Validation of a method for noninvasive prenatal testing for fetal aneuploidies risk and considerations for its introduction in the Public Health System

Objective: The aim of this study was to validate noninvasive prenatal testing (NIPT) for fetal aneuploidies by whole-genome massively parallel sequencing (MPS). Methods: MPS was performed on cell-free DNA (cfDNA) isolated from maternal plasma in two groups: a first set of 186 euploid samples and a second set of 195 samples enriched of aneuploid cases (n=69); digital PCR for fetal fraction (FF) assessment was performed on 178/381 samples. Cases with <10 x 10(6) reads (n=54) were excluded for downstream data analysis. Follow-up data (invasive testing results or neonatal information) were available for all samples. Performances in terms of specificity/sensitivity and Z-score distributions were evaluated. Results: All positive samples for trisomy 21 (T21) (n=43), trisomy 18 (T18) (n=6) and trisomy 13 (T13) (n=7) were correctly identified (sensitivity: 99.9%); 5 false positive results were reported: 3 for T21 (specificity=98.9%) and 2 for T13 (specificity=99.4%). Besides FF, total cfDNA concentration seems another important parameter for MPS, since it influences the number of reads. Conclusions: The overall test accuracy allowed us introducing NIPT for T21, T18 and T13 as a clinical service for pregnant women after 10+4 weeks of gestation. Sex chromosome aneuploidy assessment needs further validation due to the limited number of aneuploid cases in this study.