Expression of β defensins 1, 3 and 4 in chorioamniotic membranes of preterm pregnancies complicated by chorioamnionitis

Objective

To quantify the expression of human β-defensins (HBDs) 1, 3 and 4 in chorioamniotic membranes in pregnancies complicated by prematurity associated with histologic chorioamnionitis.

Study design

The study group included 23 fragments of chorioamniotic membranes with histologic chorioamnionitis from women with preterm premature rupture of membranes (PPROM) and preterm labor (PTL) with intact membranes, who had preterm deliveries. As a control group, 30 chorioamniotic membranes without chorioamnionitis at the same gestational age as those in the study group were included. Chorioamniotic membranes were collected for histopathological analyses, and HBD mRNA expression quantification was analyzed by real time PCR. Comparisons were performed using the Mann-Whitney or Kruskal-Wallis tests and all the women were informed and provided written consent.

Results

The expression of HBDs mRNA in the fetal membranes was similar in patients without histologic chorioamnionitis (HBD1: 0.7-fold, HBD3: 0.9-fold, HBD4: 0.3-fold) compared to patients with chorioamnionitis (HBD1: 1.1-fold, HBD3: 0.9-fold, HBD4: 0.4-fold; p > 0.05). Regarding the gestational complications that resulted in premature delivery, PPROM or PTL, the relative quantification of HBD1, HBD3 and HBD4 showed no statistically significant differences in either the absence or presence of chorioamnionitis. Among patients with histologic chorioamnionitis, patients with PPROM (HBD1: 2.7-fold, HBD3: 0.3-fold, HBD4: 0.7-fold) presented similar mRNA expression to those with PTL (HBD1: 0.7-fold, HBD3: 1.2-fold, HBD4: 0.13-fold; p > 0.05).

Conclusions

Chorioamniotic membranes are sources of β defensins 1, 3 and 4; however, considering the sample size and the methodology applied, mRNA concentrations were not related to the presence of histologic chorioamnionitis.     

Interleukin-33 in the human placenta

Objective: Interleukin-33 (IL-33) is the newest member of the IL-1 cytokine family, a group of key regulators of inflammation. The purpose of this study was to determine whether IL-33 is expressed in the human placenta and to investigate its expression in the context of acute and chronic chorioamnionitis. Methods: Placental tissues were obtained from five groups of patients: 1) normal pregnancy at term without labor (n = 10); 2) normal pregnancy at term in labor (n = 10); 3) preterm labor without inflammation (n = 10); 4) preterm labor with acute chorioamnionitis and funisitis (n = 10); and 5) preterm labor with chronic chorioamnionitis (n = 10). Immunostaining was performed to determine IL-33 protein expression patterns in the placental disk, chorioamniotic membranes, and umbilical cord. mRNA expression of IL-33 and its receptor IL1RL1 (ST2) was measured in primary amnion epithelial and mesenchymal cells (AECs and AMCs, n = 4) and human umbilical vein endothelial cells (HUVECs, n = 4) treated with IL-1β (1 and 10?ng/ml) and CXCL10 (0.5 and 1 or 5?ng/ml). Results: 1) Nuclear IL-33 expression was found in endothelial and smooth muscle cells in the placenta, chorioamniotic membranes, and umbilical cord; 2) IL-33 was detected in the nucleus of CD14+ macrophages in the chorioamniotic membranes, chorionic plate, and umbilical cord, and in the cytoplasm of myofibroblasts in the Wharton’s jelly; 3) acute (but not chronic) chorioamnionitis was associated with the presence of IL-33+ macrophages in the chorioamniotic membranes and umbilical cord; 4) expression of IL-33 or IL1RL1 (ST2) mRNA in AECs was undetectable; 5) IL-33 mRNA expression increased in AMCs and HUVECs after IL-1β treatment but did not change with CXCL10 treatment; and 6) IL1RL1 (ST2) expression decreased in AMCs and increased in HUVECs after IL-1β but not CXCL10 treatment. Conclusions: IL-33 is expressed in the nucleus of placental endothelial cells, CD14+ macrophages, and myofibroblasts in the Wharton’s jelly. IL-1β can induce the expression of IL-33 and its receptor. Protein expression of IL-33 is detectable in macrophages of the chorioamniotic membranes in acute (but not chronic) chorioamnionitis.     

Progesterone receptor localization and isoforms in myometrium,decidua, and fetal membranes from rhesus macaques: evidence for functional progesterone withdrawal at parturition

OBJECTIVE: It is not known whether withdrawal of progesterone (P) action is a prerequisite for parturition in women or in nonhuman primates because concentrations of circulating progesterone or progesterone receptors (PR) in myometrium and decidua do not decrease before delivery. To examine this potentially important regulatory mechanism, we determined PR isoforms, PR localization, and mRNA in myometrium, decidua, and fetal membranes from rhesus monkeys during pregnancy and in spontaneous labor at term.METHODS: Gestational tissues were obtained midpregnancy (day 80-100), late pregnancy (day 130-145), and during spontaneous labor at term (day 161-167). Samples of rhesus monkey myometrium, decidua, chorion-decidua, and amnion were collected and analyzed for total nuclear and cytosolic PR by competitive binding assay. Progesterone receptor isoforms were identified and quantified by Western blot analysis, and PR mRNA was determined by a specific ribonuclease protection assay. Nuclear PR was localized by immunohistochemistry with monoclonal anti-PR (JZB39) after microwave stabilization.RESULTS: Myometrium and decidua showed no change in total PR during pregnancy and labor. Nuclear PR was not detected in fetal membranes by binding assay but was localized in amnion epithelial and mesenchymal cells and in chorion laeve cytotrophoblasts by immunohistochemistry. Staining for PR was substantially less by serial antibody dilution in fetal membranes than in decidua. Message for PR was confirmed in all tissues analyzed. A significant (P <.05) shift in the ratio of PR isoforms (from PR-B dominance at midpregnancy to PR-A dominance in labor) was observed in myometrium but not in decidua. Both PR-A and PR-B isoforms and PR nuclear staining were nearly undetectable in amnion obtained during labor.CONCLUSION: A shift to PR-A dominance in myometrium at term together with a loss of PR in fetal membranes provides evidence for a functional progesterone withdrawal mechanism, which may facilitate the initiation of parturition in primates.     

Uterine artery Doppler in predicting pregnancy outcome in women with antiphospholipid syndrome

OBJECTIVE: To determine the expression and localization of cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), E-selectin, platelet-endothelial cell adhesion molecule (PECAM), and vascular cell adhesion molecule (VCAM) in the cervix and myometrium during pregnancy and labor. METHODS: Biopsies of myometrium and cervix were obtained from non-pregnant women and from pregnant women before and after onset of spontaneous labor at term. Cell adhesion molecule mRNA expression was quantified using Northern blotting and cell adhesion molecule protein was localized using immunohistochemistry. RESULTS: ICAM-1 mRNA was upregulated in the cervix (10-fold increase, P <.01) and myometrium (10.5-fold increase, P <.01) during labor. ICAM-1 was localized in the vascular endothelium and in leukocytes in the cervix and myometrium from all three groups of women. VCAM mRNA was upregulated in the cervix (2.5-fold increase, P <.01) during pregnancy and there was no further change during labor. VCAM localized weakly to the vascular endothelium in cervical and myometrial biopsies from pregnant and non-pregnant women. PECAM mRNA was significantly upregulated in myometrium during pregnancy (ninefold increase, P <.01) and did not change with the onset of labor. PECAM localized to the vascular endothelium in all cervical and myometrial biopsies and was identified on leukocytes. There were no significant changes in E-selectin mRNA expression in either tissue with pregnancy or parturition. CONCLUSION: Cell adhesion molecule expression changes in human cervix and myometrium during pregnancy and parturition. At least part of these changes are attributable to expression by leukocytes infiltrating these tissues.     

Differential expression pattern of genes encoding for anti-microbial peptides in the fetal membranes of patients with spontaneous preterm labor and intact membranes and those with preterm prelabor rupture of the membranes

Objective.?Increased amniotic fluid concentrations of anti-microbial peptides, components of the innate immune system, have been reported in patients with preterm labor (PTL) with intact membranes and intra-amniotic infection and/or inflammation (IAI), as well as in patients with preterm prelabor rupture of the membranes (PPROM). This study was designed to confirm these results using a targeted approach, detecting DEFA1, DEFB1, GNLY, and S100A9 gene expression in the choriamniotic membranes in pregnancies complicated with PTL and intact membranes or PPROM, with and without histologic chorioamnionitis.

Study design.?Human fetal membranes were obtained from patients in the following groups: (1) PTL with intact membranes (n?=?15); (2) PTL with intact membranes with histologic chorioamnionitis (n?=?12); (3) PPROM (n?=?17); and (4) PPROM with histologic chorioamnionitis (n?=?21). The mRNA expression of α-defensin-1, β-defensin-1, calgranulin B and granulysin in the fetal membranes was determined by qRT-PCR.

Results.?(1) The expression of α-defensin-1 mRNA in the fetal membranes was higher in patients with PTL and intact membranes with histologic chorioamnionitis, than those without chorioamnionitis (19.4-fold, p?<?0.001); (2) Among patients with histologic chorioamnionitis, patients with PTL and intact membranes had a higher α-defensin-1 mRNA expression than those with PPROM (5.5-fold, p?=?0.003); (3) Histologic chorioamnionitis was associated with a higher calgranulin B mRNA expression in the chorioamniotic membranes of patients with both PTL and intact membranes (7.9-fold, p?=?0.03) and PPROM (7.6-fold, p?<?0.0001); (4) The expression of calgranulin B mRNA in the fetal membranes was higher in patients with PTL and intact membranes without histologic chorioamnionitis than in those with PPROM without histologic chorioamnionitis (2.7-fold, p?=?0.03); (5) There were no differences in the expression of β-defensin-1 and granulysin in the chorioamniotic membranes between the study groups even in the presence of histologic chorioamnioniotis.

Conclusions.?(1) Among patients with histologic chorioamnionitis, the mRNA expression of α-defensin-1 and calgranulin B in the fetal membranes of patients with PTL and intact membranes as well as that of calgranulin B in the fetal membranes of patients with PPROM is higher than in the membranes of those without histologic chorioamnionitis; (2) histologic chorioamnionitis is associated with differences in the pattern of α-defensin-1 mRNA expression in the fetal membranes in patients with PTL and intact membranes and those with PPROM.     

Interleukin 1β regulates progesterone metabolism in human cervical fibroblasts

Progesterone plays a critical role in regulating cervical structure necessary for pregnancy maintenance. Preterm labor and early cervical ripening are often associated with localized infection. We hypothesized that proinflammatory cytokines enhance progesterone metabolism in human cervical fibroblasts (HCFs) in vitro, through the regulation of the expression of 20α-hydroxysteroid dehydrogenases (aldo-keto reductase [AKR]1C1, AKR1C2, or AKR1C3), 5α-reductase type 1 (5α-RDT1), and/or 17β-hydroxysteroid dehyrogenases (17β-HSD) type 1 and 2. The expression of both progesterone receptor (PR) and estrogen receptor α (ERα) was also studied. Human cervical fibroblasts were found to express AKR1C1, C2, and C3, with AKR1C1 exhibiting the greatest expression. These cells also expressed 5α-RDT1 and 17β-HSD1 and 2, albeit to a lesser level compared to the aldo-keto reductases. The fibroblasts also expressed both PR and ERα. Interleukin 1β (IL-1β) significantly increased the expression of AKR1C1 and C2 but not C3 but did not alter 5α-RDT1 nor 17β-HSD1 or 2 expression. Interleukin 1β treatment significantly increased progesterone metabolism by these cells. Use of specific inhibitors for aldo-keto reductases or 5α reductases confirmed that the increased progesterone metabolism was a consequence of the increased expression and/or activity of AKR1C1/2. Our results indicate that a major proinflammatory cytokine, IL-1β, can facilitate local progesterone metabolism in a cell type critical for maintaining cervical structure via regulating expression of AKR1C1 and 2.     

Expression and localization of cell adhesion molecules in human fetal membranes during parturition

There is increasing evidence to support the view that human parturition represents an inflammatory process. We have previously demonstrated that parturition is associated with leukocyte invasion and pro-inflammatory cytokine production in the cervix and myometrium. Furthermore, we have shown that several cell adhesion molecules are upregulated in these tissues during labor. In fetal membranes, previous studies have shown intercellular adhesion molecule-1 (ICAM-1) upregulation in association with labor. The role of other adhesion molecules has not been explored. The aims of this study were, therefore, to determine the expression of ICAM-1, platelet endothelial cell adhesion molecule (PECAM), vascular cell adhesion molecule (VCAM) and E-selectin in pre- and post-laboring amnion and choriodecidua and to identify cell types responsible for their expression. Biopsies of fetal membranes were obtained from pregnant women delivered by caesarean section before the onset of labor (n = 8) and following spontaneous vaginal delivery (n = 8). Cell adhesion molecules were identified using immunohistochemistry and messenger RNA expression quantified using Northern analysis. We found that following labor, ICAM-1 mRNA expression was significantly upregulated in amnion and choriodecidua (P < 0.05). PECAM mRNA expression was also increased in choriodecidua (P < 0.05). The main cell types responsible for adhesion molecule expression were leukocytes, amniotic epithelial cells and endothelial cells. The upregulation of ICAM-1 and PECAM mRNA expression in fetal membranes following labor provides further evidence that fetal membranes play an important role in the inflammatory process of parturition.     

Steroid synthetic and prostaglandin metabolizing activity is present in different cell populations from human fetal membranes and decidua

We examined whether different cell subpopulations from human fetal membranes and decidua produce steroids (estrone and progesterone) and metabolize prostaglandins (prostaglandin F2 alpha to 13, 14-dihydro-15-keto-prostaglandin F2 alpha and if these changed with labor. Amnion, chorion, and decidua were obtained at elective cesarean section at term or at spontaneous labor. Cells were dispersed with collagenase and separated by density on discontinuous Percoll gradients. At cesarean section there was a major broad band of cells from amnion and chorion. This band contained most of the estrone sulfatase (estrone sulfate to estrone) activity. The 3 beta-hydroxysteroid dehydrogenase (pregnenolone to progesterone conversion) and prostaglandin F2 alpha metabolizing activities were present in these cells and those that migrated at greater Percoll densities. Amnion and chorion obtained after spontaneous labor had two major bands of cells. Estrone sulfatase was present in cells from both hands, whereas progesterone output from pregnenolone and prostaglandin F2 alpha metabolism predominated in the second band of cells with greater density. This pattern was particularly apparent in chorion. Dispersed cells from decidua tended to migrate throughout the gradient. In general, estrone sulfate to estrone conversion predominated in lighter cells whereas progesterone output from pregnenolone and prostaglandin F2 alpha metabolism predominated in cells of greater density. The output of progesterone from pregnenolone was significantly lower in cell preparations from chorion and decidua at spontaneous labor compared with cesarean section. We conclude that human amnion, chorion, and decidua contain distinct cell subpopulations based on Percoll migration and that in the membranes these change between cesarean section and spontaneous labor. Partial separation of estrone sulfatase from 3 beta-hydroxysteroid dehydrogenase and prostaglandin F2 alpha metabolizing activities has been demonstrated, which raises the possibility of paracrine interactions in vivo.     

Expression and intracellular localization of protein phosphatases 2A and 2B, protein kinase a, A-Kinase anchoring protein (AKAP79), and binding of the regulatory (RII) subunit of protein kinase a to AKAP79 in human myometrium

OBJECTIVE: To determine the expression and intracellular localization of protein phosphatases 2A (PP2A) and 2B (PP2B), protein kinase A (PKA), and A-kinase anchoring protein (AKAP79), and expression of PKA (RII subunit) binding to AKAP79 in human postmenopausal and pregnant myometrium and to correlate their expressions to blood levels of estradiol, progesterone, and oxytocin. METHODS: Myometrial samples were taken from postmenopausal hysterectomy specimens (group 1, n = 5), from pregnant nonlaboring women (group 2, n = 7) and pregnant laboring women (group 3, n = 5) at cesarean. Western immunoblotting, immunohistochemical, and RII overlay assays were performed. Blood samples were assayed for estradiol, progesterone, and oxytocin levels. RESULTS: There were no significant differences in expression of PP2A, PKA, AKAP79, or PKA(RII) binding to AKAP79 between the three groups. Expression of PP2B was significantly greater in the nonlabor group (group 2) compared with groups 1 and 3. Protein phosphatase 2B, PKA, and AKAP79 expressions were localized in myometrial cytoplasm, but PP2A was localized in blood vessel endothelium. There was no significant correlation between the protein expression and the hormone level in the three groups. CONCLUSION: Human postmenopausal and pregnant (nonlabor and labor) myometrium expressed PP2A, PP2B, PKA, AKAP79, and PKA (RII)-AKAP79 binding. Levels of PP2A, PKA, and AKAP79 expression did not appear to be determinants of human myometrial contractility at parturition. Expression of PP2B may play a role in uterine quiescence. No association was found between protein expression and hormone level.     

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。     

不同药物对晚孕鼠子宫平滑肌及左心室心肌细胞电压依赖性钙通道-L亚型mRNA表达的影响

目的　探讨缩宫素、米索前列醇及尼莫地平 3种药物对晚孕鼠子宫平滑肌与左心室心肌细胞电压依赖性钙通道 L亚型 (VDCC L)mRNA表达的影响。方法　应用半定量逆转录聚合酶链反应 (RT PCR)技术分别测定自然分娩鼠 (自然分娩组 )、缩宫素诱导分娩鼠 (缩宫素组 )、米索前列醇诱导分娩鼠 (米索前列醇组 )及尼莫地平作用 48h后分娩鼠 (尼莫地平组 )的子宫平滑肌与左心室心肌细胞VDCC Lα1、β2 亚单位mRNA的表达。结果　 (1)自然分娩组、缩宫素组、米索前列醇组及尼莫地平组孕鼠的子宫平滑肌细胞VDCC Lα1mRNA的相对表达量依次为 0 .80 0 3± 0 .165 9、0 .863 1±0 .192 1、0 .812 0± 0 .173 4及 0 .742 6± 0 .182 6,缩宫素组与米索前列醇组虽高于自然分娩组及尼莫地平组 ,但 4组之间比较 ,差异均无显著性 (P >0 .0 5 ) ;VDCC Lβ2 mRNA的相对表达量依次为 0 .64 69± 0 .13 0 4、0 .5 0 62± 0 .1472、0 .5 0 0 5± 0 .13 5 6及 0 .492 9± 0 .12 76,自然分娩组虽高于其他用药的 3组 ,但差异无显著性 (P >0 .0 5 )。 (2 )自然分娩组、缩宫素组、米索前列醇组及尼莫地平组孕鼠的左心室心肌细胞VDCC Lα1mRNA相对表达量依次为 0 .662 5± 0 .180 1、0 .63 65± 0 .15 78、0 .5 917± 0 .1413及 0 .5 42 6±