[Management of postpartum hemorrhage (PPH): algorithm of the interdisciplinary D-A-CH consensus group PPH (Germany - Austria - Switzerland)]. / Management der postpartalen Blutung (PPH): Algorithmus der Interdisziplinären D-A-CH-Konsensusgruppe PPH (Deutschland - Österreich - Schweiz).

Postpartum hemorrhage (PPH) is one of the main causes of maternal deaths even in industrialized countries. It represents an emergency situation which necessitates a rapid decision and in particular an exact diagnosis and root cause analysis in order to initiate the correct therapeutic measures in an interdisciplinary cooperation. In addition to established guidelines, the benefits of standardized therapy algorithms have been demonstrated. A therapy algorithm for the obstetric emergency of postpartum hemorrhage in the German language is not yet available. The establishment of an international (Germany, Austria and Switzerland D-A-CH) "treatment algorithm for postpartum hemorrhage" was an interdisciplinary project based on the guidelines of the corresponding specialist societies (anesthesia and intensive care medicine and obstetrics) in the three countries as well as comparable international algorithms for therapy of PPH.The obstetrics and anesthesiology personnel must possess sufficient expertise for emergency situations despite lower case numbers. The rarity of occurrence for individual patients and the life-threatening situation necessitate a structured approach according to predetermined treatment algorithms. This can then be carried out according to the established algorithm. Furthermore, this algorithm presents the opportunity to train for emergency situations in an interdisciplinary team.

Prenatal microarray analysis as second-tier diagnostic test: single-center prospective study.

OBJECTIVE: To evaluate the usefulness of chromosome microarrays as a second-tier test in prenatal genetic testing. METHODS: We prospectively analyzed 75 high-risk pregnancies undergoing invasive prenatal genetic testing in which the karyotype either was normal or had findings other than a common non-mosaic autosomal aneuploidy. RESULTS: Chromosomal microarray analysis (CMA) was performed successfully in all cases. Pathological copy-number variations (CNVs) explaining the phenotypes were found in 11 cases (14.7%). Four cases were detected with an unbalanced translocation. In three of these cases, subsequent genetic analysis demonstrated that a parent was an unknown carrier of a balanced translocation. Among the 67 cases with normal karyo-types, submicroscopic rearrangements with pathological significance were detected in five (7.5%) and CNVs of unclear significance were detected in one (1.5%). CMA was able to discriminate correctly between true mosaicism and confined or pseudomosaicism in all six mosaic cases. CONCLUSION: CMA is a valuable second-tier test in high-risk pregnancies for which identification or further delineation of genetic aberrations is important. Its higher resolution results in a higher detection rate of aberrant cases, with a clear clinical benefit for estimation of risk of recurrence.

Oxytocin receptors and human parturition: a dual role for oxytocin in the initiation of labor.

The concentration of oxytocin receptors increased in the myometrium of pregnant women and reached maximum levels in early labor. Concentrations of oxytocin receptors were also high in the decidua and reached a maximum at parturition. In vitro, prostaglandin production by the decidua, but not by the myometrium, was increased by the addition of oxytocin. Oxytocin may therefore stimulate uterine contractions by acting both directly on the myometrium and indirectly on decidual prostaglandin production. Oxytocin receptors are probably crucial for the onset of human labor, and the stimulus for the increase in uterine prostaglandins may be oxytocin originating from the fetus.

Effects of different human chorionic gonadotrophin preparations on trophoblast differentiation.

Recent evidence from the literature suggested that hCG preparations purified from urine of pregnant women, which are widely used in in vitro studies and IVF programs, may contain contaminants such as EGF. To determine the putative biological effects of the contaminating growth factor, we here investigated distinct trophoblast differentiation processes in the presence of various hCG compounds. Western blot analyses indicated that treatment of trophoblastic SGHPL-5 cells and purified term trophoblasts with potentially EGF-contaminated hCG (hCG-A) resulted in auto-phosphorylation of the EGF receptor at tyrosine 1173 whereas supplementation of another urine-purified hCG preparation (hCG-B), recombinant holo-hCG or recombinant alphahCG had no effects. Phosphorylation was specifically blocked by the EGF receptor inhibitor PD153035. Urinary hCG-A was most effective in promoting invasion of SGHPL-5 cells through Matrigel-coated transwells, but increased invasiveness was also observed in the presence of hCG-B or recombinant holo-hCG. Similarly, the extent of syncytialisation of term trophoblasts, quantitated by nuclei in desmoplakin-negative areas, was highest upon addition of hCG-A or recombinant EGF as a control. PD153035 reduced invasion and fusion of trophoblasts supplemented with hCG-A, but did not diminish the effects provoked by hCG-B. In conclusion, the data suggest that the EGF contamination of hCG considerably affects trophoblast function. Experiments using EGF-free hCG preparations demonstrate that the hormone increases trophoblast invasion and syncytialisation.

TNFalpha-mediated induction of PAI-1 restricts invasion of HTR-8/SVneo trophoblast cells.

The pro-inflammatory cytokine TNFalpha has numerous effects on placental trophoblasts. Here, we investigated the effects of the cytokine on gene expression and function of the extravillous trophoblast cell line HTR-8/SVneo. Wound healing and Matrigel invasion assays demonstrate that TNFalpha impairs motility and invasiveness. In contrast, counting of cumulative cell numbers and FACS analyses revealed that the cytokine did neither affect proliferation nor distribution of cell cycle phases. Immunocytochemistry of the cytokeratin 18 neo-epitope suggests that TNFalpha did not induce apoptosis in HTR-8/SVneo cells. Gelatine zymography and enzyme activity assays of supernatants of TNFalpha-treated cells demonstrate elevation of the pro- and active form of MMP-9 suggesting that increased expression of the protease cannot overcome the TNFalpha-inhibitory effect on cell invasion. Semi-quantitative RT-PCR analyses suggest that the cytokine may not alter mRNA levels of uPA and tPA. However, elevated expression of PAI-1 was detected by RT-PCR, as well as by Northern and Western blot analyses. Supplementation of PAI-1-blocking antibodies restored invasion of TNF-alpha-incubated HTR-8/SVneo cells through Matrigel-coated transwells. In addition, immunocytochemistry revealed nuclear accumulation of the p65 subunit of NFkappaB in the presence of the cytokine. EMSA indicated TNFalpha-induced binding of the inflammatory transcription factor to an NFkappaB consensus sequence and to the NFkappaB recognition site located in the PAI-1 promoter. The data suggest that TNFalpha restricts trophoblast invasion mainly by increasing the expression of PAI-1. Induction of the inhibitor may involve TNFalpha-stimulated activation of NFkappaB.

Cost effectiveness of a screen-and-treat program for asymptomatic vaginal infections in pregnancy: towards a significant reduction in the costs of prematurity.

OBJECTIVE: The purpose of this investigation was to determine the cost-saving potential of a simple screen-and-treat program for vaginal infection, which has previously been shown to lead to a reduction of 50% in the rate of preterm births. STUDY DESIGN: To determine the potential cost savings, we compared the direct costs of preterm delivery of infants with a birth weight below 1900g with the costs of the screen-and-treat program. We used a cut-off birth weight of 1900g because, in our population, all infants with a birth weight below 1900g were transferred to the neonatal intensive care unit. The direct costs associated with preterm delivery were defined to include the costs of the initial hospitalization of both mother and infant and the costs of outpatient follow-up throughout the first 6 years of life of the former preterm infant. The costs of the screen-and-treat program were defined to include the costs of the screening examination and the resulting costs of antimicrobial treatment and follow-up. All calculations were based on health-economic data obtained in the metropolitan area of Vienna, Austria. RESULTS: The number of preterm infants with a birth weight below 1900g was 12 (0.5%) in the intervention group (N=2058) and 29 (1.3%) in the control group (N=2097). The direct costs per preterm birth were found to amount to EUR (euro) 60262. Overall, the expected total savings in direct costs achieved by the screen-and-treat program and the ensuing 50% reduction in the number preterm births with a birth weight below 1900g amounted to more than euro 11 million. The costs of screening and treatment were found to amount to merely 7% of the direct costs saved as a result of the screen-and-treat program. CONCLUSION: A simple preterm prevention program, consisting of screening and antimicrobial treatment and follow-up of women with asymptomatic vaginal infection, leads not only to a significant reduction in the rate of preterm births but also to substantial savings in the direct costs associated with prematurity.

Labour and Childbirth After Previous Caesarean Section: Recommendations of the Austrian Society of Obstetrics and Gynaecology (OEGGG).

The new expert recommendation from the Austrian Society of Obstetrics and Gynaecology (OEGGG) comprises an interpretation and summary of guidelines from the leading specialist organisations worldwide (RCOG, ACOG, SOGC, CNGOF, WHO, NIH, NICE, UpToDate). In essence it outlines alternatives to the direct pathway to elective repeat caesarean section (ERCS). In so doing it aligns with international trends, according to which a differentiated, individualised clinical approach is recommended that considers benefits and risks to both mother and child, provides detailed counselling and takes the patient's wishes into account. In view of good success rates (60-85 %) for vaginal birth after caesarean section (VBAC) the consideration of predictive factors during antenatal birth planning has become increasingly important. This publication provides a compact management recommendation for the majority of standard clinical situations. However it cannot and does not claim to cover all possible scenarios. The consideration of all relevant factors in each individual case, and thus the ultimate decision on mode of delivery, remains the discretion and responsibility of the treating obstetrician.

Screening of gestational diabetes mellitus in early pregnancy by oral glucose tolerance test and glycosylated fibronectin: study protocol for an international, prospective, multicentre cohort trial.

INTRODUCTION: As the accurate diagnosis and treatment of gestational diabetes mellitus (GDM) is of increasing importance; new diagnostic approaches for the assessment of GDM in early pregnancy were recently suggested. We evaluate the diagnostic power of an 'early' oral glucose tolerance test (OGTT) 75 g and glycosylated fibronectin (glyFn) for GDM screening in a normal cohort. METHODS AND ANALYSIS: In a prospective cohort study, 748 singleton pregnancies are recruited in 6 centres in Switzerland, Austria and Germany. Women are screened for pre-existing diabetes mellitus and GDM by an 'early' OGTT 75 g and/or the new biomarker, glyFn, at 12-15 weeks of gestation. Different screening strategies are compared to evaluate the impact on detection of GDM by an OGTT 75 g at 24-28 weeks of gestation as recommended by the International Association of Diabetes and Pregnancy Study Groups (IADPSG). A new screening algorithm is created by using multivariable risk estimation based on 'early' OGTT 75 g and/or glyFn results, incorporating maternal risk factors. Recruitment began in May 2014. ETHICS AND DISSEMINATION: This study received ethical approval from the ethics committees in Basel, Zurich, Vienna, Salzburg and Freiburg. It was registered under http://www.ClinicalTrials.gov (NCT02035059) on 12 January 2014. Data will be presented at international conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT02035059.

Invasive trophoblasts generate regulatory collagen XVIII cleavage products.

Endostatin, the C-terminal proteolytic fragment of the noncollagenous domain 1 (NC1) of the basement membrane protein collagen XVIII, inhibits cell proliferation and migration. Placental and decidual expression of the peptide suggested a role in angiogenesis and/or extravillous trophoblast differentiation. Here, we demonstrate that supernatants of trophoblastic SGHPL-5 cells, purified first trimester villous trophoblasts and villous explant cultures contain proteases which in vitro cleave 20kDa endostatin from purified, recombinant NC1 domains. However, supernatants of decidual and villous fibroblasts failed to generate the 20kDa endostatin fragment. Moreover, we show that recombinant endostatin inhibits invasion of SGHPL-5 cells through Matrigel invasion chambers. Since mesenchymal cells but not trophoblasts produce collagen XVIII we suspect that invasive trophoblasts may produce endostatin upon contacting the extracellular matrix deposited by decidual stromal cells. Generation of endostatin through trophoblast-derived proteases could play a role in the regulation of trophoblast invasiveness.

Tissue-specific and ubiquitous basic helix-loop-helix transcription factors in human placental trophoblasts.

Tissue-specific class B basic helix-loop-helix (bHLH) transcription factors, dimerising with ubiquitously produced class A bHLH proteins, play a major role in murine trophoblast development. Here, we investigated expression patterns of class A and B bHLH factors in the human placenta and different trophoblast culture systems. Semi-quantitative RT-PCR and RNase protection assay revealed expression of the tissue-restricted factors Hash-2, I-mfa and Stra13 in placentae of early and late pregnancy, in purified villous trophoblasts as well as in invasive trophoblasts isolated from first trimester villous explant cultures. Accordingly, RNA in situ hybridisation localised Hash-2, I-mfa and Stra13 to the trophoblast epithelium, cell columns and extravillous trophoblasts invading maternal decidua. Villous stromal cells in situ and cultivated placental fibroblasts also produced I-mfa and Stra13 but failed to express Hash-2. The widely expressed class A proteins, E12/E47 were absent from all placental cell types while ITF-2 was restricted to placental stromal cells of early and late gestation. In contrast, HEB was identified in all trophoblast cell types using RT-PCR, Western blotting and immunohistochemistry. The negative HLH-regulators Id-1 and Id-2 lacking the DNA-binding domain, were detected in villous stromal cells and different cytotrophoblast subtypes but were absent from the syncytium. The data suggest that a complex interplay of activators (Hash-2, HEB) and repressors (Stra13, I-mfa) could be involved in extravillous trophoblast differentiation whereas downregulation of Id proteins could play a role in syncytialisation.

Preeclampsia and fetal loss in women with a history of venous thromboembolism.

A higher prevalence of risk factors for venous thromboembolism (VTE) has been found in women with preeclampsia and fetal loss. We investigated whether women with a history of VTE have a higher prevalence of pregnancy-associated complications compared with control subjects. In 395 patients with a history of VTE and in 313 control women, the prevalence of complications during pregnancy and the mean birth weight of viable infants were evaluated. The prevalence of pregnancy-induced hypertension and preeclampsia was higher in patients (5.1% and 3.0%, respectively) compared with control subjects (1.3% each). The odds ratio was 4.13 for pregnancy-induced hypertension (95% CI 1.4 to 12.22, P=0.0058) and 2.43 for preeclampsia (95% CI 0.78 to 7.6, P=0.133). Stillbirth was slightly more frequent in patients (4.3%) than in control subjects (3.2%); the difference was not statistically significant. Miscarriage was equally frequent in patients (21.8%) and control subjects (21.3%). The birth weight of viable infants born to patients was, on average, 109 g lower than that of the infants born to the control subjects (P=0.014) after adjustment for the mother's body mass index. Our study demonstrates that women with a predisposition to VTE have, overall, a good chance for a successful pregnancy outcome. However, the findings from our study support the assumption that a predisposition to venous thrombosis is associated with a higher risk for complications during pregnancy and lower infant birth weight.

Promoter elements and transcription factors involved in differentiation-dependent human chorionic gonadotrophin-alpha messenger ribonucleic acid expression of term villous trophoblasts.

Differentiation of primary villous cytotrophoblasts into syncytia is associated with increasing production of alpha and beta human CG subunits, which is predominantly governed at the level of messenger RNA expression. Here, we present a detailed study on the mechanisms involved in the differentiation-dependent regulation of the trophoblast-specific CGalpha gene promoter. Site-directed mutations in each of the five DNA-elements of the composite enhancer were performed to investigate the contribution of the individual regulatory sequences to the overall transcriptional activity of the promoter at two different stages of trophoblast in vitro differentiation. We show that deletion of one cyclic AMP response element (CRE) did not affect CGalpha promoter activity in cytotrophoblasts; however, it reduced transcription by 33% in differentiating cultures. Removal of both CREs almost abolished transcription at early and later stages of in vitro differentiation. Upon mutation the enhancer elements alphaACT, JRE, and CCAAT significantly decreased luciferase reporter transcription; however their contribution to the total promoter activity did not change during in vitro differentiation. Contrary to that, mutated TSE diminished promoter activity by 19% during 12 and 48 h of cultivation but reduced luciferase expression by 78% between 48 and 84 h of differentiation. In electrophoretic mobility shift assay, the TSE interacted with activating protein (AP)-2alpha in both primary trophoblasts and choriocarcinoma cells. While CRE-interacting proteins were detectable 12 h after isolation, the TSE-binding complex did not appear before 36 h of in vitro differentiation. During syncytium formation increasing protein expression of activating transcription factor (ATF)-1, cAMP response element-binding protein (CREB)-1, and AP-2alpha was observed on Western blots. Moreover, phosphorylated CREB-1 and ATF-1 accumulated between 24 and 78 h of trophoblast cultivation. By fluorescence immunohistochemistry, we show that CREB-1 was predominantly expressed in syncytiotrophoblasts, whereas ATF-1 and AP-2alpha localized to the syncytium and some cytototrophoblasts as well as to stromal and endothelial cells of the placental villus. Phosphorylated CREB-1/ATF-1 and the coactivator protein CBP were primarily detected in syncytial nuclei, suggesting the presence of functional, cAMP-dependent transcriptional complexes in the differentiated tissue. In agreement to the in vivo situation, phosphorylated CREB-1/ATF-1 were observed in nuclei of the differentiated trophoblast cultures. The activity of the CGalpha promoter as well as CREB-1/ATF-1 phosphorylation increased upon elevation of cAMP levels and overexpression of the catalytic subunit of protein kinase A. Additionally, we demonstrate that overproduction of the enzyme enhanced protein expression and binding of AP-2alpha to the TSE. We conclude that differentiation-dependent transcription of the CGalpha gene in villous trophoblasts is mainly governed by increasing expression of AP-2alpha and PKA-dependent phosphorylation of CREB-1 and ATF-1.

Molecular cloning of the human Hand1 gene/cDNA and its tissue-restricted expression in cytotrophoblastic cells and heart.

The basic helix-loop-helix (bHLH) factor Hand1 plays a role in the developing chicken heart and is required for trophoblast giant cell differentiation and cardiac looping of mouse embryonic development. Here, we report the cloning of the human Hand1 cDNA and gene from a heart-specific cDNA library and a genomic lambda-DNA library, respectively. We present the nucleotide sequence of a 1.75kb cDNA clone, encoding the presumptive 215 amino acid human Hand1 protein, and show homology comparison of the conserved bHLH region between different species. In vitro transcription-translation of Hand1 mRNA and analysis of protein size suggest that the Hand1 polypeptide is (post)translationally modified. By Southern blot analysis we demonstrate that the isolated genomic DNA clone harbours the entire Hand1 gene and describe molecular structure and sequences of the two 799 and 938bp exons and the single 1.56kb intron. The expression pattern of the mRNA in different human tissues revealed that Hand1 transcripts are restricted to the heart, suggesting that the protein could be required for cardiac-specific gene transcription and function in adults. Hand1 transcripts were undetectable in a non-tumorigenic villous trophoblast cell line, immunopurified cytotrophoblasts undergoing in vitro differentiation, and first trimester placental tissue, suggesting that the transcription factor is not involved in the development of villous and extravillous trophoblast cell lineages. Hand1 mRNA, however, was abundantly expressed in cytotrophoblastic Jeg-3 and BeWo cells, suggesting that Hand1 could be required for early trophoblast differentiation.

Interleukin-1 stimulates tumor necrosis factor-alpha (TNF-alpha) release from cytotrophoblastic BeWo cells independently of induction of the TNF-alpha mRNA.

Constitutive tumor necrosis factor-alpha expression (TNF-alpha) has been detected in first trimester trophoblast cells and differentiated syncytiotrophoblasts. However, molecules which induce TNF-alpha release from these cells and their mechanism of action have not been defined. We show for the first time that interleukin-1 (IL-1), a regulator of trophoblast development, induces TNF-alpha expression in proliferating cytotrophoblastic cells and purified term trophoblasts. Both IL-1alpha and beta stimulate TNF-alpha release from BeWo cells and TNF-alpha mRNA was transiently expressed. In growth-arrested/differentiated BeWo cells TNF-alpha mRNA was detectable without inducer, however, in the presence of IL-1beta TNF-alpha secretion was weakly stimulated compared to proliferating cells. Cycloheximide strongly increased IL-1beta-induced TNF-alpha mRNA concentration indicating that de novo protein synthesis is not required for TNF-alpha gene expression. However, treatment with cycloheximide did not prevent IL-1beta-stimulated release of TNF-alpha, indicating that the cytokine can regulate TNF-alpha secretion at a posttranslational level, independently of TNF-alpha mRNA induction. Besides demonstration of this novel mechanism of IL-1-stimulated TNF-alpha expression, our data indicate an important role of IL-1 in TNF-alpha production of cytotrophoblastic cells.

TNF-alpha/TNFRI in primary and immortalized first trimester cytotrophoblasts.

During the first trimester of pregnancy endogenous expression of tumour necrosis factor (TNF)-alpha has been detected in villous, as well as in proliferating and invading extravillous, trophoblasts suggesting that the protein could be involved in trophoblast differentiation. To gain insights into the putative role of the TNF-alpha signalling pathway, we investigated expression of its receptors, TNFR I and II, in first trimester placentae and early trophoblasts, and studied the influence of the cytokine on cell proliferation and apoptosis. ELISA and RT-PCR revealed secretion/expression of TNFRI protein/mRNA in immortalized ED27 cells and purified first trimester cytotrophoblasts, while soluble TNFRII was undetectable in cell culture supernatants. In agreement, immunohistochemical analyses of first trimester placentae showed that TNFRI is localized to the villous cyto- and syncytiotrophoblast, to the proliferating cytotrophoblasts of the cell islands and cell columns, as well as to extravillous cells invading decidual tissue. TNFRII, however, was absent in early trophoblast populations. Interleukin (IL)-1 and phorbol 12-myristate 13-acetate (PMA) induced shedding of TNFRI from ED27 and primary cells suggesting that under inflammatory conditions the soluble receptor protein may protect from cytotoxic effects of TNF-alpha. Upon incubation with increasing amounts of TNF-alpha no significant changes in DNA-content or cell numbers were found, suggesting that the cytokine does not augment proliferation of primary cytotrophoblasts. High doses of TNF-alpha, however, provoked growth arrest in ED27 cells as evaluated by cell counting, but did not induce necrosis/apoptosis as was assessed by TUNEL assay. In first trimester cells addition of elevated amounts of TNF-alpha resulted in the appearance of TUNEL-positive cells and an increase in caspase-3 enzyme activity suggesting that the TNF-alpha-dependent apoptotic cascade is executed in a portion of the early cytotrophoblasts.

Expression pattern of collagen XVIII and its cleavage product, the angiogenesis inhibitor endostatin, at the fetal-maternal interface.

Endostatin, the C-terminal fragment of the basement membrane protein collagen XVIII regulates epithelial cell migration and impairs tumour growth by inhibiting angiogenesis. Here, we investigated the expression pattern of collagen XVIII/endostatin in human placental and decidual tissues of various ages of gestation as well as in primary villous cytotrophoblasts, trophoblast cell lines, and villous explant cultures differentiating along the invasive pathway. RT-PCR analysis revealed production of collagen XVIII mRNA in total placenta and decidua of early and late pregnancy and in SGHPL-5 and HTR-8/Svneo cells. Collagen XVIII transcripts were absent from purified extravillous trophoblasts and syncytialising trophoblast cultures. Accordingly, an antibody against a protein domain common to different collagen XVIII isoforms detected the 180 kDa protein in villous and decidual tissue and cultivated placental fibroblasts but not in the different isolated trophoblast cell types. Immunohistochemical analyses localised collagen XVIII to villous basement membranes and to the endothelium as well as to placental and decidual stromal cells. Interestingly, expression of various forms of endostatin (20 and 26 kDa) was detected in placenta and decidua using Western blot analyses. Moreover, supplementation of recombinant endostatin increased MMP-2 expression in villous explant cultures and SGHPL-5 cells suggesting that the inhibitor may modulate extravillous trophoblast differentiation.

Expression of endothelial (type III) nitric oxide synthase in cytotrophoblastic cell lines: regulation by hypoxia and inflammatory cytokines.

Expression of endothelial nitric oxide synthase (eNOS) has been localized to the villous syncytiotrophoblasts suggesting that NO release from these cells could prevent platelet adhesion and aggregation in the intervillous space. Hypoxia- or inflammation-dependent changes in the release of this vasoactive substance may result in thrombus formation and altered vascular resistance which occur in the placental bed of pre-eclamptic patients. To evaluate the influence of low-oxygen tension and inflammation on eNOS production in the trophoblast steady-state eNOS mRNA and protein levels were investigated in cytotrophoblastic BeWo and Jeg-3 cells cultured at 3.5 per cent oxygen and/or in the presence of the pro-inflammatory cytokines IL-1 and TNF-alpha. By RT-PCR and immunocytochemistry we demonstrate that BeWo cells produce eNOS mRNA and protein while eNOS polypeptide was undetectable in JEG-3 cells. In BeWo cells addition of both cytokines decreases eNOS mRNA and protein abundancies within 24 h of incubation while each substance alone had no effect. Compared to controls, the amount of eNOS transcripts was found to be elevated at low-oxygen tension, however, cNOS protein was downregulated after 24 h in the hypoxic environment, as shown by immunocytochemistry and Western blot analysis. Forskolin and methotrexate, which induce biochemical differentiation/ growth arrest in choriocarcinoma cells, stimulate eNOS mRNA and protein synthesis, but cannot overcome the decline of eNOS polypeptide levels during hypoxic incubation. It is speculated that acute hypoxia and inflammation impair eNOS/NO production of the trophoblast in vivo, which might contribute to pathological conditions of gestational diseases.

Hypoxia downregulates continuous and interleukin-1-induced expression of human chorionic gonadotropin in choriocarcinoma cells.

The effects of hypoxia on JEG-3, BeWo, and JAr cells were investigated and it was demonstrated that choriocarcinoma cells can be used as a model to study the molecular mechanism of hypoxia-mediated repression of human chorionic gonadotropin (hCG). Cells were maintained under hypoxia (3.5 per cent O2) for 72 h without loss of variability, as demonstrated by the fact that 93-98 per cent of the cells excluded trypan blue. Up to 48 h, cell growth was not significantly influenced by hypoxia, and analysis by flow cytometry did not reveal major changes in cell cycle distribution. JEG-3, BeWo, and JAr cells which were grown for 48 h under hypoxia secreted 81, 67, and 71 per cent less hCG than cells cultivated under normoxic conditions. The extent of hCG reduction was dependent on the oxygen concentration. Moreover, release of the hormone from hypoxic JAr cells was not stimulated upon addition of interleukin-1 (IL-1). Treatment of JEG-3 cells with methotrexate (MTX) led to a 4.3-fold augmentation in hCG secretion and to an increase in the amount of G0/G1 cells. However, when cells were cultured in the presence of MTX and hypoxia, hCG secretion decreased 10-fold and beta hCG mRNA declined to almost undetectable levels suggesting that downregulation of beta hCG mRNA is the major cause of diminished hCG release under hypoxic conditions.

Adjunctive antibiotic treatment in preterm labor and neonatal morbidity: a meta-analysis.

OBJECTIVE: To estimate the effect of prophylactic antibiotics on neonatal mortality and morbidity in patients with preterm labor, based on a meta-analysis of seven published randomized clinical trials. DATA SOURCES: We searched 18 medical data bases, including MEDLINE from 1964 and EMBASE from 1974, to identify all literature included under preterm or premature labor and antibiotics. We scanned all abstracts from the computer printouts, the retrieved full-text reports, the references from each retrieved report, and review articles to determine whether studies met our inclusion criteria. METHODS OF STUDY SELECTION: The following criteria were used to select studies for inclusion: article-original published report written in English; study design-randomized controlled trial; population-patients with preterm labor, defined as labor before 37 weeks' gestation; intervention-antibiotic treatment; and one or more of the following outcomes-neonatal mortality, sepsis, pneumonia, respiratory distress syndrome, intraventricular hemorrhage, and necrotizing enterocolitis. TABULATION, INTEGRATION AND RESULTS: We analyzed study patients and methods, and abstracted quantitative outcome data. For each outcome, both odds ratio (OR) and 95% confidence interval (CI) were calculated. Seven trials, published between 1989 and 1995 included a total of 795 patients. Adjunctive antibiotic therapy appeared to reduce the risk of pneumonia (OR 0.45, 95% CI 0.12-1.72) and necrotizing enterocolitis (OR 0.38, 95% CI 0.14-1.08) and to increase the risk of neonatal mortality (OR 3.25, 95% CI 0.93-11.38), but it had no effect on neonatal sepsis (OR 0.98, 95% CI 0.34-2.83), respiratory distress syndrome (OR 0.93, 95% CI 0.54-1.87), and intraventricular hemorrhage (OR 1.01, 95% CI 0.20-5.10). None of the effects observed reached a significance level of P < .05. CONCLUSION: The results of this meta-analysis do not support the routine use of adjunctive antibiotic treatment in patients with preterm labor diagnosed on the basis of subjective uterine contractions and the resulting cervical changes.

Influence of delayed staging laparotomy after laparoscopic removal of ovarian masses later found malignant.

OBJECTIVE: To determine whether delayed laparotomy after attempted laparoscopic excision of an ovarian mass later found to be malignant has an impact on the stage of disease. METHODS: A questionnaire regarding laparoscopic management of ovarian masses later found to be malignant was mailed to all gynecologic departments in Austria. Of the 70 cases reported, laparotomy was performed after laparoscopy in 48 cases. In 24 of these cases, laparotomy was performed within 17 days of laparoscopy, whereas 24 cases involved a delay of more than 17 days. Twenty-two patients in whom laparotomy was performed immediately after laparoscopy were used as controls. RESULTS: In patients with borderline tumors who underwent laparotomy more than 17 days after laparoscopy, the odds ratio (OR) for International Federation of Gynecology and Obstetrics (FIGO) stage IIB-IV disease was 5.3 (95% confidence interval [CI] 0.40, infinity), compared with patients undergoing immediate laparotomy (multivariate analysis). Patients with invasive ovarian cancer who underwent laparotomy more than 17 days after laparoscopy had an OR of 9.2 (CI 0.92, 481) for stage IIB-IV disease compared with patients undergoing immediate laparotomy (multivariate analysis). In patients with borderline tumors, multivariate analysis showed that the timing of laparotomy is an independent prognostic factor for the stage of disease. In invasive ovarian cancer, none of the factors evaluated by multivariate analysis was found to be an independent prognostic factor for the distribution of disease stage. A delay between laparoscopy and laparotomy may affect adversely the distribution of disease stage. CONCLUSION: The timing of subsequent laparotomy was found to be a factor predictive of the distribution of disease stage.