Alessandro Bertoldo

Mechanism of Human Papillomavirus Binding to Human Spermatozoa and Fertilizing Ability of Infected Spermatozoa

Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases in females and males. Precise data about the presence, mechanism of infection and clinical significance of HPV in the male reproductive tract and especially in sperm are not available. Here we show that HPV can infect human sperm, it localizes at the equatorial region of sperm head through interaction between the HPV capsid protein L1 and syndecan-1. Sperm transfected with HPV E6/E7 genes and sperm exposed to HPV L1 capsid protein are capable to penetrate the oocyte and transfer the virus into oocytes, in which viral genes are then activated and transcribed. These data show that sperm might function as vectors for HPV transfer into the oocytes, and open new perspectives on the role of HPV infection in males and are particularly intriguing in relation to assisted reproduction techniques.

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

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.

Human papillomavirus sperm infection and assisted reproduction: a dangerous hazard with a possible safe solution

BACKGROUND Human papillomavirus (HPV) infection has been demonstrated in the sperm of a large percentage of sexually active males and is associated with an impairment of sperm parameters, with a particular negative impact on sperm motility, suggesting a possible role in male infertility. Conventional sperm selection techniques have a low efficiency in removing HPV. METHODS Evaluation of sperm parameters, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling test to evaluate DNA fragmentation and fluorescence in situ hybridization or immunohistochemistry for HPV were performed on semen samples from infected patients (n= 22), control subjects (n= 13) and on pooled control sperm samples incubated with HPV16-L1 (HPV capsid), before and after direct swim-up and modified swim-up (with added Heparinase-III). Moreover, cytofluorimetry for HPV detection was performed in pooled sperm pre- and post-incubation with HPV 16-L1 before and after direct and modified swim-up. Statistical analysis was performed with a two-tailed Students t-test. RESULTS Direct swim-up reduces the number of HPV-infected sperm by ~24% (P< 0.01), while modified swim-up is able to remove completely HPV DNA both from naturally and artificially infected sperm. Enzymatic treatment with Heparinase-III tended to decrease sperm motility, viability and DNA integrity but the effects were not significant. CONCLUSIONS This study shows that Heparinase-III treatment seems not to affect spermatozoa in vitro and suggests that this treatment should be investigated further as a means of preparing sperm from patients who are infected with HPV in order to reduce the risk of HPV infection when using assisted reproduction techniques.

Treatment of polycystic ovary syndrome with spironolactone plus licorice

OBJECTIVE The aim of the study was to compare the effect of spironolactone (antagonist of mineralocorticoid and androgen receptors) versus spironolactone plus licorice (agonist of mineralocorticoid receptors and mild inhibitor of androgen synthesis) on plasma renin activity, aldosterone and androgen levels in women with polycystic ovary syndrome (PCOS). STUDY DESIGN Thirty-two women with PCOS were divided into two groups: 16 received 100 mg spironolactone and 16 spironolactone plus 3.5 g of licorice a day. Blood pressure, body mass index, serum electrolytes, plasma renin activity, plasma aldosterone and cortisol, serum testosterone, and urinary tetrahydrocortisol/tetrahydrocortisone ratio were measured before and during treatment. RESULTS Mean blood pressure was significantly reduced during spironolactone treatment, while it was unchanged in women receiving spironolactone plus licorice. Twenty percent of women treated with spironolactone and none treated with the addition of licorice complained of symptoms related to volume depletion. Consistently, the activation of the renin-aldosterone system was significantly lower during spironolactone plus licorice than with spironolactone alone. The prevalence of metrorrhagia was lower in the combined therapy. CONCLUSIONS In patients with PCOS the mineralocorticoid properties of licorice can reduce the prevalence of side effects related to the diuretic activity of spironolactone.

Influence of tumor necrosis factor α inhibitors on testicular function and semen in spondyloarthritis patients

OBJECTIVE To evaluate sperm parameters and sexual hormones in young males affected with spondyloarthritis (SpA) before and after 1 year of anti-tumor necrosis factor (TNF) α treatment. DESIGN Prospective case-control study. SETTING Rheumatology and human reproduction pathology units at a university hospital. PATIENTS Ten SpA outpatients attending the rheumatology clinic; 20 healthy control subjects attending the unit of human reproduction pathology within an infertility prevention program. INTERVENTIONS At baseline and after a 12-month treatment, disease activity was assessed and an andrologic evaluation made. MAIN OUTCOME MEASURE(S) Rheumatologists assessed anamnestic, clinical, functional, and biomarker data. Andrologists evaluated semen analysis, fluorescence in situ hybridization for chromosomes X, Y, 13, 18, and 21, FSH, LH, and T plasma levels, and testicular color Doppler ultrasound. RESULTS At baseline, SpA patients showed reduced sperm motility, higher plasma LH and FSH, and lower T levels compared with control subjects; a significant correlation between disease activity and sperm quality was found. After treatment, a statistically significant decrease in sperm aneuploidies and normal hormone levels were observed. CONCLUSIONS Although inflammation in SpA appears to be related to impaired testicular function, anti-TNF-α agents seem to be safe on testicular function and fertility.

Association, prevalence, and clearance of human papillomavirus and antisperm antibodies in infected semen samples from infertile patients.

OBJECTIVE To evaluate prevalence, association, and clearance of human papillomavirus (HPV) and antisperm antibodies (ASAs) in infected semen samples from infertile patients. DESIGN Cross-sectional clinical study. SETTING Andrology and microbiology sections at a university hospital. PATIENT(S) Three groups of subjects: 61 infertile patients with HPV semen infection, 104 noninfected infertile subjects, and 92 control subjects. INTERVENTION(S) Semen analysis, spermMar test, fluorescence in situ hybridization for sperm aneuploidy and for HPV, and immunofluorescence for HPV 16-L1 and immunoglobulins (IgA, IgG, and IgM) determination. MAIN OUTCOME MEASURE(S) Association of sperm procedures, HPV sperm infection, sperm aneuploidies, and sperm ASAs. RESULT(S) Infertile patients with HPV semen infection showed high percentages of ASAs. In these patients HPV sperm infection was associated with lower sperm motility, which was worse in subjects with ASAs. No alterations of sperm chromosomes were observed. To obtain a significant clearance of both HPV sperm infection and ASAs at least 24 months of follow-up were needed. CONCLUSION(S) Human papillomavirus has been recently suggested to have an important role in male infertility. This study demonstrated that HPV sperm infection can be long lasting and frequently associated with ASAs that may further reduce male fertility. Infertile patients with positive spermMar test results should be considered for investigation for HPV, especially if they are candidates for assisted reproduction.

Human papilloma virus in the sperm cryobank: an emerging problem?

Human papilloma virus (HPV) infection is very common worldwide, but the actual incidence and significance of HPV infection in sperm are poorly understood. In this study, we evaluated the presence of HPV in spermatozoa from thawed semen samples previously stored in our sperm bank. We performed polymerase chain reaction and in situ hybridization for HPV detection in cryovials belonging to 98 oncology patients and in 60 semen samples from healthy controls. Statistical analysis was performed by two-tailed Students t-test and Fishers exact test. The frequency of HPV semen infection was 6.1% in thawed cryovials from patients and 3.3% in semen samples from controls. Among the patients, four were found positive for high-risk HPV, one for medium-risk HPV and another for low-risk HPV. Patients had a significantly higher percentage of infected sperm than controls. In conclusion, this report shows the presence of HPV in sperm cells from cryovials of a sperm bank. It is still unclear if HPV-infected sperm are able to cross-contaminate cryovials and impair the outcome of assisted reproduction techniques or to infect partners. Further studies are needed to understand whether screening for HPV should be performed in all semen samples before sperm banking or before intra-cytoplasmic sperm injection procedures.

Twenty-four-hour monitoring of scrotal temperature in obese men and men with a varicocele as a mirror of spermatogenic function

STUDY QUESTION How do day and night scrotal temperatures, spermatogenesis parameters, sex hormones and intratesticular perfusion in obese men and men with a varicocele compare with healthy controls? SUMMARY ANSWER Compared with healthy controls, 24-h monitoring of scrotal temperature in men with a varicocele and obese men showed higher temperatures and this condition was related to a significant alteration of spermatogenesis and stasis of testicular perfusion. WHAT IS KNOWN ALREADY Several studies have shown that increased scrotal temperature has dramatic effects on spermatogenesis. Scrotal hyperthermia by exposure to sauna is able to induce a significant alteration of sperm production. STUDY DESIGN, SIZE AND DURATION In a case-control study, data were collected over a period of 2 years from 60 subjects with risk factors for testicular heating and 20 healthy subjects who consecutively attended an andrology unit as participants in an infertility prevention program. PARTICIPANTS/MATERIALS, SETTING AND METHODS Forty subjects with a left varicocele, 20 obese men and 20 healthy subjects who served as controls, were evaluated for testicular volumes, sex hormones, sperm parameters, sperm aneuploidies, mean transit time (MTT) of intratesticular blood and 24-h scrotal temperature monitoring by a cutaneous thermochip. Subjects with a varicocele were further subgrouped on the basis of normo or oligozoospermia (VN and VO). Students t-test was used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE We found a significant increase in 24-h mean scrotal temperature in obese men and men with a varicocele compared with controls (both P < 0.01). This increase in scrotal temperature was associated with impaired sperm parameters and higher FSH plasma levels compared with controls. Dynamic evaluation of scrotal temperatures showed wide fluctuations in controls, but little variation in obese men and men with a varicocele. Men with VO had left and right increase in scrotal temperatures (the right was increased also versus VN, P < 0.01) (both P < 0.001). Men with VN showed a left scrotal temperature higher than controls (P < 0.01) and a right scrotal temperature no different from controls (34.92 ± 0.53 and 34.66 ± 0.65, respectively). Mean MTT values recorded in men with VO were significantly higher than men with VN and obese men (both P < 0.001). LIMITATIONS AND REASONS FOR CAUTION Different lifestyle, diet, occupation, stress level and environmental temperatures due to seasonal conditions are major limitations of this study. WIDER IMPLICATIONS OF THE FINDINGS Our data suggested for the first time that dynamic evaluation of scrotal temperatures seems to reflect alterations of testicular function and perfusion in obese men and men with a varicocele. In these clinical conditions, spermatogenic impairment and scrotal heating seem to be related to different mechanisms. The dynamic evaluation of scrotal temperature in subjects with risk factors for testicular heating could allow the identification of subjects needing treatment or a change in lifestyle. STUDY FUNDING/COMPETING INTERESTS No external funding was sought for this study, and the authors have no conflict of interest to declare.

Human papillomavirus proteins are found in peripheral blood and semen Cd20+ and Cd56+ cells during Hpv-16 semen infection

BackgroundHuman papillomavirus (HPV) currently represents an important risk factor for cancer development and infertility in humans. Whilst binding of HPV to spermatozoa has been associated with male infertility, an investigation about the presence of HPV-DNA in non-spermatozoal semen cells is lacking. Previous findings documented the presence of HPV in peripheral blood leukocytes. The aim of this study was to investigate the expression of HPV markers in semen and blood leukocytes during HPV-16 infection.MethodsA total of 32 subjects, 16 patients affected by HPV-16 semen infection and 16 controls, were evaluated in our andrological centre and enrolled in the study. Semen non-spermatozoal cells from all subjects were isolated and evaluated for the expression of HPV-16 markers (DNA and L1, E6 proteins) and further characterized for their molecular phenotype. Analogue determination was performed on peripheral blood mononuclear cells.ResultsThe presence of HPV-DNA by FISH analysis in a round cell population from semen, confirmed to be CD45+ leukocytes, was observed. These HPV-DNA containing-cells also displayed HPV-16-E6 and HPV-16-L1 viral proteins and, upon further investigation, were found to be CD20+ and CD56+, likely phenotypes of B cells and natural killer cells (NK) respectively. In 25% of the patient group, a very small population of peripheral blood mononuclear cells was found to be positive for HPV-DNA via FISH. These cells displayed the CD20+ and CD56+ phenotype alike. None of the control subjects displayed HPV-DNA in either semen or peripheral blood.ConclusionConsidering the role of CD20+ and CD56+ cell populations in the antiviral immune response, the detection of HPV markers on leukocytes may reflect the presence of virus particles within the endosomal compartment. However, the presence of HPV markers in circulating mononuclear cells raise concerns about the risk of developing cancers to distal organs.

Semen washing procedures do not eliminate human papilloma virus sperm infection in infertile patients

OBJECTIVE To determine the effectiveness of three sperm washing protocols for removing human papillomavirus (HPV)-infected cells from semen samples of infertile patients. DESIGN Cross-sectional clinical study. SETTING Andrology and microbiology sections at a university hospital. PATIENT(S) A group of 32 infertile patients positive for semen HPV, detected with polymerase chain reaction (PCR) and in-situ hybridization in sperm and exfoliated cells. INTERVENTION(S) Semen analysis and in-situ hybridization for HPV detection were performed before and after sperm washing, discontinuous Ficoll gradients, and swim-up protocols. Statistical analysis was performed with a two-tailed Students t-test. MAIN OUTCOME MEASURE(S) Evaluation of sperm parameters and presence of HPV, performed in semen samples before and after procedures of sperm selection. RESULT(S) All native samples showed the presence of infected sperm with a mean percentage of positivity (24.7% ± 8.9%) higher than exfoliated cells (13.8% ± 4.3%). Fifteen samples had HPV DNA on sperm and exfoliated cells. Sperm washing centrifugation showed no changes in the number of infected samples and in the percentage of infected cells. Ficoll and swim-up protocols induced a slight reduction in the number of infected samples (30 and 26, respectively). CONCLUSION(S) This study demonstrated that conventional sperm selection rarely eliminates HPV sperm infection. More attention should be paid to the reproductive health of infected patients because, not only can HPV be transmitted, but it may also have a negative effect on development of the fetus.