| Abstract|| |
Introduction: Hypertensive disorder in pregnancy (HDP) represents the most common medical complication in pregnancy. It is the leading cause of maternal and perinatal mortality and morbidity. Vascular endothelial growth factor (VEGF) stimulates vascular endothelial cell growth, survival, and proliferation, and they are known to be expressed in human placenta. The aim of this study was to determine the VEGF expression in the placenta of hypertensive and normotensive patients. Materials and Methods: This is a retrospective, cross-sectional study from January 1, 2015 to December 31, 2015. A total of 30 placentae comprised of 15 hypertensive and 15 normotensive cases were assessed. VEGF expression in placenta was assessed by immunohistochemistry, and the number of syncytial knots was counted. Results: Our study showed an increased syncytial knot formation in the placenta of hypertensive mothers. VEGF expression was seen in syncytiotrophoblasts of 14 of the hypertensive cases (14/15, 93.3%), while only two of the normotensive cases were positive (2/15, 13.3%). There were no statistically significant differences in VEGF expression in other placenta cells, that is, cytotrophoblasts (P = 1.0), decidual cells (0.1394), maternal endothelial cells (0.5977), and fetal endothelial cells (P = 1.0). Conclusions: This study showed an increased number of syncytial knots is a consistent histological finding in the placenta of patient with HDP. VEGF expression was significantly increased in syncytiotrophoblasts in placenta of hypertensive group, and it could be used as a biomarker for hypertension.
Keywords: Hypertension, immunohistochemistry, placenta, syncytiotrophoblasts, vascular endothelial growth factor
|How to cite this article:|
Azliana AF, Zainul-Rashid MR, Chandramaya SF, Farouk WI, Nurwardah A, Wong YP, Tan GC. Vascular endothelial growth factor expression in placenta of hypertensive disorder in pregnancy. Indian J Pathol Microbiol 2017;60:515-20
|How to cite this URL:|
Azliana AF, Zainul-Rashid MR, Chandramaya SF, Farouk WI, Nurwardah A, Wong YP, Tan GC. Vascular endothelial growth factor expression in placenta of hypertensive disorder in pregnancy. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Aug 25];60:515-20. Available from: http://www.ijpmonline.org/text.asp?2017/60/4/515/222975
| Introduction|| |
Hypertensive disorder of pregnancy (HDP) affects about 2%–7% of pregnancies worldwide and is a major cause of maternal and fetal morbidity and mortality.,, The prevalence of HDP in Malaysia is estimated to be about 23.3/1000 live births. HDP represents the fourth most common cause of maternal mortality in Malaysia during the period 2001–2006, after obstetric embolism, associated medical conditions, and postpartum hemorrhage. The percentage of maternal death due to HDP is between 8.5% and 16.5% from 1997 to 2008.
In pregnancy, improper differentiation of maternal uterine spiral arteries results in poor trophoblast invasion. This reduces the blood flow in the placenta and causes poor oxygen and nutrient transfer between mother and fetus, which leads to endothelial dysfunction and resulted in HDP. Maynard et al. suggested that imbalances of level between pro- and anti-angiogenic factors as the implication of endothelial dysfunction. Subsequently, Yelumalai et al. found that elevated plasma level of fms-like tyrosine kinase lead to suppression of placenta growth factor and vascular endothelial growth factor (VEGF) and might have contributed in HDP.
The studies suggested in pregnancy, the uteroplacental blood flow may be decreased due to maternal vasospasm and constriction of fetal stem arteries, which resulted in fetal hypoxia and fetal death., Redline et al. found that placental examination for evidence of maternal vascular under perfusion could be reproducible. This suggests that placental examination may be used as a tool to investigate for hypoxia-related disorders such as HDP.
The placenta is a vital organ for developing fetus, and it is also an easily accessible tissue of human body. After delivery, assessment of the placenta provides a morphological record of intrapartum events of gestation and could provide insight into the prenatal health of the baby and the mother. However, it is often discarded.,,
VEGF is a protein that plays an important role in angiogenesis. It stimulates vascular endothelial cell growth, survival, and proliferation. It can also facilitates the survival of existing vessels, contribute to vascular abnormalities such as tortuousness and hyperpermeability, which may impede effective delivery of antitumor compounds, and stimulate new vessel growth.
There are seven members in the VEGF family (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor [PLGF]). VEGF and PIGF expression were found in chorionic mesenchyme, villous trophoblasts, and vascular endothelium. VEGF-A and PLGF are important molecules in regulating early placental vascular changes. They are expressed in the human placenta throughout gestation and act as regulator in placental angiogenesis and maternal spiral artery remodeling.,,
In normal pregnancies, trophoblasts invade the maternal decidua that lead to remodeling of the spiral arteries, converting them to low-resistance vessels. However, in pregnancies complicated by preeclampsia, trophoblastic cell invasion is inadequate, resulting in poor placental perfusion and fetal hypoxia.,, Hypoxia is a potent stimulus for the induction of VEGF gene expression., Baker et al. showed serum VEGF was elevated in women with pregnancies complicated by hypertensive disorders compared with normotensive women. The aim of this study was to determine the VEGF expression in placentas of hypertensive and normotensive mothers. In addition, to compare the number of syncytial knots in the two groups.
| Materials and Methods|| |
This is a retrospective, cross-sectional study of placenta of normal and hypertensive pregnancies. This study was approved by our Ethical and Research Committee, (project code: FF-2015-074). Tissue blocks were obtained from archives of the Department of Pathology of our hospital for 1 year from January 1, 2015 to December 31, 2015. There was a total of 30 cases consisted of women whose pregnancies were complicated by hypertension (n = 15) and normal healthy controls (n = 15). Hematoxylin and eosin-stained slides were reviewed, and selected paraffin-embedded tissue blocks were retrieved for microtome sectioned and subsequently for immunohistochemical analysis of VEGF expression. The demographic data of patients were obtained from the record unit.
Immunohistochemical analysis of vascular endothelial growth factor
Formalin-fixed, paraffin-embedded tissue blocks of 30 cases of placenta (15 normal pregnancies and 15 hypertensive pregnancies). Tissue blocks were sectioned at 3-μm thick by microtome. Monoclonal mouse antihuman VEGF clone VG1 (Code No. M7273, Dako Denmark, dilution of 1:50) was used for the immunohistochemical analysis. Normal human placenta tissue was used as positive control.
Staining was performed following the manufacturer protocol using EnVisionTM FLEX Mini Kit, High pH (Code No. K8023, Dako Denmark). The primary antibody was diluted to optimal concentration using Antibody Diluent, Dako (Code No. S2022, Dako Denmark). Washing steps between each reagent were performed using EnVision™ FLEX Wash Buffer 20X (Code No. K8007, Dako Denmark) diluted to a 1X working solution with deionized water. The 1X DAB-containing Substrate Working Solution was prepared by diluting the 50x concentrated EnVision™ FLEX DAB + Chromogen with Envision™ FLEX™ Substrate Buffer (Code No. K8023, Dako Denmark).
The slides were incubated on hot plate at 60°C for 30 min. An initial dewaxing step was done using 2 times Xylene (Merck Germany), and rehydration step was performed using decreasing alcohol solutions (100%, 80%, and 70%). Then, the slides were rinsed in running tap water for 3 min. The slides were subsequently incubated with EnVision™ FLEX Peroxidase Blocking Reagent (Code No. K8023, Dako Denmark) for 5 min followed by rinsing by running tap water. Antigen retrieval step was performed in the Dako PTLink (Product No. PT10126, DakoCytomation, USA) using the EnVision™ FLEX Target Retrieval Solution, high pH (Code No. K8004, Dako Denmark) with the conditions of temperature 98°C and time 30 min followed by cooling at room temperature for 20 min and rinsed with running tap water.
Then, the slides were incubated for 30 min at room temperature with primary antibody, followed by incubation with EnVisionTM FLEX HRP (Code No. K8023, Dako Denmark) for 20 min. Subsequently, the slides were incubated with 1X DAB-containing Substrate Working Solution for 10 min. Then, the slides were counterstained with Hematoxylin (REF 7231, ThermoScientific, USA) for 15 s. Finally, the slides were mounted and cover-slipped using DPX mounting medium (Cat. No.: 100579, Merck Millipore Germany).
Analysis of vascular endothelial growth factor expression
The expression of VEGF on formalin-fixed paraffin-embedded tissue sections was evaluated using Olympus microscope BX40 at ×40 magnification. The positivity and intensity of the cytoplasm staining of VEGF expression were evaluated on syncytotrophoblasts cells, cytotrophoblast cells, fetal endothelial cells, maternal endothelial cells, and decidua cells. The intensity of positive cells was scored as negative, weak, moderate, and strong staining. The status of VEGF expression was evaluated independently by two observers who were blinded from the original diagnosis.
Counting of number of syncytial knots
Syncytiotrophoblastic knots or syncytial knots are aggregates of syncytial nuclei at the surface of terminal villi. They invade the wall of the uterus to establish nutrient circulation between the embryo and the mother. Histologically, it appears multinucleated. The number of syncytial knots was counted in three high-power fields, and an average number was obtained.
Data collected was statistically analyzed using Graphpad online statistics software (http://www.graphpad.com). Chi-square or Fisher exact tests and unpaired t-test were used to determine differences between variables. Any P < 0.05 was considered statistically significant.
| Results|| |
All placentas were from the placenta of mother with singleton pregnancies. In this study, 24 cases (80.0%) were Malay, 5 cases (16.7%) were Chinese, and 1 case (3.3%) was Indian. Our cases comprised 21 primigravidas, six were in their second pregnancy, and three were in third, fourth, and six pregnancies each. The gestational age of pregnancies was between 28 and 39 weeks. Majority of them (17/30, 56.7%) are term pregnancies (37–39 weeks).
Histological study of placenta in hypertensive and normotensive mothers
The number of syncytial knots
The average number of syncytial knots (syncytiotrophoblasts) in the placenta of hypertensive cases was higher (17.2 per high-power field) [Figure 1] compared to normotensive cases (6.5 per high-power field). Fourteen (14/15, 93.3%) of the hypertensive cases have more than seven syncytial knots per high-power field. In contrast, this was observed in only two of the normotensive cases (2/15, 13.3%). The different of number of syncytial knots in placenta between hypertensive and normotensive groups is statistically significantly (P< 0.001).
|Figure 1: Increased number of syncytial knots in placenta of hypertensive group|
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Expression of vascular endothelial growth factor
The expression of VEGF was assessed in syncytiotrophoblasts, cytotrophoblasts, decidual cells, fetal endothelial cells, and maternal endothelial cells. We found that VEGF expression was seen in the fetal endothelial cells of all cases of hypertensive (15/15, 100%) and normotensive (15/15, 100%). However, the intensity of expression was higher in normotensive cases. All 15 (15/15, 100%) cases of the normotensive group were either moderate or strong staining, whereas this was observed in only eight cases (8/15, 53.3%) in hypertensive group. The difference of VEGF expression in fetal endothelial cells between hypertensive and normotensive groups is not statistically significant (P = 1.0) [Table 1].
|Table 1: Expression of vascular endothelial growth factor in fetal endothelial cells, syncytiotrophoblasts, cytotrophoblasts, decidual cells, and maternal endothelial cells|
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As for syncytiotrophoblasts, the VEGF expression was seen in 14 of the hypertensive cases (14/15, 93.3%) [Figure 2], whereas only two of the normotensive cases were positive (2/15, 13.3%). The difference is statistically significant (P< 0.0001). Furthermore, in hypertensive cases, the intensity of VEGF staining in the majority of the cases was strong (12/14, 85.7%). In contrast, the intensity in the nonhypertensive group was only moderate [Table 1].
|Figure 2: Vascular endothelial growth factor expression in syncytiotrophoblasts (a; yellow arrow), fetal endothelial cells (b; yellow arrow), cytotrophoblasts (c; yellow arrow), and d; decidual cells|
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In hypertensive group, the number of cases with positive-VEGF expression in cytotrophoblasts, decidual cells, and maternal endothelial cells was 6/15 (40.0%), 6/15 (40.0%), and 1/15 (6.7%), respectively [Figure 2]. While in nonhypertensive group, the number of cases with positive-VEGF expression in cytotrophoblasts, decidual cells, and maternal endothelial cells were 5/15 (33.3%), 11/15 (73.3%), and 3/15 (20.0%), respectively. There are no significant difference in the VEGF expression in all these cells (P = 1.0 [cytotrophoblasts], 0.1394 [decidual cells], and 0.5977 [maternal endothelial cells]).
| Discussion|| |
In HDP, there is a reduction in maternal uteroplacental blood flow., The previous study showed that the number of syncytial knots was increased in placental explant culture in a simulated hypoxic condition by exposure to reactive oxygen species. Fox described that excess syncytial knot formation occurs in generalized form, whenever the blood circulation through the villi appears to be reduced. He also showed that syncytial knot formation depends on length of duration of gestation and suggested that it might be regarded as an index of placental maturity. Our study showed a significant increase in the number of syncytial knots in the placenta of hypertensive group compared with normotensive group. Majority of the hypertensive cases had more than seven syncytial knots in one high-power field; in contrast, this was observed only two of the 15 cases in normotensive cases. Similarly, Narasimha and Vasudeva also described an increased in syncytial knots in the placenta of patients with toxemia of pregnancy. However, instead of counting the exact number of syncytial knots, they determined the ratio between syncytial knots and chorionic villi.
The histological findings of the placenta in hypertensive mothers include increase syncytial knot formation, cytotrophoblasts proliferation, proliferation of endothelial lining of capillaries, stromal fibrosis, calcification, hyalinization of villi and infarction., Studies also showed placenta of patient with HDP may have significant reduced weight, surface area, and volume. The placental weight and size were directly proportional to the birth weight of babies and degree of hypertension.,
The current widely accepted model for poor placental perfusion is that failure of proper extravillous trophoblast invasion and remodeling of maternal spiral arteries resulted in decreased fetoplacental growth, that is, small placenta and intrauterine growth retardation. VEGF and its receptors are essential for embryonic vascular development as loss of even a single-VEGF allele results in embryonic death., A characteristic feature of VEGF regulation is the induction of its mRNA by tissue hypoxia and hypoglycemia., Helske et al. showed increased VEGF receptor 1 expression in syncytiotrophoblasts of patients with severe preeclampsia. In this study, we found nearly all hypertensive cases had increased VEGF expression in syncytiotrophoblasts. In contrast, only two of the normotensive cases were positive. This finding suggests that HDP resulted in hypoxia which induced the expression of VEGF.
The previous study also showed VEGFR-2 was expressed exclusively in endothelial cells in placenta villi, and there was no different in the expression between hypertensive and normal control groups. We also found that VEGF was expressed in fetal endothelial cells of both hypertensive and normotensive groups. The expression of VEGF in cytotrophoblasts, decidual cells, and maternal endothelial cells was inconsistent in both hypertensive and normotensive groups and there was no significant difference between the two groups.
| Conclusions|| |
We found that the increased number of syncytial knots in the placenta is a consistent histological finding in HDP and more than seven syncytial knots per high-power field may be considered as the cut-off point as increased syncytial knot in the placenta. However, in the areas where syncytial knots are counted, the placenta villi should be neither too compact nor too loose. The expression of VEGF was significantly increased in syncytiotrophoblasts in the hypertensive group, which were not seen in cytotrophoblasts, decidual cells, fetal, and maternal endothelial cells. The increased number of syncytial knots along with high-VEGF expression suggests HDP is a hypoxic-related condition. VEGF could be used as a biomarker for hypertension in pregnancy.
Financial support and sponsorship
We would like to thank Universiti Kebangsaan Malaysia for providing the fund for this study. Grant number: FF-2015-074.
Conflicts of interest
There are no conflicts of interest.
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Dr. Geok Chin Tan
Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Center, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Kuala Lumpur
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]