Preimplantation Embryo Quality Following Partial Denudation of Oocytes

Sharma, Nidhi; Saravanan, Mahalakshmi; Saravanan, Lakshmanan; Narayanan, Sindhu

doi:10.31031/prm.2018.03.000553

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“…Partial removal of cumulus cells is usually sufficient for ART, but 300-500 cells may remain surrounding the oocyte (Ebner et al, 2006;Sharma, 2018). In the context of this project it was imperative that all the cumulus cells were removed prior to FISH (Bedford, 1971).…”

Section: Denuded Oocytesmentioning

confidence: 99%

Synthesis and Validation of Fluorescence in situ Hybridisation Probes (FISH) for Genotyping Mouse Models of Embryonic Lethal Aneuploidies

Derecourt¹

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Approximately 50% of all embryos produced in fertility clinics have an abnormal number of chromosomes (i.e., aneuploid)(Vera-Rodriguez et al., 2015). Of these, 25% have aneuploidies that are embryonic lethal (O’Connor, 2008) and result in spontaneous abortions in the first trimester(Chen et al., 2017). My research contributes to wider research programme that aimsto develop a non-invasive method for detecting aneuploidies in preimplantation humanembryos. For the overarching research program, several models of embryonic lethal aneuploidies must be developed and validated. One way to produce embryonic lethal aneuploidies is through mouse breeding programmes that make use of Robertsonian translocations. Robertsonian translocations (RobT) are one of the most common forms of chromosomal rearrangements in humans and can cause whole chromosome duplications/deletions(Zhuang et al., 2014). A balanced carrier of these RobT is viable but produces normal gametes, carrier gametes, as well as gametes that will result in aneuploid (trisomy or monosomy) embryos(Beyer & Willats, 2017). As these RobT mouse models are being developed in our laboratory, an accurate method for identifying the trisomic and monosomic (i.e., aneuploid) embryos produced is critical. This project aims to synthesise and validate fluorescence in situ hybridisation (FISH) point probes for mouse chromosomes 4, 10, 11, and 15 using a cost-effective, PCR-based method (Bienko et al., 2013). Herein, I present data from experiments that were conducted to validate FISH probes for mouse chromosome 4, 10, 11, and 15. I also describe a series of experiments to optimise metaphase spread preparation from two different sample types, mouse lymphocytes and cleavage stage embryos. From this study, four FISH probes (specific for chromosome 4, 10, 11, & 15) were synthesised. Based on existing metrics, these were determined to be sufficiently labelled and at a high enough concentration to be evaluated as FISH probes. However, when FISH was performed using each of these probes, no specific signal due to hybridisation was observed. This is despite attempts to identify and address the cause of this lack of binding. Despite this, a positive, pan-centromeric control was successfully validated using the optimised FISH protocol. Also, metaphase spread protocols for two different tissue types were also devised to produce a reliable sample source for FISH validation. Overall, this project has shown that these PCR based point probes are not appropriate for the genotyping of RobT induced aneuploidies and other forms of genotyping should be considered.

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Section: Denuded Oocytesmentioning

confidence: 99%

Synthesis and Validation of Fluorescence in situ Hybridisation Probes (FISH) for Genotyping Mouse Models of Embryonic Lethal Aneuploidies

Derecourt¹

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show abstract

Preimplantation Embryo Quality Following Partial Denudation of Oocytes

Cited by 1 publication

References 0 publications

Synthesis and Validation of Fluorescence in situ Hybridisation Probes (FISH) for Genotyping Mouse Models of Embryonic Lethal Aneuploidies

Synthesis and Validation of Fluorescence in situ Hybridisation Probes (FISH) for Genotyping Mouse Models of Embryonic Lethal Aneuploidies

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