Hum. Reprod. Advance Access originally published online on February 13, 2006
Human Reproduction 2006 21(6):1525-1530; doi:10.1093/humrep/del004
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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
1 Department of Obstetrics and Gynaecology, Sacco Clinical Sciences Institute, University of Milan Medical School and 2 Human Genetics Laboratory, Department of Medicine, Surgery and Dentistry, San Paolo School of Medicine, University of Milan, Milan, Italy
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Sacco Clinical Sciences Institute, University of Milan Medical School, 74 20157 Milan, Italy. E-mail: riproduzione.assistita{at}hsacco.it
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Abstract |
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BACKGROUND: To determine the presence of human immunodeficiency virus-1 (HIV-1) viral RNA/DNA in whole semen, in properly isolated seminal fractions and in spermatozoa after swim-up, by extractive nested PCR and to compare the detection of HIV DNA by in situ PCR (IS-PCR) with the results of nested PCR. METHODS: We tested HIV-1 RNA and DNA by nested PCR in semen and in seminal fractions from 55 patients. Non-spermatic cells and spermatozoa pellet fractions from 10 HIV-1-positive and five HIV-1-negative men were tested for proviral DNA by IS-PCR. RESULTS: All samples of spermatozoa recovered after sperm washing were free of HIV RNA. HIV RNA tested positive in seven (13%) seminal plasma samples and only in two (4.2%) whole semen of these same samples. Of the seven seminal plasma samples testing positive for HIV RNA, four men had elevated blood viral load and three an undetectable viraemia. HIV DNA by IS-PCR turned positive in three of five samples in semen of HIV-noninfected men. CONCLUSION: HIV RNA/DNA detection in the semen of HIV-infected men proves the efficacy of sperm washing with swim-up of spermatozoa. It is recommended that nested PCR be conducted on purified seminal compartments. IS-PCR is inadequate for detecting HIV in semen.
Key words: HIV-1/IS-PCR/nested PCR/semen wash/serodiscordant
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Introduction |
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There is no report of the birth of an infant with congenital human immunodeficiency virus-1 (HIV-1) infection conceived by a father infected with HIV-1, without maternal HIV-1 coinfection. Centres providing reproductive assistance through sperm washing and insemination to couples serodiscordant for HIV-1 because of male infection have also observed no cases of congenital infection (Semprini et al., 1992
; Marina et al., 1998; Coll et al., 1999; Kim et al., 1999; Gilling-Smith, 2000; Hanabusa et al., 2000; Pasquier et al., 2000; Bujan et al., 2002). This could be due either to the absence of HIV-1 genome in the male sperm cells or to a low viral load after sperm washing or to the inability of HIV-1-infected spermatozoa to fertilize or even to a poor follow-up of these couples.
There are several reports indicating that HIV-1 DNA cannot be found in washed spermatozoa isolated from non-spermatozoa seminal cells and seminal plasma (Borzy et al., 1988; Quayle et al., 1997; Bujan et al., 2004). In contrast to this reassuring epidemiological and laboratory background, there are reports indicating the possibility that HIV-1 virions are found attached to the sperm surface and even within its cytoplasm, by transmission electronic microscopy. Other papers report that proviral HIV-1 DNA can be detected in spermatozoa of men with acquired immune deficiency syndrome (AIDS), by extraction PCR, or found in spermatozoa of men infected with HIV-1, by in situ PCR (IS-PCR) (Bagasra et al., 1988, 1990, 1994; Anderson et al., 1990; Baccetti et al., 1990, 1991, 1994, 1998; Gobert et al., 1990; Miller and Scofield, 1990; Pudney, 1990; Mermin et al., 1991; Van Voorhis et al., 1991; 1994; Dussaix et al., 1993; Nuovo et al., 1994; Muciaccia et al., 1998; Shevchuk et al., 1998). To investigate these contradictory findings and to assess the role of sperm-washing technique in eliminating both HIV-1 RNA and HIV-1 DNA from semen infected with HIV-1, we tested the ejaculates of men infected with HIV-1 before and after processing semen into the three main seminal fractions—non-spermatozoa cells, cell-free seminal plasma and spermatozoa—by highly sensitive extractive nested PCR and by IS-PCR. The aim of this study was to determine the presence of HIV-1 viral RNA and DNA in whole semen, in properly isolated seminal fractions, and in spermatozoa after swim-up, by extractive nested PCR, and to compare the detection of HIV DNA by IS-PCR with the standard results of nested PCR.
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Patients
Fifty-five asymptomatic HIV-1-infected men (median age 36.0, range 28–43) donated their semen, and blood samples were collected after informed consent following an institutional review board-approved protocol. These consecutive patients were undergoing preliminary screening for assisted conception to reduce the risk of infection to their HIV-noninfected female partners. Forty-four men (80%) were former injected drug users, eight (15%) acquired the infection through heterosexual intercourse and two (4%) acquired the infection through contaminated blood products. Forty-one (74%) patients were on highly active antiretroviral therapy (HAART) including protease inhibitors, with 31 receiving three drugs and 10 receiving two drugs. Fourteen patients were not taking any antiretroviral drug as indicated by the Centers for Disease Control (CDC) clinical guidelines for the treatment of HIV-1 infection.
Samples
Semen samples were produced by masturbation and collected into sterile jars. Blood samples were taken into EDTA vacutainers on the same day. All samples could be processed within 2 h. Blood plasma was separated from other blood components by centrifugation and frozen in aliquots at –80°C. Peripheral blood mononuclear cells (PBMC) were prepared by gradient centrifugation over Ficoll (Biochrom KG, Berlin, Germany) and stored at –80°C as a dry pellet. A fraction of whole semen, after liquefaction, was stored at –80°C. Seminal plasma, spermatozoa and non-sperm cells (NSCs) were prepared by differential density-gradient centrifugation over 47 and 90% of PureSperm solution (Irvine Scientific, Santa Ana, CA, USA) for 30 min at 1600 g (Semprini et al., 1992). The seminal plasma was filtered using 0.20 µm filter (Sartorius, Goettingen, Germany) and stored at –80°C. The NSCs from the layer between seminal plasma and 47% PureSperm solution were washed twice in phosphate-buffered saline (PBS) and stored at –80°C as a dry pellet. The spermatozoa pellet was washed after 10 min at 1600 g in a sperm-washing solution (Irvine Scientific), then overlaid with 1 ml of sperm-washing medium and incubated at 37°C in an atmosphere of 5% CO2 for 60 min to allow spontaneous migration of spermatozoa to the surface of the culture medium. Motile spermatozoa at the upper layer of the culture medium were recovered by pipetting. The remaining spermatozoa were washed twice in PBS and stored at –80°C as a dry pellet.
HIV-1 RNA detection in blood plasma
HIV-1 RNA in blood plasma was quantified by Amplicor HIV-1 Monitor assay (Roche Diagnostic Systems) using the ultrasensitive protocol with a lower detection limit of 50 copies/ml.
HIV-1 RNA detection in whole semen and seminal plasma
RNA extraction was performed according to the Nuclisens Kit procedure (Organon Teknika, Durhan, NC, USA). Volumes ranged from 200 to 300 µl of pure or diluted (1:1, 1:5 and 1:10) seminal plasma. Each sample was tested with and without the addition of an internal control (130 000 copies/ml) to validate the extraction and the amplification steps.
HIV-1 RNA detection in non-spermatozoa cells and spermatozoa
RNA extraction was performed with a differential lysis technique: 1–2 x 106 cells were pretreated with lysis buffer [100 µg of proteinase K, 0.1 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA and 0.5% sodium dodecyl sulphate (SDS)] and incubated at 60°C for 1 h. Spermatozoa were lysed using a modified lysis buffer: 10 mM dithiothreitol (DTT) was added to allow the decondensation of DNA spermatozoa. Viral RNA was extracted by using modified acid phenol–guanidinium thiocyanate method (Chomczynski and Sacchi, 1987) (RNAzol; Bioprobe Systems, Montreuil sous bois, France), followed by isopropanol precipitation. The final resuspension for HIV-1 RNA was in 50 µl of ddH20, 20 U of RNAsin (Promega), 2 mM DTT and 5 mM Tris–HCl. Before retrotranscription of samples, the RNA was incubated with 1 U of DNAsi (Promega) at 37°C for 30 min and at 65°C for 10 min with DNAsi stop solution (Promega, Italia srl, Milan, Italy).
We amplified 
-actin RNA from semen RNA samples by PCR to verify the integrity of the cellular RNA purified from semen fractions.
HIV-1 DNA detection in whole semen and semen fractions
About 200 µl of whole semen and 106 to 2 x 106 NSCs and spermatozoa were lysed in a buffer containing proteinase K (100 µg), 0.1 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA and 0.5% SDS and incubated at 60°C for 1 h. In the whole semen and spermatozoa fraction, the lysis buffer was added with 10 mM DTT. Phenol–chloroform extraction and ethanol precipitation were then carried out as previously described (Hamed et al., 1993).
We amplified -globin DNA from semen DNA samples by PCR to verify the integrity of the cellular DNA purified from semen fractions.
Retrotranscription and nested PCR
About 15 µl of RNA solution was converted to cDNA with 1.3 U of MuLV-Reverse Transcriptase (Promega), in a 30 µl reaction mixture containing 1x PCR Buffer (Perkin Elmer, Norwalk, CT, USA), 5.8 mM MgCl2, 1 mM dNTPs, 5 µM DTT, 2.5 µM random hexamers (Perkin Elmer) and 20 U of RNAsin (Promega). About 1 µg of DNA and 10 µl of cDNA preparation were used for the nested PCR. The reaction mixture (100 µl) contained 10 mM Tri–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 1.25 U of AmpliTaq Gold DNA polymerase (Perkin Elmer) and 20 pmol of each primer. The two primer pairs used for PCR were complementary to conserved regions of the HIV-1 gag gene. The first round of PCR was performed with 1 µM primers (SK399S, 5'-ATA CCC ATG TTT TCA GCA TTA TCA GAA G-3'; SK399AS, 5'-ATC CAT CCT ATT TGT TCC TGA AGT A-3'). One-twentieth of the first PCR product was used in the second round of PCR using 1 µM primers (SK145, 5'-AGT GGG GGG ACA TCA AGC AGC CAT GCA AAT-3'; SK431, 5'-TGC TAT GTC AGT TCC CCT TGG TTC TCT-3'). The amplification was carried out at 95°C for 5 min followed by 95°C, 60 °C and 72°C for 30 s at each temperature for 35 cycles. The final PCR products were electrophoresed in 1.5% agarose gel. Each experiment was carried out twice to confirm our results. Furthermore, negative and positive controls were included in each PCR assay. The sensitivity of our PCR assay was 50 copies/ml for HIV-1 RNA and 10 copies/ml for HIV-1 DNA.
IS-PCR on PBMCs, non-spermatozoa cells and spermatozoa pellet
To perform IS-PCR (Bagasra et al., 1992, 1994; Bagasra and Pomerantz, 1993) and to detect HIV-1 provirus, cells (0.5 x 106 cells/ml) were seeded on the top of special slides composed of three separate compartments (Perkin Elmer IS-PCR glass slide) by sedimentation through gravity. The slides were air-dried and then placed sequentially, first on a heat block at 105°C for 90 s and then in 1% paraformaldehyde–phosphate-buffered saline solution (pH 7.4) for 1 h. The paraformaldehyde was inactivated by washing the slides once in 3% PBS and three times in 1% PBS. The slides were then treated with proteinase K (5 µg/ml in PBS) at 55°C for 2 h for PBMCs and NSCs and for spermatozoa at 20°C for 15 min. Proteinase K was inactivated by placing the slides on a heat block at 96°C for 2 min. The slides were washed in distilled water and air-dried. The cells were then subjected to amplification. The pair of primer used was SK145 and SK431 (see Retrotranscription and nested PCR). About 50 µl of a PCR mixture containing 10 µM of each nucleotide triphosphate, 0.5 µM of each primer, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris (pH 8.3) and 0.03 U of Taq polymerase (Promega) was added to the top of the first two compartments of each slide, and a PCR mixture lacking the primers was added to the last compartment. The slides were covered with Amplicover discs and Amplicover clips (Perkin Elmer) and placed on Gene Amp In Situ PCR System 1000 (Perkin Elmer). The amplification was carried out as described above for nested PCR.
After amplification, all the slides were washed in a 2% saline sodium citrate (SSC) buffer, and amplification products were detected by a biotinylated oligonucleotide (SK102, 5'-GAG ACC ATC AAT GAG GAA GCT GCA GAA TGG-3') inner to the amplified fragment. The hybridization mixture contained 0.03 pM of biotin-labelled probe, 15 nM DTT, 0.2% SSC, 15 µg of DNA of salmon sperm, 7.5 µM formaldehyde, 15 µg of Escherichia coli transfer RNA and 300 µg of bovine serum albumin (BSA). The mixture was applied to the slides and incubated at 92°C for 5 min and then transferred to a heated humidified chamber at 56°C for 4 h. The slides were thoroughly washed with PBS and then incubated with streptavidin–fluorescein isothiocyanate (FITC) complex (100 µg/ml in PBS, pH 7.2) at 37°C for 1 h to be examined with fluorescent microscope. Cells with yellow staining were counted as positive. Fractionated semen samples from HIV-1-negative patients were used as negative controls. To exclude the possibility of cross-reaction between NSCs and spermatozoa pellet, we mixed different cell lines: PBMCs from HIV-1-seropositive men and spermatozoa from seronegative patients at the ratio 1:2. Aspecific hybridization is a problem inherent in IS-PCR, and to reduce the possibility of false-positive results, we established a cut-off in samples from HIV-1-positive men, which was established arbitrarily as twice the number of positive cells observed in negative controls.
Statistical analyses
Nonparametric descriptive statistics was adopted after evaluating the normality of data. The Bravais–Pearson linear correlation coefficient was used to test the correlation between the viral load in blood plasma and the CD4 count. Spearman’s rank correlation coefficient was used to test the correlation between the viral load in blood plasma, the presence of the virus in semen and antiretroviral therapy. Fisher’s exact text was used to evaluate the correlation between viraemic and CD4 levels and the prevalence of seminal-fraction–positive cases.
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Results |
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HIV-1 RNA in blood
Median HIV-1 RNA copies per millilitre of blood plasma for the 55 subjects was 134 (interquartile range 79–6847; range 49–370 000 copies/ml). Mean CD4 cell count was 406 ± 32 x 105/ml. In the whole series, HIV-1 RNA viraemia was significantly and inversely correlated with CD4 counts (r = –0.33, P < 0.02). Forty-one men were on HAART (median HIV-1 RNA copies/ml blood plasma 99, interquartile range 49–6195 copies/ml). Twenty-eight patients had a viraemia below 250 copies/ml. Fourteen patients were not taking any antiretroviral therapy (median 1905 copies/ml; interquartile range 125–8222).
HIV-1 RNA testing
Table I summarizes HIV-1 RNA-positive samples in blood plasma, different seminal compartments and motile spermatozoa after sperm washing. HIV-1 RNA was not detected in any of 46 samples, available for testing, of spermatozoa after separation by gradient centrifugation and swim-up.
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HIV-1 DNA testing
Table II summarizes the results of testing for HIV-1 DNA in blood and semen. HIV-1 DNA was detected in all PBMC samples but could not be detected in any whole semen and in any spermatozoa sample both after gradient separation and washing only and after gradient separation, washing and swim-up.
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We analysed the relationship between the detection of HIV-1 nucleic acid in semen and viraemia, CD4 counts and antiretroviral treatment in the corresponding individuals (Table III). Four men had many plasma viral copies below the detection limit. Detection of HIV-1 DNA in seminal fraction correlated with detectable concentrations of HIV-1 RNA in blood plasma (r = 0.62, P < 0.000001). No significant correlation was found between the presence of the virus DNA in semen and HAART (r = 0.29, P < 0.03).
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IS-PCR in PBMCs, NSCs and spermatozoa
The results of IS-PCR in HIV-1-positive patients are summarized in Table IV. Five of 10 tested positive in NSC samples. For HIV-1-negative patients, the results of IS-PCR are summarized in Table V. Two of five HIV-1-negative controls tested positive in PBMC samples and three tested positive in washed spermatozoa. The prevalence of positive cells in the positive and negative controls was not significant (P > 0.5, Fisher’s exact test). To exclude contaminations or cross-reactions, the spermatozoa of these five negative controls were mixed with PBMCs of HIV-1-positive men: not a single case resulted in IS-PCR-positive spermatozoa.
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Discussion |
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All our males were in long-term relationship with their partners (over 90% were married), those who acquired HIV-1 through the use of injected drugs were free from addiction, those with genital tract infection were cured before producing the specimen we tested and all patients were under medical therapy or surveillance for their infection. Our data cannot therefore be directly applied to the general population of HIV-1-positive males, but our determinations of HIV-1 nucleic acid in semen before and after processing could be relevant to HIV-1-positive males who want to have a child, with or without reproductive assistance.
As expected, we found a significant negative correlation between HIV-1 viral loads and CD4 counts in treated and nontreated patients.
All samples of spermatozoa recovered after separation by gradient centrifugation and swim-up (sperm washing) were free of HIV-1 RNA above our threshold of 50 copies/ml and of proviral DNA. This confirms findings of previous reports in which nested PCR (Ohl et al., 2003; Bujan et al., 2004; Garrido et al., 2004) was used to assess the validity of sperm washing in HIV-infected semen. Although other more recent methodologies of sperm washing (Politch et al., 2004) confirm the validity of the general principle of removing the cellular component of semen, contradictory reports could be due to the inaccuracy of PCR techniques as in older studies (Marina et al., 1998) or to a too-lower threshold (one viral copy) of the PCR assay used to detect viral copies (Garrido et al., 2002, 2004; Meseguer et al., 2002) or to an improper use of the definition of sperm washing (Leruez-Ville et al., 2002), without the final swim-up of spermatozoa.
The second most important finding was that HIV-1 RNA tested positive in seven (13%) seminal plasma samples and only in two (4.2%) whole semen of these same samples. These data indicate that testing for HIV-1 RNA/DNA with the available PCR should be conducted on purified seminal compartments, seminal plasma, non-spermatic cells and spermatozoa, to limit the possibility of false-negative results. This can be due to the presence of inhibitors of PCR extraction which are eliminated by seminal processing. Alternatively, the amount of HIV-1 RNA present in seminal plasma could be diluted below the detection limit in the whole ejaculate. The clinical impact of this finding is a warning on possible false-negative results when testing unprocessed whole semen for viral nucleic acids. These findings were confirmed by HIV-1 DNA findings in seminal compartments, where eight (15%, of 52) non-spermatic cell samples tested positive, despite zero positive cases on whole semen.
Of the seven seminal plasma samples testing positive for HIV-1 RNA, six were from patients on HAART. Four men (one was untreated) had elevated blood viral load and three an undetectable viraemia. These findings confirm findings of previous reports of discrepancies between haematic and seminal HIV-1 concentrations (Bujan et al., 2004; Xu et al., 2005), either due to subtherapeutic concentrations of antiretroviral drugs in the male seminal tract or due to local production of HIV-1 RNA from localized cells which respond poorly to treatment.
The false-positive detection of HIV-1 DNA by IS-PCR in semen of HIV-1-noninfected men (Table V) confirmed that this technique is not adequate for studying the presence of provirus in semen fractions. Nonspecific hybridization inherent in the IS-PCR method could explain these false findings. To avoid false-positive results, we established cut-off at the level of twice the maximum percentage of positive cells tagged by IS-PCR in noninfected males.
Despite these restrictive criteria, two of 10 spermatozoa pellets, before swim-up, tested positive at IS-PCR (Table IV). The presence of the virus in spermatozoa pellet samples could be due to the presence of NSCs not completely eliminated during semen separation by discontinuous gradient centrifugation before swim-up. Alternatively, these could be real false-positive results due to nonspecific hybridization of IS-PCR.
Bagasra et al. (1994), Nuovo et al. (1994) and Muciaccia et al. (1998) showed the presence of provirus by IS-PCR in spermatozoa and in germ cells at all stages of differentiation, from spermatogonium to round spermatid. However, in none of these studies proper standards for IS-PCR specificity were assessed on noninfected males.
These methodological limitations of IS-PCR probably explain why in recent works this technique had been abandoned. Our experimental findings add further evidence in favour of the practical dismission of IS-PCR in this area of investigation.
In conclusion, this experimental work on HIV RNA and DNA detection in semen of HIV-infected men proves (i) the efficacy of the typical sperm-washing technique, which must include the final swim-up of spermatozoa, (ii) that testing for HIV-1 RNA/DNA with the available PCR should be conducted on purified seminal compartments and (iii) that IS-PCR is inadequate for detecting HIV in semen and seminal compartments in HIV-infected men. Despite consistent reported evidences of the validity of sperm-washing techniques, in assisted reproductive techniques, the quality of each procedure should be tested to provide an additional individual confirmation. This individual PCR check should be considered also for intracytoplasmic sperm-injection techniques as a quality assessment procedure in such a delicate medical treatment.
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References |
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Submitted on October 20, 2005; resubmitted on December 16, 2005; accepted on December 28, 2005.
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