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have an increased DNA stainability (Evenson and Melamed, 1983; Engh et al., 1992 ), which can be visualized by univariate analysis (Engh et al., 1992) as well as by the SCSA bivariate analysis. In most cases, the defects of high DNA stainability and DNA denaturation are mutually exclusive, and any single cell rarely has both defects. However, the authors have observed a limited number of human clinic cases where both defects occur within the same sample. Figure 7.13.1 illustrates how to resolve normal versus high green fluorescing cells. % HIGRN is calculated from the red versus green fluorescence cytogram, and has been discussed in a recent paper (Evenson et al., 1999). The authors do not yet have enough experience with this parameter to be clear-cut as to what percent indicates the likelihood of infertility.

COMPOt is the most important variable for fertility counselling. A recent study (Evenson et al., 1999) correlated SCSA data with accumulated fertility data. Due to high correlations and the establishment of thresholds of sperm with denaturable DNA for compatibility with fertility, the authors anticipate that the SCSA will be of most use prospectively in the human and animal clinics. According to this study, men with <15%, 15% to 30%, and >30% COMP^ would be predicted to have high fertility potential (pregnancy within 3 months), medium fertility potential (pregnancy within 4 to 12 months) and very low fertility potential (pregnancy in >1 year, defined as infertile), respectively (Table 7.13.3). A couple requesting counselling on their chances of pregnancy may be advised that their chances of pregnancy are greatly reduced if the percent sperm with abnormal chromatin is above 30%.

Figure 7.13.4 consists of cartoon illustrations of normal fluorescence populations and common variations when sperm are measured by the SCSA. Panel A is a classical SCSA cytogram with the following numbered areas:

(1) The main population of cells in a semen sample that, under the conditions imposed, remain as a coherent population. Although the DNA content in these cells is the same haploid amount, the cytogram cluster is elliptical in shape due to the optical artifact discussed in the text. This artifact is of little to no consequence to the outcome and interpretation of SCSA data.

(2) This area contains the cells outside the main population, or COMPa^ These cells typically move out and downward from the main population at a ~45° angle, showing the increase of red fluorescence at the expense of green fluorescence. Some samples show a continuous cluster ranging from just outside the main population to ones with very high red and very low green values. Alternatively, there are several discrete clusters in the COMPat population with two shown in this illustration. (3) This area represents seminal debris consisting of broken cellular components and other particulate matter stained with AO. For proper SCSA analysis, the debris signals must be resolved from the sperm signals. Since the COMPat population forms a ~45° slope, downward and to the right, an effective means to delineate between these cell clusters is to draw a 45° computer gate between them at the bottom edge of the main population. Note that this is also an active gate during acquisition so that 5000 or more sperm signals are accumulated and any debris signals excluded. This is most important in samples with a high ratio of debris to sperm so that the same number of sperm cells are statistically analyzed per sample. (4) The sperm signals appearing in areas 4 and 5 have high AO stain-ability and are termed sperm with high green stainability (% HIGRN). Chromatin in these sperm is probably not fully condensed, thus allowing a greater accessibility by intercalating DNA dyes. The percentage of HIGRN sperm is a ratio of the number of cells in areas 4 and 5 divided by the total number of cells contained in the gating region (includes sperm populations 1, 2, and 4) plus population 5 x 100. (Note that for most SCSA parameters the cytogram gating region contains the total number of sperm cells in areas 1, 2, and 4). The % HIGRN region starts at ~70%-75% of the green fluorescence scale. (5) This area contains sperm that also have higher DNA stainability, aggregates of sperm, early sperm forms, and possible somatic cells. The authors have not sorted this population for light microscopic identification; thus, a conclusion on how much of the region to include in this calculation has not been finalized.

Panel B of Figure 7.13.5 is the same as panel A except that area (6) represents a semen sample with excessive bacterial contamination. Due to random size clumping of the bacteria, a straight line of signal is seen to the left of the sperm population. When present, this population is gated out during acquisition in order to accumulate 5000 sperm signals. Note that this tends to be confounded with cells in the upper high green stainability area also.

Panel C of Figure 7.13.5 is also the same as A except that area (7) is an approximation of a sperm sample that has been compromised by excessive freeze/thaw cycles or left in a thawed state for an extended time period. This popula tion has an increased DNA stainability and shifts a bit to the right, as discussed in the text, but this compromised state is also revealed by a telltale hook usually present at the top of the population cluster. Samples showing this artifact are removed from the database. When all the samples in a box have this telltale artifact, inquiry usually confirms that a freezer failure or similar event occurred.

Panel D of Figure 7.13.5 is a composite of possible sperm populations.

Time Considerations

For fresh sperm samples, following collection of the sample, and a 30-min period for semen liquefaction, an aliquot of the sample can be measured by the SCSA, or be placed into a cryotube and plunged directly into liquid nitrogen or an ultracold freezer for storage. Since cryoprotectants are not needed, this is accomplished within a couple of minutes. Longitudinal studies of changes in sperm parameters are possible with the SCSA, as the sperm can be stored frozen until all samples are collected. The time of course depends on the length of the experimental study.

The SCSA requires paying very strict attention to time. Once a sample that has been frozen is warmed for thawing, the process is committed. As mentioned in the text, a sample may be refrozen once or more for a nonexacting measurement of SCSA parameters.

Thawing the sample takes ~30 to 60 sec and preparing the sample for flow cytometry measurement and equilibration in the flow cytometer takes 3 min. Data on 5000 cells can be collected in less than 1 min. When a flow operator becomes proficient, 5 to 6 samples measured in duplicate can be run per hour. Data analysis on each sample takes ~10 min.

Literature Cited

Aravindan, G.R., Bjordahl, J., Jost, L.K., and Even-son, D.P. 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. 236:231-237.

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. 36:915-925

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. An-drol. 9:109-115.

Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M.R. 1975. Thermal denaturation of DNA in situ as studied by Acridine Orange staining and automated cytofluorometry. Cell Res. 90:411-428.

Engh, E., Clausen, O.P.F., Scholberg, A., Tollefsrud, A., and Purvis, K. 1992. Relationship between sperm quality and chromatin condensation measured by sperm DNA fluorescence using flow cytometry. Int. J. Androl. 15:407-415.

Estop, A.M., Munne, S., Jost, L.K., and Evenson, D.P. 1993. Alterations in sperm chromatin structure correlates with cytogenetic damage of mouse sperm following in vitro incubation. J. Androl. 14:282-288.

Evenson, D.P. 1997. Sperm nuclear DNA strand breaks and altered chromatin structure: Are there concerns for natural fertility and assisted fertility in the andrology lab? Moving Beyond Boundaries: Clinical Andrology in the 21st Century. Andrology Laboratory Workshop, Postgraduate Course, Baltimore, Md.

Evenson, D.P. 1999a. Alterations and damage of sperm chromatin structure and early embryonic failure. In Towards Reproductive Certainty: Fertility and Genetics Beyond 1999. Proceedings of the 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics (R. Jannsen and D. Mortimer, eds.) pp. 313-329. Parthenon Publishing Group, New York.

Evenson, D.P. 1999b. Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects. Reprod. Fertility Dev. 11:1-15.

Evenson, D.P. and Darzynkiewicz, Z. 1990. Acridine orange induced precipitation of mouse tes-ticular sperm cell DNA reveals new patterns of chromatin structure. Exp. Cell Res. 187:328-334.

Evenson, D.P. and Jost, L.K. 1993. Hydroxyurea exposure alters mouse testicular kinetics and sperm chromatin structure. Cell Prolif. 26:147159.

Evenson, D.P. and Jost, L.K. 1994. Sperm chro-matin structure assay: DNA denaturability. In Methods in Cell Biology, Vol. 42: Flow Cy-tometry (Z. Darzynkiewicz, J.P. Robinson, and H.A. Crissman, eds.) pp. 159-176. Academic Press, Orlando, Fla.

Evenson, D.P. and Melamed, M.R. 1983. Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cy-tometry. J. Histochem. Cytochem. 31:248-253.

Evenson, D.P., Darzynkiewicz, Z., and Melamed, M.R. 1980. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 240:1131-1133.

Evenson, D.P., Klein, F.A., Whitmore, W.F., and Melamed, M.R. 1984. Flow cytometric evaluation of sperm from patients with testicular carcinoma. J. Urol. 132:1220-1225.

Nucleic Acid Analysis

100 Pregnancy Tips

100 Pregnancy Tips

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