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.
Gnrha
Manufactured by Ipsen
Sourced in France, China
GnRHa is a laboratory equipment product. It is a synthetic analogue of the naturally occurring gonadotropin-releasing hormone (GnRH). GnRHa functions to stimulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland.
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7 protocols using gnrha
1
Prolonged Ovarian Stimulation Protocol
2
Controlled Ovarian Stimulation Protocol
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After evaluation of follicular condition according to blood hormone tests (FSH, LH, E2) and vaginal ultrasound, Gn 75 U to 300 U/day was administered to stimulate follicular growth. Follicular development was evaluated using vaginal ultrasound and blood hormone tests every three to five days, and Gn dosage adjusted when necessary. GnRH antagonist was added on the fifth to seventh day after Gn stimulation began (i.e., fixed protocol), or according to the size of the dominant follicle and LH level (i.e., flexible protocol). HCG (Livon, China) 4000 U to 10,000 U or GnRHa (IPSEN) 0.2 mg was administered to induce oocyte maturation, when at least one follicle ≥18 mm or three follicles ≥ 17 mm in diameter. Oocyte recovery was performed 36 - 38 h after the trigger.
3
Controlled Ovarian Stimulation Protocol
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Long-acting GnRHa (0.93–1.25 mg intramuscular [i.m.] once; IPSEN, Paris, France) or a short-acting GnRHa (0.1 mg i.m. per day until HCG day; IPSEN) was usually administered on the 18th to 22nd days of menstrual cycle (5–7 days after ovulation) after the corpus luteum detected on the vaginal ultrasound, if necessary, we measured serum progesterone level to determine the ovulation. Two to 3 weeks later, the vaginal ultrasound and blood hormones test (FSH, luteinizing hormone [LH], estradiol [E2]) were necessary. Gonadotropin 100 to 300 U/day can be administered when the results fully reached the pituitary complete down-regulation criteria. Every 3 to 5 days, the ultrasound and blood hormones were monitored to evaluate follicular development, when at least one follicle ≥18 mm or 3 follicles ≥17 mm in diameter emerges, HCG (Livon, China) 4000 to 10,000 U or GnRHa (IPSEN) 0.2 mg can be used as a trigger. Thirty-six to 38 h later, the vaginal oocyte retrieval was performed, embryos were cultured until the 3rd to 5th day, and then the embryo transfer was performed. Corpus luteal support began from the oocyte recovery with a progesterone intramuscular injection 60 mg/day or progesterone capsules 600 mg/day trans-vaginal until the day of pregnancy test.
4
PCOS vs. Non-PCOS Patients: ICSI Outcomes
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Patients were scheduled for intracytoplasmic sperm injection (ICSI) at the Assisted Reproduction Centre of Maternal and Child Health Care Hospital of Shaanxi Province (Xi'an, China) from November 2012 to July 2013. The study population included 24 women with PCOS and 24 normal responders without PCOS (non-PCOS, controls). PCOS patients were diagnosed following the Rotterdam 2003 criteria (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004) and on the basis of the presence of oligo-or anovulation and polycystic ovaries (ultrasound imaging). The non-PCOS controls were from women undergoing ICSI only due to male factor and without any other fertility problems. Patients with Cushing's syndrome, congenital adrenal hyperplasia and other systemic diseases were excluded from this study. All patients underwent a routine luteal phase down-regulation protocol with gonadotrophin-releasing hormone (GnRH) agonist (GnRH-a; Decapeptyl, Ipsen, France) and recombinant FSH (Gonal-F Merck Serono, Switzerland or Puregon, NV Organon, The Netherlands) for controlled ovarian hyperstimulation. Each patient gave informed written consent, and the study protocol was approved by the Ethics Review Board of Maternal and Child Health Care Hospital of Shaanxi Province on 18 September 2012.
5
Evaluating the Effects of GnRHa and ST on Rat Limb Development
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All animal experiments were approved by the Medical Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University and carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. Normal healthy Sprague-Dawley rats were provided by the experimental animal center of Sun Yat-sen University and housed in a standard animal room with food and water ad libitum under controlled conditions of humidity (50%-70%) and temperature (21°C ± 2°C) and under a 12 h light/12 h dark lighting schedule.
To evaluate the effect of GnRHa and ST, 2.5mg/kg of GnRHa (Diphereline, Ipsen Pharma Biotech, France) was injected intramuscularly once every 2 weeks and 10 mg/kg of ST (Sigma-Aldrich, MO, USA) was injected once a day. PBS as a negative control. Rats were euthanized at 7 weeks of age. Hind limb specimens were dissected and fixed in 4% paraformaldehyde for histological analyses (n = 5 animals per group) (15 (link)).
To evaluate the effect of GnRHa and ST, 2.5mg/kg of GnRHa (Diphereline, Ipsen Pharma Biotech, France) was injected intramuscularly once every 2 weeks and 10 mg/kg of ST (Sigma-Aldrich, MO, USA) was injected once a day. PBS as a negative control. Rats were euthanized at 7 weeks of age. Hind limb specimens were dissected and fixed in 4% paraformaldehyde for histological analyses (n = 5 animals per group) (15 (link)).
6
Ovarian Granulosa Cell Isolation
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For ovarian stimulationFollicular aspirates were collected during oocyte retrieval following published procedures and ovarian stimulation was the use of a prolonged protocol (25, 26) . Briefly, gonadotropin-releasing hormone agonist (GnRH-a, Ipsen, Boulogne-Billancourt, France) was used in the second or three day of menstrual cycle for pituitary down-regulation. Gonadotropin stimulation were started after 28 (volume fraction) Percoll gradient (Sigma-Aldrich, Germany). They were centrifuged at 4000 rpm for 20min to purify human GCs from any red blood cells. After washing and recentrifugation, sheets of human GCs were digested with trypsinat a 1:1 ratio for 4 min to separate them. The GCs were removed using a pipette and washed with phosphate buffered saline (PBS). 10% FBS DMEM / F12 culture medium at a 2:1 ratio were added to terminate the digestion, centrifuged at 1500 rmp for 3 min. GCs were resuspended with 1xPBS at a 1:5 ratio, centrifuged at 1500rmp for 3 min. After discarding the supernatant, added 1 ml of 10% FBS DMEM / F12 culture medium, resuspended, inoculated in a dish, and placed in a 37 , 5% CO2 incubator. After observing the morphology of the granulosa under the microscope , changed the fluid per 24h. The cells at indicated time were stored at -80°C for future analysis.
7
GnRHa and Letrozole for Large Tumors
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Patients with large unresectable tumors were administered 3.75 mg diphereline (GnRHa, Ipsen Pharma Biotech, Boulogne-Billancourt-France) by intramuscular injection once every 28 days for at least 3 months before operation, which could inhibits the enlargement of tumor and reduce the size, so as to profit the operation. Patients that underwent oophorectomy and hysterectomy were administered 2.5 mg letrozole (Jiang Su HengRui Medicine Co. Ltd, Lianyungang, Jiangsu, China) orally daily for at least 6 months after surgery.
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