Extended Non‐Invasive Prenatal Testing for Chromosomal and Monogenic Disorders by Cell‐Free DNA Sequencing: A Blinded Comparison With Amniocentesis

Objective To determine whether molecular integrated non‐invasive fetal exome sequencing (iNIFS), using clinical exome sequencing of maternal‐plasma cell‐free DNA, concords per pregnancy with paired amniocentesis. Method Prospective, blinded, single center, paired sample diagnostic accuracy study in 641 consecutive pregnancies referred for clinically indicated amniocentesis at 16–18 weeks. Plasma cell‐free DNA underwent low‐pass whole‐genome sequencing for aneuploidies and CNVs and, after short‐fragment enrichment, clinical exome sequencing within a parent fetus trio design. The reference standard comprised QF‐PCR, karyotyping, array‐comparative genomic hybridization and, where indicated, trio clinical exome sequencing on amniotic‐fluid DNA. Results iNIFS was reportable in 624/641 pregnancies (97.3%); 612 (95.5%) had complete paired data and formed the analysis population. The reference established a pathogenic or likely pathogenic finding in 107/612 (17.5%). NIPT gave 106 true positives, 503 true negatives, 2 discordant positives and 1 discordant negative: sensitivity 99.1% (95% CI 94.9–99.8), specificity 99.6% (98.6–99.9), PPV 98.1% (93.5–99.5), NPV 99.8% (98.9–100.0), agreement 99.5% and Cohen's κ 0.98. The discordant negative was a maternally inherited variant, the class for which distinguishing maternal from fetal contributions is most challenging. Conclusion Molecular iNIFS showed high concordance with paired amniocentesis across chromosomal, copy‐number and monogenic findings, while retaining an explicit no call rate. Multicentre validation and invasive confirmation of positive findings remain necessary.