- Email: [email protected]
Alteration of serum semaphorin 3B levels in preeclampsia
Alteration of serum semaphorin 3B levels in preeclampsia Hanzhi Wang, Linxiang Jiang, Bo Gao, Minyue Dong PII: DOI: Reference:
S0009-8981(16)30037-7 doi: 10.1016/j.cca.2016.01.030 CCA 14263
To appear in:
Clinica Chimica Acta
Received date: Revised date: Accepted date:
27 October 2015 26 January 2016 27 January 2016
Please cite this article as: Wang Hanzhi, Jiang Linxiang, Gao Bo, Dong Minyue, Alteration of serum semaphorin 3B levels in preeclampsia, Clinica Chimica Acta (2016), doi: 10.1016/j.cca.2016.01.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Alteration of Serum Semaphorin 3B Levels in Preeclampsia
SC R
IP
T
Hanzhi Wang, Linxiang Jiang, Bo Gao, Minyue Dong
Women’s Hospital, School of Medicine, Zhejiang University
Key Laboratory of Reproductive Genetics, Ministry of Education
NU
Key Laboratory of Women’s Reproductive Health of Zhejiang Province
MA
1, Xueshi Road, Hangzhou, Zhejiang, 310006, China
D
Corresponding Author: Minyue Dong, MD/PhD, Women’s Hospital, School of Medicine,
TE
Zhejiang University, Add: 1, Xueshi Road, Hangzhou, Zhejiang, 310006, China. Tel: (+86)
CE P
571 8999 1862, Fax: (+86) 571 8706 1878, Email: [email protected],
AC
[email protected]
1
ACCEPTED MANUSCRIPT Abstract Background: Placental Semaphorin 3B (SEMA 3B) expression has been reported changed in
IP
T
precclampsia and its possible involvement of in the disease proposed. We clarified the
SC R
alterations of maternal SEMA 3B level in women suffering preeclampsia and pregnant women at gestational weeks of 16-20 before the onset of preeclampsia. Methods: Serum SEMA 3B concentration was measured with ELISA in preeclamptic women
NU
(preeclamsia) and normotensive women (control) in 3rd trimester, and also in pregnant
MA
women at gestational weeks of 16-20 who developed preeclampsia or had favorable pregnant outcome.
D
Results: Serum SEMA 3B level was significantly increased in preeclampsia compared with
TE
control (P<0.001). There was a significant difference in serum SEMA 3B between mild and
CE P
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
AC
3B was significantly higher in women who developed preeclampsia than women who had normal pregnant outcome (P<0.001). Conclusions: Maternal SEMA 3B level increased in preeclampsia before the onset of manifestations, indicating SEMA 3B plays a role in the pathogenesis of preeclampsia. Key Words: Semaphorin 3B, preeclampsia, pregnancy
2
ACCEPTED MANUSCRIPT Introduction Preeclampsia is a serious pregnancy complication affecting 2–8% of all pregnancies, and is
IP
T
one of the major causes for maternal mortality and morbidity, preterm birth, perinatal death
SC R
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
NU
etiology of the disorder is not completely understood [1-3].
MA
Placenta and its major cell component, trophoblast, have been considered play pivotal roles in the pathogenesis of preeclampsia for decades [1-7]. Defects in villous
D
cytotrophoblasts (CTBs), which lead to shadow interstitial invasion, poor placentation and
TE
deficient remodeling of spiral arterioles, have been proposed to be the initial etiology of
CE P
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
AC
involved trophoblast deficit remain largely unknown. In an attempt to explore the differentially expressed genes in villous cytotrophoblasts isolated from placenta of preeclamptic women, Zhou et al [11] found the up-regulated semaphorin 3B (SEMA 3B) in preeclampsia 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 indicates further investigation is needed. However, the alteration in maternal SEMA 3B has never been described in preeclampsia. 3
ACCEPTED MANUSCRIPT Semaphorins was first identified as axon guidance factors that direct growth cones of axons to their proper target during the formation of nervous system [13-15]. Thereafter, it has
IP
T
been realized that semaphorins play a role in many developmental processes outside the
SC R
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].
NU
Class-3 semaphorins are the only secreted vertebrate semaphorins, and display properties of
MA
angiogenesis inhibitors and tumor progression modulators. SEMA 3B, a member of class-3 semaphorins and an angiogenesis inhibitor, has been proposed to be involved in tumor
D
progression and a potential therapeutic target [13-15]. However, the roles of SEMA 3B in
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TE
pregnancy and complications remain largely unknown [11,12].
Materials and Methods
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Subjects
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 preeclampsia, 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. Preeclampsia 4
ACCEPTED MANUSCRIPT 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
IP
T
or a diastolic blood pressure of 90 mm Hg or higher on two occasions at least six hours apart
SC R
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
NU
a random urine sample or greater than 5.0 g of proteinuria over 24 h. Other evidence of
MA
severe disease included increased serum creatinine, eclampsia, pulmonary edema, oliguria (<500 ml/24 h), fetal growth restriction, oligohydramnios and symptoms suggesting
D
significant end-organ involvement (headache, visual disturbance, or epigastric or right upper
TE
quadrant pain). Women who met criteria of preeclampsia but not severe preeclampsia were
CE P
diagnosed mild preeclampsia.
To clarify if the alteration of maternal Sema 3B occurred in the second trimester of
AC
pregnancy, before the onset of preeclampsia, a retrospective nested case-control study was conducted. Blood samples were chosen from the sample bank. Blood samples were collected at 16-20 weeks of gestational age for routine Down’s syndrome screening. 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 y in preeclampsia group. Maternal age, gestational age and the date of sample collection were matched for preeclampsia group and control group. 5
ACCEPTED MANUSCRIPT All subjects were nulliparous Chinese women with singleton pregnancies. Exclusion criteria were multiple gestation, diabetes mellitus, chronic hypertension, infectious diseases
IP
T
recognized in pregnancy, premature rupture of membrane and signs of other concurrent
SC R
medical complication. 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
NU
approval of the current study was obtained from Institutional Ethical committee of Women’s
MA
Hospital, School of Medicine, Zhejiang University, and all the participants provided their
TE
Samples collection and assay
D
informed consents.
CE P
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 2000×g for 15 min to clarify serum. Serum was
AC
collected and stored at −80 ◦C until assay. Serum SEMA 3B concentration was determined with commercially purchased enzyme-linked immunosorbent assay (ELISA) kit (Uscn Life Science Inc.).
Statistic analysis The distribution of serum concentration of SEMA 3B was normal as 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. 6
The Statistical Analysis System was
ACCEPTED MANUSCRIPT used for data analysis. A p < 0.05 was considered significant.
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Results
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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
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trimester as expected. Maternal serum SEMA 3B levels were determined by ELISA. We
MA
found there was a significant difference in SEMA 3B levels between preeclamptic women (preeclampsia) and normal pregnant women (control) (Fig. 1; t=3.911, P<0.001).
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Preeclamptic women had significantly higher serum SEMA 3B level than control. The serum
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SEMA 3B levels were significantly different between mild and severe preeclampsia (Fig. 2;
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t=2.134, P=0.04). Women with severe preeclampsia had significantly higher serum SEMA 3B levels than women with mild preeclampsia.
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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 preeclampsia and control women (Fig. 3; t=4.980, P<0.001). Pregnant women who destined to have preeclampsia had significantly higher serum SEMA 3B levels than control.
Discussion 7
ACCEPTED MANUSCRIPT In the current investigation, we revealed the first time that maternal SEMA 3B levels significantly increased in preeclampsia compared with normotensive pregnant women, and
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the increase in maternal SEMA 3B occurred in 16-20 weeks of gestation before the onset of
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preeclampsia. 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
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pathology of preeclampsia and up-regulation of SEMA 3B impairs the differentiation and
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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. [11]
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To determine the alteration in gene expression by CTB in preeclampsia, Zhou et al.
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isolated villous CTB from preeclamptic placentas and placentas of preterm labor women with
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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
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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 contributed 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 observed in preeclamptic CTBs. These authors proposed that up-regulated SEMA 3B is the major driver of CTB 8
ACCEPTED MANUSCRIPT aberration and participates the pathogenesis of preeclampsia by inhibiting VEGF-induced angiogenesis via autocrine manner.
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More recently, Katuu-Lino et al. [12] reported different findings regarding SEMA 3B in
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placentas of women with severe early onset of preeclapsia. They observed 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
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trophoblast or explants to hypoxia (1% O2; 8% O2 as normoxia) induced significant
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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 [12] and Zhou et al. [11], Katuu-Lino et al. [12] considered that the high
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Katuu-Lino et al.
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expression of SEMA 3B in syncytiotrophoblast may mask the alteration specific to
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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.
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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 characteristic 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 9
ACCEPTED MANUSCRIPT 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
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well-accepted for decades that the deficit in CTB, leading to shadow interstitial invasion of
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trophoblast, poor placentation and deficient remodeling of spiral arterioles, 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
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ultrastructural and biochemical criteria as well as proliferative index [8-10].
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In summary, maternal SEMA 3B level is significantly increased in preeclampsia before the onset of manifestations; this increase is maintained through the preeclamptic pregnancy
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and SEMA 3B level is linked with disease severity. Considering the role SEMA 3B in
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angiogenesis inhibition and the change in maternal SEMA 3B in preeclampsia, it is indicated
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of preeclampsia.
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SEMA 3B is involved in the pathogenesis of preeclampsia and is a potential therapeutic target
Acknowledgements: This work was financially supported by Natural Scientific Foundation of and National Basic Research Program of China.
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ACCEPTED MANUSCRIPT References [1] Uzan J, Carbonnel M, Piconne O, Asmar R, Ayoubi JM. Pre-eclampsia: pathophysiology,
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diagnosis, and management. Vasc Health Risk Manag 2011; 7:467-474.
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[2] Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet 2010; 376:631-644.
[3] Mol BW, Roberts CT, Thangaratinam S, Magee LA, de Groot CJ, Hofmeyr GJ.
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Pre-eclampsia. Lancet 2015.
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[4] Fisher SJ. Why is placentation abnormal in preeclampsia? Am J Obstet Gynecol 2015; 213:S115-122.
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[5] Brosens IA, Robertson WB, Dixon HG. The role of the spiral arteries in the pathogenesis
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of pre-eclampsia. J Pathol 1970; 101:Pvi.
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[6] Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ. Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J Clin Invest 1993;
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91:950-960.
[7] Lim KH, Zhou Y, Janatpour M, et al. . Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia. Am J Pathol 1997; 151:1809-1818. [8] Redline RW, Patterson P. Pre-eclampsia is associated with an excess of proliferative immature intermediate trophoblast. Hum Pathol 1995; 26:594-600. [9] Arkwright PD, Rademacher TW, Dwek RA, Redman CW. Pre-eclampsia is associated with an increase in trophoblast glycogen content and glycogen synthase activity, similar to that found in hydatidiform moles. J Clin Invest 1993; 91:2744-2753. [10] Kaya B, Nayki U, Nayki C, et al. . Proliferation of trophoblasts and Ki67 expression in 11
ACCEPTED MANUSCRIPT preeclampsia. Arch Gynecol Obstet 2015; 291:1041-1046. [11] Zhou Y, Gormley MJ, Hunkapiller NM, et al. . Reversal of gene dysregulation in cultured
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cytotrophoblasts reveals possible causes of preeclampsia. J Clin Invest 2013; 123:2862-2872.
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[12] Kaitu'u-Lino TJ, Hastie R, Cannon P, et al. . Placental SEMA3B expression is not altered in severe early onset preeclampsia. Placenta 2014; 35:1102-1105.
[13] Varshavsky A, Kessler O, Abramovitch S, et al. . Semaphorin-3B is an angiogenesis
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inhibitor that is inactivated by furin-like pro-protein convertases. Cancer Res 2008;
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68:6922-6931.
[14] Luo Y, Raible D, Raper JA. Collapsin: a protein in brain that induces the collapse and
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paralysis of neuronal growth cones. Cell 1993; 75:217-227.
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[15] Neufeld G, Sabag AD, Rabinovicz N, Kessler O. Semaphorins in angiogenesis and tumor
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progression. Cold Spring Harb Perspect Med 2012; 2:a006718. [16] ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia.
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Number 33, January 2002. Obstet Gynecol 2002; 99:159-167. [17] Obstetricians ACo, Gynecologists. Diagnosis and management of preeclampsia and eclampsia. ACOG practice bulletin 2002; 33:159-167.
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ACCEPTED MANUSCRIPT
Fig. Legends
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Fig. 1: Alteration in serum SEMA 3B in preeclampsia. Serum SEMA 3B concentration was
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determined with ELISA in normotensive women (control) and preeclamptic women (preeclampsia). There was a significant difference in serum SEMA 3B between control and
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preeclampsia (t=3.911, P<0.001).
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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,
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TE
women with mild preeclampsia.
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P=0.04). Women with severe preeclampsia had significantly higher serum SEMA 3B than
Fig. 3: Alteration in serum SEMA 3B in preeclamptic women at 16-20 weeks of gestation.
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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<0.001).
13
ACCEPTED MANUSCRIPT
Table 1: Maternal and gestational ages
N
36
36
Maternal age
29.9±5.2
29.3±3.5
36.6±1.3
35.4±3.8
Significance
T
Preeclampsia
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Control
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(years)
NS
NS
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Gestational age
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Third trimester
(weeks)
D
Blood pressure
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(mmHg)
111±10
156±20
P<0.001
70±6
103±15
P<0.001
N
25
25
Maternal age
28.0± 3.0
27.4± 3.6
NS
17.1± 0.9
16.9±1.3
NS
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Diastolic
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Systolic
Second trimester
(years) Gestational age (weeks)
14
ACCEPTED MANUSCRIPT
AC
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TE
D
MA
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P<0.001
Fig. 1
15
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T
ACCEPTED MANUSCRIPT
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Fig. 2
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D
MA
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P=0.04
16
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T
ACCEPTED MANUSCRIPT
Fig. 3
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TE
D
MA
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P<0.001
17
ACCEPTED MANUSCRIPT
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Highlights
Serum SEMA 3B was increased in preeclampsia. Serum SEMA 3B was increased before preeclampsia.
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MA
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SEMA 3B may participate the pathogenesis of preeclampsia.
18
S0009-8981(16)30037-7 doi: 10.1016/j.cca.2016.01.030 CCA 14263
To appear in:
Clinica Chimica Acta
Received date: Revised date: Accepted date:
27 October 2015 26 January 2016 27 January 2016
Please cite this article as: Wang Hanzhi, Jiang Linxiang, Gao Bo, Dong Minyue, Alteration of serum semaphorin 3B levels in preeclampsia, Clinica Chimica Acta (2016), doi: 10.1016/j.cca.2016.01.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Alteration of Serum Semaphorin 3B Levels in Preeclampsia
SC R
IP
T
Hanzhi Wang, Linxiang Jiang, Bo Gao, Minyue Dong
Women’s Hospital, School of Medicine, Zhejiang University
Key Laboratory of Reproductive Genetics, Ministry of Education
NU
Key Laboratory of Women’s Reproductive Health of Zhejiang Province
MA
1, Xueshi Road, Hangzhou, Zhejiang, 310006, China
D
Corresponding Author: Minyue Dong, MD/PhD, Women’s Hospital, School of Medicine,
TE
Zhejiang University, Add: 1, Xueshi Road, Hangzhou, Zhejiang, 310006, China. Tel: (+86)
CE P
571 8999 1862, Fax: (+86) 571 8706 1878, Email: [email protected],
AC
[email protected]
1
ACCEPTED MANUSCRIPT Abstract Background: Placental Semaphorin 3B (SEMA 3B) expression has been reported changed in
IP
T
precclampsia and its possible involvement of in the disease proposed. We clarified the
SC R
alterations of maternal SEMA 3B level in women suffering preeclampsia and pregnant women at gestational weeks of 16-20 before the onset of preeclampsia. Methods: Serum SEMA 3B concentration was measured with ELISA in preeclamptic women
NU
(preeclamsia) and normotensive women (control) in 3rd trimester, and also in pregnant
MA
women at gestational weeks of 16-20 who developed preeclampsia or had favorable pregnant outcome.
D
Results: Serum SEMA 3B level was significantly increased in preeclampsia compared with
TE
control (P<0.001). There was a significant difference in serum SEMA 3B between mild and
CE P
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
AC
3B was significantly higher in women who developed preeclampsia than women who had normal pregnant outcome (P<0.001). Conclusions: Maternal SEMA 3B level increased in preeclampsia before the onset of manifestations, indicating SEMA 3B plays a role in the pathogenesis of preeclampsia. Key Words: Semaphorin 3B, preeclampsia, pregnancy
2
ACCEPTED MANUSCRIPT Introduction Preeclampsia is a serious pregnancy complication affecting 2–8% of all pregnancies, and is
IP
T
one of the major causes for maternal mortality and morbidity, preterm birth, perinatal death
SC R
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
NU
etiology of the disorder is not completely understood [1-3].
MA
Placenta and its major cell component, trophoblast, have been considered play pivotal roles in the pathogenesis of preeclampsia for decades [1-7]. Defects in villous
D
cytotrophoblasts (CTBs), which lead to shadow interstitial invasion, poor placentation and
TE
deficient remodeling of spiral arterioles, have been proposed to be the initial etiology of
CE P
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
AC
involved trophoblast deficit remain largely unknown. In an attempt to explore the differentially expressed genes in villous cytotrophoblasts isolated from placenta of preeclamptic women, Zhou et al [11] found the up-regulated semaphorin 3B (SEMA 3B) in preeclampsia 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 indicates further investigation is needed. However, the alteration in maternal SEMA 3B has never been described in preeclampsia. 3
ACCEPTED MANUSCRIPT Semaphorins was first identified as axon guidance factors that direct growth cones of axons to their proper target during the formation of nervous system [13-15]. Thereafter, it has
IP
T
been realized that semaphorins play a role in many developmental processes outside the
SC R
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].
NU
Class-3 semaphorins are the only secreted vertebrate semaphorins, and display properties of
MA
angiogenesis inhibitors and tumor progression modulators. SEMA 3B, a member of class-3 semaphorins and an angiogenesis inhibitor, has been proposed to be involved in tumor
D
progression and a potential therapeutic target [13-15]. However, the roles of SEMA 3B in
CE P
TE
pregnancy and complications remain largely unknown [11,12].
Materials and Methods
AC
Subjects
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 preeclampsia, 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. Preeclampsia 4
ACCEPTED MANUSCRIPT 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
IP
T
or a diastolic blood pressure of 90 mm Hg or higher on two occasions at least six hours apart
SC R
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
NU
a random urine sample or greater than 5.0 g of proteinuria over 24 h. Other evidence of
MA
severe disease included increased serum creatinine, eclampsia, pulmonary edema, oliguria (<500 ml/24 h), fetal growth restriction, oligohydramnios and symptoms suggesting
D
significant end-organ involvement (headache, visual disturbance, or epigastric or right upper
TE
quadrant pain). Women who met criteria of preeclampsia but not severe preeclampsia were
CE P
diagnosed mild preeclampsia.
To clarify if the alteration of maternal Sema 3B occurred in the second trimester of
AC
pregnancy, before the onset of preeclampsia, a retrospective nested case-control study was conducted. Blood samples were chosen from the sample bank. Blood samples were collected at 16-20 weeks of gestational age for routine Down’s syndrome screening. 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 y in preeclampsia group. Maternal age, gestational age and the date of sample collection were matched for preeclampsia group and control group. 5
ACCEPTED MANUSCRIPT All subjects were nulliparous Chinese women with singleton pregnancies. Exclusion criteria were multiple gestation, diabetes mellitus, chronic hypertension, infectious diseases
IP
T
recognized in pregnancy, premature rupture of membrane and signs of other concurrent
SC R
medical complication. 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
NU
approval of the current study was obtained from Institutional Ethical committee of Women’s
MA
Hospital, School of Medicine, Zhejiang University, and all the participants provided their
TE
Samples collection and assay
D
informed consents.
CE P
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 2000×g for 15 min to clarify serum. Serum was
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collected and stored at −80 ◦C until assay. Serum SEMA 3B concentration was determined with commercially purchased enzyme-linked immunosorbent assay (ELISA) kit (Uscn Life Science Inc.).
Statistic analysis The distribution of serum concentration of SEMA 3B was normal as 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. 6
The Statistical Analysis System was
ACCEPTED MANUSCRIPT used for data analysis. A p < 0.05 was considered significant.
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Results
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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
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trimester as expected. Maternal serum SEMA 3B levels were determined by ELISA. We
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found there was a significant difference in SEMA 3B levels between preeclamptic women (preeclampsia) and normal pregnant women (control) (Fig. 1; t=3.911, P<0.001).
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Preeclamptic women had significantly higher serum SEMA 3B level than control. The serum
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SEMA 3B levels were significantly different between mild and severe preeclampsia (Fig. 2;
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t=2.134, P=0.04). Women with severe preeclampsia had significantly higher serum SEMA 3B levels than women with mild preeclampsia.
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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 preeclampsia and control women (Fig. 3; t=4.980, P<0.001). Pregnant women who destined to have preeclampsia had significantly higher serum SEMA 3B levels than control.
Discussion 7
ACCEPTED MANUSCRIPT In the current investigation, we revealed the first time that maternal SEMA 3B levels significantly increased in preeclampsia compared with normotensive pregnant women, and
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the increase in maternal SEMA 3B occurred in 16-20 weeks of gestation before the onset of
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preeclampsia. 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
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pathology of preeclampsia and up-regulation of SEMA 3B impairs the differentiation and
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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. [11]
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To determine the alteration in gene expression by CTB in preeclampsia, Zhou et al.
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isolated villous CTB from preeclamptic placentas and placentas of preterm labor women with
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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
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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 contributed 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 observed in preeclamptic CTBs. These authors proposed that up-regulated SEMA 3B is the major driver of CTB 8
ACCEPTED MANUSCRIPT aberration and participates the pathogenesis of preeclampsia by inhibiting VEGF-induced angiogenesis via autocrine manner.
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More recently, Katuu-Lino et al. [12] reported different findings regarding SEMA 3B in
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placentas of women with severe early onset of preeclapsia. They observed 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
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trophoblast or explants to hypoxia (1% O2; 8% O2 as normoxia) induced significant
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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 [12] and Zhou et al. [11], Katuu-Lino et al. [12] considered that the high
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Katuu-Lino et al.
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expression of SEMA 3B in syncytiotrophoblast may mask the alteration specific to
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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.
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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 characteristic 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 9
ACCEPTED MANUSCRIPT 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
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well-accepted for decades that the deficit in CTB, leading to shadow interstitial invasion of
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trophoblast, poor placentation and deficient remodeling of spiral arterioles, 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
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ultrastructural and biochemical criteria as well as proliferative index [8-10].
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In summary, maternal SEMA 3B level is significantly increased in preeclampsia before the onset of manifestations; this increase is maintained through the preeclamptic pregnancy
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and SEMA 3B level is linked with disease severity. Considering the role SEMA 3B in
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angiogenesis inhibition and the change in maternal SEMA 3B in preeclampsia, it is indicated
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of preeclampsia.
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SEMA 3B is involved in the pathogenesis of preeclampsia and is a potential therapeutic target
Acknowledgements: This work was financially supported by Natural Scientific Foundation of and National Basic Research Program of China.
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ACCEPTED MANUSCRIPT References [1] Uzan J, Carbonnel M, Piconne O, Asmar R, Ayoubi JM. Pre-eclampsia: pathophysiology,
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diagnosis, and management. Vasc Health Risk Manag 2011; 7:467-474.
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[2] Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet 2010; 376:631-644.
[3] Mol BW, Roberts CT, Thangaratinam S, Magee LA, de Groot CJ, Hofmeyr GJ.
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Pre-eclampsia. Lancet 2015.
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[4] Fisher SJ. Why is placentation abnormal in preeclampsia? Am J Obstet Gynecol 2015; 213:S115-122.
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[5] Brosens IA, Robertson WB, Dixon HG. The role of the spiral arteries in the pathogenesis
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of pre-eclampsia. J Pathol 1970; 101:Pvi.
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[6] Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ. Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J Clin Invest 1993;
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91:950-960.
[7] Lim KH, Zhou Y, Janatpour M, et al. . Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia. Am J Pathol 1997; 151:1809-1818. [8] Redline RW, Patterson P. Pre-eclampsia is associated with an excess of proliferative immature intermediate trophoblast. Hum Pathol 1995; 26:594-600. [9] Arkwright PD, Rademacher TW, Dwek RA, Redman CW. Pre-eclampsia is associated with an increase in trophoblast glycogen content and glycogen synthase activity, similar to that found in hydatidiform moles. J Clin Invest 1993; 91:2744-2753. [10] Kaya B, Nayki U, Nayki C, et al. . Proliferation of trophoblasts and Ki67 expression in 11
ACCEPTED MANUSCRIPT preeclampsia. Arch Gynecol Obstet 2015; 291:1041-1046. [11] Zhou Y, Gormley MJ, Hunkapiller NM, et al. . Reversal of gene dysregulation in cultured
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cytotrophoblasts reveals possible causes of preeclampsia. J Clin Invest 2013; 123:2862-2872.
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[12] Kaitu'u-Lino TJ, Hastie R, Cannon P, et al. . Placental SEMA3B expression is not altered in severe early onset preeclampsia. Placenta 2014; 35:1102-1105.
[13] Varshavsky A, Kessler O, Abramovitch S, et al. . Semaphorin-3B is an angiogenesis
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inhibitor that is inactivated by furin-like pro-protein convertases. Cancer Res 2008;
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68:6922-6931.
[14] Luo Y, Raible D, Raper JA. Collapsin: a protein in brain that induces the collapse and
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paralysis of neuronal growth cones. Cell 1993; 75:217-227.
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[15] Neufeld G, Sabag AD, Rabinovicz N, Kessler O. Semaphorins in angiogenesis and tumor
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progression. Cold Spring Harb Perspect Med 2012; 2:a006718. [16] ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia.
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Number 33, January 2002. Obstet Gynecol 2002; 99:159-167. [17] Obstetricians ACo, Gynecologists. Diagnosis and management of preeclampsia and eclampsia. ACOG practice bulletin 2002; 33:159-167.
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Fig. Legends
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Fig. 1: Alteration in serum SEMA 3B in preeclampsia. Serum SEMA 3B concentration was
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determined with ELISA in normotensive women (control) and preeclamptic women (preeclampsia). There was a significant difference in serum SEMA 3B between control and
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preeclampsia (t=3.911, P<0.001).
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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,
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women with mild preeclampsia.
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P=0.04). Women with severe preeclampsia had significantly higher serum SEMA 3B than
Fig. 3: Alteration in serum SEMA 3B in preeclamptic women at 16-20 weeks of gestation.
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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<0.001).
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Table 1: Maternal and gestational ages
N
36
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Maternal age
29.9±5.2
29.3±3.5
36.6±1.3
35.4±3.8
Significance
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Preeclampsia
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Control
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(years)
NS
NS
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Gestational age
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Third trimester
(weeks)
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Blood pressure
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(mmHg)
111±10
156±20
P<0.001
70±6
103±15
P<0.001
N
25
25
Maternal age
28.0± 3.0
27.4± 3.6
NS
17.1± 0.9
16.9±1.3
NS
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Diastolic
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Systolic
Second trimester
(years) Gestational age (weeks)
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P<0.001
Fig. 1
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Fig. 2
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P=0.04
16
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Fig. 3
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P<0.001
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Highlights
Serum SEMA 3B was increased in preeclampsia. Serum SEMA 3B was increased before preeclampsia.
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SEMA 3B may participate the pathogenesis of preeclampsia.
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