and TE cells. The expression of genes of the TNF ligand and receptor family was not different in day 3 embryos and TE cells. Conversely, several genes belonging to the BCL-2, BIRC and Caspase families appeared to be differentially expressed in the two groups. Specifically, the BCL-2 family members BCL2L10, BCL2L11, and BIK, the expression of which was validated by qRT-PCR, and the BIRC family member BIRC2 were up-regulated in day 3 embryos. Caspase 6 was over-expressed in TE cells. MCL1, a gene that belongs to the BCL2 family and promotes cell survival, was strongly expressed in both day 3 embryos and TE samples. Comparison of the Gene Expression Profiles of Day 3 Embryos and TE Cells Isolated from Day 5 Blastocysts Evaluation of DNA Repair Regulation in Day 3 Embryos and TE Samples The microarrays data were also used to investigate the expression of a comprehensive list of DNA repair genes in day 3 embryos and TE samples. Of the 123 DNA damage repair genes investigated, five were up-regulated in day 3 embryos and eleven in TE cells. We then analyzed the functional relationship between the DNA damage repair genes that were differentially expressed in TE samples and day 3 embryos using the Ingenuity Pathway Analysis software. In both cases, all the DNA repair genes displayed a Transcriptome Analysis of Embryo and Trophectoderm documented functional interaction with each other, forming a tightly connected network. Dynamic Expression of Epigenetic and Metabolic Regulators During Trophoblast Development Since specification of the TE lineage during blastocyst formation involves initiation of differentiation, it is likely that epigenetic regulators may have an important role in this first developmental decision. The majority of the epigenetic regulators that were up-regulated in TE cells are associated with a repressive epigenetic status. Specifically, the expression of the DNA methyl transferases DNMT3A, DNMT3B and DNMT1 increased between 2- and 13-fold in TE cells in comparison to day 3 embryos. DNMT3L expression was 70-fold higher in TE samples than in day 3 embryos. Similarly, several transcripts coding for proteins involved in chromatin remodeling and histone modification were up-regulated between 2- and 7-fold in TE cells. Conversely, many histone deacetylases and histone acetyltransferases were down-regulated in TE cells in comparison to day 3 embryos. Another feature of the TE molecular signature was the upregulation of several metabolic genes. Specifically, genes that are involved in 485-49-4 estrogen biosynthesis and lipid metabolism were strongly up-regulated in TE cells. One of the most striking observations was the high expression of genes that are involved in steroidogenesis. Stemness Genes and Transcriptional Regulatory Networks Identified in Day 3 Embryos and TE Cells We then performed a stemness gene enrichment analysis using a previously published dataset from hESCs, in which we defined a consensus hESC stemness gene list . The key stemness factors NANOG, POU5F1 and SOX2 were enriched in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22201297 day 3 human embryos, whereas DNMT3B, LIN28, PHF17, SEPHS1 were over-represented in TE cells. Conversely, other genes, such as UGP2 and PIM2, were enriched in both day 3 embryos and 
TE samples. Bioinformatic gene pathway analysis of the day 3 embryo molecular signature showed that many genes of the NANOG signaling pathway, including NANOG, were up-regulated in day 3 human embryos, thus confirming the role of NANOG in the maintenance of pluripotency.
