Background
Reactive oxygen and reactive nitrogen species (ROS and RNS), a group of highly reactive oxidants, most of which contain unpaired electrons, are usually known as ROS or free radicals. At physiological levels they have important roles in metabolism in all aerobic organisms. However, excessive ROS production which can not be effectively controlled by antioxidants leads to oxidative stress (OS) which has been linked to many pathological processes, including male infertility [
1‐
3]. In the genital tract, low levels of ROS are necessary for normal function of human spermatozoa [
4], including their capacitation, acrosome reaction and sperm-oocyte fusion. On the other hand, excessive OS may cause lipoperoxidation of sperm membranes resulting in DNA damage and sperm apoptosis. Standard semen analysis is often inadequate to explain conception failure as the routine microscopic evaluation can not reveal subtle disorders at the molecular level which may be caused by OS [
5]. This situation often leads to diagnosis of idiopathic infertility. Moreover, there is some degree of overlap in sperm parameters between fertile and infertile males [
6].
Although the importance of seminal OS assessment has already been advocated for semen analysis by WHO Laboratory Manual 1999 [
7], only a few centers worldwide have so far developed methods for indirect measurement of ROS production in semen. ROS production in human spermatozoa was first measured in washed spermatozoa suspended in phosphate buffered saline (PBS) to the concentration of 20 millions per milliliter. The chemiluminescent signal induced by the addition of luminol was measured integrally and expressed in cpm [
2,
8,
9]. We implemented this method in the University Hospital in Olomouc in 2002, to our knowledge for the first time in the Czech Republic. The weakness of this approach is that seminal plasma with its powerful total antioxidant capacity (TAC) is removed prior to measurement. Later, as a better predictor of oxidative sperm damage, the ROS/TAC score was introduced [
10]. Unfortunately, both ROS and TAC measurements are subject to various methodological problems. ROS measurement by luminol-induced chemiluminescence in neat semen was first reported by Allamaneni
et al. [
11]. Since the beginning of 2007 we have been measuring ROS simultaneously in neat ejaculate and in spermatozoa suspension in PBS, i.e. washed semen. The aim of our study was to compare their clinical usefulness in the evaluation of the male factor infertility and to evaluate the commutability between the ROS measurement in washed and neat semen.
Discussion
Since the appearance of the first reports in the early nineteen nineties [
1,
2,
16,
17], the role of OS in the pathophysiology of male infertility has been gradually accepted. The importance of seminal ROS production has been already stressed in the WHO Manual (1999) and several methods of ROS detection in semen have been reported [
8,
9,
12,
18]. Nevertheless, reliable and reproducible methods of ROS measurement in semen for routine clinical use are still missing. Such method(s) would be a useful tool in the diagnosis of male infertility and in the selection of patients who would benefit from antioxidant treatment [
3,
18‐
22].
Measurement of the level of OS in ejaculate arising from imbalance between ROS production and the capacity of the complex antioxidant defense system is extremely difficult. The direct measurement of free radicals is practically impossible. The indirect methods have either used lucigenin or luminol mediated chemiluminescence in spermatozoa suspension [
1], or they have to rely on the detection of some stable oxidized end-products, the so called biomarkers of OS, mostly in body fluids [
23]. Luminol mediated luminescence is preferred as it can detect the sum of several important intra- and extra-cellular ROS including hydrogen peroxide, superoxide and hydroxyl radicals [
2,
9,
16].
Unfortunately, there are some limitations to ROS measurement in spermatozoa suspension in PBS. The absolute values are expressed either in count per minute (cpm) or RLU per minute, and moreover, may differ with respect to the sensitivity and type of luminometer used. ROS production by spermatozoa suspended in PBS declines with time, therefore the measurement should be performed within one hour of obtaining semen sample [
9]. There is also a possibility of an artificial increase in ROS production caused by repeated centrifugation during the preparation of the spermatozoa suspension [
14]. All this makes it difficult to compare results reported from different settings. Another, even more important drawback of the measurement of ROS in washed spermatozoa, is the fact that they are deprived of their natural antioxidant environment, seminal plasma. The ROS/TAC score [
10], thought to assess only the excess of ROS not scavenged by seminal plasma antioxidants, introduces another variable which may also be prone to analytical error. According to our experience [
24], the TAC of seminal plasma measured by the TAS Randox
® method varied to a much smaller extent in contrast to ROS production because it measured only non-enzymatic antioxidants.
The determination of ROS production in neat semen is a better solution. It avoids centrifugation and washing procedures and shortens the time lag between semen collection and ROS measurement. Moreover, it can be expected to measure only the excess ROS which are not scavenged by seminal plasma antioxidants and thus directly identify samples with OS. In our setting, the ROS levels in neat semen were lowest in samples from fertile volunteers, normozoospermic men and leukocyte-free samples from men with semen abnormalities. ROS levels were significantly higher in samples with peroxidase-positive leukocyte concentrations <0.5 × 106/ml and the highest in samples with leukocyte concentrations >0.5 × 106/ml. The corresponding results measured in spermatozoa suspension in PBS were markedly higher, in some cases up to two orders of magnitude. A significantly higher ROS production was detected only in samples from SA males with leukocyte concentrations >0.5 × 106/ml.
Our findings of a reasonable commutability of measuring ROS in washed and neat semen well agree with the first reported comparison of luminol-mediated chemiluminescent measurement of ROS production in neat semen versus spermatozoa suspensions in PBS [
11]. Allamameni
et al. evaluated semen from 34 semen donors and 44 patients with abnormal semen parameters. The ROS production in neat semen of donors was about five times lower than the respective ROS levels in spermatozoa suspension. The levels measured in neat semen of patients with abnormal semen were significantly higher than in healthy donors. A later study of Athayde
et al. included semen samples from 114 fertile Brazilian men seeking voluntary sterilization by vasectomy and 47 subfertile males [
25]. In samples without leukocytes, the threshold for normality of ROS in neat semen samples was set at 0.55 × 10
4 cpm per 20 × 10
6 spermatozoa; almost 20 times lower than that in washed spermatozoa in PBS. A recent study by this group [
26] reported the median and interquartile range of seminal ROS levels in neat semen samples of 78 vasectomy candidates younger than 40 years as 0.29 (0.18, 0.58) × 10
4 cpm per 20 × 10
6 spermatozoa, which agrees well with our findings in Czech fertile volunteers, that is 0.26 (0.12, 0.55) × 10
3 RLU/min per 20×10
6 spermatozoa, even though they were obtained on a different population and using a different luminometer.
This study, to our knowledge the first one in the Czech Republic, has proved that a significant positive correlation exists between ROS levels in neat semen and that in spermatozoa suspension in PBS, in which the antioxidant capacity of seminal plasma is absent. This suggests that the individual total antioxidant capacity of seminal plasma may vary to a lesser extent than the ROS produced by spermatozoa and/or activated leukocytes.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HF wrote the paper, IO designed the study, recruited study subjects and controls, provided clinical information and gave final approval, JN perfomed chemiluminesce measurements, JB and MS performed semen analysis, samples preparation and data collection, LR performed statistical analysis. All authors read and approved the final manuscript.