2013

http://informahealthcare.com/jmfISSN: 1476-7058 (print), 1476-4954 (electronic)

J Matern Fetal Neonatal Med, Early Online: 1–6! 2013 Informa UK Ltd. DOI: 10.3109/14767058.2013.784252

ORIGINAL ARTICLE

Analysis of three different strategies in prenatal screening for Down’ssyndrome in twin pregnancies

Pilar Prats1, Ignacio Rodrıguez1, Carmina Comas1, and Bienvenido Puerto2

1Department of Obstetrics, Gynaecology and Reproductive Medicine, Institut Universitari Dexeus, Barcelona, Spain and 2Department of Maternal

Fetal Medicine, Institut Clinic de Ginecologia, Obstetrıcia I Neonatologia, Hospital Clınic, Barcelona, Spain

Abstract

Objectives: To compare the performance of three different strategies in prenatal screeningfor Down’s syndrome in twins [nuchal translucency, the combined test, the combinedtestþductus venosus pulsatility index (DVPI)].Methods: We included 277 twin pregnancies with two cases of trisomy 21 (both dichorionic).We performed a computer simulation of Down’s syndrome NT screening, combined testscreening and the combined test with the addition of DVPI screening using the commercializedsoftware SsdwLab6. The strategies were compared using the area under the receiver operatingcharacteristic curve.Results: NT screening false-positive rate (FPR) was 10.9% (95% CI: 8.3–13.5). The combinedtest FPR was 6.2% (95% CI: 4.1–8.2%) and the combined test plus DVPI was 6% (95% CI: 4–8).FPR was higher in advanced maternal age patients. Detection rate was 100% in all cases.The area under the curve was 0.987 (95% CI: 0.972–0.994) in NT screening; 0.987 (95% CI:0.978–0.997) in the combined test and 0.983 (95% CI: 0.977–0.996) in the combined testþDVPI.Conclusions: Down’s syndrome screening is feasible in twins with low FPR. The results of thisstudy are similar to the results achieved in singletons. The combined test appears to bethe most effective. The addition of DVIP does not significantly improve the prenatal screeningfor trisomy 21.

Keywords

Combined test, Down’s syndrome, ductusvenosus, first trimester, nuchaltranslucency, screening, twins

History

Received 27 August 2012Revised 21 February 2013Accepted 7 March 2013Published online 29 April 2013

Introduction

In twin pregnancies, effective screening for trisomy 21 is

provided by the combination of maternal age and fetal nuchal

translucency (NT) thickness [1–4]. Risk assessment in twin

pregnancies with NT does provide fetus-specific risk in dizyg-

otic pregnancies. The detection rate with the use of maternal

age and NT has been described as similar to that in singleton

pregnancies, although the false-positive rate (FPR) is higher

[2].

The performance of the screening can be improved by the

addition of maternal serum biochemistry [free beta-human

chorionic gonadotrophin (free b-hCG) and pregnancy-

associated plasma protein A (PAPP-A)], but appropriate

adjustments are needed for chorionicity [5]. In dichorionic

twins at 11–13 weeks, the levels of maternal serum free

b-hCG and PAPP-A are approximately twice as high as in

singleton pregnancies, but the levels are lower in monochor-

ionic twins than in dichorionic twins [6–9]. Few reports have

been published about the benefit of the combination of

maternal age, maternal serum biochemistry (free b-hCG,

PAPP-A) and NT (combined test) for the screening for

Down’s syndrome in twin pregnancies [10–15]. All these

studies conclude that the combined test improved the results

achieved with NT screening, with low FPRs. Regarding FPRs,

there is a trend of decreasing rates in the latest published

series.

In singleton pregnancies, other highly sensitive and

specific first-trimester sonographic markers of trisomy 21

(nasal bone, reversed a-wave in the ductus venosus, tricuspid

regurgitation) can be incorporated into first-trimester com-

bined screening [16–18]. Few data are available regarding

the contribution of these markers in Down’s syndrome

screening in twins [19–21]: they highlight the difficulty of

adding new sonographic markers, such as nasal bone, due

to the difficulty in scanning twins (fetus position, fetal

movements, etc.); regarding ductus venosus and its role

in twin-to-twin transfusion syndrome (TTTS) [22] results

could be difficult to interpret in the screening of Down’s

syndrome in twins.

The performance of maternal ageþNT, combined test

and combined testþ additional sonographic marker [ductus

venosus pulsatility index (DVPI)] in twin pregnancies is

analysed in this study.

Address for correspondence: Pilar Prats, Department of Obstetrics,Gynaecology and Reproductive Medicine, Institut Universitari Dexeus,Gran Via Carlos III, 71-75, 08028 Barcelona, Spain. Tel: +34 93 227 4700. Fax: +34 93 418 78 32. E-mail: pilpra@dexeus.com

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Materials and methods

From February 2007 to June 2011, the combined test was

offered to all women carrying a twin pregnancy attending

our Unit for first-trimester Down’s syndrome screening.

During the study period, we attended 447 twin pregnancies,

and 277 were included in this study. The inclusion criteria

were that data from NT, maternal serum biochemistry and

DVPI were available, as well as the perinatal outcome.

Biochemical risk assessment for Down’s syndrome (mater-

nal serum free b-hCG and PAPP-A) was performed between

8 and 13þ 6 weeks’ gestation and NT scan was performed

between 11 and 13þ 6 weeks [crown-rump length (CRL):

45–84 mm], following the Fetal Medicine Foundation

Guidelines [23]. At the time of the NT scan, we systematically

measured the DVPI, following again the same guidelines [24].

The ultrasound exploration was transvaginal, transabdominal

or combined, when necessary. The risk assessment provided

to the women was calculated using the combined test

(maternal age, NT measurement and maternal serum levels

of free b-hCG and PAPP-A). When pregnancy was achieved

with egg donors, the age of the egg donor was used to

calculate the risk assessment.

Chorionicity was determined; it was considered mono-

chorionic in the presence of a single placenta and the absence

of the lambda sign and was considered dichorionic when the

placentas were not adjacent or the lambda sign was present

[25]. In twin pregnancies, the larger of the two CRL

measurements was used to estimate the overall gestational

age of the pregnancy. Biochemistry values were determined

using the Kryptor analyzer (BRAHMS�, Berlin, Germany).

These values were expressed as multiples of the median

(MoM) adjusted to the number of fetuses, maternal weight,

history of chromosomopathy, smoking habit, ethnicity and

chorionicity. SsdwLab6 software uses specific distribution

population parameters for every biochemical marker (mean of

the Gauss curve for unaffected and affected twins) and for

monochorionic and dichorionic twins instead of dividing the

MoM by a correcting factor [26]. Down’s Syndrome

combined test risk was performed using commercialized

software SsdwLab6, with the calculation of specific risk for

each fetus. Risk assessment of individual fetuses was provided

in dichorionic twins. In monochorionic twins, the mean risk

assessment of the two fetuses was used.

An invasive diagnostic procedure was offered when

the risk was 1:270 or over in either one of the fetuses. Fetal

chromosomal status was determined either by amniocentesis

or CVS when requested, or by phenotypic evaluation after

delivery by the attending paediatrician. Outcome variables

were retrieved from computerized medical record review. If

the pregnancy was not followed or/and delivered in our

centre, data were obtained by phone enquiry.

For the aim of this study, we recalculated the Down’s

syndrome risk of the 277 twin pregnancies included

in the study, using two more different strategies: ageþNT

and combined testþDVPI. For these calculations, we used

the same software SsdwLab6 applying different algorithms

for each case. However, the only screening test clinically

applied during the study period was the combined test.

Continuous variables were compared using Mann–Whitney

U-test. All tests are bilateral with a level of significance

a¼ 0.05.

Performance of the test was expressed as detection rate,

specificity, FPR, positive predictive value and negative

predictive value. FPRs were compared with the use of 95%

CI. Screening strategies in twins were compared using the

area under the receiver operating characteristic (ROC) curve

and its confidence interval.

Results

During the study period, 554 twin fetuses [502 (90.6%)

fetuses were dichorionic and 52 (9.3%) were monochorionic]

fulfilled the inclusion criteria. In 340 fetuses, we could not

measure the ductus venosus flow.

In the study group, the mean maternal age was 33.8� 4.8

SD years old, with 42.2% over 36 years. The mean gestational

age at the time of biochemistry sampling was 68.01� 7.99 SD

days. The mean CRL was 63.17 mm� 9.41 SD. The median

for free b-hCG expressed in MoM was 1.53 and the median

for PAPP-A expressed in MoM was 1.73. The median for NT

and DVPI expressed in MoM were 0.96 and 0.99, respect-

ively. In Table 1, we have shown the characteristics of the

patients distinguishing between monochorionic and dichor-

ionic twins.

Down’s syndrome was identified in two fetuses of two

different multiple pregnancies; both of them were dichorionic

twins and in patients older than 36 years old. In case I,

maternal age was 38 years old, the affected fetus had NT of

2.6 mm, CRL of 51 mm, DVPI of 3.07, free b-hCG concen-

tration at 8 weeks was 126.85mf/L, corrected MoM was 1.4,

PAPP-A was 301 mU/L and 0.93 corrected MoM and

calculated Down’s syndrome risk with the combined test

was 1/9. In case 2, maternal age was 37 years old, the affected

Table 1. Patients’ characteristics.

Dichorionic twins (n¼ 502) Monochorionic twins (n¼ 52) p Value

Mean maternal age (years� SD) 33.7� 4.9 34.2� 3.9 nsMean gestational age maternal serum sampling (days� SD) 67.9� 8.0 68.5� 7.6 nsMean maternal weight (kg� SD) 63.6� 11.3 58.5� 7.6 0.025Median b-hCG MoM 1.6 1.37 nsMedian PAPP-A MoM 1.79 1.32 0.0001Mean CRL (mm� SD) 63.6� 9.5 59.3� 11.2 0.012Median NT MoM 0.97 0.84 0.043Median DVPI MoM 0.99 0.92 ns

NT, nuchal translucency; MoM, multiples of the median; b-hCG, free beta-human chorionic gonadotrophin; PAPP-A, pregnancy-associated plasmaprotein A; DVPI, ductus venosus pulsatility index; SD, standard derivation; CRL, crown rump length; ns, no significance.

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fetus had NT of 3.2 mm, DVPI of 1.31, CRL of 72 mm, free-

b-hCG concentration at 8 weeks was 120.89 mf/L, corrected

MoM was 1.52, PAPP-A was 219.05 mU/L and 0.79 corrected

MoM and calculated Down’s syndrome risk with the

combined test was 1/34.

NT screening, the combined test and the combined test

with the addition of the DVPI for the detection of Down’s

syndrome results are shown in Tables 2 and 3. With NT

screening, 11.19% (62/554) had a high risk of Down’s

syndrome. Applying the combined test to our study popula-

tion, 6.49% (36/554) had a high risk of Down’s syndrome.

With the algorithm combined testþDVPI, 6.31% (35/554) of

fetuses had a high risk of Down’s syndrome.

If we focus in FPR, and we distinguish between patients

with advance maternal age or younger than 35 years old

(Table 4), the results showed a higher FPR in the group of

advanced maternal age, especially in the NT screening group.

If we distinguish between monochorionic and dichorionic

twins, the results are different depending on the test applied

(Table 5). NT screening, the combined test and the combined

test plus the addition of DVPI were effective screening

strategies in our population (Table 6).

Discussion

Different strategies for prenatal Down’s syndrome screening

in twins are now available. During the last decade, NT

screening has been the election test. Its greatest advantage is

that it provides a fetus-specific risk. Moreover, in high-order

multiple pregnancies in which biochemistry is not feasible,

NT screening can also identify the fetus at risk. The

sensitivity of NT screening is similar to that obtained in

singletons but with a higher FPR, probably due to a higher

prevalence of increased NT in fetuses from monochorionic

pregnancies [2]. In our series, NT screening identified the

two affected fetuses. In this study, the sensibility of this

marker is higher than the one reported before in the literature

(75%) [6], but this could be due to the low number of affected

fetuses in our present series. In this series, the FPR using

NT screening was 11.5 and 10.8% in monochorionic and

dichorionic twins, respectively. Median NT MoM was 0.84

and 0.97 in monochorionic and dichorionic twins, respect-

ively. In this series of twins, we did not find an increase

of NT measurement in the group of monochorionic twins;

on the contrary, in this series, it was lower than in dichorionic

twins. In the literature, other series found no differences

in NT measurements between twins and singletons [27] or

between monochorionic and dichorionic twins [4]. At follow

up, TTTS was not identified in any of the 26 monochorionic

pregnancies included in our study, but the number of cases

was too small to draw any conclusions. The estimation of two

different risks applied to each of the fetuses in monochorionic

pregnancies is not useful since, in most cases, both should be

concordant as affected or unaffected. There is some contro-

versy regarding the most appropriate approach to calculate

pregnancy risk by using the largest, the smallest or the

average of the two NT measurements [28,29]. In our study, we

used the largest one, in order to avoid a decrease in detection

rate.

In singleton pregnancies, the addition of first-trimester

biochemistry to NT assessment has been found to increase

the detection rate of trisomy 21, with low FPRs [30,31].

A study of 159 first trimester chromosomally normal twin

pregnancies using statistical modelling techniques predicted

that the addition of maternal biochemistry could increase

the detection rate by 5% without losing any of the benefits

of ultrasound screening [6]. Later on, in a prospective study,

the same author reported that the addition of first-trimester

biochemistry to NT assessment had a 75% detection rate for

9% of FPR of pregnancy and 6.8% of fetuses [13]. Madsen

et al. [15] published the largest series about the benefits of the

addition of the biochemical markers (free b-hCG and

PAPP-A) into the NT screening. They reported that adding

biochemistry into the risk assessment using a fixed risk

Table 3. NT screening, the combined test screening and the combined testþDVPI screening results, depending on the maternal age.

DR (%) FPR (%) PPV (%) NPV (%) OR (95% CI)

Maternal age (years) �35 436 �35 436 �35 436 �35 436 �35 436

MAþNTFetus – 100 2.5 22.4 – 3.7 100 100 – 963 (914–1015)Pregnancy 100 3.8 29.6 – 5.6 100 100 944 (873–1022)

Combined testFetus – 100 1.6 12.5 – 6.5 100 100 – 935 (853–1026)Pregnancy – 100 1.9 15.7 – 10 100 100 – 900 (778–1042)

Combined testþDVPIFetus – 100 1.9 11.6 – 6.9 100 100 – 931 (843–1028)Pregnancy – 100 1.9 18.3 – 8.7 100 100 – 913 (805–1036)

DT, detection rate; FPR, false-positive rate; PPV, positive predictive value; NPV, negative predictive value; OR, odds ratio;CI, confidence interval; DVPI, ductus venosus pulsatility index; NT, nuchal translucency; MA, maternal age.

Table 2. NT screening, the combined test screening and the combinedtestþDVPI global results.

�270 DR FPR PPV NPV OR (95% CI)

MAþNTFetus 62/554 100% 10.9% 3.2% 100% 968 (925–1013)Pregnancy 42/277 100% 14.5% 4.8% 100% 952 (890–1019)

Combined testFetus 36/554 100% 6.2% 5.6% 100% 944 (873–1022)Pregnancy 23/277 100% 7.6% 8.7% 100% 913 (805–1036)

Combined testþDVPIFetus 35/554 100% 6% 5.7% 100% 943 (869–1023)Pregnancy 26/277 100% 8.7% 7.7% 100% 923 (826–1031)

DT, detection rate; FPR, false-positive rate; PPV, positive predictivevalue; NPV, negative predictive value; OR, odds ratio; CI, confidenceinterval; DVPI, ductus venosus pulsatility index; NT, nuchal translu-cency; MA, maternal age.

DOI: 10.3109/14767058.2013.784252 Different strategies in screening for Downs’s syndrome in twins 3

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cut-off of 1 in 100 increased the detection rate for fetal

trisomy 21 in dizygotic twin pregnancies from 78 to 90% and

decreased the FPR from 8 to 5.9%.

In this series of 554 fetuses, the use of the combined test

for the screening of trisomy 21 enabled the detection of all

cases of Down’s syndrome with a FPR of 6.4% in dichorionic

fetuses and 3.8% in monochorionic fetuses (cut-off �1/270).

Wide CIs were observed around the FPR owing to the small

number of affected fetuses. There are few series in the

literature about the performance of the combined test in twin

pregnancies [10–15]. Our results are comparable with those

reported previously. There is a decrease in the FPR in the

latest studies: from 10.6% to 3.5%. The increase in the

specificity in the publications of Gonce et al. [10,12] and

in our present series could be due to the performance of the

combined test in two steps. We believe that the earlier

maternal serum sampling is performed, the more optimal

these markers seem to be for the screening of Down’s

syndrome [32]. The decrease in the FPR is important because

it leads to a decreased need of invasive procedure. Invasive

techniques are more difficult in twin pregnancies and are

associated with a higher risk of fetal loss.

Regarding the biochemical markers, b-hCG and PAPP-A

values in our series follow a slightly different distribution to

the ones published by Spencer [7]. We recommend that every

centre should know their own biochemical marker distribu-

tions in order to apply a correcting factor depending on the

chorionicity for the screening of Down’s syndrome in

twin pregnancies. We previously reported [9] the distribution

of the serum markers in monochorionic and bichorionic

twin pregnancies in our population. In our series, PAPP-A and

b-hCG MoM do not rise to twice that of unaffected

singletons. This deviation could be due to an earlier sampling

for biochemical markers in our two-step screening approach.

We found very few studies in the literature about the role

of additional sonography markers in trisomy 21 prenatal

screening in twins [19–21]. Maiz et al. [21] reported that in

twin pregnancies the prevalence of reversed a-wave in the

ductus venosus at 11–13 weeks of gestation is more common

in fetuses with aneuploidies and miscarriages, as previously

reported in singletons [33]. These authors also reported that

the prevalence of reversed a-wave in the ductus venosus was

higher in monochorionic twins, probably related with the

possibility of developing a TTTS. In our series, 5 fetuses

had reversed a-wave in the ductus venosus, all of them were

dichorionic twins. We did not find significant differences

in DVPI between monochorionic and dichorionic twins.

We think this might be because in our series, there are no

cases of TTTS. Studies in singleton pregnancies have shown

Table 4. False-positive rate per fetus and pregnancy by NT screening, the combined test and the combined testþDVPI screening depending on the ageof the mother.

FPR per fetus %(95% CI)

MAþNTscreening

Combinedtest

CombinedtestþDVPI

GlobalFetus 10.9 (95% CI 8.3–13.5) 6.2 (95% CI 4.1–8.2) 6 (95% CI 4.1–8.2)Pregnancy 14.5 (95% CI 10.4–18.7) 7.6 (95% CI 4.5–10.8) 8.7 (95% CI 5.4–12.1)�35 years old

Fetus 2.5 (95% CI 0.8–4.2) 1.6 (95% CI 0.2–2.9) 1.9 (95% CI 0.4–3.4)Pregnancy 3.8 (95% CI 0.8–6.7) 1.9 (95% CI 0–4) 1.9 (95% CI 0–4)

436 years oldFetus 22.4 (95% CI 17–27.8) 12.5 (95% CI 8.2–16.8) 11.6 (95% CI 7.5–15.8)Pregnancy 29.6 (95% CI 21–38) 15.7 (95% CI 8.9–22.4) 18.3 (95% CI 11.1–25.4)

FPR, false-positive rate; CI, confidence interval; DVPI, ductus venosus pulsatility index; NT, nuchal translucency; MA, maternal age.

Table 5. False-positive rate per fetus and pregnancy by NT screening, the combined test and the combined testþDVPI screening depending on thechorionicity.

FPR per fetus% (95% CI)

MAþNTscreening Combined test

CombinedtestþDVPI

GlobalFetus 10.9 (95% CI 0.83–1.35) 6.2 (95% CI 4.1–0.82) 6 (95% CI 4–8)Pregnancy 14.5 (95% CI 10.4–18.7) 7.6 (95% CI 4.5–10.8) 8.7 (95% CI 5.4–12.1)

MonochorionicFetus 11.5 (95% CI 2.6–20.5) 3.8 (95% CI 1.6–9.3) 3.8 (95% CI �1.6–9.3)Pregnancy 11.5 (95% CI 0–24.7) 3.8 (95% CI 0–11.8) 3.8 (95% CI 0–11.8)

DichorionicFetus 10.8 (95% CI 8.1–13.5) 6.4 (95% CI 4.2–8.6) 6.2 (95% CI 4.1–8.3)Pregnancy 14.9 (95% CI 10.4–19.3) 8 (95% CI 4.6–11.4) 9.2 (95% CI 5.6–12.9)

FPR, false-positive rate; CI, confidence interval; DVPI, ductus venosus pulsatility index; NT, nuchal translucency; MA, maternal age.

Table 6. ROC curve in NT screening, combined test and combinedtestþDVPI screening.

AUC 95% CI

MAþNT screening 0.987 0.972–0.994Combined test 0.987 0.978–0.997Combined testþDVPI 0.983 0.977–0.996

ROC, receiver operating characteristic curve; AUC, area under the curve;CI, confidence interval; DVPI, ductus venosus pulsatility index; NT,nuchal translucency; MA, maternal age.

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that abnormal flow in the ductus venosus is a good marker

for aneuploidies and improves the performance of first-

trimester screening for trisomy 21 provided by the combin-

ation of maternal age, fetal NT thickness, and maternal serum

free b-hCG and PAPP A [31,34–36]. In our study of twins, the

assessment of ductus venosus (DV) flow slightly improved the

prediction of Down’s syndrome provided by the combined

test: FPR decreased from 6.2 to 6%. Nevertheless, this twin

series is too small in comparison with that of singletons

previously reported, to provide an accurate prediction of the

estimated improvement in the performance of the screening

with the inclusion of ductus venosus flow.

All these three screening tests appeared to be effective in

multiple pregnancies in our series as the ROC curve shows.

However, the worse result is achieved with the addition of the

extra sonographic marker (Table 6). In view of these results,

we might conclude that the most efficient screening test for

trisomy 21 in twins is the combined test. It maintains the same

detection rate as NT screening and improves the FPR,

decreasing it approximately 6%, especially in two specific

groups: monochorionic twins, where the FPR decreases from

11.5 to 3.8% and in the advanced maternal age group, where

the FPR decreases from 22 to 12%. The addition of the

additional marker slightly improved the FPR in dichorionic

twins but not in monochorionic twins. We should not forget

that screening for DV is not easy. In our series, we could

not achieve an appropriate DVPI measure in 340 fetuses

(61.3%) and all these cases were excluded from the study.

There are some requirements clearly described by The

Fetal Medicine Foundation that need to be fulfilled in order

to incorporate the DV flow to the Down’s syndrome

screening. These requirements need some previous training

and people performing theses scans should be skilled

in prenatal ultrasound scanning [22]. Moreover, scanning is

more challenging and time consuming in twin pregnancies

than in singletons.

The small number of affected fetuses is the main limitation

of this study. Although we describe a 100% detection rate

with all three analysed screening test, we would anticipate a

lower detection rate with a larger number of affected

pregnancies. Larger studies with more affected fetuses are

needed to confirm our data.

Acknowledgements

This study was conducted under the auspices of the Catedra d’

Investigacio en Obstetrıcia i Ginecologia de la Universitat

Autonoma de Barcelona. Pau Castaneda and Jose Sabria

collaborators in the software management.

Declaration of interest

No conflict of interest declared.

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