The predictive value of sperm chromatin structure assay
Centro de Infertilidad Masculina ANDROGEN, La Coruña, Spain and Harvard Medical School, Boston, MA, USA
Email: jalvarez{at}androgen.es
Sir,
I read with great interest the Letter to the Editor by Nicopoullos et al. (2005)
entitled ‘The predictive value of sperm chromatin structure assay’, published in the March issue of Human Reproduction, and also the replies by Bungum (2005), Check and Johnson (2005) and Gandini (2005).
Perhaps, before discussing the predictive value of the sperm chromatin structure assay (SCSA) test in assisted reproductive technologies, it would be useful to review the causes and types of DNA damage found in human sperm and the different tests that can be used to assess this damage.
DNA damage in sperm can affect both mitochondrial and nuclear DNA and can be induced by five main mechanisms: (i) apoptosis during the process of spermatogenesis; (ii) DNA strand breaks produced during the remodelling of sperm chromatin during spermiogenesis; (iii) post-testicular DNA fragmentation induced by oxygen radicals, including the hydroxyl radical and nitric oxide, during sperm transport through the seminiferous tubules and epididymis; (iv) DNA fragmentation induced by endogenous endonucleases; and (v) DNA damage induced by radio- and chemotherapy. Of these five mechanisms, perhaps the one that may play a major role in causing sperm DNA fragmentation is post-testicular damage induced by oxygen radicals during sperm transport. This is supported by previous reports demonstrating that DNA fragmentation is higher in epididymal (Steele et al., 1999) and ejaculated (Ollero et al., 2001; Greco et al., 2005) compared to testicular sperm.
DNA fragmentation induced by the hydroxyl radical results in the formation of 8-OH-guanosine and 8-OH-2'-deoxyguanine in a first stage followed by double-stranded DNA fragmentation thereafter (Cui et al., 2000). While DNA damage of the first type could be repaired to some extent by the oocyte, double-stranded DNA damage is irreversible and incompatible with normal fertilization and the development of a viable pregnancy. Since DNA fragmentation values in ejaculated human sperm >10%, as assessed by TUNEL (Benchaib et al., 1993), or >30%, as assessed by the SCSA test (Evenson et al., 1999), are associated with low pregnancy rates, one would think that the remaining 90 and 70% of the sperm respectively could fertilize the oocyte and result in a viable pregnancy. However, in addition to double-stranded DNA breaks, a significant proportion of these sperm could have DNA base modifications of the 8-OH-guanosine and 8-OH-2'-deoxyguanine type. Therefore, the probability that a spermatozoon with normal DNA would fertilize the oocyte would be much lower than that expected from a DNA fragmentation value of 10 or 30% respectively. That is, in addition to the measurable 10 and 30% of sperm with DNA fragmentation, the remaining 90 and 70% of sperm would have some type of DNA damage that is not compatible with the development of a viable pregnancy. This concept has been designated as the ‘iceberg effect’ (Evenson et al., 1999). If, on the other hand, DNA damage is related to single-stranded DNA breaks, like those produced during the process of chromatin remodelling, this type of damage would not be normally expressed after sperm fertilization, since it would require dissociation of both DNA strands during sperm decondensation and male pronuclei formation. Tests such as the SCSA, DNA breakage detection (DBD)–FISH (Fernandez et al., 2000), SCD (Fernandez et al., 2003), Chromomycin A3 (Manicardi et al., 1995) or single cell gel electrophoresis (COMET) (Singh et al., 1988, 1989) require an initial denaturation step in order to detect measurable DNA fragments or potential breaks in the DNA backbone. In contrast, TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) (Gorczyca et al., 1993), in situ-nick translation (ISNT) (Gorczyca et al., 1993) or COMET under neutral pH conditions (Singh et al., 1988), do not require a denaturation step and measure actual single (ISNT, TUNEL and COMET) and double-stranded (TUNEL and COMET) DNA breaks. This may help to explain why threshold values for TUNEL in predicting pregnancy outcome in assisted reproduction treatment are significantly lower than those for the SCSA test. Since the intracellular pH of the oocyte is 
7.0, single-stranded DNA damage would be of little or no consequence for pronuclei formation, since under neutral pH the sperm DNA strands will not dissociate. Therefore, to a first approximation, two types of DNA fragmentation tests can be considered: (i) tests that measure ‘real’ DNA damage, such as TUNEL, ISNT or COMET under neutral pH conditions; and (ii) tests that measure ‘potential’ DNA damage and susceptibility to DNA denaturation, such as the SCSA, DBD-FISH, SCD, Chromomycin A3 or COMET under denaturing conditions (Singh et al., 1989). Tests that measure real DNA damage should have a higher predictive value than tests that measure potential DNA damage. This is supported by Greco et al. (2005), who showed that microinjection of sperm with TUNEL test values >15% resulted in a pregnancy rate per cycle of 5.6% whereas microinjection of sperm with TUNEL test values 
6% in a second ICSI attempt in these same couples resulted in a pregnancy rate per cycle of 44.4%.
It should be pointed out that although the TUNEL test is frequently used to determine apoptosis in cells, TUNEL positivity is not always synonymous with apoptosis, since hydroxyl radical-induced DNA damage also results in double-stranded DNA fragmentation, that can be detected by the TUNEL test (Negoescu et al., 1998).
Another important point is that since >90% of DNA is composed of non-protein-coding regions or introns, the probability that DNA damage will affect protein-coding regions or exons is very low. This would also explain, at least in part, why relatively high levels of sperm DNA fragmentation can result in viable pregnancies.
Metaphase II oocytes can repair, to some extent, DNA damage in sperm after fertilization by pre- and post-replication repair mechanisms (Brandiff and Pedersen, 1981; Genesca et al., 1992). This will depend mainly on the extent of sperm DNA fragmentation and the cytoplasmic and genomic quality of the oocyte. The latter would be expected to be higher in oocytes from younger women. Therefore, the efficiency of the oocyte to repair DNA damage in the spermatozoon would be expected to decrease with female age.
In conclusion, the presence of single-stranded DNA breaks in the paternal genome above a critical threshold, as measured by the SCSA test, may not necessarily lead to failed pregnancy after assisted reproduction treatment. However, the presence of extensive double-stranded DNA breaks and high levels of 8-OH-guanosine and 8-OH-2'-deoxyguanine, like those produced after hydroxyl radical-induced damage during sperm transport through the seminiferous tubules and the epididymis, could severely compromise embryo development. However, even if there is significant DNA damage, the probability that a viable pregnancy will ensue is going to depend on: (i) the proportion of sperm with damaged DNA, the extent of DNA fragmentation per cell and the level of 8-OH-guanosine and 8-OH-2'-deoxyguanine in sperm DNA; (ii) the DNA regions that are damaged (i.e. introns versus exons); and (iii) the ability of the fertilized oocyte to repair this damage. However, unlike DNA fragmentation, the latter cannot be measured. Therefore, the predictive value of sperm DNA fragmentation tests will always have DNA region and oocyte-derived uncertainty factors and, thus, cannot have a 100% negative predictive value, as was originally suggested in the ‘iceberg effect’ model proposed by Evenson et al. (1999). The concomitant determination of DNA fragmentation and nucleotide modifications of the 8-OH-guanosine and 8-OH-2’-deoxyguanine type in sperm DNA may significantly improve the assisted reproduction treatment outcome predictive value of tests that measure DNA fragmentation, especially those that measure double-stranded DNA breaks.
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Submitted on February 24, 2005; accepted on March 10, 2005.
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