Secondary Logo

Journal Logo


HIV-1 is undetectable in preejaculatory secretions from HIV-1-infected men on suppressive HAART

Politch, Joseph A.; Mayer, Kenneth H.; Anderson, Deborah J.

Author Information
doi: 10.1097/QAD.0000000000001130
  • Free



Semen is a major vector for the sexual transmission of HIV-1 [1–3]. Elevated peripheral blood HIV-1 viremia and genital inflammation/infections have been associated with increased levels of HIV in semen [4–6], whereas effective antiretroviral therapy (ART) that suppresses HIV in blood decreases seminal HIV-1 levels [7–10]. However, we and others have demonstrated that many men on HAART continue to shed HIV in semen [7–10]. Genital tract infections have been associated with increased seminal HIV-1 shedding in both untreated and HAART-treated men [6,10–16]. Information concerning the source of HIV in semen from men on HAART could shed light on HIV reservoirs in HIV-infected men on HAART, and could also have important implications for HIV prevention.

The human penile urethra is a primary site of HIV-1 infection [17]. This tissue contains a high number of HIV target cells [18,19], and is readily infectable with HIV in vitro[20,21]. Urethral secretions from HIV-infected men often contain HIV-1 DNA [22] and RNA [23], and urethral inflammation has been associated with high concentrations of HIV in semen [13,14,16]. Furthermore, preejaculatory fluid, a viscous urethral secretion produced by the glands of Littré, Cowper's glands and the glands of Morgagni, and released during sexual excitement [24] has been implicated in HIV transmission. Preejaculatory fluid from HIV-infected men contains HIV-1-infected white blood cells [25,26], and delayed application of condoms is a risk factor for HIV-1 transmission among men who have sex with men (MSM), providing evidence that preejaculatory fluid may be infectious [27,28]. SIV-infected nonhuman primates, like men, harbor virus in the penile urethra [29], and a recent study in male macaques demonstrated that HAART does not reduce SIV RNA levels in the urethra [30]. The purpose of the present study was to determine whether HIV persists in preejaculatory fluid from HIV-1-infected men on HAART, and whether it is associated with seminal HIV shedding.


Study participants

Study participants were a predominantly MSM cohort receiving medical care for HIV infection at Fenway Health in Boston, Massachusetts, USA. Fenway Health is the largest center caring for sexual and gender minority patients in the USA [31]. Men recruited for the study were HIV-1 infected, sexually active (i.e. having had sex in the past 6 months), and had been on a stable HAART regimen for at least 3 months. This study was approved by the Institutional Review Boards of both Fenway Health and Boston University Medical Campus.

Specimen collection and processing

Men provided preejaculatory fluid secretions and semen in a private room at Fenway Health after a minimum of 24 h of sexual abstinence. All patients were instructed to identify preejaculatory fluid (drops of viscous secretion appearing in the opening of the penile urethra during sexual excitement prior to ejaculation), and to collect preejaculatory fluid during masturbation using an ophthalmic wick [Merocel Eye Spear (Medtronic Xomed, Jacksonville, Florida, USA)] [32,33]. Wicks were made more permeable and softened by adding 100 μl sterile phosphate-buffered saline (PBS) prior to use [34], enabling the collection of more secretion (total wick volume ∼500 μl), and to reduce the possibility of tissue irritation or damage. After sample collection, swabs were placed individually in a 5-ml sterile polystyrene test tube with a snap cap (Falcon; Becton Dickinson and Co., Franklin Lakes, New Jersey, USA), and participants then used a specimen container to collect the ejaculate. In total, 10 ml of blood was collected by venipuncture in test tubes coated with ethylenediaminetetraacetic acid at the same visit. All samples were sent to the laboratory at Boston University Medical Campus, and processed within 4 h of collection.

Upon receipt of preejaculatory fluid samples by the laboratory, 1 ml PBS was added, and samples were incubated for 10 min at room temperature with intermittent vortexing. The eluate was then transferred to an Eppendorf tube and centrifuged at high speed (15 600 ×g) for 40 s; a 250-μl aliquot that contained supernatant and pelleted cells was reserved for HIV RNA-PCR. For semen specimens, volume was measured, and semen was processed as previously described [10,35,36]. In brief, semen was diluted 1 : 1 in PBS, and the Endtz test was performed to enumerate polymorphonuclear leukocytes (PMN) [36]. Seminal cells were pelleted by centrifugation at 600 × g for 10 min, and seminal plasma aliquots were taken. Semen cells were resuspended in PBS. Whole blood was centrifuged (400 ×g for 20 min) and plasma aliquots taken. Seminal plasma, semen cell, preejaculatory fluid and blood plasma aliquots were stored with TRI Reagent/PolyAcryl Carrier (Molecular Research Center, Cincinnati, Ohio, USA) at –80oC for subsequent RT-PCR.

Quantitative RT-PCR

HIV-1 RNA was quantified in seminal plasma, semen cells, blood plasma, and preejaculatory fluid (fluid and cells). The details of the HIV RT-PCR assay have been described by us previously [10]. Seminal plasma HIV RNA was extracted with silica beads from NucliSens (Biomerieux, Boxtel, the Netherlands) to exclude the possibility of PCR inhibition by seminal plasma. The lowest quantitative limit for HIV-1 RNA was 1 copy/μl RNA (equivalent to 40 copies/ml for blood plasma, preejaculatory fluid and semen cells, and 80 copies/ml for seminal plasma). Herpes simplex virus (HSV) serostatus and seminal HSV-2 DNA were determined as described by us previously [10].

Statistical analysis

GraphPad Prism (version 5.04; GraphPad Software, La Jolla, California, USA) and StatView (version 5.0.1; SAS Institute, Cary, North Carolina, USA) statistical software were used to perform the statistical computations. Categorical data were analyzed by Fisher's exact test or McNemar's test. Differences were considered to be statistically significant when P less than 0.05, the standard convention.


Study participants

The 60 men that provided samples for this study are a subset of 101 patients for whom seminal HIV data were previously reported [10]. These men were selected for the current analysis because they had provided a complete set of blood, semen, and preejaculatory fluid samples. Protein concentrations were assessed in preejaculatory fluid to confirm successful sample collection; preejaculatory fluid wick eluates contained 0.3–4.8 (median = 0.9) mg/ml of protein.

Eight of the 60 men, had detectable HIV-1 RNA in blood plasma (range: 80–640 000 copies/ml), and were removed from the principal analysis for this study. The remaining 52 patients with undetectable HIV RNA in blood plasma had been on a stable HAART regimen for at least 3 months prior to sample collection, and 40 patients (77%) had been on ART for more than 1 year. The age range of the 52 patients was 24–59 (median = 42.5), and their peripheral CD4+ cell counts ranged from 108 to 1492 cells/μl (median = 518.5 cells/μl). Fifty (96.2%) of the men self-identified as MSM, and 44.2% of the men had engaged in unprotected insertive anal sex within the past 3 months. None of the patients had a bacterial sexually transmitted infection (STI) diagnosis at the time of sample collection. However, one subject was positive for HSV-2 DNA in semen. In addition, one had urethritis of undetermined etiology and leukocytospermia (≥106 PMN/ml of semen [37]), and nine had leukocytospermia without urethritis. Thirty two (61.5%) of the 52 men were seropositive for HSV-1 and 30 (57.7%) were seropositive for HSV-2.

HIV-1 in semen and preejaculatory fluid

For the purpose of this analysis, we combined HIV RNA detection in seminal plasma and semen cells to create an overall semen HIV RNA variable which reflects potential cell-free and cell-associated HIV transmission. Preejaculate HIV detection also included both fluid and cellular components. Four of the 8 men with detectable HIV in blood had HIV RNA in semen (copy numbers ranging from 40–96 000), and one man from this group with HIV RNA in semen (96 000 copies/ml) also had HIV RNA in preejaculate (2400 copies/sample). This man did not have any signs of infection or urethritis.

Of the 52 men with undetectable HIV in blood, 10 (19%) were positive for HIV RNA in semen (range: 59–800 copies/ml), whereas none had HIV RNA in preejaculatory fluid (P = 0.004, McNemar's test). Data are summarized in Table 1. Detection of HIV RNA in semen was associated with leukocytospermia [6/10 (60%) men with leukocytospermia vs. 4/42 (9.5%) men without leukocytospermia had HIV in seminal plasma (P = 0.002, Fisher's exact test)]. The individual with nonspecific urethritis, and the individual positive for seminal HSV-2 DNA both had detectable levels of seminal HIV RNA. Detection of HIV RNA in semen was not associated with HSV-1 or HSV-2 serostatus (P > 0.10, Fisher's exact tests).

Table 1
Table 1:
HIV-1 detection in semen and preejaculatory fluid from HIV-1-infected men on HAART.


Treatment of HIV-infected men and women with antiretroviral drugs dramatically reduces the sexual transmission of HIV [38] and is being touted as an effective HIV prevention strategy [39]. However, a number of recent studies have shown that HIV persists in semen from a subset of men on stable HAART regimens. Evidence from several studies indicates that the male genital tract is a compartmentalized HIV environment, where genital infections promote localized HIV proliferation, evolution, and shedding into semen, even in men on HAART [23,40–43]. The penile urethra has been identified as an important HIV/SIV infection site, and HIV in urethral preejaculatory fluid has been implicated in the sexual transmission of HIV. The purpose of our study was to determine whether HIV is present in preejaculatory urethral fluid of men on HAART, and whether preejaculatory fluid HIV viral loads are associated with semen viral loads in men who continue to shed HIV in semen when virus is undetectable in blood. This information would provide evidence that the penile urethra is an important HIV sanctuary in men on HAART, and also that preejaculatory fluid from men on HAART may be infectious.

Whereas high levels of HIV RNA were detected in preejaculatory fluid from a man in this study with high levels of virus in semen and blood, HIV was not detected in preejaculatory fluid from 52 men on stable HAART with undetectable blood HIV viral load. A high percentage (19%) of men from this group, however, had detectable levels of HIV in semen as previously described [10]. None of the men in our study had been diagnosed with an STI, although one man had nonspecific urethritis at the time of the study, and nine more had leukocytospermia which is another index of male genital inflammation [44].

This study is the first to document high levels of HIV RNA in a urethral preejaculatory fluid sample, although the detection of HIV DNA and infected cells in preejaculatory fluid has been previously described [25,26]. Other reports have documented HIV in urethral samples, but the methodologies differed significantly from the present study. The study by Moss et al.[22] detected HIV DNA in urethral swabs from men in the pre-HAART era with a high prevalence of gonococcal urethritis. Coombs et al.[23] reported a high prevalence of HIV RNA in urethral samples from HIV-positive men, and a correlation between urethral and seminal RNA levels. However, in their study, as in the Moss study, urethral samples were collected by insertion of a wick or swab into the penile opening in nonsexually stimulated men, and all of the patients had detectable HIV in blood and semen (the majority of their patients were not on ART). In our study, we used a noninvasive sampling technique to detect HIV in urethral secretions that were naturally expressed from the penis as ‘preejaculatory fluid’ during sexual excitement, and all the men were on HAART. Our study provides the first evidence that preejaculatory sexual secretions in men on HAART, unlike those from untreated HIV-infected men, do not contain detectable HIV. Additional research is needed to determine whether HIV shedding into preejaculatory fluid is enhanced by clinical urethritis or STIs, and to determine the effect of HAART on urethral HIV in this high-risk group.

In conclusion, although HIV-1 RNA was detected in semen of men on stable HAART with undetectable blood HIV viral load, it was not detected in preejaculatory secretions. These data indicate that preejaculatory fluid may not contribute to HIV transmission in men on HAART, at least in men without genital infections.


The authors thank Frederick Bowman, BS, and Chong Xu, MD, Boston University, for their expert technical assistance, and William X. O’Brien, Rodney VanDerwarker, Marcy Gelman, and Chris Grasso, Fenway Health, for their assistance in obtaining local IRB approval for this study, recruiting participants and study coordination. The authors also express their appreciation to study participants for their participation in this demanding protocol.

Author contributions: study concept: D.J.A., K.H.M., and J.A.P. Study design: D.J.A., K.H.M., and J.A.P. Experimental procedures: J.A.P. Data analysis: J.A.P. Initial manuscript draft: J.A.P and D.J.A. Manuscript revisions: J.A.P., D.J.A., and K.H.M.

This research was supported by NIH grant R56 AI071909 (D.J.A.).

Conflicts of interest

There are no conflicts of interest.

Presented at the 12th meeting of the International Society for Immunology of Reproduction, Boston, Massachusetts, 29 May to 2 June 2013 (Abstract# P-57).


1. Ho DD, Schooley RT, Rota TR, Kaplan JC, Flynn T, Salahuddin SZ, et al. HTLV-III in the semen and blood of a healthy homosexual man. Science 1984; 226:451–453.
2. Anderson DJ, O’Brien TR, Politch JA, Martinez A, Seage GR 3rd, Padian N, et al. Effects of disease stage and zidovudine therapy on the detection of human immunodeficiency virus type 1 in semen. JAMA 1992; 267:2769–2774.
3. Chakraborty H, Sen PK, Helms RW, Vernazza PL, Fiscus SA, Eron JJ, et al. Viral burden in genital secretions determines male-to-female sexual transmission of HIV-1: a probabilistic empiric model. AIDS 2001; 15:621–627.
4. Chakraborty H, Helms RW, Sen PK, Cohen MS. Estimating correlation by using a general linear mixed model: evaluation of the relationship between the concentration of HIV-1 RNA in blood and semen. Stat Med 2003; 22:1457–1464.
5. Chan DJ, Ray JE, McNally L, Batterham M, Smith DE. Correlation between HIV-1 RNA load in blood and seminal plasma depending on antiretroviral treatment status, regimen and penetration of semen by antiretroviral drugs. Curr HIV Res 2008; 6:477–484.
6. Kalichman SC, Di Berto G, Eaton L. Human immunodeficiency virus viral load in blood plasma and semen: review and implications of empirical findings. Sex Transm Dis 2008; 35:55–60.
7. Gianella S, Smith DM, Vargas MV, Little SJ, Richman DD, Daar ES, et al. Shedding of HIV and human herpesviruses in the semen of effectively treated HIV-1-infected men who have sex with men. Clin Infect Dis 2013; 57:441–449.
8. Halfon P, Giorgetti C, Khiri H, Penaranda G, Terriou P, Porcu-Buisson G, et al. Semen may harbor HIV despite effective HAART: another piece in the puzzle. PLoS One 2010; 5:e10569.
9. Marcelin AG, Tubiana R, Lambert-Niclot S, Lefebvre G, Dominguez S, Bonmarchand M, et al. Detection of HIV-1 RNA in seminal plasma samples from treated patients with undetectable HIV-1 RNA in blood plasma. AIDS 2008; 22:1677–1679.
10. Politch JA, Mayer KH, Welles SL, O’Brien WX, Xu C, Bowman FP, et al. Highly active antiretroviral therapy does not completely suppress HIV in semen of sexually active HIV-infected men who have sex with men. AIDS 2012; 26:1535–1543.
11. Atkins MC, Carlin EM, Emery VC, Griffiths PD, Boag F. Fluctuations of HIV load in semen of HIV positive patients with newly acquired sexually transmitted diseases. BMJ 1996; 313:341–342.
12. Chan DJ, McNally L, Batterham M, Smith DE. Relationship between HIV-RNA load in blood and semen in antiretroviral-naive and experienced men and effect of asymptomatic sexually transmissible infections. Curr HIV Res 2008; 6:138–142.
13. Cohen MS, Hoffman IF, Royce RA, Kazembe P, Dyer JR, Daly CC, et al. Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research Group. Lancet 1997; 349:1868–1873.
14. Johnson LF, Lewis DA. The effect of genital tract infections on HIV-1 shedding in the genital tract: a systematic review and meta-analysis. Sex Transm Dis 2008; 35:946–959.
15. Sadiq ST, Taylor S, Copas AJ, Bennett J, Kaye S, Drake SM, et al. The effects of urethritis on seminal plasma HIV-1 RNA loads in homosexual men not receiving antiretroviral therapy. Sex Transm Infect 2005; 81:120–123.
16. Winter AJ, Taylor S, Workman J, White D, Ross JD, Swan AV, et al. Asymptomatic urethritis and detection of HIV-1 RNA in seminal plasma. Sex Transm Infect 1999; 75:261–263.
17. Anderson D, Politch JA, Pudney J. HIV infection and immune defense of the penis. Am J Reprod Immunol 2011; 65:220–229.
18. McCoombe SG, Short RV. Potential HIV-1 target cells in the human penis. AIDS 2006; 20:1491–1495.
19. Pudney J, Anderson DJ. Immunobiology of the human penile urethra. Am J Pathol 1995; 147:155–165.
20. Fischetti L, Barry SM, Hope TJ, Shattock RJ. HIV-1 infection of human penile explant tissue and protection by candidate microbicides. AIDS 2009; 23:319–328.
21. Ganor Y, Zhou Z, Bodo J, Tudor D, Leibowitch J, Mathez D, et al. The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages. Mucosal Immunol 2013; 6:776–786.
22. Moss GB, Overbaugh J, Welch M, Reilly M, Bwayo J, Plummer FA, et al. Human immunodeficiency virus DNA in urethral secretions in men: association with gonococcal urethritis and CD4 cell depletion. J Infect Dis 1995; 172:1469–1474.
23. Coombs RW, Lockhart D, Ross SO, Deutsch L, Dragavon J, Diem K, et al. Lower genitourinary tract sources of seminal HIV. J Acquir Immune Defic Syndr 2006; 41:430–438.
24. Masters WH, Johnson VE. Human sexual response. Boston: Little, Brown and Co.; 1966.
25. Ilaria G, Jacobs JL, Polsky B, Koll B, Baron P, MacLow C, et al. Detection of HIV-1 DNA sequences in preejaculatory fluid. Lancet 1992; 340:1469.
26. Pudney J, Oneta M, Mayer K, Seage G 3rd, Anderson D. Preejaculatory fluid as potential vector for sexual transmission of HIV-1. Lancet 1992; 340:1470.
27. Allman D, Xu K, Myers T, Aguinaldo J, Calzavara L, Maxwell J, et al. Delayed application of condoms with safer and unsafe sex: factors associated with HIV risk in a community sample of gay and bisexual men. AIDS Care 2009; 21:775–784.
28. Calzavara L, Burchell AN, Remis RS, Major C, Corey P, Myers T, et al. Delayed application of condoms is a risk factor for human immunodeficiency virus infection among homosexual and bisexual men. Am J Epidemiol 2003; 157:210–217.
29. Miller CJ, Vogel P, Alexander NJ, Dandekar S, Hendrickx AG, Marx PA. Pathology and localization of simian immunodeficiency virus in the reproductive tract of chronically infected male rhesus macaques. Lab Invest 1994; 70:255–262.
30. Matusali G, Dereuddre-Bosquet N, Le Tortorec A, Moreau M, Satie AP, Mahe D, et al. Detection of simian immunodeficiency virus in semen, urethra, and male reproductive organs during efficient highly active antiretroviral therapy. J Virol 2015; 89:5772–5787.
31. Das P. Kenneth Mayer: global leader in HIV prevention. Lancet 2013; 382:1479.
32. Castle PE, Rodriguez AC, Bowman FP, Herrero R, Schiffman M, Bratti MC, et al. Comparison of ophthalmic sponges for measurements of immune markers from cervical secretions. Clin Diagn Lab Immunol 2004; 11:399–405.
33. Dezzutti CS, Hendrix CW, Marrazzo JM, Pan Z, Wang L, Louissaint N, et al. Performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators. PLoS One 2011; 6:e23136.
34. Prodger JL, Hirbod T, Kigozi G, Nalugoda F, Reynolds SJ, Galiwango R, et al. Immune correlates of HIV exposure without infection in foreskins of men from Rakai, Uganda. Mucosal Immunol 2014; 7:634–644.
35. Politch JA, Wolff H, Hill JA, Anderson DJ. Comparison of methods to enumerate white blood cells in semen. Fertil Steril 1993; 60:372–375.
36. Endtz AW. A rapid staining method for differentiating granulocytes from ‘germinal cells’ in Papanicolaou-stained semen. Acta Cytol 1974; 18:2–7.
37. Anderson DJ, Politch JA. Centola GM, Ginsburg KA. White blood cells in semen and their impact on fertility. Evaluation and treatment of the infertile male. Cambridge: Cambridge University Press; 1996. 263–276.
38. Cohen MS, Chen YQ, McCauley M, Gamble T, Hosseinipour MC, Kumarasamy N, et al. Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med 2011; 365:493–505.
39. Cohen J. Breakthrough of the year. HIV treatment as prevention. Science 2011; 334:1628.
40. Kiessling AA, Fitzgerald LM, Zhang D, Chhay H, Brettler D, Eyre RC, et al. Human immunodeficiency virus in semen arises from a genetically distinct virus reservoir. AIDS Res Hum Retroviruses 1998; 14 (suppl 1):S33–S41.
41. Mestecky J, Moldoveanu Z, Smith PD, Hel Z, Alexander RC. Mucosal immunology of the genital and gastrointestinal tracts and HIV-1 infection. J Reprod Immunol 2009; 83:196–200.
42. Galvin SR, Cohen MS. Genital tract reservoirs. Curr Opin HIV AIDS 2006; 1:162–166.
43. Svicher V, Ceccherini-Silberstein F, Antinori A, Aquaro S, Perno CF. Understanding HIV compartments and reservoirs. Curr HIV/AIDS Rep 2014; 11:186–194.
44. Politch JA, Marathe J, Anderson DJ. Characteristics and quantities of HIV host cells in human genital tract secretions. J Infect Dis 2014; 210 (suppl 3):S609–S615.

HAART; HIV-1; preejaculate; semen; urethra

Copyright © 2016 Wolters Kluwer Health, Inc.