**10. Sperm motility and progression**

124 Enhancing Success of Assisted Reproduction

**8. Injection with immature sperm** 

practice [71].

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

Round spermatid nucleus injection (ROSNI) or round spermatid injection (ROSI) is a method in which precursors of mature sperm obtained from ejaculated specimens or testicular sperm extraction (TESE) are injected directly into oocytes. ROSNI is proposed as a treatment for men in whom other more mature sperm forms (elongating spermatids or sperm) cannot be identified for ICSI [72]. It is not widely performed, not as successful as ICSI and is still an experimental procedure. It should be applied only in the setting of a clinical trial approved and overseen by a properly constituted institutional review board. Accurate identification of round spermatid is a technical challenge of ROSNI. It is difficult to distinguish haploid round spermatids from diploid spermatogenic precursors and somatic cells using the standard optics present in most clinical IVF laboratories. Mouse round spermatids have increased levels of DNA fragmentation [73] that may interfere with fertilization [63]. Increased DNA damage may occur because of deficient sperm nuclear protamine to histone replacement and decreased nuclear condensation in these immature sperm allowing increased susceptibility to reactive oxygen species and other damaging agents in culture. Another major concern is genetic risk. Any genetic abnormality sufficiently severe to result in meiotic arrest during spermatogenesis may also have adverse effects on other normal cellular processes or other systemic manifestations. Occurrence of significant congenital anomalies in ROSNI-conceived pregnancies raises serious concerns [74]. ROSNI should not be performed when more mature sperm forms (elongating spermatids or sperm) can be identified and used for ICSI. Patients who may be candidates for ROSNI should receive careful and thorough pre-treatment counselling to ensure they are

clearly informed of the limitations and potential risks of the procedure [75].

When a cell, with chromosomes in MII, fuses with an inter-phase cell, the nuclear membrane of the cell in the inter-phase dissolves and its chromatin condenses. This phenomenon is called premature chromosomal condensation (PCC) [76]. Following penetration of sperm into an oocyte; oocyte activation is triggered, resulting in completion of meiosis and formation of both male and female pronuclei. Under some circumstances although the sperm is within the oocyte, fertilization fails to occur, the oocyte remains in the MII stage and the sperm head transforms into PCC, separate from the oocyte chromosomes [77, 78]. Chromatin analysis of human oocytes has revealed that sperm PCC is one of the prevalent

**9. Premature chromosomal condensation** 

causes of fertilization failure in both IVF and ICSI [77].

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

many births of healthy babies have been reported. The main concern that remains is the potential transmission to the offspring [89].

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 127

Obtaining viable spermatozoa from testicular biopsies using pentoxifylline is more effective in terms of fertilization and pregnancies than obtaining it through an HOS test [97]. The clinical use of pentoxifylline for activation of immotile ejaculatory sperm before ICSI in patients with Kartagener's syndrome improves the outcome of the treatment and reduces the need of invasive intervention such as TESE in these patients. The immotile sperm are treated for 30 min with pentoxifylline (1.76 mM) before ICSI. Some spermatozoa show minimal motion and can be used for ICSI. Fertilization rate after ICSI is about 75% [105].

A new era in the field of assisted reproduction opened after the achievement of pregnancies and births after ICSI of human oocytes [106]. In special cases of long-standing male infertility, only a few functional sperm are available. By means of ICSI, most sub-fertile men and even men previously considered sterile (those with azoospermia, extreme

Azoospermia, is the most severe form of male factor infertility. The condition is currently classified as 'obstructive' or 'non-obstructive'. Obstructive azoospermia is the result of obstruction in either the upper or lower male reproductive tract. Sperm production may be normal but the obstruction prevents the sperm from being ejaculated. Non-obstructive azoospermia is the result of testicular failure where sperm production is either severely impaired or nonexistent, although in many cases sperm may be found and surgically

Conflicting results for fertilization and pregnancy rates are available in the literature after use of ejaculated or surgically retrieved sperm. After ICSI, ejaculated or surgically extracted sperm, when motile and morphologically normal, result in similar fertilization, implantation [108, 109] and clinical pregnancy rates ([109]. The incidence of early or late spontaneous abortion and ectopic pregnancy, or malformations is also similar [108]. However, after conventional IVF, even testicular or epididymal aspirates with very good sperm concentration and motility, generally achieve low fertilization and pregnancy rates [110].

The effect of cryopreservation of sperm on ICSI outcome has been thoroughly studied. Current studies suggest that the use of fresh or frozen-thawed sperm does not appear to affect ICSI outcomes [111]. Testicular tissue and epididymal sperm can be cryopreserved successfully without markedly reducing subsequent fertilization and implantation rates and repeated

The origin of the sperm used in ICSI does not have a major influence on the early life outcomes for the offspring, but transgenerational and epigenetic effects remain unknown. From the limited information available, it appears that there is no increased risk of congenital malformations in children born from ICSI. There is, however, a small increase in both de novo and inherited chromosome abnormalities. In terms of growth and neurodevelopment, there are very few studies, and so far, no adverse outcomes have been

testicular biopsy can be avoided without the risk of any decrease in the outcome [112].

found in young children whose fathers have a sperm defect [113].

oligozoospermia or cryptozoospermia) can now father a child.

extracted directly from the testicles [107].

**11. Sperm origin** 

Whether sperm movement is slow or rapid generally has no influence on ICSI results. However, injection of immotile sperm usually results in impaired fertilization. In particular, where a non-viable immotile sperm is injected into an oocyte, normal fertilization and pregnancy rarely occurs [90, 91]. In case of immotile sperm, it is possible that the sperm may be dead. The most common practice to select viable non-motile sperm for ICSI involves the hypo-osmotic swelling (HOS) test. However, preliminary results in animal experiments (mouse and rabbit) indicate that viability of injected sperm is not an absolute pre-requisite for fertilization. Embryos derived after injecting mouse oocytes with freeze-dried and thawed sperm developed normally [92]. It appears that provided the DNA integrity of the sperm is maintained, embryos can be generated, at least in animal model, from severely damaged sperm that are no longer capable of normal physiological activity.

The identification of a viable spermatozoon amongst immotile spermatozoa for ICSI often is difficult. However, selection of birefringent spermatozoa under Polscope shows promising results in asthenozoospermic men and men undergoing testicular sperm aspiration or extraction before ICSI [93]. The other tests employed are hypo-osmotic swelling test, the stimulation of motility with pentoxyfilline and non-contact diode laser [94, 95, 96, 97, 98].

In patients with 100% immotile sperm, HOS test is a useful method to examine sperm viability. It measures the functional integrity of the sperm membrane [99]. Upon exposure of the sperm to hypo-osmotic conditions, the intact semi-permeable barrier formed by the sperm membrane allows an influx of water and results in swelling of the cytoplasmic space and curling of the sperm tail fibers. Only viable sperm react to the HOS solution since dead sperm are unable to maintain the osmotic gradient.

Sperm HOS test based on fructose and sodium citrate dihydrate is applied for identification of immotile sperm for ICSI [100]. A significantly greater fertilization and cleavage rate after injection of sperm selected using the HOS test is achieved in contrast to injection of randomly selected sperm. A modified HOS test based on NaCl solution further improves fertilization rate in patients with 100% immotile sperm [101]. In these procedures, approximately 200,000 sperm are exposed to the HOS solution for 1 hour at 37°C. A modified HOS test has been used for samples with a low sperm count such as testicular samples [102]. In this technique, individual morphologically normal sperm is aspirated by microinjection pipette and is exposed to HOS solution for a brief period to minimize the sperm membrane damage.

A mixture of 50% culture medium and 50% deionized grade water has the least delayed harmful effects on sperm vitality [103]. This mixture achieves similar implantation, pregnancy and ongoing pregnancy rates in the ejaculated and testicular non-motile sperm groups [104]. It is a simple and practical procedure and achieves acceptable and comparable pregnancy rates.

Obtaining viable spermatozoa from testicular biopsies using pentoxifylline is more effective in terms of fertilization and pregnancies than obtaining it through an HOS test [97]. The clinical use of pentoxifylline for activation of immotile ejaculatory sperm before ICSI in patients with Kartagener's syndrome improves the outcome of the treatment and reduces the need of invasive intervention such as TESE in these patients. The immotile sperm are treated for 30 min with pentoxifylline (1.76 mM) before ICSI. Some spermatozoa show minimal motion and can be used for ICSI. Fertilization rate after ICSI is about 75% [105].
