Conventional IVF and ICSI Protocols

For conventional IVF, HTF medium supplemented with 10% serum substitute (Irvine Scientific) was used as the fertilization medium.²⁶ (link) COCs were cultured with sperm (100,000 sperm/mL) at 5% CO₂ in air at 37°C. Extrusion of the second polar body was confirmed at 5 hours after insemination (day 0) following the removal of cumulus cells. The oocytes were individually cultured in EmbryoSlide (Vitrolife, Göteborg, Sweden), which is suitable for group cultures in 180 µL of medium (ONESTEP medium; Nakamedical Inc, Tokyo, Japan) under paraffin oil. In cases of ICSI, oocytes were inseminated in HEPES-buffered HTF medium.²⁸ (link) Injected oocytes were immediately placed onto EmbryoSlides and cultured individually in 180 µL of medium in paraffin oil. Embryos were cultured at 37°C (gas phase, 5% O₂, 5% CO₂, and 90% N₂) in an Embryoscope+ time-lapse incubator (Vitrolife) for 2 days. Fertilization was observed using the EmbryoViewer software (Vitrolife). Embryo vitrification and warming were performed using Cryotop (Kitazato Corporation) as described previously.²⁹ (link)