Altered proteomics profile in the amnion of patients with oligohydramnios.

In pregnancy, idiopathic oligohydramnios is an obstetrical complication that compromises maternal health with poor perinatal outcome. Effective therapeutic treatment of this condition has been hampered by the unknown etiology and lack of understanding of cellular and molecular mechanisms that underlie idiopathic oligohydramnios. Amniotic fluid volume (AFV) is determined by intramembranous (IM) transport of amniotic fluid across the amnion and this pathway is regulated to maintain AFV within the normal range. To gain understanding of the causes of idiopathic oligohydramnios, we performed proteomics analysis of the human amnion to investigate the changes in protein expression profiles of cellular transport pathways and regulators in patients with oligohydramnios. Placental amnions from five patients with normal pregnancies and five patients with oligohydramnios were subjected to proteomics experiments followed by bioinformatics analysis. Using Ingenuity Pathway Analysis (IPA) software, five categories of biological functions and multiple canonical pathways within each category were revealed. The top differentially expressed proteins that participate in mediating these pathways were identified. The functional pathways activated include: (a) cellular assembly and organization, (b) cell signaling and energy metabolism, and (c) immunological, infectious, and inflammatory functions. Furthermore, the analysis identified the category of pathways that facilitate molecular endocytosis and vesicular uptake. Under oligohydramniotic conditions, the mediators of clathrin vesicle-mediated uptake and transport as well as intracellular trafficking mediators were up-regulated. These findings suggest that idiopathic oligohydramnios may be associated with alternations in cellular organization and immunological functions as well as increases in activity of vesicular transport pathways across the amnion.

Multiomics analyses of vesicular transport pathway-specific transcripts and proteins in ovine amnion: responses to altered intramembranous transport.

Amniotic fluid volume (AFV) is determined by the rate of intramembranous (IM) transport of amniotic fluid (AF) across the amnion. This transport is regulated by fetal urine-derived stimulators and AF inhibitors. Our objective was to utilize a multiomics approach to determine the IM transport pathways and identify the regulators. Four groups of fetal sheep with experimentally induced alterations in IM transport rate were studied: control, urine drainage (UD), urine drainage with fluid replacement (UDR), and intra-amniotic fluid infusion (IA). Amnion, AF, and fetal urine were subjected to transcriptomics (RNA-Seq) and proteomics studies followed by Ingenuity Pathway Analysis. The analysis uncovered nine transport-associated pathways and four groups of differentially expressed transcripts and proteins. These can be categorized into mediators of vesicular uptake and endocytosis, intracellular trafficking, pathway activation and signaling, and energy metabolism. UD decreased IM transport rate and AFV in conjunction with enhanced expression of vesicular endocytosis regulators but reduced expression of intracellular trafficking mediators. With UDR, IM transport rate decreased and AFV increased. Energy metabolism activators increased while trafficking mediators decreased in expression. IA increased IM transport rate and AFV together with enhanced expressions of vesicular endocytosis and trafficking mediators. We conclude that IM transport across the amnion is regulated by multiple vesicular transcytotic and signaling pathways and that the mediators of intracellular trafficking most likely play an important role in determining the rate of IM transport. Furthermore, the motor protein cytoplasmic dynein light chain-1, which coexpressed in AF and fetal urine, may function as a urine-derived IM transport stimulator.

Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion.

Vascular endothelial growth factor (VEGF) has been proposed as an important regulator of amniotic fluid absorption across the amnion into the fetal vasculature on the surface of the placenta. However, the activators of VEGF expression and action in the amnion have not been identified. Using the pregnant sheep model, we aimed to investigate the presence of the retinoic acid (RA) pathway in ovine amnion and to determine its effect on VEGF expression. Further, we explored relationships between RA receptors and VEGF and tested the hypothesis that RA modulates intramembranous absorption (IMA) through induction of amnion VEGF in sheep fetuses subjected to altered IMA rates. Our study showed that RA receptor isoforms were expressed in sheep amnion, and RA response elements (RAREs) were identified in ovine RARß and VEGF gene promoters. In ovine amnion cells, RA treatment upregulated RARß messenger RNA (mRNA) and increased VEGF transcript levels. In sheep fetuses, increases in IMA rate was associated with elevated VEGF mRNA levels in the amnion but not in the chorion. Further, RARß mRNA was positively correlated with VEGF mRNA levels in the amnion and not chorion. We conclude that an RA pathway is present in ovine fetal membranes and that RA is capable of inducing VEGF. The finding of a positive relationship between amnion VEGF and RARß during altered IMA rate suggests that the retinoid pathway may play a role through VEGF in regulating intramembranous transport across the amnion.

Effects of maternal western-style diet on amniotic fluid volume and amnion VEGF profiles in a nonhuman primate model.

During pregnancy, high fat diet (HFD) induces maternal obesity, insulin resistance, and placental inflammatory responses that compromise placental and fetal development. Whether maternal HFD would adversely affect amniotic fluid volume (AFV) has not been explored. Vascular endothelial growth factor (VEGF) is expressed in the amnion and has been proposed as a regulator of AFV. Our aim was to investigate the effects of HFD on AFV and the associated changes in VEGF and soluble VEGF receptor 1 (sFlt-1) expression profiles in three amnion regions of a nonhuman primate model. Further, we examined the relationships between VEGF expression and HFD-induced changes in maternal metabolic status. Japanese macaques were maintained on control or HFD and amniotic fluid index (AFI) was measured as an ultrasonic estimate of AFV. Amniotic fluid VEGF concentrations were determined by ELISA and amnion VEGF and sFlt-1 mRNA levels by real-time RT-qPCR. HFD increased maternal plasma triglyceride while glucose levels were unchanged. Maternal weight gain was found in diet-sensitive animals whereas amniotic fluid VEGF concentration was reduced in diet-resistant animals. HFD did not alter AFI and there was no correlation between AFI and maternal weight or amniotic fluid VEGF concentrations. VEGF mRNA levels were lowest in secondary placental amnion while sFlt-1 mRNA were lowest in the primary placental amnion. HFD did not affect amnion VEGF or sFlt-1 mRNA expression. These findings suggest that although maternal HFD increased maternal weight in diet-sensitive and reduced amniotic fluid VEGF concentrations in diet-resistant phenotype, AFV as indicated by the AFI, was not significantly affected.

Regulation of amniotic fluid volume: insights derived from amniotic fluid volume function curves.

Recent advances in understanding the regulation of amniotic fluid volume (AFV) include that AFV is determined primarily by the rate of intramembranous absorption (IMA) of amniotic fluid across the amnion and into fetal blood. In turn, IMA rate is dependent on the concentrations of yet-to-be identified stimulator(s) and inhibitor(s) that are present in amniotic fluid. To put these concepts in perspective, this review 1) discusses the evolution of discoveries that form the current basis for understanding the regulation of AFV, 2) reviews the contribution of IMA to this regulation, and 3) interprets experimentally induced shifts in AFV function curves and amnioinfusion function curves in terms of the activity of the amniotic fluid stimulator and inhibitor of IMA. In the early 1980s, it was not known whether AFV was regulated. However, by the late 1980s, IMA was discovered to be a "missing link" in understanding the regulation of AFV. Over the next 25 years the concept of IMA evolved from being a passive process to being an active, unidirectional transport of amniotic fluid water and solutes by vesicles within the amnion. In the 2010s, it was demonstrated that a renally derived stimulator and a fetal membrane-derived inhibitor are present in amniotic fluid that regulate IMA rate and hence are the primary determinants of AFV. Furthermore, AFV function curves and amnioinfusion function curves provide new insights into the relative efficacy of the stimulator and inhibitor of IMA.

High-fat diet effects on amniotic fluid volume and amnion aquaporin expression in non-human primates.

Western style, high-fat diet (HFD) and associated high lipid levels have deleterious effects on fetal and placental development independent of maternal obesity and/or diabetes. Our objectives were to determine whether HFD without development of obesity would alter amniotic fluid volume (AFV) and amnion aquaporin (AQP) expression in a non-human primate model. Japanese macaques were fed either a control diet or HFD before and during pregnancy. The four quadrant amniotic fluid index (AFI) was used as an ultrasonic estimate of AFV at 120 days gestation. Amnion samples were collected at 130 days gestation by cesarean section and AQP mRNA levels were determined by quantitative RT-PCR. Similar to that in human, AQP1, AQP3, AQP8, AQP9, and AQP11 were expressed in the macaque amnion with significant differences in levels among AQPs. In macaque, neither individual AQPs nor expression profiles of the five AQPs differed between control and non-obese HFD animals. There were regional differences in AQP expression in that, AQP1 mRNA levels were highest and AQP8 lowest in reflected amnion while AQP3, AQP9, and AQP11 were not different among amnion regions. When subdivided into control and HFD groups, AQP1 mRNA levels remain highest in the reflected amnion of both groups. The HFD did not significantly affect the AFI, but AFI was positively correlated with AQP11 mRNA levels independent of diet. Collectively, these data suggest that HFD in pregnant non-obese individuals may have at most modest effects on AFV as the AFI and amnion AQP expression are not substantially altered.

Transport-associated pathway responses in ovine fetal membranes to changes in amniotic fluid dynamics.

Current evidence suggests that amniotic fluid volume (AFV) is actively regulated by vesicular transport of amniotic fluid outward across the amnion and into the underlying fetal vasculature in the placenta. Our objective was to determine whether gene expression profiles of potential stimulators, inhibitors, and mediators of vesicular transport are altered in response to changes in intramembranous absorption (IMA) rate. Samples of ovine amnion and chorion were obtained from fetal sheep with normal, experimentally reduced or increased AFVs and IMA rates. Amnion and chorion levels of target mRNAs were determined by RT-qPCR In the amnion, caveolin-1 and flotillin-1 mRNA levels were unchanged during alterations in IMA rate. However, levels of both were significantly higher in amnion than in chorion. Tubulin-α mRNA levels in the amnion but not in chorion were reduced when IMA rate decreased, and amnion levels correlated positively with IMA rate (P < 0.05). Dynamin-2 mRNA levels were not altered by experimental conditions. Vascular endothelial growth factor (VEGF164 and VEGF164b) mRNA levels increased during both increases and decreases in IMA rate, whereas soluble Flt-1 levels did not change. Neither HIF-1α nor PBEF mRNA levels in the amnion were correlated with VEGF164 expression levels and were not related to IMA rate. Collectively, our findings suggest that changes in amnion microtubule expression may be important in the regulation of transcellular vesicular transport of amniotic fluid and thus modulate IMA rate. Further, our results are consistent with the concept that the amnion is the rate-limiting layer for amniotic fluid transport.

Vesicular uptake of macromolecules by human placental amniotic epithelial cells.

INTRODUCTION: Studies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy. METHODS: Placental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%-50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1-30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris® software. RESULTS: Vesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions. CONCLUSIONS: In human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism.

Aquaporins in ovine amnion: responses to altered amniotic fluid volumes and intramembranous absorption rates.

Aquaporins (AQPs) are transmembrane channel proteins that facilitate rapid water movement across cell membranes. In amniotic membrane, the AQP-facilitated transfer of water across amnion cells has been proposed as a mechanism for amniotic fluid volume (AFV) regulation. To investigate whether AQPs modulate AFV by altering intramembranous absorption (IMA) rate, we tested the hypothesis that AQP gene expression in the amnion is positively correlated with IMA rate during experimental conditions when IMA rate and AFV are modified over a wide range. The relative abundances of AQP1, AQP3, AQP8, AQP9, and AQP11 mRNA and protein were determined in the amnion of 16 late-gestation ovine fetuses subjected to 2 days of control conditions, urine drainage, urine replacement, or intraamniotic fluid infusion. AQP mRNA levels were determined by RT-qPCR and proteins by western immunoblot. Under control conditions, mRNA levels among the five AQPs differed more than 20-fold. During experimental treatments, mean IMA rate in the experimental groups ranged from 100 ± 120 mL/day to 1370 ± 270 mL/day. The mRNA levels of the five AQPs did not change from control and were not correlated with IMA rates. The protein levels of AQP1 were positively correlated with IMA rates (r(2) = 38%, P = 0.01) while the remaining four AQPs were not. These findings demonstrate that five AQPs are differentially expressed in ovine amnion. Our study supports the hypothesis that AQP1 may play a positive role in regulating the rate of fluid transfer across the amnion, thereby participating in the dynamic regulation of AFV.

Differential expression and regional distribution of aquaporins in amnion of normal and gestational diabetic pregnancies.

The region of the amnion overlying the placenta plays an active role in fluid exchange between amniotic fluid and fetal blood perfusing the surface of the placenta, whereas little transfer occurs across the reflected amnion that contacts the membranous chorion. Because aquaporins (AQPs) facilitate rapid movement of water across cells, we hypothesized that AQP gene expression in placental amnion is higher than in reflected amnion. Furthermore, because gestational diabetes mellitus (GDM) is often associated with polyhydramnios, we hypothesized that amnion AQP gene expression is reduced when amniotic fluid volume is elevated. Human placental and reflected amnion were obtained at cesarean delivery and subjected to relative quantitation of AQP mRNA by real-time RT-qPCR and proteins by western immunoblot. Amnion mRNA levels of five AQPs differed by up to 400-fold (P < 0.001), with AQP1 and AQP3 most abundant, AQP8 least and AQP9 and AQP11 intermediately expressed. Aquaporin proteins showed a similar profile. Aquaporin mRNA abundance was higher (P < 0.001) in placental than reflected amnion, whereas protein levels were lower (P < 0.01). In GDM pregnancies, neither AQP mRNA nor protein levels were different from normal. There was no correlation between AQP mRNA or protein levels with the amniotic fluid index in normal or GDM subjects. We conclude that there is a strong differential expression profile among individual AQPs and between regions of the amnion. These findings suggest differences in contribution of individual AQPs to water transport in the two regions of the amnion. Furthermore, AQP expression in the amnion is not altered in patients with GDM.

Regulation of amniotic fluid volume: mathematical model based on intramembranous transport mechanisms.

Experimentation in late-gestation fetal sheep has suggested that regulation of amniotic fluid (AF) volume occurs primarily by modulating the rate of intramembranous transport of water and solutes across the amnion into underlying fetal blood vessels. In order to gain insight into intramembranous transport mechanisms, we developed a computer model that allows simulation of experimentally measured changes in AF volume and composition over time. The model included fetal urine excretion and lung liquid secretion as inflows into the amniotic compartment plus fetal swallowing and intramembranous absorption as outflows. By using experimental flows and solute concentrations for urine, lung liquid, and swallowed fluid in combination with the passive and active transport mechanisms of the intramembranous pathway, we simulated AF responses to basal conditions, intra-amniotic fluid infusions, fetal intravascular infusions, urine replacement, and tracheoesophageal occlusion. The experimental data are consistent with four intramembranous transport mechanisms acting in concert: 1) an active unidirectional bulk transport of AF with all dissolved solutes out of AF into fetal blood presumably by vesicles; 2) passive bidirectional diffusion of solutes, such as sodium and chloride, between fetal blood and AF; 3) passive bidirectional water movement between AF and fetal blood; and 4) unidirectional transport of lactate into the AF. Further, only unidirectional bulk transport is dynamically regulated. The simulations also identified areas for future study: 1) identifying intramembranous stimulators and inhibitors, 2) determining the semipermeability characteristics of the intramembranous pathway, and 3) characterizing the vesicles that are the primary mediators of intramembranous transport.

Regulation of amniotic fluid volume: evolving concepts.

Studies in late gestation fetal sheep have provided several new insights into the regulation of amniotic fluid (AF) volume (AFV): There are four quantitatively important amniotic inflows and outflows that include fetal urine production, lung liquid secretion, swallowing, and intramembranous absorption. Of these, AFV is regulated primarily by modulating the rate of intramembranous absorption of AF water and solutes across the amniotic epithelial cells into the underlying fetal vasculature. Modulation of the rate of intramembranous absorption depends on the presence of stimulators and inhibitors present in the AF. A stimulator of intramembranous absorption is present in fetal urine. In addition, AF contains a non-renal, non-pulmonary inhibitor of intramembranous absorption presumably secreted by the fetal membranes. Although passive bidirectional movements of water and solutes occur across the intramembranous pathway, intramembranous absorption is primarily a unidirectional, vesicular, bulk transport process mediated through VEGF activation of transcytotic transport via caveolae. Further, the stimulators and inhibitors of intramembranous absorption alter only the active, unidirectional component of intramembranous absorption while the passive components are not altered under experimental conditions studied thus far. Future progress depends on identifying the cellular and molecular mechanisms that regulate active and passive intramembranous absorption as well as their regulatory components.

Prostaglandin E2 regulation of amnion cell vascular endothelial growth factor expression: relationship with intramembranous absorption rate in fetal sheep.

We hypothesized that prostaglandin E2 (PGE2) stimulates amniotic fluid transport across the amnion by upregulating vascular endothelial growth factor (VEGF) expression in amnion cells and that amniotic PGE2 concentration correlates positively with intramembranous (IM) absorption rate in fetal sheep. The effects of PGE2 at a range of concentrations on VEGF 164 and caveolin-1 gene expressions were analyzed in cultured ovine amnion cells. IM absorption rate, amniotic fluid (AF) volume, and PGE2 concentration in AF were determined in late-gestation fetal sheep during control conditions, isovolumic fetal urine replacement (low IM absorption rate), or intra-amniotic fluid infusion (high IM absorption rate). In ovine amnion cells, PGE2 induced dose- and time-dependent increases in VEGF 164 mRNA levels and reduced caveolin-1 mRNA and protein levels. VEGF receptor blockade abolished the caveolin-1 response, while minimally affecting the VEGF response to PGE2. In sheep fetuses, urine replacement reduced amniotic PGE2 concentration by 58%, decreased IM absorption rate by half, and doubled AF volume (P < 0.01). Intra-amniotic fluid infusion increased IM absorption rate and AF volume (P < 0.01), while amniotic PGE2 concentration was unchanged. Neither IM absorption rate nor AF volume correlated with amniotic PGE2 concentration under each experimental condition. Although PGE2 at micromolar concentrations induced dose-dependent responses in VEGF and caveolin-1 gene expression in cultured amnion cells consistent with a role of PGE2 in activating VEGF to mediate AF transport across the amnion, amniotic PGE2 at physiological nanomolar concentrations does not appear to regulate IM absorption rate or AF volume.

Ovine fetal swallowing responses to polyhydramnios.

Abstract Swallowing of amniotic fluid by late gestation fetuses increases when amniotic fluid volume (AFV) is elevated. Our objectives were to quantitatively characterize fetal swallowing when AFV is elevated above normal to polyhydramniotic levels and to explore the mechanisms that mediate these changes. Late gestation fetal sheep were studied under basal conditions and during intra-amniotic infusion of lactated Ringer's solution. Control AFV averaged 631 ± 214 mL (SE, n = 6), swallowed volume was 299 ± 94 mL/day, and there were 5.7 ± 1.8 bouts/day of rapid swallowing. During intra-amniotic infusion, AFV (3065 ± 894 mL) and daily swallowed volume (699 ± 148 mL/day) increased (P < 0.05) and the number of bouts reached a maximum of 13.7 ± 2.0 bouts/day when AFV exceeded 1500 mL. Unexpectedly, the volume swallowed per bout (57.3 ± 5.8 mL, n = 102) did not vary with AFV (r = 0.023, P = 0.81). Neither the number of swallows/day nor the volume/swallow changed consistently with elevated AFV. Daily swallowed volume increases and reaches a maximum of twice normal as AFV approaches polyhydramniotic levels. Mechanistically, the increase in swallowing was achieved primarily by an increase in the number of bouts of swallowing per day rather than the expected passive increase in volume per bout. This implies changes in fetal behavior as AFV was elevated. Furthermore, swallowed volume was four times more sensitive to increases in AFV than reported previously.

Inhibitor of intramembranous absorption in ovine amniotic fluid.

Intramembranous absorption increases during intra-amniotic infusion of physiological saline solutions. The increase may be due partly to the concomitant elevation in fetal urine production as fetal urine contains a stimulator of intramembranous absorption. In this study, we hypothesized that the increase in intramembranous absorption during intra-amniotic infusion is due, in part, to dilution of a nonrenal inhibitor of intramembranous absorption that is present in amniotic fluid. In late-gestation fetal sheep, amniotic fluid volume and the four primary amniotic inflows and outflows were determined over 2-day intervals under three conditions: 1) control conditions when fetal urine entered the amniotic sac, 2) during intra-amniotic infusion of 2 l/day of lactated Ringer solution when urine entered the amniotic sac, and 3) during the same intra-amniotic infusion when fetal urine was continuously replaced with lactated Ringer solution. Amniotic fluid volume, fetal urine production, swallowed volume, and intramembranous absorption rate increased during the infusions independent of fetal urine entry into the amniotic sac or its replacement. Lung liquid secretion rate was unchanged during infusion. Because fetal membrane stretch has been shown not to be involved and because urine replacement did not alter the response, we conclude that the increase in intramembranous absorption that occurs during intra-amniotic infusions is due primarily to dilution of a nonrenal inhibitor of intramembranous absorption that is normally present in amniotic fluid. This result combined with our previous study suggests that a nonrenal inhibitor(s) together with a renal stimulator(s) interact to regulate intramembranous absorption rate and, hence, amniotic fluid volume.

Regulation of intramembranous absorption and amniotic fluid volume by constituents in fetal sheep urine.

Our objective was to test the hypothesis that fetal urine contains a substance(s) that regulates amniotic fluid volume by altering the rate of intramembranous absorption of amniotic fluid. In late gestation ovine fetuses, amniotic fluid volumes, urine, and lung liquid production rates, swallowed volumes and intramembranous volume and solute absorption rates were measured over 2-day periods under control conditions and when urine was removed and continuously replaced at an equal rate with exogenous fluid. Intramembranous volume absorption rate decreased by 40% when urine was replaced with lactated Ringer solution or lactated Ringer solution diluted 50% with water. Amniotic fluid volume doubled under both conditions. Analysis of the intramembranous sodium and chloride fluxes suggests that the active but not passive component of intramembranous volume absorption was altered by urine replacement, whereas both active and passive components of solute fluxes were altered. We conclude that fetal urine contains an unidentified substance(s) that stimulates active intramembranous transport of amniotic fluid across the amnion into the underlying fetal vasculature and thereby functions as a regulator of amniotic fluid volume.

Fetal swallowing as a protective mechanism against oligohydramnios and polyhydramnios in late gestation sheep.

UNLABELLED: Our objectives were to (1) quantify the relationship between daily swallowed volume and amniotic fluid volume (AF volume) in late gestation ovine fetuses and (2) use the resulting regression equation to explore the role of swallowing in regulating AF volume. Daily swallowed volume ranged from 36 to 1963 mL/d while experimental AF volume ranged from 160 to 6150 mL (n = 115). Swallowed volume was near zero when AF volume was far below normal, a maximum of 635 ± 41 (standard error) mL/d when AF volume was 1682 ± 31 mL and did not increase further with higher AF volumes. Computer simulations predicted that fetal swallowing would (1) return AF volume to normal in 5 to 6 days following an acute volume change in the absence of changes in other amniotic inflows or outflows and (2) stabilize AF volume in 4 to 8 days following sustained alterations in amniotic inflows or outflows other than swallowing. CONCLUSIONS: The volume of AF swallowed each day by the fetus is a strong function of AF volume and reaches a maximum when mild polyhydramnios develops. With deviations in AF volume from normal, changes in fetal swallowing protect against oligohydramnios and polyhydramnios because the changes in swallowing over time reduce the extent of the AF volume change. However, with experimental changes in AF volume stabilizing in 1 to 2 days, it appears that swallowing is not the major regulator of AF volume.

Amniotic fluid volume and composition after fetal membrane resection in late-gestation sheep.

The chronically catheterized fetal sheep is a widely used model for fetal physiologic and pathophysiologic investigations. Catheterization involves opening the amniochorion to gain access to the fetus. In the current study, we explored the role of the amnion and amniochorion in maintaining normal amniotic fluid volume (AFV) and composition and fetal blood-gas status after surgery. Fetal sheep were catheterized at 119.6 ± 0.3 (mean ± SE, n = 25) d gestation (term, approximately 147 d). An opening equal to approximately 5% of total membrane surface area was created by resecting a portion of the amnion or amniochorion during surgery. The uterine wall was closed in all animals. Compared with control sheep (AFV = 992 ± 153 mL, n = 11), resection of the amnion had no significant effect on AFV (745 ± 156 mL, n = 7) measured 5 d after surgery, whereas resection of the amniochorion resulted in extensive loss of amniotic fluid (AFV = 131 ± 38 mL, n = 7). This loss resulted from extensive entry of amniotic fluid into the space between the chorion and uterine wall. Amniotic fluid, fetal plasma, and urinary solute concentrations; arterial pH; oxygen tension; and carbon dioxide tension were unchanged. A small opening in the amnion has minimal effects on ovine AFV, whereas a small opening in the amniochorion results in oligohydramnios. In addition, the amnion appears to be the primary site that limits the rate of amniotic fluid absorption by the chorionic vasculature.

Regulation of caveolin-1 expression and phosphorylation by VEGF in ovine amnion cells.

Vascular endothelial growth factor (VEGF) has been implicated in the regulation of vesicular transport of amniotic fluid via caveolae across the amnion. This study tested the hypothesis that VEGF regulates caveolar function by stimulating caveolin-1 expression and phosphorylation in ovine amniotic epithelial cells (oAECs). Using primary cultures of oAECs, caveolin-1 was identified by immunofluorescent staining. Caveolin-1 messenger RNA (mRNA) abundance was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and protein by Western blotting. The effects of VEGF( 165) on caveolin-1 expression and phosphorylation were determined. Caveolin-1 immunoreactivity was detected in oAECs. In response to 10 ng/mL VEGF( 165), caveolin-1 mRNA levels increased whereas the protein levels were unaffected. Furthermore, VEGF stimulated caveolin-1 phosphorylation, an effect abrogated by the inhibition of c-Src protein kinase. These data suggest that VEGF upregulates caveolin-1 activity through c-Src signaling pathways. Our observations support the hypothesis that VEGF regulates amniotic fluid transport across the amnion by stimulating caveolin-1 activity to mediate caveolar function in amnion cells.

Intramembranous solute and water fluxes during high intramembranous absorption rates in fetal sheep with and without lung liquid diversion.

OBJECTIVE: To examine mechanisms that mediate increased intramembranous solute and water absorption. STUDY DESIGN: Intramembranous solute and water fluxes were measured in fetal sheep under basal conditions and after intraamniotic infusion of lactated Ringer's solution of 4 L/d for 3 days with and without lung liquid diversion. RESULTS: Intramembranous sodium, potassium, chloride, calcium, glucose, and lactate fluxes increased 2.5- to 7.9-fold, were linearly related to volume fluxes (r = 0.83-0.99), and were unaffected by lung liquid. All clearance rates, except that of lactate, increased to equal the intramembranous volume absorption rate during infusion. CONCLUSION: Under basal conditions, passive diffusion makes a minor and bulk flow a major contribution to intramembranous solute absorption. During high absorption rates, the increase in solute absorption above basal levels appears to be due entirely to bulk flow and is unaffected by lung liquid. The increased bulk flow is consistent with vesicular transcytosis.