DNA methylation influences chromatin structure and gene expression, and methylation of the cytosine residues by DNA methyltransferase is considered one of the major epigenetic mechanisms that control gene expression and imprinting 1. Hypomethylation of DNA has been shown to be associated with gene transcriptional activity, whereas DNA hypermethylation has been shown to be associated with gene silencing 2,3. In humans, the methylation pattern of the paternally imprinted gene is erased in early fetal life; remethylation is initiated as spermatogonia enter meiosis and is effectively complete by the primary spermatocyte stage of differentiation. It has been shown that sperm pathology within the testicular or the epididymal environment has the potential to disrupt the establishment of normal sperm DNA methylation patterns but spermatogenesis-specific genes can undergo late epigenetic re-programming while maturing in the epididymis 4,5.
Sperm abnormalities have been shown to play a role in the majority of epigenetic defects observed in pregnancies through in-vitro fertilization, linking sperm DNA hypomethylation with reduced pregnancy rates 6. Also, aberrant sperm DNA methylation is more commonly seen in semen samples of infertile men compared with normozoospermic ones 7,8. An underlying mechanism for abnormal sperm DNA methylation in infertile men was identified by DNA sequence variations in the gene encoding the DNA methyltransferase enzyme in infertile men associated with abnormal paternal DNA methylation. Other possible mechanisms for sperm DNA hypomethylation may exist such as defects in the folate/homocysteine pathway and oxidative stress 6. Oxidative stress is detrimental to the spermatozoa, causing damage of sperm DNA and plasma membrane through lipid peroxidation that alters sperm membrane fluidity, leading to dysfunctional metabolism and disrupting acrosome reaction reactivity 9,10.
Moreover, infertile men have substantially more damaged sperm DNA than fertile men, which adversely affects their reproductive outcomes 11,12. Sperm DNA damage is proposed to be due to testicular gonadotoxins, hyperthermia, genital tract infection, varicocele, increased oxidants, aberrant protamine expression, excessive reactive oxygen species (ROS), or abortive apoptosis during spermatogenesis 13–15. Oxidative attacks were demonstrated to form DNA base adducts such as 8-hydroxyl-2′-deoxyguanosine and O6-methylguanine, which interfere with the ability of DNA to act as a substrate for DNA methyltransferases and inhibit methylation of adjacent cytosine residues, resulting in global DNA hypomethylation 16–18.
This study aimed to assess the association of seminal S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in infertile men.
Materials and methods
In all, 95 men recruited from the Andrology Department, University Hospital, were included in this study after Institutional review board approval and informed consent. They were divided on the basis of diagnosis into asthenozoospermia (n=20), asthenoteratozoospermia (n=20), and oligoasthenoteratozoospermia (n=55). They were compared with 22 healthy fertile men as controls. Men with varicocele, leukocytospermia, exposed to radiation, consumed cytotoxic drugs, or smokers were excluded. History taking, clinical examination, and semen analysis were carried out.
Semen samples were obtained in sterile plastic containers by masturbation after 5 days of abstinence. After liquefaction, semen analysis was performed according to the WHO guidelines 19, and sperm morphology was evaluated using a phase-contrast microscope and Spermac stain (Fertio Pro, NV, Beernem, Belgium); leukocytospermia was excluded by a myeloperoxidase staining test 20. The sperm fraction was subjected to assessment of DNA fragmentation and acrosin activity and the seminal plasma was subjected to estimation of malondialdehyde (MDA), SAM, and SAH.
Sperm DNA fragmentation analysis 21
Sperm DNA fragmentation analysis was carried out using the enhanced apoptotic DNA ladder detection kit (BioVision Research Products, Mountain View, California, USA). Sperm pellet with 5–10×105 cells in a 1.5 ml microcentrifuge tube was washed with PBS and centrifuged for 5 min at 500g, and the cells were lysed with 35 μl TE lysis buffer. Five microliters of enzyme A solution was added and mixed by gentle vortexing and incubated at 37°C for 10 min; then, 5 μl of enzyme B solution was added to each sample and incubated at 50°C for 30 min. Also, 5 μl ammonium acetate solution and 50 μl isopropanol were added and mixed well. The DNA pellet was washed with 0.5 ml 70% ethanol and air dried. The DNA pellet was dissolved in 30 μl DNA suspension buffer. Fifteen to 30 μl of the sample was added on a 1.2% agarose gel containing 0.5 μg/ml ethidium bromide in both the gel and the running buffer. The gel was run at 5 V/cm for 1 h. Ethidium bromide-stained DNA was visualized by transillumination with ultraviolet light and photographed (Fig. 1).
Estimation of seminal malondialdehyde 22
The proteins of the seminal plasma were precipitated by adding trichloroacetic acid, which reacts with MDA to form a thiobarbituric acid-reactive product that was measured at 534 nm.
Seminal S-adenosylmethionine and S-adenosylhomocysteine estimation 23
Seminal plasma was mixed 1 : 2 with 0.4 mol/l HClO4 for 30 min and centrifuged at 10 000g for 15 min at 4°C and then the supernatant was filtered. Twenty microliters of the acid extract was used directly for high-performance liquid chromatography (HPLC). SAM and SAH standards (Sigma, St Louis, Missouri, USA) were dissolved in water at a concentration of 1 mmol/l and then diluted with 0.4 mol/l HClO4 to the final concentration for HPLC analysis. Twenty microliters of standard solutions containing 50–11 000 pmol were injected onto the HPLC equipped with a variable ultraviolet detector (Hewlett Packard, 1050 series, Palo Alto, California, USA) set to a wave length of 254 nm. The separation was carried out on a reversed phase column (Hibar, Merck, Darmastadt, Germany). The mobile phase consisted of 40 mmol/l NH4H2PO4, 8 mmol/l 1-heptanesulfonic acid (Sigma), and 18% (v/v) methanol (HPLC grade), pH 3.0, with HCl. HPLC analysis was conducted at a flow rate of 0.7ml/min room temperature on the basis of integration of peak areas compared with the standard calibration curves.
Determination of sperm acrosin activity 24
Gelatin-covered slides were prepared by spreading 20 μl of 5% gelatin (Merck, Darmstadt, Germany) in distilled water on the slides. The slides were then air-dried, stored at 4°C overnight, fixed, and washed in PBS. Purified spermatozoa were diluted 1 : 10 in PBS containing 15.7 mmol/l α-D-glucose. Semen samples were smeared on the prepared slides and incubated in a moist chamber at 37°C for 2 h. The halo diameter around any 10 spermatozoa was measured in phase contrast using an eyepiece micrometer. The rate of halo formation was calculated per slide as the percentage of spermatozoa showing a halo. One hundred spermatozoa were evaluated. An acrosin activity index was calculated by multiplying the halo diameter by rate of the halo formation.
Seminal α-1,4-glucosidase estimation 25
Seminal α-1,4-glucosidase was estimated using a commercial kit (Epi screen; Fertipro, Ghent, Belgium) by assessing the intensity of color change induced by the reaction between α-glucosidase and 0.125 of 0.09% Na-azide added to 0.125 ml thawed seminal plasma. The mixture was mixed well by pipetting, one diagnostic tablet (p-nitrophenyl-α-D-glucopyranoside) was then added, and the mixture was remixed, vortexed for 60 s, and incubated for 4 h at 37°C. Then, 3 ml 0.02 mol/l NaOH was added and centrifuged for 6 min at 3000g. The absorbance value, obtained by reading the supernatant against reagent 2 as a blank, was calculated at 405 nm.
Statistical analysis was carried out using SPSS program version 17 (SPSS Inc., Chicago, Illinois, USA). The parametric data were expressed as mean±SD. The nonparametric data were expressed as median and range. The Mann–Whitney test was used as a test of significance for comparison of two groups. The Spearman rank correlation coefficient (r) was calculated to study the relation between the investigated variables. P value less than 0.05 was considered as significant.
Semen parameters of the different investigated groups are presented in Table 1. Seminal SAM, SAM/SAH ratio, acrosin activity, and α-glucosidase were significantly increased, whereas seminal SAH and MDA were significantly decreased in healthy fertile men compared with the other groups. Sperm DNA fragmentation percentage showed a significant increase in asthenoteratozoospermia and oligoasthenoteratozoospermia men compared with the fertile controls.
Semen samples with sperm DNA fragmentation showed a significant decrease in sperm count, sperm motility, sperm velocity, linear velocity, linearity index, sperm normal morphology, acrosin activity, α-glucosidase, seminal SAM, and SAM/SAH ratio and a significant increase in seminal SAH and MDA compared with semen samples without sperm DNA fragmentation (Table 2).
Seminal SAM and the SAM/SAH ratio showed a significant positive correlation with sperm count, sperm motility, sperm velocity, linear velocity, linearity index, sperm normal morphology, acrosin activity, and α-glucosidase and a negative correlation with seminal SAH and MDA. Seminal SAM demonstrated the inverse significant correlations (Table 3).
Several studies have reported that spermatozoa from infertile men are more likely to express aberrant DNA methylation patterns 6–8. This study describes the abundance of seminal plasma S-adenoylated amino acids in seminal plasma with the amount of aberrations in sperm parameters. Depending on the degree of sperm aberrations in general, there was a trend toward higher levels of S-adenoylated homocysteine and lower levels of S-adenoylated methionine in infertile men compared with the fertile controls. In addition, seminal SAM and the SAM/SAH ratio were positively correlated with sperm parameters. Dhillon et al.26 showed that infertile men are more prone to inefficient folate cycle reconversion of homocysteine into methionine as polymorphisms in their methylenetetrahydrofolate reductase gene are more common. Kelly et al.27 added that the adverse effects of methylenetetrahydrofolate reductase deficiency on spermatogenesis are mediated by alterations in the transmethylation pathway.
There was a significant increase in seminal SAH and MDA in infertile men compared with healthy fertile controls. The link between oxidative DNA damage and hypomethylation was established for somatic cells reporting a link between oxidative DNA adducts and impaired DNA methyltransferase activity 28. Also, the presence of oxidative stress in a significant proportion of infertile men was believed to be a cause of sperm DNA fragmentation, which plays a role in sperm DNA hypomethylation 29,30. Tunc and Tremellen 28 added that oxidative damage to sperm DNA is responsible for sperm global DNA hypomethylation, with a significant correlation between total semen ROS production and sperm DNA methylation. This would imply that spermatozoa themselves are the primary source of ROS production interfering with the DNA methylation process and thus, biologically, intrinsic ROS production within the sperm cytoplasm is more likely to interfere with this process in the adjacent nucleus.
Also, cases with sperm DNA fragmentation were associated with decreased sperm parameters, seminal SAM, and SAM/SAH ratio. Tavalaee et al.31 and Benchaib et al.32 reported a significant negative correlation between sperm DNA fragmentation and sperm DNA methylation, suggesting that hypomethylated spermatozoa are more prone to DNA damage. As normally methylated sperm DNA is less susceptible to DNA damage, it might be hypothesized that DNA methylation protects it from apoptotic and/or oxidative damage, the two principal causes of sperm DNA damage.
Seminal epididymal marker α-glucosidase showed a positive correlation with seminal SAM and SAM/SAH ratio and a negative correlation with seminal SAH level. The epididymis plays a crucial role in the maturation of spermatozoa and their acquisition of progressive motility and fertilizing capacity, with a significant relation between α-glucosidase activity and semen parameters 33,34. Ariel et al.4 and Xie et al.35 added that sperm remethylation is a part of the process of sperm maturation that occurs in the epididymis.
Seminal SAM and SAM/SAH ratio were shown to have a positive correlation with sperm acrosin activity. Chaudhury et al.36 considered acrosin activity a sensitive biochemical marker for the clinical evaluation of unexplained male infertility, whereas Chen et al.37 correlated its activity with semen quality, reflecting aspects not diagnosed by routine semen analysis or acrosome ultrastructure. Therefore, there is an inverse correlation between affected sperm acrosin activity and deleterious factors that could affect sperm DNA integrity such as sperm hypomethylation, DNA fragmentation, oxidative stress, varicocele, or smoking 12,38–40.
A negative link was suggested between sperm DNA methylation status and the likelihood of pregnancy 6,41. A complicating factor in determining the direct effect of sperm DNA methylation on pregnancy outcome is its positive association with sperm DNA integrity but it is not possible to determine whether sperm DNA fragmentation alone or hypomethylation is primarily responsible for pregnancy outcome 42. Le Bouc et al.43 suggested that unfaithful maintenance of DNA methylation marks following fertilization involves the dysregulation of a trans-acting regulatory factor that could be altered by assisted reproductive technology. It has been reported that quality control of intracytoplasmic sperm injection sperm through detection of its epigenetic factors, such as methylated DNA, is essential for reducing its genetic and epigenetic risk 44.
A limitation in this study is that sperm DNA methylation was not measured to correlate reduced SAM or SAM/SAH levels with hypomethylation in the same samples.
It is concluded that alteration in the seminal transmethylation pathway in infertile men is associated with increased oxidative stress, sperm DNA fragmentation and decreased sperm acrosin activity, seminal α-glucosidase.
Conflicts of interest
There are no conflicts of interest.
1. Rampersaud GC, Kauwell GP, Hutson AD, Cerda JJ, Bailey LB, Genomic DNA. methylation
decreases in response to moderate folate depletion in elderly women. Am J Clin Nutr. 2000;72:998–1003
2. Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet. 2004;363:1700–1702
3. Navarro Costa P, Nogueira P, Carvalho M, Leal F, Cordeiro I, Calhaz Jorge C, et al. Incorrect DNA methylation
of the DAZL promoter CpG island associates with defective human sperm
. Hum Reprod. 2010;25:2647–2654
4. Ariel M, Cedar H, McCarrey J. Developmental changes in methylation
of spermatogenesis-specific genes include reprogramming in the epididymis. Nat Genet. 1994;7:59–63
5. Kerjean A, Dupont JM, Vasseur C, Le Tessier D, Cuisset L, Paldi A, et al. Establishment of the paternal methylation
imprint of the human H19 and MEST/PEG1 genes during spermatogenesis. Hum Mol Genet. 2000;9:2183–2187
6. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation
of imprinted loci in sperm
from oligospermic patients. Hum Mol Genet. 2007;16:2542–2551
7. Houshdaran S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ. Widespread epigenetic abnormalities suggest a broad DNA methylation
erasure defect in abnormal human sperm
. PLoS ONE. 2007;2 Art. No. e1289
8. Marques CJ, Costa P, Vaz B, Carvalho F, Fernandes S, Barros A, et al. Abnormal methylation
of imprinted genes in human sperm
is associated with oligozoospermia. Mol Hum Reprod. 2008;14:67–73
9. Stuppia L, Gatta V, Scarciolla O, Colosimo A, Guanciali Franchi P, Calabrese G, et al. The methylenetethrahydrofolate reductase (MTHFR) C677T polymorphism and male infertility
in Italy. J Endocrinol Invest. 2003;26:620–622
10. Zalata AA, Ahmed AH, Allamaneni SSR, Comhaire FH, Agarwal A. Relationship between acrosin activity of human spermatozoa and oxidative stress
. Asian J Androl. 2004;6:313–318
11. Collins JA, Barnhart KT, Schlegel PN. Do sperm
DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril. 2008;89:823–831
12. Sigman M, Zini A. Semen
analysis and sperm
function assays: what do they mean? Semin Reprod Med. 2009;27:115–123
13. Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation
, and effectiveness of antioxidants. Hum Reprod. 1998;13:1429–1436
14. Zini A, Blumenfeld A, Libman J, Willis J. Beneficial effect of microsurgical varicocelectomy on human sperm
DNA integrity. Hum Reprod. 2005;20:1018–1021
15. Aitken RJ, De Iuliis GN, Mclachlan RI. Biological and clinical significance of DNA damage in the male germ line. Int J Androl. 2009;32:46–56
16. Hepburn PA, Margison GP, Tisdale MJ. Enzymatic methylation
of cytosine in DNA is prevented by adjacent O6-methylguanine residues. J Biol Chem. 1991;266:7985–7987
17. Weitzman SA, Turk PW, Milkowski DH, Kozlowski K. Free radical adducts induce alterations in DNA cytosine methylation
. Proc Natl Acad Sci USA. 1994;91:1261–1264
18. Valinluck V, Tsai HH, Rogstad DK, Burdzy A, Bird A, Sowers LC. Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res. 2004;32:4100–4108
19. WHO laboratory manual for the examination of human semen
-cervical mucus interaction. 19994th ed Cambridge Cambridge University Press
20. Shekarriz M, Sharma RK, Thomas AJ Jr, Agarwal A. Positive myeloperoxidase staining (Endtz test) as an indicator of excessive reactive oxygen species formation in semen
. J Assist Reprod Genet. 1995;12:70–74
21. Daniel PT, Sturm I, Ritschel S, Friedrich K, Dorken B, Bendzko P, et al. Detection of genomic DNA fragmentation
during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers. Anal Biochem. 1999;266:110–115
22. Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med. 1993;15:353–363
23. She QB, Nagao I, Hayakawa T, Tsuge H. A simple HPLC method for the determination of S-adenosylmethionine and S-adenosylhomocysteine in rat tissues: the effect of vitamin B6 deficiency on these concentrations in rat liver. Biochem Biophys Res Commun. 1994;205:1748–1754
24. Henkel R, Muller C, Miska W, Schill WB, Kleinstein J, Gips H. Acrosin activity of human spermatozoa by means of a simple gelatinolytic technique: a method useful for IVF. J Androl. 1995;16:272–277
25. Roaiah MMF, Mostafa T, Salem D, El Nashar AR, Kamel II, El Kashlan MS. α-1,4-glucosidase activity in infertile oligoasthenozoospermic men with and without varicocele. Andrologia. 2007;39:28–32
26. Dhillon VS, Shahid M, Husain SA. Associations of MTHFR DNMT3b 4977 bp deletion in mtDNA and GSTM1 deletion, and aberrant CpG island hypermethylation of GSTM1 in non-obstructive infertility in Indian men. Mol Hum Reprod. 2007;13:213–222
27. Kelly TL, Neaga OR, Schwahn BC, Rozen R, Trasler JM. Infertility in 5,10-methylenetetrahydrofolate reductase (MTHFR)-deficient male mice is partially alleviated by lifetime dietary betaine supplementation. Biol Reprod. 2005;72:667–677
28. Tunc O, Tremellen K. Oxidative DNA damage impairs global sperm
in infertile men. J Assist Reprod Genet. 2009;26:537–544
29. Turk PW, Laayoun A, Smith SS, Weitzman SA. DNA adduct 8-hydroxyl-2′-deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase. Carcinogenesis. 1995;16:1253–1255
30. Tremellen K. Oxidative stress
and male infertility
– a clinical perspective. Hum Reprod Update. 2008;14:243–258
31. Tavalaee M, Razavi S, Nasr Esfahani MH. Influence of sperm
chromatin anomalies on assisted reproductive technology outcome. Fertil Steril. 2009;91:1119–1126
32. Benchaib M, Braun V, Ressnikof D, Lornage J, Durand P, Niveleau A, et al. Influence of global sperm
on IVF results. Hum Reprod. 2005;20:768–773
33. Viljoen MH, Bornman MS, van der Merwe MP, du Plessis DJ. Alpha-glucosidase activity and sperm
motility. Andrologia. 1990;22:205–208
34. Peña P, Risopatrón J, Villegas J, Miska W, Schill WB, Sánchez R. Alpha-glucosidase in the human epididymis: topographic distribution and clinical application. Andrologia. 2004;36:315–320
35. Xie W, Han S, Khan M, DeJong J. Regulation of ALF gene expression in somatic and male germ line tissues involves partial and site-specific patterns of methylation
. J Biol Chem. 2002;277:17765–17774
36. Chaudhury K, Das T, Chakravarty B, Bhattacharyya AK. Acrosin activity as a potential marker for sperm
membrane characteristics in unexplained male infertility
. Fertil Steril. 2005;83:104–109
37. Chen XJ, Long XL, Sun XF, Zhang WL, Wu XS, Luo YM, et al. Sperm
acrosin activity helps predict IVF-ET outcome. Zhonghua Nan Ke Xue. 2009;15:16–19
38. Menkveld R, Rhemrev JP, Franken DR, Vermeiden JP, Kruger TF. Acrosomal morphology as a novel criterion for male fertility diagnosis: relation with acrosin activity, morphology (strict criteria) and fertilization in vitro. Fertil Steril. 1996;65:637–644
39. El Segini Y, Schill WB, Kohn FM, Zeid SA, Kamshushy AA, Marzouk S. Assessment of sperm
functions in infertile patients with varicoceles. Andrologia. 2002;34:291–295
40. Smit M, Romijn JC, Wildhagen MF, Weber RFA, Dohle GR. Sperm
chromatin structure is associated with the quality of spermatogenesis in infertile patients. Fertil Steril. 2010;94:1748–1752
41. Cisneros FJ. DNA methylation
and male infertility
. Front Biosci. 2004;9:1189–1200
42. Zini A, Boman JM, Belzile E, Ciampi A. Sperm
DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2008;23:2663–2668
43. Le Bouc Y, Rossignol S, Azzi S, Steunou V, Netchine I, Gicquel C. Epigenetics, genomic imprinting and assisted reproductive technology [Epigénétique, empreinte génomique et aide médicale à la procréation]. Ann Endocrinol. 2010;71:237–238
44. Ge SQ, Kang XJ, Duan F. The genetic and epigenetic defect from the sperm
for intracytoplasmic sperm
injection (ICSI). Yi Chuan. 2010;32:289–294