Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies

In the 1970s, the main method of screening for trisomy 21 was by maternal age and in the 1980s it was by maternal serum biochemistry and detailed ultrasonographic examination in the second-trimester. In the 1990s the emphasis shifted to the first-trimester, when it was realized that the great majority of affected fetuses could be identified by a combination of maternal age, fetal nuchal translucency (NT) thickness, maternal serum β‑human chorionic gonadotropin (β-hCG), and pregnancy-associated plasma protein A (PAPP-A). Screening by this combined test can identify about 90% of fetuses with trisomy 21 for a FPR of 5% [6]. In many countries all over the world, like the UK, there is a national program of screening for trisomy 21, based on the combined test and the offer of invasive testing at a certain risk cut-off. However, in most countries there are no national guidelines on screening and individual practitioners offer a variety of first- and/or second-trimester methods often driven by market forces and the rules of supply and demand. Consequently, in some countries, the rate of invasive testing ranged from 20–40% before the introduction of cfDNA testing. Since 2012, there has been a rapid and widespread introduction of cfDNA testing into clinical practice, first in the private and then in the public sector, but similarly, very few countries have established national policies for offering cfDNA and those that have done so have adopted different strategies, from universal to contingent screening.

Traditionally, screening for aneuploidies has been focused on trisomy 21. However, invasive testing in the screen-positive group often leads to the detection of many additional clinically significant aneuploidies. In the case of some aneuploidies, such as trisomies 18 and 13, triploidy and Turner syndrome, their incidence in the screen-positive group for trisomy 21 is much higher than in the screen-negative group because they have a similar pattern in the expression of biophysical and biochemical markers [6‐9]. Therefore, by using the first-trimester combined test for the screening of trisomy 21, detection of other aneuploidies was given at no extra “cost”, meaning with no increase in the FPR. However, this cannot apply to cfDNA testing because for every condition we include in the analysis, we are adding its related FPR. For example, if we test for trisomy 21 alone, the FPR is only 0.04%, but if we include trisomies 18 and 13, the FPR goes up to 0.12% [2] which, although still extremely low, would continue to increase with every single new condition analyzed.

On the other hand, prenatal detection of fetal anomalies potentially associated with genetic conditions necessitates invasive diagnosis, and the use of any method of screening, regardless of its accuracy, is not an appropriate option in these cases.

Moreover, not only the lack of sufficient scientific evidence is a burden for including sex chromosome aneuploidies, rare autosomal aneuploidies or subchromosomal anomalies in routine cfDNA screening but also the difficulty in parental counselling when discussing these conditions, either because of the wide spectrum of their clinical manifestation or because of inappropriate understanding of the disease.

For all the reasons above, there are no current recommendations to include any other condition in addition to trisomies 21, 18, and 13 when requesting cfDNA testing for screening of aneuploidies, even if it is technically possible [10, 11].

A recent meta-analysis in singleton pregnancies reported that in the combined total of 1963 cases of trisomy 21 and 223,932 non-trisomy 21 singleton pregnancies, the weighted pooled detection rate (DR) was 99.7% (95% CI, 99.1–99.9%) and the FPR was 0.04% (95% CI, 0.02–0.07%). In a total of 563 cases of trisomy 18 and 222,013 unaffected pregnancies, the pooled weighted DR and FPR were 97.9% (95% CI 94.9–99.1%) and 0.04% (95% CI 0.03–0.07%) respectively, and in a total of 119 cases of trisomy 13 and 212,883 unaffected singleton pregnancies, the pooled weighted DR and FPR were 99.0% (95% CI 65.8–100%) and 0.04% (95% CI 0.02–0.07%) [2]. Similarly, a recent meta-analysis in twin pregnancies reported that in a combined total of 56 trisomy 21 and 3,718 non-trisomy 21 twin pregnancies, the pooled weighted DR and FPR were 98.2% (95% CI 83.2–99.8%) and 0.05% (95% CI 0.01–0.26%), respectively. In a total of 18 cases of trisomy 18 and 3,143 non-trisomy 18 pregnancies, the pooled weighted DR and FPR were 88.9% (95% CI 64.8–97.2%) and 0.03% (95% CI 0.00–0.33%) respectively. Although the number of twin pregnancies with trisomy 13 (n = 3) was too small for accurate assessment of the DR, the average FPR for trisomy 13 of 0.19% (5 out of 2,569) seems slightly higher than the values reported for singleton pregnancies [12]. These results show that cfDNA testing is by far the best available method for screening of these conditions.

Studies on a smaller number of confirmed cases have reported the ability of cfDNA analysis in maternal blood to detect sex chromosome aneuploidies, rare autosomal trisomies, triploidy, microdeletion and microduplication syndromes, and even monogenic disorders [5, 13‐15]. However, the exact performance and clinical utility of the test for these conditions require further investigation.

By parallel sequencing of numerous cfDNA fragments, millions of nucleotide sequences can be amplified and sequenced. This results in a large amount of data that bioinformatics have to analyze and compare with the reference genome. Two main approaches for analysis have been used in the main clinical studies assessing performance of cfDNA testing: massively parallel shotgun sequencing (MPSS), by which the whole genome is analyzed, and targeted chromosome analysis, either by next-generation sequencing, custom microarray or single-nucleotide polymorphisms (SNP) analysis, which is directed and limited only to the chromosomes of interest.

There are two main limitations of cfDNA testing in the implementation of this method of screening for aneuploidies. First, although the cost of the test is similar to that of invasive testing and karyotyping, it is considerably higher than that of the currently available screening methods. Second, there is a rate of failure of the test to provide results of about 1% [2]. An important cause of not getting a result from cfDNA testing is a low fetal fraction, which is often a consequence of maternal obesity but also secondary to a small placental mass [23, 24].

There are two possible options: first, to take the blood at 10 weeks, in which case the results of the test would be available at the time of the scheduled first-trimester ultrasound examination, which is ideally performed at 12 weeks; second, to take the blood at 12 weeks, after the first-trimester examination. The major advantage of taking the blood sample at 10 weeks is that the results of the test should be available at the time of the first-trimester scan, which will then be solely performed to diagnose major fetal defects and to evaluate the risk for pregnancy complications. In addition, it would allow the realization of a rescue first-trimester combined test in those cases in which the cfDNA test has not provided results [27]. However, this model has the disadvantage of performing many unnecessary tests for pregnancies that miscarry spontaneously before the 12th–13th week or that are diagnosed as having increased fetal NT or major defects requiring invasive testing at the time of the ultrasound [28]. By taking the blood sample after the first-trimester assessment, these problems would be overcome, but with the disadvantage of losing the possibility of performing a rescue first-trimester combined test in those cases without a cfDNA result, especially if the ultrasound was performed in week 13.

An alternative to universal screening by cfDNA testing is to offer cfDNA testing contingent on the results of first-line screening by another method, preferably the first-trimester combined test. cfDNA testing could be offered to the high-risk group as an alternative to invasive testing, aiming to reduce the invasive testing rate, or to the intermediate-risk group, aiming to increase the DR of aneuploidies [29]. The exact risk cut-offs that define the high- and intermediate-risk groups depend on the cost of cfDNA testing and therefore the proportion of the population that can be offered this test [30].

If cfDNA testing reports a high-risk for trisomy 21, 18 or 13, it does not mean that the fetus definitely has one of these aneuploidies and it is important to confirm or refute the result by invasive testing. In contrast, if cfDNA testing reports a low risk, the parents can be reassured that it is highly unlikely that the fetus has one of these aneuploidies. However, these results should always be interpreted together with a detailed ultrasound examination that has excluded increased fetal NT and major malformations. In those cases where fetal NT is above 3.5 mm or where there are major fetal defects, irrespective of the cfDNA results, parents should be offered invasive testing with array analysis, to exclude not only the three major trisomies but also other chromosomal and subchromosomal conditions.

Those cases where cfDNA testing does not provide a result must be managed individually. As explained before, the main reason why the test fails to provide a result is a low fetal fraction and the main determinants for this to occur are maternal obesity and a low placental mass. In trisomies 18 and 13, but not in trisomy 21, the fetal fraction is lower and the rate of no-results is therefore higher than in unaffected pregnancies [31]. Consequently, those pregnancies in which a result from cfDNA test is not obtained can be considered at high risk for trisomies 18 and 13, but not for trisomy 21. The management in these cases will depend essentially on the reason why the test was performed in the first place. If there was a previous screening that had already shown a low-risk result without fetal defects, it is preferable to repeat the cfDNA test, explaining to the parents that there is >60% chance that a result will be obtained in the second attempt. However, some pregnant women prefer not to undergo the test again to avoid the anxiety generated by the inconclusive result of the first one. In these cases and in those in which the test fails for the second time, it is advisable to perform a detailed ultrasound, looking specifically for fetal anomalies associated with trisomies 18 and 13 and, if these are present, an invasive test should be recommended [31]. In cases in which previous screening had already shown a high risk for these conditions but the detailed ultrasound had not detected any findings suggestive of fetal pathology, most patients choose to repeat the cfDNA test, although some prefer to undergo an invasive test directly.