Cryopreservation
Introduction. Cryopreservation is a process by which cells or tissues are preserved at -196 °C. Vitrification is a cryopreservation technique that allows human gametes and embryos to be preserved for a long time while maintaining structure and function. In the United States, a cryopreserved embryo has been successfully implanted for twenty years. Since 1987, a new technique, vitrification, has been reported as an alternative to ‘slow’ freezing. It bases its principle on the fact that the water in the cells does not have time to form ice crystals, thanks to the extreme cellular viscosity achieved by cryoprotective agents and the high speed at which the temperature is lowered. The general principle behind this procedure is that the higher the freezing or thawing speed, the lower the concentration of cryoprotectant (potentially toxic to the cell) needed to protect cells from forming ice crystals.
Indications. There are several potential applications of the oocyte cryopreservation technique. These include patients choosing to preserve their gamete heritage in order to preserve their fertility. This is the case for instance in cancer, where therapies can destroy the ovarian heritage, and in patients undergoing Assisted Fertilisation techniques. Oocytes are unique cells: their large size, spherical shape and general fragility explain many of the difficulties encountered during cryopreservation. Oocytes are particularly sensitive to cooling and are susceptible to cryogenic injuries.
How it is performed. Vitrification is now the ‘elective technique’ for cryopreservation of oocytes and embryos and has replaced (since around 2006) the slow freezing technique, providing extremely high results in terms of survival and recovery of vitrified gametes and embryos. Today, thawing survival of cryopreserved embryos reaches 98%. This is in fact a widely studied and safe methodology, since at cryogenic temperatures, i.e. below -180°C, all biochemical and physical cellular phenomena are suspended, a sort of hibernation. It is also estimated that the average life span of biological material properly stored in liquid nitrogen (-196.5°C) is approximately 1000 years.
The advent of this methodology has provided the field of Medically Assisted Reproduction with a fundamental tool as it allows both male and female fertility preservation and embryo cryopreservation to be made safe.
Cryopreservation is performed not only on supernumerary embryos but also on those subjected to trophectoderm biopsy (PGT) and those generated by heterologous fertilisation treatments.
Conclusions. Oocyte freezing survival is very high, with the use of appropriate technical adjustments, and can be in the order of 75-100%. The fertilisation rate can reach 90% and the pregnancy rate obtained after freezing of oocytes, fertilised oocytes, embryos or blastocysts is of the order of 25-55%. Several births of healthy babies have been reported in the literature since 1999. Recent data seem to reassure about the outcomes of this procedure. The scientific literature is divided on the efficacy of the method, but it has been suggested that vitrification may be less traumatic on the meiotic spindle and other cellular and embryonic structures than ‘slow’ freezing.
