REVIEW ARTICLE
Immunological Modes of Pregnancy Loss Joanne Kwak-Kim1,2, Joon Cheol Park1,3, Hyun Kyong Ahn1,4, Joon Woo Kim5, Alice Gilman-Sachs2 1
Reproductive Medicine Program, Department of Obstetrics and Gynecology, The Chicago Medical School at Rosalind Franklin University of Medicine and Science, Vernon Hills, IL, USA; 2 Department of Microbiology and Immunology, The Chicago Medical School at Rosalind Franklin University of Medicine and Science, Vernon Hills, IL, USA; 3 Department of Obstetrics and Gynecology, School of Medicine, Keimyung University, Daegu, Korea; 4 Department of Obstetrics and Gynecology, Cheil General Hospital & Women’s Healthcare Center, Kwandong University, College of Medicine, Seoul, Korea; 5 Divison of Rheumatology, Department of Medicine, Feinberg School of Medicine, North Western University, Chicago, IL, USA
Keywords Coagulation, implantation, NK, pregnancy loss, TH1 ⁄ TH2 Correspondence Joanne Kwak-Kim, MD, Director, Reproductive Medicine, Associate Prof. Department of Obstetrics and Gynecology Associate Prof. Department of Microbiology and Immunology, The Chicago Medical School at Rosalind Franklin University of Medicine and Sciences 830 West End Court, Suite 400, Vernon Hills, IL 60061, USA. E-mail:
[email protected] Submitted February 26, 2010; accepted March 1, 2010. Citation Kwak-Kim J, Park JC, Ahn HK, Kim JW, Gilman-Sachs A. Immunological modes of pregnancy loss. Am J Reprod Immunol 2010; 63: 611–623
During the implantation period, a significant portion of embryos are lost and eventually less than half of clinically established pregnancies end as full-term pregnancies without obstetrical complications. A significant portion of these pregnancy losses is associated with immune etiologies, including autoimmune and cellular immune abnormalities. Although an autoimmune etiology such as anti-phospholipid antibodies (APAs) has been reported to induce placental infarct and thrombosis at maternal– fetal interface, APAs induce inflammatory immune responses as well. Inflammatory immune responses, such as increased proportions of NK cells and Th1 ⁄ Th2 cell ratios in peripheral blood are related to recurrent pregnancy losses and multiple implantation failures. Systemic and local inflammatory immune responses seem to be induced by activation of Toll-like receptors with infectious agents, fetal cell debris, or gonadotropin-releasing hormone agonist, etc. Cellular activation of T and NK cells leads to pro-inflammatory cytokine storm and consequently, placental infarction and thrombosis. Potential application of anti-inflammatory therapeutic agents for the prevention of pregnancy losses should be explored further.
doi:10.1111/j.1600-0897.2010.00847.x
Introduction Human pregnancy is a quite inefficient process. When pregnancy loss was examined in 200 fertile couples, the maximal fecundity rate was approximately 30% per cycle in the first two cycles and this rate quickly tapered over with continuous trial.1 Even after a successful implantation, approximately 31% of pregnancies were reported to be miscarried.
A significant proportion (41%) of these pregnancy losses are reported to be occult losses. Prevalence of occult losses was reported to be 41–70% depending on the timing and sensitivity of pregnancy test.1,2 In in vitro fertilization (IVF) pregnancies, 51.4% of embryo transfers resulted in embryo implantation. Of these implantations, 33.7% resulted in pre-clinical pregnancy loss, 3.7% in biochemical pregnancy, and 14.9% in clinical miscarriage, whereas the other
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implantations (47.7%) resulted in ongoing pregnancies.3 Therefore, during the implantation period, a significant portion of pregnancies are already lost. The frequency of clinically recognized spontaneous abortion in the general population has been estimated to range between 15 and 20%.4 From cytogenetic studies of aborted products of conception, 50–60% of those pregnancies lost in the first trimester are owing to chromosomal abnormalities. In general, sporadic chromosomal errors account for approximately 30% of spontaneous pregnancy losses.4 Most of the chromosomal aberrations involved in spontaneous abortions have been presumed to be because of random events that are not necessarily repetitious. Although the expected prevalence of three or more recurrent pregnancy losses (RPL) is 0.3%, actual prevalence of RPL has been reported to be higher than expected prevalence. Approximately 1–2% of population will experience RPL three or more times, and typically these losses happen during the first trimester.5 By 20 weeks of gestation, approximately 50–60% of clinically established pregnancies remain as ongoing pregnancy.6 Even after 20 weeks of gestation, about 3.4% of all pregnant mothers past 20 weeks of gestation ended up in delivering a stillbirth; another 4.4% of the live births died before discharge from hospital. Thus, 7.9% of pregnancy losses occur after 20 weeks.7 In addition, the prevalence of low birth weight, preterm birth, and intrauterine growth restriction (IUGR) was reported to be 9.1%, 8.0%, and 8.9% respectively, and can lead to unfavorable pregnancy outcome.8 Therefore, less than half of clinically established pregnancies end as full-term pregnancies without obstetrical complications. Whether pregnancy losses or obstetrical complications occur early or late in gestation, or are a random or recurrent event, these losses instigate physical and psychological strain on affected couples and can be a major socioeconomical impact in our society. Immunological factors associated with implantation At the time of embryonic implantation, a successful pregnancy requires two major elements, a quality embryo and endometrial receptivity. Blastocysts secrete various cytokines, chemokines, and other immunologic factors, such as soluble HLA-G. HLA-G plays an important role in pre-implantation embryo
development.9 It was reported that HLA-G has a modest diagnostic predictability to achieve clinical pregnancy in women undergoing infertility treatment.10 Interestingly, the diagnostic odds ratio (12.67; 95% CI, 3.66–43.80) was even better with good quality embryos. Human embryos secrete ICAM-1 and IL-1ra commonly, but IL-6, IL-7, IL-8, IL-9, IL-13, eotaxin, IP-10, MCP-1 (MCAF), MIP-1b, PDGF-BB, RANTES, and VEGF in various degrees. However, IL-1b, IL-2, IL-4, IL-5, IL-10, IL-12 (P70), IL-15, IL-17, bFGF, G-CSF, GM-CSF, IFN-c, MIP-1a, tumour necrosis factor alpha (TNF-a) were not present or undetectable in the supernatants of embryo cultures.11 Although the embryo itself does not produce TNF-a, the number of growing follicles or embryos has been reported to be negatively associated with maternal serum TNF-a levels in women with ovulation induction.12 Hence, immunological factors play a major role in embryonic development and implantation. Endometrial receptivity is related to multiple factors including immunological factors, cytokines, chemokines, endometrial luminal changes, hormones, and growth molecules, etc.6 Hormonal challenge, such as ovarian stimulation with GnRH analogs, alters these factors and consequently, endometrial receptivity for embryonic implantation. Significantly higher concentrations of IL-1b, IL-5, IL-10, IL-12, IL-17, TNF-a, heparin-binding epidermal growth factor (HbEGF), eotaxin, and dickkopf homologue-1 are present in endometrial secretions obtained in stimulated compared with natural cycles.13 Implantation is associated with MCP-1 (P = 0.005) and IP-10 (P = 0.037) levels (negative and positive association, respectively) in endometrial secretions, and clinical pregnancy is significantly associated with IL-1b (P = 0.047) negatively and TNF-a (P = 0.023) levels positively.3 Indeed, the ability to predict clinical pregnancy by IL-1b and TNF-a levels in endometrial secretions was similar to the predictive value of embryo quality.3 It has been suggested that a delicate, complex, and stage-specific cytokine equilibrium is involved in the tissue remodeling that controls uterine receptivity and that the Th1 ⁄ Th2 cytokine ratio is one component of that balance.14 Dysregulated cytokine milieu is related to the cellular changes in endometrium. In mouse models, IFN inducers, such as IL-12, IL-15, and IL-18, are required to allow the proper activation of uterine NK (uNK) cells and control the angiogenic process American Journal of Reproductive Immunology 63 (2010) 611–623
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but are deleterious if lacking or in excess.15,16 Women with implantation failures had significantly elevated CD56 bright uNK cells when compared to normal controls and the number of CD56 bright uNK cells strongly correlated to the ratio of IL-18 ⁄ IL-18BP mRNA (r = 0.52, P < 0.0001) and IL-15 ⁄ actin mRNA (r = 0.42, P < 0.01). IL-18 was itself correlated with IL-15 and IL-12, suggesting a local control of uNK cells activation and recruitment.17 Therefore, an endometrial cytokine balance demonstrates distinct immune-related mechanisms that are involved in the broader context of inadequate uterine receptivity. These data support the notion that some implantation failures or RPL may be related to distinct local or systemic dysregulation patterns in the immune network and enhancement of endometrial receptivity via cytokine manipulation may further improve the implantation rate and reproductive outcome. Current clinical practice for infertility or RPL treatment mainly focuses either on generation of quality embryos or genetically normal embryos with ovulation induction and ⁄ or artificial reproductive technologies. However, minimum attention is paid to either endometrial receptivity or immunological factors related to embryonic and endometrial development. Further studies are needed to develop diagnostic and therapeutic approaches for the enhancement of local and systemic immunological network for embryonic and endometrial development. Inflammatory immune process and pregnancy outcome Multiple etiologies for RPL have been reported including chromosomal-6%, anatomic-1%, hormonal-5%, immunologic-65%, and unexplained-23%.18 In a recent study of Ford et al., approximately 20% of women with RPL have autoimmune etiologies and 50% of them were reported to be unknown including non-anti-phospholipid antibody-related thrombophilic tendencies.19 Therefore, approximately 65–70% of RPL can be related to immunological etiologies. Previously, we have reported that 37.1% of women with RPL have elevated peripheral blood natural killer (NK) cells.20 In addition, women with RPL or multiple implantation failures (MIF) have significantly elevated Th1 ⁄ Th2 cytokine-producing cell ratios in the peripheral blood when compared to normal controls.21 Immunopathological evaluation of the placental implantation site that terminated in a
spontaneous abortion during the first trimester revealed elevated CD57+ NK cells at the implantation site in 29.6% (P = 0.030) and thromboembolism in decidual vessels in 33.9% of cases (P = 0.025).22 Furthermore, second-trimester pregnancy losses are strongly associated with placental inflammation, and histologic abruption is likely another etiology.23 Therefore, local and systemic inflammatory processes and coagulation seem to play a major role in RPL. Inflammation is a process by which tissues respond to various insults such as ‘infectious agents’ or ‘danger signals’. It is characterized by up-regulation of chemokines, cytokines, and pattern recognition receptors that sense microbes and tissue breakdown products.24 During an implantation, an inflammatory microenvironment is required for tissue remodeling.14 Contrarily, the second trimester is the period of uterine immune senescence, and pro- and anti-inflammatory signals are in a constant state of equilibrium. During parturition, a partial immune suppression is needed. An inflammatory milieu is again elicited to induce events leading to parturition.25 An untimely manifestation or the sudden onset of abnormal inflammatory events necessary for implantation or natural parturition may initiate and intensify the cascade of inflammatory cytokine production involved in adverse pregnancy outcomes, such as implantation failure, pregnancy loss, preeclampsia, preterm labor, IUGR, and fetal inflammatory syndrome.21,26–29 Infection initiates inflammatory immune responses. During a pregnancy, microbes are introduced via transvaginal, transperitoneal, or transplacental routes. Fifteen percentage of the first-trimester losses and 66% of second-trimester losses are attributable to reproductive tract infections. Bacterial vaginosis, Listeria monocytogenes, Chlamydia trachomatis, and syphilis are the most common bacterial pathogens or infections associated with spontaneous abortion.30 Inflammation can be initiated with non-infectious conditions. ‘Danger signals’ such as tissue debris or fetal DNA can initiate inflammatory immune responses at the maternal– fetal junction.31,32 Pre-existing maternal autoimmune diseases, environmental chemicals, or nutritional insults can also initiate or aggravate inflammatory immune responses.33–35 What have we learned from animal models? The CBA ⁄ J · DBA ⁄ 2 mating combination provides a mouse model for the rejection of the semi-allogeneic fetoplacental unit.36 The abortion was thought to be
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mediated by GM1+ natural effector cells and activated macrophages.37,38 When CBA female mice mated to DBA ⁄ 2 males were treated with either polyinosinic ⁄ cytidylic acid (poly(I:C)) to boost NK activity or rabbit anti-asialo GM1 (RaASGM1) to decrease NK activity, a significant increase in aborted embryos after poly(I:C) and a marked decrease in spontaneous abortions after RaASGM1 treatment were reported. This finding suggested that spontaneous abortions may be mediated in part by the cytotoxic activity of unregulated NK cells.37 TNF-a, IFN-c, and interleukin-2 (IL-2) can, in some circumstances, increase fetal resorption rates in abortion-prone (CBA ⁄ J · DBA ⁄ 2) and non-abortion prone (CBA ⁄ J · BALB ⁄ c,C3H · DBA ⁄ 2) matings.39 Cytokines of the CSF family, including IL-3 and GM-CSF, increased the chances of fetal survival when injected into abortion-prone mice and also increased fetal and placental weight. The latter observations may be because of a direct tropic influence on placental cells, perhaps through a cytokine cascade, or an indirect effect because of inhibition of NK-like cells, or both.39 In mixed lymphocyte–placenta reactions (MLPR), the extent of stimulation of maternal strain lymphocytes in response to stimulator placental cells was much higher in the normal mating combination compared with the abortionprone mating combination. Cytokine analysis of the supernatants from MLPR indicates that there is significantly higher production of TNF-a, IFN-c, and IL-2 in supernatants from the abortion-prone combination than in supernatants from the normal combination.40 As earlier experiments established that products of activated macrophages (TNF-a and nitric oxide) were implicated in embryo loss in this model, the augmented infiltration of the decidua with maternal F4 ⁄ 80+ macrophages is an early event that precedes spontaneous abortion of the early embryo.38 In addition to innate immune cells, T cells were also reported to play a major role in this model. Th1 cytokine production has been attributed to the asialo NK cells and Vgamma1.1delta6.3+ T cells -GM1+ that infiltrate the decidua by day 6.5, during the peri-implantation period. Abortions can be prevented by a second population of V gamma 1.1 delta 6.3+ cells, which infiltrate on day 8.5 of gestation, and produce the Th2 cytokine IL-10 and Th3 cytokine transforming growth factor (TGF)-b2. In low abortion rate immune-competent mice, most of the
TGF-b2 is derived from gammadelta T cells. NK-gammadelta T cells in deciduas may be quite important in the Th1 response in early pregnancy that predisposes to abortions in CBA · DBA ⁄ 2 mating, whereas gammadelta T-only cells appear to be protective.41 Consequently, murine resorptions are characterized by focal necrosis at the junction of the fetal trophoblast with decidua, an infiltrate of polymorphonuclear leukocytes (with some lymphocytic cells) at sites of necrosis and along the walls of large vessels in decidua, and by thrombosis and hemorrhage.38,42–44 Infections or other events that induce inflammations may cause activation of cells at the Maternal–fetal interface Toll-like receptors (TLRs) are a conserved set of single-membrane spanning receptors each evolved with distinctive specificity for particular pathogen-associated molecular patterns. Antigen-presenting cells express the majority of these receptors. TLRs allow these cells to ‘forget’ pathogens to which they respond, as cellular memory is not required for innate immune response. Placenta transcripts for TLRs1-10 have been found, and human uterine NK clones are positive for TLR1, 2, 3, 4, 6, 7 and 9.45 First-trimester trophoblast cells are known to express TLR1 and TLR2 and to undergo apoptosis following exposure to their ligand, peptidoglycan (PDG) from Gram-positive bacteria. Trophoblast apoptosis can be reversed by the presence of TLR6. Furthermore, TLR2 ⁄ TLR6 responses to PDG results in trophoblast NF-jB activation and cytokine ⁄ chemokine production. These findings suggest that in the trophoblast, the expression of TLR6 is a key factor determining whether the response to PDG would be apoptosis or inflammation.46 TLR-3 stimulation with poly(I:C), which mimics viral infection, induces IL-2 and inhibits IL-10 expression in CD45+ cells from the placenta47 and uNK cell activation.48 When abortion-prone male DBA ⁄ 2J-mated CBA ⁄ J female mice were given poly I:C at gestation day 6.5, it significantly increased fetal losses at mid-gestation stage compared with control animals. Poly(I:C) injection-induced TLR3 expression markedly within deciduas basalis, and endometrial TNF-a but not IFN-c in homogenized endometrium. These findings suggest that enhanced TNF-a expression in endometrial stroma may play a critical role in inflammatory factor production and impairment of uterine spiral artery American Journal of Reproductive Immunology 63 (2010) 611–623
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remodeling in the pregnancy failure of CBA · DBA ⁄ 2 mating.49 In addition, activation of TLR-3 with poly(I:C) induces preterm labor in 31% of the animals with induction of interferon-b and RANTES in uterine tissues but not in fetal membranes.50 Toll-like receptor 9 activation with CpG-induced fetal resorption and preterm birth in IL-10() ⁄ )) mice when injected I.P. on gestational day 6 or 14, respectively. Amplification of uterine neutrophil and macrophage subpopulations followed by their migration to the placental zone was present in these mice. Furthermore, a dramatic increase in serum levels of mouse keratinocyte-derived cytokine and TNF-a production by uterine F4 ⁄ 80+ macrophages, but not uterine NK or Gr-1+ CD11b+ cells, was observed. Depletion of F4 ⁄ 80+ macrophages or neutralization of TNF-a rescued the pregnancy.51 Growing evidence clearly suggests that TLR-mediated activation through endogenous signals may indeed provide pregnancy compatible responses.52,53 On the other hand, the TLR machinery will respond to external signals with origins from intrauterine infections and inflammation, particularly when coupled with intrinsic deficiencies in molecules that are critical to normal pregnancy outcome. Two such scenarios are the bacterial and DNA breakdown products from virus or possibly other cell debris. Pro-inflammatory cytokines induce coagulation Pro-inflammatory cytokines such as IL-6, TNF-a, IFN-c, and IL-1 have been associated with pregnancy losses.54–56 It has been reported that cytokines induce fetal resorption because of ischemia as a result of the activation of vascular endothelial cell procoagulant, which causes thrombosis and inflammation in the mouse model. Spontaneous and cytokine-boosted abortions in the CBA · DBA ⁄ 2 mouse model can be blocked by antibody to fgl2 prothrombinase which is expressed by cytokinestimulated vascular endothelial cells and monocytes.55 Additionally, inhibition of intravascular thrombosis by fibrinogen depletion, in the absence of any other manipulation, unmasks NK-cell-dependent acute xenograft rejection in the mouse-to-rat heart transplantation model.57 These findings suggest that cytokine-triggered inflammatory processes and thrombosis in maternal and utero-placental blood vessels cause abortions.
It is recognized that endotoxins and other bacterial, fungal, and viral products can activate TLRs, leading to the elaboration of inflammatory cytokines58 that in turn elicit tissue factor expression to trigger the blood-clotting process in mid to small size vessels.59 Multiple mechanisms are at play including up-regulation of tissue factor leading to the initiation of clotting, amplification of the clotting process by augmenting exposure of cellular coagulant phospholipids, inhibition of fibrinolysis by elevating plasminogen activator inhibitor 1 (PAI-1), and decrease in natural anticoagulant pathways, particularly targeted toward down-regulation of the protein C anticoagulant pathway through multiple mechanisms.60,61 TNF-a and other inflammatory mediators can downregulate endothelial protein C receptor (EPCR) and thrombomodulin by inhibiting gene transcription and in the case of EPCR by promoting shedding from the endothelium.62 IL-6 can depress levels of protein S in experimental animals.63 Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury, and leukocyte extravasation in response to endotoxin, processes that are decreased by infusion of activated protein C (APC). In vitro, APC inhibits TNF-a elaboration from monocytes and blocks leukocyte adhesion to selectins. As thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation as a result of impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis. Activated protein C is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factors Va and VIIIa. Besides well-defined anticoagulant properties, APC also demonstrates anti-inflammatory, antiapoptotic, and endothelial barrier-stabilizing effects that are collectively referred to as the cytoprotective effects of APC.64 APC inhibits the release of inflammatory cytokines such as TNF-a, IL-6, and IL-8 in experimental models of endotoxin-induced inflammation as well as the expression of adhesion molecules such as ICAM-1, VCAM-1, and E-selectin in HUVECs.65,66 Inflammatory immune response and NK cells Dysregulation of NK cells has been associated with RPL, infertility, and preeclampsia.67–71 Various NK parameters have been investigated in women with RPL including absolute numbers or percentage,20
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subsets and functional activity,72 secretory cytokine profile,73 receptor or gene expression.74 It has been reported that women with RPL and unexplained infertility have increased peripheral blood NK (pNK) cells when compared to normal controls. In women with RPL, 37.3% had mild to moderate increase in CD56+ pNK cells and 14.7% had marked increase (>18%).67 Furthermore, the number of pNK cells is significantly higher in patient with APA,20,75 which is autoimmune inflammatory condition.76 Clinically, increased numbers of pNK cells were correlated with reduced gestational age at abortion in RPL patients with anti-phospholipid antibody syndrome (APS),77 and down-regulation of NK cells in women with RPL was associated with a favorable pregnancy outcome.77,78 Although Emmer et al. reported no difference in NK cell levels or cytotoxicity before pregnancy in women with RPL when compared to normal controls, a longitudinal study revealed that, compared with controls, in RPL women, higher numbers of CD56+ CD16+ cells were present during early pregnancy, paralleled by an increase in cytotoxic NK cell reactivity.79 Women with infertility of repeated IVF failures have the similar NK pathology as women with RPL. The percentages of CD56+ and CD56+ ⁄ 16+ pNK cells on the day of embryo transfer were significantly higher in women with IVF failure than in women with implantation. Additionally, increase of cytotoxic NK cells in peripheral blood and endometrium was negatively related to the IVF outcome.80 In women with repeated implantation failures after IVF ⁄ ICSI, additional intravenous immunoglobulin G (IVIg) treatment for down-regulation of elevated CD3)CD56+ CD16+ NK cells (>12%) significantly increased pregnancy outcome. Total 42% of IVIGpatient or 34.9% ⁄ embryo transfer had a live born baby, and the live born rate per embryo was 16.6%.81 High levels of pNK cells were found to predict biochemical pregnancy and spontaneous abortions with normal karyotype in the next pregnancy. Elevated CD56+ cells in pregnant women predict loss of karyotypically normal conceptus: a specificity of 87% and positive predictive value of 78%. While the specificity value of this test was reported to be high in both infertile and RPL populations, the sensitivity was 86% in RPL and only 54% in infertility women, suggesting enumeration of CD56+ pNK cells does not identify all losses among infertility women.82 The relative risk of biochemical pregnancy losses or spontaneous abortion with normal karyo-
type was reported to be 4.9 when CD56+ cells are greater than 16.4%.83 It has been reported that the modulation in the number of circulating NK cells is most likely to be a primary event rather than an active inflammation or drug-induced consequences during an inflammatory ⁄ autoimmune process, thus playing an important role in the pathogenesis of immunological infertility and pregnancy losses.20,84 NK cells express inhibitory and activating receptors on their surface. The balance between inhibitory signals from receptors specific for MHC class I molecules [killer inhibitory receptor (KIR)] and stimulatory signals mediated by a variety of activating receptors ultimately determines the outcome of NK cell target encounter. A significant increase in CD69 expression on CD56+ NK cells was demonstrated in women with RPL (P < 0.005) and infertility (P < 0.05) when compared with that of normal controls. Conversely, CD94 expression was significantly decreased in women with RPL (P < 0.005) and infertility (P < 0.05) in comparison with that of controls.68,85 Differences in NK receptor expression seem to be genetically linked. A higher prevalence of activating KIR genes was seen in women with RPL than in controls. Among women experiencing RPL, the BB genotypes were more prevalent (P < 0.0001, OR = 4.4, 95% CI = 2.89–6.69) compared with controls.86 Therefore, the balance between inhibitory and activating receptor-mediated signals present in NK cells is predisposed toward an activating state that may contribute to pregnancy loss. Although decidual NK cells are different from peripheral blood NK cells in immune-phenotypic expression,87 cytokine production, cytolytic activities etc., the %CD3) ⁄ 56+ and %CD3) ⁄ 56+ ⁄ 16+ pNK cells showed a significant correlation with mean number of CD56+ dNK cells. The number of decidual CD16+ cells was significantly higher in women with elevated pNK (>OR = 15%) than that of normal pNK (
