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.
Stampalija, T., Forcato, C., Grati, F. R., Volpe, P., De Robertis, V., Izzi, C., Bertucci, E., Fabietti, I., Novelli, A., Ornaghi, S., Pasquini, L., Bevilacqua, E., Paladini, D., Ghi, T., Lattuada, D., Dori, M., Mercatelli, D., Dal Molin, A., Buson, G., Bolognesi, C., Doffini, A., Musci, T. J., Ferrazzi, E., Giovannone, E. D., Mangano, C., Maranta, C., Amadesi, C., Brocco, A., Casadei, A., Garrì, M., Maiocchi, R., Molinaro, I., Ortolan, E., Piano, A., Iannitiello, M. C., Lisi, D., Feresin, A., Gasparini, P., Fregona, C., Rembouskos, G., Odicino, F. E., Signorelli, M., Sponzilli, A., Facchinetti, F., Vassallo, C., Nicastri, E., Di Gregorio, M. G., Orlando, V., Cozzolino, S., Verderio, M., Masini, G., Franchi, C., Tiziano, F. D., Pasquetti, D., Biancotto, G., Della Sala, F., Volpe, N., Dall'Asta, A., Silipigni, R., Catusi, I., Single-cell-based non-invasive screening for fetal pathogenic microimbalances using maternal blood: comparison with invasive prenatal diagnosis, <<ULTRASOUND IN OBSTETRICS & GYNECOLOGY>>, 2026; 67 (4): 500-509. [doi:10.1002/uog.70201] [https://hdl.handle.net/10807/336821]
Single-cell-based non-invasive screening for fetal pathogenic microimbalances using maternal blood: comparison with invasive prenatal diagnosis
Bevilacqua, Elisa;Ghi, Tullio;Tiziano, Francesco Danilo;
2026
Abstract
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.
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