**5. Slow freezing versus vitrification**

To compare slow freeze technology to vitrification, the efficiencies of cryopreservation must take into consideration several factors: (i) disparities in embryo quality between the "best" freshly transferred embryo and subsequent frozen embryos; (ii) lab-specific criteria for embryo cryopreservation may foster higher implantation rates by discarding some reproductive potential of lesser quality embryos; (iii) cryo-survival should be defined in terms of complete or partial survival and (iv) post-thaw selection criteria for the transfer of cryopreserved embryos.

Therefore, a randomized control study comparing slow freeze and vitrification protocols would require standardization of protocol under optimal conditions with sibling specimens. To add more complexity in comparative analysis are individual case variations, including age discrepancies, effects of hormonal stimulation, supplementation, and endometrial priming, all of which must be taken into account.

Despite these challenges in reviewing evidence based data, as a generality the technique of vitrification has been preferentially adopted over the more traditional approach of slow cooling.

Vitrification of oocytes [95, 96] and embryos of all stages has been shown to be superior to slow freezing [6]. A large amount of clinical data suggest that one of the major consequences of the intracellular damage to embryos from slow "conventional" freezing is decreased survival as well as diminished implantation potential and outcomes when compared to vitrification [97–99]. Despite lower survival rate, there are some data that suggest similar if not improved implantation rate with slow freeze technology with fully intact good quality day 3 embryos [100]. It is known that embryo survival is not an all or none phenomenon, and therefore, comparison should be stratified on a similar quality basis.

The lack of homogeneity in some reported data is anticipated and may be due to laboratory practice or clinic-specific differences, as with other ART procedures.

As the majority of early vitrification was with cleavage stage embryos, it was recognized that failure to develop to an expanded blastocyst stage was largely a consequence of chromosomal compromise and inability to lead to a successful outcome. A bifurcated movement to karyotype embryos through pre-gestational genetic screening and cryopreserve blastocysts rather than their cleavage stage counterparts is advantageous in the identification of embryo competence and in reducing risk of miscarriage and chromosomal defects [101, 102]. This practice is important in that common morphological parameters of blastocyst scoring are not related to chromosomal status [101] and particularly for women of advancing maternal age [103].

perfusion, due to capillary distribution and time requirement of CPA diffusion, may not be equivalent throughout a larger structure, random ice crystal growth can be lethal simply by mechanical disruption. By achieving deliberate ice growth in specific sites, the damaging effects of super-cooling and likewise intracellular ice formation can be mediated and poten-

Lastly as organisms synthesize solutes and metabolites in response to cold survival strategies (e.g., trehalose [91], glycerol [92], polyols [93, 94]), understanding how biological structures

To compare slow freeze technology to vitrification, the efficiencies of cryopreservation must take into consideration several factors: (i) disparities in embryo quality between the "best" freshly transferred embryo and subsequent frozen embryos; (ii) lab-specific criteria for embryo cryopreservation may foster higher implantation rates by discarding some reproductive potential of lesser quality embryos; (iii) cryo-survival should be defined in terms of complete or partial survival and (iv) post-thaw selection criteria for the transfer of cryopreserved

Therefore, a randomized control study comparing slow freeze and vitrification protocols would require standardization of protocol under optimal conditions with sibling specimens. To add more complexity in comparative analysis are individual case variations, including age discrepancies, effects of hormonal stimulation, supplementation, and endometrial priming, all

Despite these challenges in reviewing evidence based data, as a generality the technique of vitrification has been preferentially adopted over the more traditional approach of slow

Vitrification of oocytes [95, 96] and embryos of all stages has been shown to be superior to slow freezing [6]. A large amount of clinical data suggest that one of the major consequences of the intracellular damage to embryos from slow "conventional" freezing is decreased survival as well as diminished implantation potential and outcomes when compared to vitrification [97–99]. Despite lower survival rate, there are some data that suggest similar if not improved implantation rate with slow freeze technology with fully intact good quality day 3 embryos [100]. It is known that embryo survival is not an all or none phenomenon, and

The lack of homogeneity in some reported data is anticipated and may be due to laboratory

As the majority of early vitrification was with cleavage stage embryos, it was recognized that failure to develop to an expanded blastocyst stage was largely a consequence of chromosomal compromise and inability to lead to a successful outcome. A bifurcated movement to karyotype

therefore, comparison should be stratified on a similar quality basis.

practice or clinic-specific differences, as with other ART procedures.

interact with these mixtures may offer added benefits to current freezing regimes.

tially avoided.

148 Cryopreservation in Eukaryotes

embryos.

cooling.

**5. Slow freezing versus vitrification**

of which must be taken into account.

Over the past decade, with vitrification, it has become a standard of practice to expect a postthaw survival of >90% [104, 105] and implantation and pregnancy potentials marginally equivalent to fresh embryos [106–109].

Studies reveal longer gestational periods and heavier and healthier babies born as a result of frozen embryos compared to their fresh counterparts [110]. It is not clear whether this is related to cryotechnique or maternal factors [111]; but confirms the value of vitrification.

Reports of increased post-revitalization implantation potential over fresh counterparts [112] may be a consequence of staggered embryo transfers in which embryo procurement and implantation are performed in separate cycles. In this scenario, optimal synchronization and endometrial receptivity may be achieved in contrast to the impact of high levels of hormones present in harvesting cycles [113].

Success rates with vitrification supporting this revolutionary technology are not limited to gametes and embryos; however, extend to gonadal tissues, and non-reproductive applications including cornea [114], brain [115], heart [116], vascular [117] tissues, and cartilage [118]. The permeation of larger tissue sections and even whole organs, e.g., ovaries [119–122], shows promise in transplantation. Efficacy and potential of vitrification technologies as demonstrated through such a broad spectrum of applications justify its utility and warrant further investigation into enhanced cryopreservation potential.

Though the safety and efficacy of cryopreservation technologies is largely supported by current success rate, however, some degree of uncertainty and challenge remains.

Human embryonic stem cells (hESCs) have been established from isolated inner cell masses and more recently from single blastomeres obtained from cell stage embryos [123, 124]. The systemic reporting of chromosomal abnormalities and the recurrent manner in which they appear highlights the importance of understanding the underlying source [125]. In part, these changes are ascribed to the cryopreservation method, "adaptive pressure to" *or* "lab-specific variations in" cell culture [126–128] or are simply inherent to the cell itself [129–131]. Similarly, IVF embryos may be associated with increased risk of epigenetic abnormalities. At least in the case of hESCs, for cell line stability and quality assurance, the safety and efficacies of different cytogenetic methodologies have been assessed as they relate to genomic integrity and chromosomal stability [132]. As chromosomal instability is largely related to carcinogenesis, similar investigation into embryo cryopreservation methods may provide insight into the quality and safety of established cryopreservation protocols. Understandably, in as much as embryonic culture periods are acute in length (as compared to hESCs), still, the long-term effects of even small epigenetic changes are unknown.
