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V WORKSHOP MALE REPRODUCTIVE BIOLOGY November 2015 – São Paulo – Brazil
CLINICAL UTILITY OF SPERM DNA FRAGMENTATION TESTING IN MALE INFERTILITY TREATMENT
Dr Sandro ESTEVES Medical and Scientific Director ANDROFERT - Andrology & Human Reproduction Clinic Campinas, Brazil
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Learning Objectives
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1. Appraise the role of SDF in male infertility
2. Understand why test SDF and what tests are available
3. Update on the lessons learned from recent clinical research and their implications to practice
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Male infer*lity most common cause of infer*lity nowadays
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Infertile couples worldwide (50-80 million people)
10-15% male factor infertility
Up to 50%
seek assistance for fertility issues
1 in 8 men
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We are only making a marginal improvement in pregnancy and birth rates a;er over 35 years of
trying
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Empirical treatments
Conventional semen analysis
Conventional surgeries ART
Esteves Asian J Androl 2015; Interna0onal Commi6ee Monitoring ART Hum Reprod 2013
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History taking, physical examination, endocrine profile and sperm analysis are minimum standards
Esteves Int Braz J Urol 2014; Lewis MEFSJ 2013
A major reason is we do not understand the causes of male infer*lity at the molecular level
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Semen analysis is s*ll the most widely used biomarker to assess
male fer*lity
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Miyaoka et al. Clinics 2011; Esteves et al. IBJU 2012
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~2,000 semen specimens; recent fathers
Percen*le 5% 50% 95%
Volume (mL) 1.5 3.7 6.8 Count (x106/mL) 15.0 73.0 213.0 Total count (x106) 39.0 255.0 802.0 % Mo*le 40 61 78 % Progressive mo*lity 32 55 72 % Normal (Kruger) 4 15 44 % Alive 58 79 91
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Only 1% of sperm will eventually reach the oocyte in vivo…
so why would we expect an analysis
of the widely ranging gross parameters of the whole ejaculate to
give strong discriminatory information?
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Conven*onal semen analysis is not enough
single-strand break
mis-match
damaged base double-strand
break
inter-strand crosslink
intra-strand crosslink
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Esteves et al Int Urol Nephrol 2014; Gosálvez et al J Reprod Biotech Fertil 2015
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SDF Biological Mechanisms Protamination failure Deficient replacement of histones to protamines during spermiogenesis
Oxidative stress Epididymis transit Post-ejaculation
leukocytes, immature sperm, low levels semen antioxidants
Apoptosis During sperm maturation (testis & epididymis)
Fernández et al. 2009; Alvarez and Sakkas 2010; Agarwal et al. 2013
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Cho, Esteves & Agarwal Asian J Androl 2015 in press
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Sperm DNA fragmenta*on tes*ng associated with early
fer*lity checkpoints • Impaired fertilization • Slow early embryo development, reduced
implantation • Miscarriage and, in animal studies, birth defects
in the offspring • Childhood cancers have also been associated
with oxidative damage to sperm DNA as a consequence of paternal smoking
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Bungum et al. Hum Reprod 2007; Gosálvez et al. 2015
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19%
1.5%
Normal Elevated
Live birth rates with IUI
N=387; OR = 0.07 [95% CI: 0.01-‐0.48]
Bungum et al. Hum Reprod 2007
IUI outcome is nega*vely affected by elevated SDF
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26% 42%
IVF ICSI
Pregnancy in cases of elevated sperm DNA fragmentation
IVF outcome is nega*vely affected by elevated SDF
Robinson et al. Hum Reprod 2012
Meta-‐analysis of 16 studies; 2,969
couples:
Increased miscarriage in ART
RR = 2.16 95% CI: 1.54-‐3.03; p<0.00001
Bungum et al. Hum Reprod 2007
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SDF Main Areas of Clinical Research
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• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
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Current Collabora*ve Research
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ANDROFERT CAMPINAS, BRAZIL
AMERICAN CENTER FOR REPRODUCTIVE MEDICINE, CLEVELAND USA
GENETICS UNIT, DEPARTMENT OF BIOLOGY
UNIVERSIDAD AUTÓNOMA DE MADRID,
SPAIN
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SDF Main Areas of Clinical Research
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• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
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SDF TESTING & VALIDATION
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Fertil Steril 2014; 101(1):58-63
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Enzymatic addition of modified nucleotides to DNA breaks
Sharma et al. Urology 2010
TUNEL Terminal deoxynucleotidyl transferase dUTP
nick end labeling
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Sperm Chromatin Dispersion Test
Susceptibility of DNA to denaturation with formation of single-strand (ss) DNA from pre-existing single or double strand breaks
Protocol involves DNA denaturation and protein depletion
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Fernández et al. 2003, 2005; Gosálvez et al. 2006
Difference in loop (halo) patterns around lysed and acid treated nuclear membrane carcass reflects overall chromatin structure
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Sperm Chromatin Dispersion Test
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Correlation between SCD and TUNEL • SCD more sensitive than
TUNEL • Important to distinguish
what method is used
20.6
11.5
% SDF
SCD TUNEL
Feijo & Esteves. Fertil Steril 2014; 101(1):58-63.
P<0.01
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Diagnostic accuracy of SCD in unexplained infertility
Despite poorly correlated, SCD may discriminate men with
normal and abnormal sperm DNA damage with ~70% accuracy when compared with TUNEL
Feijo & Esteves. Fertil Steril 2014
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Frequency of abnormal SDF levels in men with unexplained infer*lity
N=987
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36%
Androfert 2014
Medium SDF
15-30%
High SDF > 30%
Normal SDF
< 15%
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Protocol, reference values and QC
Sharma, Ahmad, Esteves & Agarwal J Assist Reprod Genet, accepted
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SDF Main Areas of Clinical Research
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• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
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SDF results phenotypes
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Andrologia 2014; 46(6): 602–9
Fragmented
Degraded
Normal
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Andrologia 2014; 46(6): 602–9
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SDF Main Areas of Clinical Research
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 29 2015
ANDROFERT
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• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
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ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 30 2015
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2015; 47(9): 1471-7
G2: recurrent ART failure; G3: leukocytospermia; G4: Chlamydia infection; G5: testicular cancer
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DDSi ≥0.33 AUC: 0.94
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ANDROFERTEsteves et al. 2015; 47(9): 1471-7
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SDF Main Areas of Clinical Research
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• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
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Medical Interven*ons to Decrease
Sperm DNA Fragmenta*on
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• Oral antioxidants • Varicocele surgery • Short ejaculatory abstinence • Testicular sperm retrieval
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ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 34 2015
ANDROFERTJ Urol 2010;184:1442-6
N=242
ICSI
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Wang YJ et al. Reprod Biomed Online 2012;25:307-14.
Seven studies evaluating the effect of varicocele repair on SDF MD = -3.4% (95% CI: -4.1% to -2.6%; p<0.0001)
Varicocele surgery decreases SDF V WMRB
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147 Infer*le men with oligozoospermia (>5 & <15 M/mL) and persistent high SDF (DFI>30%) a;er oral an*oxidants
ICSI with ejaculated
sperm (N=91)
ICSI with tes*cular
sperm (N=81)
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SDF 5-‐fold lower in tes*cular sperm
40.7%
8.3%
Ejaculate Tes*s
P<0.001
Esteves et al Fertil Steril 2015
ICSI with tes*cular sperm (N=81)
Modified SCD test: dual fluorescent cocktail probe
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SDF tes*ng integral element of male infer*lity work-‐up at Androfert
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Clinical u*lity of SDF tes*ng in male infer*lity management – KEY MESSAGES
1. Conventional semen analysis limited value for fertility assessment
2. SDF testing emerged as a tool to assess male infertility at the molecular level
3. Inclusion of testing to male infertility work-up allows better clinical management
Diagnostic value in Unexplained Infertility & Varicocele Biomarker to follow up interventions’ outcome Biomarker to select best ART modality
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Thank you Obrigado
Review presenta*on at hsp://www.slideshare.net/sandroesteves