Predicting Preeclampsia PDF

PREDICTING PRE-ECLAMPSIA
Angiogenic factors and various forms of human chorionic

gonadotropin
Elina Keikkala
Faculty of Medicine
Institute of Clinical Medicine
Department of Obstetrics and Gynecology
University of Helsinki
Finland
Faculty of Medicine
Haartman Institute
Department of Clinical Chemistry
Finland
Academic dissertation
To be publicly discussed with the permission of the Medical Faculty of the University of
Helsinki on January 24th, 2014, at 12 noon.
Supervised by
Adjunct professor Piia Vuorela, MD, PhD
Finland
Emeritus professor Ulf-Hkan Stenman, MD, PhD
Department of Clinical Chemistry
Finland
Reviewed by
Adjunct professor Katri Koli, PhD
Translational Cancer Biology Program
Faculty of Medicine
Finland
Adjunct professor Eeva Ekholm, MD, PhD
University of Turku
Finland
Opponent
Adjunct professor Jukka Uotila, MD, PhD
University of Tampere
Finland
ISBN 978-952-10-9686-0 (Paperback)

ISBN 978-952-10-9687-7 (PDF)
http://ethesis.helsinki.fi
Unigrafia
Helsinki 2014
2
To all mothers
4
Contents
1 List of original publications ...................................................................................... 6
2 Abbreviations .......................................................................................................... 7
3 Abstract .................................................................................................................. 8
4 Introduction ............................................................................................................ 9
5 Review of the literature .......................................................................................... 10
5.1 Development of the placenta..................................................................................... 10
5.1.1 Maternal blood supply to the placenta ................................................................... 10
5.1.2 Origin of placental trophoblasts and the villous tree ................................................ 10
5.2 Pre-eclampsia .......................................................................................................... 13
5.2.1 Diagnosis and symptoms ..................................................................................... 13
5.2.2 Risk factors ........................................................................................................ 15
5.2.3 Pathophysiology ................................................................................................. 17
5.2.4 Intrauterine growth restriction .............................................................................. 20
5.2.5 Prevention of pre-eclampsia ................................................................................. 21
5.3 Prediction of pre-eclampsia ...................................................................................... 21
5.3.1 Maternal characteristics and Doppler ultrasound ..................................................... 22
5.3.2 PlGF and sVEGFR-1 ........................................................................................... 23
5.3.3 Angiopoietins ..................................................................................................... 26
5.3.4 PAPP-A ............................................................................................................. 28
5.3.5 hCG, hCG and hCG-h........................................................................................ 29
6 Aims of the study ................................................................................................... 33
7 Materials and methods ........................................................................................... 34
7.1 Patients and study design ......................................................................................... 34
7.2 Inclusion and exclusion criteria ................................................................................ 34
7.3 Samples ................................................................................................................... 36
7.4 Assays ..................................................................................................................... 36
7.5 Statistical methods ................................................................................................... 38
7.6 Ethics ...................................................................................................................... 38
8 Results .................................................................................................................. 39
8.1 First trimester hCG-h, hCG and PAPP-A (I) .......................................................... 39
8.2 Second trimester hCG-h, PlGF and sVEGFR-1 (II) ................................................... 42
8.3 Angiopoietins in the second trimester (III) ................................................................ 43
8.4 Angiopoietins at term pregnancy (IV) ....................................................................... 45
9 Discussion ............................................................................................................. 47
9.1 Prediction of pre-eclampsia ...................................................................................... 47
9.2 Fetal growth restriction............................................................................................ 50
9.3 Comparison of markers predictive of pre-eclampsia.................................................. 51
9.4 Angiopoietins at term (IV)........................................................................................ 53
9.5 Limitations of the study ........................................................................................... 53
9.6 Clinical implications ................................................................................................ 54
10 Summary and conclusions .................................................................................... 57
11 Acknowledgments ................................................................................................ 58
12 References ........................................................................................................... 60
13 Original publications............................................................................................ 75
1 List of original publications

This thesis is based on the following original publications, which are referred to in the text by
their Roman numerals:
I - Keikkala Elina, Vuorela Piia, Laivuori Hannele, Romppanen Jarkko, Heinonen Seppo,
Stenman Ulf-Hkan. First trimester hyperglycosylated human chorionic gonadotropin in
serum a marker of early-onset preeclampsia. Placenta 2013; 34:1059-65.
II - Keikkala Elina, Ranta Jenni, Vuorela Piia, Leinonen Reetta, Laivuori Hannele, Visnen
Sari, Marttala Jaana, Romppanen Jarkko, Pulkki Kari, Stenman Ulf-Hkan, Heinonen Seppo.
Serum hyperglycosylated human chorionic gonadotrophin at 1417 weeks of gestation does
not predict preeclampsia. Submitted
III - Leinonen Elina, Wathn Katja-Anneli, Alfthan Henrik, Ylikorkala Olavi, Andersson
Sture, Stenman Ulf-Hkan, Vuorela Piia. Maternal serum angiopoietin-1 and -2 and Tie-2 in
early pregnancy ending in preeclampsia or intrauterine growth retardation. Journal of
Clinical Endocrinology and Metabolism 2010; 95:126-33.
IV - Keikkala Elina, Hytinantti Timo, Wathn Katja-Anneli, Andersson Sture, Vuorela Piia.
Significant decrease in maternal serum concentrations of angiopoietin-1 and -2 after delivery.
Acta Obstetricia Gynecologica Scandinavica 2012; 91:917-22.
The original publications are reprinted with the permission of the copyright holders. Please
note that the name Leinonen in publication III is the maiden name of the author.
6
2 Abbreviations
ACOG
ADAM12
Ang-1
Ang-2
AT1-AA
AUC
BMI
CRP
DIC
FSH
GWAS
GWLS
hCG
hCG-h
hCG
HELLP
HUS
ICD-10
IGF
IGFBP
IgG
IUGR
kDa
LH
LBW
MAP
MoM
MW
NNS
NNT
PAPP-A
PI
PlGF
PP13
PRES
ROC
RR
sEng
SGA
sVEGFR-1
sTie-2
TSH
VEGF
American College of Obstetricians and Gynecologists

A disintegrin and metalloproteinase 12
Angiopoietin-1
Angiopoietin-2
Angiotensin II type 1 receptor agonistic antibody
Area under the curve
Body mass index
C-reactive protein
Disseminated intravascular coagulation
Follicle stimulating hormone
Genome-wide association screening
Genome-wide linkage analyses
Human chorionic gonadotropin
Hyperglycosylated human chorionic gonadotropin
Beta subunit of human chorionic gonadotropin
Hemolysis, elevated liver enzymes, low platelet count
Hemolytic-uremic syndrome
International Classification of Diseases, the 10th revision
Insulin-like growth factor
Insulin-like growth factor binding protein
Immunoglobulin G
Intrauterine growth restriction
Kilodalton
Luteinizing hormone
Low birth weight
Mean arterial pressure
Multiples of median
Molecular weight
Number needed to screen
Number needed to treat
Pregnancy-associated plasma protein-A
Pulsatility index
Placental growth factor
Placental protein 13
Posterior reversible encephalopathy
Receiver operating characteristics
Relative risk
Soluble endoglin
Small-for-gestational age
Soluble vascular endothelial growth factor receptor 1
Soluble endothelial cell-specific tyrosine kinase receptor 2
Thyroid stimulating hormone
Vascular endothelial growth factor
3 Abstract
Pre-eclampsia, defined as hypertension
and proteinuria, causes significant
maternal and perinatal complications. It
affects about 2-8% of all pregnancies and
there is no curative medication. If women
at risk could be identified, prevention or, at
least, reduction of the complications of
pre-eclampsia might be possible. When
serum biochemical markers and clinical
characteristics have been combined, the
prediction of pre-eclampsia has improved.
We studied whether hyperglycosylated
human chorionic gonadotropin (hCG-h) is
useful for identifying women at risk of
pre-eclampsia already in the first or second
trimester of pregnancy. Concentrations of
hCG, hCG-h, pregnancy-associated plasma
protein-A (PAPP-A) and the free beta
subunit of hCG (hCG) in the serum in the
first trimester and of placental growth
factor (PlGF), soluble vascular endothelial
growth factor receptor 1 (sVEGFR-1) and
of the angiogenetic factors, angiopoietin-1
(Ang-1), -2 (Ang-2) and their common
soluble endothelial cell-specific tyrosine
kinase receptor Tie-2 (sTie-2) in the
second trimester were studied as predictors
of pre-eclampsia.
For first trimester screening, 158 women
who
subsequently
developed
preeclampsia,
41
with
gestational
hypertension, 81 with small-for-gestational
age (SGA) infants and 427 controls were
selected among 12,615 pregnant women
who attended for first trimester screening
for Down's syndrome. For second
trimester screening we used serum samples
from 55 women who subsequently
developed
pre-eclampsia,
21
with
gestational hypertension, 30 who were
normotensive and gave birth to SGA
infants, and 83 controls.
For analysis of Ang-1, -2 and sTie-2 at 1215 and 16-20 weeks of pregnancy, 49
8
women who subsequently developed preeclampsia, 16 with intrauterine growth

restriction (IUGR) and 59 healthy controls
were recruited among 3240 pregnant
women attending for first trimester
screening for Down's syndrome. Another
20 healthy women were recruited at term
pregnancy for examination of the
concentrations of these markers in
maternal and fetal circulation and urine
and the amniotic fluid before and after
delivery.
The proportion of hCG-h out of total hCG
(%hCG-h) was lower in the first but not in
second trimester in women who
subsequently developed pre-eclampsia as
compared to controls. In the first trimester,
%hCG-h was predictive of pre-eclampsia,
especially of early-onset pre-eclampsia
(diagnosed before 34 weeks of pregnancy).
The predictive power improved when
PAPP-A, the mean arterial blood pressure
and parity were combined with %hCG-h.
When these four variables were used
together, 69% of the women who were to
develop early-onset pre-eclampsia were
identified with a specificity of 90%.
The concentrations of circulating Ang-2
during the second trimester were higher
and those of PlGF were lower in women
who later developed pre-eclampsia. Ang-2
was only 20% sensitive and at 90%
specific for predicting pre-eclampsia, but
PlGF performed well and was 53%
sensitive and 90% specific for predicting
early-onset pre-eclampsia. %hCG-h did
not provide independent prognostic value
in the second trimester.
In conclusion, hCG-h is a promising first
trimester marker of early-onset preeclampsia. In line with the earlier
observations, PlGF is a useful second
trimester predictive marker of early-onset
pre-eclampsia.
4 Introduction
Pre-eclampsia, defined as hypertension
and proteinuria after 20 weeks of
gestation, affects approximately 2-8% of
pregnant women and may lead to severe
maternal and neonatal complications
(Khan et al 2006, Duley 2009). The
etiology of pre-eclampsia is unknown,
although several risk factors have been
identified.
Recent studies show that
treatment with low-dose aspirin may
reduce the risk of early-onset preeclampsia, i.e., pre-eclampsia diagnosed
before 34 weeks of pregnancy, which
usually is a severe form of the disease
(Steegers et al 2010, Roberge et al 2012).
together with pregnancy-associated plasma

protein-A (PAPP-A), for screening of
Downs syndrome (Haddow et al 1998,
Stenman et al 2006). Hyperglycosylated
hCG (hCG-h) is a form of hCG containing
more complex carbohydrate structures than
the main form of hCG (Elliott et al 1997,
Valmu et al 2006). A large proportion of
hCG-h occurs in malignant trophoblastic
diseases and hCG-h is decreased in early
miscarriage
(Elliott
et
al 1997,
Kovalevskaya et al 2002a). Low
concentrations of hCG-h occur in the urine
of women who develop pre-eclampsia
(Bahado-Singh et al 2002).
Angiopoietins are angiogenic factors that

mediate vessel growth and development in
the placenta and the fetus (Thomas and
Augustin 2009). Maternal concentrations
of angiopoietin-1 (Ang-1), -2 (Ang-2) and
their common endothelial cell-specific
tyrosine kinase receptor Tie-2 change in
manifest pre-eclampsia as compared to
healthy controls (Hirokoshi et al 2005,
Nadar et al 2005, Sung et al 2011). Other
angiogenic factors, e.g., placental growth
factor (PlGF) and soluble vascular
endothelial growth factor receptor 1
(sVEGFR-1), have been widely studied as
predictors of pre-eclampsia but the results
have shown that their predictive value is
only moderate (Kleinrouweler et al 2012).
This study was performed to evaluate

whether maternal serum markers alone or
in combination are useful tools to predict
pre-eclampsia
in early pregnancy.
Identifying pregnant women at risk of preeclampsia may be of value in the future
with regard to prevention and appropriate
perinatal care.
Human chorionic gonadotropin (hCG)

occurs at high concentrations in several
forms in the maternal circulation during
pregnancy. The concentrations of these
different forms may vary in different
pathophysiological conditions (Stenman et
al 2006). hCG is a glycoprotein hormone
consisting of two noncovalently coupled
subunits, hCG and hCG (Stenman et al
2006). hCG is useful for diagnosis of
malignant trophoblastic diseases and,
5 Review of the literature

5.1 Development of the
placenta
The main function of the placenta is to
transfer gases, nutrients and metabolites
between the fetus and the mother. The
placenta has also endocrine and
immunological functions. It produces large
amounts of human chorionic gonadotropin
(hCG) and other hormones important for
maintaining pregnancy and for stimulating
fetal growth (Freemark 2010). In preeclampsia, placental development and
function are disturbed, which causes
several changes in these processes
(Steegers et al 2010).
5.1.1 Maternal blood supply to

the placenta
During pregnancy the maternal circulating
blood volume increases by 20-30%. The
cardiac output also increases, whereas
maternal blood pressure and vascular
resistance decrease (Duvekot and Peeters
1994). In the mother, renal plasma flow
increases by 50-85% and the glomerular
filtration rate by 40-65% (Conrad 2004).
Healthy pregnant women have often
moderately increased concentrations of
protein in the urine, but this is of no
clinical significance (Taylor and Davison
1997).
Oxygenated maternal blood flows via the
uterine arteries that branch into spiral
arteries and open into the placental
intervillous spaces. Already at the time of
placentation these spiral arteries have been
transformed into low-resistance vessels
that lack maternal vasomotor control. This
ensures an uninterrupted blood flow to the
placenta, irrespective of any changes in
maternal blood pressure (Huppertz 2008).
10
Deoxygenated blood leaves the placenta

via uterine venules that collect blood from
the periphery of the intervillous spaces.
The circulation in the placenta may reach
1000 ml per min (Konje et al 2001).
5.1.2 Origin of placental

trophoblasts and the villous
tree
The development of the placenta starts at
implantation of the blastocyst, an early
embryonic structure, which attaches to the
uterine epithelium. The blastocyst consists
of an inner cell mass, called the
embryoblast, and an outer trophoblast
layer (Schoenwolf et al 2009) (Figure 1A).
At the beginning of the third week of
gestation (one week after conception)
these trophoblasts invade the uterine
endometrium. They differentiate into
invasive
syncytiotrophoblasts,
multinucleated cells formed through fusion
of several cytotrophoblasts (Figure 1A).
The syncytiotrophoblasts continue to
invade
deeper
into
the
uterine
endometrium, now termed decidua, and
form lacunae, maternal fluid-filled spaces
between syncytial protrusions. Maternal
capillaries anastomose with these lacunae
and fill them with maternal blood (Figure
1B). Cytotrophoblasts remain at the
embryonic side where they act as stem
cells for the syncytiotrophoblasts. A new
structure termed the chorion is now formed
from the cytotrophoblast layer and
extraembryonic mesodermal cells. The
chorion is at the fetal surface of the
placenta and is later covered by the
amnion (Huppertz 2008) (Figure 1B and
Table 1).
At the beginning of the fourth week of
gestation the syncytial protrusions start to
form the basic structure of the placenta,

tree-like protrusions called villi (Figure 2).
Cells within the villi differentiate into
hematopoietic cells and form the first
embryonic vessels lined with endothelial
cells. By the end of the fourth week of
gestation, the villi have developed enough
to enable effective exchange of gases,
nutrients and metabolites between
maternal and fetal circulations (Huppertz
2008, Schoenwolf et al 2009).
endometrial stroma into the endometrial

spiral arteries. They replace both the
maternal endothelium and maternal
vascular smooth muscle cells. The spiral
arteries now enlarge and become lowresistance vessels independent of maternal
vasomotor control. This ascertains
sufficient blood flow to the placenta
throughout pregnancy (cf. chapter 5.1.1)
(Huppertz 2008, Schoenwolf et al 2009).
Different types of trophoblasts and their
functions
are
listed
in
Table
Also at the beginning of the fourth week of

gestation part of the cytotrophoblasts
differentiate
into
extravillous
cytotrophoblasts, which invade through the
Figure 1. Implantation of the blastocyst and early development of the placenta by the third
week of gestation. Modified from Schoenwolf et al (Schoenwolf et al 2009).
A - Trophoblasts from the
maternal side of the
blastocyst differentiate to
multinucleated
syncytiotrophoblasts and
invade the decidua.
BSyncytiotrophoblasts
form syncytial protrusions
and fluid-filled spaces,
lacunae, between them. The
chorion is formed from
cytotrophoblasts
and
extraembryonic mesodermal
cells, and forms the fetal
surface of the placenta.
11
Figure 2. Cross-section
of the villus. The villus
consists
of
a
syncytiotropholast layer,
a cytotrophoblast layer
and villous stroma. Fetal
vessels run inside the
villi and maternal blood
flushes the villi (Elina
Keikkala 2013).
Table 1. Different types of trophoblasts and their function (Huppertz 2008).

Trophoblast type
Weeks of
gestation
Function
Trophoblast
3-4 weeks
Form outer blastocyst layer
Differentiates into
Syncytiotrophoblasts
Cytotrophoblasts
Syncytiotrophoblast
From 3rd
week
Invade endometrium
(=decidua)
Formation of first lacunae

Form syncytial protrusions
From 4th
week
Cytotrophoblast
Villous
cytotrophoblast
Extravillous
trophoblast
3-4 weeks
From 5th
week
From 4th
week
From 5th
week
Form outer (=syncytial) layer

of villous tree throughout
pregnancy
Forms chorion with
extraembryonic mesodermal
cells
Forms layer under
syncytiotrophoblasts in
placental villi
Maintenance of syncytial layer
of villi by differentiation
Invade endometrial stroma
(decidua)
Villous
cytotrophoblasts
Extravillous
cytotrophoblasts
Invade through maternal spiral

arteries
Replace maternal endothelium
and smooth muscle cells in
spiral arteries
Transformation of spiral
arteries to dilated tubes
without maternal vasomotor
control
12
Throughout pregnancy the villi continue to

mature and the placenta to develop. From
the central to the distal part of the villous
tree the number of cytotrophoblasts and
the thickness of the syncytiotrophoblast
layer
decrease
(Huppertz
2008,
Schoenwolf et al 2009). During the
pregnancy, the weight of the placenta and
the
amount
of
cytoand
syncytiotrophoblasts
increase.
The
syncytiotrophoblasts remain dominant
form and the proportion in relation to
cytotrophoblasts increases as pregnancy
advances (between weeks 13 and 31)
(Mayhew et al 1999).
5.2 Pre-eclampsia
Pre-eclampsia is a major cause of infant
and maternal mortality and morbidity. It
affects 2-8% of all pregnancies worldwide
(Khan et al 2006). Of all deaths from preeclampsia, 99% occur in the developing
countries (Duley 2009). The perinatal risks
include intrauterine growth restriction
(IUGR) (see chapter 5.2.4) and
prematurity (Duley 2009). Management is
mainly symptomatic, i.e., antihypertensive
drugs and magnesium infusions, which are
used in an attempt to prevent maternal and
fetal complications (Datta 2004). Delivery
is the only curative treatment for preeclampsia (Duley 2009). Despite extensive
research its etiology remains unknown.
In Finland, the incidence of pre-eclampsia
was 2.6 % among nulliparous women
between 1997 and 2008 (Lamminp et al
2012). The lifetime incidence of preeclampsia was 5.0 % according to a large
national study conducted between 2000
and 2001, while that of gestational
hypertension was almost 19 % (Koponen
and Luoto 2004). Interestingly, a crosssectional study of 3650 women reported a
twofold risk of pre-eclampsia in northern
Finland as compared to southern Finland
(Kaaja et al 2005), but this was not
confirmed in a recent
(Lamminp et al 2012).
cohort study
5.2.1 Diagnosis and symptoms

The diagnosis of pre-eclampsia is based on
findings of high blood pressure and
proteinuria in previously normotensive
women after 20 weeks of pregnancy
(Steegers et al 2010) (Table 2). The
symptoms, clinical findings and the order
in which they occur vary. The most typical
symptoms
are
headache,
visual
disturbances, upper abdominal pain and a
general feeling of being unwell. The
condition may also be asymptomatic
(Duley 2009).
Clinical findings vary from mild blood
pressure elevation and proteinuria to
uncontrolled hypertension, anuria and
severe cerebral symptoms, such as
hyperreflexia and convulsions (Steegers et
al 2010). The severity of the disease is
defined according to clinical symptoms,
blood pressure and/or urinary protein
excretion (Table 2) (American College of
Obstetricians and Gynecologists 2002,
Steegers et al 2010). Severe manifestations
include eclampsia, hemolysis, elevated
liver enzymes, low platelet (HELLP) syndrome, disseminated intravascular
coagulation (DIC) syndrome, hemolyticuremic syndrome (HUS) and posterior
reversible
encephalopathy
(PRES)
(Zeeman 2009, Tranquilli et al 2012).
Eclampsia, defined as new tonic-clonic
seizures in women with pre-eclampsia, is
often followed by severe maternal and
fetal complications, even death (MacKay
et al 2001, Duley 2009). Classification of
pre-eclampsia into severe or mild, and
according to its time of diagnosis, is
shown in Table 2.
The onset of the disease is related to its
severity. In the US population, onset
before 32 weeks of pregnancy is related to
a 20-fold risk of maternal mortality as
13
compared to onset after 37 weeks of

pregnancy (MacKay et al 2001). Preeclampsia before 34 weeks of gestation is
classified as early and after this as lateonset disease (Tranquilli et al 2012) (Table
2). Pre-eclampsia may also occur
postpartum, usually defined as new-onset
hypertension and proteinuria within 6
weeks after delivery (Sibai 2012). Women
with chronic hypertension or hypertension
diagnosed before 20 weeks of pregnancy

and with proteinuria or with any of the
other signs or symptoms described in
Table 2 is defined as superimposed preeclampsia
(American
College
of
Obstetricians and Gynecologists 2002).
Table 2. Diagnostic criteria of pre-eclampsia and severe pre-eclampsia based on the

guidelines of the American College of Obstetricians and Gynecologists (ACOG) in 2002
(American College of Obstetricians and Gynecologists 2002).
Blood
pressure
Proteinuria
Pre-eclampsia
Severe pre-eclampsia
All criteria below
Criteria of pre-eclampsia and one or

more of the criteria below
Previously normal blood pressure

Occurs 20 weeks of pregnancy
Systolic 160 mmHg and/or

diastolic 110 mmHg
Systolic 140 mmHg and/or

diastolic 90 mmHg
Measured at least twice 6 h apart

while patient in bed rest
0.3 g in 24-h urine collection
5 g in 24-hour urine collection or

urine dipstick test 3+ in two
samples collected more than 6 h
apart
Other signs
or
symptoms
Oliguria < 500 mL/24-h

Cerebral or visual disturbances
Pulmonary edema or cyanosis
Epigastric or right upper-quadrant
pain
Fetal growth restriction
Thrombocytopenia
Impaired liver function
Onset of
preeclampsia
Early-Onset
Onset before 341 weeks of pregnancy
Late-Onset
Onset at 341 weeks of pregnancy or later
According to the statement of International Society for the Study of Hypertension in

Pregnancy in 2012 (Tranquilli et al 2012).
14
5.2.2 Risk factors

Risk factors have been identified in studies
based on national birth registries
(Skjaerven et al 2005, Lamminp et al
2012) and prospective cohort studies
(Sibai et al 1995, Knuist et al 1998, Ros et
al 1998). Some risk factors, such as first
pregnancy
(nulliparity),
are
well
established according to numerous studies
involving various populations, whereas
some factors are more controversial (Table
3).
Established risk factors
Nulliparity increases the risk of preeclampsia approximately 3-fold as
compared to multiparity (Skjaerven et al
2005, Luo et al 2007). It is assumed that
immune tolerance against the fetus is not
as well developed in the first as in later
pregnancies, which could, through
immunological maladaptation, associate
with pre-eclampsia. There is, however,
little evidence for this biological
mechanism (Luo et al 2007). Other
theories suggest that differences in the
profiles of angiogenic factors or in insulin
resistance between nulliparous and
multiparous women may be related to preeclampsia (Luo et al 2007).
A history of pre-eclampsia is a strong risk
factor for pre-eclampsia in subsequent
pregnancies: the risk of pre-eclampsia
recurring is 7-fold in the second pregnancy
(Duckitt and Harrington 2005). Also, a
family history of pre-eclampsia in firstdegree relatives almost triples the risk
(Duckitt and Harrington 2005). Cohort
studies show that the incidence of preeclampsia is also related to ethnicity:
African-American women seem to have
more pre-eclampsia than Caucasian
women (Sibai et al 1995, Knuist et al
1998). These findings indicate that there is
also genetic influence on the occurrence of
pre-eclampsia (Trogstad et al 2011).
Multiple pregnancies increase the risk of

pre-eclampsia. The risk is 3-fold when
comparing twin to singleton pregnancies,
and 3-fold when comparing triplet to twin
pregnancies (Duckitt and Harrington
2005). Here, the risk increment could be
related to the increased placental mass,
which releases high amounts of placental
cytokines and antiangiogenic factors into
the circulation causing a systemic
inflammatory response (Bdolah et al
2008). Also, exposure to paternal genetic
material may be increased in multiple
pregnancies (Trogstad et al 2011).
Obesity increases the risk of preeclampsia. A pre-pregnancy body mass
index (BMI) of more than 35 kg/m2
increases the risk 3- to 5 fold as
compared to those with a pre-pregnancy
BMI of less than 24 kg/m2 (Duckitt and
Harrington 2005). Here, maybe changes in
lipid metabolism and inflammatory
responses known to be present in obese
women are pathophysiologically related to
an enhanced risk of pre-eclampsia (Bodnar
et al 2005). In Finland, the proportion of
overweight (BMI > 30 km/m2) among
women aged 25-34 years has increased
from 8.5% to 9.4% between 1999 and
2007 (Helakorpi et al 1999, Helakorpi et al
2008). As obesity is an increasing problem
in all Western countries, the incidence of
pre-eclampsia and other severe health
consequences will probably increase
(Luoto et al 2011).
Pre-existing medical conditions such as
insulin-dependent
diabetes
and
autoimmune diseases are clear risk factors
of pre-eclampsia (Duckitt and Harrington
2005). Chronic hypertension increases the
risk of pre-eclampsia approximately 10fold as compared to controls. Women with
chronic hypertension are also at risk for
developing a severe form of the disease
(Sibai et al 1995, McCowan et al 1996).
Diastolic hypertension of 110 mmHg
15
before the 20th week of gestation increases

the risk for pre-eclampsia 5-fold, preterm
delivery (before gestational week 32) 7fold and IUGR 7-fold (McCowan et al
1996). Antihypertensive treatment has not
been shown to reduce the risk of
subsequent pre-eclampsia, IUGR or
preterm delivery (American College of
Controversial risk factors
Some studies conclude that a maternal age
above 40 years doubles the risk of preeclampsia independently of parity (Duckitt
and Harrington 2005, Trogstad et al 2011).
In these studies, however, no adjustment
was made for the effect of maternal age
with pre-existing diseases, such as chronic
hypertension or diabetes, which become
more prevalent with age and may influence
the results (Duckitt and Harrington 2005,
Trogstad et al 2011). Data on the effect of
a very young maternal age (less than 19
years) on the risk of pre-eclampsia are
inconsistent, and not well supported in the
literature (Duckitt and Harrington 2005).
Women who smoke during pregnancy
seem to have less pre-eclampsia than nonsmokers (Trogstad et al 2011). Smoking
can, however, by no means be encouraged
since it has numerous adverse effects on
pregnancy which far outweigh any
possible benefits (Andres and Day 2000).
Also pre-eclamptic women who stop
smoking have better pregnancy outcomes
than women who continue smoking
(Pipkin et al 2008).
16
Paternal factors seem also to contribute to

the occurrence of pre-eclampsia. Men who
have, in an earlier relationship, fathered a
pregnancy with pre-eclampsia or who
themselves are born from a pre-eclamptic
pregnancy, have an increased risk to have
a child born from a pre-eclamptic
pregnancy (Trogstad et al 2011).
Primipaternity may also be an interesting
risk
factor
for
pre-eclampsia,
a
phenomenon not observed in all studies,
but one which might be explained by the
usually long intervals between pregnancies
fathered by different men, as the risk of
pre-eclampsia in multiparas seems to
increase if the time from the previous
pregnancy reaches 4-5 years (Trogstad et
al 2001).
In conformity with what is known about
the risk of pre-eclampsia among
primiparous women, it has been claimed
that induced abortions might reduce the
risk of pre-eclampsia in subsequent
pregnancies (Seidman et al 1989, Sibai et
al 1995). Data on spontaneous abortions
and miscarriages are conflicting in this
respect (Seidman et al 1989, Sibai et al
1995, Xiong et al 2002). Infertility and
recurrent
spontaneous
miscarriages
increase the risk of pre-eclampsia, which,
in turn, might be due to some shared
underlying
pathophysiologic
feature
hindering a normal pregnancy (Trogstad et
al 2011).
Table 3. Risk factors for pre-eclampsia
Risk factor
Risk
Established risk factors

Pregnancy-associated factors
Nulliparity
3-fold a
Previous pre-eclampsia
7-fold b
Multiple pregnancy
3-fold b
Familial factors
Family history of pre-eclampsia
3-fold b
Ethnicity (African-American)
2-fold b, c
Pre-existing medical conditions
Obesity (BMI > 35 kg/m2)
3- to 5-fold b
Chronic hypertension
10-fold b, c
Insulin-dependent diabetes mellitus
3- to 4-fold b
Anti-phospholipid syndrome and other
10-fold b
thrombophilic conditions
Autoimmune disease
7-fold b
Renal disease
3-fold b
Controversial risk factors
Personal history
High maternal age (> 40 years)
2-fold b
Teenage pregnancy (< 19 years)
Increased/ No effectb
Smoking during pregnancy
0.5- to 0.8-fold d
Paternal factors
Men fathered a pregnancy with preIncreased/ No effect d
eclampsia
Primipaternity
Increased/ No effect d
Pregnancy-associated factors
Long (> 4-5 years) interval between
pregnancies
Induced abortion
Decreased/No effect c
Previous miscarriage
Increased/ No effect c, d
Infertility
In-vitro fertilization/ donor insemination
a
b
Skjaerven et al 2005, Duckitt and Harrington 2005, c Sibai et al 1995,
d
Trogstad et al 2001.
5.2.3 Pathophysiology
The proteinuria of pre-eclampsia results
from glomerular endotheliosis and preeclamptic hypertension and is considered
to be due to secondary endothelial
dysfunction (Roberts et al 1989). Any
other features of the pathophysiology of
pre-eclampsia are more speculative. Some
theories are listed in the following.
Placental vascular remodeling and

endothelial dysfunction
A generally accepted hypothesis for the
pathophysiology of pre-eclampsia is
impaired transformation of the spiral
arteries. This is thought to be a two-stage
process. In the first stage, the physiological
adaptation of the uterine spiral arteries is
thought to be inadequate, which leads to a
hypoxic-ischemic reperfusion circulation
17
in the placenta. In the second stage,

hypoxia is followed by the release of
several biologically active placental factors
into the maternal blood circulation. These
factors are thought to cause maternal
endothelial dysfunction and a systemic
inflammatory reaction (Redman and
Sargent 2009).
In pre-eclampsia the differentiation of
cytotrophoblasts into extravillous invasive
cytotrophoblasts is incomplete and
invasion into the maternal spiral arteries
shallow. This could explain why the spiral
arteries do not undergo the changes that
occur in healthy pregnancies (Meekins et
al 1994, Zhou et al 1997, Burton et al
2009). The maternal spiral arteries retain
their smooth muscle layer and remain
high-resistance arteries (Brosens et al
1970) (see chapter 5.1.1) (Figure 3).
Intermittently inadequate blood supply to

the placenta leads to an ischemic
reperfusion state and there are changes
between hypoxia and reoxygenation
(Redman and Sargent 2009). In vitro
studies of cultures of term placenta show
that ischemic reperfusion causes damage
similar to what occurs in pre-eclamptic
placentas (Hung et al 2001).
The structure of the placenta changes in
pre-eclampsia, especially in early-onset
pre-eclampsia
and
pre-eclampsia
accompanied with IUGR. The intervillous
spaces widen and may become lined with
thrombotic material, and the volume of the
terminal villi and surface area are reduced
(James et al 2010) (Figure 3). This is
probably
secondary
to
impaired
uteroplacental blood flow.
Figure 3. Spiral artery transformation in the pre-eclamptic and healthy placenta (Elina
Keikkala 2013).
Inflammatory system
In pre-eclampsia, the usual activation of
the inflammatory system (including
immune cells and the clotting and
complement systems) and of the
18
endothelium is exaggerated. This would

enhance synthesis of the acute phase
proteins including C-reactive protein
(CRP), several complement components
and fibrinogen (Redman and Sargent
2009).
An
exaggerated
systemic
inflammatory reaction is associated with
several findings in pre-eclampsia: with the
severity of the HELLP syndrome (Terrone
et al 2000), the degree of abnormal
hemostasis, impaired clotting and DIC
(Thachil
and
Toh
2009).
Syncytiotrophoblasts secrete also several
pro-inflammatory
factors,
including
soluble vascular endothelial growth factor
receptor 1 (sVEGFR-1), into the maternal
circulation. These factors may well be
related to maternal endothelial dysfunction
(Redman and Sargent 2009).
Cytotrophoblastic plugging
Burton et al introduced a theory of
cytotrophoblastic plugging that protects
embryos against oxidative stress. Until the
eighth week of gestation the intervillous
spaces are plugged by cytotrophoblast
layers and arterial blood cannot enter them
(Burton et al 1999). These plugs would
then be displaced at the end of the first
trimester allowing oxygenated blood to
enter the intervillous space and the
peripheral placenta. It is further thought
that failure to form these plugs might lead
to early oxidative stress of the placenta and
damage to the syncytiotrophoblast layer.
The clinical consequences would then be
miscarriage or pre-eclampsia, especially in
its early-onset form (Burton and Jauniaux
2011). This theory is supported by
histological studies on placentas from
early
miscarriage,
where
no
cytotrophoblastic plugs were observed
(Hustin et al 1990). Regarding preeclampsia, there is no direct evidence for
or against this theory.
Autoantibodies
Wallukat et al reported significant titers of
angiotensin II type 1 receptor agonist
antibodies (AT1-AA), an immunoglobulin
G (IgG) class autoantibody, in the sera of
70-95%
of
pre-eclamptic
women
(Wallukat et al 1999, Walther et al 2005,
Siddiqui et al 2010). These antibodies are
not present in the blood of non-pregnant
women, pregnant women with essential

hypertension or in women with
uncomplicated pregnancies (Wallukat et al
1999). When IgG extracted from the serum
of pre-eclamptic women was injected into
pregnant mice, typical symptoms of preeclampsia
ensued:
hypertension,
proteinuria and intrauterine growth
restriction (Zhou et al 2008). However,
AT1-AA is not specific for pre-eclampsia,
since it is also present in the serum of
women with gestational hypertension
(Wallukat et al 1999) and with renal
allograft rejection (Dragun et al 2005). The
role of AT1-AA in the pathophysiology of
pre-eclampsia needs still to be clarified.
Genetics
Pre-eclampsia is a complex disorder
caused by genetic as well as environmental
factors (Williams and Broughton Pipkin
2011). A variety of genetic changes have
been observed in association with preeclampsia, including mutations that affect
endothelial function, vasoactive proteins,
oxidative stress, the blood clotting system
and immunological factors. Screening of
the entire genome by genome-wide linkage
analyses (GWLS) has revealed three loci
that are significantly linked to preeclampsia: 2p13, 2p25 and 9p13
(Arngrimsson et al 1999, Laivuori et al
2003). Further information could be
derived by the most up-to-date method,
genome-wide
association
screening
(GWAS), which allows more exact ways
to investigate gene polymorphisms and
single candidate genes (Williams and
Broughton
Pipkin
2011).
The
heterogeneity of pre-eclampsia, genetic
variation
between
populations,
environmental interactions and agedependent effects are challenges even for
the latest genetic technologies.
19
5.2.4 Intrauterine growth

restriction
Definitions
The criteria for IUGR are an estimated
fetal weight below the tenth percentile and
evidence of placental insufficiency (Ott
1988). Low birth weight (LBW), defined
as a birth weight of less than 2500 g, is a
more explicit term that is often used in
national registries and international
databases (UNICEF 2007). In Finland,
4.2% of infants were of LBW in 2012
(Vuori and Gissler 2013). Infants smallfor-gestational-age
(SGA),
another
diagnosis used in parallel with IUGR and
LBW, are usually defined as having a
gestational-age adjusted birth weight
below the tenth percentile in the absence
of placental insufficiency. SGA was
diagnosed in 2.2% of the infants born from
singleton pregnancies in Finland in 2011
(Vuori and Gissler 2012).
Approximately 20-30% of pre-eclamptic
pregnancies are associated with IUGR, and
- vice versa - 10% of all cases of IUGR are
secondary to pre-eclampsia (Villar et al
2006). Little is known about the general
etiology of IUGR, although impaired
development of the placenta similar to
what occurs in pre-eclampsia is
occasionally seen (James et al 2010).
Maybe pre-eclampsia and IUGR are
different manifestations of the same
disease, since the placental pathology has
similar traits. Nevertheless, clinical
placental insufficiency occurs only in
some cases of IUGR and pre-eclampsia
and is by no means a universal finding in
these conditions (Villar et al 2006, James
et al 2010).
Diagnosis
The diagnosis of IUGR is based on
estimation of the fetal weight by
ultrasound and of the waveforms of the
uterine, umbilical and middle cerebral
20
arteries by Doppler velocimetry (Bamberg

and Kalache 2004). The main diagnostic
aim is to distinguish IUGR with placental
insufficiency from SGA, because IUGR is
strongly related to perinatal complications
and poor neurological outcome (Yanney
and Marlow 2004).
Causes and risk factors
The causes of IUGR and SGA are
heterogenic
and
involve
maternal
hypertension or pre-eclampsia, maternal
substance
abuse,
malnutrition,
chromosomal
abnormalities,
genetic
diseases and congenital
infections
(Sankaran and Kyle 2009). However, most
IUGR cases remain unexplained (Villar et
al 2006, James et al 2010).
IUGR and pre-eclampsia share some risk
factors, e.g., nulliparity and multiple
pregnancies (Villar et al 2006). Similar
pathological changes occur in the
formation of placental vessels: the number
and surface area of placental tertiary villi
arterioles are reduced, maternal spiral
artery formation is impaired and the fetal
cytotrophoblast layer is disorganized
(Khong et al 1986, Sankaran and Kyle
2009).
In
addition,
angiogenesispromoting
factors
affect
placental
development in a similar way in IUGR and
pre-eclampsia, and the concentrations of
placental growth factor (PlGF) in the
maternal serum are lower in IUGR and in
pre-eclampsia than in healthy controls
(Helske et al 2001, Taylor et al 2003,
svold et al 2011).
There are differences between IUGR and
pre-eclampsia. A previous infant with
LBW is a risk factor for IUGR but not for
pre-eclampsia (Villar et al 2006). Women
giving birth to IUGR infants do not usually
exhibit
the
systemic
endothelial
dysfunction and inflammation reaction that
occurs in pre-eclampsia (James et al 2010).
The studies on serum concentrations of
antiangiogenic factors, e.g., sVEGFR-1,
have yielded conflicting results. sVEGFR-
1 has been reportedly elevated in preeclampsia but not in unexplained IUGR in

some studies (Wathen et al 2006), whereas
others report similar changes in IUGR and
pre-eclampsia (svold et al 2011).
5.2.5 Prevention of pre-eclampsia

The preventive effect of dietary
supplements and lifestyle modifications
when started before symptoms of preeclampsia in risk populations has been
examined in several studies. Calcium
supplementation reduces the risk of preeclampsia only among women with low
dietary calcium intake and a high risk of
pre-eclampsia (Hofmeyr et al 2010). Other
food supplements including magnesium,
fish oil, folic acid, antioxidants and
vitamin C, D and E do not affect the risk
of pre-eclampsia. Numerous drugs have
also been studied but most of them, e.g.
anti-hypertensive
medication,
low
molecular weight heparin, nitric oxide,
progesterone and diuretics, are not
effective for prevention of pre-eclampsia
(Dekker and Sibai 2001, Thangaratinam et
al 2011).
A recent systematic review of five
randomized controlled trials in high-risk
populations showed that low-dose aspirin
started before 16 weeks of pregnancy
reduces the risk of pre-eclampsia. The
relative risk (RR) of early-onset preeclampsia (diagnosed before 37 weeks)
was 0.11 (95% confidence interval, CI,
0.04 - 0.33), but low-dose aspirin has no
effect on pre-eclampsia occurring after 37
weeks of pregnancy (Roberge et al 2012).
This has been suggested to be due to the
beneficial
effects
of
aspirin
on
placentation, although the specific
mechanism is unknown. Studies included
in the review were performed on nulliparas
or women with a history of pre-eclampsia,
IUGR or chronic hypertension (Roberge et
al 2012). In contrast, in a meta-analysis by
Askie et al comprising 31 randomized
trials, only a 10% reduction in the RR of

pre-eclampsia was observed among
women on low-dose aspirin or other
antiplatelet or anticoagulant agents
(dipyridamole or heparin). However, the
timing of treatment varied, which probably
explains the difference in results as
compared to those in the review by
Roberge et al (Askie et al 2007, Roberge et
al 2012).
5.3 Prediction of preeclampsia

Various biochemical markers related to
endothelial dysfunction, oxidative stress,
insulin resistance and immunological
function have been sought for in samples
of urine and serum for prediction of preeclampsia. Clinical data on maternal risk
factors
and
Doppler
ultrasound
measurements assessing placental blood
flow
have
been
combined
with
biochemical factors in an attempt to
improve the predictive accuracy (Steegers
et al 2010). Recent studies have focused on
prediction in the first trimester of
pregnancy. No test has yet proven to be
sensitive and specific enough for clinical
use (Giguere et al 2010, Kuc et al 2011).
Some of the most promising predictive
markers of pre-eclampsia are listed in
Table 4.
5.3.1 Maternal characteristics

and Doppler ultrasound
Some maternal characteristics may be used
to calculate the risk of pre-eclampsia.
Known risk factors are some chronic
diseases, e.g., essential hypertension,
insulin-dependent diabetes, high blood
pressure in the first or second trimester,
high BMI or weight before pregnancy or
during the first trimester, and history of
pre-eclampsia. The smoking habits are also
involved. These characteristics are used to
estimate the overall risk of pre-eclampsia
(Steegers et al 2010).
21
Table 4. Prediction of pre-eclampsia by Doppler ultrasound and serum biomarkers. Arrows

indicate the Doppler ultrasound findings or changes in the concentrations of the respective
biomarkers in women who developed pre-eclampsia as compared to controls ( elevated,
decreased, no change).
1st trimester
2nd trimester
PE
EO
LO
PE
EO PE LO
Doppler ultrasound
PE
PE
PE
Uterine artery diastolic flow

a
a
Uterine artery pulsatility index
b
b
b
Uterine artery resistance index

b
Biochemical markers
Activin A
c
Inhibin A
c
A disintegrin and metalloproteinase
c
12 (ADAM12)
Free subunit of hCG (hCG)
c
Human chorionic gonadotropin (hCG)
i
d
d
e
e
f
g,h
g,j

c
c
c
k
(PAPP-A)
Placental protein 13 (PP13)

c
c
c
Placental growth factor (PlGF)
c c
l
l
l
c
m
m
Soluble endoglin (sEng)

Soluble vascular endothelial growth
c
c
c
l
l
l
factor receptor 1 (sVEGFR-1)
PE, pre-eclampsia (onset not specified); EO PE, early-onset pre-eclampsia; LO PE, lateonset pre-eclampsia. a Harrington et al 1991, b Akolekar et al 2009, c Kuc et al 2011,
d
Giguere et al 2011, e Muttukrishna et al 2000, f Bestwick et al 2012, g Wald et al 2006,
h
Pouta et al 1998, i Morris et al 2008, j Sorensen et al 1993, k Bersinger and Odegard 2004,
l
Levine et al 2004, m Levine et al 2006.
A large prospective study involving
nulliparous women showed that a systolic
blood pressure of 120 mmHg between
13 and 27 weeks of pregnancy, a high
body weight before pregnancy (120% of
desirable weight) and the number of
previous
miscarriages
were
each
independently predictive of pre-eclampsia
(Sibai et al 1995). Maternal characteristics
alone provide risk calculations with a
sensitivity of approximately 50% and a
specificity of approximately 90% (Kuc et
al 2011).
Doppler ultrasound is used to evaluate the
uteroplacental circulation in complicated
pregnancies (Campbell et al 1983). In preeclampsia, reduced uterine artery diastolic

flow is often considered as a sign of
reduced perfusion (Staudinger et al 1985).
Reduced blood flow can be seen already
before clinical symptoms of pre-eclampsia
emerge (Harrington et al 1991) or IUGR
(Harrington et al 1997). This could be an
indirect sign of impaired placental vascular
development, i.e., impaired trophoblast
invasion and spiral artery transformation
(Harrington et al 1991). A high firsttrimester uterine artery pulsatility index
(PI) or resistance index (RI) predict preeclampsia, especially its early-onset form;
the specificity of these variables has been
22
90%, but sensitivity has varied from 29%

to 83% (Akolekar et al 2009, Kuc et al
2011). One reason for the marked degree
of variation could be related to the
technique
of
Doppler
ultrasound
examination, since its sensitivity depends
strongly on the person who performs the
examination.
5.3.2 PlGF and sVEGFR-1

PlGF
The placental growth factor (PlGF) is a
member of the vascular endothelial growth
factor (VEGF) family. It is a 46 kilodalton
(kDa) dimeric protein mainly expressed in
villous trophoblasts of the placenta
(Malone et al 1991) (Figure 4). The protein
is expressed also slightly in some other
organs (heart, lung and adipose tissue)
(Table 5). PlGF is considered to be
angiogenic (Ziche et al 1997). It has also
chemotactic properties and mobilizes
hematopoietic progenitor cells from the
bone
marrow.
The
angiogenic
characteristics of PlGF play a role in
pathological conditions like ischemia,
tumor growth and wound healing. PlGF
knockout mice are born without vascular
defects, but their responses to ischemic
insults are reduced (Ribatti 2008).

sVEGFR-1
Soluble vascular endothelial growth factor
receptor 1 (sVEGFR-1) is a circulating
receptor of PlGF and VEGF. sVEGFR-1 is
formed by alternative mRNA splicing of
the membrane-bound form of VEGFR-1 or
is released from the membrane by
proteolytic cleavage (Kendall and Thomas
1993). Therefore, its molecular size varies
between 60 and 150 kDa. In addition to
placental trophoblasts it is expressed by
vascular endothelial and smooth muscle
cells, activated mononuclear monocytes in
the peripheral blood, corneal epithelial
cells and the proximal tubular epithelial
cells of the kidneys (Figure 4 and Table 5).
The main function of sVEGFR-1 is to bind
circulating VEGF, which leaves less free
VEGF available for cell surface-bound
VEGFR-1 receptors. This, in turn, inhibits
the angiogenic function of VEGF. In
addition, sVEGFR-1 counteracts edema,
since cell membrane-bound VEGF also
induces vascular
permeability and
promotes inflammation (Wu et al 2010).
Figure 4. Expression of PlGF,

sVEGFR-1 and VEGF in the
placental villus. ST,
syncytiotrophoblasts; CT,
cytotrophoblasts; EC,
endothelial cells; Str, stroma
(Elina Keikkala 2013).
23
PlGF and sVEGFR-1 in uncomplicated

pregnancy
In normal pregnancy, PlGF and sVEGFR1 are expressed in the placenta by various
cell types (Figure 4 and Table 5) (Ahmed
et al 1995, Clark et al 1996, Vuorela et al
1997, Zhou et al 2002, Tsatsaris et al
2003). sVEGFR-1 is not expressed in
placental vascular endothelial cells, in
contrast to membrane-bound VEGFR-1
(Clark et al 1996, Ribatti 2008). sVEGFR1 is not detectable in the sera of nonpregnant women and it becomes detectable
in the maternal circulation at six weeks of
gestation (Levine et al 2004, Wathen et al
2006, Noori et al 2010). The
concentrations of PlGF increase during
pregnancy and decrease at term (Taylor et
al 2003, Levine et al 2004, Noori et al
2010) (Figure 5).
PlGF and sVEGFR-1 in pre-eclampsia
PlGF and sVEGFR-1 have been widely
studied as markers of pre-eclampsia (Kuc
et al 2011, Kleinrouweler et al 2012).
Manifest pre-eclampsia does not seem to
affect the placental expression of PlGF
(Tsatsaris et al 2003, Zhou et al 2002), but
PlGF mRNA is reduced in women who
subsequently
develop
pre-eclampsia,
according to a study on first-trimester
placental biopsies (Farina et al 2008)
(Table 5). Concentrations of PlGF in the
serum are lower in manifest and
24
subsequent pre-eclampsia as compared to

controls (Torry et al 1998, Maynard et al
2003) (Figure 5). Some studies show lower
concentrations already in the first trimester
of pregnancy of women who develop preeclampsia (Thadhani et al 2004, Akolekar
et al 2008, Noori et al 2010), but other
studies show no difference (Ong et al
2001, Taylor et al 2003, Levine et al
2004). However, all published studies
show that serum PlGF concentrations are
lower in the second trimester in women
who subsequently develop pre-eclampsia
than in controls (Tidwell et al 2001, Taylor
et al 2003, Levine et al 2004, Wathen et al
2006, Noori et al 2010, Villa et al 2013)
(Table 5 and Figure 5).
In pre-eclampsia, the expression of
sVEGFR-1 is increased especially in
invasive cytotrophoblasts (Maynard et al
2003, Tsatsaris et al 2003, Ahmad and
Ahmed 2004) and in first trimester
placental biopsies in women who later
develop pre-eclampsia (Farina et al 2008).
High concentrations of maternal serum
sVEGFR-1 may be present already before
clinical symptoms (Maynard et al 2003,
Tsatsaris et al 2003, Levine et al 2004,
Wathen et al 2006), especially in the
second trimester (Levine et al 2004,
McKeeman et al 2004, Wathen et al 2006,
Noori et al 2010) (Table 5 and Figure 5).
Table 5. Expression of VEGF, PlGF and sVEGFR-1 in normal and pre-eclamptic placentas
(Ahmed et al 1995, Clark et al 1996, Vuorela et al 1997, Zhou et al 2002, Tsatsaris et al 2003,
Farina et al 2008) ( no change, decreased, increased).
VEGF
PlGF
sVEGFR-1
Main site of
expression
Decidua
Villous
cytotrophoblasts in
distal villi
Extravillous
cytotrophoblasts
Villous stroma
Villous
cytotrophoblasts in
distal villi
Invasive
cytotrophoblasts
Placental endothelium
Decidua
Villous
cytotrophoblasts
Extravillous
trophoblasts
Villous stroma
Perivascular cells
Changes in
subsequent
pre-eclampsia
levels of mRNA in
1st trimester
levels of mRNA in
levels of mRNA
expression in
in amount of
Changes in
pre-eclampsia
extravillous
cytotrophoblasts
1st trimester
mRNA
levels of mRNA in
1st trimester
levels of mRNA
secretion in
extravillous
cytotrophoblasts
Figure 5. PlGF and sVEGFR-1 concentrations trends during pregnancy. Dotted lines indicate
concentrations in healthy pregnant women, arrows show concentration changes in women
with pre-eclampsia (PE) ( no difference, decreased, increased). Modified from Levine
et al (Levine et al 2004).
25
5.3.3 Angiopoietins
The vascular growth factors angiopoietin-1
(Ang-1) and angiopoietin-2 (Ang-2) are 75
kDa glycoproteins which both bind to the
same site of their common endothelial cellspecific tyrosine kinase receptor Tie-2
(Fiedler et al 2003, Thomas and Augustin
2009). Ang-1 is a Tie-2 agonist and thus
angiogenic, whereas Ang-2 is a Tie-2
antagonist and thus antiangiogenic
(Maisonpierre et al 1997, Thomas and
Augustin 2009). The soluble form of Tie-2
(sTie-2) is also approximately 75 kDa in
size. It is present in the circulation after it
has shed the extracellular region of the
membrane-bound Tie-2 receptor; this leads
to decreased concentrations of functional
membrane-bound receptors (Reusch et al
2001). Like the membrane-bound receptor,
sTie-2 also binds both Ang-1 and Ang-2. It
leaves less Ang-1 and Ang-2 available for
membrane-bound receptors and thus
decreases their biological activity (Findley
et al 2007, Shantha Kumara et al 2010).
Ang-1
In the placenta, Ang-1 is expressed by the
syncytiotrophoblasts and perivascular cells
(Geva et al 2002, Thomas and Augustin
2009) (Figure 6). Ang-1 is also expressed
by myocardial cells during early human
development and by perivascular cells in
adult tissues (Davis et al 1996). Ang-1 is
also expressed in some pathological

conditions, e.g. by some tumor cells, and it
is thought to interact with VEGF in tumor
angiogenesis (Saharinen et al 2011). Its
expression is decreased by hypoxia (Zhang
et al 2001).
Ang-2
Ang-2 is mainly expressed by endothelial
cells (Thomas and Augustin 2009). Its
expression is upregulated at sites of
vascular remodeling (Zhang et al 2001). In
the placenta Ang-2 is expressed by
syncytiotrophoblasts and perivascular cells
throughout gestation, but both its protein
and mRNA expression decrease towards
term (Zhang et al 2001, Geva et al 2002)
(Figure 6). Ang-2 is also expressed in the
ovaries (Sugino et al 2005). Some tumors
express Ang-2 (Saharinen et al 2011).
Contrary to Ang-1, the expression of Ang2 is strongly upregulated by hypoxia
(Mandriota and Pepper 1998).
sTie-2
In first trimester placenta, Tie-2 is
expressed by endothelial cells, decidua
(Zhang et al 2001) and cytotrophoblasts
(Kayisli et al 2006) (Figure 6). Before
term, its expression either decreases
(Zhang et al 2001) or remains stable
(Kayisli et al 2006). Placental Tie-2
expression is not influenced by hypoxia, in
contrast to the ligands Ang-1 and Ang-2
(Zhang et al 2001).
Figure 6. Expression of Ang-1, Ang2 and Tie-2 in the placental villus.

ST, syncytiotrophoblasts; CT,
cytotrophoblasts; EC, endothelial
cells; Str, stroma (Elina Keikkala
2013).
26
Angiopoietins in uncomplicated
pregnancy
Angiopoietin-1, Ang-2 and Tie-2 are all
essential for vascular growth (Demir et al
2007). Mice which lack the Ang-1 or Tie-2
genes or which overexpress Ang-2, die in
utero because of severe vascular injuries
(Sato et al 1995, Suri et al 1996). Mice
deficient in Ang-2 survive embryogenesis
but die postpartum because of lymphatic
injuries (Gale et al 2002). In human
pregnancy, Ang-1, Ang-2 and sTie-2 have
all been detected in amniotic fluid and
maternal serum throughout pregnancy
(Pacora et al 2009, Buhimschi et al 2010).
Their concentrations during pregnancy are
higher than in non-pregnant women
(Hirokoshi et al 2005, Nadar et al 2005).
With
advancing
pregnancy
the
concentrations of Ang-1 either remain
stable (Bolin et al 2009) or increase
(Buhimschi et al 2010), whereas those of
Ang-2 decrease (Bolin et al 2009, Pacora
et al 2009, Buhimschi et al 2010) and
those of sTie-2 either remain stable or
decrease slightly (Buhimschi et al 2010,
Sung et al 2011).
Angiopoietins in pre-eclampsia
Information regarding the concentration of
Ang-1 in maternal blood in pre-eclampsia
is conflicting (Nadar et al 2005, Bolin et al
2009, Kamal and El-Khayat 2011).
Preeclampsia does not seem to change the
placental expression of Ang-1 (Sung et al
2011) (Table 6).
In most studies, maternal circulating

concentrations of Ang-2 have been lower
in women with pre-eclampsia than in
controls (Hirokoshi et al 2005, Nadar et al
2005, Hirokoshi et al 2007), but in severe
pre-eclampsia
increased
Ang-2
concentrations have been reported (Han et
al 2012). A prospective longitudinal study
with a limited number of pre-eclamptic
women (n=19) showed that the
concentrations of Ang-2 or Ang-1 did not
differ from those of controls, but the ratio
of Ang-1 to Ang-2 was lower in women
who developed pre-eclampsia from
gestation week 25 onwards (Bolin et al
2009). The results concerning Ang-2
expression in third trimester placental
tissue recovered from pre-eclamptic
women have been conflicting and it is not
known if expression differs in comparison
to non-pre-eclamptic women (Zhang et al
2001, Han et al 2012, Sung et al 2011)
(Table 6).
Maternal circulating concentrations of
sTie-2 are lower in pre-eclampsia than in
normal pregnancy according to most
studies (Vuorela et al 1998, Gotsch et al
2008, Sung et al 2011). The reduction in
the concentration of sTie-2 may be
observed already at week 24, well before
any pre-eclampsia symptoms appear (Sung
et al 2011) (Table 6).
27
Table 6. Changes in concentrations of Ang-1, Ang-2 and sTie-2 and their mRNA expression
in the placenta in women with pre-eclampsia as compared to controls ( no difference,
decreased, increased).
Trimester
1st 2nd 3rd Method
Reference
mRNA

RT-PCR
Sung et al 2011
Ang-1 Serum
ELISA (R&D Systems) Kamal and El-Khayat 2011
Plasma
ELISA (R&D Systems) Bolin et al 2009
ELISA (R&D Systems) Nadar et al 2005
mRNA
RT-PCR
Zhang et al 2001

RT-PCR
Sung et al 2011

a RT-PCR
Han et al 2012
Ang-2 Serum
ELISA (R&D Systems) Hirokoshi et al 2005
ELISA (R&D Systems) Hirokoshi et al 2007

ELISA (R&D Systems) Kamal and El-Khayat 2011
Plasma
ELISA (R&D Systems) Bolin et al 2009

a ELISA (R&D Systems) Han et al 2012
mRNA

RT-PCR
Sung et al 2011
Serum
in-house IFMA
Vuorela et al 1998
sTie-2

ELISA (R&D Systems) Sung et al 2011
Plasma
ELISA (R&D Systems) Gotsch et al 2008

a
Severe pre-eclampsia; RT-PCR, reverse transcriptase-polymerase chain reaction; IFMA,
immunofluorometric assay.
5.3.4 PAPP-A
(PAPP-A) is a protease that is expressed
almost exclusively by the placenta. The
expression and secretion of PAPP-A
increase during differentiation of villous
cytotrophoblasts to syncytiotrophoblasts
(Guibourdenche et al 2003). PAPP-A is
also expressed at low levels in some other
tissues (endometrium, colon and kidney)
(Overgaard et al 1999). The biological
function of PAPP-A is not known, but it
has been postulated that it may increase
the bioavailability of insulin-like growth
factors 1 and 2 (IGF-1 and -2) by cleavage
of insulin-like growth factor binding
proteins (IGFBP) -4 and -5 (Laursen et al
2007). Thus, PAPP-A may contribute to
28
placental and fetal growth, since IGF-1

and -2 are important mediators of cell
proliferation, differentiation and migration
(Laursen et al 2007) (Table 7). Placental
expression and circulating PAPP-A
concentrations increase with advancing
pregnancy (Overgaard et al 1999). The
production rate of PAPP-A from
trophoblasts does not increase with
advancing gestational weeks, and elevated
concentrations of PAPP-A in the plasma
may reflect syncytiotrophoblast mass
(Guibourdenche et al 2003). The
concentration of PAPP-A decreases in
Down's syndrome and that is why PAPPA, along with hCGE, is used for firsttrimester screening of the syndrome
(Haddow et al 1998, Wald 2011) (Table
7).
Table 7. Pregnancy-associated plasma protein-A (PAPP-A) (Haddow et al 1998, Overgaard

et al 1999, Guibourdenche et al 2003, Laursen et al 2007, Kuc et al 2011, Wald 2011).
PAPP-A
Heterotetramer
Structure
MW 500 kDa
Villous cytotrophoblasts
Decidua
Expression
Endometrium, myometrium
Kidney, colon, breast etc.
Biological function unclear
Main
Protease
Function
Cleaves IGFBP-4 -> increase bioavailability of IGF-1 and -2 -> May
contribute to fetal growth
IUGR: decreased
Relation to
Premature delivery: decreased
pathological
Cornelia Delange syndrome: decreased
conditions
Pre-eclampsia: decreased
Clinical
Screening of Downs syndrome in 1st trimester
implications
MW, molecular weight; kDa, kilodalton; IGFBP-4, insulin-like growth factor binding
protein-4; IGF, insulin-like growth factor.
PAPP-A in pre-eclampsia
Concentrations of PAPP-A are decreased
already in the first (Ong et al 2000, Kuc et
al 2011) and second trimesters (Bersinger
and Odegard 2004, D'Anna et al 2011) in
women who develop pre-eclampsia. Most
studies indicate that PAPP-A is moderately
adequate for prediction of pre-eclampsia
(Akolekar et al 2009, Poon et al 2009) and
other studies have not found the marker
very useful at all (Foidart et al 2010,
Vandenberghe et al 2011). All the same, in
manifest pre-eclampsia the circulating and
placental concentrations of PAPP-A are
increased in comparison to healthy
controls (Hughes et al 1980, Bersinger et
al 2002).
5.3.5 hCG, hCG and hCG-h

hCG
Human chorionic gonadotropin (hCG) is a
glycoprotein consisting of two subunits,
(hCG) and (hCG). hCG is almost
identical to the -subunits of the three
pituitary glycoprotein hormones thyroid
stimulating hormone (TSH), follicle
stimulating hormone (FSH) and luteinizing
hormone (LH), whereas the -subunits are
unique to each hormone and determine the
biological activity of the hormone
(Stenman et al 2006). Determination of
hCG is used as a pregnancy test and for
other diagnostic and therapeutic purposes.
The main biological function of hCG is to
maintain progesterone synthesis in the
corpus luteum during early pregnancy, but
it also exerts other functions, e.g.,
stimulation of angiogenesis (Stenman et al
2006) (Table 8).
29
Table 8. hCG variants. Reviewed by Stenman et al (Stenman et al 2006).

hCG
Human chorionic
gonadotropin
Structure
Expression
Main function
Clinical
implications
Heterodimer: and
subunits
237 amino acids
Glycan structures: 2
attached to -subunit and
6 attached to subunit
Disulfide bonds
MW 37.5 kDa
Trophoblastic tumors
Other tumors
Binds to LH/HCG
receptor
Stimulates progesterone
production from corpus
luteum
Stimulates uterine growth
and angiogenesis
Diagnostics:
Pregnancy test
Extrauterine pregnancy
Miscarriage
Screening of Downs
syndrome at 2nd trimester
Gestational trophoblastic
disease
Testicular germ cell
tumors
Doping control
Therapy:
Ovarian stimulation in
infertility
hCG
Free hCG subunit
Homodimer: -subunit
145 amino acids
Glycan structure
Disulfide bonds
MW 23.5 kDa
Other tumors
No receptor found
May promote tumor
growth via other
factors
hCG-h
Hyperglycosylated
hCG
Heterodimer: and
subunits
237 amino acids
Glycan structures:
more complex on and
subunits than in
hCG
Disulfide bonds
MW >37.5 kDa
Extravillous and villous
cytotrophoblasts
Testicular tumors
Binds to LH/HCG
receptor
Same as in hCG
Contributes to
trophoblast invasion
Diagnostics:
Screening of Downs
syndrome at 1st
trimester
Gestational
trophoblastic disease
Testicular germ cell
tumors
kDa, kilodalton; MW, molecular weight; LH, luteinizing hormone
hCG
hCG has several structural variants
including hyperglycosylated hCG (hCG-h)
and one variant with a free -subunit of
hCG (hCG) (Stenman et al 2006) (Table
8). hCG is mainly secreted by the
placental syncytiotrophoblasts (Gaspard et
al 1980). The concentrations of hCG in
maternal serum increase rapidly during the
week 7-10 of pregnancy and decreases
30
thereafter (Stenman et al 2006). The role

of hCG in the physiological development
of the placenta is unclear as it lacks the
biological effects of hCG (Stenman et al
2006). Clinically, hCG is used for firsttrimester screening of Downs syndrome
together with maternal serum PAPP-A
concentrations and nuchal translucency by
ultrasound (Haddow et al 1998, Wald
2011). In Downs syndrome maternal
serum hCG concentrations are higher

than in controls, which might be related to
delayed placental maturation (Macintosh et
al 1994) (Table 8).
Malignant trophoblastic cells produce
large amounts of hCG and hCG. Thus, in
hydatidiform
mole,
an
abnormal
pregnancy state with trophoblastic
proliferation, and in chorioncarcinoma the
concentrations of hCG and hCG are
higher than in normal pregnancies
(Gaspard et al 1980, Stenman et al 2006,
Lurain 2010). The proportion of hCG out
of total hCG increases in these conditions,
and a proportion of hCG of more than 5%
of hCG is a strong indicator of malignant
trophoblastic disease (Stenman et al 2006).
hCG is also expressed in many
nontrophoblastic cancers. hCG promotes
tumor growth (Gillott et al 1996) and
inhibits apoptosis in cells derived from the
urinary bladder in vitro (Butler et al 2000).
An elevated concentration of hCG in
serum is associated with an adverse
prognosis for patients with any one of
several nontrophoblastic tumors (Stenman
et al 2006).
hCG-h
Glycosylation
is
an
important
posttranslational modification of proteins.
It affects their structure and function, and
different
pathological
conditions
(including cancer) give rise to different
glycosylation variants (Hakomori 2002).
Approximately one third of the molecular
weight of hCG is carbohydrate, and
carbohydrates generate great variation to
the structure, function and half-life of hCG
(Elliott et al 1997, Valmu et al 2006). A
hyperglycosylated form of hCG (hCG-h)
was first identified in urine samples from
women with chorioncarcinoma or molar
pregnancy (Elliott et al 1997). Further
mass spectrometric studies identified a
glycan structure of hCG and showed that
hCG from chorioncarcinoma, molar

pregnancy and very early pregnancy (< 7
weeks) has a more complex glycan
structure than hCG from mid- or late
pregnancy (Valmu et al 2006). The glycan
structures of hCG in early uncomplicated
pregnancy and chorioncarcinoma or molar
pregnancy are not identical, and they all
show complex glycosylation patterns.
Thus, hCG-h is defined as a spectrum of
hCG variants that contains more complex
glycan structures than hCG (Elliott et al
1997, Valmu et al 2006) (Table 8).
In early pregnancy, hCG-h is the most
prevalent form of hCG in urine and serum
(Kovalevskaya et al 2002a). It accounts for
90-100% of all hCG at 4 weeks of
gestation and for less than 3% at 20 weeks
of gestation (Stenman et al 2011) (Figure
7). hCG-h binds to the same LH/hCG
receptor as hCG and shares most of the
biological effects of hCG (Stenman et al
2006). Expression of hCG-h differs from
that of hCG: invading extravillous
cytotrophoblasts stain deeply for hCG-h by
immunocytochemistry, whereas hCG is
mainly
present
in
placental
syncytiotrophoblasts (Kovalevskaya et al
2002b, Cole et al 2006, Guibourdenche et
al 2010). hCG-h promotes trophoblast
invasion in cell cultures, and it is thought
to mediate the invasion of cytotrophoblasts
in vivo (Cole et al 2006, Guibourdenche et
al 2010).
Increased hCG-h concentrations occur in
pregnancies with Downs syndrome,
which is thought to reflect the delayed
maturation of placenta (Cole et al 1999,
Palomaki et al 2005). In addition to
gestational trophoblastic diseases, hCG-h
is a major form of circulating hCG in
nonseminomatous
testicular
cancer
(Lempiinen et al 2012) (Table 8).
31
Figure 7. Serum concentrations of hCG, hCG-h and their ratio during pregnancy in one
woman. Modified from the thesis of Lempiinen (Lempiinen 2012).
Other hCG variants

Several other variants of hCG are known.
When excreted into urine, a large part of
hCG is degraded and forms what is called
a -core fragment of hCG (hCGcf). This
structure lacks the C-terminal extension of
hCG and several amino acids in the Nterminus and in the region between amino
acids 44 - 48, and its glycan structures are
smaller. Part of hCG in the urine and
serum is also cleaved between amino acids
44 48 into what is called nicked hCG
(hCGn) or nicked hCG (hCGn). These
variants should be taken into account when
evaluating concentrations of hCG in urine
(Stenman et al 2006).
hCG and variants in pre-eclampsia
The concentration of hCG in the serum
during the second trimester is reportedly
high among women who develop preeclampsia
later
during
pregnancy
(Sorensen et al 1993, Wald et al 2006), but
the test is not appropriate for screening of
pre-eclampsia in the second (Wald et al
2006) or first trimester (Morris et al 2008).
The concentration of hCGE in maternal
serum during the first and the second
32
trimester has been studied in women who

subsequently
have
developed
preeclampsia. Most studies show no
association between concentrations and the
risk of pre-eclampsia in the first trimester
(Smith et al 2002, Dugoff et al 2004,
Goetzinger et al 2010, Kirkegaard et al
2011), while two studies have shown that
lower concentrations are associated with
subsequent pre-eclampsia (Ong et al 2000,
Ranta et al 2011). A systematic review by
Kuc et al concluded that in the first
trimester of pregnancy hCGE is not
valuable for prediction of pre-eclampsia
(Kuc et al 2011). In the second trimester,
however, high concentrations of hCGE in
the serum of women with subsequent preeclampsia have been demonstrated in two
studies (Lee et al 2000, Wald et al 2006)
but not in a third (Pouta et al 1998). The
sensitivity and specificity have been
modest for prediction of pre-eclampsia
(Lee et al 2000, Wald et al 2006). Women
who develop pre-eclampsia seem to have
low
second-trimester
hCG-h
concentrations in urine (Bahado-Singh et
al 2002).
6 Aims of the study

The aim of this study was to identify serum markers for screening of pre-eclampsia. We
analyzed the predictive value of several molecules that seem to be involved in the
pathophysiology of pre-eclampsia.
The detailed aims of the study were:
1.
To study whether pre-eclampsia can be reliably predicted by first-trimester maternal

serum concentrations of
a. hCG-h (I)
b. hCG, PAPP-A, and hCG-h combined (I).
2.
To study whether pre-eclampsia can be reliably predicted by second-trimester maternal

serum concentrations of
a. hCG-h (II)
b. PlGF, sVEGFR-1 and hCG-h combined (II),
c. Ang-1, Ang-2 or sTie-2 (III).
3.
To describe the physiological distribution of Ang-1, Ang-2 and sTie-2 in maternal serum,
umbilical serum, maternal and newborn urine and in amniotic fluid at term (IV).
33
7 Materials and methods

7.1 Patients and study design
7.1.1 First trimester hCG-h,
PAPP-A and hCG (I)
Downs syndrome. Serum samples were

collected at 12-16 and/or at 16-20 weeks
of pregnancy of 49 women who developed
pre-eclampsia, 16 women who had IUGR
and 59 healthy pregnant controls.
7.1.4 Angiopoietins at term

pregnancy (IV)
A nested case-control study was performed

in the Kuopio University Hospital region
between April 2008 and December 2010.
The study population was selected among
12,615 pregnant women participating in
combined first trimester screening for
Downs syndrome at 8 - 13 weeks of
gestation. 158 women who developed preeclampsia, 41 women with gestational
hypertension, 81 normotensive women
with SGA infants and 427 controls were
included.
A descriptive study was carried out to

determine the concentrations of Ang-1,
Ang-2 and sTie-2 in maternal serum and
urine, amniotic fluid, umbilical blood and
neonates urine in healthy term pregnancy.
Twenty women undergoing elective
cesarean delivery were enrolled in the
Helsinki University Central Hospital
between March 2008 and March 2010.
7.1.2 Second trimester hCG-h,

PlGF and sVEGFR-1 (II)
7.2 Inclusion and exclusion

criteria

in the Kuopio University Hospital between
December 2007 and May 2009. The study
population was chosen among 1470
pregnant women attending for screening of
Down's syndrome at 14-17 weeks of
gestation. 55 women who developed preeclampsia, 21 who developed gestational
hypertension, 30 normotensive women
with SGA infants and 83 controls were
included.
The inclusion criteria were a diagnosis of

pre-eclampsia according to American
College
of
Obstetricians
and
Gynecologists (ACOG), as described on
chapter 5.2.1 (American College of
Obstetricians and Gynecologists 2002), a
diagnosis of gestational hypertension (I, II,
III) or a diagnosis of SGA (I, II) or IUGR
(III). Gestational hypertension was defined
as pre-eclampsia according to the criteria
of ACOG without proteinuria, as described
on chapter 5.2.1 (American College of
SGA (I, II) was defined as birth weight
10th percentile below the national average.

IUGR was defined as an infant's
gestational age-adjusted birth weight
2
SD (III). Different definitions were used
because of the difference in the practices
of the hospitals involved. Patients for each
study were identifying on the basis of the
7.1.3 Angiopoietins in the second

trimester (III)
in the Helsinki University Central Hospital
between January 2001 and December
2002. The study population was chosen
among 3240 pregnant women attending a
routine two-phase ultrasound screening for
34
10th revision of the International

Classification of Diseases (ICD-10)
diagnostic code used in the patient records
of the hospital in question. Studies I and II
included controls that did not meet the
criteria of pre-eclampsia, gestational
hypertension or SGA, and were chosen to
match the duration of gestation at
sampling, maternal age and BMI. Healthy,
normotensive, non-proteinuric controls,
who gave birth to infants with normal birth
weight were randomly chosen for study
III. Study IV included women who
underwent elective cesarean section due to
breech presentation of the fetus, two or

more previous cesarean deliveries, fear of
vaginal delivery or a history of shoulder
dystocia. A detailed description of the
enrolled subjects is presented in Table 9.
Exclusion criteria for all studies were
multiple pregnancies and/or major fetal
anomalies. In studies III and IV women
with any history of hypertension, renal
disease, pre-existing or gestational
diabetes and/or ethnicity other than
Caucasian and in study IV cigarette
smoking were also excluded.
Table 9. Description of women enrolled and proteins studied.
Weeks
Study groups (No of women)

Pre-eclampsia
IUGR
EarlyControls
GH
or
a
All
Severe
Onseta
SGA
8-13
427
158
29
68
41
81
12-15
50
41
ND
14
11
14-17
83
55
12
23
21
30
16-20
36
31
ND
15
38-41
20
Proteins
studied
hCG
hCG-h
PAPP-A
hCG
Ang-1
Ang-2
sTie-2
hCG
hCG-h
PlGF
sVEGFR-1
Ang-1
Ang-2
sTie-2
Ang-1
Ang-2
sTie-2
Sample type
Study
No
Maternal serum
Maternal serum
III
Maternal serum
II
Maternal serum
III
Maternal serum
Maternal urine
Amniotic fluid
Umbilical serum
Neonates urine
IV
Some women may be included in both groups.

GH, gestational hypertension; IUGR, intrauterine growth restriction; SGA, small-forgestational age; ND, not determined.
35
7.3 Samples
The blood samples used in the predictive
studies (I, II, III) were collected during
routine Downs syndrome screening visits
at 8-13 weeks (I), 12-16 weeks (III), 14-17
weeks (II) or 16-20 weeks (III) of
pregnancy. Samples were allowed to clot
at room temperature, serum was separated
by centrifugation and stored at -20 C (I,
II) or at -80 C (III) until analysis
according
to
the
manufacturers
instructions. hCG in serum is stable when
frozen (Lempiinen et al 2011).
Maternal blood samples used in the

descriptive study (IV) were collected
before and after elective cesarean delivery.
Antepartum blood samples, as well as
maternal urine via a urinary catheter, were
collected before intravenous infusions and
epidural anesthesia were administered in
the operation theater. Postpartum blood
samples were collected on the first
postoperative day. Amniotic fluid was
collected immediately after rupture of the
membranes or by puncture through intact
membranes. The umbilical venous blood
sample was collected following delivery of
the placenta (Figure 8). All blood samples
were handled as described in the previous
chapter, and all samples were stored
immediately at -20 C.
Figure 8. Sampling procedure for study IV. Ang-1, Ang-2 and sTie-2 concentrations were
measured in all samples.
7.4 Assays
Commercial assays were used for analyses
of Ang-1, Ang-2, sTie-2, PlGF, sVEGFR1, hCG and hCG according to the
manufacturers instructions (Table 10).
hCG-h was analyzed by an in-house
immunofluorometric assay described in
36
Figure 9. Serum samples were diluted 100fold prior to assay of hCG-h to eliminate
interference by complement. The hCG-h
assay also recognizes hyperglycosylated
free hCG but its response is only 25 % of
that of hCG-h (Stenman et al 2011).
Because the mean proportion hCG in the
samples is only 1 % of that of hCG and its
effect on the concentration of hCG-h is
only about 0.25 %.
Table 10. Description of assays.

IntraInterassay
assay CV
Protein
CV
(%)
(%)
Detection
limit
Ang-1
<3.3
<6.4
63 pg/mL
Ang-2
<6.9
<10.4
47 pg/mL
hCG
1.8
<8.8
5 pmol/L
hCG
<3.3
<4.9
0.2ng/mL
hCG-h
PAPP-A
PlGF
sVEGFR-1
2.2
<2.4
1.4
0.6
<10.8
<4.0
<1.3
<2.7
9 pmol/L
5mU/L
10 pg/mL
15 pg/mL
sTie-2
<5.9
<8.3
156 pg/mL
Assay
ELISA
Quantikine a
ELISA
Quantikine a
Delfia b
Delfia or
AutoDelfia b
In-house IFMA c
AutoDelfia b
ECLIA d
ECLIA d
ELISA
Quantikine a
Sample
dilution
Study
no
50-fold
III, IV
5-fold
III, IV
100-fold
I, II
100-fold
5-fold
-
I, II
I
II
II
10-fold
III, IV
CV, coefficient of variation

a
R&D Systems, Inc., Minneapolis, MN, USA
b
Perkin Elmer Wallac Oy, Turku, Finland
c
In-house immunofluorometric assay (IFMA) (Stenman et al 2011)
d
Electrochemiluminescence sandwich immunoassay (ECLIA): Roche Diagnostics GmbH,
Mannheim, Germany
Figure 9. Schematic presentation of in-house immunofluorometric assay for hCG-h (I, II).
Serum hCG-h is recognized by the monoclonal antibody (MAb) 152 that binds to biantennary
core-2 glycan on serine 132 (Ser 132) in the hCG subunit. An in-house antibody 1B2 was
used to detect hCG (Stenman et al 2011).
37
7.5 Statistical methods

Table 11. Statistical methods.
Analysis
Study No.
Analysis of distribution
Kolmogorov-Smirnovs test
Corrections of multiple analysis
I, III
Bonferroni's adjustment
Dunnetts test
Comparisons of data between groups
IV
I
Dunnetts test
F2 test
Mann-Whitney's U test
One-way ANOVA
Students t test
Comparisons of data within groups
I
I, III
I, II
I, III
III
Paired -samples Students t test

III
IV
Wilcoxons signed-rank test
Correlations between studied protein concentrations and clinical characteristics
Multivariate linear regression analysis
Spearmans rank correlation
Univariate linear regression analysis
I, II
IV
III
Diagnostic test
F2 test
Logistic regression
ROC analysis
Preanalytic transformation of data
Logarithmic transformation
Calculation of multiples of median (MoM)
Power calculations
Statistical analyses were performed using

SPSS versions 14.0 (III), 17.0 (IV) and
18.0 (I, II). The comparisons between the
groups were performed using Students t
test (III), the F2 test (I, III), one-way
ANOVA (I, III), Mann-Whitney's U test (I,
II) and Dunnetts test (I). The F2 test (III),
logistic regression (I, II) and receiver
operating characteristics (ROC) curve
analysis (I, II) were used as diagnostic
tests. Kolmogorov-Smirnovs test was to
analyze
the distribution (I, III).
Prenalytical
transformations
were
performed
using
logarithmic
transformation (I, III) or calculation of
gestational age-adjusted multiples of
median (MoM) values (I, II). Correlations
between the protein concentrations and
38
III
I, II
I, II
I, III
I, II
I, II, III
clinical characteristics were studied by

univariate (III) or multivariate linear
regression analysis (I, II) and Spearmans
rank correlation (IV). A two-tailed P-value
of <0.05 was considered statistically
significant in all analyses (Table 11).
7.6 Ethics
The studies were approved by the Ethical
Research Committee of the Kuopio
University Hospital (I, II) or by the Ethics
Committee of the Helsinki University
Central Hospital (III, IV). Written
informed consent was obtained from all
participants.
8 Results
8.1 First trimester hCG-h,
hCG and PAPP-A (I)
8.1.1 %hCG-h
The proportion of hCG-h to hCG (%hCGh) was lower in women who developed
pre-eclampsia (median MoM, 95% CI;
0.92, 0.89 0.95; P < 0.001) or gestational
hypertension (0.92, 0.86 1.00, P = 0.006)
than in controls (1.01, 0.98 1.01). As
compared to controls, significantly lower
%hCG-h values were recorded for women
who developed early-onset (0.86, 0.79

0.91; P < 0.001) or severe pre-eclampsia
(0.89, 0.86 0.91; P < 0.001). No
difference in %hCG-h was observed
between women who gave birth to SGA
infants and controls (Figure 10 and Table
12).
8.1.2 hCGE
E
Concentrations of hCGE did not differ
among women who developed preeclampsia, gestational hypertension, SGA
and
controls
(Table
12).
Figure 10. Proportion of

hCG-h to hCG (%hCG-h)
in women who developed
pre-eclampsia
(n=158),
gestational hypertension
(GH) (n=41) or small-forgestational age (SGA)
(n=81) infants and earlyonset
pre-eclampsia
(n=29) as compared to
controls (n=427). Box plot
shows 10th, 25th, 50th, 75th
and 90th percentiles (Elina
Keikkala 2013).
39
Table 12. Changes in the concentrations of markers as compared to controls in 1 st and 2nd
trimester ( elevated; decreased; - no change).
1st trimester
2nd trimester
EO
SE
SGA /
EO
SE
SGA /
PE
GH
PE
GH
PE
PE
IUGR
PE
PE
IUGR
Ang-1

Ang-2
sTie-2

%hCG-h
hCGE

PlGF
sVEGFR-1
PE, pre-eclampsia (onset not specified); EO PE, early-onset pre-eclampsia; LO PE,
late-onset pre-eclampsia; SE PE, severe pre-eclampsia; GH, gestational hypertension;
SGA, small-for-gestational-age; IUGR, intrauterine growth restriction.
8.1.3 PAPP-A
The concentrations of PAPP-A were lower
in women who developed pre-eclampsia
than in controls (median MoM, 95% CI;
0.95, 0.91 0.99 vs. 1.00, 0.99 1.01; P =
Figure 11. Concentrations of

PAPP-A in women who
developed
pre-eclampsia
(n=158),
gestational
hypertension (GH) (n=41) or
small-for-gestational
age
(SGA) (n=81) infants, earlyonset pre-eclampsia (n=29) as
compared to controls (n=427).
Box plot shows 10th, 25th,
50th, 75th and 90th percentiles.
40
0.001). There were also differences in the

group early-onset pre-eclampsia (0.95,
0.86 0.98; P = 0.001) and the group
severe pre-eclampsia (0.97, 0.93 0.98; P
<0.001) but not in the groups gestational
hypertension or SGA (Figure 11, Table
12).
8.1.4 Prediction of early-onset

pre-eclampsia
For prediction of early-onset preeclampsia, the area under the curve (AUC)
value for %hCG-h was 0.757 (P < 0.001)
and for PAPP-A 0.688 (P < 0.001) giving
sensitivities of 56% and 33% at 90%
specificity. The combination of %hCG-h
and PAPP-A gave an AUC value of 0.835
(P < 0.001) with a sensitivity of 48% at
90% specificity. Maternal risk factors were
combined with %hCG-h and PAPP-A by
logistic regression to determine AUC
values for prediction of pre-eclampsia. The
AUC value for the combination of %hCGh, PAPP-A, first trimester mean arterial
pressure (MAP) and parity was 0.863 (P <
0.001) giving a sensitivity of 69% at 90%

specificity (Figure 12A).
8.1.5 Prediction of severe preeclampsia

The AUC values for prediction of severe
pre-eclampsia were 0.692 (P < 0.001) for
%hCG-h and 0.644 (P < 0.001) for PAPPA giving sensitivities of 31% and 29% at
90% specificity. The combination of these
gave an AUC value of 0.766 (P < 0.001)
with a sensitivity of 40% at 90%
specificity. The combination of %hCG-h,
PAPP-A, MAP and parity gave an AUC
value of 0.839 (P < 0.001), and a
sensitivity of 52% at 90% specificity
(Figure 12B).
Figure 12. ROC curve analysis of %hCG-h, PAPP-A, mean arterial pressure (MAP) and
parity for the prediction of A) early-onset (modified from study I with the permission of
Elsevier) or B) severe pre-eclampsia.
A
41
8.2 Second trimester hCG-h,

PlGF and sVEGFR-1 (II)
8.2.1 %hCG-h
At 14-17 weeks of gestation, %hCG-h did
not differ between women who developed
pre-eclampsia and controls (P = 0.3)
(Table 12).
Figure 13. Concentrations

of PlGF in women who
developed pre-eclampsia
(n=55),
gestational
hypertension
(GH)
(n=21),
small-forgestational age (SGA)
infants (n=30) or earlyonset
pre-eclampsia
(n=12) as compared to
controls (n=83). Box plot
shows 10th, 25th, 50th, 75th
and 90th percentiles.
8.2.3 sVEGFR-1
There were no differences in the
concentrations of sVEGFR-1 between
women who developed pre-eclampsia,
gestational hypertension or SGA as
compared to controls (Table 12).
42
8.2.2 PlGF
The concentrations of PlGF were
significantly lower in women who
developed pre-eclampsia (median MoM,
95% CI; 0.62, 0.53 0.79; P < 0.001) or
gestational hypertension (0.65, 0.44
0.90; P = 0.001) as compared to controls
(1.00, 0.93 1.06). The difference was
even greater when women with early-onset
(0.46, 0.21 0.67; P < 0.001) or severe
pre-eclampsia (0.46, 0.40 0.60; P <
0.001) were compared with controls
(Figure 13 and Table 12).
8.2.4 Prediction of pre-eclampsia

By ROC curve analysis the AUC values
for PlGF were 0.746 (P < 0.001) for
prediction of pre-eclampsia, 0.882 (P <
0.001) for early-onset pre-eclampsia and
0.890 (P < 0.001) for severe pre-
eclampsia. The corresponding sensitivities

were 19%, 53% and 65% at 90%
specificity (Figure 14). Combining PlGF
with %hCG-h or sVEGFR-1 by logistic
regression analysis did not improve the
AUC values.
Figure 14. ROC curve analysis of PlGF for the prediction of A) early-onset pre-eclampsia
and B) severe pre-eclampsia
A
B
8.3 Angiopoietins in the

second trimester (III)
8.3.1 Ang-1
At 12-15 or 16-20 weeks of pregnancy the
maternal serum concentrations of Ang-1
did not differ between women who
developed pre-eclampsia (n=41 or n=55)
and controls (n=50 or n=83), or between
women who had IUGR (n=11 or n=9) and
controls (Table 12). At 12-15 weeks of
pregnancy, the Ang-1 concentrations in the
subgroup of women who developed preeclampsia with IUGR (n=10) were higher
than in controls (median, interquartile
range; 36.5 ng/mL, 26.4 50.8 ng/mL vs.
27.8, 18.3 34.9 ng/mL; P = 0.006).
8.3.2 Ang-2
At 12-15 weeks of pregnancy maternal
serum Ang-2 concentrations did not differ
between the groups. At 16-20 weeks of
gestation the concentrations of Ang-2
among the controls was 17.7 ng/mL, 10.827.4 ng/mL. The concentrations were
higher in women who developed preeclampsia (25.0 ng/mL, 19.3 39.5
ng/mL; P = 0.006) and IUGR (31.0
ng/mL, 19.3 36.5 ng/mL; P = 0.038). In
the group of women who developed severe
pre-eclampsia (n=15), the concentrations
were also higher than in controls (29.0
ng/mL, 20.2 36.6 ng/mL; P = 0.016)
(Figure 15, Table 12). By ROC curve
analysis the AUC value was 0.692 (95%
43
CI, 0.566 0.818; P = 0.007) for Ang-2

for prediction of pre-eclampsia with a
sensitivity of 20% at 90% specificity. For
prediction of severe pre-eclampsia, the
AUC value for Ang-2 was 0.717 (0.567
0.867; P = 0.016) with a sensitivity of
17% at 90% specificity (Figure 16).
8.3.3 sTie-2
The concentrations of sTie-2 did not differ
between the groups at 12-15 or 16-20
weeks of pregnancy (Table 12).
Figure 15. Concentrations of

Ang-2 (ng/mL) at 16-20 weeks of
pregnancy. Boxplot shows 10th,
25th, 50th, 75th and 90th
percentiles. Modified from study
III with the permission of The
Endocrine Society.
Figure 16. ROC curve analysis of Ang-2 at 16-20 weeks of pregnancy for the prediction of
A) pre-eclampsia and B) severe pre-eclampsia.
A
B
44
8.4 Angiopoietins at term

pregnancy (IV)
8.4.1 Ang-1
The concentration of Ang-1 was higher in
umbilical (median, range; 46 ng/mL, 28
59 ng/mL) than in maternal serum (P <
0.0001) (Figure 17A and Table 13) and the
concentrations in the maternal serum
decreased slightly by the first day after
delivery (33 ng/mL, 25 51 ng/mL vs. 30
ng/mL, 0.6 49 ng/mL; P = 0.017). Ang1 was present in amniotic fluid and in most
of the maternal urine samples, but not in
the urine of the neonates (Table 13).
8.4.2 Ang-2
Maternal serum concentrations of Ang-2
decreased to one third (5.4 ng/mL, 1.8 - 18
vs. 1.4 ng/mL, 0.7 - 4.6 ng/mL; P <
0.0001) by the first day after delivery.

There were no differences between the
Ang-2 concentrations in umbilical and
maternal serum. Ang-2 was detectable in
amniotic fluid (1.1 ng/mL, 0.3 - 4.1
ng/mL) (Figure 17B). In maternal urine
Ang-2 was detected at low concentrations
only in 3 samples out of 20 and in none of
urine samples of the neonates (Table 13).
8.4.3 sTie-2
The maternal serum concentrations of
sTie-2 did not change between delivery
(23 ng/mL, 13 - 41 ng/mL) and the first
postpartum day (25 ng/mL, 14 - 29
ng/mL). The concentrations of sTie-2 (45
ng/mL, 21 - 71 ng/mL) in umbilical serum
were twice those in maternal serum (P <
0.0001) (Figure 17C). sTie-2 was
detectable at very low levels in amniotic
fluid (0.4 ng/mL, 0.08 - 0.9 ng/mL), but
not in the urine of the mother nor the
neonate (Table 13).
Table 13. Classification of Ang-1, Ang-2 and sTie-2 concentrations in maternal serum before
(antepartum) and one day after (postpartum) cesarean section in umbilical serum, maternal
and neonate urine and amniotic fluid (- not detectable, + low concentrations, ++ average
concentrations, +++ high concentrations)
Serum
Urine
Maternal
Maternal Umbilical Maternal Neonate
antepartum postpartum
Ang-1
++
+a
+++ a
+
a
Ang-2
++
+
++
+/sTie-2
++
++
+++ a
a
P < 0.05 as compared to maternal antepartum serum.
Amniotic
fluid
++
+
+
45
Figure 17. Serum A) Ang-1, B) Ang-2 and C) sTie-2 concentrations (ng/mL) at term pregnancy (n=20). Box plot shows 10th, 25th, 50th, 75th and
90th percentiles. Modified from study IV with the permission of John Wiley & Sons Ltd.
46
9 Discussion
9.1 Prediction of preeclampsia
9.1.1 hCG-h, hCG and hCG (I,
II)
hCG-h (I, II)
This study shows that %hCG-h was
reduced in women who developed preeclampsia in the first trimester of
pregnancy. The difference was even
greater for women with early-onset or
severe forms of the disease (I).
Interestingly, this difference was no longer
evident in the second trimester (II). This
finding could well be related to the fact
that the differentiation of villous
cytotrophoblasts into extravillous ones and
the invasion of these into the maternal
spiral arteries are especially active in the
first trimester of pregnancy (Pijnenborg et
al 1980). Since this differentiation and
invasion of extravillous cytotrophoblasts
in spiral arteries are reduced in preeclampsia (Meekins et al 1994, Zhou et al
1997) and since only extravillous
cytotrophoblasts actively secrete hCG-h
(Kovalevskaya
et
al
2002b,
Guibourdenche et al 2010), hCG-h
concentrations would be reduced in
women who develop pre-eclampsia.
Consistent with this theory is the finding
of low concentrations of hCG-h in women
with early miscarriage (Kovalevskaya et al
2002a), where trophoblastic invasion is
impaired (Jauniaux et al 1994). Since
cytotrophoblastic
differentiation
is
completed during the second trimester
(Genbacev et al 1992), the difference in
%hCG-h between pre-eclamptic women
and controls would disappear by this time.
An obvious conclusion is that %hCG-h
predicts pre-eclampsia only in the first

trimester of pregnancy (I, II).
Another possible biological mechanism
explaining reduced values of %hCG-h in
women who develop pre-eclampsia is
placental hypoxia (I). Hypoxia causes
alterations in the glycosylation of several
proteins derived from cancer cells in vitro
(Lehnus et al 2013). Hypoxia influences
glycosylation by several mechanisms,
including activation and expression of
hypoxia-inducible factor -1 (HIF-1), which
further regulates glycosylation through
other mechanisms (Shirato et al 2011). The
expression of HIF-1, in turn, is
upregulated in pre-eclamptic placentas
(Nishi et al 2004), which may also be
affected by conditions of ischemiareperfusion (Hung et al 2001). This theory
is speculative, since the regulation of hCG
glycosylation is not known, but if this
glycosylation would be regulated by
hypoxia, this would result in reduced
concentrations of hCG-h in the sera of
women who develop pre-eclampsia.
Different glycosylation patterns of hCG
are associated with distinct biological
functions. Thus, a highly glycosylated
early pregnancy-associated form of hCG
has been found to bind to the mannose
receptor of human uterine natural killer
(uNK) cells, while other forms of hCG
lack this capability (Kane et al 2009).
Activation of the mannose receptor
induces proliferation of uNK cells, which
are placental leukocytes associated with
the pathogenesis of pre-eclampsia and
important for the regulation of the
development of the placenta during early
pregnancy (Kane et al 2009, Williams et al
2009). hCG-h - but not hCG - promotes
cytotrophoblast and chorioncarcinoma cell
47
invasion in cell culture and in vivo (Cole et

al 2006, Guibourdenche et al 2010).
Therefore, glycosylation seems to play an
important role for the function of hCG.
However, the ultimate regulatory function
of hCG-h in the pathophysiology of preeclampsia is unknown, and only the
hyperglycosylated form of hCG was
associated with subsequent pre-eclampsia
in the present study (I).
hCG (I)
In our study, hCG was not associated
with subsequent pre-eclampsia (I). This
finding is in agreement with several
(Smith et al 2002, Dugoff et al 2004,
Goetzinger et al 2010, Kirkegaard et al
2011) but not all previous studies (Ong et
al 2000, Ranta et al 2011).
The physiological function of hCG is not
known. It may promote tumor growth
(Gillott et al 1996). The concentration of
hCG depends on the duration of
pregnancy:
it increases exponentially
during the first 7 weeks and decreases at 710 weeks of pregnancy (Stenman et al
2006). The concentrations of hCG in the
serum are increased between 9 and 11
weeks of gestation, if the fetus has Down's
syndrome. This is thought to reflect the
delay in placental maturation in this
condition (Macintosh et al 1994). Such
alterations in the hCG concentrations do
not occur in women who develop preeclampsia, but some studies have recorded
reduced
first-trimester
hCG
concentrations (Ong et al 2000, Ranta et al
2011). This may reflect the smaller
placental mass and the impaired placental
maturation typical of pre-eclampsia (Ong
et al 2000). On the other hand, hCG is
derived mainly from syncytiotrophoblasts
(Gaspard et al 1980), cells that are
apparently
not
as
important
pathophysiologically in pre-eclampsia as
cytotrophoblasts in the first trimester
(Meekins et al 1994, Zhou et al 1997).
This may explain why most studies,
including the present study, fail to show an
48
association
eclampsia.
between
hCG
and
pre-
hCG (I, II)

In the present study, the concentrations of
hCG did not differ between women who
developed pre-eclampsia and controls (I,
II). In contrast to this, some studies have
reported
elevated
serum
hCG
concentrations in the second trimester
(Sorensen et al 1993, Wald et al 2006),
whereas the results in the first trimester are
in line with ours (Morris et al 2008) (I, II).
hCG may be an essential regulator of fetomaternal immune tolerance, since hCG
protects the fetus from inflammatory
immune responses by attracting certain
regulative T cells in the placenta (Norris et
al 2011). Decreased populations of these T
cells occur in pre-eclamptic placentas.
hCG may also stimulate the production
interleukin-10 (IL-10), which is an
immunosuppressant
essential
for
pregnancy. Interestingly, IL-10 deficient
mice are more susceptible to pre-eclampsia
than nondeficient mice (Norris et al 2011).
9.1.2 PAPP-A (I)

The concentrations of PAPP-A in the
serum of pregnant women were reduced if
pre-eclampsia ensued (I), which is in line
with several (Ong et al 2000, Kuc et al
2011), but not all, previous studies
(Foidart et al 2010, Vandenberghe et al
2011).
PAPP-A during pregnancy is thought to
contribute to fetal and placental growth by
regulation of IGF-1 and IGF-2 by cleaving
the respective binding proteins IGFBP-4
and IGFBP-5 (Laursen et al 2007). Knockout mice deficient in PAPP-A give birth to
smaller young and the local IGF-1
signaling is reduced compared to wild-type
mice (Mason et al 2011). PAPP-A is
mainly expressed by placental villous
cyto- and syncytiotrophoblasts and is
thought to reflect placental trophoblastic
mass (Guibourdenche et al 2003). Indeed,

the first-trimester placental volume is
associated with circulating maternal
PAPP-A concentrations (Plasencia et al
2011) and, in the present study, with
placental weight (I). Logically, decreased
maternal serum PAPP-A concentrations in
women who develop pre-eclampsia may,
like the decreased hCG-h concentrations,
reflect impaired development of placental
villous structures known to occur in preeclampsia (Jackson et al 1995, Zhou et al
1997, Redman and Sargent 2009).
In
manifest
pre-eclampsia,
the
concentrations of PAPP-A are elevated
(Hughes et al 1980), but the biological role
of PAPP-A is unclear. Increased
expression of PAPP-A occurs in glomeruli
of patients with diabetic nephropathy who
present with similar glomerular defects as
those seen in pre-eclampsia. Diabetic mice
deficient in PAPP-A are resistant to
nephropathy (Roberts et al 1989, Mader et
al 2013). PAPP-A is also associated with
the acute coronary syndrome and its
expression is increased at sites with acute
vascular defects (Bayes-Genis et al 2001).
However, there is no direct evidence for
biological mechanisms associating PAPPA with pre-eclampsia.
9.1.3 PlGF and sVEGFR-1 (II)

Serum concentrations of PlGF were lower
in women who developed pre-eclampsia
than in controls at 14-17 weeks of
pregnancy (II), which is in line with
previous studies (Taylor et al 2003, Levine
et al 2004, Wathen et al 2006, Noori et al
2010, Villa et al 2013). The concentrations
of sVEGFR-1 did not differ between these
groups (II), which contrasts several earlier
studies (Maynard et al 2003, Tsatsaris et al
2003, Levine et al 2004, Wathen et al
2006). Prospective longitudinal studies
show that sVEGFR-1 concentrations in
women who develop pre-eclampsia
increase only after 18 weeks of gestation
(Levine et al 2004, McKeeman et al 2004,

Noori et al 2010, Villa et al 2013) or only
five weeks before appearance of clinical
symptoms (Levine et al 2004). We
conclude that the time frame 14-17 weeks
of pregnancy was too early to predict preeclampsia with sVEGFR-1 (II). Because
PlGF predicts pre-eclampsia earlier in
pregnancy it is a more useful marker of
pre-eclampsia than sVEGFR-1 (II).
An imbalance between PlGF and
sVEGFR-1 seems to be related to the
development
of
pre-eclampsia.
Administration of sVEGFR-1 to pregnant
rats causes hypertension, proteinuria and
glomerular endotheliosis similar to what is
seen in pre-eclampsia. This effect can be
prevented by exogenous administration of
vascular endothelial growth factor (VEGF)
or PlGF (Maynard et al 2003). In addition,
hypoxic conditions increase sVEGFR-1
and
decrease
PlGF
levels
in
cytotrophoblast culture (Nagamatsu et al
2004). The results of studies with a
primate model show that uteroplacental
ischemia increases both placental and
peripheral blood mononuclear cell
production of sVEGFR-1 mRNA and
concentrations of circulating sVEGFR-1
(Makris et al 2007).
Taken together, these studies suggest that
insufficient circulation in the pre-eclamptic
placenta, along with reduced oxygen
supply, may cause an imbalance between
PlGF and sVEGFR-1. This may further
cause
alterations
in
placental
vasculogenesis
and
endothelial
dysfunction typical of pre-eclampsia
(Maynard et al 2003). Logically, the
sVEGFR-1/PlGF ratio seems to be a better
predictor of pre-eclampsia than the
markers alone (Verlohren et al 2013, Villa
et al 2013).
49
9.1.4 Ang-1, Ang-2 and sTie-2

(III)
9.2 Fetal growth restriction
The concentrations of Ang-2 are higher

already at 16-20 weeks of pregnancy in
women who develop pre-eclampsia than in
controls - this observation has not been
reported previously (III). The difference
became obvious only after 12-15 weeks of
pregnancy (III). Data on placental villous
samples taken at 11 weeks of gestation
also failed to show any difference in Ang2 mRNA expression between women who
developed pre-eclampsia and controls
(Farina et al 2011).
Normotensive women with SGA or IUGR

and pre-eclampsia may have similar
changes in some biochemical markers
(Villar et al 2006, James et al 2010).
Another reason for including women with
IUGR in the study was to distinguish
whether any changes in biochemical
markers are specific for pre-eclampsia and
not associated with IUGR, which often
accompanies pre-eclampsia.
The results for Ang-1 and Ang-2 in

women with manifest pre-eclampsia are
conflicting. Earlier studies have reported
decreased maternal circulating Ang-2
concentrations (Hirokoshi et al 2005,
Nadar et al 2005, Hirokoshi et al 2007) or
no difference (Kamal and El-Khayat
2011). Decreased (Zhang et al 2001) as
well as increased (Han et al 2012)
placental expression of Ang-2 has been
reported. Also, since serum concentrations
of Ang-2 decrease rapidly after delivery,
the placenta is assumed to be the main
source of circulating Ang-2 (IV)
(Hirokoshi et al 2007). The conflicting
data on Ang-2 concentrations in manifest
pre-eclampsia may be due to differences in
gestational ages at the time of sampling
between the different studies.
In manifest pre-eclampsia, maternal
concentrations of Ang-1 have previously
been reported to be increased (Nadar et al
2005) and those of sTie-2 to be decreased
(Gotsch et al 2008) or similar (Hirokoshi
et al 2005) in comparison to controls. In
the present study, there were no changes in
the concentrations of Ang-1 and sTie-2
before pre-eclampsia (III).
50
9.2.1 hCG-h, hCG and PAPP-A

(I)
First-trimester concentrations of hCG-h,
hCG and PAPP-A were unchanged in
normotensive women who gave birth to a
SGA infant (I). No alterations in hCG
concentrations have previously been
reported in association with SGA or in
relation to the birth weight of the infant
(Luckas et al 1998). Findings on hCGE
have been conflicting: lower (Kirkegaard
et al 2011, Ranta et al 2011), and
unchanged (Ong et al 2000, Smith et al
2002, Dugoff et al 2004) concentrations of
hCGE have been reported in women who
had a SGA infant as compared to controls.
Contrary to our findings (I), previous
studies have reported lower PAPP-A
concentrations in women who had a SGA
infant than in controls (Ong et al 2000,
Smith et al 2002, Kirkegaard et al 2011,
Ranta et al 2011).
Women who developed both preeclampsia and SGA had lower %hCG-h
values than normotensive women who had
a SGA infant (I). Thus, %hCG-h does not
seem to be related to the development of
SGA, and would be specific for predicting
pre-eclampsia.

At 14-17 weeks of pregnancy there were
no differences in serum PlGF or sVEGFR1 concentrations between women with
SGA and controls (II), which is in line
with some other studies that show a
difference in PlGF concentrations only
later in the second trimester (Taylor et al
2003) or no difference at all (Stepan et al
2007). The concentrations of sVEGFR-1
have not been shown to differ between
women with SGA and controls in the
second trimester of pregnancy (svold et
al 2011, Stepan et al 2007, Sung et al
2011, Thadhani et al 2004). Since these
findings do not support the existence of an
association between SGA and maternal
circulating
PlGF
and
sVEGFR-1
concentrations (II), the conclusion form
our study is that PlGF and sVEGFR-1
predict pre-eclampsia but not SGA.
9.3 Comparison of markers

predictive of preeclampsia
A recent meta-analysis indicated that
treatment with low-dose aspirin started
before the 16th week of gestation by
women at the risk of pre-eclampsia can
reduce the risk of early-onset preeclampsia (Roberge et al 2012). Thus, an
optimal test for screening of pre-eclampsia
should be sensitive before the 16th weeks
of pregnancy. Since comprehensive
screening for Downs syndrome is
performed in many countries, Finland
included, between 9 and 12+6 weeks of
gestation by sonography and serum
markers, simultaneous screening for preeclampsia would be feasible, cost-effective
and comprehensive.
9.3.1 hCG-h (I)

9.2.3 Angiopoietins (III)
According to our findings, Ang-2
concentrations are higher in women with
subsequent IUGR or pre-eclampsia than in
controls at 16-20 weeks of pregnancy (III).
In contrast to this, a previous study at 1013 weeks of pregnancy showed that Ang-2
is decreased in women whose fetus
develops IUGR. However, this study did
not specify if the etiology of IUGR was
involved with pre-eclampsia or not (Wang
et al 2007). Ang-2 is antiangiogenic and
essential for vascular development of the
placenta, and its expression is upregulated
at sites of vascular remodeling (Thurston
2003, Sugino et al 2005). On the other
hand, mice overexpressing Ang-2 die
because of vascular defects (Sato et al
1995). Our observation that Ang-2 is
elevated in women with pre-eclampsia or
IUGR (III) is in agreement with the
hypothesis that pre-eclampsia and IUGR
may have a shared pathophysiology and
thus Ang-2 is not specific for predicting
pre-eclampsia.
Of all markers in the present study, first

trimester hCG-h was best at predicting preeclampsia. Sensitivity was 56% at 90%
specificity for prediction of early-onset
pre-eclampsia (I). In other studies, another
potential first trimester serum marker,
PlGF, has been 41-59% sensitive for earlyonset pre-eclampsia at 90% specificity
(Akolekar et al 2008, Foidart et al 2010,
Wortelboer et al 2010, Kuc et al 2013).
First trimester placental protein 13 (PP13)
has also an acceptable sensitivity of 36
80% at 90% specificity (Nicolaides et al
2006, Akolekar et al 2009, Khalil et al
2009, Wortelboer et al 2010), but the study
groups in these reports have been very
small (10 and 14 women) which has
yielded
wide
confidence
intervals
(Nicolaides et al 2006, Khalil et al 2009).
In the present study, the sensitivity of
hCG-h increased to 69% when it was
combined with PAPP-A, MAP and parity
(I). Earlier studies have reported a
sensitivity of 54% at 90% specificity for
prediction of early-onset pre-eclampsia
51
when PlGF is combined with PP13

(Wortelboer et al 2010). The sensitivity
increases to 70% when PlGF is combined
with PAPP-A, maternal characteristics and
MAP (Kuc et al 2013). A sensitivity of
77% has been reported for the combination
of PP13 with maternal characteristics and
Doppler ultrasound (Akolekar et al 2009).
Analytical problems may limit the use of
PP13 for screening, since the assay has had
poor reproducibility (Akolekar et al 2009).
Taken together, hCG-h seems at least
equal to other markers for the prediction of
early-onset pre-eclampsia. Of note, hCG-h
is lightly reduced also among women with
gestational hypertension compared to
controls (I), and this could reduce the
specificity of hCG-h as a predictor of preeclampsia.
9.3.2 PAPP-A (I)

PAPP-A alone was only moderately
effective for prediction of pre-eclampsia
(I), as has been reported previously
(Giguere et al 2010, Kuc et al 2011). In
earlier studies the predictive accuracy of
PAPP-A has improved when PAPP-A has
been combined with clinical risk factors
such as chronic hypertension, prepregnancy diabetes, BMI and maternal
race (Goetzinger et al 2010) or with first
trimester
Doppler
ultrasound
measurements (Poon et al 2009). First
trimester maternal serum concentrations of
PlGF have, nevertheless, been shown to be
superior to PAPP-A when PIGF and
PAPP-A are analyzed in the same setting
(Foidart et al 2010, Vandenberghe et al
2011). Several studies have also reported
that combining PAPP-A with PP13 is not
helpful (Spencer et al 2007, Akolekar et al
2009, Wortelboer et al 2010).

The utility of second-trimester serum
concentrations of sVEGFR-1 and PlGF
52
either alone or combined for prediction of

pre-eclampsia has been widely studied. A
recent systematic review included 27
studies run at 5 to 28 weeks of gestation
and found that the accuracy of sVEGFR-1
and PlGF for prediction of pre-eclampsia
is too low for clinical practice. It was
concluded that circulating maternal
concentrations of PlGF were lower if preeclampsia ensued than in controls, but
sensitivity was only 32% at 95%
specificity. The studies reviewed all types
of pre-eclampsia (Kleinrouweler et al
2012).
In the present study the results for PlGF
concentrations at 14-17 weeks of
pregnancy were even more modest sensitivity was only 19% at 90%
specificity (I). The sensitivity of PIGF to
predict early-onset pre-eclampsia was,
however, much better - sensitivity was
53% at 90% specificity (I). The same
conclusions have been drawn from two
systematic reviews (Giguere et al 2010,
Kuc et al 2011). Data on first-trimester
PlGF for prediction of pre-eclampsia is
conflicting and was not tested in our study
(Ong et al 2001, Taylor et al 2003, Levine
et al 2004, Thadhani et al 2004, Akolekar
et al 2008, Noori et al 2010). PlGF seems
to be at least a moderately effective
predictor of pre-eclampsia in the second
trimester of pregnancy. It is not affected
by SGA (II) (Stepan et al 2007).
In the systematic review by Kleinrouweler
et al, the utility of sVEGFR-1 to predict
pre-eclampsia was low (Kleinrouweler et
al 2012). Recent studies imply that
sVEGFR-1 and its ratio to PlGF seem to
be suitable for short-term prediction of the
occurrence and severity of manifest preeclampsia. Ratio of sVEGFR-1 to PlGF
showed a sensitivity of 73% at 94%
specificity for adverse outcomes which
were defined as the HELLP syndrome,
eclampsia or renal failure (Rana et al
2012).
9.3.4 Ang-2 (III)

The concentrations of Ang-2 were not of
clinical value for the prediction of the
occurrence and severity of pre-eclampsia sensitivity was 20% at 90% specificity
(III). Some improvement for the prediction
of severe pre-eclampsia was obtained as
evidenced by the AUC values, but the
sensitivity remained only at 17% at 90%
specificity (III). In addition, Ang-2 was of
value only at 16-20 weeks of pregnancy
(III),
i.e.,
at
a
time
when
pathophysiological changes of preeclampsia may already have occurred
(Harrington et al 1991, Farina et al 2008)
and the effect of low-dose aspirin seems to
be weaker than if it is started before the
16th week of pregnancy (Roberge et al
2012). An earlier study showed that a low
ratio of Ang-1 to Ang-2 may predict preeclampsia at the 25th week of pregnancy
(Bolin et al 2009). The specificity of Ang2 for prediction of pre-eclampsia may be
weaker, since its value will be affected by
IUGR (III).
9.4 Angiopoietins at term

(IV)
Ang-1, Ang-2 and sTie-2 are present at
high concentrations in umbilical cord and
maternal serum. Concentrations of Ang-1
and sTie-2 were higher in the umbilical
cord blood compared to the maternal
circulation (IV). The site of the secretion
of these angiopoietins into umbilical blood
is not known. In the maternal circulation,
however, the concentrations of Ang-1 and
sTie-2 were similar before and after
delivery but Ang-2 decreased sharply
already by the first day post partum (IV).
This indicates that the placenta might be
an important source of Ang-2, but not of
Ang-1 or sTie-2, at least in the maternal
circulation. This speculation is supported
by a previous study showing that term
villous placentas secrete more Ang-2 than
Ang-1 (Buhimschi et al 2010). Expression
of Tie-2 in the term placenta has been

demonstrated by quantitative real time
polymerase
chain
reaction
(PCR)
(Buhimschi et al 2010), but in situ
hybridization failed to show any
expression (Zhang et al 2001). Circulating
sTie-2 could be derived from other sources
than the placenta, e.g., from hematopoietic
cells (Thomas and Augustin 2009).
In agreement with earlier studies, we
detected only low concentrations of Ang-1,
Ang-2 and sTie-2 in amniotic fluid (IV)
(Pacora et al 2009, Buhimschi et al 2010).
Since these markers were not detected in
the urine of the neonate (IV), the low
concentrations in amniotic fluid implies
that these markers originate from other
sources than the kidneys, e.g., the lungs, of
the fetus or through a placental pathway
(Desforges and Sibley 2010).
Circulating sTie-2 is binds both Ang-1 and
Ang-2, which implies that the biological
activity of Ang-1 and Ang-2 is inhibited
through this mechanism (Findley et al
2007, Shantha Kumara et al 2010). In this
study, high angiogenic activity was
recorded in the fetal circulation but low in
amniotic fluid (IV). This observation
indicates that an angiogenetic effect is
redundant in amniotic fluid.
9.5 Limitations of the study

In studies I and II women who developed
pre-eclampsia were compared with
controls without evidence of preeclampsia, gestational hypertension or
SGA. The diagnosis of pre-eclampsia
relied on information documented in
individual patient records. Also for
controls, we relied on the diagnosis being
correct by scrutiny of hospital records, and
thus there might be some diagnostic errors
among the controls. However, as we found
that pre-eclampsia was overdiagnosed
among some patients, it is unlikely that
53
there were cases of pre-eclampsia among

the controls that would have been missed.
In study IV serum samples were collected
from 20 women immediately before and a
day after elective cesarean delivery. Serum
concentrations of Ang-1, Ang-2 and sTie-2
were measured only on the first day post
partum. This was due to practical reasons,
since women undergoing elective cesarean
section are followed in the hospital at least
overnight. A rapid reduction in the
concentrations of Ang-1, Ang-2 and sTie-2
were observed post partum. This finding
implies that the placenta is involved in the
synthesis or kinetics of these proteins. It
was not possible to determine the
elimination half-lives of these substances,
since this would have required repeated
sampling post partum, which was not
possible in the present study.
9.6 Clinical implications

The studies concerning prediction of preeclampsia (I, II, III) show that serum
%hCG-h combined with serum PAPP-A,
first trimester blood pressure and parity at
8-13 weeks of pregnancy provide the best
model for screening of early-onset preeclampsia (I). Early-onset pre-eclampsia is
clinically the most important form of the
disease, because it often causes preterm
delivery and severe perinatal and neonatal
complications (Duley 2009, Lisonkova and
Joseph 2013). The incidence of preeclampsia is approximately 15% among
pregnant women who give birth before 28
weeks of pregnancy (Hgberg and
Holmgren 2007). Pre-eclampsia occurred
among 22% of all women giving birth
before 32 weeks in Finland 2008-2010
(Medical Birth Register). In 2012, 0.31%
(187) of infants were born before 28 weeks
and 1.5% (914) before 34 weeks of
pregnancy (Vuori and Gissler 2013).
Interestingly, on the other hand, infants
born to pre-eclamptic women tend to have
lower mortality than infants born
54
prematurely for other causes at same

weeks of pregnancy (Hutcheon et al 2011,
Lisonkova and Joseph 2013).
We have estimated the number needed to
treat (NNT) and the number needed to
screen (NNS) to prevent one case of earlyonset pre-eclampsia based on %hCG-h,
PAPP-A, first-trimester blood pressure and
parity information. For this, different
specificity levels for the first trimester
screening test were used (Table 14). The
calculations are based on the following
assumptions:
a) The incidence of early-onset preeclampsia is 0.4% (Hernandez-Diaz et al
2009, Lamminp et al 2012, Lisonkova
and Joseph 2013).
b) Some 60.000 women would participate
in the screening annually. This would
cover all pregnant women in Finland
(Vuori and Gissler 2012).
c) Low-dose aspirin would be started
before the 16th week of pregnancy in all
test-positive women.
d) The treatment would prevent 89% of the
cases of early-onset pre-eclampsia in this
high-risk population (Roberge et al 2012).
Under these premises the NNT would vary
between 48 and 208 and the NNS between
347 and 803, depending on cut-off values.
At 90% specificity, NNT would be 98 and
NNS 407. The screening test would
recognize 69% of all women who would
proceed to early-onset pre-eclampsia.
Treatment with low-dose aspirin would
prevent 61% of all instances of early-onset
pre-eclampsia, provided that 10% of all
pregnant women are treated with low-dose
aspirin (Table 14). For comparison, the
NNS for screening of cervical cancer to
find one cervical dysplasia varies between
200 and 333 in Finland annually (Finnish
Cancer Registry 2006-2009). These
calculations show that better screening
methods for pre-eclampsia need to be
further improved.
Based on the calculations in Table 14 and

the incidence of early-onset pre-eclampsia
in Finland, prevention of 61% of instances
of early-onset pre-eclampsia would reduce
the rate of preterm delivery by 15%.
Specifically, there would be an annual
reduction from approximately 900 to 760
preterm deliveries before 34 weeks of
gestation. This would reduce the need for
neonatal intensive care, and long-term
morbidity of these infants would be
avoided (Lisonkova and Joseph 2013). In
women with early-onset pre-eclampsia,
almost all deliveries are by cesarean
section, which predisposes to surgical and
anesthetic complications of the parturient
(Hgberg and Holmgren 2007).
Treatment of pregnant women with lowdose aspirin is fairly safe during pregnancy
(James et al 2007). In recent randomized
trials, maternal bleeding complications
have not been observed (Askie et al 2007,
Roberge et al 2012), but an earlier trial
reported a slightly increased need for
blood transfusions after delivery (CLASP
1994). Aspirin crosses the placenta, but
low-dose aspirin has not been shown to be
associated with premature closure of the
ductus arteriosus or with neonatal bleeding
complications (James et al 2007).
However, some, but not all, studies have

shown that aspirin use during the first
trimester could increase the risk of
gastroschisis, a very rare closure anomaly
of the fetal abdominal wall (James et al
2007). National guidelines for the use of
low-dose aspirin for prevention of preeclampsia have not been established in
Finland. At the Helsinki University
Central Hospital, low-dose aspirin is
currently recommended for women who
have had early-onset pre-eclampsia in
previous pregnancies.
Before considering our combination model
for screening of all pregnant women at
least the following aspects need to be
considered:
a) A prospective study in a low-risk
population is needed.
b) The costs of screening and of treatment
should be evaluated from a socioeconomic
perspective.
c) The screening should be evaluated
according to criteria of the National
Institute for Health and Welfare in Finland
(http://www.thl.fi/fi_FI/web/fi/aiheet/tieto
paketit/seulonnat/seulontaohjelmat/seulont
ojen_arviointikriteerit).
Table 14. Number needed to treat (NNT) and number needed to screen (NNS) when %hCGh, PAPP-A, blood pressure and parity information are used to screen and predict early-onset
pre-eclampsia (EO PE). The numbers are based on the assumption that the test is performed
for 60.000 pregnant women, that the incidence of early-onset pre-eclampsia is 0.4%, that
low-dose aspirin is started for test-positive women by the 16th week of pregnancy and that
low-dose aspirin would prevent 89% of instances of early-onset pre-eclampsia.
Specificity
(%)
Sensitivity
(%)
No of testpositive
women
No of EO
PE cases
recognized
No of EO
PE cases
prevented
% of EO
PE cases
prevented
NNS to
prevent
one case of
EO PE
NNT to
prevent
one case of
EO PE
75
80
85
90
95
97,5
81
77
73
69
46
35
15.000
12.000
9.000
6.000
3.000
1.500
194
185
175
166
110
84
173
164
156
147
98
75
72
69
65
61
41
31
347
365
385
407
611
803
208
175
139
98
73
48
55
A first-trimester model for prediction preeclampsia has recently been become

commercially available. It is based on
determination of PlGF combined with
maternal risk factors and/or Doppler
ultrasound measurements (Akolekar et al
2013) (PerkinElmer Wallac Oy, Turku,
Finland, http://www.perkinelmer.com).
Second-trimester screening, based on the
ratio of the serum concentration of
sVEFGR-1 to PlGF, has also been
introduced (Schiettecatte et al 2010)
(Roche Diagnostics GmbH, Mannheim,
Germany, www.roche.com). We also
found that low serum PlGF concentrations
at 14-17 weeks of pregnancy predict preeclampsia, especially early-onset preeclampsia, whereas %hCG-h does not
predict pre-eclampsia at that stage of
pregnancy.
An important subject for
further study is to elucidate whether the
incidence of pre-eclampsia is reduced
among pregnant women who use these
screening methods.
Because of the rarity of early-onset preeclampsia (the incidence is only
approximately 0.4%) (Hernandez-Diaz et
al 2009, Lamminp et al 2012, Lisonkova
and Joseph 2013) it is difficult to reach
acceptable power in scientific studies. To
facilitate studies on the utility of various
screening
approaches
the
Global
Pregnancy
CoLaboratory
database
(http://pre-empt.cfri.ca/Objectives/
CoLaboratory.aspx) has been introduced.
56
10 Summary and conclusions

We studied whether the concentrations of severall serum markers predict pre-eclampsia in the
first and second trimesters of pregnancy. An optimal screening test should identify women at
risk before 16 weeks of pregnancy, and especially women at risk of early-onset preeclampsia, who are likely to benefit from low-dose aspirin therapy started before the 16th
week of gestation.
The following conclusions can be drawn:
1. Serum hCG-h is a promising first-trimester marker of early-onset pre-eclampsia. The
concentrations of hCG-h in the serum are lower in women who develop pre-eclampsia, and
especially early-onset pre-eclampsia, than in controls. The combination of hCG-h, PAPP-A
and maternal clinical factors may improve the utility of screening (I). However, hCG-h was
not useful in this purpose at 14-17 weeks of pregnancy (II).
2. Serum concentrations of Ang-2 are higher in women who turn out to have pre-eclampsia
than in controls at 16-20 weeks, but not at 12-15 weeks of pregnancy. However, serum Ang-2
is not a clinically useful marker to predict pre-eclampsia (III).
3. Serum concentrations of PlGF are lower in women who develop pre-eclampsia than in
controls at 14-17 weeks of pregnancy. Thus, PIGF is a useful marker for prediction of preeclampsia, if the sample is taken during the second trimester of pregnancy (II).
57
11 Acknowledgments
This study was carried out in 2008 2013
at the Helsinki University Central Hospital
and Biomedicum Helsinki, at the
and at the Department of Clinical
Chemistry. I thank the current and the
former heads of the departments,
Professors Juha Tapanainen and Olavi
Ylikorkala, and Professors Pirkko Vihko
and Ulf-Hkan Stenman for providing me
with a pleasant working atmosphere and
excellent working facilities. I thank
Professor Aila Tiitinen warmly for acting
as the custos at the dissertation.
I express my sincere gratitude to my
supervisors, Adjunct Professor Piia
Vuorela and Emeritus Professor Ulf-Hkan
Stenman, for their unfailing support and
abundant advices to me during this work.
Piia has always found the time for
discussing my questions and has
supervised and guided me through all
practical matters of this study. Her
constant help and optimism encouraged
me throughout this scientific journey. I am
truly thankful to Uffe for his guidance into
the world of science and for offering his
expertise in laboratory medicine. I have
been privileged to work with a mentor who
shared his extensive knowledge and
showed an amazing curiosity and new
ideas to explore.
I thank the official reviewers of the thesis,
Adjunct Professors Eeva Ekholm and Katri
Koli for their professional criticism, which
markedly improved the quality of the
thesis.
I express my warm thanks to my coauthors, Katja-Anneli Wathn, PhD,
Henrik Alfthn, PhD, Timo Hytinantti,
PhD, Jarkko Romppanen, PhD, Jenni
Ranta, PhD, Sari Visnen, PhD, Jaana
58
Marttala, PhD, and Reetta Leinonen, MD.

I was also privileged to have Professors
Sture Andersson, Seppo Heinonen, Kari
Pulkki, Olavi Ylikorkala, and Adjunct
Professor Hannele Laivuori as co-authors
whose experienced perspective and
professionalism contributed strongly to the
study.
I am grateful to my fellow collaborators,
laboratory technicians, Taina Grnholm,
Maarit Leinimaa, Marianne Niemel,
Kristiina Nokelainen, Annikki Lfhjelm
and Sari Lindn. Their expertise in
laboratory methods and kind help, with all
the technicalities, in the laboratory were a
constant source of admiration. I also thank
Ansa Karlberg for her help with
innumerable practical matters in the
laboratory.
I warmly thank my colleagues and friends
Laura Hautala, Hanna SavolainenPeltonen, Anna Lempiinen, Johanna
Mattsson, Suvi Ravela, Leena Valmu,
Hannu Koistinen, Kristina Hotakainen,
Heini Lassus, Sini Koskinen and all others
whom I have had pleasure to work with at
the research laboratory.
I thank my colleagues in Ktilopisto
Maternity Hospital and Womens Hospital,
especially the Heads of the clinics,
Adjunct Professors Jari Sjberg, Pekka
Nieminen, Mika Nuutila and Professor
Oskari Heikinheimo for their positive and
constructive attitude toward science and
for their supervision of the fascinating
clinical work. I thank all my co-residents
for creating an inspiring and pleasant
working atmosphere.
I also thank Adjunct Professor Robert Paul
for excellent language revision of the
thesis.
I direct my warm thanks to my friends

who helped me to forget science every
now and then. I thank especially my lifelong friends Essi, Heidi and Lotta for
sharing my moments for better or worse,
and my friends Hanna-Reetta, Lilli and
Maria for a long-lasting friendship which
started at the medical school.
I am most indebted to my parents, Pirjo
and Reijo, for teaching me the true value
of work and diligence and for supporting
me on my way to becoming a doctor. I
extend my gratitude to my sister Reetta,
not only for her co-authorship but also for
sharing my thoughts in medicine. She has
also given me priceless help with attending
to my son when I needed time for my
work. I warmly thank my brother Lauri for
inspiring conversations. I also thank my
mother-in-law Sirkka for encouraging me
through this project especially when it was
difficult. I thank Viivi, my sister-in-law,
for sharing with me her delightful thoughts
of life.
My husband Ville deserves my most

loving thanks. He has always been there
for me, understanding the demands of this
project and taking charge of our hectic life.
I thank our son Vertti for reminding me
everyday what is important in this life.
Verttis future sibling has brought extra
joy to my life and has also encouraged me
to complete this writing process in due
time.
This study was financially supported by
The Medical Society of Finland, Helsinki
University Central Hospital Research
Funds, The Finnish-Norwegian Medical
Foundation and The National Graduate
School of Clinical Investigation.
Helsinki, December 2013
Elina Keikkala
59
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PREDICTING PRE-ECLAMPSIA

Angiogenic factors and various forms of human chorionic

ISBN 978-952-10-9686-0 (Paperback)

1 List of original publications

American College of Obstetricians and Gynecologists

women who subsequently developed preeclampsia, 16 with intrauterine growth

together with pregnancy-associated plasma

Angiopoietins are angiogenic factors that

This study was performed to evaluate

Human chorionic gonadotropin (hCG)

5 Review of the literature

5.1.1 Maternal blood supply to

Deoxygenated blood leaves the placenta

5.1.2 Origin of placental

form the basic structure of the placenta,

endometrial stroma into the endometrial

Also at the beginning of the fourth week of

Table 1. Different types of trophoblasts and their function (Huppertz 2008).

Form outer blastocyst layer

Formation of first lacunae

Form outer (=syncytial) layer

Invade through maternal spiral

Throughout pregnancy the villi continue to

5.2.1 Diagnosis and symptoms

compared to onset after 37 weeks of

diagnosed before 20 weeks of pregnancy

Table 2. Diagnostic criteria of pre-eclampsia and severe pre-eclampsia based on the

All criteria below

Criteria of pre-eclampsia and one or

Previously normal blood pressure

Systolic 160 mmHg and/or

Systolic 140 mmHg and/or

Measured at least twice 6 h apart

0.3 g in 24-h urine collection

5 g in 24-hour urine collection or

Oliguria < 500 mL/24-h

According to the statement of International Society for the Study of Hypertension in

5.2.2 Risk factors

Multiple pregnancies increase the risk of

before the 20th week of gestation increases

Paternal factors seem also to contribute to

Table 3. Risk factors for pre-eclampsia

Established risk factors

Placental vascular remodeling and

in the placenta. In the second stage,

Intermittently inadequate blood supply to

endothelium is exaggerated. This would

women, pregnant women with essential

5.2.4 Intrauterine growth

arteries by Doppler velocimetry (Bamberg

1 has been reportedly elevated in preeclampsia but not in unexplained IUGR in

5.2.5 Prevention of pre-eclampsia

trials, only a 10% reduction in the RR of

5.3 Prediction of preeclampsia

5.3.1 Maternal characteristics

Table 4. Prediction of pre-eclampsia by Doppler ultrasound and serum biomarkers. Arrows

Pregnancy-associated plasma protein-A

pregnancies (Campbell et al 1983). In preeclampsia, reduced uterine artery diastolic

90%, but sensitivity has varied from 29%

5.3.2 PlGF and sVEGFR-1

insults are reduced (Ribatti 2008).

Figure 4. Expression of PlGF,