**4. Cleavage stage embryo**

Reports of human embryo vitrification have been more frequent. Liebermann and Tucker (2002) using either the cryoloop or the hemi-straw system (HSS) showed post-warming survival rates (after 2 hours of culture of day-3 embryos where more than half of their blastomeres were intact) from 84 to 90% which was dependent on the carrier system used. There was a reasonable further cleavage and compaction rate of 34%. This finding supports previous reports in which high survival rates of eight-cell human embryos using 40% EG were documented (Mukaida *et al*., 1998). In comparison to traditional slow-rate cryopreservation, a survival rate of cleavage stage embryos of 76% was reported with vitrification (Jericho *et al*., 2003). Recently reported successful pregnancies and deliveries after vitrification of day-3 human embryos using the OPS have been reported (El-Danasouri and Selman, 2001; Selman and El-Danasouri, 2002). Their results showed a negative correlation between stage of development and survival, eight-cell embryos showed a higher survival rate (79.2%; 62/78) than did embryos with fewer than six cells (21.1%; 11/53) after vitrification (El-Danasouri and Selman, 2001). Despite the fact, that Liebermann and Tucker (2002) achieved a promising post-warming survival rate, overall only about 34% of the surviving embryos had the developmental potential to reach the compaction stage. Recently publications on cleavage stage vitrification provided good outcome data. Loutradi *et al.* (2008) were performing a meta-analysis and systematic review by comparing traditional and vitrification protocols for cleavage stage embryos, and found a survival rate of 84.0% versus 97.0%. In addition, clinical pregnancy rates between 35 and 48%, with implantation rates between 15 to 39% have been reported (Rama Raju *et al*., 2005; Desai *et al*., 2007; Li *et al*.,

or vitrification technology (Chen *et al*., 2004; Bianchi *et al.,* 2005; Larman *et al*., 2007). The scientific literature on oocyte cryopreservation grows daily it seems. Most reports focus on clinical pregnancy rates (Boldt *et al.*, 2003; Boldt *et al*., 2006), and as such while this data is helpful to increase our confidence in the technology, it does little to research new directions

Conventional cryopreservation of pronuclear zygotes (2PN) is well established in countries such as Germany where freezing of later stage human embryos is by law or by ethical reasons not allowed. The time to complete the conventional protocol to cryopreserved zygotes is 98min. In Germany the clinical pregnancy outcomes arising from the frozen/thawed 2PN cycles is about 18%, with an implantation of around 10% per embryo transferred. The time to complete vitrification of zygotes requires approximately 12min. Recently successful vitrification of 2PN with high survival (~ 90%), cleavage rates on day-2 (>80%), and blastocyst formation of 31% and pregnancies were reported (Park *et al*., 2000; Jelinkova *et al*., 2002; Liebermann *et al*., 2002b; Al-Hasani *et. al*., 2007). Zygote vitrification implemented as a clinical setting can provide a clinical pregnancy rate of close to 30%, with an implantation rate of 17% (Al-Hasani *et al.,* 2007). The pronuclear stage appears well-able to withstand the vitrification and warming conditions, which is probably due to the significant membrane permeability changes that occur post-fertilization; such changes to the oolemma may also make it more stable and able to cope with the vagaries of the cold-shock

Reports of human embryo vitrification have been more frequent. Liebermann and Tucker (2002) using either the cryoloop or the hemi-straw system (HSS) showed post-warming survival rates (after 2 hours of culture of day-3 embryos where more than half of their blastomeres were intact) from 84 to 90% which was dependent on the carrier system used. There was a reasonable further cleavage and compaction rate of 34%. This finding supports previous reports in which high survival rates of eight-cell human embryos using 40% EG were documented (Mukaida *et al*., 1998). In comparison to traditional slow-rate cryopreservation, a survival rate of cleavage stage embryos of 76% was reported with vitrification (Jericho *et al*., 2003). Recently reported successful pregnancies and deliveries after vitrification of day-3 human embryos using the OPS have been reported (El-Danasouri and Selman, 2001; Selman and El-Danasouri, 2002). Their results showed a negative correlation between stage of development and survival, eight-cell embryos showed a higher survival rate (79.2%; 62/78) than did embryos with fewer than six cells (21.1%; 11/53) after vitrification (El-Danasouri and Selman, 2001). Despite the fact, that Liebermann and Tucker (2002) achieved a promising post-warming survival rate, overall only about 34% of the surviving embryos had the developmental potential to reach the compaction stage. Recently publications on cleavage stage vitrification provided good outcome data. Loutradi *et al.* (2008) were performing a meta-analysis and systematic review by comparing traditional and vitrification protocols for cleavage stage embryos, and found a survival rate of 84.0% versus 97.0%. In addition, clinical pregnancy rates between 35 and 48%, with implantation rates between 15 to 39% have been reported (Rama Raju *et al*., 2005; Desai *et al*., 2007; Li *et al*.,

and striking osmotic fluctuations that occur during the vitrification process.

for oocyte cryopreservation.

**4. Cleavage stage embryo** 

**3. Zygotes** 

2007; Balaban *et al*., 2008). So clearly vitrification appears to have a positive impact on overall embryo utilization. A study on the neonatal outcome of 907 vitrified/warmed cleavage stage embryos found no significant increase in the congenital birth defect rate when compared with pregnancies using fresh cleavage stage embryos (Rama Raju *et al*., 2009).
