EP300 facilitates human trophoblast stem cell differentiation.

Early placenta development involves cytotrophoblast differentiation into extravillous trophoblast (EVT) and syncytiotrophoblast (STB). Defective trophoblast development and function may result in severe pregnancy complications, including fetal growth restriction and pre-eclampsia. The incidence of these complications is increased in pregnancies of fetuses affected by Rubinstein-Taybi syndrome, a developmental disorder predominantly caused by heterozygous mutations in CREB-binding protein (CREBBP) or E1A-binding protein p300 (EP300). Although the acetyltransferases CREBBP and EP300 are paralogs with many overlapping functions, the increased incidence of pregnancy complications is specific for EP300 mutations. We hypothesized that these complications have their origin in early placentation and that EP300 is involved in that process. Therefore, we investigated the role of EP300 and CREBBP in trophoblast differentiation, using human trophoblast stem cells (TSCs) and trophoblast organoids. We found that pharmacological CREBBP/EP300 inhibition blocks differentiation of TSCs into both EVT and STB lineages, and results in an expansion of TSC-like cells under differentiation-inducing conditions. Specific targeting by RNA interference or CRISPR/Cas9-mediated mutagenesis demonstrated that knockdown of EP300 but not CREBBP, inhibits trophoblast differentiation, consistent with the complications seen in Rubinstein-Taybi syndrome pregnancies. By transcriptome sequencing, we identified transforming growth factor alpha (TGFA, encoding TGF-α) as being strongly upregulated upon EP300 knockdown. Moreover, supplementing differentiation medium with TGF-α, which is a ligand for the epidermal growth factor receptor (EGFR), likewise affected trophoblast differentiation and resulted in increased TSC-like cell proliferation. These findings suggest that EP300 facilitates trophoblast differentiation by interfering with at least EGFR signaling, pointing towards a crucial role for EP300 in early human placentation.

Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching.

Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing complex proteins PCBP1, PCBP2, and YBX1 to LINC-HELLP. In this study, by using both in vitro and ex vivo experiments, we investigate if these proteins are involved in the regulation of EVT invasion during placentation. Additionally, we study if this regulation is due to alternative mRNA splicing. HTR-8/SVneo extravillous trophoblasts and human first trimester placental explants were used to investigate the effect of siRNA-mediated downregulation of PCBP1, PCBP2, and YBX1 genes on the differentiation of EVTs. Transwell invasion assays and proliferation assays indicated that upon knockdown of PCBP2 and, to a lesser extent, YBX1 and PCBP1, EVTs fail to differentiate toward an invasive phenotype. The same pattern was observed in placental explants where PCBP2 knockdown led to approximately 80% reduction in the number of explants showing any EVT outgrowth. Of the ones that still did show EVT outgrowth, the percentage of proliferating EVTs was significantly higher compared to explants transfected with non-targeting control siRNAs. To further investigate this effect of PCBP2 silencing on EVTs, we performed whole transcriptome sequencing (RNA-seq) on HTR-8/SVneo cells after PCBP2 knockdown. PCBP2 knockdown was found to have minimal effect on mRNA expression levels. In contrast, PCBP2 silencing led to a switch in splicing for a large number of genes with predominant functions in cellular assembly and organization, cellular function and maintenance, and cellular growth and proliferation and the cell cycle. EVTs, upon differentiation, alter their function to be able to invade the decidua of the mother by changing their cellular assembly and their proliferative activity by exiting the cell cycle. PCBP2 appears to be a paramount regulator of these differentiation mechanisms, where its disturbed binding to LINC-HELLP could contribute to dysfunctional placental development as seen in the HELLP syndrome.

Circular RNA Sequencing of Maternal Platelets: A Novel Tool for the Identification of Pregnancy-Specific Biomarkers.

BACKGROUND: In the first trimester of pregnancy, the maternal platelet is directly involved in a positive feedback mechanism that facilitates invasion of the extravillous trophoblast into the maternal spiral arteries. Dysfunctional trophoblast invasion with defective deep placentation is primordial in the etiology of the "great obstetrical syndromes." METHODS: In this proof-of-concept study, using transcriptome analysis of circular RNA (circRNA) following RNA sequencing of maternal platelets, we tested whether pregnancy-specific circRNA markers could be identified in the first trimester of normal pregnancies. Differential transcript expression analysis of circRNAs, as predicted by Accurate CircRNA Finder Suite, CircRNA Identifier (version 2), and Known and Novel Isoform Explorer, was done using thromboSeq.R with variation of multiple settings. Test performance was checked for (a) de novo circRNA identification using the novel platelet-specific Plt-circR4 as a positive control, (b) complete segregation of groups (pregnant vs nonpregnant) after heat map-dendrogram clustering, (c) identification of pregnancy-specific circRNA markers at a false discovery rate (FDR) <0.05, and (d) confirmation of differentially expressed circRNA markers with an FDR <0.05 by an independent method, reverse transcription-quantitative PCR. RESULTS: Of the differentially expressed circRNAs with P values <0.05, 41 circRNAs were upregulated (logFC >2), and 52 circRNAs were downregulated (logFC less than -2) in first-trimester platelet RNA. Of these, nuclear receptor-interacting protein 1 circRNA covering exons 2 and 3 of the 5'-untranslated region was pregnancy specific with upregulation in first-trimester maternal platelets compared to nonpregnant controls. CONCLUSION: CircRNA sequencing of first-trimester maternal platelets permits the identification of novel pregnancy-specific RNA biomarkers. Future use could include the assessment of maternal and fetal well-being.

The bivariate NRIP1/ZEB2 RNA marker permits non-invasive presymptomatic screening of pre-eclampsia.

Using genome-wide transcriptome analysis by RNA sequencing of first trimester plasma RNA, we tested whether the identification of pregnancies at risk of developing pre-eclampsia with or without preterm birth or growth restriction is possible between weeks 9-14, prior to the appearance of clinical symptoms. We implemented a metaheuristic approach in the self-learning SVM algorithm for differential gene expression analysis of normal pregnancies (n = 108), affected pregnancies (n = 34) and non-pregnant controls (n = 19). Presymptomatic candidate markers for affected pregnancies were validated by RT-qPCR in first trimester samples (n = 34) from an independent cohort. PRKG1 was significantly downregulated in a subset of pregnancies with birth weights below the 10thpercentile as shared symptom. The NRIP1/ZEB2 ratio was found to be upregulated in pregnancies with pre-eclampsia or trisomy 21. Complementary quantitative analysis of both the linear and circular forms of NRIP1 permitted discrimination between pre-eclampsia and trisomy 21. Pre-eclamptic pregnancies showed an increase in linear NRIP1 compared to circular NRIP1, while trisomy 21 pregnancies did not.

High-quality human preimplantation embryos stimulate endometrial stromal cell migration via secretion of microRNA hsa-miR-320a.

STUDY QUESTION: How do high-quality human preimplantation embryos influence the endometrium to promote their own implantation? SUMMARY ANSWER: High-quality human preimplantation embryos secrete a specific microRNA (miRNA), hsa-miR-320a, which promotes migration of human endometrial stromal cells (hESCs). WHAT IS KNOWN ALREADY: We have previously shown that high-quality human preimplantation embryos excrete unknown factors that influence migration of hESCs. STUDY DESIGN, SIZE, DURATION: Embryo excreted miRNAs, specifically those excreted by high-quality embryos, were identified and their effect on hESCs was determined by measuring the migration capacity and gene expression patterns of primary isolated hESCs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryo conditioned medium (ECM) from routine ICSI procedures was used to identify embryo excreted miRNAs. miRNome analyses were performed on ECM from individually cultured embryos with high morphological quality, with low morphological quality or empty control medium. MiRNA mimics and inhibitors were then used to further study the effect of miRNAs of interest on migration and gene expression of hESCs. Migration assays were performed using hESCs that were obtained from endometrial biopsies performed on hysterectomy specimens from women that received surgery for spotting due to a niche in a cesarean section scar. MAIN RESULTS AND THE ROLE OF CHANCE: By using miRNA mimics and inhibitors, we showed that hsa-miR-320a alone can stimulate migration of decidualized hESCs, accurately resembling the response typically triggered only by high-quality embryos. Transcriptome analysis further demonstrated that this effect is very likely mediated via altered expression of genes involved in cell adhesion and cytoskeleton organization. LIMITATIONS, REASONS FOR CAUTION: The effect of hsa-miR-320a on hESCs was measured in vitro. Further studies on the in vivo effect of hsa-miR-320a are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Implantation failure is one of the major success limiting factors in human reproduction. By secreting hsa-miR-320a, high-quality human preimplantation embryos directly influence hESCs, most likely to prime the endometrium at the implantation site for successful implantation. Together, our results indicate that hsa-miR-320a may be a promising target to further increase success rates in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam University Medical Centers and the Amsterdam Reproduction & Development Research Institute. R.P.B., G.H. and S.M. have a patent on the use of hsa-miR-320a in assisted reproduction treatments pending. TRIAL REGISTRATION NUMBER: N/A.

The idiopathic preterm delivery methylation profile in umbilical cord blood DNA.

BACKGROUND: Preterm delivery is the leading cause of neonatal morbidity and mortality. Two-thirds of preterm deliveries are idiopathic. The initiating molecular mechanisms behind spontaneous preterm delivery are unclear. Umbilical cord blood DNA samples are an easy source of material to study the neonatal state at birth. DNA methylation changes can be exploited as markers to identify spontaneous preterm delivery. To identify methylation differences specific to idiopathic preterm delivery, we assessed genome-wide DNA methylation changes in 24 umbilical cord blood samples (UCB) using the 450 K Illumina methylation array. After quality control, conclusions were based on 11 term and 11 idiopathic preterm born neonates. The differentially methylated positions (DMPs) specific for preterm/term delivery, neonatal sex, use of oxytocin and mode of initiation of labor were calculated by controlling the FDR p value at 0.05. RESULTS: The analysis identifies 1855 statistically significant DMPs between preterm and term deliveries of which 508 DMPs are also attributable to clinical variables other than preterm versus term delivery. 1347 DMPs are unique to term vs preterm delivery, of which 196 DMPs do not relate to gestational age as such. Pathway analysis indicated enrichment of genes involved in calcium signalling, myometrial contraction and relaxation pathways. The 1151 DMPs that correlate with advancing gestational age (p < 0.05) include 161 DMPs that match with two previously reported studies on UCB methylation. Additionally, 123 neonatal sex specific DMPs, 97 DMPs specific to the induction of labour and 42 DMPs specific to the mode of initiation of labor were also identified. CONCLUSION: This study identifies 196 DMPs in UCB DNA of neonates which do not relate to gestational age or any other clinical variable recorded and are specific to idiopathic preterm delivery. Furthermore, 161 DMPs from our study overlap with previously reported studies of which a subset is also reported to be differentially methylated at 18 years of age. A DMP on MYL4, encoding myosin light chain 4, is a robust candidate for the identification of idiopathic preterm labour as it is identified by all 3 independent studies.

Total bile acids in the maternal and fetal compartment in relation to placental ABCG2 expression in preeclamptic pregnancies complicated by HELLP syndrome.

OBJECTIVE: To investigate total bile acid (TBA) levels in maternal (MB) and umbilical cord blood (UCB) in normotensive, preeclamptic (PE), and PE pregnancies complicated by hemolysis elevated liver enzymes and low platelets (HELLP) syndrome in the context of ABCG2 placental gene expression levels, a recently reported placental bile acid transporter. METHODS: TBA levels were determined in 83 paired MB and UCB samples of normotensive, PE and PE/HELLP pregnancies and in 22 paired arterial and venous UCB samples from uncomplicated term pregnancies. ABCG2 gene expression was measured in 104 human placentas by reverse transcriptase quantitative polymerase chain reaction. RESULTS: Overall, TBA levels in MB are higher compared to levels in UCB (p<0.0001), but this comparison looses statistical significance for the 11 PE/HELLP cases. TBA levels in maternal blood are increased in PE/HELLP compared to PE pregnancies (p=0.016). TBA levels in arterial and venous UCB from 22 normotensive pregnancies are not statistically different. ABCG2 expression is reduced in pregnancies where preeclampsia is further complicated by HELLP syndrome. ABCG2 expression in human placenta is not correlated with TBA levels in either the maternal or fetal compartment. CONCLUSION: Increased maternal TBA levels in PE/HELLP pregnancies indicate a relation between bile acids in the maternal circulation and HELLP syndrome. As overall TBA levels in maternal blood are increased compared to UCB, we conclude that the placenta partly protects the fetus from increased maternal TBA levels. This consistent difference in TBA levels between the maternal and fetal compartment is unrelated to the placental expression of ABCG2.

Resistance artery creatine kinase mRNA and blood pressure in humans.

Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

Expression of placental FLT1 transcript variants relates to both gestational hypertensive disease and fetal growth.

The recent discovery of additional alternative spliced FLT1 transcripts encoding novel soluble (s)FLT1 protein isoforms complicates both the predictive value and functional implications of sFLT1 in preeclampsia. We investigated FLT1 expression levels and splicing patterns in placentas of normotensive and preeclamptic women, and established the tissue specificity of all FLT1 transcript variants. mRNA levels of sFLT1 splice variants were determined by real-time polymerase chain reaction in 21 normal human tissues and placental biopsies from 91 normotensive and 55 preeclamptic women. Cellular localization of placental FLT1 expression was established by RNA in situ hybridization. Of all tissues investigated, placenta has by far the highest FLT1 mRNA expression level, mainly localized in the syncytiotrophoblast layer. More than 80% of placental transcripts correspond to sFLT1_v2. Compared with normotensive placenta, preeclamptic placenta has ≈3-fold higher expression of all FLT1 transcript variants (P<0.001), with a slight shift in favor of sFLT1_v1. Although to a lesser degree, transcript levels are also increased in placenta from normotensive women that deliver a small for gestational age neonate. We conclude that sFLT isoform-specific assays could potentially improve the accuracy of current sFLT1 assays for the prediction of preeclampsia. However, placental FLT1 transcript levels are increased not only in preeclampsia but also in normotensive pregnancy with a small for gestational age fetus. This may indicate a common pathway involved in the development of both conditions but complicates the use of circulating sFLT1 protein levels for the prediction or diagnosis of preeclampsia alone.

Initial characterization of C16orf89, a novel thyroid-specific gene.

BACKGROUND: Thyroid hormone is prerequisite for proper fetal and postnatal neurodevelopment, growth, and metabolism. Although much progress has been made in the characterization of genes implicated in thyroid development and function, the majority of genes involved in this process are still unknown. We have previously applied serial analysis of gene expression (SAGE) to identify novel genes preferentially expressed in the thyroid, and this has resulted in the characterization of DUOX2 and IYD (also known as DEHAL1), two genes encoding essential enzymes in the production of thyroid hormone. In the current study we characterize the gene C16orf89, which is linked to another thyroid-specific SAGE tag CCAGCTGCCT. METHODS: We establish tissue-specific expression of C16orf89 using novel tissue-specific SAGE libraries and quantitative polymerase chain reaction. In addition, we characterize the C16orf89 gene and protein, and analyze its mRNA expression in response to thyrotropin and during mouse development. RESULTS: C16orf89 is predominantly expressed in human thyroid tissue with a specificity intermediate between thyroid transcription factors and proteins involved in thyroid hormone synthesis. C16orf89 shows the same expression pattern as Nkx2-1 (thyroid transcription factor 1) from embryonic day (E) 17.5 onward in the developing mouse thyroid and lung. The developmental timing of C16orf89 mRNA expression is similar to that of the iodide transporter Slc5a5 (also known as Nis). Both transcripts are detected from E17.5 in the developing thyroid. This is clearly later than the onset of Tg mRNA expression (from E14.5), while Nkx2-1 and Iyd mRNA can already be detected in the E12.5 thyroid. In in vitro cell culture C16orf89 expression is stimulated by thyrotropin. The major splice variant encodes a 361 amino acid protein that is well conserved between mammals, contains an N-terminal signal peptide, is secreted in a glycosylated form, and does not contain any known functional domain. CONCLUSIONS: We present a novel gene highly expressed in thyroid that encodes a currently enigmatic protein.

Regulation of alpha1,3galactosyltransferase expression in pig endothelial cells. Implications for xenotransplantation.

The disaccharide galactose(alpha)1,3 galactose (the alphaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the alphaGal epitope is catalyzed by the enzyme alpha1,3-galactosyltransferase (alpha1,3GalT). To be able to control porcine alpha1,3GalT gene expression specifically, we have analyzed the upstream portion of the alpha1,3GalT gene, and identified the regulatory sequences. Porcine alpha1,3GalT transcripts were detected by 5' RACE analysis, and the corresponding genomic sequences were isolated from a phage library. The porcine alpha1,3GalT gene consists of at least 10 different exons, four of which contain 5' untranslated sequence. Four distinct promoters, termed A-D, drive alpha1,3GalT gene transcription in porcine cells. A combination of alternative promoter usage and alternative splicing produces a series of transcripts that differ in their 5' portion, but encode the same protein. Promoters A-C have been isolated, and functionally characterized using luciferase reporter gene assays in transfected porcine endothelial cells (PEC-A). Promoter preference in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quantitative PCR. More than 90% of the alpha1,3GalT transcripts in PEC-A cells originate from promoter B, which has characteristics of a housekeeping gene promoter. While promoter preference remains unchanged, alpha1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor alpha-activation of PEC-A cells. However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface alpha1,3-galactosylation.