High pressure frozen oocytes have improved ultrastructure but reduced cleavage rates compared to conventionally fixed or vitrified oocytes.

Context: Live birth rates are lower for cryopreserved oocytes than for fresh IVF cycles, indicating a need for improved methodologies.

Aims: The aim of this study was to determine if high pressure freezing (HPF) could improve both ultrastructural preservation and cryopreserved oocyte quality when compared to conventional fixation and vitrification methods.

Methods: Sheep oocytes and embryos were prepared by HPF or vitrification, with or without cryoprotectants. Frozen oocytes were prepared for transmission electron microscopy or warmed, in vitro fertilised and the recovery and cleavage rates recorded.

Key Results: Blastocyst rates were similar between fresh, HPF and vitrified embryos. HPF oocytes had improved ultrastructure compared to conventional fixation or vitrification, but had poorer survival and cleavage rates compared to vitrified oocytes. Freeze-substitution of cryopreserved oocytes and transmission electron microscopy demonstrated disruption of the oocyte ultrastructure in the presence of cryoprotectants.

Conclusions: Superior preservation of ultrastructure was observed in HPF oocytes compared to vitrification or conventional fixation methods. In the presence of CP, both embryos and oocytes could survive HPF and warming but oocytes had reduced development.

Implications: The HPF method has potential to be developed and lead to improved oocyte and embryo cryopreservation and outcomes for assisted reproduction.