Ultrasound Obstet Gynecol 2013; 42: 15–33
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/uog.12513
Non-invasive prenatal testing for aneuploidy: current status
and future prospects
P. BENN*, H. CUCKLE† and E. PERGAMENT‡
*Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA; †Department of
Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA; ‡Northwestern Reproductive Genetics, Chicago,
IL, USA
K E Y W O R D S: amniocentesis; aneuploidy; chorionic villus sampling; Down syndrome; fetal DNA; maternal plasma; screening;
sequencing; trisomy
ABSTRACT
Non-invasive prenatal testing (NIPT) for aneuploidy
using cell-free DNA in maternal plasma is revolutionizing
prenatal screening and diagnosis. We review NIPT in the
context of established screening and invasive technologies,
the range of cytogenetic abnormalities detectable, cost,
counseling and ethical issues. Current NIPT approaches
involve whole-genome sequencing, targeted sequencing
and assessment of single nucleotide polymorphism (SNP)
differences between mother and fetus. Clinical trials
have demonstrated the efficacy of NIPT for Down and
Edwards syndromes, and possibly Patau syndrome, in
high-risk women. Universal NIPT is not cost-effective,
but using NIPT contingently in women found at moderate
or high risk by conventional screening is cost-effective.
Positive NIPT results must be confirmed using invasive
techniques. Established screening, fetal ultrasound and
invasive procedures with microarray testing allow the
detection of a broad range of additional abnormalities
not yet detectable by NIPT. NIPT approaches that
take advantage of SNP information potentially allow the
identification of parent of origin for imbalances, triploidy,
uniparental disomy and consanguinity, and separate
evaluation of dizygotic twins. Fetal fraction enrichment,
improved sequencing and selected analysis of the most
informative sequences should result in tests for additional
chromosomal abnormalities. Providing adequate prenatal
counseling poses a substantial challenge given the broad
range of prenatal testing options now available. Copyright
© 2013 ISUOG. Published by John Wiley & Sons Ltd.
INTRODUCTION
The past quarter century has been witness to a series of
remarkable advances in the screening of pregnancies for
aneuploidy, particularly in the identification of Down
syndrome (trisomy 21). During the 1970s and early
1980s, advanced maternal age, defined in most localities
as over 35 years, was the only means by which the
general population was assessed as to risk of a fetal
chromosomal abnormality. Fewer than one third of
Down syndrome pregnancies were diagnosed prenatally
and of those undergoing invasive prenatal diagnosis only
about 2% had fetal karyotype abnormalities1 , a figure
comparable to the 0.5–1% chance of procedure-related
fetal loss associated with amniocentesis or chorionic villus
sampling (CVS)2 . In the late 1980s and early 1990s, the
introduction of second-trimester maternal serum markers,
in the form of ‘double’, ‘triple’ and ‘quad’ marker testing,
improved significantly the screening performance for aneuploidy. The proportion of Down syndrome pregnancies
diagnosed more than doubled and a chromosomal abnormality was found in as many as 4% of those designated
as ‘screen-positive’3 . In the late 1990s and early 2000s,
aneuploidy screening shifted to the first trimester with
the ‘combined’ test, which uses ultrasound measurement
of nuchal translucency thickness (NT) together with
maternal serum concentration of placental proteins
human chorionic gonadotropin (hCG) (free β, intact
or total) and pregnancy-associated plasma protein-A
(PAPP-A). Currently available screening protocols also
incorporate additional ultrasound markers and sequential
screening using two blood samples, one in the first
and one in the second trimester, with or without NT.
Consequently, screening performance has improved such
that more than nine-tenths of Down syndrome cases can
be diagnosed prenatally4 and the yield from invasive
testing has risen to about 6%5 .
Recently, analysis of cell-free (cf)DNA in maternal
blood for non-invasive prenatal testing (NIPT) has been
shown to be highly accurate in the detection of common
fetal autosomal trisomies. In 1997, Lo et al.6 first reported
their seminal discovery that plasma from pregnant women
contained cfDNA, including a fraction designated ‘fetal’,
although this is thought to be placental in origin, resulting
Correspondence to: Dr P. Benn, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington,
CT, USA (e-mail: benn@nso1.uchc.edu)
Accepted: 14 May 2013
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
REVIEW
16
from apoptotic trophoblasts. This and other early studies7
suggested that the ‘fetal fraction’ was only 3–6%, but
more recent studies have found that it may be closer
to 10–20%8 . Fetal cfDNA can be detected as early as
4 weeks’ gestation9 and exceeds 4% of all the cfDNA in
nearly all women from 10 weeks onward. The typical
size of the cfDNA fragments is approximately 150
basepairs10,11 and, importantly, the entire fetal genome
is represented. It has been shown that the half-life of
fetal cfDNA is very short12 ; fetal fragments are no longer
detectable very soon after birth13,14 . There is therefore
no serious concern that a prenatal cfDNA test could be
confounded by a prior pregnancy in multigravid women.
Maternal plasma cfRNA, unlike RNA extracted directly
from cells, appears to be relatively stable and can also
potentially be used in screening15 – 17 .
The commercial introduction of NIPT raises several
concerns. One major issue is the optimal integration of
NIPT into current screening practice18 . Should NIPT and
conventional screening tests be treated as related but
independent measures when assessing aneuploidy risk or
should they be complementary to one another? How can
healthcare providers be educated and prospective patients
counseled about all the prenatal testing options now
available?
In this Review we provide the information needed by
clinicians and public health providers when considering
to whom NIPT will be offered and regarding the
consequences of doing so, practical suggestions on how to
implement this new and powerful technology into routine
clinical practice, and some indications of how we expect
this testing to expand. We focus primarily on the detection
of fetal chromosomal abnormalities, recognizing that the
technology will eventually be used for testing for a broad
range of other genetic disorders.
ESTABLISHED SCREENING MODALITIES
In the current context it is important to distinguish prenatal diagnosis of aneuploidy from antenatal screening. A
diagnostic test performed on chorionic villi, amniotic fluid
or fetal blood needs to have very few false negatives (aneuploid pregnancies misdiagnosed as euploid) and false positives (euploid pregnancies misdiagnosed as aneuploid),
since the result will inform the decision as to whether to
terminate the pregnancy. In contrast, antenatal screening
does not aim to be definitive; rather, it is designed to identify women who are at sufficiently high risk of common
aneuploidies as to warrant invasive prenatal diagnosis.
Since the invasive diagnostic procedures, mainly CVS and
amniocentesis, are hazardous and expensive, only a relatively small group of women are identified as being at high
risk. Established screening programs determine the risk of
Down syndrome, many also including Edwards syndrome
(trisomy 18) and some including Patau syndrome (trisomy
13) and Turner syndrome (45,X). The model-predicted
aneuploidy detection rates (DR) for fixed false-positive
rates (FPR) of established prenatal screening protocols
has been reviewed elsewhere4 .
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
Benn et al.
Among those undergoing invasive prenatal diagnosis
because of a high risk of Down and Edwards syndromes,
a large proportion have a different chromosomal abnormality. Alamillo et al.19 reported on 10 years’ experience
of a combined test, giving risks of Down syndrome
and Edwards or Patau syndromes. The 97 cases with
chromosomal abnormalities included Down syndrome
(48%), Edwards syndrome (16%), Patau syndrome (6%),
Turner syndrome (9%), other sex chromosomal aneuploidies (4%), other aneuploidies including mosaics (9%)
and chromosomal rearrangements (6%). The California
state-wide screening program20 used a second-trimester
quad test for Down and Edwards syndromes and detected
a total of 1316 chromosomal abnormalities. Excluding
balanced translocations, these were Down syndrome
(48%), Edwards syndrome (14%), Patau syndrome
(2%), Turner syndrome (8%), triploidy (2%), Klinefelter
syndrome (47,XXY) (1%) and other abnormalities
(25%). Some of these were chance findings but many
were related to maternal age or abnormal marker profiles.
Conventional aneuploidy screening modalities also
need to be considered in the context of the substantial
number of non-chromosomal fetal abnormalities and
pregnancy complications that may be identified as a result
of this testing. Particularly important is the role of firsttrimester ultrasound, which has the potential to identify
a non-chromosomal abnormality in approximately 1%
of cases5 .
ESTABLISHED INVASIVE PRENATAL
DIAGNOSIS
Conventional cytogenetics
Chromosomal analysis of cultured chorionic villi and
amniotic fluid cells can identify fetal aneuploidy,
structural rearrangements, such as translocations and
inversions, and relatively large duplications and deletions
(generally exceeding 5 Mb in size)21 . Chromosomal
analysis of amniotic fluid cells is considered to be the
‘gold standard’ in prenatal testing because error rates are
exceedingly low, probably less than 0.01–0.02%, and
mostly appear to be due to maternal cell contamination,
laboratory error or typographic mistakes22 . Error rates
with CVS are higher than those seen with amniocentesis,
and these can be due to confined placental mosaicism
(CPM), maternal cell contamination or lower resolution
banding of chromosomes23 . In a small proportion of
cases in which CVS karyotyping is carried out there
are ambiguous results which may require resampling or
amniocentesis for clarification.
Molecular cytogenetics
Because of the time taken to obtain a complete chromosomal analysis, the occasional finding of a karyotype of
minimal or uncertain significance and cost considerations,
some centers perform a rapid aneuploidy test (RAT)
using molecular genetic techniques as an adjunct to or
Ultrasound Obstet Gynecol 2013; 42: 15–33.
17
NIPT for aneuploidy
replacement for karyotyping. Methods include the use
of fluorescence in-situ hybridization (FISH)24,25 , quantitative fluorescence polymerase chain reaction (QF-PCR)
testing26 and multiplex ligation-dependent amplification
(MLPA)27 . These tests are generally designed to test only a
restricted range of cytogenetic abnormalities28 . Although
proven to be very accurate, confirmatory testing using
conventional cytogenetics has been recommended29 .
In contrast, relative to conventional karyotyping, other
molecular approaches can now improve substantially on
the detection of clinically significant genetic imbalances.
In high-risk pregnancies with a normal chromosomal
constitution, the increased resolution of array comparative genome hybridization (aCGH) coupled with single
nucleotide polymorphism (SNP) array has the potential to
identify submicroscopic copy number variations (CNVs),
specifically microdeletions and microduplications, as well
as regions of homozygosity.
When compared to conventional chromosomal analysis, aCGH and SNP array are purported to have significant
advantages in terms of: (1) faster turnaround time;
(2) higher throughput while being less labor-intensive;
(3) higher resolution independent of the ability of the
cells to grow and/or generate good metaphase spreads;
(4) amenability to automation and quality control; and,
(5) direct mapping of aberrations to the genome sequence.
Faster turnaround time is possible because arrays can
be performed on DNA obtained from uncultured villi
and amniotic fluid cells, thus avoiding the need for tissue culture and the associated time delay required for
conventional chromosomal analysis.
A National Institute of Health (NICHD) multicenter,
prospective, blinded study30 evaluated the application
of microarrays in over 4000 high-risk pregnancies
undergoing prenatal invasive testing largely because of
advanced maternal age, a positive first-trimester screen
or the presence of an ultrasound anomaly. The use of
microarrays resulted in the identification of clinically
relevant CNVs that were unrecognizable by conventional
karyotyping in 2.5% of all cases. In those cases referred
for testing because of an ultrasound abnormality and
showing a normal karyotype, 6% were found to have
a clinically relevant CNV. These studies were performed
on low-resolution aCGH platforms; newer arrays have
much higher resolution. The use of SNP arrays has
expanded the ability to detect triploidy and significant
regions of homozygosity, thereby detecting uniparental
disomy and potentially identifying disorders associated
with consanguinity. The NIH multicenter study also
revealed the limitations of all aCGH and SNP array
platforms, the principal limitation being the identification
of variants of uncertain significance (VOUS).
Hazards of invasive testing
The principal hazard of amniocentesis is miscarriage, but
the excess risk associated with the procedure is difficult
to quantify precisely. Some 3–4% of mid-trimester
pregnancies will miscarry without amniocentesis and in
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
a particular case of fetal loss following the procedure it
is possible only rarely to attribute directly the adverse
outcome to the procedure; cases of amnionitis or chronic
amniotic fluid leakage would be attributable but these
are relatively rare consequences. In a randomized trial of
amniocentesis at a single center31 , the fetal loss rate was
0.8% higher in the amniocentesis group compared with
controls. There have been no similar randomized trials
for CVS, but a number of studies have compared late
first-trimester CVS with second-trimester amniocentesis.
A Cochrane Review32 showed that, when performed by a
skilled operator, the fetal loss rates of the two procedures
were comparable. An updated review2 concluded that the
excess miscarriage rate for either procedure is between
0.5% and 1%. An initial concern that CVS may cause
fetal limb reduction defects has not been sustained, as
the reported excesses occurred when CVS was performed
early in gestation, before 10 weeks32 .
NIPT METHODS
The fact that fetal cfDNA and cfRNA are present only
as minority components of the total cell-free nucleic acids
in maternal plasma specimens has posed a significant
technical obstacle in the development of NIPT. A
number of approaches have been suggested that allow
enrichment of the fetal component. These include taking
advantage of size differences between fetal and maternal
DNA fragments11 , using formaldehyde in the samples33 ,
taking advantage of the nucleosome binding property
of the DNA34 and immunoprecipitation of hypo- or
hypermethylated DNA35 , but none is currently being
used in clinical practice. Even without enrichment a
number of technical approaches have now been proven
to be effective. Each approach relies on the extraordinary
advances in molecular biology and sequencing achieved
in recent years. We briefly review the major methods
below, without discussing in detail all of the possible
refinements and other developments that are currently
under consideration.
Shotgun massively parallel sequencing (s-MPS)
This approach relies on the identification and counting
of large numbers of the DNA fragments in the plasma
specimen. Using massively parallel sequencing (MPS),
millions of both fetal and maternal DNA fragments can
be sequenced simultaneously and, since the entire human
genome sequence is known, each piece that maps to a
discrete locus can be assigned to the chromosome from
which it came36,37 . If fetal aneuploidy is present, there
should be a relative excess or deficit for the chromosome
in question. The difference in counts will be small; for
example, if fetal trisomy 21 is present and the fetal
fraction is 20%, the relative excess in chromosome
21 DNA fragments will be (0.8 × 2) + (0.2 × 3) = 2.2
compared with the situation for a euploid fetus
(0.8 × 2) + (0.2 × 2) = 2, i.e. a relative increase in number
of chromosome 21 counts of only 10%. To be able to
Ultrasound Obstet Gynecol 2013; 42: 15–33.
18
detect reliably these differences, large numbers of counts
are necessary, the fetal fraction needs to be appreciable
and the counts need to be compared with the expected
counts for euploid cases. The latter can be achieved either
by normalizing counts for the chromosome of interest
against other chromosomes that are expected to be
disomic within the same test run36 or by comparing the
fraction of counts assigned to a particular chromosome
against the fractions seen for a set of known euploid
cases38 . Results can be expressed as a z-score which
can be converted into a probability that a specific
chromosome result departs from the normal diploid
situation. Although it is possible to convert the z-score
to a patient-specific risk, major commercial providers
of this testing currently choose to present their results
only in terms of ‘positive’ or ‘negative’ based on z-scores
exceeding predefined thresholds. Another variation of the
statistical analysis calculates two Student t-test statistics,
one based on the hypothesis that the result is from a
euploid fetus and the other that it is from an aneuploid
fetus and then calculates a further test statistic, L,
which is the logarithmic likelihood ratio39 . Refinements
in the statistical analysis also include adjustment for
guanine-cytosine (GC) base content of the sequences40,41 .
The approach is referred to as ‘shotgun’ because it relies
on sequencing and counting all informative chromosome
regions. Sequences which do not map to a unique locus
are uninformative. In some studies, only those fragments
that have sequences identical to the reference genome
sequence are accepted in the analysis, while in others
one or two mismatches in the sequence will be accepted.
Because of costs, it is common to run multiple samples
simultaneously (‘multiplexing’) by adding a ‘bar-code’
sequence to each fragment that will allow identification
of the sample origin. The number of cases run together
can be variable and is an important consideration because
high-level multiplexing places a limitation on the total
number of sequence reads that are carried out on each
case (i.e. the ‘depth of sequencing’). The number of
fetal sequence reads will also vary according to the
fetal fraction and therefore a minimum fetal fraction,
typically 4%, is currently required. Most studies have
used Illumina sequence analyzers but there are also studies
using Solid42,43 and Helicos44 platforms.
Targeted massively parallel sequencing (t-MPS)
It is also possible to amplify selectively only those chromosomal regions that are of interest (e.g. chromosomes
21, 18 and 13) and then to assess whether there is a
departure from euploidy based on the relative number of
DNA fragment counts for this subset of chromosomes45 .
This ‘targeted’ approach has the advantage of requiring
considerably less sequencing and thereby reduces costs.
The major commercial provider of this approach includes
an adjustment to allow for the variability in the proportion of DNA that is fetal and then combines the results of
the laboratory test with maternal age to provide a patientspecific risk for Down, Edwards and Patau syndromes45 .
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
Benn et al.
In theory, results could also be combined with other factors, for example results of other screening tests or prior
history of an aneuploid pregnancy.
Single nucleotide polymorphism (SNP)-based
approaches
The feasibility of a non-invasive aneuploidy test that takes
advantage of DNA polymorphisms was first demonstrated
by Dhallan et al.46 . In their approach, the paternal,
maternal and fetal SNPs present in blood (paternal),
buffy coat (maternal) and maternal plasma (maternal
and fetal) were evaluated as bands on sequencing gels.
Quantification of band intensities of the uniquely inherited
paternal SNPs in the plasma allowed an estimation of
the fetal fraction. Comparison of the maternal plus
fetal bands with the unique fetal band intensity also
allowed estimation of the fetal chromosome 21 dosage.
A similar approach was used by Ghanta et al.47 , who
identified combinations of paired SNPs to focus on highly
informative regions and then used cycling temperature
capillary electrophoresis for evaluating fetal fraction and
dosage. The method was thought to be useful in situations
in which the fetal fraction was as low as 2%.
A more complex approach that relies on polymorphisms has been proposed by Zimmermann et al.48 . The
approach involves a multiplex amplification of 11 000
SNP sequences in a single PCR reaction carried out on
the plasma DNA followed by sequencing. Each product is
evaluated based on the hypothesis that the fetus is monosomic, disomic or trisomic. After considering the positions
of the SNPs on the chromosomes and the possibility
that there may have been recombination, a maximum
likelihood is calculated that the fetus is either normal,
aneuploid (chromosome 21, 18, 13 or sex chromosome)
or triploid, or that uniparental disomy is present.
In theory, with SNPs there should be advantages over
methods based on total sequence counting. SNPs can
provide information about parent of origin of aneuploidy,
recombination and inheritance of mutations. On the
other hand, SNPs account for only 1.6% of the human
genome and therefore enrichment for fetal DNA, deeper
sequencing or higher levels of high-fidelity amplification
may be required to identify unambiguously affected
pregnancies with small imbalances49 . When few loci are
used in the analysis, greater attention needs to be paid to
the exclusion of regions in which there are rare benign
CNVs. The SNP approach could result in the inadvertent
detection of non-paternity or perhaps consanguinity. In
the case of assisted reproduction with an egg from an
unrelated donor, SNP-based approaches would need to
be modified to take into consideration the presence of
additional maternally derived fetal alleles not present in
the surrogate mother.
The laboratory methods and data analyses used for
SNP-based NIPT are substantially different from those
used in shotgun and targeted counting approaches. In this
Review we therefore consider the emerging clinical trial
data for SNP-based testing separately.
Ultrasound Obstet Gynecol 2013; 42: 15–33.
19
NIPT for aneuploidy
Methylated DNA-based approaches
This approach takes advantage of the fact that there
will be epigenetic differences throughout the genome
which will result in the differential expression of genes,
depending on the tissue type50 . These differences can be
associated with either hyper- or hypomethylation of the
genes. Some genes present in placental cells (specifically,
trophoblasts) will therefore differ from maternal cells
in the methylation pattern. Methylated DNA can be
chemically modified, thus allowing its characterization
separate from the unmethylated DNA51 . Alternatively,
the hypermethylated DNA can be analyzed separately
following restriction enzyme digest of the hypomethylated
DNA52 . If, additionally, there are polymorphic differences
in the fetal alleles, disturbance from a 1:1 ratio can be
used to identify trisomy. This has been demonstrated for
detection of Edwards syndrome but the approach has not
been tested in clinical trials51 . Differentially methylated
regions may be relatively common53 so the method may
also be applied to detect other aneuploidies.
It is also possible to use immunoprecipitation with
antibodies specific to 5-methylcytidine to enrich for the
methylated DNA. For example, the identification of a set
of specific DNA sequences on chromosome 21 that are
hypermethylated in placental cells allows the selective
enrichment of fetal cfDNA from maternal plasma35 .
Following quantitative real-time PCR, there may be
measurable differences in the amount of chromosome 21derived DNA when fetal trisomy 21 is present, compared
with in euploid fetuses. One proof-of-principle study has
demonstrated the efficacy of such an approach to NIPT54 .
A follow-up study using a modified set of differentially
methylated chromosome 21 DNA sequences also showed
encouraging results55 . This approach is currently the
subject of further clinical trials. If an approach such
as this can be fully developed, it could be substantially
cheaper than sequencing-based methods.
Digital PCR
Another way in which the costs of sequencing can be
avoided is through the use of digital PCR. This technology
is based on the dilution of test materials such that
individual DNA fragments of interest (e.g. chromosome
21) can be amplified separately and counted56 – 58 .
Theoretical estimates of the number of amplification
events have been computed59 and this approach could
become particularly attractive if reliable fetal cfDNA
enrichment methods were developed.
RNA-based testing
Identification of genes that are expressed by trophoblasts
but not maternal cells should result in the presence of
the corresponding cfRNA species in maternal plasma,
free of contaminating maternal RNA with a similar
sequence. If the cfRNA also carries polymorphisms that
distinguish the fetal alleles, the presence of aneuploidy
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
could be deduced from departures from a normal 1:1
ratio in the relative amount of the inherited alleles60 . This
approach to NIPT showed some success in the initial
proof-of-principle studies, but unpublished clinical trial
data were disappointing. Nevertheless, with the discovery
of additional RNAs that can be analyzed, this approach
may yet play an important role in NIPT61 .
NIPT IN CLINICAL TRIALS:
NON-MOSAIC DOWN, EDWARDS AND
PATAU SYNDROMES
Proof-of-principle studies demonstrated the feasibility of
NIPT using small series of maternal plasma samples
from aneuploid and euploid pregnancies36 – 38,40 – 46,62 – 64 .
However, generally, the samples were not tested ‘blind’
to the outcome of pregnancy, ascertainment criteria were
not fully described and some samples were obtained after
an invasive diagnostic procedure which could have led
to increased transfer of fetal DNA into the maternal
circulation. There have now also been several large clinical
trials of MPS which have overcome these limitations and
potential biases39,65 – 75 . These have established NIPT as a
potentially highly effective screening test but not as a test
that could replace current invasive prenatal diagnosis. We
therefore prefer the term NIPT or cfDNA screening rather
than non-invasive prenatal diagnosis, which is misleading.
High-risk studies
There have been seven cfDNA studies involving
shotgun or targeted sequencing in women who had
invasive prenatal diagnosis because of high risk for:
Down syndrome65,67 , Down syndrome or another
aneuploidy66,68,69 or any disorder70,71 . In one study, a
screen-positive screening test was the only indication
for high risk69 , whilst in the four other studies in which
the reason for invasive testing was high risk for Down
syndrome or another aneuploidy, the indications were
mixed. The reason for invasive testing was a screenpositive screening test in 77%65 , 43%67 , 30%66 and
22%68 of cases; advanced maternal age in 68%66 , 37%
(only indication in these cases)68 and 33%67 of cases;
ultrasound in 13%66 , 11%67 and 7%68 of cases; family
history in 5%66 , 3%67 and 3%68 of cases; and multiple
reasons in 22%68 and 9%67 of cases. Five studies included
results on both Down and Edwards syndromes68 – 72 and
three also included Patau syndrome68,72,75 .
The study design details for these trials on high-risk
women are summarized in Table 1. One study designated
as ‘unclassified’ results with z-scores of 2.5–4.0 for
autosomal chromosomes and excluded them from the
DR and FPR calculations68 . Doing so is not valid76 ,
particularly when no protocol was suggested for the
unclassified group, and in this Review we have reclassified
them as negative. However, the unclassified group had a
five-fold increased likelihood of aneuploidy (3.5% (5/144)
of trisomies unclassified compared with 0.7% (9/1358) of
controls) and it could be argued that they should in fact
Ultrasound Obstet Gynecol 2013; 42: 15–33.
20
Benn et al.
Table 1 Eleven cell-free DNA studies of shotgun or targeted sequencing: methodological differences
Trial
High risk
Chiu et al.65
Ehrich et al.66
Palomaki et al.67,72
Bianchi et al.68
Sparks et al.70
Ashoor et al.69,75
Norton et al.71
Other than high risk
Lau et al.73
Nicolaides et al.74
Dan et al.39
Gil et al.78
Design
Average GA (weeks)
Method
Algorithm
Prospective and case–control
Case–control
Case–control
Case–control
Case–control
Case–control
Prospective
13
16
15
15
18
13
16
Shotgun
Shotgun
Shotgun
Shotgun
Targeted
Targeted
Targeted
z-score
z-score
z-score
z-score
Risk†
Risk†
Risk†
Prospective
Retrospective
Prospective
Prospective
14
12
20
10
Shotgun
Targeted
Shotgun
Targeted
z-score?
Risk†
z-score & L-score
Risk†
Cut-off*
3.0
2.5
3.0
4.0‡
1%
1%
1%
?
1%
2.5 or 1.0§
1%
*For autosomal chromosomes. †Includes maternal and gestational age. ‡Results with scores 2.5–4.0 were ‘unclassified’. §Positive if z < 2.5
but L > 1. GA, gestational age at testing.
be reclassified as positive, so those rates are also given in
this Review.
Table 2 summarizes the Down and Edwards syndromes
results for these seven cfDNA studies carried out in highrisk pregnancies. In total these studies compared 594
samples from pregnancies with Down syndrome with
5745 that did not, and 193 samples from pregnancies
with Edwards syndrome with 5459 that did not, although
in both cases some had other aneuploidies. The results
from the various studies, notwithstanding methodological
differences, are consistent with each other and their
combined data represent the best estimates of DR
and FPR. For Down syndrome, the overall DR of
99.3% has a 95% CI of 98.2–99.8% and FPR of
0.16% (95% CI, 0.08–0.31%). Even with the most
extreme CI values this performance exceeds, by far,
anything achievable by current Down syndrome screening
protocols. However, this performance falls short of that
for current diagnostic tests. Furthermore, reclassifying as
positive the unclassified results in the study of Bianchi
et al.68 would have increased the overall DR slightly to
99.5%, but almost doubled the FPR to 0.26%. Hence,
we can conclude that based on present evidence cfDNA
testing is not a diagnostic test of Down syndrome but
rather is a very good secondary screening test. A woman
with a positive cfDNA test will be at high risk of
Down syndrome but depending on her pretest risk it
may not be extremely high. This can be illustrated from
the combined data in Table 3, by estimating a positive
likelihood ratio (LR) using the DR divided by the FPR,
i.e. 634. For example, a woman with a pre-test risk of
1 in 270 following a combined test would have a posttest odds of 634:269 or a risk of 2 in 3. This is not
sufficiently high as to warrant termination of pregnancy
without a confirmatory invasive diagnostic test. Similarly,
the negative LR, estimated from the false-negative rate
divided by the true negative rate, is 1/148. A woman
with a pre-NIPT risk of 1 in 10 would have a 1 in 1333
risk after a negative test, which might be insufficiently
reassuring for some women. These LRs are illustrative
and cannot be used to counsel individual women because
some of the studies had already incorporated prior risk
based on maternal age into their algorithms. However,
it is reasonable to conclude that using NIPT following a
screen-positive test for any of the established screening
protocols followed by confirmatory invasive diagnostic
testing if the NIPT result is positive will probably hardly
affect the overall screening DR but will reduce the FPR
about 300-fold.
For Edwards syndrome, the overall DR is 97.4%
(95% CI, 93.7–99.0%) and the FPR 0.15% (95% CI,
0.07–0.31%), with positive LR of 665 and negative
Table 2 Down syndrome and Edwards syndrome results in seven cell-free DNA studies carried out in high-risk pregnancies
Down syndrome
Trial
Edwards syndrome
DR (n (%))
FPR (n (%))
DR (n (%))
FPR (n (%))
Chiu et al.65 *
Ehrich et al.66
Palomaki et al.67,72
Bianchi et al.68
Sparks et al.70
Ashoor et al.69
Norton et al.71
86/86 (100)
39/39 (100)
209/212 (98.6)
89/90 (98.9)
36/36 (100)
50/50 (100)
81/81 (100)
3/146 (2.1)
1/410 (0.24)
3/1471 (0.20)
0/410 (0.00)
1/123 (0.81)
0/297 (0.00)
1/2888 (0.03)
—
—
59/59 (100)
35/38 (92.1)
8/8 (100)
49/50 (98.0)
37/38 (97.4)
—
—
5/1688 (0.30)
0/463 (0.00)
1/123 (0.81)
0/297 (0.00)
2/2888 (0.06)
Total
590/594 (99.3)
9/5745 (0.16)
188/193 (97.4)
8/5459 (0.15)
*2-plex and 8-plex data were reported but only the more favorable 2-plex results are included here. DR, detection rate; FPR, false-positive
rate.
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
Ultrasound Obstet Gynecol 2013; 42: 15–33.
21
NIPT for aneuploidy
Table 3 Failure rates in 11 studies of cell-free DNA testing*
Trial
Failure rate (n (%))
Chiu et al.65
11/764 (1.4)
Ehrich et al.66
18/467 (3.8)
Palomaki et al.67
13/1696 (0.8)
Bianchi et al.68
Sparks et al.70
16/532 (3.0)
8/338 (2.4)
Ashoor et al.69
Norton et al.71
3/400 (0.8)
148/3228 (4.6)
Lau et al.73
Nicolaides et al.74
Dan et al.39
0/567 (0.0)
100/2049 (4.9)
79/11 184 (0.7)
Gil et al.78
40/997 (4.0)
Reasons for failure
Low total DNA (n = 2), low DNA library concentration (n = 8), low matched DNA sequence
reads (n = 1)
Low % fetal DNA (< 4%) (n = 7), low total DNA (< 556 copies) (n = 7), low DNA library
concentration (< 32.3 nM) (n = 15), low number unique DNA sequence counts (< 3
million) (n =11); some failed more than one criteria
Low % fetal DNA (< 4%) (n = 6), other QC parameters (n = 7): low DNA library
concentration (< 25 nM) and low matched DNA sequence reads (< 12.5 million)
No fetal DNA
Low % fetal DNA (< 3%), low DNA sequence counts, evidence from SNPs of non-singleton
pregnancy
Amplification and sequencing
Low % fetal DNA (< 4%) (n = 57), assay failure (n = 91): inability to measure % fetal, high
variation in DNA counts and failed sequencing
—
Low % fetal DNA (< 4%) (n = 46), assay failure (n = 54)
Quality of separation, extraction, sample preparation and sequencing: low peak DNA library
size, low library concentration (< 30 nM) and low matched DNA sequence reads (< 2
million)
Low % fetal DNA (< 4%) (n = 23), assay failure (n = 17)
*Excluding tests rejected because of inadequate sample quality.
LR 1/38. Including the unclassified results in the study
of Bianchi et al.68 as positive would have increased
the DR to 98.4% and the FPR to 0.20%. Since a
cfDNA test is unlikely to be aimed at detecting Edwards
syndrome alone, these false-positives should be regarded
as additional to those generated by cfDNA testing for
Down syndrome. There are no data with which to assess
whether there is any tendency for those testing falsely
positive for one type of aneuploidy to be falsely positive
for another. Assuming these are uncorrelated, the FPR for
both aneuploidies together is 0.30%, or 0.46% if those
unclassified by Bianchi et al.68 are included.
Combining the results from the three studies which
included Patau syndrome68,72,75 gives a DR of 30/38
(78.9%; 95% CI, 65.9–91.9%) and FPR of 17/4112
(0.41%; 95% CI, 0.22–0.61%), with a positive LR of
191 and a negative LR of 1/4.7. Including the unclassified
results as positive, the DR increased to 80.0% with no
increase in the FPR. On the basis of these results, an MPS
test for all three trisomies would have a FPR of 0.7–0.9%,
depending on the inclusion of the unclassified results.
Initial prospective clinical experience has been reported
for a commercial laboratory, Verinata Inc, offering
tests to high-risk women77 . While follow-up information
was incomplete, among 5974 samples there were false
positives for Down, Edwards and Patau syndromes and
false negatives for Down and Edwards syndromes, with
rates compatible with those seen in the trial data.
Non-high risk
There have been four NIPT studies involving shotgun
or targeted sequencing in which the subjects were not
specifically selected because they had invasive prenatal
diagnosis. Their methods are summarized in Table 1.
Lau et al.73 reported prospective cfDNA screening
results of 567 patients, 49% of whom were tested
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
at 12–13 weeks’ gestation. These women comprised
179 (32%) who were screen-positive by conventional
testing, 194 (34%) who were screen-negative or awaiting
results and 194 (34%) who were unscreened. There was
insufficient follow-up to report DRs reliably, but eight
cases of Down syndrome and one of Edwards syndrome
were detected and there were no false positives. Seven of
the detected cases were among the screen-positive women
(1 in 25) and two, both Down syndrome, were among
the remainder (1 in 194).
Nicolaides et al.74 carried out a retrospective study
using stored plasma samples from women who had a
combined test at 11–13 weeks’ gestation. After excluding
non-viable pregnancies, those terminated for reasons
other than Down or Edwards syndromes and cases lost
to follow-up, 1949 were successfully tested for cfDNA,
including eight with Down syndrome and two with
Edwards syndrome. All aneuploidies were detected and,
while there were no false-positive Down syndrome cases
among 1939 tested, there were two (0.05%) false-positive
Edwards syndrome cases among 1937 tested. Invasive prenatal diagnosis had been carried out in 4.3% (86/2049) of
those selected, including all of the cases with aneuploidy.
A large prospective study of cfDNA screening was
carried out in China39 . A total of 11 105 pregnancies
were tested successfully, including 4522 (41%) that
were screen-positive following conventional screening and
2770 (25%) with risk factors such as advanced maternal
age, ultrasound abnormalities and family history. All
of the known Down syndrome (n = 140) and Edwards
syndrome (n = 42) cases were detected by the cfDNA
test. However, approximately one third of the 10 915
with negative test results were lost to follow-up; 2818
(26%) had invasive testing, a further 70 (< 1%) were
terminated, and follow-up was available for 4524 (41%).
The protocol included an insurance scheme whereby the
family would be compensated in the event of unpredicted
Ultrasound Obstet Gynecol 2013; 42: 15–33.
22
delivery of either a Down or an Edwards syndrome
baby. This is likely to be a considerable incentive to
report false-negative results so the lack of follow-up may
not be a major problem. There were two proven falsepositive cfDNA results, one each for Down and Edwards
syndromes. However, a further eight positive cases did
not have a karyotype following fetal loss or termination
and therefore the possibility of additional false positives
cannot be excluded.
Gil et al.78 carried out a prospective study of cfDNA
screening at 10 weeks’ gestation in 997 women with
median age 37 years; combined tests were also carried
out. A total of 15 cases of aneuploidy were detected by
cfDNA and confirmed by invasive testing (10 cases of
Down syndrome, four of Edwards syndrome and one
of Patau syndrome) and there was one false positive
(for trisomy 18). One case was positive for trisomy 21
but miscarried before invasive confirmation and no false
negatives had emerged at the time of writing.
These studies provide some, albeit limited, evidence
to suggest that cfDNA screening may be as effective
in the general population as it is in those already
scheduled for invasive testing: the FPR is not dissimilar
to that in the high-risk studies; all aneuploidies appear
to have been detected, although most of them were in
pregnancies which were already candidates for invasive
testing. Moreover, among high-risk women, performance
does not appear to be related to the indication for invasive
testing. Palomaki et al.67 found that the DRs and FPRs
were very similar according to indication: screen-positive
99% (96/97) and 0.2% (1/637), respectively; advanced
maternal age or family history 100% (24/24) and 0.3%
(2/587); ultrasound 100% (51/51) and 0% (0/130);
and multiple reasons 95% (37/39) and 0% (0/112).
Nevertheless, until much larger general population studies
are published, the limited direct evidence of efficacy needs
to be supplemented by indirect evidence. The separation
in the distributions of z-scores or risks between aneuploid
and euploid pregnancies is necessarily dependent on the
fetal fraction and it is therefore important to consider
whether this might be lower in low-risk women.
Several covariables for fetal fraction have been
investigated, including indication for invasive prenatal
diagnosis, maternal age, screening marker level and
estimated aneuploidy risk. In one published series of
pregnancies at high risk of Down syndrome or other
aneuploidy67 , there was no significant difference in mean
z-score, in either Down syndrome or euploid pregnancies,
according to indication or correlated with maternal age,
but there was a significant negative correlation with
maternal weight. The latter effect can be explained by
there being a fixed mass of fetal DNA, from the placenta,
mixing with an increasing mass of maternal DNA; such
an effect is also seen in conventional maternal serum
markers. In another published series of pregnancies at
high risk of Down syndrome69 , a multivariate regression
analysis was carried out which confirmed that maternal
weight was a covariable but maternal age was not79 . In
addition it showed that there was a significant positive
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
Benn et al.
correlation between fetal fraction and both PAPP-A and
free β-hCG, which might be related to placental volume,
although there was no association with gestational age in
either study. Since in Down syndrome pregnancies PAPPA is reduced on average and free β-hCG is increased, the
correlations with fetal fraction may cancel each other out.
In Edwards syndrome pregnancies, both serum analyte
markers are reduced, consistent with reduced placental
volume80 . There could therefore be a somewhat increased
chance for cfDNA test failure in affected pregnancies.
On the other hand, those cases with higher PAPP-A and
β-hCG which might not be detected by the combined test
may show higher fetal fractions and these may therefore be
more amenable to detection by a cfDNA-based NIPT. In a
further published series of those having invasive prenatal
diagnosis due to high risk generally71 , the mean fetal
fraction was computed for deciles of maternal age, NT
and aneuploidy risk81 . There was no significant difference
in means between the highest and lowest deciles. The
highest decile for risk comprised 100 women with risk
greater than 1 in 10 and a mean fetal fraction of 11.4%
compared with 135 women in the lowest decile with risk
less than 1 in 6500 and a mean fetal fraction of 10.8%.
Failed results
Several of the MPS studies discussed above specified the
number of pregnancies not considered for cfDNA testing
because of sample quality. Overall, among 13 260 eligible
pregnancies there were 814 (6.1%) untested. This rate
ranged from 0.8% to 9.9% between the studies and may
reflect the stringent standards required when carrying
out a research project. Of more concern is the 2.0% of
pregnancies (436/22 222) in which a cfDNA test was
performed but failed because of insufficient fetal DNA
or other technical reasons (Table 3). The failure rate
may be even higher in those resampled after an initial
failure: it was 32% (13/40) in one study78 . Quality
control criteria varied between the studies but in those
that included a minimum acceptable percentage of fetal
DNA, about half the failures were due to this not being
met. In the prospective laboratory experience reported
by Futch et al.77 , a result was not possible in 2.4% of
cases. This is relevant to comparisons with conventional
screening, which rarely reports failure to obtain a result.
Three prospective studies of women not specifically
selected because they had invasive prenatal diagnosis
provide information on the extent to which a successful
test result was achieved following an initial failure. In one
study73 , among 567 successful cfDNA tests, four (0.7%)
had required a repeat blood sample and no other tests had
failed during that period (T.K. Lau, pers. comm.). The
time from the initial blood draw to the cfDNA report was
greater than 14 days for 16 (2.8%) tests, including those
requiring a repeat sample. In another study39 , among
11 105 tests, 97 (0.9%) needed resampling. In this study
as a whole, the turnaround time was within 10 working
days for 95% of pregnancies and within 15 for 99%;
those requiring resampling took a further 10–15 days to
Ultrasound Obstet Gynecol 2013; 42: 15–33.
23
NIPT for aneuploidy
produce a report. In the third study82 , a request for a
patient redraw was made in 1.9% of referrals and among
those who did undergo a redraw there was sufficient fetal
cfDNA in the second sample in 56% of cases. The success
rate was strongly dependent on maternal weight.
the fetoplacental remains of the non-viable fetus could
interfere with the cfDNA result; there is a relatively high
chance that the fetal loss would have been associated
with aneuploidy and this could lead to a false-positive
result77 .
SNP studies
COST OF NIPT
Following the initial proof-of-concept study using polymorphisms and maximum LRs for test interpretation48 ,
the results of one small clinical trial have been published83
and additional information has been presented at scientific meetings in the form of an abstract84 and a poster85 .
The study of Nicolaides et al.83 involved 242 singleton
pregnancies with results available for 229 (95%). All 32
aneuploid and 210 euploid cases were identified correctly.
The abstract84 provided information on 407 pregnancies including 44 with aneuploidies and the results were
100% correct among samples that passed quality control.
In addition to the maternal plasma, paternal blood or
buccal mucosa samples were genotyped. Whilst this was
not an absolute requirement (paternally inherited alleles
in the fetus can be deduced), the failure rate was lower
when a paternal sample was available. In the poster85 ,
there were 763 samples, of which 45 (5.9%) failed to pass
quality control, and among the remaining 717 (including
47 Down, 15 Edwards, 7 Patau and 12 Turner syndromes)
samples all except one result, for Turner syndrome, was
correct.
Currently, the cost of a cfDNA test is high, ranging from
about $800 to $2000 in the USA, and from $500 to
$1500 elsewhere. Whilst this may not necessarily deter
individuals from being tested, it is likely to determine
whether public health planners provide the testing and
will be the basis for whether health insurance schemes
reimburse those tested. We have published a sensitivity
analysis90 in which the factors contributing to these
overall costs are elucidated for four cfDNA screening
policies: (1) cfDNA screening for those screen-positive on
combined test; (2) universal cfDNA screening, replacing
all current screening modalities; (3) contingent cfDNA
screening, for the 10–20% of women with the highest
risk based on a combined test; (4) cfDNA screening for
women of advanced maternal age and younger women
with a positive combined test90 .
Restricting cfDNA testing to screen-positive cases leads
to a small reduction in detection, a massive reduction in
false positives and is the least expensive way in which
to utilize cfDNA testing. With this policy the average
cost per Down syndrome birth avoided is dependent only
on the difference in unit cost of a cfDNA test and of
invasive prenatal diagnosis. For a combined test with
FPR of 3%, the average cost would be reduced by using
secondary cfDNA testing, if the unit cost was less than or
equal to three-quarters that of invasive testing. Even at a
higher unit cost of cfDNA, the average cost per fetal loss
prevented would be relatively low.
For other policies in which the aim of cfDNA testing
is to increase detection, the most important public health
consideration will be not the total or average cost but
the ‘marginal’ cost compared with the existing screening
provision. The marginal cost is the average cost per Down
syndrome birth avoided or loss prevented over and beyond
that which would have been avoided by the existing
service. This is shown in Table 4 for fixed unit costs for
the combined test and invasive testing and with variable
unit costs of cfDNA. The table also shows screening
performance.
Universal cfDNA screening yields both a very high DR
and a small FPR but the marginal cost is very high,
exceeding the lifetime cost to the system of a Down
syndrome birth91 – 94 . With universal screening the cost
per fetal loss prevented when replacing conventional
screening is also very high. Contingent protocols, in which
all women receive the combined test and the 10–20% with
the highest risk receive cfDNA testing, are efficient and
can reduce massively the marginal cost compared with
universal screening. The average cost of preventing a fetal
loss is also several times lower than that for universal
cfDNA testing. A policy of cfDNA testing only for older
Multiple pregnancies
There is a potential problem with using cfDNA to
screen for aneuploidy in multiple pregnancies. As with
biochemical screening, in twins discordant for trisomy the
excess in fragments from a specific chromosome in the
affected twin may be masked by the normal proportion
of those fragments in the euploid cotwin. This would also
be the case for higher order multiple pregnancies. This
problem might be overcome by using SNPs to measure
the variations in the fetal fraction between genomic
regions86,87 . The method allows determination of zygosity
and, in dizygous twins, examination of the distribution of
fetal fragments for each fetus.
At present, only two small multiple-pregnancy cfDNA
series have been published, using the same method as
for singletons88,89 . The first series was derived from one
of the high-risk studies67 and included three discordant
twins (two with Down and one Patau syndrome), five
concordant Down syndrome twins, 17 euploid twins and
two euploid triplets88 ; there were no false-positive or falsenegative cfDNA test results. The second series was from
one of the studies of women not selected because they
were at high risk73 and included only one discordant
twin with Down syndrome and 11 euploid twins89 ;
all pregnancies were correctly classified by the cfDNA
test.
In a singleton pregnancy that had had a vanishing twin,
it is theoretically possible that apoptosis of cells from
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
Ultrasound Obstet Gynecol 2013; 42: 15–33.
24
Benn et al.
Table 4 Four cell-free (cf)DNA policies*: performance, marginal cost per Down syndrome (DS) birth avoided and average cost per fetal loss
prevented, according to unit cost of cfDNA testing†
Policy
Combined
cfDNA
None
All
All
Contingent:
10%
15%
20%
≥ 35 years
≥ 35 years or positive
on combined test
< 35 years
< 35 years
Marginal cost per DS birth
avoided ($ million) for
unit cost cfDNA of:
Performance
Average cost per fetal loss
prevented ($ million) for
unit cost cfDNA of:
DR (%)
FPR (%)
OAPR
$1500
$1000
$500
$1500
$1000
$500
99.3
0.16
1:1
5.84
3.63
1.42
9.47
5.89
2.31
90.4
92.8
94.4
85.0
84.7
0.016
0.024
0.032
2.1
0.017
7:1
5:1
4:1
1:19
6:1
1.11
1.39
1.67
2.90
3.69
0.65
0.86
1.05
1.73
2.02
0.19
0.32
0.44
0.57
0.37
0.82
1.33
1.85
2.35
0.81
0.48
0.82
1.16
1.41
0.44
0.14
0.30
0.48
0.46
0.08
*Three reported in a modeling analysis90 and the fourth, cfDNA restricted to older women, using the same model. †Combined test,
false-positive rate (FPR) = 3%, unit cost = $150; invasive prenatal diagnosis, unit cost = $1000, iatrogenic fetal loss rate = 0.5%. DR,
detection rate; OAPR, odds of being affected given a positive result.
women was not substantially more effective than was
the combined test and is more costly than contingent
screening. A hybrid policy of offering cfDNA testing to
all those of advanced maternal age plus all combined test
screen-positive women was better as it reduced the FPR
massively but it was less effective and more expensive
than was a contingent testing policy.
The detection rate for a contingent policy can be
increased by including additional serum and ultrasound
markers. Nicolaides et al.95 suggest markers which are
also of value in screening for pre-eclampsia and fetal
cardiac abnormalities. Cost-benefit analyses for such
variants have not yet been carried out.
Two other published studies have estimated costs when
cfDNA is applied to women with positive conventional
screening results, using the estimated NIPT DR and FPR
from their own single study. Palomaki et al.67 considered
total costs following screening in 100 000 screen-positive
women, with an average risk of 1 in 33 for a Down
syndrome fetus. They concluded that if the unit cost of
cfDNA was less than 96% of the unit cost of invasive
testing, there could be a net saving (4% of costs need
to be assigned to test failures and confirmatory invasive
testing following cfDNA positive results). The second
study also considered 100 000 screen-positive women,
modeled as a mixture of combined test or second-trimester
serum screening96 . They estimated total costs including
both the conventional screen and subsequent testing and
concluded that with a unit cost of cfDNA $1200, CVS
$1700 and amniocentesis $1400, there would be a 1%
cost reduction. One other study has estimated costs
for cfDNA screening in older women and in younger
women with screen-positive conventional screening tests,
using DR and FPR estimates from the literature97 . The
authors claimed that this policy, in the USA, would have
lower total cost compared with conventional screening.
However, this conclusion appears to have been reached
following incorrect assumptions about the DR and FPR in
younger women and a very low uptake of invasive testing
for women screen-positive by conventional screening
compared with cfDNA screening90 .
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
FUTURE PROSPECTS: NIPT FOR
ADDITIONAL CYTOGENETIC
ABNORMALITIES
Other autosomal abnormalities
With the widely anticipated rapid improvements in
sequencing, it may soon be possible to screen for
other autosomal trisomies in situations in which the
fetal fraction is very low, thereby allowing detection of
aneuploidy even earlier in pregnancy. Early pregnancy
is associated with very high levels of chromosomal
abnormality and although nearly all abnormal cases
will spontaneously abort, early identification could be
beneficial to women. First-trimester autosomal trisomy
can involve any chromosome, but some occur much more
frequently than others22 . Detection of these through NIPT
will be facilitated by the larger size of the additional
chromosome in the non-viable trisomies. Early studies
suggested that there was greater variability in the fragment
counts for some of the larger chromosomes36 , but this
could be overcome by GC correction and selective use of
the most consistent regions of chromosomes37,62 .
Screening for a large number of potential abnormalities
together does require very high specificity for each
component so that the aggregate FPR for all chromosomes
combined is low.
Sex-chromosome abnormality
Establishing NIPT for sex-chromosome imbalances is
desirable because some specific sex-chromosomal abnormalities are associated with a sufficiently severe phenotype
as to fully justify prenatal diagnosis: for example, many
cases of Turner syndrome, most of which will not survive to full term, gain of multiple X chromosomes and
some unbalanced structural rearrangements of the Xchromosome. Collectively, unbalanced sex-chromosomal
abnormalities constitute over a third of the unbalanced
karyotypes seen in amniocenteses performed in women
under 35 years of age and nearly a quarter of those in
older women98 . There is mosaicism in approximately one
Ultrasound Obstet Gynecol 2013; 42: 15–33.
25
NIPT for aneuploidy
third of sex chromosomal aneuploidies detected through
amniocentesis98 .
One commercial laboratory that provides NIPT,
Sequenom Inc, was routinely providing information on
the presence or absence of Y-chromosome DNA in their
reporting. Based on a series of 1639 samples, the DR
for a Y-chromosome was 828/833 (99.4%), and the
accuracy for the prediction of a female fetus was 803/806
(99.6%)99 . More recently, they have been providing
NIPT for Turner syndrome, Klinefelter syndrome,
47,XXX and 47,XYY based on a study by Mazloom
et al.100 , with DRs of 83% (25/30), 85% (11/13), 83%
(5/6) and 75% (3/4), respectively, for each of the these
abnormalities. The FPR for Turner syndrome was 0.2%
(4/1945) and there was one other false positive (47,XXX)
in the series100 . Of the normal cases in which gender was
interpreted, it was inconsistent with the karyotype result
in 0.7% (13/1814)100 . Bianchi et al.68 developed a complex algorithm for evaluating sex-chromosome-derived
cfDNA in plasma. Their study derived z-scores for the
X-chromosome (zX ) and Y-chromosome (zY ). A result was
classified as positive for Turner syndrome if zX < −4.0 and
zY < 2.5; it was classified as 46,XX or 46,XY or 47,XXX
or 47,XXY or 47,XYY using different cut-offs, and all
others were unclassified. Among the non-mosaic Turner
syndrome cases the DR was 75% (15/20) and the FPR was
0.2% (1/462), with 49 remaining unclassified (including
four affected and 45 controls). Among the mosaic cases
with a 45,X cell line, the DR for the monosomy was
29% (2/7). For the other non-mosaic sex chromosomal
abnormalities the DRs were: XXX, 75% (3/4); XYY,
100% (3/3); and Klinefelter syndrome, 67% (2/3). Robust
estimates for the efficacy of sex chromosomal aneuploidy
detection are therefore not provided, although Verinata
Inc. does offer testing on the basis of this study.
There are several factors that could confound the
development of highly effective NIPT for X- and Ychromosomal imbalances. First, many fetal sex chromosomal aneuploidies are mosaic, with either a normal cell
line or an additional abnormal cell line. This includes
situations in which one abnormal cell line may mask
another when cfDNA is analyzed. For example, fetal
45,X/46,XXX mosaicism may show approximately normal proportions of X-chromosome cfDNA if the proportions of the two cell types are similar. Second, although
the detection of Y-chromosome-specific DNA is relatively
simple and reliable in establishing gender101 , finding sufficient Y-chromosome loci that are informative for copy
number quantification may be difficult. The potentially
informative euchromatic part of the Y-chromosome is
relatively small, it contains many sequences that show
polymorphic CNVs, and some segments show partial
homology with X-chromosomal sequences102 . A third
difficulty is that normal adult females show an age-related
loss of X-chromosomes103 . In women at normal reproductive age, the proportion of maternal cells with a missing
X-chromosome can be comparable to the proportion of
cfDNA in a maternal plasma specimen and there may be
some women with unusually high levels of 45,X cells104 .
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
The age-related loss of the X-chromosome is thought
to involve preferentially the inactive X-chromosome105 .
Some women may also show cells that have gained an
X-chromosome106 . Clonal populations of maternal cells
with X-chromosome gain or loss in hematological cells
(which are believed to be the source maternal cfDNA in
plasma), maternal constitutional mosaicism or even a nonmosaic X-chromosomal abnormality such as 47,XXX are
also possible107 . The fetal X-chromosomal dosage therefore has to be identified against a background of maternal
X-chromosomal DNA that is likely to be quantitatively
more variable than is seen for the autosomes. These considerations suggest that NIPT based on simply counting
the total X- and Y-chromosomal sequences in maternal
plasma and comparing them with the expected normal
karyotype values may be less effective than that which is
achievable for autosomal gain or loss. Furthermore, it may
be misleading to quote a DR that is based on all sex chromosomal abnormalities combined for genetic counseling.
Approaches based on quantification of SNPs present
in the plasma DNA may be informative for at least some
fetal sex chromosomal aneuploidies. Y-chromosome
gain, most cases of Turner syndrome108 , approximately
half of cases with Klinefelter syndrome109 and some other
imbalances are attributable to erroneous segregation of
a paternally derived sex chromosome. These cases could
therefore be identified in a maternal plasma specimen
through the over- or under-representation of paternal Xand Y-chromosomal SNPs that differ from the mother’s
SNPs. On the other hand, most cases of 47,XXX110 ,
and the remaining cases of Turner syndrome, Klinefelter
syndrome and others are attributed to erroneous segregation of a maternally derived chromosome. The reliable
quantification of cfDNA with these fetal X-chromosomal
SNPs, against a background of the identical maternal
SNPs, is more difficult.
Mosaicism
Two of the NIPT clinical trials have specifically addressed
the ability to detect mosaic cytogenetic abnormalities in
the fetus68,111 . In one study68 , of 11 cases in which a noncomplex mosaicism was known to be present based on
karyotyping, five were called affected and six unaffected.
In additional studies involving deeper sequencing,
four mosaic abnormalities recognized by conventional
chromosomal analysis were not detected by NIPT112 . In
the second study involving five cases111 , the abnormal line
was detected in one case and not detected in four cases.
Approximately 14% of all cytogenetic abnormalities
identified through karyotyping of amniotic fluid cells are
mosaic98 . For CVS, the proportion showing mosaicism
is much higher; approximately 45%23 . This excludes
cases in which there is pseudomosaicism (possible cell
culture artifact) and includes cases in which there is a
discrepancy between CVS direct or short-term culture
preparation results (cytotrophoblasts) and longer term cell
culture results (mesenchymal tissue). Discrepancy usually
involves abnormal cells in cytotrophoblasts with normal
Ultrasound Obstet Gynecol 2013; 42: 15–33.
26
mesenchyme, but the reverse situation can also be present.
An abnormal cell line can also be present in both direct and
long-term cultures but be undetectable in the fetus. Longer
term cell culture is usually considered to be a more reliable
indicator of the true karyotype of the fetus compared
with direct preparations. Overall, approximately 1% of
all CVS specimens will show a second cell line that is
not detectable in the fetus, a situation termed ‘confined
placental mosaicism’ (CPM)113 . This may sometimes
result in spontaneous loss, low birth weight or preterm
delivery, particularly when it involves both trophoblastic
and mesenchymal cell lineages114 and/or when it involves
specific karyotype abnormalities such as trisomy 16115 .
However, in most pregnancies in which CPM is detected
the pregnancy outcome is normal116 .
The frequency and significance of CPM is relevant to
NIPT because cfDNA is believed to originate primarily
from non-viable trophoblasts. CPM could result in either
false-positive or false-negative NIPT results. It has been
suggested that viable pregnancies with Edwards or Patau
syndrome may have a substantial euploid cell line in
the trophoblasts117,118 and therefore NIPT could fail to
detect some viable trisomic cases in which there is a
substantial population of abnormal cells in the fetus but
not in the placenta. This would lead to an apparent falsenegative result by NIPT. Conversely, the trophoblasts
may be mostly abnormal and the fetal cells normal,
leading to an apparent false-positive result. Moreover, if
chromosomally abnormal trophoblasts were more likely
to undergo apoptosis, NIPT could preferentially identify
cases in which the proportion of abnormal cells in the
placenta is low.
A number of cases have already been described which
illustrate how differences in the distribution of normal
and abnormal cells may lead to test result discrepancies
between NIPT and karyotyping. Faas et al.43 described the
identification of Turner syndrome based on NIPT when
the abnormal cell line was confirmed by cytogenetic analysis of short-term CVS cultures but the abnormality was
not seen in the long-term CVS cultures or in the newborn
blood. Choi et al.119 described a case involving possible
trisomy 22 detected by cfDNA testing. The trisomy 22
cell line was confirmed by analyzing the term placenta
but the abnormality was not detected in a blood specimen
from the dysmorphic baby. Hall et al.120 describe a
case with a positive Patau syndrome cfDNA test, mosaic
47,XY,+13/46,XY result for CVS, normal result for amniotic fluid cells, normal result for cord blood, but trisomy
13 mosaicism confirmed in the placenta. These examples
can each be interpreted as CPM although the presence of
low-level true fetal mosaicism cannot be excluded.
Early trials on chromosomal analysis following CVS
provide an indication as to how often CPM might lead to
an apparent false-positive or false-negative result. Because
milder abnormalities such as sex chromosomal aneuploidy
or mosaicism involving abnormalities not typically seen
in livebirths may not have been fully pursued in followup studies, this analysis needs to be confined to Down,
Edwards and Patau syndrome. Ledbetter et al.23 noted six
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
Benn et al.
cases (four involving chromosome 13, one chromosome
18 and one chromosome 21) interpreted as a false-positive
CPM result and one false-negative result (chromosome
18) among 6560 cases that had analysis of a direct CVS
preparation. Similarly, Smith et al.121 reported nine cases
(two involving chromosome 13, four chromosome 18 and
three chromosome 21) interpreted as a false-positive CPM
result and five false-negative results (two chromosome 18,
three chromosome 21) in approximately 18 195 cases
that had direct preparation analysis. Combining these
data, for chromosome 21, 18 or 13, the incidence of
false positives was 1 in 1650 and the incidence of false
negatives was 1 in 4126. These estimates are crude because
the number of cells analyzed on direct CVS preparation
is often small, some women receiving an abnormal result
terminated their pregnancy without confirmation, and
low-level true fetal mosaicism may have gone undetected.
It should be noted that most cases of CPM do not
involve chromosomes 21, 18 and 13. Therefore, as NIPT
tests are expanded to include additional chromosomes or
chromosomal regions, the issue of erroneous results due
to CPM is likely to become increasingly important.
For those cases that do show true fetal mosaicism, the
phenotype can be highly variable but will generally be less
severe than that present with the non-mosaic abnormal
karyotype22 . Although the proportion of abnormal cells
in amniotic fluid or chorionic villi can be a poor
guide to phenotype in individual cases, in general, a
high proportion of abnormal cells is associated with
adverse pregnancy outcome122 . NIPT tests that only
present a result categorically as either ‘positive’ or
‘negative’ obviously cannot provide any information
about the proportion of abnormal cells or clinical severity.
Furthermore, since some of the clinical trials have
specifically excluded mosaic cases and optimized their
z-score cut-off on the basis of non-mosaic cases, testing is
presumably biased towards not detecting mosaic cases. It
is not yet clear whether patients with z-scores close to the
cut-off should be offered invasive testing to help rule out
mosaic trisomy. To present this problem appropriately
to clinicians, and also to allow better integration of the
results with other screening and diagnostic techniques,
we recommend that NIPT reports should be formatted
so that they provide patient-specific risks for each nonmosaic trisomy, the z-score and the fetal fraction, rather
than just a categorical positive or negative finding.
The possibility of maternal mosaicism also needs to be
considered. This includes the rare situation in which the
abnormal maternal cfDNA is derived from a malignant
cell population123 . Maternal mosaicism could potentially
be recognized using SNP-based NIPT methodology.
Translocations
Unbalanced translocations, in theory, should be identifiable in NIPT as a result of the partial trisomy and
monosomy that is present. In a retrospective analysis of
six plasma specimens from pregnant women with known
fetal imbalances, Srinivasan et al.112 were able to identify
Ultrasound Obstet Gynecol 2013; 42: 15–33.
27
NIPT for aneuploidy
the imbalances in maternal plasma in all six cases. In two
of their cases the karyotype showed additional material of
unknown origin and the NIPT analysis was able to provide
a more precise characterization of the extra chromosomal
material. The ability to detect routinely smaller imbalances would require deeper sequencing and improvements
in bioinformatic analysis of sequencing data41,124 – 126 .
For SNP-based approaches to NIPT, detection of small
imbalances requires identification of sufficient informative
polymorphisms in the region of interest.
Conventional chromosomal analysis does allow detection of most apparently balanced translocations and large
inversions. Prenatally identified de-novo rearrangements
are associated with an increased risk for abnormality127 .
It would therefore be desirable to detect these noninvasively. Paired-end sequencing of DNA fragments that
span a chromosomal breakpoint can identify at least some
translocations through the analysis of cellular DNA. For
example, Schluth-Bolard et al.128 used a whole-genome
paired-end protocol to identify the specific sequence disruptions in four cases with apparently balanced translocations and abnormal phenotypes. Breakpoints were mostly
in repeat sequences and most breakpoints were associated
with gain or loss of some basepairs. Talkowski et al.129
used sequencing to refine breakpoints and establish a
diagnosis of CHARGE syndrome based on disruption of
the CHD7 gene by analyzing amniotic fluid cells. These
studies involved the sequencing of larger pieces of DNA
and were carried out in cases in which the karyotype
information was used to help identify candidate DNA
fragment sequences that spanned the breakpoints.
Although the routine detection of de-novo rearrangements using cfDNA, without knowledge of where to look,
would not seem to be technically achievable at this time,
it is potentially possible. The challenge is similar to the
detection of other mutations130 – 132 , but is made simpler with the presence of two related reciprocal events.
By sequencing cfDNA it should be possible to construct
the haplotype sequence in which one end matches perfectly to a chromosome, the other end matches perfectly
a second chromosomal location and there are similar
hybrid sequences present that correspond to the reciprocal translocation product(s). Improved sequencing fidelity,
deeper sequencing and the development of advanced biometric algorithms that specifically look for the hybrid
sequences would be needed.
Inherited rearrangements are generally considered
benign, but their detection through karyotyping following CVS or amniocentesis is considered advantageous
because it identifies a risk factor for future pregnancies.
When a balanced translocation is already known to be
segregating in a family and the phase of SNPs on the
chromosomes in the region of interest can be established,
the presence or absence of the breakpoint can be inferred
from the SNPs in the cfDNA in a plasma specimen. In the
case of a paternally inherited translocation, the presence
of the translocation chromosomal SNPs and absence of
the normal homolog SNPs identifies the translocation in
the conceptus. In maternal inheritance, detection of the
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
translocation relies on the identification of a relative excess
of the translocation chromosomal SNPs and relative deficiency of the normal homolog SNPs in the plasma sample.
Triploidy
Triploid pregnancies can arise as a result of an
additional set of maternal (digynic) or paternal (diandric)
chromosomes. Most recognized cases show a 69,XXX
or 69,XXY karyotype; 69,XYY forms are thought to
undergo early first-trimester miscarriage133 . Most cases
of triploidy will be identified by ultrasound abnormality
and characteristic first- or second trimester-maternal
serum marker levels134 – 136 . It is exceptional for these
pregnancies to survive into the third trimester.
Since all chromosomes are proportionately represented
in a 69,XXX pregnancy, NIPT based on the counting
of all chromosomal sequences would yield a normal
result. Pregnancies with either a 69,XXY or a 69,XYY
karyotype might be identifiable through unexpected sex
chromosomal sequence ratios. Digynic triploidy might
be particularly difficult to detect by NIPT because the
placenta is very small and the fetal fraction may therefore
be reduced137 . Consistent with this, Bianchi et al.68
reported that of nine cases of fetal 69,XXX triploidy, there
was insufficient fetal cfDNA for analysis in three (33%).
Using the approach that takes into consideration the
SNPs present in the cfDNA, diandric triploidy could be
identified through the presence of two different paternally
inherited SNPs at some loci. Digynic cases with sufficient
fetal cfDNA for analysis might be recognized through
the quantitative excess of maternally inherited fetal DNA
fragments relative to paternally inherited fragments.
CNVs
Small duplications and deletions have the potential to be
detected through NIPT. Examples that have been detected
include a 4-Mb deletion on chromosome 12138 , and two
cases in which 3-Mb 22q11.2 deletions were present139 .
In a series of 11 prenatal plasma specimens, Srinivasan
et al.112 identified small gains or losses not reported in
the clinical karyotypes in eight cases, including four with
two such variations. However, it is not clear whether they
were true or false positives.
A variety of strategies have been proposed to detect the
small deletions, insertions, inversions, and duplications
that are common in the human genome140 . Much of
the analysis is contingent on the ability to identify
departures in the location and orientation of sequenced
DNA fragments from where they are expected to be found
in reference mapped genomes. The ability to do this type
of analysis in a non-invasive test is a long-term goal in
molecular cytogenetics.
Overview of future testing
Currently, only methods based on MPS have been the
subject of clinical trials and can be viewed as being
Ultrasound Obstet Gynecol 2013; 42: 15–33.
28
suitable for clinical use. An optimal NIPT for fetal
aneuploidy will be accurate, simple, cheap, applicable
early in pregnancy and fully compatible with existing
prenatal screening methods so that risks developed using
the different approaches can be combined. Many of the
methods under consideration can be refined to allow for
the detection of other chromosomal imbalances. MPS
refinements that will aid this process include selection
of optimal sequences for analysis, adjustments for base
composition and the use of sequencing platforms that
provide higher read accuracy.
Sequencing also provides opportunities to detect the
presence or absence of single gene disorders130 . The
methods can even be applied to the construction of an
entire fetal genotype131,132 . On the other hand, methods
that do not require sequence information from the parents
or which do not generate unwanted additional genotypic
status of the parents (for example resulting in information
about paternity or other disease risks) should also be
considered advantageous. The optimal technologies may
ultimately be those which are most amenable to answering
only those clinical questions that are being asked, rather
than those which provide the most information.
COUNSELING
The internationally accepted approach to dealing with
patients’ diverse ethical, religious and cultural values is to
provide individual non-directive counseling, testing and
other clinical information at each step in the screening and
diagnosis process. This model, which maximizes patient
autonomy in reproductive decision making, has been
adopted by diverse populations throughout the world.
Counseling of women who are considering NIPT is
challenging. The range of cytogenetic abnormalities currently detectable through cfDNA testing is smaller than
that detectable by conventional karyotyping and substantially less than that achievable through microarray testing.
In the absence of public health policies that define testing
strategies, each high-risk woman who previously would
have made a decision about amniocentesis or CVS and
karyotyping, will now need to choose between no testing,
NIPT, invasive testing with conventional cytogenetics or
invasive testing with microarray testing. These women will
need to be counseled carefully regarding the complex benefits, hazards and trade-offs associated with each option.
In part, these decisions will be determined by the
indication for testing and perceived risk, but they will also
be determined by the available alternative testing services.
For example, in a situation in which routine invasive
testing is based on RAT for a limited set of disorders, the
choice of NIPT that detects the same range of aneuploidies
might seem preferable. On the other hand, offering array
technology may provide much more valuable information
concerning fetal wellbeing than can current NIPT. This
benefit has to be weighed against the risk of losing a
normal pregnancy following an invasive procedure or the
heightened anxiety caused by the presence of a CNV of
unknown significance, poorly defined penetrance and/or
Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.
Benn et al.
variable expressivity. In addition, the testing could expose
consanguinity or non-paternity. Given these realities, the
challenge now facing those providing pretest counseling
cannot be understated. The National Society of Genetic
Counselors recognizes that due to limited resources,
pretest counseling cannot always be provided by a
genetic counselor and this information will therefore also
need to be communicated by other qualified healthcare
providers141 .
The availability of professional guidelines, such
those from the American College of Obstetricians and
Gynecologists, together with the Society for MaternalFetal Medicine142 , the Society of Obstetricians and
Gynaecologists of Canada143 , the National Society of
Genetic Counselors144 , the American College of Medical
Genetics and Genomics145 and the International Society
for Prenatal Diagnosis (ISPD)146 , can greatly facilitate
the process by clarifying which processes are scientifically
valid and clinically acceptable standards of care.
ETHICAL ISSUES
The promise of NIPT is that it will reduce the number of
women with an indication for invasive testing and only
those women who are at very high risk for defined disorders such as Down syndrome will then need to deal with
the ethical challenges associated with procedure-related
loss and pregnancy termination. However, the ease of
providing NIPT will potentially result in substantially
more prenatal diagnoses and terminations of affected
pregnancies18 . The issue is confounded by an inability to
provide adequate counseling for all women who might be
considering NIPT147 . There may also be a shift in patients’
and/or healthcare providers’ views on prenatal diagnosis,
with a greater expectation for pregnancy termination
as the usual response to an abnormal diagnosis. This
has been referred to as ‘normalization’ of testing and
termination148 . Potential consequences could include a
sharply reduced incidence of Down syndrome neonates,
greater emphasis on prevention and less on support and
accommodation of their disabilities, and altered attitudes
to individuals with Down syndrome and their parents
who chose not to test or terminate an affected pregnancy.
The currently available NIPT for aneuploidy is the
consequence of a massive investment by private companies
and venture capital, and so far is only provided by private,
for-profit companies. Aspects of the testing are the subject
of contested patents and other proprietary ownership.
Claims of exclusive intellectual property ownership can
inflate costs, restrict access to test protocols and limit
further test development. Although there is currently
no direct-to-consumer testing, there are press releases,
patient information materials and extensive marketing to
healthcare providers, many of whom may have a poor
understanding of the true performance of the tests.
In the future, NIPT may also be encouraged by governments or insurance carriers who view genetic disorders as
an economic burden149 . The concern is that these financial
pressures will be the dominating factor in determining
Ultrasound Obstet Gynecol 2013; 42: 15–33.
NIPT for aneuploidy
who has access to testing, without adequately considering
medical need. Changes in financial support for testing,
counseling and clinical services can also indirectly
compromise individual patient autonomy when deciding
whether to continue or terminate an affected pregnancy.
It is also worth noting that the early history of NIPT
shows how easily test quality can be compromised when
development and introduction is left entirely to unregulated free market forces150,151 . ISPD, in its latest Position
Statement on NIPT has strongly emphasized the importance of adoption of laboratory standards for NIPT146 .
DISCLOSURES
H. Cuckle is a consultant to PerkinElmer Inc, Ariosa
Diagnostics Inc, Natera Inc, and director of Genome Ltd;
E. Pergament is a consultant to PerkinElmer Inc and
Natera Inc, and director of Northwestern Reproductive
Genetics Inc.
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