Sandro Esteves, MD, PhD

Director, ANDROFERT Center for Male Reproduction and Infertility

Campinas, BRAZIL

“Meet the Expert” – May 2012

What is in it for me?

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Lecture Overview

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Antioxidants: To whom and how

00,5

11,5

22,5

Fertile Infertile

Seminal Reactive Oxygen

Species (ROS) (Log ROS + 1; cpm)

Pasqualotto et al., Fertil Steril 2000

Evidence-based Use of Antioxidants in Male Infertility

Author Antioxidant Agent Results Geva et al., 1996 Vit E 200 mg Increased fertilization in IVF Suleiman et al, 1996 Vit E 100 mg Decreased ROS; increased

spontaneous PR Wong et al., 2002 Folic acid 5 mg + Zinc

66 mg Increased total sperm count

Greco et al., 2005 Vit C 1.0 g + E 1.0 g Improved sperm DNA integrity Greco et al., 2005 Vit C 1.0 g + Vit E 1.0 g Increased CPR and IR in ICSI

cycles Tremellen et al., 2007

Menevit® (vit C + E; zinc 25 mg; selenium 26

mcg; lycopene 6 mg)

Increased IR/PR in IVF/ICSI cycles

Boxmeer et al., 2009 Decreased folate in seminal plasma

Increased sperm DNA fragmentation

Antioxidant Treatment Cochrane Review 2011

Outcome N studies

N participants

Effect size (OR; 95% CI)

Live birth 3 214 4.85 [1.92, 12.24]

Pregnancy rate 15 964 4.18 [2.65, 6.59]

DNA fragmentation 1 64 -13.80 [-17.50, -10.10]

Miscarriage, sperm count, sperm motility

6-16 242-700 No effect

Adverse effects 6 426 No effect

Improve the outcomes of live birth and pregnancy rate for subfertile couples undergoing ART cycles

Showell MG, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. Cochrane Database of

Systematic Reviews 2011, Issue 1. Art. No.: CD007411. DOI: 10.1002/14651858.CD007411.pub2.

Antioxidants in Male Infertility

To whom? Everyone

How? q.d. Vitamic C 500mg Vitamin E 400 UI Folic acid 2 mg Zinc 25 mg Selenium 26 mcg

How long?

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Esteves et al. What the gynecologist should know about male infertility: an update. Arch Gynecol Obstet 2012; Epub March 6

From Initiation of Sperm Production to Ejaculation

Misell LM et al.: A stable isotope-mass spectrometric method for measuring human spermatogenesis kinetics in vivo.

J Urol. 2006; 175: 242-6.

Old concept ~80 days

New concept ~60 days

Semen analysis: New WHO Standards

Semen Parameter WHO 1999 WHO 20101

Volume (mL) ≥2.0 1.5 Count (x106/mL) ≥20 15 Total sperm number per ejaculate ≥40 39 Motility (%) ≥50 (a+b) 32 (a+b) Vitality (%) ≥75 58 Morphology (%)2 (14) 4 Leukocytes (x106/mL) <1.0 <1.0

1Lower Limit (5% percentile), Recent fathers; 2Strict criteria Grade a = rapid progressive motility; Grade b = slow/sluggish progressive motility

Centiles 5% 50%* 95% Volume (mL) 1.5 3.7 6.8 Sperm count per mL (x106) 15.0 73.0 213.0 Sperm count per ejaculate (x106) 39.0 255.0 802.0 % Motility (total) 40 61 78 % Motility (progressive) 32 55 72 % Normal (strict criteria) 4 15 44 % Alive (eosin-nigrosin staining) 58 79 91

Cooper et al: World Health Organization reference values for human semen characteristics. Hum Reprod Update 16: 231-245, 2010

Percentile distribution of semen characteristics values of recent fathers (1,953) whose partners had a TTP ≤ 12

months, used to establish the reference limits in the 2010 WHO manual

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New WHO standards Should we adopt them?

Critical Appraisal of the WHO New Reference Values for Human Semen and Impact on Diagnosis

and Treatment of Subfertile Men

Sandro Esteves, BRAZIL Armand Zini, CANADA Nabil Aziz, UNITED KINGDOM Juan Alvarez, SPAIN Edmund Sabanegh, USA Ashok Agarwal, USA

Urology 2012 Jan;79(1):16-22.

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New WHO standards Critical Appraisal - Summary

Reasons for lower cutt-off

Merits Demerits

Different way of generating data: • Method for semen

analysis (higher QC standards; strict morphology)

• Population studied

Controlled studies No systematic review of fertile populations: • Not representative of

global fertile male population

Recent fathers with known TTP

Standardized semen analysis

Morphology using different criteria

Single semen specimen of each individual

Critical Appraisal of the WHO New Reference Values for Human Semen and Impact on Diagnosis and Treatment of Subfertile Men

Esteves, Zini, Aziz et al, Urology, in press

Columbia, MinneapolisUSA

Melbourne Australia

Turku Finland

Oslo Norway

Edimburgh UK

Paris France

Copenhagen Denmark

New WHO Standards: Implications • Reclassification of semen analysis reports

Abnormal

results WHO 1999

Reclassified as “Normal” WHO 2010

(38.7%)

Couples (N=987) with infertility duration > 12 months

Source: ANDROFERT, Brazil

Morphology results accounted for 53% of reclassification

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New WHO Standards Not accurate to discriminate fertile and

infertile men

A comprehensive infertility workup,

including sperm function testing, is crucial to

assess the male fertility potential

Esteves, Miyaoka & Agarwal. An update on the initial assessment of the infertile male.

CLINICS 2011; 66:1-10.

New Diagnostic Tests • Beyond routine semen analysis

• Sperm DNA Integrity Testing

• Y Chromosome Microdeletion Screening

Esteves SC & Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37:5-15.

Sperm DNA Integrity Testing

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• Normal sperm chromatin essential for paternal genetic transmission Background

• Infertility • Recurrent pregnancy loss • Poor outcomes in IUI and IVF

Sperm DNA Damage

• Quantification of sperm DNA strand breaks Principle

• Semen Specimen

• Nuclear dyes (Acridine orange, SCSA) • Direct assessment (TUNEL, COMET) • Nuclear matrix assays (SCD)

Techniques

Sperm DNA Integrity Testing & ART

0%

5%

10%

15%

20%

DFI ≤30% DFI >30%

Pregnancy by Sperm DNA Integrity Results in

IUI

Live birth (%)

OR 0.07 (0.01-0.48)

0,00%

10,00%

20,00%

30,00%

40,00%

50,00%

IVF ICSI

Pregnancy by Sperm DNA Integrity Results in IVF and

ICSI

DFI>30%

* <.05

*

Esteves

19 Bungum et al. Sperm DNA integrity assessment in prediction of assisted

reproduction technology outcome. Hum Reprod 2007; 22: 174-9.

Y Chromosome Microdeletion

• Deletions linked to spermatogenic failure Background

• Severe oligozoospermia and NOA To whom?

• PCR of the long arm of Y-chromosome Principle

• Peripheral blood Specimen

• Diagnosis and predictive value for sperm retrieval

Clinical Significance

80%

5% 10% 5%

Genetic Causes of Male Infertility

Klinefelter Syndrome (47,XXY)Y-chromosome microdeletionCongenital Vas Absence

Esteves SC & Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37:5-15.

Esteves SC & Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37:5-15.

AZFa deletion

No retrievable sperm

AZFc deletion

Sperm retrieved in 70% of cases

AZFb deletion

No retrievable sperm

Predictive Value of Yq Microdeletion Screening for Sperm

Retrieval in NOA

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Fertility Restoration Spontaneous Pregnancy

Surgical Treatment of Varicocele

Varicocelectomy for Fertility Restoration

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Fertil Steril 2007;88:639–48.

Fertility Improvement

Sperm Retrieval in

Azoospermia

Fertility Improvement

ICSI

Outcomes Fertility Restoration Spontaneous Pregnancy

Surgical Treatment of Varicocele • It can improve success of ART

Varicocele Repair Before ICSI

Microsurgical varicocele repair prior

to ICSI (N=80)

ICSI in the presence of varicocele (N=162)

6.7

15.4

Total Number of Motile Sperm (x106)

Pre-op Post-op

P<0.01

Clinical Outcome of Intracytoplasmic Sperm Injection in Infertile Men With Treated and Untreated Clinical Varicocele

SC Esteves, FV Oliveira, RP Bertolla. ANDROFERT, Center for Male Reproduction, Campinas, BRAZIL and Division of Urology, São Paulo Federal

University, São Paulo, BRAZIL.

The Journal of Urology Vol. 184,1442-1446, October 2010

78%*

46%*

22%

66%

31% 31%

Varicocele and ICSI Outcomes

Treated Varicocele Untreated Varicocele

Fertilized Eggs (%2PN)

Live Birth (%)

*P<0.05

Miscarriage (%)

Esteves SC, Oliveira FV, Bertolla RP. Clinical Outcome of ICSI in Infertile Men with Treated and Untreated Clinical Varicocele. J Urol 2010;184:1442-1446

Odds ratio 1.87 0.43 95% CI 1.08 - 3.25 0.22 – 0.84 P-value 0.03 0.01

Varicocele Repair Before Sperm Retrieval Sperm Retrieval and Intracytoplasmic Sperm Injection in Men With Nonobstructive Azoospermia, and Treated and

Untreated Varicocele K Inci, M Hascicek, O Kara et al. Department of Urology, School of

Medicine, Hacettepe University, Ankara, Turkey.

The Journal of Urology Vol. 182,1500-1505, October 2009

53% 30%

Successful Sperm Retrieval Rate

Treated VaricoceleUntreated Varicocele

OR: 2.63 (95% CI: 1.05-6.60; P=0.03)

Microsurgical varicocele repair prior to sperm retrieval ICSI

(N=66)

Sperm Retrieval in the presence of varicocele

(N=30)

Azoospermia • It is not a synonymous of sterility

• Normal sperm production

• Mechanical blockage • Vasectomy, Post-

infectious, Congenital

Obstructive

• Sperm production deficient or absent

• Cryptorchidism, Radiation, Chemotherapy, Trauma, Genetic, Orchitis, Varicocele, Gonadotoxins, Unexplained

Non-obstructive

Obstructive Azoospermia

• Microsurgical reconstruction

• TURED Potentially treatable

• Epididymis • Testis • Simple and

effective

Sperm retrieval for ART

Esteves, Miyaoka & Agarwal. Surgical Treatment of Male Infertility in the ICSI Era. CLINICS 2011; 66:1463-77.

Watch this video at http://androfert.com.br/videos

Esteves SC, et al. Success of percutaneous sperm retrieval and intracytoplasmic sperm injection (ICSI) in obstructive azoospermic (OA) men

according to the cause of obstruction. Fertil Steril. 2010;94 (Suppl):S233.

Non-obstructive Azoospermia

• Sperm production reduced or absent

• Geographic location unpredictable

Sperm Retrieval for ART

Untreatable condition

TESA

TESE

Non-obstructive Azoospermia

TESA vs. TESE

Watch this video at http://androfert.com.br/videos

OR = 1.63 (95% CI: 1.32 – 2.01)

41% 53%

TESE Micro-TESE

Schlegel 1999

Amer et al. 2000

Okada et al. 2002

Okubu et al. 2002

Tsujimura et al. 2002

Ramon et al. 2003

Esteves et al. 2011

Micro-TESE vs TESE Success Rates in Controlled Series

Sperm Retrieval Live Birth

97.9%

38.2% 55.2% 25.0%

Obstructive (N=142)Non-obstructive (N=172)

Odds ratio 43.0 1.86 95% CI 10.3 – 179.5 1.03 – 2.89 P-value <0.01 0.03

Sperm Retrieval and Reproductive Potential of Men with OA and NOA with ICSI

Prudencio C, Seoul B, Esteves SC. Reproductive potential of azoospermic men undergoing intracytoplasmic sperm injection is dependent on the type of azoospermia.

Fertil Steril 2010; 94(4):S232-3.

Take-home Messages (1) Antioxidants helpful to decrease oxidative

stress. Treatment effect is noted > 60 days later.

New WHO standards have several shorcomings. Caution to interpret results.

Sperm DNA integrity and Y-chromosome microdeletion testing have prognostic

value in ART.

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Take-home Messages (2)

Treatment of Clinical Varicoceles prior to ICSI may be beneficial for patient

subgroups of severe oligozoospermia and NOA.

Most azoospermic men are not sterile. Sperm retrieval and reproductive

potential is dependent on the type of azoospermia.

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