Clinical Intracytoplasmic Sperm Injection in Human Assisted Reproduction: Guidelines for Customization and Best Practice of the Procedure

Oocyte Denudation Process

Oocyte denudation process i.e. the removal of any cumulus or corona cells surrounding the oocyte, is mandatory before the ICSI procedure. The process combines chemical and mechanical means, such as the use of the enzyme hyaluronidase and mechanical pipetting of the cumulus-oocyte-complexes (COC) by the embryologist. Two key factors in this process may influence the ICSI result. Firstly, the hyaluronidase used need to be recombinant and not bovine-derived. Evison, et al. [2] found that the use of recombinant hyaluronidase in COC denudation significantly decreased oocyte degeneration compared to the bovine-derived one. Secondly, a complete denudation of the ova from the surrounding cells may increase oocyte survival after ICSI, since Ebner, et al. [3] reported that incomplete oocyte denudation produced a significantly higher degeneration rate.

Oocyte Degeneration after ICSI

The major problem puzzling all embryologists performing ICSI, is the reduction of the percentage of degenerated/destroyed oocytes due to the invasiveness of the procedure. The biological basis of oocyte degeneration after ICSI is not that clear. Oocyte maturity at ICSI time, day 3 Follicular Stimulating Hormone (FSH) levels and estradiol levels on hCG triggering day, have been shown to be predictors of degeneration rate [4]. Other factors include excessive manipulation and movements of the injecting pipette while inside the ooplasm and thick/hard zona pellucida (ZP). The latter results in oocyte deformation during injection that eventually causes the cytoplasm to lyse. Careful hormonal monitoring of the stimulation cycle, increased oocyte maturity level and correct microsurgical treatment of the ovum by the operating embryologist are the key factors of reducing oocyte degeneration post-ICSI. In the last case- scenario of thick/hard ZP the best option is to pre-treat the ova with 1.48μm diode Laser creating a small opening of about 5μm on the ZP. The technique is known as Laser- Assisted ICSI and has been shown to significantly reduce oocyte degeneration rate, while increases fertilization, embryo quality, blastulation and clinical pregnancy rates [5]. Finally, the use of ultra-thin injection micropipette with 4.5μm internal diameter could significantly reduce oocyte degeneration resulting in higher survival and fertilization rates (personal data, not shown).

Immature Metaphase I ova: To inject or not?

Quite often the ICSI embryologist has to face the problem of a high incidence nuclear immaturity in the oocytes of a patient. These Metaphase I (MI) oocytes are known to maintain meiotic arrest. The use of MI oocytes could be advantageous especially in patients with low oocyte yield after oocyte retrieval. The intervention here would be a short culture (2-3 hours) of these oocytes in the commercial culture medium used by each clinic, in order these ova to achieve nuclear maturation and extrude the first polar body. A recent study [6] revealed, in concordance with previous studies, that the use of in vitro cultured MI oocytes for ICSI resulted in very poor fertilization rates (37%), while the transfer of embryos derived from such ova did not produce any clinical pregnancies at all. The chromosomal status of embryos produced by MI-MII in vitro matured oocytes has been found to be highly abnormal (80.6%) [7], posing the question of introducing high percentage of culture- derived aneuploidies if such embryos are to be used in embryo transfers. Based on the available literature, the widespread use of MI oocytes should be avoided in ICSI and if it is deemed necessary such as in cases of no other option, aneuploidy screening by preimplantation genetic diagnosis employing embryo biopsy is mandatory before any clinical use of the embryos created.

Time to ICSI after Oocyte Retrieval

Another major factor that can affect ICSI outcome is considered to be the best time to perform ICSI after oocyte collection. It is important to mention that the oocytes biologically are characterized by the nuclear maturation evidenced by the presence of the first polar body in their perivitelline space and the cytoplasmic maturation that involves reorganization of cytoplasmic organelles in the cytoskeleton, and the synthesis of maternal mRNAs and proteins [8]. Both types of maturation are necessary to occur in order to achieve normal fertilization. In stimulated cycles though these two types of maturation appear to be asynchronous [9]. In

order to achieve the maximum ICSI results, oocyte denudation needs to be performed 1 hour post-oocyte retrieval, while ICSI needs to performed between 37 and 39 hours post-hCG administration [10].

ICSI and Fertilization Failure

Fertilization failure post-ICSI is a rare phenomenon present in about 1-3% of ICSI cases, even in the presence of normal spermatozoa [11]. The main reason contributing substantially in fertilization failure is oocyte activation deficiency (40-70% of failed ICSI cases) [12].

In order to salvage such cases, artificial oocyte activation (AOA) mostly by exposing the oocytes in calcium ionophores immediately after ICSI has been proposed as a rescue technique. The use of calcium ionophore could restore fertilization in otherwise known failed fertilization ICSI outcomes in azoospermic or cryptozoospermic men [13] or in globozoospermia cases [14].

Interestingly, calcium ionophore A23187 has been used to rescue fertilization failure of unexpected cases of aged unfertilized oocytes 18 hours post-ICSI [15]. In such cases the combination of calcium ionophore A23187 and granulocyte-macrophage colony-stimulating factor (GM- CSF) could restore fertilization, while the producing embryos could develop into euploid morphologically nice blastocysts with potential clinical use.