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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