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In the Netherlands, prenatal screening for trisomy 21 in the first trimester of pregnancy for singletons is conducted through a combined test based on maternal age, nuchal translucency measurement and maternal serum free β-hCG and PAPP-A. In our clinic risk calculations in twins are currently based on the NT of both fetuses instead of the combined test. In this study we looked at differences in early first-trimester free β-hCG and PAPP-A between mono- and dichorionic twins.A total of 202 twin pregnant women participated in the study and agreed to donate first-trimester serum for research.The data of 200 twins with normal outcome were used for setting up reference values for free β-hCG and PAPP-A. Trisomy 21 was identified in the two remaining pregnancies. The overall median weight-corrected MoM was 1.99 for free β-hCG, and 2.14 for PAPP-A in all twins. Monochorionic twins have a significantly lower free β-hCG weight-corrected MoM (1.53 vs 2.11; Mann-Whitney U, p = 0.002) and a significantly lower PAPP-A weight-corrected MoM (1.59 vs 2.40; Mann-Whitney U, p = 0.003) compared to dichorionic.This study strengthened the need to make a distinction between mono- and dichorionic twins for the risk calculation in Down syndrome screening as biochemical markers are significantly lower in monochorionic than in dichorionic twins.