Eugene Pergament

Non‐invasive prenatal testing for aneuploidy: current status and future prospects

Non‐invasive prenatal testing (NIPT) for aneuploidy using cell‐free DNA in maternal plasma is revolutionizing prenatal screening and diagnosis. We review NIPT in the context of established screening and invasive technologies, the range of cytogenetic abnormalities detectable, cost, counseling and ethical issues. Current NIPT approaches involve whole‐genome sequencing, targeted sequencing and assessment of single nucleotide polymorphism (SNP) differences between mother and fetus. Clinical trials have demonstrated the efficacy of NIPT for Down and Edwards syndromes, and possibly Patau syndrome, in high‐risk women. Universal NIPT is not cost‐effective, but using NIPT contingently in women found at moderate or high risk by conventional screening is cost‐effective. Positive NIPT results must be confirmed using invasive techniques. Established screening, fetal ultrasound and invasive procedures with microarray testing allow the detection of a broad range of additional abnormalities not yet detectable by NIPT. NIPT approaches that take advantage of SNP information potentially allow the identification of parent of origin for imbalances, triploidy, uniparental disomy and consanguinity, and separate evaluation of dizygotic twins. Fetal fraction enrichment, improved sequencing and selected analysis of the most informative sequences should result in tests for additional chromosomal abnormalities. Providing adequate prenatal counseling poses a substantial challenge given the broad range of prenatal testing options now available. Copyright

A Randomized Comparison of Transcervical and Transabdominal Chorionic-Villus Sampling

BACKGROUND Chorionic-villus sampling is done in early pregnancy to obtain fetal cells for the prenatal diagnosis of genetic and chromosomal defects. Transcervical chorionic-villus sampling has been shown to be safe and effective in national trials. Recently, an alternative transabdominal technique has been suggested as potentially easier and safer. METHODS From April 1987 through September 1989, we prospectively compared transcervical and transabdominal chorionic-villus sampling in 3999 women with singleton pregnancies in whom the risk of a genetically abnormal fetus was increased. Women between 7 and 12 weeks of gestation underwent ultrasonographic evaluation of placental and uterine position. Those with active vaginal infections, active bleeding, or cervical polyps were excluded. If the obstetrician thought either sampling procedure was acceptable, the woman was asked to consent to random assignment to one of the two procedures. Both groups were followed to determine the outcome of pregnancy and the rate of spontaneous fetal loss after chorionic-villus sampling. RESULTS Among the 3999 women who entered the study, sampling was attempted in 3873 (97 percent), 1944 of whom had been assigned to undergo transcervical sampling and 1929 to undergo transabdominal sampling. Of these 3873 women, sampling was eventually successful in 3863. Sampling was successful after a single insertion of the sampling instrument in 94 percent of the transabdominal procedures and 90 percent of the transcervical procedures. Among the women with cytogenetically normal pregnancies who had sampling because of maternal age, the rate of spontaneous fetal loss through 28 weeks of pregnancy was 2.5 percent in the transcervical-sampling group and 2.3 percent in the transabdominal-sampling group (difference, 0.26 percent; 95 percent confidence interval, -0.5 to 1.0 percent). CONCLUSIONS Transabdominal and transcervical chorionic-villus sampling appear to be equally safe procedures for first-trimester diagnosis of fetal abnormalities.

Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort.

OBJECTIVE: To estimate performance of a single-nucleotide polymorphism–based noninvasive prenatal screen for fetal aneuploidy in high-risk and low-risk populations on single venopuncture. METHODS: One thousand sixty-four maternal blood samples from 7 weeks of gestation and beyond were included; 1,051 were within specifications and 518 (49.3%) were low risk. Cell-free DNA was amplified, sequenced, and analyzed using the Next-generation Aneuploidy Test Using SNPs algorithm. Samples were called as trisomies 21, 18, 13, or monosomy X, or euploid, and male or female. RESULTS: Nine hundred sixty-six samples (91.9%) successfully generated a cell-free DNA result. Among these, sensitivity was 100% for trisomy 21 (58/58, confidence interval [CI] 93.8–100%), trisomy 13 (12/12, CI 73.5–100%), and fetal sex (358/358 female, CI 99.0–100%; 418/418 male, CI 99.1–100%), 96.0% for trisomy 18 (24/25, CI 79.7–99.9%), and 90% for monosomy X (9/10, CI 55.5–99.8%). Specificity for trisomies 21 and 13 was 100% (905/905, CI 99.6–100%; and 953/953, CI 99.6–100%, respectively) and for trisomy 18 and monosomy X was 99.9% (938/939, CI 99.4–100%; and 953/954, CI 99.4–100%, respectively). However, 16% (20/125) of aneuploid samples did not return a result; 50% (10/20) had a fetal fraction below the 1.5th percentile of euploid pregnancies. Aneuploidy rate was significantly higher in these samples (P<.001, odds ratio 9.2, CI 4.4–19.0). Sensitivity and specificity did not differ in low-risk and high-risk populations. CONCLUSIONS: This noninvasive prenatal screen performed with high sensitivity and specificity in high-risk and low-risk cohorts. Aneuploid samples were significantly more likely to not return a result; the number of aneuploidy samples was especially increased among samples with low fetal fraction. This underscores the importance of redraws or, in rare cases, invasive procedures based on low fetal fraction. LEVEL OF EVIDENCE: II

Cytogenetic results of chorionic villus sampling: high success rate and diagnostic accuracy in the United States collaborative study.

Cytogenetic results of first-trimester chorionic villus sampling are reported from seven U.S. medical centers. For 6033 patients who had a successful chorionic villus sampling procedure, the rate for obtaining a cytogenetic diagnosis was 99.6% with the direct method, long-term culture, or both. There were no incorrect sex predictions and no diagnostic errors involving trisomies 21, 18, or 13, sex chromosome aneuploidies, or structural abnormalities. There were no cases of normal cytogenetic diagnosis followed by birth of a cytogenetically abnormal infant. Three cases of unusual aneuploidies (tetraploidy, trisomy 16, and trisomy 22) detected by the direct method only were not confirmed by cytogenetic follow-up. Mosaic cytogenetic abnormalities were observed in 0.83% of all cases in which chorionic villus sampling was done but were confirmed by amniocentesis or in fetal tissues in only 7 of 30 cases (23.3%). Maternal cell contamination occurred in 1.9% of long-term cultures, although this did not present any cytogenetic diagnostic difficulties. Overall, a very high degree of laboratory success and diagnostic accuracy was observed with either cytogenetic method, although fewer predictive errors were observed with the long-term culture method and none were observed when both methods were used.

Maternal cfDNA screening for Down syndrome – a cost sensitivity analysis

This study aimed to determine the principal factors contributing to the cost of avoiding a birth with Down syndrome by using cell‐free DNA (cfDNA) to replace conventional screening.

Sequential pathways of testing after first-trimester screening for trisomy 21

OBJECTIVE: To evaluate the performance and use of second-trimester multiple-marker maternal serum screening for trisomy 21 by women who had previously undergone first-trimester combined screening (nuchal translucency, pregnancy-associated plasma protein A, and free β-hCG), with disclosure of risk estimates. METHODS: In a multicenter, first-trimester screening study sponsored by the National Institute of Child Health and Human Development, multiple-marker maternal serum screening with alpha-fetoprotein, unconjugated estriol, and total hCG was performed in 4,145 (7 with trisomy 21) of 7,392 (9 with trisomy 21) women who were first-trimester screen-negative and 180 (7 with trisomy 21) of 813 (52 with trisomy 21) who were first-trimester screen-positive. Second-trimester risks were calculated using multiples of the median and a standardized risk algorithm with a cutoff risk of 1:270. RESULTS: Among the first-trimester screen-negative cohort, 6 of 7 (86%) trisomy 21 cases were detected by second-trimester multiple-marker maternal serum screening with a false-positive rate of 8.9%. Among the first-trimester screen-positive cohort, all 7 trisomy 21 cases were also detected in the second trimester, albeit with a 38.7% false-positive rate. CONCLUSION: Our data demonstrate that a sequential screening program that provides patients with first-trimester results and offers the option for early invasive testing or additional serum screening in the second trimester can detect 98% of trisomy 21–affected pregnancies. However, such an approach will result in 17% of patients being considered at risk and, hence, potentially having an invasive test. LEVEL OF EVIDENCE: II-2

Late First-Trimester Invasive Prenatal Diagnosis: Results of an International Randomized Trial

OBJECTIVE: To assess, in a randomized trial, the safety and accuracy of amniocentesis and transabdominal chorionic villus sampling (CVS) performed at 11–14 weeks of gestation, given that this time frame is increasingly relevant to early trisomy screening. METHODS: We compared amniocentesis with CVS from 77 to 104 days of gestation in a randomized trial in a predominantly advanced maternal age population. Before randomization, the feasibility of both procedures was confirmed by ultrasonography, and experienced operators performed sampling under ultrasound guidance; conventional cytogenetic analysis was employed. The primary outcome measure was a composite of fetal loss plus preterm delivery before 28 weeks of gestation in cytogenetically normal pregnancies. RESULTS: We randomized 3,775 women into 2 groups (1,914 to CVS; 1,861 to amniocentesis), which were comparable at baseline. More than 99.6% had the assigned procedure, and 99.9% were followed through delivery. In contrast to previous thinking, in the cytogenetically normal cohort (n = 3,698), no difference in primary study outcome was observed: 2.1% (95% confidence interval 1.5, 2.8) for CVS and 2.3% (95% confidence interval, 1.7, 3.1) for amniocentesis. However, spontaneous losses before 20 weeks and procedure-related, indicated terminations combined were increased in the amniocentesis group (P = .07, relative risk 1.74). We found a 4-fold increase in the rate of talipes equinovarus after amniocentesis (P = .02) overall and in week 13 (P = .03, relative risk = 4.65), but data were insufficient to determine this risk in week 14. CONCLUSION: Amniocentesis at 13 weeks carries a significantly increased risk of talipes equinovarus compared with CVS and also suggests an increase in early, unintended pregnancy loss. LEVEL OF EVIDENCE: I

The clinical application of interphase FISH in prenatal diagnosis.

Fluorescence in situ hybridization (FISH) for five chromosomes (13, 18, 21, X and Y) detected 87 of 107 (81%) of the chromosome aberrations identified by conventional chromosome analysis applied to fetal interphase cells obtained by chorionic villus sampling or amniocentesis. The choice of FISH was solely determined by prospective parents after formal genetic counselling concerning the advantages and disadvantages of FISH analysis. Excluding known familial chromosome aberrations, if FISH analysis revealed normal signals, there was an overall residual risk of 1 in 149 for an undetectable chromosome aberration. This risk varied according to the indication for prenatal diagnosis: 1 in 177 for women of advanced maternal age; 1 in 60 for women at increased risk for Down syndrome based on maternal serum screening; and, 1 in 43 for women whose ultrasound examination revealed fetal anomalies. There were 20 cases of discordance between the FISH results and standard karyotype analysis: four were the outcome of a failure to apply the appropriate FISH probe; 16 were not detectable by the available FISH probes. Of these 16, nine were chromosome abnormalities with clinical significance and seven were familial. If FISH is to become a standard part of prenatal genetic diagnosis, genetic counselling that is sensitive to patient health needs must be based on accurate information about the biological and obstetrical implications of the results of FISH analysis. Copyright

Pregnancy-associated Plasma Protein A, Free β-hcg, Nuchal Translucency, and Risk of Pregnancy Loss

OBJECTIVE: To estimate the likelihood of clinical early and late pregnancy loss as a function of first-trimester maternal serum analytes and fetal nuchal translucency measurements. METHODS: Study subjects were recruited for a National Institute of Child Health and Human Development–sponsored multicenter cohort study initially designed to study the detection of Down syndrome during the first trimester of pregnancy. The cohort consisted of women who had a live fetus between 10 and 14 weeks of gestation and had no significant vaginal bleeding. Women with prior fetal trisomy (T21/18) and those with structural or chromosomal abnormalities in the index pregnancy were excluded. First-trimester screening consisted of pregnancy-associated plasma protein A (PAPP-A), free β-hCG, and nuchal translucency. Pregnancy loss rates in women with various levels of PAPP-A, free β-hCG, or nuchal translucency (less than 1st, less than 5th, more than 95th, and more than 99th percentile) were compared with losses in women with normal values (5th to 95th percentile). RESULTS: The mean gestational age at screening of 7,932 women meeting study criteria was 12.1 weeks. Loss rates were only 0.36% at less than 20 weeks after normal free β-hCG, PAPP-A, and nuchal translucency. Conversely, low levels of PAPP-A and free β-hCG as well as increased nuchal translucency were individually associated with increased early loss. These associations persisted after controlling for maternal age and race using logistic regression analysis. CONCLUSION: Normal values of PAPP-A, free β-hCG, and nuchal translucency are associated with a very low risk of pregnancy loss at less than 20 weeks. LEVEL OF EVIDENCE: II-2

Cell-free DNA screening for fetal aneuploidy as a clinical service ☆

Non-invasive prenatal testing (NIPT) through the analysis of cell free (cf)DNA is revolutionizing prenatal screening for fetal aneuploidy. Current methods used in clinical practice include shotgun massively parallel sequencing (s-MPS); targeted (t-MPS); and an approach that takes advantage of single nucleotide polymorphism (SNP) differences between mother and fetus. Efficacy of cfDNA testing for the common autosomal trisomies far exceeds that of conventional screening. Depending on the methodology used, reasons for discordancy between cfDNA results and fetal karyotype can include true fetal mosaicism, confined placental mosaicism, presence of a maternal karyotype abnormality, insufficient counting due to low fetal fraction, and a vanishing twin. Among the possible cfDNA strategies a Primary test has the highest performance but is expensive, while a Contingent cfDNA test can achieve high performance at a relatively low cost. Practicalities to be considered in the provision of testing include pretest counseling about the scope and accuracy of the testing, the interpretation of results when there is a low fetal fraction and follow-up studies for positive test results. The role of first trimester nuchal translucency measurement and conventional biochemical testing needs to be reassessed in the context of the use of cfDNA.