Hum. Reprod. Advance Access originally published online on March 5, 2008
Human Reproduction 2008 23(5):1235-1236; doi:10.1093/humrep/den053
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LETTERS TO THE EDITOR |
Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive of the outcome of ART
1 Institute Marques, Barcelona, Spain 2 Harvard Medical School, Boston, MA, USA 3 Queen's University, Belfast, UK
4 Correspondence address. E-mail: juan.alvarez{at}institutomarques.com
We read with great interest the article recently published by Bungum et al., in Human Reproduction (Bungum et al., 2008
) entitled, ‘Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive of the outcome of ART’.
Two years ago I, Alvarez, published a letter to the editor in this journal commenting on the predictive value of the SCSA test in ART. In that article, I pointed out that DNA damage in sperm can affect both mitochondrial and nuclear DNA and that it can be induced by six main mechanisms: (i) apoptosis during the process of spermatogenesis; (ii) DNA strand breaks produced during the remodelling of sperm chromatin during the process of spermiogenesis; (iii) post-testicular DNA fragmentation induced mainly 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; (v) DNA damage induced by radio and chemotherapy and (vi) damage induced by environmental toxicants. Of these six mechanisms, the one that appears to play a major role in causing sperm DNA fragmentation is post-testicular damage during sperm transport through the epididymis. This is supported by previous reports that demonstrate that DNA fragmentation is higher in epididymal and ejaculated (Ollero et al., 2001; Greco et al., 2005) compared with testicular spermatozoa. More recent reports have confirmed this hypothesis (Suganuma et al., 2005).
In that letter, I also pointed out that, 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 (n-COMET); 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. Tests that measure real DNA damage should have a higher predictive value than tests that measure potential DNA damage.
The main question that arises from the report by Bungum et al. is why DNA fragmentation levels in the pellet of the gradient, as measured by the SCSA test, are not predictive of pregnancy outcome, if these are the actual sperm used in ART? One explanation could be that the actual DNA damage that interferes with embryo implantation and/or the development of a viable pregnancy is related to a DNA property that the SCSA test does not measure. As pointed out above, the SCSA test measures ‘susceptibility’ to DNA denaturation. But, in fact, even DNA fragmentation values in neat semen are not predictive of pregnancy outcome after IVF or ICSI according to the present report by Bungum et al. In contrast, the results reported by Borini et al. (2006) and by Duran et al. (2002), cited by Bungum et al. in the present article under discussion, provide strong evidence for the predictive value of DNA fragmentation values in ART in sperm from the gradient pellet, as measured by the TUNEL test. This is even more significant in the report by Duran et al., where the predictive value of TUNEL was applied to IUI cycles, where a limited number of oocytes are available compared with IVF. That is, while in IVF the probability that a mature oocyte be fertilized by a spermatozoon with intact DNA or that a spermatozoon with damaged DNA fertilize an oocyte with a high DNA repair capacity is relatively high, given the high number of oocytes usually obtained after oocyte retrieval, this probability is much lower in IUI where usually 1 to 2 oocytes are available. But, why may TUNEL test values in the gradient pellet be predictive of pregnancy outcome and not SCSA's? One of the main modes of post-testicular sperm DNA damage is most likely that induced by oxidative stress via the hydroxyl radical resulting in the formation of 8-OH-guanine and 8-OH-2'-deoxyguanosine (8-OHdG) 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 and/or the embryo, double-stranded DNA damage is virtually irreversible and incompatible with the development of a viable pregnancy. The TUNEL and n-COMET tests measure single- and/or double-stranded DNA fragmentation and, therefore, should provide more meaningful biological information concerning embryo's implantation potential than the SCSA test. In addition, DNA fragmentation values, as measured by COMET (Agbaje et al., 2008) and TUNEL are associated to DNA damage of the 8-OH-guanine and 8-OHdG type.
In fact, as predicted by the study of Ollero et al. (2001), the levels of 8-OH-guanine and 8-OHdG in sperm DNA should correlate with the levels of oxidative damage in sperm from the gradient pellet, since ROS-producing immature sperm, isolated from the lower density layers, could induce oxidative damage of the mature sperm, isolated from the gradient pellet, during co-migration through the epididymis. On the other hand, Drevet (2006) has recently postulated that the risk of post-testicular oxidative damage in the epididymis increases from the caput to the cauda and that this damage may be related, at least in part, to glutathione peroxidase content in the lumen and epithelial cells of the epididymis and in spermatozoa, and to the oxygen radical recycling equilibrium beyond the caput epididymis related to sperm protamine and flagellar protein disulfide crosslinking, which also increases from the caput to the cauda epididymis. Those sperm with a lower degree of disulfide crosslinking would be more vulnerable to sperm DNA damage.
In conclusion, additional studies are required to evaluate the predictive value of sperm DNA damage levels in the gradient pellet in ART, but using tests that measure real DNA damage, such as TUNEL by flow cytometry. In addition, in order to give a more complete picture, we should also include measures of strand break precursor damage, e.g. 8-OHdG levels in these sperm. This is especially important in disease conditions, such as diabetes, where oxidative stress is overtly manifested and where this type of oxidative DNA damage may lead to double-stranded DNA fragmentation (Agbaje et al., 2008).
References
Agbaje IM, McVicar C, Schock BC, McClure N, Atkinson AB, Rogers D, Lewis SEM. Increased concentrations of the oxidative DNA adduct 7,8-dihydro-8-oxo-2'-deoxoguanosine in the germ-line of men with type 1 diabetes. Reprod BioMed Online (2008) 16. advance access online.
Bungum M, Spanò M, Humaidan P, Eleuteri P, Rescia M, Giwercman A. Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART. Hum Reprod (2008) 23:4–10.
Borini A, Tarozzi N, Bizarro D, Bonu MA, Fava L, Flamigni C, Coticchio G. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod (2006) 21:2876–2881.
Cui J, Holmes EH, Greene TG, Liu PK. Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain. FASEB J (2000) 14:955–967.
Drevet JR. The antioxidant glutathione peroxidase family spermatozoa: a complex story. Mol Cell Endocrinol (2006) 250:70–79.[CrossRef][Web of Science][Medline]
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Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Franco G, Anniballo N, Mendoza C, Tesarik J. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod (2005) 20:226–230.
Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, Evenson D, Thomas AJ Jr, Alvarez JG. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod (2001) 16:1912–1921.
Suganuma R, Yanagimachi R, Meistricht M. Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI. Hum Reprod (2005) 20:3101–3108.
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