Association of abnormal morphology and altered gene expression in human preimplantation embryos  Dagan Wells, Ph.D., Mercedes G. Bermúdez, M.Sc., Nury Steuerwald, Ph.D., Henry E. Malter, Ph.D., Alan R. Thornhill, Ph.D., Jacques Cohen, Ph.D.  Fertility and Sterility  Volume 84, Issue 2, Pages 343-355 (August 2005) DOI: 10.1016/j.fertnstert.2005.01.143 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Results of hierarchic cluster analysis, revealing embryos that have similar patterns of gene expression for the nine genes assessed during this study. Each line of nine colored squares represents the gene expression data from a single embryo. The color of each square represents the relative number of transcripts of a specific gene for a single embryo. Green = low expression; black = intermediate expression; red = high expression. Embryos of identical developmental stage have similar gene expression profiles and tend to cluster together. Additionally, embryos with certain abnormal morphologies are also grouped together, indicating that patterns of gene expression may be correlated with morphology. Wells. Gene expression and embryo morphology. Fertil Steril 2005. Fertility and Sterility 2005 84, 343-355DOI: (10.1016/j.fertnstert.2005.01.143) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Gene expression in a 3-cell embryo of abnormal morphology, compared to typical expression profiles for oocytes and morphologically normal 3-cell embryos. Data on transcript copy number has been normalized so that the expression of each gene is shown on a scale ranging from zero to one. The abnormal 3-cell embryo displayed much higher levels of gene expression than usually observed in embryos at this stage of development. Indeed, the pattern of expression observed was reminiscent of that typically observed in oocytes. Abnormalities of the polar bodies and pronuclei and a highly asymmetric first cleavage division suggest that this embryo may have failed to fully activate the cellular pathways regulating postfertilization events. Wells. Gene expression and embryo morphology. Fertil Steril 2005. Fertility and Sterility 2005 84, 343-355DOI: (10.1016/j.fertnstert.2005.01.143) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Image of two day-1 zygotes displaying “condensed organelles,” a morphologic feature characterized by concentration of cellular structures (organelles, etc,) toward the center of the cell (arrow) and correlated with poor implantation rates. Peripheral regions appear to contain little subcellular material. Wells. Gene expression and embryo morphology. Fertil Steril 2005. Fertility and Sterility 2005 84, 343-355DOI: (10.1016/j.fertnstert.2005.01.143) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Three graphs illustrating the relationship between expression of ATM, BRCA1, and TP53 and the proportion of embryo volume occupied by cellular fragments. Expression levels were assessed based on the number of transcripts per embryo, divided by the number of blastomeres (i.e., average number of transcripts per cell). The three embryos expressing the highest levels of BRCA1 were not included in this figure but contained 0%, 15%, and 20% fragmentation and had transcript numbers ranging from 2,800 to 35,240 per cell. Wells. Gene expression and embryo morphology. Fertil Steril 2005. Fertility and Sterility 2005 84, 343-355DOI: (10.1016/j.fertnstert.2005.01.143) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 [1] Low expression levels of all genes in 2 and 3 cell embryos; [2] upon activation of the genome, embryos containing cells with DNA damage express genes responsible for DNA repair, including ATM, BRCA1, BRCA2, and TP53. The ATM protein is an upstream regulator of DNA repair pathways and consequently ATM is one of the first genes expressed. BRCA1 and BRCA2 proteins interact to repair the damaged DNA. Additionally, BRCA1 stabilizes the p53 protein and directs its transcription factor activity toward the stimulation of DNA repair and away from apoptosis; [3] p53 protein begins to accumulate and acts to downregulate BRCA1 transcription; (4) as BRCA1 levels fall, p53 is able to activate apoptotic pathways. Upregulation of TP53 maintains high levels of p53 protein, despite the loss of BRCA1-mediated stabilization. Blastomeres that have not successfully repaired their DNA by this stage may undergo apoptosis, perhaps leading to fragmentation. Wells. Gene expression and embryo morphology. Fertil Steril 2005. Fertility and Sterility 2005 84, 343-355DOI: (10.1016/j.fertnstert.2005.01.143) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions