Pluto Bioinformatics

GSE47019: Developmentally Abnormal Human Embryos Trigger a Maternal ER Stress Response

Bulk RNA sequencing

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study were to compare signals from competent and abnormal human embryos impacted differently on the expression of endometrial receptivity genes in mouse uteri using transcriptome profiling (RNA-seq).; Methods: Immature female (25 d C57BL/6) mice were given a hormone treatment of a single dose 1 mg progesterone and 10 mg/kg/day -estradiol for a total of 3d to prime the uterus for embryo transfer. The uterine horns of control and study mice were injected with an equal volume (50 l) of either unconditioned embryo culture medium (ECM), serving as controls, or pooled conditioned media from competent (n = 9) or arresting embryos (n = 18). Uterine mRNA profiles of 25-day-old wild-type (WT) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000 platform. The sequence reads that passed quality filters were analyzed with the following methods: Bowtie Alignment followed by TopHat (splice juntions mapper) and Cufflinks (transcript abundance). qRTPCR validation was performed using SYBR Green assays.; Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the uteri WT and Nrl/ mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRTPCR. RNA-seq data had a linear relationship with qRTPCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl/ retina, with a fold change 1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRTPCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.; Conclusions: Our study represents the first detailed analysis of maternal uterine reponse to signals from competent and abnormal human embryos, with biological replicates, generated by RNA-seq technology. SOURCE: Jan,J,Brosens (j.j.brosens@warwick.ac.uk) - Division of Reproductive Health University of Warwick