Understanding the Impact of Assisted Reproductive Technologies (ART) on Embryo Health, Child Health and Disease and Longevity in Later Life
2015
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 impacts
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