G1 plus medium

This study was performed at the first IVF/Intracytoplasmic Sperm Injection (ICSI) cycles after injection of 3.75 mg triptorelin for prolonged pituitary downregulation in the follicular phase of the menstrual cycle. Ovarian stimulation with exogenous gonadotropins promoted the growth of follicles. When two or more leading follicles reached 18 mm, ovulation was induced with 10,000 IU human chorionic gonadotropin (hCG). Oocyte retrieval was performed at 35 h post-hCG administration. Cumulus-enclosed oocytes were separated from the follicular fluid, placed in a medium, and incubated at 37°C incubated d Cumulus-encl₂ atmosphere for 2 h. Routine IVF or ICSI was performed based on sperm quality. The embryos were placed in droplets of G-1 PLUS medium (Vitrolife, Göteborg Sweden) in AMP-30D incubators (Bioz, Los Altos, CA, USA) in a 6.0% CO₂ and 5% O₂ balance N₂ atmosphere at 37°C.

Human embryos were obtained from the Karolinska University Hospital, Huddinge and from the Carl von Linné Clinic, Uppsala, either frozen at embryonic day (E) 2 or from preimplantation genetic diagnosis testing at E4, with informed consent from donating couple and with ethical approval for these experiments to F.L. from the Regional Ethics Board, Stockholm (2012/1765-31/1). Frozen embryos were thawed with ThawKit Cleave (Vitrolife) into G-1 Plus medium (Vitrolife) covered with Ovoil (Vitrolife) and from E3 embryos were cultured in G-2 Plus medium until E6-7 in 5% O₂, 5% CO₂ at 37°C.

We had access to 344 samples of G1 Plus medium (Vitrolife, Sweden) from day 3 of embryo culture. The concentration of sHLA-G (U/ml) in the medium was tested with a sandwich enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer’s protocol (Exbio/Biovendor, Czech Republic). The standard curve measured the concentration of sHLA-G1 and sHLA-G5 isoforms from 3.91 to 125 U/ml. The detection limit of sHLA-G in this assay was 0.6 U/ml.

Mice were housed in a temperature-controlled environment under a 12 h light: 12 h dark cycle. All animal procedures complied with the Animal Care and Use Committee of Huazhong Agriculture University (HZAUMO-2016-031). Fertilized embryos were obtained from C57BL/6 females mated with DBA/2. For SCNT, metaphase II (MII) oocytes were collected from 8–10 weeks old female B6D2F1 (C57BL/6×DBA/2) mice. SCNT was performed as previously described [22 (link)]. Briefly, the spindle of oocyte was removed by the Piezo-driven (Prime Tech, Japan) enucleation pipette on the Olympus inverted microscope, and the nuclei of cumulus cells were directly injected into the enucleated oocytes. The reconstructed oocytes were cultured in CZB medium for 1 h before activation treatment. The cloned constructs were activated in Ca²⁺-free CZB medium supplemented with 10 mM SrCl₂ (Sigma, 255521) and 5 μg/ml cytochalasin B (Sigma, C-6762) for 6 h. Cloned and fertilized embryos were cultured in G1-plus medium (Vitrolife, 10132) at 37 °C in an atmosphere of 5% CO₂ in air.