It is interesting that during the process of pregnancy, the immune system of the mother accepts an allogeneic fetus and does not reject it, but why? More than 35 years ago, for the first time, the TH1/TH2 hypothesis was propounded . In this concept, type 1 CD4+ T helper cells (TH1), which release pro-inflammatory cytokines such as IL-2 and IFN-γ, have a role in cellular immunity, inflammation, and rejection process. On the contrary, type 2 CD4+ T helper cells (TH2) are known as the mediators of humoral immunity, which secrete anti-inflammatory cytokines like IL-10, IL-5, IL-4, and so on and act in the opposite way to TH1-type cytokines . For many years, it was hypothesized that in the course of healthy pregnancy, a slight shift occurs from TH1-type to TH2-type immunity. However, later findings illustrated that the levels of both pro-inflammatory and inflammatory cytokines differ in various stages of pregnancy . In animal autoimmune studies such as experimental autoimmune encephalomyelitis (EAE), many questions emerged that did not follow TH1/TH2 solid dichotomy of immune balance . A subpopulation of CD4+ T cells, namely TH17 cells, was a main point to solve the puzzle of autoimmune diseases. In addition to autoimmune diseases, TH17 cell studies have been extended in various fields such as host defense, metabolic dysfunctions, allergic disease, allograft rejection, and cancer immunology [22, 23].
We presumed that the overexpression of TH17 cells has a considerable role in pathogenesis of RSA. To determine this probability, we evaluated IL-6 and IL-17 concentrations as well as RORγt expression in peripheral blood of women with RSA and control group. A significant increase was observed in the expression of RORγt and in the concentration of pro-inflammatory cytokines such as IL-6 and IL-17 among women with RSA. Although the frequency of TH17 cells was not determined by flow cytometric assay due to financial constraints, our results that demonstrate the enhancement of TH17-related agents are in line with previous studies regarding TH17 cells. To obtain accurate results, recent studies have investigated TH17 cells in all aspects, including total number, receptor presentation, signaling pathway, cytokine production and gene expression in both peripheral blood and decidua. A number of studies in this field have reported the increase in proportion of TH17 cells, IL-17 and IL-23 cytokines as well as overexpression of RORγt as well as and the decrease of Treg in peripheral blood and/or decidua of women with RSA [24, 25]. Activation of IL-17 might increase NF-κB expression, which reduces the quantity of progesterone receptors and weakens its function. Consequently, progesterone cannot bind a sufficient number of progesterone receptors, leading to decidua dysplasia and inadequate embryonic nutrition and finally resulting in miscarriage . In another study, the association of IL-17 F gene polymorphism with a high risk of RSA among Iranian women has been reported . A link has also been reported between polymorphisms in the genes of cytokines IL-1, TNF-α, and IL-17 with early pregnancy loss . By producing pro-inflammatory cytokines, TH17 cells may enhance TH1 and NK cell activities, finally leading to abortion and preterm labor [29, 30]. In this regard, the enhancement of NK cells number and activity has been demonstrated in women with RSA and those with IVF failure . Besides, the increase of TCD8+ cells has been reported in women with repeated miscarriage . Disturbance in immune regulation between CD8+ T cells and NK cells could increase NK cell activity, increasing the chance of reproductive failure and leading to RSA .
All of these findings imply that the increase of TH17 cells in women with RSA may augment the production of inflammatory cytokines and reinforce the function of effector cells such as NK cells and CD8+ T-cells. Thus, the synergistic effect of TH17 cells, NK cells, CD8+ T-cells and pro-inflammatory products may expand exaggerated inflammatory environment and finally lead to fetal rejection and reproductive failure.
On the contrary, Treg cells that modulate the functions of effector T-cells such as TH1, TH2, and TH17 have a significant role in maintaining peripheral tolerance. Treg cells perform their regulatory function through the secretion of anti-inflammatory cytokines such as IL-10 and TGF-β. TGF-β can elevate FoxP3 expression by inducing the expansion of CD4+CD25− T-cells into Treg cells . However, the presence of IL-6 or activated dendritic cells may induce the differentiation of Treg cells into TH17 cells. Therefore, it can be concluded that inflammatory conditions predispose to switching of Treg cells into TH17 cells . FoxP3 expression in humans is mainly restricted to CD4+CD25+ T cells, but other subpopulations of T-cells such as CD4+CD25− and CD8+ T cells express it, too. Thus, it is supposed that FoxP3-expressing T-cells have suppressive and regulatory functions . It is also known that Treg cells play a crucial role in restoring fetal maternal tolerance. The passive transfer of pregnancy-induced CD4+CD25+ Treg therapy has contributed to success of pregnancy and reduced the rate of spontaneous abortion among abortion-prone mice . Decrease of CD4+CD25+ and FoxP3+ Treg cells has been reported in decidua and/or peripheral blood of women with RSA and missed abortion. The expression of full-length FOXP3 protein was reduced in women with RSA compared to control group . Our findings were in line with previous studies because we demonstrated the reduction of CD4+CD25bright T-cells, FoxP3 expression and TGF-β concentration in peripheral blood of women with RSA .However, some studies have also reported that no significant difference is observed between the patient and control groups .
Our study was performed at secretory phase of menstrual cycle. The enhancement of IL-6 and IL-17 secretion, the reduction of TGF-β, and no difference in IL-10 level were found at this phase among women with RSA. In contrast to our study, some researchers reported the increase of IL-17 at both proliferative and secretory phases and the decrease of TGF-β and IL-10 only at proliferative phase among RSA group . These controversies in studies may depend on variations in sample size and type (decidua or peripheral blood), time of sampling (secretory or proliferative phase, pregnancy or non-pregnancy conditions), and time elapsed from the last abortion.
Seminal fluid is a strong source of prostaglandin E and TGF-β, which induce the expansion of Treg cells after coitus for embryo implantation . In the course of pregnancy, development of Treg cells in human and mice protects the invasive trophoblast cells containing fetal antigens against maternal immune system attack . In animal models, the depletion of Treg cells could increase inflammation and lead to aberrant uterine artery function in mice . Recent investigations in mouse models have indicated that adoptive transfer of Tregs can prevent pregnancy loss in these models by improving maternal tolerance. Hence, passive cell therapy by autologous Tregs could be a potential novel therapeutic approach for cell-based immunotherapy for women with repeated spontaneous abortion .