Sperm DNA Fragmentation from the Male Infertility Specialist's Perspective

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


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


Why semen analysis is not enough

Sperm Chromatin Compaction


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 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


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


DNA Damage

SDF in Different Male Infertility Etiologies


Gosálbez et al. 2013

Frequency of Elevated SDF in Men with Normal Semen Analysis (WHO 2010)


36%

Androfert 2013; N=860; SCD test; cutoff value of 20%

SDF and Infertility: Why bother?


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


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


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


•  Repair likely to occur at pronuclei stage (prior syngamy)

•  Low levels breaks can be repaired (especially ss-DBs) •  Repair ability decrease with female age


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.


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


Which is the best method for SDF?

Labor-intensive

Expensive equipment

Analysis Subjectivity

Validation & Standardization

TUNEL ++++ +++ ++ ++ SCSA ++ ++++ + ++++ Comet ++++ ++ +++ ++ SCD + + ++ +++


Esteves et al. Int Urol Nehrol 2014

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


Comparison Between SDF Methods

Fernández et al. 2003, 2005; Gosálvez et al. 2006


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


TUNEL Terminal deoxynucleotidyl transferase dUTP

nick end labeling

Correlation between SCD and TUNEL


•  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


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


•  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


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


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


Henkel R et al, AJA 2007; Alvarez et al. Fertil Steril 2002

25% 34% 39%

Normal Abnormal Abnl & Leukocytospermia

% DNA Damage (SCSA)


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)


Miyaoka & Esteves. Adv Urol 2012 Agarwal, Esteves, Hamada. Nature Urol Rev 2013;

Wang YJ et al. Reprod Biomed Online. 2012.


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)



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)


Effect of Varicocele Surgery on SDF


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


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


Difference in SDF between Testicle and Ejaculate

Laboratory Management

l Abstinence period l Iatrogenic damage l Sperm selection techniques


Abstinence Period


Gosálbez et al. Fertil Steril 2011

Serial ejaculation every 24h for 4 days: 25% reduction SDF

Sperm Processing by DGC


Gosálbez et al. Fertil Steril 2011

Density gradient centrifugation: 22%-44% reduction in SDF

Dynamic Nature of SDF Iatrogenic damage


Zeta method

Modified HOST

IMSI PICSI

Sperm head birefringence

Electrophoretic selection

Magnetic- activated cell sorting

Sperm Selection Techniques & SDF


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


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


Oral antioxidants, life-style modifications (quit smoking, weight loss)


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


Post-processing incubation should not exceed 4h

Sperm processing by DGC

Management Strategies: Laboratory Key Messages

Sperm DNA Fragmentation from the Male Infertility Specialist's Perspective

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