Ole Bjarne Christiansen1, 2 , Astrid Marie Kolte3, Elisabeth Clare Larsen1 and Henriette Svarre Nielsen1
(1)
Fertility Clinic 4071, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark
(2)
Department of Obstetrics and Gynaecology, Aalborg University Hospital, Aalborg, Denmark
(3)
Recurrent Pregnancy Loss Unit, Fertility Clinic 4071, University Hospital Copenhagen, Rigshospitalet, Copenhagen, Denmark
Ole Bjarne Christiansen
Email: obchr@post5.tele.dk
Keywords
AutoantibodiesNatural killer cellsHLA-GHY-antigensPregnancy lossRecurrent miscarriageRecurrent pregnancy loss
Introduction
Recurrent pregnancy loss (RPL) is in Europe defined as three or more consecutive pregnancy losses prior to gestational week 22 [1] and affects approximately 2–3 % of all women aiming to get a child. In RPL women like in other women approximately half of the pregnancy losses are due to embryonal aneuploidy probably occurring by chance but with increased incidence with increased maternal age [2]. In the vast majority of couples no documented cause of pregnancy loss can be found, although a series of risk factors for pregnancy loss have been identified.
In this chapter we do not refer to “causes ” of pregnancy loss since the only documented cause of pregnancy loss is severe embryonal malformation, often caused by chromosomal aberration. Biomarkers associated with pregnancy loss or RPL will be entitled “risk factors” if they have been strongly associated with RPL or pregnancy outcome in case–control studies and/or in well-designed prospective studies. The main focus in RPL research has been on biomarkers relating to endocrinologic, thrombophilic, and immunological dysfunctions in the women suffering from RPL.
We provide an overview of the scientific evidence for immune aberrations being involved in the pathogenesis of RPL and we discuss which biomarkers related to immune function are candidates for further research or can already be used in clinical practice.
The feto-placental unit is often entitled the “feto-placental allograft ” as it bears similarities to the transplantation of an organ such as a kidney from an allogenic donor. In this situation the allograft can only avoid being rejected when intensive immunosuppressive therapies are implemented. A priory, it must be presumed that the maternal immune system would make efforts to reject the feto-placental unit, which carries paternal alloantigens. The previous belief that rejection is avoided because alloantigens on the fetus or placenta are separated from the maternal immune-competent cells has now been abandoned; in contrast there is plenty of documentation that recognition of paternally derived antigens on the feto-placental unit is a normal feature in pregnancy. One such proof of immune recognition of paternal antigens is the observation that antipaternal human leukocyte antigen (HLA) antibodies develop in 10–30 % of all normal pregnancies [3]. However, in a meta-analysis of relevant studies their presence did not increase the risk of early pregnancy complications such as miscarriage [4].
The research in immunological biomarkers associated with RPL has focused on measurements of autoantibodies in the blood, natural killer (NK) cells in the blood or decidual tissue, and cytokines in the blood or decidual tissue and investigations of classical and nonclassical HLA polymorphisms in patients or couples with RPL and studies of HLA protein expression on trophoblast.
Autoantibodies
Much focus has been on investigation of autoantibodies in RPL since it was early recognized that women with specific autoimmune diseases , especially systemic lupus erythematosus (SLE), hypo- and hyperthyroidism, and inflammatory bowel disease had an increased risk of pregnancy loss in early and late gestation [5]. In SLE this increased risk was associated with the presence of antiphospholipid antibodies such as lupus anticoagulant (LAC) and anticardiolipin antibodies (ACA) and in thyroid disease the risk was associated with the presence of thyroid autoantibodies. LAC and probably also high-titer ACA are associated with an increased risk of venous and arterial thrombosis and are in many studies associated with a reduced chance of live birth in RPL patients [6]. The role of antiphospholipid antibodies will not be reviewed further here as it will be dealt with in another chapter in this book.
Thyroid autoantibodies can be found in 5–15 % of women of reproductive age but in the majority of cases they are not associated with thyroid dysfunction or reproductive problems. On the other hand almost all cases of clinical hyper- or hypothyroidism are associated with the presence of thyroid autoantibodies. The most prevalent thyroid autoantibody is thyroid peroxidase (TPO) antibody. A considerable number of studies have found thyroid autoantibodies with increased prevalence in RPL and a meta-analysis including 8 case–control studies, most of them small, found that antithyroid antibodies are associated with RPL with odds ratio (OR) 2.3 (95 % CI 1.5–3.5) [7]. It remains to be elucidated whether the association between antithyroid antibodies and RPL reflects an increased risk of clinical hypothyroidism, which may lead to delayed embryonal development in pregnancy, or whether their presence is just a marker of a generally increased predisposition to an autoimmune response.
Another autoantibody, antinuclear antibody (ANA), has been extensively studied in RPL. In a review from 1996, Christiansen [8] reported that in 10 of 12 relevant case–control studies there was an increased ANA prevalence in RPL patients, though not always statistically significant. In the three relevant studies published subsequently, two found significant increased ANA prevalence in RPL compared with controls [9, 10] whereas one could not detect such an association [11]. In one study, the presence of ANA was associated with an increased risk of pregnancy loss in the next pregnancy [12].
A direct pathophysiologic link between the presence of autoantibodies in RPL and fetal death has not been convincingly documented. In our opinion, ACAs, antithyroid antibodies, ANA, and most other autoantibodies in RPL patients are most likely markers of a general breakage of autotolerance [5] or are epiphenomenons associated with carriage of specific HLA alleles such as HLA-DRB1*03. This HLA allele is associated with both production of ACA, antithyroid antibodies, and ANA and the risk of RPL [13, 14].
Cytokines
Cytokines are signaling molecules secreted from immune cells and usually bind to receptors on other immune cells resulting in stimulation or inhibition of function. One important division of cytokines is between the so-called T-helper type 1 cytokines, which promote T lymphocyte cytotoxicity and often inflammation and T-helper type 2 cytokines, which promote antibody production and anti-inflammation. Typical cytokines in the former group are interferon (IFN)-γ and interleukin (IL)-2 whereas IL-4 and IL-10 are characteristic T-helper type 2 cytokines. Tumor necrosis factor (TNF)-α is more difficult to classify but induces inflammation and apoptosis of target cells. Wegmann et al. [15] proposed the theory that normal pregnancy is characterized by a predominant production of T-helper type 2 cytokines, whereas adverse pregnancy outcomes such as RPL are characterized by a predominant T-helper type 1 cytokine production. This theory is probably too simplistic and has now been modified to include interactions between T-helper 17 cells secreting the pro-inflammatory cytokine IL-17 and T-regulatory cells [16]. It has been recognized that different cytokine profiles may be beneficial or harmful at different stages of pregnancy; for example IFN-γ, TNF-α, and other inflammatory cytokines seem to be crucial during the implantation process [17], whereas high levels of these cytokines may be harmful later in pregnancy. This fact makes research in the role of cytokines in RPL extremely difficult.
Since most cytokines display their effects at close range, interpretation of results from studies of cytokine secretion by peripheral blood lymphocytes or direct measurements of cytokines in the blood must be done with caution. Measurements of cytokines in endometrial biopsies or flushing or decidual tissue are subject to technical and methodological difficulties and will not be reviewed here.
TNF-α is one of the few cytokines with levels in the peripheral blood well above the detection limit of most assays, and since it is a typical pro-inflammatory cytokine it may be a good marker for the level of systemic inflammation. High plasma levels have been reported to increase the risk of pregnancy loss in RPL patients [18] and high TNF-α and TNF-α/IL10 ratios characterized women with euploid miscarriage compared with those with aneuploid miscarriage [19]. Kruse et al. [20] found that stimulated lymphocytes from RPL patients in very early pregnancy, who went on to miscarry, produced more TNF-α than those who gave birth. Lastly, it was reported that patients with RPL after a birth (secondary RPL) had significantly higher plasma levels of TNF-α in very early pregnancy than RPL patients with exclusively early miscarriages (primary RPL) [21]. These observations suggest that a high systemic inflammatory stage in early pregnancy increases the risk of miscarriage and that in particular secondary RPL patients are in a pro-inflammatory stage from very early pregnancy.
Mannose-Binding Lectin
Mannose-binding lectin (MBL ) is a plasma protein produced in the liver. After binding to oligosaccharides on the surface of microorganisms, it activates complement that can kill the microorganisms. Furthermore, MBL by enhancing phagocytosis can help in clearing apoptotic cells, cellular debris, and immune complexes, which would otherwise prompt inflammatory processes. The result of MBL deficiency may therefore be pro-inflammatory processes at the feto-maternal interface and MBL deficiency, which is genetically determined (see later), is therefore expected to increase the risk of pregnancy loss. In concordance with this assumption, MBL deficiency (<100 ng/ml) has been found to be associated with RPL in three case–control studies [22–24]. In the latter study, MBL deficiency was also associated with a significantly poorer prognosis in RPL patients.
Natural Killer Cells
In the search for immunological aberrations in RPL patients, there has been much focus on NK cells in the peripheral blood or decidual or endometrial tissue. NK cells are part of the innate immune system and in contrast to T lymphocytes they can recognize and react against target antigens typically on cells affected by intracellular infection or malignancy without prior sensitization. This reaction can result in killing of the cells (cytotoxicity) or secretion of an array of cytokines. The interest for NK cells in RPL and other pregnancy complications has been stimulated by three observations: (1) there is a unique composition of NK cells in the endometrium and decidual tissue. More than 90 % of lymphocytes in the endometrium in the luteal phase and in the decidual tissue in early pregnancy are low-cytotoxicity, high cytokine-producing NK cells, which carry a high density of the CD56 surface marker (CD56bright) by flow cytometry, but are negative for the CD16 marker [25]. In contrast, in peripheral blood, 90 % of the NK cells carry the CD56dimCD16 markers, which are associated with high cytotoxicity and low cytokine production; (2) the HLA molecules expressed on trophoblast subsets, HLA-G, HLA-C, and HLA-E, can all act as ligands for the three kinds of activating or inhibitory receptors found on NK cells and other cell types found in the uterus: the killer immunoglobulin-like receptors (KIRs), the CD94 receptors, and the immunoglobulin-like transcripts (ILTs) [26, 27]; (3) studies in NK- and T-cell-deficient transgenic mice with a high fetal loss rate show that restoration of NK cells by bone marrow transplantation results in a normal fetal resorption rate [28].
Investigations of NK cells in RPL can be divided into (1) studies of NK cell subsets by flow-cytometric analysis or tests of NK cytotoxicity of peripheral blood lymphocytes before or during pregnancy and (2) studies of NK cells in endometrial biopsies from before pregnancy or in decidual tissue collected from missed miscarriages and elective abortions.
Due to the easy availability, studies based on peripheral blood have been dominant. Excluding small studies (studies with <30 RPL patients), the majority of studies found that the percentage of CD56+ cells in peripheral blood taken prior to pregnancy is significantly higher in RPL women than controls [29–34]. Even so, some studies did not find any difference in percentage of CD56+,16+ cells or CD56+ cells [16, 35]. The fact that most of the investigated RPL women were nulliparous and most controls were multiparous is a methodological problem in this kind of studies [36] as a previous successful pregnancy can induce permanent changes in NK cell subsets [37]. The limitations of the immunological biomarkers tested in RPL and proposals for the research needed to clarify their clinical usefulness are listed in the table.
Cells from RPL patients collected before pregnancy have also been investigated in tests of NK cytotoxicity. In several studies in all RPL subsets [33, 38–40] or in primary RPL [37] a significantly increased NK cytotoxicity was found in patients compared with controls; however, Emmer et al. [41] in a large study did not find any difference between the two groups.
A series of studies have investigated the impact of high NK cytotoxicity on subsequent pregnancy outcome in RPL patients. Aoki et al. [42] first reported that RPL patients with high peripheral blood NK cytotoxicity before pregnancy had a significantly higher rate of pregnancy loss (71 %) in the next pregnancy than patients with lower NK cytotoxicity (20 %). Yamada et al. [43] found a significantly higher NK cytotoxicity in patients with a subsequent euploid miscarriage compared with those with live birth and Morikawa et al. [44] found a nonsignificant tendency for the same. In contrast Liang et al. [45] found similar NK cytotoxicity in RPL patients with subsequent pregnancy loss and live birth.
The strongest argument against a significant role for measurement of NK cytotoxicity in RPL patients came in a large prospective study by Katano et al. [46]. In a logistic regression analysis adjusting for recognized risk factors for miscarriage, high NK cytotoxicity before pregnancy had no impact on subsequent pregnancy loss rate.
The composition of endometrial lymphocytes fluctuates highly in the menstrual cycle with a six- to tenfold increase in the late luteal phase compared with the follicular phase [47] and as previously described the frequencies of the NK markers in the endometrium and peripheral blood vary as well. It has therefore rightly been questioned whether the endometrial NK cell subsets reflect those in the peripheral blood. A series of studies have investigated NK cells in endometrial biopsies taken in nonpregnant cycles in RPL patients and controls. Assessment of NK cell populations in these biopsies has been by immunohistochemistry or flow cytometry of homogenized tissue. The former technique is semiquantitative and subjective and the latter technique also has limitations, because the tissue undergoes enzymatic digestion, which influences marker expression. By flow cytometry, Lachapelle et al. found that the CD56bright subset was significantly lower in RPL patients than in controls [48]; using immunohistochemistry, Clifford et al. found that the frequency of CD56+ cells was significantly higher in RPL than controls [49]; Quenby et al. found that significantly more RPL patients than controls had NK cells >5 % [50]; and Tuckerman et al. reported that mean frequency of CD56+ cells was significantly higher in RPL than controls [51]. However, no relationship between CD56+ count in the endometrium and subsequent pregnancy outcome was found in the latter study: the patients who gave birth even tended to have higher NK cell numbers than those who miscarried again. Two quite small studies, using immunohistochemistry and flow cytometry, respectively, did not find any statistically significant difference in NK cell subsets in the endometrium between RPL patients and controls [52, 53].
Some studies have compared NK cell subsets in decidual tissue from missed miscarriages of RPL patients and fertile women having an elective termination and found differences in NK cell compositions between the two groups [54, 55]. Since the tissue in the former cases is often necrotic and inflamed due to the death of the fetus, whereas fresh and vital in the latter case, these kinds of studies provide limited valid information and they will not be reviewed further here.
T Regulatory Cells
T regulatory (Treg) lymphocytes have gained much attention in both general and reproductive immunology during the recent years. After being activated by tolerogenic antigen-presenting cells (APCs), Tregs can suppress the generation and the effector function of type 1T-cell-mediated immune responses, which are considered harmful to pregnancy.
Studies in T-cell-deficient transgenic mice strains have clearly demonstrated that lymphocytes with the Treg phenotype CD4+, CD25+,Foxp3+ are important for implantation and successful pregnancy in allogenic matings [56]. The role in Tregs in human pregnancy and especially in RPL is still not clear, as relevant studies are small and sparse. Kwiatek et al. recently reported that the percentage of Tregs in the peripheral blood at the time of pregnancy loss was significantly lower in women with RPL than women with normal pregnancies at the same gestation age [57]. In women without a history of RPL, Jin et al. found that those who miscarried had significantly lower frequencies of CD4+,CD25bright cells in peripheral blood and deciduas than those with normal pregnancies [58].
A working hypothesis to guide future research that integrates the current knowledge from animal and human research of the role of Tregs in normal and adverse pregnancy has been proposed by Robertson et al. [59]: increasing plasma estrogen in the late follicular phase causes the Treg pool in the blood or regional lymph nodes to expand and causes increased uterine expression of chemokines resulting in the recruitment of T cells to the uterus. Male antigens and cytokines in seminal fluid in the vagina recruit tolerogenic APCs to the uterus and regional lymph nodes that activate local Tregs, which suppress pro-inflammatory T-helper type 1 immunity towards alloantigens on the embryo and trophoblast. The hypothesis is attractive as it introduces adaptive cellular immunity in the pathogenesis of adverse pregnancy outcomes such as RPL. In contrast to NK-cell-mediated immunity, an important feature of adaptive immunity is immunological memory, which is stored in memory T cells. Clinical observations such as the rare occurrence of preeclampsia in a second pregnancy with the same husband or the negative prognostic impact of the sex of the firstborn child in women with secondary RPL (see later) can most likely be explained by mechanisms, where tolerance or harmful immunity has developed in the first ongoing pregnancy and is remembered by memory T cells.
The current knowledge about Tregs in normal pregnancy and RPL illustrates the complexity of the research required in the future. It must take into account the very dynamic nature of the Tregs, which relocate between various compartments, proliferate, and activate according to cycle-specific endocrine factors and external antigen exposures provided, e.g., by coitus.
Immunogenetic Studies
All proteins which participate in immune interactions or are parts of immune cells are encoded by genes, which are often polymorphic due to single-nucleotide polymorphisms (SNPs) or copy number variations (CNVs) of DNA sequences. These polymorphisms may give rise to decreased or increased protein production and sometimes disturbances of immune functions.
By genome-wide screening, the polymorphisms suggested to affect gene function can be assigned to specific immunological or metabolic pathways modulated by the affected genes. In a recent study, CNVs that rearranged genes in pathways of “innate immune signaling,” “complement cascade,” or “interaction of Fc gamma receptors with antigen-bound IgG” were found highly significantly more often in RPL patients than in fertile controls [60].
In the following section we concentrate on studies of a single or a restricted series of candidate genes which a priori were considered likely to play a role in the immunological interactions being important for pregnancy.
Cytokine Genes
As discussed previously, it is generally believed that cytokines characterizing a T-helper type 1 or a pro-inflammatory immune response are involved in the pathogenesis of RPL. The plasma levels or the in vitro production of many cytokines are in part determined by polymorphisms in the genes coding for the cytokines. Many studies of cytokine gene polymorphisms have been undertaken in RPL patients. A review [61] concluded that some studies have reported altered prevalence of polymorphisms in genes coding for IFN-γ, IL-10, IL-6, and IL-1B, but the findings could not be confirmed in studies by other groups. The reasons for the lack of confirmed associations may be that studies have been small, patient groups have been clinically heterogeneous, and the prevalence of the polymorphisms is very different between ethnic groups.
Mannose-Binding Lectin Genes
As previously discussed, two research groups have reported that MBL deficiency is associated with RPL and a poor prognosis in these patients. The plasma levels of MBL are determined by polymorphisms in the promoter region and exon 1 of the MBL-2 gene on chromosome 10. Specific combinations of genetic polymorphisms associated with low (<100 ng/ml) MBL levels have been reported with increased frequency in RPL patients and in particular in those with unexplained late fetal death [24, 62].
HLA
The HLA region l ocated on the short arm of chromosome 6 contains the most polymorphic genes known in the human species. Dependent on the genetic distance between the various HLA loci, alleles in each locus display stronger or weaker linkage disequilibrium, which means that alleles in different loci are inherited together more often or less often than expected by chance. This is an important feature when studies of HLA polymorphisms in RPL and other disorders are evaluated.
The HLA molecules play an important role in both the adaptive and innate immune system . In the adaptive system, CD8+ lymphocytes can exert cytotoxic reactions against cells carrying class I HLA molecules, especially HLA-A and B, which play an important role in transplantation immunology. Class II HLA molecules (HLA-DR and -DQ) are carried primarily on APCs and present antigenic peptides to T-helper (CD4+) lymphocytes, which can initiate both humeral and cellular immune reactions. An individual’s two sets of HLA class II alleles determine the repertoire of antigens that he/she can easily be immunized against, and which antigens will not give rise to an immune response. Due to this feature, HLA class II alleles are associated (sometimes strongly) with most autoimmune diseases, since these diseases are caused by an adverse reaction against one or several self-antigens.
NK cells, which belong to the innate immune system, were initially believed to react against all cells not carrying HLA molecules, but we now know that things are more complicated, as NK receptors can be inhibited or activated by HLA-C, HLA-G, and –E ligands, often dependent on the polymorphism of the HLA molecule.
Due to the different ways HLA can influence immune reactions, studies of HLA in RPL can be divided into three main categories: studies of HLA allele incompatibility (sharing) between partners with RPL; studies of HLA allele prevalences in women with RPL; and studies of HLA-C, -G, and -E alleles in couples with RPL.
All three kinds of studies have been addressed in a recent meta-analysis by Meuleman et al. [63], which provide a comprehensive review of the literature.
Increased HLA compatibility (sharing) was originally thought to decrease the probability that the mother would react immunologically adequate to the fetus and produce so-called blocking antibodies but the importance of these has never been documented. The vast majority of HLA-sharing studies is of older date, and used obsolete serological techniques for HLA determination, which can detect only broad antigen specificities of the HLA alleles. The meta-analysis [63] reported that allele sharing in the HLA-B, -DR, and -DQ loci was found with significantly higher frequency in RPL than control couples. However, these results should be interpreted with caution due to the obsolete methods used in most of these generally small studies.
Studies of HLA allele frequencies in RPL have focused on the HLA-DR or -DQ allele prevalences in RPL women and controls since these are the strongest immune response genes, as mentioned above. In the meta-analysis comprising eight case–control studies using modern polymerase chain reaction techniques , it was found that HLA-DRB1*04 and -DRB1*15 were significantly increased in RPL patients [63]. HLA-DRB1*03 was found with an OR of 1.32 (95 % CI: 0.89–1.97) in patients versus controls, which was not significant. However, we are convinced that HLA-DRB1*03 may be the strongest RPL susceptibility class II HLA allele in Caucasians. In a large case–control study [14] we found this allele to be highly significantly increased in RPL patients with increased prevalence with increased number of previous pregnancy losses. The reason why the HLA-DRB1*03 allele was not significantly increased in RPL in the meta-analysis may be due to several factors: Firstly, four of the included studies were Japanese. In Japan the HLA-DRB1*03 allele is very rare and the many Japanese patients and controls will dilute an association of HLA-DRB1*03 and RPL in the meta-analysis. Studies of associations between HLA polymorphisms and disease susceptibility should always be restricted to specific ethnic groups. Secondly, a large study with 234 patients and 360 controls [64] was excluded from the meta-analysis since the control group comprised normal blood donors rather than fertile women. This is an unjustified exclusion since individuals from an unselected population rather than individuals with no disease (fertile women) are fully accepted as controls in genetic case–control studies as long as the disease is rare (RPL prevalence 2–3 %). Third, in several of the included studies, patients (but not controls) with all kinds of autoantibodies were excluded. This will deplete the patient group for HLA-DRB1*03 positives since this allele is well known to be associated with autoantibody production [13].
In conclusion, taking the meta-analysis and our own full data set into consideration we conclude that HLA-DRB1*03, -DRB1*04, and -DRB1*15 may confer susceptibility to RPL in Caucasians. However, as all these alleles are quite frequent in a Caucasian population (combined frequency 80 %) this knowledge is not very useful in clinical practice.
Studies assessing the impact of maternal carriage of specific HLA class II alleles on future pregnancy outcome have provided information that can be more useful in clinical practice and in addition have highlighted the importance of a not previously recognized immune dysfunction in patients with RPL. Epidemiological studies have shown that among patients with RPL after a birth (secondary RPL), the birth of a boy in the pregnancy preceding the miscarriages is significantly more prevalent (61 % versus 39 %) and patients with a firstborn boy exhibit a significantly lower chance of live birth in their next pregnancy [65] and also after a period of 5 years [66]. Among RPL patients with a firstborn boy, maternal carriage of one of the three HLA-class II alleles, HLA-DRB1*15, -DQB1*0501/2, and -DRB3*0301, the chance of live birth was 22 % lower than in similar patients not carrying these alleles [67]. These alleles (HY-restricting class II HLA alleles) are known from in vitro models to present peptides derived from male-specific proteins (HY antigens) to T-helper cells and in transplantation immunology carriage of the alleles predisposes to graft-versus-host disease after sex-mismatched bone-marrow transplantation. Recently it has been reported that the HLA class II allele HLA-DRB1*07 also restricts immunity against HY antigens and in a new prospective study we confirmed that maternal carriage of this allele reduced the chance of live birth in patients with RPL after the birth of a boy [68]. Our hypothesis derived from the HLA studies and supported by a study of anti-HY antibodies in RPL patients [69] is that T-helper lymphocytes from some women carrying these HY-restricting class II HLA alleles recognize HY antigens on the placenta of their first ongoing pregnancy with a boy, which initiates a series of harmful immune reactions targeting the trophoblast in the subsequent pregnancies ultimately leading to RPL. More research confirming this mechanism of RPL is needed; in particular we need to isolate the suggested clones of HY-specific T lymphocytes that initiate or carry out the suggested immune reactions leading to RPL.
In the section about NK cells we were discussing the relationship between specific KIR receptor polymorphisms, HLA and RPL. It has been shown that feto-maternal mismatch for HLA-C alleles can induce Tregs that may promote tolerance to the pregnancy in the uterus [70]. HLA-C alleles can be divided into so-called C1 and C2 groups according to a dimorphism at position 80 of the segment of HLA-C molecule that can bind KIRs. C1 allotypes are ligands for the inhibiting KIRDL2/3 and activating KIRDS2, whereas C2 allotypes are ligands for the inhibitory KIR2DL1 and activating KIR2DS1. Hiby et al. [71] published data suggesting a role for maternal KIR polymorphisms and parental HLA-C polymorphisms in RPL. It was found that situations where the woman carries a combination of KIR genes that is primarily inhibitory (so-called AA genotype) and where the father carries C2 allotypes are more frequent among RPL couples than couples with normal fertility. Hiby et al. suggested that this combination of mainly inactivating KIR genotypes in the woman and their ligands in the parents results in a predominant decidual NK cell inhibition that may lead to insufficient secretion of specific cytokines at the feto-maternal interface, resulting in defective trophoblast proliferation and invasion and subsequent RPL. Another large study [72] in contrast found that maternal carriage of the inhibitory KIR2DL1 in combination with C2 homozygosity in both partners was found significantly more often in controls than RPL women, whereas maternal carriage of the activating KIR2DS2 in conjunction med C1 homozygosity in both partners was found to be significantly increased in RPL patients. The conclusion from this study was the opposite of the above: that receptor-ligand combinations that promote inactivation of NK cells are beneficial for pregnancy and may prevent RPL. In the meta-analysis of HLA in RPL [63] no association between parental C2 allotypes and RPL could be detected.
Another set of studies have investigated HLA-G polymorphisms in RPL. HLA-G is a so-called nonclassical HLA gene , which exhibits much less polymorphism than classical HLA genes but has the interesting feature of being highly expressed in extravillous trophoblast cells in contrast to all other HLA genes except HLA-C and -E. No polymorphism in the coding part of the HLA-G gene has repeatedly been associated with RPL whereas many studies have been undertaken regarding a 14-base pair insertion/deletion dimorphism in exon 8 of the HLA-G gene, which may affect transcription of the gene. Several studies have showed an association between low levels of soluble HLA-G in plasma and homozygosity for the HLA-G 14-base pair insertion [73]. Low levels of soluble HLA-G may in itself result in reduced immunity against the trophoblast since soluble HLA-G can modulate NK cell function via inhibition of interactions between NK cells and specific antigen-presenting dendritic cells [74]. In the meta-analysis by Meuleman et al. combing results from seven studies, the HLA-G 14-base pair insertion was nonsignificantly increased in RPL compared with controls, OR 1.38 (95 % CI: 0.85–2.26) for the insertion/insertion genotype. However, two other recent meta-analyses including 17 studies [75] and 14 studies [76] found that the HLA-G 14-base pair insertion frequency was significantly increased in RPL with ORs 1.27 (95 % CI: 1.04–1.55) and OR = 1.47 (95 % CI: 1.13–1.91), respectively. In conclusion, the HLA-G 14-base pair insertion in exon 8 seems to predispose to RPL. Since the HLA-G 14-base pair insertion is in positive linkage disequilibrium with the HLA-DRB1*03 allele [77] the question remains whether the HLA-G gene insertion or HLA-DRB*03 is the main RPL susceptibility gene.
Conclusions
Overall, the three main arguments for immunological disturbances playing a role in RPL are the following:
1.
2.
3.
Most of the non-genetic immunologic biomarkers (except autoantibody measurements) reviewed in this chapter have not been tested in assays with sufficient reproducibility or the reference values in normal women or during pregnancy have not been sufficiently established (Table 6.1). Regarding the genetic biomarkers, DNA-based tests are suggested to have high reproducibility, but in most cases their diagnostic values must be further studied in large studies of patients and controls, which are homogenous with regard to reproductive history and ethnicity.
Table 6.1
Immunological biomarkers investigated in recurrent pregnancy loss (RPL), their diagnostic value, and suggestions for further research
|
Biomarker |
Associated to RPL |
Prognostic value |
Needed research |
|
|
Autoantibodies |
Antiphospholipid antibodies |
+++ |
++ |
Standardization of assays and cutoff values Prognostic studies in untreated patients |
|
Thyroid antibodies Antinuclear antibodies |
+++ |
? |
Prognostic studies in untreated patients |
|
|
Soluble immune biomarkes |
Peripheral blood cytokines |
? |
? |
More sensitive and reliable methods |
|
Mannose-binding lectin |
++ |
+ |
More studies for further documentation |
|
|
Immune cells |
Peripheral blood NK cell subsets |
+ |
? |
Establishment of reference values in different phases of cycle or pregnancy |
|
Peripheral blood NK cytotoxicity |
+ |
? |
Establishment of reference values in different phases of cycle or pregnancy |
|
|
Endometrial NK cells |
?/+ |
? |
Establishment of reference values in different phases of cycle Standardization of methods Larger studies |
|
|
Decidual NK cells |
? |
- |
Suitable control samples (aneuploid missed miscarriages?) |
|
|
Peripheral blood Treg cells |
? |
? |
Establishment of reference values in different phases of cycle Standardization of methods Larger studies |
|
|
Genetic biomarkers |
Cytokine gene polymorphisms |
? |
− |
More and larger studies Ethnic and diagnostic homogeneity of cases and controls |
|
Mannose lectin gene polymorphism |
+ |
+ |
More studies |
|
|
HLA sharing |
? |
? |
Studies using up-to-date techniques Clear definition of criteria for allele sharing |
|
|
HLA class II |
++ |
+ |
Larger studies Studies homogeneous with regard to reproductive history and ethnicity |
|
|
HLA-C, HLA-G |
+ |
? |
Larger studies Studies homogeneous with regard to reproductive history and ethnicity |
|
The lack of tests with sufficient diagnostic value for detecting immunological causes of RPL has wide implications for the identification of patients for specific treatments or inclusion in randomized controlled trials. The failure to find significant beneficial effects of immunotherapeutic treatments such as prednisone [80] or intravenous immunoglobulin [81] in randomized controlled trials has by many researchers been attributed to the non-selection of patients for these trials due to the presence of immune biomarkers such as high NK cell cytotoxicity levels. We believe that as long as the diagnostic specificity of the immune tests is not well established, patients for treatment or participation in randomized trials must still be selected based on their reproductive history, e.g., a poor spontaneous prognosis evidenced by a high number of previous pregnancy losses.
In our view, not one but several disturbances or disruptions of pathways relating to immune interactions are probably causing many cases of RPL; the reproductive process is too important to be vulnerable to the disruption of only one immune pathway. We think that disturbances in several immune pathways (caused by SNPs or CNVs disrupting DNA sequences) in conjunction with immunizing events in previous pregnancies, such as a substantial transfer of fetal antigens (cells) into the maternal circulation [82], can promote pro-inflammatory reactions or breakage of autotolerance. This will predispose to RPL and other adverse pregnancy outcomes and ultimately lead to atherosclerotic or autoimmune disease.
This suggested complexity of the pathogenesis of RPL will be a challenge for future research in the area, which is further complicated by the fact that almost half of all pregnancy losses in RPL women are due to embryonal aneuploidy with no immunological background. We find it important for researchers to acknowledge this complexity instead of narrow-sightedly dividing the patients into subgroups, each suggested to be caused by one specific (simple) immunological or non-immunological etiology.
Large epidemiological studies have now documented that RPL patients carry a substantial risk of developing later atherosclerotic disease [78, 79], which may be due to chronic activation of pro-inflammatory pathways. Research in the immunological abnormalities associated with RPL therefore both has the potential to disclose pathways leading to pregnancy loss that may be modified by specific therapies but it may also identify biomarkers, which can be used for identifying those patients who are in the greatest risk of contracting cardiovascular disease in order that preventive measures can be initiated.
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