Human papillomaviruses (HPV) may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive HPV array. To examine whether HPV was associated with the sperm, in situ hybridization were performed with HPV-6, HPV-16 and -18, and HPV-31-specific probes. The prevalence of HPV-positive sperm donors was 16.0% and in 66.7% of these individuals high-risk types of HPV were detected. In 5.3% of sperm donors, two or more HPV types were detected. Among all identified HPV types, 61.9% were high-risk types. In situ hybridization experiments identified HPV genomes particularly protruding from the equatorial segment and the tail of the sperm. Semen samples from more than one in seven healthy Danish donors contain HPV, most of them of high-risk types binding to the equatorial segment of the sperm cell. Most HPV-positive sperm showed decreased staining with DAPI, indicative of reduced content of DNA. Our data demonstrate that oncogenic HPV types are frequent in men.

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STUDY QUESTION Do the Comet parameters of the proportions of sperm with low or high DNA damage improve the power of the test in the diagnosis of male infertility and/or prediction of IVF and ICSI live birth rates? SUMMARY ANSWER The mean Comet score and the scores for proportions of sperm with high or low DNA damage were useful in diagnosing male infertility and provided additional discriminatory information for the prediction of both IVF and ICSI live births. WHAT IS KNOWN ALREADY Sperm DNA damage impacts adversely on male fertility and IVF outcomes. STUDY DESIGN, SIZE, DURATION A retrospective study was performed involving a total of 457 participants (381 patients and 76 fertile donors). Data was collected from a fertility clinic between 2015 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 381 consecutive male partners of couples attending for ART and 76 fertile donors were included in the study. DNA fragmentation was measured by the alkaline Comet assay. Receiver operator characteristic curve analysis (area under the ROC curve (AUC)) was used to determine the value of average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose male factor infertility. In total, 77 IVF and 226 ICSI cycles were included to determine thresholds for each parameter (AUC analysis) and to compare live birth rates (LBRs) following each ART. MAIN RESULTS AND THE ROLE OF CHANCE ACS, HCS and LCS were predictive of male infertility (AUC > 0.9, P < 0.0001). IVF LBRs declined once DNA damage exceeded the threshold levels. HCS showed the sharpest decline. Following ICSI, the highest LBRs were in men whose DNA damage levels approached the fertile range. Trends differed in IVF. LBRs decreased as damage increased whereas in ICSI the LBRs decreased but then remained stable. LIMITATIONS, REASONS FOR CAUTION Since this is the first study to show the impact of sperm DNA damage on ICSI live births, a prospective study should be performed (stratifying patients to IVF or ICSI based on these thresholds) to validate this study. WIDER IMPLICATIONS OF THE FINDINGS Our study presents novel information towards elucidating the genetic basis of male infertility and secondly on relevance of the extent of DNA damage as an impending factor in both IVF and ICSI success. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Examenlab Ltd, The Lister Clinic, Cryos International and Imperial College London NHS Trust. No external funding was obtained for this study. SL and KL are employees of Examenlab Ltd, a university spin-out company with a commercial interest in sperm DNA damage. No other author has a conflict of interest to declare. TRIAL REGISTRATION NUMBER Non-applicable.

An analysis of all known human herpesviruses has not previously been reported on sperm from normal donors. Using an array-based detection method, we determined the cross-sectional frequency of human herpesviruses in semen from 198 Danish sperm donors. Fifty-five of the donors had at least one ejaculate that was positive for one or more human herpesvirus. Of these 27.3% (n = 15) had a double herpesvirus infection. If corrected for the presence of multiple ejaculates from some donors, the adjusted frequency of herpesviruses in semen was 27.2% with HSV-1 in 0.4%; HSV-2 in 0.1%; EBV in 6.3%; HCMV in 2.7%; HHV-6A/B in 13.5%; HHV-7 in 4.2%, whereas none of the samples had detectable VZV or HHV-8. Subsequently, we examined longitudinally data on ejaculates from 11 herpesvirus-positive donors. Serial analyses revealed that a donor who tested positive for herpesvirus at one time point did not necessarily remain positive over time. For the most frequently found herpesvirus, HHV-6A/B, we examined its association with sperm. For HHV-6A/B PCR-positive semen samples, HHV-6A/B could be detected on the sperm by flow cytometry. Conversely, PCR-negative semen samples were negative by flow cytometry. HHV-6B was shown to associate with sperm within minutes in a concentration dependent manner. Confocal microscopy demonstrated that HHV-6B associated with the sperm head, but only to sperm with an intact acrosome. Taken together, our data suggest that HHV-6A/B could be transported to the uterus via binding to the sperm acrosome. Moreover, we find a 10 times higher frequency of HHV-7 in semen from healthy individuals than previously detected. Further research is required to determine the potential risk of using herpesvirus-positive donor semen. Longitudinally analyses of ejaculate series indicate that implementation of quarantine for a donor shown to shed a herpesvirus is not a tenable solution.

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