Post on 22-Aug-2014
description
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
Contents
Relationship between SDF and infertility
Methods for SDF assessment
Management Strategies
Esteves, 2 ANDROFERT, Referral Center for Male Reproduction
Semen analysis
Functional Status of
Reproductive Tract
Seminal Fluid and Sperm
Central Laboratory
Investigation
Esteves, 3 ANDROFERT, Referral Center for Male Reproduction
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
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
Esteves, 6 ANDROFERT, Referral Center for Male Reproduction
Why semen analysis is not enough
Sperm Chromatin Compaction
Esteves, 7 ANDROFERT, Referral Center for Male Reproduction
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
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
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
SDF in Different Male Infertility Etiologies
Esteves, 11 ANDROFERT, Referral Center for Male Reproduction
Gosálbez et al. 2013
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%
SDF and Infertility: Why bother?
Esteves, 13 ANDROFERT, Referral Center for Male Reproduction
Esteves et al. Int Urol Nehrol 2014
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
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<0.00001 Bungum et al. Hum Reprod 2007
Esteves, 15 ANDROFERT, Referral Center for Male Reproduction
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
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
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
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
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
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
Fertility and Sterility 2014; 101(1):58-63.
Esteves, 22 ANDROFERT, Referral Center for Male Reproduction
Comparison Between SDF Methods
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;
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
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
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)
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
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
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
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
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
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
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)
Esteves, 34 ANDROFERT, Referral Center for Male Reproduction
Esteves, 35 ANDROFERT, Referral Center for Male Reproduction
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
Esteves, 37 ANDROFERT, Referral Center for Male Reproduction
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
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
Laboratory Management
l Abstinence period l Iatrogenic damage l Sperm selection techniques
Esteves, 40 ANDROFERT, Referral Center for Male Reproduc4on
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
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
Dynamic Nature of SDF Iatrogenic damage
Esteves, 43 ANDROFERT, Referral Center for Male Reproduction
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
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
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
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
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