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To compare euploid pregnancies with congenital heart defects (CHDs) to similar pregnancies without CHDs on typically collected second trimester biomarker measurements.Second trimester serum levels of alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), and unconjugated estriol were compared for 306 CHD cases and 1224 no-CHD controls drawn from a sample of singleton pregnancies without chromosomal or neural tube defects (NTDs). Logistic regression models were built comparing biomarkers for cases and controls.Regardless of the severity of defect, CHD cases were more likely to have unusually high AFP and/or hCG levels and/or unusually low hCG and/or uE3 levels [odds ratio (OR) 1.8–2.4, 95% confidence intervals (CIs) 1.2–4.0]. Cases with critical CHDs were more than twice as likely to have an AFP multiple of the median (MoM) ≥ the 95th percentile and/or an hCG and/uE3 MoM ≤ the 5th percentile (OR 2.1–3.9, 95% CIs 1.1–7.8).Abnormal levels of specific second trimester maternal serum biomarkers indicated an increased risk for CHDs among this sample of low risk pregnancies. Our data suggest that future efforts aimed at improving CHD detection in low risk pregnancies may benefit from considering serum biomarkers. Copyright © 2011 John Wiley & Sons, Ltd.