Leukocytospermia and Oxidative Stress

Chapter 23 Leukocytospermia and Oxidative Stress

Margot Flint , Ashok Agarwal , and Stefan S. du Plessis

Abstract The invasion of microorganisms and infective bacteria in the genito- urinary tract leads to the rapid increase in white blood cells, a condition referred to as leukocytospermia. This inﬂ ammatory response, aimed at killing the microorganisms via the production and release of reactive oxygen species (ROS), can result in pathologically high concentrations of ROS. When these concentrations greatly exceed the level required for normal physiological function, the natural defense system of scavenging antioxidants can be overwhelmed, resulting in oxidative stress (OS) thereby compromising the integrity of spermatozoa and functional parameters vital for successful fertilization. The complexity of OS is furthered when additional factors (e.g., smoking, varicocele) increase ROS levels in the male genito-urinary system. At present, the association between semen parameters and leukocyte concentrations is a focal point in the ﬁ eld of male reproductive science. This chapter aims at exploring the relationship between leukocytospermia, OS, the harmful effects on male reproductive potential, as well as possible treatment regimes.

Keywords Leukocytospermia • Oxidative stress • Male genitalia tract infections • Reactive oxygen species • Leukocytes • Antioxidants

M. Flint, MSc • S. S. du Plessis, PhD, MBA (*) Division of Medical Physiology , Stellenbosch University , Francie van Zijl Avenue , PO Box 19063 , Tygerberg 7505 , South Africa e-mail: [email protected] A. Agarwal, PhD Center for Reproductive Medicine , Cleveland Clinic , Lerner College of Medicine, 9500 Euclid Avenue , Cleveland , OH 44195 , USA

A. Agarwal et al. (eds.), Studies on Men’s Health and Fertility, Oxidative Stress 517 in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-776-7_23, © Springer Science+Business Media, LLC 2012 518 M. Flint et al.

23.1 Introduction

Various infective pathological conditions can result in the defensive reaction of tissues to the attack of microorganisms. An infection leads to the activation of specifi c immune cells, in particular a form of white blood cells (WBC’s) known as leukocytes. The result which arises from such defensive action is the generation of reactive oxygen species (ROS). These are chemically reactive molecules containing oxygen and are produced by the invading leukocytes. The defensive oxidative pathway that the ROS take in killing microbes can have a biopositive effect when ROS concentrations are maintained at a low level or are counterbalanced by the protective antioxidant scavenging system which maintains homeostasis [1 ] In certain conditions whereby homeostasis is disrupted, an imbalance arises between oxidants and antioxidants, in favor of the former, resulting in a condition known as oxidative stress (OS) [1, 2 ] . The harmful effect of this imbalance is OS-induced damage, which is a threat to all cellular elements, including: amino acids, carbohydrates, lipids, and nucleic acids [3 ] . OS is considered as the circumstance which underlies the etiology of various human conditions [ 4 ] . In context of this, a wide variety of diseases exist in which the pathophysiological role of ROS and OS has been implicated in the patho- genesis of the condition. Examples of these include: cancer, diabetes, and infl ammatory bowel disease [3 ] . In the male reproductive system, the harmful effect that ROS can have on sperm and their parameters has been known for a relatively long period of time. In the mid- 1980s, Professor John Aitken and his group pioneered studies into the activities of ROS in male reproductive biology [3 ] , which has continued comprehensively over the following years, in particular the Cleveland Clinic Foundation in America which has extensively researched the effects of OS in male infertility. Research into the negative infl uence that leukocytospermia can have on semen parameters introduced investigations into the role of leukocyte-produced ROS. ROS are spontaneously generated and required at a basal level for certain spermatozoal physiological functions [5 ] . It has been proven that samples considered being peroxidase-positive have higher concentrations of ROS [6 ] and these polymorphonu- cleated leukocytes (PMNL) release oxygen radicals, such as hydrogen peroxide and superoxide which are known toxic factors towards spermatozoa. Irrelevant of the concentration of leukocytes in semen, the presence of these WBC’s have been shown to be associated with OS which can negatively effect semen parameters such as sperm concentration and morphology [7 ] .

23.2 Male Genital Tract Infections

Specifi c seminal parameters and chemical components which contribute towards the ejaculate can serve as diagnostic tools in assessing if the accessory sex glands are normally functioning [ 8 ] . Diverse irregularities or deviations from the standard 23 Leukocytospermia and Oxidative Stress 519 reference values of these physical characteristics indicate a possible underlying pathophysiological condition [ 9 ] . The response of the genito-urinary tract to the invasion of microorganisms and infl ammation is an important component of the immune defense system [10 ] . The reaction is considered to be extremely similar to the reaction in other body sites [ 11] . Therefore, a semen analysis can serve as a valuable diagnostic tool in assessing possible disorders of the male genital tract and the secretory pattern of the male accessory sex glands [ 9 ] . The most common infective bacteria in mixed accessory gland infection (MAGI) are Chlamydia trachomatis (41.4%), a common sexually transmissible pathogens in sexually active young men, followed by Ureaplasma urealyticum (15.5%) and Mycoplasma hominis (10.3%) [12, 13] as well as Neisseria gonorrhoeae, an additional marker of seminal tract infection [ 8, 14 ] . In men experiencing infertility issues, the presence or colonization of U. urealyticum and M. hominis in semen is a common fi nding [14 ] and semen cultures of bacterial pathogens remain the most common diagnostic method for seminal tract infections [ 8 ] . This passive or active invasion of these bacterial strains induces a generalized or local reaction in the urogenital tract [ 1 ] . This infl ammatory response which can continue for extended periods of time leads to a pathological condition resulting in the activation of seminal WBC’s [ 15] . Inadequate treatment of an infection and eradication of bacterial pathogens can lead to a chronic bacterial infection of the male accessory sex glands [4 ] .

23.3 Leukocytospermia

Human semen is a heterogeneous fl uid which contains a variety of cellular elements beyond spermatozoa. Several immunologic factors are present in human semen, such as chemokines, immunoglobulins, and growth factors [16 ] , as well as a subset of WBC’s. The microscopic evaluation of almost all semen samples will display nonspermatozoal cells, in particular WBC’s [ 17 ] , which encompass various forms such as granulocytes, lymphocytes, and macrophages [ 13 ] . An abnormally high concentration of WBC’s in the semen is a condition called leukocytospermia, also referred to as leukospermia, pyospermia, or pyosaemia [ 18 ] . An increased concentration of leukocytes is the basic molecular defense mechanism against the detection of foreign organisms. The World Health Organization (WHO) criteria defi nes leukocytospermia as the presence of >1 × 10 6 WBC’s/mL of semen [ 18 ] . This threshold is regarded as a possible indicator of an ongoing male genital tract infection [ 18 ] . Specifi cally, the detection of pathological concentrations of leukocytospermia with the exclusion of a bladder infection or urethritis has been suggested as a basic diagnostic tool in recognizing genital tract infection [19, 20 ] . The activation of PMNL, which constitute 50–80% of the total seminal WBC count [ 8, 21 ] , results in releasing a protease by degranulation known as elastase [ 22 ] . The presence of this particular protease is considered a highly reliable and sensitive marker of an asymptomatic infection [ 23] and can be used in diagnosing 520 M. Flint et al. a clinically silent illness [ 24 ] . At present, due to the fact that the condition is asymptomatic and various sites in the reproductive system can be affected, the exact location of the origin of excess leukocytes is unknown [ 25, 26 ] . Due to the lack of understanding as to where exactly leukocytes are produced from, their release may be initially prompted by an infl ammatory response of the genital tract to a bacterial invasion and then continually produced in their absence by immuno- logical activity [ 7 ] .

23.4 Effects of Leukocytospermia

Among the increasing number of partners experiencing fertility challenges, it has been estimated that the male factor is solely responsible for 30% of the failed fertilization rates [26 ] . Symptomatic and asymptomatic urinary tract infections which produce leukocytes is a condition frequently observed in infertility clinics [ 27, 28 ] . This negative effect on the male’s fertilizing potential can be a result of the direct correlation which is found between increased concentrations of leukocytes and chromatin alterations and morphological abnormalities [6 ] . Despite the controversy which has been created from studies, the general biological and clinical conclusion is that the presence of leukocytes in semen, regardless of the concentration [ 7 ] , is associated with OS and can be of a negative inﬂ uence on certain parameters which can impair fertility [29, 30 ] .

23.5 Reactive Oxygen Species

The metabolism of oxygen results in the generation of highly reactive agents belong- ing to the class of free radicals and are termed ROS [31 ] . Free radicals are short- lived atoms or molecules that contain one or more electrons with unpaired spin [ 1, 32] . These chemical intermediates can include hydroxyl radicals, superoxide anions, hypochlorite ions, and peroxyl radicals and are natural by-products of normal physiological processes [ 3, 31, 33 ] . An important factor to consider when examining the role of ROS is the concentration at which they are found. ROS illicit a biopositive inﬂ uence when maintained at low concentrations. However, excessive generation of these oxidizing agents, accompanied by a lack of inactivation, results in damage to biomolecules [31, 32 ] . In the light of this counterbalance, spermatozoa face what is known as the “oxygen paradox.” This contradiction lies in the fact that spermatozoa require oxygen for survival; however, the metabolites it produces, for example ROS, can compromise cell survival [ 30, 32] . When this environment develops, the natural defense system of scavenging antioxidants can be overwhelmed and basic semen parameters are negatively affected [ 15 ] . The consistent production of ROS by cellular aerobic metabolism makes it challenging to prevent OS injury in spermatozoa. The result is the counterbalance and OS which can cause pathological effects [ 34 ] . The extent of the damage that OS can have on semen and spermatozoa parameters 23 Leukocytospermia and Oxidative Stress 521 is dependent on the level of ROS that is produced by the activated leukocytes and the extent by which it can overwhelm the natural antioxidant defense system [35 ] .

23.5.1 Positive Effects of ROS

Like all cells living in aerobic conditions, spermatozoa need a consistent supply of oxygen. Under controlled physiological conditions, the generation of a low level of reactive oxidants is essential for maintaining normal physiological processes such as sperm–oocyte fusion, capacitation, and hyperactivation [ 31, 32 ] . In order to maintain cellular stability, this small amount of ROS needs to be continuously inac- tivated [ 5, 31, 32 ] .

23.5.2 Negative Effects of ROS

In contrast to a benefi cial role these oxidizing agents can play in various cellular events, it must be strongly considered that all cellular components, including nucleic acids, lipids, and proteins, are potential OS targets as a result of supra-physiological concentrations of ROS [ 1, 32] . Due to the fact that free radicals predominantly attack the closest stable molecule, which subsequently turns that specifi c particle into a free radical, ROS can be involved in a cascade of reactions which can damage a wide variety of biomolecules [ 31, 36] . The action which occurs on a molecular level by the interaction of ROS is the removal of hydrogen molecules [37 ] which results in a loss of motility [ 38 ] , as well as other spermatozoa functions such as capacitation and acrosomal reactions [36 ] . Various conditions can promote the production of ROS which include: an increase in cellular metabolism, loss of antioxidant capability, and presence of infl ammatory cells, for example leukocytes [1 ] . Two approaches prevent possible cell damage from pathologically or physiologically produced ROS. The fi rst preventative step involves antioxidants which are compounds that scavenge and suppress ROS. The action of antioxidants counterbalances the damaging effect of OS. The second action is the control of the reactive oxidants in a microenvironment which limits the possible damaging effect that the ROS can have [ 1 ] .

23.6 Generation of ROS in the Male Reproductive System

In contrast to normozoospermic semen samples, teratospermic, oligospermic, and asthenospermic subjects present semen samples with higher concentrations of ROS [ 39] . These free radicals are produced in spermatozoa by two predominant systems [ 31, 32, 40, 41 ] . First, as the generation of ROS is a result of oxygen metabolism, spermatozoal mitochondria are evidently the main contributor of the free radical [ 36 ] . Through the act of cellular respiration under normal physiological conditions, 522 M. Flint et al. superoxide anions are the main ROS produced [ 36 ] . Spermatozoa require a constant source of energy to maintain motility and ROS is generated at a mitochondrial level by the nicotinamide adenine dinucleotide-dependant oxido-reductase (NADH) pathway [42 ] . Second, the sperm plasma membrane can generate ROS through the nicotinamide adenine dinucleotide phosphate-dependant oxidase system (NADPH) [35 ] . The plasma membrane is incredibly sensitive to ROS due to the high content of polyunsaturated fatty acids (PUFA), primarily docosahexaenoic acid [1, 4, 43 ] . This form of fatty acid provides ﬂ uidity to the plasma membrane with their highly spe- ciﬁ c lipid composition [36 ] . However, an unstable high concentration of ROS in an ejaculate can result in the peroxidation of these lipids [44 ] . The negative resultant effect from the peroxidative damage caused by the oxygen free radicals is increased permeability of the sperm plasma membrane [29, 44– 46 ] . This process is induced by hydrogen peroxide, the most toxic compound for spermatozoa [ 3, 35, 47 ] . An additional and third source of ROS in seminal plasma is xanthine oxidase, an enzyme crucial in the catabolism of purine [48 ] .

23.6.1 Generation of ROS by Leukocytes

Despite the fact that spermatozoa are capable of producing ROS, even in small amounts, the most predominant source of ROS formation by phagocytosis is peroxidase-positive leukocytes, mainly polymorphonuclear (PMN) neutrophilic granulocytes, which are crucial in diagnosing MAGI as they are active in the inﬂ ammatory process [ 41, 49– 51] . In comparison to the amount of ROS produced by spermatozoa at the point of capacitation, the production rate by leukocytes is 1,000 times greater [ 52 ] . Various biological arguments between research groups have been created in determining the exact level of leukocytes that produce concentrations of ROS which are harmful towards spermatozoal parameters. The general consensus is that irrelevant of the exact concentration, the mere presence of these WBC’s has been shown to be associated with OS [ 3, 39 ] . Since the presence of ROS in a semen sample can be the result of either spermatozoa in a pathological condition or leukocyte inﬁ ltration following infection, it is important in a clinical sense to accurately establish the source of seminal ROS [34 ] .

23.7 Sources of OS

23.7.1 Extracellular Sources

When considering the OS that cells experience, it must be considered that this state can result from exogenous or endogenous inﬂ uences [4 ] . The male genito-urinary system can be exposed to damaging exogenous factors, which are suggested sources 23 Leukocytospermia and Oxidative Stress 523 of ROS and promoters of infection. These include lifestyle behaviors such as smoking and external inﬂ uences such as environmental pollutants [ 35 ] , as well as clinical diagnoses such as varicocele [53, 54 ] and spinal cord injuries [31 ] . Assisted reproductive therapy (ART) is also an established source of ROS [39 ] . The removal of seminal plasma is a procedure carried out in preparation for ART. This subsequently also removes natural antioxidants in the semen [ 7] . In this context, an additional exogenous source of OS can be certain processing techniques that are used in preparing semen, such as centrifugation [37 ] .

23.7.2 Intracellular Sources

In contrast, when considering endogenous inﬂ uences in the context of the male reproductive system, the seminal ﬂ uid contains a broad variety of cellular cells which can be considered possible sources. These include epithelial and round cells, spermatozoa at varying stages of spermatogenesis, as well as leukocytes [ 3 ] . Taking this into account demonstrates that essentially all ejaculate cells are potential providers of ROS [ 34] , with seminal leukocytes and abnormal or immature spermatozoa being the predominant contributors [ 29, 41 ] . Immature spermatozoa have been postulated as being responsible for oxidative damage to their mature counterparts during the seminal migration from the seminiferous tubules to the epididymis [ 6 ] . Following an increase in the production of ROS by these two sources, the natural antioxidative capabilities are overwhelmed with the induction of OS [44 ] .

23.8 Activation of Leukocytes

Infection of the genital tract is often observed as an asymptomatic subclinical infl ammation [55, 56 ] with up to 80% of leukocytospermic semen samples showing no visual detection of microbial infection [ 26 ] . Following infi ltration of infectious agents into the genital tract, the initial immune reaction is the increase in seminal leukocytes [15 ] . This infl ammatory process aimed at killing the microorganisms results in the increase in leukocyte-produced ROS from the activated WBC’s [ 11 ] . The infi ltration and activation of these excess PMNL can cause excessively high concentrations of ROS which greatly exceed the required level for normal physiological functions. This elevation results in OS being initiated which produces a damaging prooxidant load [36 ] . Irrelevant of the concentration of leukocytes in semen, the presence of these WBC have been shown to be associated with OS and impairment in the quality of semen and sperm parameters such as concentration and morphology [ 7 ] . Two pathways are found in response to the activation of seminal leukocytes during infection. The fi rst is the increase of NADPH through the hexose mono- phosphate shunt [ 32, 57 ] . The second route is the generation of high concentrations 524 M. Flint et al. of ROS produced by a respiratory burst which acts as a protective mechanism following the infection [1, 32 ] . The result is the antioxidants in the cell being con- sumed. This oxidative burst is from the activation of the myeloperoxidase system present in both macrophages and PMNL and is controlled by various cytokines [ 1, 58 ] . The damaging effect of the infection is shown by the fact that leukocytes in an activated state are able to produce a 100-fold increase in the concentration of ROS when compared to leukocytes that are not activated [59 ] . This rapid and high increase of OS is an example of how damaging the condition of leukocytospermia can be.

23.9 Identiﬁ cation of an Infection

Biochemical evidence has shown that in males with clinically silent genital tract infections, the prostate and seminal vesicles are the organs predominantly affected and targeted by infl ammation [ 60 ] . The primary role of the seminal vesicles is to provide high concentrations of fructose to the seminal plasma and is vital to the functional integrity of spermatozoa as it is the major source of glycolytic energy in order to maintain motility [ 18 ] . The reference value for normal concentrations of fructose is 13 μ mol (2.34 mg) or more per ejaculate [18 ] . Determination of the concentration of the monosaccharide is commonly employed in laboratories for a variety of purposes including the auxiliary diagnosis of retrograde ejaculation, obstructive and nonobstructive azoospermia [ 61 ] , and as a marker to assess seminal vesicular function [ 18, 62] . Changes in seminal vesicles secretory patterns can modify the composition of products of the vesicular fl uid and of the ejaculate, affecting sperm function [63 ] . Conditions such as abnormal concentrations of zinc and fructose as well as hyperspermia, an increase in semen volume (>6 mL), and hypospermia (<2 mL) are all symptomatic of a glandular dysfunction in the seminal vesicles [ 64 ] . Infl ammation from infection can cause the secretory epithelium lin- ing the seminal vesicles to be affected, resulting in atrophy and a subsequent decrease in the seminal fructose concentration [61 ] . Biochemical evidence has shown that in males with clinically silent genital tract infections, the prostate is the organ that is predominantly affected and targeted by infl ammation [ 60] . The total output of citric acid has been stated as the strongest discriminating power in terms of biochemical markers in differentiating between semen of infected and nonin- fected infertile men [ 65 ] . The threshold level for normal citrate concentrations is >52 μ mol (9 mg) per ejaculate [18 ] . Inadequate treatment of an infection and removal of bacterial pathogens can lead to a chronic bacterial infection of these two male accessory sex glands [53 ] . An important symptomatic effect accompanying an infection is the damaging effect that the prostate and the seminal vesicles suffer from [18, 64 ] . Also, the leukocytes, predominantly PMN-granulocytes, are produced from these genito-urinary glands [ 66 ] due to the damaged tissue attracting WBC’s to the site of infection [64 ] . During the up- regulation of cell-mediated immunity in response to infection, a coordinated response allows for PMNL to release a protease by degranulation known as elastase [ 16, 22 ] . 23 Leukocytospermia and Oxidative Stress 525

The presence of this particular protease is considered to be a highly reliable and sensitive marker of an asymptomatic infection [23 ] and can be used in diagnosing a clinical infection [ 24 ] . It can also be used in clinical application as a marker of the efﬁ ciency of anti-inﬂ ammatory treatment, as well as an alternative means of monitoring the levels of seminal WBC’s [ 22 ] . An enzyme-linked immunoabsorbant assay (ELISA) can be used for the quantitative detection of PMN-elastase. PMN- elastase determination has been stated as being a useful screening method to detect leukocytospermia [67 ]

23.10 Identiﬁ cation of Leukoctyospermia

A wide variety of detection methods are available to identify leukocytes in seminal plasma. Determining the quantity of seminal ROS generation in a sample has been suggested as an important test to be performed in the clinical andrology assessment [57 ] . The identifi cation of the presence of ROS-producing cells in a semen sample can be an important step taken in establishing the underlying cause for acquired or inherent defects that may be undetected [57 ] . The tests carried out to establish the condition of leukocytospermia include immunocytology, peroxidase, polymorphonuclear-elastase, and cytology [7 ] . The traditional method and technique recommended by the WHO [ 18 ] for counting leukocytes in human semen is to use a histochemical procedure to identify the peroxidase enzyme found in the cytoplasm that characterizes PMN-granulocytes [37 ] . In the examination of a semen sample, the difference between immature germ cells and leukocytes cannot be morphologically distinguished from each other, therefore cytological identifi cation is an inaccurate method to detect leukocytes [68 ] . PMN- granulocytes and leukocytes are considered “round cells” of nonspermatogenic origin [69 ] . Due to a lack of diligence in differentiating between different categories of round cells, particularly spermatogenic germ cells and leukocytes, an overestima- tion of the number of leukocytes in a semen sample can often result [70 ] which can increase the chance of misdiagnoses. There is a signifi cant relationship between the presence of PMN granulocyte elastase and the concentration of peroxidase-positive cells in a semen sample [ 68 ] . This particular cytological staining technique is considered to be a reliable method in the detection of leukocytes as it has minimal chances of misdiagnoses [27 ] . However, it must be considered that during the infl ammatory process, degranulation and deregulation occur, resulting in the extracellular liberation of the leukocyte’s cellular contents. The quantifi cation and identifi cation of leukocytes based on this specifi c method may therefore be affected as the peroxidase compound may be undetectable [71 ] . In order to ensure the most accurate quantifi cation of leukocytes, an obvious recommendation would be to use peroxidase staining, which detects intracellular enzymatic activity [67 ] , as well as an alternative method such as the biochemical analysis of the concentration of PMN-elastase, which identifi es extracellular enzymes. 526 M. Flint et al.

23.11 Measuring ROS Production

While detecting the presence of ROS-producing cells is possible, the direct measurement of in vivo ROS concentrations is challenging due to the fact that ROS are very short-lived [ 1 ] . In the fi eld of male reproductive studies, the measurement of OS is quantifi ed only indirectly by the presence of ROS. The quantifi cation of ROS has been promoted as a valuable test to be performed in the assessment of seminal plasma that is to be used for ART as it may be benefi cial in predicting end points such as the fertilization rate [ 5 ] . The detection and quantifi cation of ROS as a means of studying the role of OS is carried out in varying ways. One of these methods is the luminol-dependant chemiluminescence assay, which indirectly measures ROS produced by spermatozoa by means of a sensitive probe, which can either be luminol or lucigen [ 31, 41] . The principle of this test is based on the concentration of released luminescence in a luminometer [ 1, 72 ] . This is the amount of light that is generated with the interaction between the ROS present in the sample and a specifi c chemiluminescent probe [ 41] . This particular assay is a repro- ducible test with high sensitivity and specifi city and allows for both the extra- and intracellular ROS to be measured [ 5, 31] . In conjunction with quantifying and identifying ROS in the semen sample by means of this particular assay, the relationship between the presence of leukocytospermia and the excessive ROS concentrations can be investigated. This can be done by determining leukocytospermia by perform- ing the Endtz test, which is a myeloperoxidase staining technique based on the peroxidase activity of PMN [41 ] . An Endtz positive semen sample has been proven as an indicator of positive chemiluminescence for ROS [41 ] . The second test that can be used to determine if ROS is generated is by the measurement of lipid peroxidation through the Lipid Peroxidation Assay (LPO) [ 1 ] . Lipid peroxidation in spermatozoa is initiated by the presence of ROS and results in the generation of lipid hydroperoxides [ 1 ] which are stable molecules under normal physiological conditions [ 44 ] . However, under conditions of elevated ROS and resultant OS, malondialdehyde is produced from the decomposition of lipid peroxides [ 44 ] . The concentrations of this particular compound can be measured in biochemical assays to assess the level of peroxidative damage [ 73 ] . An additional third test to assess the level of seminal OS is the ROS-TAC score. This particular method combines the concentrations of TAC and ROS in semen samples and is quantifi ed by using a statistical formula that allows for the optimal quantifi cation of seminal OS [ 44, 54 ] . Owing to cases where normozoospermic males suffer from infertility, which may be the result of seminal OS, the ROS-TAC score has been suggested as an advantageous means of assessing OS in comparison to ROS alone when distinguishing between fertile and infertile subjects [54 ] . Due to the clinical relevance of identifying OS due to its causative role in infertility, research has led to the development of an inexpensive assay: the photometric nitro blue tetrazolium (NBT) assay which allows for the measurement of seminal ROS production [ 74] . This fi nding serves as a hopeful indicator of what future research into OS may hold. 23 Leukocytospermia and Oxidative Stress 527

23.12 Reproductive Sytems Defense Mechanisms

In examining the negative effects of high concentrations of ROS and the resultant OS, it must be considered that a degree of protection is provided to the spermatozoa by antioxidant defense mechanisms [ 44, 75] . Due to the proven relationship existing between increased incidents of infertility and inept antioxidant functioning in the seminal plasma [48, 76 ] , determining the concentration of antioxidants may allow for the assessment of the fertility capabilities [ 31 ] . The properties of antioxidants allow for varying degrees of protection in the seminal plasma by acting as scaven- gers and creating a defense system against OS by removing the extracellular free radicals [3, 32] . This is due to the aforementioned fact that all ejaculates contain forms of cellular elements which can protect the spermatozoa from free radical toxicity [49 ] . In assessing the antioxidative capabilities of a semen sample, the pre- viously explained TAC (Total Antioxidant Capacity) score is utilized. In determining the TAC score of a semen sample, two measurement techniques can be performed: a colorimetric assay and enhanced chemiluminescence, which quantiﬁ es TAC levels according to a set equation [31 ] .

23.12.1 Antioxidants

The line of defense employed against oxidative insult consists of antioxidants which are agents that reduce the level of OS by breaking oxidative chain reactions and can be classiﬁ ed according to two subsets: enzymatic or “natural” such as superoxide dimutase, catalase, and glutathione peroxidase, and nonenzymatic or “synthetic” chain-breaking antioxidants for example pyruvate, glutathione, and carnitine, all of which can offer protection to spermatozoa against hazardous OS produced by the leukocytes [1, 2, 10, 77, 78 ] . These defensive compounds against free radical-induced OS can be regarded in two categories: preventative and scavenging antioxidants [ 43 ] In human semen, examples of the three main antioxidants present are urate, thiols, and ascorbate [77 ] . Antioxidative action effective in offering protection to spermatozoa below a critical threshold of ROS can be considered to consist of two stages. The primary step may be viewed as preventative, whereby the ROS are scavenged directly by the antioxidative compounds present in the seminal plasma [1 ] . Following this initial response initiated by antioxidative reactions is the secondary stage which encapsulates the inﬂ uence that antioxidants may have on the actions that the ROS have already initiated. This step is regarded as interception which includes interruptions of chain reactions, for example the process of lipid peroxidation [1 ] The cytoplasm of the spermatozoa also contain a low concentration of antioxidants such as catalase and superoxise dimutase, which are capable of offering additional protection against ROS [10, 79] . However, due to the small volume of cytoplasm in each spermatozoa, this defensive approach against extracellular free radicals is less effective compared to the antioxidants abundantly present in seminal plasma [80 ] . 528 M. Flint et al.

Despite the antioxidative system to provide protection against OS, this pathway can be easily overwhelmed. The rapid increase in the production of leukocyte- derived ROS can overpower the antioxidative capabilities of the seminal plasma and spermatozoa [57 ] . The effect of leukocytospermia is highly effective in causing potential DNA damage towards spermatozoa at varying stages of maturation which can decrease the chances of successful oocyte fertilization [ 6, 39 ] . Damaging effects on the sperm genome that can arise from OS include: chromatin cross-linkage, base modiﬁ cation, and strand breakages in spermatozoa [ 31 ] . These DNA damages work in a cascade-like manner [7 ] and abnormal spermatozoa from leukocytospermic samples have more than doubled levels of impaired DNA under the inﬂ uence of OS when compared to similar forms of irregular spermatozoa in samples that are not leukocytospermic [81 ] .

23.13 Future Research and Treatment Approaches

A predominant contributing factor that idiopathic male infertility has been shown to have is the imbalance between OS and the antioxidant-induced scavenging of ROS present in the semen [ 10, 82] . Numerous factors may negatively inﬂ uence semen parameters, for example posttesticular damage in the epididymis, abnormal spermatogenesis, environmental variables, or infection [ 18 ] . However, the presence of high concentrations of ROS can add to a damaging effect on the sperm parameters, such as morphology and motility [ 40] , which are essential for normal spermatozoa functioning. By maintaining low concentrations of the free radicals in semen, the chances of successful fertilization can be considerably enhanced for a number of couples facing fertility challenges [36 ] . Past studies have shown the negative inﬂ uence that elevated concentrations of ROS may elicit on the fertilizing capabilities of male subjects through decreasing spermatozoa motility and DNA damage when it was demonstrated that 40–88% of these samples had markedly high levels of ROS [77 ] .

23.13.1 Clinical Treatment

Various forms of drug therapies against the presence of ROS have been developed as a result of research showing the harmful effect of these biomolecules, the fi rst approach being drugs which can inhibit the formation of ROS [1 ] . The second is the stimulation of endogenous defense mechanisms because ROS are part of the initial protection against infl ammation [ 1, 11 ] . The use of antioxidants is regarded as the most logical approach in the clinical intervention to treat the free radical’s harmful effects [ 31 ] . It has been put forward that semen from males presenting with high concentrations of leukocytes and therefore OS may benefi t from supple- mentary treatment with selected oral antioxidants [ 10, 15] , even if subjects have 23 Leukocytospermia and Oxidative Stress 529 normal semen analysis [ 54 ] . The intake may improve the functional properties of spermatozoa such as sperm’s capability of successful oocyte fusion by providing protection from peroxidative damage [ 31, 83] . At present, the Cochrane Review provides evidence which suggests that subfertile couples involved in ART have increased chances of pregnancy and live birth rates, if the male subject receives an oral supplementation of antioxidants [84 ] . The supplementation with antioxidants during the preparation processes performed for ART such as density gradient centrifugation has been proposed and evaluated in the treatment pathway for ROS generation [31 ] . In addition, sperm– oocyte fusion can be enhanced by halting the production of ROS by immature spermatozoa and leukocytes by the addition of antioxidants to the culture media [85 ] . In addition to antioxidants, the pharmacological treatment of leukocytospermia with therapeutic concentrations of specifi c antibiotics has been shown to decrease leukocyte-produced ROS. Example of such drugs includes tetracycline and erythro- mycin, which can inhibit the actions of PMN-granulocyte [1 ] . Pharmacological fi ndings further open the avenue for the treatment of leukocytospermia with drugs to avoid a state of damaging OS. Diagnostic and prognostic tests which allow for the measurement of the OS levels could be a considerable contributing factor in the treatment of idiopathic infertility [ 38 ] . Dietary supplementation with foods containing high concentrations of antioxidants such as carotenoids, vitamin C, and the main chain-breaking antioxidant vitamin E offers an additional line of defense against OS-induced injury [82 ] . An additional avenue into the benefi cial effect that antioxidants have is shown by the cyroprotective effect that they have over sperm when added as supplements in the cyropreservation process [86 ] .

23.14 Conclusion

In order to maintain normal physiological functioning in the male reproductive tract, there is a delicate balance between the concentration of ROS and the seminal antioxidants which must be maintained. The predominant contributing factor to idiopathic male infertility has been shown to be this imbalance which is created during periods of genito-urinary infection. As covered in the chapter leukocytospermia, the infl ammatory condition resulting from the rapid increase in leukocytes during infection results in a high elevation in the concentrations of ROS and OS, which puts a powerful negative effect on various semen and spermatozoa variables [ 10, 36, 43] . The complexity of OS is furthered when additional factors which can increase ROS levels in the male genito-urinary system are considered [57 ] . Extensive growth in knowledge has been provided by a broad number of research groups which have focused on the signifi cance and effects of excessive leukocytes that have infi ltrated into the semen. The harmful infl uence that ROS can have on sperm and their parameters has been known for a relatively long period of time in the fi eld of male reproductive biology. At present, the association between semen parameters and leukocyte concentrations is still a focal point in the fi eld of male reproductive science. 530 M. Flint et al.

References

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