‘Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular sperm’

Juan G. Alvarez

Centro de Infertilidad Masculina ANDROGEN, La Coruña, Spain and Harvard Medical School, Boston, Masachussetts, USA

Email: jalvarez{at}androgen.es

Sir,

I have read with great interest the article by Greco et al., entitled ‘Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa’, published in the January issue of Human Reproduction.

The study shows that DNA fragmentation levels (assessed by the TUNEL test) in testicular sperm from normozoospermic and oligozoospermic males were significantly lower than those found in ejaculated sperm from these same males (4.8±3.6 versus 23.6±5.1%, P<0.001). More importantly, pregnancy rates in ICSI cycles using ejaculated sperm from males with DNA fragmentation levels in semen >15% were significantly lower than those obtained using testicular sperm (5.6% versus 44.4%, P<0.05). With the exception of one sample, all testicular sperm samples had TUNEL test values ≤6% (range 1–6%). In sharp contrast, all ejaculated sperm samples had TUNEL test values >15% (range 15–37%). This suggests that DNA fragmentation is, for the most part, a post-testicular event.

Previous studies have postulated that one of the mechanisms involved in sperm DNA fragmentation is ROS-induced DNA damage during co-migration of mature sperm with ROS-producing immature sperm through the seminiferous tubules and epididymis (Ollero et al., 2001Go; Alvarez, 2003Go; Alvarez et al., 2004Go). In the study reported by Ollero et al. (2001)Go, a strong correlation was found between reactive oxygen species (ROS) production by immature sperm and DNA fragmentation in mature sperm from the same semen samples. This is consistent with the observation that centrifugation of semen samples containing high levels of ROS-producing immature sperm results in significant DNA damage of mature sperm (Twigg et al., 1998Go). This is also consistent with the fact that in vitro exposure of mature sperm to high levels of ROS resulted in significant DNA damage (Aitken et al., 1998Go; Lopes et al., 1998Go). Steele et al. (1999)Go have reported that DNA fragmentation levels (as determined by the COMET assay) are significantly higher in epididymal compared to testicular sperm from patients with obstructive azoospermia.

It is well known that testicular sperm are more vulnerable to DNA damage due to the fact that the protamines of sperm chromatin are not fully cross-linked by disulphide bonds. Completion of disulphide cross-linking takes place in the epididymis (Bedford et al., 1973Go). Therefore, exposure of mature testicular sperm to ROS, produced either by immature sperm or by the epithelial cells lining the epididymis, could result in damage of these sperm before disulphide cross-linking takes place. This is consistent with the recent report by Dalzell et al. et al. (2004)Go who found that testicular sperm are more sensitive to DNA fragmentation following aerobic incubation.

All this evidence strongly suggests that DNA fragmentation could occur during sperm transport from the seminiferous tubules to the epididymis.

Although the number of patients included in the study is relatively small and the results should be independently confirmed in other studies, the data reported by Greco et al. would suggest that DNA fragmentation testing could be of great value in couples with failed pregnancy in two or more IVF cycles. Those couples with DNA fragmentation levels in semen >15% could benefit from ICSI using testicular sperm, and therefore should be offered the option of performing testicular sperm extraction or testicular sperm aspiration. This would apply to normozoospermic males, as well as to males with oligozoospermia. However, since DNA fragmentation levels in semen have been shown to vary with the spermatogenic cycles (Alvarez et al., 2004Go), DNA fragmentation testing should first be performed in several semen samples obtained from at least two consecutive spermatogenic cycles before recommending testicular biopsy. If all samples have DNA fragmentation values >15%, testicular biopsy would be indicated. If one or more of the samples evaluated have values ≤6%, then they should be cryopreserved for use in future IVF cycles.

The authors' suggestion of developing new selection techniques to enrich the sample in ‘healthy sperm’ with low or absent DNA damage is attractive but inconsistent with their hypothesis of the ‘tip of the iceberg effect’ in samples with DNA fragmentation levels >15%.

Finally, since post-testicular sperm DNA fragmentation appears to be related to ROS-induced damage during sperm transport through the seminiferous tubules and epididymis, patients with high DNA fragmentation levels could also benefit from the use of oxygen radical scavengers and antioxidants, as the authors suggested. Preliminary data from our laboratory indicate that administration of diclofenac significantly reduces DNA fragmentation in males with high DNA fragmentation values (Alvarez et al., unpublished data). Diclofenac, in addition to being an anti-inflammatory agent, is also a known scavenger of the hydroxyl radical (Aruoma and Halliwell, 1988Go) and readily crosses the blood–testis barrier.

References

Aitken RJ, Gordon E, Harkiss D et al. (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 59, 1037–1046.[Abstract/Free Full Text]

Alvarez JG (2003) DNA fragmentation in human spermatozoa: significance in the diagnosis and treatment of infertility. Minerva Ginecol 55, 233–239.[Medline]

Alvarez JG, Ollero M, Larson-Cook KL and Evenson DP (2004) Selecting cryopreserved semen for assisted reproductive techniques based on the level of sperm nuclear DNA fragmentation resulted in pregnancy. Fertil Steril 81, 712–713.[CrossRef][ISI][Medline]

Aruoma OI and Halliwell B (1988) The iron-binding and hydroxyl radical scavenging action of anti-inflammatory drugs. Xenobiotica 18, 459–470.[ISI][Medline]

Bedford JM, Bent MJ and Calvin H (1973) Variations in the structural character and stability of the nuclear chromatin in morphologically normal human spermatozoa. J Reprod Fertil 33, 19–29.[ISI][Medline]

Dalzell LH, McVicar CM, McClure N, Lutton D and Lewis SE (2004) Effects of short and long incubations on DNA fragmentation of testicular sperm. Fertil Steril 82, 1443–1445.[CrossRef][ISI][Medline]

Lopes S, Jurisicova A, Sun JG et al. (1998) Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 13, 896–900.[Abstract]

Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, Evenson D, Thomas AJ, Jr and Alvarez JG (2001) Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod 16, 1912–1921.[Abstract/Free Full Text]

Steele EK, McClure N, Maxwell RJ and Lewis SE (1999) A comparison of DNA damage in testicular and proximal epididymal spermatozoa in obstructive azoospermia. Mol Hum Reprod 5, 831–835.[Abstract/Free Full Text]

Twigg JP, Irvine DS and Aitken RJ (1998) Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod 13, 1864–1871.[Abstract]

Submitted on December 31, 2004; accepted on January 25, 2005.





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