The History and Development of ICSI Technique

Since the birth of Louise Brown in June 1978, IVF has proven to be an effective treatment for infertility due to female factors, especially tubal infertility (Edwards et al., 1980).

In the subsequent years, IVF was also successfully used in couples with unexplained infertility, male infertility, and also in endometriosis (Mahadevan et al., 1983). The characteristics of male semen were evaluated according to the criteria of the World Health Organization (WHO).

It became apparent to many research groups that the outcome of classic IVF was much less successful when the characteristics of male semen were below reference values in terms of concentration, morphology, and motility. The percentage of oocytes that were normally fertilized was much lower, resulting in the formation of much fewer embryos; this meant that there were no embryos to transfer in a considerable number of cycles (Tournaye et al., 1992).

Some couples with progressive motility < 500,000 spermatozoa ready for insemination could not be included in an IVF program. This also included patients with azoospermia. Thus, at the end of the 1980s, many assisted fertilization procedures were developed and applied to couples where classic IVF could not be used.

In a first technique - partial zona dissection, PZD - a small opening was made in the zona pellucida allowing direct contact between the sperm and the oolemma. Overall results of PZD were unstable and disappointing. Another technique that was introduced was subzonal insemination SUZI. Several motile spermatozoa were microinjected into the perivitelline space. After SUZI, almost 20% of the eggs fertilized with this technique were normally fertilized (Ng et al., 1991; Fishel et al., 1993).

The overall experience with PZD and SUZI was that the percentage of normal fertilizations was very low. As a result, only one or two embryos became available for transfer in nearly two-thirds of the patients. The percentages of pregnancies and births were too low to consider the application of PZD and SUZI in routine clinical practice.

In June 1992, the first pregnancies and births after the transfer of embryos generated by ICSI (intra-cytoplasmic sperm injection), a new procedure in assisted fertilization, were published (Palermo et al., 1992). In ICSI, a single spermatozoon was microinjected into the oocyte after passing through the zona pellucida and the oocyte membrane (oolemma).

Before this application in humans, live pups had been born after ICSI in rabbits and sheep. Two other IVF groups, one in the USA and one in Singapore, had applied ICSI to 134 oocytes. In two patients, four 2-pronuclear stage (2PN) embryos and 11 divided embryos were transferred to seven patients. However, no pregnancies resulted (Lanzendorf et al., 1988).

Initial observations with ICSI showed that fertilization was much better after ICSI than after SUZI and more embryos capable of being transferred were obtained, and since the second half of 1992, ICSI has been used in most clinics (Van Steirteghem et al., 1993a).

CURRENT ICSI PRACTICE

Indications and procedure

Many couples with severe male infertility factor can be treated with ICSI. To generate normally fertilized oocytes after ICSI, a spermatozoon containing a functional genome and centriole is needed. ICSI can be performed with sperm from the epididymis and testis in cases of excretory duct blockage. ICSI can be performed in cases of azoospermia caused by damage to spermatogenesis if sufficient sperm is obtained from testicular tissue(Craft et al., 1993; Schoysman et al., 1993; Devroey et al., 1994, 1995, 1996; Tournaye et al., 1994).

ICSI using ejaculated sperm can also be performed in the presence of oligoasthenoteratozoospermia, in cases of continuous IVF failure, in the presence of a high concentration of anti-sperm antibodies, in patients recovering from cancer where sperm was cryopreserved before chemo- and radiotherapy, in patients with spinal cord injuries, in patients with ejaculatory disorders, in patients with retrograde ejaculation, and in patients whose semen was banked before vasectomy (Lahteen Maki et al., 1995; Nagy et al., 1995; Chung et al., 1998; Benadiva et al., 1999; Nikolettos et al., 1999; Schatte et al., 2000). ICSI is also indicated in cases where pre-implantation genetic diagnosis (PGD) is applied and PCR is used (Liebaers et al., 1998).

ICSI can be performed with sperm from the epididymis in cases of obstructive azoospermia: congenital bilateral absence of the vas deferens (CBAVD), Young's syndrome, failed vaso-epididymostomy, failed vasovasostomy, and bilateral inguinal (iatrogenic) obstruction of both ejaculatory ducts (Tournaye et al., 1994).

ICSI can also be performed with testicular sperm in all cases where epididymal sperm can be used, in the presence of excessive connective tissue that does not allow the retrieval of sperm from the epididymis, in cases of testicular failure due to maturation arrest, partial aplasia of germ cells or tubular sclerosis, and special cases with necrozoospermia (Silber et al., 1995, 1996; Tournaye et al., 1996).

Epididymal sperm can be obtained by microsurgical epididymal sperm aspiration (MESA). In MESA, thousands of spermatozoa are obtained in patients with obstructive azoospermia. Excess sperm can be cryopreserved for later use (Devroey et al., 1995a). Epididymal sperm can also be obtained by percutaneous sperm aspiration (PESA) (Tsirigotis et al., 1996).

In cases of epididymal fibrosis, testicular sperm can be isolated from small pieces of testicular tissue taken by open biopsy (TESE: testicular sperm extraction) (Devroey et al., 1994). In patients with obstructive azoospermia and normal spermatogenesis, testicular biopsy can also be performed with percutaneous sperm aspiration, which means aspiration with a fine needle (FNA: fine needle aspiration) (Bourne et al., 1995; Tournaye et al., 1998).

Sperm can be found in approximately half of the patients with non-obstructive azoospermia; it may require hours of searching by the embryologist to find live spermatozoa in several biopsy pieces (Devroey et al., 1995b; Verheyen et al., 1995, 1997; Crabbé et al., 1997; Schlegel et al., 1997, 1999; Tournaye et al., 1997; Amer et al., 1999, 2002).

In all cases where possible, fresh sperm should be used, but cryopreserved-thawed sperm can also be used: ejaculated, epididymal, and testicular. This can avoid repeated surgical interventions which are not without risk (Devroey et al., 1995a; Tournaye et al., 1999; Chan and Schlegel, 2000; Gil‐Salom et al., 2000; Habermann et al., 2000; Kupker et al., 2000).

Before the first clinical application, the ICSI procedure was evaluated and approved by the Ethical Committee of the Medical Campus of the Dutch‐speaking Brussels Free University. Before starting the treatment, couples were informed about the general aspects of the treatment, the results of ICSI so far, and the known risks at that time. Patients were asked to undergo prenatal diagnosis and to participate in a prospective follow-up study of the born children (Bonduelle et al., 1994).

Reviewing thousands of ICSI procedures, ICSI could not be performed in 1-3% of the retrieved oocytes due to the absence of mature oocytes and/or sperm; the latter was particularly in cases of couples with non-obstructive azoospermia (Liu et al., 1995; Vandervorst et al., 1997). From the authors' experience, approximately 85% of the ICSI cycles were done with fresh or thawed ejaculated sperm and in 15% with fresh or thawed epididymal and testicular sperm (Bonduelle et al., 1999).

For the ICSI procedure, the oocyte is fixed using a holding pipette; and an injection pipette with an inner diameter of 6 micrometers is used to aspirate a single spermatozoon. These pipettes can be purchased from special companies or produced in the laboratory. Before aspiration, the sperm is immobilized with polyvinylpyrrolidone.

A morphologically normal spermatozoon is aspirated into the injection pipette, initially by the tail. Immobilization of the sperm can also be achieved by cutting the tail with the injection pipette. The injection pipette is passed through the zona pellucida and the oocyte membrane into the cytoplasm at a position sufficiently far from the first polar body.

After ICSI, more than two-thirds of the ejaculated sperm-injected oocytes become normally fertilized. The fertilization rate with surgically retrieved sperm in non-obstructive azoospermia was lower than that with ejaculated sperm but still > 50% (Joris et al., 1998; Van Steirteghem et al., 1998).

New developments on normally fertilized oocytes after ICSI were evaluated as a ‘trend’ for IVF. More than 80% of the normally fertilized oocytes developed further into embryos with sufficient morphological quality for transfer. For all types of sperm, the percentage of embryos transferred or frozen was between 60 and 65% of the normally fertilized oocytes. In cases of non-obstructive azoospermia, testicular tissue may be damaged after repeated surgical interventions (Schlegel and Su, 1997).

After ICSI, the absence of fertilization occurred when there were only a few oocytes, had totally immotile sperm, no sperm had an acrosome, all oocytes had abnormal morphology and were damaged by the injection itself. Fertilization occurred more often in subsequent cycles (Liu et al., 1995; Nagy et al., 1995; Staessen et al., 1995; Vandervorst et al., 1997).

However, there were also studies that reached the conclusion that ICSI did not offer any clinical advantage over classic IVF in cases with non-male factor subfertility. They supported these data on the practice that ICSI should be reserved only for cases with severe male factor problems (Bhattacharya et al., 2001; Van Steirteghem and Collins, 2003).

In conclusion, it was suggested that ICSI should be the treatment of choice in all cycles of assisted reproduction. But if this were to be introduced without further studies, this policy could have an impact on laboratory time, on medical resources, and above all perhaps on overall safety due to bypassing the natural gamete selection mechanism and due to the invasiveness of the technique itself (Oehninger, 2001).

As studies continue according to data from IVF/ICSI registries, ICSI has been applied as a fertilization procedure. It became clear worldwide that a large number of groups had left classic IVF and used ICSI as a standard procedure even in cases where sperm parameters were normal (Bhattacharya et al., 2001; Oehninger, 2001; Nygren and Anderson, 2002).

ICSI is the technique used today as part of assisted reproduction techniques (ART) and at the IVF center in the American Hospital, Tirana and Pristina, resulting in successful treatment for a good portion of infertile patients.