Sperm Chromatin Structure Assay for Fertility Assessment

Donald Evenson1, Lorna Jost1

1 South Dakota State University, Brookings, South Dakota
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.13
DOI:  10.1002/0471142956.cy0713s13
Online Posting Date:  May, 2001
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The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring. Damaged DNA in the single sperm that fertilizes the female egg can have a dramatic negative impact on fetal development. This comprehensive and detailed unit presents a rapid, reliable, practical test for DNA integrity based on staining with acridine orange. SCSA data have been conclusively shown to predict sub/infertility. This assay is ideally suited to human and animal fertility clinics to assess male sperm DNA integrity as related to fertility potential and embryo development. The authors, who have decades of experience in studying sperm viability, provide extensive commentary and methodological tips, making this unit the most detailed method for this test published to date. Keywords: flow cytometry; sperm chromatin structure assay; SCSA; DNA denaturation; acridine orange;animal and human fertility; toxicology The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring

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Table of Contents

  • Basic Protocol 1: Sperm Chromatin Structure Assay
  • Support Protocol 1: Flow Cytometer Alignment and Setup
  • Support Protocol 2: Identifying, Collecting, and Freezing a Reference Sample
  • Support Protocol 3: Acquisition and Analysis Protocol of SCSA Listmode Measurements
  • Support Protocol 4: Sonication of Sperm Cells for SCSA of Sperm Nuclei
  • Support Protocol 5: Preparing Cytoplasm‐Free Sperm Nuclei
  • Support Protocol 6: Ethanol Fixation of Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Sperm Chromatin Structure Assay

  • AO equilibration buffer (see recipe)
  • Reference sample (see protocol 3)
  • Semen or caudal epididymal or testicular aspirate sample(s)
  • 1× TNE buffer (see recipe)
  • Acid detergent solution (see recipe)
  • AO staining solution (see recipe)
  • Ethanol/bleach tubing cleanser solution (see recipe)
  • Household bleach tubing cleanser solution (see recipe)
  • Flow cytometer (see Table 7.13.1 and Fig. ) with 488‐nm excitation appropriate filters for collection of green and red fluorescence, and ≥15 to 35 mW power, interfaced to a computer with appropriate software (Table 7.13.2) for calculating SCSA parameters
  • Nonadjustable 200 µl pipettor and appropriate tips
  • 0.20 to 0.80‐ml and 0.80 to 3.0‐ml Oxford adjustable dispensers with amber glass bottles (Fisher Scientific)
  • Polystyrene 12 × 75–mm (4.5‐ml) conical tubes (Sarstedt)
  • Additional reagents and solutions for setting up and aligning the flow cytometer ( protocol 2), employing a reference sample ( protocol 3), counting sperm using a hemacytometer ( appendix 3A, and data analysis ( protocol 4)
    Table 7.3.1   Materials   Flow Cytometers that Have Successfully Been Used to Run the SCSA a   Flow Cytometers that Have Successfully Been Used to Run the SCSA

    Flow cytometer Configuration Company
    Cytofluorograf Orthogonal Ortho
    Elite Orthogonal Coulter
    FACScan Orthogonal Becton Dickinson
    ICP22A Epiillumination Ortho
    Skatron Epiillumination Bio‐Rad

     aData taken from Evenson et al. ( ), also see Fig.
CAUTION: For personnel safety against potential infectious agents (e.g., hepatitis and HIV) handle human samples using disposable gloves in a biological safety cabinet.IMPORTANT NOTE: The SCSA procedure requires that samples be thawed and processed in the immediate vicinity of the flow cytometer. Human samples are thawed and prepared in a biological safety hood near the flow cytometer. All necessary equipment (e.g., 2 to 3 ice buckets containing wet ice with reagent bottles and TNE container deeply imbedded in the ice, sample tubes, disposable gloves, automatic pipetters and tips, stopwatch, 37°C water bath, marker) should be easily accessible in the safety hood or on a nearby laboratory cart.NOTE: Although quick indications of the sperm with denatured DNA can be calculated from the red versus green fluorescence cytogram in “live‐time” or on a stand‐alone computer, the authors' laboratory calculates the SCSA data after all the samples have been measured in an experiment. The actual α t values are taken from α t frequency histogram statistics.NOTE: For this protocol, all reagents, buffers, and samples must be kept on ice (i.e., 4°C).

Support Protocol 1: Flow Cytometer Alignment and Setup

  • Sheath fluid (see recipe)
  • Standard fluorescent beads
  • Sperm sample, 1–2 × 106/ml
  • Reference sample (see protocol 3)

Support Protocol 2: Identifying, Collecting, and Freezing a Reference Sample

  • Semen donor
  • Clinical specimen jars, polystyrene, sterile (VWR Scientific)
  • 0.5‐ to 1.5‐ml polypropylene microcentrifuge tubes (Sarstedt)
  • 1.2‐ml cryogenic vials (Fisher)
NOTE: All studies with human subjects must be approved by the Institutional Review Board (IRB), which must adhere to the Office for the Protection from Research Risk (OPRR) guidelines or other applicable governmental regulations for using human subjects.NOTE: It is important to work quickly and efficiently with samples for freezing.NOTE: Quick‐frozen and cryoprotectant‐frozen sperm provide equivalent SCSA data (Evenson et al., ), which is a unique feature of mammalian sperm cells due to the highly condensed crystalline nature of the nuclei.

Support Protocol 3: Acquisition and Analysis Protocol of SCSA Listmode Measurements

  • Offline software capable of generating calculated parameters (see Table 7.13.2)
    Table 7.3.2   Additional Materials (also see protocol 1)   Additional MaterialsPC Hardware/Software Options for Collecting and Analyzing SCSA Data

    Hardware Supplier
    CICERO SYSTEM b Cytomation
    LISTVIEW Phoenix Flow Software
    WINLIST c Verity Software House
    FCS Express De Novo Software

     bThis packages allows for viewing the calculated α t parameters in live time that is very helpful when setting up the instrument and checking/maintaining system stability and alignment. The Cicero System can be interfaced with all known commercial flow cytometers.
     cThe macro for analyzing SCSA data using WINLIST software is not provided by Verity House

Support Protocol 4: Sonication of Sperm Cells for SCSA of Sperm Nuclei

  • 1–2 × 106 sperm cells in recipe1× TNE buffer
  • 2‐ml screw‐cap cryogenic vial (Corning)
  • No. 11 rubber stopper with 12‐mm hole
  • Branson Sonifier II, Model 450, coupled to a Branson Cup Horn (VWR Scientific) and linked to a Masterflex peristaltic pump (Cole‐Parmer Instrument) or equivalent
CAUTION: Because human semen samples potentially contain infectious agents such as hepatitis or HIV, sonication must be done only in a closed tube using a cup horn sonicator.IMPORTANT NOTE: Tail/cytoplasm separation varies between species and needs to be tested for each sonicator to achieve a ≥95% head/tail separation. Using a microscope, check the separation by scoring >100 sperm cells on 3 different slides (for each time/setting tested). Test the sonication unit (probe or cup horn) using several different semen samples to determine optimal time and power required for ≥95% head/tail separation. Because of the size of the tails on rat sperm used in toxicology studies, these samples must always be sonicated or the whole sperm will clog the sample filter.

Support Protocol 5: Preparing Cytoplasm‐Free Sperm Nuclei

  • 60% sucrose solution, pH 7.5 (see recipe)
  • 1× TNE buffer (see recipe)
  • Beckman T‐J6 benchtop clinical ultracentrifuge or equivalent
  • Additional reagents and equipment for sonication of sperm cells ( protocol 5)

Support Protocol 6: Ethanol Fixation of Samples

  • Semen sample
  • Hank's balanced salt solution (HBSS; Life Technologies, also see appendix 2A)
  • 80% ethanol, −20°C
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Literature Cited

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   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.
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   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 testicular 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. 26:147‐159.
   Evenson, D.P. and Jost, L.K. 1994. Sperm chromatin structure assay: DNA denaturability. In Methods in Cell Biology, Vol. 42: Flow Cytometry (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 cytometry. 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.
   Evenson, D.P., Higgins, P.H., Grueneberg, D., and Ballachey, B. 1985. Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry 6:238‐253.
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   Evenson, D.P., Darzynkiewicz, Z., Jost, L., Janca, F., and Ballachey, B. 1986b. Changes in accessibility of DNA to various fluorochromes during spermatogenesis. Cytometry 7:45‐53.
   Evenson, D.P., Baer, R.K., and Jost, L.K., 1989a. Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups. Mol. Reprod. Dev. 1:283‐288.
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   Evenson, D.P., Janca, F.C., Jost, L.K., Baer, R.K., and Karabinus, D.S., 1989c. Flow cytometric analysis of effects of l,3‐dinitrobenzene on rat spermatogenesis. J. Toxicol. Environ. Health 28:81‐98.
   Evenson, D.P., Janca, F.C., Baer, R.K., Jost, L.K., and Karabinus, D.S. 1989d. Effect of l,3‐dinitrobenzene on prepubertal, pubertal and adult mouse spermatogenesis. J. Toxicol. Environ. Health 28:67‐80.
   Evenson, D.P., Jost, L., Baer, R., Turner, T., and Schrader, S. 1991. Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod. Toxicol. 5:115‐125.
   Evenson, D.P., Emerick, R.J., Jost, L.K., Kayongo‐Male, H., and Stewart, S.R. 1993a. Zinc‐silicon interactions influencing sperm chromatin integrity and testicular cell development in the rat as measured by flow cytometry. J. Anim. Sci. 71:955‐962.
   Evenson, D.P., Jost, L.K., and Baer, R.K. 1993b. Effects of methyl methanesulfonate on mouse sperm chromatin structure and testicular cell kinetics. Environ. Mol. Mutagen. 21:144‐153.
   Evenson, D.P., Jost, L.K., and Gandy, J.G. 1993c. Glutathione depletion potentiates ethyl methanesulfonate‐induced susceptibility of rat sperm DNA denaturation in situ. Reprod. Toxicol. 7:297‐304.
   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 41:637‐651.
   Evenson, D., Jost, L., Gandour, D., Rhodes, L., Stanton, B., Clausen, O.P., De Angelis, P., Coico, R., Daley, A., Becker, K., and Yopp, T. 1995a. Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers. Cytometry 19:295‐303.
   Evenson, D., Jost, L., and Sailer, B. 1995b. Flow cytometry of sperm chromatin structure as related to toxicology and fertility. Proceedings of the Seventh International Spermatology Symposium, Cairns, Australia.
   Evenson, D.P., Jost, L.K., Zinaman, M.J., Clegg, E., Purvis, K., de Angelis, P., and Clausen, O.P. 1999. Utility of the sperm chromatin structure assay (SCSA) as a diagnostic and prognostic tool in the human fertility clinic. Hum. Reprod. 14:1039‐1049.
   Evenson, D.P., Jost, L.K., Corzett, M., and Balhorn, R. 2000a. Effect of elevated body temperature on human sperm chromatin structure. J. Andrology. Submitted for publication.
   Evenson, D.P., Jost, L.K., and Varner, D.D. 2000b. Stallion sperm nuclear protamine‐SH status and susceptibility to DNA denaturation are not strongly correlated. J. Fertility Reprod. Suppl. In press.
   Fossa, S.D., De Angelis, P., Kraggerud, S.M., Evenson, D., Theodorsen, L., and Claussen, O.P. 1997. Prediction of post‐treatment spermatogenesis in patients with testicular cancer by flow cytometric sperm chromatin structure assay. Commun. Clin. Cytometry 30:192‐196.
   Gledhill, B.L., Lake, S., Steinmetz, L.L., Gray, J.W., Crawford, J.R., Dean, P.N., and VanDilla, M.A. 1976. Flow microfluorometric analysis of sperm DNA content: Effect of cell shape on the fluorescence distribution. J. Cell Physiol. 87:367‐376.
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   Sailer, B.L., Jost, L.K., Erickson, K.R., Tajiran, M.A., and Evenson, D.P. 1995a. Effects of X‐ray irradiation on mouse testicular cells and sperm chromatin structure. Environ. Mol. Mutagen. 25:23‐30.
   Sailer, B.L., Jost, L.K., and Evenson, D.P. 1995b. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J. Androl. 16:80‐87.
   Selevan, S.G., Borkovec, L., Slott, V.L., Zudova, Z., Rubes, J., Evenson, D.P., and Perreault, S.D. 2000. Semen quality and reproductive health of young Czech men exposed to seasonal air pollution. Submitted for publication.
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Key References
   Evenson et al., 1980. See above.
  The first article correlating SCSA data and fertility.
   Evenson et al., 1999. See above.
  The most definitive paper on the SCSA and its application to human fertility clinics.
   Evenson et al., 1991. See above.
  First longitudinal study utilizing the SCSA and showing the within‐donor repeatability of samples over time and the repeatability of the SCSA when measuring the same sample several times.
   Evenson et al., 1995a. See above.
  For more details on instrumentation already used with the SCSA.
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