Sperm DNA Fragmentation in Male Infertility

Delhi & Chennai, India 2014

Sperm DNA Fragmentation
in Male Infertility
Sandro C. Esteves, MD, PhD
Director, ANDROFERT
Campinas, Brazil

Contents

Role of SDF on fertility
SDF and ART outcome
Methods for SDF assessment
Management Strategies

Esteves, 2

ANDROFERT, Referral Center for Male Reproduction

Semen analysis

Central
Laboratory
Investigation

Esteves, 3

Functional
Status of
Reproductive
Tract

Seminal Fluid
and Sperm


Why semen analysis is not enough

Esteves, 4


Sperm Function
Deliver intact DNA into
the oocyte
Intact chromatin is critical
for the production of a
viable pregnancy

DNA is a manual of
instructions

Esteves, 5


Sperm Chromatin Compaction

Esteves, 6


What are the lesions associated with
Sperm DNA Fragmentation?
Defects in DNA structure:
Single-strand DNA break (ss-DB)
Double-strand DNA break (ds-DB)
Base deletion or modification
Inter or intra-strand cross linkage
damaged base
single-strand
break mis-match

double-strand
break

intra-strand
crosslink

inter-strand
crosslink

Esteves et al 2013; Alvarez and Gosálbez 2011; Ward 2011
Esteves, 7


What are the biological
mechanisms of SDF?
Protamination Failure
Replacement of histone to protamines during
spermiogenesis

Oxidative Stress
Epididymis transit
Post-ejaculation: leukocytes, immature sperm,
abnormal levels seminal plasma antioxidants

Apoptosis
During sperm maturation (testis & epididymis)
Fernández et al. 2009; Alvarez and Sakkas 2010; Agarwal et al. 2013
Esteves, 8


What are the external factors
leading to increased SDF?
Environmental factors
Phtalate exposure, radiation,
temperature

Diseases
Varicocele, GTI, fever

Life-style
Obesity, smoking

DNA Damage

Aging
Kort et al. 2006; Rubes et al 2007; Viloria et al 2007; Esteves & Agarwal 2011
Esteves, 9


SDF and Male Infertility Etiologies

Gosálbez et al. 2013

Esteves, 10


Frequency of elevated SDF in men
with normal semen analysis

52

143
Normal semen analysis results (WHO)
Elevated SDF (36.4%)
Esteves, 11


SDF and Infertility: Why bother?

Esteves, 12


IUI Outcome and SDF
Live Birth Rates with
Intrauterine Insemination
19%

OR = 0.07
[95% CI: 0.01-0.48]

1.5%
Normal
Esteves, 13

Bungum et al. Hum Reprod 2007

Elevated

IVF Outcome and SDF
Pregnancy by Method in
Cases of Elevated Sperm DNA
Fragmentation
42%

Meta-analysis of 16 studies and
2,969 couples
Increased miscarriage in couples
undergoing IVF/ICSI with high
sperm DNA damage

26%

Risk ratio (RR) = 2.16
95% CI: 1.54-3.03; p<0.00001
IVF

ICSI

Robinson et al. Hum Reprod 2012

Bungum et al. Hum Reprod 2007

Esteves, 14


SDF and reproductive success
Points to consider (1)
Oocyte repair capability and severity
of damage
Repair likely to occur at pronuclei stage (prior syngamy)
Low levels breaks can be repaired (especially ss-DBs)
Repair ability decrease with female age
Menezo et al 2007; Genescá et al. 1992; Obe et al. 2002

Esteves, 15


SDF and reproductive success
Points to consider (2)
Site of damage
Coding DNA (exons)
represent ~3% of genome
Gosálbez et al. 2013; Dada et al. 2012

Esteves, 16


SDF and Male Infertility
Key Messages (1)
SDF
gives
diﬀerent
informa4on
than
rou4ne

semen
analysis,
and
of
be;er
prognos4c
value

SDF
is
mainly
oxida4ve-‐stress
mediated
during

sperm
transit
through
the
epididymis

Elevated
SDF
associated
with
infer4lity,
poor

ART
outcome
and
miscarriage

Reproduc4ve
outcome
related
to
oocyte
repair

capacity
as
well
as
severity
and
site
of
DNA

damage

Esteves,
17

ANDROFERT,
Referral
Center
for
Male
Reproduc4on

What are the methods for SDF
assessment?
Direct
Incorporation of probes at the site of damage
e.g. TUNEL, ISNT

Indirect
Susceptibility of DBs to denature in a acid solution
e.g. Sperm chromatin structure assay (SCSA), sperm
chromatin dispersion test (SCD), Comet assay

Chromatin compaction
Incorporation of probes to nuclear proteins
e.g. Aniline blue, toluidine blue
Gosálbez et al 2013; Esteves & Agarwal 2011; Esteves et al. 2013
Esteves, 18


Comparison Between SDF Methods

Fertility and Sterility 2014; 101(1):58-63.

Esteves, 19


TUNEL

Terminal deoxynucleotidyl transferase dUTP
nick end labeling
Enzymatic addition of modified
nucleotides to DNA breaks;

Sharma et al. 2010
Esteves, 20


Sperm Chromatin Dispersion (SCD)
Susceptibility of DNA to denaturation
with formation of single-strand (ss)
DNA from pre-existing single or
double strand breaks;
Combination of DNA denaturation used
in SCSA and protein depletion used in
the comet assay;
Difference in the pattern of forming a
loop (halo) around lysed and acid
treated nuclear membrane carcass
reflects the overall chromatin structure.
Fernández et al. 2003, 2005 Gosálvez et al. 2006
Esteves, 21


Correlation between SCD and TUNEL
SCD more sensitive than
TUNEL.
Important to distinguish
between the methods as they
differently evaluate SDF.
20.6

SCD

TUNEL
11.5
Fertil Steril 2014; 101(1):58-63

% SDF
Esteves, 22


Diagnostic accuracy of SCD in men with
unexplained infertility
Despite poorly correlated, SCD
may discriminate men with
normal and abnormal sperm DNA
damage with ~70% accuracy
when compared with TUNEL.

Fertil Steril 2014; 101(1):58-63
Esteves, 23


Which is the best method for SDF?
Laborintensive

TUNEL
SCSA
Comet
SCD

Esteves, 24

Expensive
equipment

Analysis
Subjectivity

Validation
& Standardization

++++
++
++++
+

+++
++++
+++
+

++
+
+++
++

++
++++
+
+++


SDF and Male Infertility
Key Messages (2)
Several
methods
available
to
assess
SDF

Methods
diﬀeren4ally
assess
SDF
and
cannot

determine
nature
or
e4ology
of
damage

Best
method
yet
to
be
determined

SCD
is
a
quick
and
easy
assay
to
assess
SDF

Esteves,
25

ANDROFERT,
Referral
Center
for
Male
Reproduc4on

What can we do to reduce SDF?
Antioxidants and life-style
changes
Treatment of underlying condition
Avoid iatrogenic SDF
Wong et al., 2000; Wong et al. 2002; Comhaire and Mahmoud, 2003; Agarwal and Said, 2004;
Bansal and Bilaspuri, 2010; Gosálbez et al. 2009, 2011; Esteves et al. 2011; Sánchez-Martín et al 2013

Esteves, 26


Oral Antioxidants
Outcome

No.
studies

Eﬀect
size

(OR;
95%
CI)

Live
birth

3

4.85
[1.92,
12.24]

Pregnancy
rate

15

4.18
[2.65,
6.59]

DNA
fragmenta4on

1

-‐13.80
[-‐17.50,
-‐10.10]

6-‐16

No
eﬀect

Miscarriage,
sperm

count,
sperm
mo4lity

Showell MG et al. Cochrane Database Syst Rev 2011

Esteves, 27


Oral Antioxidants
Beneficial

No effect

Kodama 1997
Dawson, 1992
Kessopoulou, 1995
Vezina, 1996
Vicari, 2001; 2002
Lenzi, 2003; 2004
Cavallini, 2004
Comhaire, 2005
Grecco 2005
Menezo 2007
Tremellen 2007

Giovenco, 1987
Moilanen, 1993
Iwanier, 1995
Rolf, 1999
Sigman, 2006

Piomboni 2008
Gil Villa 2009

Esteves, 28

l  Short-term use
appear to be safe

Detrimental
long-term use and high
doses;
increased mortality in
cancer populationbased studies.

l  Caution against
indiscriminate use of
high dosages for
long periods

Heinonen, 1994
Lonn, 2005
Bjelakovic, 2007

Oral Antioxidants
How I prescribe
Vitamin C 500mg; Vitamin E 400 mg
Folic acid 2 mg, Zinc 25 mg
Selenium 26 mcg

Minimum 2 months
From initiation of sperm production to ejaculation
Old concept ~80 days
New concept ~60 days

Misell LM et al. J Urol. 2006

Esteves & Agarwal. Novel concepts in male infertility. Int Braz J Urol 2011
Esteves, 29


Decrease No. Leukocytes in Semen
% DNA Damage (SCSA) 39%
34%

25%

granulocyte

Normal

Abnormal

macrophage

lymphocyte

Abnl &
Leukocytospermia

Endtz
test
Henkel R et al, AJA 2007; Alvarez et al. Fertil Steril 2002

Esteves, 30


Treatment of Subclinical GTI and
Associated Inflammatory Changes
Antibiotics

• Yanushpolsky et al, 1995; Erel et al., 1997
• Branigan et al., 1995

Cicloxigenase-2
Inhibitors

Antihistamines

• Oliva & Mutigner, 2006

Antioxidants

Esteves, 31

• Lackner et al., 2006
• Gambera et al., 2007

• Tremellen et al., 2007
• Piombini et al., 2008


Subclinical Male Genital Tract Infection

Anti-bacterial
properties (Zinc)

Azitromycin 1.0g single dose (couple)+ frequent ejaculation
(every 2-3 days) + Antioxidants
42% leukocytospermia resolution (N=278)
Esteves, 32


Varicocele Surgery

Twelve studies comparing SDF
in pts. with and without
varicocele:
SDF higher in varicocele
Mean difference = 9.9%
(95% CI: 9.2-10.5; p<0.0001)
Miyaoka & Esteves. Adv Urol 2012
Agarwal, Esteves, Hamada. Nature Urol Rev 2013;
Wang YJ et al. Reprod Biomed Online. 2012;25:307-14.

Esteves, 33


Effect of Varicocele Surgery on SDF

Meta-analysis of seven studies
evaluating the effect of
varicocele repair on SDF
SDF decreased after repair
Mean difference = 3.4%
(95% CI: -4.1 to -2.6; p<0.0001)

Wang YJ et al.
Reprod Biomed Online. 2012;25:307-14.

Esteves, 34


Management

Esteves, 35


TESA-ICSI and SDF

Sperm

% TUNEL +

% CPR

Ejaculated

23.6

6

Testicular

4.8

44

<0.001

<0.05

P value

Greco et al. Hum Reprod 2005
Esteves, 36


Difference in SDF between
Testicle and Ejaculate
DNA damage in
Testicular
Spermatozoa
three-fold lower
compared with
Ejaculated
Spermatozoa*

*Absolute differences between two specimens ranging from -3.3% to -56.3%.
Moskovtsev et al. Fertil Steril 2010
Esteves, 37


TESA-ICSI: ANDROFERT

Esteves, 38


TESA-ICSI: ANDROFERT
•  93 patients enrolled; Mean age: 37.5 yo.
•  SDF (Halosperm®): 39.0% ± 15.4% [range: 21%-88%]
•  53 pts. with live birth data (Sept. 2013)
80
60
40

70.5

61.5

53.6

54.8

51

p=0.21

p=0.62

20

p=0.50
25
11.7

CPR (%)

Miscarriage (%)

33.3
p=0.70

48.4
25
p=0.17

0
2PN (%)

TQE (%)

Ejaculate
Esteves, 39

LBR (%)

TESA

What can we do to decrease SDF?
Ø  Oral antioxidants
Ø Life-style modifications, including quit
smoking and weight loss
Ø Identify and treat underlying condition
(GTI and varicocele)
Ø  Consider TESA-ICSI

Esteves, 40


Dynamic Nature of SDF
Iatrogenic damage

Esteves, 41


Abstinence Period and Sperm
Processing

Serial ejaculation every 24h for 4 days:
25% reduction SDF
Density centrifugation post-3h
ejaculation: 44% reduction

Gosálbez et al. Fertil Steril 2011

One-day abstinence: Reduction in ~90% pts.
Esteves, 42

Pons et al. 2013


Annexin-V microbeads and
MACS columns
Microbeads conjugated with
Annexin-V

Esteves, 43


Sperm Selection
Annexin-V columns
Parameters

Pre-MACS

Annexin V neg.

Annexin V
pos.

Count

1 x 106/ml

600.000/ml

400.000/ml

Viability

49%

64%

0

Progressive
motility

30%

43%

0

SDF

34%

10%

70%

Cleaved
caspase-3

8%

7%

20%

Ø  Annexin-V negative fraction: IVF, ICSI, IUI
Ø  Annexin-V positive fraction: discard
Esteves, 44

Rawe et al., RBM Online 20:320, 2010


Laboratory handling of ejaculated
and testicular specimens

Esteves SC & Varghese A, J Hum Reprod Sci 2012
Esteves, 45


What we can do to avoid iatrogenic SDF
Ø Short abstinence periods (1 day) and serial ejaculation
Ø Instruct patients to deliver specimens 1- 2h before
ART is to be performed
Ø Process specimens as soon as possible
Ø Keep specimens at room T using appropriate culture
media
Ø Incubation time after processing should not exceed 4h
Ø Thaw cryopreserved specimens just before performing
ART
Esteves, 47


Take-home Messages
SDF provides information that is different
and of better prognostic value than
semen analysis.
SDF mainly occur during sperm transit through
the epididymis, and it is mediated by ROS.
Oocyte can repair ssDNA but not dsDNA
damage.
Sperm chromatin dispersion (SCD) test is
the simplest method to assess SDF in
routine clinical practice.
Esteves, 48


Take-home Messages
SDF has a negative effect on reproductive
potential, both in vivo and in vitro.
Strategies to reduce SDF includes antioxidant
therapy, treatment of subclinical GTI,
varicocele repair, and TESA-ICSI.
Avoid iatrogenic damage: short abstinence
periods, laboratory sperm selection and
proper sperm handling.
Esteves, 49


Sperm DNA Fragmentation in Male Infertility

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