To investigate the effect of human papillomavirus (HPV) and Chlamydia trachomatis (Ct) co-infection on sperm concentration, motility and morphology, in a large cohort of young heterosexual male patients with chronic prostatitis-related symptoms.
Patients and Methods
Patients with chronic prostatitis-related symptoms, attending the same centre for sexually transmitted diseases from January 2005 and December 2010, were consecutively enrolled in this cross-sectional study.
All patients underwent clinical and instrumental examination, microbiological cultures for common bacteria, DNA extraction, mucosal and serum antibodies evaluation for Ct, specific tests for HPV and semen analysis. The semen variables analysed were: volume; pH; sperm concentration; motility; and morphology.
Subjects were subdivided in two groups: group A, patients with Ct infection alone and group B, patients with Ct and HPV co-infection.
The main outcome measurement was the effect of Ct and HPV co-infection on the semen variables examined.
Of 3050 screened patients, 1003 were enrolled (32.9%) in the study. A total of 716 (71.3%) patients were allocated to group A, and 287 (28.7%) to group B.
Significant differences between the two groups were reported in terms of percentage of motile sperm (degrees of freedom [df] = 1001; t-test = 11.85; P < 0.001) and percentage of normal morphological forms (df = 1001; t-test = 7.18; P < 0.001), while no differences were reported in terms of semen volume or pH.
According to World Health Organization thresholds for normal semen, 364 (50.8%) men in group A and 192 (66.8%) men in group B were subfertile (odds ratio = 1.95; 95% confidence interval 1.46–2.60; P < 0.001).
No correlation between HPV genotype, mucosal IgA type and semen variables was found.
In a population of prostatitis-related symptoms attributable to Ct infection, co-infection with HPV has a significant role in decreasing male fertility, in particular with regard to sperm motility and morphology.
Human papillomavirus (HPV), and Chlamydia trachomatis (Ct) infections are amongst the most common sexually transmitted infections worldwide, causing considerable morbidity and socio-economic problems [1-3]. In particular, Ct is a well-established cause of symptomatic infection in the lower male genital tract and also has a significant impact on male fertility [3, 4]. In fact, Mazzoli et al.  recently showed the negative impact of chronic prostatitis resulting from Ct on male fertility, highlighting suspected immunomediated damage to germinal cells. In addition, transmission-electron microscopy has shown that Ct serovars D, H and I all attach to human spermatozoa in vitro, with a subsequent increase in DNA damage [5, 6]. In addition, HPV has previously been found to be present in semen, although the studies involved did not suggest a relationship with male infertility [7, 8]. Some authors, however, have hypothesized that HPV has a negative role in male fertility . Foresta et al.  showed a significant reduction in mean sperm motility in HPV-infected sperm samples, suggesting a role for HPV infection in idiopathic male factor infertility.
The aim of the present study was to investigate the effect of HPV and Ct co-infection on sperm concentration, motility and morphology in a large cohort of young heterosexual male patients with chronic prostatitis-related symptoms.
Materials and Methods
To evaluate the impact of Ct and HPV co-infection on male fertility compared with that of Ct infection alone, consecutive patients with a diagnosis of chronic prostatitis-related symptoms, attending a single centre for sexually transmitted diseases (STDs), between January 2005 and December 2010, were prospectively screened for this cross-sectional study, using microbiological evaluation, laboratory tests and clinical evaluation as described below.
Inclusion and Exclusion Criteria
Inclusion criteria were the presence of chronic prostatitis-related symptoms lasting for >6 months, in accordance with European Association of Urology guidelines ; men were categorized as having prostatitis-like symptoms if they reported perineal or ejaculatory pain, and had a pain score of ≥4 [12, 13]. Subjects were excluded if they: were aged <18 and >45 years; were affected by major concomitant diseases, endocrine disorders or obesity (body mass index ≥30 kg/m2); had known anatomical abnormalities of the urinary tract, had clinically and/or instrumentally demonstrated diagnosis of varicocele; had evidence of other urological diseases; had been diagnosed with genital or anal warts; were participating in an HPV vaccine study; or were homosexual. Patients positive for urothelial carcinoma at cytological urine analysis or who had previously undergone prostate surgery were also excluded. In addition, all patients who received antibiotics, NSAIDS or steroids in the 12 weeks before the study were excluded; we chose this long period to exclude any negative effects of antibiotics on semen quality. Furthermore, all patients with a positive Meares–Stamey test for common bacteria, even if in association with Ct infection, were excluded. The final study cohort comprised all patients with demonstrated Ct infection, with or without HPV co-infection, who met the inclusion criteria.
Study Schedule and Sample Collection
The local research ethical committee approved the present study. Written informed consent was obtained from all patients before enrolment. The study was conducted in line with Good Clinical Practice guidelines and the STROBE statement (http://www.strobe-statement.org). All patients agreed to donate blood and semen samples. Each patient underwent microbiological cultures for common bacteria and yeasts, DNA extraction and mucosal IgA evaluation for Ct diagnosis. All patients underwent a Meares–Stamey test, performed according to European Association of Urology guidelines using a four-glass test, with total ejaculate collection [11, 14]. Then, from each patient, genital samples were collected in accordance with the indications described by Mazzoli et al. : first-void early-morning urine (VB1), mid-stream urine (VB2), expressed prostatic secretion (EPS), post-prostate massage urine (VB3) and total ejaculate (TE). To exclude all patients with urethritis attributable to Ct or HPV infections, each patient underwent a urethral swab. The four-glass test indicates bacterial prostatitis only if the bacterial load in EPS or in VB3 is at least 1000 colony-forming units/mL and at least 10 times higher than in VB1 and VB2. The Meares–Stamey test was considered to be positive where a significant number of bacteria in EPS and/or VB3 was found to be present. All samples were collected and immediately processed to the laboratory under refrigerated conditions, analysed for cultures and aliquot for DNA extraction and PCR as described by Mazzoli et al.  and Cai et al. . In accordance with Nickel et al.  the white blood cell counts in all biological samples were obtained but not considered in the results analysis of the present study because no study has shown any correlation between WBC and human fertility . Each patient also underwent serum IgA and IgG anti-Ct analysis. The blood sample was collected on arrival at the STD centre. All enrolled subjects were further evaluated in two groups: group A, patients with prostatitis-related symptoms attributable to Ct infection; group B, patients with prostatitis-related symptoms attributable to Ct and HPV co-infection. Data from microbiological and laboratory analysis were correlated with data from semen evaluation and compared between the groups. The group A to group B ratio was ∼3:1. Data on the patients' treatment were not described, because this was a cohort rather than an interventional study.
Microbiological culture was carried out in accordance with the methods described by Motrich et al. . The DNA extraction and purification of all biological materials was performed using a DNeasy® Tissue Kit (Qiagen, Milan, Italy): 200 μL of pellet was pre-incubated overnight by proteinase k and the day after extracted and purified in agreement with the manufacturer's instructions. The presence of genital HPV was investigated in all the biological materials of the whole study population using Alpha Watch HPV (Alphagenic-Diaco-Biotechnology, Trieste, Italy). The biological materials from our patients were analysed using Inno-Lipa HPV Genotyping Extra (Innogenetics, Rome, Italy). Amplification of a fragment of the β-globin gene served as an internal quality control for each specimen. We classified the genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 as high-risk-HPV (HR-HPV), genotypes 26, 53 and 66 as probable-high-risk HPV (PHR-HPV), and genotypes 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 as low-risk HPV (LR-HPV) . Samples that were positive for HPV but that did not hybridize with any of the type-specific probes were classified as positive non-genotypable HPV (PNG-HPV). This STDs laboratory is registered by the UK National External Quality Assessment Service (NEQAS) for microbiology. All microbiological analyses were performed blindly.
Anti-Ct Antibodies Analysis (Western Blot Analysis)
Serum IgA, IgG and TE, EPS and VB3 IgA analyses were performed using the methods described previously . We quantitatively assigned a value with ‘plus’ (e.g. from 1+, 2+, to 20+) to define the level of mucosal IgA found in all samples. The ‘plus’ value was dependent on the colour intensity of intracellular inclusion caused by Ct present in the infected fibroblasts, which was used as an antigen in the method and evaluated using a microscopic reading (400X) . The blotted Ct proteins were: lipopolysaccharide (two fractions), MOMP 1 (two fractions), MOMP 2, 29 kD, 45 kD, 80 kD, HSP60 and HSP70 proteins .
Semen samples were collected in a sterile container via masturbation after 2–7 days of ejaculatory abstinence. Semen collection and analysis were performed in the same laboratory according to WHO 2010 . All semen analyses were reviewed using the 2010 WHO criteria, even though the study started in 2005. Data from semen analysis were collected from each patient. The semen variables considered in this study were: semen volume (mL), pH, sperm concentration (×106/mL), percentage of motile sperm and percentage of normal morphological forms. Sperm concentration was determined using haemocytometer chambers, in accordance with the WHO 2010 guidelines . These guidelines stipulate that, to minimize the sampling error and to obtain a maximum standard error of 7.1%, a minimum of 200 sperm per examination must be counted.
Questionnaires and Urological Examinations
The validated Italian versions of the National Institutes of Health Chronic Prostatitis Symptoms Index (NIH-CPSI)  and the IPSS  were administered to each patient.
Pearson's coefficient and Spearmen's test were used to evaluate the relationship between the presence of Ct/HPV infection and each semen variable. The differences between the groups in terms of semen analysis results were obtained by using a chi-squared or Fischer's exact tests. For the t-test (t) we reported data with the degrees of freedom (df) to evaluate the real value of P. An anova test was used to compare the mean IgA secretory levels between the two groups. Multivariable Cox proportional hazards regression analysis was then performed to determine the association between Ct and HPV co-infection and semen variables. Odds ratios (ORs) and 95% CIs were calculated to determine the significance of differences . The anova test was also used for univariate analysis and the log-rank test (Mantel–Cox) for multivariate analysis. The effect size between the means (Cohen's d) was also calculated. The variables considered for univariate and multivariate analysis were as follows: age, education level, sexual behaviour, contraceptive use, Ct positivity, HPV positivity and Ct co-infection. The number of patients and control subjects who presented at our STD centre during the study period determined the sample size. A P value <0.05 was considered to indicate statistical significance. All reported P values were two-sided. All data were prospectively collected and recorded in a dedicated database, the Advanced PROSTATitis DataBase (Microsoft Access format). The original database had been planned to evaluate the impact of chronic prostatitis attributable to Ct or other infections on male fertility. The same population was evaluated in a previous study  and the present study represents a secondary analysis of that previously published trial.
A total of 3315 patients with chronic prostatitis-related symptoms were pre-screened, of whom 3050 patients were further screened. Of the 265 patients excluded from the study, 87 did not wish to be enrolled, 75 did not complete the biological sample collection and 103 did not wish to donate total ejaculate. Of 3050 screened patients, 1003 were positive for IgA anti-Ct and/or singular Ct-DNA amplifications and were enrolled in the study (Fig. 1).
Data on demographic and socio-economic variables, medical history and clinical variables at enrolment are shown in Table 1. Patients reported a mean (range) symptom time of 19.3 (14–37) months. A total of 327 patients in group A (45.6%) and 119 in group B (41.4%) reported pain during ejaculation. The mean (sd) baseline questionnaire scores were 19.8 (6.2) and 16.1 (3.3) for NIH-CPSI and IPSS, respectively. A total of 716 patients (71.3%) were negative for HPV-DNA amplification and, thus, were assigned to group A, while 287 patients (28.7%) were positive for HPV-DNA amplifications and were assigned to group B. All microbiological results are shown in Table 1.
Table 1. Patient's sociodemographic anamnestic and clinical characteristics at time of enrolment (N = 1003)
No. of patients
Median (sd) age, years
Ct plasmidic DNA positive, n (%)
Ct secretory IgA positive, n (%)
Both Ct plasmidic DNA and secretory IgA positivities, n (%)
Ct serum IgA positive, n (%)
Ct serum IgG positive, n (%)
Both Ct serum IgA and IgG positive, n (%)
HPV DNA positive, n (%)
Educational level, n (%)
Sexually active in the past month
Sexual behaviour, n/N (%)
Contraceptive use, n/N (%)
Clinical presentation, n (%)
Dysuria + Frequency
Pain, n (%)
Chronic prostatitis symptom history, months
19.1 ± 9.7
18.9 ± 9.2
Mean (sd) symptoms score at baseline
All 716 patients in group A were positive to Ct-DNA amplification. Of these, 423 (59.1%) had detectable levels of mucosal IgA anti-Ct. Mucosal IgA levels ranged from 7+ to 20+, with a mean value of 14.30+. All patients were positive for the MOMP1 protein. A total of 297 of 423 patients (70.2%) were positive for the high-molecular-weight proteins (HSP60, HSP70; 80 kDa), while 174 patients (41.1%) were positive for anti-MOMP2 IgA. Serum IgA and IgG were positive in 28 and 52% of patients, respectively.
All 287 patients in group B had positive Ct-DNA amplification results while 119 (41.4%) had detectable levels of mucosal IgA anti–Ct. Mucosal IgA levels ranged from 3+ to 20+, with a mean value of 15.25+. All patients were positive for the MOMP1 protein. Of the 287 patients, 201 (70%) were positive for high-molecular-weight proteins (HSP60, HSP70; 80 kDa), while anti-MOMP2 IgA was positive in 103 patients (35.9%). Serum IgA and IgG were positive in 31 and 51% of patients, respectively. The HPV genotype distribution was as follows: HR-HPV, 122 patients (42.5%); PHR-HPV, 14 patients (4.9%); LR-HPV, 35 patients (12.2%); and PNG-HPV, 116 patients (40.4%). The most common HPV genotypes were: 6, 11, 16, 26, 51, 53, and 81. The prevalence of all HPV genotypes is shown in Table 2.
Table 2. Prevalence of HPV genotypes among patients in group B
HPV genotype distribution according to Munoz classification
HPV genotype distribution
HPV 16 + 18
HPV 6 + 11
HPV 16 + 31
HPV 16 + 11 + 18
The results from semen analysis are shown in Table 3. There was no difference between the groups in terms of volume and pH, but sperm motility (df = 1001; t = 11.85; P < 0.001), and normal morphology (df = 1001; t = 7.18; P < 0.001) were significantly lower in group B than in group A (Fig. 2). According to WHO 2010 thresholds for normal semen, 298 (41.6%) men in group A and 192 (66.8%) men in group B were subfertile (OR = 1.95; 95% CI 1.46–2.60; P < 0.001). Multivariate analysis identified being positive for HPV and Ct co-infection markers as independent prognostic factors (P = 0.001) for subfertility (Table 4).
Table 3. Semen variable analysis stratified by group
Group A, mean (sd)
Group B, mean (sd)
Sperm concentration, ×106/mL
Motility, % sperm
Morphology, % sperm
Table 4. Univariate and multivariate analysis results of factors negatively affecting fertility
Positivity for Ct
Positivity for Ct and HPV co-infection
A strong correlation between positivity for HSP60, HSP70 and percentage of normal morphological forms (OR = 1.31; 95% CI 1.16–1.85; P < 0.001) in both groups was found. No differences were found between patients with only HSP60 and those with only HSP70 positivity, probably because of the low percentage of patients who were only positive for high-molecular-weight proteins.
In a subanalysis of patients positive for HPV, no HPV genotype was found to have a significant negative impact on male fertility. There was no correlation between HPV genotype and HSP60 and/or HSP70 positivity, nor was there any correlation between HPV genotypes, serum IgA and/or IgG and the other semen variables, or between mucosal anti-Ct IgA levels and HPV genotypes.
A high incidence of HPV infection has been reported in sperm from sexually active men, with and without risk factors for HPV, and from infertile patients . In addition, semen infection is associated with an impairment of sperm quality, suggesting a possible role in male infertility . HPV co-infection with other STDs, such as Neisseria gonorrhoeae, Ct, Mycoplasmata and Ureaplasma has been described but no data on the impact of HPV and Ct co-infection on male fertility has been reported . We chose a cohort of young heterosexual males with chronic prostatitis-related symptoms attributable to Ct infection for the following reasons: (i) Ct infection is one of the most common STDs ; (ii) chronic prostatitis-related symptoms have been established as an important socio-economic problem; and (iii) chronic prostatitis-related symptoms resulting from Ct infection not only reduces quality of life but also seems to have a significant impact on couples' reproductive health . We found that patients with HPV and Ct co-infection had lower percentages of motile sperm and normal morphological forms when compared with those infected with Ct only. Moreover, the HPV infection was an independent risk factor for subfertile status when associated with Ct infection. This study highlights two important issues. Of 1003 patients, 287 (28.6%) were positive for HPV in one or more genital samples, which represents a high prevalence of HPV among heterosexual patients with chronic prostatitis-related symptoms attributable to Ct infection. Several authors have reported the prevalence of HPV in specific cohorts of men such as homosexual men [26, 27], but the prevalence of HPV in young heterosexual men, not participating in an HPV vaccine study, has not been well studied up to now. Foresta et al.  investigated the presence of HPV DNA sequences in semen samples of asymptomatic young adult men who had unprotected intercourse, and reported the presence of HPV semen infection in 10% of these men. Moreover, they reported that a high incidence of HPV infection in sperm was found not only in individuals with risk factors but also in asymptomatic infertile men . Recently, Vardas et al.  reported that genital HPV DNA is commonly found in heterosexual men, with a prevalence of infection of 21%. The higher prevalence in the present study is probably attributable to the fact that we analysed a specific population of patients affected by another STD, who therefore had a higher risk of coming into contact with HPV, and also to possible variation in the laboratory methods used. Secondarily, the negative role of HPV co-infection in male fertility when associated with Ct infection has not been addressed before. Foresta et al. , by using fluorescence in situ hybridization for HPV, found that the virus was localized at the sperm head . Pérez-Andino et al.  also found 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 also suggested the presence of glycosaminoglycans, 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 . Finally, Foresta et al.  clearly showed that the HPV capsid protein L1 and the glycosaminoglycan syndecan-1 co-localize in the equatorial region of the sperm head, suggesting that HPV infects sperm by the primary attachment with syndecan-1. The binding of HPV to spermatozoa might therefore reduce motility, raising the hypothesis that HPV sperm infection may also reduce fertility . In the present study, we found that patients with HPV and Ct co-infection showed a reduction in sperm motility, which was not dependent on the different HPV genotypes, which is consistent with the study by Foresta et al. . We also found that patients with HPV co-infection showed a higher frequency of abnormal forms. Bezold et al.  found that the presence of HPV DNA was associated with a significant decrease in total sperm count, and a nonsignificant trend for a lower total motile sperm count. The correlation between HPV infection and its negative impact on sperm morphology is not well understood, but we can hypothesize that it could be the result of the ability of HPV to bind to the spermatozoa head, as has been shown for Ct infection .
The present study has some limitations that should be taken into account when interpretating the results, e.g. the absence of semen biochemical indices, sperm capacitation in vitro, and the limited number of spermiograms for each patient; however, even though it is well known that assessing a limited number of sperm variables is subject to relatively large individual variations and these variables are somewhat limited in their ability to accurately predict fertility, we think that the differences observed in the present study between the two groups in terms of fertility are attributable to the HPV and Ct co-infections and are not mere statistical effects. In addition, the fact that all patients were from the same country may be a limitation in that we did not consider the effect of migration on the variation in HPV distribution.
In conclusion, in a population with chronic prostatitis-related symptoms attributable to Ct infection, co-infection with HPV has a significant negative impact on male fertility, in particular in terms of reducing sperm motility and normal morphology. This highlights the possibility of direct damage to germinal cells caused by HPV infection.
We are grateful to the staff of the STC Centre at the Santa Maria Annunziata Hospital for their technical laboratory assistance and to Prof. John Denton for language revision.
Conflict of Interest
sexually transmitted disease
degrees of freedom
first-void early-morning urine
expressed prostatic secretion
post-prostate massage urine
positive non-genotypable HPV
National Institutes of Health Chronic Prostatitis Symptoms Index