Letter to the editor |
Reply to: ‘The predictive value of the sperm chromatin structure assay (SCSA®)’—a response from the SCSA inventor
Department of Biology and Microbiology, Box 2140D, Northern Plains Biostress, South Dakota State University, Brookings, SD 57007, USA
1 To whom correspondence should be addressed. E-mail: scsa{at}brookings.net
Sir,
We have followed with great interest several Letters to the Editor and would like to thank the authors for their attention and support for the sperm chromatin structure assay (SCSA®). Several comments were made about the need for clarification regarding the predictivity of the SCSA, which we would like to take this opportunity to address. In a recently conducted meta-analysis we found that the SCSA is significantly predictive for in vivo, IUI and routine IVF methods of fertilization. The meta-analysis indicated that patients were 7.1 times (CI 3.37, 14.91) more likely to achieve a pregnancy/delivery if the DNA fragmentation index (%DFI) was <30% (n = 1962, P = 0.0001) using in vivo or IUI fertilization. When routine IVF alone was considered, couples were 
2.0 times (CI 1.10, 2.96) more likely to become pregnant if their DFI was <30% (n = 375, P = 0.02). A meta-analysis of six studies using ICSI and/or routine IVF showed a non-significant trend where patients were 1.7 times (CI 0.97, 3.14) more likely to achieve a pregnancy/delivery if the %DFI was <30% (n = 322, P = 0.06). These meta-analyses indicate to us and physicians alike that the SCSA should be part of the standard semen analysis to better manage patients’ infertility treatment. Based on current research, if the patient’s DFI remains elevated over time, IUI should probably be skipped and routine IVF or preferably ICSI should be the method of fertilization. The meta-analyses will be updated as more studies become available.
In our original human clinical study (n = 200) (Zinaman et al., 2000
; Evenson et al., 1999), we stated that no pregnancies occurred by carefully timed intercourse when the man’s DFI was >30% in the month of attempted conception. Unfortunately, this has been misquoted as saying that pregnancies were not possible when the DFI was >30%. Correctly stated, somecouples in that study became pregnant in months 4–12 when the man’s mean DFI was >30% in the first 3 months of the study. Thus, a >30% DFI was correlated with a longertime to pregnancy and a higher spontaneous miscarriage rate.
Initial assisted reproduction treatment studies by Larson-Cook et al. (2003) and Larson et al. (2000) found no pregnancies when the DFI was >30%. These studies were referenced by Bungum (2005) and Check and Johnson (2005) in their respective Letters to the Editor. These highly negative predictions of a failed pregnancy outcome with DFI >30% were amplified by a typographical error of a <1% pregnancy rate with assisted reproduction treatment (Evenson et al., 2002) when the calculated rate at that time was <10% (see Evenson and Wixon, in press).
It is important to note that the SCSA protocol has been done in our laboratory precisely the same way in every detail on >100 000 sperm samples and is run under the strictest of laboratory controls. The slightest deviation, as known to us for years and only recently published by Boe-Hansen et al. (2005a,b), may produce erroneous results. Some recent commercial entries into offering SCSA as a diagnostic service have produced results that have not always agreed with our results and at the least offer less comprehensive data than are possible with our SCSAsoft® software. It is also obvious that the success rates of infertility clinics play a vital role in circumventing elevated sperm chromatin damage and this fact certainly has a very important influence on correlations between SCSA values and pregnancy success. It is also important to remember that the classifications of sperm DNA integrity are based on SCSA-derived statistical groups. This view was obvious from data on our early work on cattle where single bulls sired thousands of progeny. Thus, human pregnancies occur in the >30% DFI group as well as not occurring in the <30% group. The SCSA is a test for infertility, not fertility, based on sperm DNA integrity. As pointed out by Alvarez (2005), there are many factors that lead to a viable pregnancy.
To further define the role of the SCSA in the infertility clinic, it is of utmost importance to report in vivo, IUI, routine IVF and ICSI fertilization procedures separately as Gandini (2005) points out. In conclusion, Gandini (2005) stated: ‘. . . no single test . . . can predict the outcome of a complicated, multi-stage process such as human reproduction’ and further, ‘all tests are therefore useful or even necessary to achieve the best description of semen quality, its fertility potential and the outcome of oocyte fertilization’. We certainly agree that, given the complexity of human reproduction, it would be an oversight not to make use of tests that may save emotional and financial costs for infertility patients.
Alvarez (2005) elucidates his understanding of the ‘iceberg effect’ first presented by Evenson et al. (1999) where the statistical DFI groups were first defined. To clarify our position, Evenson et al. (1999) stated: ‘... it is important to discuss that this does not mean that the other 70% of spermatozoa have fully normal chromatin’ and further, ‘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.’ Stronger physical DNA-inducing conditions for sperm DNA denaturation will cause higher percentages of sperm nuclei to cross that threshold (as studied extensively in developing the SCSA but not published); however, no new interpretation was gained by varying the physical conditions. That is, under current SCSA conditions, if 30% of the sperm have enough DNA damage to cross the 30% threshold, the other 70% may have DNA damage to lesser and varying degrees, e.g. ROS-damaged DNA bases (Aitken and Sawyer, 2003). This is in agreement with miscarriage rates being the highest for ICSI procedures in patients whose DFI was >30% (Virro et al., 2004; Check et al., 2005). Alvarez also indicates that the predictive value of sperm DNA fragmentation tests will not have a 100% negative predictive value. We are obviously in agreement with this statement; Evenson et al. (1999) stated, ‘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.’ However, the standard semen analysis measures generally show little correlation to SCSA data. Results from several studies have shown r values ranging from –0.42 to 0.12 for concentration of sperm and DFI values and from –0.60 to 0.21 for morphology and DFI values. Motility showed correlations ranging from –0.59 to 0.17. However, while these correlations may be statistically significant, they may have little biological meaning. The great variability of the r values may be due to variations in the measurements of the standard semen analysis. The highest correlations have usually been between motility and DFI, which which may be explained by oxidative stress damaging both cell membranes, including mitochondrial membranes, and DNA integrity.
Alvarez (2005) indicates that TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) values (10%, Benchaib et al., 1993) for predicting pregnancy outcome are significantly lower than SCSA values (30%, Evenson et al., 1999). The 10% TUNEL threshold value that Alvarez refers to from the Benchaib et al. (2003) article refers to fertilization rate, not pregnancy rate. Benchaib et al. (2003) states later in the same article, ‘No pregnancy was obtained when DNA fragmentation was higher than 20%’ TUNEL threshold value; thus, Benchaib et al. (2003) defined an absolute threshold rather than a statistical threshold. Virro et al. (2004) and Bungum et al. (2004) found no correlation between SCSA values and fertilization rate. A current search of the literature shows that TUNEL threshold values range from 12% (Duran et al., 2002) through 20% (Benchaib et al., 2003; Sergerie et al., 2005) to 36.5% (Henkel et al., 2003, 2004). Note that the latter 36.5% TUNEL threshold established by internationally recognized laboratories is close to the 30% SCSA threshold. In this case, the SCSA may have been the more sensitive assay.
The distinction between ‘real’ [TUNEL and neutral COMET (single cell gel electrophoresis)] and ‘potential’ (SCSA and others) sperm DNA damage is made by Alvarez (2005); however, he does not discuss correlation values found in the literature regarding TUNEL and SCSA data. SCSA data have been shown to be highly correlated with TUNEL data (r = 0.859, P< 0.001) (Gorczyca et al., 1993), strongly suggesting that the SCSA technique adds acridine orange fluorochrome markers at the sites of DNA strand breaks which are the same sites of the enzymatic addition of fluorochromes with the TUNEL assay. These data are supported by correlations between %DFI and %TUNEL-positive sperm from data on bulls (r = 0.78, P< 0.001) stallions (r= 0.65, P< 0.001) and rams (r = 0.84, P< 0.001) (Sailer et al., 1995). This is in agreement with Erenpreiss et al. (2004) who showed significant correlations between SCSA data and TUNEL (r = 0.63, P = 0.005). SCSA and TUNEL were positively correlated (P< 0.05) when homozygous 
-thalassaemia patients with iron overload were evaluated for sperm DNA damage (Perera et al., 2002).
Due to these very high correlations, we believe that the SCSA and TUNEL both measure the same single and double strand DNA breaks. In contrast, neutral COMET measures only double strand breaks (or very closely positioned breaks on opposite strands) whereas alkaline COMET measures single and double strand breaks. However, the alkaline COMET data are compromised by the existence of ‘alkaline-sensitive sites’ in sperm DNA that are not classical single strand breaks. Nevertheless, if a constant number of ‘alkaline-sensitive sites’ were present in all human sperm, then alkaline COMET values above that constant would likely be a measure of classical single strand breaks.
Although TUNEL and SCSA tests likely measure the same type of sperm DNA damage, the thresholds for TUNEL from various laboatories appear to differ significantly as discussed earlier. This is likely due to the observation that there have been many variations of the TUNEL protocol, whereas the Evenson SCSA protocol has had no significant variation (Perreault et al., 2003). Thus, the conclusion by a panel of international experts on methods to measure sperm DNA fragmentation (Perreault et al., 2003) appears to be the same, namely, the SCSA is the most consistent and reliable test with very high levels of statistical robustness.
We are grateful for the continued interest of researchers and clinicians in evaluating the SCSA and its role in the infertility clinic. We appreciate the interest from Nicopoullos’ group and welcome the publication of their study (Nicopoullos et al., 2005).
References
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