The relationship among sperm global DNA methylation, telomere length, and DNA fragmentation in varicocele: a cross-sectional study of 20 cases

ABSTRACT

Varicocele pathophysiology is related to increased oxidative stress, which might result in loss sperm DNA integrity as well as in genomic instability. Sperm telomere shortening and loss of global DNA methylation are the main features of genomic instability, leading to cell senescence and death, whereas sperm DNA fragmentation (SDF) characterizes the loss of chromatin integrity. We hypothesize that sperm genomic stability and DNA integrity is reduced in infertile men with moderate and large-sized varicoceles, thus being candidate markers of sperm quality in varicocele-related infertility. Here, we assessed the sperm global DNA methylation, telomere length, and SDF in men with and without clinically palpable varicoceles. While the rates of SDF and telomere length were not statistically different between varicocele patients and controls, global sperm DNA methylation seems to be lower in men with varicocele (49.7% ± 20.7%) than controls (64.7% ± 17.1%). A negative correlation between SDF and sperm motility and a positive correlation between sperm morphology and telomere length were observed. Our results suggest that varicocele may result in genomic instability, in particular, global DNA hypomethylation. However, a large sample size may confirm these findings. The understanding of the molecular mechanisms involved in the pathophysiology of varicocele-related infertility may help to better select candidates for varicocele repair.

KEYWORDS:

• Varicocele
• sperm DNA fragmentation
• DNA methylation
• telomere
• DNA stability

Introduction

Varicocele is a major cause of male infertility affecting 19–41% of men at reproductive age (Jarow 2001). The disease is characterized by an abnormal dilatation of scrotal veins in the pampiniform plexus (Miyaoka and Esteves 2012) and it can impair spermatogenesis through several distinct pathophysiological mechanisms. Current evidence supports oxidative stress as a central element in the pathophysiology of varicocele-related infertility, although other mechanisms including scrotal hyperthermia, hormonal disturbances, testicular hypoperfusion and hypoxia as well as backflow of toxic metabolites are potential contributors (Naughton et al. 2001; Agarwal et al. 2012; Sheehan et al. 2014). The semen of infertile men with clinically palpable varicoceles is often abnormal, including alterations in sperm count, motility, and morphology (Xue et al. 2012; Kadioglu et al. 2014; Agarwal et al. 2016). Furthermore, an increase proportion of sperm with damaged chromatin is commonly seen in ejaculates of such men (Zini and Dohle 2011; Wang et al. 2012; Roque and Esteves 2018), presumably due to overproduction of reactive oxygen species (ROS) and decreased seminal antioxidant capacity (Shiraishi et al. 2012). It has been also suggested that the decrease in sperm chromatin integrity may result from loss of global DNA methylation and sperm DNA fragmentation (SDF) (Tavalaee et al. 2015). DNA methylation, along with other epigenetic markers, such as histone modification, non-coding RNAs, and telomere, play a critical role in maintaining chromatin stability (Ng and Bird 1999; O’Sullivan and Karlseder 2010).

A complex molecular pathway strictly related to environmental condition seems to contribute to the development of varicocele and may aggravate this condition (Benoff and Gilbert 2001; Santana et al. 2017; Hassanin et al. 2018). DNA methylation, in particular, is important for maintaining telomere integrity since hypomethylation of subtelomeric regions relates to telomere shortening as well as cell senescence and apoptosis (Vera et al. 2008; Yehezkel et al. 2008). Telomeres are one of the most important biological markers of genomic stability, protecting against DNA damage, and ensuring the correct chromosome alignment during DNA replication (Farr et al. 1991; O’Sullivan and Karlseder 2010). Excessive telomere erosion is associated with decreased sperm concentration, motility, and vitality (Ferlin et al. 2013; Cariati et al. 2016; Rocca et al. 2016), and telomere shortening has been suggested as an important marker of male infertility (Thilagavathi et al. 2013). DNA methylation and telomere integrity are not only important for sperm genomic stability but also for normal fertilization and embryo development (Dada et al. 2012; Jenkins and Carrell 2012; Thilagavathi et al. 2013).

In this pilot study, we postulate that sperm global DNA methylation and telomere length are altered in infertile men with varicocele (Figure 1). Furthermore, we hypothesize that (epi)genomic stability lead to poor seminal quality and SDF in men with varicocele-related infertility.

Figure 1. An altered testicular environment with factors such as thermal and oxidative stress, hypoxia and toxic metabolite accumulation can lead to reduced testicular function and cause alterations in the sperm DNA, like telomere shortening and DNA methylation changes. Alterations in DNA are related to loss of genomic stability, which can directly affect seminal quality, fertilization, and correct embryonic development.

Sperm DNA fragmentation

SDF has a negative correlation with sperm concentration, motility, and morphology, leading to poor seminal quality that may result in infertility (Shen et al. 2002; Peluso et al. 2013). The loss of sperm DNA integrity may be due to genetic factors, such as gene mutation, chromosomal abnormalities, and altered gene expression. Other important modulators of DNA damage are the environmental and lifestyle, including exposure to toxins and pollutants, lifestyle (cigarette smoking, use of illicit and licit drugs with gonadotoxic potential), exposure to chemo-radiation, conditions that increase the DNA fragmentation and alter cell cycle division (Andrabi 2007; Aitken et al. 2009).

Men with palpable varicocele may have increased SDF, especially those with poor seminal quality (Saleh et al. 2003; Enciso et al. 2006; Smith et al. 2006; Blumer et al. 2008; Zini and Dohle 2011; Santana et al. 2015). The increase in SDF is usually accompanied by changes in the markers of oxidative stress and sperm function (Roque and Esteves 2018).

The main processes associated with the physiopathology of varicocele (thermal and oxidative stress, hypoxia, and increased apoptosis) are likely interconnected (Hassanin et al. 2018) and SDF seems to be their end point (Esteves and Agarwal 2016). The major sources of ROS in men with varicocele are the epididymis cells, endothelial cells of the dilated pampiniform plexus, developing germ cells, Leydig cells, macrophages, and peritubular cells (Esteves and Agarwal 2016). Excessive ROS leads to peroxidation of the sperm plasma membranes (Aitken et al. 2014) and results in a cascade of events that generates nuclear and mitochondrial DNA damage, including base modification, cross linking, and chromatin remodeling (Aitken and De Iuliis 2010; Sakkas and Alvarez 2010).

Sperm DNA methylation

DNA methylation controls gene expression by histone modification, chromatin condensation, and gene silencing (Rice and Allis 2001). DNA methylation occurs throughout the course of development in multicellular organisms and ensures the maintenance of tissue and lifetime-specific gene expression patterns (Urdinguio et al. 2015).

In mammals, DNA methylation occurs especially in cytosines followed by guanines (Cytosine-phosphate-Guanine, CpG), and consists of the covalent addition of a methyl group at position 5 of cytosine (Lister et al. 2009). DNA methyltransferases (DNMTs) mediate the transfer of the methyl group to the DNA (Lan et al. 2010; Jin et al. 2011). The loss of DNMT activity or changes in DNA sequence may lead to global hypomethylation of the genome (Tunc and Tremellen 2009).

DNA methylation is a primary mechanism of developmental processes, such as genomic imprinting (Feinberg et al. 2002), X-Chromosome inactivation (Payer and Lee 2008), silencing of transposable elements (Doerfler 1991), DNA compaction and post-meiotic gene silencing (Oakes et al. 2007). Epigenetic reprogramming during gametogenesis is essential for the fertilization process and embryo development, in which a second round of reprogramming during embryo development establish epigenetic markers for embryo growth and maintain paternal imprinting (Hammoud et al. 2009). During spermatogenesis, DNA methylation remains stable, and the sperm genome contains remnant epigenetic signatures of this process, in addition to tags that influence fertilization and embryonic development (Jenkins et al. 2017).

The sperm DNA methylation pattern is a predictor of fertilization capacity and embryonic quality (Jenkins and Carrell 2012; Aston et al. 2015). Camprubí et al (Camprubí et al. 2016) identified 696 CpG islands related to spermatogenesis differentially methylated in sperm from fertile compared to infertile men. Altered DNA methylation has been associated with decreased reproductive capacity, mainly related to low sperm count (Benchaib et al. 2003; Houshdaran et al. 2007; Kobayashi et al. 2007; Marques et al. 2008; Hammoud et al. 2010; Aston et al. 2012; Krausz et al. 2012; Jenkins et al. 2015; Urdinguio et al. 2015) and reduced rates of pregnancy (Benchaib et al. 2003, 2005).

To our knowledge, only two studies evaluated sperm global DNA methylation in men with varicocele. Bahreinian et al. (2015) found DNA hypomethylation in men with varicocele when compared to fertile individuals, which had a negative relation with SDF. The same group also investigated the sperm global DNA methylation levels before and 3 months after varicocelectomy. The authors found a tendency to higher rates of methylation after surgery, although differences were not significant (Tavalaee et al. 2015). With the categorization of individuals into subgroups, the increase in global DNA methylation appears to have been higher in oligozoospermic men (Tavalaee et al. 2015).

Telomeres

In mammals, telomeres consist of non-coding (5ʹ-TTAGGG-3 ‘) repeating DNA sequences associated with proteins, which form the ends of eukaryotic chromosomes (Zalenskaya et al. 2000). Telomeres prevent the recognition of the end of the strands as a double strand break, protecting against DNA degradation, and allowing the correct alignment of the chromosomes during the replication, playing an important role in the regulation of genomic stability (Farr et al. 1991; O’Sullivan and Karlseder 2010).

Telomeres, shortened at each cell division, are the main biological marker of aging and limit the proliferative time of cells (Bonetti et al. 2013). However, to avoid excessive loss, the maintenance of telomeres is guaranteed by an enzymatic complex, consisting of a reverse transcriptase and an RNA template, called telomerase (Blackburn 1991).

The maintenance of the telomere length involves an interaction between factors that shorten it and those that extend it. In addition to cell division, telomere shortening can be intensified by extrinsic environmental factors, such as environmental pollutants, radiation, aging, lifestyle, and oxidative stress (von Zglinicki 2000; Zhang et al. 2013; Sharma et al. 2015). The increase of the inflammatory activity is related to shortening of the telomeres and occurs due to the increase of ROS that damages the telomeric DNA (O’Donovan et al. 2011; Wong et al. 2014a).

Changes in telomere homeostasis may affect the synapse of homologous chromosomes and recombination in spermatocytes that will undergo meiosis I, so that telomere impairment may be indicative of a phenotype of sterility (Thilagavathi et al. 2013; Reig-Viader et al. 2014). Studies found that the sperm telomere length is related to sperm count since oligozoospermic men have shorter telomeres than normozoospermic men (Ferlin et al. 2013; Cariati et al. 2016). The telomere length has also been reported associated positively with progressive motility and vitality, and negatively with SDF and protamination (Rocca et al. 2016).

However, to our knowledge, there are no studies that have sought to evaluate the role of telomeres in the pathophysiology of varicocele. As telomeric shortening can generate accelerated sperm aging and might be associated with DNA fragmentation, leading to infertility, we sought to investigate this association in infertile men with varicocele. Our results might help unravel a novel pathophysiology mechanism in varicocele, and introduce a potential sperm quality biomarker in these patients.

Hypothesis

Varicocele is a major cause of male infertility as it adversely impacts semen parameters, the molecular and ultrastructural characteristics of spermatozoa, and the testicular microenvironment. However, the mechanism by which varicocele impairs fertility is not fully elucidated. Since methylation levels were related to environmental changes and DNA damage, and this epigenetic mark has a regulatory role in telomere length (Wong et al. 2014b; Udomsinprasert et al. 2016), we hypothesized that alterations in DNA methylation and telomere length may occur in men with varicocele, being related to poor seminal quality and the phenotype of infertility and this was tested in a pilot study. This study tested the hypothesis that the decrease of fertility in men with varicocele may be due to alterations in the sperm global DNA methylation and telomere length, negative consequences for seminal quality and SDF. In this pilot study, 20 men with varicocele and 20 healthy controls without the disease were investigated. The seminal quality was evaluated by conventional semen analysis and the SDF was analyzed by sperm chromatin dispersion assay. The global DNA methylation was evaluated by enzyme-linked immunosorbent assay (ELISA) and telomere content by quantitative PCR (q-PCR) as described in Supplemental File 1 Materials and Methods.

Preliminary results

Of the 20 patients with varicocele included, 10 (50%) had grade 3 left-sided varicocele; 7 (35%) had grade 2 left-sided varicocele; and 3 (15%) had bilateral varicocele. The seminal parameters, SDF, telomere length and global DNA methylation of men with varicocele and controls are presented in Table 1.

Table 1. Comparative analysis of quantitative variables measured in the varicocele and control groups.

Variable	N		Mean (SD)		Median		Q1		Q3		P
	V	C	V	C	V	C	V	C	V	C
Age (years)	19	20	28.8 (5.7)	26.4 (5.0)	31.0	26.0	26.0	22.5	32.0	28.0	0.06
Concentration (x10^6/mL)	20	20	30.9(28.5)	73.9(52.7)	21.5	66.2	6.2	32.5	48.7	88.0	<0.01
Progressive Motility (%)	20	20	24 (15)	38 (11)	25	39	13	28	33	44	<0.01
Non-progressive Motility (%)	20	20	43 (11)	37 (9)	46	36	35	30	51	44	0.07
Immotile (%)	20	20	33(18)	25 (6)	27	24	22	20	37	30	0.18
Vitality (%)	20	20	84 (11)	87 (5)	88	88	84	84	90	91	0.56
Morphology (%)	20	20	2 (2)	4 (2)	2	4	0	3	3	6	<0.01
Volume (mL)	20	20	2.8 (1.3)	2.9 (1.1)	2.5	2.5	1.8	2.1	4.0	3.4	0.85
SDF (%)	19	20	37 (20)	26 (10)	27	24	24	19	47	30	0.09
Telomere (T/S ratio)	13	15	3.6 (1.0)	3.6 (0.8)	3.6	3.4	2.9	2.8	4.2	4.1	0.82
GDM (%)	10	5	49.7 (20.7)	64.7 (17.1)	45.3	64.0	34.3	48.5	65.7	75.6	–

V: varicocele; C: control; SDF: sperm DNA fragmentation; GDM: Global DNA Methylation; N: number of patients in each group; SD: standard deviation. In bold p < 0.05.

Sperm concentration, progressive motility, and sperm morphology were significantly lower in men with varicocele than controls (Table 1). SDF rates were higher in varicocele patients than and controls, albeit of not statistical significance (37.0 ± 20.0% vs. 26.0 ± 10.0%, p = 0.09) (Figure 2(A)). Telomere length was not different between men with varicocele (3.56 ± 1.04) and controls (3.57 ± 0.8, p = 0.82) (Figure 2(B)). As for global DNA methylation analysis, we observed reduced methylation level in the varicocele group (49.74% ± 20.75%) compared to the control group (64.66% ± 7.08%) (Figure 2(C)), however we could not do statistical analysis due to limited sample size.

Figure 2. Variables analyzed in the varicocele and control groups. (A) DNA fragmentation index; (B) Telomere content (T/S ratio); (C) Percentage of global DNA Methylation (p > 0.05) .

There was no statistical difference in the outcome measures mentioned above between men with grade 2 and 3 varicoceles (Table 2).

Table 2. Comparative analysis of quantitative variables among varicocele patients according to varicocele grades 2 and 3 (G2 and G3).

Variable	N		Mean (SD)		Median		Q1		Q3		P
	G2	G3	G2	G3	G2	G3	G2	G3	G2	G3
Age (years)	8	11	31.6 (4.7)	26.8 (5.6)	31.0	29.0	29.5	21.0	32.0	32.0	0.24
Concentration (x10^6/mL)	8	12	26.3 (30.9)	34.0 (27.7)	15.7	28.2	5.0	13.4	38.2	51.0	0.56
Progressive Motility (%)	8	12	25 (15)	23 (16)	25	24	18	10	32	33	0.91
Non-progressive Motility (%)	8	12	45 (9)	42 (11)	47	44	38	32	53	51	0.64
Immotile (%)	8	12	30 (15)	35 (20)	25	28	21	22	34	40	0.56
Vitality (%)	8	12	86 (5)	82 (13)	87	88	82	85	91	90	0.85
Morphology (%)	8	12	2 (2)	2 (3)	2	2	1	0	3	3	0.81
Volume (mL)	8	12	3.1 (1.3)	2.7 (1.4)	3.2	2.3	2.1	1.7	3.8	4.1	0.51
SDF (%)	8	11	41 (27)	34 (13)	27	28	24	24	60	47	0.99
Telomere (T/S ratio)	5	8	3.5 (0.68)	3.6 (1.26)	3.6	3.7	2.9	2.7	4.2	4.4	0.99
GDM (%)	2	8	75.4 (13.8)	43.3 (17.0)	75.4	40.8	65.7	31.1	85.2	53.4	–

SDF: sperm DNA fragmentation; GDM: Global DNA Methylation; N: number of patients in each group; SD: standard deviation.

A negative correlation was observed between SDF and progressive motility (p = 0.04), whereas a positive correlation was noted between SDF and the percentage of immotile spermatozoa (p = 0.01). We also observed a positive correlation between sperm morphology and telomere length (p = 0.05). No other significant correlations were observed when evaluating other variables (Table 3).

Table 3. Spearman’s correlation matrix between the variables evaluated in the spermogram and SDF, GDM and telomere length in the group with varicocele.

	SDF		GDM		Telomere (T/S ratio)
	R	p	R	p	R	p
Concentration	−0.42	0.07	−0.14	0.70	0.51	0.07
Progressive motility	−0.46	0.04	0.35	0.32	0.22	0.47
Non progressive motility	−0.19	0.42	0.34	0.33	0.33	0.27
Immotile	0.58	0.01	−0.40	0.25	−0.03	0.92
Vitality	−0.38	0.11	0.40	0.25	0.18	0.56
Morphology	−0.18	0.45	0.30	0.40	0.56	0.05
GDM	−0.50	0.14	–	–	0.45	0.22
Telomere (T/S ratio)	−0.19	0.54	0.45	0.22	–	–

SDF: sperm DNA fragmentation; GDM: Global DNA Methylation; In bold p < 0.05.

The participants were young (mean age, 27.6 years) and within the reproductive age. Notably, the group of men with varicocele had lower sperm concentration, decreased percentage of sperm with progressive motility, and increased percentage of sperm with abnormal morphology than those without varicocele. These findings are in accordance with previous results regarding poor seminal quality in these patients (Xue et al. 2012; Kadioglu et al. 2014).

Although the effect of varicocele on conventional seminal parameters is well reported, the genetic and epigenetic alterations are still poorly understood. Recent evidence indicates that SDF is increased in sperm from varicocele, which might be related to genomic instability (Saleh et al. 2003; Smith et al. 2006; Blumer et al. 2008; Zini and Dohle 2011; Wright et al. 2014; Santana et al. 2015; Roque and Esteves 2018). Thus, the evaluation of important biological markers of genomic stability, such as SDF, telomere length, and global DNA methylation, is important to better understand the underlying mechanisms leading to infertility in men with varicocele. Telomeres were similar between varicocele and controls. However, the sperm chromatin integrity and global DNA methylation were reduced in men with varicocele, albeit not reaching statistical significance, due to the small sample size given our study was designed as a proof of concept pilot study.

The global DNA methylation is closely related to SDF because genomic hypomethylation facilitates chromosomal rearrangements that lead to DNA damage, thus making it an important epigenetic marker of genomic stability and cell viability (Jones and Baylin 2002; Deaton and Bird 2011). In our preliminary results, we noted that men with varicocele exhibited a lower percentage of global DNA methylation in sperm. Only two studies have reported similar findings, both of which reported hypomethylation in men with varicocele (Bahreinian et al. 2015; Tavalaee et al. 2015). The loss of methylation might make the sperm DNA more susceptible to damage, creating a negative correlation between global DNA methylation and the SDF (Bahreinian et al. 2015). In this study, we observed a lower percentage of DNA methylation in men with grade 3 varicocele compared with men with grade 2 varicocele, suggesting that the disease severity could have a differential impact on sperm methylation.

To our knowledge, our preliminary data is the first attempting to evaluate sperm telomere length in men with varicocele. Telomeric changes can affect the recombination of homologous chromosomes in spermatocytes undergoing meiosis and may be indicative of a sterile phenotype (Reig-Viader et al. 2014). Spermatozoa with shorter telomeres exhibit increased DNA fragmentation (Moskovtsev et al. 2010) and excessive telomere shortening is a known trigger of apoptotic DNA fragmentation (Blasco 2003). As telomere shortening can lead to accelerated testicular aging and infertility, we believe that further studies of telomere length may be crucial to better understanding the mechanisms involved in seminal quality of patients with varicocele.

Current evidence supports the concept that oxidative stress plays a key role in the pathophysiology of varicocele-related infertility (Hamada et al. 2013). Excessive ROS leads to higher rates of DNA damage, consequently leading to poor sperm quality and lower fertility rates (Koksal et al. 2003; Agarwal et al. 2012; Muratori et al. 2015; Cho et al. 2016). Therefore, the semen from patients with varicocele contains a greater proportion of spermatozoa with abnormal and immature chromatin than in that obtained from men without the disease (Saleh et al. 2003; Smith et al. 2006; Blumer et al. 2008; Zini and Dohle 2011; González-Marín et al. 2012; Wright et al. 2014; Santana et al. 2015). Although our study revealed no statistical difference between the varicocele and control groups, the higher SDF values observed in the varicocele group should be investigated further.

The relationship between varicocele grade and the degree of sperm quality deterioration is controversial. Some studies have reported that grade 3 varicocele is associated with a more pronounced decrease in sperm quality (Ishikawa and Fujisawa 2005; Al-Ali, Marszalek, et al. 2010; Al-Ali, Shamloul, et al. 2013), while others have demonstrated that disease grade does not directly affect sperm quality (Diamond et al. 2007; Zargooshi 2007; Shiraishi et al. 2010). Our findings corroborate the hypothesis that varicocele, regardless of grade, negatively affects semen quality.

Despite no differences in the genomic instability parameters evaluated, a relationship between SDF and sperm motility, including a negative correlation with progressive motility and a positive correlation with the percentage of immobile spermatozoa, was observed. This finding is in agreement with published reports (Peluso et al. 2013), and this mechanism may be due to the presence of ROS or other types of agents which compromise the energetic metabolism of gametes in men with varicocele (Armstrong et al. 1999). Therefore, the present results also point toward a positive correlation between morphology and telomere length, and to our knowledge no other reports on this matter exists. Defects in sperm maturation are mirrored in sperm morphology (Ghasemian et al. 2015), and a correlation may exist between changes in genomic stability (e.g., that caused by telomere length modifications) and the formation of sperm with morphological abnormalities.

The variability in the clinical phenotypes of varicocele suggests that genetic and/or epigenetic factors may play an important role in the disease etiology, the mechanisms of which are still not well understood (Santana et al. 2017). Global DNA hypomethylation may be a candidate factor as it has been associated with shorter telomere length (Wong et al. 2014b; Udomsinprasert et al. 2016). By contrast, global hypermethylation in the subtelomeric regions confers protection to telomeres and helps maintain genomic stability (O’Sullivan and Karlseder 2010; Deaton and Bird 2011). Disturbances in these processes may lead to DNA fragmentation and, consequently, to decreased sperm quality and fertility in men with varicocele, a phenomenon that warrants further investigation.

The main limitation of our pilot study is the small sample size. Also, a few specimens had insufficient DNA quality to perform telomere length and global DNA methylation analyzes, thus we could not evaluate these parameters in all men included in the study. We used the Mann–Whitney test to compare the variables between the groups, but since the sample size in the global DNA methylation evaluation was very small, we elected to present the results descriptively only.

Our preliminary results suggest that sperm global methylation seems to be reduced in men with clinically palpable varicocele and might affect sperm chromatin integrity. Large cohorts and well-defined phenotypes are required to reveal the true picture of the association of varicocele with the analyzed parameters. However, it should be noted that these data are the first evaluation of sperm telomere length in men with varicocele. Further studies with larger sample size are essential to confirm this hypothesis and provide a better understanding of the underlying genetic and epigenetic mechanisms associated with varicocele, which may be influencing the infertility phenotype in these men.

Acknowledgments

The authors are extremely grateful to the study participants and their families. We appreciated the technical assistance of Marilda Hatsumi Yamada Dantas and Cristiana C. Padovan Ribas.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico [478689/2013-6], Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), under the fellowship number 2012/11069-9 (CLMF), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Programa de Excelência Acadêmica (PROEX) e Programa de Apoio à Pós-Graduação (PROAP).

Notes on contributors

Rosana Maria dos Reis

Conception and experiments design: VPS, CLM-F, RMR; Measurement of SDF and semen analysis: VPS, MACV; Performance and analysis of global DNA methylation: VPS, CLM-F, MCE; Measurement of telomere content: VPS, CLM-F, DCCP; Writing of the paper: VPS, CLM-F; Contribution with reagents and materials, and critical review of the manuscript: ESR, RTC, RAF, SCE, RMR. All authors have approved the final version and submission of this manuscript.