Introduction
Recurrent Spontaneous Abortion (RSA) is defined as three or more repeated abortions before the 20^th week of pregnancy, which occurs in 5% of fertile couples [1, 2]. Several factors such as chromosomal, placenta, genetic, anatomic, endocrinology, infection, environmental and immunological abnormalities contribute to this complication [3] that 69% of causes are unknown. In the meantime, autoimmune and alloimmune factors play an important role [4]. Patients with a history of recurrent spontaneous abortion, have abortion in all their pregnancy and have no live children. Patients with early recurrent abortions and patients with at least one successful pregnancy are referred to secondary recurrent abortions [5]. Considering that immunological mechanisms and proper mother-to-embryonic immune response play a significant role in the success of pregnancy, the immunological factors involved in RSA patients have been gradually identified. Accordingly, immunological disorders along with this complication are divided into two groups of autoimmune and alloimmune factors.
During pregnancy, the semi-allogeneic embryo is not invaded by the mother`s immune system. If the adaptation becomes dominated, the fetus is rejected by the mother`s immune system. Over the years, the molecular basis of adaptation or rejection has been studied. The molecules encoded by the major histocompatibility complex (MHC) have been identified as the main regulator for the success or failure of bond [6]. The half-fetal bond is preserved due to the existence of numerous protective mechanisms at the level of contact between mother and fetus during pregnancy.
Pregnancy from an immunological point of view occurs after the development of controlled inflammation during implantation and immune tolerance during this period [7]. There are important factors in protecting the fetus from the mother`s immune system. However, an inappropriate immunological response at different stages of pregnancy leads to failure of this process and abortion.
In this study, more than 40 papers on immunological factors involved in recurrent spontaneous abortion have been studied. According to the studies in the field of recurrent spontaneous abortion immunology, there are several factors involved in this disorder, which are discussed below.
 
Autoimmune factors
Autoimmune factors are produced after the response of the mother`s immune system to its own antigens. These factors contribute to failure of self-tolerance and result in maternal-fetal immunological response that is responsible for 30% of spontaneous abortions [8]. Several autoantibodies such as anti-phospholipid antibodies (APA), anti-nucleus antibody (ANA), anti-thyroid antibodies (ATA), anti-proxin-redoxin-4 antibodies, and endothelial antimicrobial antibodies can increase the response of the mother`s immune system. The secretion of these autoantibodies is inhibited by NK cells in normal pregnancy. By detecting HLA-G molecule by its own inhibitor, these cells block the activity of the B cell to secrete autoantibodies [4].
                      
Antiphospholipid Antibody (APA)
Different types of antiphospholipid antibodies such as anti-cardiolipin (ACA), antiphsosphatidylserine, and antiphosphatidyl tinanoamine have been found in women with recurrent spontaneous abortion. This group of antibodies interfere with fetal rejection with direct effect on trophoblast cells, changes in the cytokine patterns of decidua cell cultures, increased expression and secretion of pro-inflammatory cytokines such as 1-IL-6, IL and TNF, activation of NK cells and the destruction of placenta [9]. Antiphospholipid antibody syndrome (APS) accounts for 2% to 15% of RSA cases.
 
Antinuclear antibodies (ANA)
These antibodies are seen in autoimmune diseases such as lupus and in healthy people. Antibody against histones means the mother`s immune response to DNA. However, the exact mechanism of ANA in the fetal rejection has not yet been adequately identified. The rate of ANA is prevalent in women with recurrent abortion with uncertain causes [10].
 
Anti-thyroid Antibody (ATA)
Autoantibody against thyroid peroxidase or thyroglobulin are the most common autoimmune disease that occurs in about 5% to 20% of healthy pregnant women. High levels of anti-thyroglobulin and thyroid peroxidase antibodies are associated with an increase in the number of abortion, and approximately 31% of women who have experienced RSA are positive for one or both of these antibodies. Therefore, the levels of these antibodies are commonly evaluated for these patients [11].
 
Antiproxy Redoxin Antibody 4
According to recent findings, embryonic anti-protein auto-antibody especially Prx4 which have antioxidant activity, are found in patients with a history of recurrent spontaneous abortion.
 
Alloimmune Factors
Alloimmune mechanisms represent the immunological response of the mother to the allogeneic embryo. The success of pregnancy is greatly related to the placenta, which acts as an immunological barrier. The ability of trophoblast cells to protect the mother`s immune system was initially investigated due to its non-antigenic nature. However, due to the expression of some human leukocyte antigen (HLA) on the surface of these cells, alloimmune factors are needed to maintain the immunological tolerance of the mother [4].
 
Alloantibodies
During the pregnancy process, the mother`s immune system considers paternal HLA to be a foreign antigen and induces the expression of various alloantibodies including blocking antibodies such as Ab2, MLR-Bf and APCA, which cover the embryo and protects it against the maternal cytotoxic immunity response. Reducing these antibodies can lead to RSA [4]. Also, increasing the similarity of maternal HLA with paternal HLA antigens inhibits the production of blocking antibodies by the mother`s immune system. In several studies, the reduction of these antibodies in women with RSA has been shown compared to normal women. Anti-Ab2 antibodies, HLA, detect alloantigen receptors on mother`s T-cell and inhibit the alloimmune response of maternal T-cells during normal pregnancy. The MLR-Bf factor has been also found to be 82% in normal pregnant women and 10% in RSA patients. The amount of this antibody in the first trimester of pregnancy decreases to a maximum and gradually decreases. This antibody reduces significantly in RSA, which indicates its role in preserving pregnancy [4]. The production of MLR-Bf occurs as a result of the presence of trophoblast-lymphocyte cross-reactive (TLX) which is present in the sperm and has common antigenic indexes of HLA molecules and antithyroid trophoblast epitopes. In response to these antigens, blocking antibodies of the IgG class are made which bind to the Fc receptor at the level of trophoblast cells and protect the fetus from attacking the mother`s immune cells. In the majority of patients with a history of recurrent spontaneous abortion, a low level of MLR-Bf is reported. Reducing the level of this antibody can be due to increased HLA compatibility for couples, leading to poor immune responses [12].
 
Cell activity
The activity, maturation, and proliferation of NK cells increase under the influence of multiple cytokines such as 15-IL-2, IL-18, IL and IFNy. Thus, an examination of peripheral blood-centrifuged cells increases the tendency toward Th1-type immunity. NK cells activate Th1 cells to secrete toxic trophoblast cytokines. NK cell activity is also affected by the Th1 cytokine pattern. Hence, NK cells play an important role in the defense and immunological regulation of pregnancy, and in studies, the high activity of these cells in women with RSA, has been observed, while the activity of NK peripheral blood cells in the early pregnancy decreases compared to non-pregnancy and in the third trimester of normal pregnancy [13]. The findings show that the percentage of peripheral blood NK in RSA patients is significantly higher than healthy women [14]. Also, the ratio of uNK/pNK cells in the peripheral blood of these patients is lower than that of healthy women [8].
 
Balance of Th1/Th2
Basically, the presence of Th1 in the distance between mother and fetus in women with RSA is associated with increased Th1 cytokines production and inflammatory response, and more Th1-type immunity has been observed in recurrent abortion. Also, the proportion of Th1/Th2 in RSA patients is significantly higher than that of healthy pregnant women [15]. Th1 cells produce inflammatory responses by producing2-IL, TNF, and IFNy pro-inflammatory cytokines, resulting in the death of trophoblast cells. High level of IL-12 is also observed in RSA, which induces the secretion of Th1 and inhibits the Th2-type cytokines that contribute to the development of fetal rejection. Therefore, increasing the number of Th1 cells and decreasing the amount of Th2 cells plays a significant role in RSA [4]. However, according to recent reports about the Th2 type predominance in spontaneous abortion and the role of Th17 and Treg cells in controlling Th1 and Th2 responses, the balance between Th1/Th2 cells had led to Th1/Th2/Th17 and Treg cell pattern analysis [16].
 
Cytokines
Cytokines play a vital role in all stages of pregnancy. The precise model of imbalance between Th1/Th2 cells is considered to be a prerequisite for frequent abortions through the study of secreted cytokines of T cells and NK cells. Cytokines have different functions, and studies show that the Th1/Th2 balance changes to Th1 cells in RSA. In addition, since Th17 cells accompany Th1 cells with production of cytokines such as Il-17 in the induction of inflammation and rejection of the allograft, analysis of secreted cytokines from Th17 cells are also important.
Th1 secreted cytokines including IFNy, TNF β, TNF α and IL-2 can directly damage the placenta or indirectly activate the immune cells. The TNF α factor can cause fetal excretion by creating necrosis in the embryo and increasing the contraction of the uterus. The IFNy agent inhibits the secretion of GM-CSF, which is important for differentiation and growth of trophoblast cells in normal pregnancy, and the non-secretion of GM-CSF can lead to RSA. The secreted cytokines from Th2 cells include 5-IL-4, IL and Il-13 [4]. IL-4 can inhibit Th1 and macrophages and stimulate antibody production by B cells and inhibit fetal rejection. This cytokine plays an important role in the immunology of pregnancy by inhibiting the production of TNFα, IFNγ and IL-2. As the reduction in the cytokines in RSA women has been shown in comparison to women who have experienced natural pregnancy, IL-5 also acts as anti-inflammatory cytokine. Interleukin IL-13 is anti-inflammatory cytokine that many of its activities are similar to IL-4 and play a role in the natural evolution of the placenta and regulation of trophoblast invasion during the first trimester of pregnancy [17]. IL-17 is secreted by Th 17 cells that TCD4+ cells are in the peripheral and decidua blood and induce inflammatory mediators [18]. This cytokine has a poly-tropic property that induces the expression of cytokines and precursor chemokines and ultimately tissue degradation [19]. Recent studies have shown that the levels of this cytokines in RSA women are significantly higher than healthy women [20]. Interleukin IL-10 is also secreted by Treg cells as an anti-inflammatory and inhibitory cytotoxic agent, and by decreasing the activity of uterine macrophages during pregnancy and diversion of immune responses to pregnancy to Th2, it plays an important role in immunology of pregnancy, and it is involved in controlling intrinsic immune responses and cellular immunity. Also, TGFβ as an inhibitory ctyokines secreted from regulatory T cells by controlling its immune and inflammatory responses inhibit IL-17 secretion by Th17 cells, implantation of blastocyst, placenta evolution, invasive trophoblast, and embryo formation [19].
Treg Cells (T regulatory)
The activity of T-cells such as Th1, Th2 and Th1 is regulated by Treg cells, which contribute to the maintenance of pregnancy tolerance. Patients with RSA have a low level of T-cell regulation during pregnancy and after abortion. This decrease has reversal relationship with an increase in Th17 cells in the blood and decidua. Also, in Treg cells in women with RSA, the ability to inhibit Th17 cells has decreased [19, 21]. Thus, the deficiency in the function of Treg cells in recurrent spontaneous abortion can play an important role [22]. TCD4 + CD25+ decidua and peripheral blood cells control the TCD4+CD25+ in an unresponsive (energetic) manner. The presence of intra-gastric imunoglobin in human CD4+CD25+ T cells in the culture medium significantly increases the expression of Foxp3, TGF-β and 10-IL [23]. Based on the findings, decreasing the number and function of T-regulatory cells in women with this disorder leads to pregnancy failure.
 
Common treatments for recurrent spontaneous abortion
So far, several therapies have been used experimentally for RSA patients that drug treatments such as aspirin, heparin, progesterone, steroids, as well as immunological treatments such as intravenous immunoglobulin or IVIG, anti TNF- α, G-CSF and lymphocyte therapy can be mentioned [24].
 
Medicinal Therapies
Heparin therapy is one of the primary therapies for recurrent abortion with thrombotic events. Because thrombotic disorders are associated with an increase in abortion early in pregnancy, heparin has the potentiality to improve implantation and formation of placenta, anti-inflammatory function and immunological mediation, and its use in pregnancy will not harm the mother and the fetus [25]. However, the success rate of using heparin in the treatment of recurrent spontaneous abortion as a result of coagulation problems is still controversial. On the one hand, the role of anticoagulant agents in these patients is not well defined. On the other hand, the number of patients who have been included in the clinical trials to study the effect of this intervention is not enough, and more samples are needed to provide accurate report of the results [26]. Several therapeutic approaches have been evaluated for patients with a history of RSA. Most of these cases are focused on the use of heparin alone or in combination with prednisolone, aspirin, or progesterone [27].
Like heparin, aspirin as an anticoagulant can also be used in treating RSA with thrombophilic causes. However, recent findings suggest that treatment with aspirin alone or in combination with heparin is not effective in treating RSA patients with unknown causes and does not increase the birth rate of live infants [28].
Glucocorticoids which are used as steroids in the treatment of RSA patients, play a role in reducing the inflammation and inhibition of the activity of various types of immune cells, including T cells, due to binding to the recipient and inhibiting the NF-κB. Concomitant use of prednisolone with low dose heparin and aspirin may be effective in treating these patients, possibly due to the inhibition of NK cell proliferation [24].
Progesterone is used as an important hormone for pregnancy and as a medicine for the treatment of patient with a history of RSA. Considering the role of progesterone in regulating inflammatory mediators in order to preserve pregnancy by reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines, this drug can be effective and its effect on these patients reduces the incidence of recurrent spontaneous abortion [29]. However, several clinical trials with more samples are needed to complete the results.
 
G-CSF immunological treatments
The G-CSF is a colony stimulating factor in the cytokines family that secretes in placenta and decidua cells during pregnancy, and its high concentration increases the probability of an implantation. Since it is an effective cytokine in the fetus development, it is used as a target for the treatment of patients with RSA. The results of study indicate the effect of this cytokine on the success rate of pregnancy and the reduction of abortion rates after the treatment of patients. The TNF-α factor plays a major role in trophoblast invasion and fetal development and is produced and secreted by the uterine NK cells and trophoblast cells [30].
 
Inactivated immunization with Intravenous Immunoglobulin (IVIG)
IVIG is one of the therapeutic methods for the antiphospholipid antibody syndrome and patients with a history of RSA, which is human Ig derived from combination of several normal blood plasma levels. Given than recurrent spontaneous abortion may occur following improper immunological or inflammatory responses, such as cytokine or autoimmune responses, IVG can be effective in treating this condition [31]. Antibodies in IVIG can neutralize harmful autoantibodies. In addition, these antibodies inhibit Fcy receptors on the surface of endothelial cells, B cells and NK cells, thereby reducing the function of B cell protection, antibody production and NK cell lethal activity [30]. However, findings from several clinical trials on the effectiveness of IVIG in treating patients with a history of RSA have been reported on a systematic review that shows the significant increase in success rate of pregnancy and the birth of live infant following IVIG injection compared to the control group who received placebo [32].
 
Lymphocyte Therapy
Immunization by the paternal leukocytes (PLI) or lymphocyte therapy (LIT) is widely used to treat recurrent spontaneous abortion with alloimmunity causes and sub therapy for these patients by injection of IVIG [33]. Since immunological identification of paternal antigens and the appropriate immune response to maintain a semi-allogeneic fetus, lymphocyte therapy with paternal cells or third-person cells is used to stimulate the mother`s specific immune response to paternal alloantigen [12]. In the early 1980s, immunotherapy has often been conducted with allogeneic lymphocytes and numerous clinical trials have been conducted in this area. The first randomized clinical trial was conducted by Moobery et al. They were more successful in treating women with lymphocyte therapy than treating women with their own lymphocytes [34].
Increased improvement in the outcome of pregnancy after the LIT of patients with a history of RSA has been reported in many studies [35]. However, conflicting observations suggest that the treatment is not successful, and therefore its effectiveness remains uncertain. This had led the US Food and Drug Administration (FDA) to oppose this treatment [33].
Precise lymphocytotherapy mechanisms have not yet been fully identified. However, preliminary studies about this treatment examine the similarity of HLA antigens in couples. The findings show that the high similarity of HLA in women with the history of RSA with the HLA in their husband has reduced the production of blocking antibodies and thus the fetal rejection [36]. Some researchers believe that lymphocyte therapy may act as an immunogen and increase the immune response of the mother and induces the production of multiple antibodies that can play an important role in immunologic regulation and preservation of pregnancy [35]. Alloantibodies are produced as a result of immunotherapy with the spouse`s lymphocytes that can mask the HLA paternity antigens and protect them from the attach of maternal T cells. Anti-TCR antibodies are also followed by this kind of treatment that specifically binds to TCR and inhibits maternal immune response against the fetus [37]. Therefore, the beneficial effects of this process initially led to the induction of multiple antibodies that inhibited the immunological rejection mechanisms of the fetus and contributed to the implantation and fetal development.
Also, the beneficial effects of lymphocytic therapy, including the specific and non-specific inhibition of T cells, the decrease in the level of the IL-2 receptors that leads to immune response to Th2 type, reduces the activity of NK cells and inhibits their lethal operation. However, the effectiveness of this therapy in treating recurrent spontaneous abortion is doubtful.
 
Conclusion
Regarding the fact that the cause of recurrent spontaneous abortion is unknown in about 50% of cases, immunologic factors have been implicated in these cases and several findings have been obtained about the role of immunological factors. Also, immunological therapies for recurrent spontaneous abortion is increasing. Accordingly, the identification and investigation of the role of immunological factors in recurrent spontaneous abortion is of great importance.
 
Acknowledgements
Non-declared
Ethical Permissions
Non-declared
Conflicts of interests
Non-declared
Financial Sources
Non-declared
Contribution of the Authors
Narges Romandeh (First author): author of article/methodologist/ main author or helper/statistical analysis/ author of discussion (…%); Ahad Zare (Second author), author of article /methodologist/main author or helper/Statistical analyst/ author of discussion (…%);Abo Taleb Sarem (Third author), author of article /methodologist/main author or helper/Statistical analyst/ author of discussion (…%); (Choose the role of each person in the article from among the specified items. Note that choose the appropriate role for each person and avoid choosing all items for all authors, as it will not be acceptable).