Vitrification versus slow freezing of human oocytes: Effects on ultrastructure and developmental potential

Abstract

Objective: This study compared spindles, cytoskeleton, and developmental potential between vitrified and slow-frozen oocytes using PolScope and electron microscopy. Methods: Oocytes were randomly divided into control, slow freeze-thaw, and vitrification freeze-thaw groups (0, 1, and 3 h). PolScope was used to observe spindles, angle of spindles to the first polar bodies, surface areas of oocytes, and lining and outer ret of zona pellucida. Surfaces and ultrastructure of oocytes were observed by scanning electron microscopy and transmission electron microscopy. These measures were used to characterize the impacts of two freezing methods on the developmental capacity of human oocytes. Results: The visible frequency of spindle formation was 92.4%, 56.4%, 11.2%, 24.8%, and 61.1% in control group, slow freeze-thaw group, and the three vitrification freeze-thaw groups (0, 1, and 3 h), respectively. Compared to oocytes in the slow freeze group, the angle of the spindle to the first polar body in oocytes in the 3-h vitrification freeze-thaw group was less (37.3° vs. 68°, P = 0.023). No significant differences were observed between the surface area of oocytes, or lining and outer ret of oocyte zona pellucida between freeze-thaw in these same two groups. Microvilli appeared normal. However, protrusions on oocyte surfaces were increased, and microvilli were laid down on the membrane surface in the 3-h vitrification freeze-thaw group in comparison to the slow-freeze group. Similar comparisons showed better recovery of perivitelline space and mitochondria between the 3-h vitrified and slow-frozen groups. Bipronuclear (2PN) fertilization rate observed in the slow-freeze group (65.7%) was lower than the rate seen in controls (79.2%, P = 0.041). No significant differences were observed in 2PN fertilization, cleavage, and blastocyst formation rates between the 3-h vitrification freeze-thaw and control groups. Conclusions: Results suggest that vitrification freeze-thaw for oocyte cryopreservation was a better choice than slow freeze-thaw.

Keywords

• Human Oocyte; Meiotic Spindle; Slow Freezing; Vitrification; Zona Pellucida