Decrease in Regulatory T-cells and Increase in T Helper 17 Cells in Women With Recurrent Spontaneous Abortion

Background Appearance of improper immune responses against the fetus and/or inadequate immunoregulatory mechanisms during pregnancy may lead to recurrent spontaneous abortion (RSA). T H 17 cells play a signicant role in inducing inammation, autoimmune disease, and acute transplant rejection, while regulatory T (Treg) cells moderate the function of immune system in order to retain homeostasis. This case-control study was designed to evaluate T H 17 as well as Treg cells in 25 women with RSA and 25 age-matched healthy non-pregnant women. Flow cytometric assay was performed using monoclonal antibodies to detect CD4 + CD25 + Treg cells (CD25 dim and CD25 bright ). FoxP3 and RORγt expressions were compared using real-time PCR, and pro-inammatory and anti-inammatory cytokines were measured by ELISA kits. Independent-samples T test was employed for statistical analysis.


Introduction
Pregnancy is a complex and challenging event in which all organs of the mother such as immune system should adapt to new physiological changes emerging by the growing fetus to ensure a good and successful outcome. Since the fetus expresses paternal antigens, it is considered as a semi-allograft; however, due to the immune tolerance in the course of healthy pregnancy, maternal immune system tolerates the fetus and does not reject it. Any disruption in this immune tolerance and the appearance of improper immune responses against the fetus could affect the pregnancy outcome and may lead to disorders such as spontaneous abortion, which usually occurs in the rst trimester of pregnancy [1]. While anatomic, genetic, endocrinologic, and coagulative disorders and infections have been implicated in etiology of RSA, immunological problems play a main role in this phenomenon [2]. The distribution and function of decidual immune cells have been frequently studied in normal and pathologic conditions. Uterine NK (uNK) cells, T-cells, and macrophages are principal subpopulations of leukocytes in decidua [3]. According to the traditional classi cation, T lymphocytes are categorized into T H 1 and T H 2 subsets, which produce a variety of cytokines [4].
T H 17 cells constitute an important subset of CD4 + T-cells secreting IL-17 and other pro-in ammatory cytokines, which play a signi cant role in host defense against extracellular bacterial and fungal agents, neutrophils recruitment, chronic in ammation, acute transplant rejection, and autoimmune disorders [5].
Retinoic acid-related orphan receptor γt (RORγt or RORC) has been reported to be a transcription factor involved in the development and expansion of T H 17 cells [6].
Regulatory T (Treg) cells have been recognized as the main regulators of immune system, which modulate the impact of T H 17 cells on in ammatory states [7]. Interestingly, T H 17 and Treg cells stem from a single developmental lineage; therefore, there is a complicated relationship between them because progenitor cells differentiate into Treg cells in presence of TGF-β alone, developing into T H 17 cells under the in uence of both TGFβ and IL-6 [8]. CD4 + CD25 + Treg cells have been identi ed as a subpopulation of T-cells having immunomodulatory and immunoregulatory function, which establish immune balance between the elements of immune system and enhance pregnancy outcome. Forkhead box P3 (FoxP3) has been recognized as a Treg-associated factor inducing generative, proliferative, and regulatory function of Treg cells [9]. Treg cells perform their immunomodulatory function by secreting IL-10 and TGFβ cytokines. Besides, TGF-β stimulates the differentiation of Treg cells from CD4 + CD25 − T cells by FoxP3 augmentation [10] .
The delicate balance among the various components of immune system is crucial for maintenance of pregnancy. As well as predominant T H 1-type immunity, exaggerated T H 2-type immunity has unfavorable effects, which may lead to RSA [11]. Since some of the immunological disorders could not be expounded producing cells in decidua and/or peripheral blood of women with RSA and preeclampsia disorders [12,13].
Thus, to expand and con rm prior studies, we aimed to focus on T H 17 and Treg cells and related agents in peripheral blood of women with RSA in comparison with healthy controls.

Study population and sampling
In our case-control study, a total of 50 women comprising 25 women with RSA and 25 healthy nonpregnant women were recruited. The RSA group was diagnosed by a gynecologist after exclusion criteria such as chromosomal abnormalities, anatomic disorders (uterine polyp, uterine septum, broid, pelvic adhesion), hormonal and metabolic diseases (thyroid disorders, polycystic ovarian syndrome, diabetes mellitus), immunological problems (autoimmune disease, existence of anti-phospholipid, anti-cardiolipin and anti-coagulant antibodies), coagulation dysfunction, and infection. All the selected women having RSA were in reproductive age with mean age of 29.1±0.8 years and with the history of three or more repeated miscarriages with mean miscarriage of 3.2±0.1. Also, the selected patients had a normal karyotype, their male partners were examined and had normal semen state.
For control group, 25 healthy non-pregnant women without a history of abortion or disorders during previous pregnancies were randomly selected. Healthy women were in reproductive age and had mean age of 29.9±0.7 years and a minimum of one living child with the mean number of children of 1.8±0.1. Also, their health status was assessed, and all of them had regular menstrual cycle, normal gynecological, hormonal, and anatomical conditions without any symptoms of infection.
It should be noted that at least three months had elapsed from the last miscarriage, pregnancy, or vaccination of participants, and all of them signed a written informed consent form before participation.

The ethics review board of Ahvaz Jundishapur University of Medical Sciences (AJUMS) approved the current study
At secretory phase of menstrual cycle, venous blood was collected from all participants and transferred into three different tubes: one tube for ow cytometric analysis; an EDTA-contained tube for isolation of mononuclear cells using a lymphocyte separation medium for mRNA extraction and real-time PCR; and a clot activator containing tube for evaluation of serum cytokines.
Flow cytometry CD4 + CD25 + Treg cells were determined using fresh blood and stained using FITC-conjugated anti-human CD4 and PE-conjugated anti-human CD25 antibodies (eBioscienc, California) according to manufacturer's instructions. Isotype control antibodies were used to ensure the speci city of antibodies as well as precise comparison. After eliminating RBCs by the lysis solution (DAKO cytomation, Germany) and washing with phosphate buffer saline (PBS), the samples were assessed using a FACS Calibur device (Becton Dickinson). CD4 + CD25 + (CD25 dim and CD25 bright ) Treg cells were selected within the lymphocyte gate. Flowing Software v2.4.1 was used for ow cytometric analysis.
RNA extraction, cDNA synthesis, and real-time PCR After isolating peripheral blood mononuclear cells (PBMCs) by lymphocyte separation medium (PAA, Germany), total RNA was extracted using Rneasy Mini Kits (QIAGEN, Valencia, CA) by observing the manufacturer's instructions. The quality of the extracted RNA was checked using Biophotometer (Eppendorf). cDNA synthesis was performed by QuantiTec Reverse Transcription kit (QIAGEN, Valencia, CA) according to manufacturer's guidelines. Finally, the product was stored at -80ºC until use. The ampli cation was performed in an ABI Step One (Applied Biosystem, USA) PCR system using StepOne Software v2.1 and SYBR Green kit (Takara, Japan). All samples were run in duplicate with negative controls. The primer sequences were as follows: ROR C forward 5 -AGACTCATCGCCAAAGCA-3 and reverse 5 -CCTTGTAGAGTGGAGGGAAA-3 [14] as well as FoxP3 forward 5 -CAGCACATTCCCAGAGTTCCT-3 and reverse 5 -GCGTGTGAACCAGTGGTAGAT-3 [15]. Furthermore, the housekeeping gene GAPDH primers, namely forward 5 -ACCACAGTCCATGCCATCAC- 3

Statistical analysis
Data analysis was done using SPSS statistical software (version 22; SPSS, Chicago, IL). According to Kolmogorov-Smirnov Z test, the distribution of data was normal. Thus, independent-samples t-test was employed for comparison of case and control groups. Data were presented as mean±SEM (standard error of mean), and P<0.05 was considered statistically signi cant.

Age of participants
The mean age of women with RSA and control group was 29.1±0.8 years and 29.9±0.7 years, respectively, and there was no signi cant difference in this regard (P=0.441).
RORγt and FoxP3 expression GAPDH was assumed as the reference gene with reaction e ciency of 0.97. RORγt and FoxP3 were target genes with reaction e ciencies of 1.00 and 0.97, respectively. RORγt mRNA was up-regulated 2.04fold in case group relative to healthy controls (P=0.026; Fig. 3); FoxP3 mRNA was down-regulated in women with RSA by 0.53-fold compared to healthy group (P=0.037; Fig. 3).

Discussion
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 rst time, the T H 1/T H 2 hypothesis was propounded [18]. In this concept, type 1 CD4 + T helper cells (T H 1), which release proin ammatory cytokines such as IL-2 and IFN-γ, have a role in cellular immunity, in ammation, and rejection process. On the contrary, type 2 CD4 + T helper cells (T H 2) are known as the mediators of humoral immunity, which secrete anti-in ammatory cytokines like IL-10, IL-5, IL-4, and so on and act in the opposite way to T H 1-type cytokines [19]. For many years, it was hypothesized that in the course of healthy pregnancy, a slight shift occurs from T H 1-type to T H 2-type immunity. However, later ndings illustrated that the levels of both pro-in ammatory and in ammatory cytokines differ in various stages of pregnancy [20]. In animal autoimmune studies such as experimental autoimmune encephalomyelitis (EAE), many questions emerged that did not follow T H 1/T H 2 solid dichotomy of immune balance [21]. A subpopulation of CD4 + T cells, namely T H 17 cells, was a main point to solve the puzzle of autoimmune diseases. In addition to autoimmune diseases, T H 17 cell studies have been extended in various elds such as host defense, metabolic dysfunctions, allergic disease, allograft rejection, and cancer immunology [22,23].
We presumed that the overexpression of T H 17 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 signi cant increase was observed in the expression of RORγt and in the concentration of pro-in ammatory cytokines such as IL-6 and IL-17 among women with RSA. Although the frequency of T H 17 cells was not determined by ow cytometric assay due to nancial constraints, our results that demonstrate the enhancement of T H 17-related agents are in line with previous studies regarding T H 17 cells. To obtain accurate results, recent studies have investigated T H 17 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 eld have reported the increase in proportion of T H 17 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 su cient number of progesterone receptors, leading to decidua dysplasia and inadequate embryonic nutrition and nally resulting in miscarriage .
[26] In another study, the association of IL-17 F gene polymorphism with a high risk of RSA among Iranian women has been reported [27]. A link has also been reported between polymorphisms in the genes of cytokines IL-1, TNF-α, and IL-17 with early pregnancy loss [28]. By producing pro-in ammatory cytokines, T H 17 cells may enhance T H 1 and NK cell activities, nally 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 [31]. Besides, the increase of TCD8 + cells has been reported in women with repeated miscarriage [32]. 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 [33]. can be concluded that in ammatory conditions predispose to switching of Treg cells into T H 17 cells [34].

All of these ndings imply that the increase of T H 17 cells in women with
FoxP3 expression in humans is mainly restricted to CD4 + CD25 + T cells, but other subpopulations of Tcells such as CD4 + CD25 − and CD8 + T cells express it, too. Thus, it is supposed that FoxP3-expressing Tcells have suppressive and regulatory functions [35]. 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 abortionprone mice [36]. 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 [37]. Our ndings were in line with previous studies because we demonstrated the reduction of CD4 + CD25 bright T-cells, FoxP3 expression and TGF-β concentration in peripheral blood of women with RSA .However, some studies have also reported that no signi cant difference is observed between the patient and control groups . [38] 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 [24]. 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 uid is a strong source of prostaglandin E and TGF-β, which induce the expansion of Treg cells after coitus for embryo implantation [39]. 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 [40]. In animal models, the depletion of Treg cells could increase in ammation and lead to aberrant uterine artery function in mice [41]. 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 cellbased immunotherapy for women with repeated spontaneous abortion [42].

Conclusion
In conclusion, defects in immune regulatory mechanisms such as decreased number and function of regulatory T-cells and exaggerated T H 17-mediated in ammatory responses may threaten reproductive outcome, and this immunological imbalance might lead to disorders such as RSA during pregnancy.
Moreover, adoptive Treg cell therapy could potentially be a therapeutic approach for these patients.   The relative expressions of RORγt and FoxP3 in peripheral blood mononuclear cells of RSA women (n=25) as compared to healthy controls (n=25). RORγt is upregulated signi cantly in RSA group (in comparison to control group) by a mean factor of 2.04, and FoxP3 is downregulated signi cantly in RSA group (in comparison to control group) by a mean factor of 0.53 (*P<0.05).