Expression of matrix metalloproteinases and their inhibitors at the implantation site in ampullary ectopic pregnancies.

OBJECTIVE: This study investigated the qualitative and semi-quantitative expression of metalloproteinases (MMP) and their tissue inhibitors (TIMP) in trophoblastic tissue during ampullary ectopic pregnancies and correlated that expression with the degree of tubal invasion. STUDY DESIGN: It is a prospective study that included 34 patients diagnosed with ampullary tubal pregnancy who underwent salpingectomy. A histological evaluation of the depth of trophoblastic invasion in the tubes obtained was performed. Subsequently, the expression of the MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2 and TIMP-3 markers was qualitatively and semi-quantitatively evaluated by indirect immunohistochemistry. In addition, the degree of trophoblastic invasion was correlated with the expression of each marker and with the metalloproteinase/inhibitor ratios. RESULTS: MMP-2 (11.2 %; 3.6-17.9) was the marker with greater expression at the implantation site, both in the qualitative and semi-quantitative assessment, while MMP-9 (2.23 %; 0.2-5.4) and TIMP-3 (2.53 %; 0.1-15.3) were only weakly expressed. CONCLUSION: There was wide variation in expression among the markers and metalloproteinase/inhibitor ratios studied compared to the degrees of invasion.

A multi-centre, double-blind, placebo-controlled, randomised trial of combination methotrexate and gefitinib versus methotrexate alone to treat tubal ectopic pregnancies (GEM3): trial protocol.

BACKGROUND: Tubal ectopic pregnancy (tEP) is the most common life-threatening condition in gynaecology. Treatment options include surgery and medical management. Stable women with tEPs with pre-treatment serum human chorionic gonadotrophin (hCG) levels < 1000 IU/L respond well to outpatient medical treatment with intramuscular methotrexate. However, tEPs with hCG > 1000 IU/L can take significant time to resolve with methotrexate and require multiple outpatient monitoring visits. In pre-clinical studies, we found that tEP implantation sites express high levels of epidermal growth factor receptor. In early-phase trials, we found that combination therapy with gefitinib, an orally active epidermal growth factor receptor antagonist, and methotrexate resolved tEPs without the need for surgery in over 70% of cases, did not cause significant toxicities, and was well tolerated. We describe the protocol of a randomised trial to assess the efficacy of combination gefitinib and methotrexate, versus methotrexate alone, in reducing the need for surgical intervention for tEPs. METHODS AND ANALYSIS: We propose to undertake a multi-centre, double-blind, placebo-controlled, randomised trial (around 70 sites across the UK) and recruit 328 women with tEPs (with pre-treatment serum hCG of 1000-5000 IU/L). Women will be randomised in a 1:1 ratio by a secure online system to receive a single dose of intramuscular methotrexate (50 mg/m2) and either oral gefitinib or matched placebo (250 mg) daily for 7 days. Participants and healthcare providers will remain blinded to treatment allocation throughout the trial. The primary outcome is the need for surgical intervention for tEP. Secondary outcomes are the need for further methotrexate treatment, time to resolution of the tEP (serum hCG ≤ 15 IU/L), number of hospital visits associated with treatment (until resolution or scheduled/emergency surgery), and the return of menses by 3 months after resolution. We will also assess adverse events and reactions until day of resolution or surgery, and participant-reported acceptability at 3 months. DISCUSSION: A medical intervention that reduces the need for surgery and resolves tEP faster would be a favourable treatment alternative. If effective, we believe that gefitinib and methotrexate could become standard care for stable tEPs. TRIAL REGISTRATION: ISRCTN Registry ISRCTN67795930 . Registered 15 September 2016.

Oxidative status and serum prolidase activity in tubal ectopic pregnancy.

OBJECTIVE: To determine the oxidative status and serum prolidase activity in tubal ectopic pregnancy and to see if there was any association between them. MATHODS: The cross-sectional study was conducted during 2009 and 2010 at the Departments of Obstetrics and Gynaecology and Clinical Biochemistry under the Faculty of Medicine, Harran University, Turkey. It comprised 40 patients with tubal ectopic pregnancies and 42 women with healthy pregnancies. Serum prolidase activity was measured spectrophotometrically. Oxidative status was determined using total antioxidant capacity. SPSS 11.5 was used for statistical analysis. RESULTS: Total antioxidant capacity levels were lower in the ectopic pregnancy group than the healthy group (p < 0.018), whereas total oxidant status, oxidative stress index and prolidase activity were higher (p < 0.05). CONCLUSION: Ectopic pregnancy may be associated with increased serum prolidase activity and oxidative stress, and this association may help to provide a better understanding about the pathogenesis of ectopic pregnancy.

Immunohistochemistry of adhesion molecules, metalloproteinases and NO-synthases in extravillous trophoblast of tubal pregnancy.

Trophoblast invasion in uterine pregnancy is fine-tuned for the remodelling of the uterine wall and its vascularization. Tubal pregnancy, which occurs in a limited number of patients, involves a dramatic trophoblast invasion in a context of a poor decidualization. By studying the histology of the extravillous trophoblast (EVC) in the anchoring villi, the Ki67 labelling, the location of several adhesion markers (cytokeratin-7, alpha1, alpha6, alphaV, beta1, beta4 integrin subunits and E-cadherin, V/E-cadherin), metalloproteinases (MMP-2, 9 and11), NOS2 and 3, we aimed to detect the specificity of tubal compared to intrauterine pregnancies. No difference could be observed between meso or anti-salpingial trophoblast proliferation or invasion using Ki67. Cytokeratin-7 allowed detection of spindle-shape EVCs and we identified some decidualized stromal cells. Integrins alpha1, beta1 and alphaV, and V/E-cadherin were expressed mainly in the distal EVC correspondingly to intrauterine pregnancy, with a poor expression of alpha1. Integrins alpha6 and beta4, E-cadherin were detected in the distal EVC in contrast to uterine pregnancy. MMP-2, 9, 11 were also shown in distal EVC. NOS2 and 3 labelled the perivascular EVC and NOS3 the endothelial cells of the tubal vessels. These changed distributions of adhesion molecules and MMP together with that of the basic and inducible NOS expressions could be related to mechanical effects in superficial implantation or to a failure of decidualization in tubal pregnancies.

Dynamic expression of matrix metalloproteinases (MMP-2, -9 and -14) and the tissue inhibitors of MMPs (TIMP-1, -2 and -3) at the implantation site during tubal pregnancy.

Matrix metalloproteinases (MMPs) are responsible for extracellular matrix (ECM) degradation, and their functions are regulated by tissue inhibitors of MMPs (TIMPs). The evidence for the roles of MMPs and TIMPs in implantation and placentation has remained insufficient in humans, especially during the early stages. Tubal pregnancy has some similarities to normal intrauterine pregnancy and therefore may provide a unique model for implantation studies. In the present study, the expression of MMP-2, -9 and -14, and TIMP-1, -2 and -3 at the feto-maternal interface during tubal pregnancy was examined by immunohistochemistry and in situ hybridization. We found that MMP-9 and TIMP-1, -2 and -3 are produced by all types of extravillous cytotrophoblast (EVCT) cells, while MMP-2 and -14 mainly exist in distal column cytotrophoblast (CCT) cells and invasive EVCT cells. Meanwhile, the intensity of MMP-14 and TIMP-1 and -2 increased along the invasive pathway toward maternal interstitium. In addition, MMP-2, -9 and -14 and TIMP-1, -2 and -3 were all detected in the villous CT (VCT) cells. Furthermore, both the mRNA level and immunoreactivity of MMP-9, TIMP-1 and -3 increased, while those of TIMP-2 decreased concurrent with the progression of pregnancy during weeks 3-9. The unique expression pattern of various MMPs and TIMPs at the feto-maternal interface suggests that they may have roles in regulating the controlled invasion of trophoblasts during implantation and placentation. Meanwhile, the study provides a better understanding of the mechanisms involved in cellular events during human pregnancy, especially at the initiation stage of implantation.

Expression of P450 aromatase and 17beta-hydroxysteroid dehydrogenase type 1 at fetal-maternal interface during tubal pregnancy.

Steroidogenesis in the placenta has been studied widely, but little is known about steroid metabolism in ectopic pregnancy. Previous studies have indicated that trophoblast invasion and placentation in the uterus and the fallopian tube may be controlled by similar mechanisms. As far as 17beta-estradiol (E(2)) production is concerned, it has been well demonstrated that its biosynthesis in the placenta involves the action of P450 aromatase (P450arom) and 17beta-hydroxysteroid dehydrogenase type 1 (17HSD1). The purpose of this study was to characterize the expression pattern of P450arom and 17HSD1 at the fetal-maternal interface, particularly in various trophoblast cells, in tubal pregnancy. Using in situ hybridization, P450arom mRNA was localized in syncytiotrophoblast (ST) cells, which are mainly responsible for hormone production during pregnancy, whereas no signal was detected in villous cytotrophoblast (VCT), column CT and extravillous CT (EVCT) cells. Immunohistochemical assays revealed that 17HSD1 is present in ST cells, a large portion of EVCT cells and 20% of column CT cells. On the other hand, no expression of 17HSD1 was detected in VCT cells. In addition, 17HSD1 was found in epithelial cells of the fallopian tube. Interestingly, the expression level of 17HSD1 in fallopian tube epithelium during tubal pregnancy was significantly higher than that during normal cycle. Our data provide the first evidence that normal and tubal pregnancies possess identical expression of P450arom and 17HSD1 in ST cells and therefore, similar E(2) production in the placenta. Further, the association of 17HSD1 with EVCT cells indicates that 17HSD1 perhaps play a role in trophoblast invasion. Finally, increased expression of 17HSD1 in epithelial cells of fallopian tube may lead to a local E(2) supply sufficient for the maintenance of tubal pregnancy.

Messenger RNA for membrane-type 2 matrix metalloproteinase, MT2-MMP, is expressed in human placenta of first trimester.

An intimately regulated cell surface activation of matrix metalloproteinases (MMPs) is believed to be of critical importance for the control of trophoblast invasion. A histological investigation of the expression and localization of three different MMPs, the membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP, MT2-MMP) and matrix metalloproteinase 2 (MMP-2/gelatinase A) was performed by in situ hybridization on consecutive sections from human placentae of first trimester pregnancies. Cytokeratin immunostaining identified trophoblast cells. Both normal and tubal implantation sites were studied. We observed a high degree of coexpression of MT2-MMP, MT1-MMP and MMP-2 mRNAs in single extravillous cytotrophoblasts that had invaded the endometrium and tubal wall. Furthermore, mRNAs for all three genes were also seen in cytotrophoblasts of cell islands. In contrast to this coexpression pattern, MT2-MMP expression was absent from cell columns and decidual cells, in which signals for MT1-MMP and MMP-2 mRNAs were seen. The present data on the cellular expression of MT2-MMP mRNA in placenta extend our knowledge of the proteolytic events that take place during early pregnancy. The data suggest that MT2-MMP, capable of activating MMP-2 in vitro, is involved in the invasion of extravillous cytotrophoblast, possibly related to the physiological activation of MMP-2.

Co-ordinated expression of MMP-2 and its putative activator, MT1-MMP, in human placentation.

The spatial expression of mRNA for matrix metalloproteinase 2 (MMP-2), its putative activator, the membrane-type 1 matrix metalloproteinase (MT1-MMP), and the MMP-2 substrate type IV collagen was investigated in human placentas of both normal and tubal ectopic pregnancies and in cyclic endometrium using in-situ hybridization. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. In both normal and tubal pregnancies MT1-MMP, MMP-2 and type IV collagen mRNA were highly expressed and co-localized in the extravillous cytotrophoblasts of anchoring villi, in cytotrophoblasts that had penatrated into the placental bed and in cytotrophoblastic cell islands. In addition, the decidual cells of normal pregnancies in some areas co-expressed MT1-MMP and MMP-2 mRNA, with moderate signals for both components. Fibroblast-like stromal cells in tubal pregnancies were positive for MMP-2 mRNA but generally negative for MT1-MMP mRNA. The consistent co-localization of MT1-MMP with MMP-2 and type IV collagen in the same subset of cytotrophoblasts strongly suggests that all three components co-operate in the tightly regulated fetal invasion process. The co-expression of MT1-MMP and MMP-2 mRNA in some of the decidual cells indicates that these cells are also actively involved in the placentation process.

Maternal serum creatine kinase as a biochemical marker of tubal pregnancy.

OBJECTIVE: To evaluate the role of serum creatine kinase as a possible marker for diagnosis of tubal pregnancy. METHODS: Five groups of patients were included in this prospective study: group A, 20 patients with tubal pregnancies; group B, 20 patients with missed abortions; group C, 20 patients with pelvic inflammatory disease; group D, 10 patients with acute appendicitis; group E (controls), 20 patients with normal pregnancies matched for age and gestation. Total creatine kinase levels were measured in the serum of all five groups. RESULTS: Creatine kinase levels were found to be > or =75 IU/l in all patients with tubal pregnancies, which was significantly higher than in the other four groups (P < 0.0001). CONCLUSION: Our results indicate that maternal serum creatine kinase can be an important biochemical marker in suspected tubal pregnancy.

[Tumor markers and creatine kinase in the differential diagnosis of tubal pregnancy]. / Tumormarker und Kreatinkinase zur Differentialdiagnose der Tubargravidität.

In the present study we tried to evaluate the cancer marker CA 19-9, CA 12-5, CA 15-3 as well as the enzyme creatine-kinase (CK) whether they are of any diagnostic value in cases of tubal pregnancies. 15 patients with laporoscopically verified tubal pregnancy were compared with 15 patients with normal intrauterine pregnancies of the same age. Except one case of elevated CK in a patient with tubal pregnancy all other values were within the normal range. We concluded, that tumor markers and CK are inefficient for preoperative differential diagnosis in cases of tubal pregnancies.

[Tubal pregnancy: lactate dehydrogenase as a differential diagnostic parameter]. / Tubargravidität: Laktatdehydrogenase als differentialdiagnostischer Parameter.

In a prospective study on 104 patients, who were admitted because of a suspected ectopic pregnancy (EU), Serum-lactate dehydrogenase (LDH) was evaluated. Referred to the final diagnose patients were divided in four groups: ectopic pregnancy (Group A), abortion (B), intrauterine pregnancy (C) and metrorrhagia or adnexitis (D). LDH values in groups B, C and D were within normal range, values in group A were significantly higher (p less than 0.001). Particularly the comparison "chronic EU" to incomplete abortion showed impressive results: LDH (U/l; mean +/- SD) 300.2 +/- 97.0:133.8 +/- 45.2 (p less than 0.001), Serum LDH seems to be a simple, rapid and reasonable non invasive parameter in pathologic early pregnancy classification.