Ebook Agarwal - Strategies To Ameliorate Oxidative Stress During Assisted Reproduction (2014)

SPRINGER BRIEFS IN REPRODUC TIVE BIOLOGY
Ashok Agarwal
Damayanthi Durairajanayagam
Gurpriya Virk
Stefan S. Du Plessis
Strategies to
Ameliorate
Oxidative Stress
During Assisted
Reproduction
123
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Ashok Agarwal • Damayanthi Durairajanayagam
Gurpriya Virk • Stefan S. Du Plessis
Strategies to Ameliorate
Oxidative Stress During
Assisted Reproduction
Ashok Agarwal Damayanthi Durairajanayagam
Center for Reproductive Medicine Discipline of Physiology
Cleveland Clinic Faculty of Medicine
Cleveland, OH, USA MARA University of Technology
Kuala Lumpur, Malaysia
Gurpriya Virk
Melbourne, VIC, Australia Stefan S. Du Plessis
Division of Medical Physiology
Stellenbosch University
Stellenbosch, South Africa
ISSN 2194-4253 ISSN 2194-4261 (electronic)

ISBN 978-3-319-10258-0 ISBN 978-3-319-10259-7 (eBook)
DOI 10.1007/978-3-319-10259-7
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© The Author 2014

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Preface
Assisted reproduction has become a very common treatment option for couples
dealing with infertility. Despite the successes of these in vitro technologies, it is
impossible to fully reproduce the in vivo environment. Not only are the gametes and
embryos exposed to unnatural conditions, but they are also manually manipulated.
All of these interventions can lead to the generation of excess reactive oxygen spe-
cies. Under such conditions, natural antioxidant defences cannot preclude these
pathological ROS levels. Ultimately, this leads to the development of oxidative
stress which affects gamete survival, fertilization and embryogenesis negatively. It
is therefore imperative to find and pursue solutions, such as antioxidant treatment,
that may help ameliorate this process.
Antioxidant therapies in ART are written by leaders in the field of oxidative
stress in both male and female reproductive medicine. This manuscript aims to
bridge the gap between basic research, the role of the embryologist as well as the
clinician with regard to antioxidant treatment both in vivo and in the ART labora-
tory. It eloquently covers all aspects of ROS generation (both endogenous and exog-
enous) as well as the complex interplay of oxidative stress in the ART setting.
Various antioxidants and their plausible therapeutic effects to minimize ROS and
oxidative stress are also comprehensively discussed.
Cleveland, OH, USA Ashok Agarwal

Kuala Lumpur, Malaysia Damayanthi Durairajanayagam
Melbourne, VIC, Australia Gurpriya Virk
Stellenbosch, South Africa Stefan S. Du Plessis
v
Contents
1 Introduction .............................................................................................. 1
2 Sources of ROS in ART ........................................................................... 3
2.1 In Vivo Generation of ROS ............................................................... 4
2.1.1 In Vivo Generation of ROS in the Male ................................ 4
2.1.2 In Vivo Generation of ROS in Females ................................. 7
2.2 In Vitro Generation of ROS in ART .................................................. 13
2.2.1 Cryopreservation ................................................................... 13
2.2.2 Visible Light .......................................................................... 15
2.2.3 ROS from Gamete Manipulation/ART Technique ................ 16
2.2.4 pH and Temperature .............................................................. 17
2.2.5 Centrifugation ....................................................................... 18
2.2.6 Culture Media ....................................................................... 19
2.2.7 Oxygen Concentration .......................................................... 21
3 Antioxidant Strategies ............................................................................. 23
3.1 Role of Enzymatic Antioxidants in ART .......................................... 24
3.1.1 Superoxide Dismutases ......................................................... 29
3.1.2 Catalase ............................................................................... 30
3.1.3 Glutathione Peroxidase ......................................................... 31
3.2 Role of Non-enzymatic Antioxidants in ART ................................... 31
3.2.1 Vitamin E .............................................................................. 32
3.2.2 Vitamin C .............................................................................. 33
3.2.3 Melatonin .............................................................................. 34
3.2.4 Vitamin B: Folic Acid ........................................................... 35
3.2.5 Coenzyme Q10 ....................................................................... 36
3.2.6 L-Carnitine............................................................................. 37
4 Role of Combined Antioxidants.............................................................. 39
5 Conclusion ................................................................................................ 41
References ......................................................................................................... 43
vii
List of Figures
Fig. 2.1 Sources of reactive oxygen species in assisted

reproductive technology. .................................................................. 5
Fig. 3.1 Effects of pathological levels of reactive oxygen species
in assisted reproductive technology ................................................. 24
Fig. 5.1 Sources of oxidative stress and interventions to overcome
its effects during assisted reproductive technology .......................... 42
ix
List of Tables
Table 2.1 Clinical parameters of studies examining reactive oxygen

species in follicular fluid in infertile women ................................. 9
Table 3.1 Role of antioxidants in ameliorating the effects
of oxidative stress in assisted reproduction ................................... 25
xi
Authors
Ashok Agarwal is a Professor at the Lerner College

of Medicine, Case Western Reserve University and
the Head of the Andrology Center and Director of
Research at the Center for Reproductive Medicine,
Cleveland Clinic, USA. He has researched exten-
sively on oxidative stress and its implications on
human fertility and has published over 500 original
research articles and reviews. He serves on the edi-
torial boards of several key journals in human
reproduction and has edited over 20 medical text
books/manuals related to male infertility, ART, fer-
tility preservation, DNA damage and antioxidants.
Ashok’s current research interests are the study of
molecular markers of oxidative stress, DNA fragmentation and apoptosis using pro-
teomics and bioinformatics tools, as well as fertility preservation in patients with
cancer, and the efficacy of certain antioxidants in improving male fertility.
Stefan S. du Plessis is a Professor and the Head of

Medical Physiology at Stellenbosch University,
South Africa, where he also heads the Reproductive
Research Group. His research interests include fac-
tors that influence male gamete function and he has
published more than 70 scientific articles and book
chapters. Dr. du Plessis serves as editorial board
member for two leading journals, is an NRF-rated
researcher, and is currently a Fulbright Researcher at
the Cleveland Clinic’s Center for Reproductive
Medicine.
xiii
xiv Authors
Damayanthi Durairajanayagam, Ph.D. is a Senior

Lecturer in Physiology at the Faculty of Medicine,
MARA University of Technology, Malaysia. She
received a Fulbright Scholarship for her research at
the Center for Reproductive Medicine, Cleveland
Clinic. Her research interests include oxidative stress
and the use of proteomics and bioinformatics in
studying the molecular markers of oxidative stress in
infertile males.
Gurpriya Virk has a Master’s in Clinical

Embryology from Monash University, Melbourne,
Australia. She did her research training at the
Center for Reproductive Medicine, Cleveland
Clinic. She is interested in investigating the role of
oxidative stress in assisted reproduction.
Chapter 1
Introduction
Natural conception involves a series of intricate and well-orchestrated physiological

processes in both male and female partners to ultimately culminate in a successful
pregnancy. While conceiving a baby may come naturally for the majority of cou-
ples, about 15 % of all couples face infertility and achieving a pregnancy is very
likely to require clinical intervention. Infertility is defined as the inability to con-
ceive or carry a pregnancy to full term, after a year of regular, unprotected sexual
intercourse. Assisted reproductive technology (ART) has been gaining popularity
over recent years as it offers hope and a means for couples with less than ideal
reproductive parameters or situations to conceive a baby. In vitro fertilization (IVF)
and intracytoplasmic injection (ICSI) are two of the commonly used interventions
in ART. While these interventions attempt to mimic as closely as possible the envi-
ronment and conditions under which natural fertilization takes place, the exact same
conditions cannot be recreated precisely in the laboratory setting. In fact, there are
multiple factors during the various steps in ART that can hinder its outcome. One of
the key causes of defective gametes, or non- or poorly-developing embryos in ART
is the development or presence of oxidative stress while handling the gametes or
embryos during the procedure.
Attempting to conceive through assisted reproduction is not only emotionally and
physically taxing, but also carries a large financial cost. Thus, the goal for the couple
going into the chosen technique would be to improve the quality of their gametes as
much as possible, and simultaneously minimize the effect of oxidative stress (OS)
that may be incurred during the mechanical manipulation of the gametes and
embryos during the procedure, in order to optimize the ART outcome. This could be
done by performing deliberate measures to reduce any incidental development of OS
as well as to boost the antioxidant capacity of the gamete to withstand the detrimen-
tal assault of oxidation. Some of the exogenous sources/factors that can contribute
to the generation of OS include the use of cryopreserved spermatozoa, exposure
of the gamete to visible light, centrifugation, high temperatures, culture media and
handling time, gamete manipulation, and incubator environment.
© The Author 2014 1

A. Agarwal et al., Strategies to Ameliorate Oxidative Stress During
Assisted Reproduction, SpringerBriefs in Reproductive Biology,
DOI 10.1007/978-3-319-10259-7_1
2 1 Introduction
Multiple research studies have previously investigated and reported the effects of
various enzymatic and non-enzymatic antioxidant therapies on sperm parameters,
fertilization rate, blastocyst development, embryo transfer and pregnancy outcome.
Thus, the aim of this monograph is to review the current antioxidant strategies and
how it can be used in a clinical setting to minimize the damages to the gamete and/
or embryo caused by OS during ART.
Chapter 2
Sources of ROS in ART
Reactive oxygen species (ROS) are molecules derived from oxygen, which occur as
a by-product of cellular oxidative respiration. ROS are free radical molecules
containing one or more unpaired electrons in their outer shell. They tend to remove
an electron from surrounding molecules to complete their octet. As a ROS stabilizes
itself with the addition of an electron, the molecule from which it removed the elec-
tron now becomes a free radical. In this manner, a self-propagating chain reaction that
produces ROS is generated. As ROS goes on to react with other molecules, the mol-
ecule undergoes structural and functional modifications (Sharma and Agarwal 1996).
Commonly-occurring ROS in basal conditions, such as superoxide anion (O2−),
hydrogen peroxide (H2O2) and hydroxyl radical (OH−), are powerful oxidants that
are found in low concentrations in the genital tract of both males and females
(Guerin et al. 2001; Agarwal and Allamaneni 2004). In the electron transport chain,
oxygen functions as the final electron acceptor. Most oxygen ions bind with hydro-
gen ions to form water molecules, but oxygen ions that do not undergo this reaction,
are released from the mitochondria in the form of free radicals. As levels of ROS
increase, the cell’s antioxidant defences become overwhelmed and are inadequate in
scavenging the unstable metabolites of oxygen, leading to a state of oxidative stress
(OS) (Pasqualotto et al. 2004).
A subset of ROS is the nitrogen-containing compounds, known as reactive nitro-
gen species (RNS), whose formation is catalyzed by nitric oxide synthase enzymes
(O’Bryan et al. 1998). Examples of RNS include nitric oxide •NO, nitroxyl ion HNO,
peroxynitrate anion ONOO− and nitrosyl-containing compounds. Physiological
levels of RNS helps maintain normal sperm parameters and general reproductive
functions, as well as stimulate the immune functions. However, pathological levels
of RNS contribute towards a state of nitrosative stress, which negatively impacts
sperm function and its fertilizing capacity, resulting in compromised male repro-
ductive function (Doshi et al. 2012).
In the ART setting, the effect of OS is augmented by the lack of endogenous
physiological defence mechanisms and the multiple potential sources of ROS, both

DOI 10.1007/978-3-319-10259-7_2
4 2 Sources of ROS in ART
internally [endogenously from the gametes and exogenously from the environment
and by manipulation of the gamete/embryo] and externally [in vitro factors such as
oxygen partial pressure and culture media], during the ART procedure. As the in
vitro setting during ART is unable to completely mimic physiological conditions in
vivo, the gametes used in assisted reproduction are more susceptible to the detrimen-
tal effects of oxidative stress. This negatively impacts on the ART outcome (Lampiao
2012). Among the external factors that could potentially affect the gamete/embryo
viability in vitro are cryopreservation/freeze-thaw procedure, visible light, the spe-
cific ART technique employed, pH and temperature, centrifugation, culture media,
and most importantly, oxygen concentration or partial pressure. These exogenous
ROS-inducing factors, along with endogenous sources of ROS are depicted in
Fig. 2.1 and will be discussed in the following sections.
2.1 In Vivo Generation of ROS
2.1.1 In Vivo Generation of ROS in the Male
Spermatozoa use ATP as a source of energy, which is produced through mitochon-

drial oxidative phosphorylation and glycolysis. Spermatozoa physiologically
undergo both aerobic and anaerobic metabolic processes, both of which contribute
towards the production of ROS (du Plessis et al. 2008). Under physiological condi-
tions, the primary source of ROS is the escape of activated oxygen from the mito-
chondria during oxidative respiration. Under typical conditions in vivo, ROS is
neutralized continuously to maintain the required levels of ROS to facilitate the
normal functioning of human spermatozoa. The low levels of ROS aid in the physi-
ological processes that maintains male reproductive functions such as cell signal-
ling, regulation of tight junctions, steroidogenesis, capacitation and acrosome
reaction, sperm motility, and zona pellucida binding (de Lamirande and Gagnon
1993; Doshi et al. 2012).
Besides the ROS generated intrinsically from the plasma membrane and mito-
chondria of spermatozoa (Gavella and Lipovac 1992), other cells may also produce
ROS in the male genital tract. Human spermatozoa generate O2−, which spontane-
ously dismutates to H2O2 (Alvarez et al. 1987).
Impaired spermatogenesis leads to the production of immature and
morphologically-abnormal spermatozoa, which contribute to ROS in the ejaculate.
A major extrinsic source of ROS in the human ejaculate is leukocytes. These may
be present in the ejaculate due to in vivo inflammatory processes. Leukocytes physi-
ologically produce about a 100-fold more ROS than spermatozoa (Plante et al.
1994; de Lamirande and Gagnon 1995). High levels of ROS produced during leuko-
cytospermia, play a major role in infection, inflammation and cellular defence
mechanisms against pathogens.
The highly reactive ROS combines easily with molecules causing cellular
damage (de Lamirande and Gagnon 1995; Agarwal et al. 2005b). The plasma
membrane of the spermatozoon contains a high amount of polyunsaturated fatty
2.1 In Vivo Generation of ROS 5
Fig. 2.1 Sources of reactive oxygen species in assisted reproductive technology

*MACS magnetic cell separation
acids (PUFAs), making it very susceptible to elevated ROS levels during OS.
The double bonds of membrane lipids are easily oxidized by ROS, causing a
decrease in membrane fluidity. Besides the lipid membranes, OS also affects the
structural proteins and nucleic acids of the sperm. As a result of this lipid peroxida-
tion, the motility of the spermatozoon is compromised and could ultimately lead to
immobilization of spermatozoa (Agarwal et al. 2003). OS leads to DNA fragmenta-
tion and damage in the nucleus of spermatozoa. Typically this include single- or
double-strand breaks, DNA crosslinks, chromosomal rearrangements as well as
deletions (Aitken and Krausz 2001). Damage to the sperm DNA by the increased
levels of ROS is implicated in adverse outcomes such as higher incidence of abor-
tion and childhood cancers (Baker and Aitken 2005). High ROS levels also cause a
decrease in the mitochondrial membrane potential, which is an initiating event in
the apoptosis cascade (Wang et al. 2003).
The consequences of high ROS levels are especially significant in infertile
patients seeking assisted reproduction as sperm selected from these patients are
very likely to have already been exposed to OS (Saleh et al. 2003). Increased OS in
spermatozoa have a negative impact on ART outcome, with poor fertilization rates,
embryo development and pregnancy rates (Baker and Aitken 2005).
The pro-oxidant activities of ROS are quenched by circulating antioxidants in
order to prevent the detrimental effects of OS on the male gamete. Antioxidants
function to maintain low, physiological concentrations of ROS in the cell and act as
a natural defence mechanism against OS. Antioxidants can either be enzymatic (e.g.
superoxide dismutase SOD, catalase, glutathione peroxidase GPx and glutathione
reductase GR) or non-enzymatic (e.g., vitamins C, E and A, glutathione, pyruvate,
taurine and hypotaurine, albumin and transferrin) (Sharma et al. 2004; Sikka 2004).
The mid-piece of the human sperm contains mainly enzymatic antioxidants
(SOD, GPx and GR) and the plasma membrane contains a few non-enzymatic anti-
oxidants (vitamins E and A, transferrin); while the seminal plasma contains both
enzymatic and non-enzymatic antioxidants (Agarwal and Prabakaran 2005). These
antioxidants contribute towards the total antioxidant capacity of the gamete. Under
normal conditions, seminal plasma has adequate antioxidant mechanisms to mini-
mize ROS action (Ford et al. 1997). However, during preparation of the sperm for
the assisted reproductive technique, the sperm is separated from the seminal plasma,
which decreases the spermatozoa’s natural defence mechanisms.
With time, the limited antioxidant capacity of the spermatozoa declines as the
aging sperm has reduced GPx and SOD activity (Sikka 2004). While ROS is present
in sperm from both fertile and infertile men, more infertile men tend to have exces-
sive levels of ROS levels compared to fertile men. Elevated ROS production com-
promises the structural integrity and functional capacity of the sperm, causing lipid
peroxidation, DNA fragmentation and apoptosis. Elevated RNS levels can also
cause lipid peroxidation, DNA damage, inhibition of steroidogenesis, increased cas-
pase activity ultimately leading to apoptosis. Furthermore, RNS negatively impacts
sperm parameters by reducing motility and viability, causing abnormal morphology,
decreasing capacitation and the acrosome reaction, as well as sperm-oocyte fusion
(Doshi et al. 2012).
2.1 In Vivo Generation of ROS 7
In summary, spermatozoa have increased susceptibility to OS, as there is a lack

of cytoplasm in the mature sperm compared to somatic cells, and therefore its poorer
antioxidant capacity tends to render the spermatozoa at greater vulnerability to OS.
2.1.2 In Vivo Generation of ROS in Females
The role of ROS in female infertility has been of significant interest and research
over the last decade. The presence of ROS in the fluids and organs involved in
female reproductive processes has been well documented. In the female, the sources
that generate ROS in vivo are mainly the follicular fluid, fallopian tube and uterine
environment (Pasqualotto et al. 2004; Bedaiwy et al. 2004; Guerin et al. 2001). At
these locations, ROS appear to have a physiological role in oocyte maturation, ovar-
ian steroidogenesis and ovulation, implantation and formation of fluid-filled cavity,
blastocyst, luteolysis and luteal maintenance in pregnancy. Moreover, a limited
amount of lipid peroxidation relevant to ROS in follicular fluid was found obliga-
tory for establishing pregnancy in human IVF cycles (Agarwal et al. 2005a).
Each month, a cohort of oocytes develops and matures in the ovary. However,
meiosis I will resume only in the dominant oocyte. This process could be threatened
by an increase in ROS. Conversely, high levels of ROS could be inhibited by anti-
oxidants. This implies an intricate association between ROS and antioxidants in the
ovary (Behrman et al. 2001; Agarwal et al. 2012). Inside the ovary, the follicular
fluid environment enclosing the oocyte plays an important role in the fertilization
process. Once fertilized, follicular fluid (along with the fallopian tube and endome-
trium), plays an important role in subsequent embryo development. During follicu-
lar maturation, oocytes are well-sheltered against lethal injury due to OS by
antioxidants such as catalase, superoxide dismutase, glutathione transferase GST,
paraoxonase PON, heat shock protein 27 and protein isomerase (Ambekar et al.
2013). Additionally, there are other antioxidants present such as vitamin E, caro-
tene, ascorbate, cysteamine, taurine, hypotaurine, transferrin, thioredoxin, and
dithiothreitol. The imbalance between these antioxidants and pro-oxidants in female
infertility has been postulated to alter gene expression and impair adenosine tri-
phosphate (ATP) generation (Agarwal et al. 2005a), the latter of which can affect
ovulation, oocyte quality, fertilisation, embryo development and implantation
(Agarwal et al. 2006a; Revelli et al. 2009; Das et al. 2006; Pasqualotto et al. 2009).
An excess of free radicals also play a key role in gynaecological problems such as
polycystic ovarian syndrome (PCOS), endometriosis and tubal factor infertility
(Pasqualotto et al. 2009). Numerous studies discussed in this section have demon-
strated the relationship between these disorders, OS and successful ART outcome.
The oocyte, granulosa and surrounding cells, such as the endothelial and thecal
cells constitute the follicular fluid environment. It also contains phagocytic macro-
phages, parenchymal steroidogenic and endothelial cells that generate ovarian ROS
(Halliwell and Gutteridge 1988; Agarwal et al. 2005a). The composition of follicular
fluid is a key feature for predicting a successful ART outcome in females (Agarwal
et al. 2005a). While large amounts of ROS in the follicular fluid pose a serious threat
to the success of assisted reproduction, a limited amount of ROS was found to be
obligatory for establishing pregnancy in human IVF cycles (Oral et al. 2006).
A summary of the experimental studies investigating the relationship between
follicular fluid and ART outcome is portrayed in Table 2.1.
In assisted reproduction, the assessment of ROS and RNS in the follicular fluid
of women undergoing IVF is usually obtained by aspiration of follicular fluid from
each follicle during the oocyte retrieval process. Follicular fluid samples are then
centrifuged and evaluated, most commonly by the chemiluminescence assay using
luminol (other methods include nitroblue tetrazolium staining, thiobarbituric acid-
reacting substances (TBARS), and flow cytometry) (Askoxylaki et al. 2013). In a
study by (Jana et al. 2010), the upper cut-off limit for ROS levels, beyond which
viable embryo formation is not favourable, was observed to be approximately 107
counted photons per second (cps)/40 μL of follicular fluid. This cut-off level (previ-
ously determined in infertile women with tubal factor), was validated next in women
with PCOS and endometriosis, in which they were shown to adversely affect oocyte
and embryo development as well as pregnancy outcome. ROS values above this
threshold value were shown to hinder oocyte quality, maturation, fertilization, and
embryo formation. Conversely, significantly lowered levels of ROS (<100 cps) were
linked with good embryo quality. Similar were also reported the lower and upper
ROS values as 41 and 150 cps respectively (Das et al. 2006).
A prospective study by Attaran et al. (2000) reported that ROS levels were sig-
nificantly lower in the follicular fluid of patients (with tubal disease, endometriosis
and idiopathic infertility) who failed to establish pregnancy as compared to those
who did (although the specific cut off values were not described). These results
were in accordance with Pasqualotto’s study, in which patients who became preg-
nant had higher lipid peroxidation levels and total antioxidant capacity (TAC)
(Pasqualotto et al. 2004). Despite the fact that an imbalance of pro-oxidants and
antioxidants can cause a disturbance in natural female reproductive tendencies,
however these results indicate that physiological levels of ROS within the follicular
fluid is essential for different phases of oocyte development and maturation.
However, the exact function of ROS in the follicular fluid remains unknown
(Pasqualotto et al. 2004; Oyawoye et al. 2003; Jana et al. 2010).
In a study of 63 women undergoing IVF, a total of 303 follicular aspirates were
analysed using ferric reducing antioxidant power (FRAP) assay for baseline TAC
(Oyawoye et al. 2003). The study revealed that TAC levels were significantly higher
in follicular fluid where the oocyte was successfully fertilized. In a more recent study,
the association between follicular fluid, ROS levels, TAC, ROS-TAC score and preg-
nancy following ICSI were evaluated in 138 women (Bedaiwy et al. 2012). Results
of this study illustrated that pregnancy cycles were associated with significantly
lower ROS and higher TAC. Interestingly, their study found that TAC levels were
higher in women with endometriosis. Their ROS-TAC scores were also higher and
were associated with a greater probability of having normal oocytes. In these women,
elevated TAC levels, higher ROS-TAC scores and lower ROS levels in their follicular
fluid were allied with a successful pregnancy after ICSI (Bedaiwy et al. 2012).
2.1
Table 2.1 Clinical parameters of studies examining reactive oxygen species in follicular fluid in infertile women
Sample Factors measured
Study collected Patient population/characteristics in the sample collected Outcomes measured
Attaran et al. (2000) FF Patients undergoing ovarian stimulation for OS markers: Age, number of oocytes recovered,
ART: women with tubal disease, male factor, 1. ROS percentage of oocytes fertilized,
endometriosis, idiopathic infertility, ovulatory 2. TAC achievement of pregnancy
dysfunction, pelvic adhesions
Barrionuevo et al. FF Patients undergoing IVF 1. Nitric oxide metabolites Oocyte maturation, fertilization rate,
(2000) nitrate/nitrite (NO3/NO2) embryo cleavage rate
In Vivo Generation of ROS
2. IL-1β levels
Oyawoye et al. (2003) FF Patients undergoing IVF-ET treatment 1. TAC Oocyte retrieval, fertilization rate,
embryo viability
Pasqualotto et al. (2004) FF Patients undergoing IVF: male factor OS markers: Oocyte maturity, fertilization rate,
infertility, tubo-peritoneal factors, idiopathic 1. LPO cleavage rate embryo quality, and
infertility, ovulatory factors 2. TAC pregnancy rate
Das et al. (2006) FF Patients undergoing IVF-ET treatment by OS markers: Oocyte quality and fertilization potential,
controlled ovarian stimulation (long protocol): 1. ROS embryo quality
tubal factor infertility 2. LPO
Chattopadhayay FF PCOS OS markers: Meiotic spindle formation, fertilization
et al. (2010) 1. ROS rate, number of good quality embryos,
2. LPO clinical pregnancy rates
3. TAC
Jana et al. (2010) FF Patients undergoing IVF-ET by controlled OS markers: Oocyte quality, fertilization rate, embryo
ovarian stimulation: tubal factor infertility, 1. ROS quality
endometriosis, PCOS 2. LPO
3. DNA fragmentation
4. TAC
9
(continued)
10
Table 2.1 (continued)

Borowiecka et al. (2012) FF Patients undergoing IVF Lipid and protein Pregnancy rates
peroxidation markers:
1. TBARS
2. Protein carbonyl
3. Thiol groups
Bedaiwy et al. (2012) FF Patients who had ICSI: couples with male OS markers: ROS-TAC score, number of follicles,
infertility, endometriosis, tubal disease, 1. ROS number of oocytes retrieved, oocyte
idiopathic infertility, ovulatory dysfunction, 2. TAC quality, pregnancy rate
combined male and female infertility
Otsuki et al. (2012) FF Infertile patients 1. Redox state Oocyte viability
2. Albumin
Rajani et al. (2012) FF Patients undergoing ICSI-ET: with 1. ROS levels Presence of meiotic spindle, number of
endometriosis, PCOS, tubal infertility oocytes retrieved, mature MII oocytes,
(control) fertilization rate, good embryo formation
2
rate, pregnancy rate

Singh et al. (2013) FF Patients undergoing IVF: endometriosis OS markers: Oocyte quality, embryo quality,
and tubal factor (controls) 1. ROS pregnancy rate
2. NO (nitrite/nitrate)
3. LPO
4. TAC
5. Antioxidant (enzymatic
and non-enzymatic) levels
Sources of ROS in ART
2.1
Liu et al. (2013) FF Patients undergoing IVF-ET: endometriosis, OS markers: Oocyte quality, fertilization rate
tubal factor infertility 1. ROS
2. SOD
3. Vitamin E
Seino et al. (2002) GC Patients undergoing IVF-ET treatment 1. 8-OHdg expression in Oocyte quality, fertilization rate, embryo
and ICSI: endometriosis, male factor, granulosa cells quality (good embryo rate)
tubal factor, unknown
Liu and Li (2010) GC Patients undergoing IVF-ET: tubal factor 1. MDA Number of retrieved oocytes, oocyte
infertility 2. SOD maturity, embryo quality, fertilization,
cleavage
In Vivo Generation of ROS
3. Apoptosis
4. Good embryo rate
Karuputhula GC Patients undergoing IVF-ET: endometriosis, OS markers: GC characteristics: Fertilization rate,
et al. (2013) PCOS, tubal factor infertility 1. ROS number of oocytes retrieved, oocyte
2. MMP quality, good quality embryo formation
rate, pregnancy outcome
3. DNA fragmentation
4. Apoptosis
Polak et al. (2001b) PF, Infertile women with minimal or mild 1. 4-HNE levels Oxidative stress/free radicals activity
plasma endometriosis, unexplained infertility, 2. MDA levels (lipid
samples PCOS, tubal infertility peroxide levels)
Bedaiwy et al. (2004) Culture Patients undergoing IVF/ICSI: male factor, 1. ROS in culture media Fertilization rate, cleavage rate,
media anovulation, endometriosis, tubal factor, fragmentation, embryonic fragmentation
unexplained and other factors levels, blastocyst formation rate
Sample types: 4-HNE 4-hydroxynonenal, 8-OHdG 8-hydroxy-2’-deoxyguanosine, ART assisted reproductive technology, CoQ10 Coenzyme Q10, DNA deoxyribo-
nucleic acid, EDTA ethylenediaminetetraacetic acid, FF follicular fluid, GC granulosa cells, hCG human chorionic gonadotrophin, ICSI intracytoplasmic sperm
injection, ICSI-ET intracytoplasmic sperm injection and embryo transfer, IL-1ß Interleukin-1 beta, IVF in vitro fertilization, IVF-ET in vitro fertilization and
embryo transfer, LPO lipid peroxidation, MDA malondialdehyde, MII metaphase II, MMP mitochondrial membrane potential, NO nitric oxide, OS oxidative stress,
PCOS polycystic ovarian syndrome, PF peritoneal fluid, ROS reactive oxygen species, SOD superoxide dismutase, TAC total antioxidant capacity, TBARS thiobar-
bituric acid reactive substances
11
In a much earlier study, Polak’s group demonstrated the total antioxidant status
of peritoneal fluid in infertile women (Polak et al. 2001a). The peritoneal fluid was
obtained from infertile women distressing from minimal or mild endometriosis,
unexplained infertility, tubal infertility and a few fertile women. The results of this
study demonstrated a significantly lower antioxidant status in peritoneal fluid
obtained from women with unexplained infertility.
Oocyte quality is another important determinant of IVF outcome. The follicular
fluid aspirated during oocyte retrieval from women with endometriosis and tubal
infertility undergoing IVF was measured using spectroscopy and HPLC (Singh
et al. 2013). Increased levels of ROS and NO in endometriosis and tubal infertility
were found to correlate with poor oocyte and embryo quality. Further, increased
levels of ROS, nitric oxide, lipid peroxidation, cadmium and lead were seen in
women who failed to become pregnant compared to those who did.
Borowiecka et al. (2012) evaluated the levels of lipid and protein peroxidation
markers (TBARS, protein carbonyl, and thiol groups) in the follicular fluid of
patients undergoing IVF. The OS markers were compared between the pregnancy
positive and pregnancy negative patient groups. Results demonstrate that the mean
follicular fluid TBARS level of non-pregnant women was significantly higher than
that found in pregnant women. These findings suggested that elevated follicular
fluid lipid and protein peroxidation levels may have a negative impact on IVF out-
come and also supported the idea that increased levels of OS markers in follicular
fluid may play an important role in fertility.
The association between malondialdehyde (MDA), superoxide dismutase (SOD)
and incidence of apoptosis of granulosa cells in follicular fluid was examined in
women with tubal factor infertility (Liu and Li 2010). The level of MDA and the
activity of the SOD were measured by TBARS using a chemiluminescence methods,
respectively. It was discovered that non-pregnant patients showed significantly
higher MDA levels, higher incidence of apoptosis and lower SOD levels in the gran-
ulosa cells with lower good-embryo rate as compared to the pregnant patients. In
general, OS induced apoptosis in granulosa cells and subsequently lowered oocyte
quality, leading to poor outcome of IVF-ET. The levels of 8-hydroxy-2′-
deoxyguanosine (8-OHdG) in granulosa cells were detected in Seino’s study (Seino
et al. 2002). The outcome of their study suggested that OS in granulosa cells reduced
fertilization rates and consequently reduced embryo quality. They also found that the
quality of oocytes of endometriosis patients was reduced by the presence of
8-OHdG. The outcome of this study was in agreement with that of Karuputhula’s
group. A ∼20- and 100-fold increase in granulosa cells ROS generation and MMP,
was seen in PCOS patients as compared to tubal factor patients. Significant apoptosis
was also evident in PCOS and endometriosis patients. IVF outcome parameters com-
prise fertilization rate, formation rate of good quality embryo, and pregnancy rates,
and these were badly affected in endometriosis patients (Karuputhula et al. 2013).
Another study which was performed on patients with PCOS, examined the meiotic
spindle in oocytes along with ROS levels in follicular fluid of women (Rajani et al.
2012). It was demonstrated that women with endometriosis had low ROS levels and
good spindle imaging results suggesting a possible role of endometrial receptivity
2.2 In Vitro Generation of ROS in ART 13
accounting for lower pregnancy rates in these women. Poor oocyte quality, as reflected
by higher mean ROS levels and low number of oocytes with spindle visualization,
could be the factor impeding pregnancy in women with PCOS as compared to women
with tubal block (Rajani et al. 2012). Another group examined that the effect of fol-
licular fluid OS on the formation of meiotic spindle in oocytes and the outcome in
women with PCOS (Chattopadhayay et al. 2010). Oocytes were examined to visual-
ize for the meiotic spindle using a PolScope (a polarised light microscope). From the
results of this study, it was observed that OS was responsible for the absence of the
meiotic spindle which was significantly found in the groups with low fertilization
rate, reduced number of good quality embryos and clinical pregnancy. Otsuki’s group
studied the influence of the redox state of follicular fluid on the viability of aspirated
human oocytes (Otsuki et al. 2012). The redox state of the follicular fluid and serum,
at the time of oocyte retrieval, was analysed by high performance liquid chromatog-
raphy. Their results showed that the redox state of follicular fluid that contained
degenerated oocytes had a significantly higher oxidised state compared with fluids
that capitulated normal oocytes. A prospective case-control study was conducted in
endometriosis patients who underwent IVF-embryo transfer IVF-ET. The expression
and the role of OS markers in serum and follicular fluid of the patients were investi-
gated. The results of the study reported significantly higher levels of ROS in both
serum and follicular fluid. Mature oocyte and fertilization rates were also signifi-
cantly lower than in the control group (Liu et al. 2013).
Nitric oxide concentrations in follicular fluid have been negatively associated
with low pregnancy rates. It was found that serum nitric oxide concentrations in
patients with tubal factor or peritoneal factor infertility negatively correlated. They
reported that higher concentrations of nitric oxide are associated with implantation
failure, which then result in lower pregnancy rates (Bedaiwy et al. 2004).
In summary, the findings of the studies discussed in this section highlight the
importance for the detection of ROS in the female reproductive system for a variety
of reasons and indicate how ROS levels can also be used to determine the link
between gynaecological diseases (PCOS, endometriosis, tubal factor) and OS.
2.2 In Vitro Generation of ROS in ART
2.2.1 Cryopreservation
Cryopreservation is a process whereby cells and whole tissues are conserved by

cooling to sub-zero temperatures (−196 °C) (Di Santo et al. 2012). Recent advances
in assisted reproduction and embryology have made cryopreservation an appropri-
ate method for long-term storage of human reproductive cells, embryos and gonadal
tissues to preserve and protect fecundity in cases of infertility, malignancy (Woods
et al. 2004) and in some non-malignant treatments (such as diabetes and autoim-
mune disorders that may lead to testicular injury) (Anger et al. 2003). Nonetheless,
despite highly optimised protocols appear to improve cell viability, the extreme
stress of freezing and thawing treatments can modify the structure and integrity of
the sperm plasma membrane (mainly composed of phospholipids and cholesterol)
(Giraud et al. 2000; Di Santo et al. 2012).
Various reports studied the relationship between cryopreservation and antioxidant
defence system. The occurrence of DNA fragmentation in testicular sperm of men
with obstructive azoospermia, such as in vasectomised males, those with blocked or
missing ducts, and those with an absence of the vas deferens was investigated
(Dalzell et al. 2004) The study showed a significant increase in DNA fragmentation
at 24 h after incubation of fresh testicular sperm from men with obstructive azo-
ospermia. Frozen-thawed sperm DNA was found to be significantly more damaged
than fresh testicular sperm DNA. Even after 4 and 24 h post-thaw incubation, sperm
DNA continued to become more damaged compared to fresh sperm DNA. These
findings were further confirmed by Thomson’s study, which reported increased lev-
els of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) (a biomarker of oxidative
stress), indicating an increase in sperm DNA fragmentation due to cryopreservation
of human semen (Thomson et al. 2009). Furthermore the effect of cryopreservation
on motility and viability was evaluated on spermatozoa of men struggling with infer-
tility. The results in these men showed a significant decrease in sperm motility and
viability post-cryostorage. In addition, cryopreservation/thaw significantly increased
sperm DNA fragmentation and DNA oxidative damage (Zribi et al. 2010).
In human oocytes, cryopreservation frequently leads to developmental arrest
during early cleavage stages and display aberrant patterns of cytokinesis (cytoplas-
mic division of a cell at the end of mitosis or meiosis, bringing about the separation
into two daughter cells) (Van Blerkom and Davis 1994). Various studies (Gualtieri
et al. 2009; Jones et al. 2004) revealed significant reduction of mitochondrial poten-
tial in slow cooled human oocytes resulted from changes in redox status of the cell
due to cryopreservation. However, a study by Chen et al. (2012) evaluated the
impact of vitrification on mitochondrial membrane potential (MMP) in human
metaphase II (MII) oocytes, and the changes of mitochondrial membrane potential
in thawed MII oocytes. It was observed that in the vitrification/thawing process, the
mitochondrial membrane potential of MII oocytes could have temporary dynamic
changes within a couple of hours post-thaw but would recover fully after 4 h of
culture. In another study, the effect of different vitrification protocols on ROS were
evaluated in human ovarian tissues, which were exposed to different vitrification
solutions (Rahimi et al. 2003).The intracellular redox state levels were measured
using the fluorescent dye dichlorodihydrofluorescein diacetate. After vitrification
and warming, apoptotic cells imaging was monitored by anti-caspase-3 immunola-
belling. The results showed that a slower cooling of tissue resulted in significantly
elevated ROS levels and apoptosis after warming (Rahimi et al. 2003).
Based on the outcome of multiple studies, there appears to be a clear picture
where cryopreservation is responsible for OS during ART. The higher incidence of
studies regarding the male gamete may likely confirm that spermatozoa are more
vulnerable to redox alterations than oocytes during cryopreservation. This differ-
ence in sensitivity to OS is due to the higher susceptibility of spermatozoa to lipid
peroxidation and to the limited amount of ROS scavengers available, as the antioxi-
dant defences are localised mainly in the seminal plasma (Bozhedomov et al. 2009).
2.2.2 Visible Light
Visible light, also known as visible spectrum, is the portion of the electromagnetic
spectrum that is visible to the human eye, having a wavelength in the range of
400–700 nanometres (nm)—between the infrared light with longer wavelengths and
the ultraviolet light with shorter wavelengths (Buser et al. 1992) (Fig. 2.1). In an
ART laboratory, oocytes, zygotes and embryos which are kept in an artificial
medium for assisted fertilization procedures such as IVF and ICSI, are exposed to
daylight or artificial light for variable periods before the embryo is transferred to
females.
Over the years, enormous consideration has been given to the role of visible light
in the production of ROS in animals (Takenaka et al. 2007; Moshkdanian et al. 2011).
However, the effects of light have not been studied as extensively in humans. Light
is either measured as units of intensity (lux) or by the level of irridation (W/m2).
According to an older study, rabbit oocytes when exposed to strong “cool white”
fluorescent light of 3,250 lux for 20–30 min at 37 °C developed into normal near-term
fetuses. However, this observation does not mean that visible light is undisruptive to
the gametes and embryos of humans (Bedford and Dobrenis 1989).
In an IVF laboratory, light is generated by microscopes, fluorescent lighting and
indirect sunlight. When gametes and embryos are exposed to light, it is absorbed by
intracellular chromophores, which includes plasma membrane NADPH oxidase
system consisting of flavoproteins and cytochrome b (Edwards and Silva 2001;
Eichler et al. 2005). These chromophores are photosensitizers—which means they
are able to absorb light and then transfer the energy to nearby oxygen molecules.
Electrons in the shell get excited and shift to a shorter-lived singlet state. These
excited electrons then change their spin and produce a longer-lived triplet state.
As it returns to the ground state, it releases energy. This energy is then transferred
to oxygen to generate ROS (singlet oxygen and free radicals) that mediate cellular
toxicity (Girotti 2001). This mechanism of cellular toxicity was also described ear-
lier (Hockberger et al. 1999). It was shown that violet-blue light (445–455 nm),
irradiated from an inverted fluorescence microscope, initiated photoreduction of
flavins. These activate flavin-containing oxidases in mitochondria and peroxisomes,
resulting in production of H2O2 in human foreskin keratinocytes (HK cells).
The intensity and spectral composition of light reaching embryos during IVF
was also investigated, and it was detected that microscopes, at a setting appropriate
for embryo inspection, produced light at 2,500–5,000 lux. Meanwhile, light from
other sources like natural room lighting and sunlight were more than tenfold lower,
at 200–400 lux and had little effect on cultured embryos (Ottosen et al. 2007). It was
suggested that microscope light exposure is a more hazardous source of light for
oocyte and embryo viability compared to ambient light. The light intensity and
wavelengths during embryo manipulation are important factors to maintain pre-
implantation embryos viability in vitro.
The effect of visible light on human sperm motility and hyperactivation was
demonstrated in a recent study (Shahar et al. 2011). The group evaluated the path-
ways mediating these effects. In their experiment, they irradiated human spermato-
zoa for 3 min with 40 mW/cm2 visible light (400–800 nm with maximum energy at
600 nm) and suggested that ROS was produced during incubation, and that the
production was enhanced after 1–3 min of light irradiation.
Over the years, attention has been focused towards the light microscopes used in
IVF laboratories. Scientists recommend reducing the inspection light to as low as
possible and maintaining the exposure time to as short as possible. In addition,
microscope filters (optical filters) mounted on the microscope are used in a variety
of microscopy applications for increasing contrast, obstructing ambient light, and
eliminating harmful ultraviolet or infrared light (green filters block wavelengths
below 500 nm). Moreover, to reduce ultraviolet (UV) lightwaves which are emitted
from fluorescent lights, the fluorescent light could be substituted with yellow lights
or by positioning UV light protectors over the fluorescent tubes (Ottosen et al. 2007).
2.2.3 ROS from Gamete Manipulation/ART Technique
In an in vitro environment, handling or manipulating the gametes and embryos dur-

ing ART brings forth a risk of exposing these cells to higher than physiological
levels of levels ROS (Lampiao 2012).
During ART procedures, the gametes are manipulated and prepared for various
fertilization procedures such as IVF and ICSI. During conventional IVF, the selected
samples of spermatozoa (concentration in millions) and oocytes are combined in the
fertilisation medium in a petri dish and inspected for fertilisation after 16–20 h of
incubation. During this period, ROS are generated from sources like oocytes, the
cumulus cell mass and spermatozoa in culture medium. On the other hand, during the
ICSI procedure, the cumulus cells are stripped from around the oocyte and a single
sperm is injected directly into an oocyte before incubation in culture medium. Thus
in an ICSI procedure, the oocyte and spermatozoa are the only potential sources of
ROS. Furthermore, this procedure involves a shorter incubation time, which decreases
the exposure of gametes to external environmental factors (Agarwal et al. 2006b).
However, since most ICSI cycles are done due to poor sperm characterises (which
is not the case in an IVF procedure), therefore, it is commonplace to find higher rates
of spermatozoa with greater DNA fragmentation levels in ICSI compared to IVF
cycles (Benchaib et al. 2003). In addition, it was hypothesised that during the IVF
procedure, human zona pellucida has the capacity to select against sperm with aneu-
ploidy, which was supported by a theory that IVF steps leads to a ‘natural selection
of spermatozoa’ (Van Dyk et al. 2000). The spermatozoon that is chosen for fertiliza-
tion will have normal morphology and be exceedingly motile with intact DNA
(Benchaib et al. 2003). Also with ICSI, in case of very poor sperm characteristics,
the choice of spermatozoa to be injected is done using a very imperfect criteria and
where it is tough to choose one normal motile sperm, so the risk of injecting sperma-
tozoa with ROS and impaired DNA is high (Gandini et al. 2000; Irvine et al. 2000).
It was also assumed that during the ICSI procedure, the selection of a motile and
morphologically normal spermatozoa is operator-dependant. Therefore this sperma-
tozoon has additional chances of having intact DNA, but at the same time a sperma-
tozoa can be considered as ‘normal’ and still have damaged DNA (Host et al. 2000).
Elevated ROS levels were observed in the follicular fluid of women, whereby
high ROS levels were prominent in those undergoing ICSI cycles, but not in IVF
(Lee et al. 2012). It was demonstrated that the ICSI procedure might induce stress
or shearing force on the plasma membrane of the oocyte, while the integrity of the
plasma membrane has been considered as the origin of the deleterious effects of OS
on fertilization, cleavage, or even implantation (Agarwal et al. 2003; Guerin et al.
2001). Consequently, ICSI manipulation might impair the developmental potential
of oocytes after OS injury within the follicular fluid. The results indicate that ROS
levels in follicular fluid may have a negative effect on the oocytes and its subsequent
development, causing it to be affected by the insemination procedure (Agarwal
et al. 2003). Furthermore, it has been recommended that the oocyte-handling time
during ART procedures (denuding, ICSI, during media transfer) should be kept at a
minimum and that incubation time of oocytes in the culture medium be managed
well. This is to enhance oocyte quality and consequently, the success rate of the IVF
cycle. For example, exposure time to pro-oxidant media (such as IVF medium)
(even before insemination) should be kept at a minimum, as it would be helpful to
conserve the quality of oocytes (Martin-Romero et al. 2008).
2.2.4 pH and Temperature
The in vitro environment is stressful for gametes and embryos as the temperature
and pH tends to fluctuate. The measure of acidity or basicity of a solution is defined
as pH, or hydrogen ion concentration. Intracellular pH is a vital aspect of cell
homeostasis, and is controlled by membrane potential and osmolarity. The key
intracellular processes are highly susceptible to pH, including protein synthesis,
metabolism, mitochondrial function and cytoskeletal regulation (Will et al. 2011).
In culture media, pH is an important variable that influences motility and sperm
binding, oocyte maturation and embryo development, though confounding factors
such as bicarbonate and CO2 levels exist (Will et al. 2011; Bagger et al. 1987).
Animal studies have suggested that even a little increase in external pH (pHe)
during minor manipulations outside the incubator can significantly obstruct sperm
function (Marquez and Suarez 2007), alter organelle localization(Squirrell et al.
2001; Will et al. 2011), impact the development of mouse blastocyst and hatching,
and alter gene expression profiles (Huntriss and Picton 2008). Hamster embryo
studies show that even very slight deviations of internal pH (pHi) for short periods
of time, from a set point of 7.21 (slightly raised 7.42 or lowered 6.87 pHi), can
greatly impact the developing embryo. Philips and his group employed the pH-
sensitive fluorescent dye BCECF to evaluate the pHi in human oocytes and demon-
strated that the pHi values for oocytes and embryos changes during various
developmental stages: such as at GV-intact MI 7.04, MII 7.03 and at Cleavage stage
6.98 pHi (Phillips et al. 2000).
In IVF, the most common buffer used in media is sodium bicarbonate (Will et al.
2011). In addition, the HEPES media buffer has regularly been seen to be a safe and
effective buffer for the storing and handling of spermatozoa compared to other types
of buffers. CO2 and pH have an inverse relationship; as CO2 concentration decreases,

pH increases. The media pH may be maintained provided that the levels of CO2
remain constant in the incubators; nevertheless, this may be a challenge due to fre-
quent openings/closings of incubator doors in order to observe the cell and while
performing manipulations at room atmosphere (Will et al. 2011).
In case of procedures performed in room atmosphere, such as gamete collection,
ICSI, cryopreservation, and embryo transfer, labs may choose to use handling media
with reduced levels of bicarbonate and include another pH buffer to maintain pHe
outside the incubator. Sperm medium with added bicarbonate helps with the recov-
ery of motile sperm (Mehta and Sigman 2014). Likewise, temperature is another
factor that has an important role in pH and pH buffering. pH and pKa values decrease
when the temperature increases (Ferguson et al. 1980). Temperature is measured
and maintained by the incubator’s thermostat and the incubator’s heating system. In
an IVF lab, all incubator temperatures are set at 37 °C in order to mimic in vivo
conditions (Ferguson et al. 1980). Based on the physical properties of these mem-
branes and membrane-associated processes, they may be more sensitive to tempera-
ture stress (Davidson and Schiestl 2001). Temperature stress may impair
mitochondrial functions and induce oxidative damage, causing lipid peroxidation
(Larkindale and Knight 2002).
2.2.5 Centrifugation
Sperm centrifugation is regularly done during semen processing and is a common

step used for sperm preparation in ART (Agarwal et al. 2006c). The centrifugation
process separates sperm cells from the seminal plasma, and motile sperm from non-
motile or dead sperm and cell debris. Some of the commonly used sperm prepara-
tion techniques during ART that include the centrifugation step(s) are the double
wash sperm swim-up technique, and the double density gradient separation. For
example, simple washing (which removes only the seminal plasma) and swim up
technique (which involves the further removal cellular debris and non-motile
sperm) involve centrifugation at 300–400 × g for about 10 min, while discontinuous
(density) gradient involves centrifugation twice, once at <500 × g for 20 min and
then again at 300 × g for 5–10 min. These techniques aid in the selection of sperm
with enhanced motility and are more viable. Besides these advantages, the removal
of spermatozoa from seminal plasma is important in assisted reproduction, as this
step eliminates the seminal plasma that contains leukocytes, a source of ROS.
Furthermore, in severely oligospermic semen samples, centrifugation increases
the chances for selecting better quality sperm, while in azoospermic semen sam-
ples, centrifugation increases the chances of identifying the rare sperm, if any
(Sharma et al. 1997).
However, the centrifugation process itself generates ROS (Alvarez et al. 1993;
Agarwal et al. 1994; Lampiao et al. 2010). The sperm membrane is mainly made up
of polyunsaturated fatty acids (PUFAs) and is therefore particularly susceptible to
damage by ROS. High ROS concentrations could lead to lipid peroxidation of sperm
plasma membrane, causing the loss of membrane fluidity, which impairs sperm
motility. As the concentration of progressively motile sperm is indicative of success
of ART outcome/pregnancy, poor sperm motility augurs an unfavourable ART out-
come. In addition, the excessive presence of ROS causes DNA damage, which
translates to poor embryo development.
Moreover, the g force, time (Shekarriz et al. 1995) and temperature employed
during the process of centrifugation, influences the amount of ROS produced and
thereby affects the quality of the sperm that has been processed. Centrifugation
speeds greater than 500 × g and continuous centrifugation for longer than 5–7 min
was found to compromise sperm quality (Sharma et al. 1997). In another study, both
10 and 30 min of sperm centrifugation was found to compromise sperm quality and
viability, with the centrifugation time of 30 min being more detrimental to sperm
quality compared to 10 min (Lampiao et al. 2010).
Longer centrifugation time increases the temperature during centrifugation,
which affects the quality of sperm, despite the initial quality of the sample (Henkel
and Schill 2003). The increased temperature during centrifugation also affects
sperm motility, which could compromise ART outcome.
Therefore, sperm preparation techniques in ART should ideally exclude the
centrifugation step altogether, or should at least avoid the use of prolonged periods
of centrifugation (Lampiao et al. 2010). In protocols that require sperm centrifuga-
tion, the addition of antioxidants or other ROS scavengers prior to centrifugation
may help quench the ROS produced due to the centrifugation process, and yield
processed sperm with less damage (Lampiao et al. 2010). For example, pentoxifyl-
line which is added to the sperm preparation to stimulate sperm motility, also
quenches the ROS produced by spermatozoa (McKinney et al. 1996).
2.2.6 Culture Media
There are various types of media that may be used to culture human oocytes and
pre-implantation embryos during the IVF and ICSI procedures. ART laboratories
have a variety of commercially-available culture media to choose from, and these
media may be composed of different types of ingredients, depending on the manu-
facturer. However, the type of media that is eventually used is crucial as culture
media used has an important direct bearing on the quality of the embryo produced
and subsequently, the success rate of the IVF procedure (Agarwal et al. 2006b).
Some media may contain metallic ions such as iron (Fe2+) and copper (Cu2+). These
ions can incorporate into the gametes or the developing embryo during culture,
leading to the Fenton and Haber-Weiss reactions to occur, which generates ROS
(Guerin et al. 2001). To prevent these reactions from taking place and reduce the
formation of ROS, common metal chelators such as transferrin and ethylenediamine
tetra-acetic acid (EDTA) can be added to the media (Nasr-Esfahani and Johnson
1992; Orsi and Leese 2001).
However, adding supplements to the media could end up increasing the oxidant
load. For example, media additives such as serum albumin which contains elevated
levels of amine oxidase leads to a higher generation of hydrogen peroxide, a form
of ROS (Shannon 1978; du Plessis et al. 2008). The rate with which the culture
media generates ROS varies according to its composition (Jana et al. 2010). OS
within the culture media could partially deplete the GSH content in the oocyte,
which could disrupt oocyte fertilization and viability (Martin-Romero et al. 2008).
Supplementation of culture media with antioxidants such as ascorbic acid, alpha-
tocopherol, taurine, hypotaurine, isoflavones reduced the risk of OS and its subse-
quent adverse effects on the gamete (Sikka 2004; Alvarez and Storey 1983). Lipid
peroxidation due to the presence of ROS can be deterred using vitamin E supple-
mentation (Jain et al. 2000). In mouse embryos, the addition of thioredoxin to the
media reduced apoptosis rates and enhanced hatching rates, while in porcine
embryos, supplementation of media with glutathione and thioredoxin reduced the
redox status (Ozawa et al. 2006).
In the in vitro fertilization procedure, sperm from the male partner interacts with
the oocyte from the female partner in culture media, leading to fertilization. In ICSI,
sperm prepared in culture media is injected directly into the cytoplasm of the oocyte.
As the sperm is injected into the oocyte, there is an additional risk of transferring
some of the ROS that is present in the culture media along with the sperm into the
oocyte—which would have further detrimental effects on the oocyte’s DNA mate-
rial (Shen et al. 2003).
The consequences of ROS on gametes and early embryos have been
experimentally-investigated, and multiple authors have reported increased levels of
intracellular ROS during the various stages of embryo development (Hu et al. 2001;
Bedaiwy et al. 2004).
Bedaiwy and his group examined the association of ROS levels in the culture
media on the first day (day 1 ROS) after ICSI (Bedaiwy et al. 2004). During this
experiment, fertilization as well as early cultures were done in human tubal fluid
supplemented with 5 % serum substitute and levels of ROS were monitored by a
chemiluminescence method using a luminol probe. The results illustrated that high
ROS levels in culture media on day 1 caused low blastocyst rate, low fertilization
rate, low cleavage rate, and high embryonic fragmentation in ICSI cases but not in
those of conventional IVF. However, lower pregnancy rates were observed in both
IVF and ICSI cycles with high day 1 ROS levels in the culture media. The same
group followed up the experiment on examining ROS levels in day 3 culture media
to the outcome of ICSI (Bedaiwy et al. 2010). The results confirmed that increased
levels of ROS production in day 3 embryo culture media have detrimental effects on
the embryo growth parameters as well as clinical pregnancy rates.
From the studies discussed, we can conclude that in order to improve sperm
quality, embryo development as well as clinical pregnancy rates, short co-incubation
time for gametes should be clinically applied in an attempt to improve ART out-
come. Furthermore, supplementing of the culture media with required antioxidants
to help scavenge excessive production of ROS in culture media is beneficial.
In addition, the design of the culture media used should be such that the media takes
into consideration the role of ROS generation and eliminate their contribution with-
out changing the metabolic requirements of gametes and embryos.
2.2.7 Oxygen Concentration
Gametes/embryos are exposed to oxygen tension during procedures in assisted

reproduction such as insemination, fertilization and embryo growth (Catt and
Henman 2000). The development of the human pre-implantation embryo in vitro is
influenced by the atmospheric oxygen partial pressure and dissolved oxygen con-
centration in the culture medium. In ART laboratories, cells are commonly cultured
in vitro in an atmospheric oxygen concentration of 20 % (160 mmHg) or an incuba-
tor environment of ~20 % oxygen and 5 % carbon dioxide. However, under physi-
ological conditions in vivo in the oviduct and uterus, embryos are exposed to much
lower oxygen concentrations of 2–8 % or 19–70 mmHg (Calzi et al. 2012). At
37 °C, the oxygen concentration in the medium equilibrated with atmospheric oxy-
gen was found to be 20-times higher than physiological intracellular oxygen con-
centration (Jones 1985). Thus, the use of dissolved oxygen levels in the culture
media in vitro at ~5 %, which is adopted by ART laboratories nowadays, is more
comparable to the oxygen concentration at the tissue level in vivo.
Oxygen plays an essential role in cell growth and differentiation, but in an IVF
setting, the presence of high concentrations of oxygen during incubation activates
various cellular oxidase enzymes. This in turn increases the generation of ROS—
leading to a state of OS (Cohen et al. 1997). Thus, the gametes and media in the
ART laboratory setting should be protected from exposure to high partial pressures
of oxygen in order to minimize the production of ROS during IVF procedures.
When compared to embryos cultured under atmospheric oxygen conditions, those
cultured at 5 % oxygen yielded better developed embryos and higher pregnancy
rates (even among the poor responders), during IVF and ICSI cycles (Kovacic and
Vlaisavljevic 2008; Kovacic et al. 2010). Interestingly a meta-analysis of seven
randomized controlled trials (RCTs) comparing the effects of oocyte/embryo cul-
turing at low (~5 %) and atmospheric (~20 %) oxygen concentration on assisted
reproduction outcomes such as fertilisation, implantation and ongoing pregnancy
rates showed no significant difference between the two groups (Gomes-Sobrinho
et al. 2011). Embryos transferred on days 2 or 3 had similar implantation rates
regardless of the oxygen tension used (~5 % vs. ~20 %) during culture, but for
embryos transferred on day 5 or 6 (blastocyst stage), the implantation rates of
embryos cultured at ~5 % oxygen were significantly higher than embryos cultured
at ~20 % oxygen. However, the ongoing pregnancy rates did not differ significantly
despite the different oxygen tensions used (~5 % or ~20 %) or the day of transfer
(days 2/3 or days 5/6) (Gomes-Sobrinho et al. 2011).
Results of a recent Cochrane systematic review (involving 7 studies, 2,422

participants) and meta-analysis (involving 4 studies, 1,382 participants) showed
that embryos cultured in low (5 %) oxygen concentrations developed better and
were therefore of improved quality. This resulted in higher ongoing and clinical
pregnancies rates and improved live birth rates. Thus compared to embryo culture
in atmospheric (~20 %) oxygen concentrations, the culture of preimplantation
embryos under low (~5 %) oxygen concentrations improves IVF/ICSI success rates
and results in the birth of healthier babies (Bontekoe et al. 2012).
To strengthen these positive results, larger, well-designed randomized controlled
trials are required. Results from these type of studies would provide more conclu-
sive evidence on the impact of low oxygen culture on IVF outcome. In general,
incubation of spermatozoa is usually done at 37 °C in an atmosphere of 5 % CO2 for
at least 1–2 h before ICSI or conventional insemination. In addition, these types of
preparation and incubation conditions could affect the DNA integrity of ejaculated
human spermatozoa.
Chapter 3
Antioxidant Strategies
As has been portrayed in the previous section, there is a growing body of evidence
to signify that multiple in vivo and in vitro factors could encroach on an ART setting,
leading to an increase in OS and ART outcome. Therefore in ART laboratories, it is
indispensable to scrutinize approaches that will help increase the success rate of the
procedure by limiting the potentially negative effects on successful ART outcomes
(Sikka 2004).
Elevated ROS levels need to be quelled as much as possible to suppress its detri-
mental effects on the ART procedure. The effects of high levels of ROS on the human
gametes, fertilization and embryo are summarized in Fig. 3.1. ROS are neutralized by
an intricate defence system consisting of enzymatic antioxidants and non-enzymatic
antioxidants. Preferably, an antioxidant should reach high enough concentrations and
supply the deficient vital elements required for the process to occur. Furthermore, the
antioxidant should have the aptitude to enhance the scavenging capacity and main-
tain a physiological amount of ROS, without suppressing it entirely (Zini et al. 2009).
Therefore, the first step to take would be to establish the fundamental cause of the
ROS/antioxidant imbalance and treat it (Agarwal et al. 2004).
As discussed earlier, the body contains many natural antioxidant defence systems
in vivo. These mechanisms become eradicated in the in vitro state. Thus, in order to
aid in the management of OS during ART procedures, the potential patients could be
provided with oral antioxidant supplements prior to the ART procedure. Another
option would be to add various dosages of antioxidants directly into the media
during the ART procedure in order to reduce the development and effects of OS.
Oral antioxidants have been reported to improve the quality of gametes prior to
the IVF procedure. It has also been shown to enhance fertilization and pregnancy
rates. In the following section, we will discuss several studies that support the use
of specific enzymatic antioxidants including SOD, glutathione reductase (GSH)
and catalase; some non-enzymatic oxidants including vitamins C and E, folic acid,
L-Carnitine, Coenzyme Q10 and melatonin as well as combined antioxidant strate-
gies (Lampiao 2012). Table 3.1 summarizes the mechanisms of action and effects of

DOI 10.1007/978-3-319-10259-7_3
24 3 Antioxidant Strategies
Fig. 3.1 Effects of pathological levels of reactive oxygen species in assisted reproductive
technology
antioxidants that is of importance and that are frequently-used clinically. Despite

non-enzymatic antioxidant supplementation being more common than enzymatic
antioxidants, both types of antioxidants have a significant role to play in minimizing
of OS. In the coming section, we will also indicate the exact dosage information and
methods of how each antioxidant treatment works, as well as provide recommenda-
tion for its use in ART, based on the outcome of the studies discussed.
3.1 Role of Enzymatic Antioxidants in ART
In vivo, the superoxides are released by oxidative phosphorylation and other pro-
cesses. They are transformed initially to hydrogen peroxide H2O2 and then reduced
to water. This detoxification pathway has multiple enzymes—superoxide dis-
mutases begin the catalyses, then catalases and various peroxidases remove H2O2.
The contributions of these enzymes to antioxidant defences are difficult to separate
from one another. The significance of enzymatic antioxidants in males has been
reported in various studies, and will be discussed in the following section.
Table 3.1 Role of antioxidants in ameliorating the effects of oxidative stress in assisted reproduction
Antioxidant(s) Study Treatment dosage (per treatment or day) Treatment details Parameters improved
SOD Rossi et al. (2001) SOD (100 U/mL) and catalase Added to fresh semen before • Semen parameter, especially
(100 U/mL) freezing progressive motility) (SOD and
catalase)—due to their combined and
simultaneous action on superoxide
anion and hydrogen peroxide
Catalase Li et al. (2010) Ascorbate (300 or 600 μM) and catalase Added to semen before • Reduced ROS levels and ROS-induced
(200 or 400 IU/mL) freezing damages in post-thaw spermatozoa
[ascorbate (300 μM) and catalase
(200 and 400 IU/L)]
Catalase Chi et al. (2008) Catalase (1, 10, 100 U/mL) or EDTA Added to medium used during • Sperm motility (10 μM/mL EDTA)
(1, 10, 100 μM/mL) sperm wash • Acrosome reaction rate of the
spermatozoa (catalase)
• Decreased DNA fragmentation rate of

the spermatozoa (EDTA and catalase)
Vitamin E Kalthur et al. (2011) Vitamin E 5 mM Added to cryomedia prior • Post-thaw motility
to freeze-thaw • DNA integrity
Vitamin E Taylor et al. (2009) Vitamin E 100 or 200 μmol Added to cryomedia • Post-thaw motility
Vitamin E Cicek et al. (2012) Vitamin E 400 IU orally to women Between day 3 and 5 of • Endometrial thickness on hCG day
with unexplained infertility undergoing menstrual cycle until hCG
ovarian stimulation and IUI injection
Vitamin E + Moslemi and Vitamin E 400 IU + Selenium 200 μg 100 days • Sperm motility
selenium Tavanbakhsh (2011) orally in men with idiopathic • Sperm morphology
asthenozoospermia • Spontaneous pregnancy rates
Vitamin C Akmal et al. (2006) 2,000 mg vitamin C in 2 months • Mean sperm count
oligozoospermic men • Sperm motility
• Sperm morphology
(continued)
25
26
Vitamin C + Greco et al. (2005b) Oral 1 g vitamin C and 1 g vitamin E 2 months • Reduced DNA damaged sperm
vitamin E in men with elevated sperm DNA • Implantation rates
fragmentation (15 %) and prior • Implantation rates
failed ICSI attempt
• Clinical pregnancy rates
Vitamin C Henmi et al. (2003) Oral ascorbic acid 750 mg in women 1st day of the third cycle until a • Clinical pregnancy rates
with luteal phase defects positive urinary pregnancy test
Vitamin C Crha et al. (2003) Vitamin C 500 mg in women Gradual release over 8–12 h • Improved pregnancy rates
undergoing IVF-ET
Vitamin C Branco et al. (2010) Ascorbic acid 10 mM Semen before freezing • Reduced DNA damage
Melatonin Eryilmaz et al. Melatonin 3 mg in IVF-ET patients 3rd to the 5th day of the • Mean number of retrieved oocytes
(2011) menstrual cycle until hCG • Mean number of MII oocyte counts
• G1 embryo ratio
Melatonin Tamura et al. (2008) Melatonin 3 mg in women with prior Between the 3rd and the 5th • Oocyte quality
IVF-ET failure day of the menstrual cycle • Fertilization rates
until hCG injection
Melatonin Unfer et al. (2011) Myo-inositol 4 g and melatonin 3 mg 3 months • Number of MII oocytes retrieved
and folic acid 400 mcg in women with • Total and top-quality embryos
failed IVF cycle transferred
• Fertilization rate
3
Coenzyme Q10 Nadjarzadeh CoQ10 200 mg in iOAT patients 12 weeks • Increased TAC
et al. (2011) • Reduced LPO
Coenzyme Q10 Safarinejad (2012) CoQ10 600 mg in iOAT patients 12 months • Sperm quality (concentration,
progressive motility, morphology)
• Pregnancy rates
L-Carnitine Lenzi et al. (2003) L-Carnitine 2 g 2 months • Semen quality
L-Carnitine and Cavallini et al. L-Carnitine 2 g and acetyl-L-Carnitine 1 g 6 months • Semen quality
acetyl L-Carnitine (2004) • Pregnancy rates
L-Carnitine with Wang et al. (2010) L-Carnitine 2 g and vitamin E 3 months • Percentage of forward motile sperm
Vitamin E after the treatment
• Pregnancy rate
L-Carnitine and Vicari and L-Carnitine 2 g and acetyl-L-Carnitine 1 g 3 months • Sperm forward motility
acetyl L-Carnitine Calogero (2001) • Sperm viability
• Reduced ROS production
L-Carnitine Khademi L-Carnitine 3 g in men with idiopathic 3 months • Percentile of motile and grade A sperm
et al. (2005) sperm abnormalities • Percentile of normal-shaped sperms
decreased significantly
L-Carnitine Abdelrazik L-Carnitine 0.3 and 0.6 mg/mL Added to cryomedia • Blastocyst development rate
et al. (2009) • Reduced DNA damage

Folic acid and Wong et al. (2002) Folic acid 5 mg and zinc sulphate 26 weeks • Folate concentrations in seminal plasma
zinc sulphate 66 mg in subfertile men • Total normal sperm count
Myo-inositol and Papaleo et al. (2009) Myo-inositol 4 g and 400 μg folic Continuous from the day • Reduced mean number of immature
folic acid acid twice a day of GnRH administration oocytes at pick up
• Reduced mean number of degenerated
oocytes at pick up
• Number of retrieved oocytes
maintained
Myo-inositol and Ciotta et al. (2011) Myo-inositol 4 g and 400 μg folic 3 months • Number of oocytes recovered at pick-up
folic acid acid twice a day • Reduced number of immature oocytes
(continued)
27
28
Combination Wirleitner FertilovitRMplus twice daily 2 months • Sperm motility
of antioxidants et al. (2012) • Reduced percentage of immotile
sperm cells
• Total sperm count
• Percentage of class I sperm
Combination Tunc et al. (2009) 1 Menevit capsule 3 months • Pregnancy outcome
of antioxidants
Combination Tremellen Menevit 3 months • Viable pregnancy rate
of antioxidants et al. (2007)
Combination Omu et al. (2008) Zinc 5 mg, Vitamin E + zinc 10 mg 3 months • Sperm parameters
of antioxidants and Zinc + Vitamin E + C 200 mg
Combination Rizzo et al. (2010) Myo-inositol 4 g and folic acid 200 mg Continuous from the day • Oocyte quality
of antioxidants and melatonin 3 mg of GnRH administration • Number of morphologically mature
oocytes at ovum pick up
Combination of Dashti et al. (2013) Tamoxifen 20 mg, vitamin E 400 IU, 3 months prior to IUI • Sperm concentration, motility, forward
antioxidants zinc 30 mg and selenium 200 mg progression and the percentage normal
forms
3
3.1 Role of Enzymatic Antioxidants in ART 29
3.1.1 Superoxide Dismutases
Within the cell, superoxide dismutases (SODs) constitute the first line of defence
against ROS (Alscher et al. 2002). They catalyze the breakdown of the O2− anion
into oxygen (O2) and water (H2O) (Hammadeh et al. 2008). There are three isoforms
of SODs, the intracellular mitochondrial isoform, manganese SOD (Mn SOD), and
the cytoplasmic isoform, Copper/Zinc SOD (Cu-Zn SOD), along with the extracel-
lular SOD (Fe SOD). In males, this antioxidant group is naturally found in the semi-
nal plasma (Matos et al. 2009).
Hammadeh et al. (2008) observed the effects of various enzymatic antioxidants in
the seminal plasma of males undergoing IVF and ICSI treatments. A negative correla-
tion was seen between seminal plasma ROS concentration in and membrane integrity,
normal morphology as well as fertilization rate. Furthermore a positive association
was found between SOD levels and normal sperm morphology; thus concluding that
enzymatic antioxidants definitely do play a positive role in male infertility.
The activity and relationship of SOD with MDA (as a marker of lipid peroxida-
tion), in normozoospermic and asthenozoospermic males has also been tested
(Tavilani et al. 2008). The findings indicate a protective role for seminal plasma
antioxidant enzymes against lipid peroxidation of spermatozoa in normozoosper-
mic samples. The relationship between seminal antioxidant capacity and OS mark-
ers, along with semen quality has been investigated in various studies. Studies that
looked at the sperm count and progressive motility, found a significant positive cor-
relation with SOD during IVF (Shiva et al. 2011; Marzec-Wroblewska et al. 2011;
Atig et al. 2012). Results from these studies suggest that decreased antioxidant
enzymes and increased OS may be the cause of higher risk of deteriorating semen
quality. Highly significant and positive correlations were also found between semi-
nal SOD activity and semen parameters such as sperm concentration and overall
motility in the normozoospermic group, which are regarded as the most important
criteria for normal fertilizing ability of the spermatozoa.
In women with tubal factor infertility, the association between SOD, MDA and
the incidence of apoptosis of granulosa cells in follicular fluid was investigated. It
was demonstrated that there was a significant negative correlation between MDA
and SOD levels and significant positive correlation between MDA levels and the
prevalence of apoptosis (Liu and Li 2010). It was also noted that patients who had
undergone a failed cycle of IVF-embryo transfer (IVF-ET) had a propensity to have
lower levels of SOD present in their granulosa cells. It was furthermore evident that
these patients had embryos of poorer quality compared to the group of patients who
were able to achieve pregnancy. In these patients, SOD has been reported to increase
the proportions of zygotes that undergo at least one round of cleavage while
improving cleavage past the two-cell stage and overall blastocyst development
(Liu and Li 2010).
3.1.2 Catalase
Human seminal plasma contains catalase which is principally derived from the
prostate gland. In females, it is localised in the corpus luteum and tubal fluid (Rakhit
et al. 2013). This enzyme catalyzes the breakdown of H2O2 to H2O and O2. In males,
catalase is known to improve the motility, vitality and DNA integrity of cryopre-
served human spermatozoa (Moubasher et al. 2013). In their study, Moubasher’s
group added 200 μL/mL of catalase to semen during cryopreservation, and the sam-
ple was then cryopreserved for 24 h. The post-thaw results showed a momentous
increase in the percentage of progressively motile and viable spermatozoa, while
the % of DNA damage significantly decreased in samples supplemented with cata-
lase compared to samples without catalase (either fresh or processed). The outcome
of this study was in agreement with another study which evaluated the consequence
of both SOD and catalase supplementation in human semen undergoing cryopreser-
vation (Rossi et al. 2001). From the results, it was evident that the addition of SOD
(100 U/mL) and catalase (100 U/mL) to semen prior to cryopreservation improved
the percentage recovery of progressively motile spermatozoa after thawing. These
results suggested that, SOD and catalase supplementation can contribute signifi-
cantly to the prevention of sperm membrane lipid peroxidation by ROS and thus
allow for good sperm parameter recovery after freeze-thawing procedures.
The protective effects of ascorbate and catalase on cryopreserved spermatozoa
has also been investigated (Li et al. 2010). Ascorbate (300 or 600 μM) and catalase
(200 or 400 IU/mL) were added to semen samples. Sperm viability, motility, and
mitochondrial membrane potential was a significantly lower in comparison to that
of fresh spermatozoa. Moreover, there was an increase in apoptosis [positive for
annexin V and negative for propidium iodide Ann(+)/PI(−)] and DNA damage
(olive tail movement (OTM)) in the cryopreserved spermatozoa . It was observed
that ascorbate and catalase reduced the ROS levels in post thaw spermatozoa signifi-
cantly. Therefore appropriate ascorbate or catalase supplementation to the cryopro-
tective medium can control ROS levels and minimize the resultant cryodamage.
Different concentrations of catalase (100, 10, 1 U/mL, Sigma) and ethylenedi-
aminetetraacetic acid (EDTA) (a metal chelating agent) were added to spermatozoa
while washed with Ham’s F-10 media (Chi et al. 2008). The researchers indicated
that the addition of EDTA to sperm preparation medium improved sperm motility
compared to control group, EDTA group at various concentrations and catalase
group. Interestingly, catalase significantly improved the acrosome reaction rate of
the spermatozoa. EDTA and catalase both significantly lowered the sperm DNA
fragmentation rate.
A recent study demonstrated the influence of seminal catalase activity on IVF
pregnancy rates (Zelen et al. 2010). Semen samples were collected from men
undergoing IVF and ICSI. Conventional semen parameters, sperm DNA fragmenta-
tion index (%DFI), high DNA stainability (%HDS) and seminal catalase-like activ-
ity were measured and the results correlated with outcomes of IVF and ICSI. It was
discovered that catalase activity was positively correlated with IVF pregnancy rate
3.2 Role of Non-enzymatic Antioxidants in ART 31
but not with ICSI pregnancy rate. The data suggested that couples in whom the men
have low seminal catalase activity (i.e., inadequate protection from hydrogen perox-
ide) have a higher risk of post-fertilization failure during IVF cycles and it is there-
fore advisable that ICSI should be used as alternative treatment option.
3.1.3 Glutathione Peroxidase
Glutathione peroxidase (GPX) governs one of the most important systems in the
exclusion of ROS in the seminal plasma. In the female, it is located within the glan-
dular epithelium of the endometrium. GPX catalyse the reduction of organic and
inorganic hydroperoxides, using reduced glutathione as an electron donor. Few
studies have documented the role of GPX activity in human seminal plasma. A
recent investigation studied the correlation between GPX activity levels in human
seminal plasma with standard semen parameters and spermatozoa fertilization
potential, in terms of fertilization and pregnancy rates in IVF (Crisol et al. 2012).
They found that seminal plasma GPX activity was significantly lower in patients
with abnormal sperm compared to normozoospermic individuals. These results
were in agreement with an earlier study, that determined the expression and enzy-
matic activity of GPX-1 and GPX-4, concentrations in spermatozoa from fertile and
infertile men (Garrido et al. 2004). In Garrido’s study, men with poor sperm mor-
phology had significantly lower levels of GPX activity. They suggested that sperm
GSH system components in the cell, GPX-4 and GSH, vary in infertile men. These
changes may be associated with sperm morphology. From these results, we can
conclude that GPX activity in human semen is critical for normal sperm function
and morphology (Garrido et al. 2004).
3.2 Role of Non-enzymatic Antioxidants in ART
Non-enzymatic antioxidants, also known as synthetic antioxidants or dietary sup-

plements (Agarwal et al. 2005b) involve a multitude of low molecular mass ROS
scavengers such as vitamin E, C, melatonin and many other naturally-occurring
antioxidants (Gharagozloo and Aitken 2011). Based on the credence of such scien-
tific evidence (Table 3.1), clinical experiments have been carried out to ascertain the
beneficial effects of non-enzymatic antioxidants in improving ART outcome.
Couples undergoing ART infertility may benefit from the use of these non-enzymatic
antioxidants in order to improve OS situations encountered during IVF/ICSI. These
antioxidants have been evaluated clinically either individually or in combination.
The study trials that are elaborated upon in the following sections include studies
that have been conducted in various population sizes, types, doses as well as dura-
tion of antioxidant therapy.
3.2.1 Vitamin E
Vitamin E refers to a group of eight fat-soluble compounds that consist of both

tocopherols and tocotrienols. Alpha-tocopherol is one of the most important lipid-
soluble antioxidant molecules as well as the most biologically active form of vitamin
E, located mainly in the cell membranes, (the second-most common form of vitamin
E in the diet). It acts by breaking pathological ROS-induced chain reactions. It con-
fers its protective effects by shielding sperm membrane components from oxidative
stress damage (Mora-Esteves and Shin 2013). Various studies discussed in this sec-
tion evaluated the effects of vitamin E alone or in combination with other vitamins.
In clinical trials, vitamin E supplementation has been found to increase semen
parameters and fertilization rates possibly by reducing oxidative damage and lipid
peroxidation potential during IVF/ICSI treatment (Comhaire et al. 2000; Geva et al.
1996). A randomized controlled trial was set up to investigate the action of vitamin
E in men with idiopathic infertility (Cicek et al. 2012). In this study, the group ana-
lysed vitamin E effect in patients with unexplained infertility undergoing intrauter-
ine insemination (IUI). It was hypothesized that clinical outcomes of IUI cycles
may improve with vitamin E administration. 400 IU/day of vitamin E was adminis-
tered beginning from between the 3rd to the 5th day of the menstrual cycle, until the
human chorionic gonadotropin (hCG) injection day. The results demonstrated that
vitamin E supplementation in unexplained infertile patients had beneficial effects in
improving the endometrial thickness during IUI cycles. Furthermore, higher implan-
tation and ongoing pregnancy rates were observed in the vitamin E-administered
group, and it was concluded that these improvements may be a result of the improv-
ing antioxidant effect of vitamin E on the endometrial receptivity.
The effect of addition of vitamin E to cryoprotective media prior to sperm cryo-
preservation on the post-thaw motility and DNA integrity of normozoospermic and
asthenozoospermic semen samples was also investigated (Kalthur et al. 2011). The
group noted that supplementation of 5 mM vitamin E to cryomedia significantly
improves the post-thaw motility and DNA integrity in normozoospermic and astheno-
zoospermic semen samples prior to ICSI. The results of this study were in conformity
with a previous study that investigated whether the addition of an antioxidant to cryo-
preservation medium could improve the post-thaw integrity of cryopreserved human
spermatozoa, particularly from men with abnormal semen parameters. The cryo-
preservation medium contained either 100 or 200 μmol of vitamin E. The study find-
ings report of a significant improvement of post-thaw motility (Taylor et al. 2009).
The efficacy of vitamin E in combination with selenium (Se) for improving
semen parameters and pregnancy rates were also investigated in various studies. Se,
an essential trace element occurring in organic and inorganic forms, is important for
reproductive functions such as testosterone metabolism and is a constituent of the
sperm capsule selenoprotein (Mora-Esteves and Shin 2013).
The effects of vitamin E and selenium supplementation on lipid peroxidation and
on sperm parameters were inspected by one of the earlier groups (Keskes-Ammar
et al. 2003). 400 mg of vitamin E and 225 μg of selenium was provided daily to
male patients for a period of 3 months. Findings of the study include a significant
decrease in MDA levels and an improvement in sperm motility after treatment.

These results were also in agreement with a recent study that examined the efficacy
of vitamin E in combination with selenium for improving semen parameters and
pregnancy rates (Moslemi and Tavanbakhsh 2011). Patients were assigned to fixed-
dose treatment with oral 200 μg selenium tablet (L-selenomethionine) daily in com-
bination with 400 IU of synthetic vitamin E (α-tocopherol) for 100 days. Total
enhancement in sperm motility and normal morphology, as well as in spontaneous
pregnancy were observed when compared with the controls. Overall, a beneficial
and protective effect of Vitamin E on semen quality was suggested, particularly
sperm motility. Furthermore, the authors advocated supplementation of vitamin E
and selenium for the treatment of idiopathic male infertility diagnosed with asthe-
noteratozoospermia or asthenozoospermia in semen analysis.
3.2.2 Vitamin C
Vitamin C (L-ascorbic acid, ascorbate) has long been allied with fertility. This vitamin
is a crucial water-soluble micronutrient obligatory for an array of biological func-
tions. It is an unstable, easily-oxidized acid and can be damaged by oxygen, alkali
and high temperature. In males, it is considered a major antioxidant in the testes and
highly concentrated in seminal plasma where it has also been reported to contribute
up to 65 % of its total chain breaking antioxidant capacity (Augustine et al. 2005).
Several studies have reported a positive relationship between seminal vitamin C
and sperm quality as studied in both fertile and infertile men. Infertile patients were
reported to have significantly lower vitamin C levels in their seminal plasma com-
pared to fertile men (Colagar and Marzony 2009). Their findings suggested that
idiopathic infertile men have significantly lower levels of ascorbic acid in their
seminal plasma than fertile men. Thus, the authors recommended analysis of ascor-
bic acid levels in seminal plasma of patients with idiopathic infertility as ascorbic
acid supplementation could help improve semen parameters in these patients.
A study was conducted to observe if the increased incidence of DNA fragmentation
in ejaculated spermatozoa during the IVF treatment could be reduced by addition of
vitamins C and E. The study results showed that a 2 month daily oral supplementa-
tion of vitamins C (1 g) and vitamin E (1 g) reduced the percentage of DNA-
fragmented spermatozoa (Greco et al. 2005a). For such spermatozoa DNA
fragmentation cases, The same group also discovered that oral antioxidant treatment
improves ICSI outcome (Greco et al. 2005b).
A treatment protocol consisting of daily oral antioxidant intake (vitamins C and
E, beta-carotene, zinc and selenium) caused a marked improvement in clinical preg-
nancy and implantation rates in patients with >15 % sperm DNA fragmentation
index. Menezo and his group also suggested that this oral antioxidant treatment
appears to improve ICSI outcomes in those patients with sperm DNA damage, how-
ever, the degree of sperm decondensation of these patients should also be considered
(Menezo et al. 2007). The efficacy of vitamin C supplementation in cryomedium
reported improved sperm motility preservation post thaw. The experiments involved
addition of 10 mM of ascorbic acid to semen before cryopreservation. It also reduced
DNA damage and preserved the integrity of spermatozoa in infertile men (Jenkins
et al. 2011; Branco et al. 2010).
The effect of oral supplementation of vitamin C on various semen parameters in
oligospermic, infertile men during IVF/ICSI treatment was monitored (Akmal et al.
2006). Various semen parameters, including sperm motility, sperm count, and sperm
morphology, were studied before and after the vitamin C treatment. These patients
received in an open trial of 1,000 mg of vitamin C twice daily for a maximum of 2
months. Results showed that the mean sperm count was increased. The mean sperm
motility was increased significantly and mean sperms with normal morphology
increased significantly as well suggesting that vitamin C supplementation in infer-
tile men might help improve these parameters. The impact of vitamin C supplemen-
tation was also observed in females undergoing IVF-ET procedure The influence of
vitamin C supplementation on the outcome of infertility treatment 76 women (38 of
them smokers, the remaining 38 were non-smokers) was studied (Crha et al. 2003).
The women were administered vitamin C in daily doses of 500 mg. The results
proved that vitamin supplementation had a greater impact on the number of preg-
nancies in the non-smokers’ group. The pregnancy rate was significantly higher in
non-smoking women than in smokers which appears to be a reason for asking
women to cease smoking prior to infertility treatment. Further, on the addition of
vitamin C to culture media, increase sperm motility and viability along with
decreases in MDA levels were noted (Mostafa et al. 2006).
3.2.3 Melatonin
Melatonin (N-acetyl-5-methoxytryptamine) is a multi-functional and universal anti-

oxidant (Ronnberg et al. 1990), secreted during the dark hours at night by pineal
gland, and it regulates a variety of significant central and peripheral actions related
to circadian rhythms and reproduction (Ronnberg et al. 1990). Melatonin has been
demonstrated as a powerful direct scavenger of free radicals in a growing number of
studies, and is also known for its multifunctional and universal antioxidant proper-
ties (Eryilmaz et al. 2011). Melatonin regulates ovarian function by the instruction
of gonadotropin discharge in the hypothalamus-pituitary gland axis via its specific
receptors (Eryilmaz et al. 2011).
A growing number of studies have demonstrated the role of melatonin in repro-
duction. Human follicular fluids contain higher melatonin concentrations than
plasma which increases depending on the follicular growth (Nakamura et al. 2003;
Ronnberg et al. 1990). The relationship between OS, oocyte quality and melatonin
was also investigated in patients undergoing IVF-ET (Tamura et al. 2008). 3 mg/day
melatonin was administered in women from the fifth day of the previous menstrual
cycle until the day of oocyte retrieval. Intrafollicular concentrations of 8-OHdG and
hexanoyl-lysine adduct were significantly reduced by these antioxidant treatments.
The fertilization rate was improved by melatonin treatment and suggested that
melatonin protects oocytes from OS and is likely to improve the oocyte quality
and fertilization rates. The same treatment protocol was followed up in another
study (Eryilmaz et al. 2011). The results illustrated that the mean number of
the retrieved oocytes, the mean MII oocyte counts, the G1 embryo ratio were
significantly higher in the melatonin-administered group than that of the non-
melatonin-administered group.
The efficacy of a treatment with myo-inositol plus folic acid plus melatonin com-
pared with myo-inositol plus folic acid alone on oocyte quality was investigated in
women who underwent IVF (Rizzo et al. 2010). Patients were assigned to obtain
either 2 g myo-inositol twice a day combined with 200 mg folic acid and 3 mg mela-
tonin. The data showed improvement in oocyte quality, number of morphologically
mature oocytes at ovum pick up. Results from the study revealed significantly top-
quality embryos in patients treated with melatonin. In the following year, another
group conducted a study to evaluate the pregnancy outcomes after the administra-
tion of myo-inositol combined with melatonin in women who failed to conceive in
previous IVF cycles due to poor oocyte quality (Unfer et al. 2011). Women were
treated with 4 g/day myo-inositol and 3 mg/day melatonin for 3 months and then
underwent a new IVF cycle. After treatment, the number of mature oocytes, the
fertilization rate, the number of both, total and top-quality embryos transferred were
statistically higher compared to the previous IVF cycle.
The results were in agreement with another group that evaluated the effect of
melatonin alone for improving oocyte quality in patients with previous failed IVF
treatment (Batioglu et al. 2012). 1 or 3 mg tablets of melatonin were taken orally by
the patients from the fifth day of the previous menstrual cycle to the day they were
injected with HCG. The results revealed percentage of mature oocytes (M2/oocytes
retrieved) was significantly different in melatonin-treated group. The mean number
of class 1 embryos resulted higher in the melatonin-treated group as well as clinical
pregnancy rate in women undergoing IVF or ICSI was higher.
In males, melatonin is localised in seminal fluid and membrane receptors of sper-
matozoa. The in vitro effects of melatonin on human sperm motility, concentration
and motility was evaluated (Ortiz et al. 2011). The data discovered urinary
6-sulfatoxy melatonin and total antioxidant capacity levels were positively corre-
lated with sperm concentration, motility and morphology, as well as negatively cor-
related with the number of round cells. Additionally, 30-min exposure of sperm to
1 mm melatonin improved the percentage of motile and progressively motile cells.
3.2.4 Vitamin B: Folic Acid
An additional empirically-used and an inspected agent in infertility is folic acid.

They are group B vitamins implicated in the one-carbon metabolism requisite for
purine and pyrimidine synthesis and eventually affecting DNA synthesis and integ-
rity (Joshi et al. 2001). They are also involved in re-methylation of homocysteine
(HCY) into methionine which is further metabolized into S-adenosylmethionine,
the universal methyl donor for transmethylation of DNA. Homocysteine being a
methionine catabolite and its concentration in follicular fluid, is negatively related

to oocyte maturity (Papaleo et al. 2011).
As folate plays a key role in germ cell development, consequently it is noticeable
that folate is important for reproduction (Joshi et al. 2001; Ebisch et al. 2007). In order
to justify, it was established that seminal plasma total folate concentrations reflect
folate nutriture and that it may be important for male reproductive function. It was
demonstrated that total seminal plasma folate concentrations were on average 1.5
times higher than blood plasma folate concentrations in men (Wallock et al. 2001).
It was experimented that 5 mg folic acid administration to subfertile and fertile
men for 26 weeks resulted in a significant increase of folate concentrations in semi-
nal plasma. Although no effect of this involvement was observed on sperm count or
motility of spermatozoa, however there was a 74 % increase in total normal sperm
count after a combination of folic acid and zinc sulphate in both subfertile and fer-
tile me (Wong et al. 2002).
In females, a study was conducted to evaluate the association between follicular
fluid homocysteine concentration and the degree of maturity of oocyte (Szymanski
and Kazdepka-Zieminska 2003). 40 patients were qualified for IVF-ET and under-
went folic acid supplementation. The folic acid concentration recovered from folli-
cles was measured using microparticle enzyme immunoassay (MEIA). The rationale
of the study was to establish dependencies between the concentration of homocys-
teine in follicular fluid and the quality of oocytes and it was discovered that in a
group of women with folic supplementation and lower homocysteine concentration
the percentage of oocytes in first and second degree of maturity was higher and of
better quality. The effects of myo-inositol and folic acid on oocyte quality in PCOS
patients undergoing ICSI cycles were determined (Papaleo et al. 2009). The partici-
pants received myo-inositol combined with 2 g folic acid twice a day. The results of
their experiment revealed that in patients with PCOS, treatment with myo-inositol
and folic acid reduces germinal vesicles and degenerated oocytes at ovum pick-up
compared to control. The results were in agreement with the outcome of another
study that determined the effects of myo-inositol plus folic acid on oocyte’s quality
in PCOS patients (Ciotta et al. 2011). 2 g of myo-inositol plus 200 mg of folic acid
were taken by the participants twice a day, continuously for 3 months. The results at
the end of treatment revealed that the number of oocytes recovered at the time of
pick-up were significantly greater as compared to control. Significantly reduced
number of immature oocytes (vesicles germ and degenerated oocytes) was also
investigated. Data from both the studies suggest that this treatment combination
may be useful in the management of PCOS patients.
3.2.5 Coenzyme Q10
Coenzyme Q10 (CoQ10) also commonly called ubiquinones mostly produced in

mitochondria of cells and present in human seminal fluid, exerts important meta-
bolic and antioxidant functions and shows direct correlation with seminal
parameters (count and motility). Alterations of CoQ10 content have been shown in
conditions associated with male infertility, such as asthenozoospermia and varico-
cele (Balercia et al. 2009) Coenzyme Q10 is the only lipid soluble antioxidant syn-
thesized endogenously. A recent study investigated the role of CoQ10 supplementation
on semen parameters in idiopathic oligoasthenoteratozoospermia (OAT)
(Nadjarzadeh et al. 2011). In the study a total of 47 infertile men with iOAT were
randomly assigned to receive 200 mg CoQ10 daily or placebo during a 12-week
period. The results of the study showed that concentrations of thiobarbituric acid-
reactive substances (TBARS) were significantly reduced in serum of treated groups
compared with the control. Furthermore, total antioxidant capacity of seminal
plasma significantly increased in the CoQ10 group. The results provided additional
evidence suggesting that CoQ10 supplementation is associated with alleviating OS
and suggested that CoQ10 could be taken as an adjunct therapy in cases of oligoas-
thenoteratozoospermia. The evaluation of CoQ10 supplementation on semen param-
eters and pregnancy rates in idiopathic oligoasthenoteratozoospermia (iOAT) were
also reported in the following year (Safarinejad 2012). These patients were treated
with CoQ10 300 mg orally twice daily for 12 months. The results revealed that mean
sperm concentration, sperm progressive motility, and sperm with normal morphol-
ogy improved significantly after a 12-month CoQ10 therapy with beneficial effect on
pregnancy rate. For the first time, the occurrence of Coenzyme Q10 in follicular fluid
in females and its relationship with oocyte fertilization and embryo grading was
investigated in a study that measured CoQ10 levels from 20 infertile women under-
going IVF using HPLC system (Turi et al. 2012). The results of the study showed
that CoQ10/Protein levels resulted significantly in mature versus dysmorphic
oocytes. Similarly, CoQ10/Cholesterol was significantly higher in grading I–II
versus grading III–IV embryos.
3.2.6 L-Carnitine
L-Carnitine (LC) is a small water-soluble molecule which exerts an important func-

tion in fat metabolism. L-Carnitine is concentrated in high energy demanding tissues
such as skeletal and cardiac muscles and in the epididymis. It plays a significant role
in transferring long-chain fatty acids into the mitochondria for oxidation, producing
energy (Abdelrazik et al. 2009; Ahmed et al. 2011). The role of L-Carnitine in male
infertility has been demonstrated in various studies. It was hypothesised in a study
that L-Carnitine helps in maintaining normal fertility (Ahmed et al. 2011). The study
was designed to show comparison of seminal free L-Carnitine and sperm quality.
Case-controlled convenient sampling was used to assess infertile male subjects from
fertile. A total of 61 adult males were selected by consent, and were categorized as
fertile and infertile on the basis of history and semen analysis. The results showed
that the mean values of sperm count, total motility and normal morphology of asthe-
nozoospermic and oligoasthenoteratozoospermic men were significantly lower
when compared with fertile (control). When levels of seminal free L-Carnitine were
compared among groups, the result showed that infertile subjects had significantly
lower levels when compared to fertile subjects with lowest concentration in azo-
ospermic group, overall suggesting essential role of L-Carnitine level in seminal
plasma in maintaining male fertility.
Various studies explored the effect of L-Carnitine in asthenozoospermic patients
who underwent IVF-ET. Patients with asthenozoospermia were treated with L-Car-
nitine 2 g/day. The results demonstrated significantly increased percentage of semen
quality (forward motile sperm and sperm concentration) and increased pregnancy
rates after the treatment (Wang et al. 2010; Jarow 2003; Lenzi et al. 2003). These
studies demonstrated that L-Carnitine supplementation significantly improves two
of the most important sperm parameters: concentration and motility for patients
undergoing IVF-ET.
The use of L-Carnitine before percutaneous epididymal sperm aspiration
(PESA)–ICSI in the treatment of obstructive azoospermia was also explored in a
study in which the males were provided with oral 1 g L-Carnitine for 3 months. The
result of the experiment revealed that oral medication of L-Carnitine before PESA-
ICSI can raise the number and rate of good embryos in obstructive azoospermia
patients and therefore benefit the therapeutic outcome (Lu et al. 2010). In another
study, L-Carnitine 2 g/day and acetyl-L-Carnitine 1 g/day for 3 months in patients
with prostato-vesiculo-epididymitis (PVE) and elevated ROS production, showed
that carnitines are an effective treatment in such cases. The results of the study
showed increased sperm forward motility and viability in the patients with signifi-
cant reduction in ROS production (Vicari and Calogero 2001).
Supplementation with L-Carnitine 2 g/day and acetyl-L-Carnitine 1 g/day,
appeared to benefit idiopathic infertility in men. According to the study, nearly 22 %
of infertile men with enlarged varicose veins in the scrotum who took L-Carnitine
and acetyl-L-Carnitine achieved a pregnancy with their partner within 9 months,
compared to only 2 % of men taking a placebo (Cavallini et al. 2004).
In females, the protective effect of L-Carnitine was also studied in patients under-
going IVF-ET. The oocytes obtained from the infertile patients were fertilized by
ICSI. Embryos were incubated in culture media supplemented with 0.3 mg/mL
L-Carnitine and some without L-Carnitine. The results of the study showed signifi-
cant improvement in the quality of embryos and blastocyst development rate sup-
plemented with L-Carnitine. The authors suggested use of L-Carnitine in culture
media could provide a novel approach to improve ICSI outcome in infertile couples
(Abdelrazik et al. 2008).
Chapter 4
Role of Combined Antioxidants
Evidence in the literature supported the use of combined antioxidants for the
management of free radicals and OS prevention. The effectiveness of combined
non-enzymatic antioxidants was investigated in male patients with male factor
infertility prior to their third IUI treatment cycle (Dashti et al. 2013). Prior to the
treatment, the males were advised to take a combination of tamoxifen 10 mg twice
a day, vitamin E 400 IU daily, zinc 15 mg twice a day and selenium 200 mg daily
for 3 months. The results of the study detected significant differences in overall
values for the four semen parameters (sperm concentration, motility, forward pro-
gression and the percentage normal forms) in comparison to the earlier two IUI
cycles in the same group. The grouping of the female patients according to their
Body Mass Index (BMI) showed crucial differences in pregnancy outcome. The
influence of oral antioxidant supplementation on semen quality of IVF patients
undergoing IVF/ICSI was analysed in an experiment in which the male patients
were supplemented with an oral antioxidant called FertilovitRMplus twice daily for
2 months (Wirleitner et al. 2012). The components of FertilovitRMplus included:
Vitamin C 100 mg, Vitamin E 100 mg, Folic acid 500 μg, Zinc 25 mg, Selenium
100 μg, N-acetyl-L-Cysteine 50 mg, L-Carnitine 300 mg, Citrulline 300 mg,
Glutathione red 50 mg, Lycopene 4 mg and Coenzyme Q10 15 mg. The results of
this study revealed significant reduction in the percentage of immotile sperms in the
patients. Additionally, the percentage of class I spermatozoa was significantly
higher with drastic improvement in sperm motility as well as in total sperm count.
A study by Tunc’s group added to the already growing body of evidence support-
ing the use of antioxidant combinational therapy to improve sperm DNA integrity,
especially for men undergoing IVF-ICSI treatment (Tunc et al. 2009). In their study,
a total of 50 infertile men identified with OS were administered with an oral antioxi-
dant therapy called Menevit for a period of 3 months. The components included
Lycopene 6 mg, Vitamin E 400 IU, Vitamin C 100 mg, Zinc 25 mg, Selenium 26 g,
Folate 500 g, and Garlic oil 333 g (equivalent 1 g garlic). The results of the study

DOI 10.1007/978-3-319-10259-7_4
40 4 Role of Combined Antioxidants
suggested that treatment of men with a high degree of oxidative DNA damage with
antioxidants before their partner commences IVF-ICSI therapy, may be capable of
improving pregnancy outcomes.
In a previous study, the role of Menevit antioxidant therapy was examined on
embryo quality and pregnancy outcome during IVF/ICSI treatment (Tremellen
et al. 2007). Male participants were randomly assigned to take either one capsule
per day of the Menevit antioxidant or an identical-in-appearance placebo for 3
months prior to their partner’s IVF cycle. The results of the study demonstrated a
statistically significant improvement in viable pregnancy rate compared to the
control group, In another study, zinc therapy in combination with vitamin E or with
vitamins E + C were associated with comparably improved sperm parameters with
less OS, sperm apoptosis and sperm DNA fragmentation index (DFI) (Omu et al.
2008). Men with asthenozoospermia were orally provided with Zinc 5 mg, Vitamin
E + Zinc 10 mg and Zinc + Vitamins E + C 200 mg for 3 months. The results of the
study demonstrated that zinc therapy alone, in combination with Vitamin E or with
Vitamins E + C were associated with comparably improved sperm parameters.
Overall, it was concluded that this combined therapy reduced asthenozoospermia
through several mechanisms, such as prevention of OS, apoptosis and sperm DNA
fragmentation.
Rizzo’s group evaluated the efficacy of a treatment with myo-inositol plus folic
acid plus melatonin compared with myo-inositol plus folic acid alone on oocyte
quality in women who underwent IVF (Rizzo et al. 2010). Patients were assigned to
obtain either 2 g myo-inositol twice a day combined with 200 mg folic acid and
3 mg melatonin. The data showed that although the number of oocytes retrieved did
not differ between the two treatment groups, but women co-treated with melatonin
had an improvement in oocyte quality and a higher mean number of morphologi-
cally mature oocytes at ovum pick up. Women co-treated with melatonin were also
found to have a greater number of top-quality embryos compared to those treated
only with myo-inositol plus folic acid.
Chapter 5
Conclusion
Reactive oxygen species (ROS) are present in the reproductive organs and its fluids
in the female, and is generated by immature sperm, leukocytes from seminal plasma
and by the presence of varicocele in the male. While ROS play an important physi-
ological role in the process of reproduction, however, excessive amounts of ROS
leads to a state of oxidative stress, lipid peroxidation and subsequently DNA dam-
age. Spermatozoa are especially vulnerable to the effects of ROS as it lacks antioxi-
dant defences. Oral antioxidant supplementation could potentially help quench the
increased levels of oxidative stress and improve the quality of gametes produced,
especially in the male. Antioxidant therapies used clinically as oral supplementation
include vitamin E, vitamin C, selenium, folic acid and Coenzyme Q10 either alone
or in combination.
Couples suffering from reproductive disorders and/or infertility who seek assisted
reproduction are often plighted by the harmful effects of oxidative stress on gametes
and the developing embryo, and its subsequent negative impact on the pregnancy
outcome. During assisted reproduction, oxidative stress could be generated endog-
enously by the gametes itself or exogenously during sperm or oocyte preparation,
gamete handling and transfer of the embryo. Strategies to minimize oxidative stress
during in vitro fertilization and embryo transfer include the addition of antioxidants
to the culture/handling media and mindful practices during the mechanical and lab-
oratory techniques involved, as illustrated in Fig. 5.1. Although various studies
using enzymatic and non-enzymatic antioxidants have reported improvement in
sperm parameters and ART outcome, but to date, no one antioxidant in particular or
any certain combination of antioxidants could be proposed as the antidote to oxida-
tive stress for infertile couples seeking assisted reproduction. There is a need for an
increased number of well-designed randomized controlled trials in different popula-
tions to determine which of the antioxidant treatments would present with the best
outcome in subfertile couples seeking assisted reproduction.

DOI 10.1007/978-3-319-10259-7_5
42 5 Conclusion
Fig. 5.1 Sources of oxidative stress and interventions to overcome its effects during assisted
reproductive technology
ROS reactive oxygen species, ATP adenosine triphosphate, DNA deoxyribonucleic acid
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SPRINGER BRIEFS IN REPRODUC TIVE BIOLOGY

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Cleveland, OH, USA Ashok Agarwal

Fig. 2.1 Sources of reactive oxygen species in assisted

Table 2.1 Clinical parameters of studies examining reactive oxygen

Ashok Agarwal is a Professor at the Lerner College

Stefan S. du Plessis is a Professor and the Head of

Damayanthi Durairajanayagam, Ph.D. is a Senior

Gurpriya Virk has a Master’s in Clinical

Natural conception involves a series of intricate and well-orchestrated physiological

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2.1 In Vivo Generation of ROS

2.1.1 In Vivo Generation of ROS in the Male

Spermatozoa use ATP as a source of energy, which is produced through mitochon-

Fig. 2.1 Sources of reactive oxygen species in assisted reproductive technology

In summary, spermatozoa have increased susceptibility to OS, as there is a lack

2.1.2 In Vivo Generation of ROS in Females

Table 2.1 (continued)

rate, pregnancy rate

2.2 In Vitro Generation of ROS in ART

Cryopreservation is a process whereby cells and whole tissues are conserved by

2.2.2 Visible Light

2.2.3 ROS from Gamete Manipulation/ART Technique

In an in vitro environment, handling or manipulating the gametes and embryos dur-

2.2.4 pH and Temperature

of buffers. CO2 and pH have an inverse relationship; as CO2 concentration decreases,

Sperm centrifugation is regularly done during semen processing and is a common

2.2.6 Culture Media

2.2.7 Oxygen Concentration

Gametes/embryos are exposed to oxygen tension during procedures in assisted

Results of a recent Cochrane systematic review (involving 7 studies, 2,422

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antioxidants that is of importance and that are frequently-used clinically. Despite

3.1 Role of Enzymatic Antioxidants in ART

• Decreased DNA fragmentation rate of

et al. (2009) • Reduced DNA damage

3.1.1 Superoxide Dismutases

3.1.3 Glutathione Peroxidase

3.2 Role of Non-enzymatic Antioxidants in ART

Non-enzymatic antioxidants, also known as synthetic antioxidants or dietary sup-

Vitamin E refers to a group of eight fat-soluble compounds that consist of both

decrease in MDA levels and an improvement in sperm motility after treatment.

Melatonin (N-acetyl-5-methoxytryptamine) is a multi-functional and universal anti-

3.2.4 Vitamin B: Folic Acid

An additional empirically-used and an inspected agent in infertility is folic acid.

methionine catabolite and its concentration in follicular fluid, is negatively related

3.2.5 Coenzyme Q10

Coenzyme Q10 (CoQ10) also commonly called ubiquinones mostly produced in

L-Carnitine (LC) is a small water-soluble molecule which exerts an important func-

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Abdelrazik H, El-Damen A, Badrawy H, Sharma R, Agarwal A. L-Carnitine improves embryo

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