Male infertility current concepts for reproductive specialists

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Merck-Serono Stand-alone Meeting in Reproductive Medicine …

Merck-Serono Stand-alone Meeting in Reproductive Medicine
August 2011 Cochin, India

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  • Most of the infertile patients that we see in our clinical practice are at risk of excessive Oxidative Stress. Reactive oxygen species are products of aerobic metabolism. At certain levels they are not detrimental, but several conditions induce an overproduction of ROS, causing oxidative stress. Oxidative stress, that can be measured either in the seminal plasma or directly in the sperm by different methods, are higher in infertile men than fertile ones. Regardless of its cause, the end product of OS is the oxidation of sperm structures that become dysfunctional. The impairment of sperm function caused by OS may be either the only factor causing infertility or a contributor to a recognized disease that cause infertility. Options to minimize OS include: i) the treatment of the underlying pathology, if possible, ii) the removal of risk factors, which is not always feasible, or iii) by the administration of antioxidant supplementation.


  • 1. MerckSerono Stand-alone Meeting – Kochi, India – August 2011 Sandro Esteves, MD, PhD Director, ANDROFERT Center for Male Reproduction Campinas, BRAZILEsteves, 1
  • 2. 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 practiceEsteves, 2
  • 3. 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 sterilityEsteves, 3
  • 4. Medication: when and how to use antioxidants 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 2000Esteves, 4
  • 5. Evidence-based Use of Antioxidants in Male Infertility Author Antioxidant Agent ResultsGeva et al., 1996 Vit E 200mg Increased fertilization in IVFSuleiman et al, 1996 Vit E 100mg Decreased ROS; increased spontaneous PRWong et al., 2002 Folic acid 5mg + Zinc Increased total sperm count 66mgGreco et al., 2005 Vit C 1,0g + E 1,0g Improved sperm DNA integrityGreco et al., 2005 Vit C 1,0g + E (1,0g) Increased CPR and IR in ICSI cyclesTremellen et al., 2007 Menevit® (vit C + E; Increased IR/PR in IVF/ICSI zinc 25mg; selenium cycles 26mcg; lycopene 6mg)Boxmeer et al., 2009 Decreased folate in Increased sperm DNA seminal plasma fragmentation
  • 6. 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 effectcount, sperm motilityAdverse effects 6 426 No effectImprove the outcomes of live birth and pregnancy rate for subfertile couples undergoing ART cycles Showell MG, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. Cochrane Database of Systematic Reviews 2011, Issue 1. Art. No.: CD007411. DOI: 10.1002/14651858.CD007411.pub2.
  • 7. 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
  • 8. From Initiation of SpermProduction to Ejaculation ~60 New concept days Misell LM et al.: A stable isotope-mass spectrometric method for measuring human spermatogenesis kinetics in vivo. J Urol. 2006; 175: 242-6.
  • 9. Semen analysis • New reference values by WHOSemen Parameter WHO 1999 WHO 20101Volume (mL) ≥2.0 1.5Count (x106/mL) ≥20 15Total sperm number per ejaculate ≥40 39Motility (%) ≥50 (a+b) 32 (a+b)Vitality (%) ≥75 58Morphology (%)2 (14) 4Leukocytes (x106/mL) <1.0 <1.01Lower Limit (5% percentile); Recent fathersGrade a = rapid progressive motilityGrade b = slow/sluggish progressive motility2Strict criteria
  • 10. New WHO references values How 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
  • 11. 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 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, 2010Esteves, 11
  • 12. New WHO references values Critical Appraisal FINLAND NORWAY (Turku) (Oslo) UK DENMARK (Edinburgh) (Copenhagen) USA FRANCE ? (Columbia, NYC, ? Minneapolis, LA) (Paris) ? ? ? ? AUSTRALIA (Melbourne)Esteves, 12
  • 13. Studies used to establish the new limits for human semen characteristics in the 5th ed WHO Manual Study Year Country TTP < 12 Sperm Overlapping months morphology authorship or stated evaluation collaboration criterion among authors Bonde et al. 1998 Denmark Yes David Yes Slama et al. 2002 France, Denmark, Yes David, Tygerberg Yes UK, Finland Swan et al. 2003 USA No Tygerberg Yes Haugen et al. 2006 Norway Yes Tygerberg No Stewart et al. 2009 Australia Yes Tygerberg YesEsteves, 13
  • 14. New WHO references values Critical Appraisal - Summary Reasons for lower Merits Demerits cutt-off Different way of Controlled studies No systematic review generating data: of fertile populations: • Method for semen Recent fathers with • Not representative of analysis (higher QC known TTP global fertile male standards; strict population morphology) Standardized semen Morphology using • Population studied analysis different criteria Single semen specimen of each individualEsteves, 14
  • 15. Sperm Morphology Evaluation by the Strict Criteria Head width 2.5–3.5 m Oval head, smooth shape Acrosome: 40%-70% head area No neck, mid-piece or tail defects Head length 5.0–6.0 m ‘Borderline’ forms = abnormal Mid-piece 1.5x head length Tail 45 m Sperm dimensions on Diff-Quik staining Abnormal spermatozoa Abnormal spermatozoa (head defect) (tail defect)TF Kruger et al., 1986
  • 16. Predictive Value of Normal Sperm Morphology (WHO 2010) for IUI Pregnancy rate per cycleStrict 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% 12.8% P <0.001 (64/731) (208/1628)Adapted from: J Van Waart, TF Kruger, CJ Lombard et al. Predictive value of normal sperm morphologyin intrauterine insemination (IUI): a structured literature review. Hum. Reprod. Update (2001) 7:495-500
  • 17. Predictive Value of Normal Sperm Morphology for IVF and ICSI Fertilization and Fertilization, Pregnancy and Pregnancy by Sperm Miscarriage by Sperm Morphology Subgroups Morphology Subgroups in in IVF ICSI * *p<0.05 80% 80% 60% 60% 40% * 40% 20% 20% 0% 0% ≤4% >4% ≤4% >4% Fertilization rate (%2PN) Fertilization rate (%2PN) Pregnancy rate (%) Clinical Pregnancy (%) Miscarriage (%)Adapted from Coetzee et al., Hum Reprod Update 1998 Adapted from French et al., Fertil Steril 2010
  • 18. Sperm Defects and Embryo Quality Mid-piece Acrossomeless Double tail Tapered Cytoplasmic defect droplet Genetically determined Stress-induced (Elevated OS) 18 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: 611-15Esteves
  • 19. Diagnostic Tests • Beyond the routine semen analysis1) Sperm DNA Integrity Testing2) Y Chromosome Microdeletion Screening
  • 20. Sperm DNA Integrity Testing • Normal sperm chromatin essential Background for paternal genetic transmission • Infertility Sperm DNA • Recurrent pregnancy loss Damage • Poor outcomes in IUI and IVF • Quantification of sperm DNA strand Principle breaks Specimen • Semen • Nuclear dyes (Acridine orange, SCSA) Techniques • Direct assessment of DNA breaks (TUNEL, COMET) • Nuclear matrix assays (Halosperm)Esteves, 20
  • 21. Sperm DNA Integrity Testing Unexplained Infertility • Normal semen analysis Candidates Recurrent for IUI and Pregnancy Loss IVFEsteves, 21
  • 22. Sperm DNA Integrity Testing & ART Pregnancy by Sperm Pregnancy by Sperm DNA DNA Integrity Results in Integrity Results in IVF and IUI ICSI 20% 50.00% * <.05 15% 40.00% 30.00% * 10% 20.00% 5% OR 0.07 (0.01-0.48) 10.00% 0% 0.00% DFI ≤30% DFI >30% IVF ICSI Live birth (%) DFI>30% Adapted from Bungum et al., Hum Reprod 2007 Adapted from Bungum et al., Hum Reprod 2007 22Esteves
  • 23. Y Chromosome Microdeletion • Deletions are the cause Genetic MaleBackground of spermatogenic failure Infertility • Men with non-obstructiveTo whom? azoospermia 10% 5% 5% • PCR of the long arm of Principle Y-chromosome 80%Specimen • Blood Clinical • Predict the chances of Klinefelter Syndrome (47,XXY) finding sperm on sperm Y-chromosome microdeletionSignificance retrieval techniques Congenital Vas Absence
  • 24. Y Chromosome Microdeletion AZFa deleted AZFb deleted AZFc deleted Germ cell Aplasia Maturation Arrest HypospermatogenesisNo retrievable sperm No retrievable sperm 70% chance of retrieving testicular sperm for ICSI
  • 25. Surgical Treatment of VaricoceleFertilityRestorationSpontaneousPregnancy
  • 26. Varicocelectomy for Fertility Restoration Fertil Steril 2007;88:639–48.Esteves, 26
  • 27. Surgical Treatment of Varicocele • It can improve success of ART Fertility ImprovementFertility ICSIRestoration OutcomesSpontaneousPregnancy Fertility Improvement Sperm Retrieval in Azoospermia
  • 28. Varicocele Repair Before ARTClinical 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 Total Number of Motile Microsurgical Sperm (x106) varicocele repair prior 15.4 to ICSI (N=80) P<0.01 6.7 ICSI in the presence of varicocele (N=162) Pre-op Post-op
  • 29. Varicocele and ICSI Outcomes Treated Varicocele Untreated Varicocele 78%* 66% *P<0.05 46%* 31% 31% 22% Fertilized Eggs Live Birth (%) Miscarriage (%) (%2PN)Odds ratio 1.87 0.4395% CI 1.08 - 3.25 0.22 – 0.84P-value 0.03 0.01 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
  • 30. Varicocele Repair Before Sperm Retrieval Sperm 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 Successful Sperm Retrieval Microsurgical Rate varicocele repair priorto sperm retrieval ICSI OR: 2.63 (95% CI: 1.05-6.60; P=0.03) (N=66) 53% 30%Sperm Retrieval in thepresence of varicocele (N=30) Treated Varicocele Untreated Varicocele
  • 31. Azoospermia • It is not a synonymous of sterility Non-Obstructive obstructive• Normal sperm production • Sperm production deficient or absent• Mechanical blockage • Cryptorchidism, Orchitis, Ra• Vasectomy, Post- diation, Chemotherapy, Trau infectious, Congenital ma, Genetic, Gonadotoxins, Unexplained
  • 32. Obstructive Azoospermia • MicrosurgicalPotentially reconstruction treatable • TURED Sperm • Epididymis • Testis retrieval • Simple and for ART effective
  • 33. Watch the video at
  • 34. Watch the video at
  • 35. Esteves SC, Verza S, Prudencio C, Seol B. Success of percutaneous sperm retrieval andintracytoplasmic sperm injection (ICSI) in obstructive azoospermic (OA) men according tothe cause of obstruction. Fertil Steril. 2010;94 (Suppl):S233.
  • 36. Non-obstructive Azoospermia Sperm • Sperm productionUntreatable reduced or absent Retrieval • Geographic location condition for ART unpredictableTESATESE
  • 37. Non-obstructive Azoospermia Testicular Sperm Aspiration - TESAControlled studies Fine Needle Open Biopsyfor NOA men AspirationFriedler et al., 4/37 (11%) 16/37 (43%)Human Reprod 12:1488, 1997Ezeh et al. 5/35 (14%) 22/35 (63%)Human Reprod 13:3075, 1998
  • 38. Watch the video at
  • 39. NOA 39% P=.03 Micro-TESE TESA/TESE Success RateN=131; *hypospermatogenesis excluded Esteves et al.; Fertil Steril 2010; 94:S132
  • 40. Sperm Retrieval Rates and Reproductive Potential of Azoospermic Men in ICSI 97.9% Obstructive (N=142) Non-obstructive (N=172) 55.2% 38.2% 25.0% Sperm Retrieval Live BirthOdds ratio 43.0 1.8695% CI 10.3 – 179.5 1.03 – 2.89P-value <0.01 0.03Prudencio C, Seoul B, Esteves SC. Reproductive potential of azoospermic men undergoingintracytoplasmic sperm injection is dependent on the type of azoospermia. Fertil Steril 2010; 94 (4): Suppl. S232-233.
  • 41. Key Messages Antioxidants 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