Prior to ICSI the cumulus-oophorous-complex of the oocyte is commonly remove with impure animal-derived hyaluronidase of low strength. The lower strength of the enzyme, the high concentration of impurities and the necessity of extensive time-dependent mechanical denudation damages of the oocytes, increases the risk of lysing the oocytes and the transfer of animal-derived pathogens. All this can be avoided by using a highly pure, non-toxic and effective recombinant human hyaluronidase, which requires less mechanical denudation, reduces oocyte damage, increases fertilization, embryo quality and pregnancy rates. During the ICSI procedure the sperm are subjectively selected. However, it is now possible to Backgroundly select motile sperm, via their hyaluron receptors, of a better morphology, higher chromatin stability and lower incidence of aneuploidy, which improves fertilization rates, embryo quality, blastulation and ongoing pregnancy rates -“Physiological ICSI”.

ICSI procedure improve fertilization rate in cases with male factor. But fertilization failure still occurs in 2-3% of ICSI cycles. Literature studies reveal that fertilization failure has been related to sperm, oocyte factor or ICSI technique. But the main cause of failed fertilization is failure to complete oocyte activation. Oocyte activation includes large number of well defined morphological and biochemical endpoints, some of which occur within seconds or minutes of spermoocyte plasma membrane interaction, and some occur over the course of several hours. Intracellular calcium rise plays an important role in oocyte activation. Therefore, researchers have used different methods including mechanical, electrical and chemical to mimic the calcium rise in order to activate the oocytes that will be discussed during the presentation.

The largest single defines etiology of human infertility is defective sperm function, resulting in failed fertilization. This phenotype has been rescued by IVF and ICSI and over 200, 000 births per annum are achieved through these techniques. Therefore, there is a price to be paid for this achievement, including, birth defects, reduced birth weight and etc. In addition the efficiency of fertilization and embryo development in these techniques are limited to sperm quality. Considering the heterogeneity of semen samples, to overcome these price and further to improve the effectiveness of these techniques, novel sperm selection procedures has been proposed, including; hyaluronic acid binding, sperm membrane surface charge, sperm organellar morphology, sperm surface markers, birefringence and sperm ability to respond to hypo-osmotic stress. During this presentation, we hope to discuss pros and cones of each technique.

After the first reports on pregnancies after ICSI this procedure soon replaced all other methods of assisted fertilization in humans. At the Dutch-speaking Brussels Free University, after the first observations of fertilization and embryo development after injection of a sperm cell into the cytoplasm of an oocyte, the ICSI procedure was stepwise modified to become the procedure as it is nowadays used worldwide. The modifications implemented resulted in increasing fertilization rates. As for standard IVF, the availability of more embryos influenced the chances of establishing a pregnancy. Already in the early days of ICSI, epididymal and testicular sperm cells showed to be able to fertilize and developing embryos gave rise to ongoing pregnancies. Not only patients with obstructive azoospermia but also patients with non-obstructive azoospermia can be treated successfully by ICSI. So far, the use of a fully matured viable spermatozoon seems to be necessary for successful ICSI. Despite some preliminary reports, the use of precursor cells of the spermatogenesis does not seem to be very successful. An overview of the different indications for which ICSI can be used will be discussed during the presentation. Over the years, it has been discussed whether ICSI should become the only treatment offered. Studies have shown that fertilization rates after ICSI are not higher as compared to standard IVF in cases with normal semen. Considering the complexity of the procedure, IVF should still be offered where possible. In cases with moderate semen quality, allocation of oocytes to IVF or ICSI in a first treatment cycle can be a valuable tool and prevent from a possible fertilization failure. The discussion whether the ICSI procedure itself negatively influences outcome is still ongoing. However, a recent study on about 100 cycles failed to demonstrate a negative effect on fertilization and blastocyst development in vitro of the ICSI procedure itself. Together with increasing regulatory control, efforts to increase quality of treatment and quality of products used in the laboratory are necessary. A continuous effort of all parties involved, can make that results of follow-up studies of pregnancies and children born after ICSI remain reassuring as they have been so far.

During spermiogenesis, histones are replaced by protamines (P1 and P2), resulting in sperm chromatin condensation followed by a halt in gene expression of haploid spermatids and spermatozoa. As a consequence, protamine deficiency and aberrant P1/P2 ratio have a profound effect on both fertilization and embryo development. However, reports on the effect of the P1/P2 ratio on fertilization and embryo development after intracytoplasmic sperm injection (ICSI) are contradictory among human and animal studies. The question yet to be answered is which type of protamine deficiency is most common among protamine deficient samples. The main aim of this study was to investigate the possible correlations of the P1/P2 ratio with protamine deficiency, fertilization, embryo quality, and embryo development in ICSI patients. It was carried out in 71 patients. Chromomycin A3 (CMA3) staining was used to determine protamine deficiency. Since this procedure does not indicate the type of protamine deficiency, the P1/P2 ratio was evaluated by nuclear protein extraction, acetic acid urea polyacrylamide gel electrophoresis, and analysis of protein bands with software. Polyclonal anti-P1 and anti-P2 antibodies were used to confirm P1 and P2 presence. Our results showed a statistically significant negative correlation of fertilization rate with protamine deficiency and P1/P2 ratio. However, no significant correlation was observed between protamine deficiency and P1/P2 ratio. Therefore, it can be concluded that altered P1/P2 ratio effects fertilization rate and embryo quality which subsequently may affect implantation and pregnancy outcome

ICSI has become the routine method of choice for treatment of male infertility. Even male factor is the major concern during this procedure, but still much emphasis has been directed on number and quality of oocytes recovered and just the presence of sperm has been taken as granted. However, literature study on the quality sperm reveal that in male factors, the semen contain large number of protamine deficient sperm, high number of apoptotic sperm, fragmented DNA or and high number of immature sperm. In addition these factors may directly or indirectly effect fertilization and subsequent development. Therefore, along with different sperm function tests, different sperm selection procedure has been developed mainly based on sperm surface characteristics. These include: 1) Hyaluronic acid binding test base on presence of sperm surface receptors, 2) Zeta potential, based on the surface electrical charge, 3) Magnetic sperm sorting base on apoptotic markers present on the sperm surface. The consequence of these procedures on assisted reproductive techniques will be discussed during presentation.