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VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN
0022-5347/98/1593-0820$03.oa/o
Vol. 159,820-826, March 1998 Printed in lJ.S.A
THE JOURNAL OF UROLOCY
Copyright 0 1998 by AMERICAN UROUIGICAL ASS~CIATION, INC.
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN JOHN N. KRIEGER, APICHART NIRAPATHPONGPORN, MONTCHAI CHAIYAPORN, GREGORY PETERSON, IRENA NIKOLAEVA, ROBERT AKRIDGE, SUSAN 0. ROSS AND ROBERT W. COOMBS From the Departments of Urology, Medicine and Labomtory Medicine, University of Washington School of Medicine, Seattle, Washington, and Medical and Nursing Bureau, Population and Community Development Association, Bangkok, Thailand
ABSTRACT
Purpose: Human immunodeficiency virus type 1 (HIV) is cultured more often from seminal cells than seminal plasma. Because vasectomy causes dramatic reductions in seminal cells and also eliminates secretions from proximal sites in the male reproductive tract, vasectomy may change the potential infectiousness of semen. Materials and Methods. We used polymerase chain reaction (PCR) assays to measure HIV ribonucleic acid (RNA) in seminal plasma and HIV deoxyribonucleic acid (DNA) in seminal cells from 46 asymptomatic, seropositive men before and after vasectomy. Results. HIV RNA levels in semen correlated only weakly with blood levels (r = 0.22, p = 0.03). Of 183 semen specimens assayed for cell-free HIV RNA and proviral DNA 37 (20%)were positive for HIV RNA only, 41 (22%)were positive for HIV DNA only, and 18 (10%)were positive for RNA and DNA. Thus, detection of HIV RNA in seminal plasma was not associated with detection of HIV DNA in seminal cells. HIV RNA was present in 23 of 82 specimens (28%)(mean 2.87 log copiedml.) before vasectomy and in 38 of 121 specimens (31%)after vasectomy (mean 2.81 log copiedml.). Conclusions. These findings suggest that direct measurement of HIV levels in semen is necessary to assess the potential for sexual transmission, most cell-free HIV in seminal plasma arises distal to the vas deferens, and vasectomy may have minimal impact on the infectiousness of HIV seropositive men on sexual partners. KEY WORDS: vasectomy, HIV, semen, polymerase chain reaction, transmission
Epidemiological studies implicate direct contact with semen from seropositive men as the primary route for sexual transmission of human immunodeficiency virus (HIVl.1-4 HIV has been cultured more often from seminal cells than from cell-free seminal plasma. Seminal leukocytes,5.6 germ ~ e l l s , ~testes,9 .s epididymides? prostatelo and urethral and paraurethral glands11.12 have been implicated as potential reservoirs for HIV in the male genital tract. Although we have only limited information on the relationship between systemic host factors and levels of potentially infectious HIV in the semen, the male reproductive tract appears to be distinct immunologically from the systemic immune compartment.13-24 There is a need to understand the biology of HIV within the male urogenital tract, particularly the anatomical origins and sources of HIV, and the effects of therapeutic interventions on the potential for HIV seropositive men to infect sexual partners. We reasoned that important insights into the anatomical sources of HIV in the semen could be achieved by combining a well established surgical approach, vasectomy, with molecular methods for measuring HIV viral load. Vasedomy is one of the safest, most effective, widely accepted and available methods for contraception.26 The procedure disrupts the vas deferens at a relatively distal site in the male reproductive
tract and causes marked changes to the seminal cell populations by eliminating sperm and reducing seminal leukocyte counts by approximately 20-fold.26327 Our hypothesis was that vasectomy might decrease HIV levels in semen by eliminating proximal sources of HIV from germinal cells and leukocytes as well as secretions from the testes, rete testes, epididymides and proximal vasa deferentia. In contrast, there would be no change in seminal HIV originating from more distal sites, such as seminal vesicles, prostate, urethra, and periurethral and bulbourethral glands. MATERIALS AND METHODS
Subjects, clinical procedures and samples. We recruited 46 HIV seropositive Thai men from the Family Planning Clinic at the Population and Community Development Association, the AIDS Outpatient Clinic at Pramongkutklao Army Hospital and the Royal Thai Army Military Region 11 Prison (Bangkok).Subjects were enrolled followingverbal and written informed consent (in Thai) using a protocol approved by human subjects committees at Chulalonkorn University and the University of Washington. Median participant age was 25 years (mean 27.1, standard deviation 5.2). All subjects were heterosexual, had used intravenous heroin and were asymptomatic (Centers for Disease Control stage A) with no clinical evidence or history of active sexually transmitted diseases. Participants provided a semen and blood serum specimen at the enrollment visit. A week later each subject provided a second semen specimen and then underwent "no-scalpel"va~ e c t o m y Following .~~ vasectomy 3 additional semen specimens were provided at 1-month intervals with an additional
Accepted for publication September 19, 1997. Su ported in part by Grants RO1 D9407747, R 0 3 "woo204 and R019WAI49477 from the National Institutes of Health, Bethesda, Maryland. Editor'e Note: This article is the third of 6 published in this issue for which category 1 CME credits can be earned. Instructiona for obtaining credits are given with the questions on pages 1040 and 1041. 820
VASECTOMY AND HUMAN IMMUNODEFICIENCYVIRUS TYPE 1 IN SEMEN
821
blood serum sample at the final visit. The sampling protocol was designed to maximize subject compliance and to allow Correlation between systemic viral load and H N RNA level sufficient time to eliminate sperm and reduce seminal leuko- 1,n seminal plasma. HIV RNA was detected in 60 of 203 cyte counts after vasectomy. Of the 46 subjects 41 (89%) e:valuable seminal plasma specimens (30%)and at least once completed all 5 visits. in 27 of the 46 men (59%) a t concentrations from less than Overall, 216 semen specimens and 88 blood serum samples 4LOO to 9,040 copies per ml. (mean 2.84, range less than 2.60 were collected. The semen specimens were provided by mas- t,o 3.96). In contrast, HIV RNA was detected at least once in turbation into sterile plastic containers, and then maintained 1:ilood serum of all men and in 86 of the 88 blood specimens on ice during transportation to Population and Community (98%) at concentrations from less than 200 to 93,300 HIV Development Association Headquarters for processing. Each 1XNA copies per ml. (mean 3.87, range less than 2.30 to 4.97). semen sample was divided into 0.5 to 1.0 ml. aliquots within 1JIV RNA levels in the seminal plasma correlated only 2 hours of collection. Samples were mixed with equal vol- 1weakly with the viral load in the blood serum for the entire umes of fetal calf serum, cryopreserved in liquid nitrogen and (iata set (Spearman rank correlation rho 0.215, p = 0.03). shipped to Seattle. Seminal plasma and cells were separated 1However, when analysis was limited to values above the by centrifugation (2,940 X g. for 10 minutes). i3ssay detection limit (noncensored values) this relationship Quantitative polymerase chain reaction (PCR) assays for 1was of marginal significance (Pearson correlation coefficient cell-free H N ribonucleic acid (RNA) and proviral HIV de- I3.426, p = 0.054, fig. 1). oxyribonucleic acid (DNA). Virion associated RNA was pelBecause multiple HIV viral clades are present in Thailand, leted (100,000 X g. for 1 hour) and extracted following the 2 PCR assays were used to compare HIV RNA levels in a protocol of Chomczynski and Sacchi.28The dried RNA pellet subset of seminal plasma and blood serum samples. Since the was resuspended in 50 pl. sterile ribonuclease-free water, assays used different primer sets within the HIV gag gene, and then HIV RNA was assayed by reverse transcription and they might detect viral clades with varying efficiencies. The quantitative competitive PCR.29 Briefly, a 260 bp fragment of number of samples tested by the quantitative competitive the HIV gag region was amplified using primers GAG04 and PCR was smaller than the number tested by the reverse GAGO6. Initially, reactions containing 5 p1. RNA were set up transcription PCR due to limited sample volumes. HIV RNA with varying amounts of a competitor transcript (from was detected in 34 of 36 blood serum samples (94%) evalupQPlA80, 0 to 500 copies for seminal plasma and 0 to 5,000 ated by both assays. The mean log difference between the 2 copies for blood serum specimens). RNA was reverse trans- measurements was -0.100 log copiedml. (95% confidence cribed and 30 p1. reaction mixture containing the primers interval -0.338 to 0.138, p = 0.40 by paired t test). In were added. Following amplification products were sepa- contrast, HIV RNA was detected in only 6 of 40 semen samrated by electrophoresis using a 2% Synergel-l% agarose gel, ples (15%) evaluated by both PCR assays, including 4 stained with ethidium bromide and visualized by ultraviolet samples positive by both tests. HIV RNA was detected in low illumination. copy numbers in the 2 discordant semen samples. Thus, the A second reverse transcription based PCR assay was also 2 HIV RNA assays provided comparable results in this study used to measure HIV RNA levels in seminal plasma and population. blood serum. Because 41% of seminal specimens in a previAt the start of the study we planned to culture the seminal ous study inhibited the quantitative competitive HIV PCR cells and the seminal plasma separately for each specimen. assay,3O we used a modified silica extraction protocol to re- ARer transportation to Seattle we found f b g a l contaminamove potential inhibitors in the seminal plasma sam- tion in either the seminal cell or the seminal plasma cople~.~0.31 HIV RNA copies per ml. were reported for undiluted culture from almost all 20 specimens cultured. There were no seminal plasma. For statistical analyses HIV RNA values positive cultures in this group, which may reflect the tropical below analytical sensitivity (400 copies per ml. for seminal climate in Bangkok and that almost all Thai men are uncirplasma and 200 copies per ml. for blood serum) were assigned cumcised. We tried adding antifungal agents but these values of 200 and 100 copies per ml., respectively. proved toxic to the donor cells used in the co-culture system. Proviral HIV DNA in the cellular pellet was extracted Therefore, we chose to evaluate the samples using molecular according to the protocol of Mermin et a1,6 and then 1 pg. methods. DNA was amplified in separate reactions for the gag gene of HIV and for HLA-DQ to confirm presence and amplification of DNA.32 Liquid hybridization was performed using the 32 3.8. * ' b phosphorus labeled SK19 oligonucleotide probe.32 The number of HIV DNA copies in each reaction mixture was 3.60 0 I 0 calculated by comparing standards with 0, 5, 10 and 100 0 0 3.4ACH-2 cells (containing 1HIV DNA copy per cell) that were 00 0 3.2. 0 run with each amplification.33 The analytical sensitivity of the assay was 5 or greater HIV DNA copies per pg. cellular 3. i 0 DNA. 2.8Statistical analyses. The relationship between systemic vii.-.-...-..-. 2.6: d??...........-.--..-..-. 1 ral load and HIV RNA level in seminal plasma was evaluated I using linear regression. Quantitative HIV RNA results in 2.4blood serum by the 2 PCR assays were compared usmg the 2.2paired t test. McNemar's chi-square test was used to compare 20 ~ 0 0 Q) 0 ~ 0 qualitative results of the 2 HIV RNA assays in Seminal 1.81 - . - ' . . . . . - . - . - . . c plasma and to compare detection of proviral HIV DNA seminal cells to cell-free HIV RNA in seminal plasma. HIV RNA levels in seminal plasma before and after vasectomy Were Compared using the Wilcoxon signed rank test. Seminal FIG.1. Association between blood serum and seminal plasma Proviral HIV DNA assay results before and after vasectomy RNA copies per ml. by reverse transcription PCR for 72 Were compared using the paired t test following transforma- HIV-1 specimens. Left censored HIV-1RNA values are shown for blood tion to an ordinal scale of 1-less than 5, 2-5 or greater, serum (2.3log copies per ml.) and seminal plasma (2.6 lo copies per L-greater than 10 to 100 or less, and 4 - p a t e r than 100 ml.). For noncensored values Pearson correlation coe&cient wa8 0.426 (p = 0.054). HIV-1DNA proviral copies per pg. total seminal DNA. RESULTS
.
%,"a
822
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN
Comparison of proviral H N DNA in seminal cells to cellH N R N A in seminal plasma. HIV DNA was detected in 67 of 196semen specimens (34%) from 36 of 44 subjects (82%) evaluated. Among 183samples tested for HIV RNA and HIV DNA 18 (10%)were positive for both, 37 (20%) were positive for HIV RNA only, 41 (22%)were positive for HIV DNA only and 87 (48%) contained neither HIV RNA nor HIV DNA (McNemar's chi-square 0.02, p = 0.73). Thus, cell-free HIV RNA in seminal plasma was not associated with proviral HIV DNA in seminal cells. Effect of vasectomy on seminal H N RNA and HIV DNA levels. HIV RNA levels were stable in seminal plasma and blood serum throughout the 3-month study. Cell-free HIV RNA was present in 23 of 82 specimens (28%)(mean 2.87 log copies per ml.) before vasectomy and in 38 of 121 specimens (31%) aRer vasectomy (mean 2.81 log copies per ml., fig. 2). As expected blood serum HIV RNA levels were also stable (mean 3.87 at visit 1 and 3.86 log copies per ml. at visit 5). Proviral HIV DNA was detected in 9 of 79 seminal cell specimens (11%) before vasectomy compared to 58 of 117 specimens (50%) aRer vasectomy (p <0.001, fig. 3). Thus, vasectomy had no effect on recovery of cell-free HIV RNA in seminal plasma but increased detection of proviral HIV DNA (per pg. DNA) in seminal cells.
Pre-vasectomy
free
DISCUSSION
Our study provides important insights into the anatomical sites and origins of HIV in semen. Our findings did not support the hypothesis that vasectomy could decrease HIV levels in semen by eliminating HIV from germinal cells, leukocytes and secretions from proximal sites in the male reproductive tract. To date there has been only limited information on the effect of vasectomy on HIV shedding but our quantitative assay results are consistent with those of Anderson et a1 who used a qualitative assay to detect cell associated HIV DNA in semen from 4 vasectomized men.27In our study vasectomy produced no reduction in seminal HIV levels in 46 subjects. Therefore, more distal sites, such as seminal vesicles, prostate, urethra, and periurethral and bulbourethral glands, must be considered as potentially important sources of seminal HIV in asymptomatic, seropositive men. Previous anatomical studies of men dying of acquired immunodeficiency syndrome with high viral loads may hold little rele-
4
0
0 0 0
1.75j
I
0
3
4
i
b 6?6I .
1
2
I
5
t
Study visit number
E'lc. 2. Comparison of median (25 to 75 interquartile range) HIV-1 RNA log copy number per ml. of seminal plasma by study visit. Wilcoxon signed rank for each visit indicated no significant differences between values before vasectomy and a h r vasectomy. Data are presented as box plot displa 10th (lower bar), 25th (lower margin ofbox), 50th (line withm E L x ) , 75th (up r margin of box) and 90th (u per bar) percentiles of seminal HIV-1 &A copy number at each stufy visit. Box re resents 25 to 75 interquartile range (from 25th to 75th percentile). 8utliex-a (data outside 90th percentile) are presented as mdivldual data points.
N=39
Post-vasectomy
N=40
N=39
N=40
N=38
4.5 g!
s,
4
-
5'
0
0
3 4 Study visit number
5
0
I
1
2
FIG.3. HIV-1 DNA proviral copy number plotted as median ordinal score (25 to 75 interquartile range). Mean HIV roviral DNA scores indicate that values after vasectomy were sigdcantly different from those before vasectomy (p = 0.001 paired t test). Ordinal scale is 1-leas than 5 , C or greater, - t e r , t h a n 10 to 100 or ater than 100 HIV-1 D A pronral co ies per pg. greater, and 4total seminal Dr$f;"Data are presented as box plot dis paying loth (lower bar), 25th (lower margin of box), 50th (line witiin the box), 75th (upper margin of box) and 90th (up r bar) percentiles of HIV-1 DNA proviral copy number at each sturvisit. Box represents 25 to 75 interquartile range (from 25th to 7 5 x percentile). Outliers (data outside 90th percentile) are presented as individual data points. vance to healthy asymptomatic rnen.g.lo~~~ As shown in this and other studies, such healthy, seropositive men shed HIV in the semen.21.30, 35*36 Epidemiologically this population may be important for sexual transmission of HIV. Systemic parameters, such as viral load, CD4+ cell count or antinucleoside monotherapy, might predict the potential infectiousness of semen as assessed by viral culture,37 perhaps among patients with more advanced infections. However, this study emphasizes that shedding of HIV occurs in semen of healthy seropositive men. Other studies reported that CD4+ cell counts in peripheral blood and antinucleoside therapy had minimal impact on HIV shedding in semen as assessed by viral culture21924 or qualitative PCR assays.6.36 In our study HIV RNA levels in blood serum were more than 10-fold higher than HIV RNA levels in seminal plasma. Furthermore, HIV RNA levels in blood serum correlated only weakly with HIV RNA levels in seminal plasma and did not correlate with HIV DNA levels in seminal cells. These findings suggest that measures of the HIV viral burden in blood cannot predict the potential infectiousness of semen accurately for individual men. This idea agrees with observations that the male reproductive tract is distinct immunologically from the systemic immune compartment.22 The implication is that direct measurement of HIV in genital secretions is necessary to estimate potential infectiousness. In contrast to limited effect of systemic host factors, local urogenital tract factors may influence shedding of HIV in semen. For example, epidemiological studies clearly show that cofactors, including ulcerative sexually transmitted diseases, such as syphilis,3S chancroid39.40 and genital herpes virus infe~tion,~1.42 as well as nonulcerative sexually transmitted diseases43are associated with increased transmission of HIV. Seminal shedding of cytomegalovirus has also been associated with an increased risk for HIV shedding in the semen24 and increased risk for development of acquired immunodeficiency syndrome.44.45 Active urethritis and seminal inflammation are also associated with the increased HIV levels in the semen and increased potential for sexual transmi~sion.16.4'3.~~ We limited the potential effects of these factors by excluding potential subjects with clinical evidence or risk factors for active urogenital tract infections other than HIV.
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN
823
HIV RNA levels in seminal plasma were relatively stable amount of HIV. We believe that this is unlikely based on during the 3-month study, suggesting that vasectomy did not similar results with PCR assays and independent investigaalter production of cell-free virus. Cell associated proviral tions of other specimens from Thailand (data not presented) DNA was detected more often followingvasectomy. The great and experimental data from other investigators.57-58 majority of cellular DNA in semen before vasectomy origiWe are beginning to understand the anatomical sites and nates from germinal cells. Although vasectomy results in sources of HIV in the male genital tract. Because the male substantial reduction of seminal leukocyte counts, most re- reproductive tract is distinct from the systemic immune commaining seminal cells are leukocytes. Thus, by eliminating partment, there is no reliable systemic marker for potential sperm vasectomy greatly increases the proportion of seminal infectiousness. Studies must include direct sampling of genleukocytes in the cellular fraction of the ejaculate. The result ital sites and secretions. Semen represents the compilation of is that vasectomy concentrates the number of infected sem- fluids and cells from many sources, including testes, epididinal leukocytes and, thus, the proviral HIV DNA copy num- ymides, rete testes, vasa deferentia, prostate, seminal vesiber per pg. of total DNA in the seminal cell pellet.6 Because cles, urethra, and paraurethral and bulbourethral glands. the PCR assay measures HIV DNA per pg. of total DNA in Previous investigations examined cell populations within sethe sample (predominantly sperm DNA before vasectomy men,6~7.51.59,60 yet there is no consensus on the predominant with a minor component of leukocyte DNA and almost all source of potentially infectious HIV. Based on current conleukocyte DNA after vasectomy), comparison of HIV DNA cepts of systemic infection macrophagedmonocytes and other before versus after vasectomy is difficult despite use of an lymphoid cells are likely important. Our studies indicate that optimized PCR assay. While precise measurement of proviral germinal cells are an unlikely source of HIV and that much HIV DNA before versus aRer vasectomy is imprecise, our cell-free and cell associated HIV arises distal to the vas data indicate that infected seminal cells and cell-free HIV deferens. HIV has been detected in pre-ejaculatory fluid and RNA enter the male reproductive tract distal to the urethral secretions.11.12,61Re-ejaculatory fluid is produced transected vas deferens. by the urethral and bulbourethral glands (Littr6 and CowBecause there is limited information on the relative impor- per’s glands, respectively). Granulocytes, macrophages, tance of cell-free and cell associated HIV for sexual transmis- CD4+ and CD8+ lymphocytes have been described in presion of infection, we evaluated HIV RNA and HIV DNA. ejaculatory fluid.12 Infected cells may originate from the ureAlthough assessment of cultivable virus would also be desir- thral mucosa, prostate, seminal vesicles or ejaculatory ducts. able, technical problems limited our ability t o culture HIV Comprehensive sampling of multiple anatomical sites should from specimens in this study. Unfortunately, the gold stan- improve our understanding of the sources of infected cells dard remains documenting sexual transmission of HIV infec- and cell-free HIV, and may result in new approaches to limit tion. To our knowledge no laboratory assessment of HIV has sexual transmission. been clearly shown to correlate with transmission and, thus, the adequacy of a biological measure of HIV as a surrogate CONCLUSION determinant for transmission risk remains to be deterOur study has important clinical implications. Because mined.48 Culture documents presence of viable virus while the PCR assays document presence of viral RNA or DNA that blood levels of HIV RNA correlate only poorly with HIV RNA may not be associated with infectious virus. In vitro trans- levels in the semen, direct measurement of HIV levels in mission of HIV variants,49 and correlation between H N RNA semen is necessary to assess the potential for sexual transconcentration and the infectivity of seminal cells in cul- mission. Such measurements should be considered in clinical ture30.50 suggest that cell-free HIV may represent an impor- trials of antiretroviral agents. Multiple assays may be tant infectious component of semen. Other studies suggest needed since we found no correlation between HIV RNA and that cell associated HIV might be important for transmis- HIV DNA levels in the semen, and we do not know which sion.5,7,51.52Since vasectomy eliminates germinal cells from marker correlates with infectiousness. Because cell-free HIV the semen, the observation that vasectomy produced no sig- RNA and cell associated HIV DNA arise distal to the vas nificant change in either HIV RNA levels in seminal plasma deferens, future studies should concentrate on distal sites, or HIV DNA levels in seminal cells argues strongly against such as the prostate, seminal vesicles, urethra, and parauregerminal cells as an important reservoir for HIV in the se- thral and bulbourethral glands. These results also support men. This clinical finding agrees with data generated by efforts to limit inflammation at such distal sites to reduce some6*35but not all7 other investigators who used in vitro HIV levels in the semen. Finally, vasectomy may have minmethods to separate germinal cells from other cells in the imal impact on the infectiousness of healthy, HIV seroposisemen. In our study levels of HIV RNA in the seminal plasma tive men for sexual partners. did not correlate with levels of HIV DNA provirus, further Mr. Mechai Viravaida, President of the Population and supporting the idea that much cell-free virus in the seminal Community Development Association, and its staff provided plasma arises from sources other than germinal cells and study support and encouragement; members of the Departseminal leukocytes. ment of Medicine, Neuropsychiatry Section, Pramongkutklao Presence of multiple viral clades represents a potential Army Hospital, Royal Thai Army cooperated with the study, limitation of this study. In Thailand seroprevalence in- and Joel Gibson, University of Washington, provided technicreased from 1% in 1988 to 32 to 43% among IVDUs and cal assistance. 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Microbiol., 28: D.: Pre-ejaculatory fluid as potential vector for sexual transmission of HIV-1 Detterl. Lancet, 340 1470, 1992. 495,1990. 32. Ou, C. Y., Kwok, S., Mitchell, S. W., Mack, D. H., Sninsky, J. J., 13. Holmberg, S. D., Horsburgh, C. J., Ward, J . W. and Jaffe, H. W.: AIDS commentary. Biologic factors in the sexual transmission Krebs, J. W., Feorino, P., Warfield, D. and Schochetman, G.: DNA amplification for direct detection of HIV-1 in DNA of of human immunodeficiency virus. J . Infect. Dis., 160:116, peripheral blood mononuclear cells. Science, 239 295, 1988. 1989. 14. Speck, C. E., Coombs, R. W., Koutsky, L. A., Zeh, J., Ross, S. O., 33. Folks, T. M., Clouse, K. A., Justement, J., Rabson, A., Duh, E., Kehrl, J . H. and Fauci, A. S.: Tumor necrosis factor alpha Hooton, T. M., Collier, A. C., Corey, L., Cent, A., Dragavon, J., induces expression of human immunodeficiency virus in a Lee, W., Johnson, E. J., Sampoleo, R. R. and Krieger, J. N.: chronically infected T-cell clone. Proc. Natl. Acad. Sci., 88: Risk factors for HIV-1 shedding in semen. Amer. J . Epidemiol., submitted for publication, 1997. 2365, 1989. 15. Cohen, M. S.,Hoffman, I. R., Royce, R. A., Kazembe, P., Dyer, 34. Dalton, A. D. and Harcourt, N. W. J.: The histopathology of the testis and epididymis in AIDS-a post-mortem study. J. Path., J . R., Daly, C. C., Zimba, D., Vernazza, P. L., Maida, M., 163:47, 1991. Fiscus, S. A. and Eron, J . J.: Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for 35. Hamed, K. A., Winters, M. A., Holodniy, M., Katzenstein, D. A. prevention of sexual transmission of HIV-1. Lancet, 349 1868, and Merigan, T. C.: Detection of human immunodeficiency 1997. virus type in semen: effects of disease stage and nucleoside 16. Coombs, R., Speck, C., Lee, W., Sampoleo, R., Ross, S.,Dragavon, therapy. J. Infect. Dis., 161: 798, 1993. J., Peterson, G., Hooton, T., Collier, A., Corey, L., Hughes, J., 36. Ho, D. D., Schooley, R. T., Rota, T. R., Kaplan, J. C., Flynn, T., Koutsky, L. and Krieger, J.: Association between culturable Salahuddin, S. Z., Gonda, M. A. and Hirsch, M. S.: HTLV-I11 in HIV-1 in semen and RNA levels is semen and blood. J. Infect. the semen and blood of a healthy homosexual man. Science, Dis., accepted for publication, 1997. 226 451,1984. 17. Dyer, J. R., Gilliam, B. L., Eron, J. J. J., Cohen, M. S., Fiscus, 37. Anderson, D. J., OBrien, T. R., Politch, J. A., Martinez, A, S. A. and Vernazza, P. L.: Shedding of HIV-1 in semen during Seage, G. R. 3rd, Padian, N., Horsburgh, C. R. J . and Mayer, primary infection. Aids, 11: 543, 1997. K. H.: Effects of disease stage and zidovudine therapy on the 18. Gilliam, B. L., Dyer, J. R., Fiscus, S. A., Marcus, C., Zhou, S., detection of human immunodeficiency virus type 1 in semen. Wathen, L., Freimuth, W. W., Cohen, M. S. and Eron, J. J. J.: J.A.M.A., 267: 2769, 1992. Effects of reverse transcriptase inhibitor therapy on the HIV-1 38. Seage, G. R. 3rd, Horsburgh, C. J., Hardy, A. M., Mayer, K. H., viral burden in semen. J. Acq. Immun. Def. Syn. Hum. RetroBarry, M. A., Groopman, J. E., Jaffe, H. W. and Lamb, G. A: virol., 1 5 54, 1997. Increased suppressor T cells in probable transmitters of hu19. Mellors, J. W., Munoz, A, Giorgi, J. V., Margolick, J . B., Tassoni, man immunodeficiency virus infection. Amer. J . Pub. Health, C. J., Gupta, P., Kingsley, L. A., Todd, J. A., Saah, A. J., Detels, 79 1638, 1989. R., Phair, J . P. and Rinaldo, C. R.: Plasma viral load and 39. Plourde, P. J., D’Costa, L. J., Agoki, E., Ombette, J., Ndinya, CD4+ lymphocytes as prognostic markers of HIV-1 infection. A. J . O., Slaney, L. A., Ronald, A. R. and Plummer, F. A.: A Ann. Intern. Med., 126:946, 1997. randomized, double-blind study of the efficacy of fleroxacin 20. Wood,B., Shacker, T., Krieger, J., Corey, L. and Gown, A,: versus trimethoprim-sulfamethoxazole in men with cultureMorphologic, immunophenotypic and virologic collelates of reproven chancroid. J. Infect. Dis., 166 949, 1992. cent lymph node infection by HIV in a cohort of known infec- 40. Plummer, F. A., Wainberg, M. A., Plourde, P., Jessamine, P., tion duration. Hum. Path., submitted for publication, 1997. DCosta, L. J., Wamola, I. A. and Ronald, A. R.: Detection of 21. Krieger, J. N., Coombs, R. W., Collier, A. C., Ross, S. O., human immunodeficiency virus type 1(HIV-1) in genital ulcer Cummings, D. K., Murphy, V. L. and Corey, L.: Recovery of exudate of HIV-1-infected men by culture and gene amplificahuman immunodeficiency virus type 1 from semen: Minimal tion. J. Infect. Dis., 161: 810, 1990. impact of stage of infection and current antiviral chemother- 41. Holmberg, S. D., Stewart, J. A., Gerber, A. R., Byers, R. H., Lee, apy. J. Infect. Dis., 163:386, 1991. F. K., OMalley, P. M. and Nahmias, A. J.: Prior herpes sim22. Alexander, N. and Anderson, D.: Immunology of semen. Ferti. plex virus type 2 infection as a risk factor for HIV infection. Steril., 41: 192, 1987. J.A.M.A., 259 1048, 1988. 23. Krieger, J., Coombs, R., Collier, A, Ho, D., Ross, S., Zeh, J . and 42. Hook, E. W. 111, Cannon, R. O., Nahmias, A. J., Lee, F. F., Corey, L.: Intermittent shedding of human immunodeficiency Campbell, C. H. J., Glasser, D. and Quinn, T. C.: Herpes virus in semen: implications for sexual transmission. J. Urol., simplex virus infection as a risk factor for human immunode1&4:1035. 1995. ficiency virus infection in heterosexuals. J . Infect. Dis.. 1m 251, 1992. 24. Krieger, J.,Coombs. R., Collier, A., Ross,S.,Speck, C. and Corey,
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EDITORIAL COMMENT 43. Wasserheit, J . N.: Epidemiological synergy. Interrelationships between human immunodeficiency virus infection and other The authors have performed an interesting study to evaluate the sexually transmitted diseases. Sex Transm. Dis., 19 61, 1992. effect of vasectomy on levels of HIV-1 in semen. At least 28 million 44. Demmler, G. J., Buffone, G. J., Schimbor, C. M. and May, R. A,: people worldwide are infected with HIV-1, which is primarily transDetection of cytomegalovirus in urine from newborns by using mitted through sexual intercourse.1 Interventions are being actively polymerase chain reaction DNA amplification. J. Infect. Dis., sought to control the HIVIacquired immunodeficiency syndrome ep158 1177, 1988. idemic. The authors speculate that vasectomy could reduce HIV-1 45. Detels, R., Leach, C. T., Hennessey, K., Liu, Z., Visscher, B. R., levels in semen by reducing numbers of infected cells and viral Cherry, J . D. and Giorgi, J . V.: Persistent cytomegalovirus particles in semen. White blood cells (WBCs) in the testis and epiinfection of semen increases risk of AIDS. J . Infect. Dis., 1 6 9 didymis can carry HIV-1 (reference 9 in article), and levels of these 766, 1994. cells are dramatically reduced in semen following vasectomy (refer46. Suwanagool, S., Sonjai, A., Ratanasuwan, W., Techasathit, W. ence 26 in article), although 1preliminary report demonstrated that and Chuenarom, V.: Risk factors for HIV infection among Thai some vasectomized individuals have high seminal leukocyte counts laborers during 1992-1993. J. Med. Assn. Thai, 7 6 663, 1993. and HIV-1 in the semen (reference 27 in article). There is also some 47. Moss, G. B., Overbaugh, J., Welch, M., billy, M., Bwayo, J., evidence that immature germ cells (reference 52 in article) and Plummer, F. A,, Ndinya-Achola, J . O., Malisa, M. A. and sperm (reference 7 in article) carry HIV-1, although this subject is Kreiss, J . K.: Human immunodeficiencyvirus DNA in urethral controversial.2 secretions in men: association with gonococcal urethritis and The authors describe a prospectively designed study in which CD4 cell depletion. J. Infect. Dis., 1 7 2 1469, 1995. HIV-1 levels were measured by quantitative PCR assay in semen 48. DeGruttola, V., Fleming, T. R., Lin, D. and Coombs, R. W.: and blood of 46 HIV-1 seropositive Thai men before and a t 1.2 and Surrogate markers; are we being naive? J . Infect. Dis., in 3 months after no scalpel vasectomy. Followup was excellent with 41 press. of the subjects (89%)completing all visits. Levels of HIV RNA rep49. Zhu, T., Wang, N., Carr, A., Nam, D. S., Moor, J. R., Cooper, D. A. resenting cell-free virus particles were stable in serum and seminal and Ho, D. D.: Genetic characterization of human immunode- plasma throughout the study but, surprisingly, the prevalence of ficiency virus type 1 in blood and genital secretions: evidence proviral HIV DNA in the cellular fraction of semen was significantly for viral compartmentalization and selection during sexual increased after vasectomy (11versus 50%. p <0.001). It is speculated transmission. J. Virol., 7 0 3098, 1996. that this is attributable to a concentration effect of residual WBCs in 50. Dyer, J., Billiam, B., Eron, J., Grosso, L., Cohen, M. and Fiscus, semen after vasectomy due to decreased sperm numbers. The auS.: Quantitation of human immunodeficiency virus type 1 thors conclude that most HIV in semen arises distal to the vas RNA in cell free seminal plasma: comparison of NASBA and deferens and that vasectomy would appear to have no impact on the Amplicor reverse transcription-PCR ampolification land corre- infectiousness of HIV seropositive men for their sexual partners. lation with quantitative culture. J. Virol. Meth., 60:161,1996. The authors are to be congratulated for conducting the most de51. Bagasra, O., Freund, M., Weidmann, J. and Harley, G.: Interac- finitive study to date on this subject, with conclusions well supported tion of human immunodeficiency virus with human sperm in by quantitative data. However, a few questions remain to be anvitro. J . AIDS, 1: 431, 1988. swered. Followup was for only 3 months and neither seminal WBC 52. Nuovo, G. J., Becker, J., Simsi, A., Margiotta, M., Khalife, G. and nor sperm counts were reported. Vasectomy is known to cause conShevchuk, M.: HIV-1 nucleic acids localize to the spermatogo- siderable local inflammation: which could have increased WBC and nia and their progeny. A study by polymerase chain reaction in HIV-1 levels in genital tract secretions transiently. The authors do situ hybridization. Amer. J. Path., 144: 1142, 1994. not indicate if a microscopic examination was performed to detect 53. Kunanusont, C., Foy, H. M., Kreiss, J. K., Rerks-Ngarm, S., sperm in each post-vasectomy semen specimen. It has been shown Phanuphak, P., Raktham, S., Pau, C. P. and Young, N. L.: that sperm still are present after vasectomy in the semen of 35% of HIV-1 subtypes and male-to-femaletransmission in Thailand. men 1month postoperatively, in 14 to 16% a t 2 months and in 6 to 12% a t 3 month^.^,^ Thus, it cannot be assumed that germinal or Lancet, 3 4 5 1078, 1995. 54. Ou, C. Y., Takabe, Y., Luo, C. C., Kalish, M., Auwanit, W., other testicular cells were not a source of proviral DNA in some of the Bandea, C., de la Torre, N., Moore, J. L., Schochetman, G. and semen specimens. A longer followup, for example through 1 year Yamazaki, S.: Wide distribution of two subtypes of HIV-1 in after vasectomy, would address these concerns. The other missing piece of information is whether vasectomy has Thailand. AIDS Res. Hum. Retrovir., 8 1471, 1992. 55. Anderson, R. M., Schwartslander, B., McCutchan,F. and Hu, D.: an effect on the infectious potential of HIV-1 in semen. The PCR Implications of genetic variability in HIV for epidemiology and assay used in this study measured HIV DNA and RNA transcripts but it cannot determine whether the virions are capable of infecting public health. Lancet, 341: 1778, 1996. 56. Soto-Ramirez, L. E., Renjifo, B., McLane, M. F., Marlink, R., target cells in the genital tract. A number of factors in semen, OHara, C., Sutthent, R., Wasi, C., Vithayasai, P., Vithayasai, including anti-HIV antibodies, complement, zinc, defensins and free V., Apichartpiyakul, C., Auewarakul, P., Pena, C. V., Chui, oxygen radicals, can inhibit HIV-1 infection: and little is known D. S., Osathanondh, R., Mayer, K., Lee, T. H. and Essex, M.: about the genital tract origin of some of these factors or the effects of HIV-1 Langerhans’ cell tropism associated with heterosexual vasectomy on levels of these factors in semen. The observation of increased proviral HIV-1 levels in semen after vasectomy should also transmission of HIV.Science, 271: 1291, 1996. 57. Lu, S.-D., Christopherson, C. and Wang, J.: Extended range of be followed with infectious titer studies to address the possibility non-B serotypes detected by a modified aplicor HIV-1 monitor that HIV-1 transmission could be enhanced after vasectomy through test (abstract). Presented a t the 4th Conference on Retrovi- induction of local inflammation. This study underscores the need for more research to understand ruses and Opportunistic Infections, Washington, D. C., Janubetter sites and mechanisms of HIV-1 infection of the genital tract in ary 22, 1997. 58. Robb, M. M., Birx, D. and Wang, J.: Performance of the amplicor men and women, and approaches other than vasectomy to inhibit or HIV-1 mentor test and a modified HIV-1 monitor test on HIV-1 block the sexual transmissions of this dangerous virus. Important subtypes A to F (abstract).Presented at the 4th Conference on avenues being investigated a t present include vaginal microbicides Retroviruses and ODDortunistic Infections, Washington, D. c., and mucosal vaccine^.^^* Arnold M. Belker January 22, 1997. Division of Urology 59 wolff, H. and Anderson, D. J.: Male genital tract inflarmfation Department of Surgery associated with increased numbers of potential human immuUniversity of Louisville School of Medicine nodeficiency virus host cells in semen. Andrologia, 20: 404, Louisville, Kentucky 1988. and 60. G e g e r , J. N., Coombs, R. W., Collier, A. C., Koehler, J. K., Ross, Debomh J. Anderson s. o., Chaloupka, K., Murphy, V. L. and Corey, L.: Fertility Department of Obstetrics, Gynecology Parameters in men infected with human immunodeficiency and Reproductive Bwlogv &-us. J . Infect. Dis., 1tM 464, 1991. Brigham and Women’s Hospital 61. Ilaria, G., Jacobs, J. L.,Polsky, B., KO&B., Baron, P., MacLow, Harvard Medical School c., Armstrong, D. and Schlegel, P. N.: Detection of HIV-1 DNA Boston, Massachusetts sequences in pre-ejaculabq fluid. Lancet, 340:1469,1992.
..
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN
825
EDITORIAL COMMENT 43. Wasserheit, J . N.: Epidemiological synergy. Interrelationships between human immunodeficiency virus infection and other The authors have performed an interesting study to evaluate the sexually transmitted diseases. Sex Transm. Dis., 19 61, 1992. effect of vasectomy on levels of HIV-1 in semen. At least 28 million 44. Demmler, G. J., Buffone, G. J., Schimbor, C. M. and May, R. A,: people worldwide are infected with HIV-1, which is primarily transDetection of cytomegalovirus in urine from newborns by using mitted through sexual intercourse.1 Interventions are being actively polymerase chain reaction DNA amplification. J. Infect. Dis., sought to control the HIVIacquired immunodeficiency syndrome ep158 1177, 1988. idemic. The authors speculate that vasectomy could reduce HIV-1 45. Detels, R., Leach, C. T., Hennessey, K., Liu, Z., Visscher, B. R., levels in semen by reducing numbers of infected cells and viral Cherry, J . D. and Giorgi, J . V.: Persistent cytomegalovirus particles in semen. White blood cells (WBCs) in the testis and epiinfection of semen increases risk of AIDS. J . Infect. Dis., 1 6 9 didymis can carry HIV-1 (reference 9 in article), and levels of these 766, 1994. cells are dramatically reduced in semen following vasectomy (refer46. Suwanagool, S., Sonjai, A., Ratanasuwan, W., Techasathit, W. ence 26 in article), although 1preliminary report demonstrated that and Chuenarom, V.: Risk factors for HIV infection among Thai some vasectomized individuals have high seminal leukocyte counts laborers during 1992-1993. J. Med. Assn. Thai, 7 6 663, 1993. and HIV-1 in the semen (reference 27 in article). There is also some 47. Moss, G. B., Overbaugh, J., Welch, M., billy, M., Bwayo, J., evidence that immature germ cells (reference 52 in article) and Plummer, F. A,, Ndinya-Achola, J . O., Malisa, M. A. and sperm (reference 7 in article) carry HIV-1, although this subject is Kreiss, J . K.: Human immunodeficiencyvirus DNA in urethral controversial.2 secretions in men: association with gonococcal urethritis and The authors describe a prospectively designed study in which CD4 cell depletion. J. Infect. Dis., 1 7 2 1469, 1995. HIV-1 levels were measured by quantitative PCR assay in semen 48. DeGruttola, V., Fleming, T. R., Lin, D. and Coombs, R. W.: and blood of 46 HIV-1 seropositive Thai men before and a t 1.2 and Surrogate markers; are we being naive? J . Infect. Dis., in 3 months after no scalpel vasectomy. Followup was excellent with 41 press. of the subjects (89%)completing all visits. Levels of HIV RNA rep49. Zhu, T., Wang, N., Carr, A., Nam, D. S., Moor, J. R., Cooper, D. A. resenting cell-free virus particles were stable in serum and seminal and Ho, D. D.: Genetic characterization of human immunode- plasma throughout the study but, surprisingly, the prevalence of ficiency virus type 1 in blood and genital secretions: evidence proviral HIV DNA in the cellular fraction of semen was significantly for viral compartmentalization and selection during sexual increased after vasectomy (11versus 50%. p <0.001). It is speculated transmission. J. Virol., 7 0 3098, 1996. that this is attributable to a concentration effect of residual WBCs in 50. Dyer, J., Billiam, B., Eron, J., Grosso, L., Cohen, M. and Fiscus, semen after vasectomy due to decreased sperm numbers. The auS.: Quantitation of human immunodeficiency virus type 1 thors conclude that most HIV in semen arises distal to the vas RNA in cell free seminal plasma: comparison of NASBA and deferens and that vasectomy would appear to have no impact on the Amplicor reverse transcription-PCR ampolification land corre- infectiousness of HIV seropositive men for their sexual partners. lation with quantitative culture. J. Virol. Meth., 60:161,1996. The authors are to be congratulated for conducting the most de51. Bagasra, O., Freund, M., Weidmann, J. and Harley, G.: Interac- finitive study to date on this subject, with conclusions well supported tion of human immunodeficiency virus with human sperm in by quantitative data. However, a few questions remain to be anvitro. J . AIDS, 1: 431, 1988. swered. Followup was for only 3 months and neither seminal WBC 52. Nuovo, G. J., Becker, J., Simsi, A., Margiotta, M., Khalife, G. and nor sperm counts were reported. Vasectomy is known to cause conShevchuk, M.: HIV-1 nucleic acids localize to the spermatogo- siderable local inflammation: which could have increased WBC and nia and their progeny. A study by polymerase chain reaction in HIV-1 levels in genital tract secretions transiently. The authors do situ hybridization. Amer. J. Path., 144: 1142, 1994. not indicate if a microscopic examination was performed to detect 53. Kunanusont, C., Foy, H. M., Kreiss, J. K., Rerks-Ngarm, S., sperm in each post-vasectomy semen specimen. It has been shown Phanuphak, P., Raktham, S., Pau, C. P. and Young, N. L.: that sperm still are present after vasectomy in the semen of 35% of HIV-1 subtypes and male-to-femaletransmission in Thailand. men 1month postoperatively, in 14 to 16% a t 2 months and in 6 to 12% a t 3 month^.^,^ Thus, it cannot be assumed that germinal or Lancet, 3 4 5 1078, 1995. 54. Ou, C. Y., Takabe, Y., Luo, C. C., Kalish, M., Auwanit, W., other testicular cells were not a source of proviral DNA in some of the Bandea, C., de la Torre, N., Moore, J. L., Schochetman, G. and semen specimens. A longer followup, for example through 1 year Yamazaki, S.: Wide distribution of two subtypes of HIV-1 in after vasectomy, would address these concerns. The other missing piece of information is whether vasectomy has Thailand. AIDS Res. Hum. Retrovir., 8 1471, 1992. 55. Anderson, R. M., Schwartslander, B., McCutchan,F. and Hu, D.: an effect on the infectious potential of HIV-1 in semen. The PCR Implications of genetic variability in HIV for epidemiology and assay used in this study measured HIV DNA and RNA transcripts but it cannot determine whether the virions are capable of infecting public health. Lancet, 341: 1778, 1996. 56. Soto-Ramirez, L. E., Renjifo, B., McLane, M. F., Marlink, R., target cells in the genital tract. A number of factors in semen, OHara, C., Sutthent, R., Wasi, C., Vithayasai, P., Vithayasai, including anti-HIV antibodies, complement, zinc, defensins and free V., Apichartpiyakul, C., Auewarakul, P., Pena, C. V., Chui, oxygen radicals, can inhibit HIV-1 infection: and little is known D. S., Osathanondh, R., Mayer, K., Lee, T. H. and Essex, M.: about the genital tract origin of some of these factors or the effects of HIV-1 Langerhans’ cell tropism associated with heterosexual vasectomy on levels of these factors in semen. The observation of increased proviral HIV-1 levels in semen after vasectomy should also transmission of HIV.Science, 271: 1291, 1996. 57. Lu, S.-D., Christopherson, C. and Wang, J.: Extended range of be followed with infectious titer studies to address the possibility non-B serotypes detected by a modified aplicor HIV-1 monitor that HIV-1 transmission could be enhanced after vasectomy through test (abstract). Presented a t the 4th Conference on Retrovi- induction of local inflammation. This study underscores the need for more research to understand ruses and Opportunistic Infections, Washington, D. C., Janubetter sites and mechanisms of HIV-1 infection of the genital tract in ary 22, 1997. 58. Robb, M. M., Birx, D. and Wang, J.: Performance of the amplicor men and women, and approaches other than vasectomy to inhibit or HIV-1 mentor test and a modified HIV-1 monitor test on HIV-1 block the sexual transmissions of this dangerous virus. Important subtypes A to F (abstract).Presented at the 4th Conference on avenues being investigated a t present include vaginal microbicides Retroviruses and ODDortunistic Infections, Washington, D. c., and mucosal vaccine^.^^* Arnold M. Belker January 22, 1997. Division of Urology 59 wolff, H. and Anderson, D. J.: Male genital tract inflarmfation Department of Surgery associated with increased numbers of potential human immuUniversity of Louisville School of Medicine nodeficiency virus host cells in semen. Andrologia, 20: 404, Louisville, Kentucky 1988. and 60. G e g e r , J. N., Coombs, R. W., Collier, A. C., Koehler, J. K., Ross, Debomh J. Anderson s. o., Chaloupka, K., Murphy, V. L. and Corey, L.: Fertility Department of Obstetrics, Gynecology Parameters in men infected with human immunodeficiency and Reproductive Bwlogv &-us. J . Infect. Dis., 1tM 464, 1991. Brigham and Women’s Hospital 61. Ilaria, G., Jacobs, J. L.,Polsky, B., KO&B., Baron, P., MacLow, Harvard Medical School c., Armstrong, D. and Schlegel, P. N.: Detection of HIV-1 DNA Boston, Massachusetts sequences in pre-ejaculabq fluid. Lancet, 340:1469,1992.
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1. Royce, R. A., Sena, A, Cates, W. and Cohen, M. S.: Sexual transmission of HIV. New Engl. J. Med., 336:1072,1997. 2. Quayle, A. J., Xu, C., Mayer, K. H. and Anderson, D. J.: T lymphocytes and macrophages, but not motile spermatozoa, are a significaut source of human immunodeficiency virus in semen. J. Infect. Dis., 176 960,1997. 3. Alexauder, N. J. and Anderson,D. J.: Consequences of autoimmunity to sperm antigens (ModemTrends). Fertil. Steril., 32:253,1979. 4. Sivanesaratnam, V.: Onset of azwspermia after vasectomy. New Zeal. Med. J., 98: 331, 1985.
5. Esho, J. O.,Ireland, G. W. and Cass, A. S.: Recanalization following vasectomy. Urology, 3 211, 1974. 6. Anderson, D. J.: The importance of mucosal immunology to problems in human reproduction. J. Reprod. Immunol., 31: 3, 1996. 7. Elias, C. J. and Heise, L. L.: Challenges for the development of female-controlled vaginal microbicides. AIDS, 8: 1, 1994. 8. Haynes, B. V.: HIV vaccines: where we are and where we are going. Lancet, 348: 933,1996.
Vol. 159,820-826, March 1998 Printed in lJ.S.A
THE JOURNAL OF UROLOCY
Copyright 0 1998 by AMERICAN UROUIGICAL ASS~CIATION, INC.
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN JOHN N. KRIEGER, APICHART NIRAPATHPONGPORN, MONTCHAI CHAIYAPORN, GREGORY PETERSON, IRENA NIKOLAEVA, ROBERT AKRIDGE, SUSAN 0. ROSS AND ROBERT W. COOMBS From the Departments of Urology, Medicine and Labomtory Medicine, University of Washington School of Medicine, Seattle, Washington, and Medical and Nursing Bureau, Population and Community Development Association, Bangkok, Thailand
ABSTRACT
Purpose: Human immunodeficiency virus type 1 (HIV) is cultured more often from seminal cells than seminal plasma. Because vasectomy causes dramatic reductions in seminal cells and also eliminates secretions from proximal sites in the male reproductive tract, vasectomy may change the potential infectiousness of semen. Materials and Methods. We used polymerase chain reaction (PCR) assays to measure HIV ribonucleic acid (RNA) in seminal plasma and HIV deoxyribonucleic acid (DNA) in seminal cells from 46 asymptomatic, seropositive men before and after vasectomy. Results. HIV RNA levels in semen correlated only weakly with blood levels (r = 0.22, p = 0.03). Of 183 semen specimens assayed for cell-free HIV RNA and proviral DNA 37 (20%)were positive for HIV RNA only, 41 (22%)were positive for HIV DNA only, and 18 (10%)were positive for RNA and DNA. Thus, detection of HIV RNA in seminal plasma was not associated with detection of HIV DNA in seminal cells. HIV RNA was present in 23 of 82 specimens (28%)(mean 2.87 log copiedml.) before vasectomy and in 38 of 121 specimens (31%)after vasectomy (mean 2.81 log copiedml.). Conclusions. These findings suggest that direct measurement of HIV levels in semen is necessary to assess the potential for sexual transmission, most cell-free HIV in seminal plasma arises distal to the vas deferens, and vasectomy may have minimal impact on the infectiousness of HIV seropositive men on sexual partners. KEY WORDS: vasectomy, HIV, semen, polymerase chain reaction, transmission
Epidemiological studies implicate direct contact with semen from seropositive men as the primary route for sexual transmission of human immunodeficiency virus (HIVl.1-4 HIV has been cultured more often from seminal cells than from cell-free seminal plasma. Seminal leukocytes,5.6 germ ~ e l l s , ~testes,9 .s epididymides? prostatelo and urethral and paraurethral glands11.12 have been implicated as potential reservoirs for HIV in the male genital tract. Although we have only limited information on the relationship between systemic host factors and levels of potentially infectious HIV in the semen, the male reproductive tract appears to be distinct immunologically from the systemic immune compartment.13-24 There is a need to understand the biology of HIV within the male urogenital tract, particularly the anatomical origins and sources of HIV, and the effects of therapeutic interventions on the potential for HIV seropositive men to infect sexual partners. We reasoned that important insights into the anatomical sources of HIV in the semen could be achieved by combining a well established surgical approach, vasectomy, with molecular methods for measuring HIV viral load. Vasedomy is one of the safest, most effective, widely accepted and available methods for contraception.26 The procedure disrupts the vas deferens at a relatively distal site in the male reproductive
tract and causes marked changes to the seminal cell populations by eliminating sperm and reducing seminal leukocyte counts by approximately 20-fold.26327 Our hypothesis was that vasectomy might decrease HIV levels in semen by eliminating proximal sources of HIV from germinal cells and leukocytes as well as secretions from the testes, rete testes, epididymides and proximal vasa deferentia. In contrast, there would be no change in seminal HIV originating from more distal sites, such as seminal vesicles, prostate, urethra, and periurethral and bulbourethral glands. MATERIALS AND METHODS
Subjects, clinical procedures and samples. We recruited 46 HIV seropositive Thai men from the Family Planning Clinic at the Population and Community Development Association, the AIDS Outpatient Clinic at Pramongkutklao Army Hospital and the Royal Thai Army Military Region 11 Prison (Bangkok).Subjects were enrolled followingverbal and written informed consent (in Thai) using a protocol approved by human subjects committees at Chulalonkorn University and the University of Washington. Median participant age was 25 years (mean 27.1, standard deviation 5.2). All subjects were heterosexual, had used intravenous heroin and were asymptomatic (Centers for Disease Control stage A) with no clinical evidence or history of active sexually transmitted diseases. Participants provided a semen and blood serum specimen at the enrollment visit. A week later each subject provided a second semen specimen and then underwent "no-scalpel"va~ e c t o m y Following .~~ vasectomy 3 additional semen specimens were provided at 1-month intervals with an additional
Accepted for publication September 19, 1997. Su ported in part by Grants RO1 D9407747, R 0 3 "woo204 and R019WAI49477 from the National Institutes of Health, Bethesda, Maryland. Editor'e Note: This article is the third of 6 published in this issue for which category 1 CME credits can be earned. Instructiona for obtaining credits are given with the questions on pages 1040 and 1041. 820
VASECTOMY AND HUMAN IMMUNODEFICIENCYVIRUS TYPE 1 IN SEMEN
821
blood serum sample at the final visit. The sampling protocol was designed to maximize subject compliance and to allow Correlation between systemic viral load and H N RNA level sufficient time to eliminate sperm and reduce seminal leuko- 1,n seminal plasma. HIV RNA was detected in 60 of 203 cyte counts after vasectomy. Of the 46 subjects 41 (89%) e:valuable seminal plasma specimens (30%)and at least once completed all 5 visits. in 27 of the 46 men (59%) a t concentrations from less than Overall, 216 semen specimens and 88 blood serum samples 4LOO to 9,040 copies per ml. (mean 2.84, range less than 2.60 were collected. The semen specimens were provided by mas- t,o 3.96). In contrast, HIV RNA was detected at least once in turbation into sterile plastic containers, and then maintained 1:ilood serum of all men and in 86 of the 88 blood specimens on ice during transportation to Population and Community (98%) at concentrations from less than 200 to 93,300 HIV Development Association Headquarters for processing. Each 1XNA copies per ml. (mean 3.87, range less than 2.30 to 4.97). semen sample was divided into 0.5 to 1.0 ml. aliquots within 1JIV RNA levels in the seminal plasma correlated only 2 hours of collection. Samples were mixed with equal vol- 1weakly with the viral load in the blood serum for the entire umes of fetal calf serum, cryopreserved in liquid nitrogen and (iata set (Spearman rank correlation rho 0.215, p = 0.03). shipped to Seattle. Seminal plasma and cells were separated 1However, when analysis was limited to values above the by centrifugation (2,940 X g. for 10 minutes). i3ssay detection limit (noncensored values) this relationship Quantitative polymerase chain reaction (PCR) assays for 1was of marginal significance (Pearson correlation coefficient cell-free H N ribonucleic acid (RNA) and proviral HIV de- I3.426, p = 0.054, fig. 1). oxyribonucleic acid (DNA). Virion associated RNA was pelBecause multiple HIV viral clades are present in Thailand, leted (100,000 X g. for 1 hour) and extracted following the 2 PCR assays were used to compare HIV RNA levels in a protocol of Chomczynski and Sacchi.28The dried RNA pellet subset of seminal plasma and blood serum samples. Since the was resuspended in 50 pl. sterile ribonuclease-free water, assays used different primer sets within the HIV gag gene, and then HIV RNA was assayed by reverse transcription and they might detect viral clades with varying efficiencies. The quantitative competitive PCR.29 Briefly, a 260 bp fragment of number of samples tested by the quantitative competitive the HIV gag region was amplified using primers GAG04 and PCR was smaller than the number tested by the reverse GAGO6. Initially, reactions containing 5 p1. RNA were set up transcription PCR due to limited sample volumes. HIV RNA with varying amounts of a competitor transcript (from was detected in 34 of 36 blood serum samples (94%) evalupQPlA80, 0 to 500 copies for seminal plasma and 0 to 5,000 ated by both assays. The mean log difference between the 2 copies for blood serum specimens). RNA was reverse trans- measurements was -0.100 log copiedml. (95% confidence cribed and 30 p1. reaction mixture containing the primers interval -0.338 to 0.138, p = 0.40 by paired t test). In were added. Following amplification products were sepa- contrast, HIV RNA was detected in only 6 of 40 semen samrated by electrophoresis using a 2% Synergel-l% agarose gel, ples (15%) evaluated by both PCR assays, including 4 stained with ethidium bromide and visualized by ultraviolet samples positive by both tests. HIV RNA was detected in low illumination. copy numbers in the 2 discordant semen samples. Thus, the A second reverse transcription based PCR assay was also 2 HIV RNA assays provided comparable results in this study used to measure HIV RNA levels in seminal plasma and population. blood serum. Because 41% of seminal specimens in a previAt the start of the study we planned to culture the seminal ous study inhibited the quantitative competitive HIV PCR cells and the seminal plasma separately for each specimen. assay,3O we used a modified silica extraction protocol to re- ARer transportation to Seattle we found f b g a l contaminamove potential inhibitors in the seminal plasma sam- tion in either the seminal cell or the seminal plasma cople~.~0.31 HIV RNA copies per ml. were reported for undiluted culture from almost all 20 specimens cultured. There were no seminal plasma. For statistical analyses HIV RNA values positive cultures in this group, which may reflect the tropical below analytical sensitivity (400 copies per ml. for seminal climate in Bangkok and that almost all Thai men are uncirplasma and 200 copies per ml. for blood serum) were assigned cumcised. We tried adding antifungal agents but these values of 200 and 100 copies per ml., respectively. proved toxic to the donor cells used in the co-culture system. Proviral HIV DNA in the cellular pellet was extracted Therefore, we chose to evaluate the samples using molecular according to the protocol of Mermin et a1,6 and then 1 pg. methods. DNA was amplified in separate reactions for the gag gene of HIV and for HLA-DQ to confirm presence and amplification of DNA.32 Liquid hybridization was performed using the 32 3.8. * ' b phosphorus labeled SK19 oligonucleotide probe.32 The number of HIV DNA copies in each reaction mixture was 3.60 0 I 0 calculated by comparing standards with 0, 5, 10 and 100 0 0 3.4ACH-2 cells (containing 1HIV DNA copy per cell) that were 00 0 3.2. 0 run with each amplification.33 The analytical sensitivity of the assay was 5 or greater HIV DNA copies per pg. cellular 3. i 0 DNA. 2.8Statistical analyses. The relationship between systemic vii.-.-...-..-. 2.6: d??...........-.--..-..-. 1 ral load and HIV RNA level in seminal plasma was evaluated I using linear regression. Quantitative HIV RNA results in 2.4blood serum by the 2 PCR assays were compared usmg the 2.2paired t test. McNemar's chi-square test was used to compare 20 ~ 0 0 Q) 0 ~ 0 qualitative results of the 2 HIV RNA assays in Seminal 1.81 - . - ' . . . . . - . - . - . . c plasma and to compare detection of proviral HIV DNA seminal cells to cell-free HIV RNA in seminal plasma. HIV RNA levels in seminal plasma before and after vasectomy Were Compared using the Wilcoxon signed rank test. Seminal FIG.1. Association between blood serum and seminal plasma Proviral HIV DNA assay results before and after vasectomy RNA copies per ml. by reverse transcription PCR for 72 Were compared using the paired t test following transforma- HIV-1 specimens. Left censored HIV-1RNA values are shown for blood tion to an ordinal scale of 1-less than 5, 2-5 or greater, serum (2.3log copies per ml.) and seminal plasma (2.6 lo copies per L-greater than 10 to 100 or less, and 4 - p a t e r than 100 ml.). For noncensored values Pearson correlation coe&cient wa8 0.426 (p = 0.054). HIV-1DNA proviral copies per pg. total seminal DNA. RESULTS
.
%,"a
822
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN
Comparison of proviral H N DNA in seminal cells to cellH N R N A in seminal plasma. HIV DNA was detected in 67 of 196semen specimens (34%) from 36 of 44 subjects (82%) evaluated. Among 183samples tested for HIV RNA and HIV DNA 18 (10%)were positive for both, 37 (20%) were positive for HIV RNA only, 41 (22%)were positive for HIV DNA only and 87 (48%) contained neither HIV RNA nor HIV DNA (McNemar's chi-square 0.02, p = 0.73). Thus, cell-free HIV RNA in seminal plasma was not associated with proviral HIV DNA in seminal cells. Effect of vasectomy on seminal H N RNA and HIV DNA levels. HIV RNA levels were stable in seminal plasma and blood serum throughout the 3-month study. Cell-free HIV RNA was present in 23 of 82 specimens (28%)(mean 2.87 log copies per ml.) before vasectomy and in 38 of 121 specimens (31%) aRer vasectomy (mean 2.81 log copies per ml., fig. 2). As expected blood serum HIV RNA levels were also stable (mean 3.87 at visit 1 and 3.86 log copies per ml. at visit 5). Proviral HIV DNA was detected in 9 of 79 seminal cell specimens (11%) before vasectomy compared to 58 of 117 specimens (50%) aRer vasectomy (p <0.001, fig. 3). Thus, vasectomy had no effect on recovery of cell-free HIV RNA in seminal plasma but increased detection of proviral HIV DNA (per pg. DNA) in seminal cells.
Pre-vasectomy
free
DISCUSSION
Our study provides important insights into the anatomical sites and origins of HIV in semen. Our findings did not support the hypothesis that vasectomy could decrease HIV levels in semen by eliminating HIV from germinal cells, leukocytes and secretions from proximal sites in the male reproductive tract. To date there has been only limited information on the effect of vasectomy on HIV shedding but our quantitative assay results are consistent with those of Anderson et a1 who used a qualitative assay to detect cell associated HIV DNA in semen from 4 vasectomized men.27In our study vasectomy produced no reduction in seminal HIV levels in 46 subjects. Therefore, more distal sites, such as seminal vesicles, prostate, urethra, and periurethral and bulbourethral glands, must be considered as potentially important sources of seminal HIV in asymptomatic, seropositive men. Previous anatomical studies of men dying of acquired immunodeficiency syndrome with high viral loads may hold little rele-
4
0
0 0 0
1.75j
I
0
3
4
i
b 6?6I .
1
2
I
5
t
Study visit number
E'lc. 2. Comparison of median (25 to 75 interquartile range) HIV-1 RNA log copy number per ml. of seminal plasma by study visit. Wilcoxon signed rank for each visit indicated no significant differences between values before vasectomy and a h r vasectomy. Data are presented as box plot displa 10th (lower bar), 25th (lower margin ofbox), 50th (line withm E L x ) , 75th (up r margin of box) and 90th (u per bar) percentiles of seminal HIV-1 &A copy number at each stufy visit. Box re resents 25 to 75 interquartile range (from 25th to 75th percentile). 8utliex-a (data outside 90th percentile) are presented as mdivldual data points.
N=39
Post-vasectomy
N=40
N=39
N=40
N=38
4.5 g!
s,
4
-
5'
0
0
3 4 Study visit number
5
0
I
1
2
FIG.3. HIV-1 DNA proviral copy number plotted as median ordinal score (25 to 75 interquartile range). Mean HIV roviral DNA scores indicate that values after vasectomy were sigdcantly different from those before vasectomy (p = 0.001 paired t test). Ordinal scale is 1-leas than 5 , C or greater, - t e r , t h a n 10 to 100 or ater than 100 HIV-1 D A pronral co ies per pg. greater, and 4total seminal Dr$f;"Data are presented as box plot dis paying loth (lower bar), 25th (lower margin of box), 50th (line witiin the box), 75th (upper margin of box) and 90th (up r bar) percentiles of HIV-1 DNA proviral copy number at each sturvisit. Box represents 25 to 75 interquartile range (from 25th to 7 5 x percentile). Outliers (data outside 90th percentile) are presented as individual data points. vance to healthy asymptomatic rnen.g.lo~~~ As shown in this and other studies, such healthy, seropositive men shed HIV in the semen.21.30, 35*36 Epidemiologically this population may be important for sexual transmission of HIV. Systemic parameters, such as viral load, CD4+ cell count or antinucleoside monotherapy, might predict the potential infectiousness of semen as assessed by viral culture,37 perhaps among patients with more advanced infections. However, this study emphasizes that shedding of HIV occurs in semen of healthy seropositive men. Other studies reported that CD4+ cell counts in peripheral blood and antinucleoside therapy had minimal impact on HIV shedding in semen as assessed by viral culture21924 or qualitative PCR assays.6.36 In our study HIV RNA levels in blood serum were more than 10-fold higher than HIV RNA levels in seminal plasma. Furthermore, HIV RNA levels in blood serum correlated only weakly with HIV RNA levels in seminal plasma and did not correlate with HIV DNA levels in seminal cells. These findings suggest that measures of the HIV viral burden in blood cannot predict the potential infectiousness of semen accurately for individual men. This idea agrees with observations that the male reproductive tract is distinct immunologically from the systemic immune compartment.22 The implication is that direct measurement of HIV in genital secretions is necessary to estimate potential infectiousness. In contrast to limited effect of systemic host factors, local urogenital tract factors may influence shedding of HIV in semen. For example, epidemiological studies clearly show that cofactors, including ulcerative sexually transmitted diseases, such as syphilis,3S chancroid39.40 and genital herpes virus infe~tion,~1.42 as well as nonulcerative sexually transmitted diseases43are associated with increased transmission of HIV. Seminal shedding of cytomegalovirus has also been associated with an increased risk for HIV shedding in the semen24 and increased risk for development of acquired immunodeficiency syndrome.44.45 Active urethritis and seminal inflammation are also associated with the increased HIV levels in the semen and increased potential for sexual transmi~sion.16.4'3.~~ We limited the potential effects of these factors by excluding potential subjects with clinical evidence or risk factors for active urogenital tract infections other than HIV.
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN
823
HIV RNA levels in seminal plasma were relatively stable amount of HIV. We believe that this is unlikely based on during the 3-month study, suggesting that vasectomy did not similar results with PCR assays and independent investigaalter production of cell-free virus. Cell associated proviral tions of other specimens from Thailand (data not presented) DNA was detected more often followingvasectomy. The great and experimental data from other investigators.57-58 majority of cellular DNA in semen before vasectomy origiWe are beginning to understand the anatomical sites and nates from germinal cells. Although vasectomy results in sources of HIV in the male genital tract. Because the male substantial reduction of seminal leukocyte counts, most re- reproductive tract is distinct from the systemic immune commaining seminal cells are leukocytes. Thus, by eliminating partment, there is no reliable systemic marker for potential sperm vasectomy greatly increases the proportion of seminal infectiousness. Studies must include direct sampling of genleukocytes in the cellular fraction of the ejaculate. The result ital sites and secretions. Semen represents the compilation of is that vasectomy concentrates the number of infected sem- fluids and cells from many sources, including testes, epididinal leukocytes and, thus, the proviral HIV DNA copy num- ymides, rete testes, vasa deferentia, prostate, seminal vesiber per pg. of total DNA in the seminal cell pellet.6 Because cles, urethra, and paraurethral and bulbourethral glands. the PCR assay measures HIV DNA per pg. of total DNA in Previous investigations examined cell populations within sethe sample (predominantly sperm DNA before vasectomy men,6~7.51.59,60 yet there is no consensus on the predominant with a minor component of leukocyte DNA and almost all source of potentially infectious HIV. Based on current conleukocyte DNA after vasectomy), comparison of HIV DNA cepts of systemic infection macrophagedmonocytes and other before versus after vasectomy is difficult despite use of an lymphoid cells are likely important. Our studies indicate that optimized PCR assay. While precise measurement of proviral germinal cells are an unlikely source of HIV and that much HIV DNA before versus aRer vasectomy is imprecise, our cell-free and cell associated HIV arises distal to the vas data indicate that infected seminal cells and cell-free HIV deferens. HIV has been detected in pre-ejaculatory fluid and RNA enter the male reproductive tract distal to the urethral secretions.11.12,61Re-ejaculatory fluid is produced transected vas deferens. by the urethral and bulbourethral glands (Littr6 and CowBecause there is limited information on the relative impor- per’s glands, respectively). Granulocytes, macrophages, tance of cell-free and cell associated HIV for sexual transmis- CD4+ and CD8+ lymphocytes have been described in presion of infection, we evaluated HIV RNA and HIV DNA. ejaculatory fluid.12 Infected cells may originate from the ureAlthough assessment of cultivable virus would also be desir- thral mucosa, prostate, seminal vesicles or ejaculatory ducts. able, technical problems limited our ability t o culture HIV Comprehensive sampling of multiple anatomical sites should from specimens in this study. Unfortunately, the gold stan- improve our understanding of the sources of infected cells dard remains documenting sexual transmission of HIV infec- and cell-free HIV, and may result in new approaches to limit tion. To our knowledge no laboratory assessment of HIV has sexual transmission. been clearly shown to correlate with transmission and, thus, the adequacy of a biological measure of HIV as a surrogate CONCLUSION determinant for transmission risk remains to be deterOur study has important clinical implications. Because mined.48 Culture documents presence of viable virus while the PCR assays document presence of viral RNA or DNA that blood levels of HIV RNA correlate only poorly with HIV RNA may not be associated with infectious virus. In vitro trans- levels in the semen, direct measurement of HIV levels in mission of HIV variants,49 and correlation between H N RNA semen is necessary to assess the potential for sexual transconcentration and the infectivity of seminal cells in cul- mission. Such measurements should be considered in clinical ture30.50 suggest that cell-free HIV may represent an impor- trials of antiretroviral agents. Multiple assays may be tant infectious component of semen. Other studies suggest needed since we found no correlation between HIV RNA and that cell associated HIV might be important for transmis- HIV DNA levels in the semen, and we do not know which sion.5,7,51.52Since vasectomy eliminates germinal cells from marker correlates with infectiousness. Because cell-free HIV the semen, the observation that vasectomy produced no sig- RNA and cell associated HIV DNA arise distal to the vas nificant change in either HIV RNA levels in seminal plasma deferens, future studies should concentrate on distal sites, or HIV DNA levels in seminal cells argues strongly against such as the prostate, seminal vesicles, urethra, and parauregerminal cells as an important reservoir for HIV in the se- thral and bulbourethral glands. These results also support men. This clinical finding agrees with data generated by efforts to limit inflammation at such distal sites to reduce some6*35but not all7 other investigators who used in vitro HIV levels in the semen. Finally, vasectomy may have minmethods to separate germinal cells from other cells in the imal impact on the infectiousness of healthy, HIV seroposisemen. In our study levels of HIV RNA in the seminal plasma tive men for sexual partners. did not correlate with levels of HIV DNA provirus, further Mr. Mechai Viravaida, President of the Population and supporting the idea that much cell-free virus in the seminal Community Development Association, and its staff provided plasma arises from sources other than germinal cells and study support and encouragement; members of the Departseminal leukocytes. ment of Medicine, Neuropsychiatry Section, Pramongkutklao Presence of multiple viral clades represents a potential Army Hospital, Royal Thai Army cooperated with the study, limitation of this study. In Thailand seroprevalence in- and Joel Gibson, University of Washington, provided technicreased from 1% in 1988 to 32 to 43% among IVDUs and cal assistance. Injecgreater than 50% in some heterosexual groups.46*53*54 tion drug users usually contract HIV subtype B while HIV REFERENCES subtype E is common among infected heterosexual^.^^ Some 1. Curran, J.: The epidemiology and prevention of the acquired studies suggest that subtype E is transmitted more effiimmunodeficiency syndrome. Ann. Intern. Med., 103: 657, ciently by sexual intercr~urse.55In vitro subtype E replicates 1985. more efficiently in submucosal dentritic (Langerhan’s) cells 2. Friedland, G. and Klein, R.: Transmission of the human immuthan subtype B.56 However, there are no published data on nodeficiency virus. New Engi. J. Med., 317: 1125, 1987. whether the HIV viral subtypes differ in tropism for the 3. Padian, N., Marquis, L., Francis, D. P., Anderson, R. E., genital tract or blood compartments, or whether other factors Rutherford, G. W., O’Malley, P. M. and Winkelstein, W. J.: account for the seroepidemiology of H N infection. 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J., Peterson, G., Hooton, T., Collier, A., Corey, L., Hughes, J., 36. Ho, D. D., Schooley, R. T., Rota, T. R., Kaplan, J. C., Flynn, T., Koutsky, L. and Krieger, J.: Association between culturable Salahuddin, S. Z., Gonda, M. A. and Hirsch, M. S.: HTLV-I11 in HIV-1 in semen and RNA levels is semen and blood. J. Infect. the semen and blood of a healthy homosexual man. Science, Dis., accepted for publication, 1997. 226 451,1984. 17. Dyer, J. R., Gilliam, B. L., Eron, J. J. J., Cohen, M. S., Fiscus, 37. Anderson, D. J., OBrien, T. R., Politch, J. A., Martinez, A, S. A. and Vernazza, P. L.: Shedding of HIV-1 in semen during Seage, G. R. 3rd, Padian, N., Horsburgh, C. R. J . and Mayer, primary infection. Aids, 11: 543, 1997. K. H.: Effects of disease stage and zidovudine therapy on the 18. Gilliam, B. L., Dyer, J. R., Fiscus, S. A., Marcus, C., Zhou, S., detection of human immunodeficiency virus type 1 in semen. Wathen, L., Freimuth, W. W., Cohen, M. S. and Eron, J. J. J.: J.A.M.A., 267: 2769, 1992. Effects of reverse transcriptase inhibitor therapy on the HIV-1 38. Seage, G. R. 3rd, Horsburgh, C. J., Hardy, A. M., Mayer, K. H., viral burden in semen. J. Acq. Immun. Def. Syn. Hum. RetroBarry, M. A., Groopman, J. E., Jaffe, H. W. and Lamb, G. A: virol., 1 5 54, 1997. Increased suppressor T cells in probable transmitters of hu19. Mellors, J. W., Munoz, A, Giorgi, J. V., Margolick, J . B., Tassoni, man immunodeficiency virus infection. Amer. J . Pub. Health, C. J., Gupta, P., Kingsley, L. A., Todd, J. A., Saah, A. J., Detels, 79 1638, 1989. R., Phair, J . P. and Rinaldo, C. R.: Plasma viral load and 39. Plourde, P. J., D’Costa, L. J., Agoki, E., Ombette, J., Ndinya, CD4+ lymphocytes as prognostic markers of HIV-1 infection. A. J . O., Slaney, L. A., Ronald, A. R. and Plummer, F. A.: A Ann. Intern. Med., 126:946, 1997. randomized, double-blind study of the efficacy of fleroxacin 20. Wood,B., Shacker, T., Krieger, J., Corey, L. and Gown, A,: versus trimethoprim-sulfamethoxazole in men with cultureMorphologic, immunophenotypic and virologic collelates of reproven chancroid. J. Infect. Dis., 166 949, 1992. cent lymph node infection by HIV in a cohort of known infec- 40. Plummer, F. A., Wainberg, M. A., Plourde, P., Jessamine, P., tion duration. Hum. Path., submitted for publication, 1997. DCosta, L. J., Wamola, I. A. and Ronald, A. R.: Detection of 21. Krieger, J. N., Coombs, R. W., Collier, A. C., Ross, S. O., human immunodeficiency virus type 1(HIV-1) in genital ulcer Cummings, D. K., Murphy, V. L. and Corey, L.: Recovery of exudate of HIV-1-infected men by culture and gene amplificahuman immunodeficiency virus type 1 from semen: Minimal tion. J. Infect. Dis., 161: 810, 1990. impact of stage of infection and current antiviral chemother- 41. Holmberg, S. D., Stewart, J. A., Gerber, A. R., Byers, R. H., Lee, apy. J. Infect. Dis., 163:386, 1991. F. K., OMalley, P. M. and Nahmias, A. J.: Prior herpes sim22. Alexander, N. and Anderson, D.: Immunology of semen. Ferti. plex virus type 2 infection as a risk factor for HIV infection. Steril., 41: 192, 1987. J.A.M.A., 259 1048, 1988. 23. Krieger, J., Coombs, R., Collier, A, Ho, D., Ross, S., Zeh, J . and 42. Hook, E. W. 111, Cannon, R. O., Nahmias, A. J., Lee, F. F., Corey, L.: Intermittent shedding of human immunodeficiency Campbell, C. H. J., Glasser, D. and Quinn, T. C.: Herpes virus in semen: implications for sexual transmission. J. Urol., simplex virus infection as a risk factor for human immunode1&4:1035. 1995. ficiency virus infection in heterosexuals. J . Infect. Dis.. 1m 251, 1992. 24. Krieger, J.,Coombs. R., Collier, A., Ross,S.,Speck, C. and Corey,
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EDITORIAL COMMENT 43. Wasserheit, J . N.: Epidemiological synergy. Interrelationships between human immunodeficiency virus infection and other The authors have performed an interesting study to evaluate the sexually transmitted diseases. Sex Transm. Dis., 19 61, 1992. effect of vasectomy on levels of HIV-1 in semen. At least 28 million 44. Demmler, G. J., Buffone, G. J., Schimbor, C. M. and May, R. A,: people worldwide are infected with HIV-1, which is primarily transDetection of cytomegalovirus in urine from newborns by using mitted through sexual intercourse.1 Interventions are being actively polymerase chain reaction DNA amplification. J. Infect. Dis., sought to control the HIVIacquired immunodeficiency syndrome ep158 1177, 1988. idemic. The authors speculate that vasectomy could reduce HIV-1 45. Detels, R., Leach, C. T., Hennessey, K., Liu, Z., Visscher, B. R., levels in semen by reducing numbers of infected cells and viral Cherry, J . D. and Giorgi, J . V.: Persistent cytomegalovirus particles in semen. White blood cells (WBCs) in the testis and epiinfection of semen increases risk of AIDS. J . Infect. Dis., 1 6 9 didymis can carry HIV-1 (reference 9 in article), and levels of these 766, 1994. cells are dramatically reduced in semen following vasectomy (refer46. Suwanagool, S., Sonjai, A., Ratanasuwan, W., Techasathit, W. ence 26 in article), although 1preliminary report demonstrated that and Chuenarom, V.: Risk factors for HIV infection among Thai some vasectomized individuals have high seminal leukocyte counts laborers during 1992-1993. J. Med. Assn. Thai, 7 6 663, 1993. and HIV-1 in the semen (reference 27 in article). There is also some 47. Moss, G. B., Overbaugh, J., Welch, M., billy, M., Bwayo, J., evidence that immature germ cells (reference 52 in article) and Plummer, F. A,, Ndinya-Achola, J . O., Malisa, M. A. and sperm (reference 7 in article) carry HIV-1, although this subject is Kreiss, J . K.: Human immunodeficiencyvirus DNA in urethral controversial.2 secretions in men: association with gonococcal urethritis and The authors describe a prospectively designed study in which CD4 cell depletion. J. Infect. Dis., 1 7 2 1469, 1995. HIV-1 levels were measured by quantitative PCR assay in semen 48. DeGruttola, V., Fleming, T. R., Lin, D. and Coombs, R. W.: and blood of 46 HIV-1 seropositive Thai men before and a t 1.2 and Surrogate markers; are we being naive? J . Infect. Dis., in 3 months after no scalpel vasectomy. Followup was excellent with 41 press. of the subjects (89%)completing all visits. Levels of HIV RNA rep49. Zhu, T., Wang, N., Carr, A., Nam, D. S., Moor, J. R., Cooper, D. A. resenting cell-free virus particles were stable in serum and seminal and Ho, D. D.: Genetic characterization of human immunode- plasma throughout the study but, surprisingly, the prevalence of ficiency virus type 1 in blood and genital secretions: evidence proviral HIV DNA in the cellular fraction of semen was significantly for viral compartmentalization and selection during sexual increased after vasectomy (11versus 50%. p <0.001). It is speculated transmission. J. Virol., 7 0 3098, 1996. that this is attributable to a concentration effect of residual WBCs in 50. Dyer, J., Billiam, B., Eron, J., Grosso, L., Cohen, M. and Fiscus, semen after vasectomy due to decreased sperm numbers. The auS.: Quantitation of human immunodeficiency virus type 1 thors conclude that most HIV in semen arises distal to the vas RNA in cell free seminal plasma: comparison of NASBA and deferens and that vasectomy would appear to have no impact on the Amplicor reverse transcription-PCR ampolification land corre- infectiousness of HIV seropositive men for their sexual partners. lation with quantitative culture. J. Virol. Meth., 60:161,1996. The authors are to be congratulated for conducting the most de51. Bagasra, O., Freund, M., Weidmann, J. and Harley, G.: Interac- finitive study to date on this subject, with conclusions well supported tion of human immunodeficiency virus with human sperm in by quantitative data. However, a few questions remain to be anvitro. J . AIDS, 1: 431, 1988. swered. Followup was for only 3 months and neither seminal WBC 52. Nuovo, G. J., Becker, J., Simsi, A., Margiotta, M., Khalife, G. and nor sperm counts were reported. Vasectomy is known to cause conShevchuk, M.: HIV-1 nucleic acids localize to the spermatogo- siderable local inflammation: which could have increased WBC and nia and their progeny. A study by polymerase chain reaction in HIV-1 levels in genital tract secretions transiently. The authors do situ hybridization. Amer. J. Path., 144: 1142, 1994. not indicate if a microscopic examination was performed to detect 53. Kunanusont, C., Foy, H. M., Kreiss, J. K., Rerks-Ngarm, S., sperm in each post-vasectomy semen specimen. It has been shown Phanuphak, P., Raktham, S., Pau, C. P. and Young, N. L.: that sperm still are present after vasectomy in the semen of 35% of HIV-1 subtypes and male-to-femaletransmission in Thailand. men 1month postoperatively, in 14 to 16% a t 2 months and in 6 to 12% a t 3 month^.^,^ Thus, it cannot be assumed that germinal or Lancet, 3 4 5 1078, 1995. 54. Ou, C. Y., Takabe, Y., Luo, C. C., Kalish, M., Auwanit, W., other testicular cells were not a source of proviral DNA in some of the Bandea, C., de la Torre, N., Moore, J. L., Schochetman, G. and semen specimens. A longer followup, for example through 1 year Yamazaki, S.: Wide distribution of two subtypes of HIV-1 in after vasectomy, would address these concerns. The other missing piece of information is whether vasectomy has Thailand. AIDS Res. Hum. Retrovir., 8 1471, 1992. 55. Anderson, R. M., Schwartslander, B., McCutchan,F. and Hu, D.: an effect on the infectious potential of HIV-1 in semen. The PCR Implications of genetic variability in HIV for epidemiology and assay used in this study measured HIV DNA and RNA transcripts but it cannot determine whether the virions are capable of infecting public health. Lancet, 341: 1778, 1996. 56. Soto-Ramirez, L. E., Renjifo, B., McLane, M. F., Marlink, R., target cells in the genital tract. A number of factors in semen, OHara, C., Sutthent, R., Wasi, C., Vithayasai, P., Vithayasai, including anti-HIV antibodies, complement, zinc, defensins and free V., Apichartpiyakul, C., Auewarakul, P., Pena, C. V., Chui, oxygen radicals, can inhibit HIV-1 infection: and little is known D. S., Osathanondh, R., Mayer, K., Lee, T. H. and Essex, M.: about the genital tract origin of some of these factors or the effects of HIV-1 Langerhans’ cell tropism associated with heterosexual vasectomy on levels of these factors in semen. The observation of increased proviral HIV-1 levels in semen after vasectomy should also transmission of HIV.Science, 271: 1291, 1996. 57. Lu, S.-D., Christopherson, C. and Wang, J.: Extended range of be followed with infectious titer studies to address the possibility non-B serotypes detected by a modified aplicor HIV-1 monitor that HIV-1 transmission could be enhanced after vasectomy through test (abstract). Presented a t the 4th Conference on Retrovi- induction of local inflammation. This study underscores the need for more research to understand ruses and Opportunistic Infections, Washington, D. C., Janubetter sites and mechanisms of HIV-1 infection of the genital tract in ary 22, 1997. 58. Robb, M. M., Birx, D. and Wang, J.: Performance of the amplicor men and women, and approaches other than vasectomy to inhibit or HIV-1 mentor test and a modified HIV-1 monitor test on HIV-1 block the sexual transmissions of this dangerous virus. Important subtypes A to F (abstract).Presented at the 4th Conference on avenues being investigated a t present include vaginal microbicides Retroviruses and ODDortunistic Infections, Washington, D. c., and mucosal vaccine^.^^* Arnold M. Belker January 22, 1997. Division of Urology 59 wolff, H. and Anderson, D. J.: Male genital tract inflarmfation Department of Surgery associated with increased numbers of potential human immuUniversity of Louisville School of Medicine nodeficiency virus host cells in semen. Andrologia, 20: 404, Louisville, Kentucky 1988. and 60. G e g e r , J. N., Coombs, R. W., Collier, A. C., Koehler, J. K., Ross, Debomh J. Anderson s. o., Chaloupka, K., Murphy, V. L. and Corey, L.: Fertility Department of Obstetrics, Gynecology Parameters in men infected with human immunodeficiency and Reproductive Bwlogv &-us. J . Infect. Dis., 1tM 464, 1991. Brigham and Women’s Hospital 61. Ilaria, G., Jacobs, J. L.,Polsky, B., KO&B., Baron, P., MacLow, Harvard Medical School c., Armstrong, D. and Schlegel, P. N.: Detection of HIV-1 DNA Boston, Massachusetts sequences in pre-ejaculabq fluid. Lancet, 340:1469,1992.
..
VASECTOMY AND HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN SEMEN
825
EDITORIAL COMMENT 43. Wasserheit, J . N.: Epidemiological synergy. Interrelationships between human immunodeficiency virus infection and other The authors have performed an interesting study to evaluate the sexually transmitted diseases. Sex Transm. Dis., 19 61, 1992. effect of vasectomy on levels of HIV-1 in semen. At least 28 million 44. Demmler, G. J., Buffone, G. J., Schimbor, C. M. and May, R. A,: people worldwide are infected with HIV-1, which is primarily transDetection of cytomegalovirus in urine from newborns by using mitted through sexual intercourse.1 Interventions are being actively polymerase chain reaction DNA amplification. J. Infect. Dis., sought to control the HIVIacquired immunodeficiency syndrome ep158 1177, 1988. idemic. The authors speculate that vasectomy could reduce HIV-1 45. Detels, R., Leach, C. T., Hennessey, K., Liu, Z., Visscher, B. R., levels in semen by reducing numbers of infected cells and viral Cherry, J . D. and Giorgi, J . V.: Persistent cytomegalovirus particles in semen. White blood cells (WBCs) in the testis and epiinfection of semen increases risk of AIDS. J . Infect. Dis., 1 6 9 didymis can carry HIV-1 (reference 9 in article), and levels of these 766, 1994. cells are dramatically reduced in semen following vasectomy (refer46. Suwanagool, S., Sonjai, A., Ratanasuwan, W., Techasathit, W. ence 26 in article), although 1preliminary report demonstrated that and Chuenarom, V.: Risk factors for HIV infection among Thai some vasectomized individuals have high seminal leukocyte counts laborers during 1992-1993. J. Med. Assn. Thai, 7 6 663, 1993. and HIV-1 in the semen (reference 27 in article). There is also some 47. Moss, G. B., Overbaugh, J., Welch, M., billy, M., Bwayo, J., evidence that immature germ cells (reference 52 in article) and Plummer, F. A,, Ndinya-Achola, J . O., Malisa, M. A. and sperm (reference 7 in article) carry HIV-1, although this subject is Kreiss, J . K.: Human immunodeficiencyvirus DNA in urethral controversial.2 secretions in men: association with gonococcal urethritis and The authors describe a prospectively designed study in which CD4 cell depletion. J. Infect. Dis., 1 7 2 1469, 1995. HIV-1 levels were measured by quantitative PCR assay in semen 48. DeGruttola, V., Fleming, T. R., Lin, D. and Coombs, R. W.: and blood of 46 HIV-1 seropositive Thai men before and a t 1.2 and Surrogate markers; are we being naive? J . Infect. Dis., in 3 months after no scalpel vasectomy. Followup was excellent with 41 press. of the subjects (89%)completing all visits. Levels of HIV RNA rep49. Zhu, T., Wang, N., Carr, A., Nam, D. S., Moor, J. R., Cooper, D. A. resenting cell-free virus particles were stable in serum and seminal and Ho, D. D.: Genetic characterization of human immunode- plasma throughout the study but, surprisingly, the prevalence of ficiency virus type 1 in blood and genital secretions: evidence proviral HIV DNA in the cellular fraction of semen was significantly for viral compartmentalization and selection during sexual increased after vasectomy (11versus 50%. p <0.001). It is speculated transmission. J. Virol., 7 0 3098, 1996. that this is attributable to a concentration effect of residual WBCs in 50. Dyer, J., Billiam, B., Eron, J., Grosso, L., Cohen, M. and Fiscus, semen after vasectomy due to decreased sperm numbers. The auS.: Quantitation of human immunodeficiency virus type 1 thors conclude that most HIV in semen arises distal to the vas RNA in cell free seminal plasma: comparison of NASBA and deferens and that vasectomy would appear to have no impact on the Amplicor reverse transcription-PCR ampolification land corre- infectiousness of HIV seropositive men for their sexual partners. lation with quantitative culture. J. Virol. Meth., 60:161,1996. The authors are to be congratulated for conducting the most de51. Bagasra, O., Freund, M., Weidmann, J. and Harley, G.: Interac- finitive study to date on this subject, with conclusions well supported tion of human immunodeficiency virus with human sperm in by quantitative data. However, a few questions remain to be anvitro. J . AIDS, 1: 431, 1988. swered. Followup was for only 3 months and neither seminal WBC 52. Nuovo, G. J., Becker, J., Simsi, A., Margiotta, M., Khalife, G. and nor sperm counts were reported. Vasectomy is known to cause conShevchuk, M.: HIV-1 nucleic acids localize to the spermatogo- siderable local inflammation: which could have increased WBC and nia and their progeny. A study by polymerase chain reaction in HIV-1 levels in genital tract secretions transiently. The authors do situ hybridization. Amer. J. Path., 144: 1142, 1994. not indicate if a microscopic examination was performed to detect 53. Kunanusont, C., Foy, H. M., Kreiss, J. K., Rerks-Ngarm, S., sperm in each post-vasectomy semen specimen. It has been shown Phanuphak, P., Raktham, S., Pau, C. P. and Young, N. L.: that sperm still are present after vasectomy in the semen of 35% of HIV-1 subtypes and male-to-femaletransmission in Thailand. men 1month postoperatively, in 14 to 16% a t 2 months and in 6 to 12% a t 3 month^.^,^ Thus, it cannot be assumed that germinal or Lancet, 3 4 5 1078, 1995. 54. Ou, C. Y., Takabe, Y., Luo, C. C., Kalish, M., Auwanit, W., other testicular cells were not a source of proviral DNA in some of the Bandea, C., de la Torre, N., Moore, J. L., Schochetman, G. and semen specimens. A longer followup, for example through 1 year Yamazaki, S.: Wide distribution of two subtypes of HIV-1 in after vasectomy, would address these concerns. The other missing piece of information is whether vasectomy has Thailand. AIDS Res. Hum. Retrovir., 8 1471, 1992. 55. Anderson, R. M., Schwartslander, B., McCutchan,F. and Hu, D.: an effect on the infectious potential of HIV-1 in semen. The PCR Implications of genetic variability in HIV for epidemiology and assay used in this study measured HIV DNA and RNA transcripts but it cannot determine whether the virions are capable of infecting public health. Lancet, 341: 1778, 1996. 56. Soto-Ramirez, L. E., Renjifo, B., McLane, M. F., Marlink, R., target cells in the genital tract. A number of factors in semen, OHara, C., Sutthent, R., Wasi, C., Vithayasai, P., Vithayasai, including anti-HIV antibodies, complement, zinc, defensins and free V., Apichartpiyakul, C., Auewarakul, P., Pena, C. V., Chui, oxygen radicals, can inhibit HIV-1 infection: and little is known D. S., Osathanondh, R., Mayer, K., Lee, T. H. and Essex, M.: about the genital tract origin of some of these factors or the effects of HIV-1 Langerhans’ cell tropism associated with heterosexual vasectomy on levels of these factors in semen. The observation of increased proviral HIV-1 levels in semen after vasectomy should also transmission of HIV.Science, 271: 1291, 1996. 57. Lu, S.-D., Christopherson, C. and Wang, J.: Extended range of be followed with infectious titer studies to address the possibility non-B serotypes detected by a modified aplicor HIV-1 monitor that HIV-1 transmission could be enhanced after vasectomy through test (abstract). Presented a t the 4th Conference on Retrovi- induction of local inflammation. This study underscores the need for more research to understand ruses and Opportunistic Infections, Washington, D. C., Janubetter sites and mechanisms of HIV-1 infection of the genital tract in ary 22, 1997. 58. Robb, M. M., Birx, D. and Wang, J.: Performance of the amplicor men and women, and approaches other than vasectomy to inhibit or HIV-1 mentor test and a modified HIV-1 monitor test on HIV-1 block the sexual transmissions of this dangerous virus. Important subtypes A to F (abstract).Presented at the 4th Conference on avenues being investigated a t present include vaginal microbicides Retroviruses and ODDortunistic Infections, Washington, D. c., and mucosal vaccine^.^^* Arnold M. Belker January 22, 1997. Division of Urology 59 wolff, H. and Anderson, D. J.: Male genital tract inflarmfation Department of Surgery associated with increased numbers of potential human immuUniversity of Louisville School of Medicine nodeficiency virus host cells in semen. Andrologia, 20: 404, Louisville, Kentucky 1988. and 60. G e g e r , J. N., Coombs, R. W., Collier, A. C., Koehler, J. K., Ross, Debomh J. Anderson s. o., Chaloupka, K., Murphy, V. L. and Corey, L.: Fertility Department of Obstetrics, Gynecology Parameters in men infected with human immunodeficiency and Reproductive Bwlogv &-us. J . Infect. Dis., 1tM 464, 1991. Brigham and Women’s Hospital 61. Ilaria, G., Jacobs, J. L.,Polsky, B., KO&B., Baron, P., MacLow, Harvard Medical School c., Armstrong, D. and Schlegel, P. N.: Detection of HIV-1 DNA Boston, Massachusetts sequences in pre-ejaculabq fluid. Lancet, 340:1469,1992.
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1. Royce, R. A., Sena, A, Cates, W. and Cohen, M. S.: Sexual transmission of HIV. New Engl. J. Med., 336:1072,1997. 2. Quayle, A. J., Xu, C., Mayer, K. H. and Anderson, D. J.: T lymphocytes and macrophages, but not motile spermatozoa, are a significaut source of human immunodeficiency virus in semen. J. Infect. Dis., 176 960,1997. 3. Alexauder, N. J. and Anderson,D. J.: Consequences of autoimmunity to sperm antigens (ModemTrends). Fertil. Steril., 32:253,1979. 4. Sivanesaratnam, V.: Onset of azwspermia after vasectomy. New Zeal. Med. J., 98: 331, 1985.
5. Esho, J. O.,Ireland, G. W. and Cass, A. S.: Recanalization following vasectomy. Urology, 3 211, 1974. 6. Anderson, D. J.: The importance of mucosal immunology to problems in human reproduction. J. Reprod. Immunol., 31: 3, 1996. 7. Elias, C. J. and Heise, L. L.: Challenges for the development of female-controlled vaginal microbicides. AIDS, 8: 1, 1994. 8. Haynes, B. V.: HIV vaccines: where we are and where we are going. Lancet, 348: 933,1996.