Transcript STRATEGIES TO REDUCE SPERM DNA DAMAGE

STRATEGIES TO REDUCE SPERM
DNA DAMAGE
Edited by: Talebi AR. Ph.D
Sperm chromatin/DNA remodeling
 Testicular phase (histone-protamine
substitution)
 Epididymal phase (disulfide bonds formation)
 Ejaculation phase (zinc addition)
CHROMATIN / DNA ABNORMALITIES
Excessive histone
Absence or deficiency of protamines
(P1 , P2 )
Reduction in disulfide bonds formation
 Hypostabilized chromatin due to the zinc
deficiency
 DNA fragmentation
 DNA denaturation
MECHANISMS OF CHROMATIN / DNA
ABNORMALITIES
1 - Impairement in DNA nicking / ligation and
Topoisomerase II
2 - Programmed cell death via apoptosis
3 - Oxidative stress (Reactive Oxygen Species)
most important in ejaculated spermatozoa
Etiologies of sperm DNA damages
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Age
Smoking
Varicocele
Inflammation
Hyperthermia
Febrile illness
Spinal cord injury
Testicular cancer
Environmental toxins
Drugs , chemotherapy and radiation
Hormonal factors like testosterone
STRATEGIES TO REDUCE SPERM DNA
DAMAGE
8 chief strategies regarding the problem of raised sperm DNA
damage have been recommended recently:
• Using surgically-retrieved testicular spermatozoa instead of
ejaculated ones [219],
• Using ejaculated spermatozoa after at least two months of oral
antioxidant therapy [249],
• Micro-injection (ICSI) with spermatozoa selected with the use of a
high-magnification optical system (high-magnification ICSI) [481],
• Suitable preparation of the semen samples [18,426, 482],
• In-vitro culture conditions [483]
• Selected type of ART programs [364]
• Changing the lifestyle
• Repeated ejaculation during one week before ART programs
1. Testicular Spermatozoa
• Spermatozoa spend a long period of time in the epididymis
and so, it has been suggested that oxidative stress can
probably damage male germ cells in this organ due to long
exposure of ROS.
• It was found by Greco et al (2005) that DNA breakages in
ejaculated spermatozoa assessed by the TUNEL are
significantly higher than those in testicular spermatozoa
(23% versus 4.5%). They also reported that there is a higher
pregnancy rate with using testicular spermatozoa
compared to ejaculated ones [219].
• However, it should be considered that if the origin of sperm
DNA damage is abnormalities in topoisomerase II activity or
abortive apoptosis (two other causes of DNA damage),
there will not be any advantages for testicular
spermatozoa.
2. Antioxidant Therapy…
• Because spermatozoal DNA damages are supposed to be
mainly due to high levels of ROS, an antioxidant therapy can
markedly diminish the percentage of DNA-fragmented
spermatozoa in the ejaculate.
• Several in-vivo and in-vitro studies demonstrated that
antioxidants have positive effects on oxidative-induced sperm
DNA damage
• The patients can be given oral antioxidant during 3 months (at
least one spermatogenesis cycle) before an ICSI attempt. The
subsequent ICSI cycle led to a significant increase in
implantation rates and clinical pregnancy in comparison to the
pretreatment ICSI outcomes despite of the absence of
differences in fertilization and cleavage rates or in embryo
quality.
Roles of Antioxidants
 Protect normal sperm from ROS-producing
sperm
 Protect normal sperm from WBC-derived
ROS
 Suppress premature sperm maturation
Types of Antioxidant
• In classification reviewed by Agarwal et al (2008), antioxidants
can be placed into two broad categories, enzymatic and nonenzymatic.
• Seminal plasma contains three important enzymatic
antioxidants; Superoxide dismutase (SOD), catalase and
Glutathione peroxidase/glutathione reductase (GPX/GRD)
system.
• Non-eazymadc antioxidants are including, Vitamin C, Vitamin
E and Vitamin A (carotenoids), Coenzyme Q-10, proteins like
Albumin, Transferrin, hepatoglobulin, Ceruloplasmin,
Glutathione (GSH), Pyruvate, Ubiquinol and bilirubin
Antioxidant Therapy
• Greco et al (2005) showed that daily oral adminstration of
vitamin C and vitamin E for two months reduced the
number of TUNEL positive sperm cells from 22.1% to 9.1%
• The results of an in vitro study suggested that addition of
vitamin E or vitamin C to the sperm preparation media
during density gradient sperm preparation protected
spermatozoa from DNA damage.
• It is also shown that albumin helps to neutralize lipid
peroxide-mediated damage to the sperm plasma
membrane and DNA.
• It is reported that zinc therapy in men with
asthenozoospermia resulted in significant improvement in
sperm quality with increases in sperm concentration,
progressive motility, sperm nuclear integrity and improved
conception and pregnancy rates.
3. High-Magnification ICSI
• Presence of sperm head defects and intranuclear vacuoles has
been shown to be a signal for abnormalities of sperm
chromatin packaging and incomplete nuclear remodeling
during late stages of spermiogenesis .
• Sperm cells with normal morphology at standard ICSI
magnification, may show a variety of structural
abnormalities including intranuclear vacuoles, at highmagnification ICSI system.
• The ICSI cycles which were carried out with this system have
been shown to significantly increase pregnancy rates
compared to conventional ICSI procedure in patients with high
DNA fragmentation.
4. Semen Preparation Techniques…
• Several new techniques have been introduced to
select the best spermatozoa from ejaculate. Glass
wool filtration, density gradient centrifugation
and swim-up are examples of techniques used to
prepare the semen. These methods can
significantly improve sperm DNA integrity
compared to that of native semen.
• It was reported that DNA damaged spermatozoa
dropped from 12 to 5.5 % after using swim-up
and 28 to 14 % after density gradient
preparation.
Semen Preparation Techniques …
• A new technique based on the electrophoretic separation of
sperm has recently been developed for the selection of male
germ cells for use in ART programs.
• The method is based on the fact that the negatively charged
glycocalyx which has a high level of sialic acid residues, causes
mature spermatozoa to be more electronegative.
Spermatozoa are separated by moving toward the positively
charged cathode and away from the negatively charged
anode. It was shown that electrophoretically isolated
spermatozoa have low DNA damage assessed by TUNEL
examine.
• First pregnancy and normal birth were reported after ICSI
using electrophoretically isolated spermatozoa by Ainsworth
and colleagues (2007).
Semen Preparation Techniques
• The other new method is the separation of sperm cells
by magnetic-activated cell sorting (MACS). The
technique is based on the ability of spermatozoa to
express the apoptotic marker phosphatidylserine,
which binds to Annexin-V-conjugated micro-beads.
• According to Said et al (2005), spermatozoa with signs
of apoptosis including DNA fragmentation and
phosphotidylserine externalization
could be separated by a magnetic
field to Annexin-V positive and
negative fractions and therefore,
could be used in ART programs.
5. In Vitro Culture Condition…
• In-vitro culture for 48-72 hrs at 37°C has been
shown to improve the motility, post-thaw
recovery rate and DNA integrity of testicular
spermatozoa, because:
1) The degeneration of single-stranded DNAdamaged spermatozoa
2) Development of immature spermatids with
double-stranded DNA may provide an
explanation for this occurrence
In Vitro Culture Condition
• Studies have shown that repeated cycles of
centrifugation involved in conventional sperm
preparation techniques used for ART, increase
significantly the levels of ROS production due to
elimination of seminal antioxidant capacity!!
• Oxidative stress may also damage sperm during
cryopreservation. A study conducted by Bilodeau
et al (2000) revealed that ROS generated during
freeze-thaw cycles are detrimental to sperm
function and that levels of antioxidants were
decreased during each cycle.
6. Kinds of ART Programs
• Bungum et al 2004 and 2007 showed that when the DFI value was above
30 percent, the result of ICSI treatment was more better than the result in
IVF, because:
1) One possible explanation could be that ICSI procedure eliminates the
oxidative stress to which the spermatozoa are exposed during
hyperactivation and acrosome reaction as well as zona pellucida
penetration. Therefore, ICSI cycles will lead to a better chance of
pregnancy than after IUI and/or IVF.
2) Other possible explanation could be that the zygote has a better capability
to repair sperm DNA damages after ICSI than IVF treatment.
• In conclusion, if the DFI value is below 25%, the IUI and IVF
are recommended. But if the DFI value raises above 25
percent, the chance to get pregnant by intrauterine
insemination (IUI) is approximately zero , IVF very low and it
is therefore highly recommended that the couple register for
treatment by intra cytoplasmic sperm injection.
7- Changing the lifestyle….
• Cigarette smoking causes oxidative stress
either by producing high levels of free radicals
or by decreasing the antioxidant capacity of
seminal plasma.
• Sperm DNA fragmentation, axonemal damage
and decreased sperm count show significant
positive relationship with heavy smoking.
Changing the lifestyle….
• Ethanol causes a significant decrease in the
percentage of motility, concentration and normal
morphology of spermatozoa in human semen
and also in ethanol-consuming animals. It is
believed that ROS has a critical role in alcoholinduced fertility reduction
• Talebi et al (2010) showed that alcohol causes the
production of spermatozoa with less condensed
chromatin and high DNA damage which they can
be the results of oxidative stress.
Changing the lifestyle….
• Different kinds of ROS such as hydrogen
peroxide (H2O2), superoxide anion and
hydroxyl radical can be produced by radiation
and environmental pollution. However, Rosa
and colleagues (2003) demonstrated that
traffic pollution has detrimental effects on
sperm cells and may decrease fertility
potential in young and also middle-aged men.
Radiation & sperm
DNA
X-irradiation can affect the fertility
potential of spermatozoa and
cleavage rate of embryo due to DNA
damage (Hendricks, 2010)
The type of DNA damage that is
observed depends on the dose of
irradiation and the stage of
development of the exposed germ
cells (reviewed in Kamiguchi and
Tateno, 2002).
It is likely that irradiation induces
oxidative stress; (Ishii et al., 2005).
EXPOSURE TO XENOBIOTICS
• It is demonstrated that exposure to pesticides and insecticides
has been associated with increased levels of DNA damages in
spermatozoa (Xia et al. 2005).
Exposure to xenobiotics can alter chromatin in human and
animal sperm. Abnormalities such as deficiency in protamines
(Cho et al., 2001) or in histone modification (Baarends et al., 2007)
are related to xenobiotics.
Insecticides:
• Pyrethroid, Carbaryl and Chlorpyrifos cause sperm DNA
damage (Meeker JD, 2004 and 2008).
Metals
Workers with high blood lead levels have elevated sperm DNA
damage (Hsu et al., 2009).
Lead interacts with protamine 2 to decrease its binding to DNA,
altering chromatin stability (Quintanilla-Vega et al., 2000).
• On the other hand, lead and other cations (mercury, copper)
may replace zinc in chromatin structure:
=> Failure or delay in sperm chromatin decondensation in
fertilitzation process
=> Susceptibility to DNA damage agents
There is evidence that acute iron or cadmium exposure causes
oxidative DNA damage in sperm (Wellejus et al., 2000; Manna
et al., 2008).
Mobile phone radiation induces reactive oxygen species
production and DNA damage in human spermatozoa
in vitro (Geoffry N, 2009)
Purified human spermatozoa which were exposed to
electromagnetic radiation (as the same as mobile)
have shown high levels of oxidative DNA damage biomarker, 8-OH-dG, and DNA fragmentation index.
• Also, Aitken (2008) identified high levels of sperm
DNA fragmentation following16 hours exposure to
mobile.
Drugs
• Chronic exposure to MDMA (ecstasy)
increases DNA damage in sperm (Barenys, 2009
and 2010).
• Cocaine can affect sperm DNA integrity and
cause apoptosis (Li, 1999).
• Acetaminophen and hydroxyurea alter sperm
chromatin structure in laboratory mice (Wiger,
1995)
• Antidepressants like paroxetine can induce
DNA damage in spermatozoa (New Scientist. 2008)
8. Repeated ejaculation during one
week before ART programs
• Elimination of destroyed spermatozoa
• Decrease in exposure time of
spermatozoa to ROS
Indications of sperm DNA
damage tests
1. Counselling people who are planning their first pregnancy:
2.
Counselling people planning to undergo intrauterine
insemination
3. Counseling people planning to undergo IVF or ICSI specially
IVF failures
4. All idiopathic couples
5. Men older than 40 years even if prior fertility
6. Men with known exposure to toxicans
7. Men who are at risk of DNA damages like cancer, smoking,
varicocele etc.
8. Repeated pregnancy loses
9. Spinal cord injured men
Dr. Talebi AR.
Andrology lab. Director
Research & Clinical Center For Infertility