Cryopreservation and in vitro maturation of murine germinal vesicle stage oocytes

Ruppert-Lingham, Carly Jan 2005. Cryopreservation and in vitro maturation of murine germinal vesicle stage oocytes. PhD Thesis, Cardiff University.

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Abstract

Cryopreservation of unfertilised oocytes for banking or oocyte donation would be a valuable adjunct to reproductive technology. As the mature oocyte contains a temperature-sensitive meiotic spindle, cryopreservation of immature germinal vesicle (GV) stage oocytes, which do not contain the spindle, may be a practical alternative. However, one of the major obstacles to the application of immature oocyte cryopreservation is the difficulty associated with in vitro maturation (IVM) of the thawed oocytes prior to in vitro fertilisation. The cumulus cells surrounding the oocyte are essential to oocyte maturation. Thus the aim was to assess survival and function of both oocyte and cumulus cells post-cryopreservation. Initially, culture conditions during IVM of murine GV stage cumulus-oocyte complexes (COCs) were modified. In the second part of the study, survival (morphological appearance and membrane integrity) and function (ability, in vitro, to mature, be fertilised and develop into blastocysts) of the oocytes and their associated cumulus cells was assessed following cryopreservation. An attempt was made to determine the stage of the protocol at which damage was incurred. Alterations to culture conditions had little impact on the ability of fresh GV stage oocytes to develop to blastocysts, although IVM in the presence of mixed ovarian cells was found to be detrimental. Treatment with 1.5M dimethyl sulphoxide (Me2SO) without freezing had little effect on the parameters investigated, unlike exposure to a 6M Me2SO solution. Slow-cooled/thawed or cumulus-denuded oocytes had decreased developmental potential when compared with control oocytes. Development was not improved by co-culture with fresh cumulus cells. Much of the damage caused to the cumulus cells occurred during plunging from -60 °C to -196 °C. Damage was reduced by cooling at 10 °C/min from -60 °C to -150 °C prior to plunging to -196 °C. However, embryo development was not improved. Vitrification of COCs led to substantial cumulus cell damage and very poor embryo development.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
Subjects:	R Medicine > RG Gynecology and obstetrics
ISBN:	9781303170478
Date of First Compliant Deposit:	30 March 2016
Last Modified:	25 Jul 2023 14:03
URI:	https://orca.cardiff.ac.uk/id/eprint/55591

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