Study Conduct

From March 2012 through April 2013, we enrolled pregnant women undergoing first-trimester aneuploidy screening at 35 centers in six countries. At enrollment, maternal blood was drawn, locally deidentified, and sent for risk assessment for trisomy 21 with the use of cfDNA testing (Harmony Prenatal Test, Ariosa Diagnostics). We submitted the results of cfDNA testing and standard screening to an independent data-coordinating center (Veristat). We then collected pregnancy outcomes for all participants who met the eligibility criteria and completed standard screening. The institutional review board at each participating site approved the study. Written informed consent was obtained from all the participants.

Study Oversight

The study was a collaboration between the clinical investigators and the sponsors (Ariosa Diagnostics and the Perinatal Quality Foundation). The first and last authors designed the protocol in collaboration with the sponsor. Representatives of the sponsor performed the analyses and interpretation of cfDNA data; data regarding maternal and gestational age were required. Laboratory personnel performed their analyses in a blinded fashion with respect to all other clinical data, including results of ultrasonographic and standard screening. Research staff members at the clinical sites entered clinical and laboratory data into an electronic case-report form, which was stored in a secure database. The data-coordinating center compiled and analyzed the laboratory and clinical data. Ariosa supervised data accrual, participated in the preparation of the manuscript, and approved the final version of the manuscript. Veristat performed the primary analysis; secondary analyses were conducted by Ariosa. The first author wrote the first draft of the manuscript. All the authors vouch for the accuracy of the data and fidelity of the study to the protocol (available with the full text of this article at NEJM.org) and approved the submission of the manuscript for publication. There were no confidentiality agreements among the authors, sites, or sponsor.

Study Population and Sample Collection

Eligible patients were at least 18 years of age and had a singleton pregnancy between 10.0 and 14.3 weeks of gestation at the time of the study blood-sample collection. Gestational age was determined according to the crown–rump length at the time of the measurement of nuchal translucency.

Patients were ineligible if they were outside the gestational-age window, had no standard screening result, had known maternal aneuploidy or cancer, had conceived with the use of donor oocytes, or had a twin pregnancy or an empty gestational sac that was identified on ultrasonography. Peripheral blood was collected into two Cell-free DNA BCT tubes (Streck) that were labeled with a unique patient identifier. Samples were sent to the Ariosa clinical laboratory, which is certified according to the Clinical Laboratory Improvement Amendments, without further processing. Results for cfDNA testing were not available to providers or participants.

Testing Methods

All patients underwent standard screening (including the measurement of serum pregnancy-associated plasma protein A, total or free beta subunit of human chorionic gonadotropin, and nuchal translucency) with the use of local laboratories. All providers of nuchal translucency were certified by the Nuchal Translucency Quality Review program, the Fetal Medicine Foundation, or other national quality-review programs. All measurements of nuchal translucency were performed and serum samples collected within the gestational age range required by the local laboratory.

For clinical risk assessment, we used local risk algorithms and cutoffs according to standard clinical practice. For study purposes, one of the authors used a standard algorithm5 to recalculate risk using serum multiples of the median (MoM) and measurements of nuchal translucency and crown–rump length. A positive result on standard screening was defined as a mid-trimester risk of at least 1 in 270 for trisomy 21 and at least 1 in 150 for trisomy 18 and trisomy 13, cutoffs that are commonly used by laboratories in the United States.

Details on Ariosa laboratory testing methods have been described previously.6-8 For cfDNA testing, samples were rejected if they were not collected in Cell-free DNA BCT tubes; if the tubes were broken, unfilled, or not labeled; or if the sample was grossly hemolyzed or arrived in the laboratory more than 7 days after collection. Each acceptable sample underwent plasma separation and cfDNA isolation, followed by ligation of locus-specific oligonucleotides to produce a template from selected genomic loci (Fig. S1 in the Supplementary Appendix, available at NEJM.org). We estimated the risk of aneuploidy using a previously described algorithm, including chromosome cfDNA counts, fetal fraction of cfDNA, and a priori trisomy risk based on maternal and gestational age8 (Fig. S2 in the Supplementary Appendix). A risk of 1 in 100 or higher was the laboratory-designated threshold for classifying a sample as high risk. Samples were not included in the analyses if they did not pass laboratory quality control because of a low fraction of fetal cfDNA (<4%), an inability to measure the fraction of fetal cfDNA, a high variation in cfDNA counts, or an assay failure.

Pregnancy and Newborn Outcomes

We recorded all pregnancy outcomes, including miscarriage, termination, and delivery. Results of invasive prenatal diagnostic testing and testing of products of conception (i.e., miscarriages) were collected when available. Newborn outcomes were determined by medical-record review of the physical examination at birth and any genetic testing performed. In the absence of genetic testing, a newborn with a normal physical examination was considered to be euploid. The results for women who had a miscarriage, chose to terminate the pregnancy, or had a stillbirth were included only if confirmatory genetic testing was performed; those without genetic analysis were excluded. In a blinded fashion, the first and last authors reviewed medical records of all neonates with congenital anomalies and excluded those with phenotypes suggestive of aneuploidy if no confirmatory genetic testing was performed. Results of fetal and newborn genetic testing were adjudicated by two clinical geneticists, categorized as euploid or aneuploid, and classified according to the type of abnormality.

Data Handling

We transferred the results of cfDNA testing, standardized risk scores for standard screening, and clinical data to the independent data-coordinating center for consolidation and unblinding. The primary-analysis population included all eligible participants who had results on both cfDNA testing and standard screening and a documented normal or adjudicated newborn examination or results of prenatal or postnatal genetic testing.

Study Outcomes

The primary outcome was the area under the receiver-operating-characteristic (ROC) curve (AUC) for trisomy 21 screening with cfDNA testing versus standard screening in women with complete results for the two tests. Secondary outcomes included the evaluation of cfDNA testing and standard screening to assess the risk of trisomies 18 and 13. The evaluation of the performance of cfDNA testing for trisomy 13 included only patients who were enrolled after the introduction of the analysis in September 2012. We also evaluated the performance of cfDNA testing in low-risk patients, who were defined as having a maternal age of less than 35 years or a risk of trisomy 21 of less than 1 in 270 on standard screening.

Statistical Analysis

Standard screening and cfDNA testing each produces a measured value representing the risk of each aneuploidy. The ROC curve was generated by computing sensitivity and specificity at each observed cutoff for risk score. We calculated the differences between the ROC curves, taking into account the paired nature of the data. AUC values were compared with the use of a z-test according to the method of DeLong et al.9 A P value of less than 0.05 was considered to indicate statistical significance. Confidence intervals were computed with the use of the Clopper–Pearson method. We used the exact binomial test10 for paired comparisons in sensitivity and specificity and used the generalized score statistic11 to analyze positive and negative predictive values. We compared the sensitivity, specificity, positive and negative predictive values, and likelihood ratios of standard screening and cfDNA testing for the detection of trisomy 21.

On the basis of results of previous studies12,13 and assumptions with respect to the performance of cfDNA testing, we determined that a sample size of 32 cases of trisomy 21 and 1500 negative controls would provide a power of 80% to determine the primary outcome at an alpha level of 0.05. To detect an increase to an AUC of 0.95 for cfDNA testing at a power of 80% and with a prevalence of 1 in 700 for trisomy 21, we estimated that 22,400 participants would be required. To account for loss to follow-up, we planned to enroll 25,000 participants. Using the maternal age of enrolled participants mid-trial, we revised the estimate of the prevalence of trisomy 21 at 1 in 500, and we reduced the required sample size to 18,700. Interim study outcomes were not unblinded or considered in the decision to stop enrollment before achieving the planned sample size.