Highly active antiretroviral therapy (HAART) has dramatically improved the prognosis for those infected with HIV-1 . However, with the use of long-term HAART, a variety of side-effects may occur, for example lactic acidosis , peripheral neuropathy , and probably lipo-atrophy . Nucleoside anti-retroviral agents are thought to generate either depleted  or mutated mitochondrial DNA either by inhibition of mitochondrial-specific polymerase γ or through the increased generation of free radicals . Different combinations of drug therapy are thought to have varying quantitative toxic effects on these two causes of mtDNA damage . However, research has been limited by the lack of easily obtainable tissue as a result of the invasive nature of the clinical procedure. Importantly, however, human spermatozoa are an easily obtainable clinical material that is susceptible to the generation of multiple mtDNA deletions [9,10]. In order to determine whether spermatozoa could provide clinically relevant data as to the effects of HAART on individual patients, we used Long polymerase chain reaction (PCR)  on a series of sperm samples to detect the presence of multiple mtDNA deletions.
We analysed sperm samples produced by masturbation from 10 men attending our clinic. Four of the men had never taken antiretroviral therapy whereas the others (n = 6) took a combination of HAART. For one of the men, we analysed sperm samples at three different times points over 14 months (see Table 1). From each of the sperm samples, total DNA was isolated using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA). Deletions were detected using Long PCR to amplify 10.6 kb of the mitochondrial genome with primers specific to the region of the genome most prone to mtDNA deletion (D6: 5′–TCT AGA GCC CAC TGT AAA G–3′ and R10: 5′–AGT GCA TAC CGC CAA AAG A–3′) . The resulting PCR products were resolved on 0.8% agarose gels. The results are presented in Table 1.
Patient 1 was initially treated with lamivudine/delavirdine/indinavir, which was then quickly modified to stavudine, didanosine, nevirapine didanosine/stavudine/nevirapine/hydroxyurea. After 24 weeks, treatment was again changed to zidovudine/lamivudine/nevirapine because of side-effects. Before treatment, no mtDNA deletions were detected. However, at 6 months, this patient developed multiple mtDNA deletions, which persisted and became more clearly evident at 14 months of treatment. Of the four men who had never taken antiretroviral agents when the samples were analysed, only one (patient 5) of these had observable multiple mtDNA deletions. The two patients on HAART for less than 12 months harboured no mtDNA deletions. However, three men on HAART for more than 12 months possessed multiple mtDNA deletions. Interestingly, these three patients also presented with clinically evident lipodystrophy .
The proportion of patients with mtDNA deletions was significantly greater in those patients who had taken HAART for more than 12 months compared with those who had taken HAART for less than 12 months (two-tailed Fisher's exact test P < 0.05). Those patients who harboured mtDNA deletions had taken either stavudine  or didanosine, but only one patient had taken only zidovudine/stavudine. Furthermore, none of these patients had taken protease inhibitors.
We have demonstrated the propagation of multiple mtDNA deletions as a result of long-term HAART in sperm samples. These deletions may arise through damage to the mitochondrial genome of their precursor spermatogonial cells, resulting in the amplification of these deleted molecules . De novo multiple mtDNA deletions can only occur in cells that frequently replicate mtDNA. Spermatozoa are produced approximately 60 days before ejaculation, and it is common to detect both normal and abnormal spermatozoa in semen samples . It is evident from various studies that abnormal cells will undergo apoptosis  and subsequent phagocytosis . However, abnormal spermatozoa may be the consequence of an abortive mitochondrial-mediated apoptotic event  at the stem cell level accounting for the presence of both the abnormal and normal populations of mature sperm cells in samples analysed. The results of this study strongly suggest that an analysis of sperm mtDNA should be included in further studies of HAART-mediated mitochondrial disease.
The authors would like to thank the patients and staff of Birmingham Heartlands Hospital and the Department of Andrology (University of Birmingham), especially Dr Susan Drake and Dr Matthew Tomlinson for all their help and contribution to our study.
David J. Whitea
Justin C. St Johnb
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