Embryological Data

The incubation conditions of the embryo culture were set at 6% CO₂, 5% O₂ and 37°C. The culture of the embryos was supported by the sequential media provided by Origio® (Cleavage medium and Blastocyst medium). Zygotes were scored on day 1 after ICSI, based on the presence of the 2 pronuclei (2PN) and their morphology (32). Embryo quality was assessed on the morning of day 3 according to the number of the cells, the equality of the blastomere size and the fragmentation rate. Embryos were also evaluated on day 5 according to the embryo scoring system of Gardner and Schoolcraft (33), regarding the formation and the morphology of the blastocysts. The quality of the blastocysts was characterized as top (grade 3-5AA, 3-5AB), good (3-5BA, 3-5BB) or fair (1-2AA, 1-2AB, 1-2BA, 1-2BB). The proportion of each embryo quality in every studied group is mentioned in Table 1.

The proportion of top, good, and fair quality blastocysts vitrified in the overall blastocyst number (N) per each group.

Sperm preparation and ICSI procedure were carried out as described by Van Landuyt et al. (34).

The cryopreservation of the blastocysts was conducted on the fifth or the sixth day of the culture. In the studied cases, there were no incidents of day 6 blastocysts to be included in the final vitrified blastocyst cohort. Vitrification and thawing of the cryopreserved blastocysts were performed with the Kitazato® Vitrification Kit and Kitazato® Warming Kit, respectively. The vitrification carrier used was the Cryotec straw by Cryotech® Japan.