Maternal Perfusate Research Articles

The Effects of Magnesium Sulfate on the Inflammatory Response of Placentas Perfused With Lipopolysaccharide: Using the Ex Vivo Dual-Perfused Human Single-Cotyledon Model.

Multiple mechanisms have been proposed for the neuroprotective effects of magnesium sulfate (MgSO4). We aimed to examine the effects of lipopolysaccharide (LPS) and MgSO4 on the placental expression of nuclear factor κ light chain enhancer of activated B cells (NF-κB), interleukin (IL) 6, adrenocorticotropic hormone (ACTH), and nitric oxide synthase (NOS); all known to participate in the inflammatory cascade. Placentas were obtained and selected cotyledons cannulated and dually perfused ex vivo. Placentas were perfused with 4 perfusion protocols: culture medium (M-199; controls), LPS (1 μg/mL), MgSO4 (6 g/dL), and LPS + MgSO4. Each perfusion experiment continued for 3 hours. Sixteen perfusion experiments were analyzed, 4 separate placentas were studied for each protocol. The protein levels in the perfused cotyledons were studied by Western blot analysis and compared between the groups. Interleukin 6 levels were studied in the maternal and fetal perfusate. The expression of NF-κB p65, IL-6, ACTH, and NOS proteins levels were significantly increased in placentas perfused with LPS as compared to placentas perfused with M-199, MgSO4 ( P < .01 for all). Placentas perfused with LPS+ MgSO4 had similar proteins levels as in the controls and MgSO4 groups. Lipopolysaccharide significantly increased IL-6 levels in maternal perfusate. In the human placenta, MgSO4 blocks the increase in the proteins levels of NF-κB, IL-6, ACTH, and NOS in response to inflammatory stimuli. Magnesium sulfate attenuates excessive placental inflammatory response. The decrease in placental ACTH levels following perfusion with MgSO4 may point to an additional non-anti-inflammatory mechanism of MgSO4.

O-28 Placental transfer of the immunosuppressive drug tacrolimus and its effects on placental function; relevance for renal transplant recipients?

Background The number of pregnancies in the kid-ney transplant patient population is high. During gesta-tion these women continue using immunosuppressant drugs, but knowledge about their placental disposition and toxicity is scarce. We now investigated placental transfer of the immunosuppressive drug tacrolimus (TAC) as well as the potential effects on trophoblast cell viability and barrier function. Methods Isolated dual side perfusions of human pla-cental cotyledons were performed to study disposition of TAC. Additionally, clinical data on TAC concentrations in placental tissue of kidney transplant recipients and ma-ternal whole blood concentrations were gathered. BeWo choriocarcinoma cells were used to evaluate effects on trophoblast cell viability, while interaction with placental ATP-binding cassette transporters was studied in mem-brane vesicles derived from HEK293 cells recombinantly overexpressing human Breast Cancer Resistance Protein (BCRP) or P-glycoprotein (P-gp). Results We found that maternal perfusate levels de-creased during 180 min of perfusion, while being unde-tectable in the fetal circulation. At t=180 min a concen-tration of 220±50 nM was measured in placental tissue, which is almost 100-fold higher than the maternal per-fusate concentration. Analysis of placental tissue of renal transplant recipients revealed a 13-fold higher tacrolimus concentration compared to the maternal blood concen-tration (88±7 nM and 6.8±1.1 nM, respectively). TAC did not affect BeWo cell viability up to the maximum concen-tration of 1 µM tested. In transporter studies we did find stimulation of P-gp-mediated transport and inhibition of BCRP-mediated transport, at 1 and 10 µM, respectively. Conclusion TAC demonstrates strong accumulation in placental tissue and distribution across the tissue was not homongenously. However, the tissue concentrations reached are unlikely to affect trophoblast cell viability or BCRP and P-gp transport function.

Inflammatory processes are specifically enhanced in endothelial cells by placental-derived TNF-α: Implications in preeclampsia (PE)

IntroductionThere is a consensus that factors released by the placenta to maternal circulation, including TNF-α, play a key role in activating the maternal endothelium in pregnancies with preeclampsia (PE). Dual perfusion preserves the structural organization of the placenta to a greater degree than other in vitro systems and has been used by our group and others to examine placental pathophysiology associated with PE. The objective of this study was to use the dual perfusion model to test whether TNF-α released by the placenta to maternal perfusate affects pro-inflammatory cytokine secretion by, and activation of, endothelial cells, thereby furthering our understanding of placental and endothelial dysfunction in PE. MethodWe used maternal perfusate, two endothelial cell lines (HUVECs and HEECs), and a TNF-α blocking antibody to test whether placental-derived TNF-α plays a significant role in altering the expression and secretion of pro-inflammatory cytokines in endothelial cells as well as the expression of activation markers in this cell type. ResultsThe presence of maternal perfusate significantly enhanced IL-6, IL-8, and MCP-1 secretion, levels of their mRNA, as well as mRNA levels of markers of endothelial activation (E-selectin, ICAM-1, and VCAM-1). The addition of a TNF-α blocking antibody significantly inhibited the maternal perfusate-mediated enhancement of cytokine secretion by, and expression of activation markers, in both HUVECs and HEECs. DiscussionThese results demonstrate that TNF-α significantly contributed to endothelial cell pro-inflammatory cytokine secretion and activation suggesting that blocking TNF-α action may mitigate the effects of maternal endothelial dysfunction in PE.

Transfer of gangliosides across the human placenta

ObjectivesGangliosides are structural and functional glycosphingolipids, considered to have important roles in neuronal development in fetal and neonatal development and in memory formation. In this report, we have investigated the ability of bovine milk-derived gangliosides GM3 and GD3 to cross the human placenta. Study designWe have employed the ex-vivo model of dually-perfused isolated human placental lobules. ResultsThere was significant uptake of both GD3 and GM3 from the maternal perfusate. There was significant increase of GM3 in the fetal side and a non-statistically significant trend for GD3 to increase on the fetal side. ConclusionsHence an apparent preference for GM3 release into fetal circulation. We suggest that gangliosides consumed by the mother enter her circulation, can be transferred across the placenta and may be available to the developing fetus for building neural connections.

Passage of 4-nonylphenol across the human placenta

4-nonylphenol (4-NP) is an estrogenic endocrine active chemical that is present in detergents and is known to contaminate food and drinking water. The aim of the present study was to evaluate whether 4-NP crosses the human placenta and if so, to what extent. Human placentae obtained from healthy term singleton pregnancies were utilized in a dual ex-vivo re-circulating model of placental perfusion. Six placentae were perfused for 180 min following addition of 4-NP (30 ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate the integrity of the circuits. 4-NP was measured by High Performance Liquid Chromatography coupled with Fluorescence Detection (HPLC-FD). After 180 min of perfusion the transfer percentages for antipyrine and 4-NP were 25.6 ± 1.4% (mean ± s.e.m, n = 6) and 22.75 ± 3.76% respectively and the transfer index for 4-NP was 0.8. We conclude that the intact form of 4-NP at environmentally relevant concentrations can transfer across the human placenta albeit at a slow rate.

P12. Placental gene expression analysis at the end of the first trimester of pregnancy in patients at high risk of subsequent development of preeclampsia

ments using the dual placental perfusion system. Perfusions were performed with medium only and with free Hb. Maternal perfusate was collected at four (4) time points during the dual perfusion and used for analysis of MPs from both controls and Hb perfusions. MPs were isolated using ultra-centrifugation at 10,000g and 150,000g, resulting in two pellets with different sized MPs referred to as 10 K ( 0.3 lm– 1.1 lm) and 150 K ( 0.3–0.6lm) MPs. Their protein concentration was determined spectrophotometrically using a Nanodrop. Small RNAs were extracted from the MPs and miRNA expression was determined using a miRNA specific real-time quantitative reverse transcriptase-polymerase chain reaction assay for nine selected miRNAs. Results: Placentas perfused with haemoglobin showed an increase in perfusion pressure as well as morphological changes, similar to what is seen in PE. The miRNAs; mir517b, mir-517a, mir-141 and mir-222 showed significantly decreased expression in MPs from Hb perfused placentas compared to controls. There was no difference in protein concentration in MPs from Hb and controls. The amount of total RNA per MP was higher in the 150 K compared to the 10 K MPs. Conclusion: Free Hb causes oxidative stress and PE like damage to the placenta. Our results show that the placenta specific mir-517b and mir-517a are decreased in MPs after perfusion with free Hb. Mir-517a has previously been found in exosomes from trophoblasts. We also showed that mir141 and mir-222 are down-regulated after perfusion with free Hb. Interestingly, decreased levels of mir-222 expression have been shown to increase synthesis of HbF, which would suggest mir-222 to be part of a possible mechanism behind the increased levels of HbF seen in PE. Future experiments need to focus on the relationship between miRNA expression in placental tissue and MPs in the different perfusion conditions. First Author <35 years: Yes.

Transfer of heparin across the human perfused placental lobule.

A system of dual perfusion of an isolated lobule of term human placenta was used as a model to study the transfer of heparin from maternal to foetal circulation. The metabolic viability of the system was assessed by measuring beta-HCG and alkaline phosphatase levels in both maternal and foetal perfusates. Creatinine and antipyrine were used as markers to determine juxtaposition of the maternal and foetal circulations. Results of this study indicate that following administration of a single bolus dose of heparin into the maternal circulation, its concentration declined slowly from 99.01 +/- 2.98 at 15 min to 97.23 +/- 4.12% and transfer of heparin in the foetal circulation was linear and increased from 0.10% +/- 0.05% at 15 min to 0.46 +/- 0.19% over a period of 120 min. The maternal (MAUC) and foetal (FAUC) concentration-time integrals were found to be 70160 +/- 1332 and 340 +/- 30 int. units min mL-1, respectively. Placental permeability of heparin and creatinine, calculated as the ratio of foetal concentration to the integral maternal-foetal concentration difference, was 8.65 x 10(-5) +/- 0.80 x 10(-5) and 0.033 +/- 0.006 mL min-1 g-1 of perfused placental weight, respectively. These data suggest that heparin was transferred from the maternal to the foetal circulation in small quantities.

Adapting in vitro dual perfusion of the human placenta to soluble oxygen tensions associated with normal and pre-eclamptic pregnancy

For decades, superoxic ex vivo dual perfusion of the human placental lobule has been used as a model to study the physiology and metabolism of the placenta. The aim of this study was to further develop the technique to enable perfusion at soluble oxygen concentrations similar to those in normal pregnancy (normoxia) and in pre-eclampsia (PE; hypoxia). Our design involved reducing the mean soluble oxygen tension in the maternal-side intervillous space (IVS) perfusate to 5–7% and <3% for normoxia and hypoxia, respectively, while providing a more ubiquitous delivery of perfusate into the IVS, using 22 maternal-side cannulae. We achieved quasi-steady states in [O2]fetal venous (soluble), which were statistically different between the two adaptations at t=150 to t=240 min of dual perfusion (2.1, 1.2, 2.8 and 0.4, 0.0, 1.5%; median, 25th, 75th percentiles, n=20 and 24 readings in n=5 and n=6 lobules, normoxic and hypoxic perfusion, respectively; P<0.001, Mann–Whitney U-test). Lactate dehydrogenase (LDH) levels in fetal and maternal venous outflow perfusates were unaffected by the adaptations. There was also no difference in tissue lactate release between the two adaptations. Glucose consumption from the fetal circulation and maternal-side ‘venous' pyruvate release were higher under normoxic conditions, indicative of a greater metabolic flux through glycolysis. Furthermore, there was greater release of the hypoxic-sensitive marker, macrophage inflammatory protein-1α, into the maternal venous perfusate in the hypoxic model. Also, during hypoxic perfusion, we found that fetal-side venous placental growth factor (PlGF) levels were higher compared with normoxic perfusion. We conclude that these ex vivo adapted methods of placental perfusion provide a means of studying aspects of placental metabolism in relation to normal oxygenation and hypoxia-associated pregnancy disease.

Transfer of PAMAM dendrimers across human placenta: Prospects of its use as drug carrier during pregnancy

Dendrimers offer significant potential as nanocarriers for targeted delivery of drugs and imaging agents. The objectives of this study were to evaluate the transplacental transport, kinetics and biodistribution of PAMAM dendrimers ex-vivo across the human placenta in comparison with antipyrine, a freely diffusible molecule, using dually perfused re-circulating term human placental lobules. The purpose of this study is to determine if dendrimers as drug carriers can be used to design drug delivery systems directed at selectively treating either the mother or the fetus. The transplacental transfers of fluorescently (Alexa 488) tagged PAMAM dendrimer (16 kDa) and antipyrine (188 Da) from maternal to fetal circulation were measured using HPLC/dual UV and fluorescent detector (sensitivity of 10 ng/mL for dendrimer and 100 ng/mL for antipyrine respectively). C max for the dendrimer–Alexa (DA) in maternal perfusate (T max = 15 min) was 18 times higher than in the fetal perfusate and never equilibrated with the maternal perfusate during 5.5 h of perfusion (n = 4). DA exhibited a measurable but low transplacental transport of 2.26 ± 0.12 μg/mL during 5.5 h, where the mean transplacental transfer was 0.84 ± 0.11% of the total maternal concentration and the feto-maternal ratio as percent was 0.073% ± 0.02. The biochemical and physiological analysis of the placentae perfused with DA demonstrated normal function throughout the perfusion. The immunofluorescence histochemistry confirmed that the biodistribution of DA in perfused placenta was sparsely dispersed, and when noted was principally seen in the inter-villous spaces and outer rim of the villous branches. In a few cases, DA was found internalized and localized in nuclei and cytoplasm of syncytiotrophoblast and inside the villous core; however, DA was mostly absent from the villous capillaries. In conclusion, the PAMAM dendrimers exhibited a low rate of transfer from maternal to the fetal side across the perfused human placenta, which is similar to other investigations of large macromolecules, e.g., IgG. These overall findings suggest that entry of drugs conjugated to polymers, i.e., dendrimers, would be limited in their transfer across the human placenta when compared to smaller drug molecules alone, suggesting novel methods for selectively delivering therapeutics to the pregnant woman without significant transfer to the fetus, especially since the half life of the dendrimer in blood is relatively short.

Protein composition of microparticles shed from human placenta during placental perfusion: Potential role in angiogenesis and fibrinolysis in preeclampsia

Shedding of syncytiotrophoblast microparticles (MPs) from placenta to maternal blood occurs in normal pregnancy and is enhanced during preeclampsia (PE). The syncytiotrophoblast synthesizes plasminogen activator inhibitors (PAIs) which regulate fibrinolysis, as well as soluble forms of the fms-like tyrosine kinase (sFlt-1) and endoglin, which exert anti-angiogenic actions. An increase in the ratio of PAI-1/PAI-2 and elevated levels of sFlt-1 and sEng in maternal serum are linked to placental damage and maternal endothelial cell dysfunction in PE. The goal of the current study was to determine whether MPs released to maternal perfusate during dual perfusion contain these factors associated with placental pathophysiology in PE. Initially, high levels of alkaline phosphatase activity and Annexin V binding were found in MPs isolated by sequential centrifugation of maternal perfusates at 10,000 and 150,000×g(10 K and 150 K MPs), indicating their plasma membrane origin. ELISA revealed the presence of these factors at the following relative levels: Eng>PAI-2⋙PAI-1>sFlt-1. Based on comparisons of their concentration in perfusates, MPs, and MP-free 150 K supernatants, we determined that MPs constitute a significant portion of Eng released by placenta. Flow cytometric analysis of 10 K MPs supported the levels of expression found by ELISA and indicated that Eng and PAI-2 were almost exclusively localized to the surface of MPs, a site with biological potential. These results indicate that MPs shed from the syncytial surface express factors which may alter the fibrinolytic and angiogenic balance at the maternal–fetal interface and play a role in the pathophysiology of PE.

Transplacental Transfer and Biotransformation of Genistein in Human Placenta

ObjectiveTo study transplacental transfer and biotransformation of genistein in the human placenta. Study design and outcomesHuman placentae obtained from healthy term singleton pregnancies were utilised in a dual re-circulating model of ex-vivo placental perfusion. Four placentae were perfused for 180min following addition of genistein (10ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate integrity of the circuits. Concentrations of genistein and its conjugates were determined by liquid chromatography-mass spectrometry (LC–MS). ResultsThe transfer percentage for antipyrine and genistein was 25.6±1.40% and 22.1±1.61% respectively and the transfer index for genistein was 0.90±0.04 after 180min of perfusion. 12.0±2.40% of genistein in the fetal compartment and 7.36±4.73% of genistein in the maternal compartment were in the conjugated form. ConclusionsGenistein can transfer across the human placenta at environmentally relevant levels. Placental metabolizing enzymes conjugate a small fraction of genistein into the glucuronide/sulphate form, which is devoid of estrogenic action.

Transfer of bisphenol A across the human placenta

The objective of the investigation was to study placental transfer and conjugation of bisphenol A (BPA) across the human placenta. Human placentae obtained from healthy term singleton pregnancies were utilized in a dual recirculating model of ex vivo placental perfusion. Seven placentae were perfused with BPA (10 ng/mL) added to the maternal perfusate for 180 minutes. Antipyrine and fluorescein isothiocyanate dextran were used as positive and negative controls, respectively, to validate integrity of the circuits. Concentrations of BPA and its conjugates were determined by liquid chromatography-mass spectrometry. The transfer percentage for antipyrine and BPA were 25.5 +/- 1.13% and 27.0 +/- 1.88%, respectively, and the transfer index for BPA was 1.1 +/- 0.09 after 180 minutes of perfusion. Only 3.2 +/- 1.6% of BPA in the fetal compartment was in the conjugated form. Bisphenol A at low environmentally relevant levels can transfer across the human placenta, mainly in active unconjugated form.

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.

Utero-Placental Haemodynamics in the Pathogenesis of Pre-Eclampsia

Pre-eclampsia is associated with insufficient adaptations of spiral arteries which theoretically alter haemodynamics within the intervillous space. Such changes could damage the syncytiotrophoblast and release factors which instigate maternal endothelial dysfunction. We tested this hypothesis using an in vitro dual perfusion model of the human placenta, representing putative changes in flow arising from these spiral artery maladaptations. Whilst fetal-side flow rates remained constant (6 ml/min) perfusion rates on the maternal side were increased from 14 ml/min to 45 ml/min. As well as increasing placental derived intervillous hydrostatic pressures, and changes in flow dynamics observed by colour Doppler, these elevated flow rates resulted in morphologic damage, vacuolation and shedding of the syncytiotrophoblast, focal features previously defined in pre-eclampsia. The collected maternal perfusates recovered under high flow conditions also contained significantly elevated levels of biochemical markers of syncytial damage, including lactate dehydrogenase, alkaline phosphatase and human chorionic gonadotrophin. There were also significant elevations in chemokines GROα and RANTES, compared with the low flow perfusions. The soluble components of the maternal high flow rate perfusions decreased the number and proliferation of HUVECs after 24 h exposure. These results could not be attributed to GROα or RANTES alone or in combination. This study provides evidence that alterations in intervillous flow have the potential to influence both the integrity of the syncytiotrophoblast and the liberation of potentially pathogenic soluble factors. This therefore offers a putative link between utero-placental maladaptations in pregnancy and the vascular endothelial complications of pre-eclampsia.

Transplacental Pharmacokinetics of Diclofenac in Perfused Human Placenta

The aims of this study were to evaluate the transplacental transfer properties of diclofenac and to determine the effect of L-lactic acid on the transplacental transfer of diclofenac. The maternal and fetal vessels of human placenta were perfused in a single-pass mode with a solution containing diclofenac and antipyrine. The transplacental pharmacokinetic model was fitted to the time profiles of the drug concentrations in the effluent and placenta to obtain transplacental pharmacokinetic parameters. In addition, chloride ion in the perfusate was partially replaced with L-lactic acid to see the change in the transplacental transfer properties of diclofenac. The TPT(ss) value (ratio of the rate of amount transferred across the placenta to that infused in the steady state) of diclofenac was 2.22%, which was approximately one-third that of antipyrine and was significantly reduced in the presence of L-lactic acid. The transplacental pharmacokinetic model could adequately explain the transplacental transfer of diclofenac with influx clearances from maternal and fetal perfusates to placental tissue of 0.276 and 0.0345 ml/min/g cotyledon and efflux rate constants from placental tissue to maternal and fetal perfusates of 0.406 and 0.0337 min(-1), respectively. By taking into account protein binding, the placental tissue/plasma concentration ratio in humans for diclofenac was estimated to be 0.108 ml/g of cotyledon and was smaller than that of antipyrine. In conclusion, human placental perfusion and transplacental pharmacokinetic modeling allowed us to determine the transplacental transfer properties of diclofenac quantitatively. Diclofenac may share transplacental transfer system(s) with L-lactic acid.

In Vitro Dual Perfusion of Human Placental Lobules as a Flow Phantom to Investigate the Relationship between Fetoplacental Flow and Quantitative 3D Power Doppler Angiography

Flow phantoms have been used to investigate and quantify three-dimensional power Doppler data but this is the first study to use the in vitro, dual perfused, placental perfusion model. We used this model to investigate and quantify the effect of variation in fetal-side flow rates and attenuation on 3D power Doppler angiography. Perfusion of a placental lobule was commenced within 30min of delivery and experimentation was successful in 8 of the 18 placenta obtained. Fetal and maternal perfusate was modified Earle's bicarbonate buffer which, following equilibration, was supplemented on the fetal side with whole heparinised cord blood. Imaging was performed with a Voluson-i ultrasound machine. A ‘vascular biopsy’ the thickness of the placental lobule was defined and signal quantified within using VOCAL™ (GE Medical Systems, Zipf, Austria). Three vascular indices are generated: vascularisation index (VI) defined as the percentage of power Doppler data within a volume of interest; flow index (FI), the mean signal intensity of the power Doppler information; and vascularisation flow index (VFI), a combination of both factors derived through their multiplication. Attenuation was investigated in this model with the addition of tissue mimic blocks. Our results showed a predictable relationship between flow rates and the vascular indices VI and VFI. However the FI was a less reliable predictor of flow; thus it should be interpreted with caution. The power Doppler signal was markedly affected by attenuation leading to a complete loss of information at a depth of 6cm in the model used. In conclusion this model can be adapted to provide a phantom to analyse and quantify 3D power Doppler signals and demonstrates that vascular indices within a tissue remain related to volume flow. This model provides further evidence that depth dependent attenuation of signal needs to be accounted for in any in vivo work where the probe is not in direct contact with the tissue of interest.

Inhibition of placental P-glycoprotein: impact on indinavir transfer to the foetus.

We have shown previously using the dually perfused isolated human placenta model that the maternal to foetal transfer of the antiviral protease inhibitor drug indinavir is substantially lower than the transfer in the opposite direction. This finding is not consistent with passive diffusion and indicates that a carrier-mediated mechanism is involved in retarding the movement in the maternal to foetal direction. The efflux transporter P-gp located in the apical membrane domain of the placental trophoblast cells has been implicated as the likely cause of the differential bi-directional transport. The present study also utilizes the human perfused human isolated placenta to investigate the possible inhibitory effects of the P-gp inhibitor PSC833 and the P-gp substrate/inhibitor ritonavir on the maternal to foetal transfer clearance of indinavir. The studies, which were conducted such that each placenta served as its own control, demonstrated a statistically significant increase in the maternal to foetal transfer of indinavir in the presence of PSC833 but not in the presence of ritonavir, a protease inhibitor that is often used in combination with other protease inhibitors in dual therapy. The lack of effect of ritonavir is most likely related to the relatively low inhibitory activity at the clinically relevant concentration used in this study. To investigate the effect of P-gp inhibition on the maternal to foetal transfer of indinavir. Term human placentae (n = 12) were from non-HIV infected women. Maternal to foetal transfer of indinavir was examined in the absence and presence of P-gp inhibitors PSC833 (n = 7) or ritonavir (n = 5), in the perfused human placenta. Antipyrine and [(3)H]-vinblastine were included as markers of passive diffusion and P-gp transport, respectively. These markers and indinavir were added to maternal perfusate at 0 min; PSC833 or ritonavir was added at 25 min. Steady-state maternal to foetal transfer clearance was calculated during control and inhibitor phases. Indinavir and vinblastine clearances were normalized to antipyrine clearance (clearance index). Indinavir clearance index increased between the control (0.25 +/- 0.03) and PSC833 phases (0.37 +/- 0.14) (95% CI of the difference -0.23, -0.002). Vinblastine clearance index increased from (0.25 +/- 0.08) to (0.34 +/- 0.06) in the control and PSC833 phases, respectively (95% CI of difference -0.14, -0.05). Indinavir clearance index was unchanged between control (0.34 +/- 0.14) and ritonavir phases (0.39 +/- 0.13) (95% CI of the difference -0.19, 0.08). Vinblastine clearance index increased from (0.24 +/- 0.12) to (0.32 +/- 0.12) in the control and ritonavir phases, respectively (95% CI of the difference -0.15, -0.009). Maternal to foetal transfer clearance of indinavir and vinblastine increased following P-gp inhibition. The potential role for co-administration of P-gp inhibitors with PIs to reduce perinatal HIV transmission warrants further investigation.

Kinetic Analysis of the Transport of Salicylic Acid, a Nonsteroidal Anti-inflammatory Drug, across Human Placenta

The aim of this study was to develop a pharmacokinetic model to describe the transplacental transfer of drugs, based on the human placental perfusion study. The maternal and fetal sides of human placentas were perfused with salicylic acid together with antipyrine, a passive diffusion marker. The drug concentration in the placental tissue was determined at the end of perfusion. A compartment model consisting of maternal space, fetal intravascular space, and placental tissue was fitted to the observed concentration profiles of salicylic acid in the maternal and fetal effluents. The developed model could adequately explain the concentration profiles of salicylic acid in the effluents with influx clearances from maternal and fetal perfusates to placental tissue of 0.0407 and 0.0813 ml/min/g cotyledon and efflux rate constants from placental tissue to maternal and fetal perfusates (k2 and k3) of 0.0238 and 0.176 min(-1), respectively. The kinetics of antipyrine was adequately described by assuming rapid equilibrium between fetal perfusate and placental tissue compartments. The influx plasma clearance from the maternal side (K''1) in humans was estimated by taking into account the protein binding. The K''1/k2 value of salicylic acid was 1.07 ml/g cotyledon and was larger than that of antipyrine (0.642 ml/g cotyledon). We evaluated the transplacental transfer kinetics of salicylic acid by human placental perfusion study with various perfusion protocols. Based on the data obtained, we developed a pharmacokinetic model, which should enable us to estimate the influx profile of drugs into umbilical arterial blood from the maternal plasma concentration profile.

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.

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.