Katherine Leavey

Unsupervised Placental Gene Expression Profiling Identifies Clinically Relevant Subclasses of Human Preeclampsia

Preeclampsia (PE) is a complex, hypertensive disorder of pregnancy, demonstrating considerable variability in maternal symptoms and fetal outcomes. Unfortunately, prior research has not accounted for this variability, resulting in a lack of robust biomarkers and effective treatments for PE. Here, we created a large (N=330) clinically relevant human placental microarray data set, consisting of 7 previously published studies and 157 highly annotated new samples from a single BioBank. Applying unsupervised clustering to this combined data set identified 3 clinically significant probable etiologies of PE: “maternal”, with healthy placentas and term deliveries; “canonical”, exhibiting expected clinical, ontological, and histopathologic features of PE; and “immunologic” with severe fetal growth restriction and evidence of maternal antifetal rejection. Moreover, these groups could be distinguished using a small quantitative polymerase chain reaction panel and demonstrated varying influence of maternal factors on PE development. An additional subclass of PE placentas was also revealed to form because of chromosomal abnormalities in these samples, supported by array-based comparative genomic hybridization analysis. Overall, our findings represent a new paradigm in our understanding of the origins and maternal–placental contributions to the pathology of PE. The study of PE represents a unique opportunity to access human tissue associated with a complex hypertensive disorder, and our novel approach could be applied to other hypertensive and heterogeneous human diseases.

Large Scale Aggregate Microarray Analysis Reveals Three Distinct Molecular Subclasses of Human Preeclampsia

Background Preeclampsia (PE) is a life-threatening hypertensive pathology of pregnancy affecting 3–5% of all pregnancies. To date, PE has no cure, early detection markers, or effective treatments short of the removal of what is thought to be the causative organ, the placenta, which may necessitate a preterm delivery. Additionally, numerous small placental microarray studies attempting to identify “PE-specific” genes have yielded inconsistent results. We therefore hypothesize that preeclampsia is a multifactorial disease encompassing several pathology subclasses, and that large cohort placental gene expression analysis will reveal these groups. Results To address our hypothesis, we utilized known bioinformatic methods to aggregate 7 microarray data sets across multiple platforms in order to generate a large data set of 173 patient samples, including 77 with preeclampsia. Unsupervised clustering of these patient samples revealed three distinct molecular subclasses of PE. This included a “canonical” PE subclass demonstrating elevated expression of known PE markers and genes associated with poor oxygenation and increased secretion, as well as two other subclasses potentially representing a poor maternal response to pregnancy and an immunological presentation of preeclampsia. Conclusion Our analysis sheds new light on the heterogeneity of PE patients, and offers up additional avenues for future investigation. Hopefully, our subclassification of preeclampsia based on molecular diversity will finally lead to the development of robust diagnostics and patient-based treatments for this disorder.

Rapid Elevation in CMPF May Act As a Tipping Point in Diabetes Development.

Prediabetes, a state of mild glucose intolerance, can persist for years before a sudden decline in beta cell function and rapid deterioration to overt diabetes. The mechanism underlying this tipping point of beta cell dysfunction remains unknown. Here, the furan fatty acid metabolite CMPF was evaluated in a prospective cohort. Those who developed overt diabetes had a significant increase in CMPF over time, whereas prediabetics maintained chronically elevated levels, even up to 5 years before diagnosis. To evaluate the effect of increasing CMPF on diabetes progression, we used obese, insulin-resistant models of prediabetes. CMPF accelerated diabetes development by inducing metabolic remodeling, resulting in preferential utilization of fatty acids over glucose. This was associated with diminished glucose-stimulated insulin secretion, increased ROS formation, and accumulation of proinsulin, all characteristics of human diabetes. Thus, an increase in CMPF may represent the tipping point in diabetes development by accelerating beta cell dysfunction.

Placental transcriptome in development and pathology: expression, function, and methods of analysis.

The placenta is the essential organ of mammalian pregnancy and errors in its development and function are associated with a wide range of human pathologies of pregnancy. Genome sequencing has led to methods for investigation of the transcriptome (all expressed RNA species) using microarrays and next-generation sequencing, and implementation of these techniques has identified many novel species of RNA including: micro-RNA, long noncoding RNA, and circular RNA. These species can physically interact with both each other and regulatory proteins to modify gene expression and messenger RNA to protein translation. Transcriptome analysis is actively used to investigate placental development and dysfunction in pathologies ranging from preeclampsia and fetal growth restriction to preterm labor. Genome-wide gene expression analysis is also being applied to identify prognostic and diagnostic biomarkers of these disorders. In this comprehensive review we summarize transcriptome biology, methods of isolation and analysis, application to placental development and pathology, and use in diagnostic analysis in maternal blood. Key information for analysis methods is organized into quick reference tables where current analysis techniques and tools are cited and compared. We have created this review as a practical guide and starting reference for those interested in beginning an investigation into the transcriptome of the placenta.

Mining DNA methylation alterations towards a classification of placental pathologies

Abstract Placental health is a key component to a successful pregnancy. Placental insufficiency (PI), inadequate nutrient delivery to the fetus, is associated with preeclampsia (PE), a maternal hypertensive disorder, and intrauterine growth restriction (IUGR), pathologically poor fetal growth. PI is more common in early-onset PE (EOPE) than late-onset PE (LOPE). However, the relationship between these disorders remains unclear. While DNA methylation (DNAm) alterations have been identified in PE and IUGR, these entities can overlap and few studies have analysed them separately. This study aims to utilize DNAm profiling to better understand the underlying placental variation associated with PE and IUGR. Placental samples from a discovery (43 controls, 22 EOPE, 18 LOPE, 11 IUGR) and validation cohort (15 controls, 22 EOPE, 11 LOPE) were evaluated using the Illumina HumanMethylation450 array. To account for gestational age (GA) effects, EOPE samples were compared with pre-term births of varying etiologies (GA <37 weeks). LOPE and IUGR were compared with term controls (GA >37 weeks). While 1703 sites were differentially methylated (DM) (FDR < 0.05, Δβ > 0.1) in EOPE, few changes were associated with LOPE (N = 5), or IUGR (N = 0). Of the 1703 EOPE sites, 599 validated in the second cohort. Using these 599 sites, both cohorts clustered into three distinct groups. Interestingly, LOPE samples diagnosed between 34 and 36 weeks with co-occurring IUGR clustered with the EOPE. DNAm profiling may provide an independent tool to refine clinical/pathological diagnoses into subgroups with more uniform pathology. Despite large changes observed in EOPE, there were challenges in reproducing genome-wide DNAm hits that are discussed.

Epigenetic regulation of placental gene expression in transcriptional subtypes of preeclampsia

BackgroundPreeclampsia (PE) is a heterogeneous, hypertensive disorder of pregnancy, with no robust biomarkers or effective treatments. We hypothesized that this heterogeneity is due to the existence of multiple subtypes of PE and, in support of this hypothesis, we recently identified five clusters of placentas within a large gene expression microarray dataset (N = 330), of which four (clusters 1, 2, 3, and 5) contained a substantial number of PE samples. However, while transcriptional analysis of placentas can subtype patients, we propose that the addition of epigenetic information could discern gene regulatory mechanisms behind the distinct PE pathologies, as well as identify clinically useful potential biomarkers.ResultsWe subjected 48 of our samples from transcriptional clusters 1, 2, 3, and 5 to Infinium HumanMethylation450 arrays. Samples belonging to transcriptional clusters 1–3 still showed visible relationships to each other by methylation, but cluster 5, with known chromosomal abnormalities, no longer formed a cohesive group. Within transcriptional clusters 2 and 3, controlling for fetal sex and gestational age in the identification of differentially methylated sites, compared to the healthier cluster 1, dramatically reduced the number of significant sites, but increased the percentage that demonstrated a strong linear correlation with gene expression (from 5% and 2% to 9% and 8%, respectively). Locations exhibiting a positive relationship between methylation and gene expression were most frequently found in CpG open sea enhancer regions within the gene body, while those with a significant negative correlation were often annotated to the promoter in a CpG shore region. Integrated transcriptome and epigenome analysis revealed modifications in TGF-beta signaling, cell adhesion, oxidative phosphorylation, and metabolism pathways in cluster 2 placentas, and aberrations in antigen presentation, allograft rejection, and cytokine-cytokine receptor interaction in cluster 3 samples.ConclusionsOverall, we have established DNA methylation alterations underlying a portion of the transcriptional development of “canonical” PE in cluster 2 and “immunological” PE in cluster 3. However, a significant number of the observed methylation changes were not associated with corresponding changes in gene expression, and vice versa, indicating that alternate methods of gene regulation will need to be explored to fully comprehend these PE subtypes.

The clinical heterogeneity of preeclampsia is related to both placental gene expression and placental histopathology

BACKGROUND: Preeclampsia is a life‐threatening disorder of pregnancy, demonstrating a high degree of heterogeneity in clinical features such as presentation, disease severity, and outcomes. This heterogeneity suggests distinct pathophysiological mechanisms may be driving the placental disease underlying this disorder. Our group recently reported distinct clusters of placental gene expression in preeclampsia and control pregnancies, allowing for the identification of at least 3 clinically relevant gene expression‐based subtypes of preeclampsia. Histopathological examination of a small number of samples from 2 of the gene expression–based subtypes revealed placental lesions consistent with their gene expression phenotype, suggesting that detailed placental histopathology may provide further insight into the pathophysiology underlying these distinct gene expression‐based subtypes. OBJECTIVES: The objective of the study was to assess histopathological lesions in the placentas of patients belonging to each identified gene expression–based subtype of preeclampsia, characterized in our previous study. Our goal was to further understand the pathophysiologies defining these gene expression–based subtypes by integrating gene expression with histopathological findings, possibly identifying additional subgroups of preeclampsia patients. STUDY DESIGN: Paraffin‐embedded placental biopsies from patients included in the gene expression profiling study (n = 142 of 157, 90.4%) were sectioned, hematoxylin and eosin stained, and imaged. An experienced perinatal pathologist, blinded to gene expression findings and clinical information, assessed the presence and severity of histological lesions using a comprehensive, standardized data collection form. The frequency and severity scores of observed histopathological lesions were compared among gene expression–based subtypes as well as within each subtype using using Fisher exact tests, Kruskal‐Wallis tests, and hierarchical clustering. The histological findings of the placental samples were visualized using t‐distributed stochastic neighbor embedding and phylogenetic trees. Concordance and discordance between gene expression findings and histopathology were also investigated and visualized using principal component analysis. RESULTS: Several histological lesions were found to be characteristic of each gene expression–based preeclampsia subtype. The overall concordance between gene expression and histopathology for all samples was 65% (93 of 142), with characteristic placental lesions for each gene expression–based subtype complementing prior gene enrichment findings (ie, placentas with enrichment of hypoxia‐associated genes showed severe lesions of maternal vascular malperfusion). Concordant samples were located in the central area of each gene expression–based cluster when viewed on a principal component analysis plot. Interestingly, discordant samples (gene expression and histopathology not reflective of one another) were generally found to lie at the periphery of the gene expression–based clusters and tended to border the group of patients with phenotypically similar histopathology. CONCLUSION: Our findings demonstrates a high degree of concordance between placental lesions and gene expression across subtypes of preeclampsia. Additionally, novel integrative analysis of scored placental histopathology severity and gene expression findings allowed for the identification of patients with intermediate phenotypes of preeclampsia not apparent through gene expression profiling alone. Future investigations should examine the temporal relationship between these 2 modalities as well as consider the maternal and fetal contributions to these subtypes of disease.

Placental Transcriptional and Histological Subtypes of Normotensive Fetal Growth Restriction are Comparable to Preeclampsia

BACKGROUND: Infants born small for gestational age because of pathologic placenta‐mediated fetal growth restriction can be difficult to distinguish from those who are constitutionally small. Additionally, even among fetal growth–restricted pregnancies with evident placental disease, considerable heterogeneity in clinical outcomes and long‐term consequences has been observed. Gene expression studies of fetal growth–restricted placentas also have limited consistency in their findings, which is likely due to the presence of different molecular subtypes of disease. In our previous study on preeclampsia, another heterogeneous placenta‐centric disorder of pregnancy, we found that, by clustering placentas based only on their gene expression profiles, multiple subtypes of preeclampsia, including several with co‐occurring suspected fetal growth restriction, could be identified. OBJECTIVE: The purpose of this study was to discover placental subtypes of normotensive small‐for‐gestational‐age pregnancies with suspected fetal growth restriction through the use of unsupervised clustering of placental gene expression data and to investigate their relationships with hypertensive suspected fetal growth–restricted placental subtypes. STUDY DESIGN: A new dataset of 20 placentas from normotensive small‐for‐gestational‐age pregnancies (birthweight <10th percentile for gestational age and sex) with suspected fetal growth restriction (ultrasound features of placental insufficiency) underwent genome‐wide messenger RNA expression assessment and blinded detailed histopathologic evaluation. These samples were then combined with a subset of samples from our previously published preeclampsia cohort (n=77) to form an aggregate fetal growth‐focused cohort (n=97) of placentas from normotensive small‐for‐gestational‐age, hypertensive (preeclampsia and chronic hypertensive) small‐for‐gestational‐age, and normotensive average‐for‐gestational‐age pregnancies. Gene expression data were subjected to unsupervised clustering, and clinical and histopathologic features were correlated to the identified sample clusters. RESULTS: Clustering of the aggregate dataset revealed 3 transcriptional subtypes of placentas from normotensive small‐for‐gestational‐age/suspected fetal growth–restricted pregnancies, with differential enrichment of clinical and histopathologic findings. The first subtype exhibited either no placental disease or mild maternal vascular malperfusion lesions, and, co‐clustered with the healthy average‐for‐gestational‐age control subjects; the second subtype showed more severe evidence of hypoxic damage and lesions of maternal vascular malperfusion, and the third subtype demonstrated an immune/inflammatory response and histologic features of a maternal‐fetal interface disturbance. Furthermore, all 3 of these normotensive small‐for‐gestational‐age subtypes co‐clustered with a group of placentas from hypertensive small‐for‐gestational‐age pregnancies with more severe clinical outcomes, but very comparable transcriptional and histologic placental profiles. CONCLUSION: Overall, this study provides evidence for at least 2 pathologic placental causes of normotensive small‐for‐gestational‐age, likely representing true fetal growth restriction. These subtypes also show considerable similarity in gene expression and histopathology to our previously identified “canonical” and “immunologic” preeclampsia placental subtypes. Furthermore, we discovered a subtype of normotensive small‐for‐gestational‐age (with suspected fetal growth restriction) with minimal placental disease that may represent both constitutionally small infants and mild fetal growth restriction, although these cannot be distinguished with the currently available data. Future work that focuses on the identification of etiology‐driven biomarkers and therapeutic interventions for each subtype of fetal growth restriction is warranted.

The value of DNA methylation profiling in characterizing preeclampsia and intrauterine growth restriction

Placental health is a key component to healthy pregnancy. Placental insufficiency (PI), inadequate nutrient delivery to the fetus, is associated with preeclampsia (PE), a maternal hypertensive disorder, and intrauterine growth restriction (IUGR), pathologically poor fetal growth. PI is more common in early-onset PE (EOPE) than late-onset PE (LOPE). However, the relationship between these disorders remains unclear. While DNA methylation (DNAm) alterations have been identified in PE and IUGR, these entities can overlap and few studies have analyzed these separately. This study aims to identify altered DNAm in EOPE, LOPE, and normotensive IUGR, validate these alterations, and use them to better understand the relationships between these related disorders. Placental samples from a discovery cohort (43 controls, 22 EOPE, 18 LOPE, 11 IUGR) and validation cohort (15 controls, 22 EOPE, 11 LOPE) were evaluated using the Illumina HumanMethylation450 array. To minimize gestational age (GA) effects, EOPE samples were compared to pre-term controls (GA <37 weeks), while LOPE and IUGR were compared to term controls (GA >37 weeks). There were 1703 differentially methylated (DM) sites (FDR<0.05, Δβ>0.1) in EOPE, 5 in LOPE, and 0 in IUGR. Of the 1703 EOPE sites, 599 were validated in the second cohort. These sites cluster samples from both cohorts into 3 distinct methylation clusters. Interestingly, LOPE samples diagnosed between 34-36 weeks with co-occurring IUGR clustered with the EOPE methylation cluster. DNAm profiling may provide an independent tool to refine clinical diagnoses into subgroups with more uniform pathology. The challenges in reproducing genome-wide DNAm studies are also discussed.

Associations between imprinted gene expression in the placenta, human fetal growth and preeclampsia

Genomic imprinting is essential for normal placental and fetal growth. One theory to explain the evolution of imprinting is the kinship theory (KT), which predicts that genes that are paternally expressed will promote fetal growth, whereas maternally expressed genes will suppress growth. We investigated the expression of imprinted genes using microarray measurements of expression in term placentae. Correlations between birthweight and the expression levels of imprinted genes were more significant than for non-imprinted genes, but did not tend to be positive for paternally expressed genes and negative for maternally expressed genes. Imprinted genes were more dysregulated in preeclampsia (a disorder associated with placental insufficiency) than randomly selected genes, and we observed an excess of patterns of dysregulation in preeclampsia that would be expected to reduce nutrient allocation to the fetus, given the predictions of the KT. However, we found no evidence of coordinated regulation among these imprinted genes. A few imprinted genes have previously been shown to be associated with fetal growth and preeclampsia, and our results indicate that this is true for a broader set of imprinted genes.