The details for ovarian stimulation, oocyte retrieval, IVF/ICSI procedures, and embryo culture have been described elsewhere (Huang et al., 2019 (link)). hMG (Anhui Fengyuan Pharmaceutical) was started on day 3 of the menstrual cycle. Urinary hCG (Lizhu Pharmaceutical Trading Co., China) was administered at a dose of 5,000 IU when two or more follicles measured 18 mm or more. Serum FSH, LH, E2, and P levels were analyzed on spontaneous menstrual cycle day 3 (MC3) and the day of hCG triggering. Oocytes retrieval was scheduled at 34–36 h after trigger and IVF or ICSI was performed 4–5 h after oocytes retrieval.
For IVF, collected oocytes were incubated in human tubal fluid (HTF; Irvine Scientific, United States), supplemented with 10% serum substitute supplement (SSS; Irvine Scientific, United States), and 300,000 progressively motile spermatozoa and left overnight; For ICSI, denudated oocytes were injected with a single mechanically immobilized sperm and directly thereafter cultured in fertilization medium (HTF + 10% SSS). All embryos were maintained in Continuous Single Culture of HTF (Irvine Scientific, United States) and were incubated under oil at 37°C and a 5% O2 and 6% CO2 humidified incubators with 30 μL of culture media drop throughout the entire duration of in vitro culture.
For IVF, collected oocytes were incubated in human tubal fluid (HTF; Irvine Scientific, United States), supplemented with 10% serum substitute supplement (SSS; Irvine Scientific, United States), and 300,000 progressively motile spermatozoa and left overnight; For ICSI, denudated oocytes were injected with a single mechanically immobilized sperm and directly thereafter cultured in fertilization medium (HTF + 10% SSS). All embryos were maintained in Continuous Single Culture of HTF (Irvine Scientific, United States) and were incubated under oil at 37°C and a 5% O2 and 6% CO2 humidified incubators with 30 μL of culture media drop throughout the entire duration of in vitro culture.
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