“investigation of the role of oxidative stress in male

“INVESTIGATION OF THE ROLE OF OXIDATIVE

STRESS IN MALE INFERTILITY”

OZLEM TUNC

BSc BioMed (Hons) Grad Dip. Clinical Biochemistry

Discipline of Obstetrics and Gynaecology School of Paediatrics and Reproductive Health Faculty of Health Sciences The University of Adelaide South Australia Australia

Thesis submitted for the total fulfilment for the degree of

Doctor of Philosophy (PhD)

November 2010

ii

DECLARATION

This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library catalogue, the Australasian Digital Theses Program (ADTP) and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.

……………………………… Ozlem Tunc November 2010

iii

ACKNOWLEDGMENT

It is a pleasure to finally be able to thank those that have helped me complete this project. I thank Dr. Kelton Tremellen for providing me with such a significant opportunity, for steering the project and for critically assessing this thesis. Kelton also enhanced my awareness of the effort needed to achieve quality, completeness and relevance, an invaluable lesson. I sincerely thank my co-supervisor Associate Professor Jeremy Thompson for providing the clarity needed on many aspects of this work and for his insights and sharing knowledge with patient support. I would like to thank the staff of the Department of Obstetrics and Gynaecology for their advice and help with technical and administrative issues. In particular I would like to acknowledge the assistance of David Froiland for his technical assistance in establishing sperm DNA damage measurement and LPO experiments. He taught me the techniques of using fluorescence microscopy and taking images. My gratitude also goes to the staff of Andrology Laboratory in Repromed, especially Margaret Szemis for collecting the sperm samples and her assistance in recruitment of patients. I would sincerely like to express my thanks to Michele Kolo for imparting her expertise in Endocrinology and her encouragement. Much of the work and data in this thesis was made possible through a grant from the Colin Matthews Research Fund and Faculty of Health Sciences Postgraduate Scholarship. It is gratefully acknowledged. Finally, I would like to thank my family and friends; for their unwavering support and encouragement and my daughter, Alinda, who makes my life worthwhile. I thank you for your patience and the cheer you bring to my life.

iv

TABLE OF CONTENTS Page Declaration ii

Acknowledgement iii

Table of Contents iv

Publications and Abstracts arising from this thesis vii

Abbreviations viii

Abstract x

CHAPTER 1: INTRODUCTION Page1

1.1 General Overview: Free Radicals and Reactive Oxygen Species (ROS) 1.1.1 Definition of Free Radicals

1.2 Antioxidant Defence Systems 1.3 Oxidative Stress

1.3.1 Oxidative Stress and Male Infertility 1.3.2 Physiological Role of Reactive Oxygen Species in Sperm Cells

1.4 Sources of Free Radicals in semen 1.4.1 Production of ROS by Leukocytes in semen 1.4.2 Production of ROS by Spermatozoa 1.4.3 Susceptibility of Spermatozoa to Oxidative Stress

1.5 Protection of Spermatozoa from ROS 1.5.1 Sperm Chromatin Structure 1.5.2 Antioxidant Mechanisms in semen

1.5.2.1 Enzymatic Antioxidants 1.5.2.2 Non-enzymatic Antioxidants

1.6 Oxidative Stress in Male Infertility 1.6.1 Pathophysiology of Oxidative Stress in Spermatozoa

1.7 Origins of Oxidative Stress 1.7.1. Lifestyle Factors

1.7.1.1. Smoking 1.7.1.2. Alcohol Consumption 1.7.1.3. Advanced Paternal Age 1.7.1.4. Psychological Stress

1.7.2. Infections 1.7.2.2. Male Accessory Sex Gland Infection (MAGI)

1.7.3. Environmental Factors 1.7.3.1. Toxins and Chemicals 1.7.3.2. Radiation 1.7.3.3. Drugs and Chemotherapy

1.7.4. Iatrogenic 1.7.5. Varicocele

1.8 Assessment of Seminal Oxidative Stress 1.8.1 ROS Measurement by Chemiluminescence 1.8.2 Lipid Peroxidation Markers 1.8.3 Other Markers for ROS Measurement

v

CHAPTER 2: MATERIALS AND METHODS Page 26 2.1 Introduction 2.2 Study Subjects 2.3 Sample collection and Semen Analysis 2.4 Sample Preparation

2.4.1 Sperm Swim-up Procedure 2.4.2 Isolation of Leukocytes from Peripheral Blood

2.5 Determination of Leukocytes in Semen 2.5.1 Immunocytochemistry Procedure for CD-45 2.5.2 PMN Elastase Assay 2.5.3 Determination of macrophage activity in semen (Neopterin Assay)

2.6 Measurement of Reactive Oxygen Species 2.6.1 Lipid Peroxidation Test (LPO Test) 2.6.2 Nitro Blue Tetrazolium Assay (NBT Assay)

2.6.2.1 Formazan Production for Standard Curve in NBT Assay

2.7 Detection of Defective Sperm Chromatin Packaging 2.7.1 Chromomycin A3 Assay

2.8 Assessment of DNA Damage 2.8.1 TUNEL (Tdt-mediated Terminal dUTP Nick-end Labeling) Assay

2.9 Assessment of Aberrant Apoptosis 2.9.1 Annexin V Assay

2.10 Assessment of sperm global DNA Methylation 2.10.1 Immunohistochemical 5-methylcytosine Staining

2.11 Assessment of epididymal function 2.11.1 αααα-Glucosidase assay

2.12 Assessment of testicular function (Serum Reproductive Hormones) 2.12.1 Measurement of Serum Reproductive Hormone Levels 2.12.2 Measurement of AMH (Anti Mullerian Hormone) level in serum

2.13 Serum Homocysteine assay

CHAPTER 3: Development of the NBT Assay as a marker of sperm Oxidative Stress Page 49

3.1 Abstract 3.2 Introduction 3.3 Materials and Methods

3.3.1 Subjects and Study Design 3.3.2 Sample Preparation 3.3.3 Statistical Analysis

3.4 Results 3.4.1 Standardization of assay conditions 3.4.2 Relationship between ROS production and measures of sperm health

3.5 Development of Normal Ranges 3.6 Discussion

vi

CHAPTER 4:Seminal Inflammation as a Cause of Oxidative Stress and Macrophage Activity in Infertile Men’s semen 6


4.3.1 Subjects and Study Design 4.3.2 Sample Collection and Sample Preparation

4.4 Results 4.5 Discussion

CHAPTER 5: Obesity as a Cause of Oxidative Stress Page 80


5.3.1 Subjects and Study Design 5.3.2 Sample Collection and Sample Preparation 5.3.3 Statistical Analysis


CHAPTER 6: Treatment of Oxidative DNA Damage in sperm using an Oral Antioxidant Therapy Page 91




CHAPTER 7: Oxidative Stress as a cause of sperm DNA Hypomethylation Page105




CHAPTER 8: Final Discussion and Conclusions Page 119 8.1 Final Discussion 8.2 Future Directions Bibliography Page 126

vii

PUBLICATIONS AND ABSTRACTS ARISING FROM THIS THESIS

PUBLICATIONS

1 - "Development of the NBT assay as a marker of sperm oxidative stress" Ozlem Tunc, Jeremy Thompson, Kelton Tremellen International Journal of Andrology 2010 Feb; 33(1):13-21 2 - “Improvement in sperm DNA quality using an oral antioxidant therapy” Ozlem Tunc, Jeremy Thompson, Kelton Tremellen Reproductive BioMedicine Online 2009 Jun; 18(6):761-8. 3 - “Oxidative DNA damage impairs global sperm DNA methylation in

infertile men“ Ozlem Tunc, Kelton Tremellen Journal of Assisted Reproduction and Genetics2009 Sep-Oct 26(9-10):537-44

4 - “Macrophage activity in semen significantly correlated with sperm quality in infertile Men “

Kelton Tremellen, Ozlem Tunc International Journal of Andrology 2010 Dec; 33(6):823-31

5 - “Impact of Body Mass Index on sperm oxidative stress”

Ozlem Tunc, Hassan Bakos, Kelton Tremellen Andrologia (accepted in Aug 2009, anticipated online publication in December 2010)

ABSTRACTS

“A novel assay for identification of oxidative stress related male infertility”

Poster presentation The Fertility Society of Australia 9-12 September 2007

Hobart, Tasmania

"Optimization of sperm DNA quality by using an oral antioxidant therapy”

Gene, environment, lifestyle interaction and Human reproduction 7-10

February 2008 Malmo, Sweden

“Optimization of sperm DNA quality with the use of an oral antioxidant

therapy”

Oral Presentation in the Fertility Society of Australia 19-22 October 2008

Brisbane, Australia

viii

ABBREVIATIONS

8-OHdG ………………………………………………………………8-hydroxy-20-deoxyguanosine

A.U ……………………………………………………………………………………………… Arbitary Units

AMH ……………………………………………………………………………… Anti-mullerian hormone

ANOVA …………………………………………………………………………………Analysis of Variance

ART ……………………………………………………………… Assisted Reproductive Technology

BSA………………………………………………………………………………… Bovine serum albumin

oC …………………………………………………………………………………………………Degrees celsius

CK ……………………………………………………………………………………………… Creatine kinase

DMSO………………………………………………………………………………… Dimethyl sulphoxide

DNA ………………………………………………………………………………… Deoxyribonucleic acid

DTT ………………………………………………………………………………………………… Dithiothretiol

ELISA ……………………………………………………… Enzyme-linked immunosorbent assay

FSH………………………………………………………………………… Follicle stimulating hormone

GPx ………………………………………………………………………………… Glutathione peroxidise

GSR ………………………………………………………………………………… Glutathione reductase

H2O2 ……………………………………………………………………………………… Hydrogen peroxide

HBSS …………………………………………………………………… Hanks buffered salt solution

hCG ………………………………………………………………… Human chorionic gonadotrophin

Hcy ……………………………………………………………………………………………… Homocysteine

HNE …………………………………………………………………………………………… Hydroxynonenal

HPLC ……………………………………………… High Performance Liquid Chromatography

HRP …………………………………………………………………………… Horse Radish Peroxidase

IFNγ ……………………………………………………………………………………… Interferon gamma

IU …………………………………………………………………………………………… International unit

KOH …………………………………………………………………………………… Potassium hydroxide

kg…………………………………………………………………………………………………………… Kilogram

LDH ………………………………………………………………………… Lactic acid dehydrogenase

LH……………………………………………………… ……………………………… Luteinizing Hormone

MDA ………………………………………………………………………………………… Malondialdehyde

µg …………………………………………………………………………………………………………Microgram

µl ……………………………………………………………………………………………………………Microliter

mL ………………………………………………………………………………………………………… Milliliter

mg ……………………………………………………………………………………………………… Milligram

NaCl ………………………………………………………………………………………… Sodium Chloride

ix

NAG …………………………………………………………………………… neutral alpha glucosidase

NBT ………………………………………………………………………………… Nitroblue Tetrazolium

NO …………………………………………………………………………………………………… Nitric oxide

NOS …………………………………………………………………………………… Nitric oxide synthase

PBS …………………………………………………………………………… Phosphate buffered saline

PCR …………………………………………………………………………… Polymerase chain reaction

PFA ………………………………………………………………………………………… Paraformaldehyde

PMN ………………………………………………………………… Polymorphonuclear Neutrophils

Rcf ……………………………………………………………………………… Relative centrifugal force

ROC ……………………………………………………………… Receiver operating characteristic

ROS …………………………………………………………………………… Reactive oxygen species

SD ………………………………………………………………………………………… Standard Deviation

SDS …………………………………………………………………………… Sodium dodecyl sulphate

SOD ………………………………………………………………………………… Superoxide dismutase

TAC …………………………………………………………………………… Total antioxidant capacity

TBAR …………………………………………………………………………………… Thiobarbituric acid

TBARS ………………………………………………… Thiobarbituric acid-reacting substances

TNFα ………………………………………………………………………Tumor Necrosis Factor Alpha

X …………………………………………………………………………………………………………… Xanthine

XO …………………………………………………………………………………………… Xanthine oxidase

x

ABSTRACT

In recent years, there has been some suggestion of an increase in male factor

infertility in the industrialized countries with a decline in sperm counts and a rise in

sperm pathology. Male factor infertility is a multifactorial phenomenon that is

observed in approximately half of infertile couples and affects one man in 20 in the

general population. The potential causes of male infertility arise from a number of

factors including genetic, lifestyle factors and chronic diseases. However, a high

proportion of infertile male patients have now been shown to have defective sperm

functions related to oxidative stress.

Oxidative stress in semen has been speculated as one of the major factors causing

male infertility and has been identified in 30-80% of cases of male infertility. While

oxidative stress is accepted as a significant pathology, there is currently an

inadequate knowledge of the exact mechanisms by which oxidative stress develops

in male infertility, as well as a lack of an easy and reliable method for the

measurement of seminal oxidative stress in routine clinical use.

The main objective of this doctoral thesis is to investigate the underlying causes for

oxidative stress in infertile men and the mechanisms by which oxidative stress

develops. Furthermore it will also examine the effectiveness of an oral antioxidant

therapy for treatment of seminal oxidative stress.

During these doctoral studies experiments were designed with the aims of:

• Developing a standardized protocol for the measurement of seminal oxidative

stress, that can be conducted in the average clinical laboratory with minimal

additional equipment (NBT Assay)

• Examining the causes for oxidative stress in semen. Obesity has previously

been identified as a cause of systemic oxidative stress. Therefore I examined

if obesity causes oxidative stress to sperm. Seminal inflammation and its role

in oxidative damage in semen are also investigated.

• Determination if antioxidant supplementation is an effective treatment of

oxidative sperm damage.

• Assessment of the relation between Oxidative stress and sperm DNA

methylation. Previous studies have linked male infertility with epigenetic

abnormalities of the male genome. Since oxidative stress has been shown to

interfere with somatic cell epigenetic programming I investigated the

possibility of a similar link in sperm.

It is hoped that advances outlined in this thesis will have made a significant

contribution to the diagnosis, prevention and treatment of the male infertility.

xi

CHAPTER 1

INTRODUCTION

1

1.1 General Overview: Free Radicals and Reactive Oxygen Species (ROS)

In order to understand the effects of oxidative stress in male infertility it is

essential to understand the relevant players of oxidative stress; free radicals,

reactive oxygen species (ROS) and antioxidants.

Oxygen is essential for life as it is necessary in the production of energy in the

body. A molecular basis, cellular energy is produced by oxidative

phosphorylation in mitochondria through electron transport from electron

donors to electron acceptors such as oxygen. During these reaction steps,

hydrogen is provided in the form of reducing equivalents (NADH) and energy is

produced in the form of high-energy phosphates (ATP) while four-electron

reduction of molecular oxygen to water produces free radicals and oxygen-

derived reactive species. Therefore, the generation of ROS is an essential by-

product of life giving metabolism in all individuals.

1.1.1 Definition of Free Radicals

The term free radicals refers to “any species capable of independent existence

that contains one or more unpaired electrons” (Halliwell, 1999). These short-

lived but highly reactive molecules can be formed by the loss of a single

electron from a non-radical,

X � e- + x.+

or by the gain of a single electron by a non-radical;

Y + e- � Y

.+

The term Reactive oxygen species (ROS) refers to all oxygen radicals, non-

radical derivatives of oxygen including oxidising agents and carbon based

oxygen radicals (Table 1.1).

2

Table 1.1: Most common Reactive Oxygen Species including nitrogen derived free radicals and reactive oxidants. Summarised from (Halliwell, 1999).

REACTIVE

OXYGEN SPECIES

GENERATION EFFECT

Superoxide

Anion

(O2 . _)

One electron reduction state of oxgen.

Primary form of ROS. Generated in auto-

oxidation reactions, Electron transport chain

in mitochondria/endoplasmic reticulum

Oxidising and reducing agent .

1- Dismutation to hydrogen peroxide

and Hydroxyl radicals 2-Formation of

thiyl radicals by reaction with thiol

groups

3-Generation of peroxynitrite

Hydroxyl

Radical

(OH . _)

Three-electron reduction state of oxygen.

Formed by the reaction of superoxide and

Hydrogen peroxide accompanied by metal

catalyst (Haber-Weiss Reaction);

O2 . + H2O2 � O2 + OH

. + OH

-

or free iron with Hydrogen Peroxide (Fenton

Reaction);

Fe+2 + H2O2 � Fe+3 + OH . + OH

-

Highly reactive and very short half-

life oxygen radical. The most toxic

ROS. Attacks all cellular components.

Main reactions of hydroxyl radical:

hydrogen abstraction (inititiation of

lipid peroxidation),

Addition Reaction(addition to purin

base guanine in DNA forms an 8-

hydroxyguanine),

Electron-transfer reactions.

Hydrogen

Peroxide

(H2O2)

Two electron reduced state of oxygen.

Formed by dismutation of O2 . in

mitochondria during oxidative

phosphorylation or produced as by-product of

several oxidases. It is not radical itself.

High biological diffusion, easily

transverse the plasma and nuclear

membranes. Formation of DNA

adduct, affects signalling and tissue

injury processes

Singlet

Oxygen

(1O2 )

Lowest excited state of oxygen molecule.

Dismutation of superoxide anion. In vitro,

singlet oxygen can be produced by

photosensitization.

Oxidising agent. Transfers its

excitation energy to another

molecule. Effective in carbon-carbon

double bounds, such as carotenoids

and fatty acids.

Peroxyl (ROO•)

and alkoxyl(RO•)

Radicals

Produced in the presence of oxygen during

the breakdown of organic peroxides and

reaction of carbon radicals with oxygen.

Oxidising agents.Oxidize ascorbate

and NADH. Abstract Hydrogen ion

from other molecules which is

important in lipid peroxidation.

Nitric Oxide

(NO•, NOO•)

Nitric Oxide is formed from the reaction of

amino acid L-arginine by NO synthase. NO is

not a radical but can form radicals.

Transmitter substance reacts with

superoxide anion to make

peroxynitrite (OONO-) which actively

reacts with glutathione, cysteine,

deoxyribose and thiols.

Hypochlorous acid

(HOCl)

Formed from hydrogen peroxide by

myeloperoxidase

Highly reactive and lipid soluble.

Oxidise protein components, thiol and

amino groups and methionine.

3

ROS are formed by several mechanisms in vivo. The most important source of

reactive oxygen species is the electron transport chain. During cellular

respiration process while oxygen is reduced to CO2 and ATP released as high

energy molecule, oxygen is also reduced to highly reactive superoxide anion by

leaking of single electron (Fridovich, 1970)(Figure 1).

Some enzymes produce free radicals by reducing oxygen to the superoxide

radical. Xanthine oxidase, NADPH oxidase, myeloperoxidase, thyroid

peroxidase and nitric oxide synthase are well known potential producers of ROS

in vivo (Halliwell, 1999).

Figure 1.1: The pathway of oxygen reduction during cellular respiration

The primary form of ROS, superoxide anion (O2.-), is produced by addition of

one electron to dioxygen (O2). This free radical can be converted to secondary

reactive oxygen species such as hydroxyl (0OH), peroxyl radical (ROOo),

hydrogen peroxide (H2O2), nitrous oxide, peroxynitrite, nitroxyl anion and

peroxynitrous acid.

At physiological concentrations, reactive oxygen species have biopositive

effects and take place in many chemical reactions in the cell. They also act as

a cell messenger in the regulation of cellular signalling mainly by oxidatively

4

modifying compounds (Joseph and Cutler, 1994). Free radicals also take part

in phagocytosis process which is mediated by neutrophils and macrophages

(Rosen et al., 1995).

1.2 Antioxidant Defence Systems The body has developed antioxidant defense systems by scavenging and

minimizing the formation of oxygen derived radicals to protect itself from

oxidative damage. Antioxidant defense mechanisms operate by three

mechanisms by using antioxidants; prevention, interception and repair (Sies,

1997). Antioxidant protection systems consist of dietary antioxidants,

endogenous enzymatic and non-enzymatic constituents (Table 2).

Enzymatic antioxidants catalytically remove reactive oxygen species from

biological systems. Highly reactive superoxide anion (O2 -.) is converted to

hydrogen peroxide (H2O2) by Superoxide Dismutase (SOD) (EC 1.15.1.1) in

order to prevent this radical forming highly reactive hydroxyl radical. H2O2 is

then eliminated in the presence of catalase (EC 1.11.1.6) and glutathione

peroxidase (GPX) (EC 1.11.1.9).

SOD

2 (O2-). + 2H+ --------------------------------------> H2O2 + O2

Catalase

H2O2 --------------------------------------> H2O + ½ O2

Glutathione peroxidase catalyses the reduction of hydrogen peroxide and lipid

peroxide and removes peroxyl (ROO.) radicals from various peroxides by using

reduced glutathione (GSH) as an electron donor. Glutathione reductase

regenerates reduced glutathione (GSH) from oxidized glutathione (GSSG) as

shown in the following equation;

GPX

2GSH + H2O2 -----------------------------------> GSSG + 2H2O

reductase

GSSG + NADPH + H+ -----------------------------------> 2GSH + NADP

+

Dietary antioxidants are usually present in the form of vitamin C, vitamin E,

beta-carotenes, carotenoids, and flavonoids (Sies, 1993). Metal-binding

proteins such as albumin, ceruloplasmin, metallothionein, transferrin, ferritin,

and myoglobin act as an antioxidant by inactivating pro-oxidant transition

metal ions (Makker et al., 2009).

5

Antioxidant defense system differ from tissue to tissue and cell-type to cell-

type (Halliwell, 1999). The antioxidant system and their importance in semen

will be discussed in the following sections.

Table 1.2: Most important in vivo antioxidants

ENZYMATIC ANTIOXIDANTS NON-ENZYMATIC ANTIOXIDANTS

Superoxide Dismutase (SOD) Metal Binding

Proteins

Low-molecular mass agents

Catalase (CAT) Albumin Vitamin C (Ascorbic acid)

Glutathione Peroxidase (GPX) Transferrin Vitamin E (alfa-tocopherol)

Glutathione-S-transferase (GST) Lactoferrin Melatonin

Ceruloplasmin Glutathione

Haptoglobin Cytochrome C

Coenzyme Q

Bilirubin

Uric Acid

Lipoic Acid

1.3 Oxidative Stress Oxidative stress (OS) is a situation when there is an imbalance between

production of reactive oxygen species and antioxidant defence system in favour

of the former (Sies, 1997). Oxidative stress can cause damage to all types of

biomolecules, including DNA, lipids and proteins. This oxidative damage can

result in DNA strand breakage, cell membrane lipid peroxidation and amino

acid modifications respectively (Halliwell, 1999). Oxidative stress has been

linked with many degenerative processes, diseases, syndromes and ageing

processes in human being (Cutler, 1991; Davies, 1995).

1.3.1 Oxidative Stress and Male Infertility

Excessive production of reactive oxygen species (ROS) are thought to be

critically involved in the aetiology of male infertility (Aitken, 1989) and have

been identified in 30% to 80% of infertile male’s semen (de Lamirande and

Gagnon, 1995b; Tremellen, 2008). Increasing levels of oxidative stress in

semen arise from high level of ROS generation or inadequate antioxidant

protection capabilities of the male reproductive tract or seminal plasma (Aitken

et al., 1992; Iwasaki and Gagnon, 1992).

6

1.3.2 The physiological role of Reactive Oxygen Species in sperm

cells

At physiological concentrations, ROS participates in signal transduction

mechanisms in sperm such as capacitation and the acrosome reaction (de

Lamirande et al., 1997). Studies have shown that the exposure of low levels of

reactive oxygen species to spermatozoa results in higher hyperactivation and

capacitation than the control groups (de Lamirande and Gagnon, 1993; Griveau

et al., 1994; Zini et al., 1995). Mild oxidative conditions also help promote

binding of spermatozoa to the zona pellucida via lipid peroxidation of sperm

membrane (Aitken, 1989). Hydrogen peroxide eases the sperm’s transit

through the cumulus and zona pellucida by causing tyrosine phosphorylation

and stimulating the acrosome reaction (Aitken et al., 1995b; de Lamirande et

al., 1997). Therefore it is apparent that while low level of exposure to ROS are

necessary for normal sperm function, high ROS exposure can be harmful.

1.4 Sources of Reactive Oxygen Species in semen Semen consists of several 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, peroxidase positive leukocytes and

abnormal spermatozoa are thought to be main sources of reactive oxygen

species in the ejaculate (Aitken et al., 1994; Aitken and West, 1990; Cocuzza

et al., 2007; Hendin et al., 1999; Mazzilli et al., 1994; Ochsendorf, 1999;

Whittington and Ford, 1999) .

1.4.1. Production of ROS by Leukocytes in Semen

Leukocytes in semen have been suggested to have the physiological role of

removal of dead or abnormal spermatozoa (Tomlinson et al., 1992b). There are

many different leukocyte subpopulations originating from different parts of the

male reproductive tract. The predominant leukocyte sub-types in semen are

polymorphonuclear leukocytes (50-60%), macrophages (20-30%) and T-

lymphocytes (2-5%). The former two cell groups are the main producers of

free radicals in semen (Fedder et al., 1993; Wolff, 1995; Wolff and Anderson,

1988). Due to the blood-testis barrier, leukocytes are excluded from direct

contact with sperm except in the area where the barrier disappears in the rete

testis and efferent ducts (Pollanen and Cooper, 1994). Thus the rete testis

epithelium is occupied by CD8+ suppressor lymphocytes indicating active

immune regulation for suppression of autoimmune attack on spermatozoa.

7

Lymphocytes and macrophages are mostly located in the epididymis as well as

in the epithelium of the vas deferens, yet granulocytes originate from prostate

and seminal tissue (Wolff, 1995).

Activated leukocytes are capable of producing 100-fold higher amounts of ROS

than non-activated leukocytes (Plante et al., 1994) and these activated

leukocytes produce 1000-times more reactive oxygen species than

spermatozoa (de Lamirande and Gagnon, 1995a). However, the relative

importance of leukocytes and sperm in inducing oxidative stress in male

infertility is still unclear.

1.4.2. Production of ROS by Spermatozoa

Spermatozoa were the first cell type shown to generate reactive oxygen

species in human. In 1943 MacLeod observed that, when spermatozoa were

incubated under high oxygen tension, they lost their motility capability and yet

this impairment in sperm function was blocked by adding catalase, a hydrogen

peroxide scavenger (MacLeod, 1943). Future studies in which contaminating

leukocytes were separated from sperm using density gradient centrifugation

and CD45 antibody absorption columns have confirmed that sperm themselves

are capable of ROS generation (Baker et al., 2003). Generation of ROS by

sperm cells can occur via NADH (nicotinamide adenine dinucleotide) dependent

oxidase system (NOX5) at the level of plasma membrane and the NADH

dependent oxido-reductase (diphorase) system at the mitochondrial level

(Aitken et al., 1997). Additionally lipoxygenase (Oliw and Sprecher, 1989) and

cytochrome p450 reductase (Baker et al., 2004) systems are other proposed

ROS-generating systems in sperm cells. According to a recent study, the major

source of ROS in sperm is mitochondrial, with excessive ROS production in

sperm mitochondria being significantly correlated with a decrease in motility by

inducing permanent damage in the sperm motility apparatus (Koppers et al.,

2008).

The Superoxide anion is produced by sperm cells at capacitation and the

sustained production of superoxide anion is necessary for the development of

sperm activation and capacitation (de Lamirande and Gagnon, 1995a)

The maturation development of sperm is conversely related to their production

of ROS. Immature abnormal spermatozoa with the excess cytoplasm create

intrinsic oxidative stress (Henkel et al., 2005). Abnormal retention of excess

residual cytoplasm during the final stages of spermiogenesis can result in

increase production of free radicals by the high amount of cytoplasmic enzymes

8

such as Creatine kinase (CK), lactic acid dehydrogenase (LDH) and Glucose 6-

phosphate dehydrogenase (G-6-PDH) which produce ROS from generating

NADPH (Aitken et al., 1994; Aitken et al., 1995b; Huszar and Vigue, 1993; Rao

et al., 1989).

1.4.3 Susceptibility of sperm cells to Oxidative Stress

Sperm cells are more vulnerable to ROS than somatic cells due to their unique

cell structure. Spermatozoa contain large number of polyunsaturated fatty

acids in their plasma membrane (Jones et al., 1979), as this provides fluidity

for the sperm cells membrane that is necessary for membrane fusion events

such as the acrosome reaction, sperm-egg interaction and also motility.

However, the unsaturated nature of these molecules predisposes them to free

radical attack.

Another characteristic of sperm cells that makes them vulnerable to ROS

attack is their lower amount of cytoplasmic ROS-scavenging enzymes due to

their limited cytoplasmic volume (Zini et al., 1993). This restricted cytosolic

space has limited capacity for protein synthesis and little antioxidant capacity,

which makes them deficient of antioxidant protection enzymes such as

superoxide dismutase, catalase or glutathione peroxidase in condition of

excessive ROS availability (Aitken, 1989; Zini et al., 1993).

1.5 Protection of Spermatozoa from ROS

Human sperm differs from somatic cells in terms of their structure and

composition (Lewis and Aitken, 2005). Being vulnerable to the exogenous

factors such as oxidative stress, sperm cells have to be organized by specific

protection mechanisms in order to protect their genomic integrity during

transport of the paternal genome to the female. In the following section, I will

outline these protection mechanisms of spermatozoa; including their unique

organisation of DNA packaging and intrinsic antioxidant mechanisms.

1.5.1 Sperm Chromatin Structure

Packaging of DNA in sperm is significantly different from somatic cells because

sperm have limited volume in their nucleus (Fuentes-Mascorro et al., 2000).

The spermatid chromatin structure is completely reorganized throughout

spermiogenesis and the DNA is repackaged by small proteins called

protamines. The packaging of sperm DNA by protamines enables the DNA to be

compressed into a small volume (Ward and Coffey, 1991) and this condensed

structure of DNA not only provides for transferring of the very tightly

9

packaged genetic information to the egg but also helps protect the paternal

DNA from oxidative attack (Fuentes-Mascorro et al., 2000; Ward and Coffey,

1991).

During the later stages of spermatogenesis, histone proteins are displaced with

transition proteins and then protamines are added (Poccia, 1986). The

replacement of histones by protamines results in the condensation of DNA into

compact doughnut form (Diagram 1.1). Protamines are very basic proteins

about half the size of histone proteins (Fuentes-Mascorro et al., 2000) and are

rich in arginines that permit strong DNA binding. Packaging of DNA with

protamines in place of somatic histones greatly condenses the DNA, preventing

RNA transcription, and results in lower accessibility to DNA-damaging agents

(Evenson et al., 2002). Protamines also contain a large number of cysteine

residues which provide multiple intra and inter-protamine cross-links. The DNA

strands are tightly wrapped around the protamine molecules and toroids are

cross-linked by disulphide bond formation which provide further enhancement

for the stability of the nucleus during epididymal passage and the final stages

of sperm nuclear maturation (Loir and Lanneau, 1984).

All these interactions play a regulatory role in making sperm DNA the most

condensed eukaryotic DNA (Ward and Coffey, 1991). This nuclear compaction

and stabilization is important to protect sperm genome from free radical attack.

Incomplete protamine packaging of the sperm chromatin will increase the

sperm DNA’s vulnerability to damage.

10

Diagram 1.1: Schematic diagram of condensation of DNA in somatic and sperm cells (Ward 1991)

1.5.2 Antioxidant Mechanisms in semen

Seminal plasma and spermatozoa themselves possess endogenous antioxidants

for protecting spermatozoa from oxidative damage (Gagnon et al., 1991; Zini

et al., 1993). These antioxidants are broadly divided into enzymatic and non

enzymatic groups.

1.5.2.1 Enzymatic Antioxidants

Three main enzymatic antioxidants exist in seminal plasma: superoxide

dismutase (SOD), catalase, and glutathione peroxidase/glutathione reductase

(GPX/GRD). Spermatozoa themselves predominantly possess these enzymatic

antioxidants (Zini et al., 1993).

SOD protects spermatozoa against spontaneous O2 toxicity and lipid

peroxidation (Alvarez et al., 1987). Seminal SOD and catalase remove (O2-)

which is generated by NADPH oxidase in neutrophils and may play an

a1172507

Text Box

NOTE: This figure is included on page 10 of the print copy of the thesis held in the University of Adelaide Library.

11

important role in decreasing lipid peroxidation as well as protecting

spermatozoa during genitourinary inflammation (Aitken et al., 1995b).

Glutathione peroxidase (GPx) acts as an antioxidant by converting hydrogen

peroxide to H2O using glutathione as electron donor and is found in

spermatozoa, testis, prostate and the epididymis (Vernet et al., 2004). GPX is

mostly concentrated in the mitochondria, nucleus and acrosomal domain of

differentiating spermatozoa (Vaisberg et al., 2005). There are 5 different

isoforms of GPx in the different anatomical sites (Meseguer et al., 2004). GPx1

is found in sperm and the genital tract and is related with sperm motility

(Dandekar et al., 2002). GPx4 is an selenium dependent form found in

testicular tissue and acts as a peroxidase to protect sperm from oxidative

attack (Foresta et al., 2002). Gpx4 is essential to build up the mitochondrial

capsule for midpiece structure of sperm by protamine disulfide bridging

(Foresta et al., 2002). Another form of Glutathione peroxidase ,GPx5, has also

been found in epididymis (Chabory et al.) GPx5 is particularly seen in the

caudal luminal compartment in the epididymis and the lack of expression or

secretion of this isoform causes an oxidative stress that alters integrity of

spermatozoa (Chabory et al., 2009). GPx5, unlike the other isoforms of GPx

does not require selenium for activity.

1.5.2.2 Non-enzymatic Antioxidants

Total seminal antioxidant activity is also supplemented by many non-enzymatic

antioxidants in semen (Zini et al., 1993). These include minerals, vitamins,

aminoacids and protein compounds such as zinc, vitamin E and C, albumin,

glutathione, taurine, hypotaurine, carnitine, carotenoids, urate and

prostasomes (Makker et al., 2009). These antioxidants act by directly

neutralising free radical activity chemically either by becoming oxidized

themselves or reducing free radical production directly (Tremellen, 2008).

Zinc is one of the most important antioxidant mineral in seminal plasma.

Firstly, zinc is the core constituent of Superoxide dismutase enzyme. Secondly,

zinc can also protect against oxidation by inhibition of the production of

reactive oxygen with displacing transition metals (Bray and Bettger, 1990).

Finally, zinc is bound to protamine 2 in the sperm cell and therefore it is

essential for stabilizing sperm DNA against oxidative attack by augmenting

chromatin packaging (Gatewood et al., 1990). According to some in vivo

experiments, sperm with low chromatin zinc content express improper

12

chromatin condensation (Bjorndahl and Kvist, 1985; Kjellberg et al., 1992),

with dietary zinc deficiency being responsible for altered protamine process in

sperm cells (Evenson et al., 1993).

Vitamin E (α-tocopherol) is a powerful “chain breaking ”free radical scavenger

that plays an important role in preventing sperm membrane lipid peroxidation

damage (Johnson, 1979).

Vitamin C exists in sertoli cells and spermatozoa at high amount. Vitamin C

recycles Vitamin E, helping keep it in its in active state (Yoganathan et al.,

1989). Vitamin C also neutralizes the oxidative effects of pro-oxidants such as

cadmium, arsenic and alcohol (Chang et al., 2007; Maneesh et al., 2005)

Prostasomes are secreted from the prostate into the seminal fluid and have

important effects in regulating sperm viability and function such as motility,

acrosome reaction and zona penetration. Prostasomes are rich in cholesterol

and fuse with sperm cells which affect sperm fluidity (Kravets et al., 2000).

Carotenoids (beta-carotene) and ubiquinols may also play a role in quenching

singlet oxygen and reducing lipid derived free-radicals with detrimental effects

on sperm lipid peroxidation (Sikka et al., 1995).

Lycopene is a lipophilic carotenoid with antioxidant and pro-oxidant properties

(Stahl and Sies, 1996; Young and Lowe, 2001). A recent study has shown that

lycopene supplementation in vitro can protect sperm from oxidative DNA

damage and has beneficial effects on sperm motility (Zini et al.).

Urate is another chain-breaking antioxidant existing in seminal plasma. It

prevents metal (Fe+2) driven free radical reactions by binding transition metals

and scavenge peroxyl and OH- radicals (Lewis et al., 1997).

Carnitine is an antioxidant which is biosynthesized from lysine and methionine

amino acids (Steiber et al., 2004). It is required for the utilization of long chain

fatty acids to produce energy. Carnitine is found in epididymal plasma at high

concentrations and therefore has an important role preventing the formation of

lipid peroxidation. Carnitine plays a role for the maturation of spermatozoa and

provides readily available energy within the male reproductive tract (Dokmeci,

2005). The use of carnitine for treatment of male infertility has been conducted

13

in a number of controlled and uncontrolled studies and shown beneficial effects

in improvement in sperm quality (Agarwal and Said, 2004; Lenzi et al., 2003).

1.6 Oxidative Stress in Male Infertility

1.6.1 Pathophysiology of Oxidative Stress in Spermatozoa

Increased seminal ROS production has been associated with impaired sperm

motility, (Griveau and Le Lannou, 1997; Plante et al., 1994), membrane

function including the acrosomal reaction (Sharma and Agarwal, 1996; Zalata

et al., 2004) and integrity of the sperm genome (Lewis and Aitken, 2005;

Ollero et al., 2001). (Figure 1.2)

Effects of ROS in sperm cells may vary depend on factors such as the amount

of ROS produced, oxygen tension, site of interaction with ROS and duration of

exposure to ROS, as well as surrounding environmental factors such as

activation of leukocytes, concentration of molecular components and available

antioxidative systems in the seminal plasma (Agarwal and Saleh, 2002;

Cocuzza et al., 2007).

It has been reported that a low concentration of hydrogen peroxide has not

shown any adverse effect on sperm motility but inhibits sperm-oocyte

interaction (Aitken et al., 1993a). This may explain why some patients with

normal sperm parameters can still have impaired fertility. In such patients, the

ROS levels are not high enough to impair basic semen analysis parameters but

can cause defects in other functioning which is required for fertilization.

At higher concentrations of ROS, oxidative damage to polyunsaturated fatty

acid in the sperm plasma membrane occurs and this then initiates a lipid

peroxidation cascade (Storey, 1997). When lipid peroxidation occurs, lipid

peroxides accumulate on the sperm surface and this reduces the fluidity of the

sperm phospholipid membrane making the sperm flagella work less effectively,

thereby reducing motility (Alvarez et al., 1987; Twigg et al., 1998a).

Excessive leakage of ROS from mitochondria in the midpiece may also damage

the mitochondrial power source of sperm motility hence cause decreased

motility. Oxidative stress therefore has the ability to produce infertility

impairing sperm transit through the female reproductive tract. Furthermore,

damage to the acrosomal membrane diminishes acrosin activity (Zalata et al.,

14

2004) and impedes the capacity of the sperm to fuse with the oocyte,

consequently induce poor fertilisation capacity (Aitken, 1989; Jedrzejczak et

al., 2005).

Figure 1.2: Reactive Oxygen induced damage in sperm cell

15

In addition, reactive oxygen species cause depletion of intracellular ATP. In

turn, due to insufficient protein phosphorylation, a decrease in beat frequency

and an increase in axonemal damage lead to sperm immobilization (de

Lamirande and Gagnon, 1992a; de Lamirande and Gagnon, 1992b; Griveau

and Le Lannou, 1997).

Oxidative stress can also have a harmful effect on sperm DNA integrity

(Hughes et al., 1996; Kodama et al., 1997; Lopes et al., 1998). Oxidative DNA

damage can occur through either oxidation of DNA bases primarily by direct

attack on the purine and pyrimidine bases or through causing strand breaks

and cross-linking in sperm DNA (Barroso et al., 2000; Kunsch and Medford,

1999; Olinski et al., 1992; Twigg et al., 1998a; Twigg et al., 1998c). In vitro

induced oxidative stress significantly increases DNA fragmentation,

modification in base structure, deletions and frame shifts in sperm chromatin

(Aitken et al., 1998; Dizdaroglu, 1992; Hughes et al., 1996; Kemal Duru et al.,

2000).

Mitochondrial exposure to ROS provokes apoptotic process through release of

apoptosis inducing factor (AIF) which also cause DNA fragmentation (Cande et

al., 2002). High level of ROS agitates mitochondrial membrane polarization and

activates release of the cytochrome-C protein as a trigger of apoptosis (Wang

et al., 2003)

Oxidative stress has been indirectly connected to the clinical consequences of

sperm DNA damage such as increased miscarriage risk (Benchaib et al., 2007;

Borini et al., 2006; Virro et al., 2004). Oxidative DNA damage has also been

linked with poor embryonic development (Sakkas et al., 1998) and possibly

even an increase risk in childhood cancer (Lewis and Aitken, 2005).

The effects of oxidative DNA damage in sperm on the health and wellbeing of

the offspring have not yet been fully clarified but still there is a possibility that

spermatozoa significant with DNA damage can achieve fertilization and full

term pregnancy (Ahmadi and Ng, 1999; Gandini et al., 2004; Twigg et al.,

1998c).

16

1.7 Origins of Oxidative Stress

Oxidative stress can possibly arise from a number of endogenous and

exogenous factors or interaction between each. The common causes of

oxidative stress include lifestyle and environmental factors, infection,

varicocele, autoimmune related conditions, elevated testicular temperature,

chronic diseases and drugs as summarized in Figure 1.3.

Figure 1.3 The origins of Oxidative Stress in semen

ANATOMICAL

- Varicocele

IATROGENIC

Centrifugation

Cryopreservation

INFECTIONS

Male Accessory Gland Inf.(MAGI)

Infective Inflammatory

Inflammatory –eg vasectomy

ENVIRONMENTAL

FACTORS

Toxins

Chemicals

EM Radiation

Chemotheraphy Drugs

Heat

LIFESTYLE FACTORS

Smoking

Alcohol Stress

Age

OXIDATIVE

STRESS

in Male Infertility

17

1.7.1. Lifestyle Factors

1.7.1.1. Smoking

Smoking has adverse effect on sperm quality parameters with lower sperm

counts, decreased motility and increased abnormal sperm morphology (Kunzle

et al., 2003; Saleh et al., 2002; Sofikitis et al., 1995). Studies have shown that

smokers have also higher degree of oxidative stress in their semen, as

reflected by an increase in ROS production and a reduction in seminal plasma

antioxidants (Saleh et al., 2002; Sepaniak et al., 2006). Smokers have lower

seminal plasma antioxidant capacity compare to non-smokers (Fraga et al.,

1996). The adverse effect of smoking on the developing and mature sperm

cells may be mediated by increasing seminal leukocyte derived ROS production

(Agarwal et al., 2003; Saleh et al., 2002). By increasing ROS levels, it has

been postulated that smoking damages the chromatin structure and produces

endogenous DNA strand breaks in sperm (Potts et al., 1999; Sepaniak et al.,

2004). These alterations may result in sperm DNA mutations, which predispose

offspring to greater risk of malformations, cancer and genetic diseases.

Paternal smoking has been reported to be responsible for 15% of childhood

cancer (Sorahan et al., 1997).

Oxidative DNA damage in sperm can be related to reduction in antioxidant

capacity and decreased level of vitamin C (Mostafa et al., 2006; Song et al.,

2006) and Vitamin E (Fraga et al., 1996). Smokers have also 50% of increased

level of 8-OHdG in their seminal plasma compared to non-smokers (Fraga et

al., 1996).

1.7.1.2. Alcohol Consumption

Alcohol is known as a promoter of ROS production and interferes with the

body’s antioxidant defence mechanisms especially in the liver (Wu and

Cederbaum, 2003). By stimulating the activity of cytochrome P450s, alcohol

contributes to excess ROS production and increases systemic oxidative stress

as well as causing deficiency in protective antioxidant level in the body (Koch

et al., 2004; Wu and Cederbaum, 2003).

Studies have linked excess alcohol consumption with decreased sperm motility

(Gaur et al.; Villalta et al., 1997). Although the link between alcohol intake and

oxidative damage in sperm has not been fully clarified yet, it is likely that

18

alcohol causes systemic oxidative stress, which would extend to the male

reproductive tract causing decreased sperm motility.

1.7.1.3. Advanced Paternal Age

According to many studies, systemic oxidative stress increases with age

(Junqueira et al., 2004). One study has been shown that oxidative stress is

more common in men older than 40 years of age compared to men younger

than 40 years of age (Cocuzza et al., 2008).

There is suggestive epidemiological evidence that increased paternal age is

related with the abnormal reproductive outcomes and defects in resulting

children (de la Rochebrochard and Thonneau, 2002; Tarin et al., 1998). Men of

advanced age seem to produce more sperm with certain type of gene

mutations (Wyrobek et al., 2006) and increased DNA fragmentation (Morris et

al., 2002; Moskovtsev et al., 2006; Singh et al., 2003; Spano et al., 1998).

It has been reported that there is a link between increasing paternal age and

pregnancy loss (de la Rochebrochard and Thonneau, 2002; Risch et al., 1987),

birth defects (Lian et al., 1986), gene mutations such as achondroplasia

(FGFR3 gene mutation) (Crow, 2000; Tiemann-Boege et al., 2002); various

aneuploidy and chromosomal syndromes (Sloter et al., 2004) and also the

possibility of an increased risk of prostate cancer in next generation (Zhang et

al., 1999). While these gene mutations in older men might be due to

replication errors during germ cell division, it is possible that the observed

increase in DNA mutations with increasing age are due to the concomitant

increase in oxidative stress with age (Junqueira et al., 2004).

Furthermore, chromatin packaging in spermatozoa is impaired with advanced

age, thereby increasing the susceptibility of spermatozoal DNA to oxidative

damage (Zubkova et al., 2005).

1.7.1.4. Psychological Stress

Psychological stress can also affect sperm quality. The underlying mechanism

of psychological stress related semen quality alterations has not yet been

clarified, but a recent study has reported that psychological stress can cause an

increase of NO production and a decrease of arginase activity in the L-arginine-

NO pathway (Eskiocak et al., 2006). According to these researchers there is a

decreased antioxidant protection due to loss of glutathione and free sulphydryl

19

content of seminal plasma in males with physiological stress. Also this study

showed a significant decrease in sperm motility and an increase in the

percentage of abnormal morphology during the stress period, suggesting that

oxidative stress may be the underlying cause for abnormal sperm parameters

in the stress state (Eskiocak et al., 2005).

Extreme exercise activity can also cause reduced sperm quality. This may be

due to increased oxidative stress in the general body system after exercise.

During exercise, high amounts of ROS are produced via increased aerobic

metabolism and muscle damage (Peake et al., 2007). A rodent study has

reported that increased muscle activity result in reduced sperm count and

motility with the biochemical signs of increased testicular oxidative stress

(Manna et al., 2004).

1.7.2. Infections

1.7.2.1. Male Accessory Sex Gland Infection (MAGI)

Male accessory sex gland infections (MAGI) are seen in 5-12 % of cases of

male infertility and associated with biological and biochemical changes in the

seminal plasma (Comhaire et al., 1999). These alterations can originate from

seminal white blood cells (WBC) and their secretory products which have

possible adverse effects on the motility and fertilizing potential of spermatozoa.

Leukocytospermia is an important indicator of inflammation in the male genital

tract (Krause et al., 2003). Bacteria and viruses cause an acute inflammation

and induce oxidative stress through the inflow of activated leukocytes into the

affected area (Depuydt et al., 1996). According to the WHO criteria, male

genital tract inflammation is defined if the patient has more than 1x106

leukocyte in per millilitre in the ejaculate (WHO, 2001).

Many studies have been reported that obvious decrease of semen parameters

and fertility can be seen as a result of a high level of leukocytes in semen

(Gonzales et al., 1992; Henkel et al., 2003; Hill et al., 1994) while others have

reported no correlation between leukocytospermia and semen quality (Eggert-

Kruse et al., 2001; Harrison et al., 1991). Moreover, many cases of accessory

sex gland infection are an asymptomatic and clinical symptoms are not always

correlated with the presence of leukocytes (Hochreiter, 2003).

20

Presently, the prevalence and clinical significance of leukocytes in semen is

currently a matter of controversy. As main producers of ROS, leukocytes make

a significant contribution to overall levels of ROS in human semen (Aitken and

West, 1990).Furthermore, infiltrating leukocytes can lower the antioxidant

capacity of seminal plasma by consuming antioxidants (Aitken and Baker,

1995; Sharma et al., 2001).

The leukocyte induced oxidative damage in sperm cells may depend on the

number and subtype of leukocyte, as well as their activation status. Leukocytes

and their involvement to the oxidative stress related infertility will be discussed

further in Chapter 4.

1.7.3. Environmental Factors

1.7.3.1. Toxins and Chemicals

There are many epidemiologic studies examining the reproductive effects of

environmental toxin exposure on sperm and testicular function. The existence

and increase incidence of spontaneous abortions has been linked to paternal

exposures to some chemical agents. Some of these reproductive toxins cause

sperm oxidative damage and DNA fragmentation. These chemical agents

include many commonly present occupational hazards such as anaesthetic

gases, metals (mercury, lead and cadmium), solvents, pesticides, and

hydrocarbons.

In vivo studies suggest that exposure to lead and cadmium causes generation

of ROS and alteration of antioxidant defence systems in animals and

occupationally exposed workers (Acharya et al., 2003; Benoff et al., 2000; Hsu

and Guo, 2002). Cadmium (Cd) is known to produce oxidative stress and is

mainly used in the battery, metal-coating, and alloy industries. In addition to

these industrial uses, it also presents in cigarette (Spruill et al., 2002).

Occupational exposure to lead and cadmium has genotoxic effect in somatic

cells of workers in battery manufacturing area by promoting DNA damage

(Palus et al., 2003).

It has been demonstrated that air pollution causes male-mediated infertility,

miscarriage, and other adverse reproductive outcomes which is due to

increased sperm DNA fragmentation (Evenson and Wixon, 2005; Rubes et al.,

2005)

21

1.7.3.2. Electromagnetic Radiation

An exposure to electromagnetic radiation has a negative impact on semen

quality (Agarwal et al., 2009; Aitken et al., 2005) . In vitro studies have shown

that electromagnetic radiation including using regular mobile phone use

induces reactive oxygen species production and DNA damage in human

spermatozoa (De Iuliis et al., 2009). Use of cell phones decreases motility and

vitality of sperm cells as well as the concentration depending the duration of

exposure to radiation (Agarwal et al., 2008). Electromagnetic radiation

enhances mitochondrial ROS generation, while stimulating DNA base adduct

formation (De Iuliis et al., 2009).

There is also data linking with exposure to electromagnetic radiation from

regular mobile phone use and a genotoxic effect on epididymal spermatozoa

that can cause DNA damage in male germ cells (Aitken et al., 2005).

1.7.3.3. Drugs and Chemotherapy

There are hundreds of chemicals, including chemotherapeutics (eg. fludarabine,

cyclophosphamide, busulphan) which are known to have detrimental effects on

sperm morphology, number, and motility (Chatterjee et al., 2000; Wyrobek et

al., 2005).

Commonly used drugs such as aspirin and paracetamol can cause oxidative

stress by producing excessive reactive oxygen species through increased

cytochrome p450 activity (Tremellen, 2008). Some chemotherapeutic drugs

like cyclosporine and cyclophosphamide has been linked to increased level of

oxidative stress in sperm cells and decreased testicular catalase level in rats

(Das et al., 2002; Ghosh et al., 2002). Allopurinol used to treat gout reduces

seminal plasma levels of the antioxidant uric acid and may also cause oxidative

stress

Cocaine has also been demonstrated to induce increase in apoptosis and inturn

oxidative DNA damage (Li et al., 1999).

1.7.4. Iatrogenic

The increasing use of ART (Assisted Reproductive Technologies) and handling

of sperm for insemination in in vitro conditions expose them to oxidative

damage for two reasons (Alvarez, 2003). Firstly, centrifugation and

cryopreservation during ART procedures has been reported to increase sperm

ROS production (Agarwal et al., 2006). Secondly, removal of protective

antioxidants in seminal plasma during sperm preparation can also be

22

hazardous to sperm health (Zorn et al., 2003b). It has been demonstrated that

samples prepared by swim-up semen rather than sperm cells isolated by

density gradient prepared semen has reduced levels of f ROS and sperm DNA

damage, without exhibiting any major change in sperm motility (Twigg et al.,

1998b) .

1.7.5. Varicocele

Varicocele is a dilation of the spermatic vein/pampiniform venous plexus and is

present in about 40% of the infertile male population but only 15% of the

fertile population. While varicocele are known to cause testicular dysfunction

and consequently sperm DNA damage and infertility (Saleh et al., 2003), the

exact pathogenetic mechanism for this is not conclusively known. The proposed

mechanisms are reflux of toxic metabolites of adrenal or renal origin, elevation

of testicular temperature and elevated reactive oxygen species. While several

reports has conclusively linked varicocele with elevated levels of free radicals

(Agarwal et al., 2003; Zini et al., 2000), it is not understood what activates

this increase in free radical production. Men with varicoceles have been found

to have elevated levels of sperm DNA fragmentation compared to fertile men

without varicocele (Saleh et al., 2003). Presumably the elevation in seminal

free radical levels is responsible for this varicocele related sperm DNA

fragmentation.

1.8 Assessment of Seminal Oxidative Stress

If we accept that oxidative stress is a major cause of male infertility,

measurement of ROS in semen becomes necessary for proper investigation of

male factor infertility. However, routine semen analysis is the only test that

has been used for male factor evaluation in most clinics and provides limited

information about the status of sperm oxidative stress. This is a major

deficiency in modern clinical andrology.

While there are over 30 published assays to measure oxidative stress, all are

complex and time consuming assays, often requiring expensive equipment and

therefore are not practical for the average small andrology laboratory.

Direct measurement of reactive oxygen species in vivo is difficult due to their

short life span and can not be used for clinical use since they need relatively

expensive technologies (Cocuzza et al., 2007). Direct methods such as

electron-spin resonance spectroscopy (Buettner and Mason, 1990) and pulse

23

radiolysis (Asmus, 1984) can only be performed for research purposes with the

necessity of high technical experience.

The most commonly used indirect method for measurement of ROS in sperm

cells are global ROS measurement by chemiluminescence (Sharma and

Agarwal, 1996) and membrane lipid peroxidation determination using

Malondialdehyde (MDA) and thiobarbituric acid (TBA) (Aitken et al., 1993b;

Gutteridge and Quinlan, 1983).

1.8.1 ROS Measurement by Chemiluminescence

The chemiluminescence method quantifies both intracellular and extracellular

ROS by using sensitive probes such as luminol (5-amino-2, 3, dihydro 1, 4,

phthalazinedione) and lucigenin for quantification of redox activities of

spermatozoa (Aitken et al., 2004). The reaction of these probes with ROS

results in production of a signal which is converted to an electrical signal and

measured as counted photons per minute by luminometer (Agarwal et al.,

2004). Luminol probe is an extremely sensitive and oxidisable substrate and

reacts various ROS at neutral pH, thereby allowing for the measurement of

intracellular and extracellular ROS. Lucigenin is superoxide anion sensitive and

can only measure extracellular ROS generation (McKinney et al., 1996).

Measurement of ROS using chemiluminescence is relatively sensitive and has

well established ranges in infertile and fertile population (Athayde et al., 2007;

Tremellen, 2008; Williams and Ford, 2005). On the other hand, this technique

has some disadvantages for clinical use, such as the need for expensive

equipment and difficulties in quality control due to leukocyte and seminal

plasma contamination (Aitken et al., 2004; Fingerova et al., 2009; Kobayashi

et al., 2001)

1.8.2 Lipid Peroxidation Markers

Markers for lipid peroxidation have been widely used for oxidative stress

measurement in sperm cells. Oxidative stress causes an accumulation of lipid

peroxides in the spermatozoa and generates of a variety of decomposition end-

products of unsaturated lipids such as Malondialdehyde (MDA), HNE

(Hydroxynonenal), 2-propenal (acrolein) and isoprostanes, all which can be

measured as indicators of oxidative stress (Dalle-Donne et al., 2006).

Malondialdehyde is the most common used marker for the lipid peroxidation

and can be quantitatively assessed by the thiobarbituric acid-reacting

substances (TBARS) Assay. In this assay, MDA forms a 1:2 adduct with

24

thiobarbituric acid (TBA) and produces the coloured substance which can be

measured by fluorometry or spectrophotometry (Aitken et al., 1993b). The

TBARS assay is considered to be a nonspecific marker of membrane lipid

peroxidation, likely due to the reaction of thiobarbituric acid with non-lipid

moieties (Yeo et al., 1994). MDA level can also be directly measured in both

sperm cells and seminal plasma, but the measurement of MDA in sperm cells

needs to be performed by sensitive high performance liquid chromatography

(HPLC) due to its low existency (Li et al., 2004).

The recent assays for measurement of lipid peroxidation such as isoprostane 8-

iso-PGF2a and c11-BODIPY are promising but need more research to confirm

their usefulness as a marker of sperm oxidative stress status. (Drummen et al.,

2002; Pratico et al., 2001)

1.8.3 Measurement of Oxidative DNA Damage

Free radicals attacks DNA structure by generating base and sugar modification

products (Dizdaroglu et al., 2002). The most commonly studied product for

oxidative DNA damage is 8-hydroxy-20-deoxyguanosine (8-OHdG) (Halliwell

and Whiteman, 2004). This oxidative DNA adduct has been used as an index of

oxidative DNA damage due to its high sensitivity and abundance in DNA. 8-

OHdG can be assessed by HPLC, Gas chromatography (GC) or liquid

chromatography (LC), Mass Spectrometry (GC) and antibody based techniques

(Dizdaroglu et al., 2002). The analytical methods to detect these markers are

technically challenging and artifactual DNA damage may occur during isolation

(Collins et al., 2004; Halliwell and Whiteman, 2004). Immunochemical methods

visualise of the damage but they are limited for being only semi quantitative

(Halliwell and Whiteman, 2004).

Oxidative DNA damage can be measured indirectly by assessing DNA damage

in sperm. Several tests are available for the measurement of sperm DNA

integrity such as SCSA (Sperm Chromatin Structural Assay), terminal

deoxynucleotidyl transferase-mediated nick end labeling (TUNEL Assay) and

Chromomycin A3 Assays. However, all of these assays measure DNA damage

that is not necessarily directly related to oxidative attack

1.8.4 Other Markers for ROS Measurement

Measurement of the Total Antioxidant capacity (TAC) of seminal plasma gives

an indirect measurement of oxidative stress. Total antioxidant capacity

measurement can be performed by several methods. The most commonly used

25

method for measuring TAC in seminal fluid is enhanced chemiluminescence

assay (Kobayashi et al., 2001; Sharma et al., 1999). In this assay; to produce

ROS, a signal reagent which contains luminol is mixed with Horseradish

Peroxidase-attached immunoglobulin. The amount of antioxidant in the seminal

plasma reduces the signal of chemiluminescence and the antioxidant capacity

is compared with Trolox, a water soluble tocopherol analog as a reference

standard (Said et al., 2003). Although this method is accurate, it needs

expensive instrumentation and has some technical difficulties.

The another commonly used method for measurement of antioxidant capacity

is based on colorimetric assay which uses 2,2'-azinobis-(3-ethyl-

benzothiazoline-6-sulphonic acid) (ABTS). In this assay, hydrogen peroxide

ABTS is oxidized to ABTS+ which produces a coloured substance that can be

measured spectrophotometrically. Antioxidants within the seminal plasma

compete with this reaction proportionally according to their concentration.

The research goal of this thesis can be summarised as;

1- Development of a reliable assay for measurement of sperm oxidative

stress that can be easily used in an average clinical andrology

laboratory with minimal additional equipment.

2- Investigate novel cause of oxidative stress in male infertility

3- Investigate the consequence of oxidative stress on sperm genome

function

4- Investigate the use of antioxidant therapy in male infertility

26

CHAPTER 2

MATERIALS AND METHODS

27

2.1 INTRODUCTION

In order to achieve the aims of this thesis, I utilized the following tests related

with free radical production and oxidative stress in semen;

1. Quantification of free radical production in semen was made using the

NBT assay.

2. The concentration of leukocytes was measured because leukocytes are

significant producers of free radicals in semen. For measuring leukocyte

number, an immunocytochemistry procedure (CD45) was used. The bio-

activity of leukocytes within semen was also measured using PMN

Elastase (neutrophil) and Neopterin (macrophage) measurements.

3. Measurement of free radical related peroxidative damage in sperm

membrane was analysed by LPO-586.

4. Determination of DNA damage was performed by using the Tdt-

mediated Terminal dUTP Nick-end Labeling (TUNEL) assay

5. Quantification of sperm apoptosis was measured by

immunocytochemical analysis of Annexin V assay.

6. Determination of sperm global DNA methylation was assessed by

immunohistochemical 5-methylcytosne staining.

7. Determination of the protamine packaging quality of the sperm nucleus

DNA was performed using CMA3 assay.

8. Epididymal and Testicular function was quantified by the measurement

of α-glucosidase and blood reproductive hormone levels, respectively.

28

2.2 Study Subjects

The University of Adelaide’s Ethics committee was notified of the study once

ethical clearance had been obtained from Women's and Children’s Hospital,

Human Research and Ethics Committee (HREC), as per University policy.

Semen samples were obtained from an academic affiliated assisted

reproductive technology unit in Adelaide, South Australia (REPROMED) after

providing written informed consent for the participation in the study from all

participants.

The participants in this study were drawn from two groups. The fertile control

group consisted of men in the sperm donation program at Repromed who had

proven paternity in the last twelve months, were in general good health, aged

18-50 years of age, with normal sperm parameters. The infertile patient cohort

consisted of men with abnormal routine sperm parameters who were seeking

reproductive treatment at Repromed.

All semen samples were collected and coded with a unique identifier number.

All results were collated using this unique identifier rather than the patient’s

name, so as to maintain patient confidentiality.

2.3 Sample collection and Semen Analysis

All semen samples were produced by masturbation into a clean container after

3-5 days of abstinence. Semen samples were allowed to liquefy at least for 30

minutes at room temperature before analysis.

Semen analysis was performed according to WHO guidelines (1999) to obtain

volume, pH, sperm concentration, motility, and morphology by staff in the

Andrology laboratory in Repromed (WHO, 1999a). Morphology smears were

scored using WHO criteria. Sperm concentration was expressed as 106 per

millilitre of semen, whereas motility and morphology were expressed as

percentage. The motility results reported as the sum of rapidly progressive

(WHO type “a”) and low progressive (WHO type “b”) motility. Sperm sample

was classified as normospermic if sperm concentration was ≥ 20×106 per ml,

sperm motility was ≥ 50 % and normal sperm morphology ≥ 15 %.

29

2.4 Sample Preparation

After routine semen analysis, the remaining portion of semen was divided into

different portions to analyse for research parameters (Figure 2.1). One portion

of semen was washed with Dulbecco’s Phosphate buffered solution (PBS) (JRH

Biosciences, Lenexa, Kansas, USA Cat No:59321C) and smeared on poly-L-

Lysine coated slide (PolysineTM, Menzel-Glaser GmbH&Co KG,Braunschweig,

Germany) for fixation. The other part of remaining liquefied neat semen was

centrifuged for 10 min at 3000 x g to separate sperm cells from plasma.

Seminal plasma was aspirated by pipette and aliquoted into four separate

eppendorf tubes which were stored at -70 oC until assayed. Aliquoted seminal

plasmas were used to analyse LPO, a-Glucosidase, Neopterin and PMN Elastase

assays. Fixed slides were use for quantification of leukocytes, apoptosis and

DNA fragmentation.

Figure 2.1: Steps in sample preparation

30

To conduct the sperm washing procedure of semen, 400 µL of liquefied semen

was mixed with 2000 µL of Dulbecco’s Phosphate buffered solution (PBS) and

centrifuged at 300xg for 10 minutes. After spinning, the supernatant was

discarded without disturbing the pellet by using a glass pipette. The pellet was

gently resuspended in 400 µL PBS and this mixture was centrifuged at 300 x g

for 5 minutes. This last step was applied twice. After discarding all supernatant,

pellets were resuspended in 400µL PBS, thereby keeping the original volume of

neat semen before the washing procedure.

This washed semen was divided into two parts; one part for measurement of

Annexin V and Nitroblue tetrazolium assays while the other part was used for

the preparation of slides. Before applying to the slides, the washed semen was

adjusted to a concentration of 1x106 sperm/mL by adding PBS depending on

the original sperm concentration in the neat semen to provide optimal

concentration in each slide.

For the fixation step, Poly-L-lysine coated glass slides were marked by DAKO

marker pen (Dako North America, Inc., Carpentaria, California, USA) in two

square boundaries at each slides and labelled with sample’s identifier number.

10 µL of aliquots of washed sperm suspensions were applied to squares on the

glass slides and allowed to dry at room temperature. The air dried cells were

fixed for an hour at room temperature in fixative solution (96 % Ethanol) for

CD45 immunoassay and sperm global DNA methylation assays and in 3:1

methanol (95%)/Glacial acetic acid fixative for DNA fragmentation (TUNEL) and

sperm protamination (CMA3) assays. The slides were removed from fixative

solution and allowed to dry at room temperature in a fume hood. For

subsequent analysis, the slides were wrapped in aluminium foil one by one and

stored at -70 0C until assayed.

2.4.1 Sperm Swim-up Procedure

For experiments requiring pure sperm without the presence of contaminating

leukocytes, sperm “swim-up” procedure was used. To apply this procedure, 1

mL of warmed sperm culture media (G-Sperm™, Vitrolife, Kungsbacka,

Sweden) was transferred into a 10 mL Falcon tube (Becton Dickinson

Biosciences, San Jose, CA USA). 1 mL of liquefied neat semen was placed

31

gently beneath the media and after incubation for 1h at 37 oC, the upper third

of the sperm suspension was aspirated carefully and centrifuged at 300 x g for

10 minutes.

2.4.2 Isolation of Leukocytes from Peripheral Blood

Peripheral blood leukocytes were isolated by density gradient centrifugation

with subsequent erythrocyte lysis (Kovalski et al., 1991). 2 mL of whole blood

was collected into an EDTA tube and then was mixed with 10 mL 0.87% NH4Cl

solution to produce osmotic lysis of the erythrocytes. The mixture was allowed

to stand for 20 minutes at room temperature and then the haemolysed blood

was centrifuged at 2200 rpm for 15 minutes. The pellet contains leukocytes

was resuspend in 2 mL 0.87% NH4Cl solution. The cell suspension was layered

onto a single discontinuous PercollTM Gradient (Amersham Biosciences,

Uppsala, Sweden). To prepare Percoll gradients, first 20% Percoll solution in

PBS was added to the tube and then 30%, 40% and 55% Percol solutions were

layered underneath of each layer, respectively. 2 mL of leukocytes suspension

was layered onto the top of the percoll gradient and then centrifuged at 1300 x

g for 30 minutes. Neutrophils are collected from the interface between 40%

and 55% Percoll gradient. Monocytes and lymphocytes are collected from the

interface between 30% and 40% Percoll gradient. The harvested leukocyte

suspension was washed once in phosphate buffered saline (PBS) and then

resuspended at a concentration of 1 x 106/mL.

2.5 Determination of Leukocytes in Semen

The existence of seminal leukocytes was analysed by immunohistochemical

leukocyte common antigen marker CD 45. The quantification of neutrophil

activity in semen was performed using the PMN Elastase assay.

2.5.1 Immunocytochemistry Procedure for CD-45

In this procedure, streptavidin-biotin system against the common leukocyte

antigen CD45 was used. This monoclonal antibody was applied to detect

granulocytes, lymphocytes and macrophages in semen simultaneously.

To apply fluorescence staining procedure, all cells were washed and fixed

according to the procedure described at 2.4

32

The slides were taken from freezer and stood at room temperature for thawing.

After thawing, the slides were washed in PBS for 5 minutes. From this point the

slides were not allowed to dry out and kept in a humidified container.

Protein Blocking Step:

The sections in each slide were incubated for 20 minutes with 50 µL. of 5%

normal goat serum. This protein blocking solution was freshly prepared in each

assay. To prepare the protein blocking solution (PBS/BSA Solution), 150µL. of

normal Goat Serum (Vector Laboratories, CA) was diluted in 2850µL PBS. After

incubation with protein blocking solution, the slides were washed once with

PBS for 5 min.

Avidin-Biotin Blocking Step:

Avidin/Biotin Blocking Kit (Vector Laboratories, CA) were used to block

nonspecific binding of Biotin/Avidin System reagents. This kit contains Avidin D

and Biotin Solutions and these solutions were used directly as supplied. Each

section of the slides was pre-treated with the Avidin D solution for 15 min at

room temperature firstly. After washing with PBS solution in 5 minutes, the

slides were incubated for 15 minutes with the Biotin Solution and then washed

in PBS for 5 minutes as a last step.

Primary Antibody Step:

The sections were incubated for 60 minutes with 50 µL. primary antibody (Rat

Anti-human CD45) (Serotec Ltd,UK,Code: MCA 345) at room temperature in a

humidified container. Working solution of the Primary Antibody concentration

was 4µg/mL. To achieve this concentration, 10µL primary antibody solution was

diluted in 4990µL PBS-BSA solution. This dilution was equal to 1/500. After

treatment with primary antibody, the slides were washed twice for 5 minutes in

PBS.

Secondary Antibody Step:

The sections were incubated for 30 minutes with 50 µL. diluted biotinylated

secondary antibody solution (Goat anti-rat IgG) (Jackson Immuno Research

Laboratories, Inc, USA) at room temperature in a humidified container. Stock

solution of the Secondary Antibody concentration was 2 mg/mL. To prepare

Working Solution from the stock solution, 2µL secondary antibody solution was

diluted in 4990µL PBS-BSA solution. The slides were washed 3 times for 5

minutes each wash in PBS.

Avidin Conjugate Step:

The sections were incubated for 30 min with 50µL Texas Red Avidin D

conjugate (Vector Laboratories Inc., CA) at room temperature in a humidified

container. The concentration of the Texas Red Avidin Conjugate was 25 µg/mL.

33

This stock solution was diluted by the PBS in the ratio of 1/100 before starting

experiment.

After Avidin conjugate step, slides were washed 3 times in PBS for 5 minutes in

each wash. A 1:1 mixture of ProLong® Gold anti-fade reagent (Invitrogen

Molecular Probes, Eugene, Oregon, USA) and 10% glycerol in PBS was applied

to the each section as a mounting medium and was covered by 18 x 18 mm

cover slips. Subsequently, the leukocyte cells were visualized by using on

Olympus BX51 fluorescence microscope with the 595nm filter for Texas Red

Dye. Leukocyte concentration was determined by analysing the relative ratio of

CD45 positive cells to sperm in at least 300 high power fields and expressed as

106 leukocytes per milliliter.

2.5.2 PMN Elastase Assay

PMN Elastase is a proteinase enzyme which is secreted by activated

granulocytes (Reinhardt et al., 1997). During phagocytosis PMN elastase is

excreted into the extracellular matrix to phagocytise and destroy non-natural

agents (Zorn et al., 2000). This enzyme can be used as a measure activity of

granulocytes during an inflammatory response in seminal plasma (Reinhardt et

al., 1997). Latex particles were coated with antibody fragments F (ab') 2

against human PMN elastase; changes in turbidity were measured

photometrically. The extent of turbidity was proportional to PMN elastase

concentration in the test sample.

Granulocyte elastase concentration in seminal plasma was measured by the

homogeneous immunoassay ECOLINE® PMN elastase (Merck, Darmstadt,

Germany). The kit detected PMN elastase concentrations of >4 µg/L.

To determine PMN Elastase concentration, stored seminal plasma at -70°C was

taken from the freezer and stood at room temperature until thawed. While

waiting for thawing of the samples, standard solutions were prepared. The

concentration range of these standards was 10 - 0.16 ng/mL.

Thawed seminal plasma samples were diluted 1:100 with sample diluent which

is provided by the supplier. 100 µL of each 1:100 diluted sample was added to

the designated wells, in duplicate. The microplate was covered with a plate

cover and incubated at room temperature for 1 hour. After removing the plate

cover, the microwell strips were washed four times with 300 µL wash buffer.

34

After the last wash, microwell strips were tapped on an absorbent pad to

remove excess wash buffer. The wells were not allowed to dry.

150 µL of HRP-conjugate was added to all wells and covered by a plate cover

again and incubated for 1 hour at room temperature on a rotator. Then,

microwell strips were washed four times as previously described and the

procedure were carried on to the next step immediately. 200 µL of TMB

substrate solution was added to all wells and incubated at room temperature

for 20 min on a rotator. Microplate wells were protected from direct exposure

to intense light. After the incubation, 50 µL of Stop solution which is included in

the kit was pipetted into each well to stop the enzyme reaction. Immediately

after addition of the stop solution, the colour reaction was measured

spectrophotometrically at 450 nm by using a microplate reader (Bio-Tek

Instruments, Inc. Winooski, VT, USA, Model ELx800). The concentration of PMN

Elastase in the sample was calculated against to standard curve in the assay

and expressed as ng/mL.

2.5.3 Determination of macrophage activity in semen (Neopterin

Assay)

Neopterin is a catabolic product of guanosine triphosphate (GTP), a purine

nucleotide and synthesised by macrophages undergoing immune activation.

Measurement of neopterin level provides information about the immune

activation status of macrophages.

Determination of Neopterin in seminal plasma was conducted by using a

Neopterin screening ELISA kit following the manufacturer’s instructions

(BRAHMS Aktiengesellschaft, Ennigsdorf, Germany). Neopterin ELISA

Screening kit provides quantitative determination of neopterin, based on the

basic principle of a competitive enzyme-linked immunosorbent assay.

An unknown amount of antigen in the sample and a fixed amount of

neopterine/enzyme conjugate compete for the antibody-binding sites

(polyclonal sheep-anti-neopterin) of these antibodies, thus forming an immune

complex bound to solid phase (anti-neopterin antibody / neopterin or

neopterin/enzyme conjugate). Unbound antigen is removed by washing. The

intensity of the colour developed after the substrate incubation was inversely

proportional to the amount of antigen in the sample.

35

Procedure of the assay

Thawed seminal plasma was first diluted 1/10 in PBS to allow quantification of

seminal plasma neopterin within the ELISA’s detectable range. 50 µL of

Neopterin standards (at the concentration of 2, 5, 10, 25 and 50 nmol/L) which

is provided by the manufacturer and 50 µL of diluted seminal plasma were

pipetted into the clean tubes with 150 µL of the enzyme conjugate. 150 µL of

this mixture was transferred into the neopterin-antibody coated microtitre plate

within 5 minutes and incubated for 2 hours at room temperature in the dark

using black cover sheet. After incubation, the wells were washed 4 times with

350 µL of washing solution which was prepared freshly from concentrate

washing solution by diluting 1/50. 100 µL of substrate (para-nitrophenyl

phosphate) solution was pipetted into the wells and incubated for 30 minutes

at room temperature. Then, 100 µL of stop solution (2N Sodium Hydroxide)

was added to stop enzyme reaction and the formation of colour. The optical

density of colour was measured in a microplate reader (Bio-Tek Instruments,

Inc. Winooski, VT, USA, Model ELx800) at a wavelength of 405 nm. The results

were calculated by plotting against a standard curve and expressed as nmol

Neopterin per ml seminal plasma.

2.6 Measurement of Reactive Oxygen Species

2.6.1 Lipid Peroxidation Test (LPO Test)

Production of ROS level was measured by their lipid peroxidation formation

using Bioxytech LPO-586 assay kit (Oxis ResearchJ, Oxis International, USA).

Lipid peroxidation is quantified by measuring malondialdehyde (MDA) and 4-

hydroxyalkenals, the degradation products of polyunsaturated fatty acids

(PUFA) hydroperoxides (Esterbauer et al., 1991). The LPO-586 assay is based

on the reaction of a chromogenic reagent (N-methyl-2-phenylindole) with MDA

and 4-hydroxyalkenals to yield a stable chromophore at 586 nm absorbance

spectrophotometrically.

Procedure of the LPO Test

Frozen seminal plasma samples were taken from freezer and stood on the

room temperature to allow for thawing. While waiting in this procedure,

standard solutions were prepared according to Table 1. Standards were run in

duplicate in each assay.

36

Standard Concentration (µM MDA) 0 0,5 1 2 3 4

Volume of 20 µM standard to add

(mL)

0 25 50 100 150 200

Volume of PBS used for diluting (mL) 200 175 150 100 50 0

Table 2.1: Standard Curve Dilution Volumes

200 µL of thawed seminal plasma were added to clean glass test tubes.

Samples also were run in duplicate in each assay.

Reagent 1 (N-methyl-2-phenylindole in acetonitrile), was diluted to 1/3 by the

Diluent (Ferric Iron in Methanol) which were both provided in the kit.

Depending on the number of samples in each assay, one volume of Diluent was

mixed with three volumes of Reagent 1 and this solution was prepared

immediately before use.

650 µL of diluted R1 reagent was added to each sample tube and mixed gently

by vortex. Then, 150 µL of concentrated (12 N) HCl was added to tubes and

mixed well. Tubes were tightly closed and incubated at 45°C for 60 minutes. To

obtain a clear supernatant, samples were centrifuged at 15000 x g for 10

minutes. The supernatants were transferred to the reading cuvettes and

measured at the absorbance at 586 nm by a microplate reader (Bio-Tek

Instruments, Inc. Winooski, VT, USA, Model ELx800).

By using the standard data, the net A586 for each standard was calculated by

subtracting the blank (Ao) value from each of the standard A586 values. The

concentration of analyte in each sample was calculated by using standard

curve.

2.6.2 Nitro Blue Tetrazolium Assay (NBT Assay)

A modified colorimetric Nitro Blue Tetrazolium (NBT) test was used to evaluate

reactive oxygen species (ROS) production of both leukocytes and sperm within

semen (Choi et al., 2006; Esfandiari et al., 2003).

Nitroblue tetrazolium test depends on the reaction of aromatic tetrazolium

compound with cellular superoxide ions (Baehner et al., 1976). Metabolically

active cells reduce the yellow water-soluble tetrazolium salt to blue water-

insoluble formazan derivative. The composition of formazan serves as an

estimate of contribution of spermatozoa and leukocytes toward the amount of

ROS production in semen.

37

Nitrotetrazolium Formazan

Figure 2.2: Schematic chemical formula of the NBT assay

Procedure of the NBT Assay

100 µL of washed semen, which was prepared the procedure described at 2.4,

was incubated with an equal volume of NBT Working Reagent at 37oC for 45

minutes. For each sample, the assay was run in duplicate using eppendorf

tubes away from light exposure. The NBT working solution was prepared from

0.01% NBT stock, (Sigma-Aldrich, St. Louis, MO, USA Cat No: N5514) at the

dilution of 1/10 with PBS. The stock solution was prepared by dissolving one

tablet of NBT reagent into the 1000 µL of distilled water. Following the

incubation the tubes were centrifuged at 500 x g for 10 minutes to concentrate

the formazan crystals and then washed in 1000 µL PBS twice in order to

remove all residual NBT solution. To quantify the formazan product, the intra-

cellular formazan was solubilised in 60 µL of 2 M KOH and shaken vigorously.

60 µL of Dimethyl Sulphoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA

Cat.No: D2650) was added to tubes and tubes were mixed with vortex. The

resulting colour density was measured spectrophotometrically on the aide of a

microplate reader (Bio-Tek Instruments, Inc. Winooski, VT, USA, Model

ELx800) at 630 nm, as previously reported for leukocytes (Choi et al., 2006;

Rook et al., 1985).

ROS production was expressed as µg formazan per 107 sperm, derived from a

standard curve of absorbance values for known amounts of formazan

substrate.

2.6.2.1 Formazan Production for Standard Curve in NBT Assay

A standard curve for the NBT assay was performed after producing formazan

crystals in our laboratory. Since formazan crystals are no longer commercially

available, they were produced from NBT solution by the action of superoxide

anions generated by the Xanthine/ Xanthine Oxidase system. Xanthine oxidase

with xanthine solution generates significant amounts of superoxide anions with

the resulting production of formazan crystals when added to NBT solution.

OO22..--

38

To perform the procedure, 1 mmol/L Xanthine (Sigma-Aldrich, St. Louis, USA,

Cat No: X7375) solution was prepared by dissolving in 150 mL NaOH and

subsequently in deionised water. 25 U/mL Xanthine oxidase solution (Sigma-

Aldrich, St. Louis, USA, Cat No: X2252) was prepared from 3.2 mg Xanthine

oxidase solid and aliquoted into small amounts to freeze until the next assay

run.

Before the assay, the weight of half-dozen 2mL eppendorf tubes was recorded

for the measurement of the formazan crystals after reaction with NBT reagent.

500 µL of Xanthine solution was added to tubes following the addition of 500 µL

NBT working solution. After pipetting 10 µL of Xanthine oxidase, tubes were

incubated at 37 oC for 30 minutes to produce formazan crystals. Formazan

crystals were washed twice in deionised water and centrifuged at 500 x g for

10 minutes. All supernatant was taken from tubes and let dry for overnight. To

measure how much formazan was produced; tubes were re-weighed and then

solubilised in 300 µL DMSO and KOH. A standard curve was derived by plotting

measured formazan mass versus absorbance value. This assay was repeated in

5 different days to provide accurate measurement for optimizing the test.

2.7 Detection of Defective Sperm Chromatin Packaging

2.7.1 Chromomycin A3 Assay

Principle of the Assay

Chromatin packaging assessment was quantified using the chromomycin A3

(CMA3) fluorochrome which indirectly demonstrates a decreased presence of

protamine.

CMA3 is a fluorochrome which is specific for GC-rich sequences in DNA

(Franken et al., 1999). CMA3 competes with protamines for the same binding

sites in the minor groove of DNA (Bizzaro et al., 1998). Therefore, high CMA3

fluorescence is a strong indicator of poor protamination of the mature

spermatozoa, suggesting increased vulnerability to sperm DNA fragmentation

(Bianchi et al., 1996).

Procedure of the Chromomycin A3 Assay

The slides which have been fixed in methanol-glacial acetic acid (3:1) and

frozen at – 70 0C were taken from the freezer and stood at room temperature

for thawing. After thawing, slides were washed in PBS for 5 minutes and

treated for 20 minutes with 100 µL. CMA3 solution which was prepared at the

39

concentration of 0.25 mg/mL in McIlvaine Buffer [3,53 mL of 0.2 M anhydrous

citric acid and 16,47mL. of 0.2 M anhydrous sodium phosphate dibasic

(Na2HPO4)]. The slides were rinsed in PBS buffer and mounted with buffered

glycerol (1:1 v/v PBS-glycerol). Microscopic evaluation was performed by using

fluorescent microscope with the appropriate filters (460 nm - 470nm)

A total of 200 spermatozoa were randomly evaluated by distinguishing

spermatozoa that stain bright yellow as CMA3 positive from those that stain a

dull yellow as CMA3 negative (Picture 2.1). CMA3 positive cells were identified

microscopically and quantified using IP Lab 3.6.1 software. The samples

contain more than 30 % CMA3 positive cells were assessed as pathologic.

The protamination level of sperm is given by the formula;

Protamination (%) = 100 – CMA3 result

Normally sperm have a level of protamination >85%

2.8 Assessment of DNA Damage

2.8.1 TUNEL (Tdt-mediated Terminal dUTP Nick-end Labeling) assay

Principle of the assay

DNA fragmentation induced in spermatozoa was assessed using the TdT-

mediated-dUTP nick-end labeling (TUNEL) technique. This assay identifies

single and double stranded DNA breaks by labelling free 3`-OH termini of the

DNA in an enzymatic reaction with terminal deoxynucleotidyl transferase(TdT)

Picture 2.1: Representative image of CMA3 staining under the fluorescence microscope. Bright cells represents poor protamination stained by CMA3. Normal protaminated sperm appear dull yellow

40

followed by fluorescein labelling with propidium iodide (Heatwole, 1999). In

this study, the modified microscopic TUNEL technique first described by Lopes

et al (Benchaib et al., 2003b; Gandini et al., 2000; Lopes et al., 1998) was

performed.

Procedure of the TUNEL Assay

The slides which have been fixed in methanol-glacial acetic acid (3:1) and kept

in the -700C freezer were taken out and stood at room temperature until

thawed. After soaking the slides in PBS for 5 minutes at room temperature,

sperm cells were permeabilised in permeabilisation solution (0.1% Triton X-100

in 0.1% sodium citrate) .To perform this, 100 µL permeabilisation solution was

applied to each sections in the glass slides and incubated for 30 minutes at

room temperature in a humidified chamber. After washing twice with PBS, cells

treated with 20 µL TUNEL reaction mixture which was prepared by diluting 1

part enzyme solution (FITC-labelled terminal deoxynucleotidyl transferase-TdT)

in 9 part Label solution (Nucleotide mixture), ie 10 µL enzyme solution in 90 µL

label solution for each sample. The slides were incubated for 1 hour at 370C

incubator and labelled with 50 µL Propidium Iodide (10 µg/mL) for 30 minutes

at room temperature in the dark. Slides were rinsed twice in 50 µL PBS buffer

for two minutes and mounted in a 1:1 mixture of ProLong® Gold antifade

reagent (Invitrogen Molecular Probes, Oregon, USA) and glycerol. Stained cells

were quantified on Olympus BX51 fluorescence microscope, with a minimum of

300 sperm per slide being assessed using image analysis software (MacProbe V

4.3, Perceptive Scientific Instruments, League, Texas). DNA fragmentation in

sperm cells was evaluated as negative or positive on the basis of the presence

or absence of head staining. The percentage of sperm DNA fragmentation was

calculated as the number of TUNEL positive nuclei (FITC-labeled, green) per

total number of sperm nuclei (Propidium Iodide, red) in approximately 300

cells (Picture 2.2). For a positive control sperm cells were incubated with 3 U/

µL DNAse prior to incubation with the TUNEL reagents and for a negative

control the terminal transferase was omitted from the reaction.

41

A

B

Picture 2.2: Representative examples of a typical microscopic

TUNEL result for both a low (A) and high (B) DNA fragmentation

semen sample.

2.9 Assessment of Aberrant Apoptosis

2.9.1 Annexin V Assay

Annexin V is a fluorescence compound which binds to phospholipids in the

presence of calcium and helps to detect apoptotic cells (Oosterhuis et al.,

2000). At the beginning of the apoptosis, phospholipids, which are found

normally on the internal part of the plasma membrane, are translocated to

external part of the membrane (Figure 2.3). Binding of Annexin V to

phospholipids in plasma membrane shows early apoptotic process.

42

Figure 2.3: Schematic diagram of the Annexin V assay


The procedure was conducted according to the protocol recommended by the

manufacturer. As explained in section 2.4, washed neat semen was used for

Annexin V assay. To carry out the procedure, 400µL of semen was mixed with

2000µL PBS and centrifuged at 300 x g for 10 minutes. After taking the

supernatant, the washing procedure was repeated once and the pellet was

resuspended in 400µL PBS. According to the sperm concentration of the

sample, the mixture was diluted at a concentration of 1 x 106 cell/mL with the

relevant amount of binding buffer which consists of 100 mM HEPES/NaOH

Buffer. 1µL Annexin V conjugate and 0.5µL Propidium Iodide were added to the

cell suspension. After the incubation of the tubes at room temperature for 10

minutes under light-proof conditions, the suspension was centrifuged at 400 ×

g for 5 min and the pellet was mounted on a poly-L lysine coated slide by using

an 18 x 18 mm cover slip. The cells were examined immediately with an

Olympus fluorescence microscope equipped with an Olympus digital camera. At

least 200 sperm cells were counted on per slide.

Three different patterns of fluorescence were observed:

1- ) Cells which were early in the apoptotic process were stained with the

Annexin V and seen as green colour.

2- ) Necrotic cells were stained with both Propidium Iodide and Annexin V and

seen in red and green colour respectively.

3- ) The live cells showed no staining by either Propidium Iodide or Annexin V

and were seen only by phase contrast microscopy.

The division of non-staining cells (live cells) to green coloured cells (apoptotic

cells) was evaluated as an apoptotic live cells (Picture 3.2).

43

Picture 2.3: Representative example of Annexin V assay. Cells with negative

fluorescence staining indicate alive sperm cells; Annexin V positive cells

(green labelled) are showing Apoptotic cells; and both PI positive (red labelled) and Annexin V positive cells are showing necrotic cells.

2.10 Assesment of sperm global DNA Methylation

2.10.1 Immunohistochemical 5-methylcytosine Staining

Measurement of sperm global DNA methylation was made using the

immunohistochemical 5-methylcytosine staining technique (Benchaib et al.,

2003a) which is based on labelling of methylation sites of CpG dinucleotides.


After removing slides from freezer fixation of cells with ethanol (96%) the

slides were rinsed twice in PBS containing 0.25% Triton X (Sigma-Aldrich, St.

Louis, USA,Cat No: T9284). Decondensation of sperm DNA was carried out by

decondensing buffer (1M HCL, 10 mM Tris Buffer, pH 9.5 containing

dithiotretiol) (Sigma-Aldrich, St. Louis, USA) at room temperature for 20 min.

Sperm were then washed twice in PBS / Tween 0.5 % buffer and then the

sperm DNA was denatured by 6 N HCL to ensure access to methylation bases.

The denaturation process was followed by neutralisation with 100 mM TRIS

HCL (pH 8.5) for 30 min at room temperature. Cells were then incubated with

a dilution of 1:50 monoclonal primary specific antibody against 5-

methylcytidine (5mC) (Eurogentec S.A., BI-MECY-1000, Seraig, Belgium) for 1

hour at 37C. After rinsing twice in PBS, sperm were incubated with Fluorescein

Apoptotic Cells: An. V+ PI – Necrotic Cells: An.V+ PI+ Live Cells: An.V - PI -

44

Affinipure goat anti-mouse IgG (Jackson Immunoresearch Laboratories Inc,

West Grove, PA, USA) at a dilution of 1:100 in 0.05% Tween 20 in PBS for 20

min. The slides were then rinsed in PBS buffer and mounted in ProLong® Gold

antifade reagent (Invitrogen Molecular Probes, Oregon, USA). Negative controls

were performed via the application of secondary antibody by omitting the

primary 5-methylcytidine antibody. The stained cells were visualized on

Olympus BX51 fluorescence microscope and the degree of global DNA

methylation was determined by measuring mean value of intensity of the

fluorescence and expressed as Arbitrary Unit (A.U.) on at least 300 cells. The

primary antibody step was omitted on the negative control slides.

In order to minimize qualitative error related to fading of fluorescent staining

intensity over time, all slides were captured for later image analysis within 1

hour of completion of the immunochemistry process. Furthermore, all

measurements were conducted on a single microscope at a set fluorescent light

exposure intensity. Finally, individual infertile patient’s entry and exit

methylation slides were always analysed in the same assay run, so as to

minimize skewing of results by inter-assay variation. Using these precautions

the intra and inter-assay CV was 5 and 7% respectively.

2.11 Assessment of epididymal function

2.11.1 αααα -Glucosidase assay

α-Glucosidase enzyme (EC.3.2.1.20) activity is a marker of human epididymal

secretory function (WHO, 1999b). α-glucosidase is found in two isoenzyme

forms and mostly originates from epididymis (Paquin et al., 1984). The neutral

isoenzyme form of α -glucosidase is released from the epididymis and

contributes 80% of total activity. The acid isoenzyme form of α-glucosidase

provides 20% of total activity and is secreted by the accessory reproductive

glands (Bedford, 1994).

α-glucosidase activity can be measured photometrically in seminal plasma. In

this study colorimetric neutral α-Glucosidase kit was used (Roche α-

Glucosidase Assay Kit Cat No: 11742027001, Roche Applied Science,

Mannheim, Germany).

The principle of the assay was dependent on neutral α-glucosidase activity in

the sample liberating yellow 4-nitrophenol from the substrate solution of 4-

nitrophenyl- α -D-glucopyranoside according the equation below;

45

4-Nitrophenyl- α -D-glucopyranoside

4-Nitrophenol + α -D-glucopyranoside

The kit contains the substrate and all buffers for the measurement of neutral

α -glucosidase in seminal plasma including sodium dodecyl sulfate (SDS) and

castanospermine substrate which provides the specific measurement of

epididymal origin of neutral α-glucosidase.


In a first step the stored frozen semen samples were thawed and centrifuged

at 1000 x g for 10 minutes. The clear supernatant was taken by using positive

displacement pipette for avoiding higher variances because of the possibility of

existence of high viscosity in some samples. Before the assay, the reaction

solution was prepared according to manufacturer instructions. To prepare the

reaction solution, substrate concentrate solution containing 4-nitrophenyl-α -D-

glucopyranoside was warmed to 37 0C until the content of the bottle is entirely

dissolved). For each sample, 10 µL of substrate solution was prepared by

dissolving in 90 µL reaction buffer which contains pH 6.8 phosphate buffer with

1% SDS to maintain a neutral pH.

Standard samples were prepared from 100 µM standard concentrate solution

(para-nitrophenol) by diluting with stopping buffer in serial. The end

concentrations of standard solution were 0, 20, 40, 60, 80, 1000 µM para-

nitrophenol. Standards were run in duplicate in each assay.

100 µL of reaction solution was pre-warmed at 370C and mixed with 15 µL

seminal plasma. The mixture was vortexed and incubated at 370C for 2 hours.

A blank control sample was set up by mixing the specific inhibitor

castanospermine to the sample before adding the substrate. After adding

substrate solution into the sample the reaction was started. During 2 hour

incubation at +37°C the α -glucosidase containing sample will liberate coloured

4-nitrophenol from the substrate 4- nitrophenyl- α -D-glucopyranoside.

The resulting colour reaction was stopped by adding 1ml. stop buffer. 250 µL

of aliquot was transferred into a 96 well microplate for measurement at 405

α-glucosidase

46

nm on microplate reader (Bio-Tek Instruments, Inc. Winooski, VT, USA, Model

ELx800). The final result was then derived after subtracting the

castanospermine inhibited semen blank absorbance results from each well. The

enzyme activity (mU/mL) was determined from an absorbance versus enzyme

activity standard curve.

2.12 Assessment of testicular function

2.12.1 Measurement of Serum Reproductive Hormone Levels

The assessment of testicular function was done by measuring serum

reproductive hormone levels. The level of serum testosterone and LH

measurement was applied for evaluation of Leydig cell function, while FSH,

Estradiol and AMH (Anti-Mullerian Hormone) assays were applied for Sertoli

cells function.

Venous blood samples were collected into gel separator tubes (BD Vacutainer®,

Becton-Dickinson Company, Plymouth, UK) in the morning and centrifuged at

4000rpm for 10 minutes. Serum was separated within 1 hour of collection and

frozen at – 70oC until assayed. All hormone measurements, with the exception

of AMH (see below), were conducted using the automated ADVIA Centaur

chemiluminescent immunoassay system obtained from Bayer Australia

Ltd.(Pymble, NSW, Australia).

2.12.2 Measurement of AMH (Anti Mullerian Hormone) level in serum

Serum AMH levels, a measure of sertoli cell function (Rajpert-De Meyts et al.,

1999), were measured using the Immunotech high-sensitivity immuno-

enzymetric assay kit (Beckman Coulter, Marseille, France). In the first step the

Antimullerian Hormone is captured by a monoclonal antibody bound to the

wells of a microtiter plate. In the second step a biotinylated antibody binds to

the solid phase antibody-antigen complex following streptavidin-horse radish

peroxidase (HRP) conjugate binding. After incubation and washing the wells,

the antigen complex bound to the well was detected by addition of a

chromogenic TMB (tetramethylbenzidine) substrate. In the presence of HRP,

the TMB and peroxide contained in the substrate solution react to produce a

blue byproduct. The colour intensity is proportional to the amount of HRP

activity, which in turn is related to the levels of AMH. Addition of 2 N sulfuric

acid stop solution changes the color to yellow, enabling accurate measurement

of the intensity at 450 nm using a plate reader.

47


Serial standard solution was prepared prior to the assay according to the

manufacturer’s instruction. The concentration of standards was 0, 3, 9, 27, 81

and 150 pM and provided by the manufacturer in the kit.

25 µL of serum sample and standard solutions were pipetted in duplicate into

anti-AMH coated plate following addition 100 µL of buffer solution to each well.

The wells were incubated for 2 hours at room temperature on plate shaker and

then washed with freshly prepared wash buffer three times. After adding 50 µL

of biotinylated antibody and 50 µL of conjugate, the wells were incubated for

30 minutes at room temperature with shaking. After the conjugate binding

step, the wells were washed three times with wash solution and the remain

wash solution was completely removed by firmly tapping the inverted

microplate on to the absorbent paper. After pipetting 100 µL of TMB substrate,

wells were incubated for 30 minutes at room temperature away from light on

the plate shaker. The colour reaction was stopped by adding 50 µL stop

solution to each well and the intensity of colour was measured using microplate

reader at 450 nm. The concentration of AMH was calculated using standard

curve and expressed as pmol/L.

2.13 Serum Homocysteine assay Homocysteine (Hcy) is a thiol-containing amino acid produced by the

intracellular demethylation of methionine (Finkelstein, 1990).

Homocysteine level in serum was measured by using Bio-Rad Microplate

Enzyme Immunoassay Homocysteine Kit (Bio-Rad Laboratories, Inc., Hercules,

CA, USA) which is based on competition between S-adenosyl-L-homocysteine

(SAH) in the sample and immobilized SAH bound to the walls of the microtitre

plate for binding sites on a monoclonal anti-SAH antibody.

All samples including calibrators and controls for the assay were pretreated

with pretreatment solution which contains Dithiothretiol (DTT) to reduce the

mixed sulfide and protein-bound forms of homocysteine to free homocysteine.

Homocysteine in the sample was also converted to S-adenosyl-L-homocysteine

(SAH) by the use of SAH hydrolase in pretreatment solution.


25 µL of sample and standard were pre-treated with 500 µL pretreatment

solution and incubated for 30 minutes at 370C in a tightly capped tube. 500 µL

enzyme inhibitor (<0.2% Thiomersal in phosphate buffer) and 500 µL

48

Adenosine Deaminase was added to tubes and incubated at room temperature

for 15 minutes respectively. 25 µL diluted standard and sample from the tubes

was pipetted into the SAH-coated wells accompanying with 200 µL a-SAH

antibody (Monoclonal mouse-anti-S-adenosyl-L-homocysteine antibody). After

the incubation for 30 minutes at room temperature, wells were washed with

wash buffer 4 times. After washing the wells was emptied on paper towels and

100 µL enzyme conjugate (Rabbit anti-mouse-antibody with horseradish

peroxidase-HRP) was pipetted. Then 100µL 10% N-methyl-2-pyrolidone was

added as substrate and incubated at for 10 min at room temperature following

the addition of 100 µL 0.8 M sulphuric acid as stop solution.

The colour reaction was occurred depending on horseradish peroxidase activity

after addition of substrate solution and was inversely related to homocysteine

concentration in the sample. Measurement was done spectrophotometrically in

microplate reader (Bio-Tek Instruments, Inc. Winooski, VT, USA, Model

ELx800) at the wavelength of 450 nm. The result for the homocysteine

concentration in the sample was derived according the standard curve and

expressed as µmol/L. The reference range for homocysteine was accepted as

<11.4 mmol/L at 95th percentile in folate replete men aged 20-45 years

(Selhub et al., 1999).

49

CHAPTER 3

Development of the Nitro Blue Tetrazolium (NBT)

Assay as a marker of sperm Oxidative Stress

50

3.1 Abstract

Oxidative stress is a well established cause of male infertility, with Reactive

Oxygen Species (ROS) causing infertility principally by impairing sperm motility

and DNA integrity. Currently most clinics do not test their infertile patients for

the presence of oxidative stress because the available tests are expensive or

difficult to conduct. Since antioxidant therapy may improve sperm DNA

integrity and pregnancy outcomes, it has become apparent that there is an

unmet clinical need for an inexpensive and easy to conduct assay to identify

sperm oxidative stress. The aim of this study was to develop a standardized

protocol for the conduct of a photometric Nitro Blue Tetrazolium (NBT) assay

for the measurement of seminal ROS production via production of coloured

formazan, while correlating these results with impaired sperm function (motility

and DNA integrity). Semen samples from 21 fertile and 36 male aetiology

infertile men were assessed for ROS production (NBT assay), sperm DNA

integrity (TUNEL), apoptosis (Annexin V), and sperm motility. Infertile men’s

semen contained on average four-fold higher levels of ROS than fertile men.

The production of ROS by sperm was positively correlated with sperm DNA

fragmentation and apoptosis, while being negatively correlated with sperm

motility.

Receiver-operating characteristic plot analysis established a cut-off point of 24

µg formazan / 107 sperm as having a sensitivity of 91.7% and a specificity of

81% for determining the fertility status of an individual. This study has been

successful in establishing a standardized protocol for the conduct of a

photometric seminal NBT assay that has significant clinical utility in identifying

men with impaired fertility due to oxidative stress.

51

3.2 Introduction

While oxidative stress is a common and potentially treatable pathology, the

vast majority of clinical andrology laboratories do not test for its presence.

Currently there are over 30 different assays to quantify sperm oxidative stress

(Tremellen, 2008). Chemo-luminescent assays using either Luminol or

Lucigenin are the most commonly described technique for detection of ROS

within semen. While these assays are sensitive, have relatively well established

normal ranges (Athayde et al., 2007; Williams and Ford, 2005) and are the

only assay of oxidative stress described in the WHO semen analysis manual

(WHO, 2001), they are rarely used by clinical andrology laboratories. This is

probably because of significant set up costs (purchase of a luminometer) and

difficulties with quality control created by assay confounders such as incubation

time, leukocyte contamination and the presence of seminal plasma

contamination (Kobayashi et al., 2001). Simpler, less expensive assays with

well defined normal ranges based on sperm function must be established

before clinical andrology laboratories will begin to test for the presence of

oxidative stress.

The Nitro Blue Tetrazolium (NBT) assay is a well established laboratory

technique used to quantify neutrophil function and cellular oxidative

metabolism (Baehner et al., 1976). The principal behind the NBT assay is

relatively simple. Cells incubated in a water soluble tetrazolium salt (NBT)

solution will take up NBT into their cytoplasm where it is converted by the

action of superoxide anions to a water insoluble blue formazan crystal (Baehner

et al., 1976). These crystals are trapped within the cell but can be released by

solubilisation in a solvent solution and then quantified by measuring

absorbance of the resulting purple-blue solution (Choi et al., 2006; Rook et al.,

1985). Previous NBT assay protocols had used wavelengths ranging from 530

nm (Mercado-Pichardo et al., 1981) to 630 nm (Choi et al., 2006; Rook et al.,

1985) for determination of formazan production. The NBT test has been shown

to be useful in quantifying both leukocyte (Choi et al., 2006; Rook et al., 1985)

as well as sperm ROS production (Esfandiari et al., 2003; Mercado-Pichardo et

al., 1981).

Furthermore, the measurement of formazan by histochemical staining within

sperm and seminal leukocytes has been reported to be closely related to ROS

52

quantification by the well established chemo-luminescent ROS assay

(Esfandiari et al., 2003).

While the NBT is relatively easy and inexpensive to conduct, its use within

clinical andrology laboratories is hampered by a lack of published normal

ranges. The aim of the present study was to develop standardized protocols for

the conduct of the semen NBT assay and identify the associated normal

ranges. An integral part of this study will be to analyse the correlation between

NBT quantified ROS production and features of sperm oxidative attack (reduced

motility, increase in sperm apoptosis and DNA fragmentation).

3.3 Materials and Methods

3.3.1Subjects and Study Design

Participants were recruited from men undergoing infertility assessment or

fertile sperm donors at an academic affiliated ART unit (Repromed, Dulwich,

South Australia). The only entry criteria for participants was the requirement

to have a minimum sperm concentration of 2 x 106/mL, as this was the

minimum amount of sperm needed to reliably conduct the various sperm

assays. Men with proven past paternity or men in an infertile relationship but

who had normal semen parameters (count, motility morphology) and their

partner had a well defined female cause for their infertility (anovulation,

endometriosis, tubal obstruction, diminished ovarian reserve) were considered

“fertile”. Those men in an infertile relationship who had at least one defect

either in count, motility or morphology were considered to be have male factor

infertility (‘infertile group”). A total of 21 men were classified as fertile. They

consisted of 12 sperm donors of proven fertility, 2 men who initiated infertility

investigation but whose partner then conceived without medical assistance,

and 7 men in an infertile relationship of purely female aetiology. The remaining

36 participants had at least one defect in routine sperm parameters and were

considered as having male infertility.

The study was prospectively approved by the Human Research and Ethics

Committee, Women’s and Children’s Hospital, with all participants giving

written informed consent for their involvement.

53

3.3.2 Sample Preparation

Sample preparation and the assays used in this study was discussed in Chapter

2.4

3.3.3 Statistical Analysis

Data were analysed using the statistical software Sigmastat (Systat Software

Inc, California, USA.) and presented as median values (inter-quartile ranges)

as sperm parameters were not normally distributed. The normality of the

distribution was assessed automatically by the statistical software itself.

Correlation analysis was conducted using the Spearman Rank Order correlation

test and between group comparisons were analysed using the Mann-Whitney

Rank Sum test. A P value < 0.05 was considered statistically significant.

Receiver operating characteristic (ROC) curve analysis was used to develop an

optimal “cut off” point for formazan production that would best classify

individuals as being fertile or infertile. ROC analysis and the associated

sensitivity, specificity, positive and negative predictive value calculations were

all performed using the SAS Version 9.1 statistical package (SAS Institute Inc.,

Cary, NC, USA).

3.4 Results

3.4.1 Standardization of assay conditions

In order to determine the ideal wavelength for quantifying sperm ROS

production, the absorbance of a formazan solution was measured between the

range of 240 and 1100 nm using a continuous spectrophophotometer (Model

UV-160 , Shimadzu Corporation, Kyoto, Japan). As peak absorbance occurred

between 620 and 740 nm, it was decided to make all future assay

measurements at 630 nm since this is a common wavelength filter available for

most ELISA plate readers. Furthermore, measurement of formazan absorbance

on an ELISA plate reader, rather than using a spectrometer, was favoured as

most laboratories have access to an ELISA plate reader and this technique

allowed for duplicate measurements of multiple samples and standards in a

simultaneous fashion.

54

A previous publication describing the use of the NBT assay to quantify sperm

metabolic activity had determined that NBT reduction by sperm was optimally

observed at a pH of 7.4, temperature of 37 degrees Celsius, with the reaction

being complete within 30-40 minutes (Mercado-Pichardo et al., 1981). In our

lab I measured formazan production at various concentrations of sperm

following incubation for between 5 and 120 minutes and found that formazan

production was effectively complete by 45 minutes, with very minimal

additional formazan being produced between 45 and 120 minutes (Figure 3.1).

As the aim of this study was to develop a rapid, easy to conduct assay suitable

for clinical use it was terminated all further NBT assays would be read at 45

minutes.

0.01

0.03

0.05

0.07

0.09

0.11

0.13

0.15

0 15 30 60 120

Minutes

Fo

rmazan

Ab

so

rban

ce 6

30n

m

2 x 105 sperm/mL

5 x 105 sperm/mL.

1 x 106 sperm/mL.

Figure 3.1: Formazan production at various concentrations of sperm at

different incubation period.

Initial experiments in which the NBT solution was added to neat semen proved

unsuccessful as the working solution immediately turned blue, even when using

fertile men’s semen containing presumably low levels of ROS. Follow up

experiments in which serial dilutions of seminal plasma were added to NBT

solution revealed that the seminal plasma itself had the capacity to reduce NBT

to formazan. Therefore, in order to properly quantify cellular ROS production it

55

was necessary to remove seminal plasma from the sperm/ seminal leukocyte

sample by a double wash in PBS before adding the NBT working solution.

The chemo-luminescent assays most widely used to quantify sperm ROS

production are typically analysed at a standard concentration of 20 x 106 sperm

per ml, with ROS production being expressed as 104 counted photons per

minute per 20 x 106 sperm (Athayde et al., 2007; Williams and Ford, 2005).

The use of such a high starting concentration of sperm makes determination of

ROS difficult in oligospermic men as they may have insufficient sperm to meet

the 20 x 106 concentration criteria, especially if samples are assayed in

duplicate. Furthermore, dilution of clinical samples to a standard concentration

of 20 x 106 sperm may lead to dilution errors. Therefore it was decided to

measure sperm ROS production at the sperm concentration that existed in neat

semen, but then mathematically standardize the results as µg formazan

produced per 107 sperm. Measurement of formazan production by individual

men’s sperm diluted over a range of concentrations from 2 to 500 million

sperm showed a good linear response (Figure 3.2A), supporting the utility of

such an approach.

Figure 3.2A: ROS production for a single infertile individual upon serial

dilution of a sperm suspension in the range of 2 to 500x106 sperm/mL.

ROS production is represented as absorbance values (630 nm) on the

right y axis and calculated formazan production (µµµµg) on the left y axis.

0 50 100 150 200 250 300 350 400 450 5000

200

400

600

800

1000

1200

0.000

0.200

0.400

0.600

0.800

1.000

Sperm Concentration (x106/mL)

Fo

rmazan

µµ µµg

/10

7 s

perm

Fo

rmazan

Ab

so

rban

ce

(630 n

m)

56

The chemical reaction of xanthine oxidase with xanthine solution to generate

variable amounts of superoxide anions in solution (Fridovich, 1970) was

performed to confirm the NBT assays ability to accurately quantify changes in

ROS production. As can be seen from Figure 3.2B, an increasing concentration

of Xanthine substrate reacting with a fixed concentration of Xanthine Oxidase

produced a linear increase in formazan production. The calculated intra and

inter-assay coefficients of variation for the NBT assay using Xanthine/ Xanthine

Oxidase as a ROS generator were 9.3 and 7.4 % respectively.

Figure 3.2B: Formazan production using the chemical Xanthine /

Xanthine Oxidase (X/XO) ROS production system. A fixed

concentration of Xanthine Oxidase (2µµµµL 25 U/L) was added to an increasing concentration of Xanthine solution to stimulate increasing

levels of ROS production. Mean values ± SD plotted in each graph.

0.00 0.05 0.10 0.15 0.20 0.250.000

0.200

0.400

0.600

0.800

Xanthine (mmol/L)

Fo

rmazan

Ab

so

rban

ce

(63

0 n

m)

57

Table 3.1: Summary statistics of routine semen parameters, reactive oxygen species (ROS) production and sperm DNA quality in fertile and

infertile men. Values are represented as mean ± SD and median (inter-

quartile range). Mann-Whitney Rank Sum test was used for statistical

analysis.

Fertile Group (n = 21) Median (25%-75% range)

Infertile Group (n = 36) Median (25%-75% Range)

P

Age (years) 38 (33.5 – 41.5) 38 (35 – 42) 0.904

ROS Production

(µg Formazan/107 sperm)

16 (5.5 – 22.5) 65.5 (28.0 – 99.8) <0.0001

TUNEL

positivity (%) 9.6 (8.2 – 12.6) 20.7 (14.4 – 25.9) <0.0001

Early apoptosis

(%) 12.3 (7.0 – 15.5) 22.3 (16.9 – 34.4) <0.0001

Motility (%) 52 (46 – 56) 38 (30 – 45.8) <0.0001

Morphology (%) 19.0 (15.0 – 22.0) 7.0 (2.0 – 10.8) <0.0001

Sperm

Concentration (x10 6cell/mL

107 (58.3–205.3) 26.6 (10.5 – 47.9) <0.0001

PMN Elastase (ng/mL)

86.8 (36.5–169.0) 45.5 (15.5– 202.3) 0.529

58

3.4.2 Relationship between ROS production and measures of sperm

health

The mean production of formazan by infertile men was considerably higher

than that observed for their fertile counterparts (72.6 ± 58.5 µg v 17.8 ± 12.6

µg per 107 sperm, p < 0.0001, Table 3.1), confirming a well established

observation that oxidative stress is more prominent in the infertile population.

Figure 3.3 illustrate the correlation between levels of sperm oxidative stress

(formazan production) and impairment of sperm function. A negative

correlation exists between formazan production and sperm motility (r= -0.479,

p= 0.002), suggesting that production of ROS by sperm and seminal

leukocytes does impair sperm motility (Figure 3.3A). Furthermore, a very

strong correlation was seen between initiation of apoptosis (Annexin V

staining) and increasing levels of seminal ROS production (r= 0.669,

p<0.0001, Figure 3.3B). Linked with this increase in sperm apoptosis is the

observation of a significant increase in sperm DNA fragmentation with

increasing levels of production of ROS (r = 0.591, p<0.0001, Figure 3.3C).

Seminal plasma elastase is secreted by activated neutrophils and is an

acknowledged marker of both leukocyte number and activity within semen

(Zorn et al., 2000). As neutrophils are potent producers of ROS, it was

anticipated that seminal elastase concentration would be positively correlated

with formazan production. Surprisingly, no significant correlation was observed

between seminal plasma elastase and formazan production (Figure 3.4,

r =0.006, p= 0.965). However, only 4 out of 36 participants (11%) exhibited

signs of elevated leukocyte activity (PMN elastase > 290 ng/mL), which may

have limited this studies ability to correlate seminal leukocyte activity with

formazan production.

Furthermore, while infertile men’s sperm produce less than half the amount of

formazan than peripheral leukocytes on a per cell basis (Figure 3.5), the

production of ROS by sperm appears to be the dominant determinant of net

seminal ROS production because of their vast numerical superiority over

leukocytes.

59

Figure 3.3A

Figure 3.3B

Figure 3.3C

Figure 3.3: Linear regression analysis depicting the relationship

between ROS production (µµµµg formazan per 107sperm) and sperm motility (A), early apoptosis (Annexin V staining) (B) and sperm DNA

fragmentation (TUNEL Assay) (C) for the combined fertile and infertile

groups. Spearman regression correlations and p values are recorded

for each observation.

0 100 200 3000

20

40

60

80

r = - 0.479P =0.0002

Formazan µµµµg/107 sperm

Motilit

y %

0 100 200 3000

20

40

60 r = 0.669P <0.0001

Formazan µµµµg/107sperm

Annexin

V %

0 100 200 3000

10

20

30

40

50r = 0.591P <0.0001


TUNEL p

ositiv

ity %

60

Figure 3.4: Correlation between ROS production and seminal PMN

Elastase concentrations in the combined fertile and infertile groups. No

significant relationship was observed on spearman correlation analysis

(p= 0.719).

Figure 3.5: Depiction of the relative ability of a pure “swim up” infertile

sperm preparation and peripheral blood leukocytes to reduce NBT to

formazan. This graph depicts two separate experiments at different

concentrations of cells (1x106cells and 4x106 cells/mL), with all

experiments being conducted in triplicate.

1x106

cell/mL 4x106

cell/mL

0

50

100

150

200

Sperm

LeukocyteFo

rma

zan

µµ µµg

0 100 200 3000

200

400

600

800

r = 0.006P = 0.965


PM

N E

las

tas

e(n

g/m

L.)

61

3.5 Development of normal ranges

The ROC curve illustrated in Figure 3.6 depicts changes in the NBT assay

sensitivity and specificity over the entire threshold range. From this graph two

key determinants of the NBT assay’s clinical utility can be determined. Firstly,

the optimal cut off point to maximize sensitivity and specificity is 24 µg

formazan / 107 sperm, resulting in a sensitivity of 91.7% and a specificity of

81.0%. The positive and negative predictive values for this cut off point are

89.2% and 85% respectively. The calculated area under the curve (AUC) is

0.88, suggesting that the NBT assay has an excellent ability to distinguish

between fertile and infertile individuals.

Figure 3.6: ROC (receiver operating characteristic) curve for the

assessment of ROS production in semen using NBT assay. The area

under the curve (AUC) for this assay was 0.88, indicating that the

assay was very good at distinguishing between fertile/infertile

individuals

62

3.6 Discussion

The results presented in this study confirm spermatozoa’s ability to reduce NBT

to formazan, as has been suggested by most (Esfandiari et al., 2003; Mercado-

Pichardo et al., 1981), but not all previous investigators (Armstrong et al.,

2002). The sperm cytoplasm contains glucose-6-phosphate dehydrogenase

which uses glucose to produce β-nicotinamide adenine dinucleotide phosphate

(NADPH) via the hexose monophosphate shunt. NADPH is then used to fuel the

generation of superoxide anions via NADPH oxidase contained within sperm,

which in turn converts NBT into formazan. Seminal leukocytes are also known

to contain NADPH oxidase and therefore are also responsible for some of the

formazan production within each semen sample. However, a “swim up”

generated pure sperm fraction was capable of reducing NBT to formazan,

irrefutably confirming the capacity of sperm themselves to generate formazan

even in the absence of contaminating leukocytes. This observation contradicts

Armstrong’s earlier finding that sperm could not reduce NBT to formazan, even

in the presence of PMA stimulation (Armstrong et al., 2002). It is unclear why

this discrepancy exists, yet these results are consistent with the observations

of two other groups who have confirmed sperm’s ability to reduce NBT to

formazan (Esfandiari et al., 2003; Mercado-Pichardo et al., 1981).

To the best of my knowledge, this study is the first to correlate seminal ROS

production using the NBT assay with markers of sperm quality such as sperm

DNA fragmentation and motility. The production of formazan was significantly

correlated with a decline in sperm motility. This result is consistent with

previous studies that had shown seminal ROS production, quantified by chemo-

luminescent techniques, to be negatively correlated with sperm motility

(Aitken, 1989; Whittington and Ford, 1998). It is likely that increasing

production of ROS inhibit sperm motility by two different mechanisms. Firstly,

ROS induce peroxidation of the sperm membrane, decreasing its flexibility and

tail motion. Sperm membranes are vulnerable to this type of damage as they

contain large amounts of unsaturated fatty acids. Secondly, ROS can directly

damage sperm mitochondria, thereby decreasing energy availability and

impeding motility.

63

A significant positive correlation between formazan production and an increase

in sperm DNA fragmentation was noted, consistent with the well established

capacity of ROS to damage sperm DNA (Aitken et al., 1998; Meseguer et al.,

2008; Ozmen et al., 2007; Zorn et al., 2003b). Free radicals are able to induce

sperm DNA fragmentation by two mechanisms. Firstly, ROS can directly attack

the purine and pyrimidine bases and deoxyribose backbone of sperm DNA.

Secondly, free radical damage will initiate apoptotic endonuclease

fragmentation of the sperm DNA (Moustafa et al., 2004). It has been reported

that infertile men producing high levels of ROS exhibit significantly greater

levels of sperm early apoptosis (Annexin V+, PI -) compared to infertile men

with low levels of ROS production (Moustafa et al., 2004). As formazan

production was strongly correlated with early apoptosis in this study, it is likely

that the observed increase in sperm DNA fragmentation seen with increasing

oxidative stress is the net result of both direct ROS attack and endonuclease

mediated apoptotic digestion of DNA.

Seminal plasma elastase was measured as it is a well recognised marker of

leukocyte number and activity within semen. Zorn et al have reported that a

seminal plasma elastase level > 290 ng/mL has an 80% sensitivity for

identifying leukocytospermia (Zorn et al., 2000). Since leukocytes are potent

producers of ROS, and several researchers have correlated leukocyte number

with seminal ROS production, it was anticipated that a significant positive

correlation would exist between seminal plasma elastase concentrations and

formazan production. Surprisingly, no such correlation was seen. The most

likely reason for this was that only 11 % of the infertile participants had

evidence for leukocytospermia (elastase > 290 ng/mL), with the vast majority

of subjects being non-leukocytospermic, thereby weakening the study’s ability

to correlate leukocyte activity with ROS output. Interestingly, Zorn et al. also

found no significant correlation between elastase concentration and ROS

production using chemo-luminescent techniques, with 25% of his study cohort

having an elastase concentration exceeding 290 ng/mL (Zorn et al., 2000).

Finally, seminal plasma elastase did not correlate with sperm DNA quality or

motility in the study cohort (data not shown). My interpretation of these

findings is that at least in non-leukocytospermic patients, sperm cells are by far

the dominant producer of ROS and the key determinant of sperm DNA

integrity, not seminal leukocytes. This finding is consistent with the

observations of Henkel et al. (Henkel et al., 2005) who reported a much

stronger correlation existed between sperm DNA integrity and intrinsic (sperm

64

derived) ROS production compared to extrinsic (leukocyte derived) ROS

production.

The use of Receiver-operating characteristic (ROC) plots provide a pure index

of accuracy by demonstrating a test’s ability to discriminate between

alternative states of health (fertile v infertile) over the complete spectrum of

operating conditions (Zweig and Campbell, 1993). This ROC analysis of the

NBT assay has shown the assay to have a very good ability to determine

fertility status for an individual. The sensitivity (91.7%) and specificity (81.0%)

of the NBT assay, together with the recorded ROC AUC of 0.88, are all within

the range associated as a highly useful clinical diagnostic test performance

(Zweig and Campbell, 1993). However, before definitive comment can be

passed on the clinical usefulness of the NBT assay larger scale clinical trials will

need to be conducted.

Levels of sperm DNA damage exceeding 15% have now been linked with a

reduction in embryo quality, an increase in miscarriage and a reduction in live

births on the IVF program (Benchaib et al., 2007; Chohan et al., 2006). A large

majority (84.3 %) of infertile men had levels of sperm DNA damage exceeding

15% when their NBT result exceeded the optimal cut off point of 24 µg / 107

sperm. Conversely no infertile man with an NBT result below 24 µg /107 sperm

had sperm DNA fragmentation levels exceeding the clinically relevant 15%

TUNEL threshold. Therefore it appears that the chosen a cut off point of 24 µg /

107 sperm is clinically appropriate.

Recently it has been reported that antioxidant therapies have the ability to

improve infertile men’s sperm DNA integrity (Comhaire et al., 2000; Greco et

al., 2005a; Menezo et al., 2007), while also improving pregnancy outcomes

during both IVF and natural conception (Greco et al., 2005b; Suleiman et al.,

1996; Tremellen et al., 2007). Despite this, the majority of clinicians still do

not routinely prescribe antioxidants to their infertile patients. The current lack

of availability of an inexpensive, easy to conduct assay for sperm oxidative

stress is impeding optimal clinical care as many physicians are reluctant to

offer empirical antioxidant treatments without laboratory evidence for the

existence of oxidative pathology. I believe that this study provides support for

the NBT assay to be further developed as an easy to perform assessment of

sperm oxidative stress. I acknowledge that this study is relatively small and

needs to be replicated by others, preferably in large scale multi-centred trials.

65

However, the NBT assay holds considerable promise because of its accuracy in

identifying oxidatively damaged sperm and its relatively inexpensive and ease

of conduct.

66

CHAPTER 4

Seminal Inflammation as a Cause of Oxidative

Stress and Macrophage Activity in Infertile Men’s semen

67

4.1 Abstract

The presence of leukocytes within semen has the potential to impair

sperm function. Neutrophils and macrophages make up 95% of

seminal leukocytes, with both having the ability to damage sperm

via the generation of reactive oxygen species (ROS), proteases and

the induction of apoptosis. Existing cytological techniques for

quantifying leukocyte activity within semen (peroxidase, CD45) are

less than ideal as they merely count the number of leukocytes,

rather than assess their activity.

Seminal plasma elastase effectively determines neutrophil activity,

yet gives no insight into macrophage activity. Neopterin, a molecule

released from activated macrophages, may be a useful marker for

macrophage activity in the male reproductive tract. In order to

examine this possibility a total of 63 asymptomatic subjects with

male factor infertility and 11 fertile controls provided semen samples

for measurement of various inflammatory markers. This study was

able to confirm for the first time that seminal plasma does indeed

contain neopterin and that the levels of this macrophage activity

marker are 3-fold higher in infertile than fertile men.

Furthermore, seminal plasma neopterin concentration was

significantly correlated with sperm oxidative stress, DNA

fragmentation (TUNEL) and apoptosis (Annexin V), making it a

useful marker of sperm health. In contrast, seminal plasma elastase

showed no correlation with any marker of sperm health.

68

4.2 Introduction

Currently there is considerable controversy regarding the role that seminal

leukocytes play in male infertility. Leukocytes generally represent a very small

proportion of cells within semen (Eggert-Kruse et al., 1992), yet they have the

potential to very significantly alter the function of adjacent spermatozoa

(Aitken and Baker, 1995; Aitken et al., 1995a; Fraczek et al., 2008; Henkel et

al., 2005; Plante et al., 1994). Polymorphonuclear (PMN) granulocytes account

for 50% to 60% of seminal leukocytes, macrophages 20% to 30% and T

lymphocytes for the remaining 5% (Smith et al., 1989; Wang and Holstein,

1983; Wolff, 1995). Histological analysis of the male reproductive tract

suggests that macrophages primarily originate from the testicular interstitium

and epididymis, as they are generally absent from the ejaculate of

vasectomised men (Pelliccione et al., 2008; Wang and Holstein, 1983).

Conversely, PMN granulocytes (principally neutrophils) are present in the

ejaculate of vasectomised men, suggesting a seminal vesicle or prostatic origin

for these cells (Anderson, 1990) Both neutrophils and macrophages have the

potential to produce significant damage to sperm through their generation of

reactive oxygen and nitrogen species, the release of hydrolytic enzymes and

cytotoxic peptides (defensins) and via cytokine triggered sperm apoptosis.

It is interesting to note that the majority of semen samples, even those from

fertile men with no evidence of genitourinary tract inflammation or infection, do

contain leukocytes. There is general agreement that leukocyte counts between

1-5 x 104/mL are normally present within fertile men’s semen and have no

negative effect on sperm function (Aitken et al., 1995a; Tomlinson et al.,

1993; Wolff and Anderson, 1988). It has even been postulated that low

numbers of leukocytes may actually play a beneficial role by removing

abnormal and degenerative spermatozoa, while generating hydrogen peroxide

that may assist the process of sperm capacitation (Bize et al., 1991; Tomlinson

et al., 1993). In support of such a positive role is the recent observation that

the presence of a moderate number of leukocytes within semen (0.5 -1.0 x

106/mL) is associated with improved sperm motility and epididymal function

compared with a seminal leukocyte concentration of less 0.5 x 106 (Ziyyat et

al., 2008).

While low numbers of seminal leukocytes may play a positive role, the

traditional view has been that once seminal leukocyte concentration rises

above a threshold of 1 x 106 /mL (leukocytospermia), they have significant

69

potential to damage sperm and cause infertility. This negative impact of

leukocytospermia on sperm function was suggested by studies linking the

presence of greater than 1 x 106 leukocytes per ml of semen with poor sperm

function (Berger et al., 1982; Wolff et al., 1990) and is the basis of the WHO

definition of pathological seminal leukocyte content (WHO, 1999b).

Furthermore, several studies have reported that leukocyte counts are

significantly higher in infertile men’s semen than fertile controls (Ochsendorf,

1999; Plante et al., 1994; Sharma et al., 2001; Wolff, 1995) , with up to 20%

of infertile men exhibiting WHO defined leukocytospermia (Henkel et al., 2003)

Interestingly not all investigators have identified a link between semen

leukocyte concentration and male fertility potential. For example, Aitken et al

(1995) found no link between seminal leukocyte count and sperm function

while Tomlinson et al (1993) reported no link between seminal leukocyte count

and male fertility (Tomlinson et al., 1993). Aitken and Baker (1995) suggest

that the role seminal leukocytes play in male fertility runs deeper than mere

concentration due to two significant variables. Firstly, active leukocytes are

more likely to create damage to sperm than dormant leukocytes. Secondly, the

site of contact between seminal leukocytes and sperm may play a critical role

in determining sperm function. For example, epididymal macrophages are in

close proximity to sperm for prolonged periods of time during epididymal

transit and storage, placing them in a favourable position to damage sperm

(Barratt et al., 1990; Wilton et al., 1988). Conversely, neutrophils from the

seminal vesicles and prostate only get an opportunity to damage sperm post-

ejaculation when these accessory sex gland secretions come in direct contact

with sperm. Assessment of leukocyte activity in the male reproductive tract,

rather than mere determination of leukocyte concentration using traditional

peroxidase or immunocytochemical means (Villegas et al., 2002; WHO, 1999b;

Wolff, 1998), is therefore more likely to accurately reflect the immune systems

influence on sperm function.

Currently the most widely used marker of male genital tract inflammation is

seminal plasma elastase. When neutrophils become activated by inflammatory

changes they undergo a “respiratory burst” with the release of reactive oxygen

species (ROS) and proteases such as elastase. Seminal plasma elastase levels

therefore directly reflect neutrophil inflammatory activity. The enzyme elastase

has the potential to create proteolytic cell damage and DNA fragmentation in

sperm and is easily quantifiable in seminal plasma using a widely available

70

commercial ELISA (Zorn et al., 2003a). The seminal plasma of infertile men

contains higher levels of elastase than that observed in fertile men (Jochum et

al., 1986; Reinhardt et al., 1997; Wolff and Anderson, 1988; Zopfgen et al.,

2000; Zorn et al., 2000), with increasing seminal elastase levels being

correlated with a reduction in sperm motility (Wolff et al., 1991; Wolff et al.,

1990) and DNA integrity (Zorn et al., 2000).

No study to date has investigated the link between macrophage activity and

sperm quality. This is a serious omission given that macrophages are the

second most prolific leukocyte within semen, making up almost one third of all

seminal leukocytes. Surprisingly, the WHO manuals own standard method for

determination of leukocytospermia, the peroxidase staining technique, fails to

identify macrophages and as a result significantly underestimates true

leukocyte numbers within semen (Villegas et al., 2002; WHO, 1999b). While

immunocytochemical methods can reliably quantify macrophage numbers, they

do not directly measure macrophage inflammatory activity. As several groups

have published evidence that macrophages play an important role in male

reproductive dysfunction (Haidl et al., 2008; Jones, 2004; Pelliccione et al.,

2008; Wilton et al., 1988), there is clearly an unmet need for an easily

performed non-invasive test that can quantify macrophage inflammatory

activity within the male genital tract.

Neopterin is an established marker of macrophage activity that has been

studied for over 30 years. Neopterin is a low molecular mass pteridine

molecule released from macrophages / monocytes primarily upon stimulation

by the cytokine interferon gamma, and to a lesser extent other pro-

inflammatory cytokines (Hamerlinck, 1999). There is a close relationship

between the amount of neopterin released from macrophages and their

capacity to produce reactive oxygen species (Berdowska and Zwirska-Korczala,

2001; Weiss et al., 1998). While neopterin has been reported to be present in

various biological fluids such as serum, cerebrospinal fluid, synovial fluid, urine,

saliva, ascitic fluid and pancreatic juices (Berdowska and Zwirska-Korczala,

2001; Hamerlinck, 1999), no study to date has reported the presence of

neopterin in seminal plasma. Therefore, the aim of this study was to determine

if neopterin is present in seminal plasma and if present, correlate neopterin

levels with various markers of sperm health.

71


4.3.1 Subjects and Study Design

Participants in the study were recruited from men with known male factor

infertility (“infertile subjects”) defined as the presence of abnormal WHO

semen quality criteria (WHO, 1999b) and the inability to conceive despite more

than 12 months of unprotected intercourse. “Fertile controls” were men who

were acting as sperm donors at an academic affiliated ART unit (Repromed,

South Australia) and who had past proven fertility with normal semen

parameters according to WHO criteria. None of the participants had any history

suggestive of past or present infection of the male genital tract. All fertile

sperm donors were assessed for active infection by semen culture and PCR

analysis (Chlamydia, gonorrhoea), as is legally required. However, infertile

men were not formally screened for infection our experience and that of others

(Barratt et al., 1990) have found a very low positive yield for such tests in the

asymptomatic population. At the time of producing the study semen sample no

participant was taking any antioxidant supplements or medications that could

affect their immune response.

The study was prospectively approved by the Human Research and Ethics

Committee, Women’s and Children’s Hospital, with all participants giving

written informed consent for their involvement.

4.3.2 Sample Collection and Preparation

Sample preparation and assays used in this study was discussed in Chapter 2.4

4.4 Results

As expected, standard semen parameters (concentration, motility and

morphology) were significantly inferior in the infertile than the fertile cohort

(Table 4.1). Furthermore, production of ROS (formazan) and the resulting

oxidative damage to the sperm membrane (LPO-586) was significantly higher

in the infertile than the fertile cohort. Sperm DNA damage (TUNEL) and

apoptosis (Annexin V) were also significantly higher in infertile men’s sperm.

72

Table 4.1: Sperm Quality Parameters in infertile and fertile groups.

All values are expressed as Median (inter-quartile range). Statistical analysis

was performed using Mann-Whitney Rank Sum Test.

An excellent correlation was observed between the levels of production of ROS

and resulting sperm membrane lipid peroxidation (r=0.812, p <0.001, Table

4.2), supporting the use of these assays as measures of sperm oxidative

stress. Seminal ROS production was also strongly correlated with sperm DNA

damage (TUNEL, r= 0.631, p <0.001) and apoptosis (Annexin V, r= 0.688, p <

0.001); while sperm DNA damage and apoptosis were also significantly

positively correlated (r = 0.806, p<0.001).

Infertile Group (n=63)

Fertile Group (n=11)

P

Sperm Concentration (x106 cell/ml)

25.8 (11.65–52.23) 97.5 (53 – 201) 0.0001

Motility (%)

42 (30 – 48) 55 (49 – 57) 0.0001

Morphology (%)

6 ( 2– 11) 21 (15 – 25) 0.0002

Semen Volume (mL)

2.8 (1.8 – 4.0) 3.3 (2.7 – 4.5) 0.343

ROS Production (µg Formazan/107 sperm)

64.0 (31.8 – 88.0) 11.9(4.7–20.0) <0.0001

Lipid Peroxidation (µM MDA x107/sperm)

1.3 ( 0.4– 3.5) 0.05(0.01–0.14) <0.0001

TUNEL (%)

20.2 (13.9 – 25.8) 9.6 (8.1– 13.6) 0.0001

Apoptosis (Annexin V) (%)

23.5 (18.2 – 33.9) 8.6 (5.4– 13.9) <0.0001

CD 45 (x106 cell/ml) 1.0 (0.6 – 1.9) 0.9 (0.6 – 1.2) 0.404

PMN Elastase (ng/mL)

123 (22.8–275.3) 49 (10.5–118.5) 0.042

Neopterin (ng/mL) 96.9 (53 – 186) 42 (28 - 99)

0.013

73

Seminal leukocyte concentration was not significantly correlated with ROS

production, sperm lipid peroxidation, motility, apoptosis or DNA fragmentation

(Table 4.2). Seminal plasma elastase and neopterin were present at

significantly higher concentrations in infertile than fertile men (Table 4.1). A

highly significant correlation was seen between total leukocyte concentration

(CD45) and neutrophil activity (elastase, r= 0.472, p< 0.0004), yet no

significant correlation was observed between leukocyte concentration and

macrophage activity (neopterin, r= -0.019, p > 0.05, Table 4.2). Furthermore,

no significant correlation was observed between neopterin and elastase

concentration in seminal plasma (r = 0.028, p >0.05).

While seminal plasma PMN elastase levels were significantly elevated in the

infertile cohort, elastase concentration was not significantly correlated with any

marker of semen quality aside from CD45 concentration (Table 4.2).

Conversely, seminal plasma neopterin levels were predictive of many aspects

of semen quality. Seminal plasma neopterin was significantly correlated with

sperm concentration (r=-0.309, p= 0.008), morphology (r = -0.325,

p=0.008), sperm DNA fragmentation (r=0.340, p=0.003) and apoptosis

(annexin V, r=0.481, p< 0.001). There was also a strong positive correlation

between neopterin concentration and markers of sperm oxidative stress such

as LPO and NBT assay (r = 0.425, p< 0.0001 and r = 0.477, p< 0.001,

respectively).

74

PMN Elastase

CD45

Sperm Conc.

Motility

Morphology

Volume

Formazan

LPO

TUNEL

AnnexinV

Neopterin

0.028

-0.019

-0.309t

-0.200

-0.325 t

0.0851

0.477 t

0.425 t

0.340 t

0.481 t

PMN

Elastase

0.472 t

0.079

-0.014

0.052

-0.075

0.201

0.001

0.109

0.039

CD45

0.301

0.03

0.072

-0.269

-0.113

-0.276

-0.011

-0.112

Sperm

Concentra

tion

0.472 t

0.487 t

0.083

-0.778 t

-0.733 t

-0.644 t

-0.691t

Motility

0.470 t

-0.118

-0.409 t

-0.467 t

-0.693 t

-0.602t

Morpholo

gy

-0.091

-0.548 t

-0.496 t

-0.473 t

-0.405t

Volume

-0.053

-0.024

0.002

-0.082

Formazan

0.812 t

0.631 t

0.688t

LPO

0.686 t

0.757t

TUNEL

0.806t

Table 4.2: Comparison between sperm quality between and inflammatory markers

in all groups t= statistically significant correlation

75

In order to help locate the site of macrophage activity within the infertile men’s

reproductive tract, seminal plasma neopterin concentration was analysed in

relation to markers of epididymal (neutral alpha-glucosidase- NAG) and

testicular function (testosterone, LH, FSH, AMH). Neopterin levels were not

significantly correlated with any marker of testicular or epididymal function

(Table 4.3).

Table 4.3: Correlation between macrophage activity (Neopterin) and

epididymal/testicular function

4.5 Discussion

The results of this study clearly show that genital tract inflammation is more

commonly present in infertile than fertile men. This observation is in

agreement with previous reports linking increased seminal leukocyte numbers

(Plante et al., 1994; Wolff, 1995) (Ochsendorf, 1999; Sharma et al., 2001) and

elastase concentration (Jochum et al., 1986; Reinhardt et al., 1997; Wolff and

Anderson, 1988; Zopfgen et al., 2000; Zorn et al., 2000) with infertility.

However, this study is the first to report the presence of the macrophage

activity marker neopterin in human seminal plasma.

The observation that seminal plasma neopterin levels are three-fold higher in

infertile than fertile men is consistent with the small number of histological

studies reported in the literature. Frungieri et al (2002) examined macrophage

numbers/ activity using immunocytochemistry and RT-PCR techniques in

testicular biopsy samples from men with normal and abnormal

r p

Neutral α−α−α−α−glucosidase 0.088 0.496

LH

-0.176 0.276

FSH

0.031 0.843

Testosterone

-0.090 0.559

AMH

0.004 0.976

76

spermatogenesis (Frungieri et al., 2002). They found that macrophages were

mainly localized to the testicular interstitium, with others also being located

within the wall and lumen of seminiferous tubules. The density of testicular

macrophages in men with spermatogenic defects was increased between two

and six times compared to men with normal spematogenesis, depending on the

particular nature of the spermatogenic defect.

Hussein et al (2005) compared various leukocyte densities between

azoospermic men with normal (obstructive) spermatogenesis and those with

sertoli cell-only or germ cell maturation arrest (Hussein et al., 2005). This

group also observed the presence of macrophages in the interstitium and

seminiferous tubules, with the density of macrophages in the seminiferous

tubule wall and lumen of men with abnormal spermatogenesis being double

that seen in men with normal spermatogenesis. The presence of macrophages

within the epididymis has also been confirmed by histological studies (Wang

and Holstein, 1983; Yeung and Cooper, 1994). It has been suggested that

epididymal macrophages may actually be present under normal physiological

conditions to allow for the phagocytic elimination of dead or dying sperm

(Holstein, 1978; Tomlinson et al., 1992a). Conversely, elevated numbers of

macrophages have also been described in pathological conditions such as

epididymitis and post vasectomy (Flickinger et al., 1995). As the majority of

cases of chronic epididymitis are clinically silent (Haidl et al., 2008), it is

possible that many of these infertile subjects may have experienced

epididymitis despite being totally asymptomatic.

One of the most significant findings of this study was that measurement of

macrophage activity by seminal plasma neopterin has proven to be a more

useful marker of sperm quality than the more traditionally used inflammatory

markers seminal plasma elastase and leukocyte concentration (CD45). No

correlation was observed between seminal plasma elastase and any marker of

sperm quality. This result is consistent with much of the published literature.

While some observers have linked elevated seminal plasma elastase levels with

a reduction in sperm count, motility (Wolff et al., 1991; Wolff et al., 1990) and

an increase in sperm DNA damage (Zorn et al., 2000), many other studies

have found no such link (Eggert-Kruse et al., 1997; Eggert-Kruse et al., 2009;

Henkel et al., 2003; Maegawa et al., 2001). More importantly, seminal plasma

elastase concentrations in asymptomatic males has been reported to have

77

absolutely no bearing on subsequent in vivo (Eggert-Kruse et al., 2009) or in

vitro pregnancy rates (Henkel et al., 2003). These observations and studies

failure to link seminal elastase concentration with sperm function in an

asymptomatic population should not be interpreted as suggesting that

neutrophils have no role in causing male infertility. A weak non-significant

trend was observed between elastase concentration and ROS formation (r=

0.20, p=0.089). As none of the study population had any symptoms of active

infection, it is likely that most of the neutrophils present within semen were

relatively inactive. Therefore, the presence of active infection could possibly

result in a significant correlation between seminal plasma elastase and ROS

production, resulting in impaired sperm function. What seems more certain is

that neutrophils do not appear to play an important role in impairing sperm

function in men without symptoms of active genital tract infection.

Leukocyte concentration (CD45) was only significantly linked with sperm

concentration and semen volume, showing absolutely no correlation with all

other measures of sperm function. It is possible that past infection of the

prostate and seminal vesicle glands, the two principal determinants of semen

volume, has resulted in this significant correlation. Infective damage to either

the prostate and seminal vesicle will reduce semen volume (Jequier, 2000),

increasing the sperm concentration per ml of semen, even when the rate of

sperm production remains unchanged. A history of previous genital tract

infection is a good predictor of current genital tract inflammation, but such a

history is only found in 5-14% of cases (Comhaire et al., 1995; Zorn et al.,

2003a). Furthermore, chronic genital tract inflammation is significantly more

common in infertile men and the majority of cases are clinically asymptomatic

(Haidl et al., 2008; Schuppe et al., 2008). This would appear to be the case in

my study cohort.

The finding that only macrophage (neopterin), not neutrophil activity

(elastase), was correlated with sperm functional capacity makes perfect sense

when one considers the origins of these different types of leukocytes.

Neutrophils are believed to be primarily derived from the prostate and seminal

vesicle, while macrophages have a testicular or epididymal origin. Sperm only

comes in contact with the secretions of the prostate and seminal vesicles at the

time of ejaculation, giving neutrophils very limited time to damage sperm.

Conversely, sperm are likely to be in direct contact with macrophages within

78

the seminiferous tubules and epididymis for many days during

spermatogenesis, giving these activated macrophages ample opportunity to

alter sperm function (Aitken and Baker, 1995; Barratt et al., 1990). As this

study was unable to find any correlation between seminal plasma neopterin

and markers of testicular or epididymal function, it is uncertain at which point

in the male reproductive tract that macrophages primarily impact sperm

functional capacity.

Activated macrophages are capable of harming cells such as sperm through

three principal mechanisms (Anderson, 1990). Firstly, they are capable of

producing large amounts of reactive oxygen and nitrogen species that will lead

to sperm membrane lipid peroxidation and oxidative DNA damage. Secondly,

they can destroy cells through the release of enzymes such as lysosomes and

hydrolytic enzymes or cytotoxic peptides commonly referred to as defensins.

Finally, macrophages can induce apoptosis in adjacent cells directly by Fas

ligand or indirectly through the release of cytokines such as Tumor Necrosis

Factor alpha (TNFα). Testicular macrophages have been confirmed to produce

significant amounts of TNFα (Frungieri et al., 2002); with in vitro studies

suggesting that TNFα has the ability to reduce sperm motility and genomic

integrity (Said et al., 2005). Since I observed that neopterin levels were

significantly correlated with LPO-586 and formazan production (NBT assay)

plus annexin V expression, it is likely that macrophages damage sperm by both

apoptotic and oxidative stress pathways. Furthermore, these results are in

agreement with studies conducted by Solis et al, who also reported a positive

correlation between macrophage numbers in semen and sperm DNA

denaturation (Solis et al., 2003).

The measurement of seminal plasma neopterin has created a new non-invasive

tool for measuring macrophage activity within the male reproductive tract. The

observation that seminal plasma of infertile men contains significantly

increased levels of neopterin, suggestive of an increased state of macrophage

activation, has two potential interpretations in regards to the underlying

aetiology of male infertility (Pelliccione et al., 2008). Firstly, it is possible that a

silent infection or an auto-immune response to sperm antigens, akin to that

seen following vasectomy (Flickinger et al., 1995), may lead to activation and

recruitment of macrophages into the male reproductive tract where they could

induce sperm damage. Alternatively, the activation of macrophages within the

male reproductive tract may represent a normal physiological response in

79

which macrophages are involved in the removal of dead or defective sperm

(Holstein, 1978; Tomlinson et al., 1992a), both more commonly produced by

infertile men. In this second scenario macrophages are simply associated with

defective spermatogenesis, rather than being the underlying cause. My

observation of a significant link between seminal plasma neopterin and

abnormal sperm morphology and apoptosis could support such a “physiological

clearance” role for macrophages. However, it should be understood that even if

macrophage clearance of dead and dying sperm is a normal physiological

process, it may still pose a threat to male fertility as good quality live sperm

may be damaged “in the cross fire” by proteases and ROS released from

activated macrophages. Therefore, it is possible that immuno-suppressive

therapies such as Pentoxifylline and non-steroidal anti-inflammatory drugs may

prove beneficial in improving sperm quality in a select group of infertile men

with high macrophage activity in their reproductive tract.

In summary, in an asymptomatic population the measurement of seminal

plasma neopterin appears to be a significantly better marker of sperm health

than the more traditional markers of male genital tract inflammation such as

seminal plasma elastase or leukocyte concentration. Larger studies in the

future will help develop normal “cut-off” values for seminal plasma neopterin

that best define sperm health. While it has yet to be conclusively proven that

macrophage activity is actually responsible for impaired sperm function,

elevated seminal plasma neopterin levels may prove in the future to be a

useful clinical indicator for initiating anti-inflammatory and antioxidant therapy

so as to maximize sperm genomic integrity.

80

CHAPTER 5

Obesity as a Cause of Oxidative Stress

81

5.1 Abstract

Male obesity has been linked with a reduction in sperm concentration

and motility, an increase in sperm DNA damage and changes in

reproductive hormones. Recent large observational studies have linked

male obesity with a reduced chance of becoming a father. One of the

potential underlying pathological mechanisms behind diminished

reproductive performance in obese men is sperm oxidative stress. The

primary aim of this study was to determine if sperm oxidative stress

was more common in obese/ overweight men. A total of 81 men had

their Body Mass Index (BMI) correlated with seminal Reactive Oxygen

Species (ROS) production (NBT assay), sperm DNA damage (TUNEL),

markers of semen inflammation (CD45, seminal plasma PMN elastase

and neopterin concentration) and routine sperm parameters, together

with reproductive hormones. The principal finding from this study was

that oxidative stress did increase with an increase in BMI, primarily due

to an increase in seminal macrophage activation. However, the

magnitude of this increase was small and only of minor clinical

significance as there was no associated decline in sperm DNA integrity

or sperm motility with increasing Reactive Oxygen Species (ROS)

production. Increased BMI was also found to be significantly linked with

a fall in sperm concentration and serum testosterone, and an increase

in serum estradiol.

82

5.2 Introduction

Obesity has become an increasing public health concern in Western society

over the last few decades due to the ready availability of high calorie foods and

a sedentary lifestyle. It is currently estimated that one third of adult males are

obese and a further third are overweight (Hedley et al., 2004). Obesity is now

recognized as an established cause of female sub-fertility, with increasing Body

Mass Index (BMI) being linked with anovulation and an increased risk of

miscarriage (Rich-Edwards et al., 2002; Wang et al., 2002). However, more

recent data suggests that obesity outcomes for six thousand men over 30

years concluded that even after adjustment for marital status, obese men were

22% less likely to have fathered a child than their normal weight counterparts

by the time they had reached their late 40’s (Jokela et al., 2008). Similarly, the

Norwegian Mother and Child cohort study reported that overweight men (BMI

25- 29.9 kg/m2) had an OR for infertility of 1.19 (95% CI 1.03-1.62) and obese

men (BMI > 30 kg/m2) had an OR for infertility of 1.36 (95% CI 1.12-1.62)

(Nguyen et al., 2007).

The literature examining the link between BMI and routine sperm quality

parameters is unfortunately confusing due to the presence of several

conflicting observations (Hammoud et al., 2008). While many studies have

reported a reduction in sperm concentration with increasing BMI (Fejes et al.,

2005; Jensen et al., 2004; Magnusdottir et al., 2005), others have reported no

link (Aggerholm et al., 2008; Pauli et al., 2008; Qin et al., 2007). Similarly, the

link between BMI and sperm motility is also uncertain, with some studies

reporting a significant negative correlation (Fejes et al., 2005; Kort et al.,

2006), while others report no correlation (Aggerholm et al., 2008; Jensen et

al., 2004; Pauli et al., 2008; Qin et al., 2007). No study to date has reported

any significant relationship between BMI and the percentage normal sperm

morphology (Jensen et al., 2004; Pauli et al., 2008; Qin et al., 2007).

Several studies have reported that obese men have abnormal reproductive

hormone profiles which may impair spermatogenesis (Hammoud et al., 2008).

Obese men consistently have been found to have lower serum testosterone

and higher estrogen levels than their normal weight counterparts (Aggerholm

et al., 2008; Jensen et al., 2004; Pauli et al., 2008; Schneider et al., 1979;

Strain et al., 1988). The peripheral conversion of androgens to estrogen by

aromatase action in adipose tissue is likely to be responsible for this altered

83

hormone profile. The relative high levels of estrogen and increased

concentrations of hypothalamic opioids seen in obese men are hypothesised to

create a negative feedback on the pituitary, impairing the release of LH and

FSH (Hammoud et al., 2008). However, while some studies have identified a

relative hypogonadotrophic state in obese men (de Boer et al., 2005; Giagulli

et al., 1994; Pauli et al., 2008), others have reported serum LH and FSH levels

compatible with normal weight controls (Aggerholm et al., 2008; Jensen et al.,

2004).

Testicular heat stress may play a role in obesity related impaired

spermatogenesis. The testicular temperature in obese individuals is likely to be

higher than ideal due to two factors. Firstly, with an increase in BMI there is an

increase in the deposition of fat in the suprapubic and inner thigh regions,

enveloping the scrotum and impairing normal testicular heat transfer.

Secondly, obese individuals tend to lead a more sedentary lifestyle, with the

sitting position known to produce up to a 2 degree Celsius increase in scrotal

temperature compared to a standing position (Jung et al., 2005). It is therefore

probable that obesity related heating of the testicle may reduce sperm quality

as spermatogenesis is very sensitive to increases in temperature (Jung and

Schuppe, 2007).

A final mechanism by which obesity may impair male fertility is oxidative stress

initiated damage to sperm. While no study to date has linked obesity with

sperm oxidative DNA damage, there are several reasons to suspect such a link

may exist. Firstly, obesity has been reported to create a state of systemic

oxidative stress (Furukawa et al., 2004; Ozata et al., 2002), a situation that is

likely to extend to the testicular micro-environment. Obese individuals are in a

chronic state of inflammation due to adipose tissues production of pro-

inflammatory cytokines such as IL-6 and TNFα which can stimulate leukocytes

production of ROS (Das, 2001). Secondly, the production of Leptin by adipose

tissue has been reported to increase sperm oxidative metabolism (Lampiao and

du Plessis, 2008). Therefore, the primary aim of this study was to determine if

increases in BMI above the healthy weight range (20-25 kg/m2) are associated

with changes in seminal oxidative stress and sperm DNA damage. The

secondary aims were to further analyse any possible correlations between BMI

and other markers of male reproductive health (sperm count, motility,

morphology and reproductive hormones) and signs of semen inflammation

84

(semen leukocyte count and seminal plasma elastase/ neopterin

concentration).



Study participants consisted of 81 men presenting for semen analysis at the

Adelaide Fertility Centre (Repromed) as part of their assessment of male

fertility potential. None of the men had any significant symptoms or signs of

andrological dysfunction. Patient height (meters) and weight (kilograms) were

recorded and converted into a Body Mass Index (BMI = kg/ m2). Participants

were classified according to the following published BMI ranges: normal weight

BMI 20-25 kg/m2, overweight 25.1-30 kg/m2 and obese > 30 kg/m2. The study

was prospectively approved by the Human Research and Ethics Committee,

Women’s and Children’s Hospital, with all participants giving informed written

consent for their involvement.




Data were analysed using GraphPad Software (GraphPad Software Inc., La

Jolla, CA, USA) and presented as median (inter-quartile ranges) due to the

non-Gaussian distribution of the data. Statistical comparisons between groups

(normal and high BMI groups) were made using the non-parametric Mann

Whitney U test. Analysis of the correlation between continuous variables (BMI

and various semen parameters/ reproductive hormones) was conducted using

the Pearson’s correlation coefficient test. A P value < 0.05 was considered

statistically significant.

5.4 Results

A weak, yet statistically significant positive correlation was observed between

BMI and seminal oxidative stress (r= 0.23, p=0.039, Figure 5.1). As BMI

increased beyond the normal weight range (BMI > 25 kg/m2) an increasing

number of individuals exhibited quite marked oxidative stress. While many

normal weight individuals exhibited ROS production exceeding the upper limit

of ideal (24 µg formazan per 107 sperm (Tunc et al., 2008), a disproportionate

larger number of overweight/obese individuals exhibited very high levels of

ROS production (Figure 5.1).

85

Analysis of the relationship between BMI and sperm morphology, a key

determinant of sperm ROS production (Aziz et al., 2004), identified no

significant relationship (r = - 0.08, p= 0.53). Furthermore, no significant

correlations were identified between BMI and seminal CD45 leukocyte count or

seminal plasma elastase activity. However, a weak yet statistically significant

positive correlation was observed between seminal plasma neopterin and BMI

(Figure 5.2), suggesting an enhanced state of macrophage activation with

obesity.

Figure 5.1: Correlation between ROS production (µµµµg formazan

per 107sperm) and BMI.

Figure 5.2: Correlation between seminal plasma neopterin

concentration (nmol/L) and BMI

20 25 30 35 40 450

100

200

300

400

r = 0.23p = 0.039

BMI

Fo

rmazan

µµ µµg

/10

7 s

perm

15 20 25 30 35 400

200

400

600

800

1000

r = 0.238p = 0.047

BMI

Neo

pte

rin

nm

ol/L

86

Figure 5.3 depicts the relationship between sperm DNA damage (TUNEL) and

BMI. While there appears to be the possibility of a weak positive relationship

between sperm DNA damage and BMI, this relationship was not statistically

significant (r= 0.13, p=0.26). As was found for seminal ROS production,

extremes of sperm DNA damage were much more commonly seen in the high

BMI group than those with a BMI in the normal 20-25 kg/m2 range. The

median sperm DNA fragmentation in the normal weight group was 12.4 %

compared to 17.6 % in the group with a BMI > 25 kg/m2, a result that

bordered on significant (p=0.064). When the high BMI group (BMI > 25 kg/m2)

was sub-divided into two groups depending on whether individuals had normal

or high degrees of sperm DNA fragmentation (TUNEL < or > 15%

respectively), the low DNA damage group had a median ROS output of only

23µg formazan per 107 sperm, yet the high DNA damage group produced a

median output of 68.6 µg formazan per 107 sperm (p<0.001).

Figure 5.3: Linear regression analysis between sperm DNA

damage (TUNEL positivity %) and Body Mass Index (BMI).

20 25 30 35 40 450

10

20

30

40

50

r = 0.13p = 0.260

BMI

TU

NE

L p

osit

ivit

y %

87

Figure 5.4: Correlation between sperm concentration and BMI.

A significant negative correlation was found between BMI and sperm

concentration (r= -0.33, p = 0.002, Figure 5.4), but no significant relationship

was noted between BMI and sperm motility. A significant correlation was also

seen between BMI and serum oestradiol/ testosterone concentrations. As

depicted in Figure 5, serum testosterone decreased (r =-0.29, p=0.039) and

serum oestradiol increased (r= 0.35, p=0.026) with increases in BMI. No

significant correlation was seen between BMI and gonadotrophin (LH, FSH)

concentrations.

Figure 5.5: Depiction of the correlation between BMI and serum

testosterone and estradiol concentrations.

20 25 30 35 40 450

100

200

300

400

r = -0.33p = 0.002

BMI

Sp

erm

Co

nc.

(x10

6/m

L)

20 25 30 35 400

10

20

30

r = - 0.29 p = 0.039

BMI

Testo

ste

ron

e (

nm

ol/L

)

20 25 30 35 400

50

100

150

r = 0.35p = 0.026

BMI

Estr

ad

iol (p

mo

l/L

)

88

5.5 Discussion

The results of this study suggest that sperm oxidative stress is increased in

overweight (BMI >25 kg/m2) compared to normal weight individuals. However,

the link between increasing BMI and oxidative stress is a relatively weak one

and only likely to be of limited reproductive importance. This study found no

significant correlation between increasing BMI and sperm DNA damage or

motility, both likely targets for ROS attack. A previous large study of 520 men

did observe a moderate increase in sperm DNA damage with increasing BMI

(Kort et al., 2006). It is acknowledged that this study was relatively small and

therefore it is possible that the weak positive trend observed between BMI and

sperm DNA fragmentation may have become statistically significant with a

larger sample size.

The mechanism by which obesity may increase sperm oxidative stress is now

becoming clearer. It is believed that the principal sperm source of ROS

production in infertile men comes from morphologically abnormal immature

sperm containing excess cytoplasm (Aziz et al., 2004). It is postulated that

glucose-6-phosphate dehydrogenase contained in the excess residual

cytoplasm fuels the enhanced generation of NADPH that in turn, stimulates the

production of ROS. As this study did not identify any significant increase in

abnormal sperm morphology with increasing BMI, it is uncertain how sperm

themselves could be responsible for the observed increase in seminal ROS

production associated with obesity. However, a recent report has shown that

leptin and insulin, both elevated in the obese individual, can increase sperm

metabolic activity and nitric oxide production (Lampiao and du Plessis, 2008).

It is therefore still possible that morphologically normal sperm may generate

increased amounts of ROS in obese men through mechanisms unrelated to

sperm morphology.

Traditionally neutrophils have been considered the primary leukocyte source of

ROS production in semen as they account for up to 70% of seminal leukocyte

numbers and are potent producers of ROS during infection / inflammation.

However, in this study no correlation between seminal plasma elastase

concentration and BMI, making neutrophils an unlikely source for obesity

89

related oxidative stress. Conversely, seminal plasma neopterin levels were

significantly positively correlated with BMI, suggesting that obesity creates a

state of macrophage immune activation within the male reproductive tract.

Neopterin is a low molecular mass pteridine molecule released specifically from

macrophages / monocytes primarily upon stimulation by the cytokine

interferon gamma and to a lesser extent other pro-inflammatory cytokines

(Hamerlinck, 1999). It has been well established that there is a close

relationship between the amount of neopterin released from macrophages and

their capacity to produce reactive oxygen species (Berdowska and Zwirska-

Korczala, 2001; Weiss et al., 1998). A significant relationship between seminal

macrophage activation (seminal plasma neopterin) and ROS production

(Tremellen and Tunc), supporting macrophages as an important source of

oxidative stress. While previous papers have described a link between obesity

and elevated serum neopterin levels (Ledochowski et al 1999, Brandacher et al

2006), this study is the first to describe the relationship between obesity and

elevated seminal plasma neopterin concentrations. It would therefore appear

that the previously described state of systemic immune activation seen in

obese individuals (Bozdemir et al., 2006; Das, 2001; Ledochowski et al., 1999)

also extends to the male reproductive tract, resulting in an enhanced state of

macrophage activity and seminal oxidative stress.

In regards to my secondary objectives, I identified a significant negative

correlation between sperm concentration and BMI, yet no link between BMI

and sperm motility or morphology. While the published literature contains

many conflicting reports regarding the relationship between BMI and routine

sperm parameters, the finding of a significant negative correlation between

BMI and sperm concentration is consistent with the bulk of this literature (Fejes

et al., 2005; Jensen et al., 2004; Magnusdottir et al., 2005). My observations

of a significant fall in serum testosterone and an increase in oestradiol with

increasing BMI are also entirely consistent with the published literature

(Aggerholm et al., 2008; Jensen et al., 2004; Pauli et al., 2008; Schneider et

al., 1979; Strain et al., 1988).

This study is the first to report a significant correlation between increasing BMI

and seminal oxidative stress, primarily related to an increase in macrophage

activity within the male reproductive tract. I acknowledge that this link is a

relatively weak one, with obesity likely to play only a minor role in the

aetiology of oxidative sperm damage in the majority of individuals. However,

90

as obesity has been shown to reduce sperm concentration by this and other

studies, it would appear prudent to advise all men of reproductive age to

maintain a normal body weight to maximize their reproductive potential. As

weight loss has been shown to reduce systemic oxidative stress (Melissas et

al., 2006; Uzun et al., 2004), additional studies are warranted to determine if

weight loss in obese individuals can result in a reduction in sperm oxidative

stress and an improvement in sperm concentration.

91

CHAPTER 6

Treatment of Oxidative DNA Damage in sperm using an Oral Antioxidant Therapy

92

6.1 Abstract

Oxidative stress is now recognized as a common pathology affecting

up to half of all infertile men. One of the principal mechanisms by

which oxidative stress produces infertility is by damage to sperm DNA,

either through direct oxidation of the DNA by reactive oxygen species

(ROS) or by the initiation of apoptosis.

The objective of this study was to determine if an oral antioxidant/

mineral supplement could improve sperm DNA integrity in men with

known oxidative stress. A total of 50 infertile men identified as

exhibiting oxidative stress were administered oral antioxidant therapy

for a period of 3 months. Sperm DNA integrity (TUNEL), apoptosis

(Annexin V), protamination (CMA3) and reactive oxygen species (NBT

Assay) production were assessed in all participants at entry and exit.

Sperm concentration, motility and morphology; together with

assessment of serum male reproductive hormones (LH, FSH,

Testosterone, Anti-Mullerian Hormone) was also monitored.

The principal finding that emerged from this study was that

antioxidant therapy resulted in significant improvements in sperm

DNA integrity and protamine packaging; accompanied by a reduction

in seminal ROS production and apoptosis. No significant changes in

routine sperm parameters (concentration, motility, morphology) or

male reproductive hormones were observed.

93

6.2 Introduction

Elevated levels of sperm DNA damage are associated with increased time to

natural conception (Spano et al., 2000; Loft et al. 2003), decreased IUI and

IVF/ICSI pregnancy rates (Larson et al 2000; Benchaib et al., 2003; Bengum

et al., 2004; Virro et al., 2004; Borini et al 2006), increased miscarriage risk

(Virro et al., 2004; Borini et al., 2006; Benchaib et al., 2007;) and possibly

even increases in childhood cancer (Lewis and Aitken 2005). As at least 15% of

men in infertile relationships have compromised sperm DNA integrity (Benchaib

et al 2007), the development of new treatments to improve sperm DNA quality

is of major clinical importance.

While the exact causes of sperm DNA damage have not been fully elucidated,

several inter-related mechanisms have been suggested (Tesarik 2006; Aitken

and De Iuliis 2007; Ozmen et al., 2007; Tarozzi 2007). Firstly, the most widely

accepted cause for sperm DNA fragmentation is the presence of oxidative

stress. The generation of sperm oxidative stress in vitro, either through the

direct application of hydrogen peroxide (Aitken et al., 1998) or by stimulation

of the sperm’s own intrinsic production of free radicals (Twigg et al., 1998),

has been reported to significantly increase the levels of DNA damage within

sperm.

Defective protamine packaging of sperm DNA may also make infertile men’s

sperm more susceptible to oxidative attack (Ozmen et al., 2007). During

normal spermatogenesis 85% of nuclear histones are replaced with protamines

that allow tight packaging of the sperm DNA, protecting it from oxidative

attack. Approximately 15% of infertile men have been reported to exhibit

deficient protamine packaging (Carrell and Liu 2001), making these men’s

sperm more vulnerable to oxidative DNA damage (Aoki et al., 2005; Torregrosa

et al., 2006).

The final mechanism responsible for sperm DNA fragmentation is “abortive

apoptosis” (Sakkas et al 2003). Apoptosis plays two principal roles in

spermatogenesis. Firstly, it is essential in limiting the population of germ cells

to a number that can be adequately supported by Sertoli cells, thus ensuring

normal spermatogenesis. Secondly, apoptosis is proposed to be responsible for

94

the selective depletion of abnormal germ cells (Sakkas et al 2003; Tarozzi

2007). However, even when good quality sperm are exposed to oxidative

stress, apoptotic destruction is often triggered (Moustafa et al., 2004). If

apoptosis fails to completely destroy these damaged cells (“abortive

apoptosis”), endonuclease mediated digestion of the sperm DNA will occur

within these still viable sperm. Therefore, oxidative damage to sperm DNA is

the net effect of direct oxidative damage to the purine and pyrimidine bases of

the sperm DNA, in tandem with apoptotic endonuclease digestion.

The Menevit® nutraceutical is an antioxidant preparation that has recently been

shown in a placebo controlled randomized study to improve pregnancy

outcomes when used in conjunction with IVF-ICSI treatment (Tremellen et al.,

2007).

Menevit® Antioxidant Preparation

Lycopene 6 mg.

Vitamin E 400 IU

Vitamin C 100 mg.

Zinc 25 mg.

Selenium 26 µg.

Folate 500 µg.

Garlic oil 333 µg. (equivalent 1 g. garlic)

Table 6.1: Content of the MENEVIT® Antioxidant Preparation

The seven different components of Menevit® (Table 6.1) were identified as

suitable for promoting sperm DNA heath because of their capacity to impede

oxidative attack through three overlapping mechanisms. Firstly, Vitamins C and

E, selenium, garlic and lycopene have direct anti-oxidant effects (Heber and Lu

2002; Saravanan et al., 2004; Valko et al., 2004), assisting in the

neutralization of ROS already produced by sperm or seminal leukocytes.

Secondly, lycopene (Heber and Lu 2002) and garlic (Hodge et al., 2002) have

95

been shown to have potent anti-inflammatory activity, thereby potentially

resulting in a reduction in seminal leukocyte ROS production.

Finally, zinc, folate and selenium are known to play a vital role in sperm DNA

synthesis and protamine packaging (Kvist et al., 1987; Evenson et al., 1993;

Pfeifer et al., 2001; Brewer et al., 2002;), possibly protecting sperm DNA from

oxidative stress. The aim of the ADAM study (Assessment of DNA After

Menevit®) was to determine if the Menevit® antioxidant can alter oxidative

attack, protamine packaging, sperm DNA integrity and the production of male

reproductive hormones.

6.3 Materials and methods


Participants in the ADAM study were recruited from men undergoing infertility

assessment at an academic affiliated ART unit (Repromed, Dulwich, South

Australia). To be eligible for involvement study participants were required to

meet two inclusion criteria. Firstly, the entry semen analysis had to exhibit

significant oxidative stress, as assessed by the Nitro Blue Tetrazolium (NBT)

assay. Levels of seminal ROS production exceeding the 75th percentile for

fertile donors (19 µg formazan/ 107 sperm) were considered as sufficient

evidence for oxidative stress. A previous study of 12 fertile donors and 25

randomly selected infertile men had found that 68% of infertile men had a

seminal ROS score exceeding the 75th percentile for fertile donors. Secondly,

participants were required to have a minimum sperm concentration of 1 x

106/mL, as this was the minimum amount of sperm needed to conduct the

various sperm DNA quality assays.

A total of 56 men with probable oxidative stress (poor motility, high abnormal

sperm morphology, altered semen viscosity on routine analysis- Tremellen

2008) were screened to identify the target sample of 50 men with confirmed

oxidative stress. Three men were excluded from the study due to low sperm

count and three because of inadequate levels of oxidative stress on NBT

assessment. The average age of participants was 39 ± 5.8 years (range 26 to

53 years) with an average duration of infertility of 2.5 ± 0.6 years.

96

Those participants who tested positive for oxidative stress were asked to take

one capsule of Menevit® (Bayer Australia Ltd, Sydney, Australia) per day for a

period of 3 months and to provide a semen and serum sample both at entry

and exit. Four men withdrew from the study before completing their 3 months

of antioxidant treatment due to a lack of continuing interest. One man

withdrew as he believed the treatment aggravated his symptoms of Irritable

Bowel Syndrome. The study was prospectively approved by the Human

Research and Ethics Committee, Women’s and Children’s Hospital, with all

participants giving written informed consent for their involvement.




Data were analysed using the statistical software Sigmastat (Systat Software

Inc, California, USA). As the majority of data was not normally distributed, the

results are principally expressed as median values (inter-quartile range), with

statistical analysis being performed using the non-parametric Wilcoxon Signed

Rank test. When the data was normally distributed, results are expressed as

mean ± SD and were analyzed using the paired t test. A P value < 0.05 was

considered statistically significant.

6.4 Results

After 3 months of antioxidant treatment sperm DNA integrity improved

significantly, with the median sperm DNA fragmentation level dropping from

22.2% to 18.2% (Table 6.2). Recently it has been reported that short periods

of abstinence can result in spontaneous improvements in sperm DNA integrity

(Bakos et al., 2008). No significant differences in the period of abstinence

between the entry and exit samples (mean 3.8 v 4.2 days respectively) could

account for the observed improvements in sperm DNA integrity. Furthermore,

the observed improvements in sperm DNA quality were mirrored by reductions

in early apoptosis (Annexin V positive, PI negative), and a significant fall in

seminal ROS production, suggesting that an anti-oxidant effect was primarily

responsible for the observed improvement in sperm DNA integrity.

Three months of antioxidant treatment containing zinc and selenium produced

a small but statistically significant improvement in median levels of sperm

97

protamination (69% v 73.6%, p<0.001). During normal spermatogenesis 85%

of sperm histones are replaced by protamines; with impaired reproductive

outcomes being observed once sperm protamination levels fall below 70%

(Nasar-Esfahani et al 2004). At the commencement of the study, 26 infertile

men (58%) had inadequate levels of sperm DNA protamination (< 70% sperm

protamine content), yet by study completion only 14 participants (31%)

exhibited protamine deficiency (<70% sperm protamine content).

Basic sperm parameters (count, motility, morphology, semen volume) did not

change significantly following three months of anti-oxidant treatment (Table

6.3). All 45 trial participants had at least one defect in routine sperm quality,

since only men with pre-existing male factor infertility were approached to

enter the study. However, from review of Table 6.3 it can be seen that the

average sperm parameters of the group were only moderately impaired.

Table 6.2: Sperm DNA quality during antioxidant treatment

n=45

ENTRY

Median (25%-75% range)

EXIT

Median (25%-75% range)

P

DNA fragmentation

(%)

22.2 (16.5 – 26.6) 18.2 (13.4 – 23.1) 0.002

Early Apoptosis

(%)

(Annexin V +, PI -)

27.3 (20.4 – 34.7) 22.5 (19.3 – 28.1) 0.004

Protamination

(%)

69 (63.5 – 73.1) 73.6 (69.3 – 77.5) <0.001

ROS production

(µµµµg Formazan/ 107

sperm)

66.4 (43.1 – 87.8) 44.4 (33.3 – 81.4) 0.027

98

Table 6.3 Routine semen assessment during antioxidant treatment

ENTRY EXIT P

Concentration

(106 sperm/ mL) 36.2 ± 46.2 39.4 ± 44.7 0.304 b

Motility

(%) 36.5 ± 12.3 37.1 ± 11.5 0.778 a

Normal morphology

(%) (NR > 20%)

6.7 ± 5.0

6.71 ± 4.5

0.938 a

Semen volume

(mL)

2.94 ± 1.8

2.88 ± 1.7

0.729 a

Total motile sperm

(106 per ejaculate)

38.4 ± 56.4 43.9 ± 64.5 0.337 b

All values are expressed as mean ± standard deviation. The paired t-test a and Wilcoxon signed rank test b were used for

statistical analysis.

Three months of antioxidant therapy did not appear to effect the production of

any of the reproductive hormones monitored (Table 6.4). No significant

changes in Leydig cell (serum LH and testosterone), nor Sertoli cell (serum

FSH, AMH) derived hormones were observed. Furthermore, when a sub-group

analysis examining only those participants with evidence of Sertoli or Leydig

cell dysfunction at trial entry (FSH > 10 IU/L, Testosterone < 8 nmol/L) was

performed, no significant differences in any reproductive hormones was

observed following antioxidant therapy.

99

Table 6.4 Reproductive Hormone concentrations during antioxidant

treatment

ENTRY EXIT P

LH (IU/L)

3.93 ± 1.33

4.23 ± 1.34

0.078

FSH (IU/L)

5.58 ± 3.13

5.81 ± 3.28

0.160

Testosterone (nmol/L)

14.07 ± 4.1

14.96 ± 5.5

0.073

AMH (pmol/L.)

61.8 ± 28.9

61.4 ± 28.3

0.731

Thirty seven participants in the ADAM study underwent IVF treatment while on

the Menevit® antioxidant, although this was not a criterion for entry to the

study. The average age of the female partners was 35.7 ± 4.5 years, with an

average duration of infertility 3.0 ± 1.3 years. The majority of ADAM

participants underwent IVF-ICSI (36 cycles ICSI, 1 cycle IVF), with the

observed mean fertilization rate being 68.0 ± 26.7%. Thirty six of the 37

cycles proceed to an embryo transfer, with one transfer being cancelled due to

no genetically normal embryos being available for transfer (pre-implantation

genetic diagnosis cycle). Sixteen of the partners achieved a clinical pregnancy,

with 13 of these pregnancies (81.25%) being viable on a first trimester

ultrasound. A further 3 couples experienced a biochemical pregnancy, giving an

overall biochemical pregnancy rate of 52.8% and a clinical (ultrasound

identified) pregnancy rate of 44.4% per embryo transfer. Within this viable

pregnant cohort the median sperm DNA fragmentation reduced from 22 % at

entry to 13.3 % at exit.

100

6.5 Discussion

The present data show a significant improvement in sperm DNA integrity

following 3 months of antioxidant therapy. This result is consistent with the

published literature. Firstly, a combination of Vitamin C and E has been shown

in well conducted placebo controlled RCT’s to reduce both sperm (Greco et al.,

2005) and peripheral blood leukocyte DNA damage (Moller et al., 2004). Non

placebo controlled prospective studies have also shown antioxidants to improve

sperm DNA integrity (Comhaire et al., 2000; Menezo et al. 2007).

The presence of high levels of sperm DNA damage was not used as a criterion

for entry into the ADAM study, so as to avoid any suggestion that the primary

study outcome (sperm DNA quality) was biased by the “regression to the

mean” phenomenon. Baker and Kovacs (1985) had previously highlighted that

when a group of subjects are selected for extreme results, on average their

result tend to “normalize” on re-testing- a so called “regression to the mean”.

This regression phenomenon is an important consideration when analysing

semen parameters such as sperm count or motility, which are prone to wild

intra-subject variability, but is of minimal importance when considering more

stable parameters such as sperm DNA quality. A previous longitudinal study

(Sergerie et al. 2005) has reported that sperm DNA fragmentation measured

by TUNEL analysis exhibits well within individual stability over time, allowing

for accurate assessment of baseline DNA damage from just a single entry

sample.

The observation that antioxidant therapy was able to significantly reduce

seminal ROS suggests that participants received a biologically adequate dose of

antioxidant to positively influence the sperm DNA environment. Aside from a

reduction in direct oxidative damage to the sperm, the results of this study

suggest two other important mechanisms by which the Menevit® antioxidant

may improve sperm DNA integrity. Firstly, a very significant reduction in sperm

apoptosis was observed while on antioxidant therapy. Oxidative stress is

known to initiate apoptosis (Moustafa et al., 2004), which in turn will ultimately

lead to endonuclease fragmentation of the sperm DNA. It is likely that by

reducing free radical attack on “good quality” sperm, less of these sperm will

be forced down the path of abortive apoptotic DNA fragmentation. Secondly,

the Menevit® antioxidant was shown to produce a small but significant

improvement in sperm DNA protamination, making the sperm DNA more

101

impregnable to ROS attack. To the best of my knowledge, the ADAM study is

the first of its kind to show that sperm protamination and apoptosis can be

positively influenced by the use of an oral antioxidant/ mineral supplement.

A previous study using a combinational antioxidant supplement (Vitamin C 400

mg, Vitamin E 400 mg, β-carotene 18mg, Zinc 500 µmol, Selenium 1 µmol) has

reported that antioxidants may adversely effect sperm chromatin condensation,

possibly mediated by Vitamin C interfering with inter-chain disulphide linkages

in protamines (Menezo et al. 2007). These authors warn against the use of

antioxidants in men with high degrees of sperm nuclear decondensation as

protamine deficiency is linked with sperm premature chromosomal

condensation and fertilization failure (Nasr-Esfahani 2004). It is possible that

the lower dose of Vitamin C contained in the Menevit® supplement, together

with the higher doses of zinc and selenium, may be responsible for the

observed small improvement in sperm protamination compared to those

observed in the Menezo study. A previous placebo controlled study (Tremellen

et al. 2007) has reported that the use of the Menevit® antioxidant slightly

improves ICSI fertilization rates when compared to placebo or patients own

historical control IVF-ICSI cycles, making a significant negative effect of this

antioxidant combination therapy on sperm nuclear condensation highly

improbable. This is also supported by the good fertilization rates observed in

the ADAM patients who did undergo IVF-ICSI treatment.

The active ingredients responsible for the observed improvement in sperm

protamine content are most likely to be zinc and selenium as both have been

associated with the process of sperm protamination. A link between zinc

deficiency in men and abnormal sperm DNA condensation was first noted by

Kvist et al., (1987). Similarly, rodents fed a zinc deficient diet have been

reported to have abnormal sperm chromatin packaging and a resulting increase

in sperm DNA damage (Evenson et al., 1993). Zinc enhances sperm DNA

integrity by augmenting protamine 2 binding to the sperm DNA, thereby

making the sperm less susceptible to oxidative attack (Brewer et al., 2002).

Selenium is also an important cofactor in sperm DNA protamine condensation,

with a 35 kDa seleno-protein being highly expressed late in spermatogenesis,

and is felt to play an important role in disulphide cross-linking of protamines

(Pfeifer et al., 2001). As a deficiency in zinc and selenium is not uncommon

within infertile men (Ebisch et al., 2007), one can speculate that

supplementation with zinc and selenium may have increased sperm protamine

102

related DNA condensation, thereby making sperm less susceptible to oxidative

DNA damage.

The Menevit® anti-oxidant treatment had no significant effect on sperm count,

motility or morphology. Previous small non-controlled studies have reported

that lycopene (Gupta and Kumar 2002), vitamin C and E (several studies

reviewed in Agarwal 2004), zinc and folate (Wong et al., 2002) can improve

sperm parameters such as count, motility and morphology. Four previous

double-blind RCT’s have examined the effect of anti-oxidant preparations

(vitamin C, vitamin E, selenium combinations) on sperm parameters. Two

(Greco et al., 2005, Rolf et al., 1999) found no significant effect of anti-

oxidants on sperm count, motility or morphology which is consistent with my

observations. Conversely, the remaining two RCT’s found a small but

significant improvement in sperm motility but no improvement in sperm count

or morphology (Suleiman et al., 1996; Keskes-Ammar et al., 2003).

Several researchers have identified a significant correlation between sperm

count, motility and morphology and sperm DNA integrity (Tremellen 2008).

Therefore it is uncertain why this study did not observe an improvement in

these routine sperm parameters with antioxidant treatment, when sperm DNA

integrity did improved. There are several possible explanations for this. Firstly,

it is possible that sperm DNA is more resistant to ROS attack than the sperm

membrane/ mitochondria (determinants of motility). If oxidative attack is

reduced by antioxidant therapy, yet not totally resolved, the levels of available

ROS may no longer be sufficient to damage the DNA but are still capable of

decreasing motility. Secondly, while previous studies have linked poor routine

sperm parameters (count, motility and morphology) with high levels of ROS

production, an association does not prove causation. Without a cause and

effect link, antioxidant therapy can not be expected to normalize all sperm

parameters. For example, it has been well documented that excessive residual

cytoplasm (abnormal morphology) is linked with an increase in the generation

of ROS within sperm because of the availability of more cytoplasmic glucose-6-

phosphate dehydrogenase activity (Said et al., 2005). Here abnormal

morphology causes oxidative stress. However, while the use of antioxidants will

reduce ROS within the sperm, possibly reducing DNA damage, they can not be

expected to normalize sperm morphology. While the above statements are

speculative, what is clear is that the majority of good quality in vivo studies do

not show antioxidant therapy to have any material effect on routine sperm

103

parameters (Tremellen 2008), an observation which is consistent with my

results.

Three months antioxidant treatment had no significant effect on serum

hormone levels, suggesting no change in production by either the testicular

Leydig (LH, Testosterone) or Sertoli cells (FSH, AMH). Some investigators have

speculated that oxidative damage to the Leydig cell LH receptor is responsible

for the gradual decline in serum testosterone observed with increasing male

age (Hardy and Schlegel 2004). If this is the case, one would have expected to

see some increase in serum testosterone or a drop in LH concentration, yet no

such changes were observed. This lack of effect of the Menevit® antioxidant on

reproductive hormones is consistent with the few examples within the existing

literature. Comhaire et al., (2005) reported no change in serum LH, FSH or

testosterone following 3 months therapy with a potent carotenoid antioxidant

(Astaxanthin®), despite recording a very significant fall in seminal ROS

production. Similarly, a double blind randomized, placebo controlled trial using

a combination of zinc and folate reported no significant changes in serum

testosterone or FSH (Wong et al., 2002).

In conclusion, the results of the ADAM study suggest that previously reported

improvements in pregnancy outcome during IVF-ICSI treatment using the

Menevit® antioxidant (Tremellen et al., 2007) was most likely mediated by

significant improvements in sperm DNA integrity. The current data suggest that

the observed reduction in sperm DNA fragmentation is the net effect of a

reduction in ROS attack, a reduction in DNA susceptibility to ROS damage

because of improved sperm DNA protamine packaging, and a decline in ROS

initiated apoptosis. To the best of my knowledge, the ADAM study is the first

study to describe the ability of an oral antioxidant / mineral supplement to

enhance sperm DNA protamination while reducing sperm apoptosis.

This study adds to the growing body of evidence supporting the use of

antioxidant combinational therapy to improve sperm DNA integrity (Comhaire

et al., 2000; Greco et al., 2005; Menezo et al. 2007), especially for those men

undergoing IVF-ICSI treatment. Bypassing the “quality control” of natural

fertilization by the use of ICSI enables fertilization to occur even in the

presence of severely damaged sperm DNA, placing patients at significantly

increased risk of miscarriage (Virro et al., 2004; Borini et al., 2006; Benchaib

et al., 2007). The clinical miscarriage rate observed in this study cohort was

104

similar to that reported for fertile couples with no underlying sperm DNA

quality issues (Hassold and Chiu 1985). This observation suggests that

treatment of men with high degrees of oxidative DNA damage with

antioxidants before their partner commences IVF-ICSI therapy may be capable

of improving pregnancy outcomes.

105

CHAPTER 7

Oxidative Stress as a cause of sperm DNA Hypomethylation

106

7.1 Abstract

Methylation of sperm DNA is impaired in many infertile men;

potentially adversely effecting reproductive outcomes. In somatic cells

oxidative damage to DNA and hyperhomocysteinaemia are linked with

DNA hypomethylation. The objective of this study was to investigate if

these pathologies also impair sperm DNA methylation. The

relationship between sperm DNA quality, oxidative stress and serum

homocysteine was analysed at study entry and after 3 months of

antioxidant treatment.

Overall a significant negative correlation was observed between sperm

DNA methylation and sperm DNA fragmentation, as well as seminal

reactive oxygen species (ROS) production. Sperm DNA methylation

was not significantly related to serum homocysteine concentrations.

Administration of an antioxidant supplement produced a significant fall

in seminal ROS levels and sperm DNA fragmentation, while increasing

sperm DNA methylation.

These results suggest that oxidative stress related damage to sperm

DNA impedes the process of methylation, while antioxidant

supplementation appears to have the potential to reduce DNA damage

and normalize sperm DNA methylation.

107

7.2 Introduction

It is currently widely accepted that oxidative stress is a common cause for

sperm DNA damage. However, the effect of oxidative stress on the paternal

genome epigenetic expression is currently unknown. As oxidative stress has

been linked with altered epigenetic programming in somatic cells, it was

thought to be useful to explore this possible link in sperm.

Since reports emerged linking IVF conceived pregnancies with the epigenetic

disorders Angelman and Beckwith-Wiedemann Syndromes (Cox et al., 2002;

DeBaun et al., 2003; Gosden et al., 2003; Lawrence and Moley, 2008), there

has been intense scientific interest in epigenetic processes involved in

reproduction. Disorders of genomic imprinting occur when there is a failure of

the correct pattern of DNA parental-origin-dependant monoallelic gene

expression (Lawrence and Moley, 2008). The methylation of cytosine residues

in DNA by DNA methyltransferase is considered to be one of the major

epigenetic mechanisms controlling gene expression and imprinting (Schaefer et

al., 2007). Hypomethylation of DNA is associated with gene transcriptional

activity while hypermethylation is associated with gene silencing.

While several studies have suggested that adverse embryo culture

environments are responsible for the majority of epigenetic defects observed in

IVF conceived pregnancies (DeBaun et al., 2003; Lawrence and Moley, 2008;

Thompson et al., 2002), other observations have suggested that sperm

abnormalities may also play a role. Firstly, investigators have reported that

aberrant sperm DNA methylation, principally in the form of hypomethylation, is

more commonly seen in infertile men compared to their normozoospermic

counterparts (Benchaib et al., 2003a; Houshdaran et al., 2007; Kobayashi et

al., 2007; Marques et al., 2004; Marques et al., 2008). Secondly, experimental

inhibition of sperm epigenetic programming by exposure of male rodents to

endocrine disruptors results in reduced sperm fertilization capacity, altered

embryonic gene expression, an increase in pre-implantation embryonic loss

(Doerksen et al., 2000; Doerksen and Trasler, 1996; Kelly et al., 2003; Oakes

et al., 2007; Pathak et al., 2008) and cancer in future generations (Anway et

al., 2005; Anway and Skinner, 2008). Finally, studies have also linked sperm

DNA hypomethylation in men with a reduction in IVF pregnancy rates

(Benchaib et al., 2005; Cisneros, 2004; Kobayashi et al., 2007). Therefore,

108

infertility related anomalies in sperm epigenetic programming may have

serious clinical consequences and certainly warrants further investigation.

Epigenetic programming of sperm DNA occurs at several key stages in the

spermatogenesis cycle. In rodents it is reported that DNA methyltransferase 1

(DMT1) mRNA and protein are expressed at high levels in mitotic and early

meiotic male germ cells, with the enzyme then being translationally down

regulated in pachytene spermatocytes (Jue et al., 1995; Numata et al., 1994;

Trasler et al., 1992). Human studies have demonstrated that the paternally

imprinted gene H19 has its methylation pattern erased in early fetal life; with

remethylation being initiated as spermatogonia enter meiosis and is effectively

complete by the primary spermatocyte stage of differentiation (Kerjean et al.,

2000). Testicular tissue samples taken from fertile men show DMT1 gene

expression is restricted to spermatogonia, pachytene spermatocytes and round

spermatids; with DMT1 protein only being expressed in the nucleus of

spermatogonia and in the cytoplasm of round spermatids (Omisanjo et al.,

2007). Interestingly, Ariel et al reported that spermatogenesis-specific genes

can also undergo quite late epigenetic re-programming while maturing within

the epididymis (Ariel et al., 1994). Therefore, pathology within both the

testicular and epididymal environment has the potential to disrupt the

establishment of normal sperm DNA methylation patterns.

To date only one study has reported on any underlying mechanism for

abnormal sperm DNA methylation seen in infertile men (Kobayashi et al.,

2009). This study identified DNA sequence variations in the gene encoding the

DNA methyltransferase enzyme DNMT3L in several infertile men, which inturn

was associated with abnormal paternal DNA methylation. However, studies

investigating the link between somatic cell DNA hypomethylation and cancer

have suggested other possible mechanisms for sperm DNA hypomethylation

may exist such as defects in the folate / homocysteine pathway and oxidative

stress.

Oxidative stress may predispose to epigenetic abnormality as oxidative attack

leads to the generation of DNA strand breaks and the formation of DNA base

adducts such as 8-hydroxyl-2’-deoxyguanosine (8-OH-dG) and O6-

methylguanine, both reported to interfere with the DNA’s ability to act as a

substrate for DNA methyltransferases (Franco et al., 2008). The presence of 8-

OHdG in CpG dinucleotide sequences (Turk et al., 1995; Valinluck et al., 2004;

109

Weitzman et al., 1994) or O6-methylguanine (Hepburn et al., 1991), strongly

inhibits methylation of adjacent cytosine residues and ultimately leads to global

DNA hypomethylation.

The folate/homocysteine pathway is responsible for the generation of methyl

donors and therefore is central to the process of DNA methylation for all cells

(Yi et al., 2000). Defects in the folate cycle are responsible for depleting S-

adenosylmethionine (SAM) levels and have been linked with DNA

hypomethylation in somatic cells (Jamaluddin et al., 2007; Yi et al., 2000).

Methylenetetrahydrofolate reductase (MTHFR) is a key folate-metabolizing

enzyme which catalyses the conversion of 5,10-methylene tetrahydrofolate to

5-methyl tetrahydrofolate, the later which provides methyl groups for the

methionine synthase-mediated remethylation of homocysteine to methionine.

Studies in mice have linked polymorphisms in the MTHFR gene with reduced

availability of methionine / SAM resulting in hypomethylation of testicular DNA

(Chen et al., 2001). Interestingly, while polymorphisms in the MTHFR gene

limiting its enzymatic activity are more common in infertile men (Bezold et al.,

2001; Paracchini et al., 2006; Singh et al., 2005; Stuppia et al., 2003), no

study has yet examined the link between defects in the folate pathway and

sperm DNA methylation.

Since oxidative stress and hyper-homocysteinaemia have been linked with

somatic cell DNA hypomethylation, it was proposed that similar mechanisms

may operate in sperm. The principal aim of this study was to investigate the

possible link between seminal oxidative stress related DNA fragmentation,

serum homocysteine and methylation of sperm DNA.



Participants in the study were recruited from men with known male factor

infertility (“infertile subjects”) defined as the presence of abnormal WHO

semen quality criteria (WHO 1999) and the inability to conceive despite more

than 12 months of unprotected intercourse. “Fertile controls” were men who

were acting as sperm donors at an academic affiliated ART unit (Repromed,

South Australia) and who had proven fertility within the last 12 months and

normal semen parameters according to WHO criteria. All infertile participants

110

were asked to take one capsule of Menevit® (Bayer Australia Ltd, Sydney,

Australia) per day for a period of 3 months and to provide a semen and serum

sample both at study entry and exit. Three months duration of antioxidant

therapy was considered appropriate as this would cover one full

spermatogenesis cycle (~ 70 days). Each capsule of Menevit contained 500 µg

of folate and various anti-oxidants (Vitamin C 100 mg, Vitamin E 100 IU,

Lycopene 6 mg, zinc 25 mg, selenium 26 µg, and garlic oil 333 µg). Fifty men

entered the study, with 5 withdrawing over the next 3 months due to a lack of

continuing interest (4) or perceived side effects (1). The twelve “fertile

controls” produced only a single semen sample for the purposes of this study

and were not administered any antioxidant therapy. The study was

prospectively approved by the Human Research and Ethics Committee,

Women’s and Children’s Hospital, with all participants giving written informed

consent for their involvement.




Data were analysed using GraphPad Prism (GraphPad Software Inc.,La Jolla,

CA,USA). Correlations between variables were analysed using the Spearman

Rank Order correlation test. Sperm quality parameters before and after

antioxidant therapy were expressed as mean ± SD or median (inter-quartile

ranges) and analysed using the pair t-test or Wilcoxon Signed Rank test,

depending on whether the data followed a normal distribution. A P value <

0.05 was considered statistically significant.

7.4 Results

Table 7.1 depicts sperm quality parameters for both the fertile and infertile

groups. As only men with male factor infertility were recruited into the study, it

was expected that the infertile group would have lower sperm quality than the

fertile controls. There was a statistically significantly increased level of sperm

DNA fragmentation (TUNEL) and semen reactive oxygen species production

(NBT) in the infertile group compared to the fertile men. The relationship

between sperm global DNA methylation and semen parameters such as sperm

concentration (r= -0.330, p=0.0289) and morphology (r= -0.394, p=0.008)

111

was significant, but no significant relationship was observed between levels of

DNA methylation and sperm motility (r=0.075, p=0.624) or leukocyte

concentration (r= -.21, p= 0.12).

Table 7.1 Comparison of sperm quality between the infertile and fertile

study groups

SPERM QUALITY PARAMETERS

FERTILE n= 12

INFERTILE n=45

p

Sperm Concentration (x106/mL)

122.1± 79.8 36.17± 46.2 < 0.0001

Motility (%)

52.6 ± 7.0 36.5 ± 12.3 < 0.0001

Normal Morphology (%)

21.0 ± 6.6 6.7 ± 5.0 < 0.0001

CD 45 (x106 cell/ml)

0.89 ± 0.51 1.53 ± 1.30 0.136

ROS Production (mg Formazan/mL)

15.4 ± 12.1 77.8 ± 58.9 0.0006

DNA Fragmentation (TUNEL Positive %)

10.3 ± 3.8 22.9 ± 9.1 < 0.0001

Sperm Global DNA

Methylation (au)

104.7± 11.3 93.96±30.99 0.095

All values are expressed as mean ± standard deviation and analysed using the

unpaired t-test

The results in Figure 7.1A illustrate the correlation between levels of sperm

DNA methylation and DNA fragmentation (TUNEL). It can be seen that a

significant negative correlation exists between these two parameters (r=-

0.311, p=0.02), supporting a possible link between DNA damage and impaired

capacity for methylation. The observation of a similar statistically significant

negative correlation between sperm DNA methylation and seminal ROS

production (r= -0.378, p=0.004; Figure 7.1B), together with a positive

correlation between TUNEL and NBT results (r=0.336, p=0.022) suggests that

oxidative damage to sperm DNA is at least in part responsible for modifying

sperm global DNA hypomethylation.

112

Figure 7.1A: Relationship between Sperm DNA fragmentation

(TUNEL) and Global DNA Methylation.

Figure 7.1B: Relationship between seminal ROS production and

Global DNA Methylation

0 50 100

150

200

0

10

20

30

40

50

r = -0.311p = 0.02

Global DNA methylation(au)

DN

A F

rag

men

tati

on

(T

UN

EL

Po

sit

ive %

)

0 50 100 150 2000

100

200

300

400

r = -0.378p = 0.004

Global DNA methylation(au)

RO

S P

rod

uctio

n

(F

orm

aza

nµµ µµ

g/1

07 s

perm

)

113

No significant correlation was observed between serum homocysteine and

global sperm DNA methylation in the infertile cohort (r=0.26, p=0.09).

Furthermore, sperm global DNA methylation levels did not differ significantly

between those infertile men with “normal” serum homocysteine range (<11.4

mmol/L - 95th percentile in folate replete men aged 20-45 years)(Selhub et al.,

1999) and those with hyper-homocysteinemia (mean sperm DNA methylation

91.9 ± 29.7 v 94.8 ± 35.9 a.u. respectively, p=0.378).

Three months supplementation with antioxidant produced a significant fall in

seminal ROS levels and DNA damage (TUNEL), combined with an increase in

sperm global DNA hypomethylation (Table 7.2). Sperm count, motility and

morphology did not change significantly over the 3 month supplementation

period, while serum homocysteine levels decreased a small but statistically

significant amount.

Table 7.2: Sperm quality before and after 3 months of anti-oxidant

supplementation

SPERM QUALITY

PARAMETERS n = 45

Pre-antioxidant supplementation

On anti-oxidant supplementation

P

Sperm

Concentration (x106/mL)

21.7(12.08 – 38.33) 21.4(12.75–44.1) 0.304

Motility (%)

36.5 ± 12.3 37.1 ± 11.5 0.778

Normal

Morphology (%)

6.7 ± 5.0 6.71 ± 4.5 0.938

Homocysteine (µmol/L)

10.00 (9.13–11.68) 9.38 (8.50–10.90) 0.001

ROS Production (µg Formazan/mL)

66.4 (43.5–88.0) 44.4 (33.3 – 81.4) 0.027

DNA

Fragmentation (TUNEL Positive%)

22.6 (16.5 – 28.8) 18.2 (13.4 – 23.1) 0.002

Sperm Global

DNA Methylation (a.u.)

93.96 ± 30.99 108.61± 35.54 0.0017

114

7.5 Discussion

While several papers have now reported that infertile men’s sperm are more

likely to express aberrant DNA methylation patterns (Benchaib et al., 2003a;

Benchaib et al., 2003b; Houshdaran et al., 2007; Kobayashi et al., 2007;

Marques et al., 2004; Marques et al., 2008), this study is one of the first to

report on the underlying mechanisms behind such observations. The results of

this study suggest that oxidative damage to sperm DNA integrity inhibits

methylation, while abnormalities in the methyl donor pathway do not appear to

play a significant role in sperm DNA methylation. These results are consistent

with the small body of evidence already existing. Firstly, a recent report

(Tavalaee et al., 2008) also found a statistically significant negative correlation

between sperm DNA fragmentation, as assessed by the Sperm Chromatin

dispersion Test, and sperm DNA methylation. A similar negative trend

(r = -0.45, P> 0.05) had earlier been reported between sperm DNA

fragmentation (TUNEL) and methylation (Benchaib et al., 2005). Unfortunately

neither group of researchers measured seminal ROS levels in their study,

making it impossible to confirm that oxidative stress was the underlying cause

of the observed DNA damage/hypomethylation. Interestingly, Tavalaee et al

(2009) explained their results by suggesting that hypomethylated sperm may

be more prone to DNA damage (Tavalaee et al., 2009). While it is possible that

normally methylated DNA may be less susceptible to DNA damage, I are

unaware of any evidence supporting the concept that methylation of DNA

protects it from apoptotic or oxidative damage, the two principal causes of

sperm DNA damage (Aitken, 2004). Furthermore, Tavalaee et al (Tavalaee et

al., 2008) observed no link between protamination of the sperm (CMA3

staining) and global DNA methylation, making a non specific defect in

chromatin remodelling unlikely. As oxidative stress effects a significant

proportion of infertile men (Tremellen, 2008) and is widely believed to be the

primary cause of sperm DNA fragmentation (Aitken, 2004; Aitken and De Iuliis,

2007), it is more likely that oxidative DNA damage played some role in sperm

DNA hypomethylation reported in these earlier studies.

The link between oxidative DNA damage and hypomethylation is already

established for somatic cells, with several investigators reporting a link

115

between the presences of oxidative DNA adducts in somatic cells and impaired

DNA methyltrasferase activity (Turk et al., 1995; Weitzman et al., 1994).

Furthermore, incorporation of 8-OHdG in the methyl-CpG binding protein (MBP)

recognition sequence has been reported to result in significant inhibition of MBP

binding, further impeding the process of DNA methylation (Valinluck et al.,

2004). The observation of a statistically significant negative correlation

between sperm DNA methylation and seminal ROS production, together with a

strong positive correlation between TUNEL and NBT results strongly suggests

that oxidative damage to sperm DNA is responsible for sperm global DNA

hypomethylation. Furthermore, the observed improvement in sperm DNA

methylation with 3 months antioxidant therapy also suggests that oxidative

damage to sperm impairs DNA methylation. However, the link between sperm

oxidative DNA damage and hypomethylation may best be confirmed by studies

correlating the generation of the oxidative specific DNA base adduct 8-

hydroxyl-2’-deoxyguanosine (8-OH-dG) with sperm DNA methylation.

While this study did observe a very significant correlation between total semen

ROS production and sperm DNA methylation, no significant correlation was

observed between semen leukocyte concentration and sperm DNA methylation.

This would imply that sperm themselves, not seminal leukocytes, are the

primary source of ROS production interfering with the DNA methylation

process. As none of the men in this study had any history suggestive of active

genital tract infection, it is likely that the seminal leukocytes were relatively

inactive and therefore not a dominant source of ROS production. Furthermore,

it makes biological sense that intrinsic ROS production within the sperm

cytoplasm is more likely to interfere with the process of sperm DNA

methylation in the adjacent nucleus than extrinsic ROS released into the extra-

cellular environment by leukocytes.

A weakness of this study was that it did not include a concurrent placebo

control in the infertile subgroup. Some sperm parameters such as

concentration and motility are prone to large fluctuations on different sampling

occasions, even within the same individual, and therefore have a tendency for

spontaneous “improvement” if subjects are recruited into a trial based on an

initial low result. This non-treatment related improvement in sperm quality

over time is termed “regression to the mean”. Conversely, failure to see

significant fluctuations in sperm quality in a placebo control group over time

suggests that regression to the mean is not an important determinant for that

116

particular sperm parameter. As there was not a placebo control in the infertile

group, one can not state for certain that the observed improvements in sperm

DNA methylation over three months of antioxidant therapy are not the result of

spontaneous “regression to the mean”. However, since infertile participants

were not selected for enrolment in the trial based on low initial sperm DNA

methylation results, regression to the mean is unlikely to be a major cause for

the observed improvement in sperm DNA methylation. Furthermore, the

observed significant correlation between ROS production and sperm DNA

methylation suggests a true biological cause-effect association between

oxidative stress and impaired sperm DNA methylation.

The observation of no significant correlation between serum homocysteine and

sperm DNA methylation does not support a significant role for abnormalities in

the folate/ homocysteine pathway as a cause of sperm DNA hypomethylation.

Infertile men are more prone to inefficient folate cycle reconversion of

homocysteine to methionine as polymorphisms in their MTHFR gene are more

common (Bezold et al., 2001; Dhillon et al., 2007; Lee et al., 2006; Paracchini

et al., 2006; Singh et al., 2005; Stuppia et al., 2003), resulting in an up to

70% reduction in MTHFR activity. However, the lack of a significant negative

correlation between sperm DNA methylation and homocysteine makes

mutations in the MTHFR gene and hyperhomocysteinaemia unlikely candidates

for causing sperm DNA hypomethylation in infertile men. A weakness of this

study was that it did not specifically target men with MTHFR homozygous

mutations and poor dietary folate intake who are likely to have extremely high

levels of serum homocysteine. Such extreme abnormalities in homocysteine

metabolism may still potentially be associated with alterations in sperm DNA

methylation, despite the evidence suggesting that minor elevations in

homocystine do not impact on sperm DNA methylation status.

The clinical implications of impaired sperm DNA methylation are presently

uncertain. Animal studies have shown that chemically blocking sperm DNA

methylation results in reduced sperm fertilization capacity, altered embryonic

gene expression and an increase in pre-implantation embryonic loss (Doerksen

et al., 2000; Doerksen and Trasler, 1996; Kelly et al., 2003; Oakes et al.,

2007; Pathak et al., 2008) . Furthermore, the creation of mouse embryos using

sperm with high degrees of DNA damage has been shown to result in

epigenetic abnormalities in the resulting progeny with major physical and

behavioural abnormalities later in life (Fernandez-Gonzalez et al., 2008).

117

Aitken and De Iuliis have speculated that aberrant sperm DNA methylation may

also lead to epigenetic defects that adversely affecting the health of the next

generation of children (Aitken and De Iuliis, 2007). Imprinting disorders such

as Beckwith-Wiedermann and Angelman syndromes are relatively rare, making

epidemiological linkage between infertility and the development of these

imprinting disorders extremely difficult. As such, the link between sperm DNA

methylation and the development of childhood imprinting disorders is still far

from proven. Russell-Silver Syndrome, a rare disorder characterized by growth

restriction, limb and facial anomalies and learning difficulties has been shown

to be primarily caused by hypomethylation on the paternal allele of DMR1 at

11p15 (Gicquel et al., 2005; Netchine et al., 2007) . It is interesting to note

that hypomethylation of the 11p15 DMR1 has also been reported to be more

common in oligospermic infertile men (Marques et al., 2004), and at least one

case report has linked the use of IVF-ICSI for severe male factor infertility with

the development of Russell-Silver Syndrome. However, the rare association of

IVF-ICSI treatment with any form of imprinting syndrome (Amor and Halliday,

2008) suggests that aberrant sperm DNA methylation is not a significant cause

for any classical imprinting syndrome.

The majority of human studies have suggested a negative link between sperm

DNA methylation status and chances of pregnancy (Benchaib et al., 2005;

Cisneros, 2004; Kobayashi et al., 2007), with only one study failing to report

such a link (Tavalaee et al., 2008). A complicating factor in determining the

direct effect of sperm DNA methylation on pregnancy outcome is its positive

association with sperm DNA integrity (Benchaib et al., 2005; Tavalaee et al.,

2009). Since sperm DNA fragmentation is clearly linked with pregnancy

outcome (Zini et al., 2008), it is virtually impossible to determine if sperm DNA

fragmentation alone or hypomethylation are primarily responsible for

pregnancy outcome. Future experiments correlating sperm DNA methylation

status with the embryonic epigenetic profile may shed light on the role that

sperm DNA methylation plays in early embryo development.

The results of this study suggest that antioxidant supplementation in infertile

men can result in significant improvements in sperm DNA integrity and

methylation. Improvements in sperm DNA integrity with antioxidant therapy

has been reported by many previous investigators, yet the ability of

antioxidants to boost pregnancy rates is still under considerable debate

(Tremellen, 2008). Antioxidant supplements will not be capable of normalizing

sperm DNA hypomethylation in all infertile men as in some individuals

118

hypomethylation of individual gene loci is related to mutations within the

DNMT3L gene (Kobayashi et al., 2009), not oxidative stress. Furthermore,

while the role that impaired sperm DNA integrity and methylation plays in the

health of the next generation has yet to be determined, one can speculate that

antioxidant mediated improvements in sperm DNA quality has at least the

potential to benefit reproductive outcomes (Zini et al., 2008). Large

prospective studies correlating sperm DNA quality in the insemination sample

with epigenetic profiles and the heath outcomes in the resulting children are

urgently required. If these studies do confirm a link between poor sperm DNA

quality and adverse child health outcomes, pre-conception anti-oxidant

supplements may become standard clinical practice, just as pre-conception

folate supplementation is the standard for women. Until these studies are

conducted, the absolute value of male pre-conception antioxidant

supplementation will be unknown.

119

CHAPTER 8

Final Discussion and Conclusions

120

8.1 Discussion

As seminal oxidative stress is a common pathology effecting sperm

function in many infertile men, it is important to know the underlying

causes of seminal oxidative stress as this may lead to more effective

targeted therapy. The aim of this thesis was to advance our

understanding of the causes of Oxidative stress in infertile men as well

as examine the effectiveness of antioxidant treatment to boost fertility

potential in the infertile male population.

Previous work by other investigators has identified many underlying

causes for oxidative stress such as local infection in the reproductive

tract, systemic diseases such as diabetes and life style factors such as

smoking. The research in this thesis has identified a new cause of

seminal oxidative stress, obesity. The work described in Chapter 5 has

highlighted for the first time that obesity results in an increase in

seminal ROS production and an infiltration of macrophages into the

semen. Obesity has previously been shown to be linked with activation

of macrophages in adipose tissue and the vascular system and therefore

it is not surprising that this inflammation extends to the reproductive

tract. What will be interesting to see in the future is whether loss of

weight can result in a decrease in seminal macrophage activity and ROS

production. It is our intent to conduct these studies in the future.

The studies described in this thesis outline the development of a new

method for measurement of oxidative stress in semen. While the NBT is

not a new test of cell ROS production, these studies have modified the

test to allow for quantitative assessment of seminal ROS production.

Sperm cells can generate reactive oxygen species as they contain

glucose-6-phosphate dehydrogenase in their cytoplasm. This enzyme

uses glucose to produce β-nicotinamide adenine dinucleotide phosphate

(NADPH) via the hexose monophosphate shunt and generate superoxide

radical. The NBT method developed in chapter 3 is based on the ability

of the superoxide anion to reduce yellow water-soluble tetrazolium salt

121

(NBT) to a blue water-insoluble formazan derivative, which can be

solubilized and quantified by spectrophotometrically at 630 nm. Seminal

ROS production quantified by the NBT assay has for the the first time

been shown to have a significant correlation with markers of sperm

quality such as motility and DNA fragmentation. In light of these

findings I believe that the NBT assay has the potential to be used by

many clinical andrology laboratories as a screening test for oxidative

stress related male infertility, especially as the NBT assay has been

shown to be accurate, easy to establish and low cost.

Sperm cells along with leukocytes are the main ROS producers in

semen and their contribution to the generation of oxidative stress is

dominant in the absence of infection. The studies in this thesis found no

significant correlation between seminal leukocyte markers (PMN

elastase and CD 45) and markers of oxidative stress (NBT, Lipid

Peroxidase-LPO) in infertile men’s semen. This is not surprising as none

of the men had any physical signs or symptoms of genital tract infection

that would have resulted in activation of an inflammatory response.

However, chronic low grade genital tract inflammation is significantly

more common in the infertile male population than a fertile cohort, with

many of these men being asymptomatic (Haidl et al., 2008; Schuppe et

al., 2008). In my study I was anticipating to find a significant

correlation between seminal plasma PMN Elastase activity and ROS

production, but no such correlation was observed between these two

parameters. This may result from having low numbers of

leukocytospermic patients in my study cohort or it may reflect that

neutrophils are not the primary leukocyte type contributing to ROS

production in semen. While neutrophils are the most common leukocyte

contained in semen, accounting for up to 70% of seminal leukocytes,

macrophages also are prevalent in semen (~30% of leukocytes).

Accordingly I aimed to examine if macrophages were a significant

source of ROS production in seminal plasma by correlating the

concentration of Neopterin in seminal plasma, an established marker for

macrophage activity, with ROS levels. This study was the first to report

122

the presence of the macrophage activity marker neopterin in human

seminal plasma.

Furthermore, the studies outlined in chapter 4 show for the first time

that seminal plasma neopterin levels correlated better with semen ROS

production than the more traditionally assessed seminal plasma

elastase. Neopterin levels in seminal plasma of infertile men was nearly

double that seen in the fertile cohort. Seminal plasma elastase and CD

45 levels were also present at significantly higher concentrations in the

infertile group. While seminal plasma PMN elastase levels show no

correlation with any marker of semen quality aside from leukocyte

(CD45) concentration, neopterin level was significantly correlated with

sperm concentration, morphology, sperm DNA fragmentation and

apoptosis. There was also a strong positive correlation between

neopterin concentration and markers of sperm oxidative stress such as

LPO and NBT assay. Overall this suggests that measuring macrophage

activity in seminal plasma using the neopterin assay is a more useful

marker of sperm quality than the more traditionally used inflammatory

markers seminal plasma elastase and leukocyte concentration (CD45).

Two possibilities exist for the underlying cause of a macrophage

infilitration into semen. Firstly, it could represent a normal inflammatory

response to a pathogenic stimulus such as low grade epididymal

infection. Secondly, the macrophage influx may be a beneficial

“physiological” response in which the body is attempting to remove

dead or defective sperm. The studies in chapter 4 outline for the first

time a significant correlation between seminal plasma neopterin and

sperm abnormal morphology and apoptosis which suggests this second

“physiological” role for macrophages may be dominant.

Oxidative stress is known to produce sperm DNA fragmentation which in

turn results in increased time to natural conception and miscarriage

risk. Furthermore, decreased IVF success rates and even a possible

increased risk of childhood cancer have been linked with increased

sperm DNA damage. Several inter-related mechanisms have been

123

postulated as a cause of DNA damage in sperm cells. These are

oxidative stress, abortive apoptosis and abnormal sperm protamination.

During spermatogenesis, when the apoptotic destruction of sperm cells

is failed, damaged cells can not be completely destroyed. This abortive

apoptotic state can be triggered by oxidative stress and DNA will then

be damaged by endonucleases mediated digestion. As can be seen in

Chapter 3, the link between ROS and early apoptosis with DNA damage

confirm this postulate. The infertile group had three times more ROS

production and two times more DNA damage and apoptosis than the

fertile cohort.

The replacing of nuclear histones with protamines during normal

spermatogenesis allows a tight packaging of the sperm DNA which

protects the defective sperm from oxidative attack. Infertile men more

commonly have defective protamination of their DNA, making these

sperm cells more susceptible to ROS damage. Approximately 15% of

infertile men have been reported to exhibit deficient protamine

packaging (Carrell and Liu, 2001). The results in chapter 6 show that

sperm protamination can be improved by antioxidant treatments. While

several previous studies have reported that antioxidants can reduce

sperm DNA damage, the study outlined in chapter 7 has shown for the

first time that a combination of antioxidants and minerals such as zinc

and selenium, vital to protamination processes, can also boost sperm

DNA protamine packaging, thereby improving sperm DNA integrity.

These results help explain the molecular mechanisms behind how the

Menevit antioxidant preparation improved pregnancy outcomes during

IVF/ICSI treatment in a peviously reported placebo controlled study

(Tremellen et al., 2007).

The contribution of epigenetic processes in reproduction is a subject of

considerable scientific and clinical interest at present. While oxidative

stress has already been linked with altered epigenetic programming in

somatic cells, the effect of oxidative stress on the sperm paternal

124

genome epigenetic expression was unknown before the conduct of

experiments outlined in chapter 7.

Methylation of cytosine residues in DNA by DNA methyltransferase is

considered to be one of the major epigenetic mechanisms controlling

gene expression. Hypomethylation of the sperm DNA has been reported

to be related with abnormal semen parameters and low fertilization

capability (Benchaib et al., 2003a; Houshdaran et al., 2007). Previous

reports (Benchaib et al., 2005; Tavalaee et al., 2009) have shown a

negative correlation between DNA fragmentation and DNA methylation,

but these investigators did not assess oxidative stress and therefore did

not identify sperm oxidative stress as a cause of DNA hypomethylation.

The research outlined in chapter 7 reveals for the first time a significant

negative correlation between seminal ROS production and sperm DNA

methylation. This observation, together with the fact that sperm DNA

methylation improved with antioxidant therapy, suggests that oxidative

stress is a significant cause of sperm DNA hypomethylation seen in

infertile men. While it is clear that infertile men have aberrant sperm

DNA methylation, it remains to be seen how this may affect the health

of the next generation.

8.2 Future Directions

In order to validate the NBT Assay developed in this thesis for use in

clinical Andrology laboratories there is a need to conduct larger scale

trials, preferably multi-centred. Expansion on the study sample size,

especially the fertile “normal” cohort will hopefully provide more robust

reference ranges. Since publication of my NBT paper in 2009, several

laboratories from around the world have made contact with our

research group and commenced their own testing.

125

Obesity has been shown to create oxidative stress in semen, primarily

from activation if a macrophage inflammatory response in the male

reproductive tract. In the future I would like to investigate if a weight

loss program in infertile men can result in a decrease in seminal

macrophage activity and ROS production, and a subsequent

improvement in sperm quality.

The link observed between sperm oxidative DNA damage and

hypomethylation using the NBT assay will be confirmed by a future

study correlating the generation of the oxidative specific DNA base

adduct 8-hydroxyl-2’-deoxyguanosine (8-OH-dG) with sperm DNA

methylation. Furthermore, correlation between embryonic epigenetic

profiles with sperm DNA methylation will hopefully help shed light on

the importance of sperm DNA quality to the development of the next

generation. Finally, as it has already been shown that sperm DNA

damage can produce miscarriage and possibly affect the health of the

next generation, I would like to correlate sperm DNA quality at the time

of IVF insemination with pregnancy/child health outcomes.

If sperm DNA damage or hypomethylation are conclusively linked with

adverse health outcomes in children, it would be interesting to study if

antioxidant supplementation can prevent these adverse outcomes. The

likely sample size required for such a study is several thousand cycles of

IVF, a formidable administrative task. However, as the health of the

next generation is of utmost importance, I hope that such studies can

be organised in the future.

126

BIBLIOGRAPHY

Acharya, U. R., Acharya, S., and Mishra, M. (2003). Lead acetate induced cytotoxicity in

male germinal cells of Swiss mice. Ind Health 41, 291-294.

Agarwal, A., Allamaneni, S. S., and Said, T. M. (2004). Chemiluminescence technique for

measuring reactive oxygen species. Reprod Biomed Online 9, 466-468.

Agarwal, A., Deepinder, F., Sharma, R. K., Ranga, G., and Li, J. (2008). Effect of cell

phone usage on semen analysis in men attending infertility clinic: an observational study.

Fertil Steril 89, 124-128.

Agarwal, A., Desai, N. R., Makker, K., Varghese, A., Mouradi, R., Sabanegh, E., and

Sharma, R. (2009). Effects of radiofrequency electromagnetic waves (RF-EMW) from

cellular phones on human ejaculated semen: an in vitro pilot study. Fertil Steril 92, 1318-

1325.

Agarwal, A., and Said, T. M. (2004). Carnitines and male infertility. Reprod Biomed

Online 8, 376-384.

Agarwal, A., Said, T. M., Bedaiwy, M. A., Banerjee, J., and Alvarez, J. G. (2006).

Oxidative stress in an assisted reproductive techniques setting. Fertil Steril 86, 503-512.

Agarwal, A., and Saleh, R. A. (2002). Role of oxidants in male infertility: rationale,

significance, and treatment. Urol Clin North Am 29, 817-827.

Agarwal, A., Saleh, R. A., and Bedaiwy, M. A. (2003). Role of reactive oxygen species in

the pathophysiology of human reproduction. Fertil Steril 79, 829-843.

Aggerholm, A. S., Thulstrup, A. M., Toft, G., Ramlau-Hansen, C. H., and Bonde, J. P.

(2008). Is overweight a risk factor for reduced semen quality and altered serum sex

hormone profile? Fertil Steril 90, 619-626.

Ahmadi, A., and Ng, S. C. (1999). Fertilizing ability of DNA-damaged spermatozoa. J Exp

Zool 284, 696-704.

Aitken, J., Krausz, C., and Buckingham, D. (1994). Relationships between biochemical

markers for residual sperm cytoplasm, reactive oxygen species generation, and the

presence of leukocytes and precursor germ cells in human sperm suspensions. Mol Reprod

Dev 39, 268-279.

Aitken, R. J. (1989). The role of free oxygen radicals and sperm function. Int J Androl 12,

95-97.

Aitken, R. J. (2004). Founders' Lecture. Human spermatozoa: fruits of creation, seeds of

doubt. Reprod Fertil Dev 16, 655-664.

Aitken, R. J., and Baker, H. W. (1995). Seminal leukocytes: passengers, terrorists or good

samaritans? Hum Reprod 10, 1736-1739.

Aitken, R. J., Baker, M. A., and O'Bryan, M. (2004). Shedding light on

chemiluminescence: the application of chemiluminescence in diagnostic andrology. J

Androl 25, 455-465.

127

Aitken, R. J., Bennetts, L. E., Sawyer, D., Wiklendt, A. M., and King, B. V. (2005). Impact

of radio frequency electromagnetic radiation on DNA integrity in the male germline. Int J

Androl 28, 171-179.

Aitken, R. J., Buckingham, D., and Harkiss, D. (1993a). Use of a xanthine oxidase free

radical generating system to investigate the cytotoxic effects of reactive oxygen species on

human spermatozoa. J Reprod Fertil 97, 441-450.

Aitken, R. J., Buckingham, D. W., Brindle, J., Gomez, E., Baker, H. W., and Irvine, D. S.

(1995a). Analysis of sperm movement in relation to the oxidative stress created by

leukocytes in washed sperm preparations and seminal plasma. Hum Reprod 10, 2061-2071.

Aitken, R. J., Buckingham, D. W., and West, K. M. (1992). Reactive oxygen species and

human spermatozoa: analysis of the cellular mechanisms involved in luminol- and

lucigenin-dependent chemiluminescence. J Cell Physiol 151, 466-477.

Aitken, R. J., and De Iuliis, G. N. (2007). Origins and consequences of DNA damage in

male germ cells. Reprod Biomed Online 14, 727-733.

Aitken, R. J., Fisher, H. M., Fulton, N., Gomez, E., Knox, W., Lewis, B., and Irvine, S.

(1997). Reactive oxygen species generation by human spermatozoa is induced by

exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and

quinacrine. Mol Reprod Dev 47, 468-482.

Aitken, R. J., Gordon, E., Harkiss, D., Twigg, J. P., Milne, P., Jennings, Z., and Irvine, D.

S. (1998). Relative impact of oxidative stress on the functional competence and genomic

integrity of human spermatozoa. Biol Reprod 59, 1037-1046.

Aitken, R. J., Harkiss, D., and Buckingham, D. W. (1993b). Analysis of lipid peroxidation

mechanisms in human spermatozoa. Mol Reprod Dev 35, 302-315.

Aitken, R. J., Paterson, M., Fisher, H., Buckingham, D. W., and van Duin, M. (1995b).

Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the

control of human sperm function. J Cell Sci 108 ( Pt 5), 2017-2025.

Aitken, R. J., and West, K. M. (1990). Analysis of the relationship between reactive

oxygen species production and leucocyte infiltration in fractions of human semen

separated on Percoll gradients. Int J Androl 13, 433-451.

Alvarez, J. G. (2003). Nurture vs nature: how can we optimize sperm quality? J Androl 24,

640-648.

Alvarez, J. G., Touchstone, J. C., Blasco, L., and Storey, B. T. (1987). Spontaneous lipid

peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa.

Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 8,

338-348.

Amor, D. J., and Halliday, J. (2008). A review of known imprinting syndromes and their

association with assisted reproduction technologies. Hum Reprod 23, 2826-2834.

Anderson (1990). Immunology of the male reproductive tract:implication for the sexual

transmission of human immunodeficiency virus., (New York: Oxford University Press).

128

Anway, M. D., Cupp, A. S., Uzumcu, M., and Skinner, M. K. (2005). Epigenetic

transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466-

1469.

Anway, M. D., and Skinner, M. K. (2008). Epigenetic programming of the germ line:

effects of endocrine disruptors on the development of transgenerational disease. Reprod

Biomed Online 16, 23-25.

Ariel, M., Cedar, H., and McCarrey, J. (1994). Developmental changes in methylation of

spermatogenesis-specific genes include reprogramming in the epididymis. Nat Genet 7, 59-

63.

Armstrong, J. S., Bivalacqua, T. J., Chamulitrat, W., Sikka, S., and Hellstrom, W. J.

(2002). A comparison of the NADPH oxidase in human sperm and white blood cells. Int J

Androl 25, 223-229.

Asmus, K. D. (1984). Pulse radiolysis methodology. Methods Enzymol 105, 167-178.

Athayde, K. S., Cocuzza, M., Agarwal, A., Krajcir, N., Lucon, A. M., Srougi, M., and

Hallak, J. (2007). Development of normal reference values for seminal reactive oxygen

species and their correlation with leukocytes and semen parameters in a fertile population.

J Androl 28, 613-620.

Aziz, N., Saleh, R. A., Sharma, R. K., Lewis-Jones, I., Esfandiari, N., Thomas, A. J., Jr.,

and Agarwal, A. (2004). Novel association between sperm reactive oxygen species

production, sperm morphological defects, and the sperm deformity index. Fertil Steril 81,

349-354.

Baehner, R. L., Boxer, L. A., and Davis, J. (1976). The biochemical basis of nitroblue

tetrazolium reduction in normal human and chronic granulomatous disease

polymorphonuclear leukocytes. Blood 48, 309-313.

Baker, M. A., Krutskikh, A., and Aitken, R. J. (2003). Biochemical entities involved in

reactive oxygen species generation by human spermatozoa. Protoplasma 221, 145-151.

Baker, M. A., Krutskikh, A., Curry, B. J., McLaughlin, E. A., and Aitken, R. J. (2004).

Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-

dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations.

Biol Reprod 71, 307-318.

Barratt, C. L., Bolton, A. E., and Cooke, I. D. (1990). Functional significance of white

blood cells in the male and female reproductive tract. Hum Reprod 5, 639-648.

Barroso, G., Morshedi, M., and Oehninger, S. (2000). Analysis of DNA fragmentation,

plasma membrane translocation of phosphatidylserine and oxidative stress in human

spermatozoa. Hum Reprod 15, 1338-1344.

Bedford, J. M. (1994). The status and the state of the human epididymis. Hum Reprod 9,

2187-2199.

Benchaib, M., Ajina, M., Lornage, J., Niveleau, A., Durand, P., and Guerin, J. F. (2003a).

Quantitation by image analysis of global DNA methylation in human spermatozoa and its

prognostic value in in vitro fertilization: a preliminary study. Fertil Steril 80, 947-953.

Benchaib, M., Braun, V., Lornage, J., Hadj, S., Salle, B., Lejeune, H., and Guerin, J. F.

(2003b). Sperm DNA fragmentation decreases the pregnancy rate in an assisted

reproductive technique. Hum Reprod 18, 1023-1028.

129

Benchaib, M., Braun, V., Ressnikof, D., Lornage, J., Durand, P., Niveleau, A., and Guerin,

J. F. (2005). Influence of global sperm DNA methylation on IVF results. Hum Reprod 20,

768-773.

Benchaib, M., Lornage, J., Mazoyer, C., Lejeune, H., Salle, B., and Francois Guerin, J.

(2007). Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted

reproductive technology outcome. Fertil Steril 87, 93-100.

Benoff, S., Jacob, A., and Hurley, I. R. (2000). Male infertility and environmental

exposure to lead and cadmium. Hum Reprod Update 6, 107-121.

Berdowska, A., and Zwirska-Korczala, K. (2001). Neopterin measurement in clinical

diagnosis. J Clin Pharm Ther 26, 319-329.

Berger, R. E., Karp, L. E., Williamson, R. A., Koehler, J., Moore, D. E., and Holmes, K.

K. (1982). The relationship of pyospermia and seminal fluid bacteriology to sperm

function as reflected in the sperm penetration assay. Fertil Steril 37, 557-564.

Bezold, G., Lange, M., and Peter, R. U. (2001). Homozygous methylenetetrahydrofolate

reductase C677T mutation and male infertility. N Engl J Med 344, 1172-1173.

Bianchi, P. G., Manicardi, G. C., Urner, F., Campana, A., and Sakkas, D. (1996).

Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of

hidden anomalies in normal spermatozoa. Mol Hum Reprod 2, 139-144.

Bize, I., Santander, G., Cabello, P., Driscoll, D., and Sharpe, C. (1991). Hydrogen peroxide

is involved in hamster sperm capacitation in vitro. Biol Reprod 44, 398-403.

Bizzaro, D., Manicardi, G. C., Bianchi, P. G., Bianchi, U., Mariethoz, E., and Sakkas, D.

(1998). In-situ competition between protamine and fluorochromes for sperm DNA. Mol

Hum Reprod 4, 127-132.

Bjorndahl, L., and Kvist, U. (1985). Loss of an intrinsic capacity for human sperm

chromatin decondensation. Acta Physiol Scand 124, 189-194.

Borini, A., Tarozzi, N., Bizzaro, D., Bonu, M. A., Fava, L., Flamigni, C., and Coticchio, G.

(2006). Sperm DNA fragmentation: paternal effect on early post-implantation embryo

development in ART. Hum Reprod 21, 2876-2881.

Bozdemir, A. E., Barutcuoglu, B., Dereli, D., Kabaroglu, C., Habif, S., and Bayindir, O.

(2006). C-reactive protein and neopterin levels in healthy non-obese adults. Clin Chem Lab

Med 44, 317-321.

Bray, T. M., and Bettger, W. J. (1990). The physiological role of zinc as an antioxidant.

Free Radic Biol Med 8, 281-291.

Buettner, G. R., and Mason, R. P. (1990). Spin-trapping methods for detecting superoxide

and hydroxyl free radicals in vitro and in vivo. Methods Enzymol 186, 127-133.

Cande, C., Cecconi, F., Dessen, P., and Kroemer, G. (2002). Apoptosis-inducing factor

(AIF): key to the conserved caspase-independent pathways of cell death? J Cell Sci 115,

4727-4734.

130

Carrell, D. T., and Liu, L. (2001). Altered protamine 2 expression is uncommon in donors

of known fertility, but common among men with poor fertilizing capacity, and may reflect

other abnormalities of spermiogenesis. J Androl 22, 604-610.

Chabory, E., Damon, C., Lenoir, A., Henry-Berger, J., Vernet, P., Cadet, R., Saez, F., and

Drevet, J. R. Mammalian glutathione peroxidases control acquisition and maintenance of

spermatozoa integrity. J Anim Sci 88, 1321-1331.

Chabory, E., Damon, C., Lenoir, A., Kauselmann, G., Kern, H., Zevnik, B., Garrel, C.,

Saez, F., Cadet, R., Henry-Berger, J., et al. (2009). Epididymis seleno-independent

glutathione peroxidase 5 maintains sperm DNA integrity in mice. J Clin Invest 119, 2074-

2085.

Chang, S. I., Jin, B., Youn, P., Park, C., Park, J. D., and Ryu, D. Y. (2007). Arsenic-

induced toxicity and the protective role of ascorbic acid in mouse testis. Toxicol Appl

Pharmacol 218, 196-203.

Chatterjee, R., Haines, G. A., Perera, D. M., Goldstone, A., and Morris, I. D. (2000).

Testicular and sperm DNA damage after treatment with fludarabine for chronic

lymphocytic leukaemia. Hum Reprod 15, 762-766.

Chen, Z., Karaplis, A. C., Ackerman, S. L., Pogribny, I. P., Melnyk, S., Lussier-Cacan, S.,

Chen, M. F., Pai, A., John, S. W., Smith, R. S., et al. (2001). Mice deficient in

methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased

methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet 10,

433-443.

Chohan, K. R., Griffin, J. T., Lafromboise, M., De Jonge, C. J., and Carrell, D. T. (2006).

Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J

Androl 27, 53-59.

Choi, H. S., Kim, J. W., Cha, Y. N., and Kim, C. (2006). A quantitative nitroblue

tetrazolium assay for determining intracellular superoxide anion production in phagocytic

cells. J Immunoassay Immunochem 27, 31-44.

Cisneros, F. J. (2004). DNA methylation and male infertility. Front Biosci 9, 1189-1200.

Cocuzza, M., Athayde, K. S., Agarwal, A., Sharma, R., Pagani, R., Lucon, A. M., Srougi,

M., and Hallak, J. (2008). Age-related increase of reactive oxygen species in neat semen in

healthy fertile men. Urology 71, 490-494.

Cocuzza, M., Sikka, S. C., Athayde, K. S., and Agarwal, A. (2007). Clinical relevance of

oxidative stress and sperm chromatin damage in male infertility: an evidence based

analysis. Int Braz J Urol 33, 603-621.

Collins, A. R., Cadet, J., Moller, L., Poulsen, H. E., and Vina, J. (2004). Are we sure we

know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Arch

Biochem Biophys 423, 57-65.

Comhaire, F., Zalata, A., Mahmoud, A., Depoorter, B., Huysse, L., Christophe, A., and

Depuydt, C. (1995). Diagnostic and therapeutic approach to moderate and severe male

subfertility in 1995. Hum Reprod 10 Suppl 1, 144-150.

Comhaire, F. H., Christophe, A. B., Zalata, A. A., Dhooge, W. S., Mahmoud, A. M., and

Depuydt, C. E. (2000). The effects of combined conventional treatment, oral antioxidants

131

and essential fatty acids on sperm biology in subfertile men. Prostaglandins Leukot Essent

Fatty Acids 63, 159-165.

Comhaire, F. H., Mahmoud, A. M., Depuydt, C. E., Zalata, A. A., and Christophe, A. B.

(1999). Mechanisms and effects of male genital tract infection on sperm quality and

fertilizing potential: the andrologist's viewpoint. Hum Reprod Update 5, 393-398.

Cox, G. F., Burger, J., Lip, V., Mau, U. A., Sperling, K., Wu, B. L., and Horsthemke, B.

(2002). Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J

Hum Genet 71, 162-164.

Crow, J. F. (2000). The origins, patterns and implications of human spontaneous mutation.

Nat Rev Genet 1, 40-47.

Cutler, R. G. (1991). Antioxidants and aging. Am J Clin Nutr 53, 373S-379S.

Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D., and Milzani, A. (2006).

Biomarkers of oxidative damage in human disease. Clin Chem 52, 601-623.

Dandekar, S. P., Nadkarni, G. D., Kulkarni, V. S., and Punekar, S. (2002). Lipid

peroxidation and antioxidant enzymes in male infertility. J Postgrad Med 48, 186-189;

discussion 189-190.

Das, U. B., Mallick, M., Debnath, J. M., and Ghosh, D. (2002). Protective effect of

ascorbic acid on cyclophosphamide- induced testicular gametogenic and androgenic

disorders in male rats. Asian J Androl 4, 201-207.

Das, U. N. (2001). Is obesity an inflammatory condition? Nutrition 17, 953-966.

Davies, K. J. (1995). Oxidative stress: the paradox of aerobic life. Biochem Soc Symp 61,

1-31.

de Boer, H., Verschoor, L., Ruinemans-Koerts, J., and Jansen, M. (2005). Letrozole

normalizes serum testosterone in severely obese men with hypogonadotropic

hypogonadism. Diabetes Obes Metab 7, 211-215.

De Iuliis, G. N., Newey, R. J., King, B. V., and Aitken, R. J. (2009). Mobile phone

radiation induces reactive oxygen species production and DNA damage in human

spermatozoa in vitro. PLoS One 4, e6446.

de la Rochebrochard, E., and Thonneau, P. (2002). Paternal age and maternal age are risk

factors for miscarriage; results of a multicentre European study. Hum Reprod 17, 1649-

1656.

de Lamirande, E., and Gagnon, C. (1992a). Reactive oxygen species and human

spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J

Androl 13, 368-378.

de Lamirande, E., and Gagnon, C. (1992b). Reactive oxygen species and human

spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the

inhibition of sperm motility. J Androl 13, 379-386.

de Lamirande, E., and Gagnon, C. (1993). A positive role for the superoxide anion in

triggering hyperactivation and capacitation of human spermatozoa. Int J Androl 16, 21-25.

132

de Lamirande, E., and Gagnon, C. (1995a). Capacitation-associated production of

superoxide anion by human spermatozoa. Free Radic Biol Med 18, 487-495.

de Lamirande, E., and Gagnon, C. (1995b). Impact of reactive oxygen species on

spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod 10

Suppl 1, 15-21.

de Lamirande, E., Jiang, H., Zini, A., Kodama, H., and Gagnon, C. (1997). Reactive

oxygen species and sperm physiology. Rev Reprod 2, 48-54.

DeBaun, M. R., Niemitz, E. L., and Feinberg, A. P. (2003). Association of in vitro

fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and

H19. Am J Hum Genet 72, 156-160.

Depuydt, C. E., Bosmans, E., Zalata, A., Schoonjans, F., and Comhaire, F. H. (1996). The

relation between reactive oxygen species and cytokines in andrological patients with or

without male accessory gland infection. J Androl 17, 699-707.

Dhillon, V. S., Shahid, M., and Husain, S. A. (2007). Associations of MTHFR DNMT3b

4977 bp deletion in mtDNA and GSTM1 deletion, and aberrant CpG island

hypermethylation of GSTM1 in non-obstructive infertility in Indian men. Mol Hum

Reprod 13, 213-222.

Dizdaroglu, M. (1992). Oxidative damage to DNA in mammalian chromatin. Mutat Res

275, 331-342.

Dizdaroglu, M., Jaruga, P., Birincioglu, M., and Rodriguez, H. (2002). Free radical-

induced damage to DNA: mechanisms and measurement. Free Radic Biol Med 32, 1102-

1115.

Doerksen, T., Benoit, G., and Trasler, J. M. (2000). Deoxyribonucleic acid

hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is

associated with altered testicular histology. Endocrinology 141, 3235-3244.

Doerksen, T., and Trasler, J. M. (1996). Developmental exposure of male germ cells to 5-

azacytidine results in abnormal preimplantation development in rats. Biol Reprod 55,

1155-1162.

Dokmeci, D. (2005). Oxidative stress, male infertility and the role of carnitines. Folia Med

(Plovdiv) 47, 26-30.

Drummen, G. P., van Liebergen, L. C., Op den Kamp, J. A., and Post, J. A. (2002). C11-

BODIPY(581/591), an oxidation-sensitive fluorescent lipid peroxidation probe:

(micro)spectroscopic characterization and validation of methodology. Free Radic Biol Med

33, 473-490.

Eggert-Kruse, W., Bellmann, A., Rohr, G., Tilgen, W., and Runnebaum, B. (1992).

Differentiation of round cells in semen by means of monoclonal antibodies and

relationship with male fertility. Fertil Steril 58, 1046-1055.

Eggert-Kruse, W., Boit, R., Rohr, G., Aufenanger, J., Hund, M., and Strowitzki, T. (2001).

Relationship of seminal plasma interleukin (IL) -8 and IL-6 with semen quality. Hum

Reprod 16, 517-528.

133

Eggert-Kruse, W., Rohr, G., Demirakca, T., Rusu, R., Naher, H., Petzoldt, D., and

Runnebaum, B. (1997). Chlamydial serology in 1303 asymptomatic subfertile couples.

Hum Reprod 12, 1464-1475.

Eggert-Kruse, W., Zimmermann, K., Geissler, W., Ehrmann, A., Boit, R., and Strowitzki,

T. (2009). Clinical relevance of polymorphonuclear (PMN-) elastase determination in

semen and serum during infertility investigation. Int J Androl 32, 317-329.

Esfandiari, N., Sharma, R. K., Saleh, R. A., Thomas, A. J., Jr., and Agarwal, A. (2003).

Utility of the nitroblue tetrazolium reduction test for assessment of reactive oxygen species

production by seminal leukocytes and spermatozoa. J Androl 24, 862-870.

Eskiocak, S., Gozen, A. S., Taskiran, A., Kilic, A. S., Eskiocak, M., and Gulen, S. (2006).

Effect of psychological stress on the L-arginine-nitric oxide pathway and semen quality.

Braz J Med Biol Res 39, 581-588.

Eskiocak, S., Gozen, A. S., Yapar, S. B., Tavas, F., Kilic, A. S., and Eskiocak, M. (2005).

Glutathione and free sulphydryl content of seminal plasma in healthy medical students

during and after exam stress. Hum Reprod 20, 2595-2600.

Esterbauer, H., Schaur, R. J., and Zollner, H. (1991). Chemistry and biochemistry of 4-

hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 11, 81-128.

Evenson, D. P., Emerick, R. J., Jost, L. K., Kayongo-Male, H., and Stewart, S. R. (1993).

Zinc-silicon interactions influencing sperm chromatin integrity and testicular cell

development in the rat as measured by flow cytometry. J Anim Sci 71, 955-962.

Evenson, D. P., Larson, K. L., and Jost, L. K. (2002). Sperm chromatin structure assay: its

clinical use for detecting sperm DNA fragmentation in male infertility and comparisons

with other techniques. J Androl 23, 25-43.

Evenson, D. P., and Wixon, R. (2005). Environmental toxicants cause sperm DNA

fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA). Toxicol Appl

Pharmacol 207, 532-537.

Fedder, J., Askjaer, S. A., and Hjort, T. (1993). Nonspermatozoal cells in semen:

relationship to other semen parameters and fertility status of the couple. Arch Androl 31,

95-103.

Fejes, I., Koloszar, S., Szollosi, J., Zavaczki, Z., and Pal, A. (2005). Is semen quality

affected by male body fat distribution? Andrologia 37, 155-159.

Fernandez-Gonzalez, R., Moreira, P. N., Perez-Crespo, M., Sanchez-Martin, M., Ramirez,

M. A., Pericuesta, E., Bilbao, A., Bermejo-Alvarez, P., de Dios Hourcade, J., de Fonseca,

F. R., and Gutierrez-Adan, A. (2008). Long-term effects of mouse intracytoplasmic sperm

injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol

Reprod 78, 761-772.

Fingerova, H., Oborna, I., Novotny, J., Svobodova, M., Brezinova, J., and Radova, L.

(2009). The measurement of reactive oxygen species in human neat semen and in

suspended spermatozoa: a comparison. Reprod Biol Endocrinol 7, 118.

Finkelstein, J. D. (1990). Methionine metabolism in mammals. J Nutr Biochem 1, 228-237.

134

Flickinger, C. J., Howards, S. S., and Herr, J. C. (1995). Effects of vasectomy on the

epididymis. Microsc Res Tech 30, 82-100.

Foresta, C., Flohe, L., Garolla, A., Roveri, A., Ursini, F., and Maiorino, M. (2002). Male

fertility is linked to the selenoprotein phospholipid hydroperoxide glutathione peroxidase.

Biol Reprod 67, 967-971.

Fraczek, M., Sanocka, D., Kamieniczna, M., and Kurpisz, M. (2008). Proinflammatory

cytokines as an intermediate factor enhancing lipid sperm membrane peroxidation in in

vitro conditions. J Androl 29, 85-92.

Fraga, C. G., Motchnik, P. A., Wyrobek, A. J., Rempel, D. M., and Ames, B. N. (1996).

Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res

351, 199-203.

Franco, R., Schoneveld, O., Georgakilas, A. G., and Panayiotidis, M. I. (2008). Oxidative

stress, DNA methylation and carcinogenesis. Cancer Lett 266, 6-11.

Franken, D. R., Franken, C. J., de la Guerre, H., and de Villiers, A. (1999). Normal sperm

morphology and chromatin packaging: comparison between aniline blue and chromomycin

A3 staining. Andrologia 31, 361-366.

Fridovich, I. (1970). Quantitative aspects of the production of superoxide anion radical by

milk xanthine oxidase. J Biol Chem 245, 4053-4057.

Frungieri, M. B., Calandra, R. S., Lustig, L., Meineke, V., Kohn, F. M., Vogt, H. J., and

Mayerhofer, A. (2002). Number, distribution pattern, and identification of macrophages in

the testes of infertile men. Fertil Steril 78, 298-306.

Fuentes-Mascorro, G., Serrano, H., and Rosado, A. (2000). Sperm chromatin. Arch Androl

45, 215-225.

Furukawa, S., Fujita, T., Shimabukuro, M., Iwaki, M., Yamada, Y., Nakajima, Y.,

Nakayama, O., Makishima, M., Matsuda, M., and Shimomura, I. (2004). Increased

oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 114, 1752-

1761.

Gagnon, C., Iwasaki, A., De Lamirande, E., and Kovalski, N. (1991). Reactive oxygen

species and human spermatozoa. Ann N Y Acad Sci 637, 436-444.

Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari, G., Verlengia, C.,

Dondero, F., and Lenzi, A. (2000). Study of apoptotic DNA fragmentation in human

spermatozoa. Hum Reprod 15, 830-839.

Gandini, L., Lombardo, F., Paoli, D., Caruso, F., Eleuteri, P., Leter, G., Ciriminna, R.,

Culasso, F., Dondero, F., Lenzi, A., and Spano, M. (2004). Full-term pregnancies achieved

with ICSI despite high levels of sperm chromatin damage. Hum Reprod 19, 1409-1417.

Gatewood, J. M., Schroth, G. P., Schmid, C. W., and Bradbury, E. M. (1990). Zinc-

induced secondary structure transitions in human sperm protamines. J Biol Chem 265,

20667-20672.

Gaur, D. S., Talekar, M. S., and Pathak, V. P. Alcohol intake and cigarette smoking:

impact of two major lifestyle factors on male fertility. Indian J Pathol Microbiol 53, 35-40.

135

Ghosh, D., Das, U. B., and Misro, M. (2002). Protective role of alpha-tocopherol-succinate

(provitamin-E) in cyclophosphamide induced testicular gametogenic and steroidogenic

disorders: a correlative approach to oxidative stress. Free Radic Res 36, 1209-1218.

Giagulli, V. A., Kaufman, J. M., and Vermeulen, A. (1994). Pathogenesis of the decreased

androgen levels in obese men. J Clin Endocrinol Metab 79, 997-1000.

Gicquel, C., Rossignol, S., Cabrol, S., Houang, M., Steunou, V., Barbu, V., Danton, F.,

Thibaud, N., Le Merrer, M., Burglen, L., et al. (2005). Epimutation of the telomeric

imprinting center region on chromosome 11p15 in Silver-Russell syndrome. Nat Genet 37,

1003-1007.

Gonzales, G. F., Kortebani, G., and Mazzolli, A. B. (1992). Leukocytospermia and

function of the seminal vesicles on seminal quality. Fertil Steril 57, 1058-1065.

Gosden, R., Trasler, J., Lucifero, D., and Faddy, M. (2003). Rare congenital disorders,

imprinted genes, and assisted reproductive technology. Lancet 361, 1975-1977.

Greco, E., Iacobelli, M., Rienzi, L., Ubaldi, F., Ferrero, S., and Tesarik, J. (2005a).

Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J

Androl 26, 349-353.

Greco, E., Romano, S., Iacobelli, M., Ferrero, S., Baroni, E., Minasi, M. G., Ubaldi, F.,

Rienzi, L., and Tesarik, J. (2005b). ICSI in cases of sperm DNA damage: beneficial effect

of oral antioxidant treatment. Hum Reprod 20, 2590-2594.

Griveau, J. F., and Le Lannou, D. (1997). Influence of oxygen tension on reactive oxygen

species production and human sperm function. Int J Androl 20, 195-200.

Griveau, J. F., Renard, P., and Le Lannou, D. (1994). An in vitro promoting role for

hydrogen peroxide in human sperm capacitation. Int J Androl 17, 300-307.

Gutteridge, J. M., and Quinlan, G. J. (1983). Malondialdehyde formation from lipid

peroxides in the thiobarbituric acid test: the role of lipid radicals, iron salts, and metal

chelators. J Appl Biochem 5, 293-299.

Haidl, G., Allam, J. P., and Schuppe, H. C. (2008). Chronic epididymitis: impact on semen

parameters and therapeutic options. Andrologia 40, 92-96.

Halliwell, B., and Whiteman, M. (2004). Measuring reactive species and oxidative damage

in vivo and in cell culture: how should you do it and what do the results mean? Br J

Pharmacol 142, 231-255.

Halliwell, G. J. M. C. (1999). Free Radicals in Biology and Medicine, Vol 1, Third edn

(London: Oxford University Press).

Hamerlinck, F. F. (1999). Neopterin: a review. Exp Dermatol 8, 167-176.

Hammoud, A. O., Gibson, M., Peterson, C. M., Meikle, A. W., and Carrell, D. T. (2008).

Impact of male obesity on infertility: a critical review of the current literature. Fertil Steril

90, 897-904.

Harrison, P. E., Barratt, C. L., Robinson, A. J., Kessopoulou, E., and Cooke, I. D. (1991).

Detection of white blood cell populations in the ejaculates of fertile men. J Reprod

Immunol 19, 95-98.

136

Heatwole, V. M. (1999). TUNEL assay for apoptotic cells. Methods Mol Biol 115, 141-

148.

Hedley, A. A., Ogden, C. L., Johnson, C. L., Carroll, M. D., Curtin, L. R., and Flegal, K.

M. (2004). Prevalence of overweight and obesity among US children, adolescents, and

adults, 1999-2002. Jama 291, 2847-2850.

Hendin, B. N., Kolettis, P. N., Sharma, R. K., Thomas, A. J., Jr., and Agarwal, A. (1999).

Varicocele is associated with elevated spermatozoal reactive oxygen species production

and diminished seminal plasma antioxidant capacity. J Urol 161, 1831-1834.

Henkel, R., Kierspel, E., Stalf, T., Mehnert, C., Menkveld, R., Tinneberg, H. R., Schill, W.

B., and Kruger, T. F. (2005). Effect of reactive oxygen species produced by spermatozoa

and leukocytes on sperm functions in non-leukocytospermic patients. Fertil Steril 83, 635-

642.

Henkel, R., Maass, G., Hajimohammad, M., Menkveld, R., Stalf, T., Villegas, J., Sanchez,

R., Kruger, T. F., and Schill, W. B. (2003). Urogenital inflammation: changes of

leucocytes and ROS. Andrologia 35, 309-313.

Hepburn, P. A., Margison, G. P., and Tisdale, M. J. (1991). Enzymatic methylation of

cytosine in DNA is prevented by adjacent O6-methylguanine residues. J Biol Chem 266,

7985-7987.

Hill, J. A., Anderson, D. J., Polgar, K., Abbott, A. F., and Politch, J. A. (1994). Seminal

white blood cells and recurrent abortion. Hum Reprod 9, 1180-1183.

Hochreiter, W. W. (2003). Male accessory gland infection: standardization of

inflammatory parameters including cytokines. Andrologia 35, 300-303.

Holstein, A. F. (1978). [Spermatophagia in male seminiferous tubules]. Verh Anat Ges,

543-544.

Houshdaran, S., Cortessis, V. K., Siegmund, K., Yang, A., Laird, P. W., and Sokol, R. Z.

(2007). Widespread epigenetic abnormalities suggest a broad DNA methylation erasure

defect in abnormal human sperm. PLoS ONE 2, e1289.

Hsu, P. C., and Guo, Y. L. (2002). Antioxidant nutrients and lead toxicity. Toxicology 180,

33-44.

Hughes, C. M., Lewis, S. E., McKelvey-Martin, V. J., and Thompson, W. (1996). A

comparison of baseline and induced DNA damage in human spermatozoa from fertile and

infertile men, using a modified comet assay. Mol Hum Reprod 2, 613-619.

Hussein, A., Ozgok, Y., Ross, L., and Niederberger, C. (2005). Clomiphene administration

for cases of nonobstructive azoospermia: a multicenter study. J Androl 26, 787-791;

discussion 792-783.

Huszar, G., and Vigue, L. (1993). Incomplete development of human spermatozoa is

associated with increased creatine phosphokinase concentration and abnormal head

morphology. Mol Reprod Dev 34, 292-298.

Iwasaki, A., and Gagnon, C. (1992). Formation of reactive oxygen species in spermatozoa

of infertile patients. Fertil Steril 57, 409-416.

137

Jamaluddin, M. D., Chen, I., Yang, F., Jiang, X., Jan, M., Liu, X., Schafer, A. I., Durante,

W., Yang, X., and Wang, H. (2007). Homocysteine inhibits endothelial cell growth via

DNA hypomethylation of the cyclin A gene. Blood 110, 3648-3655.

Jedrzejczak, P., Fraczek, M., Szumala-Kakol, A., Taszarek-Hauke, G., Pawelczyk, L., and

Kurpisz, M. (2005). Consequences of semen inflammation and lipid peroxidation on

fertilization capacity of spermatozoa in in vitro conditions. Int J Androl 28, 275-283.

Jensen, T. K., Andersson, A. M., Jorgensen, N., Andersen, A. G., Carlsen, E., Petersen, J.

H., and Skakkebaek, N. E. (2004). Body mass index in relation to semen quality and

reproductive hormones among 1,558 Danish men. Fertil Steril 82, 863-870.

Jequier (2000). Male Infertility: a guide for the clinician. ).

Jochum, M., Pabst, W., and Schill, W. B. (1986). Granulocyte elastase as a sensitive

diagnostic parameter of silent male genital tract inflammation. Andrologia 18, 413-419.

Johnson, F. C. (1979). The antioxidant vitamins. CRC Crit Rev Food Sci Nutr 11, 217-309.

Jokela, M., Elovainio, M., and Kivimaki, M. (2008). Lower fertility associated with

obesity and underweight: the US National Longitudinal Survey of Youth. Am J Clin Nutr

88, 886-893.

Jones, R. (2004). Sperm survival versus degradation in the Mammalian epididymis: a

hypothesis. Biol Reprod 71, 1405-1411.

Jones, R., Mann, T., and Sherins, R. (1979). Peroxidative breakdown of phospholipids in

human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action

of seminal plasma. Fertil Steril 31, 531-537.

Joseph, J. A., and Cutler, R. C. (1994). The role of oxidative stress in signal transduction

changes and cell loss in senescence. Ann N Y Acad Sci 738, 37-43.

Jue, K., Benoit, G., Alcivar-Warren, A. A., and Trasler, J. M. (1995). Developmental and

hormonal regulation of DNA methyltransferase in the rat testis. Biol Reprod 52, 1364-

1371.

Jung, A., Leonhardt, F., Schill, W. B., and Schuppe, H. C. (2005). Influence of the type of

undertrousers and physical activity on scrotal temperature. Hum Reprod 20, 1022-1027.

Jung, A., and Schuppe, H. C. (2007). Influence of genital heat stress on semen quality in

humans. Andrologia 39, 203-215.

Junqueira, V. B., Barros, S. B., Chan, S. S., Rodrigues, L., Giavarotti, L., Abud, R. L., and

Deucher, G. P. (2004). Aging and oxidative stress. Mol Aspects Med 25, 5-16.

Kelly, T. L., Li, E., and Trasler, J. M. (2003). 5-aza-2'-deoxycytidine induces alterations in

murine spermatogenesis and pregnancy outcome. J Androl 24, 822-830.

Kemal Duru, N., Morshedi, M., and Oehninger, S. (2000). Effects of hydrogen peroxide on

DNA and plasma membrane integrity of human spermatozoa. Fertil Steril 74, 1200-1207.

Kerjean, A., Dupont, J. M., Vasseur, C., Le Tessier, D., Cuisset, L., Paldi, A., Jouannet, P.,

and Jeanpierre, M. (2000). Establishment of the paternal methylation imprint of the human

H19 and MEST/PEG1 genes during spermatogenesis. Hum Mol Genet 9, 2183-2187.

138

Kjellberg, S., Bjorndahl, L., and Kvist, U. (1992). Sperm chromatin stability and zinc

binding properties in semen from men in barren unions. Int J Androl 15, 103-113.

Kobayashi, H., Gil-Guzman, E., Mahran, A. M., Rakesh, Nelson, D. R., Thomas, A. J., Jr.,

and Agarwa, A. (2001). Quality control of reactive oxygen species measurement by

luminol-dependent chemiluminescence assay. J Androl 22, 568-574.

Kobayashi, H., Hiura, H., John, R. M., Sato, A., Otsu, E., Kobayashi, N., Suzuki, R.,

Suzuki, F., Hayashi, C., Utsunomiya, T., et al. (2009). DNA methylation errors at

imprinted loci after assisted conception originate in the parental sperm. Eur J Hum Genet.

Kobayashi, H., Sato, A., Otsu, E., Hiura, H., Tomatsu, C., Utsunomiya, T., Sasaki, H.,

Yaegashi, N., and Arima, T. (2007). Aberrant DNA methylation of imprinted loci in sperm

from oligospermic patients. Hum Mol Genet 16, 2542-2551.

Koch, O. R., Pani, G., Borrello, S., Colavitti, R., Cravero, A., Farre, S., and Galeotti, T.

(2004). Oxidative stress and antioxidant defenses in ethanol-induced cell injury. Mol

Aspects Med 25, 191-198.

Kodama, H., Yamaguchi, R., Fukuda, J., Kasai, H., and Tanaka, T. (1997). Increased

oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients.


Koppers, A. J., De Iuliis, G. N., Finnie, J. M., McLaughlin, E. A., and Aitken, R. J. (2008).

Significance of mitochondrial reactive oxygen species in the generation of oxidative stress

in spermatozoa. J Clin Endocrinol Metab 93, 3199-3207.

Kort, H. I., Massey, J. B., Elsner, C. W., Mitchell-Leef, D., Shapiro, D. B., Witt, M. A.,

and Roudebush, W. E. (2006). Impact of body mass index values on sperm quantity and

quality. J Androl 27, 450-452.

Kovalski, N., de Lamirande, E., and Gagnon, C. (1991). Determination of neutrophil

concentration in semen by measurement of superoxide radical formation. Fertil Steril 56,

946-953.

Krause, W., Bohring, C., Gueth, A., Horster, S., Krisp, A., and Skrzypek, J. (2003).

Cellular and biochemical markers in semen indicating male accessory gland inflammation.

Andrologia 35, 279-282.

Kravets, F. G., Lee, J., Singh, B., Trocchia, A., Pentyala, S. N., and Khan, S. A. (2000).

Prostasomes: current concepts. Prostate 43, 169-174.

Kunsch, C., and Medford, R. M. (1999). Oxidative stress as a regulator of gene expression

in the vasculature. Circ Res 85, 753-766.

Kunzle, R., Mueller, M. D., Hanggi, W., Birkhauser, M. H., Drescher, H., and Bersinger,

N. A. (2003). Semen quality of male smokers and nonsmokers in infertile couples. Fertil

Steril 79, 287-291.

Lampiao, F., and du Plessis, S. S. (2008). Insulin and leptin enhance human sperm

motility, acrosome reaction and nitric oxide production. Asian J Androl 10, 799-807.

Lawrence, L. T., and Moley, K. H. (2008). Epigenetics and assisted reproductive

technologies: human imprinting syndromes. Semin Reprod Med 26, 143-152.

139

Ledochowski, M., Murr, C., Widner, B., and Fuchs, D. (1999). Association between

insulin resistance, body mass and neopterin concentrations. Clin Chim Acta 282, 115-123.

Lee, C. R., North, K. E., Bray, M. S., Fornage, M., Seubert, J. M., Newman, J. W.,

Hammock, B. D., Couper, D. J., Heiss, G., and Zeldin, D. C. (2006). Genetic variation in

soluble epoxide hydrolase (EPHX2) and risk of coronary heart disease: The

Atherosclerosis Risk in Communities (ARIC) study. Hum Mol Genet 15, 1640-1649.

Lenzi, A., Lombardo, F., Sgro, P., Salacone, P., Caponecchia, L., Dondero, F., and

Gandini, L. (2003). Use of carnitine therapy in selected cases of male factor infertility: a

double-blind crossover trial. Fertil Steril 79, 292-300.

Lewis, S. E., and Aitken, R. J. (2005). DNA damage to spermatozoa has impacts on

fertilization and pregnancy. Cell Tissue Res 322, 33-41.

Lewis, S. E., Sterling, E. S., Young, I. S., and Thompson, W. (1997). Comparison of

individual antioxidants of sperm and seminal plasma in fertile and infertile men. Fertil

Steril 67, 142-147.

Li, H., Jiang, Y., Rajpurkar, A., Dunbar, J. C., and Dhabuwala, C. B. (1999). Cocaine

induced apoptosis in rat testes. J Urol 162, 213-216.

Li, K., Shang, X., and Chen, Y. (2004). High-performance liquid chromatographic

detection of lipid peroxidation in human seminal plasma and its application to male

infertility. Clin Chim Acta 346, 199-203.

Lian, Z. H., Zack, M. M., and Erickson, J. D. (1986). Paternal age and the occurrence of

birth defects. Am J Hum Genet 39, 648-660.

Loir, M., and Lanneau, M. (1984). Structural function of the basic nuclear proteins in ram

spermatids. J Ultrastruct Res 86, 262-272.

Lopes, S., Jurisicova, A., Sun, J. G., and Casper, R. F. (1998). Reactive oxygen species:

potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 13, 896-900.

MacLeod, J. (1943). The role of oxygen in the metabolism and motility of human

spermatozoa. Am J Physiol 138, 512-518.

Maegawa, M., Kamada, M., Irahara, M., Yamamoto, S., Yamano, S., Ohmoto, Y., Gima,

H., Thaler, C. J., and Aono, T. (2001). Concentration of granulocyte elastase in seminal

plasma is not associated with sperm motility. Arch Androl 47, 31-36.

Magnusdottir, E. V., Thorsteinsson, T., Thorsteinsdottir, S., Heimisdottir, M., and

Olafsdottir, K. (2005). Persistent organochlorines, sedentary occupation, obesity and

human male subfertility. Hum Reprod 20, 208-215.

Makker, K., Agarwal, A., and Sharma, R. (2009). Oxidative stress & male infertility.

Indian J Med Res 129, 357-367.

Maneesh, M., Jayalakshmi, H., Dutta, S., Chakrabarti, A., and Vasudevan, D. M. (2005).

Experimental therapeutic intervention with ascorbic acid in ethanol induced testicular

injuries in rats. Indian J Exp Biol 43, 172-176.

140

Manna, I., Jana, K., and Samanta, P. K. (2004). Effect of different intensities of swimming

exercise on testicular oxidative stress and reproductive dysfunction in mature male albino

Wistar rats. Indian J Exp Biol 42, 816-822.

Marques, C. J., Carvalho, F., Sousa, M., and Barros, A. (2004). Genomic imprinting in

disruptive spermatogenesis. Lancet 363, 1700-1702.

Marques, C. J., Costa, P., Vaz, B., Carvalho, F., Fernandes, S., Barros, A., and Sousa, M.

(2008). Abnormal methylation of imprinted genes in human sperm is associated with

oligozoospermia. Mol Hum Reprod 14, 67-74.

Mazzilli, F., Rossi, T., Marchesini, M., Ronconi, C., and Dondero, F. (1994). Superoxide

anion in human semen related to seminal parameters and clinical aspects. Fertil Steril 62,

862-868.

McKinney, K. A., Lewis, S. E., and Thompson, W. (1996). Reactive oxygen species

generation in human sperm: luminol and lucigenin chemiluminescence probes. Arch

Androl 36, 119-125.

Melissas, J., Malliaraki, N., Papadakis, J. A., Taflampas, P., Kampa, M., and Castanas, E.

(2006). Plasma antioxidant capacity in morbidly obese patients before and after weight

loss. Obes Surg 16, 314-320.

Menezo, Y. J., Hazout, A., Panteix, G., Robert, F., Rollet, J., Cohen-Bacrie, P., Chapuis,

F., Clement, P., and Benkhalifa, M. (2007). Antioxidants to reduce sperm DNA

fragmentation: an unexpected adverse effect. Reprod Biomed Online 14, 418-421.

Mercado-Pichardo, E., Vilar-Rojas, C., Wens-Flores, M. D., and Bernal-Torres, A. (1981).

Reduction capacity on nitroblue tetrazolium (NBT) of the normal human spermatozoa.

Arch Invest Med (Mex) 12, 107-114.

Meseguer, M., Garrido, N., Simon, C., Pellicer, A., and Remohi, J. (2004). Concentration

of glutathione and expression of glutathione peroxidases 1 and 4 in fresh sperm provide a

forecast of the outcome of cryopreservation of human spermatozoa. J Androl 25, 773-780.

Meseguer, M., Martinez-Conejero, J. A., O'Connor, J. E., Pellicer, A., Remohi, J., and

Garrido, N. (2008). The significance of sperm DNA oxidation in embryo development and

reproductive outcome in an oocyte donation program: a new model to study a male

infertility prognostic factor. Fertil Steril 89, 1191-1199.

Morris, I. D., Ilott, S., Dixon, L., and Brison, D. R. (2002). The spectrum of DNA damage

in human sperm assessed by single cell gel electrophoresis (Comet assay) and its

relationship to fertilization and embryo development. Hum Reprod 17, 990-998.

Moskovtsev, S. I., Willis, J., and Mullen, J. B. (2006). Age-related decline in sperm

deoxyribonucleic acid integrity in patients evaluated for male infertility. Fertil Steril 85,

496-499.

Mostafa, T., Tawadrous, G., Roaia, M. M., Amer, M. K., Kader, R. A., and Aziz, A.

(2006). Effect of smoking on seminal plasma ascorbic acid in infertile and fertile males.


141

Moustafa, M. H., Sharma, R. K., Thornton, J., Mascha, E., Abdel-Hafez, M. A., Thomas,

A. J., Jr., and Agarwal, A. (2004). Relationship between ROS production, apoptosis and

DNA denaturation in spermatozoa from patients examined for infertility.

Hum Reprod 19, 129-138.

Netchine, I., Rossignol, S., Dufourg, M. N., Azzi, S., Rousseau, A., Perin, L., Houang, M.,

Steunou, V., Esteva, B., Thibaud, N., et al. (2007). 11p15 imprinting center region 1 loss

of methylation is a common and specific cause of typical Russell-Silver syndrome: clinical

scoring system and epigenetic-phenotypic correlations. J Clin Endocrinol Metab 92, 3148-

3154.

Nguyen, R. H., Wilcox, A. J., Skjaerven, R., and Baird, D. D. (2007). Men's body mass

index and infertility. Hum Reprod 22, 2488-2493.

Numata, M., Ono, T., and Iseki, S. (1994). Expression and localization of the mRNA for

DNA (cytosine-5)- methyltransferase in mouse seminiferous tubules. J Histochem

Cytochem 42, 1271-1276.

Oakes, C. C., Kelly, T. L., Robaire, B., and Trasler, J. M. (2007). Adverse effects of 5-aza-

2'-deoxycytidine on spermatogenesis include reduced sperm function and selective

inhibition of de novo DNA methylation. J Pharmacol Exp Ther 322, 1171-1180.

Ochsendorf, F. R. (1999). Infections in the male genital tract and reactive oxygen species.

Hum Reprod Update 5, 399-420.

Olinski, R., Nackerdien, Z., and Dizdaroglu, M. (1992). DNA-protein cross-linking

between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured

human cells. Arch Biochem Biophys 297, 139-143.

Oliw, E. H., and Sprecher, H. (1989). Metabolism of polyunsaturated fatty acids by an (n -

6)-lipoxygenase associated with human ejaculates. Biochim Biophys Acta 1002, 283-291.

Ollero, M., Gil-Guzman, E., Lopez, M. C., Sharma, R. K., Agarwal, A., Larson, K.,

Evenson, D., Thomas, A. J., Jr., and Alvarez, J. G. (2001). Characterization of subsets of

human spermatozoa at different stages of maturation: implications in the diagnosis and

treatment of male infertility. Hum Reprod 16, 1912-1921.

Omisanjo, O. A., Biermann, K., Hartmann, S., Heukamp, L. C., Sonnack, V., Hild, A.,

Brehm, R., Bergmann, M., Weidner, W., and Steger, K. (2007). DNMT1 and HDAC1 gene

expression in impaired spermatogenesis and testicular cancer. Histochem Cell Biol 127,

175-181.

Oosterhuis, G. J., Mulder, A. B., Kalsbeek-Batenburg, E., Lambalk, C. B., Schoemaker, J.,

and Vermes, I. (2000). Measuring apoptosis in human spermatozoa: a biological assay for

semen quality? Fertil Steril 74, 245-250.

Ozata, M., Mergen, M., Oktenli, C., Aydin, A., Sanisoglu, S. Y., Bolu, E., Yilmaz, M. I.,

Sayal, A., Isimer, A., and Ozdemir, I. C. (2002). Increased oxidative stress and

hypozincemia in male obesity. Clin Biochem 35, 627-631.

Ozmen, B., Koutlaki, N., Youssry, M., Diedrich, K., and Al-Hasani, S. (2007). DNA

damage of human spermatozoa in assisted reproduction: origins, diagnosis, impacts and

safety. Reprod Biomed Online 14, 384-395.

142

Palus, J., Rydzynski, K., Dziubaltowska, E., Wyszynska, K., Natarajan, A. T., and Nilsson,

R. (2003). Genotoxic effects of occupational exposure to lead and cadmium. Mutat Res

540, 19-28.

Paquin, R., Chapdelaine, P., Dube, J. Y., and Tremblay, R. R. (1984). Similar biochemical

properties of human seminal plasma and epididymal alpha-1,4-glucosidase. J Androl 5,

277-282.

Paracchini, V., Garte, S., and Taioli, E. (2006). MTHFR C677T polymorphism, GSTM1

deletion and male infertility: a possible suggestion of a gene-gene interaction? Biomarkers

11, 53-60.

Pathak, S., Kedia-Mokashi, N., Saxena, M., D'Souza, R., Maitra, A., Parte, P., Gill-

Sharma, M., and Balasinor, N. (2008). Effect of tamoxifen treatment on global and insulin-

like growth factor 2-H19 locus-specific DNA methylation in rat spermatozoa and its

association with embryo loss. Fertil Steril.

Pauli, E. M., Legro, R. S., Demers, L. M., Kunselman, A. R., Dodson, W. C., and Lee, P.

A. (2008). Diminished paternity and gonadal function with increasing obesity in men.


Peake, J. M., Suzuki, K., and Coombes, J. S. (2007). The influence of antioxidant

supplementation on markers of inflammation and the relationship to oxidative stress after

exercise. J Nutr Biochem 18, 357-371.

Pelliccione, F., D'Angeli, A., Cordeschi, G., Mihalca, R., Ciociola, F., Necozione, S.,

Francavilla, F., and Francavilla, S. (2008). Seminal macrophages in ejaculates from men

with couple infertility. Int J Androl.

Plante, M., de Lamirande, E., and Gagnon, C. (1994). Reactive oxygen species released by

activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal

sperm motility. Fertil Steril 62, 387-393.

Poccia, D. (1986). Remodeling of nucleoproteins during gametogenesis, fertilization, and

early development. Int Rev Cytol 105, 1-65.

Pollanen, P., and Cooper, T. G. (1994). Immunology of the testicular excurrent ducts. J

Reprod Immunol 26, 167-216.

Potts, R. J., Newbury, C. J., Smith, G., Notarianni, L. J., and Jefferies, T. M. (1999). Sperm

chromatin damage associated with male smoking. Mutat Res 423, 103-111.

Pratico, D., Lawson, J. A., Rokach, J., and FitzGerald, G. A. (2001). The isoprostanes in

biology and medicine. Trends Endocrinol Metab 12, 243-247.

Qin, D. D., Yuan, W., Zhou, W. J., Cui, Y. Q., Wu, J. Q., and Gao, E. S. (2007). Do

reproductive hormones explain the association between body mass index and semen

quality? Asian J Androl 9, 827-834.

Rajpert-De Meyts, E., Jorgensen, N., Graem, N., Muller, J., Cate, R. L., and Skakkebaek,

N. E. (1999). Expression of anti-Mullerian hormone during normal and pathological

gonadal development: association with differentiation of Sertoli and granulosa cells. J Clin

Endocrinol Metab 84, 3836-3844.

143

Rao, B., Soufir, J. C., Martin, M., and David, G. (1989). Lipid peroxidation in human

spermatozoa as related to midpiece abnormalities and motility. Gamete Res 24, 127-134.

Reinhardt, A., Haidl, G., and Schill, W. B. (1997). Granulocyte elastase indicates silent

male genital tract inflammation and appropriate anti-inflammatory treatment. Andrologia

29, 187-192.

Rich-Edwards, J. W., Spiegelman, D., Garland, M., Hertzmark, E., Hunter, D. J., Colditz,

G. A., Willett, W. C., Wand, H., and Manson, J. E. (2002). Physical activity, body mass

index, and ovulatory disorder infertility. Epidemiology 13, 184-190.

Risch, N., Reich, E. W., Wishnick, M. M., and McCarthy, J. G. (1987). Spontaneous

mutation and parental age in humans. Am J Hum Genet 41, 218-248.

Rook, G. A., Steele, J., Umar, S., and Dockrell, H. M. (1985). A simple method for the

solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human

macrophages by gamma-interferon. J Immunol Methods 82, 161-167.

Rosen, G. M., Pou, S., Ramos, C. L., Cohen, M. S., and Britigan, B. E. (1995). Free

radicals and phagocytic cells. Faseb J 9, 200-209.

Rubes, J., Selevan, S. G., Evenson, D. P., Zudova, D., Vozdova, M., Zudova, Z., Robbins,

W. A., and Perreault, S. D. (2005). Episodic air pollution is associated with increased DNA

fragmentation in human sperm without other changes in semen quality. Hum Reprod 20,

2776-2783.

Said, T. M., Agarwal, A., Falcone, T., Sharma, R. K., Bedaiwy, M. A., and Li, L. (2005).

Infliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human

spermatozoa: an in vitro model. Fertil Steril 83, 1665-1673.

Said, T. M., Kattal, N., Sharma, R. K., Sikka, S. C., Thomas, A. J., Jr., Mascha, E., and

Agarwal, A. (2003). Enhanced chemiluminescence assay vs colorimetric assay for

measurement of the total antioxidant capacity of human seminal plasma. J Androl 24, 676-

680.

Sakkas, D., Urner, F., Bizzaro, D., Manicardi, G., Bianchi, P. G., Shoukir, Y., and

Campana, A. (1998). Sperm nuclear DNA damage and altered chromatin structure: effect

on fertilization and embryo development. Hum Reprod 13 Suppl 4, 11-19.

Saleh, R. A., Agarwal, A., Sharma, R. K., Nelson, D. R., and Thomas, A. J., Jr. (2002).

Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a

prospective study. Fertil Steril 78, 491-499.

Saleh, R. A., Agarwal, A., Sharma, R. K., Said, T. M., Sikka, S. C., and Thomas, A. J., Jr.

(2003). Evaluation of nuclear DNA damage in spermatozoa from infertile men with

varicocele. Fertil Steril 80, 1431-1436.

Schaefer, C. B., Ooi, S. K., Bestor, T. H., and Bourc'his, D. (2007). Epigenetic decisions in

mammalian germ cells. Science 316, 398-399.

Schneider, G., Kirschner, M. A., Berkowitz, R., and Ertel, N. H. (1979). Increased estrogen

production in obese men. J Clin Endocrinol Metab 48, 633-638.

Schuppe, H. C., Meinhardt, A., Allam, J. P., Bergmann, M., Weidner, W., and Haidl, G.

(2008). Chronic orchitis: a neglected cause of male infertility? Andrologia 40, 84-91.

144

Selhub, J., Jacques, P. F., Rosenberg, I. H., Rogers, G., Bowman, B. A., Gunter, E. W.,

Wright, J. D., and Johnson, C. L. (1999). Serum total homocysteine concentrations in the

third National Health and Nutrition Examination Survey (1991-1994): population reference

ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med

131, 331-339.

Sepaniak, S., Forges, T., Fontaine, B., Gerard, H., Foliguet, B., Guillet-May, F., Zaccabri,

A., and Monnier-Barbarino, P. (2004). [Negative impact of cigarette smoking on male

fertility: from spermatozoa to the offspring]. J Gynecol Obstet Biol Reprod (Paris) 33, 384-

390.

Sepaniak, S., Forges, T., and Monnier-Barbarino, P. (2006). [Cigarette smoking and

fertility in women and men]. Gynecol Obstet Fertil 34, 945-949.

Sharma, R. K., and Agarwal, A. (1996). Role of reactive oxygen species in male infertility.

Urology 48, 835-850.

Sharma, R. K., Pasqualotto, A. E., Nelson, D. R., Thomas, A. J., Jr., and Agarwal, A.

(2001). Relationship between seminal white blood cell counts and oxidative stress in men

treated at an infertility clinic. J Androl 22, 575-583.

Sharma, R. K., Pasqualotto, F. F., Nelson, D. R., Thomas, A. J., Jr., and Agarwal, A.

(1999). The reactive oxygen species-total antioxidant capacity score is a new measure of

oxidative stress to predict male infertility. Hum Reprod 14, 2801-2807.

Sies, H. (1993). Strategies of antioxidant defense. Eur J Biochem 215, 213-219.

Sies, H. (1997). Oxidative stress: oxidants and antioxidants. Exp Physiol 82, 291-295.

Sikka, S. C., Rajasekaran, M., and Hellstrom, W. J. (1995). Role of oxidative stress and

antioxidants in male infertility. J Androl 16, 464-468.

Singh, K., Singh, S. K., Sah, R., Singh, I., and Raman, R. (2005). Mutation C677T in the

methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian

population. Int J Androl 28, 115-119.

Singh, N. P., Muller, C. H., and Berger, R. E. (2003). Effects of age on DNA double-strand

breaks and apoptosis in human sperm. Fertil Steril 80, 1420-1430.

Sloter, E., Nath, J., Eskenazi, B., and Wyrobek, A. J. (2004). Effects of male age on the

frequencies of germinal and heritable chromosomal abnormalities in humans and rodents.


Smith, D. C., Barratt, C. L., and Williams, M. A. (1989). The characterisation of non-

sperm cells in the ejaculates of fertile men using transmission electron microscopy.


Sofikitis, N., Miyagawa, I., Dimitriadis, D., Zavos, P., Sikka, S., and Hellstrom, W. (1995).

Effects of smoking on testicular function, semen quality and sperm fertilizing capacity. J

Urol 154, 1030-1034.

Solis, E. A., Bouvet, B. R., Brufman, A. S., Feldman, R., and Gatti, V. N. (2003). The

possible macrophage role in seminal fluid. Actas Urol Esp 27, 185-189.

Song, G. J., Norkus, E. P., and Lewis, V. (2006). Relationship between seminal ascorbic

acid and sperm DNA integrity in infertile men. Int J Androl 29, 569-575.

145

Sorahan, T., Prior, P., Lancashire, R. J., Faux, S. P., Hulten, M. A., Peck, I. M., and

Stewart, A. M. (1997). Childhood cancer and parental use of tobacco: deaths from 1971 to

1976. Br J Cancer 76, 1525-1531.

Spano, M., Kolstad, A. H., Larsen, S. B., Cordelli, E., Leter, G., Giwercman, A., and

Bonde, J. P. (1998). The applicability of the flow cytometric sperm chromatin structure

assay in epidemiological studies. Asclepios. Hum Reprod 13, 2495-2505.

Spruill, M. D., Song, B., Whong, W. Z., and Ong, T. (2002). Proto-oncogene amplification

and overexpression in cadmium-induced cell transformation. J Toxicol Environ Health A

65, 2131-2144.

Stahl, W., and Sies, H. (1996). Lycopene: a biologically important carotenoid for humans?

Arch Biochem Biophys 336, 1-9.

Steiber, A., Kerner, J., and Hoppel, C. L. (2004). Carnitine: a nutritional, biosynthetic, and

functional perspective. Mol Aspects Med 25, 455-473.

Storey, B. T. (1997). Biochemistry of the induction and prevention of lipoperoxidative

damage in human spermatozoa. Mol Hum Reprod 3, 203-213.

Strain, G. W., Zumoff, B., Miller, L. K., Rosner, W., Levit, C., Kalin, M., Hershcopf, R. J.,

and Rosenfeld, R. S. (1988). Effect of massive weight loss on hypothalamic-pituitary-

gonadal function in obese men. J Clin Endocrinol Metab 66, 1019-1023.

Stuppia, L., Gatta, V., Scarciolla, O., Colosimo, A., Guanciali-Franchi, P., Calabrese, G.,

and Palka, G. (2003). The methylenetethrahydrofolate reductase (MTHFR) C677T

polymorphism and male infertility in Italy. J Endocrinol Invest 26, 620-622.

Suleiman, S. A., Ali, M. E., Zaki, Z. M., el-Malik, E. M., and Nasr, M. A. (1996). Lipid

peroxidation and human sperm motility: protective role of vitamin E. J Androl 17, 530-

537.

Tarin, J. J., Brines, J., and Cano, A. (1998). Long-term effects of delayed parenthood. Hum

Reprod 13, 2371-2376.

Tavalaee, M., Razavi, S., and Nasr-Esfahani, M. H. (2008). Influence of sperm chromatin

anomalies on assisted reproductive technology outcome. Fertil Steril.

Tavalaee, M., Razavi, S., and Nasr-Esfahani, M. H. (2009). Influence of sperm chromatin

anomalies on assisted reproductive technology outcome. Fertil Steril 91, 1119-1126.

Thompson, J. G., Kind, K. L., Roberts, C. T., Robertson, S. A., and Robinson, J. S. (2002).

Epigenetic risks related to assisted reproductive technologies: short- and long-term

consequences for the health of children conceived through assisted reproduction

technology: more reason for caution? Hum Reprod 17, 2783-2786.

Tiemann-Boege, I., Navidi, W., Grewal, R., Cohn, D., Eskenazi, B., Wyrobek, A. J., and

Arnheim, N. (2002). The observed human sperm mutation frequency cannot explain the

achondroplasia paternal age effect. Proc Natl Acad Sci U S A 99, 14952-14957.

Tomlinson, M. J., Barratt, C. L., Bolton, A. E., Lenton, E. A., Roberts, H. B., and Cooke, I.

D. (1992a). Round cells and sperm fertilizing capacity: the presence of immature germ

146

cells but not seminal leukocytes are associated with reduced success of in vitro

fertilization. Fertil Steril 58, 1257-1259.

Tomlinson, M. J., Barratt, C. L., and Cooke, I. D. (1993). Prospective study of leukocytes

and leukocyte subpopulations in semen suggests they are not a cause of male infertility.


Tomlinson, M. J., White, A., Barratt, C. L., Bolton, A. E., and Cooke, I. D. (1992b). The

removal of morphologically abnormal sperm forms by phagocytes: a positive role for

seminal leukocytes? Hum Reprod 7, 517-522.

Trasler, J. M., Alcivar, A. A., Hake, L. E., Bestor, T., and Hecht, N. B. (1992). DNA

methyltransferase is developmentally expressed in replicating and non-replicating male

germ cells. Nucleic Acids Res 20, 2541-2545.

Tremellen, K. (2008). Oxidative stress and male infertility--a clinical perspective. Hum

Reprod Update 14, 243-258.

Tremellen, K., Miari, G., Froiland, D., and Thompson, J. (2007). A randomised control

trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-

ICSI treatment. Aust N Z J Obstet Gynaecol 47, 216-221.

Tremellen, K., and Tunc, O. Macrophage activity in semen is significantly correlated with

sperm quality in infertile men. Int J Androl. 33(6):823-31

Tunc, O., Thompson, J., and Tremellen, K. (2008). Development of the NBT assay as a

marker of sperm oxidative stress. Int J Androl. 33(1) :13-21

Turk, P. W., Laayoun, A., Smith, S. S., and Weitzman, S. A. (1995). DNA adduct 8-

hydroxyl-2'-deoxyguanosine (8-hydroxyguanine) affects function of human DNA

methyltransferase. Carcinogenesis 16, 1253-1255.

Twigg, J., Fulton, N., Gomez, E., Irvine, D. S., and Aitken, R. J. (1998a). Analysis of the

impact of intracellular reactive oxygen species generation on the structural and functional

integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness

of antioxidants. Hum Reprod 13, 1429-1436.

Twigg, J., Irvine, D. S., Houston, P., Fulton, N., Michael, L., and Aitken, R. J. (1998b).

Iatrogenic DNA damage induced in human spermatozoa during sperm preparation:

protective significance of seminal plasma. Mol Hum Reprod 4, 439-445.

Twigg, J. P., Irvine, D. S., and Aitken, R. J. (1998c). Oxidative damage to DNA in human

spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection.

Hum Reprod 13, 1864-1871.

Uzun, H., Zengin, K., Taskin, M., Aydin, S., Simsek, G., and Dariyerli, N. (2004).

Changes in leptin, plasminogen activator factor and oxidative stress in morbidly obese

patients following open and laparoscopic Swedish adjustable gastric banding. Obes Surg

14, 659-665.

Vaisberg, C. N., Jelezarsky, L. V., Dishlianova, B., and Chaushev, T. A. (2005). Activity,

substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine

reproductive organs and semen. Theriogenology 64, 416-428.

Valinluck, V., Tsai, H. H., Rogstad, D. K., Burdzy, A., Bird, A., and Sowers, L. C. (2004).

Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG

147

binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res 32,

4100-4108.

Vernet, P., Aitken, R. J., and Drevet, J. R. (2004). Antioxidant strategies in the epididymis.

Mol Cell Endocrinol 216, 31-39.

Villalta, J., Ballesca, J. L., Nicolas, J. M., Martinez de Osaba, M. J., Antunez, E., and

Pimentel, C. (1997). Testicular function in asymptomatic chronic alcoholics: relation to

ethanol intake. Alcohol Clin Exp Res 21, 128-133.

Villegas, J., Schulz, M., Vallejos, V., Henkel, R., Miska, W., and Sanchez, R. (2002).

Indirect immunofluorescence using monoclonal antibodies for the detection of

leukocytospermia: comparison with peroxidase staining. Andrologia 34, 69-73.

Virro, M. R., Larson-Cook, K. L., and Evenson, D. P. (2004). Sperm chromatin structure

assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing

pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril

81, 1289-1295.

Wang, J. X., Davies, M. J., and Norman, R. J. (2002). Obesity increases the risk of

spontaneous abortion during infertility treatment. Obes Res 10, 551-554.

Wang, X., Sharma, R. K., Sikka, S. C., Thomas, A. J., Jr., Falcone, T., and Agarwal, A.

(2003). Oxidative stress is associated with increased apoptosis leading to spermatozoa

DNA damage in patients with male factor infertility. Fertil Steril 80, 531-535.

Wang, Y. F., and Holstein, A. F. (1983). Intraepithelial lymphocytes and macrophages in

the human epididymis. Cell Tissue Res 233, 517-521.

Ward, W. S., and Coffey, D. S. (1991). DNA packaging and organization in mammalian

spermatozoa: comparison with somatic cells. Biol Reprod 44, 569-574.

Weiss, G., Thuma, P. E., Biemba, G., Mabeza, G., Werner, E. R., and Gordeuk, V. R.

(1998). Cerebrospinal fluid levels of biopterin, nitric oxide metabolites, and immune

activation markers and the clinical course of human cerebral malaria. J Infect Dis 177,

1064-1068.

Weitzman, S. A., Turk, P. W., Milkowski, D. H., and Kozlowski, K. (1994). Free radical

adducts induce alterations in DNA cytosine methylation. Proc Natl Acad Sci U S A 91,

1261-1264.

Whittington, K., and Ford, W. C. (1998). The effect of incubation periods under 95%

oxygen on the stimulated acrosome reaction and motility of human spermatozoa. Mol Hum

Reprod 4, 1053-1057.

Whittington, K., and Ford, W. C. (1999). Relative contribution of leukocytes and of

spermatozoa to reactive oxygen species production in human sperm suspensions. Int J

Androl 22, 229-235.

WHO, ed. (1999a). WHO Laboratory Manual for the examination of human semen and

sperm-cervical mucus interaction., 4th edn (Cambridge,UK: Cambridge University Press).

WHO (1999b). WHO Laboratory manual for the examination of human semen and sperm-

cervical mucus interaction., (Cambridge: Cambridge University Press).

148

WHO (2001). [Laboratory manual of the WHO for the examination of human semen and

sperm-cervical mucus interaction]. Ann Ist Super Sanita 37, I-XII, 1-123.

Williams, A. C., and Ford, W. C. (2005). Relationship between reactive oxygen species

production and lipid peroxidation in human sperm suspensions and their association with

sperm function. Fertil Steril 83, 929-936.

Wilton, L. J., Temple-Smith, P. D., Baker, H. W., and de Kretser, D. M. (1988). Human

male infertility caused by degeneration and death of sperm in the epididymis. Fertil Steril

49, 1052-1058.

Wolff, H. (1995). The biologic significance of white blood cells in semen. Fertil Steril 63,

1143-1157.

Wolff, H. (1998). Methods for the detection of male genital tract inflammation. Andrologia

30 Suppl 1, 35-39.

Wolff, H., and Anderson, D. J. (1988). Male genital tract inflammation associated with

increased numbers of potential human immunodeficiency virus host cells in semen.


Wolff, H., Bezold, G., Zebhauser, M., and Meurer, M. (1991). Impact of clinically silent

inflammation on male genital tract organs as reflected by biochemical markers in semen. J

Androl 12, 331-334.

Wolff, H., Politch, J. A., Martinez, A., Haimovici, F., Hill, J. A., and Anderson, D. J.

(1990). Leukocytospermia is associated with poor semen quality. Fertil Steril 53, 528-536.

Wu, D., and Cederbaum, A. I. (2003). Alcohol, oxidative stress, and free radical damage.

Alcohol Res Health 27, 277-284.

Wyrobek, A. J., Eskenazi, B., Young, S., Arnheim, N., Tiemann-Boege, I., Jabs, E. W.,

Glaser, R. L., Pearson, F. S., and Evenson, D. (2006). Advancing age has differential

effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm.

Proc Natl Acad Sci U S A 103, 9601-9606.

Wyrobek, A. J., Schmid, T. E., and Marchetti, F. (2005). Relative susceptibilities of male

germ cells to genetic defects induced by cancer chemotherapies. J Natl Cancer Inst

Monogr, 31-35.

Yeo, H. C., Helbock, H. J., Chyu, D. W., and Ames, B. N. (1994). Assay of

malondialdehyde in biological fluids by gas chromatography-mass spectrometry. Anal

Biochem 220, 391-396.

Yeung, C. H., and Cooper, T. G. (1994). Study of the role of epididymal alpha-glucosidase

in the fertility of male rats by the administration of the enzyme inhibitor castanospermine.

J Reprod Fertil 102, 401-410.

Yi, P., Melnyk, S., Pogribna, M., Pogribny, I. P., Hine, R. J., and James, S. J. (2000).

Increase in plasma homocysteine associated with parallel increases in plasma S-

adenosylhomocysteine and lymphocyte DNA hypomethylation. J Biol Chem 275, 29318-

29323.

Yoganathan, T., Eskild, W., and Hansson, V. (1989). Investigation of detoxification

capacity of rat testicular germ cells and Sertoli cells. Free Radic Biol Med 7, 355-359.

149

Young, A. J., and Lowe, G. M. (2001). Antioxidant and prooxidant properties of

carotenoids. Arch Biochem Biophys 385, 20-27.

Zalata, A. A., Ahmed, A. H., Allamaneni, S. S., Comhaire, F. H., and Agarwal, A. (2004).

Relationship between acrosin activity of human spermatozoa and oxidative stress. Asian J

Androl 6, 313-318.

Zhang, Y., Kreger, B. E., Dorgan, J. F., Cupples, L. A., Myers, R. H., Splansky, G. L.,

Schatzkin, A., and Ellison, R. C. (1999). Parental age at child's birth and son's risk of

prostate cancer. The Framingham Study. Am J Epidemiol 150, 1208-1212.

Zini, A., Boman, J. M., Belzile, E., and Ciampi, A. (2008). Sperm DNA damage is

associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review

and meta-analysis. Hum Reprod 23, 2663-2668.

Zini, A., de Lamirande, E., and Gagnon, C. (1993). Reactive oxygen species in semen of

infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal

plasma and spermatozoa. Int J Androl 16, 183-188.

Zini, A., De Lamirande, E., and Gagnon, C. (1995). Low levels of nitric oxide promote

human sperm capacitation in vitro. J Androl 16, 424-431.

Zini, A., Defreitas, G., Freeman, M., Hechter, S., and Jarvi, K. (2000). Varicocele is

associated with abnormal retention of cytoplasmic droplets by human spermatozoa. Fertil

Steril 74, 461-464.

Zini, A., San Gabriel, M., and Libman, J. Lycopene supplementation in vitro can protect

human sperm deoxyribonucleic acid from oxidative damage. Fertil Steril 94, 1033-1036.

Ziyyat, A., Barraud-Lange, V., Sifer, C., Ducot, B., Wolf, J. P., and Soufir, J. C. (2008).

Paradoxical increase of sperm motility and seminal carnitine associated with moderate

leukocytospermia in infertile patients. Fertil Steril 90, 2257-2263.

Zopfgen, A., Priem, F., Sudhoff, F., Jung, K., Lenk, S., Loening, S. A., and Sinha, P.

(2000). Relationship between semen quality and the seminal plasma components carnitine,

alpha-glucosidase, fructose, citrate and granulocyte elastase in infertile men compared with

a normal population. Hum Reprod 15, 840-845.

Zorn, B., Sesek-Briski, A., Osredkar, J., and Meden-Vrtovec, H. (2003a). Semen

polymorphonuclear neutrophil leukocyte elastase as a diagnostic and prognostic marker of

genital tract inflammation--a review. Clin Chem Lab Med 41, 2-12.

Zorn, B., Vidmar, G., and Meden-Vrtovec, H. (2003b). Seminal reactive oxygen species as

predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro

fertilization and intracytoplasmic sperm injection. Int J Androl 26, 279-285.

Zorn, B., Virant-Klun, I., and Meden-Vrtovec, H. (2000). Semen granulocyte elastase: its

relevance for the diagnosis and prognosis of silent genital tract inflammation. Hum Reprod

15, 1978-1984.

Zubkova, E. V., Wade, M., and Robaire, B. (2005). Changes in spermatozoal chromatin

packaging and susceptibility to oxidative challenge during aging. Fertil Steril 84 Suppl 2,

1191-1198.

“investigation of the role of oxidative stress in male

Documents