2 Review of Literature
2.5 Sperm DNA fragmentation assessment
Life offspring is built up halfway from a paternal genome provided by the spermatozoon.
Therefore, it is crucial to examine the quality of sperm DNA (deoxyribonucleic acid) in the form of fragmented DNA. It is well understood that poor quality of sperm chromatin structure is associated with male infertility and adverse effects on reproductive events (SHARMA et al.
2004; FERNÁNDEZ et al. 2005; ZINI and LIBMAN 2006). The DNA fragmentation may cause reproductive problems such as low fertilization rates, decreased preimplantation development, increased abortion rates and incidence of disease in the offspring (LEWIS and AITKEN 2005). Fecundity decreases gradually when more than 30 % of spermatozoa with DNA damage are identified (AGARWAL and SAID 2003). At present, the conventional
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analysis of semen quality which considers parameters of volume, color, odor, pH, and sperm density of the ejaculate as well as the total count, motility, morphology and viability of spermatozoa, is the most common procedure for evaluating male fertility. However, the semen analysis in routine laboratory work may be insufficient when dealing with subfertile individuals. For example, gamma irradiated spermatozoa exhibit severe DNA damage, although membranes and organelles as well as motility remain undisturbed (SILVA and GADELLA 2006). Therefore, advanced methods are needed to distinguish infertile from fertile males (ZINI and LIBMAN 2006). Spermatozoa from infertile males may show a variety of disturbances of chromatin organization and structure, such as single- or double-strand DNA-breaks, aneuploidy and/or chromosome Y-microdeletions (BARROSO et al.
2000). Therefore, the integrity of sperm DNA is accepted as a new parameter with regard to semen quality and fertility prognosis which cannot be detected by standard parameters. In order to be able to obtain a better fertilization outcome, it is essential to evaluate the sperm quality before use of spermatozoa for further purposes (SILVA and GADELLA 2006).
Sperm chromatin in mature mammalian spermatozoa is a very compact, highly stable and complex structure (AGARWAL and SAID 2003). The compact packaging of the sperm DNA filament is produced by DNA-protamine complexes (SAKKAS et al. 1999; FUENTES-MASCORRO et al. 2000). Chromatin condensation includes a replacement of histones with protamines. Protamine P1 is found in most of the mammals (QUERALT et al. 1995), and P2 is found in humans, mice and horses (reviewed by WYKES and KRAWETZ 2003). This sperm chromatin structure shields the genetic material of the spermatozoa during their transport through the male and female reproductive tract (WARD 2010). Sperm DNA damage might occur during spermatogenesis or during sperm transportation, due to oxidative stress by free-radicals (reactive oxygen species; ROS) and abortive apoptosis (SAKKAS et al. 1999;
AGARWAL and SAID 2003; ZINI and LIBMAN 2006). However, the mechanism of mammalian sperm damage has not been exactly comprehended (LEWIS and AITKEN 2005).
39 2.5.1 Evaluation of sperm DNA fragmentation
At the present time, research is focused on the influence and the involvement of sperm DNA fragmentation on male fertility, since hidden anomalies may be present at the chromatin level (KAZEROONI et al. 2009). There are various methods to evaluate sperm DNA fragmentation such as acidic aniline blue staining (HAMMADEH et al. 2001; MUKHOPADHYAY et al.
2011; MANAS et al. 2013; SELLAMI et al. 2013), toluidine blue staining (ERENPREISA et al. 2003; TSAREV et al. 2009; NAVA-TRUJILLO et al. 2011; SASIKUMAR and DAKSHAYANI 2013), acridine orange staining (CHOHAN et al. 2004; VARGHESE et al.
2009; YAGCI et al. 2010; REPALLE et al. 2013), sperm chromatin dispersion test (FERNÁNDEZ et al. 2003; SHANMUGAM et al. 2014), chromomycin A3 staining (IRANPOUR et al. 2000; NASR-ESFAHANI et al. 2009; SIMÕES et al. 2009;
ZANDEMAMI et al. 2012), DNA breakage detection-fluorescent in situ hybridization (FERNÁNDEZ et al. 1998; FERNÁNDEZ et al. 2000; NACCARATI et al. 2003; MURIEL et al. 2004), in situ nick translation assay (IRVINE et al. 2000; TOMLINSON et al. 2001), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling assay (SHARMA et al. 2010; FUNARO et al. 2013; FAURE et al. 2014; FEIJÓ and ESTEVES 2014), single-cell gel electrophoresis or COMET assay (RIBAS-MAYNOU et al.
2012; SIMON et al. 2013; RIBAS-MAYNOU et al. 2014; SARIÖZKAN et al. 2014), sperm chromatin structure assay (NIU et al. 2011; NOVOTNY et al. 2013; DUALE et al. 2014;
RAMÓN et al. 2014) and high-performance liquid chromatography (De IULIIS et al. 2009;
AITKEN et al. 2010; THOMSON et al. 2011; CAMPI et al. 2013). AGARWAL et al. (2009) summarized the different techniques as shown in Table 1. The most apparent method is the assessment of the presence of actual molecular DNA strand breaks (SHARMA et al. 2013).
However, the most often used assays for evaluating sperm DNA fragmentation are SCSA, Comet assay, TUNEL assay and SCD test (RIBAS-MAYNOU et al. 2013).
Recently, the Halomax® test kit, the latest version of the sperm DNA fragmentation tests, had been developed. It is the improved version of the SCD test, and enables better sperm chromatin quality and tail preservation (FRASER et al. 2010). The Halomax® provides the test kits for sperm DNA analysis in various species such as mouse, dog, ram, bull, donkey,
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horse and boar. In this study, Halomax® Sui was used, which is according to the manufacturer specific to boar spermatozoa. However, in pigs, the scoring criteria of sperm DNA fragmentation using the Halomax® test kit are different from human scoring criteria.
Fragmented pig sperm DNA exhibits large haloes of chromatin dispersion around the sperm head, whereas non-fragmented pig sperm DNA exhibits a compact sperm head or a small halo (PÉREZ-LLANO et al. 2010; PARILLA et al. 2012; ALKMIN et al. 2013; MEN et al. 2013).
Since a strong correlation between the improved SCD-test and the Sperm Chromatin Structure Assay (SCSA) is obvious, improved SCD is now recognized as an appropriate replacement for SCSA (EVENSON and WIXON 2005; FERNÁNDEZ et al. 2005). The measurement of SCSA is based on flow cytometry. In the study of FERNÁNDEZ et al. (2005) the very high correlation coefficient between improved SCD and SCSA (R = 0.85) has been reported. The special advantage of the improved SCD technique is that it can be used with small numbers of about 500 spermatozoa per sample, whereas SCSA needs at least 5,000 spermatozoa for an examination. As a consequence, the improved SCD is more beneficial for low numbers of sperm cells (EVENSON and WIXON 2005).
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Table 1: Various assays for evaluating DNA integrity of spermatozoa (adapted from AGARWAL et al. 2009)
Assay Parameter Method of analysis
Acidic aniline blue stain
8-OHdG, 8-hydroxy-2-deoxyguanosine; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA,
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