The aim of this thesis was to assess the effect of suboptimal handling on mouse pre-implantation embryo development, viability and gene expression of the HI9 and Igf2 imprinted genes and to investigate the effects on fctal and placental development following embryo transfers. Establishment of the expression pattern of H19 and Igf2 genes showed that H 19 was expressed only at the blastocyst stage whereas the Igf2 imprinted gene employed a gradual increase in expression from the l-cell to the blastocyst stage, with maximal tran script detection at the post-compaction stages (P< 0.05). Culture of pre-implantation embryos from 1-cell stage until the blastocyst stage of development under temperature variations as small as ± 0.5 °C impaired blastocyst development and significantly reduced blastocyst total cell numbers (P< 0.05). These blastocysts also showed effected levels of H19 and Igf2 expression. Daily exposure of pre- implantation embryos to ambient air at 37.0 •C for small amounts of time caused small fluctuations of the culture media pH between 7.20 and 7.30. This resulted in a statistically significant reduction in the first cleavage, blastocyst development and hatching rates, decreased trophectodenn and inner cell mass cell development and increased apoptotic cell index (P< 0.05). Expression of imprinted genes H 19 and Igf2 was perturbed in blastocysts (P< 0.05). Following embryo transfers, the implantation rates were effected and fetuses displayed reduced weight on day 18 of pregnancy (P< 0.05). Placentae displayed significantly reduced levels of H 19 and Jg£2 expression and in some cases the aberrant expression persisted from the blastocyst to post*implantation embryo development and was retained in placentae (P< 0.05). Vitrification of mouse 2-cell stage embryos, which are warmcd and grown to blastocysts, resulted in increased survival and blastocyst development rates. However, it was found that blastocyst vitrification at the 2-cell stage altered the expression of H 19 and Igf2 (P< 0.05). Artificially reduction of blastocoel cavities prior to vitrification using the ICSI needle or laser-pulse methods showed significantly increased survival and re-expansion rates when compared to blastocysts vitrified with intact blastocoel cavities (P< 0.05). Blastocoel reduction using these two methods prior to vitrification at the blastocyst stage did not compromise the expression of H 19 and Igf2. The results from this study clearly demonstrate that stress applied to the pre- implantation embryo during the in vitro culture and handling alters blastocyst homeostasis without compromising morphology. Even minimal micromanipulations of pre-implantation embryos during in vitro culture display a worrying domino effect. Morphologically good-looking embryos have the ability to mislead us as the negative effect mainly occurs at the global gene expression, imprinting and metabolism levels. Irreversible affects come at a cost of impaired post-implantation, post-natal development and disease in adult life. The current study underlines how suboptimal culturing and handling of embryos has long reaching effects far beyond blastocyst development and successful pregnancy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595684 |
| Date | January 2013 |
| Creators | Koustas, Georgios |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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