Intrauterine growth restriction in first half of pregnancy

F A C U L T Y O F H E A L T H S C I E N C E S

U N I V E R S I T Y O F C O P E N H A G E N

Ph.D thesisNina Gros Pedersen

Intrauterine growth restrictionin first half of pregnancy

Academic advisor: Ann Tabor, professorKaren R Wøjdemann, Ph.D

Intrauterine growth restriction in first half of pregnancy

1

ContentsPreface and aims of the Ph.D thesis.........................................................................................3

List of abbreviations ................................................................................................................... 4

Introduction...................................................................................................................................5

Background ....................................................................................................................................5

Assessing Intra Uterine Growth Restriction .....................................................................5

Fetal growth............................................................................................................................5

Diagnosis of Intra Uterine Growth Restriction.................................................................. 6

Definitions of Intra Uterine Growth Restriction ................................................................7

Customized fetal growth charts ...........................................................................................7

Determining gestational age................................................................................................ 8

Longitudinal measurements of growth .............................................................................. 8

Conditional centiles.............................................................................................................. 9

Consequences of Intra Uterine Growth Restriction....................................................... 9

Mortality ................................................................................................................................ 9

Short term morbidity........................................................................................................... 10

Long term morbidity ........................................................................................................... 10

Prematurity and growth restriction .................................................................................... 11

Growth restriction and adult disease.................................................................................. 11

Causes of Intra Uterine Growth Restriction.................................................................... 12

Trophoblast invasion ........................................................................................................... 13

Doppler ultrasound measurements .................................................................................. 15

Uterine artery Doppler ultrasound .................................................................................... 16

Unselected population ........................................................................................................ 17

High risk population............................................................................................................ 18

Severe preeclampsia / Intra Uterine Growth Restriction ................................................ 19

Stillbirth ................................................................................................................................ 19

Interpretation ....................................................................................................................... 19

Umbilical artery Doppler ultrasound .............................................................................. 20

High risk population............................................................................................................ 21

Low risk population.............................................................................................................22

Placental growth regulating hormones............................................................................22

Growth Hormone gene........................................................................................................23


2

Human Placental Lactogen.................................................................................................23

Placental Growth Hormone ............................................................................................... 24

Insulin Growth Factor I and II........................................................................................... 26

Insulin Growth Factor Binding Proteins............................................................................27

Interventions for Intra Uterine Growth Restriction .................................................... 28

Oxygen therapy, bed rest, exercise and social support ................................................... 28

Calcium, fishoil, antioxidant supplements and antiplatelet agents .............................. 28

Food supplementation and infection................................................................................ 29

Own studies: ............................................................................................................................... 30

Papers I and II .......................................................................................................................... 30

Paper III .....................................................................................................................................35

Paper IV .................................................................................................................................... 38

Conclusions and clinical implications .................................................................................... 41

Fetal growth in first half of the pregnancy and the risk of adverse outcome ......... 41

Conditional and Date centiles: Fetuses with growth rates below 2.5th

centile ............. 42

Conditional and Date centiles: Association to low birth weight and perinatal

mortality .............................................................................................................................. 42

Growth hormones and growth rate in first half of pregnancy ....................................43

Fetal growth in first half of pregnancy as a predictor of adverse outcome ............ 44

Future research .......................................................................................................................... 45

Summary ...................................................................................................................................... 47

Dansk resume ............................................................................................................................. 50

Reference List ..............................................................................................................................53

Appendix...........................................................................................................................................


3

Preface and aims of the Ph.D thesis

The present Ph.D thesis is based on work carried out during my employment as a Ph.D

student at the Department of Fetal Medicine and Ultrasound, Rigshospitalet, Denmark in

the period 2005-10. The work was supervised by Ann Tabor, Dr Med, professor,

Department of Fetal Medicine and Ultrasound, Rigshospitalet, Denmark and Karen R

Wøjdemann, Ph.D, Department of Obstetrics and Gynecology, Roskilde Sygehus,

Denmark.

The Ph.D thesis is based on the following original papers

I. Fetal growth between the first and second trimester and the risk of adverse

pregnancy outcome. NG Pedersen, KR Wøjdemann, T Scheike and A. Tabor.

Ultrasound Obstet Gynecol 2008;32:147-154

II. Early fetal size and growth as predictors of adverse outcome. NG Pedersen, F

Figueras, KR Wøjdemann, A Tabor and J Gardosi. Obstet Gynecol 2008;112:765-771

III. Maternal serum levels of placental Growth Hormone in early pregnancy, but not

of human Placental Lactogen, and Insulin Growth Factor-I are positively associated

with fetal growth in first trimester of human pregnancy. NG Pedersen, A Juul, M

Christiansen, KR Wøjdemann and A Tabor, submitted

IV. First trimester growth restriction and uterine arteries blood flow as predictors of

adverse pregnancy outcome. NG Pedersen, L Sperling, KR Wøjdemann, S Olesen

Larsen and A Tabor, in manuscript

The manuscripts are referred to in roman numerals (I-IV) in the text

The aims of the Ph.D thesis were:

1. To evaluate the association between fetal growth and size in first half of pregnancy

and subsequent adverse outcome

2. To analyze the association between growth factors in maternal blood and fetal

growth rate in first half of pregnancy

3. To investigate if fetuses with first trimester growth restriction have poorer

perfusion of the placenta, measured by ultrasound Doppler of the uterine and

umbilical arteries compared to a control group, and to investigate if first trimester

growth restriction in combination with poor flow in the uterine and umbilical

arteries can be used to predict poor outcome.


4

List of abbreviations

A. Arteria

Aa. Arteriae

ADAM 12 A disintegrin and metalloprotease 12

AGA Appropriate for gestational age

BPD Biparietal diameter

CRL Crown rump length

CTG Cardiotocography

EFW Estimated fetal weight

GH Growth hormone

hPL Human placental lactogen

IGF Insulin growth factor

IGFBP Insulin growth factor binding protein

IUGR Intra uterine growth restriction

PGH Placental growth hormone

PI Pulsatility index

PAPP-A Pregnancy associated plasma protein - A

RI Resistance index

SD Standard deviation

SGA Small for gestational age


5

Introduction

Fetal growth is of key importance in many aspect of perinatology. Impaired growth is

associated with increased neonatal morbidity and mortality, and much of routine prenatal

care focuses on detecting fetuses at increased risk of growth restriction and targeting

intensive monitoring and interventions.

In this Ph.D thesis the focus is on fetal growth in early pregnancy. We wished to elucidate

if there was an association between impaired growth in first half of pregnancy and

subsequent poor outcome. Furthermore we wished to analyze the associations between

fetal growth in first half of pregnancy and known growth factors, and finally to investigate

if the results from the first two studies could be used to improve the prediction of poor

outcome prospectively.

In the background section of the Ph.D thesis present knowledge on: Assessment of

intrauterine growth restriction (IUGR); Consequences of IUGR; Causes of IUGR; Doppler

ultrasound measurements; Placental growth regulating hormones and Interventions for

IUGR are presented. These are selected topics that are of relevance to the design and

results of our studies. The background section is followed by a presentation and discussion

of our own results.

BackgroundAssessing Intra Uterine Growth Restriction

Fetal growth

The regulation of fetal growth differs from postnatal growth regulation. Postnatal growth

is largely determined by the genetic make-up of the individual, while nutrition plays a

smaller role. Fetal growth is largely determined by the availability of nutrients to the fetus,

while it is generally thought that the genome plays a limited role1. The importance of the

maternal phenotype has been shown in cross-breeding experiments and embryo transplant

experiments in animals2 3

. In humans, in a study of pregnancies involving ovum donations,

it was found that the only factors contributing to birth weight were gestational age and the

recipient mother´s weight. The weight of the donor mother was not related to birth weight

4. These studies thus indicate that fetal growth is constrained by nutrition, and the uterine

environment is a major determinant of fetal growth.

In half-sibling studies, the influence of the mother on birth weight has been demonstrated.

There is a stronger correlation between the birth weights of half-siblings, who share the

same mother than half-siblings who share only a father5. It has been estimated that 62%


6

of the variation of human birth weight results from the intrauterine environment,

compared with 20% and 18% from maternal and paternal genes6.

Normal fetal growth involves an increase in cell number during first and second trimester

followed by an increase in cell size, which becomes dominant in third trimester7. Thus it

is especially in late gestation that the ability of the uteroplacental unit to deliver oxygen

and nutrients becomes important for fetal growth.

Diagnosis of Intra Uterine Growth Restriction

Estimation of fetal growth during pregnancy can be made in many different ways. An

estimation of fetal weight is helpful in predicting fetal survival and making management

decisions in the low birth weight group, and in managing the delivery of the large baby,

where complications may occur8. The oldest of the clinical methods used to diagnose

IUGR is abdominal palpation, using the Leopold manoeuvres’9. The diagnostic

performance of abdominal palpation is of limited value; the sensitivities reported are

between 30% and 50%10 11

. Today the most widely used screening tool for IUGR is

symphysis – fundal height tape measurements, which seems to be somewhat better at

detecting IUGR than abdominal palpation; sensitivities range from 56% to 86%12

.

When a more accurate estimation of fetal weight is necessary ultrasonically estimated fetal

weight (EFW) is used. The parameters most often used to estimate fetal weights

ultrasonically are: biparietal diameters (BPD), head circumference, mean abdominal

diameter, abdominal circumference and femur. The measurements have been combined in

several regression equations or volumetric formulae to evaluate which combination most

accurately estimates fetal weight and predicts fetal compromise. According to a Cochrane

review from 20058

where EFW was compared with birth weight, the formula by Hadlock et

al13 14

gave approximately a mean deviation between EFW and birth weight of 10%.

In all growth retarded fetuses the abdominal circumference is the first biometric

measurement to change and it is also the most sensitive single measurement for predicting

IUGR. An abdominal measurement within the normal range almost reliably excludes IUGR

15. Another early radiographic sign of IUGR is decreased amniotic fluid volume; about 85%

of IUGR infants have oligohydramnios16

EFW, abdominal circumference and amniotic fluid levels are useful for the detection of

cases with IUGR, but less useful for the management of IUGR and have to be used in

association with other techniques of antenatal surveillance such as CTG (Cardiotocogrpah),

biophysical profile and Doppler ultrasound to make decisions about for instance the timing

of delivery17

.


7

Definitions of Intra Uterine Growth Restriction

There are numerous definitions for impaired growth of the baby in pregnancy. Impaired

fetal growth is a disturbance of normal growth resulting in a baby that does not grow to its

potential. It can be difficult accurately to differentiate between the constitutionally small

baby and one with impaired growth, when compared with its potential. A baby may be of

normal birth weight but still significantly lighter than its genetic growth potential18

. SGA

(Small for Gestational Age) defines a child that hasn’t reached a specific weight at a given

gestational age, and IUGR defines a child that hasn’t reached its genetic growth potential19

.

However, it is not all literature that distinguishes between SGA and IUGR, and the

classifications are not consistently used according to the classifications above20-22

.

The current WHO criterion for low birth weight is a weight less than 2,500 g or below the

10th

percentile for gestational age23

and the classification “very low birth weight” is

commonly used for weights below the 3rd

percentile for gestational age. McIntire DD et al

found that for term infants morbidity and mortality were significantly higher among

infants, who were at or below the 3rd

percentile of weight for their gestational age24

, but

other groups define alternative thresholds as the best diagnostic cut-off for increased

neonatal mortality and morbidity25;26

, and at present there is no general agreement on the

most appropriate threshold .

In Denmark IUGR during pregnancy is most often classified as EFW that is 22% or more

smaller than the ultrasonically estimated mean for gestational age27

. This classification is

equal to an EFW below the 2.3 percentile for gestational age, and to the classification made

by Marsal et al in Sweden (IUGR= EFW that is below ultrasonically estimated mean weight

for gestational age – 2 SD)28

.

Historically, IUGR has been categorized as symmetric or asymmetric. Symmetric IUGR

refers to fetuses with equally poor growth velocity of the head, the abdomen and the long

bones. Asymmetric IUGR refers to infants whose head and long bones are spared compared

with their abdomen and viscera29

. The ponderal index, which compares birth weight with

length (birth weight (g) / birth length (cm)3)×100, is sometimes used as an assesment of

the thinness or obesity of the neonate30

. Infants with symmetric growth restriction have a

normal ponderal index, whereas those with asymmetric growth restriction have a reduced

ponderal index.

Customized fetal growth charts

Customized fetal growth charts are growth charts that are individually adjusted /

“customized” for physiological factors known to affect birth weight and growth: fetal sex,

gestational age, maternal height, maternal weight in early pregnancy, ethnicity and parity.


8

The customized growth charts have in several studies demonstrated that they are more

accurate in predicting individual birth weight31;32

and are more closely associated with

stillbirths, neonatal deaths or low Apgar scores (<4) than the unadjusted population

percentile33

, probably due to improved identification of IUGR.

Determining gestational age

Since the majority of ultrasound measurements used to define IUGR depends on

gestational age a precise estimate of gestational age is a prerequisite when screening and

diagnosing IUGR. Traditionally gestational age has been determined from the first day of

the last menstrual period and then adding 280-283 days to predict the day of delivery34

.

The conditions for Naegels’s rule are: that the pregnant woman remembers the first day in

her last period; her cycle must have been regular at least for three months with an interval

of 28±4 days; she must have had at least 3 spontaneous periods before the last menstrual

period; and no bleeding in first trimester34 35

. The conditions are only met in 30-80% of the

pregnancies36-39

, and many women have delayed ovulation in some cycles, which causes a

tendency to overestimate the length of gestation39-42

.

Ultrasound dating has in several studies been shown to be more accurate than menstrual

dating43;43-48

. Ultrasound dating of pregnancy is usually based on CRL (Crown Rump

Length) or BPD49;50

. In most studies the use of CRL in first trimester has been shown to

be more accurate than BPD in second trimester for date setting36;38;51

, and in Demark where

about 90% of the pregnant women are scanned in first trimester52

there is a consensus to

use the gestational age determined by CRL in first trimester if possible53

.

Longitudinal measurements of growth

In clinical practice it is commonplace for fetal size to be estimated more than once during

pregnancy, so routine data are often longitudinal. However, when diagnosing potential

IUGR conventionally cross sectional standards for size are used, even though it seems more

logical to use longitudinal standards for growth, when having two or more measurements.

Cross sectional standards for size give information only on size, whereas longitudinal data

may be used to produce rates of growth (changes over time in size). Since fetal growth is a

dynamic process longitudinal standards for growth could improve the prediction and the

differentiation between SGA and IUGR. It would allow early identification of the baby who

begins pregnancy on a certain size percentile within the normal range and then

experiences impaired growth. The few studies that have used longitudinal measurements of

growth, and then related them to adverse outcome show convincing results that low rates

of growth can be used as a sign for fetal demise54-56

.


9

Conditional centiles

One of the reasons that longitudinal standards for growth aren’t commonly used in the

clinic is most likely that correct statistical analysis of longitudinal fetal data is somewhat

complicated. Below is a short description of conditional centiles, which is an

acknowledged statistical method, used to estimate growth longitudinally. Conditional

centiles uses individual predictions for the expected normal range of a measurement

(conditional centiles) at the second time of measurement on the basis of the first

measurement. Each second-time measurement is thus assigned a centile, which is

conditional to the previous measurement in the same child. Conditional centiles takes into

account the normal phenomenon of regression to the mean; underweight children tend to

catch up towards median weight, while relatively heavy infants catch down57

.

Consequences of Intra Uterine Growth Restriction

Mortality

The perinatal mortality rate is higher at each gestational age in SGA-children compared

with normal weight children.

Figure 1 Risk of neonatal death at the 10th percentile relative to the 45th to 55th percentiles across gestational agefor single live births, 1996-2000.

A study from 2006 with more than 18.000.000 singleton births included showed that at 26

weeks of gestation, infants born with birth weight at the 10th

percentile or below

experienced a 3-fold risk of dying within the first 28 days of life relative to children born

with birth weights between the 45th

-55th

percentile, at 40 weeks the ratio was 1.1358

. Figure

1 with the use of data from reference58

. Other studies that use lower threshold for the


10

definition of IUGR have reported 5-10 fold increase in the rate of death among both term

and preterm IUGR children24;59-61

.

Short term morbidity

SGA-children are at delivery compromised due to longterm placental insufficiency and

chronich intrauterine hypoxia, this renders them more vulnerable after birth. Some of the

most common problems in neonatal period are described below.

Infants born at term with birth weight below 3rd

percentile for gestational age have twice

the incidence of low Apgar scores and umbilical pH 724

. This leads to higher rates of

hypoxic brain injuries and seizures; one study with more than 200,000 infants included

demonstrated that birth weight below the 10th

percentile for gestational age conferred a 5-

fold increased risk of perinatal strokes for infants born at term62

.

Because of decreased liver glycogen and fat stores growth restricted infants are more

vulnerable to hypothermia and hypoglycaemia63

, and due to a relatively hypoxic

intrauterine environment growth restricted infant have higher risk of polycythemia64

.

Although polycythemic infants often remain asymptomatic polycythemia can in some cases

be associated with hyperbilirubinemia, hypoglycemia and necrotizing enterocolitis65

.

The incidence of sepsis is increased in growth restricted children24;66

possibly caused by a

compromised immune system. Growth restriction has previously been reported to

correlate with neutropenia in infants of hypertensive mothers67

. Furthermore growth

restricted infants are prone to coagulation abnormalities, which include a reduction in

vitamin K-dependent coagulation factors68

and decreased platelet count69

. It has been

proposed that the coagulation abnormalities are caused by antenatal hypoxia affecting the

liver and hence vitamin K-dependent coagulation70

.

An intolerance of protein intake in the first postnatal weeks has been reported when

infants that are SGA are compared with infants that are appropriate for gestational age71

.

It has been suggested that intrauterine growth restriction alters a number of metabolic and

physiologic variables in the fetus that probably affect the hepatocyte function and protein

tolerance72

.

Long term morbidity

Several studies have found an association between low birth weight for gestational age and

an increased risk of cerebral palsy73-75

. Data from 10 European countries totalling 45,703

singleton children born between 1976 and 1990 found that babies born at 32-42 weeks of

gestation with a birth weight below the 10th

centile were four to ten times more likely to

have cerebral palsy than children with birth weight between the 25th

and 75th

centile76

.


11

Further more subtle neurologic findings, including alternation of tone during infancy,

increased activity and clumsiness in later childhood have been identified frequently in

children who had been growth restricted77;78

.

The specific risk attributable to intrauterine growth restriction on neurodevelopmental

outcome is difficult to isolate, confounding factors such as prematurity, maternal disease

and socioeconomic factors may often obscure the picture and alter the interpretation of the

specific risk of intrauterine growth restriction. Although some studies report no effect of

SGA status on cognitive outcome79;80

most studies find that on average the overall

intellectual performance of SGA children is lower but still within the reference range

compared to the performance of children with a normal birth weight,81;82

.

90% of babies who are born SGA for any reason will catch up in height with their peers by 2

years of age, but about 10% will continue to be two standard deviations below the average

height for their age, and these children are also leaner than their peers and have a lower

food intake compared with healthy controls83;84

.

Prematurity and growth restriction

Growth restriction is associated with an increased risk of spontaneous and iatrogenic

preterm deliveries59;85

. It was previously a commonly held clinical assumption that

growth-restricted fetuses are stressed by the unfavourable environment in utero, and as a

result it was claimed that these infants have accelerated maturation and are less likely to

suffer from prematurity complications when compared with appropriately grown infants86

.

However, more recent studies show an increased risk of complications related to

prematurity in SGA infants compared with Appropriate for Gestational Age (AGA) infants

including: respiratory distress syndrome70;87

, chronic lung disease22;60;61;88

, retinopathy of

prematurity89

and necrotizing enterocolitis70;90;91

, so it seems that when born preterm the

growth restricted child is vulnerable not only to the complications of growth restriction but

also to those of prematurity.

Growth restriction and adult disease

Birth weight is inversely related to the mortality rate of cardiovascular disease92 93

, and it

has been shown that infants born IUGR have an increased incidence of type 2 diabetes94

,

increased insulin resistance95

, cardiovascular disease96;97

and a set of metabolic

abnormalities known as sydrome X98

in adulthood. These finding has led to the hypothesis

that impaired fetal nutrition has long-term adverse impact on adult health by permanently

altering or programming the physiology and metabolism of the developing organism92 93

.

It has been hypothesized that fetal malnutrition and changes in the Insulin Growth Factor


(IGF) axis may permanently imprint the organism for further pathology. In support of the

theory, there is some evidence from animal studies that long-term programming of the IGF

axis can occur as a result of fetal events99

. Furthermore IGFs and Insulin Growth Factor

Binding Proteins (IGFBP) are nutritionally regulated in the fetus, and there are

abnormalities in the Growth Hormone (GH) - IGF axis both in growth retarded

fetuses/neonates and in many of the adult diseases that are associated with low birth

weight, including cardiovascular disease, diabetes and osteoporosis100

.

Causes of Intra Uterine Growth Restriction

Several conditions are associated with restricted growth, and growth restricted infants

represent a highly heterogeneous group in terms of etiology and severity. Growth

restriction may be the only sign of the disease, or it may come with severe abnormalities in

different organ systems. The causes of growth restriction can be divided into: extrinsic,

maternal, placental and fetal, see Figure 2. In this thesis I will concentrate on the placental

causes of growth restriction, with special focus on trophoblast invasion and the secretion of

growth regulating hormones.

Figure 2.

F

C

M

I

M

C

O

MATERNAL FACTORS

Hypertension

Preeclampsia

Chronic diseases

Inherited thrombophilia

FETAL GROWTH RES

Most common conditions and causes associated

ETAL FACTORS

hromosomal trisomy

endelian single gene disorders

nborn errors of metabolism

ultiple pregnancy

ongenital structural abnormalities

ther syndromes

PLACENTAL FACTORS

Plancental mosaicism

Abnormal placentation

Uterine abnormality

Placental infarcts

Placenta praevia

12

TRICTION

with fetal growth restriction

EXTRINSIC FATCTORS

Smoking

Alcohol

Drug abuse

Infections


Trophoblast invasion

During human pregnancy the fetus and placenta form a functional unit, with placenta

playing a key role in fetal growth. The relationship between the development of the

placenta and fetus is illustrated by the correlation between placental and fetal weight

throughout pregnancy101

. SGA neonates have significantly reduced placental weights

compared with appropriately grown neonates of the same birth weight102

. The main

structural and functional units of the human placenta are the chorionic villi. The structure

is explained in Figure 3.

Figure 3 Structure of first trimester placental villi: Villous mesenchycytotrophoblasts, which in turn are covered by a multinucleated synfeto-placental blood vessels can be seen.

Within the developed placenta there is a cavity (intervi

network of chorionic villi, which float in circulating ma

facilitate exchanges between the maternal and fetal cir

surface is 11 m2 103

. The main cells inside the chorionic

cytotrophoblasts lies the mesenchymal core, which con

various cells including fibroblasts104

. When the blastoc

the cytotrophoblasts proliferate and differentiate into v

The villous phenotype differentiates by aggregation to

multinucleated epithelium that acts as nutrient and ga

endocrine unit of the placenta106

. The extravillous phe

ability to aggressively invade maternal tissue107;108

. Ple

adapted from109

.

Mesenchymal core

Cytotropnoblasts

Syncytiotrophoblasts

Intervillous space

13

mal core surrounded by a continuous layer ofcytiotrophoblast. In the mesenchymal core

llous space) that possesses a vast

ternal blood. A large surface to

culation is created; at term the

villi are cytotrophoblasts. Below the

tains placental capillaries and

yt invades the endometrial tissue

illous or extravillous phenotypes105

.

syncytiotrophoblast, a

s exchange barrier104

, and is the

notype differentiate and acquire the

ase see Figure 4 for details. Figure

Feto-placental blood vessel


14

Figure 4 Anatomy of the maternal-fetal interface (A) Placental chorionic villi stem from the chorionic plate andlie within the intervillous space. The point of attachment between anchoring villi and the underlying tissue isreferred to as the basal plate (box B). (B) enlargement of the area in box B. Undifferentiated cytotrophoblastprogenitors in the anchoring villi give rise to invasive cytotrophoblasts that invade the uterine interstitium andthe maternal endothelium.

Some of the extravillous cells embed in the maternal stroma either individually or in small

clusters with decidual, myometrial and immune cells. A group of the extravillous cells

express surface antigens that resemble maternal endothelial cells, which enable them to

migrate up the spiral arteries110

. During the endovascular invasion the cytotrophoblasts

plug the spiral arteries, which temporarily reduces maternal flow and oxygen supply110

.

Subsequently the fetal cells replace the maternal endothelium and portions of the smooth

muscle wall109;111

, and the spiral arteries are transformed into larger low-resistance vessels

capable of transporting the increased maternal blood flow to the placenta at low

pressure112;113

. Normally this process encompasses the proportion of the uterine arterioles

that span the decidua and the inner third of the myometrium114

. The decidual part of the

spiral arteries is invaded between week 8 and 12 of gestation115

, the myometrium around

mid-pregnancy116

.

Succesfull trophoblast invasion is required to sustain fetal growth, and absence of

trophoblast induced changes in decidual or myometrial segments of the spiral arteries is a

feature of some pregnancies complicated by growth restriction117

. In women with

preeclampsia, a group susceptible to IUGR, this deep invasion of the myometrial vessels

together with the associated vascular change is seriously impaired118

.

How this deficiency of trophoblast invasion translates into preeclampsia with endothelial

dysfunction, hypertension and possible multiorgan deficiency of the mother is not

understood. It is hypothesized that the response may result from defective trophoblast


differentiation, impaired decidualization, extreme hyperinflammatory response or still

undefined maternal tissue processes119

.

Increased blood flow is essential to meet metabolic demand from the growing uterus as

well as the placenta and fetus. Total maternal blood volume120

and cardiac output increase

by approximately 40% during pregnancy103

, and the total uteroplacental blood flow

represents 25% of cardiac output112

. Uteroplacental blood flow has been shown to be

reduced by 50% in women with preeclampsia121

, and often there is a decrease in the

number and surface of terminal villi in IUGR, representing a malfunction of vascularization

in these pregnancies122

.

Doppler ultrasound measurements

Doppler measurements use the Doppler effect to assess the velocity and whether structures

are moving towards or away from the probe. By calculating the frequency shift of a

particular volume its speed and direction can be determined and visualized. The result

when examining vessels is a specification of the direction and velocity of the bloodstream

depicted as a velocity waveform. The curve reflects the speed of the erythrocytes and the

color-intensity reflects the number of erythrocytes in the vessel at a given velocity. The

shape of the velocity waveform depends on: heart action, blood velocity, vessel wall

compliance and both proximal and distal resistance to flow.

Figure 5 Doppler indices. (S)

Max

imu

mfr

eq

ue

ncy

shif

tcm

/se

c

Mean

pea

S

S/D ratio

D

k systolic frequency shift (D) end diastolic frequency sh

Time

RI = (S-D)/S

PI = (S-D)/mean

15

ift.


The vascular resistance peripheral to the site of measurement mainly affects the diastolic

part of the velocity waveform. Thus the dominant influence on the waveforms recorded

from a.umbilicalis (arteria umbilicalis) and aa.uterinae (arteriae uterinae) is exerted by

vascular resistance in the placenta. As fetal hypoxia in many instances is caused by

placental pathology analysing the waveforms can serve as a diagnostic test123

. A number of

formulas and indices have been evolved to characterize the waveforms mathematically.

Some make use of only two points on the curve e.g. the systolic to diastolic ratio (S/D ratio)

or the resistance index (RI). The most commonly used measurement is the pulsatility

index (PI) that includes the average velocity over the heart cycle in the calculation, and is

superior to the other two indices for practical purposes124

. For details of the different

indices, please see Figure 5.

The persistence of an early diastolic notch (beyond 24 weeks’ gestation) in the uterine

arteries represents another sign of high impedance to flow125

. In normal pregnancies

impedance to flow decreases in a. umbilicalis and aa. uterinae with advancing gestation126

,

therefore reference curves for flows are adjusted for gestational age.

Uterine artery Doppler ultrasound

In the uterine arteries the Doppler signals are sampled 1 cm cranial to the crossing point

with the external iliac artery. The investigation should be performed on both sides, and

preferably be done in the absence of maternal breathing. Increased resistance in the

uterine arteries can be defined as: 1) flow index (PI or RI) above 95th percentile 2) PI

above a certain cut–off 3) the absence or presence of notch. See Figure 6 for details. The

indices are often calculated as an average of the bilateral measurements. Very lateral

insertion of the placenta may affect the flow; if placental site is taken into consideration it

marginally improves the predictive ability of uterine artery Doppler127

.

Figure 6 (A) Normal flow velocity wavefowaveform from the uterine artery at 24 wediastole there is a notch and in late diasto

Failure of trophoblastic invasion h

insufficiency, and several Doppler

BA

rm from the uterine artery at 24 weeeks of gestation in a pregnancy withle there is decreased flow.

as been found to be associat

studies both in first and seco

A

16

ks of gestation. (B) Flow velocityimpaired placentation. In early

ed with uteroplacental

nd trimester have


17

Estimates for predicting preeclampsia and IUGR in an unselected population

Second trimester

Pos. LR (95% CI) Neg. LR (95% CI) No of studies Pos. LR (95% CI) Neg. LR (95% CI) No of studies

Preeclampsia Cnossen et al Papageorghiou et al*

PI 4.5 (1.7-7.3) 0.6 (0.5-0.82) 7 5.9 (5.3-6.5) 0.6 (0.5-0.6) 12

Bilateral notch 6.5 (4.3-8.7) 0.6 (0.4-0.8) 17

Unilateral notch 4.6 (1.3-7.9) 0.7 (0.5-0.9) 6

PI og notch (bi/unilateral) 7.5 (5.4-10.2) 0.6 (0.5-0.7) 1

IUGR (<10 percentil)

PI 3.4 (1.7-5.1) 0.9 (0.8-0.9) 3 3.7 (3.3-4.0) 0.8 (0.8-0.8) 13

Bilateral notch 2.8 (1.7-3.9) 0.8 (0.8-0.9) 11

Unilateral notch 2.4 (2.0-2.9) 0.9 (0.9-0.9) 2


First trimester

Pos. LR (95% CI) Neg. LR (95% CI) No of studies Pos. LR (95% CI) Neg. LR (95% CI) No of studies

Preeclampsia Cnossen et al Papageorghiou et al*

PI 5.4 (4.1-6.7) 0.8 (0.7-0.8) 3 5.4 (4.2-6.7) 0.5 (0.4-0.6) 2

Bilateral notch 3.0 (2.4-3.3) 0.1 (0.1-0.4) 1


PI* 2.7 (1.9 - 3.8) 0.9 (0.9-1.0) 1 2.2 (1.8-2.8) 0.9 (0.8-0.9) 2

Bilateral notch 1.3 (0.6-2.0) 0.6 (0.3-1.0) 3> 16 weeks gestation

< 16 weeks gestation

* Papageorghiou et al pooled different impedance measurments (S/D ratio, RI and PI) to define abnormal doppler results

demonstrated an association between increased impedance to flow in uterine arteries and

subsequent development of preeclampsia, IUGR and fetal death128-135. Studies investigating

the predictive accuracy of uterine artery Doppler for adverse outcome have revealed

considerably varied results. The varied results may be the consequence of differences in

Doppler technique for sampling and the definition of abnormal flow velocity waveform,

differences in the populations examined, the gestational age at which women were studied,

and different criteria for the diagnosis of preeclampsia and intrauterine IUGR.

Unselected population

The two most recent meta analyses that describe the diagnostic accuracy of uterine artery

Doppler for preeclampsia and IUGR were published in 2004 by Papageorghiou et al.

(20,000 pregnancies included in total)136

, and in 2008 by Cnossen et al. (79,000

pregnancies included in total)137

. The following likelihood ratios are reported in Table 1.

Second trimester

Cnossen et al.137

report that in second trimester the positive likelihood ratio for

preeclampsia in an unselected population is between 4.5 and 7.5, and the positive likehood

ratio for IUGR is between 2.4 and 9.1, depending on which Doppler measurements are

used. Papageorghiou et al.136

have pooled the different measurements for increased

impedance and report a positive likelihood ratio of 5.9 for pre-eclampsia and of 3.7 for

IUGR, which roughly seems to be the average of the different likelihood ratios reported by

Cnossen et al.


18

Estimates for predicting preeclampsia and IUGR in high risk populations

Second trimester

Pos. LR (95% CI) Neg. LR (95% CI) Pos. LR (95% CI) Neg. LR (95% CI)

Preeclampsia Cnossen et al No of studies Chien et al* No of studies

PI 1.8 (0.2-3.4) 0.8 (0.5-1.0) 4 2.8 (2.3-3.4) 0.8 (0.7-0.9) 11

Bilateral notch 3.4 (1.1-5-7) 0.7 (0.5-1.0) 8

Unilateral notch 20.2 (7.5-29.5) 0.2 (0.0-0.6) 1 20.2 (7.3-56.3) 0.2 (0.03-1.0) 1



PI 2.3 (1.0-3.6) 0.6 (0.1-1.0) 2 2.7(2.1-3.4) 0.7 (0.6-0.9) 9

Bilateral notch 3.8 (0.7-7.0) 0.8 (0.5-1.0) 4

Unilateral notch 3.0 (1.7-4.3) 0.5 (0.4-0.9) 2 2.4 (0.6-8.6) 0.9(0.7-1.1) 1

RI og notch (bi/unilateral) 4.3 (0.6-8.1) 0.6 (0.5-0.7) 4> 16 weeks gestation

* Chien et al pooled different impedance measurments (S/D ratio, RI and PI) to define abnormal doppler results

First trimester

In first trimester the positive likelihood ratio for preeclampsia is between 3.0 and 5.4

depending on the measurement used, and the positive likelihood ratio for IUGR is between

1.3 and 2.7136;137

. In conclusion and based on the limited number of studies, it seems that

uterine artery Doppler performed in first trimester has little diagnostic accuracy in

predicting preeclampsia and IUGR in an unselected population, and the diagnostic

accuracy of uterine artery Doppler performed in second trimester is superior to that

performed in first trimester.

High risk population

The diagnostic accuracy of uterine artery Doppler has also been estimated in high risk

populations such as women with a previous history of preeclampsia, IUGR, vascular

complications, poor pregnancy outcome, unfavourable biochemical profile or women with

a known disease such as diabetes mellitus, essential hypertension, systemic lupus

erythematosus etc.

Table 2 depicts the likelihood ratios from two systematic reviews for high risk populations

published by Cnossen et al in 2008 (performed as a subgroup analysis of 79,000 high and

low risk pregnancies included in total)137

and by Chien et al in 2000 (13,000 pregnancies

included in total)138

.

In high-risk populations a positive test result predicts preeclampsia with a positive

likelihood ratio between 1.8 and 21.0, depending on which of the Doppler measurements

are used. IUGR is predicted with a positive likelihood ratio between 2.3 and 2.7137;138

. The

limited sample size of only one study and subsequent uncertainty most likely explain the

very high positive likelihood ratio around 20 for predicting preeclampsia, when either the

Doppler measurements unilateral notch or PI and notch are used.137;138

.


19

Severe preeclampsia / Intra Uterine Growth Restriction

Several studies report that uterine artery Doppler is better at predicting severe rather than

mild disease. Steel et al.139

have reported that the sensitivity of uterine artery Doppler for

preeclampsia was 63%, whereas for gestational hypertension was 29%. Papargheorgiou et

al135

report that the sensitivity for preeclampsia with IUGR was 69%, while for

preeclampsia without IUGR it was 24%, and the sensitivity for IUGR defined by the 5th

centile was 19% whereas it was 16% for the 10th

centile.

The severity of preeclampsia and IUGR can also be estimated by looking at the gestational

age at which delivery is undertaken. Three studies134;135;140

, that all take the gestational age

at delivery into account, report that the overall sensitivity of uterine artery Doppler for

predicting preeclampsia is between 41-45%, whereas the sensitivity for preeclampsia for

those pregnancies, which required delivery before week 35, is between 81-90% .

Stillbirth

Two meta analyses136;138

have evaluated the diagnostic accuracy of uterine artery Doppler in

second trimester for perinatal death. The positive likelihood ratios reported for a: low risk,

unspecified risk and high risk population respectively were 1.8(95%CI 1.2-2.9); 2.4 (95%

CI1.54-3.44) and 4.0(95%CI 2.4-6.6); the negative likelihood ratios were 0.9(95%CI 0.8-1.1);

0.8(95%CI 0.68-0.93) and 0.6(95%CI 0.4-0.9). Both meta analyses were based on four

studies only, which of course entails some uncertainty for the reported likelihood ratios.

A study from 2007 with 30,519 women, who had uterine artery Doppler performed between

week 22 and 24 of pregnancy, showed that abnormal uterine artery Doppler is strongly

associated with a high risk of stillbirth early in pregnancy. Abnormal uterine artery

Doppler was a better predictor of the risk of stillbirth due to placental causes than

unexplained stillbirths. Since stillbirths due to placental dysfunction tend to occur at

earlier gestations the association between abnormal uterine artery Doppler and stillbirth

was strongest for stillbirths occurring at extreme preterm gestations141

.

Interpretation

Preeclampsia, IUGR and perinatal death all have a relatively low prevalence. As a thumb

rule a clinically useful test thus requires a high positive likelihood ratio (>10.0) and a low

negative likelihood ratio (<0.10). Moderate prediction can be achieved with likelihood

ratios of 5-10 and 0.1-0.2 whereas likelihood ratios of 1-5 and 0.2-1 generate only minimal

prediction142

. In light of these categories and the likelihood ratios reported in the studies

described above, it seems that uterine artery Doppler either performed in first or second

trimester has moderate predictive value for preeclampsia and minimal predictive value for


20

IUGR and perinatal death. It is possible that uterine artery Doppler has a better predictive

value in not yet specified high risk groups or in statistic models that in combination with

other risk factors estimate the patient-specific risk for adverse pregnancy outcome.

Umbilical artery Doppler ultrasound

The umbilical arteries direct 33% of the fetal ventricular output to the placenta at 20 weeks

of gestation143

, and the assessment of umbilical bloodflow provides information on blood

perfusion of the fetoplacental unit. The normal Doppler velocity waveforms in a.

umbilicalis have a pattern compatible with the fetoplacental circulation as a low resistance

system, displaying forward flow of blood throughout the cardiac cycle. In compromised

pregnancies an absence of flow during diastole is observed, and in severely compromised

pregnancies a reversal of flow, directed from the placenta to the fetus, can be observed.

A significant difference in resistance exist along the length of the umbilical cord, the closer

to the fetus the higher the resistance, by convention / if possible the Doppler signals should

be sampled from the midportion of the cord. Furthermore in order to avoid artefacts the

Doppler gate should be placed within the walls of the selected vessel (avoiding inclusion of

both arteries or the vein), attention should be paid to aliasing, the Doppler insonation

angle should be as close to 0 as possible, and the fetus should not be breathing or

moving123

. Abnormal Doppler velocimetry in the umbilical artery is usually quantified by

an increased PI and /or absent or even reversed end diastolic velocities. In the

Scandinavian countries a scoring system with four different Blood flow classes is often used

to classify the blood flow144

.

Blood flow classes

0 = normal umbilical artery blood flow velocity waveforms

1 = PI between 2 and 3 SD above the mean

2 = PI > +3 SD and forward flow in diastole

3 = absent end-diastolic flow or reversed flow

Figure 7 displays the changes seen in the umbilical artery waveform in a normal pregnancy

and in pregnancies with fetal growth restriction.


Normal pregnancy

Figure 7 Changes in Umbilical Dopplerrestriction

At 20 weeks gestation the umbil

145. The loss of resistance in the

substantial increase in vascularis

villi, which leads to an increase i

vasculature in the placenta has n

constricting effect146

and nitric

of the vasculature is not yet com

High risk populationA meta analysis of randomized c

artery Doppler waveform analys

significantly decreases perinatal

included, involving nearly 7000

especially if the pregnancy was c

growth, was associated with a re

inductions of labour (OR 0.8, 95

Absent end diastolic velocity

Reduced end diastolic velocity

21

waveforms in healthy pregnancies and in pregnancies with fetal growth

ical blood flow is 35ml/min, at 40 weeks it is 240 ml/min

umbilical artery throughout the pregnancy is the result of a

ation in the placenta and maturation of the tertiary stem

n the cross-sectional area for vascular outflow145

. The

o neural regulation, endothelin and prostanoid have a

oxide a dilating effect147

, but the exact humoral regulation

pletely understood.

ontrolled trials suggests that incorporation of umbilical

is into management protocols for high risk pregnancies

mortality. In a Cochrane review from 1996 11 studies were

women. The use of umbilical artery Doppler ultrasound,

omplicated with hypertension or presumed impaired fetal

duction in perinatal deaths (OR 0.7, 95% CI 0.7-0.9), fewer

% CI 0.7-0.9) and fewer admissions to hospital (OR 0.6,

Reversed end diastolic velocity


22

95% CI 0.4-0.7). In total the use of umbilical artery Doppler ultrasound in high risk

pregnancies was associated with a 29% reduction in overall perinatal mortality148

.

Low risk population

Two meta analyses149;150

both conclude that in unselected or low risk populations umbilical

artery Doppler does not result in increased antenatal, obstetric and neonatal interventions,

and no overall difference was detected for short term clinical outcomes including perinatal

mortality.

Placental growth regulating hormones

During pregnancy the placenta is an important endocrine organ that produces numerous

hormones. The placenta secretes hormones into the circulation of the mother with the

purpose of modifying maternal metabolism in ways that will benefit the fetus, and the

maternal response to the hormones evolves to conserve resources in the interest of

maternal fitness. GH and Prolactin are two ancient, structurally related molecules with

important regulatory function for human reproduction and growth.

Serum GH increases during fasting and hypoglycaemia and among the known effects of GH

are increased lipolysis, increased hepatic output of glucose and increased peripheral

resistance to insulin151;152

. GH mediates its effect either directly or via IGF-I or II that are

both secreted from the liver and regulated by GH153

.

During childhood and adolescence GH is closely linked to longitudinal growth, but during

pregnancy the production of GH is gradually suppressed, and hormones produced in the

placenta replace many of its functions. Prolactin has a much broader spectrum of activities

than GH and these are classified as reproduction, metabolism, osmoregulation,

immunoregulation and behaviour154

.

During pregnancy the placenta secretes a ligand of maternal GH receptors: placental

Growth Hormone (PGH) and a ligand of maternal prolactin receptors: human Placental

Lactogen (hPL). PGH and hPL can both be detected in the maternal circulation at 6 weeks’

gestation and increase with advancing gestation155;156

, peak values are reached at week 34-

37156-158 159

. In the third trimester serum hPL is present in mg/l whereas PGH is found in

g/l. PGH gradually replaces the maternal pituitary GH, which disappears from maternal

serum in the second half of pregnancy. In contrast to pituitary GH PGH is secreted

tonically and is not regulated by GH releasing hormone160 161

. Until recently the general

opinion was that PGH does not cross the placenta into the fetal circulation, and therefore

the effect of PGH on fetal growth is considered to be mediated via its effect on maternal


23

IGF-I161

. However a recent study has demonstrated that PGH can be detected in cord

blood, and it may therefore have a direct effect on fetal growth162

. Both the maternal and

fetal circulation contain hPL163;164

.

Growth Hormone gene

GH, PGH and hPL are members of a superfamily of protein hormones that are important in

the regulation of growth and development. In humans the GH-related genes are clustered

on chromosome 17165-167

. These include GH1 whose expression is restricted to the anterior

pituitary and encodes human GH (hGH) plus four genes expressed in the placental

syncytiotrophoblast (GH2, CSHL1, CSH1, CSH2). GH2 encodes PGH, and CSH1 and CSH2

encode hPL166

, whether the CSHL gene is expressed or if it is a pseudogene is not clarified.

All of the genes consist of five exons and four introns, and they show a high degree of

similarity (91%-99% conserved sequences)166;168

. Each gene encodes for a mature protein of

approximately 200 amino acids preceded by a signal peptide of about 25 amino acids169

.

The hormones have different affinities for GH Receptors and Prolactin receptors: GH binds

to both GH receptors and Prolactin receptors, PGH differs from GH by 13 of its constituent

191 amino acids; this reduces sevenfold its affinity for binding to prolactin receptors, but

has no effect on its affinity for GH receptors170

. hPL has high affinity prolactin receptors

but very weak affinity for GH receptors171;172

.

Human Placental Lactogen

Little is known of hPL’s physiological role in pregnancy. One study found a link between

fetal growth velocities in second half of pregnancy and changes in hPL in maternal serum

173, and it has been hypothesized that hPL along with PGH is released from the

syncytiotrophoblast into the maternal circulation to stimulate IGF-I production174

. It has

been demonstrated that hPL is unable to promote the growth of human fetal fibroblast and

myoblasts in the presence of an IGF-I antibody175

. However, other studies that have

investigated the relationship between PGH, hPL and IGF-I in human pregnancies, find that

regardless of gestational age IGF-I values are correlated with corresponding PGH but not

with hPL values. This suggests that PGH and not hPL is the primary regulator of maternal

IGF-I secretion157;158

.

It has also been hypothesised that hPL along with PGH contributes to the development of

maternal insulin resistance in second half of pregnancy. In second half of pregnancy

maternal utilization of glucose falls, despite increases in insulin production and secretion.

The insulin resistance promotes lipolysis and fasting ketonaemia as well as post-prandial

hyperglycaemia, sparing energy for the fetus. The increase of nutrients to the fetus


24

stimulates a rise in fetal insulin, which promotes fetal growth. The development of insulin

resistance in second half of pregnancy matches sharp increases in the maternal

concentrations of hPL and PGH176

, and both hormones reduce insulin sensitivity in

adipocytes and skeletal muscle cells in in vitro experiments177-179

.

The majority of studies does not show significant changes in hPL concentrations following

glucose administration or insulin-induced hypoglycemia in pregnant women180;181

, and in

vitro findings in term placentae have demonstrated no effect of high or low glucose

concentrations on the production of hPL182

. However, longterm fasting (72-90 hours prior

to therapeutic abortion) has in two studies been shown to significantly increase maternal

hPL concentrations with 30-40%183;184

, so controversies exist upon the interactions between

and hPL, insulin-sensitivity and glucose in pregnancy.

In the literature cases of hPL gene deletion have been described. Among 11 offspring of

pregnancies in which no hPL could be detected in maternal plasma five had birth weights

equal to or less than 2600g185-189

. One of the cases, was a severely growth retarded, but

otherwise normal boy (birth weight of 1.270g at 35 weeks), who developed transient

neonatal hypoglycaemia. The maternal levels of IGF-I were very low and besides the

deletion of the hPL-A and hPL-B genes a deletion of the GH-V gene was observed185

.

However, two other cases of combined hPL and GH-V gene deletions were described as

having normal fetal growth190-192

. The observations from the first severely growth retarded

case suggest that the hPL, PGH and most likely other somatogenic and lactogenic

hormones act together and supplement each other in the regulation of fetal development

and growth. This inter-dependency and the ability to supplement each other is most likely

also one of the explanations to why not all children with gene deletions in hPL and GH V

genes becomes growth retarded, and why so many of the results on the IGF-axis differ and

are difficult to interpret.

Besides regulating maternal metabolism and thereby fetal growth hPL is thought to

contribute to human reproductive behavior in other ways as well. In concert with sex

steroids it stimulates DNA synthesis in human mammary epithelial cells and growth of the

ductal epitehlium. It promotes maternal hyperphagia most likely via prolactin receptors in

the hypothalamus193

, and administration of hPL to virgin rats promotes maternal

caretaking and nesting behavior194

.

Placental Growth Hormone

PGH is one of the key regulators of IGF I during pregnancy, studies of large numbers of

normal and pathological pregnancies by different groups have revealed that IGF I values in

maternal plasma correlate with corresponding PGH values158;195;196

. The correlation is


25

supported by a study of pregnancies in women with acromegaly197

. In these cases, in spite

of high levels of pituitary GH and high basal IGF-I concentrations maternal IGF-I levels

increased gradually during pregnancy, following the pattern of PGH secretion.

The regulation of PGH secretion is very dissimilar from the regulation of pituitary GH.

During pregnancy GH releasing hormone analogues have no effect upon PGH secretion

and the levels of GH releasing hormone are stable compared to non-pregnant levels198

. In

vitro low glucose concentrations in the culture media lead to an increased liberation of

PGH from cultured placental explants182

. In vivo PGH concentrations decrease during oral

glucose tolerance test in women with gestational diabetes199

. Based on these results it has

been suggested that the syncytiotrophoblast, which is in direct contact with maternal

blood, and expressing the major glucose transporter (Glut 1) responds to rapid variations in

maternal blood glucose levels by modifying PGH secretion. Because the main fetal energy

source is glucose (80%), defective placental PGH secretion may play a role in fetal

malnutrition161

.

In transgenic mice, that over express the PGH gene, fasting insulin concentrations are four

to sevenfold higher than in control mice, and insulin sensitivity is markedly reduced152;200

.

Furthermore the over expression of PGH stimulates an 84% increases in body weight, a

56% increase in IGF I concentrations, and a 15% reduction of body fat152;200

. These

observations suggest that PGH is a major determinant of maternal insulin resistance and

stimulates the rise in maternal IGF-I concentrations in pregnancy158

.

In vitro PGH binds to intact fat cells and produces insulin-like and lipolytic responses in rat

adipose tissue in a similar way to pituitary GH151

. In vivo several studies have reported that

maternal BMI is inversely correlated to serum levels of PGH at various stages of pregnancy

156;201 202;203. During pregnancy maternal fat is accumulated with maximum increase in fat

deposits from midgestation until week 30. From week 30 and onwards fat accumulation

diminishes or even reverses, and lipolytic activity increases204;205

. The high level of PGH in

week 30 and onwards along with its lipolytic effects151

suggest PGH as a likely candidate for

these effects on maternal adipose tissue. Fetal growth during late pregnancy is dependent

on maternal substrate supply and PGH is one example of the hormonal mechanism from

placental origin, which allows adaptation of the maternal organism to this catabolic phase.

In addition to PGH’s systemic effects in the maternal organism, GH receptors in placental

tissue suggest that PGH also has autocrine or paracrine mechanisms in the placenta; in a

recent study PGH has been shown to affect the invasiveness of extravillous cytotrophoblast

206.

In conclusion the permanent secretion of PGH by villous syncytiotrophoblasts into the

maternal circulation is most likely one of the factors that alters maternal metabolism

during pregnancy. The syncytiotrophoblast replace pituitary GH with its own product,


26

PGH and thereby seems to take control of part of the maternal metabolism during

pregnancy. In the maternal liver and other organs PGH stimulates gluconeogenesis,

lipolysis and anabolism thereby increasing nutrient availability for the fetoplacental unit206

Insulin Growth Factor I and II

The IGFs are mitogenic polypetides that stimulate cellular proliferation and differentiation

207. They have the ability to influence the transport of glucose and aminoacids across the

placenta208

and are significantly influenced by fetal nutrition and insulin levels209;210

. Fetal

insulin promotes growth of the fetus, acting as a signal of nutrient availability211

. Insulin

deficiency results in a reduction in fetal growth, as the fetal tissues decrease their uptake

and utilization of nutrients212

. IGF-I is a basic peptide that shares more than 50% homology

to insulin, and act as a potent insulin sensitizer by virtue of its supression of GH secretion

as well as through direct actions on peripheral tissues213

. In the non-pregnant woman the

IGFs are secreted by the liver and regulated by GH, but during pregnancy PGH gradually

replace the maternal pituitary GH, and takes over the control of IGF secretion158;195;196

.

During pregnancy IGF I and II are produced by the placenta214

, and serum concentrations

are higher in pregnant women than in non pregnant women215

, with concentrations rising

throughout the pregnancy216

.

Gene deletion experiments provide the most convincing evidence for a vital role of the fetal

IGF system in regulating fetal growth210;217

. Homozygote IGF-I knockout mice carry fetuses

that are 60% of the size of their wild-type littermates217

, whereas inactivation of the IGF-I

receptor results in the most severe growth retardation (45% of normal birth weight)

because of the loss of both IGF-I and IGF-II action210

. Mutation in the promoter of the

IGF–II gene in mice results in morphological changes and reduced growth of the placentas,

and this translates into a reduction of fetal size218

. Children born with gene deletions

support the role for IGF-I in normal human fetal growth. One neonate, with a

homozyogous partial deletion of the gene encoding IGF-I, resulting in IGF-I deficiency, had

severe IUGR and postnatal growth failure219

. Furthermore a polymorphism in the

promoter region of the gene encoding IGF-I, which results in low serum IGF-I

concentrations is associated with birth weights that are 215g lower than those of individuals

who are homozygous for the wild-type allele220

, and IGF-I in umbilical cord blood have

been shown to correlate with birth weights221

.


27

Insulin Growth Factor Binding Proteins

The bioavailability of the IGFs is affected by the tissue expression and circulating

concentrations of the IGFBPs (Insulin Growth Factors Binding Proteins). At least six

different IGFBPs have been identified in fetal plasma and tissues209;222

.

These carrier proteins prolong IGF half-life; regulate IGF transport between intra and

extracellular space; limit the bioavailability of IGF via limiting free IGF’s; and modulate

cellular proliferation and death via IGFBP receptors independent of IGFs223

. Under most

circumstances IGFBP´s inhibit IGF’s action224

, however under specific condition some of

the IGFBPs are able to enhance IGF’s action225

. Disorders of fetal growth may also be the

consequence of abnormal IGFBP. IGFBP-1 and IGFBP-3 are produced in large quantities by

the maternal-fetal interface. Increased levels of IGFBP-1 are thought to reflect fetal

hypoinsulinemia, its level can be seen as an indicator of fetal malnutrition and it inhibits

the mitogenic effects of the IGF´s223 226

. In humans birth weight is negatively correlated

with IGFBP-1 concentrations227-229

. This is supported by observations in transgenic mice,

that over express IGFBP-1; in these animals there was no change in the levels of IGF-I, yet

fetal growth restriction was observed, thereby demonstrating that elevated levels of IGFBP-

1 are sufficient to cause alterations in fetal growth226

. The role of IGFBP-3 still remains

unclear; levels of IGFBP-3 and IGF-I have been seen to correlate positively with birth

weight in a numbers of studies230;231 232 233

, however, elevated levels of IGFBP-3 have also

been reported in SGA infants234

, similarly in offspring of mice over-expressing the human

IGFBP-3 a significant reduction in birth weight was reported235

.

Proteolysis of IGFBPs is an additional mechanism to regulate the levels of IGF during

pregnancy, and the IGFBP proteases very likely have a crucial role in determining tissue

availability of the IGF´s. Pregnancy associated plasma protein-A (PAPP-A)and ADAM12 (A

disintegrin and metalloprotease) are both placenta-derived proteins that are identified as

proteases for IGFBP 4 and 3 respectively236

. Low maternal serum levels of PAPP-A in early

pregnancy is known to increase the risk for IUGR237

. Gene knockout mice studies have

shown that disruption of the PAPP-A gene results in 40% reduction of fetal growth238

. A

large multicenter study showed that there was a greater proportion of infants with birth

weight below 2.500g if the maternal PAPP-A concentration was in the lowest 5%, all results

that support the hypothesis that PAPP-A is an important growth factor in the first

trimester, where it may increase the availability of free IGF-s by degrading their binding

proteins239

.

As illustrated by the over 29.000 publications on PubMed on the IGF axis, the system is

complex and intricate, exemplifying the multiple functions of the axis. In this very short


28

review I have tried to describe some of the most important pathways for maternal

metabolism and fetal growth, however, many of the functions still remain to be explored.

Hopefully future discoveries will reveal many of the multifaceted mechanisms in the

system, and as our knowledge becomes more sophisticated and our understanding better

we may find that idiopathic cases of IUGR are caused by alterations in the IGF-axis.

Interventions for Intra Uterine Growth Restriction

Several randomized trials that aim to reduce the risk factors associated with low birth

weight have been published, but only very few of the trials have been effectfull on lowering

the number of children born with a low birth weight240

.

Oxygen therapy, bed rest, exercise and social support

Cochrane meta-analyses that investigate the effects of oxygen therapy, bed rest, exercise

and social support for women with suspected impaired fetal growth conclude that at

present no effect on birth weight can be documented from any of these interventions241-244

.

For oxygen therapy, bed rest and exercise the number of trials and sample sizes are

according to the authors too small for any final conclusion to be drawn, and results from

further trials should be awaited. For social support the meta analysis was based on

eighteen trials, involving 12,658 women, and the final conclusion was that pregnant women

need the support of caring family members, friends, and health professionals, while

programs, which offer additional support during pregnancy are unlikely to be powerful

enough to prevent the pregnancy from resulting in a low birth weight or preterm birth, but

they may reduce the number of caesarean birth244

.

Calcium, fishoil, antioxidant supplements and antiplatelet agents

Interventions, which in theory could delay or reverse inadequate trophoblast invasion such

as: calcium, fishoil, antioxidant supplements and antiplatelet agents have been tried in

different trials. Calcium245

, fishoil246

and antioxidant supplements247

showed no effect on

the risk of having a child with a low birth weight in Cochrane reviews. Two meta analyses

both including more than 12,000 high-risk pregnancies showed an effect of prophylactic

Acetylic Acid treatment. Acetylic Acid treatment showed in the first meta analysis from

1993 a reduction in the subsequent risk of IUGR, especially if the prophylaxis was started

before 17 weeks of pregnancy248

, and in the second meta analysis from 2003; treatment

with Acetylic Acid reduced the subsequent risk of preeclampsia, spontaneous preterm

birth, perinatal death and gave a mean birth weight increase of 215g249

. However, authors


29

of other studies have come to the opposite conclusions250

, so the effect of Acetylic acid is

still disputed.

Food supplementation and infection

The majority of evidence on food supplementation and treatment of infection come from

trials conducted in developing countries or in poor communities; the relevance for women

in developed countries is less clear. In developing societies prevention of infections such as

HIV and malaria and food supplementation to undernourished women does increase birth

weight251;252

. However, when food supplementation or prophylactic prescription of

antibiotics is tried on obstetrical high-risk, but not necessarily undernourished or infected,

pregnant women in developed societies, no effect is observed on birth weight253-255

.

The lack of success for the different interventions does not necessarily mean that they are

completely ineffective. Fish oil and antioxidant supplements, does not reduce the risk of

having a child with a low birth weight, but the groups treated with fish oil and antioxidants

did have a higher mean birth weight compared to the placebo group246;247

. When social

intervention programs are explicitly designed to women, who in tests score high on

objective measurements of stress and low on objective measurements of support, the

programs leads to lower rates of tobacco, drug and alcohol use and a successive reduction

in the number of children born SGA256-265

, and when programs, that teach pregnant

women to identify and stay away from activities that provoke uterine contractions, are

specifically applied to women, who find their work physically challenging, the number of

children born SGA is reduced260;261;266-269

. Thus for an intervention to be effective it is

important not to target a whole unspecified population of high-risk pregnancies, as a large

proportion of these women probably do not have a need for the specific intervention.

Effective interventions can be seen in populations where individual specific needs are

carefully identified and then approached.


30

Own studies:Papers I and II

Our findings regarding the first aim of the Ph.D thesis “To evaluate the association

between fetal growth and size in first half of pregnancy and subsequent adverse outcome”

are described in paper I and II, which will be summarized below.

Fetal growth between the first and second trimester and the risk of adverse

pregnancy outcome

Introduction

The most common definition of IUGR is an EFW at a single point in time below the 10th

centile for gestational age. In clinical practice however, it is commonplace for fetal size to

be estimated more than once during pregnancy, so routine data are often longitudinal.

When several measurements are made it is possible to estimate the rate of change over

specified time intervals or within-subject changes. Compared with a single measurement of

fetal size at one point during gestation sonographic measurement of fetal growth rates has

been shown to be a more accurate method of identifying fetuses at risk of neonatal

morbidity and mortality56

. Longitudinal studies on growth are often based on small

numbers of fetuses and concentrate on growth rate between the second and third

trimesters56;270;271

. To our knowledge prior to our publication there was no previous studies

on adverse pregnancy outcome and early fetal growth rate i.e. growth rate between the first

and second trimesters. The aim of this paper was to relate growth rate between the first

and second trimesters to the risk of perinatal death, IUGR, macrosomia, preterm/post-term

delivery and pre-eclampsia.

Methods

Data were retrieved from the Copenhagen First Trimester Study for Down syndrome and

malformations conducted 1998-2001. Sonographic BPD measurements at 11-14 and 17-21

weeks of gestation were analyzed for 8215 singleton pregnancies. Growth rate was defined

as millimeters of growth per day between the two measurements. To facilitate

comparisons of growth rate between ultrasound examinations performed at different

gestational weeks, growth rate was standardized according to the gestational age at the

time of the scans. A Z-score was thus calculated for each fetus by subtracting the mean

growth rate for all fetuses that were scanned the same two weeks, and dividing by the SD of

the growth rate for this group. The individual Z-scores were transformed into centiles.


31

We obtained odds ratios (OR) for dichotomized adverse outcomes by comparing fetuses

with growth rate below the 2.5th

and above the 97.5th

centiles with a defined normal group

of fetuses with growth rates between 2.5th

and 97.5th

centiles, and comparing fetuses with

growth rates below the 10th

and above the 90th

centiles with a defined normal group of

fetuses with growth rates between the 10th

and the 90th

centiles.

Results

The mean growth rate for BPD varied as a function of gestational age at the time of the

scans: the lower the gestational age at the time of the first or second scan, the higher the

growth rate.

Growth rates below the 2.5th

centile were associated with IUGR: fetuses with growth rates

below the 2.5th

centile had an OR of 2.64 (95% CI, 1.51–4.62) for birth weights below the

mean – 2 SD28

. High growth rates were associated with an increased risk for macrosomia:

fetuses with growth rates above the 90th

and 97.5th

centiles had ORs of 2.53 (95% CI, 1.78–

3.60) and 2.83 (95% CI, 1.58–5.06), respectively, for birth weights above the mean + 2 SD28

.

When adjusted for gestational age at delivery, parity, mother’s height, mother’s weight,

mother being a smoker and fetal gender, growth rates below the 2.5th

centile meant a 105g

reduction in birth weight. In a similar model growth, rates below the 10th

centile meant a

63g reduction in birth weight. The combination of all factors included in the model

explained 45% of the variation in birth weight.

There was an association between very low growth rates and perinatal mortality. Fetuses

with growth rates below the 2.5th

centile were at increased risk of perinatal mortality, with

an OR of 4.79 (95% CI, 1.43–15.99). All three cases of perinatal death with growth rates

below the 2.5th

centile were stillborn before the 25th

week of gestation. Fetuses with growth

rates below the 10th

centile had a non-significant OR of 1.21 (95% CI, 0.36–4.08) for

perinatal mortality.

There was an association between high growth rates and preterm delivery. Fetuses with

growth rates above the 97.5th

centile had a significant OR of 2.30 (95% CI, 1.15–4.59) for

spontaneous delivery in weeks 34–36, while the OR of 3.27 (95% CI, 0.99–10.73) for

spontaneous delivery in weeks 22–33 approached significance. Growth rate showed no

association with pre-eclampsia.

Discussion

The central finding of our study was the significant relationship between BPD growth rate

between the first and second trimesters and adverse pregnancy outcome. Low growth rates

were associated with increased ORs for perinatal mortality and IUGR, and high growth

rates were associated with increased ORs for macrosomia and preterm delivery. These


32

findings underline the importance of focusing on early growth, potentially to prevent

irreparable damage later in pregnancy. Traditional thinking is that early growth is

principally controlled genetically and occurs at a constant exponential rate with little

biological variation272

; IUGR has been assumed to occur mainly in the later half of

pregnancy. Our study refutes this showing that fetal growth rate between the first and

second trimesters does vary between fetuses and is associated with perinatal death and

birth weight.

Early fetal size and growth as predictors of adverse outcome

Introduction

Slow fetal growth is usually inferred retrospectively from the weight reached at birth,

which can be adjusted or customized for maternal factors to distinguish between

constitutional and pathological smallness. Cases thus determined to be SGA have been

found to be strongly linked to perinatal mortality33

. Recently it has been suggested that a

low first-trimester measurement of CRL in pregnancies dated by last menstrual period is

also linked with adverse outcome such as prematurity and fetal growth restriction273

. One

explanation for this observation could be that these pregnancies are misdated; a prolonged

menstruation-conception interval has been shown to be associated with adverse

outcome271;274;275

. However CRL in the first trimester also has been found to be associated

with subsequent length of gestation and birth weight in pregnancies achieved with assisted

reproductive techniques276

.

We set out to study and compare the effects of diminished early pregnancy size and

growth, as assessed from measurements in the first and second trimesters, on subsequent

pregnancy outcome including small for gestational age-birthweight and perinatal death.

Data was analyzed using conditional centiles and customized fetal growth charts.

Conditional centiles is an acknowledged statistical method, used to estimate growth

longitudinally57

, and customized fetal growth charts are growth charts that are individually

adjusted / “customized” for physiological factors known to affect birth weight and growth.

Methods

Data were retrieved from the Copenhagen First Trimester Study for Down Sydrome and

malformations conducted 1998-2001277

. Of the 13,621 women who were invited to

participate, a total of 8,215 women with singleton pregnancies agreed and had records

stored from two ultrasound examination, at 11-14 weeks and 17-21 weeks, respectively. Of

these 8215 pregnancies, 30 ended in miscarriage, 219 were excluded because of congenital


33

anomalies, and 130 were lost to follow-up. A further 194 cases were excluded because of

one or more items of missing data, including fetal sex, parity, birthweight, gestational age

at delivery, maternal height, maternal booking weight. This left 7642 pregnancies for

analysis.

CRL were regressed against gestational age in days, calculated from the last menstrual

period, and fetal size was then assessed by gestation-specific z-scores278;279

. A small first

trimester CRL was defined as less than the 10th

centile. BPD were regressed against

gestational age in days, calculated from the first CRL, and fetal size was then assessed by

gestation-specific z-scores278;279

. Small first and second trimester BPD was defined as less

than the 10th

centile.

Early fetal growth rate was determined from the increment between the first and second

trimester BPD measurements, using conditional centiles57

. This method uses individual

predictions for the expected normal BPD range (‘conditional centiles’) during the second

trimester, on the basis of the first trimester BPD measurement. Each second trimester BPD

measurement value was thus assigned a centile which was conditional to the previous

measurement in the same pregnancy. A fetus with a conditional BPD centile below the 10th

centile was considered to have second trimester growth restriction.

To establish the normal growth velocity for BPD between first and second trimester, a

subset of all pregnancies with normal outcome was defined (n=6164), which were i) non-

smoking during pregnancy; ii) live birth iii) birthweight > 2500 g; and, iv) gestational age at

delivery 37 or more weeks, dated by first trimester CRL measurement. From this ‘normal’

subset, a sample of 300 cases was randomly selected by means of a computerized

algorithm. A linear model provided the best fit for the paired data, and the growth curve

for each fetus was described as:

Log (BDP(mm))=α + βt + γgender

where α, β and γ were the parameters characterizing the individual fetus, t the gestational

age in days determined by CRL and gender (female=0; male=1). The parameters were

calculated assuming a random effect model280

The main pregnancy outcomes assessed were prematurity, smallness for gestational age

and stillbirth. Birthweight was assessed by customised percentiles, which are individually

adjusted for maternal characteristics (booking weight, height, parity, ethnic origin)281

using coefficients derived from a Scandinavian population33

.

Results

Slow growth of the BPD <10th

and <2.5th

conditional centiles between first and second

trimesters occurred in 10.4% and 3.6% of the population, respectively. BPD growth <2.5th


34

centile was strongly associated with increased risk of perinatal death (OR 7.3, CI 2.4 – 22.2).

All deaths occurred before 34 weeks gestation, where the association was also significant at

the 10th

conditional centile cut-off for early fetal growth (OR 16.0, CI 2.9-88.7). Small BPDs,

dated by CRL did not have an increased risk of perinatal deaths. However there was a mild

increase in smallness for gestational age at birth (weight <10th

customised centile) if the

BPD was <10th

centile in the first trimester (OR 1.4, CI 1.1 – 1.7) and in the second trimester

(OR 1.3, CI 1.1 – 1.5).

We found no link between small first trimester CRL and subsequent preterm delivery,

small for gestational age, birthweight or perinatal death

Discussion

In this study we assessed the link between fetal growth in early pregnancy and subsequent

adverse pregnancy outcome, using sequential ultrasound measurements of BPD in the first

and second trimester. Previous publications on growth in early pregnancy have looked at

early fetal size in relation last menstrual period271;273;275;276;282

or early fetal size in relation to

the day of conception when assisted reproductive technology was used276

. By using two

sequential ultrasound measurements of the BPD our method becomes independent of

gestational age error or other confounders, and it appears that slow BPD growth is able

identify a subgroup of slow growing babies whom are at increased risk of perinatal death

before 34 weeks.


35

Paper III

Our findings regarding the second aim of the Ph.D thesis “To analyze the association

between growth factors in maternal blood and fetal growth rate in first half of pregnancy”

are described in paper III, which will be summarized below.

Maternal serum levels of placental Growth Hormone in early pregnancy, but not of

human Placental Lactogen, and Insulin Growth Factor-I are positively associated

with fetal growth in first trimester of human pregnancy

Introduction

Recent studies have shown that fetal growth in first half of pregnancy does vary between

fetuses272

and that decreased early fetal growth rate is associated with perinatal death, low

birth weight and preterm delivery54;55;273;283

. The regulation of fetal growth in the early half

of pregnancy has not been clarified. In postnatal life GH plays an important role, but the

role of GH in fetal growth remains uncertain.

The GH gene cluster contains a single gene expressed in the anterior pituitary that encodes

pituitary Growth Hormone (pGH) plus four genes expressed in the placenta that encodes

pGH, Placental GH (PGH) and hPL284

. PGH is a ligand of maternal GH receptors, and hPL

is a ligand of maternal prolactin receptors. hPL and PGH can be detected in the maternal

circulation at 6 weeks’ gestation and their concentrations increase with advancing

gestation; several studies have suggested a role for hPL and PGH in the control of fetal

growth156;176;285

. In second half of pregnancy it is known that the concentrations of PGH and

IGF-I in maternal serum are positively correlated156

, and in pregnancies with IUGR

maternal IGF-I serum levels are found to be significantly lower than the serum levels found

in normal pregnancies195

.

We wished to investigate if maternal levels of hPL, PGH and IGF-I were correlated to

growth rate in early pregnancy; speculating that high levels of hPL, PGH and IGF-I would

stimulate growth and lower levels would be associated with slower growth rates. No

previous studies have examined the correlation between fetal growth rate and maternal

hormones in the first half of pregnancy.

Methods

Data was retrieved from the Copenhagen First Trimester Study for Down´s syndrome and

malformations conducted 1998-2001. Of the 13,621 women who were invited to participate,

3,680 either refused or had an early miscarriage, leaving 9,941 (73%) women who had a first

trimester ultrasound examination, including 9710 singleton pregnancies. A total of 8,215 of


36

these 9,710 (85%) singleton pregnancies had a record of two ultrasound examinations, at 11-

14 weeks and 17-21 weeks, respectively. Blood samples were drawn from an antecubital

vein, on the same day as the first trimester ultrasound in gestational week 11-14.

Pregnant women were categorized by fetal growth into three groups (low, intermediate and

high fetal growth rates). We defined growth rate as millimetres of growth in BPD per day

between the two sonographic measurements. As the growth from week to 11 to 21 isn’t

linear286

growth rate was standardized according to the gestational age measured in weeks

at the time of the scans. A Z-score was thus calculated for each fetus by subtracting the

mean growth rate for all fetuses that were scanned the same two weeks and dividing by the

SD of the growth rate for this group. The individual Z-scores were transformed into

centiles, and all fetuses with growth rate below the 2.5th

centile (low growth rate, n=203)

and above the 97.5th

centile (high growth rate, n=203) were identified. As a reference group

all fetuses with growth rates from the 50th

to the 51st

centile were identified (intermediate

growth rate, n=212).

The maternal blood serum concentration of hPL, PGH and IGF-I was determined in the

three different growth rate groups. Simple linear and multiple linear regression analysis,

including adjustment for known and potential confounders were used to compare serum

levels between the groups.

Results

Simple linear regression showed a differences in serum level of log10 (PGH) between the

high and intermediate growth rate groups (Table 3, p=0.037). When adjusted for maternal

weight and CRL multiple linear regression analysis confirmed this difference. Fetuses with

high growth rates thus had a higher maternal serum level of log10 (PGH) than those with

intermediate growth rate equal to B=0.05 (95%CI: 0.01-0.09). The increase in log10 (PGH)

may be translated to a relative increase of PGH of 12% (95%CI: 2%-20%) by taking the

antilog10 of B and its CI. Fetuses with low growth rates also had higher level of PGH

compared to those with intermediate growth rate, but this did not reach statistical

significance. When the serum levels in the multiple linear regression analyses were further

adjusted for the sex of the fetus and the parity of the mother, no independent effect of sex

and parity was observed for log10(PGH); the p-value, B and its CI remained robust (p =

0.009, B=0.06(95%CI: 0.01-0.10)).

No differences in hPL and IGF-I levels between the three different growth rate groups were

found after simple and multiple linear regression analysis.


37

Discussion

In our large prospective cohort of pregnant women we observed higher maternal serum

levels of PGH in week 11-14 in women carrying fetuses with high growth rates until week 17-

20 compared to women carrying fetuses who grew at average growth rates. This result

persisted when also adjusting for CRL, maternal weight, parity and fetal sex. This

association has not previously been demonstrated.

It has been hypothesised that PGH acts to augment the supply of fatty acids and glucose to

the placenta151;152

, which would be in agreement with our present results; a high maternal

serum level of PGH would increase substrate supply to the fetus and thus promote fetal

growth. We observed a trend towards higher maternal serum levels of PGH in the low

growth rate group when compared to the intermediate group, which may seem surprising.

We have previously shown that pregnant women, who were smoking during pregnancy,

had higher PGH levels compared to non-smokers which could suggest a compensatory

mechanism by which the fetus is protected201

. In line with these findings, it has been

suggested that high maternal plasma levels of PGH in pre-eclamptic women may be a

compensatory mechanism to preserve fetal growth. In the study by Mittal et al162

patients

with preeclampsia that delivered a SGA-neonate had lower maternal serum concentrations

of PGH than patients with preeclampsia alone. Thus in line with these findings we suggest

that the trend towards higher maternal PGH levels is a compensatory mechanism to

preserve fetal growth.

In conclusion we demonstrated that maternal serum levels of PGH, but not of hPL and

total IGF-I were associated with fetal growth in early human pregnancy in a prospective

cohort of healthy pregnant women. Future studies are needed to evaluate the potential

clinical use of PGH for monitoring early fetal growth.


38

Paper IV

Our findings regarding the third aim of the Ph.D thesis “To investigate if fetuses with first

trimester growth restriction have poorer perfusion of the placenta, measured by ultrasound

Doppler of the uterine and umbilical arteries compared to a control group, and to

investigate if first trimester growth restriction in combination with poor flow in the uterine

and umbilical arteries can be used to predict poor outcome.” are described in paper IV,

which will be summarized below.

First trimester growth restriction and uterine arteries blood flow as predictors of

adverse pregnancy outcome

Introduction

Several studies have now demonstrated that fetal growth in first half of pregnancy does

vary between fetuses, and different measurements for impaired growth in first half of

pregnancy, such as smaller than expected CRL and decreased early fetal growth rates are

associated with perinatal death, low birth weight and preterm delivery54;55;273;276;283;287

. This

implies that important information on growth restriction in early pregnancy in many cases

is lost, when the gestational age for a fetus where CRL is smaller than expected, is simply

altered.

Pre-eclampsia, fetal growth restriction, placental abruption and some cases of fetal death

often result from impaired placentation in early gestation288

. In the past 30 years Doppler

ultrasonographic studies of the uteroplacental circulation have shown that high impedance

to flow in the umbilical and uterine arteries is associated with subsequent adverse neonatal

outcome129-135;289-292

In this prospective study we wished to investigate if fetuses with a smaller than expected

CRL have poorer perfusion of the placenta, measured by ultrasound Doppler of the uterine

and umbilical arteries compared to a control group, and to investigate if a smaller than

expected CRL in combination with poor flow in the uterine arterie can be used to predict

poor outcome

Methods

Women with singleton pregnancies, where the gestational age estimated by CRL at the first

trimester scan was seven days or more smaller than the gestational age estimated by last

menstrual period were identified and invited to the study. A control group of women with

singleton pregnancies, where the gestational age was either equal to or one day larger were

identified and invited to the study. Inclusion criterions were: pregnancy should be


39

spontaneously conceived; at least three spontaneous periods before last menstrual period;

maximum seven weeks discrepancy in gestational age when determined by CRL and last

menstrual period; planning to give birth at either one of the two participating hospitals;

live fetus at the time of the anomaly scan; able to understand Danish.

The study entailed the routine scans, which are Down Syndrome screening in gestational

week 11-14 and an anomaly scan in gestational week 18-21. In addition to the routine scans

the participants were offered a growth scan in gestational week 23-24, where a

questionnaire was answered. At the anomaly scan and growth scan umbilical and uterine

artery Doppler flows were measured. Following delivery the maternal and neonatal records

were reviewed for pre-defined perinatal outcomes.

Statistics: A group of “normal outcome pregnancies” was defined to include liveborn

infants, delivered week 37 – 41, without intrauterine growth restriction, and with no pre-

eclampsia. From the normal outcome group we determined the 5 and 95 % fractiles for the

various indicators for adverse outcome. Logistic regression was used to evaluate the

impact on the risk of adverse outcome of values below the 5 % fractile or above the 95 %

fractile. Differences in proportions were evaluated using chi-square tests and differences in

average values by using u-tests.

Results

The inclusion criterions were met by 357 cases and 451 controls, of these 182 cases and 230

controls were included in the study.

There was a significantly lower percentage of pregnant women in the case group with a

regular menstrual cycle compared to the control group (54% vs. 80 %, p<0.001), and a

smaller proportion of the case group had continued education after mandatory schooling

(73% vs. 85%, p=0.003). Otherwise there were no significant differences between the two

groups in maternal characteristics.

There was a significantly higher proportion of fetuses born with a birth weight below 2500g

in the case group compared to the control group (7.4% vs. 3.1%, p=0.048), otherwise no

significant differences in outcome was observed between the two groups.

At the scan performed week 18-21 the case and control group showed no significant

differences in growth characteristics such as: head circumference, abdominal

circumference and femur length, or in placental blood flow characteristics such as: number

of cases with mean uterine artery PI above 95th

percentile, number of cases with bilateral or

unilateral notch in the uterine arteries, number of cases with umbilical artery PI above 95th

percentile.

The growth and placental blood flow characteristics were repeated at the scan week 23-24,

and again no significant differences were observed between the two groups.


40

In our logistic regression model only birth weight below 2500g showed a significant

association to the case group. Having a CRL seven days or more smaller than expected

increased the risk of having a child with a birth weight below 2500g with an odds ratio of

3.29.

Discussion

In this study we found no association between smaller than expected CRL as a measure of

first trimester growth restriction and placental perfusion measured by ultrasound Doppler

of the uterine and umbilical arteries in second trimester. The number of cases and controls

with high PI or bilateral/unilateral notch in the uterine arteries or high PI in the umbilical

arteries showed no significant differences between the case and control group at the scans

performed week 18-21 and week 23-24.

Only birth weight below 2500g showed a significant association to the case group in our

logistic regression model. Having a CRL seven days or more smaller than expected

increased the risk of having a child with a birth weight below 2500g with an odds ratio of

3.3. In line with these findings fetuses with a gestational age determined by CRL seven days

or more smaller than expected had about double the risk of being born with a birth weight

below 2500g.

In conclusion we confirmed the association between first trimester growth restriction and

higher risk of delivering a low birth weight infant. We were not able to demonstrate a link

between poor placental perfusion and first trimester growth restriction, and therefore do

not recommend implementation of uterine or umbilical artery flow measurements

specifically for fetuses with at smaller than expected CRL. However, we do recognize that

a smaller than expected CRL is a risk factor for giving birth to a low birth weight neonate,

and simple re-dating of pregnancies with a smaller than expected CRL, especially if the

woman has a regular menstrual cycle and is certain of the first day in her last period, can

conceal early growth restriction. On these grounds we do recommend for pregnancies with

a smaller than expected CRL that extra attention is paid to the ultrasound measurements

obtained at the anomaly scan, and should these measurements be at the low end of the

normal range scale a follow-up scan to check fetal growth should be offered to the

pregnant woman.


41

Conclusions and clinical implicationsFetal growth in first half of the pregnancy and the risk ofadverse outcome

We have demonstrated a link between growth rate in first half of pregnancy and an

increased risk of perinatal mortality and low birth weight. Prior to our studies low

measurements of CRL and BPD in pregnancies dated by last menstrual period had been

associated to adverse outcome such as prematurity and fetal growth restriction. However,

this link could be explained by the fact that these pregnancies were misdated or had a long

prolonged menstruation-conception interval. By looking at growth rate instead of fetal size

in relation to last menstrual period, we overcame the problem of delayed ovulation and still

observed an association.

The use of longitudinal fetal measurements is somewhat complicated, and many factors

compromise the clinical value of the measurements. First there is the accuracy of the

measurements; a sonographic measurement will always contain some degree of

measurement error. The way growth rate was defined in paper I entailed that the group of

fetuses with high growth rates were the fetuses that had the lowest BPD at the first scan

and the highest BPD at the second scan; and vice versa for group of fetuses with low

growth rate. This definition of growth rate implies that some of the fetuses categorized as

having either very high or very low growth rate were most likely categorized as such with

some degree of inaccuracy due to the statistical principle of:”regression toward the mean”,

which states that if a pair of independent measurements are made from the same

distribution, samples far from the mean of the first measurement will tend to be closer to

the mean on the second one. The phenomenon occurs because each sample is affected by

random variance. In paper II we tried to overcome the problem of “regression toward the

mean” with the use of conditional centiles.

In Paper I we demonstrated that the mean growth rate for BPD varied as a function of

gestational age at the time of the scans: the lower the gestational age at the time of the first

or second scan the higher the growth rate, please see Figure 1 and 2 in Paper I for details.

To compensate for the variation in growth rate as a function of gestational age at the time

of ultrasound examination in Paper I we calculated Z-scores specific to the pair of

gestational ages at which each fetus was scanned. The conditional centiles used in Paper II

do not compensate for the variation in growth rates the same way. Thus Papers I and II

represent two different statistical approaches of standardizing longitudinal measurements

of growth in first half of pregnancy. Below we have tried to compare the two approaches to

evaluate whether they classify the same fetuses as having low growth rates, and we look at

the ability of the two approaches to predict low birth weight and perinatal mortality. As


42

Date centiles Conditional centiles

Growth rate <2.5 percentile OR (95% CI) P Value OR (95% CI) P Value

Birth weight < mean - 2 Std Dev* 2.64 (1.51-4.62) 0.002 1.93 (1.13-3.31) 0.015

Birth weight < 2.5 customized percentile† 1.94 (1.07-3.53) 0.040 1.77 (1.05-2.98) 0.030

Perinatal mortality 4.79 (1.43-15.99) 0.031 4.12 (1.62-13.73) 0.002

the centiles used in Paper I adjust for the gestational age at the time of the scans they are

referred to as date centiles.

Conditional and Date centiles: Fetuses with growth rates below 2.5th

centile

Figure 8 illustrates the number of cases with growth rates below 2.5th centile that are classified in accordance and

not in accordance by date and customized centiles respectively.

As illustrated by Figure 8, 87 fetuses were classified as having growth rates below the 2.5th

centile by both date and conditional centiles. This represents 43% of the fetuses classified


centile by date centiles and 30% of the fetuses, classified


centile by conditional centiles. Thus the two different

statistical approaches seem to differ in more than 50% when defining the group of fetuses

with a very low growth rate. Given the large discrepancy between date and conditional

centiles in defining the group of fetuses that have growth rates below the 2.5th

centile, it is

possible that a statistical model that combines the two and takes into account the variation

in growth according to gestational age, the size of the fetus at the first scan, and the

phenomenon of regression to the mean would give an even better estimation of fetuses at

risk.

Conditional and Date centiles: Association to low birth weight and perinatal

mortality

Table 3 depicts the odds ratios for low birth weight and perinatal mortality from week 20

until a week after birth for date and conditional centiles below the 2.5th

percentile.

Table 3 P values are for the comparison of between groups by a two-tailed Pearson Chi square or Fischers exacttest if expected count less than 5. * Marsal K et al28. †Gardosi J et al293.

N=116

57%

N=200

70%

N=87

43% 30%

Date centiles <2.5 Conditional centiles <2.5


43

In Table 3 the odds ratios for low birth weight and perinatal mortality seem comparable for

date and conditional centiles. Having a growth rate below the 2.5th

centile by either date or

conditional centiles produces an odds ratio between 1.77 and 2.64, depending on the

definitions of low birth weight. Odds ratios below 2 are usually considered to have

minimal clinical value, as the number of pregnancies falsely classified as being at risk is too

high compared to those actually at risk. Therefore date and conditional centiles seem to

have limited value in predicting low birth weight in the daily clinic.

Perinatal death is in Table 3 defined as stillbirth after 20 weeks of gestation plus neonatal

death through one week of life. In Table 3 conditional and date centiles below 2.5th

centile

seem equally capable of predicting perinatal mortality from week 20 until one week after

term. In Paper II perinatal mortality was defined as stillbirth after 24 weeks of gestation

plus neonatal death through 28 days of life, and in this Paper we report an odds ratio of

16(95% CI 2.9-88.7) for perinatal death before 34 weeks for fetuses with growth less than

the 10th

conditional centile. In Paper I the odds ratio for perinatal death (defined as

stillbirth after 20 weeks of gestation plus neonatal death though one week of life) is

1.21(95% CI 0.36-4.08) for fetuses with growth less than the 10th

date centile. It thus seems

that the use of conditional centiles can give us the possibility of defining a subgroup of

pregnancies with an increased risk of early fetal death that is high enough for it to have

potential clinical value. Early indications of an increased risk would allow more intensive

fetal assessment and surveillance and informed considerations on when to advise early

delivery to avoid fetal demise.

Clinical use of longitudinal measurements will require statistical processing before the

information can be of any practical use for the clinician; this could be done in already

existing computer software for ultrasound, such as ASTRAIA. The measurements could be

taken as part of the routine scans conducted in weeks 11-14 and 17-21 in many countries.

However, new studies and perhaps other statistical approaches are needed to confirm their

clinical value.

Growth hormones and growth rate in first half of pregnancy

We demonstrated that maternal serum levels of PGH, but not of hPL and total IGF-I were

associated with fetal growth in early human pregnancy. Higher maternal serum levels of

PGH were observed in women carrying fetuses with high growth rates compared to women

carrying fetuses, who grew at average growth rates, and there was a trend towards higher

maternal serum levels of PGH in the low growth rate group when compared to the

intermediate group, which we suggest is a compensatory mechanism to preserve fetal

growth. The association between PGH and early fetal growth has not previously been


44

demonstrated, and the trend towards preservation of growth in the low growth rate group

confirms the results of other studies.

As access to the fetal compartment entails risk for the fetus involved, the vast majority of

studies on fetal growth factors, including ours, are based on maternal levels of growth

factors, not fetal levels. During pregnancy the mother and the fetus are in a physiological

conflict; in case of sparse resources the hormone system of the mother is programmed to

preserve her health, and the hormone system of the fetus is programmed to continue fetal

growth, even at the cost of the mother’s health. This conflict is reflected in diverse growth

factors and different levels of the growth factors in the maternal and fetal compartment.

Today only maternal levels of Pregnancy associated plasma protein A (PAPP-A) from the

IGF-system are used to predict IUGR, and its clinical value is limited. There is a vast

clinical potential in predicting growth restriction with the help of growth factors from the

IGF-system. However before this can become a clinical reality it is necessary with a better

understanding of the intricate relations between the fetal and maternal compartments, and

an understanding of the complex interactions between the growth factors in the IGF-

system. Many of the growth factors may rise or fall to compensate for poor function in

other parts of the system. In addition IUGR is a multifactorial condition, and the

heterogeneity of the group and diversity of causes leading to growth restriction makes it

even more difficult to pin-point one growth factor that could be used as a clinical marker

for growth restriction. Most likely different patterns of more than one growth factor will

give the best prediction of growth restriction and high-risk pregnancies in the future.

Fetal growth in first half of pregnancy as a prospectivepredictor of adverse outcome

We were not able to demonstrate a link between poor placental perfusion and first

trimester growth restriction, and therefore do not recommend measurements of uterine or

umbilical artery flow at the routine scans or at an extra scan week 23/24 specifically for

fetuses with a smaller than expected CRL.

The original intention of the last study was to investigate if growth rate in first half of

pregnancy in combination with biochemical markers associated with growth could predict

adverse outcome prospectively. However, as we were setting up the study we realised that

longitudinal measurements of growth were difficult to use in a clinical setting. The

measurements require a statistical processing and interpretation before they can be used,

and this was impossible to combine with the day-to-day recruitment for a clinical trial.

In the cohort of 8215 pregnant women used for subproject I and II there were 6027 women

with a known menstrual cycle length and of these, when adjusted for cycle length, 6% had

a gestational age estimated by ultrasound (based on CRL) that was 7-21 days smaller than


45

expected(non-published data)

. The OR for IUGR defined as birth weight below mean – 2 SD was

1.94 (95%CI 1.14-3.30)(non-published data)

for this group compared to 2.64 (95%CI 1.51-4.62) for

fetuses with a growth rate below 2.5th

date centile (Paper I) while the OR was 1.2 (95%CI

0.97-1.48) for birth weight below the 10th

customized centile for fetuses with BPD growth

less than the 10th

conditional centile ( Paper II). As we wished to focus on IUGR in the

prospective study, we judged that a smaller than expected CRL could be used as surrogate

for longitudinal measurements in the study.

When originally planning the studies we had expected a stronger association between

maternal growth factors and early fetal growth rate, but when the results from paper III

became available we decided that the association between maternal growth factors and

early fetal growth were too weak to be used in a prospective study for the prediction of

poor outcome.

We were not able to demonstrate an association between poor placental flow and first

trimester growth restriction, but our study did confirm the association between low birth

weight and first trimester growth restriction. Based on this observation we recommend for

pregnancies with a smaller than expected CRL and ultrasound measurements at the lower

end of the normal range scale in second trimester, that a follow-up scan to check fetal

growth is offered.

Future research

The great challenge in the future is to determine different markers or risk factors during

pregnancy that can identify a high-risk group and provide prevention or treatment of

adverse outcome for this selected group. It seems logical that both the detection and

possible treatment shall take place in the beginning of the pregnancy, before irreparable

damage has occurred; this underlines the importance of focusing on first trimester in

future research. We are today aware of many risk factors for adverse pregnancy outcome,

however, the use of these risk factors in an efficient, and for the pregnant woman

beneficial, way seems one of the challenges of future research. In this Ph.D thesis the focus

is on: growth rate; conditional centiles; smaller than expected CRL; customized fetal

growth charts; biochemical markers for fetal growth; umbilical and uterine artery flow, all

factors that our study along with other studies suggest can be used to improve the

prediction of adverse outcome. Except for abnormal flow in the umbilical artery, none of

the risk factors focused on in this thesis has the capacity on its own to predict poor

outcome. However, if the risk factors are combined they might prove to have significant

clinical value. The transformation of the several known risk factors to a practical clinical

tool is something that perinatal care could benefit immensely from in the future.


46

Successful transformation will require a strong collaboration between, statisticians,

software-programmers and doctors, which is not always easy, but improved prediction of

high-risk pregnancies would allow clinicians to give more accurate fetal assessment and

better focus resources, which most likely would lead to a decline in perinatal morbidity and

mortality.


47

SummaryThe thesis consists of three subprojects:

First subproject

The first subproject aimed to analyze the association between growth in first half of

pregnancy and the risk of adverse outcome in a cohort of 8215 pregnant women. Data was

obtained from the Copenhagen First Trimester Study conducted in 1998-2001. Two papers

were published.

The objective of the first paper was to relate growth rate of the biparietal diameter (BPD)

between the first and second trimesters to the risk of perinatal death, intrauterine growth

restriction, macrosomia, preterm/post-term delivery and preeclampsia. We analyzed

sonographic BPD measurements at 11–14 and 17–21 weeks, and defined growth rate as

millimetres of growth per day between the two measurements. Growth rate was

dichotomized into growth rates < 2.5th

and > 97.5th

vs. 2.5th

–97.5th

centiles. Odds ratios

and 95% confidence intervals for adverse outcome were calculated. We found a significant

relationship between the growth rate of BPD from the first to the second trimester and

adverse pregnancy outcome. Low growth rates were associated with increased odds ratios

for perinatal death and intrauterine growth restriction, while high growth rates were

associated with increased odds ratios for macrosomia and preterm delivery.

The second paper was written in collaboration with Dr. Francesc Figueras and Professor

Jason Gardosi from West Midlands Perinatal Institute, Birmingham, UK. In this

collaboration data was analyzed using conditional centiles and customized fetal growth

charts. Conditional centiles are an acknowledged statistical method, used to estimate

growth longitudinally, and customized fetal growth charts are growth charts that are

individually adjusted / “customized” for physiological factors known to affect birth weight

and growth. The objective of the second paper was to determine the association between

fetal size and growth between the first and second trimesters and subsequent adverse

pregnancy outcome using conditional centiles and customized fetal growth charts for

statistical analyzes. The cohort was the same as in the first paper: 8215 pregnant women

that had fetal size estimated by ultrasound at 11-14 weeks (CRL and BPD) and again at 17-21

weeks (BPD). With the use of conditional centiles and customized fetal growth charts we

found a strong association between early fetal growth rate and perinatal death before 34

weeks (OR 16.0, CI 2.9-88.7).

Second subproject

The second subproject was done in collaboration with Professor Anders Juul, Department

of Growth and Reproduction, Rigshospitalet and Dr Michael Christiansen, Department of


48

Clinical Biochemistry, Statens Serum Institute. In this project we wished to analyze the

association between growth factors in maternal blood and fetal growth rate in first half of

pregnancy. We investigated if maternal levels of human Placental Lactogen (hPL),

Placental Growth Hormone (PGH) and Insulin Growth Factor I (IGF-I) are associated with

growth rate of the biparietal diameter (BPD) between week 11-14 and week 17-21. Again

data from 8215 singleton pregnancies from the Copenhagen First Trimester Study were

analyzed, and growth rate was defined the same way as in our first paper. Fetuses with

growth rate of the BPD below the 2.5th

centile (low growth rate) and fetuses with growth

rate above the 97.5th

centile (high growth rate) were identified. As a reference group a

similar number of fetuses with growth rate around the median were identified

(intermediate growth rate). The maternal blood serum concentration of hPL, PGH and

IGF-I was determined in the three different growth rate groups. We found that when

adjusted for maternal weight and crown rump length (CRL), maternal PGH levels were 12%

higher in women carrying fetuses with high first trimester growth rates compared to

controls. Thus, PGH may be involved in fetal growth regulation already in early human

pregnancy.

Third subproject

The third subproject is a prospective study conducted in collaboration with Dr Lene

Sperling, Department of Obstetrics and Gynaecology, Herlev Hospital. In this study we

looked at fetuses with a discrepancy in gestational age determined by crown rump length

(CRL) and last menstrual period, and used a “smaller than expected CRL” as an indicator of

first trimester growth restriction.

We wished to investigate if fetuses with a smaller than expected CRL have impaired

perfusion of the placenta, measured by ultrasound Doppler of the uterine and umbilical

arteries compared to a control group, and to investigate if a smaller than expected CRL in

combination with abnormal flow in the uterine and umbilical arteries can be used to

predict adverse neonatal outcome.

We included 182 singleton pregnancies with a gestational age set by CRL that was seven

days or more smaller than the gestational age set by last menstrual period and 230 controls

with a gestational age set by CRL that was equal to the gestational age set by last menstrual

period. The study entailed the routine scans (nuchal translucency scan in first trimester

and anomaly scan in second trimester), a growth scan in gestational week 23-24 and a

questionnaire. At the anomaly scan and growth scan umbilical and uterine artery Doppler

flows were measured.


49

We were not able to demonstrate a link between poor placental perfusion and first

trimester growth restriction, and therefore, based on the result from this study, do not

recommend measurements of uterine or umbilical artery flow specifically for fetuses with

at smaller than expected CRL.


50

Dansk resumeAfhandlingen består af tre delprojekter

Første delprojekt

Formålet var at analysere associationen mellem føtal vækst i første halvdel af graviditeten

og risikoen for dårligt graviditetsudkomme i en kohorte på 8215 gravide kvinder. Data kom

fra ”Copenhagen First Trimester Study 1998-2001”. To artikler er publiceceret i første

delprojekt.

Formålet med den første artikel var at relatere vækstrate af biparietal diameteren (BPD)

mellem første og andet trimester af graviditeten til risikoen for perinatal død, intrauterin

væksthæmning, macrosomi, præterm/postterm fødsel og præeclampsi. Vi anvendte

ultralydsmål af BPD målt i uge 11-14 og 17-21 af graviditeten. Føtal vækstrate definerede vi

som millimeter vækst pr. dag mellem de to målinger. Materialet blev dichotomiseret i

grupper med vækstrate < 2.5th

vs. 2.5th

–97.5th

percentil samt > 97.5th

vs. 2.5th

–97.5th

percentil og odds ratio og 95% confidence interval for dårligt graviditets udkomme blev

beregnet.

Vi fandt signifikante sammenhæng mellem BPD-vækstraten fra første til andet trimester

og risikoen for dårligt graviditets udkomme. Lav vækstrate var associeret med en øget odds

ratio for perinatal død og intrauterin væksthæmning og høj væksrate var associeret med

øget odds ratio for macrosomi og præterm fødsel.

Den anden artikel blev skrevet i samarbejde med Dr. Francesc Figueras og Professor Jason

Gardosi fra West Midlands Perinatal Institute, Birmingham, UK. I dette samarbejde

analyserede vi data ved brug af ”conditional centiles” og ”customized fetal growth charts”.

”Conditional centiles” er en anerkendt statistisk metode, der anvendes til at estimere vækst

målt longitudinelt og ”customized fetal growth charts” er vækstdiagrammer som er

individuelt justeret/”customized” for fysiologiske faktorer, som påvirker fødselsvægten og

væksten. Formålet med artikel nummer to var at beskrive associationen mellem føtal

størrelse og vækst mellem første og andet trimester og efterfølgende graviditetsudkomme

ved anvendelsen af ”conditional centiles” og ”customized fetal growth charts” til de

statistiske analyser.

Kohorten var den same som i den første artikel: 8215 gravide kvinder, som havde fået

foretaget ultralydsmålinger i uge 11-14 (CRL, BPD) og igen i uge 17-21 ( BPD). Ved

anvendelsen af ”conditional centiles” og ”customized fetal growth charts” fandt vi en stærk

association mellem vækst i den tidlige gravidtet og perinatal død før uge 34 (OR 16.0, CI

2.9-88.7).


51

Andet delprojekt

Andet delprojekt blev skrevet i samarbejde med Professor Anders Juul, klinik for vækst og

reproduktion, Rigshospitalet og overlæge Michael Christiansen, klinisk biokemisk afdeling,

Statens Serum Institut. I dette projekt ønskede vi at analysere associationen mellem

vækstfaktorer i maternelt blod og fosterets vækstrate mellem første og andet trimester. Vi

undersøgte om maternelle serum niveauer af human Placental Lactogen (hPL), Placental

Growth Hormone (PGH) og Insuline Growth Factor I (IGF-I) er associerede med

vækstraten af biparietal diametern mellem uge 11-14 og uge 17-21. Vi anvendte den samme

kohorte fra “ Copenhagen First Trimester Study” som i første delprojekt, ligesom

væksraten blev defineret på samme måde som i den første artikel. Fostre med BPD-

vækstrate under 2.5 percentilen (lav vækstrate) og fostre med vækstrate over 97.5

percentilen (høj vækstrate) blev identificeret. Et tilsvarende antal fostre med vækstrate

omkring medianen (intermediær vækstrate) blev anvendt som referencegruppe.

Maternelle serum koncentrationer af hPL, PGH og IGF-I blev målt i de tre forskellige

vækstrate grupper. Vi fandt at efter justering for crown rump length (CRL) og maternel

vægt er det maternelle serum niveau for PGH 12% højere hos gravide med fostre, der har en

høj vækstrate sammenlignet med den intermediære kontrol gruppe og konkluderede derfor

at PGH er involveret i føtal vækstregulering allerede tidligt i graviditeten.

Tredie delprojekt

Det tredie delprojekt er et prospektivt studie udført i samarbejde med overlæge Lene

Sperling, Obstetrisk/Gynækologisk afdeling, Herlev Hospital. I dette studie fokuserede vi

på fostre, hvor der var en forskel i gestationsalderen fastsat ved crown rump length (CRL)

og gestationsalderen fastsat ved sidste menstruation. Vi anvendte ”mindre end forventet

CRL” som en indikator for første trimester vækstretardering.

Vi ønskede at undersøge om disse små fostre har dårligere perfusion af plancenta målt ved

ultralyd Doppler i aa. Uterinae og a. Umbilicalis sammenlignet med en kontrol gruppe,

samt at undersøge om dårligt flow i aa. Uterinae og a. Umbilicalis kan anvendes til at

forudsige dårligt neonatal udkomme.

Vi inkluderede 182 singleton graviditeter, hvor gestationsalderen estimeret ved CRL i første

trimester var syv dage eller mere mindre end gestationsalderen estimeret ved sidste

menstruation og 230 kontroller hvor gestationsalderen estimeret ved CRL og sidste

menstruation var den samme. Studiet omfattede rutine skanningerne

(nakkefoldsskanningen i første trimester og anomaliskanningen i andet trimester) samt en

tilvækstskanning i uge 23-24 og et spørgeskema. Ved gennemskanningen og


52

tilvækstskanningen blev blodgennemstrømningen i aa.uterinae og a.umbilicalis målt med

ultralyd Doppler.

Vi fandt ikke en association mellem dårlig perfusion af placenta og første trimester

vækstretardering, og kan på baggrund af dettes studie derfor ikke anbefale uterin eller

umbilical arterie flow målinger specielt til fostre med en mindre end forventet CRL.


53

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Appendix

Paper I

Fetal growth between the first and second trimester and the risk of adverse pregnancy outcome. NG Pedersen,

KR Wøjdemann, T Scheike and A. Tabor. Ultrasound Obstet Gynecol 2008;32:147-154

Paper II

Early fetal size and growth as predictors of adverse outcome. NG Pedersen, F Figueras, KR Wøjdemann, A

Tabor and J Gardosi. Obstet and Gynecol 2008;112:765-771

Paper III

Maternal serum levels of placental Growth Hormone in early pregnancy, but not of human Placental Lactogen,

and Insulin Growth Factor-I are positively associated with fetal growth in first trimester of human pregnancy.

NG Pedersen, A juul, M Christiansen, KR Wøjdemann and A Tabor, submitted

Paper IV

First trimester growth restriction and uterine arteries blood flow as predictor of adverse pregnancy outcome.

NG Pedersen, L Sperling, KR Wøjdemann, S Olesen Larsen, A Tabor, in manuscript

Questionnaire used in subproject 3

Ultrasound Obstet Gynecol 2008; 32: 147–154Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/uog.6109

Fetal growth between the first and second trimestersand the risk of adverse pregnancy outcome

N. G. PEDERSEN*, K. R. WØJDEMANN†, T. SCHEIKE‡ and A. TABOR*Departments of *Fetal Medicine and Ultrasound and †Gynecology and Obstetrics, Rigshospitalet, Copenhagen University Hospital and‡Department of Biostatistics, Copenhagen University, Copenhagen, Denmark

KEYWORDS: birth weight; BPD; growth rate; IUGR; perinatal mortality; preterm delivery; ultrasound

ABSTRACT

Objectives To relate growth rate of the biparietal diam-eter (BPD) between the first and second trimesters tothe risk of perinatal death, intrauterine growth restric-tion (IUGR), macrosomia, preterm/post-term deliveryand pre-eclampsia.

Methods In this retrospective study, we analyzed sono-graphic BPD measurements at 11–14 and 17–21 weeksfrom 8215 singleton pregnancies in the Copenhagen FirstTrimester Study. Growth rate was defined as millimetersof growth per day between the two measurements and wasdichotomized into growth rates < 2.5th vs. 2.5th –97.5th

centiles, and > 97.5th vs. 2.5th –97.5th centiles. Oddsratios (OR) and 95% CIs for adverse outcome werecalculated.

Results Fetuses with growth rates < 2.5th centile had anOR of 4.79 (95% CI, 1.43–15.99) for perinatal deathand an OR of 2.64 (95% CI, 1.51–4.62) for birth weight< sonographically estimated mean fetal weight (adjustedfor gestational age) − 2 SD. Fetuses with growth rates> 97.5th centile had an OR of 2.83 (95% CI, 1.58–5.06)for birth weight > mean + 2 SD and an OR of 2.30 (95%CI, 1.15–4.59) for delivery in weeks 34–36. Growth rateshowed no association with pre-eclampsia.

Conclusions There is a significant relationship betweenthe growth rate of BPD from the first to the secondtrimester and adverse pregnancy outcome. Low growthrates are associated with an increased OR for perinataldeath and IUGR, while high growth rates are associatedwith an increased OR for macrosomia and pretermdelivery. Copyright 2008 ISUOG. Published by JohnWiley & Sons, Ltd.

INTRODUCTION

Fetal growth is of key importance in many aspectsof perinatology. Impaired growth is associated with

increased neonatal morbidity and mortality1–3. Muchroutine prenatal care focuses on detecting fetuses atincreased risk of growth restriction (IUGR) and targetingintensive monitoring and intervention. Fetal growth canbe assessed by comparing the size of a fetus of knowngestational age with reference centiles for fetal size.The most common definition of IUGR is an estimatedfetal weight at a single point in time that is belowthe 10th centile for gestational age. In clinical practice,however, it is commonplace for fetal size to be estimatedmore than once during pregnancy, so routine dataare often longitudinal. When several measurements aremade, it is possible to estimate the rate of changeover specified time intervals or within-subject changes.Compared with a single measurement of fetal size atone point during gestation, the sonographic measurementof fetal growth rates has been shown to be a moreaccurate method of identifying fetuses at risk of neonatalmorbidity and mortality4. Longitudinal studies on growthrate are often based on small numbers of fetuses andconcentrate on growth rate between the second and thirdtrimesters4–6. To our knowledge, there have been nostudies on adverse pregnancy outcome and early fetalgrowth rate, i.e. growth between the first and secondtrimesters. One study that addressed early fetal growthand birth weight examined the relationship betweenthe observed and expected first-trimester crown–rumplength (CRL) and found a clear relationship betweena CRL 2–6 days smaller-than-expected and the risk ofdelivering a baby with a birth weight below 2500 g, a birthweight below the 5th percentile and premature delivery7.Analysis of the Swedish birth registry, including data frommore than 700 000 births, demonstrated associationsbetween smaller-than-expected biparietal diameter (BPD)in gestational weeks 16–18 and stillbirth, neonatal death,birth weight < 2500 g and preterm delivery, with oddsratios (OR) of 1.18–1.878. However, the discrepancyin these studies between expected and observed size of

Correspondence to: Dr N. G. Pedersen, Department of Fetal Medicine and Ultrasound, Rigshospitalet, Copenhagen University Hospital,Afsnit 4031, Blegdamsvej 9, 2100 Copenhagen, Denmark (e-mail: [email protected])

Accepted: 25 March 2008

Copyright 2008 ISUOG. Published by John Wiley & Sons, Ltd. ORIGINAL PAPER

148 Pedersen et al.

the fetus depends to some extent on the uncertaintysurrounding the exact dating of ovulation and conception,which obscures the possibility of detecting early growthrestriction. This was addressed recently in a cohort of976 women with known date of conception as a result ofassisted reproduction, in whom it was confirmed that asmaller-than-expected CRL increased the risk of pretermbirth and of delivering a small-for-gestational-age infant9.

We speculated that measurement of early growth ratebetween gestational weeks 11–14 and weeks 17–21would produce a more precise estimation of associatedrisks later in pregnancy and at delivery. The aim of thisstudy was to relate growth rate between the first and sec-ond trimesters to the risk of perinatal death, IUGR, macro-somia, preterm/post-term delivery and pre-eclampsia.

METHODS

For this retrospective study, data were retrieved fromthe prospective Copenhagen First Trimester Study forDown Syndrome and malformations, conducted from1998 to 200110. A total of 13 621 women, referred tothe three obstetric departments in Copenhagen UniversityHospital, were invited to participate in the CopenhagenFirst Trimester Study. Participation entailed the choiceof an ultrasound examination and/or a blood sample ingestational weeks 11–14. Additionally, at all three depart-ments, routine antenatal care included the offer of oneanomaly scan performed at around 18–20 weeks’ gesta-tion. Data, including indications and diagnoses, from allscans performed during the pregnancies were recorded inViewPoint Fetal Database

TM(ViewPoint GmbH, Webling,

Germany). Outcome data were gathered from each new-born’s clinical records after delivery, using predefinedcriteria. Of the 13 621 women initially invited to theCopenhagen First Trimester Study, 8215 women with sin-gleton pregnancies underwent a first-trimester ultrasoundexamination in weeks 11–14 and a second-trimester ultra-sound examination in weeks 17–21. Written informedconsent was obtained from the participants and the sci-entific ethics committee of the cities of Copenhagen andFrederiksberg (No. (KF) 01-288/97) and the Data Protec-tion Agency approved the study protocol. The ultrasoundexaminations were performed by 13 sonographers ordoctors experienced in obstetric ultrasound and certi-fied by The Fetal Medicine Foundation in London, UK.The ultrasound examinations in weeks 11–14 were allperformed using a Logic 700 MR scanner (General Elec-tric, Milwaukee, WI, USA). Those in weeks 17–21 wereperformed using one of the following machines: AcusonAspen Advanced, Sonoline SI 400 or Sonoline Sienna(Siemens AG, Erlangen, Germany). Gestational age wasdetermined by CRL11. BPD measurements were obtainedin an axial plane at the level of the thalami, the thirdventricle and the cavum septi pellucidi, from the outerborder to the inner border of the skull.

We defined growth rate as millimeters of growth in BPDper day between the two sonographic measurements. Tofacilitate comparisons of growth rate between ultrasound

examinations performed at different gestational weeks,growth rate was standardized according to the gestationalage at the time of the scans. A Z-score was thus calcu-lated for each fetus by subtracting the mean growth ratefor all fetuses that were scanned the same 2 weeks, anddividing by the SD of the growth rate for this group:Z-score = (observed growth rate − group mean growthrate) / (group growth rate SD). The individual Z-scoreswere transformed into centiles, and fetuses with growthrates below the 2.5th or 10th centiles, or above the 90th or97.5th centiles, were identified.

We determined the ability of growth rates below the2.5th and 10th centiles (low growth rate) and that ofgrowth rates above the 90th and 97.5th centiles (highgrowth rate) to identify pregnancies with increased risk ofperinatal death, IUGR, macrosomia, preterm/post-termdelivery and pre-eclampsia. We expected that extremegrowth rates would have a high risk for adverse outcomeand therefore categorized growth rate as low, intermediateand high. Perinatal death was defined as: death of an infantunder 1 week in age or stillbirth at 20 weeks or moreof gestation; outcome was dichotomized into perinataldeath vs. live birth. IUGR was defined as birth weightbelow 2500 g; birth weight below 2500 g at 37 weeksof gestation or later; birth weight smaller than the meansonographically estimated fetal weight for gestational ageminus two SD (mean − 2 SD)12. Each of the definitionsof IUGR was dichotomized into IUGR vs. no IUGR.Macrosomia was defined as: birth weight above 4500 g;birth weight above 4500 g at 37 weeks of gestation orlater; birth weight greater than the mean sonographicallyestimated fetal weight for gestational age plus 2 SD(mean + 2 SD). Each of the definitions of macrosomiawas dichotomized into macrosomia vs. no macrosomia.Different definitions of IUGR and macrosomia were usedto allow comparisons with other studies. Very prematuredelivery was defined as spontaneous live birth at weeks22–33, premature delivery as spontaneous live birth atweeks 34–36, term delivery as spontaneous live birthat weeks 37–41, and post-term delivery as spontaneouslive birth after 41 weeks of gestation. Gestational ageat delivery was dichotomized into very premature,premature or post-term vs. term. Pre-eclampsia wasdefined according to the guidelines of the InternationalSociety for Study of Hypertension in Pregnancy13: tworecordings of diastolic blood pressure > 90 mmHg atleast 4 h apart in previously normotensive women, andproteinuria of 300 mg or more within 24 h / two readingsof at least ++ on dipstick analysis of midstream orcatheter urine specimens if no 24-h collection wasavailable. Pre-eclampsia was dichotomized into pre-eclampsia vs. pregnancies with no pre-eclampsia.

We obtained ORs for the dichotomized adverseoutcomes by comparing fetuses with growth rates belowthe 2.5th and above the 97.5th centiles with a definednormal group of fetuses with growth rates between the2.5th and 97.5th centiles, and comparing fetuses withgrowth rates below the 10th and above the 90th centileswith a defined normal group of fetuses with growth

Copyright 2008 ISUOG. Published by John Wiley & Sons, Ltd. Ultrasound Obstet Gynecol 2008; 32: 147–154.

Fetal growth rate 149

rates between the 10th and 90th centiles. Univariatecomparisons were made using the Chi-square test (morethan five observations in all cells) or Fisher’s exact test(five or fewer observations in one or more cells). P-valuesfor all hypotheses tested were two-sided and statisticalsignificance was set at P < 0.05. Data were processedusing the following software programs: Access 2000,Excel 2000 (Microsoft Corp., Redmond, WA, USA), SPSS11.0 (SPSS, Chicago, IL, USA) and SigmaPlot 10.0 (SyStatsoftware, Point Richmound, CA, USA).

RESULTS

Characteristics of the participants are presented inTable 1, and perinatal data are presented in Table 2.

Growth rate and gestational age at the first and secondultrasound examinations

The mean growth rate for BPD varied as a function ofgestational age at the time of the scans: the lower thegestational age at the time of the first or second scan,the higher the growth rate (Figures 1 and 2). Univariatevariance analysis confirmed this by showing significantdifferences in mean growth rates for fetuses with thefirst scan in week 11, 12, 13 or 14 (P < 0.001) andsignificant differences in mean growth rates for fetuseswith the second scan in week 17, 18, 19, 20 or 21(P < 0.001). Univariate variance analysis showed nosignificant interaction between gestational age at the timeof the first or second scan in accounting for variation infetal growth (P = 0.112). To compensate for the variationin growth rate as a function of gestational age at the timeof ultrasound examination, we calculated Z-scores specificto the pair of gestational ages at which each fetus wasscanned in order to standardize the growth rates.

Growth rate and adverse outcome

The number of infants delivered with very high or verylow birth weights varied as a function of growth rate:

Table 1 Characteristics of the study population (n = 8215)

Characteristic

Median(5th –95th

percentile)or n (%)

Data missing(n (%))

Maternal age at enrollment (years) 29.6 (23–37) 0 (0)Maternal height (cm) 169 (158–179) 161 (2.0)Maternal weight at enrollment (kg) 63 (51–86) 138 (1.7)Body mass index (kg/m2) 22 (19–30) 200 (2.4)Smoker 1358 (16.7) 71 (0.9)Caucasian 7854 (98.3) 223 (2.7)Parity 46 (0.6)

Nulliparous 5162 (63.2)Parous 3007 (36.8)

Conception 133 (1.6)Spontaneous 7805 (96.6)Assisted 277 (3.4)

Table 2 Characteristics and outcome of the pregnancies (n = 8215)

Characteristicn (%) or median(5th –95th centile)

Pre-eclampsia 108 (1.3)Diabetes 57 (0.7)Emergency Cesarean section 852 (10.4)Elective Cesarean section 474 (5.8)Forceps/vacuum delivery 920 (11.2)Gestational age at delivery (days) 282 (259–295)Gestational age at delivery* (weeks)

18–21 13 (0.2)22–33 100 (1.2)34–36 269 (3.3)37–41 6927 (84.3)42–43 744 (9.1)Unknown 162 (2.0)

Birth weight (g) 3543 (2654–4400)Birth weight unknown 161 (2.0)Gender

Male 4026 (49.0)Female 4026 (49.0)Unknown 163 (2.0)

Live birth 8033 (97.8)Perinatal death 28 (0.3)Spontaneous miscarriage 5 (0.1)Termination 18 (0.2)Neonatal death > 1 week postpartum 1 (0.0)Outcome unknown 130 (1.6)

*Delivery includes both spontaneous and induced births.

0.54

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m/d

ay)

0.46

0.4410 11 12

Gestational age (weeks) at first scan13 14

21

20

1918

17

15

Figure 1 Mean growth rate of biparietal diameter according togestational age at time of first ultrasound examination. The fivelines each represent a different gestational age (in weeks) at the timeof the second scan: 17 weeks ( ); 18 weeks ( ); 19 weeks ( );20 weeks ( ); 21 weeks ( ).

7.3%, 2.9% and 1.0% of the fetuses with growth ratesbelow the 2.5th centile, between the 2.5th and 97.5th

centiles and above the 97.5th centile, respectively, weredelivered with a birth weight below the mean − 2 SD(Table 3). Conversely, 0.5%, 2.4% and 6.5% of fetuseswith growth rates below the 2.5th centile, between the2.5th and 97.5th centiles and above the 97.5th centile,respectively, were delivered with a birth weight abovethe mean + 2 SD. The frequency of perinatal death also


150 Pedersen et al.

Table 3 Distribution of adverse outcomes

(n) % with a growth rate that was:

Adverse outcome < 2.5th centile < 10th centile2.5th –97.5th

centile 10th –90th centile > 90th centile > 97.5th centile

IUGRBirth weight < 2500 g (11/193) 5.70 (29/801) 3.62 (243/7660) 3.17 (212/6480) 3.27 (19/773) 2.46 (6/201) 2.99Birth weight < 2500 g at > 37 weeks* (4/183) 2.19 (10/765) 1.31 (75/7290) 1.03 (68/6169) 1.10 (1/726) 0.14 (0/187) 0.00Birth weight < mean − 2 SD† (14/193) 7.25 (33/800) 4.13 (220/7635) 2.88 (191/6460) 2.96 (12/769) 1.56 (2/201) 1.00

MacrosomiaBirth weight > 4500 g (0/193) 0.00 (20/801) 2.50 (278/7660) 3.63 (228/6480) 3.52 (43/773) 5.56 (13/201) 6.47Birth weight > 4500 g at > 37 weeks* (0/183) 0.00 (19/765) 2.48 (276/7290) 3.79 (228/6169) 3.69 (42/726) 5.78 (13/187) 6.95Birth weight > mean + 2 SD† (1/193) 0.52 (10/800) 1.25 (182/7635) 2.38 (144/6460) 2.23 (42/769) 5.46 (13/201) 6.47

Perinatal death (3/194) 1.55 (3/802) 0.37 (25/7644) 0.33 (20/6468) 0.31 (5/769) 0.65 (0/201) 0.00Spontaneous delivery‡ at:

22–33 weeks (1/138) 0.72 (4/582) 0.69 (37/5520) 0.67 (32/4690) 0.68 (5/525) 0.95 (3/139) 2.1634–36 weeks (3/140) 2.14 (16/594) 2.69 (158/5641) 2.80 (133/4791) 2.78 (21/541) 3.88 (9/145) 6.2142–43 weeks (10/147) 6.08 (33/611) 5.40 (338/5821) 5.81 (300/4958) 6.05 (21/541) 3.88 (6/142) 4.23

Pre-eclampsia (2/203) 0.99 (12/823) 1.46 (104/7808) 1.33 (86/6607) 1.30 (10/785) 1.27 (2/204) 0.98

*Excluding fetuses delivered at or before 37 weeks. †Birth weight below the sonographically estimated (US) mean for gestational age (GA)minus two SD; birth weight greater than US mean for GA plus two SD (Marsal et al.12). ‡For gestational age at delivery the denominator isdefined as the pre- or post-term group plus the term group. IUGR, intrauterine growth restriction.

0.54

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m/d

ay)

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0.4416 17 18

Gestational age (weeks) at second scan2019 21

1412

13

11

22

Figure 2 Mean growth rate of biparietal diameter according togestational age at time of second ultrasound examination. The fourlines each represent a different gestational age (in weeks) at the timeof the first scan: 11 weeks ( ); 12 weeks ( ); 13 weeks ( );14 weeks ( ).

varied as a function of growth rate: 1.6%, 0.3% and0.0% of fetuses with growth rates below the 2.5th centile,between the 2.5th and 97.5th centiles and above the97.5th centile, respectively, died perinatally. There wasa tendency for spontaneous delivery to vary as a functionof growth rate: 2.1%, 2.8% and 6.2% of fetuses withgrowth rate below the 2.5th centile, between the 2.5th and97.5th centiles and above the 97.5th centile, respectively,were delivered at weeks 34–36, whereas 6.1%, 5.8%and 4.2% of the fetuses with growth rate below the2.5th centile, between the 2.5th and 97.5th centiles andabove the 97.5th centile, respectively, were delivered atweeks 42–43. There was no systematic variation betweengrowth rate and the number of pregnancies diagnosedwith pre-eclampsia.

Growth rate and birth weight

Growth rates below the 2.5th centile were associated withIUGR (Table 4): fetuses with growth rates below the 2.5th

centile had an OR of 2.64 (95% CI, 1.51–4.62) for birthweights below the mean − 2 SD. High growth rates wereassociated with an increased risk for macrosomia: fetuseswith growth rates above the 90th and 97.5th centileshad ORs of 2.53 (95% CI, 1.78–3.60) and 2.83 (95%CI, 1.58–5.06), respectively, for birth weights above themean + 2 SD.

When adjusted for gestational age at delivery, parity,mother’s height, mother’s weight, mother being a smokerand fetal gender, growth rates below the 2.5th centilemeant a 105-g reduction in birth weight (Table 5). In asimilar model, growth rates below the 10th centile meanta 63-g reduction in birth weight. The combination ofall factors included in the model explained 45% of thevariation in birth weight.

Birth weight adjusted for gestational age at deliveryvaried as a linear function of growth rate adjusted forgestational age at the time of the first and second scans(R2 = 0.010, P < 0.001) (Figure 3). Growth rate for BPDbetween the first and second trimesters alone was able toexplain 1% of the variation in birth weight. Higher orderpolynomial models did not improve the fit.

Growth rate and perinatal mortality

There was an association between very low growth ratesand perinatal mortality (Table 6). Fetuses with growthrates below the 2.5th centile were at increased riskof perinatal mortality, with an OR of 4.79 (95% CI,1.43–15.99). All three cases of perinatal death withgrowth rates below the 2.5th centile were stillborn beforethe 25th week of gestation. Fetuses with growth rates



Table 4 Odds ratios (OR) for intrauterine growth restriction (IUGR) and macrosomia for fetuses with growth rates < 2.5th and 10th centiles,and growth rates > 90th and 97.5th centiles

Growth rate < 2.5th centile Growth rate < 10th centile

Adverse outcome OR (95% CI) P OR (95% CI) P

IUGRBirth weight < 2500 g 1.85 (0.99–3.44) 0.050 1.11 (0.75–1.65) 0.603Birth weight < 2500 g at > 37 weeks 2.15 (0.78–5.94) 0.128 1.19 (0.62–2.32) 0.612Birth weight < mean − 2 SD* 2.64 (1.51–4.62) 0.002† 1.41 (0.97–2.06) 0.071

Growth rate > 90th centile Growth rate > 97.5th centile

OR (95% CI) P OR (95% CI) P

MacrosomiaBirth weight > 4500 g 1.62 (1.56–2.26) 0.005† 1.83 (1.03–3.26) 0.035†Birth weight > 4500 g at > 37 weeks 1.60 (1.14–2.25) 0.006† 1.90 (1.07–3.38) 0.027†Birth weight > mean + 2 SD* 2.53 (1.78–3.60) < 0.0001† 2.83 (1.58–5.06) < 0.0001†

Reference groups for IUGR and macrosomia were fetuses without IUGR and macrosomia, respectively. *Marsal et al.12: mean − 2 SD,sonographically estimated fetal weight smaller than mean for gestational age minus 2 SD; mean + 2 SD, sonographically estimated fetalweight greater than mean for gestational age plus 2 SD. †Significant P-value.

Table 5 Linear regression: change in birth weight (B, in g) according to growth rate < 2.5th centile, gestational age at delivery, fetal genderand maternal characteristics

95% CI for B

B lowest highest t P

Constant 3317.69 3262.74 3372.64 118.01 0.001Growth rate < 2.5th centile −105.39 −165.06 −45.72 −4.01 0.001Gestational age at delivery (days from 280) 26.12 25.39 26.82 71.63 0.001Parous 131.56 112.38 150.73 13.56 0.001Male fetal gender 136.03 117.58 154.48 14.18 < 0.0001Smoking during pregnancy −74.09 −86.49 −61.69 −11.71 < 0.0001Maternal height (cm from 169) 9.36 7.81 10.91 11.80 < 0.0001Maternal weight (kg from 65) 5.17 4.31 6.03 11.80 < 0.0001

R2 = 0.452 (adjusted R2 = 0.452), P < 0.001.

Table 6 Odds ratios (OR) for perinatal mortality, week of delivery and pre-eclampsia for fetuses with growth rates < 2.5th and 10th centiles,and growth rates > 90th and 97.5th centiles

Growth rate< 2.5th centile

Growth rate< 10th centile

Growth rate> 90th centile

Growth rate> 97.5th centile

Adverse outcome OR (95% CI) P OR (95% CI) P OR (95% CI) P OR (95% CI) P

Perinatal mortality 4.79 (1.43–15.99) 0.031* 1.21 (0.36–4.08) 0.735 2.11 (0.79–5.64) 0.179 0.00 1.000Spontaneous delivery at:

22–33 weeks 1.08 (0.15–7.94) 0.610 1.01 (0.36–2.86) 1.000 1.40 (0.54–3.61) 0.415 3.27 (0.99–10.73) 0.07434–36 weeks 0.76 (0.24–2.41) 1.000 0.97 (0.57–1.64) 0.908 1.41 (0.89–2.26) 0.146 2.30 (1.15–4.59) 0.037*42–43 weeks 1.18 (0.62–2.27) 0.611 0.89 (0.61–1.28) 0.523 0.63 (0.39–0.99) 0.041* 0.72 (0.31–1.63) 0.425

Pre-eclampsia 0.74 (0.18–3.01) 0.737 1.12 (0.61–2.06) 0.711 0.98 (0.51–1.89) 0.948 0.73 (0.18–2.99) 0.664

Reference groups for perinatal mortality, preterm/post-term delivery and pre-eclampsia were live birth, delivery at weeks 37–41 andpregnancies without complications, respectively. *Significant P-value.


152 Pedersen et al.

4

2

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Z-s

core

: bir

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eigh

t ad

just

ed f

orge

stat

iona

l age

at

deliv

ery

−2

−4−4 −2 0

Z-score: growth rate adjusted forgestational age at first and second scans

2 4

Figure 3 Scatterplot of birth weight adjusted for gestational age atdelivery according to growth rate for biparietal diameter adjustedfor gestational age at the time of the first and second scans. Thelinear regression line is indicated: R2 = 0.010, P < 0.001,y = 0.099x.

below the 10th centile had a non-significant OR of 1.21(95% CI, 0.36–4.08) for perinatal mortality. Fetuses withgrowth rates above 90th centile were not at increased riskof perinatal mortality and no fetus with a growth rateabove the 97.5th centile died.

Growth rate and gestational age at delivery

There was an association between high growth rates andpreterm delivery (Table 6). Fetuses with growth ratesabove the 97.5th centile had a significant OR of 2.30(95% CI, 1.15–4.59) for spontaneous delivery in weeks34–36, while the OR of 3.27 (95% CI, 0.99–10.73)for spontaneous delivery in weeks 22–33 approachedsignificance.

DISCUSSION

The central finding of our study was the significantrelationship between BPD growth rate between the firstand second trimesters and adverse pregnancy outcome.Low growth rates were associated with increased ORsfor perinatal mortality and IUGR, and high growth rateswere associated with increased ORs for macrosomia andpreterm delivery. These findings underline the importanceof focusing on early growth, potentially to preventirreparable damage later in pregnancy. Traditionalthinking is that early growth is principally controlledgenetically and occurs at a constant exponential rate withlittle biological variation14; IUGR has been assumed tooccur mainly in the latter half of pregnancy. Our studyrefutes this, showing that fetal growth rate between thefirst and second trimesters does vary between fetuses and isassociated with perinatal death and low birth weight. Thisimplies that a suboptimal environment in the beginningof pregnancy limits fetal growth for the remainder of thepregnancy.

Studies have demonstrated that smaller-than-expectedultrasound measurements in the first half of pregnancyare associated with low birth weight7,15,16. It has beensuggested that this association is due to delayed ovulationrather than an association with early fetal growth.Our study demonstrated, in a healthy cohort of 8215pregnancies, that BPD growth rate between the firstand second trimesters is indeed associated with birthweight. By looking at growth rate instead of fetal sizein relation to last menstrual period, we overcame thepotential problem of delayed ovulation, and still observedan association. Bukowski et al.9 overcame the problem ofdelayed ovulation by studying pregnancies with knowndates of conception because of assisted reproduction,and they too observed an association between early fetalgrowth and birth weight. The association between lowbirth weight and growth in early pregnancy is now wellestablished, and is most likely to represent the early onsetof IUGR.

The study that investigated the relationship betweenbirth weight and smaller-than-expected embryo/fetus atfirst-trimester ultrasound reported an OR of 1.7 forbirth weights below 2500g and an OR of 2.8 for birthweights below the 5th centile for gestational age, ifthe embryo/fetus was 2–6 days smaller than expected7.The studies that looked at the relationship betweenbirth weight and smaller-than-expected fetuses at second-trimester ultrasound examination reported ORs around1.5 for birth weight below 2500g or birth weight belowthe mean − 2 SD, if the fetus was at least 7 days smallerthan expected8,15,16. We found an OR of 1.9 for birthweight below 2500g, and an OR of 2.6 for birth weightbelow the mean − 2 SD for fetuses with growth ratesbelow the 2.5th centile. The growth rate of the BPDbetween the first and second trimesters thus seems to bea better predictor for IUGR than is a single measurementof BPD in the second trimester, and a single measurementtaken in the first trimester seems to be a better predictorfor IUGR than is a single measurement taken in the secondtrimester7,9.

Growth rate from the second to the third trimestershows a very strong correlation to birth weight4, withfetuses with growth rates below the 10th centile betweensecond and third trimesters having an OR of 16.9 for birthweight below 2500 g and an OR of 36.1 for birth weightless than the 5th centile for gestational age. Although it isno surprise that fetal growth later in pregnancy can predictneonatal birth weight, little research has been conductedto evaluate the association between fetal growth rate fromthe second to the third trimester and the association withadverse pregnancy outcome4,17–19.

We found that fetuses with growth rates below the2.5th centile had an OR of 4.79 for perinatal death.Studies that describe the association between smaller-than-expected BPD in the second trimester and perinataldeath found ORs in the region of 2–3 for perinataldeath8,15,16. Accordingly, longitudinal measurements ofgrowth between the first and second trimesters seem more



sensitive for perinatal death than does a single second-trimester measurement of BPD. A recently published studywith data from 30 755 pregnancies reported that a uterineartery pulsatility index in the top decile at 22–24 weeks ofgestation was strongly associated with the subsequent riskof stillbirth. The association was stronger for stillbirth dueto placental causes than it was for unexplained stillbirths;furthermore, stillbirth due to placental causes occurredat earlier gestations than did unexplained stillbirths, withmedians of 30 and 38 weeks of gestation, respectively20.In our study, the three cases of perinatal death withgrowth rates below the 2.5th centile were all stillbornbefore the 25th week of gestation. It could be speculatedthat both the low growth rate and the early stillbirthwere caused by very poor placentation. Future studiescombining Doppler flow velocimetry with growth ratemeasurement could help to elucidate this.

We expected that low growth rate would be relatedto preterm birth, and high growth rate would be relatedto post-term birth. This would be in accordance with theknown link between IUGR and preterm birth and previousstudies that have reported associations between smaller-than-expected CRL/BPD and preterm birth or larger-than-expected CRL/BPD and post-term birth7,8,15,21. However,we observed the opposite: the higher the growth rate,the higher the risk for spontaneous preterm delivery,and the lower the growth rate, the higher the risk forspontaneous post-term delivery. The association betweenhigh growth rates and preterm delivery could be causedby early stretching of the myometrium as a result ofthe high fetal growth rate22. It is well known thatthe risk of preterm labor is higher in pregnancies withincreased intrauterine volume, such as in multifetalpregnancies23 and singleton pregnancies diagnosed withmacrosomia24 or polyhydramnios25. Fetuses with growthrates greater than the 97.5th centile had an OR of2.30 for spontaneous delivery week 34–36 and thesame group of fetuses had an OR of 2.83 for birthweight above the mean + 2 SD, suggesting an associationbetween preterm birth and macrosomia. While in thisstudy we focused on spontaneous delivery, other studiesrelating pre- and post-term birth to discrepancy ingestational age determined by last menstrual period and byultrasound7–9,15 did not distinguish between spontaneousand induced delivery; this could be one explanationfor the different results. Furthermore, gestational ageat delivery is difficult to interpret in a populationwith deviant sizes of CRL or BPD; if the gestationalage is determined sonographically, erroneous adjustmentaccording to fetal size could seriously bias the associationbetween smaller-than-expected fetuses and preterm birthand the association between larger-than-expected fetusesand post-term birth. Like birth weight, traditionallyparturition has been thought to be controlled byevents occurring in late pregnancy26. However, there isincreasing evidence supporting the possibility that thetiming of delivery is also determined predominantly earlyin gestation: low maternal pregnancy-associated plasmaprotein-A (PAPP-A) levels as early as weeks 8–14 are

associated with preterm birth27, while maternal plasmacorticotropin-releasing hormone (CRH) concentration inweeks 16–20 can identify groups of women who aredestined to experience normal term, preterm or post-term delivery28. In addition, there seems to be a geneticpredisposition to spontaneous preterm birth29.

We expected that low growth rate and pre-eclampsiawould both be a consequence of poor placentation, andtherefore low growth rate in early pregnancy couldbe a predictor for the subsequent development of pre-eclampsia. However, this study did not demonstratesuch an association. The prevalence of pre-eclampsiawas 1.3%, which is lower than the 3–5% prevalencenormally reported30. Our low prevalence probablyreflects a fairly healthy pregnant population with alow incidence of predisposing factors such as diabetes,extremes of reproductive age, hypertension and obesity31;furthermore, 98% of the population was Caucasian, anethnic group known to have a low risk of pre-eclampsia.

Growth rate varied as a function of the gestationalage at the time of the first and second ultrasoundexaminations. Variation in growth rate according togestational age in early pregnancy is in agreement with theresults of Milani et al.32; in a longitudinal growth studyincluding 130 healthy singletons followed from week12 to delivery with a minimum of five measurementsof head circumference, 5–10 weeks apart, growth ratewas described as parabolic, increasing until 17 weeks ofgestation, and then decreasing until delivery32.

In this study growth rate was adjusted only forgestational age. More complex models, additionallyincluding biochemical markers, uterine artery pulsatilityindex and adjustment for fetal gender and parentalcharacteristics, will most likely identify at-risk pregnanciesmore accurately. Such a model is outside the scope of ourstudy, but would probably be a valuable clinical tool,enabling resources to be focused more accurately on thepregnancies at highest risk.

In conclusion, we found a significant relationshipbetween the growth rate of BPD between the first andsecond trimesters and adverse pregnancy outcome. Lowgrowth rates were associated with increased ORs forperinatal mortality and IUGR and high growth rateswere associated with increased ORs for macrosomia andpreterm delivery. These findings underline the importanceof focusing on the first trimester instead of later pregnancywhen irreparable damage may already have occurred.

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3. Bernstein IM, Horbar JD, Badger GJ, Ohlsson A, Golan A.Morbidity and mortality among very-low-birth-weight neonateswith intrauterine growth restriction. The Vermont OxfordNetwork. Am J Obstet Gynecol 2000; 182: 198–206.


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9. Bukowski R, Smith GC, Malone FD, Ball RH, Nyberg DA,Comstock CH, Hankins GD, Berkowitz RL, Gross SJ,Dugoff L, Craigo SD, Timor-Tritsch IE, Carr SR, Wolfe HM,D’Alton ME. Fetal growth in early pregnancy and risk ofdelivering low birth weight infant: prospective cohort study.BMJ 2007; 334: 836–840.

10. Wojdemann KR, Christiansen M, Sundberg K, Larsen SO,Shalmi A, Tabor A. Quality assessment in prospective nuchaltranslucency screening for Down syndrome. Ultrasound ObstetGynecol 2001; 18: 641–644.

11. Robinson HP, Fleming JE. A critical evaluation of sonar‘‘crown-rump length’’ measurements. Br J Obstet Gynaecol1975; 82: 702–710.

12. Marsal K, Persson PH, Larsen T, Lilja H, Selbing A, Sultan B.Intrauterine growth curves based on ultrasonically estimatedfoetal weights. Acta Paediatr 1996; 85: 843–848.

13. Brown MA, Lindheimer MD, de Swiet M, Van Assche A,Moutquin JM. The classification and diagnosis of thehypertensive disorders of pregnancy: statement from theInternational Society for the Study of Hypertension in pregnancy(ISSHP). Hypertens Pregnancy 2001; 20: IX–XIV.

14. Deter RL, Buster JE, Casson PR, Carson SA. Individual growthpatterns in the first trimester: evidence for difference inembryonic and fetal growth rates. Ultrasound Obstet Gynecol1999; 13: 90–98.

15. Nakling J, Backe B. Adverse obstetric outcome in fetusesthat are smaller than expected at second trimester routineultrasound examination. Acta Obstet Gynecol Scand 2002;81: 846–851.

16. Nguyen T, Larsen T, Engholm G, Moller H. A discrepancybetween gestational age estimated by last menstrual periodand biparietal diameter may indicate an increased risk offetal death and adverse pregnancy outcome. BJOG 2000; 107:1122–1129.

17. Larsen T, Greisen G, Petersen S. Intrauterine growth correlation

to postnatal growth – influence of risk factors and complicationsin pregnancy. Early Hum Dev 1997; 47: 157–165.

18. Hata T, Kuno A, Akiyama M, Yanagihara T, Manabe A,Miyazaki K. Detection of small-for-gestational-age infantswith poor perinatal outcomes using individualized growthassessment. Gynecol Obstet Invest 1999; 47: 162–165.

19. Danielian PJ, Allman AC, Steer PJ. Is obstetric and neonataloutcome worse in fetuses who fail to reach their own growthpotential? Br J Obstet Gynaecol 1992; 99: 452–454.

20. Smith GC, Yu CK, Papageorghiou AT, Cacho AM, NicolaidesKH. Maternal uterine artery Doppler flow velocimetry and therisk of stillbirth. Obstet Gynecol 2007; 109: 144–151.

21. Gardosi J, Vanner T, Francis A. Gestational age and inductionof labour for prolonged pregnancy. Br J Obstet Gynaecol 1997;104: 792–797.

22. Oldenhof AD, Shynlova OP, Liu M, Langille BL, Lye SJ.Mitogen-activated protein kinases mediate stretch-induced c-fosmRNA expression in myometrial smooth muscle cells. Am JPhysiol Cell Physiol 2002; 283: C1530–C1539.

23. Gardner MO, Goldenberg RL, Cliver SP, Tucker JM, NelsonKG, Copper RL. The origin and outcome of preterm twinpregnancies. Obstet Gynecol 1995; 85: 553–557.

24. Lackman F, Capewell V, Richardson B, da Silva O, Gagnon R.The risks of spontaneous preterm delivery and perinatalmortality in relation to size at birth according to fetal versusneonatal growth standards. Am J Obstet Gynecol 2001; 184:946–953.

25. Hill LM, Breckle R, Thomas ML, Fries JK. Polyhydramnios:ultrasonically detected prevalence and neonatal outcome.Obstet Gynecol 1987; 69: 21–25.

26. Cunningham CF, Macdonald PC, Grant NF, Leveno KJ,Gilstrap LC. Partuition. In Williams Obstetrics (19th edn), Cun-ningham CF, Macdonald PC, Grant NF, Leveno KJ, GilstrapLC (eds.) Prentice Hall International: London, 1993; 297–361.

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Early Fetal Size and Growth as Predictors ofAdverse OutcomeNina Gros Pedersen, MD, Francesc Figueras, MD, PhD, Karen R. Wøjdemann, MD, PhD,Ann Tabor, MD, Prof, and Jason Gardosi, MD, Prof

OBJECTIVE: To evaluate the association between fetalsize and growth between the first and second trimestersand subsequent adverse pregnancy outcome.

METHODS: A cohort was created of 7,642 singletonpregnancies cared for in three obstetric units associatedwith Copenhagen University. Data were obtained fromultrasound measurements at 11–14 weeks (crown-rumplength, biparietal diameter) and 17–21 weeks (biparietaldiameter). Fetal size was assessed by gestation-specific zscores, and fetal growth between the first and secondtrimester was calculated individually using conditionalcentiles. The main outcome measures were preterm deliv-ery, smallness for gestational age, and perinatal death.

RESULTS: Slow growth of the biparietal diameter lessthan the 10th and less than the 2.5th conditional centilesbetween first and second trimesters occurred in 10.4%and 3.6% of the population, respectively. Biparietal di-ameter growth less than the 10th centile was associatedwith perinatal death before 34 weeks (risk 0.5% com-pared with 0.04%, odds ratio [OR] 16.0, confidenceinterval [CI] 2.9–88.7). Biparietal diameter growth lessthan the 2.5th centile was the best predictor of perinataldeath at any gestation, with a positive likelihood ratio of4.7 and an OR of 7.3 (CI 2.4–22.2). In contrast, thebiparietal diameter, dated by crown-rump length, did nothave an increased risk of perinatal death; however, therewas a mildly increased risk of small for gestational agebirth weight (less than the 10th customized centile) if thebiparietal diameter was below the 10th centile in the firsttrimester (risk 17% compared with 12%, OR 1.5, CI1.2–1.8) or in the second trimester (risk 15.8% comparedwith 12.4%, OR 1.3, CI 1.1–1.5).

CONCLUSION: Slow growth of the fetal biparietal diam-eter between the first and second trimesters of preg-nancy is a strong predictor of perinatal death before 34weeks.(Obstet Gynecol 2008;112:765–71)

LEVEL OF EVIDENCE: II

Fetal growth restriction is associated with adverseperinatal outcome,1 cerebral palsy,2 and adverse

effects in adult life.3 Recognition of intrauterinegrowth problems allows referral for investigationssuch as ultrasonography and Doppler to assess fetalwellbeing,4 and such tests have been shown to reduceperinatal mortality.5

Slow fetal growth is usually inferred retrospec-tively from the weight reached at birth, which can beadjusted or customized for maternal factors to distin-guish between constitutional and pathological small-ness. Cases thus determined to be small for gesta-tional age (SGA) have been found to be stronglylinked to perinatal mortality.6 Longitudinal studieshave shown that most of the constitutional variation inbirth weight starts in the third trimester of pregnancy.7

More recently, it has been suggested that a lowfirst-trimester measurement of crown-rump length(CRL) in pregnancies dated by last menstrual periodis also linked with adverse outcome such as prematu-rity and fetal growth restriction.8 One explanation forthis observation could be that these pregnancies aremisdated; a prolonged menstruation–conception in-terval has been shown to be associated with adverseoutcome.9–11 However, low CRL in the first trimesteralso has been found to be associated with subsequentlength of gestation and birth weight in pregnanciesachieved with assisted reproductive techniques.12

In everyday practice, the actual date of concep-tion is not likely to be known, and thus measurementof the first scan is needed to establish the gestationalage rather than the size for gestation of the embryo or

From the Department of Fetal Medicine and Ultrasound, Copenhagen UniversityHospital, Copenhagen, Denmark; the West Midlands Perinatal Institute,Birmingham, United Kingdom; and the Hospital Clinic, Barcelona, Spain.

Corresponding author: Prof. Jason Gardosi, West Midlands Perinatal Institute,Aston Cross, Birmingham B6 5RQ, UK; e-mail: [email protected].

Financial DisclosureThe authors have no potential conflicts of interest to disclose.

© 2008 by The American College of Obstetricians and Gynecologists. Publishedby Lippincott Williams & Wilkins.ISSN: 0029-7844/08

VOL. 112, NO. 4, OCTOBER 2008 OBSTETRICS & GYNECOLOGY 765

fetus. We set out to study and compare the effects ofdiminished early pregnancy size and growth, as as-sessed from measurements in the first and secondtrimesters, on subsequent pregnancy outcome, includ-ing SGA birth weight and perinatal death. We hy-pothesized that at these early gestations, growthwould be a better predictor of subsequent pregnancyoutcome than size for gestation.

MATERIALS AND METHODSData for this study were retrieved from the prospec-tive Copenhagen First Trimester Study for Downsyndrome and malformations,13 conducted from 1998to 2001 at the three obstetric departments associatedwith Copenhagen University Hospital. At all threedepartments, antenatal care included the offer of ananomaly scan at gestational weeks 18–20. Participantsin the study were offered an additional ultrasoundexamination and a serum blood sample at gestationalweeks 11–14. Written informed consent was ob-tained. The study protocol for the Copenhagen FirstTrimester Study was approved by the scientific ethicscommittee of the cities of Copenhagen and Frederiks-berg (No. KF 01–288/97) and the Danish Data Pro-tection Agency.

Of the 13,621 women who were invited to par-ticipate, 3,680 either refused or had an early miscar-riage, leaving 9,941 (73%) women who had a first-trimester ultrasound examination, including 9,710singleton pregnancies. A total of 8,215 of these 9,710(85%) pregnancies had a record of two ultrasoundexaminations, one at 11–14 weeks and another at17–21 weeks. A further 30 pregnancies ended inmiscarriage, 130 were lost to follow-up, and 219 hada congenital anomaly detected during or after preg-nancy. Finally, 194 cases were excluded because ofone or more items of missing data, including (nonex-clusively) fetal sex (n�15), parity (n�44), birth weight(n�11), gestational age at delivery (n�24), maternalheight (n�134), and maternal booking weight(n�157). This resulted in a study cohort of 7,642singleton pregnancies with complete data includingrecords of first- and second-trimester ultrasound scan.

The ultrasound examinations were performedby 13 sonographers or doctors experienced inobstetric ultrasonography and certified by the FetalMedicine Foundation in London. Ultrasound scanswere performed with Logic 700 MR (General Elec-tric, Milwaukee, WI), ACUSON Aspen Advanced(Aspen Ultrasound, Oceanside, CA), or Sonoline SI400 or Sonoline Sienna (Siemens AG, Erlangen,Germany).

Gestational age was assessed at the first ultra-sound examination by measurement of fetal CRL.14

Fetal biparietal diameter (BPD) was obtained at thefirst and second ultrasound scans by measuring in theaxial plane at the level of the thalami, the thirdventricle and the cavum septum pellucidum, from theouter border to the inner border of the skull.

Fetal size in the first trimester was assessed byCRL and BPD. Normal ranges were constructedusing previously described methodology.15 Naturallogarithmic-transformed CRL measurements were re-gressed against gestational age in days, calculatedfrom the last menstrual period. Separate linear, linear-cubic, and linear-quadratic regression models werefitted to estimate the relationship between fetal vari-ables and gestational age in days. The best-fittingmodel for each variable was selected. Standard devi-ation (SD) curves as functions of gestational age werecalculated by means of a quadratic polynomial regres-sion procedure of absolute residuals of the measure-ment of interest. The z scores ([measurement-mean]/SD) were calculated for assessing model fit. Normaldistribution of the z scores was checked with theShapiro-Francia W’ test.16 A small first-trimester CRLwas defined as less than the 10th centile. The Appen-dix, available at www.greenjournal.org/cgi/content/full/112/4/765/DC1, details the model coefficientsand SD for CRL.

For BPD, normal ranges were constructed bymeans of the same methodology described above.Natural logarithmic-transformed BPD measurementswere regressed against gestational age in days, calcu-lated from the first CRL measurement. Small first-and second-trimester BPD was defined as less thanthe 10th centile. The Appendix, available at www.greenjournal.org/cgi/content/full/112/4/765/DC1,details the model coefficients and SD for BPD.

Early fetal growth rate was estimated from theincrement between the first- and second-trimesterBPD measurements using conditional centiles.17 Thismethod uses individual predictions for the expectednormal BPD range (conditional centiles) during thesecond trimester on the basis of the first-trimesterBPD measurement. Each second-trimester BPD mea-surement value was thus assigned a centile, which wasconditional to the previous measurement in the samepregnancy. A fetus with a conditional BPD centilebelow the 10th centile was considered to have second-trimester growth restriction.

To establish the normal growth velocity for BPDbetween the first and second trimesters, a subset ofpregnancies was defined (n�6,164) that excludedsmoking, hypertension, and preexisting diabetes and

766 Pedersen et al Early Fetal Growth and Perinatal Outcome OBSTETRICS & GYNECOLOGY

resulted in a normal outcome, which included 1) livebirth, 2) birth weight more than 2,500 g, and 3)gestational age at delivery of 37 or more weeks datedby first-trimester CRL measurement. From this “nor-mal” subset, a sample of 300 cases was randomlyselected by means of a computerized algorithm (SPSS13.0, SPSS, Inc., Chicago, IL). Natural logarithmictransformation of the BPD resulted in the leastskewed distribution of residuals about the generaltrend. A linear model provided the best fit for thepaired data, and the growth curve for each fetus wasdescribed as:

Log [BDP(mm)] � � � �t � � gender

where �, �, and � were the parameters character-izing the individual fetus, t was the gestational age indays (centered at 112 days), and gender was female 0,male 1. The parameters were calculated by means ofmultilevel multiple linear regression assuming a ran-dom effect model. Repeated measurements in thesame fetus comprised level 1, and those in differentfetuses comprised level 2. Individual regression linesfor each variable were calculated for each fetus, andfrom these the regression lines for the whole groupwere derived.18 Modeling software MLwiN v 2.1(Centre for Multilevel Modeling, University of Bris-tol, Bristol, UK) was used.

The Appendix, available at www.greenjournal.org/cgi/content/full/112/4/765/DC1, depicts the for-mulae used for calculating the conditional intervals.

The main pregnancy outcomes assessed wereprematurity, SGA, and perinatal mortality. Prematu-rity (less than 37 weeks of gestation) was divided intoearly preterm (less than 34 weeks) and late preterm(34–36 weeks). Length of pregnancy was calculated indays and was based on CRL measurement at the firstultrasound scan.

Birth weight was assessed by customized percen-tiles, as previously described,19 which were individu-ally adjusted for maternal characteristics (bookingweight, height, parity, ethnic origin) using coefficientsderived from a Scandinavian population6 and avail-able as software (GROW Centile Calculator 5.12.1,2007, Gestation Network, www.gestation.net). Smallfor gestational age was defined as a customized birthweight centile less than 10.

Perinatal mortality was defined as stillbirth after24 weeks of gestation plus neonatal deaths through 28days of life.

Data from scans, including measurements andother details, were recorded in the ViewPoint FetalDatabase (ViewPoint GmbH, Webling, Germany). Out-come data were gathered from the clinical records of

each pregnancy, delivery, and neonate and entered intospreadsheet format using predefined criteria. Two-by-two tables were created to assess the association betweenearly fetal growth and pregnancy outcome, from whichsensitivity, specificity, and likelihood ratios were calcu-lated. Confidence intervals (CIs) were calculated byexact binomial method. The statistical package SPSS13.0 (SPSS, Inc.) was used for analysis.

RESULTSTable 1 details the characteristics of the 7,642 preg-nancies included in the study. There were no differ-ences in maternal characteristics between cases withor without perinatal deaths, except for a higher ma-ternal age at delivery (32.3 compared with 29.8,P�.01). Using the 10th centile as the cutoff identified9.7% of fetuses as having a low first-trimester CRL,9.8% as having a low second-trimester BPD, and10.4% as having slow growth between the first andsecond trimesters (Table 1).

The effects of small CRL or BPD (less than the10thcentile) and slow BPD growth (less than the 10th condi-tional centile) are detailed in Table 2 for length ofpregnancy, SGA at birth, and perinatal death. Low CRLhad no effect on these pregnancy outcomes, includingbirth weight less than the 10th (Table 2) as well as lessthan the 5th centile limit (odds ratio [OR] 1.17, CI0.87–1.57). Small BPD did have a modest associationwith SGA birth weight in both the first (OR 1.49, CI1.23–1.80) and second (OR 1.27, CI 1.07–1.52) trimes-ters. Neither of these parameters showed a link withperinatal mortality at any gestational age category at lessthan the 10th centile (Table 2) or at less than the 5th andless than the 2.5th centile cutoff limits.

In contrast, slow BPD growth (less than the 10thconditional centile) was a strong predictor of perinataldeath before 34 weeks (OR 16.0, CI 2.9–88.7). Fourof the seven perinatal deaths occurring before 34weeks had early fetal growth restriction, whereasnone of the 11 deaths from 34 weeks on had earlyfetal growth restriction. Early perinatal deaths werealso more likely to have a birth weight centile lessthan 10: four of seven deaths at less than 34 weekswere less than the 10th customized centile and twomore were borderline (11th and 16th centiles, respec-tively), whereas four of the 11 perinatal deaths at 34weeks or more were SGA. The maternal and preg-nancy characteristics of early BPD growth less thanthe 10th and more than the 10th conditional centileare compared in Table 3.

In Table 4, the diagnostic performance for peri-natal death at any gestation is examined for a range ofcutoff limits for early fetal growth restriction. The risk

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increases with the severity of growth restriction, witha conditional centile less than 2.5 having a positivelikelihood ratio of 4.7 and an OR of 7.3 (CI 2.4 – 22.2)for perinatal death.

DISCUSSIONIn this study, we assessed the link between fetalgrowth in early pregnancy and subsequent adversepregnancy outcome using sequential ultrasound mea-surements of BPD in the first and second trimesters.Previous publications on growth in early pregnancyhave looked at early fetal size in relation to lastmenstrual period 8,10–12,20 or early fetal size in relationto the day of conception when assisted reproductivetechnology was used.12 By using two sequential ultra-sound measurements of the BPD, our method be-comes independent of gestational-age error. It ap-pears that slow BPD growth is indicative of asubgroup of slow-growing fetuses that are at increasedrisk of perinatal mortality.

These findings have potentially important clinicalvalue. Early indication of an increased risk wouldallow more intensive fetal assessment and surveil-lance and informed considerations on when to adviseearly delivery to avoid fetal demise. Slow BPD growthbetween the first and second trimesters was moreprevalent in a subgroup that was slightly older, had ahigher body mass index, increased parity, was morelikely to be non-European, and had a higher preva-lence of diabetes. Although statistically significant,these differences were relatively small (Table 3) andare unlikely to explain the observed increased risk forperinatal death. When comparing the characteristicsof slow with normal BPD growth pregnancies thatresulted in perinatal death, only maternal age atdelivery reached significance (32.3 compared with29.8, P�.01). Much larger databases allowing mul-tivariate analyses would be needed to quantify therelative contributions of these factors to perinatalloss.

Table 1. Characteristics and Outcome of Included Pregnancies (N�7,642)

n % Median Range

Maternal age at entrance, y 29.6 18–51European 7,320 95.8Primipara 3,225 42.2Smoking 1,251 16.4

Missing data 60 0.8Maternal booking weight, kg 63 39–155Maternal height, cm 169 111–194Delivery

Emergency cesarean delivery 805 10.5Elective cesarean delivery 450 5.9Forceps or vacuum 869 11.4Missing 2 0.03

First scanGestational age, d* 91 77–104CRL less than 10th centile 725 9.7

Second scanGestational age, d 133 119–153BPD less than 10th centile 751 9.8

BPD growth between 1st and 2nd scanLess than 10th conditional centile 796 10.4

Gestational age at delivery, d† 282 175–304Less than 34 wk 85 1.134–36 wk 262 3.437 or more wk 7,295 95.5

Mean SD

Birth weight, g 3,539 550Less than 10th customized centile 978 12.8

Stillbirths 16 0.21Neonatal death 2

Less than 1 wk 11 wk or more 1

CRL, crown-rump length; BPD, biparietal diameter; SD, standard deviation.* Total of 7,472 cases with recorded last menstrual period.† Dated by CRL.


Table 2. Association Between Perinatal Outcome and Small Crown-Rump Length, Small BiparietalDiameter, and Slow Biparietal Diameter Growth

n

First-Trimester CRLLess Than 10th Centile

OR 95% CI PYes (n�725) No (n�6,747)

GA at delivery, wkLess than 34 82 9 73 0.84 0.59–1.19 .3134 to less than 37 255 29 226 1.2 0.81–1.78 .3637 or more 7,135 687 6,448 1.15 0.57–2.31 .69

SGA cust, wkLess than 34 27 2 25 0.55 0.1–2.84 .4734 or more 923 89 834 0.99 0.78–1.25 .93Total 950 91 859 0.98 0.78–1.24 .89

Perinatal death, wkLess than 34 7 0 7 — — —34 or more 11 1 10 0.93 0.12–7.26 .94Total 18 1 17 0.55 0.07–4.11 .55

First-Trimester BPDLess Than 10th Centile

Yes (n�856) No (n�6,786)


SGA cust, wkLess than 34 27 5 25 2 0.53–7.55 .334 or more 923 142 806 1.48* 1.21–1.8 �.001Total 950 147 831 1.49* 1.23–1.8 �.001

Perinatal death, wkLess than 34 7 2 5 3.5 0.6–21.08 .1534 or more 11 1 10 0.79 0.1–6.2 .82Total 18 3 15 1.58 0.46–5.5 .46

Second-Trimester BPDLess Than 10th Centile

Yes (n�751) No (n�6,891)


SGA cust, wkLess than 34 30 4 26 4.07 0.7–23.7 .134 or more 948 115 833 1.31* 1.06–1.62 .012Total 978 119 859 1.27* 1.07–1.52 .008

Perinatal death, wkLess than 34 7 1 6 2.43 0.24–24.33 24.3334 or more 11 1 10 0.9 0.12–7.15 .93Total 18 2 16 1.15 0.36–5 .85

BPD Growth Less Than 10thConditional Centile

Yes (n�796) No (n�6,846)


SGA cust, wkLess than 34 30 6 24 3.19 0.82–12.36 .0834 or more 948 111 837 1.16 0.94–1.44 .12Total 978 117 861 1.2 0.97–1.48 .09

Perinatal death, wkLess than 34 7 4 3 16.0* 2.89–88.7 �.00134 or more 11 0 11 — — —Total 18 4 14 2.46 0.81–7.51 .1

CRL, crown-rump length; OR, odds ratio; CI, confidence interval; GA, gestational age; SGA cust, small for gestational age (less than the10th centile) by customized standards; BPD, biparietal diameter.

* Statistically significant.


The BPD measurements can be taken as part ofroutine clinical practice, which usually already offersscans at approximately 11–14 and 17–20 weeks.Having two sequential measurements allows the eval-uation of early growth proper, rather than size, and isindependent of gestational age. By using conditionalcentiles, we were further able to avoid the effect ofmeasurement error and regression to the mean, whichis inherent in repeat measurements and which canattenuate the relationship between growth and ad-verse outcome.

Many of these early deaths demonstrated signifi-cant smallness for gestational age. This is consistentwith reports that preterm stillborn fetuses are morelikely to be SGA than fetuses stillborn at term.21 Inmost fetal deaths before 34 weeks in this study, agrowth deficit can be demonstrated to have startedbetween the first and second trimesters.

The study also was able to examine associationsbetween fetal size in the first and second trimesters

and pregnancy outcome. We have found no linkbetween small first-trimester CRL and subsequentpreterm delivery, SGA birth weight, or perinataldeath. This is in contrast to a previously describedassociation between a small CRL and low birthweight.8 A possible explanation for those findings isthat, instead of representing slow early growth, asmaller-than-expected CRL could simply be due toan overestimate of the age of the fetus at the time ofthe scan. This can occur either because of incorrectmenstrual dates or because of delayed ovulation inthe conception cycle. A prolonged menstruation–conception interval has been shown to be linked toadverse outcome, including prematurity, fetal growthrestriction, and stillbirth.9–11

More recently, small first-trimester CRL mea-surements in a cohort of assisted-reproduction preg-nancies with certain conception dates also haveshown an association with low birth weight.12 It isuncertain whether such findings can be extrapolated

Table 4. Diagnostic Performance for Perinatal Mortality (Any Gestation) With Biparietal DiameterGrowth by Conditional Centiles, Using Various Cutoffs

Frequency(%)

A0/N0(%)

A1/N1(%)

Se(95% CI)

Sp(95% CI) �LHR �LHR

OR(95% CI)

BPD growthConditional centile

Less than 15 1,131/7,792 (14.8) 14/6,511 (0.22) 4/1,131 (0.35) 22.2 (6.4–47,7) 85.2 (84.4–86) 1.5 0.91 1.65 (0.54–5.01)Less than 10 796/7,792 (10.4) 14/6,846 (0.2) 4/796 (0.5) 22.2 (6.4–47,7) 89.6 (88.9–90.3) 2.14 0.98 2.46 (0.81–7.51)Less than 5 458/7,792 (6.0) 14/7,184 (0.19) 4/458 (0.87) 22.2 (6.4–47,7) 94 (93.5–94.6) 3.73 0.83 4.51 (1.48–13.76)Less than 2.5 272/7,792 (3.6) 15/7,370 (0.2) 3/272 (1.1) 16.7 (3.5–41.4) 96.5 (96.1–96.9) 4.72 0.86 7.32 (2.41–22.2)

A0, cases among fetuses with normal growth during early second trimester (N0); A1, cases among fetuses with early fetal growth restriction(N1); Se, sensitivity; Sp, specificity; LHR, likelihood ratio; OR, odds ratio; CI; confidence interval; BPD, biparietal diameter.

Table 3. Population Characteristics by Early Fetal Growth

BPD Growth

Less Than 10th Centile(n�796)

10th Centile or More(n�6,846) OR (95% CI) P*

Maternal age, y, mean (SD) 30.3 (4.1) 29.7 (4.2) �.001Maternal age 35 y or older 95 (11.9) 683 (10.0) 1.22 (0.97–1.54) .08BMI (kg/m2) mean (SD) 23.4 (4) 22.8 (3.8) .001BMI 30 or higher 58 (7.3) 350 (5.1) 1.46 (1.09–1.95) .01Primiparity 300 (37.7) 2,935 (42.7) 0.81 (0.69–0.94) .006Non-European ethnicity 51 (6.4) 271 (4) 1.66 (1.22–2.26) .001Smoking† 113 (15.1) 1,138 (16.7) 0.83 (0.67–1.02) .26Male fetus 407 (51.1) 3,392 (49.5) 1.07 (0.92–1.23) .4Medical condition

Diabetes 13 (1.6) 42 (0.6) 2.69 (1.44–5.03) .001Hypertension 2 (0.3) 47 (0.7) 0.36 (0.09–1.5) .15Other‡ 32 (4) 291 (4.3) 0.94 (0.65–1.37) .76

BPD, biparietal diameter; OR, odds ratio; CI, confidence interval; SD, standard deviation; BMI, body mass index.Data are n (%) unless otherwise indicated.* T-test or Pearson �2.† Sixty cases missing.‡ Includes neurologic, cardiovascular, renal, digestive, respiratory, and immunologic diseases.


to spontaneously conceived pregnancies. As the au-thors of that study noted, even in pregnancies wherethe observed first-trimester size was equal to thatexpected, there was a higher rate of low birth weightand SGA.12 Furthermore, practical applicability ofthis assessment would be limited in spontaneouslyconceived pregnancies because the exact date ofconception is usually not known and a CRL measure-ment cannot be used simultaneously for establishinggestational age and assessing fetal size for gestation.

We also looked at the association between BPD sizeand adverse outcome, using CRL to establish gestationalage. In contrast to our findings for slow BPD growthbetween the first and second trimesters, no significantassociation with perinatal mortality was observed. How-ever, a small BPD in either the first or second trimesterdid show a significant, albeit modest, association withsubsequent SGA birth weight (Table 2). This finding isconsistent with previous reports of ultrasound measure-ments in both the first and second trimesters.10,11,20,22

However, the latter studies had to rely on menstrualdating, which always raises the question of whethersome of the effect on birth weight is due to misdating ofthe pregnancy. Because our data included a first-trimes-ter measurement of CRL to establish the dates accu-rately, our analysis can confirm and quantify the linkbetween a smaller-than-expected second-trimester BPDand subsequent low birth weight. Furthermore, we showthat this association can be demonstrated already late inthe first trimester.

We excluded congenital anomalies from the anal-ysis because the primary purpose of recruitment tothis study was to screen for congenital defects. Anom-alies are already known to have an association withearly SGA,23 and a smaller-than-expected BPD mea-surement ought to prompt more intensive investiga-tion to establish whether a structural or chromosomalabnormality is present.

Our study shows that, in pregnancies with nocongenital anomalies, early fetal growth restriction asindicated by serial BPD measurements is a significantindicator of increased perinatal mortality risk. Suchinformation can be derived easily from ultrasound in-vestigations already carried out routinely in mostpregnancies.

REFERENCES1. M Kady S, Gardosi J. Perinatal mortality and fetal growth restric-

tion. Best Pract Res Clin Obstet Gynaecol 2004;18:397–410.2. Jarvis S, Glinianaia SV, Torrioli MG, Platt MJ, Miceli M, Jouk PS,

et al. Cerebral palsy and intrauterine growth in single births:European collaborative study. Lancet 2003;362:1106–11.

3. Godfrey KM, Barker DJ. Fetal nutrition and adult disease. AmJ Clin Nutr 2000;71:1344S–52S.

4. Royal College of Obstetricians and Gynaecologists. The investiga-tions and management of small-for gestational-age fetus. RCOGGreen Top Guideline No. 31. London (UK): RCOG Press; 2002.

5. Alfirevic Z, Neilson JP. Doppler ultrasonography in high-riskpregnancies: systematic review with meta-analysis. Am JObstet Gynecol 1995;172:1379–87.

6. Clausson B, Gardosi J, Francis A, Cnattingius S. Perinataloutcome in SGA births defined by customised versus popula-tion-based birthweight standards. BJOG 2001;108:830–4.

7. de Jong CL, Gardosi J, Baldwin C, Francis A, Dekker GA, vanGeijn HP. Fetal weight gain in a serially scanned high-riskpopulation. Ultrasound Obstet Gynecol 1998;11:39–43.

8. Smith GC, Smith MF, McNay MB, Fleming JE. First-trimestergrowth and the risk of low birth weight. N Engl J Med1998;339:1817–22.

9. Gardosi J, Francis A. Early pregnancy predictors of pretermbirth: the role of a prolonged menstruation-conception inter-val. BJOG 2000;107:228–37.

10. Larsen T, Nguyen TH, Greisen G, Engholm G, Moller H. Does adiscrepancy between gestational age determined by biparietaldiameter and last menstrual period sometimes signify early intra-uterine growth retardation? BJOG 2000;107:238–44.

11. Nakling J, Backe B. Adverse obstetric outcome in fetuses that aresmaller than expected at second trimester routine ultrasoundexamination. Acta Obstet Gynecol Scand 2002;81:846–51.

12. Bukowski R, Smith GC, Malone FD, Ball RH, Nyberg DA,Comstock CH, et al. Fetal growth in early pregnancy and riskof delivering low birth weight infant: prospective cohort study.BMJ 2007;334:836.

13. Wojdemann KR, Shalmi AC, Christiansen M, Larsen SO,Sundberg K, Brocks V, et al. Improved first-trimester Downsyndrome screening performance by lowering the false-posi-tive rate: a prospective study of 9941 low-risk women. Ultra-sound Obstet Gynecol 2005;25:227–33.

14. Robinson HP, Fleming JE. A critical evaluation of sonar“crown-rump length” measurements. Br J Obstet Gynaecol1975;82:702–10.

15. Royston P, Wright EM. How to construct “normal ranges” forfetal variables. Ultrasound Obstet Gynecol 1998;11:30–8.

16. Royston P. A pocket-calculator algorithm for the Shapiro-Francia test for non-normality: an application to medicine. StatMed 1993;12:181–4.

17. Royston P. Calculation of unconditional and conditional ref-erence intervals for foetal size and growth from longitudinalmeasurements. Stat Med 1995;14:1417–36.

18. Goldstein H. Multilevel statistical models. 2nd ed. London(UK): University of London; 1995.

19. Gardosi J, Mongelli M, Wilcox M, Chang A. An adjustable fetalweight standard. Ultrasound Obstet Gynecol 1995;6:168–74.

20. Thorsell M, Kaijser M, Almstrom H, Andolf E. Expected dayof delivery from ultrasound dating versus last menstrualperiod–obstetric outcome when dates mismatch. BJOG 2008;115:585–9.

21. Gardosi J, Mul T, Mongelli M, Fagan D. Analysis of birth-weight and gestational age in antepartum stillbirths. Br J ObstetGynaecol 1998;105:524–30.

22. Rasmussen S, Kiserud T, Albrechtsen S. Foetal size and bodyproportion at 17–19 weeks of gestation and neonatal size,proportion, and outcome. Early Hum Dev 2006;82:683–90.

23. Nikkila A, Kallen B, Marsal K. Fetal growth and congenitalmalformations. Ultrasound Obstet Gynecol 2007;29:289–95.


Maternal serum levels of placental Growth Hormone in early pregnancy, but not of human

Placental Lactogen, and Insulin Growth Factor-I, are positively associated with fetal growth

in first trimester of human pregnancy

N. G. Pedersen*, A. Juul

†, M Christiansen

‡, K. R. Wøjdemann

§ and A. Tabor*

*Department of Fetal Medicine and Ultrasound, Rigshospitalet, Copenhagen University Hospital,

Denmark

†Department of Growth and Reproduction, Rigshospitalet, Copenhagen University Hospital,

Denmark

‡ Department of Clinical Biochemistry, Statens Serum Institute, Copenhagen, Denmark

§ Department of Obstetrics and Gynecology, Roskilde University Hospital, Roskilde, Denmark

Corresponding author:

Dr N.G. Pedersen, Department of Fetal Medicine and Ultrasound, Rigshospitalet, Copenhagen

University Hospital, Afsnit 4031, Blegdamsvej 9, 2100 Copenhagen, Denmark

E-mail: [email protected]

Tel: +45 35 45 37 22

Fax: +45 35 45 37 39

Short title: Fetal growth rate

Keywords: Growth rate, BPD, PGH, HPL, IGF-1, ultrasound

2

Abstract

Objective: To investigate if maternal levels of human Placental Lactogen (hPL), Placental Growth

Hormone (PGH) and Insulin Growth Factor-I (IGF-I) are associated with growth rate of the

biparietal diameter (BPD) in first half of pregnancy.

Methods: Data on 8215 singleton fetuses from the Copenhagen First Trimester Study with

measurements of BPD from ultrasound scans performed in week 11-14 and week 17-21 of

pregnancy were analysed. Growth rate was defined as millimetres of growth/day of BPD between

the two scans. Fetuses with growth rate below the 2.5th

centile (low growth rate, n=203) and above

the 97.5th

centile (high growth rate, n=203) were identified. As a reference group 212 fetuses with

growth rate around the median were identified (intermediate growth rate). Out of the 618 selected

cases in the three growth rate groups a total of 463 cases had a blood sample taken (equal to 5.6%

of the original sample size of 8215 pregnancies). The maternal blood serum concentration of hPL,

PGH and IGF-I was determined in the three different growth rate groups. Simple linear and

multiple linear regression analysis, including adjustment for known and potential confounders were

used to compare serum levels between the groups.

Results: Simple linear regression showed a differences in serum level of log10 (PGH) between the

high and intermediate growth rate groups (p=0.037). When adjusted for maternal weight and CRL

multiple linear regression analysis confirmed this difference. Fetuses with high growth rates had

when adjusted for CRL and maternal weight a 12% (95%CI: 2%-20%, p=0.009) higher maternal

serum level of PGH than those with intermediate growth rates. No differences in hPL and IGF-I

levels between the three different growth rate groups were found after simple and multiple linear

regression analysis.

Conclusion: Maternal PGH levels were higher in women carrying fetuses with high first trimester

growth rates compared to controls both in a simple unadjusted analysis and in analyses adjusted for

3

known and potential confounders. Thus, PGH may be involved in fetal growth regulation already in

early human pregnancy.

4

Introduction

Fetal growth in early pregnancy has traditionally been considered to be under tight genetic control

and to occur at a constant exponential rate with little biologic variation; intrauterine growth

restriction has been assumed to occur mainly in the latter half of pregnancy. Recent studies,

however, have shown that fetal growth in first half of pregnancy does vary between fetuses1 and

that decreased early fetal growth rate is associated with perinatal death, low birth weight and

preterm delivery 2-5

. The regulation of fetal growth in the early half of pregnancy has not been

clarified. In postnatal life growth hormone (GH) plays an important role, but the role of GH in fetal

growth remains uncertain.

The GH gene cluster contains a single gene expressed in the anterior pituitary (GH1) plus four

genes expressed in the placenta (SHL1, CSH1, GH2, CSH2). GH1 encodes pituitary Growth

Hormone (pGH), GH2 encodes Placental GH (PGH), while CSH1 and CSH2 encode hPL 6. PGH is

a ligand of maternal GH receptors, and hPL is a ligand of maternal prolactin receptors. hPL and

PGH can be detected in the maternal circulation at 6 weeks’ gestation and their concentrations

increase with advancing gestation; several studies have suggested a role for hPL and PGH in the

control of fetal growth 7,8,9

. Pregnancies complicated by intrauterine growth restriction (IUGR)

show reduced concentration of maternal and fetal serum hPL as well as reduced maternal serum

levels of PGH 10

. In second half of pregnancy it is known that the concentrations of PGH and IGF-I

in maternal serum are positively correlated 9, and in pregnancies with IUGR maternal IGF-I serum

levels are found to be significantly lower than the serum levels found in normal pregnancies 11

.

Until recently the general opinion was that PGH does not cross the placenta into the fetal

circulation, and the effect of PGH on fetal growth was considered to be indirectly mediated by

effects on maternal metabolism, possibly via IGF-I, hereby regulating substrate supply to the fetus.

5

However a recent study has demonstrated that PGH can be detected in cord blood and it may

therefore have direct effect on fetal growth 12

.

We wished to investigate if maternal levels of hPL, PGH and IGF-I were correlated to growth rate

in early pregnancy; speculating that high levels of hPL, PGH and IGF-I would stimulate growth and

lower levels would be associated with slower growth rates. No previous studies have examined the

correlation between fetal growth rate and maternal hormones in the first half of pregnancy.

Materials and methods

Study population

Data was retrieved from the Copenhagen First Trimester Study for Down´s syndrome and

malformations conducted 1998-2001 13

. A total of 13,621 women referred to three obstetric

departments in Copenhagen University Hospital were invited to participate. Participation entailed

the choice of an ultrasound examination and / or a blood sample in gestational week 11-14.

Additionally at all three departments, routine antenatal care included the offer of one anomaly scan

at around 18-20 week’s gestation. Of the 13,621 women who were invited to participate, 3,680

either refused or had an early miscarriage, leaving 9,941 (73%) women who had a first trimester

ultrasound examination, including 9710 singleton pregnancies. A total of 8,215 of these 9,710

(85%) singleton pregnancies had a record of two ultrasound examinations, at 11-14 weeks and 17-

21 weeks, respectively. Data, including indications and diagnoses, from all scans performed during

the pregnancies were recorded in ViewPoint Fetal Database (ViewPoint GmbH, Webling,

Germany).

6

Ultrasound

The ultrasound examinations were performed by 13 sonographers or doctors experienced in

obstetric ultrasound and certified by The Fetal Medicine Foundation in London, UK. The

ultrasound examinations in week 11-14 were all performed using a Logic 700 MR scanner (General

Electric, Milwaukee, WI, USA). Those in week 17-21 were performed using one of the following

machines: Acuson Aspen Advanced, Sonoline SI 400 or Sonoline Sienna (Siemens AG, Erlangen,

Germany). Gestational age was determined by CRL14

. BPD measurements were obtained in an

axial plane at the level of the thalami, the third ventricle and cavum septi pellucidi, from the outer

border to the inner border of the skull.

Growth rate

Pregnant women were categorized by fetal growth into three groups (low, intermediate and high

fetal growth rates). We defined growth rate as millimetres of growth in BPD per day between the

two sonographic measurements. As the growth from week to 11 to 21 isn’t linear 15

growth rate

was standardized according to the gestational age measured in weeks at the time of the scans. A Z-

score was thus calculated for each fetus by subtracting the mean growth rate for all fetuses that were

scanned the same two weeks and dividing by the SD of the growth rate for this group. Z-score =

(observed growth rate - group mean growth rate) / (group growth rate SD) 5. The individual Z-

scores were transformed into centiles, and all fetuses with growth rate below the 2.5th

centile (low

growth rate, n=203) and above the 97.5th

centile (high growth rate, n=203) were identified. As a

reference group all fetuses with growth rates from the 50th

to the 51st centile were identified

(intermediate growth rate, n=212). Out of the 618 selected cases in the three growth rate groups a

total of 463 cases had a blood sample taken (equal to 5.6% of the original sample size).

7

Blood samples

Blood samples were drawn from an antecubital vein, on the same day as the first trimester

ultrasound in gestational week 11-14, and centrifuged at 3000 rpm for 10 minutes. Serum was

stored at -20 Celsius until analyses. Maternal blood from the three different growth rate groups was

analysed. The number of women that had a blood sample taken was 143/203 (70%) from the group

with low growth rate, 166/212 (78%) from the group with intermediate growth rate and 154/203

(75%) from the group with high growth rate. The actual number of blood samples analysed for

hPL, PGH and IGF-I varied slightly from the above numbers, as the amount of serum left, was not

always sufficient to perform the analysis for all three biochemical markers. The exact number of

pregnancies in the three different growth rate groups that had maternal serum levels of hPL, PGH

and IGF-I measured is depicted in Table 3.

Hormone assays

PGH was determined by a solid-phase enzyme linked immunosorbent assay (hPGH ELISA

EL2017, Biocode Hycel, Liege Belgium). Standards were recombinant hPGH provided by the

manufacturer. Analytical sensitivity was 1.9 ng/ml. In our hands, intra-assay variation was 2.6%

(at 8.2 ng/ml) and 3.7% (at 38.1 ng/ml), respectively. Inter-assay variation was 5.7% (at 8.2 ng/ml)

and 3.3% (at 38.1 ng/ml), respectively.

HPL was determined by a modification of a commercially available solid-phase

enzymeimmunoassay (HPL ELISA EIA-1283, DRG Instruments GmbH, Marburg, Germany). The

sensitivity of the assay was increased by using standards (produced by diluting provided standards

in the 0-standard) at 0.02 mg/l, 0.04 mg/l, 0.16 mg/l, 0.32 mg/l, 0.64 mg/l, and 1.25 mg/l and

increasing the incubation time of samples from 30 minutes to 3 hours. The standard material was

8

provided and quantified by the assay manufacturer. The intra-asay variation was 8.3% (at 0.76

mg/l) and 3.2% (at 1.2 mg/l), respectively. The inter-assay variation was 14% (at 0.76 mg/l) and

7.1% (at 1.2 mg/l), respectively.

IGF-I was determined by a solid-phase enzyme-labelled chemiluminescent immunometric assay

(Immulite 2000, Diagnostic Products Corporation, LA, USA). Standards were calibrated towards

the WHO NIBSC IRR 87/518. Analytical sensitivity was 20 ng/ml. In our hands, intra-assay CV%

were 1.9% (at 51 ng/ml), 2.0% (at 239 ng/ml) and 2.1% at (472 ng/ml), respectively. Interassay

CV% were 8.9% (at 72 ng/ml), 5.9% (at 273 ng/ml), and 8.2% (at 395 ng/ml), respectively.

Statistical analyses

The serum values of hPL, PGH and IGF-I all showed a distribution that was skewed to the right and

the values were therefore log10 transformed to obtain approximate normality. Q-Q plots were used

to determine if the data were normally distributed.

To compare categorized characteristics of the study population between the three growth rate

groups univariate comparisons were made using the Chi-square test (more than five observations in

all cells) or Fisher’s exact test (five or fewer observations in one or more cells). Continuous

characteristics were compared by means of one-way ANOVA.

Linear regression analyses were used to compare mean serum levels of log10(hPL), log10( PGH) and

log10 (IGF-I) between the three different growth rate groups with the intermediate growth rate group

as a reference. Analyses adjusted for CRL and mother’s weight (kg) were also performed as both

these variables are known to affect serum levels of hPL16

, PGH 17

and IGF-I 9. Other characteristics

of the mother and fetus, that we expected could influence serum levels of hPL, PGH and IGF-I and

where a significant difference between the three growth rate groups (parity and fetal sex) was

9

found, were included in supplementary analyses. P values for all hypotheses tested were two-sided

and statistical significance was set at P < 0.05.

Data were processed using the software programs Access 2000, Excel 2000 (Microsoft Corp.,

Redmond, WA, USA), SPSS 11.0 (Chicago, IL. USA) and SigmaPlot 10.0 (SyStat software, Point

Richmound, Ca, USA).

Ethical considerations

Written informed consent was obtained from the participants, and the scientific ethics committee of

the cities of Copenhagen and Frederiksberg (No. (KF) 01-288/97) and the Data Protection Agency

approved the study protocol.

Results

Data on prenatal characteristics and pregnancy outcomes are shown in Table 1. Mean BPD at both

the first and the second scan was significantly different between the three growth rate groups. At

the first scan the higher the growth rate the lower the mean BPD (21.4 mm, 22.6 mm and 23.6 mm

for high, intermediate and low growth rate respectively, p<0.001). At the second scan the higher

the growth rate the higher the mean BPD (46.2 mm, 44.2 mm and 40.5 mm for high, intermediate

and low growth rate respectively, p<0.001). The group of fetuses with the highest growth rate was

significantly younger than fetuses from the two other growth rate groups at the time of the second

scan (131.0 days vs. 134.6 days and 134.4 days, p<0.001). The fetuses from the low growth rate

group were delivered earlier, had a lower birth weight and the lowest percentage of live births.

There was a predominance of female fetuses in the low and intermediate growth rate group

compared to the high growth rate group (57% and 56% vs. 30% in the high growth rate group,

p<0.001). The percentage of caesarean sections was highest in the group of fetuses with a high

10

growth rate. The individual fetal growth rates for the three different groups are illustrated in Figure

1.

Data on maternal characteristics are shown in Table 2. The percentage of nulliparous mothers was

significantly different between the three growth rate groups, the higher the growth rate the higher

the percentage of nulliparous women (75%, 62% and 59% for high, intermediate and low growth

rates, respectively, p=0.009). Otherwise the mothers of the fetuses were similar with regard to

characteristics that could influence the growth rate.

Maternal serum concentrations of hPL, PGH and IGF-I according to growth rate group are

illustrated in Figure 2.

Simple linear regression showed a differences in serum level of log10 (PGH) between the high and

intermediate growth rate groups (Table 3, p=0.037). When adjusted for maternal weight and CRL

multiple linear regression analysis confirmed this difference (Table 4). Fetuses with high growth

rates thus had a higher maternal serum level of log10 (PGH) than those with intermediate growth

rate equal to B=0.05 (95%CI: 0.01-0.09). The increase in log10 (PGH) may be translated to a

relative increase of PGH of 12% (95%CI: 2%-20%) by taking the antilog10 of B and its CI. Fetuses

with low growth rates also had higher level of PGH compared to those with intermediate growth

rate, but this did not reach statistical significance. When subdividing the analysis for PGH by fetal

sex simple linear regression showed a significant difference in maternal serum levels of log10 (PGH)

between the high and intermediate growth rate group for male fetuses (p=0.043, n=221). The

female fetus group was slightly smaller (n=197), and here we did not observe a difference. For

log10 (PGH) there was no interaction between fetal growth rate and sex (p=0.204).

No difference was found for the levels of log10(hPL) and log10(IGF-I) between the three different

growth rate groups with simple linear regression analysis or after adjustment for maternal weight

and CRL. For log10(hPL), log10 (PGH) and log10(IGF-I) the serum levels varied significantly as a

11

function of CRL, and for log10(hPL) and log10 (PGH) the serum levels varied significantly as a

function of maternal weight (Table 4). When the serum levels in the multiple linear regression

analyses were further adjusted for the sex of the fetus and the parity of the mother, no independent

effect of sex and parity was observed for log10(PGH); the p-value, B and its CI remained robust (p =

0.009, B=0.06(95%CI: 0.01-0.10)). For log10(hPL) and log10(IGF-I) there remained no significant

difference in serum levels between the three growth rate groups, when the serum levels in the

multiple linear regression analyses were further adjusted for the sex of the fetus and the parity of the

mother.

Discussion

In our large prospective cohort of pregnant women we observed higher maternal serum levels of

PGH in week 11-14 in women carrying fetuses with high growth rates until week 17-20 compared

to women carrying fetuses who grew at average growth rates. This result persisted when also

adjusting for CRL, maternal weight, parity and fetal sex. This association has not previously been

demonstrated. It has been hypothesised that PGH acts to augment the supply of fatty acids and

glucose to the placenta 18,19

, which would be in agreement with our present results; a high maternal

serum level of PGH would increase substrate supply to the fetus and thus promote fetal growth. We

observed a trend towards higher maternal serum levels of PGH in the low growth rate group when

compared to the intermediate group, which may seem surprising. We have previously shown that

pregnant women, who were smoking during pregnancy, had higher PGH levels compared to non-

smokers which could suggest a compensatory mechanism by which the fetus is protected 17

. In line

with these findings, it has been suggested that high maternal plasma levels of PGH in preeclamptic

women may be a compensatory mechanism to preserve fetal growth. In the study by Mittal et al.12

patients with preeclampsia that delivered a small for gestational age neonate had lower maternal

serum concentrations of PGH than patients with preeclampsia alone. Thus in line with these

12

findings we suggest that the trend towards higher maternal PGH levels is a compensatory

mechanism to preserve fetal growth.

In second half of pregnancy concentrations of PGH and IGF-I in maternal serum are positively

correlated 9, and in pregnancies complicated by IUGR maternal PGH and IGF-I levels has been

found to be significantly lower than levels in normal pregnant women 11

. The IGFs are mitogenic

polypetides that stimulate cellular proliferation and differentiation. They have the ability to

influence the transport of glucose and amino acids across the placenta 20

and are significantly

influenced by fetal nutrition and insulin levels 21,22

. We expected that the high maternal levels of

PGH would lead to corresponding high levels of IGF-I, but this was not the case. There was no

difference in the IGF-I concentration between the three different growth rate groups. During

pregnancy PGH gradually replaces the maternal pituitary GH, which disappears from maternal

serum in the second half of pregnancy. PGH can be detected in the maternal circulation at 6 weeks’

gestation and its concentration increases with advancing gestation 9,23

, peak values are reached at

week 34-37 9. It is possible that maternal IGF-I level in the early half of pregnancy is primarily

controlled by pituitary GH, because placental PGH secretion may be too modest to suppress

maternal pituitary secretion of GH in early pregnancy 10

, and hereby control maternal levels of IGF-

I. This would explain why higher level of PGH in our study did not lead to a corresponding high

level of IGF-I. Alternatively, the presence of GH binding protein (GHBP) in the circulation, may

modulate the function of pituitary GH and PGH, and thereby influence the level of PGH in a way

that is not accounted for in the present study. However, free and bound PGH have been shown to

correlate highly significantly 11

, so in all likelihood GHBP is not a major confounder in our present

study.

The physiological role of hPL in pregnancy remain controversial. It has been hypothesized that

hPL along with PGH is released from the syncytiotrophoblast into the maternal circulation to

13

stimulate IGF-I production 24

. This is supported by a study that demonstrated that hPL and PGH

are unable to promote the growth of human fetal fibroblast and myoblasts in the presence of an

IGF-I antibody 25

. However, other studies that have investigated the relationship between PGH,

hPL and IGF-I in human pregnancies find, regardless of gestational age, that IGF-I values are

correlated with corresponding PGH but not with hPL values, which suggest that PGH and not hPL

is the primary regulator of maternal IGF-I secretion 10,26

. Maternal blood levels of hPL are

correlated with the weight of the placenta 27-29

, and that of the fetus 30-32

. Low hPL levels have been

described in association with severe IUGR 28,33

. Furthermore a link between fetal growth velocities

in second half of pregnancy and changes in hPL in maternal serum has been demonstrated 34

. We

were not able to confirm such an association in the first half of pregnancy in our present study as no

association between fetal growth rate and maternal level of hPL was observed.

Strengths of our study

Our study was based on a very large cohort of representative pregnant women, who were recruited

from the same geographical area. In all women, ultrasonography was performed by certified

ultrasonographers minimizing methodological bias. In addition, all hormone measurements were

done at the same time using standardized assays from the same batch, hereby minimizing the

interassay variation.

Weaknesses of our study

Although hPL 35

, PGH 36,37

and IGF-I 38

show no circadian rhythm there is a minor variation over

24 hours and a single sample taken from an individual might therefore not be entirely representative

for the individual. Furthermore the use of ultrasound techniques, when taking measurements,

unavoidably encompasses some uncertainty, which could weaken a possible association between

growth rate and growth hormones. We found that the mean BPD between the different growth rate

groups was significantly different at the first and the second scan. At the first scan high growth rate

14

was associated with the lowest BPD, and at the second scan high growth rate was associated with

the highest BPD. Part of this could most likely be explained by the statistical principle: “regression

toward the mean” 39

which states that if a pair of independent measurements are made from the

same distribution, samples far from the mean on the first measurement will tend to be closer to the

mean on the second one. The phenomenon occurs because each sample is affected by random

variance. Another explanation could be that fetal growth theoretically occurs in spurts. This would

imply that the fetuses with the smallest BPD at the first scan were behind the other fetuses of the

same gestational age with larger BPD and therefore up until the second scan experienced a period

with higher growth rate than the rest.

The group of fetuses with the highest growth rate were significantly younger at the time of the

second scan than those with intermediate or low growth rate. Milani et al 15

followed a cohort of

130 pregnant women longitudinally from week 12 to delivery and onwards, and demonstrated that

peak velocities for growth of the head circumference were reached around week 17 of pregnancy at

the rate of approximately 12.5 mm / week, from then on the velocity declines towards term, where it

was around 4 mm / week. These results are in accordance with the low gestational age for fetuses

with high growth rates, as the second ultrasound measurement was performed from week 17 to 21.

We observed a predominance of male fetuses in the large growth rate group. The higher growth

rates of male fetuses are in accordance with the study from 2007 by Bukowski R et al.40

. In a

cohort of 28,000 fetuses they found that male fetuses at week eight to twelve of gestation on

average were 0.3 days larger than female fetuses 40

. Finally the percentage of nulliparous women

was higher in the high growth rate group, for which we have no explanation.

We were not able to demonstrate an association between total IGF-I and early fetal growth rate. In

the circulation, IGF-I is bound to specific IGF binding proteins (IGFBP-1 to -6), of which IGFBP-3

bind more than 90% 41

. The presence of IGFBP prolongs the half-life of IGF-I, prevents its

15

hypoglycaemic actions, and strongly influences its biological activity. In pregnancy, specific

IGFBP-3 proteases are induced, and they degrade IGFBP-3 hereby influencing the amount of free,

biologically active IGF-I in the circulation of pregnant women. We exclusively determined total

IGF-I in our present study, and therefore it cannot be ruled out that had we been able to unmask

some of the interactions within the complex IGF-system, then perhaps other links between fetal

growth rate and IGFs could be identified. Our findings encourage further and more extensive

examinations of the role of the IGF-axis (e.g. determination of free IGF-I) in controlling fetal

growth in early pregnancy.

In conclusion we demonstrated that maternal serum levels of PGH, but not of hPL and total IGF-I

were associated with fetal growth in early human pregnancy in a prospective cohort of 600 healthy

pregnant women. Future studies are needed to evaluate the potential clinical use of PGH for

monitoring early fetal growth.

16

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19

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disease. Growth Horm.IGF.Res. 2003; 13: 113-170.

Low growth rate

Gestational age (days)

60 80 100 120 140 160

BP

D (

mm

)

10

20

30

40

50

60

Figure 1

Legend to Figure 1: Growth of the BPD as a function of gestational age in fetuses with low (n=203), intermediate (n=212) and high (n=203) growth rates respectively.

Intermediate growth rate


60 80 100 120 140 160

BP

D (

mm

)

10

20

30

40

50

60

High growth rate


60 80 100 120 140 160

BP

D (

mm

)

10

20

30

40

50

60

Figure 2

Legend to Figure 2: Box and whiskers plots (median, 25

th centile, 75

th centile and range) of maternal serum

levels of hPL, PGH and IGF-I for fetuses with low, intermediate and high growth rates, ○ represents outliers.

low intermediate high p value*

n mean (SD)/percentage n mean (SD)/percentage n mean (SD)/percentage

203 212 203

143 70% 166 78% 154 75%

BPD first scan (mm) 143 23.6 (2.9) 166 22.6 (2.5) 154 21.4 (2.6) <0.001

BPD second scan (mm) 143 40.5 (2.7) 166 44.2 (2.3) 154 46.2 (3.0) <0.001

Gest age first scan (days) 143 90.1 (4.6) 166 90.7 (4.5) 154 90.3 (4.3) 0.508

Gest age second scan (days) 143 134.4 (6.7) 166 134.6 (4.9) 154 131.0 (6.2) 0.000

138 275.5 (27.1) 165 280.5 (15.8) 153 279.1 (13.9) 0.077

missing 5 1 1

135 3366.4 (641.3) 164 3518.6 (468.2) 153 3631.9 (583.7) <0.001

missing 8 2 1

female 77 57.0% 92 56.1% 46 30% <0.001

male 58 42.9% 72 43.9% 107 70%

missing 8 2 1

caesarean sectio 14 10.3% 12 7.3% 27 17.8% 0.013

vaginal 122 89.7% 152 92.7% 125 82.2%

missing 7 2 2

Outcome

live birth 133 95% 164 99.4'% 153 100.0% 0.002

not live birth† 7 5.0% 1 0.6% 0 0%

missing 3 1 1

*Categorized characteristics were compared using the Chi-square test (more than five observations in all cells) or Fisher’s exact test (five or fewer

observations in one or more cells). Continuous characteristics were compared by means of one-way ANOVA.

†Defined as neonatal death or termination of pregnancy from 18 weeks of gestation until 1 week post partum

Delivery

Cases

Cases with blood sample

Table 1

Prenatal characteristics and pregnancy outcomes of the included pregnancies

Sex

Gest age at delivery (days)

Birth weight (g)

Growth rate: low intermediate high

n mean (SD)/percentage missing n mean (SD)/percentage missing n mean (SD)/percentage missing

Cases with blood sample 143 166 154

Weight (kg) 142 65.3 (11.2) 1 164 64.6 (9.1) 2 154 65.8 (12.3) 0

Height (cm) 142 167.0 (6.3) 1 164 169.1 (6.2) 2 151 168.9 (6.6) 3

BMI 142 23.3 (3.9) 1 163 22.6 (2.9) 3 151 23.1 (4.0) 3

Ethnicity

Caucasian 135 100% 156 97% 151 98%

Other 0 0% 5 3% 3 2%

missing 8 5 0

Parity

nulliparous 83 59% 103 62% 115 75%

parous 58 41% 63 38% 39 25%

missing 2 0 0

yes 25 18% 21 13% 33 21%

no 117 82% 144 87% 121 79%

missing 1 1 0

yes 15 11% 14 9% 8 5%

no 119 89% 150 91% 144 95%

missing 9 2 2

spontaneous 134 96% 161 99% 149 97%

assisted 5 4% 1 1% 5 3%

missing 4 4 0

*Categorized characteristics were compared using the Chi-square test (more than five observations in all cells) or Fisher’s exact test (five or fewer

observations in one or more cells). Continuous characteristics were compared by means of one-way ANOVA.

†Medical conditions includes the following conditions: abruptio placenta (1 pregnancy), diabetes (4 pregnancies), sclerosis (1 pregnancy), epilepsia (1 pregnancy),

raised liver parameters (2 pregnancies), IUGR (2 pregnancies) preeclampsia levi gradu (4 pregnancies),

preeclampsia magno gradu (4 pregnancies) and other rare diseases (18 pregnancies).

Conception

Table 2

Maternal characteristics of the study population

Smoker

Medical condition†

Table 3

(growth rate around the median=reference), using simple linear regression analyses.

†n B t P

log10 hPL (mg/L) lowest highest

constant -0.52 -0,56 -0,49

low growth rate 133 0.02 -0,03 0,07 0.85 0,397

high growth rate 139 -0.01 -0,05 0,04 -0.28 0,781

intermediate growth rate* 156

llog10 PGH (ng/ml)

constant 0.52 0.49 0.55 35.10 <0.001

low growth rate 132 0.03 -0.01 0.08 1.47 0.142

high growth rate 139 0.05 0.00 0.09 2.10 0.037


log10 IGF-I (ng/ml)

constant 2,16 2,14 2,18 222,42 <0.001

low growth rate 127 0,00 -0,03 0,03 -0,26 0,798

high growth rate 134 -0,01 -0,04 0,02 -0,54 0,590


†The number of blood samples analysed for hPL, PGH and IGF-I varies, as the amount of serum left, wasn’t always

suffient to perform the analysis for all three biochemical markers.

Predicted change in serum concentration for hPL, PGH and IGF-I by growth rate group

95% CI

*This parameter is set to zero because it is the reference

Table 4

(estimated by CRL) and growth rate group (growth rate around the median=reference), using multiple linear regression analyses.

†n B t P

log10 hPL (mg/L) lowest highest

constant -0.52 -0.55 -0.50

low growth rate 132 0.03 -0.01 0.07 1.60 0.110

high growth rate 139 0.01 -0.03 0.05 0.56 0.570


CRL (mm from 68.5) 0.01 0.01 0.02 14.73 <0.001

maternal weight (kg from 65) 0.00 0.00 0.00 -2.78 0.006

log10 PGH (ng/ml)

constant 0.52 0.49 0.55

low growth rate 131 0.04 0.00 0.08 1.95 0.052

high growth rate 139 0.05 0.01 0.09 2.63 0.009


CRL (mm from 68.5) 0.01 0.00 0.01 6.01 <0.001

maternal weight (kg from 65) 0.00 0.00 0.00 -2.30 0.022

log10 IGF-I (ng/ml)

constant 2.16 2.14 2.18

low growth rate 126 -0.01 -0.03 0.02 -0.36 0.722

high growth rate 134 -0.01 -0.04 0.02 -0.68 0.499


CRL (mm from 68.5) -0.00 -0.00 0.00 -2.40 0.017

maternal weight (kg from 65) 0.00 -0.00 0.00 1.00 0.318

†The number of blood samples analysed for hPL, PGH and IGF-I varies, as the amount of serum left, wasn’t always suffient to perform the analysis for all three biochemical markers.

suffient to perform the analysis for all three biochemical markers. When records on CRL or maternal weight was

missing, the case was omitted from the analysis.

Predicted change in serum concentration for hPL, PGH and IGF-I by mother's weight (kg), gestational age of child

95% CI

*This parameter is set to zero because it is the reference

1

First trimester growth restriction and uterine artery bloodflow as predictors of adverse pregnancy

outcome

NG Pedersen*, L Sperling†, KR Wøjdemann§, S Olesen Larsen‡, A Tabor*

*Department of Fetal Medicine and Ultrasound, Rigshospitalet, Copenhagen University Hospital, Denmark

† Department of Obstetrics and Gynecology, Herlev University Hospital, Herlev, Denmark

‡ Department of Clinical Biochemistry and Immunology Serum Institute, Copenhagen, Denmark

§ Department of Obstetrics and Gynecology, Roskilde University Hospital, Roskilde, Denmark

Corresponding author:

Dr NG. Pedersen, Department of Fetal Medicine and Ultrasound, Rigshospitalet, Copenhagen University

Hospital, Department 4002, Blegdamsvej 9, 2100 Copenhagen, Denmark

E-mail: [email protected]

Tel: +45 35 45 37 22

Fax: +45 35 45 37 39

Short title: First trimester growth restriction

Key words: CRL, menstrual cycle, growth restriction, ultrasound

mailto:[email protected]

2

Abstract

Objective: To estimate the value of screening for fetal growth restriction by performing color Doppler

assessment of uterine arteries at 18-21 and 23-24 weeks for fetuses with a with a discrepancy in gestational

age determined by crown rump length (CRL) and last menstrual period crown rump length of 7 days or

more.

Methods: Women with singleton pregnancies, where the gestational age estimated by CRL at the first

trimester scan was seven days or more smaller than the gestational age estimated by last menstrual period

were identified and invited to the study, and a control group of women with singleton pregnancies, where

the gestational age was either equal to or one day larger were invited to the study. The study entailed the

routine scans, which are Down Syndrome screening in gestational week 11-14 and an anomaly scan in

gestational week 18-21. In addition to the routine scans the participants were offered a growth scan in

gestational week 23-24, where a questionnaire was answered. At the anomaly scan and growth scan

umbilical and uterine artery Doppler flows were measured.

Results: 182 cases and 230 controls were included in the study. There was a significantly higher proportion

of fetuses born with a birth weight below 2500g in the case group compared to the control group (7.4% vs.

3.1%, p=0.048), and in our logistic regression models birth weight below 2500g showed a significant

association to the case group. Having a CRL seven days or more smaller than expected increased the risk of

having a child with a birth weight below 2500g with an odds ratio of 3.29.

At the scan performed week 18-21 the case and control group showed no significant differences in

biometric ultrasound measurements or in placental blood flow characteristics. The biometric

measurements and placental blood flow characteristics were repeated at the scan week 23-24, and again

no significant differences was observed between the two groups.

Conclusion: We were not able to demonstrate a link between poor placental perfusion and first trimester

growth restriction, and therefore do not recommend implementation of uterine or umbilical artery flow

measurements specifically for fetuses with at smaller than expected CRL.

3

Introduction

There is often a discrepancy between gestational age estimated by last menstrual period and CRL.

Common practice in most Ultrasound and Obstetrical departments is to use the date set by CRL,

irrespective of the discrepancy. Several studies have now demonstrated that fetal growth in first half of

pregnancy does vary between fetuses, and different measurements for impaired growth in first half of

pregnancy, such as smaller than expected CRL and decreased early fetal growth rates are associated with

perinatal death, low birth weight and preterm delivery 1-6. This implies that important information on

growth restriction in early pregnancy in many cases is lost, when the gestational age for a fetus where CRL

is smaller than expected, is simply altered.

Pre-eclampsia, fetal growth restriction, placental abruption and some cases of fetal death are believed to

result from impaired placentation in early gestation 7. Deficient placentation is characterized by

inadequate trophoblast invasion into the maternal spiral arteries and failure to develop low-resistance

uteroplacental circulation 8. In the past 30 years Doppler ultrasonographic studies of the uteroplacental

circulation have shown that high impedance to flow is associated with subsequent pre-eclampsia, IUGR and

related complications 9.

Uterine artery volume flow rate increased by more than 3-fold during pregnancy, partly influenced by an

increased artery diameter and reduced resistance to flow 10. In women with pre-eclampsia uteroplacental

blood flow has been shown to be reduced by 50% 11 , and several Doppler studies both in first and second

trimester have demonstrated an association between increased impedance to flow in the uterine arteries

and subsequent development of pre-eclampsia, IUGR and fetal death 12-18.

The umbilical arteries direct 33% of the fetal ventricular output to the placenta at 20 weeks of gestation 19,

and the assessment of umbilical blood flow provides information on blood perfusion of the fetoplacental

unit. Doppler measurement in the umbilical artery reflects the downstream impedance and is extensively

used to identify placental compromise 20. Poor umbilical flow is associated with high risk of perinatal,

death, IUGR, preterm birth and morbidity in the newborn period 21-24.

In this prospective study we wished to investigate if fetuses with a smaller than expected CRL have poorer

perfusion of the placenta, measured by ultrasound Doppler of the uterine and umbilical arteries compared

to a control group, and to investigate if a smaller than expected CRL in combination with poor flow in the

uterine arteries can be used to predict poor outcome

Methods

This study took place at two of the obstetric departments of Copenhagen University Hospital; Herlev

Hospital and Rigshospitalet. All women attending our antenatal services are offered Down Syndrom

4

screening in gestational week 11-14 (a double test i.e. PAPP-A and βhCG and a nuchal translucency scan)

and an anomaly scan in gestational week 18-21 as part of standard antenatal care. Pregnancies where the

gestational age estimated by CRL at the first trimester scan was seven days or more smaller than the

gestational age estimated by last menstrual period were identified, and all women with a discrepancy of

seven days or more were invited to participate in the study. This group of pregnancies is referred to as the

case group. As a control group all pregnancies, where the gestational age estimated by CRL at the first

trimester scan was either equal to or one day larger than the gestational age estimated by last menstrual

period, were identified. As the numbers of pregnancies in the control group meeting these criteria were

more than that of the case group, not all women in the control group were invited. The selection of the

women in the control group was time-wise, so that the number of women in the two groups was kept

approximately equal at all times, this ensured a random selection of the women in the control group.

Inclusion criterions were: pregnancy should be spontaneously conceived; at least three spontaneous

periods before last menstrual period; maximum 7 weeks discrepancy in gestational age when determined

by CRL and last menstrual period; planning to give birth at either one of the two participating hospitals; live

fetus at the time of the anomaly scan; able to understand Danish. At Rigshopitalet we included women

that had their first trimester scan performed in the period February 2008 to February 2009, and at Herlev

Hospital we included women that had their first trimester scan performed in the period May 2008 to

December 2008.

The study entailed the routine scans, a growth scan in gestational week 23-24 and a questionnaire. At the

anomaly scan and growth scan umbilical and uterine artery Doppler flows were measured.

Left and right uterine arteries were identified with the use of color Doppler at the crossover with the

external iliac arteries. The pulse wave Doppler signals were sampled 1 cm cranial to the crossing point.

When three similar consecutive waveforms were obtained, the presence of an early diastolic notch was

noted, the pulsatility index was measured, and the mean PI of the two vessels was calculated. The Doppler

signals from the umbilical artery were sampled as close to the midportion of the cord as possible. When

three similar consecutive waveforms were obtained the pulsatility index was measured. The assessment of

the flow velocity waveform was performed only at the time of the scans. The results of the scans were

available to the patient and the treating clinician as part of standard care. While other fetal surveillance

(e.g. serial biometry for those with abnormal Doppler results etc.) was undertaken, no women received

medical interventions such as aspirin based on the results of the extra scans.

The ultrasound examinations were performed using the following machines: Alfa 10 (Aloka, Meerbusch,

Germany), Logiq 7 / 9, Voluson E8 (General Electric, Milwaukee, WI, USA). The scans were performed by 20

5

different sonographers or doctors experienced in obstetric ultrasound. Data including indications from all

scans performed during the pregnancy were recorded in ASTRAIA.

Following delivery the maternal and neonatal records were reviewed for pre-defined perinatal outcomes.

Perinatal death was defined as: death to a child under one week or a stillborn of 20 or more weeks of

gestation. Intrauterine growth restriction was defined as low birth weight (birth weight below 2500g) or

small for gestational age (birth weight two standard deviations below ultrasonically estimated mean for

gestational age) 25. Very premature was defined as spontaneous live birth week 22-33 of gestation,

premature as spontaneous live birth week 34-36; term as spontaneous live birth week 37-41; and post-

term as spontaneous live birth after 41 weeks of gestation. Pre-eclampsia was defined according to the

guidelines of International Society of Hypertension in Pregnancy. This requires two recordings of diastolic

blood pressure of more than 90mm Hg at least four hours apart in previously normotensive women, and

proteinuria of 300mg or more in 24 hours, or two readings of at least two pluses on dipstick analysis of

midstream or catheter urine specimens if no 24-hour collection is available 26.

Ethics: In accordance with guidelines from the National Health Board of Health and Danish Data Protection

Agency approval from our institutional ethics committees was given and written consent was obtained

from all the participants.

Data were processed using the following software programs: Excel 2007 (Microsoft Corp., Redmond WA,

USA), SPSS 17 (SPSS, Chicago, IL, USA), S-PLUS (4.0, MathSoft Inc, Seattle, Washington, USA).

Statistics:

A group of "normal outcome pregnancies" was defined to include liveborn infants, delivered week 37 - 41

without intrauterine growth restriction, defined as small for gestational age and with no preeclampsia, it

consisted of 338 pregnancies. From the normal outcome group we determined the 5 and 95 % fractiles

either for the observed data as such or, if there was a clear dependence on the estimated gestational age at

the time of the scan, for the deviations (residuals) from the values expected from a linear regression on the

gestational age. This was done separately for values obtained in week 18 - 21 and week 23 - 24. For PAPP-A

measured in week 11 - 14 recorded Multiple of the Medians (MoM) values were used.

Logistic regression models were used to evaluate the impact on the risk of adverse outcome of values

below the 5 % fractile or above the 95 % fractile for the various indicators. Variables entered in the logistic

regression models were: case vs. control; PAPP-A below 5th percentile vs. PAPP-A above 5th percentile

(week 11-14); bilateral or unilateral notch vs. no notch (week 18-21 and week 23-24); mean uterine artery

PI above 95th percentile vs. mean uterine artery PI below 95th percentile (week 18-21 and week 23-24);

abdominal circumference below 5th percentile vs. abdominal circumference above 5th percentile ( week 18-

21 and week 23-24); femur length below 5th percentile vs. femur length above 5th percentile week 18-21

6

and week 23-24); estimated fetal weight below 5th percentile week vs. estimated fetal weight above 5th

percentile (week 23-24). In various steps the regressions were repeated excluding variables without any

significant effect on the outcome leading to a reasonable set of explanatory variables.

Differences in proportions were evaluated using chi-square tests and differences in average values by using

u-tests. All p-values stated are two-sided, and p-values below 5 % are stated as significant.

Results

The inclusion criterions were met by 357 cases and 451 controls, of these 182 cases and 230 controls were

included in the study. 17 cases and 7 controls refused to participate, 124 cases and 198 controls were

never asked, 5 cases did not come to the growth scan in gestational week 23-24, 2 cases emigrated and 27

cases and 16 controls were not included for unknown reasons.

Characteristics of the study population are shown in Table 1. There was a significantly lower percentage of

pregnant women in the case group with a regular menstrual cycle compared to the control group (54% vs.

80 %, p<0.001), and a smaller proportion of the case group had continued education after mandatory

schooling (73% vs. 85%, p=0.003). Otherwise there were no significant differences between the two groups

in maternal characteristics.

Outcome of the study population is illustrated in Table 2. There was a significantly higher proportion of

fetuses born with a birth weight below 2500g in the case group compared to the control group (7.4% vs.

3.1%, p=0.048), otherwise no significant differences in outcome was observed between the two groups.

Although not significant the percentage of intrauterine deaths in the case group was somewhat higher than

that in the control group (1.7% vs. 0.9% p=0.786). In the case group the causes for perinatal death were 1)

Known triploidy where the parents decided to continue the pregnancy, intrauterine death was diagnosed

week 28. 2) Unknown reasons, intrauterine death was diagnosed week 20, mother had previously had two

late miscarriages also in week 20. 3) Unknown reasons, intrauterine death was diagnosed week 36. In the

control group the different causes for perinatal death were: 1) Abruptio placentae, mother was known to

have Neurofibromatosis von Recklinghausen and hypothyroid disease and the child was severely growth

restricted, intrauterine death was diagnosed week 34. 2) Unknown reasons, intrauterine death was

diagnosed week 36.

Ultrasound characteristics of the study population is illustrated in Table 3. At the scan performed week 18-

21 the case and control group showed no significant differences in growth characteristics such as: head

circumference, abdominal circumference and femur length, or in placental blood flow characteristics such

as: number of cases with mean uterine artery PI above 95th percentile, number of cases with bilateral or

unilateral notch in the uterine arteries, number of cases with umbilical artery PI above 95th percentile.

7

The growth and placental blood flow characteristics were repeated at the scan week 23-24, and again no

significant differences was observed between the two groups.

Table 4 shows the results from the logistic regression analysis. Only birth weight below 2500g showed a

significant association to the case group. Having a CRL seven days or more smaller than expected increased

the risk of having a child with a birth weight below 2500g with an odds ratio of 3.29. Doppler flow

characteristics of the uterine arteries such as bilateral or unilateral notch, and mean uterine artery PI above

95th percentile measured week 18-21 was significantly related to the risk of giving birth to a child with a

birth weight either below 2500g or diagnosed as small for gestational age, with odds ratios between 2.48

and 4.51. Abdominal circumference below 5th percentile measured week 18-21 was significantly associated

to the risk of having a child with a birth weight below 2500g, diagnosed as small for gestational age and

spontaneous birth before gestational week 34, with odds ratios between 4.01 and 16.31. Only ultrasound

measurement obtained week 23-24 showed a significant association to pre-eclampsia, measurements

obtained week 18-21 did not. The risk of pre-eclampsia was increased with an odds ratio of 6.32 if bilateral

or unilateral notches were diagnosed week 23-24, and with 2.95 if the estimated fetal weight was below

the 5th percentile in week 23-24. The adverse outcome perinatal death had too few cases for logistic

regression analysis to be performed in a meaningful way.

Discussion

In this study we found no association between smaller than expected CRL as a measure of first trimester

growth restriction and placental perfusion measured by Doppler ultrasound of the uterine and umbilical

arteries in second trimester. The number of cases and controls with high PI or bilateral/unilateral notch in

the uterine arteries or high PI in the umbilical arteries showed no significant differences between the case

and control group at the scans performed week 18-21 and week 23-24.

Only birth weight below 2500g showed a significant association to the case group in our logistic regression

model. Having a CRL seven days or more smaller than expected increased the risk of having a child with a

birth weight below 2500g with an odds ratio of 3.3. In line with these findings we confirmed that fetuses

with a gestational age determined by CRL 7 days or more smaller than expected have about double the risk

of being born with a birth weight below 2500g. The risk is in agreement with previous studies that report

odds ratios between 2 and 3 for very low birth weights, when CRL is smaller than expected1;5;6.

Besides a slightly higher percentage of fetuses with a smaller than expected CRL that were delivered week

34-36 (4% vs. 1%) our study did not exhibit a difference in the overall pattern of week of delivery between

the case and control group, and our logistic regression model did not show any association between a

smaller than expected CRL and delivery before gestational week 34. Previous studies that investigated the

8

association between smaller than expected CRL and week of delivery1;5;6 all report an increased risk for

preterm delivery with odds ratios around two. Two of the previous studies on first trimester growth

restriction and preterm birth1;6;6 are retrospective studies conducted in cohorts of 976 and 4229

pregnancies, the larger cohorts may explain why they were able to demonstrate an association.

The number of pregnancies that developed preeclampsia did not show any significant differences between

the case and the control group, and nor did we find any significant relationship between smaller than

expected CRL and preeclampsia in our logistic regression model. The findings are in line with previous

studies on preeclampsia and first trimester growth restriction, an association has never been reported

between the two1;5;6

The mortality rate for fetuses with a positive heart rate at the ultrasound scan in week 17 was in a Danish

study from 1986 reported to be 0.7% 27. The mortality rate for fetuses with a smaller than expected CRL

was 1.7% in our study. Taking into account that only pregnancies with a positive heart action at the scan in

week 18-21 were included, we find the mortality rate of 1.7% somewhat higher than expected, suggesting

an increased risk of perinatal death in the case group. Two out of the three fetuses from the case group

that suffered intrauterine demise did so before week 29 of gestation, which is in accordance with findings

from one of our previous studies; that growth restriction in first half of pregnancy is a predictor of perinatal

death taking place relatively early in pregnancy 3.

There was a lower percentage of women with a regular menstrual cycle in the case group compared to the

control group, and the length of education was also significantly lower for the case group, otherwise

maternal characteristics between the two groups did not differ. It seems unlikely that the differences in

maternal characteristics between the two groups in any way biased the associations between first

trimester growth restriction and adverse outcome.

To assess first trimester growth by difference in gestational age, when determined by last menstrual period

and CRL requires first of all that the pregnant woman remembers the first day in her last period, and for the

assessment to be as accurate as possible her cycle must have been regular with spontaneous periods the

past months before becoming pregnant. According to previous studies these conditions are only met in 30-

80% of pregnancies 28-31, which is an important weakness of the method used in our study. In addition to

that, many women have delayed ovulation in some cycles, which causes a tendency to overestimate the

length of gestation, thereby adding error to the method 31-34.

In a clinical setting it is difficult to differentiate between women with a regular and irregular cycle when

determining perinatal care. As the purpose of the study was to investigate if a smaller than expected CRL in

combination with uterine artery flow could be used in a clinical setting to predict adverse outcome, women

9

with both regular and irregular cycles were included in the study, it is possible that we would have found a

higher association to adverse outcome had we only included women, who reported a regular cycle.

In conclusion we confirm the association between first trimester growth restriction and higher risk of

delivering a low birth weight infant. We were not able to demonstrate a link between poor placental

perfusion and first trimester growth restriction, and therefore do not recommend implementation of

uterine or umbilical artery flow measurements for fetuses with at smaller than expected CRL. However,

we do recognize that a smaller than expected CRL is a risk factor for giving birth to a low birth weight

neonate, and simple re-dating of pregnancies with a smaller than expected CRL, especially if the woman

has a regular menstrual cycle and is certain of the first day in her last period, can conceal early growth

restriction. On these grounds we do recommend for pregnancies with a smaller than expected CRL that

extra attention is paid to the ultrasound measurements obtained at the anomaly scan, and should these

measurements be at the low end of the normal range scale a follow- up scan to check fetal growth should

be offered to the woman.

10

Reference List

(1) Smith GC, Smith MF, McNay MB, Fleming JE. First-trimester growth and the risk of low birth weight.N Engl J Med 1998; 339(25):1817-1822.

(2) Kallen K. Increased risk of perinatal/neonatal death in infants who were smaller than expected atultrasound fetometry in early pregnancy. Ultrasound Obstet Gynecol 2004; 24(1):30-34.

(3) Pedersen NG, Figueras F, Wojdemann KR, Tabor A, Gardosi J. Early fetal size and growth aspredictors of adverse outcome. Obstet Gynecol 2008; 112(4):765-771.

(4) Pedersen NG, Wojdemann KR, Scheike T, Tabor A. Fetal growth between the first and secondtrimesters and the risk of adverse pregnancy outcome. Ultrasound Obstet Gynecol 2008; 32(2):147-154.

(5) Mook-Kanamori DO, Steegers EA, Eilers PH, Raat H, Hofman A, Jaddoe VW. Risk factors andoutcomes associated with first-trimester fetal growth restriction. JAMA 2010; 303(6):527-534.

(6) Bukowski R, Smith GC, Malone FD, Ball RH, Nyberg DA, Comstock CH et al. Fetal growth in earlypregnancy and risk of delivering low birth weight infant: prospective cohort study. BMJ 2007;836-840.

(7) Khong TY, De WF, Robertson WB, Brosens I. Inadequate maternal vascular response to placentationin pregnancies complicated by pre-eclampsia and by small-for-gestational age infants. Br J ObstetGynaecol 1986; 93(10):1049-1059.

(8) Osol G, Mandala M. Maternal uterine vascular remodeling during pregnancy. Physiology (Bethesda) 2009; 24:58-71.

(9) Campbell S, Pearce JM, Hackett G, Cohen-Overbeek T, Hernandez C. Qualitative assessment ofuteroplacental blood flow: early screening test for high-risk pregnancies. Obstet Gynecol 1986;68(5):649-653.

(10) Thaler I, Manor D, Itskovitz J, Rottem S, Levit N, Timor-Tritsch I et al. Changes in uterine blood flowduring human pregnancy. Am J Obstet Gynecol 1990; 162(1):121-125.

(11) Lunell NO, Nylund LE, Lewander R, Sarby B. Uteroplacental blood flow in pre-eclampsiameasurements with indium-113m and a computer-linked gamma camera. Clin Exp Hypertens B1982; 1(1):105-117.

(12) van den Elzen HJ, Cohen-Overbeek TE, Grobbee DE, Quartero RW, Wladimiroff JW. Early uterineartery Doppler velocimetry and the outcome of pregnancy in women aged 35 years and older.Ultrasound Obstet Gynecol 1995; 5(5):328-333.

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(13) Martin AM, Bindra R, Curcio P, Cicero S, Nicolaides KH. Screening for pre-eclampsia and fetalgrowth restriction by uterine artery Doppler at 11-14 weeks of gestation. Ultrasound ObstetGynecol 2001; 18(6):583-586.

(14) Gomez O, Martinez JM, Figueras F, Del RM, Borobio V, Puerto B et al. Uterine artery Doppler at 11-14 weeks of gestation to screen for hypertensive disorders and associated complications in anunselected population. Ultrasound Obstet Gynecol 2005; 26(5):490-494.

(15) Campbell S. First-trimester screening for pre-eclampsia. Ultrasound Obstet Gynecol 2005;26(5):487-489.

(16) Albaiges G, Missfelder-Lobos H, Lees C, Parra M, Nicolaides KH. One-stage screening for pregnancycomplications by color Doppler assessment of the uterine arteries at 23 weeks' gestation. ObstetGynecol 2000; 96(4):559-564.

(17) Papageorghiou AT, Yu CK, Bindra R, Pandis G, Nicolaides KH. Multicenter screening for pre-eclampsia and fetal growth restriction by transvaginal uterine artery Doppler at 23 weeks ofgestation. Ultrasound Obstet Gynecol 2001; 18(5):441-449.

(18) Harrington K, Fayyad A, Thakur V, Aquilina J. The value of uterine artery Doppler in the predictionof uteroplacental complications in multiparous women. Ultrasound Obstet Gynecol 2004; 23(1):50-55.

(19) Rudolph AM HMTKBCRN. Studies on the circulation of the previable human fetus. Pediatr Res 1971;5:452-465.

(20) Alfirevic Z, Neilson JP. Doppler ultrasonography in high-risk pregnancies: systematic review withmeta-analysis. Am J Obstet Gynecol 1995; 172(5):1379-1387.

(21) Trudinger BJ, Cook CM, Giles WB, Ng S, Fong E, Connelly A et al. Fetal umbilical artery velocitywaveforms and subsequent neonatal outcome. Br J Obstet Gynaecol 1991; 98(4):378-384.

(22) Burke G, Stuart B, Crowley P, Scanaill SN, Drumm J. Is intrauterine growth retardation with normalumbilical artery blood flow a benign condition? BMJ 1990; 300(6731):1044-1045.

(23) Soothill PW, Ajayi RA, Campbell S, Nicolaides KH. Prediction of morbidity in small and normallygrown fetuses by fetal heart rate variability, biophysical profile score and umbilical artery Dopplerstudies. Br J Obstet Gynaecol 1993; 100(8):742-745.

(24) Gaziano EP, Knox H, Ferrera B, Brandt DG, Calvin SE, Knox GE. Is it time to reassess the risk for thegrowth-retarded fetus with normal Doppler velocimetry of the umbilical artery? Am J ObstetGynecol 1994; 170(6):1734-1741.

(25) Marsal K, Persson PH, Larsen T, Lilja H, Selbing A, Sultan B. Intrauterine growth curves based onultrasonically estimated foetal weights. Acta Paediatr 1996; 85(7):843-848.

(26) Brown MA, Lindheimer MD, de SM, Van AA, Moutquin JM. The classification and diagnosis of thehypertensive disorders of pregnancy: statement from the International Society for the Study ofHypertension in Pregnancy (ISSHP). Hypertens Pregnancy 2001; 20(1):IX-XIV.

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(27) Tabor A, Philip J, Madsen M, Bang J, Obel EB, Norgaard-Pedersen B. Randomised controlled trial ofgenetic amniocentesis in 4606 low-risk women. Lancet 1986; 1(8493):1287-1293.

(28) Taipale P, Hiilesmaa V. Predicting delivery date by ultrasound and last menstrual period in earlygestation. Obstet Gynecol 2001; 97(2):189-194.

(29) Kramer MS, McLean FH, Boyd ME, Usher RH. The validity of gestational age estimation bymenstrual dating in term, preterm, and postterm gestations. JAMA 1988; 260(22):3306-3308.

(30) Verburg BO, Steegers EA, De RM, Snijders RJ, Smith E, Hofman A et al. New charts for ultrasounddating of pregnancy and assessment of fetal growth: longitudinal data from a population-basedcohort study. Ultrasound Obstet Gynecol 2008; 31(4):388-396.

(31) Geirsson RT, Busby-Earle RM. Certain dates may not provide a reliable estimate of gestational age.Br J Obstet Gynaecol 1991; 98(1):108-109.

(32) Saito M, Yazawa K, Hashiguchi A, Kumasaka T, Nishi N, Kato K. Time of ovulation and prolongedpregnancy. Am J Obstet Gynecol 1972; 112(1):31-38.

(33) Walker EM, Lewis M, Cooper W, Marnie M, Howie PW. Occult biochemical pregnancy: fact orfiction? Br J Obstet Gynaecol 1988; 95(7):659-663.

(34) Guerrero R, Florez PE. The duration of pregnancy. Lancet 1969; 2(7614):268-269.

Table 1

Maternal characteristics of study population

Case group Control group

mean (SD) or n (%) mean (SD) or n (%) p-value unknown

Maternal age at enrollment (years) 29.8 (4.9) 30.4 (4.5) NS 1

Maternal weight at enrollment (kg) 68.7 (16.6) 67.8 (13.6) NS 1

Maternal height (cm) 166.6 (11.1) 167.9 (9.7) NS 1

Body Mass Index 24.5 (5.7) 24.6 (12.8) NS 1

Caucasians 219/226 (96.9) 170/180 (94.4) NS 6

Nulliparous 61/171 (35.7) 74/225 (32.9) NS 16

Smoker 16/181 (8.8) 18/230 (7.8) NS 1

Education after mandatory schooling 126/172 (73.3) 193/226 (85.4) 0.003 14

Regular menstrual cycle 69/128 (53.9) 127/159 (79.9) <0.001 125

Cases with maternal disease

Hyperthyroid disease 2/182 (1.10) 0/230 (0.00)

Hypothyroid disease 1/182 (0.55) 4/230 (1.74)

Diabetes 6/182 (3.30) 4/230 (1.74) NS 0

Non Significant (NS), Standard Deviation (SD), numbers (n)

The maternal characteristics were dichotomized into: caucasians vs. other ethnich origin; nuliparous vs. parous; smoker vs. non-smoker;

education after mandatory schooling vs. no education after mandatory schooling; regular menstrual cycle vs. unregular menstrual cycle

pre-eclampsia vs. no preeclampsia; diabetes vs. no diabetes. The overall distrubution for hyper, hypo and no thyroid disease

was compared between cases and controls

NS 0

Table 2

Outcome of study population

Case group Control group

mean (SD) or n (%) mean (SD) or n (%) p-value unknown

Intrauterine death 3/180 (1.7) 2/229 (0.9) NS 3

Birth weight (g) 3441.6 (626.23) 3508.52 (590.0) NS 9

Birth weigth < 2500g 13/176 (7.4) 7/227 (3.1) 0.048 9

Small for gestational age 11/176 (6.2) 6/225 (2.7) 0.077 11

Placental weight at birth (g) 682.5 (164.5) 684.6 (144.4) NS 65

Head circumference at birth (cm) 35.2 (2.6) 35.2 (2.4) NS 62

Length at birth (cm) 51.6 (3.4) 52.0 (2.8) NS 20

Female infant 99/177 (55.9) 105/227 (46.3) 0.067 8

Gestational age at delivery (weeks)§

22-33 1/122 (0.8) 2/159 (1.3)

34-36 5/122 (4.1) 2/159 (1.3)

37-41 114/122 (93.4) 149/159 (93.7)

42-43 2/122 (1.6) 6/159 (3.8)

Cases with pre-eclampsia 9/182 (4.9) 8/230 (3.5) NS 0

Non Significant (NS), Standard Deviation (SD), Numbers (n)

§ Only spontaneous delivery were included † 281 pregnancies were recorded as spontanous births, all of them had week of delivery registered

The outcome variables were dichtomized into: l ive birth vs. intrauterine death; birth weight < 2500g vs. birth weigth > 2500g;

birth weight < mean - 2SD vs. birth weight > mean - 2SD; cases with preeclampsia vs. cases without preeclampsia; female vs. male.

The overall distribution of spontaneous delivery week: 22-33, 34-36, 37-41 and 42-43 was compared between cases and controls.

NS †

Table 3

Ultrasound characteristics of study population

Scan week 11-14 Scan week 18-21 Scan week 23-24

n (%) p-value unknown mean (SD) or n (%) p-value unknown mean (SD) or n (%) p-value unknown

Numbers scanned

case 182/182 (100.0) 182/182 (100.0) 179/182 (98.4)

control 230/230 (100.0) 230/230 (100.0) 226/230 (98.3)Gestational age

case 86.9 (5.5) 136.8 (5.0) 168.0 (5.1)

control 89.0 (3.5) 138.4 (4.3) 168.1 (4.3)

PAPP-A below 5th centile§

case 9/179 (5.0)

control 13/228 (5.7)

Head circumference†

case 167.8 (10.2) 218.5 (11.5)

control 171.3 (8.5) 220.2 (9.4)

Abdominal circumference†

case 145.7 (11.2) 193.8 (13.7)

control 148.9 (10.2) 195.0 (10.7)

Femur length†

case 30.3 (2.7) 42.5 (2.9)

control 31.7 (2.3) 42.6 (2.2)

Estimated fetal weight (%)

case -4.2 (10.12)

control -3.2(8.5)

Mean uterine artery PI above 95th percentile†

case 10/160 (6.3) 14/178 (7.9)

control 11/214 (5.1) 12/226 (5.3)

Uterine arteries bilateral or unilateral notch‡

case 28/119 (23.5) 27/155 (17.4)

control 38/169 (22.5) 25/194 (12.9)

Umbilical artery PI above 95th percentile†

case 11/168 (6.5) 4/174 (2.3)

control 7/212 (3.3) 6/223 (2.6)

Abbreviations: Non Significant (NS), Standard Deviation (SD), Numbers (n)§ Gestational age was adjusted for with the use of MoM values

† If there was a clear dependence on gestational age P values were adjusted for gestational age with the use of linear regression

‡ 33 pregnancies week 18-21 and 52 pregnancies week 23-24 were omitted from this analysis as they had the record: "suggestion of notch"

NS NS

NS NS

91‡

7<0.001

NS

<0.0010 0

5

NS

NS

NS

NS

NS

NS

NS

1

1

NS

NS

NS

1532

38 8

7

11‡

7

1 8

13

Table 4

Prediction of adverse outcome

B SE OR 95% CI p-value

Birth weigth < 2500g

Case 1.19 0.59 3.29 1.06-10.26 0.036

Bilateral or unilateral notch (week 18-21) 0.91 0.58 2.48 0.77-7.96 0.120

Abdominal circumference below 5th percentile (week 18-21) 1.39 0.66 4.01 1.07-15.05 0.036

Femur length below 5th percentile (week 23-24) 1.39 0.66 4.40 1.10-17.64 0.033

Small for gestational age

Bilateral or unilateral notch (week 18-21) 1.11 0.63 3.02 0.86-10.64 0.079

Mean uterine artery PI above 95th percentile (week 18-21) 1.51 0.74 4.51 1.02-20.00 0.043


Spontaneous birth before gestational week 34


Pre-eclampsia

Bilateral or unilateral notch (week 23-24) 1.84 0.55 6.32 2.09-19.06 <0.001

Estimated fetal weight below 5th percentile (week 23-24) 1.08 0.72 2.95 0.70-12.59 0.134

Regression Coefficient (B), Standard Error (SE), Odds ratio (OR), Confindence Interval (CI)

SpørgeSkemaOM TIDLIGERE GRAVIDITETER & HELBRED

NAVN:CPR:FØDESTED:

1. Hvilken dato besvarer du dette spørgeskema? dato: / år:

2. Hvornår er din termin ? ( Vi ønsker datoen for den termin, du har fået fastsat ved nakkefolds skanningen) dato: / år:

3. Hvis du ikke kender din terminsdato, sæt kryds her:

4. Hvilket land er du født i:

5. Hvilket land / lande stammer dine forældre fra:

6. Hvor gammel er du? år

7. Hvor høj er du? cm

8. Hvad vejede du lige før du blev gravid ? kg

Hvis du ikke kender den vægt, du havde lige før du blev gravid, sæt kryds her 9. Hvor mange kilo havde du cirka taget på, da du i denne graviditet fik taget blodprøven, der sammen med nakkefoldsskanningen vurderer risikoen for Downs syndrom. Jeg havde taget kg på Hvis du ikke kender den vægt, du havde, da du fik taget blodprøven i denne graviditet, sæt kryds her

10. Var du op til den nuværende graviditet behandlet for barnløshed (med hormoner/IVF)? Sæt kun ét kryds JA NEJ

Læge Nina Gros Pedersen Professor, Overlæge Ann TaborKlinik for føtalmedicin og ultralydskanning Rigshospitalet

1

CRL&FLOW

DE FØLGENDE SPØRGSMÅL HANDLER OM DIN MENSTRUATION

11. Mange kvinder oplever lidt pletblødninger i trusserne før den egentlige menstruation starter, er dette tilfældet for dig ? Sæt kryds og svar ved enten JA eller NEJ neden for NEJ ◊ Hvad var datoen for første dag i din sidste menstruation ? dato: / år:

JA ◊ For dig, som har lidt pletblødninger inden den egentlige menstruation starter, er den første dag i din sidste menstruation den første af to dage i træk, hvor du har blødt begge dage, og blødningen i mindst én af dagene har været kraftigere end en pletblødning. Hvad var datoen for første dag i din sidste menstruation før du blev gravid? dato: / år:

Hvis du ikke kan huske datoen for første dag i din sidste menstruation, sæt kryds her

12. Har din cykluslængde* i de sidste tre måneder før denne graviditet været det samme antal dage? Sæt kryds og svar ved enten JA eller NEJ neden for

JA ◊ Hvor mange dage har din cykluslængde været ? dage

NEJ ◊ Den korteste cykluslængde jeg har haft inden for de sidste tre måneder var cirka dage Den længste cykluslængde jeg har haft inden for de sidste tre måneder var cirka dage

Hvis du ikke kan huske hvor mange dage din cykluslængde har været inden for de sidste tre måneder, sæt kryds her

* Hvad er en cykluslængde ? Længden af en menstruationscyklus er antallet af dage mellem første dag i en menstruation tilførste dag i næste menstruation. Har du for eksempel første dag i en menstruation den 1. august og første dag i den næstemenstruation den 30. august er cykluslængden 29 dage.

13. Passer et af de fire nedenstående udsagn på dig ? Sæt kryds ved enten JA eller NEJ neden under de fire udsagn.

1. Jeg har haft mindre end 3 menstruationer siden sidste graviditet 2. Jeg har haft mindre end 3 menstruationer siden ophør med P-piller 3. Jeg har haft mindre end 3 menstruationer siden fjernelse af spiral 4. Jeg havde P-ring, da jeg blev gravid

NEJ ◊ ingen af de fire ovenstående udsagn passer på mig

JA ◊ mindst et af de fire ovenstående udsagn passer på mig Hvis du har sat kryds ved JA, hvilket nummer har det udsagn, der passer på dig ? Udsagn nummer : 1 2 3 4 ( sæt ring om nummeret )

2CRL&FLOW

DE NÆSTE SPØRGSMÅL HANDLER OM DIN NUVÆRENDE GRAVIDITET

14. Har du haft pletblødning / blødning i din nuværende graviditet Sæt kryds og svar ved enten JA eller NEJ neden for JA ◊ gå videre til spørgsmål 15 NEJ ◊ gå videre til spørgsmål 18

15. Hvis du har blødt i din nuværende graviditet, har du så... (Sæt ét kryds, hvor det er relevant) 1. blødt før uge 12 af graviditeten 2. blødt efter uge 12 af graviditeten 3. blødt både før og efter uge 12 af graviditeten

16. Hvor mange dage har du blødt i alt ? Jeg har blødt cirka dage i alt

17. Hvor kraftig var din blødning / blødninger ? Sæt kun ét kryds 1. Der har kun været lidt pletblødning 2. Der har minimum været én blødning, der var lige så kraftig som en menstruationsblødning

DE FØLGENDE SPØRGSMÅL HANDLER OM TIDLIGERE GRAVIDITETER

18. Har du været gravid før, dette omfatter også graviditeter, som er endt med abort eller dødfødsel. Sæt kun ét kryds JA ◊ gå videre til spørgsmål 19 NEJ ◊ gå videre til spørgsmål 37

19. Hvor mange gange har du været gravid (dette omfatter også graviditeter som er endt med abort eller dødfødsel) ? Jeg har været gravid gange

20. Hvor mange levende børn har du født? Jeg har født levende børn

21. Er nogen af dine graviditeter endt med spontan abort eller dødfødsel ? Sæt kun ét kryds JA ◊ gå videre til spørgsmål 22 NEJ ◊ gå videre til spørgsmål 23

3CRL&FLOW

22. I hvilken uge af graviditeten endte graviditeten med spontan abort eller dødfødsel? Udfyld kun for de graviditeter hvor det har relevans:

1. Min første graviditet endte i abort eller dødfødsel i uge af graviditeten 2. Min anden graviditet endte i abort eller dødfødsel i uge af graviditeten 3. Min tredje graviditet endte i abort eller dødfødsel i uge af graviditeten 4. Min fjerde graviditet endte i abort eller dødfødsel i uge af graviditeten 5. Min femte graviditet endte i abort eller dødfødsel i uge af graviditeten 6. Min sjette graviditet endte i abort eller dødfødsel i uge af graviditeten

OM DIN FØRSTE FØDSEL

23. Var der nogen af følgende komplikationer ? (Sæt et kryds ved hver række) JA NEJ a. Svangerskabsforgiftning b. Forhøjet blodtryk c. For tidlig fødsel Hvis JA til punkt c, i hvilken uge af graviditeten blev barnet / børnene født: uge af graviditeten d. Andre komplikationer Hvis JA til andre komplikationer, skriv venligst hvilke

24. Fødte du tvillinger ? JA ◊ gå videre til spørgsmål 26 NEJ ◊ gå videre til spørgsmål 25

25. Hvor meget vejede barnet ? gram Hvis du ikke ved hvor meget barnet vejede, sæt kryds her

26a. Hvor meget vejede tvilling 1 ? gram Hvis du ikke ved hvor meget tvilling 1 vejede, sæt kryds her

26b. Hvor meget vejede tvilling 2 ? gram Hvis du ikke ved hvor meget tvilling 2 vejede, sæt kryds her

OM DIN ANDEN FØDSEL (Hvis du ikke har født to levende børn, gå videre til spørgsmål 37) 27. Var der nogen af følgende komplikationer ? JA NEJ a. Svangerskabsforgiftning b. Forhøjet blodtryk c. For tidlig fødsel Hvis JA til punkt c, i hvilken uge af graviditeten blev barnet / børnene født: uge af graviditeten d. Andre komplikationer Hvis JA til andre komplikationer, skriv venligst hvilke


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30. Hvor meget vejede tvilling 1 ? gram Hvis du ikke ved hvor meget tvilling 1 vejede, sæt kryds her


OM DIN TREDJE FØDSEL (Hvis du ikke har født tre levende børn, gå videre til spørgsmål 37) 32. Var der nogen af følgende komplikationer ? JA NEJ a. Svangerskabsforgiftning b. Forhøjet blodtryk c. For tidlig fødsel Hvis JA til punkt c, i hvilken uge af graviditeten blev barnet / børnene født: uge af graviditeten d. Andre komplikationer Hvis JA til andre komplikationer, skriv venligst hvilke





HER KOMMER ET PAR SPØRGSMÅL OM DIN EGEN FØDSEL

37. Hvad vejede du, da du selv blev født ? gram Hvis du ikke kender din vægt, besvar venligst spørgsmål 38

38. Jeg kender ikke min nøjagtige fødselsvægt, men jeg mener, at jeg vejede: Sæt kun ét kryds Mindre end 3000 gram Mellem 3000 og 4000 gram Mellem 4000 og 4500 gram Mere end 4500 gram Ved ikke

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DE FØLGENDE SPØRGSMÅL HANDLER OM DIT HELBRED OG MEDICIN

39. Havde du en kronisk sygdom, da du blev gravid ? Sæt kun ét kryds JA NEJ ◊ gå videre til spørgsmål 42

40. Havde du nogen af følgende sygdomme, da du blev gravid ? Sæt evt. flere kryds a. Forhøjet blodtryk f. Nyresygdom b. Hjertesygdom g. Epilepsi c. Lungesygdom/astma h. Gigt d. Sukkersyge i. Psykisk sygdom e. Stofskiftesygdom j. Anden sygdom

41. Hvis du havde en anden sygdom, skriv hvilken

42. Har du i løbet af graviditeten taget medicin oftere end 1 gang om ugen ? Sæt kun ét kryds (Se bort fra jern, vitaminer og kalk) JA NEJ ◊ gå videre til spørgsmål 45

43. Hvilken medicin har du taget oftere end 1 gang om ugen i løbet af denne graviditet ? Sæt kryds i ét eller flere felter, hvis du har taget den pågældende medicin.

43a. Medicin mod (a-l): a. Forhøjet blodtryk b. Hjertesygdom c. Lungesygdom/astma d. Sukkersyge e. Stofskiftesygdom f. Nyresygdom g. Epilepsi h. Gigt i. Psykisk sygdom . j. Infektion (ikke blærebetændelse) k. Blærebetændelse l. Hæmoroider

43b. Anden medicin (m-q): m. Smertestillende medicin n. Beroligende medicin o. Narkotiske stoffer p. Amfetamin q. Kokain

43c. Naturmedicin (r): r. Fiskeolie

44. Skriv venligst navnet/navnene og dosis på den medicin du har taget:

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HER FØLGER NOGLE SPØRGSMÅL OM UDDANNELSE OG ARBEJDE

45. Hvilken almen skolegang har du? Sæt kun ét kryds a. Går stadig i skole b. 7 års skolegang eller mindre c. 8-9 års skolegang d. 10 års skolegang e. Studenter eksamen, HF eksamen (inklusiv HHX og HTX) f. Andet

46. Har du afsluttet en uddannelse efter din almene skolegang? Sæt kun ét kryds JA ◊ gå videre til spørgsmål 47 NEJ ◊ gå videre til spørgsmål 48

47. Hvilken uddannelse har du afsluttet? Sæt kun ét kryds (Hvis du har flere uddannelser, sæt da kun kryds ved den længstvarende afsluttede uddannelse.) a. Specialarbejderuddannelse b. Handelsskolernes grunduddannelse (HG) / basisår i EFG-uddannelse c. Lærlinge-, EFG- eller HG-uddannelse d. Anden faglig uddannelse e. Kort videregående uddannelse, under 3 år f. Mellemlang videregående uddannelse 3-4 år g. Lang videregående uddannelse, over 4 år

48. Jeg har ingen afsluttet uddannelse fordi: Sæt kun ét kryds (Hvis du har afluttet en uddannelse, gå da videre til spørgsmål 49) 1. Jeg aldrig har påbegyndt en uddannelse 2. Jeg stadig er under uddannelse 3. Jeg har afbrudt en uddannelse

49. Hvad er din erhvervsmæssige stilling ? Sæt kun ét kryds a. Selvstændig erhvervsdrivende (dvs. egen virksomhed eller forretning) Skriv antal underordnede/ansatte b. Funktionær/tjenestemand (offentligt ansat) Skriv antal underordnede/ansatte c. Funktionær (privat ansat) Skriv antal underordnede/ansatte d. Faglært arbejder Skriv antal underordnede/ansatte e. Specialarbejder Skriv antal underordnede/ansatte f. Arbejdsløs g. Medhjælpende ægtefælle/sambo, hjemmegående husmor h. Lærling / efg-elev i. På bistandshjælp / A-kasse j. Studerende angiv fag: k. Skoleelev l. Barsel / forældreorlov angiv stilling, når du ikke er på orlov: m. Andet

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50. Hvad er din stilling mere nøjagtigt? ( Skriv venligst mere nøjagtigt, fx oversygeplejerske, ikke bare ansat i sundhedsvæsenet eller lærer – ikke blot ansat på skole):

HER KOMMER NOGLE SPØRGSMÅL VEDRØRENDE RYGEVANER

51. Ryger du? Sæt kun ét kryds a. Ja, dagligt b. Ja, men der er dage, hvor jeg ikke ryger c. Nej, jeg holdt op indenfor de første 3 mdr. af graviditeten, gå videre til 54 d. Nej, jeg er holdt op før graviditeten, gå videre til spørgsmål 53 e. Nej, jeg har aldrig røget, gå videre til spørgsmål 55

52. Hvis du er ryger nu: Skriv antal ud for hvor meget, du ryger. Skriv 0, hvis du ikke ryger det pågældende. a. Cigaretter: cigaretter pr. dag b. Pibe: pibestop pr. dag c. Hash: antal gange pr. uge 53. Hvis du stoppede med at ryge før du blev gravid: Hvor mange måneder før du blev gravid stoppede du med at ryge ? måneder

54. Hvis du er stoppet med at ryge før du blev gravid - eller hvis du er stoppet med at ryge inden for de 3 første måneder af graviditeten: Skriv antal ud for hvor meget, du røg den sidste måneds tid inden du stoppede. Skriv 0, hvis du ikke ryger det pågældende. a. Cigaretter: cigaretter pr. dag b. Pibe: pibestop pr. dag c. Hash: antal gange pr. uge

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HER KOMMER NOGLE SPØRGSMÅL OM KOST OG DRIKKEVARER HER I GRAVIDITETEN

55. Hvilken kost spiser du her i graviditeten ? Sæt kun ét kryds a. Almindelig varieret b. Almindelig, men ikke særlig varieret c. Vegetarisk, dog fisk og fjerkræ d. Vegetarisk, dog mælk og æg e. Kun kost fra planteriget (veganisk)

56. Drikkevarer. Hvad drikker du nu på dette tidspunkt i graviditeten? Skriv et tal på hver linje for at vise, hvad du drikker (kaffe, te osv.), skriv 0, hvis du ikke drikker det pågældende a. Antal krus kaffe dagligt krus dagligt b. Antal krus te dagligt krus dagligt c. Antal liter cola om ugen liter pr. uge d. Antal almindelig øl om ugen flasker pr. uge e. Antal stærke øl om ugen flasker pr. uge f. Antal glas vin om ugen glas pr. uge g. Antal genstande spiritus om ugen genst. pr. uge

57. Slik m.v. Spiser du på dette tidspunkt i graviditeten: Sæt kun ét kryds i hver linie: JA NEJ slik chips

58. Hvor meget slik/chips spiser du nu på dette tidspunkt i graviditeten? Sæt evt. flere kryds, hvis du spiser både slik og chips

a. Slik dagligt b. Slik 1-3 gange om ugen c. Slik sjældnere end 1 gang om ugen d. Chips dagligt e. Chips 1-3 gange om ugen f. Chips sjældnere end 1 gang om ugen

Mange tak fordi du tog dig tid til at udfylde skemaet.

Venlig hilsen

Læge Nina Gros Pedersen Professor, overlæge Ann TaborKlinik for føtalmedicin og ultralydskanningRigshospitalet

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