Oxidative stress is associated with several body cell damage which can result into cell death and eventually mortality. Its a key factor in several infertility cases and with the knowledge of its impact a lot can be known to avert its further implications.

1. 1
ROLE OF OXIDATIVE STRESS ON MALE INFERTILY
BY
ORJI CHIBUEZE FAVOUR
FEBRUARY, 2023.
6/9/2023

2. OUTLINE
 INTRODUCTION
 ETIOLOGY
 PATHOPHYSIOLOGY
 EPIDEMIOLOGY
 LABORATORY INVESTIGATIONS
 CONCLUSSION
 RECOMMENDATION
 REFERENCES
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3. 3
INTRODUCTION
The imbalance between the production of
reactive oxygen species(ROS) and a
biological systems ability to readily
detoxify the reactive intermediates or
inability to easily repair the resulting
damage is know as Oxidative
stress.(Agarwal et al., 2023)
The production of ROS is a normal physiological
process, but an imbalance creates an enhanced
levels of ROS (Reactive Oxygen Species such as
hydrogen peroxide, superoxide anion, hydroxyl
radical, nitrogen radicals from reactive nitrogen
species) in extracellular fluid of which is
detrimental to the same body producing it.
www.wjmh.org
The international Committee for Monitoring
Assisted Reproductive Technologies(ICMART),
defines infertility as the inability to conceive
after 1year of regular, unprotected
intercourse. (World J mens Health, 2023)
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4. 4
What are
even this
ROS and
how do
they
come
about ?
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5. (Al-Gubory KH et al., 2010)
ENDOGENEOUS EXOGENEOUS
Leukocytes
Obesity
Radiation
Genital heat
stress
Smoking
Alcohol
ATP PRODUCTION
Varicocele
Age
ETIOLOGY
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WHAT HAPPENS NEXT
WITH THIS ROS
PRODUCED
IN OUR BODY
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PATHOPHYSIOLOGY
Loss of
sperm
function (Sharma and Agarwal, 1996).
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Sperm maturation
Sperm capacitation and
hyperactivity
Acrosome reaction
Sperm-oocyte fusioin
Loss of sperm function
Reduction of sperm quality;
Motility
Morphology
sperm count
DNA integrity
INFERTIITY
ROLE OF OXIDATIVE STRESS IN THE MALE GERM CELL
(Saleh et al., 2012).
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9. EPIDEMIOLOGY
 WHO estimates that nearly 190 million people struggle with
infertility worldwide with at least 30 million infertile men in
the prevalence of2.5%-15% (Rowe et al., 2015).
 Among couples unable to conceive, infertility is partially a
wholly attributable to a male factor in approximately 50% of
cases.
 Infertility remains undefined in 30% - 50% of idiopathic male
factor. A similar high incidence of OS was reported in a recent
clinical trial, with 83.8%(124 of 148 cases) of idiopathic infertile
men, having positive seminal oxidation reduction potential
(ORP).
 One of the mechanisms proposed for idiopathic male infertility
is oxidative stress(OS). So many genetic factors, hypogonadism,
cryptorchidism also varicocele, can cause infertility. However,
no underlying cause can be identified for primary or secondary
infertility in approximately 25% of idiopathic infertility 9
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General statistics of infertility in Nigeria
National Institute for Health and care, 2023.
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LABORATORY INVESTIGATION.
Assessment of sperm ROS levels among infertile men can aid
in determining which individuals who may benefit for anti
oxidant therapy.
Currently, there are no infertility guidelines that
recommend routine ROS measurement and there
is still an ongoing debate on which type of
patients have to be tested for oxidative burden.
Asthenozoospermia
in a semen sample is
probably a marker
of ROS.
Hyperviscosity has also been
suggestive of increased OS
because it is attributed to
increased malondialdehyde
levels.
Increase leukocytes or
round cells which is one of
the principal sources of ROS
may suggest further testing
for OS.
Hyper-osmotic swelling test suggests
membrane damage in the sperm due
to lipid peroxidation and this might
imply higher levels of ROS in semen.
1 2 3
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Panner et al., 2021
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LABORATORY INVESTIGATION
Measurement of LPO – thiobarbituric Acid Assay
Malondialdehyde(MDA) is an end product of LPO which is
measured through thiobarbituric acid(TBA) assay TBA
reactive substances(TBARS) are mainly formed during the
determination of LPO in vitro.
The reaction between thiobarbituric acid and MDA generates
an adduct, which can be measured either colorimetrically by
using spectrophotometer or fluorometrically. Results are
expressed as nmol MDA/10⁷ sperm.
Thiobarbituric Acid- Reactive Substance Assay
Esteves et al., 2021
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Various tests have been developed to detect
seminal ROS levels which ca be classified into
direct and indirect assays.
DIRECT ASSAYS; measures cellular levels of ROS

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Oxidation-Reduction Potential Assay (ORP) Assay.
Concentration of both oxidants and
antioxidants
Provides a global overview of the redox status
of semen sample.
LABORATORY INVESTIGATION.
Is this possible ❓
This is possible by
measuring the electron
transfer during the
reductive reaction from
antioxidants to
oxidants.
Panner et at., 2021
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14. LABORATORY INVESTIGATION cont’d
– probes such as
lucigenin (Extracellular O₂) luminol (extra and intracellular)
reduced or
oxidized
Dioxetane endoperoxide.
decomposed
Light (Relative Lights Units).
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Mahfouz et al., 2009
Chemiluminescence
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Investigating large number of cells in a limited amount of time.
Profile cells by detecting the light scatter or fluorescence emitted by dyes or
monoclonal antibodies used to stain/bind selected molecules.
Intracellular ROS analysis using two dyes;
2’,7’-dichlorofluorescin diacetate(DCFDA) = peroxyl, peroxylnitrite, Alkoxyl,
NO2, carbonate(CO3-) and OH’ radicals
Dihydroethidium(DHE)= O₂
ROS-mediated dye oxidation
DCFDA
Green colored fluorescence
after binding to DNA
DHE
Ethidium bromide,
emitting red fluorescence
in 2’,7’-dichlorofluorescein
The intensity of the fluorescence depends on the intracellular
ROS concentration.
Mahfouz et al., 2009
Flow cytometry;
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Intracytoplasmic ROS can be detected by the NBT assay. This
compound is reduced by superoxide radicals and forms formazan
crystals. This crytsals are purple colored and can be detected
microscopically or spectrophotmetrically.
Results are express as in μg formazan/10⁷ sperm.
Nitroblue tetrazolium(NBT)
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INDIRECT ASSAYS;
The short lifespan and rapid reactivity with redox state
regulating component makes direct assay difficult for both
accuracy and precision.
Indirect assay examines the oxidative damage these radicals
cause to lipids, proteins, nucleic acids of the cells. It is a
promising alternative approach to access oxidative stress in
clinical samples.
Protein Damage;
Protein Carbonyl (PC) –oxidation of protein
backbones and amino acids such as Proline,
arginine, lysine, threonine by ROS molecules.
Proteins Carbonyls(PCs) + (DNPH) 2,4- dinitrophenylhydrazine
Schiff base
Dinitrophenylhydrazone
Mesquita et al., 2014
Thus, the product can be analyzed,
spectrophotmetrically at 375nm. The value
correlate with the levels of oxidized proteins

18. Laboratory diagnosis contd.
Enzyme linked immunosorbent Assay(ELISA).
METHOD: COMPETITIVE ELISA;
Determination of the antioxidant victamin c.
SAMPLE PREPARATION
Whole semen was centrifuge at 40,000g for 7minutes to yielded sperm-free seminal plasma
in two thirds of the sample centrifuged.
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Total Antioxidant Capacity assay.
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19. PRINCIPLE
 This kit is based on competitive enzyme-linked immuno-sorbent
assay technology. An antibody is pre-coated onto a 96-well plate.
Standards, test samples, and biotin-conjugated reagent are added to
the wells and incubated. A competitive inhibition reaction takes
place between the biotin-labelled VC and the unlabelled- VC on the
pre-coated antibody.
 The HRP-conjugated reagent is then added, and the whole plate is
incubated. Unbound conjugates are removed using wash buffer at
each stage. TMB substrate is used to quantify the HRP enzymatic
reaction. After TMB substrate is added, only wells that contain
sufficient VC will produce a blue coloured product, which then
changes to yellow after adding the acidic stop solution. 19
Agarwal et al., 2021
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20. PRINCIPLE CONT’D
 The intensity of the color yellow is inversely proportional to
the VC amount bound on the plate.
 The OD is measured spectrophotometrically at 450 nm in a
microplate reader, from which the concentration of VC can be
calculated.
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21. COMPETITIVE ELISA.
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Agarwal et al., 2021
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22. PROCEDURE
 Set standard, test samples and control wells.
 Aliquot 50 µl of diluted standard into the standard wells.
 Aliquot 50 µl of Standard Diluent buffer into the control
(zero) well.
 Aliquot 50 µl of diluted samples into the sample wells.
 Immediately aliquot 50 µl of Detection Reagent A to each
well. Incubate for 1 hr at 37 °C.
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Agarwal et al., 2021
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23. PROCEDURE CONT’D
 Wash 3 times.
 Aliquot 100 µl of Detection Reagent B to each well.
Incubate for 30 mins at 37 °C.
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Agarwal et al., 2021
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24. NORMAL RANGE
 493.8 ng/ml - 40000 ng/ml
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Strategies to reduce oxidative stress
Antioxidants;
Vitamin E= directly quench free radials like peroxyl and alkoxyl (ROO.)
Enzymatic oxidants Non Enzymatic oxidants
Vitamins eg, Vit. C, vit. A,
Zinc
Taurine
Hypotaurine
Glutathione
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25. CONCLUSION
 Despite the vital function of ROS during fertilization, an
increase in those free radicals causes oxidative stress and
detrimentally affects sperm function. The origin of this
increase arises from different endogenous and exogenous
sources, such as sperm metabolism or infections,
respectively. It is well known that ROS cause a reduction
in sperm count and motility, protein alterations, lipid
peroxidation, and DNA fragmentation, amongst others.
 This problem of infertility due to oxidative stress or other
reasons, can be solved by Antioxidant supplementation
such as glutathione and/ or Assisted Reproductive
Techniques, ART (Agarwal et al., 2021).
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26. RECOMMENDATION
 Infertility testing should also be considered significant as related to oxidative
stress monitoring in semen analysis.
 Chemical pathology unit in FMC Jabi should consider diagnostic methods for
general oxidative stress.
 Minimize ‘lifestyle’ that triggers oxidative stress. Such as avoid smoking,
excessive alcohol intake and improved diet,
 Minimize environmental exposure to heat, pollution and toxins.
 Vitamin and antioxidant supplements, to decrease ROS production.
 Supplementation of antioxidant such as glutathione and/ Assisted Techniques
ART to reduce oxidative stress and improve sperm motility could be useful in
management of male infertility.
 General populace awareness in educating the risk factors associated with
oxidative stress should be practice in conjunction to the adverse effect in
humans.
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Considering the following studies, I would recommend that;
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27. REFERENCE
 Banerjee P, Bhattacharya J. Impact of Oxidative stress on Infertility, with
emphasis on infertility management strategies. Global Journal of Fertility
and Research. 2019 Aug 2;4(1):010-8.
 Bisht S, Faiq M, Tolahunase M, Dada R (2017) Various factors
contributing to oxidative stress and bringing about male infertility, digital
photograph. Nature Reviews, Urology 14. 472-485
 Louis J.F., Thoma M.E., Sørensen D.N., McLain A.C., King R.B.,
Sundaram R., Keiding N., Buck Louis G.M. The prevalence of couple
infertility in the United States from a male perspective: Evidence from a
nationally representative sample. Andrology. 2013;1:741–748.
 Pereira R, Sá R, Barros A and Sousa M (2017) Causes that lead to
oxidative stress condition, digital photograph. Asian journal of andrology.
Link: http://bit.ly/332yEPr
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28. REFERENCE cont’d
 Sharma, R. K., and Agarwal, A. (1996). Role of reactive oxygen species
in male infertility,” Urology, 48, 6, 835-850.
 Slade P, O'Neill C, Simpson AJ, Lashen H. The relationship between
perceived stigma, disclosure patterns, support and distress in new
attendees at an infertility clinic. Hum Reprod. 2007;22:2309–17.
 Tremellen K. Oxidative stress and male infertility—a clinical perspective.
Human reproduction update. 2008 May 1;14(3):243-58
 Uadia PO, Emokpae AM. Male infertility in Nigeria: A neglected
reproductive health issue requiring attention. Journal of Basic and
Clinical Reproductive Sciences. 2015 Jul 21;4(2):45-53.
 Valko, M., Morris, H., and Cronin, M. T. D. (2015). Metals, toxicity and
oxidative stress, Current Medicinal Chemistry, 12, 10, 1161-1208.
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