Relative frequency of hepatitis B virus, human papilloma virus, Epstein-Barr virus, and herpes simplex viruses in the semen of fertile and infertile men in Shiraz, Iran: A cross-sectional study

Abstract Background About 8-12% of couples on reproductive age suffers from infertility worldwide. Since 1993, the role of genital tract infections by microbes, including viruses that can infect the sperm, in human infertility has been proposed. Objective To investigate the frequency of hepatitis B virus (HBV), human papilloma virus (HPV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV) infection in the semen of fertile and infertile men referred to the Mother and Child Hospital, Shiraz, Iran. Materials and Methods In this cross-sectional study, 350 men including 200 infertile and 150 fertile men were included. All semen samples were allowed to liquefy, followed by the assessment of sperm parameters. DNA was extracted using a DNA extraction kit (CinaGene, Tehran, Iran) according to the manufacturer's instructions. Detection of HBV, HPV, EBV, and HSV1/2 was done by the PCR method. Results The mean age of the participants was 36 ± 7 yr. Molecular results showed that 16 samples (8%) of infertile men and 5 (3.3%) of fertile men were positive for HBV, which was not statistically significant (p = 0.069). Only one sample of the fertile participants was positive for HPV. None of the semen samples of the infertile or fertile groups was positive for the presence of EBV or HSV1/2. Conclusion The results of this study indicated that HBV, HPV, EBV, and HSV might not be involved in men's infertility. Further studies are recommended for clarifying the role of these viruses in infertility.


Introduction
Inability to conceive after 12 months of unprotected and regular sexual intercourse defined as infertility. It has been reported that around 8-12% of couples suffer from infertility worldwide (1), and overall 50% of infertile cases are associated with men (2).
Viruses could infect the genital tract and impair the semen by various mechanisms (4,5). Several HBV is a member of the Hepadenaviridae family, which is transmitted by infected blood and semen (7). HBV DNA has been found in the semen of HBV-infected patients, but not in HBV-negative patients (4). It has been reported that HBV infection has been linked with low quality of sperm resulting increase the frequency of infertility in men (8,9). Su and colleagues stated that the risk of infertility is higher in HBV-infected men compared to non-infected ones (10). HPV is a nonenveloped double-stranded DNA virus transmitted by sexual contact (11). Gizzo and colleagues showed that HPV infection of the sperm might be involved in decreasing the fertility rate among men through different mechanisms that may influence human embryo development (12). In a review article, Foresta and colleagues reported that the prevalence of HPV semen infection in infertile men is 10-35%; it was also shown that the motility of the sperm in the infected semen sample was lower than in uninfected semen (13).
HSV is a double-stranded DNA virus from the Herpesviridae family (14). The prevalence of this virus in semen varies from 3-50%, depending on the investigation method (15). It has been reported that there is a correlation between the presence of HSV in the semen and a decreased in sperm concentration and reduced motility (4). EBV is a ubiquitous virus that replicates in the epithelial cells and lymphocytes (16). It was found that EBV DNA was present in 40% of cases in equal frequency among normal and abnormal semen (15).
Accordingly, in this study, we aimed to investigate the relative frequency of HBV, HPV, EBV, and HSV infections and their effects on the semen quality and sperm characteristics in fertile and infertile men referred to the Ghadir Mother and Child Hospital, Shiraz, southwestern Iran.

Study design and subjects
In this cross-sectional study, 350 subjects The subjects were advised to wash their hands and genital area with soap and water prior to sampling.

HBV PCR conditions
Detection of the HBV genome was done using the HBV PCR detection kit (Sinacolon, Tehran, Iran) following the manufacturer's instructions. After that, the PCR product was run on a 1.5% agarose gels.

EBV PCR conditions
Allele ID 7 software were used to designed the sequences of primers specific for BHRF1 region of the EBV genome (Table I)

HSV PCR conditions
HSV detection was performed by common HSV1-and 2-specific primers (Table I)

Statistical analysis
Data were analyzed using the IBM SPSS Statistics software version 21 (IBM Company, USA).
Chi-square and Mann-Whitney tests were used to compare different parameters. P < 0.05 was considered as statistically significant.

Demographic characteristics of the participants
Of the 350 participants included in the study, were struggling with substance use disorder, but no significant association was found (p = 0.51).
Sperm motility in the fertile group was 50 ± 8.4 and was significantly lower in the infertile group (26.7 ± 12.8, p = 0.001). A significant difference was seen between the two studied group in the semen volume < 1.5 mL (p = 0.006). Also, those with a sperm count of < 15 million/mL were significantly more frequent in the infertile group than in the fertile one (p = 0.001). Furthermore, sperm motility of < 32% and sperm morphology of < 4% were significantly higher in the semen of infertile men than in the fertile ones (p = 0.001). The frequency of HBV in the semen of fertile and infertile groups was not significantly associated with the sperm count, abnormal morphology, sperm motility, or semen volume.

The results of HBV, HPV, EBV, and HSV1 and 2 detection through PCR
Our molecular results showed that 16

Discussion
Infertility has become a major health problem and male factors are responsible for up to 50% of the cases (18). The causes of infertility in a large number of infertile men are still unknown, but it seems that urogenital infections are responsible for infertility in 6-10% of cases (19,20). Several studies have shown that viral infections including HBV, HPV, EBV, and HSV can infect semen and cause infertility by influencing sperm characteristics and semen quality (3,4,5).
It has been reported that progressive motility and percentage of normal sperm morphology in infertile men infected with were significantly lower in comparison with HPV-negative cases (21). Also, a significant association of HSV infection with a lower seminal volume and a lower mean sperm count was reported (22). Moreover, another study showed that sperm motility and normal sperm morphology were significantly negatively affected in HBV-positive men (23).
The results showed that 8% and 3.3% samples of infertile and fertile men were positive for HBV, respectively, which was not statistically significant.
Moreover, only one sample of the fertile men was positive for HPV. Furthermore, none of the semen samples of the infertile or fertile groups was positive for the presence of EBV or HSV1/ 2.
Although more semen samples of our infertile participants were positive for HBV than of the fertile group, the difference was not statistically significant. In agreement with our study, Zangeneh and colleagues reported that the frequency of HBV in infertile persons who they studied was very low and was not statistically different from fertile men (24). A study in Ahvaz also showed a very low frequency of HBsAg among infertile couples (25).
Moreover, it has been reported that none of the semen samples of infertile men who they examined were positive for HBV DNA and the mentioned that the low prevalence of HBV infection in their population might have been the cause of the negative results (26). On the other hand, a case-control study that compared men with HBV infection to those without HBV showed an increased risk of infertility in HBVinfected men (8). Also, in a systematic review, it was stated that HBV infection could cause male infertility (4). Therefore, according to the mentioned studies, it seems that the low prevalence of between the presence of HSV and infertility (33). According to these studies, it seems that HSV might be involved in infertility at least in some areas, which might be related to a high frequency of HSV infection in those populations.
The relatively small sample size was a limitation of our study. Using the conventional PCR method and not real-time PCR can be considered as another limitation of this study.
In sum, while a number of studies have shown associations between infertility and HBV, HPV, and HSV, some others do not support these findings.
This strong discrepancy may partly come from differences in factors including the sample size, different geographical distribution of the viruses, and lifestyle (sexual behavior) of the studied subjects. Moreover, technical issues including the sensitivity of detection methods (PCR or realtime PCR) as well as differences in the extraction procedures may also explain discrepancies among different studies.

Conclusion
According to our results, among the viruses investigated in this study, only HBV and HPV were detected in the semen samples; however, their frequency revealed no significant difference between infertile and fertile groups. The frequency of HBV was not significantly associated with the sperm count, abnormal morphology, sperm motility, or semen volume in either study group.