Immune Etiology of Recurrent Pregnancy Loss and Its Diagnosis

Authors

  • Kenneth D. Beaman,

    Corresponding author
    • Clinical Immunology Laboratory, Department of Microbiology & Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
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  • Evangelos Ntrivalas,

    1. Clinical Immunology Laboratory, Department of Microbiology & Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
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  • Timothy M. Mallers,

    1. Clinical Immunology Laboratory, Department of Microbiology & Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
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  • Mukesh K. Jaiswal,

    1. Clinical Immunology Laboratory, Department of Microbiology & Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
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  • Joanne Kwak-Kim,

    1. Department of Obstetrics and Gynecology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
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  • Alice Gilman-Sachs

    1. Clinical Immunology Laboratory, Department of Microbiology & Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
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Correspondence

Kenneth D. Beaman, Clinical Immunology Laboratory and Department of Microbiology & Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.

E-mail: clinlab@rosalindfranklin.edu

Abstract

Recurrent Spontaneous Abortion of Immunological Origin (RSAI) is currently diagnosed by the occurrence of 2–3 consecutive miscarriages of unknown origin. The psychological trauma incurred by these events is a serious ailment which may be potentially avoided if a method of analysis is derived which may forecast these events and in turn prevent them from occurring. This review intends to examine studies of recurrent spontaneous abortion (RSA) which use laboratory diagnosis and also studies of RSA that do not use laboratory diagnosis. We believe that when laboratory results are incorporated into the diagnosis of RSA/RSAI that treatment is highly successful whereas the absence of laboratory results severely hinders the effectiveness of treatment. It is worth noting that correlating treatment versus outcome is imprudent because of the multiple variables involved in patient cases. It is not imprudent, however, to say that incorporation of laboratory data is essential when diagnosing RSA/RSAI.

Pathology of RSAI versus laboratory diagnoses of RSAI

The diagnosis and the cause of recurrent miscarriage of immunological origin (RSAI) are often thought to be equivalent but they are not. The evolution of this confusion is beyond the scope of this review. This elegant oversimplicity has led to inevitable controversy as well as confusion. In this review, we will discuss RSA with supportive laboratory data only.

For example, medical science has shown a number of immunological disorders such as rheumatoid arthritis and systemic lupus erythematosis have important diagnostic markers that have nothing to do with the pathology of the disease. The likelihood is that diagnostic markers used to determine recurrent miscarriage may also not play a direct role in its pathology. In either case, they certainly can be of use in diagnosing and treating this RSA.

The immune diagnosis of pregnancy losses and its immunological origin is most often determined as a rule out diagnosis. A current hypothesis is that RSAI is not because of an immune imbalance that directly causes pregnancy loss but because of an inflammation imbalance that leads to the pregnancy loss.[1-3] Perhaps a better name for the recurrent miscarriage of immune origin would be recurrent miscarriage of inflammatory origin. In this chapter, we describe useful, current new, and possible future testing methods for the detection of immune measures of RSAI miscarriage.

The diagnosis of RSAI has traditionally been described as the loss of two or more consecutive pregnancies with the same partner prior to 20–28 weeks of gestation.[4] After endocrine, genetic, or microbial causes are ruled out in the diagnosis, the presumption is that the cause of pregnancy loss is RSAI. The RSAI can be either primary, which is with no live birth, or secondary, with at least one live birth preceding the RSAI losses. Another factor in identification of RSAI diagnosis is the increased probability of pregnancy loss with time and number of losses in the affected couples. Traditionally, it is thought that about 5% of couples suffer RSAI and this number increases to 40% after four pregnancy losses.[4, 5]

Current pathology

We use the term ‘RSAI' (recurrent spontaneous abortion of immunological origin) to distinguish from recurrent miscarriage of no known origin. The often used definition for recurrent spontaneous abortion is 2–3 consecutive miscarriages of unknown origin occur before 16–20 weeks of gestation. This definition has led to a platform of ambiguities. Works such as that of Fukui et al.[6] highlights these ambiguities. This is an excellent manuscript that shows the use of an immunological treatment has no effect on recurrent miscarriage of undetermined origin. In order for immunological treatment to be effective, an immunological association must be shown between the type of immunological abnormality and the treatment. The studies of Kwak-Kim,[6-8] Winger,[9, 10] Gleicher,[11] and others have clearly shown that immunological treatment of RSAI can be very successful.[12-14]

Recurrent miscarriage of immunological origin is undoubtedly because of several different causes or etiologies. An early hypothesis for RSAI was that an inappropriate transplantation response was responsible for miscarriage and immune rejection of the fetus by the mother occurred because of the presence of allogeneic antibody in the mother, which crossed the placenta and caused the spontaneous abortion. For a number of years, the typing sera used for transplantation HLA typing were obtained from multiparous women.[15] Apparently, women who had multiple children developed allogeneic anti-paternal antibodies to the paternal HLA antigens of their fetuses in utero without negative effect. The role these antibodies play in pregnancy success or pregnancy loss may still be controversial, but it is worth pointing out that clearly these women had multiple successful births. Other studies now show that HLA antigens are not present in the developing maternal-fetal unit until later in pregnancy, probably not until the second trimester.[16, 17] Several lines of evidence suggest that adaptive immunity is probably not involved in RSAI.[18, 19] Therefore, during the first 16 weeks of pregnancy, there should be no involvement of transplantation antigens such as HLA in the acceptance or rejection of the fetus because there is no HLA-A, B, C, or D at this time. To extend this observation, recent data have shown that allogeneic T cells derived from the fetus survive in the maternal environment for years and vice versa.[20]

Current autoantibodies in RSAI

Not all RSAI are identified by a rule out diagnoses. Clearly, one etiology in some but not all patients with RSAI is the presence of anti-phospholipid antibodies. These antibodies are also found in some patients diagnosed with severe active lupus erythematosis or rheumatoid arthritis. These diagnoses of RSAI are readily made and in the case of active lupus or rheumatoid disease can be determined as a possibility etiology of pregnancy loss before a pregnancy is attempted, so patients can be monitored and treated. By extension, these antibodies could play a role in some patients with pregnancy losses but more probably represent a cellular immune response not exclusive to pregnancy.[21, 22]

We believe that antiphospholipid antibody syndrome causes RSAI because of an increase in blood clot formation and the susceptibility of the developing placental and fetal vascularization (Fig. 1). This makes some sense but has never been clearly demonstrated. In fact, the laboratory diagnosis of many autoimmune diseases and the pathophysiology of the disease are not direct or at least not easily directly related to the laboratory immunology data. Unlike allergic disease where one antigen can be demonstrated to cause the allergy, the culprit in autoimmunity may not be so easily identified. The direct cause of all factors in RSAI may also not be so easily identified, but there are definitely diagnostic immunology tests and treatments that can lead to successful outcome.

Figure 1.

Factors that are thought to be associated with recurrent miscarriage and clotting abnormalities. Key among these factors is the presence of antiphospholipid antibodies. Together all factors may have additive negative effect on pregnancy outcome.

What should be the relationship between autoimmune disease and testing for autoimmune diseases? For example, rheumatoid disease and rheumatoid factor (RF) are related but clearly the anti-immunoglobulin antibodies (RF) that is used as a diagnostic marker in rheumatoid arthritis may have no discernable pathophysiology in the progression of the disease.[16, 22] Likewise, anti-double stranded DNA antibodies may or may not be found in all cases of systemic lupus erythematosis. These antibodies may result in immune complex formation, which induces the pathology found in some patients with these autoimmune diseases. Models of putative immune-related pregnancy loss and the presence of specific immune factors in these models may tend to obfuscate the true disease as well as the importance of immune markers in RSAI diagnosis and prognosis. In SLE disease, 95–98% of patients will have a positive ANA test, but the majority of people with a positive ANA test do not have SLE.[15, 21]

Antiphospholipid antibody syndrome may or may not have a direct cause and effect with the RSAI. Even the pathology of this disease is probably initiated by cell destruction but caused not directly by effect of antibodies on the fetus or placenta but their effect on clotting (Fig. 1). Various clinical diagnosis shows positive tests, which occur in some RSAI women who can be successfully treated with low-molecular weight heparin. Usually, RSAI is multi-factorial with respect to mutational analysis of inherent clotting factors. The cell destruction is probably caused by and affected by other factors than antiphospholipid antibodies, but the final effect is undoubtedly on coagulation and ultimately blood flow and nutrition to the placenta. This causes fetal demise and pregnancy loss (Fig. 1).

Current laboratory cellular immunology tests

Important in our understanding RSAI are the differences in successful pregnancy and RSAI that can be found in NK cells and their functional activity and cytokine expression of TH1:TH2 cells during the first week of pregnancy. During this time, the inflammation necessary for implantation must be curtailed and a switch from a TH1-type response to a TH2 response is necessary.[18, 19] NK cells are the major lymphocyte population in the placenta.[23] Changes in these immune cells can be measured not only in cells obtained from the placenta but also in cells, which circulate in peripheral blood.[8, 9] Previously, it was shown that during pregnancy NK cells and NK activity decrease in successful pregnancies. However, there is clearly an increase in women with RSAI in the number of NK cells as well as an increase in NK function as measured by a cytotoxicity assay.[6] NK cells present in the placenta were previously thought to possibly kill surrounding placental tissue by cytotoxicity. It is now known that these cells secrete cytokines and other molecules that assist in placentation and successful pregnancy outcome.[6, 23] Their numbers and function are reflected in blood during pregnancy. These NK cells can be accurately measured in the blood and most accurately by cytotoxicity assays from NK cells that are present in women with RSAI.

Another significant change observed in the peripheral blood cells of pregnant women is the production of TNFα and other ‘TH1' cytokines by CD4+ T cells. Early investigations measured TNFα directly in serum from pregnant and RSA patients. This was problematic because the normal levels of cytokines are below the measurable limits of most EIA tests, and assay conditions and specimen collection vary and may affect measurement. When cells capable of making TNFα are activated and then quantitated by flow cytometry, the values not only become measurable but they are statistically significant in predicting RSAI.[7, 10] Women with RSAI demonstrated significantly higher Th1/Th2 ratios of IFN-g/IL-4 (P < 0.01), TNF-a/IL-4, and TNF-a/IL-10 (P < 0.05 each) in CD4-TH1 cells than in controls. A significantly increased Th1 cytokine expression in these activated cells may be the underlying immune etiology for reproductive failures, but regardless of the relationship to the pathophysiology of the disease, the levels can surely be used to diagnose and suggest possible treatment for these patients.

Prospective tests for RSAI

Current data support the hypothesis that the immunopathology of RSAI is often initiated before the adaptive immune response and is induced by the innate immune response.[3-5, 14] In pregnancy, NK cells, which are considered part of the innate immune response system, are the most abundant lymphocyte present in the developing placenta. A second abundantly present mononuclear cell in the placenta is the macrophage, another member of the innate immune response. Both of these cells recognize non-allogeneic somatic ‘antigens' and produce unique cytokines in pregnancy.

Several tests are available for pre-pregnancy diagnosis (prognosis) of RSAI. The first is the molecular assay of HLA-C group of receptors for NK cell regulation (KIR-HLA-C combinations).[24]

KIR and pregnancy

During pregnancy, the majority of maternal lymphocytes that reside in the uterus are NK cells. These maternal NK cells encounter trophoblast cells from the fetus that express HLA-G and HLA-C alleles (from both the mother and the father). KIR receptors that are expressed on maternal NK cells interact with HLA-C ligands in trophoblast cells and, this interaction appears to block the NK cell cytotoxicity against trophoblast cells.[24-26]

Killer cell immunoglobulin-like receptors (KIRs) are molecules found mostly on natural killer (NK) cells and regulate their function.[17, 25, 27] The KIR locus contains a family of polymorphic and highly homologous genes, maps to chromosome 19q13.4 within the leukocyte receptor complex. Table 1 shows specific interaction between KIR receptors and HLA-ligands. KIRs can be either inhibitory or activating. The inhibitory KIRs have one or two immunoreceptor tyrosine inhibitory motifs in their long cytoplasmic tail. These recruit inhibitory phosphatases, which bind via their SH2 domains. The signal transduction cascade will eventually lead to inhibition of release of cytotoxic granules, which will result in inhibition of NK cell cytotoxicity.[17]

Table 1. Relationship between KIR receptors and specific HLA-ligands
KIR receptorLigand
KIR2DL1, 2DS1HLA-Cw2, 4, 5, 6 (C2)
KIR2DL2/3, 2DS2HLA-Cw1, 3, 7, 8 (C1)
KIR2DL4HLA-G
KIR2DS4HLA-A11, HLA-Cy
KIR3DL1HLA-Bw4
KIR3DL2HLA-A3, A11

KIR ligands are members of MHC class I alleles, mostly HLA-C alleles. In any NK cell, the balance of signaling between the inhibitory and activation receptors determines the final outcome. In general, the inhibitory signal dominates the activating signal, and this is how NK cells avoid killing self-cells. When both the activating and the inhibitory KIRs bind to their respective ligands, the inhibitory signal dominates and the NK cell tolerates the target cell (Fig. 2).

Figure 2.

The diagram shows the potential relationships between NK cell and trophoblasts, which can modulate successful pregnancy.

Increased NK cell inhibition can result in inadequate trophoblast invasion into the uterine spiral arteries. Specific combinations of maternal KIR genotype and phenotypes and parental HLA-C genotypes are more prevalent in women with recurrent spontaneous abortions.[27-29]

Many studies suggest that in women with implantation failures, their NK cells express lower levels of inhibitory KIRs than the normal controls. The down-regulation of these inhibitory receptors may indicate their inability to recognize their respective HLA-C ligands and as a result show elevated NK cell cytotoxicity against the trophoblast cells (Fig. 3).

Figure 3.

Interaction of inhibitory NK cell receptors with trophoblast MHC class I molecules appears to block the NK cell cytotoxicity against trophoblast cells, whereas loss of interaction can lead to pregnancy loss by increasing the cytotoxic activity of NK cells.

Newer studies have indicated the importance of the KIR genotype of the mother, along with the HLA-C genotype of the mother and the father. Correlation of maternal KIR genotypes with parental HLA-C genotypes can serve as a prognostic indicator of allo-immune pregnancy losses.

Conclusion

During the past two decades, clinicians who treat women with RSAI to determine a possible diagnosis and treatment have used biomarkers and clinical assays. Among these immunological tests are anti-nuclear antibodies assays, tests for anti-phospholipid antibodies and thrombophilia-associated genetic assays. Recently, tests for KIR receptors and HLA-C ligands, NK cell counts and NK cytotoxicity assays, and TH1:TH2 cytokine ratios have been added by some.

Depending on the age and the number of pregnancy losses in a woman with RSA, we propose a nomogram (Fig. 4) that suggests which immune diagnostic tests may be most useful for the immune diagnosis of RSAI. These assays should be performed in women who are being evaluated by their clinician, particular if multiple miscarriages have occurred. As age and the number of miscarriages increase, we suggest that additional laboratory testing other than ANA and antiphospholipid antibodies become necessary. Certainly, increased testing is preferable to increases in the number of miscarriages. By understanding the meaning and value of these immunological and genetic assays and treating patients with the appropriate therapy as indicated when the assays are abnormal, greater success in pregnancy outcome in women with RSA or in women who have infertility will result.

Figure 4.

The nomogram represents a potential algorithm for the relationship between age of the mother and potential immunological assays to be performed to assess causes of recurrent miscarriage of immunological origin (RSAI). Not all testing may be indicated. However, as the maternal age increases and the number of miscarriages also increases, more testing may be indicated earlier in the course of RSAI. One possible nomogram draws the line indicated, which goes from the minimal number of pregnancy losses used by some for the diagnosis of recurrent miscarriage and the average age of onset of menopause in some groups of women. Test groups 1, 2, and 3 could be utilized as suggested along with indicated age.

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