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Gamete Handling Prior to ICSI
Fertilization by means of micromanipulation requires denudation of oocytes (i.e., removal of the surrounding cumulus and corona cells). This allows not only precise injection of the oocytes but also the assessment of their maturity, which is of critical importance for ICSI. Cumulus and corona cells are removed using the enzymatic procedure. Microscopic observations of the denuded oocytes include assessment of the zona pellucida and the oocyte, and the presence or absence of a Germinal Vesicle (GV) or a first polar body. ICSI is only carried out on metaphase II oocytes. Denuded and rinsed oocytes are incubated until the time of microinjection.
Routinely, sperm samples for ICSI are processed by density-gradient centrifugation [using silane-coated silica particle colloid solutions, enriching the number of motile and morphologically normal sperm cells needed for assisted reproduction.
Fertilization and Embryo Cleavage after ICSI
Post-fertilization, about 90% of 2-PN oocytes obtained by ICSI enter cleavage, resulting in multicellular embryos. Cleavage characteristics of the fertilized oocytes are evaluated daily. Normally developing, good-quality embryos reach the four-cell and eight-cell stage, respectively, on day 2 and in the morning of day 3 post microinjection. Numbers and sizes of blastomeres and the presence of anucleate cytoplasmic fragments are recorded. The cleaving embryos are scored according to equality of size of the blastomeres and proportion of anucleate fragments.
Endometrial Preparation and Luteal Support
Progesterone supplementation is continued until the 12th week of pregnancy.