Study on the selection of normal sperm and the method of fusion of sperm and oocytes for micro-feritlization.

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The swim-shift (SS) methoda novel method of sperm selection, was invented. It retrieved more motile sperm than the conventional swim-up (SU) method. The PSA-column method retrieved morphologically normal sperm selectively. When SS method and PSA-column method were combined, a sperm concentration and %norma morphology were higher and %mortility was similar, compared with the SU method alone. Several procedures of subzonal insemination of sperm (SUZI) were modified to give a minimal %damage (%D) and maximal %fertilization (%F) of hamster oocytes with human sperm without use of sucrose for expansion of subzonal space, becaouse sucrose toxicity to oocytes was suggested. Pretreatment of sperm with 5 muM calcium ionphore improved %F of zona-free hamster oocytes. When the improved procedures of SUZI was applied for treatments of severe male infertility, %F and %embryo cleavage were improved significantly but %pregnancy was not, compared with the conventional procedures of SUZI.When sperm concentration of sperm droplet was diluted by 100 times to give 100,000 per ml, %F of hamster oocytes with human sperm by intracytoplasmic sperm injection (ICSI) was significantly increased. It was suggested that incision of sperm midpiece just before ICSI increased %F.A use of Piezo-manipulator decreased %D and increased %F significantly. When the improved procedures of ICSI with dilution of sperm concentration, incision of sperm midpiece, and use of Piezo-manipulator was applied for treatments of severe male infertility, numbers of fertilized oocytes and cleaved embryos and %pregnancy per cycle were significantly increased compared with conventional procedures of ICSI,resulting in 20% of ongoing pregnancy rate per cycle.