Project description:Maternal obesity alters placental tissue function and morphology with a corresponding increase in local inflammation. We and others showed that placenta size, inflammation and fetal growth are regulated by maternal diet and obesity status. Maternal obesity alters placental DNA methylation which in turn could likely impact gene transcription of of proteins critical for normal fetal development. RNA-binding motif single-stranded interacting protein 1 (RBMS1) is expressed by the placenta and likely modulates DNA replication and transcription regulation. Serum RBMS1 protein concentration is increased with maternal obesity and RBMS1 gene expression in liver tissue is induced by a high-fat diet and inflammation. However, it is not yet known whether placental RBMS1 mRNA expression and DNA methylation are altered by maternal obesity.
Project description:To assess the impact of postnatal processing on placental DNA methylation, array data from flash frozen placental tissue was compared to perfluorocarbon-immersed and formalin fixed paraffin embedded placental tissue. We observed tissue exposed to perfluorocarbon showed no significant DNA methylation differences when compared to unprocessed tissue, while formalin processing altered the quality and reliability of the data produced on the DNA methylation array platform. Placental DNA methylation allows for the study of gene-environment interactions that influence the fetal environment and development. Our study highlights that placental postprocessing techniques must be considered in the evaluation and interpretation of epigenetic studies.
Project description:In this study, we screened human placental samples for allele-specific methylation and subsequently novel imprinted genes associated with these regions. We used reduced representation bisulfite sequencing to identify partially methylated CpG islands (CGIs) in the human placental genome. We were able to delineate potential candidates for allele-specific methylation based on the calculation of a concordance statistic. Amongst the 28 regions chosen for validation based on high levels of expression, two regions were shown to exhibit allele-specific expression. Single base-resolution methylation analysis in the placental genome and RNA-Seq
Project description:Genome wide DNA methylation profiling of normal and pathological placental villous tissue samples. The Illumina EPIC Human DNA methylation Beadchip was used to obtain DNA methylation profiles.
Project description:Banking of high-quality placental tissue specimens will enable biomarker discovery and molecular studies on diseases involving placental dysfunction. Systematic studies aimed at developing feasible standardized methodology for placental collection for genomic analyses are lacking. To determine the acceptable timeframe for placental collection, we collected multiple samples from first and third trimester placentas at serial time points 0-120 minutes after delivery, simultaneously comparing the traditional snap-freeze technique to collection in commercial solutions designed to preserve RNA (RNAlaterTM, Ambion), and DNA (DNAgard®, Biomatrica). The performance of RNAlater for preserving DNA was also tested. Nucleic acid quality was assessed by determining the RNA integrity number (RIN) and genome-wide expression and DNA methylation microarray profiling. We found that samples collected in RNAlater had higher and more consistent RINs compared to snap frozen tissue, with similar RINs obtained for tissue collected in RNAlater as large (1 cm3) and small (~0.1 cm3) tissue pieces. RNAlater appeared to better stabilize the time zero gene expression pattern compared to snap freezing for first trimester placenta. Microarray DNA methylation analysis showed that overall the DNA methylation profiles remained quite stable over a two hour time period after removal of the placenta from the uterus, with the DNAgard condition being superior to both snap freezing and RNAlater. The collection of placental samples in RNAlater and DNAgard is simple, and eliminates the need for liquid nitrogen or a freezer on-site. Moreover, the quality of the nucleic acids and the resulting data from samples collected in these preservation solutions is actually higher than that from samples collected using the traditional snap-freeze method. Thus, this new approach to placental sample collection is both easier to implement in busy clinical environments and yields higher quality data. 48 samples
Project description:Banking of high-quality placental tissue specimens will enable biomarker discovery and molecular studies on diseases involving placental dysfunction. Systematic studies aimed at developing feasible standardized methodology for placental collection for genomic analyses are lacking. To determine the acceptable timeframe for placental collection, we collected multiple samples from first and third trimester placentas at serial time points 0-120 minutes after delivery, simultaneously comparing the traditional snap-freeze technique to collection in commercial solutions designed to preserve RNA (RNAlaterTM, Ambion), and DNA (DNAgard®, Biomatrica). The performance of RNAlater for preserving DNA was also tested. Nucleic acid quality was assessed by determining the RNA integrity number (RIN) and genome-wide expression and DNA methylation microarray profiling. We found that samples collected in RNAlater had higher and more consistent RINs compared to snap frozen tissue, with similar RINs obtained for tissue collected in RNAlater as large (1 cm3) and small (~0.1 cm3) tissue pieces. RNAlater appeared to better stabilize the time zero gene expression pattern compared to snap freezing for first trimester placenta. Microarray DNA methylation analysis showed that overall the DNA methylation profiles remained quite stable over a two hour time period after removal of the placenta from the uterus, with the DNAgard condition being superior to both snap freezing and RNAlater. The collection of placental samples in RNAlater and DNAgard is simple, and eliminates the need for liquid nitrogen or a freezer on-site. Moreover, the quality of the nucleic acids and the resulting data from samples collected in these preservation solutions is actually higher than that from samples collected using the traditional snap-freeze method. Thus, this new approach to placental sample collection is both easier to implement in busy clinical environments and yields higher quality data. 48 samples In this study, our objective was to identify the optimal timing and mode of collection for nucleic acids of sufficient quality to perform genome-wide RNA gene expression and DNA methylation studies for downstream molecular and functional enrichment analysis. To do this, we evaluated three different placenta collection methods: snap freezing in liquid nitrogen, RNAlaterTM, and DNAgard, over a two-hour window upon removal from the uterus, to determine: 1) the optimal collection method(s) for evaluation of mRNA expression and DNA methylation; and 2) the time period after delivery during which such optimal samples should be collected.
Project description:Genome wide placental DNA methylation profiling of full term and preterm deliveries sampled from 5 full term deliveries and 4 preterm deliveries. The Illumina HumanMethylation450 Beadchip was used to obtain DNA methylation profiles across approximately 485,577 CpGs in formalin fixed samples. Samples included 4 placental tissues from 4 women with preterm delivery and 5 placental tissues from 5 women with full term delivery. 9 women's placental DNA (4 women had perterm deliveries and 5 women had full term deliveries) were hybridised to the Illumina HumanMethylation450 Beadchip
Project description:The influence of genetics on DNA methylation (DNAme) variation is well documented, yet confounding from population stratification is often unaccounted for in DNAme association studies. Existing approaches have been developed to address confounding by population stratification by directly using DNAme data, but have not been validated in additional human populations or tissues, such as the placenta. Results: To aid future placental DNAme studies in assessing population stratification, we developed an ethnicity classifier, PLANET (placental elastic net DNAme ethnicity classifier), on combined Infinium Human Methylation 450k BeadChip array (HM450k) data from placental samples. We used data from five North American cohorts from private and public repositories (n = 509) and show that PLANET can not only accurately predict (accuracy = 0.9379, kappa = 0.8227) major classes of self-reported ethnicity/race (African: n = 58, Asian: n = 53, Caucasian: n = 389), but can also produce probabilities that are highly correlated with genetic ancestry inferred from genome-wide SNP (>2.5 million SNP) and ancestry informative markers (n=50) data. We found that PLANET’s ethnicity classification relies on 1860 DNAme microarray sites, and over half of these were also linked to nearby genetic polymorphisms (n=955). Lastly, we found our placental-optimized method outperforms existing approaches in assessing population stratification in our placental samples from individuals of Asian, African, and Caucasian ethnicities. Conclusion: PLANET outperforms existing methods and heavily relies on the genetic signal present in DNAme microarray data. PLANET can be used to address population stratification in future placental DNAme association studies, and will be especially useful when ethnicity information is missing and genotyping markers are unavailable.
Project description:The purity of tissue samples can affect the accuracy and utility of DNA methylation array analyses. This is particularly important for the placenta which is hypomethylated. Placental villous tissue from early pregnancy terminations can be difficult to separate from the non-villous tissue, resulting in potentially inaccurate results. We used several methods to identify mixed placental samples using DNA methylation array datasets from our laboratory and those contained in the NCBI GEO database, highlighting the importance of determining sample purity during quality control processes.