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Received: 15 August 2016    Revised: 1 November 2016    Accepted: 19 December 2016

DOI: 10.1002/ijgo.12087

CLINICAL ARTICLE
Obstetrics

Observational study comparing the performance of

first-­trimester screening protocols for detecting trisomy
21 in a North Indian population

Anita Kaul1* | Chanchal Singh1 | Rachna Gupta1 | Nidhi Arora1 | Abha Gupta2

1
Apollo Centre for Fetal Medicine,
Indraprastha Apollo Hospitals, New Delhi,
India
2
Department of Biochemistry, Indraprastha
Apollo Hospitals, New Delhi, India
*Correspondence
Anita Kaul, Apollo Centre for Fetal Medicine,
Indraprastha Apollo Hospitals, New Delhi,
India.
Email: anita_kaul@apollohospitalsdelhi.com
Abstract
Objective: To evaluate first-­trimester screening protocols for detecting trisomy 21 in
an Indian population.
Methods: The present prospective study collected data from women with singleton
pregnancies and a crown-­to-­rump length of 45–84 mm who presented at the fetal
medicine unit of a tertiary care center in North India between June 1, 2006, and
December 31, 2015, for combined first-­trimester screening. Maternal age, nuchal
translucency, nasal bone, and maternal serum levels of free beta human chorionic gon-
adotropin and pregnancy-­associated plasma protein A were assessed for calculating
the risk of trisomy 21. Tricuspid regurgitation and qualitative analysis of ductus veno-
sus data were available from June 2010, and were included where available. Trisomy-­21
detection rates were calculated for various screening protocols and were compared.
Results: There were 4523 women screened and 24 records of trisomy 21. Combined
screening with maternal age, nuchal translucency, nasal bone, tricuspid regurgitation,
and ductus venosus demonstrated optimal detection and false-­positive rates of 93.8%
and 1.9%, respectively. Screening using only maternal age yielded a detection rate of
37.5%; using fixed nuchal translucency cut-­off values of 2.5 and 3 mm resulted in de-
tection rates of 66.7% and 37.5%, respectively.
Conclusion: Combined first-­trimester screening performed well in an Indian popula-
tion; combining maternal age, nuchal translucency, nasal bone, ductus venosus, and
tricuspid regurgitation yielded the most accurate screening.

KEYWORDS
Combined first-trimester screening; Nuchal translucency; Prenatal screening; Trisomy 21;
Ultrasonography

1 |  INTRODUCTION
The prevalence of trisomy 21—or Down syndrome—in India is 1
in 11501 and trisomy-­21 screening is not as yet a part of national
screening programs. The disparate delivery of health care across the
country could result in enormous variations in trisomy-­21 screening
among obstetricians both within cities, and also between urban and
semi-­urban regions. Important factors that could affect screening in-
clude testing costs, and the availability of trained sonographers and
suitable laboratories. Awareness among obstetricians of the detection
rates of different screening protocols could also influence the uptake
of testing. Several multicenter studies have already demonstrated the
efficiency of first-­trimester screening protocols and have emphasized
that they must be appropriate for the population screened.2–8 The

Int J Gynecol Obstet 2017; 1–6 wileyonlinelibrary.com/journal/ijgo © 2016 International Federation of  |  1
Gynecology and Obstetrics
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2       Kaul ET AL.
prevalence of trisomy 21 appears to be similar across different ethnic
9
groups; consequently, screening methods adopted from Caucasian
populations should perform similarly in an Indian setting.
The aim of the present study was to evaluate the performance
of combined trisomy-­21 testing in an urban Indian population. In the
current era of cell-­free DNA potentially becoming the standard triso-
my-­21 screening protocol in high-­income settings, the present study
was especially relevant because the cost of cell-­free DNA screening
is too high for it to be universally adopted in resource-­constrained
countries.
2 |  MATERIALS AND METHODS
The present prospective study collected data from women present-
ing at the fetal medicine unit of a tertiary care center in North India
between July 1, 2006, and December 31, 2015. All Indian women
presenting to the clinic during the first trimester of singleton pregnan-
cies for pre-­test counseling for combined trisomy-­21 screening using
ultrasonography, maternal serum free beta human chorionic gonado-
tropin (β-­hCG), and pregnancy-­associated plasma protein A (PAPP-­A)
were considered for inclusion. Women of Indian origin with a single,
live fetus with a crown-­to-­rump length (CRL) of 45–84 mm were then
included. Patients were excluded if any pre-­ or post-­natal fetal struc-
tural anomalies were observed, and patients who underwent induced
abortion for reasons other than aneuploidy were also excluded. All
participants provided informed consent and the study was approved
by the Indraprastha Apollo Hospitals ethical committee.
Participants underwent ultrasonography examination with
6–8 MHz abdominal and 6–12 MHz vaginal transducers (Voluson
730 and Voluson E8 systems; General Electric Healthcare, Milwaukee,
WI, USA). First-­trimester ultrasonography examination included
CRL, nuchal translucency, and nasal bone measurements, and an
early anatomy survey. From June 2010 onwards, tricuspid regurgi-
tation and qualitative assessment of the ductus venosus waveform
were performed, and were subsequently included in the study. All
measurements were examined with reference to Fetal Medicine
Foundation (FMF) criteria10 and all ultrasonography examinations
were performed by FMF-­certified operators. Blood samples for the
examination of free β-­hCG and PAPP-­A levels were taken between 10
and 13+6 weeks of pregnancy, serum analyses were performed using
FMF-­accredited assays (Perkin Elmer, Rodgau, Germany, and Roche,
Manheim, Germany). The duration of pregnancy-­specific multiples
of median values were adjusted for maternal ethnicity, as described
previously.11 Demographic data, ultrasonography parameters, and
biochemistry data were recorded prospectively in a computer data-
base and a combined risk report was prepared with Astraia software
(Astraia, Munich, Germany). The assumed cut-­off values for defining
“high risk” were 1 in 250 for trisomy 21 and 1 in 100 for trisomy 18
and 13. All patients received post-­testing counseling once the com-
bined risk report was available. Women considered at high risk were
offered diagnostic testing by chorionic villus sampling or amniocente-
sis. Noninvasive prenatal screening using cell-­free DNA was offered
to patients from April 2014 onwards. Patients who elected to un-
dergo induced abortion based on ultrasonography findings were also
offered karyotyping; if karyotype was not available these patients
were excluded from the study. Patient follow-­up continued until de-
livery for all pregnancies. Outcomes were obtained from medical re-
cords or, if they were not included in medical records, attempts were
made to obtain them by telephone enquiries before including them in
the database. The study data were compared between a trisomy-­21
group and a control group (containing data from pregnancies with no
aneuploidies). The results of screening were divided into two phases
based on the addition of tricuspid regurgitation and ductus venosus
examination, the initial 4-­year period between June 1, 2006, and June
31, 2010, and the subsequent 5-­year period between July 1, 2010,
and December 31, 2015.
Continuous variables were expressed as medians and were com-
pared using the Mann-­Whitney U test. Fixed false-­positive rates of 3%
and 5% for the pre-­ and post-­ultrasonography periods, respectively,
were used to calculate combined-­screening detection rates. To com-
pare the different screening protocols, receiver operating characteris-
tic curves were prepared. All analyses were performed using Microsoft
Excel 2007 (Redmond, WA, USA) and SPSS 15.0 (SPSS, Chicago, IL,
USA), and P<0.05 was considered statistically significant.
3 | RESULTS
A total of 5251 pregnant women at between 11 weeks and 13+6 weeks
of pregnancy underwent combined first-­trimester screening during the
study period. There were 186 women with twin pregnancies and 105
women of non-­Indian origin who were excluded; 91 women who un-
derwent spontaneous abortion or induced abortion prior to 20 weeks
(for reasons other than aneuploidy) were also excluded. Structural
anomalies resulted in 12 patients being excluded from the analyses,
and 39, eight, and eight patients were excluded owing to intrauterine
fetal demise, stillbirth, and neonatal death, respectively. At the end
of the study period, there were 102 women whose pregnancies were
ongoing who were, consequently, excluded, and 177 (3.6%) patients
were lost to follow-­up. This resulted in data from 4523 patients being
included in the present study (Data S1, Table 1).
During first-­trimester screening, there were 126 (2.8%) patients
with positive screening results and chromosomal anomalies were
identified in 32 (0.7%) pregnancies. Trisomy 21 was identified in 24
pregnancies; 22 of these were confirmed in the first trimester using
chorionic villus sampling after screening identified these patients
as being at increased risk. There was one patient who had a preg-
nancy with trisomy 21 who had a negative first-­trimester screening
result before undergoing amniocentesis during the second trimes-
ter owing to a quadruple marker demonstrating increased risk for
trisomy 21. Trisomy 21 was detected postnatally for one patient.
Trisomy 18 and Turner syndrome (45,X) were the other aneuploidies
detected, observed in four patients each. There was one further
patient excluded from the study owing to concurrent trisomy 21
and gastroschisis.
Kaul ET AL. |
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T A B L E   1   Characteristics of study participants (n=4523).a 2010) were added to screening protocols as a pair and this resulted in
improvements in detection rates. Including all the investigated mark-
Variable Value
ers in a combined test demonstrated a detection rate of 95.2%; how-
Age, y 29.88±3.96
ever, this high rate was only reached with a fixed false-­positive rate
Weight, kg 62.18±10.78 of 5% (Data S2). In the analysis of receiver operating characteristic
BMI 24.23±3.99 curves, the largest area under the curve was obtained by combining all
Conception of the markers, including tricuspid regurgitation and ductus venosus,
Spontaneous 4208 (93.0) which were included after June 2010 (Figure 2).
In vitro fertilization 315 (7.0)
Duration of pregnancy at enrollment, wk
11 to 11+6 509 (11.3) 4 | DISCUSSION
+6
12 to 12 2564 (56.7)
+6 Of all the screening protocols analyzed in the present study, com-
13 to 13 1450 (32.1)
bined screening including the extended markers demonstrated the
Crown-­to-­rump length, mm 64.44±8.03
best performance with a detection rate of 93.8% and a false posi-
Karyotype
tive rate of 1.9%; these values are comparable to previously pub-
No aneuploidies 4499 (99.5)
lished data from other countries3–5,7,8,12,13,16–18,22,24 and the study
Trisomy 21 24 (0.5) was ­designed to ­reflect common screening protocols followed in the
Other aneuploidies 8b study setting (Data S3).
Abbreviation: BMI, body mass index (calculated as weight in kilograms di- The results of the present study corroborate the well researched
vided by the square of height in meters). fact that using maternal age alone is inadequate for trisomy-­21 screen-
a
Values are given as mean±SD or number (percentage).
b
ing. Previous studies have reported that only 30% of neonates with
These aneuploidies were not included in the analyses.
chromosomally anomalies can be detected using this method.13 In the
The median age of participants and nuchal translucency were present study, 62% of fetuses with trisomy 21 would not have been
higher, and the median PAPP-­A was lower, among patients in the detected if screening was based on maternal age alone. Consequently,
trisomy 21 group (Table 2). The distribution of nuchal translucency the use of maternal age alone for screening should be discarded.
based on CRL is illustrated in Figure 1. Detection rates and false pos- India is a resource-­constrained country, and it has been argued
itive rates were calculated based on a variety of screening modalities that combined testing is too expensive for this setting and that it is
(Table 3). The highest detection rate (93.8%) was observed with an ex- more practical to perform a nuchal scan only. Most sonographers in
tended ultrasonography protocol that included maternal age, nuchal this setting do not have access to risk-­calculation software, and, as a
translucency (with a cut-­off value of above the 95th percentile for fetal result, a common approach is to use fixed cut-­off values for estimating
CRL), nasal bone, ductus venosus, and tricuspid regurgitation; this pro- risk regardless of the CRL. In the present study, a fixed cut-­off value
tocol also resulted in a false-­positive rate of 1.9%. of over 2.5 mm was examined and demonstrated a detection rate of
Individually, screening using increased β-­hCG (>2.5 multiples of 66.7%. Ghaffari et al.13 reported a detection rate of 70% (with a false-­
median) or reduced PAPP-­A (<0.3 multiples of median) demonstrated positive rate of 5.9%) when using a nuchal translucency cut-­off value
very low sensitivity; a combination of the two yielded a sensitivity of of above 2.5 mm in a study comprising 13 437 pregnancies. However,
only 62.5%, and a positive predictive value of 7.1%. a detection rate of 70% is far below the expected standard for a good
In the present study, no fetuses with trisomy 21 demonstrated an screening test. A study that included a cut-­off value of above 3 mm re-
anomalous ductus venosus waveform; however, the ultrasonography ported a detection rate of 67%.14 In the present study, a nuchal trans-
markers tricuspid regurgitation and ductus venosus (collected from lucency cut-­off value of above 3 mm would only have identified 37.5%

T A B L E   2   Comparison of screening parameters.a

Parameter Trisomy 21 group (n=24) Control group (n=4499) P value

Age, y 32.5 (28.0–38.0) 30.0 (17.0–48.0) 0.001

Maternal age ≥35 y 9 (38) 575 (12.8) 0.0003
Crown-­to-­rump length, mm 66.97±8.53 64.43±.02 0.122
PAPP-­A, MoM 0.35 (0.11–6.64) 0.87 (0.15–3.85) <0.001
Free β-­hCG, MoM 1.18 (0.14–11.15) 0.97 (0.18–5.28) 0.071
Nuchal translucency, mm 2.8 (1.7–7.9) 1.7 (0.73–14.0) <0.001
Estimated risk for trisomy 21 1/4 (1/2 to 1/9622) 1/12 279 (1/2 to 1/36 452) <0.001

Abbreviations: PAPP-­A, pregnancy-­associated plasma protein A; MoM, multiples of median; β-­hCG, beta human chorionic gonadotropin.
a
Values are given as median (range), number (percentage), or mean±SD, unless indicated otherwise.
4       | Kaul ET AL.

Nuchal translucency (mm)

Crown-to-rump length (mm)

F I G U R E   1   Distribution of fetal nuchal translucency with respect to fetal crown-­to-­rump length among patients in the trisomy-­21 (red
squares) and control (blue dots) groups.

of fetuses with trisomy 21 (Table 3). In the present cohort, the 95th
percentile nuchal translucency value for a CRL of 84 mm was 2.8 mm.
Using this as a fixed cut-­off value resulted in a detection rate of only
50%. Lowering this threshold could have improved the detection rate;
however, lowering the threshold sufficiently to achieve a detection
rate of 80% would have resulted in a false-­positive rate of 60%, an un-
acceptably high value. Consequently, this method of screening should
also be discarded.
Screening using a cut-­off value of nuchal translucency above the
95th percentile for a given CRL was also examined; this improved the
detection rate to 62.5%, with a false-­positive rate of 4.0%. A recent
study in Poland15 reported a detection rate of 73% with a false-­positive
rate of 3% by applying a similar method (combined with maternal age).
This approach is superior to using a fixed nuchal translucency cut-­off
value. However, the low detection rate still makes it sub-­optimal for
use as a screening test.

T A B L E   3   Performance of different combinations of screening variables.a

False positive False-­positive rate assuming a

Screening variables Detection rate rate fixed detection rate of 80%

Maternal age ≥35 y 9/24 (38) 12.8 −71.5

Nuchal translucency >2.5 mm 16/24 (67) 4.0 40.0
b
Nuchal translucency >95th percentile 12/24 (50) 1.6 60.2
Nuchal translucency >95th percentile for fetal CRL 15/24 (63) 4.0 47.5
Nuchal translucency >3.0 mm 9/24 (38) 1.1 67.5
Absent nasal bone 9/24 (38) 1.2 65.6
Tricuspid regurgitation 8/24 (33) 0.90 71.8
Maternal age, nuchal translucency (with a cut-­off value of >95th percentile 7/8 (88) 3.20 2.5
for fetal CRL), and nasal bone
Maternal age, nuchal translucency (with a cut-­off value of >95th percentile 15/16 (94) 1.9 1.21
for fetal CRL), nasal bone, ductus venosus, and tricuspid regurgitation

Abbreviation: CRL, crown-­to-­rump length.

a
Values are given as number/number of pregnancies with trisomy 21 (percentage) or percentage.
b
The 95th centile for a CRL of 84 mm was 2.8 mm.
Kaul ET AL.
F I G U R E   2   Receiver operating characteristic curves of screening
protocols in the detection of trisomy 21. Screening by maternal age
(blue line; AUC 0.62); maternal age and nuchal translucency (fixed
cut-­off value >2.8 mm) (black line; AUC 0.74); maternal age and
nuchal translucency (above the 95th percentile for fetal CRL) (green
line; AUC 0.79); maternal age, nuchal translucency (above the 95th
percentile for fetal CRL), and nasal bone (brown; AUC 0.92); and
maternal age, nuchal translucency (above the 95th percentile for fetal
CRL), nasal bone, ductus venosus, and tricuspid regurgitation (red
line; AUC 0.95). Abbreviations: AUC, area under curve; CRL, crown-­
to-­rump length.
Studies have reported that the addition of ultrasonography markers
15–23
to nuchal translucency increases detection rates for aneuploidies.
A recent study10 has even reported a 100% trisomy-­21 detection
rate using combined screening (maternal age, β-­hCG, PAPP-­A, nuchal
translucency, nasal bone, ductus venosus, and tricuspid regurgitation).
The present findings corroborated these results; the addition of sec-
ondary ultrasonography markers (adding tricuspid regurgitation and
ductus venosus to maternal age, nuchal translucency, and the nasal
bone) not only improved detection rates, but also reduced the false-­
positive rate from 3.2% to 1.9%.
The strengths of the present study include the use of a strict
protocol including pre-­ and post-­screening counseling, and that all
ultrasonography examinations were performed by FMF-­certified
professionals and biochemistry analyses were performed using
FMF-­certified and validated systems. Additionally, to the best of our
knowledge, this is the first Indian study to report the performance of
combined screening in an Indian setting. There were also drawbacks to
the present study; the study population was limited to North India and
could be unrepresentative of the rest of the country. Additionally, the
high incidence of trisomy 21 in the study population could be due to
referral bias arising from women deemed to be at increased risk being
referred to specialized center such as the study institution. Larger,
population-­based studies involving centers across the country would
be needed to further validate the present results.
In conclusion, combined screening for trisomy 21 has been ad-
opted in high-­income countries for longer than a decade but the situa-
tion differs vastly in resource-­limited settings. The present study of an
unselected population from North India demonstrated that combined
screening based on maternal age, biochemistry, and an extended
|
      5
ultrasonography protocol was feasible, with a detection rate of 93.8%
and a false-­positive rate of 1.9%. The present study should prompt
healthcare providers in India to adopt a uniform policy of combined
first-­trimester screening for trisomy 21.
AU T HO R CO NT R I B U T I O NS
AK conceived the study, collected and analyzed the data, and wrote
and revised the manuscript. CS collected and analyzed the data, and
wrote and revised the manuscript. RG, NA, and AG collected study
data and revised the article.
AC KNOW L ED G M ENTS
Chinmayee Ratha, Tulika Tayal, Ashwani Rathi, Akshatha Sharma,
Pooja Lodha, Nikhil Gholkar, Shweta Bhandari, Nupur Shah, Savita
Dagar, Chinmay Umarji, Shreyasi Sharma are acknowledged for assist-
ing in data collection, and Seema Thakur is acknowledged for provid-
ing support and motivation for writing the manuscript.
CO NFL I C T O F I NT ER ES T
The authors have no conflicts of interest.
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S U P P O RT I NG I NFO R M AT I O N
Additional Supporting Information may be found online in the support-
ing information tab for this article.
Data S1. Flow of study participants.
Data S2. Performance of combined screening with all available vari-
ables from the two time periods of the study.
Data S3. Comparison of screening in the present study with published
literature.