1

“GR.T. POPA”

UNIVERSITY OF MEDICINE AND PHARMACY

-IASI -

Obstetrics and Gynecology Department

DOCTORATE THESIS

Contributions to early diagnosis of

fetal abnormalities

ABSTRACT

Scientific coordinators:

Academician Professor Dr. Florentina Z. Pricop

Professor Dr. Kypros H. Nicolaides (University of London)

Doctorate candidate

Oana-Teodora Chelemen

Obstetrics-Gynecology specialist dr.

Iasi - January 2011

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Contents Page

I . Introduction 5

1. Scientific and technical context 5

2. Fetal anomalies – definition, nosology 5

3. Aims of the thesis 6

4. Acknowledgements 6

II. General part 6

1. Arguments for the choice of thesis title 6

2. Updated knowledge in the field of fetal anomalies 8

2.1. Etiology 8

2.2. Mechanisms 9

2.3. Classification 10

2.4. Clinical manifestations 10

2.5. Algorithm of prenatal diagnosis 11

2.6. Prophylaxis of genetic fetal anomalies 12

III. Personal clinical research part 13

1. The protocol of research 13

2. Working stages 13

3. Research objectives 13

4. Research method and techniques used in the algorithm of early prenatal diagnosis 14

5. Clinical material studied to fulfill the research objectives; results and discussions 14

5.1. to improve the performance of first trimester screening for fetal anomalies 14

5.2. to more correctly predict early preeclampsia with fetal growth restriction 16

5.3. to define the performance of first trimester screening for euploid fetal anomalies 20

5.4. to prove the contribution of prenatal diagnosis in reducing congenital anomalies 21

6. Encountered difficulties and possible error sources 22

7. Original contributions 23

8. Results of personal research presented as scientific papers 37

9. New objectives for research 38

10. Conclusions of the thesis 39

Selective references 42

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ABBREVIATIONS

AA/S-aortic atresia/stenosis

Ac-anticorp

ADAM12-A Disintegrine And Metalloprotease

Ag-antigen

ARSA-aberrant right subclavian artery

AVSD-atrio-ventricular septal defect

αFP-alpha fetoprotein (s-in serum, AL- in amniotic liquor)

BMI-body mass index

BP-blood pressure

βhCG- beta human chorionic gonadotropin

CAH-congenital adrenal hyperplasia

cff DNA, RNA-cell free fetal nucleic acids

CI-confidence interval

CMV-cytomegalovirus

CoAo-coarctation of the aorta

CRL-crown rump length

CVS-chorionic villus sampling

DHEA-dihidroepiandrosteron

DILV-double inlet left ventricle

DNA-ADN

DORV-double outlet right ventricle

DR-detection rate

DV-ductus venosus

E3-estriol

EUROCAT-European Registry of Congenital Abnormalities and Twins

FA-fetal anomalies

FGF –fibroblast growth factor

FGR-fetal growth restriction

FHR –fetal heart rate

FISH-flurescence in situ hybridization

FMFA-fronto-maxillo-facial angle

FNR-false negative rate

FPR-false positive rate

GH-gestational hypertension

HIV-human immunodeficiency virus

HLHS-hypoplasticleft heart syndrome

Ig-immunoglobulin

IGFBP-insulin like growth factor binding protein

IT-intacranial translucency

IUGR-intrauterine growth retardation

IVF-in vitro-fertilization

KCH-King’s College Hospital

L-PI-lowest pulsatility index (in uterine arteries)

MA/S-mitral atresia/stenosis

MAP-median arterial pressure

m.c.a-middle cerebral artery

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MLPA-multiplex ligation-dependent probe amplification

MoM-Multiple of Median

MRI-magnetic resonance imaging

NB-nasal bone

NGS-next generation sequencing

NICE-National Institute for Clinical Excellence

NIPD-non-invasive prenatal diagnosis

NT-nuchal translucency

PA/S-pulmonary atresia/stenosis

PAPP-A-Pregnancy Associated Plasma Protein A

PCR-polimerase chain reaction

PDGF-R –platelet derived growth factor -receptor

PE-preeclampsia

PI-pulsatility index

PlGF-placental growth factor

PLSVC-persistent left superior vena cava

r-correlation coefficient

R2- coefficient of multiple determination

RAA-right aortic arch

ROC-receiver-operating characteristics

SD-standard deviation

Sdr.-syndrome

SEL-systemic erythematous lupus

SGA-small for gestational age

T13,T18,T21-trisomy 13,18,21

TA/D/S-tricuspid atresia/dysplasia/stenosis

TGA-transposition of the great arteries

TGFα,β-transforming growth factor

TNF-tumor necrosis factor

TNF-R1–tumor necrosis factor receptor

TOF-tetralogy of Fallot

TR-tricuspid regurgitation

TOP-termination of pregnancy

TOPFA- termination of pregnancy for fetal anomaly

TORCH-toxoplasma, rubella, CMV, herpes virus

UtAs-uterine arteries

VEGF-vascular endothelial growth factor

VSD-ventricular septal defect

WHO-World Health Organisation

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I. INTRODUCTION

1. Scientific and technical context

The pathology encountered in medical practice, through its psychological and financial consequences – on

individual, family, society - challenged the specialists, thus becoming the main field of medical research.

Research results are reflected in the continuous scientific progress (in XXIst century – in genetics,

immunology, molecular biology -101) with impact on the knowledge of risk factors and pathogenesis, on

discovering new techniques for early diagnosis, correct prognosis prediction and more efficient curative and

prophylactic therapy.

With the principal objective – to ensure maternal and fetal morphological and physiological integrity while

minimising perinatal risks (221;355), Obstetrics increasingly benefits of advanced investigation methods

(265) for pregnancy evolution surveillance including prenatal diagnosis of fetal anomalies as means of

reducing perinatal mortality and morbidity. Fetal biological characteristics determining the quality of future

children’s lives (56;392) that influences population’s health on long term, become medical research field

(265).

There is a worldwide preoccupation for the prevention of genetic diseases (83). Among genetic screening

programs there is antenatal screening for fetal anomalies (83;159). Primary prevention of genetic disorders

(82;83) comprises preconception care (232;252) of high risk couples and post conception prenatal care

(83;245;265;301;356) as components of prenatal programme (356) detecting high risk pregnancies for

adverse outcome (220;221).

2. Fetal anomalies – definition, nosology (with impact on the frequency of fetal anomalies reported in

different studies -29).

-Anomaly: pathological variation of shape, structure, position of an anatomical element, variation that is out

of the interval offered by mean+/-2SD.

-Fetus: product of conception after 8 weeks (since conception) when it starts to present human characteristics

(314).

Fetal anomalies include: structural defects (macro-/microscopic) (malformations, fetal growth restriction

cases) as well as molecular, functional and metabolic disorders of the fetus.

Congenital anomalies represent morphological anomalies present at birth.

Fetal growth restriction represents an anomaly of fetal growth and development, defined by fetal growth

below 10th

percentile (or below 2 SD from mean) for the gestational age (278). It presents two main forms:

early (before 20 weeeks’gestation) is hypoplastic; late (after 28 weeks’gestation) is hypotrophic;

intermediate, rare (between 20 and 28 weeks), is a combination of hypoplastic, hypotrophic (270).

SGA describes neonates whose birth weight is at least 2 SD below the mean for the gestational age. Some

publications define SGA as a birth weight below the 3rd, 5th, or 10th percentiles for gestational age;

however,-2 SD is likely to capture the majority of infants with impaired fetal growth (323a).

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The term intrauterine growth retardation (IUGR) or fetal growth restriction (FGR) is often used

synonymously with the term SGA. However, because IUGR implies an underlying pathological process that

prevents the fetus from achieving its growth potential, its use should be restricted to describing infants whose

small size can be attributed to a specific cause and whose poor prenatal growth has been confirmed by

several anomalous ultrasound intrauterine growth assessments (323a).

Fetal anomalies significantly determine perinatal morbidity and mortality (124;128;303;323;355) which are

also influenced by other two components of medical system:

- the quality of obstetric care at birth

- the competence of surveillance of pregnancy evolution and fetal biological qualities (56) through antenatal

care (220;221) to detect high risk pregnancies for adverse outcomes (fetal anomalies, fetal growth

restriction), intensify their monitoring and optimise the result (125;222;355).

3. Aims of the Thesis

The aims of the thesis are:

- improvement of current performance of early screening for fetal anomalies,

- more correct prediction of pregnancy evolution to complications (i.e.fetal growth restriction),

- to define the detection rates of morphopathological subgroups of euploid structural fetal anomalies in the

first trimester,

- to demonstrate the influence of prenatal diagnosis in reducing congenital anomalies.

4. Acknowledgements

To fulfill thesis objectives, the clinical research was completed in The Harris Birthright Research

Centre for Fetal Medicine, King’s College Hospital, London – renowned worldwide, under Professor Dr.

Kypros H. Nicolaides leadership, to whom I express my gratitude for support and deontological collaboration

like in Hippocratic oath “I will always have respect and gratitude for my professors”.

I especially thank to Professor Dr. Florentina Pricop, as she facilitated the continuation of my research career

(started since the third year of medical school in Physiopathology Department under Professor Dr. Veronica

Colev’s guidance) in Obstetrics field.

“Next to whom you stay in the formation period counts enormously for your future” (267).

II. GENERAL PART

The preliminary period of personal research consisted in literature search in order to know the current stage

of knowledge in Fetal Medicine, to choose the thesis title, establish its objectives, write the research protocol

according to the model (178).

1. ARGUMENTS FOR THE CHOICE OF THESIS TITLE

In choosing thesis title I was determined by at least three reasons:

1.1.The importance of fetal anomalies field for public health (263) – based on its major social and

economical impact, quantified in statistically significant indicators – such as their prevalence, perinatal

and maternal mortality and morbidity (32;128;222).

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The prevalence of malformative fetal anomalies (110;355;383) is evaluated as follows:

overall about 8% live births;

major fetal anomalies: 2-5% live births (127;237) and 2-3% children under 5 years old

(miscarriages, stillbirths, TOPFA were not included in this calculation -120; when they are included, the

frequency for major fetal anomalies becomes 12% -323) with variations depending on: registering system,

diagnosis possibilities, geographical area, ethnical and cultural particularities (355).

minor fetal anomalies: 15% live births (124) – without vital risk, they raise aesthetic or functional

issues (303). Frequently they are associated between them or with the major ones –the more minor fetal

anomalies, the more likely their association with major ones so that they can be used as markers for the major

fetal anomalies (268). Such associations are classified as poly-malformations and they account for 10-20% of

the total malformative fetal anomalies (356).

In Romania, there are 2500-3000 severely handicapped children born per year (355) out of which

1000 - 1200 have major cardiac anomalies that require neonatal surgical repair (99) possible only in about

half of them in Tg.Mures and Bucharest centres, with great social and familial impact (355). In Iasi, between

1986-1999, congenital anomalies represented 26,52%o live births (128) and in Bucharest, in 1987 they were

8,11% (410).

After nuclear accident in Cernobil (year 1986) there was an important increase of congenital malformations

frequency: in year 2000, in Iasi this was 8,82% live births whereas in Bucharest - 11,8% live births, with

alarming medical and social implications (410).

The severity of major structural fetal anomalies results from perinatal mortality (defined as number of

stillbirths and deaths in the first week of life per 1000 live births) and postnatal morbidity.

The perinatal mortality rate for major morphological fetal anomalies is significant – of about 6%

newborns with congenital anomalies (355) that can vary according to:

prenatal diagnosis through its components:

-etiology: the chromosomal fetal anomalies mainly lead to intrauterine fetal death between detection and

term (20% of trisomy 21, 80-90% of trisomies 18,13 and 100% of triploidies -360) but also postnatal

mortality (80% of Down’s syndrome cases die within first months of extrauterine life -128).

-morphopathology: the severity of fetal anomalies is influenced by topographic subgroups (central nervous

system anomalies have 27% perinatal mortality, cardiac defects - 23% perinatal mortality, mainly neonatal

deaths -356), lesions number (for poly-malformed neonates, mortality is 20%), lesions dimension

(diaphragmatic hernia, megacystis -143;159;187).

policy for termination of pregnancy for fetal anomalies (TOPFA): in EUROCAT countries, higher

perinatal mortality in Irlanda (24%) and Malta (23%) where TOPFA is illegal (110).

Pathologic entities responsible for perinatal mortality:

Malformative fetal anomalies

- In Romania, structural fetal anomalies cause 20-30% of infantile mortality – the second cause after

prematurity -53% in year 2005 (408); in Romania about 20.000 premature neonates are born every year.

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-In European Union countries, morphopathological fetal anomalies represent the principal cause of perinatal

mortality (and accounts for ¼ of total perinatal deaths - 363).

Fetal growth restriction represents the principal cause of intrauterine fetal death (60-70% - 221;265).

41% growth restricted fetuses die neonatally (74;221).

Perinatal mortality is due to six obstetrical complications: prematurity, multiple pregnancy, hypertension in

pregnancy, retroplacental hematoma, placenta praevia, breech presentation (356).

TOPFA is excluded from perinatal mortality statistics (110).

Prematurity and preeclampsia (PE) with fetal growth restriction are considered the first two causes of

perinatal mortality (218a) and therefore they have been studied in King’s College Hospital within a program

of early prediction of pregnancy complications (278;279;289;292;298).

1.2. At this moment in time, the importance of fetal anomalies field for public health and medical

research (355) is reflected in the intense research activity developed in organised structures with periodical

results published in specialty journals and communicated within Fetal Medicine World Congresses.

1.3. In the latest period, research progress influenced the quality of medical care especially in

Obstetrics (200) through continuously advancing technologies and strategies of prenatal diagnosis

(20;47;216;233;252;268;356) as well as intrauterine/neonatal treatment (98;245;362;391).

In Romania (non included in EUROCAT) there are units of prenatal diagnosis of level I, II, III (127;129;356)

only, without any Fetal Medicine Centre for intrauterine therapy. In addition, there are no antenatal screening

programs, while scans are reserved to selected high risk pregnancies (47). In year 2003, in Romania, there

were about 50% of pregnant women only who underwent ultrasound examinations (359). FISH technique for

the prenatal diagnosis of aneuploidies was introduced in 2004 in Iasi medical centre (127). Until 1999,

prenatal diagnosis for Down’s syndrome has never been performed in Iasi (128).

2. UPDATED KNOWLEDGE IN THE FIELD OF FETAL ANOMALIES

Malformative fetal anomalies have their origin in embryogenesis defects (days 13-56 of pregnancy -355) and

fetal maldevelopment. The implicated causes are divided into two groups of pathogenic factors: genetic and

exogenous, most of the cases having both categories involved (356).

2.1. Etiology of fetal anomalies:

I. for malformative fetal anomalies there are only 50% known causes, the main cause being genetic - of up

to 45% (127):

A. Genetic defects cause 15% of major structural fetal anomalies: 7% by chromosomal abnormalities and 8%

by gene mutations (323); according to other authors, aneuploidies are predominant (20;129).

B. Exogenous teratogens determine 5-10% of major fetal anomalies; they act on genetic material or interfere

with embryo/fetus morphogenesis (124;356).

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C. 25% are induced by multiple factors, when the genetic predisposition is revealed by an exogenous teratogen

action that transforms predisposition into disease (83;127). Certain metabolic diseases are diagnosed by

neonatal screening whereas many rare genetic conditions are only detected in childhood (134).

D. For the remaining 50%, the causes are unknown (323;355); de novo spontaneous gene mutations are

considered involved, thus there is a risk of fetal genetic condition for every pregnancy- the older the parents,

the higher the risk (134;355;390).

II. for fetal growth restriction (excepting the constitutional form) which has 2 groups (74;270) with known

causes:

- early growth restriction, with ovulatory causes (20-30%), mainly genetic;

- late growth restriction, with maternal circulatory causes (early preeclampsia) (70-80% ) (24;74). There are

still 10-30% of fetal growth restriction cases of unknown etiology (74).

The risk factors for fetal anomalies are (18;220):

-maternal characteristics: older age than 37 years (355;359) has high risk of meiotic nondisjunction and

aneuploidy (134;391); after 40 years of age there is a higher risk for fetal death – of 5 times greater than for

a 20-30 years old pregnant woman (220); ethnic origin (black race) and the use of ovulation inducers are

risk factors for early preeclampsia and fetal growth restriction;

-family/obstetric history of genetic defects, hypertension, preeclampsia;

-medical history with changes in hormonal intrauterine environment (ex. hypothyroidism -230, diabetes

mellitus -365), in metabolic conditions - obesity (365), malnutrition (folic acid, iodine deficiency) or in

placental circulation (chronic high blood pressure, systemic eritematous lupus, antiphospholipidic syndrome)

-pregnancy specific pathology:

- placental insufficiency with early (before 20 weeks) symmetrical fetal growth restriction of ovulatory

causes (20-30% -mainly genetic causes, rarely chorioamniotitis, implantation in a poorly vascularised uterine

area, velamentous umbilical cord insertion, single umbilical artery); late (after 28 weeks) asymmetrical fetal

growth restriction of maternal circulatory cause (70-80%) in early preeclampsia (74;270); intermediate fetal

growth restriction (20-28 weeks), rare,

-multiple pregnancy (with increasing frequency by “iatrogenic epidemy“ induced through assisted

reproduction techniques -389) has an increased risk of malformations and fetal growth restriction,

- oligohydramnios is frequently associated to fetal growth restriction and has a high risk of limbs defects

(56).

2.2. Mechanisms of fetal anomalies (124;323):

A. Malformative fetal anomalies occur predominantly during embryogenesis (critical period of pregnancy)

as a consequence of:

-genetic disorders leading to structural defects

-exogenous teratogens responsible for gene mutations resulting in enzyme deficiency and malformations.

Teratogenic effect is influenced by three factors:

a) Exposure moment: embryogenesis stage – vulnerable period of intense cell differentiation, when the

majority of fetal anomalies are induced (323). In fetal stage, the sensitivity to teratogens decreases

progressively (323).

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b) Preferential action on histological targets (the most sensitive organs); each organ has a certain

vulnerable moment (124).

c) Teratogenic effect depends on:

-teratogen type, with particular action mechanism,

-dose

B. Fetal growth restriction is an anomaly of fetal growth and development with fetal weight below the

10th

centile or less than 2 SD from mean for gestational age (270;278).

There are two main forms of fetal growth restriction:

-early (before 20 weeks), symmetrical, by mitosis inhibition, leading to reduced cell number (the

hyperplastic period is interfered) (270);

-late (after 28 weeks) (74;270), asymmetrical, by reduced transplacental uptake (hypertrophic period is

affected) resulting in cell volume reduction, with compensatory cerebral vasodilatation (i.e. brain sparing

effect) proved by Doppler studies of fetal circulation (PI for uterine, umbilical, middle cerebral arteries);

-intermediate (between 20-28 weeks), rare.

2.3. Classification of fetal anomalies (124;323) according to multiple criteria (127):

● pathogenic mechanism

1. malformation,

2. disruption,

3. deformation,

4. complex anomalies,

5. dysplasia,

6. agenesis,

7. hypotrophy/hypertrophy (e.g. late fetal growth restriction -74),

8. hypoplasia/hyperplasia (e.g. early fetal growth restriction -270),

● affected organs number: isolated and multiple;

● severity: major (lethal/disabling, requiring therapeutic intervention) and minor (no vital risk, with

functional/aesthetic consequences).

2.4. Clinical manifestations of fetal anomalies comprise:

-miscarriages: 80% occur in the first trimester (out of which 61,5% are caused by chromosomal

abnormalities with 10% risk of recurrence; e.g. the commonest ones are trisomies 13, 16, 18, 21, triploidy,

monosomy 45X) (356);

-fetal deaths (5% caused by aneuploidies -356) with stillbirths;

-live births with malformed neonates (1% caused by aneuploidies -127), fetal growth restriction;

-functional disorders: neurological (e.g. Duchenne disease), endocrine (e.g. congenital hypothyroidism),

metabolic (e.g enzymatic defects) (323;356;363).

Congenital malformations are presented according to their frequency (355):

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1.cardiovascular anomalies – the most frequent fetal anomalies -10%o (6-75%o) live births (268;323;329);

their etiology is multifactorial (90% of cases), genetic (8%o associated with facial defects in genetic

syndromes, e.g. Down’s syndrome, Di George syndrome) and induced by exogenous teratogens (2%o).

Regardless their complexity, they are compatible with intrauterine life (355) but in neonates they manifest

dramatically with 30% of total neonatal deaths.

2. central nervous system anomalies – up to 10%o live births

3. urinary tract anomalies - 5%o live births

4. defects of abdominal wall and digestive tract - 2%o live births

5. defects of limbs – up to 1%o live births (303).

2.5. Algorythm of prenatal diagnosis

A method of reducing the frequency and severity of congenital anomalies and fetal growth restriction is

represented by prenatal diagnosis (129;363) as integrative component of pregnancy evolution surveillance in

order:

-to accurately identify malformative fetal anomalies, precisely establish pregnancy prognosis for

management options (intensive ultrasound follow-up, intrauterine/neonatal treatment (29;9) - in the first

intention, or selective termination (120) - in the second, for cases of fetal anomalies) thus preventing live

births with defects (56;83),

-to correctly predict pregnancy complications such as early preeclampsia with fetal growth restriction in

order to save fetuses from intrauterine or neonatal death (29;56;83;265).

The optimum moment for prenatal diagnosis is (356):

►preconception: identification of risk factors (hereditary and exogenous) for fetal anomalies while aiming

for their primary prophylaxis,

►postconception: early prenatal diagnosis of fetal anomalies (positive results are obtained in 5% of

pregnancies when the affected couple is counselled to choose from management options according to the

severity of the defect and therapeutic possibilities - 83).

The standard algorithm of prenatal diagnosis comprises two stages:

a. Combined screening program to prospectively detect high risk pregnancies (104;220) for fetal

anomalies using:

►clinical parameters (178): maternal and gestational characteristics; familial, medical, obstetric history;

blood pressure, body mass index;

►complementary noninvasive investigations according to gestational age (268):

-biochemical tests (in maternal serum) (265):

-in the first trimester: PAPP-A, βhCG, PlGF;

-in the second trimester: α feto-protein, βhCG, estriol, inhibin A;

-ultrasound examination – with parameters that differ according to gestational age (20;36;266;355):

-in the first trimester: intrauterine/extrauterine pregnancy localization, singleton/multiple pregnancy, CRL (to

determine gestational age), fetal viability (FHR), markers for aneuploidies (NT, nasal bone, ductus venosus

and tricuspid Dopplers, fronto-maxillary-facial angle); detailed fetal and adnexal anatomical examination;

placental insertion (guide for invasive procedures); uterine/pelvic tumours; uterine arteries pulsatility index

(early screening for preeclampsia).

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-in the second trimester: fetal biometry (for detection of fetal growth restriction) and morphology; placental

and umbilical cord insertions, amniotic fluid volume; fetal movements; uterine arteries pulsatility index

(screening for preeclampsia); pelvic tumours.

-in the third trimester: fetal presentation and position, biometry, placental assessment (insertion, structure,

maturity), amniotic fluid volume, biophysical profile, Doppler velocimetry (pulsatility index and maximum

velocity) for uterine, umbilical and middle cerebral arteries, ductus venosus (with predictive value). The

obstetrician establishes delivery prognosis according to maternal and fetal parameters (301;390).

The current performance of combined screening for fetal aneuploidies is of 95% DR for fetal trisomy 21 with

5% FPR.

b. Etiologic diagnosis (in screen positive cases that opted to find out the underlying condition for fetal

defect - 268;356;391) by using:

b1. noninvasive tests:

- maternal serology for TORCH syndrome (in case of certain fetal defects, fetal growth restriction),

- genetic tests on fetal nucleic acids in maternal blood (cell free fetal DNA/RNA or DNA in fetal

erythroblasts through feto-maternal microchimerism) (114;239;358;403). Cell free fetal nucleic acids already

have two clinical applications (detection of fetal Rhesus status and of fetal gender) and they hold great

promise for the noninvasive prenatal diagnosis of aneuploidies and gene mutations to become clinically

available in the near future.

b2. invasive tests (offered to all women, but recommended when the risk for aneuploidy exceeds the risk for

miscarriage due to the procedure alone -83) to sample biological products allowing genetic and serologic

tests; according to gestational age they are:

- in the first trimester – chorionic villus sampling (CVS),

- in the second trimester – amniocentesis, cordocentesis (391).

A positive result in etiologic tests is followed by counselling for management option in a strategy of

preventing live births with defects (83;129;363).

2.6. Prophylaxis of genetic fetal anomalies (83;363) is of two types:

● primary by preconception counselling for

-prevention of de novo mutations by avoiding mutagens (e.g. reduction of parental age),

-prevention of transmission to offspring in high risk couples for genetic disorders,

-prevention of fetal anomalies in coulples with genetic predisposition by avoiding teratogens,

● secondary by prenatal diagnosis with impact on pregnancy management (termination of pregnancy for fetal

anomaly - TOPFA).

There are ethical controversies regarding TOPFA policy (13;83):

1. termination of pregnancy for fetal anomaly in cases of severely affected fetuses:

- pro-intervention argument – TOPFA is less distressful than a suffering human life,

- contra argument – fetal right for life,

2. termination of pregnancy in cases of moderately/mildly affected and possibly treatable fetuses:

-pro- intervention pleads the principle of respect for couple’s autonomy,

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-contra intervention is the fact that prenatal diagnosis is mainly performed to avoid life birth of neonates with

major congenital anomalies.

III. PERSONAL RESEARCH PART

1. The protocol of personal clinical research

The research protocol structure comprised (according to the model -178):

1. thesis title,

2. arguments for title choice,

3. research objectives,

4. rigorous research method:

- study type, used techniques,

- studied factors (prenatal diagnosis of fetal anomalies and their risk factors),

- rationale criteria (fetal anomalies, pregnancies outcomes),

5. studied clinical material:

- method of cases selection,

- inclusion criteria,

- collection of diagnosis tests results and pregnancies outcomes,

- statistical analysis of data,

- presentation of results through descriptive statistics means (tables, figures),

6. potential errors sources,

7. encountered difficulties,

8. ethical implications,

9. conclusions,

10. research results are presented as scientific articles (published or communicated),

12. budget,

13. work stages schedule,

14. references.

2. Work stages

2.1. continuously updating the knowledge in Fetal Medicine field by literature search in Medline database,

2.2. caring out the personal research using new strategies in prenatal diagnosis algorithm – accessible in

King’s College Hospital in order to further improve detection of fetal anomalies while minimising false

positive and negative results (20;265),

2.3. performing statistical analysis of data for valid results (18;38;178) used in thesis conclusions.

3. The research objectives

3.1. To supplementary improve the performance of first trimester combined screening in detecting fetal

anomalies by searching new markers and their combinations,

3.2. To more correctly predict the pregnancy evolution to complications such as early preeclampsia

with fetal growth restriction (288;292),

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3.3. To define the current performance of first trimester ultrasound in screening for euploid fetal

anomalies,

3.4. To prove the contribution of prenatal diagnosis in reducing frequency and severity of congenital

anomalies,

3.5. To compare our research with other studies while analysing the differences in methodology and results

(in order to decide which methods are the most efficient),

3.6. To respect the fundamental principles of ethical medical research,

3.7. To ensure valid results through rigorous methodology, statistical analysis of data, large study

populations.

3.8. Research results are presented as scientific articles (published and communicated) and they contributed

to an increase in the experience and specialty competence of the doctorate candidate (along with Fetal

Medicine Foundation certifications).

4. Research method and used techniques

Thesis research was carried out within The Harris Birthright Research Centre for Fetal Medicine, (King’s

College Hospital, London) that ensured the appropriate framework: space, advanced technologies, large

obstetrical population, all necessary resources, trained teams in scientific research of high standards under

direct and close supervision of Professor dr. Kypros Nicolaides.

Personal research followed the algorithm of prenatal diagnosis for fetal anomalies in two stages:

4.1. combined screening for aneuploidies, structural fetal anomalies, calculation of patient specific risk for

adverse pregnancy outcome (fetal growth restriction in early preeclampsia),

4.2. etiologic diagnosis in screen positive cases that opted for defining the underlying condition of fetal

defect.

5. Clinical material studied in order to fulfil research objectives. Results and discussions.

Research results were presented in 12 original scientific papers (seven published in international journals,

two - in the official journal of Romanian Society of Obstetrics and Gynecology, three presented in the 7th

and

8th

World Congresses of Fetal Medicine: Sorrento Italy 2008, Portoroz Slovenia 2009) performed on large

number of prospectively investigated cases.

5.1. Clinical material studied to further increase the performance of first trimester screening for fetal

anomalies.

We searched for new biological and ultrasound markers for first trimester diagnosis of fetal anomalies in

three studies:

5.1.1. Study 1. Maternal serum ADAM12 (A Disintegrin And Metalloprotease) in chromosomally

abnormal pregnancies at 11-13 weeks (282).

Objective: To investigate the potential value of ADAM12 (A Disintegrin And Metalloprotease) in first-

trimester screening for trisomy 21 and other major chromosomal abnormalities.

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Methods: This was a case-control study performed between March 2006 and March 2007. In this interval the

first trimester screening for aneuploidies was carried out on 10,641 singleton pregnancies representing the

base cohort study population, wherein the present case-control study was nested.

The maternal serum concentration of ADAM12 at 11+0

-13+6

weeks was measured in 272 euploid and 136

chromosomally abnormal pregnancies, including 49 of trisomy 21, 28 of trisomy 18, 20 of trisomy of 13, 29

of Turner syndrome and 10 of triploidy. Regression analysis was used to determine which of the factors

among maternal characteristics and fetal crown rump length (CRL) were significant predictors of ADAM12

in the euploid group and from the regression model the value in each case and control was expressed as a

multiple of median (MoM). The levels of ADAM12 MoM were compared in cases and controls and were

assessed for association with free β-hCG MoM and (pregnancy-associated plasma protein A) PAPP-A MoM.

Results: Multiple regression analysis in the euploid group demonstrated that for ADAM12 significant

contributions were provided by fetal CRL, maternal weight and ethnic origin. The median ADAM12 MoM in

trisomy 21 (0.961 MoM) was not significantly different from the euploid group but in trisomy 18 (0.697

MoM), trisomy 13 (0.577 MoM), triploidy (0.426 MoM) and Turner syndrome (0.747 MoM) the levels were

reduced. In both the euploid and aneuploid pregnancies, there was a significant association between log

ADAM12 MoM and log free ß-hCG MoM and log PAPP-A MoM.

Conclusion: Maternal serum ADAM12 concentration at 11+0

-13+6

weeks of gestation is unlikely to be useful

in first trimester screening for chromosomal abnormalities.

Of note, the literature data were not confirmed by our large study which is an example of the need of critical

selection of published information (18).

The fundamental importance of the size of study population (as large as possible, in order for the results to

be valid and possibly generalised to the source population) was mathematically demonstrated in Study 12

(see page 22) and emphasised in the rest of our studies (1-11) by comparison with other scientific papers.

5.1.2. Study 2. ADAM12 and PlGF at 11-13 weeks. Screening for aneuplodies (293).

We present PIGF data only, as those on ADAM 12 were demonstrated in the previous study.

Objectives: To investigate the potential value of maternal serum placental growth factor (PlGF) in first-

trimester screening for trisomy 21 and other major chromosomal abnormalities.

Methods: The maternal serum concentration of PlGF at 11 to 13 weeks was measured in 609 euploid and

175 chromosomally abnormal pregnancies, including 90 with trisomy 21, 28 with trisomy 18, 19 with

trisomy 13, 28 with Turner syndrome and 10 with triploidy. The levels of PlGF were compared in cases and

controls, and were assessed for association with free β-human chorionic gonadotropin (β-hCG) and

pregnancy-associated plasma protein-A (PAPP-A).

Results: Logistic regression analysis demonstrated in the euploid group that significant independent

contributions for log PlGF were provided by fetal crown–rump length, maternal weight, cigarette smoking

and ethnic origin; after correction for these variables the median multiple of the median (MoM) PlGF was

0.991. Significantly lower values were observed in pregnancies with trisomy 21 (0.707 MoM), trisomy 18

(0.483 MoM), trisomy 13 (0.404 MoM), triploidy (0.531 MoM) and Turner syndrome (0.534 MoM).

Significant contributions in the prediction of trisomy 21 were provided by maternal age, serum PlGF, PAPP-

16

A and free β-hCG, and the detection rates of screening with the combination of these variables were 70% and

80% at respective false-positive rates of 3% and 5%.

Conclusions: Maternal serum PlGF concentration at 11–13 weeks of gestation is potentially useful in

firsttrimester screening for trisomy 21 and other major chromosomal abnormalities.

5.1.3. Study 3. Contribution of ductus venosus Doppler in first trimester screening for major cardiac

defects (61)

Objective: To determine whether assessment of ductus venosus flow at 11-13 weeks’ gestation improves the

detection rate of cardiac defects achieved by screening with nuchal translucency (NT) thickness.

Methods: Prospective first-trimester screening for aneuploidies, including measurement of fetal NT and

assessment of ductus venosus flow. The performance of different combinations of increased fetal NT and

abnormal blood flow in the ductus venosus in screening for major cardiac defects was examined.

Results: The study population of euploid fetuses included 85 cases with major cardiac defects and 40,905

with no cardiac defects. The fetal NT was above the 95th

and above the 99th

centile in 30 (35.3%) and 18

(21.2%), respectively, of the fetuses with cardiac defects and in 1,956 (4.8%) and 290 (0.7%), respectively,

of those without cardiac defects. Reversed a-wave was observed in 24 (28.2%) of the fetuses with cardiac

defects and in 856 (2.1%) of those with no cardiac defects. Specialist fetal echocardiography for cases with

NT above the 99th

centile and those with reversed a-wave, irrespective of NT, would detect 38.8% of major

cardiac defects at an overall false positive rate of 2.7%.

Conclusions: Assessment of ductus venosus flow improves the performance of NT screening for cardiac

defects.

5.2. Clinical material studied for more correct first trimester prediction of fetal growth restriction in

early preeclampsia in order to accordingly adjust the intensity of the antenatal care while targeting

reduction of adverse pregnancy outcome (studies 4-9).

5.2.1. Study 4. First-Trimester Maternal Serum a Disintegrin and Metalloprotease 12 (ADAM12) and

Adverse Pregnancy Outcome (283)

Objective: To examine the possible association of maternal serum a disintegrin and metalloprotease (ADAM

12) in the first trimester of pregnancy and subsequent development of preeclampsia, delivery of small for

gestational age (SGA) neonates, and spontaneous preterm delivery.

Methods: The maternal serum concentration of ADAM 12 at 11 to 13 weeks was measured in 128 cases of

preeclampsia, 88 cases of gestational hypertension, 296 cases with SGA neonates, 58 cases of spontaneous

preterm delivery, and 570 controls. Regression analysis was used to determine which of the maternal factors

and fetal crown rump length were significant predictors of ADAM 12 in the control group, and from the

regression model the value in each case and control was expressed as a multiple of median (MoM). The

levels of ADAM12 MoM were compared in cases and controls.

Results: In the control group the concentration of ADAM 12 increased with fetal crown rump length,

decreased with maternal weight and was higher in African-American than in white women. There was a

significant association between ADAM 12 and pregnancy-associated plasma protein A (r=0.417, P<0.001)

and between each metabolite and birth weight percentile (r=0.176, P<0.001 and r=0.109, P=0.009) and

17

although in the SGA group, the median ADAM 12 concentration (0.848 MoM) was lower than controls

(P<0.001), measurement of ADAM 12 did not improve detection of SGA achieved by maternal history and

PAPP-A (DR for SGA by maternal history, ADAM 12, PAPP-A was 24.3% compared to 24.7% by history

and PAPP-A).

In pregnancies complicated by preeclampsia (0.954 MoM), gestational hypertension (1.013 MoM) and

spontaneous preterm delivery (1.048 MoM), ADAM 12 levels were not significantly different from controls

(1.011 MoM).

Conclusion: Measurement of ADAM12 does not provide useful prediction of SGA, preeclampsia or

spontaneous preterm delivery.

5.2.2. Study 5. PAPP-A, ADAM12, PIGF at 11-13 weeks. Prediction of preeclampsia (294)

We present PIGF data only, as those on ADAM 12 were demonstrated in the previous study.

Objective: To investigate the potential value of maternal serum placental growth factor (PlGF) in first-

trimester screening for pre-eclampsia (PE).

Methods: The concentration of PlGF at 11 to 13 weeks’ gestation was measured in samples from 127

pregnancies that developed PE, including 29 that required delivery before 34 weeks (early PE) and 98 with

late PE, 88 cases of gestational hypertension (GH) and 609 normal controls. The distributions of PlGF

multiples of the median (MoM) in the control and hypertensive groups were compared. Logistic regression

analysis was used to determine the factors with a significant contribution for predicting PE.

Results: In the control group significant independent contributions for log PlGF were provided by fetal

crown–rump length, maternal weight, cigarette smoking and racial origin, and after correction for these

variables the median MoM PlGF was 0.991. In the early-PE and late-PE groups PlGF (0.611 MoM and 0.822

MoM, respectively; P < 0.0001) and pregnancy associated plasma protein-A (PAPP-A) (0.535 MoM; P <

0.0001 and 0.929 MoM; P = 0.015, respectively) were reduced but in GH (PlGF: 0.966 MoM; PAPP-A:

0.895 MoM) there were no significant differences from controls. Significant contributions for the prediction

of PE were provided by maternal characteristics and obstetric history, serum PlGF and uterine artery

pulsatility index (PI) (mean value of PI for the 2 uterine arteries) and with combined screening the detection

rates for early PE and late PE were 86.2% and 49%, respectively, for a false-positive rate of 10%.

Conclusion: Effective screening for PE can be provided by a combination of maternal characteristics and

obstetric history, uterine artery PI and maternal serum PlGF at 11 to 13 weeks’ gestation.

5.2.3. Study 6. Maternal plasma P-selectin at 11 to 13 weeks of gestation in hypertensive disorders of

pregnancy (4)

Objective: To determine if development of preeclampsia (PE) is preceded by altered maternal plasma P-

selectin and if the levels are related with uterine artery pulsatility index (PI).

Methods: Plasma P-selectin and uterine artery PI were measured at 11-13 weeks in 121 cases that

subsequently developed PE, 87 cases that developed GH and 208 unaffected controls.

18

Results: In the PE group the median multiple of the median in controls (MoM) P-selectin and uterine artery

PI were significantly increased (1.2 MoM and 1.3 MoM). There was no significant association between P-

selectin and uterine artery PI (mean value of PI for the 2 uterine arteries) in either the PE or control group.

Conclusion: In pregnancies that develop PE there is evidence of platelet activation from the first trimester.

However, there is no direct link between the degree of impaired placentation and platelet activation.

5.2.4. Study 7. Maternal risk factors for hypertensive disorders in pregnancy: a multivariate approach

(292)

Objective: The study aimed to develop prediction algorithms for hypertensive disorders based on

multivariate analysis of factors from the maternal history and compare the estimated performance of such

algorithms in the prediction of early preeclampsia (PE), late-PE and gestational hypertension (GH) with that

recommended by the National Institute for Clinical Excellence (NICE).

Methods: Logistic regression analysis was used to determine which of the maternal characteristics and

history had significant contribution in predicting early-PE, late-PE and GH.

There were 37 cases with early-PE, 128 with late-PE, 140 with GH and 8061 cases that were

unaffected by PE or GH.

Results: Predictors of early-PE were Black race, chronic hypertension, prior PE and use of ovulation drugs.

Predictors of late-PE and GH were increased maternal age and body mass index, and family history or

history of PE. Additionally, late-PE was more common in Black, Indian and Pakistani women.

The detection rates of early-PE, late-PE and GH in screening by maternal factors were 37.0, 28.9 and 20.7%,

respectively, for a 5% false positive rate.

Screening as suggested by NICE would have resulted in a false positive rate of 64.1% with detection rates of

89.2, 93.0 and 85.0% for early-PE, late-PE and GH, respectively.

Conclusion: Meaningful screening for hypertensive disorders in pregnancy by maternal history necessitates

the use of algorithms derived by logistic regression analysis.

5.2.5. Study 8. Hypertensive disorders in pregnancy: Screening by systolic, diastolic and mean arterial

pressure at 11-13 weeks (295)

Objectives: To examine the performance of screening for hypertensive disorders in pregnancy and to

compare systolic blood pressure (BP), diastolic BP and mean arterial pressure (MAP) measured by validated

automated devices in a large population of pregnant women at 11-13 weeks.

Design: Prospective observational study.

Setting: Routine booking visit.

Participants: 9,149 women at 11-13 weeks of gestation.

Main Outcome Measures: Development of hypertensive disorders in pregnancy.

19

Methods: Maternal history was recorded and BP was measured by automated devices. The performance of

screening for preeclampsia (PE) and gestational hypertension (GH) by combinations of disease-specific

maternal factor-derived a priori risk with systolic BP, diastolic BP and MAP was determined.

Results: There were 8,061 (96.4%) cases that were unaffected by PE or GH, 37 that developed PE requiring

delivery before 34 weeks (early-PE), 128 with late-PE and 140 with GH. The systolic BP, diastolic BP and

MAP were significantly higher in early-PE, late-PE and GH than in the controls (p<0.0001). The systolic BP

was significantly higher in early-PE than in late-PE (p=0.008) and both systolic BP and MAP were

significantly higher in early PE than in GH (p<0.01). The best performance in screening was provided by

MAP. The detection rate of early-PE at a 10% false positive rate increased from 47% in screening by

maternal factor-derived a priori risk alone to 76% in screening by its combination with MAP. The respective

detection rates for late-PE increased from 41% to 52% and for GH increased from 31% to 48%.

Conclusion: The measurement of blood pressure can be combined with the maternal factor-derived a priori

risk to provide effective first-trimester screening for PE and GH.

5.2.6. Study 9. Prediction of preeclampsia: biophysical profile at 11-13 weeks (296)

This study presents screening performance for PE by uterine artery PI (A) and by combining uterine artery

Doppler with mean arterial pressure at 11-13 weeks (B).

A. Screening for PE by first trimester uterine artery Doppler

Objective: To examine the performance of screening for hypertensive disorders in pregnancy by a

combination of the maternal history derived a priori risk with the uterine artery pulsatility index (PI) and to

determine whether it is best in such screening to use the mean PI of the two arteries, the highest PI or the

lowest PI.

Methods: Prospective screening study for preeclampsia (PE) requiring delivery before 34 weeks (Early-PE),

late-PE and gestational hypertension (GH) in women attending for their routine first hospital visit in

pregnancy at 11-13 weeks of gestation. Maternal history was recorded and color flow Doppler was used to

measure the PI in the left and right uterine arteries. The performance of screening for PE and GH by a

combination of the a priori maternal risk factor determined in a previous study and the uterine artery PI was

determined.

Results: There were 8,061 (96.4%) cases that were unaffected by PE or GH, 37 (0.4%) that developed early-

PE, 128 (1.5%) with late-PE and 140 (1.7%) with GH. The lowest, mean and highest uterine artery PI were

significantly higher in early-PE and late-PE than in the controls (p<0.0001) and in early-PE than late-

PE.(p<0.0001). The lowest uterine artery PI was higher in GH than in controls (p=0.014). The best

performance in screening was provided by the lowest PI. The detection rate of early-PE at a 10% false

positive rate increased from 47% in screening by maternal factors alone to 81% in screening by maternal

factors and uterine artery PI. The respective detection rates for late-PE increased from 41% to 45% and for

GH increased from 31% to 35%.

Conclusion: The patient-specific risk for PE and GH can be derived by multiplying the a priori maternal risk

factor with the appropriate likelihood ratio from the lowest uterine artery PI.

B. Hypertensive disorders in pregnancy: screening by uterine artery Doppler imaging and blood

pressure at 11–13 weeks.

20

Objective: To examine the performance of screening for hypertensive disorders in pregnancy at 11-13 weeks

by a combination of the maternal history, uterine artery Doppler and blood pressure.

Methods: Prospective screening study for preeclampsia (PE) requiring delivery before 34 weeks (early-PE),

late-PE and gestational hypertension (GH) in women attending for their routine first hospital visit in

pregnancy at 11+0

-13+6

weeks of gestation. Maternal history was recorded, color flow Doppler was used to

identify the uterine artery with the lowest pulsatility index (L-PI) and automated devices were used to

measure the mean arterial pressure (MAP). The performance of screening for PE and GH by a combination

of the maternal factor-derived a priori risk, the uterine artery L-PI and MAP was determined.

Results: There were 8,061 (96.4%) cases that were unaffected by PE or GH, 165 (2.0%) that developed PE

including 37 that required delivery before 34 weeks (early-PE) and 128 with late-PE, and 140 (1.7%) that

developed GH. The MAP was higher in early-PE, late-PE and GH than in the unaffected group (p<0.0001)

and in early-PE than in GH (p=0.002). The uterine artery L-PI was significantly higher in early-PE and late-

PE than in the unaffected group (p<0.0001), in early-PE than late-PE or GH (p<0.0001) and in GH than in

the unaffected group (p=0.014). In screening by a combination of the maternal factor-derived a priori risk,

uterine artery L-PI and MAP the estimated detection rate, with 95% confidence interval, at a 10% false

positive rate was 89.2% (74.6%-96.9%) for early-PE, 57.0% (48.0%-65.7%) for late-PE and 50.0% (41.4%-

58.6%) for GH.

Conclusion: Effective screening for hypertensive disorders in pregnancy is provided by a combination of

maternal history, uterine artery Doppler and blood pressure at 11-13 weeks.

The current highest performance in screening for PE/GH by combination of maternal risk factors with

biophysical factors (mean arterial pressure, lowest/mean uterine artery PI) and different biochemical markers

is presented in Table I.

Table I. Highest detection rate (at 5% false positive rate) for early and late preeclampsia (PE), gestational

hypertension (GH) in combined screening (maternal history, biophysical factors and different biochemical

markers)

*results achieved with mean uterine artery PI

5.3. Current performance of ultrasound screening for euploid fetal anomalies at 11-13 weeks (study 10)

Study 10. Challenges in the diagnosis of fetal non-chromosomal abnormalities at 11-13 weeks (369)

Screening test Detection rate (%) at 5% false positive rate

Early PE Late PE GH Maternal history

+ MAP

+ lowest u.a. PI

+PAPP-A 83,8 42,2 35,7

+PAPP-A+PIGF

93,1*

35,7* (PIGF only)

18,3* (No biochemistry,

no uterine artery PI)

+PIGF/Activin-A+P-Selectin/ Activin-A 88,5 (PIGF only)

46,7 (PIGF, Activin A, P-Selectin)

35,3 (Activin A)

+PAPP-A+PIGF+placental protein-13+

soluble endoglin+inhibin-A+activin-A+

pentraxin-3+P-selectin

91,0* 60,9* ---------

21

Objective: To examine the performance of the 11-13 weeks scan in detecting non-chromosomal

abnormalities.

Methods: First-trimester screening for aneuploidies including basic examination of the fetal anatomy in

45,191 pregnancies. The findings were compared to those at 20-23 weeks and postnatal examination.

Results: Aneuploidies (n=332) were excluded from the analysis. Fetal abnormalities were observed in 488

(1.1%) of the remaining 44,859 cases with 213 (44%) of these detected at 11-13 weeks, 262 (53,7%) at 20-23

weeks and 13 (2,7%) postnatally. The early scan detected all cases of acrania, alobar holoprosencephaly,

exomphalos, gastroschisis, megacystis and body stalk anomaly, 77% of absent hand or foot, 50% of

diaphragmatic hernia, 50% of lethal skeletal dysplasias, 60% of polydactyly, 34% of major cardiac defects,

5% of facial clefts and 14% of open spina bifida, but none of agenesis of the corpus callosum, cerebellar or

vermian hypoplasia, echogenic lung lesions, bowel obstruction, most renal defects or talipes. Nuchal

translucency (NT) was above the 95th

centile in 34% of fetuses with major cardiac defects.

Conclusion: At 11-13 weeks some abnormalities are always detectable, some can never be and others are

potentially detectable depending on their association with increased NT, the phenotypic expression of the

abnormality with gestation and the objectives set for such a scan.

5.4. Clinical material studied to demonstrate the contribution of prenatal diagnosis in reducing the

frequency of major congenital anomalies (Study 11)

Study 11. Contribution of prenatal diagnosis in reducing congenital anomalies (63)

Objective: To demonstrate the contribution of prenatal diagnosis for fetal defects in reducing congenital

anomalies frequency.

Study design: 13457 pregnancies (at 11-13 weeks), investigated prospectively (prenatal diagnosis) and

retrospectively (pregnancy outcome), were divided into 2 groups: adverse outcome (cases), normal outcome

(controls) and epidemiologically studied (case-control) using Chi-square test as statistical analysis method.

The influence of prenatal diagnosis on pregnancy outcome was analysed.

Results: There were 12968(96,4%) controls and 489(3,6%) cases. When comparing 166 cases of euploid

fetal anomalies with 332 controls, we detected statistically significant differences for nuchal

translucency>95th

centile only (present in 20,5% cases versus 3,0% controls, p<0.001), hence its value in

predicting fetal abnormalities, mainly cardiac defects.

Prenatal diagnosis influenced pregnancy outcome through its components: etiology (aneuploid/euploid),

morphopathology (severity) of fetal anomalies, first trimester screening markers with high predictive value

(NT, ductus venosus, PAPP-A).

Adverse pregnancy outcomes were: terminations 172/489 (35,2% with 138/489 – 28,2% for fetal anomalies

– TOPFA), spontaneous pregnancy loss 200/489 (40,9%), neonatal deaths 6/489 (1,2%), newborns with

defects 111/489 (22,7%).

TOPFA reduced the rate of live births with congenital anomalies by 55,4% and avoided neonatal deaths

within euploid fetal anomalies cases.

22

Conclusion: The contribution of prenatal diagnosis in reducing congenital anomalies frequency of 55,4%

was carried out by selective termination of pregnancies for fetal defects, with 76,1% performed in the first

trimester.

In the context of previously presented 11 studies, we considered useful the demonstration of the crucial

importance of studied population size in estimation of the prevalence for fetal anomalies, in a separate study

(study 12).

Study 12. The current impact of major fetal structural abnormalities on public health. Study on

nowadays prevalence (62)

Objectives To evaluate the prevalence of major structural fetal anomalies and to demonstrate the importance

of studied population size in such an evaluation.

Method We investigated 2976 pregnancies within Centre for Fetal Medicine (tertiary centre), King’s College

Hospital, London, in January 2008. The studied lot comprised high risk obstetrical population by including

referral cases from local centres for positive results in antenatal screening for fetal defects.

The research approach was of a descriptive, transversal epidemiologic study on fetal anomalies prevalence.

The intervention method consisted of ultrasound examination and biochemical testing of pregnant women in

order to detect fetal anomalies.

Results There were 198/2976 (6,65%) major structural fetal anomalies.

The importance of the study population size for statistical analysis is proved by formula (39):

where μ=fetal anomalies frequency in the source obstetrical population, =fetal anomalies frequency in

studied population, σ/ =standard error in studied population of size n, Zα/2= variable Z on density abscissa

of probability for level of significance α/2. Thus, for n of large size, the ratio σ/ becomes almost 0, so that

becomes very close to μ and variation intervals for studied parameters will be very small (39).

Conclusions We demonstrated the importance of the size as large as possible for the study population to

ensure valid results. The prevalence of major structural fetal anomalies in our study population was almost 3

times higher than that reported by EUROCAT owing to a relative increase (high detection performance for

fetal anomalies in a tertiary centre on a high risk population) and a real increase of fetal defects due to

multiplied endogenous risk factors (more advanced maternal age) as well as exogenous risk factors (assisted

reproduction techniques, teratogens).

6. Encountered difficulties

In the statistical analysis of data I needed to collaborate with the specialist in Informatics (24).

Potential error sources:

1.incomplete history taking (indicating risk factors)

23

2.selection of study population, of cases and controls

3.in ultrasound screening – underestimating fetal anomalies by:

-limitations in accurate NT measurement (as a consequence of this, in 2010, the semiautomated NT

measurement was introduced),

-factors influencing optimal evaluation: obesity, lesion dimension less than 1cm, fetal heart with rapid

movements, scan machine resolution, operator’s experience (in examination technique and in images

interpretation).

4.in estimation of the prevalence of fetal anomalies in screening studies (studies 3,10,11) we included:

- fetal anomalies diagnosed by ultrasound without pathological investigation in cases of termination of

pregnancy, spontaneous pregnancy loss, neonatal death

- fetal anomalies diagnosed postnatally only in the first days of life, without those diagnosed well after birth.

7. Original contributions in personal research are included in those 12 studies - published and

communicated, presented within thesis.

7.1. In order to further improve the performance of first trimester screening for fetal anomalies we

searched for new markers in 3 studies:

Study 1. Maternal serum ADAM12 (A Disintegrin and Metalloprotease) in chromosomally abnormal

pregnancies at 11-13 weeks (282 )

There was some evidence provided by small studies that in pregnancies with fetal trisomy 21 the maternal

serum concentration of ADAM12 (A Disintegrin And Metalloprotease) was reduced during the first-trimester

of pregnancy but there were contradictory results as to the potential value of this metabolite in early

screening for trisomy 21.

The aim of this study was to investigate further the potential value of ADAM12 in first-trimester screening

for trisomy 21 and other major chromosomal abnormalities.

This was a case-control study performed between March 2006 and March 2007. In this interval the first

trimester screening for aneuploidies was carried out on 10,641 singleton pregnancies representing the base

cohort study population, wherein the present case-control study was nested (272 euploid and 136

chromosomally abnormal pregnancies, including 49 of trisomy 21, 28 of trisomy 18, 20 of trisomy of 13, 29

of Turner syndrome and 10 of triploidy).

The findings of this study (on more than 10,000 cases) demonstrated that at 11-13 weeks of gestation the

maternal serum ADAM12 concentration in trisomy 21 pregnancies was not significantly different from

euploid pregnancies but in trisomy 18, trisomy 13, triploidy and Turner syndrome the level was reduced.

In this study the cases and controls were matched for storage time and ADAM12 was measured by a time-

resolved fluorescent immunoassay using a monoclonal tracer antibody that recognised a stable epitope on

24

the molecule. The results were highly reproducible with low coefficients of variation and SDs of 0.13 and

0.16 for the euploid and chromosomally abnormal groups, respectively.

In previous publications either there was no statement on the method of matching cases and controls or this

was merely based on gestational age and the research assays used were based on an antibody pair (6E6 and

8F8) which was not fully optimised. The reported SD was wide (euploid group 0.28-0.43, aneuploid group

0.26-0.78) suggesting that the binding site for the antibodies used might be either degrading or altering its

configuration. Consequently, the very low levels of ADAM12 found in cases of trisomy 21 could, at least in

part, reflect the longer storage time for the cases than the controls.

In euploid pregnancies the maternal serum ADAM12 concentration was dependent on fetal CRL, maternal

weight and ethnic origin, being higher in Black than in White women. We used multiple regression analysis

to define the contribution of maternal variables that influenced the measured concentration of ADAM12 and

the interaction between these covariates, because the alternative method of sequential adjustment for each

individual parameter failed to take into account the interaction between the covariates. In previous

publications on ADAM12 adjustments were made only for gestational age.

Our study results demonstrated that, contrary to the expectations raised by previous publications,

measurement of maternal serum ADAM12 at 11-13 weeks was not useful in screening for trisomy 21.

We found that in both euploid and aneuploid pregnancies there was a strong association between the levels

of ADAM12 and both PAPP-A and free ß-hCG and therefore the potential performance of biochemical

screening by a combination of ADAM12, PAPP-A and free ß-hCG was likely to be substantially lower than

that suggested by previous publications.

In chromosomal abnormalities other than trisomy 21, the level of reduction in serum ADAM12 at 11-13

weeks was similar to that previously reported. However, in these chromosomal abnormalities the magnitude

of the reduction in ADAM12 was substantially smaller than the reduction in PAPP-A and free ß-hCG.

Furthermore, there was a strong association between the levels of ADAM12 and both PAPP-A and free ß-

hCG. Consequently, measurement of ADAM12 was unlikely to improve the performance of first-trimester

screening for these abnormalities achieved by the combination of maternal age, fetal NT, fetal heart rate and

maternal serum free ß-hCG and PAPP-A.

Study 2. ADAM12 and PlGF (Placental Growth Factor) at 11-13 weeks. Screening for aneuplodies

(293).

Four studies investigating the maternal serum concentrations of PlGF in pregnancies with fetal trisomy 21

during the first or second trimester reported that the levels were decreased, increased or the same in trisomy

21 compared with controls. One study reported that serum PlGF was reduced in trisomy 18 pregnancies.

The aims of this study were, first, to investigate further the maternal serum concentration of PlGF at 11–13

weeks of gestation in chromosomally abnormal fetuses and, second, to examine whether measurement of

PlGF could improve the performance of first-trimester biochemical screening for trisomy 21 provided by

maternal serum β-hCG and PAPP-A.

The findings of this study demonstrated that the maternal serum concentration of PlGF was decreased at 11–

13 weeks of gestation in trisomy 21 as well as other major chromosomal abnormalities. In addition,

measurement of PlGF could improve the performance of first-trimester biochemical screening for trisomy 21

provided by maternal serum free β-hCG and PAPP-A.

25

In euploid pregnancies serum PlGF increased with fetal CRL and therefore gestational age, decreased with

maternal weight, was higher in Black than in White women and in cigarette smokers than in non-smokers.

Consequently, as in the case of PAPP-A, the measured concentration of PlGF was adjusted for these

variables before comparing results with pathological pregnancies.

The results for trisomy 21 contradicted those of previous smaller studies that did not adjust the measured

values for maternal variables and reported that the levels in affected pregnancies were either increased or

not significantly different from normal control values. Other possible explanations for the discrepant results

were differences in assay methods and gestational range of the study populations.

In both the euploid and trisomy 21 pregnancies there was a significant association between serum levels of

PlGF and PAPP-A, which presumably reflects the postulated roles of these peptides in placental

development and/or their common origin from trophoblasts.

In first-trimester biochemical screening for trisomy 21 there were significant independent contributions from

maternal age and serum PlGF, PAPP-A and free β-hCG. It was estimated that screening by a combination

of maternal age and these three biochemical markers would identify 80% of affected pregnancies at

respective false positive rate of 5% (compared to 76,7% by maternal age, PAPP-A and free β-hCG) with the

significance of a potential improvement provided by serum PlGF to the established method of combined

screening by fetal NT and serum PAPP-A and free β-hCG.

The serum level of PlGF in trisomy 18, trisomy 13, Turner syndrome and triploidy was lower than in

pregnancies with euploid fetuses and lower than in those with trisomy 21. It is therefore anticipated that a

beneficial consequence of incorporating PlGF in first-trimester combined screening for trisomy 21 would be

the detection of a high proportion of the other major aneuploidies.

To conclude, in frst trimester screening for fetal aneuploidies, while serum levels for ADAM12 did not

improve the test performance, PIGF concentrations increased the detection rate achieved with maternal age,

PAPP-A and free β-hCG.

Study 3. Contribution of ductus venosus Doppler in first trimester screening for major cardiac defects

(61)

The traditional method of screening for cardiac defects, which relies on family history of such defects, maternal

history of diabetes mellitus and maternal exposure to teratogens, identifies only about 10% of affected

fetuses.

A major improvement in screening for cardiac defects came with the realisation that many affected fetuses

have increased nuchal translucency (NT) thickness at 11-13 weeks’ gestation. In the combined data from 14

first-trimester screening studies on major cardiac abnormalities in euploid fetuses the fetal NT was increased

in 73 (33.0%) of the 221 cases.

Another early sonographic marker of major cardiac defects is abnormal blood flow in the ductus venosus. In

the combined data from eight studies that examined ductus venosus waveforms in 791 euploid fetuses with

increased NT thickness, abnormal Doppler waveforms in the ductus venosus were observed in 87% of those

with cardiac defects compared to 19% in those without cardiac defects. However, there was uncertainty as to

whether abnormal flow in the ductus venosus in fetuses with major cardiac defects was confined to those

with increased NT or it was also observed in fetuses with normal NT.

26

The aim of this study was to determine whether assessment of ductus venosus flow could improve the overall

detection rate of cardiac defects achieved by screening with NT thickness alone.

The findings of our study demonstrated that in fetuses with major cardiac defects abnormal flow in the

ductus venosus was observed not only in those with increased NT but also in those with normal NT.

Consequently, the performance of early screening for cardiac defects achieved by measurement of fetal NT

was improved by assessment of flow in the ductus venosus.

The aria under ROC curves for the prediction of cardiac defects by NT alone was 0.700 (95% CI, 0.695-

0.704) and this was significantly improved by the addition of ductus venosus flow (0.757, 95% CI, 0.753-

0.761, p<0.001). The early detection rates for cardiac defects were: DR 21,2%, FPR 0,7% for NT>99th

; DR

35,3%, FPR 4,8% for NT>95th

compared to DR 38,8% , FPR 2,7% for NT>99th

and DV or DR 47,1%, FPR

6,7% for NT>95th

and DV).

The offer of specialist fetal echocardiography to pregnancies with fetal NT above the 99th

centile is now well

accepted because the number of cases is small and the prevalence of major cardiac defects is high. The

extent to which specialist fetal echocardiography should also be offered to pregnancies with NT between the

95th

and 99th

centiles, which constitute about 4% of the total population, depends on the availability of such

services.

We proved that high NT or abnormal flow in the ductus venosus was not confined to specific types of cardiac

defects in contradiction with a previous smaller study reporting that in fetuses with abnormal ductus venosus

flow and normal NT all defects were right-sided.

In this study we developed an algorithm combining fetal NT with ductus venosus flow to estimate the patient-

specific risk for major cardiac defects. The risk increased exponentially with NT and was further increased

if the a-wave was reversed and decreased if flow in the ductus venosus was normal (Figure 2).

Ultimately, the detection of major cardiac defects will depend on the proportion of the population that can be

offered specialist fetal echocardiography and our algorithm could be used to define the risk cut-off that

selects the patients requiring referral for such an examination.

A more pragmatic approach would be to select patients on the basis of ductus venosus flow and a fetal NT

cut-off. Ideally, all cases with fetal NT above the 95th

centile and those with reversed a-wave should be

offered fetal echocardiography but in the absence of sufficient resources the examination could be reserved

for those with abnormal Doppler and those with NT above the 99th

centile.

7.2. Original contribution to a more correct first trimester prediction of fetal growth restriction in

early preeclampsia in order to accordingly adjust the intensity of antenatal care while targeting

reduction of adverse pregnancy outcomes (in 6 studies):

Study 4. First-Trimester Maternal Serum a Disintegrin and Metalloprotease 12 (ADAM12) and

Adverse Pregnancy Outcome (283)

There was some evidence that in pregnancies complicated by fetal trisomies 21 and 18 and in those destined

to develop preeclampsia or deliver SGA neonates, the concentration of maternal serum ADAM12 in the first

trimester of pregnancy was reduced. Several studies documented that PAPP-A, another placenta-derived

insulin-like growth factor – binding proteins protease, was reduced at 11-13 weeks in maternal serum of

pregnancies resulting in preeclampsia, SGA and preterm delivery.

27

Epidemiologic studies reported that preterm preeclampsia was associated with SGA, whereas in term

preeclampsia, the birth weight was often normal or increased. Doppler studies of the uterine arteries

demonstrated that the prevalence of increased pulsatility index was substantially higher in cases of

preeclampsia with SGA than for preeclampsia without SGA or SGA without preeclampsia.

The aim of this study was to investigate further the levels of maternal serum ADAM12 in the first trimester of

pregnancy in cases that subsequently developed preeclampsia and/or SGA and the relation of these levels to

uterine artery pulsatility index and maternal serum PAPP-A.

The findings of this study demonstrated that the maternal serum ADAM12 concentration at 11-13 weeks of

gestation in normal pregnancies increased with fetal CRL and therefore gestational age, decreased with

maternal weight and was higher in African-American than in white women. Consequently, the measured

concentration of ADAM12 should be adjusted for these variables before comparing results with pathologic

pregnancies. The finding was not surprising, because there was a good correlation between ADAM12 and

PAPP-A, which had been shown in a previous study involving 96,803 pregnancies to also increase with

CRL, decrease with maternal weight and to be higher in African-American than in white women.

In pregnancies developing preeclampsia or gestational hypertension, the maternal serum ADAM12

concentration at 11-13 weeks of gestation was not significantly different from normotensive pregnancies.

Furthermore, there was no significant association between ADAM12 and the severity of preeclampsia,

irrespective of whether this was defined by the gestation at which iatrogenic delivery was carried out or the

coincidence of preeclampsia with SGA. The findings of our study contradicted the reported results of two

previous studies that in pregnancies developing preeclampsia the first-trimester maternal serum ADAM12

concentration was reduced and was particularly low in those cases requiring early delivery and in those with

coincidence of preeclampsia with SGA. A likely explanation for this apparent contradiction is that in both

of these studies the level of ADAM12 was adjusted only for gestational age. Reanalysis of our data without

adjustment for maternal weight and racial origin also demonstrated that the maternal serum ADAM12 in

pregnancies developing preeclampsia was significantly lower than controls (0.927 MoM compared with

1.039 MoM, P=0.016). Therefore, the conclusion that the development of preeclampsia was associated with

low levels of ADAM12 might be a simple reflection of the association between the development of

preeclampsia and increasing maternal weight.

In normal pregnancies there was a correlation between birth weight percentile and the maternal

serum concentration of ADAM12 and PAPP-A at 11-13 weeks of gestation, providing further evidence for

the possible contribution of these placental products in the control of fetal growth. The levels of ADAM12

and PAPP-A during the first trimester were low in women who subsequently delivered small neonates and

high in those delivering large neonates. However, the performance of screening for SGA using ADAM12 was

poor because low levels below the 5th percentile were observed only in about 15% of the SGA pregnancies.

There was a good correlation between the maternal serum levels of ADAM12 and PAPP-A, and consequently

the additional contribution of PAPP-A in the prediction of SGA was only about 2%. Similarly, although the

uterine artery pulsatility index at 11-13 weeks in the SGA pregnancies was significantly increased, this

measurement did not improve the prediction of SGA provided by maternal demographic characteristics and

medical history alone.

In pregnancies resulting in spontaneous preterm delivery, the maternal serum ADAM12 concentration at 11-

13 weeks of gestation was not significantly different from pregnancies delivering at term. These findings

were not surprising, because there was only a weak association between impaired placentation and

spontaneous preterm delivery. Histologic studies reported that in patients with spontaneous preterm delivery,

28

there was failure of the physiologic transformation of the maternal spiral arteries, but to a substantially lower

extent than in cases developing preeclampsia. Doppler studies of the uterine arteries demonstrated increased

pulsatility index in association with preterm delivery, but this relationship was mainly observed in

pregnancies undergoing iatrogenic delivery for preeclampsia and SGA rather than in cases of spontaneous

preterm delivery.There was evidence that in pregnancies resulting in spontaneous preterm delivery, the

maternal serum concentration of PAPP-A at 11-13 weeks was reduced, but level of PAPP-A <5th

percentile

was observed in only 10% of preterm deliveries.

In conclusion, at 11-13 weeks of gestation, there was a good correlation between the maternal serum

ADAM12 and PAPP-A concentration, and in pregnancies delivering SGA neonates, the levels of both

metabolites were reduced. However, measurement of ADAM12 did not provide useful prediction of SGA. In

pregnancies complicated by preeclampsia, gestational hypertension, and spontaneous preterm delivery, the

levels of ADAM12 were not significantly different from normal.

Study 5. PAPP-A, ADAM12, PIGF at 11-13 weeks. Prediction of preeclampsia (294)

Pre-eclampsia (PE) is associated with reduced production of the pro-angiogenic protein placental growth

factor (PlGF) and several studies reported that during the clinical phase of PE the maternal serum PlGF

concentration was reduced. These reduced levels of serum PlGF precede the clinical onset of the disease and

are evident from both the second and first trimesters of pregnancy. In addition, the maternal serum

concentration of pregnancy associated plasma protein-A (PAPP-A), which is thought to be involved in

placental growth and development, is reduced at 11-13 weeks’ gestation in pregnancies resulting in PE.

According to our previous studies, the likelihood of developing PE can be predicted by a combination of

factors in the maternal history, including black racial origin, high body mass index (BMI) and prior or family

history of PE, the measurement of uterine artery pulsatility index (PI) and the maternal serum level of PAPP-

A at 11-13 weeks.

Previous studies demonstrated that prediction of PE could be provided by uterine artery Doppler in the

second trimester of pregnancy and this could be improved by combining the Doppler findings with maternal

serum concentration of PlGF and the anti-angiogenic protein soluble fms-like tyrosine kinase 1 (sFlt-1).

Although in pregnancies developing PE, reduced levels of PlGF are evident from the first trimester,

significant increases in levels of sFlt-1 become apparent only about 5 weeks before the onset of PE.

The aim of this study was to investigate further the levels of maternal serum PlGF in the first trimester of

pregnancy in cases that subsequently developed PE, to examine the relation of these levels to uterine artery

PI and maternal serum PAPP-A levels and to estimate the potential performance of screening for PE by a

combination of maternal factors, uterine artery PI and maternal serum PAPP-A and PlGF.

The findings of this study demonstrated that the maternal serum PlGF concentration at 11-13 weeks of

gestation in normal pregnancies increased with fetal CRL and therefore gestational age, decreased with

maternal weight, and was higher in black than in white women and in cigarette smokers than in non-

smokers. Consequently, as in the case of PAPP-A, the measured concentration of PlGF must be adjusted for

these variables before comparing results with pathological pregnancies. Previous studies comparing PE with

controls either made no corrections for the measured PlGF or they corrected only for gestation.

In common with PlGF, the serum concentration of PAPP-A also increased with fetal CRL, decreased with

maternal BMI and was higher in black than in white women. However, in cigarette smokers there was an

29

apparent dissociation in the relationship between these two placental products with a decrease in serum

PAPP-A and increase in PlGF.

In pregnancies developing PE the maternal serum PlGF concentration at 11-13 weeks’ gestation was lower

than in normotensive pregnancies. Furthermore, there was a significant association between PlGF and the

severity of PE defined by both the gestation at which iatrogenic delivery was carried out and the birth-weight

centile of the neonates.

The finding of an interrelationship between serum levels of PlGF and PAPP-A with uterine artery PI is

compatible with the postulated roles of PlGF and PAPP-A in placental development and the reflection of

impaired placentation in increased impedance to flow in the uterine arteries.

Abnormalities in the biochemical and Doppler indices of placentation are substantially more common in

women developing early PE than late PE. This is particularly important because it is early rather than late

disease that is associated with increased risk of perinatal mortality and morbidity and both short-term and

long-term maternal complications.

In early screening for PE there were significant independent contributions from maternal characteristics

(black race, obesity) and history (family history of PE and personal history of chronic hypertension or PE),

uterine artery PI, maternal serum PlGF and PAPP-A. We estimated that screening by a combination of these

paramaters would identify about 90% and 50% of patients developing early PE and late PE, respectively, at

a false-positive rate of 10%.

Identification of women at high risk for PE could potentially improve pregnancy outcomes because intensive

maternal and fetal monitoring in such patients would lead to an earlier diagnosis of the clinical signs of the

disease and the associated fetal growth restriction and avoid

the development of serious complications through such interventions as the administration of

antihypertensive medication and early delivery. The proposed combined screening test could also be used

for effective identification of the high-risk group for future studies investigating the potential role of

pharmacological interventions starting from the first trimester to improve placentation and reduce the

prevalence of the disease.

To conclude, in pregnancies complicated by PE, while serum levels for ADAM12 are not significantly

different from normal, PAPP-A and PIGF concentrations are significantly reduced, especially in early PE,

hence their role in predicting this condition.

Study 6. Maternal plama P-selectin at 11-13 weeks of gestation in hypertensive disorders of pregnancy

(4)

The plasma concentration of P-selectin, a marker for platelet activation, is increased during established PE

and there is also evidence that this elevation may be evident from the first-trimester of pregnancy.

In our studies investigating the pathophysiology of PE we found that at 11-13 weeks of gestation the uterine

artery pulsatility index (PI) was increased in pregnancies that subsequently develop PE. The increase in

uterine artery PI was particularly marked in severe early onset PE requiring delivery before 34 weeks

providing support for the hypothesis that the underlying mechanism of the disease was impaired placentation,

which was evident from as early as the first trimester.

30

The aim of this study was to investigate further whether in pregnancies that subsequently developed PE the

maternal plasma concentration of P-selectin at 11- 13 weeks of gestation was increased and whether such a

possible increase was associated with uterine artery PI.

The findings of this study that at 11-13 weeks of gestation, women who subsequently developed PE had

increased maternal plasma levels of P-selectin and uterine artery PI were consistent with previous reports.

The maternal plasma levels of P-selectin were increased in patients destined to develop PE but not GH and

there were no significant differences between early- PE and late-PE in the level of this adhesion molecule.

In contrast, uterine artery PI was increased in PE but not GH and the levels in early-PE were higher than in

late-PE.

The advantage of our study compared to previous reports was that we measured P-selectin and uterine

artery PI in the same patients allowing assessment of their interrelations.

In the unaffected controls, the measured concentration of maternal plasma P-selectin was affected by

ethnicity and the mode of conception. The levels were lower in Blacks and Orientals than in White women

and in women who conceived with ovulation drugs than in women who conceived spontaneously.

Consequently, as in the case of uterine artery PI and serum PAPP-A, the measured concentration of plasma

P-selectin must be adjusted for these variables before comparing with pathological pregnancies.

Our findings of increased uterine artery PI at 11-13 weeks in pregnancies destined to develop PE and the

association between uterine artery PI and gestation at delivery provided further support for the central role

of impaired trophoblastic invasion in the pathogenesis of the disease and that early-PE was due to more

severe impairment in placentation than late-PE.

In the PE group the plasma concentration of P-selectin was increased. These findings suggested that in

women destined to develop PE there was evidence of an inflammatory process and platelet activation from as

early as the first trimester of pregnancy and these processes preceded the clinical onset of the disease by

several months. However, in the PE group there was no significant association between the level of P-

selectin and either uterine artery PI or the gestation at delivery. Consequently, our results did not provide

support for the hypothesis linking impaired trophoblastic invasion of the maternal spiral arteries with

platelet activation and endothelial dysfunction which led to the development of the clinical symptoms of the

disease.

Effective first-trimester screening for early-PE was provided by a combination of maternal factors and

uterine artery PI. Although in such pregnancies the maternal plasma P-selectin is increased, measurement of

this adhesion molecule did not improve the performance of screening for early-PE. In contrast, in the

prediction of late-PE, where the performance of uterine artery Doppler is poorer than in early-PE, the

measurement of P-selectin improved the detection of PE. The extent to which this association could be

translated into a useful pharmacological intervention to lower the incidence of the disease remains to be

determined.

Study 7. Maternal risk factors for hypertensive disorders in pregnancy: a multivariate approach (292)

In the UK, the National Institute for Clinical Excellence (NICE) issued guidelines on routine antenatal care

recommending that at the booking visit a woman’s level of risk for PE, based on factors in her history, should

be determined and the subsequent intensity of antenatal care should be based on this risk. However, we could

locate no reference on the performance of such a recommended screening strategy, which treated each of the

risk factors, such as age of 40 years or older, nulliparity, body mass index (BMI) of 30 kg/m2 or above,

31

family history or history of PE and preexisting vascular disease, as separate screening tests with additive

detection and false positive rates.

The strategy of treating each of the risk factors as a separate screening test would have falsely classified two

thirds of the obstetric population as high risk and in need of intensive monitoring

(screening suggested by NICE would have a FPR 64.1% and DR of 89.2%, 93.0% and 85.0% for early PE,

late PE and GH, respectively).

The aim of this study was to develop algorithms for hypertensive disorders in pregnancy based on

multivariate analysis of factors from the maternal history and to compare the estimated performance of such

algorithms in the prediction of early-PE, late-PE and GH with that recommended by NICE.

The factors in maternal history that contributed in estimating the risk for subsequent development of

hypertensive disorders were compatible with the results of previous studies. However, in contrast to many

previous studies, we separated the disorders into early-PE, late-PE and GH and, in addition, we performed

rigorous assessment of each case in order to establish the correct diagnosis for every affected pregnancy.

Logistic regression analysis was used to determine which of the factors amongst the maternal

characteristics, medical and obstetric history had a significant contribution in predicting early-PE, late-PE

and GH. The likelihood of developing early-PE was increased in Black compared to White women, in

women with a history of chronic hypertension, in parous women with prior PE and in those who received

ovulation drugs. The risks for developing late-PE and GH increased with maternal age and BMI and were

higher in those with a family or prior history of PE. Additionally, late-PE was more common in Black, Indian

and Pakistani women.

Some of the previously suggested risk factors, such as thrombophilia and diabetes mellitus, did not reach

statistical significance in the prediction of PE.

We found that in women with history of PE, compared with nulliparous women, there was a fourfold increase

in risk for early-PE and twofold increase in risk for late-PE, whereas in parous women with no PE, the risk

for both early and late disease was reduced by three to four times.

Most previous studies demonstrating an increased risk of recurrence of PE did not distinguish between early

and late disease, except for one study that reported similar results to our findings.

Previous studies reported a threefold increase in risk for PE in women whose mother or sister had PE. In

our study, we separated the two groups of PE in the mother and sister and found by multivariate regression

analysis that independent prediction of PE was confined to history of PE in the mother and not the sister.

Increased risk for PE was also observed in women with chronic hypertension and this was particularly true

for severe early onset disease rather than late-PE. In our study, women with chronic hypertension had a

ninefold increase of risk for early-PE but the risk for late-PE was not increased.

Assisted conception techniques increased the risk for PE but there were conflicting reports as to whether this

increase is for in vitro fertilisation, simple ovulation induction or both. In our study, ovulation induction, but

not in vitro fertilisation, was associated with substantially increased risk for early-PE. Nevertheless, there

was some evidence that in women receiving ovulation drugs, but not in vitro fertilisation, the maternal serum

concentration of pregnancy-associated plasma protein-A (PAPP-A) was reduced, and there was extensive

evidence linking low serum PAPP-A with subsequent development of early-PE.

32

Increased maternal age and BMI were associated with increased risk for late-PE and GH but not early- PE.

We proved that the risk for late-PE and GH increased by 4% for every year over the age of 32 years and by

10% for every 1 kg/m2 above 24 kg/m

2.

The performance of screening was determined by receiver operating characteristic (ROC) curves: DR for

early PE, late PE, GH was of 37, 29 and 21%, respectively, for a FPR 5%.

The approach to early screening for hypertensive disorders in pregnancy might be analogous to first

trimester screening for trisomy 21. In this condition, the patient-specific risk was derived by multiplying the

maternal age-related a priori risk by the likelihood ratio associated with fetal nuchal translucency thickness

and maternal serum free β-hCG and PAPP-A. Similarly, there was increasing evidence that effective

screening for hypertensive disorders would require a combination of maternal factors and a series of

biochemical and biophysical parameters. In pregnancies destined to develop PE, the mean arterial pressure

and uterine artery pulsatility index at 11–13 weeks were increased and the maternal serum PAPP-A and

placental growth factor were decreased. To derive the patient-specific risk for hypertensive disorders, the a

priori risk should be multiplied by the likelihood ratio associated with the biophysical and biochemical

measurements.

In conclusion, meaningful screening for hypertensive disorders in pregnancy by maternal history

necessitated the use of algorithms derived by logistic regression analysis.

This study established the methodology for deriving the a priori risk for early- PE, late-PE and GH based on

maternal age, BMI, racial origin, history of PE and chronic hypertension and method of conception. The

findings of this study demonstrated that the NICE recommendations of screening for PE by maternal

characteristics and previous history to define the intensity of antenatal care would be potentially useful only

when the various factors were incorporated into a combined algorithm derived by multivariate analysis.

Study 8. Hypertensive disorders in pregnancy: Screening by systolic, diastolic and mean arterial

pressure at 11-13 weeks (295)

Several studies examined the use of blood pressure (BP) measurement as a screening test for subsequent

development of hypertensive disorders in pregnancy. The studies reported widely contradictory results in the

performance of screening as a consequence of major methodological differences.

The aim of this prospective study was to examine and compare the performance of screening for PE and GH

(diagnosed by strict criteria) by systolic BP, diastolic BP or mean arterial pressure (MAP) (measured by

validated automated devices) in a large population of pregnant women attending for routine care at 11-13

weeks.

The findings of this study demonstrated that firstly, measurement of BP at 11-13 weeks was increased in

pregnancies that subsequently developed PE and GH and the increase was particularly marked for early-PE,

secondly, the measurement of BP could be combined with the maternal factor-derived a priori risk to provide

effective first-trimester screening for hypertensive disorders of pregnancy, and thirdly, the performance of

screening by the use of MAP appeared to be better, but not significantly so, than by systolic BP or diastolic

BP.

There wass increasing evidence from studies in non-pregnant populations that with advancing age there was

shift from diastolic BP to systolic BP as the main predictor of the adverse cardiovascular consequences of

33

hypertension. Pregnant women tended to be young and traditionally greater emphasis was given to diastolic

rather than systolic BP as the predictor of the adverse consequences of pregnancy hypertension.

The World Health Organization recommended that in the classification of PE only the diastolic BP should be

considered and that use of MAP would not only increase the possibility of errors in recording of both systolic

and diastolic BP but would also be unacceptable to physicians because they would have to calculate the

MAP.

However, these arguments became less relevant. Firstly, there had been a gradual increase in the age of

pregnant women with the proportion being 35 years or more increasing from 5% in the 1970’s to 20% in the

2000’s with a consequent expected increase in the contribution of systolic BP in pregnancy related

hypertension. Secondly, the MAP did not necessitate complex calculations by physicians but it was measured

directly by automated devices, which would in any case replace the environmentally toxic mercury based

traditional sphygmomanometers.

The advantages of this study were the prospective examination of a large population of pregnant women

attending for routine care in a well defined gestational age, the use of a validated automated devices and

appropriately trained doctors to measure BP, the application of strict criteria to define the diagnosis of

early-PE, late-PE and GH, and the use of a statistical approach that was widely accepted in screening for

trisomy 21.

All but one of previous studies did not distinguish between GH and PE or between early and late disease.

However, there is now extensive evidence that these conditions have major differences in both

pathophysiology and clinical implications for the mother and fetus. It is early-PE, rather than late-PE and

GH, which is associated with impaired trophoblastic invasion of the maternal spiral arteries, increased

impedance to flow in the uterine arteries and substantially increased risk of perinatal mortality and both

short-term and long-term maternal mortality and morbidity.

In the unaffected group, which did not develop PE or GH, the maternal BP decreased with gestation,

increased with maternal age and BMI and it was lower in cigarette smokers and in all racial groups other

than in White women. These findings are in agreement with previous reports in non-pregnant individuals. A

consequence of these results is that the measured BP must be adjusted for these variables and expressed as a

MoM in order to assess whether a given value is within the normal range or increased. This is analogous to

biochemical testing with serum pregnancy associated plasma protein-A (PAPP-A) where the measured

concentration is expressed as a MoM after adjustment for maternal characteristics.

Taking the maternal history and recording BP are the cheapest and ubiquitously accessible screening tools.

We chose 11-13 weeks as the gestation for screening because this is emerging as the first hospital visit of

pregnant women at which combined sonographic and biochemical testing for chromosomal and other major

defects is carried out. It would be easy to measure the MAP of women in this same visit and use the same

methodology to calculate the patient-specific risk for both chromosomal defects and PE.

Essentially, factors from the maternal characteristics and history are used to calculate the a priori risk which

is multiplied by the likelihood ratio associated with biophysical and biochemical measurements to derive

patient-specific risks.

DR for early PE, late PE, GH in screening by maternal risk factors was 47%, 41% and 31%, respectively,

for a FPR 10%.

34

In this study we showed that the maternal factor-derived a priori risk could be combined with the simple

measurement of the MAP to improve the detection rates to 76%, 52%, and 48%, respectively. Further

improvement in screening is likely to be achieved with the inclusion of uterine artery Doppler and serum

metabolites, such as PAPP-A and placental growth factor.

Study 9. Prediction of preeclampsia: biophysical profile at 11-13 weeks (296)

9A. First trimester screening for PE by uterine artery Doppler

In PE there is impaired trophoblastic invasion of spiral arteries and this impairment is most marked in early

PE. Indirect evidence for impaired placental perfusion in pregnancies destined to develop PE has been

provided by Doppler studies of the uterine arteries (UtAs) which showed increased pulsatility index (PI) both

during the second trimester and also in the first trimester of pregnancy.

Traditionally the PI is measured in both the UtAs and the mean of the two measurements is used in the

prediction of risk for PE in the second trimester. However, there are no prospective studies to document that

the performance of screening is best by the use of the mean rather than the lowest or highest PI.

We have demonstrated recently that the NICE recommendation of screening for PE by maternal

characteristics and previous history was potentially useful only when the various factors were incorporated

into a combined algorithm derived by multivariate analysis. Such an approach made it possible to derive the

a-priori risk for early PE, late PE and GH based on maternal age, body mass index (BMI), racial origin,

history of PE, chronic hypertension and method of conception. The estimated detection rates for early PE,

late PE and GH were about 47%, 41% and 31%, respectively, at a 10% false-positive rate.

The aim of this study was to examine the performance of screening for hypertensive disorders in pregnancy

by a combination of the maternal factor-derived a-priori risk and the UtA-PI, and to determine whether it

was best in such screening to use the mean PI of the two arteries, the highest PI or the lowest PI.

The findings of this study confirmed that UtA-PI at 11–13 weeks was increased in pregnancies that

subsequently developed PE and that the increase was particularly marked for early PE. The performance of

screening appeared to be best with the lowest PI. The lowest UtA-PI, but not the mean or highest, was also

significantly increased in pregnancies that subsequently developed GH.

In the unaffected group, which did not develop PE or GH, UtA-PI decreased with increasing gestational age

and increasing maternal BMI, and was higher in Black women than in other racial groups. Consequently, the

measured PI must be adjusted for these variables and expressed as a MoM in order to assess whether a given

value is within the normal range or increased. This is analogous to biochemical testing with serum

pregnancy-associated plasma protein-A (PAPP-A) where the measured concentration is expressed as a MoM

after adjustment for maternal characteristics.

The relationship between UtA-PI and maternal BMI is unlikely to be the consequence of enhanced

trophoblastic invasion in obese women but rather vasodilatation in the uterine circulation owing to the

increased levels of circulating estrogens associated with increased BMI.

Impedance to flow in the UtAs was lower on the side of implantation than in the non-placental site but it

decreased with increasing gestational age at both sites. The finding that in screening for hypertensive

disorders the best performance was provided by the vessel with the lowest PI was not surprising because this

was likely to be a better reflection of the degree of trophoblastic invasion of the spiral arteries.

35

The approach to early screening for hypertensive disorders in pregnancy may be analogous to first-trimester

screening for trisomy 21. In screening for early PE, late PE and GH the patient-specific risk is derived by

combining the disease specific maternal factor-derived a-priori risk with the measurement of the lowest UtA-

PI in a multivariate regression model. Significant predictors of the maternal factor-derived a-priori risk for

early PE were black race, chronic hypertension, previous PE and use of ovulation drugs, whereas predictors

of late PE and GH were increased maternal age and BMI, family or previous history of PE. Additionally, late

PE was more common in black, Indian and Pakistani women than in white women.

In screening for trisomy 21 the detection rate, for a 5% false-positive rate, was increased from about 30% in

screening by maternal age alone to 90% with combined screening by maternal age, fetal nuchal translucency

thickness, maternal serum free β-hCG and PAPP-A. Similarly, as shown in this study, in screening for early

PE, the detection rate, for a 10% false-positive rate, was increased from 47% in screening by maternal

factors alone to 81% with combined screening by maternal factors and UtA-PI. The respective detection

rates for late PE increased from 41% to 45% and those for GH increased from 31% to 35%. Further

improvement in screening is likely to be achieved with the inclusion of maternal blood pressure and serum

concentration of placental products, such as PAPP-A and placental growth factor.

9B. Hypertensive disorders in pregnancy: screening by uterine artery Doppler imaging and blood

pressure at 11–13 weeks.

Early prediction of hypertensive disorders can also be provided by measurement of the mean arterial pressure

(MAP) at 11–13 weeks. The estimated detection rate, at a 10% false-positive rate, in screening by a

combination of the maternal factor-derived a-priori risk with MAP was 79% for early PE, 52% for late PE

and 48% for GH.

The aim of this study was to examine the performance of screening for hypertensive disorders in pregnancy

by a combination of the maternal factor-derived a-priori risk with the uterine artery lowest PI (L-PI) and

MAP at 11–13 weeks.

This study demonstrated that in screening for hypertensive disorders in pregnancy the patient-specific risk

for early PE, late PE and GH could be derived by combining the disease-specific maternal factor-derived

apriori risk with the measurements of the uterine artery L-PI and MAP. Hence, the estimated detection rate

of early PE, for a 10% false-positive rate, was increased from 47% in screening by maternal factors alone to

89% with combined screening by maternal factors, uterine artery L-PI and MAP. The respective detection

rates for late PE increased from 41% to 57% and for GH increased from 31% to 50%.

This screening study for hypertensive disorders examined more than 8000 pregnancies, including more than

300 cases that developed PE or GH. We used multiple regression analysis to define the contribution of

maternal factors, MAP and uterine artery L-PI in predicting pregnancy hypertensive complications, and the

interaction between these covariates. The algorithms we derived could be used to calculate the combined a-

priori risk in future smaller case–control studies investigating the potential value of additional biophysical

or biochemical measurements.

Since the population we examined in this study was large it is likely that the data on maternal factors, MAP

and uterine artery L-PI would be more universally applicable than those derived from small case–control

studies.

36

We chose 11–13 weeks as the gestational age for screening because this is emerging as the time of the first

hospital visit of pregnant women at which combined sonographic and biochemical testing for chromosomal

and other major defects is carried out. It would be easy to measure the MAP and uterine artery PI of women

in the same visit and utilise the same methodology to calculate the patient-specific risk for both chromosomal

defects and hypertensive disorders. This methodology could be applied for improved screening in the future

with the use of additional biochemical markers, such as maternal serum PAPP-A and placental growth

factor.

PE is the most common pregnancy complication associated with serious maternal–fetal morbidity and

mortality, and at present the only effective treatment is delivery of the placenta. The ability to predict those

women at risk for PE in a very early stage of pregnancy might decrease maternal and fetal morbidity

through closer surveillance by physicians experienced or specialised in high-risk obstetrics, as well as

delivery at tertiary care centres. Centralised care of pregnancies at high-risk for PE would also lead to a

more effective concentration of research activity in an attempt to improve understanding of the

pathophysiology and treatment of the condition.

7.3. Original contribution in diagnosing euploid fetal anomalies and defining the current ultrasound

detection of their morphopathological subgroups at 11-13 weeks (1 study)

Study 10. Challenges in the diagnosis of fetal non-chromosomal abnormalities at 11-13 weeks (369)

In a systematic review of previous studies on the clinical effectiveness of the routine second trimester scan,

there were large differences between studies in detection rates which ranged from 15 to 85% and this

variation could be attributed to differences in the abnormalities included in their analysis, the rate and method

of follow up of the screened population and the protocols followed for examination of the fetuses.

Several studies on the first-trimester scan reported the diagnosis of a wide range of fetal abnormalities, but in

most studies the abnormalities were either a coincidental finding during screening for aneuploidies or they

were detected after detailed examination of euploid fetuses because of increased NT. In this respect, the

findings of reported studies do not reflect the true performance of the 11-13 weeks scan in screening for non-

chromosomal abnormalities.

The aim of this screening study in more than 45,000 singleton pregnancies was to define the current

performance of the 11-13 weeks scan in the detection of fetal non chromosomal abnormalities.

The findings of this study demonstrated that:

-11-13 weeks scan detected 44% euploid fetal anomalies whereas 20-23 weeks scan -53,7%,

-major fetal anomalies fell into essentially three groups in relation to whether they could be detected at the

11-13 weeks scan: always detectable (31%), undetectable (26%) (thus requiring rescan in the second

trimester), potentially detectable (43%) fetal anomalies whose detection depended on their association with

easily detectable markers (retronasal triangle for the facial cleft; NT, ductus venosus and tricuspid flows for

cardiac defects; brain stem for open spina bifida) and a decision policy as to the objectives of the scan and

the necessary allocation of resources for achieving such objectives.

7.4. The original component in demonstrating the contribution of prenatal diagnosis in reducing the

frequency of congenital anomalies (1 study)

Study 11. Contribution of prenatal diagnosis in reducing congenital anomalies (63)

37

This study original character consisted in demonstratig the particularity of this advantageous combination of

best parameters of efficiency in the policy for termination of pregnancy for fetal anomaly (TOPFA) (the

highest overall, early and super early TOPFA rates associated to the lowest late TOPFA rate). Such an

efficient policy is the direct consequence of the superior performance of 11-13 weeks screening for

aneuploidies and structural fetal anomalies in a strategy ultimately focused on earliest detection of fetal

defects. Consequently, first trimester rather than later TOPFA is promoted with clear clinical and

psychological benefits, while targeting major reduction in prevalence of malformed neonates.

Study 12. The current impact of major fetal structural abnormalities on public health. Study on

nowadays prevalence (62)

In this study we mathematically demonstrated the crucial importance of the size – as large as possible – of

the study population in evaluation of prevalence for fetal anomalies in order to ensure valid results.

8. The results of thesis research are presented in personal scientific papers (published articles and

communications):

1. Poon LC, Chelemen T, Minekawa R, Frisova V, Nicolaides KH. Maternal serum ADAM12 (A disintegrin

and metalloprotease) in chromosomally abnormal pregnancy at 11-13 weeks. Am. J. Obstet. Gynecol. 2009

May;200(5):508.e1-6. (282)

2. Poon LC, Akolekar R, Zaragoza E, Chelemen T, Nicolaides KH. ADAM12 & PlGF @ 11-13 weeks.

Screening for aneuploidies. Oral presentation within the 7th

World Congress of Fetal Medicine. Sorrento,

Italy, June 2008 (293)

3. Chelemen T, Sygalaki A, Maiz N, Allan L, Nicolaides KH. Contribution of ductus venosus Doppler in first

trimester screening for major cardiac defects. Fetal Diagnosis and Therapy. 2010 Dec 16 (61)

4. Poon LC, Chelemen T, Granvillano O, Pandeva I, Nicolaides KH. First-trimester maternal serum a

disintegrin and metalloprotease 12 (ADAM12) and adverse pregnancy outcome. Obstet. Gynecol. 2008

Nov;112(5):1082-90 (283)

5. Poon L, Akolekar R, Zaragoza E, Chelemen T, Nicolaides KH. PAPP-A, ADAM12 & PlGF @ 11-13

weeks. Prediction of preeclampsia. Oral presentation within the 7th

World Congress of Fetal Medicine.

Sorrento, Italy, June 2008 (294)

6. Akolekar R, Veduta A, Minekawa R, Chelemen T, Nicolaides KH. Maternal serum P-selectin at 11-13

weeks of gestation in hypertensive disorders of pregnancy. Hypertens. Pregnancy. 2010 Mar 8 (4)

7. Poon LC, Kametas NA, Chelemen T, Leal A, Nicolaides KH. Maternal risk factors for hypertensive

disorders in pregnancy: a multivariate approach. J. Hum. Hypertens. 2010 Feb;24(2):104-10 (292)

8. Poon LC, Kametas NA, Valencia C, Chelemen T, Nicolaides KH. Hypertensive Disorders in Pregnancy:

Screening by Systolic Diastolic and Mean Arterial Pressure at 11-13 Weeks. Hypertens. Pregnancy. 2010

Sep 6 (295)

38

9. Poon LC, Romero X, Chelemen T, Karagiannis G, Nicolaides KH. Prediction of preeclampsia: biophysical

profile at 11-13 weeks. Oral presentation within the 8th

World Congress of Fetal Medicine, Portoroz,

Slovenia, July 2009 (296)

10. Syngelaki A, Chelemen T, Dagklis T, Allan LD, Nicolaides KH. Challenges in the diagnosis of fetal non-

chromosomal abnormalities at 11-13 weeks. Prenat. Diagn. 2011 Jan;31(1):90-102

(369)

11. Chelemen T, Pricop F, Butureanu S, Dumitrache F, Boiculese L, Chelemen S. Contribution of prenatal

diagnosis in reducing congenital anomalies. Obstetrica Ginecologia. vol. LVIII Nr 4. Oct-Dec 2010; 241 -

248 (63)

12. Chelemen T, Pricop F, Butureanu S, Dumitrache F, Chelemen S. Impactul actual al anomaliilor fetale

structurale majore asupra sanatatatii publice. Cercetarea prevalentei actuale. Obstetrica

Ginecologia.vol.LVIII. Nr.2.Apr-Iun 2010;115-120 (62)

9. New objectives of specialty research:

Supplementary improvement in performance of first trimester screening for fetal anomalies by

inclusion of new ultrasound markers:

- brain stem in early detection of open spina bifida cases,

- hepatic artery Doppler - for aneuploidies.

Etiologic diagnosis of fetal anomalies through new non-invasive tests in prenatal diagnosis in order to

replace invasive testing for fetal karyotyping (with associated risk of miscarriage), by:

- using maternal-fetal microchimerism with detection, isolation and study of fetal DNA in

eritroblasts present in maternal blood,

- testing cell free fetal DNA in maternal blood.

Over the past decade, cell free fetal nucleic acid detection has been increasingly used to diagnose genetic

diseases and identify pregnancy complications, mainly in a research setting. Incorporation of detection and

measurement of fetal nucleic acids into routine obstetric care is likely to become a reality if future

population-based, double blind, large-scale clinical cohort trials confirm the promising results of smaller

studies (125a).

Furthermore, ready identification of uniquely fetal transcripts in maternal blood will allow an expansion of

this field from traditional prenatal diagnosis of Mendelian disorders to improvement of our overall

understanding of physiology and pathology in the living human fetus. Therefore, prenatal diagnosis is poised

to evolve from detection of aneuploidy to detection of deviation from normal development, which should

provide novel opportunities for fetal treatment (201a).

39

Analysis of the correlation between risk factors and fetal anomalies to completely define

etiopathogenesis of fetal anomalies in order to create new strategies of primary prophylaxis along

with the secondary (TOPFA) and tertiary ones (intrauterine/neonatal treatment) while aiming for

reduction of prevalence and severity of congenital anomalies.

Further investigations will determine whether in the high-risk cases for PE, pharmacological

interventions, such as low-dose aspirin, starting from the first trimester could improve placentation

and reduce the prevalence of the disease.

10. Conclusions of the Thesis

Study 1. In contrast to previous 9 small studies suggesting ADAM 12 (A Disintegrin And Metalloprotease)

as a new marker for aneuploidies, our research results (on more than 10,000 pregnancies) demonstrated that

at 11-13 weeks maternal serum concentration of ADAM 12 in cases of trisomy 21 was not significantly

different from euploids. In addition, in cases of trisomies 18, 13, Turner syndrome and triploidy, its level was

reduced but there was a strong association between ADAM 12 and PAPP-A, β-hCG levels. Therefore,

ADAM 12 did not improve the performance of first trimester biochemistry screening for aneuploidies.

Study 2. While 5 previous articles presented PIGF (Placental Growth Factor) as having low, high or

unchanged first trimester serum levels in aneuploid compared to euploid pregnancies, our study proved that

its serum level at 11-13 weeks was reduced in trisomy 21 and other chromosomal abnormalities.

Consequently, measurement of PIGF could improve the performance of first trimester biochemistry

screening for aneuploidies by β-hCG and PAPP-A.

Study 3. There were conflicting results among published first trimester studies about abnormal ductus

venosus (DV) flow in euploid fetuses with major cardiac defects as being confined to increased NT cases or

also observed in those with normal NT. However, our study on 45,191 singleton pregnancies at 11-13 weeks

demonstrated that in chromosomally normal fetuses with major cardiac defects, an abnormal DV flow was

present not only in those with increased NT, but also in normal NT cases. As a result of this, the

performance of first trimester screening for major cardiac defects provided by NT alone was improved by

DV evaluation. Furthermore, our study results showed that increased NT/abnormal DV flow was not

confined to a specific type of heart anomaly, in contradiction with a previous smaller study reporting that in

cases of normal NT and abnormal DV Doppler, all cardiac defects were right-sided.

Study 4. Previous studies reported on reduced maternal serum concentrations of ADAM 12 in first trimester

of pregnancies destined to develop preeclampsia (PE), especially in those resulting in premature/SGA

neonates. Conversely, our study (on 8,234 singleton pregnancies) demonstrated that in pregnancies

complicated by PE, GH, spontaneous premature delivery, levels of ADAM12 at 11-13 weeks were not

significantly different from normal. In addition, in pregnancies with SGA, levels of ADAM12, PAPP-A

were reduced but there was a good correlation between these 2 metabolites. As a result of this, testing

maternal serum for ADAM12 did not improve prediction for SGA by maternal history and PAPP-A.

Study 5. Previous studies reported on reduced maternal serum concentration of PIGF, PAPP-A in the first

trimester of pregnancies destined to develop PE, especially early PE. On the other hand, we previously

demonstrated that early PE could be predicted by a combination of maternal history, uterine artery PI and

serum concentration of PAPP-A at 11-13 weeks, with a DR 83,3%, FPR 5%. Moreover, our current study on

PIGF estimated that a screening by maternal history, uterine artery PI and serum concentration PIGF,

PAPP-A would identify 86% of early PE and 49% of late PE, for FPR 10%.

40

Study 6. First trimester P-selectin plasma concentration was increased in pregnancies developing PE,

according to previous studies. Similarly, uterine artery PI was increased at 11-13 weeks in pregnancies

complicated with PE, as shown in our earlier research. Hence, the current study proved that maternal plasma

concentration of P-selectin and uterine arteries PI at 11-13 weeks were increased in pregnancies

complicated with PE. Nevertheless, while P-selectin was not significantly different in early PE compared to

late PE cases, uterine arteries PI was higher in early PE than late PE. Thus, first trimester screening for early

PE by maternal factors and uterine arteries PI was not improved by measurement of P-selectin.

Study 7. The findings of this study demonstrated that the NICE recommendation for screening of PE by

maternal characteristics and previous history (using maternal risk factors as separate screening tests with

DR 89.2%, 93.0% and 85.0% for early PE, late PE and GH, respectively, FPR 64.1%) to define the intensity

of subsequent antenatal care was potentially useful only when the various factors were incorporated into a

combined algorithm derived by multivariate analysis. Such analysis proved that significant predictors for a

priori risk (derived from maternal risk factors) for early PE were black ethnic origin, history of chronic

hypertension or PE, use of ovulation inducers, while predictors for late PE and GH were advanced maternal

age, high BMI, family/personal history of PE. As a result of this, early screening for hypertensive

complications in pregnancy, based on maternal risk factors, would have DR of 47%, 41% and 31%, for

early PE, late PE, GH, respectively for a FPR 10%.

Study 8. Performance of screening for PE and GH by blood pressure measurement in the first trimester was

reported with wide variations in previous studies. In contrast, our study results demonstrated that blood

pressure at 11-13 weeks was increased in pregnancies developing PE, GH (this increase was particularly

marked in early PE) with mean arterial pressure (MAP) as a better predictor for hypertensive complications

in pregnancy than systolic/diastolic blood pressure. Accordingly, DR for early PE, late PE, GH was

improved (76%, 52%, 48% for FPR 10%) by adding MAP to a-priori risk (derived from maternal risk

factors) in a combined first trimester screening for hypertensive disorders in pregnancy.

Study 9. Our study on 8,366 pregnancies (with 305 cases of PE/GH) demonstrated increased uterine artery

PI at 11-13 weeks in pregnancies developing PE, with increase particularly marked in early PE. The best

screening performance for hypertensive disorders in pregnancy was obtained with lowest PI (compared to

mean/highest); DR was of 81%, 45%, 35% with FPR 10% for early PE, late PE, GH, respectively in first

trimester screening combining maternal risk factors and uterine artery PI. An even more efficient

screening for hypertensive disorders in pregnancy was derived from a combination of a-priori risk (based

on disease specific maternal risk factors), lowest uterine artery PI and mean arterial pressure: DR of 89%,

57%,50%, with FPR 10% for early PE, late PE, GH, respectively.

Study 10. On a lot of 45,191 singleton pregnancies, we proved the current performance of 44% of 11-13

weeks scan in diagnosing non-chromosomal fetal anomalies. Furthermore we classified major fetal

anomalies into 3 groups in relation to the possibility of their detection in the first trimester: always

detectable (31%), undetectable (26%), potentially detectable (43% -whose diagnosis depended on their

association with easily identifiable markers and a decision policy to establish the objectives for the first

trimester scan and to provide necessary resources).

Study 11. On an unselected population of 13,457 singleton pregnancies, we demonstrated that the direct

result of high efficiency in early prenatal diagnosis (by first trimester combined screening for fetal

anomalies) is the significant reduction in frequency of major congenital anomalies - of 55,4% live births

with defects. Such result was achieved through selective terminations of pregnancies for fetal

41

abnormalities with 76,1% being performed in the first trimester having the advantage of a less traumatic

procedure, both physically and psychically.

Thesis final conclusion

By improved performance of first trimester combined screening in detecting fetal anomalies and in more

correct prediction of pregnancy evolution, early prenatal diagnosis allows to individually adjust the rhythm

and the content of prenatal visits, thus more efficient in reducing maternal and perinatal mortality and

morbidity. Hence, first trimester prenatal diagnosis becomes the basis of the current antenatal care in the

UK that replaces the classic model of prenatal care (with more frequent visits as the pregnancy progresses).

Turning the pyramid of antenatal care upside down

Classic model of prenatal care Current model of antenatal care

42

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