Objective Studies in mouse models implicate complement activation as a causative factor in adverse pregnancy outcomes (APOs). We investigated whether activation of complement early in pregnancy predicts APOs in women with systemic lupus erythematosus (SLE) and/or antiphospholipid (aPL) antibodies.
Methods The PROMISSE Study enrolled pregnant women with SLE and/or aPL antibodies (n=487) and pregnant healthy controls (n=204) at <12 weeks gestation and evaluated them monthly. APOs were: fetal/neonatal death, preterm delivery <36 weeks because of placental insufficiency or preeclampsia and/or growth restriction <5th percentile. Complement activation products were measured on serial blood samples obtained at each monthly visit.
Results APO occurred in 20.5% of SLE and/or aPL pregnancies. As early as 12–15 weeks, levels of Bb and sC5b-9 were significantly higher in patients with APOs and remained elevated through 31 weeks compared with those with normal outcomes. Moreover, Bb and sC5b-9 were significantly higher in patients with SLE and/or aPL without APOs compared with healthy controls. In logistic regression analyses, Bb and sC5b-9 at 12–15 weeks remained significantly associated with APO (ORadj=1.41 per SD increase; 95% CI 1.06 to 1.89; P=0.019 and ORadj=1.37 per SD increase; 95% CI 1.05 to 1.80; P=0.022, respectively) after controlling for demographic and clinical risk factors for APOs in PROMISSE. When analyses were restricted to patients with aPL (n=161), associations between Bb at 12–15 weeks and APOs became stronger (ORadj=2.01 per SD increase; 95% CI 1.16 to 3.49; P=0.013).
Conclusion In pregnant patients with SLE and/or aPL, increased Bb and sC5b-9 detectable early in pregnancy are strongly predictive of APOs and support activation of complement, particularly the alternative pathway, as a contributor to APOs.
- systemic lupus erythematosus
- antiphospholipid syndrome
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Women with systemic lupus erythematosus (SLE) and/or antiphospholipid (aPL) antibodies are at increased risk for adverse pregnancy outcomes (APOs), including preeclampsia, fetal and neonatal death and fetal growth restriction. Although we have made progress in identifying clinical risk factors and dysregulated circulating antiangiogenic factors as predictors of APOs, identification of patients destined for complications remains challenging.1 2
Studies in mice implicate inflammation, particularly complement activation and recruitment of neutrophils, as an essential and causative factor in placental insufficiency, fetal loss and growth restriction.3–5 Activation of complement stimulates infiltrating leucocytes to release TNF-α and soluble fms-like tyrosine kinase-1 (sFlt1), a potent antiangiogenic factor, which are both associated with impaired development of the placenta and preeclampsia.5–7 Complement activation is initiated by classical, alternative and lectin pathways. The convergence of the three pathways on C3 results in generation of common effectors: anaphylatoxins, opsonins and the membrane attack complex. Mice deficient in alternative and classical pathway complement components (factor B, C4, C3 and C5) and mice treated with inhibitors of complement activation (anti-C5 mAb, anti-factor B mAb, C5a receptor antagonist peptide) are resistant to fetal injury induced by aPL,3 8 indicating that both pathways contribute to damage.
Studies in humans support the role of complement in aPL-associated pregnancy complications9–11 and in preeclampsia and growth restriction in non-autoimmune women.12 13 Complement fragment C4d, a marker of classical pathway activation, is present in placentae from women with SLE and/or antiphospholipid syndrome (APS) and from women with preeclampsia.12 14–16 Mild hypocomplementaemia has also been reported in primary APS in two studies.17 18 The presence of risk variants in complement regulatory proteins in patients with SLE and/or aPL antibodies who develop preeclampsia, and in preeclampsia patients without an associated autoimmune disease, links complement activation to disease pathogenesis.9 Furthermore, in a prospective study of non-autoimmune patients, elevated levels of the alternative pathway complement activation product Bb were strongly associated with preeclampsia.19 There have been no longitudinal studies examining complement activation in high risk pregnancies.
Accordingly, we investigated whether alterations in plasma levels of complement activation fragments before mid-second trimester predict APOs in women with SLE and/or aPL using data from the PROMISSE Study (Predictors of pRegnancy Outcome: bioMarkers In antiphospholipid antibody Syndrome and Systemic Lupus Erythematosus). PROMISSE is the largest multicentre, multiethnic and multiracial study to prospectively assess clinical and laboratory predictors of APO in women with aPL and/or SLE with inactive or mild/moderate activity at conception. We hypothesised that complement activation products are elevated early in pregnancy in patients destined for APOs.
The PROMISSE Study enrolled pregnant women between September 2003 and August 2013 from eight US sites and one Canadian site. Institutional review boards at each site approved protocols and consent forms; written informed consent was obtained from all subjects. Consecutive pregnant women referred to the study with diagnoses of SLE,20 aPL (defined in table 1) or both were recruited at <12 weeks’ gestation as previously described.21 Healthy control patients matched for ethnicity/race were also included.
Inclusion criteria were: live singleton intrauterine pregnancy confirmed by ultrasound; age 18–45 years; haematocrit >26%. Exclusion criteria to minimise confounding by known causes of APOs not specifically associated with SLE and/or aPL included: prednisone >20 mg/day; urine protein (mg)/creatinine (g) ratio ≥1000 on random sampling or 24 hour collection; erythrocyte casts on urinalysis; serum creatinine >1.2 mg/dL; type I or II diabetes mellitus; blood pressure >140/90 mm Hg.
Detailed medical and obstetrical information and serial blood samples were obtained at screening and monthly from 12 weeks’ gestation until the end of pregnancy and rheumatology assessments at each trimester.1 2
Adverse pregnancy outcomes
APOs defined for PROMISSE included one or more of the following: (1) fetal death after 12 weeks’ gestation unexplained by chromosomal abnormalities, anatomical malformation or congenital infection; (2) neonatal death before hospital discharge due to complications of prematurity and/or placental insufficiency; (3) indicated preterm delivery at less than 36 weeks due to gestational hypertension, preeclampsia or placental insufficiency and (4) small-for gestational-age (SGA) neonate (<5th percentile).
Complement levels were measured in a blinded manner by MicroVue EIA kits for Bb Plus Fragment, sC5b-9 Plus Fragment, Ba Fragment, C3a Plus Fragment, C4d Fragment, iC3b Fragment and C5a. Duplicate samples were run in two plates and averaged to obtain final results. Angiogenic factor (sFlt1 and PlGF) levels were measured as described.2
Categorical and continuous variables were compared between groups using Fisher’s exact and t-tests, respectively. Correlations were estimated using Pearson’s correlation. ORs for the associations between APO status and complement levels were estimated using logistic regression models including the following covariates selected a priori based on clinical considerations and prior studies: race/ethnicity, SLE status, lupus anticoagulant (LAC) status, history of thrombosis, screening diastolic pressure, aspirin use, antihypertensive use and body mass index (BMI). Model performance was evaluated using the Hosmer-Lemeshow goodness-of-fit test and area under the ROC curve (AUC). To address the potential for overfitting, leave-one-out cross-validation was performed. Complement levels measured at the time of or after an APO were excluded. Missing data were handled with both listwise deletion and multiple imputation (MI) using the Markov chain Monte Carlo approach. Missing data rates were 0%–6% for baseline variables and 29.5%–31.1% for 12–15 and 16–19 week complement variables. Given similarity in parameter estimates across missing data methods, MI results are provided in online supplementary table S1. Change in complement levels over time was analysed by linear mixed effects models. All analyses were conducted using SAS V.9.4 (SAS Institute).
Supplementary file 1
Study population and pregnancy outcomes
Of 770 pregnant women with SLE and/or aPL screened for PROMISSE, 487 who met study inclusion/exclusion criteria, had documented outcomes and had at least one blood sample evaluated for complement levels during pregnancy were included: 326 (66.9%) with SLE and without aPL, 60 (12.3%) with SLE and aPL and 101 (20.7%) with only aPL. APOs occurred in 100 cases (20.5%): fetal death in 27 (5.5%), neonatal death in 5 (1.0%), indicated preterm delivery for placental insufficiency or gestational hypertensive disease in 49 (10.1%) and SGA in 47 (9.7%). Also included were 204 healthy controls, 7 of whom had APOs (3.4%).
Baseline demographic and clinical risk factors
Women with SLE and/or aPL who had subsequent APOs were more likely to be positive for aPL and LAC, have higher systolic and diastolic blood pressures, higher BMI, history of thrombosis and exposure to heparin and antihypertensive medications compared with women without APOs (table 1). Race/ethnicity, age, parity, history of lupus nephritis, proteinuria, aspirin use and smoking status at enrolment were not significantly associated with pregnancy outcome in bivariate analyses.
Complement activation products: Bb and sC5b-9
We initially focused on Bb and sC5b-9, the complement measures which varied the least among the healthy controls (ie, smallest coefficients of variation at all visits). As early as 12–15 weeks, patients with APOs demonstrated significant elevations in Bb and sC5b-9 compared with patients without APO (figure 1). These differences tended to increase through week 31. Patients with SLE and/or aPL and without APO had consistently higher levels of Bb and sC5b-9 compared with the reference group, the healthy controls without APO (n=197). Bb levels were elevated at 12–15 weeks even in the subset of 138 patients with SLE with SLEPDAI ≤2 at baseline, compared with the reference group (P<0.001). In all groups, Bb decreased as pregnancy progressed, but the rate of decrease was smaller in patients with APOs compared with patients without APOs (P=0.06) and compared with the reference group (P=0.002), suggesting increased complement activation and cleavage of Bb. Patients with APO also showed less decline in sC5b-9 compared with patients without APO (P=0.02) and the reference group (P=0.002). Correlations between Bb and sC5b-9 in patients with disease ranged from 0.49 (P<0.001) at 12–15 weeks to 0.68 (P<0.001) at 36–39 weeks.
Early elevations in complement tended to persist throughout pregnancy: correlations between 12–15 and 36–39 week levels were 0.62 for Bb (P<0.001) and 0.44 for sC5b-9 (P<0.001). In ROC analysis of the 12–15 week measures, the optimal cut-points for discriminating APO from non-APO in patients with disease when sensitivity (SN) and specificity (SP) are equally weighted were >1.04 µg/mL for Bb (SN=46%, SP=75%, positive predictive value (PPV)=33%, negative predictive value (NPV)=84%; AUC=0.64) and >347 ng/mL for sC5b-9 (SN=52%, SP=68%, PPV=29%, NPV=85%; AUC=0.60). Using the two complement measures together to predict APO minimally increased the AUC under the ROC (ΔAUC=0.0035 compared with Bb alone; P=0.59) because they are significantly correlated.
Multivariable analyses of complement levels
Bb at 12–15 weeks remained significantly associated with APOs after controlling for potential confounders (table 2; ORadj=1.41 per SD increase; 95% CI 1.06 to 1.89; P=0.019). SC5b-9 at 12–15 weeks was also significantly associated with APO in logistic regression analyses (ORadj=1.37 per SD increase; 95% CI 1.05 to 1.80; P=0.022). At 16–19 weeks, ORs were ORadj=1.59 per SD increase for Bb (95% CI 1.16 to 2.17; P=0.004) and ORadj=1.26 per SD increase of sC5b-9 (95% CI 0.94 to 1.68; P=0.12). Between 20 and 31 weeks, Bb was significantly associated with APO at all visits; sC5b-9 levels at 28–31 weeks and not earlier were significant (results not shown).
Results above were based on patients with disease. Among all healthy subjects, Bb, but not sC5b-9, was significantly elevated at 12–15 weeks in the APO compared with non-APO groups (Bb: 0.91 vs 0.71 µg/mL, P=0.03; sC5b-9: 241.5 vs 226.5 ng/mL, P=0.66). To assess the independent effects of disease status and complement on APO, a separate logistic regression was fit with main effects for Bb at 12–15 weeks and disease (yes/no), based on all subjects with disease and healthy subjects. The adjusted OR for APO was 4.82 (95% CI 2.01 to 11.55; P<0.001) comparing subjects with disease and healthy subjects and for Bb was 1.61 (95% CI 1.26 to 2.06; P<0.001) per SD increase. The OR for sC5b-9 at 12–15 weeks after adjusting for disease status was 1.34 per SD increase (95% CI 1.06 to 1.70; P=0.01). Adjustment for additional covariates was not feasible due to limited APOs in the healthy group.
Relationship with angiogenic factors
Given prior evidence that complement activation stimulated release of sFlt1 by leucocytes22 and that angiogenic dysregulation early in pregnancy is associated with APOs,2 we examined the relationship between levels of complement activation products and angiogenic factors. Correlations between Bb and sC5b-9 measured at or before 12–15 weeks with sFlt1, PlGF and sFlt1/PlGF measured at the same and subsequent visits were modest and did not exceed 0.25. Adjustment for sFlt1/PlGF in multivariable analyses did not affect the association between APOs and Bb measured at 12–15 weeks (ORadj=1.39; 95% CI 1.03 to 1.88; P=0.034) or at 16–19 weeks (ORadj=1.60; 95% CI 1.14 to 2.2; P=0.006). SC5b-9 results were also minimally affected after adjusting for sFlt1/PlGF at 12–15 weeks (ORadj=1.34; 95% CI 1.00 to 1.78; P=0.047) and 16–19 weeks (ORadj=1.30; 95% CI 0.95 to 1.76; P=0.10).
Because studies in mouse models of aPL-associated APO strongly implicate complement activation and reveal complement deposition on trophoblasts,8 we analysed complement analytes in the subgroup of patients who were aPL-positive with or without SLE (n=161). Associations between Bb and APO increased for both the 12–15 week measures (ORadj=2.01 per SD increase; 95% CI 1.16 to 3.49; P=0.013) and 16–19 week measures (ORadj=2.46 per SD increase; 95% CI 1.27 to 4.76; P=0.008) after adjusting for the same variables in table 2; Bb results remained highly significant after further adjustment for heparin (P=0.01 and P=0.009, respectively). ORs for sC5b-9 in aPL-positive subjects were similar to those based on the overall study population using the 12–15 week levels (ORadj=1.35 per SD increase; 95% CI 0.86 to 2.12; P=0.19) and 16–19 week levels (ORadj=1.16 per SD increase; 95% CI 0.69 to 1.94; P=0.57).
Among patients with SLE who do not have aPL antibodies (n=326), associations between Bb with APO were weaker (12–15 weeks: ORadj=1.28 per SD increase; 95% CI 0.87 to 1.86; P=0.21; 16–19 weeks: ORadj=1.45 per SD increase; 95% CI 0.99 to 2.14; P=0.06) than corresponding results in the aPL-positive group. In contrast, associations of sC5b-9 with APOs in the patients with SLE only were comparable to those in the aPL positive group (12–15 weeks: ORadj=1.39 per SD increase; 95% CI 0.98 to 2.01; P=0.07; 16–19 weeks: ORadj=1.33 per SD increase; 95% CI 0.93 to 1.90; P=0.12). Baseline SLEPDAI scores were significantly correlated with Bb and sC5b-9 at both 12–15 and 16–19 weeks (ρ=0.22–0.30; P<0.001 for all correlations). In addition, among all patients with SLE, mean Bb was significantly elevated at both visits in those who were LAC-positive versus negative (12–15 weeks: 1.21 µg/mL vs 0.87 µg/mL; P<0.001; 16–19 weeks: 1.19 µg/mL vs 0.85 µg/mL; P<0.001); mean sC5b-9 was non-significantly elevated (12–15 weeks: 399.5 ng/mL vs 356.3 ng/mL, respectively; P=0.28; 16–19 weeks: 433.3 ng/mL vs 358.5 ng/mL; P=0.19).
Other complement factors
C5a, C4d and iC3b levels at 12–15 weeks and 16–19 weeks were not significantly associated with APOs (online supplementary figures). C3a was significantly associated with APO in bivariate analyses, but not when added to the multivariable models that included Bb or sC5b-9, whereas Bb remained significant (12–15 weeks: P=0.057; 16–19 weeks: P=0.004). Limited data were available for Ba (>40% had missing values), precluding rigorous evaluation of this analyte. However, consistent with the contribution of alternative pathway activation, Ba appeared to be a stronger predictor of APOs than sC5b-9 at both 12–15 weeks (ORadj=1.74 per SD increase; 95% CI 1.24 to 2.43; P=0.001) and 16–19 weeks (ORadj=1.47 per SD increase; 95% CI 1.06 to 2.05; P=0.02), when added to the models in table 2. It was also a stronger predictor of APOs than Bb at 12–15 weeks (ORadj=1.59 per SD increase; 95% CI 1.10 to 2.30; P=0.01) but not at 16–19 weeks (ORadj=1.25 per SD increase; 95% CI 0.89 to 1.75; P=0.20).
In our study that included nearly 500 pregnant patients with SLE and/or aPL, increased levels of Bb and sC5b-9 early in pregnancy were significantly associated with APOs. This supports the concept that complement activation, particularly the alternative pathway, contributes to abnormal placental development that leads to pregnancy complications. That the association of alternative complement activation with APO was greatest in the patients with aPL is consistent with our experiments showing that mice deficient in factor B or treated with an inhibitor of factor B activation were protected from aPL-induced fetal resorptions and growth restriction.3 23
Bb levels fell as pregnancy progressed in uncomplicated pregnancies in both subjects with disease and healthy subjects. However, in those destined for APOs, Bb levels were significantly higher throughout pregnancy, and the rate of decrease during pregnancy was lower. Lynch et al similarly showed that Bb levels decreased with gestational age in normotensive women, while dysregulation of Bb activation occurred between 10 and 20 weeks gestation in those who developed preeclampsia.24 Others have shown that elevated Bb is present in amniotic fluid of women with severe preeclampsia, supporting the concept that complement is activated at the maternal–fetal interface.25 26
We also found a significant association between elevated sC5b-9 levels and APOs, but this was not the case in a study of non-autoimmune women who developed preeclampsia.27 Nonetheless, a role for terminal pathway complement activation, as a cause or consequence of preeclampsia, is supported by reports of markedly increased urinary sC5b-9 excretion and correlations between urinary sC5b-9 and angiogenic dysregulation.28 29
Although antibodies are essential to the pathogenesis of SLE and aPL, the alternative pathway fragment Bb, rather than classical pathway components, was the strongest predictor of APO. The correlation between Bb and sC5b-9 further supports that the alternative pathway is a key driver of complement activation in pregnancy complications. Whether the complement cascade is triggered by the classical, lectin or alternative pathway, activation is amplified by the alternative pathway. Classical pathway activation depends on affinity and subclass of multiplexed antibodies, and aPL are skewed towards IgG2 subclass, relatively ineffective classical pathway activators.30 Excess deposition of non-classical pathway activating antibodies can inhibit membrane-bound complement regulators and allow unabated activation of the alternative pathway. Furthermore, cleavage of complement components by neutrophil-derived and platelet-derived proteases can bypass the classical pathway.31
In normal pregnancy, the human placenta is subjected to complement-mediated immune attack at the maternal–fetal interface.32 33 Complement activation is controlled in successful pregnancies by inhibitory proteins on trophoblast cells.34–36 In patients with APL and/or SLE, the excessive complement activation, evident in the circulation and in the placenta,12 14–16 overwhelms regulatory pathways and places the fetus at risk.
Infiltrating leucocytes recruited and activated by complement are a source of the excess antiangiogenic factor sFlt1 that impairs early placental development. Subsequent hypoperfusion of the intervillous space stimulates trophoblasts to synthesise large amounts of sFlt-1 as pregnancy progresses. Because the latter is not directly driven by complement, the modest relationship between sFlt1 levels and complement activation fragments was not surprising.
Although PROMISSE patients had quiescent, stable or mildly active disease, even those without APO had significantly higher levels of Bb and sC5b-9 at baseline (6–11 weeks) compared with healthy controls. Prolonged activation of complement, also described in non-pregnant patients with inactive SLE and those with primary APS, may lower the threshold for exaggerated, uncontrolled activation that recruits leucocytes and unleashes potent inflammatory and antiangiogenic mediators associated with placental insufficiency.17 37 38
Interestingly, baseline SLEPDAI scores correlated significantly with early measures of Bb and sC5b-9. A small study also reported that elevations of Bb and sC5b-9 accompanied disease activity in pregnant patients with SLE.39 Interpretation of complement is confounded in pregnancy because circulating complement reflects both synthesis (enhanced by oestrogen) and consumption.40 Our findings of early increased complement activation products in women destined for APOs, and our previous report that less increase in C3 levels from baseline to second trimester was predictive of APO and argues that excess complement activation is the driver rather than consequence of APOs. The presence of classical pathway complement fragment C4d deposition on placentae from women with SLE and/or APS and women with preeclampsia, and in kidneys of women with preeclampsia,12–16 provides further evidence for local activation of classical or lectin pathways by aPL or necrotic fetoplacental debris. Such activation, regardless of the source, is then augmented through the alternative pathway. Importantly, circulating levels of complement fragments may not reflect the extent of activation in the placenta.
Our study had limitations. We did not measure prepregnancy levels of complement activation products so could not differentiate whether increased complement consumption was due to SLE or placental inflammation. Sample sizes did not allow for analyses stratified by APO type. Whether patient aPL antibodies were complement-fixing was not determined, and not possible for LAC, the aPL associated with greatest risk.
In conclusion, we demonstrate that complement pathway activation is associated with APOs in patients with SLE and/or APL. Aberrant complement activation, whether initiated by immune complexes in SLE or by aPL, may trigger or amplify inflammation at the maternal–fetal interface and thereby contribute to the pathogenesis of APOs.
Supplementary file 2
Supplementary file 3
Supplementary file 4
Supplementary file 5
Supplementary file 6
We are grateful to Mary S Stephenson, Alan Peaceman and Munther Khamashta for contributing patients to the study. We thank Quidel Corporation for their guidance with the complement split product measurements and S Ananth Karumanchi for thoughtful discussions about angiogenic factors.
Handling editor Tore K Kvien
Contributors All authors made substantial contributions to study design or acquisition, analysis or interpretation of data. MYK, JES, JPB wrote the manuscript and all authors contributed to revising it critically for important intellectual content. All authors have read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work have been appropriately investigated and resolved.
Funding Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number RO1 AR49772 (PROMISSE Study, MYK, MMG, EK, CAL, MP, DWB, MDL, LRS, JTM, TFP, AS, JPB, JES), Mary Kirkland Center for Lupus Research (JES, MDL) and NIH AR43727, AR69572 (MP).
Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Competing interests DWB reports serving on the UCB Pharmaceuticals Advisory Board; JES has received an investigator-initiated grant from UCB Pharmaceuticals and consulting fees from Alnylam and Alexion.
Ethics approval The Institutional Review Board at each study site.
Provenance and peer review Not commissioned; externally peer reviewed.
Presented at Preliminary data included in this publication were reported in an abstract at the American College of Rheumatology (ACR) Meeting in 2015.