A body of evidence has demonstrated a crucial role for seminal fluid priming in eliciting the expansion of the Tregs population in early pregnancy (Figure 1) [46]. In support of this, removal of seminal vesicle gland attenuates Tregs activation and abolishes the expansion of Treg pool in mice [48–50]. In addition, prior seminal fluid contact increases implantation success in in vitro fertilization (IVF) treatment cycles [51]. Seminal fluid contains paternal alloantigens, abundant TGF-β, and TLR4-Ls as well as an array of cytokines [52]. Paternal antigens in seminal fluid in the vaginal lumen stimulate APCs such as DCs and macrophages. Moreover, TGF-β and TLR4-Ls in seminal fluid can provoke an inflammation-like response in the female reproductive tract. These processes can trigger DCs and macrophages to carry paternal alloantigens to the regional lymph nodes and present antigens to naive T cells, leading to a transformation of effector CD4⁺ cell into iTregs [16, 53] within days of conception and then in the spleen and peripheral blood. Circulating Tregs are then recruited to the implantation site in response to epithelial cell-expressed and -secreted chemokines and their receptors [46]. It is proposed that this process contributes to the establishment and maintenance of immune tolerance to fetal alloantigens during embryo implantation [46].

TGF-β is a pivotal differentiation factor for promoting conversion of peripheral CD4⁺CD25^– naive T cells to CD4⁺CD25⁺ Tregs through increasing FOXP3 expression [54]. However, in the presence of IL-6, TGF-β fails to induce the generation of FOXP3 Tregs; instead, IL-6 and TGF-β together increase the differentiation of Th17 cells from naive T cells in mice (Figure 1) [55]. These findings suggest that TGF-β-mediated induction of FOXP3⁺ Tregs depends on the state of the innate immune system and the production of inflammatory cytokines such as IL-6 [55].

Pregnancy-associated hormones, including estrogen, progesterone, and hCG, are key pregnancy hormones, playing an immune modulatory role (Figure 1). However, whether and how these hormones modulate the expansion of Tregs remains poorly understood. In this regard, Aluvihare et al. (2004) [33] found that the expansion of CD4⁺CD25⁺ FOXP3⁺ Tregs during pregnancy is alloantigen-independent in syngeneically mated female mice, suggesting the role of pregnancy hormones. Additionally, transferring CD4⁺CD25⁺ Tregs from syngeneically mated female mice can protect allogeneic fetuses from rejection [33, 56, 57]. These results suggest that Treg expansion may be associated with hormonal modulation. Corroborating studies report that prior to conception and implantation, nTregs expand systemically and accumulate in the uterus in response to increased estradiol at the time of ovulation in mice [44, 58]. In vitro and in vivo experiments have shown that estrogen treatment elicits the expansion of Tregs through binding to estrogen receptor α. In vitro studies demonstrate that estrogen enhances the conversion of CD4⁺CD25^– T cells to Tregs through elevating FOXP3 messenger RNA (mRNA) expression [59, 60]. Estrogen-induced expansion of Tregs occurs during embryo implantation [60]. Likewise, administration of progesterone not only increases the proportion of Tregs in peripheral blood, spleen, and inguinal lymph nodes, but also enhances the suppressive activity of Tregs in ovariectomized mice during midterm pregnancy [61]. Progesterone increases the expansion of Tregs through binding to the nuclear progesterone receptors [61]. On the other hand, progesterone can bind to the glucocorticoid receptor, leading to an increase in the proportion of Tregs and apoptosis of Teffs [62]. A recent study has revealed that progesterone drives the development of nTregs through the osteoclast differentiation receptor, the receptor activator of nuclear factor kappa B (NF-kB) ligand (RANK), and genetic deletion of RANK leads to reduced accumulation of nTregs in the mouse placenta and increases miscarriages [63]. In this regard, knockout of progesterone receptor on CD11c⁺ DCs in mice decreases the frequency of CD4⁺FOXP3⁺ and CD8⁺CD122⁺ Tregs, which is associated with intrauterine growth restriction [64]. The progesterone receptor membrane component 1 (PGRMC1) in the endometrial glands is decreased in women with recurrent miscarriage [65]. However, alteration of PGRMC1 expression inversely correlates with the number of infiltrated FOXP3⁺ Tregs in patients with recurrent miscarriage. This was interpreted by the authors as a compensatory response to the increased uNK endometrial infiltration [65].

hCG is a critical pregnancy hormone that can be detected during the very early days post-implantation, reaches the peak around gestational week 12, and then remains low during gestation until birth [36]. hCG is a trophoblast-produced hormone [66, 67] and has a myriad of functions in pregnancy, including the promotion of progesterone production, implantation and decidualization, angiogenesis, cytotrophoblast differentiation, and immune cell regulation [36]. Low levels of hCG and Treg numbers have been observed in women with spontaneous abortion, raising the question of whether hCG can influence Treg generation and expansion. Indeed, accumulated evidence has shown that hCG functions as a chemoattractant for recruiting Tregs at the maternal-fetal interface, contributing to immune tolerance during pregnancy [37, 68]. Administration of hCG significantly increases the frequencies of Tregs in pregnant but not nonpregnant mice [68]. In vitro studies have shown that hCG can augment the induction of CD4⁺FOXP3⁺ Tregs from CD4⁺FOXP3^– T cells, which can be inhibited by blockade of hCG [68]. In abortion-prone mice, administration of hCG during peri-implantation upregulates the accumulation of Tregs in the endometrium, thereby reducing the fetal resorption rate [37].

Interestingly, based on these observations, intrauterine administration of hCG before embryo transfer has been employed as a therapeutic strategy to improve IVF-embryo transplantation outcomes [69–73] and enhance the live birth rate in women with repeated implantation failure [69, 74–76]. Subcutaneous injection of hCG in women prior to IVF can increase the production of anti-inflammatory IL-27 and IL-10 and numbers of Tregs in the peripheral blood while decreasing the abundance of inflammatory IL-17 [77]. Likewise, intramuscular injection of immunoglobulin-combined hCG induces a decrease in the number of Th17 cells and production of IL-17 and IL-6, and an increase in the number of Tregs and abundance of IL-10 and TGF-β1 in patients with unexplained RSA [78]. A recent study reports that intrauterine infusion of hCG enhances recruitment of Tregs to the endometrium in patients with repeated implantation failure, which may be mediated by inducing the production of chemokine C-C motif chemokine ligand 2 (CCL2) [79]. Taken together, hCG plays pivotal roles in attracting Tregs to the endometrium and elevating the numbers and suppressive capacity of Tregs at the maternal-fetal interface.

PD-1 receptor and PDLs, PDL-1 and PDL-2, are expressed on hematopoietic cells, including conventional CD4⁺ and CD8⁺ T cells, nonhematopoietic cells, and cancer cells. PDL is a type-1 transmembrane protein. The binding of PDL to the inhibitory checkpoint molecule PD-1 transmits inhibitory signals that limit the immune responses of effector and cytotoxic T cells, regulate the tolerance and exhaustion of T cells, and resolve inflammation. Thus, the PD-PDL pathway plays an inhibitory role in regulating the magnitude of adaptive immune response and tolerance. Notably, blockade of PD-1 and PDL-1 has been utilized for efficacious treatment of over 20 types of cancers [80]. The role of PD-1-PDL-1 negative costimulatory pathway in the maintenance of tolerance has been described in multiple transplant models, a diabetes model, and at the maternal-fetal interface of an alloantigen-specific CD4⁺ TCR transgenic mouse model [81, 82].

Studies from Guleria et al. [81] provided for the first time that blockade of PDL-1 using anti-PDL-1 monoclonal antibody leads to an increase in the rate of spontaneous fetal resorption in allogeneic pregnancy mouse model, and the expression of PDL-1 on the surface of Tregs is essential for their suppressive function and maternal immune tolerance [83]. PDL-1 has the ability to enhance and sustain FOXP3 expression and promote iTreg development and suppressive function through downregulation of phosphor-Akt, mechanistic target of rapamycin (mTOR), and extracellular signal-regulated kinase 2 (ERK2) and concomitant with an upregulation of phosphatase and tensin homolog (PTEN) [84]. PDL-1^–/–PDL-2^–/–Rag^–/– naive CD4⁺ T cells display a marked reduction in iTreg conversion and rapid onset of a fatal inflammation [84]. A deficiency of PDL-1 induces apoptosis of Tregs, decreases the ratio of Tregs to Teffs, and promotes Th1 and Th17 cell development and expansion, leading to fetal rejection and reduction in fetal survival [82, 83]. Furthermore, neutralization of IL-17 or adoptive CD25⁺FOXP3⁺ Treg transfer restores Treg numbers, thereby rescuing the effects of PDL-1 deficiency on fetal survival rate [82]. In addition, interaction of PDL-1 with PD-1 has been shown to induce the conversion of human Th1 cells into Tregs [85].

In contrast, a most recent study demonstrates an inhibitory effect of PD-1 on Tregs in mice in which deletion of PD-1 can be selectively induced in Tregs by tamoxifen administration. Selective deletion of PD-1 in Tregs in tissues such as the thymus and spleen of mice leads to an increase in their immunosuppressive capacity. Interestingly, PD-1-deficiency in Tregs ameliorates experimental autoimmune encephalomyelitis and protects nonobese diabetic mice, the mouse model of spontaneous autoimmunity, from developing type 1 diabetes. Mechanistically, the phosphatidylinositol 3-kinase (PI3K)-AKT pathway is responsible for the increased suppressive activity of PD-1 deficient Tregs [86].

TLRs are a large family of pattern recognition receptors that are expressed both on trophoblasts and immune cells [87, 88]. TLRs expressed on CD4⁺CD25⁺ Tregs can regulate the expansion and suppression of Tregs [89, 90]. Activation of TLR9 by oligodeoxynucleotide (ODN)1826 decreases the proportion of Tregs in the decidua of abortion-prone mice [91]. In vitro experiments show that TLR9 activation inhibits the proliferation of Tregs and induces apoptosis of Tregs through caspase pathway. These TRL9-mediated effects can be abrogated by antagonist ODN [91]. Thus, TLR9-mediated signaling plays an inhibitory role in the generation of Tregs. In contrast, TLR4 knockout mice exhibit reduced pregnancy rate, increased mid-gestation fetal loss and fetal growth restriction, failed expansion of peripheral and thymic CD25⁺FOXP3⁺ Tregs, and fewer Tregs expressing Ki67 proliferation marker and suppressive function marker CTLA4. This suggests that TLR4 is a positive mediator for the generation of Tregs [92]. Thus, TLR members may play differential roles in the regulation of Tregs.

Hypoxic microenvironment in tumors has been shown to promote recruitment of Tregs, which in turn enhances immune tolerance and angiogenesis in tumors [93, 94]. Interestingly, the physiological hypoxic microenvironment also occurs at the maternal-fetal interface during early pregnancy. Emerging evidence has shown a role for hypoxia in the recruitment and expansion of Tregs during early gestation. It has been proposed that hypoxia-inducible factor-1α (HIF-1α) deficiency can reduce the recruitment of dNK cells into the maternal decidua [95], suggesting that hypoxia promotes the infiltration of dNK cells into the maternal-fetal interface. It is unclear whether a hypoxic environment directly induces recruitment of Tregs to the decidua during early gestation. However, recruited dNK cells can enhance the induction and expansion of Tregs at the maternal-fetal interface [96].

The role of mesenchymal stem cells from different origins such as bone marrow, adipose tissue, and endometrium in induction of Tregs has recently emerged. A recent study by Aleahmad et al. [97] shows that pretreatment of menstrual mesenchymal stem/stromal cells (MnSCs) with IFN-γ and IL-1β increases the ability of Tregs to inhibit the proliferation of anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs), while non-treated MnSCs decreases the frequency of Tregs. This effect is regulated through IL-6, IL-10, TGF-β, and IDO [97]. These results suggest a pivotal role of endometrial stem cells in local induction of Tregs in the endometrium.

RSA is characterized by three or more consecutive pregnancies. Although the etiology of this pregnancy adversity is still unclear, accumulating evidence has shown impaired immune tolerance as a contributing factor to RSA. In the mouse model of abortion, diminished activity of Tregs and subsequent accumulation of paternal alloantigen specific Th1 cells were observed in the decidua of abortion mice [43]. Notably, adoptive transfer of Tregs from normal pregnant mice protects against fetal rejection [43, 121]. Likewise, a significantly reduced quantity and function of Tregs was observed at the maternal-fetal interface of abortion-prone mice or mice with abortion induced by progesterone antagonist [61]. Furthermore, administration of TLR4 antagonist E5564 to abortion-prone mice increases the proportion of Tregs and expression of FOXP3 mRNA and proteins and decreases inflammatory response, thereby reducing embryo absorption rate [122].

Clinical studies have demonstrated a markedly reduced proportion and function of Tregs and an imbalance of the Th1, Th2, Th17 and Tregs in the decidua and peripheral blood from patients with spontaneous abortions or recurrent implantation failure compared to controls [30, 118, 122–126]. The expression of FOXP3, a determinant for the function of Tregs, was found to be significantly lower in both the decidua and peripheral blood from women with unexplained RSA [122, 125]. In support of this, Tregs from women with RSA exhibit reduced ability to expand during the pre-implantatory phase relative to those from fertile women [118]. In addition, increased ratios of Th1/Th2 and Th17/Tregs have been detected in peripheral blood from patients with RSA [126–128]. In vitro studies have revealed that Tregs from patients with RSA display inhibited ability to suppress the proliferation of Th17 cells and their secretion of IL-17 via cell-cell contact and mediation by IL-10 and TGF-β [118]. Additionally, higher levels of TLR1, TLR3, TLR4, TRL8, and TLR9 gene expression were observed in the decidua of patients with RSA [89]. Given that TLRs activation can inhibit the proliferation of Tregs [91], increased levels of TLRs may contribute to inhibition of Tregs expansion and suppressive activity in women with RSA. Taken together, decreased proportion and function of Tregs or their conversion to Th17 cells in response to inflammation [117] may disturb maternal immune tolerance against fetal rejection and subsequently increase inflammation, thereby leading to implantation failure and abortion.

PE is a multifactorial, pregnancy-specific syndrome characterized by new onset of hypertension at or after 20 weeks of gestation. It is a leading cause of maternal morbidity and mortality and affects 5–8% of all pregnancies. While the etiology of PE is still unclear, accumulating evidence suggests that impaired maternal immune tolerance contributes to the pathophysiology of PE. Inadequate paternal antigen-specific immune tolerance has been associated with an increased risk of PE. An epidemiological study unraveled that women inseminated with donor sperm have an increased risk of PE compared to those with their partner’s sperm [129]. Primiparous pregnancy, long interval between pregnancies, first pregnancy after changing partner, and reduced time length of exposure to seminal fluid have been linked to a substantially elevated risk of developing PE [130–132]. These clinical observations point to an important role of paternal antigen exposure-induced immune tolerance and immunological memory in the mechanisms for maintaining normal pregnancy and protecting against the development of PE during subsequent pregnancies [132].

As discussed earlier, the expansion of FOXP3⁺ Tregs is essential for maternal-fetal immune tolerance in normal pregnancy. Experimental data indicate that abnormalities of Tregs in the decidua and peripheral blood contribute to the etiology of PE [133–137]. For instance, reduction in FOXP3 expression and elevation in Th17-specific transcription factor retinoic acid related orphan receptor (ROR)ɤt and IL-17 presence have been observed in peripheral blood from patients with PE. An imbalance of Th17 cells/Tregs and anti-/pro-inflammatory cytokines illustrates a disturbed immune tolerance in PE patients [138]. In addition, many studies demonstrate that the expansion and suppressive activity of Tregs declines in PE patients [26, 136]. In vitro experiments show that a subset of CD14⁺ APCs, CD14⁺DC-specific intercellular adhesion molecule 1-grabbing nonintegrin (SIGN)⁺ APCs, from healthy pregnant women significantly increase the induction of iTregs; however, these cells from PE patients have a decreased ability to induce iTregs. Thus, these results suggest that reduced expansion of iTregs in the decidua of PE patients may be caused by decreased proportion of CD14⁺DC-SIGN⁺ APCs in the decidua [26].

FOXP3⁺ Tregs play an essential role in maintaining the balance between immune suppression and immune activation for host defense against infection at the maternal-fetal interface [12, 119]. It has been reported that influenza A virus infection in pregnant women has serious consequences as evidenced by markedly higher rates of hospitalization, morbidity and mortality [139]. It has been shown that expanded FOXP3 Tregs markedly impair host defense and increase susceptibility to intracellular bacterium Listeria monocytogenes in pregnant mice relative to nonpregnant controls at mid-gestation, and ablation of maternal Tregs beginning mid-gestation reverses the pregnancy-associated susceptibility to bacterial infection [119]. These results may explain why this bacterium has a striking predilection for disseminating infection in pregnant women [140]. Furthermore, mice containing IL-10-deficient Tregs exhibit stronger resistance to Listeria monocytogenes infection compared to controls in pregnant [119] or nonpregnant mice [141], and cell intrinsic IL-10 is required for Treg-mediated infection susceptibility [119]. Additionally, Adetunji et al. [142] reported that inactivation of Tregs using TNF receptor II (TNFR2) antagonistic antibody remarkably reduces Brucella abortus bacterial burden systemically and locally in reproductive tissues, suggesting that highly suppressive CD4⁺FOXP3⁺TNFR2⁺ Tregs are responsible for the persistent of Brucella abortus infection.

Importantly, Tregs may switch from suppressive to inflammatory phenotype in the aberrant environment. Emerging evidence has shown that nTregs can convert into Th1, Th17 and T follicular helper cells in an inflammatory milieu rich with inflammatory cytokines, such as IL-1, IL-6, and IL-23 [120]. Mechanistically, toxic conversion of nTregs may be caused by downregulation of FOXP3 expression. nTregs-converted Th17 cells can produce IL-17 and chemokine receptor CCR6 [120]. In contrast, iTregs are stable in the inflammatory milieu because they are resistant to conversion into Th1, Th2, and Th17 cells [120]. These observations suggest that nTregs and even iTregs may play distinct roles in controlling the adaptive immune response [120].

Tregs may also be subjected to transdifferentiation and become hybrid Treg-Th17 cells in response to temporal viral infections. These cells may acquire inflammatory phenotype by virtue of producing IL-17 and cause fetal brain development abnormalities. Their depletion or conversion to normal Tregs in mid-pregnancy may reduce the toxic effects caused by temporal viral infections (Jash and Sharma, unpublished results) [117].