*3.1.2.5. Multiple marker screening in the second trimester of pregnancy*

Second-trimester serum screening could be applied between 14 and 22 weeks of gestation, but usually it is carried out at 15–18 weeks of pregnancy. The serum markers could be combined in different ways but the most commonly used tests are double test (association between aFP and hCG), triple test (association between aFP, μE3 and hCG), and quadruple test (association between aFP, μE3, hCG and INH-A) [42, 44, 45]. The values obtained by these tests are combined based on a multivariate Gaussian model and finally a risk algorithm is obtained [46].

A very important parameter is the age of gestation and this must be established using an ultrasound determination of gestational age (best results with crown-rump measurement between 8 and 11 weeks) [47]. The risk algorithm must include other variables like maternal weight, ethnicity, maternal diseases (diabetes, systemic lupus erythematosus), multiple gestation, smoking, in vitro fertilization, sex of the fetus or maternal rhesus blood type [48–56]. In unaffected twins' pregnancy, the concentrations of maternal serum markers are twofold higher than that seen in unaffected singleton pregnancies and thus the algorithm needs an adjustment by using a "pseudo-risk" [51].
