Comparative transcriptomic analysis of human placentae at term and preterm delivery.

Preterm birth affects 1 out of every 10 infants in the United States, resulting in substantial neonatal morbidity and mortality. Currently, there are few predictive markers and few treatment options to prevent preterm birth. A healthy, functioning placenta is essential to positive pregnancy outcomes. Previous studies have suggested that placental pathology may play a role in preterm birth etiology. Therefore, we tested the hypothesis that preterm placentae may exhibit unique transcriptomic signatures compared to term samples reflective of their abnormal biology leading to this adverse outcome. We aggregated publicly available placental villous microarray data to generate a preterm and term sample dataset (n = 133, 55 preterm placentae and 78 normal term placentae). We identified differentially expressed genes using the linear regression for microarray (LIMMA) package and identified perturbations in known biological networks using Differential Rank Conservation (DIRAC). We identified 129 significantly differentially expressed genes between term and preterm placenta with 96 genes upregulated and 33 genes downregulated (P-value <0.05). Significant changes in gene expression in molecular networks related to Tumor Protein 53 and phosphatidylinositol signaling were identified using DIRAC. We have aggregated a uniformly normalized transcriptomic dataset and have identified novel and established genes and pathways associated with developmental regulation of the placenta and potential preterm birth pathology. These analyses provide a community resource to integrate with other high-dimensional datasets for additional insights in normal placental development and its disruption.

The CSN/COP9 signalosome regulates synaptonemal complex assembly during meiotic prophase I of Caenorhabditis elegans.

The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.

A genome scale transcriptional regulatory model of the human placenta.

Gene regulation is essential to placental function and fetal development. We built a genome-scale transcriptional regulatory network (TRN) of the human placenta using digital genomic footprinting and transcriptomic data. We integrated 475 transcriptomes and 12 DNase hypersensitivity datasets from placental samples to globally and quantitatively map transcription factor (TF)-target gene interactions. In an independent dataset, the TRN model predicted target gene expression with an out-of-sample R2 greater than 0.25 for 73% of target genes. We performed siRNA knockdowns of four TFs and achieved concordance between the predicted gene targets in our TRN and differences in expression of knockdowns with an accuracy of >0.7 for three of the four TFs. Our final model contained 113,158 interactions across 391 TFs and 7712 target genes and is publicly available. We identified 29 TFs which were significantly enriched as regulators for genes previously associated with preterm birth, and eight of these TFs were decreased in preterm placentas.

HAND1 knockdown disrupts trophoblast global gene expression.

Congenital heart disease (CHD) affects nearly 1% of births annually, and CHD pregnancies carry increased risk of developing pathologies of abnormal placentation. We previously reported significant developmental impacts of disrupting Hand1, a gene associated with CHD, expression in placenta trophoblast and endothelial cells in multiple mouse models. In this study, we aimed to build upon this knowledge and characterize the mechanistic impacts of disrupting HAND1 on human placenta trophoblast and vascular endothelial cell gene expression. HAND1 gene expression was silenced in BeWo cells, a choriocarcinoma model of human cytotrophoblasts, (n = 3-9 passages) and isolated human placental microvascular endothelial cells (HPMVEC; n = 3 passages), with HAND1 siRNA for 96 h. Cells were harvested, mRNA isolated and RNA sequencing performed using the Illumina NextSeq 550 platform. Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR packages. Statistical significance was determined using a log2 fold change of >1.0 or < -1.0 and unadjusted p-value ≤0.05. Panther DB was used for overrepresentation analysis, and String DB for protein association network analysis. There was downregulation of 664 genes, and upregulation of 59 genes in BeWo cells with direct HAND1 knockdown. Overrepresentation analysis identified disruption to pathways including cell differentiation, localization, and cell projection organization. In contrast, only seven genes were changed with direct HAND1 knockdown in HPMVECs. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not endothelial cells. This data provides further evidence that future studies on genetic perturbations in CHDs should consider the extra-embryonic tissue in addition to the fetal heart.

Characterization of methylation profiles in spontaneous preterm birth placental villous tissue.

Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls where the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples display a hypermature methylation signature than expected given their respective gestational age which likely impacts birth timing.

Using high-density exon arrays to profile gene expression in closely related species.

Global comparisons of gene expression profiles between species provide significant insight into gene regulation, evolutionary processes and disease mechanisms. In this work, we describe a flexible and intuitive approach for global expression profiling of closely related species, using high-density exon arrays designed for a single reference genome. The high-density probe coverage of exon arrays allows us to select identical sets of perfect-match probes to measure expression levels of orthologous genes. This eliminates a serious confounding factor in probe affinity effects of species-specific microarray probes, and enables direct comparisons of estimated expression indexes across species. Using a newly designed Affymetrix exon array, with eight probes per exon for approximately 315,000 exons in the human genome, we conducted expression profiling in corresponding tissues from humans, chimpanzees and rhesus macaques. Quantitative real-time PCR analysis of differentially expressed candidate genes is highly concordant with microarray data, yielding a validation rate of 21/22 for human versus chimpanzee differences, and 11/11 for human versus rhesus differences. This method has the potential to greatly facilitate biomedical and evolutionary studies of gene expression in nonhuman primates and can be easily extended to expression array design and comparative analysis of other animals and plants.

Abnormalities of placental development and function are associated with the different fetal growth patterns of hypoplastic left heart syndrome and transposition of the great arteries.

BACKGROUND: Birthweight is a critical predictor of congenital heart disease (CHD) surgical outcomes. Hypoplastic left heart syndrome (HLHS) is cyanotic CHD with known fetal growth restriction and placental abnormalities. Transposition of the great arteries (TGA) is cyanotic CHD with normal fetal growth. Comparison of the placenta in these diagnoses may provide insights on the fetal growth abnormality of CHD. METHODS: Clinical data and placental histology from placentas associated with Transposition of the Great Arteries (TGA) were analyzed for gross pathology, morphology, maturity and vascularity and compared to both control and previously analyzed HLHS placentas [1]. RNA was isolated from HLHS, TGA and control placentas and sequenced by Illumina HiSeq.Transcriptome analysis was performed using AltAnalyze. Immunohistochemistry was utilized to assess placental nutrient transporter expression in all three groups. RESULTS: Placental weight was reduced in TGA cases, and demonstrated reduced villous vasculature, immature terminal villi in the parenchyma compared to controls and reflected our previous data from HLHS placentas. However, birth weight was not reduced in TGA cases compared to controls in contrast to the HLHS cohort and birthweight:placental weight ratio was significantly increased in TGA cases but not HLHS compared to control. Transcriptomic and histologic analysis demonstrates reduced cell activity and nutrient transport capability in HLHS but not TGA placentas which appear to increase/maintain these mechanisms. CONCLUSIONS: Despite common vascular disturbances in placentas from HLHAs and TGA, these do not account for the disparities in birthweights frequently seen between these CHD subtypes, in contrast our transcriptomic and histologic analyses reveal differentially regulated mechanisms between the subtypes that may explain these disparities.

Unique transcriptomic landscapes identified in idiopathic spontaneous and infection related preterm births compared to normal term births.

Preterm birth (PTB) is leading contributor to infant death in the United States and globally, yet the underlying mechanistic causes are not well understood. Histopathological studies of preterm birth suggest advanced villous maturity may have a role in idiopathic spontaneous preterm birth (isPTB). To better understand pathological and molecular basis of isPTB, we compared placental villous transcriptomes from carefully phenotyped cohorts of PTB due to infection or isPTB between 28-36 weeks gestation and healthy term placentas. Transcriptomic analyses revealed a unique expression signature for isPTB distinct from the age-matched controls that were delivered prematurely due to infection. This signature included the upregulation of three IGF binding proteins (IGFBP1, IGFBP2, and IGFBP6), supporting a role for aberrant IGF signaling in isPTB. However, within the isPTB expression signature, we detected secondary signature of inflammatory markers including TNC, C3, CFH, and C1R, which have been associated with placental maturity. In contrast, the expression signature of the gestational age-matched infected samples included upregulation of proliferative genes along with cell cycling and mitosis pathways. Together, these data suggest an isPTB molecular signature of placental hypermaturity, likely contributing to the premature activation of inflammatory pathways associated with birth and providing a molecular basis for idiopathic spontaneous birth.

The genetics of preterm birth: Progress and promise.

Preterm birth is the single leading cause of mortality for neonates and children less than 5 years of age. Compared to other childhood diseases, such as infections, less progress in prevention of prematurity has been made. In large part, the continued high burden of prematurity results from the limited understanding of the mechanisms controlling normal birth timing in humans, and how individual genetic variation and environmental exposures disrupt these mechanisms to cause preterm birth. In this review, we summarize the outcomes and limitations from studies in model organisms for birth timing in humans, the evidence that genetic factors contribute to birth timing and risk for preterm birth, and recent genetic and genomic studies in women and infants that implicate specific genes and pathways. We conclude with discussing areas of potential high impact in understanding human parturition and preterm birth in the future.

Qualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography.

Detecting and quantifying generalized mitochondrial heteroplasmy is essential if the field of mitochondrial genetics is to advance in the arena of complex genetic disorders. The majority of techniques used to detect and quantify mitochondrial heteroplasmy focus on a known mutation or polymorphism. The necessity of knowing the mitochondrial DNA (mtDNA) change beforehand means that non-specific heteroplasmy in general cannot be assessed. In this study, we assessed the extent that denaturing high-performance liquid chromatography (dHPLC) could detect and quantify mitochondrial heteroplasmy from cerebrospinal fluid (CSF). Although we used a known polymorphism to assess reliability and sensitivity of this technique, a distinct advantage to using dHPLC for heteroplasmy detection is that the entire fragment is screened for variability and any unique fragments will be detected regardless of the placement or type of change. Our results demonstrate that dHPLC can consistently and reliably detect mitochondrial heteroplasmy in a CSF sample down to 0.01%. In addition, the level of heteroplasmy was consistent with peak height for each homoduplex, giving a reliable method to quantify level of heteroplasmy.

akirin is required for diakinesis bivalent structure and synaptonemal complex disassembly at meiotic prophase I.

During meiosis, evolutionarily conserved mechanisms regulate chromosome remodeling, leading to the formation of a tight bivalent structure. This bivalent, a linked pair of homologous chromosomes, is essential for proper chromosome segregation in meiosis. The formation of a tight bivalent involves chromosome condensation and restructuring around the crossover. The synaptonemal complex (SC), which mediates homologous chromosome association before crossover formation, disassembles concurrently with increased condensation during bivalent remodeling. Both chromosome condensation and SC disassembly are likely critical steps in acquiring functional bivalent structure. The mechanisms controlling SC disassembly, however, remain unclear. Here we identify akir-1 as a gene involved in key events of meiotic prophase I in Caenorhabditis elegans. AKIR-1 is a protein conserved among metazoans that lacks any previously known function in meiosis. We show that akir-1 mutants exhibit severe meiotic defects in late prophase I, including improper disassembly of the SC and aberrant chromosome condensation, independently of the condensin complexes. These late-prophase defects then lead to aberrant reconfiguring of the bivalent. The meiotic divisions are delayed in akir-1 mutants and are accompanied by lagging chromosomes. Our analysis therefore provides evidence for an important role of proper SC disassembly in configuring a functional bivalent structure.