Objective
We sought to assess performance of noninvasive prenatal testing for fetal trisomy in a routinely screened first-trimester pregnancy population.
Study Design
This was a cohort study of 2049 pregnant women undergoing routine screening for aneuploidies at 11-13 weeks’ gestation. Plasma cell-free DNA analysis using chromosome-selective sequencing was used. Laboratory testing on a single plasma sample of 2 mL was carried out blindly and results were provided as risk score (%) for trisomies 21 and 18.
Results
Trisomy risk scores were given for 95.1% (1949 of 2049) of cases including all 8 with trisomy 21 and 2 of the 3 with trisomy 18. The trisomy risk score was >99% in the 8 cases of trisomy 21 and 2 of trisomy 18 and <1% in 99.9% (1937 of 1939) of euploid cases.
Conclusion
Noninvasive prenatal testing using chromosome-selective sequencing in a routinely screened population identified trisomies 21 and 18 with a false-positive rate of 0.1%.
In the last 40 years, screening and diagnosis of fetal aneuploidies has shifted from second-trimester amniocentesis for advanced maternal age, with a detection rate (DR) of trisomy 21 of 30% at false-positive rate (FPR) of 5%, to first-trimester chorionic villous sampling (CVS) in the high-risk group identified by screening with fetal nuchal translucency (NT) and serum biochemistry, with DR of about 90% at FPR of 3-5%. Invasive testing with CVS or amniocentesis is diagnostic, but associated with the risk of miscarriage.
For Editors’ Commentary, see Contents
Recently, noninvasive prenatal testing (NIPT) by analysis of cell-free DNA (cfDNA) in maternal blood has shown promise for highly accurate detection of common fetal autosomal trisomies. Analysis of cfDNA has been validated in several clinical studies utilizing next-generation DNA sequencing technology. Clinical studies have primarily included women identified by prior screening, with maternal age and biochemical and/or sonographic testing in the first or second trimester of pregnancy, to be at high risk for aneuploidies. It has therefore been uncertain if the results of NIPT in such high-risk pregnancies are applicable to the general pregnancy population.
The objective of this study is to assess the performance of screening by NIPT for trisomies 21 and 18 using a chromosome-selective sequencing method of cfDNA in maternal plasma obtained from a population undergoing routine screening at 11-13 weeks’ gestation.
Materials and Methods
Study population
The data for this study were derived from analysis of stored maternal plasma obtained during prospective first-trimester combined screening for aneuploidies in women with singleton pregnancies attending for their routine first hospital visit in pregnancy. In this visit, which was held at 11 +0 -13 +6 weeks of gestation, we recorded maternal characteristics and medical history and performed an ultrasound scan to: firstly, determine gestational age from the measurement of the fetal crown-rump length; secondly, diagnose any major fetal abnormalities; thirdly, measure fetal NT thickness; and fourthly, assess the nasal bone (NB) as present or absent, the flow across the tricuspid valve as normal or regurgitant (tricuspid regurgitation [TR]), and the a-wave in the ductus venosus (DV) as normal or reversed. In addition, the maternal serum concentrations of pregnancy-associated plasma protein (PAPP)-A and free β-human chorionic gonadotrophin (hCG) were determined within 10 minutes of blood collection using automated machines (DELFIA Xpress system, PerkinElmer Life and Analytical Sciences, Waltham, MA). Biophysical and biochemical markers were combined to estimate the patient-specific risk for trisomies 21, 18, and 13. Women were given their estimated individual risk for these trisomies and those considering their risk to be high were offered CVS for fetal karyotyping. Karyotype results, obtained from genetic laboratories, and details on pregnancy outcomes, obtained from the maternity computerized records or the general medical practitioners of the women, were added into the database as soon as they became available.
Venous blood was obtained from women who gave written informed consent to provide samples for research into early prediction of pregnancy complications, which was approved by the National Research Ethics Service of the National Health Service. Blood was collected in EDTA BD Vacutainer tubes (Becton Dickinson UK Ltd, Oxfordshire, United Kingdom) and processed within 15 minutes of collection. The tubes were centrifuged at 2000 g for 10 minutes to separate plasma from packed cells and buffy coat and subsequently at 16,000 g for 10 minutes to further separate cell debris. Plasma samples were divided into 0.5-mL aliquots in separate Eppendorf tubes (Jencons Scientific Ltd, VWR International, Willard Way, Bedfordshire, UK) labeled with a unique patient identifier and stored at −80°C until subsequent analysis.
We searched our database and selected all cases with at least 2 mL of available stored plasma, collected from October 2010 through January 2011. The population consisted of 2230 singleton pregnancies. We excluded 74 pregnancies where there was no fetal karyotype and the outcome was miscarriage, stillbirth, termination (n = 28), or no follow-up (n = 46). We also excluded 7 pregnancies where CVS demonstrated that the fetal karyotype was abnormal but other than trisomy 21 or trisomy 18; 2 cases of triploidy; and 1 case each of Turner syndrome, 47,XX,+i(8)(p10), 46,XY,del(13)(q33), 46,XX,dup(10)(q26.1q24.1), and mosaic 45,X/47,XXX. There were 2149 cases after excluding for the above reasons.
Laboratory analysis
Plasma samples (4 tubes of 0.5 mL per patient) from the selected cases were sent overnight on dry ice from London, United Kingdom, to the laboratory of Ariosa Diagnostics Inc in San Jose, CA. The information provided to Ariosa Diagnostics Inc for each case was: patient unique identifier, maternal age, gestational age, date of blood collection, and fetal sex, but not fetal karyotype or birth outcome.
Prior to evaluation for fetal trisomy, Ariosa Diagnostics Inc determined 29 cases had inadequate sample volume, 1 case had tube labels that did not match the sample manifest, and 70 cases had issues of sample mixing during the manual pooling process of individual Eppendorf tubes by laboratory personnel. Therefore, the eligible study population consisted of 2049 cases.
Plasma samples from the study population were analyzed using a chromosome-selective assay (Harmony Prenatal Test, Ariosa Diagnostics Inc). Results were provided on the risk of trisomy 21 and trisomy 18 on each case to K.H.N. who then determined the correlation between the assay results with the fetal karyotype or birth outcome. The risk scores were represented as a percentage with ranges capped at >99% and <0.01%.
Statistical analyses
Descriptive data were presented in median and interquartile range for continuous variables and in numbers and percentages for categorical variables. Comparison between the outcome groups was by χ 2 or Fisher exact test for categorical variables and Mann-Whitney U test for continuous variables. In all cases we used Bonferroni correction with adjusted P value of < .025.
The statistical software package SPSS 20.0 (IBM Corp, Armonk, NY) was used for data analyses.
Results
Study population
The total study population included 2049 pregnancies. In 86 (4.3%) cases, the fetal karyotype was determined by CVS or amniocentesis and was normal in 75 cases, trisomy 21 in 8, and trisomy 18 in 3. The remaining 1963 pregnancies resulted in the live birth of phenotypically normal neonates assumed to be euploid and therefore, the total number of proven or assumed euploid pregnancies was 2038.
Maternal characteristics and results from conventional first-trimester screening with serum markers and ultrasound of the study population are presented in the Table . In trisomy 21, compared to the euploid pregnancies, the median maternal age, delta NT, and serum free β-hCG were significantly higher, and there was a higher prevalence of absent NB, TR, and reversed a-wave in DV. In trisomy 18, delta NT and serum free β-hCG were higher and PAPP-A and fetal crown-rump length were lower and similar to trisomy 21, there was a higher prevalence of absent NB, TR, and reversed a-wave in DV.
| Characteristic | Total n = 2049 | Euploid n = 2038 | Trisomy 21 n = 8 | Trisomy 18 n = 3 |
|---|---|---|---|---|
| Median maternal age, y (IQR) | 31.8 (27.7–35.4) | 31.8 (27.7–35.4) | 39.6 (33.3–41.7) a | 39.4 (23.4–42.3) |
| Median maternal weight, kg (IQR) | 65.2 (58.5–76.0) | 65.2 (58.5–76.0) | 64.5 (60.5–88.5) | 64.0 (56.0–126.0) |
| Median maternal height, cm (IQR) | 164 (160–169) | 164 (160–169) | 168 (164–170) | 165 (158–165) |
| Racial origin, n (%) | ||||
| Caucasian | 1431 (69.8) | 1423 (69.8) | 6 (75.0) | 2 (66.7) |
| African | 422 (20.6) | 419 (20.6) | 2 (25.0) | 1 (33.3) |
| South Asian | 82 (4.0) | 82 (4.0) | 0 | 0 |
| East Asian | 57 (2.8) | 57 (2.8) | 0 | 0 |
| Mixed | 57 (2.8) | 57 (2.8) | 0 | 0 |
| Cigarette smoker, n (%) | 131 (6.4) | 131 (6.4) | 0 | 0 |
| Method of conception, n (%) | ||||
| Spontaneous | 2007 (98.0) | 1996 (97.9) | 8 (100.0) | 3 (100.0) |
| Ovulation drugs | 19 (0.9) | 19 (0.9) | 0 | 0 |
| In vitro fertilization | 23 (1.1) | 23 (1.1) | 0 | 0 |
| Preexisting diabetes mellitus, n (%) | ||||
| Type 1 | 10 (0.5) | 10 (0.5) | 0 | 0 |
| Type 2 | 9 (0.4) | 9 (0.4) | 0 | 0 |
| Median fetal crown-rump length, mm (IQR) | 62.4 (57.3–67.3) | 62.4 (57.3–67.3) | 64.1 (59.5–70.4) | 47.7 (45.5–59.2) a |
| Fetal gender, n (%) | ||||
| Male | 1069 (52.2) | 1063 (52.2) | 4 (50.0) | 2 (67.7) |
| Female | 980 (47.8) | 975 (47.8) | 4 (50.0) | 1 (33.3) |
| Median PAPP-A MoM (IQR) | 1.048 (0.722–1.491) | 1.052 (0.724–1.494) | 0.700 (0.274–1.325) | 0.195 (0.152–0.300) a |
| Median free β-hCG MoM (IQR) | 1.089 (0.729–1.641) | 1.089 (0.730–1.631) | 3.809 (1.852–8.602) a | 0.339 (0.253–0.573) a |
| Median delta nuchal translucency (IQR) | 0.112 (−0.083 to 0.349) | 0.110 (−0.084 to 0.347) | 1.366 (0.797–3.590) a | 6.341 (5.872–8.721) a |
| Absent nasal bone, n (%) | 122 (6.0) | 115 (5.6) | 4 (50.0) a | 3 (100.0) a |
| Tricuspid regurgitation, n (%) | 41 (2.0) | 34 (1.7) | 4 (50.0) a | 3 (100.0) a |
| Reversed a-wave in ductus venosus, n (%) | 59 (2.9) | 51 (2.5) | 5 (62.5) a | 3 (100.0) a |
| Median fetal fraction, % (IQR) | 10.0 (7.8–13.0) | 10.0 (7.8–13.0) | 12.5 (9.2–21.3) | 9.3 (5.6–13.0) |
| Median estimated risk for trisomy 21 (range) | 1:8 469 (1:2–1:23 527) | 1:8 547 (1:2–1:23 527) | 1:2 (1:2–1:3) a | 1:6 (1:4–1:13) a |
| Median estimated risk for trisomy 18 (range) | 1:14 894 (1:2–1:47 472) | 1:14 980 (1:3–1:47 472) | 1:177 (1:2–1:1 562) a | 1:2 a |
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