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The use of the second trimester alpha-fetoprotein (AFP) along with the first trimester pregnancy-associated plasma protein-A (PAPP-A) has been found to be useful in the estimation of unfavourable pregnancy outcome. Our aim in this study was to determine the relationship between maternal PAPP-A and b-hCG and AFP concentrations in spontaneous preterm birth (sPTB). This prospective cohort study included 372 singleton pregnancies with PAPP-A, b-hCG and AFP levels in the first trimester, which were converted to multiples of the median (MoM). The predictive ability of AFP-to-PAPP-A and AFP-to-b-hCG ratios for sPTB was evaluated. The risk for sPTB ≤34 weeks increased in women with AFP-to-PAPP-A ratio >7 (OR 2.9, 95% CI 1.2-6.4). Women with AFP-to-b-hCG ratio >0.6 had a 3.5-fold higher risk for sPTB ≤32 weeks. Increased maternal AFP-to-PAPP-A or AFP-to-b-hCG ratios in the first trimester may help to predict pregnant women at high risk for sPTB, and this may be beneficial in developing management plans.Impact Statement What is already known on this subject? There is a synergistic association between the combination of low pregnancy-associated plasma protein-A (PAPP-A) in the first trimester with alpha-fetoprotein (AFP) in the second trimester with subsequent development of PTB. Maternal serum biochemical markers measured as a part of aneuploidy screening are reflective of pregnancy adverse outcomes related with placental insufficiency. PAPP-A and AFP have a low predictive ability to determine women at high risk for preterm birth. What do the results of this study add? Elevated AFP:PAPP-A or AFP:B-HCG ratio in the first trimester is associated with increased risk for sPTB. The ratios of these biochemical markers in the first trimester may be beneficial to identify women at high risk for sPTB. What are the implications of these findings for clinical practice and/or further research? The ratios may predict pregnant women at high risk for sPTB, and such risk may be helpful in the development of a management plan. Incorporation of AFP:PAPP-A or AFP:B-HCG ratios in the first trimester may help to improve the screening efficacies, and provide a simple alternative tool.