Ovarian stimulation was performed with the use of a prolonged protocol. Briefly, standard full dose of gonadotropin-releasing hormone agonist (3.75 mg, GnRH-a, Ipsen, France) was used in the second day of menstrual cycle for down regulation. Pituitary down regulation (Endometrial thickness ≤ 5 mm, serum FSH < 5 mIU/mL, LH < 5 mIU/mL, E2 < 50 pg / mL) was confirmed with transvaginal ultrasound and endocrine examination after 30 days. Then, according to the patient’s age, body mass index, serum basal FSH levels, LH levels, estradiol levels and antral follicle count, initial doses of 75–112.5 IU/d of recombinant human FSH (Merck-Serono, German) were used. The time and dose of recombinant human FSH was adjusted according to ovarian response as monitored by serum estradiol levels and vaginal ultrasound. When the dominant follicle was≥19 mm in diameter or at least 2 follicles were ≥ 18 mm in diameter, recombinant human FSH was stopped and a single injection of 6000–8000 IU of hCG (Merck-Serono, German) was administered. Oocyte retrieval was performed 36–40 h later under transvaginal ultrasound guidance.
Recombinant human fsh
Manufactured by Merck Group
Sourced in Switzerland, United Kingdom
Recombinant human Follicle-Stimulating Hormone (rh-FSH) is a laboratory product produced through recombinant DNA technology. It is a glycoprotein hormone that plays a crucial role in the regulation of the reproductive system. rh-FSH is primarily involved in the stimulation of follicular growth and development in the ovaries of females and the production of sperm in males.
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Lab products found in correlation
3 isobutylmethylxanthine, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Human insulin, by Merck Group (1 mentions)
Selenium, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Human serum albumin (hsa), by Thermo Fisher Scientific (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
24 well cell culture plate, by Corning (1 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (1 mentions)
Gnrha, by Ipsen (1 mentions)
G 1 plus, by Vitrolife (1 mentions)
G2 plus, by Vitrolife (1 mentions)
8 protocols using recombinant human fsh
1
Prolonged Ovarian Stimulation Protocol
2
Mouse Ovarian Granulosa Cell Isolation
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All experiments and analyses were in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 86‐23, revised in 1996). Experimental animal protocols were approved by the Institutional Animal Care and Use Committee of Korea Institute of Oriental Medicine (19‐019, Daejeon, Korea). Eight‐week‐old C57BL/6 female mice were obtained from Nara Biotech (Pyeongtaek, Korea) and housed under specific pathogen‐free conditions. Animals were administered intraperitoneal injections of either saline (n = 15) or Cy (n = 16, 100 mg/kg; Sigma‐Aldrich, St. Louis, MO) 6 times over 2 weeks. The body weight of mice was measured immediately after or 4 weeks after Cy cessation. Mice were sacrificed at 24 h after intraperitoneal injections of pregnant mare's serum gonadotropin (PMSG, 5 IU, ProSpec‐Tany TechnoGene, Rehovot, Israel) to synchronize the menstrual cycle affecting GCs proliferation in growing ovarian follicles. The ovaries were weighed, then placed immediately into puncture medium to isolate the GCs, containing minimum essential medium (MEM) with GlutaMAX (Gibco, Paisley, UK), 1× insulin‐transferrin‐selenium (ITS, Zen‐Bio, NC), recombinant human FSH (Sigma‐Aldrich), and 10% fetal bovine serum (Gibco). To measure AMH levels, the serum was separated from whole blood and frozen at –80°C.
3
Ovarian Tissue Culture and Evaluation
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One to three fragments were selected from each biopsy as Day 0 (D0) controls. The remaining fragments (two to three per day of culture) were incubated individually in 24-well cell culture plates (Corning B.V. Life Sciences Europe, Amsterdam) as previously described (Telfer et al., 2008 (link); McLaughlin et al., 2018 (link)). Briefly, 300 μl of culture medium was added per well [McCoy’s 5a medium with bicarbonate supplemented with HEPES (20 mM; Invitrogen Ltd), glutamine (3 mM; Invitrogen Ltd), HSA (0.1%), penicillin G (0.1 mg/ml), streptomycin (0.1 mg/ml), transferrin (2.5 μg/ml), selenium (4 ng/ml), human insulin (10 ng/ml), 1 ng/ml recombinant human FSH, and ascorbic acid (50 μg/ml) (all obtained from Sigma Chemicals, UK, unless specified)]. Fragments were cultured for up to 6 days at 37°C in humidified air with 5% CO2 with half the media being removed and replaced every second day. After 0, 2, 4, or 6 days of culture, the ovarian fragments were fixed in 10% normal buffered formalin for histological and immunohistological evaluation.
4
Antagonist Protocol for IVF with Trophectoderm Biopsy
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The standard techniques were used for in vitro fertilization (IVF) and the antagonist protocol was used for ovarian stimulation. The patient was treated with 150–225 IU/d of gonadotropin (recombinant human FSH, Merck Serono, Switzerland) at the beginning of day two of the menstrual cycle and the dose of gonadotropin was adjusted in accordance with ovarian reactivity. When the diameter of the follicles reached 12 mm, 0.25 mg of Nirek acetate (Merck Serono, Switzerland) was given daily. When the diameter of at least three dominant follicles reached 18 mm, 0.2 mg of triptorelin acetate (Ferring AG, Switzerland) and 4000 IU of Human Chorionic Gonadotropin (HCG, Merck Serrano, Switzerland) were administered by intramuscular injection, to trigger ovulation. After 36–38 h, transvaginal ultrasound-guided oocyte retrieval was performed.
All metaphase II (MII) oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Fertilization was confirmed after 16–18 h by the presence of two pronuclei (2 PN) and the second polar body (2 PB). Embryos were cultured in G1-plus and G2-plus (Vitrolife, Sweden) sequential media (Gardner and Schoolcraft, 1999 (link)). Between four and ten trophectoderm (TE) cells were biopsied from each blastocyst on day 5 or day 6 by zona drilling with a laser, and transferred into a lysis buffer for whole-genome amplification (WGA).
All metaphase II (MII) oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Fertilization was confirmed after 16–18 h by the presence of two pronuclei (2 PN) and the second polar body (2 PB). Embryos were cultured in G1-plus and G2-plus (Vitrolife, Sweden) sequential media (Gardner and Schoolcraft, 1999 (link)). Between four and ten trophectoderm (TE) cells were biopsied from each blastocyst on day 5 or day 6 by zona drilling with a laser, and transferred into a lysis buffer for whole-genome amplification (WGA).
5
Controlled Ovarian Hyperstimulation for Blastocyst Transfer
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All patients were stimulated by the long protocol. Ovarian follicular development was stimulated with recombinant human FSH (Merck Serono, Switzerland) at doses of 225-450 IU/day. Ovulation was triggered by human chorionic gonadotrophin (HCG 4000-10000 IU) (Livzon Pharmaceutical, China) when at least two follicles were 18 mm and half of the remainder were > 15 mm. Oocytes were recovered transvaginally under ultrasound guidance approximately 34.5 h later. All monitoring of controlled ovarian hyperstimulation (COH) as well as egg retrievals and embryo transfers were performed by one of ve physicians. Speci c methods refer to previous study [9] . Fertilized eggs were cultured to blastocysts. All embryos were graded microscopically according to Gardner grading standard [10] . All patients chose to transplant the embryo at the blastocyst stage on the fth day. Each patient selected the embryo with the highest morphological score for single-embryo transfer. All embryo transfers were performed using a Wallace catheter under direct ultrasound guidance 120 h after egg retrieval.
6
Controlled Ovarian Hyperstimulation for Blastocyst Transfer
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All patients were stimulated by the long protocol. Ovarian follicular development was stimulated with recombinant human FSH (Merck Serono, Switzerland) at doses of 225-450 IU/day. Ovulation was triggered by human chorionic gonadotrophin (HCG 4000-10000 IU) (Livzon Pharmaceutical, China) when at least two follicles were 18 mm and half of the remainder were > 15 mm. Oocytes were recovered transvaginally under ultrasound guidance approximately 34.5 h later. All monitoring of controlled ovarian hyperstimulation (COH) as well as egg retrievals and embryo transfers were performed by one of ve physicians. Speci c methods refer to previous study [9] . Fertilized eggs were cultured to blastocysts. All embryos were graded microscopically according to Gardner grading standard [10] . All patients chose to transplant the embryo at the blastocyst stage on the fth day. Each patient selected the embryo with the highest morphological score for single-embryo transfer. All embryo transfers were performed using a Wallace catheter under direct ultrasound guidance 120 h after egg retrieval.
7
Measurement of cAMP Accumulation in FSHR Mutant
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Forty-eight hours after transfection with the WT and D408Y FSHR cDNAs (cloned into the pSG5 plasmid), cells (in 24-well dishes) were washed with DMEM-5% fetal-calf serum and then stimulated with increasing doses of human recombinant FSH (Merck-Serono, Mexico City, Mexico) in the presence of 0.125 mM 3-isobutyl-methyl-xanthine (Sigma Aldrich, Mexico City, Mexico). At the end of the incubation period (2 h), the medium was removed and total (extracellular plus intracellular) cAMP accumulation was measured by radioimmunoassay as reported previously [29 (link)].
8
FSH-Induced cAMP Accumulation Assay
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Forty-eight hours after transfection, the medium was removed and the cells in 24-well-dishes were washed twice with DMEM-5% FCS and then stimulated with increasing doses of human recombinant FSH (Merck-Serono, Mexico D.F., Mexico) in the presence of 0.125 mM 3-isobutyl-methyl-xanthine (Sigma). At the end of the incubation period (18 h), the medium was removed and total (extracellular plus intracellular) cAMP accumulation was measured by radioimmunoassay (Zambrano et al., 1996 (link)).
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