Review article Journal of Andrological Sciences 2009;16:77-90 Report from the International Congress of Andrology (ICA) 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic” F. Lanzafame, S. La Vignera*, P. Asero* Centro Territoriale di Andrologia, Azienda Sanitaria Provinciale 8, Siracusa; * U.O.C. Andrologia ed Endocrinologia della Riproduzione, Ospedale Garibaldi (centro), Università di Catania Summary Key words Sperm DNA • Sperm epigenetic • Male infertility • Oxidative stress Several factors are related to sperm DNA damage and not all mechanism are known. Multiple sources have been proposed including abortive apoptosis, abnormal chromatin packaging during the transition from round to elongated spermatids and oxidative stress. Moreover, many enviromental conditions are related to reproductive toxicity, including structural and functional alterations of human sperm and particularly sperm DNA damage. Finally, several evidences suggest that infiammation/infection in the male reproductive tract may impair fertility leading to improve of DNA fragmentation. In the last years, several evidences showed like sperm DNA damage may to related to infertility leading to the insight that the assessment of sperm DNA integrity could be considered a potential new semen quality biomarker. Introduction As reflected by the proceedings of the recent ICA 2009 Satellite Symposium held in Rome, Italy, modern andrology takes a wider responsibility for sperm DNA damage related to male infertility. The ‘poor’ quality of sperm DNA is an important factor affecting male reproductive ability. The presence of DNA breaks in sperm chromatin may decrease the capability of sperm to fertilize both in natural and in assisted procreation 1. Sperm DNA fragmentation may lead to disorders of pregnancy and may have a harmful effect on embryo development 1. The presence of DNA breaks in paternal genome is also one of the factors increasing the risk of genetic defects in offspring 2-14. It seems that because of its extremely strong condensation and consequently its unique structure of chromatin, sperm DNA fragmentation is hardly possible. However, from many clinical and experimental studies it appears that sperm genome may be susceptible to toxic influence from many different endogenous and exogenous factors 2 4 6. The susceptibility is a result of sperm poor ability to repair damaged genetic material, as well as Corresponding author: Paola Asero, Sezione di Endocrinologia, Andrologia e Medicina Interna, Dipartimento di Scienze Biomediche, Ospedale Garibaldi, piazza S.M. Gesù, 95123 Catania, Italia – Tel. +39 340 9691968 – E-mail: [email protected] 77 F. Lanzafame, et al. its insufficient antioxidative defence preventing DNA damage caused by radical oxygen species (ROS). Undoubtly the susceptibility of sperm genome to the damage is associated with biochemical and morphological maturity of sperm, which depends on normal spermatogenesis. These conditions have been discussed during the ICA 2009 Satellite Symposium of Andrology from a panel of expert scientists who exposed the presentations, following reported: 1. environmental hormones and male reproduction (J.P. Bonde, Denmark); 2. gene environmental interaction: the impact of persistent organohalogen pollutants on sperm characteristics and genital malformations (Y. Giwercman, Sweden); 3. cryopreservation of sperm DNA (S. Lewis, Northern Ireland); 4. epigenetic control in male germ cells: the chromatoid body as an RNA-processing center (P. Sassone-Corsi, USA); 5. effect of chemotherapy and folate pathway deficiencies on the sperm epigenome (J. Trasler, Canada); 6. our genome in the male germ line: is it safe? (A. Grootegoed, The Netherlands); 7. clinical significance of sperm DNA fragmentation assays (M. Spanò, Italy); 8. sperm aneuploidy and ART offspring (L. Gianaroli, Italy); 9. Y chromosome rearrangements: their cellular origin and clinical consequences (S. Krausz, Italy); 10.mtDNA and sperm function (J. St John, UK); 11.sperm chromatin packaging and DNA methylation: relevance to ART (D. Carrel, USA); 12.defining sites susceptible to DNA damage within the sperm nucleus: the nuclear matrix connection (S. Krawetz, USA); 13.characteristic histone modifications and timing of histone to protamine switch in Drosophila sperm chromatin (A. Awe, Germany); 14.persistence of DNA damage and its consequence for utagenesis in male germ cells of OGG1 -/- Big Blue mice exposed to benzo(a)pyrene (A.K. Olsen, Norway); 15.cellular mechanism underlying the effects of partenal acrylamide-exposure on preimplantation development in mice (S. Shahzadi, Norway); 16.epydidimal glutathione peroxidase 5 contributes to the maintenance of sperm DNA integrity and to embryo viability (J. Drevet, France); 17.male-to-female sex-ratio is potentially correlated to air pollution levels (J. Hallak, Brazil); 18.analytical investigation on TUNEL/P1 assay for the determination of sperm DNA fragmentation: pitfalls and possible solutions (M. Muratori, Italy); 19.impact of environmental exposure to perfluorinated compounds on sperm DNA quality (L. Governini, Italy); 20.chromomycin A3 staining vs TUNEL assay: different prognostic value on ART outcome (M. Nadalini, Italy); 21.apoptosis and sperm DNA fragmentation in infertile patients with Chlamydia and Mycoplasms infection (S. Alvarez, Mexico); 22.inflammatory mediators induce apoptosis in ejaculated spermatozoa in in vitro conditions (M. Fraczek, Poland); 23.ROS induced damage and its clinical significance (J. Aitken, Australia); 24.diagnostic tests for monitoring scrotal hyperthermia (A. Ledda, Italy); 25.testicular hyperthermia and the pathways leading to DNA breakage (C. Wang, USA); 26.pathways of ROS generation in male germ cells: insights generated by proteomics (J. Aitken, Australia); 27.genital tract inflammation and its consequences on sperm DNA (A. Calogero, Italy); 28.antioxidants and sperm DNA damage (A. Zini, Canada). Origin of DNA damage in spermatozoa There are several mechanisms which can damage sperm DNA. Defective sperm chromatin packaging, Table I. Biological events related to chromatin condensation impairment. Chromatin damage causes References Chromatin structure alterations Evenson et al., 15 1999; Spano et al., 2000 16; Zini et al., 2001 17 DNA fragmentation Hughes et al., 1996 18; Irvine et al., 2000 19 DNA oxidation Shen et al., 2000 20 Protamine deficiency Gatewood et al., 1990 21; Carrell et al., 2001 22; Zhang et al., 2006 23 78 Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic” Table II. Consequences of sperm DNA poor integrity. 1. Fertilization impairment 2. Embryo development disorder 3. Implantation impairment 4. Spontaneous abortion 5. Risk-danger of genetic defects in blastomeres 6. Epigenetic modification errors 7. Cancers 8. Infertility apoptosis and oxidative stress are the most important aetiological factors which disrupt DNA integrity. Sperm chromatin condensation impairment Infertile men have higher levels of sperm DNA chromatin damage than fertile men. DNA chromatin damage are due to several biological events which are reported in the Table I. DNA fragmentation and sperm apoptosis Different pathological condition can lead to DNA fragmentation resulting in germ cell apoptosis. Several type of sperm DNA alteration can occur including whole and segmental-chromosomal aneuploidies, mutations 24, trinucleotide repeat-length variations 25, defects in the imprinting profiles 26 27 and unspecific DNA breaks 28. The pathological condition associated to DNA fragmentation and sperm apoptosis will be discussed following. Oxidative stress Among the principal process that lead to DNA fragmentation and sperm apoptosis there is oxidative stress (OS). In recent years, OS and the role of ROS in the physiopathology of human sperm function and male infertility have been emphasized. Indeed, spermatozoa, from the time that they are produced in the testes to ejaculation and in the female reproductive tract, are constantly exposed to oxidizing environments. They are extremely sensitive to ROS because of their high content of polyunsaturated fatty acids (PUFA) and their inability to repair DNA damages 20 29. Sperm DNA damage is due (in part) to OS. Male infertility is associated with high ROS levels and these are associated with sperm DNA damage. OS can cause both loss of motility and DNA damage in sperm 30-32. There is evidence that infertile men present substantially more sperm DNA damage than fertile men and human sperm DNA damage may adversely affect reproductive outcomes. This is particularly relevant for the couples undergoing to assisted reproduction technologies (ARTs) (these technologies often bypass the barriers to natural selection) since there is some uncertainty regarding the safety of utilizing DNA-damaged spermatozoa for these purposes. Therefore, it is important to identify strategies that may reduce sperm DNA damage. At present, there is some evidence to suggest that antioxidant may be useful for these reasons. Recent studies suggest that oral antioxidants therapies, can protect sperm and improve their function by increasing the antioxidant levels 33-35. Menezo et al. 36 have shown that although vitamins (vitamins C, E, zinc, selenium and β carotene) can reduce sperm DNA fragmentation however, it also led to an unexpected negative effect: an increase in sperm decondensation. Although in vitro studies have demonstrated a beneficial effect of antioxidant supplements in protecting sperm from oxidative DNA injury, the beneficial effect of dietary antioxidants on sperm DNA integrity has not been clearly demonstrated. Sperm DNA damage and pathological conditions Sperm DNA damage can be attributed to various pathological conditions including: some diseases, many environmental conditions, sperm preparation protocols and the last but not least is inflammation/ infection in the male reproductive tract. Some diseases and/or their treatment are related to sperm DNA damage and also cancer is among them. Most common form of cancer affect men on reproductive age. Cancer treatments, which are necessarily based on the use of DNA damaging compounds, are examples of iatrogenic induced sperm DNA damage. Today, the vast majority of childhood cancer patients are treated by chemo or radiotherapy. Among the several long-term complications of oncological treatments in cancer survivors, an important one is certainly sperm DNA damage. The knowledge of the kinetics of the induction and removal of DNA lesions would be important. Frias et al. 37 using multiprobe FISH (fluorescence in situ hybridization) techniques observed that, in Hodgkin’s disease patients, chemotherapy induced a transient increase (up to 5 times) of sperm chromosomal aneuploidy which came back to normal in the following 2-3 years. The same trend has been 79 F. Lanzafame, et al. observed by Robbins et al. 38 in testicular cancer patients, the most frequent malignant disease in young men. Testicular cancer can be treated in 90-95% of cases. Spano et al. 16 evaluated the impact of Chemotherapy (TC) on sperm DNA integrity using both SCSA (sperm chromatin structure assay) and TUNEL (TdT-mediated-dUTP nick end labeling) assay. Semen was collected at specific intervals of time up to 5 years after treatment. Compared with pretreatment values, radiotherapy induced a transient increase in DNA Fragmentation Index (DFI) 1-2 years after treatment and a following normalization after 3-5 years. After chemotherapy, the transient increase at 1 year was less pronounced and DFI was even reduced as compared to pre-treatment values, possibly because of some selective effects. The question if cancer condition per se is associated with a higher rate of defective sperm is still debatable. Among the pathological conditions related to sperm DNA damage, one of the most common is varicocele. Varicocele is one of the main cause of male infertility 39-41; afflicting about the 15% of the general popolation, about the 35% of men with primary infertility and more of the 80% of men with secondary infertility 42 43. Different studies suggest that men with varicocele, even with normal seminal parameters or with a documented previous fertility, risk a progressive loss of testicular function and fertility 44 45. In the last years, studies about the role of OS in male infertility showed that infertile men with varicocele have elevated concentration of sperm-derived ROS 46 47 and different studies showed that this population of men have an increased levels of seminal OS as indicated from elevated ROS levels and a reduced total antioxidant capacity (TAC) suggesting that the spermatic dysfunction should be also associated to the OS 46-51. Moreover, the OS affect the spermatic DNA integrity leading, with elevated frequency, to DNA single and double strand breaks, which often is found in the ejaculate of infertile men 19 52 53-54. It has been hypothisized that spermatic dysfunction shoul be associated with an increase of scrotal themperature concern to venous reflow. Human scrotal/testicular thermoregulation is a complex process that maintains the testes temperature at levels compatible with a normal spermatogenesis. Sinha Hikim et al. demonstrated that mild testicular hyperthermia induces accelerated germ cell apoptosis predominantly via the mitochondrial-depen80 dent death pathway 55 56 hyperthermia lead to DNA breakage and DNA breakage leading to germ cell apoptosis. In fact different studies, in rodents, monkey and men, showed that transient testicular hyperthermia induced DNA fragmentation resulting in germ cell apoptosis. The heat stress is mediated by the stress kinases such as p38 MAPK (Mitogen-activated protein kinase) 57 leading to phosphorylation and inactivation of the pro-survival protein BCL-2, cytochrome c and DIABLO release from the mitochondria, induction of the caspase cascade leading to DNA breakage and germ cell death 58. Cessation of heat treatment results in rapid recovery of spermatogenesis 59. Environmental conditions Recent studies suggested that exposure to particular compounds is associated with reproductive toxicity, including structural and functional alterations of human sperm and particularly sperm DNA damage. Environmental factors include a lot of organic and chemical compounds, chemical solvents, pesticides, heavy metals; polychlorinated biphenyls (PCBs) and other persistent organic pollutants (POPs). Among these agents endocrine disruptors chemicals (EDCs) may mimic, block or modulate the normal system of hormones. EDCs are hormonally active compounds, that can interfere with the endocrine system. EDCs are estrogen-like and/or anti-androgenic chemicals that have potentially effects on male reproductive axis, resulting in infertility. EDCs may mimic, block or modulate the: synthesis; release; transport; metabolism; binding; elimination of hormones 42. Much attention has focused on changing trends in male reproductive parameters in relation to EDC exposure and an association has been postulated between the global decline in semen quality and the increased exposure to these environmental chemicals 60. Among these, the perfluorinated compounds (PFCs), are suspected to play an adverse effects on human fertility. perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) are the most well know members of the PFC chemical group. PFCs are characterised by chains of carbon atoms, which are strongly bonded with fluorine atoms. These are very persistent in the environment and extremely resistent to degradation. Indeed, these are heat stable and repel both water and oil 61. PFCs have been discovered as global pollutants; they are widely used as industrial surfactants and in various commercial applications. The properties that makes PFCs so effective in industrial product are also the reason why they tend to persist in the environment. The acute toxicity of Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic” PFCs is moderate, but they have various potential health effect. We are always expose to PFCs and the levels of these compounds in our bodies may never be completely removed. Moreover, PFCs can play a potential developmental toxicity 62. These molecules infact are involved in alterations of hypotalamus-hypophisis-gonadal axis. A number of recent studies suggest possible associations of exposure to PFCs with altered functions of reproductive system. PFOS and PFOA act as endocrine disruptors with direct effects on sex hormone levels, resulting in lower testosterone and higher estradiol levels 63 64. Governini et al. 65 evaluated PFC contamination in three different organic samples: whole blood, seminal plasma and sperm cell fraction, from subfertile men. PFC contamination was present in 42.40% of subjects. Particularly, PFC contamination was significantly more frequent among men with abnormal semen parameters in comparison to normospermic subjects. The results obtained in this study suggest a negative interference of PFCs on sperm quality in accord to the study of Muratori et al. where, the results showed that PFC “positive” subjects had the highest degree of DNA fragmentation and diffuse sperm structural anomalies, mainly related to apoptosis 66. In conclusion, PFCs could induce spermatogenetic imbalances by raising the levels of sperm DNA fragmentation and decreasing sperm quality. POPs can have hormone like or antihormone like effects and can act via sex hormone receptors such as AR. AR mutations are responsible to profound effects on phenotype. AR gene polymorphisms concern CAGrepeat and GGN-repeat. Polymorphisms for this gene are frequent (> 1% of the population) and can be related to small effects on phenotype. So far, only limited evidence link GGN number to male subfertility; one study indicated an increase of infertility in males with GGN ≥ 24 67, while any correlation has been reported between CAG length and sperm concentration 68. Indeed, no correlation between POP and sperm number have been shown 69, whereas CAG length modifies the impact of POP on sperm number 68. Lichtenfels et al., demonstrate that male-to-female sex ratio is potentially correlated to air pollution levels. Increased levels of air pollution and a decrease in the human and mouse male-to-female ratio in São Paulo, Brazil has been reported 70. In conlusion, AR polymorphisms and dioxin receptor related (AHRR) genes were shown to modify the effect of POPs regard to sperm concentration as well as Y:X ratio. Another toxic environmental factor is Benzo(a)pyrene (BaP) which is believed to induce both bulky DNA adducts (NER) as well as oxidative DNA lesions. BaP is known to cause DNA adducts, and is also believed to increase ROS mediated oxidative DNA damage. The latter is well documented in vitro, but very scarce data exist from in vivo studies. The study of Olsen et al. 71 obtained specific information on the susceptibility of each spermatogenic cell stage. By exposing the mice to BaP and waiting for 120 days before sacrifice, the isolated epididymal sperm emanated from exposed stem cell spermatogonia, reflecting the susceptibility of these cells. By this procedures, have been obtained specific information about stem cell spermatogonia (120 days), differentiating spermatogonia (45 days), primary spermatocytes (31 days), round spermatids (16 days), and spermatozoa (5 days). The results achieved from this study showed that environmental agents have negative effects on spermatozoa. Agents such as BaP leads to spermatozoa containing DNA lesions and to spermatozoa containing de novo germ line mutations. These effects are seen both in the wild type, and the repair system deficient mouse line studied. Sperm with excessive DNA damage retain the ability to fertilize oocytes, but the normal development of the early embryo is often compromised, resulting in reduced fertility or developmental toxicity. Shahzadi et al. 72 studied the effects of acrylamide, a toxicant that humans are exposed to, via heat-treated starchrich food. Acrylamide is a germ cell mutagen inducing clastogenic effects in cells of the male germline. Mating acrylamide-exposed males with unexposed females has been shown to result in reduced fertility and to induce pre and post-implantation loss. In this study has been valuated the measurements of acrylamide-induced DNA damage in individual sperm and the induction of stress responses in the early embryo. This study is part of a larger project, the primary objective of which is to clarify mechanisms underlying negative effects of environmentally induced paternal DNA modifications on early embryo development. Epididymal spermatozoa were isolated at different time, following acrylamide exposure for analysis of DNA lesions by the Comet assay. The results indicate that the highest amount of damage in cauda sperm results from an exposure seven days earlier, a time point which has been associated with induction of preimplantation loss. The Comet assay showed an increased damage levels in the majority of exposed cauda sperm. In contrast to testicular cells, no Formamidopyrimidine DNA-glycosylase (fpg)-sensitive DNA lesions (representing oxidated purines) were observed in sperm following in vivo 81 F. Lanzafame, et al. acrylamide exposure. Paternal acrylamide exposure influenced the early embryo cleavage rate and induced the expression of DNA damage response proteins like γH2AX and p53. Sperm preparation protocols Sperm cryopreservation is routinely used in a variety of circumstances including assisted reproduction, preradiation or chemotherapy treatment, as “fertility insurance” for men undergoing vasectomy and for storage of donor semen until seronegativity for HIV and hepatitis is confirmed. It is also used for storage of sperm retrieved from azoospermic patients who have undergone testicular sperm extraction or percutaneous epididymal sperm aspiration to prevent the repetition of invasive biopsies. and in many other circumstances in seminology laboratories. Studies investigating the effects of cryopreservation have largely been limited to conventional parameters. Reports include changes in sperm morphology including damage to mitochondria, acrosome and flagellum. Unfortunately, the proportion of fully functional sperm that retain intact membranes, tail and mitochondrial activity after freeze-thawing is often low. Sperm motility has also been shown to be particularly sensitive to such damage but, while it is generally accepted that sperm motility is reduced by cryopreservation, the mechanism by which this occurs is, yet unclear. However, since up to 40% of post thawed sperm are still motile, these spermatozoa could be utilized. Only few trials have been performed to improve cryopreservation procedures and strategies for its limitation to more meaningful diagnostic parameters such as DNA quality. Sperm DNA is particularly susceptible to oxidative damage due to high content of PUFA acting as substrates for ROS and for its lack of repairing mechanism. Only few studies have been evaluated the effects of cryopreservation on nuclear and mitochondrial DNA. The results of these studies are often conflicting as following reported. Watson et al. found that sperm chromatin structure remains stable 73; Spano et al. and Royere et al. found chromatin alterations 74 75 and Hammadeh et al. found abnormal DNA condensation 76 77. A study lead by Donelly et al. 78 found that the DNA fragmentation percentage significatilly increase in human semen after cryopreservation (Freeze-thaw) respect to fresh semen. The increase is more evident in infertile respect to fertile men. Zribi et al. 79 demonstrated that cryopreservation increased DNA fragmentation, the obtained results 82 showed that in infertile men, following cryoinjury, the susceptibility of morphologically abnormal sperm to damage is three fold higher than normozoospermic samples. Besides DNA fragmentation correlates with abnormal morphology. Freezing process lead to intracellular ice formation and dissolution and also osmotic stresses caused by dehydration 80-82; freezing caused a GSH (glutathione) depletion (-78%) and a reduced SOD activity (-50%) 83 the addition of thiols (GSH, Cysteine, NAC) in post-thaw samples prevented the H2O2 mediated motility decrease 84 and also pyruvate, metal chelators or catalase avoided this phenomenon 85 86. Antioxidant addition, during cryopreservation have fertility benefits, infact α tocopherol and ascorbate increase viability and SOD and Catalase improve embryo numbers 87. During thawing process, The rapid warming prevents recrystallization 74. Novel methods of freezing and novel cryoprotectants can lead to reduction of cryoinjury to DNA, examples of those are vitrification and ultra rapid freezing. The processes involve solidifying liquids without crystallization, and embryo cryopreservation by vitrification 88-91 in liquid nitrogen slush 92. Isachenko et al. 93 evaluated DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification and the results showed that human spermatozoa vitrified with cryoprotectant have a lower percentage of DNA fragmentation respect to human spermatozoa vitrified without cryoprotectant. Moreover, the results demonstrated that the percentage of DNA fragmentation is lower in vitrified spermatozoa respect to slowly-frozen spermatozoa and the use of cryoprotectant lead to a reduction of DNA fragmentation in human spermatozoa after swim up, slow-frozen and vitrification procedures. Inflammation/infection in the male reproductive tract A number of evidences suggest that urogenital inflammation may impair fertility. The inflammation of the male accessory glands has been also called male accessory gland infection (MAGI) 94. MAGI are very frequent pathological conditions and this-term include: • uncomplicated forms: prostatitis; • complicated forms: prostato-vesiculitis (PV); prostato-vesiculo-epididymitis (PVE) and epididymal-orchitis (EO). Many studies investigated the effects of MAGI on Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic” sperm conventional sperm parameters (density, motility, normal form), the results of these studies are often conflicting probably because many studies published up to the 1990s have not relied on the above-mentioned, generally accepted classification of prostatic diseases 95 96 and WHO criteria for MAGI, thus making it impossible to differentiate between patients with a real inflammatory prostatic process and the so called ‘pelvic pain patients’ without inflammatory reaction in prostatic secretions. Even recent investigations are contradictory 97. The inflammatory-infective process may affect reproduction through the functional alteration of the male accessory glands because they do not produce adequate amounts of nutrients and antioxidant compound or they produce and release ROS and cytokines that alter the microenvironment where spermatozoa develop and mature. Particularly, cytokines are mediators of the host response to inflammation, that may modulate the activities of prooxidative and scavenger systems which also brings about the burst of ROS. La Vignera et al. 98 evaluated seminal plasma cytokine levels in prostatitis and prostato-vesiculitis and have been found significatively elevated levels of TNF (tumor necrosis factor)-α, IL-6 and IL-10 in prostatitis and prostato-vesiculitis respectively compared to normal controls. TNF-α and sperm function were also assessed in some other studies and the results showed that seminal plasma TNF-α concentration is higher in patients with bacterial or mycoplasma infections compared to normal controls. Seminal plasma TNF-α concentrations are increased in patients with leukocytospermia 99 while TNF-α and IL-6 significantly reduces total and progressive motility in a time and dose-dependent manner 100. Moreover, they increase nitric oxide production in a dose-dependent manner too 100. Only few studies evaluated TNF-α effects on sperm DNA integrity. Said et al. 101, measured the exposure of human spermatozoa to varying concentrations of TNF-α and infliximab. The results showed that spermatozoa quality declined following incubation with TNF-α in a dose (100, 300, 400, 500 pg/ml, and 2.5 mg/ml) and time-dependent manner. Sperm motility and DNA integrity were higher in the samples incubated with TNF-α plus infliximab than in the samples treated with TNF-α only. These results suggest that, exposing spermatozoa to pathological concentrations of TNF-α can result in significant loss of their functional and genomic integrity 101. Moreover, the inflammatory-infective process may affect reproduction also through the germ-spermatozoa interaction. The association of germs commonly observed in genital infections include Chlamydia trachomatis (C. trachomatis) and Mycoplasms. Some studies about C. trachomatis infection showed that it is associated with sperm parameters alterations 102-105. other studies did not confirmed the above mentioned results 106-109. This discrepancy could be also ascribed to the different methods used to identify the presence of C. trachomatis (cultural test and immunological test). Data obtained from Hossenzadeh 110 suggest a negative effect on sperm motility and viability. The apoptotic process related with elevated levels of ROS and the damage in the spermatic DNA have been associated with the presence of infectious agents and may have a crucial influence on fertilization process 111. In a study of Alvarez et al. 112 thirty seven patients, grouped according to the microbiological results Mycoplasms (+)/C. trachomatis (-), Mycoplasms (-)/ C. trachomatis (+) and Mycoplasms (+)/C. trachomatis (+) and a control group of eleven normozoospermic healthy men have been evaluated. The authors assessed sperm apoptosis and spermatic DNA fragmentation. High levels of DNA breaks were associated with the presence of C. trachomatis and Mycoplasms. These, in combination, shown a synergistic effect as inducers of damage to the DNA structure. There is an association between high levels of apoptosis/DNA sperm fragmentation and seminal infection by C. trachomatis and Mycoplasms. Satta et al., 113 found that Infection with C. trachomatis EB at the concentration of 300, 3000 or 30000 CFU had no effect on the percentage of sperm with PS translocation after 6 h of incubation A significant effect on this parameter was instead observed after 24 h of incubation. C. trachomatis also caused a statistically significant increase in the percentage of sperm with DNA fragmentation both after 6 and 24 h of incubation. The effect reached a statistical significance only at the highest concentration (30000 EB) of C. trachomatis EB after 6 h of incubation, whereas it was effective at 3000 EB after 24 h of incubation. The molecular mechanism by which C. trachomatis induce sperm death is still unknown, some studies showed that LGV and serovar E extracted LPS cause a marked reduction of sperm motility and a concomitant increase of sperm death; Escherichia Coli extracted LPS is about 500 times less powerful, C. trachomatis EB have a stronger effect, suggesting that other molecules may play an additional role 110. An experimental model 114 evaluated LPS (0.1 mg lipopolysaccharide (LPS)/kg body weight/day for 7 days) administered Imprinting Control Region (ICR) mice. In this study, were examined sperm concen83 F. Lanzafame, et al. tration and motility in the cauda epididymis as well as immunohistochemical localization of Fas and FasL and germ cell apoptosis. Sperm concentration and motility markedly fluctuated in LPS-treated mice. The increase of apoptotic cells was common in all post-LPS treatment groups, with a peak at 24h after LPS injection. In contrast to the lack of Fas immunoreactivity in control testes, LPS-treated groups demonstrated Fas in many germ cells, especially spermatocytes and spermatids. Moreover FasL immunoreactivity was positive for some Sertoli cells, Leydig cells and germ cells in both control and LPStreated mice. In conclusion These results suggest that the Fas/FasL system mediates apoptosis of germ cells in LPS-treated mice testes 114. Hakimi et al., 115 studied Lipid A (the toxic component of LPS) and 3-deoxy-D-manno-octulosonic acid (Kdo), a C. trachomatis LPS component. The results demonstrated that Lipid A and Kdo cause sperm death. Particularly, Lipid A and Kdo co-incubation causes apoptotic-like sperm death mediated by caspase activation 115. It is clear that the inflammatory reactions are inevitably associated with the oxidative stress phenomenon. The results obtained from Fraczek et al. 116, suggest that the bacterial invasion or local tissue damage is accompanied by infiltrating leukocytes, especially phagocytic cells connected with the production and release of large amounts of ROS and biologically active substances, such as proinflammatory cytokines. The cytokines, mediators of the host response to inflammation, may modulate the activities of the prooxidative and antioxidative systems which brings about the rush of ROS. When the ROS amount overwhelm the potential of the antioxidative defence, peroxidative damage to spermatozoa occurs, which in turn lead to sperm dysfunction that results in infertility 116. Taking into consideration that DNA fragmentation and apoptosis may result from ROS-dependent activity, and that the inflammatory process is inseparably connected with OS, Fraczek et al. 116, analyzed the effect of selected inflammatory mediators on DNA fragmentation of different sperm subpopulations and, particularly, three sperm subpopulations exposed to inflammatory mediators (leukocytes, proinflammatory cytokines, bacteria). The results indicated that, during the male reproductive tract infections, bacteria are the most important inducers of DNA fragmentation in ejaculated spermatozoa. 84 Epigenetics and genetics sperm DNA alterations As before exposed it is clear that different pathological condition lead to the increase of OS and definitely, the OS phenomenon is inevitably associated with epigenetics and/or genetics alterations. Deficiencies on sperm genome and epigenome could be related to inpairment on male reproduction. DNA methylation; chromatin modifications (acetylation or methylation of specific residues on chromatin); post-translational modifications of histone aminoterminal (histone code), are examples of epigenetic processes and all this modifications are heritable through cell division, yet reversible. The biological roles of DNA methylation are genomic imprinting; X-chromosome inactivation and repression of transposons. Altered methylation profiles are associated with human diseases, some examples of these are cancer imprinting diseases, Angelman Syndrome, Prader-Willi Syndrome; Beckwith-Wiedemann Syndrome and immunodeficiency Syndrome (ICF syndrome). The DNA methylation patterns start in germ cells and have important implications for health and disease. Some studies show that epigenetic defects can be also associated with infertility. Marques et al., found that oligozoospermia is associated with methylation of H19 and methylation of PEG1 117Kobayashi et al., found that oligozoospermia is associated with methylation of H19 and also with GTL2 SNRPN, PEG1, LIT1, ZAC 118. Epigenetic program start in gametes (prenatal and postnatal phases) can be perturbed and ‘epimutations’ can be transmitted. Altered chromatin modifications can also be associated with infertility. Protamines are sperm small nuclear basic proteins (50-57 amino acids), with a high content of: arginine (50%) and cysteine (10%) 119. In spermatozoa somatic cell histones are replaced by the protamines to yield chromatin condensation and related to compact sperm cell size and transcriptional quiescence 119. The protamines are a varied family of small argininerich proteins that are synthesized in the late-stage spermatids of many animals and plants and bind to DNA, condensing the spermatid genome into a genetically inactive state. Human sperm protamine are protamine-1 (P1) and protamine-2 (P2) Vertebrates have from one to fifteen protamine genes per haploid genome, which are clustered together on the same chromosome. Comparison of protamine gene and amino-acid sequences suggests that the family evolved from specialized histones through prot- Report from the ICA 2009 Satellite Symposium “Sperm DNA Damage: from Research to Clinic” amine-like proteins to the true protamines. Structural elements present in all true protamines are a series of arginine-rich DNA-anchoring domains (often containing a mixture of arginine and lysine residues in non-mammalian protamines) and multiple phosphorylation sites. The two protamines found in mammals, P1 and P2, are the most widely studied. P1 packages sperm DNA in all mammals, whereas protamine P2 is present only in the sperm of primates, many rodents and a subset of other placental mammals. P2, but not P1, is synthesized as a precursor that undergoes to proteolytic processing after binding to DNA and it also binds a zinc atom, the function of which is not known. P1 and P2 are soon phosphorylated after their synthesis, but after binding to DNA most of the phosphate groups are removed and cysteine residues are oxidized, forming disulfide bridges that link the protamines together. Both P1 and P2 have been shown to be required for normal sperm function in primates and many rodents. Aoki et al. 120 evaluated P1/P2 ratio distribution for fertile men and infertility patients and they showed a novel population of infertile males with a reduced P1/P2 ratio. Aberrant P1/P2 ratios arise from an abnormal concentration of P1 and/or P2, either of which is associated with male infertility. Moreover, in human spermatozoa, there are different evidences that abnormal protamine expression is associated with a low spermatozoa concentration and motility 121-124. Histone retention mainly involve three gene classes: developmental promoters; miRNAs and imprinted clusters. Particularly, the results obtained from Aoki et al. 120 about TH2B showed its high relative amount of retention in sperm (2% of genome bound to Th2B, previously hypothesized to “poise” sperm genome 124. Rangasamy et al. 125 demonstrated that H2Az too, with a low amount, is retained in spermatic DNA. In conclusion the results obtained from different studies indicate that abnormal protamination is associated with diminished sperm quality including elevated DNA damage and altered methylation of some imprinted genes. Moreover, sperm from fertile men retain about 5% of the genome bound to histones, and an incomplete protamination is associated with increased histone retention; furthermore, in fertile men, retained histones are generally associated with demethylated DNA regions in promoters of embryonic developmental genes, imprinted genes, and miRNAs. Particularly, TH2B is retained in gene groups linked to sperm biology and H2Az with pericentromeric heterochromatin. Additionally, developmental genes are generally bound with H3K4me3 and H3K4me2 activating modifications. During sper- miogenesis, human sperm chromatin undergoes replacement of nuclear histones by protamines, resulting in a highly condensed DNA. The replacement of nuclear histones by protamines has both the goals, to pack DNA tightly and to protect DNA from chemical and physical damage (i.e. ROS). One of the potential consequence of abnormal protamination is the greater susceptibility to DNA damage 126-128. Both protamine deficiency and sperm DNA damage are related to decreased reproductive ability of men, in natural as well as in assisted reproduction 128 129. Tarozzi et al. 129 carried out a study to evaluate the impact of abnormal protamination on sperm parameters, sperm DNA fragmentation, ART outcome and the link between protamine deficiency and seminal plasma antioxidant ability. A significant negative correlation was found between abnormal protamination and sperm parameters, including sperm DNA integrity (p < 0.001). The results showed a close relationship among sperm protamination, fertilization and pregnancy only in IVF procedures (p = 0.004 and p < 0.04, respectively) while ICSI demonstrated a correlation between DNA integrity and pregnancy (p = 0.031). Finally, authors found a negative correlation between chromatin underprotamination and seminal plasma antioxidant ability (p < 0.01). The results of this study underline that, despite sperm abnormal protamination and DNA fragmentation are positively correlated, they affect the reproductive outcome in different manners. Particularly authors found good prognostic value of CMA3 analysis only in IVF, whereas DNA fragmentation analysis is of prognostic value only for ICSI outcome. The results obtained, also provided data supporting the idea of a relationship between a defective antioxidant system activity and the impairment of chromatin packaging. The DNA fragmentation induced in sperm emerging from the testes as a result of aberration chromatin repackaging and aberrant free radical generation. Damage can be trasmitted because mutagenic change present in fertilizing spermatozoon lead to the failure of the oocyte to repair the DNA damage. Among them, Y chromosome deletion are frequent because Y chromosome is very susceptible to DNA deletion 130. The Y chromosome structure predisposes it to deletions, segmental duplications and to copy number variations. Three regions on the Y chromosome called azoospermia factor (AZF) regions (AZFa, AZFb, AZFc) contain genes involved in spermatogenesis 131. Their complete removal following deletions causes impairment of spermatogenesis. Therefore, they are considered clear causes of spermatogenic failure 132. Two others structural 85 F. Lanzafame, et al. variations are known on the Y chromosome with potential effect on spermatogenesis, the partial AZFc deletions/duplications and the copy number variation of the TSPY cluster. Among partial AZFc deletions the gr/gr deletions has been reported as a genetic risk factor for impaired sperm production 133. Given that the number of genes removed by the gr/ gr deletion is half that the classical AZFc deletion its effect on spermatogenesis seems to be milder. Thus we should consider it as a co-factor for spermatogenic impairment with variable penetrance. On the other hand its pathogenic effect may also be related to distinct Y-linked or non-Y genetic factors. Krausz et al. have carried out a multicenter study to investigate the contribution of Y-chromosomal factors to the extensive and puzzling phenotypic variation exhibited by gr/gr deletion carriers, which ranges from normal spermatogenesis to azoospermia 134. The genetic factors examined included the known AZFc structural variants associated with this deletion (removal of different DAZ and CDY1 gene copies), deletion followed by duplication and the more general Y chromosome background. The obtained results, showed significant geographic differences in the deletion subtypes distribution, which may affect the outcome of case control association studies in different geographic areas. However, the phenotypic variation of gr/gr carriers in men of European origin seems to be largely independent from the Y chromosomal background 134. Conclusion The integrity of the genetic material is a prerequisite for normal fertilization and transmission of paternal genetic information. Finely tuned differentiation steps of the male germ cell line, during its active lifetime, ensure this goal and any derailment is thought to be crucial, especially during spermiogenesis when repair system fade leaving DNA more vulnerable. Causes of sperm DNA damage are numerous, and not all mechanism are known. Multiple (testicular and extra-testicular) sources have been proposed including abortive apoptosis, abnormal chromatin packaging during the transition from round to elongated spermatids, and OS. Genetic defects that may be transmitted through sperm are different and include whole and segmentalchromosomal aneuploidies, mutations, trinucleotide repeat-length variations, defects in the imprinting profiles and unspecific DNA breaks. Sperm DNA damage can be associated with reduced rates of fertilization in vivo, by natural conception or intrauterine insemination. Less unequivocal informa86 tion exists regarding the link between DNA strand breaks and in vitro fertility. 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