Project description:Purpose: To chart the human myometrial transcriptomes before and after the onset of labour. Methods: Tophat splice junction mapping of paired-end reads, HTSeq to generate counts, cufflinks to track transcripts, DESeq, edgeR and baySeq to detect differentially expressed genes and principal component analysis for clustering analyses. Results: We mapped on average 14 million paired-end reads per sample (counting each end individually) to the human genome (build hg19) and covered the expressed transcriptome about 13 times with a TopHat-HTSeq workflow. We performed a comparative analysis with an analogous microarray study (Mittal et al., 2010) and found some overlap between the RNA-seq and the microarray data. Conclusions: Our study is the first RNA-seq study of the human myometrium before and after the onset of labour. We show that while microarray and RNA-seq studies may complement each other, RNA-seq has a much greater resolution. At term with and at term without labour human myometrial mRNA profiles were generated by deep sequencing, using Illumina GAIIx (five biological replicates each).
Project description:This study identifies a transciptomic myometrial profile associated with dystocia in spontanous nulliparous term labour We used microarrays to compare myometrial biopsies obtained at cesarean section from women in spontaneous term labour
Project description:This study identifies a transciptomic myometrial profile associated with dystocia in spontanous nulliparous term labour We used microarrays to compare myometrial biopsies obtained at cesarean section from women in spontaneous term labour Women in spontaneous labour undergoing cesarean section for dystocia (slow progressing labour) compared to women who had progressed in the second stage
Project description:During pregnancy, the myometrium remains quiescent but at term, switches to a state capable of producing a series of coordinated contractions for the delivery of the fetus. Myometrial contractions of labour signify the normal physiological end-point of pregnancy but the biochemical onset of labour may occur at or before term via a series of changes in expression of labour associated genes that are responsible for controlling the activity of the uterus during pregnancy and parturition. There is increasing evidence that components of the cAMP-signalling pathway are up-regulated in the human myometrium during pregnancy to promote the relaxation of the myometrium until term. Our aim was to determine which cAMP-associated genes are important during pregnancy and parturition by exposing myometrial cells to forskolin and performing an a gene array. We then plan to study the trend of the cAMP-associated genes at different stages of gestation and during labour. In this study, we used microarrays to elucidate forskolin responsive genes in human myometrium. These data may provide a broader view of gene networks and cellular functions regulated by forskolin in human myometrial cells. In our future study, this will also help us understand the role of cAMP in human parturition. Primary cultures of human myometrial cells were grown from myometrial biopsies obtained at the time of elective caesarean section at term. Cells were exposed to forskolin (100 µM) for 48 hours, and then total RNA were extracted from each culture. Two comparisons were carried out including: 1. Control 2. Forksolin
Project description:During pregnancy, the myometrium remains quiescent but at term, switches to a state capable of producing a series of coordinated contractions for the delivery of the fetus. Myometrial contractions of labour signify the normal physiological end-point of pregnancy but the biochemical onset of labour may occur at or before term via a series of changes in expression of labour associated genes that are responsible for controlling the activity of the uterus during pregnancy and parturition. There is increasing evidence that components of the cAMP-signalling pathway are up-regulated in the human myometrium during pregnancy to promote the relaxation of the myometrium until term. Our aim was to determine which cAMP-associated genes are important during pregnancy and parturition by exposing myometrial cells to forskolin and performing an a gene array. We then plan to study the trend of the cAMP-associated genes at different stages of gestation and during labour. In this study, we used microarrays to elucidate forskolin responsive genes in human myometrium. These data may provide a broader view of gene networks and cellular functions regulated by forskolin in human myometrial cells. In our future study, this will also help us understand the role of cAMP in human parturition.
Project description:Purpose: To chart the human myometrial transcriptomes before and after the onset of labour. Methods: Tophat splice junction mapping of paired-end reads, HTSeq to generate counts, cufflinks to track transcripts, DESeq, edgeR and baySeq to detect differentially expressed genes and principal component analysis for clustering analyses. Results: We mapped on average 14 million paired-end reads per sample (counting each end individually) to the human genome (build hg19) and covered the expressed transcriptome about 13 times with a TopHat-HTSeq workflow. We performed a comparative analysis with an analogous microarray study (Mittal et al., 2010) and found some overlap between the RNA-seq and the microarray data. Conclusions: Our study is the first RNA-seq study of the human myometrium before and after the onset of labour. We show that while microarray and RNA-seq studies may complement each other, RNA-seq has a much greater resolution.
Project description:Circulating progesterone (P4) levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is functional withdrawal of P4 action at the myometrial level prior to labor onset. Mifepristone is widely used to induce human labour In this study, we aimed to establish and validate a model of human myometrial explants for the study of P4 action. Myometrial biopsies obtained at Caesearean section at term were dissected into explants after a portion was immediately snap-frozen (t=0). Transcriptomic comparison of paired explants and primary myometrial cells as well as the hTert immortalized myometrial cell line demonstrated that explants more closely resemble t=0.
Project description:Circulating progesterone (P4) levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is functional withdrawal of P4 action at the myometrial level prior to labor onset. Mifepristone is widely used to induce human labour In this study, we aimed to establish and validate a model of human myometrial explants for the study of P4 action. Myometrial biopsies obtained at Caesearean section at term were dissected into explants after a portion was immediately snap-frozen (t=0). Transcriptomic comparison of paired explants and primary myometrial cells as well as the hTert immortalized myometrial cell line demonstrated that explants more closely resemble t=0. Biopsies obtained from non-laboring women at elective Caesarean section at term were divided into 3: (i) dissected and immediately snap-frozen (t=0), (ii) dissected into 3x3x3mm3myometrial explants and (iii) processed for primary cell culture. Explants, primary cells at passage 4 (the typical passage our group uses for experiments) and hTERT cells were cultured for a period of 30 hours without treatment. Total RNA was extracted and microarray analysis performed. 6 replicates were used for this study.
Project description:The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. RNA-seq was used to: 1) identify which myometrial cells retained the most similar transcriptome profile to native tissue, and 2) compare the uterine myometrial transcriptome to VSMCs in hopes of identifying a uterine-selective transcriptome that was “druggable” for tocolytic or uterotonic use. Four sources of myometrial cells were examined: 1) term pregnant human primary myometrial cells isolated from tissue biopsies obtained at the time of caesarean sections, 2) term pregnant mouse primary myometrial cells, 3) commercially-available immortalized pregnant human myometrial (PHM1) cells and 4) human telomerase immortalized myometrial (hTERT-HM) cells. Correlation analysis of aligned reads identified that the transcriptome of primary human myometrial and hTERT-HM cells showed 85% and 80% correlation, respectively, to human myometrial tissue and that the transcriptome of hTERT-HM and PHM1 cells is 90% or more correlative to human primary myometrial cells. The expression levels (fold-change) of contraction-associciated transcripts (OXTR, PTGFR, PTGS2 and GJA1) strongly correlated (r=0.93) between RNA sequencing and qRT-PCR analysis. Analysis of aligned reads among myometrial cells revealed the number of differentially expressed transcripts (fold-change≥2.0, adjusted p-value≤0.01) relative to primary human myometrial cells: hTERT-HM (946 upregulated and 2,351 downregulated), PHM1 (1,575 upregulated and 2,415 downregulated) and primary mouse myometrial cells (3,435 upregulated and 2,966 downregulated). Correlation analysis showed that the human primary myometrial cell transcriptome is over 90% similar to the transcriptome of VSMCs examined. A number of genes associated with smooth muscle contractile machinery (TPM1, TPM2, CNN1, CALD1, ACTA2 and PLN)were significantly (p≤0.01) upregulated (≥2-fold) in human primary myometrial compared to vascular SMCs. We identified 498 transcripts were identified as upregulated in human primary myometrial cells compared to all three VSMCs examined. Of these, the drug-gene interaction database identified 142 genes as druggable