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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

Understanding The Immunobiology Of Pregnancy And Applying It To Treatment Of Recurrent Pregnancy Loss

 Carolyn B. Coulam

The Center for Human Reproduction, Chicago, Illinois 60610 USA,
Tel: (312) 440-5180, Fax: (312) 440-5063, E-Mail: coulam@ais.net

Abstract 

Successful mammalian pregnancy depends upon tolerance of a genetically incompatible fetus by the maternal immune system. When tolerance is not achieved pregnancies fail. Repeated pregnancy failures are associated with high emotional and financial cost. To provide a better understanding of an approach to treatment for recurrent pregnancy loss this manuscript presents a review of current concepts of the immunobiology of successful pregnancies and the immunopathophysiology of unsuccessful pregnancies. Based upon information gleaned from these reviews an approach to diagnosis and treatment of recurrent pregnancy loss is presented.

Immunobiology of Pregnancy
The mechanisms by which the tolerance of the genetically incompatible fetus by the maternal immune system is established has been the focus of much research. It was originally proposed that the semiallogeneic conceptus was invulnerable to rejection because of lack of classical expression of classical MHC alloantigens on the trophoblastic surface that envelops the embryo to form the feto-maternal interface. However, resistance of trophoblastic cells to effectors of alloimmune rejection was evident even when paternal MNC Class I antigens were present and experimentally upregulated on mouse trophoblasts (1).

Antigen-bearing tumor cells may be afforded protection via expression of FAS ligand (FASL) (2). Expression of FASL by trophoblast could also inhibit potential effector cells (3). The relevance of FASL must be questioned, however, given that allogeneically mated MRL-lpr females which lack FAS have successful pregnancies even though FASL cannot delete MRL-lpr T cells capable of reacting with any MCH which may be expressed on trophoblast (G. Chaouat, unpublished data). Thus the relevance of anti-allograft mechanisms to survival of a being that is not an allograft remains moot. With respect to antibody-mediated rejection acting via complement activation, it is notable that a deficiency in complement inhibitor proteins has not been found in recurrent miscarriage material (4).

Furthermore, in sera of women with successful pregnancies anti-fetal, anti-placental and anti-paternal antibodies are detectable (5), clearly showing that maternal recognition of fetal antigens does not compromise pregnancy. Therefore, a conceptual model different from the fetal allograft was sought.

Tolerance is believed to depend in part on the interactions of an array of cytokines secreted by maternal and fetal cells at the site of implantation. In humans, the endometrium becomes receptive to implantation by developing into decidua under the influence of gestational hormones and stimulation of trophoblastic cells. Communication between trophoblastic and decidual cells is mediated by cytokines and cell surface receptors. Cytokines are produced by trophoblastic and lymphomyeloid cells. Lymphomyeloid cells present within the decidua include T lymphocytes (CD8+), macrophages and natural killer (NK) (CD56+) cells. As a result of intercellular communication, changes of expression of types and quantities of cytokines occur. These changes in expression include colony stimulating factors (CSF), tumor necrosis factor (TNF) alpha, interferons (INF), transforming growth factor (TGF) beta, and several interleukins (IL) (6-8). It is the balance of production of the various cytokines that dictates success or failure of a pregnancy. The interactions of cytokines stimulate placental growth and regulate invasion of trophoblastic cells within the maternal endometrium. Wegmann (9) proposed a model in which the balance of pro-inflammatory T helper (Th) 1 cytokines and anti-inflammatory Th2 cytokines is critical to normal, successful pregnancy. Local dominance of the Th2 cytokine pattern protects the immunologically foreign feto-placental unit against both adaptive cell mediated immune attack and the nonspecific innate inflammatory and phagocytic responses.

The pro-inflammatory cytokines INF gamma and TNF alpha have been shown to be embryotoxic (10). IL-1 beta and TNF alpha have been reported to regulate trophoblastic cell apoptosis (11), protease production (12), angioneogenesis (13) and biosynthesis by trophoblastic cytokines (14,15), prostaglandins (16) and hormones (17,18). Anti-inflammatory cytokines (IL-4, IL-10, TGF beta) have been proposed to play a role in preventing maternal rejection of the embryonic semi-allograft in early pregnancy (9, 19-28).

TGF beta has been shown to inhibit placental differentiation and invasion (22), extracellular matrix deposition (23), and hormone production (22,24) as well as to deactivate macrophages (25) and to inhibit monocyte cytokine production in many tissues (26). In hematopoietic cells, IL-4 inhibits prostaglandin and cytokine production by monocytes, regulates immunoglobulin G isotype switching in B cells, stimulates erythroid cell differentiation and B cell growth, and regulates the activation of monocyte and T cell cytotoxicity (27). A characteristic of these actions is the maintenance of humoral immunity and Th2 dominance, proposed hallmarks of normal human pregnancy (9). IL-10 also is believed to play a major role in directing Th0 cell differentiation toward a Th2 phenotype (28). It also has potent inhibitory effects on the production of prostaglandins (29) and cytokines (30) by macrophages and monocytes.

Thus, it is the balance of Th1 pro-inflammatory and Th2 anti-inflammatory cytokines that determines success and failure of pregnancy. An inflammatory response with production of pro-inflammatory cytokines is necessary for initial implantation with invasion of trophoblasts and induction of angioneogenesis. But shortly thereafter the potential detrimental effects of the inflammatory response must be counteracted by anti-inflammatory cytokines involving a Th1 to Th2 shift. The presence of TGF beta 2, a molecule involved in induction of tolerance to orally fed antigens, has been proposed as critical for the Th1 to Th2 shift (35,36). TGF beta 2 is a Th3 cytokine produced by CD56+ CD16- CD3- cells in the decidua. The balance between Th1 and Th2/3 cytokine production by these cells seems to be decided by CD8+ T cells and a low molecular weight factor (38). Szekeres-Bartho has shown that CD8+ T cells of pregnant women develop progesterone receptors very early in pregnancy (39). Progesterone receptor-positive lymphocytes in the presence of progesterone produce a 34kDa mediator protein – the progesterone-induced blocking factor (PIBF) (40). PIBF is responsible for the immunologic and antiabortive effects of progesterone. PIBF induces increased production of Th2 cytokines (31, 41). CD8+ cell activity seems to be present during the preimplantation and peri-implantation phase of pregnancy when Th1 cytokines are prevalent. After implantation, Th2/3 gamma delta T cells and CD56+ NK cells become active.

Mechanism of Recurrent Abortion
Recurrent spontaneous abortion is a healthcare concern occurring in approximately 7,500 couples each day worldwide. Effective treatment is needed for these couples. Before effective treatment can be instituted, the cause of pregnancy loss must be determined. While anatomic and hormonal factors have been previously reported to contribute to recurrent spontaneous abortion, genetic and immunologic factors have data supporting their roles in the mechanisms of pregnancy loss.

Chromosomal abnormalities are present in 60% of abortuses from women with a history of recurrent spontaneous abortion (42) Women with a history of a previous aneuploidic pregnancy are more likely to have an abortus with an abnormal karyotype (43). Thus karyotype analysis of the aborted tissue should be considered in all couples with recurrent spontaneous abortion. This practice will provide prognostic information for subsequent pregnancies and assessment of treatment efficacy can be determined more accurately. Results of recent research have provided insights into the mechanisms of pregnancy loss of a karyotypic normal conceptus.

At the time of implantation, trophoblasts form two layers: an inner layer of cytotrophoblast, and an outer layer of fused cytotrophoblasts called syncytiotrophoblasts. Any agent that would interfere with differentiation of cytotrophoblasts to syncytiotrophoblasts could inhibit normal development of placenta and decidua. Antiphospholipid antibodies have been shown to inhibit differentiation of cytotrophoblasts to syncytiotrophoblasts (44). In addition, antiphospholipids have been shown to have a detrimental effect on prostaglandin production by maternal decidua (45) and a thrombotic effect on both maternal (46) and fetal (47) vessels.

APA and ANA but not ATA have also been shown to have a direct effect on the pre-embryo (48). It is not clear whether ANA works by the same mechanisms as APA. The frequent association of the presence of elevated APA and ANA in the same individual can be explained by the recently proposed hypothesis that non-organ-specific autoimmune disease may be secondary to some basic cellular abnormality, such as apoptosis (49). Recent reports indicated that most of the prominent ANA (DNA, histones, Sm, SS-A, RNP) are clustered in two populations of surface structures on apoptotic cells. It has been postulated that these surface blebs of apoptotic cells as well as the phospholipids exposed on the outside of these blebs are important immunogenic particles (50), leading to enhanced or aberrant expression of APA and ANA (51).

Trophoblastic cells are resistant to lysis by cytotoxic T lymphocytes and unstimulated NK cells (52), but are susceptible to activated NK cells or LAK cells (52). Cytokines such as IL-2 and TNF alpha that convert resting NK cells to LAK cells caused abortion in mice (53). Thus it was concluded that suppression of the activation of NK to LAK cells is necessary for successful pregnancy. A number of cytokines had been shown to prevent LAK cell activation. These cytokines included IL-3, granulocyte-macrophage colony stimulating factor (GMCSF) (54), TGF beta 2 (55) and PIBF (56). However, the NK trophoblast killing variation of immunotolerance of pregnancy model failed because in decidua of women experiencing spontaneous abortions, LAK cells and lysed trophoblasts were not demonstrable (57,58). Furthermore, human trophoblasts were not susceptible to killing by LAK cells without a period of in vitro tissue culture of trophoblasts (58). Last but not least, lower NK activity has been found in women experiencing recurrent spontaneous abortion compared with controls (59).

Th1 cytokines (IL-2, TNF alpha, INF gamma) can boost and Th2 cytokines (IL-3, IL-4, IL-10) can reduce abortion rates in mice (60). But the inefficiency of NK cell, macrophage, and Th1 cytokines in killing trophoblasts led to the question as to the mechanism whereby the cytokines produced their effects. A target other than trophoblasts for cytokines was sought. A maternal vascular target was suggested by pathologic specimens of aborted material that showed hemorrhagic necrosis at the trophoblast-decidual interface. Salafia et al. (61) showed that activation of coagulation mechanisms was a distinguishing feature of abortion in women of chromosomally normal embryos. The histopathology suggested activation of prothrombinase activity resulting in increased thrombin generation. Pro-inflammatory cytokines such as IL-1, TNF alpha, and INF gamma collaborate to activate procoagulant expression in endothelial cells that are in direct contact with maternal blood. Prothrombin is converted to thrombin. Thrombin then catalyzes generation of fibrin and activates IL-8 secretion by endothelial cells. IL-8 recruits polymorphonuclear leukocytes (PMNs) which kill endothelium that has been activated by IL-1, TNF alpha and INF gamma (62). The end result of unchecked thrombin production is clot formation occluding blood supply to the embryo and it dies (63). The procoagulant responsible for prothrombinase activity in abortions has been identified as the prothrombinase called fgl2 (64). Fgl2 is present in both decidua and trophoblasts of aborted but not control tissue (63). IL-4 and IL-10 inhibit cytokine stimulation of endothelial prothrombinase activity (65). TGF beta protects ischemic endothelium from damage by PMNs (66). Thus, the balance of Th1: Th2/3 determines success or failure of the pregnancy. Another example of interference with hemostatic reactions is APA inhibition of binding of annexin V to cultured endothelial cells and trophoblasts (67). The role of immunologic factors in triggering abortions can be summarized as follows: at the time of implantation, Th1 cytokines predominate so as to promote trophoblastic cell invasion and angioneogenesis. Shortly thereafter a Th1 to Th2/3 shift occurs in successful pregnancies. If this shift does not occur and Th1 cytokines persist unchecked, Th1 cytokines are abortogenic via upregulation of fgl2 prothrombinase activity. Th2/3 cytokines prevent abortion by decreasing fgl2 expression as a result of blocking generation of Th1 cytokines, endothelial cell activation and response to PMNs. CD8+ maternal T cells play a role in adjusting Th1: Th2/3 balance so as to prevent or foster abortion. The cytokine PIBF from CD8+ cells which express progesterone receptor downregulates expression of fgl2 (63). In individuals experiencing recurrent abortion who display high circulating levels of antithyroid antibodies (ATA), the decidual T cell activity shows a different pattern. A significant increase in the endometrial T cell population has been observed in women with ATA compared to controls with no ATA (68). Moreover, HLA DR expression increased and cytokine production showed lower IL-4 and IL-10 and higher IFN gamma compared to controls (68). These observations indicate that there are activated T cells in the uteri of women with ATA and the activated T cells secrete Th1 cytokines which promote abortion.

The role of cytokines and CD56+ cells in recurrent spontaneous abortion has been studied more locally at the feto-maternal interface, but there is evidence of systemic changes during pregnancy. In humans, elevated levels of circulating NK cells and NK activity have been linked to abortion of chromosomally normal concepti (69). Th1 cytokine production by circulating blood lymphocytes in response to trophoblast is a feature of about 50% of recurrent aborters (10), perhaps the 50% who abort chromosomally normal embryos (43). In addition to embryotoxic effects of Th1 cytokines, other circulating embryotoxins have been reported (70-73). One study suggested that embryotoxicity is caused by an IgG fraction in serum (73).

Diagnosis and Treatment of Women Experiencing Recurrent Pregnancy Loss
Since women experiencing recurrent spontaneous abortion are a heterogeneous population, specific markers are necessary to identify those who will respond to various treatments. Diagnostic tests have been used to identify putative immunologic causes of recurrent spontaneous abortion including APA, ANA, ATA, NK cells and embryotoxic factors. Successful pregnancies have been reported in women with a history of reproductive failure associated with elevated serum concentrations of APA and ANA after treatment with heparin, aspirin, glucocorticoids, IVIg or combinations of these (74-77). Among women experiencing recurrent pregnancy loss associated with elevated ATA, circulating NK cells or embryotoxic factors, treatment with IVIg has led to successful pregnancies (78, 80). Both IVIg and allogeneic monocyte immunization have been used to successfully treat unexplained recurrent pregnancy loss (81,82). The continuing challenge is to appropriately identify the immunologically mediated mechanisms associated with recurrent spontaneous abortion and to appropriately treat each individual patient.

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