sperm dna fragmentation in male infertility

Sandro C. Esteves, MD, PhD Director, ANDROFERT

Campinas, Brazil

Sperm DNA Fragmentation

in Male Infertility

Delhi & Chennai, India 2014

Contents

Role of SDF on fertility

SDF and ART outcome

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



Why semen analysis is not enough

Sperm Function

Deliver intact DNA into the oocyte Intact chromatin is critical for the production of a viable pregnancy

DNA is a manual of instructions


Sperm Chromatin Compaction


What are the lesions associated with Sperm DNA Fragmentation?

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

What are the 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

What are the external factors leading to increased SDF?

Kort et al. 2006; Rubes et al 2007; Viloria et al 2007; Esteves & Agarwal 2011


DNA Damage

SDF and Male Infertility Etiologies


Gosálbez et al. 2013

Frequency of elevated SDF in men with normal semen analysis


143

52

Normal semen analysis results (WHO) Elevated SDF (36.4%)

SDF and Infertility: Why bother?


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 sperm DNA damage

Risk ratio (RR) = 2.16 95% CI: 1.54-3.03; p<0.00001

Bungum et al. Hum Reprod 2007


SDF and reproductive success 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

Gosálbez et al. 2013; Dada et al. 2012

SDF and reproductive success Points to consider (2)

Site of damage

SDF and Male Infertility Key Messages (1)

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, 17 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

What are the methods for SDF assessment?

Direct Incorporation of probes at the site of damage e.g. TUNEL, ISNT

Indirect Susceptibility of DBs 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


Comparison Between SDF Methods

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


TUNEL Terminal deoxynucleotidyl transferase dUTP

nick end labeling

Enzymatic addition of modified nucleotides to DNA breaks;

Sharma et al. 2010


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;

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

Diagnostic accuracy of SCD in men with unexplained infertility

Despite poorly correlated, SCD may discriminate men with

normal and abnormal sperm DNA damage with ~70% accuracy when compared with TUNEL.


Fertil Steril 2014; 101(1):58-63

Which is the best method for SDF?

Labor-intensive

Expensive equipment

Analysis Subjectivity

Validation & Standardization

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


SDF and Male Infertility Key Messages (2)

Several methods available to assess SDF

Methods differen4ally assess SDF and cannot determine nature or e4ology of damage

Esteves, 25 ANDROFERT, Referral Center for Male Reproduc4on

Best method yet to be determined

SCD is a quick and easy assay to assess SDF

What can we do to reduce SDF?

Antioxidants and life-style changes

Avoid iatrogenic SDF


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

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

• Yanushpolsky et al, 1995; Erel et al., 1997 • Branigan et al., 1995 Antibiotics

• Lackner et al., 2006 • Gambera et al., 2007

Cicloxigenase-2 Inhibitors

• Oliva & Mutigner, 2006 Antihistamines

• Tremellen et al., 2007 • Piombini et al., 2008 Antioxidants

Treatment of Subclinical GTI and Associated Inflammatory Changes


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;25:307-14.


Varicocele Surgery

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)

Effect of Varicocele Surgery on SDF

Wang YJ et al. Reprod Biomed Online. 2012;25:307-14.

Meta-analysis of seven studies evaluating the effect of varicocele repair on SDF

SDF decreased after repair Mean difference = 3.4% (95% CI: -4.1 to -2.6; p<0.0001)


Management


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


DNA damage in Testicular Spermatozoa three-fold lower compared with Ejaculated Spermatozoa*

*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

TESA-ICSI: ANDROFERT


•  93 patients enrolled; Mean age: 37.5 yo. •  SDF (Halosperm®): 39.0% ± 15.4% [range: 21%-88%] •  53 pts. with live birth data (Sept. 2013)

TESA-ICSI: ANDROFERT

70.5 53.6

33.3 25 25

61.5 51 54.8

11.7

48.4

0

20

40

60

80

2PN (%) TQE (%) CPR (%) Miscarriage (%) LBR (%)

Ejaculate TESA


p=0.62 p=0.70

p=0.21 p=0.17

p=0.50

What can we do to decrease SDF?

Ø  Oral antioxidants Ø Life-style modifications, including quit

smoking and weight loss Ø Identify and treat underlying condition

(GTI and varicocele) Ø  Consider TESA-ICSI


Dynamic Nature of SDF Iatrogenic damage


Abstinence Period and Sperm Processing


One-day abstinence: Reduction in ~90% pts. Pons et al. 2013

Gosálbez et al. Fertil Steril 2011

Serial ejaculation every 24h for 4 days: 25% reduction SDF Density centrifugation post-3h ejaculation: 44% reduction

Annexin-V microbeads and MACS columns

Microbeads conjugated with Annexin-V


Parameters Pre-MACS Annexin V neg. Annexin V pos.

Count 1 x 106/ml 600.000/ml 400.000/ml

Viability 49% 64% 0

Progressive motility

30% 43% 0

SDF 34% 10% 70%

Cleaved caspase-3

8% 7% 20%

Sperm Selection Annexin-V columns

Rawe et al., RBM Online 20:320, 2010


Ø  Annexin-V negative fraction: IVF, ICSI, IUI Ø  Annexin-V positive fraction: discard

Esteves SC & Varghese A, J Hum Reprod Sci 2012

Laboratory handling of ejaculated and testicular specimens


What we can do to avoid iatrogenic SDF

Ø Short abstinence periods (1 day) and serial ejaculation Ø Instruct patients to deliver specimens 1- 2h before

ART is to be performed Ø Process specimens as soon as possible Ø Keep specimens at room T using appropriate culture

media Ø Incubation time after processing should not exceed 4h Ø Thaw cryopreserved specimens just before performing

ART


Take-home Messages


Sperm chromatin dispersion (SCD) test is the simplest method to assess SDF in routine clinical practice.

SDF provides information that is different and of better prognostic value than semen analysis.

SDF mainly occur during sperm transit through the epididymis, and it is mediated by ROS. Oocyte can repair ssDNA but not dsDNA damage.

Take-home Messages


Strategies to reduce SDF includes antioxidant therapy, treatment of subclinical GTI, varicocele repair, and TESA-ICSI.

SDF has a negative effect on reproductive potential, both in vivo and in vitro.

Avoid iatrogenic damage: short abstinence periods, laboratory sperm selection and proper sperm handling.

Thank you

sperm dna fragmentation in male infertility

Health & Medicine