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The Journal of Maternal-Fetal and Neonatal Medicine, April 2008; 21(4): 267273
Whole-genome microarray and targeted analysis of angiogenesisregulating gene expression (ENG, FLT1, VEGF, PlGF) in placentas
from pre-eclamptic and small-for-gestational-age pregnancies
Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim,
Norway, 2Perinatology Research Branch, Division of Intramural Research, National Institute of Child Health and Human
Development, Detroit, Michigan, USA, 3Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen,
Norway, and 4Computer Science, Wayne State University, Detroit, Michigan, USA
(Received 27 June 2007; revised 5 August 2007; accepted 14 January 2008)
Abstract
Objective. To compare the placental pathology associated with pre-eclampsia (PE) and/or fetal growth restriction, the
transcriptomes of placental tissues from PE and small-for-gestational-age (SGA) pregnancies were explored. In addition, a
targeted analysis of angiogenesis-regulating gene expression was performed.
Methods. Whole-genome microarray analysis was performed on placental tissue from gestational age-matched PE (n 10),
SGA (n 8) and PE SGA (n 10) pregnancies. The expression of genes regulating angiogenesis (endoglin (ENG), fmsrelated tyrosine kinase 1 (FLT1), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)) was
analyzed by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR).
Results. Microarray analysis did not reveal any significant differences between groups. However, an increased expression of
ENG and FLT1 was detected by qRT-PCR in the PE SGA group.
Conclusions. The placental transcriptome did not differ between groups, although an increased anti-angiogenic gene
expression in PE SGA was observed with qRT-PCR analysis. Based on this, we conclude that although microarray
technology may represent a powerful tool in generating new hypothesis in complex fields, it may not be sensitive enough to
detect subtle changes in gene expression.
Keywords: pre-eclampsia, small-for-gestational-age, placenta, genome-wide gene expression, ENG, FLT1, angiogenesis
Introduction
The pre-eclamptic syndrome is characterized by
maternal manifestations (hypertension and proteinuria), but in some cases, the fetus may also be
involved, and pre-eclamptic women have an increased risk of delivering a small-for-gestational-age
(SGA) neonate [1]. The placental bed histopathological changes in PE and in SGA pregnancies (without pre-eclamptic manifestations) have several
shared characteristics, including abnormal trophoblast invasion and impaired spiral artery remodeling with subsequent placental hypoperfusion
[2]. Severe fetal growth restriction is usually associated with reduced placental weight, villous surface,
capillary volume and capillary surface, independent
of pre-eclamptic manifestations present [3]. However, isolated PE is generally associated with less
placental abnormalities, with the exception of the
early-onset (534 weeks) PE subgroup, which can
present extensive placental changes [4]. It has been
proposed that an excess of syncytiotrophoblast
shedding may evoke a maternal inflammatory response in PE patients. In contrast, shedding
appears to occur at physiological levels in SGA
pregnancies [5].
Correspondence: Rigmor Austgulen MD, PhD, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology
(NTNU), Medisinsk teknisk senter, 7489 Trondheim, Norway. Tel: 47 72573561. Fax: 47 72574704. E-mail: rigmor.austgulen@ntnu.no
*Toft and Lian have contributed equally to this work.
ISSN 1476-7058 print/ISSN 1476-4954 online 2008 Informa UK Ltd.
DOI: 10.1080/14767050801924118
268
J. H. Toft et al.
Methods
Study groups
The study population included singleton pregnancies
delivering at St Olavs University Hospital (Trondheim, Norway), and Haukeland University Hospital
(Bergen, Norway) from 2002 to 2006 in the
following groups: women who delivered an SGA
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Results
Study groups
The clinical characteristics of the study groups are
presented in Table I. Twenty-eight patients fulfilled
the criteria for being enrolled in the study and the
following study groups were included: (i) PE
(n 10); (ii) SGA (n 8); and (iii) PE SGA
(n 10) (Table I). As expected, patients in the
SGA and PE SGA groups had lower birthweight
than in the PE group. Blood pressure was higher
among patients in the PE and PE SGA groups than
in the SGA group.
Placental gene expression, as assessed by microarray
analysis
No significant differences in gene expression
were observed between the study groups after
correction for multiple comparisons was performed
(Table II). Of note there were several genes which
had p-values 5 0.05 prior to the multiple testing
PE
n 10
PE SGA
n 10
SGA
n8
p-value
30.3 + 5.4
34.2 + 2.5
2166.6 + 390.5
151.5 + 10.7
97.9 + 6.3
5/10
5/10
30.3 + 4.9
33.9 + 2.0
1529.7 + 341.8
148.3 + 12.0
94.1 + 7.3
5/10
5/10
34.4 + 5.0
34.5 + 3.8
1561.5 + 611.2
129.0 + 15.4
70.3 + 10.4
3/8
5/8
0.11
0.85
50.05*1
50.05*2
50.05*2
0.36
0.36
PE, pre-eclampsia; SGA, small-for-gestational-age; BP; blood pressure measured at time for diagnosis; *p 5 0.05 in the comparisons:
1
(PE vs. SGA, PE SGA vs. PE) and 2(PE vs. SGA, PE SGA vs. SGA); Data are presented as mean + SD.
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J. H. Toft et al.
Table II. Comparison between expression of angiogenesis-regulating genes, as assessed by microarray and qRT-PCR.
Microarray (p-values)
Gene name
qRT-PCR (p-values)
PE SGA vs. PE
PE vs. SGA
Global
p-value
PE SGA vs. PE
PE vs. SGA
0.999
0.999
0.999
0.999
0.859
0.867
0.940
0.909
0.952
0.930
0.941
0.956
0.033*
0.033*
0.054
0.740
0.023
0.046
0.067
0.945
0.019
0.015
0.006
0.444
0.346
0.241
0.171
0.455
ENG
FLT1
VEGF
PlGF
PE, pre-eclampsia; SGA, small-for-gestational-age; gene name, gene names are taken from Entrez-Gene; ENG, Endoglin; FLT1, fmsrelated tyrosine kinase 1, includes both soluble and membrane-bound isoforms of FLT1; VEGF, vascular endothelial growth factor; PlGF,
placental growth factor; p-values are adjusted for multiple comparisons; *p 5 0.05.
Discussion
Principal findings
First, microarray analysis did not demonstrate
significant differences in the placental transcriptome
of patients with PE, SGA and PE SGA with the
current sample size and method for data analysis
applied; Second, patients with PE SGA had
significantly higher placental expression of ENG
and FLT1 than patients in the PE and SGA groups;
Third, there were no differences in placental
VEGF and PlGF expression between the study
groups; and finally, there was a positive correlation
between the microarray results and the qRT-PCR
data.
Increased expression of anti-angiogenic genes in
PE SGA placentas
Assessment by qRT-PCR revealed that expression of
ENG and FLT1 in placental tissue from PE SGA
pregnancies was significantly higher than that detected in isolated PE or SGA pregnancies. This is
consistent with previous reports indicating the
association of high placental mRNA expression of
ENG in both isolated PE and SGA pregnancies
[6,7]. However, these reports are based on comparisons between normal placentas and placentas
obtained from pregnancies complicated by either
PE or SGA. Therefore, the data is in fact not
comparable. Similarly, FLT1 expression has also
been reported to be elevated in isolated PE and SGA
pregnancies compared to normal pregnancies, with
PE displaying the highest level of expression [9,10].
We observed a similar tendency (although not
271
Table III. Fold change expression of angiogenesis-regulating genes, as assessed by microarray and qRT-PCR.
Microarray
Gene name
ENG
FLT1
VEGF
PlGF
qRT-PCR
PE SGA vs. PE
PE vs. SGA
PE SGA vs. PE
PE vs. SGA
1.578
1.539
1.279
0.886
1.951
2.373
1.114
0.946
1.227
1.540
0.881
1.122
2.087
2.456
2.130
1.018
3.179
4.826
4.442
1.550
1.523
1.965
2.085
1.522
PE, pre-eclampsia; SGA, small-for-gestational-age; gene name, gene names are taken from Entrez-Gene; ENG, Endoglin; FLT1, fmsrelated tyrosine kinase 1, includes both soluble and membrane-bound isoforms of FLT1; VEGF, vascular endothelial growth factor; PlGF,
placental growth factor.
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J. H. Toft et al.
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