Mco 5m

For hypoxia treatment, Neuro-2a cells were incubated in an O₂/N₂/CO₂ incubator (MCO-5M, Sanyo, Japan) and exposed to 1% O₂ for the indicated time periods. The basal medium containing 25 mM glucose (control conditions, “C”) met the high metabolic requirements of neuronal cells. For high-glucose analyses(“G”), the medium was replaced by fresh medium with an additional 75 mM of glucose (Sigma,USA) to yield a total of 100 mM glucose. The osmolarities of all of media were adjusted to 100 mM by adding different amounts of mannitol (Sigma, USA).

AMSCs and BMSCs were isolated and cultured according to our previous study [26 (link)]. Briefly, AMSCs from the inguinal adipose tissue were collected and digested by type I collagenase (0.2%, Sigma, USA), filtered by 200-mesh sieve, centrifuged at 350×g for 5 min, resuspended in DME/F12 complete medium containing 15% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin/amphotericin B (Cellmaxin plus, Gendepot, USA), and plated onto 10-cm cell culture dishes at 37 °C with 5% CO₂. BMSCs were isolated from bone marrow of the femora which was flushed out with the DME/F12 complete medium. After repetitively pipetting, the bone marrow was plated onto 10-cm cell culture dishes at 37 °C with 5% CO₂. The medium was changed every 2 days and the cells (passage 0, P0) were subcultured when 80–90% confluence was reached. The P3 cells were used in the experiment.
In hypoxic condition described in our previous study [26 (link)], MSCs were cultured in DME/F12 complete medium for 7 consecutive days in a tri-gas incubator maintained 1% O₂, 5% CO₂, and 94% N₂ (MCO-5M, SANYO, Japan). Whereas in Tgf-β1 induction, MSCs were cultured in complete medium containing 10 ng/ml Tgf-β1 (Sigma, USA) for 7 consecutive days. The complete medium was changed every 2 days.

Under hypoxia condition, cells were cultured in complete medium in a trigas incubator maintained at 1% O₂, 5% CO₂, and 94% N₂ (MCO-5 M, SANYO, Japan) for 7 consecutive days. The medium was changed every 2 days. Whereas in Tgf-β1 condition, cells were cultured in complete medium containing 10 ng/ml Tgf-β1 (Sigma, USA) for 7 consecutive days. The medium containing Tgf-β1 was also changed every 2 days.

Bovine aortic endothelial cells (BAECs) were purchased from TOYOBO (Osaka, Japan) and used in passages 2–6. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako, Osaka, Japan) with 10% fetal bovine serum (FBS) and antibiotic-antimycotic mixed stock solution (Nacalai Tesque, Kyoto, Japan). Cells were maintained in 5% CO₂, 95% air at 37°C and incubated with 5.5 mM glucose DMEM. For hypoxic conditions, cells were placed in a multi-gas incubator MCO-5M (SANYO, Osaka, Japan) that was flushed with 1% O₂, 5% CO₂, balance N₂ at 37°C.
Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), rotenone, and antimycin A were from Sigma-Aldrich Japan (Tokyo, Japan). Hydrogen peroxide was from Santoku Chemical Industries (Tokyo, Japan).
BAECs were incubated for 16 h in 0.4% FBS before the experiments. During the experiments, cell survival was monitored using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Tokyo, Japan) and no changes in cell viability were observed (data not shown).

For IVF procedures, oocytes were inseminated with motile spermatozoa at a concentration of 10,000 per ml. For ICSI, oocytes were microinjected with sperm 4–6 h after oocyte retrieval. The fertilized oocytes were transferred into pre-equilibrated culture dishes (Thermo Scientific, Waltham, MA, USA) with 25 μl of culture medium (Vitrolife Sweden, Gothenburg, Sweden) covered with 1.2 ml of paraffin oil (Vitrolife Sweden). The embryos were cultured in an incubator (MCO-5 M, Sanyo, Osaka, Japan) at 37 °C with 5% O₂ and 6% CO₂ until ET on day 3.
Embryo morphology was assessed according to the guidelines of the European Society of Human Reproduction and Embryology/Alpha consensus [50 (link)]. Embryos were graded according to blastomere number, blastomere size, and degree of fragmentation, using a morphological scoring system, accounting for the regularity of the blastomeres, degree of fragmentation, and microscopic appearance of the embryos. Transferable embryos were defined by the presence of 6–10 symmetrical blastomeres and < 20% fragmentation, with no multinucleation, on day 3.