Sandro Esteves, MD, PhD

Director, ANDROFERTCenter for Male Reproduction Campinas, BRAZIL

Male Infertility:current concepts for reproductive

medicine specialists

Esteves, 1

MerckSerono Stand-alone Meeting – Kochi, India – August 2011

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

Objectives

Medication• When and how to use antioxidants

Semen analysis• New reference values by WHO

Diagnostic Tests• Beyond the routine semen analysis

Surgical Treatment of Varicocele• It can improve success of ART

Azoospermia• It is not a synonymous of sterility

Esteves, 3

Esteves, 4

Medication: when and how to use antioxidants

Fertile Infertile0

0.5

1

1.5

2

2.5

Seminal Reactive Oxygen Species (ROS)

(Log ROS + 1; cpm)

Pasqualotto et al., Fertil Steril 2000

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

Antioxidant TreatmentCochrane 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 cyclesShowell MG, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. Cochrane

Database ofSystematic Reviews 2011, Issue 1. Art. No.: CD007411. DOI: 10.1002/14651858.CD007411.pub2.

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, 7

~80 daysOld concept

~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.

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 fathersGrade a = rapid progressive motilityGrade b = slow/sluggish progressive motility 2Strict criteria

New WHO references valuesHow they were obtained

1,953 semen samples of recent fathers Time to pregnancy (TTP) ≤ 12 mo 5 studies in 7 countries on 3 continents Laboratories with QC only Morphology by strict criterion (Kruger) Progressive and non-progressive motility Lower reference limits (5th centile)

Esteves, 10

  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 (strict criteria) 4 15 44

% Alive (eosin-nigrosin staining) 58 79 91

Cooper et al: World Health Organization reference values for human semen characteristics. Hum Reprod Update 16: 231-245, 2010

Percentile distribution of semen characteristics values of recent fathers whose partners had a TTP ≤ 12

months, used to establish the reference limits in the 2010 WHO manual

Esteves, 11

Esteves, 12

New WHO references valuesCritical Appraisal

USA(Columbia, NYC, Minneapolis, LA)

AUSTRALIA (Melbourne)

NORWAY(Oslo)

FINLAND(Turku)

DENMARK (Copenhagen)

FRANCE(Paris)

UK(Edinburgh)

??

??? ?

Study Year Country TTP < 12 months stated

Sperm morphology evaluation criterion

Overlapping authorship or collaboration

among authors

Bonde et al. 1998 Denmark Yes David Yes

Slama et al. 2002 France, Denmark, UK, Finland

Yes David, Tygerberg Yes

Swan et al. 2003 USA No Tygerberg Yes

Haugen et al. 2006 Norway Yes Tygerberg No

Stewart et al. 2009 Australia Yes Tygerberg Yes

Studies used to establish the new limits for human semen characteristics in the

5th ed WHO Manual

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Esteves, 14

New WHO references valuesCritical Appraisal - Summary

Reasons for lower cutt-off

Merits Demerits

Different way of generating data:

• Method for semen analysis (higher QC standards; strict morphology)

• Population studied

Controlled studies No systematic review of fertile populations:

• Not representative of global fertile male population

Recent fathers with known TTP

Standardized semen analysis

Morphology using different criteria

Single semen specimen of each individual

Sperm Morphology Evaluation by the Strict Criteria

Head length5.0–6.0 m

Mid-piece1.5x head length

Tail 45 m

Head width 2.5–3.5 m

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)

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

Predictive Value of Normal Sperm Morphology for IVF and ICSI

Adapted from French et al., Fertil Steril 2010

≤4% >4%0%

20%

40%

60%

80%

Fertilization, Pregnancy and Miscarriage by Sperm Morphology Subgroups in

ICSI

Fertilization rate (%2PN)Clinical Pregnancy (%)Miscarriage (%)

≤4% >4%0%

20%

40%

60%

80%

Fertilization and Pregnancy by Sperm

Morphology Sub-groups in IVF

Fertilization rate (%2PN)Pregnancy rate (%)

Adapted from Coetzee et al., Hum Reprod Update 1998

*

*

*p<0.05

Mid-piece defect

Acrossomeless Double tail Tapered Cytoplasmic droplet

Genetically determined Stress-induced (Elevated OS)

Low ICSI Fertilization RatesPoor Embryo Morphology ?

Low Pregnancy Rates

Menkveld R et al. Significance of sperm morphology. AJA (2011); 13:59-68 Tesarik J et al. Paternal effect on embryo development. Hum Reprod (2004); 19: 611-15

Sperm Defects and Embryo Quality

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Diagnostic Tests• Beyond the routine semen analysis

1) Sperm DNA Integrity Testing

2) Y Chromosome Microdeletion Screening

Sperm DNA Integrity Testing

Esteves, 20

• Normal sperm chromatin essential for paternal genetic transmission

Background

• Infertility• Recurrent pregnancy loss• Poor outcomes in IUI and IVF

Sperm DNA Damage

• Quantification of sperm DNA strand breaks Principle

• SemenSpecimen

• Nuclear dyes (Acridine orange, SCSA)• Direct assessment of DNA breaks (TUNEL,

COMET)• Nuclear matrix assays (Halosperm)

Techniques

Sperm DNA Integrity Testing

Esteves, 21

Marker of Sperm Function

Unexplained Infertility• Normal

semen analysis

Candidates for IUI and

IVF

Recurrent Pregnancy Loss

Sperm DNA Integrity Testing & ART

DFI ≤30% DFI >30%0%

5%

10%

15%

20%

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

IVF ICSI0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

Pregnancy by Sperm DNA Integrity Results in IVF and

ICSI

DFI>30%

* <.05

*

Esteves

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Y Chromosome Microdeletion

• Deletions are the cause of spermatogenic failure

Background

• Men with non-obstructive azoospermiaTo whom?

• PCR of the long arm of Y-chromosome Principle

• BloodSpecimen

• Predict the chances of finding sperm on sperm retrieval techniques

Clinical Significanc

e

80%

5%10%5%

Genetic Male Infertil-ity

Klinefelter Syndrome (47,XXY)Y-chromosome microdeletionCongenital Vas Absence

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

Fertility Restoration

SpontaneousPregnancy

Surgical Treatment of Varicocele

Varicocelectomy for Fertility Restoration

Esteves, 26

Fertil Steril 2007;88:639–48.

Fertility Improvement

Sperm Retrieval in Azoospermia

Fertility Improvement

ICSIOutcomes

Fertility Restoration

SpontaneousPregnancy

Surgical Treatment of Varicocele• It can improve success of ART

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

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

Varicocele Repair Before Sperm RetrievalSperm 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)

Azoospermia• It is not a synonymous of sterility

• Normal sperm production

• Mechanical blockage • Vasectomy, Post-

infectious, Congenital

Obstructive• Sperm production

deficient or absent

• Cryptorchidism, Orchitis, Radiation, Chemotherapy, Trauma, Genetic, Gonadotoxins, Unexplained

Non-obstructive

Obstructive Azoospermia

• Microsurgical reconstruction

• TUREDPotentially treatable

• Epididymis• Testis• Simple and

effective

Sperm retrieval for ART

Watch the video at http://androfert.com.br/videos

Watch the video at http://androfert.com.br/videos

PERCUTANEOUS RETRIEVAL

Esteves SC, Verza S, Prudencio C, Seol B. Success of percutaneous sperm retrieval and intracytoplasmic sperm injection (ICSI) in obstructive azoospermic (OA) men according to the cause of obstruction. Fertil Steril. 2010;94 (Suppl):S233.

Non-obstructive Azoospermia

• Sperm production reduced or absent

• Geographic location unpredictable

Sperm Retrieval for ART

Untreatable condition

TESA

TESE

Controlled studies for NOA men

Fine Needle Aspiration

Open Biopsy

Friedler et al., Human Reprod 12:1488, 1997

4/37 (11%) 16/37 (43%)

Ezeh et al. Human Reprod 13:3075, 1998

5/35 (14%) 22/35 (63%)

Non-obstructive AzoospermiaTesticular Sperm Aspiration - TESA

Watch the video at http://androfert.com.br/videos

MICRO-TESE

Success Rate

22%

40.00%

NOA

TESA/TESE

N=131; *hypospermatogenesis excluded

Esteves et al.; Fertil Steril 2010; 94:S132

Micro-TESE39%

P=.03

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

Key MessagesAntioxidants helpful to decrease oxidative

stress.Interventions impact on semen quality 60

days later.WHO lowered semen analysis reference

values.

Sperm DNA integrity and Y-chromosome microdeletion testing are

of prognostic value.

Treatment of Clinical Varicoceles prior to ART beneficial for patient subgroups.

Sperm retrieval and reproductive potential is dependent on the type of

azoospermia.Esteves, 41