Cumulus-oocyte complexes were incubated in an incubator at 37°C in an atmosphere containing 5% CO2. CCs were dissected from cumulus-oocyte complexes using a sterile pipette and immediately transferred into a sterile tube individually. The CCs surrounding the same oocyte were taken as individual groups, and were not pooled together. The CCs were then stored at −80°C for further analyses for about 3 months [8 (link)]. The remaining CCs adjacent to the oocytes were removed from the oocytes with enzymatic and mechanical methods using a sterile pipette and hyaluronidase (Hyase, Vitrolife, Frolunda, Sweden). Oocytes were evaluated morphologically and according to their nuclear maturation.
Hyase
Manufactured by Vitrolife
Sourced in Sweden
Hyase is a laboratory reagent used in cell culture processes. It contains the enzyme hyaluronidase, which is responsible for the hydrolysis of hyaluronic acid. Hyase is used to facilitate the isolation and dissociation of cells from tissues or cell aggregates.
Automatically generated - may contain errors
10 protocols using hyase
1
Cumulus-Oocyte Complex Isolation and Analysis
2
Intracytoplasmic Sperm Injection Procedure
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The procedures for intracytoplasmic sperm injection (ICSI) have been described previously [23 (link)]. Briefly, during the ICSI processing, cumulus cells and the corona radiata of the oocytes were removed by brief exposure to hyaluronidase 2-3 h after retrieval (HYASE, Vitrolife); ICSI was performed on metaphase II oocytes as observed under an inverted microscope. All injected eggs were placed into timelapse incubator immediately (Embryoscope Plus). Then, the fertilized oocytes were continuously cultured at 5%CO2, 5%O2 and 37ºC for 2 more days (G1-plus, Vitrolife, Sweden). The culture conditions of the embryos included in this study were the same. All of the embryos were checked on the morning of day 3 after oocyte retrieval. Then the embryos were changed to blastocyst medium to continue culture until Day5/6 (G2-plus, Vitrolife, Sweden). The blastocyst biopsy is done on the Day5/6. A small hole is made in the zona pellucida using a laser just prior to biopsy, and then a mechanical cutting method is used to obtain 3-6 trophectoderm cells.
3
Oocyte Donation for Infertility Treatment
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Oocytes were donated by women undergoing infertility treatment, either as part of an egg sharing programme or following egg collection to prevent multiple pregnancy following ovarian stimulation with exogenous gonadotrophins and intra-uterine insemination. Consenting women shared if ≥6 oocytes were harvested. Women, from whom 5 or fewer oocytes were collected, did not donate eggs but still received subsidised treatment29 (link). Oocytes were collected by ultrasound-guided follicle aspiration. The surrounding cumulus cells were removed using hyaluronidase (HyAse, Vitrolife). MII oocytes were identified by the presence of the first polar body. Oocytes were incubated in G1 Plus medium (Vitrolife) at 37°C and 7% CO2 until nuclear transfer.
4
ICSI and Embryo Evaluation Protocol
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After using 80 IU of HYASE (Vitrolife) to denude the oocyte cumulus complex, mature oocytes were identified. Three hours after retrieval, ICSI was performed using a mature oocyte and sperm that had been previously prepared. The injected oocyte was cultivated in a single drop of 20 μL of G-TL (Vitrolife) covered by 3 mL of Ovoil (Vitrolife) under conditions of 6% CO2 and 5% O2. Next, 16 to 18 hours after the injection, fertilized oocytes were detected by the presence of two pronuclei. On days 2 and 5, embryos were evaluated according to the Istanbul consensus. A high-quality blastocyst was defined as possessing a densely packed inner cell mass and trophectoderm composed of many cells forming a cohesive epithelium [23 (link)].
5
Controlled Ovarian Hyperstimulation and ICSI
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Controlled ovarian hyperstimulation (COH) was performed under a flexible Gonadotropin-releasing hormone antagonist (GnRH) protocol. Gonadotropin doses were adjusted according to age, body mass index, and ovarian reserve testing. The oocytes retrieved through follicular aspiration were cultured in Life Global Total medium/HEPES (Life Global®) or GMops Plus (Vitrolife®) for two hours until decumulation using ICSIcumulase (Origio®) or Hyase (Vitrolife®). Subsequently, the ICSI was conducted, and the injected oocytes were cultured in NUNC™ plates (CN 150255 Nunc®) in Life Global Total or GTL plus (Vitrolife®) culture medium under a mineral oil layer (LiteOil; Life Global® or OVOIL; Vitrolife®), and incubated at a pH of 7.3, 5% oxygen and 8,9% CO2 in Tri-Gas incubators (K-Systems®). Until August 2018, Life Global® media were used; but since then, and following changes in the laboratory's internal protocols, Vitrolife® media is being used.
After ICSI, fertilization was assessed between 16 and 18 hours; embryo culture continued for 72, 120 or 144 hours according to the embryo development. The 72-hour embryos were classified according to their blastomere number and fragmentation percentage (ASEBIR, 2015), while 120- and 144-hour embryos were graded according to the Gardner and Schoolcraft's system (Gardner et al., 1998 (link)).
After ICSI, fertilization was assessed between 16 and 18 hours; embryo culture continued for 72, 120 or 144 hours according to the embryo development. The 72-hour embryos were classified according to their blastomere number and fragmentation percentage (ASEBIR, 2015), while 120- and 144-hour embryos were graded according to the Gardner and Schoolcraft's system (Gardner et al., 1998 (link)).
6
ICSI Procedure for Oocyte Fertilization
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Only Intracytoplastmic Sperm Injection (ICSI) procedure was used to ensure that all oocytes at the MII stage after denudation were fertilized at the same time and Time-lapse timings were then observed exactly. Cumulus-oocyte complexes (COC) were aspirated by conventional single lumen needle 17,107 (Vitrolife, Västra Frölunda, Sweden) 36–38 hours after hCG injection. The oocytes were retrieved and washed in a 35 BD-falcon dish containing 2 ml G-MOPS PLUS (Vitrolife, Västra Frölunda, Sweden), covered with 2 ml OVOIL (Vitrolife, Västra Frölunda, Sweden) and incubated at 370C. After washing, the COC was incubated in 1 ml of G-IVF PLUS (Vitrolife, Västra Frölunda, Sweden), equilibrated to 37 °C and 5.5% CO2. The COC were incubated for 2 h before denudation.
The COC were then immersed in 80 IU of HYASE (Vitrolife, Västra Frölunda, Sweden) for 1 min. These cumulus cells were removed from the oocyte by sucking and slicing in a 135 μm diameter pipette. After denudation the oocytes, they were incubated in 100 μl drops of G-IVF PLUS covered with 3 ml OVOIL for 1 h before ICSI.
The COC were then immersed in 80 IU of HYASE (Vitrolife, Västra Frölunda, Sweden) for 1 min. These cumulus cells were removed from the oocyte by sucking and slicing in a 135 μm diameter pipette. After denudation the oocytes, they were incubated in 100 μl drops of G-IVF PLUS covered with 3 ml OVOIL for 1 h before ICSI.
7
Transvaginal Oocyte Retrieval and Vitrification
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Oocyte retrieval was performed using a transvaginal needle under ultrasound guidance 36 h after the triggering of follicular maturation, under the sedation of the patients. Oocytes were incubated in fertilization medium (Global for Fertilization®, LifeGlobal, Guilford, CT, USA) under an oil (Ovoil™, Vitrolife, Gothenburg, Sweden) overlay at 37 °C and 6.6% CO2 with 5% O2 (previously equilibrated overnight). Hyaluronidase (HYASE™, Vitrolife) and pipetting were used to remove the cumulus cells surrounding the oocytes, and the maturation status of the oocytes was assessed. Only metaphase II (MII) oocytes were prepared for ICSI.
For vitrified donor oocytes, oocytes were devitrified according to the manufacturer’s instructions (Kitazato, Shizuoka, Japan).
For vitrified donor oocytes, oocytes were devitrified according to the manufacturer’s instructions (Kitazato, Shizuoka, Japan).
8
Conventional and ICSI Fertilization Protocols
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For conventional fertilization, OCC were inseminated over-night with a sperm suspension at a concentration of 100–150.103 motile spermatozoa/ml. Oocytes selected for ICSI fertilization were stripped of their cumulus using Hyase (Vitrolife, Goteborg, Sweden) 40 IU/ml. Sperm injection was performed only on metaphase II oocytes, four hours after pick up.
9
Oocyte Retrieval and ICSI Protocol
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Cumulus-oocyte complexes (COCs) were retrieved from follicular fluid and placed in preincubated fertilization medium (G-IVF™ PLUS, Vitrolife®). They were cultured at 37 °C under a gas mixture of 5% O2, 6% CO2 and 89% N2 until insemination or denudation. For intracytoplasmic sperm injection (ICSI), oocytes were denuded using mechanical (Stripper, CooperSurgical®) and enzymatic methods (hyaluronidase: HYASE®, Vitrolife). Each semen sample was collected by masturbation in a sterile container after 2 days of abstinence. Sperm was prepared by density gradient centrifugation.
10
Oocyte Maturation and ICSI Preparation
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Cumulus-oocyte complexes (COCs) were incubated in the culture medium (G-IVF-PLUS, Vitrolife) covered with mineral oil (Ovoil, Vitrolife) at 37°C in 6% CO2 for 2–3 hours. Cumulus cells were removed after exposure to G-Mops (3-N-morpholino-propane sulfonic acid) buffered medium containing 80 IU/mL hyaluronidase (Hyase, Vitrolife) for 30 seconds with the help of a glass Pasteur pipette (Humagen Fertility Diagnostics, Charlottesville, VA, USA). The nuclear status of the denuded oocytes was then assessed. Oocytes that were observed to have released the first polar body were considered mature and used for ICSI. Immature oocytes with a prominent nucleus (GVs) were assigned to the study and taken up for the prematuration culture.
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