Sandro Esteves, MD, PhD

Director, ANDROFERT

Center for Male Reproduction and Infertility

Campinas, BRAZIL

EOFF 2012, Dubai, Nov 24th

Learning Objectives

How to revert male hypogonadism and infertility by specific medical therapy

Oral antioxidant therapy is a hot topic: what you need to know in 2012

Obesity is a risk factor for male infertility: how to use aromatase inhibitors to increase sperm

production

Esteves

2

Review this lecture at:

http://www.androfert.com.br/review

Esteves, SC; EOFF 2012

Medical Therapy for Male Infertility Overview

Non-specific Therapy

Empirical treatment for

idiopathic male infertility

Specific Therapy

Genital tract infection

Endocrine disorders

Ejaculatory disorders

Antioxidants Aromatase inhibitors

Esteves

4

Medical Therapy for Male Infertility

Non-specific Therapy

Empirical treatment for

idiopathic male infertility

Androgens

hCG/HMG

FSH

Anti-estrogens

Bromocriptine

Alpha-blockers

Systemic corticosteroids No demonstrable

cause for abnormal

semen parameters;

~44% infertile men

Esteves

5

Empirical Medical Treatment

for Idiopathic Male Infertility

Guidelines on Male Infertility.

European Association of Urology 2012

In general, NOT EFFECTIVE.

Selected cases may benefit of FSH and anti-estrogens

Esteves

6

MR

I

Hypogonadotropic Hypogonadism

Clinical Features

• Absent/low virilization

• Hypotrophic testes

• Azoospermia

Endocrine Lab

• Abnormal low levels of FSH, LH and testosterone

Main Causes

• Congenital: • Kallman syndrome

• Prader-Willi

• Acquired: • Pituitary tumor

• Steroid abuse

• Testosterone replacement therapy

Esteves

7

Urinary hCG 1,000-2,000 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

Adult onset hypo-hypo:

Medical therapy aimed to restore spermatogenesis and androgenic status

Classic Treatment:

Esteves

8

Adult onset Hypo-hypo: hCG Preparations

LH activity

(IU/ampule

or vial)

Ampoule/

vial filling

method

% Protein

contamination

Source Technology Route of

administration

Urinary hCG 1,000-

10,000

Filled-by-

bioassay

<5% Urine Chemical

extraction

IM

Recombinant

hCG

250µg Filled-by-

mass

(FbM)

Negligible Transfected

CHO cells

Recombinant

DNA

SC

Esteves

9

Pretreatment

Abnormal virilization

Posttreatment

Normal hair distribution

Pretreatment

Hypotrophic scrotum/reduced testes

Posttreatment

Normal scrotum and testes

Figure 2. Photographs illustrating a patient with gigantism associated with a pituitary tumor secreting growth hormone and prolactin cured by

transsphenoidal hypophysectomy. Secondary hypo-hypo was treated with rec-hCG leading to a marked improvement in virilization and testicular

volume (with patient permission).

Baseline Posttreatment

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

Series of 11 azoospermic males with adult onset hypo-hypo

Pituitary tumor, Steroid abuse, Testosterone replacement therapy, Encephalic trauma

Once a week SC self-administration of 250µg rec-hCG (ready-to-use prefilled syringe)

Restoration of spermatogenesis and androgen production: 10/11 men; No side-effects

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

10

1. There is little scientific

evidence for an empirical

medical treatment in idiopathic

male infertility.

2. Medical treatment is

recommended in cases of

Hypogonadotropic Hypogonadism (GR-A) .

Specific and Non-Specific Medical Treatment of Male Infertility

Esteves

11

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

Oxidative Stress An emerging

explanation

for several

cases of male

infertility

30%-80% of infertile men have

elevated markers of OS Agarwal et al., Urology 2006

Esteves

12

Chemical species with unpaired electron capable to oxidize lipids, proteins and nucleic acids:

Superoxide anion (•O-2) Hydroxyl radical (•OH) Hydrogen peroxide (H2O2) Peroxyl radical (ROO•) Hypochlorite ion (OCl-)

Generated from aerobic metabolism in mitochondria.

Sources: Leukocytes (extrinsic) Spermatozoa (intrinsic)

Lampiao et al., Oxidative stress. In: Male Infertility. Parekattil & Agarwal (eds) 2012, pp.225-35

Reactive Oxygen Species (ROS)

Sperm and Seminal Plasma Antioxidants:

Enzymatic (SOD, catalase, GPX): inactivate ROS (conversion into other substances) Non-enzymatic (ascorbic acid, alpha-tocopherol, carnitine, etc.): neutralize ROS

Esteves

13

Rationale for Oral Antioxidants in Male Infertility

AO classified according to

their mechanism of action:

Catalytic: enhances the

already present antioxidant

enzymes (Glutathione, NAC).

Scavenging:

Water soluble: react with

oxidants in the cytosol

(Vitamin C)

Lipid soluble: protect cell

membrane from lipid

peroxidation (Vitamin E)

Esteves

14

Oral Antioxidants 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 et al. Antioxidants for male subfertility.

Cochrane Database Syst Rev 2011 Jan 19;(1):CD007411. Esteves

15

Sperm DNA Fragmentation and

Assisted Conception

19%

1.5%

Normal Elevated

Live Birth Rates by Intrauterine Insemination

OR = 0.07

[95% CI: 0.01-0.48]

Adapted from Bungum et al., Hum Reprod 2007

26%

42%

IVF ICSI

Pregnancy by Method in Cases of Elevated Sperm DNA

Fragmentation

P <0.05

Esteves

16

Sperm DNA Fragmentation and

Miscarriage

Robinson L et al. Hum Reprod. 2012; 27(10): 2908-17

• 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

Esteves

17

Oral Antioxidant Therapy

Controversies

Methodological weakness of antioxidants

trials make it difficult to determine “who”,

“how” and “for how long”

• Patient selection and controls

• Associated pathology

• Single or combination antioxidants

• Dosage & formulation

• Outcome measures

• Varying duration of treatment

• Lack of diagnostic markers for oxidative stress

• Presence of molecular and genetic differences

Esteves

18

Oral Antioxidants

in Male Infertility

No consensus yet.

Short-term use

appear to be safe.

Caution against

indiscriminate use

of high dosages for

long periods.

Beneficial

Kodama 1997

Dawson, 1992

Kessopoulou, 1995

Vezina, 1996

Vicari, 2001; 2002

Lenzi, 2003; 2004

Cavallini, 2004

Comhaire, 2005

Grecco 2005

Menezo 2007

Tremellen 2007

Piomboni 2008

Gil Villa 2009

No effect

Giovenco, 1987

Moilanen, 1993

Iwanier, 1995

Rolf, 1999

Sigman, 2006

Detrimental

long-term use and high doses;

increased mortality in cancer population-based studies.

Heinonen, 1994 Lonn, 2005

Bjelakovic, 2007

Esteves

19

Who are the candidates for Oral

Antioxidant Therapy?

Infertile men with OS

Diagnosis

Indirect Assessment

• Lipid Peroxidation (Malondialdehyde)

• Protein oxidation products (eg. 8-OHdG)

• Sperm DNA integrity

Direct Assessment

• Total Antioxidant Capacity

• Seminal ROS Levels

• Detection of Superoxide Anion

Esteves et al. What the gynecologist should know about male infertility:

an update. Arch Gynecol Obstet 2012; 286(1): 217-29 Esteves

20

Sperm Chromatin Dispersion Test:

● Sperm with absent “halos” have DNA strand breaks

● Semen/Spermatozoa

● Quantitative

Sperm DNA Fragmentation

Detection of Superoxide Anion:

● Reduction of Nitroblue Tetrazolium in Formazan (coloured intermediate)

● Semen

● Qualitative

Reactive Oxygen Species

Quick and Easy Tests for OS

Esteves

21

How to use Antioxidant Therapy Treatment Strategy

Ascorbic acid (Vit. C)

- tocopherol (Vit. E)

Glutathione

N-acetyl-cysteine

Carnitine

Coenzyme Q10

Lycopene

Picnogenol

Pentoxifylline

Selenium

Shao-Fu-Zhu-Yu-Tang

Astaxanthin

Lepidium meyenii

-linolenic acid and lignans

Folic acid

Zinc

Select antioxidant formulation and dosage

Testing for

Leukocytes in Semen

Differentiate between sperm and leukocyte

source of ROS

Varicocele

Genital Infection

Smoking

Medication

Drug abuse

Systemic diseases

Pollution

Radiation

Once OS is diagnosed, focus on identifying

and controlling source of increased ROS

Esteves

22

Oral Antioxidants in Male

Infertility Our formula

Vitamin C 500mg

Vitamin E 400 mg

Folic acid 2 mg

Zinc 25 mg

Selenium 26 mcg

How long: minimum 2 months

Old concept ~80 days

New concept ~60 days

From initiation of sperm production to ejaculation

Misell LM et al. J Urol. 2006

Esteves & Agarwal. Novel concepts in male infertility. Int Braz J Urol 2011; 37 (1): 5-15 Esteves

23

Oral Antioxidant Therapy

for Male Infertility

Esteves

24

1. Oxidative stress impairs sperm

function and is a risk factor for male infertility and miscarriage (GR-A) .

2. Current evidence suggest that OA

supplementation for subfertile males

improve the chances for pregnancy

and live birth for couples undergoing ART (GR-A).

3. Well-designed studies are needed to determine

the best candidates for AO therapy and which

formulation and dosages yield better results.

Esteves

25

Jensen et al. Fertil Steril 2004; 82: 863; Hammoud et al. Fertil Steril 2008; 90: 2222;

Kriegel et al. RBM Online 2009; 19: 660; Martini et al. Fertil Steril 2010; 94: 1739. Esteves

26

Esteves, 27

Esteves

28

Aromatase Inhibitors for

Infertile Obese Men

Serum Levels of Total Testosterone and Estradiol

T/E2 Ratio Normal > 10

e.g.: 4.9 =𝟑𝟓𝟎 𝒏𝒈/𝒅𝑳

𝟔𝟐 𝒑𝒈/𝒎𝑳

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

inhibitors for male infertility. J Urol 2002; 167: 624.

T/E2 <10

Aromatase Hyperactivity

Anastrozole 1 mg q1d 60-90 days

Esteves

29

Raman JD, Schlegel PN. Aromatase inhibitors for male infertility. J Urol. 2002;167:624-9

5.9

2.9

5.5

15.6

3.5

15.6

0

2

4

6

8

10

12

14

16

18

T/E ratio Ejaculate volume(mL)

Sperm Count(million/mL)

Anastrazole (1mg q1d for 3-6 months)

Pre-treatment Post-treatment

Aromatase Inhibitors for

Obesity-related Male Infertility

P <0.05

Esteves

30

Aromatase Inhibitors for Obesity-related Male Infertility

1. Obesity is a risk factor for male infertility (GR-B).

2. Peripheral androgen aromatization is enhanced in men with elevated BMI. Obese men show increased plasma estradiol and low testosterone levels (GR-B).

3. Lowering estradiol levels, by administration of AI, increases

LH and FSH levels by pituitary modulation, and increase testosterone levels (GR-A).

4. AI may stimulate sperm production (GR-C); effects upon

fertility are still to be determined.

Esteves

31