The chromosome-segregating spindle can lose its shape when human oocytes are thawed after ultrarapid freezing
The chromosome-segregating spindle can lose its shape when human oocytes are thawed after ultrarapid freezing
On August 22, a preprint reported experiments with 234 human oocytes matured in the laboratory from immature cells. The authors tracked the stage at which the chromosome-segregating structure changes during ultrarapid freezing and thawing.
Vitrification is an ultrarapid freezing method that turns the water inside a cell into a glass-like state. Before cooling, the oocyte is placed in solutions containing cryoprotectants, substances that partially replace the water inside the cell. During thawing, the cryoprotectants are removed gradually, allowing water to reenter the cell.
The authors followed the same cells using polarized light microscopy, in which the ordered protein filaments of the spindle produce a light signal. The signal remained visible while the cryoprotectants were introduced, but disappeared as the solutions were diluted during thawing. Cell staining confirmed that the protein filaments remained present but lost their stable bipolar spindle structure.
The researchers tested whether water influx alone could cause this disruption. They reduced the osmolarity of the medium, meaning the concentration of dissolved substances, by half in fresh oocytes matured in the laboratory. Water entered the cells rapidly. Within eight minutes, the width of the spindle poles increased to approximately 160–180% of the initial value at n=5, while the chromosomes remained aligned during the first few minutes. In this experiment, rapid water influx alone disrupted spindle organization.
Differences between cells were already visible before cooling. In a 2019 study by the same group, the first polar body, a small fragment of the cell released during maturation, appeared before the spindle had fully formed. The researchers therefore grouped the cells in the new experiment according to their light signal before vitrification. Oocytes with a distinct bipolar signal were more likely to retain normal spindle shape after thawing. Cells with a weak or undetectable signal were more likely to lose this shape and showed poorer recovery.
Among 13 vitrified and thawed oocytes monitored for another 16 hours, 5 developed chromosome segregation toward multiple poles and formed multiple nuclei. In these experiments, the vulnerable stage of the cryopreservation cycle was the return of water during thawing, while spindle preservation depended on how mature the spindle was before cooling.