Hum. Reprod. Advance Access originally published online on March 17, 2008
Human Reproduction 2008 23(5):1044-1052; doi:10.1093/humrep/den081
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Characterization of sperm chromatin quality in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy
1 Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada 2 Department of Urology, McGill University, Montréal, Québec, Canada 3 Department of Obstetrics and Gynecology, McGill University, Montréal, Québec, Canada 4 McGill University Health Centre, Montréal, Québec, Canada
5 Correspondence address. Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William Osler, Montréal, Québec, Canada H3G 1Y6. E-mail: bernard.robaire{at}mcgill.ca
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Abstract |
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BACKGROUND: Although the incidences of testicular cancer and Hodgkin's lymphoma have increased in young men over the past decade, combination chemotherapy has improved survival. As fertility is of importance to these patients, characterization of sperm chromatin structure is needed. We assessed sperm chromatin in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy, in comparison with control community and idiopathic infertile volunteers.
METHODS: DNA damage was assessed with the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and comet assays; reactive thiols (SH) and DNA compaction were determined with the monobromobimane (mBBr) and chromomycin A3 (CMA3) assays, respectively.
RESULTS: Both testicular cancer (37%) and Hodgkin's lymphoma (81%) patients had normospermic samples with increased DNA damage, compared with controls. Cancer patients also had higher reactive thiols and CMA3 staining, indicating low DNA compaction.
CONCLUSIONS: Sperm DNA integrity and compaction were affected in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy. Although SCSA, TUNEL and comet assays all detected DNA damage, the latter was optimal for use in cancer patients. A combination of the comet assay with tests that evaluate sperm DNA compaction, such as flow cytometry-based CMA3 and mBBr assays, is a reliable strategy to characterize sperm chromatin quality in cancer patients at the time of sperm banking.
Key words: DNA strand breaks/nuclear compaction/CMA3/SCSA/comet assay
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Introduction |
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Although there has been an increase in the incidence of testicular cancer (Garner et al., 2005
; Bray et al., 2006; Walsh et al., 2006) and Hodgkin's lymphoma (Liu et al., 2003) during the past decade, 80–90% of the patients can now be cured because of the improvement in therapeutic strategies (Huddart and Birtle, 2005; Kopp et al., 2006). These two malignancies occur in men at a young age; the high survival rate makes fertility potential of paramount importance for these patients. Although the chemotherapy used in these patients is effective, it may also have a persistent negative impact on spermatogenesis (Petersen and Hansen, 1999; Gandini et al., 2006). As a consequence, cryopreservation of seminal material before men initiate their chemotherapy is recommended to preserve their fertility potential (Magelssen et al., 2005; Edge et al., 2006). Thus, it is important to elucidate sperm chromatin quality in cancer patients prior to chemotherapy. The term ‘chromatin quality’ refers to all possible alterations that could occur in the sperm chromatin.
It is now evident that damage to the paternal genome may have detrimental effects on the production of offspring; these include a low fertilization rate, blastocyst formation rate and pregnancy rate, in addition to an increased rate of abortion and abnormal fetal development, perhaps also impacting on the health of surviving children (Zini et al., 1999; Virro et al., 2004; Marchetti and Wyrobek, 2005). Sperm chromatin is formed by the association of DNA with small basic proteins called protamines, promoting the compaction of the genetic material to be able to fit into the head of the spermatozoon (Wykes and Krawetz, 2003). The compaction of chromatin increases further during epididymal transit with the cross-linking of thiol groups in protamines to form disulfide bridges (Bedford et al., 1973). Defects in sperm chromatin structure have been associated with infertility in men (Aoki and Carell, 2003; Aoki et al., 2005; Oliva, 2006).
Both flow cytometry technology, using fluorescent probes with the ability to bind DNA or other components of the sperm chromatin, and single cell microscope-based methods are available to study the sperm chromatin structure. These approaches have been used to assess DNA integrity, and the level of compaction and protamination in spermatozoa from infertile men and cancer patients (Evenson and Jost, 2000; Virro et al., 2004; Stahl et al., 2006). The sperm chromatin structure assay (SCSA®) evaluates the susceptibility of the sperm DNA to low pH-induced denaturation in situ (Evenson et al., 1999). Single- and double-strand DNA breaks can be detected in spermatozoa by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (Sergerie et al., 2005) and comet assays (Haines et al., 1998; Codrington et al., 2004). The latter is a very sensitive microscope-based method that has been used to evaluate oligozoospermic semen samples (Morris et al., 2002). Reactive SH groups in protamines can be characterized using monobromobimane (mBBr) with flow cytometry (Seligman et al., 1994; Zubkova et al., 2005), whereas sperm DNA compaction can be assessed using chromomycin A3 (CMA3) in a flow cytometry-based assay (Zubkova et al., 2005). Thus, a battery of tests is available to assess sperm chromatin quality.
Controversy remains with respect to the utilization of these techniques (Zini et al., 1999; Evenson and Wixon, 2005, 2006; Payne et al., 2005; Makhlouf and Niederberger, 2006). Discrepancies between laboratories may be the result of differences in the instruments and protocols used, or they may be due to variations in the patient population selected. Moreover, the correlation among these techniques and with standard World Health Organization seminal parameters remains to be established (Spano et al., 2000; Virro et al., 2004; Payne et al., 2005), further complicating decision-making when clinicians must recommend a therapeutic solution to treat patients. Furthermore, most studies have evaluated sperm chromatin damage using one or two of the assays presented above; this strategy limits our ability to characterize damage to this complex sperm structure. Knowing that normal sperm chromatin is essential for embryo development, an exhaustive evaluation of its different components is necessary to assure sperm quality in infertile and cancer patients. Therefore, the aim of this study was to characterize sperm chromatin quality in cancer patients prior to chemotherapy using a battery of tests in every sperm sample from each subject.
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Materials and Methods |
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Sperm samples
Semen samples were obtained from a cohort of subjects (age 21–48 years) with advanced testicular cancer (stages II–III; n = 15) after orchiectomy or Hodgkin's lymphoma (stages II–IV; n = 15) who were to undergo chemotherapy. A cohort of subjects with idiopathic infertility (n = 21) and healthy community volunteers (n = 21) were also recruited as controls. This study was approved by the institutional ethics review board and informed consent was obtained from all subjects. Sperm concentrations, progressive motility (PM) and percentage of normal forms (NF) were determined in fresh samples according to World Health Organisation guidelines (1999). A progressive motility-normal forms index (PMNF index) [sperm concentration x (%PM + 1%) x (%NF + 1%), where %PM is the percentage of sperm with forward motility and %NF the percentage of sperm with normal morphology] was used to estimate the concentration of sperm with forward motility and normal morphology. The addition of 1% to %PM and %NF is an arbitrary adjustment used to help distinguish complete asthenospermia or complete teratospermia from azoospermia. Semen samples were stored at –80°C in the absence of cryoprotectants for subsequent sperm chromatin quality evaluation. The maximum storage time was <4 years. Recently, Edelstein et al. (2007)
showed that there is no detrimental effect on measures of DNA damage or on motility in human sperm stored for short (1–5 years) or long (9–13 years) periods of time. We are aware of the potential negative impact of reactive oxygen species on frozen/thawed spermatozoa. However, previous studies have shown that there are no differences in DNA fragmentation or chromatin compaction in spermatozoa from fresh or frozen samples (Evenson et al., 1999; Evenson and Jost, 2000; Duty et al., 2002). We avoid the addition of antioxidants because they can change the oxidation status of the thiol groups, rather than maintain the sperm chromatin in its original state; this completely modifies sperm compaction, as was observed in a recent study by Ménézo et al. (2007) in which they found that antioxidant therapy decreased sperm DNA compaction by reducing the -SS- bridges of protamines and had an adverse effect on fertility. In order to determine sperm chromatin quality, semen samples were thawed at 37°C, diluted 1:10 with phosphate buffered saline (PBS; pH 7.4), and centrifuged at 2300g for 5 min at 4°C to remove the seminal plasma. Owing to the necessity of selecting samples with sufficient sperm to run the majority of the sperm chromatin assays, sperm concentrations were usually >20 million spermatozoa/ml. The spermatozoa were fixed with 70% ethanol for 10 min, centrifuged again and resuspended in PBS. From this suspension, aliquots were taken for the acridine orange/SCSA®, TUNEL, comet, CMA3 and mBBr assays. These aliquots were kept at –80°C until further analysis for sperm chromatin structure.
Acridine orange/SCSA®
To assess the susceptibility of sperm DNA to acid-induced denaturation, the SCSA® was done using a method described previously (Evenson et al., 2002). A total of 10 000 acridine orange-labeled spermatozoa per sample were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Mississauga, ON, Canada) and the raw data were analyzed by using WinList cytometry software (Verity Software, Topsham, ME, USA). Results were expressed as mean DNA fragmentation index (DFI), standard deviation of DFI, percentage of DFI (%DFI, corresponding to the percentage of cells outside the main population) and as percentage of spermatozoa with high green fluorescence or high DNA stainability (%HDS), as an indication of sperm DNA compaction (Evenson and Wixon, 2005).
TUNEL assay
DNA strand breaks were analyzed using the Apo-DirectTM kit (BD Biosciences, Canada), according to Zubkova and Robaire (2006). An aliquot consisting of cells stained with the staining solution lacking the terminal deoxynucleotidyl transferase enzyme was included as a negative control for each sample. Fluorescence from 10 000 FITC-labeled spermatozoa was spectrophotometrically analyzed for every sample using the BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA) and was quantified using the BD FACSDiva software (BD Biosciences, USA). Results were presented as percentage of TUNEL positive cells or FITC mean fluorescence (arbitrary units).
Comet assay
DNA strand breaks present in spermatozoa were determined by the comet assay (Haines et al., 1998; Codrington et al., 2004) with modifications. Thawed spermatozoa (1 x 105 spermatozoa/ml) were mixed with melted agarose (0.5% low-melting-point grade in Mg2+ and Ca2+ free PBS, pH 7.4, at 42°C, 1:10). The slides were immersed in pre-chilled lysis buffer, containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% dimethyl sulfoxide (DMSO) and 1% Triton-X 100, for 1 h on ice, incubated in 0.05 mg/ml proteinase K in lysis buffer without DMSO and Triton-X 100 for 30 min at 37°C and finally in alkali solution (1 mM EDTA and 0.05 M NaOH, pH 12.1) for 45 min in the dark. Then, electrophoresis was done at 0.7 V/cm for 10 min (Mini-Sub Cell GT; Bio-Rad Laboratories, Inc., Mississauga, ON, Canada). Finally, slides were immersed in chilled 70% ethanol for 5 min and stored at room temperature. Sperm DNA was stained with SYBR Green (Trevigen; 1:10 000 in Tris–EDTA buffer, pH 7.5) and immediately photographed using a DAGE-MTI CCD300-RC camera (DAGE-MTI Inc., Michigan City, IN, USA) attached to an Olympus BX51 epifluorescence microscope. One hundred cells were analyzed randomly. Results were expressed as tail extent moment (tail length/fraction of tail DNA), using the KOMET 5.0 image analysis system (Kinetic Imaging Ltd, Liverpool, UK).
mBBr thiol labeling assay
Thiol (SH) labeling was done according to the method described by Zubkova et al. (2005) with modifications. Spermatozoa were incubated with or without 0.05 mM DTT. After a wash with PBS, samples were incubated in the dark for 10 min at 37°C with 0.5 mM mBBr (Calbiochem, La Jolla, CA, USA). After another wash with PBS, cells were sonicated and stored at 4°C in the dark. Analysis of spermatozoa was done at the Institut de Recherche Clinique de Montréal (IRCM) using a FACS Vantage flow cytometer (BD Biosciences, Canada). A total of 20 000 mBBr-labeled sperm was analyzed for each sample using Cellquest Pro (BD Biosciences, Canada). Results were expressed as the percentage of free thiols determined from the mean fluorescence histograms of spermatozoa incubated without DTT and the fluorescence of DTT-treated cells.
CMA3 assay
CMA3 quantification was according to Zubkova et al. (2005) and used with modifications. Spermatozoa were incubated in 0.25 mg/ml CMA3 in McIlvaine's buffer (17 ml of 0.1 M citric acid mixed with 83 ml of 0.2 M Na2HPO4 and 10 mM MgCl2, pH7.0) for 20 min at room temperature in the dark. A total of 20 000 sperm were analyzed for each sample at the IRCM using a MoFlo High Performance Cell Sorter (DakoCytomation Inc., Fort Collins, CO, USA). Results were expressed as CMA3 mean fluorescence and were obtained using Summit v.3.1 software (DakoCytomation Inc.).
Statistical analysis
Statistical analyses were done using the SigmaStat 2.03 software package (SPSS Inc, Chicago, IL, USA). Significant differences were determined using one-way analysis of variance and the Bonferroni post hoc test or Kruskal–Wallis one-way analysis of variance, as appropriate according to the data distribution. To differentiate samples with low sperm chromatin quality in cancer patients, receiver operating characteristic (ROC) curves and specificity and sensitivity values (Riffenburgh, 2006) were generated using MedCalc software (Mariakerke, Belgium). Owing to the lack of a gold standard method to assess sperm chromatin quality, we used ROC curves to analyze data from each assay by comparing community healthy volunteers with the infertile patient group. The latter was considered as a positive control with a known abnormal reproductive outcome. The ROC analysis allow us to establish the cut-off values for each assay in order to identify those samples with ‘poor sperm chromatin quality’ when they do not meet the cut-off for the specific assay. Correlation analyses were done using the Pearson's coefficient or the Spearman rank test, as appropriate. The percentages of men with low sperm chromatin quality in cancer patients were compared with those of community healthy volunteers by the Fisher exact test. Data are presented as means ± SEM. A difference is considered to be significant when the P-value is 
0.05.
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Results |
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Semen analysis
According to the WHO guidelines (World Health Organisation, 1999
), semen analysis was normal in 37% of the patients with testicular cancer and in 81% of those with Hodgkin's lymphoma. In contrast, only one patient (4%) with idiopathic infertility was classified as normospermic. The infertile and testicular cancer groups showed significantly lower sperm concentrations (Fig. 1A), PM (Fig. 1B) and percentage of NF (Fig. 1C) compared with the control group. Sperm concentrations are higher than the cut-off suggested by the WHO (20 million spermatozoa/ml) due to the necessity of selecting samples with sufficient sperm to run the majority of the sperm chromatin assays. When the PMNF index was analyzed, the infertile and testicular cancer groups showed significantly lower values compared with control (Fig. 1D). Sperm parameters in Hodgkin's lymphoma patients, according to either WHO guidelines or the PMNF index, did not differ significantly from the control values (Fig. 1).
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Sperm chromatin structure analyses
The susceptibility of spermatozoa to low-pH DNA denaturation was determined by the SCSA® assay (Fig. 2). Percentage DFI was significantly higher in infertile and testicular cancer patients, compared with the control group (Fig. 2A); the mean DFI was significantly higher in infertile, testicular cancer and Hodgkin's lymphoma patients (Fig. 2B). No statistical differences were found among the testicular cancer and Hodgkin's lymphoma groups and the control group when SD DFI was considered (Fig. 2C).
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DNA strand breaks were detected by TUNEL and comet assays. Spermatozoa from infertile patients had a significantly higher percentage of TUNEL positive cells compared with the control group (Fig. 3A). Similar information was obtained when the FITC mean fluorescence was considered (data not shown). Although not significant, a trend toward an increase in TUNEL positive cells (Fig. 3A) and FITC fluorescence (data not shown) was observed in testicular cancer patients. There were no significant differences in the number of TUNEL positive cells or in the FITC fluorescence in spermatozoa from Hodgkin's lymphoma patients compared with the control group. The comet tail extent moment was significantly higher in infertile, testicular cancer and Hodgkin's lymphoma patients, compared with the control group (Fig. 3B).
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Compared with the control group, the levels of reactive SH were significantly lower in the infertile patients, but significantly higher in Hodgkin's lymphoma patients (Fig. 4A); there were no significant differences in reactive SH groups between the controls and testicular cancer patients. No statistical differences were found between the control and the infertile group regarding the level of CMA3 mean fluorescence or the %HDS (Fig. 4B and C). However, CMA3 mean fluorescence was higher in testicular cancer patients compared with the control group (P < 0.05), and there was a trend toward an increase in Hodgkin's lymphoma patients (P = 0.05). In comparison to the control group, the %HDS was also higher in the testicular cancer patients, but only slightly higher in the Hodgkin's lymphoma patients.
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Correlations among sperm chromatin quality assays
The correlations among the different assay parameters chosen to assess sperm chromatin structure are presented in Table I. DFI was positively correlated with mean DFI, SD DFI (SCSA® assay), TUNEL positive cells, FITC mean fluorescence and comet tail extent moment, but not with reactive SH (mBBr labeling assay). There was a positive correlation between mean DFI, TUNEL positive cells and FITC mean fluorescence. However, mean DFI did not correlate with reactive SH or with the comet tail extent moment. Standard deviation of DFI was negatively and positively correlated with reactive SH and FITC mean fluorescence, respectively. There was a negative correlation between FITC mean fluorescence and reactive SH.
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ROC curves and sensitivity and specificity analysis
To assess the value of the individual assays in detecting sperm samples with poor sperm chromatin quality, we compared the SCSA®, TUNEL, comet, CMA3 and mBBr labeling data in community healthy volunteers and infertile men using ROC curve analysis. The area under the curve (AUC) measures the accuracy of the test; an AUC of 1 represents a ‘perfect’ test and an AUC of 0.5 represents a ‘worthless’ test. The AUC were 0.87, 0.92, 0.84, 0.79, 0.60, 0.61 and 0.97 for DFI (SCSA®), TUNEL positive cells, comet tail moment (comet assay), free thiol groups (mBBr assay), CMA3 mean fluorescence, HDS (SCSA®) and PMNF index, respectively. There were no statistical differences among the 95% confidence intervals of the AUC for the DFI, TUNEL positive cells, reactive SH and comet tail extent moment tests, suggesting that these assays have a similar power to predict the sperm chromatin quality of a given sample. CMA3 and HDS (SCSA®) had lower areas under the curve and thus were poorer predictors of sperm chromatin quality. There were no statistical differences in specificity and sensitivity among the assays (Table II). DFI, mean DFI and SD DFI had similar sensitivity and specificity values (data not shown). Interestingly, the PMNF index had high values of sensitivity and specificity (Table II).
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Correlations among assays and sperm parameters
Correlations among the sperm chromatin quality assays and sperm parameters from the semen analysis are presented in Table III. Sperm concentration negatively correlated with DFI and SD DFI (SCSA®). Total motility, PM and percentage of NF negatively correlated with the SCSA® assay and positively correlated with reactive SH groups. TUNEL positive cells correlated negatively only with PM. The total number of spermatozoa in the ejaculate did not correlate with any of the assays used. Moreover, only total motility correlated with the comet tail extent moment. The PMNF index correlated only with SCSA® parameters.
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Relationship between sperm parameters and low sperm chromatin quality
Although the sperm chromatin quality is of paramount importance in the preservation of the fertility potential in cancer patients, strategies to determine if sperm samples from cancer patients contain low levels of DNA damage are still elusive. We classified the semen samples from testicular cancer and Hodgkin's lymphoma patients according to the PMNF index with a cut-off of 3.9. A normal semen sample according to the PMNF index was observed in 37% and 81% of the testicular cancer and Hodgkin's lymphoma patients, respectively. However, we found that even among the testicular cancer patients with a normospermic sample, many had low sperm chromatin quality: 83% and 100% of the subjects had high mean DFI and comet tail extent moment values, respectively; these percentages were significantly higher compared with that of the community volunteers group (14% and 8% for mean DFI and comet tail moment, respectively; P < 0.05). A similar situation was observed among Hodgkin's lymphoma patients; in those with samples considered to be normospermic, 58% and 73% had high mean DFI and comet tail extent moment values, respectively. Again, these percentages were higher than those from the community volunteers group (P < 0.05; Table IV). Interestingly, a high percentage of samples from testicular cancer (67%) and Hodgkin's lymphoma (73%) patients had significantly higher levels of CMA3 staining, a parameter associated with poor sperm DNA compaction (Zubkova et al., 2005
) compared with the community healthy volunteers group (P < 0.05; Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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In this study, we compared the sperm chromatin quality in samples from testicular cancer and Hodgkin's lymphoma patients with those from healthy volunteers (control group) and infertile patients by using a complementary approach that included flow cytometry-based analysis and the comet assay. We found that although 37% and 81% of the testicular cancer and Hodgkin's lymphoma patients, respectively, were normospermic (according to the PMNF index), they did have poor sperm chromatin quality.
There is controversy regarding the possibility that spermatozoa from cancer patients have significant DNA damage (Thomson et al., 2002; Stahl et al., 2004, 2006; O'Donovan, 2005; Spermon et al., 2006). SCSA®, TUNEL and comet assays detected DNA damage when samples from the testicular cancer and Hodgkin's lymphoma patients were analyzed (Figs 2 and 3, Table IV). That these assays were significantly correlated with each other (Table I) and detected sperm samples with DNA damage with similar specificity and sensitivity in infertile men (Table II) suggests that any of these assays can be used to determine DNA damage in sperm samples from cancer patients. However, the comet assay was best to evaluate DNA damage in cancer patients, detecting DNA damage in 100% and 73% of the normospermic samples from testicular cancer and Hodgkin's lymphoma patients, respectively (Fig. 3B and Table IV). Because the comet assay requires a low number of cells, it is a useful tool to evaluate DNA damage in patients with severe oligozoospermia, a condition that frequently occurs in men prior to and after cancer chemotherapy.
Spermatozoa from infertile men have been reported to have high CMA3 fluorescence levels, suggesting decreased DNA compaction (Ramos et al., 2004; Spermon et al., 2006). However, we did not observe any statistical difference in CMA3 fluorescence between healthy volunteers and infertile patients (Fig. 4B). Since idiophatic infertility is a multifactorial disease in which the causes are not yet known, it is possible that in our infertile population, a low level of protamination might not be the main cause of infertility. We determined CMA3 fluorescence by flow cytometry in a large number of cells, whereas other investigators have used a microscope-based assay. Spermon et al. (2006), determining CMA3 fluorescence by direct observation of the labeled spermatozoa under the microscope and further quantification of fluorescence by image analysis, reported an improvement of DNA compaction after chemotherapy in testicular cancer patients. In the present study, we observed higher levels of CMA3 fluorescence in samples from cancer patients compared with controls (Fig. 4B), suggesting that spermatozoa from these patients had a low level of compaction and therefore a low sperm chromatin quality. The advantage of determining CMA3 fluorescence by flow cytometry over the slide-based assay is the possibility of performing a more rapid, sensitive and accurate analysis of high numbers of cells, thus increasing statistical relevance and reducing selection bias.
The percentage of reactive SH, determined by flow cytometric analysis of mBBr fluorescence, was higher in fertile than infertile patients (Seligman et al., 1994) (Fig. 4A). Interestingly, spermatozoa from these patients had less reactive SH in most of their proteins, including protamines. The ‘overoxidation’ of SH groups may arise from abnormal sperm maturation in the epididymis. Our findings in the infertile group are in concordance with those obtained by Seligman et al. (1994); spermatozoa from infertile patients had lower levels of mBBr fluorescence (indication of reactive SH) but similar levels of SS+SH (Seligman et al., 1994) (data not shown). The percentage of free thiols in spermatozoa was higher in samples from Hodgkin's lymphoma patients than from the control group. This finding, and the observation of higher levels of CMA3 labeling in spermatozoa from Hodgkin's lymphoma and testicular cancer patients (Fig. 4B), suggests that cancer patients also have less compacted sperm chromatin, confirming the results of Spermon et al. (2006) for testicular cancer patients.
The PMNF index increased the pressure of selection of samples since it considers the three main parameters of the semen analysis (sperm concentration, percentage of PM and percentage of NF) and showed an AUC of 0.97 when the ROC analysis was used with high sensitivity and specificity (Table II). However, this parameter failed to detect those samples with low sperm chromatin quality, since 37% and 81% of the testicular cancer and Hodgkin's lymphoma patients, considered as normospermic according to the PMNF index, had poor sperm chromatin quality (Table IV). The fact that only a few of the assays used in this study have significant but low or moderate correlation with seminal parameters (Table III) confirms what others have reported in humans using only the SCSA® (Evenson et al., 1999; Spano et al., 2000; Virro et al., 2004; Payne et al., 2005).
In some cases of idiopathic infertility, there is no detectable sperm DNA damage present in the semen samples (Verit et al., 2006); in these men, genetic abnormalities or not yet characterized damage to the sperm nucleus may play a significant role in the pathogenesis of the disease. Thus, a complementary study, similar to the one presented in this paper, may give a better indication of sperm chromatin quality by evaluating the level of DNA strand breaks, DNA compaction and redox status of the protamine thiol groups.
Human sperm chromatin is a complex structure, susceptible to damage from different sources; therefore, complementary approaches are of paramount advantage in determining causes of male infertility. The present study is the first to utilize techniques that evaluate different components of the sperm chromatin in the same sperm samples from infertile and cancer patients. There is no doubt that sperm chromatin integrity is of fundamental importance for these patients to father a child. Improvements in the evaluation of sperm samples to be used for assisted reproduction technologies are important for the next generation. Although ICSI is becoming the therapeutic strategy to achieve this goal, the results presented in the present study suggest that more research on the genetic integrity of the spermatozoa from cancer patients is needed to assure normal reproductive outcomes. Furthermore, an understanding of the specific damage that the cancer itself and the chemotherapy promote in the testis, as well as in the epididymis, will contribute to the treatment of cancer patients. In this study, we presented new data demonstrating the high incidence of DNA damage and low compaction in spermatozoa from testicular cancer and Hodgkin's lymphoma patients at the time of the diagnosis. The combination of the comet assay and tests that evaluate sperm DNA compaction, such as the flow cytometry-based CMA3 and mBBr assays, is a reliable strategy to better characterize the sperm chromatin quality in cancer patients at the time of sperm banking and after the initiation of chemotherapy, until the time when sperm chromatin integrity has recovered.
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Funding |
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These studies were funded by a grant from the Institute for Human Development, Child and Youth Health of the Canadian Institutes of Health Research.
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Acknowledgements |
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We thank Drs Eric Massicotte and Martine Dupuis from Institute de Recherches Cliniques de Montréal and Mr Ken McDonald from McGill University Life Science Complex for their assistance with the CMA3 and mBBr and TUNEL assays, respectively.
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References |
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Submitted on August 8, 2007; resubmitted on February 8, 2008; accepted on February 22, 2008.
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