Background
Over 20% of pregnancies in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (APL) result in an adverse pregnancy outcome (APO) related to abnormal placentation. The ability to identify, early in pregnancy, patients who are destined for poor outcomes would significantly impact care of this high-risk population. In nonautoimmune patients, circulating angiogenic factors are dysregulated in disorders of placentation, such as preeclampsia (PE) and fetal growth restriction.
Objective
We sought to determine whether early dysregulation of circulating angiogenic factors can predict APO in high-risk SLE and/or APL pregnancies.
Study Design
We used data and samples from the Predictors of Pregnancy Outcome: Biomarkers in APL Syndrome and SLE (PROMISSE), a multicenter prospective study that enrolled 492 pregnant women with SLE and/or APL from September 2003 through August 2013. Patients were followed through pregnancy from <12 weeks gestation. Circulating levels of soluble fms-like tyrosine kinase-1 (sFlt1), placental growth factor (PlGF), and soluble endoglin were measured monthly and subjects followed up for APO, classified as severe (PE <34 weeks, fetal/neonatal death, indicated preterm delivery <30 weeks) or moderate (PE ≥34 weeks, indicated preterm delivery 30-36 weeks, growth restriction without PE).
Results
Severe APOs occurred in 12% and moderate APOs in 10% of patients. By 12-15 weeks, sFlt1, PlGF, and soluble endoglin levels were markedly altered in women who developed severe APO. After adjusting for clinical risk factors, sFlt1 was the strongest predictor of severe APO among 12-15 week measures (odds ratio, 17.3 comparing highest and lowest quartiles; 95% confidence interval [CI], 3.5–84.8; positive predictive value [PPV], 61%; negative predictive value [NPV], 93%). At 16-19 weeks, the combination of sFlt1 and PlGF was most predictive of severe APO, with risk greatest for subjects with both PlGF in lowest quartile (<70.3 pg/mL) and sFlt1 in highest quartile (>1872 pg/mL; odds ratio, 31.1; 95% CI, 8.0–121.9; PPV, 58%; NPV, 95%). Severe APO rate in this high-risk subgroup was 94% (95% CI, 70–99.8%), if lupus anticoagulant or history of high blood pressure was additionally present. In contrast, among patients with both sFlt1 <1872 pg/mL and PlGF >70.3 pg/mL, rate of severe APO was only 4.6% (95% CI, 2.1–8.6%).
Conclusion
Circulating angiogenic factors measured during early gestation have a high NPV in ruling out the development of severe adverse outcomes among patients with SLE and/or APL syndrome. Timely risk stratification of patients is important for effective clinical care and optimal allocation of health care resources.
Introduction
Systemic lupus erythematosus (SLE) predominantly affects women and presents during their childbearing years. Pregnancy in patients with SLE, particularly those with antiphospholipid antibodies (APL), and in patients with APL alone, is associated with an increased risk for maternal and fetal morbidity due to preeclampsia (PE) and insufficient placental support of the developing fetus. PE and placental insufficiency are, in turn, associated with adverse pregnancy outcomes (APOs), including maternal complications of PE, intrauterine fetal death, and fetal growth restriction, as well as indicated preterm delivery. Given that APOs affect over one fifth of pregnancies in SLE and/or APL, the ability to identify patients early in pregnancy who are destined for poor outcomes would significantly impact care of this high-risk population.
Failure of adequate vascularization of the developing placenta leads to APOs. Inability of trophoblasts to remodel uterine spiral arteries occurs early in pregnancy, is clinically silent, and results in placental ischemia. Hypoperfusion drives placental production of the potent antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFlt1) (also known as soluble vascular endothelial growth factor [VEGF] receptor 1) and soluble endoglin (sEng). SFlt1 sequesters VEGF and placental growth factor (PlGF), antagonizing their proangiogenic actions. In the maternal circulation, excess sFlt1 produces a deficiency of angiogenic factors required for endothelial vascular homeostasis. Experiments in animals indicate that elevated circulating antiangiogenic proteins recapitulate complications characteristic of human PE. Prospective studies in otherwise healthy women demonstrate angiogenic dysregulation up to 5 weeks before clinical manifestations of PE, suggesting that angiogenic factors are useful for diagnosing PE. An imbalance between circulating antiangiogenic and proangiogenic proteins is also associated with fetal growth restriction and death in nonautoimmune patients.
Biomarkers altered early in pregnancy are needed to identify, counsel, and manage patients at high risk of APOs. Accordingly, we evaluated whether alterations in angiogenic factors before the midsecond trimester would predict subsequent APOs in women with SLE and/or APL using data from the Predictors of Pregnancy Outcome: Biomarkers in APL Syndrome and SLE (PROMISSE) study. PROMISSE is the largest multicenter, multiethnic, and multiracial study to prospectively assess clinical and laboratory predictors of APO in SLE and/or APL women with inactive or mild/moderate activity at conception.
Materials and Methods
Patient population
Pregnant subjects were enrolled into PROMISSE from September 2003 through August 2013 at 7 US sites and 1 Canadian site ( Figure 1 ). Institutional review boards at each site approved protocols and consents; written informed consent was obtained from all subjects ( ClinicalTrials.gov identifier NCT00198068 ). Consecutive pregnant women referred to the study with diagnoses of SLE (≥4 American College of Rheumatology criteria for SLE), APL (anticardiolipin antibody [IgG ≥40 GPL units; IgM ≥40 MPL units] and/or positive lupus anticoagulant [RVVT, kaolin, dilute TTI or PTT LA] and/or anti-β2 glycoprotein I [anti-β2 GPI: IgG ≥40 GPL units; IgM ≥40 MPL units] at least twice between 6 weeks and 5 years apart of which one must be during the PROMISSE pregnancy at a core laboratory, as previously described), or both were recruited at <12 weeks’ gestation. Healthy controls of similar ethnicity to patients and at low risk of APO (≥1 successful pregnancy, no fetal deaths, and <2 miscarriages <10 weeks’ gestation) were also enrolled. Consecutive healthy controls who met inclusion criteria were enrolled during their routine visits in general obstetric clinics at PROMISSE patient recruitment sites. This study focuses on women with SLE and/or APL. Clinical determinants of APOs have been previously reported.
At enrollment, all patients met inclusion criteria: live singleton intrauterine pregnancy confirmed by ultrasound; age 18-45 years; hematocrit >26%. Exclusion criteria to minimize confounding by known causes of APO not specifically associated with SLE and/or APL included: prednisone >20 mg/d; 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 1 or 2 diabetes mellitus; and blood pressure >140/90 mm Hg at screening.
Adverse pregnancy outcomes
PROMISSE was designed to identify biomarkers of APOs due to abnormal placental vascularization leading to PE and placental insufficiency. APOs defined for PROMISSE included ≥1 of the following: (1) PE at any time ; (2) fetal death >12 weeks’ gestation unexplained by chromosomal abnormality, anatomic malformation, or congenital infection; (3) neonatal death prior to hospital discharge due to complications of prematurity; (4) preterm delivery or termination of pregnancy <36 weeks due to gestational hypertension, PE, growth restriction, or placental insufficiency; (5) small for gestational age (SGA) <5th percentile at birth. Premature preterm rupture of membranes and/or spontaneous preterm birth were not included as APOs defined for PROMISSE as they are not known to be directly linked to poor placental vascularization. Details of these and other deliveries <36 weeks not counted as APOs are presented in Figure 1 .
To identify markers predictive of the most serious and costly outcomes, APOs were further classified (post hoc) as severe (PE with delivery at <34 weeks, fetal/neonatal death, indicated preterm delivery <30 weeks), and moderate (PE with delivery ≥34 weeks, indicated preterm delivery for reasons other than PE [30-36 weeks], isolated SGA <5th percentile). Outcomes were confirmed by chart review and, if necessary, diagnoses were adjudicated by study obstetricians.
Screening and follow-up visits
Screening evaluation included history, physical examination, complete blood cell count, comprehensive metabolic panel, APL measurement, urinalysis, and random or 24-hour urine for protein/creatinine ratio (if dipstick >1+) ( Table 1 ). Detailed medical and obstetrical information and serial blood samples were obtained monthly.
| Severe APO | Moderate APO | No APO | |
|---|---|---|---|
| n = 59 | n = 49 | n = 384 | |
| DEMOGRAPHICS | |||
| Race/ethnicity | |||
| Non-Hispanic white | 36 (61.0) | 21 (42.9) | 220 (57.3) |
| Hispanic white | 8 (13.6) | 8 (16.3) | 44 (11.5) |
| African American | 9 (15.3) | 12 (24.5) | 60 (15.6) |
| Asian | 4 (6.8) | 7 (14.3) | 41 (10.7) |
| Other | 2 (3.4) | 0 (0) | 12 (3.1) |
| Do not know | 0 (0) | 1 (2.0) | 7 (1.8) |
| Age, y, mean (SD) | 31.0 (4.7) | 30.2 (5.9) | 31.4 (4.8) |
| CLINICAL HISTORY | |||
| Parity | |||
| Nulliparous | 27 (45.8) | 31 (63.3) | 229 (59.6) |
| Parous without history of PE | 24 (40.7) | 15 (30.6) | 132 (34.4) |
| Parous with history of PE | 8 (13.6) | 3 (6.1) | 23 (6.0) |
| Smoking | |||
| Never | 40 (67.8) | 36 (73.5) | 290 (75.9) |
| Ever | 16 (27.1) | 10 (20.4) | 81 (21.2) |
| Current | 3 (5.1) | 3 (6.1) | 11 (2.9) |
| Missing | 0 | 0 | 2 |
| APL a /SLE b status | |||
| APL+/SLE– | 20 (33.9) | 10 (20.4) | 68 (17.7) |
| APL+/SLE+ | 13 (22.0) | 8 (16.3) | 38 (9.9) |
| APL–/SLE+ | 26 (44.1) c | 31 (63.3) | 278 (72.4) |
| Lupus nephritis | |||
| No | 40 (67.8) | 36 (73.5) | 293 (76.3) |
| Yes | 19 (32.2) | 13 (26.5) | 91 (23.7) |
| Thrombosis | |||
| No | 40 (67.8) | 43 (87.8) | 348 (90.6) |
| Yes | 19 (32.2) c | 6 (12.2) | 36 (9.4) |
| High blood pressure d | |||
| No | 43 (72.9) | 37 (75.5) | 337 (87.8) |
| Yes | 16 (27.1) e | 12 (24.5) f | 47 (12.2) |
| PHYSICAL EVALUATION | |||
| BMI, kg/m 2 | |||
| <25 | 22 (40.0) | 23 (50.0) | 225 (63.7) |
| 25–30 | 14 (25.5) | 12 (26.1) | 78 (22.1) |
| >30 | 19 (34.5) e | 11 (23.9) | 50 (14.2) |
| Missing | 4 | 3 | 31 |
| Blood pressure, mm Hg | |||
| Systolic mean (SD) | 117.9 (12.0) c | 116.2 (13.9) e | 110.3 (11.7) |
| Diastolic mean (SD) | 73.0 (9.0) c | 70.2 (11.0) f | 66.6 (8.9) |
| MEDICATIONS | |||
| Aspirin | |||
| No | 31 (52.5) | 25 (51.0) | 175 (45.6) |
| Yes | 28 (47.5) | 24 (49.0) | 209 (54.4) |
| Heparin | |||
| No | 22 (37.3) | 32 (65.3) | 255 (66.4) |
| Yes | 37 (62.7) c | 17 (34.7) | 129 (33.6) |
| Antihypertensive | |||
| No | 49 (83.1) | 39 (79.6) | 365 (95.1) |
| Yes | 10 (17.0) e | 10 (20.4) c | 19 (5.0) |
| LABORATORY VALUES | |||
| LAC status during pregnancy | |||
| Negative | 29 (49.2) | 38 (77.6) | 334 (87.0) |
| Positive | 30 (50.9) c | 11 (22.4) | 50 (13.0) |
| Proteinuria | |||
| Negative | 54 (91.5) | 42 (85.7) | 360 (94.8) |
| Positive | 5 (8.5) | 7 (14.3) | 24 (6.3) |
a Presence of anticardiolipin antibody (IgG ≥40 GPL units, IgM ≥40 MPL units) and/or positive lupus anticoagulant (LAC: RVVT, kaolin, dilute TTI or PTT LA) and/or anti-β2 glycoprotein I (anti-β2GPI: IgG ≥40 GPL units, IgM ≥40 MPL units) at least twice between 6 wk and 5 y apart, including at least once during Predictors of Pregnancy Outcome: Biomarkers in APL Syndrome and SLE (PROMISSE) pregnancy at core laboratories
b SLE is defined as ≥4 revised American College of Rheumatology criteria
c P < .001 compared to women without APO
d History of high blood pressure or taking antihypertensive medications at screening–only patients with blood pressure ≤140/90 mm Hg at screening were enrolled
e P < 0.01 compared to women without APO
Maternal angiogenic factor assay
sFlt1, PlGF, and sEng levels were measured in a blinded fashion and in duplicate using enzyme-linked immunosorbent assay kits (R&D Systems Minneapolis, MN) as previously described. For all assays, minimum detectable was 5 pg/mL and interassay coefficients of variation was <10%.
Statistical methods
Associations between categorical variables were analyzed with Fisher exact test. Student t test and analysis of variance were used for between group comparisons of continuous variables; nonnormally distributed variables were log-transformed prior to performing tests. To maximize flexibility and clinical interpretability in modeling angiogenic factor effects on different categories of APO, hierarchical (conditional log odds) multinomial logistic regression was initially used to estimate unadjusted odds ratios (OR) for: (1) severe vs nonsevere APO; and (2) moderate vs no APO (conditional on nonsevere APO). Because angiogenic profiles early in pregnancy were similar between moderate and no APO groups, subsequent analyses focused on predicting severe APO. A baseline model including only clinical and demographic variables at enrollment was first developed using logistic regression. Decisions regarding variable selection at each step of model development were based on both clinical considerations and statistical significance. When a continuous variable (eg, diastolic blood pressure), yielded similar results whether dichotomized using a clinically justified cutpoint or in the original scale, the more clinically interpretable binary form was chosen. Model performance was evaluated using the Hosmer-Lemeshow test for goodness-of-fit and area under the receiver operating characteristic curve (AUC) to assess discrimination. The incremental prognostic value of angiogenic factors measured at 12-15 weeks and 16-19 weeks was then evaluated by identifying the combination of factors at each time point that most improved model performance when added to the baseline model. The degree of improvement was assessed using both the likelihood ratio (LR) test and the change in AUC, with the corresponding 95% confidence interval (CI) estimated using DeLong et al. To address the potential for overfitting the model to the data, leave-one-out cross-validation was performed. Angiogenic factors measured at the time of or after an APO were excluded from analyses. Missing data in logistic regression analyses were handled with both listwise deletion and multiple imputation (MI) using the Markov chain Monte Carlo approach in the SAS procedure, PROC MI (SAS Institute Inc, Cary, NC). Rates of missing data were 0-8% for baseline variables and 27-29% for 12-15 and 16-19 week angiogenic variables. Given the similarity in parameter estimates across missing data methods, the MI results are provided in supplementary material. Statistical significance was defined as a 2-sided P value <.05. All analyses were conducted using SAS software version 9.4.
Materials and Methods
Patient population
Pregnant subjects were enrolled into PROMISSE from September 2003 through August 2013 at 7 US sites and 1 Canadian site ( Figure 1 ). Institutional review boards at each site approved protocols and consents; written informed consent was obtained from all subjects ( ClinicalTrials.gov identifier NCT00198068 ). Consecutive pregnant women referred to the study with diagnoses of SLE (≥4 American College of Rheumatology criteria for SLE), APL (anticardiolipin antibody [IgG ≥40 GPL units; IgM ≥40 MPL units] and/or positive lupus anticoagulant [RVVT, kaolin, dilute TTI or PTT LA] and/or anti-β2 glycoprotein I [anti-β2 GPI: IgG ≥40 GPL units; IgM ≥40 MPL units] at least twice between 6 weeks and 5 years apart of which one must be during the PROMISSE pregnancy at a core laboratory, as previously described), or both were recruited at <12 weeks’ gestation. Healthy controls of similar ethnicity to patients and at low risk of APO (≥1 successful pregnancy, no fetal deaths, and <2 miscarriages <10 weeks’ gestation) were also enrolled. Consecutive healthy controls who met inclusion criteria were enrolled during their routine visits in general obstetric clinics at PROMISSE patient recruitment sites. This study focuses on women with SLE and/or APL. Clinical determinants of APOs have been previously reported.
At enrollment, all patients met inclusion criteria: live singleton intrauterine pregnancy confirmed by ultrasound; age 18-45 years; hematocrit >26%. Exclusion criteria to minimize confounding by known causes of APO not specifically associated with SLE and/or APL included: prednisone >20 mg/d; 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 1 or 2 diabetes mellitus; and blood pressure >140/90 mm Hg at screening.
Adverse pregnancy outcomes
PROMISSE was designed to identify biomarkers of APOs due to abnormal placental vascularization leading to PE and placental insufficiency. APOs defined for PROMISSE included ≥1 of the following: (1) PE at any time ; (2) fetal death >12 weeks’ gestation unexplained by chromosomal abnormality, anatomic malformation, or congenital infection; (3) neonatal death prior to hospital discharge due to complications of prematurity; (4) preterm delivery or termination of pregnancy <36 weeks due to gestational hypertension, PE, growth restriction, or placental insufficiency; (5) small for gestational age (SGA) <5th percentile at birth. Premature preterm rupture of membranes and/or spontaneous preterm birth were not included as APOs defined for PROMISSE as they are not known to be directly linked to poor placental vascularization. Details of these and other deliveries <36 weeks not counted as APOs are presented in Figure 1 .
To identify markers predictive of the most serious and costly outcomes, APOs were further classified (post hoc) as severe (PE with delivery at <34 weeks, fetal/neonatal death, indicated preterm delivery <30 weeks), and moderate (PE with delivery ≥34 weeks, indicated preterm delivery for reasons other than PE [30-36 weeks], isolated SGA <5th percentile). Outcomes were confirmed by chart review and, if necessary, diagnoses were adjudicated by study obstetricians.
Screening and follow-up visits
Screening evaluation included history, physical examination, complete blood cell count, comprehensive metabolic panel, APL measurement, urinalysis, and random or 24-hour urine for protein/creatinine ratio (if dipstick >1+) ( Table 1 ). Detailed medical and obstetrical information and serial blood samples were obtained monthly.
| Severe APO | Moderate APO | No APO | |
|---|---|---|---|
| n = 59 | n = 49 | n = 384 | |
| DEMOGRAPHICS | |||
| Race/ethnicity | |||
| Non-Hispanic white | 36 (61.0) | 21 (42.9) | 220 (57.3) |
| Hispanic white | 8 (13.6) | 8 (16.3) | 44 (11.5) |
| African American | 9 (15.3) | 12 (24.5) | 60 (15.6) |
| Asian | 4 (6.8) | 7 (14.3) | 41 (10.7) |
| Other | 2 (3.4) | 0 (0) | 12 (3.1) |
| Do not know | 0 (0) | 1 (2.0) | 7 (1.8) |
| Age, y, mean (SD) | 31.0 (4.7) | 30.2 (5.9) | 31.4 (4.8) |
| CLINICAL HISTORY | |||
| Parity | |||
| Nulliparous | 27 (45.8) | 31 (63.3) | 229 (59.6) |
| Parous without history of PE | 24 (40.7) | 15 (30.6) | 132 (34.4) |
| Parous with history of PE | 8 (13.6) | 3 (6.1) | 23 (6.0) |
| Smoking | |||
| Never | 40 (67.8) | 36 (73.5) | 290 (75.9) |
| Ever | 16 (27.1) | 10 (20.4) | 81 (21.2) |
| Current | 3 (5.1) | 3 (6.1) | 11 (2.9) |
| Missing | 0 | 0 | 2 |
| APL a /SLE b status | |||
| APL+/SLE– | 20 (33.9) | 10 (20.4) | 68 (17.7) |
| APL+/SLE+ | 13 (22.0) | 8 (16.3) | 38 (9.9) |
| APL–/SLE+ | 26 (44.1) c | 31 (63.3) | 278 (72.4) |
| Lupus nephritis | |||
| No | 40 (67.8) | 36 (73.5) | 293 (76.3) |
| Yes | 19 (32.2) | 13 (26.5) | 91 (23.7) |
| Thrombosis | |||
| No | 40 (67.8) | 43 (87.8) | 348 (90.6) |
| Yes | 19 (32.2) c | 6 (12.2) | 36 (9.4) |
| High blood pressure d | |||
| No | 43 (72.9) | 37 (75.5) | 337 (87.8) |
| Yes | 16 (27.1) e | 12 (24.5) f | 47 (12.2) |
| PHYSICAL EVALUATION | |||
| BMI, kg/m 2 | |||
| <25 | 22 (40.0) | 23 (50.0) | 225 (63.7) |
| 25–30 | 14 (25.5) | 12 (26.1) | 78 (22.1) |
| >30 | 19 (34.5) e | 11 (23.9) | 50 (14.2) |
| Missing | 4 | 3 | 31 |
| Blood pressure, mm Hg | |||
| Systolic mean (SD) | 117.9 (12.0) c | 116.2 (13.9) e | 110.3 (11.7) |
| Diastolic mean (SD) | 73.0 (9.0) c | 70.2 (11.0) f | 66.6 (8.9) |
| MEDICATIONS | |||
| Aspirin | |||
| No | 31 (52.5) | 25 (51.0) | 175 (45.6) |
| Yes | 28 (47.5) | 24 (49.0) | 209 (54.4) |
| Heparin | |||
| No | 22 (37.3) | 32 (65.3) | 255 (66.4) |
| Yes | 37 (62.7) c | 17 (34.7) | 129 (33.6) |
| Antihypertensive | |||
| No | 49 (83.1) | 39 (79.6) | 365 (95.1) |
| Yes | 10 (17.0) e | 10 (20.4) c | 19 (5.0) |
| LABORATORY VALUES | |||
| LAC status during pregnancy | |||
| Negative | 29 (49.2) | 38 (77.6) | 334 (87.0) |
| Positive | 30 (50.9) c | 11 (22.4) | 50 (13.0) |
| Proteinuria | |||
| Negative | 54 (91.5) | 42 (85.7) | 360 (94.8) |
| Positive | 5 (8.5) | 7 (14.3) | 24 (6.3) |
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