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From the * Department of Large Animal Sciences,
Section for Veterinary Reproduction and Obstetrics and the
Section for Population Biology, The Royal
Veterinary and Agricultural University, Frederiksberg, Denmark.
| Correspondence to: Gry Brandt Boe-Hansen, The Royal Veterinary and Agricultural University, Department of Large Animal Sciences, Section for Veterinary Reproduction and Obstetrics, Dyrlaegevej 68, 1870 Frederiksberg C, Denmark (e-mail: gbh{at}kvl.dk). |
| Received for publication April 5, 2004; accepted for publication December 17, 2004. |
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Abstract |
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Key words: Flow cytometry, DNA integrity, spermatozoa, acridine orange
The structure of the sperm chromatin and the stability of the DNA in relation to the fertility potential have been widely studied. Various assays that assess different aspects of the chromatin structure and DNA integrity of sperm cells that use specific and complex interactions between compounds and DNA have been developed. These include the single-cell gel electrophoresis assay (Comet assay) (Haines et al, 1998), terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) (Sailer et al, 1995), in situ nick translation (NT) (Manicardi et al, 1995), and the sperm chromatin structure assay (SCSA) (Evenson and Jost, 2000). In the 1980s, Evenson et al (1980) carried out the pioneering work that described the flow cytometric assessment of sperm chromatin structure. The SCSA protocol has since been refined by Evenson et al (2002). This method uses the metachromatic properties of the dye acridine orange (AO) to detect the susceptibility of sperm DNA to acid–induced denaturation in situ. The SCSA protocol uses flow cytometry to detect green fluorescence from AO when intercalated into the double-stranded DNA helix and red fluorescence when the dye is bound to single-stranded nucleic acids (Darzynkiewicz et al, 1975). Although Evenson and Jost (2000) described the protocol for the SCSA in great detail, several authors have since introduced changes in the analytic procedure (Tejada et al, 1984; Golan et al, 1997; Giwercman et al, 1999; Spano et al, 1999; Acevedo et al, 2002). In most cases, these changes have not been validated against the protocol described by Evenson and Jost (2000).
Several reports emphasize the need for improvement in overall quality of semen testing within and between laboratories (Neuwinger et al, 1990; Jorgensen et al, 1997; Keel et al, 2000). However, the subjective nature of conventional semen analyses, combined with their relatively low precision due to the low number of cells assessed, leads to poor intra- and interlaboratory reproducibility; therefore, the introduction of standardized or quality controlled procedures will probably have a limited effect. The conventional analyses are used to determine whether parameters obtained from an ejaculate are within the range characterized by fertile men, and these methods can therefore provide only unclear cut-off values when used for the prediction of fertility status. Many of the advantages that accrue when using flow cytometry may, when applied to assessment of sperm cells, help overcome some of the mentioned problems found in conventional semen analysis. The SCSA is objective, fast, and precise, and the data obtained from human (Evenson et al, 1999; Larson-Cook et al, 2003) and animal (Ballachey et al, 1987; Evenson et al, 1994) studies have shown that the fragmentation of sperm DNA can be detrimental to achieving and sustaining a pregnancy. Statistical thresholds have been established for fertility prognosis when using SCSA procedures in humans (Evenson et al, 2002). The different measures obtained when using SCSA procedures have also been shown to be independent from conventional semen quality measures, and the assay therefore makes a contribution to the semen analysis profile (Evenson et al, 1991).
In the field of semen analysis, validation of a method is important because it is essential to have specific, precise, objective, and accurate laboratory tests to establish a correlation of the data with fertility or to determine the fertility potential of a semen sample correctly (Amann, 1989). Precision of a laboratory test is of great concern to the andrologist in the fertility clinic, since the results of the semen analysis are often used to advise a patient about his fertility and the prognosis for the treatment of the couple. To use established cut-off values and ensure uniform diagnosis, within and between laboratory variations should be determined and followed closely. The precision of a laboratory test is influenced by a number of factors, including the number of cells assessed, but also the human error involved in running the test and the performance or variation of the instruments used (Amann, 1989; World Health Organization, 1999). Increasing attention to the details of standard procedures and protocols should therefore increase the precision of results and reduce variation (Keel et al, 2000), both between and within laboratories.
During our first trials with SCSA protocol, described by Evenson and Jost (2000), smaller disagreements between duplicate measurements of the same sample were not an uncommon phenomenon, and it was observed that different factors in the SCSA protocol could affect the SCSA measures. The objective of the present study was to evaluate specific factors affecting the measurements obtained from the SCSA in human semen.
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Materials and Methods |
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Fluorescent Staining
The SCSA was performed according to the procedure previously described
(Evenson and Jost, 2000;
Evenson et al, 2002). After
thawing, aliquots of the thawed semen were diluted to a concentration of 2
x 106 sperm/mL with TNE buffer to a total volume of 200 µL
in a 5-mL Falcon tube (BD Biosciences, San Jose, Calif). Immediately, 400
µL of an acid detergent solution (0.08 M HCl, 0.15 M NaCl, 0.1% [vol/vol]
Triton X-100, pH 1.2) was added, and a stopwatch was started. After exactly 30
seconds, 1.20 mL of AO staining solution was added containing 6 µg of AO
(chromatographically purified; Polysciences Inc, Warrington, Pa) per
milliliter of buffer (0.037 M citric acid, 0.126 M
Na2HPO4, 1.1 mM EDTA disodium, 0.15 M NaCl, pH 6.0).
Flow Cytometric Measurements
The samples were analyzed using a FACScan (BD Biosciences) flow cytometer
with an air-cooled argon orthogonal laser operated at 488 nm at 15 mW of
power. After transiting a 560-nm short-pass dichroic mirror, the green
fluorescence (FL1) was collected through a 515- to 545-nm band-pass filter.
After transiting a 640-nm long-pass filter, the red fluorescence (FL3) was
collected through a 650-nm long-pass filter. The FACScan has been shown to be
capable of successfully measuring mammalian and avian sperm using the SCSA
(Evenson et al, 1995). The
sheath/sample was set on "low" and adjusted to a flow rate of 200
events per second when analyzing a sample with a concentration of 2 x
106 sperm/mL. Immediately after the addition of the AO staining
solution, the sample was placed in the flow cytometer and was run through the
system. Data acquisition of 5000 events was begun exactly 3 minutes after the
initiation of acid detergent treatment and was collected in list mode using BD
CellQuest Pro version 4.0 software (BD Biosciences). The laboratory technician
manually recorded the X-mean (red fluorescence) and the Y-mean (green
fluorescence) values for each sample.
Data Analysis
List mode data were analyzed with the software program SCSA-Soft (SCSA
Diagnostics Inc, Brookings, SD). From the list mode data, SCSASoft
automatically calculated the percentage of sperm with an abnormally high DNA
stainability (HDS), the level of DNA fragmentation index (DFI), and the
standard deviation of the DFI (SD-DFI). Note that SCSA terminology has
recently been changed; DFI was formerly termed COMP
t (cells
outside the main peak of t); SD-DFI was formerly termed
SDt; and what is now known as HDS was termed HIGRN (high
green fluorescence) (Evenson et al,
2002). An example of an SCSA report generated from a human semen
sample is shown in the
Figure.
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Experimental Design
Experiment 1—
To determine the effects that thawing time, incubation time on ice, and
variation caused by laboratory technician, day, and time of day had on the
samples, a factorial design was performed repeatedly for 4 days with semen
from 3 donors.
Semen straws were thawed in a water bath at 37°C for 7 or 30 seconds and then incubated on ice. After 0, 5, 10, 15, 20, and 25 minutes of incubation, an aliquot of the samples was diluted, stained, and analyzed. This was done in the morning and in the afternoon in random order by 2 laboratory technicians. A sample of AO equilibration buffer (400 µL of an acid detergent solution and 1.20 mL of an AO staining solution) was run through the flow cytometer for at least 60 seconds between every analysis. The same sheath fluid was used throughout the study (0.05% [vol/vol] Triton X-100). For each of the 3 donors, a total of 16 straws were analyzed; 4 straws per donor were analyzed each day.
Experiment 2— To determine the effects that sheath fluid, incubation time on ice, AO equilibration buffer, and variation caused by laboratory technician and day had on the samples, a factorial design was performed repeatedly for 4 days with semen from 3 donors.
Semen straws were thawed in a water bath at 37°C for 30 seconds and then incubated on ice. After 0, 5, 10, 15, 20, and 25 minutes of incubation, an aliquot of the samples was diluted, stained, and analyzed. The samples were analyzed in random order by the same 2 laboratory technicians as in experiment 1. A sample containing AO equilibration buffer was run between all analyses for 60 seconds or only before the first analysis (0 minutes). The sheath fluid used in this study was either 0.05% (vol/vol) Triton X-100 or FACSFlow (BD Biosciences). For each of the 3 donors, a total of 16 straws were analyzed; 4 straws per donor were analyzed each day.
Statistical Analysis
The statistical analyses were performed using SAS, version 8.2 (Statistical
Analysis Systems Institute, Cary, NC). The effect of laboratory factors was
evaluated in the 2 experiments by an analysis of variance using a mixed model.
The outcome variables were the X-mean, Y-mean, DFI, SD-DFI, and HDS. In the
first experiment, laboratory technician, day, and time of day were included as
random effects in the analyses. The fixed effects of laboratory factors
evaluated were the donor, thawing time, and incubation time on ice. In the
second experiment, the laboratory technician and day were included as random
effects. The fixed effects of laboratory factors evaluated were the donor,
sheath fluid, incubation time on ice, and AO equilibration buffer method.
Two-way interactions between the fixed effects were included. A backward
elimination of nonsignificant interactions and factors was used. The
assumption of normality was evaluated for each outcome by the Shapiro-Wilks
test for normality. The assumption of equal variances was evaluated by visual
inspection of residual plots. To fulfill the assumption of a normal
distribution, DFI was transformed using the ARSIN transformation. A 5%
significance level was used throughout the study.
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Results |
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DNA Fragmentation Index— The statistical analysis of the ARSIN-transformed data showed that incubation time (5, 15, 20, and 25 minutes) had a significant effect on DFI when compared with no incubation on ice (0 minutes) (P < .01), but at the 10-minute incubation, there was no significant difference (P = .08). There were no significant differences in DFI between the samples that were incubated on ice. The 2 thawing methods did not differ significantly in DFI.
Standard Deviation of DFI— Analysis of SD-DFI showed that incubation on ice (5, 10, 15, 20, and 25 minutes) had a significant effect when compared to no incubation on ice (0 minutes) (P < .01). There were no significant differences in SD-DFI between the samples that were incubated on ice. There was no significant difference between the 2 thawing methods with respect to SD-DFI.
High DNA Stainability— Analysis of HDS showed that there was no significant difference between the 6 time points (P > .05). There was a significant difference between the 2 thawing methods (P = .003).
Experiment 2
X-mean and Y-mean—
The X-mean for the samples not incubated on ice (0 minutes) was
significantly different from that for the samples incubated on ice (5, 10, 15,
20, and 25 minutes). There was a significant difference between the 2 AO
equilibration buffer methods used with respect to the X-mean (P =
.001). There was no significant difference for the X-mean between the 2 sheath
fluids (P = .47), but an effect caused by the interaction between
sheath fluid and donor (P < .001) was observed, which is shown in
Table 3. For the Y-mean, there
was a significant difference between sheath fluids (P < .001),
incubation on ice (P = .002) and AO equilibration buffer method
(P < .001). Interactions between sheath fluid and donor
(P < .001), donor and incubation time (P < .001), and
AO equilibration buffer method and incubation time (P < .001) were
detected (Table 3).
DNA Fragmentation Index— Analysis of DFI showed that the DFI at the time point immediately postthaw (0 minutes) differed significantly from that obtained for the samples incubated on ice (5, 10, 15, 20, and 25 minutes) (P < .01). The only other time points that differed with respect to DFI were 15 and 20 minutes (P = .020). The 2 types of sheath fluid were not significantly different (P = .15) with respect to DFI. An interaction between the AO equilibration buffer method and donor was found (P = .028) (Table 3).
Standard Deviation of DFI— The analysis of SD-DFI showed that there was a significant difference between the 2 used sheath fluids (P = .044). The SD-DFI immediately postthaw (0 minutes) differed significantly from that obtained from the samples incubated on ice (5, 10, 15, 20, and 25 minutes) (P < .001). There was also a significant difference between the samples incubated at 5 and 20 minutes (P = .020), 10 and 20 minutes (P = .016), and 15 and 20 minutes (P = .021). There was a significant effect caused by the AO equilibration buffer method (P = .036).
High DNA Stainability— Analysis of HDS showed that there was no significant difference between the 2 AO equilibration buffer methods (P = .24). Significant differences were detected between the 2 types of sheath fluid (P < .001) and incubation time (P < .001). Interactions between the donor and incubation time (P = .030), and the AO equilibration buffer method and sheath fluid were found (P = .040) (Table 3).
Random Effects— The variations due to random effects in the 2 experiments are shown in Tables 4 and 5. The DFI was affected by day in both experiments, but laboratory technician and time of day had little or no effect on this measure. For the SD-DFI, both laboratory technician and day had an effect, but time of day had no effect. For the HDS, both day and laboratory technician had an effect in experiment 1, but this was not the case in experiment 2.
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Discussion |
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There are few data available in the literature concerning variation related
to laboratory procedure and repeatability and reproducibility between
replicates of the SCSA. In one study, duplicate measurements from an
individual semen sample were shown to be highly repeatable, with correlations
of 0.988 for COMPt, 0.991 for SDt, 0.985
for HIGRN, 0.982 for the X-mean, and 0.973 for the Y-mean
(Evenson et al, 1999). In
another study, it was found that for an SCSA method that was slightly
modified, the intra-assay coefficients of variation for the
COMPt varied between 0.8% and 16.8%, and the corresponding
value for the SDt varied between 1.3% and 5.8%
(Giwercman et al, 1999). In a
recently published paper, the correlation between replicates measured by the
SCSA was determined to be 0.87 for SDt and 0.98 for
COMPt (Acevedo et al,
2002). One study of 2 laboratories performed a quality control
exercise in conjunction with a study designed to determine if there was a
correlation between sperm motility and the SCSA demonstrated an
interlaboratory, Pearson correlation of r = 0.90
(Giwercman et al, 2003). The 2
laboratories used 2 slightly different SCSA protocols.
In the present study, we found that a number of laboratory factors affected the outcome of the SCSA. It was shown that even a short (5 minutes) incubation time on ice postthaw had an effect on the measures X-mean, DFI, and SD-DFI in both experiments; therefore, careful protocol description should be practiced when measuring the same frozen-thawed semen sample twice in order to achieve a measurement in duplicate. It was also shown that thawing method had a significant effect on HDS. Interactions between sheath fluid and donor for the X-mean, Y-mean, and HDS and between the AO equilibration buffer method and donor for DFI were shown. According to the protocol by Evenson and Jost (2000), samples for the SCSA should be thawed in a water bath at 37°C until the last remnant of ice has disappeared, and the samples should be analyzed immediately thereafter. The remainder of the sample should be stored on ice, and a repeated staining and measurement should be performed directly after completing the first analysis. In our study, we found a significantly higher DFI and SD-DFI in the samples analyzed directly postthaw than in the same samples stored on ice for up to 25 minutes. This could be caused by a thermally induced increase of fluorescence intensity of AO-stained cells. The fluorescence intensity of AO has been described to be temperature dependent at higher temperatures (Darzynkiewicz et al, 1975). An alternative and far-fetched explanation for the decrease in DNA denaturation following incubation on ice is that the storing or thawing procedure may enhance the sperm DNA integrity in the short term. Unfortunately, a comparison of these data with SCSA results obtained from the fresh semen, before freezing, was not possible due to unavailability of sufficient amounts of data. With regard to the thawing procedure (7 vs 30 seconds), the only effect was observed for HDS, where HDS was significantly higher for the shorter thawing time. The sperm in the HDS region are characterized by increased DNA stainability and are excluded from the calculation of the DFI and SD-DFI. This population of HDS is supposedly composed of immature cells that lack chromatin condensation (Evenson et al, 1999) but may also represent doublets. The significantly higher HDS found for the 7-second thawing procedure may therefore be explained by a higher number of doublets due to clumping. The random effect of laboratory technician was also lower in experiment 2 where the thawing time was 30 seconds throughout the experiment. The recommended method of thawing semen frozen in 0.23-mL straws is therefore 37°C for 30 seconds.
We also found that sheath fluid consisting of 0.05% Triton X-100 should be used and that a sample containing an AO equilibration buffer should be run through the flow cytometer for about 60 seconds between stained aliquots to ensure saturation of the tubes in the flow cytometer, as recommended by Evenson and coworkers (Evenson and Jost, 2000). The combined use of an AO equilibration buffer and 0.05% (vol/vol) Triton X-100 resulted in the most stable results for the SCSA measurements. The interactions shown for some of the variables between donor sheath fluid and the donor and AO equilibration buffer method show the weakness of having included only 3 donors in the study. If more donors with a larger variation between the SCSA measurements had been included, perhaps the results from this part of the study would have been clearer.
It has previously been recommended that "reference samples" be used to establish instrument settings and when running a series of samples, after about every 5 samples. In this way, the reference samples should ensure stability of the instrument and quality control over all measurements (Evenson and Jost, 2000). The mean red (X) and green (Y) fluorescence values should fall within plus or minus 5 channels of an established laboratory standard, and the flow cytometer should be adjusted to accomplish this (Evenson and Jost, 2000). In the present study, it was not possible to adjust the flow cytometer so that the X- and Y-mean values stayed within plus or minus 5 channels by measuring just a single reference sample in duplicate. A larger number of reference samples would be required to obtain the settings for the X- and Y-mean values; then, adjustments should be made accordingly.
The variation due to time of day was shown to be 23.5% for the Y-mean measurement in experiment 1. This change in green fluorescence during the day has previously been observed to occur in another type of instrument when working with the SCSA (Evenson and Jost, 2000). In the present study, we found that for the 3 variables DFI, SD-DFI, and HDS, the variation due to time of day was close to 0%. We therefore agree that reference samples should be used to establish daily instrument settings and measurements between test samples. However, we cannot recommend adjusting the flow cytometer on the basis of the results determined from a single reference sample obtained while analyzing a row of samples. The flow cytometer should be adjusted according to the reference sample before running a row of test samples, but these established instrument settings should be used throughout the day without any further adjustment. If any slight deviations occur during the day, they will be recorded in the X-mean, Y-mean, DFI, SD-DFI, and HDS, for the reference samples analyzed in duplicate for every 5 test samples.
Our experience is that, even with objective flow cytometric techniques, the individual running the assay often has an influence on the outcome of the assay. In the present study, the laboratory technician accounted for up to 15% of the variation in the SCSA variables DFI, SD-DFI, and HDS. In comparison, the variations due to day and time of day were smaller for each of these 3 variables. By adopting a standardized training program in how to handle samples in a correct and uniform way in cell preparation, staining, and measuring, the effect of the laboratory technician may be markedly reduced. Useful tools that can be used when introducing a new technique into a laboratory should include careful standardization and a detailed protocol description of the technique, an extensive training program, and a follow-up in the form of an intralaboratory quality control program. For a widely used method such as the SCSA, after having established an intralaboratory quality control program, interlaboratory variation should be considered. As previously suggested, when managing interlaboratory variation for the SCSA, it may be useful to establish a quality control reference center (Evenson et al, 2002).
This variation study was performed as part of determining the immediate source of laboratory variation for the SCSA. This type of study is an important part of introducing a new technique to a laboratory before commencing further experiments or when beginning to use it in routine clinical analysis. In this study, the most stable results when using the SCSA for the analysis of human semen were achieved by thawing samples frozen in 0.23-mL straws at 37°C for 30 seconds and placing them on ice for 5 minutes before the first aliquot was diluted, stained, and analyzed. The subsequent repeated measurement of a sample (10 or 15 minutes of incubation on ice postthaw) will give a more precise estimate of the DFI and SD-DFI. Sheath fluid consisting of 0.05% (vol/vol) Triton X-100 should be used, and a sample with an AO equilibration buffer should be run through the tubes of the flow cytometer between every analysis.
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Acknowledgments |
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Footnotes |
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References |
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