G mops plus

Cumulus-oocyte complexes (COC) retrieved were washed in MOPS-buffered medium with human serum albumin (GMOPS Plus; Vitrolife; Sweden). The COC were cultured in 0.8 ml GIVF medium (Vitrolife; Sweden) under paraffin oil at 37°C, 6% CO ${}_2$ and 5% O ${}_2$ for 2 hr after retrieval. After 2 hr of incubation, COC were then exposed to hyaluronidase (80 IU Synvitro Hyadase; Medicult; Denmark) in order to remove the cumulus cells. Metaphase II oocytes were inseminated using the ICSI method 38-42 hr after the hCG injection. Prior to ICSI, each oocyte was set into a 15 μl droplet of GMOPS Plus (Vitrolife; Sweden) under paraffin oil (Ovoil; Vitrolife; Sweden). Injected oocytes were cultured in a single droplet of 50 μl GTL (Vitrolife; Sweden) until 3 days.
On the day of oocyte(s) retrieval, spouse collected the sperm samples using masturbation method. The semen samples were analyzed for concentration, motility, as well as morphology. The semen was processed and selected by using a density gradient method, layer concentration used was 45:90% SpermGrad (Vitrolife; Sweden). The solution was centrifuged at 300-600 g for 10-20 min and re-suspended with 1 ml of MOPS-buffered medium (GMOPS Plus, Vitrolife; Sweden). For ICSI purposes, a single spermatozoon was immobilized using a polyvinylpyrrolidone (PVP) solution (Medicult; Denmark).

Retrieved oocytes were first rinsed in G-MOPS ™ Plus media (G-MOPS ™ Plus, Vitrolife, Sweden) then maintained in G-IVF ™ Plus culture (G-IVF ™ Plus, VitroLife, Sweden) covered with paraffin oil (OVOIL, VitroLife, Sweden) before cumulus cell removal. The surrounding cumulus cells were removed within 2 h after retrieval by the exposure to hyaluronidase (HYASE-10 × in G-MOPS ™ Plus media, Vitrolife, Sweden) for several seconds before being transferred to G-MOPS ™ Plus media where they were mechanically dissociated from the oocyte.
The denuded oocytes were classified according to their level of maturation using a Nikon SMZ1500 stereoscope. The number of Metaphase II Oocytes (MII; identified as oocytes with the extrusion of the first polar body), Metaphase I Oocytes (MI; identified as oocytes lack the presence of both the germinal vesicle and the polar body), Germinal Vesicle Oocytes (GV; identified as oocytes with Germinal Vesicle), and Atretic Oocytes (oocytes with signs of degeneration) were documented. The Maturation Rate was calculated by dividing the number of mature (MII) oocytes by the number of retrieved oocytes. In addition, the ovarian sensitivity index (OSI) was calculated by dividing the number of retrieved oocytes by the total dose of FSH used and multiplying the results by 1000 [33 (link)].

The number of cells in the inner cell mass (ICM) and the trophectoderm (TE) of expanded and hatched blastocysts was determined using differential staining on day 7.5 post-IVF, based on previously published methods46 (link). Briefly, embryos were placed in 0.5% (w/v; 10 μL) pronase in GMOPS (Vitrolife) to remove the zona pellucida, and then washed in GMOPS-PLUS (Vitrolife) for 5 min. Embryos were subsequently incubated in 10 μL of 10 mM trinitrobenzenesulfonic acid (TNBS) in 4 mg/mL polyvinyl pyrollidone (PVP) in simple-G1 medium (Vitrolife; G1-PVP) for 20 min, washed twice in GMOPS-PLUS then transferred to 0.1 mg/mL anti-2,4 dinitrophenol-G1-PVP (10 μL) for 10 min. Following a further wash in GMOPS-PLUS, embryos were transferred to 10 μL drops of 10% v/v guinea pig serum in 25 mg/mL propidium iodide in GMOPS for 1 min, and then placed into 10 μL drops of 0.1 mg/mL bisbenzimide for 20 min. Embryos were individually mounted in 100% glycerol on a glass slide under a coverslip. Embryos were viewed under fluorescent light using a Nikon Eclipse TS100 microscope equipped with a mercury lamp (Olympus) and images of embryos were taken using the Nikon Digital Sight DS-L2 (Nikon). The number of cells in the inner cell mass (ICM) and the trophectoderm (TE) were counted using the cell-counter tool in ImageJ software (National Institute of Health).