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Introduction Functional disorders of the villous trophoblast may result in preeclampsia through the release of endothelial activating substances. Progranulin is an anti-inflammatory, pro-angiogenic cytokine with TNF-α antagonizing activity. The trophoblastic expression of progranulin is increased during preeclampsia. The aim of the study was to investigate the impact of placental progranulin synthesis on endothelial cell activation. Methods Placental progranulin expression was modified by transduction of an adenoviral vector. Primary isolated human umbilical venous endothelial cells (HUVECs) were incubated with conditioned medium of first trimester placental explants. Functional studies on HUVECs included assays for proliferation, viability, cytotoxicity and analyzes of Intercellular adhesion molecule-1 (ICAM-1) and E-selectin expression. Results Placental progranulin expression was more than 10-fold higher by using an adenoviral-mediated overexpression system (Ad.PGRN) compared to control vector (Ad.CTRL) and untreated controls. Incubation of HUVECs with conditioned placental medium revealed a dose-dependent increase of cytotoxicity, reduced cell proliferation and viability and resulted in an increase of ICAM-1 and E-selectin expression. Overexpression of progranulin (Ad.PGRN) antagonized the ICAM-1 expression induced by conditioned medium. However progranulin did not influence the effects on cell proliferation, viability, cytotoxicity and E-selectin expression in HUVECs. Discussion Regulation of gene expression in human placental explants is possible by usage of an adenoviral vector system. The increase of endothelial ICAM-1 expression following the incubation with placental conditioned medium was partly reversed by overexpression of placental progranulin. It is suggested that up-regulation of the placental progranulin expression is an endogenous anti-inflammatory mechanism that partially antagonizes the endothelial cell activation during preeclampsia.

We investigated the anticarcinogenic potential of green tea and its components epigallocatechin gallate (EGCG) and quercetin, as well as tamoxifen, on MCF-7 and MDA-MB-23 breast cancer cells. Using high-performance liquid chromatography, the quantity of EGCG and quercetin in green tea was analyzed. The receptor status of the cells was confirmed immunohistochemically. Various viability and cytotoxicity tests were later performed to investigate the effects of the substances. After incubating the cells with green tea extract, EGCG, quercetin and tamoxifen, a decrease in viability (MTT test) or proliferation (BrdU assay) was found in all cell tests with varying effects, depending on the assay used. The effects were similar in both cell lines. This work confirmed that EGCG and quercetin are contained in green tea and that both substances in pure form and as green tea have an anticarcinogenic effect on both estrogen receptor-positive and -negative breast cancer cells. This effect could also be demonstrated with tamoxifen in both cell lines (MTT and BrdU assays). These results suggest that the effects observed in these experiments are not generated only via estrogen receptor-mediated pathways.

Biological matrixes and synthetic meshes are increasingly used in implant-based breast reconstruction (IBBR). The objective was to test different materials used for internal support in IBBR in regards to biocompatibility and discuss possible limitations in a clinical context. In vitro investigations were performed on four relevant cell lines: Normal Human Dermal Fibroblasts (NHDF), Human White Preadipocytes (HWP), Endothelial cells (HDMEC) and Skeletal muscle cells (SkMC). A titanium-coated polypropylene mesh (TiLOOP® Bra), a partially resorbable mesh (SERAGYN BR®) and a porcine derived biologic matrix (Strattice™) were investigated. Test of cytotoxicity, cell proliferation and oxidative stress was performed. Real-time cell analysis was used to determine adhesion rate. Light- and scanning electron microscopy investigated cell migration. No relevant cytotoxicity was detected for any mesh or matrix. Good cell proliferation was observed in all materials with best results for NHDF and SkMC. For HWP and HDMEC decreased proliferation and adherence to the synthetic meshes and biologic matrix were observed. Real-time cell analysis of fibroblasts incubated with the corresponding material, showed increased impedance for the synthetic meshes. A morphologic cell change was observed within all materials. Scanning electron microscopy showed good cell penetration into the meshes and matrix. The material compositions did not seem to influence the clinical outcome, although the biological matrix was much thicker compared to the synthetic meshes. Biochemical examination showed good biocompatibility for the investigated meshes and matrix. All products seem to have their value in IBBR and can be recommended for IBBR.

The glycoprotein progranulin directly binds to TNF-receptors and thereby can antagonize the inflammatory effects of TNF-α. Here we analyzed the impact of both cytokines on cytotoxicity and viability of trophoblast cells. Isolated villous first trimester human trophoblast cells and the human choriocarcinoma cell line BeWo were treated with recombinant human progranulin and TNF-α. Analyses were performed by LDH- and MTT-assay and measurement of caspase-8-activity. Progranulin treatment showed some cytoprotective effects on isolated trophoblast cells. However, TNF-α-induced apoptosis was not antagonized by addition of progranulin. Effects were similar, but more pronounced in BeWo cells. The cytoprotective activity of progranulin on trophoblast cells in vitro was only weak and of doubtful biologic relevance. It was not able to antagonize TNF-α. Future studies should focus on possible paracrine activities of progranulin.

Phytoestrogens have a controversial effect on hormone-dependent tumours. Herein, we investigated the effect of the pumpkin seed extract (PSE) on estradiol production and estrogen receptor (ER)-α/ER-β/progesterone receptor (PR) status on MCF7, Jeg3, and BeWo cells. The PSE was prepared and analyzed by mass spectrometry. MCF7, Jeg3, and BeWo cells were incubated with various concentrations of PSE. Untreated cells served as controls. Supernatants were tested for estradiol production with an ELISA method. Furthermore, the effect of the PSE on ER-α/ER-β/PR expression was assessed by immunocytochemistry. The PSE was found to contain both lignans and flavones. Estradiol production was elevated in MCF7, BeWo, and Jeg3 cells in a concentration-dependent manner. In MCF7 cells, a significant ER-α downregulation and a significant PR upregulation were observed. The above results after properly designed animal studies could highlight a potential role of pumpkin seed's lignans in breast cancer prevention and/or treatment.

Flaxseeds were shown to have anticancerogenic properties on breast cancer. In this work, an extract of roots of Linum usitatissimum was tested on MCF-7 and BT20 mamma carcinoma cells in vitro.The extract was produced by an ethanolic extraction method and its chemical composition was afterwards analysed by pyrolysis field ionization mass spectrometry. The extract was tested in concentrations from 0.01 to 1,000 μg/mL. Its effects were detected by measuring the influence on cell lethality, viability and proliferation.The extract was shown to contain mainly sterols and triterpenes (21.4 %), free fatty acids (17.8 %), lignin dimers (12.2 %) and lipids (7.7 %). High concentrations of the extract caused significant cell lethality and suppression of cell vitality and proliferation.In this study, it was shown for the first time that an extract made of flaxroots caused different anticancerogenic effects on MCF-7 and BT20 cells in vitro. The extract supposably acts as a plantal multicomponent mixture, whereas the main active agents are not yet indentified and can only be suggested. Summarized, roots of flax may contain potential agents in the therapy of mamma carcinomas. Further investigations have to be carried out.

Herein we investigated the effect of elderflower extracts (EFE) and of enterolactone/enterodiol on hormone production and proliferation of trophoblast tumor cell lines JEG-3 and BeWo, as well as MCF7 breast cancer cells. The EFE was analyzed by mass spectrometry. Cells were incubated with various concentrations of EFE. Untreated cells served as controls. Supernatants were tested for estradiol production with an ELISA method. Furthermore, the effect of the EFE on ERα/ERβ/PR expression was assessed by immunocytochemistry. EFE contains a substantial amount of lignans. Estradiol production was inhibited in all cells in a concentration-dependent manner. EFE upregulated ERα in JEG-3 cell lines. In MCF7 cells, a significant ERα downregulation and PR upregulation were observed. The control substances enterolactone and enterodiol in contrast inhibited the expression of both ER and of PR in MCF7 cells. In addition, the production of estradiol was upregulated in BeWo and MCF7 cells in a concentration dependent manner. The downregulating effect of EFE on ERα expression and the upregulation of the PR expression in MFC-7 cells are promising results. Therefore, additional unknown substances might be responsible for ERα downregulation and PR upregulation. These findings suggest potential use of EFE in breast cancer prevention and/or treatment and warrant further investigation.

Manifestation of preeclampsia is characterized by an inflammatory response and altered expression of acute-phase proteins. In this study, we examined the predictive value of serum amyloid A, progranulin, transthyretin, C-reactive protein and interleukin-6. Soluble endoglin was used as control. Maternal serum levels of the putative biomarkers were measured in 49 women with a midtrimester bilateral abnormal uterine artery Doppler velocimetry. Preeclampsia developed in 26.5 %. 75.0 % had an early-onset disease (

To investigate the serum level distribution of angiogenic markers (PlGF, endoglin, sFlt-1) and acute-phase proteins (SAA, CRP) in patients with HELLP syndrome or preeclampsia (PE) including matched controls.The matching procedure revealed 46 controls for 23 HELLP cases, and 81 controls for 42 preeclamptic patients. Maternal serum concentrations were determined by immunoassays.SAA and CRP levels were significantly higher in HELLP patients compared with controls. This finding was not observed in preeclamptic subgroup. Pro-angiogenic PlGF is significantly lower in PE and HELLP syndrome. Anti-angiogenic endoglin is significantly higher in PE and HELLP syndrome. The sFlt-1 analysis supports the anti-angiogenic shift in HELLP and preeclamptic patients, but with smaller differences between subgroups. The SAA/PlGF ratio showed the highest ROC value of all tested parameters in discrimination between HELLP and HELLP controls.These findings support the concept that patients with HELLP syndrome have both an anti-angiogenic state and a pronounced inflammatory response, while patients with PE are characterized only by an anti-angiogenic shift.

Corticotropin-releasing hormone (CRH) and its receptors are expressed in human placenta. Recently, the impaired function of this system has been associated with a number of complications of pregnancy, including pre-eclampsia. The aim of the study was to test the hypothesis that CRH participates in the pathophysiology of pre-eclampsia through the induction of macrophage-mediated apoptosis of extravillous trophoblasts (EVTs). We found that the expression of CRH was increased in the EVT of the placental bed biopsy specimens from pre-eclamptic pregnancies (1.8-fold increase; P < 0.05). In addition, significantly larger numbers of apoptotic EVT were detected in pre-eclamptic placentas compared with normal ones (P < 0.05), and only in pre-eclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. In vitro studies on the effect of CRH on human macrophages suggested that CRH induced the expression of the FasL protein in human macrophages and potentiated their ability to induce the apoptosis of a Fas-expressing EVT-based hybridoma cell line in co-cultures. These findings demonstrate a possible mechanism by which the aberrant expression of CRH in pre-eclampsia may activate the FasL-positive decidual macrophages, impair the physiological turnover of EVT and eventually disturb placentation.

AIM AND SETTING: To test the effects of crude extracts from flax (Linum usitatissimum) on progesterone and estradiol and ERα and β/PR production in choriocarcinoma cell lines Jeg 3 and BeWo. Tumor trophoblast cells (Jeg 3 and BeWo) were incubated in the presence of different concentrations of the flax crude extracts. Estradiol and progesterone production was measured. Estrogen receptor α and β as well as progesterone receptor expressions were also assessed.In Jeg 3 cells, progesterone production was downregulated by flax root and leaves extract, while in BeWo cells only flax root extract did manage to downregulate progesterone production. ERβ expression was significantly downregulated by flax root and flax leaves extract in both cell lines; on the contrary, ERα expression was increased by flax leaves extract in BeWo cells. PR expression was downregulated by flax leaves extract in Jeg 3 and by flax root extract in BeWo cells.Flax extracts derived from leaves and especially from roots can modify progesterone and possibly estradiol production, while at the same time they seem to alter ERβ expression. Further studies on animal models and adequately designed retrospective epidemiological studies are imperative to clarify this role upon progesterone.

The potential of substances from elm bark extracts to affect cancer has been described in several studies. In this study, the anticancer effects of extracts from Ulmus laevis bark were tested in hormone-dependent gynecological tumours using human chorion carcinoma cell lines.The molecular-chemical composition of the bark extract was analysed by pyrolysis-field ionisation mass spectrometry. The influence of the extracts was determined on cell vitality and cytotoxicity in the human chorion carcinoma cell lines Jeg3 and BeWo in comparison with primary trophoblast cells.The elm bark extract was mainly composed of triterpenes, phytosterols, free fatty acids and suberins with lower amounts of dilignols and lipids. The elm bark extract significantly inhibited the vitality of Jeg3 and BeWo cells but increased the vitality of primary trophoblast cells.Substances extracted from elm bark might have beneficial effects for the prevention of hormone-dependent tumours.

Expression of the glycoprotein progranulin has been recently identified in rodent trophoblast cells during early embryonic development. The aim of our study was to describe the expression pattern of progranulin in human placental tissue specimens by immunostaining. We further analyzed the influence of progranulin on invasion and migration of isolated first trimester villous trophoblast cells. The effect of progranulin on cell proliferation was investigated using the human choriocarcinoma derived cell lines BeWo and Jeg-3. Cells were tested with recombinant human progranulin at various concentrations (0.1, 0.2 and 1.0 µg/ml). The strongest expression of progranulin was observed in the villous trophoblast cells, particularly in the syncytiotrophoblast. The intensity of staining in these cells was higher in the first trimester than in the third trimester. In contrast, the staining of the extravillous trophoblast cells and of the villous and decidual stroma was only weak. Using an ELISA technique, we also detected progranulin in amniotic fluid of the early second trimester. Isolated human first trimester trophoblast cells also expressed and secreted progranulin. Progranulin significantly stimulated the cell proliferation of BeWo cells, but it did not influence the amount of trophoblast cell migration and invasion in vitro. Furthermore, it did not promote the cell proliferation of Jeg-3 cells. Our results suggested that progranulin, although it is mainly synthesized and secreted by villous trophoblast cells, may not primarily act on the villous trophoblast cells in a paracrine or autocrine manner. The observed effect of progranulin on cell proliferation in BeWo cells may indicate a growth stimulating effect also on the small part of proliferating extravillous trophoblast cells during placental development.

Summary Inhibins (INHs) are dimeric glycoproteins composed of an alpha (- α ) subunit and one of two possible beta ( β -) subunits ( β A or β B). The aims of this study were to determine the frequency and distribution of INH beta ( β A and β B) subunits in normal, hyperplastic and malignant human endometrium. Endometrial tissue was obtained from normal, hyperplastic (simple, complex and atypical) and endometrioid adenocarcinoma (EC) and INH- α , - β A and - β B were labelled using immunohistochemistry and immunofluorescence. INH- β A and - β B labelling was increased significantly between the proliferative and secretory phase ( p 0.0 5 ). The lowest labelling was demonstrated in EC, being significantly lower than in secretory phase ( p 0.0 1 ) and in simple, complex and atypical hyperplastic tissue ( p 0.0 5 ). For inhibin- β B, the most intense labelling was noted in atypical hyperplasia compared to EC ( p 0.0 5 ). A strong colocalisation of inhibin- α and - β A could be demonstrated in malignant endometrial tissue, suggesting the production of inhibin A within the tumour. Additionally, only limited colocalisation of inhibin- β B with - α subunit could be observed, suggesting the synthesis of activin B rather than inhibin B in malignant endometrium. In conclusion, INH- β A and - β B were labelled in normal, hyperplastic and malignant endometrium. Hyperplastic tissue labelled more intensely than EC for the presence of INH- β A and - β B, suggesting a substantial function in endometrial pathogenesis and an important role in endometrial carcinogenesis.

Aims : Glycodelin is a glycoprotein with a molecular weight of 28 kDa. Unusual LacdiNAc structures have been identified on glycodelin A, isolated from amniotic fluid. Three major functions of this glycoprotein have been identified. Glycodelin is an immunosuppressive molecule, a marker of morphological differentiation, and a contraceptive. Because no monoclonal antibodies for glycodelin A are commercially available, our aim was to develop and characterize three monoclonal antibodies against this glycoprotein. Methods and results : Glycodelin A was purified from amniotic fluid by three chromatographic steps and its purity was checked by SDS–PAGE. Antibodies were generated from immunized BALB/c mice. Three IgG1 monoclonal antibodies detecting glycodelin A were cloned. All three antibodies recognized carbohydrate structures of glycodelin A and did not cross-react with glycodelin S. They are applicable to immunohistochemistry (frozen and paraffin sections), ELISA and Western blots. Conclusion : The new antibodies can be used for the detection of glycodelin A in frozen and paraffin-embedded decidual and endometrial tissue. One antibody (A87-B/D2) can be used for the detection of glycodelin in endometrial and ovarian tumour tissues. Because glycodelin A is a major secretory endometrial product during the luteal phase, in early pregnancy and in gynaecological tumours, the new antibodies are, potentially, valuable tools for the study of endometrial development and tumour progression.

Background: In most mammals, onset of labor is accompanied with progesterone withdrawal. In humans, cortisol blockade of progesterone is a possible mechanism involved in the initiation of labor. Therefore, aim of the study was to clarify the effect of CRH, ACTH and cortisol (prednisolone) on the release of progesterone by term trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a percoll gradient centrifugation step. Trophoblasts were incubated with CRH, ACTH as well as with prednisolone Results: The release of progesterone is decreased in CRH- and ACTH-treated trophoblast cell cultures compared to untreated trophoblast cells. Addition of prednisolone in varying concentrations leads to an increase of trophoblast progesterone production. Conclusions: The results suggest that CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production. Prednisolone on the other hand showed a stimulating effect on progesterone production in term trophoblast cells in vitro. Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that CRH and ACTH are directly involved in the auto- or paracrine regulation of this procedure.

Phytoestrogens are a diverse group of non-steroidal compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind to estrogen receptors in humans. In the present study we tested the effects of the phytoestrogens genistein and daidzein on the production of human chorionic gondaotropin (hCG) in isolated trophoblast cells of term placentas in vitro.Genistein and daidzein were incubated at different concentrations with trophoblast cells. Untreated cells were used as controls. At designated times aliquots were removed and tested for hCG production.Production of the protein hormone hCG was influenced by the phytoestrogens genistein and daidzein in trophoblast cells. We found a significant decrease of hCG production in genistein- and daidzein-treated trophoblast cells that was concentration-dependent. Compared with daidzein, genistein seems to be a more efficient inhibitor of the production of hCG.The phytoestrogens genistein and daidzein can reduce hCG production in human term trophoblasts. Both phytoestrogens belong to the group of isoflavones, which are enriched in soy-containing foods and are widely consumed by humans for putative beneficial health effects. Because both phytoestrogens have inhibitory effects on hCG production during pregnancy, exposure to these estrogen-like compounds during sensitive periods of development may have the capacity to alter the function of the reproductive system and thereby influence fertility.

The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro. Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid. The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.

The immunosuppressive protein glycodelin A (formerly named PP14) is produced by human decidua and secreted in the maternal circulation. Glycodelin A concentrations in serum have been used as indicators of endometrial function. The purpose of this study was to investigate the effect of glycodelin A on human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release by freshly isolated cytotrophoblasts (in vitro). Cytotrophoblasts have been prepared from human term placenta by the three-step trypsin-DNase dispersion method of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the isolated mononuclear trophoblasts differentiated into syncytial counterparts within 12-72 h after plating. Trophoblasts were incubated with varying concentrations (60-300 microg/ml) of glycodelin A. Glycodelin A was isolated and purified by chromatographic methods from amnion fluid. Supernatants of the trophoblast cell cultures were assayed for hCG and hPL by immunological methods. The release of hCG is increased in glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. Glycodelin A inhibits the production of hPL in vitro. Differences in Glycodelin A stimulated cells and untreated controls are statistical significant. hCG and hPL are markers for the differentiation process of trophoblast cells to syncytial trophoblasts. The results imply that glycodelin A secreted by decidualised endometrium modulates endocrine function, as well as the differentiation of trophoblasts in culture.

PURPOSE: We studied the prognostic and predictive value of immunohistochemically detected occult tumor cells (OTCs) in lymph nodes and bone marrow aspirates obtained from node-negative breast cancer patients. All were classified as distant metastases-free using conventional staging methods. PATIENTS AND METHODS: A total of 484 patients with pT1-2N0M0 breast cancer and 70 with pT1-2N1M0 breast cancer and a single affected lymph node participated in our trial. Ipsilateral axillary lymph nodes and intraoperatively aspirated bone marrow were examined. All samples were examined for OTCs using monoclonal antibodies to cytokeratins 8, 18, 19. Immunohistological findings were correlated with other prognostic factors. The mean follow-up was 54 ± 24 months. RESULTS: OTCs were detected in 180 (37.2%) of 484 pT1-2N0M0 patients: in the bone marrow of 126 patients (26.0%), in the lymph nodes of 31 patients (6.4%), and in bone marrow and lymph nodes of 23 (4.8%) patients. Of the 70 patients with pT1-2N1MO breast cancer and a single involved lymph node, OTCs were identified in the bone marrow of 26 (37.1%). The ability to detect tumor cells increased with the following tumor features: larger size, poor differentiation, and higher proliferation. Tumors of patients with OTCs more frequently demonstrated lymph node invasion, blood vessel invasion, higher urokinase-type plasminogen activator levels, and increased PAI-1 concentrations. Patients with detected OTCs showed reduced disease-free survival (DFS) and overall survival (OAS) rates that were comparable to those observed in patients who had one positive lymph node. Multivariate analysis of prognostic factors revealed that OTCs, histological grading, and tumor size are significant predictors of DFS; OTCs and grading of OAS. CONCLUSION: OTCs detected by simultaneous immunohistochemical analysis of axillary lymph nodes and bone marrow demonstrate independent metastatic pathways. Although OTCs were significantly more frequent in patients with other unfavorable prognostic factors, they were confirmed as an independent prognostic factor for pT1-2N0M0, R0 breast cancer patients.

Syndrome of hemolysis, elevated liver enzymes and low platelets (HELLP) represents a distinct subgroup of severe preeclampsia. The aim of our study was to identify differentially expressed miRNAs in sera of patients with HELLP syndrome in comparison to unaffected controls.Blood samples were obtained from patients with manifest HELLP syndrome and matched unaffected controls. The expression of 754 mature miRNAs was assessed using the TaqMan Array format (n = 12). Results of seven differentially expressed miRNAs were further validated by single quantitative real-time polymerase chain reaction (qPCR) assays.Serum miRNA analysis allowed detection of maternal and fetal miRNAs. Distinct miRNA expressions were confirmed for miR-122, miR-758 and miR-133a represented by a median up-regulation ≥ two-fold in the HELLP group. The liver specific miR-122 was 11.5-fold increased with an area under curve (AUC) of 0.82 in the receiver operating characteristic (ROC) analysis. Cluster analyses of our data uncovered subgroups of HELLP patients were associated with clinical subtypes and differences in organ manifestation.In our proof of principle study, we demonstrated that patients with HELLP syndrome showed alterations of serum miRNA expression patterns. Data analysis goes along with the hypothesis that HELLP syndrome is regarded to be a heterogeneous disease.

Aims: The expression of the anti-inflammatory glycoprotein progranulin and the hypoxia-induced transcription factor 1α (HIF-1α) in the villous trophoblast was compared between placentae from patients with preeclampsia (PE), fetal growth restriction (FGR), and normal controls. Study design: Matched pairs analysis of third trimester placentae specimens (mean gestational age 36+2) was performed by semiquantitative measurements of the immunohistochemical staining intensities for progranulin and HIF-1α expression (PE n=13, FGR n=9 and controls n=11). Further, placental progranulin mRNA expression was analyzed by qRT-PCR on term placentae (n=3 for each group). Results: Compared to controls, villous trophoblast revealed a significantly higher expression of progranulin in cases of PE (P Conclusions: Increased expression of progranulin protein in villous trophoblast cells in cases of PE and FGR may result from disturbed placental development and, therefore, may be of pathogenetic importance. The increase was correlated to HIF-1α expression. Further evaluation of this potential mechanism of regulation is required.

Phytoestrogens are plant-derived, non-steroidal phytochemicals with anticarcinogenic potential. The major structural classes are the isoflavones and lignans. The aim of this study was to compare the effect of the plant-derived lignans secoisolariciresinol and matairesinol with the human lignans enterodiol and enterolactone as well as with 17β estradiol and tamoxifen on cell proliferation of breast carcinoma cell lines. The influence of the lignans, 17β estradiol and tamoxifen on cell proliferation was determined using the BrdU test in MCF 7 and BT 20 cell lines. Enterodiol and enterolactone induced a stronger inhibition of cell growth in MCF 7 and BT 20 cells than secoisolariciresinol and matairesinol. The inhibition effects were less expressed in the BT 20 than in the MCF 7 cells. The human lignans enterodiol and enterolactone are more biologically active than their precursors secoisolariciresinol and matairesinol, and may be defined as the real drugs in cancer prevention.

Phytooestrogens are known to cause anti-cancer effects on mamma carcinoma cells. In this study, the effects of the lignan secoisolariciresinol and the isoflavone glycosides and aglycones genistein, genistin, daidzein and daidzin were tested on MCF-7 and BT20 cells in vitro. First, the cellular expression of hormone receptors was examined by immunohistochemical procedures. The effects of the phytooestrogens on the cells were detected by using three different assays measuring cell letality, viability and proliferation. The phytooestrogens were tested in concentrations of 1, 5, 10 and 50 μg/mL, respectively. 17β-oestradiol and tamoxifen were used as controls, respectively, in the same concentrations as the phytooestrogens. The immunohistochemistry showed evidence of oestrogen- and progesterone receptors at the MCF-7 cell line, whereas no expression could be seen at the BT20 cells. Among the phytooestrogens, genistein and secoisolariciresinol showed various anti-cancerogenic effects on both cell lines, respectively, but only in the highest concentration. Regarding the controls, tamoxifen showed a stronger antivital and anti-proliferative effect on BT20 than on MCF-7. Oestradiol caused sporadic anti-cancer effects on both cell lines, respectively, at its highest concentration. Genistein and Secoisolariciresinol have anti-cancer properties on MCF-7 and BT20 in vitro. There are differences in the effects of isoflavones depending on the glycolysation status. The role of the oestrogen receptors in the mechanisms of action of both the phytooestrogens and controls is of less importance. Further investigations have to be carried out, especially concerning the mechanisms of action. Phytooestrogens may be potential substances in the therapy of mamma carcinomas.

Anti-inflammatory effects of elm tree have been shown in several studies. Besides this, protective effects of components of elm bark on damaged tissue have also been described. This study was carried out to investigate the antitumour potential of an ethanolic extract isolated from Ulmus laevis in the hormone-dependent endometrial carcinoma cell line RL95-2. A range of 2.5-500 microg/ml of elm bark extract was used as standard concentrations. The molecular-chemical composition of the bark extract was analysed by pyrolysis-field ionization mass spectrometry. The influence of the bark extracts was determined on cell vitality [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test], cell proliferation (5-bromo-2-deoxyuridine test) and cytotoxicity (lactate dehydrogenase test) in the human endometrial carcinoma cell line RL 95-2. By pyrolysis-field ionization mass spectrometry, the main substance classes of the extract as a composition of sterols/triterpenes, free fatty acids and a group of phenols, lignin monomers and flavonoids was identified. Our study showed a significant inhibition of cell vitality and proliferation measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test up to 5 microg/ml extract and up to 100 microg/ml according to the 5-bromo-2-deoxyuridine test. Concentrations of 500 microg/ml induced a significant inhibition of cell vitality up to 80% and cell proliferation up to 81.5%. A significant cytotoxity was not observed. The results lead to the assumption that the bark extract from Ulmus laevis has antiproliferation and anticancer potential in hormone-dependent endometrial carcinoma cells.

Phytoestrogens are a diverse group of nonsteroidal plant compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind on estrogen receptors alpha and beta in humans. The effects of the phytoestrogens genistein and daidzein on the production of progesterone and estrogen in isolated human term trophoblast cells in vitro were tested in this study.Cytotrophoblast cells were isolated from human term placentas. Phytoestrogens genistein and daidzein were incubated in different concentrations with trophoblast cells. Untreated cells were used as controls. After 24 h aliquots were removed and tested for progesterone and estrogen production.The production of the steroid hormones progesterone and estrogen are influenced by phytoestrogens genistein and daidzein in human term trophoblast cells. A strong inhibition effect of both phytoestrogens tested in the production of progesterone was demonstrated. In addition, a significant stimulating effect on estrogen production by genistein and daidzein was observed.Results obtained with this study show that phytoestrogens (genistein and daidzein) sufficiently reduce progesterone production in human term trophoblast cells. Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that high doses of phytoestrogens at the feto-maternal interphase could play a negative role in maintenance of pregnancy. Stimulation of estrogen production by genistein and daidzein in trophoblast cells is probably due to estrogen receptor blocking effects of both phytoestrogens. Trophoblast cells seem to compensate blocking of its estrogen receptors by higher estrogen production.

Human reproduction is remarkably inefficient, with more than half of spontaneous conceptions failing to complete the first trimester. However, little is known on the molecular events that take place at the implantation site during abortion. Here, we examined the hypothesis that the expression of the proapoptotic Fas/FasL system at the implantation site is impaired in abortions. We found that, in contrast to normal pregnancy, abortive deciduas contain leukocytes that are positive for FasL and extravillous trophoblasts (EVTs), which show increased expression of Fas and increased rates of apoptosis. In addition, the neuropeptides, corticotropin-releasing hormone and urocortin, were elevated in placental material obtained from abortions. In vitro, these peptides induced the expression of FasL in decidual lymphocytes (DL) obtained from elective termination of pregnancy placentas and thus potentiated the cells' ability to induce Fas-mediated apoptosis in an EVT-based hybridoma cell line. Finally, DL from abortion sites effectively induced apoptosis of EVT without prior treatment. It is possible that these events may impede successful early placentation and thus contribute to the pathophysiology of human abortion. Mol Hum Reprod

The immunosuppressive protein glycodelin A (GdA) is secreted by a large number of gynaecological tumors. GdA has a unique glycosylation, including fucosylated LacdiNAc structures. Trophoblast tumor cells produce a variety of steroid hormones and human chorionic gonadotropin (hCG). The purpose of this study was to investigate the effect of GdA N-glycans on hCG, cortisol, estradiol and progesterone release by the chorion carcinoma cell line BeWo in vitro.Chorionic carcinoma cells of the cell line BeWo (1 x 10(6) cells/ml) were cultivated in the presence of 25 mmol/ml glycodelin A and 25 mmol/ml glycodelin A N-glycans RH-3, RH-4 and RH-5 for up to 72 h. Unstimulated BeWo cells were used as controls. After 24 h, 48 h and 72 h, 1 ml cell culture supernatant was removed, stored at -25 degrees C and replaced by fresh medium. All experiments were carried out at least in triplicates. Collected cell culture supernatants were analysed for hCG, progesterone, estradiol (E2) and cortisol concentration.The production of estradiol, cortisol and progesterone were increased in GdA N-glycan-treated cell cultures as compared to untreated BeWo cell cultures. GdA N-glycans did not stimulate hCG production in BeWo cells. A significant increase in E2 secretion was observed in BeWo cells incubated with glycodelin A and glycodelin A N-glycans (RH-3: 2.6% E2 increase compared to control, p = 0.401; RH-4: 8.9% increase, p = 0.012; RH-5: 4% increase, p = 0.017; glycodelin: 7.7% increase, p = 0.017). Although an increase in progesterone secretion was observed in BeWo cells incubated with glycodelin A (9.0%) and glycodelin A N-glycans (RH-3: 16.2%, RH-4: 2.8%, RH-5: 8.7%) compared to controls, these differences were not statistically significant.Estradiol, cortisol and progesterone are hormone markers for BeWo trophoblast tumor cells. The results suggest that GdA with its distinct glycosylation modulates the hormone production of trophoblast tumours.

Phytoestrogens seem to have estrogen-like effects in the human body as their structure is very similar to those estrogens produced in human glands. The aim of the present study was to analyse the effects of genistein and daidzein on estrogen receptor (ER)alpha- and ERbeta-mRNA and protein expression in the endometrium of premenopausal women.Glandular endometrial cells were isolated from endometrial biopsies obtained from regularly menstruating women undergoing gynaecological abrasio or hysterectomy. Cells were stimulated with single doses of genistein or daidzein. ERalpha- and ERbeta-protein expression were determined by immunocytochemical analysis. In addition ERalpha- and ERbeta-mRNA expression were determined by quantitative real-time RT-PCR. Quantification was carried out by the deltadelta C(T)-method using glyceraldehyde phosphate dehydrogenase (GAPDH) as housekeeping gene.Endometrial glandular cells responded to stimulation with genistein and daidzein by alteration of both ERalpha- and ERbeta-mRNA expression. The effects were time- and dose-dependent. Detection of ERalpha- and ERbeta-protein expression by immunocytochemistry showed a dose-dependent regulation after stimulation of isolated endometrial cells with phytoestrogens.According to our results, we suggest that ER expression in endometrial glandular cells is regulated by genistein and daidzein on the mRNA and protein levels. We could detect a decrease in ERalpha- and an increase in ERbeta-mRNA expression after stimulation with tested phytoestrogens. Our results are in line with findings that phytoestrogens act as anti-estrogens in organs expressing more ERalpha and as estrogens in ERbeta-presenting organs.

The role of glycodelin in human reproduction and gynecological malignancies has been investigated in a large number of studies in recent years. Three dominant functions of this glycoprotein were identified. Glycodelin is immunosuppressive, a morphological marker of differentiation and a contraceptive. Glycodelin is a glycoprotein with a molecular weight of 28 kDa and a carbohydrate content of 17.5%. Unusual LacdiNAc structures were identified on glycodelin A, isolated from amniotic fluid. Because no kit for glycodelin quantification is commercially available, we developed all reagents and a functional ELISA.Glycodelin A was purified from amniotic fluid by chromatographic methods. The purity of the isolated protein was checked with SDS-PAGE. Polyclonal antibodies against glycodelin were generated in rabbits. Monoclonal antibodies against glycodelin were generated from immunized BALB/c mice. Positive hybridomas were cloned and cultured. Monoclonal antibodies were isolated by immunoadsorption chromatography from culture supernatants. The glycodelin ELISA was developed in two formats, namely coating with polyclonal antibodies and the use of monoclonal antibodies.The factors of variance for the ELISA were 7% (intraassay variance) and 15% (inter-assay variance). The glycodelin ELISA was used to determine the glycodelin A concentration in sera of fertile women during the proliferative and secretory phases of the endometrium. The glycodelin A concentration was insignificantly elevated in the secretory phase compared to the proliferative phase. Significantly higher levels of glycodelin A were found in women using oral contraceptives compared to women who were not (p0.001). This is probably due to progesterone, which stimulates glycodelin production. We also found significantly increased glycodelin concentrations in the fluids of malignant ovarian cysts compared to benign ovarian tumors (p0.001). Furthermore, we tested the monoclonal and polyclonal antibodies successfully in Western blot analysis and immunoadsorption chromatography.We consider the described ELISA for the quantification of glycodelin as a useful tool for the determination of glycodelin in amniotic fluid, serum and cystic fluids. Its most promising application is expected in the diagnosis of ovarian cancer. The antibodies generated are applicable to multiple techniques.

Glycodelin A levels in fluids of benign ovarian cysts, borderline tumours and ovarian cancer, as well as serum-levels of glycodelin A, were analysed in patients with benign and malignant ovarian tumours. The aim of the study was to investigate if the level of glycodelin A in body fluids of patients with malignant ovarian tumours could be a marker for the disease. Additionally, immunohistochemical investigations of glycodelin A expression in tissue of ovarian cancer were performed.A total of 158 samples of fluids from benign ovarian cysts, borderline tumours and ovarian cancer were collected during surgery in the Department of Obstetrics and Gynaecology at the University of Rostock, Germany. Additionally, 69 samples of serum from patients with benign ovarian cysts and ovarian cancer on the day before surgery were collected. An ELISA for determination of glycodelin A was used. Immunohistochemical detection of glycodelin A expression was performed on ovarian cancer tissue from 38 patients.Malignant cystic fluids showed higher glycodelin A values (mean: 1814.4 ng/ml) compared to fluids of benign ovarian cysts (mean: 784.4 ng/ml). The results of glycodelin A determination were compared using the Mann- Whitney U-test for comparison of the means. There was a statistically significant difference between benign ovarian cysts and malignant ovarian cancer for the fluid (p0.001). In addition, serum samples of malignant ovarian tumours also showed significantly higher glycodelin A values compared to serum levels of benign tumours (p0.001). Immunohistochemical staining on ovarian cancer tissue showed a glycodelin A expression in 25-30% of the carcinoma cells.High levels of glycodelin A were found in cystic fluids of ovarian cancer. In addition, we also found higher levels of glycodelin A in the serum of patients with ovarian cancer compared to the serum of patients with benign ovarian cysts. Furthermore, ovarian cancer tissues showed intense staining with a glycodelin A antibody. Further investigations are necessary to show if glycodelin A quantification could help to diagnose ovarian cancer.

Parathyroid hormone-related peptide (PTH-rp) is a peptide initially purified from tumors with hypercalcemia, which is also produced in human endometrium. The aim of this study was to determine the frequency and tissue distribution of PTH-rp in normal, hyperplastic and malignant endometrium.Paraffin-fixed endometrial tissue was obtained randomly from women in the proliferative (n = 4), early secretory (n =5) and late secretory (n = 5) phases, as well as glandular-cystic hyperplasia (n = 5), adenomatous hyperplasia (AH) grade I (n = 5), grade II (n = 4), grade III (n = 4) and endometrioid adenocarcinoma grade I (n = 5). The PTH-rp expression was evaluated by immunohistochemical procedure. A semiquantitative analysis and a statistical evaluation was performed.Immunohistochemical reaction with PTH-rp was primarily observed in glandular and luminal epithelial cells. Stromal and myometrial cells also expressed PTH-rp. The expression of PTH-rp in glands was significantly higher during the late secretory than in the proliferative phase. The highest expression was observed during AH grade III, while the lowest reaction was detected in the proliferative phase and adenocarcinoma.PTH-rp was expressed in normal, hyperplastic and malignant endometrial tissue. A cyclical expression of PTH-rp in normal glandular epithelium was observed, being more prominent in the late secretory phase. AH grade I to III also expressed PTH-rp with higher immunostaining than adenocarcinoma. Since AH grade III can be considered as a precursor of endometrial cancer, PTH-rp could be a marker of cell transformation. Endometrioid adenocarcinoma expressed the lowest PTH-rp immunostaining, indicating either a lower expression or a reflection of an increased PTH-rp shedding of malignant transformed endometrium. Further studies are required to establish the usefuleness of PTM-rp as a marker in different endometrial pathologies.

Background: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. Objectives: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. Methods: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. Results: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. Conclusion: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

Aim: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. Results: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 mu g/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. Conclusions: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.

Inhibins (INH) are dimeric glycoproteins, composed of an alpha subunit (INH-alpha) and one of two possible beta subunits (INH-betaA or INH-betaB). They have substantial roles in human reproduction and in endocrine-responsive tumours. Therefore, the aims of this study were to determine the frequency and tissue distribution of INH-alpha, INH-betaA and INH-betaB in normal human endometrium and glandular-cystic endometrial polyps, and polyps caused by tamoxifen use.Tissue samples were obtained from women in the proliferative, early secretory and late secretory phase as well as glandular-cystic polyps and endometrial polyps associated with tamoxifen use (n = 5 each). Immunohistochemistry with specific monoclonal antibodies, a semi-quantitative analysis and statistical evaluation was performed.INH-alpha, INH-betaA and INH-betaB were primarily observed in glandular and luminal epithelial cells, with a variant staining intensity in stromal cells. INH-alpha in glands was significantly higher during the early secretory phase (p0.05) and the late secretory phase (p0.01) than in the proliferative phase with a significant difference between the early secretory and the late secretory phases (p0.01). INH-betaA expression was significantly higher during the late secretory than the proliferative phase (p0.05) and the late secretory than the early secretory phase (p0.05), with no significant differences for INH-betaB. Glandular-cystic polyps showed significantly lower expression of INH-alpha and INH-betaA than the late secretory endometria (p0.05 and p0.01 respectively). Additionally, tamoxifen-associated polyps also demonstrated a significantly lower expression of INH-alpha and INH-betaA than late secretory endometria (p0.01 and p0.01 respectively). No statistical differences were observed between tamoxifen-associated and glandular-cystic polyps.INH-alpha, INH-betaA and INH-betaB were expressed in normal endometrium and endometrial polyps. A cyclical expression of INH-alpha and INH-betaA in normal glands may reflect a functional and hormone-dependent role in human endometrium. Significant differences in staining reaction between the late secretory endometria and polyps suggest that this tissue remains in the proliferating state rather than the secretory state. Therefore, endometrial polyps may be tumours of dysregulation with mainly proliferating characteristics, being unable to synchronise with normal endometrium.

Background: Progesterone is thought to mediate immune modulator effects by regulating uterine responsiveness. The aim of the study was to clarify the effect of transferrin and glycodelin A (former name PP14) as sialyl Lewis X-expressing glycoproteins on the release of progesterone by trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a Percoll gradient centrifugation step. Trophoblasts were incubated with varying concentrations (50–300 µg/ml) of human amniotic fluid- and serum-transferrin as well as with glycodelin A. Culture supernatants were assayed for progesterone, human chorionic gonadotropin (hCG) and cortisol by enzyme immunometric methods. Results: The release of progesterone is increased in amniotic fluid transferrin- and glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. There is no relation between transferrin and the hCG or cortisol production of trophoblast cells. Conclusion: The results suggest that sialyl Lewis carbohydrate antigen-expressing amniotic fluid glycoproteins modulate the endocrine function of trophoblasts in culture by upregulating progesterone production.

Aims: During pregnancy, the placenta produces a variety of steroid hormones and proteins. Several of these substances have been shown to exert immunomodulatory effects. Progesterone is thought to mediate some of these effects by regulating uterine responsiveness. The aim of this study was to clarify the effect of amniotic fluid transferrin and its N-glycans on the release of progesterone by first trimester trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placentae by trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation and depletion of CD45 positive cells by magnetic cell sorting. Trophoblasts were incubated with varying concentrations (50-300 mug/ml) of transferrin from human amniotic fluid and serum as well as with N-glycans obtained from amniotic fluid transferrin. Culture supernatants were assayed for progesterone by enzyme-immunometric methods. Results: The release of progesterone increased in amniotic fluid transferrin- and N-glycan-treated trophoblast cell cultures compared to untreated trophoblast cells. There was no stimulating effect of serum transferrin on the progesterone production of trophoblast cells. Conclusions: The results suggest that amnion-transferrin and especially its N-glycans modulate the endocrine function of trophoblasts in culture by up regulating progesterone secretion.

Objective . Determination of the frequency and tissue distribution of inhibin-alpha (INH-α) in normal, hyperplastic and malignant endometrium. Materials and methods . Endometrial tissue was obtained from normal, hyperplastic (glandular-cystic hyperplasia), adenomatous hyperplasia (AH grade I to III) and endometroid adenocarcinoma and immunohistochemically characterized with INH-α antibody. Results . INH-α was expressed in normal, hyperplastic and malignant endometrium. Highest expression was observed during secretory phase and glandular-cystic hyperplasia compared to all groups. A continuous decline was noted from AH grade I to III with a significance between AH I and II. Discussion . A menstrual cycle associated expression of INH-α in glandular endometrial epithelium was observed. Since AH grade II and III can be considered as a precursor of endometrial cancer, INH-α could be a marker of cell transformation and an endometrial tumor-suppressor agent.

Cathepsin D (CathD), a lysosomal aspartyl protease secreted by normal and malignant cells, is considered to be involved in breakdown of the extracellular matrix. Aim of the present study was to determine the frequency and tissue distribution of CathD in normal, hyperplastic and malignant endometrium. Paraffin-fixed endometrial tissue was obtained from premenopausal women in the proliferative phase (n = 5), early secretory phase (n = 4) and late secretory phase (n = 4) as well as glandular-cystic hyperplasia (n = 5), endometrial polyps (n = 5), endometrial polyps from the use of tamoxifen (n = 5), adenomatous hyperplasia (AH) grade I (n = 5), grade II (n = 4), grade III (n = 5) and endometroid adenocarcinoma (n = 5). CathD expression was evaluated with the IRS score and ANOVA analysis was used for statistical evaluation. CathD was primarily localised in luminal and glandular epihelium with little staining in stromal cells. The expression of CathD was significantly higher during the late secretory phase than in the proliferative phase. Highest expression of CathD was observed in the late secretory phase and in glandular-cystic hyperplasia, whereas endometroid carcinoma showed no expression. A continuous increase in CathD expression was observed in AH, with a significant difference between AH grade I and III. In conclusion, CathD was found to be expressed in normal and hyperplastic endometrial tissue. CathD immunostaining in normal endometrial glands varied on the basis of the phase of the menstrual cycle, suggesting physiological functions of CathD in endometrial maturation and degradation. Adenocarcinomas did express significant lower amounts of CathD. Therefore, the prognostic value of this parameter remains uncertain. A continuous increase in CathD immunostaining was observed in AH. Since AH grade III can be considered as a precursor of endometrial cancer, CathD could be a possible parameter for assessing malignant transformation.

Inhibins are multipotent dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (betaA or betaB). Aims of this study were (a): the immunohistochemical characterisation of normal human endometrium for the inhibin-alpha subunit; (b) the assessment of the secretion and metabolism of inhibin, E2 and cortisol; (c) the evaluation of any relationship between these three substances in cell culture medium of isolated and cultivated normal human endometrial glandular cells.Samples of human endometrium were obtained from 34 premenopausal patients. Nineteen endometrial specimen (proliferative [PP] n=8; early secretory [ES] n=7; late secretory phase [LS] n=4) were brought into cell culture. Fifteen endometrial specimen (PP n=5; ES n=5; LS n=5) were paraffin-fixed and used for the immunohistochemical analysis for inhibin-alpha. Stromal and epithelial cells were separated by collagenase digestions, filtrations, sedimentations and Ficoll-gradient centrifugation. E2 and cortisol were measured with radioimmunoassay (RIA) and inhibin with enzyme-immunoassay (EIA). Statistical analysis was performed with the non-parametric Mann-Whitney rank-sum test and linear regression analysis.Inhibin-alpha showed a weak (positive) expression during proliferative phase, which increased significantly as the menstrual cycle continued. In secretory glands the mean inhibin concentration was higher than that from proliferative samples. A significant correlation was observed between inhibin and E2 (p0.001) as well as cortisol and inhibin (p0.0001) in glands from proliferative phase. Between inhibin and E2 (p0.05) as well as inhibin and cortisol (p0.002) a significant correlation in early secretory glands was also noted. In late secretory phase inhibin and E2 (r2=0.78650; p0.0001), inhibin and cortisol (r2=0.58326; p0.001) and E2 and cortisol (r2=0.52880; p0.001) showed a significant correlation.In conclusion, we found a cyclical expression of inhibin-alpha subunit in the endometrium demonstrated by immunohistochemical means. A higher in vitro secretion of inhibin from secretory glands was also observed. In addition, a significant correlation between inhibin with E2 and cortisol in PP and ES glands and a significant correlation between inhibin, E2 and cortisol in LS glands could also be demonstrated. We conclude that inhibin can be associated with E2 and cortisol metabolism, playing an important role in paracrine/autocrine mechanisms in the endometrium and possibly exerting its function through cortisol and E2. The cortisol concentration also correlates with E2, suggesting a link between these steroids in the endometrial function. The correlation of inhibin, E2 and cortisol suggest complex autocrine/ paracrine mechanisms in human endometrial glands, modulated and controlled by all these three substances.

Susceptibility genes present in both mother and fetus most likely contribute to the risk of pre-eclampsia. Placental biopsies were therefore investigated by high-density DNA microarray analysis to determine genes differentially regulated within chorionic villous tissue in pre-eclampsia and normal pregnancy. The pooled RNAs of pre-eclamptic and normotensive subjects were hybridized to the HuGeneFL array representing sequences from approximately 5600 full-length human cDNAs. The differentially expressed genes that were detected could be categorized into nine groups: adhesion molecules, obesity-related genes, transcription factors/signalling molecules, immunological factors, neuromediators, oncogenic factors, protease inhibitors, hormones and growth factor-binding proteins. Among those, the obesity-related genes included putative candidate genes associated with the pathogenesis of pre-eclampsia. One of the most up-regulated transcripts was the obese gene (43.6-fold change), and this was reflected by elevated leptin protein levels. In the case of feto-maternal contribution of polymorphic genes to pre-eclampsia, expression analysis of placental tissue has lead to numerous target genes waiting for large scale genetic linkage analyses.

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope Galbeta1-3GalNAcalpha-O-) has been known for a long time as a carcinoma-associated antigen. In normal tissues the occurrence of TF antigen is restricted to a few immunologically privileged areas. Here we report on the identification of the TF epitope and its putative carrier protein mucin 1 (MUC1) in human placental tissue, on isolated trophoblast cells in vitro and on trophoblast tumour cell lines BeWo and Jeg3. Cryosections of placental and decidual tissues of the first, second and third trimester were double stained with monoclonal antibodies directed against the TF epitope (IgM) and against MUC1 (IgG). In the first trimester of pregnancy we found strong expression of TF antigen and MUC1 at the apical side of the syncytiotrophoblast directed towards the maternal blood. This expression was consistent in the second trimester of pregnancy, and to a lesser degree in the third trimester. In addition, we found positive staining for TF antigen and MUC1 on extravillous trophoblast cells in the decidua during the first and second trimester of pregnancy. Trophoblast tumour cells of the cell line BeWo, which form a syncytium in vitro, were also positive for TF antigen and MUC1, whereas Jeg3 cells, which are unable to form a syncytium, expressed only MUC1. Freshly isolated trophoblast cells from first trimester placentas showed strong staining for MUC1; however, only a few of these cells (less than 1%) were positive for TF antigen, and might consist of digested fragments of the syncytium. In summary, TF antigen and MUC1 are expressed by the syncytiotrophoblast at the feto-maternal interface and by extravillous trophoblast cells invading the decidua, whereas villous cytotrophoblast cells in situ as well as freshly isolated trophoblast cells from first trimester placentas only express MUC1 but not TF antigen.

Highly purified fractions of isolated endometrial cells can be useful for investigating endometrial function. After a first collagenase digestion, normal human endometrial stromal and epithelial cells were separated by filtration. Glands were purified further by two collagenase digestion steps, filtration, differential sedimentations, and Ficoll gradient centrifugation. Epithelial cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandular fragments. High cell culture purity was confirmed with the positive immunohistochemical reaction against cytokeratin 7,8,18,19. Isolated human glands had a similar distribution pattern of estrogen receptor (ER) and progesterone receptor (PR) as observed in vivo, suggesting that glands have a functional hormone receptor system at the time of plating. Using a specific monoclonal antibody against glycodelin A (GdA), a characteristic cyclical expression was demonstrated during the menstrual cycle. The GdA reaction was weak in the proliferative phase, increasing significantly till the late secretory phase, suggesting a similar GdA concentration in vitro as observed in vivo glands. In conclusion, this method could be a model for studying endometrial glandular cells from different menstrual phases, endometrial cell interactions, implantation mechanisms, GdA regulation mechanisms, and pharmacological or other influences on ER and PR alteration.

Background Women with bilateral abnormal uterine artery Doppler velocimetry (UtADV) are at increased risk for an adverse pregnancy outcome. This study aimed to determine if additional assessment of midtrimester angiogenic factors improves the predictive accuracy of Doppler results for various outcome parameters. Methods Women with a bilateral abnormal UtADV, which was defined as a postsystolic incision and/or an increased pulsatility index greater than the 95th centile, and a singleton pregnancy were prospectively recruited between 19 + 0 and 26 + 6 weeks of gestation. Maternal serum levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFLT-1) were measured with a fully automated immunoassay and their ratio was calculated. Results Angiogenic factors could predict the development of preeclampsia (PE), as well as induced delivery at 95th centile ratio with a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 66.7%, 89.5%, 66.7%, and 89.5% for PE, and 85.7%, 86.1%, 50.1%, and 97.4% for induced delivery, respectively. Positive and negative likelihood ratios were 6.33 (95% CI 2.31–17.38) and 0.37 (95% CI 0.17–0.84) for PE, and 6.14 (95% CI 2.76–13.69) and 0.17 (0.03–1.02) for induced delivery, respectively. Corresponding odds ratios were 17.0 (95% CI 3.5–83.0) and 37.0 (95% CI 3.8–363.9), respectively. Conclusions Measurement of angiogenic factors improves the specificity of an abnormal UtADV for prediction of PE. Compared with prediction of PE an abnormal sFLT-1/PlGF ratio revealed higher sensitivity for prediction of induced delivery at