Aula ministrada no 5th. Dubai International Obs-Gyne & Fertility Conference & Exhibition. DIOFCE 2010.

1. Sandro Esteves, MD, PhD
Director, ANDROFERT
Center for Male Reproduction and Infertility
Campinas, BRAZIL
Esteves, 1

2. What is in it for me?
There are novel concepts in
Male Infertility that you need
to know. They will make a
difference in your clinical
practice
Esteves, 2

3. Objectives
Medication
• When and how to use antioxidants
Medication
• When and how to use antioxidants
Semen analysis
• New reference values by WHO
Semen analysis
• New reference values by WHO
Diagnostic Tests
• Beyond the routine semen analysis
Diagnostic Tests
• Beyond the routine semen analysis
Surgical Treatment of Varicocele
• It can improve success of ART
Surgical Treatment of Varicocele
• It can improve success of ART
Azoospermia
• It is not a synonymous of sterility
Azoospermia
• It is not a synonymous of sterility
Esteves, 3

4. Esteves, 4
Medication:
when and how to
use antioxidants
0
0,5
1
1,5
2
2,5
Fertile Infertile
Seminal
Reactive Oxygen
Species (ROS)
(Log ROS + 1; cpm)
Pasqualotto et al., Fertil Steril 2000

5. Evidence-based Use of
Antioxidants in Male Infertility
Author Antioxidant Agent Results
Geva et al., 1996 Vit E 200mg Increased fertilization in IVF
Suleiman et al, 1996 Vit E 100mg Decreased ROS; increased
spontaneous PR
Wong et al., 2002 Folic acid 5mg + Zinc
66mg
Increased total sperm count
Greco et al., 2005 Vit C 1,0g + E 1,0g Improved sperm DNA integrity
Greco et al., 2005 Vit C 1,0g + E (1,0g) Increased CPR and IR in ICSI
cycles
Tremellen et al., 2007 Menevit® (vit C + E;
zinc 25mg; selenium
26mcg; lycopene 6mg)
Increased IR/PR in IVF/ICSI
cycles
Boxmeer et al., 2009 Decreased folate in
seminal plasma
Increased sperm DNA
fragmentation

6. Antioxidants in Male Infertility
When?
Always
How? q.d.
Vitamic C 500mg
Vitamin E 400 UI
Folic acid 2 mg
Zinc 25 mg
Selenium 26 mcg
How long?
Esteves, 6

7. ~60
daysNew concept
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.

8. Semen analysis
• New reference values by WHO
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
Grade a = rapid progressive motility
Grade b = slow/sluggish progressive motility
2Strict criteria

9. Sperm Morphology Evaluation
by the Strict Criteria
Head length
5.0–6.0 Pm
Mid-piece
1.5x head length
Tail
45 Pm
Head width 2.5–3.5 Pm
TF Kruger et al., 1986
Sperm
dimensions on
Diff-Quik staining
Oval head, smooth shape
Acrosome: 40%-70% head area
No neck, mid-piece or tail defects
‘Borderline’ forms = abnormal
Abnormal spermatozoa
(tail defect)
Abnormal spermatozoa
(head defect)

10. Pregnancy rate
per cycle
Strict Morphology ≤4% >4%
Montanaro-Gauci et al. (2001) 2.6% 15.6%
Ombelet et al. (1997) 12.1% 16.5%
Karabinus and Gelety (1997) 6.5% 9.0%
Lindheim et al. (1996) 1.0% 19.5%
Toner et al. (1995) 7.0% 11.3%
Matorras et al. (1995) 10.9% 13.0%
Total 8.7%
(64/731)
12.8%
(208/1628)
P <0.001
Predictive Value of Normal Sperm
Morphology (WHO 2010) for IUI
Adapted from: J Van Waart, TF Kruger, CJ Lombard et al. Predictive value of normal sperm morphology
in intrauterine insemination (IUI): a structured literature review. Hum. Reprod. Update (2001) 7:495-500

11. Predictive Value of Normal Sperm
Morphology for IVF and ICSI
Adapted from French et al., Fertil Steril 2010
0%
20%
40%
60%
80%
≤4% >4%
Fertilization, Pregnancy and
Miscarriage by Sperm
Morphology Subgroups in
ICSI
Fertilization rate (%2PN)
Clinical Pregnancy (%)
Miscarriage (%)
0%
20%
40%
60%
80%
≤4% >4%
Fertilization and
Pregnancy by Sperm
Morphology Subgroups
in IVF
Fertilization rate (%2PN)
Pregnancy rate (%)
Adapted from Coetzee et al., Hum Reprod Update 1998
*
*
*p<0.05

12. Diagnostic Tests
• Beyond the routine semen analysis
1) Sperm DNA Integrity Testing
2) Y Chromosome Microdeletion
Screening

13. Sperm DNA Integrity Testing
Esteves, 13
• Normal sperm chromatin essential
for paternal genetic transmissionBackground
• Infertility
• Recurrent pregnancy loss
• Poor outcomes in IUI and IVF
Sperm DNA
Damage
• Quantification of sperm DNA strand
breaksPrinciple
• SemenSpecimen
• Nuclear dyes (Acridine orange, SCSA)
• Direct assessment of DNA breaks (TUNEL, COMET)
• Nuclear matrix assays (Halosperm)
Techniques

14. Sperm DNA Integrity Testing
Esteves, 14
Unexplained
Infertility
• Normal semen
analysis
Candidates for
IUI and IVF
Recurrent
Pregnancy Loss

15. Sperm DNA Integrity Testing
0%
5%
10%
15%
20%
DFI ≤30% DFI >30%
Pregnancy by Sperm
DNA Integrity Results in
IUI
Live birth (%)
Adapted from Bungum et al., Hum Reprod 2007
OR 0.07 (0.01-0.48)
Adapted from Bungum et al., Hum Reprod 2007
0%
10%
20%
30%
40%
50%
IVF ICSI
Pregnancy by Sperm DNA
Integrity Results in IVF and
ICSI
DFI≤30% DFI>30%
* <.05
*

16. Y Chromosome Microdeletion
• Deletions are the cause
of spermatogenic failureBackground
• Men with non-obstructive
azoospermiaTo whom?
• PCR of the long arm of
Y-chromosomePrinciple
• BloodSpecimen
• Predict the chances of
finding sperm on sperm
retrieval techniques
Clinical
Significance
80%
5%
10%
5%
Genetic Male
Infertility
Klinefelter Syndrome (47,XXY)
Y-chromosome microdeletion
Congenital Vas Absence

17. Y Chromosome Microdeletion
AZFa deleted
Germ cell Aplasia
No retrievable sperm
AZFb deleted
Maturation Arrest
No retrievable sperm
AZFc deleted
Hypospermatogenesis
70% chance of retrieving
testicular sperm for ICSI

18. Fertility
Restoration
Spontaneous
Pregnancy
Surgical Treatment of Varicocele

19. Varicocelectomy for Fertility Restoration
Esteves, 19
Fertil Steril 2007;88:639–48.

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

21. Varicocele Repair Before ART
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

22. 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

23. 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 Varicocele
Untreated 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)

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

25. Obstructive Azoospermia
• Microsurgical
reconstruction
• TURED
Potentially
treatable
• Epididymis
• Testis
• Simple and
effective
Sperm
retrieval
for ART

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

27. 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
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): Suppl. S232-233.
Sperm Retrieval Rates and Reproductive
Potential of Azoospermic Men in ICSI

28. Key Messages
Antioxidants helpful to decrease oxidative stress.
Interventions impact on semen quality 60 days later.
Antioxidants helpful to decrease oxidative stress.
Interventions impact on semen quality 60 days later.
WHO lowered semen analysis reference values.WHO lowered semen analysis reference values.
Sperm DNA integrity and Y-chromosome
microdeletion testing are of prognostic value.
Sperm DNA integrity and Y-chromosome
microdeletion testing are of prognostic value.
Treatment of Clinical Varicoceles prior to ART
beneficial for patient subgroups.
Treatment of Clinical Varicoceles prior to ART
beneficial for patient subgroups.
Sperm retrieval and reproductive potential is
dependent on the type of azoospermia.
Sperm retrieval and reproductive potential is
dependent on the type of azoospermia.
Esteves, 28