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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
Learning Objectives
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 2 2015
ANDROFERT
V WMRB 2015
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
Male infer*lity most common cause of infer*lity nowadays
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 3 2015
ANDROFERT
V WMRB 2015
Infertile couples worldwide (50-80 million people)
10-15% male factor infertility
Up to 50%
seek assistance for fertility issues
1 in 8 men
We are only making a marginal improvement in pregnancy and birth rates a;er over 35 years of
trying
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 4 2015
ANDROFERT
V WMRB 2015
Empirical treatments
Conventional semen analysis
Conventional surgeries ART
Esteves Asian J Androl 2015; Interna0onal Commi6ee Monitoring ART Hum Reprod 2013
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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 5 2015
ANDROFERT
V WMRB 2015
Semen analysis is s*ll the most widely used biomarker to assess
male fer*lity
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 6 2015
ANDROFERT
V WMRB 2015
Miyaoka et al. Clinics 2011; Esteves et al. IBJU 2012
~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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 7 2015
ANDROFERT
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?
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 8 2015
ANDROFERT
V WMRB 2015
Conven*onal semen analysis is not enough
single-strand break
mis-match
damaged base double-strand
break
inter-strand crosslink
intra-strand crosslink
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 9 2015
ANDROFERT
V WMRB 2015
Esteves et al Int Urol Nephrol 2014; Gosálvez et al J Reprod Biotech Fertil 2015
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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 10 2015
ANDROFERT
V WMRB 2015
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 11 2015
ANDROFERT
V WMRB 2015
Cho, Esteves & Agarwal Asian J Androl 2015 in press
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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 12 2015
ANDROFERT
V WMRB 2015
Bungum et al. Hum Reprod 2007; Gosálvez et al. 2015
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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 13 2015
ANDROFERT
V WMRB 2015
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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 14 2015
ANDROFERT
V WMRB 2015
SDF Main Areas of Clinical Research
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 15 2015
ANDROFERT
V WMRB 2015
• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
Current Collabora*ve Research
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 16 2015
ANDROFERT
V WMRB 2015
ANDROFERT CAMPINAS, BRAZIL
AMERICAN CENTER FOR REPRODUCTIVE MEDICINE, CLEVELAND USA
GENETICS UNIT, DEPARTMENT OF BIOLOGY
UNIVERSIDAD AUTÓNOMA DE MADRID,
SPAIN
SDF Main Areas of Clinical Research
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 17 2015
ANDROFERT
V WMRB 2015
• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
SDF TESTING & VALIDATION
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 18 2015
ANDROFERT
V WMRB 2015
Fertil Steril 2014; 101(1):58-63
Enzymatic addition of modified nucleotides to DNA breaks
Sharma et al. Urology 2010
TUNEL Terminal deoxynucleotidyl transferase dUTP
nick end labeling
V WMRB 2015
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 19 2015
ANDROFERT
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
V WMRB 2015
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 20 2015
ANDROFERT
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
V WMRB 2015
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 21 2015
ANDROFERT
Sperm Chromatin Dispersion Test
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
V WMRB 2015
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 22 2015
ANDROFERT
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
V WMRB 2015
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 23 2015
ANDROFERT
Frequency of abnormal SDF levels in men with unexplained infer*lity
N=987
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 24 2015
ANDROFERT
36%
Androfert 2014
Medium SDF
15-30%
High SDF > 30%
Normal SDF
< 15%
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 25 2015
ANDROFERT
V WMRB 2015 TUNEL using benchtop flow cytometer:
Protocol, reference values and QC
Sharma, Ahmad, Esteves & Agarwal J Assist Reprod Genet, accepted
SDF Main Areas of Clinical Research
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 26 2015
ANDROFERT
V WMRB 2015
• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
SDF results phenotypes
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 27 2015
ANDROFERT
V WMRB 2015
Andrologia 2014; 46(6): 602–9
Fragmented
Degraded
Normal
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 28 2015
ANDROFERT
Andrologia 2014; 46(6): 602–9
SDF Main Areas of Clinical Research
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 29 2015
ANDROFERT
V WMRB 2015
• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 30 2015
ANDROFERT
2015; 47(9): 1471-7
G2: recurrent ART failure; G3: leukocytospermia; G4: Chlamydia infection; G5: testicular cancer
DDSi ≥0.33 AUC: 0.94
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 31 2015
ANDROFERTEsteves et al. 2015; 47(9): 1471-7
SDF Main Areas of Clinical Research
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 32 2015
ANDROFERT
V WMRB 2015
• Tests development & validation • SDF testing results interpretation • Differential expression of SDF in
various subgroups of infertile men
• Role of interventions to decrease SDF
Medical Interven*ons to Decrease
Sperm DNA Fragmenta*on
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 33 2015
ANDROFERT
V WMRB 2015
• Oral antioxidants • Varicocele surgery • Short ejaculatory abstinence • Testicular sperm retrieval
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 34 2015
ANDROFERTJ Urol 2010;184:1442-6
N=242
ICSI
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
2015
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 35 2015
ANDROFERT
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 36 2015
ANDROFERT
V WMRB 2015
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)
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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 38 2015
ANDROFERT
V WMRB 2015
SDF tes*ng integral element of male infer*lity work-‐up at Androfert
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 39 2015
ANDROFERT
V WMRB 2015
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
ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 40 2015
ANDROFERTANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 40 2015
ANDROFERT
V WMRB 2015
Thank you Obrigado
Review presenta*on at hsp://www.slideshare.net/sandroesteves