1 Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, 2 Clinic of Urology and Pediatric Urology, University of the Saarland, Homburg/Saar, 3 Institut für Fortpflanzungsmedizin, Saarbrücken and 4 Biostatistik, Deutsches Krebsforschungszentrum, Heidelberg, Germany
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Abstract |
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Key words: AAV/fertility/HCMV/HPV/PCR
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Introduction |
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Material and methods |
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Cervical smears
Cervical swabs were obtained from 57 female partners (age range: 2142 years, mean: 32.2) participating in the fertility programme in Saarbrücken (see above). Of these women, 22 had no fertility-related clinical conditions, 18 had pathological/anatomical disorders possibly associated with infertility, 10 presented with endometriosis, and seven had hormonal problems.
Urethral smears
Smears from the urethra were available from 17 men, not related to the semen samples described above. Smears were taken in temporal distance from ejaculation to avoid contamination with semen and were from men with malignant melanoma (n = 3), benign tumour (n = 5), stone (n = 1) and adenoma of the prostate (n = 1). In seven cases, no diagnosis was available except for the notion that there was no tumour.
Testicular biopsies
Tissue samples obtained surgically prior to intracytoplasmic sperm injection (ICSI) from 38 azoospermic men (unrelated to the semen donors described above) were fixed with Bouin's solution and embedded in paraffin. Sections that had been analysed histomorphologically for grade of atrophy (Sigg, 1979) were used for AAV DNA detection. Eight tissue samples were taken from patients with prostate cancer and therapeutic orchiectomy (hormonal therapy). No data concerning status of fertility and histology for these anonymous samples were available.
DNA extraction
Samples were digested with proteinase K and Tween 20 buffer prior to DNA extraction, performed as previously described (Tobiasch et al., 1994, 1998
; Walz et al., 1997
).
Polymerase chain reaction (PCR)
For detection of AAV DNA, the primer sets pan1/pan3 and Rep78.1/Rep78.2 were used according to the method of Tobiasch et al. (Tobiasch et al., 1998). Briefly, an initial PCR using the primers Rep78.1 and pan3 was followed by a second (nested) PCR using the primers Rep78.2 and pan1 and 5 µl of the amplified product of the first PCR. The nested PCR for AAV DNA resulted in a 176-base pair product. The genomic DNA of a cell line containing AAV DNA (Walz and Schlehofer, 1992
) served as a positive control. Using this protocol, AAV types 2, 3 and 5 are detected. Samples that were found positive for AAV by nested PCR were further analysed using PCR primers specific for AAV-3 and AAV-5 respectively, followed by hybridization with a type specific oligonucleotide (Tobiasch et al., 1998
)
To detect HPV (genital types) DNA, a PCR was performed according to the method of Jacobs et al. using consensus primers for HPV followed by hybridization with virus- and type-specific oligonucleotide probes (Jacobs et al., 1995). The genomic DNAs of cells of the SiHa (HPV type 16 DNA-positive) and the HeLa (HPV type 18 DNA-positive) cell lines respectively, were used as positive controls. PCR products were separated by electrophoresis through 1% agarose gels and transferred to nylon membranes by the Southern blot technique. To confirm the specificity of the PCR products, blots were hybridized with digoxigenin-labelled oligonucleotide probes detecting HPV16/18 or 31/33 and chemiluminescent analysis was performed (CSPD; Boehringer-Mannheim, Mannheim, Germany).
HCMV DNA sequences were amplified using the nested PCR protocol as described (Arai et al., 1995; Burguete et al., 1999
), using HCMV-infected cells as a positive control (Malhomme et al., 1997
).
Statistical analysis
Prevalence of viral DNA (AAV, HCMV, HPV) in men with normal semen analyses was compared with the prevalence in abnormal diagnostic groups (asthenozoospermia, oligoasthenozoospermia, oligozoospermia or azoospermia) using the 2-tailed Fisher's exact test for contingency tables (Altman, 1991). A P value of < 0.05 was considered statistically significant.
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Results |
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As shown in Table I, AAV DNA was detected in 29/95 samples (30.5%). None of the samples was found to be positive by specific PCR for AAV-3 or AAV-5. In cases with an abnormal semen analysis, AAV DNA was detected significantly more frequently (28/73) than in ejaculates with a normal semen analysis (1/22) (P = 0.003). The presence of AAV DNA was significantly higher in oligoasthenozoospermia (18/39, P < 0.001) and oligozoospermia (2/3, P = 0.029) than in normal semen samples. Less of a difference was found between normal samples and those of asthenozoospermia (7/23, P = 0.047) and azoospermia (1/8, P = not significant).
In a second step, we tested the ejaculate samples for the presence of genital helper viruses for AAV replication (HPV, HCMV).
The presence of DNA of genital HPV types was found in 16/94 specimens tested (all samples were found to be HPV-type 16/18). Five specimens exhibited infection with both HPV and AAV (two with asthenozoospermia, three with oligoasthenozoospermia). In contrast to the findings with AAV, DNA of HPV was found at a similar frequency in samples with normal and abnormal semen analyses. HPV DNA was detected in 4/21 (19.0%) samples with a normal semen analysis, and in 12/73 samples (16.4%) with an abnormal semen analysis (P = not significant) (Table I).
Infection with HCMV was only rarely observed. Of 57 samples tested, two out of 14 (14.3%) normal semen analysis samples contained HCMV DNA, whereas among the abnormal samples, no HCMV infection was detected (0/43) (P = not significant) (Table I).
An association of specific clinical conditions with the presence of viral DNA could not be assessed since patients had many different diagnoses that could not be grouped because of small numbers (data not shown). There was no correlation of age with the detection of viral DNA (determined using the Spearman's rank correlation coefficient; data not shown).
Analysis of cervical swabs from women attending the fertility programme with their partners revealed 26.3% (15/57) to contain AAV DNA. In five cases, AAV DNA was detected in both the male and the female partner. There was no correlation between specific clinical conditions of the women and the presence of AAV DNA in cervical smears.
In order to assess if detection of AAV DNA in semen was associated with a possibly frequent infection of epithelial cells, rather than with infection of spermatozoa, we analysed urethral smears. The samples were unrelated to the sperm samples and randomly chosen from diagnostic tests performed for diseases others than infertility. In contrast to the results from semen analyses, which demonstrated AAV DNA in 30.5% (29/95) of the cases, in urethral swabs AAV DNA was detected in only 17.6% (3/17).
Since AAV DNA has been found to be associated with the spermatocyte fraction of semen samples (Rohde et al., 1999), testicular tissue was analysed in order to clarify a possible infection of spermatozoa during maturation. In testicular biopsies from azoospermic men seeking ICSI (n = 38) or from men orchidectomized for clinical reasons (n = 8), AAV DNA was detected at a similar frequency as in semen. Twelve of the 46 samples tested were found to contain AAV DNA (25.5%).
In testicular samples from the 38 infertile men with azoospermia, 10 biopsies were found to contain AAV DNA (26.3%). As shown in Table II, 3/15 with Sertoli-cell-only syndrome, 2/4 with `Bunte Atrophie' (Sigg, 1979
), 1/6 with atrophia grade 2A/2B, and 4/8 with normal spermatogenesis were AAV DNA-positive. Among the AAV DNA-positives with regular spermatogenesis, one had a successfully treated hypogonadotrope hypogonadism, one had bilateral congenital absence of the vas deferens and two had no specific symptoms (hormonally or phenotypically). Among the AAV DNA- negative samples of those with normal spermatogenesis, one patient had a hypospadia classified as coronaral and three had no specific symptoms. No statistical differences were observed between AAV DNA status and histological diagnosis.
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Discussion |
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To address the question of a possible infection of spermatozoa during maturation, we analysed testis samples from azoospermic men participating in an ICSI programme. It was found that AAV DNA is frequently present in testes. Interestingly, AAV infection was more frequently present in patients in whom a normal spermatogenesis was diagnosed (4/8) whereas in atrophy, AAV DNA was rarely found (6/30). However, it was not possible to establish clear-cut associations because of the small numbers of samples that could be tested. Furthermore, testis samples of elderly men treated for prostate cancer had a similar frequency of AAV detection (25%). Nevertheless, our data demonstrate that AAV is present in testicular tissue, and at a rather high frequency, showing that an infection of spermatozoa could be possible. This would explain the finding that in most cases the detection of AAV DNA was restricted to spermatozoa and not detected in other semen components (Rohde et al., 1999). However, the localization of AAV in testicular tissue needs to be identified using in-situ hybridization. Since AAV DNA was found in urethral swabs, AAV detection in ejaculates may be partly due to contamination with AAV-positive epithelial cells of the lower urinary tract. Whether the presence of AAV in testicular tissue is related to infertility remains unclear. However, the finding that AAV DNA was almost exclusively detected in samples from men with abnormal semen analyses points to an influence of AAV on spermatogenesis. To date, very little is known about the effect of virus infections of the testes on fertility. It seems conceivable that additional factors (e.g. hormonal factors or co-infection with other helper viruses or specific stages of maturation) could be involved in a disturbance of sperm development, together with AAV. In this context it is interesting to note that AAV has recently been shown to replicate in differentiating epithelia without the presence of a helper virus, inducing morphological changes including cell lysis (Meyers et al., 2000
). This might hint to a possible pathogenic effect in differentiating cells, and may also explain the frequent detection of AAV without the presence of a helper virus. An association between the detection of AAV DNA and specific clinical or endocrinological conditions could not be assessed due to the great diversity of clinical diagnoses. The small number of patients in the respective subgroups did not allow us to create categories for disease groups. This issue should be addressed in further investigations to determine a possible role of AAV in male infertility.
It is unclear how AAV infection of the testis could occur. The previously reported in-utero infection with this virus (Burguete et al., 1999) might allow infection of the testis of embryos, with the possibility of reactivation of the persisting virus later in life. It seems as if genital tissue might be a preferential target for AAV persistence since AAV DNA has been most frequently found in genital tissue compared with a much rarer detection in other organs. It will be necessary to determine the factors reactivating latent AAV and to analyse AAV DNA integration sites (yet to be proven) in specific cells. Possibly, the site of AAV DNA integration in latently infected cells may determine whether the presence and/or reactivation of viral functions have pathological consequences or not.
Concerning detection of AAV infection of the female partners in fertility problems, analysis of cervical swabs did not give conclusive results (26.3% AAV DNA-containing smears). A co-infection of both partners was observed in only five cases, but infection of women is certainly underestimated when analysing cervical smears: we and others had observed previously that cervical biopsies were AAV DNA-positive in up to 80% of women as opposed to 2050% AAV DNA-containing smears, depending on the number of cells obtained with the various swab techniques (Tobiasch et al., 1994; Walz et al., 1997
; K.Erles, unpublished results). Therefore, in this study, a correlation of the role of co-infection of both partners in infertility could not be assessed. Since ~60% of the women attending the fertility programme had specific conditions possibly contributing to infertility (endometriosis, hormonal or anatomical problems), a decisive role of AAV cannot be established. However, there was a significant association of AAV infection with abnormal seminal analyses.
In view of the findings on persistent AAV infection of the human genital tract, further virological and molecular studies are required to assess whether there is a role of AAV in infertility and whether AAV infection could have an influence on the outcome of assisted reproductive techniques.
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Acknowledgements |
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Notes |
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References |
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Submitted on April 18, 2001; accepted on August 9, 2001.