Understanding the Impact of Assisted Reproductive Technologies (ART) on Embryo Health, Child Health and Disease and Longevity in Later Life

Study question: Can gene expression microarray data from different stages of human embryo development be used to investigate the expression of metabolic and epigenetic pathways contributing to embryo health? Such key pathways are likely to be affected by ART factors such as maternal oocyte age, embryo cryopreservation and prolonged embryo culture. Summary answer: Several novel genetic and epigenetic regulatory pathways have been identified as important in embryonic genome activation and blastocyst formation. Expression of some of these are altered by ART factors. Further analysis into interacting partners of the novel genes reveal unique developmental networks important for healthy blastocyst formation. What is known already: Many studies have shown the relationship between the maternal environment and offspring health, using animal models and epidemiological data. Mouse embryos removed from a diabetic environment before embryo replacement have an increased risk of developing obesity, diabetes and metabolic syndrome in later life and other studies demonstrate the importance of epigenetic DNA methylation stability during embryonic development. However few studies have attempted to clarify the link between the environment/ ART technology and human embryo and future offspring health. Study design, size, duration: Gene expression microarray data and QPCR analysis of amplified cDNAs from individual embryos and isolated blastomeres from different stages of human embryo development (n = four × oocytes, four × 4 cell, three × 8 cell, eight individual 8 cell blastomeres and ten × blastocyst stage embryos). Participants/materials, setting, methods: Embryos were donated by patients undergoing IVF at St. Mary’s hospital, Manchester, UK. cDNA was amplified via polyAPCR and analysed using the Affymetrix microarray HG U133 plus 2 chip, at the Paterson cancer Institute, Manchester. Arrays undergo rigorous AQM analysis within R, statistical analysis within Partek, Cytoscape and Ingenuity. Main results and the role of chance: We identified a large epigenetic gene list which correlated with maternal age with two clear groups of gene expression profiles emerging, in embryos from women aged 35 and under, versus those aged >35. We detect a strong up-regulation of a selection of oxidative stress pathways as the embryo remains in extended culture; in particular the NRF2 mediated oxidative stress response. A more systems based network analysis resulted in the generation of novel embryonic developmental expression networks, network modules and module/gene hierarchy and causal networks. Data mining revealed many interesting genes, amongst them Zscan4, previously shown to restore developmental potency of mouse embryonic stem cells, identified as a key gene during EGA. A network of transcription factors and the epigenetic regulatory genes GATA2, TRIM8 and DNMT3L are significantly increased in the blastocyst and may represent important changes occurring during the period of in vitro culture in IVF. Limitations, reason for caution: The number of samples per developmental stage was restricted in order to focus on individual embryos. Embryos are inherently different to one another, e.g., in genetic background and quality and therefore detailed analysis needs to be applied to identify trends in gene expression. Wider implications of the findings: Understanding developmentally important pathways which contribute to human embryo health is critical for our understanding of potential risk factors to which human embryos are subject in vitro. Assessing the perturbation of such genetic pathways in response to ART is an essential part of a fully-informed risk assessment. This will ultimately lead to increased understanding of long term health outcomes for ART children and the consequent modification of risk factors will ultimately help to improve ART conditions and avoid unwanted impact