Reply: ‘Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa’
1 Centre for Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy and 2 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain
3 To whom correspondence should be addressed. Email: cmendoza{at}ugr.es
Sir,
We have read with interest the letter by Alvarez concerning our recent paper about the use of ICSI with testicular sperm in the treatment of infertility due to sperm DNA damage (Greco et al., 2005a
). This letter contains stimulating ideas for future reasearch into the mechanism, diagnosis and treatment of this pathological condition.
Sperm nuclear DNA fragmentation is known to reduce implantation and pregnancy rates after ICSI, but the resulting embryo handicap remains latent during the first 3 days after ICSI (Tesarik, 2005). Our observation that testicular sperm show a lower incidence of DNA fragmentation and a higher developmental potential than ejaculated sperm in men with elevated sperm nuclear DNA damage supports the hypothesis that the DNA damage mainly occurs after sperm release from the seminiferous tubules. Alvarez cites several other studies supporting this hypothesis and puts forward the idea that incomplete disulphide cross-linking of the protamines of sperm chromatin, a process which is normally completed during sperm passage through the epididymis, is responsible for this phenomenon. This is a plausible explanation, but it has to be underscored that other possible pathophysiological mechanisms may also come into play. These mechanisms are essentially related to functional disorders of Sertoli cells. In a previous work, in which the relationship between caspase activity and germ cell DNA fragmentation was analysed, we showed that those DNA-fragmented germ cells which remain firmly associated with Sertoli cells also display caspase activity, and their plasma membrane externalizes phosphatidyl serine (Tesarik et al., 2004). The Sertoli-associated germ cells thus appear to undergo a classical apoptotic pathway. The externalized phosphatidyl serine is known to be a surface marker of apoptotic cells to be recognized as targets for phagocytosis. In fact, fragmented DNA was detected in the cytoplasm of Sertoli cells clearly outside their own nuclei, indicating that germ cells with fragmented DNA are recognized and actively removed by Sertoli cells (Tesarik et al., 2004). In contrast, DNA-fragmented germ cells that are detached from Sertoli cells do not display caspase activity and do not externalize phosphatidyl serine (Tesarik et al., 2004). It is thus tempting to speculate that the premature detachment of germ cells makes them particularly susceptible to oxidative DNA damage which may begin during their passage through the seminiferous tubules. Because most of the Sertoli-associated germ cells undergoing the classical apoptotic pathway are probably not released from the testis, the DNA damage detected in ejaculated sperm can be supposed to result mostly from non-apoptotic oxidative DNA damage occurring in prematurely released late spermatids.
The recourse to testicular biopsy to recover sperm for ICSI makes it possible to shorten the Sertoli cell-unprotected time-period during which maturing spermatids and sperm are particularly vulnerable to DNA-damaging agents. However, the need for testicular biopsy makes this therapeutic approach more invasive and expensive than ICSI with ejaculated sperm. A search for more conservative treatment alternatives, aimed at the limitation of the extent of DNA damage in ejaculated sperm, is thus warranted. Alvarez has suggested an approach based on the use of diclofenac, a prostaglandin synthesis inhibitor and hydroxyl radical scavenger. Interestingly, earlier work has demonstrated a significant activation of dog spermatogenesis by subcutaneous application of diclofenac for 42 days (Moskovitz et al., 1987). We look forward to seeing the publication of the new data on the application of this drug in males with high sperm DNA fragmentation values announced by Alvarez.
In our clinics we have recently tested another conservative approach to the problem of sperm DNA fragmentation, based on combined oral treatment with two antioxidants, vitamins C and E. In a prospective randomized study we found that the oral treatement with these two antioxidants for 2 months significantly alleviates DNA damage to ejaculated sperm (Greco et al., 2005b). The effect of this treatment on clinical outcomes of ICSI performed with ejaculated sperm from men whose initially elevated DNA fragmentation values return to normal values after treatment is currently under study.
References
Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Franco G, Anniballo R, Mendoza C and Tesarik J (2005a) Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod 20, 226–230.
Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S and Tesarik J (2005b) Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl, in press.
Moskovitz B, Munichor M and Levin DR (1987) Effect of diclofenac sodium (Voltaren) and prostaglandin E2 on spermatogenesis in mature dogs. Eur Urol 13, 393–396.[Medline]
Tesarik J (2005) The paternal effects on cell division in the human preimplantation embryo. Reprod Biomed Online RB 2004/1538.
Tesarik J, Ubaldi F, Rienzi L, Martinez F, Iacobelli M, Mendoza C and Greco E (2004) Caspase-dependent and -independent DNA fragmentation in Sertoli and germ cells from men with primary testicular failure: relationship with histological diagnosis. Hum Reprod 19, 254–261.
Submitted on January 24, 2005; accepted on January 25, 2005.
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