P-206: Non-immersion vitrification - a viable alternative to slow freezing of blastocysts

Abstract

Slow freezing of human blastocysts is a widely used technique in ART but is often associated with a high rate of cell lysis leading to decreased embryo viability. It is time consuming and also has high equipment costs. In contrast, vitrification reduces the risk of ice crystal formation hence reducing cell lysis, has shown promising implantation rates in many species including human, is rapid and relatively inexpensive. In this ongoing study we compare the preliminary clinical results using the Cryologic Vitrification Method 1 (CVM 1) with historical data using a standard slow freezing procedure. Oocytes were fertilised and cultured in the Sydney IVF suite of sequential media (Cook IVF, Brisbane) for up to 144 hours. Day 5/6 supernumerary blastocysts produced prior to January 2006 were frozen using a programmed control rate freezer (Planer, UK) in Sydney IVF blastocyst freezing media. Slow freeze embryos were passaged through a series of ambient temperature solutions containing glycerol (9%) and sucrose (0.2M) in the final solution, transferred into embryo freeze straws, placed into a programmed freezer and then seeded manually. From January 2006 all blastocysts were vitrified In Sydney IVF Vitrification Media using the CVM 1 kit (Cryologic, Australia). Embryos were passaged through a series of vitrification solutions at 37°C, with the final solution containing trehalose (0.68 M), ethylene glycol (16%) and DMSO (16%) . Within 30 to 40 seconds in this final vitrification solution blastocysts were loaded in a 2-3 μl drop onto a fine plastic hook at the end of a fibreplug. This microdrop was vitrified by placing it onto the surface of the liquid nitrogen pre-cooled metal vitrification block and were then stored in sealable plastic straws. The blastocysts did not have direct contact with liquid nitrogen at any stage. Straws containing the slow freeze embryos were thawed in air for 30 seconds then in a water bath (30°C) for 30 seconds and then were transferred into a series of thawing solutions containing varying concentrations of sucrose at ambient temperature. Vitrified blastocysts were thawed by plunging the hook containing the vitrified embryo directly into the warming solution at 37°C. Historically, most pregnancies (55%) obtained using our slow freezing protocol were from embryos that had 90 -100% cell survival. Our 2005 data indicates that 41% (432/1047) of slow frozen embryos were in this survival band. Preliminary 2006 data indicates that a higher embryo survival rate of 79% (19/24) of vitrified embryos had cell survival of 90% or greater, (p < 0.005). To date we have obtained 10 +βhCG pregnancies from 18 embryo transfers (56%, 1.1 embryos per transfer). Comparative data from slow frozen embryos in 2005 gave a +βhCG pregnancy rate of 34% (222/658), transferring on average1.3 embryos, (p=0.095). These results indicate that non-immersion vitrification of human blastocysts could provide a safe and viable alternative to current slow freezing protocols. High post-thaw survival and pregnancy rates are necessary to encourage an increase in the proportion of cases where single embryo transfer (SET) is the preferred protocol to avoid multiple pregnancies [Fertil. Steril. 2005;84: 1620].