We examined the efficacy and effect of HAART in HIV-1-infected men confronted with assisted fertilization procedures. We showed that HAART did not always reduce the HIV-1-RNA level in blood and semen compartments, and that a significant upward shift in mitochondrial DNA was observed in spermatozoa from a HAART-treated patient group compared with spermatozoa from HAART-untreated or HIV-1-uninfected groups (P < 0.001). These findings emphasize the negative role of HAART, but not of HIV-1 infection, in determining semen alterations.
aDivision of Obstetrics, Gynecology and Reproduction Medicine, Italy
bDivision of Infectious Diseases, ASMN Reggio Emilia, Italy
cDepartment of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Italy
dDepartment of Clinical and Experimental Medicine, Microbiology Section, University of Bologna, Italy.
Received 19 September, 2006
Accepted 30 October, 2006
A number of studies have demonstrated that HIV-1-RNA levels in blood and semen compartments decrease below the detection limits during HAART [1,2]. However, the presence of latently infected cells has been a major issue for the sustained control and eradication of HIV-1 from the body.
Some studies reported that HIV-1 compartmentalization tends to differ in semen and in blood [3–5]. In particular, several lines of evidence suggest that HIV-1 in semen arises from genital tissues rather than from blood, and the HIV-1 particles in semen are further subcompartmentalized between seminal leukocytes and seminal plasma [6–8]. It remains to be determined whether the semen compartment, like the blood compartment, harbours a latent reservoir after prolonged therapy. Some studies detected replication-competent HIV-1 virus in the semen compartment in a few patients after 1–2 years of therapy, whereas others reported the opposite findings [8–11]. It is therefore still controversial as to whether the semen compartment represents a major reservoir of latently HIV-1-infected cells.
In the majority of HIV-1-infected couples the infected partner is the man. In that case, semen washing can be applied to eliminate the risk of sexual transmission of the virus during assisted fertilization procedures . The seminal viral load may decrease but is not eliminated in HIV-positive men taking HAART .
The safety of antiretroviral drug treatment is a key issue in the management of HIV-infected men belonging to HIV-1-serodiscordant couples. HAART may modify cell metabolism, and the risk of adverse effects on semen characteristics and pregnancies should be assessed. It is known that sperm DNA fragmentation associated with apoptosis can be a cause of recurrent pregnancy loss [14,15]. In particular, the role of the mitochondrial genome (mtDNA) in male infertility and an analysis of the implications of mtDNA on sperm mobility should be carefully considered . The adverse effect observed during treatment with nucleoside analogue reverse transcriptase inhibitors seems to be linked to an alteration in mitochondrial activity [17,18].
The present study examined the efficacy of reducing HIV-1 replication in semen after prolonged HAART therapy, and its effect on the genomic material of the male gamete.
Nineteen patients were enrolled in the study, 11 had been on HAART therapy for 5 years, eight of them having developed antiretroviral drug resistance. The cellular and viral characteristics of the patients are listed in Table 1 [19,20]. CD4 cell counts ranged between 25 and 704 cells/μl; blood viral loads ranged between 3.5 × 102 and 4.8 × 105 copies/ml, whereas seminal viral loads ranged between less than 50 and 1.8 × 105 copies/ml. HIV-1 was detected by HIV-1-RNA analysis at least four times during the study period. HIV-1 RNA was detectable in blood samples at baseline, whereas viral load was below the detection limits in semen (< 50 copies/ml of semen) in seven out of 19 patients during therapy. In general, viral loads in blood and semen were stable throughout therapy in patients with available longitudinal samples. The discrepancy between viral loads in blood and in semen in our study was thus probably caused by a compartmentalization process reflecting different active replication or evolution in these sites. In this connection, antiretroviral therapy did not reduce the HIV-1-RNA level in either blood or seminal plasma in some patients, whereas in others it reduced viral shedding in the semen, but not in the blood (see Table 1). HIV-1 isolates from blood and semen showed different drug resistance patterns, confirming a divergent expansion of the infection in the two sites. These data clearly demonstrate the concomitant presence of different strains generated under therapy.
The HIV-1-RNA viral load assessed in the blood to monitor HIV-1 patients during and after HAART treatment cannot be indicative of the viral load in the semen, and therefore it is not possible to evaluate the risk of HIV-1 transmission by in-vitro fertilization just by analysing HIV-1-RNA levels in the blood. Moreover, the probability of sexual transmission increases when the semen viral load is high . These findings are of particular interest for assisted reproduction in discordant couples consisting of an infected man and an uninfected woman. In this case, semen washing and in-vitro fertilization are recommended . We observed that the washing process reduced the original HIV-1-RNA semen load of the subjects by approximately three logs (data not shown), indicating that a seminal viral content below the detectable limit of molecular bioassay can be obtained only from sperm with a low HIV-1 load (< 1000 copies/ml).
The diagnosis of male infertility has primarily been based on traditional semen analysis, with a strong emphasis on the assessment of semen volume and sperm concentration, motility and morphology . This study focused on the mtDNA content in semen after HIV infection and undergoing HAART therapy. The semen changes reported in infected men, such as lower ejaculate volume and sperm counts , were confirmed in our cohort. In addition, sperm DNA fragmentation was also tested by electrophoresis . The HAART regimen and corresponding HIV-1-RNA semen load in enrolled subjects are shown in Table 1. Four patients were resistant to therapy, seven were responders, and eight, with high viral loads, were naive for HAART treatment. The determination of mtDNA/β-globin gene ratios using real-time polymerase chain reaction indicated that the mtDNA copy number in motile spermatozoa, HIV-1 negative for gag-nested polymerase chain reaction, ranged from 5.9 to 11.0 in subjects under HAART and from 1.9 to 3.6 in untreated subjects. Table 1 showed a significant upward shift of mtDNA in the sperm of the HAART-treated group with respect to controls (P < 0.001), whereas the HIV-1 untreated men presented a copy number distribution not significantly different from that of the control group. This difference was not statistically significant in relation to high or low plasma/semen viral loads. In no case was sperm DNA fragmentation associated with apoptosis detected (data not shown).
In conclusion, the level of sperm mtDNA is increased in HAART-treated subjects, thus raising the risk involved in assisted reproduction techniques. These findings emphasize the negative role of HAART, but not of HIV-1 infection, in determining semen alterations. These results are in line with the findings of May-Panloup et al.  who measured the mtDNA content in sperm samples presenting abnormal motility and morphology. Recent studies have suggested that nucleoside analogue reverse transcriptase inhibitor exposure could impair the mitochondrial energy-generating ability of spermatozoa .
This report highlights the importance of ongoing investigation into the use of HAART regimens in infected men in relation to the sexual transmission of HIV-1, and the prospect of assisted fertilization in HIV-discordant couples with seropositive men.
Sponsorship: This work was funded by Associazione Centro Studi Sterilità-Fertilità ‘Antonio Vallisneri’ of Reggio Emilia, Italy.
1. Gupta P, Mellors J, Kingsley L, Riddler S, Singh MK, Schreiber S, et al
. High viral load in semen of human immunodeficiency virus type 1-infected men at all stages of disease and its reduction by therapy with protease and nonnucleoside reverse transcriptase inhibitors. J Virol 1997; 71:6271–6275.
2. Liuzzi G, Chirianni A, Bagnarelli P, Clementi M, Piazza M. A combination of nucleoside analogues and a protease inhibitor reduces HIV-1 RNA levels in semen: implications for sexual transmission of HIV infection. Antivir Ther 1999; 4:95–99.
3. Coombs RW, Speck CE, Hughes JP, Lee W, Sampoleo R, Ross SO, et al
. Association between culturable human immunodeficiency virus type 1 (HIV-1) in semen and HIV-1 RNA levels in semen and blood: evidence for compartmentalization of HIV-1 between semen and blood. J Infect Dis 1998; 177:320–330.
4. Byrn RA, Zhang D, Eyre R, McGowan K, Kiessling AA. HIV-1 in semen: an isolated virus reservoir. Lancet 1997; 350:1141.
5. Gupta P, Leroux C, Patterson BK, Kingsley L, Rinaldo C, Ding M, et al
. Human immunodeficiency virus type 1 shedding pattern in semen correlates with the compartmentalization of viral Quasi species between blood and semen. J Infect Dis 2000; 182:79–87.
6. Levy JA. The transmission of AIDS: the case of the infected cell. JAMA 1988; 259:3037–3038.
7. Paranjpe S, Craigo J, Patterson B, Ding M, Barroso P, Harrison L, et al
. Subcompartmentalization of HIV-1 quasispecies between seminal cells and seminal plasma indicates their origin in distinct genital tissues. AIDS Res Hum Retroviruses 2002; 18:1271–1280.
8. Craigo JK, Patterson BK, Paranjpe S, Kulka K, Ding M, Mellors J, et al
. Persistent HIV type 1 infection in semen and blood compartments in patients after long-term potent antiretroviral therapy. AIDS Res Hum Retroviruses 2004; 20:1196–1209.
9. Zhang H, Dornadula G, Beumont M, Livornese L Jr, Van Uitert B, Henning K, Pomerantz RJ. Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy. N Engl J Med 1998; 339:1803–1809.
10. Nunnari G, Otero M, Dornadula G, Vanella M, Zhang H, Frank I, Pomerantz RJ. Residual HIV-1 disease in seminal cells of HIV-1-infected men on suppressive HAART: latency without on-going cellular infections. AIDS 2002; 16:39–45.
11. Liuzzi G, D'Offizi G, Topino S, Zaccarelli M, Amendola A, Capobianchi MR, et al
. Dynamics of viral load rebound in plasma and semen after stopping effective antiretroviral therapy. AIDS 2003; 17:1089–1092.
12. Semprini AE, Levi-Setti P, Bozzo M, Ravizza M, Taglioretti A, Sulpizio P, et al
. Insemination of HIV-negative women with processed semen of HIV-positive partners. Lancet 1992; 340:1317–1319.
13. Semprini AE, Fiore S. HIV and reproduction. Curr Opin Obstet Gynecol 2004; 16:257–262.
14. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, et al
. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 1995; 182:367–377.
15. Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 2003; 80:531–535.
16. Ruiz-Pesini E, Diez C, Lapena AC, Perez-Martoz A, Montoya J, Alvarez E, et al
. Correlation of sperm motility with mitochondrial enzymatic activities. Clin Chem 1998; 44:1616–1620.
17. Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA, Koopmans PP. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway. AIDS 1998; 12:1735–1744.
18. Vernazza PL, Gilliam BL, Dyer J, Fiscus SA, Eron JJ, Frank AC, Cohen MS. Quantification of HIV in semen: correlation with antiviral treatment and immune status. AIDS 1997; 11:987–993.
19. Persico T, Savasi V, Ferrazzi E, Oneta M, Semprini AE, Simoni G. Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure. Hum Reprod 2006; 21:1525–1530.
20. May-Panloup P, Chretien MF, Savagner F, Vasseur C, Jean M, Malthiery Y, Reynier P. Increased sperm mitochondrial DNA content in male infertility. Hum Reprod 2003; 18:550–556.
21. Dondero F, Rossi T, D'Offizi G, Mazzilli F, Rosso R, Sarandrea N, et al
. Semen analysis in HIV seropositive men and in subjects at high risk for HIV infection. Hum Reprod 1996; 11:765–768.
22. St John JC, Jokhi RP, Barratt CL. The impact of mitochondrial genetics on male infertility. Int J Androl 2005; 28:65–73.
23. Sergerie M, Martinet S, Kiffer N, Teule R, Pasquier C, Bujan L. Impact of reverse transcriptase inhibitors on sperm mitochondrial and genomic DNA in assisted reproduction techniques [in French]. Gynecol Obstet Fertil 2004; 32:841–849.