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Challenges in prenatal screening and diagnosis in the NetherlandsBakker, Merel

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Challenges in

Prenatal Screening

and Diagnosis

in the Netherlands

Merel Bakker

Merel Thesis.indd 3 23-12-16 18:22

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van

de rector magnificus, prof.dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

maandag 6 februari 2017 om 14.30 uur.

door

Merel Bakker

geboren op 10 augustus 1981te Purmerend

Challenges in

Prenatal Screening

and Diagnosis

in the Netherlands


Promotor

Prof. dr. C. M. Bilardo

Copromotor

Dr. E. Birnie

Beoordelingscommissie

Prof. dr. S. A. ScherjonProf. dr. A. RanchorProf. dr. I. M. van LangenProf. dr. O. B. Petersen


Voor de Wetenschap

For Science

Per la Scienza


Colofon

Bakker, M.

Challenges in Prenatal Screening and Diagnosis in the Netherlands

Dissertation University of Groningen

ISBN: 978-90-367-9297-4 (printed version)ISBN: 978-90-367-9296-7 (electronic version)

© 2016 M. Bakker. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, mechanically, by photocopying, recording, or otherwise, without the written permission of the author.

Design and lay-out: Niels de RijkIdea cover design: Merel Bakker (with premitted use of a photo made by Steven O’Connor MD, Pathologist Houston, Texas, USA)Printing: Ipskamp drukkers

This thesis was realized independently of finances from industry.


Contents

chapter 1 General introduction

chapter 2 Low uptake of the combined test in the Netherlands – which factors contribute?

Prenatal Diagnosis 2012 Dec; 32(13): 1305-12

chapter 3 Intra- and inter-operator reliability of manual and semiautomated measurement of fetal nuchal translucency: a cross-sectional study

Prenatal Diagnosis 2013 Dec; 33(13): 1264-71

chapter 4 Total pregnancy loss after chorionic villus sampling and amnio-centesis in the Netherlands: a cohort study

Ultrasound Obstetrics & Gynecology. 2016 Jun 3. doi: 10.1002/uog.15986. [Epub ahead of print]

chapter 5 Increased nuchal translucency with normal karyotype and anomaly scan: what next?

Best Practice & Research Clinical Obstetrics & Gynaecology 2014 Apr; 28(3): 355-66.

chapter 6 Targeted ultrasound examination and DNA testing for Noonan

syndrome, in fetuses with increased nuchal translucency and normal karyotype

Prenatal Diagnosis 2011 Sep; 31(9): 833-40

chapter 7 Prenasal thickness, prefrontal space ratio and other facial profile markers in first trimester fetuses with aneuploidies, cleft palate and micrognathia

Fetal Diagnosis & Therapy, 2016 Nov 18. [Epub ahead of print]

chapter 8 Is 3D technique superior to 2D in Down syndrome screening? Evaluation of six second and third trimester fetal profile markers

Prenatal Diagnosis 2015 Mar; 35(3): 207-13.

chapter 9 General discussion and future perspectives

chapter 10 English summary Dutch summary

Appendix Abstracts List of Publications

Research Institute SHARE Curriculum Vitae Dankwoord

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General introduction

M. Bakker


1General introduction

M. Bakker


12

General introduction

M. Bakker

Prenatal screening in the Netherlands: Introduction of a national screenings program

“Pregnancy care” consists of care around pregnancy and birth, starting already from the preconception period. During pregnancy future parents can opt for prenatal screening in order to let assess the risk of congenital anomalies in their fetus. A distinction should be made between prenatal screening in a low risk population and prenatal diagnosis in a high risk population. Prenatal screening does not give a definitive diagnosis but it gives an estimate of the probability that a fetal anomaly will be present. In contrast, prenatal diagnosis confirms or excludes the suspected problem in a pregnancy. Pregnant women may opt for prenatal diagnosis because in case of specific congenital anomalies (e.g. heart anomalies) it can be beneficial for the child to be born in a tertiary center, where postnatal care can be optimized for the baby. Alternatively, in case of congenital anoma-lies parents have the option of termination of the pregnancy. In the Netherlands the national prenatal screenings program was introduced in Janu-ary 2007. This includes the combined test (CT) which is on average performed at around 12-13 weeks of gestation and the structural anomaly scan performed at around 20 weeks of gestation. As a consequence all pregnant women are nowadays asked during antenatal visit by their caregiver if they want to be informed about prenatal screening. Only if they opt to be informed they will be counseled by their midwife or gynaecologist, or rarely by the general practitioner. This set-up was chosen to guarantee the right of ‘not to know’. Based on the information provided, parents are expected to be able to make an informed choice on whether or not they want to opt for prenatal screening (RIVM).1 The 20 week anomaly scan was officially introduced as screening for neural tube de-fects, although women are informed beforehand that other structural anomalies can also be encountered.2 The uptake of this scan is high, more than 95 percent, which is compa-rable to other European countries (RIVM 2011). The CT is based on the measurement of the fetal nuchal translucency (NT, see Figure 1), maternal age and maternal serum markers (ß-HCG and PAPP-A) and has a detection rate of about 90% for a false positive rate of 5%.3,4,5,6-8 The CT was introduced initially as risk-assessment for trisomy 21 only, but from May 2010 the screening was extended to include also trisomy 13 and 18.3 During this scan, besides chromosomal abnormalities, non-chromosomal abnormalities can be found. Women/couples are not systematically informed about the possibility of finding structural abnormalities at the time of the CT. Nor are they informed that an enlarged NT can also be associated with structural abnor-malities, especially cardiac defects. In contrast to the 20 week anomaly scan, the uptake of the CT in the Netherlands is low, around 25-30% and this varies among different regions. The CT uptake in recent years is even lower than the uptake of invasive prenatal diagnosis in women aged 36 or older before the national screenings program was introduced.9,10 A study performed in


Chapter 1

13

the Netherlands before the advent of the national screening program and involving the offer of NT screening free of charge to pregnant women attending a number of commu-nity midwife practices, showed a much higher uptake of the CT i.e. 86%. The large major-ity of women, including those who declined the offer, were in favor of its standard offer.9 At the start of the national screenings program an age-related reimbursement policy was introduced for the CT: women aged 36 years and older had free access to the test whilst younger women had to pay approximately 150 euros. In contrast, all women had and still have free access to the structural anomaly scan. After a plea of professional orga-nizations to abolish this age-related access to prenatal screening, all women now have to pay for the CT.

The nuchal translucency plays an important role in prenatal screening. It consists of a subcutaneous accumulation of fluid behind the neck of the fetus and is generally visible by ultrasound between 11 and 14 weeks of gestation. The NT is part of normal develop-ment and its size is influenced by gestational age.11 It is considered abnormal only when it exceeds a certain cut-off.3 Many different definitions and cut-offs for an increased NT have been used in the past.12 Although debate continues as to which cut-off should be used to offer further ultrasound investigation, the 95th or the 99th centile. Currently the second one (3.5 mm) is considered as an ultrasound abnormality. Measurement of the nuchal translucency is performed by certified and skilled sonographers, but it still remains a difficult measurement to perform accurately.13,14 To further improve the detec-tion rate of trisomies, but especially to lower the false positive rate, the ductus venosus, tricuspid valve Doppler and the nasal bone were added in (1998-2001) as additional markers to the combined test.15-23 Once a high risk of a trisomy is found after the combined test (>1:200) or when there is suspicion of a structural anomaly, women are counseled for prenatal diagnosis. Whether or not they opt for prenatal diagnosis depends for most women on the procedure related risk.24

Figure 1 — Nuchal Translucency (NT)


14

1. LOW UPTAKE OF THE COMBINED TEST

Although the CT is offered to all the women in the Netherlands, its uptake is low, and varies among different regions, suggesting that possible cultural differences in attitude towards Down syndrome and termination of pregnancy (TOP) may play a role.9,10,25 Furthermore, the difference in uptake in comparison with other European countries with a national screening program, such as Denmark (>90%) or with regional screening policies such as England and France (88%)26-29 is striking. In contrast to other screen-ing policies, the aim of the Dutch prenatal screening is not to reach the highest uptake as possible, but to make sure that future parents make an informed decision regarding screening. So it is important to find out what possible reasons are for the low uptake of the CT in the Netherlands.

2. IMPROVING THE QUALITY OF THE NUCHAL TRANSLUCENCY MEASUREMENT

The NT is the most effective marker of trisomy 21 and is able to detect about 75-80% of the affected fetuses for a false positive rate of about 3-5%.6,7 Moreover, an enlarged NT is associated with other chromosomal anomalies, genetic syndromes and structural anomalies.30 It is therefore important that the NT is measured precisely. The Fetal Medi-cine Foundation (FMF) has developed international guidelines to promote a standardized measurement technique aimed at obtaining accurate manual NT measurements. Aim of the guideline is to achieve uniformity among different operators and guarantee a valid risk assessment.31 However, the acquirement of the correct midsagittal plane, the selec-tion of the area containing the maximum NT and the placement of the calipers are still prone to error with the manual technique, which may compromise the performance of screening.13,14,32 Quality control programs performed in the setting of prenatal screening indicate that some sonographers tend to underestimate the NT-measurements, probably trying unconsciously to avoid unfavorable risk assessment results. In this respect the introduction of a technical tool aimed at standardizing the NT measurement may reduce such a measurement error. In order to minimize variability in the measurement of the NT, a semi-automated NT measurement has been developed recently (sono-NT, GE Medi-cal Systems). Standardization through semi-automated measurement is thought to lower the standard deviation (SD) of the distribution of NT measurements, increase its preci-sion, and enhance the correct discrimination of normal from trisomic fetuses, especially when the operators are less experienced.33-35,36 However, it is not yet evaluated if possible differences between the manual and semi-automated measurements are not only signifi-cant in terms of precision but also in terms of changing the individual risk status.

3. COUNSELING REGARDING THE RISK OF MISCARRIAGE AFTER INVASIVE PROCEDURES

Once an increased risk or an increased nuchal translucency is found during the com-bined test, women are counseled on the possibility of prenatal diagnostics. About 20-30% of the fetuses with an increased risk at the CT has a chromosomal abnormality.37 When the NT is increased the presence of an abnormal karyotype varies from approximately 7% for a NT between the 95th and 99th centile (3.5 mm), to 20% for a NT of 3.5-4.4 mm, 50% for a NT of 5.5-6.4 mm, and 75% for a NT of 8.5 mm or more.37


Chapter 1

15

After an increased risk parents are counseled and offered invasive prenatal diagnosis. One of the factors influencing women’s decision to opt for or decline chorionic villus sampling (CVS) or amniocentesis (AP) is the procedure related risk.24 Estimates of procedure related fetal loss rates differ considerably in literature, varying from 0.5% to 1.0% or even more, for both CVS and AP.38-52 Most of these studies are cohort studies, have mixed populations and are not recent. There are only a few randomized (controlled) trials. Tabor et al com-pared women undergoing an AP with a control group, showing a 1% higher fetal loss rate in women undergoing midtrimester amniocentesis.43 The fetal loss rate (FLR) following CVS has never been compared in a randomized controlled trial (RCT) with a control group. The FLR of CVS has been compared to AP in a RCT and the procedure related risk was found comparable.42,44,48,53 When parents are counseled regarding the procedure related risk of CVS or AP, most often 1% (or 1:100) is quoted according to the study of Tabor et al.54

It remains a challenge to estimate and report realistic valid risk figures from ‘real life’ cohort studies, taking into account all variables that influence the procedure related FLR. Furthermore, some of the above mentioned studies were performed a while ago at a time when ultrasound systems were less advanced and techniques and training in invasive procedures less standardized. A recent meta-analysis showed that accurate estimates of procedure-related risks following invasive procedures in the current clinical settings are lacking.55 Many experts believe that procedure related risks need reevaluation.56 Our national guideline published in 2000 quotes a procedure related risks of 0.5% for CVS and 0.3% for AP, and these numbers are based on an Cochrane article from 1998, which is withdrawn, and an article based on a statistical model from 1991.57,58 Recent numbers for the Netherlands are lacking.54 These numbers are important in order to prevent discrep-ancies in information given to patients among different centers.

4. COUNSELING OF PARENTS AFTER AN ENLARGED NT

When after an increased nuchal translucency, the karyotype appears to be normal this cannot be regarded as a complete reassurance, as far as the final outcome of the pregnan-cy is concerned. This is especially the case in fetuses where the NT is severely increased. At present, after exclusion of chromosomal aberrations, the most challenging part of managing pregnancies with an increased NT is to establish an adequate diagnostic work-up and provide parents with realistic and correct information about outcome, especially long term neurological outcome.59-69

Our study group has investigated at length the associations between an enlarged NT and poor pregnancy outcome with the aim of correctly informing women on the associa-tions and on the predictors of poor or favorable outcome, given an enlarged NT (like sex-related-differences, impact of the size of the enlarged NT, other anomalies present be-sides the enlarged NT, and the possibility of an underlying genetic syndrome).9,11,23,59,70-86 There is a long and still growing list of genetic syndromes presenting with increased NT.60,72 Among the genetic syndromes the most frequently reported in combination with an increased NT is Noonan syndrome (NS), with an incidence ranging from 2-6%. 65,69,87 NS is an autosomal dominant disorder caused in approximately 50% of the cases by a missense mutation in the PTPN11 gene on chromosome 12.88 Mutations in the SOS1-, RAF1-, KRAS-, BRAF-, MAP2K1/2-, NRAS- and SHOC2-genes account for a smaller percent-age of NS cases.89 In some clinically diagnosed NS cases the genetic background is still


16

unknown. Clinical diagnosis of Noonan syndrome is often challenging because of the great variability in clinical characteristics.90,91 The main facial characteristics are hyper-telorism, downslanting palpebral fissures, epicanthic fold, ptosis and low set posteriorly angulated ears. The most common cardiovascular defects are pulmonary valve stenosis and hypertrophic cardiomyopathy. Other phenotypic characteristics are short stature, broad or webbed neck and chest deformity. Associated pathologies are haematological disorders (bleeding diathesis, juvenile myelomonocytic leukemia), lymphatic vessel dys-plasias, deafness and cryptorchidism. Affected individuals show a wide range in level of intelligence, with mental retardation being present in 15-35%, usually in the mild range and mainly consisting of specific visual-constructional problems and verbal perfor-mance discrepancy.91,92

The use of 3D rendering of the fetal face in case of (subtle) anomalies after an increased NT and normal karyotype could be a valuable tool in the prenatal detection of fetuses with NS, since the facial characteristics may be even more pronounced prenatally than postnatally. A protocol for the management of pregnancies complicated by an increased NT to aid doctors in the prenatal follow-up of a fetus with an enlarged NT and a normal karyotype, especially to increase the prenatal detection rate of Noonan syndrome the most fre-quently encountered genetic syndrome, is still lacking.

5. FACIAL MARKERS IN THE FIRST TRIMESTER AND THEIR RELATIONSHIP WITH ANEUPLOIDIES AND OTHER FORMS OF ABNORMAL DEVELOPMENT

Assessment of the fetal face in the second trimester of pregnancy has become an impor-tant part of fetal evaluation, not only for the detection of facial anomalies but also in the setting of screening for trisomies, especially trisomy 21. Established second trimester profile markers for trisomies are the nasal bone length (NBL), the prenasal thickness (PNT), the ratio between the NBL and PNT and more recently the prefrontal space ratio (PFSR).22,93-95 Other second trimester profile parameters, e.g. the profile line (FP line) and maxilla-nasion-mandible angle (MNM-angle), have been studied as markers for facial anomalies including profile alterations in case of aneuploidies.96-99 These are proven reproducible markers for the diagnosis of retrognathia, maxillary alveolar ridge inter-ruption, sloping forehead, frontal bossing and flat profile. Reference values for most of these markers are available for the second trimester, however it has not yet been tried to see whether these markers can be measured in the first trimester and more importantly what their clinical significance would be. The frontomaxillary facial angle (FMF-angle) and the NBL have been introduced in the first trimester to improve screening algorithms for trisomies and to improve detection rates and decrease false positive rates.16,17,100,101 Measurement of the PNT, PNT/ NBL ratio, MNM-angle, FP line and PFSR in the first trimester could possibly further improve the detection of trisomies and/or facial abnormalities early in pregnancy. Despite the rapid availability of cell free fetal DNA (cffDNA) as screening test for tri-somies, the CT is still the standard of care in the majority of countries. Further improve-ment of detection of trisomies is still valuable in case cffDNA is not performed or when it is performed as second tier test and to enhance the first trimester detection of struc-tural anomalies which are not trisomy related.


Chapter 1

17

6. ANEUPLOIDIES AND FACIAL MARKERS IN THE SECOND TRIMESTER

Specific facial profile features of Down syndrome fetuses have been investigated and used as second and third trimester markers.22,94-99,102-105 The nasal bone length (NBL) was the first to be extensively investigated, followed by the prenasal thickness (PT). Recent studies have shown that the ratio between these two markers (PT-NBL ratio) and the pre-frontal space ratio (PFSR) yields an even better detection rate.94,105 Furthermore, we have previously investigated the maxilla-nasion-mandible (MNM) angle and fetal profile (FP) line in both euploid and pathological cases.97-99,106 Several studies have compared 2D and 3D US imaging during gestation and suggested 3D to be superior by allowing a better identification of anatomical landmarks, a higher ac-curacy and reproducibility in measurements of structures in the fetal face and profile, in-cluding the NBL. In a previous study, it was shown that 2D images judged to be midsagittal in fact are not and need 3D multiplanar correction of in average 11.9 (Y-axis) - 4.3 (Z-axis) degrees to become truly midsagittal.102 Clear landmarks to identify the exact midsagittal plane are missing when only 2D imaging is used, making it difficult to be absolutely sure to be in the exact midsagittal plane.102 However, it is not clear whether addition of 3D im-aging in a clinical setting 3D improves the detection rate when compared to 2D.

Aims of thesis

This thesis was designed to fill some of the gaps in knowledge that counselors, sonogra-phers and clinicians encounter in their daily practice when dealing with prenatal screen-ing and diagnosis. In view of the above mentioned clinical problems, we summarize the research questions as follows:

• Why has the introduction of a national policy of first trimester prenatal screening for chromosomal anomalies been so poorly utilized by pregnant women and their part-ners and what are the factors affecting the uptake of the CT in the Netherlands?

• One of the factors mentioned by women declining the CT is that the NT measurement is not accurate and this influences the reliability of the risk assessment. Quality con-trols have indicated that some sonographers tend to underestimate the NT-measure-ments. In this respect the introduction of a technical tool aimed at standardizing the way the NT is measured may reduce such a bias. The following question was therefore: can the NT-measurement be improved by a semi-automated measurement?

• One of the major determinants of a negative attitude towards the CT in the Nether-lands is the fear, in case on an increased risk, of having to undergo an invasive proce-dure that may lead to an iatrogenic abortion. We felt the need to redefine the actual risk of invasive procedures in a Dutch population since recent studies on this subject are in fact still missing. The investigated issues were: what is the total fetal loss rate and procedure-related risk for CVS and AP in the Dutch setting, and which maternal-, operator-, and procedure-related risk factors can be identified?

• At present the most challenging part of managing pregnancies with an increased NT, after exclusion of chromosomal aberrations, is to establish an adequate diagnostic work-up and provide parents with realistic and correct information about outcome, es-


18

pecially long term neurological outcome in absence of structural anomalies. Therefore the study aim was: what are various aspects that should be investigated in the setting of an increased NT?

• As Noonan syndrome is the most frequently observed genetic syndrome in fetuses with an enlarged NT and normal karyotype, and, considering the diagnosis of this con-dition can be challenging: which ultrasound characteristics can guide us in the prena-tal diagnosis of Noonan syndrome?

• Ultrasound measurement of facial markers has an increasingly important role in sec-ond trimester risk assessment and in the work-up of other facial anomalies, such as micrognathia. We wanted to investigate if these measurements can also be performed reliably in the first trimester of pregnancy. Therefore the next issue we have investi-gated was: can the PNT/NBL-ratio, MNM angle, FP line and PFSR already be measured in the first trimester of pregnancy? Are these measurements also useful markers of an abnormal development when measured in abnormal fetuses in the first trimester of pregnancy?

• The last question we wanted to answer was whether in the application of these facial markers at second trimester of pregnancy, 3D technique has an additional value with respect to the standard use of 2D technique. The question was therefore: is use of 3D technique superior to 2D technique when measuring the NB, PNT, FP line, MNM-an-gle, PNT/NB-ratio and PFSR, in Down syndrome screening?

References

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3. Nicolaides K. H., Azar G., Byrne D., Mansur C., Marks K. Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy. BMJ 1992 Apr 4; 304(6831): 867-869.

4. Pandya P. P., Snijders R. J., Johnson S. P., De Lourdes Brizot M., Nicolaides K. H. Screen-ing for fetal trisomies by maternal age and fetal nuchal translucency thickness at 10 to 14 weeks of gestation. Br J Obstet Gynaecol 1995 Dec; 102(12): 957-962.

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Nicolaides K. H. UK multicentre project on assessment of risk of trisomy 21 by maternal age and fetal nuchal-translucency thickness at 10-14 weeks of gestation. Fetal Medicine Foundation First Trimester Screening Group. Lancet 1998 Aug 1; 352(9125): 343-346.

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9. Muller M. A., Bleker O. P., Bonsel G. J., Bi-lardo C. M. Women's opinions on the offer and use of nuchal translucency screening for Down syndrome. Prenat Diagn 2006 Feb; 26(2): 105-111.

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11. Pajkrt E., Bilardo C. M., Van Lith J. M., Mol B. W., Bleker O. P. Nuchal translucency mea-surement in normal fetuses. Obstet Gynecol 1995 Dec; 86(6): 994-997.

12. Souka A. P., Von Kaisenberg C. S., Hyett J. A., Sonek J. D., Nicolaides K. H. Increased nuchal translucency with normal karyotype. Am J Obstet Gynecol 2005 Apr; 192(4): 1005-1021.

13. Kagan K. O., Wright D., Etchegaray A., Zhou Y., Nicolaides K. H. Effect of deviation of nuchal translucency measurements on the performance of screening for trisomy 21. Ultrasound Obstet Gynecol 2009 Jun; 33(6): 657-664.

14. Schmidt P., Staboulidou I., Elsasser M., Vaske B., Hillemanns P., Scharf A. How imprecise may the measurement of fetal nuchal translucency be without worsening first-trimester screening? Fetal Diagn Ther 2008; 24(3): 291-295.

15. Bilardo C. M., Muller M. A., Zikulnig L., Schipper M., Hecher K. Ductus venosus studies in fetuses at high risk for chromo-somal or heart abnormalities: relationship with nuchal translucency measurement and fetal outcome. Ultrasound Obstet Gynecol 2001 Apr; 17(4): 288-294.

16. Cicero S., Curcio P., Papageorghiou A., Sonek J., Nicolaides K. Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: an observational study. Lancet 2001 Nov 17; 358(9294): 1665-1667.

17. Cicero S., Bindra R., Rembouskos G., Trip-sanas C., Nicolaides K. H. Fetal nasal bone

length in chromosomally normal and ab-normal fetuses at 11-14 weeks of gestation. J Matern Fetal Neonatal Med 2002 Jun; 11(6): 400-402.

18. Cicero S., Longo D., Rembouskos G., Sacchi-ni C., Nicolaides K. H. Absent nasal bone at 11-14 weeks of gestation and chromosomal defects. Ultrasound Obstet Gynecol 2003 Jul; 22(1): 31-35.

19. Cicero S., Avgidou K., Rembouskos G., Ka-gan K. O., Nicolaides K. H. Nasal bone in first-trimester screening for trisomy 21. Am J Obstet Gynecol 2006 Jul; 195(1): 109-114.

20. Matias A., Gomes C., Flack N., Montenegro N., Nicolaides K. H. Screening for chromo-somal abnormalities at 10-14 weeks: the role of ductus venosus blood flow. Ultrasound Obstet Gynecol 1998 Dec; 12(6): 380-384.

21. Orlandi F., Bilardo C. M., Campogrande M., Krantz D., Hallahan T., Rossi C., Viora E. Measurement of nasal bone length at 11-14 weeks of pregnancy and its potential role in Down syndrome risk assessment. Ultra-sound Obstet Gynecol 2003 Jul; 22(1): 36-39.

22. Sonek J. D., McKenna D., Webb D., Croom C,. Nicolaides K. Nasal bone length through-out gestation: normal ranges based on 3537 fetal ultrasound measurements. Ultrasound Obstet Gynecol 2003 Feb; 21(2): 152-155.

23. Timmerman E., Oude Rengerink K., Pajkrt E., Opmeer B. C., Van der Post J. A., Bilardo C. M. Ductus venosus pulsatility index mea-surement reduces the false-positive rate in first-trimester screening. Ultrasound Ob-stet Gynecol 2010 Dec; 36(6): 661-667.

24. Bakker M., Birnie E., Pajkrt E., Bilardo C. M., Snijders R. J. Low uptake of the combined test in the Netherlands — which factors con-tribute? Prenat Diagn 2012 Dec; 32(13): 1305-1312.

25. Fransen M. P., Wildschut H. I., Mackenbach J. P., Steegers E. A., Galjaard R. J., Essink-Bot M. L. Ethnic and socio-economic differences in uptake of prenatal diagnostic tests for Down's syndrome. Eur J Obstet Gynecol Reprod Biol 2010 Aug; 151(2): 158-162.


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26. Seror V., Ville Y. Prenatal screening for Down syndrome: women's involvement in decision-making and their attitudes to screening. Prenat Diagn 2009 Feb; 29(2): 120-128.

27. Ekelund C. K., Petersen O. B., Skibsted L., Kjaergaard S., Vogel I., Tabor A., Danish Fetal Medicine Research Group. First-trimester screening for trisomy 21 in Denmark: impli-cations for detection and birth rates of tri-somy 18 and trisomy 13. Ultrasound Obstet Gynecol 2011 Aug; 38(2): 140-144.

28. Rowe R., Puddicombe D., Hockley C., Redshaw M. Offer and uptake of prenatal screening for Down syndrome in women from different social and ethnic back-grounds. Prenat Diagn 2008 Dec; 28(13): 1245-1250.

29. Shantha N., Granger K., Arora P., Polson D. Women's choice for Down's screening – a comparative experience in three district general hospitals. Eur J Obstet Gynecol Re-prod Biol 2009 Sep; 146(1): 61-64.

30. Bilardo C. M., Pajkrt E., De Graaf I., Mol B. W., Bleker O. P. Outcome of fetuses with enlarged nuchal translucency and normal karyotype. Ultrasound Obstet Gynecol 1998 Jun; 11(6): 401-406.

31. The Fetal Medicine Foundation, 2010. 32. Pandya P. P., Altman D. G., Brizot M. L., Pet-

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55. Akolekar R., Beta J., Picciarelli G., Ogilvie C., D'Antonio F. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic re-view and meta-analysis. Ultrasound Obstet Gynecol 2015 Jan; 45(1): 16-26.

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59. Bilardo C. M., Muller M. A., Pajkrt E., Clur S. A., Van Zalen M. M., Bijlsma E. K. Increased nuchal translucency thickness and normal karyotype: time for parental reassurance. Ultrasound Obstet Gynecol 2007 Jul; 30(1): 11-18.

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in chromosomally normal fetuses with increased nuchal translucency in the first trimester. Ultrasound Obstet Gynecol 2001 Jul; 18(1): 9-17.

61. Saldanha F. A., Brizot M. de L., Moraes E. A., Lopes L. M., Zugaib M. Increased fetal nuchal translucency thickness and normal karyotype: prenatal and postnatal follow-up. Rev Assoc Med Bras 2009 Sep-Oct; 55(5): 575-580.

62. Senat M. V., De Keersmaecker B., Audibert F., Montcharmont G., Frydman R., Ville Y. Pregnancy outcome in fetuses with in-creased nuchal translucency and normal karyotype. Prenat Diagn 2002 May; 22(5): 345-349.

63. Adekunle O., Gopee A., el-Sayed M., Thilaga-nathan B. Increased first trimester nuchal translucency: pregnancy and infant out-comes after routine screening for Down's syndrome in an unselected antenatal popu-lation. Br J Radiol 1999 May; 72(857): 457-460.

64. Cheng C. C., Bahado-Singh R. O., Chen S. C., Tsai M. S. Pregnancy outcomes with in-creased nuchal translucency after routine Down syndrome screening. Int J Gynaecol Obstet 2004 Jan; 84(1): 5-9.

65. Hiippala A., Eronen M., Taipale P., Salonen R., Hiilesmaa V. Fetal nuchal translucency and normal chromosomes: a long-term follow-up study. Ultrasound Obstet Gynecol 2001 Jul; 18(1): 18-22.

66. Maymon R., Jauniaux E., Cohen O., Dreazen E., Weinraub Z., Herman A. Pregnancy out-come and infant follow-up of fetuses with abnormally increased first trimester nuchal translucency. Hum Reprod 2000 Sep; 15(9): 2023-2027.

67. Senat M. V., Bussieres L., Couderc S., Roume J., Rozenberg P., Bouyer J., Ville Y. Long-term outcome of children born after a first-trimester measurement of nuchal translu-cency at the 99th percentile or greater with normal karyotype: a prospective study. Am J Obstet Gynecol 2007 Jan; 196(1): 53.e1-53.e6.

68. Van Vugt J. M., Tinnemans B. W., Van Zalen-Sprock R. M. Outcome and early childhood follow-up of chromosomally normal fetuses with increased nuchal translucency at 10-14 weeks' gestation. Ultrasound Obstet Gyne-col 1998 Jun; 11(6): 407-409.

69. Brady A. F., Pandya P. P., Yuksel B, Gree-nough A., Patton M. A., Nicolaides K. H. Out-come of chromosomally normal livebirths with increased fetal nuchal translucency at 10-14 weeks' gestation. J Med Genet 1998 Mar; 35(3): 222-224.

70. Bilardo C. M. Increased nuchal translucency and normal karyotype: coping with uncer-tainty. Ultrasound Obstet Gynecol 2001 Feb; 17(2): 99-101.

71. Bilardo C. M., Muller M. A., Pajkrt E. Out-come of fetuses with increased nuchal translucency. Curr Opin Obstet Gynecol 2001 Apr; 13(2): 169-174.

72. Bilardo C. M., Timmerman E., Pajkrt E., Van Maarle M. Increased nuchal translucency in euploid fetuses – what should we be telling the parents? Prenat Diagn 2010 Feb; 30(2): 93-102.

73. Muller M. A., Bleker O. P., Bonsel G. J., Bi-lardo C. M. Nuchal translucency screening and anxiety levels in pregnancy and puerpe-rium. Ultrasound Obstet Gynecol 2006 Apr; 27(4): 357-361.


75. Muller M. A., Clur S. A., Timmerman E., Bilardo C. M. Nuchal translucency measure-ment and congenital heart defects: modest association in low-risk pregnancies. Prenat Diagn 2007 Feb; 27(2): 164-169.

76. Muller M. A., Pajkrt E., Bleker O. P., Bonsel G. J., Bilardo C. M. Disappearance of en-larged nuchal translucency before 14 weeks' gestation: relationship with chromosomal abnormalities and pregnancy outcome. Ultrasound Obstet Gynecol 2004 Aug; 24(2):


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169-174. 77. Muller M. A., Pajkrt E., Bilardo C. M. Ultra-

sound screening for Down's syndrome early in pregnancy: nuchal translucency thick-ness. Ned Tijdschr Geneeskd 2002 Apr 27; 146(17): 793-798.

78. Pajkrt E., Bilardo C. M., Van Lith J. M., Mol B. W., Hansson K., Van Prooijen Knegt A. C., Bleker O. P. Ultrasound screening for fetal trisomies at 10-14 weeks' gestation. Early Hum Dev 1996 Dec 30; 47 Suppl: S35-6.

79. Pajkrt E., De Graaf I. M., Mol B. W., Van Lith J. M., Bleker O. P., Bilardo C. M. Weekly nu-chal translucency measurements in normal fetuses. Obstet Gynecol 1998 Feb; 91(2): 208-211.

80. Pajkrt E., Mol B. W., Bleker O. P., Bilardo C. M. Pregnancy outcome and nuchal trans-lucency measurements in fetuses with a normal karyotype. Prenat Diagn 1999 Dec; 19(12): 1104-1108.

81. Pajkrt E., Mol B. W., Boer K., Drogtrop A. P., Bossuyt P. M., Bilardo C. M. Intra- and inter-operator repeatability of the nuchal trans-lucency measurement. Ultrasound Obstet Gynecol 2000 Apr; 15(4): 297-301.

82. Pajkrt E., Mol B. W., Van Lith J. M., Bleker O. P., Bilardo C. M. Screening for Down's syndrome by fetal nuchal translucency measurement in a high-risk population. Ultrasound Obstet Gynecol 1998 Sep; 12(3): 156-162.

83. Pajkrt E., Van Lith J. M., Mol B. W., Bleker O. P., Bilardo C. M. Screening for Down's syndrome by fetal nuchal translucency mea-surement in a general obstetric population. Ultrasound Obstet Gynecol 1998 Sep; 12(3): 163-169.

84. Timmerman E., Clur S. A., Pajkrt E., Bilardo C. M. First-trimester measurement of the ductus venosus pulsatility index and the prediction of congenital heart defects. Ul-trasound Obstet Gynecol 2010 Dec; 36(6): 668-675.

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86. Timmerman E., Pajkrt E., Maas S. M., Bilardo C. M. Enlarged nuchal translucency in chro-mosomally normal fetuses: strong associa-tion with orofacial clefts. Ultrasound Obstet Gynecol 2010 Oct; 36(4): 427-432.

87. Souka A. P., Snijders R. J., Novakov A., Soares W., Nicolaides K. H. Defects and syndromes in chromosomally normal fetuses with increased nuchal translucency thickness at 10-14 weeks of gestation. Ultrasound Obstet Gynecol 1998 Jun; 11(6): 391-400.

88. Tartaglia M., Mehler E. L., Goldberg R., Zampino G., Brunner H. G., Kremer H., Van der Burgt I., Crosby A. H., Ion A., Jeffery S., Kalidas K., Patton M. A., Kucherlapati R. S., Gelb B. D. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001 Dec; 29(4): 465-468.

89. Jorge A. A., Malaquias A. C., Arnhold I. J., Mendonca B. B. Noonan syndrome and re-lated disorders: a review of clinical features and mutations in genes of the RAS/MAPK pathway. Horm Res 2009; 71(4): 185-193.

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93. Yazdi B., Riefler P., Fischmuller K., Sonek J., Hoopmann M., Kagan K. O. The frontal space measurement in euploid and aneu-ploid pregnancies at 11-13 weeks' gestation.


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Prenat Diagn 2013 Dec; 33(12): 1124-1130. 94. De Jong-Pleij E. A., Vos F. I., Ribbert L. S.,

Pistorius L. R., Tromp E., Bilardo C. M. Pre-nasal thickness-to-nasal bone length ratio: a strong and simple second- and third-trimester marker for trisomy 21. Ultrasound Obstet Gynecol 2012 Feb; 39(2): 185-190.

95. Persico N., Borenstein M., Molina F., Azu-mendi G., Nicolaides K. H. Prenasal thick-ness in trisomy-21 fetuses at 16-24 weeks of gestation. Ultrasound Obstet Gynecol 2008 Nov; 32(6): 751-754.

96. Sonek J., Molina F., Hiett A. K., Glover M., McKenna D., Nicolaides K H. Prefrontal space ratio: comparison between trisomy 21 and euploid fetuses in the second trimester. Ultrasound Obstet Gynecol 2012 Sep; 40(3): 293-296.

97. De Jong-Pleij E. A., Ribbert L. S., Manten G. T., Tromp E, Bilardo C. M. Maxilla-nasion-mandible angle: a new method to assess profile anomalies in pregnancy. Ultrasound Obstet Gynecol 2011 May; 37(5): 562-569.

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99. De Jong-Pleij E. A., Ribbert L. S., Pistorius L. R., Tromp E., Bilardo C. M. The fetal profile line: a proposal for a sonographic refer-ence line to classify forehead and mandible anomalies in the second and third trimes-ter. Prenat Diagn 2012 Aug; 32(8): 797-802.

100. Borenstein M., Persico N., Kaihura C., Sonek J., Nicolaides K. H. Frontomaxillary facial angle in chromosomally normal fetuses at

11+0 to 13+6 weeks. Ultrasound Obstet Gy-necol 2007 Oct; 30(5): 737-741.

101. Plasencia W., Dagklis T., Sotiriadis A., Boren-stein M., Nicolaides K. H. Frontomaxillary facial angle at 11+0 to 13+6 weeks' gestation-reproducibility of measurements. Ultra-sound Obstet Gynecol 2007 Jan; 29(1): 18-21.

102. De Jong-Pleij E. A., Ribbert L. S., Tromp E., Bi-lardo C. M. Three-dimensional multiplanar ultrasound is a valuable tool in the study of the fetal profile in the second trimester of pregnancy. Ultrasound Obstet Gynecol 2010 Feb; 35(2): 195-200.

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2Low uptake of the combined test in the Netherlands –

which factors contribute?

M. Bakker 1

E. Birnie 1,4

E. Pajkrt 2

C. M. Bilardo 1 R. J. M. Snijders 3

1 Department of Obstetrics and Gynecology, Fetal Medicine Unit,

University Medical Centre, Groningen, the Netherlands.2 Department of Obstetrics and Gynecology, Fetal Medicine Unit,

Academic Medical Centre, Amsterdam, the Netherlands.3 Prenatal Screening Foundation Northeast Netherlands, University

Medical Centre, Groningen, the Netherlands.4 Department of Genetics, University Medical Centre Groningen,

University of Groningen, Groningen, the Netherlands.

Published in Prenatal Diagnosis 2012; 32, 1305-1312.


26

Low uptake of the combined test in the Netherlands – which factors contribute?

M. Bakker1, E. Birnie1,4, E. Pajkrt2, C. M. Bilardo1 and R. J. M. Snijders3

1 Department of Obstetrics and Gynecology, Fetal Medicine Unit, University Medical Centre, Groningen, the Netherlands.

2 Department of Obstetrics and Gynecology, Fetal Medicine Unit, Academic Medical Centre, Amsterdam, the Netherlands.

3 Prenatal Screening Foundation Northeast Netherlands, University Medical Centre, Groningen, the Netherlands.

4 Department of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands.

Objective: Objective The aim of this study was to evaluate which of the following fac-tors affect the uptake of the combined test (CT) in the Netherlands: women’s socio-demographic background, attitude towards Down syndrome, attitude to-wards termination of pregnancy, coun-seling process, reimbursement policy, and knowledge on the aim of the CT.

Methods: Cross-sectional survey in the Northwest (NW) and the Northeast (NE) region of the Netherlands.

Results: Analyses were based on 820 question-naires (73% response rate). Women from the NW region opted more often for the CT than women from the NE

region (52.1% and 16.5%, respectively, p < 0.001). Women of 36 years and older opted more often for the CT than young-er women (59.4% and 28.2%, respec-tively, p < 0.001). Women’s socio-demo-graphic background and their attitude towards Down syndrome and termina-tion of pregnancy (TOP) had contributed independently on CT choice.

Conclusion: The uptake of the CT in this study is low. The main reason for the low uptake is the relatively positive attitude towards Down syndrome and a negative attitude towards TOP. Moreover, the perception of maternal age as strong predictor of Down syndrome risk and the inequal-ity of access to care, due to the financial threshold for younger women, are likely to affect participation in screening.

Introduction

The aim of the combined test (CT) is to identify fetuses with an increased risk for Down syndrome, thus offering parents the possibility to opt for invasive prenatal diagnosis. In case of an affected pregnancy, parents can opt for termination of pregnancy (TOP) or pre-pare themselves for the birth of a child with Down syndrome. In 2007, a national screening program has been introduced in the Netherlands with


Chapter 2

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the aim to ask all pregnant women if they want to be informed about prenatal screening. If a woman wants to receive information, she is counseled by her healthcare profession-al, and on the basis of the information provided, she decides whether or not she wants to opt for screening. The Dutch Department of Health has introduced an age-related system for reimbursement of the CT; women aged 36 years and older have free access to the test, whereas younger women have to pay approximately 150 euros. If women of 36 years and older decline screening, they can still opt for prenatal diagnosis based on their age. Before 2007, the CT was offered in the Netherlands in research settings with uptake rates ranging from 53% to 86%.1,2 An important incentive for the present study was the observation that since the start of the national screening program the uptake of the CT is low and lower than in the past, with pronounced regional differences.3,1,2 The aim of this study was to evaluate which of the following factors affects the uptake of the CT in the Netherlands: women’s sociodemographic background, attitude towards Down syn-drome, attitude towards termination of pregnancy, counseling process, reimbursement policy, and knowledge on the aim of the CT.

Methods

DESIGN

Between March and April 2010, a cross-sectional survey was conducted at 13 ultrasound clinics in the Northwest (NW) and Northeast (NE) of the Netherlands to investigate the uptake of the CT. All pregnant women who attended the ultrasound center received an information letter and a questionnaire about the CT. The questionnaires were distrib-uted at the time of the 20-week anomaly scan to ensure that knowledge on the outcome of prenatal diagnosis would not bias women’s opinions on the CT in retrospect.

STUDY INSTRUMENT

The questionnaire used in the present study was based on a questionnaire developed in France by Seror et al.,4 addressing women’s attitudes and decisions on screening for Down syndrome. Our questionnaire included four sections with a total of 53 questions (see supporting information). The first section (six questions) contained questions regarding the dating scan in this pregnancy. The second section (23 questions) addressed the counseling and reasons for accepting or declining the CT. The third section (eight questions) assessed whether or not women opted for prenatal diagnosis. The final section contained ques-tions about women’s socio-demographic characteristics, attitude towards termination of pregnancy and costs of the first trimester scan. The following demographic variables were included as follows: age, parity, educational level, income status, region, and ethnicity.

STATISTICAL ANALYSIS

To compare differences in categorical variables between women who opted for the CT and those who declined, the X2 test (or Fisher’s Exact Test, if appropriate) was used. To


28

compare differences in continuous variables between these groups, the student’s t-test was used. Comparison by age was performed because only women aged 36 years and older receive reimbursement for the CT. Comparison by region was performed because differences between regions may reflect differences in determinants or uptake. Associations between single co-variables and women’s decision to accept or decline the CT were assessed by univariate binary logistic regression and expressed in odds ra-tios (ORs, 95% confidence intervals). Next, multiple binary logistic regression analysis with stepwise backward conditional inclusion of variables was performed to evaluate the adjusted impact of co-variables. Correlations and stratified tables were used to check for confounding and interaction (income education). Multiple logistic regressions were redone accordingly, taking confounding and interaction into account. To study the relative impact of co-variables on women’s decision, we distinguished six blocks of determinants: patient characteristics, women’s opinions and attitudes, care characteristics, decision making and women’s knowledge, reimbursement policy, and region. The relative impact of each block was evaluated by adding blocks of variables suc-cessively to the multiple binary logistic regression, and the change in 2 log likelihood

Table 1 — Socio-demographic characteristics of women opting for or declining the combined test (CT)

¬ ¬ ¬ ¬ ¬ ¬ ¬ CT ¬ ¬ ¬ ¬ ¬ ¬ ¬Variables No N(%) Yes N(%) p

Age:

≤29 307 (55.1) 86 (32.7)

30-35 209 (37.5) 117 (44.5)

≥36 41 (7.4) 60 (22.8) <0.001

Education:

Low 57 (10.4) 25 (9.6)

Middle 250 (45.5) 100 (38.3)

High 243 (44.2) 136 (52.1) 0.101

Income:

<1500 euros 43 (8.1) 18 (7.2)

1500-3500 euros 375 (71.0) 129 (51.8)

>3500 euros 110 (20.8) 102 (41.0) <0.001

Parity:

Primiparous 271 (48.7) 119 (45.2)

Multiparous 286 (51.3) 144 (54.8) 0.370

Etnicity:

Caucasian 385 (83.9) 199 (84.0)

Non-Caucasian 74 (16.1) 38 (16.0) >0.99

Religion:

Important 163 (29.7) 44 (16.8)

Not important 386 (70.3) 218 (83.2) <0.001

Region:

Northeast 385 (69.1) 76 (28.9)

Northwest 172 (30.9) 187 (71.1) <0.001


Chapter 2

29

Table 2 — Information provided on the combined test (CT)

¬ ¬ ¬ ¬ ¬ ¬ ¬ CT ¬ ¬ ¬ ¬ ¬ ¬ ¬Information provided No N(%) Yes N(%) p

Who discussed the possibility of the CT with you

General physician 14 (2.6) 2 (0.3)

Midwife 444 (83.9) 192 (73.3)

Gynecologist 48 (9.1) 40 (15.3)

Sonographer 8 (1.5) 11 (4.2)

Searched autonomously for information 15 (2.8) 17 (6.5) <0.001

Was the amount of information enough

Too much information 5 (0.9) 1 (0.4)

Enough information 515 (95.9) 253 (96.2)

Too little information 17 (3.2) 9 (3.4) 0.756

Did you get written information in addition to oral information

Yes, a leaflet on Down syndrome screening 430 (80.7) 230 (87.5)

Yes, information on the Decision Aid on the internet 16 (3.0) 9 (3.4)

No, I did not receive any written information 87 (16.3) 24 (9.1) <0.022

Would you prefer to receive the written information before or after counseling

Before 213 (40.2) 159 (60.5)

After 216 (40.8) 33 (12.5)

I do not know 101 (19.1) 71 (27) <0.001

was evaluated and the proportion of cases correctly predicted. Statistical significance was defined as p < 0.05 (two-sided). All statistical analyses were conducted using SPSS 17.0.0.

Results

A total of 837 (73.0%) of 1140 women returned the questionnaire. Analyses were based on 820 women; 17 questionnaires were excluded from analysis because women had already opted for prenatal diagnosis and were aware whether or not their child was affected at the time the questionnaire was filled out; nine of these women had declined, and eight women had opted for the CT. Table 1 shows that 263 (32.1%) participants opted for the CT. Acceptors were on average older than decliners: 31.4 and 29.0 years, respectively (p < 0.001). There were no significant age differences between the NW and NE regions. Most women were counseled by their midwife (N=636, 80.4%), and the first visit took usually place at 7 to 8 weeks of gestation (N=356, 43.8%). Besides oral information, 660 (82.9%) women received the leaflet on Down syndrome screening developed by the Dutch National Screening Board, and 25 (3.1%) women used the Decision Aid on the internet. A subgroup of 111 (13.9%) women reported not to have received any written information. Most women who opted for the CT preferred to receive the information before counseling (N=159, 60.5%) in contrast to 40.2% of women who declined (N=213) (p < 0.001). More women who declined the CT indicated that they did not receive any written information (N=87, 16.3%) than women who opted for the CT (N=24, 9.1%)


30

(p < 0.022) (Table 2). The vast majority of women reported to have made the decision on CT autonomously (N=648, 81.5%) and before the counseling had taken place (N=475, 59.9%). Women who opted for the CT more frequently reported having received a positive advice from the healthcare professional (N=33, 12.8%) than decliners (N=13, 2.5%) (p < 0.001). Similarly, women of 36 years and older were more frequently advised to opt for the CT (N=14, 14.6%) than younger women (N=31, 4.7%) (p < 0.002). The majority of women reported that it had been easy to decide whether or not to opt for prenatal screening (N=601, 75.4%). More acceptors found the decision easy (N=212, 81.2%) than decliners (N=389,

Table 3 — Reasons to opt for the combined test (CT) (multiple answers possible per participant)

Reasons to opt for the CT Total acceptors = 263

Total answersN=425

<36 yearsanswers N(%)

≥36 yearsanswers N(%) p

NE regionN(%)

NW regionN(%) p

I want to know if there is an increased risk on Down syndrome

223 (84.8) 179 (88.2) 44 (73.3) 0.008 59 (77.6) 164 (87.7) 0.057

I will opt for all tests offered 26 (9.9) 25 (12.3) 1 (1.7) 0.013 6 (7.9) 20 (10.7) 0.649

I am of older age and I rather opt for the CT than for an invasive procedure

61 (23.2) 16 (7.9) 45 (75.0) <0.001 25 (32.9) 36 (19.3) 0.024

I had the CT in previous pregnancy

79 (30.0) 64 (31.5) 15 (25.0) 0.423 19 (25.0) 60 (32.1) 0.300

My midwife/family/friends advised me to do the CT

14 (5.3) 7 (3.4) 7 (11.7) 0.021 2 (2.6) 12 (6.4) 0.363

Other reasons 22 (8.4) 18 (8.9) 4 (6.7) 0.792 5 (6.6) 17 (9.1) 0.627

NW = Northwest. NE = Northeast.

Table 4 — Reasons to decline the combined test (CT) (multiple answers possible per participant)

Reasons to decline the CT Total decliners = 557

Total answersN=1101



NE regionN(%)

NW regionN(%) p

I am young and therefore the test is unnecessary

191 (34.3) 189 (36.6) 2 (4.9) <0.001 134 (34.8) 57 (33.1) 0.772

I want to minimize testing during this pregnancy

30 (5.4) 26 (5.0) 4 (9.8) 0.266 19 (4.9) 11 (6.4) 0.543

I think my risk on Down syndrome is low

138 (24.8) 133 (25.8) 5 (12.2) 0.060 100 (26.0) 38 (22.1) 0.341

I do not want to make a decision on TOP

123 (22.1) 111 (21.5) 12 (29.3) 0.245 80 (20.8) 43 (25.0) 0.271

Down syndrome is for me no reason to terminate a pregnancy

321 (57.6) 292 (56.6) 29 (70.7) 0.100 236 (61.3) 85 (49.4) 0.009

The test does not give any guarantees

16 (2.9) 14 (2.7) 2 (4.9) 0.332 11 (2.9) 5 (2.9) >0.99

I have the impression that the test is not reliable

191 (34.3) 178 (34.5) 13 (31.7) 0.864 127 (33.0) 64 (37.2) 0.336

I found the test too expensive 37 (6.6) 37 (7.2) 0 (0) 0.099 20 (5.2) 17 (9.9) 0.044

Other reasons 54 (9.7) 51 (9.9) 3 (7.3) 0.786 31 (8.1) 23 (13.4) 0.062



Chapter 2

31

72.6%) (p=0.007). Decliners reported more often that they were unaware a decision was being made (N=105, 19.6%) than ac-ceptors (N=28, 10.7%) (p=0.007). Women from the NE region were more frequently unaware a decision was being made (N=87, 19.5%) in comparison to women from the NW region (N=46, 13.1%) (p=0.040). The reasons to opt for the CT are shown in Table 3. The main reason to opt for CT was to obtain an individualized risk assess-ment on Down syndrome (N=223, 84.8%). Women of 36 years and older opted more often for the CT than younger women be-cause others advised them to opt for the CT (N=7, 11.7% vs. N=7, 3.4%; p=0.021). In the NE region, older women more often opted for the CT to avoid an invasive procedure (N=25, 32.9%) than women from the NW region (N=36, 19.3%; p=0.024). The main reason to decline the CT was that women would not consider TOP in case of Down syndrome (N=321, 57.6%) (Table 4). Younger women declined the CT more often than women of 36 years and older because they considered their age-related risk to be low (N=189, 36.6% vs. N=2, 4.9%; p < 0.001). Women from the NE region declined the CT more often because Down syndrome would not be a reason to consider TOP (N=236, 61.3% vs. N=85, 49.4%; p=0.009) and women from the NW more often declined the CT because they found it too expensive (N=17, 9.9% vs. N=20, 5.2%; p=0.044). Acceptors of the CT would consider TOP in case of Down syndrome more often than decliners (Table 5). A subgroup of 210 (39.2%) women would consider a TOP in case of severe physical anomaly. Women from the NE region would consider TOP in case of Down syndrome less often than women from the NW region (N=46, 10.3% vs. N=108, 30.6%; p < 0.001). There were no significant differences in attitude towards TOP between the two age-groups.

Tabl

e 5

— A

ttit

ude

tow

ards

term

inat

ion

of p

regn

ancy

Att

itu

de

tow

ard

s TO

P

Com

bin

ed te

st

p<3

6 ye

ars

answ

ers

N(%

)≥36 years

answ

ers

N(%

)p

NE

regi

onN

(%)

NW

regi

onN

(%)

pN

o N

(%)

Yes

N(%

)

Con

side

r te

rmin

atio

n o

f pre

gnan

cy

in c

ase

of D

own

syn

drom

e30

(5.6

)12

4 (4

7.3)

131

(18.

7)23

(23.

2)46

(10.

3)10

8 (3

0.6)

Con

sid

er te

rmin

atio

n o

f pre

gnan

cy

in c

ase

of s

ever

e st

ruct

ura

l an

omal

y21

0 (3

9.2)

74 (2

8.2)

247

(35.

3)37

(27.

4)17

3 (3

8.9)

111

(31.

4)

I d

id n

ot th

ink

term

inat

ion

of

pre

gnan

cy w

as a

n o

pti

on17

2 (3

2.1)

52 (1

9.8)

201

(28.

8)23

(23.

2)13

3 (2

9.9)

91 (2

5.8)

I w

ill a

lway

s ca

rry

to te

rm12

4 (2

3.1)

12 (4

.6)

<0.0

0112

0 (1

7.2)

16 (1

6.2)

0.57

293

(20.

9)43

(12.

2)<0

.001

TOP

= te

rmin

atio

n o

f pre

gnan

cy. N

W =

Nor

thw

est.

NE

= N

orth

east

.

Table 3 — Reasons to opt for the combined test (CT) (multiple answers possible per participant)

Reasons to opt for the CT Total acceptors = 263

Total answersN=425



NE regionN(%)

NW regionN(%) p

I want to know if there is an increased risk on Down syndrome

223 (84.8) 179 (88.2) 44 (73.3) 0.008 59 (77.6) 164 (87.7) 0.057

I will opt for all tests offered 26 (9.9) 25 (12.3) 1 (1.7) 0.013 6 (7.9) 20 (10.7) 0.649

I am of older age and I rather opt for the CT than for an invasive procedure

61 (23.2) 16 (7.9) 45 (75.0) <0.001 25 (32.9) 36 (19.3) 0.024

I had the CT in previous pregnancy

79 (30.0) 64 (31.5) 15 (25.0) 0.423 19 (25.0) 60 (32.1) 0.300

My midwife/family/friends advised me to do the CT

14 (5.3) 7 (3.4) 7 (11.7) 0.021 2 (2.6) 12 (6.4) 0.363

Other reasons 22 (8.4) 18 (8.9) 4 (6.7) 0.792 5 (6.6) 17 (9.1) 0.627


Table 4 — Reasons to decline the combined test (CT) (multiple answers possible per participant)

Reasons to decline the CT Total decliners = 557

Total answersN=1101



NE regionN(%)

NW regionN(%) p

I am young and therefore the test is unnecessary

191 (34.3) 189 (36.6) 2 (4.9) <0.001 134 (34.8) 57 (33.1) 0.772

I want to minimize testing during this pregnancy

30 (5.4) 26 (5.0) 4 (9.8) 0.266 19 (4.9) 11 (6.4) 0.543

I think my risk on Down syndrome is low

138 (24.8) 133 (25.8) 5 (12.2) 0.060 100 (26.0) 38 (22.1) 0.341

I do not want to make a decision on TOP

123 (22.1) 111 (21.5) 12 (29.3) 0.245 80 (20.8) 43 (25.0) 0.271

Down syndrome is for me no reason to terminate a pregnancy

321 (57.6) 292 (56.6) 29 (70.7) 0.100 236 (61.3) 85 (49.4) 0.009

The test does not give any guarantees

16 (2.9) 14 (2.7) 2 (4.9) 0.332 11 (2.9) 5 (2.9) >0.99

I have the impression that the test is not reliable

191 (34.3) 178 (34.5) 13 (31.7) 0.864 127 (33.0) 64 (37.2) 0.336

I found the test too expensive 37 (6.6) 37 (7.2) 0 (0) 0.099 20 (5.2) 17 (9.9) 0.044

Other reasons 54 (9.7) 51 (9.9) 3 (7.3) 0.786 31 (8.1) 23 (13.4) 0.062



32

Table 6 — Univariate and multivariate analysis – which factors influence combined test (CT) choice

Univariate logistic regression Multiple logistic regression

Variables OR (95% CI) p OR (95% CI) p

BLOCK 1 – socio-demographics

Age:

≤29 1 ¬ 1 ¬

30-35 2.00 (1.44 – 2.78) <0.001 2.00 (1.17 – 3.35) 0.011

≥36 5.22 (3.29 – 8.31) <0.001 5.61 (2.77 – 11.33) <0.001

Parity:

Primiparous 1 ¬

Multiparous 1.15 (0.85 – 1.54) <0.362

Etnicity:

Caucasian 1 ¬

Non-Caucasian 0.99 (0.65 – 1.52) 0.976

Education:

Low 1 ¬

Middle 0.91 (0.54 – 1.54) 0.731

High 1.28 (0.76 – 2.14) 0.353

Income:

<1500 1 ¬ 1 ¬

1500-3500 0.82 (0.46 – 1.48) 0.511 1.52 (0.52 – 4.50) 0.447

>3500 2.2 (1.20 – 4.08) 0.011 2.56 (0.80 – 8.22) 0.114

Ever TOP:

No 1 ¬ 1 ¬

Yes 3.25 (1.75 – 6.04) <0.001 1.81 (0.76 – 4.34) 0.183

BLOCK 2 – attitude

Opinion TOP:

TOP in case of Down 1 ¬ 1 ¬

TOP in case of anomaly 0.09 (0.05 – 0.14) <0.001 0.14 (0.07 – 0.26) <0.001

Never thought about it 0.07 (0.04 – 0.12) <0.001 0.11 (0.06 – 0.22) <0.001

Always carry to term 0.02 (0.01 – 0.05) <0.001 0.04 (0.02 – 0.09) <0.001

Positive opinion on offer national screening:

No 1 ¬

I do not know 5.83 (0.64 – 52.90) 0.117

Yes 7.54 (0.99 – 57.43) 0.051

BLOCK 3 – counseling

CT discussed by:

Midwife 1 ¬ 1 ¬

Self 2.62 (1.28 – 5.40) 0.008 3.71 (1.07 – 12.84) 0.038

General physician 0.33 (0.07 – 1.50) 0.145 0.46 (0.04 – 5.83) 0.550

Gynecologist 1.93 (1.23 – 3.03) 0.004 1.18 (0.58 – 2.39) 0.653

Sonographer 3.20 (1.26 – 8.03) 0.014 6.47 (1.20 – 35.01) 0.030

CT information:

Enough 1 ¬

Too much 0.41 (0.05 – 3.50) 0.413

Too little 1.08 (0.47 – 2.45) 0.858


Chapter 2

33

Table 6 (continued)

Univariate logistic regression Multiple logistic regression

Variables OR (95% CI) p OR (95% CI) p

Type of CT information:

No written information 1 ¬ 1 ¬

Official leaflet 1.94 (1.20 – 3.13) 0.007 3.12 (1.44 – 6.75) 0.004

Decision aid on internet 2.04 (0.80 – 5.19) 0.135 0.94 (0.19 – 4.65) 0.939

Timing of CT information:

Before counseling 1 ¬ 1 ¬

I do not know 0.44 (0.28 – 0.68) <0.001 0.42 (0.21 – 0.87) 0.019

After counseling 0.44 (0.31 – 0.62) <0.001 0.40 (0.24 – 0.67) <0.001

Advice from healthcare professional:

Informed choice 1 ¬ 1 ¬

Do not opt for CT 0.63 (0.20 – 0.1.93) 0.417 0.40 (0.09 – 1.77) 0.224

Opt for CT 5.58 (2.88 – 10.81) <0.001 14.97 (4.26 – 52.60) <0.001

Follow advice health care professional:

Decide without help 1 ¬ 1 ¬

Decide with help 0.88 (0.53 – 1.47) 0.624 0.53 (0.23 – 1.22) 0.141

Follow advice 1.96 (1.28 – 3.01) 0.002 1.72 (0.84 – 3.54) 0.136

BLOCK 4 – decision making

Decision difficulty:

Easy 1 ¬

Hard 0.92 (0.53 – 1.60) 0.759

Did not know a decision was being made

0.49 (0.31 – 0.78) 0.002

Decision made:

Before counseling 1 ¬ 1

During counseling 0.78 (0.52 – 1.17) 0.226 1.43 (0.70 – 2.91) 0.323

After counseling 0.48 (0.33 – 0.72) <0.001 0.54 (0.30 – 0.95) 0.033

BLOCK 5 – costs and knowledge

Opinion on costs:

Alright to pay 1 ¬ 1 ¬

Only opt for CT in case of reimbursement

0.26 (0.13 – 0.52) <0.001 0.29 (0.10 – 0.89) 0.030

Only opt for CT if cheaper 0.59 (0.35 – 0.99) 0.045 1.59 (0.75 – 3.35) 0.227

Knowledge on purpose of CT 1.28 (1.09 – 1.51) 0.003

BLOCK 6 – region

Region:

Northeast 1 ¬ 1 ¬

Northwest 5.51 (4.00 – 7.60) <0.001 4.86 (3.01 – 7.83) <0.001

Block 1: 2 log likelihood 739.009, correctly predicted 67.9%.Block 2: 2 log likelihood 618.411, correctly predicted 76.9%.Block 3: 2 log likelihood 547.756, correctly predicted 79.7%.Block 4: 2 log likelihood 542.302, correctly predicted 79.2%.Block 5: 2 log likelihood 534.612, correctly predicted 79.4%.Block 6: 2 log likelihood 489.050, correctly predicted 82.8%.TOP = termination of pregnancy. OR = odds ratio. CI = confidence interval.


34

Acceptors of the CT indicated in 241 (91.6%) cases that they would also opt for the CT in a future pregnancy. Of the decliners, 366 (66.2%) women indicated that they would not consider the test in a future pregnancy, 175 women (31.6%) were uncertain, and only 12 (2.2%) stated they would opt for the CT (p < 0.001). If the purpose of CT would be broad-ened to detect also severe physical anomalies, 131 decliners (23.7%, p < 0.001) reported that they would opt for the CT. The results of the multiple regression analyses, used to examine whether the decision could be predicted from background variables, are presented in Table 6. The six blocks of determinants correctly predicted participation of the CT in 82.8% of women. Women’s socio-demographic background, their attitude towards Down syndrome and TOP as well as region, had a large independent impact on CT choice.

Discussion

Although the majority of women in this study appreciated to receive information on the CT, only 32% actually opted for it. The main reason to opt for the CT corresponded with the primary aim of the screening test, that is, to obtain an individualized risk assessment on Down syndrome. Almost half of the women who opted for CT would consider TOP in case of Down syndrome. Women’s motivations to decline the CT were diverse; two thirds indicated that Down syndrome would not be a reason to terminate a pregnancy, one third indicated that they considered their prior risk to be low and a quarter of all women indicated that they had doubts about the reliability of the screening test. Exclu-sion of the 17 women who opted for prenatal diagnosis is unlikely to have biased our results. It was a small group, with heterogeneous reasons to opt for or decline the CT and heterogeneous opinions on prenatal diagnosis and TOP. Furthermore, the incidence of congenital anomalies is low. The CT uptake in this study (32%) is considerably lower than uptake rates reported in Denmark (>90%) and France (88%).4,5 In England, the uptake rates vary per region, rang-ing from 98% in the London area to 20% in Lancaster.6-10 The CT uptake in the present study is lower than uptake rates previously reported in research settings in the Nether-lands (86% and 53%, respectively) prior to introduction of the national screening pro-gram.1,2 A similar decreasing trend was seen in the UK, where uptake rates over the years decreased from 83% to 41% (1993-2005).7

What are the reasons behind this decreasing trend? Our study shows that most women declined the CT because they would not consider TOP in case of Down syndrome. More-over, 23.4% of the decliners would opt for the CT if it was not only aimed at detecting Down syndrome, and 39% of the decliners would consider TOP in case of a severe physi-cal anomaly, suggesting that disease perception also plays a role. In the Netherlands, good specialized medical care, family support, and special education contribute to a high societal acceptance of children with Down syndrome. Although this may explain the overall low uptake, it does not explain the difference in uptake between the NE and NW regions as good facilities for children with Down syndrome are available throughout the country. Our results indicate that in the Netherlands, there are substantial inter-regional differences in attitudes toward Down syndrome and TOP. Similar findings were report-ed by Shanta et al. who concluded that attitude towards the CT and TOP had a larger im-


Chapter 2

35

pact on the uptake of screening than knowledge on the CT despite similar information and counseling.9 In our study, women’s age and parity did not differ by region. These determinants are therefore unlikely to explain the regional inequalities. An Australian study related differences in uptake to inequality in access to screening for women liv-ing in remote areas.11,12 Although the NW of the Netherlands is more densely populated than the NE region, these differences do not compare with the Australian situation. In summary, not only different attitudes towards TOP but also different attitudes towards Down syndrome are likely the reasons for the low and variable uptake rate.9

Our study indicates that Dutch women still perceive maternal age as a strong and reli-able predictor of Down syndrome risk. In the national information leaflet on Down syn-drome screening, substantial emphasis is put on the age-related risk, and it is suggested that the CT performs better in older than in younger women.13 This belief is strength-ened by the age-related CT reimbursement policy which undermines equal access and may give the impression that the CT in younger women is unnecessary. In our study, one in 14 young women declined the CT because they found this test too expensive. The effect of reimbursement could be more substantial than our results suggest, because respondents probably are reluctant to mention costs as main reason to decline the CT. Furthermore, women seem to fail to make a distinction between the age-related risk and their individual risk, and fail to realize that the age-related risk is only one of the con-stituting elements of the individual risk.14,15 Only unbiased counseling can adjust these assumptions. Our results suggest that some of the healthcare professionals in the two regions may not have counseled without bias. This is unlikely to explain the difference in CT uptake because the increase in predictive power is less than 3% (see block 3, Table 6). However, on the basis of our data, it is impossible to conclude if they truly deviated from value neutrality or rather made a shared decision without affecting women’s autonomy. In order to improve counseling and informed decision making, we propose that both in the national information leaflet and during the counseling, it is emphasized that mater-nal age is part of the risk assessment and not an independent determinant of risk. More-over, a more objective explanation of the performance of the CT to younger women and abolishment of the financial threshold would stimulate equal access.

Conclusion

The uptake of the CT in this study is low, especially among younger women and women from the NE region of the Netherlands. The main reason for the low uptake is the rela-tively positive attitude towards Down syndrome and a negative attitude towards TOP. Moreover, the perception of maternal age as strong predictor of Down syndrome risk and the inequality of access to care, due to the financial threshold for younger women, are likely to affect participation in screening.

ACKNOWLEDGEMENTS

We would like to thank Mrs. S. Binnema (office manager, prenatal screening foundation, NE Netherlands) and all the participating ultrasound clinics for their contribution to this study.


36

References

1. Muller M. A., Bleker O. P., Bonsel G. J. et al. Women’s opinions on the offer and use of nuchal translucency screening for Down syndrome. Prenat Diagn 2006; 26(2): 105-11.

2. Van den Berg M., Timmermans D. R., Kle-inveld J. H. et al. Accepting or declining the offer of prenatal screening for congenital defects: test uptake and women’s reasons. Prenat Diagn 2005; 25(1): 84-90.

3. Fransen M. P., Wildschut H. I., Mackenbach J. P. et al. Ethnic and socioeconomic differ-ences in uptake of prenatal diagnostic tests for Down’s syndrome. Eur J Obstet Gynecol Reprod Biol 2010; 151 (2): 158-62.

4. Seror V., Ville Y. Prenatal screening for Down syndrome: women’s involvement in decision-making and their attitudes to screening. Prenat Diagn 2009; 29(2): 120-8.

5. Ekelund C. K., Petersen O. B., Skibsted L. et al. First-trimester screening for trisomy 21 in Denmark: implications for detection and birth rates of trisomy 18 and trisomy 13. Ultrasound Obstet Gynecol 2011; 38(2): 140-4.

6. Dormandy E., Michie S., Weinman J. et al. Variation in uptake of serum screening: the role of service delivery. Prenat Diagn 2002; 22(1): 67-9.

7. Gidiri M., McFarlane J., Holding S. et al. Maternal serum screening for Down syn-drome: are women’s perceptions changing? BJOG 2007; 114(4): 458-61.

8. Rowe R., Puddicombe D., Hockley C. et al. Offer and uptake of prenatal screening for

Down syndrome in women from different social and ethnic backgrounds. Prenat Di-agn 2008; 28(13): 1245-50.

9. Shantha N., Granger K., Arora P. et al. Wom-en’s choice for Down’s screening – a com-parative experience in three district general hospitals. Eur J Obstet Gynecol Reprod Biol 2009; 146(1): 61-4.

10. Spencer K., Spencer C. E., Power M. et al. Screening for chromosomal abnormalities in the first trimester using ultrasound and maternal serum biochemistry in a one-stop clinic: a review of three years prospective experience. BJOG 2003; 110(3): 281-6.

11. Maxwell S., Brameld K., Bower C. et al. Socio-demographic disparities in the up-take of prenatal screening and diagnosis in Western Australia. Aust N Z J Obstet Gynae-col 2011; 51(1): 9-16.

12. Muggli E. E., Collins V. R., Halliday J. L. Map-ping uptake of prenatal diagnosis for Down syndrome and other chromosome abnor-malities across Victoria, Australia. Aust N Z J Obstet Gynaecol 2006; 46(6): 492-500.

13. RIVM. Official leaflet on Down syndrome screening.

14. Marteau T. M., Kidd J., Cook R. et al. Per-ceived risk not actual risk predicts uptake of amniocentesis. Br J Obstet Gynaecol 1991; 98(3): 282-6.

15. Timmermans DRM. Prenatal screening and the communication and perception of risks. Int Congr Ser 2005; 1279(0): 234-43.


3Intra-operator and inter-operator reliability of manual and

semiautomated measurement of fetal nuchal translucency:

a cross sectional study

M. Bakker 1 P. Mulder 1 E. Birnie 1,2

C. M. Bilardo 1


University Medical Centre, Groningen, the Netherlands.2 Department of Genetics, University Medical Centre Groningen,

University of Groningen, Groningen, the Netherlands.

Published in Prenatal Diagnosis 2013; 33: 1264-1271.


38

Intra-operator and inter-operator reliability of manual and semiautomated measurement of fetal nuchal translucency: a cross sectional study

M. Bakker1, P. Mulder1, E. Birnie1,2 and C. M. Bilardo1

1 Department of Obstetrics and Gynaecology, Fetal Medicine Unit, Univer-sity Medical Centre, Groningen, the Netherlands.


Objective: The goal of this study was to examine the intra-operator and inter-operator differ-ences of the manual and semiautomated nuchal translucency (NT) measurements and to evaluate if these differences alter women’s risk status.

Methods: A cross sectional study was performed. Two operators obtainedmanual and semiautomated NT measurements of 153 NT images. The maximal acceptable difference in NT measurements within and between operators was 0.15 mm. Intra and inter-operator differences were analyzed by the paired Student’s t-test and homogeneity of variances by the Levene’s test. Intra-operator and in-ter-operator agreement were quantified with Bland and Altman’s limits of agree-ment, and changes in women’s risk sta-

tus were tested with the binomial test.

Results: Intra-operator agreement was high for each of the measurementmethods. Operator 1 had lower SDS for manual measurements. Conversely, operator 2 had lower SDS of the differences for semiautomated measurements, al-though the SD never reached the same level as operator 1. Inter-operator agree-ment was highest for the semiauto-mated measurements. Changes in risk status occurred between the manual and inner-middle method resulting in dif-ferent clinical policies in up to 1 out of 20 cases.

Conclusion: Well-trained operators do not seem to benefit from the use of the semiauto-mated measurement methods.

Introduction

The combined test (CT) offers women an individual risk assessment for trisomy 21, 13, and 18 in the first trimester of pregnancy.1,2 The test is based on the measurement of the fetal nuchal translucency (NT), maternal age and maternal serum markers (β-hCG and PAPP-A). The NT is themost effective marker of trisomy 21 and is able to detect about 75% to 80% of the affected fetuses for a false positive rate of about 3% to 5%.1,3 Moreover, an enlarged NT is associated with other chromosomal anomalies, genetic syndromes, and


Chapter 3

39

structural anomalies.4 It is therefore important that the NT is measured precisely. The Fetal Medicine Foundation (FMF) has developed guidelines to promote a standard-ized measurement technique for obtaining a valid and precise manual NT measurement. Aim of the guideline is to achieve uniformity among different operators and guarantee a reliable risk assessment.5 However, the acquirement of the midsagittal plane, the selec-tion of the area containing the maximum NT and the placement of the calipers are still prone to error with themanual technique, which may compromise the performance of screening.6-8

In order tominimize variability in themeasurement of the NT, a semiautomated NT measurement has been developed recently (sono-NT, GE Medical Systems). Standardiza-tion through semiautomated measurement is thought to lower the standard deviation (SD) of the distribution of NT measurements, increase its precision, and enhance the cor-rect discrimination of normal from trisomic fetuses. Recent studies show that semiauto-mated NT measurement indeed helps to standardize the NT assessment by lowering the SD, especially in inexperienced sonographers.9-12 The aim of this study was to examine the intra-operator and inter-operator agreement of the manual and semiautomated mea-surements, and to evaluate if differences were not only significant in terms of precision but also in terms of changed individual risk status.

Methods

DESIGN

A cross sectional study on the reliability of the manual and semiautomated measure-ments of the NT was conducted at the University Medical Centre Groningen. We se-lected NT images from singleton pregnancies obtained at 11+0 to 13+6 weeks of gesta-tion, stored between 2011 and 2012, and satisfying the FMF guidelines. All images were acquired transabdominally, with harmonics on, using a Voluson E8 equipped with a 4 to 8Hz probe (GE Medical Systems). For each image, two FMF-certified operators measured the NT using the manual first and semiautomated NT measurement technique (SONO-NT©: inner-inner and innermiddle method) second. Operator 1 is FMF-certified since May 2010 and operator 2 since October 2009. Each operator performs more than 100 NT measurements annually. Each operator performed the measurements twice, with an in-terval of 2 to 4 weeks between the first and second measurement to minimize any recall bias. Because we only used images without previous measurements, both operators were blinded for all measurement methods.

NUCHAL TRANSLUENCY MEASUREMENT USING SONO-NT©

After the midsagittal plane is acquired manually, the operator places an adjustable box over a large area of the NT containing the thickest part. The software uses the original image within this box and a corresponding ‘edge image’, that reflects the changes in brightness rather than the brightness itself, to define the echogenic lines delineating the NT that should be used for measurement. The upper caliper will be automatically placed


40

on the inner border of the upper line, and the operator then decides whether the lower caliper is placed on the inner border (inner-inner method) or on the middle of the lower echogenic line (inner-middle method). The software connects every point on one line to all possible points on the other line. For each point on the first line, the shortest distance to the other line is calculated and the maximum NT distance from all these distances is selected. For the inner-middle method, both inner and outer borders of the lower echo-genic line are detected, and the midpoint between them is calculated.9,10


Comparisons of NTS between the inner-inner method and the manual method, as well as between the inner-middle method and the manual method were described with con-ventional statistics. Intra-operator differences of the NTS measured with the manual, inner-inner and inner-middle methods were described as mean difference (SD, 95% CI) and as standard deviation of the difference between the two measurements divided by √2. Inter-operator differences of the NTS measured with the manual, inner-inner and inner-middle methods were described as mean difference (SD, 95% CI) of operator 1 to that of operator 2. Differences in SDS (homogeneity of variances) between operators were tested with the Levene’s test. Intra-operator and inter-operator agreement were quantified descriptively with Bland and Altman’s limits of agreement (LOAS) and their 95% confidence intervals. Themaximal clinically acceptable mean difference in NT measurements within and between operators was considered to be 0.15 mm7, and tested with the one-sample Student’s

Table 1 — Characteristics

N=153¬ ¬ ¬ ¬ ¬ OPERATOR 1 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ OPERATOR 2 ¬ ¬ ¬ ¬ ¬

Manual Inner-inner Inner-middle Manual Inner-inner Inner-middle

Mean [CI 95%] 1.878[1.781, 1.975]

1.799[1.696, 1.901]

2.040[1.940, 2.150]

1.857[1.759, 1.955]

1.759[1.655, 1.863]

1.970[1.870, 2.010]

Standard Deviation 0.606 0.643 0.653 0.6140 0.653 0.644

Range 0.940 – 4.840 0.800 – 4.600 1.000 – 5.000 0.660 – 4.490 0.700 – 4.700 1.000 – 5.000

Median [p25 – p75] 1.730[1.460 – 2.195]

1.700[1.300 – 2.150]

1.810[1.410 – 2.115]

1.600[1.300 – 2.100]

1.900[1.500 – 2.300]

1.900[1.500 – 2.300]

Table 2 — Intra-operator differences


SD of measurement

errorMean ∆[SD ∆] P†

SD of measurement

errorMean ∆[SD ∆] P**

Manual – manual 0.054 0.010 [0.076] <0.001 0.156 0.008 [0.220] <0.001

Inner-inner – inner-inner 0.062 -0.005 [0.088] <0.001 0.120 0.016 [0.170] <0.001

Inner-middle – inner-middle 0.086 -0.017 [0.122] <0.001 0.122 -0.012 [0.172] <0.001

Manual – inner-inner 0.122 -0.080 [0.173] <0.001 0.180 -0.098 [0.225] <0.001

Manual – inner-middle 0.123 0.163 [0.174] 0.348 0.145 0.114 [0.205] 0.032

† One-sample t-test (0.15 mm), applied to the differences between the two observations. A significant p-value implies that the H0: mean ∆ >= 0.15 mm is rejected.∆ Represents difference between measurements within operators.SD = standard deviation.


Chapter 3

41

t-test. Agreement of NT measurements within or between operators was expressed with the intraclass correlation coefficient (ICC). The NT measurements were used to estimate the associated risk of trisomy 21, in As-traia, and to classify women as low (<1 : 200) or high risk (≥1 : 200), keeping the other parameters of the risk calculation unchanged. In the Netherlands, prenatal invasive test-ing is offered when the risk estimate after the CT is ≥1 : 200. A difference in NT measure-ments was considered clinically relevant when a change of woman’s risk status occurred (from low to high risk or vice versa). The proportion of changes in women’s risk status compared to 0 was tested with the binomial test.

Results

A total of 153 NT images were selected for the study. Median crown-rump length (CRL) was 66.6 mm (IQR 58.5 – 71.4 mm) and median NT was 1.8 mm (IQR 1.5 – 2.2 mm). Table 1 shows the characteristics of the NT measurements for each operator.

INTRA-OPERATOR AGREEMENT OF MEASUREMENTS

For both operators, the lowest mean NT measurement was obtained by the inner-inner method and the highest by the inner-middle method, whereas the mean of the manual method fell in between. The lowest SD for both operators was obtained when the mea-surement was performed manually (Table 1).

Table 3 — Intra-operatorlimits of agreement

N=153¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ OPERATOR 1 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬

ICC(95% CI) RC [LOA] LOA 95% CI

Manual – manual 0.992(0.989 – 0.994)

0.149 [-0.139 – 0.159][-0.178 – 0.169]

(-0.1392 – -0.1388)(0.1588 – 0.1592)

Inner-inner – inner-inner 0.990(0.987 – 0.993)

0.173 [-0.178 – 0.169] (0.1783 – -0.1777)(0.1687 – 0.1693)

Inner-middle – inner-middle 0.982(0.975 – 0.987)

0.239 [-0.256 – 0.222] (-0.2566 – -0.2554)(0.2214 – 0.2226)

Manual – inner-inner 0.954(0.917 – 0.972)

0.339 [-0.418 – 0.260] (-0.4192 – -0.4169)(0.2589 – 0.2612)

Manual – inner-middle 0.931(0.644 – 0.974)

0.342 [-0.178 – 0.505] (-0.1792 – -0.1768)(0.5038 – 0.5062)

N=153¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ OPERATOR 2 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬

ICC(95% CI) RC [LOA] LOA

Manual – manual 0.935(0.911 – 0.952)

0.431 [-0.422 – 0.439] (-0.4239 – -0.4201)(0.4371 – 0.4409)

Inner-inner – inner-inner 0.964(0.951 – 0.974)

0.333 [-0.317 – 0.349] (-0.3181 – -0.3159)(0.3479 – 0.3501)

Inner-middle – inner-middle 0.963(0.949 – 0.973)

0.338 [-0.350 – 0.325] (-0.3511 – -0.3489)(0.3239 – 0.3264)

Manual – inner-inner 0.926(0.873 – 0.954)

0.441 [-0.540 – 0.343] (-0.5420 – -0.5381)(0.3411 – 0.3450)

Manual – inner-middle 0.932(0.853 – 0.963)

0.401 [-0.287 – 0.516] (-0.2886 – -0.2854)(0.5144 – 0.5176)

ICC = intraclass correlation coefficient. RC = repeatability coefficient. LOA = limits of agreement. CI = confidence interval.

Table 2 — Intra-operator differences


SD of measurement

errorMean ∆[SD ∆] P†

SD of measurement

errorMean ∆[SD ∆] P**

Manual – manual 0.054 0.010 [0.076] <0.001 0.156 0.008 [0.220] <0.001

Inner-inner – inner-inner 0.062 -0.005 [0.088] <0.001 0.120 0.016 [0.170] <0.001

Inner-middle – inner-middle 0.086 -0.017 [0.122] <0.001 0.122 -0.012 [0.172] <0.001

Manual – inner-inner 0.122 -0.080 [0.173] <0.001 0.180 -0.098 [0.225] <0.001

Manual – inner-middle 0.123 0.163 [0.174] 0.348 0.145 0.114 [0.205] 0.032

† One-sample t-test (0.15 mm), applied to the differences between the two observations. A significant p-value implies that the H0: mean ∆ >= 0.15 mm is rejected.∆ Represents difference between measurements within operators.SD = standard deviation.


42

Figure 1 — Bland Altman plots and their corresponding limits of agreement for differences between repeated measurements within operators.

[b][a]

[c]

[e]

[d]

[f]


Chapter 3

43

Mean intra-operator differences and SDS for the various measurement methods are shown in Table 2. Operator 1 had a lower SD for measurements performed manually than for measurements performed by the inner-inner and innermiddle method. The SD of operator 2, however, was higher using the manual method than for the inner-inner and inner-middle methods, although this difference was not significant. Figures 1 and 2 and Table 3 show Bland Altman plots and their corresponding limits of agreement (LOA) for differences between repeated measurements within operators and between measurement methods within operators. The LOAS for differences between repeated measurements were >+0.15 mm or <-0.15 mm for both operators, except for the upper limit of agreement of the repeat manual measurement of operator 1. However, the mean differences for the repeat measurements were significantly smaller than 0.15 mm for both operators as tested with the one-sample t-test, see Table 2. The LOAS for differences between measurement methods were >+0.15 mm or <-0.15

Figure 2 — Bland Altman plots and their corresponding limits of agreement for differences between measurement methods within operators.

[b][a]

[c] [d]


44

mm. The mean differences between different measurement methods were however sig-nificantly smaller than 0.15 mm for both operators, except for the difference between the manual and inner-middle method of operator 1 (Table 2). For each operator, the agreement between repeat measurements and between differ-ent measurement methods was high (Table 3). Operator 1 had the highest predictability for the repeat manual measurements (ICC 0.992). The ICC was 0.990 for the repeat innerinner measurements and 0.982 for repeat inner-middle measurements. Agreement between the different measurement methods was lower, ICC 0.954 (manual vs. inner-inner) versus ICC 0.931 (manual vs. inner-middle). For operator 2, agreement of the repeated inner-inner measurements (ICC 0.964) was higher than the repeated manual measurements (ICC 0.935) and inner-middle measure-ments (ICC 0.963). Predictability between different measurement methods was a lower, ICC 0.926 (manual vs. inner-inner) versus ICC 0.932 (manual vs. inner-middle).

INTRA-OPERATOR DIFFERENCES OF ESTIMATED RISK

Changes in estimated risk of trisomy 21 between repeated measurements were found (Table 4). For operator 1, these differences were only significant for the inner-middle method (0.048) and for the comparison between the manual and semiautomated meth-ods (manual-inner-inner: 0.017, manual-inner-middle: <0.001). For operator 2, these differences were significant for the manual method (0.048), the inner-middle method (0.048), and for the comparison between the manual and semiautomated methods (manualinner-inner: <0.001, manual-inner-middle: <0.001).

Table 4 — Intra-operator differences in risk status (risk <1 : 200 or ≥1 : 200)

¬ ¬ ¬ ¬ ¬ OPERATOR 1 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ OPERATOR 2 ¬ ¬ ¬ ¬ ¬Discordant pairs (%)

[CI 95%] p*Discordant pairs (%)

[CI 95%] p*

Manual – manual 0/153 (0.0%) [0 – 0.024] >0.99 3/153 (2.0%) [0.004 – 0.056] 0.048

Inner-inner – inner-inner 1/153 (0.7%) [0.002 – 0.036] 0.360 2/153 (1.3%) [0.002 – 0.046] 0.130

Inner-middle – inner-middle 3/153 (2.0%) [0.004 – 0.056] 0.048 3/153 (2.0%) [0.004 – 0.056] 0.048

Manual – inner-inner 4/153 (2.6%) [0.007 – 0.066] 0.017 8/153 (5.2%) [0.023 – 0.100] <0.001

Manual – inner-middle 8/153 (5.2%) [0.023 – 0.100] <0.001 7/153 (4.6%) [0.019 – 0.092] <0.001

* Binomial test.CI = confidence interval.

Table 5 — Inter-operator differences

N Mean ∆ SD of ∆Equality of variance p* p**

Manual (operator 1 – operator 2) 153 0.021 0.247 0.767 <0.001

Inner-inner (operator 1 – operator 2) 153 0.040 0.199 0.591 <0.001

Inner-middle (operator 1 – operator 2) 153 0.070 0.200 0.347 <0.001

* Levene’s Test for differences in SD - H0: mean manual (operator 1) = mean manual (operator 2). A non-significant p-value implies that H0 cannot be rejected.** One-sample t-test (0.15 mm), applied to the differences between the two observations. A significant p-value implies that the H0: mean ∆ >= 0.15 mm is rejected.∆ Represents difference between operator 1 and operator 2.SD = standard deviation.


Chapter 3

45

INTER-OPERATOR AGREEMENT OF MEASUREMENTS

Mean differences and SDS of the differences between operators for each method are shown in Table 5. The mean difference between operators was the lowest for the manual method. The SD of the differences of the manual method (0.247) was higher than the SD of the inner-inner (0.199) and inner-middle methods (0.197), but these SDS did not differ significantly (Table 4). The mean differences between operators for the different measurement methods were significantly smaller than 0.15 mm (Table 5). Agreement was the highest for the semiau-tomated measurements and the LOAS for the different measurement methods were all >+0.15 mm or <-0.15 mm (Table 6).

INTER-OPERATOR DIFFERENCES OF ESTIMATED RISK

In view of the inter-operator differences, women’s risk status altered in 2.6% to 3.3% of the cases, depending on the measurement method. All differences in estimated risk of trisomy 21 between the operators were significant (Table 7).

Discussion

In this study, we evaluated differences in intra-operator and inter-operator agreement between the manual and semiautomated measurements, and studied the clinical rel-evance of lack of agreement in terms of altered CT risk assessment. The intra-operator agreement was high for each of the measurement methods. Opera-tor 1 had lower SDS for measurements performed manually. Conversely, operator 2 had lower SDS of the differences for semiautomated measurements, although the SD of the


ICC (95% CI) RC LOA LOA (95% CI)

Manual (operator 1) – (operator 2) 0.918 (0.889 – 0.940)

0.484 [-0.463 – 0.505] (-0.4653 – -0.4607)(0.5027 – 0.5073)

Inner-inner (operator 1) – (operator 2) 0.951 (0.933 – 0.965)

0.390 [-0.350 – 0.430] (-0.3515 – -0.3485)(0.4285 – 0.4315)

Inner-middle (operator 1) – (operator 2) 0.949 (0.921 – 0.966)

0.386 [-0.316 – 0.455] (-0.3175 – 0.3145)(0.4535 – 0.4565)

ICC = intraclass correlation coefficient. RC = repeatability coefficient.LOA = limits of agreement. CI = confidence interval.

Table 7 — Inter-operator differences in risk status (risk <1 : 200 or ≥1 : 200)

Discordant pairs (%)[CI 95%] p*

Manual (operator 1) – manual (operator 2) 5/153 (3.3%) [0.019 – 0.092] 0.006

Inner-inner (operator 1) – inner-inner (operator 2) 5/153 (3.3%) [0.019 – 0.092] 0.006

Inner-middle (operator 1) – inner-middle (operator 2) 4/153 (2.6%) [0.007 – 0.066] 0.017


Table 4 — Intra-operator differences in risk status (risk <1 : 200 or ≥1 : 200)

¬ ¬ ¬ ¬ ¬ OPERATOR 1 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ OPERATOR 2 ¬ ¬ ¬ ¬ ¬Discordant pairs (%)

[CI 95%] p*Discordant pairs (%)

[CI 95%] p*

Manual – manual 0/153 (0.0%) [0 – 0.024] >0.99 3/153 (2.0%) [0.004 – 0.056] 0.048

Inner-inner – inner-inner 1/153 (0.7%) [0.002 – 0.036] 0.360 2/153 (1.3%) [0.002 – 0.046] 0.130

Inner-middle – inner-middle 3/153 (2.0%) [0.004 – 0.056] 0.048 3/153 (2.0%) [0.004 – 0.056] 0.048

Manual – inner-inner 4/153 (2.6%) [0.007 – 0.066] 0.017 8/153 (5.2%) [0.023 – 0.100] <0.001

Manual – inner-middle 8/153 (5.2%) [0.023 – 0.100] <0.001 7/153 (4.6%) [0.019 – 0.092] <0.001



N Mean ∆ SD of ∆Equality of variance p* p**

Manual (operator 1 – operator 2) 153 0.021 0.247 0.767 <0.001

Inner-inner (operator 1 – operator 2) 153 0.040 0.199 0.591 <0.001

Inner-middle (operator 1 – operator 2) 153 0.070 0.200 0.347 <0.001

* Levene’s Test for differences in SD - H0: mean manual (operator 1) = mean manual (operator 2). A non-significant p-value implies that H0 cannot be rejected.** One-sample t-test (0.15 mm), applied to the differences between the two observations. A significant p-value implies that the H0: mean ∆ >= 0.15 mm is rejected.∆ Represents difference between operator 1 and operator 2.SD = standard deviation.


46

difference never reached the same level as operator 1. It seemed that operator 2 benefit-ted from the use of the semiautomated measurements. Agreement between operators was highest for the semiautomated measurements sug-gesting that the use of semiautomated measurements results in lower variability be-tween operators (standardization), however, differences in variance were not significant. There seems to be no real benefit of the semiautomated measurements over the manual measurement in terms of measurement variability. Earlier studies have addressed the reproducibility of the manual and semiautomated NT measurement and reported on the benefit of the use of the semiautomated meth-od.9,10,12 Moratalla et al. found a between-operator SD of 0.0149 for the inner-middle method, 0.109 for the manual method, and high ICCs for both methods (0.98 for the inner-middle method and 0.85 for the manual method). Their conclusion was that the measurement of the NT is more reliable when a semiautomated approach is used rather than the manual method, because it will reduce variation between operators.9 Kagan et al. found an estimated SD of differences between operators of ~0.02 for the inner-inner method, ~0.03 for the inner-middle method and ~0.11 for the manual method.12 They concluded that especially non-experts will benefit from this method as it helps to stan-dardize caliper placement. Abele et al. found that the inter-operator variability for all measurement methods was similar for experts, and these were lower than those of less experienced sonographers. They also concluded that inexperienced operators will benefit from the semiautomated method, but they added that the most important contributor to inter-operator variability is image acquisition.10 Our results are consistent with the aforementioned studies; however, we could interpret these studies conversely and state that the semiautomated measurement does not seem to be useful in well-trained opera-tors. Although the inter-operator and intra-operator differences were larger than the 0.15 mm threshold in this study, the impact of measurement variability within and between operators on women’s risk status was small. However, these small differences translated in different clinical policies in up to 1 out of 20 cases, which we clinically cannot neglect. Both operators in our study are FMF accredited and perform the same number of NT measurements annually, except operator 2 has more years of experience as a sonogra-pher. Most studies evaluating measurement variability between the manual and semiau-tomated methods conclude that less experienced operators will benefit from the semiau-tomated measurement as it helps standardize caliper placement.10-12 Paradoxically, in our study, it was the more experienced operator 2 who seemed to benefit from the use of the semiautomated method. This suggests that more experience does not necessarily imply accurate measurements. Not only experience but also attitude as part of competence seems also prerequisite for obtaining precise NT measurements. This is reflected by the higher SD between measurements of operator 2 in comparison with operator 1. Precise measurement of the NT not only consists of the selection of the thickest part and accurate placement of calipers, but also requires the appropriate acquisition of the midsagittal plane and accurate measurement of the CRL. Use of the semiautomated mea-surement theoretically can help reducing measurement variability in the first two steps, however correct image acquisition and measurement of the CRL still remain operator dependent. Kagan et al. showed that an underestimation and overestimation of the CRL has a major impact, resulting in substantial underestimation or overestimation of those


Chapter 3

47

risks.13 Also, deviation from themidsagittal plane can have a great impact on women-specific risks, and this impact is higher than measurement variability due to caliper placement.14,7 The importance of factors influencing image quality, for example, gain and harmonics, are well known.15 From a technical point of view, the main contributor to intra-operator and inter-operator differences is thus not caliper placement, but the acquisition of the optimal NT image complying with all the FMF criteria. This means that precision of NT measurements is still largely dependent on appropri-ate training, adherence to strict criteria, coincidental circumstances (e.g. fetal position, maternal BMI, and time allocated), and, last but not least, on the operator’s personal at-titude in terms of endurance and accuracy. At this moment, there seems to be no real advantage of the semiautomated measure-ment method over the manual method to warrant its implementation on a large scale. It is unclear which operator will truly benefit from semiautomated measurements because well-trained operators do not appear to benefit from this method, and secondly, how this selection should be performed because attitude besides experiences seems to play a ma-jor role. A limitation of this study is that no more than two operators were included. Our results may be reassessed with multiple operators of various levels of experience and attitude in future studies. At present, normal ranges for NT measurements used in the FMF risk assessment al-gorithm are based on manual NT measurements. No study has evaluated the impact of the semiautomated methods on the screen positive rate. Advances that may have a larger impact in reducing inter-operator variability are software packages that provide the pos-sibility of selecting the right midsagittal plane while scanning with the aid of a 3D.16

Conclusion

Well-trained operators do not seemto benefit fromthe use of the semiautomated mea-surement methods. Manual measurement of the NT according to the FMF guidelines is mandatory and, in itself, should be sufficient to obtain a reliable risk calculation for pre-natal trisomies screening.

References

1. Kagan K. O., Etchegaray A., Zhou Y. et al. Prospective validation of firsttrimester com-bined screening for trisomy 21. Ultrasound Obstet Gynecol 2009; 34(1): 14-8.

2. Kagan K. O., Wright D., Baker A. et al. Screen-ing for trisomy 21 by maternal age, fetal nuchal translucency thickness, free beta-hu-man chorionic gonadotropin and pregnan-cy-associated plasma protein-A. Ultrasound Obstet Gynecol 2008; 31(6): 618-24.

3. Kagan K. O., Wright D., Valencia C. et al. Screening for trisomies 21, 18 and 13 by ma-ternal age, fetal nuchal translucency, fetal heart rate, free betahCG and pregnancy-associated plasma protein-A. Hum Reprod 2008; 23(9): 1968-75.

4. Bilardo C. M., Pajkrt E., De Graaf I. et al. Out-come of fetuses with enlarged nuchal trans-lucency and normal karyotype. Ultrasound Obstet Gynecol 1998; 11(6): 401-6.


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5. The fetal medicine foundation, 2010.6. Pandya P. P., Altman D. G., Brizot M. L. et

al. Repeatability of measurement of fetal nuchal translucency thickness. Ultrasound Obstet Gynecol 1995; 5(5): 334-7.

7. Kagan K. O., Wright D., Etchegaray A. et al. Effect of deviation of nuchal translucency measurements on the performance of screening for trisomy 21. Ultrasound Obstet Gynecol 2009; 33(6): 657-64.

8. Schmidt P., Staboulidou I., Elsasser M. et al. How imprecise may the measurement of fetal nuchal translucency be without wors-ening first-trimester screening? Fetal Diagn Ther 2008; 24(3): 291-5.

9. Moratalla J., Pintoffl K., Minekawa R. et al. Semi-automated system for measurement of nuchal translucency thickness. Ultra-sound Obstet Gynecol 2010; 36(4): 412-6.

10. Abele H., Hoopmann M., Wright D. et al. In-tra- and interoperator reliability of manual and semi-automated measurement of fetal nuchal translucency by sonographers with different levels of experience. Ultrasound Obstet Gynecol 2010; 36(4): 417-22.

11. Grange G., Althuser M., Fresson J. et al. Semi-automated adjusted measurement of nuchal translucency: feasibility and repro-

ducibility. Ultrasound Obstet Gynecol 2011; 37(3): 335-40.

12. Kagan K. O., Abele H., Yazdi B. et al. Intra-operator and interoperator repeatability of manual and semi-automated measurement of increased fetal nuchal translucency ac-cording to the operator’s experience. Prenat Diagn 2011; 31(13): 1229-33.

13. Kagan K. O., Hoopmann M., Baker A. et al. Impact of bias in crown-rump length mea-surement at first-trimester screening for trisomy 21. Ultrasound Obstet Gynecol 2012; 40(2): 135-9.

14. Abele H., Wagner N., Hoopmann M. et al. Effect of deviation from the mid-sagittal plane on the measurement of fetal nuchal translucency. Ultrasound Obstet Gynecol 2010; 35(5): 525-9.

15. Pasquini L., Tondi F., Rizzello F. et al. Impact of tissue harmonic imaging on measure-ment of nuchal translucency thickness. Ul-trasound Obstet Gynecol 2010; 36(4): 423-6.

16. Cho H. Y., Kwon J. Y., Kim Y. H. et al. Com-parison of nuchal translucency measure-ments obtained using Volume NT(TM) and two- and threedimensional ultrasound. Ultrasound Obstet Gynecol 2012; 39(2): 175-80.


4Total pregnancy loss after chorionic villus sampling

and amniocentesis

M. Bakker 1

E. Birnie 1,2

P. Robles de Medina 3

Krystyna Sollie 1

E. Pajkrt 3

C. M. Bilardo 1



University of Groningen, Groningen, the Netherlands.3 Department of Obstetrics and Gynecology, Fetal Medicine Unit,

Academic Medical Centre, Amsterdam, the Netherlands.

Published in Ultrasound Obstetrics & Gynecology. 2016 Jun 3.

doi: 10.1002/uog.15986.


50

Total pregnancy loss after chorionic villus sampling and amniocentesis

M. Bakker1, E. Birnie1,2, P. Robles de Medina3, Krystyna Sollie1, E. Pajkrt3 and C. M. Bilardo1

1 Department of Obstetrics and Gynaecology, Fetal Medicine Unit, University Medical Centre, Groningen, the Netherlands.


3 Department of Obstetrics and Gynecology, Fetal Medicine Unit, Academic Medical Centre, Amsterdam, the Netherlands.

Objective: To identify maternal-, operator-, and procedure-related variables affecting the procedure related pregnancy loss after transcervical (TC) and transabdominal (TA) chorion villus sampling (CVS) and amniocentesis (AC). To estimate the spontaneous and procedure-related fe-tal losses in comparable subgroups of women.

Methods: A retrospective cohort study conducted at the University Medical Centre Gron-ingen and the Academic Medical Centre Amsterdam, the Netherlands. The da-tabases of both centers were searched for singleton pregnancies that had undergone a combined test (CT) and/or an anomaly scan at around 20 weeks’ gestation, or an invasive procedure (CVS and/or AC) between January 2001 and December 2011. Maternal characteristics, obstetric history, technical aspects of the invasive procedure, ultrasound examina-tions and fetal and neonatal outcomes were available in 29.201 cases.

Results: Variables significantly associated with a higher fetal loss rate (FLR) were: for CVS (TC or TA) repeated attempts dur-

ing a procedure, the use of a TC-cannula instead of a biopsy forceps, gestational age of 13 weeks or more and pregnan-cies after assisted reproduction; for AC if the indication was a fetal anomaly or a family history of anomalies and repeated attempts during the procedure. In the group of women aged 36 years or older who did not undergo an invasive proce-dure the total spontaneous FLR after a first trimester scan was 1.40%, whereas after a TC or TA CVS the total FLR was 2.76% and 2.43%, respectively. Therefore, the additional risk of a TC - CVS was 1.36% (1:74) and this varied according to the instrument used: 0.27% for the forceps and 3.12% for the cannula. After a TA - CVS the risk was 1.03% (1:97). In women aged 36 years and older un-dergoing a 20 weeks scan the spontane-ous FLR was .63%. In the group women undergoing an AC performed solely for advanced maternal age the FLR was 1.11%. Therefore the additional risk of an AC was .48% (1:208).

Conclusion: The procedure related FLRs after a TA-CV, TC-CVS and AC appear lower than the current risks women are counselled on. All risks decreased when the level of ex-perience increased.


Chapter 4

51

Introduction

The wish of both women and doctors to avoid unnecessary procedures during preg-nancy is the driving force behind the search for optimal screening strategies and, more recently, non-invasive prenatal testing (cell free fetal DNA). The most important factor influencing uptake of invasive procedures (chorionic villus sampling (CVS) and amnio-centesis (AC)) is the procedure related fetal loss rate (FLR)1; which is the loss rate attrib-utable to the invasive procedure minus the spontaneous FLR. The latter is influenced by the maternal risk profile (age, weight, parity and obstetric history)2 and the pregnancy related risk profile (gestational age and presence of fetal anomalies).3-5 In contrast, the procedure related risk depends on the technique and instruments used, possible technical difficulties and operator experience.4,6,7 As a result of interaction between these variables the total FLR varies greatly.8

In literature procedure related FLRs range from 0.06% to 1.0% or even higher, for both CVS and AC.4,5,8-20 Most studies are observational and include mixed populations. Of the few randomized (controlled) trials Tabor et al compared women undergoing an AC with a control group, showing a 1% higher FLR in women undergoing amniocentesis.10 Lit-erature suggests that transabdominal CVS and AC seem to have smaller and comparable risks.11,12,14,21

It remains a challenge to report realistic risk figures, taking into account all variables that influence the procedure related FLR. Furthermore, some of the above mentioned studies were performed a while ago at a time when ultrasound systems were less ad-vanced and techniques and training in invasive procedures less standardized. A recent meta-analysis showed that accurate estimates of current procedure-related risks follow-ing invasive procedures are lacking.5 Many experts believe that procedure related risks need reevaluation.22 A new consensus should be reached to avoid discrepancies in infor-mation given among centers. The aim of this study was to identify maternal-, operator-, and procedure-related risk factors that modify the overall estimated risk of fetal loss and to create comparable sub-groups in order to calculate the total spontaneous FLR and the procedure-related FLR after CVS and AC.

Methods

DESIGN

This retrospective cohort study on spontaneous as well as procedure related FLRS in women undergoing CVS and AC was conducted in two University Medical Centres: the UMCG in Groningen, and the AMC in Amsterdam, the Netherlands. Data on all consecu-tive singleton pregnancies that had undergone CVS and/or AC, performed in the period between January1st, 2001 and December 31st, 2011, were retrieved from the databases of both hospitals. The same data were retrieved for women who had only undergone a com-bined test (CT) and/or an anomaly scan at 20 weeks of gestation. Both academic hospitals act as referral centers, but also have their own ‘low risk’ popu-


52

lation. Prenatal screening is offered to women in the form of the combined test and/or the 18-20 week anomaly scan. According to the national guideline women in the Neth-erlands undergoing a CVS and AC are always referred to a tertiary hospital. As a conse-quence this study includes both ‘low risk’ and referred women. The database was divided into 5 groups, depending on the examination(s) that women had undergone. The first group consisted of women who had undergone the CT (and 20 week anomaly scan), the second group of women who had only undergone a 20 week anomaly scan, the third of women who had undergone a TC or TA CVS, the fourth of women who had undergone an AC, and the last group consisted of women who had an AC after a (unsuccessful) CVS. In order to calculate the crude procedure related FLR, the spontaneous FLRS after 11-14 weeks and after the second trimester of pregnancy were calculated in two groups of women aged 36 years and older, who had undergone the CT and/or the 20 weeks anomaly scan. These spontaneous FLRS were then compared with the FLRS in three groups of women, who had undergone a TC CVS, TA CVS or AC on ma-ternal age indication only, without known a-priori risk factors. In all cases information was available on procedure related characteristics (indication, operator, technique) as well as on maternal characteristics, obstetric history and preg-nancy outcome.

DEFINITIONS

Outcome was classified into the following categories; alive, miscarriage, preterm labor, intra-uterine fetal demise (IUD), termination of pregnancy (TOP), fetal loss during labor or neonatal death. Alive was defined as a newborn showing signs of life after delivery. Miscarriage was defined as spontaneous delivery of the fetus before 24 weeks of gesta-tion. Preterm labor was defined as spontaneous delivery between 24 to 37 weeks of gesta-tion. Intra-uterine fetal demise (IUD) was defined as fetal death from 24 weeks onwards, prior to delivery. Fetal loss during labor was defined as intra-partum death from 24 weeks onwards. Neonatal death (NND) was defined as infant death before 28 days of age. The total spontaneous FLR was defined as the total of losses before 24 weeks of gestation in the group women of 36 years and older who did not underwent an invasive procedure. After 24 weeks of gestation this was the sum of preterm labor followed by loss and IUD. Procedure related FLR was defined as the total of fetal loss before 24 weeks of gestation based on the group of women undergoing an invasive procedure because of advanced maternal age, minus the background risk. Operator’s experience with performing a CVS was classified as; level 1: <50, level 2: be-tween 50-150 and level 3: >150 procedures performed in total. For AC, operator’s experi-ence was classified as; level 1: <50, level 2: 50-150, level 3: >150-500, level 4: >500 proce-dures performed in total. The number of procedures and attempts were classified as: (1) one procedure with one attempt, (2) one procedure with more than one attempt and (3) more than one procedure.


To compare differences in categorical variables between women who did or did not un-dergo CVS and/or AC, the X2 test (or Fisher’s Exact Test, if appropriate) was used.


Chapter 4

53

CVSN=4862

Figure 1 — Flowchart (crude data, no adjustment for background risk). Because of small numbers the group women who underwent a CVS and AC are not included in this flowchart.

CT = combined test. CVS = chorion villus sampling. AC = amniocentesis. TA-CVS: transabdominal chorion villus sampling. TC-CVS: transcervical chorion villus sampling. TFLR = total fetal loss rate.

CTN=9651

20 weeks scanN=6432

ACN=7970

Study PopulationN=29,201

TFLR1.64 % (N=158)

≥ 36 yearsFLR 1.91% (N=86)

TFLR0.82% (N=58)

≥ 36 yearsFLR 1.58% (N=5)

TFLR3.05% (N=243)

≥ 36 yearsFLR 1.42% (N=75)

TA-CVSTFLR 2.42%

(N=13)

TC-CVSTFLR 3.24%

(N=47)

Associations between single co-variables and outcome were assessed by univariate bina-ry logistic regression and expressed in odds ratios (ORs, 95% confidence intervals). Cor-relations were used to check for confounders and interactions. Multiple binary logistic regression analysis (backward stepwise elimination method) was performed to evaluate the adjusted impact of co-variables. Statistical significance was defined as p < 0.05 (two-sided). All statistical analyses were conducted using SPSS 17.0.0.

Results

In total 36.350 cases were available for analysis, however only cases with known out-come (N=29.201, 80.3%) were used. The population was divided into five main groups; 9.651 women had undergone a CT, 6.432 had undergone a 20 week anomaly scan, 4.862 had undergone a CVS (TA-CVS: N=1341 and TC-CVS: N=2833; of which 1787 by forceps and 1046 by a cannula and 7970 cases had undergone an AC. In 286 cases both a CVS and an AC were performed (Figure 1). Table 1 shows the baseline characteristics in all groups. Women opting for CVS or AC were on average older (<.01), and more frequently multiparous (<.01) and smokers (<.01). The results of the multiple regression analyses are presented in Table 2a and 2b. In the group of women undergoing a CVS (TC + TA) the variables significantly associated with a

ForcepsTFLR 0.21%

CannulaTFLR 3.15%


54

Table 1 — Baseline characteristics

Groups

CT (+20 weeks scan) N(%)

20 weeks scan N(%)

CVS N(%)

AC N(%)

CVS + AC N(%) p

Age: <.01**

<36 years 5140 (53.26) 2695 (89.51) 1652 (33.98) 1857 (23.30) 86 (30.07) ≥36 years 4511 (46.74) 316 (10.49) 3210 (66.02) 6113 (76.70) 200 (69.93)

Gravida - Para: <.01

Primiparous 3284 (34.03) 2401 (37.33) 1428 (29.37) 2580 (32.37) 88 (30.77) Multiparous 4999 (51.80) 2520 (39.18) 3149 (64.77) 4861 (60.99) 188 (65.73) Unknown 1368 (14.17) 1511 (23.49) 285 (5.86) 529 (6.64) 10 (3.50)

Conception: <.01

Spontaneous 3876 (40.16) 728 (11.32) 2689 (55.31) 4705 (59.03) 192 (67.13) Medication, IUI, IVF, ICSI, KID or Egg Donation

611 (6.33) 58 (.90) 134 (2.76) 301 (3.78) 5 (1.75)

Unknown 5164 (53.51) 5646 (87.78) 2039 (41.94) 2964 (37.19) 89 (31.12)

Smoking: <.01**

Yes 574 (5.95) 273 (4.24) 399 (8.21) 641 (8.04) 17 (5.94) Stopped 57 (.59) 67 (1.04) 11 (.23) 11 (.14) - No 5632 (58.36) 1743 (27.10) 3428 (70.51) 5998 (75.26) 229 (80.07) Unknown 3388 (35.11) 4349 (67.62) 1024 (21.06) 1320 (16.56) 40 (13.99)

BMI: <.01**

Underweight (<18.50) 148 (1.53) 103 (1.60) 80 (1.65) 97 (1.22) 5 (1.75) Normal range (18.50 – 24.99) 3428 (35.52) 1688 (26.24) 2360 (48.54) 2253 (28.27) 150 (52.45) Overweight (25.00 – 29.99) 1205 (12.49) 630 (9.79) 754 (15.51) 706 (8.86) 44 (15.38) Obese (≥30.00) 520 (5.39) 416 (6.47) 254 (5.22) 287 (3.60) 23 (8.04) Unknown 4350 (45.07) 3595 (55.89) 1414 (29.08) 4627 (58.06) 64 (22.38)

Year: <.01**

2001 545 (5.6) 39 (0.6) 352 (7.24) 858 (10.77) 22 (7.7) 2002 547 (5.7) 31 (0.5) 407 (8.37) 824 (10.34) 23 (8.0) 2003 745 (7.7) 32 (0.5) 394 (8.10) 787 (9.87) 15 (5.2) 2004 981 (10.2) 113 (1.8) 400 (8.23) 739 (9.27) 13 (4.5) 2005 822 (8.5) 83 (1.3) 375 (7.71) 611 (7.67) 11 (3.8) 2006 760 (7.9) 260 (4.0) 350 (7.20) 844 (10.59) 11 (3.8) 2007 1384 (14.3) 1187 (18.5) 598 (12.30) 776 (9.74) 31 (10.8) 2008 1183 (12.3) 1273 (19.8) 584 (12.01) 741 (9.30) 29 (10.1) 2009 1077 (11.2) 1063 (16.5) 508 (10.45) 682 (8.56) 52 (18.2) 2010 1096 (11.4) 1143 (17.8) 487 (10.02) 658 (8.26) 50 (17.5) 2011 511 (5.3) 1208 (18.8 407 (8.37) 450 (5.65) 29 (10.1)

* Due to rounding the numbers do not add up to 100%.** Fisher Exact Test was used.CT = combined test. CVS = chorion villus sampling. AC = amniocentesis.

higher risk of fetal loss were repeated attempts during a procedure, the use of a TC can-nula, gestational age of 13 weeks or beyond and pregnancies after assisted reproduction In the group of women undergoing an AC variables significantly associated with a higher risk of spontaneous fetal loss were repeated attempts during a procedure, the presence of an anomaly and a past history for congenital anomalies in the family. Table 3 shows the pregnancy outcome for each group, including all variables and indi-cations. The total FLR before 24 week’ gestation in the CT group was 1.21% and in the CVS group (TC + TA) 3.12%; giving a procedure related risk of 1.91% or 1:52 for CVS. This per-


Chapter 4

55

Table 2a — CVS <24 weeks of gestational age

Univariate Logistic Regression Multiple Logistic Regression

OR (95% CI) p OR (95% CI) p

Maternal Characteristics

Age:

<36 years 1.347 .083 >36 years -

Parity:

Primiparous 1.104 .597 Multiparous - Unknown 1.660 .095

Conception:

Spontaneous .405 .013 .352 .011 Assisted - - - Unknown .421 .019 .243 .002

Smoking:

Yes 1.570 .104 Stopped .000 .999 No - Unknown 1.523 .030

BMI:

Underweight (<18.50) 1.815 .298 Normal range (18.50 – 24.99) .747 .422 Overweight (25.00 – 29.99) .857 .699 Obese (≥30.00) - Unknown .897 .769

CVS Characteristics

Number of CVS procedures:

1 procedure – 1 attempt - - 1 procedure – >1 attempt 2.359 .001 2.476 .001 >1 procedure .000 .998 .000 .999

GA when procedure takes place:

≤10 weeks - - - 11 weeks 1.112 .795 1.469 .400 12 weeks 1.385 .425 1.845 .192 13 weeks 1.955 .116 3.203 .018 ≥14 weeks 3.407 .012 4.047 .027

Indication:

Maternal age - Increased risk CT 1.180 .458 (Suspicion) Anomaly 2.726 .001 Obstetric history 1.261 .504 Family history .747 .626 DNA examination .924 .821 Different reason .000 .998

Operator Characteristics

Experience .891 .371

Instrument:

TA 17-19G Needle - -


56

Table 2a — CVS <24 weeks of gestational age

TA 20 G Needle 1.663 .266 1.836 .287 TA 22G Needle 1.815 .274 2.708 .085 TV Forceps .983 .942 1.584 .114 TV Cannula 1.556 .067 3.018 .002

CVS = chorionvillus biopsy. GA = gestational age.

Table 2b — AC <24 weeks of gestational age


OR (95% CI) p OR (95% CI) p

Maternal Characteristics

Age:

<36 years .350 .001 >36 years -

Parity:

Primiparous 1.137 .504 Multiparous - Unknown .880 .749

Conception:

Spontaneous 1.589 .434 Assisted - Unknown 1.602 .431

Smoking:

Yes .613 .247 Stopped 14.410 .001 No - Unknown 1.253 .323

BMI (WHO):

Underweight (<18.50) .986 .983 Normal range (18.50 – 24.99) .502 .068 Overweight (25.00 – 29.99) .625 .278 Obese (≥30.00) - Unknown .420 .017

AC Characteristics

Number of AC procedures:

1 procedure – 1 attempt - 1 procedure – >1 attempt 2.220 .045 2.901 .017 >1 procedure – 1 attempt .000 1.000 4.185 .192 >1 procedure – >1 attempt 6.238 .014

GA when procedure takes place:

≤15 weeks - - 16 weeks .717 .172 .707 .281 17 weeks 1.938 .035 1.431 .442 18 weeks 2.331 .075 1.405 .607 19 weeks 1.844 .309 .624 .578 20 weeks 4.236 .001 .727 .593 21 weeks 1.033 .950 .078 .021 22 weeks 1.591 .439 .185 .130

Table 2a (continued)


Chapter 4

57

Table 2b — AC <24 weeks of gestational age


OR (95% CI) p OR (95% CI) p

23 weeks 1.616 .638 .000 .998 ≥24 weeks .000 .994 .000 .995

Indication:

Maternal age - - (Suspicion) Anomaly 4.017 .001 8.531 .001 Increased risk CT 1.499 .158 1.558 .229 AC after CVS .000 .999 .000 .999 Obstetric history 1.970 .195 2.713 .104 Family history 2.065 .319 4.964 .032 DNA examination 3.032 .279 5.153 .128 Different reason 1.866 .297 2.665 .191

Approach:

Via amniotic fluid - Via placenta .924 .745Amount of amniotic fluid .999 .852

Operator Characteristics

Experience .858 .088

Instrument:

TA 17-19G Needle - TA 20G Needle .499 .189 TA 21G Needle .000 .999 TA 22G Needle .395 .080

AC = amniocentesis. GA = gestational age.

centage includes all indications for an invasive irrespective of CVS technique. The total spontaneous FLR before 24 weeks’ gestation in the 20 weeks anomaly scan group was 0.31% and in the AC group 1.56%; giving a procedure related risk of 1.25% or 1:80 for AC. Univariately there was a trend between an increased level of experience and a lower fetal loss rate. These estimates included all indications. Table 4 shows the pregnancy outcome for each group, stratified for indication. The total spontaneous FLR in women of 36 years and older undergoing a CT was 1.40% (N=63). For women undergoing a TC or TA CVS, performed on advanced maternal age indication, was 2.76% (N=40) and 2.43% (N=13), respectively (Chi Square <.01). The ad-ditional risk of a TC CVS was therefore 1.36% or 1:74; the additional risk of a TA CVS was 1.03% or 1:97 (Chi Square .06), respectively. When TC CVS was performed by forceps the additional risk was 0.27% (1.67 - 1.40%); and when performed by cannula 3.12% (4.52 - 1.40%). When the number of procedures and attempts was taken into account for CVS (TC and TA) the risk was 1.07% (2.47 - 1.40%) when only one procedure and attempt had taken place, 4.48% (5.88 - 1.40%) when during one procedure more than one attempt was per-formed. No risk could be calculated for the group where more than one procedure and attempt were performed because the group was too small (N=8, 0 fetal loss). In women aged 36 years and older undergoing the 20 weeks scan the total FLR was .63% (N=2) and in women undergoing an AC 1.11% (N=60)(Chi Square .325). Therefore

Table 2b (continued)


58

Table 3 — Outcome per group

Alive Miscarriage

IUVD 24-37 weeks

IUVD >=37 weeks

Partus prematurus and deceased 24-37 weeks TOP

Deceased durante partu >=37 weeks

Neonatal Death

CT (+20 weeks scan) N 9,403 117 18 11 6 71 3 15% 97.50 1.21 .19 .11 .06 .74 .03 .16

18-20 weeks scan N 6,361 20 23 5 6 9 - 8% 98.90 .31 .36 .08 .09 .14 - .12

TA-CVS N 1,001 40 8 2 - 283 - 6% 74.70 2.99 .60 .15 - 21.12 - .45

TC-CVS Forceps N 1,445 47 4 2 3 278 - 4% 81.04 2.64 .22 .11 .17 15.59 - .22

TC-CVS Cannula N 822 43 6 - - 146 - 2% 80.67 4.22 .59 - - 14.33 - .20

AC N 7,184 124 91 21 3 449 2 89% 90.22 1.56 1.14 .26 .04 5.64 .03 1.12

CVS + AC N 226 11 - - - 48 - 1% 79.02 3.85 - - - 16.78 - .35

Total N 26,442 402 150 41 18 1284 5 125% 92.89 1.41 .53 .14 .06 4.51 .02 .44

All indications were included. CT = combined test. CVS = chorion villus sampling. AC = amniocentesis. TA-CVS = transabdominal chorion villus sampling. TC-CVS: transcervical chorion villus sampling. IUD = intra-uterine death. TOP = termination of pregnancy.

the additional risk of an AC was .48% or 1:208. In women younger than 36 years undergo-ing a midtrimester scan the spontaneous FLR decreased to .52% (p .79). When the number of procedures and attempts was taken into account for the AC, the risk was .34 (.97 - .63%), the additional risk was 1.34% (1.97 - .63%) when during one pro-cedure more than one attempt was performed, and 5.25% (5.88 - .63%) when more than one procedure and more than one attempt were performed. With increasing operator experience the FLR after a TA-CVS decreased. At level 1 the risk was 2.24% (3.64 - 1.40%), at level 2 1.65% (3.05% - 1.40%) and at level 3 0.42 (1.82 - 1.40%) (Figure 2a). The risk after TC CVS by forceps could not be calculated for level 1 and 2 due to a relative low number of women and no fetal losses. At level 3 the risk was 0.44% (1.84 - 1.40%) (Figure 2b). The risk when using the cannula was 0.68% (2.08 - 1.40%) at level 1, 1.57% (2.97 - 1.40%), at level 2, and 6.85% (8.25 - 1.40%) at level 3 (Figure 2c). The influence of operator’s experience on fetal loss rate after an AC was examined ac-cording to four levels of experience. At level 1 the additional risk was 0.82% (1.45 - .63%), at level 2 1.00% (1.63 - .63%), at level 3 .17% (0.80 - .63%) and at level 4 .52% (1.15 - .63%) (Figure 2d).

Discussion

This study shows that the FLR in women aged 36 years or older undergoing a transcervi-cal CVS on maternal age indication was 0.27% or 3.12%, depending on whether a forceps or a cannula was used, respectively. For the transabdominal approach the risk was 1.36%. Factors influencing the fetal loss rate after CVS were the use of a TC cannula (OR 3.0),


Chapter 4

59

Table 4 — Outcome according to indication

¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ Outcome ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬¬ ¬ ¬ ¬ ¬ ¬¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬

Alive Miscarriage

IUVD 24-37 weeks

IUVD >=37 weeks



Neonatal Death Total

CT

<36 years 5,010(97.55)

54(1.05)

8(.16)

2(.04)

4(.08)

47(.92)

2(.04)

9(1.18)

5,136

≥36 years 4,393(97.45)

63(1.40)

10(.22)

9(.20)

2(.04)

24(.53)

1(.02)

6(.13)

4,508

18-20 wk scan

<36 years 2,656(98.55)

14(.52)

12(.45)

1(.04)

6(.22)

4(.15)

- 2(.07)

2,695

≥36 years 307(97.15)

2(.63)

1(.32)

1(.32)

- 3(.95)

- 2(.63)

316

CVS (TA)

Maternal Age 508(94.78)

13(2.43)

- 1(.19)

- 13(2.43)

- 1(.19)

536

Increased risk CT 318(67.80)

14(2.99)

2(.43)

- - 134(28.57)

- 1(.21)

469

Anomaly 60(31.58)

10(5.26)

4(2.11)

1(.53)

- 111(58.42)

- 4(2.11)

190

Anomaly in obstetric history

68(89.47)

3(3.95)

1(1.32)

- - 4(5.26)

- - 76

Anomaly present in family member or parents

13(59.09)

- 1(4.55)

- - 8(36.36)

- - 22

DNA-research 24(66.67)

- - - - 12(33.33)

- - 36

Other 9(90.00)

- - - - 1(10.00)

- - 10

CVS Forceps


15(1.67)

2(.22)

1(.11)

- 31(3.46)

- 2(.22)

896


9(3.33)

1(.37)

- 2(.74)

64(23.70)

- - 270

Anomaly 52(33.77)

12(7.79)

1(.65)

- - 89(57.79)

- - 154

Anomaly in previous pregnancy

105(92.11)

3(2.63)

- - - 6(5.26)

- - 114

Anomaly in one of the parents or family member

53(77.94)

- - 1(1.47)

- 14(20.59)

- - 68

DNA-investigation 186(68.63)

8(2.95)

- - 1(.37)

74(27.31)

- 2(.74)

271

Other 10(100)

- - - - - - - 10

CVS Cannula


25(4.52)

2(.36)

- - 18(3.25)

- 1(.18)

553


5(3.14)

2(1.26)

- - 40(25.16)

- 1(.63)

159

Anomaly 28(35.90)

4(5.13)

1(1.28)

- - 45(57.69)

- - 78


60

Table 4 — Outcome according to indication

¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ Outcome ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬¬ ¬ ¬ ¬ ¬ ¬¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬

Alive Miscarriage

IUVD 24-37 weeks

IUVD >=37 weeks



Neonatal Death Total


78(87.64)

4(4.49)

- - - 7(7.87)

- - 89


45(73.77)

3(4.92)

1(1.64)

- - 12(19.67)

- - 61


2(2.08)

- - - 24(25.00)

- - 96

Other 10(100)

- - - - - - - 10

AC

Maternal Age 5,218(96.74)

60(1.11)

13(.24)

4(.07)

- 83(1.54)

1(.02)

15(.28)

5,394

Anomaly 625(54.82)

47(4.12)

66(5.79)

14(1.23)

3(.26)

313(27.46)

1(.09)

71(6.23)

1,140

Increased risk CT 1,011(90.92)

17(1.53)

7(.63)

2(.18)

- 74(6.65)

- 1(.09)

1,112

AC after CVS 66(85.71)

1(1.30)

- - - 10(12.99)

- - 77


197(94.71)

4(1.92)

- - - 6(2.88)

- 1(.48)

208


94(93.07)

2(1.98)

2(1.98)

1(.99)

- 2(1.98)

- - 101


1(2.38)

1(2.38)

- - 7(16.67)

- - 42

Other 161(96.41)

3(1.80)

- - - 2(1.20)

- 1(0.60)

167

* Total fetal loss includes intra-uterine death, preterm labor and miscarriage. CT = combined test. CVS = chorion villus sampling. AC = amniocentesis. TA-CVS: transabdominal chorion villus sampling. TC-CVS = transcervical chorion villus sampling.

repeated attempts (OR 2.5), gestational age of 13 weeks or beyond (OR 3.2-4.0) and preg-nancies after assisted reproduction. Pregnancy loss after an AC was 1.11%. Factors affecting fetal losses after AC were re-peated attempts (OR 2.9), the presence of fetal anomalies (OR 8.5) and a family history of anomalies (OR 5.0). Furthermore, the influence of operator’s experience on iatrogenic fetal losses was confirmed.4,23,24

This study confirms the negative effect of variables such as an enlarged NT or structural anomalies on the total FLR (including TOP) after CVS (TC or TA) and AC. These factors are by far more predisposing to pregnancy losses than advanced maternal age.5,9,25

This retrospective study shows that the previously quoted risk of fetal loss after AC of 1.0% does not reflect current practice. The risk after invasive procedures performed by experienced operators seems to be lower (.17 - .52% for the most experienced operators (level 3 for CVS and level 4 for AC)), which is in line with the meta-analysis of Akolekar et al.4,5,9,10,23,26

Strength of this study is that it includes a large population of women undergoing proce-

Table 4 (continued)


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Figure 2a — Fetal loss in percentage (x-axis) in relation to level of experience when performing a transabdominal CVS (y-axis): (level 1) <50, (level 2) between 50-150 and (level 3) >150 procedures performed in total.

Figure 2b — Fetal loss in percentage (x-axis) in relation to level of experience when performing a transcervical CVS using a forceps (y-axis): (level 1) <50, (level 2) between 50-150 and (level 3) >150 procedures performed in total.

dures for different indications and two control groups. A limitation is its retrospective nature and, in the attempt of creating similar groups and excluding possible biases, some groups had only few patients or losses. Although the only way of confirming the conclu-sions of this study would be to perform a prospective randomized study, this is nowadays ethically unfeasible.22,27

Furthermore, we were unable to retrieve the follow-up in 19% of the cases. We did not opt for the possibility of imputation, and choose to stay as close to the data as registered in first instance. In our experience frequently normal outcomes are missing as caregivers tend to report fetal losses, especially iatrogenic losses. Although we cannot be certain, it is likely that we overestimated the risk of fetal loss rather than underestimating it. Regarding first trimester procedures, it is clear that the TC CVS by forceps and the TA technique are the methods of choice. A Cochrane review on CVS did not show convincing evidence to favor TA CVS over the TC technique.6 In contrast, Chueh et al and Jensen et al showed a significant difference in risk between the two techniques, prompting abandonment of the TC CVS in their centers.11 This study shows that depending on the technique, both TA and TC CVS can have low FLR.28

In our study the TC-CVS was the technique in use in the early years. Only since the in-troduction of first trimester screening, the TA technique has become more widespread. Rationale for using TC-CVS is that it allows for a larger sample, especially useful in case of DNA analysis, and that it can be performed from 11 weeks onwards. We confirm that the


62

FLR after a TC and TA-CVS seems to be comparable, but only when the forceps is used.11,28 The higher FLR when using the cannula, irrespective of the operator’s experience, is possibly due to the need in 2008 to abandon the previous cannula which was withdrawn from the market and using a new less flexible cannula, in one of the two centers. Since 2010 this center has adopted the forceps for TC CVS also and implemented the TA -ap-proach. The advantage of being skilled in both techniques enhances the chance of suc-cessful sampling irrespective of placental localization or position of the uterus. Opera-tors performing TC CVS should preferably be trained to use the forceps. As suggested in the literature, growing experience with CVS techniques has the potential to bring down the fetal losses to similar levels of the AC.11,12,14,21

One of the concerns of comparing women of 36 years and older undergoing an AC at around 16 weeks with a mid-trimester control group was the possible effect of the 2-4 weeks difference in gestational age on the spontaneous FLR with a possible overestima-tion of the procedure related loss rate at the time of the procedure.22,27 We were therefore surprised when, after adjustment for gestational age (≥16 - <24 weeks), both groups showed the same trend of a lower loss rate after AC. The background FLR in this study (.63% for women of 36 years and older) falls within previously reported percentages, varying between 0.2 and 1.16%.20 Similarly, the low procedure related risk after AC performed by an experienced operator is in line with some recent studies with a high caseload and experienced operators.5,13,16,23

Figure 2c — Fetal loss in percentage (x-axis) in relation to level of experience when performing a transcervical CVS using a cannula (y-axis): (level 1) <50, (level 2) between 50-150 and (level 3) >150 procedures performed in total.

Figure 2d — Fetal loss in percentage (x-axis) in relation to level of experience when performing an AC (y-axis): (level 1) <50, (level 2) 50-150, (level 3) >150-500, (level 4) >500 procedures performed in total.


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1. Bakker M., Birnie E., Pajkrt E. et al. Low uptake of the combined test in the Neth-erlands – which factors contribute? Prenat Diagn 2012 Dec; 32(13): 1305-1312.

2. Dugoff L., Cuckle H. S., Hobbins J. C. et al. Prediction of patient-specific risk for fetal loss using maternal characteristics and first- and second-trimester maternal serum Down syndrome markers. Am J Obstet Gy-necol 2008 Sep; 199(3): 290.e1-290.e6.

3. Papantoniou N. E., Daskalakis G. J., Tziotis J. G. et al. Risk factors predisposing to fetal loss following a second trimester amnio-centesis. BJOG 2001 Oct; 108(10): 1053-1056.

4. Tabor A., Alfirevic Z. Update on procedure-related risks for prenatal diagnosis tech-niques. Fetal Diagn Ther 2010; 27(1): 1-7.

5. Akolekar R., Beta J., Picciarelli G. et al. Procedure-related risk of miscarriage fol-

lowing amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound Obstet Gynecol 2014 Jul 17.

6. Young C., Von Dadelszen P., Alfirevic Z. In-struments for chorionic villus sampling for prenatal diagnosis. Cochrane Database Syst Rev 2013 Jan 31; 1: CD000114.

7. Wijnberger L. D., Van der Schouw Y. T., Christiaens G. C. Learning in medicine: cho-rionic villus sampling. Prenat Diagn 2000 Mar; 20(3): 241-246.

8. Tabor A., Vestergaard C. H., Lidegaard O. Fe-tal loss rate after chorionic villus sampling and amniocentesis: an 11-year national registry study. Ultrasound Obstet Gynecol 2009 Jul; 34(1): 19-24.

9. Akolekar R., Bower S., Flack N. et al. Predic-tion of miscarriage and stillbirth at 11-13

A recent large national population based study performed in Denmark, even showed that neither TA-CVS nor AC is associated with a higher FLR in comparison with the control group. Suggesting that the procedure-related FLR is very low for both CVS and AC.29

Appropriate training of new operators under experienced supervision or by the use of training models, can minimize the learning curve effect and increase the success rate.28,30

More than one needle insertion during a procedure was associated with an increased risk of pregnancy loss. Silver et al showed that there is a direct relationship between op-erator caseload and sampling efficiency.31

With the declining numbers of CVS and AC due to the widespread use of cell ff-DNA screening, a new directive on the number of operators performing invasive procedures and a minimum caseload per operator should be defined by the professional society. In the Netherlands the Dutch society for Obstetrics and Gynecology recommends a min-imal number of 30 procedures per operator per year.32

In view of our results and declining number of invasive procedures, centralization in a few centers by experienced operators seems recommended, increasing the minimal caseload per year per operator. We suggest that in the future, similarly to the FMF audit for the NT, the development of an individual quality control program for invasive proce-dures should be considered, taking into account numbers, efficiency and safety.5,7,32

The real question is whether operator’s fetal loss rate should be mentioned when coun-seling women regarding their choices in prenatal screening methods. In conclusion, pregnancy losses after invasive procedures performed transabdominal-ly (CVS and AC) or transcervically by forceps, are lower than thought and reported in the past when performed by experienced operators.

References


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weeks and the contribution of chorionic villus sampling. Prenat Diagn 2011 Jan; 31(1): 38-45.

10. Tabor A., Philip J., Madsen M. et al. Ran-domised controlled trial of genetic amnio-centesis in 4606 low-risk women. Lancet 1986 Jun 7; 1(8493): 1287-1293.

11. Smidt-Jensen S., Permin M., Philip J. et al. Randomised comparison of amniocentesis and transabdominal and transcervical cho-rionic villus sampling. Lancet 1992 Nov 21; 340(8830): 1237-1244.

12. Multicentre randomised clinical trial of chorion villus sampling and amniocentesis. First report. Canadian Collaborative CVS-Amniocentesis Clinical Trial Group. Lancet 1989 Jan 7; 1(8628): 1-6.

13. Giorlandino C., Cignini P., Cini M. et al. An-tibiotic prophylaxis before second-trimester genetic amniocentesis (APGA): a single-cen-tre open randomised controlled trial. Prenat Diagn 2009 Jun; 29(6): 606-612.

14. Caughey A. B., Hopkins L. M., Norton M. E. Chorionic villus sampling compared with amniocentesis and the difference in the rate of pregnancy loss. Obstet Gynecol 2006 Sep; 108(3 Pt 1): 612-616.

15. Centini G., Rosignoli L., Kenanidis A. et al. A report of early (13 + 0 to 14 + 6 weeks) and mid-trimester amniocenteses: 10 years' ex-perience. J Matern Fetal Neonatal Med 2003 Aug; 14(2): 113-117.

16. Corrado F., Cannata M. L., La Galia T. et al. Pregnancy outcome following mid-trimes-ter amniocentesis. J Obstet Gynaecol 2012 Feb; 32(2): 117-119.

17. Eddleman K. A., Malone F. D., Sullivan L. et al. Pregnancy loss rates after midtrimester amniocentesis. Obstet Gynecol 2006 Nov; 108(5): 1067-1072.

18. Odibo A. O., Dicke J. M., Gray D. L. et al. Evaluating the rate and risk factors for fetal loss after chorionic villus sampling. Obstet Gynecol 2008 Oct; 112(4): 813-819.

19. Pitukkijronnakorn S., Promsonthi P., Pan-burana P. et al. Fetal loss associated with

second trimester amniocentesis. Arch Gyne-col Obstet 2011 Oct; 284(4): 793-797.

20. Towner D., Currier R. J., Lorey F. W. et al. Miscarriage risk from amniocentesis performed for abnormal maternal serum screening. Am J Obstet Gynecol 2007 Jun; 196(6): 608.e1-5; discussion 608.e5.

21. Lau K. T., Leung Y. T., Fung Y. T. et al. Out-come of 1,355 consecutive transabdominal chorionic villus samplings in 1,351 patients. Chin Med J (Engl) 2005 Oct 20; 118(20): 1675-1681.

22. Akolekar R., Beta J., Picciarelli G. et al. Reply. Ultrasound Obstet Gynecol 2015 Jun; 45(6): 755-757.

23. Mujezinovic F., Alfirevic Z. Procedure-related complications of amniocentesis and chorionic villous sampling: a systematic review. Obstet Gynecol 2007 Sep; 110(3): 687-694.

24. Roper E. C., Konje J. C., De Chazal R. C. et al. Genetic amniocentesis: gestation-specific pregnancy outcome and comparison of outcome following early and traditional am-niocentesis. Prenat Diagn 1999 Sep; 19(9): 803-807.

25. Cohen-Overbeek T. E., Hop W. C., den Ouden M et al. Spontaneous abortion rate and advanced maternal age: consequences for prenatal diagnosis. Lancet 1990 Jul 7; 336(8706): 27-29.

26. Nanal R., Kyle P., Soothill P. W. A classifica-tion of pregnancy losses after invasive pre-natal diagnostic procedures: an approach to allow comparison of units with a different case mix. Prenat Diagn 2003 Jun; 23(6): 488-492.

27. Ghidini A. Re: Risk of miscarriage following amniocentesis and chorionic villus sam-pling. Ultrasound Obstet Gynecol 2015 Jun; 45(6): 755.

28. Chueh J. T., Goldberg J. D., Wohlferd M. M. et al. Comparison of transcervical and trans-abdominal chorionic villus sampling loss rates in nine thousand cases from a single center. Am J Obstet Gynecol 1995 Oct;


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173(4): 1277-1282. 29. Wulff C. B., Gerds T. A., Rode L., Ekelund

C. K., Petersen O. B., Tabor A., Danish Fetal Medicine Study Group. Risk of fetal loss associated with invasive testing following combined first-trimester screening for Down syndrome: a national cohort of 147 987 singleton pregnancies. Ultrasound Ob-stet Gynecol 2016 Jan; 47(1): 38-44.

30. Ville Y., Cooper M., Revel A., Frydman R., Nicolaides K. H. Development of a train-ing model for ultrasound-guided invasive

procedures in fetal medicine. Ultrasound Obstet Gynecol 1995 Mar; 5(3): 180-183.

31. Silver R. K., Russell T. L., Kambich M. P., Leeth E. A., MacGregor S. N., Sholl J. S. Midtrimester amniocentesis. Influence of operator caseload on sampling efficiency. J Reprod Med 1998 Mar; 43(3): 191-195.

32. Alfirevic Z. Who should be allowed to per-form amniocentesis and chorionic villus sampling? Ultrasound Obstet Gynecol 2009 Jul; 34(1): 12-13.


66


5Increased nuchal translucency with normal karyotype and

anomaly scan: What next?

M. Bakker 1

E. Pajkrt 2

C. M. Bilardo 1


University Medical Centre, Groningen, the Netherlands.2 Department of Obstetrics and Gynecology, Fetal Medicine Unit,

Academic Medical Centre, Amsterdam, the Netherlands.

Published in Best Practice & Research Clinical

Obstetrics and Gynaecology 2014; 28: 355-366.


68

Increased nuchal translucency with normal karyotype and anomaly scan: What next?

M. Bakker1, E. Pajkrt2 and C. M. Bilardo1



Abstract: Over the years, it has become clear that increased nuchal translucency is a mark-er for chromosomal abnormalities, and it is also associated with a wide spectrum of structural anomalies, genetic syn-dromes, a higher risk of miscarriage, and intrauterine fetal death. These risks are all proportionally related to the degree of nuchal translucency enlargement. After the initial assessment of in-creased nuchal translucency, parents

should be counselled by the fetal medi-cine specialist about the possible out-comes and the value of additional karyo-typing and array comparative genomic hybridisation. A detailed late firsttrimes-ter and subsequent 20-week scan should aim at identifying structural anomalies, with special focus on the fetal heart and subtle dysmorphic features. In the absence of structural anomalies or mark-ers, the chance of a favourable outcome is high.

Introduction

In 1992, Nicolaides et al.1 proposed nuchal translucency measurement as a marker for chromosomal abnormalities in the first trimester of pregnancy. Over the years, it has become clear that an increased nuchal translucency is also associated with a wide spec-trum of structural anomalies, genetic syndromes, a higher risk of miscarriage, and in-trauterine fetal death. These risks are all proportionally related to the degree of nuchal translucency enlargement.2,3

At present, the most challenging part of managing pregnancies with increased nuchal translucency, after exclusion of chromosomal aberrations, is to establish an adequate diagnostic work up, and provide parents with realistic and correct information about outcome, especially long-term neurological outcome in the absence of structural anoma-lies.4-6

In this chapter, we provide an overview of issues relating to nuchal translucency. We subsequently suggest a protocol for managing these pregnancies to aid parental counsel-ling once a normal karyotype or genotype has been confirmed. At present, nuchal translucency measurement is offered in most countries as part of first-trimester screening for Down’s syndrome. Participation rates vary considerably per country, as its uptake is influenced by local policies, socioeconomic factors, attitude towards Down’s syndrome screening, and termination of pregnancy.7-12 When women are informed about first-trimester screening, the focus of counselling is primarily on


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the detection of Down’s syndrome. They should, however, be informed that this type of screening may detect many other chromosomal anomalies, and an increased nuchal translucency is also a powerful marker for cardiac anomalies, other structural anomalies, and genetic syndromes.13-14 Furthermore, fetuses with an increased nuchal translucency have an increased risk of adverse pregnancy outcome, such as fetal loss and developmen-tal delay.2,3,15-18

Increased nuchal translucency

Nuchal translucency is a subcutaneous accumulation of fluid behind the neck of the fe-tus and generally visible by ultrasound up to 15 weeks of gestation. The size of the nuchal translucency is influenced by gestational age and is part of normal development.19 Nu-chal translucency is considered abnormal only when it exceeds a certain cut-off.1 Many different definitions and cut-offs for increased nuchal translucency have been used in the past.20 Although debate continues about whether nuchal translucency should be regarded as an increase above the 95th or 99th centile, there is consensus that nuchal translucency above the 99th centile (3.5 mm) is definitely increased. Nuchal translucency seems to be influenced by gender. Two studies have shown that male fetuses tend to have a slightly larger nuchal translucency than females, about 0.06-0.1 mm21-24, but this finding could not be confirmed by another study.23 Timmerman et al.25 showed, that among fetuses with an increased nuchal translucency, significantly more male fetuses had a favourable outcome compared with females (adverse outcome male 20.1% compared with 35.9% in females). The favourable outcome was especially present in male fetuses, with a marginally increased nuchal translucency (between P95 and 99), suggesting that a different cut-off may be necessary in male fetuses.

Increased nuchal translucency and aetiology

The pathophysiology behind increased nuchal translucency is not yet fully understood, and many hypotheses about the cause of nuchal translucency and the pathophysiology behind an increased nuchal translucency have been forwarded.27-33 One of the possible causes for increased nuchal translucency is a congenital heart defect, but it is difficult to explain the exact mechanism behind this possible relationship, as different types of con-genital heart defects with their own corresponding haemodynamics are encountered. An alternative explanation could be heart failure,33-36 although at present the relationship between impaired cardiac function as the main cause of increased nuchaltranslucency has not yet been established by all research groups.31,32 Bekker et al.37 sug-gested that impaired endothelial development could be the link between increased nu-chal translucency and congenital heart defects. Another possibility is developmental delay of the lymphatic system. Lymphatic jugular sacs are part of the lymphatic system, and a delay in development of these sacs, could cause increased nuchal translucency owing to fluid accumulation.38 A study by De Mooij et al.39 showed that a disturbance in lymphatic endothelial differentiation is present in euploid fetuses, with increased nuchal translucency, and that this disturbance has a


70

similar phenotype as aneuploid fetuses with enlarged jugular lymphatic sacs. More re-search, however, is needed to ascertain that this is a plausible explanation for all cases of increased nuchal translucency. Changes in the extra-cellular matrix, owing to a higher concentration of hyaluronan, and as a result excessive hydration of the extracellular matrix and a perturbed function or migration of the neural crest cells, have also been suggested as plausible causes of increased nuchal translucency.40-42 The latter disturbance plays a key role in determining the craniofacial defects and cardiac abnormalities that are present in Noonan syndrome and other syndromes associated with increased nuchal translucency.43

Increased nuchal translucency and chromosomal abnormalities

About 20% of fetuses with increased nuchal translucency will have a chromosomal ab-normality.44 The incidence increases with nuchal translucency thickness from about 7% for nuchal translucency between the 95th and 99th centile (3.5 mm), to 20% for nuchal translucency of 3.5-4.4 mm, 50% for nuchal translucency of 5.5-6.4 mm, and 75% for nu-chal translucency of 8.5 mm or more.44

Submicroscopic chromosomal abnormalities generally missed by conventional karyo-typing may be responsible for, the sometimes subtle, structural anomalies or develop-mental delay later in the life of a fetus with increased nuchal translucency and apparently ‘normal’ karyotype. These submicroscopic chromosomal abnormalities may be identified using comparative genomic hybridisation (CGH) microarray. The main advantage of CGH-array is the ability to detect simultaneously aneuploidies, deletions, duplications, amplifications, or both, of any locus represented on an array. In addition, CGHarray has proven to be a powerful tool for the detection of submicroscopic chromosomal abnor-malities in individuals with idiopathic mental retardation and various birth defects. A systematic review and meta-analysis by Hillman et al.45 showed that, when conven-tional karyotyping was normal, array-CGH detected 3.6% additional genomic imbalances (regardless of referral indication). This increased to 5.2% when the referral indicationwas structural malformation on ultrasound. Leung et al.46 showed that one out of 10 fetuses with increased nuchal translucency and an apparently normal karyotype had a submicro-scopic chromosomal abnormality likely to be pathological. A challenge of the application of CGH-array prenatally is determining whether a copy number variant (CNV) is de novo and likely to be causative, or inherited and likely to be benign. In case of doubt, the CGH-array of the fetus should be compared with the CNV’s in parental blood. A disadvantage of CGH-array is that balanced rearrangements, such as translocations and inversions, cannot be identified. Furthermore, information is gained on treatable and non-treatable diseases that may develop later in life, and parents need to decide whether they wish to receive this information. Fetuses with increased nuchal translucency should undergo conventional karyotyping and also receive counselling about array-CGH.


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Increased nuchal translucency and structural abnormalities

Convincing evidence shows that increased nuchal translucency in an euploid fetus is associated with an increased risk for structural anomalies, most commonly congenital heart defects.6,20,36,47 A review by Souka et al.20 showed large differences between studies in the prevalence of major anomalies, ranging from 3% to 50%, mainly because of dif-ferences in population, differences in definition of increased nuchal translucency and varying distribution of the nuchal translucency.48 A study byWestin et al.49 showed that nuchal translucency 3 mm or more increased the likelihood of lethal or serious malfor-mation about 15-fold, nuchal translucency 3.5 mm or more about 40-fold, and nuchal translucency 4.5 mm or more about 80-fold. Major congenital heart defects are found in about 4-5% of chromosomally normal fe-tuses with increased nuchal translucency.27,47,50 The prevalence increases from 0.6 to 2.5% with nuchal translucency between 95th and 99th centile to 64% in nuchal translucency greater than 8.5 mm.35,51-53 No specific type of congenital heart defect predominantes.54

A meta-analysis by Makrydimas et al.55 showed that the 99th centile threshold captures about 30% of congenital heart defects, instead of 56%, initially suggested by Hyett et al.53 Michailidis et al.52 found that 27% and 36% of all major cardiac defects occurred within the group of chromosomally normal fetuses with nuchal translucency above the 95th and 99th centile, respectively. In contrast, Mavrides et al.51 found that only 11% and 15% of major congenital heart defects occurred in those similar groups. Muller et al.56 found similar results, with a prevalence of major congenital heart defects in fetuses with nu-chal translucency above the 99th centile of 9.5%. A meta-analysis by Sotiriadis et al.57 showed that when analysis was restricted to stud-ies with operators certified by the Fetal Medicine Foundation, the sensitivity was 40.7% using the 95th centile cut-off and 14.5% using the 99th centile cut-off. Wald et al.58 found that an enlarged nuchal translucency is especially of value in iden-tifying (duct dependent) congenital heart defects that benefit from prenatal detection. Thus far, the heterogeneity of studies prevents the assessment of the true predictive val-ue of nuchal translucency measurement in the screening for congenital heart defects. At present, however, nuchal translucency measurement is the most effective early screen-ing method for cardiac defects. Future research should focus on the role of the Doppler of the hepatic artery, ductus venosus and tricuspid valve as sonomarkers for congenital heart defects in fetuses with and without increased nuchal translucency. Besides congenital heart defects, a clear association can also be found between orofa-cial clefts and increased nuchal translucency. Timmerman et al.59 showed a 19-fold higher chance of having a facial cleft in fetuses with an increased nuchal translucency compared with fetuses with a normal nuchal translucency. A study by Bilardo et al.6 and Souka et al.20 showed that when no (subtle) structural anomalies or markers are present at the 20-week anomaly scan, the chance of a normal outcome is similar to that of the general population, around 4%, irrespective of the en-largement of the nuchal translucency. A limitation of these studies is the small number of fetuses with a large nuchal translucency.Scott et al.60 examined 120 cases of fetuses with a nuchal translucency over 6.5 mm; 74% had a chromosomal abnormality and 26% had a normal karyotype. In the group with a normal karyotype, only eight babies were liveborn, of whom seven showed no


72

abnormalities at the detailed ultrasound scan. Four of the seven babies had a structural abnormality or genetic syndrome at birth. It is difficult to draw a definite conclusion on outcome of fetuses with a large nuchal translucency, as few are alife due to a high chance of fetal demise or termination of pregnancy. Furthermore, follow up over a longer period of time is necessary as some conditions present later in childhood.

Increased nuchal translucency and genetic syndromes

In 3% of fetuses with an increased nuchal translucency, an increased nuchal fold (6 mm) will be present at the 20-week anomaly scan.6 The cause of this phenomenon is not yet clear, although many hypotheses exist.61 When an increased nuchal fold is present, a 10% risk on a genetic syndrome or fetal hydrops and possible perinatal death is present.20

A long, and still growing, list of genetic syndromes present with increased nuchal translucency.5,20 For syndromes, such as Noonan syndrome and syndromes with muta-tions in the same pathway, Smith-Lemli-Opitz syndrome, spinal muscular atrophy and other muscle-skeletal disorders, the association with increased nuchal translucency is undisputed. In sporadic syndromes, the association with an enlarged nuchal translu-cency is more difficult to prove. Some syndromes are rare, and the available information is based on case reports, implying that the association could be coincidental. In case a genetic syndrome is suspected, a clinical geneticist should be consulted to discuss addi-tional genetic testing. Prenatally, Noonan syndrome is the most frequently reported genetic syndrome in as-sociation with increased nuchal translucency, with an incidence ranging from 2 - 5%.62-64 It is an autosomal dominant disorder, and is caused in about 50% of the cases by a mis-sense mutation in the PTPN11 gene on chromosome 12.65 Mutations in the SOS1-, RAF1-, KRAS-, BRAF-, MAP2K1/2-, NRAS- and SHOC2-genes account for a small percentage of Noonan syndrome cases.66 In chromosomally normal fetuses with enlarged nuchal translucency, the prevalence of Noonan syndrome tested by PTPN11 seems to vary from 6 to 18%.63,64,67

Houweling et al.68 advocate that, given the high incidence of Noonan syndrome in fetuses with increased nuchal translucency and normal karyotype, genetic counselling and Noonan syndrome mutation detection should be offered in all cases, irrespective of additional abnormalities. It is debatable, however, if nuchal translucency measurement should be used as a screening tool for Noonan syndrome, mainly as this syndrome has a highly variable expression and most cases only have mild dysmorphic features and nor-mal neurodevelopment.69

Moreover, testing for Noonan is expensive, and the important clinical question is in which cases testing should be offered. Our group proposes a more cost-effective selec-tion of cases.We showed that the diagnosis of Noonan syndrome can be suspected prena-tally, especially in chromosomally normal fetuses with a large nuchal translucency and one or more of the following characteristics: persistent nuchal fold or cystic hygroma, hydrops fetalis, pleural effusion, cardiac anomalies, polyhydramnios, or specific facial features.70 Croonen et al.67 recommend that, in the presence of the above mentioned ul-trasound features, testing should be extended to KRAS, RAF1, BRAF, and MAP2k1 genes for mutations, in case PTPN11 is negative.


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Increased nuchal translucency and development (delay)

Fourteen original studies have reported on the long-term follow up of fetuses with in-creased nuchal translucency, normal ultrasound findings, and normal karyotype. The proportion of developmental delay in early childhood reported in these studies ranges from 0 to 8.7% (Table 1).6-17,18,62,71-80 Interpretation of the studies so far is hampered by lack of standardisation: the cutoff values used to define increased nuchal translucency range between 3 mm, 3.5 mm, 4 mm, 95th centile and the 99th centile; the age of the children at follow up ranges from 6 months to 75 months; there are different ascertainment methods for developmental delay; and only three studies use a control group.17,49,72

The heterogeneity in prenatal and postnatal studies makes information on the preva-lence of neurodevelopmental delay in euploid fetuses with increased nuchal translu-cency difficult to interpret to reach a final conclusion.21,48 In studies in which a control group was used, and having excluded for chromosomal abnormalities, structural defects and genetic syndromes, no statistically significant difference was found between fetuses with an increased nuchal translucency compared with the general population, where developmental delay is about 4-5%.17,49,72 In a systematic review by Sotiriadis et al.71 the same conclusion was reached; however, the reviewers concluded that the reassuring re-sults should be interpreted with caution. In fact, no consensus has yet been reached on

Table 1 — Postnatal follow up in chromosomally normal fetuses with increased nuchal translucency thickness

Author StudyNuchaltranslucency

Control group

Follow up (months) Follow up type

Development delay

Case Control

Van Vugt et al., 199880 P ≥3 mm No 7-75 Questionnaire (1/34) 2.9%

Brady et al., 199879 C-C ≥3.5 mm Yes 6-42 Clinical examination (1/89) 1.1% (1/302) 0.33%

Adekunle et al., 199975 P ≥4 mm No 12-38 Questionnaire (2/23) 8.7%

Maymon et al., 200077 P ≥95th centile No 12-36 Questionnaire or by telephone

(0/36) 0

Souka et al., 200174 R ≥3.5 mm No NA Information from maternity units, the patient or GP

(4/980) 0.4%

Hiippala et al., 200162 P ≥3 mm No 24-84 Clinical examination (1/50) 2%

Senat et al., 200218 R ≥4 mm No 12-72 Clinical examination (3/54) 5.6%

Cheng et al., 200476 R ≥3 mm No 8-30 Clinical examination (1/14) 7.1%

Senat et al., 200778 P/R ≥99th centile Yes 0-24 Clinical examination and ASQ

(2/162) 1.2% (?/370) ?

Bilardo et al., 20076 R ≥95th centile No 6-60 Questionnaires or by telephone

(7/425) 1.6%

Saldanha et al., 200917 P ≥95th centile No 29 days to 72 months

Questionnaires and clinical examination

(0/128) 0

Mula et al., 201273 P ≥99th centile No 24 Four paediatricconsultations or ASQ

(4/108) 3.7%

Miltoft et al., 201272 C-C ≥4 mm Yes 24 ASQ (1/80) 1.3% (6/137) 4.4%

Sotiriadis et al., 201271 SR Total≥95th centile≥99th centile>3 mm

(28/2458) 1.14%(7/669) 1.05%(15/1567) 0.96%(6/222) 2.70%

ASQ = ages and stages questionnaire. C-C = case-control study. P = prospective study. P/R = prospective and retrospective study. R = retrospective study. SR = systematic review.


74

the definition of developmental delay, and assessment is not standardised. Moreover, large and long-term follow-up studies are needed.

Increased nuchal translucency and overall pregnancy outcome

Most fetuses with nuchal translucency above the 95th centile will have a normal karyo-type.15 The chance of a favourable outcome is related to the degree of increased nuchal translucency, ranging from 90% in case of nuchal translucency between the 95th and 99th centile to around 17% in case of nuchal translucency greater than 6.5 mm.6-20

Conversely, the prevalence of miscarriage or fetal death increases with increasing nuchal translucency, from 1.4% with nuchal translucency between the 95th centile (3.4 mm), 2.0% between 3.5 (4.4 mm), 2.9% between 4.5 (5.4 mm), 8.3% (5.5-6.4 mm), to 16.9% in fetuses with nuchal translucency 6.5 mm or greater.3

Where no anomalies or subtle variations from the norm are found, parents should be counselled that the chance of a normal outcome is high and no different from the normal population.

Nuchal translucency and macrosomia

Fetuses with an increased nuchal translucency tend to have higher birth weights. Poon et al.81 showed that prediction of macrosomia is related to maternal characteristics (e.g. ethnicity, maternal height and weight, previous delivery of a macrosomic baby, smoking and history of chronic hypertension and diabetes), and also to fetal characteristics. Their results show that an increased nuchal translucency is associated with an increased risk of delivering a macrosomic baby. These results were confirmed by Timmerman et al (un-published data), who showed that normal fetuses with an increased nuchal translucency have a higher risk of being macrosomic at birth compared with fetuses with a normal nuchal translucency. Weissmann-Brenner et al.82 also showed the relation between nu-chal translucency and birth weight in singletons from non-diabetic mothers. This cor-relation was independent of gender, and the predictive effect of nuchal translucency was limited to large for gestational age only.

Algorithm for the management of pregnancies with an enlarged nuchal translucency

After initial assessment of the fetus with an increased nuchal translucency, parents should be offered array-CGH in case no numerical anomalies are found with quantitative fluorescent polymerase chain reaction. A detailed late first-trimester scan should aim at identifying structural anomalies with special focus on the fetal heart. The current con-sensus is that detailed ultrasound examination, including fetal echocardiography, should be offered in all cases in which nuchal translucency is greater than 99th centile (3.5 mm). No consensus has been reached on the group with nuchal translucency between the 95th

and 99th centile in view of the rather low positive predictive value of 2%.56 When re-


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sources are available, we would suggest using the 95th centile cut off. A possible protocol for follow up of fetuses with a nuchal translucency greater than 95th and 99th centile is presented in Figure 1. Results from several studies showthat, with improved equipment and more extensive experience, it is possible to detect about 40-70% of all serious congenital anomalies in the first trimester of pregnancy.83 This percentage is even higher in high-risk pregnan-cies, were 84% of severe anomalies and congenital heart defects can be detected by the end of the first trimester.84

One of the benefits of a detailed scan early in pregnancy is that anomalies will be de-tected at an early stage. This allows more time for follow-up scans and more time for prenatal diagnostics if indicated. Another benefit is that parents may wish to opt for ter-mination of pregnancy, which his clinically safer in the first trimester than in the second

Nuchal translucency ≥95th centile

Detailed ultrasound scan at 13-14 weeks of gestation:ductus venosus Doppler

Early fetal echocardiography,including tricuspid valve doppler

(hepatic artery Doppler)

Invasive procedureQf-PCR

Conventional karyotypeArray-CGH

Normal karyotype

No anomalies or markers

Detailed ultrasound scan at18-22 weeks of gestation,

including echocardiography

Detailed ultrasound scan at30 weeks of gestation,


Normal: reassure Abnormal

Anomalies or markers

Detailed ultrasound scan at18-22 weeks of gestation,


Testing for Noonan syndrome if suspected features are present

Follow-up scans will beplanned according to type

of anomaly

Figure 1 — Flowchart for the clinical follow-up of a fetus with an increased nuchal translucency.


76

trimester of gestation. Emotionally, termination of pregnancy in the first trimester may be less distressing for women than in the second trimester, as the relationship between the mother, embryo, or fetus deepens as pregnancy advances.85,86

Some investigators have proposed that, in fetuses with enlarged nuchal translucency and severe structural anomalies, termination of pregnancy without first carrying out karyotyping, is a cost-saving option.87 In our view, however, karyotyping is important to help parents make a decision, apart from determining the recurrence risk. Doppler of the ductus venosus and tricuspid valve should be measured in addition to carrying out an anomaly scan, as an abnormal ductus venosus flow or tricuspid regurgi-tation can be associated with poor pregnancy outcome (e.g. cardiac and other structural anomalies, miscarriage or intrauterine demise), although, in most cases the outcome will be normal.33,88-90 Furthermore, the use of these two markers in first-trimester screen-ing reduces the false-positive rate. In addition to tricuspid regurgitation and abnormal ductus venosus flow, measure-ment of lowresistance flow in the hepatic artery is an unfavourable prognostic factor, as it is associated with chromosomal abnormalities, genetic syndromes, and structural anomalies. Furthermore, lowresistance flow in the hepatic artery is associated with tri-cuspid regurgitation and abnormal ductus venosus flow.91 One of the mechanisms sug-gested as explanation for the increased hepatic artery flow is the hepatic arterial buffer response to hypoxaemia.91 Cardiac dysfunction, resulting in abnormal ductus venosus flow and secondary dilatation in the vascular tree supplied by the hepatic artery, is sug-gested as explanation for the association between increased hepatic artery flow, abnor-mal ductus venosus flow and tricuspid regurgitation.90-92

If an abnormal Doppler of the ductus venosus or tricuspid regurgitation is present, irrespective of the nuchal translucency, a detailed follow-up scan at 18-19 weeks of gesta-tion should be carried out, including a fetal echocardiography. Screening for Noonan syndrome should be discussed with the parents if nuchal oe-dema or hydrops persists in the second trimester or other subtle features are present as mentioned above. During the whole diagnostic work up, parents may benefit from psycho-social guid-ance while facing the burden of coping with the uncertainty of fetal and neonatal out-come after an enlarged nuchal translucency.90-93

In the absence of anomalies or subtle variations from the norm, parents should be counselled that the chance of a normal outcome is high and not dissimilar from the nor-mal population. In case of doubt, such as in the presence of subtle anomalies, the infant may be seen by a geneticist after birth, and the achievement of developmental milestones should be checked (Figure 1).5,94

Conclusion

Increased nuchal translucency is much more than just a marker for Down’s syndrome. It is also associated with other chromosomal abnormalities, structural anomalies, genetic syn-dromes, a higher risk of miscarriage, and intrauterine death. When no structural anoma-lies or markers are found on the detailed scans, the chance of a favourable outcome is high.


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1. Nicolaides K. H., Azar G., Byrne D. et al. Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy. BMJ 1992; 304: 867-9.

2. Bilardo C. M., Pajkrt E., De Graaf I. et al. Out-come of fetuses with enlarged nuchal trans-lucency and normal karyotype. Ultrasound Obstet Gynecol 1998; 11:401-6.

3. Souka A. P., Snijders R. J., Novakov A. et al. Defects and syndromes in chromosomally normal fetuses with increased nuchal trans-lucency thickness at 10-14 weeks of gesta-tion. Ultrasound Obstet Gynecol 1998; 11: 391-400.

4. Ville Y. Nuchal translucency in the first tri-mester of pregnancy: ten years on and still a pain in the neck? Ultrasound Obstet Gyne-col 2001; 18: 5-8.

5. Bilardo C. M., Timmerman E., Pajkrt E. et al. Increased nuchal translucency in euploid fetuses – what should we be telling the par-ents? Prenat Diagn 2010; 30: 93-102.

6. Bilardo CM, Muller MA, Pajkrt E et al. In-creased nuchal translucency thickness and normal karyotype: time for parental reas-surance. Ultrasound Obstet Gynecol 2007; 30: 11-8.

7. Seror V., Ville Y. Prenatal screening for Down syndrome: women’s involvement in decision-making and their attitudes to

screening. Prenat Diagn 2009; 29: 120-8.8. Gidiri M., McFarlane J., Holding S. et al.

Maternal serum screening for Down syn-drome: are women’s perceptions changing? BJOG 2007; 114: 458-61.

9. Ekelund C. K., Petersen O. B., Skibsted L. et al. First-trimester screening for trisomy 21 in Denmark: implications for detection and birth rates of trisomy 18 and trisomy 13. Ultrasound Obstet Gynecol 2011; 38: 140-4.

10. Maxwell S., Brameld K., Bower C. et al. Socio-demographic disparities in the up-take of prenatal screening and diagnosis in Western Australia. Aust N Z J Obstet Gynae-col 2011; 51: 9-16.

11. Bakker M., Birnie E., Pajkrt E. et al. Low uptake of the combined test in the Neth-erlands – which factors contribute? Prenat Diagn 2012; 32: 1305-12.

12. Shantha N., Granger K., Arora P. et al. Wom-en’s choice for Down’s screening: a com-parative experience in three district general hospitals. Eur J Obstet Gynecol Reprod Biol 2009; 146: 61-4.

13. Muller M. A., Bleker O. P., Bonsel G. J. et al. Women’s opinions on the offer and use of nuchal translucency screening for Down syndrome. Prenat Diagn 2006; 26: 105-11.

14. Favre R., Moutel G., Duchange N. et al. What about informed consent in first-trimester

Future perspectives

At present, non-invasive fetal karyotyping is becoming increasingly available, and will probably replace the initial purpose of nuchal translucency measurement: screening for Down’s syndrome.26,95-98 Although non-invasive fetal karyotyping may replace the combined test we believe in the need of an early risk assessment in pregnancy and in the importance of a scan at 12-13 weeks of gestation, including the nuchal translucency mea-surement as marker of normal development. Furthermore, the first trimester scan has the potential for more than only risk calculation on chromosomal and structural anoma-lies. At present, research is under way on risk calculation for pre-eclampsia, preterm labour, intrauterine growth restriction, macrosomia, and gestational diabetes in the first trimester of pregnancy.

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71. Sotiriadis A., Papatheodorou S., Makrydimas


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94. Bilardo C. M. Increased nuchal translucency and normal karyotype: coping with uncer-tainty. Ultrasound Obstet Gynecol 2001; 17: 99-101.

95. Palomaki G. E., Kloza E. M., Lambert-Messerlian G. M. et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med 2011; 13: 913-20.

96. Bianchi D. W., Platt L. D., Goldberg J. D. et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol 2012; 119: 890-901.

97. Chiu R. W., Akolekar R., Zheng Y. W. et al. Non-invasive prenatal assessment of triso-my 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 2011; 342: c7401.

98. Nicolaides K. H., Syngelaki A., Ashoor G. et al. Noninvasive prenatal testing for fetal trisomies in a routinely screened firsttri-mester population. Am J Obstet Gynecol 2012; 207: 374.


6Targeted ultrasound examination and DNA testing for Noonan

syndrome, in fetuses with increased nuchal translucency

and normal karyotype

M. Bakker 1

E. Pajkrt 2

I. B. Mathijssen 3

C. M. Bilardo 1

1 Department of Obstetrics and Gynaecology, Fetal Medicine

Unit, University Medical Centre, Groningen, the Netherlands.2 Department of Obstetrics and Gynaecology, Fetal Medicine

Unit, Academic Medical Centre, Amsterdam, the Netherlands.3 Department of Clinical Genetics, Academic Medical Centre,

Amsterdam, the Netherlands.

Published in Prenatal Diagnosis 2011; 31: 833-840.

Published on Wiley Online Library (wileyonlinelibrary.com),

27 June 2011; DOI: 10.1002/pd. 2782.


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Targeted ultrasound examination and DNA testing for Noonan syndrome, in fetuses with increased nuchal translucency and normal karyotype

M. Bakker1, E. Pajkrt2, I. B. Mathijssen3 and C. M. Bilardo1


2 Department of Obstetrics and Gynaecology, Fetal Medicine Unit, Academic Medical Centre, Amsterdam, the Netherlands.

3 Department of Clinical Genetics, Academic Medical Centre, Amsterdam, the Netherlands.

Objective: To define sonographic criteria that may improve the prenatal diagnosis of Noon-an syndrome by targeted DNA testing.

Methods: We searched our Fetal Medicine Unit re-cords for all cases with a final diagnosis of Noonan syndrome. A literature review was undertaken to identify the sono-graphic features of Noonan syndrome fetuses. Information was pooled to de-fine the most common features.

Results: In our database, we identified three cases of Noonan syndrome. The diagno-sis was suspected prenatally in two of them. Thirty-nine cases were identified

in the literature. In the presented cases we show that suspicion of Noonan syn-drome should arise when, after an in-creased nuchal translucency, ultrasound investigation in the second trimester shows a persistant nuchal fold (NF) or cystic hygroma in combination with at least one of the following features: hy-drops fetalis, pleural effusion, cardiac anomalies, polyhydramnios or specific facial abnormalities.

Conclusion: Prenatal ultrasound findings in Noonan syndrome can be subtle and aspecific, but when specific characteristics are present additional targeted DNA analysis is indicated.

Introduction

Measurement of the nuchal translucency (NT) at 11-13 weeks 6 days of gestation is an established screening method for fetal aneuploidy (Snijders et al., 1998). Chromosom-ally normal fetuses with an increased NT (above the 95th centile for gestational age) are at increased risk of adverse pregnancy outcome. Moreover, an increased NT thick-ness has also been associated with a wide range of structural abnormalities and genetic syndromes involving neurodevelopmental delay (Pajkrt et al., 1999; Souka et al., 2005; Bilardo et al., 2007; Senat et al., 2007). Among the genetic syndromes the most frequently


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reported is Noonan syndrome. Noonan syndrome is an autosomal dominant disorder with a prevalence between 1 : 1000 and 1 : 2500 live births (Nora et al., 1974; Allanson, 1993). The majority of postnatal diagnosis concern de novo mutations; however, an affected parent is found in 30-75% of families (Van Huizen et al., 2005). Diagnosis of Noonan syndrome is often challenging because of the great variability in clinical characteristics (Allanson, 1993; Noonan, 1994). The main facial characteristics are hypertelorism, downslanting palpebral fissures, epi-canthic fold, ptosis and low set posteriorly angulated ears. The most common cardiovas-cular defects are pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). Other phenotypic characteristics are short stature, broad or webbed neck and chest de-formity. Associated pathologies are hematological disorders (bleeding diathesis, juvenile myelomonocytic leukemia), lymphatic vessel dysplasias, deafness and cryptorchidism. Affected individuals show a wide range in level of intelligence, with mental retardation being present in 15-35%, usually in the mild range and mainly consisting of specific vi-sual-constructional problems and verbal performance discrepancy (Sharland et al., 1992; Allanson, 1993; Van der Burgt et al., 1999; Van der Burgt, 2007). Besides the variability in expression, the facial phenotype changes with age resulting in less pronounced features in adults (Allanson, 1993). At present a simple and accurate scoring system, proposed by Van der Burgt et al. in 1994, is used for the (postnatal) diag-nosis of Noonan syndrome (Van der Burgt, 2007). Here we report our experience with ultrasound findings in three cases of Noonan syn-drome, two of which were diagnosed prenatally, following ultrasonographic evaluation of an increased NT and normal karyotype. These data, together with a detailed review of the published literature will serve as a useful aid to facilitate targeted DNA testing and parental counseling. A special focus is set on the role that 3D ultrasound may play in the diagnostic work-up of these pregnancies.

Results

CASE 1

A 24-year-old primigravida was referred to our Fetal Medicine Unit (FMU) because of an increased NT of 9.6 mm at 12+6 weeks of gestation (Figure 1a). Detailed first trimester US examination revealed a hypoplastic nasal bone. Brachycephaly, generalized edema and a ventricular septal defect were suspected. Ductus venosus flow showed a reversed a-wave and a pulsatility index for veins (PIV) of 4.00 (Figure 1b). Moreover, low resistance and high velocity hepatic artery flow were observed. No other structural anomalies were detected. Due to the increased risk of aneuploidy, chorionic villus sampling (CVS) was performed and demonstrated a normal male karyotype (Table 1). The ultrasound scan was repeated at 14+4 weeks of gestation. The NT was still 6.8 mm and bilateral distended jugular lymphatic sacs (JLS) were noted (Figure 1c). Follow-up sonography at 20+5 weeks of gestation showed a nuchal skin fold of 8 mm, bilateral distended JLS and brachycephaly (Figures 1d and 2a). Fetal echocardiography showed a structural and functional normal heart. The rest of the fetal biometry was nor-


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mal and no other structural anomalies were detected. At 23 weeks 4 days of gestation three-dimensional (3D) ultrasound of the fetus showed facial features typical for Noonan syndrome (hypertelorism, low set ears, broad nose and lips) and the parents were counseled about the possibility of Noonan syndrome (Figure 2b and c). The patient continued prenatal care in her local hospital, only to be referred back at 36 weeks 3 dyas with severe bilateral hydrothorax, mild generalized edema and polyhy-dramnios (AFI 31.4 cm). The pulsatility index (PI) of the umbilical artery was increased and there was redistribution in the middle cerebral artery (MCA). The next day all Dop-pler parameters deteriorated and labor was induced. A boy weighing 3000 g was born by caesarean section, performed for fetal distress, with Apgar scores of 4 and 5 at 1 and 5 min, respectively, and an umbilical artery pH of 7.14 with a base excels (BE) of -8. The baby presented with the following dysmorphic features: downward palpebral slant, epi-canthus, telecanthus, low set posteriorly angulated ears, low posterior hairline, broad nose, increased distance between the nipples, tendency to clinodactyly, less pronounced palmar grooves and cryptorchidism. Echocardiography showed poly-valvular disease (‘nodular compaction of the aortic and pulmonary valve, long chorda tendinae of the mi-

Figure 1 — (a) First trimester sagittal view of case 1 showing an increased nuchal translucency. (b) Ductus Venos Doppler measured in the first trimester showing a reversed a-wave. (c) First trimester transverse view showing the increased nuchal translucency and distended lymphatic jugular sacs. (d) Second trimester transverse view of the skull showing a brachycephaly.


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tral valve, tricuspid valve insufficiency and bidirectional shunt over the ductus arterio-sus, good left ventricular function and mild hypertrophy). A chest X-ray confirmed a chy-lothorax with a right-sided pneumothorax. Newborn hearing screening was sufficient on the left, but insufficient on the right.

Table 1 — Prenatal and postnatal findings in the three Noonan syndrome cases

Prenatal and postnatalfindings in Noonan syndrome Case 1 Case 2 Case 3

Age of the mother (years) 24 28 33Primigravida/multigravida Primigravida Primigravida Primigravida

Prenatal findings:

Increased NT (mm) 9.6 4.4 6.0

Distended JLS Yes, still present at23+4 weeks

No Yes

Ductus venosus Reversed a-wave Not performed Reversed a-wave

Hepatic artery Low resistance flow Not performed Low resistance flow

Nasal bone Hypoplastic Hypoplastic Normal

Increased NF second trimester Yes Yes No

Edema At 36 weeks 3 days; severe bilateral hydrothorax and mild generalized edema

TOP No

Facial features Hypertelorism, low set ears, broad nose and lips, brachycephaly

Low set ears with uplifted earlobes, small nose, sloping forehead, brachycephaly

Unknown

Cardiac anomalies No Malalignment VSD,deviation of the heart axis, mild TR, right ventricular dysfunction, ericardial effusion

Suspicion of a small VSD

Renal anomalies No Bilateral pyelectasis No

Amniotic fluid At 36 weeks 3 days;polyhydramnios

Normal At 27 weeks 5 days;polyhydramnios

Short femur No No No

Other anomalies No Mild ventriculomegaly No

Postnatal findings:

Dysmorphic facial features Downward palpebral slant,epicanthus, telecanthus, low set posteriorly angulated ears, broad nose, low posterior hairline

Low set posteriorlyangulated ears, road nose,brachycephaly

Downward palpebral slant,telecanthus, low set ears

Other dysmorphic features Increased distance betweenthe nipples, tendency toclinodactyly, reducedpalmar grooves,

Redundant nuchal skin,muscular physique

Short stature, pectuscarinatum, asymmetricalthorax

Cardiac anomalies Poly-valvular disease Left ventricularhypertrophy, subaortalstenosis, perimembrousVSD

No

Edema Chylothorax Generalized skin edema No

DNA mutation PTPN11 (c.124A>G(p.Thr42Ala)), de novo

RAF1 (c.770C>T(p.Ser257Leu)), de novo

PTPN11 (c.417G>C,p.Glu139Asp), de novo

NT = nuchal translucency. JLS = jugular lymphatic sacs. VSD = ventricular septal defect. TR = tricuspid regurgitation.


88

As both prenatal and postnatal findings were suggestive of Noonan syndrome, DNA test-ing was performed and showed a PTPN11 mutation (c.124A>G (p.Thr42Ala)), confirming the diagnosis. Both parents are not carriers of the mutation. Two weeks after birth a bone marrow biopsy was performed in view of a monocytosis, leukocytosis and hepatomegaly. An acute monocytic reaction, suspicious for juvenile monocytic myelogenous leukemia (JMML), was diagnosed. As a germ-line mutation on exon 2 was found, the diagnosis of JMML was unlikely (differential diagnosis transient leukaemoid reaction). The baby received a low dosed Cytarabine (ARA-C) treatment for 5 days and was doing well after treatment.

CASE 2

A 28-year-old primigravida was referred to our FMU because of an increased NT of 4.4 mm at 11 weeks 4 days of gestation. A hypoplastic nasal bone and a cyst in the poste-rior fossa were also observed. No other structural anomalies were detected. Due to the increased risk of aneuploidy, CVS was performed and demonstrated a normal female karyotype. An ultrasound examination at 15 weeks in her local hospital reported no structural

Figure 2 — (a) Second trimester transverse view of the skull showing a brachycephaly. (b) Three-dimensional (3D) view of fetal face showing hypertelorism, broad nose and thick lips. (c) 3D view of fetal face showing hypertelorism, broad nose and thick lips.


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anomalies, besides mild cardiac disproportion and because of this finding the patient was referred back to us for the 20-week scan. Follow-up sonography at 19+5 weeks of ges-tation showed a nuchal skin fold of 7.7 mm (Figure 3a and b), wide anterior and normal posterior horns (9.6 and 8.8 mm), small nose, sloping forehead, brachycephaly (Figure 3c and d) and bilateral pyelectasis. Fetal echocardiography showed a heart axis deviated to the left, mild pericardial effusion, a small subaortic malalignment ventricular septal defect (VSD), moderate tricuspid regurgitation and right ventricular dysfunction. Fetal biometry was normal and no other structural anomalies were detected (Table 1). At 20 weeks 3 days of gestation an MRI was performed showing normal intracranial structures, normal gyri and mild ventriculomegaly. Follow-up at 21 weeks 3 days of gesta-tion demonstrated a nuchal skin fold of 10.6 mm and low set ears with uplifted earlobes(Figure 4a and b). As above-mentioned findings were suggestive of Noonan or Costello syndrome, the parents were counseled as such. They decided to terminate the pregnancy. A female baby of 590 g was born at 22 weeks 1 day of gestation. Examination by the clinical geneticist confirmed the classical facial features and muscular physique suggestive of Noonan syn-drome. Autopsy ascertained the presence of low set ears, generalized skin edema, redun-

Figure 3 — (a) Second trimester sagittal view showing an increased nuchal fold. (b) Second trimester transverse view of the skull, showing the increased nuchal fold. (c) Second trimester transverse view of the skull, showing a brachycephaly. (d) Second trimester sagittal view showingthe profile of the fetus with a thickened prenasal thickness.


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dant nuchal skin, left ventricular hypertrophy, subaortal stenosis and a perimembrane-ous VSD. Placenta pathology was normal and X-ray showed no abnormalities. DNA analysis showed a RAF1 mutation (c.770C>T(p.Ser257Leu)) making a definitive diagnosis of Noonan syndrome. Both parents are not carriers of this mutation.

CASE 3

A 33-year-old primigravida was referred to our FMU because of an increased NT of 6.0 mm at 12 weeks 4 days of gestation. No other structural anomalies were detected. Ductus venosus showed a reversed a-wave and the PIV was 3.40. Due to the increased risk of an-euploidy, CVS was performed demonstrating a normal female karyotype. Ultrasound scanning was repeated at 14+4 weeks’ gestation. Although the NT was al-most normalized, bilateral distended JLS were observed. The PIV of the ductus venosus was increased (1,32), but with a positive a-wave (Table 1). Follow-up sonography at 20 weeks 6 days of gestation showed no structural abnormali-ties and normal fetal biometry. Fetal echocardiography showed a structural and func-tional normal heart. At 27 weeks 5 days, 29 weeks 6 days, 32 weeks 6 days, 36 weeks 6 days of gestation so-nography demonstrated a progressive polyhydramnios (AFI 33.0-37.1). Normal stomach- and bladder-filling were present. Besides the suspicion of a small VSD, no other abnor-malities were seen. At 39 weeks 3 days of gestation, after an uncomplicated vaginal delivery, a girl of 3350 g was delivered. Apgar scores were 8 and 10 at 1 and 5 min, respectively. At 19 months of age, because of dysmorphic features (short stature (-2SD), telecan-thus, downward palpebral slant, low set ears, pectus carinatum, asymmetrical thorax,

Figure 4 — (a) Three-dimensional (3D) view of fetal face showing the low set ears. (b) 3D view of fetal face showing the thick lips and low set ears.


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increased distance between the nipples) and possible motor delay were suggestive of Noonan syndrome, DNA testing was performed, which showed a PTPN11 mutation (c.417G>C, p.Glu139Asp). Both parents are not carriers of this mutation.

Discussion

In this report we have demonstrated that the diagnosis of Noonan syndrome can be made prenatally when the pattern of anomalies is recognized, especially in case of subtle dysmorphic features in fetuses after increased NT and normal karyotype. Three-dimen-sional investigation may be helpful in defining the diagnosis and attention should be focused on the nose, mouth, ears and profile of the fetus. Diagnosis of Noonan syndrome is important as the prognosis for individuals may vary. In a previous study we reported that one out of five chromosomally normal fetuses with increased NT has an adverse pregnancy outcome (Bilardo et al., 2007). As genetic syndromes are diagnosed in around 5% of the fetuses, additional investigations should be considered (Bilardo et al., 2007). Noonan syndrome is the most frequently reported genetic syndrome in association with an increased NT, with a prenatal incidence ranging between 1 and 3% (Brady et al., 1998; Souka et al., 1998; Hiippala et al., 2001). Approximately 50% of Noonan syndrome cases are caused by missense mutations in the PTPN11 gene on chromosome 12 (Tartaglia et al., 2001). PTPN11 encodes the non-receptor protein tyrosine phosphatase SHP-2. The mutations associated with Noonan syndrome result in a gain of function of SHP-2. This protein participates in a wide variety of intracellular signal cascades elicited by a number of growth factors, cytokines and hor-mones, and is required in several developmental processes (Tartaglia et al., 2001, 2002). Mutations in the SOS1-, RAF1-, KRAS-, BRAF-, MAP2K1/2-, NRAS- and SHOC2-gene have been described to account for a small percentage of Noonan syndrome cases (Jorge et al., 2009). The above-mentioned genes, especially mutations in the RAS/MAPK pathway, are not only involved in the pathogenesis of Noonan syndrome but also in four syndromes with clinical features overlapping with Noonan syndrome; Leopard syndrome, Cardiofa-cio-cutaneous syndrome, Costello syndrome and Neurofibromatosis type 1 (Schubbert et al., 2007). In the presented cases we show that suspicion of Noonan syndrome should arise when, after an increased NT, ultrasound investigation in the second trimester shows a persistant NF or cystic hygroma in combination with at least one of the following fea-tures: hydrops fetalis, pleural effusion, cardiac anomalies, polyhydramnios or specific facial abnormalities (Table 1) (Witt et al., 1987; Benacerraf et al., 1989; Izquierdo et al., 1990; Sonesson et al., 1992; Nisbet et al., 1999; Achiron et al., 2000; Bradley et al., 2001; Menashe et al., 2002; Witters et al., 2002; Eccles et al., 2003; Gandhi et al., 2004; Ragavan et al., 2005; Schluter et al., 2005; Becker et al., 2007; Bekker et al., 2007; Kiyota et al., 2008; Gonzalez-Huerta et al., 2010; Houweling et al., 2010). Heart anomalies are found in 60-70% of the postnatal cases (mostly pulmonary steno-sis, ASDS and hypertrophic obstructive cardiomyopathy) and will be one of the major causes requiring medical attention (Sharland et al., 1992; Allanson, 1993). HCM is pres-ent in 10-20% of the cases and the clinical course varies from asymptomatic to rapidly progressive heart failure in infancy (Allanson, 1987; Van der Burgt, 2007). Above-men-


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tioned cardiac anomalies are often missed prenatally as they are typical examples of late onset malformations which may appear during the third trimester of pregnancy or even after birth (Achiron et al., 2000; Menashe et al., 2002), this should be discussed with the parents. In our series and in the literature a CHD was diagnosed prenatally in 16 of the 42 Noonan syndrome cases (38.1%), which is significantly lower than diagnosed postna-tally in Noonan syndrome infants. As above-mentioned findings are mild and not specific, being common to other syn-dromes and sometimes also present in normal fetuses it is necessary to define which sonographic findings should prompt targeted prenatal DNA diagnostics for Noonan syn-drome (Nisbet et al., 1999; Schluter et al., 2005). Houweling et al. (2010) advocate that given the high incidence of Noonan syndrome in fetuses with increased NT and normal karyotype, genetic counseling and Noonan syn-drome mutation detection should always be offered, even in the absence of additional

Table 2 — Prenatal findings in Noonan syndrome in the literaturea

Prenatal findings in Noonan syndrome

Our cases Literature TotalN % N % N %

Total cases 3 100 39 100 42 100

Increased NT/cystic Hygroma 3 100 12 30.8 15 35.7

Distended JLS 2 66.7 5 12.8 7 16.7

Increased NF/cystic hygromasecond trimester

2 66.7 19 48.7 21 50

Edema:

Pleural effusion 1 33.3 16 41 17 40.5

Ascites 0 0 6 15.4 6 14.3

Scalp/skin edema 1 33.3 13 33.3 14 33

Facial features:

Brachycephaly 2 66.7 1 2.6 3 7.1

Hypertelorism 2 66.7 0 0 2 4.8

Low set ears 2 66.7 5 12.8 7 16.7

Broad nose 1 33.3 3 7.7 4 9.5

Full lips 1 33.3 3 7.7 4 9.5

Cardiac anomaliesb 1 33.3 15 38.5 16 38.1

Renal anomaliesc 1 33.3 9 23.1 10 23.8

Amniotic fluid:

Polyhydramnios 2 66.7 19 48.7 21 50

Oligohydramnios 0 0 1 2.6 1 2.4

Short femur 0 0 4 10.3 4 9.5

Other anomalies 2 66.7 3 7.7 5 11.9

NT = nuchal translucency. JLS = jugular lymphatic sacs.a Witt et al. (1987), Benacerraf et al. (1989), Izquierdo et al. (1990), Sonesson et al. (1992), Nisbet et al. (1999), Achiron et al. (2000), Bradley et al. (2001), Menashe et al. (2002), Witters et al. (2002), Eccles et al. (2003), Gandhi et al. (2004), Ragavan and Vause (2005), Schluter et al. (2005), Becker et al. (2007), Bekker et al. (2007), Kiyota et al. (2008), Gonzalez-Huerta et al. (2010), Houweling et al. (2010).b (Malalignment) VSD (5), ASD (1), AVSD (1), AV canal (1), pericardial effusion (3), ventricular dysfunction (1), pulmonary stenosis (5), aortic stenosis (1), cardiomyopathy (6), supraventricular extrasystoles (1).c Pyelectasia; bilateral in most cases, in three cases unilateral.


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abnormalities. We do not believe this strategy should be pursued, as it will not be cost-effective. More importantly, it will cause unnecessary anxiety in the majority of patients, given the fact that most fetuses with normal chromosomes and absence of structural anomalies will be absolutely fine at birth. Lee et al. suggest that the use of prenatal PTPN11 DNA testing based on selected ultrasonographic findings will identify Noonan syndrome in a significant proportion of fetuses. PTPN11 testing based on prenatal ul-trasound abnormalities resulted in detection of a mutation in 16 and 2% of fetuses with cystic hygroma and increased NT, respectively (Lee et al., 2009). Based on our experience and on data from the literature we suggest that prenatal DNA testing is justified in case of increased NT, increased NF or cystic hygroma in the second trimester in combination with one or more of the characteristics mentioned in Table 2. Prenatal DNA testing can aid physicians in counseling parents, planning management options and in optimizing perinatal care. A PTPN11 gene mutation on chromosome 12 is found in about 50% of the cases (Tartaglia et al., 2001). In all other cases genetic investigation should be extended to mutations in other genes involved in the pathogenesis of Noonan syndrome, such as the SOS1, RAF1, KRAS and NRAS genes. In case of prenatal features suggestive of Noonan syndrome, the parents should also be genetically examined, in view of the autosomal dominant inheritance (Van Huizen et al., 2005). Bearing in mind that the diagnosis of Noonan syndrome can be challenging in adult due to the high variability of the clinical characteristics and change in phenotype with age (Allanson, 1993). In case of doubt, asking the parents for childhood pictures may reveal more pro-nounced Noonan syndrome features. Based on the presented cases we suggest that they may be even more pronounced prenatally. Therefore, use of 3D rendering of the fetal face in case of subtle anomalies after an increased NT and normal karyotype, can be a valuable tool in the prenatal assessment of these fetuses. In conclusion, prenatal ultrasound findings in Noonan syndrome can be subtle and aspecific, but when above-mentioned characteristics are present (Table 1), additional tar-geted DNA analysis is indicated.

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29. Snijders R. J., Noble P., Sebire N., Souka A., Nicolaides K. H., 1998. UK multicentre proj-ect on assessment of risk of trisomy 21 by maternal age and fetal nuchal-translucency thickness at 10-14 weeks of gestation. Fe-tal Medicine Foundation First Trimester Screening Group. Lancet 352(9125): 343-346.

30. Sonesson S. E., Fouron J. C., Lessard M., 1992. Intrauterine diagnosis and evolution of a cardiomyopathy in a fetus with Noon-an’s syndrome. Acta Paediatr 81(4): 368-370.

31. Souka A. P., Snijders R. J., Novakov A., Soares W., Nicolaides K. H., 1998. Defects and syn-dromes in chromosomally normal fetuses with increased nuchal translucency thick-ness at 10-14 weeks of gestation. Ultrasound Obstet Gynecol 11(6): 391-400.

32. Souka A. P., Von Kaisenberg C. S., Hyett J. A., Sonek J. D., Nicolaides K. H., 2005. Increased nuchal translucency with normal karyotype. Am J Obstet Gynecol 192(4): 1005-1021.

33. Tartaglia M., Mehler E. L., Goldberg R. et al., 2001. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 29(4): 465-

468.34. Tartaglia M., Kalidas K., Shaw A. et al., 2002.

PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 70(6): 1555-1563.

35. Van der Burgt I., 2007. Noonan syndrome. Orphanet J Rare Dis 2: 4.

36. Van der Burgt I., Thoonen G., Roosenboom N. et al., 1999. Patterns of cognitive func-tioning in school-aged children with Noon-an syndrome associated with variability in phenotypic expression. J Pediatr 135(6): 707-713.

37. Van Huizen M. E., Pighetti M., Bijlsma E. K., Knegt A. C., Bilardo C. M., 2005. Increased nuchal translucency thickness: a marker for chromosomal and genetic disorders in both offspring and parents. Ultrasound Obstet Gynecol 26(7): 793-794.

38. Witt D. R., Hoyme H. E., Zonana J. et al., 1987. Lymphedema in Noonan syndrome: clues to pathogenesis and prenatal diagno-sis and review of the literature. Am J Med Genet 27(4): 841-856.

39. Witters I., Spitz B., Van H. C., Devriendt K., Fryns J. P., Verbek K., 2002. Resolution of non-immune hydrops in Noonan syndrome with favorable outcome. Am J Med Genet 110(4): 408-409.


96


7First Trimester Facial Markers for Abnormal Development – Feasibility,

reproducibility and normal ranges of the PNT, MNM angle, PL-distance,

PNT/NBL-ratio & PFSR

M. Bakker 1

M. Pace 1

E. de Jong-Pleij 3

E. Birnie 1,2

K. O. Kagan 4

C. M. Bilardo 1

1 Department of Obstetrics and Gynaecology, Fetal Medicine Unit,


University of Groningen, Groningen, the Netherlands.3 Department of Obstetrics and Gynecology, St. Antonius Hospital,

Nieuwegein, Utrecht, the Netherlands.4 Department of Obstetrics and Gynecology,

University of Tuebingen, Germany.

Published in Fetal Diagnosis & Therapy, 2016 Nov 18.


98

First Trimester Facial Markers for Abnormal Development – Feasibility, reproducibility and normal ranges of the PNT, MNM angle, PL-distance, PNT/NBL-ratio & PFSR

M. Bakker1, M. Pace1, E. de Jong-Pleij3, E. Birnie1,2, K.O. Kagan4 and C. M. Bilardo1



3 Department of Obstetrics and Gynecology, St. Antonius Hospital, Nieuwe-gein, Utrecht, the Netherlands.

4 Department of Obstetrics and Gynecology, University of Tuebingen, Germany.

Objective: To investigate the feasibility and repro-ducibility of the prenasal-thickness-to-nasal-bone-ratio (PNT/NBL ratio), mandibular-nasion-maxilla (MNM) angle, facial profile (FP) line, profile line (PL-)distance and prefrontal-space-ratio (PFSR) in the first trimester of preg-nancy, to develop normal ranges, and to evaluate these markers in abnormal fetuses.

Methods: In this cross sectional study measure-ments were performed by two opera-tors. Feasibility was tested between op-erators with the X2 test. Inter-operator agreement was quantified descriptively by Bland and Altman’s limits of agree-ment, repeatability coefficient and in-traclass coefficient. Prediction intervals, indicating the interval in which 95% of the individual prediction would lie for a given CRL value, were calculated for all measurements.

Results: Feasibility within and between op-erators was good. The NBL, PNT, PFSR and MNM angle increased with CRL, whereas the PNT/NBL ratio and the PL-distance decreased. All parameters, except the PL-distance and the PFSR had high inter-operator agreement. The PNT and PNT/NBL ratio were increased in most trisomic cases. The MNM-angle was increased in case of micrognathia and complete bilateral cleft. The PFSR and the PL-distance showed large inter-observer variation.

Conclusion: The PNT, PNT/NBL ratio and MNM an-gle can already be measured in the first trimester of pregnancy. The PNT and the PNT/NBL ratio are abnormal in more than a third of the trisomic fetuses. The MNM-angle is increased in fetuses with micrognathia and facial clefts.


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Introduction

Assessment of the fetal face in the second trimester of pregnancy has become an im-portant part of fetal evaluation, not only for the detection of facial anomalies but also in screening for trisomies, especially trisomy 21. Established second trimester profile markers for trisomies are the nasal bone length (NBL), the prenasal thickness (PNT), the ratio between the NBL and PNT and more recently the prefrontal space ratio (PFSR).1-4 Other second trimester profile parameters, e.g. the profile line (FP line) and maxilla-nasion-mandible angle (MNM-angle), have been studied as markers for facial anomalies including profile alterations in case of aneuploidies.2,3,5-7 These are proven reproducible markers for the diagnosis of retrognathia, maxillary alveolar ridge interruption, sloping forehead, frontal bossing and flat profile.6,7 Reference values for these markers are avail-able for the second trimester, however normal ranges for the PNT/NBL ratio, PL-distance, MNM-angle and PFSR in the first trimester are still missing. The frontomaxillary facial angle (FMF-angle) and the NBL have been introduced in the first trimester to improve screening algorithms for trisomies and as a result improve detection rates and decrease false positive rates.8-11 Measurement of the PNT, PNT/ NBL ratio, MNM-angle, FP line and PFSR in the first trimester could possibly further improve the detection of trisomies and/or facial abnormalities early in pregnancy. Although cell free fetal DNA (cffDNA) is becoming rapidly available worldwide and in some countries has replaced the CT as screening test for trisomies, the CT is still the stan-dard of care in the majority of countries. Further improvement of detection of trisomies is still valuable in case cffDNA is not performed or when it is performed as second tier test and to enhance the first trimester detection of structural anomalies which are not trisomy related. Aim of this study was to investigate whether the PNT/NBL-ratio, MNM angle, FP line and PFSR can already be measured in the first trimester of pregnancy (feasibility); to study the inter-operator agreement (reproducibility); and to develop normal ranges for these measurements. Furthermore, we preliminarily explored the performance of these new potential first trimester markers in a number of abnormal fetuses.

Methods

DESIGN

A retrospective cross sectional study on the measurement of PNT/NBL-ratio, MNM angle, FP line, PL-distance and PFSR in the first trimester and early second trimester of pregnancy was undertaken at the University Medical Centre Groningen, the Nether-lands. We selected images stored between 2011 and 2013 from singleton pregnancies between 11-14 weeks of gestation, satisfying the FMF guidelines and with known follow-up. Only images with a true midsagittal plane were used. All images were acquired trans-abdominally, using a Voluson E8 equipped with a 4-8 Hz probe (GE Medical Systems). For each image two experienced operators independently measured the PNT/NBL-ratio, MNM angle, FP line, PL-distance and PFSR.


100

ABNORMAL CASES

Stored first trimester pictures of fetuses with chromosomal anomalies or other genetic or structural anomalies diagnosed during pregnancy were retrieved from the FMU databas-es of the UMC Groningen and the University of Tuebingen. Only pictures with a clear pro-file image where the NBL, the PT and the bony landmarks of the fetal profile were clearly discernable were selected and measured following the methodology described above.

PARAMETERS AND MEASUREMENTS

The NBL was measured from the proximal to the distal end of the white ossification line in midsagittal view of the profile. The PNT was measured as the shortest distance be-tween the nasion and the frontal skin. Since the nasion is not yet fully formed, we took the lowest point of the frontal bone. Calipers were placed on the outermost borders of the skin or bone. The MNM angle objectifies the anteroposterior position of the jaws and is defined as the angle between the lines maxilla-nasion and mandible-nasion in the midsagittal plane (see Figure 1).7

The PFSR was established after drawing a line from the anterior aspect of both the mandibula and maxilla which was extended in front of the fetal forehead (MM-line), as the distance of the leading edge of the frontal bone to prenasal skin (d1) divided by the distance of the prenasal skin to the point where the MM-line is intercepted (d2).1 When the MM-line crossed behind the edge of the prenasal skin, the d1 measurement was still taken between the frontal bone and skin but the d2 measurement was then taken be-tween the MM-line and skin and multiplied by -1 (see Figure 1). The FP line was defined as the line that passes through the middle point of the anterior

Figure 1 — (a) Measurement of the MNM-angle: defined as the angle between the lines maxilla-nasion and mandible-nasion in the midsagittal plane. (b) Measurement of the PFSR: established after drwaing a line from the anterior aspect of both the mandibula and maxilla which was extended in front of the fetal forehead (MM-line), as the distance of the leading edge of the frontal bone to prenasal skin (d1) divided by the distance of the prenasal skin to the point where the MM-line is intercepted (d2). When the MM-line crossed behind the edge of the prenasal skin, the d1 measurement was stilll taken between the frontal bone and skin, but the d2 measurement was then taken between the MM-line and skin and multiplied by -1.(c) Measurement of the PL-distance: perpendicular distance from the FP line to the fetal skin.


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border of the mandible and the nasion. When the FP line passed the frontal bone ante-riorly its position was called ‘negative’. When it passed lengthwise through the frontal bone this was called ‘zero’, and when it passed the frontal bone posteriorly, its position was called ‘positive’.6 The PL-distance is the perpendicular distance from the FP line to the fetal skin (see Figure 1).


Conventional descriptive statistics were applied to all measured parameters. Feasibil-ity for each measure was expressed as the proportion of images that could be measured relative to the total number of images. The difference in feasibility for each measure be-tween the two operators was tested with the X2 test (or Fisher’s Exact Test, if appropriate). Inter-operator agreement was quantified descriptively with Bland and Altman’s limits of agreement (LOAs) and their 95% confidence intervals, and the bias or mean difference (SD, 95% CI) of operator 1 to that of operator 2. The repeatability coefficient (RC), or the value below which the absolute differences between two measurements would lie with-in 0.95 probability, was calculated. Correlations between the operators’ measurements were expressed using the intraclass correlation coefficient (ICC, two way mixed, single measures). Prediction intervals, indicating the interval in which 95% of the individual prediction would lie for a given CRL value, were calculated for all measures. Values of the NBL, PNT, MNM-angle, FP line, PL-distance, PNT/NBL-ratio and PFSR of the individual anomaly cases were also expressed as multiples of the expected value for CRL (MoM). A p-value <0.05 (two-sided) was considered a statistically significant difference.

Table 1 — Characteristics

¬ ¬ ¬ ¬ ¬ Normal N=148 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ Abnormal N=77 ¬ ¬ ¬ ¬ ¬

Mean [CI 95%] Std. deviationMedian

[p25-p75] Mean [CI 95%] Std. deviationMedian

[p25-p75]

CRL 65.66[64.17 – 67.15]

9.17 66.60[58.43 – 73.08]

69.12[66.74 – 71.50]

10.42 68.90[61.78 – 76.68]

NT 1.90[1.80 – 1.99]

.59 1.80[1.50 – 2.19]

4.29[3.80 – 4.77]

2.12 3.60[2.80 – 5.30]

Gestational age 12.76[12.65 – 12.87]

.69 12.71[12.14 – 13.29]

13.14[12.95 – 13.33]

.78 13.24[12.60 – 13.71]

CRL = crown rup length, NT = nuchal transluceny

Table 2 — Feasibility and inter-operator differences in obtaining measurements

N=148 Operator 1 (%) Operator 2 (%) Operator 1 and operator 2

p (operator 1 - operator 2)

NBL 110 (74.32) 118 (79.73) 107 (72.30) .269

PNT 98 (66.22) 117 (79.05) 93 (62.84) .013

PNT/NBL ratio 81 (54.73) 105 (70.95) 78 (52.70) .004

MNM angle 112 (75.68) 117 (79.05) 108 (72.97) .487

PL distance 101 (68.24) 109 (73.65) 97 (65.54) .306

PFSR 93 (62.84) 114 (77.03) 91 (61.49) .008

NBL = nasal bone length. PNT = prenasal thickness. PNT/NB ratio = prenasal thickness/nasal bone length ratio. MNM angle = maxilla-nasion-mandible angle. PL distance = profile line distance. PFSR = prefrontal space ratio.


102

Results

In total 225 fetal profile images were included in the study; 148 from normal fetuses and 77 from fetuses with a (chromosomal) anomaly (trisomy 21: N=63, trisomy 18: N=4, mi-crognathia: N=5, bilateral cleft: N=2,22q11.21 duplication: N=1, Roberts syndrome: N=1, and Turner syndrome: N=1). Table 1 shows the characteristics of both groups. Feasibility, expressed as the proportion of how often a marker could be measured, as well as the difference and overlap in feasibility between operators, is shown in Table 2. Feasibility was the highest for the NBL and the MNM angle and the lowest for the PNT/NBL ratio. Table 3 shows the means and medians of NBL, PNT, PNT/NBL ratio, MNM-angle and PFSR for each operator separately. The mean NBL, PNT and PFSR measured by operator 1 were larger than in operator 2. Operator 2 had a larger mean for the MNM-angle and PL-distance. Both operators had the lowest SD for the PNT/NBL ratio and the

Table 4 — Agreement and correlation between operators

Mean ∆ SD of ∆ ICC (95% CI) SE RC LOA LOA95% CI

NBL (operator 1-2)

.140 .240 .835(.655 – .910)

.030 .470 [-.330 – .620] Lower limit (-.400 – -.270)Upper limit (.550 – .680)

PNT (operator 1-2)

.080 .220 .706(.559 – .805)


MNM angle (operator 1-2)

-.194 2.111 .746(.649 – .819)

.301 4.138 [-4.333 – 3.944] Lower limit (-4.922 – -3.743)Upper limit (3.355 – 4.533)

PL distance (operator 1-2)

-.090 .400 .596(.450 – .711)


D1 (part of PFSR (operator 1-2))

.080 .210 .700(.548 – .801)


D2 (part of PFSR (operator 1-2))

.030 .790 .655[.519 – .758]

.110 1.550 [-1.579 – 1.520] Lower limit (-1.800 – -1.360)Upper limit (1.300 – 1.730)

NBL = nasal bone length. PNT = prenasal thickness. PNT/NB ratio = prenasal thickness/nasal bone length ratio. MNM angle = maxilla-nasion-mandible angle. PL distance = profile line distance. PFSR = prefrontal space ratio. d1 = distance of the leading edge of the frontal bone to prenasal skin. d2 = distance of the prenasal skin to the point where the mm-line is intercepted (d2).

Table 3 — Mean, medians and SD per operator

¬ ¬ ¬ ¬ ¬ Operator 1 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ Operator 2 ¬ ¬ ¬ ¬ ¬



[p25-p75]

NBL 1.91[1.81 – 2.00]

.500 1.80[1.60 – 2.20]

1.77[1.69 – 1.85]

.440 1.70[1.50 – 2.00]

PNT 1.44[1.38 – 1.50]

.290 1.40[1.20 – 1.63]

1.32[1.26 – 1.38]

.330 1.30[1.10 – 1.50]

PNT/NBL ratio .770[.724 – .816]

.210 .750[.625 – .914]

.756[.711 – .138]

.232 .750[.590 – .865]

MNM angle 4.566[4.006 – 5.126]

2.990 4.300[2.375 – 5.983]

4.760[4.243 – 5.286]

2.849 4.300[2.815 – 5.955]

PL distance 2.08[1.99 – 2.17]

.463 2.00[1.70 – 2.30]

2.17[2.08 – 2.26]

.490 2.20[1.80 – 2.50]

PFSR .067[-.069 – .202]

.656 0[-.335 – .512]

-.065[-.213 – .084]

.802 0[-0.555 – 0.374]



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Table 5 — Description abnormal cases other than trisomy 21

N=63 Measured % Mean MoM Range MoM >p95 % (N)

NBL 49.2* .97 .27 – 1.75 -

PNT 95.2 1.43 .76 – 2.52 33.3 (20/60)

PNT/NBL ratio 46.0 1.63 .55 – 7.13 37.9 (11/29)

MNM angle 96.8 0.98 .13 – 3.33 9.7 (6/61)

FP line 95.2 - - -

PL distance 93.7 1.16 .66 – 2.61 16.9 (10/59)

PFSR** 88.9 - - -

* In 50.8% (32/63) of the trisomy 21 cases the nasal bone was absent and as a consequence could not be measured. ** No MoMs could be calculated. NBL = nasal bone length. PNT = prenasal thickness. PNT/NB ratio = prenasal thickness/nasal bone length ratio. MNM angle = maxilla-nasion-mandible angle. PL distance = profile line distance. PFSR = prefrontal space ratio.

Table 6 — Description abnormal cases other than trisomy 21

N=63 NBL(MoM)

PNT(MoM)

PNT/NBL ratio (MoM)

MNM angle(MoM)

PL distance(MoM)

PFSR* FP line

1 Duplication 22q11.21 with palatoschisis and micrognathia

- 1.4 (1.07) - 5.66 (0.96) 2.0 (.95) .43 0

2 Roberts syndrome with micrognathia, club feet, and short upper arms

3.3 (1.75) 1.7 (1.30) .52 (.66) 15.90 (2.69) 1.7 (.81) 1.47 0

3 Bilateral complete cleft, normal karyotype

2.7 (1.43) 1.8 (1.38) .67 (.85) 21.74 (3.68) 1.8 (.85) 0

4 Bilateral cleft, normal karyotype

- 2.0 (1.53) - 30.40 (5.15) 2.0 (.95) 3.50 0

5 Micrognathia 3.6 (1.91) 2.8 (2.14) .78 (.99) 18.60 (3.15) 3.0 (1.42) .65 Neg

6 Micrognathia 2.5 (1.33) 2.4 (1.83) .96 (1.22) 19.80 (3.35) 2.5 (1.18) 1.72 0

7 Micrognathia - .17 (1.30) - 13.50 (2.29) .30 (1.42) .88 Pos

8 Micrognathia .17 (.90) .16 (1.22) .94 (1.20) 14.80 (2.51) .16 (.76) .53 0

9 Micrognathia .16 (.85) .15 (1.15) .94 (1.20) 16.60 (2.81) .17 (.81) 1.00 0

10 Trisomie 18 - 1.8 (1.38) - 6.80 (1.15) 2.3 (1.09) 1.00 Pos

11 Trisomie 18 1.5 (.80) 1.9 (1.45) 1.27 (1.62) 9.40 (1.59) 2.4 (1.14) .39 0

12 Trisomie 18 1.1 (.58) 2.1 (1.60) 1.91 (2.43) 4.30 (0.73) 2.3 (1.09) 1.16 0

13 Trisomie 18 - 1.7 (1.30) - 4.78 (0.81) 1.8 (.85) -.50 0

14 Turner 2.0 (1.06) - - 10.20 (1.73) 2.9 (1.37) -.12 -

* No MoMs could be calculated. NBL = nasal bone length. PNT = prenasal thickness. PNT/NB ratio = prenasal thickness/nasal bone length ratio. MNM angle = maxilla-nasion-mandible angle. PL distance = profile line distance. PFSR = prefrontal space ratio.

highest for the MNM-angle. Agreement and correlation between operators for each measurement are shown in Table 4. The mean difference between operators was the lowest for the measurement of d2 (.030 mm) and the highest for the MNM angle (-.194). The lowest SD was found for the measurements of d1 and the PNT (.210 and .220). The LOAs were the smallest for d1 [-.340 – .500] and the PNT [-.340 – .510]. Correlation between operator 1 and operator 2 was high for the measurement of the

Table 3 — Mean, medians and SD per operator

¬ ¬ ¬ ¬ ¬ Operator 1 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ Operator 2 ¬ ¬ ¬ ¬ ¬



[p25-p75]

NBL 1.91[1.81 – 2.00]

.500 1.80[1.60 – 2.20]

1.77[1.69 – 1.85]

.440 1.70[1.50 – 2.00]

PNT 1.44[1.38 – 1.50]

.290 1.40[1.20 – 1.63]

1.32[1.26 – 1.38]

.330 1.30[1.10 – 1.50]

PNT/NBL ratio .770[.724 – .816]

.210 .750[.625 – .914]

.756[.711 – .138]

.232 .750[.590 – .865]

MNM angle 4.566[4.006 – 5.126]

2.990 4.300[2.375 – 5.983]

4.760[4.243 – 5.286]

2.849 4.300[2.815 – 5.955]

PL distance 2.08[1.99 – 2.17]

.463 2.00[1.70 – 2.30]

2.17[2.08 – 2.26]

.490 2.20[1.80 – 2.50]

PFSR .067[-.069 – .202]

.656 0[-.335 – .512]

-.065[-.213 – .084]

.802 0[-0.555 – 0.374]



104

Figure 2(a-f) — Shows the reference ranges for the NBL, PNT, PNT/NBL ratio, MNM-angle, PL-distance and PFSR in relation to CRL (from 45 - 84 mm for all measurements) in normal fetuses and in a cohort abnormal fetuses. (c) Measurement of the PL-distance: perpendicular distance from the FP line to the fetal skin.


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NBL (ICC .84), MNM-angle (ICC .746), PNT (ICC .71) and d1 (ICC .70) (Table 4). Correla-tion for d2 and the PL-distance was moderate, with an ICC of .66 and .60 respectively.Figures 2a-f show the reference ranges for NBL, PNT, PNT/NBL ratio, MNM-angle, PL-distance and PFSR in relation to CRL (from 45 to 84 mm for all measurements) in nor-mal fetuses. The mean NBL increased with CRL from 1.3 mm to 2.6 mm. Mean PNT also increased with CRL, from 1.2 mm to 2.0 mm. The mean PNT/NBL ratio decreased slightly with CRL, from .69 to .44. The mean MNM-angle increased with CRL, from 0.98 degrees to 7.76 degrees. The mean PFSR increased with CRL, from -.97 to .74. The PL was positive in 13/148 fetuses (0.088, 95%CI [.048 – .146]). Figures 2a-f also show the measurements of the markers in relation to CRL in the ab-normal cases in comparison to the normal ones. Table 5 shows the characteristics of the trisomy 21 cases. Absence of the nasal bone, an increased MoM of the PNT and PNT/NB ratio were most common in trisomy 21 cases. Table 6 shows the characteristics of the abnormal cases other than trisomy 21. In the cases of bilateral cleft and micrognathia the MNM-angle MoMs were all increased above the p95.

Discussion

This study demonstrates that second trimester profile parameters can be measured reli-ably in the first trimester with a high inter-operator agreement, especially for the NBL, the PNT and the MNM-angle. Success in obtaining a measurement was operator and marker-dependent. A possible explanation for this is that the selected images were not taken with the purpose of mea-suring the fetal facial markers, but rather the NT. Furthermore, operators may interpret certain landmarks of a marker differently, implying that adherence to strict criteria would be necessary in order to minimize these differences. In normal fetuses the NBL and the PNT increase with CRL2-4,12-15, whereas the PNT/NBL ratio decreases, suggesting that the growth of the NBL in early pregnancy exceeds that of the PNT. Conversely, in the second and third trimester of pregnancy NBL and PNT seem to grow at a similar rate, resulting in a constant ratio in both normal and abnormal fetus-es.2 Although operators had a different success rate in obtaining the PNT measurement, the PNT/NBL ratio had the lowest SD between operators, meaning that it can be already reliably measured in the first trimester of pregnancy. The MNM-angle and PFSR increase with CRL, whereas in the second and third trimes-ter the MNM-angle and PFSR remain constant.1,57 The increase of the MNM-angle in the first trimester can be explained by a more distinct growth and forward displacement of the maxilla compared to the mandibula, which is only connected to the cranium through the temporomandibular joint and facial muscles and as a consequence the forces producing the development of the mid face with forward displacement of the maxilla could have a lesser effect on the mandibular growth.16,17 The increase of the PFSR can be explained by forward displacement and growth of the maxilla, with a growth rate that seems to exceed that of the prenatal thickness. The mea-


106

surement of PFSR proved to be challenging: incorrect placement of the MM-line of only a few tenths of a millimeter has a large impact on the measured angle and consequently on the measurement of d2. In spite of a recent adaptation of the PFSR to facilitate its mea-surement in the first trimester, its detection rate for trisomies remained still about half of that in the second and third trimester.18

The PL-distance decreases with increasing CRL; this seems to be the result of forward movement of the maxilla and a decrease of convexity of the forehead in the first trimes-ter.16 Inter-operator agreement was low, possibly because a slightly different start of the line at the mandible or a different interpretation of the nasion has a major impact on the measured PL distance. We speculate that due to the small dimension of the fetal face in the first trimester and the high susceptibility to inaccuracies in placing lines with different landmarks, the PFSR and the PL-distance cannot be measured with sufficient reliability. In abnormal fetuses the evaluation of the facial markers revealed that prenasal edema can already be present in the first trimester. The PNT was increased and the NB was ab-sent in the majority of the trisomy 21 and 18 cases in which it could be measured, which is in line with the literature.10,19 The PNT/NBL ratio can therefore also be a marker for trisomies in the first trimester.14 Also in abnormal cases the PFSR, the FP and the PL distance line showed limitations, showing inconsistent trends. An explanation for the heterogenous results might be that in some fetuses with trisomy 21 the PNT is increased, whereas the growth and forward displacement of the maxilla lags behind, resulting in a smaller d2 and decreased PFSR. In cases where the PNT is normal or mildly increased, but d2 markedly increased because of severe midfacial hypoplasia, the PFSR will be increased. The FP line showed no clear trends in trisomic fetuses, although a negative FP line seems indicative of pathology.6 At variance with Yazdi1 et al, who found an abnormal PL-distance in 41.1% of the tri-somy 21 cases, we found an abnormal PL-distance in 18.6% (11/59), with a mean MoM of 1.16. This could be explained by the different measurement of the PL-distance where Yazdi et al use the maxilla instead of the nasion as second landmark. In view of the high inter-operator variability and different causes of an increased PL-distance (e.g. skin ede-ma, smaller jaw, or measurement error) we do not envisage a role of the PL-distance in the first trimester of pregnancy. As in the second trimester, the MNM-angle was increased in the bilateral cleft, micro/retrognathia, 22q11.21 duplication and Roberts syndrome cases and in one case of triso-my 18 with a suspicion of micrognathia suggesting that this could be used as an accurate and reproducible early marker for micrognathia and facial clefts. Limitation of the study is that measurements were performed on first trimester pic-tures originally taken with the purpose of measuring the NT and not specifically to visu-alize facial landmarks. As a consequence feasibility only relates to the success in obtain-ing measurements in the preselected images. At this moment, where the future of first trimester ultrasound screening for chromosomal anomalies is uncertain, it is difficult to speculate on the additional role of the facial markers. Their most important role may be found in the setting of early ultrasound screening for fetal anomalies, as objective tool to assist in the detection of abnormal facial feature isolated or as part of genetic syndromes. Due to the limited number of abnormal cases and to the retrospective design of the


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References

1. Yazdi B., Sonek J., Oettling C., Hoopmann M., Abele H., Schaelike M., Kagan K. O. Pre-frontal space ratio in second- and third-tri-mester screening for trisomy 21. Ultrasound Obstet Gynecol 2013 Mar; 41(3): 262-266.

2. De Jong-Pleij E. A., Vos F. I., Ribbert L. S., Pistorius L. R., Tromp E., Bilardo C. M. Pre-nasal thickness-to-nasal bone length ratio: a strong and simple second- and third-trimester marker for trisomy 21. Ultrasound Obstet Gynecol 2012 Feb; 39(2): 185-190.

3. Sonek J. D., McKenna D., Webb D., Croom C., Nicolaides K. Nasal bone length through-out gestation: normal ranges based on 3537 fetal ultrasound measurements. Ultrasound Obstet Gynecol 2003 Feb; 21(2): 152-155.

4. Persico N., Borenstein M., Molina F., Azu-mendi G., Nicolaides K. H. Prenasal thick-ness in trisomy-21 fetuses at 16-24 weeks of gestation. Ultrasound Obstet Gynecol 2008 Nov; 32(6): 751-754.

5. Sonek J., Molina F., Hiett A. K., Glover M., McKenna D., Nicolaides K. H. Prefrontal space ratio: comparison between trisomy 21 and euploid fetuses in the second trimester. Ultrasound Obstet Gynecol 2012 Sep; 40(3): 293-296.

6. De Jong-Pleij E. A., Ribbert L. S., Pistorius L. R., Tromp E., Bilardo C. M. The fetal profile

line: a proposal for a sonographic refer-ence line to classify forehead and mandible anomalies in the second and third trimes-ter. Prenat Diagn 2012 Aug; 32(8): 797-802.

7. De Jong-Pleij E. A., Ribbert L. S., Manten G. T., Tromp E., Bilardo C. M. Maxilla-nasion-mandible angle: a new method to assess profile anomalies in pregnancy. Ultrasound Obstet Gynecol 2011 May; 37(5): 562-569.

8. Plasencia W., Gonzalez Davila E., Tetilla V., Padron Perez E., Garcia Hernandez J. A., Gonzalez Gonzalez N. L. First-trimester screening for large-for-gestational-age in-fants. Ultrasound Obstet Gynecol 2012 Apr; 39(4): 389-395.

9. Cicero S., Curcio P., Papageorghiou A., Sonek J., Nicolaides K. Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: an observational study. Lancet 2001 Nov 17; 358(9294): 1665-1667.

10. Cicero S., Bindra R., Rembouskos G., Trip-sanas C., Nicolaides K. H. Fetal nasal bone length in chromosomally normal and ab-normal fetuses at 11-14 weeks of gestation. J Matern Fetal Neonatal Med 2002 Jun; 11(6): 400-402.

11. Borenstein M., Persico N., Kaihura C., Sonek J., Nicolaides K. H. Frontomaxillary facial angle in chromosomally normal fetuses at

study, it is still premature to draw firm conclusions on the possible role of the facial markers in first trimester screening.

Conclusion

The PNT and PNT/NBL ratio are already abnormal in a great proportion of first trimester trisomic fetuses, however, as other strong first trimester ultrasound markers for aneu-ploidies are available, it is unlikely these will be of use in improving existing first trimes-ter screening algorithms. The MNM angle seems to be an easy and reproducible marker to assist in first trimester detection of micrognathia and facial clefts. Facial markers may especially play a role as adjunct in the early diagnosis of structural anomalies and genetic syndromes other than trisomies.


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11+0 to 13+6 weeks. Ultrasound Obstet Gy-necol 2007 Oct; 30(5): 737-741.

12. Kagan K. O., Cicero S., Staboulidou I., Wright D., Nicolaides K. H. Fetal nasal bone in screening for trisomies 21, 18 and 13 and Turner syndrome at 11-13 weeks of gesta-tion. Ultrasound Obstet Gynecol 2009 Mar; 33(3): 259-264.

13. Kanellopoulos V., Katsetos C., Economides D. L. Examination of fetal nasal bone and repeatability of measurement in early preg-nancy. Ultrasound Obstet Gynecol 2003 Aug; 22(2): 131-134.

14. Miron J. P., Cuckle H., Miron P. Prenasal thickness in first-trimester screening for Down syndrome. Prenat Diagn 2012 Jul; 32(7): 695-697.

15. Senat M. V., Bernard J. P., Boulvain M., Ville Y. Intra- and interoperator variability in fe-tal nasal bone assessment at 11-14 weeks of gestation. Ultrasound Obstet Gynecol 2003 Aug; 22(2): 138-141.

16. Trenouth M. J. Shape changes during hu-man fetal craniofacial growth. J Anat 1984

Dec; 139 (Pt 4)(Pt 4): 639-651. 17. Som P. M., Naidich T. P. Illustrated Review

of the Embryology and Development of the Facial Region, Part 2: Late Development of the Fetal Face and Changes in the Face from the Newborn to Adulthood. AJNR Am J Neu-roradiol 2013 Mar 14.

18. Yazdi B., Riefler P., Fischmuller K., Sonek J., Hoopmann M., Kagan K. O. The frontal space measurement in euploid and aneu-ploid pregnancies at 11-13 weeks' gestation. Prenat Diagn 2013 Dec; 33(12): 1124-1130.

19. Orlandi F., Bilardo C. M., Campogrande M., Krantz D., Hallahan T., Rossi C., Viora E. Measurement of nasal bone length at 11-14 weeks of pregnancy and its potential role in Down syndrome risk assessment. Ultra-sound Obstet Gynecol 2003 Jul; 22(1): 36-39.

20. Cicero S., Longo D., Rembouskos G., Sacchi-ni C., Nicolaides K. H. Absent nasal bone at 11-14 weeks of gestation and chromosomal defects. Ultrasound Obstet Gynecol 2003 Jul; 22(1): 31-35.


8Is 3D technique superior to 2D in Down syndrome screening?

Evaluation of six second and third trimester fetal profile markers

F. I. Vos 1

M. Bakker 1

E. A. P. de Jong-Pleij 2

L. S. M. Ribbert 2

E. Tromp 3

C. M. Bilardo 1

1 Fetal Medicine Unit, University Medical Centre Groningen,

Groningen, the Netherlands.2 Department of Obstetrics and Gynecology, St. Antonius Hospital,

Nieuwegein, the Netherlands.3 Department of Statistics, St Antonius Hospital,

Nieuwegein, the Netherlands.

Published in Prenatal Diagnosis 2015 Mar; 35(3): 207-13.


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Is 3D technique superior to 2D in Down syndrome screening? Evaluation of six second and third trimester fetal profile markers

F. I. Vos1, M. Bakker1, E. A. P. de Jong-Pleij2, L. S. M. Ribbert2, E. Tromp3 and C. M. Bilardo1

1 Fetal Medicine Unit, University Medical Centre Groningen, Groningen, the Netherlands.2 Department of Obstetrics and Gynecology, St Antonius Hospital, Nieuwegein, the Netherlands.3 Department of Statistics, St Antonius Hospital, Nieuwegein, the Netherlands.

Objective: The objective of this article is to inves-tigate whether in the clinical setting of second trimester ultrasound (US) in-vestigations, 3D multiplanar correction prior to the measurement of Down syn-drome (DS) facial markers (nasal bone length, prenasal thickness, fetal profile line, maxilla-nasion-mandible angle, prenasal thickness to nasal bone length ratio, and prefrontal space ratio) is supe-rior to subjective judgment of a correct midsagittal plane by 2D technique.

Methods: Measurements were performed on 2D images and 3D volumes (corrected to the midsagittal plane), acquired during the same scanning session.

Results: All six markers were measured in 105

datasets (75 of euploid fetuses and 30 of DS fetuses). The maxilla-nasion- man-dible angle measured on 2D images was significantly larger than on 3D volumes (p < 0.01). In all other markers, there was no significant difference between mea-surements performed on 2D images or 3D volumes. No statistical difference was found for any marker between measure-ments performed on images acquired by either 2D or 3D US in their ability to discriminate between normal and DS fetuses.

Conclusion: Nasal bone length, prenasal thickness, fetal profile line, prenasal thickness to nasal bone length ratio, and prefrontal space ratio can be confidently used as DS markers in second trimester US exami-nations performed by 2D US.

Introduction

Specific facial profile features of Down syndrome (US) fetuses have been investigated and used as second and third trimester markers.1-13 The nasal bone length (NBL) was the first to be extensively investigated, followed by the prenasal thickness (PT). Recent studies have shown that the ratio between these two markers (PT-NBL ratio) and the pre-frontal space ratio (PFSR) yields an even better detection rate.6,10 Furthermore, we have previously investigated the maxilla-nasion-mandible (MNM) angle and fetal profile (FP)


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line in both euploid and pathological cases.11,14-16 In countries such as the Netherlands, where participation in first trimester screening is low17 and many DS fetuses remain undetected until the 20weeks scan, these markers may be of importance. Several studies have compared 2D and 3D US imaging during gestation and sug-gested 3D to be superior by allowing a better identification of anatomical landmarks,18 a higher accuracy and reproducibility in measurements of structures in the fetal face and profile,13,19,20 including the NBL.9,21 In a previous study,13 we have shown that 2D images judged to be midsagittal in actual fact are not and need 3D multiplanar correction of in average 11.9 degrees (Y-axis) to 4.3 degrees (Z-axis) to become truly midsagittal. Clear landmarks to identify the exact midsagital plane are missing when only 2D imaging is used, making it difficult to be absolutely sure to be in the exact midsagittal plane. How-ever, it is not clear whether in a clinical setting, 3D imaging has an additional value in terms of an improved detection rate when compared with 2D. The aim of this study is to compare the differences in 2D and 3D technique in the mea-surement and detection rate of facial markers in the second and third trimester.

Methods

Eligible cases were collected from the databases of the Fetal Medicine Unit of the Uni-versity Medical Centre Groningen, which acts as a referral center, and of the Ultrasound Unit of the Saint Antonius Hospital in Nieuwegein, which performs US investigations of high-risk patients. Images were obtained by a Voluson 730 Expert US machine or E8 system equipped with a RAB4-8L probe (GE Medical Systems, Kretz Ultrasound, Zipf, Austria). Three-D volumes of euploid fetuses were retrieved from an available dataset used for a previous study13 of nonsmoking, healthy, low-risk, pregnant Caucasian women with a singleton and uncomplicated pregnancy. The dataset was collected prospectively; after 2D images of the FP were obtained, judged to represent the midsagittal plane and with the fetus facing the transducer, 3D volumes of the fetal face were acquired. Databases of participating centers were searched for second and third trimester DS fetuses of Caucasian parents, collected between January 2006 and July 2013. All cases had been confirmed by karyotyping. The images were collected during clinical investiga-tions and therefore were, in contrast to the images of the euploid fetuses, gathered in a less systematic fashion. For this study, cases with both a midsagittal 2D image of the FP and a 3D volume, acquired separately in the same scanning session, were included. We excluded 2D images that were obviously not midsagittal by systematically assessing all components of the profile. Images that showed a body of the mandible, a retrognathic appearance of the chin, a nostril, odd appearance of the nose, a frontal process of the maxilla, a sharp or blunt angle between the nasal and frontal bones, a bossing or sloping appearance of the forehead, os sphenoidale or a lateral ventricle, or plexus chorioideus were excluded. Visibility of the vomeral bone was considered a very strong indication of the exact midsagittal plane. A square shape of the mandible, normal appearance of lips, philtrum and nose, a flat or only slightly curved forehead, and visibility of the corpus cal-losum were indications for a good midsagittal plane. In order to avoid bias, all the measurements on 2D images were performed first. This


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was followed by multiplanar correction of the 3D volumes to the exact midsagittal plane with subsequent measurements. To assess the interobserver and intraobserver variability of the measurement error, markers were remeasured after a 1-week interval. Markers were measured by two exam-iners (F. I. V. and E. J. P.), who were blinded to gestational age and to previous measure-ments, but not to karyotype. The NBL, PT, FP line, MNM angle, PT-NBL ratio, and PFSR were all measured as de-scribed in previous studies of euploid and DS fetuses6,10,14,15,22 (Figure 1). Data were com-pared with the reference values derived from previous reports on euploid fetuses:6,10,14,22 the NBL22 and PT22 increased with gestation from 3.3 mm at 15 weeks’ gestation to 9.6 mm at 33 weeks (NBL = -6.927 + (0.83 ◊ GA) - (0.01 ◊ GA2)) and from 2.3 mm at 15 weeks to 6.1 mm at 33 weeks (PT = 0.212 ◊ GA - 0.873), respectively. The MNM angle,14 PT-NBL6 ratio, and PFSR10 were stable throughout gestation, with a mean of 13.5 degrees (95th centile = 16.9), 0.61 degree (95th centile = 0.80), and 0.97 degree (5th centile = 0.55), re-spectively. Measurements below the 5th centile (for NBL and PFSR) or above the 95th centile (for MNM angle, PT, and PT-NBL ratio) of the reference ranges were considered abnormal. An FP line that was not ‘zero’ was considered abnormal.15

The difference between the 2D and 3D measurement was analyzed in each individual fetus, of which a mean difference was calculated. Differences between measurements

Figure 1 — Ultrasound images of the markers in DS fetuses. a. FP line position ‘zero’; b. FP line position ‘positive’; c. MNM angle; d. NBL (d, A), PT (d, B), PT-NBL ratio (d, B/A), PFSR (d, C/B). FP = fetal profile line. MNM angle = maxilla-nasion-mandibula angle. NBL = nasal bone length. PT = prenasal thickness. PT-NBL ratio = prenasal thickness to nasal bone length ratio. PFSR = prefrontal space ratio.


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were calculated for the whole group, and a separate analysis was performed in the group of DS fetuses, in order to assess if the use of one of the two techniques (2D vs. 3D) had an impact on the detection rate.

Figure 2 — Box plot showing mean difference (black bars) and 95% limits of agreement (boxes) with their confidence intervals (whiskers), for intraobserver variability and interobserver variability in 2D and 3D measurements. The nasal bone length (NBL) and prenasal thickness (PT) are expressed in millimeters and the maxilla- nasion-mandible (MNM) angle in degrees. PT-NBL ratio = prenasal thickness to nasal bone length ratio. PFSR = prefrontal space ratio.

Figure 3 — Nasal bone length (NBL) measurements performed on 2D images and 3D volumes in euploid and Down syndrome (DS) fetuses, plotted on normal ranges (mean, 5th centile and 95th centile).


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Table 1 — Mean values of 2D and 3D measurements (N=105) in a combined cohort of euploid and Down syndrome fetuses.

¬ ¬¬¬ ¬ Mean ¬ ¬ ¬¬¬2D 3D Mean difference (LOA) ICC

NBL (mm) 6.29 6.20 0.08 (-2.48 – 2.57) 0.84

FP Line * ** - 0.68

MNM (degrees) 14.77 13.75 1.03 (-5.14 – 6.13)*** 0.40

PT (mm) 5.41 5.09 0.33 (-1.50 – 1.83) 0.83

PT-NBL ratio 0.91 0.89 0.03 (-0.45 – 0.49) 0.83

PFSR 1.04 1.09 -0.06 (-0.87 – 0.79) 0.68

Corresponding mean difference with limits of agreement (LOA) and intra class correlation coefficients (ICCs) are reported. It was not possible to analyze mean differences between measurements of the FP line, as the outcome was noncontinues.* FP line in 2D: 77.5% positive, 22.5% negative.** FP line in 3D: 79.8% positive, 20.2% negative.*** Significant difference between 2D and 3D measurements in the MNM angle (p < 0.01).MNM = maxilla-nasion-mandible angle. PT = prenasal thickness. PT-NBL ratio = prenasal thickness to nasal bone length ratio. PFSR = prefrontal space ratio.


Intraclass correlation coefficients (ICCs) were calculated, and Bland–Altman analysis was performed to analyze intraobserver and interobserver variability. The student’s t-test was used to analyze differences between measurements. A p-value of less than 0.05 was con-sidered statistically significant. Data were analyzed using the statistical software SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, USA) and Excel for windows 2000.

Results

In the analysis, a total of 105 datasets were included: 75 of euploid fetuses (median gesta-tional age 24, range 15-32 weeks) and 30 of DS fetuses (median gestational age 24, range 17-34 weeks). Originally, 32 datasets of DS fetuses were available, but two were excluded, as the 2D images were judged not to be truly midsagittal. Mean values, mean differences between 2D and 3D measurements, and corresponding ICC of 2D and 3D measurements in a combined cohort of euploid and DS fetuses (N=105) are shown in Table 1. It was not possible to analyze mean differences between measure-ments of the FP line as the outcome was noncontinuous (positive or zero; no FP line was negative). For the MNM angle, 2D measurements were significantly larger (p < 0.01), although the mean difference was small (1.0 degree). For the other markers (NBL, PT, FP line, PT-NBL ra-tio, and PFSR), there was no significant difference in measurements performed in either 2D or 3D US. Intraobserver and interobserver variability in 2D and 3D US for each marker (except


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Table 2 — Mean values for 30 datasets of Down syndrome fetuses with their corresponding detection rate.

¬ ¬¬¬ ¬ Mean ¬ ¬ ¬¬¬ ¬ ¬ Detection rate ¬ ¬2D 3D 2D(%) 3D(%)

NBL (mm) 4.37 3.91 83.3 82.6

FP Line - - 37.0 41.4

MNM (degrees) 14.35 14.07 3.7 10

PT (mm) 6.31 6.15 83.3 86.7

PT-NBL ratio 1.40 1.55 96.7 100

PFSR 0.60 0.51 50 56.7

No statistical significant differences were observed between 2D and 3D measurements. MNM = maxilla-nasion-mandible angle. PT = prenasal thickness. PT-NBL ratio = prenasal thickness to nasal bone length ratio. PFSR = prefrontal space ratio.

for the FP line), with their corresponding limits of agreement (LOA) and 95% confidence interval, is shown in Figure 2. LOAs were smaller for all 3D measurements, except for the MNM angle. In the separate analysis of DS fetuses only, no statistical difference was found for any marker between measurements performed in images acquired by either 2D or 3D US in



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their ability to discriminate between normal and DS fetuses (Table 2, Figures 3-8).

Discussion

This study demonstrates that when strict criteria are applied, subjective judgment of a good mid-sagittal plane on 2D images is sufficient to ensure a good performance of facial markers for DS. In a cohort of euploid and DS fetuses, no significant difference was found in NBL, PT, FP line, PT-NBL ratio, and PFSR, measured in midsagittal images obtained by 2D or 3D US. Only for the MNM angle, 2D measurements were slightly, but significantly, larger. Both 2D and 3D technique can perform equally well in identifying DS fetuses, without signifi-cant difference between measurements. The clinical implication of these findings is that these markers can be used effectively in routine screening settings relying on 2D technique using strict criteria, without miss-ing out on the additional benefit of 3D US. This finding has great implications in a mo-ment of worldwide financial constraints and growing medico-legal problems, where the general opinion is that 3D US is superior to 2D US.13,18–21

In literature, the role of 3D technique in the measurement of fetal facial biometrical parameters has been underlined by many studies. A volume obtained starting from an oblique scanning plane can be corrected to the exact midsagittal plane, allowing accurate and reproducible measurements. Moreover, a stored 3D volume can be analyzed offline retrospectively, possibly shortening the time of investigation. Suggested disadvantages of using 3D volume corrections are, that it requires costly


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equipment, specialized personnel, and it may be more time-consuming.23 However, other studies found no difference in time13,24 or found 3D to be even faster.25-27

Following our previous report,13 this is the first study that evaluates the use of 2D ver-sus 3D acquired images in the evaluation of profile markers. Benoit and Chaoui21 demonstrated that, in case of suspicion of an absent nasal bone on 2D images, the nasal bone can be better visualized in 3D volumes. Persico et al.9 showed that 3D NBL measurements tend to be larger when the scanning plane is not exactly mid-sagittal, which decreased the detection rate for DS. In our previous study,13 we found no difference between measurement modality but reported narrower LOA in 3D performed measurements. A possible criticism of this study is that for comparison, we performed a selection of 2D pictures likely to represent the true midsagittal plane. All 2D US measurements are taken on planes subjectively judged as correct according to anatomical landmarks. However, in a previous study,13 we showed that after volume acquisition, even when the image on the A plane was subjectively judged to be midsagittal, variable degrees of cor-rection by multiplanar mode were required in order to obtain the true midsagittal plane. Based on the results of the present study, the measurements of facial markers in the initial 2D im-age are highly comparable with those measured in 3D corrected planes. Limitations of this study are its retrospective nature and the fact that examiners were not blind-ed to the karyotype. An ideal repeatability and reproducibility study would require reacqui-sition of the same images by two observers. Because of the retrospective nature of this study, this was not possible; however, reproducibility of the markers is established in the



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1. Hansmann M., Trisomy 21 in the mid tri-mester: sonographic phenotyping of the fetus is the key. Ultrasound Obstet Gynecol 2004; 23: 531-4.

2. Bromley B., Lieberman E., Shipp T. D., Bena-cerraf B. R. Fetal nose bone length: a marker

for Down syndrome in the second trimes-ter. J Ultrasound Med 2002; 21(12): 1387-94.

3. Cicero S., Sonek J. D., McKenna D. S. et al. Nasal bone hypoplasia in trisomy 21 at 15-22 weeks’ gestation. Ultrasound Obstet Gyne-col 2003; 21(1): 15-8.

original publications.6,14,22

Influence of circumstances such as reduced amniotic fluid or the fetus facing down was not taken into account; however, these circumstances would equally affect 2D and 3D performance.13,28

We expressed differences in measurements in ICC, as the markers are quantified by different metric parameters (degrees, mm, and ratios). The MNM angle, FP line and PFSR had a relatively low ICC when 2D and 3D measurements were compared. One explana-tion may be that the aforementioned markers, in contrast to the PT and NBL, require multiple landmarks that may enhance the variability in the measurement. For the MNM angle, 2D performed measurements were significantly larger, the ICC of 2D versus 3D measurements was low, and LOAs of intraobserver and interobserver vari-ability were relatively wide. This could be because the reproducibility of the MNM angle is in general more challenging and that, especially in this case, the bony structures used as a landmark for the measurement are better identified by 3D US. Conversely, the PFSR has a suboptimal ICC when 2D and 3D measurements are com-pared, but the mean difference between 2D and 3D measurements is not significant. The LOAs of the PFSR are larger when compared with that of the PT-NBL ratio (also expressed as a ratio), especially in 2D. These findings show that the reproducibility of the PFSR (in 2D) is lower, but the actual measurement is not influenced by the technique of image acquisition. In spite of these findings, no impact of acquisition modality could be found in detec-tion rate and measurements performed in DS fetuses. This is reassuring, as the goal of these measurements is to discriminate between DS and euploid cases. The best moment for DS screening is undisputable the late first trimester. However, uptake of first trimester screening varies among countries. In the Netherlands, for in-stance, where the combined test is only covered by insurance beyond 36 years of age, the uptake is low.17 In contrast, uptake of second trimester US screening for structural anomalies, which is covered for all women,29 is over 95%.30 This means that a consid-erable number of DS pregnancies remain undetected. Also when late termination of pregnancy is not available, a late diagnosis of a chromosomal anomaly is important to prepare future parents and establish the appropriate obstetrical management. In conclusion, we have shown that, with exception of larger MNM angles in 2D im-ages, no significant differences were found between 2D and 3D imaging in a number of facial DS markers. In particular, NBL, PT, FP line, PT-NBL ratio, and PFSR can be confi-dently used as DS markers in US examinations performed by 2D US, provided the mark-ers are measured in a midsagittal image, acquired according to strict criteria.

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22. Vos F. I., De Jong-Pleij E. A., Ribbert L. S. et al. Three-dimensional ultrasound imaging and measurement of nasal bone length, pre-nasal thickness and frontomaxillary facial


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28. Turner G. M., Twining P. The facial profile in the diagnosis of fetal abnormalities. Clin Radiol 1993; 47: 389-95.

29. RIVM (Dutch National Institute for Public Health and the Environment). A brief history of the 20-week ultrasound, 2014. Available at: http://www. rivm.nl/Onderwerpen/T/Twintig_wekenecho/Voor_professionals/ Achtergronden/Korte_geschiedenis [Ac-cessed on January 2013].

30. Ensing S., Kleinrouweler C. E., Maas S. M., Bilardo C. M., Van der Horst C. M. Influ-ences of the introduction of the 20 weeks fetal anomaly scan on prenatal diagnosis and management of fetal facial clefts. Ultra-sound Obstet Gynecol 2014; 44(2): 154-9.


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General discussion and future perspectives

M. Bakker


9General discussion and future perspectives

M. Bakker


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General discussion and future perspectives

M. Bakker

In 2007, a prenatal screenings program was introduced in the Netherlands. This pro-gram included the combined test (CT) which is on average performed at around 12-13 weeks of gestation, and the structural anomaly scan performed around 20 weeks of ges-tation. All women are offered the possibility to opt for prenatal screening. Once an increased risk (>1:200) or an increased nuchal translucency (>p99) is found during the combined test, or there is a suspicion on an anomaly during the structural anomaly scan, women are counseled on the possibility of prenatal diagnostics. One of the factors influencing women’s decision to opt for or decline chorionic villus sampling (CVS) or amniocentesis (AP) is the procedure related risk, which is lower than previously counseled 1%, in experienced hands.

LIMITATIONS

Limitations of Chapter 3, 7 and 8 are that measurements were performed on preselected images. As a consequence in Chapter 3 and 7 feasibility only relates to the success in ob-taining measurements in the preselected images and not to the ability of obtaining the correct images, which might positively have influenced our results. In Chapter 8 this is also the case and during that study there was more time for postproduction measure-ments than normally in the clinic would be the case. The importance of factors influencing image quality, e.g. gain and harmonics, are well known.1 From a technical point of view, the main contributor to intra- and inter-oper-ator differences is the acquisition of the optimal image.2 This means that precision of measurements is still largely dependent on appropriate training, adherence to strict cri-teria, coincidental circumstances (e.g. fetal position, maternal BMI, time allocated) and, last but not least, on the operator’s personal attitude in terms of endurance and accuracy.

UPTAKE

In Chapter 2 we addressed the uptake of the CT and its determinants. The uptake of the CT since its introduction is around 30%, especially among women under the age of 36 years. This is considerable lower than the > 90% uptake of the 20 week anomaly scan.3 One of the criteria to evaluate the success of a national screenings program is its uptake.4 High uptake as success criterion is less obvious when it comes to prenatal screening: there is no effective treatment for the screened conditions that represent important health prob-lems like trisomy 21, 18 and 13. Parents are offered choices that are supposed to increase their reproductive autonomy. Therefore it is important to allow women/parents to make an informed choice rather than to achieve an as high as possible uptake.5 Making an in-formed choice depends mainly on two things, the decision should be based on relevant information and it should be consistent with the decision makers’ values.6 So women should be aware of the option of prenatal screening, they should be adequately coun-


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seled, and when they take a decision it should be an informed one. In Chapter 2 we also addressed the fact that the CT uptake is currently lower than up-take rates previously reported in research settings in the Netherlands (86% and 53% re-spectively).7,8 Like the Netherlands, a similar decreasing trend was seen in the UK, where uptake rates over the years decreased from 83% to 41% (1993-2005).9 Do women nowa-days more often make informed choices? Shanta et al. concluded that attitude towards the CT and TOP had a larger impact on the uptake than knowledge on the CT.10 However, uptake rates in the UK are still higher than in the Netherlands, so cultural, societal or health system characteristics may also play a role.10 This brings us to the possible deter-minants of the low uptake of the CT in the Netherlands.

DETERMINANTS OF UPTAKE

Chapter 2 also addresses the determinants or reasons for women to decline the CT. These were mainly a positive attitude towards Down syndrome, a negative attitude towards termination of pregnancy, doubts about the reliability of the CT as screening test and fear for iatrogenic pregnancy loss when invasive diagnostics are indicated. Chapter 2 also gives the impression that Dutch women still perceive maternal age as a strong and reliable predictor of Down syndrome risk. Women of 36 years and older were almost five times more likely to opt for the CT than women below that treshold, and over one third of the younger women declined the CT because they considered their age-related risk to be low. According to literature, women’s perceptions of maternal age as strong indicator of Down syndrome risk and the inequality of access to care due to the financial threshold for younger women which existed until January 2015, has likely been influencing partici-pation in screening.11,12 Both aspects, perceptions of maternal age and the perception of age-related copayment, will be discussed below.

DETERMINANTS: PERCEPTION OF MATERNAL AGE

The perception of women that their age is a strong predictor of Down syndrome risk can be understood from the prenatal screenings history of the Netherlands, which is unique in Europe. The 2003 advice of the Ministry of Health (Kamerstukken, 2003, 29 323, nr.1) reads that the risk of trisomy 21 increased exponentially after a pregnant woman turned 36 years, justifying an invasive procedure. Although the age-related relationship may be justified from a descriptive point of view, the prognostic impact of maternal age in individual decisions is less obvious. It has long been known that maternal age as single risk factor is an inadequate prognostic model of screening for trisomy 21 (detection rate based on maternal age only: 30%) with the inherent risk of iatrogenic miscarriage, in comparison to the more comprehensive prognostic model of the CT (detection rate 90%).13 As a result, until 2015, advanced maternal age (≥ 36 years) was used as an accepted indication for direct invasive prenatal diagnostics in the Netherlands.

DETERMINANTS: COPAYMENT

Inequality of access to care was also present due to an age-related threshold for the reim-bursement of the CT for women younger than 36 years (until 2015), discouraging them


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to opt for this test.11 The 2003 advice of to the Ministry of Health (Kamerstukken, 2003, 29 323, nr.1), mentioned two other relevant aspects. Firstly, that the CT would perform less well (in terms of predictive value) in younger women than in women of 36 years, and that it would lead to unnecessary medicalization of younger, supposedly low-risk, pregnant women, implying indirectly that this was an inferior test for this age group and therefore should not be recommended. However, later research has shown that the CT performs equally well in younger as in older women.14 It is likely that the age-related threshold, in combination with women’s biased perception of their age-related risk influenced their risk perception and, consequently, their decision to participate in prenatal screening.11,12

Recently, the Ministry of Health decided that the CT should be accessible to all women irrespective of their age from the 1st of January 2015. Since then however, all pregnant women in the Netherlands have to pay for the CT, while the structural anomaly scan is free of charge. Studies have shown that personal costs or co-payment may play a signifi-cant role in the decision whether or not to opt for the CT.12,15 Research among women accepting or declining the CT showed that co-payment in itself was not per se a reason to accept or decline, but women who declined the CT perceived it as extra confirmation that the test was not necessary whereas women accepting the CT thought it would dis-courage others from taking the test causing inequality of care.11 As a consequence, this financial threshold may now also discourage women of 36 years and older to opt for this test. Access to prenatal screening should be free of charge for all women so their decision is based on willing to know their individual risk for a trisomy in this pregnancy and not on whether or not they want to pay or are able to pay for this test. It seems that we have substituted the age-related threshold for a financial one.

DETERMINANTS: COUNSELING

Besides access to care, the quality of counselling is also likely to affect women’s decision to opt for or decline prenatal screening. A study by Crombag et al. showed that women who intended to decline prenatal screening often referred to their low a priori risk of tri-somy 21 according to the age-related risk tables in the information leaflet and perceived support for their decision to decline by their midwife.11 The national information leaf-let states that ‘The mother’s age affects the likelihood of a child with Down syndrome’, but does neither reveal if the mother’s age is the single determinant, nor how large the impact on the likelihood of Down syndrome is. Alternatively, a clearer statement could be that maternal age is part of the risk assessment and not the only determinant of risk, as we already suggested in Chapter 2. To avoid over-emphasis on maternal age, the age-related risk table could be removed from the leaflet, since this table can be interpreted in different ways (e.g., comparing age-related risks, interpreting the average age-related risk as an individual risk) and is prone to comparing risks between age-groups. Women often fail to make a correct distinction between their average age-related risk and their own individual risk.16 Moreover, a more objective explanation of the age-related and estimated risk to younger women could stimulate equal access. It is not only the women who think that their age is the strongest predictor of their Down syndrome risk, but there is evidence that some healthcare professionals think the same and counsel accord-ingly.16,17,18


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LEARNING CURVE AND EXPERIENCE

Chapter 4 shows that pregnancy losses after invasive procedures performed transabdomi-nally or transcervically (by forceps) are lower than previously thought, provided they are performed by experienced operators. The fear of losing a wanted pregnancy is still one of the most important factors influencing women’s decision to opt or decline CVS or AP. Chapter 4 confirms the impact of operator and procedure related characteristics on the procedure related risk of fetal loss, with fewer losses by experienced operators. This is in line with the study of Wijnberger et al. that emphasized the importance of the level of operator and center experience, irrespective of the technique used.17 Appropriate train-ing of new operators under experienced supervision or by the use of training models, can minimize a learning curve effect.20 Furthermore there is a direct and positive rela-tionship between operator caseload and sampling efficiency.21 This calls for centraliza-tion in only few centers by experienced operators, increasing the minimal caseload per year per operator. In the Netherlands, the Dutch society for Obstetrics and Gynecology has recommended a minimal number of 30 procedures per operator per year.22 However, there is no clear evidence that this number of procedures is sufficient to maintain and develop experience. Nor is it clear how many operators will still meet this requirement in view of the introduction of the cell free-DNA test and, consequently, the rapidly de-clining numbers of CVS and AP. This prompts for reconsideration of our national quality-norm. However it remains difficult to establish what the minimal number of procedures per year must be. One should be aware that operator’s competence is more than his/hers experience in terms of numbers performed alone and also depends on individual opera-tor’s skills (e.g., the amount of repeated attempts). The RCOG states that an operator’s competence should be reviewed when fetal loss rates appear high and an audit should occur when the loss rate exceeds 4/100 consecutive amniocenteses or 8/100 consecutive CVS, but these criteria are based on expert opinion.23 Increasing the minimal number of procedures per year per operator will most likely positively influence operator competency and patient safety, but it also compromises training of new specialists as well as the numbers of procedures needed to maintain op-erator competency. As a consequence, the development of an individual quality control program for invasive procedures could be considered, taking into account individual operator’s numbers, and safety. In other fields of medicine, especially in minimal inva-sive surgery, cumulative summation (CUSUM) analysis is used to assess the learning curve for certain procedures.24 CUSUM is a quantitative assessment of consecutive pro-cedures and their outcome performed over a certain time interval, with reference to an agreed standard (for example, an expected complication rate). This could be an appropri-ate method to monitor learning curves for invasive procedures and set a standard for the minimal caseload per year per operator, but also to monitor the performance of experi-enced operators. Alternatively, the use of simulation models could be an effective strat-egy to train new operators and refresh and maintain experience of trained operators.20

CURRENT PRENATAL SCREENINGS PROGRAM AND INTRODUCTION OF NIPT

In Chapter 3, 5 and 7 we examined the possibility to improve the detection rate of chro-mosomal and structural abnormalities in the first trimester of pregnancy. We show that


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an increased NT is much more than just a marker for Down syndrome, since increased NT is also associated with other chromosomal abnormalities, structural anomalies, genetic syndromes, a higher risk of miscarriages and intrauterine death. Furthermore, facial markers can help to detect chromosomal and structural abnormalities in the first trimester. However, the role of first trimester ultrasound as screening test for Down syn-drome and other chromosomal and genetic abnormalities, as described in Chapter 2-7, is likely to change with the recent preliminary decision of the Ministry of Health to make NIPT available as first tier screening for all women as part of the TRIDENT II Study.25

A study by Tamminga et al. showed that most healthcare professionals in the Nether-lands (72%) think that NIPT should replace the CT, but almost half (43%) feels that the possibility of NT-measurement should be maintained.26 Internationally, the percentage of healthcare professionals in favor of maintaining the NT-measurement is even higher, 71%.27 In the Netherlands NIPT was introduced in April 2014, as part of the TRIDENT-I na-tional implementation study. Dutch pregnant women were eligible for NIPT only when they had an increased risk after the CT (≥1:200), except when the NT was >p99, or when they had an increased risk based on their obstetric history. When it comes to detecting trisomy 21, NIPT has better screening characteristics than the CT, with a high sensitivity of 99% and a low false positive rate of < 0.1.28 As a result, the future of the CT as screening test for Down syndrome is uncertain since the test characteristics of the NIPT are by far better, it can be carried out from 10 weeks of gestation and onwards, and preliminary results of the TRIDENT-I study show that the number of invasive procedures is declining. The place NIPT is going to take in the Dutch prenatal screening program, as first or second tier test, is still a matter of debate. For TRIDENT II, the successor of the TRIDENT-I study, the Ministry of Health has granted a permission in July 2016, within the Popula-tion Screening Act, to carry on with NIPT and to extend the offer to all pregnant women as a first tier test.29 As a result, in the near future all women are allowed to choose be-tween NIPT or the CT as first-tier screening test for trisomy 21, 18 and 13. In the TRIDENT II study, women who opt for the CT as first-tier test, can still later opt for NIPT when an increased risk is present (TRIDENT-I). When women opt for NIPT as first-tier test, the CT will be replaced (TRIDENT-II). Since NIPT has a higher detection rate for trisomy 21 and a lower false positive rate than CT, the likelihood of an unwanted invasive procedure is much lower. As a result, many Dutch women who were not eligible to the TRIDENT inclusion criteria, travelled abroad to undergo NIPT in centers offering NIPT commercially. This situation was the main incentive for the liberalization by the Ministry of Health, since it created a dispar-ity between well informed women with a good financial situation and others in less fortunate circumstances. Despite the granted permission of the Ministry of Health, the financial aspects (who will be responsible for the costs; the women, the government or insurance companies) and the implications for equal access to the NIPT are unresolved at this moment. The Netherlands would be the first European country offering this new form of screening as a first tier test, implying that all women may choose to have it as first form of screening, followed by a confirmatory invasive procedure in case of a posi-tive test. Other European countries use NIPT mainly as second tier test.30,31 One draw-back of offering NIPT as first tier test, besides associated health care costs,30 is that it may result in a poorer detection of trisomy 13, trisomy 18, triploidy and sex chromosome


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anomalies. Which are more efficiently detected by a contingent screening approach, i.e. CT followed by NIPT in case of high-risk.31,32

While the Dutch laboratories are preparing to accommodate the expected rise in de-mand for NIPT, a Commission Prenatal Screening of the Health Council is working on a proposal for a future broad screening offer in pregnancy, not only confined to congenital anomalies, but also for other conditions, such as pre-eclampsia and premature labor. One of the arguments against making NIPT available as 1st tier test to all women is the concern, shared by both healthcare professionals and women, that pregnant women would opt for the test too easily without having thought about the possible consequenc-es.26 Interestingly, a Dutch study showed that 54% of pregnant women did not think that NIPT would lead to more women feeling obliged or coerced to participate in screen-ing.33 However to facilitate informed decision making regarding NIPT, as with the CT, adequate counseling is and remains a key-issue. In the Netherlands counseling is mainly performed by community midwives, and research suggests that 74% of the counseled women make an informed choice regarding the CT.34 At this moment, counseling for NIPT is mainly performed by trained healthcare professionals in academic hospitals. When NIPT will be adopted as first-tier test, the counseling in primary care will mainly be performed by community midwives. To guarantee the quality of counseling for this more complex screening test, counselors will have to be trained. The National Center for Screening is already planning mandatory training workshops for 2017. Counseling has to cover the differences between NIPT and the CT, the predictive value of a positive NIPT test and the possibility of unexpected fetal or maternal findings. The difference between the micro array’s coverage and NIPT, in case of an increased NT > 3,5 mm or structural anomalies, should also be discussed. With increasing complexity of counseling, the ex-pectation is that only a limited number of midwives specialized in prenatal counseling will perform the counseling. Also other options should be explored, like a decision aid or web-counseling.35

Another concern is that when NIPT as 1st tier test is performed early in pregnancy, women become aware of an increased risk on a trisomy in early pregnancy, and they could be more likely to terminate the pregnancy. Some women and also some healthcare professionals think this could have a negative impact on the current social view on people with a handi-cap in society, in that they are less accepted in comparison to people without a handicap. However, reasons to terminate a pregnancy can be very diverse and may be independent from a woman’s view on existing handicapped people in society. When the pregnancy is wanted, women always experience the decision to terminate as a difficult one.36

An important incentive whether or not the NIPT becomes a first or second-tier test is the impact on health care costs. A Dutch study by Beulen et al. showed that implementa-tion of NIPT as second-tier test was the strategy with the lowest cost per case of trisomy 21 diagnosed, in comparison to current clinical practice or when NIPT was used as first-tier test, but this partially depended on the cost per NIPT-test.37 If the costs per NIPT-test de-creased, it became feasible, in terms of health care costs, to introduce it as a first-tier test.. In case the Dutch laboratories will be able to offer the test at a lower cost, there will be no financial barrier to introduce NIPT as first tier test, although other than financial barriers may still exist. However, that leaves still three issues that need further consid-eration: The first one is that NIPT shows excellent performance for trisomy 21, relatively


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good performance for trisomy 18, but definitely suboptimal performance for trisomy 13, Turner and Triploidy in comparison to the CT.30-32 The second one is that the Ministry of Health has agreed to counsel pregnant women whether they want to be informed on coincidental findings that may indicate other kind of pathologies, for example a still unknown maternal neoplastic disease.29,38 Although use of filters can eliminate these unexpected findings,29 above mentioned issues will make counseling of women even more challenging if all the nuances, limitations and coincidental findings have to be un-derstood in light of making an informed decision, for both the caregiver and the women. Thirdly, removal of the first trimester scan at 11-13 weeks from the prenatal screenings program and only including a dating scan before 10 weeks of gestation, prior to NIPT, would also forego the opportunity of early detection, since the first trimester scan has the potential to be more than just a risk calculation for chromosomal anomalies in the form of the CT (e.g. early detection of anencephaly, holoprosencephaly, hydrops, om-phalocele, gastroschisis, body-stalk anomalies).39 This calls for a serious reconsideration of the role of first trimester ultrasound and the need of a possible early anomaly assess-ment, including the NT as marker of abnormal development.

In the Netherlands, 2-3% of the children are born with a congenital anomaly, and 10% of these children will have a chromosomal abnormality. At this moment NIPT is able to detect trisomies 21, 18 and 13, together around 50-60% of all the numerical chromosomal anomalies. The majority of congenital fetal abnormalities, however, are structural anom-alies40 that can be detected by first and/or second trimester ultrasound and not by NIPT. Beulen et al. showed that NIPT is not a valid alternative to conventional karyotyping or CGH-array for the evaluation of the etiology of structural anomalies, as microscopic aber-ration responsible for malformations cannot yet be picked-up by NIPT.41 Since women in the Netherlands value screening for structural anomalies, we sug-gest all women should be counseled on the possibility of early diagnosis of structural anomalies by a scan around 12-13 weeks. NT-measurement should be part of this scan, in view of its value as marker of structural (especially congenital heart defects) and genetic disorders (especially Noonan syndrome), requiring referral to a Fetal Medicine Center and counseling on a genetic test with a broader coverage than NIPT.39,42 This is shown in Chapter 5 and 6. This possibility should be offered to all women, irrespective of their choice for NIPT.

Our research group has conducted a study on the 12-13 week anomaly scan with the pri-mary aim to study which percentage of congenital anomalies are detected by ultrasound in the first trimester of pregnancy. Preliminary yet unpublished data indicate that a first trimester anomaly scan, performed by trained sonographers at the time of the CT, can identify about 45-50% of the anomalies in an unselected population and 100% of the trisomies. This is in line with previously published data.39 Especially severe anomalies in which parents should decide on the termination of pregnancy in more than 50% of the cases, are detected. Furthermore, there is increasing evidence that at the first trimester scan individual risks can be predicted for a number of conditions such as pre-eclampsia, IUGR, preterm labor and gestational diabetes.43-45 Our research group is performing a study where we evaluate the ability to predict preeclampsia (PE) and growth restriction not associated with PE (GR) by using a risk assessment model based on existing algo-


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rithms. The aim is to see whether early risk prediction for pre-eclampsia is feasible and valid, and if it is indeed, an implementation study could follow to see whether this could work as part of the national screening program in the Netherlands. The results of the ASPRE Study, an international multicenter study on the yield of first trimester screen-ing for PE and consequent use of low-dose aspirin in the high-risk group, will shortly be known.46 It may well be that the first trimester assessment will lose its character of primarily screening for chromosomal anomalies and become, as already suggested in the literature for some time, the first and most important moment of a general risk as-sessment (care for low risk women by the midwife, or high risk women under care of the gynecologist) in pregnancy.45,47 Based on the recent discussion on the general risk assessment during pregnancy in our Dutch system, characterized by midwives and gynecologists sharing the care of pregnant women, this new role of the first trimester assessment may revolutionize the model of care.

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36. Korenromp M. J., Page-Christiaens G. C., Van den Bout J., Mulder E. J., Hunfeld J. A., Potters C. M., Erwich J. J., Van Binsbergen C. J., Brons J. T., Beekhuis J. R., Omtzigt A. W., Visser G. H. A prospective study on paren-tal coping 4 months after termination of pregnancy for fetal anomalies. Prenat Diagn 2007 Aug; 27(8): 709-716.

37. Beulen L., Grutters J. P., Faas B. H., Feenstra I., Van Vugt J. M., Bekker M. N. The conse-quences of implementing non-invasive pre-natal testing in Dutch national health care:

a cost-effectiveness analysis. Eur J Obstet Gynecol Reprod Biol 2014 Nov; 182: 53-61.

38. Bianchi D. W., Chudova D., Sehnert A. J., Bhatt S., Murray K., Prosen T. L., Garber J. E., Wilkins-Haug L., Vora N. L., Warsof S., Goldberg J., Ziainia T., Halks-Miller M. Non-invasive Prenatal Testing and Incidental Detection of Occult Maternal Malignancies. JAMA 2015 Jul 14; 314(2): 162-169.

39. Syngelaki A., Chelemen T., Dagklis T., Allan L., Nicolaides K. H. Challenges in the diag-nosis of fetal non-chromosomal abnormali-ties at 11-13 weeks. Prenat Diagn 2011 Jan; 31(1): 90-102.

40. EUROCAT - European Surveillance of Con-genital Anomalies.

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43. Syngelaki A., Pastides A., Kotecha R., Wright A., Akolekar R., Nicolaides K. H. First-Trimes-ter Screening for Gestational Diabetes Mel-litus Based on Maternal Characteristics and History. Fetal Diagn Ther 2015; 38(1): 14-21.

44. O'Gorman N., Wright D., Syngelaki A., Akolekar R., Wright A., Poon L. C., Nicolaides K. H. Competing risks model in screening for preeclampsia by maternal factors and biomarkers at 11-13 weeks gestation. Am J Obstet Gynecol 2016 Jan; 214(1): 103.e1-103.e12.

45. Nicolaides K. H. A model for a new pyramid of prenatal care based on the 11 to 13 weeks' assessment. Prenat Diagn 2011 Jan; 31(1): 3-6.

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Summary - English

Chapter 1 provides a general introduction and describes the aims and outlines of this the-sis. The uptake of the combined test in the Netherlands is low.

In Chapter 2 we evaluate which of the following factors affect the uptake of the combined test in the Netherlands: women’s socio-demographic background, attitude towards Down syndrome, attitude towards termination of pregnancy, counseling process, reim-bursement policy, and knowledge on the aim of the CT. We found that the main reason for the low uptake is the relatively positive attitude towards Down syndrome and a negative attitude towards TOP. Moreover, the perception of maternal age as strong predictor of Down syndrome risk and the inequality of access to care, due to the financial threshold for younger women, are likely to affect participa-tion in screening also.

In Chapter 3 we examine the intra-operator and inter-operator differences of the manual and semi-automated nuchal translucency measurements and we evaluate if these differ-ences alter women’s risk status. We concluded that well-trained operators do not seem to benefit from the use of the semi-automated measurement methods. Furthermore, changes in risk status occurred between the manual and inner-middle method resulting in different clinical policies in up to 1 out of 20 cases.

In Chapter 4 we estimate the total and procedure-related fetal loss rate of CVS and AC in the Dutch setting, the background and procedure-related fetal loss rates in different subgroups, and we identify maternal-, operator-, and procedure-related risk factors that modify the overall estimated risks of fetal loss. Maternal, operator and procedure related factors influence iatrogenic fetal losses. Besides these factors, the procedure related loss rate of a TV CVS 1:74 and of a TA CVS is 1:97. Looking at the instruments used during the transcervical procedure the risk was 1:476 when using the forceps, and 1:32 when using the canulla. The procedure related loss rate after AC was 1:208. Pregnancy losses after invasive procedures performed transabdominally (CVS and AC) or transcervically by for-ceps, are lower than thought and reported in the past when performed by experienced operators.

In Chapter 5 we discuss that, over the years, it has become clear that increased nuchal translucency is a marker for chromosomal abnormalities, and it is also associated with a wide spectrum of structural anomalies, genetic syndromes, a higher risk of miscar-riage, and intrauterine fetal death. These risks are all proportionally related to the degree of nuchal translucency enlargement. After the initial assessment of increased nuchal translucency, parents should be counselled by the fetal medicine specialist about the pos-


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sible outcomes and the value of additional karyotyping and array comparative genomic hybridisation. A detailed late first trimester and subsequent 20-week scan should aim at identifying structural anomalies, with special focus on the fetal heart and subtle dysmor-phic features. In the absence of structural anomalies or markers, the chance of a favor-able outcome is high.

In Chapter 6 we define sonographic criteria that may improve the prenatal diagnosis of Noonan syndrome by targeted DNA testing. We searched our Fetal Medicine Unit re-cords for all cases with a final diagnosis of Noonan syndrome and a literature review was undertaken to identify the sonographic features of Noonan syndrome fetuses. Informa-tion was pooled to define the most common features. In our database, we identified three cases of Noonan syndrome. The diagnosis was suspected prenatally in two of them. Thirty-nine cases were identified in the literature. In the presented cases we show that suspicion of Noonan syndrome should arise when, after an increased nuchal translucency, ultrasound investigation in the second trimester shows a persistant nuchal fold (NF) or cystic hygroma in combination with at least one of the following features: hydrops fetalis, pleural effusion, cardiac anomalies, polyhydram-nios or specific facial abnormalities.We concluded that although prenatal ultrasound findings in Noonan syndrome can be subtle and aspecific, when specific characteristics are present additional targeted DNA analysis is indicated.

In Chapter 7 we investigate whether the PNT/NBL-ratio, MNM angle, PL, PL-distance and PFSR could be measured in the first trimester of pregnancy. We evaluated the inter-oper-ator agreement and developed normal ranges. Furthermore, we explored these markers in a number of abnormal fetuses. We concluded that feasibility was the highest for the NBL (74.3 - 79.7%) and the MNM angle (75.7 - 79.05%), correlation was good for the NBL, the PNT and the MNM angle (ICC .706 - .835) and the mean difference between operators was the lowest for the PNT and PFSR (0.03 - 0.08). Measurements in abnormal fetuses showed that the majority of trisomy 21 fetuses had either an absent NB or a shorter NBL. The PNT and PNT/NBL ratio were above the 97.5th centile in one third of the cases. Fetuses with facial clefts or mi-crognathia showed on average a large MNM angle (MoM .96 - 5.15). However, as other strong first trimester ultrasound and serum markers for aneuploidies are available, it is unlikely that above mentioned parameters will improve existing first trimester screen-ings’ algorithms. Facial markers may especially have a role as adjunct in the early diagno-sis of structural anomalies and genetic syndromes other than trisomies.

In Chapter 8 we evaluated whether the 3D ultrasound technique is superior to the 2D technique in imaging and measurement of the NBL, PNT, PNT/NBL ratio, PFSR, MNM angle and FP line in screening for Down syndrome. With the exception of measurement of larger MNM angles with the 2D technique, no significant differences were found in the rest of the facial parameters between the 2D and 3D technique. Thus the NBL, PNT, FP line, PNT/NBL ratio and PFSR can be confidently used by the 2D technique as Down syn-drome screening markers, provided the markers are measured in a midsagittal image, acquired according to strict criteria.


138

Samenvatting - Nederlands

Hoofdstuk 1 geeft een algemene inleiding en beschrijft de doelstellingen en hoofdlijnen van dit proefschrift.

De deelname aan de combinatietest in Nederland is laag. In Hoofdstuk 2 evalueren we welke van de volgende factoren de deelname beïnvloeden: socio-demografische ach-tergrond, houding ten opzichte van het syndroom van Down, houding ten aanzien van zwangerschapafbreking, counseling, kosten, en kennis van het doel van de CT. Wij vonden dat de belangrijkste reden voor de lage opname de relatief positieve hou-ding ten opzichte van het syndroom van Down en een negatieve houding ten opzichte van zwangerschapsafbreking is. Bovendien is de perceptie van vrouwen van hun leeftijd als sterkste voorspeller van het Downsyndroom risico en de ongelijke toegang tot de combinatie test vanwege de financiële drempel voor jongere vrouwen, ook van invloed op de deelname aan de combinatietest.

In Hoofdstuk 3 beoordelen we de intra- en inter-operator verschillen van de handma-tige meting en semi-automatische meting van de nekplooimeting, en evalueren we of eventuele aanwezige verschillen veranderingen geven in het combinatietest risico. We concludeerden dat goed opgeleide operators niet lijken te profiteren van het gebruik van de semi-automatische meetmethoden. Bovendien zijn er verschillen tussen de meetme-thoden aanwezig welke resulteren in een ander klinische beleid in maximaal 1 op de 20 gevallen.

In Hoofdstuk 4 beoordelen we het totale miskraam percentage en het miskraam per-centage dat toegeschreven kan worden aan de vlokkentest danwel vruchtwaterpunctie in een Nederlands cohort, bekijken we het totale miskraam percentage en het achter-grondrisico in verschillende subgroepen, en identificeren we maternale, operator en procedure gerelateerde risico factoren welke van invloed zijn op het miskraampercen-tage. We concluderen dat het procedure gerelateerde miskraamrisico 1:97 is voor een transabdominale vlokkentest, 1:74 voor de transcervicale vlokkentest (wanneer gekeken wordt naar de gebruikte instrumenten is dat 1:467 wanneer een forceps en 1:32 wanneer een canule wordt gebruikt) en 1:208 voor de vruchtwaterpunctie. Het miskraam risico na transabdominale danwel trancervicale (forceps) procedures is lager, dan eerder gedacht en gerapporteerd in de literatuur, wanneer de procedure wordt uitgevoerd door iemand met ervaring.

In Hoofdstuk 5 bespreken we dat in de loop der jaren duidelijk is geworden dat een ver-dikte nekplooi niet alleen een marker is voor chromosomale afwijkingen, maar ook is geassocieerd met een breed spectrum van structurele afwijkingen, genetische syndro-men, een verhoogd risico op een miskraam en intrauterine foetale dood. Deze risico’s neme toe naarmate de nekplooidikte toeneemt. Wanneer een verdikte nekplooi wordt geconstateerd, moeten ouders worden gecounseld over de mogelijke uitkomsten en de waarde van karyotypering en array-comparatieve genomische hybridisatie. Een gede-tailleerde beoordeling van de anatomie in het eerste trimester en de daaropvolgende 20 weken echo moeten gericht zijn op het identificeren van structurele afwijkingen, met


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speciale aandacht voor het foetale hart en subtiele dysmorfe kenmerken. Wanneer er geen structurele afwijkingen of markers worden gevonden is de kans op een gunstige uitkomst hoog.

In Hoofdstuk 6 definiëren we echografische criteria die de prenatale diagnose van het syndroom van Noonan door gericht DNA-onderzoek kunnen verbeteren. We zochten in onze database naar alle gevallen met een definitieve diagnose van Noonan syndroom en tevens verrichtten we een literatuurstudie om prenatale echografische kenmerken van het syndroom van Noonan te identificeren. In onze database hebben we drie gevallen van Noonan syndroom geïdentificeerd. De diagnose werd overwogen in twee prenatale casussen. Negenendertig casussen werden in de literatuur gevonden. We vonden dat een vermoeden op het Noonan syndroom zou moeten ontstaan nadat na een verdikte nekplooi, bij echo onderzoek in het tweede trimester sprake is van een verdikte nuchal vouw of cystic hygroma in combinatie met ten minste een van de volgende afwijkingen: hydrops foetalis , pleurale effusie, cardiale anomalieën, polyhydramnios of specifieke gelaatskenmerken. Wij concludeerden dat, hoewel prenatale echografische kenmerken in Noonan syndroom subtiel en aspecifiek kunnen zijn, wanneer deze kenmerken aanwezig zijn, gerichte DNA-analyse is aangewe-zen.

In Hoofdstuk 7 evalueren we of de PNT/NBL-ratio, MNM angle, PL, PL-distance en PFSR gemeten kunnen worden in het eerste trimester van de zwangerschap. Daarbij is geke-ken naar de inter-operator agreement en zijn er normaalwaarden ontwikkeld. Tevens zijn deze markers in een aantal casussen, waarbij sprake was van een structurele danwel chromosomale afwijking, bekeken. We concludeerden dat de feasibilty het hoogst was voor de NBL (74,3 - 79,7%) en de MNM angle (75,7 - 79,05%), de correlatie goed was voor de NBL, de PNT en MNM angle (ICC 0,706 - 0,835) en het gemiddelde verschil tussen de operators het laagst was voor de PNT en PFSR (0,03 - 0,08). De meerderheid van trisomie 21 foetussen hadden of een afwe-zige NB of een kortere NBL. De PNT en PNT/NBL ratio waren boven de 97,5e percentiel in eenderde van de gevallen. Foetussen met een schisis of micrognathie bleken gemid-deld een vergrootte MNM angle te hebben (MoM 0,96 - 5,15). Aangezien de bestaande eerste trimester echo en serum markers al een hoge detectie van aneuploidieën oplevert tegen een laag percentage fout positieven, is het onwaar-schijnlijk dat bovengenoemde parameters de bestaande algoritmen voor eerste trimester screening zullen verbeteren. Echter markers van het aangezicht kunnen een rol spelen bij de vroege detectie van structurele afwijkingen en genetische syndromen.

In Hoofdstuk 8 evalueren wij of de 3D echo-techniek superieur is aan de 2D techniek in het in beeld brengen en nauwkeurig meten van de NBL, PNT, PNT/NBL ratio, PFSR, MNM-angle en FP line in screening naar het Down syndroom. Met uitzondering van de MNM angle, die met de 2D techniek groter wordt gemeten, zijn er geen significante ver-schillen gevonden tussen de twee technieken in het meten van de faciale parameters. De NBL, PNT, FP line, PNT/NBL ratio en PFSR kunnen dus nauwkeurig met de 2D techniek worden gemeten in de screening naar het Down syndrome, mits zij worden gemeten in het midsagittale vlak.


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Appendix

Abstracts

List of Publications

Reasearch Institute SHARE

Curriculum Vitæ

Dankwoord


AppxAppendix

Abstracts


Reasearch Institute SHARE

Curriculum Vitæ

Dankwoord


142

Abstracts

OP 18.03 - First trimester screening in the Netherlands: why is the uptake so low?

M. Bakker, E. Pajkrt, R. J. S. Snijders, K. Bouman, C. M. Bilardo Short Oral Presentation at the 21th World Congress on Ultrasound in Obstetrics and Gynecology, September 18-22, 2011, Los Angeles, USA.

Objective: The combined test (CT) for Down syndrome screening was implemented in the Neth-erlands in 2007. After introduction of the CT the uptake of screening was much lower than uptakes reported in the UK and Denmark. Purpose of this study was to identify determinants which may explain this relatively low uptake.

Methods: 1140 women were invited to fill out a questionnaire at 20 weeks of gestation. Recruit-ment took place at 12 ultrasound clinics in the Northeast (NE) and Northwest (NW) of the Netherlands. The questionnaire was derived from a questionnaire developed by Seror et al in France which addressed women’s decisions on first trimester screening and invasive testing for Down syndrome.

Results: 837 (73%) women returned the questionnaire; 816 of these were filled out complete and used for analysis. The uptake of the CT in the NE of the Netherlands was signifi-cantly lower (N=77; 17%, 12% <36 years and 46% >36 years) than in the NW of the Neth-erlands (N=194; 52%, 49% <36 years and 75% >36 years). The majority of participants (95%) appreciated being informed about the CT. Of these women 66% did not opt for the CT; however 25% would opt for the test if the aim was to detect major congenital malformations. This would result in a participation rate of 50% instead of 33%.

Conclusion: The uptake of the CT in the Netherlands is low compared to other European countries. One of the reasons is that the CT is offered exclusively as Down syndrome screening and little or no information is given on the fact that the scan may reveal major congen-ital malformations. This study shows that counseling should include this information and needs improvement.


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143

Inter-operator reliability of manual and semi-automated measurement (SONO-NT) and Manual and semi-automated measurement of the nuchal translucency – are there any clinical significant differences?

Oral Presentation at the 11th World Congress in Fetal Medicine, June 24-28, 2012, Kos, Greece.

Objective: Are the differences between the manual and semi-automated NT measurement clinically relevant?

Patients and methods: Cross-sectional study on singleton pregnancies between 11+0 - 13+6 weeks of gestation. Two FMF-accredited operators obtained manual and semi-automated NT measurements of 99 NT-images. The maximal acceptable difference in NT measurements within and between operators was 0.15 mm. Intra and inter-operator differences were analyzed by the paired Student’s t-test and homogeneity of variances by the Levene’s test. Intra and inter-operator agreement were quantified with Bland and Altman’s limits of agreement and changes in women’s risk status were tested with the binomial test.

Results: Intra-operator agreement.

Table — Differences in measurement

¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ OPERATOR 1 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ OPERATOR 2 ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬ ¬Mean ∆ SD R2 Mean ∆ SD R2

Manual .0116* .07824 0.985 .0581* .17618 0.928

Inner-inner .0000* .08452 0.985 .0162* .10371 0.976

Inner-middle -.0162* .11755 0.973 -.0109* .12462 0.966

*T-test: p < .001

Table — Difference in risk calculation (risk <1:200 or ≥1:200)

OPERATOR 1 OPERATOR 2N(%) N(%)

Manual (1)Manual (1) 0 0

Inner-inner (1)Inner-inner (2) 0 0

Inner-middle (1)Inner-middle (2) 2 (2%) 2 (2%)

ManualInner-inner 3 (3%) 4 (4%)

ManualInner-middle 5 (5%)# 3 (3%)

*McNemar p < .05 and #p=0.063


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Conclusion:Intra-operator variability: High R2 for all 3 measurement-methods. Mean ∆ + SD of SONO-NT: = or ↑ than manual method for operator1 and ↓ for operator 2. Difference in Risk Calculation: up to 5% difference in risk calculation. Inter-operator: High R2 for all 3 measurement-methods. Mean ∆ + SD ↓ using SONO-NT. Difference in Risk Calculation: max. 2% difference in risk calculation. Manual measurement according to the FMF guidelines is sufficient for reliable NT measurements. Less experienced operators will benefit from the semi-automated SO-NO-NT (mean ∆ and SD ↓). However, experience lies not only in number of cases… also in precision of image acquisition!


Mean ∆ SD R2

Manual (R1)Manual (R2) -.0285* .18678 0.919

Inner-inner (R1)Inner-inner (R2) .0505* .15477 0.949

Inner-middle (R1)Inner-middle (R2) .0756* .16850 0.942

* T-test: p < .001

Table — Difference in risk calculation

N (%)

Manual (R1) - Manual (R2) 2 (2%)

Inner-inner (R1) - Inner-inner (R2) 1 (1%)

Inner-middle (R1) - Inner-middle (R2) 2 (2%)

*McNemar significant


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OP 07.03 - Manual and semi-automated measurement of the nuchal translucency – are there any clinical significant differences?

M. Bakker, P. B. Mulder, E. Birnie, C. M. Bilardo Short Oral Presentation at the 22th World Congress on Ultrasound in Obstetrics and Gynecology, September 9-12, 2012, Copenhagen, Denmark.

Objectives: Are the differences between the manual and semi-automated measurement of the nu-chal translucency (NT) clinically relevant?

Methods: Retrospectively 100 NT images from singleton pregnancies were selected, obtained at 11+0 to 13+6 weeks of gestation. All images had been acquired trans-abdominally using a Voluson E8 equipped with a 4-8 Hz probe (GE Medical Systems). Only images without measurements were used. For each image two trained operators obtained the manual measurements (according to FMF guidelines) and the semi-automated NT measurements (SONONT: inner-inner and inner-middle method). The respective NT measurements and the associated risk on trisomy 21, calculated in Astraia, were trans-formed into a low (<1:200) or high risk (≥1:200) category. A change in risk status was considered a clinically relevant difference and tested with the McNemar’s test.

Results: The misclassification rate of operator 1 was 3.3% (CI [0.007 – 0.092], p=.99) between the manual and inner-inner method; 5.4% (CI [0.018 – 0.122], p=.06) between the manual and inner-middle method; and 4.3% (CI [0.012 – 0.108), p=.13) between the inner-inner and inner-middle method. For operator 2, the misclassification rates were 4.3% (CI [0.012 – 0.108], p=.63), 3.3% (CI [0.007 – 0.092], p=.25) and 5.5% (CI [0.018 – 0.122], p =.06) respectively. Between the manual measurements of the two operators, two cases were discordant (2.2%, CI [0.003 – 0.076], p=.500).

Conclusions: There are no significant differences in classification between the manual measure-ment and SONO-NT measurements. In our opinion manual measurement according to the FMF guidelines is sufficient for a valid risk calculation for Down syndrome.


Mean ∆ SD R2

Manual (R1)Manual (R2) -.0285* .18678 0.919

Inner-inner (R1)Inner-inner (R2) .0505* .15477 0.949

Inner-middle (R1)Inner-middle (R2) .0756* .16850 0.942

* T-test: p < .001

Table — Difference in risk calculation

N (%)

Manual (R1) - Manual (R2) 2 (2%)

Inner-inner (R1) - Inner-inner (R2) 1 (1%)

Inner-middle (R1) - Inner-middle (R2) 2 (2%)

*McNemar significant


146

P 06.07 - Inter-operator reliability of manual and semi-automated measurement (SONO-NT)

M. Bakker, P. B. Mulder, E. Birnie, C. M. Bilardo Poster at the 22th World Congress on Ultrasound in Obstetrics and Gynecology, September 9-12, 2012, Copenhagen, Denmark.

Objectives: To compare the inter-operator reliability of: manual and semi-automated nuchal trans-lucency (NT) measurements.

Methods: Retrospectively 100 NT images of singleton pregnancies were selected, obtained at 11+0 to 13+6 weeks of gestation. All had been acquired trans-abdominally using a Voluson E8 equipped with a 4-8 Hz probe (GE Medical Systems). Only images with-out measurements were used. For each image, two operators obtained the manual measurements (according to FMF guidelines) and semi-automated NT measurements (SONO-NT: inner-inner and inner-middle method). Inter-measurement reliability within operators for the inner-inner and inner-middle measurement was compared to the operators’ manual measurement. Inter-operator reliability of the manual, inner-inner and inner-middle measurements was assessed by comparing the measurement of operator 1 to the same measurement of operator 2. The maximal clinically acceptable difference was considered to be 0.1 mm (using t-tests and R2).

Results: Compared to the operators’ manual measurement, the R of operator 1 was 0.975 for inner-inner and 0.972 for inner-middle measurements; and 0.951 and 0.955 respec-tively for operator 2. The inter-operator reliability coefficient R was 0.918 for manual, 0.941 for inner-inner and 0.933 for inner-middle measurements. The mean difference between the operators’ manual measurements was -0.02 mm (CI [-0.061 – 0.017]), 0.06 mm between inner-inner (CI [0.027 – 0.099]) and 0.09 mm between inner-middle measurements (CI [0.048 – 0.126]). The manual and inner-inner mean difference did not deviate significantly when the clinically accepted difference of 0.1 mm was taken into account. The inner-middle mean differences did however.

Conclusions: The inter-observer reliability for both the SONO-NT measurements and manual mea-surements is high. Mean difference between operators is lowest for the manual mea-surements. Manual measurement according to FMF guidelines is sufficient for reliable NT measurements.


Appendix › List of Publications

147


First Author

- Targeted ultrasound examination and DNA testing for Noonan syndrome, in fetuses with increased nuchal translucency and normal karyotype. Bakker M., Pajkrt E., Mathijssen I. B., Bilardo C. M. Prenat Diagn 2011 Sep; 31(9): 833-40.

- Low uptake of the combined test in the Netherlands – which factors contribute? Bakker M., Birnie E., Pajkrt E., Bilardo C. M., Snijders R. J. Prenat Diagn 2012 Dec; 32(13): 1305-12.

- Intra-operator and inter-operator reliability of manual and semiautomated measure-ment of fetal nuchal translucency: a cross sectional study. Bakker M., Mulder P., Birnie E., Bilardo C. M., Prenat Diagn 2013 Dec; 33(13): 1264-71.

- Increased nuchal translucency with normal karyotype and anomaly scan: what next? Bakker M., Pajkrt E., Bilardo C. M. Best Pract Res Clin Obstet Gynaecol. 2013 Dec 3; pii: S1521-6934(13)00157-0.

- Total pregnancy loss after chorionic villus sampling and amniocentesis in the Neth-erlands: a cohort study. UOG 2016 Jun - Ac-cepted.

- Prenasal thickness, prefrontal space ratio and other facial profile markers in first tri-mester fetuses with aneuploidies, cleft pal-ate and micrognathia. Submitted.

Co-author

- Is 3D technique superior to 2D in Down syndrome screening? A review of six second and third trimester fetal profile markers. Vos F. I., Bakker M., De Jong-Pleij E. A. P., Ribbert L. S. M., Tromp E., Bilardo C. M. Pre-nat Diagn. 2015 Mar; 35(3): 207-13.

- Nasal bone length, prenasal thickness, prenasal thickness-to-nasal bone length ratio and prefontrol space ratio in second and third trimester fetuses with Down syn-drome. Vos F. I., De Jong-Pleij E. A. P., Bakker M., Kagan O. K., Ribbert L. S. M., Tromp E., Bilardo C. M. Fetal Diagnosis and Therapy, 2015 Jan 30. [Epub ahead of print]

- Trends in serial measurements of five ul-trasound markers measured in second and third trimester Downsyndrome fetuses. Vos F. I., De Jong-Pleij E. A. P., Bakker M., Tromp E., Bilardo C. M. Fetal Diagnosis and Therapy, 2015; 38(1): 48-54.

- Fetal facial profile markers of Down syn-drome in the second and third trimester of pregnancy. Vos F. I., De Jong-Pleij E. A. P., Bakker M., Tromp E., Kagan O. K., Bilardo C. M. Ultrasound Obstet Gynecol. 2015 Aug; 46(2): 18-73.

- Fetal profile markers in second and third trimester fetuses with trisomy 18. Vos F. I., De Jong-Pleij E. A. P., Bakker M., Tromp E., Manten G. T., Bilardo C. M. Ultrasound Ob-stet Gynecol. 2015 Jul; 46(1): 66-72.

- Premaxillary protrusion assessment by the maxilla-nasion-mandible angle in fetuses with facial clefts. De Jong-Pleij E. A. P., Pis-torius L. R., Ribbert L. S., Breugem C. C., Bakker M., Tromp E., Bilardo C. M. Prenat Diagn. 2013 Apr; 33(4): 354-9.

Oral and Poster Presentations

- OP 18.03 - First trimester screening in the Netherlands: why is the uptake so low? Bak-ker M., Pajkrt E., Snijders R. J. S., Bouman K., Bilardo C. M. Short Oral Presentation at the 21th World Congress on Ultrasound in Ob-stetrics and Gynecology, 18-22 September 2011, Los Angeles, USA.


148

- Inter-operator reliability of manual and semi-automated measurement (SONO-NT) and Manual and semi-automated measure-ment of the nuchal translucency – are there any clinical significant differences? Oral Presentation at the 11th World Congress in Fetal Medicine, 24-28 June 2012, Kos, Greece.

- OP 07.03 - Manual and semi-automated measurement of the nuchal translucency – are there any clinical significant differ-ences? Bakker M., Mulder P. B., Birnie E., Bilardo C. M. Short Oral Presentation at the 22th World Congress on Ultrasound in Obstetrics and Gynecology, 9-12 September 2012, Copenhagen, Denmark.

- P 06.07 - Inter-operator reliability of manual and semi-automated measurement (SONO-NT). Bakker M., Mulder P. B., Birnie E., Bilardo C. M. Poster at the 22th World Congress on Ultrasound in Obstetrics and Gynecology, 9-12 September 2012, Copenha-gen, Denmark.

- OP 28.11 - Premaxillary protrusion in fetuses with facial clefts. De Jong-Pleij E., Ribbert L. S., Pistorius L. R., Bakker M., Breugem C.,

Tromp E., Bilardo C. M. Oral Presentation at the 22th World Congress on Ultrasound in Obstetrics and Gynecology, 9-12 September 2012, Copenhagen, Denmark.

- OP 18.06 - First things first: preconditions to reliably estimate the risk of fetal trisomy. Snijders R., Bakker M., Pajkrt E., Muller-Kobolt A., Sturk G., Bilardo C. Oral Presenta-tion at the 21th World Congress on Ultra-sound in Obstetrics and Gynecology, 18-22 September 2011, Los Angeles, USA.

- OP 18.07 - Pre- and postnatal diagnosis of fetal trisomy in the north-east of the Neth-erlands. Bouman K., Snijders R., De Walle H., Bakker M., Bilardo C. Oral Presentation at the 21th World Congress on Ultrasound in Obstetrics and Gynecology, 18-22 Sep-tember 2011, Los Angeles, USA.

- OP 12.10 - Diagnosing fetal long QT syn-drome (LQTS) using tissue Doppler imag-ing (TDI), preliminary report. Clur S. B., Bakker M., Ottenkamp J., Bilardo C., Kuipers I., De Bruin-Bon R. Oral Presentation at the 20th World Congress on Ultrasound in Obstetrics and Gynecology, 10-14 October 2010, Prague, Czech Republic.


Appendix › Research Institute SHARE

149

This thesis is published within the Research Institute SHARE (Science in Healthy Ageing and healthcaRE) of the University Medical Center Groningen / University of Groningen.Further information regarding the institute and its research can be obtained from our website: http://www.share.umcg.nl/.

More recent theses can be found in the list below ((co-)supervisors are between brackets).

2016

> Bonvanie I. J. - Functional somatic symptoms in adolescence and young adults; personal vulnerabilities and external stressors

(prof. J. G. M. Rosmalen, prof. A. J. Oldehin-kel, dr. K. A. M. Janssens)

> De Greeff J. W. - Physically active academic lessons: effects on physical fitness and executive functions in primary school children

(prof. C. Visscher, prof. R. L. Bosker, dr. E. Hartman, dr. S. Doolaard)

> Van Dijk L. - The reality of practice; an action systems approach to serious gaming

(prof. C. K. van der Sluis, dr. R. M. Bongers)

> Smit R. - Health economics of tick-borne diseases

(prof. M. J. Postma, prof. K. Poelstra)

> Norder-Kuper L. - Common mental disorders; prediction of sickness absence durations and recurrences

(prof. U. Bültmann, prof. J. J. L. van der Klink, dr. C. A. M. Roelen)

> Kamstra J. I. - Trismus seconday to head and neck cancer; risk factors and exercise therapy

(prof. P. U. Dijkstra, prof. J. L. N. Rooden-burg, dr. H. Reintsema)

> Bruins J. - Metabolic risk in people with psy-chotic disorders; no mental health without physical health

(prof. G. H. M. Pijnenborg, prof. E. R. van den Heuvel, dr. F. Jorg, dr. R. Bruggeman)

> Holtman G. A. - Diagnostic strategies in chil-dren with chronic gastrointestinal symptoms in primary care

(prof. M. Y. Berger, dr. Y. Lisman-van Leeu-wen, dr. P. F. van Theenen)

> Lopez Angarita A. - Self-compassion; a closer look at its assessment, correlates and role in psychological wellbeing

(prof. R. Sanderman, dr. M. J. Schroevers)

> Zandstra A. R. E. - Psychosocial adversity and adolescents’ mental health problems; moderat-ing influences of basal cortisol, resting heart rate and Dopamine Receptor D4

(prof. J. Ormel, dr. C. A. Hartman)


150

> Armbrust W. - The impact of juvenile idio-pathic arthritis; moving beyond the joint

(prof. P. J. J. Sauer, prof. J. H. B. Geertzen, prof. N. M. Wulffraat)

> Roy A. - The development of depression in chil-dren and adolescents with ADHD

(prof. A. J. Oldehinkel, dr. C. A. Hartman)

> Holubcikova J. - Eating habits, body image and health and behavioural problems of ado-lescents; the role of school and family context

(prof. S. A. Reijneveld, dr. J. P. van Dijk, dr. A. Madarasova-Geckova, dr. P. Kolarcik)

> Nguyen T. P. L. - Health economics of screening for hypertension in Vietnam

(prof. M. J. Postma, dr. C. C. M. Schuilinga-Veninga, dr. T. B. Y. Nguyen, dr. E. P. Wright)

> Mihajlovic J. - Health economics of targeted cancer therapies; a comparative analysis for Serbia and the Netherlands

(prof. M. J. Postma, dr. P. Pechlivanoglou)

> Darvishian M. - Real-world influenza vaccine effectiveness; new designs and methods to ad-just for confounding and bias

(prof. E. Hak, prof. E. R. van den Heuvel)

> Berm E. J. J. - Optimizing treatment with psychotropic agents through precision drug therapy; it is not about the mean

(prof. B. Wilffert, prof. E. Hak, dr. J. G. Maring)

For more theses from 2016 and earlier please visit our website.


Appendix › Curriculum Vitæ

151

Curriculum Vitæ

Merel Bakker werd op 10 augustus 1981 geboren te Purmerend waar zij in 1999 haar diploma behaalde aan het atheneum “het Da Vinci College”. In datzelfde jaar begon zij aan de studie Medische Biologie nadat zij was uitgeloot voor de studie Geneeskunde. In 2000, nadat zij haar propedeuse had behaald, werd zij alsnog ingeloot voor de studie Geneeskunde aan de Universiteit van Amsterdam. Tijdens deze periode heeft zij onder andere onderzoek gedaan naar Lepra, in Makassar, te Indonesië. Na het afronden van de studie Geneeskunde is zij als AGNIO in het Kennemer Gast-huis in Haarlem gaan werken. Aansluitend is zij begonnen als arts prenatale diagnostiek in het AMC waar de basis voor haar proefschrift is gelegd. Dit traject heeft zij voortgezet in het UMCG toen zij meeging met prof. dr. Bilardo naar Groningen. Hier werkte zij als arts prenatale diagnostiek en in deeltijd aan haar promotie. Tevens gaf zij trainingen en onderwijs in het verrichten van echoscopisch onderzoek in het eerste en tweede trimes-ter van de zwangerschap en heeft zij gedurende 3 maanden gewerkt aan de Fetal Medici-ne Unit van de Stellenbosch Universiteit in Kaapstad, Zuid-Afrika. In 2014 is zij met veel plezier gestart met de opleiding tot gynaecoloog in het Deventer Ziekenhuis en heden werkzaam in het UMCG te Groningen.


152

Dankwoord

Katia, cara Katia, wat is het een turbulente maar zeer waardevolle rit geweest! Door alles wat we samen in de afgelopen jaren hebben meegemaakt ben je veel meer voor mij dan ‘alleen’ mijn promotor. Bedankt voor je blinde vertrouwen in mij, voor de vrije hand die ik heb gekregen, voor je stimulatie om alles eruit te halen wat er in zit, om naar congressen en symposia te gaan en steeds nieuwe dingen aan te blijven pakken. Ik ben door dit alles ver gekomen. Ik bewonder je onuitputtelijke passie voor het vak, je kennis, je gedrevenheid, je eerlijkheid, je non verbale communicatie en je warme persoonlijkheid. Zowel tij-dens het onderzoek als op de werkvloer was je altijd bereikbaar, zelfs wanneer je op vakantie was! We blijven in de toekomst samenwerken, questo è certo.

Erwin, de (statistische) rots in de branding, dankjewel voor alles. Je wist mij altijd te stimuleren en het beste in mij naar boven te brengen. Hoewel ik de Kappa nooit meer zal durven gebruiken. Ik zal onze vrijdagmiddagbespreking, met chocolade, erg gaan missen. Ik hoop dan ook dat we in de toekomst blijven samenwerken.

Beste Eva, bedankt voor je nuchtere en heldere blik op zaken. De werkplek die je creëerde in het AMC zorgde ervoor dat ik ook daar fijn kon werken. Ik bewonder je gedrevenheid in het vak. We zullen elkaar in de toekomst ongetwijfeld vaak blijven tegenkomen!

Dr. Lips, beste Jos, ik moest in het begin wel even aan je wennen op de werkvloer, maar zonder jou zou ik hier nu niet hebben gestaan. Het juiste zetje in de rug heb jij gegeven, bedankt!

De promotiecommissie, Prof. dr. S. A. Scherjon, Prof. dr. A. Ranchor, Prof. dr. I. M. van Langen en Prof. dr. O. B. Petersen wil ik graag bedanken voor hun aandacht aan het manuscript.

Mijn paranimfen, Martine en Eline, vanzelfsprekend. Lieve Martine, wat hebben wij een hoop meegemaakt in 15 jaar tijd! Leuke en min-der leuke dingen, maar ik had het voor geen goud willen missen. Waar zou ik soms zijn zonder jou?! Dank je voor luisterend oor, je gevatheid, je humor en warme per-soonlijkheid. We moeten dat boek maar eens gaan schrijven.

Lieve Eline, lief Lientje, promoveren gaat niet over rozen, daar weten wij alles van. Dank je voor alle steun, gezelligheid, humor en inzichten tijdens deze periode. Dat we daar nog maar vele jaren aan vast mogen plakken! Met wijn!


Appendix › Dankwoord

153

Lieve Els en Fedja, wat was en is het heerlijk samenwerken met jullie! Els ik bewon-der je op vele vlakken en ik deel je passie voor het foetale aangezicht, dat we nog maar vele congressen samen mogen bezoeken. Fedja, ook voor jou heb ik veel be-wondering! Wat heb je dat laatste stuk van je promotie vlot en kundig afgemaakt, tijdens je zwangerschap nog wel, klasse! Ik zie jullie snel weer. Pascale, lieve Pascale, mijn AMC-tijd zou niet hetzelfde zijn geweest zonder jou. Ik waardeer je eerlijkheid, je humor en gastvrijheid (een letterlijke open-deur-policy). Mijn echomaatje waar ik eindeloos tegen aan kon kletsten over 3D en 4D echogra-fie… en dan alsnog de tutorial kon sturen ;).

Lieve Sally, ik heb zo ontzettend veel van je geleerd! Ik zou een abonnement op je hartenspreekuur willen hebben. Elke keer weer deed ik daar nieuwe inspiratie op. Ik vind je een prachtig mens en ik ben blij dat we elkaar niet alleen in Nederland maar ook in Zuid-Afrika hebben leren kennen.

Lieve kamergenootjes, Aniek, Ellen, Ninke, Irene, Violetta, Anne, Jelmer, Marco, Catarina en collega’s, Kim, Elsbeth, Janna, Ineke, Welmoed, Ingrid, wat is het ontzettend gezel-lig geweest! Bedankt voor de brainstorm sessies, de hulp, gedeelde frustraties, het theeleuten, Wordfeud-sessies en de borrels! Zonder jullie had ik mij deze rit niet kunnen voorstellen.

Dear Catarina, thank you for all your wise words, your clear vision and Italian/Portu-gese lunches. Hope we do that coffee soon!

Lieve Petra, bedankt voor je gezelligheid en je tijd die je voor mij vrij wilden maken om op de meest gekke tijdstippen metingen te verrichten (je zal me vast wel eens achter het behang hebben kunnen plakken).

Lieve Laurien, Aukje en Maaike bedankt voor jullie inzet voor alle onderzoeken die we op de afdeling hadden en hebben lopen!

Lieve Anja, Cora, Wilma en Karin ontzettend bedankt voor alles wat jullie hebben geregeld en in goede banen hebben geleid op het secretariaat. Zonder jullie was dat vast anders gelopen.

Dear Lucia and Margherita, thank you for your help and our awesome time together, in and outside of this hospital. You are dear friends and thanks to you the Italian Cuisine is my religion.

Dear Karin, Christine, Elzabe, and Shannon, I had an amazing time in South Africa be-cause of you guys. I came back with loads of new inspiration, knowledge and most important of all, new friends. Suid-Afrika sal altyd in my hart wees.


154

Lieve Niels, het was vanzelfsprekend dat jij je met de vormgeving van dit boekje ging bemoeien! Niemand anders had dat mogen doen. Ik vind het nog steeds bij-zonder dat we al zo ontzettend lang vrienden zijn, daar plakken we nog eens 25 jaar aan vast!

Lieve Eveline, je bent een van mijn liefste vriendinnetjes. Daar verandert geen lands-grens iets aan. Dank je voor alles wat je voor me hebt gedaan en ik kom je snel weer opzoeken in Duitsland.

Lieve familie en vrienden, bedankt dat jullie altijd voor mij klaar staan.

Pap en Mam, dank voor alle kansen die jullie mij hebben gegeven en het eindeloze vertrouwen in wat ik ook maar uitspookte! Jullie hebben mij aangemoedigd om alles eruit te halen wat er in zit, waar dat ook ter wereld was. Bedankt dat jullie er altijd voor mij zijn.


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