All patients used long or short protocols for ovarian stimulation. Oocytes were retrieved through the vagina 36 h after human chorionic gonadotropin (hCG) injection and fertilized using conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Normal zygotes with two pronuclei were cultured in G1 medium (Vitrolife, Sweden) at 37°C with 5% O2 and 6% CO2 in incubators until ET or vitrification on day 3. We vitrified at maximum of four good-quality embryos on day 3 after transfer per patient, and the remaining embryos were placed in extended culture media. For day 3 embryos, we scored embryos on their blastomere shape, blastomere number, and fragmentation rate. An embryo was defined as grade 1 when it had an even blastomere shape and <10% fragmentation, grade 2 when it had uneven blastomeres and 10–25% fragmentation, grade 3 when it had uneven blastomeres and 25–35% fragmentation, and not recommended for transfer or cryopreservation when the fragmentation >35%. Embryos with grade 1-3 were defined as transferrable embryos.
G1 medium
Manufactured by Vitrolife
Sourced in Sweden, United States
G1 medium is a laboratory culture medium developed by Vitrolife for the in vitro culture of human embryos. It is a complex, well-defined culture medium formulated to support the growth and development of human embryos from the time of fertilization up to the blastocyst stage.
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Lab products found in correlation
G2 medium, by Vitrolife (9 mentions)
G ivf medium, by Vitrolife (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Desktop incubator, by Cook Medical (2 mentions)
G morpholinepropanesulfonic acid mops medium, by Vitrolife (2 mentions)
G in vitro fertilization plus medium, by Vitrolife (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Recombinant bfgf, by R&D Systems (1 mentions)
Recombinant activin a, by Thermo Fisher Scientific (1 mentions)
Gonal f, by Merck Group (1 mentions)
Repli g single cell kit, by Qiagen (1 mentions)
Decapeptyl, by Ferring (1 mentions)
Calcium ionophore, by Merck Group (1 mentions)
G mops, by Vitrolife (1 mentions)
Human serum albumin (hsa), by Vitrolife (1 mentions)
Te2000 u, by Nikon (1 mentions)
Liquid paraffin, by Merck Group (1 mentions)
Dibutyryl camp, by Merck Group (1 mentions)
33 protocols using g1 medium
1
Ovarian Stimulation and Embryo Cryopreservation
2
Time-Lapse Oocyte Culturing Protocol
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After injection, the oocytes were individually cultured in a culture dish containing pre-equilibrated G-1 medium (Vitrolife) covered with sterile mineral oil and placed in the time-lapse microscope (Primo Vision Time-lapse Embryo Monitoring System, Goteborg, Sweden) in a triple-gas incubator for 3 days.
3
Derivation of human embryonic pluripotent stem cells
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Human pre‐implantation embryos were donated from patients who had completed their family after assisted reproduction treatment. Written informed consents were obtained from all the donors recruited and the study protocols were approved by the Institutional Review Boards of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB number: UW 18‐017) and the Council of Reproductive Technology, Hong Kong (research license number: R5004). Cryopreserved day 2 human pre‐implantation embryos were thawed and cultured in G‐1 medium (Vitrolife) for 1 day before transferred to G‐2 medium (Vitrolife) and cultured till morula or early blastocyst stages when the zona pellucida of the embryos was removed using acid tyrode solution (Sigma Aldrich). The human embryos were then placed on the STO feeder cells in hEPSC medium supplemented with 10 ng mL−1 recombinant bFGF (R&D), 20 ng mL−1 recombinant activin A (PeproTech), and 5% FBS (Thermo Fisher Scientific). The embryos showed outgrowth after several days. The resultant cell colonies were dissected into small clusters using glass pipettes and transferred onto new STO feeder cells. After several passages, the colonies were digested with 0.05% Trypsin (Thermo Fisher Scientific) and passaged like other hEPSCs.
4
IVF with Single-Cell Biopsy
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This study was approved by the Medical Institutional Review Board of the International Peace Maternity and Child Health Hospital (IPMCH), Shanghai Jiao Tong University School of Medicine. The patient was treated with FSH (Gonal-F; Serono) after pituitary function was downregulated by treatment with a gonadotrophin-releasing hormone (GnRH) agonist (GnRH-a, Decapeptyl; Ferring). Follicular development was monitored by serial vaginal ultrasound imaging and measurement of serum E2 levels. Oocytes were retrieved at 36 h after human chorionic gonadotropin injection under ultrasound guidance and were subsequently fertilized by intracytoplasmic sperm injection (ICSI). The fertilized zygotes were cultured in G1 medium (Vitrolife, Sweden) at 37°C in a humidified atmosphere with 6% CO2. Single-cell biopsy was performed on day 3 after fertilization, and the DNA was amplified using the REPLI-g Single Cell Kit (Qiagen) according to the instructions. For each biopsied cell, a blank control was prepared from the final wash drop.
5
Calcium Ionophore-Assisted Sperm Activation for ICSI
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AGT was carried out by exposing spermatozoa and post-ICSI oocytes to calcium ionophore (19657, Sigma-Aldrich, Saint Louis, MO, USA) as previously described [20 (link)]. Before ICSI injection, ejaculated spermatozoa processed by density gradient or centrifugation were briefly exposed to 50 μM calcium ionophore in G-MOPS™ media (Vitrolife, Göteborg, Sweden) enriched with human serum albumin (G-MM™, Vitrolife, Göteborg, Sweden). During the ICSI procedure, spermatozoa were aspirated individually from the media drop containing calcium ionophore and immobilized in a separate polyvinylpyrrolidone (PVP) drop. Then, a small portion of calcium ionophore was aspirated into the micropipette and injected into the oocyte with the spermatozoa. Next, post-ICSI oocytes were exposed to 50 μM calcium ionophore in G-1 medium (Vitrolife, Göteborg, Sweden) for 10 min at 37 °C, and then rinsed and placed in fresh G-1 medium [20 (link)].
6
In Vitro Fertilization Protocol
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IVF patients were treated with controlled ovarian hyperstimulation (COH) by gonadotropin-releasing hormone (GnRH) agonist or GnRH antagonist, as previously described [14 (link), 15 (link)]. When two dominant follicles reached 17–18 mm in diameter, oocytes were retrieved transvaginally 36–38 h after a human chorionic gonadotropin (hCG) trigger. Embryos were cultured in G1 medium (Vitrolife, Sweden) after fertilization and transferred on day 3. Subsequently, biochemical pregnancy was ascertained by a positive serum hCG 2 weeks after embryo transfer with serial elevation. Clinical pregnancy was defined as a gestational sac and active fetal heartbeat on ultrasound demonstration 8 weeks after embryo transfer.
7
Oocyte Retrieval and Embryo Culture
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Ovulation induction protocols include a gonadotrophin-releasing hormone (GnRH) antagonist protocol, or a short or long GnRH analogue suppression protocol combined with recombinant FSH. Oocyte cumulus complexes retrieval was performed 36 h after the administration of hCG and fertilized by conventional IVF or ICSI. Normal fertilized zygotes were cultured in G1 medium (Vitrolife, Sweden) to cleavage embryos for fresh embryo transfer, vitrification or blastocyst culture. The embryos transferred to G2 medium (Vitrolife, Sweden) were cultured to the blastocyst stage for fresh embryo transfer or vitrification. Garnder scoring system were used to evaluated blastocyst morphology based on the inner cell mass and trophectoderm grading [12 (link)].
8
Ovarian Stimulation and Embryo Culture
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Ovarian stimulation was conducted by using a standard protocol with gonadotropin dose adjustment for the ovarian reserve. Oocyte maturation was triggered when leading follicles were ≥ 17 mm and oocyte retrieval was conducted 36 h later. Intracytoplasmic sperm injection (ICSI) was used for insemination. All the embryos were cultured to the blastocyst stage in 6% CO2 and 5% O2. All embryos were transferred into G1 medium (Vitrolife, 10,128) from day 1 to day 3 and subsequently transferred into G2 medium (Vitrolife, 10,029) from day 3 to day 6.
9
Pronuclear Checking and Embryo Evaluation
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Pronuclear checking was performed using an inverted microscope (TE2000-U, Nikon, Japan) at 19–20 h post-fertilization. Concomitantly, the numbers of pronuclear and cleaved embryos were recorded.
The cleavage staged embryos were singularly cultured in 50 μl microdrops containing G1 medium (Vitrolife) supplemented with 5% HSA (Vitrolife), at 37 °C and 6% CO2. On the morning of Day 3 of culture, embryos were observed using an inverted microscope and scored according to the Peter cleavage-stage embryo Scoring system18 . Abnormal embryos following fertilization, such as those with no obvious pronucleus (0PN), one pronucleus (1PN), three pronuclei or higher (3PN and ≥4PN) and prematurely cleaved embryos (2Cell), were collected for further culture in vitro. In addition, poor quality embryos developed from normal fertilization (2PN) on Day 3, such as embryos with fewer than five blastomeres or with higher than 50% fragments, were also collected as part of this study (Supplementary Figure S1 ).
The cleavage staged embryos were singularly cultured in 50 μl microdrops containing G1 medium (Vitrolife) supplemented with 5% HSA (Vitrolife), at 37 °C and 6% CO2. On the morning of Day 3 of culture, embryos were observed using an inverted microscope and scored according to the Peter cleavage-stage embryo Scoring system18 . Abnormal embryos following fertilization, such as those with no obvious pronucleus (0PN), one pronucleus (1PN), three pronuclei or higher (3PN and ≥4PN) and prematurely cleaved embryos (2Cell), were collected for further culture in vitro. In addition, poor quality embryos developed from normal fertilization (2PN) on Day 3, such as embryos with fewer than five blastomeres or with higher than 50% fragments, were also collected as part of this study (
10
Vitrification and Warming Embryo Protocol
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The vitrification was done according to Vitrolife kit (Vitrolife Inc., Gothenburg, Sweden). Briefly, the embryos were equilibrated into the Vitri 1 Cleave solution for 5 min, and in Vitri2 Cleave solution for 2 min. finally the embryo were placed into Vitri 3 Cleave solution for 30 sec and loaded on the top of cryotop. The cryotop was submerged directly to liquid nitrogen and store it. For warming, the top of cryotop was submerged to Warm1 solution and then placed in Warm2 and 3 . Finally washed in Warm4 solution and culture in G1 medium (Vitrolife Inc., Gothenburg, Sweden).
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