Fertilization and embryo evaluation
Semen samples were prepared by the swim-up procedure. About 2 hours after oocyte retrieval, each oocyte was inseminated with approximately 20,000–30,000 motile spermatozoa. If the total number of motile sperm was <105 after washing or normal morphology was <1%, intracytoplasmic sperm injection (ICSI) was performed. Oocytes were decoronated and checked for the presence of two pronuclei to confirm fertilization. Embryos were graded on day 3 after retrieval as grade one to grade six according to the evenness of each blastomere and the percentage of fragmentation. Embryos of 6-8 cells and of grade one or two were regarded as top quality embryos. Some non-top-quality embryos were placed in extended culture until they reached the blastocyst stage.