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
ADVERSE PREGNANCY OUTCOMES
Danish thesis defense .................................................................................. 23
Macrosomia in diabetic pregnancies - prediction .......................................... 19
Paternal age ................................................................................................... 23
Pyramids inverted or otherwise ...................................................................... 18
Screening for ectopic pregnancy – potential markers ........................... 26
Screening in early pregnancy ........................................................................ 32
DNA/RNA
Free fetal DNA - recent developments ........................................................... 16
Non-invasive prenatal diagnosis - ethics ........................................................ 16
DOWN’S SYNDROME
Phenotype and micro RNA ............................................................................. 8
EPIDEMIOLOGY & AETIOLOGY
Trisomy 21 origin – germ cells ........................................................................ 29
IPSG
9th IPSG Congress. Preliminary program ...................................................... 6
9th IPSG Congress & ISPD Education Day ................................................... 6
Anne M Summers (1954-2009) ...................................................................... 10
Barcelona link ..................................................................................... 24
Co-ordination ...................................................................................... 15
Editorial .............................................................................................. 3
Membership form ................................................................................ 3
PhDs from the Netherlands ........................................................................... 32
Position statement ......................................................................................... 26
Publishing honour for Aubrey Milunsky ......................................................... 12
Robert Cocciolone (1957-2010) ...................................................................... 14
INFORMATION
Bibliography - 219 references .............................................................. 39
KNOWLEDGE & ATTITUDES
Healthy carrier - not strictly true .................................................................... 31
Informed decision-making - prenatal screening ............................................. 34
MEETINGS & COURSES
World Congress on MFM, Rhodes June 2010 .............................................. 28
15th ISPD Conference, Amsterdam July 2010 ............................................. 28
METHODOLOGY & POLICY ISSUES
First among equals ......................................................................................... 7
Innovations in DS screening ........................................................................... 33
International Society for Prenatal Diagnosis – new directions ............... 24
Second trimester combined testing ................................................................ 8
US professional guidelines - screening .......................................................... 9
NT & NASAL BONE
Semi-automated NT measurement - progress and caution .......................... 17
Prenatal Screening Perspectives is sponsored by
PSP 2011 VOL 16 PerkinElmer
Page 2
Page
OTHER ANEUPLOIDY
Sex chromosome trisomies ............................................................................. 4
Trisomies 13 and 18 - prevalence ................................................................... 31
Trisomy 16 - first trimester markers ................................................................ 31
OTHER DISORDERS
Cystic Fibrosis ................................................................................................. 10
Fragile X syndrome (FRAX) screening ............................................................ 15
Joubert syndrome - genes found .................................................................... 18
Mendelian disorders - universal carrier testing ............................................... 4
NTD prevention - randomised trial ................................................................. 12
Spina bifida - first trimester markers .............................................................. 18
Spinal Muscular Atrophy (SMA) population screening .................................. 29
PRENATAL DIAGNOSIS
CGH versus karyotyping .................................................................................. 7
PROSPECTIVE SCREENING & NATIONAL ACTIVITY
Self-reporting outcome - influence on detection rate ...................................... 20
Community Genetics Network ............................................................. 24
DS screening in Europe .................................................................................. 5
DS screening in the US ................................................................................... 5
DS screening in the UK ................................................................................... 5
Ethnic differences in DS screening ................................................................. 33
Maternal serum screening in the West Bank ................................................. 36
Publishing prospective studies of DS screening ............................................ 27
Spinal Muscular Atrophy (SMA) – prospective screening results .................. 4
QUALITY MANAGEMENT
Serum tests quality control ................................................................... 30
REFINEMENTS & ADJUSTMENTS
Anomalous marker values .............................................................................. 8
Chorionicity – importance for twin risks .......................................................... 21
Extreme results ............................................................................................... 9
Maternal age – affect on screening performance ........................................... 11
PIGF – Marker for aneuploidy and pre-eclampsia ................................ 25
Repeat measures ................................................................................ 27
SOFTWARE
Chromosome 21 balanced translocation - warning on risks .......................... 15
Correlation of NT between twin fetuses ......................................................... 27
ULTRASOUND
Fetal size and dating ....................................................................................... 7
Fetal Ductus Venosos - marker for cardiac defects ........................................ 25
Genetic sonogram – how to combine results .................................................. 17
Crown-rump length and gestational age ......................................................... 38
VARIOUS
Health Technology Assessment (HTA) - current activity .............................. 13
Re-inverting the wheel: again!.............................................................. 22
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

Page 3 PSP 2011 VOL 16


Sex
chromosome
trisomies
XXX, XXY and XXY
are not associated with
dysmorphic features and fetal loss but are
occasionally prenatally identified following screening.
A modest excess over that expected by chance is
expected because XXX and XXY are more frequent in
older mothers and maternal age is a component of
all aneuploidy screening protocols. Based on
EUROCAT databases, Boyd et al (European J Human
Genet 2010) concluded that only about 12% of the
cases expected to be present at birth are actually
prenatally identified. This is probably an over-
estimate because their estimate for the total
expected number of cases in the population was
based on rates established in older consecutive
newborn studies at which time maternal ages were
lower than they are today.
Among women aged <35 and receiving a
diagnosis of a sex chromosome trisomy, 41% chose
to terminate the pregnancy (50% for XXX, 40% for
XXY and 32% for XYY). Significantly less (33%)
women aged ≥35, terminated a sex chromosome
trisomy pregnancy (23% for XXX, 41% for XXY, and
28% for XYY).
Spinal Muscular Atrophy (SMA)
screening – Prospective results
A massive prospective evaluation of antenatal screening
for SMA has been completed in Taiwan (Su et al, PLoS
One 2011;6(2):e17067). A sequential approach was
taken using a method that identifies women with one
copy of the SMN1 gene, then testing their partners, only
offering invasive prenatal diagnosis to carrier couples.
Over 100,000 women were screened and 2,262 SMA
carriers were identified, an observed carrier frequency of
1 in 48. Among the 2,038 partners tested 47 were
carriers (1 in 43). Prenatal diagnosis was carried out in
43 couples, 12 affected fetuses were diagnosed and 11
pregnancies were terminated. The false-negative rate is
not known but carriers will be missed because of (1) two
copies of the SMN1 gene on one allele and none on the
other or (2) mutations rather than deletion of the SMN1
gene, and a proportion of cases are de novo.
Nevertheless, this is an impressive study and it might just
encourage some other countries to introduce routine
screening for this terrible disorder.
PSP 2011 VOL 16
Mendelian
disorders:
Universal carrier
testing
For $349, Counsyl Inc
now offers carrier testing for more
than 100 single gene disorders screening for over 500
mutations (www.counsyl.com). The panel includes sickle
cell disease, beta thalassemia, the nine disorders in the
ACMG recommended Ashkenazi Jewish panel, spinal
muscular atrophy, and coverage of 109 mutations in the
cystic fibrosis gene. The company claims >99.9%
sensitivity and specificity for the targeted mutations. The
testing is carried out on a saliva sample sent to Counsyl’s
laboratory and needs to be ordered through a clinician.
On-line patient reports contain residual risk of being a
carrier and the post-test risk for an affected child.
For some disorders, the analyses only detects a
small proportion of the mutations present in the
population and some of the disorders are extremely rare
so even when a carrier is identified, the risk of an affected
child is relatively small. Initial clinical data indicates that
as many as 35% of patients may be identified as carriers
for at least one disorder (Srinivasen et al, Reprod Biomed
Online, 2010;21(4):537-51). Tandem testing of both
parents at the same time is, therefore, optimal. Some of
the mutations will be of uncertain clinical significance in
compound heterozygotes. There will also be some
situations where the results could have potential clinical
significance for the individual being tested, for example,
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
to offer carrier screening for an even larger number of
recessive childhood diseases through next generation
sequencing technology. The project is being developed
in conjunction with the Beyond Batten Disease
Foundation. In an initial study, they sequence 437 genes
that represent 448 disorders with an average of 160-fold
target coverage (Bell et al, Science Translational
Medicine 12 January 2011 http://stm.sciencemag.org/
content/3/65/65/ra4.full). They report an approximately
95% detection rate for mutations in these genes. Based
on 104 unrelated individuals, they detected an average of
2.9 (range 0-7) recessive mutations per individual.
Although the cost of testing is projected to be low ($400
for large volume testing) the authors acknowledge that
there are considerable costs associated with
interpretation, reporting, genetic counseling, and data
management. It will be some considerable time before
there is sufficient information to interpret the clinical
significance of many of the mutations/polymorphisms that
will be identified but they still hope to make the testing
clinically available before the end of 2011 (News report.
Couzin-Frankel; Science 331;130-1).
Peter Benn (benn@nso1.uchc.edu)
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.

Peter Benn (benn@nso1.uchc.edu)

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).

Page 5 PSP 2011 VOL 16

 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

PSP 2011 VOL 16 Page 6

 CGH v Karyotyping
Comparative genomic hybridization (CGH) using
microarrays to identify copy number changes is now
recommended as the first-line test for the evaluation of
individuals with multiple anomalies not specific to a well
delineated syndrome, developmental delays, intellectual
disabilities, and autism spectrum disorders (1,2). The
indications for this testing also include better definition of
imbalances partially characterized through karyotyping.
The approach would also seem to already be appropriate
when there are ultrasound findings indicative of major
malformation (3). The preliminary results of a broad US
multicenter prospective study to evaluate CGH in prenatal
diagnosis indicates that additional abnormalities will be
identified but there are some significant challenges
associated with counseling when there is a copy number
variant of unknown significance (4,5).
Widespread use in prenatal diagnosis will involve
balancing the additional detection of significant
imbalances, the level of reassurance the testing provides,
turn around time, availability of resources and cost. It may
also require some new difficult decisions about what
information is worth diagnosing and/or communicating.
1) Miller et al, Am J Hum Genet 2010;86:749-64. 2)
Manning et al, Genet Med 2010;12:742-5. 3) Faas et al,
Prenat Diagn 2010;30:S27. Abstract 11-3. 4) Wapner &
Jackson. Prenat Diagn 2010;30:S3. Abstract 2-2. 5)
Wapner & Jackson. Prenat Diagn 2010;30:S31. Abstract
12-6.
Peter Benn (benn@nso1.uchc.edu)
Fetal size
and dating
The British Medical
Ultrasound Society
has issued a ‘Biochemical and ultrasound screening for Down's
recommended
conversion between
crown rump length
(CRL) measured at
6-13 weeks and gestational age (GA, days). In
conjunction with the Fetal Medicine Foundation and the
NHS National Screening Program, the conversion
formula has been adopted for prenatal aneuploidy
screening programs:
GA=8.052 x √(CRL x1.037) +23.72
The report also provides gestational age conversions for
13-25 week head circumference (BPD is not
recommended) and femur lengths. Estimating expected
fetal size (head circumference, abdominal
circumference, and femur length) when gestational age
is known can be determined from additional formulae
provided in this report.
Loughna et al, Ultrasound 2009;17(3):161-7.
Page 7
First among equals
I have recently been asked for the earliest reference to
suggest the ‘Combined’ test. I think Prenatal Screening
Perspectives readers will know that in the past I wasn’t a
big fan of naming tests Double, Triple, Quadruple and
Combined as the names might inhibit the development of
new 2, 3 or 4 marker protocols or other combinations of
serum and biochemical markers. Still, these shorthands
have proved useful and these days I use them myself.
I’m not a big fan of firsts either. Often in our field adding
one or more new marker to existing markers has been a
logical next step. The idea was in the air and anyone could
have been first. Even the great breakthroughs of science
have been characterised by this phenomenon. The real leap
forward has been conceptual developments like crunching
information from multiple markers into a single entity, the
risk. Of course, even conceptual leaps can be in the air.
Leibnitz and Newton simultaneously invented the calculus
while resident in different countries and without
knowledge of each others ideas on the subject.
Well back to the Combined test. The earliest reference I
can find is from an article I wrote in 1994 for the Royal
College of Obstetricians and Gynaecologists journal The
Diplomate (1(2):104-109) entitled ‘Multimarker Screening
for Down's Syndrome’. Having described the available
first trimester biochemical and ultrasound markers,
including NT, I said “Ideally both biochemical and
ultrasound markers could be combined but there is as yet
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
the College's Diploma in Obstetrics and Gynaecology
(DRCOG), but was also available for sale. By 1996 the
idea of combining was certainly established and
promoted in my ‘Opinion’ editorial piece for Ultrasound
in Obstetetrics and Gynecology (7:236-8) entitled
syndrome: rivals or partners?’. In this I said “A move to
earlier screening could provide the opportunity to plan a
more rational service based on the use of both serum and
ultrasound markers in combination. Much attention has
recently been paid to nuchal translucency (NT), which is
both a powerful first trimester ultrasound marker of
aneuploidy, and is largely uncorrelated with free β-hCG,
one of the most discriminatory biochemical markers. A
screening policy could be envisaged whereby the general
practitioner takes a blood sample at, say, 10 weeks
gestation and schedules an ultrasound examination for 11
weeks. The blood marker levels would be available at the
time of the scan and could readily be combined with the NT
and other ultrasound markers to derive the best estimate of
Down's syndrome risk.”
If anyone knows of an earlier reference please let us
know.
Howard Cuckle (hsc2121@columbia.edu)
PSP 2011 VOL 16
A n om a l o us m ar k er
values
Heterophilic antibodies (HAb)
are antibodies that can be present in
p a ti e nts’ se ru m tha t bi nd to
immunoglobulins from other species.
These can interfere with an immunoassay thereby giving an
erroneous value for a marker concentration.
HAb have been noted to cause incorrect measures of
hCG (1) and INH-A (2). A recent case report nicely
documents the same phenomenon with uE3 (3). A 36 year-
old women received “Integrated” screening with NT=1.69
MoM, PAPPA=0.41 MoM, AFP=0.79 MoM, hCG=3.2 MoM
and uE3 >5.0 MoM (but truncated to 2 MoM for risk
calculation purposes). Her term risk was calculated using
SURUSS statistical parameters and it was 1:1500 for all
markers but 1:3 when uE3 was omitted from the risk
assessment. The fetus was subsequently found to have a
47,XY,+21 karyotype.
There are simple methods to block HAb and this
would seem to be indicated when there is a single
unexplained extreme value. However, it is currently unclear
just how commonly this problem arises, whether there are
specific patients who are particularly likely to carry HAb, and
what criteria should be used to reflex to a blocking
technique.
…and anomalous risk calculation
In the example cited above, four markers together
with age suggested an increased risk (1:3) and yet a single
additional marker, uE3, was sufficient to drastically reduce
the risk (1:1500). Why did the uE3 result carry so much
weight that it negated all other information and provided a
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
correlation coefficients for unaffected pregnancies. The
observed full combination of results was unlikely for both an
affected and an unaffected pregnancy but the constraints
imposed by the affected pregnancy correlation coefficients
actually made this full set of results much more likely for an
unaffected pregnancy. . This effect becomes most evident
when we calculate a risk for an extremely unlikely set of test
results. As noted elsewhere in this issue of Perspectives
(article on Repeat Measures) the SURUSS standard
deviations and correlation coefficients do appear to overly
restrict the allowable multi-dimensional space occupied by
the affected and perhaps also the normal distributions.
As well as test interferences such as HAb, there are
multiple other reasons for highly atypical test result
combinations (for example, presence of an open neural
tube defect, pregnancy complications, genetic disorders
such as steroid sulfatase deficiency, etc) and in each of
these situations the Down syndrome risk algorithm will could
potentially provide misleading information. Ways to deal
with these relatively common situations would be to impose
truncation limits that are based on large disparities in the
paired results and to omit a specific marker or use
alternative correlation coefficients in such cases.
1.Butler and Cole. Clin Chem 2001;47:1332-3. 2. Lambert-
Messerlian et al, Cin Chem 2007;53:800-1. 3. Dennis et al,
Prenat Diagn. 2010;30:165-7.
Peter Benn (benn@nso1.uchc.edu)
PSP 2011 VOL 16
Second trimester combined testing
Although the world is rapidly moving to the first trimester
as the best time for aneuploidy screening we have to face
the fact that a sizable proportion of women do not contact
the medical services until the second trimester. Such
women are usually offered, at best, the Quad test but it is
inadequate. Surely we should improve detection for them
by combining the Quad markers with ultrasound markers?
There are lots of candidates: nuchal skinfold (NF), nasal
bone length (NBL), femur length (FL), humerus length
(HL) and various more subjective ‘soft’ markers – those
incorporated in the standard 18-20 week anomaly scan or
genetic sonogram.
The most discriminatory ultrasound marker is pre-nasal
thickness (PT). There have now been 5 published series
including a total of 105 Down syndrome cases (Maymon et
al, Prenat Diagn 2005;25:906-911; Maymon et al,
Ultrasound Obstet Gynecol 2006;27:290-295; Persico et
al, Ultrasound Obstet Gynecol 2008;32:751-754;
Maymon et al, Ultrasound Obstet Gynecol 2009;34:629-
633; Miguelez et al, J Ultrasound Med. 2010;29(12):1741-
7). In the combined data the median was 1.33 MoM with a
tight log10 standard deviation of 0.0772 in cases and 0.0752
in controls.
Modeling predicts that routinely adding PT to Quad
markers would increase the detection rate for a 5% false-
positive rate from 71% to 85% (Miguelez et al, 2010).
And adding two other ‘facial profile’ markers, NF and
NBL would increase it to 93%.
Howard Cuckle (hsc2121@columbia.com)
Chromosome 21 codes for approximately 500 genes
including five micro RNAs (miRNAs). miRNAs are post
transcriptional regulators that reduce expression levels.
The chromosome 21 miRNA could therefore have an
important role in determining the Down syndrome
phenotype.
Kuhn et al, (J Biol Chem 2010;285:1529-43) report that
the chromosome 21 miRNA are overexpressed in fetal
heart and brain from Down syndrome specimens
compared to normal. There are several thousand proteins
that could be affected but the authors focused on the
methyl-CpG-binding protein, MeCP2, which is mutated in
Rett syndrome. They noted reduced MeCP2 expression
and also aberrant expression of two genes, MEF2C/Mef2c
and CREB1/Creb1, that are affected by the reduced
expression of MeCP2. These genes are involved in
neuronal development.
There are obvious phenotypic differences between
Down syndrome and Rett syndrome and there are many
other transcripts that could be the target of the
chromosome 21 encoded miRNA. Nevertheless, the
possibility that aspects of trisomy 21 phenotype might be
caused by miRNA levels is of considerable importance
because there is a potential to develop therapeutics that
lower the miRNA levels to that present in disomic cells.
Peter Benn (benn@nso1.uchc.edu)
Page 8
 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

We often get enquiries from IPSG members to see if others
have any experience of pregnancy outcome when the
screening marker profile is extreme. If the pregnancy in
question is ongoing they would value a rapid response but
we don’t usually have time to broadcast IPSG for an
opinion. However, now that Prenatal Screening
Perspectives is an online publication there may be an
opportunity to do so in the future. Meanwhile, we’d still
like to hear from you about two recent cases:
Case1
This woman had a maternal serum AFP level of 160 MoM.
A repeat test (weeks later, albeit tested in a different
laboratory yielded a level of 18 MoM. MRI failed to find a
maternal cause in the adnexa, abdomen or mediastinum,
or a fetal cause. Targeted ultrasound of the placenta and
fetus was normal. A Kleihauer-Betke (KB) test, which might
indicate fetal-maternal transfusion was negative. Liver
function tests were normal. Currently, the pregnancy has
reached 36 weeks and fetal growth is normal.
Case 2
This 29 year old woman had first trimester serum screening
with a raised free β-hCG (2.29 MoM) and absent PAPP-A (ie
below the limit of detection for the assay). Both markers
were repeated and there was no change. The PAPP-A level
was checked again in another laboratory that uses an
entirely different type of antibody and again the level was
zero.
Later second trimester markers were measured: AFP 1.04
MoM, free β-hCG 2.32 MoM and uE3 1.19 MoM. PAPP-A
was also measured and again the level was zero. A genetic
sonogram (anomaly scan) at 18 weeks revealed no
abnormalities. Amniocentesis showed a normal karyotype.
The first trimester serum sample was later mixed (1:1) with
the first trimester serum from another woman who had
normal marker levels. The free β-hCG level was halved and
the PAPP-A level was zero. This implies the presence of an
interfering substance that blocks contact between
antibody and antigen in the PAPP-A assay. Currently, the
pregnancy is at 31 weeks gestation and everything appears
to be fine. A scheduled third trimester scan has not yet
been carried out.

Page 9 PSP 2011 VOL 16

 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
It is now nearly 10 years since ACMG/ACOG first
issued recommendations for population-based carrier
testing for cystic fibrosis (CF). Strom et al (Genet Med
2011;13;166-72) have published their experience for a large
reference laboratory that has provided nearly 3 million
carrier tests.
Using a 32-mutation panel, they estimate that they
detect 86% of the CF-causing mutations in the White
population, 88% for Ashkenazi Jewish, 75% for Hispanics,
63% for African Americans and 43% for Asian Americans.
The rate for the Ashkenazi Jewish population is somewhat
lower than expected (94%) while the rate for Hispanics
appears to slightly higher than expected (72%),
excluding common mutations), the authors estimate that the
use of an expanded CF panel with 97 mutations (instead of
the currently recommended 23 mutation panel) would only
increase the overall detection of newborns with CF by 0.7%.
Among patients with atypical or mild CF who
received sequencing, they found that 64% had no
detectable mutations, 10% had two mutations and 27% had
only a single detectable mutation. This latter result
suggests that some heterozygotes may show symptoms
perhaps due to modifier genes or environmental factors.
There also appear to be occasional cases of atypical CF
where there are two ACOG/ACMG panel mutations present
that would normally be expected to be associated with a
typical CF phenotype.
Peter Benn (benn@nso1.uchc.edu)

PSP 2011 VOL 16 Page 10

 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.

Page 11 PSP 2011 VOL 16

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.

PSP 2011 VOL 16 Page 12


Health Technology Assessment (HTA)
Here are some of the relevant UK HTA (www.hta.ac.uk)
projects started or completed in the last year:
Cervix cancer
Title: A modelled analysis using data from the ARTISTIC
trial of the potential effectiveness and cost-effectiveness of
HPV testing in cervical cancer screening.
Background: The HTA funded ARTISTIC trial studied
24,000 women aged 20-64 who underwent primary
cervical screening and were followed over 2 screening
rounds 3 years apart between 2001 and 2007. The trial
cohort was randomised to combined screening with liquid
based cytology (LBC) and human papillomavirus (HPV)
testing, or screening only with LBC (though HPV tests
were performed and the results concealed). The trial
found LBC to be as effective and less costly than LBC
combined with HPV testing for primary screening.
Furthermore when 3 scenarios were compared for costs,
both cytology triaged with HPV for low grade
abnormalities, and HPV primary testing triaged with
cytology were less costly than the current standard
strategy of repeat cytology for low grade abnormalities.
The costs of HPV testing with cytology triage is sensitive to
the number of colposcopies performed, in cytology
negative 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
strategies will begin with either cytology or HPV testing,
which would then be triaged by the other and will also
incorporate HPV genotyping as this, HPV-16 in particular,
could increase the specificity of HPV testing and be a
clinically valuable approach.
Completion: Late 2011
Recurrent miscarriage
Title: First trimester progesterone therapy in women with a
history of unexplained recurrent miscarriages: A
randomised, double-blind, placebo-controlled, multi-centre
trial [The PROMISE (PROgesterone in recurrent
MIScarriage) Trial].
Design: Women with a history of recurrent miscarriages
are referred to and seen in a recurrent miscarriage clinic
(standard practice), where appropriate baseline
investigations are performed (standard clinical practice). A
woman entering the trial is given instructions to notify the
research nurse by telephone as soon as she conceives (a
positive urinary pregnancy test). The research nurse will
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
Page 13
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.
Haemoglobinopathies (published Dormany et al, HTA
2010;14(20))
Title: Antenatal screening for haemoglobinopathies in
primary care: a cohort study and cluster randomised trial to
inform a simulation model. The Screening for
Haemoglobinopathies in First Trimester (SHIFT) trial.
Design: A population-based cohort study, cluster
randomised trial and refinement of a published decision
model. The units of randomisation were 25 general
practices in two inner city boroughs with a high proportion
of residents from minority ethnic groups. Practices were
allocated to screening in primary care with parallel father
testing (test offered to mother and father simultaneously;
n=8 clusters, 1010 participants); screening in primary care
with sequential father testing (test offered to father only if
mother identified as carrier; n=9 clusters, 792 participants);
and screening in secondary care with sequential father
testing (standard care; n = 8 clusters, 619 participants).
Conclusions: Offering antenatal screening for sickle cell
disease and thalassaemia as part of pregnancy-
confirmation consultations significantly increased the
proportion of women screened before 10 weeks, from 2%
in standard care to between 16% and 27% in primary care,
but additional resources may be required to implement
this. There was no evidence to support offering fathers
screening at the same time as women.
Gestational diabetes (published Waugh et al, HTA 2010;14
(45))
Title: Screening for hyperglycaemia in pregnancy: a rapid
update for the National Screening Committee.
Design: A systematic review and meta-analysis of the
literature updating a 2002 HTA report to include among
others data from the Australian Carbohydrate Intolerance
Study in Pregnant Women, the Maternal and Fetal
Medicine Units Network trial and the Hyperglycemia and
Adverse Pregnancy Outcomes (HAPO) study. Review data
on recent trends in maternal age at birth and on the
prevalence of overweight and obesity and the effect on
prevalence.
Conclusions: There is now good evidence for treatment
of oral drugs instead of insulin and it looks increasingly as
if fasting plasma glucose could be the test of choice.
However some key uncertainties remain to be resolved,
which can be done by further analysis of the already
collected HAPO data and by using the UK model used in
developing the NICE guidelines to assess the cost-
effectiveness of intervention in each of the seven HAPO
categories.
PSP 2011 VOL 16
 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

PSP 2011 VOL 16 Page 14

 Chromosome 21 balanced
translocation
Recently, I received an email from a
Genetics Department in the UK “We have
had several cases of carriers of
Robertsonian translocations involving 21
who have been given risks that do not reflect their a priori
10% risk. Their laboratory’s software does not cope with
this and has almost led to misdiagnosis in one case.”
Readers, check your software! Does it allow entry of
Down’s syndrome carrier status? If it only has the
possibility of entering 'previous non-inherited Down’s
syndrome' what would you do about a previous inherited
case?
Actually there are a number of situations related to family
history of Down’s syndrome which may or may not alter
the a priori risk, including:
(1) mother had previous standard Down’s syndrome
pregnancy
(2) father had previous standard Down’s syndrome
pregnancy
(3) previous de novo translocation Down’s syndrome
pregnancy
(4) previous translocation Down’s syndrome pregnancy,
mother balanced translocation carrier
(5) previous translocation Down’s syndrome pregnancy,
father balanced translocation carrier
(6) no previous Down’s syndrome pregnancy, but mother
known to be balanced translocation carrier
(7) no previous Down’s syndrome pregnancy, but father
known to be balanced translocation carrier
Risk software will definitely deal with (1) under 'previous
non-inherited Down’s syndrome' by using a published
formula, such as adding a fixed percentage to the
maternal age-specific risk expressed as a percentage.
Risk calculation for the others is tricky and will depend on
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
Amniocentesis. In: Genetic Disorders and the Fetus:
Diagnosis, Prevention and Treatment - 6th edition. (Ed A
Milunsky & JM Milunsky), Johns Hopkins University Press
Baltimore, 2010; pp 194-272. For example, with the
most common balanced Robertsonian translocation, rob
(14q21q), the Down’s syndrome (unbalanced
translocation) risk for a carrier mother is 15% and for a
carrier father only about 1%. With parental reciprocal
translocations, if the ascertainment was a term affected
pregnancy the risk of an unbalanced translocation is
several times higher for cases where the carrier status
resulted from an affected birth compared with other
reasons for testing such as recurrent miscarriage.
Howard Cuckle (hsc2121@columbia.edu)
Page 15
Fragile-X
Fragile-X syndrome in males with a full mutation is
common (about 1 in 4,000) and is a serious medical condition.
The disorder in females is variably expressed with an
estimated 1 in 5,000-8,000 individuals affected. The frequency
of premutation carrier females has been estimated to be 1 in
158 to 1 in 549 and these individuals are not only risk for
children with the full mutation, they themselves are at
increased premature ovarian failure. Both males and females
with the premutation, but especially males, are also at risk for
late onset tremor/ataxia syndrome (FXTAS).
ACOG have recently revised their opinion on carrier
screening (1). They now endorse screening for women with a
family history of a fragile-X related disorder, unexplained
mental retardation or developmental delay, autism, premature
ovarian insufficiency, unexplained elevated FSH prior to age
40, or if a patient requests the test. But counseling is
problematic because the screening will identify women with a
full mutation, individuals at risk for FXTAS, premutations of
uncertain risk for expansion, and the results can have complex
implications for additional family members who may be
carriers.
The possibility of providing the testing as part of
newborn screening has recently been reviewed by Hill et al (2)
with commentary by Coffee (3). Such screening could be
advantageous with early definitive diagnosis and potential for
early educational intervention. But the diagnosis of late-onset
FXTAS, increased risk for ovarian failure, and unintended
diagnosis of sex chromosome aneuploidy would still be
problematic. Newborn screening is often provided without
consent (or only with an opt-out provision) and for some
parents these screening results may not be wanted. A
compromise is to provide the screening at age 1-2 following
counseling and informed consent from the parents.
Screening pre-conception adults, following counseling
and consent, would probably be the least controversial. But,
based on the experience with screening for other inherited
disorders, this is also the hardest to implement.
1) Committee on Genetics. Obstet Gynecol 2010;116:1008-
10. 2) Hill et al, Genet Med 2010;12:396-410. 3) Coffee.
Genet Med 2010;12:411-2.
Peter Benn (benn@nso1.uchc.edu)
Co-ordination
The European Down Syndrome Screening Group (EDSSG)
begat the International Down Syndrome Screening Group
(IDSSG) which begat the International Prenatal Screening
Group (IPSG). Having co-ordinated the Group in all its
guises for nearly 20 years, initially with Bent Norgaard-
Pedersen, I think its time for me to step down especially as
for the next two years I’m President of the International
Society for Prenatal Diagnosis (ISPD). Luckily, the two
Peters – Benn and Schielen – have agreed to take over co-
ordination with me in the background advising and pitching
in as and when needed although I draw the line at any
more begetting! So please help the new team by sending
in pieces for Prenatal Screening Perspectives.
Howard Cuckle (hsc2121@columbia.edu)
PSP 2011 VOL 16
 Free fetal DNA;
recent developments
Following the two proof-of-
principle reports (1,2) that
shotgun sequencing of cell-free
DNA in maternal circulation
can potentially be used for
no n - i n va s i ve d i a g n o s i s,
refinements have been reported.
Chiu et al (3) consider an alternative sequencing method
(sequencing by ligation, Applied Biosystems/Life
Technologies, Solid 3 system) from that originally used
(sequencing by synthesis, Genome Analyzer, Illumina
Applied Biosystems). They were able to correctly
distinguish between 5 cases of trisomy 21 and 6 controls.
They noted that, as with the previous methodology, the
GC content of the sequences imposes a limitation on
determining genomic representation for some
chromosomes or chromosome regions.
Fan and Quake (4) describe a method of removing the
GC bias in the sequence reads. They conclude that
distinguishing relative genomic representation is only
limited by the counting statistics and therefore small
genomic imbalances could be identified provided there
was deep enough (large number) sequencing.
Liao et al (5) consider the effect of enriching for DNA
fragments present in a sample. Enrichment would
potentially allow for more informative sequence reads
from a sample. They show that a specific enrichment
method (SureSelect Target Enrichment System, Agilent),
did not result in a bias in proportions of maternal and fetal
alleles.
Tong et al (6) continue to develop a non-invasive
diagnostic approach through looking for epigenetic
differences between maternal and fetal (trophoblast)
DNA. They report on methylation differences in a
chromosome 21 gene (HLCS) promoter that can be used
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
contributions, but also analyze maternal contributions by
a new method referred to as relative haplotype dosage
(RHDO). Using this method, closely linked SNPs are
cumulatively counted until it is established that there is a
relative excess of one maternal haplotype over the over.
The excess thereby defines the maternal contribution to
the fetal DNA in the sample. They demonstrate the
method for the non-invasive diagnosis of beta-
thalassemia.
Although currently extraordinarily expensive, this study
demonstrates the potential for non-invasive diagnosis of
genetic disorders on an unprecedented scale.
In yet another important paper, Dennis Lo’s group (8),
perform DNA sequencing on maternal plasma samples
PSP 2011 VOL 16
using a multiplex protocol that involved either two (2-
plex), or eight (8-plex), samples sequenced together by
adding a unique identifying set of bases to each piece of
DNA (barcode). Based on 86 Down syndrome affected
and 571 unaffected samples, the 8-plex protocol had a
detection rate of 79.1% and a false-positive rate of 1.1%.
The 2-pex protocol (which involves sequencing more
fragments), was performed on 86 affected and 146
unaffected pregnancies and it had a 100% detection rate
and 2.1% false-positive rate. In an interview with BBC
News (accessed January 13, 2011), Kypros Nicolaides
who co-authored this study suggests that routine use of
the approach may not be for 10 years. Based on the
extraordinary pace of developments in this technology,
this would seem to be too conservative?
Another trial has just been published (9). This used a
multiplex approach on 480 samples. There were 13
samples with insufficient DNA and 18 that did not meet
quality control standards. Of the remaining 449, the
Down syndrome detection rate was 39/39 (100%) and the
false-positive rate 1/410 (0.3%).
There are two new useful reviews. Chiu et al (10)
summarize various approaches to non-invasive prenatal
diagnosis and Lo et al (11) present the current state of
the art for the diagnosis of hemoglobinopathies.
1. Fan et al, Proc Natl Acad Sci USA.
2008;105:16266-71.
2. Chiu et al, Proc Natl Acad Sci USA.
2008;105:20458-63.
3. Chiu et al, Clin Chem 2010;56:459-63.
4. Fan & Quake. PLoS ONE2010;5(5):e10439.
5. Liao et al. Clin Chem 2010;57:in press.
6. Tong et al. Clin Chem 2010;56:90-8.
7. Lo et al. Sci Transl Med 2010;2:61ra91 .
8. Chiu et al. BMJ 2011;342:c7401
9. Ehrich et al. Am J Obstet Gynecol 2011;204
(3):205.e1-205.e11.
10. Chiu et al. Trends Genet 2009;25:324-31.
11. Lo & Chiu. Hematol Oncol Clin N Am
2010;24:1179-86.
********
Non-invasive diagnosis: ethics
The expectation that non-invasive prenatal diagnosis will
soon be introduced for fetal aneuploidy, disorders with a
Mendelian pattern of inheritance and other conditions raises
some challenging ethical issues. Ethicists and potential
providers are concerned about the adequacy of information
and informed consent process prior to the test, accuracy and
reliability of the testing, use for paternity and gender
identification, commercial and direct-to-consumer
promotion, and increased pregnancy termination rates (1-3).
Skotko considers the potential for a substantially lower
incidence for Down syndrome (4). De Jong et al (5) suggest
that the greater moral challenge lies in the expanded rage of
disorders that will be potentially be diagnosable. This would
Page 16
include adult onset conditions where the “right not to know”
will need to be considered. A survey of pregnant women
carried out in the Netherlands indicated enthusiasm for NIPD
but also that women may find difficulty with the
consequences and choices associated with the new
technology (6).
1. Benn PA, Chapman AR. JAMA 2009;301:2154-6.
2. Benn PA, Chapman AR. Curr Opin Obstet Gynecol 2010:
22;128-34.
3. Hall A, Bostanci A, Wright CF. Public Health Genomics
2010;13:246-55.
4. Skotko BG. Arch Dis Child 2009;94:823-6.
5. de Jong A et al. Europ J Hum Genet 2010;18:272-7.
6. Kooij L et al. Prenat Diagn 2009;29:164-8.
Peter Benn (benn@nso1.uchc.edu)
The “Genetic
Sonogram”
A series of second trimester ultrasound markers have
variable utility in screening for fetal Down syndrome.
Relative to second trimester serum triple or quadruple
screening, they are less effective at distinguishing between
affected and unaffected pregnancies (Benn & Egan Prenat
Diagn 2008;28:230-235). However, it has become common
practice to use this “genetic sonogram” or “anomaly scan”
contingently; i.e. women at high or intermediate risks often
will receive the genetic sonogram and risks are modified
prior to a decision about amniocentesis.
Aagaard-Tillery et al (Obstet Gynecol 2009;114:1189-96)
show that the approach also has value for women who have
received first trimester combined screening. Using
prospectively gathered data from the FASTER trial, they
developed likelihood ratios for each of the commonly used
second trimester ultrasound markers. For any particular
patient, each likelihood ratio is multiplied together based on
the presence or absence of the marker. i.e. the markers are
considered to be independent.
The efficacy of the combined test, quadruple test, and
integrated test are each improved with the addition of the
“genetic sonogram”. However, using the genetic sonogram
as a replacement for the second trimester serum markers in
a sequential or contingent approach (i.e. combined first
trimester screening followed by the genetic sonogram) is
less effective than retaining the quadruple test.
In the approach used by Aagaard-Tlillery et al, nuchal fold
thickness and femur or humerus length were used as
categorical variables (i.e. either above or below specific cut-
offs). There is the potential for considerable enhancement
for the genetic sonogram by converting the measures into
MoMs and using the data as continuous variables (Benn et
al, Obstet Gynecol 2002;100:1168-76; Borell et al, Obstet
Gynecol 2007;30:941-5).
Page 17
Semi-automated NT
measurement – Progress and
caution
It is widely acknowledged that of the commonly used Down’s
syndrome screening markers, NT is both the most
discriminatory and the most difficult to obtain and maintain
quality. External schemes have been established to train,
certify and monitor NT performance, principally the Fetal
Medicine Foundation (www.fetalmedicine.com) and the Nuchal
Translucency Education and Quality Review (NTQR) program
(www.ntqr.org). Both schemes promote a standardised
approach to measurement: clear NT edges; mid-sagittal plane;
the fetus occupying more than half the image; the fetal head in
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
computer technology to make the actual NT measurement [1].
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
tissue overlying the cervical spine and identifies the largest
vertical distance between the two lines. Whist this does
produce more reliable results than the manual method there is
still a need to capture the correct image [1-3]. Thus NTQR
have now made the following statement on their website:
Appropriate education, NT credentialed physicians and
sonographers, adequate equipment and counseling resources
are essential for practices offering first trimester risk
assessment includ ing the nuchal translucen cy
measurement. NT measurements require the ability to image
the fetus in the correct plane, to select appropriate images or
conversely to discard suboptimal images, to optimize settings,
to select the appropriate area for measurement, and to place
the calipers correctly on the NT. Automated NT functions
available on some ultrasound machines may facilitate uniform
placement of calipers. This technology does not address
optimal position of the fetus or substitute for other essential
factors. The NTQR committee provides education and ongoing
quality review for participants reporting NT measurements.
Obtaining the NT through automation is acceptable when the
credentialed provider confirms the image and automated
caliper placement meet all NTQR criteria. Additional research
on the efficacy of automated NT measurements is
recommended.
1. Moratalla J, Pintoffl K, Minekawa R, Lachmann R, Wright D,
Nicolaides KH. Semi-automated system for measurement of
nuchal translucency thickness. Ultrasound Obstet Gynecol
2010;36(4):412-6.
2. Abele H, Hoopmann M, Wright D, Hoffmann-Poell B,
Huettelmaier M, Pintoffl K, Wallwiener D, Kagan KO. Intra-
and interoperator reliability of manual and semi-automated
measurement of fetal nuchal translucency by sonographers
with different levels of experience. Ultrasound Obstet Gynecol
2010;36(4):417-22.
3. Grangé G, Althuser M, Fresson J, Bititi A, Miyamoto K,
Tsatsaris V, Morel O. Semi-automated adjusted measurement
of nuchal translucency: feasibility and reproducibility.
Ultrasound Obstet Gynecol 2011;37(3):335-40.
PSP 2011 VOL 16
Pyramids inverted
or otherwise
The special issue of Prenatal
Diagnosis published in January
2011 (31(1)) is devoted to Kypros
Nicolaides idea that the current
practice of obstetrics is weighted
towards the end of pregnancy and
that with properly designed programs more could be achieved by
reversing the process and providing most care at 11-13 weeks.
The reviews in the issue make the case not only for aneuploidy
screening at this stage in pregnancy but also screening for structural
fetal abnormalities and adverse outcomes of pregnancy including
pre-eclampsia, pre-term delivery, growth restriction and fetal loss.
Much of the data presented comes from Nicolaides group at Kings
College Hospital (KCH), London.
For example, spina bifida can be detected at this time, not by
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
brain stem (BS) diameter and the BS to occipital bone diameter.
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
centile in 29 (97%), the BS to occipital bone diameter was below the
5th percentile in 26 (87%) and the ratio between them was above the
95th percentile in all cases.
There are now some impressive predicted detection rates for adverse
outcomes. On the basis of KCH data in the issue the model
predicted detection rate of early pre-eclampsia (ie requiring delivery
before 34 weeks) for a 5% false-positive rate is 78% when using
four markers – serum PlGF & PAPP-A, blood pressure and uterine
artery Doppler – together with information on prior risk factors
(Akolakar et al, 66-74). An even higher prediction of 88% comes
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,
although this has a wide confidence interval (Buyold et al, Obstet
Gynecol 2010;116:402–14).
The EU funded a research initiative entitled “Development of Early
Non-Invasive Biomarkers and Means for the Diagnosis and
Progression Monitoring of Preeclampsia and Tailoring Putative
Therapies” with the acronym PREGENESYS. The research group
held a Consensus Meeting in Cambridge in July 2009 to review the
state of the art and consider issues that may need to be addressed in
future recommendations. This has now been published (Cetin et al,
Placenta 2011;32:S4-16).
When modelling detection of other adverse outcomes one runs into
the problem of double counting. For example, PAPP-A and PlGF
are markers of both aneuploidy and pre-eclampsia; moreover the
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
have a common patho-physiology can be seen in a recent massive
Swedish study (Wikström et al, Am J Obstet Gynecol
2010;203:Epub ahead of print).
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 &
d, etc.
PSP 2011 VOL 16
Spina bifida – first trimester markers
AFP screening at 16-18 weeks for spina bifida is passé.
Higher detection rates can be obtained by routine fetal
anomaly scanning at 18-20 weeks or during a BPD scan
as early as 14 weeks looking for the ‘lemon’ and ‘banana’
signs.
With the shift in aneuploidy screening to the first trimester
the obvious question is whether spina bifida can be
detected at the time of the NT scan. Some advocates of
an early – trans-vaginal – anomaly scan at this time have
reported detection of some cases. Now there are
suggestions that examination of the fetal brain stem at
this time could detect open spina bifida cases with the
Arnold-Chiari syndrome. Compression of the fourth
ventricle leading to small (in relation to gestational
norms) or absent intracranial translucency has been
reported.
So far, in the literature there are only a few spina bifida
cases with these measurements, but watch this space:
1) Chaoui et al, Assessment of intracranial translucency
(IT) in the detection of spina bifida at the 11-13-week
scan. Ultrasound Obstet Gynecol. 2009;34(3):249-52.
2) Chaoui and Nicolaides. From nuchal translucency to
intracranial translucency: towards the early detection of
spina bifida. Ultrasound Obstet Gynecol. 2010;35(2):133-
8.
3) Egle et al, Appearance of the fetal posterior fossa at
11+3 to 13+6 gestational weeks in transabdominal
ultrasound examination. Ultrasound Obstet Gynecol.
[Epub ahead of print].
JOUBERT SYNDROME
Joubert Syndrome affects the cerebellum, which is responsible
for controlling balance and coordination of skeletal muscles.
Infants born with this condition demonstrate decreased muscle
tone, difficulty in swallowing, jerky eye movements and an
inability to coordinate voluntary muscle movements. As the
children grow, they may suffer from mental retardation and
develop kidney failure
Dor Yeshorim, an organisation that promotes genetic testing in
ultra-Orthodox Jews had observed an increasing number of
children born with the syndrome in recent years, and asked
Hadassah University Medical Center in Jerusalem to
investigate.
The disease has previously been mapped to the centromeric
region of chromosome 11. Using homozygosity mapping in 13
patients from eight Ashkenazi Jewish families, the Hadassah
group identified a homozygous mutation, R12L, in the
TMEM216 gene, in all affected individuals (Edvardson et al, Am
J Hum Genet 2010;86(1):93-7; erratum 86(2):294). Based on
these initial findings, researchers examined the DNA of 2,766
healthy Ashkenazi Jews and discovered that one out of 92
carried the mutation.
Page 18
 Predicting macrosomia in diabetic
pregnancies from serum markers.
A study was carried out to evaluate this for free β-hCG
and PAPP-A - Sylvia Kuc
Despite major obstetric care and strict glycemic management,
pregestational type 1 and type 2 diabetes mellitus (PGDM) is
strongly associated with increased fetal morbidity and
mortality compared to the general pregnant population1, 2, 3.
Diabetes-induced metabolic disorders are thought to interfere
with embryonic organogenesis and hyperglycaemia is thought
to trigger excessive fetal growth4. Currently, macrosomia at
birth (birthweight above 90th centile) is one of the most
concerning complications of a PGDM pregnancy. During
labor, macrosomic infants suffer higher rates of shoulder
dystocia, brachial plexus injuries and intrapartum asphyxia
compared to non-macrosomic infants1, 2, 3. After delivery,
these infants are at increased risk for hypoglycaemia, infant
respiratory distress syndrome (IRDS) and cardiomyopathy2.
Furthermore macrosomia at birth predisposes to childhood
obesity and to increased morbidity including insulin
resistance, hypertension and diabetes5.
The first-trimester screening test for Down syndrome (DS) is
now a part of obstetric care worldwide. The DS risk is
calculated on the basis of two markers in maternal serum: free
β-human chorionic gonadotrophin (fβ-hCG) and pregnancy
associated plasma protein-A (PAPP-A) together with the
nuchal translucency measurement (NT) and maternal age.
Numerous studies report predictive value of first-trimester
serum markers for macrosomia at birth in non-diabetic
pregnancies6, 7.
Serum samples were collected at the Dutch National Institute
for Public Health and the Environment (RIVM) between 2005
and 2007 as part of the national first-trimester Down
syndrome (DS) screening program. Serum samples were
collected between 8 and 14 weeks of gestation age (GA) and
analysis of serum concentrations fß-hCG and PAPP-A was
performed. For all women taking part in the screening sample
date, maternal age, maternal weight, smoking and medication
were recorded by the requestor. Pregnancy outcome,
including pregnancy duration, date of birth, pregnancy
complications, chromosomal disorders, child gender and
weight, were collected after delivery. From this cohort, serum
samples from women with PGDM were selected and retrieved
from storage. Women were classified as having PGDM if they
had been registered as using insulin in the first-trimester of
pregnancy. One control serum from an uncomplicated
singleton pregnancy was matched to each PGDM case, for
the same day of GA at sampling (±1 week), maternal weight
(±5 kg), maternal age (±1 year) and for sample date (±6
months). Serum marker levels were expressed as multiples of
the gestation-specific normal median (MoMs). The
correlations between marker concentrations and birthweight
centiles were assessed by Pearson correlation coefficients.
Differences with P-values < 0.05 were considered statistically
significant. Growth charts corrected for gestational age, sex
and parity according to the Dutch Perinatal Registry were
used to calculate the exact birthweight centiles8. Macrosomia
was defined as birth weight above the 90th centile8. PGDM
and control groups were subdivided in non-macrosomic
(control non-macrosomic; PGDM non-macrosomic) and
macrosomic (control macrosomic; PGDM macrosomic)
subgroups.
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
Page 19
available in 100% (n = 186) of the control group and in 91%
(n=162) of the PGDM group. Median birthweight centiles were
significantly higher in the PGDM group than in the control
group (89th and 72nd centile respectively; p < 0.0001) and
significantly more PGDM infants were macrosomic (n = 69
(42.6%) and n = 34 (18.3%), respectively; p < 0.0001).
The median MoMs of both markers were not significantly
different between the PGDM and control group. However,
when the PGDM and control groups were divided according to
the macrosomia at birth, the median PAPP-A MoM was
noticeably lower in the non-macrosomic PGDM subgroup
compared to the other subgroups. Median fβ-hCG MoMs were
comparable in all four subgroups.
The screening performance for macrosomia at birth in the
PGDM group with PAPP-A provided a detection rate (DR) of
18% for a 5% false positive rate (FPR) and a DR of 30% for a
10% FPR.
So it seems that the first-trimester DS serum markers fβ-hCG
and PAPP-A were comparable between PGDM and control
groups, but subgroup analyses according to macrosomia at
birth showed that levels of PAPP-A were significantly lower
only in the non-macrosomic PGDM subgroup. There was no
difference in PAPP-A concentrations between the control
population and the PGDM macrosomic subgroup. Thus,
PAPP-A seems to distinguish between non-macrosomic and
macrosomic subgroups in PGDM pregnancies (estimated DR
of 30% at 10% FPR) and is therefore a promising predictor of
macrosomia at birth in PGDM pregnancies. Currently our
group is evaluating PAPP-A in combination with the other
known first-trimester serum markers (A Desintegrin And
Metalloproteinase 12 – ADAM12, Placental Protein 13 –PP13,
Placental Growth Factor – PlGF) for the prediction of
macrosomia at birth in this particular high-risk population.
1. Persson M, Norman M, Hanson U. Obstetric and perinatal
outcomes in type 1 diabetic pregnancies: A large, population-
based study. Diabetes Care 2009;32:2005-2009
2. Evers IM, de Valk HW, Visser GHA. Risk of
complications of pregnancy in women with type 1 diabetes:
nationwide prospective study in the Netherlands. BMJ
2004;328:915
3. Balsells M, Garcia-Patterson A, Gich I, Corcoy R.
Maternal and fetal outcome in women with type 2 versus type
1 diabetes mellitus: a systematic review and metaanalysis. J
Clin Endocrinol Metab 2009;94:4284-4291
4. Allen VM, Armson BA, Wilson RD, Allen VM, Blight C,
Gagnon A et al. Society of Obstetricians and Gynecologists.
Teratogenicity associated with pre-existing and gestational
diabetes. J Obstet Gynaecol Canada 2007;29:927-944
5. Rijpert M, Evers IM, de Vroede MA, de Valk HW,
Heijnen CJ, Visser GHA. Risk factors for childhood
overweight in offspring of type 1 diabetic women with
adequate glycemic control during pregnancy: Nationwide
follow-up study in the Netherlands. Diabetes Care
2009;32:2099-2104
6. Tul N, Pusenjak S, Osredkar J, Spencer K, Novak-
Antolic Z. Prediciting complications of pregnancy with first-
trimester maternal serum free-betahCG, PAPP-A and inhibin-
A. Prenat Diagn 2003;23(12):990-6
7. Poon LC, Karagiannis G, Stratieva V, Syngelaki A,
Nicolaides KH. First-trimester prediction of macrosomia.
Fetal Diagn Ther 2010; [Epub ahead of print]
8.Visser GHA, Eilers PH, Elferink-Stinkens PM, Merkus HM,
Wit JM. New Dutch reference curves for birthweight by
gestational age. Early Hum Dev 2009;85:737-744
Sylwia.Kuc@rivm.nl
PSP 2011 VOL 16
Audit of self-reporting of pregnancy outcome:
influence on detection rate of first-trimester
Down syndrome screening.
An audit was carried out to see if this influenced a pro-
gram with apparently low detection - Nathalie Poierrié,
Peter Schielen & Wendy Koster
Flowchart – Schematic overview of numbers of Down
syndrome cases in the database. First level: cases are
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
after verification of the outcome in the ‘low risk’ group.
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.
The performance of the first-trimester combined test
depends on (1) technical aspects such as the quality of
NT measurements or biochemical assays; (2) accurate
measurements and registration of gestational age,
maternal weight or maternal age; (3) the existence of
confounding biological factors such as diabetes, vaginal
blood loss, smoking, IVF, medication use or obstetric
history. There are indications that for some of these
confounders a correction factor should be applied,
however, this is not standard policy in the Netherlands.
Subsequently, for all DS cases in the ‘low risk’ group (n
= 39), data on previous aneuploidy, gestational age at
sampling, diabetes, mode of conception (e.g. IVF or
ICSI), first trimester vaginal blood loss, medication, twin
pregnancy, smoking status and maternal weight were
verified and, if necessary, completed based on the
original patient charts. Strikingly, after data-collection at
hospitals and obstetric practices 17 pregnancies in this
group appeared not to represent a DS pregnancy.
Therefore the initial DR of 71% (93 of 146 DS cases
detected) changed to 83% (107 of 129 DS cases
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%)

PSP 2011 VOL 16 Page 20


Surprisingly, the most striking result of this retrospective
analysis was that a significant number of registered DS
cases appeared to be no case at all. For this dataset
this means that the actual DR is 13% higher than
initially thought. Thus, it resembles more closely
international reports on screening performances.
However, we have not yet confirmed that pregnancies
in the ‘high risk’ are indeed all DS cases.
Errors in our database of a total of 17
pregnancy outcomes were either incorrectly reported by
the patients or were administrative input errors at our
laboratory. However, a random check of received
follow-up forms indicates that the latter is quite unlikely.
Suggested other reasons for these wrongful
reports could be: (1) women do not quite understand
what “Down syndrome (trisomy 21)” means and report
DS in case of other chromosomal or structural defects
of the child; (2) women accidentally report a previous
DS pregnancy; (3) communicational problems due to
not mastering the Dutch language.
To overcome wrongful reports of DS cases in
the future one could argue to clarify the terminology of
‘Down syndrome’ on the post-partum forms. However,
since DS is an internationally accepted and well known
condition we do not think this will entirely solve this
issue. Ongoing digitalization of the entire screening
process in the future may also allow for outcome
reporting by health professionals, warranting both
correctness and completeness.
1
Valinen Y, Rapakko K, Kokkonen H, Laitinen P, Tekay
A, Ahola T, Ryynanen M. Clinical First-trimester routine
screening for Down syndrome in singleton pregnancies
in northern Finland. Am J Obstet Gynecol 2007;196
(3):278e1-5.
2
Wojdemann KR, Shalmi AC, Christiansen M, Larsen
SO, Sundberg K, Brocks V, Bang J, Norgaard-
Pedersen B, Tabor A. Improved first-trimester Down
syndrome screening performance by lowering the false-
positive rate: a prospective study of 9941 low-risk
women. Ultrasound Obstet Gynecol 2005;25(3):227-
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
for trisomy 21in 75821 pregnancies: result and
estimation of the potential impact of individual risk-
oriented two-stage first-trimester screening. Ultrasound
Obstet Gynecol 2005;25:221-6.
4
Spencer K, Souter V, Tul N, Snijders R, Nicolaides
KH. A screening program for trisomy 21 at 10-14
weeks using fetal nuchal translucency, maternal serum
free β-human chorionic gonadotrophin and pregnancy-
associated plasma protein A. Ultrasound Obstet
Gynecol 1999;13:231-237.
5
Wortelboer EJ, Koster MP, Stoutenbeek P, ELvers LH,
Loeber JG, Visser GH, Schielen PC. First trimester
Down screening performance in the Dutch population;
how to achieve further improvement? Prenat Diagn
2009;29(6):588-92.
6
Schielen PCJI, Koster MPH, Elvers LH, Loeber JG.
Downsyndroom kansbepaling met de eerste trimetsre
combinatietest 2006-2008. Rapport RIVM
230083001/2010 (Dutch).
Page 21
Im por ta nc e of
chorionicity for risk
assessment in
twins
On the basis of 4843 first trimester
blood samples from unaffected twin
pregnancies it has been shown that
PAPP-A levels are much lower (about 0.4 MoM on average) in
monochorionic (MC) pregnancies in comparison to those with
dichorionic (DC) placentae (Madson et al, Ultrasound Obstet
Gynecol 2011; 37: 38–47). Moreover, for both types of twins MoMs
increase on average substantially during the first trimester. Levels
of free β-hCG were lower for MC compared with DC twins but this
was a far weaker effect than with PAPP-A, and was only statistically
significant before 10 weeks. There was a strong tendency for
MoMs to increase with gestation but only among MC twins.
It is common practice to estimate gestational age in twins from the
largest CRL rather than the average since, it is argued, there are
reasons for fetuses in early pregnancy to be growth restricted
whilst growth enhancement is rare. Nevertheless taking the largest
is a type of bias and may have contributed to the results in this
paper. If any readers have a large series of twins please let us
know if it makes a difference to the trend in first trimester serum
marker MoMs when the average CRL is used compared to the
largest CRL.
The paper also reported 47 affected twins, 6 MC and 41 DC; for
both markers levels were lower in MC.
The rest of the paper describes and assesses a model of risk
calculation for twins but having read it through a few times I can’t
understand what they have done. In my simple minded view the
gestation- and chorionicity- specific normal median MoMs they
report for PAPP-A and free β-hCG are all we need. They can be
used to modify the already well described model. For example, at
10 weeks the PAPP-A median for DC twins is 1.7756 (Table 3) and
from the literature mean at this gestation for singleton Down’s
syndrome pregnancies is 0.40 MoM. In the model the Gaussian
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
To add to the confusion the authors conclude “Whether risk
calculation based on chorionicity-specific twin medians is superior
to the ‘pseudo risk’ approach in gestational weeks 12–14 is
unknown…”. Pseudo risk was proposed (by me) in the early days of
screening in twins as a stopgap until we knew something about the
marker median MoMs in affected pregnancies. The idea was to
ensure that the false-positive rate was the same as for singletons,
accepting that the detection rate, in discordant twins, was low. This
was decades ago and the approach should have been abandoned
everywhere by now. We don’t need pseudo risks because we can
estimate actual risks. The mean MoMs for Down’s syndrome can
be adequately derived indirectly as described above and these
values are close to the directly observed values in the literature
(Spencer K. (2005) Non-invasive screening tests. In: Blickstein I &
Keith LG (eds.) Multiple Pregnancy, Epidemiology, Gestation and
Perinatal Outcome, Parthenon Publishing, London, pp.368-384).
Howard Cuckle (hsc2121@columbia.edu)
PSP 2011 VOL 16

RE-INVENTING THE
WHEEL: AGAIN!
- a new regular opinion column by
Mark I Evans
In the 1960’s “modern” concepts of
quality control and business
processes were vestigial by today’s
standards. W. Edward Deming was
a Detroit auto efficiency business analyst who tried unsuccessfully
to get the major American automotive manufacturers to adopt
certain principles for business operations and efficiency including
labor – management interactions and methods for quality control
(QC). (1) His ideas were mostly ignored – in part because there was
little competition in the industry, and cost inefficiencies could just
be passed onto consumers who essentially had no alternatives. In
Japan, however, his ideas were adopted enthusiastically turning
around a culture in which “made in Japan” had been a synonym in
the 1950’s for cheap and poor quality into one by the 1970’s of
meticulous attention to detail and high quality. Overall, Deming
articulated 14 “principles” meant to apply to industry and the usual
job/factory functions. Some are outside the bounds of the
professional setting, but many apply equally well to medical
practice.
Some of the relevant principles are paraphrased here and include:
1. Create constancy of purpose for continual improvement
2. Cost alone is not the whole driver of decisions
3. Improve every process
4. Continual on the job training
5. The purpose of leadership is to help everyone improve
6. Drive out fear of communication up and down the ranks
7. Break down barriers (end silo thinking across groups)
Throughout my entire medical training, teaching was focused on
learning to do the job correctly, but it was never presented through
the rubric of anything systematic such as the above. Physicians
were taught to be stalwart individuals trying to care for their
patients. Seldom were they indoctrinated into a team concept of
feeling as being one component of a bigger process. Emergency
rooms, code teams, and MASH units would be notable clinical
exceptions. When it came to surgical procedures or interpretation
of clinical or radiological data, the idea of consistency across
providers was a very seldom mentioned concept. In contrast, the
laboratory was long expected to do daily quality control
monitoring, and the principles of statistical performance and rigor
were clearly in place by the 1970’s.
What does this have to do with Nuchal Translucency (NT)
screening? Actually, lots! Early arguments over the validity of NT
screening were solved with standardization of the approach (2-4).
In fact NT development was a complete replay of the issues of AFP
screening in which in the 1970s and 80’s, it was believed that each
city had to have their own medians and curves because of
population differences. (5) It was only with standardization of
laboratory methods that it became clear that such “population
differences” were very minor, and reported variations were
actually almost exclusively methodology differences.
Recently we have shown that the curve of measurements in the
United States is shifted to the left as compared to FMF data from
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
and specificity of screening in the United States. While it is
PSP 2011 VOL 16
impossible today that anyone would commit the resources for a
true nationwide outcomes study, we have modeled that a 0.5mm
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
consistent with previously published patient data that showed poor
technical performance lead to diminished sensitivity.
It has been a gospel of the technology assessment literature, that
there are two phases of new clinical and laboratory approaches.
First there is a phase of development in which a small number of
investigators, commonly but not always at academic medical
centers, pioneer a new technique, publish on it, demonstrate its
utility, and create a demand for such services.(8) Eventually others
get into the act and the technology “diffuses” out among the
community. Repeatedly, for clinical procedures, the data show
vast increases in utilization accompanied by dramatic drops in
performance and increased complications.
The purpose of quality assessment programs which were created -
such as the Fetal Medicine Foundation worldwide and the Nuchal
Translucency Quality Review program in the USA - was to minimize
the negative impact of new providers trying to mimic the
performance of the developers of the new technology. In many
parts of the world, the concept that “simple ultrasound
measurements” had to be certified or even reviewed were met
with scorn by some sonographers who stated that ultrasound was
an art. Unfortunately, if ultrasound measurements are to be used
in laboratory algorithms, then experience says they need to have
the same QC as the laboratory uses. Published and unpublished
data have clearly shown that failure to adhere to stringent
methodology control procedures produces skewed distribution
curves with most inexperienced sonographers tending to under
measure. Even in well controlled studies such as BUN and FASTER,
gradual increases in provider curves were well documented over
the years of the studies.
In future “pieces” I will lay out how this disconnect between
obtaining simple measurements and the public health implications
of poor performance must be addressed for quality health care for
women to be provided across the board.
1. Demming WE. Out of the Crisis. MIT Press, Cambridge 1986
2. Snijders RJM, Noble P, Sebire N, Souka A, Nicolaides KH. UK
multicentre project on assessment of risk of trisomy 21 by
maternal age and fetal nuchal translucency thickness at 10–
14 weeks of gestation. Lancet 1998;351:343–6.
3. Haddow JE, Palomaki GE, Knight GJ, Williams J, Miller WA,
Johnson A. Screening of maternal serum for fetal Down's
syndrome in the first trimester. N Engl J Med 1998;338:955-
61.
4. Monni G, Zoppi MA, Ibba RM, Floris M. Results of
measurement of nuchal translucency before and after
training. Lancet 1997;350:1631.
5. Evans MI, Cuckle HS: Biochemical Screening for Aneuploidy
Expert Rev Obstet Gynecol 2007;2:765-774
6. Evans MI, Krantz DA, Hallahan TW, Sherwin JE:
Undermeasurement of nuchal translucencies: implications for
screening. Obstet Gynecol 2010;116:815-818
7. Evans MI, Van Decruyes H, Nicolaides KH: Nuchal
translucency (NT) measurements for 1st trimester screening:
the “price” of inaccuracy. Fetal Diagn Ther 2007;22:401-404
8. Cohen AB, Hanft RS. Technology in American health care:
policy directions for effective evaluation and management.
Ann Arbor, MI: University of Michigan Press. 2004
Mark Evans (evans@compregen.com)
Page 22
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
conducted in co-operation with the Department of
Obstetrics and Gynecology at Holbæk Hospital. The
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
delivery. The serum markers considered were
pregnancy-associated plasma protein A (PAPP-A), free
beta-subunit of human chorionic gonadotropin (beta-
hCG), a disintegrin and metalloprotease 12 (ADAM12),
the proform of eosinophil major basic protein (proMBP)
and pregnancy-specific beta-1-glycoprotein (SP1). The
studies were based on merging of registry data from
ASTRAIA, the Danish Cytogenetic Central Registry, the
Danish Newborn Screening Registry and the Danish
Birth Registry. Furthermore, serum samples from
Holbæk Hospital routinely forwarded to Statens Serum
Institut for analysis (PAPP-A and beta-hCG) and storage
in the Prenatal Screening Biobank were used for
biochemical analysis. The first study demonstrated a
quality control model for the first trimester combined
screening program for DS. The model was used to
establish a study cohort for the following studies. A
retrospective cohort study was conducted confirming
that PAPP-A is a significant marker of SGA. Three case-
control studies were conducted for the evaluation of
ADAM12, proMBP and SP1. Serum samples were
retrieved from storage and analyzed manually using
different immunoassay techniques. ADAM12 was
significantly reduced in pregnancies with SGA. ProMBP
was consistently reduced in all of the outcomes
The fresh PhD, listening to the laudatio of his supervisor.
Peter Schielen (peter.schielen@rivm.nl)
Paternal Age
Sartorius and Nieschlag (Hum Reprod Update; 2010;16
(1):65-79) provide an updated review of the risks associated with
advanced paternal age. The authors note that
increasing male age is associated with
decreasing androgen levels, decreased sexual
activity, alterations in testicular morphology
and deterioration in semen quality (volume,
motility and morphology). As well as the
well known correlation between age and
autosomal dominant disorders, there appears
to be also a paternal age effect in many
mulitifactorial disorders. Miscarriage, pre-
eclampsia, preterm birth, Caesarian section rates may all be more
common for older fathers.
But the news is not all bad. The authors conclude that at the
current time advanced paternal age, per se, is not a reason for
invasive diagnostic testing. With the exception of some sex
chromosome abnormalities, there remains no evidence for increased
aneuploidy. Also, interestingly, telomere repeat lengths (which are
usually shorter with approaching cell senescence and aging) are
longer in the sperm from older males.
So, we can continue to take encouragement from the lives
of elderly celebrity fathers who seem to defy aging. Also from
Metheselah (Noah's grandfather) who is said to have fathered a son at
age 187 years.
Peter Benn (benn@nso1.uchc.edu)

Page 23 PSP 2011 VOL 16


IPSG m em bers may consider joining
the Community Genetics Network, which puts out a monthly
newsletter. The network has 927 members in 73 countries
presently. The aim is to facilitate communication between all
those working in the field of community genetics (and
genomics). Members of the network are all those who have
expressed their wish to become a member by e-mail
(commgennet@gmail.com).
The newsletter lists references to papers by members with a
hyperlink to the abstract. In the March issue 52 papers are
listed broken down into categories: Complex Conditions,
Congenital Disorders, Consanguinity & Inbreeding, Economic
Issues, Ethical Issues, Family History, Genetic Counseling,
Genetic Disorders, Genetics Education and Literacy, Genetics
in Primary Care, Genetic Screening, Genetic Testing,
Genomic Applications, Guidelines for Specific Disorders,
Health Worker Perspective, Miscellaneous, Patient
Perspective, Pharmacogenetics and Pharmacogenomics,
Population History, Preimplantation Genetic Diagnosis,
Psychosocial Issues in Genetic Testing, Public Attitudes,
Public Awareness, Public Health Genetics and Genomics,
Specific Disorders or Mutations in Specific Communities and
Statistical issues.
Under Genetic Screening there 4 papers:
Standardizing newborn screening results for health
information exchange. Abhyankar S, Lloyd-Puryear MA,
Goodwin R, Copeland S, Eichwald J, Therrell BL, Zuckerman
A, Downing G, McDonald CJ. AMIA Annu Symp Proc
2010;2010:1-5.
Health benefits and cost-effectiveness of primary genetic
screening for Lynch syndrome in the general population. Dinh
TA, Rosner BI, Atwood JC, Boland CR, Syngal S, Vasen HF,
Gruber SB, Burt RW. Cancer Prev Res (Phila). 2011;4(1):9-
22. Epub 2010 Nov 18.
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]
Long-term Consequences of the Early Treatment of Children
with Congenital Hypothyroidism Detected by Neonatal
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
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
conferences (22, in Australia, Belgium, Canada, Cuba,
France, Germany, Greece, India, Japan, the Netherlands,
Portugal, Spain, Turkey and USA).
It also lists position opportunities, academic, post-doc,
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
months at CSG in Nijmegen or one of its affiliated research
groups spread around the country”.
PSP 2011 VOL 16
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.
See page 28 for an account of the 15th Conference in
Amsterdam last June; the 16th Conference will be at the
Loews Miami Beach, 3–6 June 2012. These meetings
are a good opportunity to catch up with what’s new
prenatal diagnosis, but it is a rapidly changing field.
Thus it has been decided to have international meetings
devoted to education in the years between the
Conferences. The first such ISPD Education Day will
be in Barcelona immediately preceding the IPSG
Congress (see page 6) with a shared infrastructure and
local organisation led by Tony Borrell. Most of the
teaching will be through the special interest groups
(SIGs) which are being coordinated by Brigitte Faas.
Eventually it is hoped that the SIGs will go on the road
worldwide to provide educational services outside the
context of meeting per se.
Opinion leaders have an important role in the transfer of
research findings into routine clinical part. But opinions
differ and practice tends to become chaotic. Some
professional bodies have for a long time tried to use
their authority to modulate the extremes of practice (see
p9) although they do have a tendency to be
conservative and promote the lowest common
denominator. Against this background ISPD have
decided to issue their own ‘position statements’ in order
to give rational direction to relevant new developments.
And the first entitled “Aneuploidy Screening: a Position
Statement from a Committee on Behalf of the Board of
the International Society for Prenatal Diagnosis” will be
published soon in Prenatal Diagnosis. The committee
was chaired by our very own Peter Benn and comprises
many IPSG members.
IPSG 9th International Congress
School of Biology, University of Barcelona,
20th & 21st June 2011
The bells are ringing out....
El canto vuela....
They're calling us together....
Guiding us forever
Barcelona!
Freddie Mercury & Montserrat Caballe
http://www.youtube.com/watch?v=Ztqcuawqi34
Page 24
FETAL DUCTUS VENOSUS AS A MARKER
FOR CARDIAC DEFECTS
Most of papers on the ductus venosus (DV) Doppler studies
have assessed its role as a marker in the prenatal detection of
chromosomal anomalies or signaling fetal acidemia in fetal
wellbeing assessment. Recently a couple of papers have
investigated its role in the prenatal detection of cardiac
defects in chromosomally normal fetuses. Although previous
studies showed that DV assessment was redundant to the use
of nuchal translucency (NT), both studies show now that DV
improves the performance of NT screening for cardiac
defects, and that a 40% detection rate may be achieved by the
combined use of both markers.
The first paper from the Barcelona group (Martinez et al,
Ultrasound Obstet Gynecol 2010;35:267–272) evaluated the
independent contribution of DV at 11–13+6 weeks’ gestation
to the prediction of cardiac defects in chromosomally normal
fetuses, irrespective of the value of the NT thickness.
Abnormal DV blood flow was defined as either absent or
reversed flow during atrial contraction, and fetal
echocardiography was performed in all these fetuses, as well
as in NT above the 99th centile.
Among 6,120 consecutive singleton scanned pregnancies,
there were 145 chromosomally normal fetuses with abnormal
DV Doppler and 45 chromosomally normal fetuses with a
cardiac defect. Eleven affected fetuses presented with an
abnormal DV, giving a 24% detection rate, a 7.6% positive
predictive value and 9.8 odds ratio. In 6 of these 11 fetuses
(with both a cardiac defect and abnormal DV) an increased
NT above the 99th centile was also observed. Thus, the group
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
of 8.5. Interestingly, right-heart anomalies were predominant
in those cases with isolated abnormal DV, but no specific
pattern was found in those with increased NT.
The authors concluded that in experienced hands, abnormal
DV blood flow in the first trimester is an independent
predictor of fetal cardiac defects and should constitute an
indication for early echocardiography. In this study, the use
of DV blood flow assessment increased early detection of
cardiac defects by 11% with respect to the use of NT
measurement alone (rising from 29% to 40%).
The second paper, from Nicolaides group (Cheleman et al,
Fetal Diagn Ther 2011;29:127–134) recently appeared,
sought to determine whether assessment of DV improved the
detection rate of cardiac defects achieved by screening with
NT thickness. The study population of euploid fetuses at 11-
13 weeks included 85 cases with major cardiac defects and
40,905 with no cardiac defects. The fetal NT was above the
99th centile in 18 (21%) of the fetuses with cardiac defects.
Abnormal DV, defined as reversed a-wave, was observed in
24 (28%) of the fetuses with cardiac defects and in 2.1% of
those with no cardiac defects. Specialist fetal
echocardiography for cases with NT above the 99th centile
and those with abnormal DV, irrespective of NT, would
detect 39% of major cardiac defects at an overall false
positive rate of 2.7%.
Page 25
Placental growth factor
PAPP-A is a marker of both aneuploidy and pre-eclampsia but
to really get started with combined first trimester biophysical
(uterine Doppler) and biochemical screening for early pre-
eclampsia additional serum markers are needed. One
candidate is placental growth factor (PlGF) and it has the big
advantage over other such markers in that it is also a marker
of aneuploidy. Hence, screening for pre-eclampsia can
increase aneuploidy detection.
The published median or mean MoM results so far are:
(1) Down’s syndrome 0.71 (90 cases); Edwards’ syndrome
0.48 (28); Patau’s syndrome 0.40 (19); triploidy 0.53 (10);
Turner’s syndrome 0.53 (28). Zaragoza E, Akolekar R, Poon
LC, Pepes S, Nicolaides KH. Maternal serum placental growth
factor at 11-13 weeks in chromosomally abnormal
pregnancies. Ultrasound Obstet Gynecol 2009;33(4):382-6.
(2) Down’s syndrome 0.76 (70). Cowans NJ,
Stamatopoulou A, Spencer K. First trimester maternal serum
placental growth factor in trisomy 21 pregnancies. Prenat
Diagn 2010;30(5):449-53.
(3) Down’s syndrome 0.80 (151). Koster MP,
Wortelboer EJ, Stoutenbeek P, Visser GH, Schielen PC.
Modeling the Down syndrome screening performance using
first trimester serum markers. Ultrasound Obstet Gynecol
2010 Nov 12. [Epub ahead of print]
Second trimester results are also available:
(4) Down’s syndrome 0.67 (24). Debieve F, Moiset A,
Thomas K, Pampfer S, Hubinont C. Vascular endothelial
growth factor and placenta growth factor concentrations in
Down's syndrome and control pregnancies. Mol Hum Reprod
2001;7(8):765-70.
But one paper on early first trimester samples goes in the
opposite direction:
(5) Down’s syndrome 1.3 (37). Cowans NJ,
Stamatopoulou A, Tørring N, Spencer K. Early first-trimester
maternal serum placental growth factor (PlGF) in trisomy 21
pregnancies. Ultrasound Obstet Gynecol 2010 Nov 23. [Epub
ahead of print]
Simultaneously screening for different outcomes raises
another issue, whether to report the risk for each as though
they are independent. This may not be the case for Patau’s
syndrome (trisomy 13). Boyd et al. (Lancet 1987;2
(8556):425-7) reported severe pre-eclampsia in 5 out of 14
trisomy 13 cases, all nulliparous, and none of 28 age and
parity matched controls. No such association was seen for 7
Down’s syndrome and 4 Edwards’ syndrome pregnancies. A
similar case-control study found pre-eclampsia in 6/25
trisomy 13, 1/38 Edwards’ syndrome and 1/50 controls
(Tuohy JF, James DK. Br J Obstet Gynaecol 1992;99(11):891-
4). Both studies found the association higher in primigravids.
PSP 2011 VOL 16
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
was about to leave for a long
flight from the UK to China, an
obstetrician friend of mine
collapsed whilst scanning a patient in the hospital. She knew it was
very serious because they were transfusing her with blood products
that were cold – too urgent for even the shortest delay. It was an
ectopic pregnancy. Presentation is not always as dramatic or life
threatening but even when abdominal pain and other symptoms lead
to an earlier diagnosis tubular damage may already have been done
with consequent reduced fertility - although this is a chicken and
egg (ho-ho) situation and not easy to unravel the cause and effect.
At any rate there is a need, both in those at increased risk and also
generally, for an early screening method, apart from ultrasound.
Various serum markers have been reported and recently a group in
Philadelphia tested 12 of the most promising on sera from 200
symptomatic women – 100 with ectopic and 100 uterine
pregnancies (Rausch et al., Obstet Gynecol 2011;117(3):573-82).
ROC analysis indicated that five were reasonably discriminatory
with area under the curve exceeding 0.7: inhibin A, progesterone,
activin A, VEGF and SP1.
The authors then tried to combine markers using a method which
shows that they are not from the screening world. They used
something called Classification and Regression Trees (CART),
which is nothing but sequentially
using one marker to make a first
cut to select out a definitely
ectopic pregnancy group and in
the remainder using a second marker to select another group etc etc
and doing the same to select out uterine pregnancy groups. The
results are poor – for example the best combination was inhibin A,
progesterone and VEGF which detected 49% of ectopic and 41% of
uterine pregnancies. But they claim high sensitivity (98%) and
specificity (95%) with just one ectopic and two uterine pregnancies
being misdiagnosed. They are clearly not using these terms in the
same way as we would in the screening field. Very misleading!!!
So the question is still open as to whether some combination of
serum markers could efficiently identify those at high risk of extra-
uterine pregnancies.
The search for markers continues in Philadelphia. Beer et al. (J
Proteome Res 2011;10(3):1126-38) used a number of proteomic
methods to identify 70 candidate markers which they whittled down
to 12 biomarkers for further study, including ADAM12 and five
isoforms of SP1. The ADAM12 finding was subsequently been
confirmed in another series (Rausch et al., Fertil Steril 2011;95
(4):1373-8).
Howard Cuckle (hsc2121@columbia.edu)

PSP 2011 VOL 16 Page 26

Correlation of NT between
twin fetuses
When NT is measured in twins the
recommended approach is to consider
each fetus like a singleton and calculate a
fetus-specific risk from the NT MoM based on the fetal CRL.
However, the NT measurements in the two fetuses are not
independent with published correlation coefficients: 0.34
(Wøjdemann et al, Prenat Diagn 2006;26(3):218-20), 0.45
(Cuckle and Maymon, Prenat Diagn 2010;30(9):827-33), 0.43
(Maymon et al, Prenat Diagn 2011;31:Epub online) and 0.34
(Wright et al, Prenat Diagn 2011;31:16-21). Therefore, the
Down’s syndrome risk in an individual fetus is dependent on
both the specific NT and that of the co-twin.
An algorithm has been published to calculate the fetal
specific risk from an NT and CRL (Cuckle and Maymon, 2010).
There are four computational steps: (1) prior probability of
Down’s syndrome in fetus 1 only, fetus 2 only or both fetuses
according to maternal age and zygocity; (2) likelihood ratios of
discordant Down’s syndrome in fetus 1 only, fetus 2 only or
concordant Down’s syndrome, from the two NT MoMs; (3)
proportion of dizygotic (DX) and monozygotic (DZ) twins given
chorionicity, gender, whether assisted reproduction or
spontaneous, maternal age and ethnicity; and (4) multiply the
prior probabilities by likelihood ratios and take the weighted
average using the zygocity distribution. The weights are
crucial: all monochorionic twins are MZ and all dichorionic
unlike gender twins are DZ but the zygocity distribution
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
the mixture model (Wright et al, 2011). They made the
simplifying assumption that all dichorionic twin are DZ (sic) and
used a mixture model rather than the simple Gaussian model
for the NT distribution. Whilst one or other of these
approaches is necessary to provide a correct risk for the
individual women (both demonstrate with examples clinically
important differences in risk compared with the naïve
assumption of independence between the fetuses), like so
many refinements in aneuploidy screening it will not have a
large effect on detection rates. Maymon et al, (2011) have
estimated the magnitude of the effect using modelling. For
example, at 11 weeks gestation using NT as the single marker,
the predicted detection rate for a 5% false-positive rate is 78%
without taking account of the correlation between fetuses and
82% when this is allowed for using their algorithm. In these
calculations a ‘positive’ was taken to be where at least one of
the fetuses has a risk exceeding the cut-off. Wright et al,
(2011) applied their model directly to the dichorionic twins
studied (5530 unaffected and 65 with Down’s syndrome) and
found no effect on the detection rate although it is
underpowered to prove an effect.
When maternal serum markers are also used in
addition to NT the appropriate likelihood ratio for twins (see
page 21) can then be applied to each fetal specific risk
calculated from the above algorithms.
Page 27
P u b l i sh i n g
prospective
studies of
D o w n ’ s
s yn d r om e
screening
At least 25 large
prospective studies
of second trimester serum screening, Double, triple
or Quad, and at least 20 prospective studies of first
trimester screening, NT alone or Combined, have
been published. Consequently, it is now difficult
to publish further studies using these protocols
unless the screened population is extremely large
or special in some way. Nevertheless a small
study may be a useful baseline for further
developments in a specific country. To overcome
this problem Prenatal Screening Perspectives is
willing to include such study reports from IPSG
members, and in the current issue we have a report
from Mutaz Akkawi and his colleagues from Al-
Quds and Birzeit Universities on screening in the
West Bank (see page 36)
REPEAT MEASURES
REPEAT MEASURES
REPEAT MEASURES
Based on SURUSS statistical parameter estimates,
Wright and Bradbury (BJOG 2005;112:80–3) pointed out
that that first and second trimester measures of the same
serum analyte, notably PAPP-A, could potentially be highly
effective in Down syndrome screening. Following up on this,
Wright et al (Health Technology Assessment 2010;14:No.
33) have carried out an evaluation of the strategy using
FASTER samples and additional cases from North York
Hospital, Canada. Disappointingly, the addition of repeat
measures resulted in poorer screening performance when
the risks were calculated using an algorithm based on the
SURUSS statistical parameters.
The authors point out that their affected patient
samples showed a set of statistical parameters that departed
substantially from the SURUSS estimates and that
determinants of SURUSS covariance matrices were
unrealistically small. Parameters based on pooled affected
and unaffected pregnancies actually provided better
screening performance.
When a set of statistical parameters was developed
from the FASTER and North York pooled affected and
unaffected cases, repeat measures of PAPP-A was shown to
be beneficial if the first sample was drawn at 11 weeks. A
prospective study would be needed to confirm this.
PSP 2011 VOL 16
 Rhodos-Amsterdam;
the 2010 FMF and ISPD
congresses
The 2010 (9th) world congress on
fetal medicine was held in
Rhodes, Greece from June 20-
24. As a first-timer I needed to adapt to the typical
world congress-schedules, implicating that the
programme would automatically push lunch to late
afternoon and dinner to late-at-night. This was all
to no surprise for the experienced visitors, that are
used to the extensive discussions on every paper,
usually moderated by Prof. Kypros Nicolaides
himself. For the readers of PSP, especially the
session on screening for fetal aneuploidies was of
interest. There was a number of presentations on
the quality of the NT measurement and the quality
assurance of the screening test in general. Dave
Wright presented data on an exceptionally large
series of twin pregnancies, presenting detection
rates and showing differences in serum marker
levels between monochorionic and dichorionic
pregnancies. A number of new sonographic and
biochemical markers for aneuploidies was
presented as well. Wendy Koster modeled new
and common markers to show elevated DR for
aneuploidies. Moreover, there were two
presentations on high-end detection proteomic
expeditions for yet-undiscovered aneuploidy
markers.
Prediction of fetal and maternal health become
more and more prominent themes-the FMF
congress hosted sessions on the predictability of
preterm birth, early diagnosis of fetal defects, as
well as pre-eclampsia. There is growing evidence
that with the proper combination of maternal
history, serum markers and sonographic markers,
DR pushing 90 % for a 5-10 % FPR may be
achievable to detect pre-eclamptic pregnancies.
Especially in the domain of biochemical markers,
proteomics techniques may be a suitable tool to
identify useful markers.
The award for most presentations in this congress
went to Julien Stirneman, who appeared no less
than five times.
A number of the presentations of the 9th FMF
congress are published on the website of the FMF
( h t tp :/ / w w w .f et a l me d i c ine . co m /f mf /o n l in e -
education/07-lectures-in-fetal-medicine/). Amongst
others, you will find papers on non-invasive
diagnosis of aneuploidies (Diana Bianchi), Fetal
DNA in maternal blood (Lyn Chitty) and intra-
uterine growth restriction (Gerard Visser).
PSP 2011 VOL 16
As always, the congress was well-cared for. The
dinner and party at the harbour of Rhodos-city
were a delight, as was the visit to the ancient city
of Lindos.
15th International
Conference on Prenatal
Diagnosis and Therapy,
Amsterdam (Netherlands),
July 11-14 2010.
The fact that this years ISPD
congress was situated in
Amsterdam was particularly welcomed by my head
of department; my travel-expenses were only 35
euros, enough to travel the 40 km between
Maarsen, where I live, and Amsterdam-RAI
railway station for a couple of days.
The 2010-congress was preceded by a number of
courses, organized by the Special Interest Groups,
that were established at the Vancouver-meeting
(2008). Tak Yeung Lueng was chairman of the
course entitled ‘Advances, refinements and the
future of maternal screening’. Mark Evans lectured
on QC and QA for NT and other ultrasound and
serum markers. Wendy Koster reported on new
serum markers for aneuploidy and demonstrated
increased screening performance after modeling
these new markers. Prof Richard Choy presented
on the diagnosis of rare disorders identified
through screening. Howard Cuckle gave an update
on the screening of pre-eclampsia and Dave Wright
lectured on repeat measures and highly correlated
markers. Finally, Prof Lueng himself gave a
summary on the possibilities of non-invasive
prenatal diagnosis being able to replace screening.
In the main conference, this theme was also
extensively covered by e.g. the lecture of Dennis
Lo and others. The course was attended by about
50 congregates, and as judged from the course
evaluation and post-course comments, very well
received. During the post-course debate, future
themes of interest were identified, among which
were standards for risk estimation and a position
paper on aneuploidy screening.
In the main event, among numerous interesting
presentations, especially the controversies, in
which two contesters defended opposing views on
a specific theme, were interesting and fascinating.
Thus, Christine Mummery and Magnus Westgren
battled on the theme; ‘is stem cell therapy ready for
human fetuses’ and Beryl Benacerraf and Sicha
Page 28
Yagel explored the pros and cons of cell-free
nucleic acids in maternal blood versus ultrasound
in prenatal diagnosis. The-Hung Bui and Lisa
Shaffer debated whether conventional analysis
(karyotyping and FISH) is really necessary in the
post array CGH.
Dennis Lo, in his lecture, gave an overview of the
progress on the use of fetal DNA and RNA for
non-invasive diagnosis of Down syndrome. While
the tests are currently still very expensive, and the
experimental procedures tedious and vulnerable,
Lo was quite confident that fetal DNA and RNA
will replace conventional invasive tests in the
future. Whether it would also be a screening tool
however, is less certain. Finally the growing
interest for the role of placental microRNAs and
cellular mRNA in e.g. pre-eclampsia was a novelty
on this years programme.
Almost all presentations can be found on the
websit e of t he ISPD ( link: ht t p://
www.ispdhome.org/pres2010.asp).
Peter Schielen (peter.schielen@rivm.nl)
Trisomy 21 origin
A study involving fluorescence
in situ hybridization (FISH) with two
chromosome 21 probes hybridized to
the ovarian cells from eight 14-22 week
fetuses indicated that all were low level
mosaics(1). The percentage of cells
with an extra chromosome 21 was low
(average 0.54%) but the use of two probes should result
in a very low false-positive rate. The finding has
important implications in understanding the origins of
Down syndrome. It had been widely accepted that the
extra chromosome 21 arose as a meiotic non-disjunction
event which would only occur once female meiosis
completed i.e. at the time of ovulation and fertilization.
The authors suggested that higher levels of trisomy 21
ovarian mosaicism may be present in some females
placing them at higher risk for a Down syndrome
pregnancy or a higher risk of recurrence.
In a follow-up study (2), the same group
observed no such trisomy 21 cells in the germ cells from
4 male fetuses. They have also suggested that trisomy
21 germ cells may lag in maturation (relative to those
with disomy) accounting for the maternal age effect (3).
Also, the trisomy 21 germ cells could be at a selective
advantage during the normal attrition processes that
reduce the number of germ cells present in females
during development.
1. Hultén et al, Molec Cytogenetics 2008;1:21.
2. Hultén et al, Molec Cytogenetics 2010;3:4.
3. Hultén et al, Reproduction 2010;139:1-9.
Page 29
Population Screening for
Spinal Muscular Atrophy
Spinal muscular atrophy (SMA) is a
relative common fetal neuromuscular
genetic disorder caused by the
degeneration of motor neurons in the
anterior horn of the spinal cord. SMA is prevalent world-wide,
with an estimated incidence of 1:6,000 to 1:10,000 and a
carrier frequency of 1/38 to 1/50 (Feldkotter et al, 2002,
Ongio et al, 2004). About 93% of childhood SMA patients
have a homozygous deletion of exon 7 of the survival motor
neuron 1 (SMN1) gene. The remaining patients carry a
deletion on one allele, and an intragenic mutation on the
other allele. Given the severity of this disease, the relatively
high carrier frequency rate, and the current lack of effective
treatment, population-based carrier screening for SMA may
be justified. Indeed the American College of Medical Genetics
(ACMG) recently recommended that genetic SMA carrier
screening should be made available to all US couples.
Conversely the American College of Obstetricians and
Gynecologists (ACOG) stated in their Committee Opinion
(2009) that "preconception and prenatal screening for SMA is
not recommended in the general population at this time".
We have recently performed a study on large-scale
population based screening for SMA (Ben-Shachar et al,
Genetics in Medicine, in press). The study included 6394
Israeli individuals without family history of SMA, who
underwent screening by multiplex ligation-dependent probe
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
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
not be detected by MLPA or similar quantitative methods.
Given this limitation and the fact that some individuals have a
point mutation that cannot be detected by MLPA, we estimate
that SMA screening has a detection rate of about 90%. In
addition, we analyzed the acceptance level of this newly
introduced SMA screening test among women undergoing
testing for fragile X syndrome and cystic fibrosis. During the
study period, the acceptance rate increased from 77% to 99%
within 29 months, with an overall acceptance rate of 93%
during the study period. Given the severity of SMA, the
relatively high carrier frequency and the estimated detection
rate, we may conclude that SMA population based screening
is feasible and acceptable.
Shay Ben-Shachar (shayb@tasmc.health.il), Yuval Yaron
(yyaron@tasmc.health.gov.il)
References
1) Feldkotter M, Schwarzer V, Wirth R, Wienker TF, Wirth B.
Quantitative analyses of SMN1 and SMN2 based on real-time
lightCycler PCR: fast and highly reliable carrier testing and
prediction of severity of spinal muscular atrophy. Am J Hum
Genet 2002;70:358-68. 2) Ogino S, Wilson RB, Gold B.
New insights on the evolution of the SMN1 and SMN2 region:
simulation and meta-analysis for allele and haplotype
frequency calculations. Eur J Hum Genet 2004;12:1015-23.
3) Spinal Muscular Atrophy. ACOG committee opinion,
No.432. Americam College of Obstetricians and
Gynecologist. Obstet Gynecol 2009;113:1194-6. 4) Ben-
Shachar S, Orr-Urtreger A, Bardugo E, Shomrat R, Yaron Y.
Large scale population screening for spinal muscular
atrophy. Genet in Med, in press.
PSP 2011 VOL 16
Serum
Tests
Quality
Control
Considerable attention has
been paid to the quality control and
quality assurance of NT measurement.
Surprisingly, at least in the US, much
less attention is paid to the accuracy of
serum marker test results.
In the US, the assays used for serum markers for
aneuploidy screening are not approved by the Food and Drug
Administration (FDA) and, although some are actively marketed
for screening, manufacturers of kits make no claims about their
suitability. It is up to individual laboratories to validate the tests
but there are no specific requirements for the components to this
validation (1). For validation of a new test format, it is common
to demonstrate a high correlation of concentrations with an
established form of the test. But such studies usually do not fully
establish the suitability of tests for prenatal screening because
they generally do not include a substantial series of affected
pregnancies, evaluate the data in MoMs, and validate all statistical
parameters.
For example, a recent report by Lambert-Messerlian et
al (2) evaluated the new Beckman Access® PAPPA assay by
comparing it to Perkin Elmer’s AutoDELFIA PAPPA. Although
concentrations for a series of 194 normal pregnancies were very
highly correlated, there was a small but significant systematic
difference in the assays identifiable through a Bland-Altman plot
of the concentrations. The authors noted that another pair of
PAPPA assays also showed biases relative to each other and this
resulted in a considerable difference in the affected pregnancy
median MoM for the two assays. For different assay formats,
there also appears to be a very wide range in the estimates of the
PAPPA standard deviation for normal pregnancies and it seems
reasonable to think that the performance of the various available
assays could contribute to this. Some PAPPA assays may be very
reproducible at higher concentrations but much poorer for lower
values. Therefore, any added variance associated with a particular
methodology will not necessarily be the same for affected and
unaffected pregnancies. Similar considerations apply to all
markers and equivalent performance should not be assumed for
the various available manufacturers’ formats. Most labs currently
use the published parameter sets in their screening algorithm
without considering the possibility that their assay performance
might not in fact match.
Nearly all laboratories in the US receive oversight from
the College of Pathologists (CAP). CAP uses a peer-review
process for on-site inspections with requirements defined in
checklists. The checklists contain few specifics relating to
maternal serum screening although a recent revision (June, 2010)
introduced requirements for NT measurement quality assurance,
and new lot verification. There are some requirements for test
requisitions, establishing medians, reporting, and quality
assurance through monitoring median MoMs but no specific
requirements for the analytes most suitable for use, quality control
materials or test run acceptance criteria, validation of test
statistical parameters, appropriate covariable adjustments, etc.
The inspection process is part of CAP’s broader ‘special
chemistry’ category and generally the inspectors are not
specialists in prenatal screening.
CAP also provides second trimester four serum marker
proficiency testing through the distribution of test materials but
PSP 2011 VOL 16
the MoMs (except for AFP), risks, and interpretation are all not
graded ‘educational’ challenges and there is no penalty for poor
performance. The range of risks reported by different laboratories
for a second trimester quadruple test sample will typically cover
several orders of magnitude (3). In part, this could reflect matrix
effects associated with the use of artificial proficiency test media
rather than actual patient sera. But it is hard to explain why there
is so much variability in MoMs for peer groups using the same
method.
A voluntary first trimester proficiency test is also
available, the First Trimester Inter-laboratory Comparison
Program (FTICP), from Women an Infants Hospital, Providence,
RI (4). Proficiency in establishing NT MoMs from a set of raw
measurement data is stressed. There are currently approximately
26 participating laboratories (including manufacturer’s
development labs and overseas participants) with 18 producing
sequential risk estimates. Again, there is considerable
heterogeneity in results generated by the participating labs. Some
US labs may participate in State proficiency programs (e.g. New
York) or other external programs (e.g. UK’s National External
Quality Assessment Service (NEQAS)).
Most prenatal screening laboratories will run their tests
in batches with acceptance of the run based on the performance of
quality control materials that have well established expected
values. High quality laboratories will pay considerable attention
to this and use a variety of rules to evaluate their control data. A
commonly used set of criteria has been defined by Westgard (5,6)
and, correctly used, these can provide an early warning of testing
problems. Because test errors are compounded in multiple marker
testing (7), it is very important that all tests are highly
reproducible. Overall detection rates and false-positive rates are
rather insensitive to differences in test performance but individual
patient risk figures can be substantially changed (8).
A further unfortunate practical reality is that, for economic
reasons, a screening program may be forced to accept a test
format that is compatible with other areas of the laboratories’
automated activities rather than use testing that is judged most
well suited to aneuploidy risk assessment [see insert box]. There
are core clinical chemistry laboratory directors who may be
unaware or, worse, unwilling to provide the high level of quality
control oversight needed in prenatal screening or they may
compromise the quality control evaluation rules to make the
prenatal screening tests compatible with other areas. There is
little purpose in ensuring that NT medians are being measured
within narrowly defined criteria (for example median MoM 0.9-
1.1) when the results are being combined with batches of serum
marker results that may have been accepted based on a
substantially lower standard.
Perhaps the strongest force for quality in the US is actual or,
fear of, litigation. But this is not always a positive influence
because it often requires conformity to common practice
standards. As noted above, these practices may be mediocre or
lack solid evidence-based validation. It can also be extremely
costly.
Peter Benn (benn@nso1.uchc.edu)
1. Palomaki et al Genet Med 2009;11:669-81. 2. Lambert-
Messerlian et al, J Med screen 2010;17:109-13. 3. CAP.
Maternal Serum Survey Summary Reports. Palomaki et al, Arch
Pathol Lab Med. 2010;134:1685-91. 4. Westgard et al, Clin
Chem 1981;27:493-501. 5. Westgard. www.westgard.com/
westgard-rules-and-multirules.htm. 6. Benn & Collins. Ann
Clin Biochem, 2001;38:28-36. Benn et al, Clin Chem 2006;
52:2087-94.
Page 30
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
Based on the data from nine prenatal and postnatal
congenital anomaly registers, curves have now been
developed for maternal age specific prevalence of
trisomy 13 and trisomy 18 (Saava et al; Prenat Diagn
2010;30:67-64). The registries included 975 diagnoses
of trisomy 13 and 2,254 cases of trisomy 18.
Prevalence of trisomy 13 = 1/{1 + exp(9.53-(3.25/(1+exp
(-0.332*(age-37.5)))}
and
Prevalence of trisomy 18 = 1/{1 + exp(9.11-(4.27/(1+exp
(-0.324*(age-38.9)))}
An earlier publication provided values for the expected
fetal loss rates Morris and Saava (Am J Med Genet Part
A. 2008;146A;827-32). From 12 weeks to term an
estimated 49% of trisomy 13 and 72% for trisomy 18
pregnancies end in misscarrage or stillbirth, and, from 18
weeks the corresponding rates are 42% and 65%,
respectively.
Trisomy 16
Pregnancies with trisomy 16 mosaicism usually show
second trimester elevated MSAFP, grossly elevated
hCG and INH-A and low/normal uE3 (Neiwanger et al;
Prenat Diagn 2006;26:454-61; Benn 2010 In Genetic
Disorders of the Fetus; 6thEd. Ed Milunsky & Milunsky).
But, so far, there has been little information available
about first trimester markers.
Two reports involving 4 cases with apparently confined
placental trisomy 16 mosaicism all had very low PAPPA
(Verwoerd-Dikkeboom et al, Fertil Steril 2008;
90;2017.e19-2017.e22; Petracchi et al Prenat Diagn
2009;29;1175-6). Surprisingly, only one case had
elevated free beta hCG. NT was unremarkable.
Does anyone have more cases?
true
When DNA screening for recessive
conditions is offered to individuals or
couples there is an almost implicit
assumption that the information on carrier
status has no direct health implications for
the screenee in addition to any past or
future offspring. Indeed the term ‘healthy
carrier’ sometimes appears in the patient
information leaflets. However, this can be
misleading.
One way in which an apparent carrier might be medically
affected is if they are not actually a carrier but a
compound heterozygote. This happens with CF where
someone is a proven carrier for one of the multiplexed
series of relatively common mutations sought in the
screening test, but they are also a carrier of a rare and
possibly mild mutation. This combination may not
present clinically with typical CF but they may have mild
symptoms.
Duchenne/Becker Muscular Dystrophy is not recessive
but X-linked and about 10% of female carriers are
symptomatic. This is largely due to ‘skewed’ X
inactivation favouring the mutated dystrophin gene.
There seems to be a lack of consensus over whether
these women need long term heart monitoring.
With Fragile X syndrome, which is neither recessive not
X-linked but hereditably unstable, female carriers are at
risk of premature ovarian failure. Males would not
normally be screened but prenatal diagnosis in
pregnancies to carrier women may reveal a carrier male.
Such individuals are at increased risk of spinocerebellar
ataxia in later life.
In short those offering screening may need to make
patients aware, before testing, that their carrier status
will not necessarily be benign.

Page 31 PSP 2011 VOL 16

 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

PSP 2011 VOL 16 Page 32

Innovations in Down syndrome screening-
Wendy Koster
Dr. Koster was tremendously productive in only 3.5 years,
publishing 11 papers as a first author. Her thesis covers some
of the very basic issues in prenatal screening but she ends
with DS markers no one has ever heard of.
To start with the basics, Dr. Koster showed that the current
screening for Down syndrome in the Netherlands can be
improved by accurately measuring the crown-rump length of
the foetus for the determination of gestational age, by
improving the quality of nuchal translucency (NT)
measurements through monitoring and training of
sonographers and by drawing the serum sample early in the
first trimester (before 11 weeks).
Secondly, she determined that by adapting the screening
algorithm for trisomy 13 and 18, it is possible, specifically
calculated for the population of the reference laboratory, to
identify approximately 80% of foetuses with trisomy 18 or
trisomy 13, with only 0.2% extra false positive test results.
Wendy investigated the concentrations of the common and
some new (ADAM-12, PP13) biochemical markers in a quite
large series of monochorionic and dichorionic twin
pregnancies. None of the marker concentrations showed a
difference between monochorionic and dichorionic twins.
Remarkably, median ADAM-12 and PP13 were only about 1,5
times the MoM in singleton pregnancies. A review of the
available literature is ambiguous on this issue. A recent paper
of Wright et al shows that especially early in pregnancy the
biomarker concentrations in monochorionic twins are lower.
In addition to the work on early sampling, Wendy showed
that, to increase the Down syndrome screening performance,
it would be advisable to draw two serum samples during the
first trimester; one sample before 11 weeks to measure PAPP-
A and one sample after 11 weeks to measure fβ-hCG. At
these points in time, the biochemical screening markers are
most distinctive between Down syndrome and unaffected
pregnancies. Logistically it would be best to draw the second
serum sample at the time of the NT measurement.
Next, Wendy showed that adding potential biochemical
screening markers, such as ADAM12, PP13, PlGF and thCG, to
the first-trimester combined test increases the detection rate
for aneuploidies, but also allows screening for other
pregnancy complications. Given the potential of new
biomarkers for DS screening, and given that proteomics
techniques are now available for the identification of new
biochemical screening markers for Down syndrome, Wendy
set out on a quest for undiscovered markers. She found EGF,
EN-RAGE and CA19-9 to be promising screening markers,
showing concomitantly that initial experiments produce
impressive numbers of new biomarker candidates, but the
majority does not survive additional selection in follow-up
validation experiments. EGF, EN-RAGE and CA19-9 will be
tested in large-scale validation experiments now. Bead-based
multiplexed immunoassays and antibody microarrays are
high-throughput platforms that allow for multiple marker
analyses. Therefore, these techniques can be used for Down
syndrome screening purposes in the future. Wendy showed
that in principle, both analytical platforms can be put to use
for high throughput screening. Wendy’s thesis also concluded
with a five-year view, suggesting a screening test that will
focus on several foetal abnormalities and maternal
complications using a broad panel of biochemical markers,
which can be analyzed simultaneously, in one screening test.
Page 33
Ethnic differences in prenatal screening for
Down syndrome-Mirjam Fransen
Objective
The main aim of the research was to assess ethnic variations in
pregnant women’s decision-making on participation in prenatal
screening for Down syndrome in the Netherlands.
Methods
Pregnant women from Dutch, Turkish and Surinamese ethnic
origin were recruited from community midwifery practices in
Rotterdam and the outpatient clinic of Erasmus MC. In total 270
women were personally interviewed in the language they
preferred, a mean of 3 weeks after booking for prenatal care. We
assessed source and type of received information, knowledge on
Down syndrome and prenatal screening, attitude towards
participating in prenatal screening, and considerations whether or
not to participate in prenatal screening. Actual (non-)
participation was assessed several months after the interview. In
addition, 57 midwives filled in a web-based questionnaire to
assess ethnic variations in providing information on prenatal
screening. In order to assess ethnic difference in participation of
the prenatal screening programme in a larger study population,
we performed a register-based study in 2009, in the Southwest
of the Netherlands.
Results
Most women reported that the midwife is the prime source of
information about prenatal screening for Down syndrome, and
that most of them received oral and/or written information at
booking for prenatal care. However, women from Turkish and
Surinamese ethnic origin had significantly less knowledge about
Down syndrome and prenatal screening than Dutch women. In
total 5% of the women from Turkish origin, 26% of the women
from Surinamese origin and 71% of the women from Dutch
origin made an informed decision whether or not to participate in
prenatal screening. Differences in language skills and educational
level contributed most to these variations.
Midwives reported that they do not always offer information
when pregnant women from a non-Western ethnic background
hardly speak and understand Dutch. Only a few midwives
reported to use translated materials or professional interpreters in
case of language barriers.
The register-based study showed that the overall participation in
the prenatal screening programme was 26%. Compared to Dutch
women, those from Turkish, Moroccan, Aruban/Antillean and
other non-Western ethnic origin were less likely to participate in
the prenatal screening programme. The differences between
women from Dutch origin versus women from Moroccan and
Turkish origin remained significant after adjustment for socio-
economic background and age. No significant differences were
found between women from Surinamese and Dutch ethnic origin.
Discussion
The ethnic variations we found in pregnant women’s knowledge,
decision-making and uptake of prenatal screening demonstrate
that the goal of the prenatal screening programme to enable all
pregnant women to make an informed decision whether or not to
participate in prenatal screening for Down syndrome is certainly
not achieved, and indicate that there are ethnic differences in
access and quality of prenatal screening. The interventions to
improve access and quality of prenatal screening require efforts
by the Dutch government, the Central Agency of prenatal
screening, regional prenatal screening centres and healthcare
professionals.
Acknowledgements
The work presented in the thesis was conducted at Erasmus MC,
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.
PSP 2011 VOL 16
Informed decision-making about prenatal screening-
Marleen Schoonen
Schoonen, H.M.H.J.D., Wildschut, H.J., Essink-Bot, M.L.,
Steegers, E.A.P., de Koning, H.J.
Schoonen, Marleen, MSc. (Corresponding Author)
Department of Public Health, Erasmus MC, University
Medical Center Rotterdam, Room AE-206, P.O Box 2040,
3000 CA Rotterdam, The Netherlands. Telephone: 31-10-463
8460 / 31-10-704 3732 / Fax: 31-10-703 8474 / E-mail:
h.schoonen@erasmusmc.nl
Wildschut, Hajo, MD PhD. Department of Obstetrics and
Gynaecology, Division of Obstetrics and Prenatal Medicine,
Erasmus MC, University Medical Center Rotterdam,
Rotterdam, The Netherlands
Essink-Bot, Marie-Louise, MD PhD. Associate Professor,
Department of Social Medicine, Academic Medical Center,
Amsterdam, The Netherlands
Steegers, Eric, MD PhD. Professor, Department of Obstetrics
and Gynaecology, Division of Obstetrics and Prenatal
Medicine, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands
de Koning, Harry, MD PhD. Professor, Department of Public
Health, Erasmus MC, University Medical Center Rotterdam,
Rotterdam, The Netherlands
Introduction
First trimester prenatal screening for Down’s
syndrome aims at informing pregnant women about the
likelihood of having a Down’s syndrome-affected child in a
timely manner, in order to allow them the opportunity to act,
that is prepare for the birth of a child with Down’s syndrome
or termination of the pregnancy if the condition is diagnosed1.
The Dutch prenatal screening programme, organised in its
present form since 2007, makes use of the combined test to
estimate the individual probability of being pregnant of a
child with Down’s syndrome.
Second trimester ultrasound screening for fetal
anomalies (or fetal anomaly scan) primarily aims at the
detection of structural malformations when termination of
pregnancy is still legal2. In the Netherlands, as in most other
Western countries, second trimester ultrasound screening for
fetal anomalies has become a standard part of prenatal
care3,4, 5.
The Dutch prenatal screening programme is
characterised by an active, standard information offer to
every pregnant woman6, 7. The goal of this information offer
is informed decision-making. Facilitating informed choice on
participation is a fundamental part of international guidelines
on prenatal screening8-13. An informed choice is based on
relevant knowledge. In addition, actual behaviour (in this
case, participating or non-participating in the prenatal
screening programme) should be consistent with decision-
maker’s attitudes14.
What knowledge is relevant for informed decision-making
about prenatal screening for Down’s syndrome?
Many Western countries have guidelines or
recommendations for prenatal screening for Down’s
syndrome, in which either women of advanced age are
offered invasive diagnostic testing (chorionic villus sampling
or amniocentesis), or in which women, irrespective of their
age, are offered non-invasive risk-assessment tests in the first
trimester pregnancy (nuchal scan, often combined with
maternal biochemistry). The latter screening tests provide an
PSP 2011 VOL 16
individual risk estimation of carrying a child with Down’s
syndrome, and may be followed by diagnostic testing to
confirm whether or not the fetus is affected4, 15.
We determined the content of relevant knowledge
needed to make an informed decision about (non-)
participation in prenatal screening for Down’s syndrome, in
order to develop a knowledge questionnaire to be used for
large scale evaluations of informed decision-making. A
generic list of content domains for knowledge about
screening was extracted from the literature. Items reflecting
specific knowledge domains were constructed. An expert
group of professionals and pregnant women expressed
whether domains and items represented decision- relevant
information.
All presented domains were scored as (very)
important. Options when receiving an ‘increased probability
for Down’s syndrome test result, the meaning of this result,
the aim of the screening, and voluntary nature of the test were
scored as most important. The condition being screened for,
prevalence, and the screening procedure were scored as
relatively less important, with a high amount of expert
consensus16.
What knowledge is relevant for informed decision-making
about the fetal anomaly scan?
Second trimester screening for fetal abnormalities and
first trimester screening for Down’s syndrome have one goal
in common; the detection of fetal anomalies. However, these
screening programs are quite different entities and have
different characteristics; Down’s syndrome screening – with
its (seemingly) straightforward purpose – and 20- week
screening with its myriad of possible syndromes, its soft
markers and its assessment of structures other than the fetus,
such as the placenta and the umbilical cord. In addition to
these different technical aspects and test-characteristics of
second trimester screening for fetal abnormalities, compared
with first trimester screening for Down’s syndrome, women
attribute different goals to these screening programs and
perceive them substantially differently as well17. Pregnant
women often wish to have ultrasounds for non-medical
reasons such as seeing the baby, making the pregnancy seem
more real, and knowing the sex of the baby 17-19. Hence,
women expect ultrasound to be a positive and pleasant event2,
5, 17, 19
. In contrast, pregnant women show ambivalence
towards screening for Down’s syndrome20. They associate
participating in prenatal screening for Down’s syndrome with
abortion and often cite unwillingness to have an abortion as a
reason for not participating in the screening program21. To
summarize, it seems that first trimester screening for Down’s
syndrome is perceived by pregnant women as being primarily
focused on the detection of ‘abnormalities’, whereas the
second trimester ultrasound is perceived as aiming to confirm
‘normality’17, 19
Similar to determining decision-relevant knowledge
about prenatal screening for Down’s syndrome, we also
determined the content of relevant knowledge needed for
informed decision-making in second trimester ultrasound
screening for fetal anomalies. In addition, we compared the
content of decision-relevant knowledge for second trimester
ultrasound screening with the content of decision-relevant
knowledge for first trimester screening for Down’s syndrome
using the combined test.
The meaning of an abnormal test result, the
disorders being screened for, and the purpose of the screening
were regarded as very important for informed decision-
Page 34
making about the fetal anomaly scan, along with the
voluntary nature of the test. All knowledge domains were
included in the final questionnaire. The relationship between
the ranking of the domains according to relevance for first
trimester and second trimester screening was 0.71 (Pearson’s
r). The domain ‘consequences of a positive test result’
received a higher expert ranking in first trimester screening
than in second trimester screening22.
Evaluating the information provision process about
prenatal screening for Down’s syndrome, and about
ultrasound screening for fetal anomalies
Informed decision-making about prenatal screening
for Down’s syndrome is considered an indicator of adequate
information provision. Currently, we investigate the process
of providing information about prenatal screening for Down’s
syndrome. In addition, we investigate whether the
information provision is adequate, using informed decision-
making as an indicator. Regarding the process of providing
information, questions we address are: Are all pregnant
women offered information about the screening programme?
How many women accept the information offer and, of these
women, how many actually receive the information?
Furthermore, we address the following questions
regarding the quality of information provision: What is the
knowledge on Down’s syndrome (screening), what are the
attitudes, are pregnant women willing to participate in the
screening programme and what are their reasons to (not)
participate? To what extent is decision-making regarding
participation in prenatal screening for Down’s syndrome
based on an informed decision? What are the determinants of
informed decision-making? In a separate study, the same
questions are addressed for prenatal screening with the fetal
anomaly scan, and compared with the results for prenatal
screening for Down’s syndrome.
Data are obtained in an open, unselected population
in the Southwest region of the Netherlands, from pregnant
women (both participants as well as non-participants in the
screening) and from midwives. Results of these studies are
expected soon.
References
1. Health Council of the Netherlands: Committee Genetic
screening. Genetic Screening 1994;22.
2. Bricker L, Garcia J, Henderson J, Mugford M, Neilson J,
Roberts T, et al. Ultrasound screening in pregnancy: a
systematic review of the clinical effectiveness, cost-
effectiveness and women's views. Health Technol Assess
2000;4(16):i,vi, 1-193.
3. A brief history of the 20-week ultrasound. Available at:
http://www.rivm.nl/pns_en/down_ultrasound/history/ .
Accessed 03/05, 2010.
4. Boyd PA, Devigan C, Khoshnood B, Loane M, Garne E,
Dolk H, et al. Survey of prenatal screening policies in
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);
Down's syndrome, neural tube defects. Health Council of the
Netherlands 2004;2004/06.
Page 35
7. Health Council of the Netherlands. Population screening
act: inition of a national programme for prenatal screening:
Down's syndrome and neural tube defects [Wet
Bevolkingsonderzoek: aanzet voor een landelijk programma
voor prenatale screening]. 2006;2006/03.
8. General Medical Council. Seeking patients' consent: the
ethical considerations. London: GMC; 1999.
9. World Health Organization. Statement of WHO expert
advisory group on ethical issues in medical genetics. ; 1998.
10. American College of Obstetricians and Gynecologists.
Screening for fetal chromosomal abnormalities. Washington
(DC): American College of Obstetricians and Gynecologists;
2007.
11. Joint Working Party of the Royal College of Obstetricians
and Gynaecologists and the Royal College of Paediatrics and
Child Health. Fetal abnormalities- guidelines for screening,
diagnosis and management: report of a joint working party of
the Royal College of Obstetricians and Gynaecologists and
the Royal College of Paediatrics and Child Health. RCOG
Press 1997;.
12. Royal College of Obstetricians and Gynaecologist.
Amniocentesis and chorionic villus sampling. London: ;
2005.
13. National Screening Committee. Second report of the UK
National Screening Committee. London: Department of
Health; 2000.
14. Marteau TM, Dormandy E, Michie S. A measure of
informed choice. Health Expect 2001;4(2):99-108.
15. EUROCAT. Special Report: Prenatal Screening Policies
in Europe. EUROCAT Publications on Prenatal Diagnosis;
2010.
16. Schoonen HM, van Agt HM, Essink-Bot ML, Wildschut
HI, Steegers EA, de Koning HJ. Informed decision-making
in prenatal screening for Down's syndrome: What knowledge
is relevant?. Patient Educ Couns 2010; Aug 25;.
17. Santalahti P, Aro AR, Hemminki E, Helenius H,
Ryynanen M. On what grounds do women participate in
prenatal screening?. Prenat Diagn 1998;18(2):153-65.
18. Stephens MB, Montefalcon R, Lane DA. The maternal
perspective on prenatal ultrasound. J Fam Pract 2000;49
(7):601-4.
19. Ahman A, Runestam K, Sarkadi A. Did I really want to
know this? Pregnant women's reaction to detection of a soft
marker during ultrasound screening. Patient Educ Couns
2010;81(1):87-93.
20. Vassy C. How prenatal diagnosis became acceptable in
France. Trends Biotechnol 2005;23(5):246-9.
21. Green JM, Hewison J, Bekker HL, Bryant LD, Cuckle
HS. Psychosocial aspects of genetic screening of pregnant
women and newborns: a systematic review. Health Technol
Assess 2004;8(33):iii, ix,x, 1-109.
22. Schoonen HMHJD, Essink-Bot ML, van Agt HME,
Wildschut HI, Steegers EAP, de Koning HJ. Informed
decision-making about the fetal anomaly scan: What
knowledge is relevant. Ultrasound Obstet Gynecol In Press;.
PSP 2011 VOL 16

Maternal serum screening
in the West Bank
An audit of Palestinian and non-Palestinian women having
Triple tests - Mutaz Akkawi, Rula Ghandour, Abdullatif
Husseini, Lamia Halasseh, Qasem Abo Remeleh
Abstract
Objectives The aim of this second phase of study was to
detect or confirm any change in the screening results of Triple
tests carried out on Palestinian women living in the West Bank
during the period 2004-2010 if compared to that done
between 2000-2003.
Methods Data on 1354 pregnant Palestinian and non-
Palestinian women living in the West Bank (Filipino women)
who were offered the Triple test during the period 2004-2010
were analyzed statistically using the Statistical Package for
Social Sciences (SPSS) version 17
Results In this study, out of 1354 subjects, 1267 were eligible
for the final analysis. The mean expected age at delivery ± SE
(standard error) of the Palestinian women was 27.02±0.17
years compared with 25.5 ±0.2 years in the first study. The
AFP results of the Palestinian women tested using AFP MoM
cutoff of 2.5 showed that 2.7% had high concentrations
compared with only 1.3% in the first study. Filipino women
showed the same result for AFP. Our study showed that
women with high risk of Down’s syndrome were 5% compared
with 2.8% in the first phase of study.
Conclusion Our study showed that within about six years
there is increase in both the percentage of women with high
AFP results and with those with high risk of Down’s syndrome
which becomes similar to that obtained in the international
community. This may be due to direct result of the increase in
the mean age of women.
Introduction
Antenatal testing describes procedures performed during
pregnancy to detect health problems in the growing fetus.
These tests can provide a non-invasive screening for Down’s
syndrome (trisomy 21) and other congenital anomalies such
as spina bifida and anencephaly (neural tube defects – NTDs)
through the assessment of maternal serum markers and
ultrasonography .1, 2
Down’s syndrome (trisomy 21) is the most common
cause of severe learning disability in children with an
incidence of 1 in 800 live births .3 The incidence of Down’s
syndrome in some Arab countries exceeds 1.7 per 1000
compared to incidences of 1.0-1.4 per 1000 for other Arab
populations .4 As a result of this high incidence, screening
and diagnostic tests should be offered. In our community the
importance of such tests has been recognized and the first
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
ordered between 11-13 weeks of pregnancy. This test
measures the levels of two serum markers, free β-human
chorionic gonadotropin (free β-hCG) and Pregnancy
Associated Plasma Protein-A (PAPP-A) in addition to a
special ultrasound scan that measures the nuchal
translucency (NT) thickness, a space at the back of the baby’s
neck .7 These tests cannot evaluate the fetus's risk of
developing NTDs. First trimester screening is not common in
the Palestinian community and efforts are exerted to
encourage women to do it in the near future. The most
common test done by Palestinian pregnant women is the
Triple test. 5
The Triple and Quadruple tests are used to screen in
PSP 2011 VOL 16
the second trimester of pregnancy between 14 weeks and 20
weeks of pregnancy. It is ordered to help evaluate the risk
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-
fetoprotein (α-FP), Human Chorionic Gonadotropin (hCG) and
unconjugated Estriol (uE3). 9 When Inhibin A is added to
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
of the three serum markers can suggest the type of
abnormality present. 11
A number of important factors can influence the results
of the Triple test. These include: maternal age, race, weight,
number of fetuses and gestational age. 3, 12 Women who
have a positive screening test result for Down’s syndrome are
usually referred for genetic counseling and further testing.
In this article we describe the results of Triple tests
carried out on Palestinian and non-Palestinian women who
came to perform this test in different areas of the West Bank
during the period 2004-2010.
Subjects and materials
Laboratory methods
All tests were performed in Zer laboratories in Jerusalem
utilizing standardized techniques for analysis. Both AFP and
hCG were performed using immunoradiometric assay (IRMA).
AFP kit was produced by Byk-Santgtec Diagnostica-Germany,
while the hCG kit was produced by ICN-USA. For uE3 the
radioimmunoassay (RIA) kit used was a product of DSL-USA.
ALPHA, which is special software for risk calculation was
utilized in this study and produced by Logical Medical
Systems, UK. The cut-off point utilized in this analysis was
1:380 at term risks.
Variables used in the study
The following variables were used: age at expected date of
delivery, weight in kilograms, gestational age in weeks, in vitro
fertilization (IVF) status, twins status, AFP levels in IU/mL and
multiples of the normal gestation-specific median (MOM),
hCG in IU/mL and MOM, uE3 in ng/mL and MOM. The
variables were used to calculated risk for age alone and for
the Triple test.
Statistical analysis
All data were entered into Statistical Package for Social
Sciences (SPSS) version 17. Data cleaning was conducted
and outliers were excluded, those included 29 twins
pregnancies, 5 IVF, 1 unknown origin, and 52 Africans. A total
1267 cases were included in the final analysis. Basic
descriptive statistics including means, medians, modes,
standard deviation, and standard error were calculated.
Frequencies were also calculated for relevant variables. Cross
tabulations and Chi square test were used to explore the
relation between different variables.
Results
Characteristics of the subjects
A total 1267 cases were investigated of which 88.4 % (1120)
were Palestinian Arabs residing in the West Bank, while
11.6% (147) were non-Arabs (Filipinos). The mean expected
age at delivery ± SE (standard error) of all subjects was
27.54±0.17 years while it was 27.02±0.17 years for
Palestinian women and 31.47 ±0.45 years for Filipino women.
11.8% (150) of the participants were 35 years old or above.
The mean weight for women at the time of testing was 65.79 ±
0.34 kilograms. The range of gestational age when the test
was performed was 15 to 21 weeks, with a mean of 17.12
±0.02.
Page 36
 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)

Page 37 PSP 2011 VOL 16

References
Cuckle HS, Wald NJ. Principles of screening. In: Wald NJ,
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Cuckle H. Biochemical screening for Down syndrome. Eur J
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Crown-Rump Length and
Gestational Age
“The optimal gestational age for measurement of fetal
NT is 11 weeks to 13 weeks 6 days. The minimum fetal
crown-rump length should be 45mm and the maximum
84mm.” (Nicolaides KH, www.fetalmedicine.com/fmf//
FMF-English.pdf)
Although not explicitly stated, this equivalency of crown
rump length (CRL) and gestational age (GA) appears to
be based on the formula of Robinson and Fleming (1975)
because this appears to give the closest match among
the most commonly used conversion formulae available
(see Table). But many MFM centers often do use the
alternative conversions - including the newly
recommended equation of Loughna et al (2009). The
Table illustrates that there are differences for the various
available formulae and these differences are most
apparent for higher CRL pregnancies. The curve of
Daya (1993) appears to be an outlier at higher CRL
values but the other curves also vary by up to 4 days.
PSP 2011 VOL 16
≥ 45mm ≤ 84mm
≥ weeks + days ≤ weeks + days
Loughna et al, 2009 11 + 2 14 + 1
Kuhn et al, 1995 11 +2 14 + 0
Daya 1993 11 + 3 13 + 3
Hadlock et al 1992 11 + 3 14 + 2
5.
Nelson 1981 11 + 1 14 + 3
Drumm 1977 11 + 2 14 + 2
Robinson and Fleming, 1975 (a) 11 + 1 13 + 6
Robinson and Fleming, 1975 (b) 11 + 0 14 + 0
(a) Based on the “point estimate“ formula
(b) Based on a modified formula as described by Pedersen
(1982)
The use of alternative conversions from CRL to GA
poses several problems. First, it creates an ambiguity
as to which patients are within an acceptable range for
NT or serum test measurement. Should we use 45-84
mm or 11-13 weeks 6 days? If the latter, which curve
should we use? Second, imprecise risk estimates can
arise because the serum test normal median values
(and sometimes NT medians) are based on GA rather
than CRL. A laboratory may construct its medians
based on data from heterogeneous conversions of CRL
to GA derived from multiple MFM practices. It will then
convert patients’ results into MoMs on the basis of that
heterogeneous data. But an individual woman’s GA was
based on a single method for dating that may depart
from the consensus. Therefore, the MoMs can be
inaccurate. Third, the statistical parameters in the risk
calculation algorithm are also GA dependent reflecting
the differences in efficacy of the screening at different
times. An individual risk calculation could therefore also
be based on statistical parameters that are not optimal
for that particular patient. These difficulties will mostly
affect cases that are screened close to the upper CRL/
GA limit.
The problems could be minimized if normal median
values and statistical parameters were routinely
developed from a regression of the measurements
against CRL rather than GA. It would also be very
helpful if the Fetal Medicine Foundation FMF) and the
Nuchal Translucency Quality Review (NTQR) program
would provide a single recommendation for CRL/GA
conversion and redefine (or reiterate) the recommended
range for NT measurement.
1) Daya. 1993. Am J Obstet Gynecol. 168:903-8
2) Drumm 1977. BJOG 84;2-5.
3) Hadlock et al,1992. Radiology. 182;501-505.
4) Kuhn et al, 1995. Am J Obstet Gynecol. 172;32-5.
5) Loughna et al, 2009. Ultrasound. 17;161-7.
6) Nelson 1981. J Clin Ultrasound. 9;67-70.
7) Pedersen 1982. BJOG. 89;926-30.
8) Robinson and Fleming, 1975. BJOG 82;702-10.
Peter Benn (benn@nso1.uchc.edu)
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PRENATAL SCREENING PERSPECTIVES
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Prenatal Screening Perspectives
Newsletter for the International Prenatal Screening Group
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Email: info@ipsgroup.org.uk
PSP 2011 VOL 16
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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
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The DELFIA Xpress instrument and DELFIA Xpress screening assays are not available in the USA.

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