Clinica Chimica Acta 455 (2016) 60–63

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Clinica Chimica Acta

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Alteration of serum semaphorin 3B levels in preeclampsia

Hanzhi Wang, Linxiang Jiang, Bo Gao, Minyue Dong ⁎
a
Women's Hospital, School of Medicine, Zhejiang University, 1, Xueshi Road, Hangzhou, Zhejiang 310006, China
b
Key Laboratory of Reproductive Genetics, Ministry of Education, 1, Xueshi Road, Hangzhou, Zhejiang 310006, China
c
Key Laboratory of Women's Reproductive Health of Zhejiang Province, 1, Xueshi Road, Hangzhou, Zhejiang 310006, China

a r t i c l e i n f o a b s t r a c t

Article history: Background: Placental Semaphorin 3B (SEMA 3B) expression has been reported changed in preeclampsia and its
Received 27 October 2015 possible involvement of in the disease proposed. We clarified the alterations of maternal SEMA 3B level in
Received in revised form 26 January 2016 women suffering preeclampsia and pregnant women at gestational weeks of 16–20 before the onset of
Accepted 27 January 2016 preeclampsia.
Available online 29 January 2016
Methods: Serum SEMA 3B concentration was measured with ELISA in preeclamptic women (preeclampsia) and
normotensive women (control) in 3rd trimester, and also in pregnant women at gestational weeks of 16–20
Keywords:
Semaphorin 3B
who developed preeclampsia or had favorable pregnant outcome.
Preeclampsia Results: Serum SEMA 3B level was significantly increased in preeclampsia compared with control (P b 0.001).
Pregnancy There was a significant difference in serum SEMA 3B between mild and severe preeclampsia (P = 0.04).
Women with severe preeclampsia had significant serum SEMA 3B than women with mild preeclampsia. At
gestational weeks of 16–20, serum SEMA 3B was significantly higher in women who developed preeclampsia
than women who had normal pregnant outcome (P b 0.001).
Conclusions: Maternal SEMA 3B level increased in preeclampsia before the onset of manifestations, indicating that
SEMA 3B plays a role in the pathogenesis of preeclampsia.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction In an attempt to explore the differentially expressed genes in villous

Preeclampsia is a serious pregnancy complication affecting 2–8% of
all pregnancies, and is one of the major causes for maternal mortality
and morbidity, preterm birth, perinatal death and intrauterine growth
restriction [1–4]. This pregnancy complication is a multisystem disorder
affecting the liver, kidneys and cardiovascular and clotting systems
[1–3]. These manifestations are alleviated after the delivery of the
fetus and the placenta, however, the etiology of the disorder is not
completely understood [1–3].
Placenta and its major cell component, trophoblast, have been
considered to play pivotal roles in the pathogenesis of preeclampsia
for decades [1–7]. Defects in villous cytotrophoblasts (CTBs), which
lead to shadow interstitial invasion, poor placentation and deficient
remodeling of spiral arterioles, have been proposed to be the initial
etiology of preeclampsia [1–7]. On the other hand, hyper-regeneration
of trophoblast has also been reported in preeclampsia [8–10]. However,
the molecular mechanisms behind and the genes involved trophoblast
deficit remain largely unknown.
⁎ Corresponding author at: Women's Hospital, School of Medicine, Zhejiang University,
1, Xueshi Road, Hangzhou, Zhejiang 310006, China.
E-mail addresses: mydong.cn@hotmail.com, dongmy@zju.edu.cn (M. Dong).
cytotrophoblasts isolated from placenta of preeclamptic women, Zhou
et al. [11] found the up-regulated semaphorin 3B (SEMA 3B) in pre-
eclampsia and thought the up-regulated SEMA 3B played a role in the
development of preeclampsia. Later, Kaituu-Lino et al. [12] reported
that placental SEMA 3B expression was not significantly altered in
severe early-onset preeclampsia compared with control. These findings
regarding the expression of SEMA 3B in preeclampsia indicate that
further investigation is needed. However, the alteration in maternal
SEMA 3B has never been described in preeclampsia.
Semaphorins were first identified as axon guidance factors that di-
rect growth cones of axons to their proper target during the formation
of nervous system [13–15]. Thereafter, it has been realized that
semaphorins play a role in many developmental processes outside the
nervous system, in particular as a regulators of cell migration, immune
responses, and organogenesis [13–15]. The semaphorin family contains
more than 30 genes divided into seven subfamilies, all of which are
characterized by the presence of a sema domain [13–15]. Class-3
semaphorins are the only secreted vertebrate semaphorins, and display
properties of angiogenesis inhibitors and tumor progression modula-
tors. SEMA 3B, a member of class-3 semaphorins and an angiogenesis
inhibitor, has been proposed to be involved in tumor progression and
a potential therapeutic target [13–15]. However, the roles of SEMA 3B
in pregnancy and complications remain largely unknown [11,12].

http://dx.doi.org/10.1016/j.cca.2016.01.030
0009-8981/© 2016 Elsevier B.V. All rights reserved.
 H. Wang et al. / Clinica Chimica Acta 455 (2016) 60–63 61

2. Materials and methods 2.3. Statistic analysis

2.1. Subjects The distribution of serum concentration of SEMA 3B was normal as

Firstly, a cross-sectional observation was conducted to determine
the changes of maternal Sema 3B level in preeclamptic women in
third trimester of pregnancy in Women's Hospital, School of Medicine,
Zhejiang University. Thirty-six women with preeclampsia were
recruited and 36 normal pregnant women who were matched for
both maternal age and gestational age served as control. Among
the preeclamptic women, 27 were diagnosed with severe pre-
eclampsia, and 9 mild. Pregnancy was diagnosed upon positive
human chorionic gonadotropin test after missed menstruation.
Gestational age was calculated by menstrual dating. Ultrasound
was performed to confirm pregnancy and gestational age. Pre-
eclampsia were diagnosed and classified according to the criteria
recommended by American College of Obstetrics and Gynecologist
(ACOG) [16]: a systolic blood pressure of 140 mm Hg or higher or a
diastolic blood pressure of 90 mm Hg or higher on two occasions at
least 6 h apart after 20 weeks of gestation in a pregnant woman with
previously normal blood pressure and detectable urinary protein
(≥1 + by dipstick or ≥0.3 g/24 h) [17]. Severe preeclampsia was defined
as a blood pressure ≥ 160/110 mm Hg with either a urine dipstick
showing 3 + or 4 + in a random urine sample or greater than 5.0 g of
proteinuria over 24 h. Other evidence of severe disease included
increased serum creatinine, eclampsia, pulmonary edema, oliguria
(b 500 ml/24 h), fetal growth restriction, oligohydramnios and
symptoms suggesting significant end-organ involvement (headache,
visual disturbance, or epigastric or right upper quadrant pain).
Women who met criteria of preeclampsia but not severe preeclampsia
were diagnosed mild preeclampsia.
To clarify if the alteration of maternal Sema 3B occurred in the
second trimester of pregnancy, before the onset of preeclampsia,
a retrospective nested case–control study was conducted. Blood sam-
ples were chosen from the sample bank. Blood samples were collected
at 16–20 weeks of gestational age for routine Down's syndrome screen-
ing. The preeclampsia group consisted of 25 pregnant women who
subsequently developed severe preeclampsia, and the control group
consisted of 25 normal pregnant women who had a normal pregnancy
outcome. Among the preeclamptic women, 23 developed severe
preeclampsia and 2 mild. Maternal ages ranged from 22 to 34 years in
control groups and from 22 to 35 years in preeclampsia group. Maternal
age, gestational age and the date of sample collection were matched for
preeclampsia group and control group.
All subjects were nulliparous Chinese women with singleton
pregnancies. Exclusion criteria were multiple gestation, diabetes
mellitus, chronic hypertension, infectious diseases recognized in
pregnancy, premature rupture of membrane and signs of other con-
current medical complications. The subjects for control had no signs
of gestational complications and fetal distress and all gave birth to
healthy neonates of appropriate size for gestational age.
Clinical data and demographic data were collected according to
the medical records. The approval of the current study was obtained
from Institutional Ethical committee of Women's Hospital, School of
Medicine, Zhejiang University, and all the participants provided their
informed consents.
2.2. Sample collection and assay
Fasting blood samples were taken and stood at room temperature
for at least 30 min for the blood to clot and then was centrifuged at
tested by Kolmogorov–Smirnov test, and thus serum SEMA 3B data
are presented as mean with standard deviation (SD) and compared
with the Student's t-test. Chi-square test was used for the analysis of a
number of cases with proteinuria. The Statistical Analysis System was
used for data analysis. A p b 0.05 was considered significant.
3. Results
As shown in Table 1, there were no significant differences in maternal
age, gestational age at sampling, either in the third trimester or the
second trimester, between preeclampsia and control; however, there
were significant differences in blood pressure in the third trimester as
expected. Maternal serum SEMA 3B levels were determined by ELISA.
We found that there was a significant difference in SEMA 3B levels
between preeclamptic women (preeclampsia) and normal pregnant
women (control) (Fig. 1; t = 3.911, P b 0.001). Preeclamptic women
had significantly higher serum SEMA 3B level than control. The serum
SEMA 3B levels were significantly different between mild and severe
preeclampsia (Fig. 2; t = 2.134, P = 0.04). Women with severe
preeclampsia had significantly higher serum SEMA 3B levels than
women with mild preeclampsia.
To determine if maternal SEMA 3B levels is altered in preeclamptic
women in the second trimester of pregnancy, serum SEMA 3B levels
were determined in pregnant women who subsequently developed
preeclampsia (preeclampsia) and pregnant women who had favorable
pregnant outcome (control). There was a significant difference in
serum SEMA 3B levels between pregnant women who developed pre-
eclampsia and control women (Fig. 3; t = 4.980, P b 0.001). Pregnant
women who destined to have preeclampsia had significantly higher
serum SEMA 3B levels than control.
4. Discussion
In the current investigation, we revealed the first time that maternal
SEMA 3B levels significantly increased in preeclampsia compared with
normotensive pregnant women, and the increase in maternal SEMA
3B occurred in 16–20 weeks of gestation before the onset of preeclamp-
sia. Since maternal age and gestational age are comparable in both the
third and the second trimesters, the differences in SEMA 3B are due
to the presence of preeclampsia. Given that enhanced expression of
SEMA 3B by placental cytotrophoblast is an important pathology of
preeclampsia and up-regulation of SEMA 3B impairs the differentiation
and invasion of CTBs as well as VEGF signaling leading to the inhibition
of angiogenesis, increased maternal SEMA 3B may have a role in the
pathogenesis of preeclampsia.
To determine the alteration in gene expression by CTB in preeclamp-
sia, Zhou et al. [11] isolated villous CTB from preeclamptic placentas and
Table 1
Maternal and gestational ages.
Control Preeclampsia Significance
Third trimester
N 36 36
Maternal age (years) 29.9 ± 5.2 29.3 ± 3.5 NS
Gestational age (weeks) 36.6 ± 1.3 35.4 ± 3.8 NS
Blood pressure (mm Hg)
Systolic 111 ± 10 156 ± 20 P b 0.001
Diastolic 70 ± 6 103 ± 15 P b 0.001

2000 × g for 15 min to clarify serum. Serum was collected and stored Second trimester
at −80 °C until assay. Serum SEMA 3B concentration was determined N 25 25
with commercially purchased enzyme-linked immunosorbent assay Maternal age (years) 28.0 ± 3.0 27.4 ± 3.6 NS
Gestational age (weeks) 17.1 ± 0.9 16.9 ± 1.3 NS
(ELISA) kit (Uscn Life Science Inc.).
62 H. Wang et al. / Clinica Chimica Acta 455 (2016) 60–63
Fig. 1. Alteration in serum SEMA 3B in preeclampsia. Serum SEMA 3B concentration was
determined with ELISA in normotensive women (control) and preeclamptic women
(preeclampsia). There was a significant difference in serum SEMA 3B between control
and preeclampsia (t = 3.911, P b 0.001).
placentas of preterm labor women with no signs of infection, and used
global transcriptional profiling to explore mRNA changes underlying
CTB defects in preeclampsia. They found a series of differentially
expressed genes in CTB of preeclamptic placentas and focused on SEMA
3B that was highly expressed in CTB and up-regulated in preeclampsia
as confirmed by binding of 32P-SEMA 3B probe, northern hybridization,
in situ hybridization, western blotting and immunohistochemistry.
Hypoxia (2% O2; 20% O2 as normoxia) up-regulated the SEMA 3B protein
in chorionic villous explants from normal second trimester (18 and
20 weeks of gestation) placentas. The autocrine action of SEMA 3B con-
tributed to the phenotypic alterations including inhibited differentiation
and invasion that are the hallmark of preeclampsia, and down-regulated
VEGF signaling through the PI3K/AKT and GSK3 pathways that were ob-
served in preeclamptic CTBs. These authors proposed that up-regulated
SEMA 3B is the major driver of CTB aberration and participates the
pathogenesis of preeclampsia by inhibiting VEGF-induced angiogenesis
via autocrine manner.
More recently, Katuu-Lino et al. [12] reported different findings
regarding SEMA 3B in placentas of women with severe early onset of
Fig. 2. Serum SEMA 3B levels in mild and severe preeclapsia. There was a significant
difference in serum SEMA 3B levels between mild and severe preeclampsia (t = 2.134,
P = 0.04). Women with severe preeclampsia had significantly higher serum SEMA 3B
than women with mild preeclampsia.
Fig. 3. Alteration in serum SEMA 3B in preeclamptic women at 16–20 weeks of gestation.
There was a significant difference in serum SEMA 3B between women who developed
preeclampsia (preeclampsia) and women who had favorable pregnancy outcomes
(control) (t = 4.980, P b 0.001).
preeclapsia. They observed that SEMA 3B relative expression and copy
number were not significantly changed in preeclamptic placentas, and
confirmed this at the protein level with western blotting. In addition,
exposure of term trophoblast or explants to hypoxia (1% O2; 8% O2
as normoxia) induced significant down-regulation of SEMA 3B
mRNA and remarkable increase in SEMA 3B protein (approximately
1.6 folds). Since the patient characteristics were similar in studies by
Katuu-Lino et al. [12] and Zhou et al. [11], Katuu-Lino et al. [12] consid-
ered that the high expression of SEMA 3B in syncytiotrophoblast may
mask the alteration specific to cytotrophoblast observed by Zhou et al.
[11]. However, Katuu-Lino et al. also pointed to the importance of
assessing gene and protein alterations in large cohorts.
Herein, we observed the elevation of maternal SEMA 3B in
preeclampsia, and confirmed this occurred before the clinical onset
of preeclampsia. Given the belief that placental hypoxia is a characteris-
tic of preeclampsia [1–3] and the findings that hypoxia up-regulates
SEMA 3B in placental explants [11,12], the increase in serum SEMA
3B may be due to the hypoxia-induced up-regulation in placentas. In
combination of the mechanical effects of SEMA 3B on trophoblast and
angiogenesis as clarified by Zhou et al. [11], our findings point to the
importance of SEMA 3B in the development of preeclampsia and
potential target for the prevention and therapy of this disease.
The difference in SEMA 3B expression between CTBs and placentas in
preeclampsia points to the complexity in the pathology of preelampsia,
and indicates the possible differences in the preeclampsia-associated
pathologies between CTBs and STBs. It has been well-accepted for
decades that the deficit in CTB, leading to shadow interstitial invasion
of trophoblast, poor placentation and deficient remodeling of spiral arte-
rioles, is the key pathology in preeclampsia [1–8]. However, regeneration
hyperplasia of trophoblast has been proposed on the basis of immature
and proliferative properties of trophoblast observed by ultrastructural
and biochemical criteria as well as proliferative index [8–10].
In summary, maternal SEMA 3B level is significantly increased
in preeclampsia before the onset of manifestations; this increase is
maintained through the preeclamptic pregnancy and SEMA 3B level is
linked with disease severity. Considering the role SEMA 3B in angiogen-
esis inhibition and the change in maternal SEMA 3B in preeclampsia, it is
indicated SEMA 3B is involved in the pathogenesis of preeclampsia and
is a potential therapeutic target of preeclampsia.
Acknowledgments
This study was funded by Natural Science Foundation of China
(81170572 and 81370726) and National Basic Key Research Pro-
gram of China (2012CB944903).
 H. Wang et al. / Clinica Chimica Acta 455 (2016) 60–63
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