Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation

The demand for cryopreservation of human oocytes is increasing in assisted reproduction clinics and yet remains an experimental procedure. Surprisingly, little is known about the effects of cryopreservation on spindle-chromosome interactions and the recovery of meiotic spindle functionality. The goal of these studies was to evaluate the process of meiotic spindle reassembly and chromosome alignment in cryopreserved human metaphase II oocytes. Unfrozen control oocytes were compared with frozen oocytes fixed at 0, 1, 2 and 3 h after thawing. Oocytes were analysed by confocal microscopy and subjected to 3-dimensional image analysis to evaluate spindle integrity. Freezing resulted in a loss of spindle bipolarity and chromosome alignment. One hour following thawing, most oocytes recovered spindle bipolarity and equatorial chromosomal alignment. However, between 2 and 3 h, a progressive loss of chromosome alignment was observed. Further analysis revealed a positive correlation between spindle length and number of displaced chromosomes following freezing. This time-dependent redistribution of chromosomes involved outward displacement from the equatorial plate and retention at the surface of the meiotic spindle. Spindle disassembly incurred by cryopreservation is rapidly reversed and is coordinated with chromosome alignment within 1 h but is not sustained at later times.