Expression of natural antimicrobials by human placenta and fetal membranes

Preterm birth associated with infection is a major clinical problem. We hypothesized that this condition is associated with altered expression of natural antimicrobial molecules (beta-defensins (HBD), elafin). Therefore, we examined expression of these molecules and their regulation by proinflammatory cytokines in placentae and fetal membranes from term pregnancy. HBD1-3 and elafin were localized by immunohistochemistry in fetal membranes and placenta. Real-time quantitative PCR was used to examine mRNA expression in primary trophoblast cells treated with inflammatory molecules. HBD1-3 and elafin were immunolocalized to placental and chorion trophoblast layers of fetal membranes and placenta. Immunoreactivity was also observed in amnion epithelium and decidua. No differences were noted between samples from women who were not in labour compared to those in active labour. In in vitro cultures of primary trophoblast cells, HBD2 and elafin mRNA expression was upregulated by the proinflammatory cytokine, IL-1beta. These results suggest that the chorion and placental trophoblast layers may be key barriers to the progression of infection in the pregnant uterus. Natural antimicrobial expression may be altered in response to inflammatory mediator expression associated with the onset of labour and/or uterine infection, providing increased protection when the uterus may be particularly susceptible to infection.     

The content of placental protein 12 in decidua and fetal membranes is greater than in placenta

Summary. Radioimmunoassay, gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis were used to study the content and properties of placental protein 12 (PP12) in the placenta, decidua and fetal membranes. The tissues were obtained from early pregnancy in 12 cases, and after normal term delivery in eight cases in seven of which chorion and amnion laeve were also studied. There was more PP12 in decidua and fetal membranes than in placenta. The decidua/placenta ratio of PP12 content ranged from 2 to 1154 (mean 193, SEM 66). These results suggest that PP12 is a decidual rather than placental protein.     

The content of placental protein 12 in decidua and fetal membranes is greater than in placenta

Radioimmunoassay, gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis were used to study the content and properties of placental protein 12 (PP12) in the placenta, decidua and fetal membranes. The tissues were obtained from early pregnancy in 12 cases, and after normal term delivery in eight cases in seven of which chorion and amnion laeve were also studied. There was more PP12 in decidua and fetal membranes than in placenta. The decidua/placenta ratio of PP12 content ranged from 2 to 1154 (mean 193, SEM 66). These results suggest that PP12 is a decidual rather than placental protein.     

Immunocytochemical localization and messenger ribonucleic acid concentrations for human placental lactogen in amnion, chorion, decidua, and placenta

Human placental lactogen is one of the major hormones secreted by the placental syncytiotrophoblast and detected in the maternal circulation. Other sources of this hormone in intrauterine tissues at term have been sought by means of immunohistochemistry and northern analysis. Avidin-biotin immunoperoxidase staining with a specific polyclonal antibody to human placental lactogen showed this hormone to be present in groups of cells at the interface between chorionic cytotrophoblast and decidua parietalis and in some cells of the basal plate in addition to the classic source, the syncytiotrophoblast. Hybridization of polyadenylic-(+)ribonucleic acid extracted from amnion, chorion, decidua parietalis, basal plate, and placental trophoblast with a radiolabeled 48 mer oligonucleotide and a 540 base pair complementary deoxyribonucleic acid probe to human placental lactogen showed the placental trophoblast to be the major source of human placental lactogen and the extravillous chorion and basal plate to be additional minor sources.     

Vitamin D3 metabolites stimulate prostaglandin production by human fetal membranes and placenta in vitro

In the present study we examined whether the vitamin D3 metabolites, 25-hydroxyvitamin D and 1-25-dihydroxycholecalciferol affected the production of the prostaglandins PGE2 and PGF2 alpha in human fetal membranes and placenta in vitro. Human amnion, chorion, decidual, and placental cells were maintained in primary monolayer culture. Treatment with the vitamin D3 metabolites resulted in an increase in PGE2 and PGF2 alpha production by amnion, decidua, and placental cells; however, these effects varied with time and were different between tissues. Although there was no significant increase in the production of PGE2 and PGF2 alpha by chorion cells in vitro, there was a significant increase in the production of prostaglandin F metabolites after treatment with the vitamin D3 metabolites. The data suggest that the vitamin D3 metabolites may increase free calcium availability and the conversion of arachidonic acid to the prostaglandins. The data do not, however, exclude the possibility that the vitamin D3 metabolites act at other points of arachidonic acid metabolism. These findings raise the possibility of a paracrine role for the vitamin D3 metabolites in the modulation of prostaglandin production within the human fetal membranes and placenta.     

水通道蛋白3、9在特发性羊水过多产妇胎盘和胎膜中的表达变化及意义

目的 探讨水通道蛋白(AQP)3、9在特发性羊水过多产妇胎盘和胎膜中的表达、分布及其在特发性羊水过多发病巾的作用.方法 2006年6月至2008年3月,选择在温州医学院附属第二医院产科住院并分娩的21例特发性羊水过多的足月产妇为研究组,同期分娩的30例羊水量正常的足月产妇为对照组.采用实时荧光定量PCR技术,检测两组产妇胎盘、胎膜中AQP3、9 mRNA的表达及分布,采用免疫组化链霉菌抗生物素蛋白.过氧化物酶(SP)连接法枪测两组产妇胎盘、胎膜中AQP3、9蛋白的表达及分布.结果 (1)两组产妇羊膜、绒毛膜、胎盘中均可检测到AQP3、9 mRNA的表达.AQP3、9主要表达于羊膜上皮细胞、绒毛膜滋养细胞、胎盘滋养细胞.(2)研究组羊膜上皮细胞AQP3、9 mRNA的表达分别为对照组的5.00倍和3.25倍,而研究组绒毛膜滋养细胞AQP3、9 mRNA的表达分别为对照组的2.03倍和2.08倍,分别比较,差异均有统计学意义(P<0.01);但是研究组胎盘滋养细胞AQP3、9 mRNA的表达均明显低于对照组,差异也有统计学意义(P<0.01).(3)AQP3、9蛋白在研究组羊膜上皮细胞的表达强度为7.5±2.0、11.1±1.8,对照组为5.3 ±1.6、5.6±2.3,两组比较,差异有统计学意义(P<0.05);AQP3、9蛋白在研究组绒毛膜滋养细胞的表达强度为7.5±2.0、10.0±1.6,对照组为5.4±2.2、5.6±2.1,分别比较,差异也有统计学意义(P<0.05),但是AQP3、9蛋白在胎盘滋养细胞的表达强度(3.5±1.4、4.0 ±2.5)较对照组(5.6±1.3、7.1±2.9)明显下调(P<0.05).结论 AQP3、9在特发性羊水过多产妇胎盘和胎膜中的表达变化可能为羊水增加后的代偿性反应,其调节机制尚待进一步研究.     

Expression of Aquaporin 1 and Aquaporin 3 in Fetal Membranes and Placenta in Human Term Pregnancies with Oligohydramnios

ObjectiveTo explore the pathophysiology of oligohydramnios, the association between the expression of aquaporin 1 and aquaporin 3 in fetal membranes and placenta and oligohydramnios was investigated.MethodsSixty patients underwent elective cesarean sections at term were studied, 30 patients with isolated oligohydramnios and the other 30 with normal amniotic fluid volume (AFV). Real-time polymerase chain reaction and immunohistochemistry were employed to determine expression and localization of aquaporin 1 and aquaporin 3 in amnion, chorion and placenta, respectively.ResultsThe expression of aquaporin 1 and aquaporin 3 was detected in amnion, chorion and placenta using real-time RT-PCR. By immunohistochemistry, aquaporin 1 and aquaporin 3 protein expressions in amnion epithelia and chorion cytotrophoblasts were identified. In placenta, aquaporin 1 was detected in placental vessels, while aquaporin 3 was found in trophoblast cells. In comparison to normal AFV group, there was a significant decrease of aquaporin 1 expression in amnion in oligohydramnios group, but no significant difference in chorion and placenta between the two groups. The expression of the aquaporin 3 in amnion and chorion in oligohydramnios group was significantly decreased, while expression in placenta was significantly increased compared with that in normal AFV group.ConclusionsAlteration of aquaporin 1 and aquaporin 3 expression in fetal membranes and placenta may be important in the pathophysiology of isolated oligohydramnios.     

Localization and distribution of adrenomedullin receptor in the human placenta: changes with gestational age

OBJECTIVE: To examine the distribution and localization of adrenomedullin (AM) receptor (AM-R) in human placenta and fetal membranes to assess any change during pregnancy or with labor. STUDY DESIGN: Immunohistochemistry was performed by the avidin/biotin immunoperoxidase method using an antibody specific to AM-R on intrauterine tissues collected from 7-41 weeks of gestation (n=73). RESULTS: AM-R was localized in the placenta and fetal membranes in all 3 trimesters. The distribution of AM-R in the villous and extravillous trophoblast cells of the placenta and in chorion and decidua cells of the fetal membranes changed with gestational age but not with labor. CONCLUSION: AM is secreted by decidua and trophoblast cells that also possess AM-R, suggesting that placental tissues function in both the synthesis and action of AM. Changes in AM-R in the placenta during pregnancy may reflect changes in AM function throughout gestation.     

Perfusion with lipopolysaccharide differently affects the secretion of interleukin-1 beta and interleukin-1 receptor antagonist by term and preterm human placentae

The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the secretion of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) and of its natural inhibitor interleukin-1 receptor antagonist (IL-1Ra), by perfused human term and preterm placental tissue. Eight term and eight preterm placentae were collected immediately after delivery; four term and four preterm placentae were perfused with control medium (without LPS) and the other four term and four preterm placentae were perfused with medium containing LPS. The release of IL-1beta into the maternal compartment by term placenta was significantly higher than the release by preterm placenta (p<0.001). However, there were no significant differences between IL-1beta levels released into the fetal compartments of term and preterm placentae. No significant differences were observed in the release of IL-1Ra into the maternal and fetal compartments of term placenta, when compared to preterm placenta. Exposure to LPS significantly decreased the capacity of term placenta to release IL-1beta into the maternal compartment (p<0.001) and increased the capacity of term placenta to release IL-1Ra into the maternal and fetal compartments (p<0.001 and p=0.017, respectively). However, the capacity of preterm placentae to release IL-1beta and IL-Ra into the maternal and fetal compartments was not affected by LPS. IL-1beta was expressed by both term and preterm placentae before and after perfusion (+/- LPS), by epithelial cells of the amnion, chorion, by syncytiotrophoblast and stromal cells of villous tissue and by the decidua. IL-1Ra in term and preterm placentae was expressed before perfusion mainly in epithelial cells of the amnion. After perfusion of term placentae (+/- LPS), additional IL-1Ra expression was seen in epithelial cells of the amnion and in syncytiotrophoblast and stromal cells of villous tissue and by the decidua. However, perfusion of preterm placentae (+/- LPS) did not affect IL-1Ra expression. The localization of IL-1beta and IL-1Ra in both term and preterm human placental tissue suggests a their physiologic role. The data presented indicates that the IL-1 system in term and preterm placentae seems to be differently affected by LPS. Down-regulation in the release of the pro-inflammatory cytokine IL-1beta and the up-regulation of its antagonist (IL-1Ra) may be a part of the inflammatory response to infection in human term, but not preterm, placentae. The IL-1 system in term and preterm placentae seems to be differently affected by LPS.     

水通道蛋白9在人胎盘和胎膜中的表达

目的 检测正常人胎盘与胎膜中水通道蛋白9（aquaporin 9，AOP9）的表达。方法 收集5例足月剖宫分娩的胎盘和胎膜样本，运用RT—PCR方法从mRNA水平检测AQP9在胎盘与胎膜的表达；运用免疫组织化学和Western印迹方法检测AQP9蛋白在胎盘与胎膜中的表达。结果 RT—PCR结果显示AQP9mRNA在胎盘和胎膜均有表达；Western印迹显示两条带在相对分子量为30kD及45kD左右；免疫组织化学显示AQP9表达于胎盘的合体滋养细胞、羊膜上皮细胞及平滑绒毛膜滋养细胞。结论 AQP9在母胎液体交换、胎儿代谢物的排出及羊水平衡等的分子机制中可能发挥重要作用。     

Spatial and temporal patterns of expression of messenger RNA for insulin-like growth factors and their binding proteins in the placenta of man and laboratory animals

To better understand the role of the insulin-like growth factors (IGF-I and -II) and their binding proteins (IGFBPs 1-6) in placental development and function, it is important to review similarities and differences between species in expression of the respective mRNAs. In human placenta, IGF-II mRNA is expressed in chorionic mesoderm and first trimester villous cytotrophoblast, but not in syncytiotrophoblast. In contrast, in rhesus monkey placenta, IGF-II mRNA is expressed in syncytiotrophoblast but not in chorionic mesoderm. IGFBP-3 mRNA is present in the chorionic mesoderm of placental villi from both these species and may modulate IGF-II action through a paracrine mechanism. In rodent placentae, IGF-II mRNA is expressed both in fetal mesoderm and in the trophoblast of the placental labyrinth. In guinea pig, where IGFBP-5 mRNA is expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth, interaction between IGF-II and IGFBP-5 mRNA may be involved in vascularization of the placenta by fetal vessels. In sheep placenta, IGF-II mRNA is expressed, not in the trophoblast layer, but in the fetal mesoderm immediately adjacent to it. In the basal plate of human, rhesus monkey and baboon placentae, extravillous trophoblasts express IGF-II mRNA and uterine decidual cells IGFBP 1-6 mRNAs. The inference is that there is interaction between IGF-II and IGFBPs at the maternal-fetal interface of the primate placenta during trophoblast invasion and decidualization. IGFBP-1 expressed by the decidua may also interact with alpha(5)beta(1)integrin expressed by the extravillous trophoblast. The placentae of rodents are also of the invasive type. Glycogen cells of the mouse placenta are analogous with human extravillous trophoblast and express IGF-II mRNA. However, expression of IGFBP mRNAs in the mouse, as in the guinea pig, is confined to non-decidualized endometrium and myometrium. IGF-II mRNA is strongly expressed by trophoblasts invading uterine vessels in human and guinea pig placentae. Interactions probably occur between IGF-II expressed by these trophoblasts and IGFBPs expressed in the vessel walls. However, it is possible that IGFBPs expressed by maternal vessels are associated with processes that are independent of trophoblast invasion. Thus, IGFBP-3 mRNA is highly expressed in the maternal blood vessels of the non-deciduate sheep placenta. Findings to date highlight the diversity in the expression of the IGF system among placentae of man and different laboratory animals, and even between closely related species. Comparative studies will continue to be required to understand the functional role of IGFs and IGFBPs in each species.     

Ontogeny of aquaporins 1 and 3 in ovine placenta and fetal membranes

A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP(3)) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145-150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP(3)were found to be the trophoblast cells. Some AQP(3)was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP(1)was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP(3)expression is consistent with known ovine placental permeabilities to water, glycerol and urea.     

Localization of prostaglandin H synthase, prostaglandin dehydrogenase, corticotropin releasing hormone and glucocorticoid receptor in rhesus monkey fetal membranes with labor and in the presence of infection

Prostaglandins (PGs) play a central role in primate parturition by their actions on uterine contractility and on cervical ripening. Rhesus monkey placentation is hemochorial and the endocrine events surrounding parturition are qualitatively similar to human pregnancy. Although there is an increase in PG production before the onset of labor, little is known about the cellular localization of the PGH synthase (PGHS) or the 15-hydroxy PG dehydrogenase (PGDH) in the fetal membranes of nonhuman primates and whether it changes at term in spontaneous labor or during preterm labor associated with infection. Placental corticotropin releasing hormone (CRH) and the glucocorticoid receptor (GR) have also been implicated as mediators in parturition by virtue of their roles in PG production. We utilized immunohistochemical methods to localize the inducible isoform PGHS-2, PGDH, GR and CRH in rhesus monkey amnion, chorion and attached decidua. Tissues were obtained at cesarean section during late pregnancy, in spontaneous labor at term and in premature labor induced by Group B streptococcal intraamniotic infection. Specific staining for immunoreactive (ir)-PGHS-2 was observed in amnion epithelial and mesenchymal cells and to a lesser extent in chorion and decidua. In contrast, ir-PGDH was localized primarily to the extravillous trophoblast layer of chorion. GR was localized to both the cytoplasm and nucleus of amnion epithelial cells, subepithelial fibroblasts, chorion trophoblasts and in decidua. Immunostaining for CRH was found in amnion and in scattered decidual cells but was most intense in the chorion trophoblast layer. There was no demonstrable change in this overall pattern of immunostaining in association with the onset of labor at term except for a decrease in staining for ir-PGDH in chorion. Experimental Group B streptococcal chorioamnionitis resulted in preterm labor and extensive necrosis of extravillous trophoblast cells with subsequent loss of chorionic ir-PGDH and relative sparing of ir-PGHS-2 in amnion epithelium which favors the net production of PGs. The expression pattern of these effectors in the rhesus monkey fetal membranes points to a functional role of PGs and glucocorticoids in the process of term and preterm parturition which is similar to that in human pregnancy.     

Changes in expression of the cyclooxygenase gene in human fetal membranes and placenta with labor.

OBJECTIVE: Our objective was to examine the expression of the gene coding for cyclooxygenase, the central enzyme in prostaglandin synthesis, in human placenta and fetal membranes during pregnancy and before and after labor at term. STUDY DESIGN: Expression of the gene for cyclooxygenase was examined with Northern hybridization to ribonucleic acid from human placenta throughout pregnancy and human amnion and chorion decidua in the late third trimester. RESULTS: Expression was undetectable in trophoblast during the first and second trimesters. Expression in amnion and trophoblast increased 3.5- and 2.5-fold, respectively, in association with labor. CONCLUSIONS: Our results suggest that the increase in prostaglandin synthesis within the uterus that is seen with the onset of labor is associated with an increase in the expression of the gene cyclooxygenase.     

The use of laser capture microdissection (LCM) and quantitative polymerase chain reaction to define thyroid hormone receptor expression in human 'term' placenta

Term 'villous' placenta consists of a heterogeneous mix of different cell types comprising the trophoblast layers, villous stroma and fetal blood vessels. The importance of using techniques which allow investigation of pure populations of cells has been increasingly recognised. We demonstrate the use of laser capture microdissection (LCM) in combination with quantitative RT-PCR (QPCR) to assess the relative expression of mRNAs encoding the major thyroid hormone receptor (TR) isoforms (alpha1, alpha2 and beta1) in trophoblasts (syncytiotrophoblast and cytotrophoblast layers) compared with stromal cells in human term placenta. Highly reproducible results for each gene were obtained for each placental sample studied (n = 6). There was significantly less mRNA encoding TRalpha1 (68%; p = 0.05) and TRbeta1 (40%; p = 0.03) in the trophoblast layer compared to the heterogeneous stromal cells. However, there was no significant difference in TRalpha2 mRNA expression between the two groups of cells. CONCLUSION: LCM with QPCR can precisely locate and accurately quantify mRNA expression in specific cell populations from placental tissue.     

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.     

Scavenger receptor for hemoglobin in preterm prelabor rupture of membranes pregnancies complicated by histological chorioamnionitis

Objective: We sought to evaluate the distribution of scavenger receptor for hemoglobin positive (CD163⁺) cells in the placenta and fetal membranes from pregnancies complicated by preterm prelabor rupture of membranes with respect to the presence and absence of histological chorioamnionitis. Methods: Sixty-two women with singleton pregnancies with a gestational age between 24+0 and 36+6 weeks were included in a prospective cohort study. CD163 was evaluated by immunohistochemistry in the placenta and fetal membranes. The number of CD163⁺ cells and neutrophils was counted in the following locations: fetal membranes’ amnion, chorion, and decidua, as well as the placenta’s amnion, chorionic plate, subchorionic fibrin, stem villi, terminal villi, and decidua. Results: CD163⁺ cells were found in all compartments of the placenta and the fetal membranes regardless of the inflammatory status. A positive correlation between the number of CD163⁺ cells and neutrophils in the subchorionic fibrin and the chorionic plate was found. The number of CD163⁺ cells was higher in the placental subchorionic fibrin and chorionic plate when histological chorioamnionitis was present. Conclusion: The presence of histological chorioamnionitis affected the number of CD163⁺ cells in the placental chorionic plate and in the subchorionic fibrin but not in the fetal membranes.     

Corticotrophin-releasing factor in human placenta: localization, concentration and release in vitro

Corticotrophin-releasing factor (CRF) immunoreactivity was demonstrated by immunohistochemical staining in the cytotrophoblast of the early pregnancy placenta, in the decidua and in the amnion. This localization is different from that of adrenocorticotrophic hormone (ACTH) and beta-endorphin, which are present in the syncytiotrophoblast. The release of immunoreactive CRF was demonstrated from both early and term placental tissues in vitro. The mean amounts of CRF in the early and term pregnancy placental/decidual extracts were 0.99 +/- 0.5 ng/g and 19.7 +/- 3.1 ng/g, respectively. A slightly greater amount of CRF was found in extracts from term placentae and in cord venous plasma collected after spontaneous vaginal delivery than in those collected at elective caesarean section performed before the beginning of labour.