lymphocyte infiltration generally disrupts the immunological equilibrium. Since the potential role of Treg cells in maintaining homeostasis in ectopic endometrium and decidua has not been precisely established in the literature, from a clinical standpoint, both endometriosis and ectopic pregnancy constitute unresolved problems. For this reason, we have chosen the specific type of ectopic endometrium and decidua found at the beginning of decidualization as a research model in order to demonstrate the potential role of Treg cells in the development of the suppressive profile of the endometrium. Thus to reiterate, the aim of our present study has been to evaluate the subpopulations of Treg cells in ectopic endometrium and decidua.

CD25+CD4+FOXP3+ T cells were found in all the examined endometriosis tissue samples. These same cells were observed in 72% of the eutopic endometrium tissue samples but were present in only 29% of the tissue samples taken from patients who had ectopic pregnancies (Figure 1).

We did not observe any differences over the course of the menstrual cycle in the percentage of FOXP3+ positive cells within the subpopulation of CD4+T lymphocytes in ectopic endometrium in the tissue samples from the group of patients with ovarian endometriosis. The median for OE during the proliferative and secretory cycle phases were 8.5 (16.7) and 6.3 (5.9) respectively.

The percentage of FOXP3+ cells within the subpopulation of CD4+ lymphocytes was statistically significantly higher in the tissue samples from the patients with endometriosis as compared with the percentage found in the tissue samples from the patients who had developed ectopic pregnancies. Likewise, the percentage of FOXP3+ cells within the subpopulation of CD4+ lymphocytes was higher than in the endometrium from the control group, but these differences were not statistically significant. The percentages of these cells observed in the endometrium from the control group were, however, statistically significantly higher than those found in the ectopic deciduas (Figure 2).

Similar results were obtained from analysing FOXP3 positive cells within the subpopulation of CD4+CD25+ lymphocytes (Figure 3).

In our study, the percentages of FOXP3+ cells in the subpopulation of CD4+ T lymphocytes found in the decidua of the patients treated for Fallopian tube pregnancy were statistically significantly lower than both those observed in the ovarian endometriosis samples and those found in the secretory eutopic endometrium of the control group.

Single statements derived from the literature on the subject indicate that Treg lymphocytes are present within ovarian endometrial tissue [8, 21]. Furthermore, it has been demonstrated that the percentage of FOXP3 positive lymphocytes found within the subpopulation of T cells in the peripheral blood of women suffering from ovarian endometriosis does not differ from that observed in healthy women [8]. Berbic et al. previously demonstrated the presence of Treg cells within peritoneal ectopic endometrium [21]. In that study the authors demonstrated that there was an alteration in the FOXP3 expression within the eutopic endometrium that was derived from women suffering from peritoneal endometriosis during the secretory cycle phases when compared to what was observed in the tissue taken from healthy women [21]. For fertile women the significant decrease in the Treg cell population in the peripheral blood typically occurs just after ovulation [15]. By contrast, the women suffering from recurrent spontaneous abortion (RSA) were characterized by an absence of fluctuation in the number of Treg cells [15]. The increase in the number of Treg cells depends upon estrogen levels [22], and estrogens are also responsible for the increase in the immune suppressive potential of these cells [22]. Estrogen levels generally seem to be linked with a decrease in TH1 response [22]. Furthermore, a local increase in estrogen levels is characteristic of patients with ovarian endometrioma lesions. In our study the percentages of FOXP3+ cells in the subpopulation of CD4+ T lymphocytes found in eutopic endometrium were slightly lower than those found in ovarian endometrioma tissue. Additionally, no differences in the percentage of Treg cells within the T lymphocyte subpopulation were observed over the course of the menstrual cycle in the ovarian endometriosis tissue samples. Most likely, the absence of Treg cell fluctuation can be linked to an immune defect arising with the development of endometriosis. Although in 95% of cases the endometrial cells migrate along with retrograde menstruation from the endometrial cavity to the peritoneal cavity, only 15% of them actually survive in ectopic localization despite the host immune system response [23]. Since they are able to regulate the activity of immune cells, such cells themselves evade immune surveillance. The immunomodulatory activity of endometrial cells is crucial for the proper course of reproductive processes. Under normal physiological conditions the intensity of the immune suppressive activity of the endometrium depends on the menstrual cycle phase and differs significantly between the proliferative and secretory phases (as has been observed by Arruvito et al. in a study on the percentage of Treg cells in the late proliferative and early secretory cycle phases [15] or in different studies concerning the expression of the factors responsible for immune cell suppression, such as RCAS1 expression within endometrial cells) [24]. In patients with endometriosis, however, the suppressive activity of the endometrium does not fluctuate. In our previous studies we have demonstrated that both the expression of RCAS1 and HLA-G in ovarian endometriosis and its concentration in the peripheral blood does not differ between the proliferative and secretory cycle phases [25, 26]. Both protein RCAS1 and HLA-G are important immunosuppressive factors [27‐30]. The suppressive profile of the endometrium therefore depends not only on the absence of physiological changes--as has been observed from the Treg cell population--but also on the over-expression of suppressive factors within ectopic endometrium. For example, the ovarian endometriosis tissue samples were characterized by an over-expression of mRNA for FOXP3 in comparison to normal endometrial tissue [8]. Additionally, women experiencing primary unexplained infertility have significantly lower FOXP 3 expression within the endometrium than do fertile women [31]. These studies thus show how important the proper regulation of immune cell activities is for maintaining homeostasis during pregnancy. Sasaki et al. have reported that about 7% of CD4+ cells in the decidua derived from patients suffering spontaneous abortion are CD4+CD25^bright cells and that this percentage is lower than in the tissue of women experiencing normal pregnancies [9]. Furthermore, Shansham et al. have shown that patients suffering unexplained spontaneous abortion were typified by a smaller proportion of CD4+CD25+FOXP3+ lymphocytes within decidua than women experiencing normal pregnancies. Recently, Arruvito et al have demonstrated that the Treg lymphoctes of women suffering RSA typically exhibit lower than normal suppressive activity [32]. Thus the decrease in the level of Treg cell infiltration within decidua may result in an increase in both cytotoxic T lymphocyte and NK cell activity. During pregnancy Treg cells are recruited to the decidua [10], but the percentage of Treg cells in both the decidua and the peripheral blood increases only until spontaneous labor begins [14]. When recruited to the endometrium, Treg cells are directly able to suppress the immune response to both fetus-specific and fetus non-specific antigens [14, 33]. The beginning of spontaneous labor, however, is associated with a decrease in the percentage of Treg cells within decidua along with a subsequent increase in the immune response [34‐37], as has also been observed during spontaneous abortion [17]. The activity of immune cells within the uterus is precisely controlled by many factors, and the appearance of such pathology as preeclampsia, abortion, placental abruption, and retained placental tissue results from a disturbance in the regulation of maternal immune cells [38‐40]. Just such a situation has also been observed during the development of ectopic Fallopian tube pregnancy where the tubal rupture is seen as a consequence of the increase in maternal immune cell activity [6, 41]. In our previous study we have demonstrated that the development of Fallopian tube pregnancy is related both to the accumulation of cytotoxic immune cells and NK cells within ectopic decidua and to a continued increase in the activity of these cells within the tubal wall [6, 41]. In our current study, however, we observed that the Treg cell accumulation within the tubal wall was not as dense as it was within the eutopic endometrium during the secretory cycle phase. This is not unlike what occurs in eutopic endometrium with Arias Stella reaction where the Treg cell population decreases in comparison to that of eutopic endometrium during the secretory cycle phases [42]. The decrease in the accumulation of Treg cells in ectopic decidua within the Fallopian tube wall observed both in the Schumacher et al.[16] study and in our current one would therefore seem to be associated with an increase in the activity of the immune cells infiltrating these tissues that leads finally to tubal rupture. This contrasts with the conditions linked to ectopic endometrium; ovarian endometrioma development, for example, is associated with an immune defect. This observation would seem to accord with our own results showing that the percentage of the Treg cells within the overall cell population was the highest in the ovarian endometrioma tissue samples.

The proper balance between immune cell activity and the intensity of the suppressive profile of the endometrium or decidua is what enables the proper course of physiological reproductive processes. The correct balance is a result of the accumulation of Treg lymphocytes within the endometrium and decidua respectively. Such processes as the development of ectopic pregnancy and ovarian endometrioma are associated with a disturbance in the suppressive profile of the endometrium resulting from the alteration in the Treg cell population.

The disturbance in the immunological equilibrium observed in ectopic endometrium and decidua is associated with the alteration in the Treg cell population that occurs in these ectopic tissues.