Search

BACKGROUND Preeclampsia (PE) is a hypertensive disorder during pregnancy with endotheliosis leading to occlusion of renal perfusion with an impact on the glomerular filtration barrier. We therefore analyzed the role of intrarenal resistance indices in the renal interlobular arteries measured by Doppler ultrasound as a diagnosis of PE. METHODS Women with preeclampsia (n = 24; mean blood pressure/24 h = 145 ± 11/ 93 ± 7 mm Hg; mean proteinuria = 5.63 ± 1.0 g/24 h) were compared against a group of healthy pregnant women (n = 24). All patients underwent a Doppler ultrasound of the intrarenal arteries between the 24th week of gestation and the 5th week postpartum. Several risk factors for PE, as well as the arterial resistive indices of the Arteriae uterinae and the Arteria umbilicalis, were monitored in parallel. RESULTS The intrarenal resistive index (mean ± SD = 0.63 ± 0.05 in women with preeclampsia vs. 0.59 ± 0.056 in healthy pregnant women; P < 0.003), the pulsatile index (mean ± SD = 1.15 ± 0.19 in women with preeclampsia vs. 0.92 ± 0.13 in healthy pregnant women; P < 0.0001), and the end diastolic flow velocity (mean ± SD = 14.16 ± 4.75 cm/s in women with preeclampsia vs. 10.67 ± 2.68 cm/s in healthy pregnant women, P < 0.006) were elevated in patients with PE, as were the arterial resistive indices of the Aa. uterinae and A. umbilicalis. The intrarenal resistive indices correctly classified 84.2% of the women as having PE. CONCLUSIONS Intrarenal resistive indices are a significant classifier of PE, providing the possibility to predict nephropathy. They could be a prognostic tool for cardiovascular comorbidity in PE patients even after delivery.

Early molecular interaction between embryo and mother, involving chemoattractants, especially chemokine CXC-motif ligand 1 (CXCL1)(2), determines the pregnancy outcome. So far nothing is known about the signalling cascades of CXCL1 expression in human decidua. The aim of the study was to identify signalling cascades mediating the CXCL1 expression in human decidua incubated with IL-1β as a major secretion product of the embryo. Therefore, decidualised endometrial stromal cells were incubated with IL-1β in a concentration- and time-dependent manner. The specificity of the IL-1β induced CXCL1 expression was verified by application of the IL-1 receptor antagonist, which opposed the binding of IL-1β to its receptor, leading to a dose dependent diminished to complete CXCL1 elimination. IL-1β signalling was investigated using inhibitors for MAPK, STAT3 and JNKinase cascades. The CXCL1 secretion of decidualised endometrial cells was measured by ELISA. The MAPK signalling cascade was explored by western blot analysis of ERK and NFκB p65(3) as well as phospho ERK and pp65 activation by IL-1β in detail. A statistical significant increase in CXCL1 mRNA- and protein-expression after incubation with 0.1ng/ml IL-1β after 48h was detected. CXCL1 protein secretion could be completely prevented by IL-1 receptor antagonist treatment. Only inhibition of the MAPKinase pathway resulted in a statistically significant decrease of CXCL1 protein secretion. Initiation of the MAPK pathway depicted by phospho ERK activation started as early as 2min after coincubation of decidualised endometrial stromal cells with IL-1β. Activation of NFκB p65 could be measured within 15min of IL-1β incubation in decidualised endometrial stromal cells. CXCL1 is a target for the embryos' secretion product IL-1β in decidualised endometrial stromal cells during the peri-implantation period. IL-1β's rapid effect on CXCL1 synthesis is uniquely mediated via the MAPK-signalling cascade and the activation of CXCL1s' transcription factor NFκB p65.

Fragestellung: Bei der Endometriose handelt es sich um eine benigne hormonabhangige Erkrankung der Frau im reproduktionsfahigen Alter. Ziel einer operativen Therapie ist die chirurgische Entfernung der Endometrioseherde. Medikamentose Therapieansatze zielen ab auf eine Suppression des Ostrogens auf ein postmenopausales Hormonniveau, was durch den Einsatz von Gonadotropin-Releasing-Hormon-(GnRH-)Analoga erreicht werden kann. Der therapeutische Nutzen einer systemischen Endometriosetherapie ist jedoch limitiert und mit signifikanten Nebenwirkungen vergesellschaftet. Aus diesem Grund besteht ein groser Bedarf an der Entwicklung einer spezifischen und zielgerichteten Therapie der Endometriose. Ziel dieser Untersuchung war die Identifikation geeigneter Zielstrukturen. Material und Methodik: Vergleichende Untersuchungen der Expression von VEGF, Midkine, Angiopoietin-2 und Heparanase erfolgten in Endometriose-Stroma und ‐drusenzellen, eutopem Endometrium und makroskopisch unauffalligen Peritonealgewebeproben mittels immunhistochemischer Untersuchungen und RT‐PCR. Anschliesend wurde die Expressionsstarke von VEGF und Heparanase in oberflachlichen peritonealen Endometrioseherden und tief infiltrierenden Endometrioseknoten immunhistochemisch untersucht. Zur Untersuchung des therapeutischen Einsatzes wurde ein VEGF-gesteuertes rekombinantes Adenovirus (AdVEGFE1) als Vektor eingesetzt. Untersucht wurden die spezifische Induktion von Apoptose in vitro sowie die Biodistribution in einem Mausmodell. Ergebnisse: VEGF und Heparanase werden in Endometriosezellen signifikant starker exprimiert als in eutopem Endometrium oder makroskopisch unauffalligen Peritonealgewebeproben. Weiterhin zeigen die korrespondierenden Promotoren beider Gene eine starke Expression in peritonealen Endometrioseherden. Eine Korrelation der Expressionsstarke mit dem Schweregrad der Erkrankung lies sich jedoch nur fur VEGF nachweisen. In Untersuchungen zur therapeutischen Anwendung eines VEGF-gesteuerten adenoviralen Vektors zeigt AdVEGFE1 in vitro eine Induktion von Apoptose in aufgereinigten kultivierten Endometriosezellen von Patientinnen. Das rekombinante konditional replizierende Adenovirus Ad5VEGFE1 zeigt eine effiziente Apoptoseinduktion in vitro und im Biodistribution-Mausmodell eine nur schwache Infektion des eutopen Endometriums und der Leber. Schlussfolgerung: VEGF ist eine geeignete Zielstruktur zur Entwicklung einer zielgerichteten Therapie der Endometriose.

Despite advances in therapy, metastatic breast carcinoma maintains a disappointing 5-year survival rate of 20.4 %. Hence, there is a need for alternative and complementary therapeutic strategies. In this regard oncolytic virotherapy is a promising approach that uses mainly conditionally replicative viruses (CRAds) to selectively kill tumor cells by exploiting the lytic property of virus replication. Adenovirus-based vectors have emerged as leading candidates for in vivo virotherapy. However intravenously injected CRAds have shown only limited antitumor efficacy due mainly to low viral infectivity and insufficient viral delivery to the sites of tumor. Based on recent findings that systemically administered human mesenchymal stem cells (hMSCs) home to breast cancer metastasis of the lung, our investigation assesses the possibility of using hMSCs as cellular virus carriers to pulmonary metastases of breast cancer. HMSCs were transduced with CRAds. In a SCID mouse xenograft model we established breast cancer pulmonary metastatic disease to examine the effects of intravenously injected CRAd-loaded hMSCs versus CRAds alone. In mice treated with CRAd-loaded hMSCs the transduced stem cells homed to the metastatic lesions and led to reduced tumorgrowth and extended survival compared to mice treated with CRAds alone. We concluded that systemically administered hMSCs transduced with CRAds have the ability to home in on and kill metastatic breast cancer cells; these findings validate the feasibility of using hMSCs as cellular vehicles for the targeted therapy of metastatic breast cancer with CRAds.

Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.

Endometrial epithelial cells are known to undergo apoptosis during trophoblast invasion. We postulate that the cell surface molecule Syndecan-1 which is expressed on endometrial cells and syncytiotrophoblast is important for implantation in general and especially for induction of maternal cell apoptosis during trophoblast invasion because Syndecan-1's influence on apoptotic susceptibility of cancer cells is already described in the literature. Using the human endometrial epithelial cell line RL95-2, a new stable cell line with Syndecan-1 knockdown was generated. Via antibody array analysis, a significant decrease in the expression of anti-apoptotic proteins like inhibitors of apoptosis, Clusterin, heme oxygenase (HO-2), heat shock protein (HSP)27 and -70 and Survivin due to the Syndecan-1 knockdown was discovered. Correspondingly, active Caspase-3 as an indicator for apoptosis was increased more severely in these cells compared with unmodified RL95-2 after treatment with implantation-related stimuli, which are the cytokines interleukin-1β, interferon-γ, tumor necrosis factor-α and transforming growth factor-β1 and an anti-Fas antibody. Furthermore, a treatment with a combination of all factors caused a higher Caspase-3 induction compared with each single treatment. These results demonstrate that Syndecan-1 is involved in the control of apoptosis in RL95-2 cells and therefore may affect the fine tuning of apoptosis in endometrial epithelium regulating the embryo's invasion depth as a crucial step for regular implantation followed by successful pregnancy.

Purpose In early pregnancy the dialogue between maternal endometrium and embryo is a key process in establishing a receptive decidua and placental network. Decidual ISG15 induction is thought to promote pregnancy maintenance and development. ISG15 is involved in RNA splicing, cytoskeletal organization, stress response and further intracellular processes. Methods ISG15 expression was examined immunohistologically in paraffin-embedded human placental and decidual tissue samples of all pregnancy trimesters on adjacent sections (first trimester n = 5, second n = 5, third n = 3). Samples were processed using a protocol applying a rabbit polyclonal ISG15 antibody. A mouse monoclonal cytokeratin seven antibody was utilized to identify the different placental departments and decidual glands. Staining results and anatomical features were evaluated blindly with strict rating criteria. Results ISG15 expression was identified in first and second trimester tissue samples. ISG15 localized especially to the extravillous cytotrophoblasts in the maternal wall and in maternal blood vessel. Expression was detected in cytotrophoblast progenitor cells in the placental villi and the cell column with a maximum in the first trimester. The syncytial layer stained positive in first and second trimester samples. Third trimester samples showed no expression of ISG15 at all. Conclusions ISG15 abundance in the human placenta is an interesting finding, with implications for placental development, fetal growth and potential defense mechanism against infections. The maximal expression of ISG15 in the first and second trimester of pregnancy suggests that ISG function is needed when placental and embryo development is enormous and embryo susceptibility to external influences is high.

Background. The aim of this paper was to determine the influence of differentin vitroculture media on mRNA expression of Hedgehog genes,il-6,and important genes regarding reactive oxygen species in single mouse embryos.Methods. Reverse transcription of single embryos either culturedin vitrofrom day 0.5 until 3.5 (COOK’s Cleavage medium or Vitrolife’s G-1 PLUS medium) orin vivountil day 3.5post coitum. PCR was carried out forβ-actinfollowed by nested-PCR forshh, ihh, il-6, nox, gpx4, gpx1,andprdx2.Results. The number of murine blastocysts cultured in COOK medium which expressedil-6, gpx4, gpx1,andprdx2mRNA differed significantly compared to thein vivogroup. Except fornox, the mRNA profile of the Vitrolife media group embryos varied significantly from thein vivoones regarding the number of blastocysts expressing the mRNA ofshh, ihh, il-6, gpx4, gpx1andprdx2.Conclusions. The present study shows that differentin vitroculture media lead to different mRNA expression profiles during early development. Even the newly developedin vitroculture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

Fragestellung: Die rupturierte Extrauteringraviditat (EUG) ist nach wie vor eine der haufigsten Todesursachen junger Frauen. Fruhere Entzundungen des Unterleibs als Hauptursache fur die Entstehung ektoper Schwangerschaften pradisponieren uber postinfektiose, mechanische Hindernisse im Eileiter. In jungerer Zeit wird dazu noch evident, dass auch eine Kinderwunschbehandlung im Sinne einer in-vitro Fertilisation (IVF) das Risiko fur eine EUG erhoht. Entzundungsmediatoren wie CXCL8 (IL-8) lassen sich beim Menschen durch proinflammatorische Zytokine wie IL-1β induzieren, das nicht nur einen Infektionsmarker, sondern auch ein Hauptsekretionsprodukt des Embryos darstellt. Unsere Arbeitsgruppe konnte bereits zeigen, dass im menschlichen Eileiter der Sekretionsphase die Expression von CXCL8 im Gegensatz zur Dezidua herunterreguliert ist (Hess et al., 2011, in Begutachtung). Wir postulieren daher einen direkten hormonellen Einfluss der Chemokinexpression auf den regelrechten Transport und die Implantation des Embryos und vermuten daruber hinaus eine hormonelle Komponente bei der Entstehung der EUG. Da menschliche Eileiter fur Forschungszwecke kaum zur Verfugung stehen, ist es unser Ziel, in primaren Eileiter-Epithelzellen des Schweins die biochemischen Bedingungen einer EUG nachzuahmen und in diesem System mogliche hormonelle Auswirkungen einer IVF- Behandlung auf den Eileiter zu untersuchen. Methodik: Primare porcine Eileiter-Epithelzellen wurden aus Schlachthofmaterial isoliert und uber 5 Tage in Ham's F12-Medium kultiviert. Die Epithelzellen wurden nach Vorbehandlung mit einer im IVF-Verfahren ublichen Estradiolkonzentration von 3000 pg/mL uber 48 Stunden mit porcinem IL-1β inkubiert und anschliesend der Kulturuberstand gewonnen. Analog hierzu wurde mit hormonfrei kultivierten Kontrollen verfahren. Die CXCL8-Konzentration im Uberstand wurde durch einen kommerziell erhaltlichen ELISA quantifiziert. Ergebnisse: CXCL8 lies sich sowohl auf mRNA-Ebene als auch immunhistochemisch in den porcinen Eileiter-Epithelzellen nachweisen. Fur das sezernierte Chemokin konnte daruber hinaus eine signifikant gesteigerte Expression nach Inkubation mit IL-1β im Zellkulturuberstand mittels ELISA detektiert werden. Die Vorinkubation mit einer erhohten Estradiolkonzentration ergab jedoch keine weitere Steigerung der CXCL8-Expression gegenuber den hormonfrei kultivierten Kontrollen. Schlussfolgerung: Die signifikante Expressionssteigerung von CXCL8 in Eileiter-Epithelzellen des Schweins nach Inkubation mit IL-1β weist auf eine direkte Reaktion des Epithels auf die Anwesenheit des embryonalen Surrogatmarkers hin. Da die erhohte Estradiolkonzentration im Medium keine Auswirkung auf die CXCL8-Expression hatte, sind daruber hinaus weitere Versuche erforderlich, um die Ursachen einer erhohten Rate ektoper Schwangerschaften nach IVF-Behandlungen zu ergrunden. Die gute Verfugbarkeit von Eileitern des Schweins und der vergleichbare Sexualzyklus von Mensch und Schwein machen die primare Zellkultur von porcinem Eileiter-Epithel jedoch zu einem wertvollen Modell zur Erforschung tubarer Pathologien.

A successful implantation of a mammalian embryo into the maternal endometrium depends on a highly synchronized fetal–maternal dialogue involving chemokines, growth factors, and matrix-modifying enzymes. A growing body of evidence suggests an important role for proteinases playing a role in matrix degeneration and enhancing the embryo's invasive capacity and influencing the mother's immunological status in favor of the conceptus. This study focused on the expression of cathepsin S (CTSS) and its inhibitors in the murine fetal–maternal interface as well as the detection of the cellular sources of either proteinase and inhibitors. Nested RT-PCR for detection of embryonic mRNAs, immunohistochemistry of maternal and fetal tissues in B6C3F1 mice, and FACS analysis for determination of immunocompetent cell population were applied. This study shows that the cysteine proteinase CTSS is upregulated in the stroma of the implantation site, and that pregnancy induces an influx of CTSS-positive uterine natural killer cells. Compared to maternal tissues, the CTSS inhibitors cystatin F and C, but not the proteinase itself, are expressed in blastocysts. In conclusion, CTSS underlies a hormonal regulation in the maternal tissue and therewith most likely supports the embryonic implantation. The invading embryo regulates the depth of its own invasion through the expression of the cathepsin inhibitors and furthermore, interleukin-6 to activate CTSS in maternal tissues. Additionally, the observed decrease in CD3+cells leads to the hypothesis that cells of the cytotoxic T-cell group are down-regulated in the decidua to support the implantation and ensure the survival of the embryo.

Objective To report the case of a Nigerian patient who suffered from sterility and underwent abdominal laparotomy to remove an adnexal tumor. Histologic analysis revealed Schistosoma haematobium ova. Design Case report. Setting Multidisciplinary group practice and teaching hospital. Patients One patient who underwent an abdominal laparotomy to remove an adnexal tumor. Intervention(s) In vitro fertilization and adnexal tumor resection via laparotomy. Main Outcome Measure(s) Treatment of the adnexal tumor affecting the reproductive health. Result(s) Histologic analysis performed on the tissue removed at surgery revealed fibrous patches around aggregates of calcified Schistosoma haematobium eggs in the fallopian tube wall. Conclusion(s) Schistosomiasis needs to be considered as a differential diagnosis of female infertility and sterility.

Based on the reported tocolytic action of the hormone relaxin (RLX) in rodents, locally produced in reproductive tissues and the corpus luteum in mammals, the present study aimed to evaluate the influence of RLX on contraction-mediating cyclooxygenases-1 and -2 (COX) and the contractile prostaglandin PGE(2) in human myometrial and decidual cells. Primary cultured cells were obtained from uteri and placentas of term and preterm women undergoing elective caesarean section.In vitro culture of primary myometrial and decidual cells, immunocytochemistry, reverse transcription and real-time PCR, Western blot, ELISA.We demonstrate for the first time an activating effect of RLX for human COX-1 and COX-2 in primary myometrial and decidual cells in vitro.These effects might potentially contribute to birth-associated induction of contractions in vivo.

International audience; The human fallopian tube provides the environment for the first 5 days of embryonic development . The IL-1 system is involved in human embryo implantation. This study aimed to investigate IL-1β, IL-1ra and IL-1R tI expression within the length of the human fallopian tube on mRNA- and protein-level in samples from proliferative versus secretory phase, postmenopause samples (PMP) and samples from intra- (IUP) and extrauterine (EUP) pregnancies to examine possible spatial and hormonal induced changes (fimbrial, ampullary and isthmic tube segments). On mRNA-level, IL-1β was expressed in all samples except in PMP. IL-1R tI could be detected in all samples whereas IL-1ra was only expressed in secretory phase and the IUP sample. Immunohistochemically we could detect IL-1β and IL-1R t1 protein in all proliferative and secretory phase samples with maximum intensity in secretory phase samples whereas IL-1ra was expressed in secretory phase samples only. Overall no spatial but temporal differences possibly due to hormonal changes could be observed suggesting a precise regulation of the IL-1 system, especially for IL-1ra and moreover a stable molecular architecture within the full length of the fallopian tube.

Human embryo invasion and implantation into the inner wall of the maternal uterus, the endometrium, is the pivotal process for a successful pregnancy. Whereas disruption of the endometrial epithelial layer was already correlated with the programmed cell death, the role of apoptosis of the subjacent endometrial stromal cells during implantation is indistinct. The aim was to clarify whether apoptosis plays a role in the stromal invasion and to characterize if the apoptotic susceptibility of endometrial stromal cells to embryonic stimuli is influenced by decidualization and Syndecan-1. Therefore, the immortalized human endometrial stromal cell line St-T1 was used to first generate a new cell line with a stable Syndecan-1 knock down (KdS1), and second to further decidualize the cells with progesterone. As a replacement for the ethically inapplicable embryo all cells were treated with the embryonic factors and secretion products interleukin-1β, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1 and anti-Fas antibody to mimic the embryo contact. Detection of apoptosis was verified via Caspase ELISAs, PARP cleavage and Annexin V staining. Apoptosis-related proteins were investigated via antibody arrays and underlying signaling pathways were analyzed by Western blot. Non-decidualized endometrial stromal cells showed a resistance towards apoptosis which was rescinded by decidualization and Syndecan-1 knock down independent of decidualization. This was correlated with an altered expression of several pro- and anti-apoptotic proteins and connected to a higher activation of pro-survival Akt in non-differentiated St-T1 as an upstream mediator of apoptotis-related proteins. This study provides insight into the largely elusive process of implantation, proposing an important role for stromal cell apoptosis to successfully establish a pregnancy. The impact of Syndecan-1 in attenuating the apoptotic signal is particularly interesting in the light of an already described influence on pregnancy disorders and therefore might provide a useful clinical tool in the future to prevent pregnancy complications provoked by inadequate implantation.

Lymph node biopsy is not only important as a prognostic factor, but also influences therapy. However, axillary lymphadenectomy is often accompanied by high morbidity. The sentinel lymph node biopsy (SLN) should reduce the morbidity, but give the same prognostic value. In 97.5% cases metastasis occurs in lymph nodes (LN) of levelI first, and only less than 3% directly in LN level II. The first draining LN can be identified either by radioactive material or colouring technique.

Background Successful embryonic implantation depends on a synchronized embryo-maternal dialogue. Chemokines, such as chemokine ligand 1 (CXCL1), play essential roles in the maternal reproductive tract leading to morphological changes during decidualization, mediating maternal acceptance towards the semi-allograft embryo and induction of angiogenesis. Chemokine binding to their classical G-protein coupled receptors is essentially supported by the syndecan (Sdc) family of heparan sulfate proteoglycans. The aim of this study was to identify the involvement of Sdc-1 at the embryo-maternal interface regarding changes of the chemokine and angiogenic profile of the decidua during the process of decidualization and implantation in human endometrium. Methods A stable Sdc-1 knock-down was generated in the immortalized human endometrial stromal cell line St-T1 and was named KdS1. The ability of KdS1 to decidualize was proven by Insulin-like growth factor binding 1 (IGFBP1) and prolactin (PRL) confirmation on mRNA level before further experiments were carried out. Dot blot protein analyses of decidualized knock-down cells vs non-transfected controls were performed. In order to imitate embryonic implantation, decidualized KdS1 were then incubated with IL-1beta, an embryo secretion product, vs controls. Statistical analyses were performed applying the Student's t-test with p < 0.05, p < 0.02 and p < 0.01 and one way post-hoc ANOVA test with p < 0.05 as cut-offs for statistical significance. Results The induction of the Sdc-1 knock-down revealed significant changes in cytokine and angiogenic factor expression profiles of dKdS1 vs decidualized controls. Incubation with embryonic IL-1beta altered the expression patterns of KdS1 chemokines and angiogenic factors towards inflammatory-associated molecules and factors involved in matrix regulation. Conclusions Sdc-1 knock-down in human endometrial stroma cells led to fulminant changes regarding cytokine and angiogenic factor expression profiles upon decidualization and imitation of embryonic contact. Sdc-1 appears to play an important role as a co-receptor and storage factor for many cytokines and angiogenic factors during decidualization and implantation period, supporting proper implantation and angiogenesis by regulation of chemokine and angiogenic factor secretion in favour of the implanting embryo.