Chapter 20 – DNA Damage: Sperm Chromatin Structure Assay




Abstract




The year 2020 is the fortieth anniversary of the introduction of the concept of “sperm DNA fragmentation” as related to human and animal male factor fertility. This concept was introduced by Donald Evenson in a Science article (1980) that also introduced the first test for its detection, the Sperm Chromatin Structure Assay (SCSA®). Sperm DNA fragmentation is defined as sperm single and double DNA strand breaks. Experiments on bulls, boars and stallions clearly show that the SCSA test identifies the highest fertile animals. Thousands of measurements on clinical human semen samples also clearly show that when more than 25 percent of sperm (DFI pecentage) in an ejaculate have measurable DNA fragmentation the probability of live birth pregnancy is significantly diminished. SCSA data on percentage DFI and Mean DFI are the same, meaning that the SCSA test measures the total of sperm DNA strand breaks detected with acridine orange staining.





Chapter 20 DNA Damage: Sperm Chromatin Structure Assay Sperm Chromatin Structure Assay Test on its Fortieth Anniversary



Don Evenson



20.1 Introduction


The year 2020 is the fortieth anniversary of the introduction of the concept of “sperm DNA fragmentation” as related to human and animal male factor fertility. This concept was introduced by Donald Evenson in a Science article (1980) that also introduced the first test for its detection, the Sperm Chromatin Structure Assay (SCSA®). Sperm DNA fragmentation is defined as sperm single and double DNA strand breaks. Experiments on bulls, boars and stallions clearly show that the SCSA test identifies the highest fertile animals. Thousands of measurements on clinical human semen samples also clearly show that when more than 25 percent of sperm (DFI pecentage) in an ejaculate have measurable DNA fragmentation the probability of live birth pregnancy is significantly diminished. SCSA data on percentage DFI and Mean DFI are the same, meaning that the SCSA test measures the total of sperm DNA strand breaks detected with acridine orange staining.



20.2 Sperm Chromatin Structure Assay History 1980–2020


In 2011 John Aitken wrote the Forward to the book Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction, edited by A. Zini and A. Agarwal. Aitken wrote: “The impetus to study the composition and integrity of sperm chromatin from a clinical perspective can be traced back to the pioneering studies of Don Evenson, who not only initiated research in this area long before it became fashionable but also pioneered one of the major analytical techniques used in assessment of sperm chromatin, the Sperm Chromatin Structure Assay (SCSA). This assay has now become the industry standard against all other techniques.”


Going forward to 2017 for an assessment on the value of our anniversary finding there is a quote from C. O’Neill and G. Palermo writing: “Many assays have been introduced into male infertility testing over the past two decades regarding varying aspects of spermatozoal competence. However, none have provided more clinically relevant data and insight into male fertility potential than the study of DNA fragmentation in the male gamete” [1].


Going to PubMed and entering our coined term “Sperm DNA fragmentation” yields 2400 manuscripts. Entering “Sperm Chromatin Structure Assay” yields 804 manuscripts. Our pioneering SCSA clinical paper [2] is among the top three papers ever cited from Human Reproduction. The evidence is clear, the SCSA test has a highly significant impact on the outcome for patients struggling with infertility.


The SCSA test has been extensively evaluated and validated for biochemical and fertility outcome soundness. Sperm from many hundreds of animals exposed to a variety of agents and environmental conditions have been analyzed to understand the SCSA test. On this fortieth anniversary of the SCSA test it can be solidly stated that the SCSA test is the most statistically robust assay for understanding the concepts of sperm DNA fragmentation.



20.3 The Sperm Chromatin Structure Assay Test


The original SCSA test [3] used heating of sperm (1000C/five minutes) to open the sperm nuclear DNA strands at sites of single (ss) and double (ds) DNA breaks. These DNA strand breaks are captured by staining with fluorescent acridine orange (AO). AO intercalated into ds DNA fluoresces green while AO stained ss DNA collapses into a crystal that upon exposure to 488 nm light has a metachromatic shift from green to red fluorescence as seen in the Science cover (Figure 20.1).





Figure 20.1 Cover of Science (1980) showing AO stained heated bull sperm.


While many have tried to quantitate sperm DNA fragmentation with light microscopy of AO stained sperm [4], it is now clear that due to many technical problems this can not reliably by done by light microscopy [5] but is, however, very accurately accomplished by flow cytometry [2, 6].


This Science article [3] showed the increased shift from green to red fluorescence in sperm from sub-infertile bulls and humans. Sub-fertile bulls and infertility clinic patients had 2.6x and 1.6x increased mean alpha t (Mean DFI) respectively, relative to highly fertile bulls and men. These data were very encouraging for the prospect of using the SCSA test in the human infertility clinic; however, before using this new test in the highly sensitive area of human infertility, this test was evaluated in numerous ways including toxicology, reproductive biology and animal fertility experiments.



20.4 The Sperm Chromatin Structure Assay Test Protocol


Switching from heating of sperm to the new acid denaturation protocol is done as per the following and has been described [57]. Individual frozen semen samples are thawed in a 370C water bath just until all ice has melted and then immediately diluted with TNE buffer (0.01M TRIS, 0.15 M NaCl, 1 mM EDTA, 4°C) to a final concentration of 1–2 × 106 sperm/mL. A 200 µL sperm suspension is admixed with 400 µL acid solution (0.1 percent Triton X-100, 0.15 M NaCl, and 0.08 N HCI, pH 1.20, 4°C) for 30 seconds followed by addition of 1.20 mL of acridine orange (AO) staining solution (containing 6 µg chromatographically purified AO (Polysciences Inc., Warrington, Pennsylvania) per mL of AO buffer (370 mL of stock 0.1 M citric acid, 630 mL of stock 0.2 M Na2HPO4 disodium hydrogen phosphate , 1 mM disodium EDTA. 0.15 M NaCl, pH 6.0, 4°C) as previously described in detail [6, 7]. Individual samples are placed into a flow cytometer (for our lab an Ortho Diagnostics L30 flow cytometer (FCM)) and after ~2 minutes of hydrodynamic equilibration of sample and sheath flow, 5000 sperm are measured at rates of ≤250 cells/second. All samples are measured independently twice. The mean values of the two independent measurements are then calculated. These mean data are processed through SCSAsoft® software (or equivalent) to produce a clinical report. These reports are sent back to the clinics via a secured WEB address.


Figure 20.2 shows SCSA raw data obtained by the acid denaturation protocol and processed though our SCSAsoft software.





Figure 20.2 SCSA test data. A) Raw data from a flow cytometer showing each of 5000 sperm as a single dot on a scattergram. Y axis = green fluorescence with 1024 gradations (channels) of DNA stainability. X axis = red fluorescence with 1024 gradations of red fluorescence (ss DNA). Axes shown are 1024/10. Dotted line at Y = 75 marks the upper boundary of DNA staining of normal sperm chromatin; above that line are sperm (dots) with partially uncondensed chromatin allowing more DNA stainability (HDS sperm). Bottom left corner shows gating out of seminal debris. B) Raw data from left panel are converted by SCSAsoft® software (or equivalent) to red/red + green fluorescence. This transforms the angled sperm display in left panel to a vertical pattern that is often critical for accurately delineating the percentage of sperm with fragmented DNA. Y axis = total DNA stainability versus X axis = red/red + green fluorescence (DFI). C) Frequency histogram of data from middle panel showing computer gating into percentage DFI and Mean DFI. Bottom box. SCSAsoft calculations of mean of two independent measures of: percentage DFI, percentage HDS, mean DFI and SD DFI. Note: Mean DFI are presented here with range from 0 to 1024 flow cytometer channels. Some studies have shown this in a range from 0 to 1; e.g. 0.22.


Very importantly, all semen samples are measured by exactly the same strict protocol. Prior to measurements, laser focus is accomplished by maximizing red and green fluorescence values of fluorescent polystyrene beads [7]. Also, a positive (high percentage DFI) and a negative (low percentage DFI) semen sample are measured to verify the results as previously established. Then a reference semen sample from a set made up with hundreds of samples of small aliquots stored in LN2 are used to set the red and green photomultipliers tubes to the same (±5/1024 channels) X, Y coordinates; approximately 540/1024 green versus 130/1024 red. This setting allows the capture of high DNA stainable (HDS) sperm. When one set of reference samples is nearly depleted, another set is made, even if from a different individual with different mean red (X) and green (Y) values. This is accomplished by first measuring the previous reference sample, then the new reference semen sample is measured at the same red/green photomultiplier gains and noting the new mean red and green fluorescence values. In this fashion, samples measured years ago can be measured again with the new reference sample and obtain the near exact same results [7].



20.5 Examples of Sperm Chromatin Structure Assay Validation Experiments



20.5.1 Toxicology


Triethylenemelamine, a trifunctional alkylating agent, has a highly negative effect on mammalian spermatogenesis as seen in Figure 20.3. For the purpose of this chapter, two important factors were learned about the SCSA test from our study on TEM treated mice [8].




  1. 1. Fresh sperm and frozen/thawed sperm produce the same near exact results.



  2. 2. Measurements made of freshly isolated sperm at repeated times following toxicant exposure provided the same results as when aliquots of the frozen and stored samples were measured months later; thus showing that the flow cytometer variables can be repeated with exacting results.





Figure 20.3 Epididymal sperm from mice treated with triethylenemelamine (TEM) and over 45 weeks harvested with one fresh sample measured at the time of isolation and frozen/thawed aliquots measured months later.



20.5.2 Human Air Pollution


Since the 1950s the residents of Teplice, Czech Republic, had been exposed to high levels of air pollution generated from the combustion of high-sulfur coal used for local industry and home heating. The air pollution was severe during the winter when climate inversion smog conditions existed. Infertility and miscarriage were a significant problem. The Czech Republic government and the US EPA came in to evaluate the problem. The study protocol had semen sample testing of young men over two years that included both winter-time smog and summer-time clean air exposure. Figure 20.4 shows that the Teplice donors had very poor SCSA percentage DFI data during wintertime smog exposure [9]. While the great majority of semen samples should normally have been in the 5–10 percent DFI range, the majority of these samples were above that level, with 20 percent being in the pathological range above 30 percent DFI. Interestingly, semen samples from Teplice men exposed to the winter-time air pollution had an increase of sperm motility.





Figure 20.4 Percent of men with extent of sperm percentage DFI fragmentation. Twenty percent of men had pathological (>30 percent DFI) levels of DNA fragmentation.



20.5.3 Reproductive Biology


Analysis of monthly semen samples obtained over eight consecutive months from donor men [10] showed that although SCSA data are heterogeneous among men, individual men provide highly repeatable SCSA data from month to month as seen in Figure 20.5.





Figure 20.5 Sperm Chromatin Structure Assay cytograms of semen samples from four donors over eight consecutive months of collections.


Experimental studies on animal reproduction have some great advantages over human studies that require more ethical standards. One of the best means to evaluate the fertility potential of sperm from different animals is done with heterospermic insemination. For example, equal numbers of motile sperm can be mixed from e.g. a black and white bull, and that mixture used to inseminate 100 cows. If 80 black and 20 white calves are born that would clearly show the superiority of the sperm from the black bull. Figure 20.6 shows that SCSA data of percentage DFI and SD DFI are highly correlated with the known competitive index of bulls [11].





Figure 20.6 Bull heterospermic competitive index versus SCSA SD DFI and SCSA percentage DFI of bulls with different phenotypes.


Another study [12] with boars provided data showing that SCSA data are significantly correlated with the number of successful pregnancy outcomes. Also, boars with poorer percentage DFI had fewer pigs/litter likely due to death of embryos as seen in Table 20.1. Semen from 18 sexually mature boars with known fertility information was bred to 1867 females. Boar fertility was defined by farrow rate (FR) and average total number of pigs born (ANB) per litter of gilts and sows mated to individual boars. Fertility data were compiled for 1867 matings across the 18 boars (Table 20.1).


May 5, 2021 | Posted by in GYNECOLOGY | Comments Off on Chapter 20 – DNA Damage: Sperm Chromatin Structure Assay
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