Cuadra et al., (2000) evaluated the effect of sildenafil on sperm motility. Spermatozoa were exposed to sildenafil at either 0 nmol/L, 0.4 nmol/L, 4.0 nmol/L, or 40 nmol/L. The investigators observed increased sperm motility parameters in the presence of 0.4 nmol/L sildenafil compared with the control sample four hours after the exposure to sildenafil. However, the motility parameters decreased 48 hours after the exposure to sildenafil. Spermatozoa exposed to higher concentration of sildenafil (40 nmol/L) showed decreased sperm motility parameters. cGMP regulates calcium entry into microdomains along the sperm flagellum affecting sperm motility. Since PDE5 hydrolyzes cGMP, the authors have suggested that inhibition of PDE5 by sildenafil citrate enhances the effects of cGMP on sperm motility. The data provided by the authors (Cuadra et al., 2000) suggest that there is a stimulatory effect on sperm motility when PDE5 is moderately inhibited; however,

Another group of researchers (Glenn et al., 2007) attempted to determine the influence of sildenafil on sperm motility. Semen samples from 57 unselected men with asthenozoospermic profiles were prepared and then exposed to 0.67 μmol/L of sildenafil which is equivalent to the plasma concentration of sildenafil, one hour after oral ingestion of 100 mg of sildenafil (Glenn et al., 2007). The authors found that both the number and the velocity of progressively motile spermatozoa were significantly increased. The investigators have suggested that the elevated levels of cGMP, attributable to the inhibitory effect of sildenafil, may affect calcium transport into spermatozoa which potentially affects the

The effects of tadalafil on human sperm motility in vitro have been investigated. Mostafa (2007) assessed the effects of tadalafil on human sperm motility in 70 asthenozoospermic semen specimens. The semen samples were exposed to three different concentrations of tadalafil (4.0, 1.0, 0.5 mg/mL) and it was found that sperm samples treated with 4 mg/mL tadalafil solution demonstrated a significant decrease in sperm motility compared with the control samples. On the other hand, sperm samples treated with 1.0 or 0.5 mg/mL tadalafil solution demonstrated a significant increase in sperm progressive forward motility. The authors have suggested that the concentration of tadalafil is an important factor in such

Alternatively, the effect of tadalafil on sperm motility may be related additionally to the inhibitory effect of this compound on PDE11. In fact, PDE11 is highly expressed in the testis, prostate, and developing sperm cells even if its physiological role is not known (Table 1). Wayman et al., (2005) in an effort to investigate the role of PDE11 in spermatozoa physiology, retrieved spermatozoa from PDE11 knockout mice (PDE11-/-). The authors found a reduced sperm concentration, decreased forward motility, and lower percentage of alive spermatozoa in the latter animals which suggest a role for PDE11 in spermatogenesis

Taken together the results of the above ex vivo studies, we may postulate a dose dependent effect of sildenafil and tadalafil on sperm motility. In fact this effect seems to be enhanced at

suggesting an inhibitory action on a PDE that is different to PDE5.

extensive inhibition of PDE5 appears to lead to decreased sperm motility.

sperm motion.

studies.

and fertilization potential.

Jarvi et al., (2008) performed a randomized, double-blind, placebo controlled, parallel group, multicenter study investigating the effect of vardenafil and sildenafil on sperm characteristics. A total of 200 men with or without erectile dysfunction, able to produce semen samples without therapy for erectile dysfunction, 25 to 64 years old, were randomized to daily treatment with vardenafil, sildenafil or placebo for 6 months. Vardenafil or sildenafil had no effect vs. placebo on the percentage of patients with 50% or greater decrease in sperm concentration (Jarvi et al., 2008). Additionally, vardenafil or sildenafil did not affect any sperm parameters or peripheral serum gonadotropin profiles (Jarvi et al., 2008).

#### **6.2 The development of in vitro studies**

After the introduction of sildenafil in the market, several studies have evaluated the in vitro effects of this compound on sperm parameters. Burger et al., (2000) in an ex vivo study investigated the effect of sildenafil on the motility and viability of human spermatozoa. The above spermatozoal parameters were evaluated in sperm samples of both healthy donors (n=6) and clinically infertile men (n=6). Separate sperm aliquots were incubated for 0 h, 1 h and 3 h in the absence or presence of sildenafil (125 ng/mL, 250 ng/mL, and 750 ng/mL), PTX (as a positive control), or Ham's medium (as a reagent control). The authors have reported no statistically significant effect of sildenafil on sperm viability, sperm motility, and sperm forward progression after incubation of spermatozoa with various doses of sildenafil.

Similarly, in another study, the group of Andrade et al., (2000) attempted to evaluate a direct effect of sildenafil or phentolamine on sperm motility. Using samples of either unwashed or washed spermatozoa, the investigators added directly to the sperm samples sildenafil at a concentration of 20 mg/mL or phentolamine in various doses and incubated the samples for 30 minutes. The authors demonstrated a dose-related inhibition of sperm motility in sperm samples treated with phentolamine, whereas sildenafil (at a concentration of 200 µg/mL) did not adversely affect sperm motility either in unwashed or washed sperm. In contrast the highest dose of sildenafil (2000 μg/mL) reduced the sperm motility approximately at a level of 50% of the original value. However, it should be emphasized that at this concentration sildenafil caused a marked acidification of the medium which may be the reason for the reduced sperm motility (Su & Vacquier, 2006).

In an experimental study Su and Vacquier (2006) cloned and characterized a sea urchin spermatozoal PDE (suPDE5) which is an ortholog of human PDE5. The authors have found that phospho-suPDE5 localizes mainly on sperm flagella and that the PDE5 phosphorylation increases when spermatozoa contact the jelly layer that surrounds the eggs. Since the in vitro dephosphorylation of suPDE5 decreases its activity, the authors have suggested that PDE5 inhibitors such as sildenafil may inhibit the activity of suPDE5 and increase sperm motility.

A concentration-dependent stimulatory effect of sildenafil on sperm motility was also demonstrated by Mostafa (2007) when 85 semen specimens from asthenozoospermic patients were exposed to different concentrations of sildenafil (4.0 mg/mL, 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL, and 0.1 mg/mL). However, the evaluation of sperm motility in that study was performed 3 hours only after the spermatozoal exposure to sildenafil.

Jarvi et al., (2008) performed a randomized, double-blind, placebo controlled, parallel group, multicenter study investigating the effect of vardenafil and sildenafil on sperm characteristics. A total of 200 men with or without erectile dysfunction, able to produce semen samples without therapy for erectile dysfunction, 25 to 64 years old, were randomized to daily treatment with vardenafil, sildenafil or placebo for 6 months. Vardenafil or sildenafil had no effect vs. placebo on the percentage of patients with 50% or greater decrease in sperm concentration (Jarvi et al., 2008). Additionally, vardenafil or sildenafil did not affect any sperm parameters or peripheral serum gonadotropin profiles

After the introduction of sildenafil in the market, several studies have evaluated the in vitro effects of this compound on sperm parameters. Burger et al., (2000) in an ex vivo study investigated the effect of sildenafil on the motility and viability of human spermatozoa. The above spermatozoal parameters were evaluated in sperm samples of both healthy donors (n=6) and clinically infertile men (n=6). Separate sperm aliquots were incubated for 0 h, 1 h and 3 h in the absence or presence of sildenafil (125 ng/mL, 250 ng/mL, and 750 ng/mL), PTX (as a positive control), or Ham's medium (as a reagent control). The authors have reported no statistically significant effect of sildenafil on sperm viability, sperm motility, and sperm forward progression after incubation of spermatozoa with various doses of

Similarly, in another study, the group of Andrade et al., (2000) attempted to evaluate a direct effect of sildenafil or phentolamine on sperm motility. Using samples of either unwashed or washed spermatozoa, the investigators added directly to the sperm samples sildenafil at a concentration of 20 mg/mL or phentolamine in various doses and incubated the samples for 30 minutes. The authors demonstrated a dose-related inhibition of sperm motility in sperm samples treated with phentolamine, whereas sildenafil (at a concentration of 200 µg/mL) did not adversely affect sperm motility either in unwashed or washed sperm. In contrast the highest dose of sildenafil (2000 μg/mL) reduced the sperm motility approximately at a level of 50% of the original value. However, it should be emphasized that at this concentration sildenafil caused a marked acidification of the medium which may

In an experimental study Su and Vacquier (2006) cloned and characterized a sea urchin spermatozoal PDE (suPDE5) which is an ortholog of human PDE5. The authors have found that phospho-suPDE5 localizes mainly on sperm flagella and that the PDE5 phosphorylation increases when spermatozoa contact the jelly layer that surrounds the eggs. Since the in vitro dephosphorylation of suPDE5 decreases its activity, the authors have suggested that PDE5 inhibitors such as sildenafil may inhibit the activity of suPDE5 and increase sperm

A concentration-dependent stimulatory effect of sildenafil on sperm motility was also demonstrated by Mostafa (2007) when 85 semen specimens from asthenozoospermic patients were exposed to different concentrations of sildenafil (4.0 mg/mL, 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL, and 0.1 mg/mL). However, the evaluation of sperm motility in that

study was performed 3 hours only after the spermatozoal exposure to sildenafil.

be the reason for the reduced sperm motility (Su & Vacquier, 2006).

(Jarvi et al., 2008).

sildenafil.

motility.

**6.2 The development of in vitro studies**

Lefièvre et al., (2000) have investigated whether PDE5 is present in human spermatozoa and whether sildenafil affects sperm function. The authors have demonstrated that this PDE5 inhibitor stimulates human sperm motility with an increase in intracellular cAMP suggesting an inhibitory action on a PDE that is different to PDE5.

Cuadra et al., (2000) evaluated the effect of sildenafil on sperm motility. Spermatozoa were exposed to sildenafil at either 0 nmol/L, 0.4 nmol/L, 4.0 nmol/L, or 40 nmol/L. The investigators observed increased sperm motility parameters in the presence of 0.4 nmol/L sildenafil compared with the control sample four hours after the exposure to sildenafil. However, the motility parameters decreased 48 hours after the exposure to sildenafil. Spermatozoa exposed to higher concentration of sildenafil (40 nmol/L) showed decreased sperm motility parameters. cGMP regulates calcium entry into microdomains along the sperm flagellum affecting sperm motility. Since PDE5 hydrolyzes cGMP, the authors have suggested that inhibition of PDE5 by sildenafil citrate enhances the effects of cGMP on sperm motility. The data provided by the authors (Cuadra et al., 2000) suggest that there is a stimulatory effect on sperm motility when PDE5 is moderately inhibited; however, extensive inhibition of PDE5 appears to lead to decreased sperm motility.

Another group of researchers (Glenn et al., 2007) attempted to determine the influence of sildenafil on sperm motility. Semen samples from 57 unselected men with asthenozoospermic profiles were prepared and then exposed to 0.67 μmol/L of sildenafil which is equivalent to the plasma concentration of sildenafil, one hour after oral ingestion of 100 mg of sildenafil (Glenn et al., 2007). The authors found that both the number and the velocity of progressively motile spermatozoa were significantly increased. The investigators have suggested that the elevated levels of cGMP, attributable to the inhibitory effect of sildenafil, may affect calcium transport into spermatozoa which potentially affects the sperm motion.

The effects of tadalafil on human sperm motility in vitro have been investigated. Mostafa (2007) assessed the effects of tadalafil on human sperm motility in 70 asthenozoospermic semen specimens. The semen samples were exposed to three different concentrations of tadalafil (4.0, 1.0, 0.5 mg/mL) and it was found that sperm samples treated with 4 mg/mL tadalafil solution demonstrated a significant decrease in sperm motility compared with the control samples. On the other hand, sperm samples treated with 1.0 or 0.5 mg/mL tadalafil solution demonstrated a significant increase in sperm progressive forward motility. The authors have suggested that the concentration of tadalafil is an important factor in such studies.

Alternatively, the effect of tadalafil on sperm motility may be related additionally to the inhibitory effect of this compound on PDE11. In fact, PDE11 is highly expressed in the testis, prostate, and developing sperm cells even if its physiological role is not known (Table 1). Wayman et al., (2005) in an effort to investigate the role of PDE11 in spermatozoa physiology, retrieved spermatozoa from PDE11 knockout mice (PDE11-/-). The authors found a reduced sperm concentration, decreased forward motility, and lower percentage of alive spermatozoa in the latter animals which suggest a role for PDE11 in spermatogenesis and fertilization potential.

Taken together the results of the above ex vivo studies, we may postulate a dose dependent effect of sildenafil and tadalafil on sperm motility. In fact this effect seems to be enhanced at

The Role of PDE5 Inhibitors in the Treatment of Testicular Dysfunction 93

The expression of PDEs has been documented in several regions of the male reproductive tract. Increasing evidence based on several studies tend to suggest that PDE5 inhibitors may have a therapeutic effect in some disorders of semen parameters. Moreover, PDE5 inhibitors may promote sperm capacitation process. In addition PDE5 inhibitors may affect sperm transfer through the male reproductive tract by affecting the contractility of the tunica albuginea and the epididymis. However, the current evidence needs further confirmation by additional studies that are necessary to suggest unequivocally a therapeutic role of PDE5

**Type of cell/ Tissue PDE Species Relative References**

Human

PDE1C Rat Dousa (1999)

PDE11A Human D'Andrea et al., (2005)

PDE2 Rat Swinnen et al., (1989)

PDE11 Human Baxendale et al., (2001)

PDE11 Mouse Baxendale et al., (2001)

Foresta et al., (2008) Baxendale et al., (2005) Dousa et al., (1999) Michibata et al., (2001) Fawcett et al., (2000) Kotera et al., (1999) Yuasa et al., (2001) D'Andrea et al., (2005) Horvath et al., (2006)

Mouse Baxendale et al., (2005)

PDE5 PDE8A PDE8B PDE1A PDE11A PDE11A2 PDE11A3

PDE1A PDE1B PDE1C PDE3B PDE4A PDE4B PDE4C PDE4D PDE5A PDE6γ' PDE7A PDE8A PDE10A

PDE1

inhibitors in the management of defects in testicular or epididymal function.

**9. Conclusions**

**Testis**

**Germ cells**

**Spermatogonia**

low doses of PDE5 inhibitors but it may be reduced at high concentrations. Definitely further investigations are required to evaluate the mechanisms mediating the effects of PDE5 selective inhibitors on sperm motility.

#### **7. The effect of PDE5 inhibitors on sperm capacitation and acrosome reaction process**

Lefièvre et al., (2000) investigated the potential effect of PDE5 inhibitor sildenafil on spermatozoal ability to undergo capacitation process. The authors showed that sildenafil at 30 μmol/L, 100 μmol/L, and 200 μmol/L triggered the capacitation process of washed spermatozoa. Capacitated spermatozoa underwent an acrosome reaction when challenged with lysophosphatidylcholine (LPC) alone or LPC plus IBMX (a non selective PDE inhibitor), but not with sildenafil alone. The authors have suggested that PDE inhibitors by themselves cannot initiate the acrosome reaction nor can they potentiate the acrosome reaction of capacitated spermatozoa.

Cuadra et al., (2000) investigated the effect of sildenafil on sperm acrosomal reaction. Spermatozoa were exposed to different doses of sildenafil (from 0 nmol/L to 40 nmol/L). The authors reported that sildenafil affected the sperm acrosomal reaction with an increase in the percentage of acrosomally reacted spermatozoa of almost 50% compared to the control samples. It is known that cGMP directly opens cyclic nucleotide-gated channels for calcium entry into the spermatozoa initiating the acrosome reaction (Biel et al., 1998). Since PDE5 hydrolyzes cGMP, inhibition of PDE5 by sildenafil citrate enhances the effects of cGMP on sperm acrosome reaction.

Glenn et al., (2007) evaluated the influence of sildenafil on sperm acrosomal reaction. The study included fifty-seven unselected men with asthenozoospermic profiles who provided semen samples which were further processed for sperm isolation. Then spermatozoa were exposed to 0.67 μmol/L of sildenafil. The investigators (Glenn et al., 2007) noticed that sildenafil caused a significant increase in the proportion of acrosome-reacted spermatozoa. In a case report, sildenafil was administered for semen collection for ART purposes. The investigators failed to fertilize oocytes despite the intracytoplasmic injection of the sperm (Tur-Kaspa et al., 1999). Although this fertilization failure was attributed to the advanced age of oocytes due to the delay in obtaining the semen sample, a deleterious effect of sildenafil on spermatozoal function can not be excluded.

#### **8. The effects of PDE5 inhibitors on sperm functional assays**

Several studies have evaluated the in vitro effects of PDE5 inhibitors on sperm functional assays. Burger et al., (2000) performed an in vitro study investigating and comparing the effects of sildenafil on the membrane integrity, and functional sperm capacity between healthy donors and clinically infertile men. The authors noted a marked decrease of sperm membrane integrity in spermatozoa of infertile patients treated with sildenafil (Burger et al., 2000). This observation may be taken into consideration when treatment with sildenafil is planed in subfertile couples with a male factor infertility (Burger et al., 2000). However, sperm penetration assay data has suggested that there is neither a beneficial nor a detrimental effect of sildenafil on its outcome (Burger et al., 2000).

### **9. Conclusions**

92 Male Infertility

low doses of PDE5 inhibitors but it may be reduced at high concentrations. Definitely further investigations are required to evaluate the mechanisms mediating the effects of

Lefièvre et al., (2000) investigated the potential effect of PDE5 inhibitor sildenafil on spermatozoal ability to undergo capacitation process. The authors showed that sildenafil at 30 μmol/L, 100 μmol/L, and 200 μmol/L triggered the capacitation process of washed spermatozoa. Capacitated spermatozoa underwent an acrosome reaction when challenged with lysophosphatidylcholine (LPC) alone or LPC plus IBMX (a non selective PDE inhibitor), but not with sildenafil alone. The authors have suggested that PDE inhibitors by themselves cannot initiate the acrosome reaction nor can they potentiate the acrosome

Cuadra et al., (2000) investigated the effect of sildenafil on sperm acrosomal reaction. Spermatozoa were exposed to different doses of sildenafil (from 0 nmol/L to 40 nmol/L). The authors reported that sildenafil affected the sperm acrosomal reaction with an increase in the percentage of acrosomally reacted spermatozoa of almost 50% compared to the control samples. It is known that cGMP directly opens cyclic nucleotide-gated channels for calcium entry into the spermatozoa initiating the acrosome reaction (Biel et al., 1998). Since PDE5 hydrolyzes cGMP, inhibition of PDE5 by sildenafil citrate enhances the effects of

Glenn et al., (2007) evaluated the influence of sildenafil on sperm acrosomal reaction. The study included fifty-seven unselected men with asthenozoospermic profiles who provided semen samples which were further processed for sperm isolation. Then spermatozoa were exposed to 0.67 μmol/L of sildenafil. The investigators (Glenn et al., 2007) noticed that sildenafil caused a significant increase in the proportion of acrosome-reacted spermatozoa. In a case report, sildenafil was administered for semen collection for ART purposes. The investigators failed to fertilize oocytes despite the intracytoplasmic injection of the sperm (Tur-Kaspa et al., 1999). Although this fertilization failure was attributed to the advanced age of oocytes due to the delay in obtaining the semen sample, a deleterious effect of

Several studies have evaluated the in vitro effects of PDE5 inhibitors on sperm functional assays. Burger et al., (2000) performed an in vitro study investigating and comparing the effects of sildenafil on the membrane integrity, and functional sperm capacity between healthy donors and clinically infertile men. The authors noted a marked decrease of sperm membrane integrity in spermatozoa of infertile patients treated with sildenafil (Burger et al., 2000). This observation may be taken into consideration when treatment with sildenafil is planed in subfertile couples with a male factor infertility (Burger et al., 2000). However, sperm penetration assay data has suggested that there is neither a beneficial nor a

**7. The effect of PDE5 inhibitors on sperm capacitation and acrosome**

PDE5 selective inhibitors on sperm motility.

reaction of capacitated spermatozoa.

cGMP on sperm acrosome reaction.

sildenafil on spermatozoal function can not be excluded.

**8. The effects of PDE5 inhibitors on sperm functional assays**

detrimental effect of sildenafil on its outcome (Burger et al., 2000).

**reaction process**

The expression of PDEs has been documented in several regions of the male reproductive tract. Increasing evidence based on several studies tend to suggest that PDE5 inhibitors may have a therapeutic effect in some disorders of semen parameters. Moreover, PDE5 inhibitors may promote sperm capacitation process. In addition PDE5 inhibitors may affect sperm transfer through the male reproductive tract by affecting the contractility of the tunica albuginea and the epididymis. However, the current evidence needs further confirmation by additional studies that are necessary to suggest unequivocally a therapeutic role of PDE5 inhibitors in the management of defects in testicular or epididymal function.

 Phosphodiesterase 5 inhibitors in rapid ejaculation: potential use and possible mechanisms of action. *Drugs*, Vol. 64, No. 1, pp. 13-26, ISSN 0012-6667 (Print) Ali, S.T. & Rakkah, N.I. (2007). Neurophysiological role of sildenafil citrate (Viagra) on

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### **10. Abbreviations**

AC = adenylate cyclase ANP = atrial natriuretic peptide ART = assisted reproductive technology cAMP = cyclic adenosine monophosphate cGMP = cyclic guanosine monophosphate CNP = C-type natriuretic peptide CO = carbon monoxide eNOS = endothelial nitric oxide synthase GC = guanylate cyclase GC-B = membrane-bound GC HO-1= heme oxygenase-1 ICSI = intracytoplasmic sperm injection IVF = in vitro fertilization LPC = lysophosphatidylcholine NO = nitric oxide NOS = nitric oxide synthase PDEs = phosphodiesterases PKA = protein kinase PTX = pentoxifylline SCs = spontaneous phasic contractions sGC = soluble guanylate cyclase SNP = sodium nitroprusside suPDE5= sea urchin spermatozoal PDE

#### **11. References**

96 Male Infertility

PDE3A PDE4A PDE4B PDE4C PDE4D PDE5A PDE6 PDE7A PDE8A PDE9A PDE11A

**Endothelial cells** PDE11A Human

Table 1. Expression of phosphodiesterases PDEs in the male reproductive tract.

 Abbreviations**  AC = adenylate cyclase

CO = carbon monoxide

GC = guanylate cyclase GC-B = membrane-bound GC HO-1= heme oxygenase-1

IVF = in vitro fertilization LPC = lysophosphatidylcholine

NOS = nitric oxide synthase PDEs = phosphodiesterases PKA = protein kinase PTX = pentoxifylline

NO = nitric oxide

ANP = atrial natriuretic peptide

CNP = C-type natriuretic peptide

ART = assisted reproductive technology cAMP = cyclic adenosine monophosphate cGMP = cyclic guanosine monophosphate

eNOS = endothelial nitric oxide synthase

ICSI = intracytoplasmic sperm injection

SCs = spontaneous phasic contractions sGC = soluble guanylate cyclase SNP = sodium nitroprusside

suPDE5= sea urchin spermatozoal PDE

**Corpus cavernosum**

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*Urology*, Vol. 45, No. 3, pp. 440-446, ISSN 0090-4295 (Print)

**5**

*Italy*

**Effectiveness of Assisted Reproduction**

Sandrine Chamayou and Antonino Guglielmino *Unità di Medicina della Riproduzione – Via Barriera del Bosco*

*Sant'Agata Li Battiati (CT)*

**Techniques as an Answer to Male Infertility**

The assisted reproductive technologies (ART) are of huge help in the resolution of couple infertility especially for male factor. For an evaluation of male fertility, the semen exam is the first analysis requested. All the procedures for an objective exam and parameters of normality have been worldwide established and described in a manual written by the

In the application of ART to obtain the pregnancy of healthy children, there are different levels of invasiveness that take in account both male and female factors. For moderate alteration of semen values, the ART called *intra* uterine insemination (IUI) is easy to perform because fertilization occurs *in vivo.* In case of intermediate alteration of semen values, metaphase II oocytes are incubated in a solution of motile spermatozoa in a process called *in vitro* fertilization (IVF). One spermatozoon penetrates by itself the oocyte membranes and fertilizes it. The development of embryos generated in the laboratory is followed until embryos are *in utero* transferred or stored in liquid nitrogen. Intra-cytoplasmic sperm injection (ICSI) is a variation of IVF in which the fertilization process is assisted. The elective field of ICSI application is a severe male factor. ICSI can be performed with fresh or thawed, motile or immotile, ejaculated or surgically retrieved (microsurgical epididymal sperm aspiration-MESA, sperm aspiration from the epididymis-PESA or testicle-TESA) spermatozoa. Recently, a new technique called IMSI (morphologically selected sperm injection) was introduced in the clinically assisted reproduction. In this case, the spermatozoon to micro-inject in the ooplasm is accurately chosen according to its morphology and particularly to the number and volume of vacuoles in the head of the cell. Numerous studies related *in vivo* and *in vitro* ART outcomes with male infertility and semen characteristics. The aim of this chapter is to present the effectiveness of ART as an answer to male infertility according to semen alterations (number, motility, morphology), the extraction of the spermatozoa (ejaculated/surgically extracted) and the process it underwent (cryopreservation). Furthermore, it has to be underlined that spermatozoa cryopreservation combined with ART represents a huge opportunity of fertility preservation for cancer patients. The genetic analysis of cells from biopsied embryos produced after ICSI (preimplantation genetic diagnosis-PGD) is an alternative mode of reproduction for couples

**1. Introduction**

World Health Organization (WHO).

with high risk in the transmission of male genetic disorder.

men or cryptozoospermic men participating in an assisted reproduction programme. *Andrologia*, Vol. 32, No. 3, pp. 131-138

## **Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility**

Sandrine Chamayou and Antonino Guglielmino *Unità di Medicina della Riproduzione – Via Barriera del Bosco Sant'Agata Li Battiati (CT) Italy*

#### **1. Introduction**

106 Male Infertility

Yan, C., Zhao, A.Z., et al. (2001). Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis. *Biol Reprod*, Vol. 64, No. 6, pp. 1746-1754 Yovich, J.M., Edirisinghe, W.R., et al. (1988). Preliminary results using pentoxifylline in a

Yovich, J.M., Edirisinghe, W.R., et al. (1990). Influence of pentoxifylline in severe male factor

Yuasa, K., Kanoh, Y., et al. (2001). Genomic organization of the human phosphodiesterase

Yuasa, K., Omori, K., et al. (2000). Binding and phosphorylation of a novel male germ cell-

Yunes, R., Fernandez, P., et al. (2005). Cyclic nucleotide phosphodiesterase inhibition

Zaccolo, M. & Pozzan, T. (2002). Discrete microdomains with high concentration of cAMP in

Zhang, C., Yeh, S., et al. (2006). Oligozoospermia with normal fertility in male mice lacking

Zhao, A.Z., Yan, C., et al. (1997). Recent advances in the study of Ca2+/CaM-activated

programme. *Andrologia*, Vol. 32, No. 3, pp. 131-138

infertility. *Fertil Steril*, Vol. 53, No. 4, pp. 715-722

human spermatozoa. *Biocell*, Vol. 29, No. 3, pp. 287-293

Vol. 103, No. 47, pp. 17718-17723, ISSN 0027-8424 (Print)

*Phosphoprotein Res*, Vol. 31, pp. 237-251, ISSN 1040-7952 (Print)

*Fertil Steril*, Vol. 50, No. 1, pp. 179-181

*Eur J Biochem*, Vol. 268, No. 1, pp. 168-178

ISSN 1095-9203 (Electronic)

(Print)

men or cryptozoospermic men participating in an assisted reproduction

pronuclear stage tubal transfer (PROST) program for severe male factor infertility.

PDE11A gene. Evolutionary relatedness with other PDEs containing GAF domains.

specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha. *J Biol Chem*, Vol. 275, No. 7, pp. 4897-4905, ISSN 0021-9258

increases tyrosine phosphorylation and hyper motility in normal and pathological

stimulated rat neonatal cardiac myocytes. *Science*, Vol. 295, No. 5560, pp. 1711-1715,

the androgen receptor in testis peritubular myoid cells. *Proc Natl Acad Sci U S A*,

phosphodiesterases: expression and physiological functions. *Adv Second Messenger*

The assisted reproductive technologies (ART) are of huge help in the resolution of couple infertility especially for male factor. For an evaluation of male fertility, the semen exam is the first analysis requested. All the procedures for an objective exam and parameters of normality have been worldwide established and described in a manual written by the World Health Organization (WHO).

In the application of ART to obtain the pregnancy of healthy children, there are different levels of invasiveness that take in account both male and female factors. For moderate alteration of semen values, the ART called *intra* uterine insemination (IUI) is easy to perform because fertilization occurs *in vivo.* In case of intermediate alteration of semen values, metaphase II oocytes are incubated in a solution of motile spermatozoa in a process called *in vitro* fertilization (IVF). One spermatozoon penetrates by itself the oocyte membranes and fertilizes it. The development of embryos generated in the laboratory is followed until embryos are *in utero* transferred or stored in liquid nitrogen. Intra-cytoplasmic sperm injection (ICSI) is a variation of IVF in which the fertilization process is assisted. The elective field of ICSI application is a severe male factor. ICSI can be performed with fresh or thawed, motile or immotile, ejaculated or surgically retrieved (microsurgical epididymal sperm aspiration-MESA, sperm aspiration from the epididymis-PESA or testicle-TESA) spermatozoa. Recently, a new technique called IMSI (morphologically selected sperm injection) was introduced in the clinically assisted reproduction. In this case, the spermatozoon to micro-inject in the ooplasm is accurately chosen according to its morphology and particularly to the number and volume of vacuoles in the head of the cell.

Numerous studies related *in vivo* and *in vitro* ART outcomes with male infertility and semen characteristics. The aim of this chapter is to present the effectiveness of ART as an answer to male infertility according to semen alterations (number, motility, morphology), the extraction of the spermatozoa (ejaculated/surgically extracted) and the process it underwent (cryopreservation). Furthermore, it has to be underlined that spermatozoa cryopreservation combined with ART represents a huge opportunity of fertility preservation for cancer patients. The genetic analysis of cells from biopsied embryos produced after ICSI (preimplantation genetic diagnosis-PGD) is an alternative mode of reproduction for couples with high risk in the transmission of male genetic disorder.

volume.

Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility 109

with tails). Defective spermatozoa (pinheads and tailless heads) should also be counted but recorded separately. If no spermatozoa is detected by microscopy, the entire sample should be centrifuged at 3000*g* for 15 minutes. If no spermatozoa is found after the complete analysis of the centrifuged sample, the semen is qualified as 'azoospermic'. The total sperm number refers to the total number of spermatozoa in the entire ejaculate. This number is obtained by multiplying the sperm concentration by the semen

The grade of progressive motility of the spermatozoa is correlated with the pregnancy rate (Jouannet *et al.* 1988; Larsen *et al.* 2000; Zinaman *et al.* 2000). The sperm motility valuation should be performed close to the liquefaction of the sample, between 30 minutes and one hour after sperm collection. At least five microscopic fields are assessed in a systematic way to classify 200 spermatozoa. The motility of each spermatozoon is graded as 'progressive',

The vitality is estimated by assessing the membrane vitality of the cells. It is routinely determined for the samples with less than 40% of progressively motile spermatozoa. The percentage of viable cells normally exceeds that of motile cells. The presence of a large proportion of vital but immotile spermatozoa may be indicative of structural defects in the flagellum (Cheme and Rawe, 2003); a high percentage of immotile and non-viable spermatozoa (necrozoospermia) may indicate epididymal pathology (Wilton *et al.* 1988;

The term of 'Agglutination of spermatozoa' means that motile spermatozoa stick to each other head-to-head, tail-to tail or in a mixed way, e.g. head-to-tail. The presence of agglutination is suggestive, but not sufficient evidence for, an immunological cause of

The spermatozoa morphology is determined after cell staining. The most widely recommended staining recommended method used is the Papanicolaou stain. For a spermatozoa to be normal, the sperm head, neck, midpiece and tail must be normal. The head should be oval in shape. Allowing for the slight shrinkage induced by fixation and staining, the length of the head should be 4.0-5.0 um and the width 2.5-3.5 um. The lengthto-width ratio should be 1.50 to 1.75. There should be a well-defined acrosomal region comprising 40-70% of the head area. The midpiece should be slender, less than 1 um in width, about one and a half times the length of the head, and attached axially to the head. The morphological sperm defects should be noted as head defects, neck and midpiece defects, tail defects and cytoplasmic droplets. Abnormal spermatozoa generally have a lower fertilizing potential. Morphologically defects are associated with chromosomal

In the microscopic analysis the non- nemaspermic cellular components are also valuated. There are referred as 'round cells' and compound of leukocytes, spermatogenic cells,

displacing the head, or when only a flagellar beat can be observed;

'non-progressive', 'immotile', according to whether it shows:

circle, regardless of speed;

Correa-Perrez *et al.* 2004).

infertility.

abnormalities.

#### **2. Semen characteristics**

The semen analysis is the basic exam in the determination of male fertility or infertility as it has been early determined a positive correlation between normal semen parameters and male infertility potential (WHO, 1987, 1992).

The semen is a mixture of spermatozoa suspended in secretions from the testis and epididymis which are combined with seminal liquid. The seminal liquid is secreted in 90% by the prostate and the seminal vesicles, and a small proportion is secreted by bulb urethral glands. The final semen is a viscous fluid that comprises the ejaculate. The total number of spermatozoa reflects the sperm production by the testes and the patency of the posttesticular duct system. The volume of the sample, produced by the accessory glands reflect the secretary activity of the glands. Other important parameters of the sperm function are represented by the vitality, the motility, the morphology and composition of the semen. The semen analysis must be standardized and objective. To emphasize the standardization of analysis procedures and to establish reference values, the WHO wrote a manual where the procedure of semen collection, analysis and the parameters of normality are described. Nowadays the fifth edition is available where the criteria of male infertility has been revised (WHO, 2010).

#### **2.1 Semen analysis**

This paragraph resumes the procedures for a correct semen collection and laboratory analysis as described by the WHO manual (2010).

Commonly, the semen sample is obtain by masturbation. The semen sample should be collected after a minimum of 48 hours but no longer than seven days of sexual abstinence. The semen sample collection should be completed and two samples should be collected for initial evaluation within an interval of time that should not be less than 7 days or more than 3 weeks. In case of markedly differences between two assessments, additional samples should be analyzed.

The semen analysis is divided in macroscopic analysis and microscopic analysis.

The macroscopic parameters are the semen aspect, the viscosity, liquefaction, the volume and the pH.

The microscopic parameters are the sperm concentration calculated per millilitre (ml), the total number of spermatozoa per ejaculate, the percentage of motile spermatozoa, the sperm vitality, the agglutination of spermatozoa, the spermatozoa morphology and the presence of cellular elements other than spermatozoa. It is recommended to execute all microscopic examinations using a phase-contrast microscope.

The semen analysis must be carried out on a fixed 20 um depth preparation. Depths less than 20 um constrain the rational movement of spermatozoa. Since sperm motility is highly dependent on temperature, the assessment of the motility should be performed at 37°C.

The concentration of spermatozoa should be determined using the haemocytometer chamber as the 'Newbauer haemocytometer' on a diluted preparation of semen covered with a coverslip. The sperm count should be made of complete spermatozoa (heads with tails). Defective spermatozoa (pinheads and tailless heads) should also be counted but recorded separately. If no spermatozoa is detected by microscopy, the entire sample should be centrifuged at 3000*g* for 15 minutes. If no spermatozoa is found after the complete analysis of the centrifuged sample, the semen is qualified as 'azoospermic'. The total sperm number refers to the total number of spermatozoa in the entire ejaculate. This number is obtained by multiplying the sperm concentration by the semen volume.

The grade of progressive motility of the spermatozoa is correlated with the pregnancy rate (Jouannet *et al.* 1988; Larsen *et al.* 2000; Zinaman *et al.* 2000). The sperm motility valuation should be performed close to the liquefaction of the sample, between 30 minutes and one hour after sperm collection. At least five microscopic fields are assessed in a systematic way to classify 200 spermatozoa. The motility of each spermatozoon is graded as 'progressive', 'non-progressive', 'immotile', according to whether it shows:

108 Male Infertility

The semen analysis is the basic exam in the determination of male fertility or infertility as it has been early determined a positive correlation between normal semen parameters and

The semen is a mixture of spermatozoa suspended in secretions from the testis and epididymis which are combined with seminal liquid. The seminal liquid is secreted in 90% by the prostate and the seminal vesicles, and a small proportion is secreted by bulb urethral glands. The final semen is a viscous fluid that comprises the ejaculate. The total number of spermatozoa reflects the sperm production by the testes and the patency of the posttesticular duct system. The volume of the sample, produced by the accessory glands reflect the secretary activity of the glands. Other important parameters of the sperm function are represented by the vitality, the motility, the morphology and composition of the semen. The semen analysis must be standardized and objective. To emphasize the standardization of analysis procedures and to establish reference values, the WHO wrote a manual where the procedure of semen collection, analysis and the parameters of normality are described. Nowadays the fifth edition is available where the criteria of male infertility has been revised

This paragraph resumes the procedures for a correct semen collection and laboratory

Commonly, the semen sample is obtain by masturbation. The semen sample should be collected after a minimum of 48 hours but no longer than seven days of sexual abstinence. The semen sample collection should be completed and two samples should be collected for initial evaluation within an interval of time that should not be less than 7 days or more than 3 weeks. In case of markedly differences between two assessments, additional samples

The macroscopic parameters are the semen aspect, the viscosity, liquefaction, the volume

The microscopic parameters are the sperm concentration calculated per millilitre (ml), the total number of spermatozoa per ejaculate, the percentage of motile spermatozoa, the sperm vitality, the agglutination of spermatozoa, the spermatozoa morphology and the presence of cellular elements other than spermatozoa. It is recommended to execute all microscopic

The semen analysis must be carried out on a fixed 20 um depth preparation. Depths less than 20 um constrain the rational movement of spermatozoa. Since sperm motility is highly dependent on temperature, the assessment of the motility should be performed

The concentration of spermatozoa should be determined using the haemocytometer chamber as the 'Newbauer haemocytometer' on a diluted preparation of semen covered with a coverslip. The sperm count should be made of complete spermatozoa (heads

The semen analysis is divided in macroscopic analysis and microscopic analysis.

**2. Semen characteristics**

(WHO, 2010).

**2.1 Semen analysis**

should be analyzed.

and the pH.

at 37°C.

male infertility potential (WHO, 1987, 1992).

analysis as described by the WHO manual (2010).

examinations using a phase-contrast microscope.

The vitality is estimated by assessing the membrane vitality of the cells. It is routinely determined for the samples with less than 40% of progressively motile spermatozoa. The percentage of viable cells normally exceeds that of motile cells. The presence of a large proportion of vital but immotile spermatozoa may be indicative of structural defects in the flagellum (Cheme and Rawe, 2003); a high percentage of immotile and non-viable spermatozoa (necrozoospermia) may indicate epididymal pathology (Wilton *et al.* 1988; Correa-Perrez *et al.* 2004).

The term of 'Agglutination of spermatozoa' means that motile spermatozoa stick to each other head-to-head, tail-to tail or in a mixed way, e.g. head-to-tail. The presence of agglutination is suggestive, but not sufficient evidence for, an immunological cause of infertility.

The spermatozoa morphology is determined after cell staining. The most widely recommended staining recommended method used is the Papanicolaou stain. For a spermatozoa to be normal, the sperm head, neck, midpiece and tail must be normal. The head should be oval in shape. Allowing for the slight shrinkage induced by fixation and staining, the length of the head should be 4.0-5.0 um and the width 2.5-3.5 um. The lengthto-width ratio should be 1.50 to 1.75. There should be a well-defined acrosomal region comprising 40-70% of the head area. The midpiece should be slender, less than 1 um in width, about one and a half times the length of the head, and attached axially to the head. The morphological sperm defects should be noted as head defects, neck and midpiece defects, tail defects and cytoplasmic droplets. Abnormal spermatozoa generally have a lower fertilizing potential. Morphologically defects are associated with chromosomal abnormalities.

In the microscopic analysis the non- nemaspermic cellular components are also valuated. There are referred as 'round cells' and compound of leukocytes, spermatogenic cells,

Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility 111

Total sperm number (106 per ejaculate) 39 (33-46) Sperm concentration (106 per ml) 15 (12-16) Total motility (PR+NP, %) 40 (38-42) Progressive motility (PR, %) 32 (31-34) Vitality (live spermatozoa, %) 58 (55-63) Sperm morphology (normal form, %) 4 (3.0-4.0)

Peroxidase-positive leucocytes (106 per ml) < 1.0

Aspermia No ejaculate

Hemospermia (Hematospermia) Presence of leukocytes in the ejaculate

Asthenozoospermia Less than the reference value for motility Asthenoteratozoospermia Less than the reference values for motility and

Azoospermia No spermatozoa in the ejaculate

Cryptozoospermia No spermatozoa is observed in the fresh preparation

Necrozoospermia Low percentage of live and high percentage of

Normozoospermia Normal ejaculate as defined by the reference values

Oligoteratozoospermia Less than the reference values for concentration or

Teratozoospermia Less than the reference value for morphology

Oligozoospermia Sperm concentration (or total number) less than the

morphology

but only after centrifuged pellet

Presence of leukocytes in the ejaculate above the threshold value

immotile spermatozoa in the ejaculate

Signifies disturbance of all three variables (concentration or total number, motility, morphology)

total number and morphology

reference value

MAR test (motile spermatozoa with

Immunobead test (motile spermatozoa

intervals)

Leukospermia (Leukocytospermia, pyospermia)

Oligoastenoteratozoospermia

Table 2. Nomenclature related to semen variations.

**Parameter Lower reference limit**

Semen volume (ml) 1.5 (1.4-1.7)

pH > 7.2

bound particles, %) < 50

with bound beads, %) < 50 Seminal zinc (umol/ejaculate) > 2.4 Seminal fruttose (umol/ejaculate) > 13 Seminal neutral glucosidase (mU/ejaculate) > 20 Table 1. Lower reference limits of semen characteristics (5th centiles, 95% confidence

epithelium cells, red cells, prostate cells. A normal ejaculate should not contain more than 5 x 106 round cells/ml.

The semen analysis can be computer-assisted (Computeur-assisted semen analysis-CASA) (Yeung *et al.* 1997). The advantage of a computer assisted system for sperm analysis is the ability to obtain precise quantitative data as wells as the potential for the standardization of semen analysis procedures. Nevertheless, the main disadvantages are the lack of standardization procedures and the significant expense of equipment requirement. CASA is a valuable tool for research but the manual semen analysis remains the reference (Amann *et al.* 2004). Other tests can be made on semen such as the detection of sperm antibodies, sperm vitality, hypo-osmotic swelling test, assays of the sperm acrosome, sperm penetration assay, hemizona assay, mannose binding assay and assays of sperm DNA integrity and fragmentation.

Fig. 1. Spermatozoon with normal morphology (a) and spermatozoa with vacuoles into the head (b).

#### **2.1.1 Reference values of semen analysis and semen variables**

Tables 1 and 2 summarise the lower references limits of semen characteristics as defined in the fifth edition of WHO manual, and the nomenclature of semen variables.

epithelium cells, red cells, prostate cells. A normal ejaculate should not contain more than

The semen analysis can be computer-assisted (Computeur-assisted semen analysis-CASA) (Yeung *et al.* 1997). The advantage of a computer assisted system for sperm analysis is the ability to obtain precise quantitative data as wells as the potential for the standardization of semen analysis procedures. Nevertheless, the main disadvantages are the lack of standardization procedures and the significant expense of equipment requirement. CASA is a valuable tool for research but the manual semen analysis remains the reference (Amann *et al.* 2004). Other tests can be made on semen such as the detection of sperm antibodies, sperm vitality, hypo-osmotic swelling test, assays of the sperm acrosome, sperm penetration assay, hemizona assay, mannose binding assay and assays of sperm DNA integrity and

Fig. 1. Spermatozoon with normal morphology (a) and spermatozoa with vacuoles into the

Tables 1 and 2 summarise the lower references limits of semen characteristics as defined in

**2.1.1 Reference values of semen analysis and semen variables**

the fifth edition of WHO manual, and the nomenclature of semen variables.

5 x 106 round cells/ml.

fragmentation.

head (b).

Table 1. Lower reference limits of semen characteristics (5th centiles, 95% confidence intervals)

Table 2. Nomenclature related to semen variations.

Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility 113

thawed) into the vagina using a catheter. With the development of new sperm preparation and the separation of the motile spermatozoa from the secretive part (Mortimer, 1994*)*, the IUI could be attempted. Concentrated preparation of sperm were attempted directly into the Fallopian tubes, peritoneal cavity, or ovarian follicles of the women, before to reach the consensus that IUI was the most effective method, providing a maximum pregnancy rate with a minimum of complications (Sacks and Simon, 1991). Although IUI can be performed in the natural cycle, nowadays it is combined with ovarian stimulation to increase the

The clinical indications for IUI are couples with unexplained infertility, mild or moderate male-factor infertility. This technique can be applied in certain female factors infertility such as antisperm antibodies or hostile cervical environment. In case of male infertility, the presence of ten million of motile spermatozoa is a prerequisite but good results can be

From a technical point of view, on the day of IUI, the patient is asked to produce sperm into a sterile cup. After liquefaction and semen analysis, the sample is treated as previously described to select a high concentration of good motility spermatozoa. The final suspension of selected spermatozoa is concentrated in a maximal final volume of 500 ul of culture medium. IUI can be performed on natural cycle or on female patient that underwent a mild ovarian stimulation. Around the moment of pharmacologically monitored ovulation, the spermatozoa are deposited into the uterus without neither local anesthetic nor antibiotic prophylaxis using polyethylene catheter attached to a plastic syringe. After IUI, the female

The success rates of IUI depends on the female patient age (De Sutter *et al.* 2005), the presence of comorbid conditions, ovulation method and the male factor (Burr *et al.* 1996). Abnormal spermatozoa morphology is associated with poor IUI outcomes on natural cycles and IUI with clomiphene or gonadotropin stimunation (Van Waart *et al.* 2001). Furthermore, abnormal sperm morphology has been associated with higher IUI complication rates such as miscarriage. Motility and concentration play also an important role in the pregnancy rate expectancy (Montanaro *et al.* 2001; Ombelet *et al.* 1997). Patients with percentage of a sperm motility superior to 30 percent have a four time increase cumulative pregnancy rate than

In conclusion, IUI is the most commonly performed ART procedure, a low cost technique

The first world birth after IVF was obtained but Steptoe and Edwards in 1978 (Steptoe and Edwards, 1978). In 2006, it was estimated that more than three million of babies had been born using this procedure (ESHRE, 2006). The indications for IVF treatment include longstanding infertility due to tubal disease, endometriosis, unexplained infertility, infertility

The principle of IVF is the following: the female partner is given stimulatory drugs (gonadotrophin) in the weeks leading up to the procedure and in a view to trigger the development of several follicules. The follicular fluid are collected in a tube and under

obtained with 5 and even one million of motile spermatozoa injected in uterus.

patients with sperm motility less than 30 percent (Yalti *et al.* 2004).

pregnancy rate.

patient can resume normal activity.

and straightforward to perform.

involving male factor.

**3.2 IVF**

#### **2.1.2 Sperm preparation techniques**

In a view to apply diagnostic tests of functions or to use the spermatozoa for therapeutic recovery for intra-uterine insemination (IUI) or *in vitro* ART, the spermatozoa must be separated from the seminal liquid. While the seminal liquid support the cervical mucus penetration in natural intercourse, the elimination of part of its compounds is necessary to perform IUI or *in vitro* fertilization (IVF). In the separation of spermatozoa from the seminal liquid, the final preparation concentrates morphologically normal and motile spermatozoa, free from debris, non-germ cells and dead spermatozoa. The separation and concentration of those spermatozoa is done performing a sperm preparation. Three methods are mainly used. The choice of a method is leaded by the semen characteristics. These methods are the simple washing, the direct swim-up and the density gradients. In all the sperm preparations, balanced salt solutions supplemented with proteins and containing an appropriate buffer are used as culture medium.

The 'Sperm washing' is a simple sperm preparation, performed for good quality semen, often used for IUI. The principal is in the washing of the semen to remove the secretive part (WHO, 2010).

The direct swim-up select the spermatozoa on their ability to swim out of the seminal liquid to the culture medium. It is the preferred method for the extraction of motile spermatozoa especially when their percentage is low e.g. for IVF and intracytoplasmic sperm injection (ICSI) (WHO, 2010).

The discontinuous density gradient consists in the centrifugation of the semen on a column of colloidal silica coated with silane which separate the cells according to their density. Furthermore, the motile spermatozoa swim through the gradients and concentrate at the bottom of the tube. This method selects a high fraction of motile spermatozoa and is selective against the debris, the leukocytes, non-germ cells and degenerating germs cells. The discontinuous density gradient is mainly used for IVF and ICSI (WHO, 2010).

If the patient is infected by the human immunodeficiency virus (HIV), the viral RNA and proviral DNA can be found in the seminal liquid and in non-sperm cells. In a view to prevent female partner contamination, a combination of density-gradient centrifugation followed by swim-up can be performed (Gilling-Smith *et al.* 2006; Savasi *et al.* 2007). With this combination of procedures, the motile spermatozoa are separated from the seminal liquid and virus-affected non-sperm cells.

#### **3. ART and results**

In the IUI practice, the female patient is monitored to determine the moment of ovulation; the semen is collected and prepared to select the best spermatozoa to transfer *in utero*. The fertilization process is *in vivo*. In IVF and ICSI protocols, the fertilization process is followed and *in vitro* assisted. Embryos are produced in the laboratory and are subsequently transferred *in utero*.

#### **3.1 IUI**

IUI is one of the earliest technique of assisted conception to be applied. It derives from intracervical insemination in which the principle was to deposit a sample of semen (fresh or thawed) into the vagina using a catheter. With the development of new sperm preparation and the separation of the motile spermatozoa from the secretive part (Mortimer, 1994*)*, the IUI could be attempted. Concentrated preparation of sperm were attempted directly into the Fallopian tubes, peritoneal cavity, or ovarian follicles of the women, before to reach the consensus that IUI was the most effective method, providing a maximum pregnancy rate with a minimum of complications (Sacks and Simon, 1991). Although IUI can be performed in the natural cycle, nowadays it is combined with ovarian stimulation to increase the pregnancy rate.

The clinical indications for IUI are couples with unexplained infertility, mild or moderate male-factor infertility. This technique can be applied in certain female factors infertility such as antisperm antibodies or hostile cervical environment. In case of male infertility, the presence of ten million of motile spermatozoa is a prerequisite but good results can be obtained with 5 and even one million of motile spermatozoa injected in uterus.

From a technical point of view, on the day of IUI, the patient is asked to produce sperm into a sterile cup. After liquefaction and semen analysis, the sample is treated as previously described to select a high concentration of good motility spermatozoa. The final suspension of selected spermatozoa is concentrated in a maximal final volume of 500 ul of culture medium. IUI can be performed on natural cycle or on female patient that underwent a mild ovarian stimulation. Around the moment of pharmacologically monitored ovulation, the spermatozoa are deposited into the uterus without neither local anesthetic nor antibiotic prophylaxis using polyethylene catheter attached to a plastic syringe. After IUI, the female patient can resume normal activity.

The success rates of IUI depends on the female patient age (De Sutter *et al.* 2005), the presence of comorbid conditions, ovulation method and the male factor (Burr *et al.* 1996). Abnormal spermatozoa morphology is associated with poor IUI outcomes on natural cycles and IUI with clomiphene or gonadotropin stimunation (Van Waart *et al.* 2001). Furthermore, abnormal sperm morphology has been associated with higher IUI complication rates such as miscarriage. Motility and concentration play also an important role in the pregnancy rate expectancy (Montanaro *et al.* 2001; Ombelet *et al.* 1997). Patients with percentage of a sperm motility superior to 30 percent have a four time increase cumulative pregnancy rate than patients with sperm motility less than 30 percent (Yalti *et al.* 2004).

In conclusion, IUI is the most commonly performed ART procedure, a low cost technique and straightforward to perform.

#### **3.2 IVF**

112 Male Infertility

In a view to apply diagnostic tests of functions or to use the spermatozoa for therapeutic recovery for intra-uterine insemination (IUI) or *in vitro* ART, the spermatozoa must be separated from the seminal liquid. While the seminal liquid support the cervical mucus penetration in natural intercourse, the elimination of part of its compounds is necessary to perform IUI or *in vitro* fertilization (IVF). In the separation of spermatozoa from the seminal liquid, the final preparation concentrates morphologically normal and motile spermatozoa, free from debris, non-germ cells and dead spermatozoa. The separation and concentration of those spermatozoa is done performing a sperm preparation. Three methods are mainly used. The choice of a method is leaded by the semen characteristics. These methods are the simple washing, the direct swim-up and the density gradients. In all the sperm preparations, balanced salt solutions supplemented with proteins and containing an

The 'Sperm washing' is a simple sperm preparation, performed for good quality semen, often used for IUI. The principal is in the washing of the semen to remove the secretive part

The direct swim-up select the spermatozoa on their ability to swim out of the seminal liquid to the culture medium. It is the preferred method for the extraction of motile spermatozoa especially when their percentage is low e.g. for IVF and intracytoplasmic sperm injection

The discontinuous density gradient consists in the centrifugation of the semen on a column of colloidal silica coated with silane which separate the cells according to their density. Furthermore, the motile spermatozoa swim through the gradients and concentrate at the bottom of the tube. This method selects a high fraction of motile spermatozoa and is selective against the debris, the leukocytes, non-germ cells and degenerating germs cells.

If the patient is infected by the human immunodeficiency virus (HIV), the viral RNA and proviral DNA can be found in the seminal liquid and in non-sperm cells. In a view to prevent female partner contamination, a combination of density-gradient centrifugation followed by swim-up can be performed (Gilling-Smith *et al.* 2006; Savasi *et al.* 2007). With this combination of procedures, the motile spermatozoa are separated from the seminal

In the IUI practice, the female patient is monitored to determine the moment of ovulation; the semen is collected and prepared to select the best spermatozoa to transfer *in utero*. The fertilization process is *in vivo*. In IVF and ICSI protocols, the fertilization process is followed and *in vitro* assisted. Embryos are produced in the laboratory and are subsequently

IUI is one of the earliest technique of assisted conception to be applied. It derives from intracervical insemination in which the principle was to deposit a sample of semen (fresh or

The discontinuous density gradient is mainly used for IVF and ICSI (WHO, 2010).

**2.1.2 Sperm preparation techniques**

appropriate buffer are used as culture medium.

liquid and virus-affected non-sperm cells.

(WHO, 2010).

(ICSI) (WHO, 2010).

**3. ART and results**

transferred *in utero*.

**3.1 IUI**

The first world birth after IVF was obtained but Steptoe and Edwards in 1978 (Steptoe and Edwards, 1978). In 2006, it was estimated that more than three million of babies had been born using this procedure (ESHRE, 2006). The indications for IVF treatment include longstanding infertility due to tubal disease, endometriosis, unexplained infertility, infertility involving male factor.

The principle of IVF is the following: the female partner is given stimulatory drugs (gonadotrophin) in the weeks leading up to the procedure and in a view to trigger the development of several follicules. The follicular fluid are collected in a tube and under

Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility 115

in terms of oocyte fertilization rate, number of embryos produced and implantation rates

ICSI can be applied in extremely low sperm counts, impair of motility, poor morphology (oligo-astheno-teratozoospermia) or azoospermia due to impaired testicular function or obstructed excretory ducts. ICSI can also be applied with non progressive motility or immotile spermatozoa. ICSI is the ultimate and only option for successful treatment of male

(Palermo *et al.* 1993; Van Steirteghem AC *et al.* 1993a; Van Steirteghem *et al.* 1993b).

infertility due to obstruction of the excretory ducts and impair of testicular functions.

Table 3 shows the indications for ICSI.

High titers of antisperm antibodies

Asthenozoospermia (caveat for 100% immotile spermatozoa)

Autoconserved frozen sperm from cancer patients in remission Ejaculatory disorders (e.g. electroejaculation, retrograde ejaculation)

Repeated fertilization failure after conventional IVF

Congenital bilateral absence of the vas deferens

Failure of epididymal sperm recovery because of fibrosis

Table 3. Indications for clinical application of ICSI.

Teratozoospermia (even globozoospermia and acrosomeless spermatozoa)

Azoospermia caused by testicular failure (maturation arrest, germ-cell aplasia)

The clinical pregnancy rates by use of ICSI are similar to those obtained with IVF, 26.5% of pregnancy rate according to egg collection and 28.7% according to embryo-transfer (Andersen *et al.* 2007). High fertilization and pregnancy rates can be obtained from motile spermatozoa (Nagy *et al.* 1995) while immotile and dead spermatozoa result in lower

As for the IVF procedure, the patient is stimulated and the follicle fluids containing mature (Metafase II) oocytes are aspirated. The retrieved oocytes are incubated in 5%CO2 at 37°C. The difference between IVF and ICSI lays in fertilization method. In a view to perform micro-injection, the cumulus and corona cells surrounding the oocytes must be removed using a combination of enzymatic and mechanical procedures. The assessment of the zona pellucida and the presence or absence of a germinal vesicle and of the first polar body are

Ejaculated spermatozoa Oligozoospermia

Epidydimal spermatozoa

Failed vaso-epididymosotomy

Obstruction of both ejaculatory ducts

All indications for epididymal sperm

fertilization rate (Tournaye *et al*. 1996).

Young syndrome

Necrozoospermia (Van Steirteghem, 2007)

**3.3.2 ICSI protocol**

Failed vasovasostomy

Testicular spermatozoa

sedation. The follicular fluid is carefully read in the IVF laboratory and the oocytes that have grown inside the follicules are isolated and incubated. On the same day, the mature oocytes are incubated with an aliquot of fresh (or frozen) motile partner 'spermatozoa. The testtubes or dishes containing the oocytes in the solution of spermatozoa are incubated up to 18 hours at 37°C and 5% CO2. A reduced concentration of O2 (5-6%) can also be applied. Between 16 and 18 hours, the fertilization process is checked with the observation of two pronuclei. Tipically, between 60 and 70% of the oocytes are fertilized. In the following hours, a large majority of fertilized oocytes (now called 'zygotes') reach the embryo-stage with the first cell division. The embryo quality is evaluated on the base the number of cells according to the time of incubation, the absence of extra-cellular fragments, the capacity of the cells to compact at the morula stage and the blastocyst to expanse. The embryos can be maintained in *in vitro* culture until 5-6 days. Within this period, any embryo can be in utero transferred, discarded or frozen for a successive *in utero* transfer.

Oehninger *et al.* (1988) studied the correlation between normal sperm morphology and insemination concentration. By increasing the insemination concentration in samples of severe teratozoospermia, the fertilization rate can be improved.

The couples with male factor and undergoing conventional IVF have a lower fertilization and pregnancy rate expectancy. On the same trend, very low pregnancy rates are obtained with surgical retrieved sperm (Hirsh *et al.* 1994). As a consequence and if a sufficient number of progressive motility spermatozoa is not available, the assisted reproductive technology called 'Intra-cytoplasmic sperm injection' (ICSI) must be applied as alternative and in a view to assist the fertilization process. The clinical pregnancy rate of IVF across Europe is 26.1% per egg collection and 29.6% per embryo-transfer (Andersen *et al.* 2007), although the success rates decrease according to female age over 35 (HFEA, 2005).

Fig. 2. Embryo development. (a)Time of micro-injection: 0 hour (h); (b) zygote at 17+1 h; (c) 2 cells at 23+1 h ; (d) 4 cells at 44+1 h; (e) 8 cells at 68+1h; (f) compacting morula at 92+2h; (g) blastocyst at 116+2h post ICSI or IVF.

#### **3.3 ICSI**

#### **3.3.1 ICSI: History and indications**

On the process of micro-manipulation history that lead to ICSI, the techniques that were previously applied were the partial zona dissection (PZD) to facilitate the sperm penetration (Cohen *et al.* 1989), the subzonal insemination (SUZI) where the motile spermatozoa were micro-injected under the peri-vitelline space and were in direct contact with the oolemma (Laws-King *et al.* 1987). Subsequently ICSI of which the principle is the direct micro-injection of a single preferably with good morphology spermatozoon into the ooplasma was invented. The first pregnancies and births was obtained in 1992 (Palermo *et al.* 1992). Since the beginning, ICSI appeared a superior technique due to higher resutls compared to SUZI in terms of oocyte fertilization rate, number of embryos produced and implantation rates (Palermo *et al.* 1993; Van Steirteghem AC *et al.* 1993a; Van Steirteghem *et al.* 1993b).

ICSI can be applied in extremely low sperm counts, impair of motility, poor morphology (oligo-astheno-teratozoospermia) or azoospermia due to impaired testicular function or obstructed excretory ducts. ICSI can also be applied with non progressive motility or immotile spermatozoa. ICSI is the ultimate and only option for successful treatment of male infertility due to obstruction of the excretory ducts and impair of testicular functions.

Table 3 shows the indications for ICSI.

114 Male Infertility

sedation. The follicular fluid is carefully read in the IVF laboratory and the oocytes that have grown inside the follicules are isolated and incubated. On the same day, the mature oocytes are incubated with an aliquot of fresh (or frozen) motile partner 'spermatozoa. The testtubes or dishes containing the oocytes in the solution of spermatozoa are incubated up to 18 hours at 37°C and 5% CO2. A reduced concentration of O2 (5-6%) can also be applied. Between 16 and 18 hours, the fertilization process is checked with the observation of two pronuclei. Tipically, between 60 and 70% of the oocytes are fertilized. In the following hours, a large majority of fertilized oocytes (now called 'zygotes') reach the embryo-stage with the first cell division. The embryo quality is evaluated on the base the number of cells according to the time of incubation, the absence of extra-cellular fragments, the capacity of the cells to compact at the morula stage and the blastocyst to expanse. The embryos can be maintained in *in vitro* culture until 5-6 days. Within this period, any embryo can be in utero

Oehninger *et al.* (1988) studied the correlation between normal sperm morphology and insemination concentration. By increasing the insemination concentration in samples of

The couples with male factor and undergoing conventional IVF have a lower fertilization and pregnancy rate expectancy. On the same trend, very low pregnancy rates are obtained with surgical retrieved sperm (Hirsh *et al.* 1994). As a consequence and if a sufficient number of progressive motility spermatozoa is not available, the assisted reproductive technology called 'Intra-cytoplasmic sperm injection' (ICSI) must be applied as alternative and in a view to assist the fertilization process. The clinical pregnancy rate of IVF across Europe is 26.1% per egg collection and 29.6% per embryo-transfer (Andersen *et al.* 2007),

Fig. 2. Embryo development. (a)Time of micro-injection: 0 hour (h); (b) zygote at 17+1 h; (c) 2 cells at 23+1 h ; (d) 4 cells at 44+1 h; (e) 8 cells at 68+1h; (f) compacting morula at 92+2h; (g)

On the process of micro-manipulation history that lead to ICSI, the techniques that were previously applied were the partial zona dissection (PZD) to facilitate the sperm penetration (Cohen *et al.* 1989), the subzonal insemination (SUZI) where the motile spermatozoa were micro-injected under the peri-vitelline space and were in direct contact with the oolemma (Laws-King *et al.* 1987). Subsequently ICSI of which the principle is the direct micro-injection of a single preferably with good morphology spermatozoon into the ooplasma was invented. The first pregnancies and births was obtained in 1992 (Palermo *et al.* 1992). Since the beginning, ICSI appeared a superior technique due to higher resutls compared to SUZI

although the success rates decrease according to female age over 35 (HFEA, 2005).

transferred, discarded or frozen for a successive *in utero* transfer.

severe teratozoospermia, the fertilization rate can be improved.

blastocyst at 116+2h post ICSI or IVF.

**3.3.1 ICSI: History and indications**

**3.3 ICSI**

(Van Steirteghem, 2007)

Table 3. Indications for clinical application of ICSI.

The clinical pregnancy rates by use of ICSI are similar to those obtained with IVF, 26.5% of pregnancy rate according to egg collection and 28.7% according to embryo-transfer (Andersen *et al.* 2007). High fertilization and pregnancy rates can be obtained from motile spermatozoa (Nagy *et al.* 1995) while immotile and dead spermatozoa result in lower fertilization rate (Tournaye *et al*. 1996).

#### **3.3.2 ICSI protocol**

As for the IVF procedure, the patient is stimulated and the follicle fluids containing mature (Metafase II) oocytes are aspirated. The retrieved oocytes are incubated in 5%CO2 at 37°C. The difference between IVF and ICSI lays in fertilization method. In a view to perform micro-injection, the cumulus and corona cells surrounding the oocytes must be removed using a combination of enzymatic and mechanical procedures. The assessment of the zona pellucida and the presence or absence of a germinal vesicle and of the first polar body are

Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility 117

When all of the oocytes of the dish have been microinjected, the dish is removed from the microscope and the oocytes are incubated in non-buffered medium in 5% of CO2 at 37°C. Differences in the oolemma breakage have been observed. The immediate break of the oolemma without any aspiration is associated with lower oocyte survival rates (Palermo *et*

As in the IVF protocol, the fertilization process is checked 16-18 hours after microinjection and embryo culture is prolonged until the destiny of each embryo is decided (transferred, frozen or discarded). As in IVF, abnormal fertilization can be observed with the formation of one pronucleus (about 3% of the micro-injected oocytes), and the finding of three pronuclear (3PN-about 4% of micro-injected oocytes) after the micro-injection of one single spermatozoon can be the consequence of extrusion failure of the second polar body at the time of fertilization (Staessen *et al.* 1997). No embryo generated from 1PN and 3PN oocytes must be transferred. About 90% of the 2PN-oocytes cleave and

Fig. 3. Intracytoplasmic sperm injection procedure. The oocyte and the micro-injection pipette with the spermatozoon are positioned (a). The micro-injection pipette with the spermatozoon at its end goes through the zona pellucida, the peri-vitellin space (b) and penetrates the oolemma (c). The micro-injection pipette is removed after that the spermatozoon has been

released into the ooplasma (d) (with the kind collaboration of C. Ragolia).

immobilized spermatozoa is moved slowly to the oocyte. - The spermatozoa is positioned at the end of the injection pipette.

aneuploidies.

o'clock position.

*al.* 1996).

become embryos.

of the sudden oolemma break. - The aspiration is immediately stopped.

orientation but the meiotic spindle. During the oocyte denudation, the polar body can move within the peri-vitellin space. As a consequence, the position of the polar body could not correspond to the meiotic spindle position and the micro-injection can disturb the chromosomes alignment on the equatorial plane of the meiotic spindle and generate

verified at the microscopic observation. The presence and orientation of the meiotic spindle can be assessed when exposed to polarized light. 3.9% of the intact oocytes are at the metaphase I stage because they underwent germinal vesicle (GV) breakdown but they have not expelled the first polar; 10.3% of the intact oocytes are at the GV stage, and around 85.8% of the intact oocytes are in the metaphase II stage, reaching the haploid stage and therefore available for micro-injection (Bonduelle *et al.* 1999). Matured, denuded and rinsed oocytes are incubated until the time of micro-injection. ICSI can be performed with spermatozoa that have been ejaculated, extracted from epipydimus or testis. As it has been described previously, the sperm is prepared to select motile and good morphology spermatozoa.

The micro-injection of single spermatozoa inside the ooplasm is performed under an inverted microscope equipped with micro-manipulators, microinjectors and heating stage to maintain the temperature at 37°C. The magnification of 400X is recommended for the selection of moving spermatozoa. Micromanipulators give the possibility to move the two microinjectors in the 3 directions. The microinjectors can be filled with air or mineral oil and a micrometer controls the plunger. Generally, the holding is fixed to the microinjector on the left of the operator, and the injection pipette is fixed on the right. The oocyte can be fixed, positioned and released by the holding pipette, the spermatozoa is immobilized, injected and released using the injection pipette. It is recommended to position the microscope on a vibration-proof table.

For the micro-injection setting, the gametes (metaphase II oocytes to be micro-injected and spermatozoa to micro-inject) are deposed within micro-droplets of buffered medium and on the same plastic micro-injection dish. The micro-droplets are covered by mineral oil. The micro-drop containing the spermatozoa is a mixture of spermatozoa plus PVP. The PVP slows down the spermatozoa motility and facilitates the manipulation and the control of the fluids. For ejaculated spermatozoa, they should be deposited on a single micro-drop, while extracted spermatozoa from testis or epididymis can be deposited on several microdroplets. Each denuded oocytes is placed on a single micro-drop-droplet (usually eight micro-droplets contain eight oocytes on the micro-injection dish). After the preparation of the dish with the gametes to use, the ICSI is performed. Step by step, ICSI can be described as follows:

orientation but the meiotic spindle. During the oocyte denudation, the polar body can move within the peri-vitellin space. As a consequence, the position of the polar body could not correspond to the meiotic spindle position and the micro-injection can disturb the chromosomes alignment on the equatorial plane of the meiotic spindle and generate aneuploidies.

116 Male Infertility

verified at the microscopic observation. The presence and orientation of the meiotic spindle can be assessed when exposed to polarized light. 3.9% of the intact oocytes are at the metaphase I stage because they underwent germinal vesicle (GV) breakdown but they have not expelled the first polar; 10.3% of the intact oocytes are at the GV stage, and around 85.8% of the intact oocytes are in the metaphase II stage, reaching the haploid stage and therefore available for micro-injection (Bonduelle *et al.* 1999). Matured, denuded and rinsed oocytes are incubated until the time of micro-injection. ICSI can be performed with spermatozoa that have been ejaculated, extracted from epipydimus or testis. As it has been described previously, the sperm is prepared to select motile and good morphology

The micro-injection of single spermatozoa inside the ooplasm is performed under an inverted microscope equipped with micro-manipulators, microinjectors and heating stage to maintain the temperature at 37°C. The magnification of 400X is recommended for the selection of moving spermatozoa. Micromanipulators give the possibility to move the two microinjectors in the 3 directions. The microinjectors can be filled with air or mineral oil and a micrometer controls the plunger. Generally, the holding is fixed to the microinjector on the left of the operator, and the injection pipette is fixed on the right. The oocyte can be fixed, positioned and released by the holding pipette, the spermatozoa is immobilized, injected and released using the injection pipette. It is recommended to position the microscope on a

For the micro-injection setting, the gametes (metaphase II oocytes to be micro-injected and spermatozoa to micro-inject) are deposed within micro-droplets of buffered medium and on the same plastic micro-injection dish. The micro-droplets are covered by mineral oil. The micro-drop containing the spermatozoa is a mixture of spermatozoa plus PVP. The PVP slows down the spermatozoa motility and facilitates the manipulation and the control of the fluids. For ejaculated spermatozoa, they should be deposited on a single micro-drop, while extracted spermatozoa from testis or epididymis can be deposited on several microdroplets. Each denuded oocytes is placed on a single micro-drop-droplet (usually eight micro-droplets contain eight oocytes on the micro-injection dish). After the preparation of the dish with the gametes to use, the ICSI is performed. Step by step, ICSI can be described

compress the sperm flagellum. The shape of the tail should be maintained.

spermatozoa.

vibration-proof table.

as follows:

motility.

micropipette.

When all of the oocytes of the dish have been microinjected, the dish is removed from the microscope and the oocytes are incubated in non-buffered medium in 5% of CO2 at 37°C.

Differences in the oolemma breakage have been observed. The immediate break of the oolemma without any aspiration is associated with lower oocyte survival rates (Palermo *et al.* 1996).

As in the IVF protocol, the fertilization process is checked 16-18 hours after microinjection and embryo culture is prolonged until the destiny of each embryo is decided (transferred, frozen or discarded). As in IVF, abnormal fertilization can be observed with the formation of one pronucleus (about 3% of the micro-injected oocytes), and the finding of three pronuclear (3PN-about 4% of micro-injected oocytes) after the micro-injection of one single spermatozoon can be the consequence of extrusion failure of the second polar body at the time of fertilization (Staessen *et al.* 1997). No embryo generated from 1PN and 3PN oocytes must be transferred. About 90% of the 2PN-oocytes cleave and become embryos.

Fig. 3. Intracytoplasmic sperm injection procedure. The oocyte and the micro-injection pipette with the spermatozoon are positioned (a). The micro-injection pipette with the spermatozoon at its end goes through the zona pellucida, the peri-vitellin space (b) and penetrates the oolemma (c). The micro-injection pipette is removed after that the spermatozoon has been released into the ooplasma (d) (with the kind collaboration of C. Ragolia).

Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility 119

is used to identify and open the epidydimal tubule to carry out the epididymal sperm aspiration. In 90 percent of obstructive azoospermia cases, spermatozoa were retrieved from epididymis (Silber *et al.* 1990, Holden *et al.* 1997; Patrizio 2000). Nowadays this technique has

The patients with obstructive azoospermia and non-obstructive azoospermia are successfully treated with ICSI using surgically retrieved spermatozoa from epididymis or testis. Numerous literature report ICSI with spermatozoa recovered from epididymis (Silber *et al.* 1994; Tournaye *et al.* 1994; Mansour *et al.* 1996) or testis (Schyosman *et al.* 1993; Silber *et al.* 1995). The fertilization and pregnancy rates from obstructive azoospermic patients are comparable to those obtained from patients with ejaculated sperm (Aboughar *et al.* 1997). In those patients, only the etiology can influence ICSI outcomes. The patients with acquired causes of obstructive azoospermia have higher fertilization (Nicopoullos *et al.* 2004a). The patients with non-obstructive azoospermia have a statistically significant decrease of fertilization and pregnancy rate when compared to osbstructive azoospermic patients

Beyond the male factor, the efficacy of all assisted reproductive technique is directly correlated with female age with a significant pregnancy expectancy over 40 years old

With ICSI, lower rated in terms of fertilization and pregnancy rates can be expected when

It is now an overall consensus that the health of the children born from ART is the most important outcome parameter that has to be considered (Wennerholm *et al.* 2004). Pregnancy complications, major malformations, possible reasons for adverse outcome as well as the increase of multiple ART pregnancies must be taken under consideration. According to Jackson *et al.* (2004) and when compared to spontaneous conceived singleton children, the IVF and ICSI pregnancies are associated with higher odds of each perinatal mortality, pre-term delivery, low birth weight, very low birth weight and small gestation age. In twin pregnancies, the perinatal mortality is lower in assisted conception compared to spontaneous twins conceptions (Helmerhost *et al.* 2004). There is an overall increase for major congenital malformations after IVF and ICSI compared to spontaneously conceived children (Hansen *et al.* 2005) but no significant differences when IVF and ICSI are compared among them (Rimm *et al.* 2004). The possible causes of malformations are the abnormal caryotypes of infertile patients and properly in abnormal spermatozoa (Bonduelle *et al.* 1999). A significative 2.1% of *de novo* anomalies are observed when sperm concentrations used for ICSI was below 20 million/ml (Bonduelle *et al.* 2002). It is possible that ART is associated with a higher risk of imprinting disorder, especially Angelman syndrome and Beckwith-Wiedemann syndrome. However, children born with ART are healthy and develop similar to children spontaneously conceived. General health, growth, and mental and psychomotor development of IVF children do not differ from spontaneously conceived children, even if lower birth weight and prematurity may contribute to some health

been substitute by the less invasive PESA method.

**3.5 ICSI Results from surgical retrieved sperm**

(Nicopoullos *et al*. 2004b).

**3.6 IVF and ICSI results**

(Oehninger *et al.* 1995; Devroey *et al.* 1996).

no motile spermatozoa are available.

problems observed (Ludwig *et al.* 2006)*.*

#### **3.4 ICSI with surgical spermatozoa**

In patients that appear to be azoospermic after semen analysis, the surgical sperm retrieval is the last option to collect spermatozoa. The different techniques are percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA), testicular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MESA). The choice of the technique depends on the type of patient and of the diagnosis.

In all surgical sperm retrieval, ICSI is the method of choice to obtain a pregnancy using fresh or frozen/thawed spermatozoa.

#### **3.4.1 PESA**

PESA is the least invasive surgical sperm retrieval technique. The patient is in conscious sedation and/or spermatic cord blockage with marcaine or lidocaine. The testis is immobilized and the aspiration is carried out with a 25-G butterfly needle. The fluid is aspirated and examined under microscope to check for motile spermatozoa. The recovered spermatozoa can be subsequently cryopreserved. The percentage of motile spermatozoa retrieval is 93% (Ramos *et al.* 2004).

#### **3.4.2 TESA, TESE and micro-TESE**

Testicular sperm retrieval can be performed either on open surgical biopsy or as a percutaneous procedure. The term 'Testicular sperm extraction' (TESE) qualifies the open biopsy, and the term 'testicular sperm aspiration' (TESA) qualifies the percutaneous retrieval (Schlegel *et al.* 1998). The open surgical biopsy provides a higher quantity of tissue for harvesting spermatozoa than as aspiration procedure does. Nevertheless, TESA is generally sufficient for spermatozoa retrieval in patients with obstructive infertility and normal spermatogenesis. TESA is performed under local anesthesia. The transcutaneous aspiration uses a needle of 19-21 gauge directly into the testicular parenchyma and using negative pressure. In case of severely crompromised spermatogenesis, TESE should be the preferred method for harvesting spermatozoa.

TESE is performed after spermatic cord anesthesia with marcaine and a transversal 1 cm incision through the scrotum and tunica vaginalis down to the tunica albuginea and tissue from the mid anterior surface of the testis.

Microscopic TESE is a conjunction between open biopsy and it is conceptually similar to multibiopsy TESE, with the following differences: a single long testis incision is made to expose a large area of the testicular parenchyma and to access germ cells tubules, and a microscope is used to select the seminiferous tubules to be excised (Schlegel 1999). Since the number of spermatozoa obtained from micro-TESE is low, this procedure is generally performed the same day as eggs retrieval during ICSI.

#### **3.4.3 MESA**

The microsurgical epididymal sperm aspiration (MESA) is an advance in the therapy of non-reconstructable obstructive azoospermia (Schroeder-Printzen *et al.* 2000). The epididymis is exposed to a 12 mm incision made in the scrotal skin, an operating microscope is used to identify and open the epidydimal tubule to carry out the epididymal sperm aspiration. In 90 percent of obstructive azoospermia cases, spermatozoa were retrieved from epididymis (Silber *et al.* 1990, Holden *et al.* 1997; Patrizio 2000). Nowadays this technique has been substitute by the less invasive PESA method.

#### **3.5 ICSI Results from surgical retrieved sperm**

The patients with obstructive azoospermia and non-obstructive azoospermia are successfully treated with ICSI using surgically retrieved spermatozoa from epididymis or testis. Numerous literature report ICSI with spermatozoa recovered from epididymis (Silber *et al.* 1994; Tournaye *et al.* 1994; Mansour *et al.* 1996) or testis (Schyosman *et al.* 1993; Silber *et al.* 1995). The fertilization and pregnancy rates from obstructive azoospermic patients are comparable to those obtained from patients with ejaculated sperm (Aboughar *et al.* 1997). In those patients, only the etiology can influence ICSI outcomes. The patients with acquired causes of obstructive azoospermia have higher fertilization (Nicopoullos *et al.* 2004a). The patients with non-obstructive azoospermia have a statistically significant decrease of fertilization and pregnancy rate when compared to osbstructive azoospermic patients (Nicopoullos *et al*. 2004b).

#### **3.6 IVF and ICSI results**

118 Male Infertility

In patients that appear to be azoospermic after semen analysis, the surgical sperm retrieval is the last option to collect spermatozoa. The different techniques are percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA), testicular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MESA). The choice of

In all surgical sperm retrieval, ICSI is the method of choice to obtain a pregnancy using fresh

PESA is the least invasive surgical sperm retrieval technique. The patient is in conscious sedation and/or spermatic cord blockage with marcaine or lidocaine. The testis is immobilized and the aspiration is carried out with a 25-G butterfly needle. The fluid is aspirated and examined under microscope to check for motile spermatozoa. The recovered spermatozoa can be subsequently cryopreserved. The percentage of motile spermatozoa

Testicular sperm retrieval can be performed either on open surgical biopsy or as a percutaneous procedure. The term 'Testicular sperm extraction' (TESE) qualifies the open biopsy, and the term 'testicular sperm aspiration' (TESA) qualifies the percutaneous retrieval (Schlegel *et al.* 1998). The open surgical biopsy provides a higher quantity of tissue for harvesting spermatozoa than as aspiration procedure does. Nevertheless, TESA is generally sufficient for spermatozoa retrieval in patients with obstructive infertility and normal spermatogenesis. TESA is performed under local anesthesia. The transcutaneous aspiration uses a needle of 19-21 gauge directly into the testicular parenchyma and using negative pressure. In case of severely crompromised spermatogenesis, TESE should be the

TESE is performed after spermatic cord anesthesia with marcaine and a transversal 1 cm incision through the scrotum and tunica vaginalis down to the tunica albuginea and tissue

Microscopic TESE is a conjunction between open biopsy and it is conceptually similar to multibiopsy TESE, with the following differences: a single long testis incision is made to expose a large area of the testicular parenchyma and to access germ cells tubules, and a microscope is used to select the seminiferous tubules to be excised (Schlegel 1999). Since the number of spermatozoa obtained from micro-TESE is low, this procedure is generally

The microsurgical epididymal sperm aspiration (MESA) is an advance in the therapy of non-reconstructable obstructive azoospermia (Schroeder-Printzen *et al.* 2000). The epididymis is exposed to a 12 mm incision made in the scrotal skin, an operating microscope

the technique depends on the type of patient and of the diagnosis.

**3.4 ICSI with surgical spermatozoa**

or frozen/thawed spermatozoa.

retrieval is 93% (Ramos *et al.* 2004).

**3.4.2 TESA, TESE and micro-TESE**

preferred method for harvesting spermatozoa.

performed the same day as eggs retrieval during ICSI.

from the mid anterior surface of the testis.

**3.4.1 PESA**

**3.4.3 MESA**

Beyond the male factor, the efficacy of all assisted reproductive technique is directly correlated with female age with a significant pregnancy expectancy over 40 years old (Oehninger *et al.* 1995; Devroey *et al.* 1996).

With ICSI, lower rated in terms of fertilization and pregnancy rates can be expected when no motile spermatozoa are available.

It is now an overall consensus that the health of the children born from ART is the most important outcome parameter that has to be considered (Wennerholm *et al.* 2004). Pregnancy complications, major malformations, possible reasons for adverse outcome as well as the increase of multiple ART pregnancies must be taken under consideration. According to Jackson *et al.* (2004) and when compared to spontaneous conceived singleton children, the IVF and ICSI pregnancies are associated with higher odds of each perinatal mortality, pre-term delivery, low birth weight, very low birth weight and small gestation age. In twin pregnancies, the perinatal mortality is lower in assisted conception compared to spontaneous twins conceptions (Helmerhost *et al.* 2004). There is an overall increase for major congenital malformations after IVF and ICSI compared to spontaneously conceived children (Hansen *et al.* 2005) but no significant differences when IVF and ICSI are compared among them (Rimm *et al.* 2004). The possible causes of malformations are the abnormal caryotypes of infertile patients and properly in abnormal spermatozoa (Bonduelle *et al.* 1999). A significative 2.1% of *de novo* anomalies are observed when sperm concentrations used for ICSI was below 20 million/ml (Bonduelle *et al.* 2002). It is possible that ART is associated with a higher risk of imprinting disorder, especially Angelman syndrome and Beckwith-Wiedemann syndrome. However, children born with ART are healthy and develop similar to children spontaneously conceived. General health, growth, and mental and psychomotor development of IVF children do not differ from spontaneously conceived children, even if lower birth weight and prematurity may contribute to some health problems observed (Ludwig *et al.* 2006)*.*

Effectiveness of Assisted Reproduction Techniques as an Answer to Male Infertility 121

only 20 to 50 percent of these men eventually recover spermatogenesis (Shin *et al.* 2005). For these reasons, sperm banking is highly recommended for all patients with malignant disease

Sperm banking can be applied before contraceptive measure (vasectomy), for precautionary measure of fertility because of high risk occupation or activity such as military forces, to prevent infertility due to surgeries for recurrent varicocele or hydrocele (Anger *et al.* 2003), or in a view to ensure a sample available the day of *in vitro* treatment. The patients that need special collection such as assisted ejaculated patients with spinal injury, spermatozoa from retrograde ejaculation in urine or surgical collection from the genital tract, found in sperm banking the possibility to cryopreserve spermatozoa for clinical and reproductive

For all the patients that are at risk in losing the fertility due to pre-cited reasons, sperm banking gives a significant psychological support in the hope of future paternity. As a consequence, all males requiring chemo- or radiotherapy, including adolescents should