Diabetes and Sperm DNA Damage: Efficacy of Antioxidants
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Diabetes mellitus (DM) represents one of the major threats to human health all over the world, affecting almost every system of the body. Its prevalence is escalating globally and is associated with reproductive impairment in both males and females. Reports on male infertility due to DM is lacking since most affected individuals are unaware of their infertility condition due to the late onset of DM. Glucose metabolism is an imperative occasion in spermatogenesis, causing adverse effects on male fertility, principally on sperm DNA quality, motility, and ingredients of seminal plasma. DM is coupled with an increased oxidative stress (OS), causing sperm nuclear and mitochondrial DNA damage. Reactive oxygen species (ROS) hassles the fluidity of sperm plasma membrane, decreases sperm motility and ability to fuse with oocyte as well as altering the sperm DNA integrity. Lamentably, spermatozoa cannot repair the damage initiated by excessive ROS as they do not have the cytoplasmic enzymes required to achieve the repair. Diabetes may impact the epigenetic change during spermatogenesis which may be inherited through male gamete to more than one generation increasing the risk of diabetes in offspring. Administration of antioxidants in male infertility has started to pull in significant intrigue. Many studies have shown that antioxidants amazingly reduce the oxidative stress markers and boost the antioxidant enzymes. Currently treatment strategies are aimed at lowering ROS levels to maintain normal cell function. This review highlights the potential impact of DM on sperm DNA integrity, epigenetic dysregulation and efficacy of antioxidant therapy.
KeywordsAntioxidants Diabetes mellitus Epigenetic dysregulation Oxidative stress Sperm DNA integrity
Diabetes mellitus (DM), characterized by chronic hyperglycemia is a metabolic disorder of numerous etiologies, causing disturbance in carbohydrate, fat, and protein metabolism due to defects in insulin secretion and insulin action or both . The DM could provoke long-standing dysfunctions, damages, and failures of numerous organs. Many reviews in both human and animals have affirmed the malicious impact of diabetes on sexual functions for example, semen parameters, sperm DNA integrity, and chromatin quality [2, 3, 4]. Worldwide rates of male infertility extend from 2.5 to 12% implying that 30 million men globally are infertile. Even though oxygen is imperative for aerobic metabolism of spermatogonia, it may have adverse effects on cells through generation of ROS . ROS are free radicals that assume a noteworthy part in a significant number of the sperm physiology, namely, capacitation, hyperactivation, and sperm-oocyte fusion [6, 7, 8]. Studies have shown that DNA integrity of spermatozoa is essential for normal fertilization and inheritance of paternal gene in offspring [9, 10]. As spermatozoa lack cytoplasmic defenses, they are sensitive to oxidative stress (OS) [11, 12]. In addition, the sperm plasma membrane has lipids as polyunsaturated fatty acids, which are susceptible to assault by ROS resulting in lipid peroxidation [13, 14, 15]. Epigenetic modifications such as DNA methylation, remodeling of nucleosomes, histone modifications, and non-coding RNAs and higher-order chromatin reorganization are essential during spermatogenesis. Environmentally induced epigenetic modifications during embryonic gonadal development and germline differentiation might become stable in epigenome of germline leading to epigenetic transgenerational inheritance. In this paper, we first illustrate the role of glucose metabolism and oxidative stress in spermatogenesis and the impact of diabetes on male fertility. We enumerate the epigenetic dysregulation and unique features of sperm DNA integrity. In addition, the rationale for antioxidant therapy will be critically reviewed.
Chromatin Structure of Human Spermatozoa
Diabetes Mellitus and Male Infertility
The incidence of DM is increasing worldwide leading to impairment in reproductive function in both sexes. DM influence male reproduction at various stages by affecting the endocrine control of spermatogenesis and sperm maturation and by causing impairment in penile erection and ejaculation. It has been claimed that the common causes of infertility among diabetics are ejaculatory dysfunction (retrograde and antegrade) and endocrine control. Reports on men with DM and infertility are limited and not well studied. A major reason to this could be due to the age factor. In most cases, men with infertility issues due to DM are not identified since DM usually develops during the later stages of life [32, 33]. A study conducted by Loghmani demonstrates that prolonged rise in blood sugar levels is linked with micro- and macrovascular complications resulting in heart diseases, kidney diseases, blindness, and stroke. Strikingly, diabetic men who exhibited typical semen parameters indicated fundamentally more elevated amounts of defect in sperm mitochondrial and nuclear DNA, presumably coming about because of supra-physiological glucose levels and associated oxidative anxiety . Apart from hyperglycemia, various other factors also play a vital role in pathology of DM, namely, oxidative stress and hyperlipidemia ending in major risk of complications . In addition, advanced glycation end product’s (AGEs) resulting from oxidative damage have been identified in elevated levels in seminal plasma, reproductive tract and sperm of diabetic males suggesting their role in ROS induced damage in sperm DNA [35, 36, 37]. In many circumstances, chromosomal disorders contribute to the underlying cause of reproductive outcome . A study conducted by a group of authors narrated the sequestered and uninterrupted effect of elevated glucose levels on male gamete by incubating spermatozoa for 2 days supplemented with PBS-medium mimicking in vivo conditions [39, 40, 41].
Glucose Metabolism in Sperm
Except few metabolites like citrate and lactate, sperm principally consume sugars as vitality source including glucose, fructose, and mannose. Oxidative phosphorylation and anaerobic glycolysis are the two primary metabolic pathways required in energy production . As reported in earlier findings, male reproductive well-being depends primarily on glucose intake and metabolism by testicular cells. Polar molecules like glucides rich in –OH groups gets transported across the lipidic bilayer in a passive and inactive manner. Therefore, carriers are essential for their utilization by cells . Two family of membrane proteins transport glucose across the blood-testis barrier, namely, sodium-dependent glucose transporters (SGLT) or glucose transporters (GLUT) that can actively and passively transport hexoses across the lipidic bilayer [44, 42]. Different GLUTs in sperms has the adaptability to adjust to variations in the environment, metabolic prerequisites, and so on. Conversely, an unfavorable situation like diabetes can bring about dysfunction in nutrient exchange, therefore prompting reduced fertility and poor pregnancy outcomes [45, 46].
Oxidative Stress in Diabetes Mellitus
Sperm glucose take-up and metabolism are basic for male fertilizing capacity. Sperm cells utilizes few substrates for energy requirements, hence the dysregulation in glucose take-up and metabolism in hyperglycemia may produce negative effect on sperm quality . Oxidative stress induced by hyperglycemia is currently perceived as the main impetus for the advancement of diabetic complications . A study conducted by Ayepola has identified that free radicals play a vital role during the onset and progression of late diabetic complication, as they have the capacity to damage proteins, lipids, and DNA . In DM, in addition to oxidative stress, insulin dysfunction and glucose availability may lead to severe alterations in testicular cells, along with mitochondrial and nuclear DNA fragmentation. Studies have shown that oxidative stress developed during sperm metabolism affects the sperm function [50, 51]. Cells undergo apoptosis or necrosis in an oxidative environment and is the main pathway leading to sperm DNA breaks in sperm. Apoptosis and necrosis are likely triggered by an impairment of chromatin maturation in the testis during the transit in the male genital tract and occurs during spermatogenesis or after spermiation or both. These are relevant for the development of new drugs to rectify sperm DNA fragmentation (sDF) and OS in infertile men . Hyperglycemia produces more ROS, as well as diminishes antioxidative mechanism by hunting enzymes and substances . Lactate is fundamental for germ cells as it is by all accounts a dynamic metabolite that serves as energy for their advancement . Past clinical and exploratory reviews proposed that DM modifies glucose take-up from Sertoli cells and its oxidation to pyruvate and lactate [55, 56]. The disruption of its synthesis could lead to abnormal seminal parameters such as concentration, motility, viability, and morphology. It is trusted that oxidative stress assumes critical part in the progress of vascular complications especially in type 2 diabetes . Variation in the levels of enzymes superoxide dismutase (SOD), catalase (CAT-enzymatic/non-enzymatic), and glutathione peroxidase (GSH–Px) makes the tissues vulnerable to oxidative insult resulting in the development of vascular complications . Here, oxidative stress brings about incitement of polyol pathway, development of advanced glycation end products (AGE), enactment of protein kinase C (PKC), and ensuing materialization of reactive oxygen radicals [59, 60]. Mitochondria is the chief source of oxidative stress in diabetes. In mitochondria during oxidative metabolism, a part of the consumed oxygen is condensed to water and the residual oxygen is converted into oxygen free radical (O−) which is an essential ROS that is changed over to different reactive species, namely, ONOO−, OH, and H2O2 .
Reactive Oxygen Species in Semen
Oxidative stress (OS) is a state related with an increased cellular damage induced by oxygen and their derived oxidants, namely, ROS . Under normal conditions, spermatozoa are capable of producing ROS and it is the ROS that helps in the penetration of spermatozoa into the oocyte through the zona pellucida. During abnormal conditions (atypical sperms), ROS is produced in excess causing an imbalance in the level of antioxidants leading to oxidative stress [63, 64]. ROS provoke harm to cells by transient unpaired electron ensuing oxidation of molecules and cell components . Further, it also reflects the inequity between ROS and the body’s ability to actively detoxify the end products of OS or to repair the damages produced by it [66, 67]. Cells undergo apoptosis or necrosis in oxidative environment, on the other hand reducing environment leads to cell survival . Both intrinsic and extrinsic sources of ROS exist in semen. Intrinsic sources are activated leukocytes due to infection and inflammation [69, 70, 71, 72], immature spermatozoa with abnormal morphology and cytoplasmic retention , and abnormal and defective spermatozoa due to compromised spermatogenesis [74, 75]. Hipler et al. have demonstrated that even Sertoli cells in semen have the capacity to produce ROS . Various other intrinsic causes are aging, varicocele, testicular torsion, and cryptorchidism [77, 78]. Extrinsic sources associated with increased seminal and/or testicular ROS levels are alcohol consumption , environmental toxins , cigarette smoking , and exposure to radiation [80, 81]. A wide range of air pollutants, such as O3, NO2, and particles, also can produce an excessive production of ROS. They resulted in damage to DNA, lipids, and proteins, altering enzymatic systems and cell apoptosis and, ultimately it also led to decreasing semen quality . This study finds a robust association between exposure to air pollution and a low percentage of sperm normal morphology in reproductive-age men .
Sperm DNA Damage
Over the previous decade, there has been a developing collection of research exploring the part of sperm DNA integrity in male infertility. Physical and chemical environmental change may influence reproductive function . In our atmosphere, we are constantly exposed to minimal doses of ionizing radiation . Study conducted by Balachandar et al. discussed about the adverse effect of ionizing radiations leading to stable and unstable alterations in chromosome and in turn may probably lead to DNA damage . Sperm chromatin damage is also found in 8% of males with normal semen parameters . It has been proposed that sperm DNA integrity might be a superior indicator of male fertility than routine semen investigation. Sperm DNA damage has been related to elevated amounts of ROS . Elevated levels of defective sperm DNA are often associated with poor seminal factors like decreased count, movement, or abnormal forms [88, 89, 90]. There is confirmation that sperm of infertile men encompass more DNA impairment than fertile men, and this defect may negatively affect fertilizing capability of these patients . At minimal quantity, ROS assume a vital part in sperm development and roles, like acrosome reaction and capacitation . Sperm chromatin has a very consolidated and sorted out structure that shields it from oxidative insult . However, when compactness is reduced and chromatin protamination is deficient, sperm DNA is more susceptible to ROS. As examined already, in the course of spermiogenesis, sperm chromatin experiences a critical stride in restructuring in which histones are supplanted by protamines. This chromatin rebuilding is enabled by the synchronized slackening of chromatin structures by histone hyper-acetylation and in addition the compound DNA topoisomerase II (topo II) which produces brief scratches in the sperm DNA to release torsional strain subsequent to supercoiling [94, 95, 96]. Moreover, these transitory scratches are then regularly repaired by this same compound, before completion of spermiogenesis and discharge. If these nicks are not mended, spermatozoa with fragmented DNA might be available in the ejaculate .
Plasma of seminal fluid contains antioxidants which secure sperm DNA . When an over the top measure of ROS is created past the antioxidant limit of male reproductive tract and seminal plasma, the pathogenic outcome is habitually DNA and cellular damage . DNA damage triggers apoptosis , and in instances of more serious defect of spermatozoa, apoptosis brings about decreased sperm count distinctive of idiopathic male factor infertility . ROS influences nuclear or mitochondrial DNA of sperm at an amino acid or sub-atomic level, by base change (particularly guanine), assaulting the phosphodiester backbones and generating base free sites, deletions, point mutations, frame shifts, polymorphisms, strand breaks, rearranging chromosomes, translocations, and chromatin cross-links [100, 101, 102]. Defective sperm DNA may decline fertilizing capacity, increase pregnancy wastages and tendency for congenital anomalies, decrease the implantation, and finally weakens embryonic development [103, 104]. An additional hypothesis of sperm DNA defect is through failed apoptosis. Generally, apoptosis occurs in testicles to inhibit increased generation of germ cells and to specifically obliterate harmed germ cells . Sertoli cells boost a set number of germ cells in the testis. Clonal extension of germ cells is in abundance, and along these lines apoptosis is important to restrain the measure of the germ cell populace to one which Sertoli cells can bolster . Increase in the sexual abstinence period influences sperm quality. This indicates an important correlation between the duration of ejaculatory abstinence and semen parameter variation. It highlights the deleterious effect of increased abstinence on DNA damage, which is most likely associated with ROS .
Another study which provides solid evidence for vascular endothelial growth factor (VEGF) becoming a therapeutic target in type 2 diabetes mellitus (T2DM) related male infertility. It demonstrates that T2DM can reduce testicular VEGF expression, which results in testicular microcirculation impairment, and then induces testicular morphological disarrangement and functional disorder. These actions are triggered by PI3K/Akt pathway. The decrease of testicular VEGF is related to the testicular cells apoptosis, damage of rat Sertoli cells, and inactivation of PI3K/Akt pathway triggered by long-term hyperglycemia. Here they strongly believe, that the VEGF as a good therapeutic target for T2DM induced male infertility. These indicated that VEGF had a protective effect on hyperglycemia-induced sperms damage in DNA level .
Impact of Epigenetic Regulation in Spermatogenesis and Male Infertility
During spermatogenesis, epigenetic mechanisms acts as critical regulators for gene expression, which in turn have control on male reproductive function [109, 110, 111]. In male reproductive function, epigenetic regulation plays a major role by controlling germ cell growth and maintenance [111, 112, 113, 114, 115]. DNA methylation is a kind of epigenetic alteration that can efficiently endorse gene silencing. Development of germ cell is a well-organized process originating during the fetal growth and ending in adult stage. Therefore, epigenetic changes happening in gametes are vital for function of gametes and for embryonic growth after fertilization . Gonocytes are the only germ cells found in prenatal testis which undergo proliferation for a short period and then decline. These gonocytes turn out to be spermatogonia, as they reach the basement membrane after birth and then recommences proliferation. They undergo mitotic cell division—and constitute the group of stem cells, giving way for meiosis and spermatogenesis. Preceding spermatogenesis, gonocytes and spermatogonia transposable elements (TEs) are silenced . A study has revealed that there exists a considerable difference in epigenomes of somatic cells and sperm cells through genome-wide methylation studies. Further they identified that sperm epigenome is very much related to that of embryonic stem cells [118, 119]. In spermatozoa, the promoters especially of developmental genes are greatly hypomethylated. MEST/PEG1 are paternally expressed human genes, they are demethylated in fetal stage and stay unmethylated during the entire process of spermatogenesis . In this manner, unusual imprinting as an after effect of DNA methylation dysregulation might be connected with male infertility. Moreover, many research studies have reported that altered spermatogenesis is associated with imprinting errors . Men with low sperm count showed the event of intergenic-DMR and hypomethylation of H19 or hypermethylation at a few maternal DMRs, particularly when the sperm concentration is < 10 × 106/ml [122, 123, 124]. Methylation was obviously reduced in men diagnosed with oligoasthenoteratozoospermia at all CpGs, achieving statistical importance especially in patients with count < 10 × 106/ml. Therefore, these discoveries recommend that atypical DNA methylation intervened genomic imprinting is connected with oligozoospermia and oligoasthenoteratozoospermia [125, 126, 127, 128].
Parental Diabetes and Epigenetic Regulation
Gestational diabetes exerts adverse effect on the organization and function of testicular structures and endocrine system of reproductive organs equally. This harmful alteration takes place during early embryonic stage and lasts until postnatal life, which was observed previously by Jelodar et al. in diabetic adult male rats . The methylation enabled by DNA methyltransferase (DNMT) 3 L is responsible for silencing of TEs in testis. Gonocytes express DNMT3L when universal DNA methylation occurs at 14–18 days after fertilization. Experimental study conducted in adult mice has suggested undeniably that the lack of TEs silencing leads to meiotic arrest and infertility in adult mice [130, 131]. Establishment of paternal imprinting of genes occurs approximately about 15.5 days after fertilization in gonocytes and carried out through spermatogonia. In male germ cells, these genes are methylated and subjected to silencing. Factors, namely, BORIS (brother of the regulator of imprinted sites) and the DNMT, DNMT3A, DNMT3B with its closely related DNMT3L, are involved in establishing the paternal imprints in male gametes. Loss of methylation was observed in paternally imprinted regions due to deletion of Dnmt3L. Sperm cells lacking Dnmt3a and Dnmt3b revealed disparities in methylation patterns at paternally imprinted regions . In addition, elevated blood glucose levels in males alters the general methylation patterns in spermatogonia with an expanded portion of differentially methylated genes intersecting with pancreatic islets in fetus implying that paternal prediabetic condition may increase the risk of diabetes in progeny through epigenetic alterations in gametes .
Role of Antioxidants
Types of antioxidants in semen
Superoxide dismutase (SOD)
Dietary antioxidants constitute the scavenger type of antioxidant. They play vital role in antioxidant defense system of humans. Fruits, vegetables, and dietary supplements act as potential source of numerous antioxidants. Chain-breaking antioxidants like vitamin E and vitamin C can be used to limit oxidative stress . Cochrane meta-analysis on the use of oral antioxidants in male infertility found that these agents significantly improved pregnancy rates and live births and decreased sperm DNA damage .
A study found that Trolox protected the Wi-Fi-exposed semen in vitro from the damage of electromagnetic radiation-induced OS. They concluded that 2.45 GHz Wi-Fi could impair human sperm in vitro through the effects of OS on sperm DNA and mitochondria. Trolox could mitigate the harmful effects of 2.45 GHz Wi-Fi electromagnetic radiation by inhibiting OS damage to sperm DNA and mitochondria. They offer supportive evidence for clinical use of Trolox to prevent the adverse effects of Wi-Fi exposure on the male reproductive system .
Mechanism of action and effect of various antioxidants
Mechanism of action
Glutathione reductase and glutathione peroxidase
Act as scavenging antioxidants in the epididymis and testes
Protects lipid constituents in membrane and improve sperm membrane characteristics
Preserve sperm viability and motility
Mora-Esteves and Shin 2013 
Superoxide dismutase and catalase
Catalyzing the conversion of superoxide into oxygen and H2O
Prevents lipid peroxidation
Agarwal et al. 2004 
Detoxifies both intracellular and extracellular H2O2 to water and oxygen, activates nitrous oxide (NO)-
Prevents lipid peroxidation, induce sperm capacitation
Improve sperm membrane characteristics, improves fertilization
Neutralize H2O2 Quench free radicals prevents lipid peroxidation
Improve sperm membrane characteristics and activity of other antioxidants
Fight OS in the seminal plasma (Upto 65%)
Protects sperm viability and motility
Water soluble dietary antioxidant
Assist free fatty acid utilization, prevent lipid peroxidation
Prevent sperm DNA damage, maintains sperm viability and motility
Carotenoids Single molecular oxygen quenchers
Sies 1993 
Prevents lipid peroxidation in sperm membrane
Improves semen quality
Basnal and Bilaspuri 2011 
Prevents lipid peroxidation in seminal plasma
Improves semen quality
Lampiao 2012 
Precursors of intracellular GSH
Increase amount of GSH synthesis, GSH scavenges oxidants, prevents oxidative damage
Prevents oxidative damage to cell membrane and DNA
Basnal and Bilaspuri 2011 
Competitive phosphodiesterase inhibitor
Prevents breakdown of intracellular camp, suppress synthesis of tumor necrosis factor-α (TNF-α) and leukotrienes
Decreases inflammation, sperm motility preserved
Mora-Esteves and Shin 2013 
Parental nutritional status and metabolism are essential factors for determining the health status of offspring in adult stage. Due to massive increase in metabolic disease like diabetes, there is an alarming necessity to monitor the adverse environmental influence on the germ cells. Decline in fertility rate is observed in male with diabetes either type I or type II. Molecular studies with sperm from diabetics are still scarce since most men are unaware of their condition due to the development of DM due the later stages of life. Most of the mechanisms through which sperm manage to attain their energy metabolism in diabetic men remain to be disclosed. The studies deliberated in this review validate that glucose metabolism is very essential for spermatogenesis. Therefore, there is an urgent need for more studies focused on the molecular mechanisms beyond glucose transport in sperm of diabetic men. Diabetes could ensure detrimental effects on sperm motility, sperm quality, sperm DNA integrity, and seminal plasma ingredients. In a healthy condition, there exists a balance between ROS generation and antioxidant activity in male reproductive system. In diabetes, due to excess production of ROS in semen, sperm, and seminal plasma antioxidant, defense mechanism is affected which leads to oxidative stress. However, there is a more profound lack of literature concerning the effect of DM in the functioning of testicular cells. In addition, diabetes may produce alterations in epigenetic modifications during spermatogenesis, these changes might be carried over through male gamete and transmitted to many generations, resulting in increased risk of diabetes in progeny. Studies explaining the mechanism behind epigenetic regulation of DNA methylation during spermatogenesis are still in preliminary stage. There also seems to be an association between DNA methylation and male infertility. In the future, the research should be focused to identify the molecular mechanism underlying the potential impact of diabetes on epigenetic dysregulation during spermatogenesis, and its transgenerational inheritance. Many studies states that antioxidant therapy may improve sperm quality and in turn male fertility. Further studies should be done to ascertain the effectiveness of antioxidants to nullify the effects of ROS, and to prevent the oxidative damage.
The authors would like to thank all the research scholars in our lab for providing support to complete this review.
Compliance with Ethical Standards
The authors declare that they have no competing interests.
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