STRATEGIES TO REDUCE SPERM DNA DAMAGE - PowerPoint Presentation

Slide1

STRATEGIES TO REDUCE SPERM DNA DAMAGE

Edited by: Talebi AR.

Ph.DSlide2

Sperm chromatin/DNA remodeling

Testicular phase (

histone-protamine

substitution)

Epididymal

phase (disulfide bonds formation)

Ejaculation phase (zinc addition)Slide3
Slide4
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Slide6
Slide7
Slide8

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

denaturationSlide9

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 spermatozoaSlide10

Etiologies of sperm DNA damages

Age

Smoking

Varicocele

Inflammation

Hyperthermia

Febrile illness

Spinal cord injury

Testicular cancer

Environmental

toxins

Drugs

, chemotherapy and radiation

Hormonal factors like testosteroneSlide11

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 Slide12

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. Slide13

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.Slide14

Roles

of Antioxidants

Protect normal sperm from ROS-producing sperm

Protect normal sperm from WBC-derived ROS

Suppress premature sperm maturationSlide15

In classification reviewed by

Agarwal

et al (2008), antioxidants can be placed into two broad categories,

enzymatic and non-enzymatic.

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

Types of AntioxidantSlide16

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.Slide17
Slide18

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 high-magnification 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.Slide19

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.

Slide20

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

).Slide21

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.Slide22

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:

The

degeneration of single-stranded

DNA-

damaged spermatozoa

Development

of immature

spermatids

with double-stranded DNA may provide an explanation for this

occurrence Slide23

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.Slide24

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. Slide25

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. Slide26

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 alcohol-induced 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. Slide27

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. Slide28

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).Slide29

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).Slide30

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).Slide31

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 bio-marker, 8-OH-dG, and

DNA fragmentation

index.

Also,

Aitken

(2008)

identified high levels of sperm DNA fragmentation following16 hours exposure to mobile. Slide32

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)

DrugsSlide33

8. Repeated ejaculation during one week before ART programs

Elimination of destroyed spermatozoa

Decrease in exposure time of spermatozoa to ROSSlide34

Indications

of

sperm DNA

damage tests

Counselling

people who are planning their first pregnancy

:

Counselling

people planning to undergo intrauterine insemination

Counseling people planning to undergo IVF or ICSI specially IVF failures

All idiopathic couples

Men older than 40 years even if prior fertility

Men with known exposure to

toxicans

Men who are at risk of DNA damages like cancer, smoking,

varicocele

etc.

Repeated pregnancy loses

Spinal cord injured menSlide35

Thanks for your attention

Dr. Talebi AR.

Andrology

lab. Director

Research & Clinical Center For Infertility