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,
From the * Family and Sexual Health Center;
Latvian Medical Academy; and
Latvian University Biomedicine Center, Riga,
Latvia.
| Correspondence to: Juris Erenpreiss, Family and Sexual Health Center, Saharova Str. 16, Riga, LV-1021, Latvia (e-mail: family.center{at}latnet.lv). |
| Received for publication October 3, 2001; accepted for publication April 23, 2002. |
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Abstract |
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Key words: Sperm quality, DNA integrity, leukocytes, oxidative stress
Controversial reports have detailed the relationship between DNA integrity and conventional semen parameters, and whether measures of DNA integrity can differentiate the sperm of fertile and infertile men. Evenson and coworkers (1999), using SCSA on a large number of samples, have established a threshold prognosis infertility value of greater than 30% of sperm cells with impaired DNA integrity (DNA abnormality). In addition, an association was found between levels of DNA abnormality and the amount of time to achieve pregnancy in fertile couples (Evenson et al, 1999). The authors also report that although high values of DNA abnormality predict infertility, conversely lower rates do not predict fertility. Recent research undertaken by Larson et al (2000) on a group of 24 men has shown that no pregnancies resulted from intracytoplasmic sperm injection when the proportion of sperm with impaired DNA integrity was more than or equal to 27%. These authors concluded that a weak association exists between SCSA parameters with conventional semen parameters, or that there may be no association whatsoever. Hughes et al (1996, 1999), using sensitive COMET and ELISA, could not find a difference in baseline DNA damage between normozoospermic fertile and asthenozoospermic infertile men, which they determined to be around 20%. Contrary to this, the correlation between poor quality semen and higher levels of DNA damage detected as DNA fragmentation using the COMET assay, has been recently reported by Irvine et al (2000).
Therefore, whether the widely used conventional tests for DNA abnormality are sensitive enough to reveal differences in baseline DNA integrity in fertile and infertile semen, and whether such a difference is actually present, remains unclear. In addition, the designation of fertility versus subfertility or infertility in cases of secondary infertility may be differently interpreted in these comparative studies. Therefore, careful analysis and objective parameters of semen are needed.
The effect of leukocytospermia on sperm DNA integrity is similarly unclear. Aitken et al (1989a) have found that human spermatozoa produce reactive oxygen species (ROS). Physiological concentrations of ROS likely promote sperm capacitation and the acrosome reaction (De Lamirande and Gagnon, 1995). However, particularly high levels of ROS were found in about 40%-50% of cases of oligoasthenozoospermia (Aitken et al, 1989b; Iwasaki and Gagnon, 1992) and were proposed as a central cause of DNA damage in sperm cells that were unable to repair it. Indeed, ROS produce both plasma membrane and dose-dependent DNA damage (Twigg et al, 1998; Barroso et al, 2000; Krzyzosiak et al, 2000). Kodama et al (1997) found a 1.5-fold increase in the level of oxidated DNA derivatives in infertile men compared with fertile men. However, Aitken and West (1990) and others (Sukcharoen et al, 1995; Sharma et al, 2001) have suggested that the main source of sperm ROS is provided not by sperm cells themselves, but by activated polymorphonuclear leukocytes in cases of leukocytospermia. In these cases, ROS concentration appears to exhibit a powerful negative effect on fertilization rate.
In an ongoing study, we have found that 17% of young Latvian army recruits have leukocytospermia (n = 103; unpublished results). However, in sterile Latvian couples (n = 579) studied from 1998 to 2000, 78% of men had abnormal semen parameters and 43% had leukocytospermia (Erenpreiss et al, 2002). A high prevalence of leukocytospermia in infertile men has also been reported in several countries, although the values are different. It is interesting that data from Kuwait (Omu et al, 1999) are similar to those of Latvia.
Therefore, the aims of this study were 1) to evaluate the effect of standard measures of sperm quality on DNA integrity, and 2) to evaluate the effect of leukocytospermia on sperm DNA integrity in normal and abnormal semen samples.
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Materials and Methods |
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All reagents were purchased from Sigma Chemical Company (St. Louis, Mo). Sperm DNA integrity was detected by an in situ DNA denaturation test using AO staining on slides as detailed previously (Erenpreiss et al, 2001), and which has been previously applied to detect apoptosis in somatic cells (Erenpreisa et al, 1997). After semen liquefaction at 37°C for 30 minutes, samples were pelleted by low-speed centrifugation (1000 x g for 5 minutes) and resuspended in a smaller portion of supernatant at approximately 5 x 108 cells/mL. Cell smears were prepared and air-dried for 1 hour. Cells were then fixed in freshly made ethanol:acetone (1:1) at 4°C for 0.5-24 hours. Fixed samples were not air-dried before staining, but were rehydrated in 96°C ethanol for 5 minutes, 70°C ethanol for 5 minutes, and 30°C ethanol for 3 minutes, all at room temperature. Washing for 5 minutes in phosphate-buffered saline was followed by treatment with 1 N HCl at 60°C for 1 minute. Slides were then rinsed 3 times in distilled water for 2 minutes and dipped in McIlvain phosphate-citrate buffer (pH 4) for 5 minutes before staining with AO (10-4 M) in the same buffer for 15 minutes. The AO working solution was freshly prepared from a 7.6 mg/mL of AO stock stored in the dark at 4°C. Slides were then rinsed 3 times for 5 minutes in the same McIlvain buffer containing freshly diluted AO (10-6 M). A coverslip was mounted on this AO solution, blotted at the margins, and sealed with nail polish (moviol was not added to the mounting medium). Preparations were kept in the dark at 4°C and were assessed within 24 hours after staining. Using this method, AO fading does not occur, which allows scoring at a later time, as established in our previous study (Erenpreiss et al, 2001).
The percentage of sperm cells with abnormal DNA integrity (ADI) was determined by analyzing 300 cells per sample with a Nikon TE 300 fluorescence microscope (Nikon GmbH, Dusseldorf, Germany), with a mercury lamp (excitation band-pass filter, 420-490 nm; emission was observed through a suppression filter with a 515 nm limit). Sperm cells with normal DNA integrity have green fluorescence, those with abnormal DNA integrity fluoresce orange-red. Intraassay variation was assessed as 9%, and interassay variation as 11%.
In the second part of the study, 79 normal and 40 abnormal semen samples from the previous set were analyzed for leukocytospermia. This smaller set was chosen because leukocytes had been determined in this set by the same method over a small period of time (4 months). The formula for normalization of sperm quality of this set was the same as described above (with the exception that fast + slow progressive, or group A + group B motility according to WHO guidelines, was considered). In addition, these samples were classified as abnormal if one or more parameters contained the following WHO criteria: sperm concentration, 20 million/mL; motility (A + B), 50%; and normal morphology, 20%. The method of estimating DNA integrity was the same as that described above. Leukocytospermia was considered if leukocytes, as detected by the peroxidase method (WHO, 1999), comprised more than 1 million per milliliter of ejaculate. Leukocyte counts ranged from 2 to 13 (mostly from 2 to 6) million/mL.
Statistical analysis of the results was performed with Microsoft Excel 97 using power analysis and the two-tailed Student's t-test.
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Results |
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Analysis of the relationship between DNA abnormality and the 3 individual parameters of semen quality for the 187 samples is presented in Figure 2 (the figure also shows maximum values for this set). Inverse correlation coefficients are demonstrated for the following parameters: sperm concentration, r = 0.58; morphology, r = .48; and motility A, r = .42. These separate graphs also show the same phenomenon of wide dispersion and a tendency for ADI values to increase in the poorest quality sperm. This phenomenon is dependent on lower sperm concentration, particularly <50 million/mL (Figure 2a). In addition, only sperm with critical A motility (<25%) show ADI values higher than 30% (Figure 2c).
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A subset of 119 samples designated as normal and abnormal according to WHO
criteria were further analyzed for leukocytospermia. The routine sperm
parameters of these samples and corresponding ADI data are presented in the
Table. Average values of sperm DNA abnormality for normal and abnormal samples
in this set, regardless of leukocytospermia, were 18% ± 2.8% and 36%
± 5.8% (P < .05), respectively, setting a threshold for
possible subfertility or infertility at 30% ADI with 95%
confidence.
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When samples with leukocytospermia were removed from the analysis, the difference in average ADI values between normal and abnormal semen samples appeared to be much smaller; however, they were statistically significant (P = .023), constituting 17% ± 2.4% and 22% ± 4.6%, respectively (see also the Table). Although leukocytospermia did not significantly affect DNA integrity in the normal semen group, it more than doubled the ADI values in the abnormal semen group, with values of 18% ± 2.4% and 50% ± 10.7% (P < .01), respectively.
Such a small baseline difference between ADI of normal and abnormal samples, which can presumably categorize fertile and subfertile or infertile sperm, respectively, is at odds with many other reports (some reviewed above). In order to better understand the cause of this discrepancy, we plotted the ADI values against the normalized summed sperm quality values, arranged in a discontinuous row, including both normal and abnormal samples. For this analysis, standard normalized sperm quality was scored in one scale for all groups. Maximum parameter values, which were assigned 10 arbitrary units each, consisted of sperm concentration, 200 million/mL; A + B motility, 84%; and normal morphology, 42%.
The results of such an assessment are presented in Figure 3, which shows that in the set without leukocytospermia (Figure 3a), the majority of samples, both normal and abnormal by WHO criteria, are positioned in the intermediate region of normalized sperm quality (between 10 and 20), and have similar overlapping ADI values, mostly between 10% and 27%. Very good quality samples (with quality consisting of more than 20 arbitrary units) had a lower ADI (mostly under 22%-20%), whereas very poor quality samples (between 0-10 arbitrary units of quality) possessed ADI values above the infertility threshold of 30%-42%. Therefore, in the absence of leukocytospermia, the difference in ADI is significant only between semen of very good and very poor qualities, whereas it is insignificant for samples of intermediate quality, which are either normal or abnormal according to WHO criteria. The entire relationship is moderately inverse and can be equally well described as linear (r = .58) or as a polynomial of the second power (r = .60), with similar statistical significance (P < .01).
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For samples with leukocytospermia, the same analytical approach gives quite a different distribution and is presented in Figure 3b. Here, a clear separation of normal and abnormal samples is observed, with DNA integrity greatly impaired in abnormal semen. Moreover, the majority of abnormal samples of intermediate quality (between 10 to 15 arbitrary units), which were indistinguishable from normal samples with respect to ADI without leukocytospermia, now have dramatically elevated ADI. Thus, in Figure 3a, 85% of abnormal samples had ADI values <30%; whereas in Figure 3b, 84% exceed this threshold, and many display very high ADI (>60%). Comparing Figure 3a and b, we see another peculiarity. The distributions of ADI according to sperm quality in Figure 3b seem to have a parabolic form. In cases of leukocytospermia, the peak of ADI for abnormal sperm reaches values as high as 80%-90% or more at 10 arbitrary units of quality, with the tendency for ADI to decrease in the sperm of worst quality.
Therefore, the relationship between sperm quality and ADI in the samples with leukocytospermia are best described by a polynomial of the 4th power (not shown), and this reveals a tighter association between ADI and sperm quality (r = .71, P < .01) than any other factor. Additional analysis revealed that this parabolic relationship is more dependent on sperm morphology and motility than on sperm concentration (not shown). ADI was weakly associated with the number of leukocytes in sperm in this set (r = .32; P = .1).
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Discussion |
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In the 187 samples assessed, a moderate inverse relationship between standard sperm quality and DNA abnormality was found, in particular with sperm concentration. This result is similar to that of Irvine et al (2000), who used the objective parameters of sperm quality and various DNA fragmentation detection methods. Our data are also in accord with the data of Tejada et al (1984), who noted the importance of both DNA normality and sperm concentration for infertility prognosis, and who introduced the integrated parameter of "effective sperm concentration." Some authors (Liu and Baker, 1994; Larson et al, 2000) have concluded that DNA integrity parameters, which are predictive of infertility, are independent of standard sperm quality assessments. The data reporting no baseline difference in sperm DNA integrity between semen groups of fertile and infertile men obtained by Hughes et al (1996, 1999) add yet more confusion to the discussion.
One source of these contradictions can be inferred from Figure 1, where it can be seen that the effect of sperm quality on DNA abnormality is in fact evident only at the poles of sperm quality. In medium-quality sperm, ADI varies very little. In addition, ADI values for poor-quality sperm, as presented in Figure 1, are variable and dispersed around the trend line.
To understand the possible contribution of leukocytospermia to DNA abnormality, a subset of 119 samples sorted for leukocytospermia was analyzed. As seen in Figure 3a, in which normal and abnormal semen samples without leukocytospermia are labeled, both are mostly concentrated in the region of intermediate quality with overlapping ADI values. This explains a small baseline difference of ADI in our normal and abnormal semen sets averaged in the Table. Thus, the average results may be highly dependent on the given data set, sample numbers, etc.
Also in this case, as in Figure 1, ADI values showed a moderate inverse, nearly linear correlation with sperm quality. However, no high dispersion of ADI around the imaginary trend line could be found. On the contrary, in the set of abnormal sperm samples with leukocytospermia, a huge dispersion with high "jumps" of ADI were observed (Figure 3b), which accounts for the more than twofold increase in average ADI in this group (Table). Notably, a response to leukocytes provoked potential DNA damage and caused high ADI values in those abnormal samples that possessed moderately poor sperm quality and which, without leukocytospermia, were indistinguishable from normal samples with ADI values under 30%. Thus, the normal and abnormal fractions became clearly separated by leukocytospermia. However, in this case, ADI did not show a linear relationship with sperm quality. Instead, it displayed a clear tendency to dramatically increase with decreasing sperm quality, and with even worse sperm quality, to decrease. This parabolic distribution suggests involvement of 2 different mechanisms affecting the relationship between sperm quality and DNA integrity in abnormal leukocytospermic semen.
From this analysis, we conclude that high dispersion of ADI values around the trend line in abnormal-quality sperm in our material is mostly due to leukocytospermia. It seems that leukocytospermia may be increasing the ADI of abnormal semen very stringently, in a cascadelike manner, but that it is somehow less effective in the poorest quality semen.
Given that leukocytospermia may affect sperm DNA integrity in a cascade-like manner, the mechanism of the action may occur via oxidative stress. It can therefore be hypothesized that abnormal sperm is susceptible to oxidative stress, which subsequently produces extensive DNA damage, whereas normal sperm is only poorly susceptible to such damage. Oxidative stress is considered an important and key factor in the etiology of male infertility regardless of its origin (Pasqualloto et al, 2000) and its negative effect on fertility is recognized by andrologists (Comhaire et al, 1999).
This hypothesis is likely because leukocytospermia is already known to produce oxidative stress sufficient to damage DNA in sperm cells (Sharma et al, 2001). Furthermore, in our research, the average percentage of cells with impaired DNA was more than doubled in abnormal sperm with leukocytospermia compared with abnormal sperm without leukocytospermia.
An interesting aspect of this phenomenon revealed by our study is the seemingly weaker response of extremely poor quality sperm to DNA damage from leukocytes. These sperm may contain primary DNA damage, and therefore, it may be hypothesized that this paradox is due to the rapid removal of these apoptotic sperm cells by leukocytes. Irrespective of this conjecture, this mechanism is clearly opposing the increase in ADI in abnormal sperm samples, and these conflicting mechanisms result in the great variability of ADI in abnormal leukocytic semen samples.
What is the nature of the susceptibility of abnormal sperm to oxidative stress? It is interesting that weakness of the plasma membrane is a characteristic of abnormal sperm (Barroso et al, 2000) and ROS are known to be detrimental to both plasma membranes and DNA (Twigg et al, 1998; Krzyzosiak et al, 2000). Impairment of the plasma membrane probably decreases the group A motility of sperm with high ADI (under putative oxidative stress) and can be inferred from Figure 2c. In turn, the increased DNA vulnerability in abnormal sperm may, for example, be due to natural alkali-labile DNA sites being unprotected from ROS by poor chromatin packaging in immature sperm.
Regardless of the reason for high susceptibility of abnormal sperm to oxidative stress, one must take account of it when using sperm for assisted conception. Although leukocytospermia may be infrequent in some human populations, when present, it is clearly harmful for subfertile men. In addition, oxidative stress in sperm may be further exacerbated by factors other than leukocytes, either natural or during manipulations with sperm.
Three important clinical implications follow: 1) infections must be removed to prevent damage to DNA integrity in subfertile and infertile men; 2) antioxidants may be useful in decreasing the negative effects of ROS and hence DNA abnormality; and 3) the genetic risk of microassisted fertilization using genetic material from infertile men must be considered, and if fertilization is performed, use of antioxidants during manipulations may be of a paramount importance.
In summary, our analysis shows that the relationship between sperm quality and DNA abnormality is complex. Normal semen has low ADI and is resistant to the DNA damage associated with leukocytospermia. However, at least 3 factors affect the net DNA integrity in abnormal quality leukocytospermic samples: 1) primary DNA damage, which is moderately inverse to the sperm quality, in particular to sperm concentration; 2) the effect of leukocytes increasing and provoking potential DNA damage in a cascade-like manner, particularly in sperm of moderately poor morphology and motility; and 3) a decreasing proportion of cells with damaged DNA in semen of the worst quality.
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Acknowledgments |
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