DOI: 10.1111/tog.
12534 2019;21:51–7
The Obstetrician & Gynaecologist
http://onlinetog.org
Evolution in screening for Down syndrome
a,
Ghalia Ashoor Al Mahri MBBS MD, * KH Nicolaides
a
Specialist Trainee in Obstetrics and Gynaecology Trainee, London Deanery and Out Of Programme for Training at Corniche Hospital, Abu Dhabi,
United Arab Emirates
b
Director of Fetal Medicine, Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London SE5 9RS, UK
*Correspondence: Ghalia Ashoor Al Mahri. Email: ghalia@doctors.org.uk
Accepted on 1 August 2018.
Key content
 This article describes how screening for fetal trisomy 21 has
evolved from advanced maternal age, with a detection rate of
30% at a false positive rate (FPR) of 5%, to second trimester
serum biochemistry, with a detection rate of 60–75% at an FPR
of 5%, and then to the first trimester combination of fetal nuchal
translucency and serum biochemistry, with a detection rate of
90% at an FPR of 5%.
 The introduction of cell-free DNA analysis of maternal blood is
outlined. This method has improved detection rate to 99% with a
reduction in FPR to 0.1%.
 As this test is currently expensive, it may be difficult to offer it to
all patients as a first-line method of screening. A feasible approach
to the introduction of the cell-free DNA test is discussed, which is
to offer it contingent on the results of first-line screening with the
combined test.
Please cite this paper as: Ashoor Al Mahri G, Nicolaides KH. Evolution in screening for Down syndrome. The Obstetrician & Gynaecologist 2019;21:51–7. https://
doi.org/10.1111/tog.12534
Introduction
Chromosomal abnormalities are an important cause of
perinatal death and childhood disability. Currently, the
most common reason for invasive testing (amniocentesis
or chorionic villus sampling) is for the diagnosis of
chromosomal aneuploidies. However, because of the
associated risk of miscarriage, this is only carried out in
pregnancies that are at high risk for such aneuploidies.
In 1866, the physician John Langdon Down first described
the phenotype of Down syndrome.1 In 1956, Tijo and Levan2
established that the normal number of chromosomes in
humans is 46. Soon after, in 1959, Lejeune et al.3 and Jacobs
et al.4 established that Down syndrome is the result of an
extra chromosome 21. However, it was only feasible to
diagnose Down syndrome as trisomy 21 prenatally by
karyotyping of cultured amniotic fluid cells in 1966,
100 years after the original description of the phenotype by
Langdon Down.5
The critical factors in a screening test are the ability to
discriminate between affected and unaffected individuals,
and this is expressed in terms of the detection rate and the
ª 2019 Royal College of Obstetricians and Gynaecologists
Reviews
b
FRCOG
Learning objectives
 To understand the different methods available for screening for
fetal trisomy 21.
 To learn about the performance of different methods of screening.
 To be able to describe the new method of screening by cell-free
DNA testing.
 To understand the issues related to clinical implementation of cell-
free DNA testing.
Ethical issues
 Should the cell-free DNA test be offered solely based on health
economic considerations or on individual patient interests?
 Should screening be carried out for conditions other than
trisomy 21?
Keywords: cell-free DNA testing / Down syndrome / non-invasive
prenatal testing / screening / trisomy 21
false positive rate (FPR). The detection rate is the ability of a
test to give a positive result in individuals who have the
condition being screened for. The screen-positive rate is the
proportion of affected and unaffected individuals yielding a
positive result. The FPR is the proportion of unaffected
individuals yielding a positive result. In screening for
chromosomal defects, the term screen positive is usually
replaced with false positive because the vast majority of
screen-positive cases are actually unaffected.
Screening by maternal age
Screening for Down syndrome was initially introduced in the
1970s, based on an observation that risk of the condition
increases with maternal age. Women considered high risk on
screening were initially all women aged 40 years or older,
since prenatal diagnosis could not be offered to the entire
population because of a known risk of miscarriage of
amniocentesis and its cost implications. Later, when
amniocentesis became more common and its miscarriage
risk was identified as low, the cut-off for screening was
changed to all pregnant women aged 35 years or older.
51
 Screening for Down syndrome
At that time, this constituted 5% of pregnant women and
included 30% of affected fetuses.6
Since the 1980s, women in developed countries have
gradually postponed having children until later in life;
currently more than 20% of pregnant women are at least
35 years of age and this group includes 50% of the total
number of fetuses with Down syndrome.7
Screening by maternal serum biochemistry
in the second trimester
Pregnancies with fetal trisomy 21 are associated with altered
maternal serum concentrations of various fetoplacental
products, including low levels of alphafetoprotein and
unconjugated estriol and high levels of free b-human
chorionic gonadotrophin (b-hCG) and inhibin A.8–11
In the 1980s and 1990s, screening using a combination of
maternal age and various combinations of these biomarkers
improved the detection rates of trisomy 21. At an FPR of 5%,
the detection rate increased from 30% using maternal age
alone to 60–65% using the double test (a combination of
maternal age and serum alphafetoprotein and free b-hCG), to
65–70% using the triple test (double test with the addition of
unconjugated estriol) and 70–75% using the quadruple test
(triple test with the addition of inhibin A).7
Screening using ultrasound and
biochemistry in the first trimester
In the 1990s, it was discovered that in fetuses with trisomy 21
there is an increase in the accumulation of subcutaneous
fluid behind the fetal neck (nuchal translucency) that can be
assessed by ultrasonography at 11–13 weeks of gestation.6,12
It was subsequently found that in trisomy 21 pregnancies at
11–13 weeks, the maternal serum concentration of free b-
hCG is about twice as high and pregnancy-associated plasma
protein A (PAPP-A) is reduced to half compared with
euploid pregnancies. Maternal age was combined with fetal
nuchal translucency and serum biochemistry to develop a
screening test with a detection rate of about 90% at an
FPR of 5%.13
Screening in twin pregnancies
Since the 1990s, the rate of twin pregnancies has increased,
predominantly as a result of the widespread use of assisted
reproduction technology, as well as the increase in maternal age of
pregnant women. This increase mostly constitutes dizygotic twins.
The detection rate of trisomy 21 of the first trimester
combined test in twin pregnancies is similar to that in
singleton pregnancies.14 However, in monochorionic twin
pregnancies, the FPR is twice as high as in singletons, because
an increase in nuchal translucency in one fetus could be
52
either an early manifestation of twin-to-twin-transfusion
syndrome or a marker of chromosomal abnormality.15 In
dichorionic twins an individual risk is given for each fetus, but
in monochorionic twins the risk is calculated for each fetus and
an average of the two is given for the whole pregnancy.
Additional ultrasound markers
In addition to high nuchal translucency, ultrasound markers
for Down syndrome that are highly sensitive and specific
have been described: absence of nasal bone, increased
resistance to flow in the ductus venosus and tricuspid
regurgitation.16–18 These three markers can be incorporated
into the first trimester combined screening test that includes
maternal age, fetal nuchal translucency and serum-free
b-hCG and PAPP-A to improve the detection rate to more
than 95% and decrease the FPR to less than 3%.19 However,
the national screening programme has not implemented
these markers because of difficulties in nationwide training.
Screening for trisomies 18 and 13
A favourable by-product of screening for Down syndrome is
the earlier identification of fetuses affected by trisomy 18
and 13 (Edwards’ and Patau’s syndrome, respectively), the
second and third most common chromosomal abnormalities.
At the 11–13-week assessment, the relative prevalence of trisomy
18 and 13 to trisomy 21 is one to three and one to seven,
respectively.19 Therefore, for every three babies with
trisomy 21 there is one baby with trisomy 18, and for every
seven babies with trisomy 21 there is one baby with trisomy
13.
All three aneuploidies are associated with increased maternal
age, increased fetal nuchal translucency and decreased maternal
serum PAPP-A. However, in Down syndrome serum-free b-
hCG is increased, whereas in Edwards’ and Patau’s syndromes
b-hCG is decreased. In addition, Patau’s syndrome is associated
with fetal tachycardia, with more than 80% of those affected
having a heart rate above the 95th centile of euploid fetuses.19
In screening for all three aneuploidies, the detection rates are
about 90% for Down syndrome and 95% for Edwards’ and
Patau’s syndromes at an FPR of 5%.19
Screening using cell-free DNA in
maternal blood
Cell death results in the release of DNA fragments from the
nucleus to the plasma, and in the 1990s increased quantities of
these fragments were identified in the plasma of patients with
malignancies. In 1997, Lo et al.20 demonstrated the presence of
cell-free fetal DNA in the plasma of pregnant women.
With the development of sequencers in the last few years, it
has become possible to characterise and quantify the many
ª 2019 Royal College of Obstetricians and Gynaecologists
 Ashoor Al Mahri and Nicolaides

millions of cell-free DNA fragments in maternal plasma.

Table 1. Comparison of different methods of screening for fetal
Since the completion of the Human Genome Project, the trisomy 21
chromosomal origin of each fragment can be identified and
the amount of DNA fragments originating from any given False
Method of Detection positive
chromosome can be quantified. Cell-free DNA in maternal screening rate (%) rate (%)
plasma is a mixture of DNA fragments arising from dying
cells in the mother and placenta. The proportion of fetal to Maternal age 30 5
total cell-free DNA, referred to as the fetal fraction, is usually Second trimester
about 10%.21 In pregnancies with fetal trisomies the number serum biochemistry:
- Double test 60–65 5
of fragments derived from the extra fetal chromosome, as a (AFP, free b-hCG)
proportion of all sequenced molecules, will be higher than - Triple test 65–70 5
in diploid pregnancies. (AFP, free b-hCG, uE3)
- Quadruple test 70–75 5
The fetal fraction is vital to our ability to detect the small
(AFP, free b-hCG,
increase in the amount of a given chromosome in maternal uE3, inhibin A)
plasma in a trisomic pregnancy compared with a disomic one. First trimester 90 5
For example, when the fetal fraction is 20%, this means that if combined test
Cell-free DNA test 99 0.1
there were 100 units of maternal cell-free DNA fragments of
chromosome 21, then there would be 20 units of Key: AFP = alphafetoprotein; b-hCG = b-human chorionic
chromosome 21 from the fetus and 80 units from the gonadotrophin; uE3 = unconjugated estriol.
mother. However, in a trisomic pregnancy there would be
30 units from the fetus and 80 units from the mother, prior risk for the pregnancy, as well as the fetal fraction,
therefore there would be a total of 110 units of since the accuracy of cell-free DNA testing is inherently
chromosome 21 compared with 100 units of all the other dependent on the proportion of fetal to total DNA
chromosomes (110 versus 100). Therefore, the cell-free DNA in maternal plasma.26
technology depends on its ability to detect the increase in
the total amount of cell-free DNA fragments from Failed cell-free DNA tests
one chromosome compared with the other chromosomes In 1–5% of singleton pregnancies, no result is given from the
without necessarily distinguishing which is fetal DNA and cell-free DNA test after first sampling, mainly because of low
which is maternal DNA. However, if the fetal fraction is only fetal fraction. On repeat sampling, a result is obtained in
4%, the relative increase would be 102 versus 100 units, which around 60% of cases.21,27 The main determinants of low fetal
is more difficult to detect. Current methods of cell-free fraction are maternal obesity and small placental mass.21,27 In
DNA testing necessitate that the minimum fetal fraction Edwards’ and Patau’s syndromes, the placenta is small and
should be 4%.21 poorly functioning, which is evidenced by the low b-HCG
and PAPP-A levels in maternal blood; this also explains the
Performance of screening for fetal trisomies low fetal fraction and high failure rates in these pregnancies.
Since 2012, several clinical validation or implementation Therefore, pregnancies that result in a failed cell-free DNA
studies, in both high-risk and low-risk pregnancies, have result are inherently at increased risk of Edwards’ and Patau’s
been published.22–24 A meta-analysis of these studies reported syndromes but not Down syndrome.
in 2015 that the weighted pooled detection rate and the FPR There are various factors that determine how a failed cell-
were 99.2% and 0.09% for trisomy 21, 96.3% and 0.13% for free DNA result should be managed. This includes the initial
trisomy 18, and 91.0% and 0.13% for trisomy 13.25 reason why the test was performed and the cost of repeating
Consequently, this screening method is by far superior to the test, although most laboratories currently will repeat the
all previous methods (Table 1). test at no extra cost. At the time of writing, the majority of
tests are performed for extra reassurance for women with
Presentation of results maternal anxiety with a low risk on the combined test; these
Cell-free DNA analysis of maternal blood is a screening test women would generally opt to repeat the test. In those
and not a diagnostic test. Therefore, laboratories provide women that would choose not to repeat the test due to
results in the form of a risk assessment for trisomies, with increased anxiety, as well as those with a failed second
the majority reporting results for each chromosome as sample, a detailed ultrasound scan assessing for features of
either low risk (usually less than one in 10 000) or high Edwards’ and Patau’s syndromes would be recommended,
risk (more than 99%). It would be preferable if patient- and, if any features are identified, an invasive test would be
specific risks are reported, which would take into account considered. If, however, the background risk is high and an
the results from a previous screening test, also called the ultrasound scan did not demonstrate any features of

ª 2019 Royal College of Obstetricians and Gynaecologists 53

 Screening for Down syndrome
aneuploidy, then the majority would still opt to repeat the cell-
free DNA test and a few would proceed with an invasive test.
Conditions that can be screened with cell-free
DNA testing
Conventional prenatal screening for aneuploidies initially
focused on trisomy 21. This has been expanded to include
trisomies 18 and 13 because this extra information can be
provided at no extra cost. With cell-free DNA testing it is also
possible to screen for fetal sex chromosome aneuploidies and
some microdeletions, such as DiGeorge syndrome. However,
it may not be appropriate to offer pregnant women screening
for these abnormalities just because it is feasible to do so.
When it comes to screening for microdeletions, there is
insufficient evidence on the performance of cell-free DNA
testing, including the detection rate, FPR and failure rate.
Furthermore, given that sex chromosome aneuploidies are
typically mild, without physical and intellectual disability, the
rationale for screening for sex chromosome aneuploidy is not
strong, with a relatively low detection rate of 90% for a high
FPR of 0.4% and high failure rate.25 In addition, sex
chromosome aneuploidy has a high rate of fetal mosaicism
of up to 50%; moreover, the test may reveal maternal
aneuploidy such as 47XXX, where 90% of women would be
unaware of it.25 However, the lethal form of 45XO, which is
associated with a very high nuchal translucency in the first
trimester and cystic hygromas/hydrops in the second
trimester, needs to be investigated with an invasive test not
only for karyotype, but also to include sub-chromosomal
analysis with microarray rather than cell-free DNA.
Twin pregnancies
The prevalence of twin pregnancies is approximately 3% of live
births,28 with dizygotic twins being more common than
monozygotic twins at a ratio of approximately 70:30, excluding
those pregnancies conceived by assisted reproductive
technology.29 Cell-free DNA testing in twin pregnancies is
slightly more challenging, since there are two fetuses that could
either be genetically identical in monozygotic twins or genetically
different in dizygotic twins. Ultrasound assessment can identify
chorionicity but not zygosity;30 therefore, dichorionic twins on
ultrasound can either be dizygotic or monozygotic, whereas
monochorionic twins are always monozygotic. In a
monochorionic pregnancy (monozygotic/identical twins), both
fetuses release the same amount of genetic DNA into the maternal
circulation and therefore this would not affect the cell-free DNA
analysis for aneuploidy risk.
Nevertheless, the majority of dichorionic twins on
ultrasound are non-identical, and these fetuses will
contribute a discordant amount of cell-free DNA into
maternal blood. Studies using ultra-deep sequencing
demonstrated that this discordance can vary by two-fold.31
Therefore, if one of the twins was affected by aneuploidy and
54
possibly contributing a fetal fraction below 4% for satisfactory
DNA testing and the other euploid fetus was contributing a
higher fetal fraction, this could lead to an erroneous cell-free
DNA result. This is because the contribution of the euploid
fetus would dilute the contribution of the trisomic fetus in the
total fetal fraction. Therefore, a policy was proposed that the
fetal fraction used to give a successful cell-free DNA result in
dichorionic twin pregnancies should be the lower fetal fraction
of the two fetuses and not the total fetal fraction.32 This would,
however, result in an increase in failure rate by three-fold
compared with singleton pregnancies.33 The performance of
screening for trisomic twin pregnancies cannot yet be provided
as the numbers are still too small.
Therefore, cell-free DNA testing can safely be offered in
monochorionic twin pregnancies with expected performance
of screening as high as in singleton pregnancies. The test can
also be offered in dichorionic pregnancies, but parents should
be warned that data on accuracy are inadequate.
Options for clinical implementation
The cell-free DNA test can be introduced into clinical
practice either by offering it to all pregnant women or by
selecting a subgroup of women based on the first trimester
combined test, which is the current established screening
method offered by the UK’s National Health Service (NHS).
The first option would therefore use the cell-free DNA test
as the primary screening method for trisomies 21, 18 and 13
in all pregnant women. If that was the approach taken, then
testing maternal blood at 10–11 weeks of gestation would
provide patients and clinicians maximum information at the
earliest gestation currently possible. This approach would
provide the results of the cell-free DNA test at the time of the
first trimester combined test, which is ideally performed at
12 weeks of gestation.34 This would allow for screening for
both the trisomies and fetal defects, as well as assessing the
risk of pregnancy complications, all within the first trimester
of pregnancy.35 If the cell-free DNA result provided a high-
risk assessment for trisomies then the option of chorionic
villus sampling at 12 weeks is still feasible versus opting for
amniocentesis at a later gestation. On the other hand, if the
cell-free DNA result provided was low risk for trisomies, then
the parents can be reassured that it is very unlikely that the
fetus is affected. In addition, the option of doing the
first trimester combined test after a possible failed result
from the cell-free DNA test would reassure patients, as well as
those providing care, that a risk assessment can be provided
at the 12-week visit. However, this approach would obviously
have financial implications to the UK’s NHS because of the
cost of performing both the cell-free DNA test and the first
trimester combined test on all pregnant women.
The second option would retain the current established
primary screening method for trisomies and would provide
the cell-free DNA test to a subgroup of women based on the
ª 2019 Royal College of Obstetricians and Gynaecologists
 Ashoor Al Mahri and Nicolaides

Table 2. Modelled detection rates of trisomy 21 and trisomies 18 Combined screening at 11−13 weeks
or 13, and false positive rates in first trimester combined screening by (age, fetal NT, serum β-hCG and PAPP-A)
maternal age, fetal NT and serum-free b-hCG and PAPP-A32

Risk cut-off False positive Detection rate Detection rate

(1:x) rate (%) for T21 (%) for T18/T13 (%)
High risk ≥1:10 Intermediate risk Low risk
100 2.2 87.0 91.8
200 3.9 90.4 94.3
300 5.4 92.1 95.5
400 6.7 93.2 96.2
cfDNA test
500 7.9 94.0 96.7
1000 13.0 96.1 97.9
1500 17.2 97.0 98.5
2000 20.8 97.6 98.8
2500 23.9 98.0 99.0 Positive Negative
3000 26.6 98.3 99.1
3500 29.0 98.5 99.2
4000 31.3 98.7 99.3
5000 35.2 98.9 99.4 Invasive test Nothing else
Key: b-hCG = b-human chorionic gonadotrophin; NT = nuchal
translucency; PAPP-A = pregnancy-associated plasma protein A. Figure 1. Cell-free DNA testing contingent on the results of first-line
screening by the first trimester combined test.
Key: b-hCG = b-human chorionic gonadotrophin; cfDNA = cell-free
result of the combined test. This would increase the detection DNA; NT = nuchal translucency; PAPP-A = pregnancy-associated
rate of the current first trimester combined test and reduce plasma protein A.
the FPR at a much reduced cost than if the cell-free DNA test
was offered to every pregnant woman. One method of the majority that it is highly unlikely the fetus is affected (if
contingent screening is to offer cell-free DNA testing to the they were screen-negative by the cell-free DNA test) and
high-risk group as an alternative to invasive testing. In the determine a small subset of women who would need an
UK, the National Screening Committee has recommended invasive test (if they were screen-positive by the cell-free DNA
that the risk cut-off from the combined test for offering cell- test). Defining who would be included in the intermediate-risk
free DNA testing should be 1:100.36 However, this group group would then depend on the cost of the test and funding
constitutes only about 3% of the population and contains available.36 This would also reduce the number of women
only 87% of fetuses with trisomy 21 and will therefore not undergoing an invasive test unnecessarily, since the cell-free
improve the currently achieved performance of screening DNA test is a more accurate test for the common trisomies.
(Table 2).36 If the desired detection rate for trisomy 21 was
about 94%, then cell-free DNA testing should be offered for Contingent screening method
those with a risk of at least 1:500 from the combined test, and The contingent screening method described above was
this population would constitute about 8% of the total. If a implemented in 11 692 women in two NHS hospitals in
risk cut-off for offering cell-free DNA testing was 1:1000, the UK.37 The high-risk group was defined as having a risk of
then the detection rate would increase to 96%, and the cell- at least one in 100, and the intermediate-risk group was
free DNA test should be carried out in about 13% of defined as having a risk between 1 in 101 and 1 in 2500.
the population.36 Women who were considered at high risk were offered the
The preferred alternative in contingent screening is to divide option of having an invasive test, a cell-free DNA test or no
the population into three groups based on the results of the further testing, while those who were in the intermediate-risk
combined test (Figure 1). In the very high-risk group (risk of category were given the option of a cell-free DNA test or no
at least 1:10), which constitutes only about 1% of the further testing. If every women in the high-risk category had
population, invasive testing should be considered, because chosen to do an invasive test then the detection rate for
this group contains the majority of trisomies and a high trisomy 21 in that group would be 87% at 3.4% FPR. On the
proportion of other chromosomal abnormalities and genetic other hand, if every woman in both the high-risk and
syndromes that would not be detected by the currently intermediate-risk group opted to have the cell-free DNA test
available cell-free DNA test. However, the cell-free DNA test then the detection rate for trisomy 21 would be 98% at
would be more useful in the intermediate-risk group, to 0.25% FPR. However, only 38% of women in the high-risk
further screen those who are not completely reassured by their group opted to have the invasive test and 60% opted to have
risk assessment from the first trimester combined test. the cell-free DNA test, whereas 92% of those in the
Therefore, in that group an additional test would reassure intermediate-risk group opted to have the cell-free DNA

ª 2019 Royal College of Obstetricians and Gynaecologists 55

 Screening for Down syndrome
test. Therefore, prenatal diagnosis of trisomy 21 was only made
in 92% of affected pregnancies because many parents chose not
to do any further tests or chose to terminate the pregnancy,
resulting in live births of 32% of affected pregnancies.
Conclusion
The study described above shows that it is feasible to
incorporate the option of cell-free DNA testing into the
current established first trimester combined screening test.37
This would improve the detection rate and reduce invasive
testing rates, however, the extent of the improvement in
accuracy of screening would also depend on parental
choices. Therefore, clinical implementation of cell-free
DNA testing contingent on the results of the first
trimester combined test may only have a modest impact
on improving the prenatal detection of trisomy 21 and on
reducing both the invasive testing rate or live births
with trisomy 21.
Disclosure of interests
The authors have no conflict of interest to disclose.
Contribution to authorship
GA published the validation studies of the cell-free DNA
data in high impact factor journals and drafted the
manuscript. KHN is the principal investigator for the
majority of studies outlined in the article and has revised
the article, approving its content. All authors approved the
final version.
Acknowledgements
The study was supported by a grant from The Fetal Medicine
Foundation (UK Charity No: 1037116).
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