Monocytes and macrophages are major representatives of the innate immune system in the cycling and pregnant mammalian uterus. Several studies provide evidence that monocyte recruitment, differentiation into macrophages, and function in the reproductive tract is modulated by pregnancy-associated hormones (33). Hormonal influence may be achieved by directly binding to the appropriate hormone receptors expressed on human and murine macrophages (16, 34, 35) or indirectly by modulating the levels of cytokines and growth factors that target the resident macrophages and influence their secretory profile. Hunt and colleagues reported that P4 reduced macrophage migration into the murine uterus (36), while Kitzmiller and colleagues showed that E2, P4, and hCG did not affect macrophage migration in guinea pigs (37). Differentiation of monocytes into macrophages was hindered by glycodelin A, a P4 mediator, by induction of apoptosis in human monocytes. However, after differentiation glycodelin A was not able to alter phagocytic capacity of macrophages (11). Macrophages are important regulators of trophoblast activity that promote tissue remodeling and angiogenesis (38). In this regard, E2, hCG, and LH have been demonstrated to enhance the production of the vascular endothelial growth factor (VEGF) in human macrophages (39, 40), supporting vessel formation in the placenta. In addition, P4 impairs the ability of human and murine macrophages to produce potent effector molecules such as nitric oxide and IL-1 proven to be detrimental for successful pregnancy outcome (36, 41, 42). Moreover, P4 suppresses toll-like receptor-triggered activation of murine macrophages by regulating miR-155 expression (43). Menzies and colleagues recently suggested an involvement of P4 in the regulation of genes associated with alternative macrophage activation (44). By contrast, hCG treatment of human and murine IFN-γ-primed macrophages resulted in increased production of nitric oxide, reactive oxygen species, IL-6 and IL-12p40, and enhanced phagocytosis of apoptotic cells (45, 46). However, hCG treatment of murine IFN-γ-primed macrophages did not affect the induction of allogeneic T cell proliferation (45). Interestingly, macrophages regulate excess of hCG known to be teratogenic to fetal tissues. Here, human macrophages are proposed to incorporate and degrade hCG in a time-dependent manner that protect fetal gonadogenesis from excess hCG (47, 48). More precisely, Katabuchi and colleagues recently demonstrated that hCG induces transient vacuole formation in human monocytes, morphologically mimicking Hofbauer cells. The authors suggest that Hofbauer cells and especially their vacuoles are involved in the protection of fetal tissue from high amounts of maternal hCG (49). Besides an effect of steroid hormones and gonadotropins on monocytes and macrophages, AFP is suggested to have an influence on both innate immune cell types. It has been demonstrated that AFP significantly suppresses the production of TNFα and IL-1β and induces a rapid down-regulation of surface MHC class II expression in a stimulated human monocyte cell line (29, 31). Moreover, Lu and colleagues showed that AFP inhibits the cell surface expression of Ia antigens on macrophages but does not affect macrophage viability (50). Hormonal effects on macrophages are summarized in Figure 1.

NK cells and, in particular, uterine NK (uNK) cells are of special interest when analyzing mechanisms underlying normal pregnancy. This becomes obvious when taking into account that uNK cells are the predominant lymphocyte population in the late secretory phase of the menstrual cycle and in the early pregnant uterus representing circa 70% of all leukocytes in decidual tissue. uNK cells differ from peripheral NK cells in the expression of their receptor repertoire and in the expression of some genes induced by the hormonal environment. The main function of uNK cells is to regulate maternal uterine vasculature remodeling (51). Therefore, it has been demonstrated in the murine system that uNK cells produce proangiogenic factors such as VEGF and growth factors and provide local IFN-γ for initiation of spiral artery formation (52–54). Their origin or expansion remains a matter of discussion. They may migrate from the periphery, differentiating from NK cell progenitors under the control of different factors, including steroid hormones (55), and/or by recruitment of peripheral NK cells into the uterus (56–58) or expand in situ after pregnancy was established (59). Qu and colleagues demonstrated a P4-dependent osteopontin expression in human decidual stroma cells and human uNK cells and proposed a role for osteopontin in uNK cell accumulation in uterine tissue (60). Moreover, human uNK cell recruitment from the peripheral blood into the uterus seems to be favored by rising E2 and LH levels and restricted by increasing amounts of P4 (61). Interestingly, mature human and rodent uNK cells do not express steroid receptors (55, 62–64). Thus, it is suggested that, at least for P4, effects are mediated through the glucocorticoid receptor (GR), proven to be expressed on murine uNK cells (65). In addition to the lack of steroid receptors, uNKs also miss the classical LH/CG receptor. Thus, hCG was suggested to induce human uNK cell proliferation through the mannose receptor (66). Regarding the function of uNK cells, they have been shown to contain high amounts of perforin but only display low cytotoxic activity. Several studies indicated that P4 and its mediator PIBF inhibit human NK cell activity via a block of degranulation (67–70). In agreement, E2 increased human and murine NK cell number but reduced their cytotoxicity (71, 72). By contrast Kitaya and colleagues as well as Kurashige and colleagues revealed that neither P4 nor E2 had significant effects on the proliferation, cytolytic activity, and cytokine secretion of human endometrial NK cells (73, 74). This was also true for AFP (73). In contrast to these results, it was shown that hCG and LH applied to virgin mice resulted in an enhanced NK cell activity (75, 76) suggesting that pregnancy hormones differently regulate NK cell function. Moreover, contradictory results between humans and mice regarding an influence of pregnancy hormones on different immune cell populations depict the limitations of animal models in understanding mechanisms unique to human pregnancy. Finally, hCG not only influences NK cells but is also produced and secreted by them during pregnancy (77). Hormonal effects on NK cells are summarized in Figures 1 and 2.

Mast cells (MCs) are best known for their effector function in allergic diseases. After binding of allergen-specific IgE to its receptor (FcεRI), preformed and newly synthesized mediators stored within the MCs are released to induce inflammatory immune responses (78). Beside this well-documented function of MCs, recent data suggest MCs as critical regulators of adaptive immune responses (79). Additionally, we recently uncovered a critical role for MCs in pregnancy success. To study the importance of MCs in murine pregnancy, we took advantage of a MC-deficient mouse model. We found uterine MCs (uMCs) to have a unique phenotype. uMCs increase in number every time a female becomes receptive and rapidly expand after pregnancy occurs. In the absence of MCs implantation is severely impaired and spiral artery remodeling has shown to be insufficient. This is suggested to result in fetuses that are growth-retarded (80). We also proposed a role for uMCs in trophoblast survival, placentation, and fetal growth. MC salutary role in murine pregnancy is mediated at least in part by Galectin-1 (80). Oscillations in the number of uMCs during the reproductive cycle in humans (81) and mice (82) seem to be, at least partially, hormone regulated. Several studies demonstrated that P4 and E2 influence rat and mouse MC density in different tissues, including mammary glands and uterine tissue (82–85). We additionally suggested a function for both hormones in the recruitment of murine MCs from the periphery into the uterus as well as an impact on MC activity (86). In agreement, several studies confirmed a major effect of P4 and E2 on rat, mouse, and human MC activation (83, 87–90). By contrast, two other studies did not observe alterations in the number of granulated or degranulated rat and mouse MCs after P4 or E2 treatment (91, 92). Hunt and colleagues revealed that E2 influences the expression of iNOS and TNF-α in murine uMC (93). It can be assumed that the observed effects of P4 and E2 on MCs are in part of direct nature as MCs from different species have been proven to express P4 and E2 receptors (85–87, 89, 94). Studies analyzing the impact of hCG on MCs are rare to find in the literature. One study investigated the histamine content in MCs in a model of ovarian hyperstimulation induced in rabbits. However, hCG application seemed not to change the histamine content in MCs (95). Hormonal effects on MCs are summarized in Figures 1 and 2.

As professional antigen-presenting cells, DCs are at the interphase between the innate and the adaptive immune system; hence their activation and modulation is critical for the outcome of the immune response. Dependent on their activation status, DCs either secrete pro- or anti-inflammatory cytokines, thereby inducing immune responses or suppressing them, respectively. During normal pregnancy, the majority of human and murine decidual DCs presents an immature (tolerogenic) phenotype and mainly produce IL-10, thus contributing to a fetus-friendly local environment (96, 97). In line with this, spontaneous abortions in humans and mice are associated with an increased number of mature, IL-12-producing DCs (98, 99). Moreover, the importance of DCs for a proper decidualization and implantation has been nicely shown by Plaks and colleagues in a mouse model (100). Data regarding the influence of pregnancy-associated hormones on DC function are widespread and inconsistent. DCs derived from bone-marrow precursors or monocytes as well as DCs from spleen or decidua have been shown to react differentially to hormonal stimulation. This may offer a possible explanation how the endocrine system supports the pregnant immune system in tolerating the semi-allogeneic fetus while at the same time being aware of pathogens; these cells being pleiotropic and it should not come as a surprise that they have the machinery to respond differently depending on the situation. DCs are highly susceptible to hormonal stimulation by expressing receptors for P4, E2, hCG, and LH (101–103). Hormonal stimulation of activated bone-marrow derived DCs (BMDCs) resulted in the majority of studies in an impaired up-regulation of MHCII molecules and co-stimulatory molecules associated with a reduced capability to secrete pro-inflammatory cytokines (101, 104). This would imply that upon hormonal stimulation they acquire a rather pregnancy-friendly phenotype. The concrete impact of P4 on IL-10 secretion by activated rat and mouse BMDCs was differentially discussed and has to be further evaluated (101, 104, 105). In line with the hormonal-mediated induction of a tolerogenic phenotype in activated BMDCs, their T-cell stimulatory capacity was reduced. This suggests that hormone-treated DCs have a pregnancy-protective effect by suppressing alloreactive T cell responses (101, 104, 105). However, results obtained after the addition of P4, E2, and hCG to monocyte-derived DCs (moDCs) from human peripheral blood are different from the results obtained after hormonal stimulation of BMDCs. Here, two studies showed that combinations of P4, E2, and hCG did not change the expression of maturation markers of moDCs or their T cell stimulatory capacity (106, 107). Segerer and colleagues demonstrated, at least for hCG, a pregnancy-positive effect on HLA-DR expression associated with a significant reduction in the ability to stimulate T cells (108). Of great interest is the fact that most of the published studies observed a significant up-regulation of IL-10 production by human DCs after treatment with pregnancy hormones (106, 107, 109). Uemura and colleagues additionally proposed an induction of T cell differentiation into Th2 (107), a phenotype that is reportedly pregnancy-friendly. Analysis of hormonal effects on splenic DCs has been performed in several mouse models, including models of autoimmune diseases and pregnancy. In a murine model for multiple sclerosis, namely experimental autoimmune encephalomyelitis (EAE), E2 treatment has shown to be protective. Here, E2 did not affect the expression of activation markers and co-stimulatory molecules of DCs but inhibit their ability to stimulate T cell proliferation and secretion of Th1 and Th2 cytokines. The reduced T cell stimulatory capacity was suggested to be due to an increased expression of indoleamine 2,3-dioxygenase (IDO) in DCs after E2 treatment (110, 111). Accordingly, hCG was also proven to up-regulate IDO in DCs in a murine model of autoimmune diabetes (112). As for E2, it was reported that P4 seems to affect the ratio of Th1-promoting DEC-205⁺ DCs and Th2-promoting 33D1⁺ DCs during pregnancy, favoring the dominance of 33D1⁺ DCs. The need of 33D1⁺ DCs for pregnancy success results from the observation that depletion of this specific DC subset during the perinatal period in mice caused substantial fetal loss probably mediated through Th1 up-regulation via transient IL-12 secretion (113). We have recently investigated the influence of hCG and LH on the number and phenotype of peripheral and local DCs in a murine model of disturbed tolerance to the semi-allogeneic fetus. We found that the in vivo application of both hormones prevented fetal rejection and this was associated with a reduced number of total and mature DCs both in the periphery and in decidua. Furthermore, we proved that hCG-treated decidual DCs had an elevated capacity to induce regulatory T (Treg) cells (103). This confirms the pregnancy-protective impact of both gonadotropins via modulation of adaptive immune responses. Effects of AFP on different DC subsets during pregnancy are almost unknown. Evidence for an AFP-mediated induction of tolerogenic DCs came from a tumor model where AFP has been demonstrated to induce tolerogenic DC capable of suppressing tumor-specific CD8⁺ cytotoxic T lymphocytes within the tumor (114). Hormonal effects on DCs are summarized in Figures 1 and 2.

B lymphocytes are allocated to the adaptive immune system and they are best known for their capability to secrete antibodies. However, B cells do more than producing antibodies. They efficiently present antigens and modulate the function of T cells and DCs by producing cytokines (115, 116). During pregnancy, various B cell subsets are proposed to differently affect pregnancy outcome [reviewed in Ref. (117)]. Basically, B cells can be divided in two main populations, namely B1 and B2 B cells. These two populations differ in their developmental origin, surface marker expression and function (118). B1 B cells are then further subdivided in B1a and B1b B cells based on the expression of the surface marker CD5 (119). Interestingly, B1a B cells produce a specific type of antibody, the so-called natural antibodies (120). Due to their poly-reactive nature, natural antibodies are suggested to induce autoreactivity and thus are involved in the onset of autoimmune diseases (121). Even during pregnancy, B1a B cells producing natural antibodies are proposed to have detrimental effects on pregnancy outcome. We recently showed that the number of B1a B cells significantly decrease in the third trimester of normal pregnant women while remain elevated in pre-eclamptic patients (122). As patients suffering from pre-eclampsia have augmented serum hCG levels compared to normal pregnant women, we assumed that hCG may be responsible for the increased B1a B cell number. This assumption is further underlined by the fact that almost all B1a B cells express the LH/CG receptor and expand in vitro upon hCG stimulation (122). It was however shown that hCG inhibited antibody formation of murine B cells (123, 124). However, these two mentioned studies did not focus their analysis on B1a B cells but in total B cells. It is tempting to speculate that hCG may influence cell phenotype and antibody production differently in different B cell subsets. In contrast to the detrimental effect proposed for natural antibodies on pregnancy outcome, asymmetric antibodies (AABs), due to their structural anomaly, favor pregnancy success by reducing alloreactive immune responses (125). AABs increase during pregnancy and their lack is associated with pregnancy failure in humans (126, 127). Secretion of AABs seems to be, at least partially, hormone regulated. P4 but not E2 provoke AAB secretion (128). Hereby, P4 mediates its function via induction of PIBF (129). Beside an influence on antibody formation and secretion, pregnancy hormones also regulate B cell development and cytokine secretion. Administration of E2 alone, or in combination with P4, preferentially suppressed IL-7 responding cells and their progeny in bone-marrow (130). In contrast to P4 and E2, human B cell stimulation by hCG seems to be highly dependent on hCG doses and purity of hCG preparations (131–133). Recently, we proposed another pregnancy-protective effect of hCG, namely its capacity of increasing the regulatory function of human regulatory B cells (Breg or B10) shown to contribute to fetal survival. Breg express the LH/CG receptor and increase their IL-10 production in response to hCG treatment (134). Hormonal effects on B cells are summarized in Figure 3.

T cells are key regulators of both the antibody and the cell-mediated arms of the adaptive immune system and can be divided into two subcategories, CD4 expressing T helper cells and cytotoxic T cells expressing CD8. According to their cytokine secretion pattern, they are usually further subdivided in Th1, Th2, and Th3 cells although it is well known that this classification is oversimplified as further subsets as Th9 and Th17 has been described. T cells mediate their function either by direct cell–cell contact or indirectly by the secretion of cytokines defining the local environment as a pro-inflammatory or anti-inflammatory one. Normal pregnancy is associated with a pro-inflammatory Th1 profile at early and late pregnancy stages being important for a proper blastocyst implantation and initiation of labor, respectively. At midgestation, an anti-inflammatory Th2 profile guarantees tolerance of the foreign fetal antigens. Imbalances in cytokine profiles were associated with human pregnancy complications (135), suggesting a major part for T cells in fetal tolerance by regulating the local cytokine milieu. Steroid hormones are reportedly involved in modulating cytokine secretion by different T cell subsets. P4 and E2 are proposed to influence the Th1/Th2 balance favoring a Th2 predominance at the fetal–maternal interface in humans and mice (136–144). Hormonal effects are mainly mediated via their classical receptors expressed by human and murine T cells (16, 103, 143, 145, 146). However, Chien and colleagues provide evidence that P4 may act through non-classical steroid receptors to cause immune modulation and suppression of T cell activation during pregnancy (147). In addition to regulate the cytokine secretion of T cells, steroid hormones and their mediators (e.g., glycodelin A) induce apoptosis of effector T cells (148–150) and increase the expression of pregnancy-protective molecules such as the leukemia inhibitory factor (LIF) known to modulate immune responses during early human and murine pregnancy (151, 152). In line with this, hCG is proposed to support fetal survival by modulating murine T cell activity and function (153, 154). More precisely, Khil and colleagues demonstrated that hCG prevented the development of the disease in a murine model of autoimmune diabetes. Here, hCG treatment efficiently suppressed IFN-γ production, but increased IL-10 and TGF-β production in splenocytes. hCG application also suppressed TNF-α production (155). In contrast, AFP apparently has no direct effect on T lymphocytes. T cells stimulated with AFP retain their full capacity to respond in mixed lymphocytes culture (MLC) and cell-mediated lympholysis. However, AFP provokes major changes in the functional status of monocyte-enriched, MLC-stimulating cell population. This monocyte-enriched population induces T suppressor cells while at the same time, suppresses generation of cytotoxic T cells (156–158).

Pregnancy-associated hormones not only affect conventional T cells but have also been shown to support the generation and function of Treg cells. Treg cells represent a specialized subset within the T cell compartment with unique properties. During pregnancy, they are fundamentally involved in the suppression of alloreactive immune responses and, thus their absence results in worse pregnancy outcome both in humans (159) and mice (160). The presence of Treg cells in the uterus before pregnancy seems to be very relevant as well. A diminished endometrial expression of their major transcription factor Foxp3 is associated with infertility in women (161) while depletion of Treg cells in a murine model derived in a hostile uterine microenvironment hindered the implantation of the embryo (162). During the reproductive cycle, Treg cells fluctuate (162–164) and this was suggested to be hormone-driven (165). This could be interpreted as hormones preparing the mother and particularly the tissue for pregnancy. Already in the 80s hCG was assumed to induce human and murine suppressor T lymphocytes (166–168). Nowadays, steroid hormones and gonadotropins are proposed to regulate several aspects of Treg cell biology, including generation, expansion, migration, and suppressive function both in humans and mice (103, 145, 169–176). However, some studies provide evidence that P4 and E2 induce a reduction of Treg cells or did not provoke changes in the number of Treg cells (177–179). Thus, these contradictory findings may depend on the markers used to define Treg cells, the time point of analysis and the model used. Hence, more research is needed to understand at which time point and through, which mechanisms pregnancy hormones influence Treg cell function. Hormonal effects on T cells are summarized in Figures 2 and 3.