Study period and patients
This study was a comparative laboratory study carried out in 2018 at Afzalipur Infertility Center in Kerman. A total of 368 germinal vesicles (GV) oocytes were obtained from 184 patients (18–46 years old) who practised an ICSI cycle. All the patients underwent evaluation by the Kerman Medical University’s Ethical Committee and were admitted to Afzalipour Hospital of Kerman, Iran.
Ovarian stimulation was attained by long protocol via administration of a combination of (GnRH) gonadotropin-releasing hormone and follicular stimulating hormone (FSH). Following follicular growth via transvaginal ultrasound, when adequate matured follicle was reached, injection of human chorionic gonadotropin (hCG) (Switzerland, IBSA Co) 10,000 IU was started; 36 h later, the oocyte collection was carried out through laparoscopy .
Cumulus oocyte complexes (COCs) were picked up and transferred in G-IVF (Vitrolife, Sweden) culture medium under mineral oil (Vitrolife, Sweden) for 2–3 h in an incubator. The COCs were denuded via mechanical pipetting dissection using 80 IU hyaluronidase (Sigma Co, USA). Nuclear maturity of the denuded oocytes was assessed under the dissecting microscope, according to that be the presence of the first Polar Body (PB); accordingly, oocytes were classified as immature (GV or MΙ) or mature (MII). MII oocytes were used for IVF or ICSI procedures and GV oocytes, vitrified or fresh, were cultured in vitro .
Germinal vesicle oocytes were studied in six groups after being denuded. For the first two groups, 1: fresh GV oocytes were matured in three vitro maturation mediums called (fIVM). 2: GV oocytes were vitrified and then maturated after thawing (vIVM). Both of them were maturated in three vitro maturation mediums.
Three types of IVM culture mediums were used in this study
(Medium 1) as the based control medium: is Alpha Minimum Essential Medium (α-mem) .
(Medium 2) α-mem + supernatants derived from human bone marrow mesenchyme stem cells (B.M) α-mem + supernatants derived from human bone marrow mesenchyme.
(Medium 3) α-mem + ovarian factor (O.F) including: [3 ng/ml of BDNF, 100 ng/ml of IGF-I, 1 mg/ml of estradiol, 30 ng/ml of GDNF, 10 ng/ml of leptin, 0.5 ng/ml of FGF2] .
The germinal vesicle oocytes were put in the first medium containing Ham’s F10 medium + 20% human serum albumin (HSA) (Plasbumin Co, USA). Next, they were transferred in an equilibration solution (ES) containing 7.5% dimethyl sulphoxide (DMSO) with 7.5% ethylene glycol (EG) at room temperature for 10 min. Finally, these oocytes were removed to the vitrification solution (VS) containing 0.5 mol/l sucrose with 15% DMSO and 15% EG for 50–60 s at room temperature. Then the vitrified oocytes were placed on the cryotops (Vitrolife, Sweden) and placed into the fluid nitrogen storage tank instantly for reserving several months .
The thawing process of the vitrified oocytes was done in the thawing solution [Ham’s F10 medium containing 20% (HSA) as a based medium] through locating these oocytes in different mediums in four steps: first step: 1.0 mol/l sucrose for 50–60 s, second step: 0.5 mol/l sucrose for 3 min, the third step: 0.25 mol/l sucrose for 3 min and finally these oocytes were located in Ham’s F10 medium with 20% (HAS) for 3–5 min. Consequently, the oocytes were placed randomly in one of the three IVM mediums for 48 h in an incubator. Then the oocyte viability was assessed by stereomicroscope [4, 22].
MSC isolation and culture
Human bone marrow mesenchymal stem cells were provided from Afzalipour Kerman Medical University (Kerman-Iran) (α-mem) as the based control medium. These cells were cultured and Vitrified according to Ling’s method ; human bone marrow mesenchymal stem cells were washed with phosphate buffered saline (PBS) containing 100 mg/ml streptomycin (Gibco), 100 U/ml penicillin (Gibco) and collagenase I (Sigma), then, these cells cultured in α-mem medium (α-mem, Gibco) supplemented with 10% FBS (Gibco), 100 mg/ml streptomycin (Gibco) and 100 U/ml penicillin (Gibco), in the incubator. The medium was changed, and they were trypsinised after attaining complete cell confluency. After changing the medium and gathering supernatant, it was filtered with a 0.2 µm membrane for immediate use .
To examine the oocyte ultrastructure by transmission electron microscopy (TEM), 10 numbers IVM matured oocytes (MII) from each medium were compared with in-vivo MII oocytes from patients that were cancelled ICSI. For the TEM study, the oocytes were prepared according to Nottola et al. method . The oocytes were fixed in a solution of glutaraldehyde 1.5% (Sigma, USA) within 0.1 M phosphate-buffered saline (PBS) for 2–5 days at 4 °C; these oocytes were washed by PBS buffer for 10 min; next, the oocytes were fixed in a solution of osmium tetroxide 1% (Agar, UK) within PBS buffer away from the light, then the oocytes washed again in PBS buffer. They were put in small thin blocks of agar 1% (Sigma, USA) for facilitating oocyte removal. The oocytes were dehydrated in rising ethanol concentrations, immersed in propylene oxide for solvent replacement and completely embedded in Epon 812 resin (Agar, UK). Semi-thin sections (0.5–1 μm thickness) were stained with toluidine blue for light microscopy evaluation (Zeiss, Germany). Ultrathin sections (60–80 nm thickness) were provided, and then uranyl acetate (7 min) and lead citrate (13 min) were stained. Finally, these sections were photographed at 80 kV by TEM (Zeiss, Germany) . The oolemma integrity, zona pellucida (ZP), perivitelline space (PVS), quality of the cytoplasmic organelles and presence of polar body were assessed by light microscopy and TEM .
Differences in the ultra-structural parameters in matured oocytes between the groups were compared using the χ2 test in SPSS software (Version 21, USA). A p-value of < 0.05 was considered statistically significant.