Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development

Human Reproduction Vol.20, No.5 pp. 1339–1348, 2005 doi:10.1093/humrep/deh778 Advance Access publication February 10, 2005 Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development D.Wells1,2,3,6, M.G.Bermudez2, N.Steuerwald2,3, A.R.Thornhill4, D.L.Walker4, H.Malter3, J.D.A.Delhanty5 and J.Cohen3 1 Department of Obstetrics & Gynecology, Yale University Medical School, 333 Cedar Street, New Haven, CT 06520, 2Reprogenetics LCC, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052, 3Tyho-Galileo Research Laboratories LLC, 101 Old Short Hills Rd, Suite 501, West Orange, NJ 07052, 4Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota 55905 and 5Department of Obstetrics and Gynaecology, University College London, 86 –96 Chenies Mews, London WC1E 6HX, UK 6 To whom correspondence should be addressed at: Department of Obstetrics & Gynecology, Yale University Medical School, 333 Cedar Street, New Haven, CT 06520, USA. E-mail: BACKGROUND: Appropriate gene expression is vital for the regulation of developmental processes. Despite this fact there is a remarkable paucity of information concerning gene activity during preimplantation development. METHODS: We employed reverse transcription and real-time fluorescent PCR to quantify the expression of nine genes (BRCA1, BRCA2, ATM, TP53, RB1, MAD2, BUB1, APC and b-actin) in oocytes and embryos. A full characterization of all genes was achieved in 42 embryos and four oocytes. The genes analysed have a variety of important cellular functions. RESULTS: Oocytes displayed relatively high levels of mRNA transcripts, while 2 –3-cell embryos were seen to contain very little mRNA from any of the genes examined. Recovery of expression levels was not seen until the 4-cell stage or later, with the presumptive activation of the embryonic genome. Some genes displayed sharp increases in expression in embryos composed of 4 – 8 cells, but, for most, maximum expression was not achieved until the blastocyst stage. CONCLUSIONS: Our data show that it is possible to define characteristic gene expression profiles for each stage of human preimplantation development. The identification of genes active at defined preimplantation phases may provide clues to the cellular pathways utilized at specific stages of development. Expression of genes that function in DNA repair pathways indicate that DNA damage may be common at the cleavage stage. We suggest that specific patterns of gene expression may be indicative of embryo implantation potential. Key words: apoptosis/cell cycle checkpoint/IVF/PGD/RT –PCR Introduction Gene expression has a fundamental role in the coordination of homeostatic and metabolic mechanisms throughout life. During development the activation or repression of specific genes underlies the regulation of key events, such as establishing polarity and body patterning and initiating the differentiation of tissues. Precise control of gene activity during the preimplantation phase of development is of particular significance. It is at this time that the first cellular differentiation occurs and the embryo transfers from a reliance on maternal RNA derived from the oocyte to expression of its own genome. Considering the importance of gene activity, it is remarkable that so little is known concerning gene expression in human preimplantation embryos. Relatively few key players have been identified, with most of the genes responsible for governing early developmental events remaining obscure. Many human preimplantation embryos generated using assisted reproductive techniques fail to form a viable pregnancy; only , 25% implant following transfer 2 days after IVF. It is likely that a high rate of loss also exists among naturally conceived embryos, as evidenced by the high prevalence of early pregnancy failure in women (Short, 1979; Edmonds et al., 1982). Given the complex and critical nature of the processes occurring at the preimplantation stage, it is perhaps unsurprising that many embryos fail to negotiate this challenging phase of development. Knowledge of the genes expressed at early stages should highlight the pathways that must be activated in order to produce an embryo capable of successfully implanting and initiating a clinically viable pregnancy. As well as the scientific importance of studying gene activity in early human embryos, there is growing interest in q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. 1339 For Permissions, please email: D.Wells et al. how information on gene expression could be clinically applied in order to improve the success rates of assisted reproductive techniques. Indeed it is conceivable that particular patterns of gene expression may be indicative of embryo viability per se. If such patterns could be assessed prior to embryo transfer they could assist embryologists and clinicians in deciding which embryos to transfer to the uterus. Current evaluations of embryo viability are based on morphology, in some cases supplemented with information on chromosomal status via preimplantation genetic diagnosis (PGD) (e.g. Munné et al., 1993, 2003; Verlinsky et al., 1996; Gianaroli et al., 1997) or non-invasive assessment of metabolic activity (Gardner et al., 2001). We have set out to examine the expression of nine genes throughout the preimplantation phase of development. The genes tested have significance in a range of important cellular processes, including cell cycle regulation, DNA repair, apoptosis, maintenance of accurate chromosomal segregation and construction of the cytoskeleton. The number of transcripts derived from each gene was assessed using real-time RT – PCR, a highly accurate method for quantification of nucleic acids. In this way, we aimed to provide an indication of each gene’s activity during different stages of preimplantation development. Materials and methods Samples consisted of human metaphase II oocytes (n ¼ 6) that had failed to fertilize following in vitro insemination and embryos at various stages of preimplantation development (n ¼ 79) that were surplus from assisted reproductive techniques. Samples were obtained from The Institute for Reproductive Medicine and Science of Saint Barnabas, New Jersey and Mayo Clinic, Rochester, Minnesota, USA. In both cases oocytes or embryos were utilized for research only after obtaining written consent from the patients and Institutional Review Board approval. Every embryo analysed had undergone a detailed morphological assessment each day, beginning at the time of oocyte retrieval. Details of the parents’ ages, cause(s) of infertility and response to ovarian stimulation were also available for each embryo/oocyte analysed. Embryos from The Institute for Reproductive Medicine and Science of Saint Barnabas were classified n (...truncated)