Laboratory assessment of oxidative stress in semen

Peer review under responsibility of Arab Association of Urology.

Figures & data

Table 1 Advantages and disadvantages of direct measurement of ROS in semen.

Assays	Advantages	Disadvantages
Chemiluminescence [7]	Robust, high sensitivity and specificity	Time-consuming, large and expensive equipment Interfering variables Requires high sample volume
Nitroblue tetrazolium (NBT) [8,9]	Cost-effective, user friendly Detect neutrophils at a concentration of 0.5 × 10^6/mL or higher	Subjective interpretation of a positive vs negative neutrophils
Cytochrome c reduction test [10]	Quantify O2 ^− released during the respiratory burst of neutrophils or by isolated enzymes Good for high level of ROS production	Relatively insensitive to detect NADPH oxidase activity, if enzymatic activity is low Cannot detect intracellular O2^−
Fluorescein isothiocyanate (FITC)-labelled lectins [11,12]	Detect acrosome status	Difficult to distinguish true and false acrosomal reactions Impossible to detect sperm viability and acrosomal status in one picture Fluorescent signal fades at times
Electron spin resonance [10,13]	Broad usages such as observations of free radicals, analysis of free radical characteristics, quantitative analysis of free radicals, and kinetic analysis Good for high level of ROS production	Limitation if a free radical reacts immediately with a molecule other than the spin-trapping agent Inference factors such as possible neutralisation

Table 2 Advantages and disadvantages of indirect measurement of ROS in semen.

Assays	Advantages	Disadvantages
Myeloperoxidase or Endtz test [9]	Clearly distinguishes WBCs especially ROS producing granuloctyes from other immature germ cells in semen	Cannot be used to detect ROS generation by spermatozoa
Lipid peroxidation levels [8]	Malondialdehyde is a coloured substance that can be measured by fluorometry or spectrophotometry Low sperm concentration of malondialdehyde can be measured through sensitive HPLC equipment or spectrofluorometric measurement of iron-based promoters	Not widely used in clinical practice at this time
Chemokines [14,15]	Produced as a result of ROS induced inflammation	Requires a large amount of biological material (>0.5 L of culture supernatants)
Antioxidants, micronutrients, vitamins (vitamin E, vitamin C) [8]	Cofactor of essential enzymatic reactions of ROS	Assesses an end state occurring secondary to other unknown pathological processes.
Antioxidants – TAC [16,17]	Rapid colorimetric method Total antioxidants in seminal plasma measured	Does not measure enzymatic antioxidants, or individual antioxidants Requires expensive assay kit and microplate reader.
DNA damage [18,19]	Robust and sensitive method Multiple methods are available to measure DNA fragmentation such as sperm chromatin structure assay (SCSA), sperm chromatin dispersion, TUNEL, comet, sperm chromatin dispersion assay, nuclear protein composition, sperm nuclear maturity test, and 8-OHdG	Accessibility of the DNA Inter-observer, intra-observer + inter-assay and intra-assay variability. Lack of standardised reference Technique itself (i.e. 8-OHdG) has potential to cause DNA oxidation, which will interfere with the basal level

WBC, white blood cell; 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

Fig. 1 External view of the luminometer connected to the monitor for real-time display of ROS production on the monitor screen.

Fig. 2 Setting up of the tubes for ROS measurement by chemiluminescence assay. Preparation of blank, negative control, test sample, and positive controls.

Fig. 3 Luminometer showing the (A) external and (B) internal view.

Fig. 4 Components of the TAC kit used in colorimetric measurement of total antioxidants. It comprises a standard assay buffer, Trolox standard, chromogen, and metmyoglobin.

Fig. 5 Distribution of TAC levels in healthy controls and infertile patients.

Fig. 6 Measurement of ORP by the MiOXSYS system. (A) MiOXSYS analyser showing the sensor socket and the sensor module and (B) sensor showing the reference cell and the sample port where the sample is loaded.

Fig. 7 MiOXSYS system (A) placing of the sensor strip on the sensor socket (B-C) inserting the sensor in the sensor port.

Fig. 8 Loading the semen sample on the sample port of the sensor strip.

Table 3 Advantages and disadvantages of ORP in semen.

Test	Advantages	Disadvantages
ORP [24^[25]^[26]–27]	Provides redox balance in real time with all known and unknown oxidants and antioxidants Less time-consuming and requires little or no expertise Measures both fresh and frozen specimens ORP threshold value used to identify normal and abnormal semen quality: 1.36 mV/10^6  sperm/mL ORP threshold value used to identify fertile and infertile patient: 1.41 mV/10^6  sperm/mL	Affected by viscosity of the sample

Table 4 Comparisons between chemiluminescence and ORP.

Variable	Chemiluminescence	ORP
Type of sample [7,21]	Only fresh sample Whole seminal ejaculate in an unprocessed or processed sample; sperm preparation by swim-up procedure and density gradient	Can measure in both fresh and frozen samples
Time to complete the test [7,21]	Up to 60 min from start to finish. 15 min of actual run time and another 30 min to prepare the samples (control, blank, negative, test and positive sample), enter the information, print and calculate final ROS value.	Up to 4 min from start to finish
Sample volume [7,21]	Minimum of 800 µL of sample in duplicate	High reproducibility; requires only 30 µL A single aliquot is adequate
Costs [7,21]	Around $30,000	Ranges around $30 per sensor (sample)
Confounding variables [7,21]	The specific type of luminometer instrumentation; single vs multiple tube, semen age, viscosity, presence of leucocytes and other nonspecific interference	Severe oligozoospermia (<2 × 10^6  sperm/mL) and viscous samples