**7.1. Sperm structural defects**

Normal sperm ultrastructure correlates with positive IVF results [39]. Single structural defects involving the totality of ejaculated sperm are among rare cases of untreatable human male infertility. This form of infertility is of genetic origin and is generally transmitted as an autosomal recessive trait. Numerous defective genes are potentially involved in human isolated teratozoospermia but such defects have not been defined at the molecular level in most cases [40]. An in-depth evaluation of sperm morphology by transmission electron microscopy (TEM) can improve the diagnosis of male infertility and can give substantial information about the fertilizing competence of sperm [41, 42]. The TEM evaluation of sperm can also identify potentially inheritable genetic disorders (for example primary ciliary dyskinesia, Kartagener's syndrome), providing valuable information for couples contemplating ICSI [43].

Abnormal spermatozoa with head vacuoles account for the patient infertility. Sperm head vacuoles are easily detectable in human spermatozoa under the electron microscope. A sperm head vacuole is considered abnormal when it exceeds 20% of the head's crosssectional area. In rare cases, primary spermatozoa deformity is 100% vacuolated head [44]. There is a strong correlation between high relative vacuole area to sperm head and poor sperm morphology [45]. No correlation is observed between DNA defect and sperm-head morphology [46]. However, macrocephalic and large-headed spermatozoa are commonly associated with a low chance of pregnancy, mainly in relation to meiotic abnormalities during spermatogenesis. Enlarged-head spermatozoa are linked to sperm chromatin condensation dysfunction with no major meiotic dysfunction [47].

Acrosome agenesis is most often associated with a spherical shape of the head and is defined as "round head defect" or "globozoospermia". The underlying causes of the syndrome remain to be elucidated [48]. The genetic contribution has been postulated as well [49]. An additional case report [50] supports it. Studies show that the pathogenic genes associated with globozoospermia include SPATA16, PICK1, GOPC, Hrb, Csnk2a2 and bs [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 condensation also causes fertilization failure [55].

#### **7.2. Sperm DNA damage**

122 Enhancing Success of Assisted Reproduction

**7. Sperm related factors** 

contemplating ICSI [43].

**7.1. Sperm structural defects** 

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

Normal sperm ultrastructure correlates with positive IVF results [39]. Single structural defects involving the totality of ejaculated sperm are among rare cases of untreatable human male infertility. This form of infertility is of genetic origin and is generally transmitted as an autosomal recessive trait. Numerous defective genes are potentially involved in human isolated teratozoospermia but such defects have not been defined at the molecular level in most cases [40]. An in-depth evaluation of sperm morphology by transmission electron microscopy (TEM) can improve the diagnosis of male infertility and can give substantial information about the fertilizing competence of sperm [41, 42]. The TEM evaluation of sperm can also identify potentially inheritable genetic disorders (for example primary ciliary dyskinesia, Kartagener's syndrome), providing valuable information for couples

Abnormal spermatozoa with head vacuoles account for the patient infertility. Sperm head vacuoles are easily detectable in human spermatozoa under the electron microscope. A sperm head vacuole is considered abnormal when it exceeds 20% of the head's crosssectional area. In rare cases, primary spermatozoa deformity is 100% vacuolated head [44]. There is a strong correlation between high relative vacuole area to sperm head and poor sperm morphology [45]. No correlation is observed between DNA defect and sperm-head morphology [46]. However, macrocephalic and large-headed spermatozoa are commonly associated with a low chance of pregnancy, mainly in relation to meiotic abnormalities during spermatogenesis. Enlarged-head spermatozoa are linked to sperm chromatin

Acrosome agenesis is most often associated with a spherical shape of the head and is defined as "round head defect" or "globozoospermia". The underlying causes of the syndrome remain to be elucidated [48]. The genetic contribution has been postulated as well [49]. An additional case report [50] supports it. Studies show that the pathogenic genes associated with globozoospermia include SPATA16, PICK1, GOPC, Hrb, Csnk2a2 and bs

effects on embryo quality [38], probably due to oocyte ageing.

condensation dysfunction with no major meiotic dysfunction [47].

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 spermatozoa [65].

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.

Other tests of sperm nuclear DNA integrity include *in situ* nick translation and the comet assay. The toluidine blue and sperm chromatin dispersion test are potential new assays [69]. At present, there are two major strategies that may be considered for the treatment of men exhibiting high levels of DNA damage in their sperm: (i) selective isolation of relatively undamaged sperm and (ii) antioxidant treatment [70]. The lack of consensus in defining a clinically relevant standard DNA fragmentation test with a meaningful cut-off level brings challenges in implementing the routine use of sperm DNA integrity assessment in daily practice [71].

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 125

It is not yet fully understood how the sperm activates the oocytes. The failure of fertilization after ICSI may result from either the lack or deficiency of activating factors in sperm or from the lack of ooplasmic factors triggering sperm chromatin decondensation [79, 80]. Several pieces of evidence point to PLCζ being the physiological agent of oocyte activation and is detectable in different localities within the sperm head: the equatorial segment and

During normal spermiogenesis, 85% of histones are replaced with protamines [82], which results in sperm chromatin condensation. A sperm with a condensed nucleus is in the G1 stage when entering an MII oocyte and is protected from PCC because an active maturationpromoting factor (MPF) is not capable of reacting with protamine-associated DNA. Once sperm nuclear decondensation factors from the ooplasm enter the sperm, the sperm head swells and sperm associated oocyte activating factor is released. This results in MPF inactivation [83], the completion of meiosis 2 and the oocyte enters the G1 stage. During this time, protamines are slowly replaced by histones and cell cycle synchronization takes place. Under some circumstances, the oocyte fails to activate and remains arrested at MII. Because of the presence of an active MPF, sperm chromatin transforms into condensed chromatin.

Sperm PCC has been associated with the type of ovarian stimulation protocol. Some protocols, such as clomiphene citrate and human menopausal gonadotropin stimulation may tend to recruit immature oocytes with immature cytoplasm [84]. Immature cytoplasm is believed to make sperm susceptible to a high incidence of PCC after insemination because of the inability of these immature oocytes to undergo oocyte activation [85]. The incidence of sperm PCC reported in the literature ranges from 10.1 to 85 % [86, 87], with higher values noted in cases of round headed sperm injection as they fail to activate the oocyte. Furthermore, other studies suggest a correlation between fertilization outcome post-ICSI and percentage of sperm with protamine deficiency [88]. The effect of sperm protamine deficiency on fertilization rate emphasizes the need for accurate sperm selection during ICSI as protamine-deficient sperm, in the form of slightly amorphous head, may find the chance

Defective sperm tail is the principal cause of sperm motility disorders. There are two main forms of tail disorders with different phenotypic characteristics and consequences for male fertility: non-specific tail anomalies and various genetic disorders including primary ciliary diskinesia and the dysplasia of the fibrous sheath [89]. In non-specific tail anomalies, ICSI has good prognosis and does not pose additional risks in view of the lack of recognized genetic components in this Disorder. Significant sperm abnormalities of proven or suspected genetic origin are rare conditions responsible for extreme asthenozoospermia or total sperm immotility. Affected patients complain of male infertility and chronic respiratory disease, alterations caused by abnormal function of sperm flagella and respiratory cilia. In patients with tail genetic disorders, ICSI results in normal rates of fertilization and implantation, and

acrosomal/post-acrosomal region [81].

Sperm with excessive histones are prone to PCC.

of being injected due to inappropriate sperm selection [88].

**10. Sperm motility and progression** 
