Aneuploidy is an abnormality in the number of chromosomes. Among live births, Down syndrome is the most common chromosomal abnormality. Fetal nuchal translucency refers to the ultrasound detection of subcutaneous edema in the neck of the fetus. Measurement is taken at the maximal thickness of the sonolucent zone between the inner aspect of the fetal skin and the outer aspect of the soft tissue overlying the cervical spine or the occipital bone. In the early 1990s, screening studies of pregnant individuals reported an association between increased fetal nuchal translucency and an increased risk of chromosomal defects, most commonly Down syndrome and trisomy 18. At that time, it was standard of care to perform aneuploidy screening during the second trimester of pregnancy (16 weeks).
Fetal nuchal translucency, in combination with specific maternal serum markers and maternal age, is measured to detect Down syndrome in the first trimester of pregnancy (10-14 weeks). The methodology and analytic methods of published studies reflect that fetal nuchal translucency interpretation was standardized and incorporated into appropriate statistical models, which were then combined with maternal serum markers that have known associations with Down syndrome. If accurately done, first-trimester screening using fetal nuchal translucency and maternal serum assessment can provide vital information about the fetus well before the traditional second-trimester screening.
Amniocentesis and chorionic villus sampling (CVS) are the only established methods during pregnancy for the definitive diagnosis of aneuploidy, but they are invasive procedures that increase the risk of miscarriage. Before biochemical screening was developed, amniocentesis or CVS was generally only offered to individuals 35 years or older, for whom the risk of the procedure approximated the risk of Down syndrome. However, as the majority of children with Down syndrome are born to individuals younger than 35 years of age, less invasive screening methods were developed.
The commonly used second-trimester triple screening (i.e., alpha-fetoprotein [AFP], beta subunit of human chorionic gonadotropin [B-hCG], and unconjugated estriol [E3]) identifies approximately 70 percent of Down syndrome pregnancies at a false-positive rate of five percent. The false-positive rate refers to the proportion of all tests that are falsely positive (i.e., the screening test is positive but the abnormality is not present) at the cutoff that produces that particular value of sensitivity. Among individuals who test positive on the second-trimester triple screening, only about two percent actually have a fetus with Down syndrome. A fourth biochemical marker, inhibin-A, which has recently been added to the screening panel, may boost detection of Down syndrome to 80 percent at a false-positive rate of five percent.
First-trimester triple screening includes ultrasound measurement of nuchal translucency (NT), serum measurement of pregnancy-associated plasma protein-A (PAPP-A), and at least one form of beta-hCG (free b-hCG, total b-hCG, or invasive trophoblast antigen [ITA]). In a meta-analysis of studies related to first trimester Down syndrome screening, Evans et al (2007) suggested that free b-hCG outperforms intact hCG (synonymous with total b-hCG) in a multimarker protocol. Although free b-hCG has been studied more than other forms of b-hCG and performs better as an individual marker in pregnancies affected by Down syndrome, there is support for the efficacy of free b-hCG, total b-hCG, and ITA when performed in combination with NT and PAPP-A. First and Second Trimester Evaluation for Aneuploidy Risk (FASTER) trial and the BUN (Blood, Ultrasound and Nuchal Translucency) study group have presented findings that support the efficacy of free b-hCG in combined screening. However, there are data – including the Serum Urine and Ultrasound Screening Study (SURUSS) by Wald et al (2003) – which highlight an appreciable level of efficacy when total or intact b-hCG is used in combined screening with NT and PAPP-A during the first trimester. A number of small studies, including Palomaki et al. (2005), have proposed the role of ITA in the combined screening panel. Furthermore, The American College of Obstetricians and Gynecologists (ACOG), The American College of Medical Genetics (ACMG), and the guidelines from The National Institute for Clinical Excellence (NICE) either recommend or accept the role of beta-hCG, without making a distinction as to its forms, when this marker is combined with NT and PAPP-A in first-trimester screening. All of the studies that analyzed free b-hCG, intact b-hCG, and PAPP-A in the same sample set included in the meta-analysis by Evans and colleagues (2007) preceded the recommendations by ACOG, ACMG, and NICE. First-trimester triple screening has a detection rate for Down syndrome and other aneuploidies that is comparable to second-trimester quadruple screening (AFP, b-hCG, E3, and inhibin-A), with the advantage of providing information about the fetus earlier in the course of pregnancy.
First-trimester time-resolved fluorescence immunoassay from maternal dried-blood spot or maternal serum sample, with or without qualitative assessment of Y-chromosome, using a proprietary algorithm to determine risk scores for fetal trisomies 13/18, 21, and preeclampsia, such as those offered by NTD Eurofins Clinical Diagnostics, LLC, lack clinical validity and utility data for the assessment of safety and efficacy. These tests have not effectively demonstrated the added clinical value of performing first trimester screen with additional parameters, as there has been no comparison of this testing to standard screening practice. Available evidence is comprised of case-control studies, which claim that this testing paradigm yields improved performance of first-trimester screening when including the use of additional biochemical markers (AFP, placental growth factor [PlGF], and dimeric inhibin A [DIA]) to the standard protocol. Studies cited in an available peer-reviewed article (Carmichael et al., 2017) reporting improved diagnostic performance with additional serum markers in expanded first trimester screening also suffer experimental design issues (e.g., retrospective and case control designs, large variance in sizes of control groups). In order to assess the added clinical value of performing a first trimester screen with additional parameters, it would be best to see a direct comparison, as in a randomized controlled trial, of this proposed method with the traditional practice. Moreover, the proposed algorithm has not been validated externally. These methods use a lower risk cut-off than the conventional screening paradigm, which, although boosting sensitivity, makes the test less specific. As reported in the literature, this method calculated risk based upon an overall median multiple of median value for the trisomy 21 cases, instead of using a regression model to incorporate all factors, because most cases were at 12 weeks gestation.
Cell-free DNA (cfDNA) testing can also be done to screen for a variety of fetal conditions by evaluating short segments of fetal DNA in maternal blood. The current ACOG and Society for Maternal-Fetal Medicine (SMFM) Guidance states that nuchal translucency measurement for aneuploidy risk is not necessary at the time of cfDNA screening in the first trimester. Furthermore, individuals who have a negative screening test result should not be offered additional screening tests for aneuploidy because this will increase their potential for a false-positive test result (Level A).
In multifetal gestations, the risk of fetal aneuploidy is affected by the number of fetuses and the zygosity of the pregnancy; however, data regarding the risk of aneuploidy are more limited in multiple gestations compared with singleton pregnancies. In dizygous twin pregnancies, each fetus carries a risk of aneuploidy generally similar to the pregnant individual’s age-adjusted risk, but the pregnant individual carries an increased risk of having a fetus with aneuploidy because there is more than one fetus. Typically, monozygous twins will have the same karyotype, with neither or both fetuses being affected; the risk of carrying aneuploid fetuses is similar to the pregnant individual’s age-adjusted risk. No method of aneuploidy screening is as accurate in twin gestations as it is in singleton pregnancies. Nuchal translucency measurement allows each fetus in a multifetal pregnancy to be screened independently and, therefore, can be used in twin or high-order multifetal gestations.
If a definitive diagnosis is desired after a positive screening, CVS can then be performed.
Fetal nasal bone examination by ultrasound has been proposed as another potential first-trimester marker for Down syndrome. A first-trimester ultrasound done between 11 and 14 weeks that determines the absence of fetal nasal bone is considered a positive test result, indicating an increased risk of Down syndrome. Although studies have found a high rate of successful imaging of fetal nasal bone and an association between absent nasal bone and the presence of Down syndrome in high-risk populations, there is insufficient evidence on the performance of fetal nasal bone assessment in average-risk populations. Also, it has been reported that the difficulty in performing first-trimester nasal bone sonography consistently in the general population may limit its usefulness as a screening technique.
Small differences in fetal nuchal translucency measurements can significantly impact the accuracy of risk prediction of Down syndrome and other aneuploidies in the fetus. Therefore, ultrasonographer training and ongoing quality assurance are essential to maintain the integrity of this screening method.
