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

2. Learning Objectives
Overview of Male Infertility Conditions
Subjected to Specific Medical Treatment
Conventional Specific Medical Treatment
Novel Specific Medical Treatment
Life-style and its Effect on Male Fertility
Esteves, 2

4. 8% of men at
reproductive age
seek medical
assistance for
fertility-related
problems
Vital and Health Statistics, series 23, no. 26, CDC 2002; www.cdc.gov
Esteves, 4

5. US Census Bureau Estimates, 2004

6. Male Infertility Etiology
Categories
2,383
subfertile
males
19%
candidates
for medical
treatment
Esteves et al.
Esteves, 6
An update on the initial assessment of the infertile male. CLINICS 2011; 66:1-10.

7. Specific Medical Treatment
Overview
Conventional Novel
Subclinical Excessive
Male Genital Oxidative
Tract Infection Stress
Obesity-
Endocrine
related Male
Disorders
Infertility
Ejaculatory
Disorders
Esteves, 7

8. Specific Medical Treatment
Overview
Conventional
Subclinical
Male Genital Subclinical
Tract Infection GTI
Endocrine Hypogonadotropic
Disorders Hypogonadism
Ejaculatory
Disorders
Esteves, 8

9. Subclinical Male Genital
Tract Infection
Bacteria
E. Coli
N. Gonorrhoeae
C. Trachomatis Prevalence: 10-20%
U. Urealyticum
M. hominis Primary target organs:
Virus Epididymis
HPV
Herpes simplex 2 Prostate
Epstein-Barr
CMV, HIV Seminal Vesicles
hepatitis B
Significant cause of idiopathic
Protozoa
T. vaginalis male infertility
T. gondii
Esteves, 9

10. Subclinical Male Genital Tract Infection
Gram-negative Neisseria Chlamydia Ureaplasma Mycoplasma
bacilli gonorrhoeae trachomatis urealyticum hominis
Culture, nucleic Culture, direct
acid fluorescent Immunoassay,
Culture,
Culture amplification antibodies, NAAT,
NAAT
tests (NAAT) immunoassay, Culture
NAAT
penicillin, azithromycin, azithromycin,
azithromycin,
fluorquinolones fluorquinolones, doxycycline, doxycycline
doxycycline
cefalosporins ofloxacin
Anti-bacterial
properties
Esteves, 10

11. Endtz Test
substrate Simple diagnostic method
+
H 2O 2
+
semen
Peroxidase-positive
Granulocytes
Peroxidase-negative

12. Subclinical Male Genital Tract Infection
Leukocytospermia
>1.0 x106 leukocytes per milliliter of semen
marker of reproductive
granulocyte macrophage lymphocyte
tract inflammation
Yanushpolsky et al 1996, Erenpreiss et al 2002,
Sharma et al 2002, Saleh & Agarwal 2002, Aziz et al 2004

13. Henkel R et al, AJA 2007; Alvarez et al. Fertil Steril 2002

14. Treatment of Subclinical Male Genital
Tract Infection and Associated
Inflammatory Changes
• Yanushpolsky et al, 1995; Erel et al., 1997
Antibiotics • Branigan et al., 1995
Cicloxigenase-2 • Lackner et al., 2006
Inhibitors • Gambera et al., 2007
Antihistamines • Oliva & Mutigner, 2006
• Tremellen et al., 2007
Antioxidants • Piombini et al., 2008
Esteves, 14

15. Treatment of Subclinical Male Genital
Tract Infection and Associated
Inflammatory Changes
Azitromycin 1.0g single dose (couple)+ frequent
ejaculation (every 2-3 days) + Antioxidants
110
90
P=0.04 P=0.58 N = 278
70
Androfert 1999-2009
50 42% leukocytospermia
30 P=0.001 P=0.50 resolution
Max
10
Min
75th %
-10 25th %
LEUC O _PR MO T_PRE MO RF_PRE VIT_PRE
LEUC O _PO MO T_PO S MO RF_PO S VIT_PO S Median

16. Specific Medical Treatment
Overview
Conventional
Subclinical
Male Genital Subclinical
Tract Infection GTI
Endocrine Hypogonadotropic
Disorders Hypogonadism
Ejaculatory
Disorders
Esteves, 16

17. Hypogonadotropic
Hypogonadism
Features
Low levels of FSH, LH,
testosterone
MRI
Absent/low virilization,
hypotrophic testes, azoospermia
Main Causes:
● Congenital:
Kallmann syndrome
Prader-Willi
● Acquired:
Pituitary tumor
Pituitary radiation
Steroid abuse
Testosterone replacement therapy

18. Adult onset Hypo-hypo:
Treatment to restore spermatogenesis and
androgenic status
Standard Treatment:
hCG 1000-2000 UI IM injections; twice or t.i.w;
minimum 12 weeks
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Maintenance
Esteves, 18

19. Adult onset Hypo-hypo:
hCG Preparations
hCG Brand Manufacturer LH activity Ampoule/ % Protein Source Technology Route of
preparations names (IU/ampule vial filling Contamination used administration
or vial) method
Urinary hCG Pregnyl Schering- 10,000 Filled-by- <5% Urine Chemical IM
Plough; bioassay extraction
Organon
Choragon Ferring 5,000 Filled-by- <5% Urine Chemical IM
bioassay extraction
Choriomon IBSA 5,000 Filled-by- <5% Urine Chemical IM
bioassay extraction
Corion, Win-Medicare, 1,000; Filled-by- Unknown Urine Chemical IM
Choriolife, Life-Medicare, 2,000; bioassay extraction
Pubergen, Sun Pharmac., 5,000
LG IVF C, Uni-Sankyo,
Origen, LG, etc
etc.
Recombinant Ovidrel, MerckSerono 250µg Filled-by- Negligible Transfected Recombinant SC
hCG Ovitrelle mass CHO cells DNA
(FbM)
Esteves, 19

20. Evolution of hCG Preparations
Urine-derived
Recombinant
technology
Esteves, 20

21. Clinical Efficacy, Safety and Tolerability of Recombinant hCG
to Restore Spermatogenesis and Androgenic Status of
Hypogonadotropic Hypogonadism Males
Esteves SC, Papanikolaou V; Fertil Steril 2011; Vol 96: S230
Case Series of 11 azoospermic males
Adult onset hypo-hypo
Causes:
Pituitary tumor; n=7
Steroid abuse; n=1
Testosterone replacement therapy; n=2 Once a week SC self-
Cranioencephalic trauma; n=1 administration of
250µg rec-hCG using
Mean ± SD hormone levels (mUI/mL) a ready-to-use prefilled
FSH: 0.46 ± 0.28; LH: 0.39 ± 0.32 syringe
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Evaluation
Esteves, 21

22. Recombinant hCG to Treat Men with Hypo-hypo
Esteves SC, Papanikolaou V; Fertil Steril 2011; Vol 96: S230
Restoration of spermatogenesis and androgen production: 10/11 men
Side-effects not reported in men who responded to therapy
Baseline Posttreatment
647.5
33
41.3 36.0
24
0
Mean Total
Testosterone Mean Combined
(ng/dL) Testis Volume Mean Sperm
(cm3) Count (x106/mL)
Baseline Posttreatment
Esteves, 22

23. Specific Medical Treatment
Overview
Conventional Novel
Subclinical
Male Genital Excessive
Tract Infection Oxidative Stress
Endocrine
Disorders Obesity-related
Male Infertility
Ejaculatory
Disorders
Esteves, 23

24. Excessive
Oxidative Stress
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, 24

25. How Oxidative Stress Can be
Measured?
Indirect Assessment
• Lipid Peroxidation (Malondialdehyde)
• Protein oxidation products (eg. 8-OHdG)
• Sperm DNA integrity
Direct Assessment
• Total Antioxidant Capacity
• Seminal ROS levels
Esteves et al. What every GYN should know about male infertility.
Esteves, 25 Arch Gynecol Obstet 2012, Epub March 6

26. DNA Damage is the Main
Expression of ROS Production
and Oxidative Stress
• Quantification of sperm
Principle DNA strand breaks
Specimen • Spermatozoa
• Nuclear dyes (Acridine orange, SCSA)
Techniques • Direct assessment (TUNEL, COMET)
• Sperm Chromatin Dispersion (SCD)
Esteves, 26

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

28. 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

29. 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 et al. Antioxidants for male subfertility.
Cochrane Database Syst Rev 2011 Jan 19;(1):CD007411.

30. Antioxidants in Male
Infertility
To whom?
Men at risk of eOS
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, 30 an update. Arch Gynecol Obstet 2012; Epub March 6

31. 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.

32. Empirical Medical Treatment
for Idiopathic Male Infertility
No demonstrable cause for altered semen parameters
Androgens
hCG/HMG
FSH
Anti-oestrogens
Bromocriptine
Alpha-blockers
Systemic corticosteroids
Magnesium supplementation Guidelines on Male Infertility.
European Association of Urology 2012

33. Specific Medical Treatment
Overview
Novel
Obesity-related
Male Infertility
Esteves, 33

34. Obesity in Men at Reproductive Age
Esteves, 34
WHO: Global database on BMI, 2011

35. Esteves, 35

36. Jensen et al. Fertil Steril 2004; 82: 863; Hammoud et al. Fertil Steril 2008; 90: 2222;
Kriegel et al. RBM Online 2009; 19: 660;
Esteves, 36 Martini et al. Fertil Steril 2010; 94: 1739.

37. Esteves, 37

38. Esteves, 38

39. Serum Levels of Total
Testosterone and Estradiol
Normal > 10
T/E2 Ratio
350 𝑛𝑔/𝑑𝐿
Eg.: 4.9 =
62 𝑝𝑔/𝑚𝐿
T/E2 <10
Aromatase Hyperactivity
T/E2 Ratio Normalization and Anastrozole 1 mg
Sperm Count Improvement q1d 60 days
Zumoff et al. Reversal of the hypogonadotropic hypogonadism of obese men by administration of
the aromatase inhibitor testolactone. Metabolism 2003; 52: 1126. Raman & Schlegel Aromatase
Esteves, 39
inhibitors for male infertility. J Urol 2002; 167: 624.

40. Medical Treatment for Klinefelter
Syndrome Men Seeking Fertility
Karyotype: Sperm Retrieval Medical
Management
Klinefelter
Seeking Fertility
47,XXY and ICSI Treatment prior
47,XXY/46,XY to Sperm
Retrieval
Features:
Azoospermia (>90%)
Small testes
Elevated FSH
Low Testosterone
Foci of sperm
Hypogonadism
production (~40%)
Elevated testis Sciurano et al., Hum
expression Reprod. 2009
aromatase CYP19
Esteves et al. Surgical treatment of male infertility in the era of ICSI – new insights.
Esteves, 40 CLINICS 2011; 66:1463-77.

41. Klinefelter Syndrome
Medical Treatment Prior to Sperm Retrieval
 Series of 68 non-mosaic KS Men with Azoospermia
 Medical Treatment: Aromatase inhibitor, hCG, CC
 Positive Response: TT increase of >100ng/dL from baseline
72%
Positive Response to Therapy No response
330 P = 0.03
SRR: 72%
220
180
150 SRR: 55%
Baseline Testosterone Post-treatment TT
Ramasamy et al., J Urol. 2009; 182: 1108-13.
Esteves, 41

42. Specific Medical Treatment
Overview
Novel
Excessive
Oxidative Stress Life-
style
Obesity-related
Male Infertility Habits
Esteves, 42

43. Smoking
• Live birth delivery reduced by 3.7X
Alcohol
• Live birth delivery reduced by 5.5X
• Miscarriage increased by 2.7X
Stress
• Conflicting results
Caffeine
• No effect
Klonoff-Cohen H. Human Reproduction Update, Vol.11, No.2 pp. 180–204, 2005

44. Common Toxicants to Male
Reproductive Health
Ilicit Drugs Cell Phone
Licit Drugs Anabolic Steroids,
Cigarette smoking, Marijuana, Cocaine, etc.
Electromagnetic
Alcohol Radiation
Environmental/Nutritional Medication
Endocrine disruptors (xenoestrogens Antidepressants (SSRI), Antipsychotics
[Polychlorinated biphenyls, bisphenol (Lithium), Antihypertensives (calcium
A, Phtalates]), Heavy Metals, channel blockers),
Pesticides Cimetidine, Ketoconazole, Finasteride,
Antibiotics

45. Subclinical infections are treated with antibiotics
and leukocyte-induced inflammation can be
alleviated by oral antioxidants administration.
Medical treatment of adult onset hypo-hypo with
hCG is highly effective.
Antioxidant prescription to subfertile men is
recommended, especially in cases of eOS.
Sperm DNA damage marker of eOS.
Esteves, 45

46. Medical treatment (AI) is recommended for
obese/overweight subfertile men with aromatase
hyperactivity.
KS men with NOA are candidates to medical
treatment. AI, hCG and CC boost testosterone
production and may improve chances of SSR.
Re-evalualtion of life-style habits is of utmost
importance to optimize male reproductive health.
Esteves, 46