Prenatal Screning

Prenatal
Screening
Perspectives
Incorporating Down’s Screening News
Volume 16, 2011
* IPSG 2011, Barcelona

* Free Fetal DNA
* Informed decision making
* Sex Chromosome Trisomies
* Spina Bifida
* Twins
* Serum Tests QC
* Phenotype & Micro RNA
* CRL & Gestational Age
Newsletter of the International Prenatal Screening Group
Leeds Screening Centre, Gemini Park, Sheepscar Way, Leeds LS7 3JB, UK
Tel: +44(0)113 284 9230

Fax: +44(0)113 262 1658
www.ipsgroup.org.uk
Page 1 PSP 2011 VOL 16

PSP 2011 Vol 16 CONTENTS
Page Page
ADVERSE PREGNANCY OUTCOMES OTHER ANEUPLOIDY
Danish thesis defense .................................................................................. 23 Sex chromosome trisomies ............................................................................. 4
Macrosomia in diabetic pregnancies - prediction .......................................... 19 Trisomies 13 and 18 - prevalence ................................................................... 31
Paternal age ................................................................................................... 23 Trisomy 16 - first trimester markers ................................................................ 31
Pyramids inverted or otherwise ...................................................................... 18 OTHER DISORDERS
Screening for ectopic pregnancy – potential markers ........................... 26 Cystic Fibrosis ................................................................................................. 10
Screening in early pregnancy ........................................................................ 32 Fragile X syndrome (FRAX) screening ............................................................ 15
Joubert syndrome - genes found .................................................................... 18
DNA/RNA Mendelian disorders - universal carrier testing ............................................... 4
Free fetal DNA - recent developments ........................................................... 16 NTD prevention - randomised trial ................................................................. 12
Non-invasive prenatal diagnosis - ethics ........................................................ 16 Spina bifida - first trimester markers .............................................................. 18
Spinal Muscular Atrophy (SMA) population screening .................................. 29
DOWN’S SYNDROME
Phenotype and micro RNA ............................................................................. 8 PRENATAL DIAGNOSIS
CGH versus karyotyping .................................................................................. 7
EPIDEMIOLOGY & AETIOLOGY
Trisomy 21 origin – germ cells ........................................................................ 29 PROSPECTIVE SCREENING & NATIONAL ACTIVITY
Self-reporting outcome - influence on detection rate ...................................... 20
IPSG Community Genetics Network ............................................................. 24
9th IPSG Congress. Preliminary program ...................................................... 6 DS screening in Europe .................................................................................. 5
9th IPSG Congress & ISPD Education Day ................................................... 6 DS screening in the US ................................................................................... 5
Anne M Summers (1954-2009) ...................................................................... 10 DS screening in the UK ................................................................................... 5
Barcelona link ..................................................................................... 24 Ethnic differences in DS screening ................................................................. 33
Co-ordination ...................................................................................... 15 Maternal serum screening in the West Bank ................................................. 36
Editorial .............................................................................................. 3 Publishing prospective studies of DS screening ............................................ 27
Membership form ................................................................................ 3 Spinal Muscular Atrophy (SMA) – prospective screening results .................. 4
PhDs from the Netherlands ........................................................................... 32
Position statement ......................................................................................... 26 QUALITY MANAGEMENT
Publishing honour for Aubrey Milunsky ......................................................... 12 Serum tests quality control ................................................................... 30
Robert Cocciolone (1957-2010) ...................................................................... 14
REFINEMENTS & ADJUSTMENTS
INFORMATION Anomalous marker values .............................................................................. 8
Bibliography - 219 references .............................................................. 39 Chorionicity – importance for twin risks .......................................................... 21
Extreme results ............................................................................................... 9
KNOWLEDGE & ATTITUDES Maternal age – affect on screening performance ........................................... 11
Healthy carrier - not strictly true .................................................................... 31 PIGF – Marker for aneuploidy and pre-eclampsia ................................ 25
Informed decision-making - prenatal screening ............................................. 34 Repeat measures ................................................................................ 27
MEETINGS & COURSES SOFTWARE

World Congress on MFM, Rhodes June 2010 .............................................. 28 Chromosome 21 balanced translocation - warning on risks .......................... 15
15th ISPD Conference, Amsterdam July 2010 ............................................. 28 Correlation of NT between twin fetuses ......................................................... 27
METHODOLOGY & POLICY ISSUES ULTRASOUND

First among equals ......................................................................................... 7 Fetal size and dating ....................................................................................... 7
Innovations in DS screening ........................................................................... 33 Fetal Ductus Venosos - marker for cardiac defects ........................................ 25
International Society for Prenatal Diagnosis – new directions ............... 24 Genetic sonogram – how to combine results .................................................. 17
Second trimester combined testing ................................................................ 8 Crown-rump length and gestational age ......................................................... 38
US professional guidelines - screening .......................................................... 9
VARIOUS
NT & NASAL BONE Health Technology Assessment (HTA) - current activity .............................. 13
Semi-automated NT measurement - progress and caution .......................... 17 Re-inverting the wheel: again!.............................................................. 22
Prenatal Screening Perspectives is sponsored by

PSP 2011 VOL 16 PerkinElmer
Page 2
A note from your editor …..
Such of lot of changes in my life and for the Screening Group! First of all you will notice that our web address has changed as have
the contact details. This is because the web site will no longer be hosted on the Leeds University server after July but will now be
under the auspices of the International Prenatal Screening Group. Prenatal Screening Perspectives will be available online and in
due course, I will add back issues of DSNews. This edition is the first to be published entirely online - please let me know what you
think. We are always open to suggestions and any positive (or negative) thoughts will be gratefully received. At the moment,
there are no plans to produce a printed edition as printing and distribution costs are prohibitive. However, we can always revisit
this decision in the future.
When I was going through our membership list it became apparent that much of the contact information, particularly email
addresses were either missing or out of date. I would be grateful if you would forward web details of PSP (www.ipsgroup.org.uk/
psp) to anyone you know who hasn’t received it and also ask them to contact me in order to receive future issues.
Peter Benn and Peter Schielen have always been much appreciated contributors to PSP (and DSNews before it) and they have now
taken over from Howard as co-ordinators of this newsletter (see page 15). We wish them well and look forward to many more
issues and lots more articles. We are also delighted that Mark Evans has agreed to do a regular op-ed piece and the first is in this
issue on page 22.
In June, Barcelona will host the 9th International Congress and we are hoping that this will be as informative and exciting as in the
past. Contact details are on page 6 as is the preliminary programme.
Another useful plus is that we can use colour! Its such a luxury and I have to keep my creative streak in check. You will also notice
that I have extended the bibliography section (page 39) which many members have found so useful in the past. That is one of the
benefits of an online edition where we are not bound by the constraints of economics.
I am especially grateful to all those who have contributed and I hope you will enjoy our new edition.
Phillipa Bloom
Please print and fax/mail if you want to join the group or if your current details are
incorrect or go to www.leeds.ac.uk/idssg/membership.
International Prenatal Screening Group
Title: _________________ Name: ____________________________________________________________
Department:_________________________________________________________________________________
Address:____________________________________________________________________________________
Telephone:_________________________________________ Fax:____________________________________
Email: ________________________________________________
IPSG, Leeds Screening Centre, Gemini Park, Sheepscar Way, Leeds LS7 3JB, UK
Tel: +44(0)113 2849230 Fax: +44(0)113 2621658 Email: info@ipsgroup.org.uk http://www.ipsgroup.org.uk

Mendelian
Sex disorders:
chromosome Universal carrier
trisomies testing
For $349, Counsyl Inc
XXX, XXY and XXY now offers carrier testing for more
are not associated with than 100 single gene disorders screening for over 500
dysmorphic features and fetal loss but are mutations (www.counsyl.com). The panel includes sickle
occasionally prenatally identified following screening. cell disease, beta thalassemia, the nine disorders in the
A modest excess over that expected by chance is ACMG recommended Ashkenazi Jewish panel, spinal
expected because XXX and XXY are more frequent in muscular atrophy, and coverage of 109 mutations in the
older mothers and maternal age is a component of cystic fibrosis gene. The company claims >99.9%
all aneuploidy screening protocols. Based on sensitivity and specificity for the targeted mutations. The
testing is carried out on a saliva sample sent to Counsyl’s
EUROCAT databases, Boyd et al (European J Human
laboratory and needs to be ordered through a clinician.
Genet 2010) concluded that only about 12% of the On-line patient reports contain residual risk of being a
cases expected to be present at birth are actually carrier and the post-test risk for an affected child.
prenatally identified. This is probably an over- For some disorders, the analyses only detects a
estimate because their estimate for the total small proportion of the mutations present in the
expected number of cases in the population was population and some of the disorders are extremely rare
based on rates established in older consecutive so even when a carrier is identified, the risk of an affected
newborn studies at which time maternal ages were child is relatively small. Initial clinical data indicates that
lower than they are today. as many as 35% of patients may be identified as carriers
Among women aged <35 and receiving a for at least one disorder (Srinivasen et al, Reprod Biomed
diagnosis of a sex chromosome trisomy, 41% chose Online, 2010;21(4):537-51). Tandem testing of both
to terminate the pregnancy (50% for XXX, 40% for parents at the same time is, therefore, optimal. Some of
the mutations will be of uncertain clinical significance in
XXY and 32% for XYY). Significantly less (33%)
compound heterozygotes. There will also be some
women aged ≥35, terminated a sex chromosome situations where the results could have potential clinical
trisomy pregnancy (23% for XXX, 41% for XXY, and significance for the individual being tested, for example,
28% for XYY). Gaucher, cystic fibrosis and hemochromatosis gene
mutations. The approach is therefore associated with a
significant amount of genetic counseling and follow-up
clinical referrals.
The National Center for Genomic Research

(NCGR), a nonprofit private research institute, has plans
Spinal Muscular Atrophy (SMA) to offer carrier screening for an even larger number of
screening – Prospective results recessive childhood diseases through next generation
sequencing technology. The project is being developed
in conjunction with the Beyond Batten Disease
A massive prospective evaluation of antenatal screening Foundation. In an initial study, they sequence 437 genes
for SMA has been completed in Taiwan (Su et al, PLoS that represent 448 disorders with an average of 160-fold
One 2011;6(2):e17067). A sequential approach was target coverage (Bell et al, Science Translational
taken using a method that identifies women with one Medicine 12 January 2011 http://stm.sciencemag.org/
copy of the SMN1 gene, then testing their partners, only content/3/65/65/ra4.full). They report an approximately
offering invasive prenatal diagnosis to carrier couples. 95% detection rate for mutations in these genes. Based
Over 100,000 women were screened and 2,262 SMA on 104 unrelated individuals, they detected an average of
carriers were identified, an observed carrier frequency of 2.9 (range 0-7) recessive mutations per individual.
1 in 48. Among the 2,038 partners tested 47 were Although the cost of testing is projected to be low ($400
carriers (1 in 43). Prenatal diagnosis was carried out in for large volume testing) the authors acknowledge that
43 couples, 12 affected fetuses were diagnosed and 11 there are considerable costs associated with
pregnancies were terminated. The false-negative rate is interpretation, reporting, genetic counseling, and data
not known but carriers will be missed because of (1) two management. It will be some considerable time before
copies of the SMN1 gene on one allele and none on the there is sufficient information to interpret the clinical
other or (2) mutations rather than deletion of the SMN1 significance of many of the mutations/polymorphisms that
gene, and a proportion of cases are de novo. will be identified but they still hope to make the testing
Nevertheless, this is an impressive study and it might just clinically available before the end of 2011 (News report.
encourage some other countries to introduce routine Couzin-Frankel; Science 331;130-1).
screening for this terrible disorder.
Peter Benn (benn@nso1.uchc.edu)
PSP 2011 VOL 16 Page 4

USA and UK
A survey of Maternal-Fetal
Medicine physicians in the
USA carried out in 2001 and
again in 2007 demonstrates
A
summary report from the
National Down Syndrome
Cytogenetic Registry (NDSCR)
s ig ni f ic a nt s h ift s i n t he for England and Wales
approaches to prenatal screening documents trends for prenatal
(Fang et al, Am J Obstet Gynecol diagnosis from 1989 to 2008
(Morris and Alberman, BMJ 2009;339)
2009;201:97.e1-5.) The number of both affected and unaffected
births has remained relatively steady throughout the time
2001 (%) 2007 (%) period with a birth prevalence of Down syndrome
approximately 1.08/1000 in 2007/8. However, over the
Perform first trimester 43.1 97.3 nearly two decades, there has been a major change in
screening the number of women delaying or extending their years
of reproduction and in the absence of any prenatal
screening and diagnosis the number of affected births
Use first trimester 27.9 94.9 would have been 48% higher. Among women 37 and
PAPPA and hCG tests older, the proportion of affected pregnancies that are
prenatally diagnosed has remained approximately
Measure NT 48.6 96.6 constant at 70% while for younger women, the rate has
increased from 3% to 43%.
The report does not indicate how many women
Provide second trimester 73.6 10.3 do receive screening and what screening protocols were
Triple test in place. For some regions, it seems that uptake is low.
Provide second trimester 8.5 85.6 For example, Irving et al report a utilization rate of only
35% for Northern England (Irving et al, Europ J Human
Quad test Genet. 2008;16;1336-40). It remains unclear whether
earlier screening and diagnosis is improving acceptance.
Use second trimester 65.9 85.1 National registries such as NDSCR provide invaluable
ultrasound to adjust risk data for assessing the impact of screening, identifying
service gaps, and determining where long-term
healthcare and education resources will be needed.

Another indication of the changes in prenatal
counseling, screening and diagnostic testing in the
US comes from an analysis of trends among DS Detection in Europe
women aged 35 or more and referred to Genzyme
Genetics Inc for counseling (Naketa et al, Prenat
Proportion of Down syndrome cases
Diagn 2010;30:198-206). prenatally identified in 10 European
countries
From 2001 to 2008, there was a decline in AMA
patients receiving counseling, especially in 2008
following the revised ACOG guidelines that Country <35 years ≥ 35 years
removed AMA as a separate group to be offered Switzerland 88 94
invasive testing. Uptake of first trimester France 80 94
screening also showed a dramatic increase from Spain 67 88
Germany 60 83
2007 (51%) to 2008 (85%). Italy 56 85
Austria 54 73
Genzyme began offering the “Integrated” test in Belgium 52 66
2005 at which time 28% of AMA patients were UK 48 70
offered this option. In 2006, sequential screening Denmark 38 87
Poland 4 4
was available and by 2008, 48% were offered this
while Integrated had declined to just 7%.
Source: European Surveillance of Congenital Abnormalities (EUROCAT)
database. Boyd et al, European J Human Genet 2010;1-4 (legend to Fig 3).

INTERNATIONAL PRENATAL SCREENING GROUP
9th International Congress
School of Biology, University of Barcelona, 20th & 21st June 2011
Preliminary program (28 March 2011)

Monday, 20 June, 2011 Session VII. Screening for adverse
Simultaneous carrier screening for more pregnancy outcome
Session I. Aneuploidy screening than 400 disorders
Steven Kingsmore (USA) The ‘Inverted Pyramid’ of prenatal care
Contingency Test completed in the first Kypros Nicolaides (UK)
A universal carrier screen for Mendelian
trimester
disease Preliminary results on routine fetal RHD
Antoni Borrell (Spain)
Balaji Srinivasan (USA) genotyping
Risk calculation in trisomies other than +21, Antoni Borrell (Spain)
TBA
+18 and +13
Yuval Yaron (Israel) First trimester adipocytokines in pre-
Eugene Pergament (USA)
eclampsia
Weighing the possibilities of DNA and 12:30-13:30. Lunch. Michael Christiansen (Denmark)
protein for fetal and maternal health
First trimester screening for adverse
screening Session III. Structural abnormalities
obstetric outcomes
Peter Schielen (Netherlands)
Lorraine Dugoff (USA)
Screening for fetal structural anomalies in
French National Program: switching to the
the first trimester
first trimester 12:30-13:30. Lunch.
Antoni Borrell (Spain)
Françoise Muller (France)
Incidence of anencephaly in twins following Session VIII. General
Correct use of Biochemical markers in twins
IVF
Kevin Spencer (UK)
Ron Maymon (Israel) Prenatal gender selection
Cost-effectiveness of Down syndrome Peter Benn (USA)
screening paradigms 15:00-15:30. Coffee break.
Advantages of dried blood spots compared
Anthony Odibo (USA)
with whole blood in screening
Session IV. Free communication.
Massively parallel sequencing in maternal Kevin Spencer (UK)
plasma
Holland's Next Top Model: trisomy 21 risk
Brigitte Faas (Netherlands)
Tuesday, 21 June, 2011 estimation models in the Netherlands".
TBA Wendy Koster (Netherlands)
Tak Yeung Leung (Hong Kong) Session V. Breakfast debate. Chair:
Brigitte Faas (Netherlands) 15:00-15:30. Coffee break.
First trimester dried blood spot screening
results The introduction of non-invasive prenatal
Session IX. Free communication
David Krantz (USA) diagnosis is imminent
In favour List of speakers
10:30-11:00. Tea break.
TBA
Peter Benn, Antoni Borrell, Michael
Session II. Mendelian and other Against
Christiansen, Howard Cuckle, Lorraine
genetic disorders Peter Schielen (Netherlands)
Dugoff, Brigitte Faas, Steven Kingsmore,
Wendy Koster, David Krantz, Tak Yeung
A new concept in Fragile X syndrome Session VI. Refinements and cofactors
Leung, Ron Maymon, Françoise Muller,
screening in screening
Kypros Nicolaides, Anthony Odibo, Eugene
Howard Cuckle (UK)
Pergament, Mack Schermer, Peter Schielen,
Ultrasound markers of aneuploidy in multiple
Kevin Spencer, Balaji Srinivasan, David
A new method of Fragile X syndrome carrier pregnancies
Wright, Yuval Yaron
testing Howard Cuckle (UK)
Mack Schermer (USA)
Correlations across pregnancies: implications
Fragile X syndrome carrier frequency and for screening
ethnicity testing David Wright (UK)
TBA
10:30-11:00. Tea break.
To submit abstracts or for further details please contact Mercè Sabaté mesabate@clinic.ub.es at the Secretariat
http://www.meetingpharma.com/ipsg.ispd.barcelona2011/home.html

CGH v Karyotyping First among equals
Comparative genomic hybridization (CGH) using I have recently been asked for the earliest reference to
microarrays to identify copy number changes is now suggest the ‘Combined’ test. I think Prenatal Screening
recommended as the first-line test for the evaluation of Perspectives readers will know that in the past I wasn’t a
individuals with multiple anomalies not specific to a well big fan of naming tests Double, Triple, Quadruple and
delineated syndrome, developmental delays, intellectual Combined as the names might inhibit the development of
disabilities, and autism spectrum disorders (1,2). The new 2, 3 or 4 marker protocols or other combinations of
indications for this testing also include better definition of serum and biochemical markers. Still, these shorthands
imbalances partially characterized through karyotyping. have proved useful and these days I use them myself.
The approach would also seem to already be appropriate
when there are ultrasound findings indicative of major I’m not a big fan of firsts either. Often in our field adding
malformation (3). The preliminary results of a broad US one or more new marker to existing markers has been a
multicenter prospective study to evaluate CGH in prenatal logical next step. The idea was in the air and anyone could
diagnosis indicates that additional abnormalities will be have been first. Even the great breakthroughs of science
identified but there are some significant challenges have been characterised by this phenomenon. The real leap
associated with counseling when there is a copy number forward has been conceptual developments like crunching
variant of unknown significance (4,5). information from multiple markers into a single entity, the
Widespread use in prenatal diagnosis will involve
risk. Of course, even conceptual leaps can be in the air.
balancing the additional detection of significant
imbalances, the level of reassurance the testing provides,
Leibnitz and Newton simultaneously invented the calculus
turn around time, availability of resources and cost. It may while resident in different countries and without
also require some new difficult decisions about what knowledge of each others ideas on the subject.
information is worth diagnosing and/or communicating.
Well back to the Combined test. The earliest reference I
1) Miller et al, Am J Hum Genet 2010;86:749-64. 2) can find is from an article I wrote in 1994 for the Royal
Manning et al, Genet Med 2010;12:742-5. 3) Faas et al, College of Obstetricians and Gynaecologists journal The
Prenat Diagn 2010;30:S27. Abstract 11-3. 4) Wapner & Diplomate (1(2):104-109) entitled ‘Multimarker Screening
Jackson. Prenat Diagn 2010;30:S3. Abstract 2-2. 5) for Down's Syndrome’. Having described the available
Wapner & Jackson. Prenat Diagn 2010;30:S31. Abstract first trimester biochemical and ultrasound markers,
12-6. including NT, I said “Ideally both biochemical and
ultrasound markers could be combined but there is as yet
Peter Benn (benn@nso1.uchc.edu) insufficient information on the extent of any correlation
between them.” The journal, now defunct, is not cited by
PubMed and was aimed at members of the Royal College
of Obstetricians and Gynaecologists who had completed
Fetal size the College's Diploma in Obstetrics and Gynaecology
and dating (DRCOG), but was also available for sale. By 1996 the
idea of combining was certainly established and
The British Medical promoted in my ‘Opinion’ editorial piece for Ultrasound
Ultrasound Society in Obstetetrics and Gynecology (7:236-8) entitled
has issued a ‘Biochemical and ultrasound screening for Down's
recommended syndrome: rivals or partners?’. In this I said “A move to
conversion between earlier screening could provide the opportunity to plan a
crown rump length more rational service based on the use of both serum and
(CRL) measured at ultrasound markers in combination. Much attention has
6-13 weeks and gestational age (GA, days). In recently been paid to nuchal translucency (NT), which is
conjunction with the Fetal Medicine Foundation and the both a powerful first trimester ultrasound marker of
NHS National Screening Program, the conversion aneuploidy, and is largely uncorrelated with free β-hCG,
formula has been adopted for prenatal aneuploidy one of the most discriminatory biochemical markers. A
screening programs: screening policy could be envisaged whereby the general
practitioner takes a blood sample at, say, 10 weeks
GA=8.052 x √(CRL x1.037) +23.72 gestation and schedules an ultrasound examination for 11
weeks. The blood marker levels would be available at the
The report also provides gestational age conversions for
time of the scan and could readily be combined with the NT
13-25 week head circumference (BPD is not
recommended) and femur lengths. Estimating expected and other ultrasound markers to derive the best estimate of
fetal size (head circumference, abdominal Down's syndrome risk.”
circumference, and femur length) when gestational age
is known can be determined from additional formulae If anyone knows of an earlier reference please let us
provided in this report. know.
Loughna et al, Ultrasound 2009;17(3):161-7. Howard Cuckle (hsc2121@columbia.edu)

A n om a l o us m ar k er
Second trimester combined testing
values
Although the world is rapidly moving to the first trimester
Heterophilic antibodies (HAb) as the best time for aneuploidy screening we have to face
are antibodies that can be present in the fact that a sizable proportion of women do not contact
p a ti e nts’ se ru m tha t bi nd to the medical services until the second trimester. Such
immunoglobulins from other species. women are usually offered, at best, the Quad test but it is
These can interfere with an immunoassay thereby giving an inadequate. Surely we should improve detection for them
erroneous value for a marker concentration. by combining the Quad markers with ultrasound markers?
HAb have been noted to cause incorrect measures of There are lots of candidates: nuchal skinfold (NF), nasal
hCG (1) and INH-A (2). A recent case report nicely bone length (NBL), femur length (FL), humerus length
documents the same phenomenon with uE3 (3). A 36 year-
(HL) and various more subjective ‘soft’ markers – those
old women received “Integrated” screening with NT=1.69
MoM, PAPPA=0.41 MoM, AFP=0.79 MoM, hCG=3.2 MoM
incorporated in the standard 18-20 week anomaly scan or
and uE3 >5.0 MoM (but truncated to 2 MoM for risk genetic sonogram.
calculation purposes). Her term risk was calculated using The most discriminatory ultrasound marker is pre-nasal
SURUSS statistical parameters and it was 1:1500 for all thickness (PT). There have now been 5 published series
markers but 1:3 when uE3 was omitted from the risk including a total of 105 Down syndrome cases (Maymon et
assessment. The fetus was subsequently found to have a
al, Prenat Diagn 2005;25:906-911; Maymon et al,
47,XY,+21 karyotype.
There are simple methods to block HAb and this
Ultrasound Obstet Gynecol 2006;27:290-295; Persico et
would seem to be indicated when there is a single al, Ultrasound Obstet Gynecol 2008;32:751-754;
unexplained extreme value. However, it is currently unclear Maymon et al, Ultrasound Obstet Gynecol 2009;34:629-
just how commonly this problem arises, whether there are 633; Miguelez et al, J Ultrasound Med. 2010;29(12):1741-
specific patients who are particularly likely to carry HAb, and 7). In the combined data the median was 1.33 MoM with a
what criteria should be used to reflex to a blocking tight log10 standard deviation of 0.0772 in cases and 0.0752
technique. in controls.
Modeling predicts that routinely adding PT to Quad
…and anomalous risk calculation markers would increase the detection rate for a 5% false-
positive rate from 71% to 85% (Miguelez et al, 2010).
In the example cited above, four markers together
with age suggested an increased risk (1:3) and yet a single
And adding two other ‘facial profile’ markers, NF and
additional marker, uE3, was sufficient to drastically reduce NBL would increase it to 93%.
the risk (1:1500). Why did the uE3 result carry so much
weight that it negated all other information and provided a Howard Cuckle (hsc2121@columbia.com)
risk estimate that was seemingly implausible?
The SURUSS parameters for affected pregnancies
include much stronger correlations between uE3 and the
other serum markers, compared to the corresponding Chromosome 21 codes for approximately 500 genes
correlation coefficients for unaffected pregnancies. The including five micro RNAs (miRNAs). miRNAs are post
observed full combination of results was unlikely for both an transcriptional regulators that reduce expression levels.
affected and an unaffected pregnancy but the constraints The chromosome 21 miRNA could therefore have an
imposed by the affected pregnancy correlation coefficients
important role in determining the Down syndrome
actually made this full set of results much more likely for an
unaffected pregnancy. . This effect becomes most evident phenotype.
when we calculate a risk for an extremely unlikely set of test Kuhn et al, (J Biol Chem 2010;285:1529-43) report that
results. As noted elsewhere in this issue of Perspectives the chromosome 21 miRNA are overexpressed in fetal
(article on Repeat Measures) the SURUSS standard heart and brain from Down syndrome specimens
deviations and correlation coefficients do appear to overly compared to normal. There are several thousand proteins
restrict the allowable multi-dimensional space occupied by that could be affected but the authors focused on the
the affected and perhaps also the normal distributions.
As well as test interferences such as HAb, there are
methyl-CpG-binding protein, MeCP2, which is mutated in
multiple other reasons for highly atypical test result Rett syndrome. They noted reduced MeCP2 expression
combinations (for example, presence of an open neural and also aberrant expression of two genes, MEF2C/Mef2c
tube defect, pregnancy complications, genetic disorders and CREB1/Creb1, that are affected by the reduced
such as steroid sulfatase deficiency, etc) and in each of expression of MeCP2. These genes are involved in
these situations the Down syndrome risk algorithm will could neuronal development.
potentially provide misleading information. Ways to deal There are obvious phenotypic differences between
with these relatively common situations would be to impose
Down syndrome and Rett syndrome and there are many
truncation limits that are based on large disparities in the
paired results and to omit a specific marker or use other transcripts that could be the target of the
alternative correlation coefficients in such cases. chromosome 21 encoded miRNA. Nevertheless, the
possibility that aspects of trisomy 21 phenotype might be
1.Butler and Cole. Clin Chem 2001;47:1332-3. 2. Lambert- caused by miRNA levels is of considerable importance
Messerlian et al, Cin Chem 2007;53:800-1. 3. Dennis et al, because there is a potential to develop therapeutics that
Prenat Diagn. 2010;30:165-7. lower the miRNA levels to that present in disomic cells.
Peter Benn (benn@nso1.uchc.edu) Peter Benn (benn@nso1.uchc.edu)

US Professional Guidelines
The American College of Obstetrics and Gynecology (ACOG) periodically issue practice
guidelines and committee opinions for prenatal screening and these are published in Obstetrics and
Gynecology (“the Green Journal”). Similarly, the American College of Medical Genetics provides practice
guidelines and policies which are to be found in Genetics and Medicine.
As the Table shows, with the exception of the jointly developed policy for cystic fibrosis carrier
screening, there is often disagreement. Genetic counselors, clinical geneticists and obstetricians each
look to their own professional group for guidance. Further compounding the problem, both commercial
and hospital labs often offer expanded menus of disorders or mutation panels.
Consistent professional policies could reduce both legal liability and testing that is of marginal clinical value.
Screening ACMG ACOG Comments

Aneuploidy All women All women No specific protocols recommended
Cystic fibrosis 23 mutations 23 mutations 23-109 mutations often provided
Spinal muscular atrophy Yes No
Ashkenazi Jewish panel 9 disorders: 4 disorders: Also sometimes provided:
Familial dysautonomia Familial dysautonomia Familial hyperinsulism
Tay-Sachs Tay-Sachs Glycogen storage disease 1a
Canavan Canavan Maple syrup urine disease
Cystic Fibrosis Cystic Fibrosis Nemaline myopathy
Fanconi Anemia, group C Usher types IF, III
Niemann-Pick type A
Bloom syndrome
Mucolipidosis IV
Gaucher type 1
Hemoglobinopathies African, Asian and Mediterranean
descent
Fragile-X No (1) Family history of a fragile-X
disorder, unexplained cognitive
impairment, autism.
Premature ovarian failure
Patient request
(1) Testing, but not screening, for cognitive impairment, family history of fra(X), premature ovarian failure, tremor/ataxia
Case 2
This 29 year old woman had first trimester serum screening
We often get enquiries from IPSG members to see if others with a raised free β-hCG (2.29 MoM) and absent PAPP-A (ie
have any experience of pregnancy outcome when the below the limit of detection for the assay). Both markers
screening marker profile is extreme. If the pregnancy in were repeated and there was no change. The PAPP-A level
question is ongoing they would value a rapid response but was checked again in another laboratory that uses an
we don’t usually have time to broadcast IPSG for an entirely different type of antibody and again the level was
opinion. However, now that Prenatal Screening zero.
Perspectives is an online publication there may be an
opportunity to do so in the future. Meanwhile, we’d still Later second trimester markers were measured: AFP 1.04
like to hear from you about two recent cases: MoM, free β-hCG 2.32 MoM and uE3 1.19 MoM. PAPP-A
Case1 was also measured and again the level was zero. A genetic
sonogram (anomaly scan) at 18 weeks revealed no
This woman had a maternal serum AFP level of 160 MoM. abnormalities. Amniocentesis showed a normal karyotype.
A repeat test (weeks later, albeit tested in a different The first trimester serum sample was later mixed (1:1) with
laboratory yielded a level of 18 MoM. MRI failed to find a the first trimester serum from another woman who had
maternal cause in the adnexa, abdomen or mediastinum, normal marker levels. The free β-hCG level was halved and
or a fetal cause. Targeted ultrasound of the placenta and the PAPP-A level was zero. This implies the presence of an
fetus was normal. A Kleihauer-Betke (KB) test, which might interfering substance that blocks contact between
indicate fetal-maternal transfusion was negative. Liver antibody and antigen in the PAPP-A assay. Currently, the
function tests were normal. Currently, the pregnancy has pregnancy is at 31 weeks gestation and everything appears
reached 36 weeks and fetal growth is normal. to be fine. A scheduled third trimester scan has not yet
been carried out.

Dr Anne M Summers MD, FRCPC, FCCMG: 1954 –2009
Dr Anne Summers was one of the pioneers of prenatal screening in Canada. She first became the director of the Maternal Serum
Alpha Fetoprotein Program at North York General Hospital in Toronto in 1989 and later assumed various leadership roles for
prenatal screening in the province of Ontario. Anne headed the hospital’s Maternal Serum Screening Laboratory and the Ontario
Maternal Serum Screening Database. She co-chaired a committee which coordinated and advised Ontario’s prenatal screening
practice for many years. She sat on the Maternal Serum Screening Committee of Quality Management Program-Laboratory
Services in Ontario between 1996 and 2006, first as a member and then as the chair from 2004.
Anne worked tirelessly for all women in Ontario to have access to quality prenatal screening services, especially those residing in
remote locations. She was a driving force behind Ontario’s enhanced prenatal screening program. The program provides women
with an informed choice between first trimester combined screening (FTS), integrated prenatal screening (IPS), second trimester
screening (Quad) and serum integrated prenatal screening (SIPS). As a result of implementing this enhanced strategy in Ontario,
the uptake and screening performance have substantially improved.
Anne authored many publications in the area of prenatal screening including a summary of Ontario’s extensive experience in
triple marker screening1, a prospective study of Toronto’s experience with IPS and FTS2 and a clinical practice guideline on
prenatal screening for fetal aneuploidy for the Society of Obstetricians and Gynecologists of Canada3. She was an excellent
speaker and frequently presented at rounds, as well as national and international scientific meetings.
Anne was born in Greenford, UK, and early in life, immigrated to Ontario with her family. She received her undergraduate
education at the University of Western Ontario and did a year of graduate work at the University of Toronto, where she later
attended medical school. This was followed by a residency in Paediatrics and a fellowship in Clinical Genetics at The Hospital for
Sick Children in Toronto.
Following a short stint as a general paediatrician, Anne joined her colleagues in Genetics at North York General Hospital in 1988,
where she became Chief of Genetics in 2003. She acted as a consultant and advisor to several other genetics clinics in the
province, especially to the Northern Regional Genetics Program in North Bay, Ontario. Anne was an Assistant Professor in the
Department of Paediatrics at the University of Toronto.
In addition to prenatal genetics, Anne’s clinical and research interests included familial melanoma, Huntington disease and
medical ethics. She sat on numerous provincial and national committees, and was asked by the Ministry of Health and Long
Term Care, Ontario to chair the newly formed Ontario Provincial Advisory Committee on Predictive Genetic Testing in 2000.
Anne was devoted to her husband Dr Peter Karalis and two children, John Michael and Kate. In her spare time she often
pursued her love of language through reading; she also enjoyed gardening and listening to classical music.
Anne passed away on March 14, 2009 after a year-long neurological illness. She will be greatly missed by friends and colleagues
everywhere.
References:
1. Summers AM, Farrell SA, Huang T, Meier C, Wyatt PR. Maternal serum screening in Ontario using the triple marker test. J
Med Screen 2003;10:107-111
2. Okun N, Summers AM, Hoffman B, Huang T, Winsor E, Chitayat D et al. Prospective experience with integrated prenatal
screening and first trimester combined screening for trisomy 21 in a large Canadian urban center. Prenat Diagn 2008;28:987-92
3. Summers AM, Langlois S, Wyatt P, Wilson RD; Society of Obstetricians and Gynaecologists of Canada. Prenatal screening
for fetal aneuploidy. J Obstet Gynaecol Can 2007;29:146-79
Wendy S. Meschino and Tianhua Huang, Genetics Program, North York General Hospital, Toronto, On, Canada.
Based on sequencing, of newborns with CF (after

Cystic Fibrosis excluding common mutations), the authors estimate that the
use of an expanded CF panel with 97 mutations (instead of
It is now nearly 10 years since ACMG/ACOG first the currently recommended 23 mutation panel) would only
issued recommendations for population-based carrier increase the overall detection of newborns with CF by 0.7%.
testing for cystic fibrosis (CF). Strom et al (Genet Med Among patients with atypical or mild CF who
2011;13;166-72) have published their experience for a large received sequencing, they found that 64% had no
reference laboratory that has provided nearly 3 million detectable mutations, 10% had two mutations and 27% had
carrier tests. only a single detectable mutation. This latter result
Using a 32-mutation panel, they estimate that they suggests that some heterozygotes may show symptoms
detect 86% of the CF-causing mutations in the White perhaps due to modifier genes or environmental factors.
population, 88% for Ashkenazi Jewish, 75% for Hispanics, There also appear to be occasional cases of atypical CF
63% for African Americans and 43% for Asian Americans. where there are two ACOG/ACMG panel mutations present
The rate for the Ashkenazi Jewish population is somewhat that would normally be expected to be associated with a
lower than expected (94%) while the rate for Hispanics typical CF phenotype.
appears to slightly higher than expected (72%),

The effect of maternal age on screening performance
A study was carried out to directly compute Down syndrome detection and false-positive rates according to age - Wendy
Koster, Esther Wortelboer & Peter Schielen
The performance of the first-trimester combined test has been described extensively and varies between 70% and 90%
detection rate (DR) at an approximate 5% false positive rate (FPR). These rates highly depend on the age distribution of the
studied population [1]. The incidence of Down syndrome (DS) increases with maternal age [2]. Since maternal age is, besides
the biochemical parameters (pregnancy associated plasma protein-A; PAPP-A and the free beta subunit of human chorion
gonadotropin; fβ-hCG) and the NT measurement, an important factor in the risk estimation DR and FPR will also be influenced
by maternal age.
The database of the Dutch National Institute for Public Health and the Environment (RIVM), covering first-trimester combined
tests performed between 2003 and 2009, contained 261 DS cases of singleton pregnancies. All first-trimester combined tests
from unaffected singleton pregnancies between 2007 and 2009 (n = 20,707) were used as a control population. Multiple of the
gestation-specific medians (MoMs) and the risk of having a child with DS were calculated using the risk calculation software
Lifecycle (version 2.2, PerkinElmer, Turku, Finland). PAPP-A and fβ-hCG MoMs were corrected for maternal weight by
reciprocal linear regression. Test results were considered screen positive if the calculated risk for DS was at least 1:200 at the
moment of testing. The dataset was then divided into four age classes (<30 years, 30-34 years, 35-39 years and ≥40 years).
DR, FPR and the odds of being affected giving a positive result (OAPR [3]) were calculated for each age class.
Agegroup n fb-hCG MoM PAPP-A MoM NT MoM

<30 11 1.40 (0.84 – 2.15) 0.52 (0.39 – 0.79) 2.67 (1.64 – 3.86)
30-34 48 2.14 (1.18 – 3.17) 0.59 (0.30 – 0.71) 1.41 (1.11 – 2.39)
35-39 147 1.66 (1.11 – 2.79) 0.46 (0.28 – 0.66) 1.59 (1.16 – 2.10)
≥40 55 1.80 (1.35 – 2.93) 0.50 (0.26 – 0.93) 1.71 (1.30 – 2.58)
Table - Median MoMs (interquartile range) of PAPP-A, fβ-hCG and NT for all Down syndrome pregnancies between 2003 and
2009 in each age class.
Median MoMs for PAPP-A, fβ-hCG and NT in DS cases for each age class are shown in the table. For all markers MoMs were
comparable between age classes. The median gestational age in the groups differed 2 days at most. Overall, 51 DS cases
were not detected. For these cases the median MoM values were 0.70 for PAPP-A, 1.25 for fβ-hCG and 1.06 for NT.
Using the calculated Down syndrome risks DR and FPR were determined for every age class (see figure). Analysis of the age
classes showed an increase in both DR and FPR as the maternal age rises. Similar trends of increasing DR and FPR have
been shown for both first- and second trimester DS screening in previously conducted modeling studies [4, 5].
100
DR
90 FPR
80
70
60
50
40
30
20
10
<30 30-34 35-39 ≥40
Figure – Detection rates (DR) and false positive rates (FPR) with 95% confidence intervals in four different maternal age groups.
A conspicuous detail is the low DR in the 30-34 age class. Only 62.5% of the DS cases were detected in this group, a
phenomenon for which we have no obvious explanation. Based on the calculated DR and FPR an OAPR for each age class
was calculated. This OAPR was 1:11, 1:10, 1:11 and 1:12 for age classes <30 years, 30-34 years, 35-39 years and ≥40 years
respectively and thus remained constant with maternal age.

Starting January 1st 2007, every pregnant woman in the Netherlands is being informed about first trimester combined
screening for DS. Earlier, screening was only offered on a pregnant woman’s specific request. For counseling purposes it is
important for both pregnant women and health care professionals to understand that the risk of DS increases with age as well
as the DR and FPR. A commonly heard misconception among pregnant women and health care professionals alike is that DS
screening has no use for women over 40 years since they will always end up with a high-risk test result. Here, we illustrate this
belief to be not necessarily true; FPR is 18% at most in the ≥40 years age category. This study shows a relatively low DR for
women between 30-34 years of age, seemingly suggesting that DS screening is failing for this age group. However,
considering the screening performance in terms of OAPR shows similar results in all maternal age groups. In other words, DS
screening is sufficient for every pregnant woman, despite her age. This should be emphasized in counseling sessions by
midwives or gynaecologists.
References
1. Koster MP. Detection for false positive rate - Depends on age distribution. Prenatal Screening Perspectives 2009;15:24.
2. Cuckle H, Wald N, Thompson S. Estimating a woman’s risk of having a pregnancy associated with Down’s syndrome using
her age and serum alpha-fetoprotein. Br J Obstet Gynaecol 1987;94:387-402.
3. Wald NJ, Kennard A, Hackshaw A, McGuire A. Antenatal screening for Down's syndrome. J Med Screen 1997;4(4):181-
246.
4. Reynolds TM, Nix AB, Dunstan FD, Dawson AJ. Age-specific detection and false-positive rates: an aid to counseling in
Down syndrome risk screening. Obstet Gynecol 1993;81:447-450.
5. Wildschut HI, Peters TJ, Weiner CP. Screening in women's health, with emphasis on fetal Down's syndrome, breast cancer
and osteoporosis. Hum Reprod Update. 2006;12(5):499-512.
M.P.H. Koster, E.J. Wortelboer, P.C.J.I. Schielen (peter.schielen@rivm.nl)
NTD prevention
It appears that as many as one-third of NTD pregnancies are
not preventable by folic acid (FA) prophylaxis. In these FA
resistant cases there is evidence to suggest that inositol may
be effective. The evidence includes:
1. Inositol deficiency is the only ‘vitamin’ deficiency that
leads to NTDs in mice (Cockroft, Teratology
1998;38:281-90).
2. Significantly lower inositol concentration is present in
the blood of mothers carrying NTD fetuses than in
normal pregnancies (Groenen et al, Am J Obstet
Gynecol 2003; 189:1713-9.).
Peter Schielen and Howard Cuckle (in academic gown) 3. Inositol supplementation in pregnant mice significantly
at the reception following the defense of her thesis by reduces the frequency of NTDs (Greene and Copp
Wendy Koster. Nature Med 1997;3:60-6; Cogram et al, Hum Reprod
2002;17:2451-8).
4. In a single case study, a woman took 0.5 g inositol per
day in the first trimester of her third pregnancy, after
Congratulations to IPSG Member two previous pregnancies were terminated because of
NTDs (she took FA in both). The third pregnancy was
Aubrey Milunsky uneventful, and a normal baby was born (Cavalli and
Copp J Med Genet 2002;39:e5).
In the 2010 British Medical
Association Book Awards This evidence has prompted Copp and colleagues at the
Institute of Child Health in London to carry out a
u n d er the c at e g or y randomised trial: all women with a history of NTD to
Obstetrics and Gynaecology receive 5 mg/day folic acid with half additionally receiving
“Genetic Disorders and the 1g/day inositol and half receiving a placebo.
th
Fetus (6 edition)” edited by Aubrey Milunsky
For more details www.ucl.ac.uk/ich/research-ich/neural-
and Jeff M. Milunsky (Wiley-Blackwell, development/ponti_study.
December 2009) was ‘Highly commended’.
To enter your patients into the trial, contact: Victoria
Shepherd, Trial Co-ordinator at +44 20 7905 2822, +44
7772 258 243 or ponti@ich.ucl.ac.uk.

start the trial intervention on the day it is received, and
continue until 12 completed weeks of gestation. The first
outcome assessment will be an ultrasound scan performed
between 6-8 weeks of gestation. Further assessments will
be at 12-14 weeks, delivery and 28 days post-partum.
Completion: Late 2013.
Health Technology Assessment (HTA)

Haemoglobinopathies (published Dormany et al, HTA
Here are some of the relevant UK HTA (www.hta.ac.uk) 2010;14(20))
projects started or completed in the last year:
Title: Antenatal screening for haemoglobinopathies in
Cervix cancer primary care: a cohort study and cluster randomised trial to
inform a simulation model. The Screening for
Title: A modelled analysis using data from the ARTISTIC Haemoglobinopathies in First Trimester (SHIFT) trial.
trial of the potential effectiveness and cost-effectiveness of
HPV testing in cervical cancer screening. Design: A population-based cohort study, cluster
randomised trial and refinement of a published decision
Background: The HTA funded ARTISTIC trial studied model. The units of randomisation were 25 general
24,000 women aged 20-64 who underwent primary practices in two inner city boroughs with a high proportion
cervical screening and were followed over 2 screening of residents from minority ethnic groups. Practices were
rounds 3 years apart between 2001 and 2007. The trial allocated to screening in primary care with parallel father
cohort was randomised to combined screening with liquid testing (test offered to mother and father simultaneously;
based cytology (LBC) and human papillomavirus (HPV) n=8 clusters, 1010 participants); screening in primary care
testing, or screening only with LBC (though HPV tests with sequential father testing (test offered to father only if
were performed and the results concealed). The trial mother identified as carrier; n=9 clusters, 792 participants);
found LBC to be as effective and less costly than LBC and screening in secondary care with sequential father
combined with HPV testing for primary screening. testing (standard care; n = 8 clusters, 619 participants).
Furthermore when 3 scenarios were compared for costs,
both cytology triaged with HPV for low grade Conclusions: Offering antenatal screening for sickle cell
abnormalities, and HPV primary testing triaged with disease and thalassaemia as part of pregnancy-
cytology were less costly than the current standard confirmation consultations significantly increased the
strategy of repeat cytology for low grade abnormalities. proportion of women screened before 10 weeks, from 2%
The costs of HPV testing with cytology triage is sensitive to in standard care to between 16% and 27% in primary care,
the number of colposcopies performed, in cytology but additional resources may be required to implement
negative women. this. There was no evidence to support offering fathers
screening at the same time as women.
Design: The dataset from the ARTISTIC trial will be used
to model a number of novel cervical screening strategies to
test potential effectiveness and cost effectiveness. The Gestational diabetes (published Waugh et al, HTA 2010;14
strategies will begin with either cytology or HPV testing, (45))
which would then be triaged by the other and will also
incorporate HPV genotyping as this, HPV-16 in particular, Title: Screening for hyperglycaemia in pregnancy: a rapid
could increase the specificity of HPV testing and be a update for the National Screening Committee.
clinically valuable approach.
Design: A systematic review and meta-analysis of the
Completion: Late 2011 literature updating a 2002 HTA report to include among
others data from the Australian Carbohydrate Intolerance
Study in Pregnant Women, the Maternal and Fetal
Recurrent miscarriage Medicine Units Network trial and the Hyperglycemia and
Adverse Pregnancy Outcomes (HAPO) study. Review data
Title: First trimester progesterone therapy in women with a on recent trends in maternal age at birth and on the
history of unexplained recurrent miscarriages: A prevalence of overweight and obesity and the effect on
randomised, double-blind, placebo-controlled, multi-centre prevalence.
trial [The PROMISE (PROgesterone in recurrent
MIScarriage) Trial]. Conclusions: There is now good evidence for treatment
of oral drugs instead of insulin and it looks increasingly as
Design: Women with a history of recurrent miscarriages if fasting plasma glucose could be the test of choice.
are referred to and seen in a recurrent miscarriage clinic However some key uncertainties remain to be resolved,
(standard practice), where appropriate baseline which can be done by further analysis of the already
investigations are performed (standard clinical practice). A collected HAPO data and by using the UK model used in
woman entering the trial is given instructions to notify the developing the NICE guidelines to assess the cost-
research nurse by telephone as soon as she conceives (a effectiveness of intervention in each of the seven HAPO
positive urinary pregnancy test). The research nurse will categories.
dispatch the trial intervention pessaries (either
progesterone or placebo) to the woman within 72 hours of
the telephone call via courier. The woman is expected to

Robert Cocciolone (1957-2010)
Head, Antenatal Screening, Women’s and Children’s Hospital,

Genetics & Molecular Pathology Directorate, South Australia Pathology.
Robert Cocciolone was the highly respected Scientist in Charge of the South Australian Maternal Serum
Screening Program between 1994 and 2010 before his untimely death in March 2010. Rob graduated with a
Bachelor of Applied Science (Medical Laboratory Science) in 1980 from the University of South Australia
and commenced work in the Biochemistry Department of the Queen Victoria Hospital in 1981. For a short
time between 1988 and 1989, he worked as the Territory Manager for Boehringer Mannhein Australia in
South Australia and the Northern Territory. In 1989, he was appointed as a Scientist in the Department of
Chemical Pathology at the Women’s and Children’s Hospital (Adelaide) where he worked with Richard Ryall.
The Department has a long history with antenatal screening. Maternal serum screening for neural tube
defects was begun in 1978 and second trimester screening for Down syndrome in 1990. In 1994, Rob took
over responsibility for managing the South Australian Maternal Serum Screening Program (SAMSAS). In
September 2000 under Rob’s direction, first trimester screening was introduced and later in April 2009,
integrated screening was commenced for the of benefit women who were undecided on how to proceed
following their first trimester combined screen. Rob was a careful and diligent scientist. He took great pride,
ensuring that the laboratory service ran efficiently. He gave great attention to detail, always concerned that
the results that went out from the laboratory were as accurate and reliable as possible.
Rob was always innovative in the application of new technology. In the face of staff and space shortages, he
maintained output by fully automating his laboratory systems well before this was happening in other
laboratories. He co-ordinated SAMSAS program that extended across South Australia, Northern Territory
and Tasmania and he also provided support to the Western Australian PathWest screening program. Rob
audited the performance of the SAMSAS program, and compiled an extensive resource of maternal serum
screening data. Importantly, Rob spent many hours maintaining and improving the algorithm used in
antenatal screening to maximise its performance and discriminatory power. He foresaw the need and then
set up and managed a Quality Assurance Program to improve the quality of the nuchal translucency
measurements and more recently, he began to investigate the integration of first and second trimester
screening data.
Among his peers, Rob was recognised for his professional skills and was frequently invited to talk at
scientific and educational meetings. Rob always shared his experience and knowledge of screening with
characteristic generosity and consideration of the views of others. When he gave advice or opinion, it was
always firmly based on his understanding of the data accumulated in his screening program. His most recent
project had focused on the evaluation of ADAM 12 as a biomarker in antenatal screening. In retrospect, it is
a great joy to be remember Rob’s presentation “ADAM 12 – a useful addition for first or second trimester
screening?” at the Sydney meeting on first trimester screening - new ways to assess risks for adverse
obstetric outcomes, in December 2009.
Colleagues welcomed his professional contributions on prenatal screening for fetal anomalies and
pregnancy wellbeing. Those of us who knew Rob from his presentations at the Human Genetics Society of
Australasia and through the Royal Australian College of Obstetrics and Gynaecology Nuchal Translucency
Ultrasound, Education and Monitoring Program appreciated his meticulous attention to scientific rigor in the
context of a clinical service environment. Rob Cocciolone will be missed as a firm friend and an admired
colleague.
Peter O'Leary

Chromosome 21 balanced
translocation Fragile-X
Recently, I received an email from a Fragile-X syndrome in males with a full mutation is
common (about 1 in 4,000) and is a serious medical condition.
Genetics Department in the UK “We have
The disorder in females is variably expressed with an
had several cases of carriers of estimated 1 in 5,000-8,000 individuals affected. The frequency
Robertsonian translocations involving 21 of premutation carrier females has been estimated to be 1 in
who have been given risks that do not reflect their a priori 158 to 1 in 549 and these individuals are not only risk for
10% risk. Their laboratory’s software does not cope with children with the full mutation, they themselves are at
this and has almost led to misdiagnosis in one case.” increased premature ovarian failure. Both males and females
with the premutation, but especially males, are also at risk for
Readers, check your software! Does it allow entry of
late onset tremor/ataxia syndrome (FXTAS).
Down’s syndrome carrier status? If it only has the ACOG have recently revised their opinion on carrier
possibility of entering 'previous non-inherited Down’s screening (1). They now endorse screening for women with a
syndrome' what would you do about a previous inherited family history of a fragile-X related disorder, unexplained
case? mental retardation or developmental delay, autism, premature
ovarian insufficiency, unexplained elevated FSH prior to age
Actually there are a number of situations related to family 40, or if a patient requests the test. But counseling is
problematic because the screening will identify women with a
history of Down’s syndrome which may or may not alter
full mutation, individuals at risk for FXTAS, premutations of
the a priori risk, including: uncertain risk for expansion, and the results can have complex
implications for additional family members who may be
(1) mother had previous standard Down’s syndrome carriers.
pregnancy The possibility of providing the testing as part of
(2) father had previous standard Down’s syndrome newborn screening has recently been reviewed by Hill et al (2)
with commentary by Coffee (3). Such screening could be
pregnancy
advantageous with early definitive diagnosis and potential for
(3) previous de novo translocation Down’s syndrome early educational intervention. But the diagnosis of late-onset
pregnancy FXTAS, increased risk for ovarian failure, and unintended
(4) previous translocation Down’s syndrome pregnancy, diagnosis of sex chromosome aneuploidy would still be
mother balanced translocation carrier problematic. Newborn screening is often provided without
(5) previous translocation Down’s syndrome pregnancy, consent (or only with an opt-out provision) and for some
father balanced translocation carrier parents these screening results may not be wanted. A
compromise is to provide the screening at age 1-2 following
(6) no previous Down’s syndrome pregnancy, but mother
counseling and informed consent from the parents.
known to be balanced translocation carrier Screening pre-conception adults, following counseling
(7) no previous Down’s syndrome pregnancy, but father and consent, would probably be the least controversial. But,
known to be balanced translocation carrier based on the experience with screening for other inherited
disorders, this is also the hardest to implement.
Risk software will definitely deal with (1) under 'previous
1) Committee on Genetics. Obstet Gynecol 2010;116:1008-
non-inherited Down’s syndrome' by using a published
10. 2) Hill et al, Genet Med 2010;12:396-410. 3) Coffee.
formula, such as adding a fixed percentage to the Genet Med 2010;12:411-2.
maternal age-specific risk expressed as a percentage.
Risk calculation for the others is tricky and will depend on Peter Benn (benn@nso1.uchc.edu)
the type of translocation, reciprocal or Robertsonian, and
the mode of ascertainment of the index case.
For an overview of the available data on this see Benn P.

Prenatal Diagnosis of Chromosomal Abnormalities through
Co-ordination
Amniocentesis. In: Genetic Disorders and the Fetus:
Diagnosis, Prevention and Treatment - 6th edition. (Ed A The European Down Syndrome Screening Group (EDSSG)
Milunsky & JM Milunsky), Johns Hopkins University Press begat the International Down Syndrome Screening Group
Baltimore, 2010; pp 194-272. For example, with the (IDSSG) which begat the International Prenatal Screening
most common balanced Robertsonian translocation, rob Group (IPSG). Having co-ordinated the Group in all its
(14q21q), the Down’s syndrome (unbalanced guises for nearly 20 years, initially with Bent Norgaard-
translocation) risk for a carrier mother is 15% and for a Pedersen, I think its time for me to step down especially as
carrier father only about 1%. With parental reciprocal for the next two years I’m President of the International
translocations, if the ascertainment was a term affected Society for Prenatal Diagnosis (ISPD). Luckily, the two
pregnancy the risk of an unbalanced translocation is Peters – Benn and Schielen – have agreed to take over co-
several times higher for cases where the carrier status ordination with me in the background advising and pitching
resulted from an affected birth compared with other in as and when needed although I draw the line at any
reasons for testing such as recurrent miscarriage. more begetting! So please help the new team by sending
in pieces for Prenatal Screening Perspectives.
Howard Cuckle (hsc2121@columbia.edu)
Howard Cuckle (hsc2121@columbia.edu)

Free fetal DNA; using a multiplex protocol that involved either two (2-
plex), or eight (8-plex), samples sequenced together by
recent developments adding a unique identifying set of bases to each piece of
DNA (barcode). Based on 86 Down syndrome affected
Following the two proof-of- and 571 unaffected samples, the 8-plex protocol had a
principle reports (1,2) that detection rate of 79.1% and a false-positive rate of 1.1%.
shotgun sequencing of cell-free The 2-pex protocol (which involves sequencing more
DNA in maternal circulation fragments), was performed on 86 affected and 146
can potentially be used for unaffected pregnancies and it had a 100% detection rate
no n - i n va s i ve d i a g n o s i s, and 2.1% false-positive rate. In an interview with BBC
refinements have been reported. News (accessed January 13, 2011), Kypros Nicolaides
who co-authored this study suggests that routine use of
Chiu et al (3) consider an alternative sequencing method the approach may not be for 10 years. Based on the
(sequencing by ligation, Applied Biosystems/Life extraordinary pace of developments in this technology,
Technologies, Solid 3 system) from that originally used this would seem to be too conservative?
(sequencing by synthesis, Genome Analyzer, Illumina
Applied Biosystems). They were able to correctly Another trial has just been published (9). This used a
distinguish between 5 cases of trisomy 21 and 6 controls. multiplex approach on 480 samples. There were 13
They noted that, as with the previous methodology, the samples with insufficient DNA and 18 that did not meet
GC content of the sequences imposes a limitation on quality control standards. Of the remaining 449, the
determining genomic representation for some Down syndrome detection rate was 39/39 (100%) and the
chromosomes or chromosome regions. false-positive rate 1/410 (0.3%).
Fan and Quake (4) describe a method of removing the There are two new useful reviews. Chiu et al (10)
GC bias in the sequence reads. They conclude that summarize various approaches to non-invasive prenatal
distinguishing relative genomic representation is only diagnosis and Lo et al (11) present the current state of
limited by the counting statistics and therefore small the art for the diagnosis of hemoglobinopathies.
genomic imbalances could be identified provided there
was deep enough (large number) sequencing. 1. Fan et al, Proc Natl Acad Sci USA.
2008;105:16266-71.
Liao et al (5) consider the effect of enriching for DNA 2. Chiu et al, Proc Natl Acad Sci USA.
fragments present in a sample. Enrichment would 2008;105:20458-63.
potentially allow for more informative sequence reads 3. Chiu et al, Clin Chem 2010;56:459-63.
from a sample. They show that a specific enrichment 4. Fan & Quake. PLoS ONE2010;5(5):e10439.
method (SureSelect Target Enrichment System, Agilent), 5. Liao et al. Clin Chem 2010;57:in press.
did not result in a bias in proportions of maternal and fetal 6. Tong et al. Clin Chem 2010;56:90-8.
alleles. 7. Lo et al. Sci Transl Med 2010;2:61ra91 .
8. Chiu et al. BMJ 2011;342:c7401
Tong et al (6) continue to develop a non-invasive 9. Ehrich et al. Am J Obstet Gynecol 2011;204
diagnostic approach through looking for epigenetic (3):205.e1-205.e11.
differences between maternal and fetal (trophoblast) 10. Chiu et al. Trends Genet 2009;25:324-31.
DNA. They report on methylation differences in a 11. Lo & Chiu. Hematol Oncol Clin N Am
chromosome 21 gene (HLCS) promoter that can be used 2010;24:1179-86.
to distinguish between trisomy 21 and normal
pregnancies.
Lo et al (7) have further shown the extraordinary potential ********

power of deep sequencing for non-invasive diagnosis.
Using a plasma sample, they performed nearly 4 billion
reads, equivalent to 65-fold coverage of a human
genome. They were not only able to detect paternal allele Non-invasive diagnosis: ethics
contributions, but also analyze maternal contributions by
a new method referred to as relative haplotype dosage The expectation that non-invasive prenatal diagnosis will
(RHDO). Using this method, closely linked SNPs are soon be introduced for fetal aneuploidy, disorders with a
cumulatively counted until it is established that there is a Mendelian pattern of inheritance and other conditions raises
relative excess of one maternal haplotype over the over.
some challenging ethical issues. Ethicists and potential
The excess thereby defines the maternal contribution to
the fetal DNA in the sample. They demonstrate the providers are concerned about the adequacy of information
method for the non-invasive diagnosis of beta- and informed consent process prior to the test, accuracy and
thalassemia. reliability of the testing, use for paternity and gender
identification, commercial and direct-to-consumer
Although currently extraordinarily expensive, this study promotion, and increased pregnancy termination rates (1-3).
demonstrates the potential for non-invasive diagnosis of Skotko considers the potential for a substantially lower
genetic disorders on an unprecedented scale. incidence for Down syndrome (4). De Jong et al (5) suggest
that the greater moral challenge lies in the expanded rage of
In yet another important paper, Dennis Lo’s group (8), disorders that will be potentially be diagnosable. This would
perform DNA sequencing on maternal plasma samples

include adult onset conditions where the “right not to know”
will need to be considered. A survey of pregnant women Semi-automated NT
carried out in the Netherlands indicated enthusiasm for NIPD measurement – Progress and
but also that women may find difficulty with the
consequences and choices associated with the new caution
technology (6).
1. Benn PA, Chapman AR. JAMA 2009;301:2154-6.

It is widely acknowledged that of the commonly used Down’s
2. Benn PA, Chapman AR. Curr Opin Obstet Gynecol 2010: syndrome screening markers, NT is both the most
22;128-34. discriminatory and the most difficult to obtain and maintain
3. Hall A, Bostanci A, Wright CF. Public Health Genomics quality. External schemes have been established to train,
2010;13:246-55. certify and monitor NT performance, principally the Fetal
4. Skotko BG. Arch Dis Child 2009;94:823-6. Medicine Foundation (www.fetalmedicine.com) and the Nuchal
Translucency Education and Quality Review (NTQR) program
5. de Jong A et al. Europ J Hum Genet 2010;18:272-7.
(www.ntqr.org). Both schemes promote a standardised
6. Kooij L et al. Prenat Diagn 2009;29:164-8. approach to measurement: clear NT edges; mid-sagittal plane;
the fetus occupying more than half the image; the fetal head in
Peter Benn (benn@nso1.uchc.edu) a neutral position; amnion separated from the NT line; ‘+’
callipers; placed on-on; perpendicular to the long axis of the
fetus; and measurement at the widest space of NT. However,
there is a degree of subjectivity for virtually all these aspects.
To some degree this can be reduced by the use of new

The “Genetic computer technology to make the actual NT measurement [1].
Sonogram” The operator places an adjustable box over the relevant area
of the fetal neck, the software draws a line through the centre
of the nuchal membrane and another at the edge of the soft
A series of second trimester ultrasound markers have tissue overlying the cervical spine and identifies the largest
variable utility in screening for fetal Down syndrome. vertical distance between the two lines. Whist this does
Relative to second trimester serum triple or quadruple produce more reliable results than the manual method there is
screening, they are less effective at distinguishing between still a need to capture the correct image [1-3]. Thus NTQR
affected and unaffected pregnancies (Benn & Egan Prenat have now made the following statement on their website:
Diagn 2008;28:230-235). However, it has become common Appropriate education, NT credentialed physicians and
practice to use this “genetic sonogram” or “anomaly scan” sonographers, adequate equipment and counseling resources
are essential for practices offering first trimester risk
contingently; i.e. women at high or intermediate risks often
assessment includ ing the nuchal translucen cy
will receive the genetic sonogram and risks are modified measurement. NT measurements require the ability to image
prior to a decision about amniocentesis. the fetus in the correct plane, to select appropriate images or
conversely to discard suboptimal images, to optimize settings,
Aagaard-Tillery et al (Obstet Gynecol 2009;114:1189-96) to select the appropriate area for measurement, and to place
show that the approach also has value for women who have the calipers correctly on the NT. Automated NT functions
received first trimester combined screening. Using available on some ultrasound machines may facilitate uniform
prospectively gathered data from the FASTER trial, they placement of calipers. This technology does not address
developed likelihood ratios for each of the commonly used optimal position of the fetus or substitute for other essential
factors. The NTQR committee provides education and ongoing
second trimester ultrasound markers. For any particular
quality review for participants reporting NT measurements.
patient, each likelihood ratio is multiplied together based on Obtaining the NT through automation is acceptable when the
the presence or absence of the marker. i.e. the markers are credentialed provider confirms the image and automated
considered to be independent. caliper placement meet all NTQR criteria. Additional research
on the efficacy of automated NT measurements is
The efficacy of the combined test, quadruple test, and recommended.
integrated test are each improved with the addition of the
“genetic sonogram”. However, using the genetic sonogram 1. Moratalla J, Pintoffl K, Minekawa R, Lachmann R, Wright D,
as a replacement for the second trimester serum markers in Nicolaides KH. Semi-automated system for measurement of
nuchal translucency thickness. Ultrasound Obstet Gynecol
a sequential or contingent approach (i.e. combined first
2010;36(4):412-6.
trimester screening followed by the genetic sonogram) is 2. Abele H, Hoopmann M, Wright D, Hoffmann-Poell B,
less effective than retaining the quadruple test. Huettelmaier M, Pintoffl K, Wallwiener D, Kagan KO. Intra-
and interoperator reliability of manual and semi-automated
In the approach used by Aagaard-Tlillery et al, nuchal fold measurement of fetal nuchal translucency by sonographers
thickness and femur or humerus length were used as with different levels of experience. Ultrasound Obstet Gynecol
categorical variables (i.e. either above or below specific cut- 2010;36(4):417-22.
offs). There is the potential for considerable enhancement 3. Grangé G, Althuser M, Fresson J, Bititi A, Miyamoto K,
for the genetic sonogram by converting the measures into Tsatsaris V, Morel O. Semi-automated adjusted measurement
of nuchal translucency: feasibility and reproducibility.
MoMs and using the data as continuous variables (Benn et
Ultrasound Obstet Gynecol 2011;37(3):335-40.
al, Obstet Gynecol 2002;100:1168-76; Borell et al, Obstet
Gynecol 2007;30:941-5).

Pyramids inverted Spina bifida – first trimester markers
or otherwise AFP screening at 16-18 weeks for spina bifida is passé.
The special issue of Prenatal Higher detection rates can be obtained by routine fetal
Diagnosis published in January anomaly scanning at 18-20 weeks or during a BPD scan
2011 (31(1)) is devoted to Kypros as early as 14 weeks looking for the ‘lemon’ and ‘banana’
Nicolaides idea that the current signs.
practice of obstetrics is weighted
towards the end of pregnancy and With the shift in aneuploidy screening to the first trimester
that with properly designed programs more could be achieved by the obvious question is whether spina bifida can be
reversing the process and providing most care at 11-13 weeks.
detected at the time of the NT scan. Some advocates of
The reviews in the issue make the case not only for aneuploidy an early – trans-vaginal – anomaly scan at this time have
screening at this stage in pregnancy but also screening for structural reported detection of some cases. Now there are
fetal abnormalities and adverse outcomes of pregnancy including suggestions that examination of the fetal brain stem at
pre-eclampsia, pre-term delivery, growth restriction and fetal loss. this time could detect open spina bifida cases with the
Much of the data presented comes from Nicolaides group at Kings Arnold-Chiari syndrome. Compression of the fourth
College Hospital (KCH), London. ventricle leading to small (in relation to gestational
norms) or absent intracranial translucency has been
For example, spina bifida can be detected at this time, not by reported.
visualising the lesion but because of the associated Arnold-Chiari
malformation which is caused by pressure changes in the open
neural tube. This abnormality manifests itself in the first trimester
So far, in the literature there are only a few spina bifida
brain stem (BS) diameter and the BS to occipital bone diameter. cases with these measurements, but watch this space:
The study reported in the issue (Lachman et al, 103-6) included 30
cases of open spina bifida. The BS diameter was above the 95th 1) Chaoui et al, Assessment of intracranial translucency
centile in 29 (97%), the BS to occipital bone diameter was below the (IT) in the detection of spina bifida at the 11-13-week
5th percentile in 26 (87%) and the ratio between them was above the scan. Ultrasound Obstet Gynecol. 2009;34(3):249-52.
95th percentile in all cases. 2) Chaoui and Nicolaides. From nuchal translucency to
intracranial translucency: towards the early detection of
There are now some impressive predicted detection rates for adverse spina bifida. Ultrasound Obstet Gynecol. 2010;35(2):133-
outcomes. On the basis of KCH data in the issue the model
predicted detection rate of early pre-eclampsia (ie requiring delivery 8.
before 34 weeks) for a 5% false-positive rate is 78% when using 3) Egle et al, Appearance of the fetal posterior fossa at
four markers – serum PlGF & PAPP-A, blood pressure and uterine 11+3 to 13+6 gestational weeks in transabdominal
artery Doppler – together with information on prior risk factors ultrasound examination. Ultrasound Obstet Gynecol.
(Akolakar et al, 66-74). An even higher prediction of 88% comes [Epub ahead of print].
from a model using parameters derived by meta-analysis (Cuckle,
Placenta 2011;32:S42-8). Whatever the exact figure, there is now a
rational to start screening. A trials meta-analysis has been published
which shows that if low dose aspirin is used starting before 16
weeks gestation the risk of pre-eclampsia can be approximately
halved and the risk of severe pre-eclampsia reduced 10-fold, JOUBERT SYNDROME
although this has a wide confidence interval (Buyold et al, Obstet
Gynecol 2010;116:402–14).
Joubert Syndrome affects the cerebellum, which is responsible
The EU funded a research initiative entitled “Development of Early for controlling balance and coordination of skeletal muscles.
Non-Invasive Biomarkers and Means for the Diagnosis and Infants born with this condition demonstrate decreased muscle
Progression Monitoring of Preeclampsia and Tailoring Putative tone, difficulty in swallowing, jerky eye movements and an
Therapies” with the acronym PREGENESYS. The research group inability to coordinate voluntary muscle movements. As the
held a Consensus Meeting in Cambridge in July 2009 to review the children grow, they may suffer from mental retardation and
state of the art and consider issues that may need to be addressed in
future recommendations. This has now been published (Cetin et al,
develop kidney failure
Placenta 2011;32:S4-16).
Dor Yeshorim, an organisation that promotes genetic testing in
ultra-Orthodox Jews had observed an increasing number of
When modelling detection of other adverse outcomes one runs into children born with the syndrome in recent years, and asked
the problem of double counting. For example, PAPP-A and PlGF Hadassah University Medical Center in Jerusalem to
are markers of both aneuploidy and pre-eclampsia; moreover the
investigate.
latter not only leads to low birthweight through early iatrogenic
delivery because of a threat to the mother but can result in growth
restriction and stillbirth. Evidence that these adverse outcomes can The disease has previously been mapped to the centromeric
have a common patho-physiology can be seen in a recent massive region of chromosome 11. Using homozygosity mapping in 13
Swedish study (Wikström et al, Am J Obstet Gynecol patients from eight Ashkenazi Jewish families, the Hadassah
2010;203:Epub ahead of print). group identified a homozygous mutation, R12L, in the
TMEM216 gene, in all affected individuals (Edvardson et al, Am
So the inverted pyramid approach may require risks to be reported in
the form 1 in x for outcome a given that there is no outcome b, c &
J Hum Genet 2010;86(1):93-7; erratum 86(2):294). Based on
d, etc. these initial findings, researchers examined the DNA of 2,766
healthy Ashkenazi Jews and discovered that one out of 92
carried the mutation.

Predicting macrosomia in diabetic available in 100% (n = 186) of the control group and in 91%
(n=162) of the PGDM group. Median birthweight centiles were
pregnancies from serum markers. significantly higher in the PGDM group than in the control
group (89th and 72nd centile respectively; p < 0.0001) and
A study was carried out to evaluate this for free β-hCG significantly more PGDM infants were macrosomic (n = 69
and PAPP-A - Sylvia Kuc (42.6%) and n = 34 (18.3%), respectively; p < 0.0001).
The median MoMs of both markers were not significantly
Despite major obstetric care and strict glycemic management, different between the PGDM and control group. However,
pregestational type 1 and type 2 diabetes mellitus (PGDM) is when the PGDM and control groups were divided according to
strongly associated with increased fetal morbidity and the macrosomia at birth, the median PAPP-A MoM was
mortality compared to the general pregnant population1, 2, 3. noticeably lower in the non-macrosomic PGDM subgroup
Diabetes-induced metabolic disorders are thought to interfere compared to the other subgroups. Median fβ-hCG MoMs were
with embryonic organogenesis and hyperglycaemia is thought comparable in all four subgroups.
to trigger excessive fetal growth4. Currently, macrosomia at The screening performance for macrosomia at birth in the
birth (birthweight above 90th centile) is one of the most PGDM group with PAPP-A provided a detection rate (DR) of
concerning complications of a PGDM pregnancy. During 18% for a 5% false positive rate (FPR) and a DR of 30% for a
labor, macrosomic infants suffer higher rates of shoulder 10% FPR.
dystocia, brachial plexus injuries and intrapartum asphyxia
compared to non-macrosomic infants1, 2, 3. After delivery, So it seems that the first-trimester DS serum markers fβ-hCG
these infants are at increased risk for hypoglycaemia, infant and PAPP-A were comparable between PGDM and control
respiratory distress syndrome (IRDS) and cardiomyopathy2. groups, but subgroup analyses according to macrosomia at
Furthermore macrosomia at birth predisposes to childhood birth showed that levels of PAPP-A were significantly lower
obesity and to increased morbidity including insulin only in the non-macrosomic PGDM subgroup. There was no
resistance, hypertension and diabetes5. difference in PAPP-A concentrations between the control
population and the PGDM macrosomic subgroup. Thus,
The first-trimester screening test for Down syndrome (DS) is PAPP-A seems to distinguish between non-macrosomic and
now a part of obstetric care worldwide. The DS risk is macrosomic subgroups in PGDM pregnancies (estimated DR
calculated on the basis of two markers in maternal serum: free of 30% at 10% FPR) and is therefore a promising predictor of
β-human chorionic gonadotrophin (fβ-hCG) and pregnancy macrosomia at birth in PGDM pregnancies. Currently our
associated plasma protein-A (PAPP-A) together with the group is evaluating PAPP-A in combination with the other
nuchal translucency measurement (NT) and maternal age. known first-trimester serum markers (A Desintegrin And
Numerous studies report predictive value of first-trimester Metalloproteinase 12 – ADAM12, Placental Protein 13 –PP13,
serum markers for macrosomia at birth in non-diabetic Placental Growth Factor – PlGF) for the prediction of
pregnancies6, 7. macrosomia at birth in this particular high-risk population.
Serum samples were collected at the Dutch National Institute 1. Persson M, Norman M, Hanson U. Obstetric and perinatal
for Public Health and the Environment (RIVM) between 2005 outcomes in type 1 diabetic pregnancies: A large, population-
and 2007 as part of the national first-trimester Down based study. Diabetes Care 2009;32:2005-2009
syndrome (DS) screening program. Serum samples were 2. Evers IM, de Valk HW, Visser GHA. Risk of
collected between 8 and 14 weeks of gestation age (GA) and complications of pregnancy in women with type 1 diabetes:
analysis of serum concentrations fß-hCG and PAPP-A was nationwide prospective study in the Netherlands. BMJ
performed. For all women taking part in the screening sample 2004;328:915
date, maternal age, maternal weight, smoking and medication 3. Balsells M, Garcia-Patterson A, Gich I, Corcoy R.
were recorded by the requestor. Pregnancy outcome, Maternal and fetal outcome in women with type 2 versus type
including pregnancy duration, date of birth, pregnancy 1 diabetes mellitus: a systematic review and metaanalysis. J
complications, chromosomal disorders, child gender and Clin Endocrinol Metab 2009;94:4284-4291
weight, were collected after delivery. From this cohort, serum 4. Allen VM, Armson BA, Wilson RD, Allen VM, Blight C,
samples from women with PGDM were selected and retrieved Gagnon A et al. Society of Obstetricians and Gynecologists.
from storage. Women were classified as having PGDM if they Teratogenicity associated with pre-existing and gestational
had been registered as using insulin in the first-trimester of diabetes. J Obstet Gynaecol Canada 2007;29:927-944
pregnancy. One control serum from an uncomplicated 5. Rijpert M, Evers IM, de Vroede MA, de Valk HW,
singleton pregnancy was matched to each PGDM case, for Heijnen CJ, Visser GHA. Risk factors for childhood
the same day of GA at sampling (±1 week), maternal weight overweight in offspring of type 1 diabetic women with
(±5 kg), maternal age (±1 year) and for sample date (±6 adequate glycemic control during pregnancy: Nationwide
months). Serum marker levels were expressed as multiples of follow-up study in the Netherlands. Diabetes Care
the gestation-specific normal median (MoMs). The 2009;32:2099-2104
correlations between marker concentrations and birthweight 6. Tul N, Pusenjak S, Osredkar J, Spencer K, Novak-
centiles were assessed by Pearson correlation coefficients. Antolic Z. Prediciting complications of pregnancy with first-
Differences with P-values < 0.05 were considered statistically trimester maternal serum free-betahCG, PAPP-A and inhibin-
significant. Growth charts corrected for gestational age, sex A. Prenat Diagn 2003;23(12):990-6
and parity according to the Dutch Perinatal Registry were 7. Poon LC, Karagiannis G, Stratieva V, Syngelaki A,
used to calculate the exact birthweight centiles8. Macrosomia Nicolaides KH. First-trimester prediction of macrosomia.
was defined as birth weight above the 90th centile8. PGDM Fetal Diagn Ther 2010; [Epub ahead of print]
and control groups were subdivided in non-macrosomic 8.Visser GHA, Eilers PH, Elferink-Stinkens PM, Merkus HM,
(control non-macrosomic; PGDM non-macrosomic) and Wit JM. New Dutch reference curves for birthweight by
macrosomic (control macrosomic; PGDM macrosomic) gestational age. Early Hum Dev 2009;85:737-744
subgroups.
Sylwia.Kuc@rivm.nl
Serum from 186 PGDM and 186 control pregnancies was
selected for analysis. Eight samples from the PGDM group
were excluded because there was not enough serum for
analysis. Post-partum information on birthweight was

Audit of self-reporting of pregnancy outcome: Flowchart – Schematic overview of numbers of Down
influence on detection rate of first-trimester syndrome cases in the database. First level: cases are
Down syndrome screening. divided into “high risk’, ‘low risk’ and ‘unknown risk’
group. Second level: missing data was retrieved to
calculate the unknown risks which were subsequently
regrouped. Third level: number of cases in each group
An audit was carried out to see if this influenced a pro-
after verification of the outcome in the ‘low risk’ group.
gram with apparently low detection - Nathalie Poierrié,
Peter Schielen & Wendy Koster
1 46 D S
(2 0 0 6 -2 0 0 9 )
9 3 ‘h ig h r is k ’ 3 8 ‘lo w r is k ’
1 5 ‘ u n k n o w n r is k ’
( = 1 :2 0 0 ) (> 1 :2 0 0 )
The first-trimester combined test for Down syndrome
(DS) screening, which includes the serum markers + 1 4 ‘ h i g h r is k ’ + 1 ‘ l o w r is k ’
PAPP-A and fb-hCG in combination with a NT
measurement and maternal age, has been reported to 1 0 7 ‘h ig h r is k ’ 3 9 ‘lo w r is k ’
provide a detection rate (DR) of 85-90% at a false 22 D S +

positive rate (FPR) of 5%1-4.In the Netherlands, the DR 17 D S -
is 70-75% at a 3.5% FPR, which is relatively low

compared to other countries5,6. Subsequently, for all DS cases in the ‘low risk’ group (n
= 39), data on previous aneuploidy, gestational age at
The performance of the first-trimester combined test sampling, diabetes, mode of conception (e.g. IVF or
depends on (1) technical aspects such as the quality of ICSI), first trimester vaginal blood loss, medication, twin
NT measurements or biochemical assays; (2) accurate pregnancy, smoking status and maternal weight were
measurements and registration of gestational age, verified and, if necessary, completed based on the
maternal weight or maternal age; (3) the existence of original patient charts. Strikingly, after data-collection at
confounding biological factors such as diabetes, vaginal hospitals and obstetric practices 17 pregnancies in this
blood loss, smoking, IVF, medication use or obstetric group appeared not to represent a DS pregnancy.
history. There are indications that for some of these Therefore the initial DR of 71% (93 of 146 DS cases
confounders a correction factor should be applied, detected) changed to 83% (107 of 129 DS cases
however, this is not standard policy in the Netherlands. detected).
Between 2006 and 2009 a total of 146 DS pregnancies After all requested variables were included or corrected
were registered at our laboratory. This information was in the database, the characteristics of the ‘low risk’ and
recorded by questionnaires and collected through self- ‘high risk’ risk group were compared. The results are
reporting of women participating in the first-trimester shown in a table. Median gestational age at sampling
screening program after they gave birth. This way, showed a difference of 6 days between both groups.
approximately 80% of all follow-up data can be There was no such difference for maternal age or
registered. Gathering this information through the health weight. Furthermore, this analysis showed no obvious
professionals managing the pregnancy is not allowed differences between groups for the potential correction
under Dutch privacy legislation. factors (e.g. diabetes, IVF/ICSI or smoking), however,
numbers were very small. When risks were recalculated
Concentrations of PAPP-A and fβ-hCG were measured based on small differences in variables such as
with AutoDelfia (Perkin Elmer, Turku, Finland).The gestational age or maternal weight (from the patient
quality of the NT measurements was assured by the charts) the screening performance did not change.
quality guidelines of the Dutch Centre for Population
Research. Combined risks were calculated using Table – Overview of pregnancy characteristics at the
Lifecycle (version 2.2; PerkinElmer, Turku, Finland). time of the first-trimester combined test in both the ‘high
risk’ and ‘low risk’ Down syndrome cases. Data are
All DS cases were divided into three groups: ‘low risk’ if presented as medians (interquartile range) or numbers
the combined risk was lower than 1:200, ‘high risk’ if the (percentages). Since numbers are small, no statistical
combined risk was over or equal to 1:200 and ‘unknown comparison was performed.
risk’ if no combined risk had been calculated (mostly High risk Low risk
because of missing data from the NT measurement). As (n = 107) (n = 22)
shown in the flowchart, of the 146 DS pregnancies a Gestational age (days) 82 (73-87) 88 (77-90)
total of 93 pregnancies had a high combined risk, 38
Maternal age (years) 37 (35-39) 37 (34-39)
had a low combined risk and for 15 pregnancies the
combined risk was unknown at our laboratory. After Maternal weight (kg) 68 (61-80) 71 (64-79)
retrieving the data on missing NT measurements from
Previous Down 0 (0%) 0 (0%)
hospitals and obstetric practices a combined risk was
calculated for the third group after which 14 Diabetes 0 (0%) 1 (5%)
pregnancies were reassigned to the ‘high risk’ and one IVF/ICSI 6 (6%) 0 (0%)
pregnancy to the ‘low risk’ group respectively. Vaginal blood loss 3 (3%) 0 (0%)
Twin pregnancy 3 (3%) 0 (0%)
Smoking 4 (4%) 2 (9%)

Im por ta nc e of
Surprisingly, the most striking result of this retrospective
analysis was that a significant number of registered DS chorionicity for risk
cases appeared to be no case at all. For this dataset assessment in
this means that the actual DR is 13% higher than twins
initially thought. Thus, it resembles more closely
international reports on screening performances. On the basis of 4843 first trimester
However, we have not yet confirmed that pregnancies blood samples from unaffected twin
in the ‘high risk’ are indeed all DS cases. pregnancies it has been shown that
Errors in our database of a total of 17 PAPP-A levels are much lower (about 0.4 MoM on average) in
pregnancy outcomes were either incorrectly reported by monochorionic (MC) pregnancies in comparison to those with
the patients or were administrative input errors at our dichorionic (DC) placentae (Madson et al, Ultrasound Obstet
laboratory. However, a random check of received Gynecol 2011; 37: 38–47). Moreover, for both types of twins MoMs
follow-up forms indicates that the latter is quite unlikely. increase on average substantially during the first trimester. Levels
Suggested other reasons for these wrongful of free β-hCG were lower for MC compared with DC twins but this
reports could be: (1) women do not quite understand was a far weaker effect than with PAPP-A, and was only statistically
what “Down syndrome (trisomy 21)” means and report significant before 10 weeks. There was a strong tendency for
DS in case of other chromosomal or structural defects MoMs to increase with gestation but only among MC twins.
of the child; (2) women accidentally report a previous
DS pregnancy; (3) communicational problems due to It is common practice to estimate gestational age in twins from the
not mastering the Dutch language. largest CRL rather than the average since, it is argued, there are
To overcome wrongful reports of DS cases in reasons for fetuses in early pregnancy to be growth restricted
the future one could argue to clarify the terminology of whilst growth enhancement is rare. Nevertheless taking the largest
‘Down syndrome’ on the post-partum forms. However, is a type of bias and may have contributed to the results in this
since DS is an internationally accepted and well known paper. If any readers have a large series of twins please let us
condition we do not think this will entirely solve this know if it makes a difference to the trend in first trimester serum
issue. Ongoing digitalization of the entire screening marker MoMs when the average CRL is used compared to the
process in the future may also allow for outcome largest CRL.
reporting by health professionals, warranting both
correctness and completeness. The paper also reported 47 affected twins, 6 MC and 41 DC; for
both markers levels were lower in MC.
1
Valinen Y, Rapakko K, Kokkonen H, Laitinen P, Tekay
A, Ahola T, Ryynanen M. Clinical First-trimester routine The rest of the paper describes and assesses a model of risk
screening for Down syndrome in singleton pregnancies calculation for twins but having read it through a few times I can’t
in northern Finland. Am J Obstet Gynecol 2007;196 understand what they have done. In my simple minded view the
(3):278e1-5. gestation- and chorionicity- specific normal median MoMs they
2
Wojdemann KR, Shalmi AC, Christiansen M, Larsen report for PAPP-A and free β-hCG are all we need. They can be
SO, Sundberg K, Brocks V, Bang J, Norgaard- used to modify the already well described model. For example, at
Pedersen B, Tabor A. Improved first-trimester Down 10 weeks the PAPP-A median for DC twins is 1.7756 (Table 3) and
syndrome screening performance by lowering the false- from the literature mean at this gestation for singleton Down’s
positive rate: a prospective study of 9941 low-risk syndrome pregnancies is 0.40 MoM. In the model the Gaussian
women. Ultrasound Obstet Gynecol 2005;25(3):227- distribution mean for unaffected twins is 1.7756 and for discordant
Down’s syndrome twins is estimated by (1+0.40)*1.7766/2 or 1.24
33.
3 MoM and for concordant twins (0.40+0.40)*1.7766/2 or 0.71 MoM.
Nicolaides KH, Spencer K, Avgidou K, Faiola S,
Falcon O. Multicenter study of first-trimester screening
To add to the confusion the authors conclude “Whether risk
for trisomy 21in 75821 pregnancies: result and
calculation based on chorionicity-specific twin medians is superior
estimation of the potential impact of individual risk-
to the ‘pseudo risk’ approach in gestational weeks 12–14 is
oriented two-stage first-trimester screening. Ultrasound
unknown…”. Pseudo risk was proposed (by me) in the early days of
Obstet Gynecol 2005;25:221-6. screening in twins as a stopgap until we knew something about the
4
Spencer K, Souter V, Tul N, Snijders R, Nicolaides marker median MoMs in affected pregnancies. The idea was to
KH. A screening program for trisomy 21 at 10-14 ensure that the false-positive rate was the same as for singletons,
weeks using fetal nuchal translucency, maternal serum accepting that the detection rate, in discordant twins, was low. This
free β-human chorionic gonadotrophin and pregnancy- was decades ago and the approach should have been abandoned
associated plasma protein A. Ultrasound Obstet everywhere by now. We don’t need pseudo risks because we can
Gynecol 1999;13:231-237. estimate actual risks. The mean MoMs for Down’s syndrome can
5
Wortelboer EJ, Koster MP, Stoutenbeek P, ELvers LH, be adequately derived indirectly as described above and these
Loeber JG, Visser GH, Schielen PC. First trimester values are close to the directly observed values in the literature
Down screening performance in the Dutch population; (Spencer K. (2005) Non-invasive screening tests. In: Blickstein I &
how to achieve further improvement? Prenat Diagn Keith LG (eds.) Multiple Pregnancy, Epidemiology, Gestation and
2009;29(6):588-92. Perinatal Outcome, Parthenon Publishing, London, pp.368-384).
6
Schielen PCJI, Koster MPH, Elvers LH, Loeber JG.
Downsyndroom kansbepaling met de eerste trimetsre Howard Cuckle (hsc2121@columbia.edu)
combinatietest 2006-2008. Rapport RIVM
230083001/2010 (Dutch).

impossible today that anyone would commit the resources for a
RE-INVENTING THE true nationwide outcomes study, we have modeled that a 0.5mm
WHEEL: AGAIN! decrement in measurements would lead to an 18% loss of
sensitivity.(7) With 4 million births per year in the USA, certainly
over 100 Down Syndrome cases alone would be missed. This is
- a new regular opinion column by consistent with previously published patient data that showed poor
Mark I Evans technical performance lead to diminished sensitivity.
In the 1960’s “modern” concepts of It has been a gospel of the technology assessment literature, that
quality control and business there are two phases of new clinical and laboratory approaches.
processes were vestigial by today’s First there is a phase of development in which a small number of
standards. W. Edward Deming was investigators, commonly but not always at academic medical
a Detroit auto efficiency business analyst who tried unsuccessfully centers, pioneer a new technique, publish on it, demonstrate its
to get the major American automotive manufacturers to adopt utility, and create a demand for such services.(8) Eventually others
certain principles for business operations and efficiency including get into the act and the technology “diffuses” out among the
labor – management interactions and methods for quality control community. Repeatedly, for clinical procedures, the data show
(QC). (1) His ideas were mostly ignored – in part because there was vast increases in utilization accompanied by dramatic drops in
little competition in the industry, and cost inefficiencies could just performance and increased complications.
be passed onto consumers who essentially had no alternatives. In
Japan, however, his ideas were adopted enthusiastically turning The purpose of quality assessment programs which were created -
around a culture in which “made in Japan” had been a synonym in such as the Fetal Medicine Foundation worldwide and the Nuchal
the 1950’s for cheap and poor quality into one by the 1970’s of Translucency Quality Review program in the USA - was to minimize
meticulous attention to detail and high quality. Overall, Deming the negative impact of new providers trying to mimic the
articulated 14 “principles” meant to apply to industry and the usual performance of the developers of the new technology. In many
job/factory functions. Some are outside the bounds of the parts of the world, the concept that “simple ultrasound
professional setting, but many apply equally well to medical measurements” had to be certified or even reviewed were met
practice. with scorn by some sonographers who stated that ultrasound was
an art. Unfortunately, if ultrasound measurements are to be used
Some of the relevant principles are paraphrased here and include: in laboratory algorithms, then experience says they need to have
the same QC as the laboratory uses. Published and unpublished
1. Create constancy of purpose for continual improvement data have clearly shown that failure to adhere to stringent
2. Cost alone is not the whole driver of decisions methodology control procedures produces skewed distribution
3. Improve every process curves with most inexperienced sonographers tending to under
4. Continual on the job training measure. Even in well controlled studies such as BUN and FASTER,
5. The purpose of leadership is to help everyone improve gradual increases in provider curves were well documented over
6. Drive out fear of communication up and down the ranks the years of the studies.
7. Break down barriers (end silo thinking across groups)
In future “pieces” I will lay out how this disconnect between
Throughout my entire medical training, teaching was focused on obtaining simple measurements and the public health implications
learning to do the job correctly, but it was never presented through of poor performance must be addressed for quality health care for
the rubric of anything systematic such as the above. Physicians women to be provided across the board.
were taught to be stalwart individuals trying to care for their
patients. Seldom were they indoctrinated into a team concept of 1. Demming WE. Out of the Crisis. MIT Press, Cambridge 1986
feeling as being one component of a bigger process. Emergency 2. Snijders RJM, Noble P, Sebire N, Souka A, Nicolaides KH. UK
rooms, code teams, and MASH units would be notable clinical multicentre project on assessment of risk of trisomy 21 by
exceptions. When it came to surgical procedures or interpretation maternal age and fetal nuchal translucency thickness at 10–
of clinical or radiological data, the idea of consistency across 14 weeks of gestation. Lancet 1998;351:343–6.
providers was a very seldom mentioned concept. In contrast, the 3. Haddow JE, Palomaki GE, Knight GJ, Williams J, Miller WA,
laboratory was long expected to do daily quality control Johnson A. Screening of maternal serum for fetal Down's
monitoring, and the principles of statistical performance and rigor syndrome in the first trimester. N Engl J Med 1998;338:955-
were clearly in place by the 1970’s. 61.
4. Monni G, Zoppi MA, Ibba RM, Floris M. Results of
What does this have to do with Nuchal Translucency (NT) measurement of nuchal translucency before and after
screening? Actually, lots! Early arguments over the validity of NT training. Lancet 1997;350:1631.
screening were solved with standardization of the approach (2-4). 5. Evans MI, Cuckle HS: Biochemical Screening for Aneuploidy
In fact NT development was a complete replay of the issues of AFP Expert Rev Obstet Gynecol 2007;2:765-774
screening in which in the 1970s and 80’s, it was believed that each 6. Evans MI, Krantz DA, Hallahan TW, Sherwin JE:
city had to have their own medians and curves because of Undermeasurement of nuchal translucencies: implications for
population differences. (5) It was only with standardization of screening. Obstet Gynecol 2010;116:815-818
laboratory methods that it became clear that such “population 7. Evans MI, Van Decruyes H, Nicolaides KH: Nuchal
differences” were very minor, and reported variations were translucency (NT) measurements for 1st trimester screening:
actually almost exclusively methodology differences. the “price” of inaccuracy. Fetal Diagn Ther 2007;22:401-404
8. Cohen AB, Hanft RS. Technology in American health care:
Recently we have shown that the curve of measurements in the policy directions for effective evaluation and management.
United States is shifted to the left as compared to FMF data from Ann Arbor, MI: University of Michigan Press. 2004
much of the world, and that there is a specific bulge at the bottom
percentiles.(6) The net effect of this shift is to lower the sensitivity Mark Evans (evans@compregen.com)
and specificity of screening in the United States. While it is

Danish thesis defense Kasper Pihl considered whereas SP1 only was significantly reduced
in cases with spontaneous preterm delivery and SGA.
The best screening performance was found for severe
SGA (birth weight <2.5th percentile) with the combination
On March 18th 2011, I had the pleasure of being the
of PAPP-A, SP1 and smoking status – yielding a 43%
opponent at Kasper Pihls thesis defense at the Statens
detection rate for a 10% false positive rate. The
Serum Institut in Copenhagen, Denmark. Kasper’s
screening performance was in general moderate and not
supervisors were Michael Christiansen (Department of
yet clinically useful. This is most likely explained by the
Clinical Biochemistry and Immunology (Statens Serum
heterogeneity of the outcomes considered.”
Institut) and Torben Larsen and Lone Krebs (both at the
Department of Gynecology and Obstetrics, University of
Copenhagen, Holbæk Hospital). The examining
committee consisted also of Prof Ann Tabor and Dr
Karen Wojdemann.
A Danish PhD exam consists of a 4-5 pages written
assessment of the thesis and a public oral examination,
shared by Karen Wojdemann and me. In a Dutch PhD
defense, every opponent (usually seven or eight) is
allowed a single question. Examining for 45 minutes, as
is Danish custom, was much more than I was used to
but due to Kaspers outstanding skills, we engaged in a
lively debate on the various aspects of his thesis. He
was of course granted the academic degree.
Congratulations Kasper!
Below is the outline of his thesis, entitled: First trimester
screening for adverse pregnancy outcome - the role of
maternal serum PAPP-A, beta-hCG, ADAM12, proMBP
and SP1.
“This PhD thesis is based on five studies conducted in

the period 2007-2009 during my employment at
Department of Clinical Biochemistry and Immunology,
Statens Serum Institut, Copenhagen. All studies were
The fresh PhD, listening to the laudatio of his supervisor.
conducted in co-operation with the Department of
Obstetrics and Gynecology at Holbæk Hospital. The
Peter Schielen (peter.schielen@rivm.nl)
overall aim for this thesis was to examine the potential of
first trimester maternal serum markers in screening for
the adverse pregnancy outcomes small-for-gestational
age (SGA), preeclampsia (PE) and spontaneous preterm Paternal Age
delivery. The serum markers considered were
Sartorius and Nieschlag (Hum Reprod Update; 2010;16
pregnancy-associated plasma protein A (PAPP-A), free (1):65-79) provide an updated review of the risks associated with
beta-subunit of human chorionic gonadotropin (beta- advanced paternal age. The authors note that
hCG), a disintegrin and metalloprotease 12 (ADAM12), increasing male age is associated with
the proform of eosinophil major basic protein (proMBP) decreasing androgen levels, decreased sexual
and pregnancy-specific beta-1-glycoprotein (SP1). The activity, alterations in testicular morphology
studies were based on merging of registry data from and deterioration in semen quality (volume,
ASTRAIA, the Danish Cytogenetic Central Registry, the motility and morphology). As well as the
Danish Newborn Screening Registry and the Danish well known correlation between age and
Birth Registry. Furthermore, serum samples from autosomal dominant disorders, there appears
to be also a paternal age effect in many
Holbæk Hospital routinely forwarded to Statens Serum
mulitifactorial disorders. Miscarriage, pre-
Institut for analysis (PAPP-A and beta-hCG) and storage eclampsia, preterm birth, Caesarian section rates may all be more
in the Prenatal Screening Biobank were used for common for older fathers.
biochemical analysis. The first study demonstrated a But the news is not all bad. The authors conclude that at the
quality control model for the first trimester combined current time advanced paternal age, per se, is not a reason for
screening program for DS. The model was used to invasive diagnostic testing. With the exception of some sex
establish a study cohort for the following studies. A chromosome abnormalities, there remains no evidence for increased
retrospective cohort study was conducted confirming aneuploidy. Also, interestingly, telomere repeat lengths (which are
that PAPP-A is a significant marker of SGA. Three case- usually shorter with approaching cell senescence and aging) are
longer in the sperm from older males.
control studies were conducted for the evaluation of
So, we can continue to take encouragement from the lives
ADAM12, proMBP and SP1. Serum samples were of elderly celebrity fathers who seem to defy aging. Also from
retrieved from storage and analyzed manually using Metheselah (Noah's grandfather) who is said to have fathered a son at
different immunoassay techniques. ADAM12 was age 187 years.
significantly reduced in pregnancies with SGA. ProMBP
was consistently reduced in all of the outcomes Peter Benn (benn@nso1.uchc.edu)

International Society for Prenatal Diagnosis
ISPD is moving in two new directions, both of them with
contribution from IPSG.
The biannual International Conference is well attended.

IPSG m em bers may consider joining See page 28 for an account of the 15th Conference in
the Community Genetics Network, which puts out a monthly Amsterdam last June; the 16th Conference will be at the
newsletter. The network has 927 members in 73 countries
presently. The aim is to facilitate communication between all Loews Miami Beach, 3–6 June 2012. These meetings
those working in the field of community genetics (and are a good opportunity to catch up with what’s new
genomics). Members of the network are all those who have prenatal diagnosis, but it is a rapidly changing field.
expressed their wish to become a member by e-mail Thus it has been decided to have international meetings
(commgennet@gmail.com). devoted to education in the years between the
Conferences. The first such ISPD Education Day will
The newsletter lists references to papers by members with a be in Barcelona immediately preceding the IPSG
hyperlink to the abstract. In the March issue 52 papers are Congress (see page 6) with a shared infrastructure and
listed broken down into categories: Complex Conditions,
local organisation led by Tony Borrell. Most of the
Congenital Disorders, Consanguinity & Inbreeding, Economic
Issues, Ethical Issues, Family History, Genetic Counseling, teaching will be through the special interest groups
Genetic Disorders, Genetics Education and Literacy, Genetics (SIGs) which are being coordinated by Brigitte Faas.
in Primary Care, Genetic Screening, Genetic Testing, Eventually it is hoped that the SIGs will go on the road
Genomic Applications, Guidelines for Specific Disorders, worldwide to provide educational services outside the
Health Worker Perspective, Miscellaneous, Patient context of meeting per se.
Perspective, Pharmacogenetics and Pharmacogenomics,
Population History, Preimplantation Genetic Diagnosis, Opinion leaders have an important role in the transfer of
Psychosocial Issues in Genetic Testing, Public Attitudes, research findings into routine clinical part. But opinions
Public Awareness, Public Health Genetics and Genomics,
differ and practice tends to become chaotic. Some
Specific Disorders or Mutations in Specific Communities and
Statistical issues. professional bodies have for a long time tried to use
their authority to modulate the extremes of practice (see
Under Genetic Screening there 4 papers: p9) although they do have a tendency to be
conservative and promote the lowest common
Standardizing newborn screening results for health
denominator. Against this background ISPD have
information exchange. Abhyankar S, Lloyd-Puryear MA,
Goodwin R, Copeland S, Eichwald J, Therrell BL, Zuckerman decided to issue their own ‘position statements’ in order
A, Downing G, McDonald CJ. AMIA Annu Symp Proc to give rational direction to relevant new developments.
2010;2010:1-5. And the first entitled “Aneuploidy Screening: a Position
Statement from a Committee on Behalf of the Board of
Health benefits and cost-effectiveness of primary genetic
the International Society for Prenatal Diagnosis” will be
screening for Lynch syndrome in the general population. Dinh
TA, Rosner BI, Atwood JC, Boland CR, Syngal S, Vasen HF,
published soon in Prenatal Diagnosis. The committee
Gruber SB, Burt RW. Cancer Prev Res (Phila). 2011;4(1):9- was chaired by our very own Peter Benn and comprises
22. Epub 2010 Nov 18. many IPSG members.
Genetic screening for Krabbe disease: Learning from the past
and looking to the future. Macarov M, Zlotogora J, Meiner V,
Khatib Z, Sury V, Mengistu G, Bargal R, Shmueli E, Meidan B,
Zeigler M. Am J Med Genet A. 2011 Feb 22. doi: 10.1002/
ajmg.a.33815. [Epub ahead of print]
IPSG 9th International Congress
Long-term Consequences of the Early Treatment of Children
with Congenital Hypothyroidism Detected by Neonatal School of Biology, University of Barcelona,
Screening in Nanjing, China: a 12-year Follow-up Study. Sun
Q, Chen YL, Yu ZB, Han SP, Dong XY, Qiu YF, Sha L, Guo 20th & 21st June 2011
XR. J Trop Pediatr. 2011 Feb 4. [Epub ahead of print]
The newsletter also lists upcoming courses (three in the

March issue in the Netherlands, UK and USA) and
The bells are ringing out....
conferences (22, in Australia, Belgium, Canada, Cuba, El canto vuela....
France, Germany, Greece, India, Japan, the Netherlands,
Portugal, Spain, Turkey and USA). They're calling us together....
It also lists position opportunities, academic, post-doc, Guiding us forever
doctorates, masters degrees and others (eg the March issue
advertises “Visiting Scholar Programme of the Centre for
Society and Genomics (CSG) provides researchers based
outside the Netherlands with the opportunity to spend 2-3 Barcelona!
months at CSG in Nijmegen or one of its affiliated research
groups spread around the country”. Freddie Mercury & Montserrat Caballe
http://www.youtube.com/watch?v=Ztqcuawqi34

FETAL DUCTUS VENOSUS AS A MARKER
FOR CARDIAC DEFECTS Placental growth factor
Most of papers on the ductus venosus (DV) Doppler studies PAPP-A is a marker of both aneuploidy and pre-eclampsia but
have assessed its role as a marker in the prenatal detection of to really get started with combined first trimester biophysical
chromosomal anomalies or signaling fetal acidemia in fetal (uterine Doppler) and biochemical screening for early pre-
wellbeing assessment. Recently a couple of papers have eclampsia additional serum markers are needed. One
investigated its role in the prenatal detection of cardiac candidate is placental growth factor (PlGF) and it has the big
defects in chromosomally normal fetuses. Although previous advantage over other such markers in that it is also a marker
studies showed that DV assessment was redundant to the use of aneuploidy. Hence, screening for pre-eclampsia can
of nuchal translucency (NT), both studies show now that DV increase aneuploidy detection.
improves the performance of NT screening for cardiac
defects, and that a 40% detection rate may be achieved by the The published median or mean MoM results so far are:
combined use of both markers.
(1) Down’s syndrome 0.71 (90 cases); Edwards’ syndrome
The first paper from the Barcelona group (Martinez et al, 0.48 (28); Patau’s syndrome 0.40 (19); triploidy 0.53 (10);
Ultrasound Obstet Gynecol 2010;35:267–272) evaluated the Turner’s syndrome 0.53 (28). Zaragoza E, Akolekar R, Poon
independent contribution of DV at 11–13+6 weeks’ gestation LC, Pepes S, Nicolaides KH. Maternal serum placental growth
to the prediction of cardiac defects in chromosomally normal factor at 11-13 weeks in chromosomally abnormal
fetuses, irrespective of the value of the NT thickness. pregnancies. Ultrasound Obstet Gynecol 2009;33(4):382-6.
Abnormal DV blood flow was defined as either absent or
reversed flow during atrial contraction, and fetal (2) Down’s syndrome 0.76 (70). Cowans NJ,
echocardiography was performed in all these fetuses, as well Stamatopoulou A, Spencer K. First trimester maternal serum
as in NT above the 99th centile. placental growth factor in trisomy 21 pregnancies. Prenat
Diagn 2010;30(5):449-53.
Among 6,120 consecutive singleton scanned pregnancies,
there were 145 chromosomally normal fetuses with abnormal (3) Down’s syndrome 0.80 (151). Koster MP,
DV Doppler and 45 chromosomally normal fetuses with a
Wortelboer EJ, Stoutenbeek P, Visser GH, Schielen PC.
cardiac defect. Eleven affected fetuses presented with an
Modeling the Down syndrome screening performance using
abnormal DV, giving a 24% detection rate, a 7.6% positive
first trimester serum markers. Ultrasound Obstet Gynecol
predictive value and 9.8 odds ratio. In 6 of these 11 fetuses
2010 Nov 12. [Epub ahead of print]
(with both a cardiac defect and abnormal DV) an increased
NT above the 99th centile was also observed. Thus, the group
Second trimester results are also available:
of fetuses with isolated abnormal DV blood flow (with
normal NT) contained 5 cardiac defects, for a detection rate
of 11%, a positive predictive value of 5.5% and an odds ratio (4) Down’s syndrome 0.67 (24). Debieve F, Moiset A,
of 8.5. Interestingly, right-heart anomalies were predominant Thomas K, Pampfer S, Hubinont C. Vascular endothelial
in those cases with isolated abnormal DV, but no specific growth factor and placenta growth factor concentrations in
pattern was found in those with increased NT. Down's syndrome and control pregnancies. Mol Hum Reprod
2001;7(8):765-70.
The authors concluded that in experienced hands, abnormal
DV blood flow in the first trimester is an independent But one paper on early first trimester samples goes in the
predictor of fetal cardiac defects and should constitute an opposite direction:
indication for early echocardiography. In this study, the use
of DV blood flow assessment increased early detection of (5) Down’s syndrome 1.3 (37). Cowans NJ,
cardiac defects by 11% with respect to the use of NT Stamatopoulou A, Tørring N, Spencer K. Early first-trimester
measurement alone (rising from 29% to 40%). maternal serum placental growth factor (PlGF) in trisomy 21
pregnancies. Ultrasound Obstet Gynecol 2010 Nov 23. [Epub
The second paper, from Nicolaides group (Cheleman et al, ahead of print]
Fetal Diagn Ther 2011;29:127–134) recently appeared,
sought to determine whether assessment of DV improved the Simultaneously screening for different outcomes raises
detection rate of cardiac defects achieved by screening with another issue, whether to report the risk for each as though
NT thickness. The study population of euploid fetuses at 11- they are independent. This may not be the case for Patau’s
13 weeks included 85 cases with major cardiac defects and syndrome (trisomy 13). Boyd et al. (Lancet 1987;2
40,905 with no cardiac defects. The fetal NT was above the (8556):425-7) reported severe pre-eclampsia in 5 out of 14
99th centile in 18 (21%) of the fetuses with cardiac defects. trisomy 13 cases, all nulliparous, and none of 28 age and
Abnormal DV, defined as reversed a-wave, was observed in parity matched controls. No such association was seen for 7
24 (28%) of the fetuses with cardiac defects and in 2.1% of Down’s syndrome and 4 Edwards’ syndrome pregnancies. A
those with no cardiac defects. Specialist fetal similar case-control study found pre-eclampsia in 6/25
echocardiography for cases with NT above the 99th centile trisomy 13, 1/38 Edwards’ syndrome and 1/50 controls
and those with abnormal DV, irrespective of NT, would (Tuohy JF, James DK. Br J Obstet Gynaecol 1992;99(11):891-
detect 39% of major cardiac defects at an overall false 4). Both studies found the association higher in primigravids.
positive rate of 2.7%.

Position Statement
The International Society for Prenatal Diagnosis’s Position Statement on Aneuploidy Screening will soon be published in Prenatal
Diagnosis. The Statement is a refinement and expansion of an earlier statement developed by the IPSG (PSP, 2009, Vol 15,p43-45).
The box contains the key recommendations for aneuploidy screening. In addition to these recommendations, the Statement provides
an ethical framework for the testing by defining the goal of screening and indicating the need for pre-test patient information and
counseling. The Statement recognizes that there are diverse approaches to the delivery of patient services. Expected detection
rates and false-positive rates of the various first and second trimester tests, alone and in combination, are also provided.
1. Ultrasound nuchal translucency at 11-13 completed weeks combined with serum markers at 10-13 weeks.
2. Extending (1) to include other first trimester sonographic markers, provided performance has been prospectively validated by
the ultrasound center where the screening is to be performed.
3. A ‘contingent’ test whereby women with borderline risks from (1) have (2) at a specialist center and risk is subsequently
modified.
4. Four maternal serum markers (quadruple test) at 15-19 weeks, for women who first attend after 13 weeks 6 days.
5. Combining (1) and (4) in either a stepwise or contingent protocol - provided that all screening test data is included in the final
risk assessment. Integrated screening can be offered when CVS is not available.
6. Contingent second trimester ultrasound to modify risks for aneuploidy (sometimes referred to as the ‘anomaly scan’ or ‘genetic
sonogram’) for women having (1), (4) or (5). Performance must be prospectively validated by the ultrasound center where the
screening is performed.
The International Statement should be useful to policy makers in those countries that may be developing aneuploidy screening pro-
grams or looking to update their programs. For the United States, the document is helpful because existing guidelines from ACOG
and ACMG , although recognizing the value of screening, do not provide any guidance as to which test should be done, and when.
As well as setting minimum standards, guideline documents can be an aid in securing funding or adequate reimbursement, particu-
larly for the newer medical technologies.
Looking ahead, it will be important to keep the Statement updated; obsolete guidance can do more harm than good. We are at a time
when there will be rapid expansion in the scope of prenatal screening and diagnosis. Developing guidelines in other areas can help
patients, keep us focused on the medically necessary tests, maintain an ethical position, and ensure the highest technical standards.
Hopefully, the Aneuploidy Statement will be a model.
The development of the Statement also provided a valuable opportunity for the committee members to appreciate each other’s inter-
national perspectives and opinions. I thank the members for these very valuable insights and thanks also to ISPD for making this
possible.
Peter Benn, Chair, ISPD Guidelines Committee for Aneuploidy Screening (benn@nso1.uchc.edu)
Many years ago, just before she which is nothing but sequentially
was about to leave for a long using one marker to make a first
flight from the UK to China, an cut to select out a definitely
obstetrician friend of mine ectopic pregnancy group and in
collapsed whilst scanning a patient in the hospital. She knew it was the remainder using a second marker to select another group etc etc
very serious because they were transfusing her with blood products and doing the same to select out uterine pregnancy groups. The
that were cold – too urgent for even the shortest delay. It was an results are poor – for example the best combination was inhibin A,
ectopic pregnancy. Presentation is not always as dramatic or life progesterone and VEGF which detected 49% of ectopic and 41% of
threatening but even when abdominal pain and other symptoms lead uterine pregnancies. But they claim high sensitivity (98%) and
to an earlier diagnosis tubular damage may already have been done specificity (95%) with just one ectopic and two uterine pregnancies
with consequent reduced fertility - although this is a chicken and being misdiagnosed. They are clearly not using these terms in the
egg (ho-ho) situation and not easy to unravel the cause and effect. same way as we would in the screening field. Very misleading!!!
At any rate there is a need, both in those at increased risk and also So the question is still open as to whether some combination of
generally, for an early screening method, apart from ultrasound. serum markers could efficiently identify those at high risk of extra-
Various serum markers have been reported and recently a group in uterine pregnancies.
Philadelphia tested 12 of the most promising on sera from 200
The search for markers continues in Philadelphia. Beer et al. (J
symptomatic women – 100 with ectopic and 100 uterine
Proteome Res 2011;10(3):1126-38) used a number of proteomic
pregnancies (Rausch et al., Obstet Gynecol 2011;117(3):573-82).
methods to identify 70 candidate markers which they whittled down
ROC analysis indicated that five were reasonably discriminatory
to 12 biomarkers for further study, including ADAM12 and five
with area under the curve exceeding 0.7: inhibin A, progesterone,
isoforms of SP1. The ADAM12 finding was subsequently been
activin A, VEGF and SP1.
confirmed in another series (Rausch et al., Fertil Steril 2011;95
The authors then tried to combine markers using a method which (4):1373-8).
shows that they are not from the screening world. They used
something called Classification and Regression Trees (CART), Howard Cuckle (hsc2121@columbia.edu)

Correlation of NT between P u b l i sh i n g
twin fetuses prospective
When NT is measured in twins the studies of
recommended approach is to consider D o w n ’ s
each fetus like a singleton and calculate a
fetus-specific risk from the NT MoM based on the fetal CRL. s yn d r om e
However, the NT measurements in the two fetuses are not screening
independent with published correlation coefficients: 0.34
(Wøjdemann et al, Prenat Diagn 2006;26(3):218-20), 0.45
At least 25 large
(Cuckle and Maymon, Prenat Diagn 2010;30(9):827-33), 0.43
(Maymon et al, Prenat Diagn 2011;31:Epub online) and 0.34 prospective studies
(Wright et al, Prenat Diagn 2011;31:16-21). Therefore, the of second trimester serum screening, Double, triple
Down’s syndrome risk in an individual fetus is dependent on or Quad, and at least 20 prospective studies of first
both the specific NT and that of the co-twin. trimester screening, NT alone or Combined, have
An algorithm has been published to calculate the fetal been published. Consequently, it is now difficult
specific risk from an NT and CRL (Cuckle and Maymon, 2010). to publish further studies using these protocols
There are four computational steps: (1) prior probability of unless the screened population is extremely large
Down’s syndrome in fetus 1 only, fetus 2 only or both fetuses
according to maternal age and zygocity; (2) likelihood ratios of
or special in some way. Nevertheless a small
discordant Down’s syndrome in fetus 1 only, fetus 2 only or study may be a useful baseline for further
concordant Down’s syndrome, from the two NT MoMs; (3) developments in a specific country. To overcome
proportion of dizygotic (DX) and monozygotic (DZ) twins given this problem Prenatal Screening Perspectives is
chorionicity, gender, whether assisted reproduction or willing to include such study reports from IPSG
spontaneous, maternal age and ethnicity; and (4) multiply the members, and in the current issue we have a report
prior probabilities by likelihood ratios and take the weighted from Mutaz Akkawi and his colleagues from Al-
average using the zygocity distribution. The weights are
Quds and Birzeit Universities on screening in the
crucial: all monochorionic twins are MZ and all dichorionic
unlike gender twins are DZ but the zygocity distribution
West Bank (see page 36)
changes markedly with age since MZ incidence is not age
related whilst DZ incidence increases rapidly with age.
A risk calculator based on the Cuckle and Maymon
algorithm is available online at www.screeninfo.co.uk.
Another algorithm has recently been published using REPEAT MEASURES
the mixture model (Wright et al, 2011). They made the REPEAT MEASURES
simplifying assumption that all dichorionic twin are DZ (sic) and
used a mixture model rather than the simple Gaussian model REPEAT MEASURES
for the NT distribution. Whilst one or other of these
Based on SURUSS statistical parameter estimates,
approaches is necessary to provide a correct risk for the
Wright and Bradbury (BJOG 2005;112:80–3) pointed out
individual women (both demonstrate with examples clinically that that first and second trimester measures of the same
important differences in risk compared with the naïve serum analyte, notably PAPP-A, could potentially be highly
assumption of independence between the fetuses), like so effective in Down syndrome screening. Following up on this,
many refinements in aneuploidy screening it will not have a Wright et al (Health Technology Assessment 2010;14:No.
large effect on detection rates. Maymon et al, (2011) have 33) have carried out an evaluation of the strategy using
estimated the magnitude of the effect using modelling. For FASTER samples and additional cases from North York
example, at 11 weeks gestation using NT as the single marker, Hospital, Canada. Disappointingly, the addition of repeat
measures resulted in poorer screening performance when
the predicted detection rate for a 5% false-positive rate is 78%
the risks were calculated using an algorithm based on the
without taking account of the correlation between fetuses and SURUSS statistical parameters.
82% when this is allowed for using their algorithm. In these The authors point out that their affected patient
calculations a ‘positive’ was taken to be where at least one of samples showed a set of statistical parameters that departed
the fetuses has a risk exceeding the cut-off. Wright et al, substantially from the SURUSS estimates and that
(2011) applied their model directly to the dichorionic twins determinants of SURUSS covariance matrices were
studied (5530 unaffected and 65 with Down’s syndrome) and unrealistically small. Parameters based on pooled affected
found no effect on the detection rate although it is and unaffected pregnancies actually provided better
underpowered to prove an effect. screening performance.
When a set of statistical parameters was developed
When maternal serum markers are also used in from the FASTER and North York pooled affected and
addition to NT the appropriate likelihood ratio for twins (see unaffected cases, repeat measures of PAPP-A was shown to
page 21) can then be applied to each fetal specific risk be beneficial if the first sample was drawn at 11 weeks. A
calculated from the above algorithms. prospective study would be needed to confirm this.

Rhodos-Amsterdam; As always, the congress was well-cared for. The
dinner and party at the harbour of Rhodos-city
the 2010 FMF and ISPD were a delight, as was the visit to the ancient city
congresses of Lindos.
The 2010 (9th) world congress on 15th International

fetal medicine was held in Conference on Prenatal
Rhodes, Greece from June 20- Diagnosis and Therapy,
24. As a first-timer I needed to adapt to the typical
Amsterdam (Netherlands),
world congress-schedules, implicating that the
programme would automatically push lunch to late July 11-14 2010.
afternoon and dinner to late-at-night. This was all The fact that this years ISPD
to no surprise for the experienced visitors, that are congress was situated in
used to the extensive discussions on every paper, Amsterdam was particularly welcomed by my head
usually moderated by Prof. Kypros Nicolaides of department; my travel-expenses were only 35
himself. For the readers of PSP, especially the euros, enough to travel the 40 km between
session on screening for fetal aneuploidies was of Maarsen, where I live, and Amsterdam-RAI
interest. There was a number of presentations on railway station for a couple of days.
the quality of the NT measurement and the quality
assurance of the screening test in general. Dave The 2010-congress was preceded by a number of
Wright presented data on an exceptionally large
courses, organized by the Special Interest Groups,
series of twin pregnancies, presenting detection
rates and showing differences in serum marker that were established at the Vancouver-meeting
levels between monochorionic and dichorionic (2008). Tak Yeung Lueng was chairman of the
pregnancies. A number of new sonographic and course entitled ‘Advances, refinements and the
biochemical markers for aneuploidies was future of maternal screening’. Mark Evans lectured
presented as well. Wendy Koster modeled new on QC and QA for NT and other ultrasound and
and common markers to show elevated DR for serum markers. Wendy Koster reported on new
aneuploidies. Moreover, there were two serum markers for aneuploidy and demonstrated
presentations on high-end detection proteomic increased screening performance after modeling
expeditions for yet-undiscovered aneuploidy these new markers. Prof Richard Choy presented
markers. on the diagnosis of rare disorders identified
through screening. Howard Cuckle gave an update
Prediction of fetal and maternal health become
more and more prominent themes-the FMF on the screening of pre-eclampsia and Dave Wright
congress hosted sessions on the predictability of lectured on repeat measures and highly correlated
preterm birth, early diagnosis of fetal defects, as markers. Finally, Prof Lueng himself gave a
well as pre-eclampsia. There is growing evidence summary on the possibilities of non-invasive
that with the proper combination of maternal prenatal diagnosis being able to replace screening.
history, serum markers and sonographic markers,
DR pushing 90 % for a 5-10 % FPR may be In the main conference, this theme was also
achievable to detect pre-eclamptic pregnancies. extensively covered by e.g. the lecture of Dennis
Especially in the domain of biochemical markers, Lo and others. The course was attended by about
proteomics techniques may be a suitable tool to 50 congregates, and as judged from the course
identify useful markers.
evaluation and post-course comments, very well
The award for most presentations in this congress received. During the post-course debate, future
went to Julien Stirneman, who appeared no less themes of interest were identified, among which
than five times. were standards for risk estimation and a position
paper on aneuploidy screening.
A number of the presentations of the 9th FMF
congress are published on the website of the FMF In the main event, among numerous interesting
( h t tp :/ / w w w .f et a l me d i c ine . co m /f mf /o n l in e - presentations, especially the controversies, in
education/07-lectures-in-fetal-medicine/). Amongst which two contesters defended opposing views on
others, you will find papers on non-invasive a specific theme, were interesting and fascinating.
diagnosis of aneuploidies (Diana Bianchi), Fetal Thus, Christine Mummery and Magnus Westgren
DNA in maternal blood (Lyn Chitty) and intra-
battled on the theme; ‘is stem cell therapy ready for
uterine growth restriction (Gerard Visser).
human fetuses’ and Beryl Benacerraf and Sicha

Yagel explored the pros and cons of cell-free Population Screening for
nucleic acids in maternal blood versus ultrasound Spinal Muscular Atrophy
in prenatal diagnosis. The-Hung Bui and Lisa
Shaffer debated whether conventional analysis Spinal muscular atrophy (SMA) is a
(karyotyping and FISH) is really necessary in the relative common fetal neuromuscular
genetic disorder caused by the
post array CGH. degeneration of motor neurons in the
anterior horn of the spinal cord. SMA is prevalent world-wide,
Dennis Lo, in his lecture, gave an overview of the with an estimated incidence of 1:6,000 to 1:10,000 and a
progress on the use of fetal DNA and RNA for carrier frequency of 1/38 to 1/50 (Feldkotter et al, 2002,
Ongio et al, 2004). About 93% of childhood SMA patients
non-invasive diagnosis of Down syndrome. While have a homozygous deletion of exon 7 of the survival motor
the tests are currently still very expensive, and the neuron 1 (SMN1) gene. The remaining patients carry a
experimental procedures tedious and vulnerable, deletion on one allele, and an intragenic mutation on the
other allele. Given the severity of this disease, the relatively
Lo was quite confident that fetal DNA and RNA high carrier frequency rate, and the current lack of effective
will replace conventional invasive tests in the treatment, population-based carrier screening for SMA may
future. Whether it would also be a screening tool be justified. Indeed the American College of Medical Genetics
(ACMG) recently recommended that genetic SMA carrier
however, is less certain. Finally the growing screening should be made available to all US couples.
interest for the role of placental microRNAs and Conversely the American College of Obstetricians and
cellular mRNA in e.g. pre-eclampsia was a novelty Gynecologists (ACOG) stated in their Committee Opinion
on this years programme. (2009) that "preconception and prenatal screening for SMA is
not recommended in the general population at this time".
Almost all presentations can be found on the We have recently performed a study on large-scale
websit e of t he ISPD ( link: ht t p:// population based screening for SMA (Ben-Shachar et al,
www.ispdhome.org/pres2010.asp). Genetics in Medicine, in press). The study included 6394
Israeli individuals without family history of SMA, who
underwent screening by multiplex ligation-dependent probe
Peter Schielen (peter.schielen@rivm.nl) amplification (MLPA), designed to detect SMN1 exon 7 copy
number. A total of 159 individuals were found to carry a
SMN1 heterozygous exon 7 deletion for a carrier frequency of
Trisomy 21 origin 1:40. About 10.8% of individuals were found to carry 2 or
more SMN1 exon 7 copies on the same chromosome (cis
configuration). This implies that some deletion carriers may
A study involving fluorescence not be detected by MLPA or similar quantitative methods.
in situ hybridization (FISH) with two Given this limitation and the fact that some individuals have a
chromosome 21 probes hybridized to point mutation that cannot be detected by MLPA, we estimate
the ovarian cells from eight 14-22 week that SMA screening has a detection rate of about 90%. In
fetuses indicated that all were low level addition, we analyzed the acceptance level of this newly
mosaics(1). The percentage of cells introduced SMA screening test among women undergoing
with an extra chromosome 21 was low testing for fragile X syndrome and cystic fibrosis. During the
(average 0.54%) but the use of two probes should result study period, the acceptance rate increased from 77% to 99%
in a very low false-positive rate. The finding has within 29 months, with an overall acceptance rate of 93%
important implications in understanding the origins of during the study period. Given the severity of SMA, the
Down syndrome. It had been widely accepted that the relatively high carrier frequency and the estimated detection
extra chromosome 21 arose as a meiotic non-disjunction rate, we may conclude that SMA population based screening
is feasible and acceptable.
event which would only occur once female meiosis
completed i.e. at the time of ovulation and fertilization.
Shay Ben-Shachar (shayb@tasmc.health.il), Yuval Yaron
The authors suggested that higher levels of trisomy 21 (yyaron@tasmc.health.gov.il)
ovarian mosaicism may be present in some females
placing them at higher risk for a Down syndrome References
pregnancy or a higher risk of recurrence. 1) Feldkotter M, Schwarzer V, Wirth R, Wienker TF, Wirth B.
In a follow-up study (2), the same group Quantitative analyses of SMN1 and SMN2 based on real-time
observed no such trisomy 21 cells in the germ cells from lightCycler PCR: fast and highly reliable carrier testing and
4 male fetuses. They have also suggested that trisomy prediction of severity of spinal muscular atrophy. Am J Hum
21 germ cells may lag in maturation (relative to those Genet 2002;70:358-68. 2) Ogino S, Wilson RB, Gold B.
with disomy) accounting for the maternal age effect (3). New insights on the evolution of the SMN1 and SMN2 region:
Also, the trisomy 21 germ cells could be at a selective simulation and meta-analysis for allele and haplotype
advantage during the normal attrition processes that frequency calculations. Eur J Hum Genet 2004;12:1015-23.
3) Spinal Muscular Atrophy. ACOG committee opinion,
reduce the number of germ cells present in females
No.432. Americam College of Obstetricians and
during development.
Gynecologist. Obstet Gynecol 2009;113:1194-6. 4) Ben-
Shachar S, Orr-Urtreger A, Bardugo E, Shomrat R, Yaron Y.
1. Hultén et al, Molec Cytogenetics 2008;1:21. Large scale population screening for spinal muscular
2. Hultén et al, Molec Cytogenetics 2010;3:4. atrophy. Genet in Med, in press.
3. Hultén et al, Reproduction 2010;139:1-9.

Serum the MoMs (except for AFP), risks, and interpretation are all not
graded ‘educational’ challenges and there is no penalty for poor
Tests performance. The range of risks reported by different laboratories
Quality for a second trimester quadruple test sample will typically cover
several orders of magnitude (3). In part, this could reflect matrix
Control effects associated with the use of artificial proficiency test media
rather than actual patient sera. But it is hard to explain why there
Considerable attention has is so much variability in MoMs for peer groups using the same
been paid to the quality control and method.
quality assurance of NT measurement.
Surprisingly, at least in the US, much A voluntary first trimester proficiency test is also
less attention is paid to the accuracy of available, the First Trimester Inter-laboratory Comparison
serum marker test results. Program (FTICP), from Women an Infants Hospital, Providence,
RI (4). Proficiency in establishing NT MoMs from a set of raw
In the US, the assays used for serum markers for measurement data is stressed. There are currently approximately
aneuploidy screening are not approved by the Food and Drug 26 participating laboratories (including manufacturer’s
Administration (FDA) and, although some are actively marketed development labs and overseas participants) with 18 producing
for screening, manufacturers of kits make no claims about their sequential risk estimates. Again, there is considerable
suitability. It is up to individual laboratories to validate the tests heterogeneity in results generated by the participating labs. Some
but there are no specific requirements for the components to this US labs may participate in State proficiency programs (e.g. New
validation (1). For validation of a new test format, it is common York) or other external programs (e.g. UK’s National External
to demonstrate a high correlation of concentrations with an Quality Assessment Service (NEQAS)).
established form of the test. But such studies usually do not fully
establish the suitability of tests for prenatal screening because Most prenatal screening laboratories will run their tests
they generally do not include a substantial series of affected in batches with acceptance of the run based on the performance of
pregnancies, evaluate the data in MoMs, and validate all statistical quality control materials that have well established expected
parameters. values. High quality laboratories will pay considerable attention
to this and use a variety of rules to evaluate their control data. A
For example, a recent report by Lambert-Messerlian et commonly used set of criteria has been defined by Westgard (5,6)
al (2) evaluated the new Beckman Access® PAPPA assay by and, correctly used, these can provide an early warning of testing
comparing it to Perkin Elmer’s AutoDELFIA PAPPA. Although problems. Because test errors are compounded in multiple marker
concentrations for a series of 194 normal pregnancies were very testing (7), it is very important that all tests are highly
highly correlated, there was a small but significant systematic reproducible. Overall detection rates and false-positive rates are
difference in the assays identifiable through a Bland-Altman plot rather insensitive to differences in test performance but individual
of the concentrations. The authors noted that another pair of patient risk figures can be substantially changed (8).
PAPPA assays also showed biases relative to each other and this
resulted in a considerable difference in the affected pregnancy A further unfortunate practical reality is that, for economic
median MoM for the two assays. For different assay formats, reasons, a screening program may be forced to accept a test
there also appears to be a very wide range in the estimates of the format that is compatible with other areas of the laboratories’
PAPPA standard deviation for normal pregnancies and it seems automated activities rather than use testing that is judged most
reasonable to think that the performance of the various available well suited to aneuploidy risk assessment [see insert box]. There
assays could contribute to this. Some PAPPA assays may be very are core clinical chemistry laboratory directors who may be
reproducible at higher concentrations but much poorer for lower unaware or, worse, unwilling to provide the high level of quality
values. Therefore, any added variance associated with a particular control oversight needed in prenatal screening or they may
methodology will not necessarily be the same for affected and compromise the quality control evaluation rules to make the
unaffected pregnancies. Similar considerations apply to all prenatal screening tests compatible with other areas. There is
markers and equivalent performance should not be assumed for little purpose in ensuring that NT medians are being measured
the various available manufacturers’ formats. Most labs currently within narrowly defined criteria (for example median MoM 0.9-
use the published parameter sets in their screening algorithm 1.1) when the results are being combined with batches of serum
without considering the possibility that their assay performance marker results that may have been accepted based on a
might not in fact match. substantially lower standard.
Nearly all laboratories in the US receive oversight from Perhaps the strongest force for quality in the US is actual or,
the College of Pathologists (CAP). CAP uses a peer-review fear of, litigation. But this is not always a positive influence
process for on-site inspections with requirements defined in because it often requires conformity to common practice
checklists. The checklists contain few specifics relating to standards. As noted above, these practices may be mediocre or
maternal serum screening although a recent revision (June, 2010) lack solid evidence-based validation. It can also be extremely
introduced requirements for NT measurement quality assurance, costly.
and new lot verification. There are some requirements for test
requisitions, establishing medians, reporting, and quality Peter Benn (benn@nso1.uchc.edu)
assurance through monitoring median MoMs but no specific
requirements for the analytes most suitable for use, quality control 1. Palomaki et al Genet Med 2009;11:669-81. 2. Lambert-
materials or test run acceptance criteria, validation of test Messerlian et al, J Med screen 2010;17:109-13. 3. CAP.
statistical parameters, appropriate covariable adjustments, etc. Maternal Serum Survey Summary Reports. Palomaki et al, Arch
The inspection process is part of CAP’s broader ‘special Pathol Lab Med. 2010;134:1685-91. 4. Westgard et al, Clin
chemistry’ category and generally the inspectors are not Chem 1981;27:493-501. 5. Westgard. www.westgard.com/
specialists in prenatal screening. westgard-rules-and-multirules.htm. 6. Benn & Collins. Ann
Clin Biochem, 2001;38:28-36. Benn et al, Clin Chem 2006;
CAP also provides second trimester four serum marker 52:2087-94.
proficiency testing through the distribution of test materials but

Laboratory X: Provides first and second trimester screening in a core chemistry laboratory setting. Each day, before running patient
samples, it runs 2-4 quality control materials per marker that have CVs (sd/mean, expressed as %) up to 8%. Similar to other chemistry
testing provided using the same instrumentation, the laboratory recalibrates the instrument if a control result departs by 3 sd, or more, from
its mean. It also has a policy that if two controls (either from consecutive days, or at different concentration levels on the same day) are 2-3
sd out, the instrument is recalibrated. If one control is 2-3 sd out, the control is repeated and if it is within range, the laboratory proceeds
with the run.
Comment: Due to regression to the mean, the repeat test of a single control that is initially 2-3 sd out would nearly always be within the
normal range- even if there had been a small systematic change in performance. By routinely repeating single controls and accepting the
second, in-range result, Lab X would not see instances where, on consecutive days, two controls were out by 2-3 sd. Furthermore, instances
where two controls at different concentration levels are 2-3 sd out would be very rare because it requires the two combinations of random
and systematic error to fall between a narrow interval and systematic changes are rarely proportionate across concentrations. Therefore,
when Lab X had systematic errors, these would often not come to attention until the combination of systematic and random errors exceeded 3
sd (which could be 24%, or more) at a particular control level.
Laboratory Y: Provided first and second trimester screening in a specialized genetics laboratory setting. It initially selected assays based on
their reproducibility with all control CVs 3-5%. It used similar control materials to Lab Y but applied multiple Westgard rules when
evaluating the run performance (5,6). If one control was 2-3 sd out, it served as a warning for extra caution before releasing patient results
and selected cases with borderline risks would receive repeat testing. The lab reviewed their overall test statistics and compared them to
those within the algorithm, carefully reviewed median MoMs, screen-positive rates and also collected pregnancy outcome information.
Comment: Due to the added quality control and quality assurance, Lab Y’s costs (but not reimbursements) were somewhat higher than Lab
X and Lab Y was therefore considered to be inefficient.
Healthy carrier – Not strictly

Trisomy 13 and trisomy 18 true
When DNA screening for recessive
Based on the data from nine prenatal and postnatal conditions is offered to individuals or
congenital anomaly registers, curves have now been couples there is an almost implicit
developed for maternal age specific prevalence of assumption that the information on carrier
trisomy 13 and trisomy 18 (Saava et al; Prenat Diagn status has no direct health implications for
2010;30:67-64). The registries included 975 diagnoses the screenee in addition to any past or
of trisomy 13 and 2,254 cases of trisomy 18. future offspring. Indeed the term ‘healthy
Prevalence of trisomy 13 = 1/{1 + exp(9.53-(3.25/(1+exp carrier’ sometimes appears in the patient
(-0.332*(age-37.5)))} information leaflets. However, this can be
and misleading.
Prevalence of trisomy 18 = 1/{1 + exp(9.11-(4.27/(1+exp
(-0.324*(age-38.9)))} One way in which an apparent carrier might be medically
An earlier publication provided values for the expected affected is if they are not actually a carrier but a
fetal loss rates Morris and Saava (Am J Med Genet Part compound heterozygote. This happens with CF where
A. 2008;146A;827-32). From 12 weeks to term an someone is a proven carrier for one of the multiplexed
estimated 49% of trisomy 13 and 72% for trisomy 18 series of relatively common mutations sought in the
pregnancies end in misscarrage or stillbirth, and, from 18 screening test, but they are also a carrier of a rare and
weeks the corresponding rates are 42% and 65%, possibly mild mutation. This combination may not
respectively. present clinically with typical CF but they may have mild
symptoms.
Trisomy 16 Duchenne/Becker Muscular Dystrophy is not recessive
but X-linked and about 10% of female carriers are
Pregnancies with trisomy 16 mosaicism usually show symptomatic. This is largely due to ‘skewed’ X
second trimester elevated MSAFP, grossly elevated inactivation favouring the mutated dystrophin gene.
hCG and INH-A and low/normal uE3 (Neiwanger et al; There seems to be a lack of consensus over whether
Prenat Diagn 2006;26:454-61; Benn 2010 In Genetic these women need long term heart monitoring.
Disorders of the Fetus; 6thEd. Ed Milunsky & Milunsky).
But, so far, there has been little information available With Fragile X syndrome, which is neither recessive not
about first trimester markers. X-linked but hereditably unstable, female carriers are at
risk of premature ovarian failure. Males would not
Two reports involving 4 cases with apparently confined normally be screened but prenatal diagnosis in
placental trisomy 16 mosaicism all had very low PAPPA pregnancies to carrier women may reveal a carrier male.
(Verwoerd-Dikkeboom et al, Fertil Steril 2008; Such individuals are at increased risk of spinocerebellar
90;2017.e19-2017.e22; Petracchi et al Prenat Diagn ataxia in later life.
2009;29;1175-6). Surprisingly, only one case had
elevated free beta hCG. NT was unremarkable. In short those offering screening may need to make
patients aware, before testing, that their carrier status
will not necessarily be benign.
Does anyone have more cases?

of early placentation. Therefore, Dr Wortelboer described the
Three brand new PhD from the relationship between biomarkers of early placental function and
Netherlands (and one on the way)! fetal growth in PGDM pregnancies. She also investigated the
relationship between six first-trimester markers and macrosomia at
Drs Mirjam Fransen, Wendy Koster and Esther Wortelboer birth (together with Sylwia Kuc-elsewhere in this issue). Although
defended their theses recently at the Utrecht and Rotterdam the association between fetal macrosomia and PGDM in pregnancy
University (the Netherlands). Prof. Gerard Visser and Howard is widely recognized, little is known about the role of placental
Cuckle were the supervisors of Wendy’s thesis and Gerard Visser function in determining macrosomia. In particular, the contribution
was the supervisor of Esther’s as well. Prof. Mackenbach was the of early placentation is unknown. The present increase in fetal
supervisor of Mirjam Fransen. Finally, Marleen Schoonen here macrosomia might be related to a better glycemic control around
presents the outline of her thesis, to be defended at Rotterdam conception and during the first trimester of pregnancy and to a
University. better early placentation. Macrosomia at birth in PGDM
pregnancies may be predicted by normal levels of PAPP-A,
All theses are in English and are published as quite beautifully laid- ADAM12, PP13 and PlGF already in the first-trimester of
out, handsome booklets. Together, they comprise some 25 papers pregnancy. While the relationship between various markers and
recently published in the scientific literature. If you are interested, their associations with different fetal and maternal disease states is
please order a copy by sending an email to wendy.koster@rivm.nl, increasingly clear, we still experienced a lack of information on the
e.wortelboer@umcutrecht.nl or m.p.fransen@amc.uva.nl. normal, physiological behaviour of these markers, and their
various interrelationships. That forced Dr Wortelboer to go back to
Screening in early pregnancy; more than Down the very basic work of investigating the physiological
syndrome alone-Esther Wortelboer concentrations and values of these markers in early pregnancy.
Therefore, she performed a longitudinal study into the levels of
The aim of Dr. Wortelboers thesis was 1) How can we improve the serum markers (PAPP-A, PlGF, ADAM12, fβ hCG) and maternal
performance of the screening in the Netherlands?2) Can we screen blood pressure and the pulsatility index of the uterine artery, and
for more than just Down syndrome? Should the screening test for thus to provide a baseline of normal values for future investigation
Down syndrome be adapted to detect other pregnancy and risk assessment.
complications in early first-trimester, and 3) With respect to
questions 1) and 2): what biochemical and sonographic markers In the discussion to her thesis Esther presented a new view on the
can be applied and what is their physiology? Dutch prenatal screening, based on the new insights of her work
The work summarizes the history of the Dutch Down syndrome and heavily inspired on Kypros Nicolaides’ concept of turning the
screening (starting with the triple test in the 90’s and the first pyramid of prenatal care upside down’. For an overview, see figure
trimester combined test since 2002. It deals with the initial 1.
difficulties of the first trimester programme (the quality of the NT
measurement) and the challenges ahead. In the second part of her Figure 1: Flowchart prenatal care (from: thesis Esther Wortelboer)
thesis she investigates the capacities of markers to improve the DS
screening, in retrospective cohorts of the serumbank of the
screening laboratory of the Dutch National Institute of Public
Health and the Environment. She describes the efficacy of
ADAM12, PP13 and PlGF as markers for T21, T18 and T13,
showing that ADAM12 works well for DS very early in
pregnancy. PP13 is not a good DS marker but works as a marker
for T13 and T18. The latter is an interesting finding in light of the
planned introduction of T13/18 screening in the
Netherlands,January 2011. Both in the Netherlands and abroad
however, there is growing awareness that the first-trimester test
can also be put to use for a broader screening for foetal and
maternal health, including trisomy 13 and 18, preeclampsia,
premature birth, growth restriction, fetal death and pregnancy-
related diabetes. Such a screening offers the possibility of early
risk selection and therapeutic options to improve maternal and fetal
health. Identifying pregnant women at risk for preeclampsia is still
one of the most important challenges in prenatal care, since
preeclampsia is a serious complication of pregnancy that affects
approximately 1-2% of all pregnant women worldwide.
Identification of women at risk, as early as the first-trimester of
pregnancy, enables intensified antenatal surveillance
As a first step, Dr Wortelboer investigated the predictive value of
maternal serum PAPP-A, fβ-hCG, ADAM12, PP13 and PlGF, for
first-trimester identification of early-onset preeclampsia, again in
cohorts of the above laboratory. It was demonstrated that lower
maternal serum first-trimester PP13 and PlGF levels are associated
with preeclampsia (/HELLP syndrome). Possibly, combined with
other variables such as blood pressure, uterine artery Doppler flow
velocity waveforms and maternal history, they may have some
value in the prediction of early preeclampsia in a low risk
population. Therefore, addition of PP13 to the current screening
test could be valuable to make a proper distinction between
normal, aneuploid and preeclamptic pregnancies.
Fetal growth and birth weight in pre-gestational type 1 and type 2

diabetes mellitus (PGDM) pregnancies is partly related to markers

Innovations in Down syndrome screening- Ethnic differences in prenatal screening for
Wendy Koster Down syndrome-Mirjam Fransen
Dr. Koster was tremendously productive in only 3.5 years, Objective
publishing 11 papers as a first author. Her thesis covers some The main aim of the research was to assess ethnic variations in
of the very basic issues in prenatal screening but she ends pregnant women’s decision-making on participation in prenatal
with DS markers no one has ever heard of. screening for Down syndrome in the Netherlands.
Methods
To start with the basics, Dr. Koster showed that the current Pregnant women from Dutch, Turkish and Surinamese ethnic
screening for Down syndrome in the Netherlands can be origin were recruited from community midwifery practices in
improved by accurately measuring the crown-rump length of Rotterdam and the outpatient clinic of Erasmus MC. In total 270
the foetus for the determination of gestational age, by women were personally interviewed in the language they
improving the quality of nuchal translucency (NT) preferred, a mean of 3 weeks after booking for prenatal care. We
measurements through monitoring and training of assessed source and type of received information, knowledge on
sonographers and by drawing the serum sample early in the Down syndrome and prenatal screening, attitude towards
first trimester (before 11 weeks). participating in prenatal screening, and considerations whether or
Secondly, she determined that by adapting the screening not to participate in prenatal screening. Actual (non-)
algorithm for trisomy 13 and 18, it is possible, specifically participation was assessed several months after the interview. In
calculated for the population of the reference laboratory, to addition, 57 midwives filled in a web-based questionnaire to
identify approximately 80% of foetuses with trisomy 18 or assess ethnic variations in providing information on prenatal
trisomy 13, with only 0.2% extra false positive test results. screening. In order to assess ethnic difference in participation of
Wendy investigated the concentrations of the common and the prenatal screening programme in a larger study population,
some new (ADAM-12, PP13) biochemical markers in a quite we performed a register-based study in 2009, in the Southwest
large series of monochorionic and dichorionic twin of the Netherlands.
pregnancies. None of the marker concentrations showed a Results
difference between monochorionic and dichorionic twins. Most women reported that the midwife is the prime source of
Remarkably, median ADAM-12 and PP13 were only about 1,5 information about prenatal screening for Down syndrome, and
times the MoM in singleton pregnancies. A review of the that most of them received oral and/or written information at
available literature is ambiguous on this issue. A recent paper booking for prenatal care. However, women from Turkish and
of Wright et al shows that especially early in pregnancy the Surinamese ethnic origin had significantly less knowledge about
biomarker concentrations in monochorionic twins are lower. Down syndrome and prenatal screening than Dutch women. In
In addition to the work on early sampling, Wendy showed total 5% of the women from Turkish origin, 26% of the women
that, to increase the Down syndrome screening performance, from Surinamese origin and 71% of the women from Dutch
it would be advisable to draw two serum samples during the origin made an informed decision whether or not to participate in
first trimester; one sample before 11 weeks to measure PAPP- prenatal screening. Differences in language skills and educational
A and one sample after 11 weeks to measure fβ-hCG. At level contributed most to these variations.
these points in time, the biochemical screening markers are Midwives reported that they do not always offer information
most distinctive between Down syndrome and unaffected when pregnant women from a non-Western ethnic background
pregnancies. Logistically it would be best to draw the second hardly speak and understand Dutch. Only a few midwives
serum sample at the time of the NT measurement. reported to use translated materials or professional interpreters in
Next, Wendy showed that adding potential biochemical case of language barriers.
screening markers, such as ADAM12, PP13, PlGF and thCG, to The register-based study showed that the overall participation in
the first-trimester combined test increases the detection rate the prenatal screening programme was 26%. Compared to Dutch
for aneuploidies, but also allows screening for other women, those from Turkish, Moroccan, Aruban/Antillean and
pregnancy complications. Given the potential of new other non-Western ethnic origin were less likely to participate in
biomarkers for DS screening, and given that proteomics the prenatal screening programme. The differences between
techniques are now available for the identification of new women from Dutch origin versus women from Moroccan and
biochemical screening markers for Down syndrome, Wendy Turkish origin remained significant after adjustment for socio-
set out on a quest for undiscovered markers. She found EGF, economic background and age. No significant differences were
EN-RAGE and CA19-9 to be promising screening markers, found between women from Surinamese and Dutch ethnic origin.
showing concomitantly that initial experiments produce Discussion
impressive numbers of new biomarker candidates, but the The ethnic variations we found in pregnant women’s knowledge,
majority does not survive additional selection in follow-up decision-making and uptake of prenatal screening demonstrate
validation experiments. EGF, EN-RAGE and CA19-9 will be that the goal of the prenatal screening programme to enable all
tested in large-scale validation experiments now. Bead-based pregnant women to make an informed decision whether or not to
multiplexed immunoassays and antibody microarrays are participate in prenatal screening for Down syndrome is certainly
high-throughput platforms that allow for multiple marker not achieved, and indicate that there are ethnic differences in
analyses. Therefore, these techniques can be used for Down access and quality of prenatal screening. The interventions to
syndrome screening purposes in the future. Wendy showed improve access and quality of prenatal screening require efforts
that in principle, both analytical platforms can be put to use by the Dutch government, the Central Agency of prenatal
for high throughput screening. Wendy’s thesis also concluded screening, regional prenatal screening centres and healthcare
with a five-year view, suggesting a screening test that will professionals.
focus on several foetal abnormalities and maternal Acknowledgements
complications using a broad panel of biochemical markers, The work presented in the thesis was conducted at Erasmus MC,
which can be analyzed simultaneously, in one screening test. in a collaboration between the department of Public Health and
the Department of Obstetrics and Gynaecology, and supervised
by Dr. M.L.Essink-Bot, Dr. H.I.J. Wildschut, Prof.dr. J.P.
Mackenbach and Prof. dr. E.A.P. Steegers.

Informed decision-making about prenatal screening- individual risk estimation of carrying a child with Down’s
Marleen Schoonen syndrome, and may be followed by diagnostic testing to
confirm whether or not the fetus is affected4, 15.
Schoonen, H.M.H.J.D., Wildschut, H.J., Essink-Bot, M.L., We determined the content of relevant knowledge
Steegers, E.A.P., de Koning, H.J. needed to make an informed decision about (non-)
Schoonen, Marleen, MSc. (Corresponding Author) participation in prenatal screening for Down’s syndrome, in
Department of Public Health, Erasmus MC, University order to develop a knowledge questionnaire to be used for
Medical Center Rotterdam, Room AE-206, P.O Box 2040, large scale evaluations of informed decision-making. A
3000 CA Rotterdam, The Netherlands. Telephone: 31-10-463 generic list of content domains for knowledge about
8460 / 31-10-704 3732 / Fax: 31-10-703 8474 / E-mail: screening was extracted from the literature. Items reflecting
h.schoonen@erasmusmc.nl specific knowledge domains were constructed. An expert
Wildschut, Hajo, MD PhD. Department of Obstetrics and group of professionals and pregnant women expressed
Gynaecology, Division of Obstetrics and Prenatal Medicine, whether domains and items represented decision- relevant
Erasmus MC, University Medical Center Rotterdam, information.
Rotterdam, The Netherlands All presented domains were scored as (very)
Essink-Bot, Marie-Louise, MD PhD. Associate Professor, important. Options when receiving an ‘increased probability
Department of Social Medicine, Academic Medical Center, for Down’s syndrome test result, the meaning of this result,
Amsterdam, The Netherlands the aim of the screening, and voluntary nature of the test were
Steegers, Eric, MD PhD. Professor, Department of Obstetrics scored as most important. The condition being screened for,
and Gynaecology, Division of Obstetrics and Prenatal prevalence, and the screening procedure were scored as
Medicine, Erasmus MC, University Medical Center relatively less important, with a high amount of expert
Rotterdam, Rotterdam, The Netherlands consensus16.
de Koning, Harry, MD PhD. Professor, Department of Public
Health, Erasmus MC, University Medical Center Rotterdam, What knowledge is relevant for informed decision-making
Rotterdam, The Netherlands about the fetal anomaly scan?
Second trimester screening for fetal abnormalities and
Introduction first trimester screening for Down’s syndrome have one goal
First trimester prenatal screening for Down’s in common; the detection of fetal anomalies. However, these
syndrome aims at informing pregnant women about the screening programs are quite different entities and have
likelihood of having a Down’s syndrome-affected child in a different characteristics; Down’s syndrome screening – with
timely manner, in order to allow them the opportunity to act, its (seemingly) straightforward purpose – and 20- week
that is prepare for the birth of a child with Down’s syndrome screening with its myriad of possible syndromes, its soft
or termination of the pregnancy if the condition is diagnosed1. markers and its assessment of structures other than the fetus,
The Dutch prenatal screening programme, organised in its such as the placenta and the umbilical cord. In addition to
present form since 2007, makes use of the combined test to these different technical aspects and test-characteristics of
estimate the individual probability of being pregnant of a second trimester screening for fetal abnormalities, compared
child with Down’s syndrome. with first trimester screening for Down’s syndrome, women
Second trimester ultrasound screening for fetal attribute different goals to these screening programs and
anomalies (or fetal anomaly scan) primarily aims at the perceive them substantially differently as well17. Pregnant
detection of structural malformations when termination of women often wish to have ultrasounds for non-medical
pregnancy is still legal2. In the Netherlands, as in most other reasons such as seeing the baby, making the pregnancy seem
Western countries, second trimester ultrasound screening for more real, and knowing the sex of the baby 17-19. Hence,
fetal anomalies has become a standard part of prenatal women expect ultrasound to be a positive and pleasant event2,
5, 17, 19
care3,4, 5. . In contrast, pregnant women show ambivalence
The Dutch prenatal screening programme is towards screening for Down’s syndrome20. They associate
characterised by an active, standard information offer to participating in prenatal screening for Down’s syndrome with
every pregnant woman6, 7. The goal of this information offer abortion and often cite unwillingness to have an abortion as a
is informed decision-making. Facilitating informed choice on reason for not participating in the screening program21. To
participation is a fundamental part of international guidelines summarize, it seems that first trimester screening for Down’s
on prenatal screening8-13. An informed choice is based on syndrome is perceived by pregnant women as being primarily
relevant knowledge. In addition, actual behaviour (in this focused on the detection of ‘abnormalities’, whereas the
case, participating or non-participating in the prenatal second trimester ultrasound is perceived as aiming to confirm
screening programme) should be consistent with decision- ‘normality’17, 19
maker’s attitudes14. Similar to determining decision-relevant knowledge
about prenatal screening for Down’s syndrome, we also
What knowledge is relevant for informed decision-making determined the content of relevant knowledge needed for
about prenatal screening for Down’s syndrome? informed decision-making in second trimester ultrasound
Many Western countries have guidelines or screening for fetal anomalies. In addition, we compared the
recommendations for prenatal screening for Down’s content of decision-relevant knowledge for second trimester
syndrome, in which either women of advanced age are ultrasound screening with the content of decision-relevant
offered invasive diagnostic testing (chorionic villus sampling knowledge for first trimester screening for Down’s syndrome
or amniocentesis), or in which women, irrespective of their using the combined test.
age, are offered non-invasive risk-assessment tests in the first The meaning of an abnormal test result, the
trimester pregnancy (nuchal scan, often combined with disorders being screened for, and the purpose of the screening
maternal biochemistry). The latter screening tests provide an were regarded as very important for informed decision-

making about the fetal anomaly scan, along with the 7. Health Council of the Netherlands. Population screening
voluntary nature of the test. All knowledge domains were act: inition of a national programme for prenatal screening:
included in the final questionnaire. The relationship between Down's syndrome and neural tube defects [Wet
the ranking of the domains according to relevance for first Bevolkingsonderzoek: aanzet voor een landelijk programma
trimester and second trimester screening was 0.71 (Pearson’s voor prenatale screening]. 2006;2006/03.
r). The domain ‘consequences of a positive test result’ 8. General Medical Council. Seeking patients' consent: the
received a higher expert ranking in first trimester screening ethical considerations. London: GMC; 1999.
than in second trimester screening22. 9. World Health Organization. Statement of WHO expert
advisory group on ethical issues in medical genetics. ; 1998.
Evaluating the information provision process about 10. American College of Obstetricians and Gynecologists.
prenatal screening for Down’s syndrome, and about Screening for fetal chromosomal abnormalities. Washington
ultrasound screening for fetal anomalies (DC): American College of Obstetricians and Gynecologists;
Informed decision-making about prenatal screening 2007.
for Down’s syndrome is considered an indicator of adequate 11. Joint Working Party of the Royal College of Obstetricians
information provision. Currently, we investigate the process and Gynaecologists and the Royal College of Paediatrics and
of providing information about prenatal screening for Down’s Child Health. Fetal abnormalities- guidelines for screening,
syndrome. In addition, we investigate whether the diagnosis and management: report of a joint working party of
information provision is adequate, using informed decision- the Royal College of Obstetricians and Gynaecologists and
making as an indicator. Regarding the process of providing the Royal College of Paediatrics and Child Health. RCOG
information, questions we address are: Are all pregnant Press 1997;.
women offered information about the screening programme? 12. Royal College of Obstetricians and Gynaecologist.
How many women accept the information offer and, of these Amniocentesis and chorionic villus sampling. London: ;
women, how many actually receive the information? 2005.
Furthermore, we address the following questions 13. National Screening Committee. Second report of the UK
regarding the quality of information provision: What is the National Screening Committee. London: Department of
knowledge on Down’s syndrome (screening), what are the Health; 2000.
attitudes, are pregnant women willing to participate in the 14. Marteau TM, Dormandy E, Michie S. A measure of
screening programme and what are their reasons to (not) informed choice. Health Expect 2001;4(2):99-108.
participate? To what extent is decision-making regarding 15. EUROCAT. Special Report: Prenatal Screening Policies
participation in prenatal screening for Down’s syndrome in Europe. EUROCAT Publications on Prenatal Diagnosis;
based on an informed decision? What are the determinants of 2010.
informed decision-making? In a separate study, the same 16. Schoonen HM, van Agt HM, Essink-Bot ML, Wildschut
questions are addressed for prenatal screening with the fetal HI, Steegers EA, de Koning HJ. Informed decision-making
anomaly scan, and compared with the results for prenatal in prenatal screening for Down's syndrome: What knowledge
screening for Down’s syndrome. is relevant?. Patient Educ Couns 2010; Aug 25;.
Data are obtained in an open, unselected population 17. Santalahti P, Aro AR, Hemminki E, Helenius H,
in the Southwest region of the Netherlands, from pregnant Ryynanen M. On what grounds do women participate in
women (both participants as well as non-participants in the prenatal screening?. Prenat Diagn 1998;18(2):153-65.
screening) and from midwives. Results of these studies are 18. Stephens MB, Montefalcon R, Lane DA. The maternal
expected soon. perspective on prenatal ultrasound. J Fam Pract 2000;49
(7):601-4.
19. Ahman A, Runestam K, Sarkadi A. Did I really want to
References know this? Pregnant women's reaction to detection of a soft
1. Health Council of the Netherlands: Committee Genetic marker during ultrasound screening. Patient Educ Couns
screening. Genetic Screening 1994;22. 2010;81(1):87-93.
2. Bricker L, Garcia J, Henderson J, Mugford M, Neilson J, 20. Vassy C. How prenatal diagnosis became acceptable in
Roberts T, et al. Ultrasound screening in pregnancy: a France. Trends Biotechnol 2005;23(5):246-9.
systematic review of the clinical effectiveness, cost- 21. Green JM, Hewison J, Bekker HL, Bryant LD, Cuckle
effectiveness and women's views. Health Technol Assess HS. Psychosocial aspects of genetic screening of pregnant
2000;4(16):i,vi, 1-193. women and newborns: a systematic review. Health Technol
3. A brief history of the 20-week ultrasound. Available at: Assess 2004;8(33):iii, ix,x, 1-109.
http://www.rivm.nl/pns_en/down_ultrasound/history/ . 22. Schoonen HMHJD, Essink-Bot ML, van Agt HME,
Accessed 03/05, 2010. Wildschut HI, Steegers EAP, de Koning HJ. Informed
4. Boyd PA, Devigan C, Khoshnood B, Loane M, Garne E, decision-making about the fetal anomaly scan: What
Dolk H, et al. Survey of prenatal screening policies in knowledge is relevant. Ultrasound Obstet Gynecol In Press;.
Europe for structural malformations and chromosome
anomalies, and their impact on detection and termination
rates for neural tube defects and Down's syndrome. BJOG
2008;115(6):689-96.
5. Garcia J, Bricker L, Henderson J, Martin MA, Mugford M,
Nielson J, et al. Women's views of pregnancy ultrasound: a
systematic review. Birth 2002;29(4):225-50.
6. Health Council of the Netherlands. Prenatal Screening (2);
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Netherlands 2004;2004/06.

the second trimester of pregnancy between 14 weeks and 20
Maternal serum screening weeks of pregnancy. It is ordered to help evaluate the risk
in the West Bank that a fetus has certain abnormalities, including Down’s
syndrome and NTDs.7, 8 The Triple test measures the levels
of three markers in the serum of the pregnant women: Alpha-
An audit of Palestinian and non-Palestinian women having fetoprotein (α-FP), Human Chorionic Gonadotropin (hCG) and
Triple tests - Mutaz Akkawi, Rula Ghandour, Abdullatif unconjugated Estriol (uE3). 9 When Inhibin A is added to
Husseini, Lamia Halasseh, Qasem Abo Remeleh these three biochemical markers, the
detection rate of the test increases by 10% to about 75% and
is called the Quadruple test. 10 Increased or decreased levels
Abstract of the three serum markers can suggest the type of
abnormality present. 11
Objectives The aim of this second phase of study was to A number of important factors can influence the results
detect or confirm any change in the screening results of Triple of the Triple test. These include: maternal age, race, weight,
tests carried out on Palestinian women living in the West Bank number of fetuses and gestational age. 3, 12 Women who
during the period 2004-2010 if compared to that done have a positive screening test result for Down’s syndrome are
between 2000-2003. usually referred for genetic counseling and further testing.
Methods Data on 1354 pregnant Palestinian and non- In this article we describe the results of Triple tests
Palestinian women living in the West Bank (Filipino women) carried out on Palestinian and non-Palestinian women who
who were offered the Triple test during the period 2004-2010 came to perform this test in different areas of the West Bank
were analyzed statistically using the Statistical Package for during the period 2004-2010.
Social Sciences (SPSS) version 17
Results In this study, out of 1354 subjects, 1267 were eligible Subjects and materials
for the final analysis. The mean expected age at delivery ± SE
(standard error) of the Palestinian women was 27.02±0.17 Laboratory methods
years compared with 25.5 ±0.2 years in the first study. The All tests were performed in Zer laboratories in Jerusalem
AFP results of the Palestinian women tested using AFP MoM utilizing standardized techniques for analysis. Both AFP and
cutoff of 2.5 showed that 2.7% had high concentrations hCG were performed using immunoradiometric assay (IRMA).
compared with only 1.3% in the first study. Filipino women AFP kit was produced by Byk-Santgtec Diagnostica-Germany,
showed the same result for AFP. Our study showed that while the hCG kit was produced by ICN-USA. For uE3 the
women with high risk of Down’s syndrome were 5% compared radioimmunoassay (RIA) kit used was a product of DSL-USA.
with 2.8% in the first phase of study. ALPHA, which is special software for risk calculation was
Conclusion Our study showed that within about six years utilized in this study and produced by Logical Medical
there is increase in both the percentage of women with high Systems, UK. The cut-off point utilized in this analysis was
AFP results and with those with high risk of Down’s syndrome 1:380 at term risks.
which becomes similar to that obtained in the international
community. This may be due to direct result of the increase in Variables used in the study
the mean age of women. The following variables were used: age at expected date of
delivery, weight in kilograms, gestational age in weeks, in vitro
Introduction fertilization (IVF) status, twins status, AFP levels in IU/mL and
multiples of the normal gestation-specific median (MOM),
Antenatal testing describes procedures performed during hCG in IU/mL and MOM, uE3 in ng/mL and MOM. The
pregnancy to detect health problems in the growing fetus. variables were used to calculated risk for age alone and for
These tests can provide a non-invasive screening for Down’s the Triple test.
syndrome (trisomy 21) and other congenital anomalies such
as spina bifida and anencephaly (neural tube defects – NTDs) Statistical analysis
through the assessment of maternal serum markers and All data were entered into Statistical Package for Social
ultrasonography .1, 2 Sciences (SPSS) version 17. Data cleaning was conducted
Down’s syndrome (trisomy 21) is the most common and outliers were excluded, those included 29 twins
cause of severe learning disability in children with an pregnancies, 5 IVF, 1 unknown origin, and 52 Africans. A total
incidence of 1 in 800 live births .3 The incidence of Down’s 1267 cases were included in the final analysis. Basic
syndrome in some Arab countries exceeds 1.7 per 1000 descriptive statistics including means, medians, modes,
compared to incidences of 1.0-1.4 per 1000 for other Arab standard deviation, and standard error were calculated.
populations .4 As a result of this high incidence, screening Frequencies were also calculated for relevant variables. Cross
and diagnostic tests should be offered. In our community the tabulations and Chi square test were used to explore the
importance of such tests has been recognized and the first relation between different variables.
local study on this subject was published in 2005. 5
Screening tests for Down’s syndrome are either done in
the first or second trimester. 3, 6 First trimester test is usually Results
ordered between 11-13 weeks of pregnancy. This test
measures the levels of two serum markers, free β-human Characteristics of the subjects
chorionic gonadotropin (free β-hCG) and Pregnancy A total 1267 cases were investigated of which 88.4 % (1120)
Associated Plasma Protein-A (PAPP-A) in addition to a were Palestinian Arabs residing in the West Bank, while
special ultrasound scan that measures the nuchal 11.6% (147) were non-Arabs (Filipinos). The mean expected
translucency (NT) thickness, a space at the back of the baby’s age at delivery ± SE (standard error) of all subjects was
neck .7 These tests cannot evaluate the fetus's risk of 27.54±0.17 years while it was 27.02±0.17 years for
developing NTDs. First trimester screening is not common in Palestinian women and 31.47 ±0.45 years for Filipino women.
the Palestinian community and efforts are exerted to 11.8% (150) of the participants were 35 years old or above.
encourage women to do it in the near future. The most The mean weight for women at the time of testing was 65.79 ±
common test done by Palestinian pregnant women is the 0.34 kilograms. The range of gestational age when the test
Triple test. 5 was performed was 15 to 21 weeks, with a mean of 17.12
The Triple and Quadruple tests are used to screen in ±0.02.

Table 2: Median serum concentration (MoM) and mean log10 for
the 3 markers of the triple test *
Individual
Table 1: Mean serum concentrations of the test results Arabs Non Arabs
three markers of the triple test. (Palestinians) (Filipinos)
N=1120 N=147
Marker
Concentration Median MoM Mean log10 Median MoM Mean log10
(Mean ± SE (Interquartile MoM(SD) (Interquartile MoM(SD)
Characteristics Range) Range)
Arabs Non Arabs
(Palestinians) (Filipinos) 1.00 1.16
N=1120 N=147 AFP 0.01 (0.22) 0.08 (0.20)
(0.76-1.29) (0.91-1.48)
AFP 40.88±0.59 52.01±1.97 0.99 1.04
hCG -0.01 (0.29) 0.02 (0.25)
(0.65-1.46) (0.73-1.52)
hCG 27.78±0.66 32.37±1.8
1.06 1.28
uE3 1.27±0.07 1.50±0.05 uE3 0.02 (0.16) 0.09 (0.16)
(0.84-1.31) (1.01-1.60)
* SD= standard deviation
Table 3: Triple test results for 1267 women and calculated risk by age group
and origin
Calculate
d Risk
Elevated Elevated Reduc Reduc
(%)* P-value
Age (years) AFP hCG ed hCG ed uE3
(age+ (Risk)
(%) (%) (%) (%) Discussion
triple
test) Palestinian pregnant women started showing
interest in screening for Down’s syndrome in
15-24
n=448
2.5 3.6 0.9 0.2 2.2 the late 1990s. This is the second study
performed in the Palestinian community in the
Age 25-34 last decade. In the period between 2004-2010,
2.5 2.7 1.2 0.0 3.7
Arabs Group n=561 0.000 a group of Palestinian pregnant women were
>35 screened for Down’s syndrome using the Triple
4.5 2.7 0.0 0.0 22.5
n=111 test.
Total(N=1120) 2.7 3.0 1.0 0.0 5.0 Neural tube defects (NTDs) are birth defects of
the brain or spinal cord. In Jordan where a
15-24 large number of Palestinians present for
0.0 0.0 0.0 0.0 0.0
n=17
medical care - the NTD incidence was
Age 25-34 estimated to be 1.1 per 1000 births. 13
Filipino Group n=91 3.3 3.3 1.1 0.0 2.2
0.001 Screening for NTDs using maternal serum
s
>35 alpha-fetoprotein is used to identify women at
2.6 5.1 0.0 0.0 17.9 increase risk of carrying of fetus with an
n=39
NTD.14, 15 The most commonly used AFP
Total (N=147) 2.7 3.4 0.7 0.0 6.1
MoM cutoff in the United States is 2.5 MoM,
yielding initial screen-positive rate of 1% to 3%.
3 In this study, the AFP results of the
Palestinian women tested using AFP MoM cutoff of 2.5 showed that 2.7% had high concentrations.
Filipino women showed the same result for AFP. These two results were in agreement with the initial screen-positive rate
given for pregnant women in the United States.
The AFP results of this study were higher than those shown for Palestinian women in our first study published in 2005. 5 in
which the proportion with high levels was 1.3% among a population of 943.
The Triple test was projected to detect about 60% of the Down’s syndrome pregnancies by identifying 5% of all pregnant
women as having a screen-positive test result, performance based on pregnancies being dated via LMP. 3 Our study showed that the
total with high risk of Down’s syndrome was 5%.
Comparing this result with that published in 2005, 5 where the proportion with high risk was 2.8%, there was a noticeable
increase in this percentage. This may be due at least in part to difference in the mean expected age at delivery ± SE (standard error)
which was 27.02±0.17 years compared with 25.5 ±0.2 years in the first study. This study showed also that for Filipino women the
proportion with high risk of Down’s syndrome was 6.1%. This higher value could also be attributed to age factor where the mean
expected age at delivery of the Filipinos women sample was 31.47±0.45 years.
When comparing age groups (< 35 and ≥35) with risk, it was found that there was almost 7 folds increase in risk and it was
statistically significant (P<0.00). This association stayed strong after controlling for ethnic origin (7 folds for Palestinian women and 9
folds for Filipinos).
When comparing the risk percentages for Down’s syndrome of the total sample tested of both the Palestinian and Filipinos, there was
no statistically significant difference in risk between them.
Acknowledgment
We are grateful to Prof. Howard Cuckle for his helpful discussions and insightful comments.
Mutaz Akkawi (makkawi2002@yahoo.com)

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PRENATAL SCREENING PERSPECTIVES

Editor: Phillipa Bloom
Leeds Screening Centre, Gemini Park,

Sheepscar Way, Leeds LS7 3JB, UK
Tel: +44 (0)113 284 9230 Fax: +44 (0)113 262 1658 Email: info@ipsgroup.org.uk

Prenatal Screening Perspectives
Newsletter for the International Prenatal Screening Group
www.ipsgroup.org.uk

© 2011 PerkinElmer, Inc. 2011. All trademarks or registered trademarks are the property of PerkinElmer, Inc. and/or its subsidiaries.
SCREENING
FOR EARLY Come and discuss with us
PRE-ECLAMPSIA at IPSG in Barcelona!
For use on the DELFIA® Xpress platform, DELFIA PlGF is the

first placental growth factor (PlGF) assay for early pre-eclampsia
screening. Ideal as part of routine first-trimester testing, the assay
provides results suitable for use with Prof. Howard Cuckle’s risk
calculation service.
DELFIA Xpress PlGF The DELFIA Xpress PlGF assay provides the performance benefits
assay now available! of the DELFIA chemistry, and complements the full range of first
and second trimester aneuploidy screening assays already available
for DELFIA Xpress.
The DELFIA Xpress instrument and DELFIA Xpress screening assays are not available in the USA.
www.perkinelmer.com

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Prenatal

Volume 16, 2011

* IPSG 2011, Barcelona

Newsletter of the International Prenatal Screening Group

Tel: +44(0)113 284 9230

Page 1 PSP 2011 VOL 16

MEETINGS & COURSES SOFTWARE

METHODOLOGY & POLICY ISSUES ULTRASOUND

Prenatal Screening Perspectives is sponsored by

International Prenatal Screening Group

Title: _________________ Name: ____________________________________________________________

Page 3 PSP 2011 VOL 16

The National Center for Genomic Research

PSP 2011 VOL 16 Page 4

Peter Benn (benn@nso1.uchc.edu)

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Preliminary program (28 March 2011)

PSP 2011 VOL 16 Page 6

Loughna et al, Ultrasound 2009;17(3):161-7. Howard Cuckle (hsc2121@columbia.edu)

Page 7 PSP 2011 VOL 16

PSP 2011 VOL 16 Page 8

Screening ACMG ACOG Comments

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Based on sequencing, of newborns with CF (after

PSP 2011 VOL 16 Page 10

Agegroup n fb-hCG MoM PAPP-A MoM NT MoM

<30 30-34 35-39 ≥40

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M.P.H. Koster, E.J. Wortelboer, P.C.J.I. Schielen (peter.schielen@rivm.nl)

PSP 2011 VOL 16 Page 12

Completion: Late 2013.

Health Technology Assessment (HTA)

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Head, Antenatal Screening, Women’s and Children’s Hospital,

PSP 2011 VOL 16 Page 14

For an overview of the available data on this see Benn P.

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Lo et al (7) have further shown the extraordinary potential ********

PSP 2011 VOL 16 Page 16

1. Benn PA, Chapman AR. JAMA 2009;301:2154-6.

To some degree this can be reduced by the use of new

Page 17 PSP 2011 VOL 16

PSP 2011 VOL 16 Page 18

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provide a detection rate (DR) of 85-90% at a false 22 D S +

is 70-75% at a 3.5% FPR, which is relatively low

PSP 2011 VOL 16 Page 20

Page 21 PSP 2011 VOL 16

PSP 2011 VOL 16 Page 22

“This PhD thesis is based on five studies conducted in

Page 23 PSP 2011 VOL 16

The biannual International Conference is well attended.

The newsletter also lists upcoming courses (three in the

PSP 2011 VOL 16 Page 24

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PSP 2011 VOL 16 Page 26

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The 2010 (9th) world congress on 15th International

PSP 2011 VOL 16 Page 28

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Title: _ Name: ____________________________________________