The role of fibrin matrices and tissue factor in early-term trophoblast proliferation and spreading.

INTRODUCTION: Fibrin deposition in placenta is a common phenomenon which can be triggered by villous injury and coagulation activation. Fibrin abnormalities (hypo/dysfibrinogenemia) and factor XIII deficiency are associated with infertility and pregnancy loss. While trophoblasts are known to grow on fibrin matrices, the role of this protein in trophoblast repair processes remains unclear. We hypothesize that fibrin may have an essential role in trophoblast remodeling. METHODS: Morphology and spreading of primary early-term human trophoblasts and villi explants were investigated on various fibrin components. Cross-linking of matrices was evaluated by D-dimer assay. TF procoaguant activity, protein and mRNA levels in cells and villi were determined by chromogenic assay, ELISA, immunohistochemistry and reverse-transcription PCR (RT-PCR). RESULTS: Fibrin but not fibrinogen, thrombin or fibronectin caused increased trophoblast proliferation and spreading. Trophoblasts cultured on factor XIII (FXIII) depleted fibrin caused their increased proliferation and spreading, associated with cross-linking. FXIII addition further increased this effect, while cell culturing on active FXIII without fibrin retained cellular proliferation. Decreased TF activity, antigen and RNA expression were demonstrated in fibrin-cultured trophoblasts and villi explants, compared to matrigel explants. CONCLUSION: Results obtained demonstrate distinct mechanisms underlying fibrin cross-linking, which can affect trophoblast proliferation. The excess of fibrin deposits may be limited by the decrease in TF levels, thus enabling adequate placental perfusion. These findings demonstrate fibrin importance for placental repair and may partly explain poor pregnancy outcome associated with certain fibrinogen/fibrin abnormalities and FXIII deficiency.

Presence of Integrin alpha(IIb)beta 3 in early gestation human trophoblasts: possible involvement of fibrin as a matrix ligand.

INTRODUCTION: Various integrins are expressed on trophoblast membrane and participate in placenta anchoring, cell adhesion , migration and vascular development. Although integrin alpha IIb beta 3 is known to be associated mainly with megakaryocytes and platelets, here we show for the first time its presence in human trophoblasts and in ex-vivo placental villi. METHODS: Trophoblasts were isolated and cultured from early term placentas (9-13 weeks of gestation). The presence of alpha IIb beta 3 integrin was demonstrated in the cells by means of immunofluorescence , flow cytometry and Western blot analysis and in villous placentas by immunohistochemistry. Interaction of trophoblasts with fibronectin and fibrin was evaluated by adhesion assays. RESULTS: Expression of alpha IIb protein in trophoblasts decreased with time in culture and was more prominent when cells were cultured on fibrin as compared to fibronectin. Presence of alpha IIb on villous trophoblasts was documented in placenta sections of 8 and 15 weeks of gestation but not in term placentas. Trophoblasts adhesion to fibrin was reduced by 45% in the presence of blocking antibodies for alpha IIb, but only by 10% to fibronectin, suggesting fibrin as a ligand for trophoblast alpha IIb beta 3. CONCLUSIONS: Our finding demonstrate the transient presence and participation of alpha IIb beta 3 in the orchestrated adhesion molecules of trophblasts and villi. The increased adhesion and expression of alpha IIb in trophoblasts on fibrin suggest its involvement as a ligand in the extracellular milieu of the early fetal placenta.

Involvement of Heparanase in early pregnancy losses.

BACKGROUND: Heparanase cloned from and abundant in the placenta is implicated in cell invasion, tumor angiogenesis and metastasis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Heparanase was found to up-regulate tissue factor (TF) expression (Nadir et al., JTH, 2006) and interact with tissue factor pathway inhibitor (TFPI) on the cell surface, leading to dissociation of TFPI from the cell membrane resulting in increased cell surface coagulation activity (Nadir et al., TH, 2008). Herein, we investigated the role of heparanase in the placenta, focusing on its effect on TF, TFPI, TFPI-2, and vascular endothelial growth factor (VEGF)-A. METHODS: Twenty formalin embedded placenta samples of abortions (weeks 6-10) were studied applying real time RT-PCR and immunostaining. Ten cases were miscarriages of women with thrombophilia and recurrent fetal losses, and ten control cases were pregnancy terminations. JAR (human choriocarcinoma trophoblasts) cells were transfected with full-length heparanase cDNA or incubated with active (50+8 kDa) recombinant heparanase and the effects on TF, TFPI, TFPI-2 and VEGF-A were examined using real time RT-PCR and immunoblotting. RESULTS: Sections obtained from miscarriages revealed increased (2-3-folds) levels of heparanase, VEGF-A and TFPI-2 compared to placentas from controls in maternal as well as in fetal placenta elements. JAR cells overexpressing heparanase or incubated with exogenous recombinant heparanase exhibited a 2-3-fold increase in TFPI and TFPI-2 in cell lysates both at the protein and mRNA levels, with no detectable effect on VEGF-A and TF levels. Accumulation of TFPI and TFPI-2 in the cell culture medium was increased 4-6-folds, exceeding the observed induction of TFPI and TFPI-2 gene transcription. CONCLUSIONS: These results indicate a regulatory effect of heparanase on TFPI and TFPI-2 in trophoblasts, suggesting a potential involvement of heparanase in early miscarriages.

Diagnosis of pregnancy-associated uterine venous plexus thrombosis on the basis of transvaginal sonography.

OBJECTIVE: To describe the sonographic signs of uterine venous plexus thrombosis. METHODS: Four pregnant patients had a diagnosis of uterine venous plexus thrombosis in the first half of gestation. The diagnosis was based on transvaginal sonography only in 3 cases, and the fourth had magnetic resonance imaging corroboration. RESULTS: All 4 patients had similar sonographic features of uterine venous plexus thrombosis on transvaginal sonographic examination. The thrombi within the dilated veins were shown as elongated echogenic structures along the lumen that appeared round on transverse views of the affected veins. They showed swinging movements provoked by gentle transducer pressure. Power and color Doppler sonography enhanced the uterine venous plexus thrombosis diagnosis by showing blood flow around the thrombi. There were no signs of thromboembolic disease. Sonographic findings in deep leg veins and iliac veins were normal in all cases. Complete thrombophilia studies did not reveal any abnormalities. The uterine venous plexus thrombosis could not be detected on transabdominal sonography and was shown better by transvaginal sonography compared with magnetic resonance imaging. During 3 months of anticoagulation therapy, the thrombi gradually disappeared in all cases. CONCLUSIONS: Focusing on the pelvic veins while performing a transvaginal sonographic study during pregnancy may reveal important findings, which may have clinical implications. The therapeutic treatment of uterine venous plexus thrombosis is controversial and still empirical.

Cervical Shirodkar cerclage may be the treatment modality of choice for cervical pregnancy.

BACKGROUND: Our objective was to evaluate the use of cervical suture in cervical pregnancy. METHODS AND RESULTS: All cases of cervical pregnancy diagnosed and treated in the gynaecological department at the Sheba Medical Center between 1994-2000 were included in the study. Eight such cases were diagnosed. The first four cases were treated medically. The last four cases (the study group) of cervical pregnancy, including one case of heterotopic pregnancy, were treated successfully with placement of Shirodkar cerclage. CONCLUSION: Cervical cerclage may be considered as the treatment of choice in cases of cervical pregnancies. It may be the only therapy in cases of heterotopic pregnancies (intrauterine and cervical pregnancy).