Immature oocytes are incubated in a culture dish containing maturation medium. The maturation medium is supplemented with 75 mIU/mL of FSH and LH. The oocytes are cultured at 37°C in an atmosphere of 5% carbon dioxide and 95% air with high humidity. Oocytes are checked for maturity 24 and 48 hours after culture. The oocytes are denuded of granulosa cells, and mature oocytes (detected by the presence of an extruded polar body) are fertilized by ICSI. ICSI is performed for in vitro matured oocytes because it reduces the risk of unexpected poor fertilization as compared with IVF. However, it has been demonstrated that ICSI may not always be essential for the fertilization of in vitro matured human oocytes collected from unstimulated ovaries when the sperm parameters are normal (61). After
ICSI, the oocytes are transferred into 1 mL of IVF medium in a tissue culture dish. Fertilization is assessed 18 hours after ICSI by examining the oocytes for the appearance of two distinct pronuclei and two polar bodies.
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