|Year : 2018 | Volume
| Issue : 1 | Page : 3-9
Understanding the central tenet and plausible consequences of oxidative DNA damage in the male gamete
V Dinesh Kumar
Department of Anatomy, Pondicherry Institute of Medical Sciences, Puducherry, India
|Date of Submission||26-Aug-2017|
|Date of Acceptance||20-Jan-2018|
|Date of Web Publication||25-May-2018|
V Dinesh Kumar
Pondicherry Institute of Medical Sciences, Puducherry
Source of Support: None, Conflict of Interest: None
One of the major causes of defective sperm function is sperm DNA damage induced by oxidative stress. The quasicrystalline state of sperm chromatin and lack of cytoplasm make it vulnerable to DNA damage induced by oxidative attack. Supraphysiological levels of reactive oxygen species result in damage to sperm DNA and therefore might provide a common underlying etiology of male infertility and recurrent pregnancy loss, in addition to childhood cancers in children fathered by men with defective sperm cells. This review aims to throw light on the causative links between oxidative stress and sperm DNA damage. Integral to this discussion is an abundance of evidence indicating that sperm DNA damage might have a profound impact in determining the outcome of assisted reproductive technologies (ARTs). Despite a growing body of evidence, routine testing for oxidative stress is not indicated in clinical practice. This calls for cost-effective, accurate, and simple diagnostic tools that can be routinely used when evaluating men with idiopathic infertility. Encouragingly, the involvement of oxidative stress in the etiology of male infertility has opened up new opportunities for therapeutic interventions involving judicious administration of antioxidants. The topic is of paramount importance in the current scenario where extensive use of ART achieves pregnancies in vitro which would have been prevented in vivo by nature, ensuring that the defective gametes not participating in generative process.
Keywords: Idiopathic male infertility, oxidative stress, sperm DNA damage, sperm morphology
|How to cite this article:|
Kumar V D. Understanding the central tenet and plausible consequences of oxidative DNA damage in the male gamete. J Curr Res Sci Med 2018;4:3-9
|How to cite this URL:|
Kumar V D. Understanding the central tenet and plausible consequences of oxidative DNA damage in the male gamete. J Curr Res Sci Med [serial online] 2018 [cited 2018 Oct 21];4:3-9. Available from: http://www.jcrsmed.org/text.asp?2018/4/1/3/233195
| Introduction|| |
Although the current treatment of infertility is dominated by the breakthrough of newer technologies, barriers to infertility treatment do exist. In developing countries like India, accessibility, economic cost, and cultural/societal factors  create an insurmountable obstacle to optimal reproductive health care. Assisted reproductive techniques (ARTs) fail in substantial percentage of the longing couples and the psychological trauma inflicted at those instances can be made out by their personal testimonies.
From a cynical point, it can be said that researches in the field of clinical andrology slowed to a grind in the past three decades  as the scientists felt that “few motile sperm” are enough for successful pregnancy. This stagnation of researches in male gamete, which contributes to half of the DNA content, has curbed the live birth rate utilizing intracytoplasmic sperm injection (ICSI) as a treatment for male infertility at 30% despite the advancement in ART over the years. Identification of the underlying defects and restoring its function by targeted treatment constitutes the scientific approach. This calls for comprehensive evaluation of male factor infertility to improve the outcomes of ART.
| Understanding Fertilization – the Role of Sperm|| |
Technically, male infertility can be described as absence or inability of functional motile spermatozoa to successfully fertilize an oocyte. Advent of micromanipulation procedures has allowed enhanced chances of fertilization even in cases of oligoasthenozoospermia (men with low functional sperm count). ICSI bypasses the sequential steps of natural fertilization such as penetration of cumulus and zona pellucida and fusion of oolemma. Integrity of the spermatozoon is quintessential to ordain the normal chromosomal segregation , as serious defects harboring the spermatozoon might impair the further development of the zygote. In a study, when sperm heads were severed from the flagellum and injected into the oocyte, oocytes that survived the injection with isolated sperm heads yielded 66.7% (44/66) fertilization rate. It can be said that sperm head, by virtue of putative activating factor  present in it, can still activate the oocyte and result in fertilization.
Case series , reports significant deleterious effects in clinical pregnancy rates following ICSI in men with impaired semen parameters. This calls for assessing the sperm morphology under higher magnification (motile sperm organelle morphology examination) to select the ideal sperm. This procedure of selecting sperm devoid of morphological defects for usage in morphologically selected ICSI yields better outcomes compared to conventional ICSI.
| Sperm DNA – a Vulnerable Target|| |
Unlike the relatively loose structure of chromatin (DNA and nuclear proteins) in somatic cells, sperm chromatin is highly compact because of the unique association with highly basic proteins (the protamines). Sperm DNA is a highly crystalline and compact toroid. During the later stages of spermatogenesis, the spermatid nucleus is remodeled and condensed, which is associated with the displacement of majority of histones (85%) by transition proteins and then by protamines. The histone-bound DNA sequences have less degree of compaction and are peripherally located in the nucleus, and it is thought that these DNA sequences or genes may be involved in fertilization and early embryo development., An excess of nuclear histones (>15%) results in poorer chromatin compaction and a subsequent increased susceptibility to environmental stresses.
Normal sperm DNA features a doughnut-shaped configuration that can prevent DNA damage during sperm transport. The sperm cells, because of their cytological differentiation, lack DNA repair activities. The repair of the paternal DNA alterations occurs after fertilization when the sperm nucleus remodels into male pronucleus.
Assessing the sperm DNA damage and correlating it with the outcomes of assisted reproductive techniques (ARTs) has always been one of the research areas of interest for the clinical andrologists. A meta-analysis  reports that sperm DNA damage predicts poor clinical pregnancy rates after in vitro fertilization (IVF) and/or ICSI (odds ratio – 1.68; 95% confidence interval – 1.49–1.89; P < 0.0001). A study  shows that infertile men having morphologically normal spermatozoa possess higher rates of sperm DNA fragmentation (20%–60%). Another study  involving 203 couples undergoing IVF found that live birth rates with IVF fell from 33% in those with DNA fragmentation <25% to 3% in those with DNA fragmentation >50%. This can be due to the late paternal effect of abnormal DNA fragmentation on embryonic development leading to miscarriage or birth defects in the offspring.
| on the Possible Consequences of Sperm Dna Damage|| |
The nucleosomal fraction of DNA contains genes of developmental importance (e.g. HOX and WNT) and telomeric DNA which are most vulnerable to environmental insults and oxidative damage. The integrity of the somatic genome is crucial as the nuclear DNA forms the blueprint for all cellular RNAs and proteins. Any damage incurred in this fraction of DNA is irreversible and irreplaceable. The outcome of DNA damage to the male germ line is determined by various factors such as: (1) type of damage (e.g., lesions that affect both the DNA strands such as double-strand breaks or interstrand cross-links are difficult to repair), (2) frequency of lesions (e.g., the presence of extensive DNA damage would make it difficult to be all repaired in the first embryo cleavage), (3) when the damage was induced, (4) region of the genome affected, and (5) ability of the embryo to repair it before the initiation of S-phase of the cell cycle.,
A two-step hypothesis has been proposed regarding the DNA damage in the germ line. According to this hypothesis, the first step in the DNA damage cascade has its origins in spermiogenesis when the DNA is being remodeled before condensation. Defects in the chromatin-remodeling process result in the production of spermatozoa that are characterized by an overall reduction in the efficiency of protamination, an abnormal protamine 1-to-protamine 2 ratio, and relatively high nucleohistone content. These defects in the chromatin-remodeling process create a “state of vulnerability,” whereby the spermatozoa become susceptible to further oxidative damage. In the second step of this DNA damage cascade, the chromatin is attacked by free radicals.
Both of these processes greatly destabilize the DNA structure and may ultimately result in the formation of DNA strand breaks. There are three possible outcomes for DNA strand breaks: (1) if the damage is too extensive to be repaired by the embryo, it would cause aneuploidies ending up in pregnancy loss, (2) if the breaks are sufficiently repaired, the integrity is recovered resulting in live birth, and (3) if the repair is inadequate, then there would be a few DNA alterations that could lead to diseases in the offspring, particularly if the alterations are mutagenic.
The repair of these DNA damages require the co-operation between the sperm and oocyte. Sperm chromatin possesses one of the base excision repair enzymes, namely, oxoguanine glycosylase 1 (OGG 1) but lacks the other enzymes involved in the mechanism. If the damages are not repaired by the rest of the enzymes present in the oocyte in the postfertilization phase, they are directly contributed to the zygote, which can affect its development and also make the offspring vulnerable to diseases such as obesity and metabolic syndrome.
In ARTs, such as ICSI where the natural selection process of spermatozoa is bypassed, couples of men with higher DFI can be forewarned/explained about risk of impaired blastocyst formation, pregnancy losses, and comorbidities in offspring. It has been said that the chance of spontaneous conception declines at sperm DNA fragmentation index values above 20% and approaches zero for values over 30%–40%. In a study  where DNA damage was measured using sperm chromatin structure assay (this technique evaluates the sperm chromatin susceptibility to acid denaturation, and the DNA fragmentation is measured as DNA fragmentation index), 64% infertile men had higher DFI (>30%). The appropriate place of sperm DNA fragmentation index in the clinical management of couples with subfertility is yet to be clearly elucidated which entitles the andrologists to adopt empirical therapeutic strategies. The entire moiety of paternally mediated reproductive risk is largely dependent on the fact that spermatozoa, which are perfectly normal in terms of function and appearance, may harbor damages in the DNA. These spermatozoa if selected can still fertilize oocytes and initiate development. The link between childhood cancers and paternal exposure to toxicants might also involve a similar temporal association between sperm DNA damage and an increased genetic/epigenetic mutational load in the offspring. There are numerous assays available that give a direct/indirect evaluation of sperm DNA nuclear integrity, and a comprehensive discussion to expose the specificity, merits, demerits, or default of these assays is beyond the scope of this review. Nevertheless, screening male patients in appropriate clinical conditions as a matter of “best practice” might provide information which help us to design the best possible management strategies.
| Oxidative Stress – a Serious Offender|| |
Oxidative stress in sperm is a result of imbalance between reactive oxygen species (ROS) generation and scavenging potential of antioxidants. For reasons not well understood at the molecular level, most cell types can develop an early onset of oxidative stress phenomenon or a “state of predisease.” Sperm, like any other aerobic cell, constantly faces the “oxygen paradox.” In addition, sperm is deficient in enzymatic antioxidants due to the lack of cytoplasm. The scavenging potential in gonads, seminal fluid, and sperm is normally maintained by adequate levels of antioxidants, superoxide dismutase, catalase, and glutathione peroxidase and reductase. In recent years, the supraphysiological levels of ROS in the male reproductive tract has become a real concern because of their potential toxic effects at high levels on sperm quality and function [Figure 1]. With the change in the lifestyle and ever-increasing exposure to environmental factors, the levels of free radicals in our body are also increasing.
|Figure 1: Effect of oxidative stress at cellular level. Increased production of ROS leads to mitochondrial and nuclear DNA damage. Damage to mitochondria causes a general decline in its function that in a vicious cycle like manner leads to oxidative stress|
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It can be generated from various sources such as (1) leukocytes as a consequence of male genital tract infections; (2) electromagnetic radiation, including heat or radiofrequency radiation in the mobile phone range; (3) redox cycling metabolites or xenobiotics, such as catechol estrogens or quinones; (4) electron leakage from the sperm mitochondria; and (5) deficiency in the antioxidant protection afforded to these vulnerable cells during their transit through the male reproductive tract.,
Free radicals could break the DNA helix backbone directly resulting in either single- or double-strand breaks, leading to the formation of base adducts particularly 8-hydroxy-2′-deoxyguanosine (8OHdG). This can be explained by the fact that high levels of ROS have been detected in the semen of 25% of infertile men. Free radicals can also inflict damage to the spermatids during their transit in the male genital tracts, by triggering apoptosis in live cells. This process of “abortive apoptosis,” whereby some of the spermatozoa with DNA damage have initiated and subsequently escaped apoptosis, usually harbors single-stranded DNA nicks. ROS generation ultimately precipitates a state of apoptosis which is characterized by rapid motility loss of sperm, mitochondrial ROS generation, caspase activation in the cytosol, annexin V binding to the cell surface, and oxidative DNA damage. This is different from conventional apoptosis in somatic cells where the mitochondria located in the cytoplasm initiate apoptotic cascade, as sperm mitochondria are entirely conserved in its mid-piece. Thus, it can be concluded that the main pathway leading to sperm DNA damage is a process of apoptosis triggered by testicular conditions (due to impairment of chromatin maturation) and by oxidative stress during the transit (self-perpetuating ROS generation by immature spermatozoa) in the male genital tract. Age-dependent increase in sperm DNA damage can also be observed because of two reasons: (1) linear increase in mutational load carried by progeny across the paternal age (leading to increase in the rate of miscarriages and enhanced risk of neuropathology in the offspring) [Figure 2] and (2) downregulation of base excision repair enzymes with age.
|Figure 2: Oxidative stress damages nuclear DNA associated with the formation of oxidative base adduct, 8-hydroxy-2′-deoxyguanosine. In case of excessive damage, oocytes fail to complete the base excision repair leading to persistence of DNA damage in each and every cell of the embryo after fertilization. Thus, fertilization of sperm carrying DNA damage possibly with the help of assisted reproduction technology such as intracytoplasmic sperm injection has the potential to impact upon the normal embryogenesis and health trajectory of the offspring|
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| Oxidative Stress – implications in Clinical Andrology|| |
Despite of the growing body of evidence, routine testing of oxidative stress is not indicated in clinical andrology practice owing to the unavailability of laboratory settings, complexity in measurement, and lack of single standardized technique which is universally accepted. The assays used can be broadly classified into two subdivisions  (1) direct assays, which assess the damage created by free radicals against the sperm lipid membrane. They measure the net biological effect between two opposing forces, i.e., ROS production and total antioxidant capacity (e.g., chemiluminescence assays, nitroblue tetrazolium test, and flow cytometry) and (2) indirect assays, which estimate the detrimental effects of oxidative stress, such as DNA damage or lipid peroxidation level (e.g., myeloperoxidase test, oxidation reduction potential, and total antioxidant capacity).
Once an individual has been identified as having oxidative stress-related subfertility, treatment should be aimed at identifying the underlying cause and ameliorating it before considering empirical antioxidant supplementation. Oxidative stress is a key element in the pathophysiology of varicocele-related infertility, and various hypotheses such as heat stress, hypoxia, and reflux of adrenal and renal metabolites are postulated to explain the pathophysiological effect of varicocele on testicular function. In a study, the chances of spontaneous conception were found to be 2.8-fold higher in the varicocelectomy group compared to the group of patients who received either no treatment or only medication. Another study  reports that varicocele repair is effective for decreasing sperm DNA fragmentation in most patients.
Smoking was associated with approximately 48% increase in seminal leukocyte concentrations, a 107% ROS level increase, and a 10-point decrease in ROS-total antioxidant capacity score. Another study  found a significant increase in levels of 8-OHdG and decreased level of antioxidants such as Vitamin E within smoker's seminal plasma. Higher levels of DNA strand breaks in men who smoke have also been identified. It can be hypothesized that smoking can cause increased infiltration of leukocytes which, in turn, increases the level of free radicals in the semen and depletes level of antioxidants in the seminal plasma. DNA strand breaks may resultant from the free radicals in the semen or mutagens (e.g., cadmium inhibit OGG 1, involved in base excision repair mechanism) present in the smoke. It has been observed that  paternal smoking is associated with a 13.3-fold increase in risk of developing retinoblastoma in the offspring (de novo germ line mutations with a postzygotic “second hit” in retinal cells). Lifestyle-based interventions in addition to empirical treatment enable reversal of oxidative stress, telomerase activity, and oxidative DNA damage.
Inflammation related to accessory sex glands such as prostatitis and seminal vesicle inflammation recruit leukocytes which produce 1000 times more ROS than spermatozoa. It is hypothesized that ROS release by activated leukocytes rapidly increases to around 100 times that of the inactivated leukocytes in the presence of infection-related stimuli (“respiratory burst of leukocyte”). In men with accessory gland infections, it was found that after 3 months of antibiotic therapy, there was a significant fall in the seminal leukocytes, ROS production, and a significant improvement in sperm motility and spontaneous conception.
| Sperm Morphology Examination – a Feasible Surrogate Marker in Poor-Resource Settings|| |
Abnormal sperm morphology can be an indicator of one or more defects including damaged DNA, chromosomal abnormalities, and centriole deficiency., Data suggest that morphologically abnormal sperm and/or DNA fragmented sperm have a negative impact on fertilization and embryo quality, even when ICSI is performed. Poor sperm morphology correlates with increased formation of ROS as well as the frequencies of single- and double-strand DNA breaks observed in the spermatozoa of infertile men.
Cytoplasmic droplet, a specific morphological defect of human sperm, has been correlated with sperm ROS production [Figure 3]. When spermatogenesis is impaired, the cytoplasmic extrusion mechanisms are defective, and spermatozoa are released from the germinal epithelium carrying surplus residual cytoplasm. Sperm deformity index (SDI) is the score of number of defective spermatozoa to the total number of spermatozoa counted. It is a novel quantitative expression of sperm morphological quality and is a more powerful predictor of sperm function and the outcome of oocyte fertilization in vitro than either the normal morphology or multiple anomalies index.
|Figure 3: Photomicrograph (×60) of hematoxylin- and eosin-stained slides showing cytoplasmic droplets (residues in the mid-piece suggestive of “immaturity”) of spermatozoa. When spermatogenesis is impaired, the cytoplasmic extrusion mechanisms are defective, and spermatozoa are released from the germinal epithelium carrying surplus residual cytoplasm. Right panel, a schematic representation of sperm nucleus and mid-piece. Mid-piece, in immature spermatozoa, generates free radicals which attack sperm head containing protamine-rich DNA|
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Increased levels of ROS were observed in samples with high proportions of sperm abnormalities such as amorphous heads, damaged acrosomes, mid-piece defects, cytoplasmic droplets, tail defects, and high SDI scores. A positive correlation has been demonstrated between SDI scores and early and late markers of sperm apoptosis, loss of the integrity of the mitochondrial membrane potential, and activated caspase-3.
In a study, which compared the ROS levels and sperm morphology of men with idiopathic infertility, it was found that SDI scores showed significant positive correlation with the sperm ROS production both in cases (r = 0.72; P= 0.00) and in controls (r = 0.495; P= 0.001) suggesting it as a single most important predictor for ROS-mediated sperm damage. Furthermore, regression studies attributed 63% of the variation in the ROS levels with morphological defects in infertile men (mainly cytoplasmic droplets and mid-piece defects). Thus, a comprehensive morphology examination of spermatozoa can serve as a useful tool in identifying men with elevated ROS levels in poor-resource settings. Based on the level of seminal oxidative stress and clinical presentation, an individualized treatment approach can be administered with adjustments in dose and type of antioxidants.
| Conclusion|| |
The integrity of sperm DNA is vital not only for the successful fertilization but also for the subsequent health trajectory of the offspring. Defective arrangement of chromatin in the sperm DNA makes it vulnerable to the oxidative attack. In this context, it is clearly imperative that a gamut of factors (lifestyle and pathological) can be responsible for inducing high levels of oxidative DNA damage in the male germ line. The purpose of this brief review is to throw light on the plausible mechanism of oxidative DNA damage which can impact the genetic profile of progeny. The tremendous development in the diagnostics of infertile men is not yet widely applied clinically. This insight could also open the way for alternative therapeutic strategies before ART that aims at reducing the level of DNA damage when possible.
I gratefully acknowledge Dr. Rima Dada, my guide and mentor, who kindled my curiosity in this field and Dr. Rajprasath R, my colleague, for helping me out in generating required images for the manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]