Sperm DNA Fragmentation - Fertility Hospital in Jaipur

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Sperm DNA Fragmentation
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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

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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 

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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.

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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.

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•  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

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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 

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• 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.

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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

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INTRINSIC FACTORS
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• 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.

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• 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.

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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 - )
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• 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 

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• 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.

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• 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).

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• 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.

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• 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.

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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
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Reactive Oxygen Species (ROS) in human semen
determination of a reference rangeJ 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?lt?24.1 RLU/sec/106 sperm
  is acceptable for seminal ROS 

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• 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.

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EXTRINSIC FACTORS
Sperm DNA fragmentation mechanisms of origin,
impact on reproductive outcome, and
analysis Denny Sakkas, fert and steril, 2010
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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

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2 STEP HYPOTHESIS

• Faulty spermatogenesis?
• defective remodeling ?
• DNA more susceptible to
• stress factors.

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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
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• 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.

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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.

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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

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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.

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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 

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• 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.

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• (a) Human ejaculate stained with toluidine blue
  (1) mature sperm heads are light blue (2)
  immature are violet. (b) DNA breakage
  detectionfluorescence in situ hybridization
  (DBDFISH) 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 .

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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

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TUNEL Terminal deoxynucleotidyl transferase dUTP
nick end labeling

• Enzymatic addition of modified
• nucleotides to DNA break

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TUNELLabels SS and DS breaks Measures percent
cells with labeled DNA

• ADVANTAGES
• 1. Fresh or frozen samples
• 2. Can be used for testicular retrieved sperms.
• 3. Can be performed on few sperm
• 4. High repeatability
• 5. Quick and simple ( ?uorescence microscopy)

• DISADVANTAGES
• 1. Variable protocols
• 2. Unclear thresholds
• 3. Not available in commercial kits

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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

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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. 

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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

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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.

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• 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.

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• 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

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SCD

• Advantages
• Commercial assay available
• Interpretation does not depend on fluorescence or
  flow cytometry

• Disadvantages
• Indirect assay only detects ssDNA breaks
• Involves acid denaturation.

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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

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• 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.

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SCSA For single-stranded DNA

• 1. High reproducibility
• 2. Established clinical
• thresholds
• 3. Many cells rapidly
• examined
• 4. Fresh or frozen samples
• 5. Has extensive body of literature and
  established clinical thresholds

• 1. Not available in commercial kits
• 2. Expensive equipment
• 3. only detects ssDNA breaks

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COMPARISION OF DIFFERENT DFI TESTS

• The COMET and Sperm Chromatin Dispersion tests
  were introduced as light microscope tests that
  dont require a flow cytometer. Since these tests
  measure only 50200 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 

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• 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)).

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• 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.

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INTERPRETATION

• Percentage of spermatozoa with fragmented DNA
• 15 good fertility potential
• 15-30 - average
• gt 30 - poor fertility potential

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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.

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Measuring Sperm DNA Fragmentation and Clinical
Outcomes of Medically Assisted Reproduction A
Systematic Review and Meta-AnalysisMaartje
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.

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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 lt 0.00001). A subgroup analysis showed that
  the miscarriage association is strongest for the
  TUNEL assay (RR 3.94 (2.45, 6.32), P lt
  0.00001).

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CURRENT PRACTICE

• Unexplained infertility , normal semen parameters
  with mild or treatable female infertility
• DFI gt 30 - direct IVF/ICSI DFI lt 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 lt15 -female lt35 years, no pathology
  spontaneous pregnancy can be tried
• DFIgt15 - treatable female subfertility
  treatment of the female

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MANAGEMENT OF HIGH DFI

• Oral antioxidants
• Lifestyle modifications(Quit smoking/ weight
  reduction)
• Treatment of underlying condition/ infections
• Consider TESA/TESE ICSI

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• 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

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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. 

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Esteves et al, Int Braz J Urol 2011
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EFFECT OF VARICOLELECTOMY

•  A varicocelectomy can improve sperm
• DNA integrity, with a mean difference of
• -3.37 (95 CI -4.09 to - 2.65 Plt0.00001)
• Meta analysis of seven studies evaluating
• the effect of varicocelectomy repair on SDF
• Wang YJ et al, Reprod Biomed Online. 2012 

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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.

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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

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SPERM SELECTION FOR SAMPLES WITH HIGH DFI
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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)

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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

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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.

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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

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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
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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.

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Jaipur Address -
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