Original Article
Placental Expression of proBNP/NT-proBNP and Plasma
Levels of NT-proBNP in Early- and Late-Onset Preeclampsia
Katja Junus,1 Anna-Karin Wikström,1 Anders Larsson,2 and Matts Olovsson1
background
Levels of plasma N-terminal pro B-type natriuretic peptide (NT-proBNP)
are elevated in preeclampsia. In this study, the possibility that the pla-
centa produces and releases proBNP/NT-proBNP was explored. Plasma
levels of NT-proBNP in early- and late-onset preeclampsia were also
measured.
methods
Placental proBNP mRNA in early-onset preeclampsia (n = 7), late-onset
preeclampsia (n = 8), and controls of similar gestational age (n = 10)
was assessed by quantitative real-time polymerase chain reaction.
ProBNP/NT-proBNP protein was studied in placental samples with
immunohistochemistry (n = 8) and tissue culture (n = 2). Plasma levels
of NT-proBNP were measured in early-onset preeclampsia (n = 18), late-
onset preeclampsia (n = 20), and relevant controls (n = 36).
results
Transcripts of proBNP mRNA were found in 20 out of 25 samples,
there were no differences in expression between the groups. ProBNP/
Affecting 2%–8% of all pregnancies,1 preeclampsia is a major
global health problem and one of the leading causes of both
maternal and fetal mortality and morbidity worldwide.
Diagnosis of preeclampsia is based on proteinuria and new
onset of hypertension after 20 weeks of gestation. Despite
extensive research, the etiology of preeclampsia is not
completely understood. The 2-stage model of preeclamp-
sia (i.e., poor placentation and consequent oxidative stress,
leading to the release of placental factors into the maternal
circulation where they cause the clinical disease)2 is widely
accepted. However, a single underlying mechanism for the
disease may not exist, and subgroups of preeclampsia may
have different causes with a similar clinical presentation.3
For example, early-onset preeclampsia, but not late-onset
preeclampsia, has a strong association with placental dys-
function. Thus, in recent studies, subgrouping of preeclamp-
sia cases based on factors such as gestational age at disease
manifestation or disease severity is becoming more frequent.
Plasma levels of B-type natriuretic peptide (BNP) and
inactive amino-terminal pro-BNP (NT-proBNP), the 2
cleavage products of proBNP, are increased in women with
pregnancy-induced hypertension and preeclampsia4–7 and
Correspondence: Katja Junus (katja.junus@kbh.uu.se).
Initially submitted October 28, 2013; date of first revision November
17, 2013; accepted for publication January 29, 2014; online publication
March 8, 2014.
American Journal of Hypertension  27(9)  September 2014  1225
NT-proBNP protein was observed in maternal spiral arteries and in syn-
cytiotrophoblasts in all placental samples. After placental tissue culture,
there were measurable amounts of NT-proBNP in the culture media.
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Women with both early- (365 [14-9815] pg/ml) and late-onset preec-
lampsia (176 [33-2547] pg/ml) had higher levels of NT-proBNP than
their controls (P < 0.001). There was a tendency toward higher levels
of NT-proBNP in women with early-onset preeclampsia than in women
with late-onset preeclampsia (P = 0.057).
conclusion
The results indicate possible placental production and release of
proBNP/NT-proBNP into the maternal circulation.
Keywords: B-type natriuretic peptide; blood pressure; early-onset
preeclampsia; hypertension; late-onset preeclampsia; NT-proBNP;
­
placenta.
doi:10.1093/ajh/hpu033
have been reported to be especially high in women with
severe or early onset disease.8,9 Both BNP and NT-proBNP
are released from the ventricles of the heart in response to
cardiac overload and stretching of myocytes and are used as
biomarkers of chronic heart failure in nonpregnant popula-
tions. In preeclampsia, elevated levels have been attributed
to strain on the heart due to cardiac overload.
However, considering that the placenta is an endo-
crine organ and one of its major functions is to synthesize
and secrete hormones that enable pregnancy and support
the growing fetus, we hypothesized that there is a placen-
tal origin of proBNP and that the elevated levels of BNP/
NT-proBNP seen in preeclampsia may partly be a result of
placental release of these peptides.
In this study, the possibility that the placenta is a source
of proBNP was explored. Protein levels of proBNP/
NT-proBNP in the placenta were assessed with immuno-
histochemistry and tissue culturing, and placental levels of
mRNA were assessed by measuring the proBNP transcript
NPPB. We also studied the plasma levels of NT-proBNP,
which is more stable than BNP,10 in early- and late-onset
preeclampsia.
1Department of Women’s and Children’s Health, Uppsala University,
Uppsala, Sweden; 2Department of Medical Sciences, Biochemical
Structure and Function, Uppsala University, Uppsala, Sweden.
© American Journal of Hypertension, Ltd 2014. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Junus et al.
METHODS
The study was approved by the Regional Ethics Review
Board, Uppsala, Sweden, and informed consent was obtained
from each woman included in the study.
Samples from both case patients and control subjects used in
the plasma and protein analysis were collected at the University
Hospital in Uppsala. Preeclampsia was defined as new-onset
hypertension (≥140/90 mm Hg) observed on at least 2 separate
occasions ≥6 hours apart, combined with proteinuria (≥2 on a
dipstick, or a 24-hour urine sample measuring ≥300 mg).
Case patients and control subjects
Case patients included in the study were divided into
two groups: early-onset preeclampsia (diagnosed at 31
completed weeks of gestation or earlier; n = 18) and late-
onset preeclampsia (diagnosed at 35 completed weeks of
gestation or later; n = 20). Two control groups were also
included in the study: an “early” control group of women
recruited during a routine visit to an antenatal clinic at
25–31 completed weeks of gestation (n = 22; only women
whose pregnancies continued normally and resulted in
full-term delivery of a healthy child of normal weight
remained in this group) and a “late” control group of
healthy pregnant women who delivered at 35–40 com-
pleted weeks of gestation (n  =  14). Women with multi-
ple pregnancies, ongoing upper urinary tract infection,
chronic hypertension, diabetes mellitus, or renal disease
were not included in any of the study groups.
Plasma NT-proBNP analysis
Plasma samples were collected in EDTA tubes. Among
the case patients and subjects in the late control group,
samples were collected before onset of active labor, 1–12
hours before delivery. In subjects in the early control
group, samples were collected at the time of recruitment.
The samples were immediately put into a refrigerator
and were centrifuged for 10 minutes at 1,500 × g within
2 hours of collection. The plasma samples were then
stored at −70 °C until analyzed. Levels of NT-proBNP
were determined with a sandwich immunoassay using a
Modular E170 analyzer.
Quantitative real-time polymerase chain reaction
Placenta samples from patients with early-onset preec-
lampsia (n = 8) and late-onset preeclampsia (n = 7) and sub-
jects in the early control group (n = 4) and late control group
(n  =  6) were used and have been described elsewhere.11
Complementary DNA was synthesized from RNA with
SuperScript III First-Strand Synthesis SuperMix (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions.
Relative mRNA levels of NPPB, the gene encoding natriu-
retic peptide B, were measured by quantitative real-time
polymerase chain reaction as previously described.12 The
primer (CyberGene AB, Stockholm, Sweden) was com-
plementary to an exon boundary to avoid amplification of
genomic DNA and designed according to guidelines from
1226  American Journal of Hypertension  27(9)  September 2014
Applied Biosystems. Melt curve analysis was carried out to
ensure specific amplification.
The genes for TATA box-binding protein (TBP), tyros-
ine 3-monooxygenase/tryptophan 5-monooxygenase
activation protein, zeta polypeptide (YWHAZ), and
succinate dehydrogenase complex subunit A  (SDHA)
were used as reference genes and are stable in preec-
lamptic placental tissue.13 LinRegPCR software (version
11.3; Heart Failure Research Center, Amsterdam, the
Netherlands) was used for Ct (threshold cycle) extraction
and efficiency calculations.14 Mean normalized expres-
sion (MNE) was calculated by means of the following
equation:
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3 (E Ctref 1,mean )(E Ctref 2 ,mean )(E Ctref 3 ,mean )
MNE = Ctt arg et ,mean
(E )
where E is the mean experimental efficiency of the primer
and Ct is the mean Ct of triplicate reactions.
Immunohistochemistry
Eight randomly selected villous placental samples were
used: 3 from early-onset preeclampsia patients, 3 from
late-onset preeclampsia patients and 1 each from the early
and late control groups. Human heart tissue was used as a
positive control. Placental tissue samples (n = 2–3) approxi-
mately 1 cm3 in size were collected from different cotyledons
from a central part of each placenta immediately after deliv-
ery. The samples were rinsed in physiological saline solution
(NaCl, 9 mg/ml), fixed in 4% paraformaldehyde for approx-
imately 24 hours, and stored in 70% ethanol for a few days
until being embedded in paraffin wax according to routine
procedures. Serial sections (5  μm) of paraformaldehyde-
fixed, paraffin-embedded placental tissue were prepared
as previously described.15 Briefly, sections were deparaffi-
nized, rehydrated, and washed. This was followed by incu-
bation in citrate buffer. Nonspecific binding was blocked,
and the sections were thereafter incubated overnight at 4 °C
with mouse monoclonal anti-proBNP (1:1,000 and 1:5,000;
15C4, ab51789; Abcam, Cambridge, UK), which reacts with
human proBNP and NT-proBNP. After incubation, the sec-
tions were washed, and biotinylated anti-mouse immuno-
globulin G (1:300; Vector Laboratories, Burlingame, CA)
was added. After 1 hour at room temperature, the sections
were washed again, and streptavidin-conjugated horserad-
ish peroxidase complex (Vector Laboratories) was added.
The sections were then rinsed, exposed to chromogen solu-
tion (liquid DAB substrate kit; DAKO, Stockholm, Sweden),
and counterstained with Mayer’s hematoxylin. Using light
microscopy, the brown reaction product was observed.
Negative control staining was done by omitting the primary
antibody.
Tissue culture
Placental villous tissue was collected from 2 women, not
included in the plasma, protein, or mRNA analysis, after
uncomplicated single pregnancies. They were both vaginally
 NT-proBNP and preeclampsia

delivered and gave birth to a healthy child at term. The women
had no ongoing upper urinary tract infection, chronic hyper-
tension, diabetes mellitus, or renal disease. Samples were
collected from the placental midlayer at 5 different but cen-
tral locations within each placenta to avoid variation due to
sampling site. The samples were thoroughly rinsed in sterile
phosphate-buffered saline, cut into pieces of approximatelyy
1 mm in diameter, and thereafter cultured for 24 hours at
37  °C in culture medium. The medium was then collected,
filtered, and centrifuged at 400 × g for 5 minutes. The super-
natant was collected and stored at −20 °C. Samples and fresh
culture medium (negative control) were then assayed for
NT-proBNP using a sandwich immunoassay with a Modular
were 2-tailed, and P  <  0.05 was considered statistically
significant.
RESULTS
Background characteristics
Baseline characteristics of the case patients and control
subjects are presented in Table 1. Women with early-onset
preeclampsia had higher systolic blood pressure and were
more often on hypertensive treatment than women with
late-onset preeclampsia. Glomerular filtration rate was
lower in women with preeclampsia than control subjects,
and lower in patients with late-onset preeclampsia than in

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E170 analyzer.
Statistics
All statistical analyses were performed using an SPSS
12.0 (IBM, Armonk, NY) for Windows software package.
Background variables in Table 1 are presented as mean and
SD or number and percentage. Comparisons between con-
tinuous background variables were carried out with analy-
sis of variance for overall comparisons of mean and Tukey’s
test for multiple pair-wise comparisons. For categorical
background variables, the χ2 test or Fisher’s exact test was
used. Comparisons of plasma levels of NT-proBNP were
done with the Kruskal–Wallis test followed by the Mann–
Whitney U test for independent samples with Bonferroni
correction between early- and late-onset preeclampsia;
early-onset preeclampsia and early controls; and late-onset
preeclampsia and late controls. Values of NT-proBNP
(Table  1) are expressed as median and range. All tests
patients with early-onset preeclampsia. Body mass index at
the time the women registered with their maternity clinics
was higher in women with early-onset preeclampsia than
in subjects in the early control group. Gestational age at the
time of sampling did not differ between women with early-
onset preeclampsia and subjects in the early control group or
between women with late-onset preeclampsia and subjects
in the late control group.
Plasma levels of NT-proBNP
Both women with early-onset preeclampisa
(median = 365; range = 14–9,815 pg/ml) and those with late-
onset preeclampsia (median  =  176; range  =  33–2,547 pg/
ml) had higher levels of NT-proBNP than control subjects
(P < 0.001) (Table 1). There was a tendency toward higher
levels of NT-proBNP in women with early-onset preeclamp-
sia than in women with late-onset preeclampsia (P = 0.06)
(Figure 1).

Table 1.  Characteristics of the women included in the study

Early-onset
Early control Late control preeclampsia Late-onset preeclampsia
group (n = 22) group (n = 14) group (n = 18) group (n = 20) P value

Maternal age, y 31 ± 3.7 33 ± 5.7 31 ± 4.2 30 ± 5.8 0.42

Nulliparous 9 (41) 4 (29) 12 (67) 11 (55) 0.14
Systolic pressure,a,b,c mm Hg 124 ± 16 124 ± 12 164 ± 17 148 ± 14 <0.001
Diastolic pressure,a,b mm Hg 80 ± 11 79 ± 12 99 ± 6 99 ± 7 <0.001
Hypertensive treatment at samplinga,b,c 0 (0) 0 (0) 15 (83) 10 (50) <0.001
Glomerular filtration rate,a,b,c ml/min/1.73m2 81 ± 10 63 ± 12 57 ± 12 45 ± 13 <0.001
BMI at registration with maternity clinic,a kg/ m2 23 ± 3 25 ± 3 27 ± 5 27 ± 6 0.01
BMI before delivery, kg/ m2 — — 31 ± 7 33 ± 5 0.38
Gestational age at sampling,c d 197 ± 13 277 ± 11 198 ± 24 265 ± 11 <0.001
Plasma levels of NT-proBNP,a,b pg/ml 48 (10–170) 41 (14–172) 365 (14–9,815) 176 (33–2,547) <0.001

Continuous variables are expressed as mean ± SD or median (range), and categorical variables are expressed as number (%). Comparisons
between continuous background variables were carried out with analysis of variance for overall comparisons of mean and Tukey’s test for
multiple pair-wise comparisons. For categorical background variables, the χ2 test or Fisher exact test was used. A value of P < 0.05 denotes a
significant difference.
Abbreviations: BMI, body mass index; NT-proBNP, N-terminal pro B-type natriuretic peptide.
aSignificant difference between early control group vs. early-onset preeclampsia group.
bSignificant difference between late control group vs. late-onset preeclampsia group.
cSignificant difference between early-onset preeclampsia group vs. late-onset preeclampsia group.

American Journal of Hypertension  27(9)  September 2014  1227

Junus et al.

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b

Figure  1.  Plasma concentrations of N-terminal pro B-type natriuretic

peptide (NT-proBNP) in each study group. Comparisons between the
groups were done with the Kruskal–Wallis test followed by the Mann–
Whitney U test with Bonferroni correction. Results are presented as scat-
ter-plots showing group medians. Values of P < 0.05 were considered to
denote a statistically significant difference. ***P < 0.001.

Figure 2.  Mean normalized expression (MNE) of placental natriuretic

peptide B (NPPB) mRNA in each study group. Comparisons between the
groups were done with the Kruskal–Wallis test. Results are presented as
scatter-plots showing group medians. There were no statistically signifi-
cant differences between the groups.

mRNA levels of NPPB

With the exception of delivery mode, where all women
with early-onset preeclampsia and one woman with late-
onset preeclampsia had a caesarian section, background
characteristics of the case patients and control subjects did
not differ between the groups.11 Transcripts of NPPB were
Figure  3.  Immunohistochemical staining of placental tissue from a
pregnancy complicated by preeclampsia. (a) Immunostaining with anti
pro B-type natriuretic peptide (anti-proBNP)/ N-terminal pro B-type
natriuretic peptide (NT-proBNP). (b) Negative control. (c) Positive con-
trol where human heart muscle was immunostained with anti-proBNP/
NT-proBNP.

1228  American Journal of Hypertension  27(9)  September 2014


found in 20 of 25 samples. No amplification was found in
1 sample from each group, except for the late-onset group,
where 2 samples showed no amplification. No differences in
NPPB expression between the groups were found (Figure 2).
Protein expression
Immunohistochemical analysis showed proBNP/
NT-proBNP protein in all placental samples, both in case
patients and control subjects. Staining was especially pro-
found in maternal spiral arteries, but staining was also visible
in the syncytiotrophoblast (Figure  3). Nonspecific staining
was not detected.
Tissue culture
Placental samples from 2 uncomplicated pregnancies were
used. After 24 hours of culture, NT-proBNP concentrations
in culture media were 7 pg/ml and 6 pg/ml. No NT-proBNP
(<5.0 pg/ml) was found in fresh culture medium.
Discussion
The placenta as a source of circulating BNP/NT-proBNP
in early- and late-onset preeclampsia was explored in this
study. We found proBNP mRNA and protein in placental
tissue, but there were no differences between women with
preeclampsia and those with normal pregnancies.
Transcripts of the gene encoding proBNP were found in
20 of 25 placental samples. All 4 study groups were repre-
sented in the 5 placental samples with no proBNP mRNA.
The absence of mRNA may be a result of lack of transcrip-
tion or transcription at too low of a level to be detected.
Protein expression of proBNP/NT-proBNP was found in
the syncytiotrophoblast. The origin of syncytiotrophoblast
proBNP/NT-proBNP is unknown; it could be produced
in the placenta itself, or it may result from uptake from
the maternal blood surrounding the placental villi. When
culturing villous placental tissue, we found measurable
amounts of NT-proBNP in the culture medium, indicat-
ing release, leakage, or secretion of NT-proBNP/proBNP
from the placental tissue. It is unlikely that remains of
blood or blood plasma could result in measurable amounts
of NT-proBNP in the culture media; the pieces of placen-
tal tissue were very small compared with the relatively
large volume of culture media, and the biopsies had been
thoroughly rinsed before they were transferred to the
culture media.
Our data confirm findings in earlier studies reporting
higher levels of circulating NT-proBNP in women with
preeclampsia than in healthy pregnant women.4–9 A  ten-
dency toward higher levels of circulating NT-proBNP in
early-onset preeclampsia vs. late-onset preeclampsia was
observed, but in contrast with the results of the study by
Rafik Hamad et al.,9 the difference was not significant. It is
likely that a larger sample size would have resulted in a sig-
nificant difference between those 2 groups.
There was a discrepancy between NT-proBNP in plasma,
where there were higher levels in preeclamptic patients than
American Journal of Hypertension  27(9)  September 2014  1229
NT-proBNP and preeclampsia
in control subjects, and the proBNP mRNA levels, where we
saw no difference in expression between the groups. This
could be explained by poor association between transcrip-
tion levels and protein levels. There is also the possibility that
there is a difference in placental proBNP mRNA in preec-
lamptic pregnancies and healthy pregnancies but our sample
size was too small to detect this.
The view of preeclampsia as a syndrome with hetero-
geneous disease mechanisms is becoming increasingly
established. Therefore, to study such disease mechanisms
it is important to use well-characterized preeclampsia case
patients. Here we separated the preeclamptic women into
subgroups consisting of the 2 entities, early-onset preec-
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lampsia and late-onset preeclampsia. Early-onset preec-
lampsia is often defined as preeclampsia that develops
before 34 weeks of gestation, and late-onset preeclampsia
develops during or after week 34.16 However, the use of
such a distinct definition may result in misclassification of
some case patients as a result of overlapping of early and
late disease mechanisms. To circumvent this, we excluded
women diagnosed with preeclampsia at weeks 32–34 of
gestation.
There are some limitations to the study. The sample
size was low, and therefore it is difficult to draw firm con-
clusions from the results. All plasma samples were taken
before the onset of active labor to avoid potential changes
in NT-proBNP due to uterine contractions. However, it is
possible that some of the women were in the latent phase at
sampling and may have had uterine contractions to a various
degree, which we have not been able to take into account in
this study.
B-type natriuretic peptide binds to natriuretic peptide
receptors. Their physiological properties include effects on
blood pressure regulation and fluid homeostasis.17 When
comparing preeclamptic and healthy pregnant women,
Borghi et al.5 found that elevated levels of BNP were associ-
ated with increases in left ventricular mass and left ventricu-
lar end-systolic and end-diastolic volumes and significant
reductions in the left ventricular ejection fraction. In addi-
tion, high levels of NT-proBNP reflect strain on the heart
caused by high afterload.7 This may indicate that the high
levels of NT-proBNP in cases of preeclampsia are secondary
to an increased cardiac load in these women. It is also pos-
sible that the increased cardiac load in these women and the
higher levels of NT-proBNP/BNP reflect different aspects of
the heterogeneous syndrome preeclampsia and that some of
the circulating NT-proBNP/BNP is released from the pla-
centa. What effect these high levels of BNP may have is not
known. Increased capillary permeability in women with
pregnancy-induced hypertension is associated with a reduc-
tion in plasma volume, and it is possible that high levels of
BNP impair plasma volume expansion in these women. It is
not known whether natriuretic peptides have any physiolog-
ical role associated with reproductive functions. High levels
of BNP are present in amniotic fluid in the first and second
trimester, and BNP is also secreted from cultured human
amnion cells.18 Also, biologically active natriuretic peptide
receptors are present in human placental tissue,19,20 indicat-
ing direct physiological effects of natriuretic peptides on the
placenta. Placental expression of the BNP-related C-type
Junus et al.
natriuretic peptide mRNA is decreased, whereas myome-
trial C-type natriuretic peptide mRNA is increased, in preg-
nancies complicated by intrauterine growth restriction and
preeclampsia.20,21 Moreover, in mouse placenta, BNP mRNA
is expressed in the peripheral margin of the decidual layer,
indicating a role for BNP in regulating maternal blood sup-
ply to the developing embryo.22 All of this evidence points
toward a role for natriuretic peptides in reproductive physi-
ology and in control of placental function.
In conclusion, our results indicate probable placental pro-
duction and release of NT-proBNP into the maternal circula-
tion. It is not known, however, whether placental production
of NT-proBNP causes elevated plasma levels of NT-proBNP
in preeclamptic women and whether BNP has a role in the
pathophysiology of preeclampsia.
Acknowledgments
This work was supported by grants from the Erik, Karin
and Gösta Selanders Foundation. We would also like to
thank Mrs. Eva Davey for substantial support during the
investigation.
DISCLOSURE
The authors declared no conflicts of interest.
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