Novel Early Pregnancy Multimarker Screening Test for Preeclampsia Risk Prediction

Abstract

Preeclampsia (PE) is a common pregnancy-linked disease, causing preterm births, complicated deliveries, and health consequences for mothers and offspring. We have previously developed 6PLEX, a multiplex assay that measures PE-related maternal serum biomarkers ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 in a single test tube. This study investigated the potential of 6PLEX to develop novel PE prediction models for early pregnancy. We analyzed 132 serum samples drawn at 70-275 gestational days (g days) from 53 pregnant women (PE, n = 22; controls, n = 31). PE prediction models were developed using a machine learning strategy based on the stepwise selection of the most significant models and incorporating parameters with optimal resampling. Alternative models included also placental FLT1 rs4769613 T/C genotypes, a high-confidence risk factor for PE. The best performing PE prediction model using samples collected at 70-98 g days comprised of PTX3, sFlt-1, and ADAM12, the subject's parity and gestational age at sampling (AUC 0.94 [95%CI 0.84-0.99]). All cases, that developed PE several months later (onset 257.4 ± 15.2 g days), were correctly identified. The model's specificity was 80% [95%CI 65-100] and the overall accuracy was 88% [95%CI 73-95]. Incorporating additionally the placental FLT1 rs4769613 T/C genotype data increased the prediction accuracy to 93.5% [AUC = 0.97 (95%CI 0.89-1.00)]. However, 6PLEX measurements of samples collected at 100-182 g days were insufficiently informative to develop reliable PE prediction models for mid-pregnancy (accuracy <75%). In summary, the developed model opens new horizons for first-trimester PE screening, combining the easily standardizable 6PLEX assay with routinely collected antenatal care data and resulting in high sensitivity and specificity.

Keywords: 6PLEX; PTX3; Preeclampsia; biomarkers; early pregnancy; multiplex immunoassay; risk prediction; statistical modeling.

Figure 1

Dynamics of preeclampsia-related biomarkers throughout gestation in healthy and preeclamptic pregnancies. Luminex^® 6PLEX assay measurement data of concentrations of ADAM12, PTX3, PlGF, sENG, and sFlt-1 in 132 serum samples collected from pregnant women. The early pregnancy group was comprised of 14 samples drawn at 70–98 g days from women that later developed PE and 20 samples from controls collected at 76–96 g days. The mid-pregnancy sample set comprised 18 PE and 21 control pregnancy sera, sampled at 100–180 and 109–182 g days, respectively. Data of late pregnancy samples representing 28 PE (206–275 g days) and 31 control sera (210–274 g days) were obtained from our recent study (15). All women, irrespective of the pregnancy outcome (PE or no-PE), were normotensive at blood draw. The whiskers on the plot show the median with an interquartile range. The statistical difference in biomarker distributions between PE and control cases was compared using the Mann-Whitney U-test, ^* representing P < 0.05 and ^** P < 0.0001. ADAM12, ADAM Metallopeptidase Domain 12; g days, gestational days; PlGF, placental growth factor; PTX3, Pentraxin3; sENG, endoglin; sFlt-1, soluble fms-like tyrosine kinase 1.

Figure 2

Distribution of maternal serum biomarkers measured with the Luminex^® 6PLEX assay during early pregnancy, stratified by the later onset of preeclampsia (PE). Samples from women that later developed PE (n = 14, drawn at 70–98 gestational days) were compared to samples representing uncomplicated gestations until term (n = 20, 76–96 gestational days; Table 1). The whiskers on the plot show the median with an interquartile range. The statistical difference in biomarker distributions between PE and control cases was compared using the Mann-Whitney U-test, * representing P<0.05. ADAM12, disintegrin and metalloproteinase domain-containing protein 12; PlGF, placental growth factor; PTX3, pentraxin-3; sENG, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase-1.

Figure 3

ROC curves, AUC values, and the formulas of the best performing preeclampsia (PE) prediction models were developed based on the Luminex^® 6PLEX assay data. The best PE prediction models are based on the analysis of serum samples collected during either (A) 70th-98th or (C) 100th−182nd g days. Early pregnancy model 1A incorporated data from three biomarkers (sFlt-1, ADAM12, and PTX3) and model 3C four markers (sFlt-1, sEng, ADAM12, and PTX3), whereas mid-pregnancy models 4 and 5 are based on only PlGF measurements. Model 3C and model 5 additionally included placental genotype data of the FLT1 rs4769613 T/C variant. All models were adjusted for gestational days (gest. days) at sampling and maternal parity (nulliparity or multiparity). (B) PE prediction formulae for models 1A and 3C. Details are presented in Tables 4, 5 and Supplementary Table S3. AUC, the area under the curve; ROC, receiver operating characteristics.

Figure 4

Spearman's rank correlation plot between sENG and sFlt-1 measurements of sera drawn from pregnant women within 70 and 98 gestational days. Gray around the linear regression line (y = 0.7651x + 1,931.4) indicates the 95% confidence region. sENG, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase-1.