Perfusion Of Placenta Research Articles

Influence of the magnesium aspartate hydrochloride administration to the maternal circuit on the aspartate concentration of the fetal circuit under in vitro perfusion of human placenta

Objectives Magnesium aspartate hydrochloride (Magnesiocard ®, Mg–Asp–HCl) is proposed as a substitute of magnesium sulfate for the treatment of preeclampsia and premature labor. After an i.v. administration of a dose equivalent to that used in the treatment of preeclampsia to nonpregnant volunteers, a 10-fold increase of aspartic acid (Asp) over the physiological level was observed. Animal experiments have demonstrated that highly increased fetal levels of acidic amino acids such as Asp could be associated with neurotoxic damage in the fetal brain. The influence of such an elevation of Asp concentration in the maternal circuit on the fetal level, using the in vitro perfusion model of human placenta, was investigated. Study design After a control phase (2 h), a therapeutic dose of Mg combined with Asp (Magnesiocard ®, Mg–Asp–HCl) was applied to the maternal circuit approaching 10 times the physiological level of Asp. The administration was performed in two different phases simulating either a peak of maximum concentration (bolus application, 2 h) or a steady state level (initially added, 4 h). Results In four experiments, during experimental phases (6 h) a slow increase in concentration in the fetal circuit was seen for Mg, AIB (alpha-aminoisobutyric acid, artificial amino acid) and creatinine confirming previous observations. In contrast, no net transfer of Asp across the placenta was seen. A continuous decrease in the concentration of Asp on both maternal and fetal side suggests active uptake and metabolization by the placenta. Viability control parameters remained stable indicating the absence of an effect on placental metabolism, permeability and morphology. Conclusion Elevation of Asp concentration up to 10 times the physiological level by the administration of Mg–Asp–HCl to the maternal circuit under in vitro perfusion conditions of human placenta has no influence on the fetal level of Asp suggesting no transfer of Asp from the maternal to fetal compartment. Therefore, the administration of Mg–Asp–HCl to preeclamptic patients would be beneficial for the patients without any impact on placental or fetal physiology.

OC080: Assessment of embryonic and placental blood flow & placental ultrastructure changed with contrast agent (SonoVueTM) in late pregnant rat

The study was to investigate the ability of the contrast pulse sequencing technology (CPS) and SonoVueTM (a new contrast agent) to delineate placental angioarchitecture and their influence on placental function, circulation and ultrastructure. sixty late pregnant Wistar rats (median weight, 300 g; range, 260 g to 330 g; median gestational age, 19 days) were selected and divided into three experimental groups, i.e. control group (n=20) injected with 2.0 ml/kg physiologic saline solution, high dose group (n=20) injected with 10 mg/ml SonoVueTM 2.0 ml/kg (Bracco S.p.A., Milan, Italy), and low dose group (n=20) injected with 0.01 mg/ml SonoVueTM 2.0 ml/kg. One of placentas was randomly selected for examination by contrast enhanced ultrasonography. Maternal and embryonic heart rate (HR) and resistance index (RI), pulsatility index (PI), and systolic-diastolic ratio (S/D) in the embryonic middle cerebral artery (MCA), umbilical artery (UA), and uterine artery (UTA) were measured before and after administration of contrast agent or physiologic saline solution. In each group, fifteen rats were cesarean to take the placenta immediately after exam, other five did after 12 hours of exam. The ultrastructural changes of the placenta were observed by electron microscope. Intravascular injection of SonoVueTM was capable of delineating the vascular architecture of the villous tree of rat. No enhancement was seen after saline solution injections. There were no significant changes of resistance index (RI), pulsatility index (PI), and systolic-diastolic ratio (S/D) in the embryonic middle cerebral artery, umbilical artery, and uterine artery after administration of contrast agent or physiologic saline solution. The maternal and embryonic heart rate significantly decreased. However, differences among three groups were not statistically significant; In low dose group, there were no obvious changes of helicine artery in rats's placenta taken immediately and after 12 hours of examination. In high dose group, taken immediately, the helicine artery wall were uneven, there are blood platelet aggregate and accrete around the thinningz; draw it after 12 hours, the vessel wall were thickening and swell, to be missing normal architecture of the blood vessel wall, the endothelial cell exfoliate in Lumen of blood vessel, we can also see apoptotic body in it. Contrast-enhanced ultrasonography limpidly presents the characteristics of perfusion of placenta in a rat model and does not affect the embryonic hemodynamic parameters. Low dose contrast agent does not impact ultrastructural changes of placent. High dose contrast agent can influence on ultrastructural of helicine artery wall.

ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of 14C-PhIP (2 μM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72 ± 0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of 14C-PhIP from maternal to fetal circulation (FM ratio 0.90 ± 0.08 at 6 h, p < 0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75 ± 0.10, p = 0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of 14C-PhIP ( R = − 0.81, p < 0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP ( R: − 0.11, p = 0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of 14C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarinoma cells.

Placental Passage of Benzoic acid, Caffeine, and Glyphosate in an Ex Vivo Human Perfusion System

Ex vivo perfusion of the human term placenta is a method to study placental transfer without extrapolation from animal to human and with no ethical concerns for mother and child. However, ex vivo placenta perfusion has a limited potential within chemical screening and testing as the method is time-consuming. This study was an attempt to construct data needed to develop quantitative structure–activity relationship (QSAR) models that are able to predict placental transfer of new compounds. Placental transfer is a biological activity that statistically may be related to the physiochemical properties of a given group of compounds. Benzoic acid, caffeine, and glyphosate were chosen as model compounds because they are small molecules with large differences in physiochemical properties. Caffeine crossed the placenta by passive diffusion. The initial transfer rate of benzoic acid was more limited in the first part of the perfusion compared to caffeine, but reached the same steady-state level by the end of perfusion. The transfer of glyphosate was restricted throughout perfusion, with a lower permeation rate, and only around 15% glyphosate in maternal circulation crossed to the fetal circulation during the study period.

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-1β (IL-1β) 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-1β 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-1β 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-1β 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-1β and IL-Ra into the maternal and fetal compartments was not affected by LPS. IL-1β 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-1β 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-1β 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.

Increased maternofetal calcium flux in parathyroid hormone‐related protein‐null mice

The role of parathyroid hormone-related protein (PTHrP) in fetal calcium homeostasis and placental calcium transport was examined in mice homozygous for the deletion of the PTHrP gene (PTHrP−/− null; NL) compared to PTHrP+/+ (wild-type; WT) and PTHrP+/− (heterozygous; HZ) littermates. Fetal blood ionized calcium was significantly reduced in NL fetuses compared to WT and HZ groups at 18 days of pregnancy (dp) with abolition of the fetomaternal calcium gradient. In situ placental perfusion of the umbilical circulation at 18 dp was used to measure unidirectional clearance of 45Ca across the placenta in maternofetal (CaKmf) and fetoplacental (CaKfp) directions; CaKfp was < 5% of CaKmf for all genotypes. At 18 dp, CaKmf across perfused placenta and intact placenta (CaKmf(intact)) were similar and concordant with net calcium accretion rates in vivo. CaKmf was significantly raised in NL fetuses compared to WT and HZ littermates. Calcium accretion was significantly elevated in NL fetuses by 19 dp. Placental calbindin-D9K expression in NL fetuses was marginally enhanced (P < 0.07) but expression of TRPV6/ECaC2 and plasma membrane Ca2+-ATPase (PMCA) isoforms 1 and 4 were unaltered. We conclude that PTHrP is an important regulator of fetal calcium homeostasis with its predominant effect being on unidirectional maternofetal transfer, probably mediated by modifying placental calbindin-D9K expression. In situ perfusion of mouse placenta is a robust methodology for allowing detailed dissection of placental transfer mechanisms in genetically modified mice.

Role of the Multidrug Transporter Proteins ABCB1 and ABCC2 in the Diaplacental Transport of Talinolol in the Term Human Placenta

Placental syncytiotrophoblasts are known to express the efflux transporter proteins P-glycoprotein (ABCB1) and multidrug resistance-associated protein 2 (ABCC2), which are supposed to be a functional part of the human placental barrier. With advancing gestational age, expression of ABCB1 decreases progressively, whereas ABCC2 is more expressed. To evaluate to which extent they contribute to placental barrier function at term, permeability of talinolol, a substrate of both carriers, was measured using a validated human placenta perfusion model. We identified in randomized, crossover experiments a unidirectional transfer of talinolol in the fetomaternal direction because the maternofetal transfer was significantly lower (0.663 +/- 0.188 versus 0.394 +/- 0.067 relative to creatinine permeability, p = 0.012). Maternofetal permeability was increased by the ABCC2 inhibitor probenecid (0.59 +/- 0.15 versus 0.68 +/- 0.13, p = 0.028) and the nonspecific inhibitor verapamil (0.53 +/- 0.09 versus 0.66 +/- 0.16, p = 0.028) but was not influenced by the ABCB1 inhibitor valspodar (PSC833) (0.48 +/- 0.11 versus 0.46 +/- 0.09, p = 0.345). Genetic polymorphisms of ABCB1 and ABCC2 lacked significant influence on expression of the carriers and permeability of talinolol, respectively. In conclusion, maternofetal transfer of talinolol is restricted by a unidirectional process that is influenced by inhibitors of ABCC2.

Participation in environmental health research by placenta donation – a perception study

BackgroundMuch environmental health research depends on human volunteers participating with biological samples. The perception study explores why and how people participate in a placenta perfusion study in Copenhagen. The participation implies donation of the placenta after birth and some background information but no follow up.MethodsNineteen semi-structured qualitative interviews were conducted with participants in the placenta perfusion study after donation of placenta. Observation studies were made of recruitment sessions.ResultsThe interviewed participants are generally in favour of medical research. They participated in the placenta perfusion study due to a belief that societal progress follows medical research. They also felt that participating was a way of giving something back to the Danish health care system. The participants have trust in medical science and scientists, but trust is something which needs to be created through "trust-work". Face-to-face interaction, written information material and informed consent forms play important parts in creating trusting relationships in medical research.ConclusionMedical research ethics do not only amount to specific types of written information material but should also be seen as a number of trust making performances involving researchers as well as research participants.

Decrease in lipid levels of syncytiotrophoblast micro-particles reduced their potential to inhibit endothelial cell proliferation

Preeclampsia is characterized by damage to the maternal endothelium that has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. Previously, we have shown that STBM, prepared by three different methods: mechanical dissection, in vitro placental explants culture and perfusion of placenta, can inhibit endothelial cell proliferation. Only mechanically prepared STBM induced apoptosis in the endothelial cells. Now, we have examined lipid levels in the three STBM preparations and their differential responses on endothelial cells. We examined the lipid levels in the three STBM preparations using thin layer chromatography. Furthermore, the effects of reduced lipid levels in the three STBM preparations using the pharmacological agent methyl-beta-cyclodextrin were examined on endothelial cell proliferation and apoptosis. Among the three STBM preparations, mechanical STBM contained highest levels of lipids. The reduction in lipid levels in mechanical STBM reduced their potential to inhibit human umbilical vein endothelial cells (HUVEC) proliferation and blocked their potential to induce apoptosis. No similar effect was observed following lipid reduction in the two other STBM preparations. As it has been suggested that mechanically derived STBM may more closely resemble placental micro-particles generated in preeclampsia, our data suggest that lipid content may play a role in the anti-endothelial defects present in this disease.

Potential Roles of Angiotensin Receptor-Activating Autoantibody in the Pathophysiology of Preeclampsia

Preeclampsia affects ≈7% of first pregnancies and is one of the leading causes of maternal and neonatal mortality and morbidity in the United States and the world.1–3 The clinical hallmarks of the disorder include hypertension, proteinuria, hypercoagulability, edema, and placental abnormalities. In advanced stages, clinical symptoms include cerebral edema, renal failure, and the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. The clinical management of preeclampsia is hampered by the lack of reliable diagnostic tests and effective therapy for the disorder. In some cases, termination of pregnancy is the only available option to prevent further deterioration of the fetus and mother. Fifteen percent of all preterm births are indicated early deliveries for preeclampsia.3 The resulting preterm births and the associated increased infant morbidity and mortality are especially disheartening consequences of preeclampsia. Despite being one of the leading causes of maternal death and a major contributor to maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are poorly understood. Roberts and colleagues4,5 were among the first to propose that alterations in endothelial cell function by activating agents produced by the placenta initiate the clinical syndromes of preeclampsia. Circulating factors, such as inflammatory cytokines, endothelin, and soluble vascular endothelial growth factor receptor termed soluble fms-like tyrosine kinase-1 (sFlt-1), are elevated in preeclamptic women and are proposed to be important links between placental ischemia and endothelial dysfunction.5–9 In particular, recent studies have shown that preeclampsia is associated with the presence of maternal autoantibodies capable of binding to and activating the angiotensin (Ang) receptor type-1 (AT1).10–15 AT1 receptor agonistic antibodies, herein termed AT1-AA, are rarely seen in normotensive pregnant women.10,11 Since the initial discovery of these autoantibodies, considerable evidence supporting a pathophysiological role of AT1-AA in preeclampsia has …

Transplacental Transfer of Monomethyl Phthalate and Mono(2-ethylhexyl) Phthalate in a Human Placenta Perfusion System

The transplacental passage of monomethylphtalate (mMP) and mono (2-ethylhexyl) phthalate (mEHP) was studied using an ex vivo placental perfusion model with simultaneous perfusion of fetal and maternal circulation in a single cotyledon. Umbilical cord blood and placental tissue collected both before and after perfusion were also analyzed. Placentas were obtained immediately after elective cesarean section and dually perfused in a recirculation system. mMP or mEHP was added to maternal perfusion medium to obtain concentrations at 10 and 25 microg/L, respectively. The placental transfer was followed analyzing samples from fetal and maternal perfusion media by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). Four perfusions with mMP indicated a slow transplacental transfer, with a feto-maternal ratio (FM ratio) of 0.30 +/- 0.03 after 150 min of perfusion. Four perfusions with mEHP indicated a very slow or nonexisting placental transfer. mEHP was only detected in fetal perfusion media from two perfusions, giving rise to FM ratios of 0.088 and 0.20 after 150 min of perfusion. Detectable levels of mMP, mEHP, monoethylphthalate (mEP), and monobutylphthalate were found in tissue. Higher tissue levels of mMP after perfusions with mMP compared to perfusions with mEHP suggest an accumulation of mMP during perfusion. No tendency for accumulation of mEHP was observed during perfusions with mEHP compared to perfusions with mMP. Detectable levels of mEHP and mEP were found in umbilical cord plasma samples. mMP and possibly other short-chained phthalate monoesters in maternal blood can cross the placenta by slow transfer, whereas the results indicate no placental transfer of mEHP. Further studies are recommended.

Unusual maternal vasculature in the placental periphery leading to the diagnosis of abdominal pregnancy at 25 weeks' gestation

Abdominal pregnancy is a rare condition in which the fetus and placenta are located within the peritoneal cavity. Sonographic findings include visualization of the fetus separate from the uterus, failure to visualize the uterine wall between the fetus and urinary bladder, close approximation of fetal parts to the maternal abdominal wall, eccentric position or abnormal fetal attitude, and visualization of extrauterine placental tissue. We present an unusual case in which mid-trimester transabdominal color Doppler sonographic findings depicted unusual maternal vasculature in the placental periphery leading to the diagnosis of abdominal pregnancy. Postpartum maternal angiography confirmed these vessels as abnormal maternal arterial perfusion of the extrauterine placenta emanating from the uterine arteries and inferior epigastric arteries. Systematic review of the literature confirms that this is the first report of such sonographic manifestations of an abdominal pregnancy.

HCG reduces leptin release in the dual in vitro placenta perfusion model

Background: High maternal hCG levels were found in preeclampsia and in conditions of fetal growth retardation. Since leptin may be involved in the pathophysiology of preeclampsia and fetal growth we are interested in the effect of hCG on placental leptin release and general placental function.

Phthalate monoesters in perfusate from a dual placenta perfusion system, the placenta tissue and umbilical cord blood

Fetal exposure to phthalates may be associated with adverse reproductive effects, including cryptorchidism and decreased semen quality. Information about human placental transfer is needed to qualify the hypotheses. A dual recirculating placenta perfusion system to monitor concentrations of eight phthalate monoesters in fetal and maternal perfusates was established. In addition to perfusate background measures of phthalate monoesters, the concentrations in umbilical cord plasma and placenta tissue were measured. Monomethyl phthalate (mMP), monoethyl phthalate (mEP), monobutyl phthalate (mBP), and mono (2-ethyl-hexyl) phthalate (mEHP) were detected in both maternal and fetal perfusate, demonstrating a release of compounds from tissue or blood to perfusates. The distribution of compounds between perfusate, umbilical cord plasma, and tissue was in accordance with the physical–chemical properties of the compounds. Results from the present study of compounds residing in the tissue are essential before studying human transplacental transfer, storage, and metabolism of selected phthalate monoesters.

Experimental methods to study human transplacental exposure to genotoxic agents

Human placenta differs more than any other organ between species. This is the primary reason to develop models utilizing human tissue to study placental functions. There are no major ethical restrictions using human placenta for scientific studies. Also, the size of human placenta enables a great number of different parameters to be studied in one placenta. The most important cell types considering transplacental transfer, are the trophoblasts differentiating into syncytiotrophoblasts facing maternal circulation, and endothelial cells of fetal vessels. Primary trophoblasts are difficult to culture and do not grow in monolayer thus inhibiting studies on the polarized functions of transport. Several cell lines originating from trophoblasts have been developed, of which BeWo cells seem most useful for transport studies, because they grow in a tight monolayer. Placental tissue can also be retained as explant cultures, although the trophoblast viability is very restricted despite of culture conditions. Cotyledons of human placenta can be retained viable in an isolated organ perfusion. Perfused placental tissue stays viable longer than placental tissue in tissue culture. Although human placental perfusion is the most tedious experimental method to study placental functions, there are several good reasons to develop it further: transplacental transfer and molecular mechanisms of genotoxic compounds can be studied. Placental perfusion is the only experimental method that retains fully the structure of placenta for polarized transport. Furthermore, perfusion of placentas from mothers, who smoke, use illegal drugs or have a disease, allows studies on the impact of such factors on fetal exposure to genotoxic agents.

Differential Release of Plasminogen Activator Inhibitors (PAIs) During Dual Perfusion of Human Placenta: Implications in Preeclampsia

Plasminogen activator inhibitors (PAIs) play critical roles in regulating cellular invasion and fibrinolysis. An increase in the ratio of PAI-1/PAI-2 in placenta and maternal serum is suggested to result in excessive intervillous fibrin deposition and placental infarction in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). In the current study we used dual (maternal and fetal) perfusion of human term placentas to examine the release of PAIs to the intervillous space. ELISA revealed a significant time-dependent increase in total PAI-1 levels in maternal perfusate (MP) between 1 and 7 h of perfusion. Conversely, PAI-2 levels decreased resulting in a 3-fold increase in the PAI-1/PAI-2 ratio in MP. Levels of PAI-1, but not PAI-2, in placental tissue extracts increased during perfusion. In perfusions carried out with xanthine and xanthine oxidase (X + XO), compounds used to generate reactive oxygen species (ROS), no time-dependent increase in total PAI-1 levels was observed. In addition, X + XO treatment promoted a 3-fold reduction in active PAI-1 levels in MP, indicating that ROS decrease PAI-1 release to MP. The finding of a time-dependent change in patterns of PAI expression and response to ROS indicates the utility of dual perfusion as a model to dissect mechanism(s) promoting aberrant fibrinolysis in pregnancies complicated by PE and IUGR.

Correlations of placental perfusion and PlGF protein expression in early human pregnancy

The purpose of this study was to investigate temporal correlations between maternal serum placenta growth factor levels and placental perfusion in early human pregnancies. Systolic umbilical artery Doppler blood flow velocity indices at fetal and placental insertion sites were measured between 7 and 22 weeks of gestation from normal singleton pregnancies. Maternal serum placenta growth factor levels were determined by enzyme-linked immunosorbent assay. Maternal serum placenta growth factor levels showed an exponential increase at approximately 14 weeks of gestation. Placenta perfusion, as estimated by systolic Doppler blood flow indices, significantly increased with gestational age (P < .0001). There was a close association between placenta growth factor expression levels and evidence of increased placenta perfusion (P < .033). The significant increase in serum placenta growth factor coincides with the increased perfusion of the maternal/fetal interface at approximately 12 to 14 weeks of gestation. Correlation of placenta growth factor expression and placental perfusion suggests that placenta growth factor may contribute to assuring adequate vascular development/function of the placenta early in gestation.

Perfusion studies of glyburide transfer across the human placenta: Implications for fetal safety

Gestational diabetes affects up to 5% of women. Oral hypoglycemics have been avoided because of the assumption that their placental transfer may cause fetal-neonatal hypoglycemia. A recent randomized trial could not show measurable glyburide levels in umbilical blood despite maternal treatment with regular doses of glyburide. The mechanism underlying this phenomenon is not known. The objective of our study was to document, using a human placenta perfusion model, whether glyburide is actively effluxed from the fetal to the maternal circulation. In vitro perfusion studies of human cotyledon were performed to quantify placental transfer of glyburide. Using close circle experiments and introducing glyburide to both maternal and fetal circulations at 200 ng/mL, we looked for evidence of transport against concentration gradient from the fetus to the mother. In parallel experiments, the P-glycoprotein inhibitor verapamil was used in an attempt to inhibit transplacental glyburide movement. There was highly significant transfer of glyburide against concentration gradient from the fetal to the maternal circulation. Fetal-to-maternal concentration ratio was 0.92 +/- 0.23 at the start of the experimental period and 0.31 +/- 0.47 3 hours later (P = .01) (n = 5). Verapamil did not modify glyburide transport. This is the first direct evidence of active glyburide transport from the fetus to the mother and, in general, of any medicinal drug used during pregnancy. These experiments suggest that glyburide is actively efflux by a transporter other than P-glycoprotein. Alternatively, it is possible that a minority of glyburide is carried by P-glycoprotein, but most of the fetal load is pumped to the mother by a yet-unidentified placental transport system.

Artificial Perfusion of the Fetal Circulation of the In situ Mouse Placenta: Methodology and Validation

Here we present methodology and validation (including measurement of unidirectional maternofetal clearance (Kmf) of (45)Ca and (14)C-mannitol) for in situ perfusion of the mouse placenta. On day 18 of gestation (term=19 days) mice were anaesthetised and the uterus delivered into a saline bath (40 degrees C). A fetus was selected, the umbilical artery and vein catheterised and perfused with Krebs Ringer (pH 7.4) at 60 microl/min. (45)Ca/(14)C-mannitol (2 microCi/5 microCi in 50 microl saline) was injected via maternal tail vein. Perfusate samples were collected every 5 min for 45 min. Maternal carotid artery pressure was monitored throughout perfusion. A terminal maternal cardiac blood sample was taken and analysed. Placentas were immersion fixed and processed for electron microscopy. Kmf for (45)Ca and (14)C-mannitol was calculated as perfusate [(45)Ca or (14)C-mannitol] x perfusion rate/maternal plasma [(45)Ca or (14)C-mannitol]xplacental weight. Maternal cardiac blood chemistry at termination (n=8-15, mean+/-SEM) was as follows: pH 7.153+/-0.016, PCO(2) 45.48+/-2.06 mmHg, PO(2) 66.47+/-7.10 mmHg, Na(+) 151.4+/-1.2 mmol/l, K(+) 5.54+/-0.17 mmol/l, Ca(2+) 1.15+/-0.03 mmol/l, glucose 7.2+/-0.5 mmol/l, and lactate 1.76+/-0.77 mmol/l. A successful 45 min perfusion in which perfusate recovery was >95% occurred in >50% of animals. Perfusion did not alter placental morphology or carotid pressure. Kmf (microl/min/g placenta) for (45)Ca (66.0+/-8.4 (n=7)) was significantly higher than Kmf for (14)C-mannitol (20.0+/-2.4 (n=5)) (p<0.01). These data demonstrate physiological perfusion of the mouse placenta in situ and its usefulness for measurement of solute transfer.

18 Ultrastructural Alterations of Placental Tissue After 6H of Hypoxic Dual in vitro Placenta Perfusion

Background: Peripartal asphyxia affects the foetus and the placenta with its functions. This can be studied by the dual in vitro placenta perfusion model enabling the separated ex vivo perfusion of maternal and foetal circulation and simulating asphyxia.Aim: Examination of placental villous ultrastructure by electron microscopy before and after 6h of dual in vitro placenta perfusion with defined hypoxia to study whether morphological alterations could indicate a loss of placental function.Methods: Placentae (n=20) after normal pregnancies were studied. Written informed consent was obtained. Villous tissue was sampled before and after 6h of normoxic (pO2 80–90mmHg, n=10) or hypoxic (pO2= 20–30mmHg, n=10) placenta perfusion. Fixation, embedding, preparation of semi- and ultrathin sections. Systematic investigation by electron microscopy of terminal villous syncytio- and cytotrophoblast with its substructures mitochondria, rough and smooth endoplasmatic reticulum (ER), nucleus, golgi-apparatus and microvilli. Glucose consumption, lactate production, creatinine and antipyrin permeability as well as leptin- and hCG-release were used as control parameters during perfusion.Results: Terminal villi of all examined placentae showed intact substructures before and after normoxic perfusion. After 6h of hypoxic placenta perfusion the terminal villi demonstrated morphological alterations: villous stroma was oedematous, cell organelles (mitochondria, ER) swollen or completely degenerated, syncytio- and cytotrophoblast interspersed with multiple vacuoles and microvilli slenderly extended or partly cut off. The foetal blood vessels appeared narrow with luminal orientated endothelial cells nuclei. The morphological results correlated with the functional data.Conclusion: 6h of hypoxic dual in vitro placenta perfusion induces degenerative alterations of terminal villi which may impair the placental barrier and placental function.