All women were stimulated with follitropin alpha (Hemeiqi®, Ferring, Germany), with daily injections of 150–300 IU. Pituitary suppression was performed with a GnRH antagonist (0.05 mg of Cetrorelix acetate, Tianxing®, Chengdu Tiantaishan Pharmaceutical Co., Ltd, China) administered daily from day 16 of stimulation. When three or more follicles of ≥18 mm of diameter were observed, final oocyte maturation was triggered with a dose of 6000-10000 IU of the human chorionic gonadotrophin (hCG, Livzon Pharmaceutical Group Inc, China). Oocyte collection was performed 36 hr later by means of ultrasound-guided transvaginal follicular aspiration.
Individual MII human oocytes were exposed to hyaluronidase (80 units/ml; Sigma-Aldrich, USA) and manipulated with a Stripper (Origio, USA) tip to remove all remaining cumulus cells, and then washed three times in Dulbecco's phosphate buffered saline with 0.01% polyvinylpyrrolidone (Sigma-Aldrich, USA). Samples were stored in PicoPure RNA extraction buffer (10 μl), heated to 42°C for 30 min, and subsequently stored at −80°C until total RNA purification as per manufacturer's protocol (Thermo Fisher Scientific, USA).
Individual MII human oocytes were exposed to hyaluronidase (80 units/ml; Sigma-Aldrich, USA) and manipulated with a Stripper (Origio, USA) tip to remove all remaining cumulus cells, and then washed three times in Dulbecco's phosphate buffered saline with 0.01% polyvinylpyrrolidone (Sigma-Aldrich, USA). Samples were stored in PicoPure RNA extraction buffer (10 μl), heated to 42°C for 30 min, and subsequently stored at −80°C until total RNA purification as per manufacturer's protocol (Thermo Fisher Scientific, USA).