Men in the reproductive age group may experience qualitative and quantitative defect in sperm
production, but there are men with normal sperm parameters who are infertile, such cases are classified with idiopathic infertility. Men with idiopathic infertility generally present with significantly higher seminal ROS levels and lower antioxidant potential than fertile controls [1
Male factor accounts for 20% cases of infertility
, female factor for 38 % cases and in 27% cases of infertility both partners are involved. Traditionally, the diagnosis of male infertility is based upon macroscopic and microscopic assessment and analysis of sperm concentration, motility and morphology as routine indicators of semen quality. These indicators provide fundamental information about sperm production upon which clinicians base their initial diagnosis [2
]. However, even with appropriate quality assurance, traditional semen parameters provide a limited degree of prognostic and diagnostic information. The semen parameters assessed by traditional methods provide modest information regarding fertilizing capacity of sperm. Sperm chromatin integrity is essential in the process of fertilization, implantation and proper embryonic development and birth of healthy off spring [3
]. Sperm and ova carry all the necessary information required for fertilization and embryonic development. Any form of damage to the paternal (sperm DNA) or maternal (ova) can have serious consequences in the form of pre and post implantation losses, impaired embryonic development, childhood morbidity and even cancer [4
]. Thus DNA damage assessment may be a diagnostic measure in cases with idiopathic infertility especially in men with normal sperm parameters [5
]. The major forms of DNA damage in the male gamete include chromosomal aberrations (mostly deletions and aneuploidies), epigenetic modifications, mutations, base oxidation and sperm DNA fragmentation (SDF). As compared to ova (which develops in a relatively hypoxic environment in the ovarian cortex), sperm exists in a state of oxygen paradox. By virtue of it being transcriptionally inert, lacking cytosolic antioxidants, being endorsed with high polyunsaturated fatty acid content and deficient in DNA damage detection and repair mechanisms sperm is most vulnerable to oxidative damage by both external and internal insult [6
]. Several factors like morphologically abnormal sperm, infection, varicocele, and sedentary life style, exposure to organic pollutants, psychological stress, high temperature, smoking, and obesity can lead to oxidative stress [7
]. As sperm has a limited DNA damage repair mechanism and a highly truncated basic repair mechanisms, preventing or minimizing exposure to free radicals may be the only option to prevent DNA damage.
SDF can originate in the testis, after spermiation and during storage and transit in the male genital
tract, when they undergo final maturation in the epididymis During the process of IVF after ejaculation, when the sperm cells are separated from the seminal plasma and incubated in vitro sperm are more vulnerable to oxidative damage as protective antioxidants are removed due to sperm washing [8
]. These insults to the sperm DNA may pose problems in the process and outcome of ART. Fragmentation of the sperm DNA also occurs during spermatogenesis in the testis as part of the apoptotic process (which is known as the abortive apoptosis) or during chromatin compaction, and also during replacement of histones by protamines [9
]. Presence of sperm DNA damage is an indicator of cell lethality and cellular death. Such sperm would be rejected in vivo and would not be able to successfully bypass various barriers of fertilization, however use of such sperm with compromised DNA in vitro in assisted conception may affect foetal well-being. Unlike oocyte which is vulnerable to segregation anomalies, DNA damage in the form of mutations accumulates in sperm as sperm cells undergo several rounds of replication and each replication event is a source of mutations.
Unlike somatic cell, sperm is enriched with arginine and cysteine-rich protamines that helps in formation of strong disulphide bonds to maintain sperm DNA integrity. In order to reduce the volume of the sperm chromatin and the sperm as well to make the sperm head more hydrodynamic for its smooth movement and function there occurs vigorous compaction the sperm chromatin in the form of replacement of histones by transition proteins followed by protamines [10
]. Apart from these anatomical and physiological factors there are several physical (environmental exposures, radiations, food habits, life style factors) and chemical agents (pesticides, chemotherapy, persistent organic pollutants) that alter the sperm DNA and introduce damage in the form of mutations, deletions and base modifications. Sperm genome is not merely a vector of paternal DNA, but transfers at time of fertilization
not only DNA, but also transcripts and miRNA that are critical for early embryonic development. Therefore, understanding the nature of sperm DNA damage and its impact on the normal functions of the sperm cells and hence the fertilization capacity and outcomes is pivotal in the management of infertility [11
Increased reactive oxygen species (ROS) levels are the chief cause of DNA damage and previous studies have shown that high seminal ROS levels are implicated in infertile men with normal and abnormal semen parameters. One of the leading causes of increased free radical production by mitochondria is disruption of OXPHOS electron transport pathway by electrophilic aldehydes (accumulate as a result of peroxidation of lipids). This arises due to dietary deficiency, age, varicocele
, life style factors and low antioxidant levels. Hence it is essential to assess sperm DNA damage in the infertility management which is not included in the normal, traditional methods of semen assessment. Oxidative stress is thought to be the main mechanism responsible for the occurrence of DNA fragmentation and DNA base oxidation. Oxidative stress oxidizes the DNA bases preferentially guanine. Sperm has a highly truncated BER mechanism and thus OGG1 removes oxidized Guanine and create basic sites. These are repaired only by oocyte at fertilization. However ageing oocyte and presence of extensive sperm DNA damage may overwhelm the oocyte repair mechanism with persistence of these mutagenic bases. This can have deleterious impact on foetal health and may manifest as infertility, pre and post implantation losses, children with congenital malformation and even cancers. In an ongoing study in our laboratory we have found very high levels of sperm 8-hydroxy 2-deoxyguanosine levels and DNA damage in fathers of children who developed Retinoblastoma by 2 years of age.
Men with abnormal semen parameters (sperm motility and morphology) have high levels of DNA fragmentation [14
]. Sperm DNA quality is important in the process of fertilization and fertility outcomes both in vivo and in vitro. However, sperm DNA quality assessment is still not included in the routine assessment of infertility. It is important in clinical practice to take a threshold value with discriminatory power to distinguish DNA damage in fertile and infertile population; routine semen analysis is not able to do so. As DNA fragmentation approaches 20, the chances to sustain conception decreases and beyond DFI of 30 it is nearly impossible to achieve conception. Recent studies from our lab have shown that a DFI threshold of 26 results in pre and post implantation losses [16
]. Concomitant with increased oxidative DNA damage in sperm there is accumulation of highly mutagenic oxidized bases with 8-OH2dG. This preferentially occurs in Telomeres causing telomere shortening (due to OGG1 action) and thus accumulates Single strand and double strand breaks. This rapid telomeric attrition leads to chromosomal instability, rearrangements, aneuploidies during segregation and also genomic instability. This may be the common underlying factor by which sperm DNA damage leads to idiopathic infertility, idiopathic RSA, congenital malformation and even childhood cancer.
As mentioned earlier oxidative stress is the major cause of DNA damage. Oxidative stress is the condition when the free radicals and reactive oxygen species production exceeds the antioxidant
capacity of the cell. These free radicals and reactive oxygen species are produced in various physiological processes, disease conditions and various pathologies. These oxidants when cross the physiological levels cause oxidative damage to DNA and cell membranes along with many other biomolecules like proteins and lipids. Oxidative damage to DNA can contribute to mutations, histone and base modifications and hamper the sperm capacity in fertilization [17
]. Although many different oxidative DNA damage products have been identified, the guanine-derived lesion 8OHGua (8-hydroxyguanine) and its corresponding deoxynucleoside 8-OHdG (8-hydroxy 2-deoxyguanosine) have been the subject of intensive study. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the best-characterized oxidized bases [20
]. 8-OHdG in DNA could lead to mis-incorporation of adenines opposite the 8-OHdG lesion thus inducing G:C to T:A transversions in genomic DNA.
Ongoing studies from our laboratory have documented that oxidative
DNA damage may be the cause of accelerated testicular ageing which manifests as oligo and azoospermia due to shorter sperm telomere. Telomere shortening may also impair cleavage and lead to pre and post implantation losses [16
]. Thus evaluation of DNA damage, oxidized DNA adducts and detecting the cause of DNA damage are not only important diagnostic marker but are invaluable for appropriate management of such couples.