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ObjectiveThe adipogenic PPARG-encoded PPARγ nuclear receptor also displays essential placental functions. We evaluated the metabolic, reproductive, and perinatal features of patients with PPARG-related lipodystrophy.MethodsCurrent and retrospective data were collected in patients referred to a National Rare Diseases Reference Centre.Results26 patients from 15 unrelated families were studied (18 women, median age 43 years). They carried monoallelic PPARG variants except a homozygous patient with congenital generalized lipodystrophy. Among heterozygous patients aged 16 or more (n = 24), 92% had diabetes, 96% partial lipodystrophy (median age at diagnosis 24 and 37 years), 78% hypertriglyceridaemia, 71% liver steatosis, and 58% hypertension. The mean BMI was 26 ± 5.0 kg/m2. Women (n = 16) were frequently affected by acute pancreatitis (n = 6) and/or polycystic ovary syndrome (n = 12). Eleven women obtained one or several pregnancies, all complicated by diabetes (n = 8), hypertension (n = 4), and/or hypertriglyceridaemia (n = 10). We analysed perinatal data of patients according to the presence (n = 8) or absence (n = 9) of a maternal dysmetabolic environment. The median gestational age at birth was low in both groups (37 and 36 weeks of amenorrhea, respectively). As expected, the birth weight was higher in patients exposed to a foetal dysmetabolic environment of maternal origin. In contrast, 85.7% of non-exposed patients, in whom the variant is, or is very likely to be, paternally-inherited, were small for gestational age.ConclusionsLipodystrophy-related PPARG variants induce early metabolic complications. Our results suggest that placental expression of PPARG pathogenic variants carried by affected foetuses could impair prenatal growth and parturition. This justifies careful pregnancy monitoring in affected families.

The human placenta is a transitory organ essential for fetal development, whose functions can be disrupted by xenobiotics in maternal blood. Benzo-[a]-pyrene (B[a]P) is a carcinogenic, mutagenic and reprotoxic pollutant as well as an endocrine disruptor that can be internalized in the human body by respiratory exposure. Cerium dioxide nanoparticles (CeO2 NP) have been introduced into our environment, mainly for their catalytic properties and share the same emission sources as B[a]P (cigarette smoke and diesel engine exhaust). The aim of our study is to determine the impact of these two atmospheric pollutants on the human placenta in concomitant exposure, in order to get closer to the environmental reality. Chorionic villi and villous cytotrophoblasts (VCT) from human placentas at term of pregnancy were exposed in vitro to the pollutants. The internalization of the pollutants was observed by confocal and Raman microscopy. Cytotoxicity was assessed using WST-1 test and extracellular LDH assay. Placental endocrine activity was assessed by ELISA. The signaling pathways impacted have been studied by Western Blot, RT-qPCR and IHC. CeO2 NP and B[a]P can be internalized within the chorionic villi. B[a]P alone or in co-exposure with CeO2 NP activate the metabolic pathway of the aryl hydrocarbon receptor (AhR), causing DNA damage. B[a]P also stabilizes the stress transcription factor p53 and its transcriptional target p21. Although these two pollutants do not cause major toxicity on term human trophoblasts after in vitro exposures, cellular stress markers are induced.

International audience; Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle®, was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCGα glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCGα was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle®. The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon®, and 30 FSHα glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations.

Human placenta is a multifunctional interface between maternal and fetal blood. Studying the impact of pollutants on this organ is crucial because many xenobiotics in maternal blood can accumulate in placental cells or pass into the fetal circulation. Benzo(a)pyrene (BaP) and cerium dioxide nanoparticles (CeO2 NP), which share the same emission sources, are found in ambient air pollution and also in maternal blood. The aim of the study was to depict the main signaling pathways modulated after exposure to BaP or CeO2 NP vs. co-exposure on both chorionic villi explants and villous cytotrophoblasts isolated from human term placenta. At nontoxic doses of pollutants, BaP is bioactivated by AhR xenobiotic metabolizing enzymes, leading to DNA damage with an increase in γ-H2AX, the stabilization of stress transcription factor p53, and the induction of its target p21. These effects are reproduced in co-exposure with CeO2 NP, except for the increase in γ-H2AX, which suggests a modulation of the genotoxic effect of BaP by CeO2 NP. Moreover, CeO2 NP in individual and co-exposure lead to a decrease in Prx-SO3, suggesting an antioxidant effect. This study is the first to identify the signaling pathways modulated after co-exposure to these two pollutants, which are common in the environment.

Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during human cytomegalovirus (hCMV) congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of hCMV infection on sEVs production, composition and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from either anex vivomodel of infected histocultures from early placenta or from the amniotic fluid of patients naturally infected by hCMV at the beginning of pregnancy. Based on these findings, we propose that placental sEVs could be key actors favoring viral dissemination to the fetal brain during hCMV congenital infection.Significance StatementHuman cytomegalovirus (hCMV) infection is a major issue during pregnancy, affecting 1% of births in western countries. Despite extensive research, the pathophysiology of this congenital infection remains unclear. Recently, increasing evidence point to the key role of placental small extracellular vesicles (sEVs) in materno-fetal communication during pregnancy. Here, we examined the impact of hCMV infection on the protein composition and function of placental sEVs. We observe that hCMV infection leads to major changes in placental sEV protein content. Functional studies show the ability of sEVs produced by placental infected cells to facilitate further infection of naive recipient fetal cells, notably human neural stem cells. Our study demonstrates that placental sEVs are key players of hCMV pathophysiology during congenital infection.

First-trimester placenta (2 pressure (12–14 GW), the activities of the antioxidant enzymes SOD1, catalase and GPX1 are increased. The redox-sensitive MAPK pathways show increased phosphorylated-p38 expression, but no variation in the phosphorylation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) during first trimester, suggesting a physiological redox adaptation, whilst ERK1/2 phosphorylation is higher after 12 GW. Nox is the major superoxide source in early pregnancy (

Human epidemiological and animal-model studies suggest that separate exposure to stress or serotonin-selective reuptake inhibitor (SSRI) antidepressants during pregnancy increases risks for neurodevelopmental disorders in offspring. Yet, little is known about the combined effects of maternal stress and SSRIs with regard to brain development in utero. We found that the placenta is highly permeable to the commonly prescribed SSRI (±)-citalopram (CIT) in humans and mice, allowing rapid exposure of the fetal brain to this drug. We investigated the effects of maternal chronic unpredictable stress in mice with or without maternal oral administration of CIT from embryonic day (E)8 to E17. We assessed fetal brain development using magnetic resonance imaging and quantified changes in serotonergic, thalamocortical, and cortical development. In utero exposure to maternal stress did not affect overall fetal brain growth. However, serotonin tissue content in the fetal forebrain was increased in association with maternal stress; this increase was reversed by maternal CIT. In utero exposure to stress increased the numbers of deep-layer neurons in specific cortical regions, whereas CIT increased overall cell numbers without changing the proportions of layer-specific neurons to offset the effects of stress on deep-layer cortical development. These findings suggest that stress and SSRI exposure in utero differentially impact serotonin-dependent fetal neurodevelopment such that CIT reverses key effects of maternal gestational stress on offspring brain development.

International audience; Preeclampsia, a hypertensive disorder occurring during pregnancy, is characterized by excessive oxidative stress and trophoblast dysfunction with dysregulation of soluble Fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) production. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (Nox) is the major source of placental superoxide in early pregnancy and its activation with the subsequent formation of superoxide has been demonstrated for various agents including Transforming Growth Factor beta-1 (TGF-β1), a well-known p38 MAPK pathway activator. However, the bridge between Nox and sFlt-1 remains unknown. The purpose of this study was to explore the possible signaling pathway of TGF-β1/Nox/p38 induced sFlt-1 production in human chorionic villi (CV).Methods: Human chorionic villi from first trimester placenta (7-9 Gestational Weeks (GW)) were treated with TGF-β1 or preincubated with p38 inhibitor, SB203580. For NADPH oxidase inhibition, CV were treated with diphenyleneiodonium (DPI). The protein levels of phospho-p38, p38, phospho-Mothers Against Decapentaplegic homolog 2 (SMAD2), and SMAD2 were detected by Western blot. The secretion of sFlt-1 and PlGF by chorionic villi were measured with Electrochemiluminescence Immunologic Assays, and NADPH oxidase activity was monitored by lucigenin method.Results: We demonstrate for the first time that NADPH oxidase is involved in sFlt-1 and PlGF secretion in first trimester chorionic villi. Indeed, the inhibition of Nox by DPI decreases sFlt-1, and increases PlGF secretions. We also demonstrate the involvement of p38 MAPK in sFlt-1 secretion and Nox activation as blocking the p38 MAPK phosphorylation decreases both sFlt-1 secretion and superoxide production. Nevertheless, TGF-β1-mediated p38 activation do not seem to be involved in regulation of the first trimester placental angiogenic balance and no crosstalk was found between SMAD2 and p38 MAPK pathways.Conclusions: Thus, the placental NADPH oxidase play a major role in mediating the signal transduction cascade of sFlt-1 production. Furthermore, we highlight for the first time the involvement of p38 activation in first trimester placental Nox activity.

Protease Inhibitors (PI e.g., ritonavir (RTV) and lopinavir (LPV)) used to treat pregnant mothers infected by HIV induce prematurity and endocrine dysfunctions. The maintenance of pregnancy relies on placental hormone production (human Chorionic Gonadotrophin (hCG) and progesterone (P4)). Those functions are ensured by the villous trophoblast and are mainly regulated by the Unfolded Protein Response (UPR) pathway and mitochondria. We investigated, in vitro, if PI impair hCG and P4 production and the potential intracellular mechanisms involved. Term villous cytotrophoblast (VCT) were cultured with or without RTV or LPV from 6 to 48 h. VCT differentiation into syncytiotrophoblast (ST) was followed measuring hCG and P4 secretion. We evaluated the expression of P4 synthesis partners (Metastatic Lymph Node 64 (MLN64), cholesterol side-chain cleavage (P450SCC), Hydroxy-delta-5-Steroid Dehydrogenase and 3 Beta-and steroid delta-isomerase 1 (HSD3B1)), of mitochondrial pro-fusion factors (Mitofusin 2 (Mfn2), Optic Atrophy 1 (OPA1)) and of UPR factors (Glucose-Regulated Protein 78 (GRP78), Activating Transcription Factor 4 (ATF4), Activating Transcription Factor 6 (ATF6), spliced X-box Binding Protein 1 (sXBP1)). RTV had no significant effect on hCG and P4 secretion, whereas lopinavir significantly decreased both secretions. LPV also decreased P450SCC and HSD3B1 expression, whereas it increased Mfn2, GRP78 and sXBP1 expression in ST. RTV has no effect on the endocrine placenta. LPV impairs both villous trophoblast differentiation and P4 production. It is likely to act via mitochondrial fusion and UPR pathway activation. These trophoblastic alterations may end in decreased P4 levels in maternal circulation, inducing prematurity.

International audience; Pregnant women are still considered as drug orphans. Developing new medications for pregnancy complications is an urgent need. Nanomedicines seem to be a promising approach to control the biodistribution of drugs to ensure both the mother’s and the fetus’ safety. Understanding the interaction between nanoparticles and the placental barrier is a key factor to the success of the development of nanomedicines for pregnant women. In this study, we evaluated the behavior of fluorescent PEGylated liposomes and lipoplexes in human placental tissue using in vitro and ex vivo models, BeWo cell culture and suspended villous placental explants, respectively. Fluorescent based analytical tools such as Fluorescence activated cells sorting (FACS), confocal microscopy and HPLC coupled to fluorescence detection were used to assess liposomes penetration and their endocytosis mechanisms in the placenta. First, no influence of the PEGylation density was observed on the cellular internalization of liposomal formulations using both models. The comparison between neutral and cationic liposomes exhibits a significant higher internalization of the cationic formulation compared to the neutral ones. In addition, the HPLC quantification of the fluorescent liposomes in human villous explants demonstrated an increase of cationic liposomes uptake with increasing incubation concentrations. Similar uptake of cationic liposomes and lipoplexes, containing the same cationic lipid, the DMAPAP but with an overall neutral surface charge, was observed and evidenced the higher effect of composition than charge surface on trophoblast penetration. Moreover, both cationic liposomes and lipoplexes exhibited an endocytosis mechanism of internalization via pathways implicating dynamin. These data highlight the key role of the liposome’s lipid composition and the possibility to modulate their internalization in the placenta by adjusting their design

Introduction To date, we have only an incomplete understanding of how gene expression in the human placenta changes at the genome-wide scale from very early in gestation to term. Our aim was to investigate the dynamic changes in gene expression throughout placentation. Methods In our study, gene expression profiles were collected of human placentas from 4 to 40 gestational weeks of age. Simple linear regression and weighted correlation network analysis were applied to identify genes of interest. Analyses of gene enrichment, including gene ontology and pathways from the Kyoto Encyclopedia of Genes and Genomes, were performed using clusterProfiler. Finally, dynamic changes in the expression of individual genes were represented using line graphs of scaled and adjusted gene expression. Results Our results highlighted a total of 5173 genes that are involved in different periods of placentation. Downstream annotation of these genes revealed the biological processes and pathways involved, from which we chose to further investigate the PPAR signaling pathway. We were able to detect changes over time in many genes involved in lipid storage/metabolism, including members of the FABP family and LPL. These patterns were corroborated by lipid staining of placental sections, which revealed a significant decrease in lipid droplet content in placentas from early in the first trimester to term. Conclusion Our study provides detailed information on the dynamics of biological processes and pathways across human placentation. These findings give us new clues for deciphering the normal functions of placentation and the ways in which the mis-regulation of these pathways may be linked to pregnancy-related diseases. As an example, our results show that the PPAR signaling pathway mediates a constant decrease in placental lipid content over the course of pregnancy.

SUMMARYHuman trophoblast stem cells (hTSC) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the human placenta. However, access to human embryos and first trimester placentas is limited thus preventing the establishment of hTSC from a variety of genetic backgrounds associated with placental disorders. In the present study, we show that hTSC can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (I) somatic cell reprogramming of adult fibroblasts using the Yamanaka factors, and (II) cell fate conversion of naive and extended pluripotent stem cells. The resulted induced and converted hTSC (hiTSC/hcTSC) recapitulated hallmarks of hTSC including long-term self-renewal, expression of specific transcription factors, transcriptome-side signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSC and show that these cells resemble post-implantation NR2F2+ cytotrophoblasts (day 8-10). Altogether, hTSC lines of diverse genetics origins open the possibility to model both placental development and diseases in a dish.HighlightsReprogramming of human somatic cells to induced hTSC with OSKMConversion of naive and extended hPSC to hTSCGenetic diversity of hTSC linesDevelopmental matching of hTSC in the peri-implantation human embryo

International audience; Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis ® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota.

Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle®, was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCGα glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCGα was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle®. The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon®, and 30 FSHα glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations. Graphical abstract.

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGβ), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 μm, 150 A), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 μL at 1 mg mL−1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.

Trophoblasts, as the cells that make up the main part of the placenta, undergo cell differentiation processes such as invasion, migration, and fusion. Abnormalities in these processes can lead to a series of gestational diseases whose underlying mechanisms are still unclear. One protein that has proven to be essential in placentation is the peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in the nuclei of extravillous cytotrophoblasts (EVCTs) in the first trimester and villous cytotrophoblasts (VCTs) throughout pregnancy. Here, we aimed to explore the genome-wide effects of PPARγ on EVCTs and VCTs via treatment with the PPARγ-agonist rosiglitazone. EVCTs and VCTs were purified from human chorionic villi, cultured in vitro, and treated with rosiglitazone. The transcriptomes of both types of cells were then quantified using microarray profiling. Differentially expressed genes (DEGs) were filtered and submitted for gene ontology (GO) annotation and pathway analysis with ClueGO. The online tool STRING was used to predict PPARγ and DEG protein interactions, while iRegulon was used to predict the binding sites for PPARγ and DEG promoters. GO and pathway terms were compared between EVCTs and VCTs with clusterProfiler. Visualizations were prepared in Cytoscape. From our microarray data, 139 DEGs were detected in rosiglitazone-treated EVCTs (RT-EVCTs) and 197 DEGs in rosiglitazone-treated VCTs (RT-VCTs). Downstream annotation analysis revealed the similarities and differences between RT-EVCTs and RT-VCTs with respect to the biological processes, molecular functions, cellular components, and KEGG pathways affected by the treatment, as well as predicted binding sites for both protein-protein interactions and transcription factor-target gene interactions. These results provide a broad perspective of PPARγ-activated processes in trophoblasts; further analysis of the transcriptomic signatures of RT-EVCTs and RT-VCTs should open new avenues for future research and contribute to the discovery of possible drug-targeted genes or pathways in the human placenta.

Placental syncytiotrophoblast (ST) is considered as the main placental endocrine tissue secreting progesterone, a steroid essential for maintenance of pregnancy. However, each step of progestins production has been poorly investigated in villous cytotrophoblast (VCT) regarding ST formation. We aimed to characterize progestins production during human differentiation of VCT into ST. VCTs were isolated from term placenta and cultivated, with or without forskolin (FSK), to stimulate trophoblast differentiation. Secreted progestins concentrations were determined by immuno-assay and Gas Chromatography-tandem mass spectrometry. Intracellular expression of cholesterol transporter and enzymes involved in steroidogenesis were studied by immunofluorescence, western-blot, and RT-qPCR. Progesterone and pregnenolone are produced by VCT and their secretion increases with VCT differentiation while 17-hydroxyprogesterone concentration remains undetectable. HSD3B1 enzyme expression increases whereas MLN64, the cholesterol placental mitochondrial transporter and P450SCC expressions do not. FSK induces progestins production. Progestins placental synthesis is effective since VCT and increases with ST formation thanks to mitochondria.

Understanding the interplay between the structure, composition and opto-electronic properties of semiconductor nano-objects requires combining transmission electron microscopy (TEM) based techniques with electrical and optical measurements on the very same specimen. Recent developments in TEM technologies allow not only the identification and in-situ electrical characterization of a particular object, but also the direct visualization of its modification in-situ by techniques such as Joule heating. Over the past years, we have carried out a number of studies in these fields that are reviewed in this contribution. In particular, we discuss here i) correlated studies where the same unique object is characterized electro-optically and by TEM, ii) in-situ Joule heating studies where a solid-state metal-semiconductor reaction is monitored in the TEM, and iii) in-situ biasing studies to better understand the electrical properties of contacted single nanowires. In addition, we provide detailed fabrication steps for the silicon nitride membranes crucial to these correlated and in-situ measurements., Comment: This is an author-created, un-copyedited version of a topical review published in Nanotechnology. IOP Publishing Ltd. is not responsible for any errors or omissions in this version of the manuscript or any version derived from it. The Version of Record is available online at https://doi.org/10.1088/1361-6528/ab99f0

The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl®.

Human chorionic gonadotropin (hCG) is one of the first hormonal messages produced by the placenta toward the mother. Detectable in maternal blood 2 days after implantation, hCG behaves like a superagonist of LH inducing the secretion of progesterone by the corpus luteum until the placenta itself acquires the ability to produce progesterone. hCG also has a role in quiescence of the myometrium and in local immune tolerance. Specific to humans, hCG is a complex glycoprotein composed of two highly glycosylated subunits. The α-subunit is identical to the pituitary gonadotrophin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene (CGA). The β-subunits are distinct in each of the gonadotropin hormones and confer receptor and biological specificity. hCGβ subunit is encoded by a cluster of genes (CGB), and the protein contains two sites of N-glycosylation and four sites of O-glycosylation. The expression of hCG and its glycosylation state vary with the stage of pregnancy, the source of production, and the pathology. The syncytiotrophoblast (ST) is the placental barrier between maternal and fetal blood that allows exchanges in nutrients and gases and also represents the endocrine tissue of the human placenta. Indeed, hCG is mainly secreted by the ST into maternal blood from about a week after fecundation reaching a peak around 8–10 weeks of gestation. Extravillous trophoblasts (EVTs), involved in chorionic villi anchoring, modulation of the uterine maternal immune system, and uterine artery remodeling, also secrete hCG. In particular, EVT produces hyperglycosylated forms of hCG (hCG-H) like in choriocarcinoma. In contrast to hCG, hCG-H is found elevated during early first trimester and then decreases, while hCG peaks. In addition to its endocrine role, hCG has autocrine and paracrine roles. It promotes the formation of the ST and angiogenesis through the LH/CG receptor but had no effect on trophoblast invasion. In contrast, hCG-H enhances trophoblast invasion and angiogenesis by interacting with the TGFβ receptor 2. hCG is largely used in antenatal screening and hCG-H represents a serum marker of implantation and early physiological trophoblast invasion. In conclusion, hCG is the main pregnancy hormone, whose maternal concentration and glycan structure change throughout pregnancy. Depending on its source of production, glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome.

International audience; Introduction The use of paclitaxel in pregnant cancer patients is feasible in terms of fetal safety, but little is known about the effects of paclitaxel on the placenta. Using three experimental models, we aimed to assess the effects of paclitaxel on the expression of placental drug transporters. Methods In the in vitro model (human primary trophoblast culture), trophoblasts were isolated from normal term placentas and subsequently exposed to paclitaxel. The transcriptional regulation of 84 genes encoding for drug transporters, and the protein expression of ABCB1/P-gp and ABCG2/BCRP were assessed. In the in vivo model, placental tissues isolated from pregnant cancer patients treated with paclitaxel were analyzed to assess the protein expression of ABCB1/P-gp and ABCG2/BCRP. The same parameters were assessed in extracts from human placental cotyledons perfused ex vivo with paclitaxel. Results In the in vitro model, the expression of twelve drug-transporters genes was found to be significantly down-regulated after exposure to paclitaxel, including ABCC10, SLC28A3, SLC29A2, and ATP7B (involved in the transport of taxanes, antimetabolites, and cisplatin, respectively). The protein expression of ABCB1/P-gp increased by 1.3-fold after paclitaxel administration. Finally, the protein expression of ABCB1/P-gp and ABCG2/BCRP was higher in cotyledons from mothers treated with multiple doses of paclitaxel during pregnancy than in cotyledons perfused with a single dose of paclitaxel. Discussion Paclitaxel modulates the expression of placental drug transporters involved in the disposition of various anticancer agents. Further studies will be needed to assess the impact of repeated or prolonged exposure to paclitaxel on the expression and function of placental drug transporters.

Objective To evaluate the association of active renin concentration (ARC), plasma aldosterone concentration (PAC) and aldosterone-to-renin ratio (ARR) with the risk of preeclampsia (PE), in particular according to the status of obesity. Design This retrospective case-control study involved 90 women with PE (mean gestation 35.6 ± 3.6 weeks) and 90 age-matched control women with uncomplicated pregnancies (mean gestation 38.5 ± 2.5 weeks). ARC and PAC were measured by radioimmunoassay; ARR calculated as PAC to ARC ratio. PE cases were stratified into 5 percentiles groups, and analyzed in multivariate logistic regression. Results Women with PE had significantly lower median ARC and PAC than control women, which were confirmed by percentiles analysis. Spearman correlation demonstrated negative correlation of ARC with body mass index, systolic/diastolic blood pressure. PAC correlated negatively with systolic/diastolic blood pressure, but positively with baby weight, ARC and ARR. On the other hand, ARR positively correlated with BMI and PAC, but negatively with ARC. Lower PAC was associated with PE, irrespective of body weight, while ARC levels were significantly lower in non-obese PE cases vs. control women. ARR was not significantly different between PE cases and control women, when stratified according to obesity. Conclusion Low ARC and PAC in third trimester are more strongly associated with preeclampsia respectively in non-obese and obese women.

International audience; Preeclampsia is a serious pregnancy disorder characterized by the onset of high blood pressure and proteinuria. Although the understanding of the disease is increasing, it remains without treatment, other than the delivery of the baby and the placenta. This review sets out to discuss some new developments and strategies in the treatment of preeclampsia. We briefly review the current knowledge on the preeclamptic pathophysiology. We then examine the recent trends in preeclampsia treatment and, in particular, the tracks of potential therapeutic targets. Finally, we focus on the possibilities nanocarriers could offer in the management of preeclampsia. Indeed, nanocarriers could help to prevent transplacental passage and promote placental-specific drug delivery, thereby enhancing efficacy and improving safety. Tendencies are then drawn from the available studies on the optimal characteristics of a nanocarrier to deliver drugs to the placenta.

Certaines pathologies semblent avoir une origine développementale et, aujourd’hui, le microbiote apparaît comme un déterminant de l’état de santé de l’homme et son établissement, une étape importante chez le nouveau-né. Des variations dans sa constitution, incluant un déséquilibre (ou dysbiose), ont ainsi été associées à de nombreuses pathologies. De récents travaux suggèrent qu’une colonisation bactérienne de l’utérus, du liquide amniotique ou encore du placenta, des sites auparavant pensés stériles, existerait. Durant les phases de son développement, le foetus pourrait ainsi rencontrer des bactériesin utero. Elles contribueraient à l’établissement de son microbiote avant même l’accouchement et donc avant la rencontre avec les microorganismes des microbiotes vaginal, fécal et cutané, ceux-ci variant selon les modes d’accouchement (voie basse ou césarienne). Les premières études sur l’existence d’un microbiotein utero, qui se caractérise par une faible biomasse, sont cependant controversées.

The human placenta is a transient organ essential for pregnancy maintenance, fetal development and growth. It has several functions, including that of a selective barrier against pathogens and xenobiotics from maternal blood. However, some pollutants can accumulate in the placenta or pass through with possible repercussions on pregnancy outcomes. Cerium dioxide nanoparticles (CeO2 NPs), also termed nanoceria, are an emerging pollutant whose impact on pregnancy is starting to be defined. CeO2 NPs are already used in different fields for industrial and commercial applications and have even been proposed for some biomedical applications. Since 2010, nanoceria have been subject to priority monitoring by the Organization for Economic Co-operation and Development in order to assess their toxicity. This review aims to summarize the current methods and models used for toxicology studies on the placental barrier, from the basic ones to the very latest, as well as to overview the most recent knowledge of the impact of CeO2 NPs on human health, and more specifically during the sensitive window of pregnancy. Further research is needed to highlight the relationship between environmental exposure to CeO2 and placental dysfunction with its implications for pregnancy outcome.