CLINICAL OBSTETRICS AND GYNECOLOGY Volume 59, Number 3, 509–523 Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. Immunologic Abnormalities, Treatments, and Recurrent Pregnancy Loss: What Is Real and What Is Not? NATHALIE F. WANG, MD,* ASTRID M. KOLTE, MD,* ELISABETH C. LARSEN, MD, PhD,* HENRIETTE S. NIELSEN, MD, DMSc,* and OLE B. CHRISTIANSEN, MD, DMScw *Recurrent Pregnancy Loss Clinic, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark; and w Department of Obstetrics and Gynecology, Aalborg University Hospital and Clinical Institute, Aalborg University, Aalborg, Denmark Abstract: Recurrent pregnancy loss, depending on the definition, affects 1% to 3% of women aiming to have a child. Little is known about the direct causes of recurrent pregnancy loss, and the condition is considered to have a multifactorial and complex pathogenesis. The aim of this review was to summarize the evaluation and the management of the condition with specific emphasis on immunologic biomarkers identified as risk factors as well as current immunologictreatmentoptions.Thereviewalsohighlightsand discusses areas in need of further research. Key words: autoantibodies, antiphospholipid antibodies, NK cells, HLA, miscarriage, recurrent pregnancy loss Correspondence: Ole B. Christiansen, MD, DMSc, Recurrent Pregnancy Loss Clinic, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: email@example.com The authors declare that they have no disclosures. CLINICAL OBSTETRICS AND GYNECOLOGY / Introduction Recurrent pregnancy loss (RPL) is traditionally defined as 3 or more consecutive pregnancy losses before gestational week 20,1 althoughitis sometimes defined as only 2 consecutive losses. Depending on the definition used, it affects 1% to 3% of all couples aiming to have a child. Approximately half of the pregnancy losses are due to embryonal aneuploidy, often occurring as a random event, but with increased incidence with advanced maternal age.2 In the vast majority of the cases, no documented cause of miscarriage can be found although a series of endocrine, thrombophilic, and immunologic risk factorsforpregnancyloss have beenidentified. VOLUME 59 / NUMBER 3 / SEPTEMBER 2016 www.clinicalobgyn.com | 509 Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. 510 Wang et al In this review, we will not use the term ‘‘causes’’ of pregnancy loss as the only documented cause of pregnancy loss is lethal embryonal malformations often caused by chromosomal abnormality. Biomarkers significantly associated with pregnancy loss or RPL will instead be entitled ‘‘risk factors’’ reflecting our conviction that RPL in most instances is a multifactorial condition, which occurs only if a woman (or couple) has several risk factors.3 In this article, we will provide an overview of the scientific evidence for immune aberrationsinvolvedinthepathogenesisof RPL. We will discuss about which biomarkers related to immune function are candidates for further research or can already be used in clinical practice. Furthermore, we will review the current status of immunologic treatments used for patients with RPL. Proposed mechanisms of action and documentation of efficacy will be highlighted. Diagnostic Tests Research on immunologic biomarkers associated with RPL has focused on measurements of autoantibodies, natural killer (NK) cells in the blood or the uterus, cytokines in the blood or the decidual tissue, and investigations of classic and nonclassic human leukocyte antigen (HLA) polymorphisms in patients or couples with RPL. An assessment of the clinical value of these tests is provided in Table 1. AUTOANTIBODIES It has previously been shown that women with various autoimmune diseases have an increased risk of pregnancy loss, especially in early and late gestation.3 In systemic lupus erythematosus (SLE), this increased risk is associated with the presence of antiphospholipid antibodies (APLs) such as lupus anticoagulant (LAC) and anticardiolipin antibodies (ACA). APLs are also associated with an increased risk of venous and arterial thrombosis. APLs are often found in RPL patients without clinical signs of SLE and are associated with an increased thrombosis risk and a highriskoffurtherpregnancylosses.4 Only high titers of ACA, especially IgG, seem to be important. APLs are often classified as acquired thrombophilia factors due to the increased thrombosis risk. In this review, APLs will be considered primarily as a sign of breakage of the immunologic autotoleranceofthewoman.Thisisemphasizedby the observation of numerous other autoantibodies in many women with APLs. Clinical hyperthyroidism or hypothyroidism is strongly associated with the presence of thyroid autoantibodies. Thyroid autoantibodies are found in 5% to 15% of women of reproductive age, but in the majority of these cases, they are not associated with thyroid dysfunction. The most prevalent thyroid autoantibody is thyroid peroxidase antibody. A considerable number of studies have found an increased prevalence of thyroid autoantibodies in RPL patients and a metaanalysis of relevant case-control studies found that the antibodies are associated with RPL with an odds ratio (OR) of 2.3 (95% CI, 1.5-3.5).5 Antinuclear antibodies (ANA) can be detected in a series of autoimmune diseases including SLE. In a review from 1996,6 10 out of 12 case-control studies found an increased prevalence of ANA in RPL patients. In the 4 relevant studies published subsequently, 2 found a significantly increased prevalence of ANA in RPL patients compared with controls.7,8 However, in most studies, a positive ANA does no predict the pregnancy outcome. It remains to be elucidated whether the association between APLs, antithyroid antibodies, and ANA and RPL reflects a directly harmful impact of the autoantibodies on placental development and vascularization and embryonic development orwhethertheirpresenceisratheramarker www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. Immunology and Recurrent Pregnancy Loss TABLE 1. 511 Biomarkers With Putative Diagnostic Value in RPL Associated With RPL Biomarker Autoantibodies Antiphospholipid +++ antibodies Thyroid antibodies +++ Antinuclear antibodies + + Soluble immune biomarkers Peripheral blood + cytokines Mannose-binding lectin + + Immune cells Peripheral blood NK-cell + subsets Peripheral blood NK + cytotoxicity Endometrial NK cells ? Prognostic Value Needed Research ++ + ? Standardization of assays and cut-off values Prognostic studies in untreated patients Prognostic studies in untreated patients Prognostic studies in untreated patients ? More sensitive and reliable methods + More studies ? Establishment of reference values in different phases of the menstrual cycle or pregnancy Establishment of reference values in different phases of the menstrual cycle or pregnancy Establishment of reference values in different phases of the menstrual cycle Standardization of methods Suitable control samples (aneuploid missed miscarriages?) Establishment of reference values in different phases of the menstrual cycle Standardization of methods ? ? Decidual NK cells ? – Peripheral blood Treg cells ? ? + + More studies ? ? HLA class II ++ + HLA-C, HLA-G + ? Studies using up-to-date techniques Clear definition of criteria for allele sharing Larger studies Studies homogenous with regard to reproductive history and ethnicity Larger studies Studies homogenous with regard to reproductive history and ethnicity Genetic biomarkers Mannose-binding lectin gene polymorphism HLA sharing HLA indicates human leukocyte antigen; NK, natural killer; RPL, recurrent pregnancy loss; ?, undetermined. of an increased predisposition to breakage of immunologic autotolerance. A direct pathophysiological link between the presenceofautoantibodiesinRPLpatientsand fetal death has not yet been documented convincingly. The production of autoantibodies may be a result of RPL in geneticallypredisposedwomensinceintrauterine retention of necrotic tissue in case of a missed miscarriage may expose hidden (cryptic) autoantigens on fetal or trophoblast cells to the mother’s immune system. A known genetic predisposing factor is the HLA-DRB1*03 allele, which is associated both with the production of ACA, antithyroid antibodies, and ANA and with the risk of RPL.9,10 Screening for LAC, ACA should be undertaken in RPL patients primarily because there may be treatments available for these patients although there is some controversy. Screening for thyroid peroxidase antibodies, ANA, and anti-ds-DNA should be undertaken in RPL patients primarily because the presence of the antibodies impacts the prognosis negatively. It is also a sign of breakage of general autotolerance pointing toward an immunologic background for pregnancy losses. www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. 512 Wang et al CYTOKINES Cytokines are signal molecules secreted from immune cells (especially T lymphocytes), which bind to receptors on other cells, resulting in the stimulation or the inhibition of function. Cytokines are often categorized as either T-helper (Th) type 1 cytokines, which promote T-lymphocyte cytotoxicity and inflammation, and Th type 2 cytokines, which promote antibody production and decrease inflammation. Typical Th1 cytokines are tumor necrosis factor (TNF)-a, interferon (IFN)-g, and interleukin (IL)-2, whereas IL-4 and IL-10 are characteristic Th2 cytokines. An imbalance in Th1/Th2 cytokines has been associated with adverse pregnancy outcomes including RPL.11 Research into the role of cytokines in RPL is complicated by several factors: (1) levels and functions of cytokines change from day to day during implantation and early pregnancy; (2) blood lymphocyte cytokine secretion or cytokine concentrations in the blood may be completely different from that in the uterus; and (3) the measurement of cytokines in endometrial biopsies or washings or in decidual tissue is subject to technical and methodological difficulties. TNF-a is a potent proinflammatory cytokine and may be a good marker for systemic inflammation. High plasma TNF-a levels are reported to increase the risk of miscarriage in RPL patients,12 and high TNF-a and TNF-a/IL-10 ratios characterize women with euploid compared with aneuploid miscarriage.13 Also, lymphocytes from RPL patients who later go on to miscarry produce more TNF-a than those of women who have live births.14 Finally, patients with RPL after a birth (secondary RPL patients) have significantly higher plasma levels of TNF-a in earlypregnancythanpatientswithnoprior live birth (primary RPL patients).15 These observations suggest that a high systemic inflammatory state in early pregnancy increases the risk of miscarriage and that secondary RPL is particularly associated with a proinflammatory state. Plasma levels or the in vitro production of many cytokines are partly determined by polymorphisms in the genes encoding the cytokines and their promoters. Therefore, if abnormal cytokine profiles play a role in RPL, it may be reflected in the cytokine gene polymorphisms carried by the women. However, a review concluded that no cytokine gene polymorphism is convincingly associated with RPL.16 The measurement of cytokines in plasma or endometrial biopsies is in our view useful only in the context of research, and still has no place in clinical practice. MANNOSE-BINDING LECTIN (MBL) MBL is a plasma protein produced in the liver. Plasma levels exhibit large interindividualvariationsthataremainlygenetically determined. MBL is a part of the so-called lectin-dependent pathway of complement activation. Furthermore, by enhancing phagocytosis, MBL can aid in clearing apoptotic cells, debris, and immune complexes that would otherwise prompt inflammatory processes. The result of MBL deficiency in pregnancy may therefore be enhanced proinflammatory processes at the fetomaternal interface and is thus expected to increase the risk of miscarriage. Indeed, MBL deficiency was found to be associated with RPL in previous casecontrol studies17,18 and was also associated with a significantly poorer prognosis.18 Plasma levels of MBL are determined by polymorphisms in several loci of the MBL2 gene. Genetic polymorphisms associated with low (<100 ng/L) MBL levels have been reported with increased frequency in RPL patients and in particular in those experiencing unexplained late fetal death.19 The measurement of MBL levels or the related genetic polymorphism is still uncommon in the RPL setting, but we believe that there is sufficient evidence to consider it in the screening program as low levels are associated with a poor www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. Immunology and Recurrent Pregnancy Loss prognosis,18 and it is often a relief for the patientstobeinformedthattheyhavearisk factor for which they cannot be blamed. NK CELLS In the search for immunologic aberrations in RPL patients, there has been much focus on NK cells. NK cells are a part of the innate immune system, and in contrast to T lymphocytes, they can recognize and react against cells infected by intracellular microorganisms or cells undergoing malignant transformation without prior sensitization against target antigens. This reaction can result in the killing of the cells (cytotoxicity) or in the secretion of specific cytokines. There has been considerable interest in NK cells in RPL due to 3 observations: (1) the composition of NK cells in the endometrium and the decidual tissue is unique: >90% of the lymphocytes in the luteal-phase endometrium and early pregnancydecidualtissuearelow-cytotoxicity, high-cytokine-producing NK cells carrying a high density of the CD56 surface marker (CD56bright),20 whereas in the peripheral blood, almost all NK cells carry the CD16 surface marker, which is associated with high cytotoxicity and low cytokine production; (2) the major HLA molecules expressed on trophoblast subsets, HLA-G and HLA-C, can act as ligands for the 3 kinds of activating or inhibitory receptors found on NK cells in the uterus21,22; and (3) studies on NK-celldeficient and T-cell-deficient transgenic mice with a high fetal loss rate show that therestorationofNKcellsbybonemarrow transplantation results in a normal fetal loss rate.23 Investigations of NK cells in RPL can be divided into flow-cytometric analyses or tests of NK cell cytotoxicity of peripheral blood lymphocytes before or during pregnancy and studies of NK cells in prepregnancy endometrial biopsies or in decidual tissues from missed miscarriages and elective abortions. 513 Because of the easy availability, studies on the peripheral blood are dominant. The majority of the larger studies found that the percentage of CD56+ cells in the peripheral blood taken before pregnancy is significantly higher in RPL women than in controls.24–27 Other studies did not confirm this observation.28,29 A flaw in many of the studies is that most of the women with RPL women were nulliparous and most controls were multiparous.30 A previous successful pregnancy can induce permanent changes in lymphocyte subsets including NK cells.31 Most of the studies on prepregnancy blood samples26,32–34 found significantly increased NK-cell cytotoxicity in RPL patientscomparedwithcontrols.Onelarge study, however, did not find such a difference.35 Aoki et al36 reported that RPL patients with high peripheral blood NK-cell cytotoxicity before pregnancy had a significantly higher rate of miscarriage in the next pregnancy compared with those with lower NK-cell cytotoxicity (71% vs. 20%). Subsequent smaller studies found higher or similar NK-cell cytotoxicity in patients with a subsequent euploid miscarriage compared with those with a live birth. Importantly, in a recent large prospective study, high NK-cell cytotoxicity before pregnancy had no impact on subsequent miscarriage rates after adjustment for recognized risk factors for miscarriage.37 It has been questioned whether endometrial NK-cell subsets reflect those in the peripheral blood. This is due to the fact that frequencies of NK-cell subsets in the endometrium and the peripheral blood are extremely different. A series of studies investigated NK cells in endometrial biopsies taken in nonpregnant cycles by immunohistochemistry or flow cytometry of homogenized tissue. The former technique is prone to subjective evaluation and the latter is flawed because the tissue undergoes enzymatic digestion, which influences marker expression.Furthermore,endometriallymphocyte www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. 514 Wang et al numbers fluctuate hugely in the the menstrual cycle, and therefore the exact timing of biopsies is extremely crucial,38 but this has rarely been reported. Lachapelle et al39 found that the CD56bright NK-cell subset was significantly lower in RPL patients than in control, whereas other studies40–42 found that the frequency of CD56+ (or unspecified NK cells) cells was significantly higher in RPL than in controls. In 2 studies,43,44 no difference was found in NK-cell subsets in the endometrium between RPL patients and controls. Importantly, no relationship between the CD56+ NK-cell count in the endometrium and the subsequent pregnancy outcome was found in a study by Tuckerman et al.42 Some studies compared NK-cell subsets in the decidual tissue from missed miscarriages of RPL patients with normal women having an elective termination and reported differences in the NK-cell composition between the 2 groups.45,46 As the tissue in the former case is often necrotic and inflamed and the tissue in the latter is fresh and vital, these studies provide limited valid information. Because peripheral blood NK-cell subsets do not reflect conditions in the uterus and the largest studies found no prognostic significance of NK-cell cytotoxicity, the benefitofmeasuringperipheralbloodNKcell parameters in clinical practice is limited and it cannot be used to select RPL patientsfor immunologictreatments. The measurement of uterine NK cells, although in theory a better approach, is also unfit for clinical practice due to methodological problems. T-REGULATORY (TREG) CELLS The fetoplacental unit is often entitled the fetoplacental allograft because it is similar to the transplantation of an organ from an allogeneic donor. In case of a transplantation, the allograft can avoid rejection only by intensive immunosuppressive therapy. A priori, it must be presumed that the maternal immune system would make efforts to reject the fetoplacental unit that carries paternal alloantigens. One of the mechanisms thought to be important for establishing immunologic tolerance to alloantigens and autoantigens and the fetoplacental allograft is Treg lymphocytes. After being activated by tolerogenic antigen-presenting cells (APCs), Tregs can suppress the formation and the effector function of T-cell-mediated immune responses that could otherwise induce alloimmunity and autoimmunity and rejection of pregnancy tissues. Studies on T-cell-deficient transgenic mice strains demonstrate that lymphocytes with the Treg phenotype CD4+ , CD25+ , Foxp3+ are important for implantation and successful pregnancy in allogeneic crossings.47 In an elegant study, the elimination of CD4+ , CD25+ cells induced fetal resorption in allogeneic, but not in syngeneic, mated mice.48 The role Tregs play in human pregnancy and especially in RPL is still not clarified as relevant studies are small and sparse. Kwiatek etal49 recentlyreportedthatthepercentage of Tregs in the peripheral blood at the time of miscarriage is significantly lower in women with RPL than in women with normalpregnanciesatthesamegestational age. In women without RPL, those who miscarried had significantly lower frequencies of CD4+ , CD25+ cells in the peripheral blood and decidual tissue than those with normal pregnancies.50 A hypothesis that integrates data from animal and human research regarding the role of Tregs in normal and adverse pregnancy is intriguing.51 Increasing plasma estrogen levels in the follicular phase cause the Treg pool in the blood or regional lymph nodes to expand. It also causes increased uterine expression of chemokines, resulting in the recruitment of T cells to the uterus. Male antigens and cytokines in the seminal fluid in the vagina recruit tolerogenic APCs to the uterus and regional lymph nodes. Here, the APCs activate local Tregs that suppress harmful www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. Immunology and Recurrent Pregnancy Loss T-cell immunity toward alloantigens on the embryo and trophoblast. The hypothesis is attractive because it introduces adaptive cellular immunity to the pathogenesis of adverse pregnancy outcomes such as RPL. In contrast to NK-cellmediated immunity, an important feature of adaptive immunity is immunologic memory, which is stored in memory T cells. Clinical observations such as the traditionally claimed rarer occurrence of preeclampsia in a second pregnancy with the same husband or the negative prognostic impact of a first-born boy in women with secondary RPL52 can potentially be explained by mechanisms where tolerance or harmful immunity has developed in the first ongoing pregnancy and is remembered by memory T cells. The measurement of Tregs in adverse pregnancies in humans is still in its infancy, and should for the time being be undertaken only in the context of research. Although there is significant potential in measuring Tregs, there is a severe obstacle for truly evaluating their impact on the pregnancy outcome. Subsets of most importance are probably (as in mice) located in regional lymph nodes draining the uterus, which are difficult to access in humans. IMMUNOGENETIC BIOMARKERS All proteins directly participating in immune interactions or expressed on immune cells are coded by genes that are often polymorphic. This may occur as singlenucleotide polymorphisms or as small additions or deletions of chromosomes that may include several genes, termed copy-number variations (CNVs). Such polymorphisms may give rise to decreased orincreasedproteinproduction,whichcan lead to alterations in immune interactions that can sometimes lead to disease. Using genome-wide screening, gene alterations that affect gene function can be assigned to specific immunologic or metabolic pathways. In a recent study, CNVs 515 that rearrange genes in pathways of ‘‘innate immune signaling,’’ ‘‘complement cascade,’’ or ‘‘interaction of Fc g receptors with antigen-bound IgG’’ were found more often in RPL patients than in fertile controls.53 The result was highly significant. This suggests that genetic disruption of the immune function may be important in the pathogenesis of RPL. Genome-wide screening studies are important for research purposes, but their value is still limited when assessing the backgroundforRPLinindividualcouples. The investigation of specific genetic polymorphisms of importance for immune function with a documented impact on the risk of RPL or new pregnancy loss after RPL could theoretically be useful. However, do such genetic polymorphisms exist? In a previous section, we discussed the association between genetic polymorphisms in the MBL2 gene associated with MBL deficiency and RPL. This association has been documented in several independent studies. We believe that investigation of MBL genetics will be potentially helpful, especially in cases with second-trimester RPL or extreme preterm birth.19,54 Numerous studies have investigated HLAs and RPL. The HLA region comprises several genetic loci located on chromosome 6 and it contains the most polymorphic genes known in humans. Depending on the genetic distance between the various HLA loci, the alleles of the genes in each locus display a stronger or weaker linkage disequilibrium. This means that alleles in different loci are inherited together more or less often than expected by chance. This is an important concept when studies of HLA polymorphisms in RPL and other disorders are evaluated. HLA molecules play an important role in the adaptive and the innate immune system. In the adaptive immune system, CD8+ T lymphocytes can exert cytotoxic www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. 516 Wang et al reactions against cells carrying class I HLA molecules, especially HLA-A and HLA-B, both of which play an important role in transplantation immunology. Class II HLA molecules (HLA-DR and HLADQ) carried primarily on APCs present antigenic peptides on Th (CD4+) lymphocytes, which can initiate both humeral and cellular immune reactions. An individual’s 2 sets of class II HLA alleles determine the repertoire of antigens that he/she can be immunized against easily. Class II alleles are therefore (sometimes strongly) associated with most autoimmune diseases as these diseases are caused by adverse reactions against self-antigens. NK cells, which belong to the innate immune system, react against all cells not carrying HLA molecules. They can also react against or interact with cells carrying specific HLA molecules; NK receptors can be inhibited or activated by HLA-C and HLA-G ligands. Because of the different ways in which HLA can influence immune reactions, studies of HLA in RPL can be divided into 3 main categories: studies of HLA allele incompatibility (sharing) between partners with RPL; studies of HLA allele prevalence in women with RPL; and studies of HLA-C and HLA-G alleles in couples with RPL. All 3 kinds of studies have been evaluated in a recent metaanalysis.55 Increased HLA compatibility between spouses was originally thought to decrease the probability of the mother adequately reacting immunologically to the fetus and producing so-called blocking antibodies. The meta-analysis55 reported that allele sharing in the HLA-B, HLA-DR, and HLA-DQ loci was more frequent in RPL than in control couples. However, the results should be interpreted with caution due to the obsolete serological techniques used for HLA determination in most of these older studies. Studies of HLA allele frequencies in RPL patients and controls have focused on alleles in HLA class II loci: HLA-DRB1 or HLA-DQB1. A meta-analysis of relevant case-controlstudies,allusingmodernPCR techniques, found that HLA-DRB1*04 and HLA-DRB1*15 alleles were significantly increased in RPL patients. HLADRB1*03 also tended to be found more often in patients than in controls.55 However, we are convinced that HLADRB1*03 may be the strongest HLA class II RPL susceptibility allele in whites. In a large case-control study,10 which was only partly included in the meta-analysis, we found that this allele was highly significantly increased in RPL patients. Furthermore, the prevalence increased with an increased number of previous miscarriages. A reason why the HLA-DRB1*03 allele was not significantly increased in RPL patients in the meta-analysis55 might be that 4 of the included studies were in Japanese women. In Japan, the HLADRB1*03 allele is very rare. Studies of associations between HLA polymorphisms and disease susceptibility should always be restricted to specific ethnic groups. Studies assessing the impact of maternal carriage of specific HLA class II alleles on future pregnancy outcomes provide information that can be useful in counseling regarding the prognosis. Among patients with RPL after a birth (secondary RPL), the birth of a boy before the miscarriages is significantly more prevalent than the birth of a girl (61% vs. 39%). Patients with a first-born boy also prospectively exhibit a significantly lower chance of a second live birth.52 Among RPL patients with a first-born boy, maternal carriage of one of the alleles, HLA-DRB1*15, HLADQB1*0501/2, and HLA-DRB3*0301, reduces the chance of a live birth by 22% compared with patients not carrying these alleles.56 These alleles (HY-restricting class II HLA alleles) are known to present peptides derived from male-specific proteins (HY antigens) to Th cells. In transplantation immunology, carriage of the www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. Immunology and Recurrent Pregnancy Loss alleles predisposesonetograft-versus-host disease after sex-mismatched bone-marrow transplantation. On the basis of these data56 and supported by the study of antiHY antibodies in RPL patients,57 our hypothesis is that Th lymphocytes from women carrying HY-restricting class II HLAalleles mayrecognizeHYantigenson the fetoplacental unit in their first ongoing pregnancy with a boy. This initiates a series of harmful immune reactions targeting the fetoplacental unit in the subsequent pregnancies, ultimately leading to RPL. More research confirming this mechanism of RPL is still needed. In the section about NK cells, we discussed the relationship between specific KIR receptor polymorphisms, HLA, and RPL. HLA-C alleles can be divided into C1 and C2 groups according to a genetic dimorphism of the segment of the HLAC molecule that can bind KIRs. C1 allotypes are ligands that inhibit KIR2DL2/3 and activate KIR2DS2, whereas C2 allotypes are ligands for the inhibitory KIR2DL1 and activating KIR2DS1. Hiby et al58 reported that the combination of a woman carrying the KIR genes that are primarily inhibitory and a man carrying C2 allotypes is more frequent among RPL than among control couples. It was suggested that this combination of mainly inactivating KIR genes in a woman and their ligands in the parents results in decidual NK-cell inhibition. This may lead to insufficient secretion of specific cytokines at the fetomaternal interface, resulting in defective trophoblast proliferation and invasion and subsequent RPL. In contrast, another large study found that maternal carriage of the inhibitory KIR2DL1 in combination with C2 homozygosity in both partners was found significantly more often in controls than in RPL women, whereas maternal carriage of the activating KIR2DS2 in conjunction with C1 homozygosity in partners was found significantly more often in RPL patients.59 Thus, the 2 studies yielded 517 completely opposite results regarding the role of maternal KIR genes and parental HLA-C allotypes. In several studies, no association between parental C2 allotypes and RPL was detected.55,60 Another set of studies have investigated HLA-G polymorphisms inRPL.HLA-Gis a nonclassic HLA gene, which exhibits much less polymorphism than classic HLA genes. It is highly expressed in extravillous trophoblast cells in contrast to all other HLA genes. It can also be detected in plasma in the form of soluble HLA-G. In RPL research, there has been considerable interest in a 14-base-pair-long insertion/ deletion dimorphism in exon 8 of the HLAG gene, which may affect the transcription of the gene. There seems to be an association between low levels of soluble HLA-G in the plasma and homozygosity for the HLA-G14 bp insertion.61 Soluble HLA-G can modulate NK-cell function in vitro.62 Two recent meta-analyses found that the HLA-G14 base-pair insertion frequency was significantly increased in RPL with ORs 1.27 (1.04-1.55) and 1.47 (1.13-1.91), respectively.63,64 As the HLA-G14 basepair insertion is in strong positive linkage disequilibrium with the HLA-DRB1*03 allele,65 it remains unclear as to whether the HLA-G gene insertion or HLA-DRB *03 is the main RPL susceptibility gene. More research into the role of classic and nonclassic HLA genes in RPL should be carried out. As the association between the pregnancy outcome and HLA polymorphisms in women is weak and results of KIR/ HLA investigations are indecisive, HLA determination in couples with RPL still has no place in clinical practice. An exception may be women with RPL after a first-born boy, where HLA class II investigations may be useful when counseling patients. Immunologic Treatments As reviewed in previous sections, there is considerable evidence that immune aberrations play a role in the pathogenesis of www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. 518 Wang et al RPL. As a consequence, it is logical to test various immunologic treatment options in these patients. In this article, we focus on treatments directly targeting immune factors or interactions such as allogeneic lymphocyte immunization (ALT), intravenous immunoglobulin (IvIg), prednisone, and growth factors. In contrast, we will not evaluate heparin, aspirin, and progesterone, which are also used in RPL treatment and may have immunomodulatory properties. These treatments are reviewed elsewhere in this symposium. Immunomodulatory treatments have been tested in RPL patients selected (1) due to RPL and positive tests for 1 or more immunologic biomarkers suggested to identify patients with a specific immune etiology and (2) RPL, but negative testing for immunologic and thrombophilia biomarkers (referred to as idiopathic RPL). It is important to note that the chance of live birth without any treatment is at least 55%after3and46%after4miscarriages.66 Accordingly, success rates for treatments should be substantially above these percentages to prove efficient. Furthermore, TABLE 2. it cannot be ruled out that any intervention will improve the pregnancy success rate due to a placebo effect. Therefore, only prospective studies with random allocation to treatment against placebo (optimal) or no treatment or treatment as usual (less optimal) are informative, and only such studies will be reviewed here (Table 2). Several treatment studies have been performed in RPL patients selected due to positivity for autoantibodies or high NK-cell numbers, but only 2 were randomized, including a placebo-treated group. Laskin et al67 carried out a large placebo-controlledRCTofprednisone andlowdose aspirin in women with RPL and positivity for ACA, LAC, ANA, antiDNA, or antilymphocyte antibodies. This is a subset of patients with serological signs of breakage of immune tolerance, and they may, in theory, benefit the most from immunomodulation therapy. A 9% higher live birth rate was found in the treatment group, but this was not significantly different from that of controls. The treated patients had a significantly higher risk of preterm birth (62% vs. 12%) and a higher risk of diabetes and hypertension. The Immunologic Treatments Tested in RPL Treatment EffectonLiveBirth Rate Adverse Effects Suggested Research Prednisone Nonsignificant increase Allogeneic lymphocyte therapy Nonsignificant increase Large studies administering moderate doses of prednisone Better studies with attention toward methodology and adverse effects. Must include long-term followup Intravenous immunoglobulin G-CSF Preterm birth, diabetes, and hypertension Neonatal alloimmune thrombocytopenia Production of red blood cell antibodies Transfer of infectious agents Hematological malignancies? Borderline significant Headache and skin rash increase in secondary RPL Significant increase Flu-like symptoms Leukocytemia More studies especially in secondary RPL More studies G-CSF indicates granulocyte colony-stimulating factor; RPL, recurrent pregnancy loss. www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. Immunology and Recurrent Pregnancy Loss authors therefore concluded that prednisone should not be given to RPL patients. It is worth remembering that very high doses of prednisone (40 to 50 mg/d) were used for the duration of the pregnancy, which explains the high rate of preterm birth. In an RCT of RPL patients selected due to endometrial NK-cell density Z5%, Tang et al68 found a 20% higher live birth rate in patients allocated to 8 weeks of prednisone in early pregnancy compared with placebo. This was, however, not significant due to small numbers. Although prednisone did not increase the live birth rate significantly, the 2 RCTs point toward a potential beneficial effect. A new study administering lower doses of prednisone to RPL patients before pregnancy and in the first trimester (optimally selected due to the presence of biomarkers suggesting immune activation) should be carried out. A larger number of RCTs have been conducted in RPL patients with idiopathic RPL. Two immunomodulatory treatments, ALT and IvIg, have been tested in a sufficient number of RCTs to allow the performance of systematic reviews and meta-analyses. The theory for using ALT was that RPL patients lack antipaternal antibodies or blocking antibodies that protect the fetus against rejection. Therefore, ALT and the subsequent production of these antibodies could be beneficial.69 The updated Cochrane systematicreviewontheefficacyofALTfound the OR for live birth in ALT-treated patients to be 1.23 in RCTs using paternal lymphocytes and 1.39 in RCTs using third-party lymphocytes compared with placebo.70 Combining results from all ALT trials irrespective of the source of the lymphocytes, the OR for live birth after ALT was 1.34 (95% CI, 0.83-2.15).71 Many of the included RCTs do not meet the current criteria for methodological quality, the impact of antipaternal antibodies is unclear,72 and, last but not least, treatment with allogeneic cells raises serious safety concerns. There is a 519 substantial risk of neonatal alloimmune thrombocytopenia and production of red blood cell antibodies,73 some risks of transferring infectious agents, and in theory an increased long-term risk of hematological malignancies. Therefore, we cannot recommend that ALT should be used in clinical practice. If further RCTs on ALT are carried out, they should be conducted using strict methodological rigor, precautions to diminish risks of adverse alloimmunization, and they should include long-termfollow-up of mothers and babies. IvIg is known to reduce symptoms in many autoimmune and inflammatory diseases. IvIg has been tested in 11 RCTs involving idiopathic RPL patients; 9 of them comparing IvIg with placebo and 2 RCTs comparing IvIg with other treatments. The most recent systematic review and meta-analysis of IvIg in RPL74 included all relevant RCTs and found in all included patients an RR of 0.92 (95% CI, 0.75-1.12) for no live birth after IvIg. In patients with secondary RPL, a subset that in previous RCTs seemed to benefit the most from IvIg,75 the RR for no live birth after IvIg was 0.77 (0.58-1.02; P = 0.06), which can be translated into a borderline significant benefit of IvIg in secondary RPL. IvIg is not labeled for use in RPL and must still be considered investigational for the purpose. Adverse events such as headache and skin rash were more frequent in IvIgtreated patients compared with placebotreated patients. There was no difference in the incidence of severe adverse events. A trial sequential analysis in the review concluded that even with meta-analyses, studies are underpowered. More RCTs are needed to make definitive conclusions about the efficacy of IvIg in RPL. Given the expense and the potential for adverse events, IVIG cannot be recommended for clinical use in women with RPL. Growth factors such as granulocytemacrophage colony-stimulating factor and granulocyte colony-stimulating factor www.clinicalobgyn.com Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved. 520 Wang et al (G-CSF) may promote trophoblast growth and improve the pregnancy outcome. One small RCT tested the efficacy of G-CSF injections before pregnancy and in early pregnancy in patients with 4 or more miscarriages, with a pregnancy loss despite IvIg treatment.76 There was a significantly increased live birth rate in the G-CSFtreated patients compared with placebo. OthersimilarRCTsareneededtoassessthe efficacy of this treatment before implementation in clinical practice. Conclusions Overall, there are 3 main arguments for immunologicdisturbancesplayingarolein RPL: (1) the general knowledge that whenever allogeneic tissue is introduced into an organism, immune reactions develop, and if not abolished, rejection will occur; (2) the observation that a series of autoimmune diseasesandautoantibodiesarefoundwith increased prevalence in RPL; and (3) the observation that women with RPL have an increased risk of atherosclerotic cardiovascular diseases probably attributable to a chronic proinflammatory state.77,78 In our view, not one but several disturbances or disruptions of pathways relatingtoimmuneinteractionscontributeto many cases of RPL.3 The reproductive process is too importantto be vulnerableto the disruption of only 1 immunologic pathway. We think that disturbances in severalimmunepathways(causedbySNPs or CNVs) in conjunction with immunizing events in previous pregnancies, such as a substantial transfer of fetal antigens (cells) into the maternal circulation, can promote proinflammatory reactions or breakage of autotolerance. This will predispose one to RPL and other adverse pregnancy outcomes and ultimately lead to atherosclerotic or autoimmune disease.77,78 The complexity of the pathogenesis of RPL is a challenge for future research in the area. It is further complicated by the fact that almosthalfofallmiscarriagesinRPLwomen are due to embryonal aneuploidy. We find it importantthatresearchersacknowledgethis complexity when carrying out future research on immunologic and nonimmunologic risk factors of RPL. It may also help if the management of RPL is centralized in a few dedicated clinics to improve patient care and possibilities for research. References 1. Jauniaux E, Farquharson RG, Christiansen OB, et al. 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