Oocyte cryopreservation - a cost effective method

Abstract

Embryo cryopreservation is a widely used and relatively well-established procedure. By contrast unfertilized mature metaphase (M il) oocyte freezing technique has not been developed as yet. The oocyte's surface to volume ratio, single membrane, temperature sensitive spindle and zona, susceptibility to parthenogenetic activation and chill injury have been proposed as contributory factors for the failure in attempts to freeze them. Embryo cryopreservation for human IVF programmes has raised a lot o f ethical issues making it necessary to develop oocyte freezing technology. Female albino mice aged 8-10 weeks were super ovulated by 5IU o f Pregnant Mare Serum Gonadotrophin (PMSG) followed by human Chorionic Gonadotrophin(hCG) at 46- 48 hours. 18-20 hours after the hCG injection, cumulus masses were retrieved and collected into M2 culture media. In the experimental group, mouse oocytes were cryopreseved with different concentrations o f DMSO, EG, glycerol with different concentrations o f sucrose. These oocytes with cumulus masses were placed in 0.5ml paillets which were cooled at 0°C for 20 minutes and transferred into liquid nitrogen vapour (LN2) for 5 minutes and were immersed in LN2 slowly before storing in LN2 tanks for one week. These experimental frozen oocytes were thawed and inseminated with sperms. Fresh oocytes with cumulus masses were used as controls and were inseminated with sperms. Both groups were incubated at 37° C with 5% CO2 for six hours. Presence o f two polar bodies or 2 pronuclei were taken as confirmation o f fertilization. Chi-Square test was used to compare the fertilization rates (FR) o f experimental with the control group. The FR for the control group was 72.74%. The highest FR o f 71.4% was obtained for the experimental oocytes cryopreserved in 3.5MDMSO with 0.2 5M sucrose. The best protocol giving a 71.4%) success rate at fertilization was by 3.5M DMSO with 0.25M sucrose, which is comparable to the control.