Sperm DNA

Fragmentation
 POINTS TO DISCUSS
1. Human Sperm Cell
2. Structure of Human Sperm Chromatin
3. Causes of Sperm DNA Damage
4. Type of DNA Damage
5. Effect on Reproductive Outcomes
6. Tests for Diagnosis
7. Usefulness of the Tests
8. Management strategies
8. Guidelines for Current Practice
 THE SPERM CELL
• Sperm cell is different from other cells in the body
• Small size –at the expense of cytoplasm –cell mass
• Reduced Cell mass – Impaired production of enzymes required
for genetic repair
• Chromatin in somatic cells – Relatively loose structure
• Chromatin in sperm because of small size – Very tightly
compacted- haploid genome must adapt to a volume 40 times
less than a somatic cell
 SPERM CHROMATIN STRUCTURE

• Fundamental packaging unit of sperm chromatin is a

toroid which has 50-60 kb of DNA
• Toroids are cross linked and further compacted by di -
sulphide bonds.
 SPERM CHROMATIN STRUCTURE

• During later stages of Spermatogenesis – spermatid nucleus

remodelled and condensed
• During spermiogenesis, sperm chromatin undergo a series of
modifications in which histones are lost and replaced with
transition proteins and subsequently with protamines.
• Protamines are approximately half the size of histones .
• The DNA strands are highly condensed by these protamines
and form the basic packaging unit of sperm chromatin, a
toroid.
• The toroids are further compacted by the intramolecular and
intermolecular disulfide cross-links between cysteine residues
present in protamine
 SPERM CHROMATIN STRUCTURE
• Sperm nuclear proteins are predominantly composed of
Protamines - 85 %
Histones- 15%
• When the strands are not packed well - long DNA strands
susceptible to damage leading to Sperm DNA fragmentation
• Somatic cell nuclear DNA is wrapped around an octamer of
histones and packaged into nucleosomes and then further
coiled into a solenoid. This type of packaging adds histones,
which increase chromatin volume.
 CAUSES OF SPERM DNA DAMAGE
• Intrinsic factors
 Remodeling and Packaging Problems
Damage by ROS
 Abortive Apoptosis
• Extrinsic factors
Chemotherapy
Cigarette smoking
Genital tract inflammation
Testicular hyperthermia
Varicoceles
 Xenobiotics
INTRINSIC FACTORS
Remodelling & Packaging Problems:
• Stage-specific transient DNA strand breaks are introduced
during Spermiogenesis.
• These physiological, temporary breaks if not repaired – lead to
DNA fragmentation or genetic mutations in the ejaculate.
• Reactive oxygen species:
• Free radicals are a group of atoms or molecules that are highly
reactive due to having one or more unpaired electrons . As a
result of having an incomplete outer valance shell, these
molecules attempt to react with other molecules in their
vicinity in order to gain one or more electrons. However, once
a molecule loses an electron to a free radical, a chain reaction
is created, as now the former molecule becomes a free radical
itself.
Reactive oxygen species Reactive nitrogen species

Superoxide anion (O 2 •− ) Nitric oxide (NO • )

Hydrogen peroxide (H 2 O 2 ) Nitric dioxide (NO 2 • )

Hydroxyl radical (OH • ) Peroxynitrite (ONOO − )
Excess ROS levels:
• ROS have an important physiological role in modulating gene &
protein activities vital for sperm proliferation.
• Physiological amounts are controlled by seminal antioxidants
• Excess – generated by morphologically defective sperms
(residual cytoplasm in particular) and semen leukocytes lead to
DNA damage
• Plasma membrane of the spermatozoa is rich in
polyunsaturated fatty acids(PUFA). Because their cytoplasm
contains low concentrations of scavenging enzymes, they are
particularly susceptible to the damage induced by excessive
ROS.
• Excess ROS can damage DNA in spermatozoa, induce cell
apoptosis, and cause lipid peroxidation, which leads to
morphological abnormalities, decrease in fertility, and
increased sperm membrane permeability.
• The seminal plasma, however, contains two different types of
antioxidants to minimize free radical-induced damage:
enzymatic and nonenzymatic antioxidants.
• Enzymatic antioxidants are: superoxide dismutase, catalase,
glutathione reductase, and peroxidase.
• Nonenzymatic antioxidants are comprised of vitamins (vitamin
C, E), proteins (albumin, transferrin, haptoglobin, and
ceruloplasmin), and other molecules (glutathione, pyruvate,
and ubiquinol).
• There are several methods to measure seminal ROS in the
clinical setting, most notably among them is the
chemiluminescence assay. This technique measures the global
ROS, i.e. both the intra- and extracellular ROS.
• The two major probes used to measure ROS generation in the
chemiluminescence assay are luminol and lucigenin.
• Luminol reacts with a variety of reactive oxygen species (H2O2
O2-, OH) and allows both intra- and extracellular ROS to be
measured.
• Lucigenin, however, yields a chemiluminescence that is more
specific for superoxide anions released extracellularly.
Autolumat 953 plus luminometer used in the measurement of ROS by chemiluminescence
assay. ( a ) External view and ( b ) internal view. Multiple tubes can be loaded simultaneously
for measuring ROS. ( c ) The luminometer can be connected with the computer and a monitor and
all the steps can be observed on the screen
 Reactive Oxygen Species (ROS) in human semen:
determination of a reference range
J Assist Reprod Genet. 2015
• For measuring ROS in semen, 10 μl luminol working solution was
added to 400 μl liquefied whole semen. All samples are mixed
gently immediately.
• Chemiluminescence is reported as Relative Light Units per second
(RLU/sec). RLU/sec is measured at 1 min intervals after addition of
luminol, over a total period of 10 min and then averaged for each
sample. This value is adjusted for sperm concentration and ROS is
reported as RLU/sec/106 sperm.
• The reference value of < 24.1 RLU/sec/106 sperm is acceptable for
seminal ROS
Abortive Apoptosis:
• Apoptosis of testicular germ cells occurs throughout life
• Some sperms have initiated but escaped apoptosis - abortive
apoptosis because of deficient cytoplasm and organelles.
 EXTRINSIC FACTORS

Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis
Denny Sakkas, fert and steril, 2010
 EXTRINSIC FACTORS
• Radiotherapy, chemotherapy and environmental toxins –
induce sperm DNA damage
• Can be direct effect on DNA or indirect effect by changing the
endocrine milieu of the testis or epididymis
• Leads to lowered activity of the testosterone-dependent DNA
enzyme topoisomerase
• Reduced production of antioxidants by epididymis
2 STEP HYPOTHESIS
• Faulty spermatogenesis→
• defective remodeling →
• DNA more susceptible to
stress factors.
Lesions Associated with Sperm DNA Damage
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

single-strand break damaged base double-strand break

• SSB is easy to repair and better prognosis
• SSB are mainly due to due to unrepaired DNA nicks and ROS
• DSB is caused by abortive apoptosis, action of caspases and
endonucleases & ROS.
• DSB may lead to gross alteration to chromosomal structure
and more serious and deleterious impact on development.
 EFFECT ON REPRODUCTIVE OUTCOME
• Oocytes and early embryos have been shown to repair sperm
DNA damage.
• The biological effect of abnormal sperm chromatin structure –
is the combined effect of sperm chromatin damage and the
capacity of the oocyte to repair the damage.
• Fertilization is independent of DNA damage.
• Post fertilization development is affected by improper repair by
the oocyte which may lead to implantation failure, early
miscarriages, diseases in the offspring.
 DNA FRAGMENTATION - TESTS

DIRECT
• TUNEL (terminal deoxynucleotydil transferase mediated
deoxyuridin triphosphate- nick-end labelling assay )
• Comet Assay at neutral pH (single cell gel electrophoresis)
• Dye tests

INDIRECT (need denaturation of DNA)

• SCSA(sperm chromatin structure assay)
• SCD (sperm chromatin dispersion test, Halosperm Assay)
• Comet Assay at acid or basic pH
 DIAGNOSTIC TESTS
• All these tests label single or double stranded DNA breaks
• Dye, Comet & TUNEL tests detect actual DNA strand breaks –
measure existing damage.
• SCD and SCSA measure the susceptibility of DNA to
denaturation – formation of single stranded DNA from native
double stranded DNA – hence includes potential future
damage.
 Acridine orange test

• Acridine orange test (AOT) is a simple microscopic procedure

based on acid conditions to denature DNA followed by staining
with metachromatic acridine orange.
• Acridine Orange fluoresce green when it binds to native DNA
and red when it binds to the fragmented DNA.
• However, issues of indistinct colors, rapid fading, and the
heterogeneous staining can cause difficulties during visual
interpretation
Toluidine blue
• Toluidine blue (TB) is a basic dye used to evaluate sperm
chromatin integrity.
Aniline blue
• Aniline blue is an acidic dye which is used to evaluate sperm
chromatin integrity.
• (a) Human ejaculate stained with toluidine blue: (1) mature sperm heads are light
blue; (2) immature are violet. (b) DNA breakage detection–fluorescence in situ
hybridization (DBD–FISH) labeling with a whole genome probe (red fluorescence),
demonstrating extensive DNA breakage in those nuclei that are intensely labeled. (c)
Acridine orange (AO) stain to native DNA fluoresces green (1); whereas denatured
DNA fluoresces red .
 TUNEL
• The terminal deoxynucleotidyl transferase-mediated (TdT)
deoxyuridine triphosphate (dUTP) nick end labeling assay
(TUNEL) is a direct quantification of sperm DNA breaks.
• dUTP is incorporated at single-stranded and double stranded
DNA breaks in a reaction catalyzed by the enzyme TdT.
• The DNA breaks based on the incorporated dUTP are then
labeled and can be measured using bright field or fluorescent
microscopy as well as flow cytometry.
• TUNEL is sensitive for both single and double stranded breaks
TUNEL Terminal deoxynucleotidyl transferase dUTP nick
end labeling
• Enzymatic addition of modified
nucleotides to DNA break
 TUNEL
Labels SS and DS breaks
Measures percent cells with labeled DNA
ADVANTAGES: DISADVANTAGES:
1. Fresh or frozen samples 1. Variable protocols
2. Can be used for testicular retrieved 2. Unclear thresholds
sperms.
3. Not available in commercial kits
3. Can be performed on few sperm
4. High repeatability
5. Quick and simple ( fluorescence
microscopy)
 COMET
• Decondensed sperm are suspended in an agarose gel, subjected to
an electrophoretic gradient, stained with fluorescent DNA-binding
dye, and then imaged with imaging software.
• Low-molecular weight DNA, short fragments of both single-
stranded and double-stranded DNA, will migrate during
electrophoresis giving the characteristic comet tail.
• High-molecular weight intact segments of DNA will not migrate and
remain in the head of the “comet.”
• Imaging software is then used to measure comet tail length and tail
fluorescent intensity, which are increased in sperm with high levels
of DNA strand breaks
 The two-tailed (TT) comet assay

• The de-proteinized sperm is first subjected to an electrophoretic field

under non-denaturing conditions to mobilize isolated free discrete DNA
fragments produced from DSBs
• This is then followed by a second electrophoresis running perpendicular
to first one but under alkaline unwinding conditions to produce DNA
denaturation exposing SSBs on the same linear DNA chain. This
procedure results in a two dimensional comet tail emerging from the
core where two types of original DNA affected molecule can be
simultaneously discriminated within the same cell.
 Comet
For single and double stranded breaks
• 1. Inexpensive
• 2. High sensitivity
• 3. Fresh samples only
• 4. Correlates with seminal
parameters
• 5. Small number of cells required
• 6. Versatile (alkaline or neutral)
• Can detect both SSB and DSB in
same sperm
• 1. Variable protocols
• 2. Unclear thresholds
• 3. Not available in commercial
kits
• 4. Time and labor intensive
• 5. Small number of cells assayed
• 6. Subjective
• 7. Requires special imaging
software
 SPERM CHROMATIN DISPERSION TEST

• The sperm chromatin dispersion (SCD) test is based on induced

condensation which is directly linked with sperm DNA
fragmentation.
• Intact sperm are immersed in an agarose matrix on a slide, treated
with an acid solution to denature, and then treated with a lysis
buffer to remove sperm membranes and proteins giving rise to
nucleoids with a central core and a peripheral halo of dispersed
DNA loops.
• Sperm can be stained with Giemsa or Wright's stain for
visualization under bright field microscopy or an appropriate
fluorescent dye for visualization under fluorescent microscopy.
• The SCD test is a “simple” method in kit form.
• Unlike all the other tests, it measures
the absence of damage rather than
the damaged DNA in sperm.
It does not rely on either color or
fluorescence intensity making the
test simple to use with light microscopy.
• During the SCD, processing of agarose embedded sperm
remove the protamine molecules. This removal leads to
breakage of disulfide bonds in the otherwise tightly looped and
compact sperm genome. As the disulfide bonds break, the
loops of DNA relax, forming haloes around the residual nuclear
central structure. Spermatozoa with fragmented DNA showed
evidence of restricted DNA loop dispersions, showing very
limited haloes or absence of them, unlike the sperm with non-
fragmented DNA
 SCD
Advantages Disadvantages
• Commercial assay available • Indirect assay only detects ssDNA
• Interpretation does not depend breaks
on fluorescence or flow • Involves acid denaturation.
cytometry
Sperm Chromatin Structure Assay (SCSA)

• This assay is based on the concept that DNA

in sperm with abnormal chromatin
structure is more prone to acid or heat
denaturation.
• Using the metachromatic properties of
acridine orange (AO), SCSA measures
susceptibility of sperm DNA to acid-induced
denaturation in situ.
• By quantifying this metachromatic shift of
AO from green to red after acid treatment
using flow cytometry, the extent of DNA
denaturation is determined
• Both SCSA and Acridine Orange Test measure the susceptibility
of sperm nuclear DNA to acid-induced conformational
transition in situ by quantifying the metachromatic shift of AO
fluorescence from green (native DNA) to red (denatured or
relaxed DNA). Compared to visual counting of red and green
cells in AO test, in SCSA the red-green fluorescence is detected
using a flow cytometer.
 SCSA
For single-stranded DNA
• 1. High reproducibility • 1. Not available in commercial
• 2. Established clinical kits
thresholds • 2. Expensive equipment
• 3. Many cells rapidly • 3. only detects ssDNA breaks
examined
• 4. Fresh or frozen samples
• 5. Has extensive body of
literature and established clinical
thresholds
 COMPARISION OF DIFFERENT DFI TESTS
• The COMET and Sperm Chromatin Dispersion tests were
introduced as light microscope tests that don’t require a flow
cytometer. Since these tests measure only 50–200 sperm per
sample, they suffer from the lack of the statistical robustness of
flow cytometric measurements.
• Only the SCSA test has an exact standardization of a fixed protocol.
The many variations of the other tests make it very difficult to
compare data and thresholds for risk of male factor infertility
Anim Reprod Sci. 2016
• The TUNEL test requires the TdT enzyme to
add dUTP to broken DNA ends. However,
due the high degree of compaction of
sperm chromatin, its requirement for TdT
almost certainly restricts its access to a
limited fraction of the in situ DNA, most
likely only to the toroid linker regions .
• In contrast, the SCSA test requires only the
very small AO molecule that detects lesions
in a broader fraction of the compact sperm
chromatin
(Gawecka et al., 2015, Hum Reprod. 2015 Dec; 30(12)).
• TUNEL is sensitive for both single and double stranded breaks
• In the modified TUNEL assay the use of dithiothreitol (DTT) was
suggested which breaks the disulphide linkage between
adjacent protamine molecules, relaxing the chromatin and
thereby allowing the TdT to access the DNA strand breaks
within sperm nucleus.
 INTERPRETATION
Percentage of spermatozoa with fragmented DNA
• ≤ 15 – good fertility potential
• 15-30% - average
• > 30% - poor fertility potential
 CLINICAL/ PRACTICAL UTILITY OF DFI
Disadvantages
• Most assays do not differentiate between clinically significant and
insignificant DNA damage
• Some DNA nicking occurs as a normal process during winding and
unwinding of DNA; current assays do not differentiate physiologic
from pathologic nicking.
• Assays do not evaluate the genes that may be affected by the
fragmentation.
• Fragmentation in areas containing certain genes may be more
detrimental than fragmentation in relatively inactive regions of the
genome.
Measuring Sperm DNA Fragmentation and Clinical Outcomes of
Medically Assisted Reproduction: A Systematic Review and
Meta-Analysis
Maartje Cissen, 2016

• Out of 658 unique studies, 30 had extractable data and were

thus included in the meta-analysis.
• At this moment, there is insufficient evidence to recommend
the routine use of sperm DNA fragmentation tests in couples
undergoing MAR both for the prediction of pregnancy and for
the choice of treatment.
The effect of sperm DNA fragmentation on miscarriage
rates: a systematic review and meta-analysis.
Robinson L et al Hum Reprod. 2012

MAIN RESULTS AND THE ROLE OF CHANCE: We identified 16 cohort

studies (2969 couples), 14 of which were prospective. Eight studies used
acridine orange-based assays, six the TUNEL assay and two the COMET
assay. Meta-analysis showed a significant increase in miscarriage in
patients with high DNA damage compared with those with low DNA
damage [risk ratio (RR) = 2.16 (1.54, 3.03), P < 0.00001)]. A subgroup
analysis showed that the miscarriage association is strongest for the
TUNEL assay (RR = 3.94 (2.45, 6.32), P < 0.00001).
 CURRENT PRACTICE

Unexplained infertility , normal semen parameters with mild or treatable

female infertility
• DFI > 30% - direct IVF/ICSI• DFI < 30% - treatment of female- spontaneous
pregnancy can be tried.

Minor impairment of semen parameters: Concentration, Motility, Morphology -

below reference range and short period of infertility
• DFI <15% -female <35 years, no pathology – spontaneous pregnancy can be
tried
• DFI>15% - treatable female subfertility – treatment of the female
 MANAGEMENT OF HIGH DFI

• Oral antioxidants
• Lifestyle modifications(Quit smoking/ weight reduction)
• Treatment of underlying condition/ infections
• Consider TESA/TESE + ICSI
• For patients with elevated levels of sperm DNA fragmentation,
we advocate the use of antioxidants and will also proceed with
testicular sperm retrieval for use in ICSI for couples with
recurrent pregnancy loss using ejaculated sperm with elevated
sperm DNA fragmentation.

Basic Clin Androl. 2016

 Antioxidants for male subfertility
Showell MG et al , Cochrane database, 2011

• There is low quality evidence from only three small

randomised controlled trials suggesting that antioxidant
supplementation in subfertile males may improve live birth
rates for couples attending fertility clinics. Low quality
evidence suggests that clinical pregnancy rates may increase.
Esteves et al, Int Braz J Urol 2011
 EFFECT OF VARICOLELECTOMY
• A varicocelectomy can improve sperm
DNA integrity, with a mean difference of
-3.37% (95% CI -4.09 to - 2.65; P<0.00001)

• Meta analysis of seven studies evaluating

the effect of varicocelectomy repair on SDF

Wang YJ et al, Reprod Biomed Online. 2012

Comparison of reproductive outcome in oligozoospermic men with high
sperm DNA fragmentation undergoing intracytoplasmic sperm injection
with ejaculated and testicular sperm.
Esteves SC, et al. Fertil Steril. 2015.

CONCLUSIONS: ICSI outcomes were significantly better in the

group of men who had testicular sperm used for ICSI compared
with those with ejaculated sperm.
SDF was significantly lower in testicular specimens compared
with ejaculated counterparts.
Our results suggest that TESTI-ICSI is an effective option to
overcome infertility when applied to selected men with
oligozoospermia and high ejaculated SDF levels.
 IATROGENIC SDF - SOLUTIONS
• Short abstinence period and serial ejaculation
• Process specimen as soon as possible
• Keep samples at room temperature using appropriate culture
media
• Incubation time post processing should not exceed 4 hours.
• Thaw cryopreserved specimen just before performing ART
 SPERM SELECTION FOR
SAMPLES WITH HIGH DFI
 IMSI
MSOME
• (Motile Sperm Organelle Morphology Examination)
• Examination performed in real time on living SP
• Inverted light microscope
• Equipped with high-power Nomarski optics instead of Hoffman
Modulation Contrast
• Enhanced by digital imaging to achieve a magnification up to 6300
• More accurate examination of spermatozoa
(Bartoov et al., 2002)
 PICSI

• PICSI device makes it possible to

select a functionally competent
sperm, indicated by its ability to
bind to hyaluronan.
• Hyaluronic acid binding by human
sperm indicates cellular maturity,
viability and un-reacted status of
sperm acrosome
 SPERM SELECTION WITH POLSCOPE
• Two types of birefringence pattern
1) sperm with partial birefringent
head/acrosome-reacted
2) sperm with total birefringent
head localized in post acrosomal
area/acrosome-non-reacted
spermatozoa
• Abnormal pattern- absence of
birefringence, presence of vacoule
like structure or small area of
birefringence located in nucleus or
acrosomal area.
 SPERM BIREFRINGENCE AND OUTCOME

• Sperms with partial head

birefringence ( acrosomally
reacted spermatozoa ) resulted
in improved LBR than total head
birefringence ( acrosomal non
reacted sperm).

• Birefringent sperms are

associated with low DFI.
Crippa A, et al HR 2009
 MACS- MAGNETIC ACTIVATED CELL SORTING

One of the early markers of apoptosis is the loss of membrane

integrity, which leads to phospholipid phosphatidylserine
externalization (a molecule with a high affinity for annexin V).
Therefore, annexin V (used as an apoptotic sperm marker)
conjugated with magnetic microspheres, which are exposed to a
magnetic field in an affinity column, can separate apoptotic from
non-apoptotic sperm. This procedure is called magnetic activated
cell sorting (MACS).
Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction: a systematic
review and meta-analysis, Monica Gil J Assist Reprod Genet, 2013
THE CLINICAL UTILITY OF SPERM DNA INTEGRITY TESTING: A
GUIDELINE THE PRACTICE COMMITTEE OF THE AMERICAN
SOCIETY FOR REPRODUCTIVE MEDICINE’ 2013

Sperm DNA damage is more common in infertile men and may

contribute to poor reproductive performance. However, current
methods for assessing sperm DNA integrity do not reliably
predict treatment outcomes and cannot be recommended
routinely for clinical use.
 Contact Us

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