It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum data set required for the accurate description of embryo development.

The ability to sequence whole genomes has taught us that our knowledge with respect to gene function is rather limited with typically 30-40% of open reading frames having no known function. Thus, within the life sciences there is a need for determination of the biological function of these so-called orphan genes, some of which may be molecular targets for therapeutic intervention. The search for specific mRNA, proteins, or metabolites that can serve as diagnostic markers has also increased, as has the fact that these biomarkers may be useful in following and predicting disease progression or response to therapy. Functional analyses have become increasingly popular. They include investigations at the level of gene expression (transcriptomics), protein translation (proteomics) and more recently the metabolite network (metabolomics). This article provides an overview of metabolomics and discusses its complementary role with transcriptomics and proteomics, and within system biology. It highlights how metabolome analyses are conducted and how the highly complex data that are generated are analysed. Non-invasive footprinting analysis is also discussed as this has many applications to in vitro cell systems. Finally, for studying biotic or abiotic stresses on animals, plants or microbes, we believe that metabolomics could very easily be applied to large populations, because this approach tends to be of higher throughput and generally lower cost than transcriptomics and proteomics, whilst also providing indications of which area of metabolism may be affected by external perturbation.

This study contributes to the evidence of DNA damage within sperm. High loads of DNA damage measured by the Comet assay were predictive of failure of embryo development after ICSI. As it is likely that sperm with DNA damage contributed to successful fertilization and in-vitro development, potential adverse effects remain to be clarified.

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95-97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

Mouse blastocysts undergo cell death in the inner cell mass (ICM) as a normal feature of development, but little is known as to how this event is regulated or as to the possible role of survival factors in preimplantation development. The observation that growth factors, which can influence preimplantation development, can act as survival factors in other cell types led us to investigate the effects of culture in vitro, embryo density during culture, and transforming growth factor alpha (TGF alpha) on cell death in the blastocyst. Mouse blastocysts cultured singly from the 2-cell stage in 25 microl of medium KSOM + amino acids showed a approximately 3-fold increase in the incidence of cell death, predominantly in the ICM, relative to blastocysts formed in vivo. Increasing the density of embryo culture to 30 embryos per 25 microl of culture medium accelerated development, increased final blastocyst cell number, and partially (approximately 50%) reduced the increase in cell death induced by culture in vitro. Addition of 0.1 pM TGF alpha to the medium of singly cultured embryos also partially (33%) reduced this increase in cell death without accelerating development or increasing final cell number. Culturing isolated ICMs for 24 h in the presence of 0.1 pM TGF alpha also partially (33%) reduced the increase in cell death. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of whole blastocysts confirmed that cell death as detected by fragmented nuclei was apoptotic, as defined by endonuclease activation. Results of these experiments suggest that endogenously produced growth factors may function as cell survival factors during preimplantation development.

Non-invasive assay of amino acid turnover has the potential to improve significantly the prospective selection of the most viable embryos, or single embryo, for replacement in an IVF cycle.

The occurrence of cell death by apoptosis was examined in blastocyst and preblastocyst stage bovine embryos. Zygotes were obtained by in vitro maturation and in vitro fertilization of oocytes from abattoir derived ovaries. Two-cell to hatched blastocyst stage embryos were stained with propidium iodide to label all nuclei and by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labelling (TUNEL) to label apoptotic nuclei, and were analysed by epifluorescent and confocal microscopy. Apoptosis was first observed at the 9-16-cell stage of development, decreasing at the morula stage before increasing at the blastocyst stage. Apoptotic dead cell index in day 7 blastocysts was negatively correlated with the total number of cells; the percentage of dead cells ranged from approximately 1 to 10% and occurred predominantly within the inner cell mass. In addition, apoptotic dead cell index was significantly higher (P < 0.05) in blastocysts cultured (from the two-cell stage) in the presence of 10% fetal bovine serum compared with those developed in serum-free medium. Embryos selected for early cleavage at < 29 h after fertilization and cultured together until the blastocyst stage showed a significantly lower rate of apoptosis (P < 0.01) compared with slower cleaving embryos.

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