257. Addition of glycine to vitrification solutions protects oocyte and embryo physiology and health

Abstract

Cryopreservation procedures for oocytes result in a significant reduction in viability. Although cryopreservation procedures cause dehydration and therefore osmotic stress, the role of osmolytes in solutions has not been considered and they have therefore not been included for routine use. The aim of this study was to assess the effects of the addition of the osmolyte glycine to vitrification solutions on the health and developmental competence of mouse oocytes. Oocytes were collected from F1 female mice and cryopreserved using cryoloop vitrification with or without glycine, with fresh oocytes examined as controls (n = 2086). Mitochondrial distribution and membrane potential as well as the morphology of the spindles and chromosomes were assessed. Oocytes were fertilised to assess their ability to develop into blastocysts, which were then assessed for their expression of Glut1, Glut3 and IGF2 by real-time RT-PCR. Statistical analysis was performed using a generalised linear model followed by multiple comparisons using an LSD test. Vitrification without glycine perturbed mitochondrial distribution (mean pixel intensity of outer region:inner region, 1.58±0.20, P<0.01) and mitochondrial membrane potential (mean pixel intensity 0.56±0.01, P<0.01) compared to control oocytes (2.34±0.24 and 0.52±0.01, respectively). The addition of glycine prevented these changes (1.97±0.16 and 0.53±0.01, respectively). Vitrification without glycine resulted in 52% of spindles and chromosomes appearing normal while this was increased to 69% with the addition of glycine, however in both treatments these abnormalities appeared to recover after culture for 2 h. Vitrification did not affect fertilisation and blastocyst development however expression of Glut3 was decreased 2.9 fold in blastocysts resulting from oocytes vitrified in the absence of glycine (P<0.01). The data presented suggests that the addition of glycine results in fewer perturbations in oocyte physiology and gene expression of the subsequent blastocysts and should therefore be considered for routine inclusion in solutions for the cryopreservation of oocytes.