The Journal of Maternal-Fetal & Neonatal Medicine
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Serum angiogenic profile in abnormal placentation Ebru Biberoglu, Ayse Kirbas, Korkut Daglar, Kutay Biberoglu, Hakan Timur, Canan Demirtas, Erdem Karabulut & Nuri Danisman To cite this article: Ebru Biberoglu, Ayse Kirbas, Korkut Daglar, Kutay Biberoglu, Hakan Timur, Canan Demirtas, Erdem Karabulut & Nuri Danisman (2016): Serum angiogenic profile in abnormal placentation, The Journal of Maternal-Fetal & Neonatal Medicine, DOI: 10.3109/14767058.2015.1118044 To link to this article: http://dx.doi.org/10.3109/14767058.2015.1118044
Published online: 10 Feb 2016.
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Date: 03 March 2016, At: 04:21
http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2016 Taylor & Francis. DOI: 10.3109/14767058.2015.1118044
ORIGINAL ARTICLE
Serum angiogenic profile in abnormal placentation Ebru Biberoglu1, Ayse Kirbas1, Korkut Daglar1, Kutay Biberoglu2, Hakan Timur1, Canan Demirtas3, Erdem Karabulut4, and Nuri Danisman1
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1
Department of Perinatology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey, 2Department of Obstetrics and Gynecology, Gazi University Medical School, Ankara, Turkey, 3Department of Biochemistry, Gazi University Medical School, Ankara, Turkey, and 4 Department of Biostatistics, Hacettepe University Medical School, Ankara, Turkey Abstract
Keywords
Objective: To evaluate the circulating soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) levels in women with abnormal placentation and to compare the data with the results of women with normal pregnancy. Material and methods: Serum biomarkers of angiogenesis and maternal and perinatal characteristics of 68 pregnant women, all in the third trimester, who were diagnosed to have vaginal bleeding due to complete placenta previa with and without concomitant placenta accreta, increta and percreta as the study group and 30 pregnant women without any placentation abnormality who eventually delivered at 37 weeks of gestational age as the control group were evaluated. Results: There was no statistical difference in the maternal serum values of sFlt1, PlGF, sFlt1/PlGF ratio and VEGF in groups with placental abnormality as compared to controls. Not even a single case of preeclampsia and intrauterine fetal growth restriction was encountered in the study group. Conclusion: We demonstrated that regardless of the localization and the degree of the myometrial invasion of the placenta in the uterus, the circulatory biomarkers of angiogenesis and vascularization were comparable.
Angiogenesis, abnormally invasive placenta, placental growth factor, placenta previa, soluble Fms-like tyrosine kinase 1, vascular endothelial growth factor History Received 19 October 2015 Revised 4 November 2015 Accepted 5 November 2015 Published online 27 January 2016
Introduction In normal pregnancy, decidualization and vascular remodeling are essential for implantation and for providing the embryo with adequate nutrient and substrate supply. Several examples of adverse pregnancy outcomes, such as early pregnancy loss and ischemic placental diseases, including preeclampsia, intrauterine fetal growth restriction and premature delivery are associated with disturbed vascular growth regulation in the fetomaternal unit [1]. The endometrium, decidua and placenta are rich sources of angiogenic substances, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), which is another member of VEGF family. While both have synergistic effects in angiogenesis, soluble fms-like tyrosine kinase 1 (sFlt1) and soluble endoglin (sEng) prevent VEGF and PlGF irreversibly, binding their endothelial cell receptors, thereby exhibit antiangiogenic effect leading to endothelial dysfunction [2,3]. During implantation and placentation process, the highly invasive nature of trophoblasts is kept in control by the Address for correspondence: Ayse Kirbas, Department of Perinatology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. Tel: +90 533-642-3162. Fax: +90 312-312-4931. E-mail: [email protected];[email protected]
decidua that requires regulation and coordination with VEGF, PlGF and sFlt1 expressions [4]. Although serum sFlt1 have been demonstrated to rise 50-fold at term in normal pregnancy compared to nonpregnant state [5], the source of overexpression at the placental level and the physiologic role of sFlt1 are unknown. It can be presumed that sFlt1 may be preparing the placenta for seperation at delivery by regulating placental invasion. When the placenta is not restricted within the decidua but abnormally attached to the uterine wall as a consequence of trophoblastic invasion into the myometrium, within the context of the present study, we preferred to choose the lately adopted term abnormally invasive placenta (AIP), instead of using placenta accreta as the general term recommended by The Publications Committee of The Society for Maternal-Fetal Medicine [6,7]. Depending on the depth of the villous tissue, from the most superficial to the deepest in the myometrium, a further subdivision into accreta, increta and percreta can be introduced [7]. A more commonly seen placentation abnormality is placenta previa in which the placenta is inserted in the lower uterine segment. The incidence of both placenta previa and AIP has increased and seems to parallel the increasing cesarean delivery rate. It has been estimated that in women with placenta previa,
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3%, 40% and 60% of women at the first, second and third previous cesarean sections develop AIP, respectively [8–10]. Although the pathogenesis of placenta previa is not known, higher parity, advanced age and previous low segment cesarean section or uterine surgery are blamed for the higher risk. It has been suggested that endometrial scarring at the sites of prior surgery or in parous women at prior placental attachments result in lower placental implantation associated with decreased uteroplacental blood flow [8–12]. Similar to placenta previa, the risk factors for AIP are well established, but the underlying mechanisms leading to abnormal placentation are less well understood [10,13]. In relation to angiogenesis during placentation, we considered placenta previa as having defective decidual vascularization and AIP with deep trophoblastic and vascular infiltration as the opposite ends of the spectrum. Nevertheless, since the most optimal site to implant for the placenta is the highly vascularized fundal portion of the uterus, the lower uterine segment implantation would offer suboptimal blood supply to the placenta unless the trophoblasts invade the deep myometrium. Given that VEGF and PlGF have potent angiogenic and sFlt1 has antiangiogenic potential, we hypothesized that an imbalance in their production might play a critical role at the trophoblast decidual interface in controlling the trophoblastic invasion. The scarcity of adequate research in the medical literature in this area allowed us to speculate further; thus, we hypothesized that the implantation in areas with less blood supply as in the setting of isolated placenta previa, the hypoxic placenta would release sFlt1 into maternal circulation which would lead to suppressed serum levels of VEGF and PlGF. On the other hand, there is also in vitro evidence that hypoxia could upregulate VEGF, promoting angiogenesis and decrease PlGF expression in trophoblast [14]. Contrarily, in case of placenta accreta/increta and particularly in percreta with highly vascularized trophoblastic invasion through the myometrium, a different scenario could have been set with decreased sFlt1 and increased VEGF as to the serum levels in women with normal pregnancy. Therefore, we aimed to evaluate the circulating sFlt1, PlGF and VEGF levels in women with isolated placenta previa totalis and in women with placenta acreta/increta and percreta and to compare the data with the results of women with normal pregnancy.
Material and methods This case–control study was conducted between May 2014 and July 2015 at the Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. Each participant provided written informed consent to donate biological specimens for this study. The local Institutional Review Board of the hospital approved the study. The universal principles of the Helsinki Declaration were applied. Eighty-seven pregnant women, all in the third trimester, who were admitted with a diagnosis of vaginal bleeding due to complete placenta previa with and without concomitant placenta accreta, increta and percreta as the study group and 30 consecutive pregnant women without any placentation abnormality who applied to the maternity ward and eventually delivered at 37 weeks of gestational age as the control
J Matern Fetal Neonatal Med, Early Online: 1–5
group were enrolled prospectively. Women with vaginal bleeding related to other causes, multiple gestation, any systemic disease including cardiovascular, endocrinological, metabolic, inflammatory and autoimmune disorders, prepregnancy obesity (BMI 30 kg/m2) and who smoke or on any drug other than iron medication were excluded from the study group. Accordingly, six pregnant women who declined to sign the informed consent form, 4 who had emergency transfusion before blood samples were taken for the study, three with twins, two with thyroid disfunction, two with gestational diabetes, one obese and one smoker were discarded from the study groups. In the end, a total of 68 patients with complete placenta previa, 33 of whom without any evidence of AIP (group 1), 17 with concomitant placenta accreta/increta (group 2), 18 with placenta percreta (group 3) and another 30 women with normal pregnancy (group 4), all together 98 pregnant women were included in the study. The localization of the placental implantation and the depth of myometrial invasion were identified on admission with transabdominal and transvaginal 2D/3D gray scale and Doppler sonography by Voluson 730 Expert scanner (GE Medical systems, Kretztechnik GmbH & OHG, Zipf, Austria). We considered the ultrasonographic findings of irregularity or loss of the retroplacental echolucent area between the uterus and the placenta, thinning of the myometrium to less than 1 mm at the site of placental bed, protrusion of the placenta into the bladder, increased vascularity of the uterine serosa and the bladder wall interface, irregular intraplacental vascularization and the presence of turbulent placental lacunae with highvelocity flow, diagnostic of AIP. The depth and extent of the hypervascularized areas were also assessed. In the cases of placenta previa without AIP, no vascularization of the uterine serosa–bladder interface was observed. In none of the cases, magnetic resonance imaging (MRI) was needed for the diagnosis. The intraoperative diagnosis of the placental abnormality was confirmed at the time of cesarean section and also in histopathologic evaluation in postpartum hysterectomized patients. All blood samples were retrieved by venipuncture at the time of admission prior to delivery or administration of betamethasone to accelerate fetal lung maturation or blood transfusion. The samples were centrifuged at 5000 g at 4 C for 20 minutes within 10 min of blood sampling and frozen immediately at 80 C until the analyses were carried out. Unbound serum levels of VEGF and PlGF were quantified using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s (USCN life science inc., Wuhan, Hubei 430056, P.R.China) protocols. The detection limits for each of the VEGF A (Cat No: SEA143Hu, Lot No: L150303051) and the PlGF (Cat No: SEA114Hu, Lot No: L141209236) measurements were 15.6–1000 picograms per milliliter (pg/ml) with intra- and interassay coefficients of variation of 510% and 512%, respectively. Similarly, ELISA kit (Cat No: AB119567, Lot No: GR193965–1, ELISA Kit, abcam, UK.) was used in the quantification of VEGF R1 (sFlt1) in serum sample with detection limit of 40.03 nanograms per mililiter (ng/ml) with the intra- and interassay coefficients of variation of 55.5% and 55.1%, respectively. The rest of the blood analyses other than the biomarkers of angiogenesis were carried out within 2 h of blood sampling,
Serum angiogenic profile in abnormal placentation
DOI: 10.3109/14767058.2015.1118044
using a hematology analyzer (GEN-S; Beckman-Coulter Inc., Brea, CA) at the central laboratories of Zekai Tahir Burak Women’s Hospital. The investigators of the study were unaware of the serum levels of the angiogenesis biomarkers in relation to the clinical characteristics of each women, including age, gravidity, parity, number of previous cesarean sections, body mass index (BMI), gestational week at delivery, birth weight, 5-min apgar score, neonatal intensive care unit (NICU) admission and perinatal mortality until the study was ended.
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Statistical analysis Categorical data were expressed as frequency (%), and numerical data were expressed as mean ± SD or median (min–max). The extension of Fisher’s exact test for r c crosstables was used to examine the association between categorical variables. Shapiro–Wilk’s test was used to test the normality assumption. One-way analysis of variance (ANOVA) with the post-hoc Dunnett test and the Kruskal– Wallis test with post hoc Dunn’s test were used normally and non-normally distributed variables, respectively.
Results The patient characteristics in the isolated placenta previa (group 1), placenta accreta/increta (group 2), placenta percreta (group 3) and the control (group 4) pregnant groups are presented in Table 1. Women in the normal pregnancy group (control) were significantly younger (24 versus 32 years), had lower gravidity (2 versus 3) with also lower parity (except in women with placenta previa) than the women in the other three groups with placentation abnormality. The number of previous cesarean section in obstetric history was significantly higher in women with placenta accreta/increta and placenta percreta groups as compared to those in the control group. BMI values were comparable among all four groups. As expected, all 68 women underwent emergency cesarean section. Women with abnormal placentation delivered at significantly less advanced gestational age (35 versus 38 weeks) with newborns’ birthweights lower (2700 versus 3200 g) than the the women with normal pregnancy. The 5-min Apgar scores and the number of newborns who were admitted to the NICU were comparable
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in all four groups of women. We lost only one baby in the isolated placenta previa group in the peripartum period due to meconium aspiration syndrome. To combat the intraoperative bleeding from the uterus, intraoperative packing, placement of compressive sutures or intrauterine tamponade balloon of Bakri were the methods tried. Transportation from the operating room for intraoperative arterial embolization was not feasible, therefore never used. Cesarean hysterectomy and/or postpartum hysterectomy were required to control life-threatening hemorrhage in a total of 24 women, 18 in percreta and five in accreta/increta. One patient with isolated placenta previa experienced intractable postpartum vaginal bleeding and underwent hysterectomy when conservative methods failed to control the bleeding from the lower uterine segment. The histological examination of the hysterectomy specimen revealed no feature of invasive placenta. We experienced bladder injury in seven patients, bowel injury in one and combined bladder and bowel injury in one patient, all of which were repaired primarily during the initial surgery. Fortunately, we had no maternal mortality. As shown in Table 2, there was no statistical difference in the maternal serum values of sFlt1, PlGF, sFlt1/PlGF ratio and VEGF in groups with placental abnormality as compared to controls.
Discussion In normal pregnancy, extravillous cytotrophoblasts migrate through and invade the decidua, anchoring the placenta to the uterus and remodeling the maternal spiral arteries by replacing maternal vascular endothelium into low-resistant vessels that are generally unresponsive to vascular control [15]. Excessive invasion of trophoblast through the decidua into the myometrium is relatively common when the placenta is located over the uterine scars where vascularization is presumably compromised as in the previous cesarean section [8–10,13,16]. Abnormally invasive placenta is encountered in 5–15% of pregnant women with placenta previa. The most frequent risk factor for both is previous cesarean section [10,12]. It could be that oxygen tension may be compromised in the lower uterine segment, especially in the presence of scar, thus as a compensatory mechanism, trophoblasts could infiltrate into the myometrium in an uncontrolled manner [17,18].
Table 1. The comparison of patient characteristics in the isolated placenta previa (group 1), placenta accreta/increta (group 2), placenta percreta (group 3) and the control (group 4). p value (groups) Parameters Age (year) Gravida Para Number of prior cesarean BMI kg/m2 Gestational weeks at delivery Birth weight (gram) 5-min Apgar NICU admission
Group 1 (n ¼ 33)
Group 2 (n ¼ 17)
Group 3 (n ¼ 18)
Group 4 (n ¼ 30)
p value
1 versus 4
2 versus 4
3 versus 4
31.46 ± 4.63 3 (1–8) 1 (0–3) 0 (0–2) 27.29 ± 4.26 35.7 ± 2.4 2692.7 ± 553.6 9 (5–10) 5 (15.2%)
31.47 ± 3.87 3 (2–6) 2 (0–5) 1 (0–2) 28.42 ± 3.27 35.2 ± 2.4 2668.2 ± 533.3 9 (0–10) 3 (17.6%)
32.94 ± 3.99 3 (2–4) 2 (1–3) 1.5 (0–3) 30 ± 3.19 35.9 ± 0.9 2828.9 ± 321.2 9 (8–9) 1 (5.6%)
24.67 ± 5.01 2 (1–7) 0 (0–3) 0 (0–0) 28.46 ± 3.77 38.4 ± 1.5 3199.3 ± 393.3 9 (5–9) 1 (3.3%)
50.001 0.004 50.001 50.001 0.114 50.001 50.001 0.069 0.256
50.001 0.032 0.061 0,139 – 50.001 50.001 – –
50.001 0.041 0.001 50.001 – 50.001 0.001 – –
50.001 0.013 50.001 50.001 – 50.001 0.026 – –
Data were expressed as mean ± SD, median (min-max) . The mean difference is significant at the 0.05 level.
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J Matern Fetal Neonatal Med, Early Online: 1–5
Table 2. Circulating levels of free sFlt-1, PlGF, sFlt-1/PlGF ratio and VEGF in the placenta previa (group 1), placenta accreta/increta (group 2), placenta percreta (group 3) and the control (group 4). Parameter sFlt-1(ng/ml) PlGF(pg/ml) sFlt-1/PlGF ratio VEGF(pg/ml)
Group 1 (n ¼ 33)
Group 2 (n ¼ 17)
Group 3 (n ¼18)
Group 4 (n ¼ 30)
p value
1.422 (1.332–1.707) 95.307 ± 36.153 0.014 (0.009–0.277) 6.405 ± 2.819
1.427 (1.315–1.689) 77.802 ± 37.101 0.017 (0.009–0.079) 4.885 ± 1.48
1.403 (1.299–1.607) 93.986 ± 49.098 0.015 (0.008–0.153) 6.34 ± 2.341
1.387 (1.312–1.666) 81.498 ± 29.522 0.016 (0.009–0.045) 5.454 ± 2.541
0.196 0.276 0.151 0.131
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Data expressed as mean ± SD, median (min–max). The mean difference is significant at the 0.05 level.
We hypothesized that suboptimally vascularized, placenta implanted in the lower segment in the case of isolated placenta previa and deeply infiltrated, highly vascularized placenta in placenta accreta/increta/percreta would alter the release of antiangiogenic and angiogenic substances differently as compared to normal fundal placentation. Although few studies have investigated VEGF, PlGF and sFlt1 serum levels in women with AIP, we were unable to find any studies examining the association between the levels of these factors and placenta previa with and without any myometrial trophoblastic invasion, specifically. Furthermore, we also studied the angiogenic markers in women with placenta accreta/increta and percreta seperately, to see whether the depth of the myometrial invasion mattered regarding angiogenesis. Thus, our study is the first to indicate the role of angiogenic markers comparatively in specific placentation abnormalities. Since several studies have reported better diagnostic accuracy as compared to individual markers, we also measured the serum sFlt1/PlGF ratio in evaluation [19–21]. Prior cell culture and immunohistochemistry studies have already demonstrated that placentation process is orchestrated by coordinated and fine balance among sFlt1, PlGF and VEGF secretions [4,14,22]. Augmented expression of VEGF in trophoblastic tissue has been suggested to promote growth, angiogenesis and invasion leading to the development of AIP [23]. In fact, VEGF expression was found to be correlated with trophoblast invasion in human placentas; furthermore, blocking VEGF signaling was shown to lead to the interception of cytotrophoblast invasion by the same group [15,16,24]. Supporting the role for antiangiogenic protein, sFLT-1 in modulating trophoblast invasion, lower sFlt1 protein at the maternal–fetal interface has been demonstrated in invasive as compared to normal placentation [25]. Contrarily, as recently reported by Wehrum et al, in a group of pregnant women with isolated placenta previa, sFlt1, PlGF and VEGF serum concentrations were comparable with the serum levels of women with normal pregnancy [16]. This is in accord with our study results. Similarly, we detected no difference in the serum levels of the three analytes among women with and without placenta previa. In the present study, on the contrary to what we have expected and hypothesized, again the serum levels of all three of the angiogenic factors were comparable among women with placenta accreta, increta and percreta and women with normal pregnancy. Again in Wehrum’s recent publication, in women with placenta previa complicated with excessive myometrial invasion, maternal serum levels of VEGF was even lower than in control cases, as an unexpected finding [16].
The data in this study suggested that the balance between antiangiogenic and angiogenic substances were comparable and normal in pregnancies complicated by either placenta previa or AIP when compared to normal pregnancies without placentation abnormality. Our data further indicate that the placenta and the fetus of women with both placenta previa and AIP succeed to reach the increased oxygen demands during pregnancy. In support of this, we encountered no case with preeclampsia (PE) and intrauterine fetal growth restriction (IUGR) in our study groups suggesting normal placental angiogenesis. This is inconsistent with the previous reports claiming that placenta previa and AIP are independent risk factors for PE and IUGR and consistent with the ones which suggested no association or even protective on the risk of PE [26–28]. One of the strength of our study is the homogeneity of the characteristics of the women in the study groups. The patients had either complete placenta previa without AIP or were diagnosed to have placenta accreta and/or increta and placenta percreta in addition to previa, as seperate study groups, each diagnosed correctly and accurately by ultrasonography, confirmed by intra- and postoperative evaluation. None of the patients had any associated comorbidities including PE or IUGR and all delivered in the third trimester. One of the shortcomings of this study could be that instead of studying the local levels at the decidual placental interface, we measured the serum concentrations of the angiogenic biomarkers. Since VEGF, as an example, mostly binds to plasma membrane and extracellular matrix after secretion and is known to function in a paracrine fashion, the local levels within the uterus and placenta could have been more relevant [29]. In the present study, we checked the unbound circulating levels of the angiogenic biomarkers. It has been suggested that measuring free or bound circulating levels also could lead to inconsistencies in the results of different studies [3]. In conclusion, we demonstrated that regardless of the localization and the degree of the myometrial invasion of the placenta in the uterus, the circulatory levels of sFlt1, PlGF and VEGF; therefore, angiogenesis and vascularization were compatible and in harmony with the normal physiology. Our conclusion is also supported clinically by the absence of any case with preeclampsia and/or IUGR in our cohort. Nevertheless, our findings should be confirmed by other larger scale studies to strengthen our conclusion.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
DOI: 10.3109/14767058.2015.1118044
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16. Wehrum MJ, Buhimschi IA, Salafia C, et al. Accreta complicating complete placenta previa is characterized by reduced systemic levels of vascular endothelial growth factor and by epithelialtomesenchymal transition of the invasive trophoblast. Am J Obstet Gynecol 2011;204:411. 17. Genbacev O, Zhou Y, Ludlow JW, Fisher SJ. Regulation of human placental development by oxygen tension. Science 1997;277: 1669–72. 18. Tantbirojn P, Crum CP, Parast MM. Pathophysiology of placenta creta: the role of decidua and extravillous trophoblast. Placenta 2008;29:639–45. 19. Kim SY, Ryu HM, Yang JH, et al. Increased sFlt-1 to PlGF ratio in women who subsequently develop preeclampsia. J Korean Med Sci 2007;22:873–7. 20. Hanita O, Alia NN, Zaleha AM, Nor Azlin MI. Serum soluble FMS-like tyrosine kinase 1 and placental growth factor concentration as predictors of preeclampsia in high risk pregnant women. Malays J Pathol 2014;36:19–26. 21. Ohkuchi A, Hirashima C, Suzuki H, et al. Evaluation of a new and automated electrochemiluminescence immunoassay for plasma sFlt-1 and PlGF levels in women with preeclampsia. Hypertens Res 2010;33:422–7. 22. Girling JE, Rogers PA. Recent advances in endometrial angiogenesis research. Angiogenesis 2005;8:89–99. 23. Tseng JJ, Chou MM. Differential expression of growth-, angiogenesis- and invasion-related factors in the development of placenta accreta. Taiwan J Obstet Gynecol 2006;45:100–6. 24. Zhou Y, McMaster M, Woo K, et al. Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am J Pathol 2002;160:1405–23. 25. McMahon K, Karumanchi SA, Stillman IE, et al. Does soluble fmslike tyrosine kinase-1 regulate placental invasion? Insight from the invasive placenta. Am J Obstet Gynecol 2014;210:68.e1–4. 26. Rosenberg T, Pariente G, Sergienko R, et al. Critical analysis of risk factors and outcome of placenta previa. Arch Gynecol Obstet 2011; 284:47–51. 27. Hasegawa J, Sekizawa A, Farina A, et al. Location of the placenta or the umbilical cord insertion site in the lowest uterine segment is associated with low maternal blood pressure. BJOG 2011;118: 1464–9. 28. Adam I, Haggaz AD, Mirghani OA, Elhassan EM. Placenta previa and pre-eclampsia: analyses of 1645 cases at medani maternity hospital, Sudan. Front Physiol 2013;4:32. 29. Ozawa CR, Banfi A, Glazer NL, et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest 2004;113:516–27.
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Serum angiogenic profile in abnormal placentation Ebru Biberoglu, Ayse Kirbas, Korkut Daglar, Kutay Biberoglu, Hakan Timur, Canan Demirtas, Erdem Karabulut & Nuri Danisman To cite this article: Ebru Biberoglu, Ayse Kirbas, Korkut Daglar, Kutay Biberoglu, Hakan Timur, Canan Demirtas, Erdem Karabulut & Nuri Danisman (2016): Serum angiogenic profile in abnormal placentation, The Journal of Maternal-Fetal & Neonatal Medicine, DOI: 10.3109/14767058.2015.1118044 To link to this article: http://dx.doi.org/10.3109/14767058.2015.1118044
Published online: 10 Feb 2016.
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Date: 03 March 2016, At: 04:21
http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2016 Taylor & Francis. DOI: 10.3109/14767058.2015.1118044
ORIGINAL ARTICLE
Serum angiogenic profile in abnormal placentation Ebru Biberoglu1, Ayse Kirbas1, Korkut Daglar1, Kutay Biberoglu2, Hakan Timur1, Canan Demirtas3, Erdem Karabulut4, and Nuri Danisman1
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1
Department of Perinatology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey, 2Department of Obstetrics and Gynecology, Gazi University Medical School, Ankara, Turkey, 3Department of Biochemistry, Gazi University Medical School, Ankara, Turkey, and 4 Department of Biostatistics, Hacettepe University Medical School, Ankara, Turkey Abstract
Keywords
Objective: To evaluate the circulating soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) levels in women with abnormal placentation and to compare the data with the results of women with normal pregnancy. Material and methods: Serum biomarkers of angiogenesis and maternal and perinatal characteristics of 68 pregnant women, all in the third trimester, who were diagnosed to have vaginal bleeding due to complete placenta previa with and without concomitant placenta accreta, increta and percreta as the study group and 30 pregnant women without any placentation abnormality who eventually delivered at 37 weeks of gestational age as the control group were evaluated. Results: There was no statistical difference in the maternal serum values of sFlt1, PlGF, sFlt1/PlGF ratio and VEGF in groups with placental abnormality as compared to controls. Not even a single case of preeclampsia and intrauterine fetal growth restriction was encountered in the study group. Conclusion: We demonstrated that regardless of the localization and the degree of the myometrial invasion of the placenta in the uterus, the circulatory biomarkers of angiogenesis and vascularization were comparable.
Angiogenesis, abnormally invasive placenta, placental growth factor, placenta previa, soluble Fms-like tyrosine kinase 1, vascular endothelial growth factor History Received 19 October 2015 Revised 4 November 2015 Accepted 5 November 2015 Published online 27 January 2016
Introduction In normal pregnancy, decidualization and vascular remodeling are essential for implantation and for providing the embryo with adequate nutrient and substrate supply. Several examples of adverse pregnancy outcomes, such as early pregnancy loss and ischemic placental diseases, including preeclampsia, intrauterine fetal growth restriction and premature delivery are associated with disturbed vascular growth regulation in the fetomaternal unit [1]. The endometrium, decidua and placenta are rich sources of angiogenic substances, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), which is another member of VEGF family. While both have synergistic effects in angiogenesis, soluble fms-like tyrosine kinase 1 (sFlt1) and soluble endoglin (sEng) prevent VEGF and PlGF irreversibly, binding their endothelial cell receptors, thereby exhibit antiangiogenic effect leading to endothelial dysfunction [2,3]. During implantation and placentation process, the highly invasive nature of trophoblasts is kept in control by the Address for correspondence: Ayse Kirbas, Department of Perinatology, Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. Tel: +90 533-642-3162. Fax: +90 312-312-4931. E-mail: [email protected];[email protected]
decidua that requires regulation and coordination with VEGF, PlGF and sFlt1 expressions [4]. Although serum sFlt1 have been demonstrated to rise 50-fold at term in normal pregnancy compared to nonpregnant state [5], the source of overexpression at the placental level and the physiologic role of sFlt1 are unknown. It can be presumed that sFlt1 may be preparing the placenta for seperation at delivery by regulating placental invasion. When the placenta is not restricted within the decidua but abnormally attached to the uterine wall as a consequence of trophoblastic invasion into the myometrium, within the context of the present study, we preferred to choose the lately adopted term abnormally invasive placenta (AIP), instead of using placenta accreta as the general term recommended by The Publications Committee of The Society for Maternal-Fetal Medicine [6,7]. Depending on the depth of the villous tissue, from the most superficial to the deepest in the myometrium, a further subdivision into accreta, increta and percreta can be introduced [7]. A more commonly seen placentation abnormality is placenta previa in which the placenta is inserted in the lower uterine segment. The incidence of both placenta previa and AIP has increased and seems to parallel the increasing cesarean delivery rate. It has been estimated that in women with placenta previa,
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3%, 40% and 60% of women at the first, second and third previous cesarean sections develop AIP, respectively [8–10]. Although the pathogenesis of placenta previa is not known, higher parity, advanced age and previous low segment cesarean section or uterine surgery are blamed for the higher risk. It has been suggested that endometrial scarring at the sites of prior surgery or in parous women at prior placental attachments result in lower placental implantation associated with decreased uteroplacental blood flow [8–12]. Similar to placenta previa, the risk factors for AIP are well established, but the underlying mechanisms leading to abnormal placentation are less well understood [10,13]. In relation to angiogenesis during placentation, we considered placenta previa as having defective decidual vascularization and AIP with deep trophoblastic and vascular infiltration as the opposite ends of the spectrum. Nevertheless, since the most optimal site to implant for the placenta is the highly vascularized fundal portion of the uterus, the lower uterine segment implantation would offer suboptimal blood supply to the placenta unless the trophoblasts invade the deep myometrium. Given that VEGF and PlGF have potent angiogenic and sFlt1 has antiangiogenic potential, we hypothesized that an imbalance in their production might play a critical role at the trophoblast decidual interface in controlling the trophoblastic invasion. The scarcity of adequate research in the medical literature in this area allowed us to speculate further; thus, we hypothesized that the implantation in areas with less blood supply as in the setting of isolated placenta previa, the hypoxic placenta would release sFlt1 into maternal circulation which would lead to suppressed serum levels of VEGF and PlGF. On the other hand, there is also in vitro evidence that hypoxia could upregulate VEGF, promoting angiogenesis and decrease PlGF expression in trophoblast [14]. Contrarily, in case of placenta accreta/increta and particularly in percreta with highly vascularized trophoblastic invasion through the myometrium, a different scenario could have been set with decreased sFlt1 and increased VEGF as to the serum levels in women with normal pregnancy. Therefore, we aimed to evaluate the circulating sFlt1, PlGF and VEGF levels in women with isolated placenta previa totalis and in women with placenta acreta/increta and percreta and to compare the data with the results of women with normal pregnancy.
Material and methods This case–control study was conducted between May 2014 and July 2015 at the Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. Each participant provided written informed consent to donate biological specimens for this study. The local Institutional Review Board of the hospital approved the study. The universal principles of the Helsinki Declaration were applied. Eighty-seven pregnant women, all in the third trimester, who were admitted with a diagnosis of vaginal bleeding due to complete placenta previa with and without concomitant placenta accreta, increta and percreta as the study group and 30 consecutive pregnant women without any placentation abnormality who applied to the maternity ward and eventually delivered at 37 weeks of gestational age as the control
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group were enrolled prospectively. Women with vaginal bleeding related to other causes, multiple gestation, any systemic disease including cardiovascular, endocrinological, metabolic, inflammatory and autoimmune disorders, prepregnancy obesity (BMI 30 kg/m2) and who smoke or on any drug other than iron medication were excluded from the study group. Accordingly, six pregnant women who declined to sign the informed consent form, 4 who had emergency transfusion before blood samples were taken for the study, three with twins, two with thyroid disfunction, two with gestational diabetes, one obese and one smoker were discarded from the study groups. In the end, a total of 68 patients with complete placenta previa, 33 of whom without any evidence of AIP (group 1), 17 with concomitant placenta accreta/increta (group 2), 18 with placenta percreta (group 3) and another 30 women with normal pregnancy (group 4), all together 98 pregnant women were included in the study. The localization of the placental implantation and the depth of myometrial invasion were identified on admission with transabdominal and transvaginal 2D/3D gray scale and Doppler sonography by Voluson 730 Expert scanner (GE Medical systems, Kretztechnik GmbH & OHG, Zipf, Austria). We considered the ultrasonographic findings of irregularity or loss of the retroplacental echolucent area between the uterus and the placenta, thinning of the myometrium to less than 1 mm at the site of placental bed, protrusion of the placenta into the bladder, increased vascularity of the uterine serosa and the bladder wall interface, irregular intraplacental vascularization and the presence of turbulent placental lacunae with highvelocity flow, diagnostic of AIP. The depth and extent of the hypervascularized areas were also assessed. In the cases of placenta previa without AIP, no vascularization of the uterine serosa–bladder interface was observed. In none of the cases, magnetic resonance imaging (MRI) was needed for the diagnosis. The intraoperative diagnosis of the placental abnormality was confirmed at the time of cesarean section and also in histopathologic evaluation in postpartum hysterectomized patients. All blood samples were retrieved by venipuncture at the time of admission prior to delivery or administration of betamethasone to accelerate fetal lung maturation or blood transfusion. The samples were centrifuged at 5000 g at 4 C for 20 minutes within 10 min of blood sampling and frozen immediately at 80 C until the analyses were carried out. Unbound serum levels of VEGF and PlGF were quantified using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s (USCN life science inc., Wuhan, Hubei 430056, P.R.China) protocols. The detection limits for each of the VEGF A (Cat No: SEA143Hu, Lot No: L150303051) and the PlGF (Cat No: SEA114Hu, Lot No: L141209236) measurements were 15.6–1000 picograms per milliliter (pg/ml) with intra- and interassay coefficients of variation of 510% and 512%, respectively. Similarly, ELISA kit (Cat No: AB119567, Lot No: GR193965–1, ELISA Kit, abcam, UK.) was used in the quantification of VEGF R1 (sFlt1) in serum sample with detection limit of 40.03 nanograms per mililiter (ng/ml) with the intra- and interassay coefficients of variation of 55.5% and 55.1%, respectively. The rest of the blood analyses other than the biomarkers of angiogenesis were carried out within 2 h of blood sampling,
Serum angiogenic profile in abnormal placentation
DOI: 10.3109/14767058.2015.1118044
using a hematology analyzer (GEN-S; Beckman-Coulter Inc., Brea, CA) at the central laboratories of Zekai Tahir Burak Women’s Hospital. The investigators of the study were unaware of the serum levels of the angiogenesis biomarkers in relation to the clinical characteristics of each women, including age, gravidity, parity, number of previous cesarean sections, body mass index (BMI), gestational week at delivery, birth weight, 5-min apgar score, neonatal intensive care unit (NICU) admission and perinatal mortality until the study was ended.
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Statistical analysis Categorical data were expressed as frequency (%), and numerical data were expressed as mean ± SD or median (min–max). The extension of Fisher’s exact test for r c crosstables was used to examine the association between categorical variables. Shapiro–Wilk’s test was used to test the normality assumption. One-way analysis of variance (ANOVA) with the post-hoc Dunnett test and the Kruskal– Wallis test with post hoc Dunn’s test were used normally and non-normally distributed variables, respectively.
Results The patient characteristics in the isolated placenta previa (group 1), placenta accreta/increta (group 2), placenta percreta (group 3) and the control (group 4) pregnant groups are presented in Table 1. Women in the normal pregnancy group (control) were significantly younger (24 versus 32 years), had lower gravidity (2 versus 3) with also lower parity (except in women with placenta previa) than the women in the other three groups with placentation abnormality. The number of previous cesarean section in obstetric history was significantly higher in women with placenta accreta/increta and placenta percreta groups as compared to those in the control group. BMI values were comparable among all four groups. As expected, all 68 women underwent emergency cesarean section. Women with abnormal placentation delivered at significantly less advanced gestational age (35 versus 38 weeks) with newborns’ birthweights lower (2700 versus 3200 g) than the the women with normal pregnancy. The 5-min Apgar scores and the number of newborns who were admitted to the NICU were comparable
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in all four groups of women. We lost only one baby in the isolated placenta previa group in the peripartum period due to meconium aspiration syndrome. To combat the intraoperative bleeding from the uterus, intraoperative packing, placement of compressive sutures or intrauterine tamponade balloon of Bakri were the methods tried. Transportation from the operating room for intraoperative arterial embolization was not feasible, therefore never used. Cesarean hysterectomy and/or postpartum hysterectomy were required to control life-threatening hemorrhage in a total of 24 women, 18 in percreta and five in accreta/increta. One patient with isolated placenta previa experienced intractable postpartum vaginal bleeding and underwent hysterectomy when conservative methods failed to control the bleeding from the lower uterine segment. The histological examination of the hysterectomy specimen revealed no feature of invasive placenta. We experienced bladder injury in seven patients, bowel injury in one and combined bladder and bowel injury in one patient, all of which were repaired primarily during the initial surgery. Fortunately, we had no maternal mortality. As shown in Table 2, there was no statistical difference in the maternal serum values of sFlt1, PlGF, sFlt1/PlGF ratio and VEGF in groups with placental abnormality as compared to controls.
Discussion In normal pregnancy, extravillous cytotrophoblasts migrate through and invade the decidua, anchoring the placenta to the uterus and remodeling the maternal spiral arteries by replacing maternal vascular endothelium into low-resistant vessels that are generally unresponsive to vascular control [15]. Excessive invasion of trophoblast through the decidua into the myometrium is relatively common when the placenta is located over the uterine scars where vascularization is presumably compromised as in the previous cesarean section [8–10,13,16]. Abnormally invasive placenta is encountered in 5–15% of pregnant women with placenta previa. The most frequent risk factor for both is previous cesarean section [10,12]. It could be that oxygen tension may be compromised in the lower uterine segment, especially in the presence of scar, thus as a compensatory mechanism, trophoblasts could infiltrate into the myometrium in an uncontrolled manner [17,18].
Table 1. The comparison of patient characteristics in the isolated placenta previa (group 1), placenta accreta/increta (group 2), placenta percreta (group 3) and the control (group 4). p value (groups) Parameters Age (year) Gravida Para Number of prior cesarean BMI kg/m2 Gestational weeks at delivery Birth weight (gram) 5-min Apgar NICU admission
Group 1 (n ¼ 33)
Group 2 (n ¼ 17)
Group 3 (n ¼ 18)
Group 4 (n ¼ 30)
p value
1 versus 4
2 versus 4
3 versus 4
31.46 ± 4.63 3 (1–8) 1 (0–3) 0 (0–2) 27.29 ± 4.26 35.7 ± 2.4 2692.7 ± 553.6 9 (5–10) 5 (15.2%)
31.47 ± 3.87 3 (2–6) 2 (0–5) 1 (0–2) 28.42 ± 3.27 35.2 ± 2.4 2668.2 ± 533.3 9 (0–10) 3 (17.6%)
32.94 ± 3.99 3 (2–4) 2 (1–3) 1.5 (0–3) 30 ± 3.19 35.9 ± 0.9 2828.9 ± 321.2 9 (8–9) 1 (5.6%)
24.67 ± 5.01 2 (1–7) 0 (0–3) 0 (0–0) 28.46 ± 3.77 38.4 ± 1.5 3199.3 ± 393.3 9 (5–9) 1 (3.3%)
50.001 0.004 50.001 50.001 0.114 50.001 50.001 0.069 0.256
50.001 0.032 0.061 0,139 – 50.001 50.001 – –
50.001 0.041 0.001 50.001 – 50.001 0.001 – –
50.001 0.013 50.001 50.001 – 50.001 0.026 – –
Data were expressed as mean ± SD, median (min-max) . The mean difference is significant at the 0.05 level.
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Table 2. Circulating levels of free sFlt-1, PlGF, sFlt-1/PlGF ratio and VEGF in the placenta previa (group 1), placenta accreta/increta (group 2), placenta percreta (group 3) and the control (group 4). Parameter sFlt-1(ng/ml) PlGF(pg/ml) sFlt-1/PlGF ratio VEGF(pg/ml)
Group 1 (n ¼ 33)
Group 2 (n ¼ 17)
Group 3 (n ¼18)
Group 4 (n ¼ 30)
p value
1.422 (1.332–1.707) 95.307 ± 36.153 0.014 (0.009–0.277) 6.405 ± 2.819
1.427 (1.315–1.689) 77.802 ± 37.101 0.017 (0.009–0.079) 4.885 ± 1.48
1.403 (1.299–1.607) 93.986 ± 49.098 0.015 (0.008–0.153) 6.34 ± 2.341
1.387 (1.312–1.666) 81.498 ± 29.522 0.016 (0.009–0.045) 5.454 ± 2.541
0.196 0.276 0.151 0.131
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Data expressed as mean ± SD, median (min–max). The mean difference is significant at the 0.05 level.
We hypothesized that suboptimally vascularized, placenta implanted in the lower segment in the case of isolated placenta previa and deeply infiltrated, highly vascularized placenta in placenta accreta/increta/percreta would alter the release of antiangiogenic and angiogenic substances differently as compared to normal fundal placentation. Although few studies have investigated VEGF, PlGF and sFlt1 serum levels in women with AIP, we were unable to find any studies examining the association between the levels of these factors and placenta previa with and without any myometrial trophoblastic invasion, specifically. Furthermore, we also studied the angiogenic markers in women with placenta accreta/increta and percreta seperately, to see whether the depth of the myometrial invasion mattered regarding angiogenesis. Thus, our study is the first to indicate the role of angiogenic markers comparatively in specific placentation abnormalities. Since several studies have reported better diagnostic accuracy as compared to individual markers, we also measured the serum sFlt1/PlGF ratio in evaluation [19–21]. Prior cell culture and immunohistochemistry studies have already demonstrated that placentation process is orchestrated by coordinated and fine balance among sFlt1, PlGF and VEGF secretions [4,14,22]. Augmented expression of VEGF in trophoblastic tissue has been suggested to promote growth, angiogenesis and invasion leading to the development of AIP [23]. In fact, VEGF expression was found to be correlated with trophoblast invasion in human placentas; furthermore, blocking VEGF signaling was shown to lead to the interception of cytotrophoblast invasion by the same group [15,16,24]. Supporting the role for antiangiogenic protein, sFLT-1 in modulating trophoblast invasion, lower sFlt1 protein at the maternal–fetal interface has been demonstrated in invasive as compared to normal placentation [25]. Contrarily, as recently reported by Wehrum et al, in a group of pregnant women with isolated placenta previa, sFlt1, PlGF and VEGF serum concentrations were comparable with the serum levels of women with normal pregnancy [16]. This is in accord with our study results. Similarly, we detected no difference in the serum levels of the three analytes among women with and without placenta previa. In the present study, on the contrary to what we have expected and hypothesized, again the serum levels of all three of the angiogenic factors were comparable among women with placenta accreta, increta and percreta and women with normal pregnancy. Again in Wehrum’s recent publication, in women with placenta previa complicated with excessive myometrial invasion, maternal serum levels of VEGF was even lower than in control cases, as an unexpected finding [16].
The data in this study suggested that the balance between antiangiogenic and angiogenic substances were comparable and normal in pregnancies complicated by either placenta previa or AIP when compared to normal pregnancies without placentation abnormality. Our data further indicate that the placenta and the fetus of women with both placenta previa and AIP succeed to reach the increased oxygen demands during pregnancy. In support of this, we encountered no case with preeclampsia (PE) and intrauterine fetal growth restriction (IUGR) in our study groups suggesting normal placental angiogenesis. This is inconsistent with the previous reports claiming that placenta previa and AIP are independent risk factors for PE and IUGR and consistent with the ones which suggested no association or even protective on the risk of PE [26–28]. One of the strength of our study is the homogeneity of the characteristics of the women in the study groups. The patients had either complete placenta previa without AIP or were diagnosed to have placenta accreta and/or increta and placenta percreta in addition to previa, as seperate study groups, each diagnosed correctly and accurately by ultrasonography, confirmed by intra- and postoperative evaluation. None of the patients had any associated comorbidities including PE or IUGR and all delivered in the third trimester. One of the shortcomings of this study could be that instead of studying the local levels at the decidual placental interface, we measured the serum concentrations of the angiogenic biomarkers. Since VEGF, as an example, mostly binds to plasma membrane and extracellular matrix after secretion and is known to function in a paracrine fashion, the local levels within the uterus and placenta could have been more relevant [29]. In the present study, we checked the unbound circulating levels of the angiogenic biomarkers. It has been suggested that measuring free or bound circulating levels also could lead to inconsistencies in the results of different studies [3]. In conclusion, we demonstrated that regardless of the localization and the degree of the myometrial invasion of the placenta in the uterus, the circulatory levels of sFlt1, PlGF and VEGF; therefore, angiogenesis and vascularization were compatible and in harmony with the normal physiology. Our conclusion is also supported clinically by the absence of any case with preeclampsia and/or IUGR in our cohort. Nevertheless, our findings should be confirmed by other larger scale studies to strengthen our conclusion.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
DOI: 10.3109/14767058.2015.1118044
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