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Background The phosphodiesterase-5 inhibitor (PDE5) sildenafil has emerged as a promising treatment for preeclampsia (PE). However, a sildenafil trial was recently halted due to lack of effect and increased neonatal morbidity. Methods Ex vivo dual-sided perfusion of an isolated cotyledon and wire-myography on chorionic plate arteries were performed to study the effects of sildenafil and the non-selective PDE inhibitor vinpocetine on the response to the NO donor sodium nitroprusside (SNP) under healthy and PE conditions. Ex vivo perfusion was also used to study placental transfer of sildenafil in 6 healthy and 2 PE placentas. Furthermore, placental mRNA and protein levels of eNOS, iNOS, PDE5 and PDE1 were quantified. Findings Sildenafil and vinpocetine significantly enhanced SNP responses in chorionic plate arteries of healthy, but not PE placentas. Only sildenafil acutely decreased baseline tension in arteries of both healthy and PE placentas. At steady state, the foetal-to-maternal transfer ratio of sildenafil was 0·37 ± 0·03 in healthy placentas versus 0·66 and 0·47 in the 2 PE placentas. mRNA and protein levels of PDE5, eNOS and iNOS were comparable in both groups, while PDE1 levels were lower in PE. Interpretation The absence of sildenafil-induced NO potentiation in arteries of PE placentas, combined with the non-PDE-mediated effects of sildenafil and the lack of PDE5 upregulation in PE, argue against sildenafil as the preferred drug of use in PE. Moreover, increased placental transfer of sildenafil in PE might underlie the neonatal morbidity in the STRIDER trial. Fund This study was funded by an mRACE Erasmus MC grant.

Introduction Parabens are a group of chemicals widely used as preservatives in daily consumer products such as cosmetics, food items, pharmaceuticals and household commodities. They have been broadly detected in human samples indicating a general human exposure, and concerns arose from their potential endocrine disrupting effect. Especially the exposure to parabens during pregnancy is concerning, as the time of fetal development is a particularly vulnerable period. The aim of this study was to investigate the transport and metabolism of four commonly used parabens: methyl-, ethyl-, propyl- and butylparaben (MeP, EtP, PrP and BuP) and the metabolite para-hydroxybenzoic acid (PHBA) across the human placenta. Methods An ex-vivo human placental perfusion model was used. The test compounds were added in the maternal compartment (with initial concentrations of 1 mM or 0.1 mM). Placental transport was evaluated by fetal-maternal concentration ratios (FM-ratio), transport index (TI) and indicative permeability (IP). Results Information about parabens kinetics was taken from 10 perfusions and PHBA from 7 perfusions. Paraben metabolism was not detected. The placental transport of MeP, EtP, PrP, BuP and PHBA revealed a transfer from maternal to fetal circulations with FM120 of 0.86±0.27 (MeP), 0.98±0.28 (EtP), 1.00±0.28 (PrP), 1.12±0.59 (BuP) and 0.82±0.37 (PHBA). The test substances accumulated in the perfused tissue in some degree. The average kinetic parameters FM-ratio, TI and IP were not different between chemicals. Discussion The present study shows that the placenta barrier is permeable to all four parabens and the metabolite, which implies potential fetal exposure. IntroductionParabens are a group of chemicals widely used as preservatives in daily consumer products such as cosmetics, food items, pharmaceuticals and household commodities. They have been broadly detected in human samples indicating a general human exposure, and concerns arose from their potential endocrine disrupting effect. Especially the exposure to parabens during pregnancy is concerning, as the time of fetal development is a particularly vulnerable period. The aim of this study was to investigate the transport and metabolism of four commonly used parabens: methyl-, ethyl-, propyl- and butylparaben (MeP, EtP, PrP and BuP) and the metabolite para-hydroxybenzoic acid (PHBA) across the human placenta.MethodsAn ex-vivo human placental perfusion model was used. The test compounds were added in the maternal compartment (with initial concentrations of 1 mM or 0.1 mM). Placental transport was evaluated by fetal-maternal concentration ratios (FM-ratio), transport index (TI) and indicative permeability (IP).ResultsInformation about parabens kinetics was taken from 10 perfusions and PHBA from 7 perfusions. Paraben metabolism was not detected. The placental transport of MeP, EtP, PrP, BuP and PHBA revealed a transfer from maternal to fetal circulations with FM120 of 0.86 ± 0.27 (MeP), 0.98 ± 0.28 (EtP), 1.00 ± 0.28 (PrP), 1.12 ± 0.59 (BuP) and 0.82 ± 0.37 (PHBA). The test substances accumulated in the perfused tissue in some degree. The average kinetic parameters FM-ratio, TI and IP were not different between chemicals.DiscussionThe present study shows that the placenta barrier is permeable to all four parabens and the metabolite, which implies potential fetal exposure.

Highlights • Placental perfusion yields large amounts of STBEV. • Differential centrifugation isolates enriched preparations of STBMV and STBEX. • fl-NTA can be optimised using an EV ‘standard’. • fl-NTA reliably detects STBMV using the specific marker PLAP., The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n = 8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet – 10KP; NTA modal size 395 ± 12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet – 150KP; NTA modal size 147 ± 6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of ‘standard’ 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n = 8) were 54.5 ± 5.7% (range 17.8–66.9%) and 30.6 ± 5.6% (range 3.3–51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.

This review summarizes the potential and also some limitations of using human placentas, or placental cells and structures for toxicology testing. The placenta contains a wide spectrum of cell types and tissues, such as trophoblast cells, immune cells, fibroblasts, stem cells, endothelial cells, vessels, glands, membranes, and many others. It may be expected that in many cases the relevance of results obtained from human placenta will be higher than those from animal models due to species specificity of metabolism and placental structure. For practical and economical reasons, we propose to apply a battery of sequential experiments for analysis of potential toxicants. This should start with using cell lines, followed by testing placenta tissue explants and isolated placenta cells, and finally by application of single and dual side ex vivo placenta perfusion. With each of these steps, the relative workload increases while the number of feasible repeats decreases. Simultaneously, the predictive power enhances by increasing similarity with in vivo human conditions. Toxic effects may be detected by performing proliferation, vitality and cell death assays, analysis of protein and hormone expression, immunohistochemistry or testing functionality of signaling pathways, gene expression, transport mechanisms, and so on. When toxic effects appear at any step, the subsequent assays may be cancelled. Such a system may be useful to reduce costs and increase specificity in testing questionable toxicants. Nonetheless, it requires further standardization and end point definitions for better comparability of results from different toxicants and to estimate the respective in vivo translatability and predictive value.

ProblemThe pregnancy-associated disease preeclampsia is related to the release of syncytiotrophoblast extracellular vesicles (STBEV) by the placenta. To improve functional research on STBEV, reliable and specific methods are needed to quantify them. However, only a few quantification methods are available and accepted, though imperfect. For this purpose, we aimed to provide an enzyme-linked sorbent assay (ELSA) to quantify STBEV in fluid samples based on their microvesicle characteristics and placental origin.Method of StudyEx vivo placenta perfusion provided standards and samples for the STBEV quantification. STBEV were captured by binding of extracellular phosphatidylserine to immobilized annexin V. The membranous human placental alkaline phosphatase on the STBEV surface catalyzed a colorimetric detection reaction.Results and ConclusionThe described ELSA is a rapid and simple method to quantify STBEV in diverse liquid samples, such as blood or perfusion suspension. The reliability of the ELSA was proven by comparison with nanoparticle tracking analysis.