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Title: Sperm DNA Fragmentation - Fertility Hospital in Jaipur


1
Sperm DNA Fragmentation
2
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

3
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 

4
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.

5
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.

6
  •  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

7
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 

8
  • 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

12
INTRINSIC FACTORS
13
  • 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.

14
  • 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.

15
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 - )
16
  • 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 

17
  • 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.

18
  • 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).

19
  • 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.

20
  • 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.

21
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
22
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 

23
  • 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.

24
EXTRINSIC FACTORS
Sperm DNA fragmentation mechanisms of origin,
impact on reproductive outcome, and
analysis Denny Sakkas, fert and steril, 2010
25
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

26
2 STEP HYPOTHESIS
  • Faulty spermatogenesis?
  • defective remodeling ?
  • DNA more susceptible to
  • stress factors.

27
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
28
  • 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.

29
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

33
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.

34
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 

35
  • 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.

36
  • (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 .

37
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

38
TUNEL Terminal deoxynucleotidyl transferase dUTP
nick end labeling
  • Enzymatic addition of modified
  • nucleotides to DNA break

39
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

40
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

41
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

46
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.

47
  • 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.

48
  • 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.

51
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

52
  • 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.

53
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

54
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 

55
  • 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)).

56
  • 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

59
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.

60
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.

61
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).

62
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

63
MANAGEMENT OF HIGH DFI
  • Oral antioxidants
  • Lifestyle modifications(Quit smoking/ weight
    reduction)
  • Treatment of underlying condition/ infections
  • Consider TESA/TESE ICSI

64
  • 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. 

67
Esteves et al, Int Braz J Urol 2011
68
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 

69
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.

70
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

71
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)

73
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

74
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.

75
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

76
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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Contact Us
Vasundharafertility.com/jaipur/
Hospital Website -
Facebook Page -
Facebook.com/vasundharahospitaljaipur
Jaipur Address -
B-9 (D), Govind Marg, Adarsh Nagar, Jaipur
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