Abstract
Nowadays, human oocytes/embryos are cryopreserved via slow freezing or vitrification. The aim of this study was to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification. This was a prospective study on 216 sibling oocytes randomized for either conventional rapid thawing or rapid warming with vitrification warming solution. The primary endpoint was morphological assessment of survival at 2h. Surviving oocytes were divided into two subgroups: (i) parthenogenetically activated; and (ii) fixed and observed for spindle/chromosome configuration. Secondary endpoints were parthenogenetic development and spindle/metaphase configuration. Survival rate with rapid warming was higher (92/102, 90.2%) than with rapid thawing (85/114, 74.6%; P=0.005), and after 3d of culture the rapidly warmed parthenotes had more blastomeres compared with those rapidly thawed (P=0.042). Meiotic spindle and chromosomal configuration were not significantly influenced by rapid warming or rapid thawing. The finding of this study allows IVF centres to increase the efficiency of oocyte slow freezing, enabling survival rates comparable to vitrification protocols, and potentially to optimize costs by using the same warming protocol for both slow-frozen and vitrified reproductive cells.Nowadays, human oocytes/embryos are cryopreserved via slow freezing or vitrification. Due to the high survival rate guaranteed by vitrification, this procedure is increasingly applied worldwide. Nevertheless, to date, perhaps millions of slow-frozen oocytes/embryos have already been stored in IVF cryobanks. The aim of this study is to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification in order to optimize the slow freezing survival rate and reduce costs by using the same solutions for both slow freezing and vitrification warming. Between December 2012 and January 2013, 216 slow-frozen oocytes donated for research were randomized for the rapid thawing conventionally used for slow-frozen oocytes, or rapid warming as for vitrification protocols. We observed that rapid warming significantly increased the survival rate of slow-frozen oocytes; furthermore, slow-frozen oocytes warmed via rapid warming had better developmental competence than their sibling counterparts thawed with conventional rapid thawing. The application of this warming protocol allows us to increase the efficiency of oocyte slow freezing procedure, enabling survival rates comparable with that reported in previous vitrification studies. This finding redefines the scenario of slow-frozen and vitrification, confirming the pivotal role of warming and indicating that the results obtained until now with slow-frozen oocytes/embryos can be improved; this could be the biggest breakthrough in human oocyte cryopreservation since the introduction of vitrification. Furthermore, this finding allows IVF centres to reduce costs by using the same ‘universal warming protocol’ for both slow-frozen and vitrifed reproductive cells.
Highlights
Nowadays, in assisted reproduction laboratories, human oocytes and embryos are cryopreserved by two main methods: vitrification or slow freezing.During vitrification, the cells are converted to a glassy state without ice-crystal formation by using a viscous medium with a very high concentration of cryoprotectants (Rall and Fahy, 1985; Yavin and Arav, 2007)
The aim of this study is to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification, to optimize the survival rate and reduce costs by using the same solutions for both slow freezing and vitrification warming
Survival rate was 90.2% (92/102) in the rapid warming group versus 74.6% (85/114) in the rapid thawing group (P = 0.005)
Summary
In assisted reproduction laboratories, human oocytes and embryos are cryopreserved by two main methods: vitrification or slow freezing.During vitrification, the cells are converted to a glassy state without ice-crystal formation by using a viscous medium with a very high concentration of cryoprotectants (Rall and Fahy, 1985; Yavin and Arav, 2007). During slow freezing due to the progressive permeation of some cryoprotectants as the ice crystals form in the cryopreservation solution, a glassy/vitrified state is obtained within the cell throughout the cooling procedure (Vanderzwalmen et al, 2013) For this reason, since both vitrified and slow-frozen oocytes have a vitrified cytoplasm, it can be postulated that the same warming protocol can be used for both. In most vitrification protocols or commercial kits, the cryoprotectant concentration in the first warming solution is approximately 1 mol/l (The Alpha Consensus Meeting, 2012), which is higher than the cryoprotectant concentration in both the freezing solution and the first thawing solution for rapid thawing of slow-frozen oocytes or embryos (e.g. for oocytes: 0.2–0.3 mol/l in freezing solution, 0.3–0.5 mol/l in thawing solution; Bianchi et al, 2007; Boldt et al, 2006; Fabbri et al, 2001; Parmegiani et al, 2008a). This suggests that vitrification warming solution could be used for thawing slow-frozen oocytes/embryos
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