**6. The advantage of blastocyst cryopreservation**

Activation of the embryonic genome occurs after the 8-cell stage (3 days postoocyte retrieval) is reached (Braude *et al.,* 1988). If the activation does not occur, the embryo will not survive further. Therefore, the improvement of human IVF outcomes requires identification of embryos that will progress beyond the 8-cell stage. Blastocyst culture (5 days postoocyte retrieval) allows for the transfer of embryos that clearly have an activated embryonic genome. This requires that the elimination of embryos in extended culture from day 3 to day 5 should depend solely on their inherited survival potential and not be a consequence of an adverse effect exerted by the sequential media used for culture beyond day 3. Additional advantages in cryopreserving at the blastocyst stage are: 1) At this stage a lower numbers of embryos can be transferred in fresh cycles, resulting in less high order multiple pregnancies, 2) The same is true for cryopreserved blastocysts showing higher pregnancy rates and implantation per thawed embryo transferred, 3) Approximately 120 hours (day five) into development the healthy human embryo should be at the blastocyst stage comprised of some 50 to 150 cells, of which about 20 to 30% make up the inner cell mass (ICM), the remainder making up the trophectoderm (TE), 4) the higher cell number allows better compensation for cryo-injuries, which results in greater viability and faster recovery, 5) the cytoplasmatic volume of the cells is lower, thus the surface-volume ratio is higher, and that in turn makes the penetration of the cryoprotectant faster, and 6) on average fewer embryos per patient were frozen-stored, but each one when thawed has a greater potential for implantation.

Both natural and hormone replacement cycles seem to provide comparable levels of receptivity in naturally cycling women, though they differ in level of convenience. Regardless of the day of cryopreservation of the embryo (whether day 5, 6 or 7), at thawing/warming blastocysts should be treated as if they had been frozen on the fifth day of development. Vitrification of blastocysts has been undertaken utilizing an "open system" (Cryotop; Kitazato Bio Pharma Co. Ltd., Fuji-shi, Japan), and since 2007 on a "closed system" (HSV [High Security Vitrification Kit]; CryoBio System, L'Aigle, France) after a two-step loading with cryoprotectant agents at 24°C. Briefly, blastocysts were placed in equilibration solution, which is the base medium (Hepes-buffered HTF with 20% Serum Supplement Substition (SSS) containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO). After 5-7 min, the blastocysts were washed quickly in vitrification solution, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, and 0.5M sucrose, for 45-60sec and transferred onto the Cryotop or HSV using a micropipette. Immediately after the loading of

Vitrification of Oocytes and Embryos 177

When the vitrified-warmed blastocysts were divided into day 5 and day 6 groups, the following data was gather (Table 3 & 4). In 1265 FETs transferring day 5 blastocysts, the survival, implantation, and cPR were 97.6%, 34.8%, and 48.3% compared to 97.2%, 25.3%,

Patient's Age < 35 35-37 38-40 > 40 Donor Total Ø Age 30.8±2.6 35.8±0.8 38.8±0.8 42.8±2.0 43.5±4.7 34.6±5.2 Day 5 Cycles 678 248 157 74 112 1269 Day 5 Transfers 677 247 155 74 112 1265

Embryos survived (%) 97.5% 96.9% 98.7% 96.0% 97.7% 97.6
