Basic screening tests are performed on both partners and the women are stimulated with injectable medications to develop multiple follicles. Blood and ultrasound testing are done every 1-3 days to monitor the development of the follicles in the ovaries.
HCG priming prior to oocyte collection increases the maturation rate of oocytes in vitro and we administer 250mcg/0.5ml (Ovitrelle) of r-DNA HCG 36 hours prior to collection.
Oocyte Retrieval
Oocyte retrieval is performed under good analgesia to give a satisfactory pain relief with rapid onset, rapid recovery. It is also important that it is safe and has no toxic effect on the oocytes. Transvaginal Ultrasound-guided Oocyte Retrieval is the gold standard for oocyte aspiration in assisted reproductive technology (ART)..
Oocyte retrieval is done under short general anaesthesia or intravenous sedation. Retrieval is performed under ultrasound guidance with a 17-19 G aspiration needle. The follicular fluid is collected in culture tubes containing a flushing medium. The procedure is repeated until all follicles seen are aspirated from another ovary. ICSI requires only one spermatozoon for the fertilization of each oocyte. Indications for ICSI are not restricted to impaired morphology of the spermatozoa, but also include low sperm counts and impaired kinetic quality of the sperm cells. ICSI can also be used with spermatozoa from the epididymis or testis when there is an obstruction in the excretory ducts. Azoospermia caused by testicular failure can be treated by ICSI if enough spermatozoa can be retrieved in testicular tissue samples.
Testicular sperm recovery may not always be successful in all azoospermia patients. Cryopreservation of supernumerary spermatozoa recovered from the epididymis or the testis is an important issue because microinjection of cryo-thawed sperm cells can avoid repeated surgery in future ICSI cycles
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.
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.
These embryos cannot be used for the transfer to the patients. Embryos in the former three categories (type A, B, and C) are eligible for transfer.
Progesterone supplementation is continued until the 12th week of pregnancy.