Since the first report of fetal DNA detected in maternal blood just over 15 years ago, researchers have sought better ways to detect and diagnose a syndromic fetus as early in the pregnancy as possible, but without compromising the safety of the developing fetus.1 Fetal genetic material has been found to be present in the maternal blood as early as four to five weeks.2 With the aid of molecular amplification technology, chromosomal fragments of specific clinical interest may be detected with a high degree of sensitivity and specificity.3,4 During the last 15 years, we have seen rapid developments with this technology as it applies to antenatal screening for specific diseases, such as trisomies 13, 18 and 21 (Down syndrome).
Until recently, antenatal screening for fetal aneuploidy has relied on several tests with multiple components. Gestational and maternal age at the time of the test, ultrasound determinants such as nuchal thickness, and serum analytes have been incorporated to produce screening tests of varying sensitivities. This has led to a fairly complicated panel of options that an obstetrician may choose from. From the clinician’s perspective, most would prefer to offer their patients a simple, reliable test which provides results as early in the pregnancy as possible.5 The information gained from this early test would then be used to counsel the patient as to the need for further, more invasive testing. Studies have found that patients are more willing to wait, and would prefer to have tests performed later in the pregnancy if this translates to fewer false positives.6
In the quest for the perfect screening test (i.e., the highest sensitivity and specificity, with the lowest false-positive rate, as well as strong positive and negative predictive values), several options have been proposed. One of the most straightforward approaches is to assess the thickness of the fetal neck (nuchal translucency) on ultrasound, measure maternal serum levels of pregnancy-associated plasma protein A (PAPP-A) and human chorionic gonadotropin (hCG), and then offer a diagnostic test to those patients with positive screening results. This approach, known as the first trimester screen, has been shown to detect 82-87% fetuses with trisomy 21 and is typically performed around 11 to 13 weeks.7
More complicated protocols incorporate both first and second trimester screening tests as an integrated test, which detects 94% to 96% of cases. A major problem with performing the integrated screening test (both a first and a second trimester screen on the same patient) is that the false positive rates of each of these tests are additive, giving an overall false positive rate of 11% to17%.8 One method for preventing this is to combine the results of each test and give the final outcome as an integrated result. In an attempt to preserve the ability to provide the patient with an early screening result, some have recommended performing a sequential screen, in which a first trimester screen is performed. If this is positive, an invasive diagnostic test is recommended. If this first trimester screen is negative, however, then a second trimester screen is performed. This allows for an algorithmic approach in which first trimester result either leads to a diagnostic test (for a screen positive) or an additional second trimester test (if the first trimester screen is negative). From this brief description of screening options, it’s easy to see why both patients and physicians have long desired a simpler method for detecting fetal aneuploidy.
During the last couple of years, researchers have developed ways to reliably detect fetal DNA present in maternal serum. This breakthrough has led to tremendous gains in the detection of fetal aneuploidy. Through nucleic acid amplification strategies, cell-free fetal DNA may be isolated and amplified from a relatively small aliquot of maternal blood. This cell-free fetal DNA, which is believed to derive from placental tissue, is present in maternal blood and comprises 3% to 13% of the total cell-free maternal DNA.1 Currently, one of the most common methods for detecting cell-free DNA, known as noninvasive prenatal testing (NIPT), is through a technique described as massively parallel genomic sequencing (MPS) in which millions of DNA fragments are isolated and amplified.9 Several retrospective studies have shown a sensitivity of 98% for the detection of trisomies 13, 18, and 21, with a false-positive rate of 0.5%.9 The test is typically performed after 10 weeks gestation, and requires one to two weeks to process. Currently, the test is recommended only for pregnancies which are at high risk for fetal aneuploidy. Characteristics which would place one at high risk for having a fetus with trisomy 13, 18, or 21 include the following: advanced maternal age (greater than 34 years of age); abnormal findings on ultrasound suggestive of fetal aneuploidy; having a previous child affected by aneuploidy; or one of the parents having a known chromosomal abnormality, such as a balanced translocation, providing increased risk of trisomy 13 or 21.9
In a large systematic review, NIPT was found to perform extremely well in detecting fetal aneuploidy in high-risk pregnancies.10 Of the various techniques employed, MPS and digital analysis of selected regions (DANSR) have been found to offer the highest sensitivity and specificity. With these methods, sensitivities and specificities as high as 100% have been reported.11 Earlier studies which relied on alternate methods for amplifying the fetal DNA found a slightly poorer performance in detecting trisomy 21, and only a slight reduction in the sensitivity and specificity allowed for a significant impact on the positive predictive value of the test. In a report by Chiu et al., the positive predictive value when this test was applied to a high-risk population (1:200 risk of Down syndrome) was 19.7%.12 If this same test was applied to a low-risk population (1:1500 risk of Down syndrome), the positive predictive value was found to be 3.1%.10 This has important implications when considering applying a test to low-risk populations, as a false-positive result may be found in a large number of patients, resulting in unnecessary exposure to invasive diagnostic testing.10
With test performance parameters nearing 100% sensitivity and specificity, many healthcare providers are understandably anxious to apply the test to a broader population of patients. While these two parameters are important characteristics of screening tests, they are not as easily influenced by the prevalence of disease in a population. Large-scale prospective studies are needed to adequately assess the performance of NIPT in low-risk populations before it is applied in this setting. As described above, Mersey et al. have shown that a test may have an acceptable positive predictive value when applied to a population in which disease is more prevalent (e.g., trisomy 21 and a woman 35 years of age or older), but when the population shifts to a low-risk group, the test’s performance may be significantly reduced.10 Already this year, a study was performed on a small number of patients (N=289) with a low risk of fetal aneuploidy (risk score prior to first-trimester screening was < 0.01%). NIPT was found to perform very well when compared to first trimester screening (Nuchal translucency, PAPP-A and AFP), but it may be more likely that the sample size used for this study was too low to make an informed decision regarding the true performance of this test.13 Indeed, Mersey et al. caution that a sample size on the order of 10 to 20 thousand is needed to reliably determine the performance of NIPT in low-risk populations.10 The authors of this underpowered study boldly state that based on their findings, they are now offering NIPT as first-line screening to all women after 10 weeks gestation.
Both the American College of Obstetricians and Gynecologists (ACOG) and the American College of Medical Genetics and Genomics (ACMG) propose clear guidelines regarding the use of NIPT.9,14 They state that NIPT is a screening test in high-risk populations, and is not a diagnostic test. Until large-scale studies examining its performance in low-risk populations are carried out, it should not be used for patients at low risk of fetal aneuploidy. Furthermore, both ACOG and ACMG clearly condone direct invasive testing for fetal aneuploidy if the patient strongly desires, after adequate counseling. Limits of NIPT are outlined by ACMG, and are significant: unbalanced translocations, deletions, duplications, and single-gene mutations will not be detected by NIPT; increased body mass index (BMI) has been shown to correlate inversely with the amount of fetal DNA recovered from maternal serum, and this has been shown to negatively affect the test result; the time required to obtain a result from NIPT is significantly longer than for routine first and trimester screening tests. It is important to remember that NIPT does not screen for neural tube defects, and that an AFP must still be ordered between 15 and 20 weeks. Furthermore, an abnormal first trimester screen, in the absence of a true trisomy, has been found to be associated with adverse pregnancy outcomes, such as spontaneous miscarriage, fetal demise, low birth weight, or preterm birth.9
So where do we go from here? We now have a test with nearly 100% sensitivity and specificity in detecting trisomy 21, and yet both ACOG and ACMG caution against its use as a first-line screening test. Clinicians will continue to search for the perfect test for their patients, so that an informed decision may be made regarding whether or not to undergo an invasive diagnostic test as early as possible. Patients too will continue to search for the perfect test, allowing them to come to a decision regarding whether or not to proceed with invasive diagnostic testing with a minimal chance of having a false-positive screen. Until NIPT has been tested on a large number of low-risk patients, however, it will continue to be reserved for patients at high-risk for fetal aneuploidy.
- Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350(9076):485-487.
- Lo YM, Hjelm NM, Fidler C, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med. 1998;339(24):1734-1738.
- Bianchi DW, Platt LD, Goldberg JD, Abuhamad AZ, Sehnert AJ, Rava RP. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012;119(5):1-13.
- Palomaki GE, Deciu C, Kloza EM, et al. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study. Genetics in Medicine. 2012;14(3):296-305.
- Bishop AJ, Marteau TM, Armstrong D, et al. Women and health care professionals’ preferences for Down’s Syndrome screening tests: a conjoint analysis study. BJOG. 2004;111(8):775-779.
- Hill M, Fisher J, Chitty L, Morris S. Women’s and health professionals’ preferences for prenatal tests for Downs syndrome: a discrete choice experiment to contrast noninvasive prenatal diagnosis with current invasive tests. Genet Med. 2012;14(11):905-913.
- Malone F, Canick JA, Ball RH, et al; First- and second-trimester evaluation of risk (FASTER) research consortium. First-trimester or second-trimester screening, or both, for Down’s syndrome. N Engl J Med. 2005;353(19):2001-2011.
- Platt LD, Greene N, Johnson A, et al; First trimester maternal serum biochemistry and fetal nuchal translucency screening (BUN) study group. Sequential pathways of testing after first trimester screening for trisomy 21. Obstet Gynecol. 2004;104(4):661-666.
- The American College of Obstetricians and Gynecologists Committee on Genetics, The Society for Maternal-Fetal Medicine Publications Committee. Committee Opinion 545: Noninvasive Prenatal Testing for Fetal Aneuploidy. Obstet Gynecol. 2012;120(6):1532-1534.
- Mersy E, Smits LJM, van Winden LAAP, et al. Noninvasive detection of fetal trisomy 21: systematic review and report of quality and outcomes of diagnostic accuracy studies performed between 1997 and 2012. Human Repro Update. 2013. doi: 10.1093/humupd/dmt001
- Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet G. Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol. 2012;207(5):374, e1-6.
- Chiu RW, Akolekar R, Zheng YW, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ. 2011;342:c7401.
- Fairbrother G, Johnson S, Musci TJ, Song K. Clinical experience of noninvasive prenatal testing with cell-free DNA for fetal trisomies 21, 18, and 13, in a general screening population. Prenat Diagn. 2013;33:1-5.
- Gregg AR, Gross SJ, Best RG, et al. ACMG statement on noninvasive prenatal screening for fetal aneuploidy. Genet Med. 2013 Apr 4 [Epub ahead of print]