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Human sperm deoxyribonucleic acid fragmentation by specific types of papillomavirus
Human sperm deoxyribonucleic acid fragmentation by specific types of papillomavirus Diane A. Connelly, MD, Philip J. Chan, PhD, HCLD, William C. Patton, MD, and Alan King, MD Loma Linda, California OBJECTIVE: Human papillomaviruses are present in up to 64.3% of patients’ sperm. The objectives were (1) to determine human papillomavirus deoxyribonucleic acid effects on sperm deoxyribonucleic acid integrity and (2) to assess human papillomavirus differential effects on the sperm cell. STUDY DESIGN: Two-layer colloid washed sperm were exposed to E6-E7 deoxyribonucleic acid fragments generated from human papillomavirus types 16, 18, 31, 33, 6/11, or control (DQA1) for 24 hours. The motility parameters were measured and analyzed. Pilot studies were performed to develop a fixed sperm comet assay to assess deoxyribonucleic acid fragmentation. RESULTS: Significant sperm deoxyribonucleic acid fragmentation occurred after exposure to deoxyribonucleic acid of human papillomavirus types 16 and 31. Human papillomavirus deoxyribonucleic acid fragment size was not a factor. Human papillomavirus types 18, 33, and 6/11 did not compromise sperm deoxyribonucleic acid integrity. Washed sperm motility was higher in the presence of human papillomavirus deoxyribonucleic acid except for type 6/11. Amplitude of head displacement was lower for human papillomavirus types 16 and 6/11. Sperm linearity was increased for all human papillomavirus types except type 18. CONCLUSION: Human papillomavirus type 16 and 31 deoxyribonucleic acid caused deoxyribonucleic acid breakages characteristic of apoptotic but not necrotic sperm. The data suggest that these human papillomavirus types may adversely affect subsequent embryonic development after fertilization. Sperm deoxyribonucleic acid appears to resist human papillomavirus types 18, 33, and 6/11 or repairing mechanisms occurred. Although enhanced motility was found in human papillomavirus–exposed sperm, important velocity parameters were decreased, suggesting impaired sperm function. (Am J Obstet Gynecol 2001;184:1068-70.)
Key words: Human papillomavirus, spermatozoa, comet assay, single-cell gel electrophoresis
Several reports have demonstrated the presence of human papillomavirus (HPV) in sperm.1-3 The deoxyribonucleic acid (DNA) of HPV types 11 and 16 was detected in 22% and 33% of unwashed semen specimens, respectively.2 HPV DNA from types 18 and 16 was also detected in 38.1% and 64.3% of postwashed sperm,3 with a greater incidence in asthenozoospermic patients.4 The presence of HPV is a concern because these DNA viruses are associated with the etiology of different cancers.5 Sperm may become infected with HPV in the male reproductive tract or in the female cervix. The null hypothesis was that condensed sperm chromatin was unaffected by HPV. The objectives were (1) to determine HPV DNA effects on sperm DNA fragmentation with the comet assay6, 7 and (2) to assess HPV differential effects on the sperm cell. DNA from HPV types 6, 31, and 33 was also evaluated in addition to types 11, 16, and 18 because these types
From the Departments of Gynecology and Obstetrics, Physiology, and Pharmacology, Loma Linda University School of Medicine. Reprint requests: Diane Connelly, MD, Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, 11234 Anderson St, Suite 3401, Loma Linda, CA 92350. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/1/115226 doi:10.1067/mob.2001.115226
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have been found in the reproductive tract and are associated with cervical cancer. The information obtained will help clinicians develop a better understanding of HPV infection of male gametes and transmissibility to the embryos. Material and methods Semen (n = 6) derived from anonymous discarded specimens of infertility patients were randomly pooled into two batches to reduce effects resulting from prior HPV infections. The study was approved by the institutional review board. The semen was washed (Isolate; Irvine Scientific, Santa Ana, Calif), and each motile sperm fraction was resuspended in 2 mL of HEPES (N-[2-hydroxyethyl]piperazine-N ´-[2-ethanesulfonic acid])– based human tubal fluid (HTF, Irvine Scientific) medium supplemented with 5% human serum albumin. The washed sperm were divided and incubated for 24 hours at 37°C in tubes containing DNA fragments of approximately 1 µg/µL synthesized by polymerase chain reaction with primers targeting either HPV type 16 (98 base pair [bp]), 18 (80 bp), 31 (162 bp), 33 (103 bp), 6/11 (157 bp), or control nonviral gene (242 bp, GH27 and GH26; Perkin Elmer Cetus, Norwalk, Conn) from the histocompatibility linked antigen DQA1 exon 2 region of
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human cervical condyloma cells. All HPV primers targeted the E6-E7 open-reading frames region, a conserved region shown to remain intact during virus-host genome DNA integration.8 All amplified products were verified with use of 5% acrylamide gel electrophoresis and ethidium bromide stain in ultraviolet light. After incubation, aliquots (10 µL) were removed and kinematic (motility) parameters were analyzed with use of the Hamilton Thorn (HTM-C; Hamilton Thorn Research, Danvers, Mass) motility analyzer, as previously described.3 Analyses were done in duplicate. A minimum of 100 sperm cells were analyzed each time. The comet assay was chosen to assess DNA integrity because it permitted the evaluation of DNA in each sperm, the assay was inexpensive, and background information supporting its versatility was available. Several preliminary experiments were carried out to develop the comet assay for fixed sperm.7 The established procedure consisted of air-drying sperm smears, fixing in methanol (Fixative Solution, Diff-Quik, Dade Behring, Newark, Del) for 15 seconds and air-drying and staining for 5 minutes in acridine orange solution prepared by dissolving 0.2 mg of acridine orange (United States Biochemical, Cleveland, Ohio) in 1.0 mL of purified water.6 The entire procedure was carried out in diffuse room lighting. The slides were washed, sticker labels were applied, and 0.8% warm (45°C) agarose (catalog No. 15510-019; Life Technologies, GIBCO BRL, Grand Island, NY) was pipetted over the slides to form a minilayer. The slides were immersed in 4°C alkaline lysis buffer (1% N-lauroylsarcosine and 1.0-mol/L tris[hydroxymethyl]aminomethane hydrochloride, pH 7.5; plus 0.5mol/L ethylenediaminetetra-acetic acid [EDTA] and 0.3-mol/L mercaptoethanol; pH adjusted to >10 with sodium hydroxide pellets) for 1 hour to release and unwind the sperm DNA. The slides were rinsed, submerged in 1× TBE (13.5 g Tris hydrochloride, pH 7.5; 6.8 g boric acid; and 5 mL of 0.5-mol/L EDTA in 1250 mL of purified water) for 10 minutes followed by electrophoresis at 50 V for 40 minutes. The intensity of the sperm head DNA was evaluated with an ultraviolet fluorescent microscope at ×500 magnification, and grayscale digital images were processed with an image analysis program.7 Lower head intensity (in pixels) represented greater DNA fragmentation. The data were expressed as mean ± SEM, and significance was determined with use of the Student t test statistic. A value of P < .05 was considered significant. Results DNA fragmentation occurred in sperm exposed to DNA of HPV types 16 and 31 (Table I). HPV DNA fragment size was not a factor in the severity of DNA fragmentation. It is interesting that exogenous DNA from HPV types 18, 33, and 6/11 did not cause sperm DNA
Table I. Comet assay detection of genomic DNA fragmentation in washed human sperm cells exposed to DNA from different types of HPV in vitro for 2 hours at 37°C HPV type
DNA size (bp)
Sperm head density (pixels, mean ± SEM)
No. analyzed
Control (DQA1) HPV 16 HPV 18 HPV 31 HPV 33 HPV 6/11
242 98 80 162 103 157
167.3 ± 2.4 125.9 ± 3.8* 163.2 ± 3.3 154.0 ± 3.5* 166.7 ± 3.7 168.0 ± 2.8
91 66 76 82 91 68
*Different from control (HLA-DQA1 second exon), P < .05.
fragmentation. The intra-assay and interassay coefficients of variation for the fixed sperm comet assay were 0.6% and 5.7%, respectively, and were judged to be reasonable. Sperm motility was higher after 24 hours of incubation in all HPV treatments with the exception of type 6/11 (Table II). However, in type 6/11 the velocity parameters (average path velocity and curvilinear velocity) were lower, whereas types 16 and 6/11 showed a lower amplitude of lateral head displacement compared with the control. Although beat cross-frequency (a measure of metabolism) was numerically increased for all HPV types, only that for type 33 was significant. The linearity parameter was increased in all HPV treatment groups except type 18. Comment The study demonstrated the fragmentation of DNA by HPV DNA E6-E7 gene sequences. Only HPV 16 and 31 caused genomic DNA breakages detectable by the comet assay. The comet tails had features of apoptotic and not necrotic sperm.6, 7 The data suggested that HPV types 16 and 31 might lead to failed embryonic development through sperm apoptosis. It is interesting that a higher percentage of spontaneously aborted products of conception9 had more HPV type 16 (29%) than type 18 (15%) detected mainly in syncytiotrophoblasts.10 HPV types 18, 33, and 6/11 did not affect sperm DNA integrity either because they formed episomes5 or because DNA repair mechanisms had occurred. HPV types 16, 18, 31, and 33 are associated with highrisk anogenital malignancies, whereas types 6 and 11 are linked to low-risk genital warts.5 In this study HPV DNA from the E6-E7 regions were used becuase E6-E7 expression proteins cause cell transformations whereas E1, E2, or L1 genes are for viral gene transcription, DNA replication, or encoding of capsid proteins.5 The study also showed enhanced sperm motility in the presence of most of the HPV types. However, the velocity and amplitude of lateral head displacement parameters were lower for some of the HPV types.4 A study carried
1070 Connelly et al
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Table II. Computer-aided sperm analyses of motility parameters in presence of DNA fragments from HPV type 16, 18, 31, 33, or 6/11 or control HLA-DQA1 exon 2 region after in vitro incubation at 37°C for 2 hours HPV type Parameter Total motility (%) Progression (%) Average path velocity (µm/s) Curvilinear velocity (µm/s) Amplitude of head displacement (µm) Beat cross-frequency (Hz) Hyperactivation (%) Linearity (%) No. analyzed
16
18
31
33
6/11
Control DQA1
48.0 ± 0.2* 5.5 ± 0.2 28.0 ± 0.1 38.5 ± 0.3 1.8 ± 0* 10.4 ± 0.1 1.0 ± 0.1 56.0 ± 0.2* 200
47.5 ± 0.1* 11.0 ± 0.2 36.0 ± 0.5 55.5 ± 0.7 2.7 ± 0 10.3 ± 0 1.0 ± 0.1 47.5 ± 0.2 209
55.0 ± 0.5* 14.5 ± 0.1 30.5 ± 0.1 45.5 ± 0.3 2.8 ± 0.1 11.6 ± 0 0±0 52.5 ± 0.5* 205
48.5 ± 0.7* 13.0 ± 0.3 28.5 ± 0.3 42.5 ± 0.4 2.7 ± 0 13.8 ± 0.1* 2.0 ± 0 52.5 ± 0.2* 224
36.5 ± 0.1 6.5 ± 0.1 23.0 ± 0* 31.5 ± 0.1* 1.8 ± 0* 12.1 ± 0 0±0 57.0 ± 0.2* 233
37.5 ± 0.2 11.0 ± 0.4 31.5 ± 0.1 47.5 ± 0.2 3.1 ± 0 9.8 ± 0.1 2.0 ± 0 49.0 ± 0.4 262
*Different from control, P < .05.
out in HPV 16– or 18–infected individuals also showed decreased values for these parameters. Decreased sperm velocity and head displacement have been linked to reduced sperm-fertilizing capacity.4 The data suggested an association between HPV presence and decreased sperm function leading to infertility. More studies are needed to determine whether the HPV DNA from the E1, E2, or L1 regions also affect sperm DNA integrity. REFERENCES
1. Ostrow RS, Zachow KR, Niimura M, Okagaki T, Muller S, Bender M, et al. Detection of papillomavirus DNA in human semen. Science 1986;31:731-3. 2. Green J, Monteiro E, Bolton VN, Sanders P, Gibson PE. Detection of human papillomavirus DNA by PCR in semen from patients with and without penile warts. Genitourin Med 1991; 67:207-10. 3. Chan PJ, Su BC, Kalugdan T, Seraj IM, Tredway DR, King A. Human papillomavirus gene sequences in washed human sperm deoxyribonucleic acid. Fertil Steril 1994;61:982-5. 4. Lai YM, Lee JF, Huang HY, Soong YK, Yang F-P, Pao CC. The ef-
5. 6.
7.
8.
9.
10.
fect of human papillomavirus infection in sperm cell motility. Fertil Steril 1997;67:1152-5. Alani RM, Münger K. Human Papillomaviruses and associated malignancies. J Clin Oncol 1997;16:330-7. Östling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual cells. Biochem Biophys Res Commun 1984;123:291-8. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chan SR, King A. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zonafree hamster oocytes. Fertil Steril 2001;75:1-7. Sarkar FH, Crissman JD. Detection of human papillomavirus DNA sequences by polymerase chain reaction. Biotechniques 1990;9:180-5. Malhomme O, Dutheil N, Rabreau M, Armbruster-Moraes E, Schlehofer JR, Dupressoir T. Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA. J Gen Virol 1997;78:1957-62. Hermonat PL, Han L, Wendel PJ, Quirk JG, Stern S, Lowery CL, et al. Human papillomavirus is more prevalent in first trimester spontaneously aborted products of conception compared to elective specimens. Virus Genes 1997;14:13-7.
Key words: Human papillomavirus, spermatozoa, comet assay, single-cell gel electrophoresis
Several reports have demonstrated the presence of human papillomavirus (HPV) in sperm.1-3 The deoxyribonucleic acid (DNA) of HPV types 11 and 16 was detected in 22% and 33% of unwashed semen specimens, respectively.2 HPV DNA from types 18 and 16 was also detected in 38.1% and 64.3% of postwashed sperm,3 with a greater incidence in asthenozoospermic patients.4 The presence of HPV is a concern because these DNA viruses are associated with the etiology of different cancers.5 Sperm may become infected with HPV in the male reproductive tract or in the female cervix. The null hypothesis was that condensed sperm chromatin was unaffected by HPV. The objectives were (1) to determine HPV DNA effects on sperm DNA fragmentation with the comet assay6, 7 and (2) to assess HPV differential effects on the sperm cell. DNA from HPV types 6, 31, and 33 was also evaluated in addition to types 11, 16, and 18 because these types
From the Departments of Gynecology and Obstetrics, Physiology, and Pharmacology, Loma Linda University School of Medicine. Reprint requests: Diane Connelly, MD, Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, 11234 Anderson St, Suite 3401, Loma Linda, CA 92350. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/1/115226 doi:10.1067/mob.2001.115226
1068
have been found in the reproductive tract and are associated with cervical cancer. The information obtained will help clinicians develop a better understanding of HPV infection of male gametes and transmissibility to the embryos. Material and methods Semen (n = 6) derived from anonymous discarded specimens of infertility patients were randomly pooled into two batches to reduce effects resulting from prior HPV infections. The study was approved by the institutional review board. The semen was washed (Isolate; Irvine Scientific, Santa Ana, Calif), and each motile sperm fraction was resuspended in 2 mL of HEPES (N-[2-hydroxyethyl]piperazine-N ´-[2-ethanesulfonic acid])– based human tubal fluid (HTF, Irvine Scientific) medium supplemented with 5% human serum albumin. The washed sperm were divided and incubated for 24 hours at 37°C in tubes containing DNA fragments of approximately 1 µg/µL synthesized by polymerase chain reaction with primers targeting either HPV type 16 (98 base pair [bp]), 18 (80 bp), 31 (162 bp), 33 (103 bp), 6/11 (157 bp), or control nonviral gene (242 bp, GH27 and GH26; Perkin Elmer Cetus, Norwalk, Conn) from the histocompatibility linked antigen DQA1 exon 2 region of
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human cervical condyloma cells. All HPV primers targeted the E6-E7 open-reading frames region, a conserved region shown to remain intact during virus-host genome DNA integration.8 All amplified products were verified with use of 5% acrylamide gel electrophoresis and ethidium bromide stain in ultraviolet light. After incubation, aliquots (10 µL) were removed and kinematic (motility) parameters were analyzed with use of the Hamilton Thorn (HTM-C; Hamilton Thorn Research, Danvers, Mass) motility analyzer, as previously described.3 Analyses were done in duplicate. A minimum of 100 sperm cells were analyzed each time. The comet assay was chosen to assess DNA integrity because it permitted the evaluation of DNA in each sperm, the assay was inexpensive, and background information supporting its versatility was available. Several preliminary experiments were carried out to develop the comet assay for fixed sperm.7 The established procedure consisted of air-drying sperm smears, fixing in methanol (Fixative Solution, Diff-Quik, Dade Behring, Newark, Del) for 15 seconds and air-drying and staining for 5 minutes in acridine orange solution prepared by dissolving 0.2 mg of acridine orange (United States Biochemical, Cleveland, Ohio) in 1.0 mL of purified water.6 The entire procedure was carried out in diffuse room lighting. The slides were washed, sticker labels were applied, and 0.8% warm (45°C) agarose (catalog No. 15510-019; Life Technologies, GIBCO BRL, Grand Island, NY) was pipetted over the slides to form a minilayer. The slides were immersed in 4°C alkaline lysis buffer (1% N-lauroylsarcosine and 1.0-mol/L tris[hydroxymethyl]aminomethane hydrochloride, pH 7.5; plus 0.5mol/L ethylenediaminetetra-acetic acid [EDTA] and 0.3-mol/L mercaptoethanol; pH adjusted to >10 with sodium hydroxide pellets) for 1 hour to release and unwind the sperm DNA. The slides were rinsed, submerged in 1× TBE (13.5 g Tris hydrochloride, pH 7.5; 6.8 g boric acid; and 5 mL of 0.5-mol/L EDTA in 1250 mL of purified water) for 10 minutes followed by electrophoresis at 50 V for 40 minutes. The intensity of the sperm head DNA was evaluated with an ultraviolet fluorescent microscope at ×500 magnification, and grayscale digital images were processed with an image analysis program.7 Lower head intensity (in pixels) represented greater DNA fragmentation. The data were expressed as mean ± SEM, and significance was determined with use of the Student t test statistic. A value of P < .05 was considered significant. Results DNA fragmentation occurred in sperm exposed to DNA of HPV types 16 and 31 (Table I). HPV DNA fragment size was not a factor in the severity of DNA fragmentation. It is interesting that exogenous DNA from HPV types 18, 33, and 6/11 did not cause sperm DNA
Table I. Comet assay detection of genomic DNA fragmentation in washed human sperm cells exposed to DNA from different types of HPV in vitro for 2 hours at 37°C HPV type
DNA size (bp)
Sperm head density (pixels, mean ± SEM)
No. analyzed
Control (DQA1) HPV 16 HPV 18 HPV 31 HPV 33 HPV 6/11
242 98 80 162 103 157
167.3 ± 2.4 125.9 ± 3.8* 163.2 ± 3.3 154.0 ± 3.5* 166.7 ± 3.7 168.0 ± 2.8
91 66 76 82 91 68
*Different from control (HLA-DQA1 second exon), P < .05.
fragmentation. The intra-assay and interassay coefficients of variation for the fixed sperm comet assay were 0.6% and 5.7%, respectively, and were judged to be reasonable. Sperm motility was higher after 24 hours of incubation in all HPV treatments with the exception of type 6/11 (Table II). However, in type 6/11 the velocity parameters (average path velocity and curvilinear velocity) were lower, whereas types 16 and 6/11 showed a lower amplitude of lateral head displacement compared with the control. Although beat cross-frequency (a measure of metabolism) was numerically increased for all HPV types, only that for type 33 was significant. The linearity parameter was increased in all HPV treatment groups except type 18. Comment The study demonstrated the fragmentation of DNA by HPV DNA E6-E7 gene sequences. Only HPV 16 and 31 caused genomic DNA breakages detectable by the comet assay. The comet tails had features of apoptotic and not necrotic sperm.6, 7 The data suggested that HPV types 16 and 31 might lead to failed embryonic development through sperm apoptosis. It is interesting that a higher percentage of spontaneously aborted products of conception9 had more HPV type 16 (29%) than type 18 (15%) detected mainly in syncytiotrophoblasts.10 HPV types 18, 33, and 6/11 did not affect sperm DNA integrity either because they formed episomes5 or because DNA repair mechanisms had occurred. HPV types 16, 18, 31, and 33 are associated with highrisk anogenital malignancies, whereas types 6 and 11 are linked to low-risk genital warts.5 In this study HPV DNA from the E6-E7 regions were used becuase E6-E7 expression proteins cause cell transformations whereas E1, E2, or L1 genes are for viral gene transcription, DNA replication, or encoding of capsid proteins.5 The study also showed enhanced sperm motility in the presence of most of the HPV types. However, the velocity and amplitude of lateral head displacement parameters were lower for some of the HPV types.4 A study carried
1070 Connelly et al
May 2001 Am J Obstet Gynecol
Table II. Computer-aided sperm analyses of motility parameters in presence of DNA fragments from HPV type 16, 18, 31, 33, or 6/11 or control HLA-DQA1 exon 2 region after in vitro incubation at 37°C for 2 hours HPV type Parameter Total motility (%) Progression (%) Average path velocity (µm/s) Curvilinear velocity (µm/s) Amplitude of head displacement (µm) Beat cross-frequency (Hz) Hyperactivation (%) Linearity (%) No. analyzed
16
18
31
33
6/11
Control DQA1
48.0 ± 0.2* 5.5 ± 0.2 28.0 ± 0.1 38.5 ± 0.3 1.8 ± 0* 10.4 ± 0.1 1.0 ± 0.1 56.0 ± 0.2* 200
47.5 ± 0.1* 11.0 ± 0.2 36.0 ± 0.5 55.5 ± 0.7 2.7 ± 0 10.3 ± 0 1.0 ± 0.1 47.5 ± 0.2 209
55.0 ± 0.5* 14.5 ± 0.1 30.5 ± 0.1 45.5 ± 0.3 2.8 ± 0.1 11.6 ± 0 0±0 52.5 ± 0.5* 205
48.5 ± 0.7* 13.0 ± 0.3 28.5 ± 0.3 42.5 ± 0.4 2.7 ± 0 13.8 ± 0.1* 2.0 ± 0 52.5 ± 0.2* 224
36.5 ± 0.1 6.5 ± 0.1 23.0 ± 0* 31.5 ± 0.1* 1.8 ± 0* 12.1 ± 0 0±0 57.0 ± 0.2* 233
37.5 ± 0.2 11.0 ± 0.4 31.5 ± 0.1 47.5 ± 0.2 3.1 ± 0 9.8 ± 0.1 2.0 ± 0 49.0 ± 0.4 262
*Different from control, P < .05.
out in HPV 16– or 18–infected individuals also showed decreased values for these parameters. Decreased sperm velocity and head displacement have been linked to reduced sperm-fertilizing capacity.4 The data suggested an association between HPV presence and decreased sperm function leading to infertility. More studies are needed to determine whether the HPV DNA from the E1, E2, or L1 regions also affect sperm DNA integrity. REFERENCES
1. Ostrow RS, Zachow KR, Niimura M, Okagaki T, Muller S, Bender M, et al. Detection of papillomavirus DNA in human semen. Science 1986;31:731-3. 2. Green J, Monteiro E, Bolton VN, Sanders P, Gibson PE. Detection of human papillomavirus DNA by PCR in semen from patients with and without penile warts. Genitourin Med 1991; 67:207-10. 3. Chan PJ, Su BC, Kalugdan T, Seraj IM, Tredway DR, King A. Human papillomavirus gene sequences in washed human sperm deoxyribonucleic acid. Fertil Steril 1994;61:982-5. 4. Lai YM, Lee JF, Huang HY, Soong YK, Yang F-P, Pao CC. The ef-
5. 6.
7.
8.
9.
10.
fect of human papillomavirus infection in sperm cell motility. Fertil Steril 1997;67:1152-5. Alani RM, Münger K. Human Papillomaviruses and associated malignancies. J Clin Oncol 1997;16:330-7. Östling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual cells. Biochem Biophys Res Commun 1984;123:291-8. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chan SR, King A. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zonafree hamster oocytes. Fertil Steril 2001;75:1-7. Sarkar FH, Crissman JD. Detection of human papillomavirus DNA sequences by polymerase chain reaction. Biotechniques 1990;9:180-5. Malhomme O, Dutheil N, Rabreau M, Armbruster-Moraes E, Schlehofer JR, Dupressoir T. Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA. J Gen Virol 1997;78:1957-62. Hermonat PL, Han L, Wendel PJ, Quirk JG, Stern S, Lowery CL, et al. Human papillomavirus is more prevalent in first trimester spontaneously aborted products of conception compared to elective specimens. Virus Genes 1997;14:13-7.