Progestogens and immunology

Fifty percent of fetal antigens are of paternal origin. These are recognized by the maternal immune system, thereby resulting in lymphocyte activation and the induction of progesterone receptors (PRs) in immune cells. Upon binding of progesterone to PRs on lymphocytes, a downstream mediator called progesterone-induced blocking factor (PIBF) is produced. The full-length PIBF is a 90 kDa protein; however, because of alternative splicing, several smaller isoforms are also produced. While the 90 kDa molecule plays a role in cell cycle regulation, the small isoforms are localized in the cytoplasm, and after secretion, they bind to their receptors on other cells and act in a cytokine-like manner. The communication between the embryo and the maternal immune system is established through PIBF-containing extracellular vesicles. PIBF induces an increased production of Th2 cytokines and inhibits degranulation of NK cells, and by regulating the maternal immune response, it contributes to successful implantation and maintenance of pregnancy.     

Activating T regulatory cells for tolerance in early pregnancy — the contribution of seminal fluid

A state of active tolerance mediated by T regulatory (Treg) cells must be functional from the time of embryo implantation to prevent the conceptus from maternal immune attack. Male seminal fluid and ovarian steroid hormones are implicated in regulating the size and suppressive function of the Treg cell pool during the peri-implantation phase of early pregnancy. Evidence that antigens and cytokine signals in seminal fluid regulate the maternal immune response includes the following: (1) the Treg cell-inducing cytokine TGFβ and male alloantigens are present in seminal fluid; (2) seminal fluid delivery at coitus is sufficient to induce a state of active immune tolerance to paternal alloantigen, even in the absence of conceptus tissue; (3) female dendritic cells can cross-present seminal fluid antigens to activate both CD8⁺ and CD4⁺ T cells, and (4) mating events deficient in either sperm or seminal plasma result in diminished CD4⁺ CD25⁺ Foxp3⁺ Treg cell populations at the time of embryo implantation. Ongoing studies indicate that the cytokine environment during priming to male seminal fluid antigens influences the phenotype of responding T cells, and impacts fetal survival in later gestation. Collectively, these observations implicate factors in the peri-conceptual environment of both male and female origin as important determinants of maternal immune tolerance. Defining the mechanisms controlling tolerance induction will be helpful for developing new therapies for immune-mediated pathologies of pregnancy such as miscarriage and pre-eclampsia.     

Fetal alloantigen is responsible for the expansion of the CD4(+)CD25(+) regulatory T cell pool during pregnancy

Increasing evidence suggests that CD4(+)CD25(+) regulatory T cells (Tregs) participate in the development of maternal tolerance to the fetus during pregnancy; however, the factors controlling the activities of Tregs are poorly understood. In the present study, CD4(+)CD25(+) Tregs were analyzed in syngeneically pregnant mice (BALB/cxBALB/c), allogeneically pregnant mice (BALB/cxC57), ovariectomized mice and pregnant women to investigate the influences of fetal alloantigens and pregnancy-related hormones on the activities of Tregs. It was demonstrated that the frequencies of CD4(+)CD25(+) Tregs increase more in allogeneically than in syngeneically pregnant mice, which contributes to a lowered alloreactivity against paternal antigens in allogeneically compared with syngeneically pregnant mice. The increased Tregs are most likely to be induced in peripheral lymphoid tissues, rather than develop in thymus. Allogeneically mated mice and humans share similar dynamic changes in Treg frequencies, markedly increasing during early pregnancy and progressively decreasing from mid-gestation onwards to return to non-pregnant levels at term. Induction of labor in humans appears to be associated with a decrease of CD4(+)CD25(high) Tregs and increase of CD4(+)CD25(low) T cells. Neither estrogen or progesterone alone, nor their combination, shows an impact on the frequencies of Tregs in ovariectomized mice. These results suggest that fetal alloantigen is responsible for the increase of Tregs during pregnancy, and the expansion of the Treg population is of importance for the allogeneic fetus to evade immune attack from the mother.     

CD4(+)CD25+ T regulatory cells in murine pregnancy

Mammalian pregnancy is thought to be a state of immunological tolerance and immunological pregnancy complications may result from incomplete allo-tolerance. We reported recently a higher frequency of Th1 cytokine-producing T cells specific against paternal antigens in abortion-prone mice compared to normal pregnant mice. Since Th2 cells were shown to be not essential for normal pregnancy; alloreactive Th1 cells must be differently regulated. In this context, T regulatory cells (Treg) were proposed to play an essential role. Normal pregnant mice show an expansion of CD4(+)CD25+ and IL-10+ Treg cells at the periphery compared to non-pregnant animals. Further, we reported significantly lower frequencies of Treg in abortion-prone mice. Interestingly, CD4(+)CD25+ Treg cells from normal pregnant mice were able to prevent fetal rejection. Accordingly, down-regulated levels of Treg were also reported during human miscarriage. The putative mechanisms involved in Treg-induced tolerance in mice and humans are discussed in this review.     

Maternal PD-1 regulates accumulation of fetal antigen-specific CD8 T cells in pregnancy

The failure to reject the semi-allogeneic fetus suggests that maternal T lymphocytes are regulated by potent mechanisms in pregnancy. The T cell immunoinhibitory receptor, Programmed Death-1 (PD-1), and its ligand, B7-H1, maintain peripheral tolerance by inhibiting activation of self-reactive lymphocytes. Here, we investigated the role of the PD-1/B7-H1 pathway in maternal tolerance of the fetus. Antigen-specific maternal T cells both proliferate and upregulate PD-1 in vivo at mid-gestation in response to paternally inherited fetal antigen. In addition, when these cells carry a null deletion of PD-1, they accumulate excessively in the uterus-draining lymph nodes (P < 0.001) without a concomitant increase in proliferation. In vitro assays showed that apoptosis of antigen-specific CD8⁺ PD-1^−/− cells was reduced following peptide stimulation, suggesting that the accumulation of these cells in maternal lymph nodes is due to decreased cell death. However, the absence of neither maternal PD-1 nor B7-H1 had detectable effects on gestation length, litter size, or pup weight at birth in either syngeneic or allogeneic pregnancies. These results suggest that PD-1 plays a previously unrecognized role in maternal–fetal tolerance by inducing apoptosis of paternal antigen-specific T cells during pregnancy, thereby controlling their abundance.     

Fetus specific T cell modulation during fertilization, implantation and pregnancy

Recently there is an increasing interest in aspects of a more specific immunoregulation during pregnancy. Understanding these mechanism might have a broader application not only for reproductive immunology but also in general for biology and medicine. Especially the induction, already before conception, of feto-specific T cells with a possibly regulatory function gives a biological explanation of local immunotolerance at the maternal fetal interface, supporting the epidemiological evidence of a feto/paternal-specific immuneregulation. Understanding the expression of specific HLA-classes on trophoblast and the crosstalk of these antigens with various cell types, specifically modulated in the decidua, resulting in the secretion of cytokines and (angiogenic) chemokines has given us a more and more detailed understanding of this regulation. This regulation could be induced by fetal cells circulating in the mother (microchimerism) and from the interaction with fetal subcellular fractions as exosomes, but also from paternal antigens present in seminal fluid. Molecular interaction between paternal and fetal antigens and receptors in endometrium and the decidua are discussed. This review highlights besides uNK cells, especially the function of CD4+ and CD8+ T cells with a regulatory function in the context of recurrent miscarriage and pre-eclampsia. Besides HLA, also male-specific minor histocompatibility antigens and the genetic background for these pregnancy complications are discussed.     

Co-ordinate expression of Th1/Th2 phenotypes in maternal and fetal blood: evidence for a transplacental nexus

If maternal atopy and environmental exposure affect prenatal Th cell development, the maternal and fetal immune systems should display common Th1/Th2 phenotypes. To test this hypothesis, we studied maternal and neonatal blood samples from mothers with total serum IgE <300 IU/mL. Basal levels of IFN-γ, IL-4, and eotaxin in paired maternal and fetal sera were tightly correlated. Polyclonal T cell activation in vitro by Staphylococcal exotoxin B induced co-ordinate IFN-γ production from paired maternal and fetal mononuclear cells, accompanied by co-ordinate increases in activated CD4+CD69+ cells that display the CCR4+Th2 and CXCR3+ Th1 phenotypes. Maternal and fetal CD4+CXCR3+ T cells were subsequently identified as the major producers of IFN-γ. The data established that a transplacental nexus exists during normal pregnancy and that fetal Th cell responses may be biased by the maternal immune system.     

The alloantibody response of pregnant women and its suppression by soluble HLA antigens and anti-idiotypic antibodies.

The aim of this study was to investigate the time course of maternal allosensitization to fetal HLA antigens during normal human pregnancy and to explore mechanisms of suppression of anti-HLA alloantibodies. We found that the mother produces antibodies against some but not all of the mismatched HLA antigens of the fetus as early as the 8th week of pregnancy. These antibodies (Ab1), however, are often complexed with soluble HLA alloantigens and become detectable when immune complexes are dissociated. Soluble HLA antigens of fetal origin are present in the maternal circulation throughout the entire pregnancy beginning at 8 weeks. In some women the production of anti-anti-HLA antibodies (Ab2) became evident as early as the first trimester, while in others Ab2 was documented during the second or third trimester. Analysis of antibody specificity showed that some healthy primipara develop antibodies reactive with self HLA antigens. Although the allo- and autoantibody responses appear to be modulated by soluble HLA antigens, cyclic variations in the level of alloantibodies, as well as the mother's selective response to some, but not all, paternal HLA antigens, are best explained by the development of anti-idiotypic antibodies.     

Immunoregulatory effects of human cord blood T lymphocytes on mixed lymphocyte reaction

Fetuses of outbred mammalian species can survive despite disparities of participants' histocompatibility antigens. During pregnancy, maternal immune response is supposed to be suppressed by various immune suppressive mechanisms. In order to know whether fetal lymphocytes can suppress the maternal immune reaction, we investigated the suppressive effect of human newborns' T lymphocytes. Newborn T cells were isolated from the umbilical cord blood by means of the sheep red blood cell rosette forming method and treated with mitomycin C. The newborn T cells were added as regulator cells to one way mixed lymphocyte reaction (MLR) using maternal lymphocytes, paternal lymphocytes and unrelated donor's lymphocytes. In a control study, adult T cells were treated in the same manner. In any combination of the MLR, the response was substantially suppressed by the addition of cord T cells in comparison with the response by the addition of normal adult T cells. The suppression was dose-related with an increasing number of regulator cord T cells. The suppressive activity of cord T cells showed radiation resistance until 1,000 rads irradiation. In the kinetics study, cord T cells were found to suppress the recognition phase of MLR. These results suggest that cord T lymphocytes suppress the mother's immunological reaction to the fetal antigens.     

Review: Fetal antigens--identity, origins, and influences on the maternal immune system

Pregnancy induces priming of the maternal cellular and humoral immune systems. The paternally-inherited fetal antigens that influence maternal T and B cells include both major and minor histocompatibility antigens - the same antigens that are problematic in allotransplantation. Animal models have facilitated our understanding of the lymphocyte responses to fetal antigens, and our appreciation of the parallel response in pregnant women is increasing. The physiologic properties of the placenta as well as trafficking of cells between mother and fetus allow ample opportunity for sampling of fetal proteins by the maternal immune system. Here, the current state of knowledge of fetal antigen-specific lymphocyte responses in pregnancy is reviewed.     

NK cells and pre-eclampsia

The immunological interaction between the mother and fetus has classically been thought of as one between paternal antigen and maternal T cells. However, the MHC antigen expression on human trophoblast and the immune cell populations present in the decidua suggest that this interaction primarily involves decidual NK cells rather than T cells, and this is supported by new functional studies. It is becoming apparent also that the maternal systemic immune response in pregnancy (Th1/Th2 shift) primarily involves NK cells. Aberrant NK cell activation both locally in the decidua and systemically in the maternal blood may be the cause of pre-eclampsia.     

Maternal cell-mediated sensitisation to paternal HLA may occur, but is not a regular event in normal human pregnancy

Maternal cell-mediated sensitisation to paternally-inherited fetal HLA was studied in 10 primigravid and 9 multigravid women. One woman in each group was found to have circulating cytotoxic effector cells specific for paternal lymphocytes, but both had a normal pregnancy outcome. These results show that sensitisation is not a regular event in pregnancy and that there must therefore be a block in the afferent (recognition) phase of the mother's immune response. Where sensitisation does occur there must also be a block on the efferent (effector) arm of the response to prevent rejection of the fetus. The restricted distribution of major histocompatibility complex antigens on trophoblast and the function of the placenta as a barrier between maternal and fetal immune compartments can explain the occurrence of both types of blockade.     

Over-expression of heme oxygenase-1 by adenoviral gene transfer improves pregnancy outcome in a murine model of abortion

Mammalian pregnancy is a complex phenomenon allowing the maternal immune system to support its allogeneic fetus. Physiological pathways protecting the fetus from rejection are thought to be comparable with those leading to allograft acceptance. Heme oxygenase (HO)-1 is known to protect locally against rejection in transplantation models due to its anti-oxidant, anti-inflammatory and cytoprotective functions. Based on previous data on low HO-1 levels in placenta from mice undergoing abortion, we hypothesized that an up-regulation of HO-1 during pregnancy would avoid fetal rejection in the murine abortion combination CBA/J x DBA/2J, using BALB/c-mated CBA/J as normal controls. We injected pregnant mice undergoing abortion with 1 x 10(5) PFU of an adenoviral vector containing HO-1 and GFP (AdHO-1/GFP), and compared the pregnancy outcome with PBS- or 1 x 10(5) AdEGFP-treated abortion-prone mice and with PBS-treated normal pregnant mice. The abortion rate diminished significantly after adenoviral gene transfer of AdHO-1/GFP. The systemic and local IL-4/IFN-gamma ratio was augmented in AdHO-1-treated mice compared to abortion-prone mice. Interestingly, the HO-1 treatment up-regulated the ratio IL-10/TNF-alpha in spleen but not in decidual lymphocytes. HO-1-treated mice further showed diminished apoptosis rate and increased Bag-1 mRNA levels at the materno-fetal interface. Thus, we propose HO-1 as a key regulator of pregnancy success. HO-1 would exert its action by locally up-regulating the Th2/Th1 cytokine ratio and by further protecting tissues from apoptosis.     

Adoptive transfer of CD4+CD25+ regulatory T cells for prevention and treatment of spontaneous abortion

Objectives

The purpose of this study was to examine whether adoptive transfer with in vitro expanded CD4⁺CD25⁺ regulatory T cells (Tregs) could prevent immune response-mediated spontaneous abortion in mice.

Study design

Female CBA/J mice were mated with male Balb/c as the control with normal pregnancy or with DBA/2J mice as a model of spontaneous abortion. The CBA/J mice mated with DBA/2J were treated intravenously with freshly isolated or in vitro expanded Tregs on day 1 or 4 of pregnancy, respectively. The numbers of surviving and reabsorbed fetuses in the different groups of mice were counted on day 14 of pregnancy, and the concentrations of cytokines in individual sera and the supernatants of cultured Tregs were measured by ELISA.

Results

Adoptive transfer with freshly isolated Tregs only slightly reduced the fetal resorption rate, which was not significantly different from that of the mice without Treg treatment, regardless of treatment at early stage and implementation of pregnancy. In contrast, adoptive transfer with in vitro expanded Tregs significantly reduced the fetal resorption rates, particularly for treatment at early stage of pregnancy (P < 0.05). Furthermore, adoptive transfer with in vitro expanded Tregs at early stage of pregnancy significantly increased the levels of serum IL-10, TGF-β1, and the ratios of IL-10 to IFN-γ.

Conclusions

Our data clearly indicated that adoptive transfer with in vitro expanded Tregs at early stage of pregnancy protected fetuses from spontaneous abortion by re-establishing immune tolerance in mice.     

Failure of in vitro-expanded hyperimmune cytotoxic T lymphocytes to affect survival of mouse embryos in vivo

This study was designed to address the question; does expression of paternal histocompatibility antigens by fetal cells make them susceptible to immune attack in vivo during normal pregnancy? The experimental design was based on the rationale that, if alloantigens are presented by trophoblasts or other fetal cells in a manner which allows accessibility, in vitro-generated immune effector cells of combined helper/cytotoxic phenotype should produce fetal rejection of abortion. Similarly generated effector cells are capable of accelerating skin graft rejection and, when combined with IL-2 in vivo, are capable of causing regression of antigenic, but operationally non-immunogenic, tumors. The alloimmune effector cells generated in vitro during the current study were highly cytotoxic against normal adult target cells, whereas placental cells were completely resistant to cytolysis and fetal cells were only slightly susceptible. Adoptive transfer of effector cells to mice at different stages of gestation had no apparent effect on pregnancy outcome. In vivo administration of IL-2 and/or indomethacin, which expand effector cell numbers in vivo and block PGE2-mediated immune suppression, respectively, failed to potentiate the cellular effect. The data provide additional evidence that paternal histocompatibility antigens are not expressed in a format which allows susceptibility to immune attack during pregnancy. The data are discussed with respect to the role of the trophoblast in protecting developing embryos.     

Valve disease in pregnancy

Maternal cardiac disease is a major cause of non-obstetric morbidity and accounts for 10–25% of maternal mortality. Valvular heart disease may result from congenital abnormalities or acquired lesions, some of which may involve more than one valve. Maternal and fetal risks in pregnant patients with valve disease vary according to the type and severity of the valve lesion along with resulting abnormalities of functional capacity, left ventricular function, and pulmonary artery pressure. Certain high-risk conditions are considered contraindications to pregnancy, while others may be successfully managed with observation, medications, and, in refractory cases, surgical intervention. Communication between the patient?s obstetrician, maternal–fetal medicine specialist, obstetrical anesthesiologist, and cardiologist is critical in managing a pregnancy with underlying maternal cardiac disease. The management of the various types of valve diseases in pregnancy will be reviewed here, along with a discussion of related complications including mechanical prosthetic valves and infective endocarditis.     

Angiogenesis in implantation

Problem Implantation failure and early pregnancy loss are common following natural conceptions and they are particularly important clinical hurdles to overcome following assisted reproduction attempts. The importance of adequate vascular development and maintenance during implantation has recently become a major focus of investigation. Materials and methods Review of current published literature was undertaken to summerize the cells and cell products that regulate tissue vascularity during implantation. Results Vascular development at the maternal fetal interface can be regulated by a number of different cell types; two principal candidates are trophoblast and natural killer cells. A wide range of soluble factors, some with well established angiogenic functions as well as other more novel factors, can contribute to vascular development and maintenance at the maternal–fetal interface. Conclusions Robust vascular development occurs during implantation and early placentation of normal pregnancies. Studies to define the extent and mechanisms by which defects in vascularity contribute to human implantation failure and early miscarriage need to be undertaken. Vascular development during implantation is mediated by numerous cell types and cell products and aberrant vascularity likely contributes to implantation failure and early pregnancy loss.     

Impact of paternal age on IVF and pregnancy outcomes with only normal sperm parameters

ObjectivePrevious reports on advanced paternal age effects on assisted reproductive technology (ART) vary considerably and those on frozen–thawed embryo transfer (FET) are rare. We investigated whether paternal age affects in vitro fertilisation (IVF) and FET pregnancy outcomes.Materials and methods1657 IVF cycles performed from January 2014 to May 2018 were retrospectively investigated excluding cases of poor semen parameters. Paternal and maternal ages were categorised into groups, namely, <35, 35–39 and ≥ 40 years, to compare normal fertilisation (2 PN (pronuclei)) and high-quality blastocyst rates. Furthermore, 741 FET cycles were investigated and pregnancy, live birth and miscarriage rates were compared.ResultsFor the maternal age group (35–39), the 2 PN rate was significantly higher with paternal age group of <35 than groups of 35–39 and ≥ 40 (median%, <35 vs. 35–39 vs. ≥40 = 100.0 vs. 71.4 vs. 77.7; P = 0.005). The miscarriage rate was significantly higher with paternal age group of ≥40 than that of <35 and 35–39 when maternal age was <35 (median %, <35 vs. 35–39 vs. ≥40 = 13.1 vs. 7.8 vs. 33.3; P = 0.038).ConclusionOur findings show that when maternal age was <35, advanced paternal age reduces the normal fertilisation rate and increases the FET miscarriage rate when maternal age was 35–39.     

Immunomodulation in Recurrent Miscarriage

There are many etiological factors responsible for recurrent abortions. However, no explanation can be identified in approximately 40–50 % of women with recurrent miscarriage (RM). Several studies demonstrated that successful pregnancy is dependant on shifting of maternal immune response from (proinflammatory) Th1 toward (anti-inflammatory) Th2 phenotypes. It was suggested that unexplained RM might be due to immunologic factors. Recently, there is improved understanding regarding the role of the different immune cells and proteins that are important at each stage of a normal pregnancy. Various immune-based therapies with variable clinical evidences have been reported in women with RM with variable efficacy. Still there is lack of information about the mode of action and possible adverse effects of the treatment and a reliable marker for patient selection for immunopotentiation. Adequately powered placebo-controlled studies are required to study and treat couples with the so-called idiopathic recurrent miscarriage.     

Inadequate tolerance induction may induce pre-eclampsia

The fetus is semi-allograft to the maternal host; therefore, a system of tolerance must be present during pregnancy. Epidemiological findings support a relationship between pre-eclampsia and the failure of tolerance induction. For induction of major histocompatibility complex (MHC) class I-specific tolerance, long-term exposure to seminal fluid, which contains paternal soluble MHC class I antigens, may induce paternal MHC class I-specific tolerance. Furthermore, soluble HLA-G1, which induces the deletion of CD8(+) T-cells, and the combination of maternal killer-immunoglobulin-like receptors (KIR) on NK cells and fetal HLA-C, which affects the balance between inhibition and activation signals of NK cells, regulatory CD8(+) T cells, and regulatory NK cells, may play very important roles in the induction of MHC class I-specific tolerance. On the other hand, exposure to sperm, which express paternal HLA-DR, and trophoblastic debris, which contain intracellular fetal HLA-DR, may induce paternal MHC class II-specific tolerance. In this process, CD4(+)CD25(+) regulatory T (Treg)-cells play central roles. In this review, we discuss the relationship between the risk of pre-eclampsia and tolerance induction.