First-Trimester Cell-Free DNA Screening for Common Trisomies Using Rolling-Circle Amplification: A Prospective Study

To evaluate cell-free DNA screening for trisomies 21, 18 and 13 using rolling circle replication in an unselected routine first-trimester screening population.

This study enrolled 3025 women undergoing first-trimester screening. Excluding unavailable samples, missing outcome data, pregnancy loss and twin gestation, 2729 pregnancies with follow-up remained; 2689 had cell-free DNA results for performance analysis. Cell-free fetal DNA was assessed by rolling circle replication and digital fluorescence detection without PCR or sequencing. Performance was confirmed with cytogenetic results or postnatal outcomes.

Among 2729 eligible singleton pregnancies with follow-up, valid cfDNA results were obtained in 2689 cases, corresponding to an overall no-call rate of 40/2729 (1.47%). For trisomy 21, sensitivity was 14/14 (100%; 95% CI, 76.8-100), specificity was 2666/2675 (99.7%; 95% CI, 99.4-99.8), and PPV was 14/23 (60.9%; 95% CI, 38.5-80.3). For trisomy 18, sensitivity was 1/2 (50.0%; 95% CI, 1.3-98.7), specificity was 2681/2687 (99.8%; 95% CI, 99.5-99.9), and PPV was 1/7 (14.3%; 95% CI, 0.4-57.9). No confirmed trisomy 13 cases were observed. Fetal sex was correctly assigned in 2657/2688 cases (98.8%). Testing before 10 weeks' gestation and in IVF pregnancies appeared technically feasible, but these subgroup findings were limited by small numbers.

Rolling circle replication-based cell-free DNA screening showed high accuracy for trisomy 21 and fetal sex determination in this unselected routine first-trimester screening population. However, the small number of trisomy 18 and trisomy 13 cases limits conclusions for these aneuploidies.