Department of Infectious Diseases, Marselisborg Hospital, Aarhus University Hospital, P.P. Oerumsgade 11, DK-8000 Aarhus, Denmark
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Abstract |
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Key words: CMV/donor/human herpes virus 8/PCR/semen
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Introduction |
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The prevalence of HHV-8 in the general population remains controversial (Blackbourn and Levy, 1997). The frequency of HHV-8 detection in semen from healthy individuals ranges from non-detectable (Corbellino et al., 1996
; Howard et al., 1997
) to 23% (Lin et al., 1995
) and 91% (Monini et al., 1996a
). This might in part be explained by geographical and methodological differences, although the divergence seems overwhelming.
If HHV-8 is ubiquitous, it may share other characteristics with the known herpes viruses, such as establishment of a latent infection and reactivation with intermittent shedding. This raises important implications as to the use of donor semen in assisted conception procedures. HHV-8 may be transmitted to the mother and even vertically to the unborn child (He et al., 1998).
The purpose of the present study was to determine if HHV-8 is present in semen from Danish donors.
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Materials and methods |
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Donors with a history of syphilis or treatment with blood products were excluded. Ninety-eight were white, native Danish males while two donors were immigrants from Iran.
Donors were tested for: syphilis using Wasserman's reaction, a serological test; gonorrhoea by culturing from the semen and serological test if the donor had received antibiotic treatment recently; Chlamydia trachromatis by culturing from urethral swabs or polymerase chain reaction (PCR) from urine; HIV-I and -II, human T-cell lymphotrophic virus (HTLV)-I and II, hepatitis B surface-antigen (HbsAg) and anti-hepatitis C virus (HCV) every 3 months. Semen was released for use 6 months after the first tests. Blood type and male karyotype were determined.
Semen was stored in liquid nitrogen and for shorter periods at 70°C. DNA was extracted from 250 µl semen, equivalent to 5x106 highly motile spermatozoa, according to the description for Puregene® DNA Isolation Kit (Gentra Systems Inc, Minneapolis, MN, USA). The concentration and purity of DNA were estimated by measuring optical densities at 260 and 280 nm, using a Gene Quant II spectrophotometer (Amersham Pharmacia Biotech Europe GmBH, Freiburg, Germany).
No attempts were made to separate semen into fractions since HHV-8 might be present preferentially in the fraction containing urothelial cells and to a lesser degree in sperm heads (Monini et al., 1996a).
All primers used in our study were manufactured by DNA Technology ApS, Aarhus, Denmark. To assure the quality of the extracted DNA, all samples were analysed using PCR with a primer pair specific for the human gene glyceraldehyde-3-phosphate dehydrogenase (GADPH): GADPH1 (5'GAGTCAACGGATTTGGTCGT3') and GADPH2 (5'GGTGCCATGGAATTTGCCAT 3'). These primers amplified a 157 bp fragment of human DNA.
To detect HHV-8 DNA, simple as well as nested PCR was done. The primers used included: the inner pair of primers KS1 (5'AGCCGAAAGGATTCCACCAT3') and KS2 (5'TCCGTGTTGTCTACGTCCAG 3') amplifying a 233 bp fragment of ORF 26 (Chang et al., 1994); and the outer primers KS4 (5'GACTCTTCGCTGATGAACTGG 3') and KS5 (5'AGCACTCGCAGGGCAGTACG 3') amplifying a 700 bp fragment of HHV-8 DNA (Boshoff et al., 1995
).
Furthermore two sets of non-overlapping primers were used: KS10 (5'AGATGTTCCTGTATGTTGTCTGCAG3') and KS11 (5'ATGTAATCCAAATGCTCTGTCGATG 3') amplifying a 270 bp hypervariable region of ORF K1 (Kasolo et al., 1998), and KS12 (5'TCGTGGGATCCAC-GGAGCATACACC3') and KS13 (5'ATATAGGATACGCTGGCAGGTGGGCG 3') from ORF 75, a putative tegument protein of HHV-8 (Russo et al., 1996
). PCR was performed with 100 ng to 500 ng of DNA (equivalent to DNA extracted from 17 µl of semen) in a 21µl PCR-mixture containing 0.5U AmpliTaq Gold® (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA) 2 µl MgCl2 25 mmol/l (Perkin Elmer), 2 µl dNTP 500 µmol/l, 2 µl PCR Buffer II (Perkin Elmer) and 20 pmol of each primer. The amplification conditions were as follows: `Hot start' 10 min at 95°C followed by 39 cycles of alternating denaturation for 1 min at 94°C, primer annealing for 1 min at 55°C and primer extension for 2 min at 72°C, terminated by a 10 min extension at 72°C. Higher annealing temperatures could have been used (and were used in some control experiments) but to ensure a high sensitivity we used a relatively low annealing temperature. In the case of nested PCR, 1 µl of the PCR product was further amplified with the inner primers under the same conditions as mentioned above. One positive and one negative sample were included for every nine semen samples. The positive controls were derived from paraffin-embedded biopsies of Kaposi's sarcoma and from the cell line BCP-1, a commercially available (ATCC) body cavity-based lymphoma (BCBL) cell line, each cell harbouring 150 copies of HHV-8.
The PCR products were visualised on 2% agarose gels containing ethidium bromide.
In addition we tested the samples for cytomegalovirus (CMV) DNA with an unnested PCR procedure using the primer pairs CMV1 (5'CAAGCGGCCTCTGATAACCAAGC'3) and CMV2 (5'CTCTTCCTCTGGGGCAACTTCCTC'3) amplifying a 421 bp fragment. The amplification was performed with 35 cycles of alternating denaturation for 1 min at 94°C, primer annealing for 1 min at 65°C, and primer extension for 1 min at 72°C. Each extension time was increased by 10 s on each cycle (Hansen et al., 1994).
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Results |
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The four primer sets for HHV-8 gave strong bands on ethidium bromide gels using DNA extracted from Kaposi's sarcoma and BCP-1. But the 100 semen samples were consistently negative for HHV-8 DNA by the simple as well as the nested PCR procedures. As to detection of CMV DNA, we found only one CMV-positive donor. The samples were tested twice for each primer set by different operators.
In a control experiment HHV-8 negative semen was spiked with HHV-8 from BCP-1 cells and serially diluted in semen. When compared to dilution series of HHV-8 in water, there was no indication that semen inhibited the PCR reactions.
The negative outcome concerning HHV-8 was not altered either by increasing the number of amplification cycles to 45 (Monini et al., 1996a), nor by doubling the amount of DNA, decreasing the annealing temperature to 48°C or varying the concentration of MgCl2 in the interval 1.253.5 mmol/l.
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Discussion |
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These findings were supported by studies in which HHV-8 was detected in 13% (6/45) of Sicilian men, thereby showing a roughly uniform picture of the prevalence of HHV-8 (Viviano et al., 1997). All the above mentioned studies used a nested PCR procedure, only yielding positive signals after the second step of amplification.
These findings were challenged (Corbellino et al., 1996; Howard et al., 1997a) by studies which failed to confirm the presence of HHV-8 in semen from 115 English semen donors and 20 Italian semen donors respectively. Nested PCR was used in both of the studies.
In our study all 100 semen specimens remained negative for HHV-8 DNA, after having used both nested and unnested PCR procedures. According to our experience, different primer sets may lead to unexplained differences in PCR sensitivity. We therefore used two pairs of non-overlapping primers in excess of the nested primers.
No study detected HHV-8 DNA in semen from healthy men by the use of a simple unnested PCR procedure. The nested PCR presumably improves the detection level to a few HHV-8 DNA copies per µg of DNA. Several authors have argued that HHV-8 may establish a latent infection in otherwise healthy individuals, shedding virus in a low number of copies. It is still a matter of debate, whether the comparatively high detection rates of HHV-8 in some studies may be due to contamination of the samples, a problem often observed when using nested PCR (Howard and Bahadur 1997b; Moore, 1998).
At present the most sensitive method for detection of CMV in semen is PCR (Mansat et al., 1997). We only detected CMV in one out of 100 donors. This frequency corresponds to earlier studies of donor populations 4/115 (Howard et al., 1997a) and 5/97(Mansat et al., 1997
). As CMV was detectable in semen from one donor, herpes viruses seem to be recovered by the extraction method used in this study. The handling of samples prior to PCR is of unquestionable importance. In our study, semen samples were put into liquid nitrogen within the first hour after ejaculation and only stored at 70°C for shorter periods. As shown in previous studies (Howard et al., 1997a) cryopreservation of semen does not affect PCR sensitivity of HHV-8 detection.
Geographical differences may have contributed to the different detection rates but it is more likely that the variation is due to the obvious differences in enrolment criteria.
Based on lifestyle questionnaires, the semen donors in our study had an exclusively heterosexual background and a rather limited number of sexual partners. This rules out most of the significant epidemiological risk factors for acquisition of HHV-8. The two studies (Corbellino et al., 1996; Howard et al., 1997
) reporting negative results used authorized semen donors from fertility clinics while the three studies with positive results seem to have recruited semen donors from a potentially more promiscuous part of the male population, e.g. men with a known history of sexually transmitted disease.
In Denmark strict biochemical and microbiological criteria are used in combination with an intensive interview to select semen donors. As HHV-8 was not demonstrated in any of the semen samples analysed, we conclude that the frequency of HHV-8 in semen from Danish donors is very low.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 22, 1999; accepted on June 9, 1999.