Safe and effective oocyte cryopreservation will have a considerable impact on clinical practice in assisted reproduction. Great improvements have been made in recent years to oocyte vitrification procedures, although further controlled trials are necessary to ensure safety, and it is necessary to know more about pregnancy and live birth outcomes. This study aims to validate various methods of oocyte vitrification as assessed by comparative target gene analysis, hence contributing to information available to clinicians advising women about fertility preservation options before cancer treatment. Target genes investigated were: the maternal effect genes Deleted in Azoospermia Like (DAZL), Maternal Antigen That Embryos Require (MATER/NLRP5) and Zygote Arrest 1 (ZAR1); three genes involved in cell cycle progression and cell death, tumour suppressor protein 53 (p53), B-cell lymphoma 2 (BCL2), BCL2-Associated X Protein (BAX); three genes known to affect spindle and chromatin structure, oocyte-specific histone 1 (H1FOO), kinesin family member 11 (KIF11) and mitotic arrest deficient 2 (MAD2); together with Factor In the GermLine, Alpha (FIGLalpha) which regulates zona pellucida proteins, octamer-binding transcription factor 4 (OCT4/POU5F1) which is associated with pluripotency and oocyte developmental competence, and superoxide dismutase 2, mitochondrial (SOD2) which responds to oxidative stress in the mitochondria. These genes may be useful indicators of oocyte quality following vitrification. Lysis, complementary DNA (cDNA) amplification, polyadenylic acid polymerase chain reaction (polyA PCR) and quantitative polymerase chain reaction (QPCR) were used to investigate gene expression patterns in failed-to-fertilize non-vitrified, vitrified and slow frozen human MII oocytes. Comparative gene analyses included oocytes vitrified using closed and open carriers, and two different media.Results indicate that the impact of vitrification varies by gene and oocyte variability, highlighting the importance of studies based on single oocytes and the need for caution in interpretation of generalised findings. OCT4 and also β-actin were significantly affected by all methods investigated, while FIGLalpha, MAD2, ZAR1 and DAZL were affected by some methods. Oocyte survival rate after thawing and the number of genes expressed by individual oocytes were higher with media incorporating dimethyl sulfoxide (DMSO) and Dextran Serum Supplement (DSS) and first-step warming in a larger volume. All methods led to altered expression of target genes, most noticeably when the second media was used. Further quantitative studies of the impact of OCT4, FIGLalpha and β-actin should be conducted, together with clinical comparisons between media and a longitudinal multi-centre study regarding outcomes arising from different vitrification methods.
|Date of Award||31 Dec 2018|
- The University of Manchester
|Supervisor||Daniel Brison (Supervisor) & Susan Kimber (Supervisor)|
- Oocyte vitification, target genes, Vitrifcation , cryopreservation