Extracellular vesicles yield predictive pre-eclampsia biomarkers

Abstract

Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population.

Keywords: Exosomes; PAI1; PIGF; TIMP1; annexin V; biomarkers; cholera toxin B chain; pre-eclampsia.

Figure 1.

a, b, Concentration of GH, PCT, in PE and healthy pregnant women. Prospectively banked plasma samples collected at 34–36 weeks of gestation from 10 pre-eclampsia and 20 matched healthy pregnant patients were assayed for Growth Hormone (GH) and Procalcitonin (PCT), in the plasma, plasma CTB-EV and plasma AV-EVs.

Figure 2.

a, b, c Concentration of PAI1, PIGF, TIMP1 in PE and healthy pregnant women. Prospectively banked plasma samples collected at 34–36 weeks of gestation from 10 pre-eclampsia and 20 matched healthy pregnant patients were assayed for Plasminogen Activator Inhibitor; Type I (PAI1), Placental Growth Factor (PlGF), and Tissue Inhibitor Of Metalloproteinases 1 (TIMP1) in the plasma, plasma CTB-EV and plasma AV-EVs.

Figure 3.

a, b, c Concentration of PAI1, PlGF and TIMP1 in healthy pregnant women of 3 different races. The concentration of PAI1, TIMP1, and PlGF in plasma, plasma CTB-EV and plasma AV-EVs from 20 Chinese, 20 Malay and 20 Indian healthy pregnant patients at 34–39 weeks of gestation were assayed.

Figure 4.

ROC curves for CTB-TIMP, AV-PAI and plasma PlGF. Univariate and multivariate receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) were calculated for CTB-TIMP, AV-PAI, plasma PlGF and the combination of all three biomarkers by presetting sensitivity at 100%.