In Vitro Follicle Culture with NT4

Basic culture media consisted of αMEM (Gibco, Carlsbad, CA, USA) containing 6% SPS, 1 μl/ml insulin–transferrin–selenium (ITS), 1 mg/ml fetuin, and 10 mIU/ml rhFSH (Gonal-f, MERCK, Darmstadt, Germany). Follicles from each patient were randomly assigned to two study groups and transferred to the corresponding culture media: the control group with basic culture media, and the NT4 group supplemented with 100 ng/ml human NT4 (PeproTech, Rocky Hill, NJ, USA) (Application number of national invention patent of China: 202211330660.7). The chosen concentration of 100 ng/ml was based on a previous study in mice, which was shown to be superior in improving follicle diameter and oocyte maturation in secondary follicle IVG (Guo et al., 2021 (link)). Experiments for the two groups were conducted in parallel. Each follicle was individually incubated in the wells of round-bottom ultra-low attachment microplates (Catalog #7007, Corning, New York, NY, USA) in 200 μl of pre-equilibrated and pre-warmed culture medium at 37 °C and 5% CO₂ for 4–6 weeks. Medium refreshment was performed every other day by replacing half of the corresponding culture media. The spent medium was collected and stored at −80°C for subsequent assays.