All women included for the study underwent GnRH antagonist protocol controlled ovarian hyperstimulation. Patients received recombinant human follicle stimulating hormone (Puregon; MSD Sharp & Dohme GMBH) for 5 days with doses according to age, weight, sAMH, and hormonal status [16 (link), 17 (link)]. Trans-vaginal sonography was performed after 5 days and on the day of oocyte retrieval. Ultrasonographical measurement was performed using a RIC 5-9-D 4D intravaginal probe of a GE Voluson E8 BT09 ultrasound machine (both from GE Healthcare Austria GmbH). GnRH antagonist (Cetrotide, Merck KGaA) was injected to avoid premature ovulation. Triggering was initiated 35 h before the punction, administered with 5000–10,000 IU human chorionic gonadotropin (hCG) subcutaneously (Pregnyl, N.V. Organon), with dose according to body weight of the patient [16 (link)].
Pregnyl
Manufactured by Organon
Sourced in Netherlands, Australia, Germany, United States
Pregnyl is a laboratory product used for the measurement of human chorionic gonadotropin (hCG) levels. It is a highly purified form of hCG, a naturally occurring hormone produced during pregnancy. Pregnyl can be used as a reference standard or calibrator in assays designed to detect and quantify hCG in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Gonal f, by Merck Group (17 mentions)
Cetrotide, by Merck Group (8 mentions)
Cetrorelix, by Merck Group (7 mentions)
Ovitrelle, by Merck Group (5 mentions)
Ganirelix, by Organon (3 mentions)
Puregon, by Organon (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Ric 5 9 d 4d, by GE Healthcare (2 mentions)
Ge voluson e8 bt09, by GE Healthcare (2 mentions)
Menopur, by Ferring (2 mentions)
Decapeptyl, by Ferring (2 mentions)
Ovidrel, by Merck Group (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Roche (1 mentions)
Dnase type 1, by Merck Group (1 mentions)
Menogon, by Ferring (1 mentions)
Cobas e 411 instrument, by Roche (1 mentions)
Pregnant mare serum gonadotropin, by Merck Group (1 mentions)
Cleavage medium, by Cook Medical (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
58 protocols using pregnyl
1
Controlled Ovarian Hyperstimulation with GnRH Antagonist
2
Maternal Estrogen Levels in Mice
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All animal protocols were reviewed and approved by the Zhejiang University Animal Care and Use Committee. The imprinting control region (ICR) mice were housed in 12/12 hours light/dark cycle at 25 ± 0.5 °C and 50–60% of humidity, and fed with a standard diet and water ad libitum. ICR females received intraperitoneal (i.p.) injection of 10 IU PMSG (pregnant mare serum gonadotropin; Pregnyl, Organon, the Netherlands) and followed by an injection (i.p.) of 10 IU hCG (Pregnyl) 46–48 hours after administration of PMSG, then mated with ICR males and checked for a vaginal plug the morning following mating. The day that the vaginal plug was first seen was designated as day 0.5. The blood samples of pregnant mice at day 0.5, day 3.5, day 6.5, day 9.5 and day 12.5 were collected in OS and NC group to detect E2 levels by Beijing North Institute of Biological Technology (Beijing, China), 5–10 cases per group at each time point (day 0.5: NC n = 7, OS n = 5; day 3.5: NC n = 5, OS n = 5; day 6.5: NC n = 5, OS n = 5; day 9.5: NC n = 9, OS n = 5; day 12.5: NC n = 8, OS n = 5; the sample numbers were varied because it was difficult to get the blood of mouse at some time points). After the blood collection for E2 measurement, the mice were killed and the fetal livers of day 12.5 were obtained from 10 different pregnant mice each group.
3
Controlled Ovarian Stimulation Protocol
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Multiple follicular stimulation was achieved with human menopausal gonadotrophin (Diclair-HP HMG, R Germany, or Menogon, Germany R). A sliding scale regimen based on the age of the patient was used for human menopausal gonadotrophin administration. Women <34 years of age received 225 IU daily; 35-38 years, 300 IU daily; and > 38 years, 450 IU daily. Follicular development was monitored with transvaginal ultrasonography (SonoAceR7, Sansung Medison) done on day 6 of gonadotrophin stimulation and follow-up on day 8. The mean maximal follicular diameter was obtained from measuring the leading follicle in two planes at 90° to each other. The 10,000 IU of human chorionic gonadotrophin (HCG) (Pregnyl ® , Organon, The Netherlands) was administered when the leading follicle is at least 18 mm in mean diameter, and at least two follicles are larger than 16 mm. If the above-described criteria are not met by the follicular measurements, then a further scan was performed after 48 h, or a projected follicular growth of ~2 mm/day was employed to estimate the time of HCG injection. Transvaginal ultrasound guided-retrieval of oocytes was done 34-36 h after HCG injection with a 17gauge aspiration needle.
4
Ovarian Stimulation and Oocyte Retrieval Protocol
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Ovarian stimulation was carried out using the long
protocol. Briefly, gonadotropin-releasing hormone
(GnRH) agonist (Superfact, Aventis Pharma, Germany)
was adminstered on day 21 of the menstrual cycle.
rFSH (Gonal-F, Merck Serono, Germany) was injected
subcutaneously each day (150–300 IU/day) after the third
day of menstrual bleeding for a duration of five days.
For triggering ovulation, intramuscular administration
of 10000 IU units of human chorionic gonadotropin (hCG)
(Ovitrelle, Merck Serono Europe; Pregnyl, Organon) was
performed when one of the follicles reached >18 mm in
size as viewed by ultrasound. Transvaginal oocyte pickup via ultrasound guidance was carried out 36–38 hours
following the hCG injection.
After oocyte retrieval, the oocytes were denuded by
brief exposure to hyaluronidase (LifeGlobal) and frequent
pipetting. Then, oocytes were evaluated under an inverted
microscope for nuclear maturation assessment: i. GV
stage showed a germinal vesicle in the cytoplasm, ii.
meiosis I (MI) stage did not show any germinal vesicle in
the ooplasm and first polar body (PB) in the perivitelline
space, and iii. MII stage showed the presence of the first
PB in the perivitelline space.
protocol. Briefly, gonadotropin-releasing hormone
(GnRH) agonist (Superfact, Aventis Pharma, Germany)
was adminstered on day 21 of the menstrual cycle.
rFSH (Gonal-F, Merck Serono, Germany) was injected
subcutaneously each day (150–300 IU/day) after the third
day of menstrual bleeding for a duration of five days.
For triggering ovulation, intramuscular administration
of 10000 IU units of human chorionic gonadotropin (hCG)
(Ovitrelle, Merck Serono Europe; Pregnyl, Organon) was
performed when one of the follicles reached >18 mm in
size as viewed by ultrasound. Transvaginal oocyte pickup via ultrasound guidance was carried out 36–38 hours
following the hCG injection.
After oocyte retrieval, the oocytes were denuded by
brief exposure to hyaluronidase (LifeGlobal) and frequent
pipetting. Then, oocytes were evaluated under an inverted
microscope for nuclear maturation assessment: i. GV
stage showed a germinal vesicle in the cytoplasm, ii.
meiosis I (MI) stage did not show any germinal vesicle in
the ooplasm and first polar body (PB) in the perivitelline
space, and iii. MII stage showed the presence of the first
PB in the perivitelline space.
5
In Vitro Maturation (IVM) Protocol for Oocyte Retrieval
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Patients underwent an IVM cycle according to accepted IVM protocols (Fadini et al., 2009) . On day 3 of a spontaneous menstrual cycle, women underwent a baseline transvaginal ultrasound assessment to determine ovarian morphology, AFC, and endometrial thickness and basal blood concentration of oestradiol and progesterone. Following that, 150 IU/day recombinant FSH (rFSH) were administered to the patients for 3 days. A second evaluation was performed on day 6. An injection of 10,000 IU human chorionic gonadotrophin (HCG; Pregnyl; Organon, Oss, Holland) was administered subcutaneously when the leading follicle was 12 mm. Oocyte retrieval was performed 36 h later by transvaginal ultrasound-guided needle aspiration.
The follicular fluid was collected in culture tubes containing flushing medium with heparin (MediCult prod. no. 10760125, Denmark). In several cases due to time limitation, IVM was performed without the routine preparation. In these cases patients did not receive HCG. Several cases were performed during the luteal phase. Data regarding patients' distribution according to fertility preservation methods is presented in Figure 1.
The follicular fluid was collected in culture tubes containing flushing medium with heparin (MediCult prod. no. 10760125, Denmark). In several cases due to time limitation, IVM was performed without the routine preparation. In these cases patients did not receive HCG. Several cases were performed during the luteal phase. Data regarding patients' distribution according to fertility preservation methods is presented in Figure 1.
6
Investigating cAMP Signaling in Leydig Cells
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The animals underwent two different in vivo treatments mimicking acute and chronic manipulation of intracellular cAMP level: (1) treatment of intact male rats with agonist of LH receptors in order to increase cAMP signaling in Leydig cells, (2) experimental model of hypogonadotropic hypogonadism in order to downregulate reproductive axis and lower cAMP signaling in Leydig cells. In the first approach, a group of adult male rats were treated with a subcutaneous injection of Pregnyl (Organon, Holland, active component is human chorionic gonadotropin (hCG)), 40 IU/50 μL per 100 g of animal weight (a dose that effectively increases cAMP in rat Leydig cells [6 (link)]. The control group received the same amount of 0.9% NaCl. Animals were decapitated 2 h and 6 h after treatment. In the second approach, adult male rats were injected intramuscularly with the long-lasting GnRH analog diphereline (PharmaSwiss, Belgrade, Serbia; 0.29 mg/50 μL/100 g). Control rats were injected with the same amount of vehicle. After one month, animals were sacrificed in the morning. In both experimental approaches, trunk blood and testes were collected and used for further analysis.
7
Controlled Ovarian Hyperstimulation Protocol
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Women underwent controlled ovarian hyperstimulation with a GnRH short antagonist (cetrorelix; Merck Serono, Geneve, Switzerland; ganirelix; Organon, Oss, the Netherlands) protocol in the majority of the cases. For stimulation, rFSH was mainly used (Puregon; Organon; Gonal-F; Merck Serono, Germany). About 36 h before oocyte recovery, HCG was administered (Pregnyl; Organon). Estradiol serum levels were assayed at the day of HCG or 1 day before.28 (link)29 (link)
8
Controlled Ovarian Stimulation Protocol
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In order to controlled induce ovarian stimulation (COS), daily subcutaneous injection of recombinant human FSH (rFSH, Gonal F®; Serono Pharma, Switzerland) was started from the second day of the cycle. Starting dose of rFSH was adjusted individually depending on patients response measured by transvaginal ultrasonography, antral follicle count (AFC), levels of serum estradiol (E2) and AMH. A GnRH antagonist-cetrorelix (Cetrotide®, Merck Serono, Germany) was administered subcutaneously when at least two ovarian follicles reached 14 mm in diameter. The protocol consisted of daily subcutaneous injections of Cetrotide 0.25 mg, until the criteria for human chorionic gonadotropin (hCG) administration were met. For final oocyte maturation, when the dominant follicle reached ≥18 mm in diameter with the following two follicles ≥16 mm and E2 levels between 1000-4000 pg/mL, an intramuscular injection of 10.000 IU hCG (Pregnyl®, Organon, Holland) or subcutaneous injection of 250 μg hCG (Ovitrelle®, Merck Serono, France) was given.
9
In Vitro Oocyte Maturation and IVF
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On Day 9 of follicle culture, GM was replaced with IVM medium. Maturation medium consisted of GM supplemented with 4 ng/ml epidermal growth factor (EGF, Roche, Vilvoorde, Belgium) and 1.2 IU/ml hCG (Pregnyl, Organon, Brussels). Following 16–18 h of maturation, cumulus oocyte complexes (COCs) were used for IVF or denuded with hyaluronidase (200 IU/ml, Merck Life Science) for 1 min and evaluated for their maturation status. For the control oocytes (in vivo-grown group), we recruited metaphase II (MII) oocytes after stimulation of B6D2 female mice (8–12 weeks old). An injection of 7.5 IU pregnant mare’s serum gonadotrophin (Folligon, MSD AH, Brussels, Belgium) was given, followed by a second injection with 7.5 IU hCG (Chorulon, MSD AH) 48 h apart. Following 12–14 h of the Chorulon injection, mice were euthanized by cervical dislocation and COCs were collected from the ovarian ampulla. Complexes were either used for IVF or denuded with hyaluronidase and used at the MII stage.
10
Oocyte Isolation for In Vitro and In Vivo Assessment
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In this experimental study, for the in vitro group, the
oocytes were obtained from female mice B6D2F1
aged between 6 and 8 weeks old and purchased from
Pasteur Institute, Tehran, Iran. Animals were kept under
controlled light/dark and temperature conditions with
free access to water and food. They received 10 IU of
pregnant mare serum gonadotropin (PMSG). Forty-eight
hours later, mice ovaries were excised, and cumulus
oocyte complexes (COCs) were collected by aspiration
of ovaries, using a 28-gauge needle, in tissue culture
medium (TCM)-199-HEPES supplemented with 5%
foetal bovine serum (FBS). For the in vivo group, the
mice were super-ovulated as described previously (22 (link)).
Briefly, the female mice were intraperitoneally injected
with human chorionic gonadotrophin (hCG, Pregnyl®,
Organon) 48 hours after PMSG injection. After 12-14
hours, the mice were killed, and the COCs were isolated
from the oviducts and put into HTCM medium, which
contained 100 IU/ml hyaluronidase, to separate cumulus
cells from the oocytes. The oocytes were applied in the
following experiments.
oocytes were obtained from female mice B6D2F1
aged between 6 and 8 weeks old and purchased from
Pasteur Institute, Tehran, Iran. Animals were kept under
controlled light/dark and temperature conditions with
free access to water and food. They received 10 IU of
pregnant mare serum gonadotropin (PMSG). Forty-eight
hours later, mice ovaries were excised, and cumulus
oocyte complexes (COCs) were collected by aspiration
of ovaries, using a 28-gauge needle, in tissue culture
medium (TCM)-199-HEPES supplemented with 5%
foetal bovine serum (FBS). For the in vivo group, the
mice were super-ovulated as described previously (22 (link)).
Briefly, the female mice were intraperitoneally injected
with human chorionic gonadotrophin (hCG, Pregnyl®,
Organon) 48 hours after PMSG injection. After 12-14
hours, the mice were killed, and the COCs were isolated
from the oviducts and put into HTCM medium, which
contained 100 IU/ml hyaluronidase, to separate cumulus
cells from the oocytes. The oocytes were applied in the
following experiments.
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