Transworld Research Network 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India
Handbook of Chemiluminescent Methods in Oxidative Stress Assessment, 2008: 195-218 ISBN: 978-81-7895-334-2 Editors: I. Popov and G. Lewin
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Oxidative stress measurement in patients with male or female factor infertility Ashok Agarwal, Marcello Cocuzza, Hussein Abdelrazik and Rakesh K. Sharma Center for Reproductive Medicine, Glickman Urological and Kidney Institute and Obstetrics and Gynecology and Women’s Health Institute Cleveland Clinic, Cleveland, Ohio, USA
Abstract Both male and female infertility is associated with poor sperm or oocyte quality. Oxidative stress has been shown to be involved in the pathophysiology of infertility. It is caused by an imbalance between the formation of reactive oxygen species (ROS) and the ability of the antioxidants to scavenge them. It is important to understand the nature of these species as they are necessary for the many physiological functions but can be harmful when produced in excess. Correspondence/Reprint request: Dr. Ashok Agarwal, Director, Center for Reproductive Medicine, Desk A19.1 Glickman Urological and Kidney Institute and Obstetrics and Gynecology and Women’s Health Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA. E-mail:
[email protected]
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The measurement of ROS in seminal ejaculates as well as in follicular fluid is explained in detail. Other markers that are helpful in evaluating oxidative stress are also described. Andrology laboratories can standardize these protocols and further aid our understanding of the causes of both male and female infertility as well as help design strategies for reducing the harmful effects of ROS and thereby improve fertilization and pregnancy rates.
Introduction Infertility is a problem with a large magnitude. Free oxygen radicals and other reactive oxygen species (ROS) affect both male and female gametes. ROS influence spermatozoa and oocytes as well as their local environments. Excessive production of ROS results in oxidative stress. Oxidative stress, in turn, affects spermatozoa quality, fertilization, early embryo development and implantation and ultimately, pregnancy rates. Oxidative stress affects both natural and assisted fertility. Therefore, understanding how ROS are produced and how they affect various functions is important. Also important is the ability to accurately measure them and establish reference values, which may help in identifying the possible causes leading to poor fertilization and subsequent stages of implantation and pregnancy. This will also be necessary to develop strategies that will help reduce oxidative stress especially during assisted reproductive techniques. This chapter aims to better our understanding on what free radicals are, their importance and how they can be measured accurately in a laboratory setting.
1. What are free radicals? Free radicals are a group of highly reactive chemical molecules that have one or more unpaired electrons and can oxidatively modify biomolecules that they encounter. This causes them to react almost instantly with any substance in their vicinity [1]. Generally, free radicals attack the nearest stable molecule, "stealing" its electron (Figure 1). When the "attacked" molecule loses its electron, it becomes a free radical itself, beginning a chain reaction. Once the process is started, it can cascade and ultimately lead to the disrupting of living cells.
2. Types of free radicals ROS represent a broad category of molecules that indicate the collection of radicals and non-radical oxygen derivatives. In addition, there is another class of free radicals that are nitrogen derived called reactive nitrogen species (RNS) (2). These reactive species are readily converted into reactive non-radical species by enzymatic or nonenzymatic chemical reactions that in turn can give rise to new radicals (Table 1).
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Figure 1. A schematic representation showing the formation of a free radical. Table 1. Examples of free radicals. Reactive oxygen species •-
Superoxide anion (O2 ) Hydrogen peroxide (H2O2) •
Hydroxyl radical (OH )
Reactive nitrogen species •
Nitric oxide (NO ) •
Nitric dioxide (NO2 ) Peroxynitrite (ONOO-)
3. Generation of free radicals in the seminal ejaculate Human semen consists of different types of cells such as mature and immature spermatozoa, round cells from different stages of the spermatogenic process, leukocytes and epithelial cells. Of these, leukocytes (neutrophils and macrophages) and immature spermatozoa are the two main sources of ROS [3,4]. Reactive oxygen species are produced by spermatozoa when a defect occurs during spermatogenesis that results in the retention of cytoplasmic droplets [5]. The retention of excess residual cytoplasm is the link between poor sperm quality and elevated ROS. Spermatozoa carrying cytoplasmic droplets are thought to be immature and functionally defective [6]. There is a strong positive
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correlation between immature spermatozoa and ROS production, which in turn is negatively correlated with sperm quality. Furthermore, as the concentration of immature spermatozoa in the human ejaculate increases, so does the concentration of mature spermatozoa with damaged DNA [7]. Peroxidase-positive leukocytes are believed to be the main source of ROS in semen. Reports suggest that positive peroxidase staining may be an accurate indicator of excessive ROS even at concentrations below the World Health Organization (WHO) cutoff value for leukocytospermia (concentration > 1x106 peroxidase positive leukocytes/mL semen) [8,9]. The extent of damage caused by ROS resulting in sperm cell dysfunction depends on the nature, amount and duration of ROS exposure in addition to temperature, oxygen tension, concentration of ions, proteins and ROS scavengers [10].
4. Antioxidant defense mechanisms Spermatozoa produce small amounts of ROS that are essential to many of the physiological processes such as capacitation, hyperactivation, and spermoocyte fusion [11,12]. Spermatozoa plasma membranes contain large quantities of polyunsaturated fatty acids (PUFA). Because their cytoplasm contains low concentrations of scavenging enzymes, they are particularly susceptible to the damage induced by excessive ROS [13,14]. The seminal plasma, however, contains two different types of antioxidants to minimize free radical-induced damage: enzymatic and non-enzymatic antioxidants (Table 2). Antioxidant defense mechanisms include three levels of protection: prevention, interception and repair. Prevention of ROS formation is the first line of defense against an oxidative insult. One example is the binding of metal ions, iron and copper ions in particular, which prevents them from initiating a chain reaction [12]. When transition metals become loosely bound to ROS, they can produce more reactive oxidants, particularly OH- [13]. Table 2. Different classes of antioxidants that scavenge ROS in seminal plasma. Enzymatic Antioxidants
Non-Enzymatic Antioxidants
Superoxide dismutase
Vitamin C, vitamin E and vitamin A (carotenoids)
Catalase
Proteins like Albumin, Transferrin, Haptoglobulin, Ceruloplasmin
Glutathione peroxidase Glutathione reductase
Glutathione (GSH) Pyruvate Ubiquinol
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Free radicals have a tendency toward triggering a chain reaction. The interception of this reaction to prevent further damage is the process of deactivation, which leads to a nonradical end product formation [12]. Alphatocopherol, a chain-breaking antioxidant, inhibits lipid peroxidation by scavenging peroxyl (RO-) and alkoxyl (ROO-) radicals. The ability of αtocopherol to maintain a steady-state rate of peroxyl radical reduction in the plasma membrane depends on the recycling of α-tocopherol by external reducing agents such as ascorbate or thiols. In this way, α-tocopherol is able to function again as a free radical chain-breaking antioxidant even though its concentration is low [14]. In most cases, free-radical induced damage can be repaired. Unfortunately, spermatozoa are unable to repair the damage induced by ROS because they lack the cytoplasmic enzyme systems required to accomplish this [15,16]. The pathological levels of ROS detected in the semen of infertile men are more likely caused by increased ROS production than by reduced antioxidant capacity of the seminal plasma [11].
5. What is oxidative stress? Oxidative stress (OS) is the term applied when oxidants outnumber antioxidants [17]. It is a common condition caused by biological systems in aerobic conditions such that antioxidants cannot scavenge the free radicals. This causes an excessive generation of ROS, which damages cells, tissues and organs [18,19]. Evidence suggests that OS induced by ROS such as superoxide anion (O2-), hydroxyl radicals (OH-) and a range of lipid peroxyl radicals produced in vascular cells is involved in the pathogenesis of a wide range of diseases of the reproductive system such as varicocele, endometriosis and infection [4,20].
6. ROS and infection Sperm damage from ROS produced by leukocytes can occur when seminal plasma is removed during sperm preparation for assisted reproduction or when the seminal leukocyte concentration is abnormally high such as in leukocytospermia [21,22]. Both leukocytospermia and elevated ROS levels have been observed in patients with accessory gland infection [16]. In these clinical settings, impairment of sperm function is attributed to abnormal lipid peroxidation caused by elevated ROS [23]. The ability of polymorphonuclear neutrophils and macrophages to produce large amounts of ROS suggests that the ROS may be ultimately responsible for the leukocyte-induced sperm dysfunction [17,19,24]. During an infection, an imbalance of pro-oxidants and antioxidants favoring the former results in OS, which impairs the sperm functions
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mentioned as well as motility and fertilization. Reactive oxygen species produced during infections of the testis and epididymis is especially harmful to spermatozoa due to the longer contact time and the lack of antioxidant protection [25]. In the final ejaculate, very high numbers of ROS-producing leukocytes are detrimental to sperm functions. An infectious injury involving ROS in the prostate gland, seminal vesicles or epididymis could impair sperm functions indirectly. Pro- and antioxidative properties of therapeutics are currently receiving more attention as part of anti-infectious therapies [18]. Leukocytes may have a role in stimulating ROS production by spermatozoa, but the exact mechanisms underlying such stimulation may involve direct sperm-leukocyte contact or may be mediated by soluble products released by the leukocytes [26].
7. Measurement of free radicals Measurement of ROS is a helpful tool in the initial evaluation and followup of infertile male patients because high levels of OS seem to be strongly correlated with reduced fertility [17]. Numerous assays for ROS measurement have been introduced recently [20] (Table 3; Fig. 2).
Figure 2. Flow diagram showing the various methods to measure seminal oxidative stress.
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Table 3. Currently available tests for detection of reactive oxygen species (direct) or their oxidized products (indirect).
Probe
Extracellular/ Intracellular
Tetrazolium nitroblue [24,27]
Ferricytochrome C
Extracellular
Chemiluminescence [25,28,29]
Luminol Lucigenin
Both Extracellular
Lipid peroxidation levels [27,30]
Thiobarbituric acid reactive substances
Measures oxidized component in the body fluids
Antioxidants, micronutrients, vitamins [31,32]
High-performance liquid chromatography
Serum and seminal plasma
Ascorbate [33]
High-performance liquid chromatography
Seminal plasma
Assay Direct Measurement
Indirect Measurement
Antioxidants enzymes [34-36] Chemokines [34,37] Antioxidant-prooxidant status [11]
Superoxide dismutase Catalase Glutathione peroxidase Glutathione reductase ELISA Total antioxidant capacity
Seminal plasma Seminal plasma Spermatozoa Spermatozoa Seminal Plasma Low molecular chainbreaking antioxidants
The chemiluminescence method is the most commonly used technique for measuring ROS produced by spermatozoa [38]. This assay quantifies both intracellular and extracellular ROS. Depending on the probe used, this method can differentiate between the production of superoxide and hydrogen peroxide by spermatozoa.
8. Luminometers A variety of luminometers can be used to measure the light intensity resulting from the chemiluminescence reaction. Although all luminometers utilize photomultiplier tubes to detect photons, they differ in the processing of
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signal input. Two different processing designs are presently found in luminometers. Photon counting luminometers count individual photons whereas direct current luminometers measure electric current that is maintained by, and is proportional to, the photon flux passing through the photomultiplier tube. The results are expressed as relative units (RLU), counted photons pre minute (cpm) or milivolts/sec. Various models of luminometers are available and they differ in price, design and features (Table 4). When comparing models, it is important to check their coefficient of variation and the lower limit of detection. Three types Table 4. Some commercially available luminometer models: price and manufacturers [38].
Model
Type
TD 20/20*
Single tube
FB-12*
Single tube
Traithler
Sensitivity and dynamic range
Price Manufacturer (US $)
0.1 fg luciferase, 5,250 >5 orders 1000 molecules of 5,350 luciferase, 6 orders
Turner biosystems Inc., Sunnyvale, CA, USA Zylux Corporation, Oak Ridge, TN, USA
Single tube
1-10 pg ATP, 7 orders
Bioscan, Washington, D.C., USA
Zylux FB 15*
Single tube
1000 molecules of luciferase, >6 7,450 orders
Optocomp-2*
Multiple tube
0.1 pg ATP, 6 orders
Autolumat LB 953**
Multiple tube
5 amol of ATP, 6 orders
MicroLumi XS*
Microplate
0.1 fg luciferase, >6 orders
Luminoskan*
Microplate
