- Ovarian stimulation.
- Oocyte retrieval.
- Embryo culture in a laboratory.
- Embryo transfer.
A baseline scan is performed between days 2 and 5 of the menstrual cycle. If the patient is amenorrheic, a withdrawal bleed with progestogens is induced. At the baseline scan, ovarian volume, ovarian stromal blood flow velocity, number of antral follicles, size of the follicles, endometrial thickness, and any ovarian or uterine abnormality are recorded. However, the antral follicle count is the only significant predictor. A second scan is performed on days 6–8 of the cycle to repeat all the above-described measurements.
Basic screening tests are performed on both partners and the women is stimulated with injectable medications to develop multiple follicles. Blood and ultrasound testing is done every 1-3 days to monitor the development of the follicles in the ovaries.
Priming with hCG
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 is supposed to be physically the most painful part of the IVF procedure, and various methods of analgesia have been tried over the years. A good analgesic method for oocyte retrieval has to give a satisfactory pain relief with rapid onset, rapid recovery, and ease of administration and monitoring. It is also important that it is safe and has no toxic effect on the oocytes. Transvaginal Ultrasound-guided Oocyte Retrieval (TVOR) is the gold standard for oocyte aspiration in assisted reproductive technology (ART). For more than 20 years, oocyte aspiration has been performed under the guidance of transvaginal ultrasound. The technique has proved to be a simple and safe procedure that is well accepted by the patients even if it is performed under local anesthesia with conscious sedation. It can thus be performed as an outpatient procedure.
Oocyte retrieval is done under short general anaesthesia or intravenous sedation using propofol or fentanyl and midazolam (1–2mg). Intravenous fentanyl or propofol is administered at intervals of 15–20min up to a total dose of 150–200 mg. Retrieval is performed under ultrasound guidance with a 17-19 G, double lumen aspiration needle. The follicular fluid is collected in culture tubes containing flushing medium. Because the aspiration pressure is low and a small-gauged needle is used, the injury to the ovaries is minimum. The procedure is repeated until all follicles seen are aspirated from other 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.
ICSI with ejaculated spermatozoa can be used successfully in patients with fertilization failures after conventional IVF and also in patients with too few morphologically normal and progressive motile spermatozoa present in the ejaculate. High fertilization and pregnancy rates can be obtained when a motile spermatozoon is injected. Injection of only immotile or probably non-vital spermatozoa results in lower fertilization rates. In cases where only non-vital sperm cells are present in the ejaculate, the use of testicular sperm is indicated. Other semen parameters, such as concentration, morphology (except for globozoospermia), and high titers of antisperm antibodies do not influence the success rates of ICSI. Any form of infertility due to obstruction of the excretory ducts can be treated by ICSI with spermatozoa microsurgically recovered from either the epididymis or the testis. Obstructive azoospermia can result from congenital bilateral absence of the vas deferens, failed vasectomy reversal, or vaso-epididymostomy. When no motile spermatozoa can be retrieved from the epididymis due to epididymal fibrosis, testicular spermatozoa can be isolated from a testicular biopsy specimen.
Testicular sperm recovery may not always be successful in all azoospermic 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
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 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. Ninety-five percent of the retrieved cumulus–oocyte complexes usually contain an intact oocyte. Approximately 10.3% of the intact oocytes are at the GV stage, and about 85.8% of them are in the metaphase II stage, showing the presence of the first polar body . ICSI is only carried out on metaphase II oocytes. Denuded and rinsed oocytes are incubated until the time of microinjection.
For microinjection, spermatozoa from three different origins are processed: ejaculated sperm and surgically retrieved sperm from the epididymis or the testis. For all three categories, ICSI in combination with sperm cryopreservation is currently used successfully.
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.
Testicular biopsy specimens, usually obtained by means of surgical excisional biopsy, are shredded into small pieces with sterile microscope slides on the heated stage of a stereomicroscope. The presence of spermatozoa is assessed with an inverted microscope, which determines whether the surgical procedure can be stopped or whether extra biopsy pieces need to be taken. The pieces of biopsy tissue are removed, and the medium is centrifuged at 300g for 5 minutes. The pellet is then re-suspended for the ICSI procedure. It is well known that it is not always possible to retrieve testicular spermatozoa from biopsy specimens in patients.
Fertilization and Embryo Cleavage After ICSI
After the injection procedure, oocytes are rinsed and cultured in micro-droplets covered with lightweight paraffin oil. The conditions are similar to those employed for IVF inseminated oocytes: the oocytes are kept at37°C in an atmosphere of 5% O2 5%CO2, and 90% N2. Injected oocytes are examined for integrity and fertilization about 16–18 hours after ICSI. An average damage rate of approximately 9% of the injected oocytes can be expected, irrespective of the origin of the sperm used. Oocytes are considered normally fertilized when two individualized or fragmented polar bodies are present together with two clearly visible pronuclei (2-PN) that contain nucleoli. The fertilization rate after ICSI is usually expressed per number of injected oocytes and ranges from 57% to 67% according to the sperm origin. 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.
- Type A (excellent quality) embryos do not contain anuclear fragments.
- Type B (goodquality) embryos have a maximum of 20% of the volume of the embryo filled with anucleate fragments.
- Type C (fair quality) embryos, anucleate fragments represent 21% to 50% of the volume of the embryo.
- Type D (poor-quality) embryos have anucleate fragments present in more than 50% ofthe volume of the embryos. These embryos cannot be used for transfer to the patients. Embryos in the former three categories (type A, B, and C) are eligible for transfer.
Endometrial Preparation and Luteal Support
Progesterone supplementation is continued until the 12th week of pregnancy.