Stripper

Cumulus-oocyte complexes (COCs) were retrieved from follicular fluid and placed in preincubated fertilization medium (G-IVF™ PLUS, Vitrolife®). They were cultured at 37 °C under a gas mixture of 5% O₂, 6% CO₂ and 89% N₂ until insemination or denudation. For intracytoplasmic sperm injection (ICSI), oocytes were denuded using mechanical (Stripper, CooperSurgical®) and enzymatic methods (hyaluronidase: HYASE®, Vitrolife). Each semen sample was collected by masturbation in a sterile container after 2 days of abstinence. Sperm was prepared by density gradient centrifugation.

Oocyte retrieval was performed 35.5 h after hCG administration, with transvaginal ultrasound‐guided aspiration. Oocytes were collected from the follicular fluid and washed in freshly equilibrated human tubal fluid medium with HEPES (Multipurpose Handling Medium; Irvine Scientific, CA, USA). They were then incubated in an insemination medium (NAKA medical inc, Tokyo, Japan) covered with mineral oil (Light Mineral Oil for Embryo Culture; Irvine, Cal., USA) for 2–4 h with 6.5% CO₂/5% O₂/88.5% N₂ at 37°C in an incubator (MINC^™, COOK MEDICAL INC, IN, USA).
The oocytes were denuded of the cumulus oophorus by a brief exposure to 40 IU/mL hyaluronidase (ICSI Cumulase; Cooper surgical, Trumbull, USA), and a pipette (STRIPPER; Cooper surgical, Trumbull, USA) was used to mechanically remove the oocytes from the surrounding cumulus cells.
Denuded oocytes were incubated in single‐step medium (ONE STEP Medium; NAKA medical inc, Tokyo, Japan) covered with mineral oil.