Seminars in Fetal & Neonatal Medicine 19 (2014) 9e14

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Seminars in Fetal & Neonatal Medicine

journal homepage: www.elsevier.com/locate/siny

Non-invasive prenatal testing for Down syndrome

Philip Twiss a, Melissa Hill a, Rebecca Daley a, Lyn S. Chitty a, b, *
a
b

NE Thames Regional Genetics Service, Great Ormond Street Hospital for Children NHS Foundation Trust, 37 Queen Square, London WC1N 3BH, UK
UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK

s u m m a r y
Keywords:
Cell-free fetal DNA
Down syndrome
Fetal aneuploidy
Non-invasive prenatal testing
Prenatal diagnosis

Prenatal screening and diagnosis of Down syndrome and other major aneuploidies may be transformed
following the identication of cell-free fetal DNA in maternal plasma at the end of the last millennium.
Next generation sequencing has enabled the development of tests that accurately predict the presence of
fetal trisomies by analysis of cell-free DNA in maternal blood from as early as 10 weeks of gestation.
These tests are now widely available in the commercial sector but are yet to be implemented in publicly
led health services. In this article we discuss the technical, social, and ethical challenges that these new
tests bring.
2013 Elsevier Ltd. All rights reserved.

Introduction
Down syndrome (DS) or trisomy 21 is the most frequently
observed aneuploidy associated with long-term survival, with an
incidence of 1 in 800 live births. Antenatal screening for DS is
routinely offered to all pregnant women in the UK and many parts
of the developed world. This is usually performed using a combination of maternal age, ultrasound, and maternal serum biomarkers to estimate a pregnancy-specic individual risk of carrying
a DS fetus [1]. Traditionally, denitive diagnosis in pregnancies
identied as being at high risk is then offered by amniocentesis or
chorionic villus sampling (CVS), both of which are invasive procedures and have miscarriage risk rates of around 0.5e1% [2]. Over
the last few decades research has focused on identifying a less
invasive approach to prenatal diagnosis. This was initially based on
the isolation of fetal cells in the maternal circulation [3] but,
following the identication of cell-free fetal DNA (cffDNA) in
maternal plasma [4], efforts to develop non-invasive prenatal
testing (NIPT) turned towards the analysis of cell-free DNA (cfDNA)
[5].
Cell-free fetal DNA is a useful potential source of fetal genetic
material to use for prenatal diagnosis as it is present in the maternal
circulation from early in pregnancy and is rapidly cleared from
maternal plasma shortly after delivery [6], making it pregnancy
specic. However, the majority of cell-free DNA in a mothers blood
is maternal in origin [7], which makes analysis of cffDNA

* Corresponding author. Address: NE Thames Regional Genetics Service, York

House, 37 Queen Square, London WC1N 3BH, UK. Tel.: 44 (0)207 813 8533.
E-mail address: l.chitty@ucl.ac.uk (L.S. Chitty).
1744-165X/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.siny.2013.10.003

challenging. Early clinical applications for cffDNA included the

polymerase chain reaction (PCR)-based detection or exclusion of
paternally inherited alleles for fetal sex determination in pregnancies at high risk of sex-linked genetic disorders [8], fetal RHD
typing in RhD-negative mothers [9] and, more recently, for the
diagnosis of some single gene disorders, which have arisen de novo
or are paternally inherited [10,11].
NIPT for DS poses different challenges because of the high
background levels of maternal chromosome 21 cfDNA. It is not
feasible to use the PCR methods described above, as these are not
sufciently sensitive to detect the relatively small changes in level
of chromosome 21 when the fetus has DS as most of the cfDNA is
maternal in origin. Detection and quantication of this small difference requires either the analysis of targets that are free from
maternal background interference, i.e. are fetal specic, or the use
of technologies that enable extremely accurate copy number
counting. Initial attempts at NIPT for DS targeted a fetal-specic
marker in mRNA in maternal plasma rather than cffDNA. This
approach was based on testing cell-free mRNA from PLAC4, a gene
located on chromosome 21, which is expressed in the placenta but
not in maternal blood (i.e. it is fetal specic) [12]. By extracting
cfRNA (rather than cfDNA) from maternal plasma and testing a
single nucleotide polymorphism (SNP, a common sequence variation found in the normal population) located in the PLAC4 fetal
mRNA sequence, the chromosome 21 allelic ratio was determined
to infer chromosome 21 dosage [12]. Named the RNA-SNP method,
this represented the rst demonstration of NIPT for DS, achieving a
sensitivity and specicity of 90% and 96.5%, respectively. However,
a major drawback to SNP-based approaches is the reliance on
polymorphisms within the DNA carrying the placenta-specic
expression, thus limiting their use to families where parents carry

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P. Twiss et al. / Seminars in Fetal & Neonatal Medicine 19 (2014) 9e14

different alleles and whose fetuses are therefore heterozygous. For

the PLAC4 RNA-SNP method this was estimated to apply to around
40% of pregnancies [12], rendering this approach impractical for
routine clinical use.
Alternative approaches to NIPT for DS have been based on
epigenetic differences between the maternal and fetal DNA, fetal
DNA from the placenta being hypermethylated compared to
maternal DNA, which is hypomethylated. The basis of this approach
is that by using methylation-sensitive restriction enzymes, hypomethylated maternal sequences can be digested leaving only
hypermethylated fetal sequences available for analysis by real-time
PCR. Papageorgiou and colleagues published a set of fetal-specic
epigenetic markers for all common aneuploidy chromosomes
[13], and subsequently reported accurate NIPT for DS using methylated DNA immunoprecipitation (MeDIP) real-time PCR [14].
However, to date no large-scale validation study has been reported
using this method. Hypermethylation of the HLCS gene promoter
on chromosome 21 has also been successfully reported for NIPT of
T21 by comparing to dosage of the ZFY gene. However, this test is
restricted to male-bearing pregnancies [15]. NIPT based on differential methylation has yet to nd a place in clinical practice, not
least because the use of epigenetic markers is limited by relatively
labour-intensive and time-consuming bisulphite conversion or restriction enzyme digestion, making them less practical for use in a
routine service laboratory.
The introduction of technologies such as digital PCR (dPCR) and
next generation sequencing (NGS) have enabled accurate estimation of very small differences in chromosome-specic sequences in
maternal blood, thus delivering an approach to NIPT that can be
used in clinical practice.

reproduced these ndings in large validation studies (Table 1) with

similarly high detection levels across a range of sequencing instruments and chemistries, using a variety of bioinformatics algorithms to open the door to the wider application of NGS for the NIPT
of DS [18e28].
Two approaches to NIPT for DS using NGS are now in common
use in the USA, Asia, and some parts of Europe [29]. The rst uses
the whole genome MPS approach described above (Table 1), which
requires sequencing of many millions of DNA fragments in order to
generate sufcient reads to detect differences in level of reads from
chromosome 21, which constitutes around 1.5% of sequenced
fragments. The alternative approach has been to target the
sequencing to selected genomic loci on the chromosome of interest,
e.g. chromosome 21 for NIPT for DS (Table 1) [30e36]. This significantly reduces the amount of sequencing required and is primarily
aimed at reducing costs while increasing throughput and test
performance. Regardless of the approach taken, the sensitivity and
specicity of these methods are high, ranging from 98.6 to 100%
and from 99.7 to 100%, respectively (Table 1). False-negative results
may be related to low fetal fraction of cffDNA and have been shown
to be more common in obese women [37,38], where it is thought
that there is a higher than average level of circulating maternal
cfDNA because of increased release of cfDNA from adipose tissue.
The small, but regularly reported, false-positive rate results from a
variety of factors and reects the fact NIPT analyses both maternal
and fetal cfDNA, and that the cffDNA emanates from the placenta.
Thus, the aetiology of reported discordant results includes conned
placental mosaicism [39,40], maternal chromosome abnormalities
[41], and the presence of maternal malignancy [42].
NIPT for other common chromosomal abnormalities

Non-invasive prenatal testing for DS using NGS

Two seminal proof-of-principle experiments published in 2008
[16,17] demonstrated that massively parallel shotgun sequencing
(MPSS) of cfDNA in maternal plasma had the potential to be an
effective method for fetal T21 detection. In brief, whole genome
cfDNA extracted from maternal plasma is sequenced to generate
millions of short sequence reads or tags. These tags are then
aligned and uniquely mapped to the reference human genome
sequence. Individual uniquely mapped reads to chromosome 21 are
then counted, and compared to the number of counts obtained
from a reference euploid sample. Fan et al. [16] successfully classied all nine trisomy 21 cases in a cohort of 18 samples, and Chui
et al. [17] correctly detected all 14 T21 cases in a cohort of 28, with
neither study producing false-positive results. Other groups have

NIPT for other common aneuploidies, trisomies 13 and 18, have

been reported with lower detection rates, which some reports
suggest is because of the larger chromosome size and higher GC
content [20,28,31e33,43]. Combined data from ve studies report a
sensitivity of 97.4% (188/193) for trisomy 18 (Edwards syndrome)
[20,28,31e33]. However, only three of these studies [20,28,43]
include data for trisomy 13 (Patau syndrome) and report a lower
sensitivity of 83.3% (30/38). However, more recent studies report
improved detection rates [22,23], although the outcome data are
not always reported in detail. False-positive rates are broadly
similar, with trisomy 18 being consistent with those seen for trisomy 21, and slightly higher for NIPT of trisomy 13 (0.41%), but as
the numbers reported are small, it is difcult to draw denitive
conclusions at this time.

Table 1
Studies reporting the use of next generation sequencing for non-invasive prenatal testing for Down syndrome.
Type of approach

MPS whole genome

Enrich et al. [18]
Palomaki et al. [19]
Bianchi et al. [20]
Dan et al. [21]
Futch et al. [22]
Liang et al. [23]
Targeted MPS
Sparks et al. [30]
Sparks et al. [31]
Ashoor et al. [32]
Norton et al. [33]
Nicolaides et al. [34]
Zimmerman et al. [35]
Nicolaides et al. [36]

Test results

Sensitivity

Specicity

TP

FN

TN

FP

Total

95% CI

95% CI

39
209
89
139
154
40

0
3
0
0
2
0

409
1468
404
2819
5515
372

1
3
0
1
1
0

449
1683
493
2959
5672
412

100
98.6
100
100
98.7
100

89e100
95.9e99.5
95.9e100
97.3e100
95.5e99.7
91.2e100

99.7
99.8
100
99.96
99.98
100

98.5e99.9
99.4e99.9
99.1e100
99.8e99.99
99.9e100
98.98e100

39
36
50
81
8
11
25

0
0
0
0
0
0
0

252
123
297
2887
1939
126
197

0
0
0
1
0
0
0

291
159
347
2969
1947
137
222

100
100
100
100
100
100
100

91.0e100
90.4e100
92.9e100
95.5e100
67.6e100
74.1e100
86.7e100

100
100
100
99.97
100
100
100

98.5e100
97.0e100
98.7e100
99.8e99.99
99.8e100
97.0e100
98.1e100

TP, true positive; FN, false negative; TN, true negative; FP, false positive; CI, condence interval; MPS, massively parallel sequencing.

P. Twiss et al. / Seminars in Fetal & Neonatal Medicine 19 (2014) 9e14

Transition to clinical practice

The series of successful validation studies has paved the way for
the clinical implementation of NIPT for DS. However, the low falsepositive rate means that NIPT is not considered fully diagnostic and
is considered to be a highly accurate screening test where invasive
testing is recommended to conrm a positive NIPT result [44,45].
Nonetheless, the clinical implementation of NIPT for DS has moved
rapidly, being wholly commercially driven. It was rst launched
through commercial providers in the USA and China/Hong Kong in
2011. Other companies have been quick to follow and several
different providers are currently offering tests for trisomies 21, 18,
13 and sex aneuploidies, with more companies likely to enter the
market in the near future [46]. Currently, NIPT is only available in
the private sector in most countries, with public sector implementation awaiting further evaluation in medium or low-risk
pregnancies. There is ongoing debate as to how NIPT may best t
into existing care pathways for the routine screening and diagnosis
of DS. The initial validation studies were performed in high-risk
populations, and the current approach supported by a variety of
professional bodies worldwide is to offer NIPT to women considered at high risk because of past history or other screening results.
Studies showing clinical efcacy in average and low-risk populations are now being published [34], which may inuence decisions on implementation, including whether NIPT should be
offered as a rst-line screening test to all pregnant women, thus
replacing current screening, or as a second tier screening test,
contingent on the results of a traditional screening test. Regardless
of the ultimate method of delivery, for national- or state-based
prenatal screening programmes, consideration needs to be given
to issues such as accuracy in the population being tested, test uptake, costs and benets derived from existing care pathways, and
maintenance of informed parental choice [47].

11

is an appropriate approach until costs of NIPT fall [28,48,51,53].

However, an accurate evaluation of economic costs and benets not
only depends on the cost testing and mode of implementation, but
also on the uptake of, and reasons for, testing and thus further
evaluation following implementation is required.
Uptake of NIPT for DS
NIPT reduces the risk of miscarriage associated with invasive
diagnostic testing, and this is reported by women to be one of the
greatest advantages of NIPT. Studies based on hypothetical scenarios of offering NIPT for DS have indicated that uptake will be
high [54], and are likely to be signicantly greater than the uptake
of current of DS screening and invasive testing. It is also clear from
some of these studies that an additional group of women, who
would decline current screening and diagnosis because of the risk
of miscarriage, will undergo NIPT to plan and prepare for the birth
of an affected child. Both uptake and clinical utility will clearly
inuence the economic aspects of implementation. The rst reports
of clinical experience and uptake following the implementation of
NIPT in the private sector in the USA are now beginning to appear
[55e57]. These conrm the positive attitude towards NIPT, but they
indicate that uptake is not as great as hypothetical studies have
suggested [55e57]. It appears that cost was an important factor in
test uptake in all studies, as insurance coverage is variable; some
people had out-of-pocket expenses for NIPT but not other types of
testing [55e57]. Other factors thought to contribute to NIPT uptake
in these studies include having certainty and additional information from invasive testing (e.g. pathogenic chromosomal rearrangements, microdeletions, etc.), later gestational age at the time
of being offered the test, ethnic background, and opportunities for
ultrasonography [55,56].
Potential impact of patents and licensing

Counselling issues
Regardless of potential changes in the care pathway, in view of
the false-positive rate described above, those offering post-test
counselling following positive results should ensure that the need
for a conrmatory invasive diagnostic test is discussed. Interpretation of results in the absence of denitive conrmation by
traditional testing of amniocytes or chorionic villi obtained after
amniocentesis or chorionic villus sampling may be complicated,
particularly in the absence of ultrasound abnormalities indicative
of the specic chromosomal abnormality in question.
Economic considerations for implementation
Cost is a major factor in determining how NIPT should t into
existing care pathways, especially for state-funded healthcare
systems. Several studies have undertaken model-based cost and
outcome analyses of offering NIPT in a variety of ways from both a
US [28,48e51] and UK [52,53] perspective. Morris et al. [52] have
evaluated NIPT as a contingent screening test and as a rst-line
screening test with reference to current screening pathways in
the UKs National Health Service (NHS). Contingent screening gave
favourable clinical outcomes in terms of the number of DS cases
detected and fewer procedure related-miscarriages. It was also
cheaper than existing pathways if NIPT was to cost less than 500.
When used as a rst-line test, NIPT detects more cases of DS and
has fewer procedure-related miscarriages, but it is considerably
more expensive than current screening even if NIPT was to cost just
50. Other studies have also favoured contingent screening in terms
of costs, cases detected, and miscarriages avoided compared to
rst-line testing, leading to suggestions that contingent screening

Patents and licensing is a complex and contentious area for NIPT

implementation that has yet to be resolved, but which may impact
signicantly on both the cost and type of NIPT offered. There are
more than 100 patents and applications relating to cffDNA testing,
with the basic technologies for isolating and genetic analysis of
cffDNA being patented or licensed to just a small number of companies or institutions [46]. Several lawsuits are currently in progress regarding enforcement and infringement of patents and
licensing, and until these are resolved it is difcult to assess what
the impact will be. If the current claims are upheld and a single
company holds all intellectual property relating to NIPT, there are
concerns that costs of tests will increase, access to alternative tests
will be limited, test improvement and quality assurance measures
will be restricted, and that there will be obstacles imposed on
research and development. All of this could result in reduced
quality of care for patients and limitations on access to tests [46]. A
detailed explanation of the commercial and patenting issues is
beyond the scope of this review, but Agarwal et al. [46] provide an
overview of the patenting landscape in the USA.
Formal regulation and guidance of testing is essential for successful
implementation
In their recent review, Hahn et al. [58] suggest that the implementation of NIPT through multiple companies competing for
marketplace share raises concerns that NIPT has come into practice
too rapidly and that appropriate safeguards for accuracy, clinical
utility, and patient awareness have not been met. Guidance for offering prenatal testing and regulation of laboratories conducting
NIPT will vary widely between countries. Formal guidance from

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P. Twiss et al. / Seminars in Fetal & Neonatal Medicine 19 (2014) 9e14

professional bodies and other expert groups has an important role to

play in the successful and ethically sound implementation of NIPT
[59]. Health professionals in both the USA [60] and the UK [61] are
reported to value formal guidance on how best to offer NIPT. There is
some evidence that, although implementation of NIPT has been
driven by commercial providers, guidance from professional bodies
has played a major part in how testing has been implemented,
particularly in the USA [62]. For example, guidelines describing NIPT
as an advanced screening test that needs to be conrmed by invasive
testing and support for tests being offered through health care
providers to promote informed choice [44,45] are being upheld by
commercial companies offering NIPT in the USA.
Addressing ethical issues through appropriate counselling and
support
There is ongoing debate about how best to circumvent key
ethical concerns associated with offering NIPT, including difculties
facilitating informed choice, increased pressure to take up prenatal
testing, and equity of access [63,64]. Concerns around informed
choice are not new for prenatal testing for DS, as research indicates
that women do not always make informed decisions when undergoing screening [65]. However, it has been suggested that
because NIPT for DS generates a highly accurate screening result, it
is especially important that we address this issue and ensure that
informed consent is obtained through detailed pre-test counselling
[64]. Yet, two of the key clinical benets of NIPT e that it is easy to
perform and that it carries no risk of miscarriage e are also the
factors that make offering NIPT susceptible to difculties in safeguarding informed choice. For example, parents may not give full
consideration to the implications of results, as NIPT is just a blood
test, [63] especially if it is offered as one of many other antenatal
blood tests. Similarly, without the miscarriage risk, parents may see
no downside to testing, and NIPT could be viewed as routine or
expected [63], which makes the possibility of informed decisions
less likely than when tests are viewed as optional [65]. There is also
evidence that both health care professionals and women view NIPT
much more like a screening test than an invasive test in terms of a
need for written consent or time for reection about testing, and
consequently both parties may be less concerned about reaching
informed consent than they would with an invasive diagnostic test.
Increased societal pressure for testing and termination as tests
become easier to access, safer, and easier to perform is a concern that
has been raised by health care professionals, pregnant women, and
the general public [54]. There is also concern that any increases in
testing and terminations from NIPT have the potential to result in a
reduction in support structures for children with disabilities and a
society that may be less tolerant in their attitudes to people who
have children with DS [54], all of which have the potential to result in
women feeling more pressure to undertake testing. In addition,
without the risk of miscarriage women may feel they cannot justify
their decision not to have a test, and clinicians need to be sensitive
and aware that some women will prefer not to have prenatal testing.
Another issue of major concern is equity of access. In the USA,
the costs of NIPT are highly variable and coverage by insurers differs, so out-of-pocket expenses vary widely. In the UKs private
sector the cost of NIPT is also variable. This is a major drawback of
testing being offered solely through commercial providers, as the
associated costs prevent equity of access with low-income families
being unable to afford testing [46].
Conclusions
There is no doubt that NIPT for DS and other major trisomies is
highly accurate when using NGS. NIPT is currently restricted to the

major trisomies and sex chromosome anomalies. Recent reports of

the detection of subchromosome abnormalities[66,67] indicate
that it may become more comprehensive over time, particularly as
sequencing costs fall. Current application is limited to the private
sector, but it is hoped that more widespread implementation into
public sector health care will follow soon. Many thousands of tests
have now been carried out across the globe and factors affecting
test performance are becoming clearer. What is certain is that
routine implementation of NIPT will require health care professional and patient education, with new strategies for pre- and posttest counselling in order to maintain ethically-sound levels of
informed parental choice and to ensure that parents understand
the implications and limitations of results and have personalised
support and information for making decisions about the next steps.
NIPT is an exciting and rapidly developing area which is set to
transform antenatal care. The challenge now is to translate this
technology into an accurate and affordable test that can be made
available to all women within current healthcare budgets.

Practice points
 NIPT using NGS is a highly accurate screening test for the
most common fetal trisomies (21, 18 and 13) that can be
performed anytime after 10 weeks of gestation.
 As NIPT is not diagnostic, invasive testing should be
offered to confirm a positive NIPT result.
 As NIPT does not test for all possible aneuploidies or
chromosomal rearrangements, invasive diagnostic
testing should be considered when fetal structural abnormalities are identified by ultrasound.
 NIPT is currently only available in the commercial sector
and is not yet being offered in public health services.
 Formal regulation, professional guidance and parent education packages will be essential for successful
implementation.
 Parents should be supported to make informed decisions
about prenatal testing.
p Information describing the benefits and limitations of
screening, NIPT and invasive testing should be
provided.
p Health professionals should not present NIPT as a
routine test and discussion should aim to circumvent
feelings of pressure to have testing.
p Post-test counselling is essential, particularly in the
event of an NIPT result that predicts the fetus to be
aneuploid.

Research directions
 Continued validation of NIPT in average- and low-risk
women is needed prior to widespread use of NIPT in
these populations.
 Current economic evaluations based on hypothetical
models suggest that NIPT as a contingent screening test
will be more cost-effective than NIPT as a first-line
screening test. To confirm these findings, further economic evaluations in real-world settings that consider
test uptake, clinical benefits and costs are required.
 Exploration of service users and providers experiences
is essential following test implementation to ensure that
the service is meeting the needs of all stakeholders.
 In the future NIPT may be used to detect subchromosomal abnormalities. Further research is required
to explore these possibilities and large-scale trials performed to validate test accuracy.

P. Twiss et al. / Seminars in Fetal & Neonatal Medicine 19 (2014) 9e14

Conict of interest statement

None declared.
Funding sources
P.T. is funded by the NIHR Biomedical Research Centre at Great
Ormond Street Hospital, and L.S.C. is partially funded by the Great
Ormond Street Hospital Childrens Charity and the NIHR Biomedical Research Centre at Great Ormond Street Hospital. The views
expressed are those of the authors and not necessarily those of the
NHS, the NIHR or the Department of Health.
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