Your SlideShare is downloading. ×
Delhi & Chennai, India 2014

Sperm DNA Fragmentation
in Male Infertility
Sandro C. Esteves, MD, PhD
Director, ANDROFERT
Ca...
Contents

Role of SDF on fertility
SDF and ART outcome
Methods for SDF assessment
Management Strategies

Esteves, 2

ANDRO...
Semen analysis

Central
Laboratory
Investigation

Esteves, 3

Functional
Status of
Reproductive
Tract

Seminal Fluid
and S...
Why semen analysis is not enough

Esteves, 4

ANDROFERT, Referral Center for Male Reproduction
Sperm Function
Deliver intact DNA into
the oocyte
Intact chromatin is critical
for the production of a
viable pregnancy

D...
Sperm Chromatin Compaction

Esteves, 6

ANDROFERT, Referral Center for Male Reproduction
What are the lesions associated with
Sperm DNA Fragmentation?
Defects in DNA structure:
Single-strand DNA break (ss-DB)
Do...
What are the biological
mechanisms of SDF?
Protamination Failure
Replacement of histone to protamines during
spermiogenesi...
What are the external factors
leading to increased SDF?
Environmental factors
Phtalate exposure, radiation,
temperature

D...
SDF and Male Infertility Etiologies

Gosálbez et al. 2013

Esteves, 10

ANDROFERT, Referral Center for Male Reproduction
Frequency of elevated SDF in men
with normal semen analysis

52

143
Normal semen analysis results (WHO)
Elevated SDF (36....
SDF and Infertility: Why bother?

Esteves, 12

ANDROFERT, Referral Center for Male Reproduction
IUI Outcome and SDF
Live Birth Rates with
Intrauterine Insemination
19%

OR = 0.07
[95% CI: 0.01-0.48]

1.5%
Normal
Esteve...
IVF Outcome and SDF
Pregnancy by Method in
Cases of Elevated Sperm DNA
Fragmentation
42%

Meta-analysis of 16 studies and
...
SDF and reproductive success
Points to consider (1)
Oocyte repair capability and severity
of damage
Repair likely to occur...
SDF and reproductive success
Points to consider (2)
Site of damage
Coding DNA (exons)
represent ~3% of genome
Gosálbez et ...
SDF and Male Infertility
Key Messages (1)
SDF	
  gives	
  different	
  informa4on	
  than	
  rou4ne	
  
semen	
  analysis,	...
What are the methods for SDF
assessment?
Direct
Incorporation of probes at the site of damage
e.g. TUNEL, ISNT

Indirect
S...
Comparison Between SDF Methods

Fertility and Sterility 2014; 101(1):58-63.

Esteves, 19

ANDROFERT, Referral Center for M...
TUNEL

Terminal deoxynucleotidyl transferase dUTP
nick end labeling
Enzymatic addition of modified
nucleotides to DNA brea...
Sperm Chromatin Dispersion (SCD)
Susceptibility of DNA to denaturation
with formation of single-strand (ss)
DNA from pre-e...
Correlation between SCD and TUNEL
SCD more sensitive than
TUNEL.
Important to distinguish
between the methods as they
diff...
Diagnostic accuracy of SCD in men with
unexplained infertility
Despite poorly correlated, SCD
may discriminate men with
no...
Which is the best method for SDF?
Laborintensive

TUNEL
SCSA
Comet
SCD

Esteves, 24

Expensive
equipment

Analysis
Subject...
SDF and Male Infertility
Key Messages (2)
Several	
  methods	
  available	
  to	
  assess	
  SDF	
  
Methods	
  differen4al...
What can we do to reduce SDF?
Antioxidants and life-style
changes
Treatment of underlying condition
Avoid iatrogenic SDF
W...
Oral Antioxidants
Outcome	
  

No.	
  studies	
  

Effect	
  size	
  	
  
(OR;	
  95%	
  CI)	
  

Live	
  birth	
  

3	
  
...
Oral Antioxidants
Beneficial

No effect

Kodama 1997
Dawson, 1992
Kessopoulou, 1995
Vezina, 1996
Vicari, 2001; 2002
Lenzi,...
Oral Antioxidants
How I prescribe
Vitamin C 500mg; Vitamin E 400 mg
Folic acid 2 mg, Zinc 25 mg
Selenium 26 mcg

Minimum 2...
Decrease No. Leukocytes in Semen
% DNA Damage (SCSA) 39%
34%

25%

granulocyte

Normal

Abnormal

macrophage

lymphocyte

...
Treatment of Subclinical GTI and
Associated Inflammatory Changes
Antibiotics

• Yanushpolsky et al, 1995; Erel et al., 199...
Subclinical Male Genital Tract Infection

Anti-bacterial
properties (Zinc)

Azitromycin 1.0g single dose (couple)+ frequen...
Varicocele Surgery

Twelve studies comparing SDF
in pts. with and without
varicocele:
SDF higher in varicocele
Mean differ...
Effect of Varicocele Surgery on SDF

Meta-analysis of seven studies
evaluating the effect of
varicocele repair on SDF
SDF ...
Management

Esteves, 35

ANDROFERT, Referral Center for Male Reproduction
TESA-ICSI and SDF

Sperm

% TUNEL +

% CPR

Ejaculated

23.6

6

Testicular

4.8

44

<0.001

<0.05

P value

Greco et al....
Difference in SDF between
Testicle and Ejaculate
DNA damage in
Testicular
Spermatozoa
three-fold lower
compared with
Ejacu...
TESA-ICSI: ANDROFERT

Esteves, 38

ANDROFERT, Referral Center for Male Reproduction
What can we do to decrease SDF?
Ø  Oral antioxidants
Ø Life-style modifications, including quit
smoking and weight loss
...
Dynamic Nature of SDF
Iatrogenic damage

Esteves, 41

ANDROFERT, Referral Center for Male Reproduction
Abstinence Period and Sperm
Processing

Serial ejaculation every 24h for 4 days:
25% reduction SDF
Density centrifugation ...
Annexin-V microbeads and
MACS columns
Microbeads conjugated with
Annexin-V

Esteves, 43

ANDROFERT, Referral Center for Ma...
Sperm Selection
Annexin-V columns
Parameters

Pre-MACS

Annexin V neg.

Annexin V
pos.

Count

1 x 106/ml

600.000/ml

400...
Laboratory handling of ejaculated
and testicular specimens

Esteves SC & Varghese A, J Hum Reprod Sci 2012
Esteves, 45

AN...
What we can do to avoid iatrogenic SDF
Ø Short abstinence periods (1 day) and serial ejaculation
Ø Instruct patients to ...
Take-home Messages
SDF provides information that is different
and of better prognostic value than
semen analysis.
SDF main...
Take-home Messages
SDF has a negative effect on reproductive
potential, both in vivo and in vitro.
Strategies to reduce SD...
Thank you
Sperm DNA Fragmentation in Male Infertility
Sperm DNA Fragmentation in Male Infertility
Upcoming SlideShare
Loading in...5
×

Sperm DNA Fragmentation in Male Infertility

2,024

Published on

Published in: Health & Medicine
2 Comments
6 Likes
Statistics
Notes
No Downloads
Views
Total Views
2,024
On Slideshare
0
From Embeds
0
Number of Embeds
3
Actions
Shares
0
Downloads
168
Comments
2
Likes
6
Embeds 0
No embeds

No notes for slide

Transcript of "Sperm DNA Fragmentation in Male Infertility"

  1. 1. Delhi & Chennai, India 2014 Sperm DNA Fragmentation in Male Infertility Sandro C. Esteves, MD, PhD Director, ANDROFERT Campinas, Brazil
  2. 2. Contents Role of SDF on fertility SDF and ART outcome Methods for SDF assessment Management Strategies Esteves, 2 ANDROFERT, Referral Center for Male Reproduction
  3. 3. Semen analysis Central Laboratory Investigation Esteves, 3 Functional Status of Reproductive Tract Seminal Fluid and Sperm ANDROFERT, Referral Center for Male Reproduction
  4. 4. Why semen analysis is not enough Esteves, 4 ANDROFERT, Referral Center for Male Reproduction
  5. 5. Sperm Function Deliver intact DNA into the oocyte Intact chromatin is critical for the production of a viable pregnancy DNA is a manual of instructions Esteves, 5 ANDROFERT, Referral Center for Male Reproduction
  6. 6. Sperm Chromatin Compaction Esteves, 6 ANDROFERT, Referral Center for Male Reproduction
  7. 7. What are the lesions associated with Sperm DNA Fragmentation? 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 damaged base single-strand break mis-match double-strand break intra-strand crosslink inter-strand crosslink Esteves et al 2013; Alvarez and Gosálbez 2011; Ward 2011 Esteves, 7 ANDROFERT, Referral Center for Male Reproduction
  8. 8. What are the 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, 8 ANDROFERT, Referral Center for Male Reproduction
  9. 9. What are the external factors leading to increased SDF? Environmental factors Phtalate exposure, radiation, temperature Diseases Varicocele, GTI, fever Life-style Obesity, smoking DNA Damage Aging Kort et al. 2006; Rubes et al 2007; Viloria et al 2007; Esteves & Agarwal 2011 Esteves, 9 ANDROFERT, Referral Center for Male Reproduction
  10. 10. SDF and Male Infertility Etiologies Gosálbez et al. 2013 Esteves, 10 ANDROFERT, Referral Center for Male Reproduction
  11. 11. Frequency of elevated SDF in men with normal semen analysis 52 143 Normal semen analysis results (WHO) Elevated SDF (36.4%) Esteves, 11 ANDROFERT, Referral Center for Male Reproduction
  12. 12. SDF and Infertility: Why bother? Esteves, 12 ANDROFERT, Referral Center for Male Reproduction
  13. 13. IUI Outcome and SDF Live Birth Rates with Intrauterine Insemination 19% OR = 0.07 [95% CI: 0.01-0.48] 1.5% Normal Esteves, 13 Bungum et al. Hum Reprod 2007 Elevated ANDROFERT, Referral Center for Male Reproduction
  14. 14. IVF Outcome and SDF Pregnancy by Method in Cases of Elevated Sperm DNA Fragmentation 42% Meta-analysis of 16 studies and 2,969 couples Increased miscarriage in couples undergoing IVF/ICSI with high sperm DNA damage 26% Risk ratio (RR) = 2.16 95% CI: 1.54-3.03; p<0.00001 IVF ICSI Robinson et al. Hum Reprod 2012 Bungum et al. Hum Reprod 2007 Esteves, 14 ANDROFERT, Referral Center for Male Reproduction
  15. 15. SDF and reproductive success Points to consider (1) Oocyte repair capability and severity of damage Repair likely to occur at pronuclei stage (prior syngamy) Low levels breaks can be repaired (especially ss-DBs) Repair ability decrease with female age Menezo et al 2007; Genescá et al. 1992; Obe et al. 2002 Esteves, 15 ANDROFERT, Referral Center for Male Reproduction
  16. 16. SDF and reproductive success Points to consider (2) Site of damage Coding DNA (exons) represent ~3% of genome Gosálbez et al. 2013; Dada et al. 2012 Esteves, 16 ANDROFERT, Referral Center for Male Reproduction
  17. 17. SDF and Male Infertility Key Messages (1) 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   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   Esteves,  17   ANDROFERT,  Referral  Center  for  Male  Reproduc4on  
  18. 18. What are the methods for SDF assessment? Direct Incorporation of probes at the site of damage e.g. TUNEL, ISNT Indirect Susceptibility of DBs 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, 18 ANDROFERT, Referral Center for Male Reproduction
  19. 19. Comparison Between SDF Methods Fertility and Sterility 2014; 101(1):58-63. Esteves, 19 ANDROFERT, Referral Center for Male Reproduction
  20. 20. TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling Enzymatic addition of modified nucleotides to DNA breaks; Sharma et al. 2010 Esteves, 20 ANDROFERT, Referral Center for Male Reproduction
  21. 21. Sperm Chromatin Dispersion (SCD) Susceptibility of DNA to denaturation with formation of single-strand (ss) DNA from pre-existing single or double strand breaks; Combination of DNA denaturation used in SCSA and protein depletion used in the comet assay; Difference in the pattern of forming a loop (halo) around lysed and acid treated nuclear membrane carcass reflects the overall chromatin structure. Fernández et al. 2003, 2005 Gosálvez et al. 2006 Esteves, 21 ANDROFERT, Referral Center for Male Reproduction
  22. 22. Correlation between SCD and TUNEL SCD more sensitive than TUNEL. Important to distinguish between the methods as they differently evaluate SDF. 20.6 SCD TUNEL 11.5 Fertil Steril 2014; 101(1):58-63 % SDF Esteves, 22 ANDROFERT, Referral Center for Male Reproduction
  23. 23. Diagnostic accuracy of SCD in men with unexplained infertility Despite poorly correlated, SCD may discriminate men with normal and abnormal sperm DNA damage with ~70% accuracy when compared with TUNEL. Fertil Steril 2014; 101(1):58-63 Esteves, 23 ANDROFERT, Referral Center for Male Reproduction
  24. 24. Which is the best method for SDF? Laborintensive TUNEL SCSA Comet SCD Esteves, 24 Expensive equipment Analysis Subjectivity Validation & Standardization ++++ ++ ++++ + +++ ++++ +++ + ++ + +++ ++ ++ ++++ + +++ ANDROFERT, Referral Center for Male Reproduction
  25. 25. SDF and Male Infertility Key Messages (2) Several  methods  available  to  assess  SDF   Methods  differen4ally  assess  SDF  and  cannot   determine  nature  or  e4ology  of  damage   Best  method  yet  to  be  determined   SCD  is  a  quick  and  easy  assay  to  assess  SDF   Esteves,  25   ANDROFERT,  Referral  Center  for  Male  Reproduc4on  
  26. 26. What can we do to reduce SDF? Antioxidants and life-style changes Treatment of underlying condition Avoid iatrogenic SDF 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 Esteves, 26 ANDROFERT, Referral Center for Male Reproduction
  27. 27. Oral Antioxidants 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]   6-­‐16   No  effect   Miscarriage,  sperm   count,  sperm  mo4lity   Showell MG et al. Cochrane Database Syst Rev 2011 Esteves, 27 ANDROFERT, Referral Center for Male Reproduction
  28. 28. Oral Antioxidants Beneficial No effect Kodama 1997 Dawson, 1992 Kessopoulou, 1995 Vezina, 1996 Vicari, 2001; 2002 Lenzi, 2003; 2004 Cavallini, 2004 Comhaire, 2005 Grecco 2005 Menezo 2007 Tremellen 2007 Giovenco, 1987 Moilanen, 1993 Iwanier, 1995 Rolf, 1999 Sigman, 2006 Piomboni 2008 Gil Villa 2009 Esteves, 28 l  Short-term use appear to be safe Detrimental long-term use and high doses; increased mortality in cancer populationbased studies. l  Caution against indiscriminate use of high dosages for long periods Heinonen, 1994 Lonn, 2005 Bjelakovic, 2007 ANDROFERT, Referral Center for Male Reproduction
  29. 29. 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 From initiation of sperm production to ejaculation Old concept ~80 days New concept ~60 days Misell LM et al. J Urol. 2006 Esteves & Agarwal. Novel concepts in male infertility. Int Braz J Urol 2011 Esteves, 29 ANDROFERT, Referral Center for Male Reproduction
  30. 30. Decrease No. Leukocytes in Semen % DNA Damage (SCSA) 39% 34% 25% granulocyte Normal Abnormal macrophage lymphocyte Abnl & Leukocytospermia Endtz test Henkel R et al, AJA 2007; Alvarez et al. Fertil Steril 2002 Esteves, 30 ANDROFERT, Referral Center for Male Reproduction
  31. 31. Treatment of Subclinical GTI and Associated Inflammatory Changes Antibiotics • Yanushpolsky et al, 1995; Erel et al., 1997 • Branigan et al., 1995 Cicloxigenase-2 Inhibitors Antihistamines • Oliva & Mutigner, 2006 Antioxidants Esteves, 31 • Lackner et al., 2006 • Gambera et al., 2007 • Tremellen et al., 2007 • Piombini et al., 2008 ANDROFERT, Referral Center for Male Reproduction
  32. 32. Subclinical Male Genital Tract Infection Anti-bacterial properties (Zinc) 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. Varicocele Surgery 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) Miyaoka & Esteves. Adv Urol 2012 Agarwal, Esteves, Hamada. Nature Urol Rev 2013; Wang YJ et al. Reprod Biomed Online. 2012;25:307-14. Esteves, 33 ANDROFERT, Referral Center for Male Reproduction
  34. 34. Effect of Varicocele Surgery on SDF Meta-analysis of seven studies evaluating the effect of varicocele repair on SDF SDF decreased after repair Mean difference = 3.4% (95% CI: -4.1 to -2.6; p<0.0001) Wang YJ et al. Reprod Biomed Online. 2012;25:307-14. Esteves, 34 ANDROFERT, Referral Center for Male Reproduction
  35. 35. Management Esteves, 35 ANDROFERT, Referral Center for Male Reproduction
  36. 36. TESA-ICSI and SDF Sperm % TUNEL + % CPR Ejaculated 23.6 6 Testicular 4.8 44 <0.001 <0.05 P value Greco et al. Hum Reprod 2005 Esteves, 36 ANDROFERT, Referral Center for Male Reproduction
  37. 37. Difference in SDF between Testicle and Ejaculate DNA damage in Testicular Spermatozoa three-fold lower compared with Ejaculated Spermatozoa* *Absolute differences between two specimens ranging from -3.3% to -56.3%. Moskovtsev et al. Fertil Steril 2010 Esteves, 37 ANDROFERT, Referral Center for Male Reproduction
  38. 38. TESA-ICSI: ANDROFERT Esteves, 38 ANDROFERT, Referral Center for Male Reproduction
  39. 39. TESA-ICSI: ANDROFERT •  93 patients enrolled; Mean age: 37.5 yo. •  SDF (Halosperm®): 39.0% ± 15.4% [range: 21%-88%] •  53 pts. with live birth data (Sept. 2013) 80 60 40 70.5 61.5 53.6 54.8 51 p=0.21 p=0.62 20 p=0.50 25 11.7 CPR (%) Miscarriage (%) 33.3 p=0.70 48.4 25 p=0.17 0 2PN (%) TQE (%) Ejaculate Esteves, 39 LBR (%) TESA ANDROFERT, Referral Center for Male Reproduction
  40. 40. What can we do to decrease SDF? Ø  Oral antioxidants Ø Life-style modifications, including quit smoking and weight loss Ø Identify and treat underlying condition (GTI and varicocele) Ø  Consider TESA-ICSI Esteves, 40 ANDROFERT, Referral Center for Male Reproduction
  41. 41. Dynamic Nature of SDF Iatrogenic damage Esteves, 41 ANDROFERT, Referral Center for Male Reproduction
  42. 42. Abstinence Period and Sperm Processing Serial ejaculation every 24h for 4 days: 25% reduction SDF Density centrifugation post-3h ejaculation: 44% reduction Gosálbez et al. Fertil Steril 2011 One-day abstinence: Reduction in ~90% pts. Esteves, 42 Pons et al. 2013 ANDROFERT, Referral Center for Male Reproduction
  43. 43. Annexin-V microbeads and MACS columns Microbeads conjugated with Annexin-V Esteves, 43 ANDROFERT, Referral Center for Male Reproduction
  44. 44. Sperm Selection Annexin-V columns Parameters Pre-MACS Annexin V neg. Annexin V pos. Count 1 x 106/ml 600.000/ml 400.000/ml Viability 49% 64% 0 Progressive motility 30% 43% 0 SDF 34% 10% 70% Cleaved caspase-3 8% 7% 20% Ø  Annexin-V negative fraction: IVF, ICSI, IUI Ø  Annexin-V positive fraction: discard Esteves, 44 Rawe et al., RBM Online 20:320, 2010 ANDROFERT, Referral Center for Male Reproduction
  45. 45. Laboratory handling of ejaculated and testicular specimens Esteves SC & Varghese A, J Hum Reprod Sci 2012 Esteves, 45 ANDROFERT, Referral Center for Male Reproduction
  46. 46. What we can do to avoid iatrogenic SDF Ø Short abstinence periods (1 day) and serial ejaculation Ø Instruct patients to deliver specimens 1- 2h before ART is to be performed Ø Process specimens as soon as possible Ø Keep specimens at room T using appropriate culture media Ø Incubation time after processing should not exceed 4h Ø Thaw cryopreserved specimens just before performing ART Esteves, 47 ANDROFERT, Referral Center for Male Reproduction
  47. 47. Take-home Messages SDF provides information that is different and of better prognostic value than semen analysis. SDF mainly occur during sperm transit through the epididymis, and it is mediated by ROS. Oocyte can repair ssDNA but not dsDNA damage. Sperm chromatin dispersion (SCD) test is the simplest method to assess SDF in routine clinical practice. Esteves, 48 ANDROFERT, Referral Center for Male Reproduction
  48. 48. Take-home Messages SDF has a negative effect on reproductive potential, both in vivo and in vitro. Strategies to reduce SDF includes antioxidant therapy, treatment of subclinical GTI, varicocele repair, and TESA-ICSI. Avoid iatrogenic damage: short abstinence periods, laboratory sperm selection and proper sperm handling. Esteves, 49 ANDROFERT, Referral Center for Male Reproduction
  49. 49. Thank you

×