Effects of vitrification and slow freezing on ultrastructure and developmental potential of human oocytes

Abstract

Objective To compare the the spindles, cytoskeleton and the developmental potential of human oocytes between vitrification and slow freezing approaches by polscope and electron microscopy. Methods The immature human oocytes were randomly divided into control, slow freezing, and vitrification freezing-thawing groups (0 h, 1 h, 3 h after thawing). The spindle, the angle of spindle to the first polarbody, the surface area of oocytes and the lining and outer retardance of zona pellucida were observed by Polscope. The surface and ultrastructure of oocytes were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Finally, the influences of two freezing methods on developmental capability of human oocytes were analyzed. Results The visible rate of spindle was 92.4%, 56.4%, 11.2%, 24.8%, 61.1% in control group, slow freezing-thawing group, and vitrification freezing-thawing after 0 h, 1 h and 3 h, respectively. Compared with slow freezing, the angle of the spindle to the first polar body in vitrification freezing-thawing after 3 h group was smaller (37.3°, 68°, P=0.023). No significant differences were observed in the surface area of oocytes, the lining and outer ret of oocytes zona pellucida between vitrification freezing-thawing after 3 h group and slow freezing group. The protrusions of oocyte surface were increased, the microvilli were normal, and laid down on the membrane surface in vitrification freezing-thawing after 3 h group than slow freezing group, and similar results of better recovery of perivitelline space and mitochondria were obtained. The 2 pronucleus (PN) fertilization rate in slow freezing group (65.7%) was decreased compared with control group (79.2%, P=0.041). No significant differences were observed between vitrification freezing-thawing after 3 h group and control group in 2PN fertilization rate, cleavage rate and blastocyst formation rate. Conclusion Preliminary results suggest that the vitrification freezing-thawing for oocyte cryopreservation is a better choice than slow freezing-thawing. Key words: Vitrification; Slow freezing; Human oocyte; Meiotic spindle; Oocyte zona pellucida