Role of Sperm Dr.dicky

Male Infertility

Male Infertility: Prevalence

15% of couples suffer from infertility

A male factor is responsible for the couple infertility in 30-50% of cases

~5-10% of males are infertile or sub-fertile

Male Reproduction:Anatomy & Physiology 1. Sperm Production

2. Sperm Transport

Etiology of Male Infertility

Multi-factorial PrevalenceVaricocele 35%Idiopathic 25%Infection – genito-urinary tract 10%Genetic 10%Endocrine 1 - 5%Immunologic 1 - 5%Obstruction 1 - 5%Developmental 1 - 5%Lifestyle: smoking, diet, heat ???%


Multi-factorialVaricocele IdiopathicInfection Genetic Endocrine Immunologic Obstruction Developmental Lifestyle: smoking, diet, heat

Dilated testicular veinsMechanisms of Injury:

Increased HeatVenous Stasis


Multi-factorial PrevalenceVaricocele 35%Idiopathic 25%Infection – genito-urinary tract 10%Genetic 10%Endocrine 1 - 5%Immunologic 1 - 5%Obstruction 1 - 5%Developmental 1 - 5%Lifestyle: smoking, diet, heat ???%

Semen Analysis:World Health Organization Guidelines

Parameters Normal range Volume 1.5 - 5 mL

Sperm conc. >20 million/mL Sperm motility >50% Sperm morphology >30% normal

forms Leukocyte density <1 million/mL

Need at least 2 S/As (parameters are highly variable)

Male Infertility: Evaluation

– History (Questionnaire)– Physical examination

– Standard semen analysis– Hormonal evaluation– Genetic counseling and evaluation– Imaging studies

Oxidative Stress in Male InfertilityMulti-factorial

Varicocele Idiopathic Infection Oxidative stress is due to the elaboration Genetic ofEndocrine ROS (reactive oxygen species)Immunologic Obstruction Developmental Lifestyle

de Lamirande et al, Fertil Steril, 1995 Wesse et al, J Urol, 1993 Hendin et al, J Urol, 1999 Mazzilli et al, Fertil Steril, 1994 Vicari et al, Hum Reprod, 1999

Reactive Oxygen Species (ROS) and Male Infertility25% infertile men have high semen ROS levels

n + Semen ROS Infertile 166 25% Fertile 12 0

Semen ROS levels correlate negatively with sperm motility

Iwasaki & Gagnon, Fertil Steril, 1992 Zini et al, Int J Androl, 1993

Biology of ROS

ROS are ubiquitous in aerobic biologic systems

Physiologic: diverse cellular functions and cellular differentiation

Pathologic: cellular aging (enzyme inactivation, DNA breaks, lipid peroxidation), tumorogenesis

Grisham & McCord, Phys Oxygen Radicals, 1986

Biology of ROS

Pathology stems from imbalance between production and scavenging

Production Degradation

ROS and ROS Reactivity

t½ Strength Potential Targets

.O2- seconds + SHgps, NADH

H2O2 >> seconds ++ DNA, SHgps

.OH << seconds ++++ Lipids,DNA,SHgps

NO. seconds ++ Metals,SHgps,ROS

Grisham & McCord, Phys Oxygen Radicals, 1986

Stamler et al, Science, 1992

Source of High ROS in Semen

Leukocytes (WBCs)

Spermatozoa

Source of ROS in Semen: WBC

WBC concentration correlates with ROS levels in whole semen

“Outlying” points represent samples with significant sperm-derived ROS production

Aitken & West, Int J Androl, 1990

Kovalski et al, Fertil Steril, 1991

Aitken et al, Hum Reprod, 1995

Source of ROS in Semen: Spermatozoa

The generation of ROS by human spermatozoa occurs spontaneously under aerobic conditions

Defective sperm function (e.g. motility) is associated with the accumulation of lipid peroxides MacLeod, Am J Physiol, 1943 Alvarez et al, J Androl, 1987 Aitken et al, Biol Reprod, 1989

Source of ROS in Semen: Spermatozoa

Retained cytoplasm (RC)– midpiece area correlates

with ROS production

– midpiece area correlates with sperm dysfuntion (motility, fertilization in vitro)

Gomez et al, J Androl, 1996

Keating et al, J Reprod Fertil, 1997

Zini et al, Urology, 1998

ROS and Sperm DysfunctionMechanism of ROS-induced sperm dysfunction:– Peroxidation of sperm membrane lipids

(accumulation of lipid peroxides)– ATP depletion – Oxidation of proteins/SH-groups– DNA oxidation/fragmentation

Alvarez et al, J Androl, 1987

Aitken et al, Biol Reprod, 1989

de Lamirande & Gagnon, J Androl, 1992

Mammoto et al, Biol Reprod, 1996

Twigg et al, Hum Reprod, 1998


ROS and Sperm Dysfunction: Fertility in Vivo

High levels of semen ROS are associated with low pregnancy rates in vivo Aitken et al, Am J Obst Gynecol, 1991

No ROS

High ROS

Intermediate

ROS and Sperm DysfunctionMechanism of ROS-induced sperm dysfunction:– Peroxidation of sperm membrane lipids

(accumulation of lipid peroxides)– ATP depletion– Oxidation of proteins/SH-groups– DNA oxidation/fragmentation

Alvarez et al, J Androl, 1987


de Lamirande & Gagnon, J Androl, 1992

Mammoto et al, Biol Reprod, 1996

Twigg et al, Hum Reprod, 1998


Successful IVF/ICSI with Damaged Sperm DNA: Implications

DNA oxidation may cause errors in DNA replication and repair after successful fertilization

Kuchino et al, Nature, 1987

Potential for de novo Genetic mutations

Drugs thought to induce male infertility

– Anti-androgens : Spironolactone, cimetidine, flutamide– Androgen suppressors :Ketoconazole & leuprolide– Oestrogens & hormones : Oestrogen agonists, growth hormone, & anabolic

steroids– Drugs of abuse : Anabolic steroids, alcohol, marijuana, cocaine & nicotine.– Psychoactive agents : Tricyclic antidepressants, amphetamine, tranquillisers

& phenytoin.– CVS agents : Propanolol, digoxin & Ca2+ channel antagonists.– GIT & antibiotics :.Sulphasalazine & nitrofurantoin

Role of Antioxidants in Semen

Function– Protect normal sperm from ROS-producing

sperm– Protect normal sperm from WBC-derived ROS– Suppress premature sperm maturation

Site of Action– Male reproductive tract– Female reproductive tract

Antioxidant Activity in Semen

Enzymatic & Non-Enzymatic Antioxidants– SOD– Catalase– Glutathione peroxidase– Taurine / Hypotaurine– Vitamin C / E– Urate – Lycopene Alvarez et al, J Androl, 1987 Alvarez & Storey, Gamete Res, 1989 Jeulin et al, Gamete Res, 1989 Holmes et al, J Androl, 1992 Zini et al, Int J Androl, 1993

Palan & Naz Arch Androl 1996

ROS and Normal Sperm Function

Low levels of oxidative stress in vitro enhance:– sperm hyperactivation– sperm capacitation– acrosome reaction– sperm-egg binding– sperm-egg fusion– Fertilization

Bize et al, Biol Reprod, 1991 de Lamirande et al, Int J Androl, 1993 Griveau et al, Int J Androl, 1994, 1995 Zini et al, J Androl, 1995 Kodama et al, J Androl, 1996 Aitken et al, Biol Reprod, 1998

Role of Antioxidants in Semen

Function– Protect normal sperm from ROS-producing

sperm– Protect normal sperm from WBC-derived ROS– Suppress premature sperm maturation

Site of Action– Male reproductive tract– Female reproductive tract

Etiology of Oxidative Stress: ROS Production or Antioxidant Deficiency?

Production Degradation

Etiology of Oxidative Stress: ROS Production or Antioxidant Deficiency?

Primary oxidant excess? Studies Aitken et al, Hum Reprod, 1995 Gomez et al, J Androl, 1996

Primary antioxidant deficiency? Studies Zini et al, Int J Androl, 1993 Smith et al, Hum Reprod, 1996 Lewis et al, Fertil Steril, 1995 Sanocka et al, J Androl, 1996 Alkan et al, J Urol, 1997

WBC

ROS

Treatment of Oxidative Stress-Mediated Infertility

1. Disease-Specific TherapyAntibiotic therapy: for genital tract infectionVaricocelectomy: to improving sperm morphology and

decrease the % of spermatozoa with retained cytoplasm

Vasquez-Levin et al, J Urol, 1997

Zini et al, Hum Reprod, 1999

Lifestyle Changes: to reduce oxidants (e.g. cigarette)

2. Antioxidant Therapy (in vitro)

Beneficial Effect of Antioxidants (in vitro) on Sperm Motility

Antioxidants may be effective in preserving sperm motility in aerobic conditions– Albumin– Taurine – Hypotaurine

Alvarez & Storey, Biol Reprod, 1983 Twigg et al, Hum Reprod, 1998

CTL

AOX1

AOX2,3

Treatment of Oxidative Stress-Mediated Infertility

1. Disease-Specific TherapyAntibiotic therapy: for genital tract infectionVaricocelectomy: to improving sperm morphology and

decrease the % of spermatozoa with retained cytoplasm

Vasquez-Levin et al, J Urol, 1997Zini et al, Hum Reprod, 1999

Lifestyle Changes: to reduce oxidants (e.g. cigarette)

2. Antioxidant Therapy (in vitro)

3. Antioxidant Therapy (oral)

Oral Antioxidant Therapy:Vitamins C & E

Study Agent Rx Placebo Outcome Comment

Fraga, ‘91 Vit C 28 --- DNA oxidatn No placebo

Dawson, ‘92 Vit C 50 25 spz quality ?abstinence

Kessopoulou,‘95 Vit E 15 15 egg-binding otherwise

neg Suleiman, ‘96 Vit E 52 35 motil, pregn Drop-out

(25%)

Rolf, ‘99 Vit C/E 15 16 negative small

*

Oral Antioxidant Therapy:Zinc, Folic Acid, Selenium

Study Agent Rx Placebo Outcome

Wong,‘02 Zn +/- FA 75 25 spz quality with combined

Rx

Scott,‘98 Se 23 23 spz motility

Vezina,’96 Se + Vit E 9 --- spz motility, morphology

*

Lycopene and Male Infertility: Overview

1. Male Infertility

2. Oxidative Stress and Male Infertility

3. Antioxidant Therapy for Male Infertility

4. Lycopene and Male Infertility

Lycopene Rx in Male Infertility:Rationale1. Oxidative stress is important in Male Reproduction

25% of infertile men have high levels of semen ROS

Spermatogenesis is very sensitive to oxidative stress

Lipid & DNA oxidation/damage are key pathologic events

2. Lycopene protects against lipid and DNA oxidation

Lycopene Rx reduces cellular damage in vitro and in vivo

3. Lycopene is highly concentrated in testis

Testis Lycopene levels: ~20 nmol per g tissue

Serum Lycopene levels: ~0.5 nmol per mlStahl & Sies, Arch Biochem Biophys 1996

Lycopene Rx for Male Infertility:Rationale1. Oxidative stress is common in Male infertility



Lipid & DNA oxidation are key pathologic events






Lycopene as an Antioxidant

1. Lycopene supplementation in vivo & in vitro protects cells from induced oxidative damage

Lipid peroxidation (TBARS) is reduced by 80%

DNA oxidation (8-oxodGuo) is reduced by 75%

Matos et al, Arch Bioch Biophys 1999

Matos et al, Arch Bioch Biophys 2000

2. Oral Lycopene supplementation protects against ex vivo induced lymphocyte DNA oxidation

DNA fragmentation (COMET assay) is reduced by 40%

Riso et al, Am J Clin Nutr 1999

Lycopene Rx for Male Infertility:Rationale1. Oxidative stress is common in Male infertility



Lipid & DNA oxidation are key pathologic events






Lycopene Rx in Male Infertility:Additional Evidence

1. Semen Lycopene Levels Palan & Naz detected reduced semen lycopene levels in infertile

compared to fertile men (19 vs. 42 ng/ml) Palan & Naz, Arch Androl 1996

2. Lycopene Supplementation Study Rx Plac. Outcome Comment Gupta, 2002 30 0 spz quality 2 mg/d x 3 months Mohanty, 2001 50 0 spz quality 8 mg/d x 3-6 months Gupta & Kumar, Int Urol Neph 2002

Mohanty et al, 2001 (personal communic.)

Lycopene and Male infertility:Future Directions

To Evaluate:1. Lycopene levels and markers of oxidative stress (e.g. lipid peroxidation) in the sperm & semen of fertile and infertile men.

-Detectable levels of lycopenes are found in human spermatozoa-Correlation between lycopene levels and markers of oxidation in

progress

2. The correlation between sperm & semen lycopene levels and dietary intake of lycopenes

-Study is currently in progress

3. Effect of lycopene supplementation on semen lycopene levels and sperm parameters in infertile men.

Zini & Rao, unpublished observations

Assisted Conception

IUI: intrauterine insemination IVF: in vitro fertilization ICSI: intracytoplasmic sperm injection GIFT: gamete intrafallopian transfer ZIFT: zygote intrafallopian transfer PESA: percutaneous epididymal sperm aspiration

Sperm Preparation

Select PMNS Remove seminal plasma, WBC, and bacteria Sperm capacitation

– Coating of sperm with seminal plasma proteins – Allow sperm to become fertile

– In vivo or in test tube

Role of Sperm Dr.dicky

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