Hum. Reprod. Advance Access published online on October 14, 2008
Human Reproduction, doi:10.1093/humrep/den296
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Decidual vascularization and the expression of angiogenic growth factors and proteases in first trimester spontaneous abortions
1 Department Biomedical Research, Gaubius Laboratory TNO-Quality and Life, PB Box 2215, Leiden 2301 CE, The Netherlands 2 Department of Gynaecology and Reproductive Medicine, Leiden University Medical Center, PO Box 9600, Leiden 2300 RC, The Netherlands 3 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Centre, van der Boechorststraat 7, Amsterdam 1081 BT, The Netherlands
4 Correspondence address. Fax: +31-71-5248181; E-mail: m.plaisier{at}lumc.nl
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Abstract |
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BACKGROUND: Decidual vascular development is important for implantation. This study analysed decidual vascular adaptation to implantation in correlation with miscarriage in decidual secretory endometrium (DSE), decidua parietalis (DP) and decidua basalis (DB) of miscarriage patients and matched controls.
METHODS: Decidua was obtained during first trimester termination of pregnancy (controls) and vacuum aspiration in case of missed abortion (cases). Vascularization and the expression of VEGF-A, placental growth factor, Flt-1, KDR, angiopoietin (Ang)-1, Ang-2, TIE-2, and membrane-type matrix metalloproteinases MT1-, MT2-, MT3- and MT5-MMP were determined at mRNA and protein level. Uterine natural killer cells (CD56), macrophages (CD68), proliferation (Ki67) and apoptosis (activated caspase-3) were evaluated in consecutive sections.
RESULTS: Decidual vascularization showed differences between cases and controls, i.e. fewer vessels with larger circumference in cases. This correlated with the differential expressions of various factors at mRNA/antigen level and with increased endothelial flt1, KDR, MT2- and MT5-MMP expression in miscarriage patients. The differences between cases and controls were probably not based on altered proliferation and/or apoptosis, since Ki67 and active Caspase-3 showed comparable expression levels in both groups. Although DB of cases and controls showed similar amounts of CD56- and CD68-positive cells, the case group did show elevated levels of CD56 in DSE (P < 0.05) and of CD68 in DP compared with the control group (P < 0.05).
CONCLUSIONS: The differences in vascularization and in the expression of angiogenic factors and proteases between groups suggest a correlation between decidual vascularization and the occurrence of miscarriages.
Key words: decidua/vascularization/angiogenic factors/missed abortion/uterine natural killer cells
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Introduction |
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Decidual vascular adaptation to implantation is important for the success of pregnancy and already starts during the receptive secretory phase of the menstrual cycle. Decidualization, vascular remodelling and invasion of immune cells are prominent processes in the first weeks after fertilization (Bulmer et al., 1991
; Smith, 2000; Salamonsen et al., 2003). Immune cells increase from 8% of total stromal cells during the menstrual cycle up to 30% during the first trimester. Approximately 70% of these leucocytes are uterine natural killer (uNK) cells and 10% are macrophages (Bulmer et al., 1991).
Vascular adaptation includes (pseudo-) vasculogenesis, arterial remodelling, and angiogenesis, the formation of new vessels out of existing ones (Pijnenborg et al., 1983; Burton et al., 1999). Angiogenesis is characterized by increased vascular permeability, endothelial cell proliferation and migration, and is regulated by various growth factors, including vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and angiopoietins (Ang), and proteases such as the membrane-type matrix metalloproteinases (MT-MMP; Pepper, 2001; Carmeliet, 2003). Disturbances in vascular development may play a role in the pathogenesis of miscarriages (Vailhe et al., 1999; Vuorela et al., 2000; Zygmunt et al., 2003).
The angiogenic growth factors VEGF-A and PlGF are extensively studied in the vascular development of the placenta (Vuorela et al., 1997; Smith, 2000; Plaisier et al., 2007). VEGF is one of the earliest genes activated during preimplantation embryo development and is produced by maternal decidual cells and by the invading blastocyst and trophoblast (Jackson et al., 1994; Kapiteijn et al., 2006; Plaisier et al., 2007). VEGF is a potent inducer of angiogenesis and interacts with the VEGF receptor-1, Flt-1 (VEGFR-1) and KDR (VEGFR-2) to promote endothelial cell proliferation, migration and vascular permeability. KDR is generally recognized as the central VEGF receptor in angiogenesis, while Flt-1 plays a supporting role. During pregnancy, a soluble variant of Flt-1 (sFlt-1) is also formed, which quenches VEGF and thereby limits its activity (Maynard et al., 2003).
PlGF shares biochemical and functional features with VEGF, but only interacts with Flt-1. PlGF and VEGF have synergistic effects regarding angiogenesis, however, PlGF-induced vessels are suggested to be more mature and stable than vessels induced by VEGF alone (Carmeliet et al., 2001). PlGF is abundantly expressed in human placenta and may be an important paracrine regulator of decidual angiogenesis and an autocrine mediator of trophoblast function (Sherer and Abulafia, 2001; Plaisier et al., 2007).
The Ang and their receptor, TIE-2, are also known for their regulating capacities regarding angiogenesis. The two ligands bind with equal affinity to TIE-2 but have different functions. Ang-1 maintains vessel integrity and probably plays a role in the later stages of vascular remodelling (Geva and Jaffe, 2000). Ang-2 is a functional antagonist of Ang-1 and leads to loosening of cell/cell interactions and allows access to angiogenic inducers like VEGF (Geva and Jaffe, 2000).
Ang-1, Ang-2 and TIE-2 are detected in various maternal cells, endovascular trophoblasts and (syn-) cytotrophoblasts during human first trimester pregnancy. These findings suggest an additional role for Ang in regulating trophoblast behaviour in the development of utero-placental circulation (Dunk et al., 2000; Zhang et al., 2001; Plaisier et al., 2007). An association of the Ang with miscarriage has not been described, but reduced endothelial TIE-2 expression has been linked to the occurrence of miscarriage (Vuorela et al., 2000).
The membrane-associated localization of MT-MMPs makes them suited for peri-cellular proteolysis (Hotary et al., 2000; van Hinsbergh et al., 2006). We studied the transmembrane-spanning MT-MMPs, MT1- (MMP-14), MT2- (MMP-15), MT3- (MMP-16) and MT5-MMP (MMP-24), since these MT-MMPs have the proteolytic potential to induce capillary tube formation. The GPI-anchored MT4- and MT6-MMP were unable to do so (Hotary et al., 2000).
MT1-MMP is the best known MT-MMP and promotes cell migration, angiogenesis and tumour metastasis (Visse and Nagase, 2003). MT2- and MT3-MMP are less well studied but are also involved in cell migration and invasion. MT1-, MT2- and MT3-MMP induce angiogenesis in vitro and MT2- and MT3-MMP may even be potential regulators of endometrial angiogenesis in vivo (Hotary et al., 2000; Plaisier et al., 2004, 2006). MT5-MMP is known for the induction of embryonic brain development and axonal growth (Llano et al., 1999).
MT1- and MT2-MMP RNA and protein expression are described in decidual extracts, stromal cells and the extra-villous trophoblast (EVT) (Nawrocki et al., 1996; Bjorn et al., 2000; Curry and Osteen, 2003) and are assumed to regulate trophoblast invasion during implantation (Salamonsen et al., 2003). The extent to which migration of other cell types, e.g. immune and endothelial cells, is also regulated by MT-MMPs remains to be seen.
Vascular adaptation to implantation has mainly been studied with regard to physiology (Craven et al., 1998; Kam et al., 1999). Recently, the potential regulation of decidual vascularization by both the EVT and pregnancy-induced hormones has been described. Moreover, the differential expression of VEGF-A, the Ang, PlGF, Flt-1, MT2- and MT3-MMP suggested their role in regulating the vascular adaptive process in uncomplicated first trimester pregnancies (Plaisier et al., 2007, 2008). Few studies have addressed the question of whether vascularization and its regulatory factors are involved in the pathogenesis of first trimester complications, like miscarriage. A higher decidual vessel density was correlated with missed abortions (Vailhe et al., 1999). Also, differential antigen expression of VEGF and its receptors has been correlated to recurrent miscarriage (Vuorela et al., 2000). Moreover, elevated uterine MMP and reduced tissue inhibitor of metalloproteinase (TIMP) expression has been linked to failed implantation (Jokimaa et al., 2002).
The present study determined decidual vascular adaptation to implantation in correlation with failure of implantation, i.e. missed abortion. Vascularization pattern and the expression of the above described angiogenic factors and proteases were determined in three first trimester decidual tissues, decidual secretory endometrium (DSE), decidua parietalis (DP) and decidua basalis (DB) of miscarriage patients (case) and matched controls. The comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages.
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Materials and Methods |
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Study group
The study was approved by the ethics committee of the Leiden University Medical Centre and informed consent was provided by all study subjects.
Decidua samples in the case group were obtained during vacuum aspiration from missed abortion patients (n = 11). Missed abortion was diagnosed based on ultrasound examination and absence of clinical miscarriage symptoms. Aspirated tissue was analysed by an independent pathologist and samples with pathologic abnormalities were excluded. Patients with pathological conditions known to interfere with implantation, like haemostatic abnormalities, auto-antibodies, etc, were also excluded. Gestational age at fetal death was determined by ultrasound.
Decidua samples in the control group were obtained from women (n = 16) with a healthy, viable intrauterine gravidity, undergoing vacuum aspiration in case of a legal voluntarily abortion. Fetal cardiac activity and gestational age were confirmed by ultrasound. Women with symptoms of miscarriage, with underlying illnesses, and cases with discrepancy between the ultrasound-determined gestational age and last day of menstruation were excluded in both control and case group.
Cases and controls were matched based on maternal and gestational age. These parameters and number of previous pregnancies and miscarriages did not differ significantly between the cases and the controls. Patient characteristics of the study groups are given in Table I.
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Tissue samples
Decidua samples were obtained from the aspirated tissue (vacuum aspiration), fixed in formaldehyde overnight and embedded in paraffin. Haematoxylin Phloxin Safrane (HPS) and anti-cytokeratin staining were performed. Haematoxylin (50 g potassium aluminium sulphate, 1 g hematox, 500 mg citric acid, 25 g chloralhydrate, 200 mg NaJO3 in 1000 ml aqua dest) stains nuclei and calcium blue. Phloxin (0.25 g phloxin in 100 ml aqua dest) stains erythrocytes, cytoplasm, fibrin and muscle red. Safrane (3 g safrane in 1000 ml 100% alcohol) stains calcium-free bone, cartilage and collagen yellow.
The HPS staining was used to differentiate between decidua and DSE, which microscopically resembles secretory endometrium. DB and DP were differentiated by the presence or absence of EVT using an anti-cytokeratin staining (see section immunohistochemistry). DSE, DP and DB were obtained from the same curettement and therefore originate from the same depth and area of the uterine wall. Only subjects with at least two complete sets of DSE, DB and DP were included, n = 11 in the missed abortion group and n = 16 in the control group. Serial sections of the paraffin-embedded tissue samples were used for all experiments and all parameters were compared between tissues within subjects.
RNA isolation and cDNA synthesis
RNA was extracted from paraffin-embedded tissue samples (Plaisier et al., 2007). Several samples per tissue per patient were used and these samples contained a proportionate amount of cells and cell types. In short, 5 µm sections were mounted on RNase-free glass slides. The first and last sections were used to verify the presence of the tissues of interest. The other sections were deparaffinized and the tissues of interest, DSE, DP or DB (without villous tissue), were dissected and dissolved in 190 µl lysis buffer (Tris 20 mM pH 7.4, EDTA 1 mM pH 8.0, 2% sodium dodecyl sulphate) and 10 µl proteinase K (20 mg/ml proteinase K, Life-Technologies Gibco BRL, Gaithersburg) for 18 h at 60°C. Subsequently, 400 µl Solution D (4 M guanidium isothiocyanate, 0.75 M sodium citrate, 10% sarkosyl and 2-mercapto-ethanol) was added and RNA was isolated (Plaisier et al., 2007).
RNA quantity and quality were analysed in a spectrophotometer (Nanodrop ND-1000). The OD260/280 of the RNA samples ranged between 1.85 and 2.00. Reverse transcription was performed with 1 µg total RNA, random primers and complementary DNA (cDNA) synthesis kit according to the manufacturer's protocol and the obtained 32 µl cDNA was diluted 1:3 (Ready-to-go You-Prime first strand beads, Amersham Biosciences, Buckinghamshire, UK).
Real-time RT–PCR
The mRNA levels were quantified according to the Taqman real-time PCR method using validated primer and probe (FAM/TAMRA double-labelled) sets for VEGF-A, VEGF-R1 (Flt-1), VEGF-R2 (KDR), PlGF, Ang-1, Ang-2, TIE-2, MT1-MMP, MT2-MMP, MT3-MMP and MT5-MMP. Glycerylaldehyde-3-phosphate dehydrogenase (GAPDH, primers/VIC-labelled probe) was used as an endogenous reference gene. β-actin, β2-microglobulin and cyclophilin were also used as reference genes and showed comparable results (data not shown, all primer/probe sets purchased from Applied Biosystems, Foster City, CA, USA).
RT–PCR reactions for target gene/GAPDH pairs were performed in 12.5 µl, containing 2.5 µl cDNA solution, using mastermix (RT-QP2X-03, Eurogentec, Maastricht, The Netherlands), DNAse-free water and the above primer/probe sets. Reactions were performed in duplicate, expressed in cycle threshold (Ct) and quantified into ng/µl using a standard curve of total RNA. The RNA expression level of DSE was set at 1.0 to compare DSE with DP and DB and the gene expression of DP was set at 1.0 to compare DP with DB. Water and negative-RT samples, obtained by the omission of the reverse transcriptase enzyme in the cDNA reaction, were used as negative controls.
Immunohistochemistry
Serial sections were deparaffinized, endogenous peroxidase was quenched with 3% H2O2/methanol and non-specific binding was reduced by incubation with 5% bovine serum albumin (BSA). Antigen retrieval in trypsin was used for the detection of CD34, TIE-2, MT2- and MT5-MMP. Heat retrieval in 0.1 M citrate buffer was used for the detection of cytokeratin, Ang-2, Ki67, CD56 and CD68.
The primary antibodies are described in Table II. Polyclonal rabbit anti-MT1-MMP was produced and characterized as previously described (Plaisier et al., 2004). The following second antibodies were used: biotinylated horse anti-mouse antibody (1:300, BA-2000, Vector), biotinylated donkey anti-rabbit antibody (1:300, RPN1004, Amersham Biosciences) and biotinylated rabbit anti-goat (1:300, E-0466, DakoCytomation).
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Primary antibodies were applied overnight at 4°C followed by one hour incubation with biotinylated secondary antibody. Antibody binding was visualized using StreptABComplex/HRP, a streptavidin complexed with biotinylated peroxidase (K0377, DakoCytomation, Glostrup, Denmark) and NovaREDTM substrate (SK-4800, Vector, Burlingame, USA) according to the manufacturer's protocol. Only CD56 immunohistochemistry was stained with diaminobenzidine. All incubations were performed in 1% BSA in phosphate-buffered saline.
Sections were counterstained with Mayer's haematoxylin. Specificity of the immunohistochemical reaction was verified by the omission of the first antibody as well as using non-immune mouse immunoglobulin (Ig)G1, concentration adjusted to primary IgG1 concentration, and goat and rabbit serum instead of primary antibody. To evaluate the expression in EVT, cytokeratin and target protein staining were performed on 3 µm serial sections.
Evaluation of the vascularization pattern
CD34-stained decidual sections were used to scan sequential fields (Vailhe et al., 1999). Two to four samples of each type of tissue of each study subject were analysed and six fields per sample were scanned at x100 magnification. The area within the stained vessels and the number of vessels per field were analysed using image analysis software (Qwin, Leica Microsystems) and these data were used to calculate the number of vessels per mm2, total vascular surface per mm2 (mm2/mm2) and the luminal surface (µm2/vessel).
Evaluation of immunohistochemical staining
In order to avoid intensity differences in the staining we analysed control samples (16 donors) simultaneously with the samples of 11 cases.
Immunostaining of Ki67, active Caspase-3 (aCasp-3), CD56 and CD68 was evaluated by counting the number of positive cells and total stromal cells in ten 16 µm2 fields per tissue per patient. In this way, the mean percentages of positive cells per stromal cells and per µm2 were determined. For Ki67 and aCasp-3, the expression in endothelial and epithelial cells was determined similarly.
Immunostaining of the other antigens was evaluated by calculating a staining index (SI): proportion of stained cells x staining intensity. The proportion of stained cells was expressed as 0, 1, 2 or 3, which marks positive staining signal in 0, <10, 10–50 or >50%, respectively, of the cells of a particular cell population. The intensity of staining was expressed as 1, 2 or 3 (weak, moderate or strong staining, respectively). The minimum score was 0 and the maximum score 9 (Plaisier et al., 2007). The average score of two independent observers was used to calculate the mean and total SI (mean and total SI). The inter-observer variability was 6% for VEGF-A, 7% for PlGF, 11% for Flt-1, 10% for KDR, 5% for Ang-1, 6% for Ang-2, 9% for TIE-2, 4% for MT1-MMP, 14% for MT2-MMP, 8% for MT3-MMP and 7% for MT5-MMP (mean variability 8%).
The mean SI represents the protein expression per studied cell type. The total SI represents the total protein expression per tissue and was calculated as the sum of the mean SI of endothelial cells, peri-vascular smooth muscle cells, glandular epithelium and stromal cells in DSE and DP samples. The mean SI of EVT was also included in the total SI of DB samples.
Statistical analysis
Data are expressed as mean ± SEM or mean ± SD. All parameters were compared between DSE and DP, between DSE and DB and between DP and DB within subjects. A general linear model for repeated measurements, repeated measures analysis of variance (ANOVA), was performed to analyse the double paired data within the case and control group as well as to compare data between the case and control group. Where appropriate, the Friedman's test for non-parametric investigations of correlated observations and the Wilcoxon test for paired samples were used to study differences within the two groups (Satistical Package for the Social Sciences 11.5). The statistical analyses used are described in legends and table footnotes. P-values of <0.05 were considered significant.
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Results |
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The vascularization pattern in cases and controls
Intense CD34 staining of vascular structures was observed in all decidual tissues of controls and missed abortion cases. Vessel density as well as total and luminal surfaces were quantified in all controls (n = 16) and cases (n = 11).
Controls
Vessel density was significantly elevated in DSE (116.1 ± 8.5 vessels/mm2) compared with DP (39.1 ± 3.3) and DB (30.9 ± 3.8); vessel density in DP and DB also differed significantly (all P < 0.01, Fig. 1A). The total vascular surface was significantly smaller in DSE (0.021 ± 0.002 mm2/mm2) compared with DP (0.040 ± 0.004) and DB (0.055 ± 0.007). The total surface in DP differed significantly from that in DB (all P < 0.01, Fig. 1B). The luminal surface was significantly smaller in DSE compared with DP (204 versus 1576 µm2/vessel) and DB (2928 µm2/vessel); this parameter was also significantly smaller in DP compared with DB (all P < 0.01, Fig. 1C). Thus, from DSE to DP to DB the vessels become larger and fewer.
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Cases versus controls
The vessel density was significantly lower in DP and DB of cases compared with the controls; 29.6 versus 39.1 vessels/mm2 in DP and 19.8 versus 30.9 vessels/mm2 in DB, respectively (both P < 0.05, Fig. 1A). The luminal surface was significantly higher in DP and DB of cases versus controls; 3604 µm2/vessel versus 1576 in DP and 5661 versus 2928 in DB, respectively (both P < 0.04, Fig. 1C). The total vascular surface and all the parameters in DSE did not differ between cases and controls.
Angiogenic factors and proteases in cases and controls: mRNA levels
The expression of various angiogenic growth factors and MT-MMPs were evaluated at the mRNA level and are given for the three decidual tissues in Tables III and IV and their relative content in cases versus controls in Table V.
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The data of the 16 controls on mRNA levels of angiogenic factors and proteases are given in Table III. They were highly comparable to previous data on control samples of 25 different women (Plaisier et al., 2007
, 2008).
Cases versus controls
The mRNA content in DSE of cases (see Table IV for mRNA content values) was markedly increased for KDR (3.5), Flt-1 (3.5), TIE-2 (2.7), MT1-MMP (2.4) and MT3-MMP (2.6) when compared with their control counterparts (1.0) (P < 0.01, Table V). Regarding the mRNA levels in DP, VEGF-A (2.1), KDR (4.4), Flt-1 (3.8), Ang-2 (2.1), and TIE-2 (3.6) mRNAs were significantly elevated and MT3-MMP expression was reduced in the case group versus control (Table V).
Finally, the mRNA levels in DB showed up-regulation of all angiogenic factors, except PlGF, in decidua of the cases. On the other hand, none of the proteases were differentially expressed (Table V).
Angiogenic factors and proteases in cases and controls: immunohistochemistry
The presence and cellular localization of the various antigens indicated in Table VI were determined in serial sections of DSE, DP and DB. The studied proteins were detectable in all decidual tissues and their presence was graded as mean SI per cell type and total SI based on the sum of the cell type-dependent indices (Table VI, Figs 2 and 3).
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Controls
First, the total expression (total SI) of the studied antigens in DP and DSE were compared. This showed a more abundant total expression of Flt-1, TIE-2 and MT1-MMP in DP (Table VI, P < 0.05). Then, the staining patterns in DB and DSE were compared; the total SI's of PlGF, Flt-1, TIE-2 and MT1-MMP were increased, whereas the total SI of Ang-1 was decreased in DB. Finally, the comparison between DP and DB showed an increased PlGF total SI and a decreased Ang-1 total SI in DB.
The antigen expression was most abundant in glandular epithelial cells, followed by peri-vascular smooth muscle and stromal cells. Endothelium displayed the expression of all angiogenic factors, except Ang-1 and MT5-MMP. TIE-2 showed the highest expression in endothelium. The endothelial protein expression of PlGF and Flt-1 showed significant differences between the three decidual tissues and MT2-MMP appeared less abundantly expressed in DB compared with DP and DSE (Table VI, Fig. 3). All angiogenic factors, except Ang-1 and MT5-MMP very dimly, were also detected in EVT; PlGF, VEGF, Flt-1 and TIE-2 were moderately expressed, whereas KDR and Ang-2 expression were weak.
Cases versus controls
The comparison of the antigen expression between cases and controls is described in Table VI as well. First, comparing protein expression in DSE of cases and controls showed a reduced total SI for KDR and increased total SI for MT5-MMP in DSE of cases (Table VI, P < 0.05). With regard to DP, the total expression of Flt-1 was elevated in the case group (P < 0.05). The proteases showed differential expression as well, reflected by a reduced total MT3-MMP and induced total MT5-MMP expression in missed abortion (case) decidua.
The angiogenic factors in DB were differentially expressed; the total SI of Flt-1 and Ang-1 expression were elevated in missed abortion DB (P < 0.05), in particular in peri-vascular smooth muscle cells and stromal cells. In addition, reduced total MT3-MMP and elevated total MT2- and MT5-MMP were detected in DB of cases (P < 0.05, Fig. 2).
The endothelial protein expression of KDR, Flt-1, MT2- and MT5-MMP showed differential expression between cases and controls (Fig. 3, Table VI).
Finally, the total Ang-1 expression was significantly elevated and the Ang-2 expression appeared decreased in DP and DB of cases compared with controls. This trend was also detected in DSE tissues. Consequently, the Ang-2/Ang-1 ratio was decreased in all tissues of cases compared with controls (P < 0.02 for DB and DP, P < 0.05 for DSE).
The expression of proliferation (Ki67) and apoptosis (aCasp-3) markers
To evaluate whether cell proliferation and apoptosis contributed to the observed changes, we evaluated the presence of aCasp-3 and the proliferation marker Ki67. The expression of aCasp-3 in epithelial (range 17–25%), endothelial (range 0.0–0.1%) and stromal (range 1.6–7.2%) cells was comparable in both groups. The differences between the decidual tissues within each group were similar, except for the expression in stromal cells where the controls showed a significantly decreased percentage of stromal aCasp-3 expression in DSE and DP compared with DB and the cases displayed the same trend (Fig. 4A).
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The expression of proliferation marker Ki67 in epithelial (range 0.3–1.9%), endothelial (range 0.3–2.3%) and stromal (range 2.6–8.1%) cells was comparable in cases and controls. The endothelial and stromal expression appeared to be decreased in DSE and DP compared with DB in both cases and controls (Fig. 4B).
The expression of uNK cell (CD56) and macrophage (CD68), markers
Because decidualization is accompanied by a large influx of immune cells, which can influence angiogenesis, we determined the number of uNK cells and macrophages in the tissues of controls and cases. The CD56 staining showed non-homogeneous staining, mainly situated surrounding vessels and glands, which was corrected for by analysing various samples per patient. DSE showed a significantly increased number of uNK cells in the case group of 15 ± 2% positive cells per total number of stromal cells versus 9 ± 2% in the control group. No significant differences were detected between the control and case groups, respectively, for DP (18 ± 6 versus 26 ± 4%) and DB (21 ± 4 versus 20 ± 4%, Fig. 4C).
The CD68 staining showed scattered positive cells throughout the tissues (range 0.4–3.2%). No significant differences were detected in DSE and DB between the two groups. The number of macrophages was significantly increased in DP of cases compared with controls (Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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The present study showed that the vascularization pattern varied in DSE, DP and DB of missed abortion cases and matched controls, i.e. fewer vessels but with larger circumference, were present in DP and DB of cases. These differences correlated with the differential expression of several angiogenic molecules at the mRNA and antigen level, including the endothelial protein expression of Flt1, KDR, MT2-MMP, MT3-MMP and MT5-MMP. These findings might indicate a correlation between decidual vascularization and the occurrence of miscarriages.
Maternal vascular adaptation to implantation and its regulation has been subject to discussion, but has mainly been studied with regard to arterial remodelling and villous vascularization (Craven et al., 1998; Kam et al., 1999). Recently, the potential regulation of decidual vascularization by the EVT, immune cells and pregnancy-induced hormones has been described (Plaisier et al., 2007, 2008). The present study compared the vascularization pattern and the expression of angiogenic factors and proteases in miscarriage cases and matched controls, which enabled hypothesizing about their correlation with the occurrence of miscarriages. All methods were performed on serial paraffin sections, which allowed studying all parameters in the same tissue. Karyotyping was not carried out. However, in previous studies no differences in the number of leucocytes in general, in uNK cells and macrophages, and in decidual architecture were detected between chromosomally normal and abnormal pregnancies (Quack et al., 2001; Greenwold et al., 2003; Shimada et al., 2006). Thus, the lack of karyotyping will probably not significantly bias our observations.
The vascularization pattern showed increasing vascular and luminal surfaces and decreasing vessel density from DSE to DP to DB in both uncomplicated and complicated first trimester pregnancies. Vailhe et al. (1999) reported a decreased vessel density in DB compared with DP, but did not study DSE. The authors described a vessel density ranging between 20 and 30 vessels/mm2, which is comparable to our findings. The changes in vascular pattern from DSE to DP to DB may be related to mechanical factors and/or proteins regulating angiogenesis. For instance, the lower vessel density and the enlargement of the vascular surface in DB may be associated with the increased blood flow in the proximity of the implantation site (Gibbons and Dzau, 1994; Kam et al., 1999; Plaisier et al., 2007). Increased luminal size might also be induced by a relatively low oxygen level (Charnock-Jones et al., 2002).
The decreased vessel density and increased vascular surface in DB and DP might also be due to an altered balance between angiogenesis regulating factors. Changes in the relative content of VEGF, angiopoietins and their receptors have been associated with the formation of dilated or enlarged microvessels (Nagy et al., 2003; Adams and Alitalo, 2007). Whether the altered contents of Ang-1 (higher) and MT-MMPs (lower) in smooth muscle cells of cases reflect an altered interaction between pericytes and endothelial cells contributing to changes in vasculature is yet unknown and requires further studies.
Comparison of cases and controls showed that the total vascular surface did not differ between the two groups. However, the arrangement of the vessels did differ in DB and DP, i.e. a significant decreased vessel density and increased luminal surface in DB and DP of cases. This is not in concordance with the previously described increased vessel density in DP of missed abortions tissue specimens (Vailhe et al., 1999).
The differences in vascularization patterns between controls and cases resemble the differences between early and late first trimester vascularization, respectively, suggesting that too fast a maturation of the vasculature is associated with the pathogenesis of miscarriages (Plaisier et al., 2007). The premature ripening may allow access of maternal blood in the intervillous space too early in the development of pregnancy, which was also demonstrated in vivo by Doppler ultrasound imaging (Greenwold et al., 2003; Jauniaux and Burton, 2005). This might result in increased oxygen levels with subsequent excessive oxidative stress, which is able to modulate the architecture of vasculature and the expression of peri-cellular proteases and angiogenic factors (Kingdom and Kaufmann, 1997; Burton et al., 1999; Sharkey et al., 2000; Zhang et al., 2001).
Vascularization was determined on the basis of staining of CD34, which is profoundly present on the endothelium of arteries, veins and capillaries and is widely used as an endothelial marker. CD34 has also been encountered in lymphatic endothelial cells of various tumours, but was absent in the lymphatic endothelial cells of unaffected tissues (Fiedler et al., 2006). In addition, Rogers et al. (2008) concluded that endometrial lymphatic endothelial cells were negative for CD34. Therefore, it is likely that our data reflect mainly blood vessel endothelial cells. However, the possibility that some CD34-positive lymphatic endothelial cells were present cannot be excluded completely without co-staining with a specific marker for lymphatic endothelium.
A second point of caution regards a contribution of remodelling of the existing vascular bed. In addition to the endothelium, smooth muscle cells play a role in vascular adaptation to pregnancy (Kohnen et al., 2000; Harris et al., 2006). The mural smooth muscle cells are not only involved in the stabilization of newly formed vessels (Armulik et al., 2005), their apoptotic death is also essential during remodelling of spiral arteries in pregnancy, a process that is facilitated by trophoblasts (Harris et al., 2006). One may hypothesize that remodelling may explain the large vascular structures observed in our decidual tissue, rather than angiogenesis. However, no increased staining for aCasp-3, a marker for apoptosis, was observed around the vessels. In addition, one would expect to observe the casts of collagen and elastin matrix around the CD34-positive vessels, even after the smooth muscle cells had disappeared. Such structures were not observed in the HE/HPS-stained tissue slices. Therefore, we favour the view that angiogenesis is predominantly involved, although we cannot completely rule out involvement of remodelled spiral arteries. Future studies using specific smooth muscle cell and matrix markers may shed further light on this uncertainty.
Steroid hormones, the EVT, and immune cells are able to modulate vascularization directly or indirectly via angiogenic factors (Hyder and Stancel, 1999; Zygmunt et al., 2002; Simoncini et al., 2003; Hanna et al., 2006). We confirm the previously described potential role for VEGF-A (via KDR), the angiopoietins (via TIE-2) and PlGF (via Flt-1) in the regulation of decidual vascularization (Plaisier et al., 2007). Additionally, their possible association with the differential vascularization pattern in the miscarriage group has been evaluated. The closer the tissue is located to the implanting embryo, the more angiogenic factors show increased expression in cases compared with controls. PlGF, the most abundantly regulated factor in uncomplicated first trimester decidua (Plaisier et al., 2007), is not differentially expressed and appears not to be involved in the pathogenesis of missed abortions. The way in which the increased expression of angiogenic factors is related to the fewer but larger vessels remains to be elucidated in future research.
While several growth factors and MT-MMPs showed marked differences between cases and controls, as summarized in the ratios of Table III, the differences observed at the antigen level were less obvious. This may, in part, be due to the fact that the stoichiometry between changes in mRNA and protein can be different. VEGF mRNA ratio between cases and controls was highest in DB (4.9), while at the protein level it was also highest in DB, but only at 1.5. Also the expression of Ang-1 tended to increase in cases when compared with controls, both at the mRNA and protein level. In contrast, the increase in Ang-2 mRNA was accompanied by a reduction in Ang-2 antigen. As Ang-2 is stored in storage organelles in endothelial cells, one may speculate that this is related to release of Ang-2, but the present data do not permit to draw a conclusion. The increase in all decidual tissues of MT5-MMP in cases is not reflected by an increase in their mRNAs in DSE and DP. The reduction of MT3-MMP antigen is found in all decidual tissues, while at the mRNA level this was only observed in DP and DB. Differences in translation and protein degradation may contribute to the observed differences. Furthermore, expression levels in specific types of cells may differently correlate with protein expression. Alternatively, methodological aspects may influence the protein data. The evaluation of antigens reflects both the extent of expression (number of cells that produce the antigen) and the intensity (actual level in a cell). Particularly when the shape of cells becomes altered, the quantification may be influenced. However, the alternative approach of extracting the protein from the tissue might be biased by extraction efficiency and, above all, would loose all information regarding individual cell types.
Only the endothelial and total expression of Flt-1 and KDR antigens and the total expression of Flt-1 and Ang-1 were differentially expressed. Thus, the receptors of VEGF and PlGF, and not the growth factors themselves, seem to be regulated at the protein level. It should be noted that our assays did not differentiate between the expression of Flt-1 and sFlt-1, which is known to have a counter regulatory role in VEGF signal transduction during early pregnancy (Maynard et al., 2003).
Decidual tissues of controls showed an Ang-2/Ang-1 ratio in favour of Ang-2, whereas decidual tissues of cases showed a ratio in favour of Ang-1. This altered ratio might be based on different oxygen levels in both groups, since hypoxia enhances Ang-2 transcription and destabilizes Ang-1 (Zhang et al. 2001). The higher Ang-1 in cases might in part explain the observed decreased vessel density with increased circumference, since Ang-1 promotes the generation of a more complex vascular network and vessels of increased luminal size (Asahara et al., 1998; Geva and Jaffe, 2000). In addition, less complex and fewer large arteries are reported in Ang-1–/– mice (Suri et al., 1996).
The expression of peri-cellular acting proteases was assessed in decidua of both groups. MT1-MMP did not show differential expression between cases and controls. The differential expression at protein but not RNA level suggests that MT2- and MT5-MMP are regulated at the translational and not at transcriptional level. The differential endothelial antigen levels of MT2, MT3- and MT5-MMP may be partially responsible for the differences in vascular pattern in DB and DP, especially since we know that MT2- and MT3-MMP are likely to be regulators of decidual angiogenesis (Hotary et al., 2000; Plaisier et al., 2004, 2006). The way in which MT5-MMP is involved in decidual vascularization and possibly other decidual remodelling processes is unknown.
Conclusions regarding cause of miscarriage based on histopathological studies of abortion tissue are often questioned since fetal death may occur days before evacuation, allowing post-mortem inflammatory or apoptotic reactions during this ‘retention time’. No effect has been described of retention time on the outcome of vascular parameters (Meegdes et al., 1988; Nelen et al., 2000; Lisman et al., 2004). Therefore, this most likely excludes a contribution of ‘retention time’ to the observed vascularization differences between cases and controls. Nevertheless, inflammatory, proliferation and apoptotic processes were analysed.
The expression of proliferation and apoptosis markers suggests an increased cellular turnover from DSE to DP to DB in both cases and controls. The level of proliferation and apoptosis was comparable in cases and controls, suggesting that our data were not seriously biased by differences in proliferation and apoptosis in response to fetal loss. The amount and distribution of apoptosis is comparable with earlier reports and the elevated apoptosis level in DB compared with DP has been described in uncomplicated pregnancies (Marx et al., 1999; Joswig et al., 2003; Von Rango et al., 2003). No reference data were available regarding proliferation in the various decidual tissues, although moderate Ki67 expression has been described in DB (Korgun et al., 2006).
Similar numbers of uNK cells were demonstrated in DP and DB of cases and controls, but their number was significantly increased in DSE of cases. Reports on uNK cells and miscarriages describe increased uNK cells in receptive endometrium of patients with recurrent miscarriages (Quenby et al., 1999; Tuckerman et al., 2007). The abundant presence of uNK cells in DSE samples may reflect these observations. The difference between cases and controls were levelled in DP and DB, possibly by hormone-regulated uNK cell migration to normal values (Van den Heuvel et al., 2005).
The number of macrophages has been reported not to be related to miscarriages (Hill et al., 1995; Quack et al., 2001). We detected increased macrophages in DP of cases and not in DSE and DB. Strikingly, the number of uNK cells and macrophages at the actual implantation site did not differ between the two groups.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Vascular adaptation to implantation is important in the development of a healthy pregnancy. Here, the involvement of decidual vascularization was studied in relation to miscarriages. Differences were observed in vascularization pattern between decidual tissues of cases and controls. Strikingly, the vascular differences correlated with the differential expression of several angiogenic molecules at mRNA and antigen level. Furthermore, the endothelial antigen levels of MT3-MMP were decreased while Flt1, KDR and MT2- and MT5-MMP were enhanced in DP and DB of cases. These findings might indicate a correlation between decidual vascularization and the occurrence of miscarriages.
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Submitted on October 3, 2007; resubmitted on May 15, 2008; accepted on May 22, 2008.
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