A detailed protocol for minimal stimulation with CC has been reported previously.21 (link), 22 , 23 (link) Briefly, CC (50–100 mg/d) (Fuji Pharma Co, Ltd, Tokyo, Japan) was administered orally on an extended regimen from day 3 of the retrieval cycle to the day before induction of final oocyte maturation. EMT was measured using an ultrasound on the day of the trigger. When the dominant follicle developed to a size >18 mm, ovulation triggering was performed using a nasal spray containing the gonadotropin-releasing hormone agonist buserelin (Suprecur; Mochida Pharmaceutical Co, Ltd, Tokyo, Japan; or Buserecur, Fuji Pharma Co, Ltd).24 (link)
At 34 to 36 hours after triggering, oocyte retrieval was performed using a 21-gauge needle (Kitazato Corporation, Shizuoka, Japan), generally without anesthesia or follicular flushing.25 (link) Cumulus-oocyte complexes (COCs) were collected, washed, and transferred to a human tubal fluid (HTF) medium (Kitazato Corporation) with paraffin oil at 5% CO2 in air at 37°C for culturing. Conventional IVF was performed 3 hours later. In cases of intracytoplasmic sperm injection (ICSI), denudation was performed at 4 hours after oocyte retrieval.26 (link),27 (link) For ICSI, cumulus cells surrounding the oocytes were removed, and the denuded oocytes were cultured in the HTF medium covered with paraffin oil for 1 hour before ICSI.
Sperm samples were collected by ejaculation and washed by centrifugation through 70% and 90% density gradients (Isolate; Irvine Scientific, Santa Ana, CA). The prepared sperm were cultured in HTF medium at 5% CO2 in air at 37°C until use.
At 34 to 36 hours after triggering, oocyte retrieval was performed using a 21-gauge needle (Kitazato Corporation, Shizuoka, Japan), generally without anesthesia or follicular flushing.25 (link) Cumulus-oocyte complexes (COCs) were collected, washed, and transferred to a human tubal fluid (HTF) medium (Kitazato Corporation) with paraffin oil at 5% CO2 in air at 37°C for culturing. Conventional IVF was performed 3 hours later. In cases of intracytoplasmic sperm injection (ICSI), denudation was performed at 4 hours after oocyte retrieval.26 (link),27 (link) For ICSI, cumulus cells surrounding the oocytes were removed, and the denuded oocytes were cultured in the HTF medium covered with paraffin oil for 1 hour before ICSI.
Sperm samples were collected by ejaculation and washed by centrifugation through 70% and 90% density gradients (Isolate; Irvine Scientific, Santa Ana, CA). The prepared sperm were cultured in HTF medium at 5% CO2 in air at 37°C until use.
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