Expression of the T regulatory cell transcription factor FoxP3 in peri-implantation phase endometrium in infertile women with endometriosis

Endometriosis (EM) is a common and benign gynecological disorder that is highly associated with infertility. It affects approximately 10% to 15% of women of reproductive age and 25% to 50% of women with infertility. Moreover, 30% to 50% of women with EM are infertile [1]. Although the mechanisms underlying EM-associated infertility include abnormal folliculogenesis, elevated oxidative stress, altered immune function and hormonal milieu in the follicular and peritoneal environments, and reduced endometrial receptivity, the precise mechanism of pathogenesis remains controversial. The combination of these factors leads to poor oocyte quality and impaired fertilization and implantation [2, 3]. Recent studies have demonstrated that endometrial molecular defects during the implantation window might be a cause of EM-associated infertility. Increasing evidence suggests that EM patients have an impaired endometrium and/or an abnormal endometrial environment which make them functionally unfavorable for implantation and pregnancy progression [2, 4].

CD4⁺CD25⁺FoxP3⁺T regulatory cells (T_regs) are a specialized subpopulation of T cells that control and suppress a range of immune responses, including T-cell proliferation and activation, macrophage, B cell, DC and NK cell function, mast cell degranulation, cell proliferation, and cytokine release. Forkhead box protein 3 (FoxP3) is a member of the forkhead-box/winged-helix transcription factor family. It is a unique marker of T_regs. FoxP3 has been reported to be an essential controlling gene for the development and function of naturally occurring T_reg populations.

Accumulating evidence from both experimental and clinical studies indicates that a balance between regulation and deletion of responder T cells is an effective strategy to control immune responsiveness after organ or cell transplantation [5]. Furthermore, FoxP3⁺ T_regs are critical for the maintenance of maternal immune tolerance as well as the prevention of autoimmunity and transplantation rejection. Recent studies have demonstrated links between impaired function or diminished T_reg cell populations and complications during pregnancy due to defective implantation or placental insufficiency [6]. In miscarriage, reduced responsiveness to pregnancy associated expansion of T_reg cell populations, due to numerically fewer T_regs as well as T_reg functional deficiency, may underpin reduced immunosuppressive capability [7]. Compared to women with induced abortion, patients experiencing spontaneous abortion exhibit fewer decidual and peripheral blood CD4⁺CD25^high T cells recovery. Women experiencing repeated miscarriage have been shown to have a reduced number of T_regs within the peripheral blood CD4⁺ pool and reduced suppressive capacity. Primary unexplained infertility is also associated with reduced expression of FoxP3 mRNA in endometrial tissue during mid-secretory phase of the menstrual cycle, suggesting that impaired differentiation and/or recruitment of uterine T_regs, even prior to conception, might affect patients’ ability to establish pregnancy [8].

A recent study demonstrated that eutopic endometrial FoxP3 was up-regulated in women with EM, suggesting that FoxP3 plays a pathogenic role in the formation of EM [9]. However, very little is known about the role of FoxP3⁺ T_regs in the pathophysiologic mechanism of EM-associated infertility and the changes of FoxP3⁺ T_reg population in different EM stages. In this study, FoxP3 expression in the endometrium during the peri-implantation phase was investigated by comparing infertile women with different stages of EM to normal fertile women. The purpose is to elucidate the pathogenesis of infertility in EM.

The mechanisms by which EM impairs fertility remain poorly understood. Accumulating evidence indicates that the eutopic endometrium of women with EM differs from that of women without EM [12], which may contribute to failure of implantation. A meta-analysis of in-vitro fertilization and embryo transfer (IVF-ET) trials showed that women with EM have similar ovulation and embryo formation rates compared to patients scheduled for IVF treatment without EM (e.g. blocked fallopian tubes). However, the implantation rates in women with EM are 50% lower than those achieved in patients being treated for other causes of infertility [13]. These results indicate a receptivity defect within the eutopic endometrium in women with EM that affects fertility regardless of other causes of infertility in EM (e.g. adhesions). Gene array studies have established aberrant gene expression in the endometrium of women with EM compared to those without EM during the implantation window [14]. Furthermore, studies indicate that an abnormal inflammatory environment is present, not only in pelvic endometriotic lesions, but also in the eutopic endometrium of patients with EM. Therefore, the decrease in fertility experienced by these women might be caused by inflammatory processes, which in turn, affecting ovulation and implantation.

Successful embryo implantation is a dynamic process, requiring dialog between the blastocyst and a receptive endometrium [15]. Although implantation is primarily regulated by the steroid hormones, a host of local immune cells, cytokines, growth factors and adhesion molecules have been identified that mediate the apposition, adhesion and invasion of the blastocyst [16, 17]. Maintenance of an optimal pro- and anti-inflammatory state at the feto-maternal interface is necessary for successful implantation. The leukocyte population in the endometrial environment at the time of implantation includes uterine natural killer (uNK), macrophages, T cells and B cells [18‐20]. Dysregulation in the production of these factors may lead to aberrant implantation. As one of these factors, T_regs play a crucial role in regulation and suppression of local immune response during implantation phase.

Recently, studies in reproductive immunology show that T_regs play an important role in maternal tolerance of the conceptus. Their suppressive actions are exerted even prior to embryo implantation. T_regs are enriched at the fetal-maternal interface, showing a suppressive phenotype. Inadequate numbers of T_regs or their functional deficiency might be linked with miscarriage, pre-eclampsia, infertility and the failure of embryo implantation. Several studies have reported an association between T_regs and implantation failure or recurrent spontaneous miscarriage in humans. Women experiencing repeated miscarriage were shown to have a reduced frequency of T_regs within peripheral blood, and reduced suppressive capacity, compared to normal fertile women [7, 21]. Primary unexplained infertility has also been associated with reduced expression of Foxp3 mRNA in endometrial tissue in the mid-secretory phase of the menstrual cycle [8]. These studies suggest that reduction in the size and functional impairment of the T_reg population and/or insufficient migration of T_regs to decidual tissue at the feto–maternal interface induce implantation failure in embryo implantation or recurrent spontaneous abortion in humans.

In contrast to other leukocytes, T_regs play the most crucial roles in controlling, suppressing and modulating a vast variety of immune responses in the development of endometriosis. Endometriosis is an inflammatory condition, associated with highly dysregulated immune response at both uterine and peritoneal levels. Recent evidence suggests that dysregulated immune response in EM is likely to originate within the eutopic endometrium [22]. Berbic et al. found that FoxP3⁺ cells in the eutopic endometrium of women with EM remained highly up-regulated during the secretory phase of the menstrual cycle, while at this time their expression was significantly down-regulated in women without EM [21]. They propose that FoxP3⁺ cells in eutopic endometrium in women with EM decrease the ability of newly recruited immune cell populations to effectively recognize and target endometrial antigens shed during menstruation, allowing their survival and ability to implant in ectopic sites [9]. T_regs are likely to be linked to pathogenesis and progression of EM. Basta et al. demonstrated that the disturbance in the immunological equilibrium observed in ectopic endometrium and deciduas would seem to be related to the alteration in the T_reg cell population that occurs in these ectopic tissues. Additionally, no differences in the percentage of T_regs within the T lymphocyte subpopulation were observed over the course of the menstrual cycle in the ovarian endometriosis. They hypothesized that the absence of T_regs fluctuation can be linked to an immune defect arising with the development of endometriosis [23]. In another genetic marker research,André GM et al. first evaluated the association between FOXP3 polymorphisms in infertile women with and without EM. They suggest that the FOXP3 polymorphisms can be associated with risk of idiopathic infertility (rs2280883 and rs2232368) and EM (rs3761549) in Brazilian women [24]. In addition, recent studies have implicated T_regs in inducing tolerance to tumours. In Prieto’s reviews, the proposed hypothesis predicts that local expansion of T_regs might suppress anti-tumour responses and facilitate the progression of EM to ovarian cancer in susceptible women [25].

Very few human studies of FoxP3 expression in the peri-implantation endometrium have been reported. FoxP3 expression in the endometrium of infertile patients with EM compared to healthy fertile women remains to be elucidated. Therefore, this study aimed to study the difference of FoxP3⁺ expression in endometrial tissue during the peri-implantation window between patients with EM-associated infertility and healthy fertile women. Furthermore, FoxP3 expression in the endometrium of infertile patients with different stages of EM and the role of T_regs in the etiology of infertility in women with EM were investigated as well. Analysis of FoxP3 mRNA expression by quantitative real-time RT-PCR revealed that infertile women with EM have higher levels of FoxP3 mRNA in eutopic endometrium than the control group. Further analysis based on the extent of EM revealed that infertile women with advanced EM have higher levels of FoxP3 mRNA in eutopic endometrium than women with mild EM and the control group. The results of this study conflict with those reported in 2006 by Jasper et al. [8], which demonstrated an association between unexplained infertility and reduced FoxP3 mRNA expression in endometrial tissue. However, the results of this study are consistent with Berbic’s report, which demonstrated upregulation of FoxP3 expression in eutopic endometrium in women with EM in the secretory phase [9]. Therefore, it is hypothesized that FoxP3⁺ T_regs in the peri-implantation endometrium participate in the pathogenesis of EM while they are not directly involved in the pathogenesis of advanced EM associated infertility.

Additionally, our study showed that the expression of FoxP3 mRNA in the infertile women with mild EM was significant lower than patients with advanced EM, but it was similar to the control group. It is suggested by this result that the uterine immune status in peri-implantation endometrium among infertile patients with mild EM is different from that of the advanced EM. This kind of difference might be involved in the failure of embryo implantation and the pathogenesis of infertility in the mild EM. Although there was no statistically significant difference between the mild EM group and the control group, FoxP3 mRNA expression in the infertile women with mild EM was slightly lower. It is not clear whether the inadequate numbers of FoxP3⁺ T_regs in the peri-implantation endometrium in sub-fertile women with mild EM is related to the pathogenesis of infertility and unsuccessful embryo implantation. This needs to be examined in future studies with large sample size.

T_regs, which comprise only 5-10% of CD4⁺T cells in human,are few in eutopic endometrium and are periodically regulated by 17-β-estradiol [21]. T_regs will increase and exert full suppressive function when they expose to alloantigen, such as embryo, sperm, and inflammation [26, 27]. Since our study excluded the effect of alloantigen, the level of FoxP3⁺ cells by semiquantitative immunohistochemical staining was relatively low. Although there was no statistically significant difference, FoxP3⁺ expression in the advanced EM group was higher than the mild EM group and the control group. This result was consistent with the findings in the quantitative real-time PCR analysis. Because it is unethical to investigate human embryo implantation process in vivo, future studies may focus on the changes of T_regs during peri-implantation phase in the eutopic endometrium of infertile women with EM in vitro.

From the above, our findings suggest that FoxP3⁺ T_regs in the peri-implantation endometrium might participate in the pathogenesis of advanced EM. However, they are not directly involved in the pathogenesis of advanced EM associated with infertility. The differential expression of FoxP3 in infertile women with mild EM and advanced EM implicates that notable differences in the uterine immune status are likely involved in the pathogenesis of mild EM associated with infertility in the peri-implantation endometrium.