A Novel Oocyte Cryopreservation Method using High Pressure Freezing

Abstract

Oocyte cryopreservation (egg freezing) involves freezing a female’s oocytes in order to preserve her fertility. These oocytes can later be thawed and fertilised in vitro when she is ready to begin fertility treatment. Women rely on this technology to preserve their fertility before undergoing gonadotoxic cancer treatment. Oocyte cryopreservation can also be used to delay parenthood so that women can pursue higher education and build their careers. This technique can also allow the storage and transportation of donor oocytes, making donor eggs accessible to more infertile couples.

The current method used for oocyte cryopreservation is termed vitrification. Assisted reproduction with vitrified oocytes is inefficient and results in significantly fewer pregnancies and live births than those which are achieved using fresh (non-frozen) oocytes. Moreover, vitrification causes damage to oocytes, and may result in premature zona hardening. This is thought to prevent normal fertilisation and creates a need for fertilisation by intra cytoplasmic sperm injection rather than standard in vitro fertilisation which increases costs and can have risks. Oocytes are generally vitrified at the mature stage following a two-week hormone treatment cycle that stimulates them to mature in vivo. This hormone treatment is contraindicated in some patients, including cancer patients with hormone sensitive cancers. Immature cumulus oocyte complexes (COCs) can be aspirated directly from the ovaries without hormone treatment, however, these COCs must first be stripped of their cumulus cells due to the size limitations of vitrification. Vitrification of immature oocytes without their cumulus cells is not very successful because the cumulus cells are required for successful maturation and fertilisation of these oocytes.

High pressure freezing (HPF) is a cell preservation technique that has been shown to provide superior ultrastructural preservation when compared to conventional vitrification methods and has a larger upper size limit that would allow immature COCs to be frozen with their cumulus cells intact. This research aimed to determine whether HPF could provide a superior oocyte cryopreservation method compared to vitrification and allow immature COCs to be frozen and retain their developmental capacity.

Immature and in vitro matured sheep oocytes were frozen using either HPF (N = 164) or a conventional vitrification method (N = 270). Groups of frozen oocytes were then thawed, in vitro matured (where appropriate), in vitro fertilised, and cultured for 8 days to compare oocyte recovery after thawing, oocyte cleavage, and embryo development. Another group of oocytes were thawed, fertilised, and stained using DAPI to visualise pronuclei and sperm attached to the zona in order to compare fertilisation status and determine whether the zona reaction had occurred prematurely or not. Finally, the remaining oocytes were thawed, in vitro matured, fixed, embedded, and sectioned to observe their ultrastructure under a transmission electron microscope.

Recovery rates after thawing were significantly lower in HPF oocytes compared with vitrified oocytes (P=0.001), as were cleavage rates (P<0.001). DAPI staining showed that neither HPF nor vitrified oocytes were affected by polyspermy as no extra nuclei were present after fertilisation. Furthermore, all fertilised oocytes had sperm bound within their zona pellucida, indicating that the zona reaction had not occurred prematurely. Ultrastructural investigation using transmission electron microscopy showed that there were no striking differences between the two groups of oocytes, however, all oocytes showed signs of cryopreservation-related damage including an absence of cortical granules and evidence of vesicle coalescence and enlargement. To conclude, high pressure freezing did not improve oocyte developmental capacity or ultrastructural preservation after freezing, however, further research is required to determine whether a more rapid thawing method could improve oocyte cryopreservation outcomes after HPF.