Studies of normal and prematurely ruptured human amniotic membranes: low calcium and magnesium in prematurely ruptured membranes

Prematurely ruptured amniotic membranes at term, and membranes from normal term pregnancies were studied. The membranes were analyzed for neutral sugars, hexosamines, sialic acid, uronic acid, DNA, RNA, cholesterol, phospholipids, calcium, magnesium, sodium and potassium. The only significant difference found between the normal and the prematurely ruptured membranes was a significantly lower concentration of calcium and magnesium in the latter group. (Calcium 0.32 +/- 0.07, vs. 0.24 +/- 0.07, p less than 0.05; Magnesium 0.30 +/- 0.19 vs. 0.15 +/- 0.04, p less than 0.05; in mg/100 mg protein). The possible metabolic effects of low calcium and magnesium may be of relevance to the pathogenesis of premature rupture of the amniotic membranes.     

Plasminogen binding by human amniochorion. A possible factor in premature rupture of membranes

Forty-six human fresh amniochorion membranes obtained at cesarean section were found to contain from 0 to 5% dead cells in the amniotic epithelial layer by direct counting and spectrophotometric analysis of trypan blue extracts. With the use of a double marker system it was discovered that many of the dead cells failed to bind the DNA-chelating fluorochrome propidium iodide but reacted with fluorescein isothiocyanate-labeled antibodies to human plasminogen. In addition, both fresh and cultured human amniotic epithelial cells that had plasma membranes damaged by either cryogenic shock or cytocentrifugation specifically bound plasminogen from serum, plasma, or amniotic fluid to cytoplasmic structures. Binding did not occur in other control proteins, but plasminogen was bound from very dilute solutions, suggesting specific and tight binding inside the cell. We propose that such plasminogen can be activated to plasmin within the cell by either plasminogen activators or lysosomal proteases and that this sets into motion a progression of events that are potentially damaging for the amniochorion, perhaps being of relevance in the pathophysiology of premature rupture of the membranes in human pregnancy.     

Activity of 72-kDa and 92-kDa matrix metalloproteinases in placental tissues of cows with and without retained fetal membranes

This study compared the activity of matrix metalloproteinases (MMP-2 and MMP-9) in retained and non-retained bovine placenta. The activities of MMPs and their zymogens were measured in fetal and maternal placental tissues from control cows (group B) and animals affected with retention of fetal membranes (group A) using a zymography technique on 10 per cent SDS polyacrylamide gels. The activity of proMMP-9 detected only in the maternal part of the placenta was lower in group A than in group B. ProMMP-2 activity was higher in group A than in group B in both tissues. The active forms of MMP-2 were observed in the maternal and fetal part of placenta in group B, but only the 68-kDa form was detected in the placental tissues of group A. The differences in enzyme activity between the groups and the lack of 64- and 60-kDa active forms of MMP-2 in the maternal and fetal parts of the retained placenta may have influenced the hydrolysis of collagen and the proper release of fetal membranes.     

Soluble tumor necrosis factor receptor II and soluble cell adhesion molecule 1 as markers of tumor necrosis factor-alpha release in preeclampsia

BACKGROUND: The purpose of this case-controlled study was to investigate whether plasma concentrations of TNF-receptors I and II and tumor necrosis factor-alpha-induced cell adhesion molecule 1 VCAM-1 could serve as more sensitive markers of tumor necrosis factor-alpha release in preeclamptic women than a direct measurement of circulating tumor necrosis factor-alpha. METHODS: Plasma concentrations of soluble tumor necrosis factor receptor I and II, immunoreactive tumor necrosis factor-a and soluble cell adhesion molecule VCAM-1 were determined in 21 patients with severe proteinuric preeclampsia (23-35 weeks' gestation) and 21 gestational age-matched normotensive controls by enzyme-linked immunoassays. Concentrations of bioactive tumor necrosis factor-alpha were assessed by the WEHI 164 bioassay. Data were statistically evaluated by Wilcoxon's rank sum and sign tests, and Spearman's test was used to evaluate clinical and biochemical correlations. RESULTS: Bioactive tumor necrosis factor-alpha was detected in 19 of 21 preeclamptic and 18 of 21 normotensive women, with no difference in plasma concentrations between both groups. Immunoreactive tumor necrosis factor-alpha, soluble TNF-receptors and soluble cell adhesion molecule VCAM-1 were significantly increased in plasma of preeclamptic patients, and a statistically significant positive correlation was observed between immunoreactive tumor necrosis factor-alpha and TNFRII. In preeclamptic patients a statistically significant negative correlation was observed between TNFRII and platelet count, and between soluble cell adhesion molecule VCAM-1 and birthweight ratio. CONCLUSION: These results show that plasma concentrations of soluble tumor necrosis factor receptor II and soluble cell adhesion molecule VCAM-1 reflect the release of tumor necrosis factor-alpha and provide sensitive markers of excessive release of this cytokine in preeclampsia.     

人胎膜组织中E26转录因子1的表达及其与胎膜早破的关系

目的 检测人胎膜组织中E26转录因子1(Ets-1)的表达及定位,探讨其与胎膜早破发生的关系.方法 选择2007年2-11月在重庆医科大学附属第一医院住院分娩的产妇100例为研究对象,按足月与否、有无胎膜早破、分娩方式将其分为早产临产、未足月胎膜早破(PPROM)、足月临产、足月胎膜早破(TPROM)组,以足月择期刮宫产产妇为对照组,每组各20例,分娩后采集其胎膜组织,用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(SP)法检测Ets-1蛋白的表达水平及定位,每组选取6例以逆转录(RT)-PCR技术检测Ets-1 mRNA的表达水平,并进行半定量分析.结果 (1)各组胎膜组织中Ets-1 mRNA的表达:早产临产、PPROM、足月临产、TPROM、对照组胎膜组织中Ets-1mRNA表达水平分别为0.342±0.016、0.603±0.027、0.325±0.013、0.582±0.075、0.139±0.012,PPROM组和TPROM组均高于对照组.分别比较,差异均有统计学意义(P<0.05);早产临产组与足月临产组,PPROM组与TPROM组比较,差异均无统计学意义(P>0.05).(2)Ets-1蛋白在胎膜组织的定位:Ets-1蛋白集中在羊膜和绒毛膜的间质层、滋养层细胞的胞质和胞核中表达;细胞内可见清晰的棕黄色颗粒.在羊膜上皮细胞中未见Ets-1蛋白表达.(3)各组胎膜组织中Ets-1蛋白的表达:早产临产、PPROM、足月临产、TPROM、对照组胎膜组织中Ets-1蛋白表达水平分别为0.552±0.018、2.853±0.174、0.538±0.042、2.731±0.090、0.214±0.013,PPROM组与TPROM组均高于对照组,分别比较,差异均有统计学意义(P<0.05);但早产临产组与足月临产组,PPROM组与TPROM组胎膜组织分别比较,差异均无统计学意义(P>0.05).结论 Ets-1在人类胎膜组织中有表达,且在胎膜早破时表达水平升高.     

Ets-1和MMP-9在胎膜早破中的表达

目的研究转录因子Ets-1和基质金属蛋白酶-9（MMP-9）在胎膜早破中的表达和作用。方法胎膜早破（PROM）患者67例,胎膜未破裂组70例,分娩后取破口处胎膜组织,采用免疫组织化学法染色,并使用病理影像多媒体图文操作系统对Ets-1和MMP-9阳性表达物的面积积分光密度（AIOD）进行半定量分析。结果①Ets-1在胎膜滋养层细胞核及胞浆中均有表达,且细胞核表达明显;MMP-9在胎膜滋养层细胞中的表达,主要位于细胞浆中;②Ets-1在胎膜早破组（0.0373±0.0119）中高表达,与胎膜未破裂组（0.0062±0.0089）比较,差异有高度统计学意义（P〈0.001）;③MMP-9在胎膜早破组（0.0928±0.0453）中高表达,与胎膜未破裂组（0.0345±0.039）比较,差异有高度统计学意义（P〈0.001）。结论Ets-1和MMP-9在人类胎膜上均有表达,且在胎膜早破中高表达且具显著相关性,表明Ets-1可能通过上调MMP-9的基因表达量使胎膜细胞外基质（ECM）的降解加速致胎膜紧张度降低,引发胎膜早破。     

肿瘤坏死因子对妊高征的致病作用及与内皮素的关系

目的：探讨肿瘤坏死因子在妊高征发病中的作用及与内皮素的关系。方法：采用放射免疫法测定了４６例妊高征患者（妊高征组）和２０例正常晚期妊娠妇女（对照组）血浆肿瘤坏死因子（ＴＮＦ）和内皮素（ＥＴ－１）的质量浓度。结果，妊高征组中，重度患者ＴＮＦ和ＥＴ－１质量浓度明显高于对照且和轻度患者（Ｐ〈０．０５及Ｐ〈０．０１）；产后７２小时两者质量浓度均明显下降。对照组血浆ＴＮＦ与ＥＴ－１质量浓度无相关性，而妊高征     

Use of insulin like growth factor binding protein-1 in the diagnosis of ruptured fetal membranes

OBJECTIVE: The benefit of insulin like growth factor binding protein-1 (IGFBP-1) in diagnosing ruptured fetal membranes in cases in which the diagnosis is clinically doubtful, is investigated. DESIGN: A total of 83 patients with clinically doubtful rupture of fetal membranes were included, and treated as usual. The clinical diagnosis, the amniotic fluid crystallization test, and IGFBP-1 detection were performed on all patients and compared with the defined gold standard. The sensitivity, specificity, positive predictive value, and negative predictive value of each test were calculated. RESULTS: In all, 27 patients (33%) had ruptured fetal membranes at the time of inclusion. The clinical diagnosis of the attending obstetrician showed a higher predictive value of both a positive and negative test result than both IGFBP-1 detection, and the amniotic fluid crystallization test. CONCLUSIONS: As the clinical diagnosis showed the highest sensitivity and specificity, IGFBP-1 detection has no additional benefit to ascertain the diagnosis of ruptured fetal membranes in cases in which the diagnosis was clinically doubtful.     

Levels of tumor necrosis factor-alpha and interleukin-2 in sera of twins delivered prematurely and at term

The concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2) was estimated in first day of live in sera of premature (n = 8 pair) and in term delivered twins (n = 12 pair). One of premature delivered newborns was born vaginally 48 hours after first twin. Concentrations of TNF-alpha were approximately 4-times higher in sera of premature delivered in comparison to term delivered twins and a highest, more than 5-times higher level, was detected in serum of premature newborn born a 48 hours after first twin. Concentrations of IL-2 in sera of premature delivered were approximately 2.5-times higher than in term delivered twins and not significantly variations were observed in delayed delivered twin.     

Expression of receptors for tumor necrosis factor in human placenta at term.

The biological effects of tumor necrosis factor (TNF) are mediated through its interaction with high affinity receptors on target cells. Secretion of soluble cytokine receptors has been suggested as a mechanism of regulating cytokine activity in vivo. In a previous study we detected soluble TNF receptors (TNFRs) in amniotic fluid and urine samples from pregnant women, suggesting that secretion of soluble TNFRs may provide a mechanism for protection of the fetus against TNF action during pregnancy. In the present study, TNFR containing cells in cryostat sections from normal placentas at term were evaluated by monoclonal antibodies against the 55 kD--and the 75 kD TNFR in an indirect immunofluorescence technique. The 55 kD TNFR was expressed by the villous syncytiotrophoblasts, by vascular endothelial cells, by some decidual cells and by occasional cells in the placental stroma. Staining for the 75 kD TNFR was confined to the vascular endothelial cells, a relatively small number of stromal cells and decidual cells, whereas the villous syncytiotrophoblasts were negative. The abundant expression of TNFRs in placental tissue suggests: 1. That a considerable number of the placental cells are receptive to the regulatory activities of TNF; 2. That placental cells may be the cellular origin of soluble TNFRs secreted during pregnancy.     

Release of tumor necrosis factor alpha by human peritoneal macrophages in response to toxic shock syndrome toxin-1

We examined the release in vitro of tumor necrosis factor-alpha (TNF-alpha) by peritoneal macrophages and peripheral blood monocytes following incubation with toxic shock syndrome toxin-1 (TSST-1). We obtained peritoneal macrophages from 22 women at laparoscopy and peripheral blood monocytes from four healthy women during both the midfollicular and midluteal phases of the menstrual cycle. The samples were incubated for 24 hours at 37 C with 10(-2)-10(4) ng/mL of TSST-1 or 10(4) ng/mL of bacterial endotoxin. Tumor necrosis factor-alpha activity was determined with a bioassay using an actinomycin D-sensitized WEHI-164 murine fibrosarcoma cell line. Twenty-four-hour incubation with TSST-1 resulted in a dose-dependent release of TNF-alpha by both peritoneal macrophages (maximal response 554 +/- 97 U of activity) and peripheral blood monocytes (maximal response 478 +/- 81 U of activity). We observed enhanced TNF-alpha release by peritoneal macrophages from women with endometriosis, compared with those without endometriosis, at a concentration of 10(4) ng/mL of TSST-1 (704 +/- 134 versus 354 +/- 103 U of activity; P less than .05). These data support the theory that the metabolic and physiologic derangements of perimenstrual toxic shock syndrome may be partially mediated by TNF-alpha released by peritoneal macrophages as a result of exposure to TSST-1.     

Inhibitory effect of interferon and tumor necrosis factor on human luteal function in vitro.

OBJECTIVE: To investigate whether immunological mechanisms may be involved in human luteal function. DESIGN: The effects of the cytokines, interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) on steroidogenesis by human luteal cells were examined in vitro. The dispersed human luteal cells, obtained from a total of 17 women at laparotomy, were cultured separately in the presence or absence of human chorionic gonadotropin (hCG) and IFNs/TNF-alpha with the medium being replaced at 48 hours. The medium was collected at 48 and 96 hours for steroid assays. RESULTS: The IFN-alpha had no significant effect on the production of estradiol or progesterone (P), whereas a dose-related inhibition of basal, as well as hCG-stimulated P formation, was observed after the addition of IFN-gamma (10 to 1,000 U/mL). Progesterone production was inhibited to about 45% of the control at 48 hours and even lower at 96 hours (n = 6, P < 0.001). The combination of IFN-gamma and low doses of TNF-alpha induced a further significant inhibition, whereas there was no effect of TNF-alpha alone. This inhibitory effect of IFN-gamma could be completely neutralized with a monoclonal antibody to IFN-gamma. Incubation with the antibody alone increased the production of P from luteal cells in culture, suggesting a local tonic inhibitory action of endogenous IFN-gamma. CONCLUSION: Interferon-gamma and TNF-alpha, whose function classically is known as antiviral, also may play a role in human luteal regression by inhibiting luteal P production.     

Vaginal fluid prolactin: a reliable marker for the diagnosis of prematurely ruptured membranes. Comparison with vaginal fluid alpha-fetoprotein and placental lactogen

The aim of this study is the evaluation of the reliability of vaginal fluid (VF) prolactin (PRL) for detecting prematurely ruptured membranes (PROM) and the comparison of this marker with vaginal fluid alpha-fetoprotein (AFP) and placental lactogen (HPL). In 21 pregnant women with recent or prolonged PROM from 20 to 41 weeks' gestation, in whom intact membranes were never found subsequently VF- and MS-PRL, -AFP and -HPL were measured by enzyme immunoassays, which are sensitive and very rapid. The same markers were also measured in MS, VF and urine samples (U) in 12 pregnant women of the same gestational age, without PROM, in whom the membranes were ruptured later during labor. In PROM, independently of prematurity and duration of PROM VF-PRL levels were significantly higher (2-10-fold) than the paired MS-PRL (p less than 0.0001) and ranged from 130 to 2315 ng/ml. In contrast, VF-PRL and urine PRL concentrations in pregnancies without PROM were very low or undetectable (range: 0-5 ng/ml and 0.15-1 ng/ml, respectively). Vaginal fluid AFP values in PROM from 20th to the 33rd week of pregnancy were significantly higher (5-50-fold) than the paired MS-AFP (p less than 0.01) and ranged from 103 to 5500 ng/ml. In PROM after the 33rd week of pregnancy, VF-AFP values were either lower (1/3), or equal to, or even higher (up to 2-fold) than MS-PRL. On the contrary in pregnancies with intact membranes, VF-AFP were always less than 9 ng/ml and urine AFP was undetectable (range: 0.2-1.1 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene expression and protein localization of TLR-1, -2, -4 and -6 in amniochorion membranes of pregnancies complicated by histologic chorioamnionitis

Objectives

To determine whether histologic chorioamnionitis is associated with changes in gene expression of TLR-1, -2, -4 and -6, and to describe the localization of these receptors in fetal membranes.

Study design

A total of 135 amniochorion membranes with or without histologic chorioamnionitis from preterm or term deliveries were included. Fragments of membranes were submitted to total RNA extraction. RNA was reverse transcribed and the quantification of TLRs expression measured by real time PCR.

Results

All amniochorion membranes expressed TLR-1 and TLR-4, whereas 99.1% of membranes expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expressions were significantly higher in membranes with histologic chorioamnionitis as compared to membranes without chorioamnionitis in preterm pregnancies (p = 0.003 and p < 0.001, respectively). Among the membranes of term pregnancies there were no differences in the expressions of such receptors regardless of inflammatory status. Regarding TLR-4 and TLR-6 expression, there was no difference among membranes with or without histologic chorioamnionitis, regardless gestational age at delivery. TLR-1, TLR-2, TLR-4 and TLR-6 expressions were observed in amniotic epithelial, chorionic and decidual cells.

Conclusion

Amniochorion membranes express TLR-1, TLR-2, TLR-4 and TLR-6 and increased expression of TLR-1 and TLR-2 is related to the presence of histologic chorioamnionitis in preterm pregnancies. This study provides further evidence that amniochorion membranes act as a mechanical barrier to microorganisms and as components of the innate immune system.     

Differential induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand in human ovarian carcinoma cells

OBJECTIVES: In this study, we examine the sensitivity of a panel of ovarian carcinoma cells, which includes four primary ovarian cancer cell samples, and four normal ovarian epithelium samples to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We also examine the intracellular regulation of TRAIL-mediated apoptosis. METHODS: The sensitivity to TRAIL was determined by short-term survival assays on seven ovarian carcinoma cell lines, four primary samples of ovarian cancer, and four normal ovarian epithelium samples. We assessed the activation of the apoptotic pathway in TRAIL-resistant and -sensitive tumor cells. The expression of TRAIL receptors was determined by flow cytometry. The protein expression of FADD, XIAP, caspase-8, caspase-3, BAX, and c-FLIP were determined by immunoblot analyses. RESULTS: We show that ovarian cancer cells display variable sensitivity to TRAIL-induced apoptosis although most cell lines have similar sensitivity to cisplatin. Normal ovarian epithelium samples were mostly sensitive to TRAIL. In sensitive cells, TRAIL induced caspase-8-dependent apoptosis, which subsequently led to activation of caspase-3. Both sensitive and resistant cells expressed caspase-8, caspase-3, FADD, XIAP, and c-FLIP at similar levels. A significant enhancement in cell death was observed in TRAIL-resistant cells when c-FLIP(L) levels were downregulated by RNA interference. CONCLUSIONS: These data suggest that sensitivity to TRAIL and chemotherapy does not necessarily correlate in human ovarian cancer cells. Cancerous cells isolated from patients with ovarian cancer show variable sensitivity to TRAIL but most normal ovarian epithelial cells are sensitive. In human ovarian cancer cells, c-FLIP(L) may participate to the regulation of the TRAIL signaling cascade.