Expression of aquaporin 9 in human chorioamniotic membranes and placenta

OBJECTIVE: Aquaporin 9 (AQP9) is one of the recently identified water channels that is also permeable to neutral solutes including urea. To investigate the molecular mechanism of intramembranous pathway of amniotic fluid regulation, we sought to determine whether AQP9 is expressed, and the cellular localization of AQP9 expression in human fetal membranes. STUDY DESIGN: Fetal membranes from 5 normal term human pregnancies were studied. Northern analysis was used to determine the tissue AQP9 messenger RNA (mRNA) expression. In situ hybridization and immunohistochemical staining with specific anti-AQP9 antibody was used for cellular AQP9 localization in the human fetal membranes. RESULTS: Northern analysis detected AQP9 mRNA expression in human amnion, chorion, and placenta. In situ hybridization revealed AQP9 mRNA expression in epithelial cells of the amnion, chorion cytotrophoblasts, and syncytiotrophoblasts and cytotrophoblasts of placenta. Further immunohistochemical study confirmed the AQP9 protein expression in these cell types of fetal membranes. CONCLUSION: This study demonstrated the expression of AQP9 mRNA and protein in human chorioamniotic membranes and placenta. The AQP9 expression in fetal membranes suggests that AQP9 may be an important water channel in intramembranous amniotic fluid water regulation.     

Prolonged hypoxia upregulates vascular endothelial growth factor messenger RNA expression in ovine fetal membranes and placenta

OBJECTIVE: In ovine fetuses, 4 days of hypoxia resulted in a large increase in urine flow, without the development of polyhydramnios, which suggests that intramembranous absorption of the amniotic fluid was enhanced. Because vascular endothelial growth factor is speculated to be a regulator of intramembranous absorption through increases of membrane vascularity and fluid transport, we hypothesized that hypoxia upregulated vascular endothelial growth factor gene expression in the fetal membranes. STUDY DESIGN: Five near-term ovine fetuses that were subjected to 4 days of hypoxia and 5 age-matched time controls were studied. On day 4, the amnion, chorion, and placenta were collected for cellular localization and quantification of vascular endothelial growth factor messenger RNA and for the determination of vascular endothelial growth factor molecular forms that were expressed. The data were analyzed statistically with the use of t tests and 2-factor analyses of variance. RESULTS: Vascular endothelial growth factor messenger RNA was expressed in the fetal membranes localized to the amniotic epithelium and chorionic cytotrophoblast, and to the villous cytotrophoblast of the placenta. In hypoxic fetuses, vascular endothelial growth factor messenger RNA levels in these cell layers were significantly increased compared with the controls. Five vascular endothelial growth factor molecular forms were identified with vascular endothelial growth factor(164) being the most abundant form expressed. The pattern of expression of the forms was not altered by hypoxia. CONCLUSION: In the near-term ovine fetus, hypoxia induced vascular endothelial growth factor messenger RNA expression in the amnion, chorion, and placenta. This was associated with an increase in intramembranous absorption of amniotic fluid. We speculate that the increased intramembranous absorption was mediated by a vascular endothelial growth factor-induced increase in the transport of amniotic fluid into the fetal membranes.     

Hypoxia modulation of caveolin-1 and vascular endothelial growth factor in ovine fetal membranes

During normal pregnancy, amniotic fluid is absorbed from the amniotic compartment into fetal blood through the intramembranous blood vessels in the fetal membranes. It has been hypothesized that this transport process is mediated by transcytosis of caveolae-like vesicles. Because fetal hypoxia increases intramembranous absorption, the authors explore the effects of hypoxia on the gene expression of caveolin-1, a structural protein of caveolae, in ovine fetal membranes and cultured amnion cells. Near-term ovine fetuses were rendered hypoxic for 4 days. Caveolin-1 mRNA and protein levels were significantly reduced in the amnion and chorion but not in the placenta. In cultured ovine amnion cells incubated in 2% oxygen for 24 hours, hypoxia did not significantly alter caveolin-1 mRNA or protein expression. Vascular endothelial growth factor mRNA levels were increased in response to hypoxia in the fetal membranes as well as in cultured amnion cells. The results indicate that hypoxia does not augment but instead down-regulates or has no effect on caveolin-1 gene expression in the amnion and chorion, suggesting that caveolin-1 may play a role as a negative regulator of amnion transport function under hypoxic conditions.     

Vascular endothelial growth factor activation of intramembranous absorption: a critical pathway for amniotic fluid volume regulation

OBJECTIVE: The purpose of this review is to propose a critical role for vascular endothelial growth factor (VEGF) in mediating the transfer of amniotic fluid from the amniotic compartment through the fetal membranes and fetal surface of the placenta into fetal blood. METHODS: Experimental findings in humans and animal models on the action of VEGF in mediating fluid transfer are reviewed and interpreted in order to postulate a proposed mechanism for VEGF regulation of amniotic fluid absorption through the fetal membranes and placenta. RESULTS: Recent scientific advances suggest that up-regulation of VEGF gene expression in the amnion and chorion is associated with increased transfer of amniotic fluid into fetal blood. The possible mechanisms of action for VEGF appear to involve regulation of intramembranous blood vessel proliferation and membrane transport via passive permeation as well as nonpassive transcytotic vesicular movement of fluid. CONCLUSION: Currently evolving concepts suggest that amniotic fluid volume is regulated through modulation of the rate of intramembranous absorption of amniotic fluid by both passive and nonpassive mechanisms. The permeability factor VEGF appears to be a critical regulator of amniotic fluid transport in the fetal membranes.     

Regulation of amniotic fluid volume by intramembranous absorption in sheep: role of passive permeability and vascular endothelial growth factor

OBJECTIVE: During long-term intravascular fluid infusion in the ovine fetus, a large increase in fetal urinary flow rate occurs while amniotic fluid volume increases only slightly because of increased intramembranous absorption. The current study tested the hypotheses that passive intramembranous permeability increases in response to fetal intravascular saline solution infusion and that the increased intramembranous absorption occurs in parallel with an increase in vascular endothelial growth factor gene expression in the amnion, chorion, and placenta. STUDY DESIGN: Chronically catheterized fetal sheep that average 126 +/- 1 (SE) days of gestation either were infused intravascularly with 7 L of normal saline solution over 3 days (n = 8 sheep) or served as time controls (n = 6 sheep). Amniotic fluid volume and fetal urinary flow rate were measured daily. Intramembranous diffusional permeability was estimated daily as being equal to the clearance of intra-amniotically injected technetium 99m. Vascular endothelial growth factor messenger RNA abundance in the amnion, chorion, and placenta was determined by Northern blot analysis. Statistical analyses included analysis of variance. RESULTS: In the infused fetuses, amniotic fluid volume and urinary flow increased (P <.01) by 891 +/- 144 mL and 3488 +/- 487 mL per day, respectively, on infusion day 3 compared with no changes over time in the control fetuses. In the infused fetuses, estimated intramembranous absorption increased by 4276 +/- 499 mL during the 3-day infusion. Intramembranous technetium 99m permeability was similar over time in the two groups. In the infused group, vascular endothelial growth factor messenger RNA levels in the amnion, chorion, and placenta increased 2- to 4-fold compared with the control group (P <.001). CONCLUSION: The up-regulation of vascular endothelial growth gene expression may mediate the increase in the intramembranous absorption that is induced by volume-loading diuresis; however, this does not occur by passive mechanisms. We speculate that vascular endothelial growth mediates the increased intramembranous absorption by increasing vesicular transport.     

Regulation of amniotic fluid volume

Water arrives in the mammalian gestation from the maternal circulation across the placenta. It then circulates between the fetal water compartments, including the fetal body compartments, the placenta and the amniotic fluid. Amniotic fluid is created by the flow of fluid from the fetal lung and bladder. A major pathway for amniotic fluid resorption is fetal swallowing; however, in many cases the amounts of fluid produced and absorbed do not balance. A second resorption pathway, the intramembranous pathway (across the amnion to the fetal circulation), has been proposed to explain the maintenance of normal amniotic fluid volume. Amniotic fluid volume is thus a function both of the amount of water transferred to the gestation across the placental membrane, and the flux of water across the amnion. Water flux across biologic membranes may be driven by osmotic or hydrostatic forces; existing data suggest that intramembranous flow in humans is driven by the osmotic difference between the amniotic fluid and the fetal serum. The driving force for placental flow is more controversial, and both forces may be in effect. The mechanism(s) responsible for regulating water flow to and from the amniotic fluid is unknown. In other parts of the body, notably the kidney, water flux is regulated by the expression of aquaporin water channels on the cell membrane. We hypothesize that aquaporins have a role in regulating water flux across both the amnion and the placenta, and present evidence in support of this theory. Current knowledge of gestational water flow is sufficient to allow prediction of fetal outcome when water flow is abnormal, as in twin-twin transfusion syndrome. Further insight into these mechanisms may allow novel treatments for amniotic fluid volume abnormalities with resultant improvement in clinical outcome.     

Amniotic fluid water dynamics

Water arrives in the mammalian gestation from the maternal circulation across the placenta. It then circulates between the fetal water compartments, including the fetal body compartments, the placenta and the amniotic fluid. Amniotic fluid is created by the flow of fluid from the fetal lung and bladder. A major pathway for amniotic fluid resorption is fetal swallowing; however in many cases the amounts of fluid produced and absorbed do not balance. A second resorption pathway, the intramembranous pathway (across the amnion to the fetal circulation), has been proposed to explain the maintenance of normal amniotic fluid volume. Amniotic fluid volume is thus a function both of the amount of water transferred to the gestation across the placental membrane, and the flux of water across the amnion. Membrane water flux is a function of the water permeability of the membrane; available data suggests that the amnion is the structure limiting intramembranous water flow. In the placenta, the syncytiotrophoblast is likely to be responsible for limiting water flow across the placenta. In human tissues, placental trophoblast membrane permeability increases with gestational age, suggesting a mechanism for the increased water flow necessary in late gestation. Membrane water flow can be driven by both hydrostatic and osmotic forces. Changes in both osmotic/oncotic and hydrostatic forces in the placenta my alter maternal-fetal water flow. A normal amniotic fluid volume is critical for normal fetal growth and development. The study of amniotic fluid volume regulation may yield important insights into the mechanisms used by the fetus to maintain water homeostasis. Knowledge of these mechanisms may allow novel treatments for amniotic fluid volume abnormalities with resultant improvement in clinical outcome.     

Expression of Erythropoietin mRNA, Protein and Receptor in Ovine Fetal Membranes

Erythropoietin and its receptor have been identified in human, murine and ovine placentas. Based on the common embryonic origin of the placenta and fetal membranes, we postulated that erythropoietin is similarly expressed in the fetal membranes. Using in situ hybridization and immunohistochemistry, we tested the hypothesis that ovine fetal membranes are sites of erythropoietin production and action. At 86, 103 and 138 days gestation, erythropoietin mRNA and protein were present in the amnion localized to the cell layer consisting largely of amniotic epithelium and in the chorion localized to the chorionic columnar cells consisting of cytotrophoblasts. Binucleate cells, differentiated cytotrophoblasts known to produce hormones, were identified in the chorion in the region of erythropoietin expression but were not observed in amniotic tissue. The erythropoietin receptor protein was present in the amnion and chorion at 103 and 138 days gestation but was not observed in either tissue at 86 days. In summary, erythropoietin appears to be produced as well as utilized within the ovine amnion and chorion. Within the amnion, the amniotic epithelial cells express the erythropoietin gene whereas, within the chorion, either the cytotrophoblasts or the binuclear cells may be the source. Due to the presence of the receptor, we speculate that the erythropoietin produced in the membranes may mediate fetal membrane function and/or growth through an autocrine and/or paracrine mechanism. Further, the fetal membranes may be the source of erythropoietin in the amniotic fluid.     

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。     

Pre-B-cell colony-enhancing factor,a novel cytokine of human fetal membranes

OBJECTIVE: Our purpose was to determine whether pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes during normal gestation and parturition in the absence of infection and to show its effects on the expression of interleukin (IL)-6 and IL-8. STUDY DESIGN: PBEF was immunolocalized in the fetal membranes from early pregnancy, at preterm, and at term. Its expression was quantitated by Northern analysis in separated uninfected amnion, chorion, decidua, and placenta of patients at term before labor and in full-thickness membranes before and after spontaneous labor at preterm and at term. Amnion-like epithelial (WISH) cells and fetal membrane explants were treated with recombinant PBEF (rhPBEF), and the expression of IL-6 and IL-8 was quantitated. RESULTS: PBEF was immunolocalized throughout gestation in the amniotic epithelium and mesenchymal cells as well as the chorionic cytotrophoblast and parietal decidua. Northern analysis showed significantly more (P <.01) PBEF expressed in the amnion than in either chorion or placenta. Its expression increased after labor at both preterm and term and correlated with that of IL-8 (r = 0.87). rhPBEF treatment of WISH cells significantly increased IL-6 (P <.05) and IL-8 (P <.01) gene expression after 4 hours and of IL-8 protein after 24 hours (P <.01); similar 4-hour treatment of fetal membrane explants significantly increased IL-6 (P <.01) and IL-8 (P <.05) gene expression. CONCLUSION: PBEF is a novel cytokine constitutively expressed by the fetal membranes during pregnancy. It increased the expression of IL-6 and IL-8 and may be important in both normal spontaneous labor and infection-induced preterm labor.     

Localization of the Fas-Fas ligand system in human fetal membranes

OBJECTIVE: To determine if fetal membranes might be one of the sources of Fas and Fas ligand in amniotic fluid. STUDY DESIGN: Human fetal membranes from elective cesarean section (n = 6) were fixed in paraformaldehyde. Rolls of paraffinembedded fetal membranes were cut into 5-micron sections. After blocking with horse and goat sera, sections were incubated overnight with primary antibodies followed by the appropriate secondary antibodies. Avidin-biotin complex and diaminobenzidine were used for immunoperoxidase localization. Expression of Fas and Fas ligand was read by light microscopy. RESULTS: Both Fas and Fas ligand were localized in amnion, chorion and decidual layers. In amnion, Fas and Fas ligand were expressed predominantly in epithelial cells and fibroblasts, while there was no immunostaining in the subepithelial compact connective tissue. In the chorion, the expression was mainly in the chorionic trophoblast, with inconsistent expression in the reticular layer. In the decidua, the expression of Fas and Fas ligand was less prominent than in amnion and chorion. CONCLUSION: Localization of Fas and Fas ligand in human fetal membranes suggests that fetal membranes could be one of the sources of soluble Fas and Fas ligand in amniotic fluid.     

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.     

水通道蛋白1和水通道蛋白3在羊水过少孕妇胎盘和胎膜的表达及其意义

目的 研究水通道蛋白1(AQP1)和AQP3在羊水过少孕妇胎盘、胎膜中的表达及分布,探讨其在羊水平衡途径中的作用. 方法 选取30例孤立性羊水过少的足月孕妇为研究组,同期分娩的30例正常羊水量的足月孕妇为对照组.分别采用实时荧光定量PCR和免疫组织化学SP法,检测两组胎盘、胎膜组织中AQP1和AQP3 mRNA和蛋白的表达及分布. 结果 AQP1mRNA在研究组羊膜组织中的表达为对照组的0.31,AQP1蛋白在研究组羊膜组织中的表达为0.14±0.02,较对照组的0.25±0.03明显下调(P<0.05).而在胎盘、绒毛膜中的分布及表达强度两组间差异无统计学意义(P>0.05).AQP3 mRNA在研究组羊膜和绒毛膜中的表达分别为对照组的0.31和0.37,而胎盘中的表达为对照组的7.36.AQP3蛋白在研究组羊膜和绒毛膜中的表达分别为0.18±0.05和0.18±0.04,均较对照组的0.26±0.03和0.29±0.06明显下调(P<0.05),而在胎盘组织中的表达研究组为0.47±0.09,较对照组0.28±0.01明显上调(P<0.05). 结论 AQP1和AQP3在羊水过少孕妇胎盘、胎膜组织中表达的改变为羊水减少后的代偿性反应.     

Release and lubricating properties of amniotic surfactants and the very hydrophobic surfaces of the amnion, chorion, and their interface

Surface hydrophobicity of 17 fresh human chorioamniotic membranes was measured as the contact angle (theta) subtended when a drop of saline is placed upon any non-wettable surface. The contact angle averaged 75.5 +/- 4.2 degrees and 76.8 +/- 5.6 degrees on the epithelial surfaces of the amnion and chorion, respectively. The interface proved to be particularly hydrophobic, averaging 108.2 +/- 8.7 degrees on the amnionic side and 121.7 +/- 4.2 degrees on the chorionic side, especially when compared with 108 degrees for Teflon. High surface hydrophobicity implies good boundary (solid-to-solid) lubrication, good release from neighboring tissues, and water repellency, which is a possible factor enabling the chorioamniotic membrane to retain amniotic fluid. Good release (68 to 71%) and boundary lubrication (coefficient of kinetic friction = 0.24 +/- 0.072) were obtained from oriented monolayers of the phospholipid extracted from samples of human amniotic fluid obtained from term patients by amniocentesis. These results support the concept that the amnionic and chorionic membrane surfaces exhibit good release and boundary lubrication probably imparted by adsorbed surfactant.     

Expression, localisation and activity of ATP binding cassette (ABC) family of drug transporters in human amnion membranes

Placental ATP-binding cassette (ABC) transporters limit fetal exposure to xenobiotics by regulating transplacental passage into the fetal circulation; their expression and function in fetal membranes, however, has not been studied. In the present study the expression, localisation and function of ABC transporters in human amnion was examined to explore their potential role in modulating amniotic fluid drug disposition in pregnancy. Single-assay oligo-microarrays were used to profile amnion gene expression, and drug transporters expressed at significant levels were identified and selected for further studies. The expression of ABCG2/breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRP) 1 (ABCC1), 2 (ABCC2) and 5 (ABCC5) was detected on the arrays, and verified by RT-PCR and immunoblotting. On confocal microscopy of fetal membrane cryosections, MRP1 and MRP5 were immunolocalised to both apical and basolateral surfaces of the amniotic epithelium, while MRP2 was expressed at low levels only in the apical membrane. BCRP in contrast showed cytoplasmic staining throughout the amniotic epithelium. In addition to the amnion, MRP1 and BCRP immunostaining was observed in the chorion and the decidua. Cell accumulation studies using selective MRP and BCRP inhibitors showed the transporters to be functionally active in amnion epithelial monolayer cultures. In contrast, transwell transport studies using intact amnion membranes did not show significant vectorial transport. These findings identify the amnion as a novel site of ABC drug transporter expression. Functional studies indicate that they may act primarily to prevent cellular xenobiotic accumulation, rather than to confer fetal protection through reduced accumulation in amniotic fluid.