Objective
We sought to characterize serum angiogenic factor profile of women with complete placenta previa and determine if invasive trophoblast differentiation characteristic of accreta, increta, or percreta shares features of epithelial-to-mesenchymal transition.
Study Design
We analyzed gestational age-matched serum samples from 90 pregnant women with either complete placenta previa (n = 45) or uncomplicated pregnancies (n = 45). Vascular endothelial growth factor (VEGF), placental growth factor, and soluble form of fms-like-tyrosine-kinase-1 were immunoassayed. VEGF and phosphotyrosine immunoreactivity was surveyed in histological specimens relative to expression of vimentin and cytokeratin-7.
Results
Women with previa and invasive placentation (accreta, n = 5; increta, n = 6; percreta, n = 2) had lower systemic VEGF (invasive previa: median 0.8 [0.02-3.4] vs control 6.5 [2.7-10.5] pg/mL, P = .02). VEGF and phosphotyrosine immunostaining predominated in the invasive extravillous trophoblasts that coexpressed vimentin and cytokeratin-7, an epithelial-to-mesenchymal transition feature and tumorlike cell phenotype.
Conclusion
Lower systemic free VEGF and a switch of the interstitial extravillous trophoblasts to a metastable cell phenotype characterize placenta previa with excessive myometrial invasion.
When the placenta is inserted into the lower uterine segment and covers partially or entirely the cervix, a diagnosis of placenta previa is established. Placenta previa complicates approximately 0.3-0.8% of pregnancies and represents one of the most frequent causes of painless bleeding during the second half of gestation. Risk factors for placenta previa include a history of cesarean delivery (CD), uterine surgery, termination of pregnancy, smoking, advanced maternal age, multiparity, drug abuse, and multiple gestations. Although the risk factors for placenta previa are well defined, much less is known about its etiology. Furthermore, the underlying cause of the excessive myometrial penetration that characterizes placenta accreta, increta, or percreta in the presence of placenta previa remains largely unknown. In normal pregnancy, development of the vascular system is essential for providing the embryo and fetus with an adequate supply of nutrients and oxygen. A variety of regulatory molecules play functional roles in controlling the process of trophoblast invasion and development during implantation and placentation. These include vasoactive and cell surface proteins, proteases, cytokines, chemokines, and growth factors.
Data derived from animal and human studies demonstrate that the signaling components of the vascular endothelial growth factor (VEGF) family including VEGF-A, placental growth factor (PlGF), and their receptors VEGFR-1 (fms-like-tyrosine-kinase [Flt]-1) and VEGFR-2 (Flk-1/kinase-insert domain receptor [KDR]) are present in the decidua and play crucial roles in the normal development of the fetoplacental vascular network. KDR is recognized as the central VEGF receptor in angiogenesis, while Flt-1 plays a supporting role. Engagement of Flt-1 and Flk-1/KDR receptors by both VEGF and PlGF leads to various downstream activations that are responsible for endothelial cell proliferation, migration, and survival. Presence of phosphotyrosine (P-Tyr) was proposed as an immunohistochemical marker of VEGF-mediated receptor activation. Importantly, an alternatively spliced soluble form of Flt (sFlt)-1 is a modulator of the VEGF and PlGF activity. There is general consensus that the process of normal trophoblast invasion and placentation requires fine coordination among VEGF, PlGF, and sFlt-1 and that oxygen tension has a key role in regulating their expression.
Excessive trophoblast invasion is a frequent occurrence in pregnancies complicated by complete placenta previa (CPP). While the pathogenesis of this phenomenon is still unknown, a possible explanation may be related to differences in oxygen tension in the lower uterine segment or in the uterine scar. Alternatively, the aforementioned risk factors for abnormal placentation may be responsible for the decreased proportion of the normally remodeled blood vessels in women with placenta accreta. Therefore, consistent with the concept of trophotropism, the unchecked trophoblast invasion of the myometrium may be the result of compensatory mechanisms aimed to meet the metabolic and oxygen requirements of the developing fetus. Alternatively, an abnormal trophoblast differentiation process with acquisition of an excessively invasive phenotype is possible. This paradigm is supported by the shared molecular pathways between normal trophoblasts and cancer cells. The observation that a transcription factor characteristic of metastatic epithelial cancers is up-regulated in placenta previa accreta supports this view. Yet, the specific signaling pathways lost or acquired during the process of abnormal trophoblast differentiation remain largely elusive.
Epithelial-to-mesenchymal transition (EMT) is a process characterizing invasive tumor cells that lose the epithelial phenotype to acquire mesenchymal features. As a result of EMT, cells change their shape, and lose their polarity and the cell-cell contact to manifest a motile phenotype. A characteristic feature of EMT is that cytoskeletal intermediate filaments of epithelial cells initiate the expression of vimentin. This is a significant observation given that under normal conditions the cytoskeletal intermediate filaments of epithelial cells express keratin only. Due to vimentin’s direct role in mediating cell motility and thus tumors’ invasive and metastatic potential, coexpression of vimentin with cytokeratin is considered a hallmark of EMT and of a metastable phenotype.
The defective development of the villous vascular tree as well as the abnormal secretion of placental VEGF, PlGF, and sFlt-1 are now considered to be implicated in the development of several pathologic states of pregnancy such as preeclampsia and hypoxia-induced intrauterine fetal growth restriction. This body of knowledge and the evidence that hypoxia regulates the transcription of VEGF, PlGF, and sFlt-1 led us to test the hypothesis that women with CPP have an abnormal serum angiogenic profile that is further altered in the setting of the life-threatening placental invasive myometrial processes accompanying placenta accreta, increta, or percreta. As a corollary to our hypothesis we proposed that the trophoblast in accreta may share pathological features of EMT to explain its abnormal invasive phenotype.
Materials and Methods
Study design and patient population
In a case-control study design we analyzed maternal blood serum samples from 90 women enrolled prospectively at Yale–New Haven Hospital between May 2005 and January 2010. Our study group consisted of 45 consecutive singleton patients (gestational age [GA] median, 31; interquartile range, 28–34 weeks) who were admitted with a diagnosis of vaginal bleeding due to CPP. Blood specimens from 45 healthy women (GA, 31; interquartile range, 28–33 weeks) pregnant with singletons were matched for GA and served as controls. All women provided signed informed consent under protocols approved by the Human Investigation Committee of Yale University. A detailed description of the exclusion criteria and clinical management of the patients following admission is available in the Appendix .
Ultrasonographic evaluation and diagnosis of the placenta previa
The prenatal diagnosis of CPP was based on gray-scale and Doppler sonography. A detailed method of ultrasonographic diagnosis of CPP is available in the Appendix .
Blood collection, storage, and immunoassay procedures
All maternal blood samples were retrieved by venipuncture. For the study group, blood collection was performed at the time of admission prior to steroid therapy or blood transfusion. Blood was allowed to clot. Serum samples were spun at 3000 g at 4°C for 20 minutes, the supernatant aliquoted and immediately stored at –80°C until VEGF, PlGF, and sFlt-1 levels were measured using specific immunoassays.
Enzyme-linked immunosorbent assays for human unbound VEGF, PlGF, and sFlt-1 were performed according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) by investigators unaware of the diagnosis. A detailed description of the immunoassay procedures is available in the Appendix .
Histology and immunohistochemistry
Following delivery, placental tissues of the placenta previa cohort were examined by a perinatal pathologist. In those patients requiring hysterectomy (n = 14), 13 pathology reports documented the presence and degree of placental invasion (accreta n = 5; increta n = 6; percreta n = 2). An additional woman with CPP but no accreta developed postpartum uterine atony that required hysterectomy for hemostatic purpose. For all CPP cases complicated by abnormal invasion of the myometrium we evaluated a full-thickness biopsy of the placenta with the underlying uterine wall as well as a biopsy of the uterine wall, opposite to the placental insertion site. For comparison, placental bed biopsies (decidua basalis) were retrieved from 5 women who had an uncomplicated gestation and an elective CD at GA of 37 [37-38] weeks. These tissues were used as the best possible tissue controls given that performance of a placental insertion site biopsy in women with healthy pregnancies is neither possible nor ethically acceptable at 35 [34-36] weeks, which is the median GA at delivery for the CPP group. Additional descriptions of clinical characteristics, details on antibodies, and immunostaining techniques are presented in Appendix .
Statistical analysis
Data were tested for normality using the Kolmogorov-Smirnov test. Comparisons among groups were performed using Student paired t tests, Wilcoxon signed rank test, McNemar, and Mann-Whitney tests. Data are reported as median and interquartile range or average ± SEM, as appropriate. For immunoassay results, logarithmic transformations were applied before statistical comparisons were performed. Relationships between variables (correlations) were explored using Spearman rank order correlations. We used SigmaStat version 2.03 (SPSS Inc, Chicago, IL) and MedCalc (MedCalc Software bvba, Broekstraat, Belgium) software. P < .05 was considered to indicate statistical significance. Sample size calculations were based on our prior data on serum levels of angiogenic factor concentration in women with preeclampsia. It was estimated that 10 patients in each group would be necessary to detect differences equal to the SD in serum concentrations of angiogenic factors in women with placenta previa compared to controls (80% power, α = 0.05, paired t test). Additional subjects were enrolled to facilitate a finer comparison between groups required to account for confounders and possible effect modifiers such as maternal age, parity, and/or race.
Results
Clinical characteristics of women
We present the demographic, clinical, and pregnancy outcome characteristics of our cohort in Table . Women with CPP were significantly older, were of significantly higher gravidity and parity, and had a significantly higher number of prior CDs when compared to controls. The vast majority of the women with CPP experienced at least one episode of vaginal bleeding during pregnancy (episodes of bleeding: 1 [1-2]). The fetuses of both CPP and control women were of appropriate growth for GA. Women with CPP more often received antenatal steroids and blood transfusions, and were delivered at an earlier GA compared to controls. As expected, the frequency of CD, hysterectomy, and histologically confirmed placenta accreta, increta, or percreta was higher in the group of women with CPP. In multivariate analysis, the only variables significantly associated with the occurrence of CPP were increased parity ( P =.027) and advanced maternal age ( P = .002), both known as risk factors for placenta previa.
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