Conventional agonist or antagonist stimulation protocols were used for ovarian stimulation as previously described (Cai et al., 2017 (link)). The initial and ongoing dosage was determined according to patients’ age, antral follicle count (AFC), BMI, and ovarian response. An intramuscular injection of human chorionic gonadotropin (4000–6000 IU, hCG; Livzen, China) or a subcutaneous injection of recombinant human chorionic gonadotropin (250 μg, Ovidrel, Merck-Serono, Switzerland) was administrated for final triggering when at least one follicle reached a mean diameter of 18 mm. Ovum puncture under transvaginal ultrasound guidance for oocyte retrieval was performed 34–36 h after hCG injection.
Routine IVF protocol in our center was carried out (Jiang et al., 2022 (link)). Cumulus-oocyte complexes were co-cultured with approximate 1.5–3 X 105 progressively motile spermatozoa in pre-equilibrated fertilization culture medium (K-SIFM, Cook) under mineral oil in traditional incubators (C200, Labotect) at 37°C, 6% CO2 and 5% O2 in a humidified atmosphere. After 4 h co-culture, oocytes were denuded and cultured individually in preequilibrated Cleavage Medium (K-SICM, Cook). The culture system and the procedure of semen preparation were kept unchanged in the period of study. Fertilization was determined according to the presence of two pronuclei (2 PN) about 17 h post insemination. It should be confirmed 2 h later if no obvious pronuclei could be observed.
Routine IVF protocol in our center was carried out (Jiang et al., 2022 (link)). Cumulus-oocyte complexes were co-cultured with approximate 1.5–3 X 105 progressively motile spermatozoa in pre-equilibrated fertilization culture medium (K-SIFM, Cook) under mineral oil in traditional incubators (C200, Labotect) at 37°C, 6% CO2 and 5% O2 in a humidified atmosphere. After 4 h co-culture, oocytes were denuded and cultured individually in preequilibrated Cleavage Medium (K-SICM, Cook). The culture system and the procedure of semen preparation were kept unchanged in the period of study. Fertilization was determined according to the presence of two pronuclei (2 PN) about 17 h post insemination. It should be confirmed 2 h later if no obvious pronuclei could be observed.
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