Insemination, embryo quality assessment and cryopreservation
Fertilization was assessed 16-18 h after IVF/ICSI by the presence of two pronuclei, and further on, embryo development was evaluated daily until the cryopreservation of either cleavage-stage embryos (Day 3) or blastocysts (Day 5 and 6). Cryopreservation was performed by means of vitrification using a closed vitrification device with high-security straws (CBS-ViT-HS®; Cryobiosystems) using a combination of dimethyl sulfoxide and ethylene glycol as cryoprotectants (Irvine Scientific Freeze Kit®; Irvine Scientific). Day 3 embryos were evaluated based on the number and symmetry of their blastomeres, percentage of fragmentation, vacuolization, granulation and multinucleation. Based on all these parameters, an EQ score was assigned to all normally fertilized embryos using a predefined algorithm, which is divided into four categories: excellent, good, moderate, or poor. These four categories were used as defined by Racca et al. [The following embryos were considered eligible for cryopreservation: day 3 embryos with ≥ 6 blastomeres and ≤ 50% fragmentation; day 5 and 6, fully expanded or hatching blastocysts with a type A/B/C ICM and type A/B TE. Only transfers of single day 5 embryos of excellent quality were included as part of this study. Vitrified blastocysts were evaluated (Table 