predicting preeclampsia.pdf

8/16/2019 predicting preeclampsia.pdf

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PREDICTING PRE-ECLAMPSIA

Angiogenic factors and various forms of human chorionic

gonadotropin

Elina Keikkala

Faculty of MedicineInstitute of Clinical Medicine

Department of Obstetrics and GynecologyUniversity of Helsinki

Finland

Faculty of MedicineHaartman Institute

Department of Clinical ChemistryUniversity of Helsinki

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.


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Supervised by

Adjunct professor Piia Vuorela, MD, PhDDepartment of Obstetrics and Gynecology

University of HelsinkiFinland

Emeritus professor Ulf-Håkan Stenman, MD, PhDDepartment of Clinical Chemistry

University of HelsinkiFinland

Reviewed by

Adjunct professor Katri Koli, PhDTranslational Cancer Biology Program

Faculty of MedicineUniversity of Helsinki

Finland

Adjunct professor Eeva Ekholm, MD, PhD

Department of Obstetrics and GynecologyUniversity of TurkuFinland

Opponent

Adjunct professor Jukka Uotila, MD, PhDDepartment of Obstetrics and Gynecology

University of TampereFinland

ISBN 978-952-10-9686-0 (Paperback)ISBN 978-952-10-9687-7 (PDF)http://ethesis.helsinki.fi

UnigrafiaHelsinki 2014


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To all mothers


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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


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1 List of original publications

This thesis is based on the following original publications, which are referred to in the text bytheir Roman numerals:

I - Keikkala Elina, Vuorela Piia, Laivuori Hannele, Romppanen Jarkko, Heinonen Seppo,Stenman Ulf-Håkan. First trimester hyperglycosylated human chorionic gonadotropin inserum – a marker of early-onset preeclampsia. Placenta 2013; 34:1059-65.

II - Keikkala Elina, Ranta Jenni, Vuorela Piia, Leinonen Reetta, Laivuori Hannele, Väisänen

Sari, Marttala Jaana, Romppanen Jarkko, Pulkki Kari, Stenman Ulf-Håkan, Heinonen Seppo.Serum hyperglycosylated human chorionic gonadotrophin at 14–17 weeks of gestation doesnot predict preeclampsia. Submitted

III - Leinonen Elina, Wathén Katja-Anneli, Alfthan Henrik, Ylikorkala Olavi, AnderssonSture, Stenman Ulf-Håkan, Vuorela Piia. Maternal serum angiopoietin-1 and -2 and Tie-2 inearly pregnancy ending in preeclampsia or intrauterine growth retardation. Journal ofClinical Endocrinology and Metabolism 2010; 95:126-33.

IV - Keikkala Elina, Hytinantti Timo, Wathén 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. Pleasenote that the name Leinonen in publication III is the maiden name of the author.


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2 Abbreviations

ACOG American College of Obstetricians and GynecologistsADAM12 A disintegrin and metalloproteinase 12Ang-1 Angiopoietin-1Ang-2 Angiopoietin-2AT1-AA Angiotensin II type 1 receptor agonistic antibodyAUC Area under the curveBMI Body mass indexCRP C-reactive proteinDIC Disseminated intravascular coagulationFSH Follicle stimulating hormoneGWAS Genome-wide association screening

GWLS Genome-wide linkage analyseshCG Human chorionic gonadotropinhCG-h Hyperglycosylated human chorionic gonadotropinhCG Beta subunit of human chorionic gonadotropinHELLP Hemolysis, elevated liver enzymes, low platelet countHUS Hemolytic-uremic syndromeICD-10 International Classification of Diseases, the 10th revisionIGF Insulin-like growth factorIGFBP Insulin-like growth factor binding proteinIgG Immunoglobulin GIUGR Intrauterine growth restriction

kDa KilodaltonLH Luteinizing hormoneLBW Low birth weightMAP Mean arterial pressureMoM Multiples of medianMW Molecular weight

NNS Number needed to screen NNT Number needed to treatPAPP-A Pregnancy-associated plasma protein-API Pulsatility indexPlGF Placental growth factorPP13 Placental protein 13PRES Posterior reversible encephalopathyROC Receiver operating characteristicsRR Relative r isksEng Soluble endoglinSGA Small-for-gestational agesVEGFR-1 Soluble vascular endothelial growth factor receptor 1sTie-2 Soluble endothelial cell-specific tyrosine kinase receptor 2TSH Thyroid stimulating hormoneVEGF Vascular endothelial growth factor


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3 Abstract

Pre-eclampsia, defined as hypertensionand proteinuria, causes significant

maternal and perinatal complications. Itaffects about 2-8% of all pregnancies andthere is no curative medication. If womenat risk could be identified, prevention or, atleast, reduction of the complications of

pre-eclampsia might be possible. Whenserum biochemical markers and clinicalcharacteristics have been combined, the

prediction of pre-eclampsia has improved.

We studied whether hyperglycosylated

human chorionic gonadotropin (hCG-h) isuseful for identifying women at risk of

pre-eclampsia already in the first or secondtrimester of pregnancy. Concentrations ofhCG, hCG-h, pregnancy-associated plasma

protein-A (PAPP-A) and the free betasubunit of hCG (hCG) in the serum in thefirst trimester and of placental growthfactor (PlGF), soluble vascular endothelialgrowth factor receptor 1 (sVEGFR-1) andof the angiogenetic factors, angiopoietin-1(Ang-1), -2 (Ang-2) and their commonsoluble endothelial cell-specific tyrosinekinase receptor Tie-2 (sTie-2) in thesecond trimester were studied as predictorsof pre-eclampsia.

For first trimester screening, 158 womenwho subsequently developed pre-eclampsia, 41 with gestationalhypertension, 81 with small-for-gestational

age (SGA) infants and 427 controls wereselected among 12,615 pregnant womenwho attended for first trimester screeningfor Down's syndrome. For secondtrimester screening we used serum samplesfrom 55 women who subsequentlydeveloped pre-eclampsia, 21 withgestational hypertension, 30 who werenormotensive and gave birth to SGAinfants, and 83 controls.

For analysis of Ang-1, -2 and sTie-2 at 12-15 and 16-20 weeks of pregnancy, 49

women who subsequently developed pre-eclampsia, 16 with intrauterine growth

restriction (IUGR) and 59 healthy controlswere recruited among 3240 pregnantwomen attending for first trimesterscreening for Down's syndrome. Another20 healthy women were recruited at term

pregnancy for examination of theconcentrations of these markers inmaternal and fetal circulation and urineand the amniotic fluid before and afterdelivery.

The proportion of hCG-h out of total hCG(%hCG-h) was lower in the first but not insecond trimester in women whosubsequently developed pre-eclampsia ascompared 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 whenPAPP-A, the mean arterial blood pressureand parity were combined with %hCG-h.When these four variables were usedtogether, 69% of the women who were todevelop early-onset pre-eclampsia wereidentified with a specificity of 90%.

The concentrations of circulating Ang-2during the second trimester were higherand those of PlGF were lower in womenwho later developed pre-eclampsia. Ang-2was only 20% sensitive and at 90%

specific for predicting pre-eclampsia, butPlGF performed well and was 53%sensitive and 90% specific for predictingearly-onset pre-eclampsia. %hCG-h didnot provide independent prognostic valuein the second trimester.

In conclusion, hCG-h is a promising firsttrimester marker of early-onset pre-eclampsia. In line with the earlierobservations, PlGF is a useful second

trimester predictive marker of early-onset pre-eclampsia.


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5 Review of the literature

5.1 Development of the

placenta

The main function of the placenta is totransfer gases, nutrients and metabolites

between the fetus and the mother. The placenta has also endocrine andimmunological functions. It produces largeamounts of human chorionic gonadotropin(hCG) and other hormones important for

maintaining pregnancy and for stimulatingfetal growth (Freemark 2010). In pre-eclampsia, placental development andfunction are disturbed, which causesseveral 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%. Thecardiac output also increases, whereasmaternal blood pressure and vascularresistance decrease (Duvekot and Peeters1994). In the mother, renal plasma flowincreases by 50-85% and the glomerularfiltration rate by 40-65% (Conrad 2004).Healthy pregnant women have oftenmoderately increased concentrations of

protein in the urine, but this is of no

clinical significance (Taylor and Davison1997).

Oxygenated maternal blood flows via theuterine arteries that branch into spiralarteries and open into the placentalintervillous spaces. Already at the time of

placentation these spiral arteries have beentransformed into low-resistance vesselsthat lack maternal vasomotor control. Thisensures an uninterrupted blood flow to the

placenta, irrespective of any changes inmaternal blood pressure (Huppertz 2008).

Deoxygenated blood leaves the placentavia uterine venules that collect blood fromthe periphery of the intervillous spaces.The circulation in the placenta may reach1000 ml per min (Konje et al 2001).

5.1.2 Origin of placental

trophoblasts and the villous

tree

The development of the placenta starts atimplantation of the blastocyst, an earlyembryonic structure, which attaches to theuterine epithelium. The blastocyst consistsof an inner cell mass, called theembryoblast, and an outer trophoblastlayer (Schoenwolf et al 2009) (Figure 1A).

At the beginning of the third week ofgestation (one week after conception)these trophoblasts invade the uterine

endometrium. They differentiate intoinvasive syncytiotrophoblasts,multinucleated cells formed through fusionof several cytotrophoblasts (Figure 1A).The syncytiotrophoblasts continue toinvade deeper into the uterineendometrium, now termed decidua, andform lacunae, maternal fluid-filled spaces

between syncytial protrusions. Maternalcapillaries anastomose with these lacunaeand fill them with maternal blood (Figure

1B). Cytotrophoblasts remain at theembryonic side where they act as stemcells for the syncytiotrophoblasts. A newstructure termed the chorion is now formedfrom the cytotrophoblast layer andextraembryonic mesodermal cells. Thechorion is at the fetal surface of the

placenta and is later covered by theamnion (Huppertz 2008) (Figure 1B andTable 1).

At the beginning of the fourth week ofgestation the syncytial protrusions start to


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form the basic structure of the placenta,tree-like protrusions called villi (Figure 2).Cells within the villi differentiate intohematopoietic cells and form the firstembryonic vessels lined with endothelial

cells. By the end of the fourth week ofgestation, the villi have developed enoughto enable effective exchange of gases,nutrients and metabolites betweenmaternal and fetal circulations (Huppertz2008, Schoenwolf et al 2009).

Also at the beginning of the fourth week ofgestation part of the cytotrophoblastsdifferentiate into extravillouscytotrophoblasts, which invade through the

endometrial stroma into the endometrialspiral arteries. They replace both thematernal endothelium and maternalvascular smooth muscle cells. The spiralarteries now enlarge and become low-

resistance vessels independent of maternalvasomotor control. This ascertainssufficient blood flow to the placentathroughout pregnancy (cf. chapter 5.1.1)(Huppertz 2008, Schoenwolf et al 2009).Different types of trophoblasts and theirfunctions are listed in Table

Figure 1. Implantation of the blastocyst and early development of the placenta by the thirdweek of gestation. Modified from Schoenwolf et al (Schoenwolf et al 2009).

A - Trophoblasts from thematernal side of the

blastocyst differentiate tomultinucleatedsyncytiotrophoblasts and

invade the decidua.

B - Syncytiotrophoblasts

form syncytial protrusionsand fluid-filled spaces,lacunae, between them. Thechorion is formed fromcytotrophoblasts andextraembryonic mesodermalcells, and forms the fetalsurface of the placenta.


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Figure 2. Cross-sectionof the villus. The villusconsists of asyncytiotropholast layer,

a cytotrophoblast layerand villous stroma. Fetalvessels run inside thevilli and maternal bloodflushes the villi (ElinaKeikkala 2013).

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

Trophoblast typeWeeks of

gestationFunction Differentiates into

Trophoblast 3-4 weeks Form outer blastocyst layerSyncytiotrophoblasts

Cytotrophoblasts

SyncytiotrophoblastFrom 3rd

week

Invade endometrium(=decidua)

Formation of first lacunae

-

From 4thweek

Form syncytial protrusions

Form outer (=syncytial) layerof villous tree throughout

pregnancy

-

Cytotrophoblast 3-4 weeksForms chorion withextraembryonic mesodermalcells

Villouscytotrophoblasts

Villouscytotrophoblast

From 5th

week

Forms layer undersyncytiotrophoblasts in

placental villi

Maintenance of syncytial layer

of villi by differentiation

Syncytiotrophoblasts

Extravillouscytotrophoblasts

Extravilloustrophoblast

From 4th

week

Invade endometrial stroma(decidua)

Invade through maternal spiralarteries

-

From 5th

week

Replace maternal endotheliumand smooth muscle cells inspiral arteries

Transformation of spiralarteries to dilated tubeswithout maternal vasomotorcontrol

-


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Throughout pregnancy the villi continue tomature and the placenta to develop. Fromthe central to the distal part of the villoustree the number of cytotrophoblasts and

the thickness of the syncytiotrophoblastlayer decrease (Huppertz 2008,Schoenwolf et al 2009). During the

pregnancy, the weight of the placenta andthe amount of cyto- andsyncytiotrophoblasts increase. Thesyncytiotrophoblasts remain dominantform and the proportion in relation tocytotrophoblasts increases as pregnancyadvances (between weeks 13 and 31)(Mayhew et al 1999).

5.2 Pre-eclampsia

Pre-eclampsia is a major cause of infantand maternal mortality and morbidity. Itaffects 2-8% of all pregnancies worldwide(Khan et al 2006). Of all deaths from pre-eclampsia, 99% occur in the developingcountries (Duley 2009). The perinatal risksinclude intrauterine growth restriction(IUGR) (see chapter 5.2.4) and

prematurity (Duley 2009). Management ismainly symptomatic, i.e., antihypertensivedrugs and magnesium infusions, which areused in an attempt to prevent maternal andfetal complications (Datta 2004). Deliveryis the only curative treatment for pre-eclampsia (Duley 2009). Despite extensiveresearch its etiology remains unknown.

In Finland, the incidence of pre-eclampsiawas 2.6 % among nulliparous women between 1997 and 2008 (Lamminpää et al2012). The lifetime incidence of pre-eclampsia was 5.0 % according to a largenational study conducted between 2000and 2001, while that of gestationalhypertension was almost 19 % (Koponenand Luoto 2004). Interestingly, a cross-sectional study of 3650 women reported atwofold risk of pre-eclampsia in northern

Finland as compared to southern Finland(Kaaja et al 2005), but this was not

confirmed in a recent cohort study(Lamminpää et al 2012).

5.2.1 Diagnosis and symptoms

The diagnosis of pre-eclampsia is based onfindings of high blood pressure and

proteinuria in previously normotensivewomen after 20 weeks of pregnancy(Steegers et al 2010) (Table 2). Thesymptoms, clinical findings and the orderin which they occur vary. The most typicalsymptoms are headache, visualdisturbances, upper abdominal pain and ageneral feeling of being unwell. Thecondition may also be asymptomatic(Duley 2009).

Clinical findings vary from mild blood pressure elevation and proteinuria touncontrolled hypertension, anuria andsevere cerebral symptoms, such ashyperreflexia and convulsions (Steegers etal 2010). The severity of the disease isdefined according to clinical symptoms,

blood pressure and/or urinary proteinexcretion (Table 2) (American College ofObstetricians and Gynecologists 2002,Steegers et al 2010). Severe manifestationsinclude eclampsia, hemolysis, elevatedliver enzymes, low platelet (HELLP) -syndrome, disseminated intravascularcoagulation (DIC) syndrome, hemolytic-uremic syndrome (HUS) and posteriorreversible encephalopathy (PRES)(Zeeman 2009, Tranquilli et al 2012).

Eclampsia, defined as new tonic-clonicseizures in women with pre-eclampsia, isoften followed by severe maternal andfetal complications, even death (MacKayet al 2001, Duley 2009). Classification of

pre-eclampsia into severe or mild, andaccording to its time of diagnosis, isshown in Table 2.

The onset of the disease is related to itsseverity. In the US population, onset

before 32 weeks of pregnancy is related toa 20-fold risk of maternal mortality as


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compared to onset after 37 weeks of pregnancy (MacKay et al 2001). Pre-eclampsia before 34 weeks of gestation isclassified as early and after this as late-onset disease (Tranquilli et al 2012) (Table

2). Pre-eclampsia may also occur postpartum, usually defined as new-onsethypertension and proteinuria within 6weeks after delivery (Sibai 2012). Womenwith chronic hypertension or hypertension

diagnosed before 20 weeks of pregnancyand with proteinuria or with any of theother signs or symptoms described inTable 2 is defined as superimposed pre-eclampsia (American College of

Obstetricians and Gynecologists 2002).

Table 2. Diagnostic criteria of pre-eclampsia and severe pre-eclampsia based on theguidelines of the American College of Obstetricians and Gynecologists (ACOG) in 2002(American College of Obstetricians and Gynecologists 2002).

Pre-eclampsia Severe pre-eclampsia

All criteria below Criteria of pre-eclampsia and one ormore of the criteria below

Blood pressure

Previously normal blood pressure

Occurs 20 weeks of pregnancy

Systolic 140 mmHg and/ordiastolic 90 mmHg

Systolic 160 mmHg and/ordiastolic 110 mmHg

Measured at least twice 6 h apartwhile patient in bed rest

Proteinuria 0.3 g in 24-h urine collection 5 g in 24-hour urine collection orurine dipstick test 3+ in twosamples collected more than 6 hapart

Other signsorsymptoms

Oliguria < 500 mL/24-h

Cerebral or visual disturbances

Pulmonary edema or cyanosis

Epigastric or right upper-quadrant pain

Fetal growth restriction

ThrombocytopeniaImpaired liver function

Onset of pre-eclampsia

Early-Onset

Onset before 341 weeks of pregnancy

Late-Onset

Onset at 341 weeks of pregnancy or later1According to the statement of International Society for the Study of Hypertension inPregnancy in 2012 (Tranquilli et al 2012).


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5.2.2 Risk factors

Risk factors have been identified in studies based on national birth registries(Skjaerven et al 2005, Lamminpää et al2012) and prospective cohort studies(Sibai et al 1995, Knuist et al 1998, Ros etal 1998). Some risk factors, such as first

pregnancy (nulliparity), are wellestablished according to numerous studiesinvolving various populations, whereassome factors are more controversial (Table3).

Established risk factors

Nulliparity increases the risk of pre-eclampsia approximately 3-fold ascompared to multiparity (Skjaerven et al2005, Luo et al 2007). It is assumed thatimmune tolerance against the fetus is notas well developed in the first as in later

pregnancies, which could, throughimmunological maladaptation, associatewith pre-eclampsia. There is, however,little evidence for this biologicalmechanism (Luo et al 2007). Other

theories suggest that differences in the profiles of angiogenic factors or in insulinresistance between nulliparous andmultiparous women may be related to pre-eclampsia (Luo et al 2007).

A history of pre-eclampsia is a strong riskfactor for pre-eclampsia in subsequent

pregnancies: the risk of pre-eclampsiarecurring is 7-fold in the second pregnancy(Duckitt and Harrington 2005). Also, a

family history of pre-eclampsia in first-degree relatives almost triples the risk(Duckitt and Harrington 2005). Cohortstudies show that the incidence of pre-eclampsia is also related to ethnicity:African-American women seem to havemore pre-eclampsia than Caucasianwomen (Sibai et al 1995, Knuist et al1998). These findings indicate that there isalso 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 whencomparing twin to singleton pregnancies,and 3-fold when comparing triplet to twin

pregnancies (Duckitt and Harrington2005). Here, the risk increment could berelated to the increased placental mass,which releases high amounts of placentalcytokines and antiangiogenic factors intothe circulation causing a systemicinflammatory response (Bdolah et al2008). Also, exposure to paternal geneticmaterial may be increased in multiple

pregnancies (Trogstad et al 2011).

Obesity increases the risk of pre-eclampsia. A pre-pregnancy body massindex (BMI) of more than 35 kg/m2

increases the risk 3- to 5 –fold ascompared to those with a pre-pregnancyBMI of less than 24 kg/m2 (Duckitt andHarrington 2005). Here, maybe changes inlipid metabolism and inflammatoryresponses known to be present in obese

women are pathophysiologically related toan enhanced risk of pre-eclampsia (Bodnaret al 2005). In Finland, the proportion ofoverweight (BMI > 30 km/m2) amongwomen aged 25-34 years has increasedfrom 8.5% to 9.4% between 1999 and2007 (Helakorpi et al 1999, Helakorpi et al2008). As obesity is an increasing problemin all Western countries, the incidence of

pre-eclampsia and other severe healthconsequences will probably increase

(Luoto et al 2011).

Pre-existing medical conditions such asinsulin-dependent diabetes andautoimmune diseases are clear risk factorsof pre-eclampsia (Duckitt and Harrington2005). Chronic hypertension increases therisk of pre-eclampsia approximately 10-fold as compared to controls. Women withchronic hypertension are also at risk fordeveloping a severe form of the disease(Sibai et al 1995, McCowan et al 1996).Diastolic hypertension of 110 mmHg


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before the 20th week of gestation increasesthe risk for pre-eclampsia 5-fold, pretermdelivery (before gestational week 32) 7-fold and IUGR 7-fold (McCowan et al1996). Antihypertensive treatment has not

been shown to reduce the risk ofsubsequent pre-eclampsia, IUGR or

preterm delivery (American College ofObstetricians and Gynecologists 2012).

Controversial risk factorsSome studies conclude that a maternal ageabove 40 years doubles the risk of pre-eclampsia independently of parity (Duckittand Harrington 2005, Trogstad et al 2011).In these studies, however, no adjustmentwas made for the effect of maternal agewith pre-existing diseases, such as chronichypertension or diabetes, which becomemore prevalent with age and may influencethe results (Duckitt and Harrington 2005,Trogstad et al 2011). Data on the effect ofa very young maternal age (less than 19years) on the risk of pre-eclampsia areinconsistent, and not well supported in theliterature (Duckitt and Harrington 2005).

Women who smoke during pregnancyseem to have less pre-eclampsia than non-smokers (Trogstad et al 2011). Smokingcan, however, by no means be encouragedsince it has numerous adverse effects on

pregnancy which far outweigh any possible benefits (Andres and Day 2000).Also pre-eclamptic women who stopsmoking have better pregnancy outcomesthan women who continue smoking

(Pipkin et al 2008).

Paternal factors seem also to contribute tothe occurrence of pre-eclampsia. Men whohave, in an earlier relationship, fathered a

pregnancy with pre-eclampsia or whothemselves are born from a pre-eclamptic

pregnancy, have an increased risk to havea child born from a pre-eclamptic

pregnancy (Trogstad et al 2011).Primipaternity may also be an interestingrisk factor for pre-eclampsia, a

phenomenon not observed in all studies, but one which might be explained by theusually long intervals between pregnanciesfathered by different men, as the risk of

pre-eclampsia in multiparas seems toincrease if the time from the previous

pregnancy reaches 4-5 years (Trogstad etal 2001).

In conformity with what is known aboutthe risk of pre-eclampsia among

primiparous women, it has been claimedthat induced abortions might reduce therisk of pre-eclampsia in subsequent

pregnancies (Seidman et al 1989, Sibai etal 1995). Data on spontaneous abortionsand miscarriages are conflicting in thisrespect (Seidman et al 1989, Sibai et al1995, Xiong et al 2002). Infertility andrecurrent spontaneous miscarriagesincrease the risk of pre-eclampsia, which,in turn, might be due to some sharedunderlying pathophysiologic featurehindering a normal pregnancy (Trogstad etal 2011).


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Table 3. Risk factors for pre-eclampsiaRisk factor Risk Established risk factors

Pregnancy-associated factors

Nulliparity 3-fold a

Previous pre-eclampsia 7-foldMultiple pregnancy 3-fold

Familial factors

Family history of pre-eclampsia 3-foldEthnicity (African-American) 2-fold , c

Pre-existing medical conditions

Obesity (BMI > 35 kg/m ) 3- to 5-foldChronic hypertension 10-fold , c

Insulin-dependent diabetes mellitus 3- to 4-foldAnti-phospholipid syndrome and other

thrombophilic conditions 10-fold b

Autoimmune disease 7-foldRenal disease 3-fold b

Controversial risk factors

Personal history

High maternal age (> 40 years) 2-foldTeenage pregnancy (< 19 years) Increased/ No effectSmoking during pregnancy 0.5- to 0.8-fold

Paternal factors

Men fathered a pregnancy with pre-

eclampsia Increased/ No effect

d

Primipaternity Increased/ No effectPregnancy-associated factors

Long (> 4-5 years) interval between pregnancies

Increased/ No effect d

Induced abortion Decreased/No effect c

Previous miscarriage Increased/ No effect c,

Infertility Increased/ No effectIn-vitro fertilization/ donor insemination Increased/ No effect

a 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 resultsfrom glomerular endotheliosis and pre-eclamptic hypertension and is consideredto be due to secondary endothelialdysfunction (Roberts et al 1989). Anyother features of the pathophysiology of

pre-eclampsia are more speculative. Sometheories are listed in the following.

Placental vascular remodeling and

endothelial dysfunction

A generally accepted hypothesis for the pathophysiology of pre-eclampsia isimpaired transformation of the spiralarteries. This is thought to be a two-stage

process. In the first stage, the physiologicaladaptation of the uterine spiral arteries is

thought to be inadequate, which leads to ahypoxic-ischemic reperfusion circulation


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in the placenta. In the second stage,hypoxia is followed by the release ofseveral biologically active placental factorsinto the maternal blood circulation. Thesefactors are thought to cause maternal

endothelial dysfunction and a systemicinflammatory reaction (Redman andSargent 2009).

In pre-eclampsia the differentiation ofcytotrophoblasts into extravillous invasivecytotrophoblasts is incomplete andinvasion into the maternal spiral arteriesshallow. This could explain why the spiralarteries do not undergo the changes thatoccur in healthy pregnancies (Meekins etal 1994, Zhou et al 1997, Burton et al2009). The maternal spiral arteries retaintheir smooth muscle layer and remainhigh-resistance arteries (Brosens et al1970) (see chapter 5.1.1) (Figure 3).

Intermittently inadequate blood supply tothe placenta leads to an ischemicreperfusion state and there are changes

between hypoxia and reoxygenation(Redman and Sargent 2009). In vitro

studies of cultures of term placenta showthat ischemic reperfusion causes damagesimilar 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-eclampsiaaccompanied with IUGR. The intervillousspaces widen and may become lined withthrombotic material, and the volume of theterminal villi and surface area are reduced(James et al 2010) (Figure 3). This is

probably secondary to impaireduteroplacental blood flow.

Figure 3. Spiral artery transformation in the pre-eclamptic and healthy placenta (ElinaKeikkala 2013).

Inflammatory system

In pre-eclampsia, the usual activation ofthe inflammatory system (includingimmune cells and the clotting andcomplement systems) and of the

endothelium is exaggerated. This wouldenhance synthesis of the acute phase

proteins including C-reactive protein(CRP), several complement componentsand fibrinogen (Redman and Sargent


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2009). An exaggerated systemicinflammatory reaction is associated withseveral findings in pre-eclampsia: with theseverity of the HELLP syndrome (Terroneet al 2000), the degree of abnormal

hemostasis, impaired clotting and DIC(Thachil and Toh 2009).Syncytiotrophoblasts secrete also several

pro-inflammatory factors, includingsoluble vascular endothelial growth factorreceptor 1 (sVEGFR-1), into the maternalcirculation. These factors may well berelated to maternal endothelial dysfunction(Redman and Sargent 2009).

Cytotrophoblastic plugging

Burton et al introduced a theory ofcytotrophoblastic plugging that protectsembryos against oxidative stress. Until theeighth week of gestation the intervillousspaces are plugged by cytotrophoblastlayers and arterial blood cannot enter them(Burton et al 1999). These plugs wouldthen be displaced at the end of the firsttrimester allowing oxygenated blood toenter the intervillous space and the

peripheral placenta. It is further thoughtthat failure to form these plugs might leadto early oxidative stress of the placenta anddamage to the syncytiotrophoblast layer.The clinical consequences would then bemiscarriage or pre-eclampsia, especially inits early-onset form (Burton and Jauniaux2011). This theory is supported byhistological studies on placentas fromearly miscarriage, where nocytotrophoblastic plugs were observed

(Hustin et al 1990). Regarding pre-eclampsia, there is no direct evidence foror against this theory.

Autoantibodies

Wallukat et al reported significant titers ofangiotensin II type 1 receptor agonistantibodies (AT1-AA), an immunoglobulinG (IgG) class autoantibody, in the sera of70-95% of pre-eclamptic women(Wallukat et al 1999, Walther et al 2005,

Siddiqui et al 2010). These antibodies arenot present in the blood of non-pregnant

women, pregnant women with essentialhypertension or in women withuncomplicated pregnancies (Wallukat et al1999). When IgG extracted from the serumof pre-eclamptic women was injected into

pregnant mice, typical symptoms of pre-eclampsia ensued: hypertension,

proteinuria and intrauterine growthrestriction (Zhou et al 2008). However,AT1-AA is not specific for pre-eclampsia,since it is also present in the serum ofwomen with gestational hypertension(Wallukat et al 1999) and with renalallograft rejection (Dragun et al 2005). Therole of AT1-AA in the pathophysiology of

pre-eclampsia needs still to be clarified.

Genetics

Pre-eclampsia is a complex disordercaused by genetic as well as environmentalfactors (Williams and Broughton Pipkin2011). A variety of genetic changes have

been observed in association with pre-eclampsia, including mutations that affectendothelial function, vasoactive proteins,oxidative stress, the blood clotting system

and immunological factors. Screening ofthe entire genome by genome-wide linkageanalyses (GWLS) has revealed three locithat are significantly linked to pre-eclampsia: 2p13, 2p25 and 9p13(Arngrimsson et al 1999, Laivuori et al2003). Further information could bederived by the most up-to-date method,genome-wide association screening(GWAS), which allows more exact waysto investigate gene polymorphisms and

single candidate genes (Williams andBroughton Pipkin 2011). Theheterogeneity of pre-eclampsia, geneticvariation between populations,environmental interactions and age-dependent effects are challenges even forthe latest genetic technologies.


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5.2.4 Intrauterine growth

restriction

DefinitionsThe criteria for IUGR are an estimatedfetal weight below the tenth percentile andevidence of placental insufficiency (Ott1988). Low birth weight (LBW), definedas a birth weight of less than 2500 g, is amore explicit term that is often used innational registries and internationaldatabases (UNICEF 2007). In Finland,4.2% of infants were of LBW in 2012(Vuori and Gissler 2013). Infants small-for-gestational-age (SGA), anotherdiagnosis used in parallel with IUGR andLBW, are usually defined as having agestational-age adjusted birth weight

below the tenth percentile in the absenceof placental insufficiency. SGA wasdiagnosed in 2.2% of the infants born fromsingleton 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 aresecondary to pre-eclampsia (Villar et al2006). Little is known about the generaletiology of IUGR, although impaireddevelopment of the placenta similar towhat occurs in pre-eclampsia isoccasionally seen (James et al 2010).Maybe pre-eclampsia and IUGR aredifferent manifestations of the same

disease, since the placental pathology hassimilar traits. Nevertheless, clinical placental insufficiency occurs only insome cases of IUGR and pre-eclampsiaand is by no means a universal finding inthese conditions (Villar et al 2006, Jameset al 2010).

DiagnosisThe diagnosis of IUGR is based onestimation of the fetal weight by

ultrasound and of the waveforms of theuterine, umbilical and middle cerebral

arteries by Doppler velocimetry (Bambergand Kalache 2004). The main diagnosticaim is to distinguish IUGR with placentalinsufficiency from SGA, because IUGR isstrongly related to perinatal complications

and poor neurological outcome (Yanneyand Marlow 2004).

Causes and risk factorsThe causes of IUGR and SGA areheterogenic and involve maternalhypertension or pre-eclampsia, maternalsubstance abuse, malnutrition,chromosomal abnormalities, geneticdiseases and congenital infections(Sankaran and Kyle 2009). However, mostIUGR cases remain unexplained (Villar etal 2006, James et al 2010).

IUGR and pre-eclampsia share some riskfactors, e.g., nulliparity and multiple

pregnancies (Villar et al 2006). Similar pathological changes occur in theformation of placental vessels: the numberand surface area of placental tertiary villiarterioles are reduced, maternal spiral

artery formation is impaired and the fetalcytotrophoblast layer is disorganized(Khong et al 1986, Sankaran and Kyle2009). In addition, angiogenesis-

promoting factors affect placentaldevelopment in a similar way in IUGR and

pre-eclampsia, and the concentrations of placental growth factor (PlGF) in thematernal serum are lower in IUGR and in

pre-eclampsia than in healthy controls(Helske et al 2001, Taylor et al 2003,

Åsvold et al 2011).

There are differences between IUGR and pre-eclampsia. A previous infant withLBW is a risk factor for IUGR but not for

pre-eclampsia (Villar et al 2006). Womengiving birth to IUGR infants do not usuallyexhibit the systemic endothelialdysfunction and inflammation reaction thatoccurs in pre-eclampsia (James et al 2010).The studies on serum concentrations of

antiangiogenic factors, e.g., sVEGFR-1,have yielded conflicting results. sVEGFR-


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1 has been reportedly elevated in pre-eclampsia but not in unexplained IUGR insome studies (Wathen et al 2006), whereasothers report similar changes in IUGR and

pre-eclampsia (Åsvold et al 2011).

5.2.5 Prevention of pre-eclampsia

The preventive effect of dietarysupplements and lifestyle modificationswhen started before symptoms of pre-eclampsia in risk populations has beenexamined in several studies. Calciumsupplementation reduces the risk of pre-eclampsia only among women with lowdietary calcium intake and a high risk of

pre-eclampsia (Hofmeyr et al 2010). Otherfood supplements including magnesium,fish oil, folic acid, antioxidants andvitamin C, D and E do not affect the riskof pre-eclampsia. Numerous drugs havealso been studied but most of them, e.g.anti-hypertensive medication, lowmolecular weight heparin, nitric oxide,

progesterone and diuretics, are not

effective for prevention of pre-eclampsia(Dekker and Sibai 2001, Thangaratinam etal 2011).

A recent systematic review of fiverandomized controlled trials in high-risk

populations showed that low-dose aspirinstarted before 16 weeks of pregnancyreduces the risk of pre-eclampsia. Therelative risk (RR) of early-onset pre-eclampsia (diagnosed before 37 weeks)

was 0.11 (95% confidence interval, CI,0.04 - 0.33), but low-dose aspirin has noeffect on pre-eclampsia occurring after 37weeks of pregnancy (Roberge et al 2012).This has been suggested to be due to the

beneficial effects of aspirin on placentation, although the specificmechanism is unknown. Studies includedin the review were performed on nulliparasor women with a history of pre-eclampsia,IUGR or chronic hypertension (Roberge et

al 2012). In contrast, in a meta-analysis byAskie et al comprising 31 randomized

trials, only a 10% reduction in the RR of pre-eclampsia was observed amongwomen on low-dose aspirin or otherantiplatelet or anticoagulant agents(dipyridamole or heparin). However, the

timing of treatment varied, which probablyexplains the difference in results ascompared to those in the review byRoberge et al (Askie et al 2007, Roberge etal 2012).

5.3 Prediction of pre-

eclampsia

Various biochemical markers related toendothelial dysfunction, oxidative stress,insulin resistance and immunologicalfunction have been sought for in samplesof urine and serum for prediction of pre-eclampsia. Clinical data on maternal riskfactors and Doppler ultrasoundmeasurements assessing placental bloodflow have been combined with

biochemical factors in an attempt toimprove 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 besensitive and specific enough for clinicaluse (Giguere et al 2010, Kuc et al 2011).Some of the most promising predictivemarkers of pre-eclampsia are listed inTable 4.

5.3.1 Maternal characteristics

and Doppler ultrasound

Some maternal characteristics may be usedto calculate the risk of pre-eclampsia.Known risk factors are some chronicdiseases, e.g., essential hypertension,insulin-dependent diabetes, high blood

pressure in the first or second trimester,high BMI or weight before pregnancy orduring the first trimester, and history of

pre-eclampsia. The smoking habits are alsoinvolved. These characteristics are used to

estimate the overall risk of pre-eclampsia(Steegers et al 2010).


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

Doppler ultrasoundPE EO

PELOPE

PE EO PE LOPE

Uterine artery diastolic flow a a

Uterine artery pulsatility index Uterine artery resistance index

Biochemical markers

Activin A c e

Inhibin A c e

A disintegrin and metalloproteinase12 (ADAM12) c

Free subunit of hCG (hCG) c g,

Human chorionic gonadotropin (hCG) g,

Pregnancy-associated plasma protein-A(PAPP-A)

c c c

Placental protein 13 (PP13) c c

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

Soluble vascular endothelial growth

factor receptor 1 (sVEGFR-1)

c c c

PE, pre-eclampsia (onset not specified); EO PE, early-onset pre-eclampsia; LO PE, late-onset 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 involvingnulliparous women showed that a systolic

blood pressure of 120 mmHg between

13 and 27 weeks of pregnancy, a high body weight before pregnancy (120% ofdesirable weight) and the number of

previous miscarriages were eachindependently predictive of pre-eclampsia(Sibai et al 1995). Maternal characteristicsalone provide risk calculations with asensitivity of approximately 50% and aspecificity of approximately 90% (Kuc etal 2011).

Doppler ultrasound is used to evaluate theuteroplacental circulation in complicated

pregnancies (Campbell et al 1983). In pre-eclampsia, reduced uterine artery diastolicflow 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-eclampsiaemerge (Harrington et al 1991) or IUGR(Harrington et al 1997). This could be anindirect sign of impaired placental vasculardevelopment, i.e., impaired trophoblastinvasion and spiral artery transformation(Harrington et al 1991). A high first-trimester uterine artery pulsatility index(PI) or resistance index (RI) predict pre-eclampsia, especially its early-onset form;the specificity of these variables has been


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90%, but sensitivity has varied from 29%to 83% (Akolekar et al 2009, Kuc et al2011). One reason for the marked degreeof variation could be related to thetechnique of Doppler ultrasound

examination, since its sensitivity dependsstrongly on the person who performs theexamination.

5.3.2 PlGF and sVEGFR-1

PlGF

The placental growth factor (PlGF) is amember of the vascular endothelial growthfactor (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 proteinis expressed also slightly in some otherorgans (heart, lung and adipose tissue)(Table 5). PlGF is considered to beangiogenic (Ziche et al 1997). It has alsochemotactic properties and mobilizeshematopoietic progenitor cells from the

bone marrow. The angiogeniccharacteristics of PlGF play a role in

pathological conditions like ischemia,tumor growth and wound healing. PlGFknockout mice are born without vasculardefects, but their responses to ischemic

insults are reduced (Ribatti 2008).

sVEGFR-1Soluble vascular endothelial growth factorreceptor 1 (sVEGFR-1) is a circulating

receptor of PlGF and VEGF. sVEGFR-1 isformed by alternative mRNA splicing ofthe membrane-bound form of VEGFR-1 oris released from the membrane by

proteolytic cleavage (Kendall and Thomas1993). Therefore, its molecular size varies

between 60 and 150 kDa. In addition to placental trophoblasts it is expressed byvascular endothelial and smooth musclecells, activated mononuclear monocytes inthe peripheral blood, corneal epithelialcells and the proximal tubular epithelialcells of the kidneys (Figure 4 and Table 5).The main function of sVEGFR-1 is to bindcirculating VEGF, which leaves less freeVEGF available for cell surface-boundVEGFR-1 receptors. This, in turn, inhibitsthe angiogenic function of VEGF. Inaddition, sVEGFR-1 counteracts edema,since cell membrane-bound VEGF alsoinduces 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).


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PlGF and sVEGFR-1 in uncomplicated

pregnancy

In normal pregnancy, PlGF and sVEGFR-1 are expressed in the placenta by variouscell types (Figure 4 and Table 5) (Ahmed

et al 1995, Clark et al 1996, Vuorela et al1997, Zhou et al 2002, Tsatsaris et al2003). sVEGFR-1 is not expressed in

placental vascular endothelial cells, incontrast to membrane-bound VEGFR-1(Clark et al 1996, Ribatti 2008). sVEGFR-1 is not detectable in the sera of non-

pregnant women and it becomes detectablein the maternal circulation at six weeks ofgestation (Levine et al 2004, Wathen et al2006, Noori et al 2010). Theconcentrations of PlGF increase during

pregnancy and decrease at term (Taylor etal 2003, Levine et al 2004, Noori et al2010) (Figure 5).

PlGF and sVEGFR-1 in pre-eclampsia

PlGF and sVEGFR-1 have been widelystudied as markers of pre-eclampsia (Kucet 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), butPlGF mRNA is reduced in women whosubsequently develop pre-eclampsia,according to a study on first-trimester

placental biopsies (Farina et al 2008)(Table 5). Concentrations of PlGF in theserum are lower in manifest and

subsequent pre-eclampsia as compared tocontrols (Torry et al 1998, Maynard et al2003) (Figure 5). Some studies show lowerconcentrations already in the first trimesterof pregnancy of women who develop pre-

eclampsia (Thadhani et al 2004, Akolekaret al 2008, Noori et al 2010), but otherstudies show no difference (Ong et al2001, Taylor et al 2003, Levine et al2004). However, all published studiesshow that serum PlGF concentrations arelower in the second trimester in womenwho subsequently develop pre-eclampsiathan in controls (Tidwell et al 2001, Tayloret al 2003, Levine et al 2004, Wathen et al2006, Noori et al 2010, Villa et al 2013)(Table 5 and Figure 5).

In pre-eclampsia, the expression ofsVEGFR-1 is increased especially ininvasive cytotrophoblasts (Maynard et al2003, Tsatsaris et al 2003, Ahmad andAhmed 2004) and in first trimester

placental biopsies in women who laterdevelop pre-eclampsia (Farina et al 2008).High concentrations of maternal serum

sVEGFR-1 may be present already beforeclinical symptoms (Maynard et al 2003,Tsatsaris et al 2003, Levine et al 2004,Wathen et al 2006), especially in thesecond trimester (Levine et al 2004,McKeeman et al 2004, Wathen et al 2006,

Noori et al 2010) (Table 5 and Figure 5).


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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 ofexpression

Decidua


Villouscytotrophoblasts indistal villi

Extravillouscytotrophoblasts

Villous stroma


Villouscytotrophoblasts indistal villi

Invasivecytotrophoblasts

Placental endothelium

Decidua


Villouscytotrophoblasts

ExtravilloustrophoblastsVillous stroma

Perivascular cells

Changes in

subsequent pre-eclampsia

levels of mRNA in1st trimester



Changes in pre-eclampsia

levels of mRNA expression inextravillouscytotrophoblasts

in amount ofmRNA

levels of mRNA

secretion inextravillouscytotrophoblasts

Figure 5. PlGF and sVEGFR-1 concentrations trends during pregnancy. Dotted lines indicate

concentrations in healthy pregnant women, arrows show concentration changes in womenwith pre-eclampsia (PE) ( no difference, decreased, increased). Modified from Levineet al (Levine et al 2004).


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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 thesame site of their common endothelial cell-specific tyrosine kinase receptor Tie-2(Fiedler et al 2003, Thomas and Augustin2009). Ang-1 is a Tie-2 agonist and thusangiogenic, whereas Ang-2 is a Tie-2antagonist and thus antiangiogenic(Maisonpierre et al 1997, Thomas andAugustin 2009). The soluble form of Tie-2(sTie-2) is also approximately 75 kDa insize. It is present in the circulation after ithas shed the extracellular region of themembrane-bound Tie-2 receptor; this leadsto decreased concentrations of functionalmembrane-bound receptors (Reusch et al2001). Like the membrane-bound receptor,sTie-2 also binds both Ang-1 and Ang-2. Itleaves less Ang-1 and Ang-2 available formembrane-bound receptors and thusdecreases their biological activity (Findleyet al 2007, Shantha Kumara et al 2010).

Ang-1In the placenta, Ang-1 is expressed by thesyncytiotrophoblasts and perivascular cells(Geva et al 2002, Thomas and Augustin2009) (Figure 6). Ang-1 is also expressed

by myocardial cells during early humandevelopment and by perivascular cells inadult tissues (Davis et al 1996). Ang-1 is

also expressed in some pathologicalconditions, e.g. by some tumor cells, and itis thought to interact with VEGF in tumorangiogenesis (Saharinen et al 2011). Itsexpression is decreased by hypoxia (Zhang

et al 2001).

Ang-2

Ang-2 is mainly expressed by endothelialcells (Thomas and Augustin 2009). Itsexpression is upregulated at sites ofvascular remodeling (Zhang et al 2001). Inthe placenta Ang-2 is expressed bysyncytiotrophoblasts and perivascular cellsthroughout gestation, but both its proteinand mRNA expression decrease towardsterm (Zhang et al 2001, Geva et al 2002)(Figure 6). Ang-2 is also expressed in theovaries (Sugino et al 2005). Some tumorsexpress Ang-2 (Saharinen et al 2011).Contrary to Ang-1, the expression of Ang-2 is strongly upregulated by hypoxia(Mandriota and Pepper 1998).

sTie-2In first trimester placenta, Tie-2 isexpressed by endothelial cells, decidua(Zhang et al 2001) and cytotrophoblasts(Kayisli et al 2006) (Figure 6). Beforeterm, its expression either decreases(Zhang et al 2001) or remains stable(Kayisli et al 2006). Placental Tie-2expression is not influenced by hypoxia, incontrast to the ligands Ang-1 and Ang-2(Zhang et al 2001).

Figure 6. Expression of Ang-1, Ang-2 and Tie-2 in the placental villus.ST, syncytiotrophoblasts; CT,cytotrophoblasts; EC, endothelialcells; Str, stroma (Elina Keikkala2013).


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Angiopoietins in uncomplicated

pregnancy

Angiopoietin-1, Ang-2 and Tie-2 are allessential for vascular growth (Demir et al2007). Mice which lack the Ang-1 or Tie-2

genes or which overexpress Ang-2, die inutero because of severe vascular injuries(Sato et al 1995, Suri et al 1996). Micedeficient in Ang-2 survive embryogenesis

but die postpartum because of lymphaticinjuries (Gale et al 2002). In human

pregnancy, Ang-1, Ang-2 and sTie-2 haveall been detected in amniotic fluid andmaternal serum throughout pregnancy(Pacora et al 2009, Buhimschi et al 2010).Their concentrations during pregnancy arehigher than in non-pregnant women(Hirokoshi et al 2005, Nadar et al 2005).With advancing pregnancy theconcentrations of Ang-1 either remainstable (Bolin et al 2009) or increase(Buhimschi et al 2010), whereas those ofAng-2 decrease (Bolin et al 2009, Pacoraet al 2009, Buhimschi et al 2010) andthose of sTie-2 either remain stable ordecrease slightly (Buhimschi et al 2010,

Sung et al 2011).Angiopoietins in pre-eclampsia

Information regarding the concentration ofAng-1 in maternal blood in pre-eclampsiais conflicting (Nadar et al 2005, Bolin et al2009, Kamal and El-Khayat 2011).Preeclampsia does not seem to change the

placental expression of Ang-1 (Sung et al2011) (Table 6).

In most studies, maternal circulatingconcentrations of Ang-2 have been lowerin women with pre-eclampsia than incontrols (Hirokoshi et al 2005, Nadar et al

2005, Hirokoshi et al 2007), but in severe pre-eclampsia increased Ang-2concentrations have been reported (Han etal 2012). A prospective longitudinal studywith a limited number of pre-eclampticwomen (n=19) showed that theconcentrations of Ang-2 or Ang-1 did notdiffer from those of controls, but the ratioof Ang-1 to Ang-2 was lower in womenwho developed pre-eclampsia fromgestation week 25 onwards (Bolin et al

2009). The results concerning Ang-2expression in third trimester placentaltissue recovered from pre-eclampticwomen have been conflicting and it is notknown if expression differs in comparisonto non-pre-eclamptic women (Zhang et al2001, Han et al 2012, Sung et al 2011)(Table 6).

Maternal circulating concentrations ofsTie-2 are lower in pre-eclampsia than innormal pregnancy according to moststudies (Vuorela et al 1998, Gotsch et al2008, Sung et al 2011). The reduction inthe concentration of sTie-2 may beobserved already at week 24, well beforeany pre-eclampsia symptoms appear (Sunget al 2011) (Table 6).


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Table 6. Changes in concentrations of Ang-1, Ang-2 and sTie-2 and their mRNA expressionin the placenta in women with pre-eclampsia as compared to controls ( no difference, decreased, increased).

Trimester

1st 2

nd 3

rd Method Reference

mRNA RT-PCR Sung et al 2011Ang-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 ELISA (R&D Systems) Nadar et al 2005 a ELISA (R&D Systems) Han et al 2012

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

sTie-2 ELISA (R&D Systems) Sung et al 2011Plasma ELISA (R&D Systems) Gotsch et al 2008

ELISA (R&D Systems) Nadar et al 2005a Severe pre-eclampsia; RT-PCR, reverse transcriptase-polymerase chain reaction; IFMA,immunofluorometric assay.

5.3.4 PAPP-A

Pregnancy-associated plasma protein-A(PAPP-A) is a protease that is expressedalmost exclusively by the placenta. Theexpression and secretion of PAPP-Aincrease during differentiation of villouscytotrophoblasts to syncytiotrophoblasts(Guibourdenche et al 2003). PAPP-A is

also expressed at low levels in some othertissues (endometrium, colon and kidney)(Overgaard et al 1999). The biologicalfunction of PAPP-A is not known, but ithas been postulated that it may increasethe bioavailability of insulin-like growthfactors 1 and 2 (IGF-1 and -2) by cleavageof insulin-like growth factor binding

proteins (IGFBP) -4 and -5 (Laursen et al2007). Thus, PAPP-A may contribute to

placental and fetal growth, since IGF-1and -2 are important mediators of cell

proliferation, differentiation and migration(Laursen et al 2007) (Table 7). Placentalexpression and circulating PAPP-Aconcentrations increase with advancing

pregnancy (Overgaard et al 1999). The production rate of PAPP-A fromtrophoblasts does not increase withadvancing gestational weeks, and elevated

concentrations of PAPP-A in the plasmamay reflect syncytiotrophoblast mass(Guibourdenche et al 2003). Theconcentration of PAPP-A decreases inDown's syndrome and that is why PAPP-A, along with hCG, is used for first-trimester screening of the syndrome(Haddow et al 1998, Wald 2011) (Table7).


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Table 7. Pregnancy-associated plasma protein-A (PAPP-A) (Haddow et al 1998, Overgaardet al 1999, Guibourdenche et al 2003, Laursen et al 2007, Kuc et al 2011, Wald 2011).PAPP-A

Pregnancy-associated plasma protein-A

Structure HeterotetramerMW 500 kDa

Expression

SyncytiotrophoblastsVillous cytotrophoblasts

Decidua

Endometrium, myometrium

Kidney, colon, breast etc.

Main

Function

Biological function unclear

ProteaseCleaves IGFBP-4 -> increase bioavailability of IGF-1 and -2 -> May

contribute to fetal growth

Relation to pathological

conditions

IUGR: decreased

Premature delivery: decreased

Cornelia Delange syndrome: decreasedPre-eclampsia: decreased

Clinicalimplications

Screening of Down’s syndrome in 1st trimester

MW, molecular weight; kDa, kilodalton; IGFBP-4, insulin-like growth factor binding protein-4; IGF, insulin-like growth factor.

PAPP-A in pre-eclampsiaConcentrations of PAPP-A are decreasedalready in the first (Ong et al 2000, Kuc etal 2011) and second trimesters (Bersingerand Odegard 2004, D'Anna et al 2011) inwomen who develop pre-eclampsia. Moststudies indicate that PAPP-A is moderatelyadequate for prediction of pre-eclampsia(Akolekar et al 2009, Poon et al 2009) and

other studies have not found the markervery useful at all (Foidart et al 2010,Vandenberghe et al 2011). All the same, inmanifest pre-eclampsia the circulating and

placental concentrations of PAPP-A areincreased in comparison to healthycontrols (Hughes et al 1980, Bersinger etal 2002).

5.3.5 hCG, hCG and hCG-h

hCG

Human chorionic gonadotropin (hCG) is aglycoprotein consisting of two subunits, (hCG) and (hCG). hCG is almostidentical to the -subunits of the three

pituitary glycoprotein hormones thyroidstimulating hormone (TSH), follicle

stimulating hormone (FSH) and luteinizinghormone (LH), whereas the -subunits areunique to each hormone and determine the

biological activity of the hormone(Stenman et al 2006). Determination ofhCG is used as a pregnancy test and forother diagnostic and therapeutic purposes.The main biological function of hCG is tomaintain progesterone synthesis in thecorpus luteum during early pregnancy, butit also exerts other functions, e.g.,

stimulation of angiogenesis (Stenman et al2006) (Table 8).


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Table 8. hCG variants. Reviewed by Stenman et al (Stenman et al 2006).hCG hCG hCG-h

Human chorionic

gonadotropinFree hCG subunit

Hyperglycosylated

hCG

Structure

Heterodimer: and –

subunits237 amino acidsGlycan structures: 2attached to -subunit and6 attached to –subunitDisulfide bondsMW 37.5 kDa

Homodimer: -subunit145 amino acidsGlycan structureDisulfide bondsMW 23.5 kDa

Heterodimer: and –

subunits237 amino acidsGlycan structures:more complex on and –subunits than inhCGDisulfide bondsMW >37.5 kDa

Expression

SyncytiotrophoblastsTrophoblastic tumorsOther tumors

SyncytiotrophoblastsTrophoblastic tumorsOther tumors

Extravillous and villouscytotrophoblastsTrophoblastic tumors

Testicular tumors

Main function

Binds to LH/HCGreceptorStimulates progesterone

production from corpusluteumStimulates uterine growthand angiogenesis

No receptor foundMay promote tumorgrowth via otherfactors

Binds to LH/HCGreceptorSame as in hCGContributes totrophoblast invasion

Clinicalimplications

Diagnostics:

Pregnancy testExtrauterine pregnancy

MiscarriageScreening of Down’ssyndrome at 2nd trimesterGestational trophoblasticdiseaseTesticular germ celltumorsDoping controlTherapy:

Ovarian stimulation ininfertility

Diagnostics:Screening of Down’ssyndrome at 1sttrimesterGestationaltrophoblastic diseaseTesticular germ celltumors

kDa, kilodalton; MW, molecular weight; LH, luteinizing hormone

hCG

hCG has several structural variantsincluding hyperglycosylated hCG (hCG-h)and one variant with a free -subunit ofhCG (hCG) (Stenman et al 2006) (Table8). hCG is mainly secreted by the

placental syncytiotrophoblasts (Gaspard etal 1980). The concentrations of hCG in

maternal serum increase rapidly during theweek 7-10 of pregnancy and decreases

thereafter (Stenman et al 2006). The roleof hCG in the physiological developmentof the placenta is unclear as it lacks the

biological effects of hCG (Stenman et al2006). Clinically, hCG is used for first-trimester screening of Down’s syndrometogether with maternal serum PAPP-Aconcentrations and nuchal translucency by

ultrasound (Haddow et al 1998, Wald2011). In Down’s syndrome maternal


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serum hCG concentrations are higherthan in controls, which might be related todelayed placental maturation (Macintosh etal 1994) (Table 8).

Malignant trophoblastic cells producelarge amounts of hCG and hCG. Thus, inhydatidiform mole, an abnormal

pregnancy state with trophoblastic proliferation, and in chorioncarcinoma theconcentrations of hCG and hCG arehigher than in normal pregnancies(Gaspard et al 1980, Stenman et al 2006,Lurain 2010). The proportion of hCG outof total hCG increases in these conditions,and a proportion of hCG of more than 5%of hCG is a strong indicator of malignanttrophoblastic disease (Stenman et al 2006).hCG is also expressed in manynontrophoblastic cancers. hCG promotestumor growth (Gillott et al 1996) andinhibits apoptosis in cells derived from theurinary bladder in vitro (Butler et al 2000).An elevated concentration of hCG inserum is associated with an adverse

prognosis for patients with any one of

several nontrophoblastic tumors (Stenmanet al 2006).

hCG-h

Glycosylation is an important posttranslational modification of proteins.It affects their structure and function, anddifferent pathological conditions(including cancer) give rise to differentglycosylation variants (Hakomori 2002).Approximately one third of the molecular

weight of hCG is carbohydrate, andcarbohydrates generate great variation tothe structure, function and half-life of hCG(Elliott et al 1997, Valmu et al 2006). Ahyperglycosylated form of hCG (hCG-h)was first identified in urine samples fromwomen with chorioncarcinoma or molar

pregnancy (Elliott et al 1997). Furthermass spectrometric studies identified aglycan structure of hCG and showed that

hCG from chorioncarcinoma, molar pregnancy and very early pregnancy (< 7weeks) has a more complex glycanstructure 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 allshow complex glycosylation patterns.Thus, hCG-h is defined as a spectrum ofhCG variants that contains more complexglycan structures than hCG (Elliott et al1997, 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 for90-100% of all hCG at 4 weeks ofgestation and for less than 3% at 20 weeksof gestation (Stenman et al 2011) (Figure7). hCG-h binds to the same LH/hCGreceptor as hCG and shares most of the

biological effects of hCG (Stenman et al2006). Expression of hCG-h differs fromthat of hCG: invading extravillouscytotrophoblasts stain deeply for hCG-h by

immunocytochemistry, whereas hCG ismainly present in placentalsyncytiotrophoblasts (Kovalevskaya et al2002b, Cole et al 2006, Guibourdenche etal 2010). hCG-h promotes trophoblastinvasion in cell cultures, and it is thoughtto mediate the invasion of cytotrophoblastsin vivo (Cole et al 2006, Guibourdenche etal 2010).

Increased hCG-h concentrations occur in

pregnancies with Down’s syndrome,which is thought to reflect the delayedmaturation of placenta (Cole et al 1999,Palomaki et al 2005). In addition togestational trophoblastic diseases, hCG-his a major form of circulating hCG innonseminomatous testicular cancer(Lempiäinen et al 2012) (Table 8).


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6 Aims of the study

The aim of this study was to identify serum markers for screening of pre-eclampsia. Weanalyzed 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 maternalserum 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 maternalserum concentrations ofa. 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).


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7 Materials and methods

7.1 Patients and study design

7.1.1 First trimester hCG-h,

PAPP-A and hCG (I)

A nested case-control study was performedin the Kuopio University Hospital region

between April 2008 and December 2010.The study population was selected among

12,615 pregnant women participating incombined first trimester screening forDown’s syndrome at 8 - 13 weeks ofgestation. 158 women who developed pre-eclampsia, 41 women with gestationalhypertension, 81 normotensive womenwith SGA infants and 427 controls wereincluded.

7.1.2 Second trimester hCG-h,

PlGF and sVEGFR-1 (II)

A nested case-control study was performedin the Kuopio University Hospital betweenDecember 2007 and May 2009. The study

population was chosen among 1470 pregnant women attending for screening ofDown's syndrome at 14-17 weeks ofgestation. 55 women who developed pre-eclampsia, 21 who developed gestationalhypertension, 30 normotensive womenwith SGA infants and 83 controls wereincluded.

7.1.3 Angiopoietins in the second

trimester (III)

A nested case-control study was performedin the Helsinki University Central Hospital

between January 2001 and December2002. The study population was chosenamong 3240 pregnant women attending aroutine two-phase ultrasound screening for

Down’s syndrome. Serum samples werecollected at 12-16 and/or at 16-20 weeksof pregnancy of 49 women who developed

pre-eclampsia, 16 women who had IUGRand 59 healthy pregnant controls.

7.1.4 Angiopoietins at term

pregnancy (IV)

A descriptive study was carried out to

determine the concentrations of Ang-1,Ang-2 and sTie-2 in maternal serum andurine, amniotic fluid, umbilical blood andneonates’ urine in healthy term pregnancy.Twenty women undergoing electivecesarean delivery were enrolled in theHelsinki University Central Hospital

between March 2008 and March 2010.

7.2 Inclusion and exclusion

criteria

The inclusion criteria were a diagnosis of pre-eclampsia according to AmericanCollege of Obstetricians andGynecologists (ACOG), as described onchapter 5.2.1 (American College ofObstetricians and Gynecologists 2002), adiagnosis of gestational hypertension (I, II,III) or a diagnosis of SGA (I, II) or IUGR

(III). Gestational hypertension was definedas pre-eclampsia according to the criteriaof ACOG without proteinuria, as describedon chapter 5.2.1 (American College ofObstetricians and Gynecologists 2002).SGA (I, II) was defined as birth weight 10th percentile below the national average.IUGR was defined as an infant'sgestational age-adjusted birth weight 2SD (III). Different definitions were used

because of the difference in the practices

of the hospitals involved. Patients for eachstudy were identifying on the basis of the


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10th revision of the InternationalClassification of Diseases (ICD-10)diagnostic code used in the patient recordsof the hospital in question. Studies I and IIincluded controls that did not meet the

criteria of pre-eclampsia, gestationalhypertension or SGA, and were chosen tomatch the duration of gestation atsampling, maternal age and BMI. Healthy,normotensive, non-proteinuric controls,who gave birth to infants with normal birthweight were randomly chosen for studyIII. Study IV included women whounderwent elective cesarean section due to

breech presentation of the fetus, two ormore previous cesarean deliveries, fear ofvaginal delivery or a history of shoulderdystocia. A detailed description of theenrolled subjects is presented in Table 9.

Exclusion criteria for all studies weremultiple pregnancies and/or major fetalanomalies. In studies III and IV womenwith any history of hypertension, renaldisease, pre-existing or gestationaldiabetes and/or ethnicity other thanCaucasian and in study IV cigarettesmoking were also excluded.

Table 9. Description of women enrolled and proteins studied.Study groups (No of women)

Weeks Controls

Pre-eclampsia

GH

IUGR

or

SGA

Proteins

studied

Sample type Study

NoAll

Early-Onseta

Severea

8-13 427 158 29 68 41 81

hCGhCG-h

PAPP-AhCG

Maternal serum I

12-15 50 41 ND 14 0 11Ang-1Ang-2

sTie-2

Maternal serum III

14-17 83 55 12 23 21 30

hCGhCG-hPlGF

sVEGFR-1

Maternal serum II

16-20 36 31 ND 15 0 9Ang-1Ang-2sTie-2

Maternal serum III

38-41 20 0 0 0 0 0Ang-1Ang-2

sTie-2

Maternal serumMaternal urineAmniotic fluid

Umbilical serum Neonates’ urine

IV

a Some women may be included in both groups.GH, gestational hypertension; IUGR, intrauterine growth restriction; SGA, small-for-gestational age; ND, not determined.


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7.3 Samples

The blood samples used in the predictivestudies (I, II, III) were collected duringroutine Down’s syndrome screening visitsat 8-13 weeks (I), 12-16 weeks (III), 14-17weeks (II) or 16-20 weeks (III) of

pregnancy. Samples were allowed to clotat room temperature, serum was separated

by centrifugation and stored at -20 °C (I,II) or at -80 °C (III) until analysisaccording to the manufacturers’instructions. hCG in serum is stable whenfrozen (Lempiäinen et al 2011).

Maternal blood samples used in thedescriptive study (IV) were collected

before and after elective cesarean delivery.Antepartum blood samples, as well asmaternal urine via a urinary catheter, were

collected before intravenous infusions andepidural anesthesia were administered inthe operation theater. Postpartum bloodsamples were collected on the first

postoperative day. Amniotic fluid wascollected immediately after rupture of themembranes or by puncture through intactmembranes. The umbilical venous bloodsample was collected following delivery ofthe placenta (Figure 8). All blood sampleswere handled as described in the previouschapter, and all samples were storedimmediately at -20 °C.

Figure 8. Sampling procedure for study IV. Ang-1, Ang-2 and sTie-2 concentrations weremeasured in all samples.

7.4 Assays

Commercial assays were used for analysesof Ang-1, Ang-2, sTie-2, PlGF, sVEGFR-1, hCG and hCG according to themanufacturers’ instructions (Table 10).

hCG-h was analyzed by an in-houseimmunofluorometric assay described in

Figure 9. Serum samples were diluted 100-fold prior to assay of hCG-h to eliminateinterference by complement. The hCG-hassay also recognizes hyperglycosylatedfree hCG but its response is only 25 % ofthat of hCG-h (Stenman et al 2011).Because the mean proportion hCG in thesamples is only 1 % of that of hCG and its

effect on the concentration of hCG-h isonly about 0.25 %.


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Table 10. Description of assays.

Protein

Intra-

assay

CV

(%)

Inter-

assay CV

(%)

Detection

limitAssay

Sample

dilution

Study

no

Ang-1


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7.5 Statistical methods

Table 11. Statistical methods.Analysis Study No.

Analysis of distribution Kolmogorov-Smirnov’s test I, IIICorrections of multiple analysis

Bonferroni's adjustment IVDunnett’s test I

Comparisons of data between groups

Dunnett’s test I

2 test I, IIIMann-Whitney's U test I, IIOne-way ANOVA I, IIIStudent’s t test III

Comparisons of data within groups

Paired -samples Student’s t test IIIWilcoxon’s signed-rank test IV

Correlations between studied protein concentrations and clinical characteristics

Multivariate linear regression analysis I, IISpearman’s rank correlation IVUnivariate linear regression analysis III

Diagnostic test

2 test IIILogistic regression I, II

ROC analysis I, IIPreanalytic transformation of dataLogarithmic transformation I, IIICalculation of multiples of median (MoM) I, II

Power calculations I, II, III

Statistical analyses were performed usingSPSS versions 14.0 (III), 17.0 (IV) and18.0 (I, II). The comparisons between thegroups were performed using Student’s ttest (III), the 2 test (I, III), one-way

ANOVA (I, III), Mann-Whitney's U test (I,II) and Dunnett’s test (I). The 2 test (III),logistic regression (I, II) and receiveroperating characteristics (ROC) curveanalysis (I, II) were used as diagnostictests. Kolmogorov-Smirnov’s test was toanalyze the distribution (I, III).Prenalytical transformations were

performed using logarithmictransformation (I, III) or calculation ofgestational age-adjusted multiples of

median (MoM) values (I, II). Correlations between the protein concentrations and

clinical characteristics were studied byunivariate (III) or multivariate linearregression analysis (I, II) and Spearman’srank correlation (IV). A two-tailed P-valueof


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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 (%hCG-h) 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). Ascompared 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). Nodifference in %hCG-h was observed

between women who gave birth to SGAinfants and controls (Figure 10 and Table12).

8.1.2 hCG

Concentrations of hCG did not differ

among women who developed pre-eclampsia, gestational hypertension, SGAand controls (Table 12).

Figure 10. Proportion ofhCG-h to hCG (%hCG-h)in women who developed

pre-eclampsia (n=158),gestational hypertension(GH) (n=41) or small-for-gestational age (SGA)(n=81) infants and early-onset pre-eclampsia(n=29) as compared tocontrols (n=427). Box plotshows 10th, 25th, 50th, 75th

and 90th percentiles (ElinaKeikkala 2013).


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Table 12. Changes in the concentrations of markers as compared to controls in 1st and 2nd

trimester ( elevated; decreased; - no change).1

st trimester 2

nd trimester

PEEO

PE

SE

PEGH

SGA /

IUGRPE

EO

PE

SE

PEGH

SGA /

IUGR

Ang-1 - - - - - -Ang-2 - - - sTie-2 - - - - - -%hCG-h - - - - - -hCG - - - - -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 lowerin women who developed pre-eclampsiathan in controls (median MoM, 95% CI;0.95, 0.91 – 0.99 vs. 1.00, 0.99 – 1.01; P =

0.001). There were also differences in thegroup early-onset pre-eclampsia (0.95,0.86 – 0.98; P = 0.001) and the groupsevere pre-eclampsia (0.97, 0.93 – 0.98; P


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8.1.4 Prediction of early-onset

pre-eclampsia

For prediction of early-onset pre-eclampsia, 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) givingsensitivities of 56% and 33% at 90%specificity. The combination of %hCG-hand PAPP-A gave an AUC value of 0.835( P < 0.001) with a sensitivity of 48% at90% specificity. Maternal risk factors werecombined with %hCG-h and PAPP-A bylogistic regression to determine AUCvalues for prediction of pre-eclampsia. TheAUC value for the combination of %hCG-h, 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 pre-

eclampsia

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 PAPP-A giving sensitivities of 31% and 29% at90% specificity. The combination of thesegave 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 asensitivity 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 ofElsevier) or B) severe pre-eclampsia.

A B


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8.2 Second trimester hCG-h,

PlGF and sVEGFR-1 (II)

8.2.1 %hCG-h

At 14-17 weeks of gestation, %hCG-h didnot differ between women who developed

pre-eclampsia and controls ( P = 0.3)(Table 12).

8.2.2 PlGF

The concentrations of PlGF weresignificantly lower in women whodeveloped pre-eclampsia (median MoM,

95% CI; 0.62, 0.53 – 0.79; P < 0.001) orgestational hypertension (0.65, 0.44 –0.90; P = 0.001) as compared to controls(1.00, 0.93 – 1.06). The difference waseven 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).

Figure 13. Concentrationsof PlGF in women whodeveloped pre-eclampsia(n=55), gestationalhypertension (GH)(n=21), small-for-gestational age (SGA)infants (n=30) or early-onset pre-eclampsia

(n=12) as compared tocontrols (n=83). Box plotshows 10th, 25th, 50th, 75th

and 90th percentiles.

8.2.3 sVEGFR-1

There were no differences in theconcentrations of sVEGFR-1 betweenwomen who developed pre-eclampsia,

gestational hypertension or SGA ascompared to controls (Table 12).


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8.2.4 Prediction of pre-eclampsia

By ROC curve analysis the AUC valuesfor PlGF were 0.746 ( P < 0.001) for

prediction of pre-eclampsia, 0.882 ( P


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CI, 0.566 – 0.818; P = 0.007) for Ang-2for prediction of pre-eclampsia with asensitivity of 20% at 90% specificity. For

prediction of severe pre-eclampsia, theAUC value for Ang-2 was 0.717 (0

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