02 Whole

“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
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
ii
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.
iii
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
iv
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 α-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
v
CHAPTER 4:Seminal Inflammation as a Cause of Oxidative
Stress and Macrophage Activity in Infertile Men’s semen Page 66
4.1 Abstract
4.2 Introduction
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

5.1 Abstract
5.2 Introduction
5.4 Results
5.5 Discussion
CHAPTER 6: Treatment of Oxidative DNA Damage in sperm

using an Oral Antioxidant Therapy Page 91
6.1 Abstract
6.2 Introduction
6.4 Results
6.5 Discussion
CHAPTER 7: Oxidative Stress as a cause of sperm DNA

Hypomethylation Page105
7.1 Abstract
7.2 Introduction
7.4 Results
7.5 Discussion
CHAPTER 8: Final Discussion and Conclusions Page 119

8.1 Final Discussion
8.2 Future Directions
Bibliography Page 126
vi
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
vii
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
o
C …………………………………………………………………………………………………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
viii
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
ix
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.
x
CHAPTER 1
INTRODUCTION
xi
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).
1
Table 1.1: Most common Reactive Oxygen Species including nitrogen derived
free radicals and reactive oxidants. Summarised from (Halliwell, 1999).
REACTIVE GENERATION EFFECT
OXYGEN SPECIES
Superoxide One electron reduction state of oxgen. Oxidising and reducing agent .
Primary form of ROS. Generated in auto- 1- Dismutation to hydrogen peroxide
Anion
oxidation reactions, Electron transport chain and Hydroxyl radicals 2-Formation of
._
(O2 ) in mitochondria/endoplasmic reticulum thiyl radicals by reaction with thiol
groups
3-Generation of peroxynitrite
Hydroxyl Three-electron reduction state of oxygen. Highly reactive and very short half-
Formed by the reaction of superoxide and life oxygen radical. The most toxic
Radical
Hydrogen peroxide accompanied by metal ROS. Attacks all cellular components.
._
(OH ) catalyst (Haber-Weiss Reaction); Main reactions of hydroxyl radical:
. . hydrogen abstraction (inititiation of
O2 + H2O2 O2 + OH + OH-
lipid peroxidation),
or free iron with Hydrogen Peroxide (Fenton Addition Reaction(addition to purin
Reaction); base guanine in DNA forms an 8-
. - hydroxyguanine),
Fe +2
+ H2O2 Fe +3
+ OH + OH
Electron-transfer reactions.
Hydrogen Two electron reduced state of oxygen. High biological diffusion, easily
. transverse the plasma and nuclear
Peroxide Formed by dismutation of O2 in
membranes. Formation of DNA
(H2O2) mitochondria during oxidative adduct, affects signalling and tissue
phosphorylation or produced as by-product of injury processes
several oxidases. It is not radical itself.
Singlet Lowest excited state of oxygen molecule. Oxidising agent. Transfers its
Dismutation of superoxide anion. In vitro, excitation energy to another
Oxygen
singlet oxygen can be produced by molecule. Effective in carbon-carbon
(1O2 ) photosensitization. double bounds, such as carotenoids
and fatty acids.
Peroxyl (ROO•) Produced in the presence of oxygen during Oxidising agents.Oxidize ascorbate
the breakdown of organic peroxides and and NADH. Abstract Hydrogen ion
and alkoxyl(RO•)
reaction of carbon radicals with oxygen. from other molecules which is
Radicals
important in lipid peroxidation.
Nitric Oxide Nitric Oxide is formed from the reaction of Transmitter substance reacts with
amino acid L-arginine by NO synthase. NO is superoxide anion to make
(NO•, NOO•)
not a radical but can form radicals. peroxynitrite (OONO-) which actively
reacts with glutathione, cysteine,
deoxyribose and thiols.
Hypochlorous acid Formed from hydrogen peroxide by Highly reactive and lipid soluble.
myeloperoxidase Oxidise protein components, thiol and
(HOCl)
amino groups and methionine.
2
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
3
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).
4
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 Low-molecular mass agents

Proteins
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).
5
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.
6
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
7
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
8
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.
9
NOTE:
This figure is included on page 10
of the print copy of the thesis held in
the University of Adelaide Library.
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
10
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
11
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
12
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.,
13
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
14
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).
15
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.
LIFESTYLE FACTORS
Smoking
Alcohol
Stress
Age
ENVIRONMENTAL
FACTORS
ANATOMICAL Toxins
- Varicocele Chemicals
EM Radiation
Chemotheraphy
Drugs
Heat
OXIDATIVE
STRESS
in Male Infertility
IATROGENIC INFECTIONS
Centrifugation Male Accessory Gland

Cryopreservation Inf.(MAGI)
Infective Inflammatory
Inflammatory
–eg vasectomy
Figure 1.3 The origins of Oxidative Stress in semen
16
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
17
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
18
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).
19
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)
20
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
21
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
22
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
23
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
24
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
25
CHAPTER 2
MATERIALS AND METHODS
26
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.
27
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 %.
28
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
29
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
30
o
gently beneath the media and after incubation for 1h at 37 C, 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
TM
onto a single discontinuous Percoll 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
31
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.
32
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.
33
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.
34
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.
35
Standard Concentration (µM MDA) 0 0,5 1 2 3 4
Volume of 20 µM standard to add 0 25 50 100 150 200

(mL)
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.
36
Nitrotetrazolium Formazan
-
O2.
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.
37
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
o
incubated at 37 C 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
38
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%
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
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)
39
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.
40
A
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.
41
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).
42
Apoptotic Cells: An. V+ PI –
Necrotic Cells: An.V+ PI+
Live Cells: An.V - PI -
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
43
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 α -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;
44
4-Nitrophenyl- α -D-glucopyranoside
α-glucosidase
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
45
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
o
frozen at – 70 C 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.
46
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
47
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).
48
CHAPTER 3
Development of the Nitro Blue Tetrazolium (NBT)
Assay as a marker of sperm Oxidative Stress
49
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.
50
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
51
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).
3.3.1Subjects 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.
52
3.3.2 Sample Preparation
Sample preparation and the assays used in this study was discussed in Chapter
2.4

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.
53
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.
0.15
Formazan Absorbance 630nm
0.13
0.11
0.09
0.07
0.05
0.03
0.01
0 15 30 60 120
2 x 105 sperm/mL Minutes

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
54
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.
1200 1.000
Formazan Absorbance (630 nm)

Formazan µg/107 sperm
1000
0.800
800
0.600
600
0.400
400
0.200
200
0 0.000
0 50 100 150 200 250 300 350 400 450 500
Sperm Concentration (x106/mL)
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 (µg) on the left y axis.
55
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.
Formazan Absorbance (630 nm)
0.800
0.600
0.400
0.200
0.000
0.00 0.05 0.10 0.15 0.20 0.25
Xanthine (mmol/L)
Figure 3.2B: Formazan production using the chemical Xanthine /

Xanthine Oxidase (X/XO) ROS production system. A fixed
concentration of Xanthine Oxidase (2µL 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.
56
Fertile Group Infertile Group
(n = 21) (n = 36) P
Median Median
(25%-75% range) (25%-75% Range)
Age (years) 38 (33.5 – 41.5) 38 (35 – 42) 0.904
ROS Production
(µg
16 (5.5 – 22.5) 65.5 (28.0 – 99.8) <0.0001
Formazan/107
sperm)
TUNEL
9.6 (8.2 – 12.6) 20.7 (14.4 – 25.9) <0.0001
positivity (%)
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 107 (58.3–205.3) 26.6 (10.5 – 47.9) <0.0001
(x10 6cell/mL
PMN Elastase
86.8 (36.5–169.0) 45.5 (15.5– 202.3) 0.529
(ng/mL)
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.
57
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.
58
80
60 r = - 0.479
Motility %
P =0.0002
40
20
Figure 3.3A
0
0 100 200 300
7
60 r = 0.669
P <0.0001
Annexin V %
40
20
Figure 3.3B
0
0 100 200 300
7
50
r = 0.591
P <0.0001
TUNEL positivity %
40
30
20
10
0 Figure 3.3C
0 100 200 300
Formazan µg/10 7sperm
Figure 3.3: Linear regression analysis depicting the relationship

between ROS production (µg 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.
59
800
600 r = 0.006
PMN Elastase
P = 0.965
(ng/mL.)
400
200
0
0 100 200 300
Formazan µg/107sperm
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).
200
150
Formazan µ g
100
Sperm
Leukocyte
50
0
1x106 cell/mL 4x106 cell/mL
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.
60
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
61
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.
62
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
63
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.
64
However, the NBT assay holds considerable promise because of its accuracy in
identifying oxidatively damaged sperm and its relatively inexpensive and ease
of conduct.
65
CHAPTER 4
Seminal Inflammation as a Cause of Oxidative

Stress and Macrophage Activity in Infertile Men’s
semen
66
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.
67
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
68
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
69
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.
70

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.
71
Table 4.1: Sperm Quality Parameters in infertile and fertile groups.
Infertile Group Fertile Group P

(n=63) (n=11)
Sperm Concentration
25.8 (11.65–52.23) 97.5 (53 – 201) 0.0001
(x106 cell/ml)
Motility
42 (30 – 48) 55 (49 – 57) 0.0001
(%)
Morphology
6 ( 2– 11) 21 (15 – 25) 0.0002
(%)
Semen Volume
2.8 (1.8 – 4.0) 3.3 (2.7 – 4.5) 0.343
(mL)
ROS Production
64.0 (31.8 – 88.0) 11.9(4.7–20.0) <0.0001
(µg Formazan/107 sperm)
Lipid Peroxidation
1.3 ( 0.4– 3.5) 0.05(0.01–0.14) <0.0001
(µM MDA x107/sperm)
TUNEL
20.2 (13.9 – 25.8) 9.6 (8.1– 13.6) 0.0001
(%)
Apoptosis
23.5 (18.2 – 33.9) 8.6 (5.4– 13.9) <0.0001
(Annexin V) (%)
CD 45
1.0 (0.6 – 1.9) 0.9 (0.6 – 1.2) 0.404
(x106 cell/ml)
PMN Elastase
123 (22.8–275.3) 49 (10.5–118.5) 0.042
(ng/mL)
Neopterin 0.013
96.9 (53 – 186) 42 (28 - 99)
(ng/mL)
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).
72
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).
73
Table 4.2: Comparison between sperm quality between and inflammatory markers
in all groups t= statistically significant correlation
TUNEL LPO Formazan Volume Morpholo Motility Sperm CD45 PMN Neopterin
gy Concentra Elastase
tion
PMN Elastase
0.028
CD45
0.472 t -0.019
Sperm Conc.
0.301 0.079 -0.309t
Motility
0.472 t 0.03 -0.014 -0.200
Morphology
0.470 t 0.487 t 0.072 0.052 -0.325 t
Volume
-0.091 -0.118 0.083 -0.269 -0.075 0.0851
Formazan
-0.053 -0.548 t -0.409 t -0.778 t -0.113 0.201 0.477 t
LPO
0.812 t -0.024 -0.496 t -0.467 t -0.733 t -0.276 0.001 0.425 t
TUNEL
0.686 t 0.631 t 0.002 -0.473 t -0.693 t -0.644 t -0.011 0.109 0.340 t
74
AnnexinV
0.806t 0.757t 0.688t -0.082 -0.405t -0.602t -0.691t -0.112 0.039 0.481 t
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
r p
Neutral α−glucosidase
α− 0.088 0.496
LH -0.176 0.276
FSH 0.031 0.843
Testosterone -0.090 0.559
AMH 0.004 0.976
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
75
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
76
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
77
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
78
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.
79
CHAPTER 5
Obesity as a Cause of Oxidative Stress
80
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.
81
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
82
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
83
(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).
84
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.
400
Formazan µ g/107 sperm
300
200
r = 0.23
100 p = 0.039
0
20 25 30 35 40 45
BMI
Figure 5.1: Correlation between ROS production (µg formazan
per 107sperm) and BMI.
1000
Neopterin nmol/L
800
600
400 r = 0.238
p = 0.047
200
0
15 20 25 30 35 40
BMI
Figure 5.2: Correlation between seminal plasma neopterin
concentration (nmol/L) and BMI
85
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).
50
TUNEL positivity %
40
30
20
r = 0.13
p = 0.260
10
0
20 25 30 35 40 45
BMI
Figure 5.3: Linear regression analysis between sperm DNA

damage (TUNEL positivity %) and Body Mass Index (BMI).
86
400
Sperm Conc. (x106/mL) 300
200
r = -0.33
100 p = 0.002
0
20 25 30 35 40 45
BMI
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.
30 150
Testosterone (nmol/L)
Estradiol (pmol/L)
20 100
r = - 0.29
r = 0.35
10 50
p = 0.039 p = 0.026
0
20 25 30 35 40
0
20 25 30 35 40
BMI
BMI
Figure 5.5: Depiction of the correlation between BMI and serum
testosterone and estradiol concentrations.
87
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
88
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,
89
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.
90
CHAPTER 6
Treatment of Oxidative DNA Damage in sperm using
an Oral Antioxidant Therapy
91
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.
92
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
93
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
94
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.
95
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
96
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
ENTRY EXIT
P
n=45 Median Median
(25%-75% range) (25%-75% range)
DNA fragmentation
22.2 (16.5 – 26.6) 18.2 (13.4 – 23.1) 0.002
(%)
Early Apoptosis
27.3 (20.4 – 34.7) 22.5 (19.3 – 28.1) 0.004
(%)
(Annexin V +, PI -)
Protamination
69 (63.5 – 73.1) 73.6 (69.3 – 77.5) <0.001
(%)
ROS production
µg Formazan/ 107
(µ 66.4 (43.1 – 87.8) 44.4 (33.3 – 81.4) 0.027
sperm)
97
Table 6.3 Routine semen assessment during antioxidant treatment
ENTRY EXIT P
Concentration
b
36.2 ± 46.2 39.4 ± 44.7 0.304
6
(10 sperm/ mL)
Motility
a
36.5 ± 12.3 37.1 ± 11.5 0.778
(%)
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

b
38.4 ± 56.4 43.9 ± 64.5 0.337
(106 per ejaculate)
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.
98
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.
99
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
100
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
101
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
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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
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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.
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CHAPTER 7
Oxidative Stress as a cause of sperm DNA
Hypomethylation
105
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.
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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,
107
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;
108
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
109
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)
110
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
FERTILE INFERTILE p
SPERM QUALITY n= 12 n=45
PARAMETERS
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 104.7± 11.3 93.96±30.99 0.095
(au)
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.
111
50
(TUNEL Positive %)
DNA Fragmentation
40
30
20 r = -0.311
p = 0.02
10
0
0
50
0
10
15
20
Global DNA methylation
(au)
Figure 7.1A: Relationship between Sperm DNA fragmentation

(TUNEL) and Global DNA Methylation.
400
(Formazan µ g/107 sperm)
ROS Production
300
200
r = -0.378
100 p = 0.004
0
0 50 100 150 200
Global DNA methylation
(au)
Figure 7.1B: Relationship between seminal ROS production and

Global DNA Methylation
112
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 Pre-antioxidant On anti-oxidant P

QUALITY supplementation supplementation
PARAMETERS
n = 45
Sperm
Concentration 21.7(12.08 – 38.33) 21.4(12.75–44.1) 0.304
(x106/mL)
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
22.6 (16.5 – 28.8) 18.2 (13.4 – 23.1) 0.002
(TUNEL Positive%)
Sperm Global
DNA Methylation
93.96 ± 30.99 108.61± 35.54 0.0017
(a.u.)
113
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
114
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
115
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).
116
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
117
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.
118
CHAPTER 8
Final Discussion and Conclusions
119
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
120
(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
121
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
122
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
123
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.
124
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.
125
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.
126
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).
127
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.
128
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.
129
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
130
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.
131
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.
132
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.
133
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.
134
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.
135
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.
136
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.
137
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.
138
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.
139
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.
140
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.
141
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.
142
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.
143
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.
144
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
145
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
146
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).
147
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.
148
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.
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“INVESTIGATION OF THE ROLE OF OXIDATIVE

STRESS IN MALE INFERTILITY”

Thesis submitted for the total fulfilment for the degree of

It is a pleasure to finally be able to thank those that have helped me complete

My gratitude also goes to the staff of Andrology Laboratory in Repromed,

CHAPTER 1: INTRODUCTION Page1

1.8 Assessment of Seminal Oxidative Stress

CHAPTER 3: Development of the NBT Assay as a marker of

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

CHAPTER 6: Treatment of Oxidative DNA Damage in sperm

CHAPTER 7: Oxidative Stress as a cause of sperm DNA

CHAPTER 8: Final Discussion and Conclusions Page 119

Bibliography Page 126

2 - “Improvement in sperm DNA quality using an oral antioxidant therapy”

3 - “Oxidative DNA damage impairs global sperm DNA methylation in

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

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

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

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

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

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

1.1.1 Definition of Free Radicals

Figure 1.1: The pathway of oxygen reduction during cellular respiration

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

1.2 Antioxidant Defence Systems

The body has developed antioxidant defense systems by scavenging and

Glutathione peroxidase catalyses the reduction of hydrogen peroxide and lipid

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).

will be discussed in the following sections.

Table 1.2: Most important in vivo antioxidants

Superoxide Dismutase (SOD) Metal Binding Low-molecular mass agents

1.3 Oxidative Stress

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

1.3.1 Oxidative Stress and Male Infertility

1.4 Sources of Reactive Oxygen Species in semen

1.4.1. Production of ROS by Leukocytes in Semen

1.4.2. Production of ROS by Spermatozoa

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

1.4.3 Susceptibility of sperm cells to Oxidative Stress

1.5 Protection of Spermatozoa from ROS

1.5.1 Sperm Chromatin Structure

Diagram 1.1: Schematic diagram of condensation of DNA in somatic and sperm

1.5.2 Antioxidant Mechanisms in semen

1.5.2.1 Enzymatic Antioxidants

SOD protects spermatozoa against spontaneous O2 toxicity and lipid

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

1.5.2.2 Non-enzymatic Antioxidants

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

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

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

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

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

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

Carnitine is an antioxidant which is biosynthesized from lysine and methionine

1.6 Oxidative Stress in Male Infertility

1.6.1 Pathophysiology of Oxidative Stress in Spermatozoa

At higher concentrations of ROS, oxidative damage to polyunsaturated fatty

Figure 1.2: Reactive Oxygen induced damage in sperm cell

Mitochondrial exposure to ROS provokes apoptotic process through release of