Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

Sperm DNA fragmentation seems to affect embryo post-implantation development in ICSI procedures: high sperm DNA fragmentation can compromise 'embryo viability', resulting in pregnancy loss.

The formation of mature spermatozoa is a unique process involving a series of meioses and mitoses, changes in cytoplasmic architecture, replacement of somatic cell-like histones with transition proteins and the final addition of protamines, leading to a highly packaged chromatin (Kumaroo et al., 1975; Goldberg et al., 1977; Poccia, 1986). Mature mammalian spermatozoa contain high percentages of protamines, for example, human and mouse sperm nuclei contain more than 85% and 95% protamines in their nucleoprotein component, respectively (Gatewood et al., 1987; Bellvé et al., 1988; Debarle et al., 1995). In mice, protamines allow the mature sperm nuclei to adopt a volume 40 times less than that of normal somatic nuclei (Ward and Coffey, 1991).In many mammals, spermatogenesis leads to the production of spermatozoa that appear highly homogeneous in form and function. However, in humans, it is apparent that there are large differences between the form and function of spermatozoa among males and within the ejaculate of an individual. Classically, analyses of the differences in spermatozoa among men have been measured by examining sperm concentration, motility and morphology. Although this analysis gives a broad clinical insight, it does not explain why and where differences originate.For a number of years, many laboratories have concentrated on analysing differences in sperm populations by examining chromatin structure. These studies have shown that the major factor affecting chromatin packaging in ejaculated human spermatozoa appears to be linked to faulty or incomplete protamine deposition during spermiogenesis. In numerous studies, spermatozoa from infertile men were found to exhibit sperm chromatin anomalies related to the deposition of protamines (Balhorn, 1982; Foresta et al., 1992; Belokopytova et al., 1993; de Yebra et al., 1993). These anomalies range from altered ratios of protamine 1 and 2 (Balhorn et al., 1988; Belokopytova et al., 1993) to the complete absence of protamine (de Yebra et al., 1993).During the 1990s, several groups have analysed the sperm nucleus further by examining the integrity of the DNA in mature human spermatozoa. This review summarizes the accumulated knowledge concerning DNA damage in mature human spermatozoa and how this may be related to male infertility. Furthermore, we will speculate on how and why DNA damage may originate in certain males and how it influences the genetic project of a mature spermatozoon. DNA packaging in mammalian spermatozoaThe chromatin contained in the nuclei of mature mammalian spermatozoa is an extremely compact and stable structure. Sperm DNA must be organized in a specific manner (Fig. 1), which differs substantially from that of somatic cells, to achieve this unique condensed state (Poccia, 1986; Ward and Coffey, 1991). This DNA organization not only permits transfer of the very tightly packaged genetic information to the egg, but also ensures that the DNA is delivered in such a physical and chemical form that the developing embryo can access the ...

In this study we investigated whether morphology and chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We examined unfertilized oocytes, using the flnorochrome Hoechst 33342, to determine whether a relationship exists between failure of fertilization and sperm chromatin quality. Sperm chromatin packaging quality was assessed using the chromomydn A 3 (CMA 3 ) fluorochrome, and the presence of DNA damage in spermatozoa, using in-situ nick translation. Normal males present sperm parameters with a normal morphology of >20%, CMA 3 fluorescence of <30% and exhibit endogenous nicks in <10% of their spermatozoa. When patients were separated according to these values no difference was observed hi their fertilization rates after ICSL When the unfertilized ICSI oocytes were examined, we found that patients with CMA 3 fluorescence of <30% and nicks in <10% of their spermatozoa had only 17.5 and 21.6% respectively of their unfertilized oocytes containing spermatozoa that remained condensed. In contrast, patients with higher CMA 3 and nick values had a significantly higher number, 412 and 48.9%, of their unfertilized oocytes containing condensed spermatozoa. Sperm morphology did not show any such pattern. The percentage of spermatozoa which had initiated decondensation in unfertilized oocytes was not influenced by morphology, CMA 3 fluorescence or nicks. In light of these results we postulate that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation after ICSI and result in failure of fertilization.