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Seminal plasma S-adenosylmethionine and S-adenosylhomocysteine associations in infertile men

Zalata, Adela; El-Baz, Aymana; Othman, Gamala; Hassan, Ashrafb; Mostafa, Taymourc

doi: 10.1097/01.XHA.0000407148.53472.9a
Original articles

Purpose To assess seminal plasma S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) association in infertile men.

Materials and methods In all, 95 infertile men were investigated and classified in terms of diagnosis into asthenozoospermia, athenoteratozoospermia and oligoathenoteratozoospermia compared with 22 healthy fertile controls. They were subjected to semen analysis, assessment of seminal SAM, SAH, malondialdehyde (MDA), acrosin activity, α-glucosidase, and sperm DNA fragmentation.

Results In healthy fertile men, seminal SAM, SAM/SAH ratio, α-glucosidase, and acrosin activity were significantly increased, whereas seminal SAH, MDA, and sperm DNA fragmentation percentage were significantly decreased compared with other investigated infertile groups. Seminal SAM and SAM/SAH ratio showed a significant positive correlation with semen parameters, acrosin activity, and α-glucosidase and a significant negative correlation with seminal MDA.

Conclusion 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.

aDepartments of Medical Biochemistry

bDermatology & Andrology, Faculty of Medicine, Mansoura University, Mansoura, Egypt

cDepartment of Andrology & Sexology, Faculty of Medicine, Cairo University, Cairo, Egypt

Correspondence to Dr Taymour Mostafa, MD, Department of Andrology & Sexology, Faculty of Medicine, Cairo University, Cairo 11562, Egypt Tel: +20 105 150 297; fax: +2 23654133; e-mail:

Received August 12, 2011

Accepted September 24, 2011

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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.

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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.

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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).

Figure 1

Figure 1

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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.

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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.

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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.

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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.

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Statistical analysis

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.

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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.

Table 1

Table 1

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).

Table 2

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).

Table 3

Table 3

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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.

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Conflicts of interest

There are no conflicts of interest.

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DNA fragmentation; male infertility; methylation; oxidative stress; semen; sperm

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