Identification of multiple HPV types on spermatozoa from human sperm donors

Abstract

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.

Figure 1. HPV frequency and type distribution in Danish donor semen, analyzed using a sensitive HPV type-specific array.

(A): Frequency of twenty high-risk types, given as percent of all tested donors. (B): Frequency of fifteen low-risk types, given as percent of all tested donors.

Figure 2. Longitudinal analysis of HPV-positive sperm samples.

HPV is not continuously shed in semen. Circles on a relative time scale image series of ejaculates from 6 different donors, occasionally positive for an HPV. Filled circles represent HPV positive samples; open circles represent HPV negative samples. Starting points are given by the first analyzed ejaculate for each donor. Series of ejaculates from six different donors were found positive for HPV type 84; type 53, 31, and 52; type 62; type 61; type 54; and type 52, respectively.

Figure 3. In situ hybridization on human sperm using HPV type-specific probes.

Specific hybridizations are indicated by arrows. (A–C): In situ hybridization on an HPV-16 positive sperm sample using a probe specific for HPV-16. The sample in picture A was simultaneously background stained with hematoxylin. (D–I): In situ hybridization on an HPV-6 positive sperm sample using a probe specific for HPV-6. (J–L): In situ hybridization on an HPV negative sperm sample using a probe specific HPV-6. This figure is a representative of three independent in situ hybridization experiments using two different HPV-6 positive samples and two different HPV-16 positive samples.

Figure 4. Binding pattern of HPV to sperm using fluorescence in situ hybridization with HPV type-specific probes.

(A–C): Close-up picture of sperm of which one is positive for HPV type 18. Picture B shows the sperm present in picture A, captured by differential interference contrast (DIC). Picture C shows an overlay of A and B. (D): Close-up picture of HPV-negative and HPV-18 positive sperm cell from an HPV-18-positive semen sample stained for HPV (red) and DNA (blue). Note the lack of DNA staining characteristic of the majority of HPV positive sperm. Lack of or decreased DNA staining was seen in all HPV-positive sperm.

Figure 5. Frequency of HPV-positive sperm using fluorescence in situ hybridization with HPV type-specific probes.

Low magnification pictures illustrating the quantity of HPV in positive semen samples. Samples are positive for HPV-6, -16, -18, and -31, respectively. Samples are stained for HPV with PE-conjugated streptavidin (red) and stained for DNA with DAPI (blue). Pictures are representatives of coincidental spots of the preparation slides, which are representatives of HPV positive semen samples from 9 donors (HPV-6, -16, -18, and -31 represented by 2–3 donors each).