The effect of slow-freeze versus vitrification on the oocyte: an animal model

Abstract

The goal of this study was to determine whether there is a deleterious effect on dynamic events in the nucleus and cytoplasm of oocytes in an animal model. Prospective experimental study. Bovine oocytes were matured in media supplemented with 75mIU/ml of a gonadotropins in vitro for 24 h and randomly frozen by either slow-freezing, SF, or vitrification with DMSO(V1) or vitrification with PROH(V2). Fresh oocytes comprised the control group(CL). Nuclear maturation status, spindle and chromosomal configurations, dynamic changes of the cortical granules(CGs) and mitochondria were evaluated by immunostaining and confocal laser scanning microscope. After thawing/warming, mitochondria formed larger clusters and migrated more centrally in all three cryo groups; CGs became more dispersed in the cytoplasm. Significant reduction in normal spindle and chromosomal configurations, was observed in all three cryo-groups as compared with CL. Global DNA methylation levels were significantly reduced in SF and V1 groups as compared to CL; however, methylation levels were significantly increased in the V2. Proportion of severely apoptotic oocytes was dramatically increased in all three cryo-groups, compared with CL.Table 1Dynamic changes of cytoplasm and nucleus of oocytes from slow-freeze and vitrificationParametersCLSFV1V2Dispersed CGs (%)35.6 ± 4.136.5 ± 4.246.7 ± 5.7∗P<0.0532.1 ± 9.0Clustered Mt(%)40.5 ± 5.373.0 ± 12.3P<0.01 (vs. Fresh).56.2 ± 7.9∗P<0.0552.6 ± 7.3∗P<0.05Normal Spindle (%)73.4 ± 7.655.7 ± 6.2P<0.01 (vs. Fresh).51.7 ± 7.9P<0.01 (vs. Fresh).39.3 ± 7.9P<0.01 (vs. Fresh).DNA Methylation (density)7.1 ± 0.54.6 ± 0.5P<0.01 (vs. Fresh).2.7 ± 0.3P<0.01 (vs. Fresh).11.4 ± 0.7P<0.01 (vs. Fresh).Oocyte Apoptosis (%)9.6 ± 0.624.1 ± 5.8P<0.01 (vs. Fresh).32.8 ± 5.8P<0.01 (vs. Fresh).44.1 ± 4.0P<0.01 (vs. Fresh).∗ P<0.05∗∗ P<0.01 (vs. Fresh). Open table in a new tab Overall, results demonstrate V1 is better in preserving cellular and nuclear integrity of the oocyte. V2 method makes the oocyte more vulnerable to increased DNA methylation, which is associated with imprinting disorders. In summary, V1 is recommended as the superior method to cryo-preserve oocytes clinically.