1 Department of Chemistry and Biochemistry and 2 Department of Animal and Range Sciences, Box 2170, South Dakota State University, Brookings, SD 57007, USA, 3 Department of Obstetrics and Gynecology, Loyola Medical Center, Maywood, IL 60153, USA, 4 Reproductive and Developmental Toxicology, Environmental Protection Agency, Washington DC, USA and 5 Institute for Pathology, National Hospital, Oslo, Norway 6 Department of Chemistry and Biochemistry and
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: chromatin structure/human fertility/pregnancy loss/SCSA
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Computer-interfaced flow cytometry (FCM) provides a powerful advantage over light microscopy techniques in terms of speed, multiple parameters/cell measured, objectivity, lack of bias in sample selection, and thousands of cells measured per sample, and thus provides a very high degree of statistical power (Shapiro, 1995). Additionally, FCM can potentially sort out unique cells that comprise <1% of the total population for further study and/or use in assisted reproduction treatment, e.g. sexing sperm (Johnson et al., 1987
, 1989
).
This study utilized the sperm chromatin structure assay (SCSA), first described by Evenson et al. (1980), and since refined for both animal and human spermatozoa (Evenson and Jost, 1994; Spano et al., 1998
). The SCSA utilizes the metachromatic properties of acridine orange (AO) to distinguish between low pH- or heat-denatured (red fluorescence = single-stranded) and native (green fluorescence = double-stranded) DNA in sperm chromatin. As first discussed by Evenson et al. (1980), human and bull sperm nuclear DNA was more resistant in fertile than subfertile subjects. Data from subsequent animal heterospermic insemination experiments showed strong correlations between SCSA data and fertility ranking in bulls (Ballachey et al., 1988
; r = 0.94, P < 0.01) and boars (Evenson et al., 1994
; r
0.93, P < 0.01), thus providing strong evidence that mammalian sperm chromatin structure was highly correlated with pregnancy outcome.
Other investigators (Tejada et al., 1984) have used a modified SCSA procedure by treating smears of human spermatozoa on glass microscope slides with acid, staining with AO, and observing sperm nuclei under fluorescence light microscopy. The sperm nuclei were generally scored as fluorescing green or red; in some cases, cells have also been scored as yellow, being intermediate between the red and green [see cover of Science (Evenson et al., 1980
)]. Although this method provides a general picture of the status of the sperm DNA denaturation susceptibility, it is limited to two or three classifications rather than the 1024 discrete channel levels of red and green fluorescence/cell by FCM. Also, since the metachromatic staining of AO is strictly dependent on exact equilibrium conditions (Darzynkiewicz et al., 1975
), artefacts exist in the light microscopy method due to the unevenness of the surface on glass microscope slides providing differential microenvironment staining conditions. Fluorescence fading presents an additional problem, as well as varied time intervals between staining and scoring (D.P.Evenson, unpublished observation). Nevertheless, Tejada et al. (1984) reported a strong correlation between human fertility and the percentage of spermatozoa which fluoresced green. In addition, others (Sterik et al., 1989
) have reported solid correlations between the percentage of green-fluorescing sperm and fertility potential, thus providing support to our FCM studies.
In a study by Ibrahim et al. (1988), AO staining was the most discriminatory test (P = 0.0001) in a study of three groups (unexplained infertility, habitual abortions and normal fertile donors) when compared with the zona-free hamster egg penetration test and conventional semen analyses. The frequency of sperm chromatin heterogeneity as detected by AO red fluorescence was highest in habitual abortion (39.4%), followed by unexplained infertility (16.4%) and fertile donors (9.4%). The percentage of penetration was highest in habitual abortion (50.7%), followed by fertile donors (43.1%) and unexplained infertility (33.9%). Conventional semen tests (concentration, motility, morphology and vitality) were the least reliable in discriminating between the three groups.
Human SCSA data are more constant over time than the classical measures of spermatozoa, as shown in a study of 45 men who were unexposed to environmental insult and who provided monthly semen samples over a 9-month period (Schrader et al., 1988; Evenson et al., 1991
). The coefficient of variation of SCSA variables within a man were much lower than for the classical measures (Schrader et al., 1988
; Evenson et al., 1991
; Spano et al., 1998
). Thus, for cross-sectional, or single sample analysis of a patient, SCSA data can be considered to characterize one component of semen quality with a higher degree of certainty than the classical measures. Human spermatozoa obtained from cancer patients (Evenson et al., 1984
) or those with high fever (D.P.Evenson, unpublished data) show increased SCSA values, followed by either a return to normal (D.P.Evenson, unpublished data) or continued abnormality (Evenson et al., 1984
; Fossa et al., 1997
). Numerous animal toxicology studies have consistently shown the same very high level of repeatability of SCSA measures and convincing biological doseresponse interpretations (Evenson et al., 1989
, 1993a
,, b
). All human studies to date have indicated that SCSA data are poorly correlated with the classical semen measures; thus, SCSA data are considered independent variables that are of diagnostic and prognostic value in the human andrology clinic, as shown in these studies (Evenson et al., 1991
; Spano et al., 1998
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Study II:
115 consecutive men appearing at the Andrology Clinic, National Hospital, Oslo, Norway, for fertility counselling provided a semen sample by masturbation following an instructed abstinence period of 3 days. One sample per patient was collected in the clinic, and analysed by routine criteria and the SCSA.
Sample handling
In both studies, 200 µl aliquots of semen were admixed with 300 µl TNE buffer (0.15 M NaCl, 0.01 M TrisHCl, 0.001 M EDTA, di-sodium pH 7.4 and 10% glycerol) and frozen directly at 80°C. (N.B. current protocol freezes raw semen directly without cyroprotectants with the same data resulting.) Study I samples were shipped on dry ice to South Dakota State University for SCSA analysis, while Study II samples were measured in the flow cytometry laboratory in the Institute for Pathology, the National Hospital, Oslo, Norway.
Sperm quality measures
Study I:
the semen quality measures of volume, sperm concentration, motility by CASA and morphology by WHO and Kruger strict criteria were performed for all samples following liquefaction. These data will be the subject of a separate manuscript (E.Clegg et al., in preparation).
Study II:
following liquefaction at room temperature for 30 min, an aliquot of spermatozoa was diluted and immobilized in 5% chloramine-T (1:10) and counted using a Makler chamber (Sefi Medical Instruments Ltd, Haifa, Israel). Additional aliquots (50 µl) of the samples were subjected to vital staining using eosin Y and nigrosin, as well as haematoxylin, for the assessment of morphology. Microcephalic, macrocephalic, bicephalous and bicaudal spermatozoa, or cells possessing a coiled tail or a deformed or abnormally small acrosome were all classified as abnormal (WHO, 1987). The total numbers of spermatozoa with head and tail abnormalities in the ejaculate were computed. Sperm motility was assessed using a Hamilton Thorn sperm motility analyser (Danvers, MA, USA) which, in addition to other motility parameters, enabled the percentage of spermatozoa exhibiting any form of movement (>10 µm/s) and the percentage of progressive spermatozoa with movement (velocity >25 µm/s) to be computed.
SCSA
Frozen aliquots of semen were placed in a 37°C water bath until just thawed, after which samples were diluted with TNE buffer to 12x106 sperm cells per ml. 0.20-ml aliquots of diluted samples were mixed with 0.40 ml of aciddetergent solution (0.08 M HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2). After 30 s, the cells were stained by adding 1.2 ml acridine orange (AO) stain solution containing 6 µg AO (chromatographically purified; Cat. # 04539, Polysciences Inc., Warrington, PA, USA) per ml buffer [0.037 M citric acid, 0.126 M Na2HPO4, 0.0011 M EDTA (di-sodium), 0.15 M NaCl, pH 6.0 (Darzynkiewicz et al., 1976; Evenson et al., 1985
)]. At 3 min after the staining procedure started, fluorescence measurements were collected on 5000 cells per sample. Both flow laboratories used Cytofluorograf II flow cytometers (Ortho Diagnostics Inc., Westwood, MA, USA) equipped with ultrasense optics. The Oslo instrument used a Coherent 95, 5 W laser operated at 200 mW, and the South Dakota instrument used a Lexel 100 mW argon ion laser operated at 35 mW; both used an excitation wavelength of 488 nm. Histogram files were transferred via MULTLINK software (Phoenix Flow Systems, San Diego, CA, USA) to a PC for analysis with MULTI2D software (Phoenix Flow Systems) which included the calculation of alpha t (
t) parameters. The South Dakota State University (SDSU) Ortho Cytofluorograf is currently interfaced to a Cicero System (Cytomation, Inc., Fort Collins, CO, USA).
The extent of DNA denaturation was quantified by the calculated parameter t [
t = red/(red + green) fluorescence; Darzynkiewicz et al., 1975]. Normal, native chromatin remains structurally sound and produces a narrow
t distribution. DNA in spermatozoa with abnormal chromatin structure has increased red fluorescence (Evenson et al., 1980
, 1985
) which yields an
t distribution which is usually broader, having a higher mean channel (X
t) and a larger percentage of cells outside the main population of cells (COMP
t). Standard deviation of
t (SD
t) describes the extent of chromatin structure abnormality within a population. Mean green fluorescence reflects DNA content and/or degree of sperm chromatin condensation, the latter because it excludes DNA stainability.
Statistical analysis
Statistical tests used are described in the corresponding results section.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The couple represented in cytograms B1 and B2 conceived in month 1 of the study, and also had a normal pregnancy. This example was an exception to the high month-to-month repeatability of SCSA values (Evenson et al., 1991). Sample B1, with a COMP
t value of 13.3%, was obtained within 13 days of conception. The COMP
t value for the following month (B2) was 39.5%, which is considered not compatible with fertility. This type of dramatic deterioration has previously been seen after exposure to high fever (D.Evenson, unpublished observations) or prescription drugs (Evenson et al., 1991
).
The couple represented in cytograms C1 and C2 experienced a miscarriage after conceiving in month 1. The COMPt value of 34.0% (C1) was just over our current threshold of 30.0%, and considered not compatible with successful pregnancy. Otherwise, this sample had a low amount of debris and a high repeatability between the two months.
Cytograms D1, D2 and D3 were obtained from a couple who conceived in month 3. There was a dramatic change in sample quality, as shown by COMPt values from months 1 to 3, as very poor (64.4%), improved (34.4%) and excellent (8.0%). The month 3 sample would have been a full spermatogenic cycle away from a possible biostress event that may have caused the very low quality in month 1. Note that the main population had essentially the same level of quality, the only difference between the samples being the percentage of cells with denatured DNA. Note also that the average COMP
t was 35.5%, a mean value not considered compatible with good fertility, though SCSA data obtained near the conception date were highly compatible with our fertility threshold.
The couple represented in cytograms E did not conceive throughout the 12 months of the study. Note the high month-to-month repeatability in cluster arrangements, with moderately high COMPt values of 19.7, 22.7 and 20.0% for months 1, 2 and 3 respectively.
Cytograms F1 to F3 represent a couple who became pregnant in month 5. Samples from months 13 would be characterized as of good quality, with respective COMPt values of 13.1, 18.8 and 15.0%. There was a high degree of repeatability, i.e. the COMP
t region had two populations of cells with approximately the same number of cells in each and whose positions were essentially identical. This implied that these abnormal cells were produced at a steady rate, and were the likely progeny of the same stem cells.
Couple G did not conceive. Monthly COMPt values between months 1 and 3 were 25.4, 22.5 and 19.0% respectively, with an overall mean of 22.3%, considered of moderate quality on that characteristic alone. However, a second abnormality was present, namely, a population with high DNA stainability (HGRN). These two characteristics are often seen to be mutually exclusive; however, this is an example where both were present and repeatable over time.
Couple H experienced an occult pregnancy which was detected 13 days before the semen sampling in month 3, the month with the highest COMPt value near the end of month 2. No later pregnancies occurred during months 3 to 12. There was a high month-to-month repeatability, and COMP
t values were 13.0, 14.5 and 19.1% in months 1, 2 and 3 respectively.
Couple I did not conceive. This series of cytograms showed an increasing relative proportion of bacteria (arrow) to spermatozoa from month to month.
Study I
SCSA data were collected from duplicate measurements on semen samples collected monthly. Of the 165 couples with initial semen samples, 163 contributed a second sample one month later, and 84 provided a sample in month 3; only 402 of these 412 samples were available for SCSA measurement.
Each of 402 semen samples from 165 men were thawed independently and sequentially measured twice (total of 804 measurements). The number of monthly samples contributed by each man depended on pregnancy outcome. Throughout the course of these sample measurements, 136 separately frozen and thawed aliquots of a single reference sample were measured in duplicate (n = 272 independent measurements) for quality control. This reference sample was not characterized as fertile or infertile, but was used only for quality control. As shown previously (Evenson et al., 1991), sonication of random samples produced the same data as on whole cells (data not shown).
The descriptive statistics for SCSA measurements for overall means are shown in Table I, which also includes reference sample data. Table II
shows data for within-couple means. Each observation was assigned to one of four pregnancy outcome groups: group 1 = normal pregnancy in months 13; group 2 = miscarriage; group 3 = normal pregnancy in months 412; and group 4 = no pregnancy in months 112. SCSA values obtained by measuring the 148 semen samples from the 73 couples achieving pregnancy within months 13 (group 1) are shown in row 1; these values serve as the `fertile' reference standard for the other data in both Studies I and II.
|
|
Least-squares means by pregnancy outcome group were calculated for selected flow cytometry parameters by mixed linear model methods using the MIXED procedure of the SAS® system software (Littell et al., 1996). This procedure takes into account the covariance structure among repeated measurements from a given subject. Observations for this analysis were the within-couple means (averaged across duplicates and months). The least-squares mean for pregnancy outcome group 1 was compared with the means of each of the other groups, using the MIXED procedure. The MIXED model least-squares analysis was repeated using the log of flow cytometer values, as the data had non-normal distribution (Table III
).
|
|
|
|
|
|
|
The range of sperm quality criteria was much broader than observed in Study I, where the couples were selected for no known risk of infertility. As shown in Table X, these samples ranged from near-azoospermic to very low motility to nearly all with abnormal morphology. Note in Table XI
that there were no biologically meaningful correlations between classical criteria of sperm quality and SCSA parameters; for example, a correlation of r = 0.23, P < 0.05 between percentage of cells showing DNA denaturation (COMP
t) and progressive motility practically stated that dead cells were not synonymous with cells having abnormal chromatin structure; these data agree with other studies with human spermatozoa (Schrader et al., 1988
; Evenson et al., 1991
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Study I, using the SCSA values from the couples conceiving during the first 3 months as a `standard' for `highly fertile' individuals, the increased SCSA values obtained from individuals conceiving during the next 9 months were significantly different (P < 0.01). More dramatically, those individuals not conceiving over the 12-month study yielded SCSA values that had greater significant differences (P < 0.001) from the `standard' group, providing strong evidence that sperm chromatin structure was reflective of fertility potential. This viewpoint is strengthened by SCSA values from Study II, in which the quality of semen samples from men of couples suspected of possible infertility were significantly (P < 0.001) different from those of the standard fertile group. Even so, in Study II and previous studies (Schrader et al., 1988; Evenson et al., 1991
) the SCSA data were not well correlated with classical semen parameters, emphasizing that SCSA data are independent to a significant degree. Of greatest importance to the andrology clinic are those cases where the classical criteria are within normal ranges, but the SCSA values are poor, and probably not compatible with good fertility (COMP
t
30%). For many reasons, before embarking on extensive assisted reproduction treatment, it would be of great value to the patient and the clinic to know the quality of the sperm chromatin, not measurable by any other efficient method.
In Study I, we had postulated that a significant correlation would be found between sperm chromatin quality and occult pregnancy. There were not sufficient numbers (n = 3) to detect differences, and only a trend was noted between occult pregnancy and reduced total DNA stainability, probably due to chromatin hypercondensation factors and/or hyperoxidation of cysteine -SH groups.
High-quality SCSA data are not directly predictive of good fertility potential, since natural fertility requires many other positive traits such as motility, morphology, acrosome integrity, etc. The inverse, i.e. poor SCSA data, are predictive of subfertility/infertility. For the purposes of intracytoplasmic sperm injection (ICSI), there is ample justification for an SCSA measurement since there is generally poor correlation between SCSA measures and motility, morphology and viability. Thus, in those cases reported in the literature where there is a concern whether to utilize an ejaculate with little or no motility or to obtain a testicular sample in hope of better quality spermatozoa, if the SCSA data are good then it would be instructive to consider using the ejaculate for ICSI. On the other hand, if the SCSA data are of poor quality, even in the face of `normal' motility and morphology, then serious consideration should be given not to utilize that sample for fertilization. In cases where the testis may have been exposed to a perturbing agent, e.g. high fever (D.P.Evenson, unpublished observations), and the resulting data are of poor quality, it is suggested that another sample be evaluated by the SCSA after one spermatogenic cycle.
For some time we have wrestled with the question of what percentage of spermatozoa with denatured DNA is the cut-off level for considering the sample as compatible or possibly non-compatible with fertility. From data in this study and other unpublished data, that threshold is currently set at 30%. However, it is important to discuss that this does not mean that the other 70% of spermatozoa have fully normal chromatin. This value means only that, given the physical conditions imposed on the spermatozoa to induce DNA denaturation, that 30% of the spermatozoa crossed that threshold. As has been discussed previously (Evenson and Jost, 1994), stronger inducing conditions for DNA denaturation will cause a higher percentage of sperm nuclei to cross that threshold. It is important to note, however, that the shift in different samples is proportional, i.e. the doseresponse curves are parallel, and thus no new valuable information is obtained by varying physical conditions. Thus, an exact set of conditions as detailed in Materials and methods for treatment of spermatozoa and measurement by flow cytometry (Evenson and Jost, 1994
) strictly defines the SCSA, and all sample data can be related to other samples.
Furthermore, whereas the variable COMPt describes only the percentage of cells demonstrating DNA denaturation, SD
t describes the variation from the mean. In other studies, e.g. effects of smoking on sperm head morphometry (Rubes et al., 1998
), the variation around the actual mean values rather than the mean values was indicative of smoke-induced changes. The SD
t has been the parameter most highly correlated with rodent reproductive toxicants, dose and time parameters. A high SD
t is further suggestive that damaged chromatin exists in the `main population', and even though any single cell may not have crossed the `threshold' to be counted as a DNA denatured (COMP
t) cell, the extent of damage may be sufficient to have negative fertility/pregnancy outcome factors.
What is the physical damage in sperm chromatin that is directly or indirectly being detected by the SCSA? A recent study (Aravindan et al., 1997) selected 23 human sperm samples with COMP
t values ranging from 5% to 95%. Aliquots of these samples were analysed for DNA strand breaks by two independent methods: (i) flow cytometric TUNEL assay for 3'-OH broken ends of DNA (Gorzcyca et al., 1993
); and (ii) DNA fragments by single cell gel electrophoresis analysis (modified COMET assay; Singh et al., 1988
). Correlations between the percentage of cells demonstrating denatured DNA by SCSA (COMP
t) were very strong with TUNEL data (r = 0.859, P < 0.001) and COMET data (r = 0.973, P < 0.001). The latter assay, being the more sensitive to DNA strand breaks, showed a near 1:1 relationship between the percentage of cells with denatured DNA and percentage of cells with `comets' consisting of DNA fragments. These data provide strong evidence that the SCSA data are showing DNA strand breaks which, by definition, would be considered negative for fertility and pregnancy outcome. The origin of these strand breaks is not clear; however, recent studies in our laboratory show that oxidative stress agents, e.g. exposure to H2O2, produce the same SCSA pattern in a doseresponse relationship (Larson et al., 1998
), and we currently favour the idea that most damage is due to oxidative stress to DNA. Also, consideration has been given to the idea that they may represent an altered form of apoptosis where the DNA fragments can exist in cells that are otherwise `healthy' from the standpoint of mitochondrial function (motility) and membrane viability (viable) (Gorzcyca et al., 1993
; Aravindan et al., 1997
). Other studies are in progress to evaluate this possibility.
In summary, these two studies, as well as previous studies, provide a solid rationale for andrology clinics to measure SCSA in all or selected semen samples before counselling patients on fertility issues, especially expensive assisted reproduction procedures.
How useful is the SCSA?
As discussed by Matson (1997), for a test to be useful, it must first be reproducible such that similar results are obtained each time a man is tested, assuming that all other factors are unchanged. Two aspects of this are: (i) the robustness of the method in performing to the same level each time; and (ii) the stability of the parameter being measured. The SCSA is among the best of semen assays with regard to both of these aspects. For the first, as shown in this studyand in measurements of thousands of other mammalian sperm samplesthe repeatability is invariably in the 0.98 to 0.99 range. Correlations between measurements of frozenthawed aliquots of the same samples on six different flow cytometers at six different institutions and times were also in the 0.97 to 0.98 range; this even included using both orthogonal laser-driven FCM versus mercury arc epi-illumination FCM. Furthermore, the use of different computer software for calculations of SCSA variables showed correlation coefficients of the same variable from different programs to all be 0.98 or higher (P < 0.001; Evenson et al., 1995). These results far exceed the repeatability of many tests, even computer-driven CASA systems, done on different instruments and in different laboratories. With regard to the second aspect, i.e. stability of the parameter being measured and also biological variability, SCSA variables were the most stable over 8 months in an unexposed population (Schrader et al., 1988; Evenson et al., 1991
). It is noted, however, that biological and physical stress to mammalian testes does produce dramatic shifts in SCSA variables which have been shown to be highly reproducible and dose/time responsive.
The technical variability of the SCSA is practically negligible; repeatability of the same samples in this study was in the range of 0.980.99. More importantly, the repeatability of the reference sample frozen in many aliquots and used repeatedly throughout the weeks of measurements was 0.98 (P < 0.001), thus showing excellent internal quality control. External quality assurance has been addressed above, with aliquots of the same sample being measured in different laboratories with different instruments, and by different technicians.
The whole purpose of performing a diagnostic test in the evaluation of the male partner of a suspected infertile couple is to learn whether his fertility is impaired (Matson, 1997). The test must then have a threshold above and below which it will provide discrimination and predictive capabilities, and with little overlap between fertile and infertile men. As an example of this problem, a `normal' sperm concentration is regarded as being >20x106/ml (WHO, 1992); however, this was derived from a study (Macleod and Gold, 1951
) where the median sperm concentrations in the fertile and infertile groups were 90x106/ml and 74x106/ml respectively. Obviously, this criterion has little power to identify prospectively infertile men. In sharp contrast, in this study, men with 30% or more spermatozoa showing DNA denaturation were subfertile/infertile, thus showing reasonable sensitivity. With regard to specificity, i.e. the proportion of normal individuals correctly predicted to have a fertility problem and who actually experienced a fertility problem was 52% (27/52). If however, SCSA data had been based on a single measurement taken within several days of attempted conception, rather than on a mean of several monthly samples, the predictive power would have significantly increased.
Using a combination of selected cut-off values for percentage spermatozoa with denatured DNA and/or increased DNA stainability (HGRN), the SCSA predicted 39% of miscarriages (seven of 18; six from COMPt and one from HGRN) (Tables V and VI
). Since over 50% of miscarriage problems are likely to be attributed to the female, this result was strongly related to the prediction. Again, these results were based on within-couple mean values for SCSA parameters. Inspection of individual (monthly) samples can be more informative. For example, some had poor values in month 1, followed by improved values in later months. Even though the overall mean COMP
t was >15%, the value at the time of pregnancy was <15%.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aravindan, G.R., Bjordahl, J., Evenson, D.P. et al. (1997) Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp. Cell Res., 236, 231237.[ISI][Medline]
Ballachey, B.E., Hohenboken, W.D. and Evenson, D.P. (1987) Heterogeneity of sperm nuclear chromatin structure and its relationship to fertility of bulls. Biol. Reprod., 36, 915925.[Abstract]
Ballachey, B.E., Saacke, R.G. and Evenson, D.P. (1988) The sperm chromatin structure assay: relationship with alternate tests of sperm quality and heterospermic performance of bulls. J. Androl., 9, 109115.
Darzynkiewicz, Z., Traganos, F., Sharpless, T. et al. (1975) Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp. Cell Res., 90, 411428.[ISI][Medline]
Darzynkiewicz, Z., Traganos, F., Sharpless, R. et al. (1976) Lymphocyte stimulation: a rapid multiparameter analysis. Proc. Natl Acad. Sci. USA, 73, 28812884.[Abstract]
Evenson, D.P. and Jost, L.K. (1994) Sperm chromatin structure assay: DNA denaturability. In: Darzynkiewicz, Z., Robinson, J.P. and Crissman, H.A. (eds), Methods in Cell Biology, Vol. 42, Flow Cytometry, Second Edition. Academic Press, Inc., Orlando, pp. 159176.
Evenson, D. and Jost, L. (1998) Utility of the sperm chromatin structure assay in the infertility clinic. XX Congress of the International Society for Analytical Cytology, March 16, Colorado Springs, CO.
Evenson, D.P. and Melamed, M.R. (1983) Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J. Histochem. Cytochem., 31, 248253.[ISI][Medline]
Evenson, D.P., Darzynkiewicz, Z. and Melamed, M.R. (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science, 240, 11311133.
Evenson, D.P., Klein, F.A., Whitmore, W.F. et al. (1984) Flow cytometric evaluation of sperm from patients with testicular carcinoma. J. Urol., 132, 12201225.[ISI][Medline]
Evenson, D.P., Higgins, P.H., Grueneberg, D. et al. (1985) Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry, 6, 238253.[ISI][Medline]
Evenson, D.P., Baer, R.K. and Jost, L.K. (1989) Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry. Environ. Mol. Mutagen., 14, 7989.[ISI][Medline]
Evenson, D.P., Jost, L.K., Baer, R.K. et al. (1991) Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod. Toxicol., 5, 115125.[ISI][Medline]
Evenson, D.P., Jost, L.K. and Baer, R.K. (1993a) Effects of methyl methanesulfonate on mouse sperm chromatin structure and testicular cell kinetics. Environ. Mol. Mutagen., 21, 144153.[ISI][Medline]
Evenson, D.P., Jost, L.K. and Gandy, J.G. (1993b) Glutathione depletion potentiates ethyl methanesulfonate-induced susceptibility of rat sperm DNA denaturation in situ. Reprod. Toxicol., 7, 297304.[ISI][Medline]
Evenson, D.P., Thompson, L. and Jost, L. (1994) Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Theriogenology, 41, 637651.[ISI]
Everson, D., Jost, L., Gandour, D. et al. (1995) Comparative sperm chromatin structure assay measurement on epiillumination and orthagonal axes flow cytometers. Cytometry, 19, 295303.[ISI][Medline]
Fossa, S.D., De Angelis, P., Kraggerud, S.M. et al. (1997) Prediction of post-treatment spermatogenesis in patients with testicular cancer by flow cytometric sperm chromatin structure assay. Commun. Clin. Cytom., 30, 192196.
Gorzcyca, W., Gong, J. and Darzynkiewicz, Z. (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res., 53, 945951.
Ibrahim, M.E., Moussa, M.A.A. and Pedersen, H. (1988) Sperm chromatin heterogeneity as an infertility factor. Arch. Androl., 21, 129133.[ISI][Medline]
Johnson, L.A., Flook, J., Look, M. and Pinkel, D. (1987) Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Res., 16, 19.[ISI][Medline]
Johnson, L.A., Flook, J. and Hawk, H. (1989) Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting. Biol. Reprod., 41, 199203.[Abstract]
Larson, K., Jost, L. and Evenson, D. (1998) Detection of oxidative stress in mammalian sperm DNA. XX Congress of the International Society for Analytical Cytology, March 16, Colorado Springs, CO.
Littell, R.C., Milliken, G.A., Stroup, W.W. et al. (1996) SAS® System for Mixed Models. SAS Institute Inc., Cary, NC.
Macleod, J. and Gold, R. (1951) Male factors in fertility and infertility: spermatozoon counts in 1000 men of known fertility and in 1000 cases of infertile marriage. J. Urol., 66, 436449.[ISI]
Matson, P.L. (1997) Clinical value of tests for assessing male infertility. Baillière's Clin. Obstet. Gynaecol., 11, 641654.[ISI][Medline]
Rubes, J., Lowe, X., Moore, D. et al. (1998) Cigarette-smoking lifestyle is associated with increased sperm disomy in teenage men. Fertil. Steril., 70, 715723.[ISI][Medline]
SAS Institute Inc. (1985) SAS® Users Guide: Statistics, Version 5 Edition. SAS Institute Inc., Cary, NC.
Schrader, S.M., Turner, T.W., Breitenstein, M.J. et al. (1988) Longitudinal study of semen quality of unexposed workers. I. Study overview. Reprod. Toxicol., 2, 183190.[Medline]
Shapiro, H. (ed.) (1995) Practical Flow Cytometry. 3rd edn. Wiley-Liss, New York.
Singh, N.P., McCoy, M.T., Tice, R.R. et al. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184191.[ISI][Medline]
Spano, M., Kolstad, A.H., Larsen, S.B. et al. (1998) The application of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum. Reprod., 13, 24952505.[Abstract]
Sterik, K., Rosenbusch, B., Sasse, V. et al. (1989) The acridine orange test. A new parameter in assessing the fertilizing capacity of spermatozoa. Zentralbl. Gynakol., 111, 13611367.[ISI][Medline]
Tejada, R.I., Mitchell, J.C., Norman, A. et al. (1984) A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil. Steril., 42, 8791.[ISI][Medline]
World Health Organization (1987) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. 2nd edn. Cambridge University Press, Cambridge.
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. 3rd edn. Cambridge University Press, Cambridge.
Zinaman, M.J., Clegg, E.D., Brown, C.C. et al. (1996) Estimates of human fertility and pregnancy loss. Fertil. Steril., 65, 503509.[ISI][Medline]
Submitted on September 2, 1998; accepted on December 23, 1998.