Understanding bovine embryo elongation: a transcriptomic study of trophoblastic vesicles.

Background: During the process of elongation, the embryo increases in size within the uterus, while the extra-embryonic tissues (EETs) develop and differentiate in preparation for implantation. As it grows, the ovoid embryo transforms into a tubular form first and then a filamentous form. This process is directed by numerous genes and pathways, the expression of which may be altered in the case of developmental irregularities such as when the conceptus is shorter than expected or when the embryo develops after splitting. In bovines, efforts to understand the molecular basis of elongation have employed trophoblastic vesicles (TVs)-short tubular EET pieces that lack an embryo-which also elongate in vivo. To date, however, we lack molecular analyses of TVs at the ovoid or filamentous stages that might shed light on the expression changes involved. Methods: Following in vivo development, we collected bovine conceptuses from the ovoid (D12) to filamentous stages (D18), sectioned them into small pieces with or without their embryonic disc (ED), and then, transferred them to a receptive bovine uterus to assess their elongation abilities. We also grew spherical blastocysts in vitro up to D8 and subjected them to the same treatment. Then, we assessed the differences in gene expression between different samples and fully elongating controls at different stages of elongation using a bovine array (10 K) and an extended qPCR array comprising 224 genes across 24 pathways. Results: In vivo, TVs elongated more or less depending on the stage at which they had been created and the time spent in utero. Their daily elongation rates differed from control EET, with the rates of TVs sometimes resembling those of earlier-stage EET. Overall, the molecular signatures of TVs followed a similar developmental trajectory as intact EET from D12-D18. However, within each stage, TVs and intact EET displayed distinct expression dynamics, some of which were shared with other short epithelial models. Conclusion: Differences between TVs and EET likely result from multiple factors, including a reduction in the length and signaling capabilities of TVs, delayed elongation from inadequate uterine signals, and modified crosstalk between the conceptus and the uterus. These findings confirm that close coordination between uterine, embryonic, and extra-embryonic tissues is required to orchestrate proper elongation and, based on the partial differentiation observed, raise questions about the presence/absence of certain developmental cues or even their asynchronies.

Single-cell profiling reveals immune disturbances landscape and HLA-F-mediated immune tolerance at the maternal-fetal interface in preeclampsia.

Background: Preeclampsia is a pregnancy-specific disorder that always causes maternal and fetal serious adverse outcome. Disturbances in maternal immune tolerance to embryo at the maternal-fetal interface (MFI) may be associated with preeclampsia onset. Recent studies have revealed the reduced expression pattern of HLA-F at the MFI in preeclampsia, while the mechanism of it mediating maternal fetal immune tolerance has not been revealed. Methods: Single-cell RNA sequencing on placental decidua was performed to reveal the immune disturbances landscape at the MFI in preeclampsia. Human Jar cells and NK-92MI cells were employed to study the role of HLA-F in trophoblasts and lymphocyte. Results: A total of 101,250 cells were classified into 22 cell clusters. Disease-related IGFBP1+SPP1+ extracellular villus trophoblast (EVT) was identified in the preeclamptic placental decidua, accompanied by newly discovered immune cellular dysfunction such as reduced ribosomal functions of NK populations and abnormal expression of antigen-presenting molecules in most cell clusters. Certain genes that are characteristic of the intermediate stage of myeloid or EVT cell differentiation were found to have unexplored but important functions in the pathogenesis of preeclampsia; specifically, we detected enhanced cell cross-talk between IGFBP1+SPP1+ EVT2 or SPP1+M1 cells and their receptor cell populations at the MFI of PE patients compared to controls. With respect to HLA-F, mIF staining confirmed its reduced expression in PE samples compared to controls. Over-expression of HLA-F in Jar cells promoted cell proliferation, invasion, and migration while under-expression had the opposite effect. In NK-92MI cells, over-expression of HLA-F increased the secretion of immunoregulation cytokines such as CSF1 and CCL22, and promoted adaptive NKG2C+NK cell transformation. Conclusions: We revealed the immune disturbance landscape at the MFI in preeclampsia. Our findings regarding cellular heterogeneity and immune cellular dysfunction, as revealed by scRNA-seq, and the function of HLA-F in cells provide new perspectives for further investigation of their roles in the pathogenesis of preeclampsia, and then provide potential new therapeutic target.

IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases.

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that block many viruses from entering cells. High levels of type I interferon (IFN) are associated with adverse pregnancy outcomes, and IFITMs have been shown to impair the formation of syncytiotrophoblast. Here, we examine whether IFITMs affect another critical step of placental development, extravillous cytotrophoblast (EVCT) invasion. We conducted experiments using in vitro/ex vivo models of EVCT, mice treated in vivo with the IFN-inducer poly (I:C), and human pathological placental sections. Cells treated with IFN-ß demonstrated upregulation of IFITMs and reduced invasive abilities. Transduction experiments confirmed that IFITM1 contributed to the decreased cell invasion. Similarly, migration of trophoblast giant cells, the mouse equivalent of human EVCTs, was significantly reduced in poly (I:C)-treated mice. Finally, analysis of CMV- and bacterial-infected human placentas revealed upregulated IFITM1 expression. These data demonstrate that high levels of IFITM1 impair trophoblast invasion and could explain the placental dysfunctions associated with IFN-mediated disorders.

Benzo(a)pyrene and Cerium Dioxide Nanoparticles in Co-Exposure Impair Human Trophoblast Cell Stress Signaling.

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.

Perinatal, metabolic, and reproductive features in PPARG-related lipodystrophy.

OBJECTIVE: The 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. METHODS: Current and retrospective data were collected in patients referred to a National Rare Diseases Reference Centre. RESULTS: 26 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. CONCLUSIONS: Lipodystrophy-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.

Novel fluorescent and secreted transcriptional reporters for quantifying activity of the xenobiotic sensor aryl hydrocarbon receptor (AHR).

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that plays a critical role in diverse biological processes, including xenobiotic metabolism, carcinogenesis, and physiological functions such as regulation of the immune system and cell differentiation. To improve studies of AHR activity, we constructed two new reporter genes: a fluorescent GFP-tagged histone 2B (XRE-H2B-eGFP) and a secreted nanoluciferase (XRE-pNL1.3[secNluc]). Here, we demonstrate how these reporters can be used to monitor AHR activity in different types of cells, including human primary trophoblasts and cell lines, following incubation with a strong AHR ligand, benzo[a]pyrene (B[a]P), or an AHR inhibitor (CH223191). Compared to vehicle control cells, a significant increase in AHR activity was observed in cells treated with 0.5 and/or 2 µM B[a]P and a significant decrease was detected in response to treatment with 3 µM CH223191. These new plasmids have great potential for use in a variety of applications, such as screening for endogenous or exogenous ligands of AHR.

C1431T Variant of PPARγ Is Associated with Preeclampsia in Pregnant Women.

Peroxisome proliferator-activated receptor γ (PPARγ) is essential for placental development, whose SNPs have shown increased susceptibility to pregnancy-related diseases, such as preeclampsia. Our aim was to investigate the association between preeclampsia and three PPARγ SNPs (Pro12Ala, C1431T, and C681G), which together with nine clinical factors were used to build a pragmatic model for preeclampsia prediction. Data were collected from 1648 women from the EDEN cohort, of which 35 women had preeclamptic pregnancies, and the remaining 1613 women had normal pregnancies. Univariate analysis comparing preeclamptic patients to the control resulted in the SNP C1431T being the only factor significantly associated with preeclampsia (p < 0.05), with a confidence interval of 95% and odds ratio ranging from 4.90 to 8.75. On the other hand, three methods of multivariate feature selection highlighted seven features that could be potential predictors of preeclampsia: maternal C1431T and C681G variants, obesity, body mass index, number of pregnancies, primiparity, cigarette use, and education. These seven features were further used as input into eight different machine-learning algorithms to create predictive models, whose performances were evaluated based on metrics of accuracy and the area under the receiver operating characteristic curve (AUC). The boost tree-based model performed the best, with respective accuracy and AUC values of 0.971 ± 0.002 and 0.991 ± 0.001 in the training set and 0.951 and 0.701 in the testing set. A flowchart based on the boost tree model was constructed to depict the procedure for preeclampsia prediction. This final decision tree showed that the C1431T variant of PPARγ is significantly associated with susceptibility to preeclampsia. We believe that this final decision tree could be applied in the clinical prediction of preeclampsia in the very early stages of pregnancy.

Age and Sex-Related Changes in Human First-Trimester Placenta Transcriptome and Insights into Adaptative Responses to Increased Oxygen.

Physiological oxygen tension rises dramatically in the placenta between 8 and 14 weeks of gestation. Abnormalities in this period can lead to gestational diseases, whose underlying mechanisms remain unclear. We explored the changes at mRNA level by comparing the transcriptomes of human placentas at 8-10 gestational weeks and 12-14 gestational weeks. A total of 20 samples were collected and divided equally into four groups based on sex and age. Cytotrophoblasts were isolated and sequenced using RNAseq. Key genes were identified using two different methods: DESeq2 and weighted gene co-expression network analysis (WGCNA). We also constructed a local database of known targets of hypoxia-inducible factor (HIF) subunits, alpha and beta, to investigate expression patterns likely linked with changes in oxygen. Patterns of gene enrichment in and among the four groups were analyzed based on annotations of gene ontology (GO) and KEGG pathways. We characterized the similarities and differences between the enrichment patterns revealed by the two methods and the two conditions (age and sex), as well as those associated with HIF targets. Our results provide a broad perspective of the processes that are active in cytotrophoblasts during the rise in physiological oxygen, which should benefit efforts to discover possible drug-targeted genes or pathways in the human placenta.

DietSee: An on-hand, portable, strip-type biosensor for lipolysis monitoring via real-time amperometric determination of glycerol in blood.

Glycerol is a clinical biomarker of lipolysis that is mainly produced by adipose tissues. Blood glycerol content increases in pathological conditions such as metabolic and cardiovascular diseases or cancer cachexia, but also in response to energetic stress such as physical exercise. Accurate glycerol monitoring is therefore important in a range of healthcare contexts. However, current methods available for the quantification of glycerol are expensive, time-consuming, and require the extraction of plasma from blood, from which blood glycerol content is then extrapolated. Here, we report the development of a new point-of-care glycerometer device, DietSee, based on a strip-type biosensor that enables the quantification of glycerol directly from whole blood in 6 s. The performance of the biosensor was first evaluated using buffer solutions and spiked human and mouse plasma samples, and its response was compared with that of the gold-standard colorimetric method. The results obtained using DietSee correlated strongly with those from the reference method and demonstrated a linear response to glycerol levels across a wide range of concentrations (40-750 µM) that were representative of those in the human body. Next, the biosensor was validated using spiked human blood samples over a range of 30-55% hematocrit; it also demonstrated a strong correlation with reference measurements under these conditions (R2 = 0.97). In addition, the biosensor was only minimally affected by a variety of potential interferents (endogenous and exogenous) and was highly stable in storage (more than 2 years when strips were stored dry at 4 °C). Finally, we investigated the application of the biosensor to real-time monitoring of lipolysis and found that the DietSee is well adapted for this purpose in both human and mouse samples. To conclude, the novel DietSee glycerometer is a sensitive, selective, and rapid tool that enables characterization of the metabolic status of an individual by measuring the glycerol concentration from a single fingertip blood drop.

Mining of combined human placental gene expression data across pregnancy, applied to PPAR signaling pathway.

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.

Peroxisome proliferator-activated receptor gamma-ligand-binding domain mutations associated with familial partial lipodystrophy type 3 disrupt human trophoblast fusion and fibroblast migration.

The transcription factor peroxisome proliferator-activated receptor gamma (PPARG) is essential for placental development, and alterations in its expression and/or activity are associated with human placental pathologies such as pre-eclampsia or IUGR. However, the molecular regulation of PPARG in cytotrophoblast differentiation and in the underlying mesenchyme remains poorly understood. Our main goal was to study the impact of mutations in the ligand-binding domain (LBD) of the PPARG gene on cytotrophoblast fusion (PPARGE352Q ) and on fibroblast cell migration (PPARGR262G /PPARGL319X ). Our results showed that, compared to cells with reconstituted PPARGWT , transfection with PPARGE352Q led to significantly lower PPARG activity and lower restoration of trophoblast fusion. Likewise, compared to PPARGWT fibroblasts, PPARGR262G /PPARGL319X fibroblasts demonstrated significantly inhibited cell migration. In conclusion, we report that single missense or nonsense mutations in the LBD of PPARG significantly inhibit cell fusion and migration processes.

Comparative Study of PPARγ Targets in Human Extravillous and Villous Cytotrophoblasts.

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 Overexpression of Soluble CORIN in Preeclampsia.

Preeclampsia (PE) is a hypertensive disease of pregnancy associated with substantial maternal and fetal morbidity and mortality. CORIN is a transmembrane type II serine protease expressed in cardiomyocytes that converts pro-atrial natriuretic peptide into atrial natriuretic peptide, a cardiac hormone that regulates blood pressure. High levels of soluble CORIN have been reported in PE and are supposed to be cardiac in origin. We hypothesized that during pregnancy soluble CORIN is released by the syncytiotrophoblast and that increased levels of soluble CORIN in preeclampsia originate from placenta. A total of 375 patients (181 PE patients and 194 controls) were analyzed. High levels of soluble CORIN were confirmed in maternal blood from preeclamptic pregnancies compared with controls. Differentiated primary villous cytotrophoblasts showed that CORIN was expressed (mRNA and protein levels) and secreted by trophoblastic cells, mostly by the syncytiotrophoblast. Finally, placental explants showed a significant increase in CORIN production and secretion in PE cases compared with controls. This study showed that CORIN is secreted by trophoblastic cells and that high levels of soluble CORIN in preeclampsia have a placental origin.

IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise.

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

The Role of Peroxisome Proliferator­Activated Receptor Gamma (PPARγ) in Mono(2-ethylhexyl) Phthalate (MEHP)-Mediated Cytotrophoblast Differentiation.

BACKGROUND: Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. Recently, exposure to phthalates has been associated with preterm birth, low birth weight, and pregnancy loss. There is limited information about the possible mechanisms linking maternal phthalate exposure and placental development, but one such mechanism may be mediated by peroxisome proliferator­activated receptor γ (PPARγ). PPARγ belongs to the nuclear receptor superfamily that regulates, in a ligand-dependent manner, the transcription of target genes. Studies of PPARγ-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARγ is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast. OBJECTIVES: The goal of this study was to investigate the action of mono(2-ethylhexyl) phthalate (MEHP) on PPARγ activity during in vitro differentiation of VCTs. METHODS: We combined immunofluorescence, PPARγ activity/hCG assays, western blotting, and lipidomics analyses to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10 µM) on cultured VCTs isolated from human term placentas. RESULTS: Doses of 0.1 and 1 µM MEHP showed significantly lower PPARγ activity and less VCT differentiation in comparison with controls, whereas, surprisingly, a 10 µM dose had the opposite effect. MEHP exposure inhibited hCG production and significantly altered lipid composition. In addition, MEHP had significant effects on the mitogen-activated protein kinase (MAPK) pathway. CONCLUSIONS: This study suggests that MEHP has a U-shaped dose­response effect on trophoblast differentiation that is mediated by the PPARγ pathway and acts as an endocrine disruptor in the human placenta. https://doi.org/10.1289/EHP3730.

Chemotherapy in pregnancy: exploratory study of the effects of paclitaxel on the expression of placental drug transporters.

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.

Different pre-implantation phenotypes of bovine blastocysts produced in vitro.

Embryo transfer in cattle is performed with blastocysts produced in vivo or in vitro using defined media. However, outdated systems such as those that use serum and co-culture remain of interest for research purposes. Here, we investigated the effect of additional culture time on in vitro-produced embryos. Specifically, we compared embryos that formed a blastocoel at different times after fertilisation to those that stayed in culture for up to two additional days with respect to their development in vivo after temporary transfer to oestrus-synchronised recipients. A pre-transfer set (D6, D6+1, D6+2, D7, D7+1, D8) was examined using microarray analyses and correlated with a post-transfer set that included two different days of transfer (D6-T6, D6+2-T8, D7+1-T8, D8-T8). All surviving conceptuses reached primitive-streak stages and filamentous sizes similarly to in vivo (D18) or in vitro controls (D7/T7). The recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25%). With an intermediate survival rate (33%), the D6 embryos with two additional days in culture produced nine times more IFN-tau (IFNT) at D18 than the D6 embryos that were immediately transferred. At the end of culture, D6 and D6+2 embryos displayed the highest number of gene expression differences. Despite a mortality of 40­60%, no signature was detectable in any of the transferred groups that would account for the embryos' fates. Initially reputed to be beneficial in producing more blastocysts, our culture system of B2 medium plus serum and co-culture generated blastocysts that were distinct from those developed in vivo (D7).

Expression, Localization, and Activity of the Aryl Hydrocarbon Receptor in the Human Placenta.

The human placenta is an organ between the blood of the mother and the fetus, which is essential for fetal development. It also plays a role as a selective barrier against environmental pollutants that may bypass epithelial barriers and reach the placenta, with implications for the outcome of pregnancy. The aryl hydrocarbon receptor (AhR) is one of the most important environmental-sensor transcription factors and mediates the metabolism of a wide variety of xenobiotics. Nevertheless, the identification of dietary and endogenous ligands of AhR suggest that it may also fulfil physiological functions with which pollutants may interfere. Placental AhR expression and activity is largely unknown. We established the cartography of AhR expression at transcript and protein levels, its cellular distribution, and its transcriptional activity toward the expression of its main target genes. We studied the profile of AhR expression and activity during different pregnancy periods, during trophoblasts differentiation in vitro, and in a trophoblast cell line. Using diverse methods, such as cell fractionation and immunofluorescence microscopy, we found a constitutive nuclear localization of AhR in every placental model, in the absence of any voluntarily-added exogenous activator. Our data suggest an intrinsic activation of AhR due to the presence of endogenous placental ligands.

Use of GATA3 and TWIST1 Immunofluorescence Staining to Assess In Vitro Syncytial Fusion Index.

In human placenta, the multinucleated syncytiotrophoblast (ST) allows all the exchanges between the maternal and fetal circulation and is also the site of placental hormonal functions. Absence or disturbances of ST formation are associated with a defect or pathologies of pregnancy such as preeclampsia (PE) and intrauterine growth retardation (IUGR). All along pregnancy, the ST is regenerated by fusion of underlying mononucleated villous cytotrophoblasts (VCT). The protocol described here provides details on how GATA3 or TWIST1 immunostaining and analysis can be used to easily assess the in vitro differentiation of human placental cytotrophoblast.

Fetal-sex determination of human placental tissues.

It is now demonstrated that the sex-specific maternal-placental-fetal interaction plays an important role in placental functions and pathologies. Determination of fetal-sex may therefore be an important consideration in studies using placenta samples. In this present study, we describe a simple, fast, and cheap protocol, which allows the fetal-sex determination of placental tissues from various starting materials (villi or formalin-fixed, paraffin-embedded (FFPE) tissues, isolated cytotrophoblasts or cellular debris from whole cell lysates, and cDNA) by a single duplex PCR reaction followed by agarose gel electrophoresis.