This document summarizes a presentation on sperm DNA fragmentation from the perspective of a male infertility specialist. It discusses the relationship between sperm DNA fragmentation and infertility, methods for assessing sperm DNA fragmentation, and strategies for managing elevated sperm DNA fragmentation levels. The key points are that sperm DNA fragmentation provides different and more prognostically useful information than a semen analysis, is mainly caused by oxidative stress during sperm transit through the epididymis, and is associated with infertility, poor assisted reproductive technology outcomes, and miscarriage.

1. Sandro C. Esteves, MD., PhD.
Director, ANDROFERT
Campinas, Brazil
Sperm DNA Fragmentation from
a Male Infertility Specialist’s
Perspective
Centre for Reproductive Health, Daresbury, United Kingdom
July 2014

2. Contents
Relationship between SDF and infertility
Methods for SDF assessment
Management Strategies
Esteves, 2 ANDROFERT, Referral Center for Male Reproduction

3. Semen analysis
Functional
Status of
Reproductive
Tract
Seminal Fluid
and Sperm
Central
Laboratory
Investigation
Esteves, 3 ANDROFERT, Referral Center for Male Reproduction

4. New WHO Reference Values
Caution to Interpret Results
ANDROFERT
androfert.com.br
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION
S ESTEVES, 4
2014 APRIL
ANDROFERT

5. Volume (mL)
1.5
Sperm count (x106/mL)
15.0
Total count (x106)
39.0
% Motile (total)
40
% Motile (progressive)
32
% Normal (strict criteria)
4
%Alive
58
Cooper et al. Hum Reprod Update 2010
WHO 2010: Recent fathers TTP ≤ 1 year
Percentiles
5% 50%*
95%
3.7
6.8
73.0
213.0
255.0
802.0
61
78
55
72
15
44
79
91
ANDROFERT
androfert.com.br
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION
S ESTEVES, 5
2014 APRIL
ANDROFERT

6. Esteves, 6 ANDROFERT, Referral Center for Male Reproduction
Why semen analysis is not enough

7. Sperm Chromatin Compaction
Esteves, 7 ANDROFERT, Referral Center for Male Reproduction

8. Lesions Associated with Sperm DNA
Damage
single-strand
break mis-match
damaged base
double-strand
break inter-strand
crosslink
intra-strand
crosslink
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
Esteves, 8 ANDROFERT, Referral Center for Male Reproduction
Esteves et al 2013; Alvarez and Gosálbez 2011; Ward 2011

9. 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, 9 ANDROFERT, Referral Center for Male Reproduction

10. Environmental factors
Phtalate exposure, radiation,
temperature
Diseases
Varicocele, GTI, fever
Life-style
Obesity, smoking
Aging
External factors leading to
increased SDF
Kort et al. 2006; Rubes et al 2007; Viloria et al 2007; Esteves & Agarwal 2011
Esteves, 10 ANDROFERT, Referral Center for Male Reproduction
DNA Damage

11. SDF in Different Male Infertility
Etiologies
Esteves, 11 ANDROFERT, Referral Center for Male Reproduction
Gosálbez et al. 2013

12. Frequency of Elevated SDF in Men with
Normal Semen Analysis (WHO 2010)
Esteves, 12 ANDROFERT, Referral Center for Male Reproduction
36%
Androfert 2013; N=860; SCD test;
cutoff value of 20%

13. SDF and Infertility: Why bother?
Esteves, 13 ANDROFERT, Referral Center for Male Reproduction
Esteves et al. Int Urol Nehrol 2014

14. 19%
1.5%
Normal Elevated
Live Birth Rates with
Intrauterine Insemination
OR = 0.07
[95% CI: 0.01-0.48]
Bungum et al. Hum Reprod 2007
IUI Outcome and SDF
Esteves, 14 ANDROFERT, Referral Center for Male Reproduction

15. 26%
42%
IVF ICSI
Pregnancy by Method in
Cases of Elevated Sperm DNA
Fragmentation
IVF Outcome and SDF
Robinson et al. Hum Reprod 2012
Meta-analysis of 16 studies
and 2,969 couples
Increased miscarriage in
couples undergoing IVF/ICSI
with high SDF
Risk ratio (RR) = 2.16
95% CI: 1.54-3.03;
p<0.00001Bungum et al. Hum Reprod 2007
Esteves, 15 ANDROFERT, Referral Center for Male Reproduction

16. SDF and Reproductive Outcome
Points to consider (1)
Oocyte repair capability and severity
of damage
Menezo et al 2007; Genescá et al. 1992; Obe et al. 2002
Esteves, 16 ANDROFERT, Referral Center for Male Reproduction
• Repair likely to occur at pronuclei stage (prior
syngamy)
• Low levels breaks can be repaired (especially ss-DBs)
• Repair ability decrease with female age

17. Esteves, 17 ANDROFERT, Referral Center for Male Reproduction
Coding DNA (exons)
represent ~3% of genome
Esteves et al. 2014; Dada et al. 2012
SDF and Reproductive Outcome
Points to consider (2)
Site of damage

18. Viable pregnancies can be obtained in
cases of high SDF but…
l SDF is promutagenic
mutations could rise after fertilization as the
oocyte attempts to repair damage before
first cleavage
l Mutations will be fixed in the germline
associated with infertility, childhood cancer
in the offspring and for a higher risk of
imprinting diseases
Pang MG et al Hum Reprod 2005
Burrello et al Cytogenet Genome Res 2005.
Esteves, 18 ANDROFERT, Referral Center for Male Reproduction

19. Relationship between SDF and Infertiity
Key Messages
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
Esteves,
19
ANDROFERT,
Referral
Center
for
Male
Reproduc4on
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

20. Methods of SDF Assessment
Direct
Incorporation of probes at the site of damage
e.g. TUNEL, ISNT
Indirect
Susceptibility of DsDNA 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, 20 ANDROFERT, Referral Center for Male Reproduction

21. Which is the best method for SDF?
Labor-
intensive
Expensive
equipment
Analysis
Subjectivity
Validation
& Standardization
TUNEL ++++ +++ ++ ++
SCSA ++ ++++ + ++++
Comet ++++ ++ +++ ++
SCD + + ++ +++
Esteves, 21 ANDROFERT, Referral Center for Male Reproduction
Esteves et al. Int Urol Nehrol 2014

22. Fertility and Sterility 2014; 101(1):58-63.
Esteves, 22 ANDROFERT, Referral Center for Male Reproduction
Comparison Between SDF Methods

23. Fernández et al. 2003, 2005; Gosálvez et al. 2006
Esteves, 23 ANDROFERT, Referral Center for Male Reproduction
Sperm Chromatin Dispersion (SCD)
Susceptibility of DNA to denaturation
with formation of single-strand (ss)
DNA from pre-existing single or
double strand breaks;
Difference in the pattern of forming a
loop (halo) around lysed and acid
treated nuclear membrane carcass
reflects the overall chromatin structure.
Combination of DNA denaturation used
in SCSA and protein depletion used in
the comet assay;

24. Enzymatic addition of modified
nucleotides to DNA breaks;
Sharma et al. 2010
Esteves, 24 ANDROFERT, Referral Center for Male Reproduction
TUNEL
Terminal deoxynucleotidyl transferase dUTP
nick end labeling

25. Correlation between SCD and TUNEL
Esteves, 25 ANDROFERT, Referral Center for Male Reproduction
• SCD more sensitive than
TUNEL
• Important to distinguish
between the methods as they
differently evaluate SDF
20.6
11.5
% SDF
SCD TUNEL
Fertil Steril 2014; 101(1):58-63.
P<0.01

26. Several
methods
available
to
assess
SDF
Methods
differen4ally
assess
SDF
and
cannot
determine
nature
or
e4ology
of
damage
Esteves,
26
ANDROFERT,
Referral
Center
for
Male
Reproduc4on
Best
method
yet
to
be
determined
Standardiza4on
and
EQC
is
needed
Key Messages (2)

27. What can we do about SDF?
• Antioxidants and life-style changes
• Avoid iatrogenic SDF during lab sperm
handling
Esteves, 27 ANDROFERT, Referral Center for Male Reproduction
• Treatment of underlying condition
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
• Short abstinence period
• Sperm selection techniques

28. 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]
Miscarriage,
sperm
count,
sperm
mo4lity
6-­‐16
No
effect
Oral Antioxidants
Showell MG et al. Cochrane Database Syst Rev 2011
Esteves, 28 ANDROFERT, Referral Center for Male Reproduction

29. Oral Antioxidants
l Short-term use
appear to be safe
l Caution against
indiscriminate
use of high
dosages for long
periods
Beneficial
Kodama 1997
Dawson, 1992
Kessopoulou, 1995
Vezina, 1996
Vicari, 2001; 2002
Lenzi, 2003; 2004
Cavallini, 2004
Comhaire, 2005
Grecco 2005
Menezo 2007
Tremellen 2007
Piomboni 2008
Gil Villa 2009
No effect
Giovenco, 1987
Moilanen, 1993
Iwanier, 1995
Rolf, 1999
Sigman, 2006
Detrimental
long-term use and high
doses;
increased mortality in
cancer population-
based studies.
Heinonen, 1994
Lonn, 2005
Bjelakovic, 2007
Esteves, 29 ANDROFERT, Referral Center for Male Reproduction

30. 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
Old concept ~80 days
New concept ~60 days
From initiation of sperm production to ejaculation
Misell LM et al. J Urol. 2006
Esteves & Agarwal. Novel concepts in male infertility. Int Braz J Urol 2011
Esteves, 30 ANDROFERT, Referral Center for Male Reproduction

31. Henkel R et al, AJA 2007; Alvarez et al. Fertil Steril 2002
25%
34%
39%
Normal Abnormal Abnl &
Leukocytospermia
% DNA Damage (SCSA)
Esteves, 31 ANDROFERT, Referral Center for Male Reproduction
Decrease No. Leukocytes in Semen
granulocyte macrophage lymphocyte
Endtz
test

32. Anti-bacterial
properties (Zinc)
Subclinical Male Genital Tract Infection
Azitromycin 1.0g single dose (couple)+ frequent
ejaculation (every 2-3 days) + Antioxidants:
• 42% leukocytospermia resolution (N=278)
Esteves, 32 ANDROFERT, Referral Center for Male Reproduction

33. Miyaoka & Esteves. Adv Urol 2012
Agarwal, Esteves, Hamada. Nature Urol Rev 2013;
Wang YJ et al. Reprod Biomed Online. 2012.
Esteves, 33 ANDROFERT, Referral Center for Male Reproduction
Varicocele
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)

34. Esteves, 34 ANDROFERT, Referral Center for Male Reproduction

35. Esteves, 35 ANDROFERT, Referral Center for Male Reproduction

36. Wang YJ et al.
Reprod Biomed Online. 2012;25:307-14.
Seven studies evaluating
the effect of varicocele
repair
SDF decreased after repair
Mean difference = 3.4%
(95% CI: -4.1 to -2.6;
p<0.0001)
Esteves, 36 ANDROFERT, Referral Center for Male Reproduction
Effect of Varicocele Surgery on SDF

37. Esteves, 37 ANDROFERT, Referral Center for Male Reproduction

38. Sperm % TUNEL + % CPR
Ejaculated 23.6 6
Testicular 4.8 44
P value <0.001 <0.05
Greco et al. Hum Reprod 2005
TESA-ICSI and SDF
Esteves, 38 ANDROFERT, Referral Center for Male Reproduction

39. SDF three-
fold lower in
testicular
sperm*
*Absolute differences between two specimens ranging from -3.3% to -56.3%.
Moskovtsev et al. Fertil Steril 2010
Esteves, 39 ANDROFERT, Referral Center for Male Reproduction
Difference in SDF between
Testicle and Ejaculate

40. Laboratory Management
l Abstinence period
l Iatrogenic damage
l Sperm selection techniques
Esteves,
40
ANDROFERT,
Referral
Center
for
Male
Reproduc4on

41. Abstinence Period
Esteves, 41 ANDROFERT, Referral Center for Male Reproduction
Gosálbez et al. Fertil Steril 2011
Serial ejaculation every 24h
for 4 days: 25% reduction SDF

42. Sperm Processing by DGC
Esteves, 42 ANDROFERT, Referral Center for Male Reproduction
Gosálbez et al. Fertil Steril 2011
Density gradient
centrifugation: 22%-44%
reduction in SDF

43. Dynamic Nature of SDF
Iatrogenic damage
Esteves, 43 ANDROFERT, Referral Center for Male Reproduction

44. Zeta method
Modified HOST
IMSI
PICSI
Sperm head
birefringence
Electrophoretic
selection
Magnetic- activated
cell sorting
Sperm Selection Techniques & SDF
Esteves, 44 ANDROFERT, Referral Center for Male Reproduction

45. Current non-invasive sperm
selection techniques are limited
in their ability to select
chromatin-intact spermatozoa
None of the available techniques
can directly assess sperm DNA
fragmentation
Dyes are needed to reach the
nucleus, using fixed specimens
Sperm Selection Techniques & SDF
Esteves, 45 ANDROFERT, Referral Center for Male Reproduction

46. SDF and Embryo Quality
Reference Assay N ART Findings
Larson et al., 2000 SCSA 24 IVF; ICSI No impact (D3)
Gandini et al., 2004 SCSA 34 IVF; ICSI No impact (D3)
Payne et al., 2006 SCSA 100 IVF; ICSI No impact (D3)
Sun et al., 1997 TUNEL 236 IVF ↓ Cleavage (D3)
Morris et al., 2002 Comet 60 ICSI ↓ Cleavage;
Morphology (D3)
Virro et al., 2004 SCSA 249 IVF; ICSI ↓ Blastulation rate
Nasr-Esfahani et al., 2005 Comet,
CMA3
28 ICSI ↓ Blastocyst
development
Esteves, 46 ANDROFERT, Referral Center for Male Reproduction

47. Oral antioxidants, life-style
modifications (quit smoking,
weight loss)
Esteves,
47
ANDROFERT,
Referral
Center
for
Male
Reproduc4on
TESA-ICSI for men with high
SDF enrolled in ART
Identify and treat underlying
condition such as GTI and
clinical varicocele
Management Strategies: Clinical
Key Messages

48. Short abstinence (1 day) and
frequent ejaculation
Esteves,
48
ANDROFERT,
Referral
Center
for
Male
Reproduc4on
Post-processing incubation
should not exceed 4h
Sperm processing by DGC
Management Strategies: Laboratory
Key Messages