**11. Sperm origin**

126 Enhancing Success of Assisted Reproduction

potential transmission to the offspring [89].

sperm are unable to maintain the osmotic gradient.

sperm membrane damage.

pregnancy rates.

many births of healthy babies have been reported. The main concern that remains is the

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

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 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

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

damaged sperm that are no longer capable of normal physiological activity.

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 oligozoospermia or cryptozoospermia) can now father a child.

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 extracted directly from the testicles [107].

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

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