Before the initiation of treatment, menstrual day-2 serum E 2 and FSH levels were assessed. Patients with acceptable parameters (E 2 <75 and FSH <13.5) underwent controlled ovarian hyperstimulation using injectable gonadotropins (Follitropin beta; Schering Plough; Serono Pharmaceuticals; or menotropins, Ferring Pharmaceuticals), with LH suppression achieved using either a gonadotropin-releasing hormone agonist (leuprolide acetate; TAP Pharmaceuticals) or antagonist (ganirelix acetate, Organon; cetrorelix, Serono). Oocyte maturation was induced when R2 follicles reached R17 mm in diameter; ultrasound-guided transvaginal oocyte retrieval was performed 34 to 36 hours later.
Ganirelix acetate
Manufactured by Organon
Sourced in France
Ganirelix acetate is a pharmaceutical product used in assisted reproductive technology procedures. It is a synthetic decapeptide that acts as a gonadotropin-releasing hormone (GnRH) antagonist. Ganirelix acetate helps regulate the ovarian stimulation process by preventing premature luteinizing hormone (LH) surges.
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6 protocols using ganirelix acetate
1
Controlled Ovarian Hyperstimulation Protocol
2
Controlled Ovarian Stimulation and Oocyte Retrieval
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Before initiation of treatment, menstrual day 2 or 3 serum E2 and FSH levels were assessed. Patients with acceptable parameters (E2 < 75 and FSH <13.5) were stimulated using injectable gonadotropins (Follitropin beta, Schering Plough, NJ; Serono Pharmaceuticals, Rockland, MA; Menotropins, Ferring Pharmaceuticals, Parsippany, NJ), with LH suppression achieved using a GnRH antagonist (ganirelix acetate, Organon; cetrorelix, Serono). Ovulation was triggered when ≥ 2 follicles reached ≥ 17 mm in diameter, and ultrasound-guided trans-vaginal oocyte retrieval was performed 34–36 hours later.
3
IVF Protocols for Oocyte Retrieval and Embryo Transfer
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Protocols for COH, oocyte retrieval, IVF, and embryo transfer were conducted according to the previously outlined practice (25) . Briefly, the patients were treated with gonadotropins (Follistim, Merck; Gonal-F, EMD-Serono; and/or Menopur, Ferring) until criteria for pituitary suppression with a GnRH antagonist (0.25mg Ganirelix acetate, Organon) were met (26) . The hCG (Pregnyl, Merck), GnRH agonist trigger (leuprolide), or dual trigger (a combination of hCG and GnRH agonist, depending on physicians' preference) were used for final oocyte maturation when the two lead follicles reached a mean diameter >17 mm. Ultrasound-guided transvaginal oocyte retrieval after 35 hours after final oocyte maturation was performed based on our standard practice (26) . One day after retrieval, luteal progesterone supplementation with intramuscular progesterone commenced. Fresh embryo transfer was performed on day 3 or day 5 using a Wallace catheter (Marlow/Cooper Surgical). The number of embryos transferred was based on the patient's age, her previous cycles, and clinical criteria (27) .
4
Prepubertal Mouse Ovarian Stimulation
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Ten μg of Ganirelix acetate (Orgalutran®, N.V. Organon, Puteaux, France) or saline was subcutaneously injected twice on prepubertal female mice, at 12 and 13 dpn. In addition to Ganirelix, 13 dpn mice received an intraperitoneal injection of either 5 IU of human Chorionic Gonadotropin (hCG) (N.V. Organon), or 5 IU of equine Chorionic gonadotropin (eCG) (N.V.Organon), or saline. Injections (less than 12.5 μl) were performed with Hamilton syringes connected with catheters and needles (Phymep, Paris, France). Mice were then dissected at 14 dpn to collect blood and ovaries. For the superovulation procedure, mice received 5 IU of equine chorionic gonadotropin (eCG) followed 48 hours later by a 5-IU injection of hCG. Females were killed 20 hours later. Their oviducts were retrieved and placed in hyaluronidase in order to visualize any oocyte removed from the ampulla. For BrdU incorporation studies, females received a single injection of 100 mg/kg BrdU the day before dissection.
5
Controlled Ovarian Hyperstimulation and IVF Protocol
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Protocols for controlled ovarian hyperstimulation, oocyte retrieval, in vitro fertilization, and embryo transfer were conducted according to the previously outlined practice. 24 Briefly, the patients were either downregulated with the use of gonadotropin-releasing hormone agonist (Lupron, Abbott Pharmaceuticals, Worcester, MA, USA) followed by stimulation with gonadotropins (Follistim, Merck, Whitehouse Station, NJ, USA; Gonal-F, EMD-Serono, Rockland, MA, USA; and/or Menopur, Ferring, Parsippany, NJ, USA) or were treated with gonadotropins until criteria for pituitary suppression with a gonadotropin-releasing hormone antagonist (0.25 mg Ganirelix acetate, Organon) were met. 25 Human chorionic gonadotropin (Pregnyl, Merck) or a combination of human chorionic gonadotropin and gonadotropin-releasing hormone agonist was used as the ovulatory trigger when the two lead follicles reached a mean diameter > 17 mm. Ultrasound-guided transvaginal oocyte retrieval was performed after 35-37 hours following the ovulatory trigger based on our standard practice. 24
6
Prepubertal Ganirelix Treatment in Mice
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Studies were conducted on C57BL/6JRj mice aged 7 to 17 days postnatal born at the animal facility from genitors purchased at Janvier Labs (Le Genest St Isle, France). Mice were maintained under controlled conditions (12h light/12h dark cycle) with food (Scientific Animal Food and Engineering (SAFE), A03-10) and water available ad libitum. The day of birth was designed as 0 dpn. Mice were anesthetized with a mix of ketamine (Imalgene® 1000) and xylazine (Rompun® 2%) and then killed by cervical dislocation. Pituitaries were collected, frozen in liquid nitrogen and stored at -80°C for RNA and protein extraction. Ten μg of Ganirelix acetate (Orgalutran®, N.V. Organon, Puteaux, France) or saline was subcutaneously injected twice on prepubertal female mice at 12 and 13 dpn, following a procedure previously published [37 (link)]. Experiments were performed in accordance with standard ethics guidelines and were approved by Institutional Animal care and Use committee of the University Paris Diderot and by the French Ministry of Agriculture (agreement #04015.01).
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