Indications for ICSI and technique of ICSI
Until recently, the clinical application of direct injection of a single sperm into the cytoplasm of an oocyte during IVF was not shown to be feasible. The demonstration of fertilization and live births by Palermo et al. in 1993 was the first successful application of ICSI. Since that time, ICSI has been performed extensively in multiple centers to treat patients with severe male factor infertility. To date, the success of ICSI procedures has been related to several factors: (1) the viability of the spermatozoon, (2) the quality of the oocyte, (3) effective activation of the oocyte, and (4) ability of the oocyte to tolerate intracytoplasmic manipulation. Application of this treatment is described below. To date, rigorous indications for ICSI have not yet been defined. Most clinical series report on using ICSI in cases where standard IVF is highly unlikely to succeed, that is, in patients with less than 500,000 motile sperm present in the ejaculate, or less than 4% normal forms with strict criteria evaluation. In addition, couples who have failed to fertilize any oocytes in a prior IVF cycle are considered appropriate candidates for IVF-ICSI.
We have proposed the following indications for ICSI:
a) sperm concentration < 2 x 106
b) sperm motility < 5 %
c) strict criteria normal morphology < 4 %
d) use of surgically retrieved spermatozoa
e) failure of fertilization in a previous IVF cycle
Although fertilization and pregnancy rates with ICSI are similar or better than those achieved with normal sperm in other couples undergoing IVF concurrently at the same center, couples with only minor semen abnormalities have not been routinely treated with IVF-ICSI. Given the relatively brief history of ICSI, and its potential effects on progeny, it would seem prudent to avoid over-application of this new technology. Therefore, ICSI should not be recommended to couples for whom there is no documented benefit, since unknown risk may exist.
Technique of ICSI
1. Oocyte processing: Oocytes are prepared by removing the cumulus mass and corona radiata with hyaluronidase. The oocytes are then examined under the inverted microscope to assess the maturation stage by observing the presence of a germinal vesicle, germinal vesicle breakdown, and the extruded first polar body. Metaphase II oocytes are identified by the presence of the extruded first polar body. Intracytoplasmic sperm injection is performed on all metaphase II oocytes. Metaphase II oocytes have their diploid complement of chromosomes delicately arranged on the metaphase plate near the polar body. Mechanical disruption of the metaphase plate can occur by injury from the injection pipette or the presence of a motile sperm in the oocyte cytoplasm. Each oocyte is placed in a droplet of medium surrounding the central droplet which contains the spermatozoa. The droplets are covered with lightweight paraffin oil and the petri dish is placed on a heated stage of the microscope. The microscope is equipped with two hydraulic micromanipulators which are fitted to two tool holders for the micropipettes. During the injection procedure oocytes are stabilized with a holding micropipette, and injected with an injection pipette.
2. Microinjection: Details of the preparation of microtools and protocols for ICSI are described in detail elsewhere. The holding and injection pipettes are made by drawing glass capillary tubes with a pipette puller and further processed on microgrinder and microforges. The outer and inner diameters of the holding and injection pipettes are, respectively, 60 and 20 �m, and 7 and 5 �m. The injection pipette has a bevel angle of 50� and a sharp spike to assist penetration through the oolemma. Washed sperm are prepared on a discontinuous mini-Percoll gradient. The sperm h-action is washed with T6 medium containing 5 mM CaCl2 just prior to the injection procedure. The sperm pellet is resuspended and transferred with a polyvinylpyrrolidone solution in HEPESbuffered Earle’s medium. From a 3-5 �l sperm-PVP droplet covered with lightweight paraffm oil, a single sperm is aspirated into an injection micropipette. A metaphase II oocyte is immobilized with slight negative pressure on the holding pipette. The polar body is held at the 12 or 6 o’clock position and the injection micropipette containing the single sperm is pushed through the zona pellucida and oolemma into the cytoplasm of the oocyte at the 3 o’clock position. A single sperm is injected head first into the ooplasm with 1-2 pl of medium. The injection pipette is withdrawn gently and the oocyte is released from the holding pipette. Further handling of injected oocytes is similar to that for oocytes in standard IVF.
