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Theoretically, repeated sampling of free β-human chorionic gonadotropin (hCGβ) and pregnancy associated plasma protein-A (PAPP-A) in the first trimester of pregnancy might improve performance of risk assessment of trisomy 21 (T21). To assess the performance of a screening test involving repeated measures of biochemical markers, correlations between markers must be estimated. The aims of this study were to calculate the autocorrelation and cross-correlation between hCGβ and PAPP-A in the first trimester of pregnancy and to investigate the possible impact of gestational age at the first sample and time between sampling on the correlation.A prospective study was conducted including 3891 unaffected singleton pregnancies. Two measurements of hCGβ and PAPP-A were obtained during the first trimester in each pregnancy. Correlations between the four parameters, hCGβ first, hCGβ second, PAPP-A first and PAPP-A second, were estimated and presented in terms of Pearson's r coefficients. Furthermore, the correlation between paired samples as a function of time between samples was investigated.The study demonstrated high correlation between first and second samples of hCGβ and PAPP-A with a correlation coefficient of 0.80 and 0.79, respectively. By contrast, the correlations between hCGβ and PAPP-A were low. In addition, the study demonstrated that the correlation between paired samples of hCGβ and PAPP-A decreases with earlier gestational age at the first sample and with increasing time between samples.We have developed a parameter set in terms of correlations between biochemical markers, which can be incorporated into a T21 screening algorithm based on repeated measures within the first trimester.