Transcriptome Analysis during Human Trophectoderm Specification Suggests New Roles of Metabolic and Epigenetic Genes

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts

Day 3 embryo and TE molecular signatures:

<p>Heat map of the molecular signatures in six day 3 embryos and five TE samples. Each horizontal line represents a gene and each column represents a single sample. The color intensity indicates the level of gene expression (red for up-regulation and blue for down-regulation) “see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039306#pone.0039306.s006" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039306#pone.0039306.s007" target="_blank">S2</a>”.</p

Gene Ontology (GO) annotations of the day 3 embryo and TE molecular signatures.

<p>We compared the GO annotations of genes specifically over-expressed in day 3 embryos and in TE cells by using the Babelomics web tool (<a href="http://babelomics.bioinfo.cipf.es/" target="_blank">http://babelomics.bioinfo.cipf.es/</a>). Histograms show the percentage of genes with a specific GO annotation in day 3 embryos (white) or in TE samples (black). Only GO categories which differed significantly (<i>p</i> value <0.01) between the two groups are shown.</p

List of the 100 genes with the highest fold change in TE samples in comparison to day 3 embryos.

<p>List of the 100 genes with the highest fold change in TE samples in comparison to day 3 embryos.</p

Up-regulated genes that are related to the <i>NANOG</i> pathway, or to metabolic and epigenetic functions in day 3 human embryos and TE samples.

<p>(A) The interaction network was generated with the Ingenuity software and shows that many genes from the <i>NANOG</i> pathway are up-regulated (red) in day 3 embryos. (B) Top-ranked functional networks in which are involved transcription factors (<i>GATA2</i> and <i>GATA3</i>), or genes that regulate steroidogenesis (including <i>HSD1B3</i>), DNA repair (<i>TDG</i> and <i>BRCA1</i>) or epigenetic modifications (including <i>DNMT3L)</i> and that are up-regulated in TE samples. The color intensity indicates their degree of up-regulation. Uncolored genes were identified as not differentially expressed by our analysis, but were, nevertheless, integrated into the computationally generated networks on the basis of the evidence stored in the IPA knowledge memory indicating a relevance to this network. In each network, nodes indicate genes, a plain line indicates direct interaction, a dashed line indicates indirect interaction; a line without arrowhead indicates binding only; a line with an arrowhead indicates “acts on”.</p

List of the 100 genes with the highest fold change in day 3 human embryos in comparison to TE samples.

<p>List of the 100 genes with the highest fold change in day 3 human embryos in comparison to TE samples.</p

Differential expression of apoptosis and stemness-related genes in day 3 embryos and TE samples.

<p>(A) Histograms show the microarray signal values for apoptosis-related genes in day 3 embryos (black) and TE samples (white). (B) The mean expression level of 48 stemness genes in six day 3 embryos and five TE samples was plotted on a logarithmic scale in a radar graph. Asterisks indicate a statistically significant difference (<i>P</i><0.05) between TE and day 3 embryos (Mann-Whitney test).</p