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Measurement of cell-free fetal DNA (cffDNA) is an indispensable process for non-invasive prenatal screening (NIPS). According to recent studies, cffDNA in maternal plasma can be enriched for various lengths of fragments, and a sufficient amount of cffDNA can effectively eliminate background interference on the part of maternal DNA. Therefore, we developed a simple and effective separation method, improved NIPS (iNIPS), that enriches the fetal fraction and improves the accuracy of NIPS for fetal aneuploid detection. We adopted a novel strategy to achieve enrichment of 125–135 bp cell-free DNA (cfDNA) by e-gel electrophoresis. To evaluate clinical performance, we compared NIPS and iNIPS results from 2153 retrospective clinical samples. Of the 22 samples with NIPS results of “no call”, 17 samples were reclassified as “unaffected” (9 cases of chr13, 5 cases of chr18, and 3 cases of chr21); 2 samples remained classified as “no call” (1 case of chr18 and 1 case of chr21); and 3 samples were identified as T21 by iNIPS. The average increase in abundance of cfDNA fragments of 125–135 bp was 2.5 times, and the average decrease in maternal background interference was 1.3 times. On this basis, the detection of fetal aneuploidy was highly improved with the fetal fraction as low as 2%; iNIPS achieved 100% sensitivity and 99.90% specificity in retrospective samples.

Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference