1. Dr. Sandro Esteves presented on updates to WHO standards for semen analysis, new diagnostic tests for male infertility, and treatments including antioxidant supplementation and sperm retrieval techniques. 2. The WHO has updated reference values for semen analysis, lowering the cut-off levels based on a study of recent fathers. This may reclassify some previously abnormal results as normal. 3. New diagnostic tests beyond routine semen analysis include sperm DNA integrity testing, reactive oxygen species assessment, and Y chromosome microdeletion screening. 4. Oral antioxidant supplementation has been shown to improve live birth and pregnancy rates for couples undergoing assisted reproduction based on meta-analyses.

1. Meet the Expert Evening Meeting, Nov 2012
(Jordan, Lebanon, Qatar, Bahrain, Kuwait)
Sandro Esteves, M.D., Ph.D.
Director, ANDROFERT
Center for Male Reproduction and Infertility
Campinas, BRAZIL

2. Learning Objectives
WHO has updated reference limits for semen
analysis results. Understand how to adopt and
interpret the new standards.
New diagnostic tests go far beyond routine
semen analysis. Learn which tests are
available and how to use them.
Antioxidant supplementation is a hot issue in
male infertility. Understand the rationale of its
use and how it can benefit your patients.
Learn what is available for azoospermic men
seeking fertility. Biological fatherhood is
2
possible even in the worst scenarios.
Esteves

3. Esteves SC, 2012
Review this lecture at:
http://www.androfert.com.br/review

4. Spermatogenesis
Where do we stand compared to our relatives?
Chimpanzee Human Gorilla
45 Kg 80 Kg 270 Kg
64 cc 20 cc 14 cc
>1 ? 5
billion/mL million/mL
4
Esteves

5. Semen Characteristics Values
of 1,953 Recent Fathers
(whose partners had a TTP* ≤ 12 months)
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 Morphology (strict criteria) 4 15 44
% Alive (eosin-nigrosin staining) 58 79 91
*TTP: time to pregnancy
5
Cooper et al: World Health Organization reference values for human semen
characteristics. Hum Reprod Update 16: 231-245, 2010
Esteves

6. 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
1LowerLimit (5% percentile), Recent fathers; 2Strict criteria
Grade a = rapid progressive motility; Grade b = slow/sluggish progressive motility
6
Esteves

7. New WHO standards
Caution to Adopt and Interpret Results
7
Esteves

8. Do they represent global semen characteristics of fertile men?
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
8 Esteves, Zini, Aziz et al, Urology, in press
Esteves

9. New WHO standards
Reasons for Lower Cut-off Values
Different way of generating data
• Method for semen analysis (QC standards)
• Adoption of strict criterion for morphology
• Single specimen of each individual
Population Studied
• Recent fathers with known TTP
• Selection bias
9
Esteves et al. Critical Appraisal of the WHO New Reference Values for Human Semen
and Impact on Diagnosis and Treatment of Subfertile Men. Urology 2012; 79(1):16-22.
Esteves

10. New WHO Standards: Implications
• Do we need to recall previous semen
analysis reports ?
Couples (N=987) with infertility
duration > 12 months
Abnormal
results
WHO 1999
Reclassified
as “Normal” Morphology by strict
WHO 2010 criterion accounted for
(38.7%)
53% of reclassification
10
Esteves
Source: ANDROFERT, Brazil

11. New WHO Standards: Interpretation
Focus on the 5th percentile
Laboratories seeking
to adopt the new
standard should
determine the strategy
that would aid the
clear communication
of the clinical
significance of the
results.
11
Esteves & Agarwal Impact of the new WHO guidelines on diagnosis and practice of
male infertility. Open Reprod Sci J 2011; 3: 7-15.
Esteves

12. New WHO Standards
Not accurate to discriminate fertile and
infertile men
Male infertility workup
goes far beyond a simple
semen analysis. History
taking, physical examination
and laboratory and sperm
function testing are
minimum standards.
Esteves, Miyaoka & Agarwal. An update on the
initial assessment of the infertile male.
12
CLINICS 2011; 66:1-10.
Esteves

13. New Diagnostic Tests
• Beyond routine semen analysis
Sperm DNA Integrity
Reactive Oxygen Species
Y Chromosome
Microdeletion Screening
13 Esteves et al. What the gynecologist should know about male infertility:
an update. Arch Gynecol Obstet 2012; 286(1): 217-29
Esteves

14. Sperm DNA Integrity
Testing
• Normal sperm chromatin essential for
Background
paternal genetic transmission
• Unexplained infertility
Sperm DNA
Fragmentation • Recurrent pregnancy loss
• Poor outcomes in IUI and IVF
• Quantification of sperm DNA strand
Principle
breaks
Specimen • Semen, spermatozoa
• Nuclear dyes (Acridine orange, SCSA)
Techniques • Direct assessment (TUNEL, COMET)
• Nuclear matrix assays (SCD)
14 Esteves SC & Agarwal A. Novel concepts in male infertility.
Esteves Int Braz J Urol 2011; 37:5-15.

15. Sperm DNA Fragmentation and
Assisted Conception
Live Birth Rates by Pregnancy by Method in Cases
Intrauterine Insemination of Elevated Sperm DNA
Fragmentation
42%
19%
P <0.05
26%
OR = 0.07
[95% CI: 0.01-0.48]
1.5%
Normal Elevated IVF ICSI
15 Adapted from Bungum et al., Hum Reprod 2007
Esteves

16. Sperm DNA Fragmentation and
Miscarriage
• Population: Meta-analysis of 16 cohort studies (2,969 couples),
14 prospective.
• Techniques for DNA integrity:
Acridine orange-based assays, TUNEL and COMET.
• Findings:
Significant increase in miscarriage rates in patients undergoing
IVF/ICSI with high sperm DNA damage compared with those
with low DNA damage.
Risk ratio (RR) = 2.16 (1.54, 3.03), P < 0.00001).
Miscarriage rates are positively correlated
with sperm DNA damage levels
16 Robinson L et al. Hum Reprod. 2012; 27(10): 2908-17
Esteves

17. Quick and Easy Test for Sperm
DNA Fragmentation Assessment
Sperm Chromatin Sperm with intact DNA show “halos”:
Dispersion (SCD) Represent dispersed chromatin without
breaks
Sperm with fragmented DNA do not
show “halos” :
Represent DNA strand breaks
Semen/Spermatozoa
Quantitative:
Normal: <30% cells with fragmented DNA
17 Esteves et al. What the gynecologist should know about male
infertility: an update. Arch Gynecol Obstet 2012; 286(1): 217-29
Esteves

18. Y Chromosome Microdeletion
Screening
Genetic Causes of
• Deletions linked to Male Infertility
Background spermatogenic failure
5%
• Severe oligozoospermia 10%
Who? and NOA 5%
• PCR of the long arm of
Principle Y-chromosome
80%
Specimen • Peripheral blood
Klinefelter Syndrome (47,XXY)
Clinical • Diagnosis and
Y-chromosome microdeletion
predictive value for
Significance sperm retrieval Congenital Vas Absence
18
Esteves SC & Agarwal A. Novel concepts in male infertility.
Int Braz J Urol 2011; 37:5-15.
Esteves

19. 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
19
Esteves SC & Agarwal A. Novel concepts in male infertility.
Int Braz J Urol 2011; 37:5-15.
Esteves

20. Oral Antioxidant Therapy
for Male Infertility
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
20 Showell MG et al. Antioxidants for male subfertility.
Esteves Cochrane Database Syst Rev 2011 Jan 19;(1):CD007411.

21. Rationale for Antioxidants
in Male Infertility:
Oxidative Stress
30%-80% of infertile men
have elevated markers of OS
Agarwal et al., Urology 2006
Seminal
Reactive Oxygen Species (ROS)
(Log ROS + 1; cpm)
2,5
2
1,5
1
0,5
0
Fertile Infertile
21 Pasqualotto et al., Fertil Steril 2000
Esteves

22. Quick and Easy Test for
Reactive Oxygen Species
Assessment
Detection of Superoxide Anion Principle:
reduction of nitroblue
tetrazolium into
formazan (coloured
intermediate)
Semen or seminal
plasma
Qualitative
colorimetric
22 Tunc et al. Development of the NBT assay as a marker of sperm oxidative stress.
Esteves Int J Androl 2010; 33(1): 13-21.

23. How to use Antioxidants
Treatment Strategy
Once OS is diagnosed,
Differentiate between
focus on identifying Select antioxidant
sperm and leukocyte
and controlling source formulation and dosage
source of ROS
of increased ROS
Varicocele Ascorbic acid (Vit. C)
Testing for α- tocopherol (Vit. E)
Genital Infection Glutathione
Leukocytes in Semen N-acetyl-cysteine
Smoking
Carnitine
Medication Coenzyme Q10
Drug abuse Lycopene
Picnogenol
Systemic diseases Pentoxifylline
Pollution Selenium
Shao-Fu-Zhu-Yu-Tang
Radiation Astaxanthin
Lepidium meyenii
α -linolenic acid and lignans
Folic acid
23
Zinc
Esteves

24. Oral Antioxidants in Male
Our formula Infertility
Vitamin C 500mg
Vitamin E 400 mg
Folic acid 2 mg
Zinc 25 mg
Selenium 26 mcg
How long: minimum 2 months
From initiation of sperm production to ejaculation
Old concept ~80 days
New concept
~60 days Misell LM et al. J Urol. 2006
24
Esteves & Agarwal. Novel concepts in male infertility. Int Braz J Urol 2011; 37 (1): 5-15
Esteves

25. Azoospermia
• It is not a synonymous of sterility
• Complete absence of sperm in the ejaculate
• 1-3% male population; ~10-15%% male infertility
Non-
Obstructive obstructive
• Sperm production deficient or
• Normal sperm production
absent
• Mechanical blockage
• Cryptorchidism, Radiation,
• Vasectomy, Post-infectious, Chemotherapy, Trauma,
Congenital Genetic, Orchitis, Varicocele,
Gonadotoxins, Unexplained
25
Esteves

26. Obstructive Azoospermia
• Microsurgical
Potentially reconstruction
treatable • TURED
Sperm • Epididymis
retrieval • Testis
for ART • Simple and
effective
26
Esteves, Miyaoka & Agarwal. Surgical Treatment of Male Infertility in the ICSI Era.
CLINICS 2011; 66:1463-77.
Esteves

27. Sperm Retrieval and ICSI Outcome
Obstructive
Azoospermia
265 277
250
CBAVD Post-vasectomy Post-infection
34.4 32.2 36.4 35.8 37.0 35.5
% Live birth Gestational age (wks) Birth weight
(gramsx10)
27 Esteves et al. J Urol 12013; 189: http://dx.doi.org/10.1016/j.juro.2012.08.084
Esteves

28. Non-obstructive Azoospermia
Sperm • Sperm production
Untreatable reduced or absent
Retrieval
condition • Geographic location
for ART unpredictable
TESE
Micro-TESE
Conventional
TESA
• 15%- • 20%- • 40%-
50% 63% 67%
SRR SRR SRR
28 Friedler et al. 1997; Ezeh et al. 1998; Schlegel 1999; Amer et al. 2000; Okada et al. 2002; Okubu et al.
Esteves 2002; Tsujimura et al. 2002; Ramon et al. 2003; Verza Jr. & Esteves 2011.

29. 29 Available at http://androfert.com.br/videos
Esteves

30. 30
Esteves Adapted from: Verza Jr & Esteves. Atlas of Human Reproduction, SBRH 2012

31. Sperm Retrieval in Testicular Failure
Novel Sperm
Retrieval
Methods for
Identification of Confocal fluorescence
microscopy
Spermatogenic (in vivo; murine model)
Sites
Multi-photon microscopy
(ex vivo; human model)
Full-field optical coherence
tomography
(ex vivo; rat model)
Najari et al, J Urol 2012; Smith et al J Urol 2012; Ramasamy et al., J Pathol Inform 2012

32. Sperm Retrieval and Reproductive
Potential of Azoospermic Men in ART
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.
32
Fertil Steril 2010; 94(4):S232-3.
Esteves

33. Key Messages (1)
WHO references for semen analysis
have been lowered.
Exercise caution to interpret results for
they have important shortcomings.
New diagnostic tests allow assessment
to sperm DNA integrity, seminal ROS
and Y-chromosome genetic infertility.
Tests results aid in the clinical
management and/or have prognostic
value in ART.
33
Esteves

34. Key Messages (2)
Oxidative stress is a risk factor for
male infertility and miscarriage.
Antioxidant supplementation improve
the chances for pregnancy and live
birth for couples undergoing ART.
Azoospermia is not synonymous of
sterility.
Treatment options include microsurgery
and sperm retrieval coupled with ICSI.
Reproductive potential in ART is
34 dependent on the type of azoospermia.
Esteves