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

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