1. Insight’12 – Lite, Coimbatore, India – May 2012
Sandro Esteves, MD, PhD
Director, ANDROFERT
Center for Male Reproduction and Infertility
Campinas, BRAZIL

2. What is in it for me?
Esteves, 2

3. Lecture Overview
Esteves, 3

5. Antioxidants:
To whom and how
Seminal
Reactive Oxygen
Species (ROS)
(Log ROS + 1; cpm)
2,5
2
1,5
1
0,5
0
Fertile Infertile
Pasqualotto et al., Fertil Steril 2000
Esteves, 5

6. 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 Increased total sperm count
66 mg
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., Menevit® (vit C + E; Increased IR/PR in IVF/ICSI
2007 zinc 25 mg; selenium 26 cycles
mcg; lycopene 6 mg)
Boxmeer et al., 2009 Decreased folate in Increased sperm DNA
seminal plasma fragmentation

7. Antioxidant Treatment
Cochrane Review 2011
Outcome N N Effect size
studies participants (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 6-16 242-700 No effect
count, sperm motility
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.

8. 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?
Esteves et al. What the gynecologist should know about male infertility:
Esteves, 8 an update. Arch Gynecol Obstet 2012; Epub March 6

9. From Initiation of Sperm
Production to Ejaculation
Old concept ~80 days
New concept ~60 days
Misell LM et al.: A stable isotope-mass spectrometric method for measuring
human spermatogenesis kinetics in vivo.
J Urol. 2006; 175: 242-6.

10. 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

11. 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
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
Esteves, 11

12. 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 Urology 2012
Juan Alvarez, SPAIN Jan;79(1):16-22.
Edmund Sabanegh, USA
Ashok Agarwal, USA

13. New WHO standards
Critical Appraisal - Summary
Reasons for lower Merits Demerits
cutt-off
Different way of Controlled studies No systematic review
generating data: of fertile populations:
• Method for semen Recent fathers with • Not representative of
analysis (higher QC known TTP global fertile male
standards; strict population
morphology)
Standardized semen Morphology using
• Population studied analysis different criteria
Single semen
specimen of each
individual
Esteves, 13

14. Oslo
Turku
Norway
Finland
Columbia,
Minneapolis Edimburgh
USA UK
Copenhagen
Paris Denmark
France
Melbourne
Australia
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

15. New WHO Standards: Implications
• Reclassification of semen analysis reports
Couples (N=987) with infertility
duration > 12 months
Abnormal
results
WHO 1999
Reclassified
as “Normal”
WHO 2010
Morphology results
(38.7%) accounted for 53%
of reclassification
Esteves, 15 Source: ANDROFERT, Brazil

16. 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.
Esteves, 16 CLINICS 2011; 66:1-10.

17. 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.

18. Sperm DNA Integrity Testing
• Normal sperm chromatin essential for
Background paternal genetic transmission
• Infertility
Sperm DNA • Recurrent pregnancy loss
Damage • Poor outcomes in IUI and IVF
• Quantification of sperm DNA strand
Principle breaks
Specimen • Semen
• Nuclear dyes (Acridine orange, SCSA)
Techniques • Direct assessment (TUNEL, COMET)
• Nuclear matrix assays (SCD)
Esteves, 18

19. Sperm DNA Integrity Testing & ART
Pregnancy by Sperm Pregnancy by Sperm DNA
DNA Integrity Results in Integrity Results in IVF and
IUI ICSI
20% 50,00% * <.05
15% 40,00%
30,00% *
10%
20,00%
5% OR 0.07 (0.01-0.48)
10,00%
0% 0,00%
DFI ≤30% DFI >30% IVF ICSI
Live birth (%) DFI>30%
Bungum et al. Sperm DNA integrity assessment in prediction of assisted
19
reproduction technology outcome. Hum Reprod 2007; 22: 174-9.
Esteves

20. Y Chromosome Microdeletion
• Deletions linked to Genetic Causes of
Background spermatogenic failure Male Infertility
• Severe oligozoospermia 5%
To whom? 10%
and NOA 5%
• PCR of the long arm of
Principle Y-chromosome
80%
Specimen • Peripheral blood
• Diagnosis and Klinefelter Syndrome (47,XXY)
Clinical predictive value for Y-chromosome microdeletion
Significance sperm retrieval Congenital Vas Absence
Esteves SC & Agarwal A. Novel concepts in male
infertility. Int Braz J Urol 2011; 37:5-15.

21. Predictive Value of Yq
Microdeletion Screening for Sperm
Retrieval in NOA
AZFa AZFb AZFc
deletion deletion deletion
No retrievable No retrievable Sperm retrieved in
sperm sperm 70% of cases
Esteves SC & Agarwal A. Novel concepts in male infertility.
Esteves, 21 Int Braz J Urol 2011; 37:5-15.

22. Surgical Treatment of Varicocele
Fertility
Restoration
Spontaneous
Pregnancy

23. Varicocelectomy for Fertility Restoration
Fertil Steril 2007;88:639–48.
Esteves, 23

24. Surgical Treatment of Varicocele
• It can improve success of ART
Fertility
Improvement
Fertility ICSI
Restoration Outcomes
Spontaneous
Pregnancy Fertility
Improvement
Sperm Retrieval in
Azoospermia

25. Varicocele Repair Before ICSI
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
Total Number of Motile
Microsurgical Sperm (x106)
varicocele repair prior
15.4
to ICSI (N=80) P<0.01
6.7
ICSI in the presence of
varicocele (N=162)
Pre-op Post-op

26. Varicocele and ICSI Outcomes
Treated Varicocele Untreated Varicocele
78%*
66% *P<0.05
46%*
31% 31%
22%
Fertilized Eggs Live Birth (%) Miscarriage (%)
(%2PN)
Odds ratio 1.87 0.43
95% CI 1.08 - 3.25 0.22 – 0.84
P-value 0.03 0.01
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

27. 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
Successful Sperm Retrieval
Microsurgical
Rate
varicocele repair prior
to sperm retrieval ICSI OR: 2.63 (95% CI: 1.05-6.60; P=0.03)
(N=66) 53%
30%
Sperm Retrieval in the
presence of varicocele
(N=30) Treated Varicocele
Untreated Varicocele

28. Azoospermia
• It is not a synonymous of sterility
Non-
Obstructive obstructive
• Normal sperm production • Sperm production deficient
or absent
• Mechanical blockage
• Cryptorchidism, Radiation,
• Vasectomy, Post- Chemotherapy, Trauma,
infectious, Congenital Genetic, Orchitis,
Varicocele, Gonadotoxins,
Unexplained

29. Obstructive Azoospermia
• Microsurgical
Potentially reconstruction
treatable • TURED
Sperm • Epididymis
• Testis
retrieval • Simple and
for ART effective
Esteves, Miyaoka & Agarwal. Surgical Treatment of Male Infertility in the
ICSI Era. CLINICS 2011; 66:1463-77.

31. 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.

32. Non-obstructive Azoospermia
Sperm • Sperm production
Untreatable reduced or absent
Retrieval
condition • Geographic location
for ART unpredictable
TESA
TESE

33. Non-obstructive Azoospermia
TESA vs. TESE

35. Micro-TESE vs TESE
Success Rates in Controlled Series
Schlegel 1999
Amer et al. 2000
Okada et al. 2002
Okubu et al. 2002 53%
41%
Tsujimura et al. 2002
Ramon et al. 2003 TESE Micro-TESE
Esteves et al. 2011 OR = 1.63 (95% CI: 1.32 – 2.01)

36. Sperm Retrieval and Reproductive Potential
of Men with OA and NOA with ICSI
97.9% Obstructive (N=142)
Non-obstructive (N=172)
55.2%
38.2%
25.0%
Sperm Retrieval Live Birth
Odds ratio 43.0 1.86
95% CI 10.3 – 179.5 1.03 – 2.89
P-value <0.01 0.03
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.

37. 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.
Esteves, 37

38. 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.
Esteves, 38