Keywords

Introduction

Sperm Chromatin Structure

The ultimate goal of any given spermatozoon is to deliver the paternal genetic information into a mature oocyte during fertilization. To accomplish this essential task for any species, a round cell spermatogonium must go through several divisions and transformations in the testis to become a fully formed spermatozoon (Kerr et al. 2006). Then the spermatozoon leaves the testis to start a journey through the epididymis, a long, single, and highly convoluted tube, to complete its maturation, acquiring some elements needed for fertilization and the ability to move (Robaire et al. 2006). Finally, it must reside in the female reproductive tract, specifically the oviduct, to achieve fertilizing ability, be able to recognize the oocyte, and undergo the acrosome reaction needed to go through the zona pellucida that surrounds the oocyte. This process is called capacitation and involves a series of biochemical and morphological changes that prepare the spermatozoon for fertilization (Yanagimachi 1994; de Lamirande and O’Flaherty 2008, 2012).

To accomplish this long journey through the testicular, epididymal, and female tract environments, the sperm chromatin is transformed into a complex structure, involving the association of DNA with basic proteins called protamines and other elements, forming a toroid structure; this transformation aims to avoid potential damage to the genomic material (Fig. 8.1). During spermatogenesis, histones are replaced by protamines, allowing a tighter compaction of the sperm DNA compared to somatic cells (Aoki and Carrell 2003; Govin et al. 2004). Histones are replaced first by transitional protein (Meistrich et al. 2003) and then by protamines 1 and 2 (P1 and P2) in human spermatids (Aoki and Carrell. 2003; Govin et al. 2004) during spermiogenesis (Kerr et al. 2006). In vitro studies suggest that hyperacetylation, an epigenetic modification of histones, allows the replacement of histones by protamines (Oliva and Mezquita 1982; Oliva et al. 1987, 1990). A cycle of phosphorylation-dephosphorylation occurs in protamines before binding to DNA and during nucleosome maturation (Marushige and Marushige 1978; Papoutsopoulou et al. 1999). Protamines have a high number of positively charged residues, allowing the formation of a highly condensed complex with the sperm DNA that has strong negative charge (Retief et al. 1993; Wouters-Tyrou et al. 1998; Lewis et al. 2003; de Mateo et al. 2011; Martinez-Heredia et al. 2006).

Fig. 8.1
figure 00081

Organization of sperm chromatin structure. The intimate interaction between the DNA strands and protamine and the formation of disulfide bridges (−SS-) among protamines and the incorporation of zinc (Zn2+) make for a tightly compacted structure

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Despite this massive protein exchange, promoting an 85–95 % association of sperm DNA with protamines, 5–15 % remains associated with histones (Gatewood et al. 1987; Zalensky et al. 2002; Balhorn 2007). Several histone isoforms (e.g., H2A, H2B, H3, and H4) and isoform variants are present in human spermatozoa, with histone H 2B being the predominant isoform (Gatewood et al. 1990a); the increased levels of histones or histone variants are associated with abnormal DNA compaction and DNA damage in astenozoospermic infertile men (Zini et al. 2008). Similarly, the change in the P1/P2 ratio due to an increase in P2, together with increased levels of the pre-P2, is associated with sperm DNA fragmentation in infertile men (de Yebra et al. 1998; Torregrosa et al. 2006), and a low P1/P2 ratio has been associated with low pregnancy rates (de Mateo et al. 2008).

The stabilization of sperm chromatin is accomplished, in part, by the addition of zinc (Zn2+) to the sperm nucleus at the initiation of nuclear compaction (Gatewood et al. 1990a; Bjorndahl and Kvist 2009; Kerr et al. 2006). This micronutrient is important for fertility as Zn2+ deficiency induces arrest at spermiogenesis, a decrease in germ cell proliferation, and impairment of sperm motility in different species including humans (Yamaguchi et al. 2009; Croxford et al. 2011). Zn2+ contributes to the stabilization of sperm chromatin by binding to free thiol (−SH) groups and forming Zn2+ bridges among protamines (Gatewood et al. 1987, 1990a; Bianchi et al. 1992; Bench et al. 2000). The stabilization of sperm chromatin is completed by the formation of disulfide (−SS-) bridges among protamines during epididymal maturation (Bedford et al. 1973; Marushige and Marushige 1975; Seligman and Shalgi 1991); in normal human spermatozoa, less than 1.5 % of cysteines are found as reactive –SH (Rousseaux and Rousseaux-Prevost 1995). Increased (Zini et al. 2001) or decreased (Seligman et al. 1994; O’Flaherty et al. 2008; Ramos et al. 2008) levels of free –SH has been observed in infertile men, indicating that under- or overoxidation of –SH is associated with abnormal sperm function. This alteration is due to the improper oxidation of –SH groups in most sperm proteins, including protamines, indicating abnormal epididymal maturation (Bedford et al. 1973; Bedford and Calvin 1974). Our current understanding of the players and the intrinsic mechanisms that induce sperm chromatin condensation during spermiogenesis is poor. It is possible that this task is performed by the nuclear isoform of glutathione peroxidase (nGPX4), which is necessary for protamine thiol cross-linking during sperm maturation (Pfeifer et al. 2001). Mice lacking the nuclear isoform of glutathione peroxidase 4 (nGPX4) have spermatozoa with increased DNA decondensation; however, these animals are viable and fertile (Conrad et al. 2005). More research is needed to reveal the other players in the sperm DNA condensation mechanisms to understand better how the sperm chromatin is shaped during sperm maturation.

Alterations of Sperm Chromatin Structure

The sperm chromatin can be altered and therefore is susceptible to damage at different stages of sperm production and maturation. Starting in the testis, it could be affected by apoptosis during spermatocytogenesis or during chromatin remodeling at the time of spermiogenesis (Kerr et al. 2006). Approximately 50 % of germ cells that enter into meiosis become apoptotic and are removed by Sertoli cells. Sometimes, this process is not as efficient as required and some defective germ cells continue developing and can be found in the ejaculate (Sakkas et al. 1999; Sakkas and Alvarez 2010). It is then possible that spermatozoa carrying apoptotic markers such as Fas, caspase activities, p53, and annexin-V can found in semen (Glander and Schaller 1999; Sakkas et al. 2002; Cayli et al. 2004; Said et al. 2006; Mahfouz et al. 2009). Although there are correlations between apoptotic markers and poor semen quality (Glander and Schaller 1999; Sakkas et al. 2002; Cayli et al. 2004; Said et al. 2006; Mahfouz et al. 2009), some infertile men have spermatozoa with normal morphology and good motility, and it is impossible to discriminate between affected and healthy sperm cells (Lee et al. 2010). Many strategies are being investigated to overcome this problem, but they will not be discussed further in this chapter.

As mentioned earlier, the sperm chromatin remodeling during spermiogenesis is another stage where this structure is susceptible to damage. The replacement of histones by protamines requires nuclease activity that creates DNA nicks to provide relief from torsional tension. This helps to achieve the necessary chromatin arrangement during histone replacement in spermatids in many species, including humans (McPherson and Longo 1992, 1993; Marcon and Boissonneault 2004). A deregulation of this process may promote abnormal chromatin packaging or DNA fragmentation, which can be detected in the ejaculated spermatozoa.

After spermiation, spermatozoa enter into the epididymis to acquire the ability to move and fertilizing ability (Yanagimachi 1994; Robaire et al. 2006). During the journey through the epididymis, which varies among species but in humans is 5–6 days, spermatozoa can be damaged and can show significant DNA fragmentation (Ollero et al. 2001; Greco et al. 2005). This is the result of reactive oxygen species (ROS) action or ROS-modified metabolites; for instance, it is known that hydrogen peroxide produces DNA fragmentation in human spermatozoa (Aitken et al. 1998) and that the radical hydroxyl can attack DNA bases, promoting oxidation and an increase in 8-oxoguanosine (8-oxoG) among other DNA metabolites. Another consequence of ROS attack is the production of abasic sites, which destabilize the double helix and can result in strand breaks (Nakamura et al. 2000).

Drugs and other chemicals can affect human sperm chromatin (Fossa et al. 1997; Tempest et al. 2007; O’Flaherty et al. 2010, 2012). Common lesions found associated with agents are the presence of interstrand cross-linking and chemical adducts. These are highly toxic DNA lesions that prevent translation and replication by inhibiting DNA strand separation (Hurley 2002; Deans and West 2011). This property is convenient when the goal is to eliminate cancerous cells; however, chemotherapy severely affects germ cells (Petersen and Hansen 1999; Gandini et al. 2006). Animal studies suggest that spermatogonia (Marcon et al. 2010, 2011) and Sertoli cells (Meistrich et al. 1982; Marcon et al. 2008) are deeply affected by chemotherapeutic agents.

DNA methylation and histone modifications (e.g., phosphorylation, methylation, and acetylation) are epigenetic changes that occur during spermatogenesis in the spermatozoa; they are important in assuring the development of the future embryo. Thus, any changes in the programming epigenetic modifications during spermatogenesis by different causes (e.g., drugs, diseases) may promote male infertility (Trasler 2009; Hammoud et al. 2011).

Evaluation of Sperm Chromatin Structure

Different assays have been developed to determine sperm DNA or other components of the sperm chromatin; interestingly, each of them measures a certain type of damage, thus having limited use in characterizing the entire sperm chromatin structure. This is important to mention because only a multi-assay approach will allow a proper characterization of this complex structure; however, this type of analysis is limited by the amount of spermatozoa in a given sample (O’Flaherty et al. 2008).

Assays to Determine Sperm DNA Damage

Acridine Orange/Sperm Chromatin Structure Assay (SCSA)

Acridine orange (AO) is a metachromatic probe that binds to single or double strands of DNA, giving a red or green fluorescence, respectively, when it is excited at 470–490 nm (Evenson and Wixon 2005). This property is useful for identifying spermatozoa with denaturated (single-stranded) DNA. Sperm DNA can then be analyzed by fluorescence microscopy or flow cytometry (Evenson et al. 1980; Kosower et al. 1992).

This assay determines the susceptibility of sperm DNA to acid denaturation (pH 1.20); the low pH induces the opening of the sperm DNA strands at the sites of DNA breaks. Then sperm are incubated with AO and the red and green fluorescence is acquired using a flow cytometer (Ballachey et al. 1988; Evenson et al. 2002).

The SCSA is one of the most well-tested assays for studying the sperm chromatin structure and has been used by various laboratories worldwide. Three main sperm populations can be distinguished after analysis of the acquired data: (1) sperm with no DNA damage, (2) sperm with moderate or high DNA damage, and (3) sperm with high AO DNA stainability. Based on these sperm populations, data are expressed as the mean DNA fragmentation index (DFI), as the standard deviation of DFI (SD DFI), as a percentage of DFI (%DFI, corresponding to the percentage of cells outside the main population), and as a percentage of spermatozoa with high green fluorescence or high DNA stainability (%HDS), as an indication of sperm DNA compaction (Evenson and Wixon 2005). The DFI and HDS are the most widely used SCSA parameters for characterizing the sperm chromatin, but the mean DFI and SD DFI are also powerful parameters to determine the presence of sperm DNA damage. For instance, significant sperm DNA fragmentation can be found with the DFI, mean DFI, and SD DFI parameters in semen samples from infertile men, whereas the mean DFI indicated significant DNA damage in samples from Hodgkin’s lymphoma patients with a DFI value similar to that of healthy controls (O’Flaherty et al. 2008).

Comet Assay

The single-cell gel electrophoresis assay, or comet assay, is a common tool in male reproductive toxicology for studying DNA fragmentation in spermatozoa in humans and in animal models (McKelvey-Martin et al. 1997; Haines et al. 1998, 2002; Codrington et al. 2004; O’Flaherty et at. 2008). This assay is based on the electrophoretic migration of DNA fragments from the core of chromatin after a sperm suspension is treated with a buffer with neutral or alkaline pH. These fragments originate from single- and double-strand breaks of sperm DNA. Then the slides are stained with a DNA labeling dye (e.g., propidium iodide, SBYR green), and individual pictures of spermatozoa are taken using a fluorescence microscope. The extension of the sperm DNA damage can be determined using a software that provides the following parameters: percentages of the DNA in the head or the tail of the comet, the tail length, and the tail extent moment (O’Flaherty et al. 2008). It is a very reliable assay to determine sperm DNA damage in humans, particularly in severe olizoospermic samples from infertile men (O’Flaherty et al. 2008) or cancer survivors (O’Flaherty et al. 2010).

TUNEL Assay

Another way to detect single- and double-strand DNA breaks is the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (Sergerie et al. 2005). The 3’-OH openings of single- and double-strand breaks are labeled by the addition of FITC-labeled deoxyuridine triphosphate nucleotides in a reaction catalyzed by deoxynucleotidyl transferase (Schmid et al. 1994; Telford et al. 1994). Labeled spermatozoa are then analyzed by fluorescence microscopy or flow cytometry (Gandini et al. 2000; Muratori et al. 2000, 2003; O’Flaherty et al. 2008). Both the microscopy- and flow-cytometry-based methods have been extensively used to determine sperm DNA damage in human spermatozoa (Gandini et al. 2000; Muratori et al. 2000, 2003; Weng et al. 2002; Marchiani et al. 2007; O’Flaherty et al. 2008).

Sperm DNA Oxidation Determination

The oxidation of sperm DNA generates the formation of 8-hydroxy-2′ deoxyguanosine (8-OHdG) promoting single- and double-strand DNA breaks; this damage is associated with oxidative stress, which is a major component in the pathophysiology of male infertility (Gagnon et al. 1991; de Lamirande and Gagnon 1995; Agarwal et al. 2006; Aitken and Baker 2006; Tremellen 2008; Aitken and Curry 2011; Gong et al. 2012). Therefore, the determination of the 8-OHdG levels in spermatozoa should be an important part of the semiology in infertile men. Immunocytochemistry- and flow-cytometry-based techniques using antibodies or binding proteins that recognize 8-OHdG were used to determine sperm DNA oxidation in spermatozoa from different species, including humans (Chabory et al. 2009; Aitken et al. 2010; Paul et al. 2011). Recently, Cambi et al. (2013) developed a flow-cytometry-based method using an anti-8-OHdG moiety antibody that overcomes the problem of false positives sometimes given by methods using binding proteins (Cambi et al. 2013). In this study, the researchers found negative correlations between levels of 8-OH-G and sperm parameters in a cohort of 94 infertile men, thus demonstrating the clinical relevance of using the detection of 8-OHdG in combination with standard semen analysis for the diagnosis of male infertility.

Sperm Chromatin Dispersion Test

The sperm chromatin dispersion (SCD) assay was developed to determine sperm DNA damage for the diagnosis of male infertility (Fernandez et al. 2003). This is a microscope-based assay that detects sperm DNA fragmentation in samples that were previously acid-denatured. The treated sperm are then stained with a probe that binds to DNA (e.g., DAPI), and pictures are taken to quantify the dispersion area of the DNA using image analyzer software (Fernandez et al. 2003, 2005). The SCD detected significantly more sperm DNA fragmentation in infertile men with varicocele than in fertile controls, thus showing its potential for use in the clinic. It also has the potential advantage of being combined with other assays that could detect other abnormalities in the sperm chromatin (e.g., aneuploidy, DNA oxidation, DNA methylation) (Gosalvez et al. 2011); although this strategy is very appealing, more work must be done to confirm whether these combinatory techniques are possible for studying sperm chromatin structure in humans.

Assays to Evaluate Sperm DNA Compaction

Monobromobimame (mBBr) Thiol Group Labeling

As mentioned earlier, sperm DNA is tightly compacted due to specific interactions among components of the sperm chromatin. The –SH groups present in protamines are important elements in the maintenance of chromatin closely compacted in sperm nuclei (Bedford et al. 1973; Bedford and Calvin 1974). The amount of –SH groups can be determined by labeling them with the fluorescent probe mBBr (Kosower and Kosower 1987). It is important to mention that –SH groups are present not only in protamines within the sperm nucleus but also in the tail; thus it is essential to separate the head from the tail by sonication before labeling the spermatozoa with mBBr (O’Flaherty et al. 2008). The intensity of the mBBr labeling in the sperm head is then determined by flow cytometry (Zubkova and Robaire 2006; O’Flaherty et al. 2008). Parallel samples previously treated with dithiothreitol (DTT) are necessary to determine the percentage of free –SH ((fluorescence of sample/fluorescence of sample with DTT)*100). A high fluorescence intensity value corresponds to a high percentage of free –SH present in the sample and therefore an indication of less sperm DNA compaction.

Chromomycin A3 Labeling

Chromomycin A3 (CMA3) is a fluorochrome that specifically binds to guanosine-cytosine-rich sequences where the protamines are prone to bind; it was observed that CMA3 competes with sperm protamines for binding to the minor groove of DNA (Bianchi et al. 1993; Bizzaro et al. 1998). Thus, an increased CMA3 labeling is an indication of lower protamination in spermatozoa and lower DNA compaction (Sakkas et al. 1995; Bianchi et al. 1996; Lolis et al. 1996; Manicardi et al. 1998; Esterhuizen et al. 2002; O’Flaherty et al. 2008). CMA3 labeling can be determined by either microscopy or flow cytometry, and although some studies have associated increased CMA3 labeling with a high percentage of abnormal spermatozoa (Hammadeh et al. 2001; Franco et al. 2012), others have reported the presence of high levels of CMA3 labeling in morphologically normal spermatozoa (Bianchi et al. 1996; O’Flaherty et al. 2008). This discrepancy could be based on the method used to determine CMA3 labeling (e.g., microscopy or flow cytometry) or teratozoospermia (e.g., strict criteria).

Aniline Blue and Toluidine Blue Assays

The aniline blue (AB) and the toluidine blue (TB) assays are based on the property of these dyes to bind to different components of the sperm chromatin and can be performed with light microscopy, thus with minimum costs at a clinical settings. The AB assay detects histones, which are rich in lysine, and binds to them at low pH (Auger et al. 1990). The AB assay showed a significant correlation between high content of histone, abnormal sperm chromatin, and male infertility. However, the correlation of AB results with other sperm parameters is inconsistent and controversial (Foresta et al. 1992).

TB is a basic planar nuclear dye useful for staining the sperm chromatin. It binds to DNA phosphate residues of sperm DNA in nuclei with loosely packed chromatin or impaired DNA, providing a metachromatic shift due to dimerization of the dye molecules from light blue to purple–violet color (Erenpreiss et al. 2001, 2004; Marchesi et al. 2010; Tsarev and Erenpreiss 2011). Recently, it was suggested that the TB assay could be a complementary tool in semen analysis to diagnose male infertility (Tsarev et al. 2009).

Both AB and TB are simple and inexpensive assays that can be performed with a light microscope and using previously prepared and stored smears. The main disadvantage is that, as a microscope-based assay, the number of spermatozoa to be counted is limited compared to assays based on flow cytometry.

High DNA Stainability

Another parameter that can be obtained with the AO/SCSA is the percentage of spermatozoa with high DNA stainability (HDS), which is associated with low sperm DNA compaction (Evenson et al. 1999; Evenson and Wixon 2005). Accessibility to the double-stranded DNA by AO is increased when sperm DNA is not well compacted, and thus these spermatozoa will show an increased AO green labeling. This sperm population can be visualized on top of a green versus red fluorescence scatter plot (Fig. 8.2). It is noteworthy that those spermatozoa showing high HDS values do not have increased DNA damage and are rather considered to be immature cells due to the high amount of histones retained (Evenson and Wixon 2005).

Fig. 8.2
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Sperm chromatin structure assay scatter plot from fertile and infertile men. Three main sperm populations can be distinguished: (1) main population with normal DNA (green oval), (2) spermatozoa from outside the main population with moderate to high DNA fragmentation (red oval), and (3) spermatozoa with normal DNA showing high DNA stainability (blue box). DFI = DNA fragmentation index, HDS = high DNA stainability

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The assays described previously were used by several laboratories to study human sperm chromatin with the goal of associating their outcome with regular semen analysis (Zini et al. 1999; Spano et al. 2000; Virro et al. 2004; Evenson and Wixon 2005, 2006; Makhlouf and Niederberger 2006; Payne et al. 2005). Different results were obtained in these studies, and there is still controversy as to which is the best approach to evaluate human sperm chromatin. A multiassay approach appears to be more useful for characterizing the sperm chromatin structure in a given semen sample than an individual test (O’Flaherty et al. 2008, 2012).

Sperm Chromatin and Cancer Treatments

There is growing evidence showing the detrimental effects that chemo- and radiation therapy have on male reproduction and, particularly, in the sperm chromatin structure (Fossa et al. 1997; Thomson et al. 2002; Tempest et al. 2007, 2009; O’Flaherty et al. 2008, 2010, 2012; Kenney et al. 2012). The field of oncology has advanced with the design of chemotherapeutic agents and new drug combinations that target cancerous cells with minimal toxic effects on normal cells; but although there are high survival rates over 5-years (80–96 % of cases) in young adults for some malignancies such as testicular cancer and Hodgkin’s lymphoma (Huddart and Birtle 2005; Kopp et al. 2006; Theis et al. 2006; Aben et al. 2012), cancer survivors must suffer another burden, which is the possibility of infertility (Petersen et al. 1994, 1998; Petersen and Hansen 1999; Aben et al. 2012). Moreover, depending on the type of cancer and treatment, childhood cancer survivors may have severe oligozoospermia or azoospermia in adult life (Romerius et al. 2011).

As mentioned earlier, chemotherapeutic agents promote a variety of damage to the sperm chromatin; depending on the cancer treatment, differential changes in components of the sperm chromatin structure may be observed. Patients successfully treated for testicular cancer or Hodgkin’s lymphoma have spermatozoa with normal DNA compaction but with significantly elevated levels of sperm DNA damage 24 months after the end of chemotherapy (Tempest et al. 2007; O’Flaherty et al. 2010, 2012). These findings are an indication that germs cells might have been irreversibly affected due to the treatment, although some components of the sperm chromatin return to normal in new generations of spermatozoa (O’Flaherty et al. 2010, 2012). It is important to mention that there is individual variability among patients in terms of how fast chromatin integrity will be restored. For instance, patients with advanced testicular cancer or Hodgkin’s lymphoma showed high sperm DNA damage, determined by the alkaline comet assay, over a period of 2 years after the end of chemotherapy (O’Flaherty et al. 2010). Concurrently, levels of HDS (SCSA) were higher in cancer patients compared to those of healthy donors, suggesting that poor DNA compaction was still present in sperm from cancer patients during this period of time (O’Flaherty et al. 2012). It is possible that insufficient Zn2+ bridges were formed to stabilize the sperm chromatin (Bjorndahl and Kvist 2009), making this structure still susceptible to acid denaturation in spermatozoa from cancer patients. However, the level of protamination (as measured by CMA3 labeling) and of free –SH in sperm of cancer patients was similar to that in sperm of healthy donors at 18 months after chemotherapy (O’Flaherty et al. 2012). Overall, these data indicate that some components of the sperm chromatin are repaired over time after chemotherapy; however, there is still significant sperm DNA damage and low compaction even in normozoospermic samples from cancer survivors. These findings stress the need to use a complementary approach to evaluate sperm chromatin quality; a single assay may not be sufficient to determine whether spermatozoa from cancer survivors have good chromatin quality (O’Flaherty et al. 2008, 2012).

Sperm Chromatin and Environmental Toxicants

Increasing evidence supports the hypothesis that the exposure to environmental toxicants, including pesticides and air pollution (products from engine combustion and waste incineration), is a cause of high sperm DNA fragmentation (Oh et al. 2005; Rubes et al. 2005). Nonoccupational exposure to pesticides or their metabolites present in the environment, such as 3-phenoxybenzoic acid (3-PBA), the pyrethroid metabolite with the highest detected rate in the general population, is worrisome (Kimata et al. 2009; Couture et al. 2009; Sams and Jones 2012; Fortes et al. 2013). High levels of sperm DNA fragmentation were associated with high levels of urinary 3-PBA in infertile men (Ji et al. 2011). Similar associations were found also with other metabolites of the pesticide chlorpyrifos present in the urine of infertile men (Meeker et al. 2004).

Animal studies suggest that pesticides affect the sperm chromatin structure, promoting a variety of alterations; diazinon, an organophosphorus pesticide (OP), promotes increased DFI (sperm DNA fragmentation) and of CMA3 labeling (low protamination) values along with phopshorylation of protamines in spermatozoa recovered from treated male mice after 8 days following the end of treatment (Piña-Guzman et al. 2005). This study suggests that late spermatids are affected by diazinon, resulting in alteration of the sperm chromatin including increased DNA decondensation, low protamination, and DNA damage. Similar toxic effects were observed in humans; spermatozoa treated with different OPs showed increased levels of DNA fragmentation (Salazar-Arredondo et al. 2008). It is noteworthy that the toxicity of OPs is not equal among them, and in some cases the oxon metabolite is ten times more toxic than the original compound (Salazar-Arredondo et al. 2008).

Air pollutants were associated with low sperm chromatin quality at a level that can be associated with male infertility (Rubes et al. 2005). In this study, men exposed to high levels of air pollution have normal semen analysis (Rubes et al. 2005). This evidence, along with that from other studies in different cohorts of men (i.e., infertile men, cancer patients) showing low or no correlation between sperm chromatin assays and semen analysis (Evenson et al. 1999; Spano et al. 2000; Virro et al. 2004; Rubes et al. 2005; Payne et al. 2005; O’Flaherty et al. 2008), supports the need for a complementary analysis of sperm chromatin quality to better characterize the health of human spermatozoa.

Toxicants that are increasingly looking like potential detrimental compounds for male reproduction are plasticizers and bisphenols, compounds used in the plastic industry. Since the discovery of the properties of these compounds, humans have been progressively more exposed to these compounds, which can leak from plastic containers into food, water, and other liquids. Urinary levels of different phthalate (plasticizer) metabolites suggest that exposure to these compounds is much higher and common that previously suspected (Blount et al. 2000) and they might be associated with sperm abnormalities including poor chromatin quality (Pant et al. 2008). Although other studies suggest low or no association between phthalate metabolites and impairment of sperm quality in humans, there is a need for further research with appropriate epidemiological studies (Meeker 2010) to rule out the possibility that these compounds are responsible for alterations of sperm chromatin quality and other sperm parameters that might explain cases of male infertility.

Conclusions and Future Directions

Based on basic and clinical studies, it is now evident that the human sperm chromatin can be altered by different agents or conditions, some of them still difficult to control. Damaged chromatin may impact negatively on the development of the embryo, inducing miscarriages. Therefore, it is necessary to develop tools to better analyze sperm chromatin quality to assure that the paternal genome is not altered, especially at the time of sperm selection for assisted reproductive technologies (ARTs). Although many tests exist for evaluating individual aspects of the sperm chromatin, there is still a limitation in characterizing its structure using a more global approach. The combination of two or more assays would make it possible to overcome this difficulty. More research is needed to improve the tools that we have today to evaluate this vital element of the spermatozoon. Moreover, large-scale epidemiological studies are necessary to help understand the extent of the exposure to drugs and environmental toxicants. The combination of knowledge harvested by basic and clinical research and data generated by epidemiological studies will serve to design better strategies to detect and possibly isolate sperm samples carrying significant sperm chromatin damage, which will help obtain semen samples that are safe for use in ARTs.