We recruited patients who underwent routine IVF/ICSI-ET at Wuhan Tongji Reproductive Medicine Hospital and Wuhan Union Hospital. After fertilization assessment, the zygotes were washed extensively in G-1 PLUS (Vitrolife, Göteborg, Sweden) and cultured in 30 μL G-1 PLUS droplets until day 3 of development. After cleavage assessment, the day 3 embryos were washed extensively in equilibrated G-2 PLUS (Vitrolife, Göteborg, Sweden) and transferred to G-2 PLUS culture droplets for further culture to blastocyst stage. After another two days in culture, the development and quality of the day 5 blastocysts were evaluated according to the blastocyst scoring system. All the embryos were cultured under 6% CO2, 5% O2, and balance N2 at 37 °C in a tri-gas incubator (Cook, Bloomington, IN, USA). Culture media were prospectively collected from the in vitro cultured embryos for miRNA sequencing and validation. The miRNA profiles of embryo culture media collected from samples with successful and failed pregnancy were compared to identify the differentially expressed miRNAs. All the participants were performed with single-embryo transfer.
G 1 plus
Manufactured by Vitrolife
Sourced in Sweden, Australia, United States
The G-1 PLUS is an embryo culture medium developed by Vitrolife for use in assisted reproductive technology. It is a formulation of nutrients, energy substrates, and other components designed to support the in vitro culture of human embryos.
Automatically generated - may contain errors
Lab products found in correlation
G2 plus, by Vitrolife (18 mentions)
G ivf plus, by Vitrolife (9 mentions)
Ovoil, by Vitrolife (5 mentions)
G mops plus, by Vitrolife (5 mentions)
Hyaluronidase, by Vitrolife (2 mentions)
G 1 plus g 2 plus, by Vitrolife (2 mentions)
Heracell 150 incubator, by Thermo Fisher Scientific (2 mentions)
Inverted microscope, by Nikon (2 mentions)
Primo vision dish 9 well or 16 well, by Vitrolife (2 mentions)
Primo vision, by Vitrolife (2 mentions)
Fertilization medium, by Cook Medical (1 mentions)
Transferman 4r, by Eppendorf (1 mentions)
G gamete, by Vitrolife (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Embryoscope time lapse incubator, by Vitrolife (1 mentions)
Embryoscope culture dish, by Vitrolife (1 mentions)
A23187, by Merck Group (1 mentions)
G series media, by Vitrolife (1 mentions)
Hyase, by Vitrolife (1 mentions)
Paraffin oil, by Vitrolife (1 mentions)
43 protocols using g 1 plus
1
Blastocyst Culture and miRNA Profiling
2
Oocyte Activation Protocol for ICSI
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Oocytes were incubated for 15 min in 30 μL drops of A23187 (GM508 CultActive, Gynemed, Germany) immediately after the microsphere injection. Oocytes were then washed twice in 30 μL drops of MOPS-and HEPES-free culture medium (GM501 Cult, Gynemed, Germany). Finally, the oocytes were placed in an Embryoslide® and cultured in an Embryoscope® TL system in standard conditions (37°C, 6% CO2, and 95% RH).
Ca 2+ ionophore ionomycin (Strepromyces conglobatus, Ionomycin, I9657, Sigma Aldrich) was diluted in DMSO (Sigma-Aldrich) to a concentration of 0.7 mg/mL. This solution was further diluted in G1™ culture medium (Vitrolife, Sweden) at a final concentration of 10 μM and then equilibrated at 37°C, 6% CO2, and 95% RH overnight. AOA was performed according to Heindryckx et al. (2008) with some modifications. Oocytes were allowed to recover for 30 min in G1™ PLUS (Vitrolife, Göteborg, Sweden) after ICSI and were incubated in an ionomycin solution (10 μmol/L in G1™ PLUS) for 7 min. Next, oocytes were washed 10 times in G1™ PLUS and then incubated for 30 min in G1™ PLUS. The oocytes were subsequently exposed to the ionomycin solution for two more rounds, first for 7 min followed by a 30-min wash in G1™ PLUS. The oocytes were then placed in the Embryoslide® for TL culture to monitor embryo and parthenote development.
Ca 2+ ionophore ionomycin (Strepromyces conglobatus, Ionomycin, I9657, Sigma Aldrich) was diluted in DMSO (Sigma-Aldrich) to a concentration of 0.7 mg/mL. This solution was further diluted in G1™ culture medium (Vitrolife, Sweden) at a final concentration of 10 μM and then equilibrated at 37°C, 6% CO2, and 95% RH overnight. AOA was performed according to Heindryckx et al. (2008) with some modifications. Oocytes were allowed to recover for 30 min in G1™ PLUS (Vitrolife, Göteborg, Sweden) after ICSI and were incubated in an ionomycin solution (10 μmol/L in G1™ PLUS) for 7 min. Next, oocytes were washed 10 times in G1™ PLUS and then incubated for 30 min in G1™ PLUS. The oocytes were subsequently exposed to the ionomycin solution for two more rounds, first for 7 min followed by a 30-min wash in G1™ PLUS. The oocytes were then placed in the Embryoslide® for TL culture to monitor embryo and parthenote development.
3
Mouse Sperm Capacitation and In Vitro Fertilization
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Male ICR mice aged 10-12 weeks were killed by cervical vertebrae dislocation. Both cauda
epididymis was removed and placed in 1 ml of fertilization medium (Cook). Capacitation was
allowed to proceed for 30 minutes in an atmosphere of 6% CO2, 5% O2,
and 89% N2 at 37°C. The spermatozoa were transferred to COCs drops for
insemination at a final motile sperm concentration of 2.5x105 /ml. All
experiment groups from the same hydrosalpinx sample were used spermatozoa obtained from
the same male. Two hours later, MII oocytes were transferred to culture in 10 µl
drop of cleavage medium (G1-plus; Vitrolife, Sydney, Australia) under mineral oil
(IrvineScientific). The fertilization rate was determined the next day by counting the
number of two-cell embryos. Seventy-two hours post insemination, the embryos were
transferred to blastocyst medium (G2-plus; Vitrolife, Sydney, Australia) under mineral oil
(Irvine Scientific) and cultured in similar conditions for 48 hours. Embryo development
was evaluated under an inverted microscope every 24 hours until completion of 120 hours.
Mouse blastocysts were classified as early, partial, full, expanding, hatching and hatched
blastocysts, using the criteria proposed byGardner
et al. (2000) for human blastocyst development.
epididymis was removed and placed in 1 ml of fertilization medium (Cook). Capacitation was
allowed to proceed for 30 minutes in an atmosphere of 6% CO2, 5% O2,
and 89% N2 at 37°C. The spermatozoa were transferred to COCs drops for
insemination at a final motile sperm concentration of 2.5x105 /ml. All
experiment groups from the same hydrosalpinx sample were used spermatozoa obtained from
the same male. Two hours later, MII oocytes were transferred to culture in 10 µl
drop of cleavage medium (G1-plus; Vitrolife, Sydney, Australia) under mineral oil
(IrvineScientific). The fertilization rate was determined the next day by counting the
number of two-cell embryos. Seventy-two hours post insemination, the embryos were
transferred to blastocyst medium (G2-plus; Vitrolife, Sydney, Australia) under mineral oil
(Irvine Scientific) and cultured in similar conditions for 48 hours. Embryo development
was evaluated under an inverted microscope every 24 hours until completion of 120 hours.
Mouse blastocysts were classified as early, partial, full, expanding, hatching and hatched
blastocysts, using the criteria proposed by
et al. (2000)
4
Superovulation and Embryo Culture
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All mice used in this study were housed according to the guidelines of EMBL Laboratory Animal Resources. Mice were maintained in individually ventilated plastic cages (Tecniplast) in an air-conditioned (temperature 22 ± 2 °C, humidity 50 ± 10%) and light-controlled room (illuminated from 7:00 to 19:00). Mice were fed 1318P autoclavable diet (Altromin) ad libitum. Mouse embryos were collected from superovulated (injection of 7.5 international units (IU) pregnant mare serum followed by injection of 7.5 IU humanchorionic gonadotropin 42–50 h later) 8- to 24-week-old female mice according to the guidelines of EMBL Laboratory Animal Resources. Embryos were cultured in 30-μl drops of G1 PLUS (Vitrolife) covered by mineral oil (Ovoil, Vitrolife). Embryos were isolated from C57BL/6J × C3H/He F1 females.
5
Intracytoplasmic Sperm Injection Protocol
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Microinjections were performed at ×400 magnification on a 37 °C heated stage inverted Nikon Eclipse Ti2 (Nikon, Tokyo, Japan). A Petri dish containing a microdroplet of ICSI ™ media in the center (ICSI ™, VitroLife, Sweden) under paraffin oil (OVOIL, VitroLife, Sweden) was used for sperms selection and immobilization. On the same dish, a microdroplet of G-Gamete ™ culture medium (G-Gamete ™, Vitrolife, Sweden) was used for placing the oocytes for microinjection. A single sperm was mechanically immobilized using the tip of the microinjection needle (Origio, USA) and then was aspirated inside the needle. The oocyte was held in place using a 35-degree angle holding micropipette (Origio, USA) with the polar body in the 6 or 12 o'clock position. Injection of a single spermatozoon within the oocyte cytoplasm was performed by using a micromanipulator (TransferMan® 4r, eppendorf, Germany). After ICSI, injected oocytes were cultured in G1-Plus ™ medium (G1-Plus ™, VitroLife, Sweden). Fertilization was confirmed by the presence of two pronuclei and the extrusion of the second polar body approximately 16–18 h after ICSI. The Fertilization Rate was calculated by dividing the number of obtained zygotes (2 PN) by the number of injected oocytes.
6
Comprehensive Blastocyst Evaluation Protocol
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Fertilisation will be considered normal when two pronuclei are present between 16 and 18 hours after ICSI or IVF. Normal fertilisation rate will be calculated as the number 2PN over the number of MII in ICSI patients or the number 2PN over the number of COCs for conventional IVF. All zygotes will be cultured in cleavage medium (G-1plus, Vitrolife, USA) for further 48–52 hour after fertilisation. Cleavage embryonic development will be assessed according to the developmental stage and degree of cytoplasmic fragmentation. All day 3 embryos will be cultured up to 2–3 days (G-2 plus, Vitrolife, USA). From day 5 to day 6, those embryos achieving the blastocyst stage will be evaluated morphologically using Gardner’s grading system.
For all participants, a freeze-only blastocyst transfer strategy will be applied. All usable blastocysts (embryos that can grow to expanded or hatching blastocysts earn a score above grade CC) will be cryopreserved by vitrification methods.
For all participants, a freeze-only blastocyst transfer strategy will be applied. All usable blastocysts (embryos that can grow to expanded or hatching blastocysts earn a score above grade CC) will be cryopreserved by vitrification methods.
7
ICSI on Heated Microscope Plate
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ICSI was performed on the heated plate (37˚C) of an inverted microscope (IX73; Olympus Corp.) with a micromanipulator system (Narishige). MII oocytes were selected for ICSI according to the method described in the Textbook of Assisted Reproductive Technologies, Third Edition (12 ). The injected oocytes were cultured in embryo culture medium (G-1 plus; Vitrolife) and in tissue culture dishes (cat. no. 353001; Falcon; BD Biosciences) with one oocyte per 30 µl medium.
8
Assisted Reproductive Procedures and Pregnancy Outcomes
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After the sperm injection, all oocytes were individually placed in 30µl
droplets of G1 plus (Vitrolife - Göteborg, Sweden) and kept at
36.8°C in a conventional incubator (6% CO2). Every 16 or 18 hours the
oocytes were checked for fertilization and classified regarding the formation of
normal zygote. For cleavage and embryo development, these pre-embryos were
washed and kept in low O2Trigas incubators (6%CO2-5%O2-89%N) (Minc
Benchtop Cook Medical) in G1 plus medium. Over the following days
cleavage and embryo quality were analyzed. The pre-embryos were taken to an
extended culture to the blastocyst stage, and were transferred from on D3 to
G2 plus (Sequential culture system) (Vitrolife - Göteborg,
Sweden).
βeta-HCG was measured from the 10th day to the 13th day after ET, in order
to confirm chemical pregnancy. Ultrasound exams were carried out from the
6th to the 8th week for the confirmation of pregnancy
through the visualization of a gestational sac (SG) and fetal heartbeat.
droplets of G1 plus (Vitrolife - Göteborg, Sweden) and kept at
36.8°C in a conventional incubator (6% CO2). Every 16 or 18 hours the
oocytes were checked for fertilization and classified regarding the formation of
normal zygote. For cleavage and embryo development, these pre-embryos were
washed and kept in low O2Trigas incubators (6%CO2-5%O2-89%N) (Minc
Benchtop Cook Medical) in G1 plus medium. Over the following days
cleavage and embryo quality were analyzed. The pre-embryos were taken to an
extended culture to the blastocyst stage, and were transferred from on D3 to
G2 plus (Sequential culture system) (Vitrolife - Göteborg,
Sweden).
βeta-HCG was measured from the 10th day to the 13th day after ET, in order
to confirm chemical pregnancy. Ultrasound exams were carried out from the
6th to the 8th week for the confirmation of pregnancy
through the visualization of a gestational sac (SG) and fetal heartbeat.
9
Embryo Culture and Blastocyst Formation
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Following ICSI, each ova was placed in a micro-culture well Embryoscope Culture Dish (Vitrolife, Sweden) containing 25 µl G-1 PLUS (REF. No. 10029, Vitrolife, Sweden) culture medium and then into Embryoscope Time-Lapse Incubator (Vitrolife, Sweden). At day 3 the culture medium was removed and changed into G-2 PLUS (REF. No. 10132, Vitrolife, Sweden) and it was cultured to day 5 until the formation of blastocyst49 (link).
10
Controlled Ovarian Hyperstimulation Protocols
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Controlled ovarian hyperstimulation (COH) protocols were determined by experienced physicians depending on the patients’ baseline data and willing. Follicle growth was initiated by either recombinant follicle stimulating hormone or human menopausal gonadotropin in general. Ovulation was induced by human chorionic gonadotropin (hCG) at a dose of 4000–10,000 IU. Oocyte retrieval was performed 36 hours after hCG administration. The time interval between removing granule cells and performing ICSI in non-AOA cycles was same as that in AOA cycles. Routinely, the time interval ranges from 0.5 h to 2 h, which depends on the maturation rate of the obtained oocytes and the number of ICSI patients on the day of oocyte retrieval. The ICSI procedure was identical in both non-AOA and AOA cycles and performed by experienced operators. Sperm used for ICSI were all fresh sperm and most were ejaculated sperm. During calcium ionophore oocyte activation cycles, MII oocytes were exposed to A23187 (5 μM, Sigma, St. Louis, MO, USA) for 15 minutes after 15 minutes of ICSI, and then rinsed for three times. The base solution for oocyte activation is cleavage-stage medium, which is G1-plus (Vitrolife) in our center.
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