Sperm DNA Fragmentation - IVF Treatment 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.

11. 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.

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 < 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.

32. 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, HalospermAssay) • 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 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 .

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 DISADVANTAGES: 1. Variable protocols 2. Unclear thresholds 3. Not available in commercial kits 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 ( fluorescence microscopy)

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. 

45. CometFor 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

50. SCD Advantages • Commercial assay available • Interpretation does not depend on fluorescence or flow cytometry Disadvantages • Indirect assay only detects ssDNA breaks • Involves acid denaturation.