4 well dishes

Zygotes were placed in pools in 4-well dishes (Thermo Scientific, Roskilde, Denmark), and embryos were cultured in pre-equilibrated Cleavage Medium (Cook) overlain with mineral oil using incubators (Minc, COOK, Bloomington, IN, USA) in a gas phase consisting of 5% O₂, 6–7% CO₂, balanced with N₂.
All cleaved embryos were morphologically evaluated under a conventional stereomicroscope using the integrated morphology cleavage score (IMCS) by Holte [17 (link)].
Embryo(s) transfer was performed using a soft catheter under ultrasound guidance. According to the policy of our IVF unit during the time period under study, one or two embryos were transferred on day 2/3 post-fertilization. Only embryos reaching the blastocyst stage were vitrified and thawed in subsequent cycles. The luteal phase was supported by administering 180 mg/day natural progesterone (Crinone 8fi, Merck, Darmstadt, Germany) for 15 days.
Pregnancy was assessed by serum hCG assay 15 days after embryo transfer (ET) and then confirmed if at least one gestational sac was visualized on transvaginal US after two further weeks.
Live birth was defined as the delivery of a live-born infant (>24 weeks of gestation). The cumulative live birth rate (CLBR) was defined as live deliveries (at least one live birth) per women including both fresh and frozen/thawed embryo transfers obtained from the same cycle.

Controlled ovarian stimulation was performed using a long, short, or gonadotropin‐releasing hormone (GnRH) antagonist protocol depending on the patient as described previously.¹⁶, ¹⁷, ¹⁸ A GnRH analogue acetate (Fuji Pharma Co., Tokyo, Japan), human menopausal gonadotropin (ASKA Pharmaceutical Co., Tokyo, Japan), and a GnRH antagonist (Cetrotide; Merck KGaA, Darmstadt, Germany) were administered as dictated in these established protocols. When at least 2 follicles reached 18–20 mm in diameter (as determined by transvaginal ultrasonography), 5000 IU of hCG (Fuji Pharma Co.) was administered. Oocyte retrieval was performed 36 h after hCG injection. The cumulus‐oocyte complexes were placed into 4‐well dishes (Thermo Fisher Scientific, Waltham, MA, USA) containing HFF99 medium (Fuso Pharmaceutical Industries, Osaka, Japan) with 10% serum protein substitute (Kitazato Corporation, Shizuoka, Japan) and cultured for 2 to 3 h until insemination.

The COCs were washed three times in maturation medium (TCM199 with Earle salts; Gibco, USA) supplemented with 10% FBS, 2 IU/mL FSH (Solarbio, Beijing, China), 0.5 IU/mL LH (Solarbio, China), 1 μg/mL 17β-estradiol (Sigma, USA), 0.3 mM sodium pyruvate, 100 µM cysteamine, and 50 μg/mL gentamicin. A group of 40–50 COCs were then transferred into 4-well dishes (Thermo, Waltham, MA, USA) containing 600 μL maturation medium covered with mineral oil (Sigma, USA). All dishes were prewarmed for at least 2 h in an incubator at 38.5 °C under 5% CO₂ in air before use. Then, the COCs were cultured in the medium for 24 h under the same conditions.