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Merck-Serono Stand-alone Meeting in Reproductive MedicineAugust 2011 Cochin, India
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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
Esteves, 13
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
Esteves
18
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
22
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