Sperm viral infection and male infertility: focus on HBV, HCV, HIV, HPV, HSV, HCMV, and AAV

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Sperm viral infection and male infertility: focus on HBV, HCV, HIV, HPV, HSV, HCMV, and AAV Andrea Garolla a , Damiano Pizzol a , Alessandro Bertoldo a , Massimo Menegazzo a , Luisa Barzon b , Carlo Foresta a,∗ a Department of Molecular Medicine, Section of Clinical Pathology & Unit for Human Reproduction Pathology, University of Padova, Via Gabelli 63, 35121, Padova, Italy b Department of Molecular Medicine, Section of Microbiology and Medical Biotechnologies, University of Padova, Italy

a r t i c l e

i n f o

Article history: Received 17 October 2012 Received in revised form 20 March 2013 Accepted 20 March 2013 Keywords: Ejaculate alteration Male infertility Sperm infection Sperm parameters Urogenital infections Viral sexually transmitted disease

a b s t r a c t Chronic viral infections can infect sperm and are considered a risk factor in male infertility. Recent studies have shown that the presence of HIV, HBV or HCV in semen impairs sperm parameters, DNA integrity, and in particular reduces forward motility. In contrast, very little is known about semen infection with human papillomaviruses (HPV), herpesviruses (HSV), cytomegalovirus (HCMV), and adeno-associated virus (AAV). At present, EU directives for the viral screening of couples undergoing assisted reproduction techniques require only the evaluation of HIV, HBV, and HCV. However, growing evidence suggests that HPV, HSV, and HCMV might play a major role in male infertility and it has been demonstrated that HPV semen infection has a negative influence on sperm parameters, fertilization, and the abortion rate. Besides the risk of horizontal or vertical transmission, the negative impact of any viral sperm infection on male reproductive function seems to be dramatic. In addition, treatment with antiviral and antiretroviral therapies may further affect sperm parameters. In this review we attempted to focus on the interactions between defined sperm viral infections and their association with male fertility disorders. All viruses considered in this article have a potentially negative effect on male reproductive function and dangerous infections can be transmitted to partners and newborns. In light of this evidence, we suggest performing targeted sperm washing procedures for each sperm infection and to strongly consider screening male patients seeking fertility for HPV, HSV, and HCMV, both to avoid viral transmission and to improve assisted or even spontaneous fertility outcome. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Sexually transmitted diseases (STDs) represent major health, social, and economic problems worldwide. Despite the discovery of antibiotics and vaccines, and the development of disease prevention and control programs, STDs remain a significant cause of acute and chronic diseases with possible involvement in pregnancy complications and infertility (Ochsendorf, 2008). In 1993 the World Health

∗ Corresponding author. Tel.: +39 049 8218517; fax: +39 049 8218520. E-mail address: [email protected] (C. Foresta).

Organization (WHO) established the role of genital tract infections in human infertility (WHO, 1993). Most male genital tract infections may induce infertility and previous studies reported that 15–20% of infertile subjects are affected by semen infection (Weidner et al., 1999). Back in 2001, Dejucq and Jégou (2001) stressed the need to study and understand the role of viruses in fertility and since then progress has been made in this direction. Based on different sites of infection, various pathogenic mechanisms have been described:

(a) Systemic acute or chronic infections can result in transient or permanent infertility, impairing hormones,

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testicular function, and spermatogenesis (La Vignera et al., 2011). (b) Testicular involvement by orchitis directly impairs sperm production (Weidner et al., 1999). (c) Male accessory gland (epididymis, prostate, and seminal vesicles) and urethral infections have been identified as playing a negative role in male reproductive function and fertility owing to obstruction or sub-obstruction, altered secretory function, and release of inflammatory mediators (La Vignera et al., 2011). Semen infections are frequently present even in asymptomatic males, and they are often associated with poor sperm quality (Bezold et al., 2007). Moreover, seminal leucocytes can be indicators of male genital tract infection, but they can be completely lacking when a high load of infectious agents are present. Actually, the relevance of chronic viral infections as an etiologic factor of male infertility is believed to be underestimated (La Vignera et al., 2011). Heading the list of chronic viral sperm infections are: human immunodeficiency virus (HIV), hepatitis B (HBV), and hepatitis C (HCV) viruses. In particular, HIV infection has been demonstrated to be significant for chronic low genitourinary tract inflammation, sperm infection, and reduced fertility (Cardona-Maya et al., 2011). Recent studies have shown that the presence of HBV or HCV in semen adversely affects sperm parameters and in particular reduces forward motility (Lorusso et al., 2010). Furthermore, viral semen infection has been associated with an increased frequency of sperm aneuploidy and DNA fragmentation (Moretti et al., 2008). Little is known about HPV semen infection; however, growing evidence suggests that this virus might play a major role in male infertility (Perino et al., 2011). In fact, HPV has been recently demonstrated to have a negative influence on sperm parameters, the fertilization process and the abortion rate (Foresta et al., 2010a,b, 2011a; Pérez-Andino et al., 2009; Perino et al., 2011). Other common sexually transmitted viruses able to colonize semen and thus of major concern for reproductive specialists are herpesviruses (HSV), human cytomegalovirus (HCMV), and adeno-associated virus (AAV). At present, EU directives for the viral screening of couples undergoing assisted reproduction techniques (ART) require evaluation of HIV, HBV, and HCV (Wingfield and Cottell, 2010). Besides the risk of horizontal or vertical transmission to a partner or neonate (Englert et al., 2004), the impact of viruses on male reproductive function concerns in particular the ability of these infections to induce an impairment of general health and thereafter infertility. Furthermore, both the possible presence of viral DNA or RNA at the sperm level and the treatment of patients with antiviral and antiretroviral therapies able to induce testicular damage may have a further detrimental effect on sperm parameters (Lorusso et al., 2010). This review attempts to focus on the interactions between defined sperm viral infections and their association with male fertility disorders. Data were acquired by a study of medical literature using the following search terms: ejaculate alteration, male infertility, sperm infection, sperm parameters, urogenital infections, and viral sexually transmitted disease.

2. HBV sperm infection During HBV infection, the virus can be found in many secretions, semen, and other tissues beyond the liver and blood. HBV is not only able to pass through the blood–testis barrier and enter the male germ cells, but also integrate into their genomes. Several methods have been developed for the detection of HBV DNA in semen. Qian et al. (2005) successfully used quantitative real-time PCR to investigate the viral load in the semen of HBV-infected patients who were seeking assisted reproduction. Using fluorescence in situ hybridization (FISH) Huang et al. (2003) showed that HBV DNA can be integrated into the sperm chromosomes of HBV carriers and may be vertically transmissible via the germ cells. Moreover, they demonstrated that HBV infection may have mutagenic effects on sperm chromosomes, which are nonspecific and multi-sited, leading to genome instability. This finding suggested that HBV sperm infection can produce hereditary effects, inducing chromosome aberrations. 2.1. HBV sperm infection and male fertility Regarding the impact of HBV on seminal parameters, it is well known that HBV infection may cause male infertility by damaging spermatozoa (Table 1). Lorusso et al. (2010) found that sperm concentration, motility, morphology, and viability were significantly impaired in HBV-seropositive patients. Other studies showed that subjects with HBV chronic infection had alteration of sperm parameters (Moretti et al., 2008; Vicari et al., 2006) and in particular a trend toward a negative correlation with the viral load was found. By in vitro studies, Kang et al. (2012) demonstrated that co-incubation of human spermatozoa with hepatitis B virus S protein induced a loss of sperm mitochondrial membrane potential, thus reducing sperm motility. Moreover, they showed that HBs exposure induced oxidative stress in sperm cells up to the stage of apoptosis, as revealed by phosphatidylserine externalization, caspase activation, and DNA fragmentation. In view of these results, it is possible to conclude that HBV-infected males have a significantly impaired sperm quality compared with that of control men. 2.2. HBV sperm infection and assisted reproduction Contamination of patients during artificial insemination has been described for HBV over the last 20 years (Englert et al., 2004). In vitro studies showed that when zona-free hamster oocytes were inseminated using human sperm carrying an HBV DNA plasmid, HBV genes were able to replicate and be expressed in two-cell embryos (Ali et al., 2006). These results suggest that human sperm cells might integrate the HBV DNA, acting as vectors for the transmission of HBV genes during IVF as well as ICSI procedures. Moreover, Zhou et al. (2011) recently reported that HBV infection, besides its association with poor sperm parameters, is associated with poorer ICSI and embryo transfer outcomes. In contrast, Lee et al. (2010) reported no adverse effect of HBV infection on the assisted reproduction results. Although in assisted reproduction, HBV

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Table 1 Review of the literature on the influence of HBV semen infection on sperm parameters, apoptosis, and chromosomes.

Huang et al., 2003 Kang et al., 2012 Lorusso et al., 2010 Moretti et al., 2008 Vicari et al., 2006 Zhou et al., 2011

Sperm count

Motility

Normal morphology

Apoptosis

Aneuploidy

N.E. N.E. ↓ = ↓ ↓

N.E. ↓ ↓ = ↓ ↓

N.E. N.E. ↓ ↓ ↓ ↓

N.E. ↑ N.E. ↑ N.E. N.E.

↑ ↑ N.E. ↑ N.E. N.E.

N.E.: not evaluated; ↑: increased; ↓: decreased; =: not affected.

transmission is a risk for the baby and also for technicians and non-infected partners, many European centers suggest that these patients should be given the opportunity to have children. Measures can be taken to prevent the uninfected partner and the future child from acquiring hepatitis B by vaccination programs. Moreover, in male HBV patients, sperm washing has been shown to effectively reduce the risk of vertical transmission and to prevent the introduction of HBV into the oocyte in the case of ICSI. However, the risk of infected sperm cells acting as vectors in male HBV carriers is not different in IVF compared with ICSI (Lutgens et al., 2009). 3. HCV sperm infection Very few studies with contradictory results have been performed to evaluate the presence of HCV-RNA in seminal fluid and spermatozoa. Debono et al. (2000) were not able to detect HCV-RNA in the semen of infected males with a high blood viral load, either by PCR or by branched DNA or in situ hybridization. In contrast, Levy et al. (2002) showed that HCV-RNA can be found in semen of infected males when Taq inhibitors have been suppressed by dilutions, thus explaining the previous contradictory results. Other authors confirmed this finding detecting HCV RNA in the semen of one-third of HCV viremic men (Bourlet et al., 2002). They reported that although the seminal viral load was low, its ability to transmit infection should not be underestimated. Also, a direct HCV negative effect on the spermatogenesis has been postulated by Durazzo et al. (2006) and an improvement in semen quality after antiviral therapy was reported. 3.1. HCV sperm infection and male fertility Many reports have linked HCV infection to altered sperm parameters such as reduced motility and abnormal

morphology (Table 2) (Durazzo et al., 2006; Hofny et al., 2011; Lorusso et al., 2010). In particular, Levy et al. (2002) demonstrated semen alteration in 30% of HCV-infected males before antiviral treatment for high serum viral load. Moreover, these studies showed that when HCV RNA was present in seminal fluid, alongside sperm alteration, fertility was also very poor. Moretti et al. (2008) reported not only a lower fertility index, but also higher sperm diploidy in those subjects with HCV semen infection, suggesting that sperm apoptosis and necrosis play a major role in these patients. These findings were further supported by a recent study (La Vignera et al., 2012) showing that mitochondrial membrane potential, chromatin compaction, and DNA fragmentation were significantly altered in patients with chronic HCV infection. Finally, these subjects had higher seminal levels of reactive oxygen species, and viral replication correlated with a worsening of all functional sperm parameters.

3.2. HCV sperm infection and assisted reproduction Although a small risk of viral infection via semen has been reported, HCV transmission during IVF procedures is possible (Clarke, 1999). Because there is no vaccine for HCV prevention, it is essential to employ any measure of risk reduction during assisted reproduction with infected patients. In addition, they should be counseled about the risks of transmission to their partner, children, and their healthcare team. When the male partner is infected, sperm washing can reduce the viral load in semen and it is recommended to reduce the risk of transmission (Pasquier et al., 2000). New methods of separating non-infected sperm have been proposed, which demonstrated the effectiveness of a sperm washing device that uses a double tube and a gradient in excluding most viral particles from the motile sperm fraction (Mencaglia et al., 2005). However, the HCV RNA RT-PCR method used to test sperm before

Table 2 Review of the literature on the influence of HCV semen infection on sperm parameters, apoptosis, and chromosomes.

Durazzo et al., 2006 Goldberg-Bittman et al., 2008 Hofny et al., 2011 La Vignera et al., 2012 Lorusso et al., 2010 Mencaglia et al., 2005 Moretti et al., 2008 Safarinejad et al., 2010

Sperm count

Motility

Normal morphology

Apoptosis

Aneuploidy

= N.E ↓ ↓ ↓ = = ↓

↓ N.E. ↓ ↓ ↓ = = ↓

↓ N.E. ↓ ↓ ↓ N.E. ↓ ↓

N.E. ↑ N.E. ↑ N.E. N.E. ↑ N.E.

N.E. ↑ N.E. ↑ N.E. N.E. ↑ ↑

N.E.: not evaluated; ↑: increased; ↓: decreased; =: not affected.

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IUI or IVF showed that this procedure is not risk-free, as small numbers of viral particles may be undetected. Therefore, patients have to be informed about the efficacy of sperm-washing in reducing the viral load of sperm, but also about the residual albeit low risk of viral presence in selected sperm. Moreover, if one partner is chronically infected, therapy has to be considered prior to fertility treatment in order to reduce the viral load. Furthermore, because experimental studies performed on animal species showed that antiviral treatments may induce teratogenic and embryocidal effects, pregnancy should be deferred for at least six months after the conclusion of therapy (Foster, 2004). In cases of chronic HCV infection, reproductive units using best practice IVF procedures reported having no viral transmission in large series of serodiscordant couples (Mencaglia et al., 2005). Nevertheless, only a few thousand cycles have been performed worldwide and it is still too early to demonstrate that these techniques are completely risk-free. Finally, no significant difference in the clinical pregnancy rate has been reported between infected males and controls undergoing ART procedures. However, fertilization and cleavage rates were significantly lower in males with HCV (Prisant et al., 2010).

interstitial tissue; infection of spermatogonia, but not more mature spermatogenic cells, was also observed (ShehuXhilaga et al., 2007). In humans, spermatozoa are probably mainly passive carriers of the virus, attached to the cell surface. In this regard, expression of the beta-chemokine receptors CCR5 and CCR3 was demonstrated on ejaculated spermatozoa from healthy subjects. CCR5 was detected in the peri-acrosomal region of the sperm surface, whereas CCR3 was also present in the post-acrosomal cap. Both CCR5 and CCR3 expressed on the sperm cell surface may be involved in HIV-1 adhesion to spermatozoa, thus allowing these cells to act as virion cellular carriers during sexual transmission of HIV-1 infection (Muciaccia et al., 2007). In addition, heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1 and in its transmission to dendritic cells (DCs), macrophages, and T cells (Ceballos et al., 2009). Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa, but also results in the phenotypic maturation of DCs, which play an important role in the sexual transmission of HIV infection (Ceballos et al., 2009). 4.1. HIV sperm infection and male fertility

4. HIV sperm infection Sexual transmission plays a major role in the spread of HIV-1. The semen of infected men may contain high levels of HIV-1, and virus can be recovered from seminal cells or seminal fluid. Seminal cells are mixtures of spermatozoa, precursors of germ cells, T lymphocytes, macrophages and epithelial cells, and HIV-1 proviral DNA has been detected in several types of these cells, mainly lymphocytes, monocytes, and macrophages (Ceballos et al., 2009; Muciaccia et al., 2007). In HIV-1-infected men who are receiving highly active antiretroviral therapy (HAART) and who have no detectable levels of viral RNA in plasma, the virus may be present in seminal cells and therefore may be capable of being transmitted sexually (Zhang et al., 1998). In a serodiscordant couple the risk of acquiring HIV is dependent on the viral load in semen (Chakraborty et al., 2001). Whether the virus also infects spermatozoa is controversial (Muciaccia et al., 2007). In the macaque model of SIV infection, viral RNA and antigens were detected in macrophages, dendritic cells, and T cells in the testicular

Semen parameters are within the normal range in asymptomatic HIV-positive men (van Leeuwen et al., 2008), while normal sperm morphology and motility are impaired with progression of the disease (Table 3) (Dondero et al., 1996). In AIDS patients, grossly abnormal sperm and leukocytospermia have been reported (Pavili et al., 2010). Sperm alterations found today are attributed to the effects of antiretroviral therapy, and these alterations include lower ejaculate volume and less progressive and more abnormally shaped spermatozoa (Kehl et al., 2011). In patients treated with nucleoside analog reverse transcriptase inhibitors with known mitochondrial toxicity, the number of mtDNA copies in spermatozoa was decreased (Pavili et al., 2010). 4.2. HIV sperm infection and assisted reproduction Depending on the semen quality, spermatozoa can be safely used for intrauterine insemination, in vitro fertilization or ICSI (Nicopoullos et al., 2004). The semen has to

Table 3 Review of the literature on the influence of HIV semen infection on sperm parameters, apoptosis, and chromosomes.

Bujan et al., 2007 Dondero et al., 1996 Kehl et al., 2011 Lorusso et al., 2010 Melo et al., 2008 Mencaglia et al., 2005 Muciaccia et al., 2007 Nicopoullos et al., 2004 Pavili et al., 2010 van Leeuwen et al., 2008

Sperm count

Motility

Normal morphology

Apoptosis

Aneuploidy

= ↓ ↓ ↓ = = = ↓ ↓ =

↓ ↓ ↓ = = ↓ ↓ ↓ ↓ ↓

= ↓ ↓ = N.E. N.E. = N.E. ↓ =

N.E. N.E. N.E. N.E. N.E. N.E. ↑ N.E. N.E. N.E.

N.E. N.E. N.E. N.E. N.E. N.E. N.E. N.E. ↑ N.E.

N.E.: not evaluated; ↑: increased; ↓: decreased; =: not affected.

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be washed free of HIV using the gradient technique and the success of this procedure has to be controlled prior to use (Melo et al., 2008). The use of antitretroviral therapy decreases HIV load in semen and thus improves the outcome of intrauterine insemination/sperm-washing procedures in HIV-positive men (Nicopoullos et al., 2004). 5. HPV sperm infection Human papillomavirus is frequently detected in semen and urethral swabs from asymptomatic men. In the past, HPV semen infection was always considered transient and without clinical consequences. In a recent study, nested polymerase chain reaction (PCR) showed the presence of HPV DNA sequences in 10% of semen samples from asymptomatic young adult males who had had unprotected intercourse (Foresta et al., 2010a). In addition, fluorescence in situ hybridization (FISH) for HPV conducted on infected samples showed that the virus was localized at the sperm head and in particular on about 25% of entire sperm population. A later study, considering the prevalence of HPV semen infection in patients with risk factors for the virus (subjects with genital warts, partners of HPVpositive females, and infertile patients) showed that in infected semen samples, HPV can be localized at different levels: in sperm, in exfoliated cells or in both sites. Curiously, infertile patients had both a prevalent infection in sperm and a higher percentage of infected sperm, while exfoliated cells were significantly more affected in patients with other risk factors. In fertile control subjects HPV infection was never found in sperm cells (Foresta et al., 2010b). In addition, HPV semen infection has also been reported in 6.1% of cryopreserved samples from a cohort of patients who banked sperm because of testicular cancer (Foresta et al., 2011b). A recent study performed by live fluorescent microscopy, analyzed semen samples after incubation with HPV-16 capsids (Pérez-Andino et al., 2009). Authors showed that the virus was present on the surface of sperm cells, located at two distinct binding sites along the equator of the sperm head. These authors suggested also the presence of glycosaminoglycans (GAGs), or soluble factors of similar chemical structure on the sperm surface, and proposed a role for these molecules in the interaction and binding between HPV and sperm. Another study, using flow cytometry and immunofluorescence analyses of infected native sperm and sperm exposed to HPV16 capsid, demonstrated that sperm express syndecan-1 most exclusively in the equatorial region of the head. These data clearly showed that the HPV capsid protein L1 and the glycosaminoglycan syndecan-1 co-localize in the equatorial region of the

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sperm head, suggesting that HPV infects sperm by primary attachment with syndecan-1 (Foresta et al., 2011a). 5.1. HPV sperm infection and male fertility Few studies have reported the effects of HPV infection on sperm parameters, and the available data are conflicting (Table 4) (Rintala et al., 2004; Lai et al., 1997). In the past, some authors showed an association between HPV semen infection and decreased sperm motility or reduced pH of seminal plasma, while other authors observed no clinically significant alteration of these parameters. Recently, we analyzed the relation between the presence of HPV at the semen level and the abnormalities of each parameter, observing no difference in seminal volume, viscosity, pH count, viability, and normal morphology in HPV-infected and non-infected semen samples. In contrast, a significant reduction of mean sperm motility was found in those subjects with infected semen (Foresta et al., 2010a,b). According to data from other authors, our results suggested that HPV, inducing an alteration of sperm motility, may play a major role in cases of idiopathic asthenozoospermia and thus in male infertility. However, it is not known whether HPV-infected sperm are able to fertilize, to transfer viral DNA to oocytes, and if infected oocytes are able to produce normal embryos. 5.2. HPV sperm infection and assisted reproduction In natural conception, the rate of spontaneous abortions and major birth defects appears not to be greater in HPV-exposed couples than in unexposed ones (Dana et al., 2009). However, this aspect is of crucial importance in relation to in vitro fertilization techniques. A recent study highlighted that HPV can be detected in the placenta and that this infection may occur not only through ascending infection from the cervix, but also via infected sperm at fecundation (Weyn et al., 2006). Additionally, a clinical study performed in women undergoing in vitro fertilization (IVF) reported a significant reduction of pregnancies in the presence of HPV cervical infection compared with no infection (Spandorfer et al., 2006). Although the possible consequences of fetal exposure to HPV are not well defined, in vitro experiments have shown that HPVtransfected trophoblast cells have an increased rate of apoptosis and a reduced placental invasion into the uterine wall compared with controls. Recently, we performed a study (Foresta et al., 2011a) using the hamster egg penetration test (HEPT) with human infected and non-infected sperm. HEPT showed that human HPV-infected sperm were

Table 4 Review of the literature on the influence of HPV semen infection on sperm parameters, apoptosis, and chromosomes.

Bezold et al., 2007 Foresta et al., 2010a Foresta et al., 2010b Garolla et al., 2012 Lai et al., 1997 Rintala et al., 2004

Sperm count

Motility

Normal morphology

Apoptosis

Aneuploidy

↓ = = = = =

↓ ↓ ↓ ↓ ↓ =

N.E. = = = N.E. N.E.

N.E. N.E. N.E. = N.E. N.E.

N.E. N.E. N.E. = N.E. N.E.

N.E.: not evaluated; ↑: increased; ↓: decreased; =: not affected.

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able to penetrate hamster oocytes, even if the mean number of penetrated sperm per oocyte was lower compared with the control. Moreover, oocytes penetrated by transfected sperm expressed the viral genes, suggesting an active transcription of viral genes by the infected oocyte. Despite the high relevance of these data, it is unknown whether these in vitro findings might apply to oocytes in vivo. In a recent prospective study, Perino et al. (2011) investigated the role of HPV infection in infertile couples undergoing ART cycles. These authors showed a reduced pregnancy rate and an increased abortion rate in couples with HPV infection compared with those not infected. Particularly, the risk was increased when HPV DNA testing was positive in the female partner, but it was even higher when sperm samples were infected. Considering that HPV appears on the sperm surface, sperm washing protocols might reduce the infectiousness of semen before ART procedures. We tested the effectiveness of conventional procedures of sperm selection for removing HPV infection. However, we observed a significant persistence of infected sperm after sperm washing (Foresta et al., 2011c). To overcome the tenacity of HPV DNA binding to sperm and to reduce the risk of HPV infection during ART, a modified swim-up with the addition of heparinase III has been recently proposed. This method was able to completely eliminate HPV DNA from semen and did not show any significant alteration of sperm quality and DNA integrity (Garolla et al., 2012).

of the reporter gene HSV-1 thymidine kinase (HSV-TK) have demonstrated male infertility with degeneration of spermatogenic cells, failure of Sertoli–germ cell interaction, and loss of germ cells owing to apoptosis (Cai et al., 2012). However, this transgenic model does not reproduce the mechanism and effect of HSV infection in sperm; instead it demonstrates the toxicity of HSV-TK overexpression in sperm cells. However, the involvement of the enzyme HSV-TK in the damage of spermatogenic cells in patients with HSV infection cannot be excluded.

6. HSV sperm infection

The prevalence of human cytomegalovirus (HCMV) DNA in the genital tract and in the semen of fertile and infertile HCMV-seropositive men has been reported to vary widely from 8% to 65% in different series (Eggert-Kruse et al., 2009; Neofytou et al., 2009; Bresson et al., 2003), but studies based on HCMV cultures revealed that recovery from infectious virus is low and occurs in less than 5% of seropositive donors (Naumenko et al., 2011). In the study by Bresson et al. (2003), the most strongly HCMV-positive semen sample belonged to an initially seronegative donor who seroconverted during the quarantine period, representing a case of primary acute infection. Sexual transmission does not seem to be a frequent route of HCMV infection (Eggert-Kruse et al., 2009). Oral and respiratory spreads appear to be the dominant routes of transmission during childhood and probably adulthood as well. Young infants and children with subclinical infection appear to be a major source of infection in pregnant women. In a recent study in male mice experimentally inoculated intratesticularly with murine cytomegalovirus, infection was demonstrated in testicular endothelial and Leydig cells, peritubular cells were severely damaged, and spermatogenesis affected, but neither germ cells nor Sertoli cells were infected. Following the mating of infected males with uninfected females, neither infectious virus nor viral DNA were found in spermatozoa recovered from uterine fluid, fertilized oocytes, blastocysts, fetal tissues or newborn animals, demonstrating that cytomegalovirus harbored in the male genital organs was not transmitted to the offspring, even when mating occurred during the acute phase of CMV disease (Tebourbi et al., 2001). In humans, in a study on HIV patients, HCMV was detected in semen

Data on the prevalence of herpesvirus infection in semen are variable among different studies (Neofytou et al., 2009). The DNA of herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) has been detected in the semen of 2–4% to 50% of men, with no significant difference between fertile and infertile subjects (Neofytou et al., 2009). Experimental infection of ejaculated sperm with a high load of HSV-2 demonstrated that a few viral particles adhered to the sperm membrane in the presence of seminal plasma, while more viral particles stuck to the membrane of the sperm head or the flagella in the absence of seminal fluid. Therefore, the presence of seminal fluid seemed to impede the adherence of HSV-2 to sperm (Pallier et al., 2002). No viral particles were detected inside the sperm while a slight difference in the percentage of motile forms, with increased mean amplitude of lateral head displacement and linear acceleration, was observed when seminal fluid-free sperm were incubated with HSV-2 (Pallier et al., 2002). 6.1. HSV sperm infection and male fertility The presence of HSV DNA in semen has been associated with a decrease in sperm concentration and reduced motility and has been considered to be responsible for some cases of male infertility (Neofytou et al., 2009). Moreover, the frequency of HSV detection in the sperm samples of partners of women with repeated miscarriages was found to be higher than in the controls (Bocharova et al., 2007). Transgenic rats with spermatid-specific ectopic expression

6.2. HSV sperm infection and assisted reproduction Experimental studies of in vitro incubation of semen with HSV-2 demonstrated that the virus does not interact with sperm, but remains in the seminal fluid (Pallier et al., 2002). In this condition, which mimics infection occurring in the adnexal glands in vivo, the techniques used in assisted reproduction to eliminate seminal fluids should also eliminate viral particles. However, if sperm is collected from the testis, there is an increased risk of sperm coming into contact with high local concentrations of HSV2 because of contamination from blood or interstitial tissues, in the presence of virus reactivation or acute infection. In this case, the washing procedures might not completely eliminate viral particles (Pallier et al., 2002). 7. HCMV sperm infection

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CD45+ cells, but not in mature spermatozoa (Rasmussen et al., 1995), while a recent study on HCMV-infected organotypic cultures identified the presence of viral antigens and viral particles in spermatogonia, spermatocytes, and in spermatids, producing lytic effects (Naumenko et al., 2011). These infected cells could develop to mature HCMVcarrying spermatozoa, while mature spermatozoa did not seem to support productive HCMV infection (Naumenko et al., 2011). However, these findings require further investigation for confirmation. 7.1. HCMV sperm infection and male fertility Human cytomegalovirus infection does not seem to be a significant cause of infertility (Naumenko et al., 2011; Eggert-Kruse et al., 2009). The presence of HCMV in semen has not been reported to significantly affect semen quality, including the functional capacity of sperm, local antisperm Ab, or seminal WBC (Eggert-Kruse et al., 2009). Experimental infection of ejaculated sperm was also performed with HCMV and adherence of viral particles to the sperm membrane was demonstrated, although the relationship appeared weaker for HCMV than for HSV-2 (Pallier et al., 2002). In addition, no effect on sperm motility was observed in semen incubated with HCMV in vitro (Pallier et al., 2002). 7.2. HCMV sperm infection and assisted reproduction Experimental studies of semen incubated in vitro with HCMV demonstrated that the virus does not interact with sperm (Pallier et al., 2002). The washing procedures used for assisted reproduction techniques are expected to eliminate viral particles or reduce viral load, but the risk of virus transmission cannot completely be excluded, especially in cases of ICSI (Pallier et al., 2002). To prevent potential HCMV transmission by sperm donation, HCMV serology testing could be useful to identify primary acute infection by detecting IgM-positive donors or those presenting IgG and/or IgM seroconversion during quarantine. 8. AAV sperm infection Adeno-associated virus (AAV) DNA was demonstrated for the first time in male semen by Rohde et al. (1999). In a subsequent study in AAV DNA-positive testis samples from two patients, the authors demonstrated that AAV DNA was present in an integrated form in testis tissue within sequences of the so-called AAVS1 region on chromosome 19 (Mehrle et al., 2004). Semen infection might also occur after delivery of engineered AAV vectors for gene therapy, as suggested for the first time by the detection of AAV vector DNA in the semen of patients enrolled in a gene therapy trial for hemophilia B based on the intramuscular delivery of AAV vectors carrying the factor IX gene (Manno et al., 2006). Following this adverse event, which raised concerns regarding the risk of germline transduction (Marshall, 2001), investigators analyzed the risk of germline transmission of AAV vectors in different species of male animals, including mouse, rat, rabbit, and dog (reviewed by Barzon et al., 2004). These studies

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showed that the risk of vector dissemination in semen was higher following intravascular delivery than after intramuscular injection, and that AAV vector sequences were transiently detected and disappeared in dose- and timedependent fashion (Arruda et al., 2001; Schuettrumpf et al., 2006; Favaro et al., 2009). Long-term follow-up spanning hundreds of spermatogenesis cycles showed that there was no recurrence of detectable vector sequences in the semen of rabbits, suggesting that AAV (at least AAV-2 and AAV8) probably presents a low risk of germline transmission for humans as well (Arruda et al., 2001; Schuettrumpf et al., 2006; Favaro et al., 2009). By using vasectomized rabbits, AAV vector sequences were demonstrated to reach the semen in the absence of germ cells (Favaro et al., 2009). Attempts to transduce isolated murine spermatogonia and mouse mature sperm directly with AAV vector were unsuccessful (Arruda et al., 2001; Cuoto et al., 2004), while transduction of mouse male germline stem cells resulted in transgene transmission after germ cell transplantation (Honaramooz et al., 2008). Another study showed that, after intraperitoneal administration of AAV vectors to newborn mice or intravascular delivery into mouse fetuses, the vectors were detected for over one year in the gonads, but were not detectable in isolated sperm DNA from the treated animals or in their offspring (Jakob et al., 2005). Taken together, these data indicate that the risk of inadvertent germline transmission following AAV delivery is very low. Anyway, the precautions proposed for clinical studies of AAV vector-based gene therapy, i.e., the use of barrier contraception until semen tests become negative for vector sequences, and the banking of sperm prior to vector injection, are advisable. These recommendations should also be applied when other potentially integrating viral and nonviral vectors are used for gene delivery (Barzon et al., 2004). 8.1. AAV sperm infection and male fertility The presence of AAV DNA in semen samples from infertile men has been reported to be significantly more frequent than in normal semen samples (20–40% vs. 0–5% respectively) (Rohde et al., 1999; Erles et al., 2001; Schlehofer et al., 2012). In infertile couples, the presence of AAV DNA was detected in 14.9% of cervical swabs and 19.9% of semen samples, but only 3.8% of couples were AAV DNA positive in both semen and endocervical specimens, suggesting that there is generally a low risk of infection by AAV in genital samples (Schlehofer et al., 2012). In this recent study (Schlehofer et al., 2012), the presence of AAV DNA in semen was not significantly related to semen quality, including the functional capacity of sperm or local antisperm antibodies ASAs, at variance with previous studies by the same authors that found an association with oligoasthenozoospermia and oligospermia (Rohde et al., 1999; Erles et al., 2001). 8.2. AAV sperm infection and assisted reproduction To our knowledge, there are no data in the literature on the effect of AAV infection of semen on the outcome of assisted reproduction, while several reports on the

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presence of AAV in amniotic fluid and material from spontaneous abortion indicate that AAV infection might be associated with adverse reproductive outcomes, including spontaneous abortion, gestational trophoblastic disease, spontaneous preterm birth, and severe pre-eclampsia (Tobiasch et al., 1994; Burguete et al., 1999; Kiehl et al., 2002; Arechavaleta-Velasco et al., 2006, 2008). In animal models, AAV was found to be lethal for embryos at early stages of gestation (Botquin et al., 1994), to cause transplacental infection (Lipps and Mayor, 1980), and to impair placental invasion of the uterine wall (Koi et al., 2001). 9. Conclusions Sperm viral infection by HBV, HCV, HIV, HPV, and HSV, but not by HCMV and AAV, induces male infertility through multiple patho-physiological mechanisms. These pathogens, which may cause incurable and even fatal infections, can also impair sperm parameters and function, particularly when localized at the semen level. Although thousands of cycles with best practice ART procedure have demonstrated no contamination using sperm from virally infected patients, transmission is possible, both to mothers and newborns. In the light of these observations it is clear that sperm alone, independently of any sexual contact, can transmit the viral infection with very similar frequencies to occasional sexual intercourse. With this review we aimed to underline the following key points: (a) Although screening for HPV and HSV is not considered by EU guidelines before ART, they may affect sperm parameters and function, thus reducing male fertility. (b) As for semen infection with HIV, HBV, and HCV, these viruses and HCMV may have a potentially negative effect on assisted reproduction outcome and they can be transmitted to partner and newborn. (c) In particular, sperm infection with HPV has been demonstrated to reduce sperm motility, sperm–egg interaction, the pregnancy rate, and to the increase abortion rate. (d) Besides the risk of horizontal and vertical viral transmission, infected sperm may be carriers of aneuploidy and fragmented DNA. (e) Sperm infection with HPV, HSV, and HCMV should be investigated before ART, even in the presence of normal sperm parameters. (f) The significance of sperm alteration induced by HIV, HBV, and HCV is frequently confounded by concomitant treatments able to affect spermatogenesis. (g) More studies are needed to evaluate the effect of antiretroviral and antiviral drugs for extended periods on sperm chromosomes and male fertility. (h) Although a number of cycles have been performed in the world using semen infected with HIV, HBC, and HBV without contamination, it is still too early to conclude that these procedures are fully safe. (i) In some cases with negative blood viral load, a viral load in semen can be detected even after sperm washing procedures. (j) More research is needed to define standardized procedures of sperm washing specific to each virus, and

standardized and sensitive methods of viral detection after sperm selection are needed. (k) In those cases of viral semen infection we suggest always considering techniques able to select sperm without infection. In conclusion, this review summarized the available studies on the influence of viral semen infection on sperm parameters, male fertility, and outcome of ART. Current EU guidelines suggest screening for HBV, HCV, and HIV before ART procedures. However, most of the viruses considered in this article have a potentially negative effect on male reproductive function and dangerous infections can be transmitted to partners and newborns. In the light of this review, we suggest performing specific sperm washing procedures for each semen infection and to strongly consider screening male patients seeking fertility for HPV, HSV, and HCMV, both to avoid transmission to mother and newborn and to improve assisted or even spontaneous fertility outcomes.

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Please cite this article in press as: Garolla, A., et al., Sperm viral infection and male infertility: focus on HBV, HCV, HIV, HPV, HSV, HCMV, and AAV. J. Reprod. Immunol. (2013), http://dx.doi.org/10.1016/j.jri.2013.03.004