Relevance of Sperm DNA

Fragmentation

Marianne Moser
Landes-Frauen- und Kinderklinik
Li
Linz, A
Austria
ti
 Relevance of Sperm
p DNA
Fragmentation
1. Sperm DNA peculiarity
2. Etiology of DNA fragmentation (DF)
3. Test methods
4. Influence of laboratory techniques
5
5. DF and outcome in infertility treatments
6. Conclusions
1 Sperm DNA peculiarity
1.

During spermiogenesis
spermatids repackage
their DNA with
protamines, a small
residue of histone-bound
DNA is retained (15%).
 protamines
• Are proteins with a high content of positively charged
amino acids (48% arginine)

• Form a highly condensed complex with the sperm DNA

(DNA has a strong negative charge)

• Incorporate cysteins

• Cysteins allow the formation of disulphide bonds

between the protamines

• Therefore strongly stabilize the nucleoprotamine

complex
In mature sperm the DNA is
• 85% protamine bound
• 15 % remains histone bound

protamine deficiency: more susceptible for ROS

Infertile men have a higher histone:protamine ratio than fertile men

(Oliva 2006, Zhang 2006)

5-10% of infertile men have a complete protamine deficiency

 Fig. 1: The human sperm

Zini, A. et al. CMAJ 2006;175:495-500

Copyright ©2006 Canadian Medical Association or its licensors

2. Etiology of DNA Damage
the etiology of sperm DNA damage is
multifactorial
• In the testis during the process of
spermatogenesis:
– Apoptosis: screening mechanisms
mechanisms,
that mark individual apoptotic
sperms which causes phagocytosis
off these
h cells,
ll ffailed
il d (d
(double
bl sb)
b)
– during remodeling of sperm
chromatin the DNA is unwound
through the induction of strand
breaks (single sb), sperms with
unrepaired strand breaks →semen.
→semen
 Etiology of Sperm DNA Damage
• post-testicular:
– during sperm transport through the
seminiferous tubules and the epididymis
– Varicocele
– Genital tract infections (leukocytes)
– Immature sperms (cytoplasmic droplet)
– radio- and/or chemotherapy
– Lifestyle factors (obesity, cell phones,
nicotine), environmental toxicants
– Laboratory factors

• Majority of DNA damage is associated with

ROS (Aitken et al., 2010)
 ROS

• Small levels are essential for normal sperm

p
functions (capacitation, acrosome reaction,
sperm-oocyte
p y fusion ((Sikka et al,, 1995))
• Balance between ROS production and
scavenging system is important
• in 25% of infertile men high ROS levels have
been detected in their semen
semen.
levels of ROS may fluctuat
within a fertile man but do not affect
sperm concentration and motility.
This may be possible due to the
presence of adequate
p q antioxidant
defense mechanismsin the present
healthy individual.

this variations may have physiologic,

seasonal, or lifestyle-related causes
3. DNA Fragmentation Tests

DFI: DNA Fragmentation Index %

 DNA fragmentation - Tests
• DIRECT
– TUNEL (terminal
(t i lddeoxynucleotydil
l t dil ttransferase-
f
mediated deoxyuridin triphosphate- nick-end labeling
assay ) (s&dSB)
– ISNT (in situ-nick translation)
– Comet Assay at neutral pH (sdSB) (single cell gel
electrophoresis
• INDIRECT (need denaturation of DNA)
– SCSA (sperm chromatin structure assay)
– SCD (sperm chromatin dispersion test test, Halosperm
Assay)
– Comet Assay at acid or basic pH (sdSB)
 TUNEL assay

Labels single and double strand breaks, quantifies the incorporation of flourescent dUTP
 SCSA Test
sperm chromatin structure assay

acid-induced denaturation of DNA followed by staining with AO

AO.
is a flow-cytometric method, measures the metachromatic shift of
AO fluorescence from green to red
– green (native DNA)
– red (denatured DNA).
 SCD or Halo Test

The SCD is based on the principle

that sperm with fragmented DNA fail
to produce the dispersed DNA loops
after acidic denaturation (halos)

Normal sperm produce a halo

Ebner et al,
al 2011
Observed a strong relationship between the tests.

AO only coincided on values >30%, (technical problems of instinct colours, rapid fading,
heterogenous staining of slides, also reported in other studies)
4. Influence of lab procedures
storage and sperm DFI
Unselected group

Our data indicate that any

sample that will not be
analyzed within 4 hours of
collection should be frozen to
preventt increasing
i i DNA
damage.

Among different methods of

freezing, there was no statistically
significant difference in the
resultant amount of DNA
fragmentation
 The potential detrimental effect of density gradient centrifugation
on sperm DNA integritiy is related to initial semen quality (Zini)

Sperms of infertile patients are more susceptible to external

influences
Sperm processing and DFI

• DFI Levels immediately following

processing were significantly lower for
swim-up,
swim up, density gradient and DG
&SU than for fresh and washed semen
samples.
90% of patients showed no strandbreaks after processing
Ebner et al, 2010
 a b

Swim-up for maximum 2 hours

Ebner et al, 2010

 39 patients
with male subfertility
(51% isolated
teratozoospermiai
(WHO 1999)).

Halo test

After
Raw
a se
semen
e After
e Zech-selector
ec se ec o
d
density
it gradient
di t

Ebner et al, 2011

 DFI motility
Raw semen 15.8 ± 7.8% 36.6 ± 19.4 %
a and b
Density gradient 14.2 ± 7.0% not
investigated
est gated
Zech Selector 0.4 ± 1.1% 100% a
• M
Motility
tilit seems tto b
be th
the utmost
t t ffactor,
t since
i the
th
selector separates spermatozoa according to
their motilty and not to their morphology
morphology.
• This is supported by the literature Ramos and
Wetzels 2001
Wetzels, 2001, Van den Berg et al 1998) and by
our own observation, that the sperm swimming
close to the surface of the supernatant show
significantly reduced rates of DF after density
centrifugation
g and swim-up.p
• Simply overlaying a sperm sample with medium,
((w/o centrifugation)
g ) could lead to similar results.
 Why do fast progressive sperms show no sign of DF?

• Both nuclear and mitochondrial DNA can be harmed by strand

breaks
• Mitochondrial DNA could cause alterations in ATP production,
which is a prerequisite for optimal sperm motilityy
• Deletions within mitochondrial DNA have been associated with
reduced sperm motility (Ozmen et al, 2007)

• It could be possible, that grade a spermatozoa could be neither

harmed by nuclear or mitochondrial DNA damage
• In the selector grade b spermatozoa cannot overcome the
capillary
p yggap
p between the outer and the inner ring
g in 2 hours
due to their reduced energy and forward movement.
Correlation of sperm parameters
and DFI

Yes No

Oligozoospermia Burallo et al 2004 Gandini et al. 2000

Host et al 1999
T li
Tomlinson 2001

Teratozoospermia Host et al 1999 Chan et al. 2001

M t i ett all 2003
Muratori D
Donnely
l ett all 2001
Trisini et al 2004
Tomlinson 2001
Asthenozoospermia Giwercman et al 2003 ??
Irvine et al 2000
Mahfouz 2010
Varghese ez al 2009
5. Is DFI testing relevant for
f tilit assessmentt
fertility
DFI and IUI

• with a DFI < 27% the chances for

pregnancy are significantly higher than in
patients with a DFI > 27%
Fertility potential
potential, threshold levels

Evenson et al (1999), Spano (2000):

If >30% sperm have abnormal chromatin: fertility is
hampered independent of sperm number, morphology
and motility.
y
Evenson et al (2002):
Fertility potential according to DFI fraction:
Excellent <15%
Good 15-24%
Fair 25-30%
poor >30%
A hi
Achievement
t off pregnancy:

– 84% of men with DFI of <15% achieved a

pregnancy during the first 3 months

– 10% of men with a DFI<30% achieved a

pregnancy during
d i monthsth 44-12
12

– 20% of men with a DFI > 30% never

achieved a pregnancy
• in the general population:
1-4% of men have a DFI > 30%.
• Infertility
f patients
– a DFI > 30% have
• IUI patients: 7%
• IVF patients:
p 16%
%
• ICSI patients: 33%
Retrospective study on 282 patients
23 papers that investigated the influence of DNA damage on

Fertilization Embryo Pregnancy Pregnancy

quality l
loss
Total nr.of 19 18 21 12
papers
YES 8 8 12 3
NO 11 10 9 9
DFI and pregnancy loss
CONCLUSIONS
sperm DNA damage is associated with a significantly increased
risk of pregnancy loss after IVF and ICSI.

The data provide a clinical indication for the evaluation of sperm DNA
damage prior to IVF or ICSI and a rationale for further investigating the
association between sperm DNA damage and pregnancy loss.
Let´s
Let s not forget the oocyte
When oocytes from infertile patients were employed, DF had a statistically
significant negative impact on chance of pregnancy.
F every 10% iincrease iin DF
For DF, th
the probability
b bilit off nott achieving
hi i pregnancy
increased by 1.31.

When donated
Wh d d oocytes were employed,
l d DF did not h
have a statistically
i i ll
significant effect
 Conclusions I
• Infertile men have higher DF than fertile men
• Sperms of infertile men are more susceptible to
damage
da age ((ROS)
OS)
• sperm with damaged DNA can successfully
fertilize - but may cause de novo mutations in
the offspring. (despite the ability of the oocyte
and embryo to repair some damage)
→ causes concern about the safety of ICSI
• Extensive
E t i DF mighti ht nott be
b overcome by b oocyte
t
repair mechanisms.
• Oocyte repair capacity cannot be measured.
 Conclusions II
• S
Smallll b
butt significant
i ifi t association
i ti b between
t DF
and pregnancy – but- no indication for routine
use in male evaluation (Collins)
• ? Indication in failed IVF, several pregnancy
losses, several failed IUI´s
• More studies needed to identify subgroups that
would merit from DF tests
• Apply careful sperm processing method, so not
to enhance DFI
• ICSI: use methods to pick out sperms with
reduced risk for DF
 Manuela
Thomas
Me R
Renate
t

Thank you for your attention

 (infertile patients!)

Infertile Patients

HDS: high DNA stainability: a measure of nuclear chromatin compaction

Sperm head abnormalities may in part be due to incomplete sperm chromatin

condensation
Is sperm dna damage associated with IVF embryo quality?
A systematic
y review.
Zini A, Jamal W, Cowan L, Al-Hathal N.
Division of Urology, Department of Surgery, McGill University, Montreal, Quebec, Canada,
[email protected].

28 studies (8 IVF, 12 ICSI and 8 mixed IVF-ICSI studies) that evaluated the
relationship between sperm DNA damage and embryo quality.

3226 treatment cycles (1033 IVF and 873 ICSI, 1320 mixed IVF-ICSI cycles)

CONCLUSIONS: This systematic review indicates that the evaluable studies

are heterogeneous and that overall, there is no consistent relationship
between sperm DNA damage and embryo quality and/or development.