Clinical Utility of Sperm DNA Fragmentation Testing in Male Infertility Treatment

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

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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

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

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

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

~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

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

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

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

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 TESTING & VALIDATION


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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

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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

V WMRB 2015


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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

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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

V WMRB 2015


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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 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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2015; 47(9): 1471-7

G2: recurrent ART failure; G3: leukocytospermia; G4: Chlamydia infection; G5: testicular cancer

DDSi ≥0.33 AUC: 0.94


ANDROFERTEsteves et al. 2015; 47(9): 1471-7



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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


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


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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)

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


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

Clinical Utility of Sperm DNA Fragmentation Testing in Male Infertility Treatment

Health & Medicine

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