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PubMed2003

Progesterone represses interleukin-8 and cyclo-oxygenase-2 in human lower segment fibroblast cells and amnion epithelial cells.

Labor is preceded by cervical ripening through upregulation of interleukin (IL)-1beta, IL-8, and increased prostaglandin synthesis via inducible type 2 cyclooxygenase (COX-2). Progesterone maintains myometrial quiescence during pregnancy. In this study, we examined the effects of IL-1beta and progesterone on IL-8 and prostaglandin E2 (PGE2) synthesis and IL-8 and COX-2 mRNA and promoter activity in amnion cells and lower segment fibroblast (LSF) cells. In both cell types, progesterone had no effect on basal IL-8 or PGE2 synthesis. In LSF cells, IL-1beta significantly increased IL-8 and PGE2 synthesis and COX-2 and IL-8 mRNA expression, but progesterone significantly attenuated these effects. In prelabor amnion cells, IL-1beta also increased IL-8 and PGE2 synthesis and both COX-2 and IL-8 mRNA and promoter expression; however, progesterone significantly attenuated these effects on IL-8 and PGE2 synthesis and COX-2 expression. In postlabor amnion cells, IL-1beta increased IL-8 and PGE2 synthesis and COX-2 expression, but progesterone did not attenuate the effect of IL-1beta upon IL-8 synthesis. Progesterone repression of IL-8 and COX-2 in LSF cells suggests that IL-8 and COX-2 have similar regulatory mechanisms in LSF cells and that progesterone may play a role in maintenance of cervical competence. The lack of effect of progesterone on IL-8 in postlabor cells may be the result of downregulation of the progesterone receptor during labor.

Amnion↗
PubMed1982

Characterization of specific steroid binding in human amnion at term.

In human fetal membranes (amnion and chorion) the appearance, during the last few weeks of pregnancy, of a protein which binds progesterone has been suggested as playing an important role in the inset of labor. The purpose of the present study was to quantitatively characterize specific binding in fetal membranes. Amnion, rather than chorion, was studied because it could be obtained free from contamination by other tissues. The cytosol (105,000 X g supernatant) was used after being extracted for 1 h with dextran-coated charcoal. The specific binding of cortisol in the cytosol, determined with a charcoal assay, was stable after storage at -20 degrees C or -60 degrees C and after heating for 1 h at 37 degrees C. It was reversible, and showed high-affinity, KD = 1 +/- 0.5 nM (mean +/- SEM, n = 6) and a large number of sites were found, 1497 +/- 666 fmoles/mg protein (mean +/- SEM, n = 6). In contrast, progesterone binding in the cytosol had a 10-fold lower affinity, KD = 12 +/- 5 nM (mean +/- SEM, n = 5) with 802 +/- 246 fmol bound/mg protein (mean +/- SEM, n = 5). A number of steroids were examined for their ability to compete with cortisol binding. The following were found to express affinities relative to cortisol which was considered to be 100: corticosterone (100), progesterone (10), cortisone (8), 5 alpha-pregnane-3, 20-dione (2), and 20 alpha-hydroxy-4-pregnen-3-one (less than 1). No competition was found for dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, R5020, medroxyprogesterone acetate, estrone or estradiol. These studies indicate that the steroid binding protein associated with human amnion at term exhibits a higher affinity for a cortisol and corticosterone than progesterone. Thus it is perhaps involved in the metabolism of glucocorticoids by the membranes, rather than in the local withdrawal of progesterone, as proposed by previous investigators.

Amnion↗
PubMed1992

Prostaglandin synthesis regulation in human amnion tissue: involvement of protein kinase C and dependence on ribonucleic acid and protein synthesis.

The role of protein kinase C (PKC) in the control of prostaglandin production by the human amnion was studied. Amnion membranes delivered spontaneously at term were minced and treated with phorbol esters, protein kinase inhibitors, cycloheximide, and actinomycin D; prostaglandin E2 (PGE2) output then was determined. Untreated tissue produced 3.97 +/- 1.13 ng PGE2/micrograms DNA/14 h (mean +/- SEM, n = 19). Phorbol dibutyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated PGE2 output up to 20-fold in a concentration-dependent manner with potencies corresponding to their efficacy as PKC activators. Four-beta-phorbol and 4-methoxy-TPA, which do not stimulate PKC, did not affect PGE2 output. Stimulation by TPA was blocked by staurosporine (IC50 = 57 nM) and H7; however, these PKC inhibitors did not decrease basal prostaglandin production. Cycloheximide inhibited basal and TPA-promoted PGE2 production and amino acid incorporation. Actinomycin D abolished TPA stimulation without decreasing unstimulated prostaglandin synthesis. These results show that amnion PGE2 production after labor is not maintained by PKC action, but PKC activation in this tissue causes a protein synthesis-dependent and RNA synthesis-dependent increase of PGE2 output. However, basal PGE2 production is dependent upon protein synthesis which, presumably, utilizes pre-existing mRNAs.

Amnion↗
PubMed1995

Interleukin 4 production by human amnion epithelial cells and regulation of its activity by glycosaminoglycan binding.

The pro-inflammatory molecules, tumor necrosis factor alpha (TNF alpha), interleukin 1 (IL-1), interleukin 6 (IL-6), and prostaglandin E2 (PGE2), are postulated to have a role in human pregnancy and parturition. The ability of interleukin 4 (IL-4) to suppress the production of TNF alpha, IL-1, IL-6, and PGE2 by activated monocytes prompted us to investigate a possible regulatory role for IL-4 in human gestation. Immunohistochemical techniques were used to show that human amnion epithelium stained positively for IL-4. Tissue from both the first (n = 5) and third (n = 46) trimester expressed immunoreactive IL-4, which was detected by the use of four antihuman IL-4 monoclonal antibodies. Analysis of mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA extracts of amnion epithelial cells indicated that they were the source of IL-4. One of the anti-IL-4 antibodies used stained IL-4 protein associated with the basement membrane of the amnion epithelium. The mechanism of this association was investigated. IL-4 was shown to be a heparin-binding cytokine, which would enable it to bind to components of the extracellular matrix. Thus, this study identified a previously undescribed cellular source of IL-4, implicating a role for IL-4 in human gestation. Additionally, glycosaminoglycan binding may regulate IL-4 activity in vivo.

Amnion↗
PubMed1999

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term.

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.

Amnion↗
PubMed2002

Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta.

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.

Amnion↗
PubMed1987

Use of human amnion for microvascular interpositional grafts.

A major problem associated with vascular grafting employing an artificial graft is the inflammatory response provoked by the graft and subsequent complications of classical acute rejection phenomena when the graft is implanted subcutaneously into human volunteers. The favorable results obtained by a preliminary study of subcutaneous implantation of amnion in our laboratory have led us to a prospective study to determine its value as a vascular graft. Tubed conduits of glutaraldehyde-treated amnion were hand constructed of varying diameters and lengths. They were employed as segmental interpositional grafts in experimentally created femoral and aortic arterial defects in Sprague-Dawley rats. Patency rates varied from 60 to 90 percent, with all grafts showing remarkable reendothelialization within 3 to 4 weeks postoperatively. Morphology, antigenic reaction, blood flow, and patency of the different experimental amnion grafts were evaluated and compared to appropriate controls.

Amnion↗
PubMed1985

Expression of c-fos in parietal endoderm, amnion and differentiating F9 teratocarcinoma cells.

The expression of the cellular proto-oncogene, c-fos, in extra-embryonic tissues of the mouse was investigated using a v-fos DNA probe and an affinity-purified antiserum raised against a C-terminal synthetic peptide. At 13.5 days of development, parietal endoderm--a tissue not previously studied using these methods--was found to express c-fos RNA at a higher level than the amnion or placenta. The previously reported dramatic increase in c-fos RNA levels in extra-embryonic membranes during gestation was found to be confined to the amnion. The antipeptide serum specifically recovered proteins with Mr values of 46,000 and 39,000 from extracts of parietal endoderm and amnion cells labelled for 15 min with 35S-methionine. On sodium-dodecyl-sulphate/polyacrylamide gel electrophoresis these proteins co-migrated with proteins immunoprecipitated using serum from rats inoculated with FBJ-MuSV-transformed cells (tumour-bearing rat serum). Pulse-chasing and 32P-labelling experiments showed that the protein with an Mr of 46,000 was rapidly converted into higher-molecular-weight phosphorylated derivatives. F9 teratocarcinoma stem cells differentiated into parietal-endoderm-like cells in response to treatment with retinoic acid and dibutyryl cyclic AMP. However, this differentiation was not accompanied by any large transient increase in c-fos RNA expression.

Amnion↗
PubMed2006

Amnion graft following hysteroscopic lysis of intrauterine adhesions.

AIM: To evaluate the safety and efficacy of amnion grafting after hysteroscopic lysis of intrauterine adhesions. METHODS: In a pilot study involving 25 patients with moderate or severe intrauterine adhesions, hysteroscopic adhesiolysis was followed by intrauterine application of a fresh amnion graft over an inflated balloon of a Foley's catheter for 2 weeks. Follow-up hysteroscopy was performed after 4 months. Outcome measures included recurrence of adhesions, achievement of normal menstrual flow, and improvement in the uterine length. RESULTS: Moderate (group A) and severe (group B) adhesions were found in 12 and 13 subjects, respectively. Uterine perforation occurred in two patients in group B, one treated conservatively and the other via laparoscopy. No clinical evidence of infection was observed, and spontaneous expulsion of the balloon occurred within days in three patients, with easy removal after 2 weeks in the rest of the subjects. Significant improvement in uterine length was found in both groups. Despite improvement, failure to achieve normal menstrual flow was found in 16.7% in group A versus 23.1% in group B. Follow-up hysteroscopy revealed adhesion reformation in 48%, all belonging to group B, all with minimal adhesions. Moderate adhesions were found in only two subjects with previous tuberculous endometritis. CONCLUSION: Hysteroscopic lysis of intrauterine adhesions with amnion grafting seems to be a promising procedure for decreasing recurrence of adhesions and encouraging endometrial regeneration. Randomized comparative studies are needed to validate its benefits, including reproductive outcome.

Adult↗
PubMed1996

Messenger RNA encoding thiol protein disulphide isomerase in amnion, chorion and placenta in human term and preterm labour.

OBJECTIVE: To investigate levels of messenger RNA (mRNA) encoding thiol protein disulphide isomerase, in human amnion, chorion and placenta during pregnancy and in relation to term and preterm labour. DESIGN: Amnion, chorion and placenta from 33 women delivered between 24 and 41 weeks of gestation were used in the study. SETTING: Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge Clinical School, Rosie Maternity Hospital, Cambridge. RESULTS: Women who were delivered spontaneously before 30 weeks of gestation had higher levels of mRNA encoding thiol protein disulphide isomerase in placenta and chorion than those who were delivered spontaneously after this time (placenta (P < 0.01, chorion P < 0.01) and compared with those who were delivered by elective caesarean section before 30 weeks of gestation (placenta (P < 0.01, chorion P < 0.05). In the group in whom spontaneous labour occurred, at all gestations studied, there were increased levels of mRNA encoding thiol protein disulphide isomerase in the placenta (P < 0.001) and chorion (P < 0.001) compared with the amnion. CONCLUSION: Changes in the steady state level of mRNA encoding thiol protein disulphide isomerase may play a role in the onset of preterm labour before 30 weeks of gestation.

Adolescent↗
PubMed1962

Actions of some anticholinesterases on the smooth muscle of the chick amnion.

The actions of both reversible and irreversible anticholinesterase drugs on the nerve-free smooth muscle of the chick amnion are described. The tertiary compound physostigmine and the irreversible inhibitor diisopropylfluorophosphonate were found to be active in causing contractions, whereas the quaternary compound neostigmine and the irreversible inhibitor Mipafox were only slightly active in this respect. Manometric studies showed that all four compounds were highly active, however, in inhibiting the cholinesterase found in the chick amnion. The effects produced by these compounds in the organ bath are thought to be due to the accumulation of endogenously produced acetylcholine within the tissue. Evidence for an intracellular action of endogenously produced acetylcholine in the smooth muscle of the chick amnion is discussed.

Acetylcholine↗
PubMed1983

Prostaglandin synthesis by human amnion is dependent upon extracellular calcium.

Prostaglandin (PG) E and F output was studied in collagenase-dispersed amnion cells to determine the effect of extracellular Ca2+ upon PG synthesis. In the presence of 2.5 mM CaCl2, PGE and PGF output (picograms per 10(5) cells per 3 h) by cells obtained at term prior to labour following elective cesarean section (CS) was 183 +/- 39 and 127 +/- 23, respectively. This increased to 435 +/- 111 (p less than 0.025) and 241 +/- 49 (p = 0.056) from cells obtained after spontaneous labour and delivery at term (SL). Exclusion of CaCl2 from the medium (plus 0.1 mM EGTA) significantly reduced (p less than 0.025) PGE output in CS and SL cells (83 +/- 22 and 183 +/- 47, respectively) and PGF output in CS cells (70 +/- 17). PGE output in both CS and SL cells was unchanged when CaCl2 concentrations in the medium were decreased from 2.5 to 0.25 mM, but significantly attenuated (p less than 0.01) when extracellular CaCl2 was decreased from 0.25 to 0 mM. The voltage-sensitive Ca2+ channel blocker, D-600, decreased PGE output in the presence of (2.5 mM) CaCl2 to levels observed in the absence of CaCl2. Ionophore A23187 restored PGE output in the presence of D-600 and Ca2+. PGE output from CS amnion cells was stimulated by A23187 and elevated extracellular K+ (40 mM). In each case, exclusion of CaCl2 from the medium eliminated the response. These results suggest that PG output by human amnion is dependent, in part, upon the presence of extracellular Ca2+ and that Ca2+ may enter the cell via a potential-sensitive mechanism.

Amnion↗
PubMed1988

The action of epidermal growth factor on human amnion prostaglandin E2 output.

The mechanism of stimulatory action of epidermal growth factor on term human amnion prostaglandin E2 production was studied. Monolayer cultures of amnion epithelial cells from spontaneous vaginal deliveries were preincubated for 24 h with serum-free media and treated with epidermal growth factor, calcium ionophore A23187 (4.5 microM), and arachidonate. Cumulative prostaglandin E2 output was not stimulated by epidermal growth factor (less than or equal to 200 ng/mL) or A23187 alone or the two added together. Pretreating the cells with epidermal growth factor for at least 2 h followed by A23187 or arachidonate (in the continuing presence of epidermal growth factor), however, stimulated prostaglandin E2 output up to 14-fold. The maximum effect of epidermal growth factor was attained at 1-10 ng/mL, while the EC50 was 0.2-0.32 ng/mL. Ionophore- or arachidonate-promoted prostaglandin E2 output was not stimulated by pretreatment with platelet-derived growth factor, fibroblast growth factor, and beta-transforming growth factor. Cycloheximide added before, at the same time as, or up to 30-60 min after epidermal growth factor completely abolished the stimulation. Epidermal growth factor did not affect [14C]arachidonate incorporation into cells or cell lipids. These results suggest that epidermal growth factor promotes, specifically and in a protein synthesis dependent manner, the conversion of arachidonate to prostaglandin E2. The provision of exogenous or endogenously liberated arachidonate is also necessary for enhanced amnion prostaglandin E2 production.

Amnion↗
PubMed1995

The controversial role of cAMP on amnionic prostaglandin release: effect of adenylate cyclase inhibition.

The suggested role of cAMP in the regulation of amnionic prostaglandin release was investigated using two adenylate cyclase inhibitors, MDL 12330A and SQ 22536. These substances exhibited a dose-dependent inhibitory effect on both amnionic enzyme and cAMP levels, but they did not influence prostaglandin E (PGE) release. In addition forskolin and IBMX (3-isobutyl-1-methylxanthine), two drugs known to increase cAMP levels, did not affect PGE output, while dibutyryl cyclic cAMP showed a dose-dependent inhibitory effect. On the basis of our data, the suggested role of amnionic adenylate cyclase in triggering prostaglandin release is not confirmed, and the pathway of phospholipase A2 activation at the onset of labor remains to be elucidated.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Mechanical stretching increases prostaglandin E2 in cultured human amnion cells.

Mechanical stretching increases synthesis and release of prostaglandin E2 (PGE2) in cultured amnion cells. The maximum level of PGE2 in stretched amnion cells is three times higher than that in nonstretched amnion cells. The maximum level of PGE2 in stretched cell medium is nine times higher than the maximal level of PGE2 in nonstretched cell medium. 3H-thymidine incorporation into DNA is 211 +/- 25 cpm/10(5) cells in nonstretched groups; that in stretched groups is 582 +/- 94 cpm/10(5) cells. There is a significant difference between the two groups. These results suggest that stretch stimulation facilitates prostaglandin production in fetal membranes, which may contribute to uterine contraction in labor.

Amnion↗
PubMed1993

Synthesis of oxytocin in amnion, chorion, and decidua may influence the timing of human parturition.

Despite the widespread clinical use of oxytocin (OT) as a potent and specific stimulant of labor, previous research data have not supported a role for OT in the physiology of normal human parturition. We have demonstrated synthesis of OT mRNA in amnion, chorion, and decidua using Northern blot analysis, ribonuclease protection assays, and in situ hybridization. Probes directed towards both the 3' and 5' ends of the gene have been used. Levels were highest in decidua with considerably less in chorion and amnion and very low levels in placenta. The transcript size in decidua appears to be 60-80 nucleotides smaller than the transcripts in amnion and chorion. OT gene expression in chorio-decidual tissues increased three- to fourfold around the time of labor onset. Estradiol stimulated synthesis of OT mRNA during in vitro incubation. These results support the hypothesis of a paracrine system involving OT and sex steroids within intrauterine tissues wherein significant changes could occur without being reflected in the maternal circulation. Such a paracrine system could rationalize a long-sought role for oxytocin in the physiology of human labor. These data may lead to novel approaches towards prevention or treatment or preterm labor.

Amnion↗
PubMed1991

Isolation and characterization of a human amnion epithelial cell line that expresses the pregnancy-specific beta 1-glycoprotein gene.

Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of 72K, 64K, 62K, and 54K. Together with the carcinoembryonic antigen, they form new members of the immunoglobulin superfamily. To study the molecular mechanisms that regulate expression of the PSG gene, we established a human amnion cell line, HAA58OD-8C, immortalized with an origin-defective simian virus-40 (SV40) temperature-sensitive A58 mutant virus. HAA58OD-8C cells were temperature sensitive for maintenance of transformation and expressed genes encoding PSG and the alpha- and beta-subunits of hCG. At the permissive temperature (33 C; transformed phenotype), they expressed low levels of PSG, hCG alpha, and hCG beta mRNAs and synthesized low levels of a 48K PSG polypeptide. At the nonpermissive temperature (39.5 C), HAA58OD-8C cells exhibited a differentiated phenotype, expressed increased levels of PSG, hCG alpha, and hCG beta mRNAs, and produced high levels of PSG polypeptides of 72K and 48K. Sodium butyrate induced PSG mRNA expression, and in the presence of butyrate, HAA58OD-8C cells produced high amounts of PSG polypeptides of 72K, 62K, and 48K. Ribonuclease protection analysis indicated that similar PSG transcripts were expressed by HAA58OD-8C cells and human term placenta. However, these amnion cells expressed selectively a certain population of PSG transcripts. Our results show that this amnion cell line provides a suitable model for studies of PSG gene expression and regulation.

Amnion↗