Sperm DNA Fragmentation


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Causes and Guidelines for Treatment - A compilation by Dr. Sasikala - Scientific Director

Sperm DNA Fragmentation

  1. 1. Sperm DNA Fragmentation -Causes & Guidelines for Current Treatment Dr. Sasikala Natarajamani MBBS,MMEDSCI(ART) EMB(ACE www.creaconceptions.com
  2. 2. Flow1. Human Sperm Cell2. Structure of Human Sperm Chromatin3. Causes of Sperm DNA Damage4. Type of DNA Damage5. Effect on Reproductive Outcomes6. Tests for Diagnosis7. Usefulness of the Tests8. Guidelines for Current Practice www.creaconceptions.com
  3. 3. The Sperm Cell• Sperm cell 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 www.creaconceptions.com
  4. 4. Sperm Chromatin Structure• Fundamental packaging unit of sperm chromatin is a toroid which has 50-60 kb of DNA• Individual Toroids represent dna loop-domains highly condensed by protamines fixed at nuclear matrix• Toroids are cross linked and further compacted by di - sulphide bonds• Each chromosome is a garland of Toroids www.creaconceptions.com
  5. 5. Sperm Chromatin Structure• During later stages of Spermatogenesis – spermatid nucleus remodelled and condensed• Histones displaced by transition proteins and then by Protamines which are more basic and much smaller than histones (Steger et al, 2000).• Unique associations between the DNA and Protamines.• DNA strands are tightly wrapped around the Protamine molecules forming tight and highly organized loops (Brewer et al, 1999).• Intermolecular and intra-molecular disulphide cross-links between Protamines lead to compaction and stabilization of the sperm nucleus• Nuclear compaction - protect the sperm genome from external stresses (Kosower et al, 1992). www.creaconceptions.com
  6. 6. Sperm Chromatin Structure• Sperm nuclear proteins are predominantly Protamines, to a lesser extent histones- about 15%• In many species – Protamines form 95% of nuclear proteins but in humans – only 85% is Protamine bound and 15% DNA is packaged by histones at specific DNA sequences• Histone bound DNA are less tightly compacted• When the strands are not packed well - long DNA strands susceptible to damage – Sperm DNA fragmentation www.creaconceptions.com
  7. 7. Sperm Chromatin StructureSource : http://molehr.oxfordjournals.org/content/16/1/30/F1.expansion www.creaconceptions.com
  8. 8. Causes of Sperm DNA Damage• Intrinsic factors – Remodeling and Packaging Problems – Protamine Deficiency – Excess ROS • Damage by ROS – Abortive Apoptosis• Extrinsic factors – Chemotherapy – Cigarette smoking – Genital tract inflammation – Testicular hyperthermia – Varicoceles www.creaconceptions.com
  9. 9. Intrinsic Factors www.creaconceptions.com
  10. 10. Remodelling & Packaging Problems• Stage-specific transient DNA strand breaks are introduced during Spermiogenesis• DNA breaks are needed for transient relief of torsional stress, favouring the histone replacement with protamines during the final maturation from round to elongated spermatozoa• These physiological, temporary breaks if not repaired – lead to DNA fragmentation or genetic mutations in the ejaculate (Marcan and Boissoneault 2004) www.creaconceptions.com
  11. 11. Protamine Deficiency• Infertile men – ↑ histone to protamine ratio ( Cho et al 2001, Aoki et al 2005,2006)• Complete lack of protamines in some ( zhang et al 2006)• Deficiency → defective chromatin compaction → ↑ susceptibility• Transgenic animal models with targeted protamine deficiency – show link between protamine deficiency, DNA damage, fertilizing capacity at IVF – (Cho et al 2001, 2003)• Probably linked to defective Spermiogenesis – when protamines are deposited ( Steger et al 2000) www.creaconceptions.com
  12. 12. Excess ROS levels• ROS – Important Physiological role – Modulate Gene & Protein activities vital for Sperm Proliferation• Physiological amounts are controlled by seminal antioxidants – imbalance between ROS production and ability of antioxidants to scavenge• Excess ROS levels - pathological ( Zini et al 1993, Irvine et al 2000)• Excess – generated by morphologically defective sperms (residual cytoplasm in particular) and semen leukocytes → DNA damage www.creaconceptions.com
  13. 13. Damage by ROS• Spermatozoa are prone to damage by free oxygen radicals due to the abundance of poly unsaturated fatty acids which are essential for fertilization• Free oxygen radicals activate caspases and endonucleases which are also induced by physiochemical factors including heat exposure – leading to DNA fragmentation www.creaconceptions.com
  14. 14. Causes - Oxidative Stress www.creaconceptions.com
  15. 15. Abortive Apoptosis• Apoptosis of testicular germ cells occurs throughout life• Prevents over proliferation and selectively aborts abnormal forms (Sinha Hikin and Swerdolff 1999)• Some sperms have initiated but escaped apoptosis - abortive apoptosis ( Sakka et al 2003) www.creaconceptions.com
  16. 16. Extrinsic Factors www.creaconceptions.com
  17. 17. 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 www.creaconceptions.com
  18. 18. 2 Step HypothesisFaulty spermatogenesis→ defective remodeling → DNAmore susceptible to stress factors Aitken & De Iullius 2010 www.creaconceptions.com
  19. 19. Type of DNA damage• Both single stranded breaks as well as double stranded breaks are possible• SSB is easy to repair and better prognosis• SSB – due to unrepaired DNA nicks and ROS• DSB – abortive apoptosis, action of caspases and endonucleases & ROS• DSB – unrepaired – gross alteration to chromosomal structure• DSB – more serious and deleterious impact on development of progeny www.creaconceptions.com
  20. 20. 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 → implantation failure, early miscarriages, diseases in the offspring www.creaconceptions.com
  21. 21. Diagnostic Tests• Comet –single cell gel electrophoresis• Tunnel –terminal deoxy nucleotide transferase mediated UTP nick end-labeling• Sperm chromatin dispersion• Sperm chromatin structure assay www.creaconceptions.com
  22. 22. Diagnostic tests• All these tests label single or double stranded DNA breaks• Comet & Tunel – detect actual DNA strand breaks – measure existing damage• Others measure the susceptibility of DNA to denaturation – formation of single stranded DNA from native double stranded DNA – hence includes potential future damage• SCSA– flow cytometry test - measures the susceptibility of DNA to acid induced denaturation ( Evanson and Jost 1994, Evanson et al ., 2002)• So far only SCSA is the only test with clear and clinically useful cut off levels for calculating male fertility potential www.creaconceptions.com
  23. 23. Diagnostic tests• Most studies define an upper normal level of the percentage of cells with DNA fragmentation.• Unit of measurement – DFI ( DNA fragmentation Index)• Samples with assay results above this threshold percentage are considered to have high DNA fragmentation.• Percentage of spermatozoa with fragmented DNA ≤ 15 – good fertility potential 15-25% - average > 25% - poor fertility potential www.creaconceptions.com
  24. 24. Usefulness of Tests -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.• Assays simply determine the amount of DNA fragmentation - based on the belief that more DNA nicking or fragmentation is pathologic. www.creaconceptions.com
  25. 25. Usefulness of Tests• No conclusive evidence - studies give conclusions ranging from “very useful” to “ not useful” or “more research is needed”• Confusion partly due to lack of standardization of the methodology for dna testing• Low number of patients for studies• Majority of studies on ART do not report on the methods used to achieve a pregnancy( IUI, IVF ICSI) www.creaconceptions.com
  26. 26. Usefulness of tests• Lack of consistency in findings of different investigators – uncertain significance of high DFI Speyer et al.,2010• ASRM practice committee 2006: tests of DNA damage are insufficiently predictive of pregnancy outcome• Meta analysis of Evanson and Wixon 2006 – High DFI has less effect on ICSI than IVF• Meta analysis of Lei et al.,2006 – Tunel assay- high DFI showed significant effect on outcome of IVF not ICSI• SCSA – no effect on IVF of ICSI www.creaconceptions.com
  27. 27. What are the relevant questions?• Do the DNA damage tests predict the chances of achieving a spontaneous pregnancy?• Do they help to choose the appropriate method of ART?• Are these tests immune to intra and individual variation? www.creaconceptions.com
  28. 28. DNA integrity & Spontaneous Pregnancy• Very few studies• Spontaneous pregnancy chances reduce when DFI exceeds 30%• In half of the cases of “unexplained infertility” sperm DNA damage is a contributing factor• In patients with oligo astheno terato zoospermia chance of increased chromatin damage is high www.creaconceptions.com
  29. 29. DNA Testing & Selection of ART• In couples with DFI >27% no pregnancy can be achieved regardless of type of ART Larson-Cook et al., 2003• 2004 – 3 independent studies –DFI >27% -compatible with pregnancy and delivery - ART compensates for poor chromatin quality Virro et al.,2004,Gandini et al., 2004, Bungum et al., 2004 www.creaconceptions.com
  30. 30. Variations in Tests• Normal S/A – inter and intra individual variations• Similarly even in DFI- such variations exist - Keel et al., 2006• Biological background – not clearly known but lifestyle factors play a major role• Individuals with specific genetic polymorphisms may be more susceptible to these environmental pollutants www.creaconceptions.com
  31. 31. Current Practice - Guidelines• Unexplained infertility , normal semen parameters with mild or treatable female infertility• DFI > 25-30% - direct IVF/ICSI• DFI < 30% - treatment of female- spontaneous pregnancy can be tried. – A. Giwercman et al .,2011• Minor impairment of semen parameters• Concentration, Motility, Morphology - below reference range and short period of infertility• DFI <10% -female <35 years, no pathology – spontaneous pregnancy can be tried• DFI>10% - treatable female subfertility – treatment of the female www.creaconceptions.com
  32. 32. Current practice -Guidelines• Couples referred for IUI with standard semen parameters• DFI > 25-30% - direct IVF/ICSI• DFI> 30% - ICSI considered www.creaconceptions.com
  33. 33. Current Practice -GuidelinesGiwercman et al 2011 www.creaconceptions.com
  34. 34. Role of DNA Damage Tests• Role in clinical practice is still widely debated• Proven role in reproductive and environmental research• Majority studies –small patient cohorts• Most studies focus on difference between fertile and infertile patients in percentage of sperms with DNA damage• Do not focus on predictive value of these tests• Powerful indicator of in vivo fertility• Between IVF and ICSI more related to IVF than ICSI• Clinical threshold values established only for SCSA – methodology highly standardized ( low inter and intra laboratory variation) www.creaconceptions.com
  35. 35. Conclusions• Successful human reproduction depends on the inherent integrity of the sperm DNA.• There appears to be a threshold of sperm DNA damage beyond which embryo development and subsequent pregnancy outcome are impaired.• Clinical evidence shows that, sperm DNA damage is detrimental to some reproductive outcomes and that the spermatozoa of infertile men possess substantially more DNA damage than that of fertile men.• Our understanding of the etiology of sperm DNA damage, and the full impact of this sperm defect on reproductive outcomes in humans, still remains rudimentary• Therapies for impaired DNA integrity need to be developed and subsequent improvements in sperm DNA integrity from such therapies need to be correlated with improved reproductive outcomes www.creaconceptions.com.
  36. 36. Thank Youwww.creaconceptions.com