Fetal chromosomal abnormalities occur in approximately 1 in 160 live births. Most fetal chromosomal abnormalities are aneuploidies, defined as an abnormal number of chromosomes. The trisomy syndromes are aneuploidies involving 3 copies of 1 chromosome. The most important risk factor for trisomy syndromes is maternal age, with an approximate risk of 1 in 1500 in young women that increases to nearly 1 in 10 by age 48.
T21 (Down syndrome) is the most common cause of human birth defects and provides the impetus for current maternal serum screening programs. Other trisomy syndromes include T18 (Edwards syndrome), and T13 (Patau syndrome), which are the next most common forms of fetal aneuploidy in fetuses that survive to birth. The prevalence of these other aneuploidies is much lower than the prevalence of T21, and identifying them is not currently the main intent of prenatal screening programs. Also, the clinical implications of identifying trisomy 18 and 13 are unclear, as most fetuses with trisomy 18 and 13 do not survive to term.
Sex chromosome aneuploidies (e.g., 45,X [Turner syndrome]; 47,XXY, 47,XYY) occur in approximately 1 in 400 live births. These aneuploidies are typically diagnosed postnatally, sometimes not until adulthood, such as during an evaluation of diminished fertility. Alternatively, sex chromosome aneuploidies may be diagnosed incidentally during invasive karyotype testing of pregnant women at high risk for Down syndrome. The net clinical value of prenatal diagnosis of sex chromosome aneuploidies is unclear.
Potential benefits of early identification such as the opportunity for early management of the manifestations of the condition, must be balanced against potential harms that can include stigmatization and distortion of a family’s view of the child.
Current national guidelines recommend that all pregnant women be offered screening for fetal aneuploidy (referring specifically to T21, T18, and T13) before 20 weeks of gestation, regardless of age. Combinations of maternal serum markers and fetal ultrasound done at various stages of pregnancy are used, but there is not a standardized approach. The detection rate for various combinations of noninvasive testing ranges from 60% to 96% when the false-positive rate is set at 5%. When tests indicate a high risk of a trisomy syndrome, direct karyotyping of fetal tissue obtained by amniocentesis or chorionic villous sampling (CVS) is required to confirm that T21 or another trisomy is present. Both amniocentesis and CVS are invasive procedures and have an associated risk of miscarriage. A new screening strategy that reduces unnecessary amniocentesis and CVS procedures and increases detection of T 21, T18, and T13 has the potential to improve outcomes.
Commercial, noninvasive, sequencing-based testing of maternal serum for fetal trisomy syndromes has recently become available and has the potential to substantially alter the current approach to screening. The test technology involves detection of fetal cell-free DNA fragments present in the plasma of pregnant women. As early as 8 to 10 weeks of gestation, these fetal DNA fragments comprise 6% to 10% or more of the total cell-free DNA in a maternal plasma sample. The tests are unable to provide a result if fetal fraction is too low, that is, below about 4%. Fetal fraction can be affected by maternal and fetal characteristics. For example, fetal fraction was found to be lower at higher maternal weights and higher with increasing fetal crown-rump length.
Sequencing-based tests use 1 of 2 general approaches to analyzing cell-free DNA. The first category of tests uses quantitative or counting methods. The most widely used technique to date uses massively parallel sequencing (MPS; also known as next-generation or “next gen” sequencing). DNA fragments are amplified by polymerase chain reaction; during the sequencing process, the amplified fragments are spatially segregated and sequenced simultaneously in a massively parallel fashion. Sequenced fragments can be mapped to the reference human genome in order to obtain numbers of fragment counts per chromosome. The sequencing-derived percent of fragments from the chromosome of interest reflects the chromosomal representation of the maternal and fetal DNA fragments in the original maternal plasma sample. Another technique is direct DNA analysis, which analyzes specific cell-free DNA fragments across samples and requires approximately a tenth the number of cell-free DNA fragments as MPS. The digital analysis of selected regions (DANSR™) is an assay that uses direct DNA analysis.
The second general approach is single nucleotide polymorphism (SNP)--based methods. These use targeted amplification and analysis of approximately 20,000 SNPs on selected chromosomes (e.g., 21, 18, 13) in a single reaction. A statistical algorithm is used to determine the number of each type of chromosome.
Noninvasive prenatal testing analyzing cell-free fetal DNA in maternal serum is a potential complement or alternative to conventional serum screening.
To be clinically useful, the technology must be sensitive enough to detect a slight shift in DNA fragment counts among the small fetal fragment representation of a genome with a trisomic chromosome against a large euploid maternal background. Whether sequencing-based assays require confirmation by invasive procedures and karyotyping depends on assay performance. However, discrepancies between sequencing and invasive test results that may occur for biological reasons could make confirmation by invasive testing necessary at least in some cases, regardless of sequencing test performance characteristics.
Assessment of a diagnostic technology such as maternal plasma DNA sequencing tests typically focuses on 3 parameters: (1) analytic validity; (2) clinical validity (i.e., sensitivity and specificity) in appropriate populations of individuals; and (3) demonstration that the diagnostic information can be used to improve individual health outcomes (clinical utility).
ANALYTIC VALIDITY
Although all currently available commercially available tests use MPS, actual performance and interpretive procedures vary considerably. Clinical sequencing in general is not standardized or regulated by FDA or other regulatory agencies, and neither the routine quality control procedures used for each of these tests, nor the analytic performance metrics have been published.
CLINICAL VALIDITY
Data from the available published studies consistently reported a very high sensitivity and specificity of maternal plasma DNA sequencing-based tests for detecting T21 in high-risk women with singleton pregnancies. There are fewer data on the diagnostic performance of sequencing-based tests for detecting T13, T18 and sex chromosome aneuploidies. The available data suggest that diagnostic performance for detecting these other fetal aneuploidies is not as high as it is for detection of T21.
The available prospective studies in general population samples (which include both high- and average-risk women), including the large Norton et al 2015 study, have found high sensitivity and specificity rates, similar to that seen in high-risk women. In the Norton study, although PPV was lower in the subsample of low-risk women than in the general population PPV of cell-free DNA testing was much higher than standard screening.
For women with multiple gestation pregnancies, there is insufficient evidence to draw conclusions about the diagnostic accuracy of sequencing-based tests for detecting fetal aneuploidies.
CLINICAL UTILITY
There is no published direct evidence that managing individuals using sequencing-based testing improves health outcomes compared with standard screening. Modeling studies using published estimates of diagnostic accuracy and other parameters predict that sequencing-based testing as an alternative to standard screening will lead to an increase in the number of T21 cases detected and, when included in the model, a large decrease in the number of invasive tests and associated miscarriages. The number of T18 and T13 cases detected is similar or higher with sequencing-based testing, although this is more difficult to estimate because of the lower prevalence of these aneuploidies, especially with T13. The impact of screening for sex chromosome aneuploidies has not been modeled in published studies.
The evidence for noninvasive prenatal screening (NIPS) using cell-free DNA to detect microdeletions in individuals with singleton, twin, or multiple pregnancies includes several studies on analytical and clinical validity. Relevant outcomes are test accuracy and validity, other test performance measures, morbid events, resource utilization, and treatment-related morbidity. The available studies on clinical validity have limitations (e.g., missing data on confirmatory testing, false-negatives) and the added benefit of NIPS compared with current approaches is unclear. Moreover, the clinical utility of NIPS for microdeletions remains unclear and has not been evaluated in published studies. The evidence is insufficient to determine the effects of the technology on health outcomes.
AMERICAN COLLEGE OF MEDICAL GENETICS AND GENOMICS
In 2013, the American College of Medical Genetics and Genomics (ACMG) published a statement on noninvasive prenatal screening for fetal aneuploidy that addresses challenges in incorporating noninvasive testing into clinical practice. Limitations identified by the organization include that chromosomal abnormalities such as unbalanced translocations, deletions and duplications, single-gene mutations and neural tube defects cannot be detected by the new tests. Moreover, it currently takes longer to obtain test results than with maternal serum analytes. ACMG also stated that pretest and post-test counseling should be performed by trained personnel.
AMERICAN COLLEGE OF OBSTETRICIANS AND GYNECOLOGISTS AND SOCIETY FOR MATERNAL-FETAL MEDICINE
In November 2012, the American College of Obstetricians and Gynecologists (ACOG) released a committee opinion on noninvasive testing for fetal aneuploidy The Committee Opinion was issued jointly with the Society for Maternal-Fetal Medicine Publications Committee. ACOG recommended that maternal plasma DNA testing be offered to individuals at increased risk of fetal aneuploidy. They did not recommend that the test be offered to women who are not at high risk or to women with multiple gestations. ACOG further recommended that women be counseled before testing about the limitations of the test and recommended confirmation of positive findings with CVS or amniocentesis. The document noted that the content reflected emerging clinical and scientific advances and is subject to change as additional information becomes available. The Committee Opinion did not include an explicit review of the literature.
In a 2015 committee opinion, the American College of Obstetricians and Gynecologists (ACOG) recommends that all patients receive information on the risks and benefits of various methods of prenatal screening and diagnostic testing, including the option of no testing. False-positive findings have been found to be associated with factors including placental mosaicism, vanishing twins and maternal malignancies. In its 2015 committee opinion, ACOG recommended diagnostic testing to confirm positive cell-free DNA tests, and that management decisions not be based solely on the results of cell-free DNA testing. ACOG further recommends that patients with indeterminate or uninterpretable (i.e., “no call”) cell-free DNA test results be referred for genetic counseling and offered ultrasound evaluation and diagnostic testing because “no call” findings have been associated with an increased risk of aneuploidy.
In sum, published studies on all commercially available tests, and meta-analyses of these studies, have consistently demonstrated very high sensitivity and specificity for detecting Down syndrome (trisomy 21 [T21]) in singleton pregnancies. Most of the studies included only women at high risk of T21. For average risk women, available studies suggest test performance similar to that reported in high-risk women.
Direct evidence of clinical utility is not available. A 2013 Blue Cross Blue Shield Association (BCBSA) Technology Evaluation Center (TEC) Assessment modeled comparative outcomes based on the published data on test performance, published estimates of standard screening performance, individual uptake of confirmatory testing, and miscarriage rates associated with invasive procedures. For each comparison and in each risk population, sequencing-based testing improved outcomes, i.e., increased the rate of Down syndrome detection and reduced the number of invasive procedures and procedure-related miscarriages. In the modeling, the negative predictive value of testing approached 100% across the range of aneuploidy risk, while the positive predictive value varied widely according to baseline risk. The variable positive predictive value highlights the possibility of a false positive finding and thus testing using karyotyping is necessary to confirm a positive result.
There is less published evidence on the diagnostic performance of sequencing-based tests for detecting T18, T13 and sex chromosome anomalies, and most of the available studies were conducted in high-risk pregnancies. Meta-analyses of available data suggest high sensitivities and specificities, but the small number of cases, especially for T13, makes definitive conclusions difficult. The findings of a decision analysis study included in the 2014 BCBSA TEC Assessment suggest similar rates of T13 and T18 detection to standard noninvasive screening; the analysis assumed that T13 and T18 screening would be done in conjunction with T21 screening. Due to the low survival rate, the clinical benefit of identifying trisomy 18 and 13 are unclear. The clinical utility of early sex chromosome aneuploidy detection is also unclear.
As noted in the 2015 ACOG committee opinion, cell-free DNA screening does not assess risk of anomalies such as neural tube defects. Patients should continue to be offered ultrasound or maternal serum alpha-fetoprotein screening, regardless of the type of serum screening selected. This opinion also states that routine cell-free DNA screening for microdeletion syndromes should not be performed. Furthermore, this opinion communicates that given the performance of conventional screening methods, the limitations of cell-free DNA screening performance, and the limited data on cost-effectiveness in the low-risk obstetric population, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population.
