Hum. Reprod. Advance Access published online on September 12, 2007
Human Reproduction, doi:10.1093/humrep/dem288
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HIV-1 viral DNA is present in ejaculated abnormal spermatozoa of seropositive subjects
1 Department of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy 2 Department of Medical Physiopathology, Sapienza University of Rome, Rome, Italy 3 National Institute for Infectious Disease Spallanzani (INMI), Rome, Italy
4 Correspondence address. Tel: +39(0)6 4976 6570; Fax: +39(0)6 4462 854; E-mail: mario.stefanini{at}uniroma1.it
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BACKGROUND: Semen is the major vehicle for HIV-1 infection as it contains free and cell-associated virions and infected cells. However, the presence of HIV-1 in spermatozoa has been a matter of debate, since the sperm cell fraction may contain somatic infected cells that jeopardize the attribution of the detected virus to the spermatozoa.
METHODS: Spermatozoa from 12 HIV-1 seropositive subjects were purified by multilayered Percoll gradient followed by osmotic shock. Residual presence of non-seminal cells (NCS) in purified spermatozoa, was then evaluated by cytometric and molecular analysis. HIV-1 DNA was revealed by nested PCR and in situ PCR after sperm chromatin decondensation. DNA-fragmented ejaculated spermatozoa in semen of infected subjects were detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) analysis.
RESULTS: Purification procedure adopted allowed complete removal of NCS. On purified sperm cells, HIV-1 DNA was detected in 5 out of 12 subjects by nested-PCR. On crude semen of 10 out of 12 subjects, HIV-1 DNA was in situ detected in a small percentage of abnormal spermatozoa with a wide range of structural alterations. TUNEL analysis revealed an increased percentage of DNA-fragmented ejaculated spermatozoa in semen of infected subjects.
CONCLUSIONS: We report molecular evidence demonstrating that HIV-1 infected subjects can ejaculate small amounts of HIV-1 DNA-positive abnormal spermatozoa. Their possible role in HIV-1 sexual transmission remains to be clarified.
Key words: osmotic shock/sperm chromatin decondensation/in situ PCR/HIV-1 nested PCR/Alu-LTR PCR
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Viral shedding in the male genital tract is a well known and common phenomenon (for a review seeDejucq and Jegou, 2001
), and different viruses such as HIV-1 (Dussaix et al., 1993; Baccetti et al., 1994; Bagasra et al., 1994; Scofield et al., 1994; Bourlet et al., 2002; Meseguer et al., 2002), HHV8 (Huang et al., 1997), HBV (Hadchouel et al., 1985), HSV (Kotronias and Kapranos, 1998), HPV (Lai et al., 1997) CMV (Sheth et al., 2006) and HCV (Bourlet et al., 2002; Levy et al., 2002; Meseguer et al., 2002) have been observed in the semen of infected subjects. In adults, HIV-1 infection is transmitted predominantly by sexual routes (Gupta and Klasse, 2006). HIV-1 virus is present in semen shortly after primary infection (Tindall et al., 1992; Pilcher et al., 2001) and at all stages of the disease (Gupta et al., 2000). Semen is in fact considered to be the major vehicle for sexual transmission, as it contains free and/or cell-associated virions and/or HIV-1 infected cells (Borzy et al., 1988; Krieger et al., 1991; Mermin et al., 1991; Van Voorhis et al., 1991; Vernazza et al., 1994,1996), mostly lymphocytes and macrophages (Quayle et al., 1997; Paranjpe et al., 2002). The virus has also been detected in spermatozoa of HIV-1 infected subjects by in situ PCR (Baccetti et al., 1994; Bagasra et al., 1994), electron microscopy (Baccetti et al., 1991,1994; Dussaix et al., 1993) and more recently by atomic force microscopy (Barboza et al., 2004), and in spermatozoa infected in vitro (Bagasra et al., 1988, Bagasra et al., 1994). However, the presence of HIV-1 in sperm has become a matter for debate (for reviews see Alexander, 1998; Piomboni and Baccetti, 2000) due to the possibility that non-seminal cells (NSC)—a variable source of infected somatic cells—may be present in the semen sample to be tested, thus jeopardizing attribution of the detected virus to sperm (Quayle et al., 1997; Pudney et al., 1999; Debono et al., 2000).
The presence of ‘contaminant’ NSC at a mean frequency of 1/1000 sperm cells has been reported in semen samples, even after purification by discontinuous Percoll gradient centrifugation (Dulioust et al., 1998; Marina et al., 1998; Tachet et al., 1999; Leruez-Ville et al., 2002). In addition, viral particles adhering to the sperm plasma membrane may be responsible for the HIV-1 and HCV positivity of ejaculated sperm. In fact, sperm washing is recommended as a useful approach to reduce the risk of HIV-1 and/or HCV transmission in serodiscordant couples with an infected male partner who want to have a child (Mencaglia et al., 2005). Adequate purification by Percoll gradient and swim-up has been claimed to almost completely remove HIV-1 and HCV RNAs from the motile sperm fraction (Kim et al., 1999; Hanabusa et al., 2000; Pasquier et al., 2000,2006a; Kato et al., 2006) and it has been recently reported that the efficiency of sperm washing in removing cell-free HIV-1 virions varies according to the seminal viral load (Fiore et al., 2005). In line with this concept, no medically assisted procreation can be performed in France if the HIV-1 seminal viral load is 
10 000 RNA copies/ml (Pasquier et al., 2006b). Results obtained by soluble phase PCR are extremely variable, especially in relation to the procedures used to process the semen samples and to detect HIV-1 DNA and RNA by PCR analysis (Meseguer et al., 2002; Kato et al., 2006; Pasquier et al., 2006a), as revealed by a recent multicenter quality control study of HIV-1 genome detection in semen before medically assisted procreation, in which 11 different European laboratories had taken part (Pasquier et al., 2006a). Several laboratories failed to detect low copy number of HIV-1, thus giving false negative results, whereas in a few cases, false positive results were obtained in the absence of HIV-1 (Pasquier et al., 2006a,b). Clearly, false negatives may be due to the low sensitivity of the technical procedures used to detect HIV-1; no commercial assay is available to detect HIV-1 in seminal plasma or semen cells and fertility centers usually use modified commercial kits for blood viral detection (Pasquier et al., 2006a,b).
Recently, a very sensitive method for same-day validation of HIV-1 RNA in processed semen has been developed. This method allows detection of low viral load and controls for possible PCR inhibitors. By this approach two motile spermatozoa preparations, obtained after Percoll gradient centrifugation followed by swim-up, were found to be HIV-1 RNA positive (Lesage et al., 2006). We have developed experimental strategies to remove ‘contaminating’ NSC from the semen, monitor any residual presence in the purified sperm fractions and verify the presence of HIV-1 proviral DNA by nested PCR, with a detection limit of 4 HIV-1 DNA copies/106 spermatozoa. In addition, we have developed an in situ PCR hybridization procedure to amplify human sperm DNA, overcoming the difficulties in accessing the highly condensed chromatin typical of these cells. Finally, sperm DNA fragmentation of HIV-1 seropositive subjects was also evaluated by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP nick-end labeling (TUNEL) technique. Herein we report the molecular evidence, obtained by in situ and soluble PCR, demonstrating the presence of proviral DNA in ejaculated spermatozoa with abnormal morphologies obtained from HIV-1 seropositive subjects.
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Subjects
The study was approved by Sapienza University of Rome Hospital Ethics Committee and conducted in accordance with the Declaration of Helsinki. A group of 12 HIV-1 seropositive—male subjects (not drug users), aged 31–49 (37.2 mean age) attending the Medical Centre for Infectious Diseases of the Umberto I General Hospital, Sapienza University of Rome and the Spallanzani National Institute for Infectious Diseases (INMI) of Rome, were studied. Five were naïve subjects and seven were receiving highly active anti-retroviral therapy (HAART). Specimens from 15 normozoospermic HIV-1-seronegative healthy subjects, aged 28–46 (35.3 mean age) attending the Laboratory of Seminology of the Department of Medical Physiopathology, Sapienza University of Rome were used as HIV-1 negative control samples.
Seminal and virological parameters
Semen specimens were collected after 3–5 days of sexual abstinence by masturbation into sterile plastic jars and processed within 60 min of ejaculation. For each subject, ejaculate volume, sperm concentration, total sperm count, forward motility and morphology were evaluated according to WHO guidelines (World Health Organization, 1999). In addition, sperm DNA fragmentation, blood and seminal plasma viral loads and CD4+ blood count were evaluated in the HIV-seropositive subjects studied. The number of HIV-1 genome copies in seminal and blood plasma was quantified by the branched DNA method with a limit of detection of 50 copies/ml (HIV-1 RNA 3.0 Assay bDNA from Bayer Diagnostics, Norwood, MA, USA) (Liuzzi et al., 1995).
Purification of the sperm cell fractions
Semen samples were allowed to liquefy for 60 min at 37°C. Aliquots of seminal samples were set aside and defined as ‘crude semen’ whereas the remaining part were further processed as follows. Samples were diluted 1:2 with Biggers–Whitten–Whittingham (BWW) buffer and centrifuged for 10 min at 600 g; the remaining pellets were re-suspended with 1 ml of BWW, layered over a multilayered Percoll gradient column (Pharmacia, Uppsala, Sweden) at dilutions of 30, 35, 40, 45, 50, 60, 70, 80, 90 and 100% and centrifuged for 25 min at 800 g at 18°C. Fractions 90–100% which contained motile and viable spermatozoa (Gandini et al., 1999) were recovered, counted and further processed. To remove residual contaminating NSC, 90–100% sperm fractions were subject to osmotic shock treatment. To this end, sperm pellets were suspended in bidistilled RNase-free water, mixed and incubated for 20 min on ice. At the end of incubation, sperm cells were centrifuged twice at 3000 g at 4°C for 10 min in phosphate-buffered saline. These samples were defined as ‘purified sperm cells’.
Evaluation of sperm purification
The presence of NSC in semen samples before and after purification (respectively, named crude semen and purified sperm cells) was evaluated by cytometric analysis and RT–PCR analysis.
Fluorescence-activated cell sorting
Samples from HIV-seronegative subjects (control samples) were stained with anti-human-CD4-PE and anti-human-CD45-PERCP monoclonal antibodies (Becton Dickinson, Italy). CD4 and CD45 are leukocyte antigens absent in sperm cells. Stained samples were analysed with a fluorescence-activated cell sorter (Epics XL Beckman Coulter, CA, USA) using an Argon laser at an excitation wavelength of 488 nm. Peripheral blood mononuclear cells (PBMC) from healthy donors were also used as positive controls and for fluorescence compensation. This analysis was repeated three times on different semen samples.
RNA isolation and RT–PCR analysis
In all HIV-seropositive and negative subjects studied, sperm purification procedure was further evaluated by molecular analysis of specific somatic and sperm cell transcripts, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and protamine-2 (PRM-2) transcripts, respectively. GAPDH is not expressed in germ cells that are known to express a unique spermatogenic-GADPH gene (Bunch et al., 1998; Welch et al., 2000; Welch et al., 2006). Conversely, PRM-2 is specifically expressed in germ cells (Oliva, 2006). Total RNA was extracted from crude and purified sperm cells using a High pure RNA isolation kit (Roche, Italy) according to the manufacturer's guide and treated with DNAse I (Invitrogen-Life Technologies, Italy) to prevent DNA genomic contamination. Total RNAs were reverse-transcribed using Sensiscript Reverse Transcriptase (Qiagen, Italy), a highly sensitive enzyme recommended for very small RNA quantities (<50 ng). cDNAs were then tested for PRM-2 and GAPDH expression levels. For PRM-2, primers were chosen to discriminate between genomic DNA and sperm mRNA, and amplified a 310 bp and a 168 bp bands, respectively (Miller et al., 1994). PCR conditions were: 94°C for 30', 54°C for 40', 72°C for 30' for 50 cycles. For the GAPDH gene, two different primer sets were designed (GenBank accession number DQ894744
[GenBank]
). The outer set (forward 5'-ctg cac cac caa ctg ctt ag -3' and reverse 5'-agg tcc acc act gac acg tt-3') amplifies a 282 bp band. The inner set (forward 5'-ggc caa ggt cat cca tga ca-3'; reverse 5'-gaa ggc cat gcc agt gag c-3') amplifies a 219 bp band. To establish the detection limit of GAPDH mRNA derived from NSC, fresh PBMC were serially diluted in purified sperm samples. PCR conditions were: 94°C for 30 s, 55°C for 45 s, 72°C for 45 s for 50 cycles, for the first round; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s for 30 cycles, for the second round. Control reaction tubes, omitting the RT enzyme, were also included to test possible DNA genomic contamination. PCR products were separated on agarose gels and visualized by ethidium bromide staining. The detection limit of GAPDH mRNA was 5 GAPDHpos cells/106 spermatozoa.
HIV-1 proviral DNA detection limits by PCR analysis
Discordant HIV-1 DNA PCR results on ejaculated spermatozoa from HIV-1 infected subjects could be related to the sensitivity threshold of the commercial kits used (Pasquier et al., 2006b). To overcome this problem we developed ‘bespoke’ PCR protocols, which are able to detect a low number of HIV-1 DNA copies in sperm samples (<20copies/106 cells).
HIV-1 DNA nested PCR
High-molecular weight genomic DNA (up to 50 Kb) from purified sperm cells of HIV-seronegative subjects was extracted using the QIAamp (Qiagen, Italy) protocol adapted for semen samples, as previously described (Muciaccia et al., 2005b). To establish a detection limit of HIV-1 proviral DNA in sperm cells, serial dilutions of 8E5/LeAS genomic DNA—a cell line which carries only one integrated copy of proviral DNA per cell (Folks et al., 1986)—were added to genomic sperm DNA. Briefly, genomic DNA from 8E5/LAS cells extracted using QIAamp following the manufacturer's protocol was serially diluted to obtain tubes containing 50, 25, 5, 1, 0.5 HIV-1 copies in 2.5 x 105 sperm cell equivalent DNA. HIV-1 proviral DNA (HIV-1 gag region) was then detected by two successive rounds of PCR, using SK40–SK41 (Muciaccia et al., 1998a) and then SK38–SK39 primers (Ou et al., 1988). Each sample was first denatured at 94°C for 10 min and then amplified with SK40–SK41 primers at 94°C for 40 s, at 55°C for 40 s and at 72°C for 40 s for 50 cycles using Taq DNA polymerase (Roche, Italy). Aliquots of the first PCR reaction were further amplified using the inner primers SK38–SK39 for 50 cycles using the above conditions. Tubes without template and with HIV-1-negative template were included as negative controls. Finally, the absence of seminal PCR inhibitors in the samples (Ball et al., 1999) was assessed by amplification for the 
-globin gene (primers PC04/GH20:Bauer et al., 1991). PCR reactions were separated on agarose gels and visualized by ethidium bromide staining. DIANA software (Raytest, Germany) was used to acquire ethidium bromide gel fluorescence. Densitometric analysis was performed using the AIDA2.1 program. The limit of detection for viral DNA was one copy of HIV-1 DNA/PCR tube, corresponding to 4 HIV-1 DNA copies/106 sperm cell DNA equivalents. This experiment was repeated twice with essentially the same results.
Integrated HIV-1 DNA Alu-LTR PCR assay
High-molecular weight genomic DNA (up to 50 Kb) from purified sperm cells of HIV-seronegative subjects was extracted using the QIAamp protocol adapted for semen samples, as previously described (Muciaccia et al., 2005b). The quantity and quality of high-molecular weight DNA fragments (up to 50 Kb) were assessed by electrophoresis on agarose gel. The quality of genomic DNA templates was further assessed by amplification of 15 kb DNA fragment of the tissue plasminogen activator (tPA) gene, using the Expand Long Template PCR System and tPA Control primer set (Roche, Italy). To establish the detection limit of Alu-LTR PCR, 8E5/LAS DNA was serially diluted and tubes containing 1000, 500, 100, 50, 10, 5, 2.5 and 1 HIV-1 copies in 2.5 x 105 sperm cell equivalent DNA were obtained. The first round of PCR amplification was carried out using a forward primer specific for human genomic Alu sequences and a reverse primer specific for conserved viral LTR sequences. Aliquots of PCR products were then analysed in nested PCR by two successive rounds of PCR amplification using NI-2 and NI-3 primer sets, respectively, (Chun et al., 1997; Ibanez et al., 1999). The first PCR amplification reaction was performed using the Expand Long Template PCR System, whereas Taq DNA polymerase (both from Roche) was used for the second and third round of amplifications. Cycle profiles were: first amplification 94°C for 40 s, 66°C for 40 s, 68°C for 5 min, for 35 cycles; second and third amplifications 94°C for 30 s, 65°C for 30 s, 72°C for 40 s, for 50 cycles. PCR products from the third amplification round were also analysed by Southern blotting using a 32-P-end labeled internal NI hybridization probe 5'-gga tgg tgc ttc aag ita gta cc-3' (Chun et al., 1997). The detection limit was 10 HIV-1 copies/PCR tube, corresponding to 40 HIV-1 copies/1 x 106 sperm cell DNA equivalents. This experiment was repeated twice with essentially the same results.
HIV-1 DNA detection in purified sperm cells
High-molecular weight DNA (up to 50 Kb) from purified sperm samples of HIV-1 seropositive subjects was extracted using the QIAamp protocol adapted for semen samples, as previously described (Muciaccia et al., 2005b). The quantity and quality of extracted DNA were analysed on agarose gels. Absence of PCR inhibitors was assessed by PCR for human -globin gene (PC04/GH20 primers; Bauer et al., 1991). Feasibility of long DNA fragment amplification was assessed by amplification of 15 kb DNA fragment of the tPA gene. HIV-1 DNA detection was then assessed by nested PCR and Alu-LTR PCR as detailed above, using purified DNA, corresponding to 2.5 x 105/tube equivalents of purified spermatozoa. Nested PCR analysis for the HIV-1 pol gene (Larder et al., 1993) was performed to confirm HIV-1 proviral detection in sperm samples positive for the gag gene. To estimate HIV-1 DNA copy number, an experimental standard curve of 8E5/LAS cell line genomic DNA (see above) was obtained in parallel under the same PCR conditions. PCR specificity and contamination controls were performed using: (i) tube with HIV-1 negative sperm DNA template from seronegative subjects (control samples); (ii) tube with all PCR reagents and no template. After electrophoresis, gel images were acquired using the DIANA program (Raytest) and densitometric analysis of PCR products was performed using the AIDA2.1 program (Raytest). Quantification of HIV-1 DNA copy number was obtained by comparison with the experimental standard curve. For each sample, nested PCR and Alu-LTR PCR were repeated at least three times with essentially the same results.
Detection of HIV-1 RNA in purified sperm cells
Total RNA from purified sperm cells was isolated as described above. In order to detect HIV-1 viral RNA, primers specific for the gag region (SK40–SK41 and SK38–SK39) were used. To distinguish the kind of viral RNA present, US (unspliced) primers, which amplify for full-length RNA molecules, and MS (multiply-spliced) primers specific for multiply-spliced tat/rev HIV-1 mRNA were employed. US primers amplify a 160 bp band, whereas MS primers amplify a 131 bp band (Saksela et al., 1993,1994). DNA genomic contamination was monitored in control reactions, where the RT enzyme was omitted during cDNA synthesis.
In situ PCR hybridization
HIV-1 proviral DNA PCR products were detected in situ as previously described (Muciaccia et al., 1998a). Experiments were performed on a crude semen sample from each HIV-1 seropositive subject and on purified sperm cell samples from two of them (Subjects 6 and 12). Briefly, 5 x 105 sperm cells were spotted onto sylanized slides (Perkin-Elmer In situ PCR glass slide, CA, USA), fixed with cold acetone and subjected to the sperm chromatin decondensation treatment with 10 mM Tris pH 8, 5 mM DTT and 1% Triton-X for 30 min at 37°C (Vidal et al., 1993; Muciaccia et al., 1998a). Hot-Start in situ PCR amplification was performed using a GeneAmp In situ PCR System 1000 (Perkin-Elmer). Primer pairs SK68/SK69, amplifying a 141 bp region of the HIV env-gene (Ou et al., 1988), and SK40/SK41 (Muciaccia et al., 1998a) amplifying a 171 bp region of the HIV gag-gene, were used. In each experiment, the following controls were performed: (i) specificity control with heterologous primers for the prokaryotic lac-z gene; (ii) amplification control in the absence of either Taq polymerase or primers; (iii) sensitivity control using primers for the endogenous -globin gene (PC04/GH20 primer set:Bauer et al., 1991); (iv) negative control using sperm samples from HIV-1 seronegative subjects (control samples). PCR products were detected by in-situ hybridization (ISH) with the internal corresponding digoxigenin-labeled oligo probes as described (Muciaccia et al., 1998a,b). For each sample, in situ PCR was performed in duplicate and repeated at least three times. Quantitative analysis was performed by light microscopy at x63 magnification with immersion oil. For each sample, at least 5 x 103 sperm cells were counted.
Detection of sperm DNA fragmentation
In situ detection of DNA fragmentation in ejaculated sperm from HIV-1 seropositive and seronegative subjects was performed by the TUNEL technique according to the manufacturer's guide (In situ Cell Death Detection Kit, POD by Roche). Briefly, 5 x 105 sperm cells were spotted onto polylysine coated slides, air dried and fixed in methanol at room temperature for 10 min. After washing, cells were permeabilized in 0.1% Triton X-100/0.1% sodium citrate and then incubated with TUNEL reaction mixture (Tdt enzyme and nucleotide label solution) in the dark for 60 min at 37°C in a humidified chamber to avoid evaporation loss. After washing, slides were incubated with Converter-POD (anti-fluorescein antibody conjugated with peroxidase) for 30 min at 37°C and a colorimetric reaction was developed by adding DAB substrate. Cells were not counterstained. For each sample, TUNEL staining was performed in duplicate and repeated at least three times. For each sample, at least 0.5 x 103 sperm cells were counted. Positive and negative controls were performed by treating sperm cells with DNAsi I and by omitting TdT enzyme from the TUNEL mixture. In addition, sperm cells from HIV-1 seronegative subjects and the 8E5/LAS cell line were processed in parallel for comparison.
Statistical analysis
Statistical analysis was performed using SPSS statistical software, version 13 (SPSS, Illinois, USA). The statistical differences between sperm concentration versus the percentage of TUNEL-positive sperm cells and abnormal sperm forms were investigated using linear regression analysis.
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HIV-1 RNA detection in blood and seminal plasma
Table 1 reports the blood and seminal plasma parameters for the 12 HIV-1 seropositive subjects studied. CD4 counts were <200 in one subject, <500 in eight, <1000 in two and >1000 in one subject, with a mean CD4 cell count of 506 x 106 cells/l and a range of 119–1387 x 106 cells/l. Six of 12 had detectable HIV-1 RNA levels in blood (range 51–31 000 copies/ml). In four subjects, HIV-1 RNA detection in seminal plasma was impossible, due to either insufficient seminal plasma volume (Subjects 7 and 11) or the presence of PCR inhibitors (Subjects 6 and 10). Three of these had detectable HIV-1 RNA levels in their paired blood plasma sample. Among the other eight, seminal plasma HIV-1 RNA was detected only in one subject, naïve to antiretroviral therapy (Subject 9). This subject, who provided a seminal sample before and after 2 months of HAART therapy, showed a higher seminal viral load than the corresponding paired blood plasma viral load, at both time points. In this case shedding of HIV-1 in semen was not associated with a concurrent increase in seminal leukocytes (0.3–0.4 x 106 cell/ml, see Table 2).
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Sperm parameters
Sperm parameters of the 13 semen samples collected from the 12 HIV-1 seropositive subjects are shown in Table 2. The mean total sperm count was 181 x 106 cells/ml (range 15–480 x 106 cells/l); 9 of the 12 HIV-1 seropositive subjects had >40 x 106 sperm/ejaculate. The mean volume was 2.5 ml (range 0.5–5), mean motility was 31.5% (range 10–50), mean abnormal sperm cells were 66.7% (range 58–80) and the mean seminal leukocyte count was 0.4 x 106 cells/ml (range 0.2–0.7), suggesting no concomitant genital tract inflammation. The subjects with the highest percentage of abnormal forms had the lowest sperm cell count (Subjects 1, 2, 3, 7 and 11). Sperm parameters of at least 9 of the 12 infected subjects studied were compatible with a normal fertilizing ability. To further evaluate sperm cell quality, sperm cell DNA fragmentation was evaluated by TUNEL technique in all HIV-1 seropositive subjects studied (Table 2), as well as in 5 HIV-1 seronegative healthy controls. This technique allows immunohistochemical detection of DNA strand breaks at the level of the individual cell. In line with our previous observations (Gandini et al., 2000
), all TUNEL-positive spermatozoa from both HIV-1 positive and negative subjects showed abnormal morphology (data not shown). However, in HIV-1 infected subjects the mean TUNEL- positive sperm cell value was 21.2% (range 9.5–35.4%; see Table 2), higher than the 2.5% (range 0.9–4.4%) for healthy subjects (data not shown and Gandini et al., 2000). A linear regression analysis was performed, where the dependent variable was the percentage of TUNEL-positive cells or abnormal forms and the independent variable was sperm cell concentration. Statistically significant negative correlations were observed between sperm concentration and percentages of TUNEL-positive sperm cells (r2 = 0.59; P = 0.003; coefficient = –0.183) and abnormal forms (r2 =0.68; P = 0.001; coefficient = –0.188).
Effective removal of NSC from HIV-1 seropositive purified sperm cells
The multilayer Percoll gradient column enabled a high concentration of viable, motile and well-structured spermatozoa together with non-viable, immotile and abnormal spermatozoa to be obtained in the 90–100% Percoll fractions (Gandini et al., 1999). However, morphological analysis revealed the presence of rare NSC that were completely removed along with their nuclei, by osmotic shock treatment (data not shown). Morphological observations were confirmed by cytometric analysis: NSC were detected in crude semen (Fig. 1A) but not in purified sperm cells (Fig. 1B) as revealed on the basis of CD4 and CD45 fluorescence signals. The efficiency of the sperm purification procedure was also evaluated by molecular analysis for specific NSC transcripts. GAPDH is expressed in NSC but not in germ cells, which are known to express a unique spermatogenic-GADPH gene (Bunch et al., 1998; Welch et al., 2000,2006). In all subjects studied, GAPDH mRNA was always detected in crude semen but not in purified sperm cells (detection limit 5 GAPDHpos cells/106 sperm cells) (Fig. 1C). PRM-2 mRNA, which is specifically present only in spermatozoa, was always amplified (Fig. 1D), thus suggesting that the purification procedure did not affect the ability to recover sperm mRNAs.
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HIV-1 DNA and RNA detection in purified sperm cells
HIV-1 DNA detection in purified sperm cells was performed by nested PCR which detects both unintegrated and integrated proviral DNA. In our assay, the detection limit was 4 HIV-1 DNA copies/106 sperm cell, as indicated by HIV-1 gag gene amplification of HIV-1 DNA experimental standard curve obtained from 8E5/LAS cell line (see Materials and Methods section). Five of the 12 HIV-1 seropositive subjects had detectable HIV-1 DNA assessed by gag and pol gene amplification (Table 3). The number of HIV-1 DNA copies in purified sperm cells ranged from 5 to 35 copies/106 cells. Alu-LTR PCR assay, which detects only integrated proviral DNA, gave negative results in all subjects (detection limit: 40 HIV-1 proviral DNA copies/106 cells). Only one (Subject 9) of the 12 HIV-1 seropositive subjects studied was positive for the HIV-1 full-length RNA genome in addition to proviral HIV-1 DNA, as indicated by RT–PCR analysis for the gag gene and LTR-gag regions (US primer set). RT–PCR performed with MS primers to reveal productive HIV-1 infection gave a negative result. In this subject, HAART therapy induced a 2 log reduction in the seminal viral load (Table 1) and the disappearance of HIV-1 DNA and HIV-1 RNA in the purified sperm cell fraction (Table 3).
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In situ HIV-1 DNA detection in crude semen and purified sperm cells
HIV-1 DNA was detected in the crude sperm cells of 10 (83%) of 12 HIV-1 seropositive subjects by in situ PCR hybridization (ISH-PCR) after chromatin decondensation. The HIV-1 DNA-positive spermatozoa were abnormal as showed by a wide range of structural alterations (Fig. 2). Positive spermatozoa showed clear staining over the whole area (Fig. 2A, D and F) or localized all over the sperm head or in the most anterior (Fig. 2C) or posterior region (Fig. 2B and E). Occasionally, HIV-1 staining was observed in the sperm neck or middle piece (Fig. 2G). The percentage of HIV-1 DNA-positive sperm in semen samples from all infected subjects studied are reported in Table 4. Semen purification decreased the number of in situ HIV-positive sperm cells. In two subjects where HIV-1 detection was carried out in paired crude and purified semen samples, a reduction in HIV-1 DNA positive sperm cells from 1.48 to 0.48% (67.5% reduction) and from 0.29 to 0.08% (73% reduction) was observed (Table 4). Sperm cells from HIV-1 seronegative subjects were always negative for HIV-1 in situ detection (Fig. 2H), whereas
-globin DNA was successfully amplified in all sperm cells (Fig. 2I).
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Detection of HIV-1 in sperm cells by PCR analysis has become a matter for debate (for reviews see Alexander, 1998
; Piomboni and Baccetti, 2000), due to the presence of infected NSC that may be present in sperm samples (Quayle et al., 1997; Pudney et al., 1999; Debono et al., 2000). In the present study, we developed an experimental strategy to purify ejaculated spermatozoa that, by avoiding swim-up selection, were representative of the sperm population as a whole. The procedure also enabled removal of NSC, quantification of their residual presence by high-sensitivity detection methods (i.e. cytometric analysis and nested RT–PCR analysis) and the detection of very low numbers of HIV-1 DNA copies. Sperm purification was obtained by centrifugation on a discontinuous Percoll gradient prepared with numerous dilutions (10 layers: from 30 to 100%). This multilayered gradient is used to separate sperm cells from the majority of NSC (Gandini et al., 1999) and to remove free and cell-adherent viral particles (Fiore et al., 2005). The sperm fraction recovered is representative of the overall population, as in addition to high-quality spermatozoa, it contains non-viable, immotile and atypical spermatozoa, whose percentage depends on the semen quality of the subject studied. This is particularly important in HIV-1 seropositive subjects, as their semen may contain few motile sperm cells, immotile and abnormal sperm and numerous NSC (Muller et al., 1998; Bujan et al., 2007). Multilayered Percoll gradient centrifugation followed by osmotic shock yielded a high degree of sperm purification, up to the complete disappearance of NSC, as demonstrated by cytometric analysis for CD4 and CD45 antigens and RT–PCR analysis for GAPDH mRNA transcript, both performed on crude semen and purified sperm cell fractions. CD4 and CD45 are leukocyte antigens absent in spermatozoa, whereas the GAPDH gene is expressed in all somatic cells but not in sperm (Bunch et al., 1998; Welch et al., 2000,2006). GAPDH provides a broad-spectrum marker for somatic cell contamination. Osmotic shock is a well-known approach to separate cells with different osmotic resistances and is routinely used to remove round immature germ cells from primary Sertoli cell cultures (Galdieri et al., 1981). Our data suggested that osmotic shock did not induce major sperm cell molecular damage as detection of sperm specific mRNAs, such as PRM-2 transcripts, was still possible by RT–PCR. Percoll followed by osmotic shock treatment has been used to obtain pure sperm RNA preparations (Miller, 1997; Miller et al., 2005) and hypo-osmotic swelling test of human spermatozoa performed with distilled water is indeed employed to assess their functional competence (Fuse et al., 1993). On the basis of our experimental strategy, we could therefore investigate the presence of HIV-1 on sperm samples form HIV-1 seropositive subjects devoid of any NSC contamination.
Our data demonstrate the presence of HIV-1 DNA in small amounts of ejaculated abnormal spermatozoa of HIV-1 infected subjects. Interestingly, in these subjects a high percentage of ejaculated spermatozoa had abnormal morphologies (range 58–80%) and the percentage of spermatozoa with fragmented DNA (range 9.5–35.4%) greatly exceeded normal values (range 0.9–4.4%) (Gandini et al., 2000). For technical reasons, we could not evaluate the percentage of TUNEL-positive cells among the HIV-1 positive sperm cells. However, we observed that HIV-1 DNA-positive sperm cells clearly show abnormal morphologies. The reliability of HIV-1 DNA detection by in situ PCR hybridization was confirmed by the numerous specificity and sensitivity controls and by amplifying for two different viral genes (gag and env). Localization of HIV-1 DNA in the sperm head was observed only after sperm chromatin decondensation, thus suggesting that the virus could either be ‘trapped’ in the sperm nucleus or integrated into the sperm genome. When in situ PCR hybridization was performed without prior chromatin decondensation, the presence of HIV-1 was revealed only in the cytoplasm of the spermatozoon (neck and middle regions) by us (data not shown) as well as by other authors (Bagasra et al., 1994). Data on in situ HIV-1 PCR hybridization have been obtained previously only on non-decondensed sperm cells (Bagasra et al., 1994; Persico et al., 2006). In our opinion, this must be considered an inadequate procedure, as a specific treatment with reducing agents is needed to gain access to the highly condensed sperm chromatin (Bernardini et al., 1997; Guttenbach et al., 1997). Detection of the virus was recently denied even when 0.4–0.5% of spermatozoa in 2 of 10 samples analysed before swim-up were found to be positive for HIV-1 DNA by in situ PCR (Persico et al., 2006). In this case, it was stated that the viral presence in spermatozoa pellet samples might be due to residual NSC or alternatively, to false positives due to non-specific hybridization of in situ PCR (Persico et al., 2006). Both explanations seem unlikely, as unequivocal sperm identification is guaranteed by its specific morphological features, and in situ PCR, when correctly performed along with positive and negative controls, is considered a powerful, highly reliable technique for the detection of viral DNA in human tissue sections and cells (Marziliano et al., 2005; Trincado et al., 2005; Comar et al., 2006; Nuovo, 2006; Tanji et al., 2006).
HIV-1 DNA was revealed in 10 and 5 out of 12 subjects by in situ PCR hybridization and by nested PCR analysis, respectively. Technical reasons could account for this discrepancy: (i) in situ PCR was performed on unfractionated crude semen, whereas nested PCR on purified sperm cells (i.e. after multilayered Percoll gradient and osmotic shock). This purification procedure reduces the number of HIV-1 positive cells, as demonstrated by drastic reduction in the percentage of HIV-1 positive cells when in situ PCR experiments were performed on the same semen sample before and after purification (
70%); (ii) infected subjects showed HIV-1 DNA positive sperm with abnormal morphology and an increased percentage value of fragmented DNA sperm (i.e. Tunel-positive cells). It is therefore conceivable that HIV-1 DNA positive sperm also carried chromatin damage. In this study, fragmented DNA was not recovered during the isolation of high-molecular weight DNA from purified sperm cells. This may also explain the discrepancy obtained in two subjects (Subjects 6 and 12) whose purified sperm cells tested positive with in situ HIV-1 DNA detection but not with nested PCR analysis.
The number of DNA viral copies detected by nested PCR ranged between 5 and 35 copies/106 spermatozoa whereas Alu-PCR, performed to evaluate the possible integration of HIV-1 into the sperm genome, gave negative results in all cases analysed. However, DNA proviral integration into the sperm genome cannot be excluded, as the Alu-PCR limit of detection was higher (limit of detection 40 HIV-1 DNA copies/106 cells) than the HIV-1 DNA copies detected by nested PCR (detection limit 4 HIV-1 DNA copies/106 cells).
Detection of HIV-1 DNA by soluble PCR in the sperm pellet has been denied by many authors (Quayle et al., 1997; Pudney et al., 1999; Kim et al., 1999; Persico et al., 2006) and confirmed by others (present study) (Baccetti et al., 1994; Bagasra et al., 1994; Scofield et al., 1994; Dulioust et al., 1998; Marina et al., 1998; Leruez-Ville et al., 2002). Several considerations may explain these discrepancies. The technical procedures used to detect HIV-1 in semen by laboratories, involved in medically assisted reproduction programs, have been based on modified commercially available PCR kits, whose sensitivity and specificity were found in a multicenter study to vary greatly (Pasquier et al., 2006a); when a low number of HIV-1 RNA/DNA copies were added to the standard sample, frequent false negatives were obtained (Pasquier et al., 2006a,b). In most studies (Marina et al., 1998; Kim et al., 1999; Pasquier et al., 2000), sperm fractions obtained by Percoll gradient followed by swim-up were used to test for HIV-1; this procedure enables only motile spermatozoa with specific structural characteristics such as normal morphology, intact membranes and normal DNA packing to be recovered (Erel et al., 2000). The remaining semen fractions containing immotile and abnormal spermatozoa have therefore been overlooked and rarely tested for HIV-1. When positive results have been obtained, they have always been attributed to the presence of contaminating infected NSC or to PCR contaminations (Dulioust et al., 1998; Marina et al., 1998; Tachet et al., 1999; Leruez-Ville et al., 2002), even in the absence of adequate controls to support these interpretations. In our opinion, adequate experimental strategies can overcome these misleading evaluations of the results obtained, as: (i) the progressive and possibly total elimination of somatic cells during sperm purification may be obtained and demonstrated by monitoring for the presence of mRNAs specific for somatic cells (present study and Miller et al., 1994); (ii) false PCR positives may be easily revealed by adequate controls and by replicating the PCR assay.
The presence of HIV-1 DNA in testicular germ cells of seropositive and AIDS subjects has been previously reported (Nuovo et al., 1994; Muciaccia et al., 1998a,b; Shevchuk et al., 1998), and more recently, productively infected testicular spermatogonia have been observed, after intravenously or intrarectally SIV/SHIV experimental infection of juvenile macaques (Shehu-Xhilaga et al., 2007). It is well known that chronic orchitis with progressive hypogonadism arises during HIV-1 infection (for a review seeLo and Schambelan, 2001). In the present study, a low sperm concentration was often associated with high percentages of sperm with abnormal morphologies and TUNEL-positive staining, thus suggesting that spermatogenesis is also impaired in these subjects. HIV-1 viral load was undetectable in the seminal plasma of all but one subject with HIV-1 DNA positive spermatozoa, thus making it less probable that sperm infection had occurred in the genital tract. It is therefore conceivable that abnormal spermatozoa carrying HIV-1 DNA were of testicular origin. The presence of HIV-1 RNA in the purified sperm fraction was detected by RT–PCR in only 1 of the 12 subjects studied. We observed the presence of viral full length RNA and not the multiply spliced viral RNA, thus suggesting that HIV-1 virus had only just penetrated into the spermatozoa. This was in a naïve subject with a high viral seminal load. It is therefore conceivable that sperm viral infection was a recent event, occurring during sperm transit along the genital tract. RT–PCR for HIV-1 performed on the purified sperm fraction of this subject after 2 months of therapy, gave negative results and the amount of virus in the seminal plasma was greatly reduced. In the other 11 cases, which were all negative for HIV-1 RNA in the purified spermatozoa, the seminal viral load was undetectable or very low.
In conclusion, our data demonstrate that HIV-1 infected subjects can ejaculate small amounts of HIV-1 DNA-positive abnormal spermatozoa. It should be pointed out that these findings may be irrelevant for medically assisted reproduction in serodiscordant couples (Savasi et al., 2007; van Leeuwen et al., 2007). In these cases, the current procedures for assisted reproduction, such as sperm washing by Percoll gradient followed by swim-up, are sufficient to remove abnormal spermatozoa. The possibility that in vitro infected spermatozoa penetrate human oocytes has been provided (Baccetti et al., 1994). However, generation of a HIV-1 infected child following assisted reproduction treatments of HIV-1 serodiscordant couples has never been reported. HIV-1 DNA-positive spermatozoa, carrying a wide range of structural alterations, may be unable to fertilize and/or to generate viable embryos. The possible role of abnormal spermatozoa carrying HIV-1 DNA in the sexual transmission of HIV-1 as well as re-infection of the host remains to be clarified. The role of sperm cells as HIV-1 carriers must be re-evaluated, considering the recent evidence of the presence of CCR5 and 3 (Muciaccia et al., 2005a,b) on the surface of the human sperm head of normozoospermic, healthy subjects.
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This work was supported by grants from: Istituto Superiore di Sanità: AIDS National Research Program to M.S.; Ministero dell'Università e della Ricerca, to M.S.; Fondazione Pasteur-Cenci Bolognetti, to M.S.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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We are grateful to Dr. Ivano Mezzaroma, Center for Infectious Diseases Sapienza University of Rome for his clinical collaboration, to Dr. Antonio Petrone, Sapienza University of Rome for the statistical analysis and to Marie-Hélèn Hayles for English language editing.
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Submitted on January 21, 2007; resubmitted on July 27, 2007; accepted on August 21, 2007.
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