The Association of Human Cytomegalovirus Infection of Male Genital Tract and Male Infertility

Background: The human cytomegalovirus (HCMV) is a widely distributed virus among humans and is included within the sexually transmitted viruses. Because its low detection rate in semen, the impact of HCMV on sperm formation and its vertical transmission within the sperm cells has not been studied sufficiently. Objectives: To investigate the association of viral infections present in the lower genital tract of males and relationship among males infertility. Methods: ELISA and RT-PCR technique of 100 semen and serum specimens, collected over 8 months from males investigated for infertility, were retrospectively assessed, by screening anti-human Herpes Simplex Virus (HCMV) IgM and IgG in the serum and detection of HSV DNA in seminal fluid. Results: One hundred seminal fluid and blood samples were taken from men and investigated for infertility over a period of 8 months from July 2016 to February 2017. The seminal fluids and serum of patients mentioned to the laboratory from the fertility clinics of Kamal AL-samarai hospital and outer clinics. The investigations were conducted to find the HCMV detection rate in the seminal fluid of infertile men and to evaluate the impact of the viral infection on the major sperm parameters. Blood and seminal fluid were collected from 100 infertile males. The results were shown that 24/100 (24.0%) were positive for IgM and 86/100 (86.0%) were positive for IgG of HCMV. In case of control group, results were (1.0%) positive for anti-HCMV IgM antibodies, while (20.0%) positive for anti-HCMV IgG antibodies in control group, using ELISA test. The PCR showed that out of 100 samples 24 (24.0%) gave positive results for HCMV patients, while 7 (7.0%) positive for HCMV in control group. Conclusions: Using sensitive procedures for assays, seminal samples of asymptomatic infertile patients showed unexpectedly high incidence of the sexually transmitted pathogens. 1Introduction In the reproductive medicine field, the sexually transmitted infections (STIs) are of major concern to researchers and doctors. Many STI pathogens usually cause incurable and even fatal diseases, and have been shown to be transmitted via insemination procedures (1). Furthermore, many of these pathogenic microorganisms can be transmitted from infected mothers to their fetuses or children (2). Over time, men can also harbor the subclinical infection in their genital tract. Many viruses, such as cytomegalovirus (CMV) and herpes simplex virus type 2 (HSV-2) were detected in the seminal fluid of asymptomatic men (3). The importance of genital tract pathogens as causative factors of male infertility is still a controversial topics. This study aims to determine the prevalence of many commonly sexually transmitted pathogens in healthy and infertile male persons (4). Like other herpesviruses, after primary infection HCMV has the ability to establish latency in various different types of cells (5). HCMV infection may occur in individual without prior exposure to the virus (primary infection), or in those with previous exposure (recurrent infection) (6). Primary or recurrent infection (active infection) may be asymptomatic or cause mild or sever illness (7). HCMV circulate worldwide without seasonal variation and commonly infects people of all ages, races, and those from a variety of socioeconomic, cultural, and geographic Journal of University of Babylon for Pure and Applied Sciences (JUBAS) by University of Babylon is licened under a Creative Commons Attribution 4.0 International License. 2018.


1-Introduction
In the reproductive medicine field, the sexually transmitted infections (STIs) are of major concern to researchers and doctors. Many STI pathogens usually cause incurable and even fatal diseases, and have been shown to be transmitted via insemination procedures (1). Furthermore, many of these pathogenic microorganisms can be transmitted from infected mothers to their fetuses or children (2). Over time, men can also harbor the subclinical infection in their genital tract. Many viruses, such as cytomegalovirus (CMV) and herpes simplex virus type 2 (HSV-2) were detected in the seminal fluid of asymptomatic men (3). The importance of genital tract pathogens as causative factors of male infertility is still a controversial topics. This study aims to determine the prevalence of many commonly sexually transmitted pathogens in healthy and infertile male persons (4). Like other herpesviruses, after primary infection HCMV has the ability to establish latency in various different types of cells (5). HCMV infection may occur in individual without prior exposure to the virus (primary infection), or in those with previous exposure (recurrent infection) (6). Primary or recurrent infection (active infection) may be asymptomatic or cause mild or sever illness (7). HCMV circulate worldwide without seasonal variation and commonly infects people of all ages, races, and those from a variety of socioeconomic, cultural, and geographic backgrounds (8). Seroprevalence of HCMV in developing countries is higher than seroprevalence in developed countries (9). HCMV viruses have been isolated from oropharyngeal secretions, stool, urine, seminal fluid, blood, cervical & vaginal secretions as well as milk (10). The virus can be discharged in the seminal fluid before coming into contact with the sperms during its presence in the accessory glands. The presence of these viruses in the seminal fluid indicates a genital tract infection, and the seminal fluid can be a viral propagation vector (11,12). CMV is the leading cause (up to 10%) of neonatal deaths in Iraq (13), In the current study, HCMV was the target of investigation in order to elucidate any relationship between this virus infection of genital tract and idiopathic male infertility.

2-Materials and methods
One hundred seminal fluid and serum specimens from infertile men attending Kamal AL-Samarai hospital were investigated for infertility and 100 fertile men as a control group were collected over a period from July 2016 to February 2017 were analyzed. ELISA technique was used to measure anti-HCMV IgM and IgG antibodies ( Human, Gesellschaft for Biochemical and diagnostical mbH, Germany).
After (3)(4)(5) days of sexual abstinence, semen samples were collected in the laboratory by masturbation. The ejaculate was deposited in sterile wide mouth, screw capped, and plastic container. The seminal fluid was examined according to world health organization (WHO) 2010 criteria of seminal fluid. The ejaculate was deposited in s sterile wide mouth , screw capped , and plastic container. The seminal fluid examination included ejaculate pH, volume, viscosity and sperm concentration in (millions / ml), which have been determined by using the Makler Chamber, while sperm motility has been determined as motile spermatozoa percentage, and morphology as percentage of sperms with normal shapes. Measuring of the ejaculate volume was performed by placing the whole specimen in a graduated pipette, and measurement of the total sperm count was done by multiplying the ejaculate volume by the sperm concentration. To study the morphology of sperms, seminal smears were put on slides, and allowed for drying at room temperature, then stained with eosin and in accordance with WHO criteria, they were examined for normal or abnormal sperm morphology (14). Using a light microscope, motility of sperms was examined after putting a drop of semen on a slide and evaluation of 100 sperms in each specimen. Sperm motility was defined pursuant to the World Health Organization as the following: type 1 (fast sperms with straight forward motility); type 2 (fast sluggish moving sperms); type 3 (locally shaking sperms); and type 4 (immotile or non-motile sperms) (14).
The reference points of a normal seminal fluid were reported in accordance with the WHO criteria as: Sperm concentration (≥20 millions sperms/ml), morphology (≥30% sperms), motility (≥50% sperms with forward motility category a and b or ≥25% sperms with category a) (14).
The seminal fluid samples with abnormal results were examined on two different occasions at eight week intervals. The average of the two readings was estimated. All samples were tested for the presence of HCMV DNA by real-time PCR method (genetic PCR solutions TM, spain). Real time PCR instrument used in this work was STRATAGENE MxPro QPCR (Agilent Technologies, USA). The thermal protocol for HCMV dtec-qPCR Test F-100/F300 format Kit is composed of an initial denaturation for activation the HotStarTaq DNA Polymerase, amplification cycle and a terminal hold as shown in table (1). The real-time data is collected at the second step of the amplification cycle. Before starting a Real-Time PCR reaction, the following steps were completed: Choosing the correct filters (FAM( Identifying and labeling of unknown samples, standards, positiveand negative controls, and assigning the quantitative values for the standards.The correct thermal protocol selected. In order to avoid sample to sample contamination and PCR product carryover, standard precautions were taken. DNA was extracted from HMCV strain, and infected Vero cells were used as positive controls, while a mixture without DNA template served as a negative control.

3-Results
In the present study, the principal finding was the age, which ranged between 20-50 years.The highest prevalence of infection was age 26-30 years than other age classes. The lowest prevalence of infection was at age 45-50 year, statistical analysis showed there were significant difference (P value=0.043) in distribution of genital tract infections among age groups as shown in table (2). No similar seropositivite results regarding anti-CMV IgM and IgG antibodies were found in both patient men and controls. There was a significant association (p<0.05) between infertility and the high levels of anti-CMV IgG and IgM antibodies, as shown in tables (3) and (4).
The current study revealed that the seroprevalence of anti-CMV IgM and IgG was 24 (24.0%) and 86 (86%) antibodies in infertile patients respectively, tables 1 and 2, and was compared with fertile control antibody 1 (1.0%) for IgM and 20 (20.0%) for IgG. The relative risk (RR) for having male infertility was 2.21 and 5.45 for positive HCMV IgM and IgG, respectively (P<0.05).

4-Discussion
Studies suggest that reduced semen volume, sperm motility and/or sperm morphology are all associated with individual's age, while sperm concentration is less affected (15)(16). Many recent studies also reveal that sperm quality in men decreases with age (17,18,19). Several study results support the finding that sperm motility decreases with advancing age (20,21).Our study also found a significant reduction in sperm motility with aging.
The presence and persistence of Cytomegalo virus (CMV) in seminal fluid samples, which is a herpesvirus family member, has been always reported previously (22). In this study, HCMV seroprevalence and HCMV infection of semen was all significantly associated with male infertility. However, a lower level of CMV IgM was detected in serum than IgG reflecting a logical data showing recent infections of the tested individuals. No significant relationship between CMV DNA and seminal parameters was revealed in this study, although a trend for low motile sperms and αglucosidase levels was seen in the CMV-infected study group, which may indicate a modest impact of CMV infection on seminal quality, probably by affecting the function of epididymis. No association between CMV infection and a reduction in the seminal fluid parameters was reported in the previous studies (23). Our study is consistent with (24).HCMV may be infectious in the seminal fluid (25), and the virus has also been isolated from endometrial cells of humans (26), indicating a probable direct infection mechanism of endometrial cells by HCMV which is carried by sperms. Moreover, active HCMV infections may be seen in human endometrial stromal cells, (27). The anti-CMV IgG antibody prevalence, which is reported in this study, has been also found consistent with other studies reported in Turkey and other developing countries. CMV seroprevalence has been recorded to be (84.3%) in patients from Nigeria, Afyon and Lagos. (28) found a 84.2% seroprevalence in infertile men.
Results of the current study disagreed with (29) who revealed 96.0% of seroprevalence of CMV IgG. In Northern Turkey, (30) reported a 1.0% and 97.3% seropositivity for anti-CMV IgM and anti-CMV IgG antibodies respectively. Seroprevalence of CMV has been also demonstrated to be age dependent in consistency with the study of (31) , he recorded that 58.7% of persons aged ≥ 80 years had positive CMV infection. An association was detected between the high CMV concentrations in ejaculate and a transient decrease in the sperm motility, and a reduction in the sperm cell concentration in seminal fluid samples of CMV infected men has been also found (32). However, no effect of CMV infection on the quality of sperms has been detected by most researchers (33).
Previously, different and contradictory studies reported the following results: 0% (culture and hybridization) among 60 males, including 30 CMV positive versus 32.7% (PCR) among 217 males from a 92% seropositive population who were referred for infertility (34).
Unlike (35), who found no positive specimens in seronegative patients, our results found that about 1.4% of initially seronegative people were HCMV DNA positive when the ejaculate PCR was used. This result was in agreement with that of (36), who indicated that several PCR positive results were reported in the seminal samples of seronegative males, some of whom had probable seroconvertion, while other men showed persistent seronegative results for several months. Similar results have been detected in other types of tissues (37). On the other hand, it has been shown by Eggert et al that the genital tract CMV infection in sub-fertile people is considerable, and seems to have a little role in infertility (38).
Another study by (22) reported the detection of DNA of sexually transmitted pathogen in the semen of large numbers of asymptomatic infertile men, which has been correlated with poor quality of seminal fluid (39). We may attribute this high prevalence of HCMV to the poor socioeconomic condition, poor hygienic practices and poor living conditions (40).

CONFLICT OF INTERESTS
There are no conflicts of interest.