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From the * Andrology and IVF Laboratories and the
Departments of Surgery and
Physiology, University of Utah School of
Medicine, Salt Lake City, Utah; the Andrology
Laboratory, Department of Pathology and Laboratory Medicine, Mount Sinai
Hospital, Toronto, Ontario, Canada; and the ¶
Department of Obstetrics and Gynecology,
University of Utah School of Medicine, Salt Lake City, Utah.
| Correspondence to: Douglas T. Carrell, PhD, University of Utah IVF and Andrology Laboratories, 675 Arapeen Drive, Suite 205, Salt Lake City, UT 84108 (e-mail: douglas.carrell{at}hsc.utah.edu). |
| Received for publication March 21, 2005; accepted for publication May 31, 2005. |
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Abstract |
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2 analysis
revealed a significant increase in the incidence of marked DNA fragmentation
in patients with diminished levels of either P1 or P2. The present study is
the first to report that human sperm protamine content is significantly
related to DNA fragmentation. In particular, sperm P1 and P2 concentrations
inversely correlate with DNA fragmentation, indicating a protective role of
the protamines against sperm DNA damage. In light of recent studies
highlighting the negative effect of sperm DNA damage on ART outcomes, these
findings indicate a possible clinical significance for human sperm protamine
levels.
Key words: Chromatin, DFI, DNA damage
In humans there are 2 forms of sperm protamine: protamine-1 (P1) and protamine-2 (P2), which occur in a strictly regulated 1-to-1 ratio (Corzett et al, 2002). Sperm protamine deficiency has been implicated in male infertility (Chevaillier et al, 1987; Balhorn et al, 1988; Belokopytova et al, 1993; de Yebra et al, 1993, 1998; Carrell and Liu, 2001; Aoki and Carrell, 2003; Aoki et al, 2005). In particular, aberrant P1/P2 ratios significantly relate to fertility status. The studies conducted by Yebra et al (1998) and Carrell and Liu (2001) describe a population of infertile males with undetectable sperm P2. Recently, P1 deficiency has also been identified in a population of subfertile males (Aoki et al, 2005).
It has been postulated that protamine deficiency is related to DNA damage in human sperm. A number of recent studies have focused on the relationship between sperm DNA damage and male infertility (Evenson et al, 2002; Tomsu et al, 2002; Virant-Klun et al, 2002; Seli et al, 2004). Although the biological significance of sperm DNA damage remains unclear, it appears to be detrimental to fertility in humans and has been linked to lower embryo quality (Tomsu et al, 2002; Virant-Klun et al, 2002), blastulation rates (Seli et al, 2004), and in vitro fertilization (IVF) pregnancy rates (Evenson et al, 2002; Bungum et al, 2004; Henkel et al, 2004; Virro et al, 2004). Mice that are haplo-insufficient for either P1 or P2 are sterile and have increased levels of sperm cell apoptosis, DNA damage, and embryonic arrest (Cho et al, 2001, 2003). However, relatively little is known about sperm DNA integrity in protamine-deficient human males.
The objective of this study was to examine the relationship between DNA integrity and protamines in human sperm. Specifically, the DNA fragmentation index (DFI) was used as a measure of DNA integrity. Protamine levels (P1/P2 ratio, P1, P2, and total protamine concentrations) are compared to DFI levels in the sperm of male infertility patients.
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Materials and Methods |
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Institutional Review Board approval was obtained before initiation of this study. Semen was collected and evaluated from 149 randomly selected male patients presenting for infertility assessment. A single semen sample was used for all assays, including the DNA Integrity Assay and protamine protein extraction and quantification. Samples with a sperm concentration of less than 3 x 106/mL were excluded because they offered insufficient material. After semen analysis and within 1 hour of the time of ejaculation, aliquots of raw semen were frozen at -80°C for later analysis.
DNA Integrity Assay
The DNA Integrity Assay was used to measure the DNA fragmentation index
(DFI) and was performed as previously described
(Evenson et al, 2002; Fischer et al, 2003). At the
time of analysis, semen samples were thawed on ice and diluted with TNE buffer
(0.01 M Tris-HCl, 0.15 M NaCl, and 1 mM ethylenediaminetetraacetic acid
[EDTA], pH 7.4) to 1-2 x 106 cells/mL. Two hundred-microliter
aliquots of diluted sample were mixed with 400 µL of a low-pH (pH 1.2)
detergent solution containing 0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl
for 30 seconds; this was followed by staining with 1.2 mL of 6 µg/mL
chromatographically purified acridine orange (AO) in a phosphate citrate
buffer (pH 6.0).
Three minutes after the staining procedure started, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif) equipped with an air-cooled argon laser. Measurements were collected in duplicate on 5000 cells per sample, and 2 aliquots were analyzed for each semen specimen. Under these conditions, AO intercalated with double-stranded DNA emits green fluorescence, and AO associated with single-stranded DNA emits red fluorescence. To avoid instrument drift, reference samples were used to set the red and green photomultiplier tube voltages. A new reference sample was run every 6 to 10 samples. FCS Express Version 2 (De Novo Software, Thornhill, Canada) was used for off-line analysis of the flow cytometric data.
DNA denaturation was expressed as the DFI, which represents the ratio of red to red plus green fluorescence intensity (Figure 1). This is similar to the SCSA definition of DFI as red (F > 630 nm)/red + green (F515-530 band pass). Based on a previously published categorization system (Evenson et al, 2002), 3 levels of DNA fragmentation were reported: low (<15% DFI), moderate (15%-30% DFI), and high (>30% DFI). These levels correspond to excellent, good, and fair-to-poor fertility potential, respectively.
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Semen aliquots with a known number of sperm (5.0-20 x 106 cells) were centrifuged at 500 x g for 5 minutes at 4°C. The pellet was washed in 1 mM phenylmethylsulfonylfluoride (PMSF) in distilled water, centrifuged at 500 x g for 5 minutes at 4°C, and the pellet was resuspended in 100 µL of 100 mM Tris buffer containing 20 mM EDTA and 1 mM PMSF (pH 8.0). One hundred microliters of 6 M guanidine and 575 mM dithiothreitol were added and mixed, followed by addition of 200 µL 522 mM sodium iodoacetate.
The suspension was maintained at room temperature for 30 minutes while being protected from light. To this suspension, 1.0 mL of 100% ethanol at 4°C was added and maintained for 1 minute before centrifugation at 12 000 x g for 10 minutes at 4°C. The ethanol wash was repeated and the pellet was resuspended in 0.5 M HCl (0.8 mL), incubated for 15 minutes at 37°C, and centrifuged at 10 000 x g for 10 minutes.
The supernatant was retained and the nuclear proteins were precipitated by the addition of 100% trichloroacetic acid (TCA) to a final concentration of 20% TCA. The solution was incubated at 4°C for 5 minutes and centrifuged at 12 000 x g for 10 minutes. The pellet was washed twice in 1% 2-mercaptoethanol in acetone (500 µL). The final pellet was dried and stored at -20°C until gel electrophoresis analysis.
Preparation of the Human Protamine Standard
A human protamine standard was prepared as previously described
(Mengual et al, 2003). Twenty
semen samples were pooled to extract and quantitate a highly purified sperm
protamine sample. Briefly, sperm were washed twice with 0.5 M HCl before
protamine extraction to remove other acid-extracted proteins. After acid
treatment, the protamines were extracted as described above. The protein
extract contained highly purified protamine, as verified by gel
electrophoresis and Western blot. The final protamine concentration was
determined using the RC DC protein assay kit (Bio-Rad). The protamine extract
was run using acid-acrylamide gel electrophoresis to determine the ratio of P1
to P2.
The final concentrations of P1 and P2 were calculated from the percent composition of each of the protamines in the total protamine standard. Subsequently, 1.52, 0.76, 0.38, and 0.19 µg of human sperm protamine standard were loaded in each gel and a standard regression curve was made to calculate the amount of protamine in each of the patient samples. The r2 value of the regression curve was 0.98 or better for each gel run in this study.
P1/P2 Quantification
Acetic-acid urea gel electrophoresis was performed as previously described
(Aoki et al, 2005). Briefly,
the separating gel contained 20% acrylamide, 0.1% bisacrylamide, 0.9 N acetic
acid, and 2.5 M urea. The stacking gel was comprised of 7.5% acrylamide, 0.2%
bisacrylamide, 2.5 M urea, and 0.375 M potassium acetate (pH 4.0). Gels were
stained with Coomassie blue using standard techniques and scanned using an
Umax-SE scanner with the SilverFast scanning software package (Umax
Technologies, Dallas, Tex).
Band intensities corresponding to P1 and P2 were quantified using National Institutes of Health Image-J software. P1 and P2 quantity were calculated against the standard curve generated from the human protamine standard (above). Protein quantity is reported as ng protein/106 sperm. Identity of protamine bands were established using Western blot analysis, as reported in a previous study (Carrell and Liu, 2001).
Protamine Quantification Quality Control
We used 2 measures of quality control to ensure our protamine
quantification protocol was valid and reproducible. First, aliquots of 20
x 106 sperm were made from a common semen sample taken from a
pool of 20 semen samples. One of these aliquots was run with each round of
extractions (n = 13). The resulting mean P1/P2 ratio (0.84 ± 0.01), P1
concentration (443.2 ± 5.1 ng/106 sperm), and P2
concentration (525.2 ± 6.1 ng/106 sperm) showed little
sample-to-sample variation (coefficient of variation [CV]; 1.19%, 1.15%, and
1.16%, respectively) and ensured reproducible results within individual
samples (Table 1).
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Second, to evaluate variations in the P1/P2 ratio, [P1], and [P2] between ejaculates from the same individual, in another study we analyzed the semen from 2 different ejaculates (obtained 6 months apart) in 42 individuals (Aoki et al, 2005). Results indicate no significant differences between ejaculates with respect to the P1/P2 ratio (1.03 ± 0.04 vs 1.11 ± 0.08), P1 concentration (560.4 ± 42.2 vs 571.9 ± 49.6 ng/106 sperm), or P2 concentration (535.5 ± 30.9 vs 527.1 ± 37.2 ng/106 sperm), as assessed by a paired Student's t test. The CV in the estimation of all sperm cell concentrations was within acceptable standards (3.9%).
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Patients were also identified who displayed a significant underexpression of the protamine proteins (both P1 and P2). The normal ranges of P1 and P2 concentrations in fertile men were established in a previous study (Aoki et al, 2005). The critical values defining the concentrations at which P1 and P2 were significantly underexpressed are used in this study (483 ng P1/106 sperm and 474 ng P2/106 sperm, respectively). Chi-square analysis was used to detect differences in the incidence of significantly elevated DNA damage among patients underexpressing P1 or P2.
Spearman's correlation coefficient was used to evaluate the correlation between sperm protamine levels (P1/P2 ratios, P1, P2, and total protamine concentrations) and DNA damage (DFI).
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Results |
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White blood cells (range, 0.0-0.4 x 106/mL) were present in 7.5% of the semen samples. None of the specimens qualified as leukocytospermic according to the WHO classification. Evaluation of semen quality parameters with respect to P1/P2 ratio groups revealed that sperm cell concentrations were significantly reduced in the low (34.7 ± 4.0, n = 37) and high (41.4 ± 15.3, n = 13) P1/P2 ratio groups compared with the normal group (69.7 ± 5.2, n = 97, P < .001). Sperm motility was significantly decreased in the low (17.6 ± 2.5, n = 17) and high (16.3 ± 3.9, n = 13) P1/P2 groups compared to the normal group (39.5 ± 1.7, n = 66, P < .0001). Normal sperm head morphology was significantly reduced in the low (14.1 ± 1.9, n = 17) and high (6.0 ± 3.5, n = 13) ratio groups compared to the normal group (25.5 ± 1.3, n = 66, P < .05; Table 2).
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Relationship of Semen Quality to DFI
Evaluation of semen quality parameters with respect to DFI categories
revealed that sperm cell concentrations were significantly reduced in the
moderate (54.0 ± 5.8, n = 48) and high (45.1 ± 7.6, n = 53) DFI
groups vs the low DFI group (70.9 ± 7.4, n = 48, P < .05;
Table 2). Progressive motility
significantly declined with increasing DFI group. The high DFI group (20.2
± 2.7, n = 34) had significantly lower progressive motility than the
moderate DFI group (29.6 ± 2.5, n = 33). In turn, the moderate DFI
group had significantly lower progressive motility than the low DFI group
(42.9 ± 2.6, n = 34, P < .001;
Table 2). Normal head
morphology was also significantly reduced in the moderate (19.1 ± 2.3,
n = 33) and high (17.3 ± 1.6, n = 34) DFI groups vs the low DFI group
(28.1 ± 2.0, n = 34, P < .005;
Table 2).
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A negative relationship was observed between the explanatory variables (P1/P2 ratio, P1, P2, and total protamine concentrations) and the DFI outcome variable (Figure 3). Spearman's correlation coefficient revealed a significant inverse correlation between DFI and the P1/P2 ratio (Rs, -0.18, P < .05). A greater significant inverse correlation was observed between DFI and P1 concentration (Rs, -0.29, P < .001), P2 concentration (Rs, -0.24, P < .005), and total protamine concentration (Rs, -0.28, P < .001).
A group of 70 patients were identified to show significant P1 underexpression. Chi-square analysis revealed a significant increase in the frequency of patients with abnormal DFI within this group compared to the 79 patients that expressed P1 normally (P < .005; Table 3). Additionally, 79 patients were identified with significant P2 underexpression. A significant increase in the frequency of abnormal DFI patients was observed in the group underexpressing P2 vs in patients expressing P2 normally (P < .01; Table 3). Therefore, there was a significant increase in DNA fragmentation observed in patients with diminished levels of either P1 or P2. Meanwhile, in patients with a normal P1/P2 ratio, there was a significant increase in DNA fragmentation observed in patients underexpressing both P1 and P2 vs in those normally expressing P1 and P2 (P < .01; Table 3).
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Discussion |
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In the present study, direct quantitative measurements of sperm protamine concentrations revealed that P1, P2, and total protamine quantity inversely correlated with DNA fragmentation. Furthermore, patients who under-expressed either P1 or P2 displayed elevated DNA damage much more frequently than patients who expressed the protamines normally. This increase in DNA damage was also observed in patients with a normal P1/P2 ratio who underexpressed both P1 and P2. These data are consistent with those obtained in studies using CMA3 staining that indicate that protamine deficiency is significantly related to increases in DNA damage. In addition, our data highlight the fact that both P1 and P2 contribute to this relationship.
The P1/P2 ratio was also correlated with DNA fragmentation. Numerous studies have implicated aberrant P1/P2 ratios in human male infertility (Balhorn et al, 1988; de Yebra et al, 1993, 1998; Khara et al, 1997; Carrell and Liu, 2001; Mengual et al, 2003; Aoki et al, 2005). The semen quality data presented here are consistent with those from these studies and show significantly reduced sperm counts, motility, and head morphology. In particular, patients with abnormally low P1/P2 ratios (<0.8) are reported to have severely affected sperm quality (Aoki et al, 2005), consistent with the present observation that DNA fragmentation is elevated in low-P1/P2 ratio patients. Although there is no fertile control group in the present study, a number of reports have established that protamine abnormalities and DNA damage are rare in men of known fertility (Evenson et al, 1999; Evenson and Jost, 2000; Carrell and Liu, 2001; Aoki et al, 2005).
Numerous studies have related sperm DNA integrity to semen quality. Many of these studies indicate that sperm motility, concentration, viability, and morphology correlate with DNA fragmentation (Evenson et al, 1991; Sun et al, 1997; Irvine et al, 2000; Muratori et al, 2000; Tomlinson et al, 2001; Zini et al, 2001; Sharma et al, 2004). Our data are consistent with those of these studies and show progressively diminished counts, motility, and morphology across the low, moderate, and high DFI categories. However, others have reported no meaningful correlation between DNA integrity and standard semen parameters (Evenson and Jost, 2000; Larson et al, 2000; Evenson et al, 2002).
The results of this study indicate that normally expressed sperm protamines may serve a protective function against DNA damage. Elegant studies in mice have established that protamine haplo-insufficiency is a direct causative factor involved in sperm DNA damage induction (Cho et al, 2003). Although a number of reports have established a link between sperm protamine content and chromatin structure, in humans the current data fall short of proving this hypothesis.
The protamines are critical for proper sperm chromatin packaging (Balhorn et al, 2000). Therefore, it is not surprising that sperm protamine concentrations correlate with DNA integrity. The protamine proteins are rich in cysteine content, which facilitates both inter- and intraprotamine cross-links via disulphide bond formation (Fuentes-Mascorro et al, 2000). Several studies have demonstrated that sperm chromatin stability is dependent on the quantity of these disulphide cross-links (Dadoune, 1995; Fuentes-Mascorro et al, 2000). Experimental evidence indicates that a reduction in disulphide bonding may alter sperm chromatin structure and reduce nuclear integrity (Kosower et al, 1992; Love and Kenney, 1999). However, the data remain conflicting on this issue, as one study demonstrated no meaningful correlation between sperm protamine thiol status and DNA denaturation in stallions (Evenson et al, 2000).
Additionally, the use of protamine-deficient human sperm for intracytoplasmic sperm injection results in sperm premature chromatin decondensation, indicating a less stable sperm chromatin in these patients (Nasr-Esfahani et al, 2004). Taken together, these studies indicate that protamine-deficient sperm adopt a less-stable chromatin structure, which may possibly arise as a consequence of decreased inter- and intraprotamine interactions, rendering them more susceptible to DNA damage. Future human studies are needed to test this hypothesis and to evaluate the relationship between sperm protamine levels, thiol content, and DNA damage.
In addition to absolute protamine quantity, the P1/P2 ratio also appears to be critical for chromatin stability. Although the P1/P2 ratio varies between genera, within a species it is highly conserved (Corzett et al, 2002). In humans, aberrations in the 1-to-1 ratio of P1 to P2 correlate significantly with male infertility, and the results of this study indicate that patients with abnormally low P1/P2 ratios have significantly elevated DNA fragmentation (Chevaillier et al, 1987; Balhorn et al, 1988; Belokopytova et al, 1993; de Yebra et al, 1993, 1998; Carrell and Liu, 2001; Aoki and Carrell, 2003; Aoki et al, 2005). However, patients with abnormally high P1/P2 ratios presented with DNA fragmentation levels comparable to those patients with normal P1/P2 ratios. These data are surprising considering the correlations between abnormal semen quality and high P1/P2 ratios. DNA fragmentation differences in these high-P1/P2 ratio patients may not have been elucidated because of the low number of patients identified. Further studies are needed that evaluate DNA fragmentation in a larger group of high-P1/P2 ratio patients.
The effects of aberrant protamine ratios on human sperm chromatin structure have not yet been elucidated. Clues may lie in studies using sophisticated atomic force microscopy and proton-induced X-ray emission spectroscopy, which provide insights into the details of protamine-DNA binding interactions (Hud et al, 1994; Bench et al, 1996; Balhorn et al, 2000). First, P1 and P2 are not randomly distributed along the DNA; instead, they tend to form characteristic clusters within species (Balhorn et al, 2000; Corzett et al, 2002). Thus, aberrations in the species-specific P1/P2 ratio may disrupt this characteristic protamine-DNA binding, possibly influencing secondary chromatin structure.
Second, we now have a description of the length of DNA that must be covered by each P1 and P2 molecule to facilitate their relative binding. Based on amino acid sequencing, P2 requires a longer stretch of DNA (approximately 15 bp) than P1 (approximately 11 bp) for proper chromatin incorporation (Bench et al, 1996). This may provide evidence for abnormal chromatin packaging when P2 is more abundant that P1, as is the case in patients with significantly reduced P1/P2 ratios. Given this scenario, physical size constraints dictate that there would be an overall reduction in the amount of protamine bound to the DNA, thereby reducing the overall number of disulphide cross-links.
Alternatively, chromatin structure aberrations and perturbations in the P1/P2 ratio may themselves be unrelated and may simply reflect generalized problems during spermiogenesis. Further studies are critical for our understanding of these proposed chromatin structure changes in patients with aberrant P1/P2 ratios.
Additional studies are necessary to fully understand how these various protamine aberrations influence abnormal DNA integrity. We suggest human studies to evaluate the protective role protamines play against sperm DNA damage. Additionally, it would be useful to evaluate the chromatin structure in patients with aberrant P1/P2 ratios to detect abnormal protamine-DNA interactions and perturbations of normal disulfide cross-linking. In the present study, only a limited number of severely oligospermic patients were included as a result of cell concentration requirements for the DNA integrity test. Therefore, a larger population of patients with severe oligospermia should be evaluated for protamine content and DNA integrity.
To conclude, we provide the first study investigating the relationship between DNA damage and direct quantitative measures of protamine levels in human sperm. Protamine concentrations (P1, P2, and total protamine) inversely correlate with the DNA fragmentation index. Additionally, patients with low P1/P2 ratios have markedly increased DNA damage. In light of recent reports highlighting reduced assisted reproductive technique (ART) fecundity in cases using DNA-damaged sperm, these data indicate that the sperm protamines may be of clinical significance in ARTs.
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  C. O'Flaherty, F. Vaisheva, B.F. Hales, P. Chan, and B. Robaire Characterization of sperm chromatin quality in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy Hum. Reprod., May 1, 2008; 23(5): 1044 - 1052. [Abstract] [Full Text] [PDF]
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K. Steger, J. Wilhelm, L. Konrad, T. Stalf, R. Greb, T. Diemer, S. Kliesch, M. Bergmann, and W. Weidner Both protamine-1 to protamine-2 mRNA ratio and Bcl2 mRNA content in testicular spermatids and ejaculated spermatozoa discriminate between fertile and infertile men Hum. Reprod., January 1, 2008; 23(1): 11 - 16. [Abstract] [Full Text] [PDF]
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 C. Ravel, S. Chantot-Bastaraud, B. El Houate, I. Berthaut, L. Verstraete, V. De Larouziere, D. Lourenco, A. Dumaine, J.M. Antoine, J. Mandelbaum, et al. Mutations in the protamine 1 gene associated with male infertility Mol. Hum. Reprod., July 1, 2007; 13(7): 461 - 464. [Abstract] [Full Text] [PDF]
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 J. de la Torre, C. Lopez-Fernandez, M. Pita, J. L. Fernandez, S. D. Johnston, and J. Gosalvez Simultaneous Observation of DNA Fragmentation and Protein Loss in the Boar Spermatozoon Following Application of the Sperm Chromatin Dispersion (SCD) Test J Androl, July 1, 2007; 28(4): 533 - 540. [Abstract] [Full Text] [PDF]
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 D. T. Carrell, B. R. Emery, and S. Hammoud Altered protamine expression and diminished spermatogenesis: what is the link? Hum. Reprod. Update, May 1, 2007; 13(3): 313 - 327. [Abstract] [Full Text] [PDF]
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A.M. Codrington, B.F. Hales, and B. Robaire Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa Hum. Reprod., May 1, 2007; 22(5): 1431 - 1442. [Abstract] [Full Text] [PDF]
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S. Singleton, A. Zalensky, G.F. Doncel, M. Morshedi, and I.A. Zalenskaya Testis/sperm-specific histone 2B in the sperm of donors and subfertile patients: variability and relation to chromatin packaging Hum. Reprod., March 1, 2007; 22(3): 743 - 750. [Abstract] [Full Text] [PDF]
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D. Miller and G.C. Ostermeier Towards a better understanding of RNA carriage by ejaculate spermatozoa Hum. Reprod. Update, November 1, 2006; 12(6): 757 - 767. [Abstract] [Full Text] [PDF]
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V. W. Aoki, B. R. Emery, L. Liu, and D. T. Carrell Protamine Levels Vary Between Individual Sperm Cells of Infertile Human Males and Correlate With Viability and DNA Integrity J Androl, November 1, 2006; 27(6): 890 - 898. [Abstract] [Full Text] [PDF]
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E. V. Zubkova and B. Robaire Effects of ageing on spermatozoal chromatin and its sensitivity to in vivo and in vitro oxidative challenge in the Brown Norway rat Hum. Reprod., November 1, 2006; 21(11): 2901 - 2910. [Abstract] [Full Text] [PDF]
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N. Torregrosa, D. Dominguez-Fandos, M. I. Camejo, C. R. Shirley, M. L. Meistrich, J. L. Ballesca, and R. Oliva Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients Hum. Reprod., August 1, 2006; 21(8): 2084 - 2089. [Abstract] [Full Text] [PDF]
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R. Oliva Protamines and male infertility Hum. Reprod. Update, July 1, 2006; 12(4): 417 - 435. [Abstract] [Full Text] [PDF]
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A. A. Makhlouf and C. Niederberger DNA Integrity Tests in Clinical Practice: It Is Not a Simple Matter of Black and White (or Red and Green) J Androl, May 1, 2006; 27(3): 316 - 323. [Full Text] [PDF]
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