Human serum albumin (hsa)

Freshly isolated PBEos (10⁶ cells/100 µL) were pre-incubated in media (RPMI-1640 [ATCC; Manassas, VA, USA] supplemented with 25 mM HEPES and 0.1% human serum albumin [Irvine Scientific; Santa Ana, CA, USA]) for 30 min followed by a 1-hour treatment with media, the cholesterol chelator MβCD (5 mg/mL), or soluble cholesterol (MβCD+Chol, 5 mg/mL), which is MβCD pre-loaded with cholesterol (or concentrations indicated in Figure 1 legend). All incubations were at 37°C.

Pre-patterned wt (XM001) and NES-mScarlet VM/mDA NSCs/NPCs grown in 6-well dishes at d11 and d23 of the modified differentiation protocol were harvested and dissociated in Accutase to obtain a single-cell suspension. Cells were resuspended in 2% human serum albumin (#9988; Irvine Scientific, Santa Ana, CA, USA) in DPBS (w/o Ca, Mg). Flow cytometry and fluorescence-activated cell sorting of mScarlet⁺ cells were performed with a BD FACSMelody™ Cell Sorter (BD Biosciences, Franklin Lakes, NJ, USA). The population of interest was identified through gating for singlets and cell size and granularity, based on the forward and side scatter of the wt control. A total of 30,000 events were acquired for each sample. Data were plotted with FlowJo™ software (LLC/BD Biosciences).

Vaginal ultrasound-guided aspiration of oocyte−cumulus complex (OPU) was performed 35 h after human chorionic gonadotrophin administration (HCG 10,000 IU, Gonasi: AMSA, Rome, Italy). Oocyte denudation was performed 2 h after oocyte retrieval. ICSI was performed 1 h after oocyte denudation on fresh oocytes and 1 h after warming and in vitro culture on vitrified oocytes with the same sample of partner’s freshly ejaculated spermatozoa sample as previously described [22 (link)].
After ICSI, in vitro culture was carried out in 25 μl of continuous single culture complete medium with human serum albumin (Irvine Scientific, Santa Ana, USA) under mineral oil and in automated incubators with 5% CO2, 5% O2 at 37 °C, fitted with time-lapse imaging acquisition (Embryoscope, Unisense, Aarhus, Denmark). The entire embryo development has been followed and analyzed using morphokinetic parameters [23 (link)].

COH was performed with the GnRH antagonist protocol. Recombinant FSH (150–300 IU, Gonal-F; Serono) and/or hMG (75–150) IU; (Merional; IBSA) was administered on day 2 of the menstrual period. Starting on the sixth day of controlled ovarian stimulation, the ovarian response was monitored by serial transvaginal ultrasound (TV-USG) and by measuring serum E₂ and P₄ levels. When the leading follicle exceeded 13 mm in diameter, 0.25 mg of GnRH antagonist (Cetrotide; Serono) was started daily until the day of the last trigger. When at least two follicles reached 18 mm in diameter, patients were administered 250 μg of human chorionic gonadotropin (hCG; Ovitrelle, Serono) or 0.2 mg of triptorelin (Gonapeptyl, Ferring), and oocyte retrieval was scheduled 35 hours after the trigger administration [16 (link)].
The oocyte retrieval, denudation, and ICSI procedures were performed as described previously by Serdarogullari et al. [17 ]. ICSI was the fertilization method in all of the cycles included in this study. After microinjection, oocytes were cultured individually in a special pre-equilibrated culture dish. In our study, single-step media, namely, Continuous Single Culture Complete (CSCM-C) with Human Serum Albumin (Irvine Scientific) was used for embryo culture throughout the culture period.

Ovaries were collected, fixed, and embedded in paraffin. Tissues were serially sectioned (5-um thickness) and stained with hematoxylin and eosin. Images were captured with the Nikon Ni-E microscope. To assess the ovulation function, pregnant mare serum gonadotropin (PMSG, 7.5 I.U.) was intraperitoneally injected to stimulate the ovaries of the mice, followed by injection of 7.5 I.U. human chorionic gonadotropin (hCG) after 48 h to induce super-ovulation. After 12–14 h, the mice were sacrificed and the oviductal ampullae were broken to obtain the cumulus-oocyte complexes (COCs). Subsequently, COCs were pipetted in the in-vitro fertilization (IVF) medium (Vitrolife Sweden AB) containing 5% human serum albumin (Irvine Scientific) and 0.1% hyaluronidase (Sigma) to remove the cumulus cells. The number of ovulated oocytes was counted under a stereoscopic microscope (Nikon SMZ800N, Tokyo, Japan).