Supplementary Material for: Early Prediction of Hypertensive Disorders of Pregnancy Using Cell-Free Fetal DNA, Cell-Free Total DNA, and Biochemical Markers

<br><strong><em>Objective:</em></strong> To evaluate the predictive value of separate and combined tests using cell-free fetal DNA (cffDNA), cell-free total DNA (cfDNA), and biochemical markers for the early detection of pregnancies with hypertensive disorders. <b><i>Methods:</i></b> A nested case-control study was conducted with 135 singleton pregnancies including 17 gestational hypertension cases, 34 preeclampsia (PE) cases, and 84 controls. We performed real-time quantitative PCR to measure levels of <i>DSCR3</i> and <i>RASSF1A</i> as cffDNA markers and <i>HYP2</i> as a cfDNA marker in the first and early second trimesters. Levels of pregnancy-associated plasma protein A (PAPP-A), α-fetoprotein, β-human chorionic gonadotropin, unconjugated estriol, and inhibin A were also determined. <b><i>Results:</i></b> Compared with controls, the median levels and multiples of the median (MoM) values of <i>HYP2</i> were significantly higher in the PE and hypertensive disorders of pregnancy (HDP) groups at 6-14 and 15-23 weeks. Frist-trimester PAPP-A MoM was significantly lower in PE and HDP than in controls. For PE and HDP, the best model included the first-trimester <i>DSCR3</i>, <i>HYP2</i>, and PAPP-A MoM values achieving detection rates of 67 and 58% at a fixed 10% false-positive rate, respectively [area under the receiver operating characteristic curve 0.832 (95% CI 0.689-0.928) for PE; 0.751 (0.607-0.863) for HDP]. <b><i>Discussion:</i></b> The study demonstrates the potential utility of combined first-trimester cffDNA, cfDNA, and PAPP-A for the early prediction of PE.