Sperm DNA Fragmentation from the Male Infertility Specialist's Perspective

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Sperm DNA Fragmentation from the Male Infertility Specialist's Perspective

  1. 1. Sandro C. Esteves, MD., PhD. Director, ANDROFERT Campinas, Brazil         Sperm DNA Fragmentation from a Male Infertility Specialist’s Perspective Centre for Reproductive Health, Daresbury, United Kingdom July 2014
  2. 2. Contents Relationship between SDF and infertility Methods for SDF assessment Management Strategies Esteves, 2 ANDROFERT, Referral Center for Male Reproduction
  3. 3. Semen analysis Functional Status of Reproductive Tract Seminal Fluid and Sperm Central Laboratory Investigation Esteves, 3 ANDROFERT, Referral Center for Male Reproduction
  4. 4. New WHO Reference Values Caution to Interpret Results ANDROFERT androfert.com.br ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 4 2014 APRIL ANDROFERT
  5. 5. Volume (mL) 1.5 Sperm count (x106/mL) 15.0 Total count (x106) 39.0 % Motile (total) 40 % Motile (progressive) 32 % Normal (strict criteria) 4 %Alive 58 Cooper et al. Hum Reprod Update 2010 WHO 2010: Recent fathers TTP ≤ 1 year Percentiles 5% 50%* 95% 3.7 6.8 73.0 213.0 255.0 802.0 61 78 55 72 15 44 79 91 ANDROFERT androfert.com.br ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 5 2014 APRIL ANDROFERT
  6. 6. Esteves, 6 ANDROFERT, Referral Center for Male Reproduction Why semen analysis is not enough
  7. 7. Sperm Chromatin Compaction Esteves, 7 ANDROFERT, Referral Center for Male Reproduction
  8. 8. Lesions Associated with Sperm DNA Damage single-strand break mis-match damaged base double-strand break inter-strand crosslink intra-strand crosslink 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 Esteves, 8 ANDROFERT, Referral Center for Male Reproduction Esteves et al 2013; Alvarez and Gosálbez 2011; Ward 2011
  9. 9. Biological Mechanisms of SDF Protamination Failure Replacement of histone to protamines during spermiogenesis Oxidative Stress Epididymis transit Post-ejaculation: leukocytes, immature sperm, abnormal levels seminal plasma antioxidants Apoptosis During sperm maturation (testis & epididymis) Fernández et al. 2009; Alvarez and Sakkas 2010; Agarwal et al. 2013 Esteves, 9 ANDROFERT, Referral Center for Male Reproduction
  10. 10. Environmental factors Phtalate exposure, radiation, temperature Diseases Varicocele, GTI, fever Life-style Obesity, smoking Aging External factors leading to increased SDF Kort et al. 2006; Rubes et al 2007; Viloria et al 2007; Esteves & Agarwal 2011 Esteves, 10 ANDROFERT, Referral Center for Male Reproduction DNA Damage
  11. 11. SDF in Different Male Infertility Etiologies Esteves, 11 ANDROFERT, Referral Center for Male Reproduction Gosálbez et al. 2013
  12. 12. Frequency of Elevated SDF in Men with Normal Semen Analysis (WHO 2010) Esteves, 12 ANDROFERT, Referral Center for Male Reproduction 36% Androfert 2013; N=860; SCD test; cutoff value of 20%
  13. 13. SDF and Infertility: Why bother? Esteves, 13 ANDROFERT, Referral Center for Male Reproduction Esteves et al. Int Urol Nehrol 2014
  14. 14. 19% 1.5% Normal Elevated Live Birth Rates with Intrauterine Insemination OR = 0.07 [95% CI: 0.01-0.48] Bungum et al. Hum Reprod 2007 IUI Outcome and SDF Esteves, 14 ANDROFERT, Referral Center for Male Reproduction
  15. 15. 26% 42% IVF ICSI Pregnancy by Method in Cases of Elevated Sperm DNA Fragmentation IVF Outcome and SDF Robinson et al. Hum Reprod 2012 Meta-analysis of 16 studies and 2,969 couples Increased miscarriage in couples undergoing IVF/ICSI with high SDF Risk ratio (RR) = 2.16 95% CI: 1.54-3.03; p<0.00001Bungum et al. Hum Reprod 2007 Esteves, 15 ANDROFERT, Referral Center for Male Reproduction
  16. 16. SDF and Reproductive Outcome Points to consider (1) Oocyte repair capability and severity of damage Menezo et al 2007; Genescá et al. 1992; Obe et al. 2002 Esteves, 16 ANDROFERT, Referral Center for Male Reproduction •  Repair likely to occur at pronuclei stage (prior syngamy) •  Low levels breaks can be repaired (especially ss-DBs) •  Repair ability decrease with female age
  17. 17. Esteves, 17 ANDROFERT, Referral Center for Male Reproduction Coding DNA (exons) represent ~3% of genome Esteves et al. 2014; Dada et al. 2012 SDF and Reproductive Outcome Points to consider (2) Site of damage
  18. 18. Viable pregnancies can be obtained in cases of high SDF but… l SDF is promutagenic mutations could rise after fertilization as the oocyte attempts to repair damage before first cleavage l Mutations will be fixed in the germline associated with infertility, childhood cancer in the offspring and for a higher risk of imprinting diseases Pang MG et al Hum Reprod 2005 Burrello et al Cytogenet Genome Res 2005. Esteves, 18 ANDROFERT, Referral Center for Male Reproduction
  19. 19. Relationship between SDF and Infertiity Key Messages SDF  gives  different  informa4on  than  rou4ne   semen  analysis,  and  of  be;er  prognos4c  value   SDF  is  mainly  oxida4ve  stress-­‐mediated  during   sperm  transit  through  the  epididymis   Esteves,  19   ANDROFERT,  Referral  Center  for  Male  Reproduc4on   Elevated  SDF  associated  with  infer4lity,  poor   ART  outcome  and  miscarriage   Reproduc4ve  outcome  related  to  oocyte  repair   capacity  as  well  as  severity  and  site  of  DNA   damage  
  20. 20. Methods of SDF Assessment Direct Incorporation of probes at the site of damage e.g. TUNEL, ISNT Indirect Susceptibility of DsDNA to denature in a acid solution e.g. Sperm chromatin structure assay (SCSA), sperm chromatin dispersion test (SCD), Comet assay Chromatin compaction Incorporation of probes to nuclear proteins e.g. Aniline blue, toluidine blue Gosálbez et al 2013; Esteves & Agarwal 2011; Esteves et al. 2013 Esteves, 20 ANDROFERT, Referral Center for Male Reproduction
  21. 21. Which is the best method for SDF? Labor- intensive Expensive equipment Analysis Subjectivity Validation & Standardization TUNEL ++++ +++ ++ ++ SCSA ++ ++++ + ++++ Comet ++++ ++ +++ ++ SCD + + ++ +++ Esteves, 21 ANDROFERT, Referral Center for Male Reproduction Esteves et al. Int Urol Nehrol 2014
  22. 22. Fertility and Sterility 2014; 101(1):58-63. Esteves, 22 ANDROFERT, Referral Center for Male Reproduction Comparison Between SDF Methods
  23. 23. Fernández et al. 2003, 2005; Gosálvez et al. 2006 Esteves, 23 ANDROFERT, Referral Center for Male Reproduction Sperm Chromatin Dispersion (SCD) Susceptibility of DNA to denaturation with formation of single-strand (ss) DNA from pre-existing single or double strand breaks; Difference in the pattern of forming a loop (halo) around lysed and acid treated nuclear membrane carcass reflects the overall chromatin structure. Combination of DNA denaturation used in SCSA and protein depletion used in the comet assay;
  24. 24. Enzymatic addition of modified nucleotides to DNA breaks; Sharma et al. 2010 Esteves, 24 ANDROFERT, Referral Center for Male Reproduction TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
  25. 25. Correlation between SCD and TUNEL Esteves, 25 ANDROFERT, Referral Center for Male Reproduction •  SCD more sensitive than TUNEL •  Important to distinguish between the methods as they differently evaluate SDF 20.6 11.5 % SDF SCD TUNEL Fertil Steril 2014; 101(1):58-63. P<0.01
  26. 26. Several  methods  available  to  assess  SDF   Methods  differen4ally  assess  SDF  and  cannot   determine  nature  or  e4ology  of  damage   Esteves,  26   ANDROFERT,  Referral  Center  for  Male  Reproduc4on   Best  method  yet  to  be  determined   Standardiza4on  and  EQC  is  needed   Key Messages (2)
  27. 27. What can we do about SDF? •  Antioxidants and life-style changes •  Avoid iatrogenic SDF during lab sperm handling Esteves, 27 ANDROFERT, Referral Center for Male Reproduction •  Treatment of underlying condition Wong et al., 2000; Wong et al. 2002; Comhaire and Mahmoud, 2003; Agarwal and Said, 2004; Bansal and Bilaspuri, 2010; Gosálbez et al. 2009, 2011; Esteves et al. 2011; Sánchez-Martín et al 2013 •  Short abstinence period •  Sperm selection techniques
  28. 28. Outcome   No.  studies   Effect  size     (OR;  95%  CI)   Live  birth   3   4.85  [1.92,  12.24]   Pregnancy  rate     15   4.18  [2.65,  6.59]   DNA  fragmenta4on   1   -­‐13.80  [-­‐17.50,  -­‐10.10]   Miscarriage,  sperm   count,  sperm  mo4lity   6-­‐16   No  effect   Oral Antioxidants Showell MG et al. Cochrane Database Syst Rev 2011 Esteves, 28 ANDROFERT, Referral Center for Male Reproduction
  29. 29. Oral Antioxidants l Short-term use appear to be safe l Caution against indiscriminate use of high dosages for long periods Beneficial Kodama 1997 Dawson, 1992 Kessopoulou, 1995 Vezina, 1996 Vicari, 2001; 2002 Lenzi, 2003; 2004 Cavallini, 2004 Comhaire, 2005 Grecco 2005 Menezo 2007 Tremellen 2007 Piomboni 2008 Gil Villa 2009 No effect Giovenco, 1987 Moilanen, 1993 Iwanier, 1995 Rolf, 1999 Sigman, 2006 Detrimental long-term use and high doses; increased mortality in cancer population- based studies. Heinonen, 1994 Lonn, 2005 Bjelakovic, 2007 Esteves, 29 ANDROFERT, Referral Center for Male Reproduction
  30. 30. Oral Antioxidants How I prescribe Vitamin C 500mg; Vitamin E 400 mg Folic acid 2 mg, Zinc 25 mg Selenium 26 mcg Minimum 2 months Old concept ~80 days New concept ~60 days From initiation of sperm production to ejaculation Misell LM et al. J Urol. 2006 Esteves & Agarwal. Novel concepts in male infertility. Int Braz J Urol 2011 Esteves, 30 ANDROFERT, Referral Center for Male Reproduction
  31. 31. Henkel R et al, AJA 2007; Alvarez et al. Fertil Steril 2002 25% 34% 39% Normal Abnormal Abnl & Leukocytospermia % DNA Damage (SCSA) Esteves, 31 ANDROFERT, Referral Center for Male Reproduction Decrease No. Leukocytes in Semen granulocyte macrophage lymphocyte Endtz test
  32. 32. Anti-bacterial properties (Zinc) Subclinical Male Genital Tract Infection Azitromycin 1.0g single dose (couple)+ frequent ejaculation (every 2-3 days) + Antioxidants: •  42% leukocytospermia resolution (N=278) Esteves, 32 ANDROFERT, Referral Center for Male Reproduction
  33. 33. Miyaoka & Esteves. Adv Urol 2012 Agarwal, Esteves, Hamada. Nature Urol Rev 2013; Wang YJ et al. Reprod Biomed Online. 2012. Esteves, 33 ANDROFERT, Referral Center for Male Reproduction Varicocele Twelve studies comparing SDF in pts. with and without varicocele: SDF higher in varicocele Mean difference = 9.9% (95% CI: 9.2-10.5; p<0.0001)
  34. 34. Esteves, 34 ANDROFERT, Referral Center for Male Reproduction
  35. 35. Esteves, 35 ANDROFERT, Referral Center for Male Reproduction
  36. 36. Wang YJ et al. Reprod Biomed Online. 2012;25:307-14. Seven studies evaluating the effect of varicocele repair SDF decreased after repair Mean difference = 3.4% (95% CI: -4.1 to -2.6; p<0.0001) Esteves, 36 ANDROFERT, Referral Center for Male Reproduction Effect of Varicocele Surgery on SDF
  37. 37. Esteves, 37 ANDROFERT, Referral Center for Male Reproduction
  38. 38. Sperm % TUNEL + % CPR Ejaculated 23.6 6 Testicular 4.8 44 P value <0.001 <0.05 Greco et al. Hum Reprod 2005 TESA-ICSI and SDF Esteves, 38 ANDROFERT, Referral Center for Male Reproduction
  39. 39. SDF three- fold lower in testicular sperm* *Absolute differences between two specimens ranging from -3.3% to -56.3%. Moskovtsev et al. Fertil Steril 2010 Esteves, 39 ANDROFERT, Referral Center for Male Reproduction Difference in SDF between Testicle and Ejaculate
  40. 40. Laboratory Management l Abstinence period l Iatrogenic damage l Sperm selection techniques Esteves,  40   ANDROFERT,  Referral  Center  for  Male  Reproduc4on  
  41. 41. Abstinence Period Esteves, 41 ANDROFERT, Referral Center for Male Reproduction Gosálbez et al. Fertil Steril 2011 Serial ejaculation every 24h for 4 days: 25% reduction SDF
  42. 42. Sperm Processing by DGC Esteves, 42 ANDROFERT, Referral Center for Male Reproduction Gosálbez et al. Fertil Steril 2011 Density gradient centrifugation: 22%-44% reduction in SDF
  43. 43. Dynamic Nature of SDF Iatrogenic damage Esteves, 43 ANDROFERT, Referral Center for Male Reproduction
  44. 44. Zeta method Modified HOST IMSI PICSI Sperm head birefringence Electrophoretic selection Magnetic- activated cell sorting Sperm Selection Techniques & SDF Esteves, 44 ANDROFERT, Referral Center for Male Reproduction
  45. 45. Current non-invasive sperm selection techniques are limited in their ability to select chromatin-intact spermatozoa None of the available techniques can directly assess sperm DNA fragmentation Dyes are needed to reach the nucleus, using fixed specimens Sperm Selection Techniques & SDF Esteves, 45 ANDROFERT, Referral Center for Male Reproduction
  46. 46. SDF and Embryo Quality Reference Assay N ART Findings Larson et al., 2000 SCSA 24 IVF; ICSI No impact (D3) Gandini et al., 2004 SCSA 34 IVF; ICSI No impact (D3) Payne et al., 2006 SCSA 100 IVF; ICSI No impact (D3) Sun et al., 1997 TUNEL 236 IVF ↓ Cleavage (D3) Morris et al., 2002 Comet 60 ICSI ↓ Cleavage; Morphology (D3) Virro et al., 2004 SCSA 249 IVF; ICSI ↓ Blastulation rate Nasr-Esfahani et al., 2005 Comet, CMA3 28 ICSI ↓ Blastocyst development Esteves, 46 ANDROFERT, Referral Center for Male Reproduction
  47. 47. Oral antioxidants, life-style modifications (quit smoking, weight loss) Esteves,  47   ANDROFERT,  Referral  Center  for  Male  Reproduc4on   TESA-ICSI for men with high SDF enrolled in ART Identify and treat underlying condition such as GTI and clinical varicocele Management Strategies: Clinical Key Messages
  48. 48. Short abstinence (1 day) and frequent ejaculation Esteves,  48   ANDROFERT,  Referral  Center  for  Male  Reproduc4on   Post-processing incubation should not exceed 4h Sperm processing by DGC Management Strategies: Laboratory Key Messages

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