First Trimester Screening for

Aneuploidy
Key Points
Nuchal translucency measurement in the first trimester is the most powerful marker for
fetal Down syndrome.
Combination of nuchal translucency with serum markers in the first trimester detects up
to 87% of
cases of Down syndrome for a 5% false-positive rate.
Septated cystic hygroma, or simple nuchal translucency of 3.0 mm or greater, are
indications for chorionic villus sampling (CVS) without need to await serum marker
results.
First trimester absence of the nasal bones, reversal of flow in the ductus venosus, and
tricuspid
regurgitation may have a limited role as second-line screening tests for select high-risk
patients by expert sonologists.
These second-line screening tests are unlikely to have any value for routine general
population screening.

INTRODUCTION

The ideal time to screen for fetal aneuploidy is now during the first trimester of
pregnancy. This evolution in screening policy is due to the significant advances that have
been made in serum and sonographic markers for fetal chromosomal abnormalities over
the past 20 years.

FETAL NUCHAL TRANSLUCENCY

The single most powerful marker available today for differentiating Down syndrome from
euploid pregnancies is the first trimester sonographic measurement of the fetal nuchal
translucency space. Nuchal translucency refers to the normal subcutaneous fluid-filled
space between the back of the fetal
neck and the overlying skin (Figure 2-1). Figure 2-2 demonstrates an increased nuchal
translucency observed in a fetus subsequently shown to have Down syndrome. By
adhering to a standard ultrasonographic technique, it is possible to obtain accurate
measurements of this area in the vast majority of fetuses between 10 and 14 weeks
gestation.When performing nuchal translucency sonography, it is absolutely essential to
ensure optimal technique, which can be attained by focusing
on the following criteria (Abuhamad, 2005):
_ Fetus should be imaged in the midsagittal plane, ideally with the fetal spine down.
_ The image should be adequately magnified so that only the fetal head, neck, and upper
thorax fill the viewable area.
Figure 2-1 Optimizing the technique for first trimester nuchal translucency sonography: Nuchal
translucency measurement in a normal fetus at 12 weeks gestation. Components of a good
sonographic screening protocol are evident, including adequate image magnification, midsagittal
plane, neutral fetal neck position,
and correct caliper placement.

Figure 2-2 Increased nuchal translucency measurement of 3.7 mm in a fetus at 12 weeks

gestation with Down syndrome.
_ Fetal neck should be neutral, with care being taken to avoid measurements in the
hyperflexed or hyperextended positions.
_ The skin at the fetal back should be clearly differentiated from the underlying amniotic
membrane, either by visualizing separate echogenic lines or by noting that the skin line
moves with the fetus.
_ Measurement calipers should be placed on the inner borders of the echolucent space,
and should be perpendicular to the long axis of the fetus.
_ Ultrasound and transducer settings should be optimized to ensure clarity of the image
and of the borders of the nuchal space in particular. This may require transvaginal
sonography in certain situations.
There is a direct correlation between increasing nuchal translucency
measurement and risk for Down syndrome, other aneuploidies, major structural
malformations, and adverse pregnancy outcome (Malone et al., 2000, 2005b; Nicolaides
et al., 2002;Wald et al., 2003b). Possible etiologies for the development of this increased
fluid-filled space include cardiac failure secondary to structural malformation,
abnormalities in the extracellular matrix, and abnormal or delayed development of the
lymphatic system (Moscoso, 1995).

NUCHAL TRANSLUCENCY SCREENING FOR DOWN SYNDROME

Nuchal translucency sonography is the most powerful marker for general population
screening for Down syndrome (Malone and DAlton, 2003). The largest study of this form
of screening was performed by the Fetal Medicine Foundation based in London (Snijders
et al., 1998). This prospective
study of 96,127 unselected patients at 22 centers required nuchal translucency
sonography to be performed between 10 and 14 weeks gestation by specially trained
sonographers who utilized a standardized technique. The overall Down syndrome
detection rate was 77%, for a 5% false-positive
rate. Another large prospective study from the United Kingdom that evaluated the role of
nuchal translucency sonography in general population screening was the SURUSS Trial
(Wald et al., 2003b). A total of 39,983 patients hadnuchal translucency sonography
obtained between 10 and 14 weeks gestation, and the Down syndrome detection rate
was 63%, for a 5% false-positive rate. The largest prospective trial of all forms of Down
syndrome screening yet performed, the FASTER Trial, has also validated the important
role of nuchal translucency sonography. This was amulticenter prospective study from15
centers in theUnited States in which 36,306 patients from the general population had
first trimester nuchal translucency sonography performed (Malone et al., 2005b). The
detection rate for nuchal translucency with maternal age ranged from70%to64%at 11
and 13weeks gestation respectively, for a 5% false-positive rate. These large studies
from varied centers and geographic locations confirm that nuchal translucency
sonography must be a key component of first trimester screening programs for fetal
Down syndrome.

COMBINED FIRST TRIMESTER SERUM AND SONOGRAPHIC

SCREENING

Together with the clear role of nuchal translucency sonography, research in first trimester
screening has consistently shown that pregnancies with fetal Down syndrome are
associated with altered levels of certain maternal serum markers, including elevated
levels of total human chorionic gonadotropin (hCG) and of the free subunit of hCG (with
a median multiple of the median [MoM] of 1.83 in affected cases) and lower levels of
pregnancy-associated plasma protein A (with a median MoM of 0.38 in affected cases)
(Canick andKellner, 1999). Studies of the combination of free subunit of hCG,
pregnancy-associated plasma protein A, and maternal age uniformly demonstrate a
sensitivity for Down syndrome of approximately 60% with a 5% falsepositive rate.
These first trimester serum markers are largely independent of nuchal
translucency, which has resulted in the development of a combined serum and
sonographic screening protocol that would be more effective for screening than
either component alone. The FASTER study evaluated 38,033 patients and demonstrated
very high Down syndrome detection rates, and showed that performance varied
significantly by gestational age. For a 5% false-positive rate, the Down syndrome
detection rates using combined serum and sonographic screening were 87%, 85%, and
82% at 11, 12, and 13 weeks gestation respectively (Malone et al., 2005b). For a 1%
false-positive rate, the Down syndrome detection rates were 73%, 72%, and 67% at 11,
12, and 13 weeks gestation respectively.
Figure 2-3 summarizes the comparative performance of the various screening
options from the FASTER Trial, and demonstrates that first trimester combined screening
has very similar performance to second trimester Quad marker serum screening (Malone
et al., 2005b). Only when performed at 11 weeks gestation does first trimester combined
screening have a significantly better performance than second trimester Quadmarker
serumscreening(87%vs.81%Downsyndrome detection, respectively, at a 5%false-positive
rate). Additionally, the combined first trimester screening test has the lowest false-
positive rate, compared with either first trimester component used alone or compared
with second trimester serum screening. It is also important to realize that only the
combination of first trimester nuchal translucency sonography with first trimester serum
markers comes close to the performance
of second trimester Quad marker serum screening. Nuchal translucency alone, without
being combined with serum markers, has significantly inferior performance
characteristics.
Figure 2-3 A. Comparison of first and second trimester screening for Down syndrome: Detection
rates of combined first trimester screening at 11, 12, and 13 weeks gestation, compared with
second trimester Quad marker serum screening performed at 15 to 18 weeks gestation (each at
5% false-positive rate). B. Comparison of first and second trimester screening for Down syndrome:
False-positive rates of first trimester nuchal translucency alone, PAPP-A and f_hCG alone, and first
trimester combined screening at 12 weeks gestation, compared with second trimester Quad
marker serum screening performed at 15 to 18 weeks gestation (each for 85% detection rate).

ENLARGED NUCHAL TRANSLUCENCY AND CYSTIC HYGROMA IN THE

FIRST
TRIMESTER

It is now clear that a subset of fetuses with very large nuchal translucency
measurements can be effectively identified in the first trimester that have an extremely
high risk of fetal aneuploidy or other adverse pregnancy outcomes. This finding has been
described as septated cystic hygroma, and is present when the nuchal translucency
space is enlarged extending along the entire length of the fetus, and in which septations
are clearly visible (Figure 2-4)(Malone et al., 2005a). Septated cystic hygroma is seen in
more than 1 in 300 first trimester pregnancies. In a recent prospective study of routine
first
trimester sonographic screening, septated cystic hygromawas shown to have a 50%
chance of being associated with fetal aneuploidy, with most cases being Down syndrome,
as well as cases of Turner syndrome and trisomy 18. Additionally, among cystic hygroma
cases confirmed as being euploid, approximately 50% had a major structural fetal
malformation, with most cases including cardiac malformations, as well as other
malformations such as skeletal dysplasias.When compared with simple increased nuchal
translucency, septated cystic hygroma cases were 5 times more likely to be
aneuploid, 12 times more likely to have cardiac malformations, and 6 times more likely to
result in fetal or neonatal demise.
The practical benefit of being able to counsel patients in the first trimester
following the identification of septated cystic hygroma is that there is no need to delay
decision-making while awaiting serum marker results or using computerized risk
calculation algorithms. When faced with a 50% chance of fetal aneuploidy, it is
reasonable to offer such patients the immediate option of CVS, and if fetal aneuploidy
has been excluded, a detailed fetal anatomical evaluation, including fetal
echocardiography, should be performed at 18 to 20 weeks gestation.
Debate has also occurred regarding the differentiation between septated cystic
hygroma and simple enlarged nuchal translucency, with some investigators believing
that they are both part of a spectrum of a similar sonographic feature (Molina et al.,
2006). However, the FASTER Trial, a prospective study of more than 38,000 pregnancies,
has also now demonstrated that whenever a simple nuchal translucency measurement of
3.0 mm or greater is noted, CVS should be offered immediately because of aminimumrisk
of aneuploidy of 1 in 6 (Comstock, 2006). With such nuchal translucency measurements,
there is no role for delaying decision-making while awaiting serummarker results, as such
additional information does not meaningfully alter the original aneuploidy risk. This
finding therefore suggests that debating the presence or absence of septations in cases
of enlarged nuchal translucency is a moot point, as the risk of aneuploidy either when a
nuchal translucency measurement of 3.0 mm or greater is noted, or when a septated
cystic hygroma is noted, is sufficiently high to warrant immediate diagnostic testing.
Figure 2-4 A. Septated cystic hygroma at 12 weeks of gestation: midsagittal sonographic view of
a fetus with septated cystic hygroma, demonstrating increased nuchal translucency space
extending along the entire length of the fetus. B. Septated cystic hygroma at 12 weeks of
gestation: transverse view through the fetal neck of the same fetus demonstrating obvious
septations (arrows). (From Malone FD, Ball RH, Nyberg DA, et al. First trimester septated cystic
hygroma: prevalence, natural history, and pediatric outcome. Obstet Gynecol. 2005a;106:288-294.)
Figure 2-5 A. First trimester ultrasound examination of a fetus at 13 weeks gestation, with normal
nasal bones. Features of good nasal bone imaging technique are evident, including midsagittal
plane, fetal profile facing upward, adequate magnification, and visualization of two parallel lines at
the level of the fetal nose, one representing fetal skin (short arrow) and the deeper line
representing nasal bones. B. First trimester ultrasound examination of a fetus at 13 weeks
gestation, with absent nasal bones and enlarged nuchal translucency thickness, in which karyotype
was confirmed as trisomy 18.

NASAL BONE SONOGRAPHY IN THE FIRST TRIMESTER

There appears to be a clear association between the absence of the fetal nasal bones on
first trimester ultrasound examination and Down syndrome. In a study conducted by
Cicero et al. (2001), 701 fetuses with increased nuchal translucency were evaluated for
the presence or absence of the nose bones during first trimester ultrasonography. The
fetal nasal bones could not be visualized in 73% of Down syndrome fetuses (43 of 59)
and in only 0.5% of unaffected fetuses (3 of 603). The authors also felt that the absence
of the fetal nose bone was not related tonuchal translucency thickness and therefore
could be combined into a single ultrasound screening modality, with a predicted
sensitivity of 85% for a 1% false-positive rate. This study was subsequently expanded to
a larger series of 3829 high risk, first trimester pregnancies, in which the detection rate
for Down syndrome using absent nasal bones was 67%, for a 2.8% false-positive rate
(Cicero et al., 2003).
Adequate imaging of the fetal nasal bones can be technically challenging in the
first trimester, and careful attention to correct technique should therefore be paid to
ensure consistency in technique. The nasal bones should be visualized on ultrasound
along the midsagittal plane with a perfect fetal profile. The fetal spine should be down,
with slight neck flexion. Two echogenic lines at the fetal nose profile should be visualized;
the superficial echogenic line is the nasal skin, and the deeper echogenic line represents
the nasal bones. This deeper echogenic line representing the nasal bones should be more
echolucent at its distal end (Figure 2-5A). Care should be taken not to perform this
evaluation with the ultrasound beam parallel to the plane of the nasal bones, because
this might erroneously lead to the conclusion of absent nasal bones (Figure 2-5B).
While several other studies evaluating the role of first trimester nasal bone
sonography as a screening test for fetal Down syndrome have also been published, all
were limited by being derived from high-risk patient populations or lacking adequate
pregnancy outcome ascertainment. The largest study of first trimester fetal nasal bone
sonography published to date, and in which an unselected general patient population has
been evaluated, did not confirma useful role for this formof screening(Malone et al.,
2004). In a prospective study of 6324 patients having nasal bone sonography by trained
and experienced sonographers, adequate views of the fetal profile were obtained in
only76%of cases, and none of the 11 cases of fetal Down syndrome were found to have
absent nasal bones. Two further studies of the role of first trimester nasal bone
sonography as a screening tool in the general population have also recently been
published, and have confirmed these findings that there is only a limited role of this
formof screening. One study screened 7116 unselected patients from the general
population including 12 cases of Down syndrome, and screened a further 510 high-risk
patients including 23 cases of Down syndrome(Prefumo et al., 2006).Absent nasal bones
in the first trimester was noted in only17%of cases in the general population and in 48%
of cases in the high-risk population. A further study screened 1800 consecutive patients
from the 2 of 7 (29%) of Down syndrome cases (Ramos-Corpas et al., 2006). While it is
possible that there may be a role for first trimester fetal nasal bone sonography in the
hands of select experts as a second-line screening tool in high-risk patients, current data
suggest that this formof sonography should not be used as a general population
screening tool.
FIRST TRIMESTER DUCTUS VENOSUS SONOGRAPHY

First trimester Doppler sonographic evaluation of ductus venosus blood flow has been
described as an adjunctive test for fetal aneuploidy screening. Forward triphasic pulsatile
ductus venosus flow, as illustrated in Figure 2-6, is normal, while reversed flow at the
time of the atrial contraction has been associated with aneuploidy and fetal cardiac
malformations (Matias et al., 1998). In a series of early studies evaluating this
association, between 59% and 93% of aneuploid fetuses had abnormal first trimester
ductus venosus flow velocities (Malone and DAlton, 2003). Abnormal ductus venosus
flow velocities were also found in as few as 3% or as many as 21% of normal fetuses. It
may therefore be possible that fetal ductus venosus flow velocity waveform analysis may
be useful to modify a patients final risk for aneuploidy following completion of the nuchal
translucency measurement. This could be used to either improve the detection rate or
alternatively to reduce the false-positive rate.
Figure 2-6 Pulsed Doppler sonography image of a normal ductus venosus wave form at 12 weeks
gestation. Of note, the ductus venosus can be identified using color flow as a small vessel with
turbulent flow. The nadir of flow during the a wave remains forward flowing (arrow).
While it appears that there is some association between abnormal ductus venosus
flow studies and aneuploidy in the first trimester, there are several pitfalls that must be
considered. The ductus venosus vessel itself may be as small as 2 mmat 10 to 14 weeks,
making it difficult to obtain accurate
flow velocity waveforms from such a tiny vessel without contamination of the waveform
from neighboring vessels. For example, if the Doppler gate is placed too proximally near
the umbilical sinus, the normal continuous venous flow from the umbilical vein may
obscure absence of flow during the atrial contraction in the ductus venosus.
Alternatively, placement of the Doppler gate too far distally, near the insertion of the
ductus venosus into the inferior vena cava may lead to the erroneous
diagnosis of reversal of flow at the atrial contraction, as such reversal of flow is
commonly seen in the inferior vena cava. The current consensus appears to be that first
trimester ductus venosus Doppler flow studies should be performed only as a secondary
screening test in the hands of experienced sonologists at referral centers, either to
modify the final risk for aneuploidy or to help predict the prognosis of fetuses with normal
chromosomes and an increased nuchal translucency measurement (Hecher, 2001;
Nicolaides et al., 2005).

FIRST TRIMESTER TRICUSPID REGURGITATION EVALUATION

An association has been suggested between fetal aneuploidy and abnormal tricuspid
regurgitation noted during first trimester sonography (Nicolaides et al., 2005). To perform
this assessment, the fetus should be oriented so that the chest wall is anterior and the
fetal heart should be insonated parallel to the ventricular septum. A pulsed Doppler gate
of approximately 3 mm size is then placed across the tricuspid valve, with care to ensure
that the angle to the direction of flow is as close to zero as possible (Figure 2-7).
Significant tricuspid regurgitation is considered to be present if a regurgitant jet
of at least 60 cm/s is noted extending to more than half of systole (Falcon et al., 2006).
In a series of 1557 high-risk pregnancies at 11 to 13 weeks gestation, significant
tricuspid regurgitation was noted in only4%of chromosomally normal fetuses, but was
present in 77 of 114 cases (68%) ofDown syndrome and 14 of 42 cases (33%) of trisomy
18 (Falcon et al., 2006). These investigators were able to obtain adequate Doppler
waveforms of the tricuspid valve in 99% of cases and there was low interobserver
variability of measurements.
While these data are encouraging regarding an association between first trimester
tricuspid regurgitation and chromosomal abnormalities, like ductus venosus assessment,
it is unclear whether this form of screening will have any role in general population
screening. Extrapolation of the results of a screening test from a high-risk population,
performed in the hands of experts, toimplementation in the general population will likely
overstate the true performance of the test. It islikely that first trimester sonography for
tricuspid regurgitation may have a role as a second-line test, following an initial high-risk
nuchal translucency and serum screen. In this setting, it may have a role in reducing the
false-positive rate of screening. In one series of 75,821 first trimester pregnancies,
addition of tricuspid regurgitation as a second-line screening test following nuchal
translucency and serum screening resulted in a Down syndrome detection rate of 92%
for a false-positive rate of only 2.7% (Nicolaides et al., 2005).

Figure 2-7 First trimester sonographic evaluation for tricuspid regurgitation in a fetus at 11 weeks
gestation. Note the pulsed Doppler gate placed across the tricuspid valve, with the angle to the
direction of flow close to zero. There is no regurgitation
greater than 60 cm/s.
NUCHAL TRANSLUCENCY SCREENING IN MULTIPLE GESTATIONS

Prenatal risk assessment for Down syndrome in multiple gestation pregnancies has been
quite limited before the advent of nuchal translucency-based screening.Maternal serum
screening has not been widely utilized in the setting of multiple gestations because of
the potential for discordancy between twins and the impact of different placentas on the
various analytes. Nuchal translucency measurements are broadly similar between
singleton and twin pregnancies, implying that theDownsyndromedetection rates should
be similar. The false-positive rate of nuchal translucency screening might be higher in
monochorionic twins because some complications unique to monochorionic gestations,
such as twinto- twin transfusion syndrome, might present with increased nuchal
translucency measurement (Sebire et al., 2000).
Options for first trimester screening for Down syndrome therefore include
providing either a fetus-specific risk based on nuchal translucency alone, or providing an
overall pregnancy risk based on combined serum and sonographicmarkers. Inone series
of 448 twin pregnancies, nuchal translucency yielded an 88% detection rate for a 7%
false positive rate (Sebire et al., 2000). In another series of 206 twin pregnancies
screened using nuchal translucency and first trimester serum markers, the Down
syndrome detection rate was estimated to be approximately 75% for a 5% false-positive
rate (Spencer and Nicolaides, 2003). To provide a pregnancy-specific risk in twins, a
likelihood ratio is calculated for each fetus nuchal translucency measurement; these two
likelihood ratios are then added together, and this is then multiplied by the biochemistry
likelihood ratio to provide a final pseudorisk calculation (Wald et al., 2003a). Although
additional research on the efficacy of this combined first trimester screening in multiple
gestations is still needed, nuchal translucency measurement should at least represent an
improvement over serumscreening inmultiple gestations. Currently, some centers use
nuchal translucency sonography
to assist in selecting fetuses for reduction in higher order multiple gestations.