In this section, the strengths and the limitations of the different screening methods for chromosomal abnormalities will be highlighted. We will discuss not only common trisomies, but the entire spectrum of chromosomal disorders. Screening based on maternal age alone will not be addressed as it should not be used as such due to poor test performance [
3]. However, maternal age should be included in risk assessment that uses other screening parameters.
First trimester screening (FTS)
The combined FTS computes individual risks for trisomies 21, 18, and 13 based on maternal age, fetal nuchal translucency (NT) measurement, and maternal serum markers, most commonly free beta-hCG and PAPP-A. Other ultrasound parameters such as the fetal nasal bone, tricuspid valve flow, and blood flow in the ductus venosus can be included in the risk calculation and have been shown to improve the test performance [
7‐
13].
Common trisomies
Santorum et al. investigated the test performance of combined FTS in screening for trisomies 21, 18, and 13 in approximately 110,000 pregnancies. For a false-positive rate of 4.6%, the detection rates were 92.1%, 96.4%, and 92.9%, respectively [
7]. Median marker levels in euploid and aneuploid pregnancies are listed in Table
1.
Table 1
Typical FTS marker profile of euploid and aneuploid fetuses [
10]
Normal | 2.0 | 1.0 | 1.0 |
Trisomy 21 | 3.4 | 2.0 | 0.5 |
Trisomy 18 | 5.5 | 0.2 | 0.2 |
Trisomy 13 | 4.0 | 0.5 | 0.3 |
The combined use of FTS with additional ultrasound markers such as the nasal bone, ductus venosus, and tricuspid valve flow increases the detection rate for trisomy 21 to about 95% and halves the false-positive rate [
8,
12‐
14]. It should be noted, however, that these additional markers represent dichotomous variables that can strongly influence the risk; therefore, they should be used only by those operators who have sufficient expertise to do so [
15].
Other chromosomal abnormalities
Increased nuchal translucency
It has been shown that the risk of genetic and structural defects increases with increasing NT measurements [
16,
17]. In a recent study, Bardi et al. reported on the outcome of 1901 pregnancies with a fetal NT above the 95th percentile [
18]. 43.0% of these fetuses were classified as abnormal. 23.9% had trisomies 21, 18, and 13, and 5.4% had other chromosomal abnormalities detectable by karyotyping alone. Single-gene disorders or abnormalities that can be detected only by microarray analysis were found in 2.0% of the cases each. Structural malformations in the absence of a genetic abnormality were detected in 9.3% of the fetuses. Based on this study, it is predicted that a policy that focuses only on trisomies 21, 18, and 13, such as cfDNA screening, would miss 44.0% of all abnormalities.
These data indicate that the option of a diagnostic test needs to be discussed with patients with a fetus that has an increased NT. However, the cut-off for NT thickness remains controversial. If there are additional defects, there is a broad consensus that an amniocentesis or a CVS should be offered [
19]. However, this is less clear for an isolated increased NT. Thresholds of 3.0 mm, 3.5 mm, and the 95th centile have been proposed. Thus far, most study groups have used the 99th percentile, which corresponds to approximately 3.5 mm across the gestational ages when FTS is performed [
6,
20,
21]. Regardless of which cut-off is used, the discussion about an appropriate threshold presupposes that the examination is done correctly by trained personnel so that the NT measurement can be accurately compared to the reference range [
22]. Table
2 gives an overview of the risk for chromosomal and structural defects stratified according to the NT thickness.
Table 2
Risk of chromosomal and structural defects according to the NT thickness (submicroscopic defects = those defects not detectable by routine karyotyping)
95th–3.4 | 13 | 1 | 1 | 1 | 6 |
3.5–4.9 | 25 | 3 | 3 | 1 | 11 |
5.0–6.4 | 44 | 13 | 4 | 6 | 11 |
6.5–7.9 | 51 | 9 | 3 | 3 | 17 |
> 8.0 | 34 | 22 | 1 | 7 | 14 |
In a large retrospective study that included more than 81,000 pregnancies, Hui et al. investigated the outcome of fetuses with an NT measurement above 3.0 mm and above the 99th percentile [
23]. The rate of atypical chromosomal defects detected only by microarray analysis was 2.1% for the NT range between 3.0 and 3.4 mm and 21.5% for measurements ≥ 3.5 mm. However, in this study, the presence or absence of major structural defects was not taken into account.
Maya et al. performed a study, which was confined to fetuses with an isolated NT enlargement [
24]. They compared the proportion of chromosomal defects and pathogenic mutations in three groups: NT of less than 3.0 mm, NT between 3.0 and 3.4 mm and NT of 3.5 mm or more. The prevalence of chromosomal defects was 1.7%, 6.5%, and 13.8%, respectively. Pathogenic mutations that could be detected only by microarray analysis and not by karyotyping or by cfDNA analysis were found in 0.9%, 1.8%, and 2.2% of cases, respectively. As a consequence, the authors recommended that microarray analysis should be performed if the NT thickness is 3.0 mm or more. This is in agreement with the American College of Obstetrics and Gynecology, which makes the same recommendation [
6].
Mellis et al. investigated whether exome sequencing (ES) is more appropriate in cases of isolated increase in NT thickness in the first trimester [
25]. The study included 213 fetuses with a NT thickness of 3.5 mm or more and where karyotyping and microarray analysis were normal, and subsequently underwent ES. Diagnostic variants (defined in this study as either pathogenic or likely pathogenic) were considered significant. Structural malformations were found in 54 fetuses, of which 22.2% had a diagnostic variant. The remaining cases with apparently isolated increased NT were stratified in three groups. In 37 fetuses, isolated increased NT was initially diagnosed, but structural abnormalities became apparent at a later date. The proportion of fetuses with a diagnostic variant in this group was 32.4%. 111 fetuses with an increased NT did not have structural defect identified even after the first trimester. Two (1.8%) fetuses in this group had an abnormality that could only be detected by exome analysis: one fetus had a uniparental disomy 15 and the other one a mutation in the RERE gene. In seven cases, the pregnancy ended before further screening was carried out. In this group, two (28.6%) fetuses had a pathogenic variant.
In summary, amniocentesis or CVS should be considered if the NT thickness is more than 3.0–3.5 mm. It is clear that diagnostic testing has a high yield in cases where fetal structural defects are identified. However, in recognition of the fact that first trimester fetal anatomic survey is limited by fetal size and that structural abnormalities may not become evident until later, offering diagnostic testing is always a prudent approach to a pregnancy where the nuchal translucency measurement exceeds 3.0–3.5 mm. Microarray analysis is the method of choice as it addresses a larger range of genetic abnormalities than karyotyping alone. However, ES may be useful in those cases where fetal structural anomalies are present, and the first-line testing is negative.
Abnormal serum markers
Increased risk for chromosomal abnormalities other than the common trisomies is also seen in cases where the deviation from normal in maternal serum marker levels (free beta-hCG and PAPP-A) is extreme. Therefore, diagnostic testing is recommended if either one of these serum markers falls below 0.2 MoM or if free beta-hCG exceeds 5.0 MoM. In these cases, a microarray analysis should be offered [
26].
The basis for this recommendation comes from two Danish studies [
27,
28]. Petersen et al. examined retrospectively 193, 638 pregnancies, 1122 of which had an abnormal fetal karyotype. Of those, 262 (23.4%) would have been missed by NIPT alone [
28]. The study cohort included 936 and 227 pregnancies where either PAPP-A or free beta-hCG, levels, respectively, were below 0.2 MoM. A fetal chromosomal abnormality was present in 21.4% and 56.6% of the cases, respectively. Out of these chromosomal abnormalities, 23.5% and 37.2% were classified as atypical. In the cohort where free beta-hCG level was ≥ 5.0 MoM, 10.9% of the fetuses were found to be chromosomally abnormal. Of these, 21.1% were labelled as atypical.
Wijngaard et al. also highlighted the importance of the serum markers [
29]. This study group included 877 pregnancies examined by microarray analysis and in which the results of the combined FTS was available. The risk for chromosomal abnormalities other than the common trisomies increased by 2.6 and 2.2 times for a free beta-hCG concentration of less than 0.37 MoM or a NT thickness above 3.5 mm, respectively.
Increased risk after combined FTS
Vogel et al. investigated whether the risk for other chromosomal abnormalities can be assessed based on the FTS risk for common trisomies [
30]. In cases where the trisomy 21 risk was between 1:50 and 1:100, 2.7% of abnormal findings were identifiable only by microarray analysis. In a large dataset of more than 100,000 first trimester screening examinations, Lindquist et al. looked for markers for other chromosomal abnormalities [
31]. About a quarter of all chromosomal abnormalities were classified as atypical, and the overall prevalence of these abnormalities was 0.1%. The prevalence increased to 4.6% in the group of pregnancies with a combined FTS risk for trisomy 21 of 1:10 or more. The authors also emphasized the importance of the serum markers and highlighted the cut-off of 0.2 MoM.
In summary, a specific FTS risk-based approach in screening for chromosomal abnormalities other than the common trisomies has not been established. However, if the FTS risk is 1:10 or more, it is reasonable to offer diagnostic testing. However, one should keep in mind that the reason for an increase in the FTS risk of this magnitude will be either due to an increase in NT thickness or abnormal serum markers, both of which are considered markers for other chromosomal abnormalities.
Useful combinations of FTS and cfDNA analysis
As shown above, both FTS and cfDNA analysis play important and additive roles in screening for chromosomal disorders. Therefore, several study groups have assessed the combined use of the two techniques. Usually, the screening algorithm starts with FTS followed by cfDNA testing in a subgroup, which is selected based on the FTS results. Miltoft et al. investigated such a two-stage approach. Combined FTS was performed in all pregnant women and cfDNA screening for trisomies 21, 18, and 13 was performed if the FTS risk fell between 1:100 and 1:1,000 [
53]. If the risk was less than 1:1000, no further tests were offered; if the risk was greater than 1:100, or if the cfDNA analysis was abnormal, a diagnostic test was performed. This model was compared with the use of combined FTS alone, at a single threshold of 1:300. All pregnancies affected by trisomy 21 were detected by both screening policies. However, the false-positive rate of the two-stage model was 1.2% while it was 3.0% with the classical approach. Gil et al. offered cfDNA screening to women with an intermediate FTS risk between 1:101 and 1:2,500 [
54]. Women in the high-risk group (risk of ≥ 1:100), were asked to choose between a diagnostic and a cfDNA test. The approach resulted in a real detection rate of 91.5% for trisomy 21, with only 38% of women with a risk above 1:100 opting for a diagnostic test. In the intermediate risk group (1:101–1:2,500), 91.5% of pregnant women opted for cfDNA testing. Overall, an amniocentesis or a CVS was performed in 2.7% of cases.
In a study from 2015, Kagan et al. used prospectively collected FTS results from nearly 87,000 pregnancies with a risk calculation based on fetal NT and ductus venosus flow [
55]. There were 324 fetuses with trisomy 21. The assumption was made that cfDNA testing would be used in women at risk of 1:100 to 1:2,500, and that the detection and false-positive rates of cfDNA screening would be 99.0% and 0.08%, respectively. Using such an approach, the detection and false-positive rates were calculated to be 96.0% and 2.3%, respectively. If the upper risk threshold is raised to 1:10, the detection rate remains almost unchanged, but the false-positive rate drops to 0.8%. In the studies mentioned above, the proportion of women in the intermediate risk group who would be offered cfDNA screening range from 11.4 to 29.9%.
In a prospective study, the Tübingen research group investigated the test performance of cfDNA screening for all pregnancies after a detailed ultrasound examination in the first trimester [
20,
21]. In cases of increased NT or fetal defects, a diagnostic test instead of cfDNA screening was carried out. The aim of our study was to compare the false-positive and invasive testing rate of such a policy with classical combined FTS. In 2.0% of the cases, the ultrasound examination was abnormal. In the group where the first trimester ultrasound did not reveal any structural abnormalities, cfDNA screening resulted in a false-positive and invasive testing rate of 0% and 0.3%, respectively. In the FTS group, the rates were 2.5% and 1.7%, respectively.
Role of the early anomaly scan at 11–13 weeks’ gestation in screening for chromosomal abnormalities
Several studies have addressed the test performance of an early anomaly scan in screening and diagnosis of fetal structural defects [
56]. In a meta-analysis by Karim et al. the authors showed that in a low-risk population the detection rate for all anomalies was 32.4% and was 46.1% for severe malformations [
57]. The overall detection rate in a high-risk cohort was 61.2%. The detection rate for cardiac defects was 55.8% [
58]. In a large study by Syngelaki et al. which was not included in the above meta-analysis, the overall detection rate of fetal structural anomalies in the first trimester was 27.6% [
59]. In a recent single center study, which included more than 50,000 low-risk pregnancies, the overall detection rate was 43%. The authors emphasized the importance of a rigid examination protocol that specifies the structures to be examined [
60].
The benefits of assessing the fetal sonomorphology even before a cfDNA test was summarized in a recent editorial [
61]. The following are the main points regarding the first trimester ultrasound evaluation:
-
Common trisomies constitute a relatively small proportion of the spectrum of fetal defects. Ultrasound plays an important role in screening and diagnosis of other chromosomal defects, genetic and non-genetic syndromes, and fetal structural defects.
-
Early anomaly scan may increase the overall detection of fetal defects in pregnancy.
-
In some cases, especially in obese women, an early anomaly scan performed transvaginally can provide better visualization than the standard second trimester examination.
-
In cases of cfDNA test failure, FTS as part of the early anomaly scan can provide a reliable risk assessment for trisomy 21.
-
Extended genetic analysis such as exome sequencing takes time and may be initiated based on the results of an anatomic scan at 11–13 weeks. Consequently, the patient still had the full range of reproductive options available when the results are known.
-
If a fetal problem is found on a comprehensive evaluation early in pregnancy, termination can also be done early in pregnancy. This is a safer procedure and tends to be less traumatic for the patient.
-
An early fetal anatomic ultrasound, including NT measurement, is an essential component of a comprehensive fetal evaluation, which provides the patient reliable information regarding the fetus at an earliest possible time in pregnancy. It is self-evident that this is what most patients desire.