1 Fertility Centre, 2 Department of Urology, Scanian Andrology Centre (EAA accredited), and 3 Department of Clinical Chemistry, Malmö University Hospital, Malmö, Sweden, 4 Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia, Rome, Italy and 5 Department of Medical Sciences, Clinical Chemistry, University Hospital, Uppsala, Sweden
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Abstract |
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Key words: accessory sex glands/SCSA/sperm chromatin/testosterone/zinc
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Introduction |
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The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which measures the susceptibility of sperm nuclear DNA to denaturation in situ (Evenson et al., 1980). The SCSA can provide an objective assessment of human sperm chromatin integrity within a semen sample, rapidly identifying and evaluating the fraction of cells showing DNA damage due to strand breaks or increased DNA stainability due to altered DNAprotamine interactions (Gorczyca et al., 1993
; Sailer et al., 1995
; Aravindan et al., 1997
; Evenson et al, 2000
, 2002
; Zini et al., 2001a
,b
).
It has been shown previously that the SCSA parameters appear to be generally weakly correlated with the semen parameters used routinely for conventional semen quality assessment (Evenson et al., 1991, 1999
; Spanò et al., 1998
, 2000
). Moreover, the SCSA has, in comparison with traditional measures of semen quality, an advantage of low within-person variability and intra/inter-assay variation (Evenson et al., 1991
, 1999
; Giwercman et al., 1999
; Zini et al., 2001c
). The SCSA can provide independent and complementary measurements of semen quality.
Recently, the SCSA has begun to be applied to a variety of large-scale epidemiological studies comparing semen quality in exposed and unexposed groups of men (Larsen et al., 1998, 1999
; Jühler et al., 1999
; Kolstad et al., 1999
; Lemasters et al., 1999
; Grajewski et al., 2000
; Selevan et al., 2000
; Bonde et al., 2002
). In clinical applications, the SCSA showed that sperm chromatin structure was compromised in patients with febrile illness (Evenson et al., 2000
) and testicular cancer (Evenson et al., 1984
; Fossa et al., 1997
; Kobayashi et al., 2001
). In general, infertile men have also been shown to have a higher fraction of sperm with chromatin abnormalities as detected by the SCSA than fertile controls (Zini et al., 2001a
,c
). Notably, it has been shown that the SCSA is an independent predictor of the human fertility potential both in vivo and in vitro (Evenson et al., 1999
, 2002
; Larson et al., 2000
; Spanò et al., 2000
). Thus, the SCSA analysis has proved its efficiency in toxicological and environmental studies, and may also become a powerful tool in clinical evaluation of the infertile male.
Some of the confounders that might impact upon the results provided by the SCSA are being identified (Spanò et al., 1998); these include the age of the donor, the duration of sexual abstinence, the presence of leukocytes and immature germ forms in the ejaculate (Ollero et al., 2001
), and smoking habits (Potts et al., 1999
). However, information regarding the exact nature of the mechanisms determining the variability of sperm chromatin structure, as assessed by this method, is still very limited. Although it is believed that the transition of intranuclear proteins from histones to protamines may be an essential factor regulating the stability of the sperm chromatin, other post-meiotic events may also be of importance. Furthermore, sperm chromatin structure increases its packaging level during passage through the epididymis (Golan et al., 1996
) and after mixing with the prostatic and seminal vesicle fluids, mainly because of the availability of zinc ions that further stabilize the final sperm DNA-nucleoprotein assembly (Evenson et al., 1993
).
A better understanding of the factors affecting sperm chromatin structure, as assessed by the SCSA, is necessary for this marker to be used in epidemiological studies and as a clinical tool. The aim of the current study was, therefore, to investigate the impact of the function of the testes, epididymis, prostate and seminal vesicles on sperm chromatin stability as assessed by the SCSA assay. Such knowledge might add to possibilities of developing cause-related treatment modalities for male infertility. The study was based on a population of 278 military conscripts who were representative of the general population of young Swedish males.
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Materials and methods |
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Hormone analyses
Circulating serum levels of FSH, LH, sex hormone-binding globulin (SHBG), testosterone and estradiol were measured using an automated fluorescence detection system (Autodelfia®; Wallac Oy, Turku, Finland) at a routine clinical chemistry laboratory (Uppsala University Hospital). Intra- and total-assay variation was <4.0 and <7.5% respectively. Free testosterone was calculated using a published method (Vermeulen et al., 1999), based on the known concentrations of testosterone and SHBG. Inhibin B levels were assessed using a specific immunometric assay (Groome et al., 1996
), with a detection limit of 15 ng/l and intra- and total-assay variation coefficients <7%. Reference levels for the hormone analyses are listed in Table I
.
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Semen analysis
Each subject provided, in a room at the laboratory, a semen sample by masturbation into a wide-mouthed plastic cup. The men were asked to maintain an abstinence period of at least 48 h, but the length of the actual abstinence period was recorded.
Sperm concentration was analysed according to World Health Organization (1999) recommendations using a modified Neubauer chamber and positive displacement pipettes for correct dilution of the ejaculate. The analyses of ejaculates were performed by only three laboratory assistants, and the inter-observer coefficient of variation for concentration assessment was 8.5%. This laboratory participates in an external Quality Control Programme organized by the Nordic Association of Andrology and ESHRE.
After 30 min of liquefaction, 0.2 ml aliquots of undiluted semen were coded and frozen in cryotubes at 80°C for later FCM SCSA analysis. In addition, 450 µl of ejaculate was mixed with 50 µl benzamidine to arrest degradation of the seminal components. The mixture was centrifuged for 20 min at 4500 g, after which the seminal plasma was decanted and stored at 20°C until analysed for concentrations of neutral -glucosidase (NAG), prostate-specific antigen (PSA), zinc and fructose.
Analysis of biochemical seminal markers
Seminal plasma NAG was measured using a commercially available Kit (Episcreen®; Fertipro, Gent, Belgium) according to the manufacturer's instructions. The test is based on measuring the intensity of a colour change evoked by the reaction between -glucosidase and 125 µl of reagent 1 (0.09% Na-azide) added to 125 µl of thawed seminal plasma. The mixture was mixed well by pipetting, one diagnostic tablet (p-nitophenyl-
-D-glucopyranoside) was added, after which the mixture was remixed, vortexed for 60 s and then incubated for 4 h at 37°C. After incubation, 3 ml of reagent 2 (0.02 mol/l NaOH) was added to the solution, which was then centrifuged for 6 min at 3000 g. The absorbance value, obtained by reading the supernatant against reagent 2 as a blank, was measured spectrophotometrically at 405 nm. This value was plotted on a standard curve and the corresponding total
-glucosidase activity was read on the abscissa. The NAG concentration was estimated by use of the corresponding table provided by the manufacturer.
The concentration of PSA in seminal plasma was determined with the PROSTATUSTM kit (Wallac Oy, Finland). This is a DelfiaTM (i.e. fluoroimmunometric) method, and uses three monoclonal antibodies against PSA. The coefficient of variation was 12% for control samples, with a mean PSA concentration of 660 mg/l.
The concentration of zinc in seminal plasma was determined colorimetrically (Makino et al., 1982). Proteins in the sample were precipitated with trichloroacetic acid, the supernatant mixed with a water-soluble pyridylazo dye, and the absorbance measured at 560 nm. The coefficient of variation was 7% for control samples with a mean zinc concentration of 2.0 mmol/l.
The concentration of fructose in seminal plasma was determined spectrophotographically, essentially as described previously (Wetterauer and Heite, 1976), using a Beckman Synchron LX20 instrument. Proteins in the sample were precipitated with perchloric acid and the absorbance of the supernatant was measured. After addition of phosphoglucose isomerase (which resulted in the conversion of fructose to glucose), the absorbance was re-measured. The absorbance difference corresponded to the concentration of fructose in the sample. The coefficient of variation was 5.0% for control samples with a mean fructose concentration of 12.7 mmol/l.
FCM SCSA
Ejaculates for SCSA analysis were available from 278 of the remaining 301 men. This subgroup was randomly selected from the total group of participants, and the 23 subjects excluded from the SCSA analysis did not differ significantly from the remaining 278 with regard to main seminal and hormonal parameters.
The SCSA was applied following the procedure described elsewhere (Spanò et al., 2000; Evenson et al., 2002
). On the day of analysis, the samples were quickly thawed in a 37°C water bath and used immediately. A portion of cell suspension (12x106 cells) was treated with a low-pH (1.2) detergent solution containing 0.1% Triton X-100, 0.15 mol/l NaCl and 0.08 N HCl for 30 s, and then stained with 6 mg/l purified acridine orange (AO) (Molecular Probes, Eugene, OR, USA) in a phosphate-citrate buffer, pH 6.0. Cells were analysed using a FACSort flow cytometer (Becton Dickinson, San Jose, CA, USA), equipped with an air-cooled argon ion laser. A total of 5000 events was accumulated for each measurement. Under these experimental conditions, when excited with a 488 nm light source, AO which is intercalated with double-stranded DNA emits green fluorescence, while AO associated with single-stranded DNA emits red fluorescence. Thus, sperm chromatin damage can be quantified by FCM measurements of the metachromatic shift from green (native, double-stranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red (fragmented DNA) versus green (DNA stainability) fluorescence intensity cytogram patterns. Adopting the guidelines described in a recent publication (Evenson et al., 2002
), the extent of DNA denaturation was expressed in terms of DNA fragmentation index (DFI, formerly termed
T function), which is the ratio of red to total (red plus green) fluorescence intensity, by using the ListView software (Phoenix Flow Systems, San Diego, CA, USA). This conversion was necessary to calculate correctly the percentage of spermatozoa with non-detectable DFI (formerly termed the main or normal population of cells), and detectable (moderate and high) DFI (formerly collectively termed Cells Outside the Main Population or COMP). The DFI value was derived for each sperm cell in a sample, and the detectable DFI values were calculated on the resulting DFI frequency histogram. These detectable DFI percentages were also used to compare the present results with COMP values reported elsewhere. The fraction of high DNA stainable (HDS) cells [formerly the highly green-stained (HGRN) fraction] was also considered. The percentage of HDS cells was calculated by setting an appropriate gate on the scattergram (abscissa: green fluorescence, Native DNA stainability; ordinate: red fluorescence, Fragmented DNA) and considering as immature spermatozoa those events which exhibit a green fluorescence intensity higher than the upper border of the main cluster, and which represent the sperm population with non-detectable DFI (Figure 1
). Consequently, the results were presented relative to the proportion of spermatozoa with increased levels of fragmented DNA and to the proportion of immature spermatozoa as both parameters are currently being considered in infertility investigation (Evenson et al., 2002
). For the flow cytometer set-up and calibration, aliquots were used from a normal human ejaculate sample retrieved from the laboratory repository.
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Bivariate associations between the length of the abstinence period, serum hormone levels, the length of the CAG repeat, biochemical seminal markers and DFI as well as HDS, were evaluated using Pearson's correlation coefficient.
For assessing potential confounding factors, similar calculations were performed for the correlations between the seminal biochemical markers, the length of the abstinence period and the sperm concentration. The effects of these factors on DFI and HDS were evaluated by multiple linear regression models, adjusting for the effect of the confounders, when appropriate.
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Results |
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Bivariate analyses showed statistically significant (but relatively weak) positive correlation between DFI and the length of the abstinence period and seminal levels of zinc and of fructose. There was a borderline statistically significant negative correlation with the sperm concentration (Table II). The negative correlations between this SCSA parameter and serum levels of estradiol and free testosterone were also weak, though statistically significant. The correlations with other hormonal, genetic or seminal parameters did not reach the level of statistical significance (Table II
).
In the bivariate model, the HDS parameter also showed a significant, albeit negative, correlation with the sperm concentration and duration of abstinence time. A similar relationship was found between HDS and seminal levels of zinc and PSA, whereas the correlation with fructose was positive. With the exception of the correlation between HDS and sperm concentration (r = 0.513, P < 0.0001) all these associations were relatively weak. There was no significant correlation with any other of the tested parameters (Table II).
The levels of biochemical markers in seminal fluid are known to be strongly correlated to each other, and also to the sperm concentration and duration of abstinence (Sorensen et al., 1999). Therefore, a multiple regression analysis was performed with sperm concentration, abstinence time, NAG, PSA, zinc and fructose as covariates and DFI and HDS respectively as the dependent variables. The negative correlation between the sperm concentration and DFI and the negative associations between this parameter and abstinence period as well as levels of zinc and fructose remained statistically significant in the multiple regression analysis (Table III
).
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Discussion |
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With regard to the other SCSA parameter, HDS, the correlations with sperm concentration, abstinence time, PSA, zinc and fructose were statistically significant in the bivariate model, but only the association with sperm concentration and with the level of PSA remained significant in the multiple regression model. HDS did not correlate with any of the endocrine parameters included in this study.
Although there are no clear explanations of the underlying mechanism correlating the levels of testosterone with the final sperm nuclear assembly, it is suspected that low testosterone levels can be associated to disturbances in sperm chromatin structure. Some clues can be obtained from the recently published observation made on FSH receptor knock-out (FORKO) mice (Krishnamurthy et al., 2000; Sairam and Krishnamurthy, 2001
), whereby mutant FORKO males exhibited low levels of testosterone while their elongated spermatids showed a variety of sperm chromatin defects as evaluated by propidium iodide binding and SCSA, eventually leading to reduced fertility. Furthermore, it has recently been demonstrated (also in rodents) that testosterone acts as a positive regulator of the DNA topoisomerase II gene expressionthe key enzyme which breaks and ligates DNA during spermatogenesis (Bakshi et al., 2001
). DFI was shown to increase in line with the age of an individual (Spanò et al., 1998
), and it has been speculated that this phenomenon might be due to an age-dependent decrease in testicular androgen production (Vermeulen, 2000
).
By contrast, in the present study no correlation was found between the length of the CAG repeat sequence in exon 1 of the AR gene and the SCSA parameters. The length of the CAG repeat was shown, both in vitro (Tut et al., 1997) and in vivo (Zitzmann et al., 2001
), to be an important regulator of the functional capacity of the receptor, for androgen levels comparable with those found in the serum of normal males. However, as intratesticular testosterone levels are 100-fold higher than in the serum, at that concentration level the functional status of the receptor may be more or less independent of the CAG repeat length.
The finding of a positive correlation between the seminal fluid levels of fructose and DFI is in accordance with a previous report indicating that an abnormally high contribution of seminal vesicular fluid to sperm-rich fractions of the ejaculate creates a risk of depleting chromatin zinc and thereby impairing zinc-dependent chromatin stability (Bjorndahl and Kvist, 1990). Zinc can bind to sperm nuclear proteins, specifically protamine 2 which is a zinc-finger protein (Bal et al., 2001
), and the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin (Bench et al., 2000
). It is also known from animal studies with zinc-controlled diets that a zinc deficiency is likely to disrupt the normal sperm chromatin quaternary structure in which zinc plays a role by providing stability and resistance to DNA denaturation in situ (Evenson et al., 1993
). It has also been demonstrated that the zinc content of sperm chromatin can be reduced by the action of zinc ligands of seminal vesicular origin (Bjorndahl and Kvist, 1990
). The weak positive correlation between the seminal zinc levels and the DFI, might represent the effect of transfer of chromatin-bound zinc into the seminal compartment during the period of liquefaction (Malm et al., 2000
). With regard to HDS, this parameter exhibited in the multivariate model a weak negative correlation with PSA levels, whereas the association with zinc concentration was positive though not statistically significant. However, the secretion of PSA and zinc from the prostate are strongly correlated to each other, and the effect of prostatic secretion on HDS might rather be due to the effect of zinc than to PSA, even though this was not confirmed by statistical analysis.
In accordance with previous studies, in the present investigation DFI was found to be positively correlated with the duration of sexual abstinence (Spanò et al., 1998, 2000
; Bonde et al., 2002
). It appears as though the longer the mature gamete stays in the epididymis, the greater the probability of its undergoing a deterioration process, eventually leading to increased susceptibility of chromatin to acid denaturation in situ. A possible explanation of this phenomenon might be attributable to changes in the thiol-disulphide status of the protamines during epididymal storage leading to a higher instability of the chromatin (Seligman et al., 1994
).
A negative correlation was also identified with sperm concentration for DFI as well as DHS. Sperm concentration is a good marker of the state of spermatogenesis, and the negative correlation between this parameter and the percentage of spermatozoa with abnormal chromatin structure may mirror the impact of spermatogenic function on sperm chromatin structure; hence, the quantitative and qualitative aspects of spermatogenesis are closely interrelated.
The mean DFI value for the present population of 278 young Swedish males was 14 ± 9.0%, and this agrees very well with other mean DFI values reported elsewhere relative to normal, non-infertile or non-exposed populations (Larsen et al., 1998, 1999
; Rubes et al., 1998
; Spanò et al., 1998
, 1999
, 2000
; Evenson et al., 1999
; Jühler et al., 1999
; Kolstad et al., 1999
; Lemasters et al., 1999
; Grajewski et al., 2000
; Kobayashi et al., 2001
; Bonde et al., 2002
). Only one other study considered this parameter on a comparably large population of 18-year-old men; this involved a semen analysis assessment on a population of 266 young men living in rural and industrially polluted area of the Czech republic (Selevan et al., 2000
; Evenson et al., 2001
). The mean DFI value found by these authors was 20.2 ± 14.0, and somewhat higher than that of the present study. This discrepancy might also be appreciated when subjects are stratified according to a DFI level as having either a low (<15%), moderate (1530%) or high (>30%) fraction of sperm with abnormal chromatin. In the case of the Czech population, the subgroups were 46% for low, 35% for moderate, and 19% for high DFI. By contrast, in the present study values of 72% low, 22% moderate and only 6% high DFI were obtained. Therefore, the somewhat higher values reported for the Czech cohort might be attributable to differences in the environment and lifestyle habits compared with their fellow Swedish young men.
We believe that it is fair to assume that the cohort of 305 young men included in the present study is representative of this age group of men in Southern Sweden, despite the fact that only 13.5% of the eligible subjects agreed to provide a semen sample. It cannot be expected that 18- to 21-year-old men have any knowledge of their reproductive capability, and the low participation rate should not imply any selection bias with respect to fertility. In a recent study (Andersen et al., 2000), a similar protocol was used for the recruitment of military conscripts in which the participation rate was similar (17%). However, 80% of the men delivered blood samples for hormone analysis, and no difference in levels of reproductive hormones was found when the groups of men who agreed to deliver a semen sample were compared with those who refused. Although we believe, for the above-given reasons, that our cohort is representative of the general population of men in this age group, some selection bias cannot be excluded. However, this should not influence the correlations between the reproductive markers and SCSA parameters as reported herein.
Although we have only recently begun to understand which confounders might impact upon the SCSA results (Spanò et al., 1998), knowledge of the possible role played by other important factors, such as key biomarkers of testicular and accessory gland function, remains largely unknown. In this respect, the results of the present study are the first to tackle a phenomenological description of this issue.
The available literature provides compelling evidence that the SCSA is a useful technique for human semen analysis offering a robust tool for a more complete evaluation of male fertility potential (Evenson et al., 1999; Spanò et al., 2000
). Here, we have identified some of the seminal and hormonal factors that correlate with sperm chromatin structure integrity, as evaluated by the SCSA. Although the correlations found were generally rather weak, they provide the first indication of the impact of accessory sex gland function on sperm chromatin structure as assessed by the SCSA. These findings might constitute the starting point for further research aimed at investigating the complex molecular mechanisms underlying male sperm genome packaging. Knowledge of the impact of chromatin structure on the fertilizing capacity of spermatozoa, as well as an identification of the factors that disturb this biological process, may add to our understanding of the pathophysiology of male infertility and lead to the development of more targeted therapeutic modalities.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 13, 2002; resubmitted on July 19, 2002; accepted on August 15, 2002.