Single‐cell‐based non‐invasive screening for fetal pathogenic microimbalances using maternal blood: comparison with invasive prenatal diagnosis

Objective Pathogenic or likely pathogenic copy-number variants (p/lpCNVs) are a significant cause of perinatal morbidity and mortality. Current prenatal screening based on cell-free DNA (cfDNA) fails to detect the majority of microimbalances (microdeletions/microduplications), leaving a significant residual risk of undetected chromosomal abnormalities. This study evaluated the clinical performance of a novel single-cell-sequencing-based non-invasive prenatal testing (scsbNIPT) method utilizing circulating extravillous trophoblasts (cEVTs) for the detection of fetal p/lpCNVs, particularly microimbalances < 8 Mb. Methods This was a prospective, blinded, observational multicenter cohort study of 1390 high-risk pregnant women undergoing prenatal invasive diagnostic testing between November 2021 and December 2023. A 20-mL maternal blood sample was collected from each subject between 11 + 0 and 22 + 6 weeks' gestation prior to invasive sampling. cEVTs were isolated and subjected to whole-genome sequencing, using a proprietary workflow. scsbNIPT results were compared with standard invasive prenatal diagnostic results obtained by karyotyping and/or chromosomal microarray analysis. Results scsbNIPT showed a sensitivity of 92.9% (95% CI, 76.5-99.1%) and a specificity of 98.2% (95% CI, 97.0-99.0%) for the detection of genome-wide microimbalances measuring >= 300 kb to < 8 Mb. The sensitivity for p/lpCNVs >= 300 kb in pregnancies screened at 11 + 0 to 14 + 6 weeks was 100% (95% CI, 83.9-100%). For trisomy 21, the sensitivity of scsbNIPT was 98.0% (95% CI, 92.9-99.8%) and the specificity was 99.7% (95% CI, 99.0-99.9%). Conclusions This study demonstrates the scientific validity and clinical utility of scsbNIPT for the non-invasive detection of genome-wide fetal p/lpCNVs, particularly microimbalances, with high sensitivity and a resolution comparable to that of chromosomal microarray analysis. scsbNIPT may offer more complete screening for genome-wide p/lpCNVs, markedly lowering the residual risk early in pregnancy compared with existing cfDNA-based methods.