In-vitro secretion of proinflammatory cytokines by human amniochorion carrying hyper-responsive gene polymorphisms of tumour necrosis factor-alpha and interleukin-1beta.

The identification of polymorphisms in genes encoding proinflammatory cytokines that affect transcription or the secretion rate has opened new ways to understand the variation in responses to infection during pregnancy. In this study, human amniochorion carrying hyper-responsive alleles of tumour necrosis factor-alpha (TNF-alpha: TNF*2 at -308) and interleukin-1beta (IL-1beta: IL-1*2 at +3953) were stimulated in vitro with bacterial lipopolysaccharide (LPS) and compared with tissues carrying the common alleles (TNF*1 and IL-1*1). Fetal membranes carrying the TNF*1 allele displayed an identical dose-response pattern to tissues carrying a TNF*2 allele, except at the highest dose of LPS tested (50 ng/ml) there was a significantly greater production of TNF-alpha in the presence of a TNF*2 allele. Membranes carrying the IL-1*2 polymorphism secreted IL-1beta in a dose-response curve that was different from IL-1* tissues when challenged with 5, 10 and 50 ng/ml LPS. These observations support the hypothesis that reproductive tissues carrying hyper-responsive proinflammatory cytokine genes may over-respond to intrauterine infection secreting higher amounts of cytokines, which in turn, may lead to adverse pregnancy outcomes.

Association of polymorphism within the promoter of the tumor necrosis factor alpha gene with increased risk of preterm premature rupture of the fetal membranes.

OBJECTIVE: The rarer allele of a polymorphism within the promoter region at position -308 of the gene for tumor necrosis factor alpha is associated with increased gene transcription. In this study we tested the hypothesis that this rarer allele is associated with spontaneous preterm birth. STUDY DESIGN: We conducted a case-control study of women admitted to our labor and delivery unit. To assess data from a single racial group with a high incidence of preterm birth we restricted our analysis to African American women, who contributed 73.6% of the samples collected during the study period. Case patients (n = 55) were defined as women who were delivered before 37 weeks' gestation after idiopathic preterm labor or preterm premature rupture of membranes. Control subjects (n = 110) included women who were delivered after 37 weeks' gestation and had no history of preterm delivery. We also performed subgroup analyses of women with idiopathic preterm labor and delivery (n = 29) and women who were delivered preterm after preterm premature rupture of the fetal membranes (n = 26). RESULTS: Although carriers (homozygotes plus heterozygotes) of the rarer allele of the polymorphism at position -308 in the gene for tumor necrosis factor alpha were not significantly more common among women who were delivered preterm (n = 24/55, 44%) than among control subjects (n = 33/110, 30%, P =.08, odds ratio 1.81, 95% confidence interval 0.92-3.54), carriers of the rarer allele were more common among women who were delivered preterm after preterm premature rupture of membranes (n = 15/26, 58%) than among control subjects (P =.008, odds ratio 3.18, 95% confidence interval 1.33-7.83). CONCLUSIONS: Our results demonstrate an association between allelic variants of the polymorphism at position -308 in the gene for tumor necrosis factor alpha and preterm birth after preterm premature rupture of the fetal membranes. We hypothesize that host susceptibility to environmental factors, such as hyperresponsiveness of the gene for tumor necrosis factor alpha to genital tract infection, may promote preterm premature rupture of the fetal membranes and subsequent preterm delivery.

Hydrosalpinx fluid enhances human trophoblast viability and function in vitro: implications for embryonic implantation in assisted reproduction.

OBJECTIVE: To assess the effects of hydrosalpinx fluid on human cytotrophoblast viability and function in vitro. DESIGN: Human cytotrophoblasts obtained from third-trimester placentas were cultured in vitro with hydrosalpinx fluid, and cell viability and protein production were assayed. SETTING: A university hospital. PATIENT(S): Ten hydrosalpinx fluid samples obtained from seven women with clearly diagnosed hydrosalpinges. INTERVENTION(S): Recovery of hydrosalpinx fluid by transvaginal aspiration or at the time of surgery. MAIN OUTCOME MEASURE(S): Cell viability was assessed by the XTT assay. Secretion of trophoblast oncofetal fibronectin (tropho-uteronectin) and beta-hCG by cultured trophoblasts was determined by Western blot and ELISA of the culture media. RESULT(S): With increasing concentrations of hydrosalpinx fluid from 0% to 20%, there was a significant increase in trophoblast cell viability (1.63-fold increase in 20% hydrosalpinx fluid). Likewise, both Western blot and ELISA assays demonstrated a significant increase in tropho-uteronectin production by trophoblasts with increasing hydrosalpinx fluid concentrations (3.76-fold increase in 20% hydrosalpinx fluid). beta-Human chorionic gonadotropin production also increased significantly in the presence of hydrosalpinx fluid (3.31-fold increase in 20% hydrosalpinx fluid). CONCLUSION(S): These findings suggest that hydrosalpinx fluid improves human trophoblast viability in vitro and enhances the production of tropho-uteronectin and beta-hCG by these cells.

Fibronectinase activity in cultured human trophoblasts is mediated by urokinase-type plasminogen activator.

OBJECTIVE: Human trophoblast proteolytic activity is believed to have implications for early implantation events and maintenance of chorionic structural integrity later in gestation. Abnormal release of chorion-derived extracellular matrix proteins such as fibronectin may identify patients at risk for preterm labor and delivery. The aim of this study was to characterize the enzyme(s) potentially responsible for trophoblast-mediated proteolysis of fibronectin. STUDY DESIGN: Human term cytotrophoblasts were analyzed for their capacity to cleave fibronectin into discrete proteolytic fragments. Selective protease inhibitors were used to characterize trophoblast-derived enzymes with fibronectinase activity. Analysis and quantitation of fibronectin fragment release was determined by Western immunoblots and enzyme-linked immunosorbent assays. RESULTS: Fibronectinase activity in trophoblast cultures was found to be both cell mediated and secreted, with the release of discrete fibronectin fragments into the media. Cell-mediated proteolytic activity could be partially inhibited by serum, whereas conditioned media containing fibronectinase activity was completely inhibited by serum, a serine protease inhibitor, and a selective inhibitor of urokinase-type plasminogen activator. Digestion of fibronectin with pure urokinase produced a similar pattern of fibronectin fragments compared with fibronectinase-generated fragments. Immunodepletion of urokinase from trophoblast media abolished fibronectinase activity. CONCLUSIONS: Trophoblast-derived urokinase-type plasminogen activator has significant proteolytic activity in vitro with the capability of cleaving fibronectin into discrete fragments. In early pregnancy this activity could be part of the enzymatic cascade leading to uterine extracellular matrix remodeling and implantation. Later in pregnancy trophoblast derived urokinase could promote normal or inflammation-induced changes in the chorionic extracellular matrix.

Monoclonal antibody X18A4 identifies an oncofetal fibronectin epitope distinct from the FDC-6 binding site.

OBJECTIVE: Oncofetal fibronectin reactive with antibody FDC-6 has been associated with trophoblastic implantation and chorion structural stability. Abnormal release of this fibronectin into cervical and vaginal secretions has identified patients at risk for preterm labor and delivery. The aim of this study was to determine whether trophoblast-derived oncofetal fibronectin contains other novel epitopes distinct from the FDC-6 binding site. STUDY DESIGN: Antitrophoblast fibronectin hybridomas were generated and screened by comparative immunoassays. One specific monoclonal antibody, X18A4, was identified and compared with antibody FDC-6 by immunocytochemical and immunoblot analyses. Both antibodies were also evaluated in "sandwich"-type double monoclonal immunosorbent assays. RESULTS: X18A4 and FDC-6 bind avidly and noncompetitively to distinct epitopes within oncofetal fibronectin. They exhibit similar immunohistochemical staining of the extracellular matrix within placental tissue, ovarian epithelial tumors, and cultured trophoblasts. However, in contrast to FDC-6, X18A4 has no detectable binding activity to human plasma fibronectin, and its binding to oncofetal fibronectin was unaffected by enzymatic deglycosylation. Immunoblot analyses of oncofetal fibronectin proteolytic digests suggest that X18A4 binds near or within the alternatively spliced type III connecting segment domain. CONCLUSIONS: X18A4 identifies and binds with high affinity to a new epitope within oncofetal fibronectin, distinct from the FDC-6 binding site. Because X18A4 displays no detectable binding to plasma fibronectin, it could be used as an important adjunctive antibody for enhancing the specificity of clinically based oncofetal fibronectin diagnostic assays.

[Role of metalloproteases of extracellular matrix in the premature rupture of fetal membranes: a novel physiopathogenic model]. / Participación de las metaloproteasas de matriz extracelular en la ruptura prematura de membranas fetales: un modelo fisiopatogénico novedoso.

Experimental proofs are presented, that involve the participation of the metalloproteinases family, of extracellular matrix in the genesis of premature rupture of membranes. The expression of this group of enzymes, which presents in normal conditions of labor appears in the RPM without relation with gestational age. This phenomenon is not presented as an isolated example of participation of metalloproteinases in events related to labor and delivery, as is very well known its participation in maturation of uterine cervix preceding product expulsion, and that consists in similar mechanisms to the ones now chosen for the maturation of fetal membranes. The working hypothesis in pathologic conditions, implies, then, the activation of a normal system in an inadequate moment that results in premature rupture of membranes.