**6. Timing of ICSI**

Both nuclear and cytoplasmic maturation of oocytes have to be completed to ensure optimal conditions for fertilization. Oocytes are retrieved prior to ovulation for IVF or ICSI procedures. In the pre-ovulatory phase, meiotic division of the oocyte must progress to metaphase II which is considered nuclear maturation and is evident by extrusion of first polar body. The oocyte also must develop the capacity to attain fertilization and initiate embryo development which is considered cytoplasmic maturation [29, 30]. Cytoplasmic maturity is thought to be asynchronous with nuclear maturity in stimulated cycles [31, 32] and the fertilizing ability of an oocyte with a mature nucleus is not necessarily at its maximum potential. Thus, preincubation of oocytes prior to IVF or ICSI may induce cytoplasmic maturation that could eventually increase fertilization and also pregnancy rates. The human oocytes progressively develop the ability for full activation and normal development during the MII arrest stage [33].

The ICSI technique is generally similar among different centres but the time intervals from retrieval to denudation and from denudation to ICSI varies. Very few studies have addressed this aspect, with discrepancies in the conclusions [34, 35]. The preincubation period between oocyte retrieval and injection improves the percentage of mature oocytes [36, 37], the fertilization rate [35, 37], and the embryo quality [35]. The appropriate incubation time for mature oocytes before ICSI is 5–6 h. This time improves embryo quality and pregnancy rate in ICSI cycles. The maximum clinical pregnancy rate is observed when ICSI is performed 5 h after oocyte retrieval. The clinical pregnancy rate dropped significantly when ICSI was performed 6 hrs after oocyte retrieval (Falcone et al., 2008). A longer oocyte pre-incubation (9– 11 hours) prior to ICSI is thought to have detrimental effects on embryo quality [38], probably due to oocyte ageing.

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 123

[51]. Globozoospermia results from perturbed expression of nuclear proteins or from an altered golgi-nuclear recognition during spermiogenesis. The sperm show both gross and ultrastructural abnormalities, including the complete lack of an acrosome, abnormal nuclear membrane and mid-piece defects. Depending on the severity of the defect, the fertilization rate after ICSI with round headed sperm ranges from 0% to 37% [52, 53]. Successful pregnancies have been reported after ICSI in patients with globozoospermia with or without oocyte activation [54, 53, 55]. The most likely cause for failed fertilization after ICSI using round-head sperm is inability of sperm to activate the oocyte. In some forms of globozoospermia, arrest of nuclear decondensation and/or premature chromosome

DNA damage in the male germ line is associated with poor fertilization rates following IVF, defective pre-implantation embryonic development and high rates of miscarriage and morbidity in the offspring, including childhood cancer [56, 57]. Activation of embryonic genome expression occurs at the four to eight-cell stage in human embryos [58], suggesting that the paternal genome may not be effective until that stage. Therefore, a lack of correlation between elevated DNA strand breaks in sperm and fertilization rates may occur before the four to eight-cell stage [59, 60]. Many published articles indicate that DNA strand breaks are clearly detectable in ejaculated sperm and their presence is heightened in the ejaculates of men with poor semen parameters [61, 62]. Nuclear DNA damage in mature sperm includes single strand nicks and double strand breaks that can arise because of errors in chromatin rearrangement during spermiogenesis, abortive apoptosis and oxidative stress [63, 64]. In the same individuals, testicular samples show a significantly lower DNA damage compared to ejaculated spermatozoa (14.9%±5.0 vs. 40.6%±14.8, P<0.05), but significantly higher aneuploidy rates for the five analyzed chromosomes (12.41%±3.7 vs. 5.77%±1.2, P<0.05). While testicular spermatozoa appear favourable for ICSI in terms of lower DNA damage, this potential advantage could be offset by the higher aneuploidy rates in testicular

Two tests are most commonly reported as indicators of sperm nuclear integrity; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and sperm chromatin structure assay (SCSA). The TUNEL technique labels single or double-stranded DNA breaks, but does not quantify DNA strand breaks in a given cell. The SCSA, a quantitative and flowcytometric test, measures the susceptibility of sperm nuclear DNA to acid-induced DNA denaturation *in situ*, followed by staining with acridine orange [66]. The SCSA accurately estimates the percentage of sperm chromatin damage expressed as DNA fragmentation index (DFI) with a cut-off point of 30% to differentiate between fertile and infertile samples [67]. A statistically significant difference is seen between the outcomes of ICSI versus IVF when DFI is >30% [68]. The biological explanation behind the superior results of ICSI in cases of high DFI needs to be elucidated. One possibility may be that women undergoing ICSI, on average, produce healthier oocytes with a better DNA repair capacity than women undergoing IVF, as in the ICSI group infertility is mainly caused by male factor.

condensation also causes fertilization failure [55].

**7.2. Sperm DNA damage** 

spermatozoa [65].
