Because the assays that are used to identify OS, DNA damage and apoptosis are complex, testing should be limited to cases where an abnormality is suspected. Routine semen analysis remains the backbone of evaluating male infertility. Most infertile men do not require advanced and sometimes expensive tests that diagnose defects at the molecular level.
Because OS is the result of an imbalance between ROS levels and the total antioxidant capacity (TAC) of seminal plasma, it is important to assess both variables when evaluating the OS status of a given sample. The chemiluminescence assay is one of the most commonly used methods to detect free radicals. The assay is accurate and reliable when the sperm concentration is >1 × 106/mL and the samples are analysed within the first hour after specimen collection . Values of >1 × 106 counted photons per minute are considered high.
Although accurate for measuring TAC in seminal plasma, the enhanced chemiluminescence assay is cumbersome and time-consuming, and requires expensive instrumentation (e.g. a luminometer). However, colorimetry is currently gaining considerable acceptance . Currently, the characterization of average TAC values is lacking. With no clear demarcation between fertile and infertile patients, assessing TAC does not currently appear to be of high value.
Although several methods are currently used to assess apoptosis and sperm DNA damage, establishing a threshold between normal levels in the average fertile population and the minimal levels of sperm DNA integrity required for achieving pregnancy remains extremely challenging. All methods currently lack a threshold, except for the sperm chromatin structure assay; this assesses the ability of the DNA to resist denaturation by acid or heat, using flow cytometry, and the damage is expressed as the DFI . In clinical applications, the DFI not only distinguished fertile men from those who were infertile, but also identified samples that were compatible with in vivo and in vitro pregnancy (<28–30%) .
At present, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling and Comet assays for apoptosis are commonly used in research applications. Both assays correlate well with fertility and in vitro fertilization  but the subjectivity and variability of their results does not currently allow their use as distinctive identifiers of samples with impaired fertility.
Oral: Levels of ROS can be reduced by augmenting the scavenging capacity of the seminal plasma with antioxidants . Combined therapy is much more beneficial in managing infertile men, because antioxidants act by different mechanisms on different free radicals. Patients diagnosed with male accessory gland infections may benefit from carnitines (l-carnitine 1 g and acetyl-carnitine 0.5 g) twice daily for 3 months, as it results in a significant reduction in ROS levels in semen samples. Nevertheless, treatment with antioxidants in these cases is mainly an adjunctive therapy and does not replace proper antibiotic regimens. Other combinations of vitamins A and E with N-acetyl-cysteine may also be used.
A few clinical trials report the positive effects of antioxidant administration on sperm DNA integrity. When given for 2 months, vitamin C (200 mg), combined with vitamin E (200 mg) and glutathione (400 mg), significantly decreased 8-OH-dG levels, considered as a marker of OS-induced sperm DNA damage. Similarly, N-acetyl-cysteine and/or a mixture of essential fatty acids and natural vitamins A and E reduced levels of 8-OH-dG .
When the molecular framework of apoptosis is identified, specific apoptotic inhibitors may have a role in promoting germ-cell survival. Current research is underway to identify agents that may rationally manipulate the apoptotic machinery for therapeutic benefits. Sphingosine-1-phosphate is an example of an apoptotic inhibitor; at 1 and 10 µmol/L it suppressed apoptosis in germ cells extracted from testicular tissue by 30%. Similarly, N-acetyl-cysteine is a potential regulator of germ-cell death, mainly because it is a well established inhibitor of physiological cell death in several systems, and because it is a compound known to act on human semen as a survival factor. When given in concentrations of 125, 100, 50 and 25 mmol/L, N-acetyl-cysteine suppressed germ cell death in a dose-dependent manner . These agents have been evaluated mainly in vitro; their efficacy still remains to be validated in vivo.
Sperm preparation methods are used during assisted reproduction techniques (ART) to recover a selected healthy population of cells. The ‘swim-up’ method, glass-wool filtration and density-gradient centrifugation all help to ensure that semen samples contain DNA-intact spermatozoa .
During sperm preparation techniques, media may be supplemented with a variety of antioxidant(s) to guard against OS. Adding different concentrations of vitamin C (300 and 600 µmol/L) and vitamin E (40 and 60 µmol/L) to sperm preparation medium significantly reduced hydrogen peroxide. The superoxide anion can be also reduced by 29–72% by adding 10 mmol/L pentoxifylline .
The effects of antioxidants in vitro are more marked in samples that were originally characterized by high levels of ROS. N-acetyl-cysteine (0.1, 1 and 5 mg/mL) has a dose-dependent effect in reducing ROS levels; the reduction was greater in patients with high levels of ROS than in those with low levels .
ARTs, which include intrauterine insemination, in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), currently have a major role in treating infertility. Success rates depend on a variety of factors, but most important is the structural and functional integrity of the gametes used. Therefore, the exact nature of the infertility, and its cause and pathogenesis, should be considered before the treatment is started. Unidentified factors may adversely affect the end result, and add to the financial, social and emotional problems of the patients.
Semen samples that contain high levels of DNA damage are often associated with decreased fertilization rates and/or embryo cleavage after IVF and ICSI, and may be linked to early embryo death. Although the most normal-appearing and motile spermatozoa are selected during ART, there is always a chance that sperm containing varying degrees of DNA damage may be used. The miscarriage rate is highest after ICSI, which possibly reflects the fact that genomically compromised spermatozoa are sometimes used and lead to irreparable DNA damage in the embryo .
In patients with obstructive azoospermia, spermatozoa are often surgically retrieved from the epididymis or testicular tissue. In general, surgically extracted spermatozoa tend to have higher percentages of DNA strand breaks. However, the use of testicular spermatozoa is preferred over epididymal spermatozoa, because the former have less DNA damage and a better developmental potential .
Sperm cryopreservation is extensively used in ART programmes. Despite various advances in cryopreservation methods, the recovery rate of functional spermatozoa after thawing remains unsatisfactory. ROS are produced during the freezing and thawing of spermatozoa, which may be the cause for the decrease in sperm function after cryopreservation . Cryopreservation induces many changes in sperm cells, including membrane disorders and cell death; it also acts as an inducer of apoptosis in sperm cells and in turn, it clearly facilitates DNA damage.
Whether the cryopreservation of raw semen samples or prepared (after washing) samples yield the best results is a matter of controversy. Sperm that is frozen unprepared in seminal fluid may be more resistant to freezing damage because of the presence of protective seminal plasma. In our opinion, separating the mature healthy spermatozoa from leukocytes and immature spermatozoa is more important, as using media supplements in vitro can always compensate for the antioxidant properties of the seminal plasma.
It is extremely important for natural and assisted conception to reduce the levels of seminal OS. Minimizing the interaction between ROS-producing cells in semen (sperm with cytoplasmic droplets/leukocytes) and the mature spermatozoa may help to protect spermatozoa that have the potential to fertilize from the detrimental effects of ROS. Any underlying causal factor such as inflammation, infection or cigarette smoking should be managed properly.
The sperm preparation techniques used for ART (e.g. density gradient and swim-up) can be used to separate the mature spermatozoa with the best ability to fertilize and to decrease their interaction with ROS-producing cells. However, during these techniques, the semen samples are repeatedly centrifuged and the antioxidant-rich seminal plasma is removed, which may result in increased ROS levels. Shortening the duration of the centrifugation may reduce the risk of OS-induced injury to the sperm.
Currently, the presence of spermatozoa in media appears to promote sperm DNA integrity. In vitro culture of testicular spermatozoa for 72 h reduces the proportion of spermatozoa containing single-stranded DNA, thereby increasing the availability of double-stranded DNA spermatozoa for ICSI use . Moreover, the immediate addition of sperm-wash media after collecting semen samples or ejaculating in media, may also decrease the incidence of sperm DNA damage .