Human Placenta Perfusion Research Articles

Evaluation of Various Approaches to Estimate Transplacental Clearance of Vancomycin for Predicting Fetal Concentrations using a Maternal-Fetal Physiologically Based Pharmacokinetic Model.

Evaluating drug transplacental clearance is vital for forecasting fetal drug exposure. Ex vivo human placenta perfusion experiments are the most suitable approach for this assessment. Various in silico methods are also proposed. This study aims to compare these prediction methods for drug transplacental clearance, focusing on the large molecular weight drug vancomycin (1449.3g/mol), using maternal-fetal physiologically based pharmacokinetic (m-f PBPK) modeling. Ex vivo human placenta perfusion experiments, in silico approaches using intestinal permeability as a substitute (quantitative structure property relationship (QSPR) model and Caco-2 permeability in vitro-in vivo correlation model) and midazolam calibration model with Caco-2 scaling were assessed for determining the transplacental clearance (CLPD) of vancomycin. The m-f PBPK model was developed stepwise using Simcyp, incorporating the determined CLPD values as a crucial input parameter for transplacental kinetics. The developed PBPK model of vancomycin for non-pregnant adults demonstrated excellent predictive performance. By incorporating the CLPD parameterization derived from ex vivo human placenta perfusion experiments, the extrapolated m-f PBPK model consistently predicted maternal and fetal concentrations of vancomycin across diverse doses and distinct gestational ages. However, when the CLPD parameter was derived from alternative prediction methods, none of the extrapolated maternal-fetal PBPK models produced fetal predictions in line with the observed data. Our study showcased that combination of ex vivo human placenta perfusion experiments and m-f PBPK model has the capability to predict fetal exposure for the large molecular weight drug vancomycin, whereas other in silico approaches failed to achieve the same level of accuracy.

Variability in perfusion conditions and set-up parameters used in ex vivo human placenta models: A literature review

The ex vivo human placenta perfusion model has proven to be clinically relevant to study transfer- and fetal exposure of various drugs. Although the method has existed for a long period, the setup of the perfusion model has not been generalized yet. This review aims to summarize the setups of ex vivo placental perfusion models used to examine drug transfer across the placenta to identify generalized properties and differences across setups. A literature search was carried out in PubMed September 26, 2022. Studies were labeled as relevant when information was reported, between 2000 and 2022, on the setups of ex vivo placental perfusion models used to study drug transfer across the placenta. The placenta perfusion process, and the data extraction, was divided into phases of preparation, control, drug, and experimental reflecting the chronological timeline of the different phases during the entire placental perfusion process. 135 studies describing an ex vivo human placental perfusion experiment were included. Among included studies, the majority (78.5%) analyzed drug perfusion in maternal to fetal direction, 18% evaluated bi-directional drug perfusion, 3% under equilibrium conditions, and one study investigated drug perfusion in fetal to maternal direction. This literature review facilitates the comparison of studies that employ similar placenta perfusion protocols for drug transfer studies and reveals significant disparities in the setup of these ex vivo placental perfusion models. Due to interlaboratory variability, perfusion studies are not readily comparable or interchangeable. Therefore, a stepwise protocol with multiple checkpoints for validating placental perfusion is needed.

Safety assessment of the SGLT2 inhibitors empagliflozin, dapagliflozin and canagliflozin during pregnancy: An ex vivo human placenta perfusion and in vitro study

Although uncontrolled hyperglycaemia during pregnancy can cause complications for both the mother and her offspring, pharmacological treatment options for gestational and type 2 diabetes in pregnancy are still limited. Empagliflozin (EMPA), dapagliflozin (DAPA) and canagliflozin (CANA) are three sodium glucose co-transporter 2 (SGLT2) inhibitors, a newer group of oral antidiabetics that are well established in the treatment of type 2 diabetes mellitus in non-pregnant patients. To date, no data regarding their placental transfer and safety in pregnant women are available. We performed ex vivo human placental perfusions (n = 4, term placentas, creatinine and antipyrine as connectivity controls) to evaluate the transplacental transfer of EMPA, DAPA and CANA across the placental barrier and assessed their influence on the secretion of two placental peptide hormones, leptin and β-human chorionic gonadotropin (β-hCG). We discovered that all three SGLT2 inhibitors cross the placental barrier and attained maximal foetal to maternal concentration ratios of 0.38 ± 0.09 (EMPA), 0.67 ± 0.05 (DAPA) and 0.62 ± 0.05 (CANA) within the tested 360 min. A moderate but statistically significant decrease in placental leptin – but not β-hCG – secretion was observed during perfusions with SGLT2 inhibitors, which was confirmed in experiments performed with human placental BeWo cells. SGLT2 inhibitors are able to cross the human placental barrier and seem to interfere with placental leptin production. These observations should be considered in the ongoing discussion on the optimal treatment for gestational diabetes and type 2 diabetes mellitus in pregnancy.

Placental transfer of tofacitinib in the ex vivo dual-side human placenta perfusion model

Tofacitinib is a small molecule Janus kinase (JAK) inhibitor, introduced to the European market in 2017, for the treatment of rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. In the treatment of women with autoimmune diseases, pregnancy is a relevant issue, as such diseases typically affect women in their reproductive years. Currently, there is limited data on the use of tofacitinib during pregnancy. To estimate the extent of placental transfer in the absence of clinical data, we conducted ex vivo dual-side perfused human placental cotyledon perfusions. Term placentas were perfused for 180 min with tofacitinib (100 nM, added to the maternal circuit) in a closed-closed configuration. At the end of the perfusions, drug concentrations in the maternal and fetal reservoirs were near equilibrium, at 35.6 ± 5.5 and 24.8 ± 4.7 nM, respectively. Transfer of tofacitinib was similar to that observed for the passive diffusion marker antipyrine (100 µg/mL, added to the maternal reservoir). Final antipyrine maternal and fetal concentrations amounted to 36.9 ± 3.0 and 36.7 ± 1.3 µg/mL, respectively. In conclusion, in the ex vivo perfused placenta tofacitinib traverses the placental barrier rapidly and extensively. This suggests that substantial fetal tofacitinib exposure will take place after maternal drug dosing.

Transplacental migration of maternal natural killer and T cells assessed by ex vivo human placenta perfusion

IntroductionThe transplacental passage of cells between a mother and her fetus, known as microchimerism, is a less studied process during pregnancy. The frequency of maternal microchimeric cells in fetal tissues in physiological pregnancies and mechanisms responsible for transplacental cell trafficking are poorly understood. This study aimed to evaluate the placental trafficking of maternal peripheral blood mononuclear cells (PBMC) using human ex vivo placenta perfusion. MethodsTen placentas and maternal PBMC were obtained after healthy pregnancies. Flow cytometry was used to characterize PBMC subtypes. They showed a higher percentage of CD3+ T cells compared to CD56+ NK cells. The isolated PBMC were stained with a fluorescent dye and perfused through the maternal circuit of the placenta in an ex vivo perfusion system. Subsequent immunofluorescence staining for CD3+ T cells and CD56+ NK cells was performed on placental tissue sections, and the number of detectable PBMC in different tissue areas was counted using fluorescence microscopy. ResultsThe applied method allowed discrimination of perfused autologous maternal cells from cells resident in the placenta before perfusion. Further, it allows additional immunohistochemical labelling and distinction of immune cell subsets. Perfused PBMC were detected in all analyzed placentas, mostly in contact to the syncytiotrophoblast. CD3+ T cells were identified more frequently than CD56+ NK cells and some CD3+ T cells were found inside fetoplacental tissues and vasculature. The results indicate that also other PBMCs than T or NK cells adhere to or enter villous tissue, but they have not been specified in this analysis. DiscussionPrevious studies have detected maternal cells in the fetal circulation which we could mimick in our ex vivo placenta perfusion experiments with fluorescence labelled autologous maternal PBMC. The applied experimental settings did not allow comparison of transmigration abilities of PBMC subsets, but slight modifications of the model will permit further studies of cell transfer processes and microchimerism in pregnancy.

An update on placental drug transport and its relevance to fetal drug exposure.

Pregnant women are often complicated with diseases that require treatment with medication. Most drugs administered to pregnant women are off-label without the necessary dose, efficacy, and safety information. Knowledge concerning drug transfer across the placental barrier is essential for understanding fetal drug exposure and hence drug safety and efficacy to the fetus. Transporters expressed in the placenta, including adenosine triphosphate (ATP)-binding cassette efflux transporters and solute carrier uptake transporters, play important roles in determining drug transfer across the placental barrier, leading to fetal exposure to the drugs. In this review, we provide an update on placental drug transport, including in vitro cell/tissue, ex vivo human placenta perfusion, and in vivo animal studies that can be used to determine the expression and function of drug transporters in the placenta as well as placental drug transfer and fetal drug exposure. We also describe how the knowledge of placental drug transfer through passive diffusion or active transport can be combined with physiologically based pharmacokinetic modeling and simulation to predict systemic fetal drug exposure. Finally, we highlight knowledge gaps in studying placental drug transport and predicting fetal drug exposure and discuss future research directions to fill these gaps.

Prediction of Maternal and Fetal Doravirine Exposure by Integrating Physiologically Based Pharmacokinetic Modeling and Human Placenta Perfusion Experiments.

Background and ObjectiveDoravirine is currently not recommended for pregnant women living with human immunodeficiency virus because efficacy and safety data are lacking. This study aimed to predict maternal and fetal doravirine exposure by integrating human placenta perfusion experiments with pregnancy physiologically based pharmacokinetic (PBPK) modeling.MethodsEx vivo placenta perfusions were performed in a closed–closed configuration, in both maternal-to-fetal and fetal-to-maternal directions (n = 8). To derive intrinsic placental transfer parameters from perfusion data, we developed a mechanistic placenta model. Next, we developed a maternal and fetal full-body pregnancy PBPK model for doravirine in Simcyp, which was parameterized with the derived intrinsic placental transfer parameters to predict in vivo maternal and fetal doravirine exposure at 26, 32, and 40 weeks of pregnancy. The predicted total geometric mean (GM) trough plasma concentration (Ctrough) values were compared with the target (0.23 mg/L) derived from in vivo exposure–response analysis.ResultsA decrease of 55% in maternal doravirine area under the plasma concentration–time curve (AUC)0–24h was predicted in pregnant women at 40 weeks of pregnancy compared with nonpregnant women. At 26, 32, and 40 weeks of pregnancy, predicted maternal total doravirine GM Ctrough values were below the predefined efficacy target of 0.23 mg/L. Perfusion experiments showed that doravirine extensively crossed the placenta, and PBPK modeling predicted considerable fetal doravirine exposure.ConclusionSubstantially reduced maternal doravirine exposure was predicted during pregnancy, possibly resulting in impaired efficacy. Therapeutic drug and viral load monitoring are advised for pregnant women treated with doravirine. Considerable fetal doravirine exposure was predicted, highlighting the need for clinical fetal safety data.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-022-01127-0.

Effects of aldosterone on the human placenta: Insights from placental perfusion studies

IntroductionIn pregnancy, aldosterone is linked to maternal plasma volume expansion, improved fetal and placental growth/angiogenesis and reduced maternal blood pressure. Aldosterone levels are low in women with pre-eclampsia. Given the placental growth properties of aldosterone in pregnancy, we hypothesised that increased aldosterone improves placental function ex vivo. We applied aldosterone in the dual human placenta perfusion model and analysed specific regulatory markers. MethodsA single cotyledon was perfused using a trimodal perfusion setup consisting of a control phase (CP; basic perfusion medium (BPM) alone) and two consecutive experimental phases (EP1/EP2; BPM supplemented with 1.5 x 10−9M and 1.5 x 10−7M aldosterone, respectively). CP and EP1/EP2 were conducted in closed circuits lasting 2 h each. Quality/time control perfusions using BPM alone were performed for 360 min to distinguish time-dependent effects from aldosterone-related effects. Perfusates were assessed for control parameters (pH/pO2/pCO2/glucose/lactate/creatinine/antipyrine). Maternal perfusates were analysed for placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α) using ELISAs. mRNA expression of abovementioned factors was measured by qPCR in post-perfusion tissue. ResultsData from quality/time control perfusions indicated that TNF-α and IL-10 release continuously increased over time. Contrary, in the trimodal perfusion setup the application of aldosterone decreased TNF-α secretion (P < 0.05, EP1/EP2 vs CP, 120 min) and increased PlGF release (P < 0.05, EP1 vs CP, 90/120 min) into the maternal perfusates. mRNA expression followed similar trends, but did not reach significance. DiscussionOur ex vivo placental perfusion data suggest that increasing aldosterone promotes anti-inflammatory and pro-angiogenic factors, which could positively contribute to healthy pregnancy outcomes.

Addressing microchimerism in pregnancy by ex vivo human placenta perfusion.

The physical connection of mother and offspring during pregnancy allows the bi-directional exchange of a small number of cells through the placenta. These cells, which can persist long-term in the recipient individual are genetically foreign to it and therefore fulfill the principle of microchimerism. Over the last years, pioneer research on microchimeric cells revealed their role in immune adaptation during pregnancy and priming of tolerogenic responses in the progeny. However, the mechanisms involved in cell transfer across the placenta barrier remain poorly investigated. In this review, we summarize the evidence of fetomaternal microchimerism, propose a mechanism for cell trafficking through the placenta and discuss the different models and techniques available for its analysis. Likewise, we aim to generate interest in the use of ex vivo placenta perfusion to investigate microchimerism in physiological and pathological settings.

Chiral Transplacental Pharmacokinetics of Fexofenadine: Impact of P-Glycoprotein Inhibitor Fluoxetine Using the Human Placental Perfusion Model.

Fexofenadine is a well-identified in vivo probe substrate of P-glycoprotein (P-gp) and/or organic anion transporting polypeptide (OATP). This work aimed to investigate the transplacental pharmacokinetics of fexofenadine enantiomers with and without the selective P-gp inhibitor fluoxetine. The chiral transplacental pharmacokinetics of fexofenadine-fluoxetine interaction was determined using the ex vivo human placenta perfusion model (n = 4). In the Control period, racemic fexofenadine (75ng of each enantiomer/ml) was added in the maternal circuit. In the Interaction period, racemic fluoxetine (50ng of each enantiomer/mL) and racemic fexofenadine (75ng of each enantiomer/mL) were added to the maternal circulation. In both periods, maternal and fetal perfusate samples were taken over 90min. The (S)-(-)- and (R)-(+)-fexofenadine fetal-to-maternal ratio values in Control and Interaction periods were similar (~0.18). The placental transfer rates were similar between (S)-(-)- and (R)-(+)-fexofenadine in both Control (0.0024 vs 0.0019min-1) and Interaction (0.0019 vs 0.0021min-1) periods. In both Control and Interaction periods, the enantiomeric fexofenadine ratios [R-(+)/S-(-)] were approximately 1. Our study showed a low extent, slow rate of non-enantioselective placental transfer of fexofenadine enantiomers, indicating a limited fetal fexofenadine exposure mediated by placental P-gp and/or OATP2B1. The fluoxetine interaction did not affect the non-enantioselective transplacental transfer of fexofenadine. The ex vivo placental perfusion model accurately predicts in vivo placental transfer of fexofenadine enantiomers with remarkably similar values (~0.17), and thus estimates the limited fetal exposure.

Ethical and Regulatory Considerations of Placental Therapeutics

PurposePlacental therapeutics aim to treat placental disease; however, ethical and regulatory issues should be considered if the drug also potentially affects the fetus. Drugs that might transfer or edit genes carry a specific challenge because currently fetal gene editing and fetal gene therapy are considered unethical. MethodsThis article reviews the literature on ethical and regulatory considerations for placental therapeutics. FindingsProposals for maternal gene therapy, directed to the maternal side of the placenta, have been discussed with patients and stakeholders. No absolute ethical, legal, or regulatory barriers to this potential treatment were identified. Patients who have experienced placental disease, such as fetal growth restriction, are interested in these therapies; some would participate in first-in-human trials. Such trials need careful regulatory considerations, such as the steps required to indicate tolerability and efficacy in preclinical models and the optimal animals for reproductive toxicology studies. Ex vivo dual human placenta perfusion experiments and villous explant in vitro studies allow drugs to be tested in normal and diseased human placenta, providing short-term tolerability and toxicologic assessment. Testing drugs in nonhuman primates is an option but carries ethical and feasibility considerations. Selection of inclusion and exclusion criteria for clinical trial participants is important to ensure that the most suitable patients are exposed to a first-in-human drug. These patients will almost certainly be pregnant women with a high risk of perinatal loss and/or perinatal and maternal morbidity. Criteria should identify sufficient numbers of patients to make a trial feasible as well as a phenotype that will respond to the mechanism of action. How to dose escalate and to capture information on adverse events are also key to optimal clinical trial design. ImplicationsDeveloping placental therapeutics requires input from scientists, practitioners, and regulators and close liaison with patients to ensure that new drugs are tested as carefully as possible.

Enrichment and characterization of extracellular vesicles from ex vivo one-sided human placenta perfusion.

Extracellular vesicles (EVs) released by the placenta are packed with biological information and play a major role in fetomaternal communication. Here, we describe a comprehensive set-up for the enrichment and characterization of EVs from human placenta perfusion and their application in further assays. Human term placentas were used for 3h ex vivo one-sided perfusions to simulate the intervillous circulation. Thereafter, populations of small (sEVs) and large EV (lEVs) were enriched from placental perfusate via serial ultracentrifugation. Following, EV populations were characterized regarding their size, protein concentration, RNA levels, expression of surface markers as well as their uptake and miRNA transfer to recipient cells. The sEV and lEV fractions from an entire perfusate yielded, respectively, 294±32µg and 525±96µg of protein equivalents and 2.6±0.5µg and 3.6±0.9µg of RNA. The sEV fraction had a mean diameter of 117±47nm, and the lEV fraction presented 236±54nm. CD63 was strongly detected by dot blot in sEVs, whereas only traces of this marker were found in lEVs. Both EV fractions were positive for the trophoblast marker PLAP (placental alkaline phosphatase) and annexin A1. EV internalization in immune cells was visualized by confocal microscopy, and the transfer of placental miRNAs was detected by quantitative real-time PCR (qPCR). Enriched EV populations showed characteristic features of sEVs and lEVs. EV uptake and transfer of miRNAs to recipient cells demonstrated their functional integrity. Therefore, we advocate the ex vivo one-sided placenta perfusion as a robust approach for the collection of placental EVs.

Nanoparticle mediated increased insulin-like growth factor 1 expression enhances human placenta syncytium function

IntroductionPlacental dysfunction is an underlying cause of many major obstetric diseases and treatment options for complications like fetal growth restriction (FGR) are limited .We previously demonstrated nanoparticle delivery of the human insulin-like growth factor 1 (hIGF1) transgene under control of the trophoblast-specific PLAC1 promoter maintains normal fetal growth in a surgically-induced FGR mouse model. However, uptake by human placental syncytiotrophoblast has yet to be determined. MethodsAn ex vivo human placenta perfusion model, term placenta villous fragments, and other in vitro syncytiotrophoblast models were used to determine nanoparticle uptake, transgene expression, and functional responses under oxidative stress conditions. ResultsIn the ex vivo perfusion, fluorescence from a Texas-Red conjugated nanoparticle increased in maternal perfusate upon nanoparticle addition and declined by the conclusion of the experiment (P < 0.001. Fluorescent histology confirmed localization in the syncytiotrophoblasts. No Texas-Red fluorescence was detected in the fetal perfusate. Transgene expression of hIGF1 in differentiated BeWo cells, isolated primary trophoblasts and fragments was increased compared to untreated (55,000-fold, P = 0.0003; 95-fold, P = 0.003; 400-fold, P < 0.001, respectively). Functionally, increased hIGF1 expression in villous fragments resulted in translocation of glucose transporter 1 to the syncytiotrophoblast cell membrane and under conditions of oxidative stress in BeWo cells, protected against increased cell death (P < 0.01) and decreased mitochondrial activity (P < 0.01). ConclusionThe current study confirms that our nanoparticle is capable of uptake in human placental syncytium which results in enhanced transgene expression, functional changes to cellular activity and protection against increased oxidative stress.

Ex Vivo Human Placenta Perfusion, Metabolic and Functional Imaging for Obstetric Research-A Feasibility Study.

Placenta metabolism is closely linked to pregnancy outcome, and few modalities are currently available for studying the human placenta. Here, we aimed to investigate a novel ex vivo human placenta perfusion system for metabolic imaging using hyperpolarized [1-13C]pyruvate. The metabolic effects of 3 different human placentas were investigated using functional and metabolic magnetic resonance imaging. The placenta glucose metabolism and hemodynamics were characterized with hyperpolarized [1-13C]pyruvate magnetic resonance imaging and by dynamic contrast-enhanced (DCE) imaging. Hyperpolarized [1-13C]pyruvate showed a decrease in the 13C-lactate/13C-pyruvate ratio from the highest to the lowest metabolic active placenta. The metabolic profile was complemented by a more homogenous distributed hemodynamic response, with a longer mean transit time and higher blood volume. This study shows different placenta metabolic and hemodynamic features associated with the placenta functional status using hyperpolarized magnetic resonance ex vivo. This study supports further studies using ex vivo metabolic imaging of the placenta alterations associated with pregnancy complications.

Transplacental transport of paracetamol and its phase II metabolites using the ex vivo placenta perfusion model

In Europe, 50–60% of pregnant women uses paracetamol (PCM), also known as acetaminophen. While it was considered to be safe, recent studies have shown an association between prenatal exposure to PCM and increased incidences of autism, cryptorchidism, asthma and ADHD. In this study the transplacental transfer of PCM and its metabolites was investigated using an ex vivo human placenta perfusion model (closed circuit; n = 38). Maternal-to-foetal (M-F) and foetal-to-maternal (F-M) transplacental transfer was determined at a concentration correlating with the maximum and steady state concentration in normal clinical use. Antipyrine (AP) was added as reference compound. Samples of the foetal and maternal perfusion medium were taken until 210 (PCM) or 360 min (paracetamol sulphate (PCM-S) and paracetamol glucuronide (PCM-G). PCM and AP concentrations reached an equilibrium between foetal and maternal compartments within the duration of the perfusion experiment and irrespective of the transfer direction. The percentage placental transfer of PCM was 45% (M-F and F-M). For PCM-S, transfer was 39% (M-F) and 28% (F-M), while the PCM-G transfer was 34% (M-F) and 25% (F-M). During placenta perfusions with the metabolites slight conversion (3.5–4.1%) to PCM was observed. In conclusion, PCM crosses the placental barrier rapidly via passive diffusion. Differences in flow rate and villous placental structure explain the significantly faster M-F transfer than F-M transfer of PCM. The larger and more hydrophilic molecules PCM-S and PCM-G cross the placenta at a significantly lower rate. Moreover, their F-M transport is about 40% slower than M-F transport, suggesting involvement of a transporter.

Evaluation of Sildenafil and Tadalafil for Reversing Constriction of Fetal Arteries in a Human Placenta Perfusion Model.

Fetal growth restriction resulting from reduced placental blood perfusion is a major cause of neonatal morbidity and mortality. Aside from intense surveillance and early delivery, there is no treatment for fetal growth restriction. A potential treatment associated with placental vasoconstriction is the class of PDE5 (phosphodiesterase type 5) inhibitors such as sildenafil, which is known to cross the placenta. In contrast, tadalafil, a more potent and selective PDE5 inhibitor has not been studied in pregnancy or experimental models of fetal growth restriction. Therefore, we compared the efficacy of these 2 PDE5 inhibitors for reversing vasoconstriction in an ex vivo human placental model and evaluating molecular and physiological responses. Sildenafil and tadalafil were infused into the intervillous space in a preconstricted human placental dual cotyledon, dual perfusion assay for the comparison of arteriole pressures and molecular indicators of drug inhibition. Results indicate a decrease arterial pressure with sildenafil citrate compared with controls, whereas tadalafil showed no difference. PDE5 and endothelial nitric oxide synthase activity were altered with sildenafil but not tadalafil. Sildenafil citrate improved preconstricted placental arterial perfusion in a human placental model, whereas tadalafil showed no response. It is possible that tadalafil did not cross the human placental barrier or was degraded by trophoblasts. This study supports human clinical trials exploring sildenafil as a potential treatment for improving fetal blood flow in fetal growth restriction associated with vasoconstriction.

Translocation of silver nanoparticles in the ex vivo human placenta perfusion model characterized by single particle ICP-MS.

With the extensive use of silver nanoparticles (AgNPs) in various consumer products their potential toxicity is of great concern especially for highly sensitive population groups such as pregnant women and even the developing fetus. To understand if AgNPs are taken up and cross the human placenta, we studied their translocation and accumulation in the human ex vivo placenta perfusion model by single particle ICP-MS (spICP-MS). The impact of different surface modifications on placental transfer was assessed by AgNPs with two different modifications: polyethylene glycol (AgPEG NPs) and sodium carboxylate (AgCOONa NPs). AgNPs and ionic Ag were detected in the fetal circulation in low but not negligible amounts. Slightly higher Ag translocation across the placental barrier for perfusion with AgPEG NPs and higher AgNP accumulation in placental tissue for perfusion with AgCOONa NPs were observed. Since these AgNPs are soluble in water, we tried to distinguish between the translocation of dissolved and particulate Ag. Perfusion with AgNO3 revealed the formation of Ag containing NPs in both circulations over time, of which the amount and their size in the fetal circulation were comparable to those from perfusion experiments with both AgNP types. Although we were not able to clarify whether intact AgNPs and/or Ag precipitates from dissolved Ag cross the placental barrier, our study highlights that uptake of Ag ions and/or dissolution of AgNPs in the tissue followed by re-precipitation in the fetal circulation needs to be considered as an important pathway in studies of AgNP translocation across biological barriers.

PP-6 Integration and validation of the ex vivo human placenta perfusion model

Background Pregnant women and their fetuses are or-phan populations with respect to knowledge on safety and efficacy of drugs. It is estimated that over 90% of pregnant women uses over-the-counter or prescription medication.1 Albeit, data on transplacental transfer or fetal effects are still lacking for most of the medicines and food supplements. The ex vivo human placenta perfusion model is an effective and non-invasive method to study transplacental passage of drugs and environmental com-pounds in humans.2 It is the only method that retains the full structure of a full term human placenta, making it possible to study transplacental passage without harm-ing the foetus or the mother.3 Due to many challenges and its high complexity it remains difficult to incorporate it routinely into laboratories. Methods A step-by-step protocol for the implementa-tion and validation of a closed-closed ex vivo perfusion model was developed. Different quality controls were implemented to ensure the integrity, viability and func-tionality of the method: (i). Antipyrine is a small drug molecule that does not bind to proteins and that passes the placental barrier by passive diffusion; It was used here to determine ‘overlap’ (solute exchange) between foetal and maternal circulation; (ii) the pressure and the flow rate in the foetal circulation as a marker for leakage; (iii) pH and glucose consumption were implemented as a marker for tissueviability. Results In total 89 placentas were collected of which 34 placentas were successfully perfused with antipyrine and fulfilled all quality control measurements. A foe-tal/maternal antipyrine concentration ratio of 0.75 was reached within 89±21 min, while 210 min were required to achieve equilibrium. The foetal pressure remained un-der 70 mmHg during the entire experiment. The end foe-tal flow was 98% of the foetal starting flow. The average glucose consumption was 0.30±0.15 µmol/min/g. Every 30 min the maternal pH declined to 7.29±0.06 and was adjusted to 7.4. The foetal pH stayed stable at 7.30±0.05. Conclusion Based on the multiple quality control mea-surements, the described method of a closed human ex-vivo placenta perfusion model was validated. The success rate (38%) was more than twice the success rate reported in literature (15%).

O-17 Transplacental transport of paracetamol and its metabolites using the ex-vivo human placenta perfusion model

Background In Europe, 50%–60% of pregnant women uses paracetamol (Dafalgan). While its use in pregnan-cy was considered safe, recent studies show an associ-ation between prenatal exposure to paracetamol and increased incidences of autism, cryptorchidism, asthma and attention deficit hyperactivity disorder in a dose and duration dependent manner1-3. Data on transplacental transfer and metabolism of paracetamol are limited. Methods In an ex vivo placenta perfusion model (closed circuit) (n=38), maternal-to-fetal and fetal-to-maternal transplacental transfer of paracetamol (PCM) and its me-tabolites, paracetamol sulfate (PCM-S) and paracetamol glucuronide (PCM-G), was determined at a concentration corresponding to the maximum (PCM: 30 µg/ml; PCM-S: 10 µg/ml; PCM-G: 25 µg/ml) and steady state (PCM: 10 µg/ml; PCM-S: 5 µg/ml; PCM-G: 12.5 µg/ml) plasma con-centrations in normal clinical use. Antipyrine 100 µg/mL was added as internal control. PCM, PCM-S, PCM-G and antipyrine concentrations in perfusion medium and pla-cental tissue were determined using HPLC and LC-MS. Samples were taken at 0, 3, 6, 10, 15, 20, 30 min then ev-ery 15 min until 150 min followed by every 30 min until 210 (PCM) or 360 min (PCM-S and PCM-G). Fetal-to-ma-ternal and maternal-to-fetal ratios were normalised for antipyrine for each time point. Tissue accumulation and recovery of the compounds was calculated. Statistical dif-ferences were assessed using ANOVA. Results The maternal-to-fetal as fetal-to-maternal trans-port of PCM was 44%–48%. For PCM-S, transplacental trans-fer was 38%–40% for maternal-to-fetal transfer and 28% for fetal-to-maternal transfer. PCM-G had a transfer of 31%–36% for maternal-to-fetal and 25% for fetal-to-maternal transfer. An equilibrium between the maternal and fetal concentrations was reached for PCM after 210 min for perfusion from maternal-to-fetal circulation. Fetal-to-ma-ternal transport of PCM-S and PCM-G was significantly slower then maternal-to-fetal transport. Extrapolation of maternal-to-fetal transport data till 360 min predict-ed equilibrium at 7.5 hour (PCM-S) and 9.5 hour (PCM-G). For fetal-to-maternal transport extrapolation of data till 210 min (PCM) and 360 min (PCM-S and PCM-G) predict-ed equilibrium for PCM after 270 min, PCM-S 36 hour and PCM-G 44 hour. PCM-S and PCM-G were converted to PCM by the placenta during the perfusions. Conclusion This study shows that PCM rapidly crosses the placental barrier via passive diffusion for both mater-nal-to-fetal and fetal-to-maternal transplacental transfer. PCM-S and PCM-G, larger and more hydrophilic mole-cules, cross the placenta at a significantly lower rate. For PCM-S and PCM-G fetal-to-maternal transport is signifi-cantly slower than maternal-to-fetal transport.

Integration and validation of the ex vivo human placenta perfusion model

IntroductionThe ex vivo human placenta perfusion model is an effective and non-invasive method to study transplacental passage of drugs and environmental compounds in humans. Due to many challenges and its high complexity it remains difficult to incorporate it routinely into laboratories. MethodsThis article describes a step-by-step protocol for the implementation and validation of a closed-closed ex vivo perfusion model. Antipyrine, a small molecule that passes the placental barrier by passive diffusion, was used as a measurement of overlap between foetal and maternal circulation. The pressure and the flow rate in the foetal circulation, glucose consumption and pH were implemented to ensure the integrity, viability and functionality of the method. ResultsIn total 89 placenta were collected of which 34 placentas were successfully perfused with antipyrine and fulfilled all quality control measurements. A foetal/maternal antipyrine concentration ratio of 0.75 was reached within 89±21 min, while 210 min were required to achieve equilibrium. The foetal pressure remained under 70 mmHg during the entire experiment. The end foetal flow was 98% of the foetal starting flow. The average glucose consumption was 0.30±0.15 μmol/min/g. Every 30 min the maternal pH declined to 7.29±0.06 and was adjusted to 7.4. The foetal pH stayed stable at 7.30±0.05. DiscussionBased on the assessment of multiple quality control measurements, the described method of a closed human ex-vivo placenta perfusion model was validated. The success rate (38%) was more than twice the success rate reported in literature (15%).