An Update on SPERM DNA FRAGMENTATION TEST in MALE INFERTILITY

1. An Update on
SPERM DNA FRAGMENTATION
TEST in MALE INFERTILITY
DR. SHARDA JAIN
DR. JYOTI AGARWAL
Dr. Rashmi Jain

2. MALE
reproductive
dysfunction is the
sole or
contributory
cause in half of
couples
presenting for
treatment of
infertility

3. CAUSES OF MALE INFERTILITY
50%
30%
15% 5%
Disorders of Spermatogenesis Obstruction of VAS
Disorders Sperm Motility Physiological

4. Systematic clinical and laboratory
evaluation of the male, and the
application of cost-effective
management strategies tailored to
the individual patient’s need are vital
components of fertility practice.
What is the DICTUM in male infertility

5. Dictum has been –’’We treat the gametes
and not the individual’’.
This is because in male infertility, gamete
manipulation followed by ART is more
rewarding than treating the person with
hormones, antioxidants, vitamins or
surgical correction.

6. There are a few unique characteristics
of human spermatozoa; the
important ones are: Adult sperm cell
do not grow or divide; unilike other
somatic cell ..sperm head has a
large nucleus and lacks large
cytoplasm.
SPERM

7. Normal Values of Semen Variables:
WHO Guidelines
1999 2010
Volume 2.0 mL or more 1.5 ml (1.4–1.7);
pH 7.2 to 8.0
Sperm concentration 20 million or more 15 million per ml (12–16);
Total sperm count : 40 million or more 39 million per ejaculate (33–46);
Motility : 50% or more with forward
progression or 25% or more with
rapid progression
progressive motility, 32% (31–34);
total (progressive + non-progressive)
motility, 40% (38–42
Morphology 30% or more with normal forms 4.0% (3.0–4.0).
White blood cells Less than 1 million
Immunobead MAR<50% bound/adherent
7

8. INTERPRETING SEMEN ANALYSIS
Normal semen
parameters
• Despite looking
normal, sperms
may still be
damaged
• RO species
damage  DNA
fragmentation
Poor semen
parameters
• Oligospermia
• Asthenospermia
• Teratozoospermia
Azoospermia
• Obstructive
• Non obstructive

9. Advance knowledge of ART in male
infertility investigations …is opening up
new Pandora box
• Semen
• Genetic & hormonal test
•DFI
• Magnetic activity cell shorting

10. What is DNA fragmentation?
Separation or breaking of DNA strands into pieces.
Causes of DF?
• Testicular Event
• Post testicular Event
DFI – DNA FRAGMENTATION INDEX

11. DFI – DNA FRAGMENTATION INDEX
What is DFI test?
It is an effective method for measuring % of
sperms with fragmented DNA in the
ejaculate

13. SPERM DNA INTEGRITY
Is other very powerful
tool to know the health
of SPERM

14. HEALTH OF SPERMS
• Human sperms play an extensive role that
extends beyond early stages of
fertilization to include abnormal
embryogenesis leading to implantation
failure and aneuploidy

15. DNA FRAGMENTATION
Most common cause of poor sperm health is
DNA fragmentation
–DNA fragmentation can be
• Single strand DNA break
• Double strand DNA break
• Base deletion or modifications
• Inter and intra-strand cross linkage

16. BASICS OF SPERM DNA
FRAGMENTATION TEST
• Head nuclear protein consisting of histone is
replaced by protamine- producing a tightly
compacted nucleus.
• Protamine is a stronger DNA compared to
histone. Unlike all other somatic cell of the
body where histone is the DNA in the
nucleus, spermatozoon is the only cell which
contains protamine to offer compactness of
the sperm head nuclear DNA.

17. BASICS OF SPERM DNA INTEGRITY TEST
• Chromatin condensation during
spermatogenesis result in DNA occupying nearly
70% of the total volume of sperm nucleus
(somatic cell- only 5%).
• The adverse effect of displacement of histone
and replacement by protamine may result in
haploid genome damage (after secondary
spermatocyte ,the sperm}

18. BASICS OF SPERM DNA INTEGRITY TEST
• Repair capabilities during
spermiogenesis phase is limited.
• As a consequences of massive changes
during Spermiogenesis there may be
tremendous load on ‘haploid’
spermatozoa, leading to germ cell arrest or
blockage – thereby causing infertility in many
individuals

19. What is Significance of sperm
DNA fragmentation?
• Sperm DNA damage is a useful biomarker for
– Male infertility diagnosis (better marker than conventional semen
analysis)
– prediction of assisted reproduction outcomes
• Sperm DNA fragmentation is associated with
– Reduced fertilization rates
– Poorer embryo quality
– Poorer pregnancy rates
– Higher rates of spontaneous miscarriage
– Higher incidence of childhood diseases.
Schulte RT, Ohl DA, Sigman M, Smith GD. Sperm DNA damage in male infertility:
etiologies, assays, and outcomes. Journal of Assisted Reproduction and Genetics.
2010;27(1):3-12.

20. WHO IS GOING TO BENEFIT FROM
SPERM DNA ASSAY?
• Couples with unexplained infertility
• Couples with history of unsuccessful ART
• Couples with history of miscarriages
• Men over 30 years of age
• Men at higher risk of Oxidative Stress
– Men with diabetes
– Men with history of drug abuse
– Men who have been treated for cancer

21. What causes sperm DNA
fragmentation?
INTRINSIC FACTORS
• Protamination failure
• Oxidative stress during
transit or after
ejaculation
• Apoptosis during sperm
maturation inside the
tubules
EXTRINSIC FACTORS
• Life style – Smoking/
Obesity
• Varicocele
• GT Infections
• Radiation exposure
• Exposure to toxins such
as Lead
Denny Sakkas and Juan G. Alvarez. Sperm DNA fragmentation:
mechanisms of origin, impact on reproductive outcome, and analysis.
Fertility and Sterility Vol. 93, No. 4, Pages 1027-36.

22. Methods to diagnose DNA
fragmentation
• Comet – single cell gel electrophoresis
–Detects actual DNA strand breaks and
measures
existing damage
• Tunnel –terminal deoxy nucleotide
transferees mediated UTP nick end-labeling
–Detects actual DNA strand breaks and
measures existing damage

23. Methods to diagnose DNA
fragmentation
• Sperm chromatin dispersion (SCD) ->
“halo sperm” test
• (normal sperms have a halo)
–Measures the susceptibility of DNA to
denaturation –> formation of single
stranded DNA from native double
stranded DNA
–Estimates potential future damage

24. METHODS TO DIAGNOSE DNA
FRAGMENTATION
• Sperm chromatin structure assay (SCSA)
– Measures the susceptibility of DNA to acid
induced denaturation
– Estimates potential future damage
– SCSA is the only test with clear and clinically
useful cut off levels for calculating male fertility
potential

25. Sperm DNA fragmentation assays
• TUNEL assay -> Blue sperm are
TUNEL negative while green sperm
are TUNEL positive indicating DNA
fragmentation.
• Sperm Chromatin Dispersion Test ->
The two sperm in the center with
non-fragmented DNA form large
halos, while the sperm in the upper
right hand corner has no halo
indicating DNA fragmentation.
Results from both assays are expressed as percentage of
sperm demonstrating DNA fragmentation.

26. DNA Fragmentation Index
• Most studies define an upper normal level of the
percentage of cells with DNA fragmentation
• Samples with assay results above this threshold
percentage are considered to have high DNA
fragmentation
• Interpretation of values
– Spermatozoa with fragmented DNA
≤ 15% –> good fertility potential
– Spermatozoa with fragmented DNA
15-25% -> average fertility potential
– Spermatozoa with fragmented DNA
> 25% -> poor fertility potential

27. Management of sperm DNA
fragmentation
• Lifestyle modifications (empirical use)
– Cessation of smoking and alcohol intake
– Weight management for obese men
• Antioxidants – should be used at least for
2-3 months for effect (empirical use only)
– Vit C (500 mg/ day)
– Vit E (200 mg/ day)
– Folic Acid (2 mg/ day)
– Zinc (25 mg/ day)
– Selenium (26 mcg/ day)

28. Treatment of underlying
pathological conditions
• Varicocele
– GU Infections
• Avoid environmental exposure to toxins and
judicial medical use of radiations
• Reducing the abstinence period or serial
ejaculation every 24 hours reduces SDF by up
to 25%
• In cases with uncorrected DNA fragmentation
– consider surgical sperm retrieval

29. But success lies
In Selection of normal efficient sperm
• Prior to ART
• During semen Preparation
• During ICSI
The Treatment
Of abnormal Sperm DNA fragmentation
Test in Male Infertility is ICSI

30. ICSI remains the gold standard
embryology procedure to tackle
Male Infertility
• No major breakthrough after ICSI to tackle male
infertility and improve outcome of ART
• Current target of research is to further improve
outcome of treatment is to find techniques which will
identify sperms with best fertilization potential

31. Sperm DNA fragmentation - Evidence
Authors Study design Outcome
Zini et al
(2008)
Meta analysis Embryos with high sperm DNA damage are associated with
early pregnancy loss
Zini and
Sigman
(2009)
Meta analysis Modestly increased pregnancy chance after IVF (OR 1.7, 95%
CI 1.3–2.2) in cases with low level of DNA-damaged sperms
Dumoulin et al
(2010)
In ICSI sperms are injected into the optimal environment of
the ooplasm within a few hours of ejaculation, thus reducing
chance of lab-induced damage to sperms
Simon et al
(2011)
In IVF, couples with low levels of sperm DNA fragmentation
(<25%) had LBR of 33% and the ones with high levels of
sperm DNA fragmentation (>50%) had LBR of 13%

32. Sperm DNA fragmentation - Review
Authors Study design Outcome
Zini (2011) RCT Sperm DNA damage not found to be predictive
for ICSI treatment
Aitken et al
(2012)
Sperms from up to 40% of infertile men have high
levels of ROS
Robinson et
al (2012)
High levels of sperm DNA damage are associated with
increased risk of pregnancy loss (OR 2.5, 95% CI 1.5–4.0)
regardless of the in-vitro technique applied
Simon et al
(2013)
OR of 76 (95% CI 8.7–1700) for clinical pregnancy when the
mean DNA fragmentation per spermatozoon was < 52%
Osman et al
(2015)
Meta analysis LBR was lower for high sperm DNA fragmentation in IVF cycles.
But, there was no difference in LBR between low and high sperm
DNA fragmentation with ICSI

33. Take home messages
•Normal semen parameters has gone beyond semen
analysis
•Poor ART outcome can be correlated with high DNA
fragmentation in sperms
•Healthy lifestyle & correction of underlying pathology
for healthier sperms
•Newer modalities of sperms selection like TESA or
IMSI, PICSI and MACS will benefit a carefully selected
segment of patients with failed ICSI

34. This sperm D.F.I. test now is available in Delhi
At our centre for male infertility
CHERISH

35. IT IS THE TEAM.. that matters.. In
infertility

36. ISO 14001:2004 (EMS)
…..Caring hearts, healing hands
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