Firsttrimester screening for chromosomal ... - Wiley Online Library

Ultrasound Obstet Gynecol 2012; 39: 528–534 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/uog.10051

First-trimester screening for chromosomal abnormalities by integrated application of nuchal translucency, nasal bone, tricuspid regurgitation and ductus venosus flow combined with maternal serum free β-hCG and PAPP-A: a 5-year prospective study S. R. GHAFFARI*†‡§#, A. R. TAHMASEBPOUR*#, A. JAMAL*¶, S. HANTOUSHZADEH*§¶, L. ESLAMIAN*¶, V. MARSOOSI*¶, F. FATTAHI*, M. RAJAEI*, S. NIROOMANESH*¶, S. BORNA*¶§, A. BEIGI*, S. KHAZARDOOST*§¶, S. SALEH-GARGARI*, F. RAHIMI-SHARBAF*¶, B. FARROKHI*, N. BAYANI*, S. E. TEHRANI*, K. SHAHSAVAN*, S. FARZAN*, S. MOOSSAVI*, F. RAMEZANZADEH§, J. DASTAN‡ and M. RAFATI*†§ *Iranian Fetal Medicine Foundation, Hope Generation Foundation, Tehran, Iran; †Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran; ‡Gene Clinic, Tehran, Iran; §Vali-e-Asr Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran; ¶Perinatology Division, Department of Obstetrics and Gynecology, Tehran University of Medical Sciences, Tehran, Iran

K E Y W O R D S: chromosomal abnormalities; ductus venosus flow; first-trimester screening; nasal bone; nuchal translucency; tricuspid regurgitation

ABSTRACT Objective To investigate the performance of firsttrimester screening for chromosomal abnormalities by integrated application of nuchal translucency thickness (NT), nasal bone (NB), tricuspid regurgitation (TR) and ductus venosus (DV) flow combined with maternal serum free β-human chorionic gonadotropin (fβ-hCG) and pregnancy-associated plasma protein-A (PAPP-A) at a one-stop clinic for assessment of risk (OSCAR). Methods In total, 13 706 fetuses in 13 437 pregnancies were screened for chromosomal abnormalities during a period of 5 years. Maternal serum biochemical markers and maternal age were evaluated in combination with NT, NT+NB, NT+NB+TR, and NT+NB+TR+DV flow data in 8581, 242, 236 and 4647 fetuses, respectively. Results In total, 51 chromosomal abnormalities were identified in the study population, including 33 cases of trisomy 21, eight of trisomy 18, six of sex chromosome abnormality, one of triploidy and three of other unbalanced abnormalities. The detection rate and false-positive rate (FPR) for trisomy 21 were 93.8% and 4.84%,

respectively, using biochemical markers and NT, and 100% and 3.4%, respectively, using biochemical markers, NT, NB, TR and DV flow. Conclusion While risk assessment using combined biochemical markers and NT measurement has an acceptable screening performance, it can be improved by the integrated evaluation of secondary ultrasound markers of NB, TR and DV flow. This enhanced approach would decrease the FPR from 4.8 % to 3.4 %, leading to a lower number of unnecessary invasive diagnostic tests and subsequent complications, while maintaining the maximum level of detection rate. Pre- and post-test genetic counseling is of paramount importance in either approach. Copyright  2012 ISUOG. Published by John Wiley & Sons, Ltd.

INTRODUCTION Chromosomal abnormalities are a leading cause of developmental delay in children. Trisomies 21, 18 and 13 and sex chromosome aberrations are the most frequently occurring chromosomal abnormalities. Definitive prenatal

Correspondence to: Dr S. R. Ghaffari, Iranian Fetal Medicine Foundation, Hope Generation Foundation, Tehran, Iran (e-mail: [email protected]) #S.R.G. and A.R.T. contributed equally to this work. Accepted: 6 July 2011

Copyright  2012 ISUOG. Published by John Wiley & Sons, Ltd.

ORIGINAL PAPER

First-trimester screening for chromosomal abnormalities diagnosis currently requires invasive sampling followed by chromosome analysis. However, invasive tests are costly and pose an inherent risk of procedure-related complications including miscarriage. Therefore, following appropriate screening, invasive tests should be performed only in high-risk pregnancies. Screening based on advanced maternal age alone or in combination with second-trimester serum analyses has been established previously with detection rates of 30% and 60–84% for a false-positive rate of 5%, respectively1,2 . However, late diagnosis of chromosomal abnormalities and pregnancy termination at an advanced gestational age could incur unnecessary anxiety for families. In addition, women prefer to undergo firsttrimester screening if the choice is available3 . First-trimester risk assessment of common chromosomal aneuploidies is based on a combination of maternal age, maternal serum-free β-human chorionic gonadotropin (fβ-hCG), pregnancy-associated plasma protein-A (PAPP-A) and fetal nuchal translucency thickness (NT) at 11 to 13 + 6 weeks. In various prospective studies the detection rate ranges from 74% to 93% for a fixed false-positive rate of 5%2,4 – 10 . Additional firsttrimester ultrasound markers, namely absent nasal bone (NB), reversed ductus venosus (DV) flow and tricuspid regurgitation (TR), have separately been found to increase the effectiveness of trisomy 21 screening11 – 13 . In two-stage risk-oriented first-trimester screening, assessment of NB, TR or DV flow in the group of women with intermediate risk according to maternal age, NT and maternal serum biochemistry resulted in detection rates of 92%, 91.7% and 94.2%, respectively, and false-positive rates of 2.1%, 2.7% and 2.7%, respectively14 . The combined application of all four of these ultrasound markers together with maternal age and maternal serum biochemistry in first-trimester screening has not yet been reported. However, Geipel et al.15 performed screening with a set of four markers in the early second trimester. In their study, increased NT, absent or hypoplastic NB, TR and reversed DV flow were observed in 32.4%, 45.9%, 27% and 24.3% of trisomy 21 fetuses, respectively. The combination of all four markers allowed detection of 75.7% of Down syndrome cases with a false-positive rate of 10.8%15 . Here we report on a 5-year prospective study to assess the performance of first-trimester screening for chromosomal abnormalities using secondary ultrasound markers (NB, TR and DV flow) in combination with conventional biochemical (fβ-hCG and PAPP-A) and ultrasound (NT) markers.

METHODS This prospective interventional study was performed between February 2006 and October 2010. The study was approved by the Ethics Committee of Tehran University of Medical Sciences. A total of 13 437 pregnant women were referred to our center by their obstetricians. The first antenatal visit took place at 11 to 13 + 6 weeks’


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gestation according to last menstrual period (LMP). At the first visit, women received prescreening genetic counseling and an information booklet. They were informed of the limitations of the screening method and informed consent was obtained. Maternal details were entered into an internally networked database (Astraia, Munich, Germany). A clotted blood sample was obtained. The serum was separated and PAPP-A and fβ-hCG were measured on the same day using a random access immunoassay analyser (Kryptor, Brahms Diagnostica GmbH, Berlin, Germany (formerly CIS)) in 13 354 cases and electrochemoluminescence (Elecsys 2010, Roche Diagnostics, Indianapolis, IN, USA) in 83 cases. The ultrasound examination took place at 11 to 13 + 6 weeks and was performed by operators certified by The Fetal Medicine Foundation (FMF) (11 obstetricians, three radiologists and one technician). In all pregnancies a scan was performed to rule out major anomalies, and crown–rump length (CRL) and NT were measured according to FMF (London) guidelines16 . Fetuses with a CRL measurement of 45–84 mm were included in the study. A satisfactory NT result was obtained in all but four cases and these were excluded from the final analysis. Nasal bone status, tricuspid regurgitation and DV flow (qualitative assessment) were investigated when at least one operator was certified for assessment. When possible, women were assessed for all four ultrasound markers unless they decided otherwise. In total, NB, TR and DV flow were investigated in 4647 pregnant women using standard methods17 – 19 . Doppler ultrasound examination was conducted by FMF-certified operators and strictly according to FMF (London) protocols. Briefly, we followed the ALARA (as low as reasonably achievable) principle. During Doppler ultrasound examination, both mechanical and thermal indices were displayed on the monitor. In all cases, thermal index for soft tissue (TIS) and mechanical index (MI) were kept below 1.0 and 0.5, respectively. Triplet or higher-order multiple pregnancies were excluded from subsequent biochemical analysis and risk assessment. Results of ultrasound findings and maternal serum biochemical analysis were entered into the database. Biochemical results were converted into multiples of the median (MoM) values using software and were adjusted for maternal weight, parity, ethnicity, smoking status and mode of conception. The Feto-maternal module of the Astraia software (version 1.18.0 88) was employed for first-trimester risk assessment according to the FMF (London) algorithm. In twin pregnancies, risk calculation was also based on maternal age, serum biochemistry and ultrasound findings. In dichorionic twins, risk was separately calculated for each fetus, whereas in monochorionic (monoamniotic or diamniotic) twins, an average risk was generated for both fetuses. The software underwent minor changes during the study according to the FMF algorithm upgrade. Adjusted risk was based on existing ultrasound markers for each woman. A calculated risk of equal to or higher than 1 : 300 was defined as ‘high-risk’. Risk assessment was done on the day of visit and post-screening counseling with a clinical geneticist

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was scheduled 1–2 days later for couples with high-risk pregnancies. A perinatologist was consulted if indicated and amniocentesis or chorionic villus sampling was offered. The invasive test was performed by the women’s referring obstetricians. An acknowledgment of receipt of the information was obtained from all women. The results of chromosome analysis using rapid aneuploidy detection tests (quantitative fluorescent polymerase chain reaction (QF-PCR) or multiplex ligation-dependent probe amplification (MLPA)) and conventional karyotyping were available in 2–3 days and 2–3 weeks, respectively. The women with high-risk pregnancies who declined diagnostic tests were followed up for outcome of pregnancy by contacting their obstetricians. The group of women with low-risk pregnancies consented to report the occurrence of an affected child at birth. Since early pregnancy loss and spontaneous miscarriage can be due to chromosomal abnormalities, we karyotyped the aborted material whenever possible. The descriptive analysis of results was performed using Microsoft Office Excel 2007.

RE SULTS Overall, 13 437 pregnant women in the first trimester were screened (13 168 singleton and 269 twin pregnancies). In total 13 706 fetuses were evaluated for risk of chromosomal abnormalities. Median maternal age was 29 (range, 14–52) years and 2824 (20.60%) women were 35 years of age or older. Maternal age distribution in the study population and in the general Tehran population (2009–2010)20 is presented in Figure 1. Demographic characteristics of the study population are summarized in Table 1. Overall, 653 (4.76%) fetuses had an estimated risk equal to or more than 1/300 (633 singleton, 10 twin). In 325 (49.77%) high-risk cases the maternal age was 35 years or older. In this interventional study we identified 51 cases of chromosomal abnormality: 33 of trisomy 21, eight of trisomy 18, three of Turner syndrome (or Turner

Table 1 Demographic characteristics of studied population (13 437 women, 13 706 fetuses) Variable

Median (range) or n (%)

Maternal age ≥ 35 years ≥ 39 years Mode of conception* Spontaneous Assisted Not reported Smoking status* Smoker Non-smoker Not reported Ethnicity* Caucasian Other Previous affected pregnancy*† GA at screening 11 12 13 14 Crown–rump length (mm)

29 (14–52) 2824 (20.60) 809 (5.90) 11 125 (81.17) 1427 (10.41) 1154 (8.42) 77 (0.56) 13 578 (99.07) 51 (0.37) 13 694 (99.91) 12 (0.09) 71 (0.52) 2172 (15.85) 6471 (47.21) 4752 (34.67) 311 (2.27) 64 (45.0–84.0)

*Variables incorporated in risk estimation algorithm. †Includes 61 cases of trisomy 21, nine cases of trisomy 18 and one case of trisomy 13. GA, gestational age.

Table 2 Prevalence of various chromosomal abnormalities in 51 aneuploid fetuses and number estimated to be at high risk of having a chromosomal abnormality

Chromosomal abnormality

n

Risk ≥ 1 : 300 (n)

Trisomy 21 Trisomy 18 Turner syndrome (or variant)* Sex chromosomal abnormalities† Triploidy Other

33 8 3 3 1 3

32 8 3 3 1 3

Total

51

50

*45,X(26),46,X,+mar(3)/46,X,i(Xq)(1). †47XXY; 47XXX; 46XX(26), 47XX+mar(3)/45X(1).

40 35

Proportion (%)

30 25 20 15 10 5 0

< 20

20–24

25–29 30–34 35–39 Maternal age (years)

≥ 40

Figure 1 Maternal age distribution in the study population ( ) and the general population of Tehran ( )20 .


variant), one of triploidy, three sex chromosomal abnormalities and three other abnormalities (Table 2). Overall, 29 out of 51 (56.80%) identified chromosomal abnormalities were found in women over 35 years of age: 21/33 trisomy 21 cases and 8/8 trisomy 18 cases. Demographic and ultrasound features of normal pregnancies compared to affected pregnancies are presented in Table 3. Absent or hypoplastic NB, TR and reversed a-wave of the DV were reported in 58.82%, 43.75% and 53.33% of trisomy 21 cases, respectively. One case of trisomy 21 had an estimated risk of 1/562 when maternal age, maternal serum biochemistry and NT measurement data were combined. Thus, it was not identified as high-risk in this study. The maternal age was 43 years, NT thickness was 1.70 mm, fβ-hCG


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Table 3 Demographic and ultrasound features of normal and affected pregnancies Chromosomal abnormality Characteristic Maternal age (years, median (range)) Median GA (weeks) Median CRL (mm) Caucasian (%) Median NT (mm) Absent nasal bone (n (%)) Tricuspid regurgitation (n (%)) Reversed DV flow (n (%))

Trisomy 21 (n = 33)

Trisomy 18 (n = 8)

Other (n = 10)

Euploid (n = 13 655)

36.0 (22–45) 12 + 3 65.9 100.0 3.5 10/17 (58.82) 7/16 (43.75) 8/15 (53.33)

38.0 (28–45) 12 + 2 58.8 100.0 3.1 1/2 (50.0) 1/2 (50.0) 1/2 (50.0)

32.0 (23–45) 12 + 2 64.9 100.0 3.0 2/2 (100.0) 1/2 (50.0) 1/2 (50.0)

29.0 (14–52) 12 + 3 64.0 99.91 1.7 88/5104 (1.72) 58/4863 (1.19) 48/4628 (1.04)

CRL, crown–rump length; DV, ductus venosus; GA, gestational age; NT, nuchal translucency thickness. Table 4 Detection rates and false-positive rates of individual biochemical and ultrasound markers in the present study Detection rate (n (%)) for: Screening mode MA ≥ 35 years MA ≥ 39 years fβ-hCG ≥ 2.5 MoM PAPP-A < 0.45 MoM NT ≥ 2.5 mm Abnormal nasal bone Tricuspid regurgitation Reversed DV flow

n

Risk ≥ 1 : 300 (n)

Trisomy 21

Trisomy 18

FPR (%)

2824 809 850 886 824 101 67 58

325 171 168 204 294 71 39 30

21/33 (63.64) 10/33 (30.30) 12/33 (36.36) 7/33 (21.21) 23/33 (69.70) 10/17 (58.82) 7/16 (43.75) 8/15 (53.33)

5/8 (62.50) 4/8 (50.0) 0 8/8 (100.0) 5/8 (62.50) 1/2 (50.0) 1/2 (50.0) 1/2 (50.0)

20.47 5.81 6.11 6.33 5.92 1.72 1.19 1.04

DV, ductus venosus; fβ-hCG, free beta human chorionic gonadotropin; MA, maternal age; NT, nuchal translucency thickness; PAPP-A, pregnancy-associated plasma protein-A. Table 5 Effectiveness of first-trimester screening using ultrasound markers in combination with maternal age and biochemical serum markers Detection rate (n (%)) for: US marker NT NT and NB NT and NB and TR NT and NB and TR and DV

n

Risk ≥ 1 : 300 (n)

Trisomy 21

8581 242 236 4647

440 28 8 177

15/16 (93.75) 1/1 (100) 1/1 (100) 15/15 (100)

Trisomy 18

Other chr. abn.

6/6 (100) 0 0 2/2 (100)

8/8 (100) 0 0 2/2 (100)

FPR (%) 4.84 11.20 2.97 3.44

chr. abn., chromosomal abnormality; DV, ductus venosus; FPR, false-positive rate; NB, nasal bone; NT, nuchal translucency thickness; TR, tricuspid regurgitation; US, ultrasound.

MoM was 1.54 and PAPP-A MoM was 1.09. A second case of trisomy 21 with the risk of 1/8 was identified postnatally because the mother declined invasive testing. There was no case of unidentified chromosomal abnormality in the group of women in which the full set of ultrasound markers was incorporated in the risk assessment strategy. With a total of 2824 (20.60%) women 35 years or older and, according to this cut-off of maternal age alone, 21/33 (63.64%) cases of trisomy 21 would be detected with a false-positive rate of 20.47%. Detection and false-positive rates change to 30.30% and 5.81%, respectively, using a maternal age cut-off of 39 years. In the studied population, NT thickness measurement of ≥ 2.5 mm would


detect 23/33 (69.70%) cases of trisomy 21 with a falsepositive rate of 5.92% (Table 4). Combined risk estimation based on maternal age, NT thickness and biochemical markers allowed detection of 15/16 (93.75%) cases of trisomy 21, 6/6 (100%) of trisomy 18 and 8/8 (100%) of other chromosomal abnormalities with a false-positive rate of 4.84%. Integrating NB status, TR and DV flow in the risk estimation algorithm allowed detection of 15/15 (100%) cases of trisomy 21, 2/2 (100%) of trisomy 18 and 2/2 (100%) of other abnormalities with a false-positive rate of 3.44% (Table 5). It is noteworthy that fewer miscellaneous chromosomal abnormalities were detected in the group in which risk estimation was based on the full set of


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ultrasound markers. Risk estimation was based on the addition of NB status in 242 and NB + TR in 236 women with the detection rate of 1/1 cases of trisomy 21 in each group and no other abnormalities, with false-positive rates of 11.20% and 2.97%, respectively. In the pooled data analysis, we identified 32/33 cases of trisomy 21 and 18/18 other abnormalities with a false-positive rate of 4.45%. Analyzing each marker separately: absent or hypoplastic NB, TR and reversed DV flow had detection rates of 58.82%, 43.75% and 53.33% for trisomy 21 with falsepositive rates of 1.72%, 1.19% and 1.04%, respectively.

DISCUSSION In this prospective study of first-trimester screening for chromosomal abnormalities by a combination of maternal serum biochemical markers and ultrasound markers in 13 706 fetuses, 51 chromosomal abnormalities including 33 cases of Down syndrome, eight of trisomy 18 and 10 of other chromosomal aberrations were identified, with a detection rate of 98.0% and false-positive rate of 4.45%. The total prevalence of Down syndrome at the time of amniocentesis in the study population was 24.08/10 000 (1/416.6). As it is estimated that approximately 30% of cases of trisomy 21 will be spontaneously lost after 16 weeks21 , the adjusted prevalence of liveborn fetuses with Down syndrome in the studied population without screening would be 16.85/10 000 (1/593). The reported crude live birth rate of fetuses with this syndrome is 1/826 which is lower than our finding22 . This difference can be explained by the fact that the over 35-year-old population in our study was approximately twice that of the general population. As women were referred to our clinic by their obstetricians, it is likely that obstetricians are biased in referring older women. Using our screening protocol, there was one falsenegative diagnosis of Down syndrome, in a 43-year-old woman that could have been detected if amniocentesis

had been performed according to the old protocol of invasive testing in all mothers of more than 35 years of age. A policy of screening by maternal age would have resulted in an FPR of 20.5%, detected 21/33 cases of Down syndrome and resulted in the miscarriage of 28 normal fetuses (presuming a 1% miscarriage risk with amniocentesis). With a fixed false-positive rate of c. 5% (maternal age > 39 years), these figures would change the rate to 10/33 cases of Down syndrome, leaving 23 affected fetuses undetected. An overall detection rate of 96.97% and false positive rate of 4.45% for trisomy 21 was achieved using combined first-trimester screening tests (93.8% and 4.84% using biochemical markers and NT and 100% and 3.4% using biochemical markers, NT, NB, TR and DV). In this study, absent or hypoplastic NB, TR and reversed DV flow was observed in 58.82, 43.75 and 53.33% of cases of Down syndrome and 1.72%, 1.19% and 1.04% of unaffected pregnancies, respectively. In previous studies absent or hypoplastic NB, TR and reversed DV flow was observed in 59.8–66.9%, 55.7–67.5% and 58.7–71% of cases of trisomy 21 and 0.6–2.8%, 0.9–8.5% and 3.2–16% of normal fetuses, respectively12,13,18,19,23 – 30 . Previous studies have shown that incorporating other first-trimester ultrasound markers, i.e. NB11,23 , TR13,31 and DV flow12,32 in the risk estimation strategy would enhance the detection rate while reducing the falsepositive rate. This is the first report of integrating all three additional first-trimester ultrasound markers in the risk assessment of chromosomal abnormalities. Incorporating the full set of NB, TR and DV flow in the risk assessment strategy in this study increased the detection rate for trisomy 21 to 100% while reducing the false-positive rate to 3.44%, leading to a decreased number of invasive tests. However, the 100% detection rate in this study should be interpreted with caution and future studies investigating more pregnancies are required to produce more precise estimation of the screening performance of this protocol.

Table 6 Comparison of screening performance of our study compared with that in nine other major studies Reference Bindra et al.7 (2002) Spencer et al.8 (2003) Wald et al.2 (2003) Cicero et al.26 (2003) Kagan et al.23 (2009) Borrell et al.32 (2005) Maiz et al.12 (2009) Falcon et al.31 (2006) Kagan et al.13 (2009) This study

n

GA (weeks)

15 030 12 399 47 053 500 19 800 3087 19 800 309 19 800 8581

11–14 10 + 4 to 13 + 6 10–14 11–14 11–14 10–14 11–13 11–14 11–14 11–14

4647

11–14

13 706

US marker NT NT NT NT, NB NT, NB NT, DV NT, DV NT, TR NT, TR NT NT, NB, TR, DV All groups

T21 DR (%)

FPR (%)

91.5 92 85 97.1 91 92 96 95 96 93.8 80* 100 90* 96.97 84.3*

5 5 6 5 2.5 5 3 5 3 4.84 3 3.44 3 4.45 3

*Detection rate for a fixed false-positive rate of 3%. DR, detection rate; DV, ductus venosus; FPR, false-positive rate; GA, gestational age; NB, nasal bone; NT, nuchal translucency thickness; T21, trisomy 21; TR, tricuspid regurgitation; US, ultrasound.



First-trimester screening for chromosomal abnormalities Several cases of other chromosomal abnormalities were also detected, suggesting that this method of screening may be applicable in screening for such abnormalities. The results of this study are compared with previously published reports in Table 6. As shown, compared to other studies, an acceptable detection rate and false-positive rate were achieved when combining first-trimester screening with NT measurement. However, when secondary ultrasound markers were added, an improvement in both detection rate and false-positive rate (DR, 100%and FPR, 3.44%) was obtained. Risk assessment was based on maternal age, maternal serum biochemistry, NT and NB in 242 women and on NT, NB and TR in 236 women. We detected 1/1 (100%) case of trisomy 21 in each group with falsepositive rates of 11.20% and 2.97%, respectively. Because of the limited number of women and cases of trisomy 21 in these groups, the results should be interpreted with caution. Analysis of detection rate and FPR of each individual biochemical and/or ultrasound marker are shown in Table 4. Aggregated data demonstrate that NT thickness (> 2.5 mm) is the single most effective marker with a detection rate of near 70% (and a false-positive rate of 5.9%). While individual biochemical markers have low detection rate, combined biochemical markers yield a DR of 69% at a FPR of 5.9%. This finding suggests that standard NT measurement can be applied when other complementary infrastructures are not available. In conclusion, although the standard first-trimester screening approach using combined biochemical markers and NT measurement has an acceptable performance, it can be improved by the integrated application of secondary ultrasound markers (NB, TR and DV). This approach would decrease the false-positive rate from 4.8% to 3.4%, leading to a lower number of unnecessary invasive diagnostic tests and consequent complications, while maintaining the maximum detection rate. Pre- and post-test genetic counseling is of paramount importance in either approach.

ACKNOWLEDGMENT We thank The Fetal Medicine Foundation (London) and Professor K. H. Nicolaides for providing training, risk assessment software and external audit of the work.

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