Fetal Circuit Research Articles

In vitro studies of ferric carboxymaltose on placental permeability using the dual perfusion model of human placenta

An in vitro perfusion model of human placenta was used to study the transplacental passage of iron applied in the form of the drug compound ferric carboxymaltose (FCM) which had been radio-labelled with 59Fe. In four placental perfusion experiments, two simulated circuits for the maternal and fetal sides of the placenta were set up with two experimental phases each lasting 3 h. FCM was added to the maternal circuit at the beginning of each phase to a final iron concentration of 11 mM, which is at least 10 times higher than the maximal predicted level in blood after an administration of 200 mg iron as FCM. The effects of adding transferrin at a physiological concentration of 1.67 mg/ ml were also tested. The concentration profiles of 59Fe showed a 10% decrease within the first 30 min of perfusion on the maternal side. Thereafter the radioactivity levels remained unchanged. The addition of transferrin had no effect on the tissue uptake of 59Fe-FCM. No transferred iron radioactivity could be detected in the fetal circuit. Despite a loss of approximately 10% of the radio-labelled iron observed on the maternal side, only 0.5-2% of the radioactivity was detected in the placental tissue after perfusion. No free iron could be detected at the end of perfusion on the maternal side using ultrafiltration or acid precipitation methods. In addition, the production of transferrin receptor remained unchanged, with similar concentrations in placental tissue before and after perfusion. No effects of FCM on placental viability were observed in terms of energy metabolism (glucose consumption and lactate production), hormone release or placental permeability (assessed by the transfer rates of creatinine and antipyrine). However, two additional observations were made: firstly, a significant reduction in the rate of cell death compared to control conditions was observed in the presence of FCM; secondly, the integrity of the fetal capillary system was improved on the fetal side of the perfusion system. It is concluded that the iron compound FCM does not cross the placenta and may increase the integrity of placental tissue (at least under in vitro conditions), but this latter observation needs further investigation.

Transplacental Transfer and Metabolism of Buprenorphine in Preterm Human Placenta

We sought to determine whether gestational age affects the transplacental transfer and metabolism of buprenorphine (BUP). Transfer of BUP (10 ng/mL) and its [ (3)H]-isotope was determined across placentas of 30 to 34 weeks of gestation utilizing the technique of dual perfusion of placental lobule. Concentration of the drug in trophoblast tissue and in maternal and fetal circuits was determined by liquid scintillation spectrometry. Microsomes prepared from placentas of 17 to 37 weeks of gestation were divided into three groups: late second, early third, and late third trimesters. Antibodies raised against human cytochrome P450 (CYP) isoforms were utilized to identify the enzyme(s) catalyzing BUP biotransformation by preterm placental microsomes. The amount of norbuprenorphine formed was determined by liquid chromatography-mass spectrometry (LC-MS). BUP transfer across the placentas of 30 to 34 weeks of gestation was similar to those at term. CYP19 antibodies caused 60% inhibition of BUP metabolism by microsomes of late second and early third trimesters and 85% by microsomes of late third trimester. The developmental changes occurring in human placenta between 30 weeks of gestation through term do not affect the transfer of BUP across human placenta. CYP19 is the major enzyme responsible for biotransformation of BUP beginning at 17 weeks of gestation until term.

Transplacental transfer and metabolism of bupropion

Objective. In order to evaluate the potential use of bupropion as smoking cessation therapy during pregnancy, the aim of this investigation was to determine transplacental transfer and metabolism of bupropion and its distribution among placental tissue and maternal and fetal circuits of the dually perfused placental lobule.Methods. Placentas obtained from healthy term pregnancies were perfused with bupropion at two concentrations 150 ng/ml and 450 ng/ml, along with the marker compound antipyrine 20 μg/ml. Radioactive isotopes of the two drugs were co-transfused to enhance their detection limits. Concentrations of bupropion and its metabolite were determined by liquid chromatography and liquid scintillation spectrometry.Results. The fetal/maternal concentration ratio of bupropion was 1.07 ± 0.22. Following 4 h of its perfusion, 48 ± 6% of bupropion was retained by placental tissue, 32 ± 5% remained in the maternal circuit, and 20 ± 6% was transferred to the fetal circuit. A metabolite of bupropion, threohydrobupropion, was identified.Conclusions. Bupropion was transferred from the maternal to fetal circuit and was biotransformed by placental tissue enzymes to its metabolite threohydrobupropion. Bupropion and its metabolite did not affect placental tissue viability or functional parameters. These data suggest that bupropion has the potential of being used for smoking cessation during pregnancy and should be further investigated for its safety and efficacy.

Antibody-Dependent Transplacental Transfer of Malaria Blood-Stage Antigen Using a Human Ex Vivo Placental Perfusion Model

Prenatal exposure to allergens or antigens released by infections during pregnancy can stimulate an immune response or induce immunoregulatory networks in the fetus affecting susceptibility to infection and disease later in life. How antigen crosses from the maternal to fetal environment is poorly understood. One hypothesis is that transplacental antigen transfer occurs as immune complexes, via receptor-mediated transport across the syncytiotrophoblastic membrane and endothelium of vessels in fetal villi. This hypothesis has never been directly tested. Here we studied Plasmodium falciparum merozoite surface protein 1 (MSP1) that is released upon erythrocyte invasion. We found MSP1 in cord blood from a third of newborns of malaria-infected women and in >90% following treatment with acid dissociation demonstrating MSP1 immune complexes. Using an ex vivo human placental model that dually perfuses a placental cotyledon with independent maternal and fetal circuits, immune-complexed MSP1 transferred from maternal to fetal circulation. MSP1 alone or with non-immune plasma did not transfer; pre-incubation with human plasma containing anti-MSP1 was required. MSP1 bound to IgG was detected in the fetal perfusate. Laser scanning confocal microscopy demonstrated MSP1 in the fetal villous stroma, predominantly in fetal endothelial cells. MSP1 co-localized with IgG in endothelial cells, but not with placental macrophages. Thus we show, for the first time, antibody-dependent transplacental transfer of an antigen in the form of immune complexes. These studies imply frequent exposure of the fetus to certain antigens with implications for management of maternal infections during pregnancy and novel approaches to deliver vaccines or drugs to the fetus.

Comparison of placental transfer of local anesthetics in perfusates with different pH values in a human cotyledon model

We aimed to investigate the placental transfer of local anesthetics in perfusates with different pH values, using a dual-perfused human cotyledon model. The dual-perfused human cotyledon model was prepared from placentas obtained following cesarean delivery (n = 5). Protein-free solution was perfused through both maternal and fetal arteries. Four amide-type local anesthetics (mepivacaine [Mep]; lidocaine [Lid]; bupivacaine [Bup]; and ropivacaine [Rop]) were added to the maternal perfusate at 1 microg ml(-1). Three conditions were tested (stage 1, maternal pH 7.4, fetal pH 7.4; stage 2, maternal pH 7.4, fetal pH 6.9; and stage 3, maternal pH 6.9, fetal pH 6.9). Venous blood samples were collected from the fetal circuit after stabilization. The fetal vein/maternal artery concentration ratio (F/M ratio) of the local anesthetics was used as an index of placental transfer. The concentration of human chorionic gonadotropin (hCG) in the maternal vein was measured at the end of each stage. The F/M ratios in all stages were in the order of: Mep > Lid > Bup [symbol: see text] Rop. The F/M ratios of Mep were significantly higher than those of the other local anesthetics in all stages. The F/M ratios of Lid were higher than those of Rop in stages 2 and 3. The F/M ratios of Lid and Rop were higher in stage 2 than in stage 3. However, the differences between the F/M ratios in the three stages were not as large as expected from the basic uncharged ([B]) condition and pH gap. The concentration of hCG showed a time-dependent decrease with increasing stage (stage 1, 81.0 +/- 58.9 mIU ml(-1); stage 2, 57.4 +/- 31.8 mIU ml(-1); stage 3, 32.1 +/- 19.7 mIU ml(-1)). Our data clearly show that it is the basic uncharged concentration that mainly determines the placental transfer of amide-type local anesthetics with protein-free perfusate. This finding suggests that Rop and Bup can be used more safely than Mep in terms of placental transfer.

Insulin glargine safety in pregnancy: a transplacental transfer study.

OBJECTIVEInsulin glargine (Lantus) is an extended-action insulin analog with greater stability and duration of action than regular human insulin. The long duration of action and decreased incidence of hypoglycemia provide potential advantages for its use in pregnancy. However, the placental pharmacokinetics of insulin glargine have not been studied. Therefore, the objective of this study was to determine whether insulin glargine crosses the human placenta using the human perfused placental lobule technique.RESEARCH DESIGN AND METHODSPlacentae were obtained with informed consent after elective cesarean section delivery of noncomplicated term pregnancies. Insulin glargine, at a therapeutic concentration of 150 pmol/l (20 μU/ml) was added to the maternal circulation. Additional experiments were carried out at insulin glargine concentrations 1,000-fold higher than therapeutic levels (150, 225, and 300 nmol/l). A subsequent perfusion for which the maternal circuit remained open and insulin glargine was continuously infused at 150 pmol/l was completed for further confirmation of findings. The appearance of insulin glargine in the fetal circulation was analyzed by a chemiluminescence immunoassay.RESULTSResults from perfusions carried out at therapeutic concentrations (150 pmol/l) of insulin glargine showed no detectable insulin glargine in the fetal circuit. After perfusion with very high insulin glargine concentrations of 150, 225, and 300 nmol/l, the rate of transfer remained low at 0.079 ± 0.01, 0.14, and 0.064 pmol · min−1 · g tissue−1, respectively.CONCLUSIONSInsulin glargine, when used at therapeutic concentrations, is not likely to cross the placenta.

Transplacental transfer and metabolism of bupropion

In order to evaluate the potential use of bupropion as smoking cessation therapy during pregnancy, the aim of this investigation was to determine transplacental transfer and metabolism of bupropion and its distribution among placental tissue and maternal and fetal circuits of the dually perfused placental lobule. Placentas obtained from healthy term pregnancies were perfused with bupropion at two concentrations 150 ng/ml and 450 ng/ml, along with the marker compound antipyrine 20 microg/ml. Radioactive isotopes of the two drugs were co-transfused to enhance their detection limits. Concentrations of bupropion and its metabolite were determined by liquid chromatography and liquid scintillation spectrometry. The fetal/maternal concentration ratio of bupropion was 1.07 +/- 0.22. Following 4 h of its perfusion, 48 +/- 6% of bupropion was retained by placental tissue, 32 +/- 5% remained in the maternal circuit, and 20 +/- 6% was transferred to the fetal circuit. A metabolite of bupropion, threohydrobupropion, was identified. Bupropion was transferred from the maternal to fetal circuit and was biotransformed by placental tissue enzymes to its metabolite threohydrobupropion. Bupropion and its metabolite did not affect placental tissue viability or functional parameters. These data suggest that bupropion has the potential of being used for smoking cessation during pregnancy and should be further investigated for its safety and efficacy.

Effect of plasma proteins on buprenorphine transfer across dually perfused placental lobule

Objective. The aim of this investigation is to determine the effect of human serum albumin (HSA) and α-acid glycoprotein (AAG) on buprenorphine (BUP) transplacental transfer and distribution.Methods. The technique of dual perfusion of placental lobule (DPPL) was utilised. BUP was co-perfused with the marker compound antipyrine (AP). In each experiment, the radiolabelled isotopes [3H]-BUP and [14C]-AP were added to enhance their detection limits. Human plasma proteins, HSA and AAG, were added to both the maternal and fetal circuits separately and in combination at their physiological concentrations in maternal and fetal circulations close to term.Results. Transplacental transfer of BUP, in absence of plasma proteins, is a two-step process: the first is its uptake by the syncytiotrophoblast from the maternal circuit, and the second is its transfer/release from the tissue to the fetal circuit. The addition of HSA to the perfusion medium affected only the second step of BUP transfer, but AAG affected both steps. The combined effect of HSA and AAG was not different from that observed in presence of the latter alone.Conclusions. Binding of BUP to circulating AAG could have an important role in the transfer of the drug from the maternal to fetal circulation.

49: Continuous infusion with with 17-Hydroxyprogesterone Caproate (17-P) into the fetal or maternal arterial circuits of the ex vivo placental cotyledon model attenuates vasoconstriction by thromboxane and angiotensin-II

49: Continuous infusion with with 17-Hydroxyprogesterone Caproate (17-P) into the fetal or maternal arterial circuits of the ex vivo placental cotyledon model attenuates vasoconstriction by thromboxane and angiotensin-II

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.

Transplacental transfer and metabolism of 17-α-hydroxyprogesterone caproate

Determine transplacental transfer and metabolism of 17-alpha-hydroxyprogesterone caproate and its distribution between the tissue and the maternal and fetal circuits of the dually perfused placental lobule. 17-alpha-Hydroxyprogesterone caproate (21 ng/mL) and its dual-labeled isotope, 17-alpha-hydroxy-[(3)H] progesterone [(14)C] caproate were added to the maternal circuit. The concentrations of the drug and its metabolite in trophoblast tissue and both circuits were determined by high performance liquid chromatography and liquid scintillation spectrometry. 17-alpha-Hydroxyprogesterone caproate was transferred from the maternal to fetal circuit. After a 4-hour perfusion period, a metabolite of 17-alpha-hydroxyprogesterone caproate that retained both progesterone and caproate moieties was identified in the tissue and the maternal and fetal circuits. Neither 17-alpha-hydroxyprogesterone caproate nor its metabolite, at the concentrations tested, had adverse effect on determined viability and functional parameters of placental tissue. 17-alpha-Hydroxyprogesterone caproate was metabolized by term placental lobule during its perfusion and both parent compound and its metabolite(s) transferred to the fetal circuit.

Effect of albumin on transplacental transfer and distribution of rosiglitazone and glyburide

Objective. The aims of this investigation were (i) to determine the rate and extent of rosiglitazone transfer across term human placenta, and (ii) to determine the effect of human serum albumin (HSA) on rosiglitazone and glyburide transfer and distribution.Methods. These aims were achieved by utilizing the technique of dual perfusion of placental lobule (DPPL). Each hypoglycemic drug was coperfused with the marker compound antipyrine (AP). In each experiment, the [3H]-isotope of the hypoglycemic drug and the [14C]-isotope of AP were added to enhance the detection limits of each drug. Human serum albumin (HSA) was added to both the maternal and fetal circuits in the experiments in which it was investigated.Results. Transplacental transfer of rosiglitazone and glyburide from the maternal to fetal circuits in media devoid of HSA was similar. However, the addition of HSA to the maternal and fetal circuits had different effects on the transfer and distribution of the two drugs, though their binding to HSA (99.8%) was almost identical. HSA increased the maternal (M) to fetal (F) transfer of rosiglitazone, as revealed by an increase in its F/M concentration ratio from 0.17 ± 0.01 (in the absence of albumin) to 0.33 ± 0.07 (p < 0.001). Moreover, the addition of albumin decreased the amount of rosiglitazone retained by placental tissue from 539 ± 148 to 60 ± 8 ng/g (p < 0.001). Conversely, the addition of HSA to the perfusion media resulted in a decrease in glyburide transfer, as revealed by the change of its F/M concentration ratio from 0.09 ± 0.02 (in the absence of albumin) to 0.03 ± 0.01 (p < 0.01). However, similar to rosiglitazone, glyburide retention by the tissue decreased from 103 ± 26 to 19 ± 6 ng/g (p < 0.001).Conclusions. These data indicate that the binding of the two drugs to albumin, though similar, is only one of the factors that could affect their placental transfer and distribution.

113: Metabolism of 17-hydroxyprogesterone caproate by the dually perfused human placental lobule

We previously reported that 17α-hydroxyprogesterone caproate (17-P) was transferred across the dually perfused human placenta from the maternal to fetal circuit, and was not hydrolyzed to 17HP and caproate during its perfusion. Therefore, the objective of this investigation is to determine whether 17-P is metabolized during its perfusion

Dual In Vitro Perfusion of an Isolated Cotyledon as a Model to Study the Implication of Changes in the Third Trimester Placenta on Preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-α, IL-1ß, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2α 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group.Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group.Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1β of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1β at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue.Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1β only the 17kD band was detected.In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Human placental transfer of 17-hyrdoxyprogesterone caproate

SARAH HEMAUER, SVETLANA PATRIKEEVA, GARY HANKINS, MAHMOUD AHMED, TATIANA NANOVSKAYA, University of Texas Medical Branch at Galveston, Obstetrics and Gynecology, Galveston, Texas OBJECTIVE: 17a-hydroxyprogesterone caproate (17HPC) results in 30% reduction in the rate of recurrent preterm deliveries. However, the mechanism and site of action of 17HPC as well as its pharmacokinetics are yet to be elucidated. Investigations in our laboratory focus on the transplacental transfer and metabolism of drugs used in treatment of the pregnant patient. The objective of this investigation is to determine the in vitro transplacental transfer and distribution of 17HPC between placental tissue, maternal and fetal circuits. STUDY DESIGN: The technique of dual perfusion of term placental lobule was utilized. The placentas were perfused with 17HPC added to the maternal reservoir at a concentration of 21ng/ml, which is equivalent to its peak levels in maternal circulation following 1000 mg intramuscular injection, together with its dual labeled radioactive isotope (H-hydroxyprogesterone and Ccaproate). The amounts of the drug in the tissue and both circuits were determined by liquid scintillation spectrometry. RESULTS: A biphasic decline in concentration of the drug in the maternal circuit was observed, initially rapid then slow. At the end of the perfusion period, a concentration gradient of the drug between the tissue and the two circuits was observed with the following ratios: tissue/maternal circuit, 2.23 G 0.70; tissue/fetal circuit, 2.89 G 1.07 and fetal/maternal circuit 0.79 G 0.07. The drug retained in the tissue did not have any adverse effects on the determined placental tissue viability and functional parameters. CONCLUSION: 17 HPC readily crosses the placenta from the maternal to fetal circuit, is retained by placental tissue and does not affect the in vitro determined placental functions. Supported by the Obstetrics Pharmacology Research Network, NICHD.

Effect of human serum albumin on transplacental transfer of glyburide

Glyburide is a second-generation sulfonylurea hypoglycemic drug used for the treatment of select women with pregestational and gestational diabetes mellitus (GDM). In vitro and in vivo investigations demonstrated its very low transplacental transfer to the fetal circulation. However, the factors influencing its low transfer across the human placenta remain unclear. Therefore, the goal of the current investigation was to determine the effect of human serum albumin (HSA) on the transfer and distribution of glyburide across the human placenta. To achieve this goal, the technique of dual perfusion of the placental lobule was utilized. The effect of HSA on the transfer of glyburide was determined at the range of glyburide to HSA molar ratios of 1:2–1:100. The transfer rate of free/unbound glyburide to the fetal circuit was 73 ± 10% of the freely diffusible marker compound antipyrine (AP). Data obtained indicates the dependence of glyburide transfer and its retention by the placental tissue on the concentration of HSA.

Transfer of metformin across the dually perfused human placental lobule

The purpose of this study was to determine the transfer characteristics of metformin across placentas that were obtained from uncomplicated pregnancies and from patients with gestational diabetes mellitus. The technique of dual perfusion of placental lobule was used. Metformin, 5 microg/mL and its [14C]-isotope were co-transfused with the marker compound antipyrine, 20 microg/mL and its [3H]-isotope from the maternal to fetal circuit. The drug content in both circulations and placental tissue was determined by liquid scintillation spectrometry. The transfer of metformin across term human placentas that were obtained from uncomplicated pregnancies was rapid and was not different from that in placentas that were obtained from patients with gestational diabetes mellitus. Metformin is distributed between the maternal and fetal circuits, with reliable amounts retained by the tissue. In an ex vivo model system, metformin was transferred readily from the maternal to fetal circuit across placentas that were obtained from uncomplicated pregnancies and pregnancies with gestational diabetes mellitus.

Effects of nicotine-specific antibodies, Nic311 and Nic-IgG, on the transfer of nicotine across the human placenta

The adverse effects of smoking during pregnancy on fetal development are, in part, due to nicotine. These effects may be due to the actions of nicotine in fetal circulation or on placental functions. In pregnant rats, vaccination with a nicotine immunogen reduces the transfer of nicotine from the maternal to fetal circulation. However, extrapolation of these results to pregnant women might not be valid due to the well-recognized differences between human and rat placentas. In the current investigation, the effects of nicotine-specific antibodies on the transfer of nicotine from the maternal to fetal circuit of the dually perfused human placental lobule were determined. Two types of nicotine-specific antibodies were investigated; nicotine-specific mouse monoclonal antibody (Nic311, K d for nicotine 60 nM) and IgG from rabbits vaccinated with a nicotine immunogen (Nic-IgG, K d 1.6 nM). Transfer of the antibodies from maternal to fetal circuits was negligible. Both rabbit Nic-IgG and, to a lesser extent, mouse monoclonal Nic311 significantly reduced nicotine transfer from the maternal to fetal circuit as well as the retention of the drug by placental tissue. These effects were mediated by a substantial increase in the protein binding of nicotine and a reduction in the unbound nicotine concentration. Therefore, the data cited in this report suggest that the use of nicotine-specific antibodies might reduce fetal exposure to the drug, and that antibody affinity for nicotine is a key determinant of the extent of nicotine transfer.

C-Reactive Protein Production in Term Human Placental Tissue

C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Role of P-glycoprotein in transplacental transfer of methadone

Methadone is the therapeutic agent of choice for treatment of the pregnant opiate addict. However, little is known on the factors affecting its concentration in the fetal circulation during pregnancy and how it might relate to neonatal outcome. Therefore, a better understanding of the function of placental metabolic enzymes and transporters should add to the knowledge of the role of the tissue in the disposition of methadone and its relation to neonatal outcome. We hypothesized that the expression and activity of the placental efflux transporter P-glycoprotein (P-gp) would affect the transfer of methadone to the fetal circulation. Data obtained utilizing dual perfusion of placental lobule and monolayers of Be–Wo cell line indicated that methadone is extruded by P-gp. Transfer of methadone to the fetal circuit was increased by 30% in the presence of the P-gp inhibitor GF120918 while the transfer of paclitaxel, a typical substrate of the glycoprotein, was increased by 50%. In the Be–Wo cell line, methadone and paclitaxel uptake was also increased in the presence of the P-gp inhibitor cyclosporin A. Moreover, the expression of P-gp in placental brush–border membranes varied between term placentas. Taken together, these data strongly suggest that the concentration of methadone in the fetal circulation is affected by the expression and activity of P-gp. It is reasonable to speculate that placental disposition of methadone affects its concentration in the fetal circulation. If true, this may also be directly related to the incidence and intensity of neonatal abstinence syndrome (NAS).