Abstract
Background It is possible to replicate the natural process of twinning by means of artificially spitting early-stage embryos in the laboratory. This method has resulted in applications relevant to agricultural and sporting animals. Embryo splitting, or in vitro twinning, has been successfully conducted in various livestock species. Human embryo splitting has been previously reported. The results were, however, inconsistent. Hypothesis The quality of the human embryos generated by twinning in vitro is comparable to the quality of the embryos created by fertilisation.Experimental Approach A total of 176 twin embryos created by splitting of 88 human embryos from either early (2 – 5 blastomeres, n = 43) or late (6 – 10 blastomeres, n = 45) cleavage stages were analysed in terms of morphokinetics and developmental competence. Data was compared to the morphometrics of embryos created by fertilisation and leading to pregnancy and live birth following single blastocyst transfer (n = 42). Furthermore, comparative analyses of the expression patterns of early lineage-specific markers (n=21 pairs) of twin blastocysts and non-manipulated Day 5 - 6 blastocysts using immunocytochemistry and human embryonic stem cells (hESCs) derivation was attempted (n = 5 pairs). Finally, comparative analyses of micor RNA (miRNA) profiles in spent blastocyst medium (SBM) of human twin embryos created by blastomere biopsy (n = 7 pairs) and SBM of blastocysts that led to a healthy pregnancy and live birth following embryo transfer (n = 7) were also conducted.
Results Morphokinetic data indicated that human preimplantation development is subject to strict temporal control according to a set ‘developmental clock’. The size of twin embryos generated in the study was directly proportional to the starting cell number of the embryos used in their genesis. Furthermore, the first commitment decision in terms of cell fate was delayed, with the inner cell mass (ICM) becoming distinguishable later in the study group than in the normal control blastocysts. The ICM, if present at all, was small in size and of low quality. Furthermore, most cells in the twin embryos concurrently expressed both ICM and trophectoderm (TE) markers. Finally, the nature of miRNA secretion in SBM consistently varied between the twin and control embryos.
Conclusion Taken together the data suggested that human twin embryos created in vitro by embryo splitting are unsuitable not only for clinical use but also for research purposes.
| Date of Award | 2017 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Dusko Ilic (Supervisor) |