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BackgroundMicroRNAs (miRNAs) are increasingly seen as important regulators of placental development and opportunistic biomarker targets. Given the difficulty in obtaining samples from early gestation and subsequent paucity of the same, investigation of the role of miRNAs in early gestation human placenta has been limited. To address this, we generated miRNA profiles using 96 placentas from presumed normal pregnancies, across early gestation, in combination with matched profiles from maternal plasma. Placenta samples range from 6–23 weeks’ gestation, a time period that includes placenta from the early, relatively low but physiological (6–10 weeks’ gestation) oxygen environment, and later, physiologically normal oxygen environment (11–23 weeks’ gestation).ResultsWe identified 637 miRNAs with expression in 86 samples (after removing poor quality samples), showing a clear gestational age gradient from 6–23 weeks’ gestation. We identified 374 differentially expressed (DE) miRNAs between placentas from 6–10 weeks’ versus 11–23 weeks’ gestation. We see a clear gestational age group bias in miRNA clusters C19MC, C14MC, miR-17∼92 and paralogs, regions that also include many DE miRNAs. Proportional change in expression of placenta-specific miRNA clusters was reflected in maternal plasma.ConclusionThe presumed introduction of oxygenated maternal blood into the placenta (between ∼10–12 weeks’ gestation) changes the miRNA profile of the chorionic villus, particularly in placenta-specific miRNA clusters. Data presented here comprise a clinically important reference set for studying early placenta development and may underpin the generation of minimally invasive methods for monitoring placental health.

Refereed/Peer-reviewed The search for novel microRNA (miRNA) biomarkers in plasma is hampered by haemolysis, the lysis and subsequent release of red blood cell contents, including miRNAs, into surrounding fluid. The biomarker potential of miRNAs comes in part from their multicompartment origin and the long-lived nature of miRNA transcripts in plasma, giving researchers a functional window for tissues that are otherwise difficult or disadvantageous to sample. The inclusion of red-blood-cell-derived miRNA transcripts in downstream analysis introduces a source of error that is difficult to identify posthoc and may lead to spurious results. Where access to a physical specimen is not possible, our tool will provide an in silico approach to haemolysis prediction. We present DraculR, an interactive Shiny/R application that enables a user to upload miRNA expression data from a short-read sequencing of human plasma as a raw read counts table and interactively calculate a metric that indicates the degree of haemolysis contamination. The code, DraculR web tool and its tutorial are freely available as detailed herein.

The abundance of cell-free microRNA (miRNA) has been measured in many body fluids, including blood plasma, which has been proposed as a source with novel, minimally invasive biomarker potential for several diseases. Despite improvements in quantification methods for plasma miRNAs, there is no consensus on optimal reference miRNAs or to what extent haemolysis may affect plasma miRNA content. Here we propose a new method for the detection of haemolysis in miRNA high-throughput sequencing (HTS) data from libraries prepared using human plasma. To establish a miRNA haemolysis signature in plasma we first identified differentially expressed miRNAs between samples with known haemolysis status and selected miRNA with statistically significant higher abundance in our haemolysed group. Given there may be both technical and biological reasons for differential abundance of signature miRNA, and to ensure the method developed here was relevant outside of our specific context, that is women of reproductive age, we tested for significant differences between pregnant and non-pregnant groups. Here we report a novel 20 miRNA signature (miR-106b-3p, miR-140-3p, miR-142-5p, miR-532-5p, miR-17-5p, miR-19b-3p, miR-30c-5p, miR-324-5p, miR-192-5p, miR-660-5p, miR-186-5p, miR-425-5p, miR-25-3p, miR-363-3p, miR-183-5p, miR-451a, miR-182-5p, miR-191-5p, miR-194-5p, miR-20b-5p) that can be used to identify the presence of haemolysis, in silico, in high throughput miRNA sequencing data. Given the potential for haemolysis contamination, we recommend that assay for haemolysis detection become standard pre-analytical practice and provide here a simple method for haemolysis detection.

Vitamin D and calcium are essential micronutrients for reproductive success. Vitamin D deficiency during pregnancy is associated with increased risk of pregnancy complications including pre-eclampsia and preterm birth (PTB). However, inconsistencies in the literature reflect uncertainties regarding the true biological importance of vitamin D but may be explained by maternal calcium intakes. We aimed to determine whether low dietary consumption of calcium along with vitamin D deficiency had an additive effect on adverse pregnancy outcome by investigating placental morphogenesis and foetal growth in a mouse model.Female mice were randomly assigned to one of four diets: control-fed (+Ca+VD), reduced vitamin D only (+Ca−VD), reduced calcium only (−Ca+VD) and reduced calcium and vitamin D (−Ca−VD), and sacrificed at gestational day (GD) 18.5. Maternal serum 25-hydroxy vitamin D (25(OH)D3) levels were lower in each reduced diet group when compared with levels in +Ca+VD fed mice. While the pregnancy rate did not differ between groups, in the −Ca−VD-fed group, 55% (5 out of 9 pregnant of known gestational age) gave birth preterm (

De novo and inherited mutations of X-chromosome cell adhesionmolecule protocadherin 19 (PCDH19) cause frequent, highly variable epilepsy, autism, cognitive decline and behavioural problems syndrome. Intriguingly, hemizygous nullmales are not affected while heterozygous females are, contradicting established X-chromosome inheritance. The disease mechanism is not known. Cellular mosaicism is the likely driver. We have identified p54nrb/NONO, amultifunctional nuclear paraspeckle protein with known roles in nuclear hormone receptor gene regulation, as a PCDH19 protein interacting partner. Using breast cancer cells we show that PCDH19-NONO complex is a positive co-regulator of ERa-mediated gene expression. Expression of mutant PCDH19 affects at least a subset of known ERα-regulated genes. These data are consistent with our findings that genes regulated by nuclear hormone receptors and those involved in themetabolism of neurosteroids in particular are dysregulated in PCDH19-epilepsy girls and affected mosaicmales. Overall we define and characterize a novelmechanism of gene regulation driven by PCDH19, which is mediated by paraspeckle constituent NONO and is ERα-dependent. This PCDH19-NONO-ERa axis is of relevance not only to PCDH19-epilepsy and its comorbidities but likely also to ERα and generally nuclear hormone receptor-associated cancers. Refereed/Peer-reviewed

DEPDC5 mutations have recently been shown to cause epilepsy in humans. Evidence from in vitro studies has implicated DEPDC5 as a negative regulator of mTORC1 during amino acid insufficiency as part of the GATOR1 complex. To investigate the role of DEPDC5 in vivo we generated a null mouse model using targeted CRISPR mutagenesis. Depdc5 homozygotes display severe phenotypic defects between 12.5-15.5 dpc, including hypotrophy, anaemia, oedema, and cranial dysmorphology as well as blood and lymphatic vascular defects. mTORC1 hyperactivity was observed in the brain of knockout embryos and in fibroblasts and neurospheres isolated from knockout embryos and cultured in nutrient deprived conditions. Heterozygous mice appeared to be normal and we found no evidence of increased susceptibility to seizures or tumorigenesis. Together, these data support mTORC1 hyperactivation as the likely pathogenic mechanism that underpins DEPDC5 loss of function in humans and highlights the potential utility of mTORC1 inhibitors in the treatment of DEPDC5-associated epilepsy.