1. How to Manage Truly and Virtually Azoospermic Men for IVF-ICSI? Paul J. Turek M.D. Emeritus Professor and Endowed Chair Department of Urology, University of California San Francisco Director, The Turek Clinic, San Francisco, CA

3. 17. I read in one of the papers that couples can get pregnant with sex. Is that true? A patient, 1998

4. Obstructive Non-obstructive The Problem of Azoospermia 5% of infertile men History Physical Exam Hormones Semen Analysis Repair ICSI ICSI

5. Case #2 There once was a couple who wanted children. Partner is 37 y.o. “ never” pregnant 38 y.o. with no prior paternity. FSH, T normal. Testes: 20mL Testis biopsy: normal Why am I doing this ? I hope he doesn’t find out…..

6. Case 2 35 yo healthy man with 2 years of unprotected sex. Partner 38 years old and no prior pregnancies. His exam reveals atrophic testes bilaterally. Has a grade III left varicocele FSH 20 (1-8 normal); Testosterone 330 (>300) Normal volume azoospermia; Pellet: no sperm. Testis biopsy: Sertoli cell only

7. Infertility M.D. Provider (Male or Female) Genetic Counseling PROGENI  Medical Evaluation Y microdeletions Karyotype CFTR/5T Post-Test Counseling Treatment Plan

8. When to do Genetic Testing? Sperm <5 million/mL No sperm/testis failure Idiopathic Obstruction, absent vas deferens Other syndromes Scenario Y Delet. Karyotype Cyst. Fibr X Whatever fits! X X X X Case #1 40% Case #2 30%

9. FNA mapping or Microdissect. Negative Azoospermia Examine semen pellet Sperm absent Palpable vas deferens? Sperm present Evaluate Oligospermia Vas not palpable CBAVD MESA, TESE Genetic Eval: CF Mutations Vas palpable FSH, Testosterone FSH elevated Nonobstructive Azoospermia Genetic Eval: Y-del; karyotype Diagn. Testis Biopsy +/- cryopreservation +Sperm - Sperm TESE FSH normal FSH, LH Testosterone low Serum prolactin Diagnostic Testis biopsy Sperm absent Sperm present Normal Elevated Obstruct. azoospermia. Genetic Eval: CF Vasography, VV, EV MRI pituitary for prolactinoma Donor sperm IUI Adopt Tumor visible No tumor Transphenoidal resection Dopamine agonist Evaluate hypog. Hypogonadism (ACTH, TSH, GH) Azoospermia Algorithm Turek PJ. Nat Clin Pract. 2005; 2:1

10. Azoospermia: The Centrifuged Pellet Jaffe et al. J Urol. 1998; 159: 1548-50 (NS, n=17/group) (600-1000 x g for 15 minutes) TYPE # Pts % Sperm Variability Obstructive 70 18.6% 41% Non obstructive 70 22.8% 12%

11. Intermittent Complete Azo-Asthenospemia: Chance of Sperm at IVF-ICSI (n=15) Zenke et al. ASRM 2003 History Physical exam Semen analysis, pellet FNA Map IVF-ICSI At ICSI: Motile ejaculated sperm? TESA/TESE Yes No

12. Does Sperm Origin Affect ICSI Fertilization Rates? (n=314 ICSI cycles in 264 couples at UCSF) Shen et al, ASRM 2003 High 2PN rates with ICSI are possible regardless of sperm source.

13. Does Motile vs. Nonmotile Sperm Make a Difference? Bachtell et al. Hum Reprod. 1999, 14:101 Biologically, it appears to….. Motility *Viability Fresh Thawed Fresh Thawed Testis, NOA 5% 0.2% 86% 46% Epididymis, OA 22% 7% 57% 24% Vas deferens, fertile 71% 38% 91% 51%

14. Does Motile vs. Nonmotile Sperm Make a Difference? Clinically….. Park YS et al. Fert Steril 2003, 526:30 (n=160 patients with obstruction) FRESH THAWED Motile Nonmotile Motile Nonmotile testis testis testis testis sperm sperm sperm sperm 2PN Fert Rate 77% 29% 70% 51% Pregnancy Rate 44% 20% 34% 27%

15. What About “Delayed Fresh” Sperm Retrieval? Morris et al. J Urol. 2007, 178:2087-91 Time After Sperm Retrieval % Motility Testis sperm motility

16. What About “Delayed Fresh” Sperm Retrieval? Wood et al. J Androl. 2003, 24: 67-72 ICSI Outcomes FRESH FRESH Epididymal Epididymal Testis Testis < 4 hrs 1-2 days < 4 hrs 1-2 days # ICSI cycles 22 7 35 38 Egg age 33 yrs 33 yrs 32 yrs 32 yrs Mean # Oocytes 8.1 11 9 8 2PN Fert Rate 67% 56% 62% 71% Clin. Pregn. 4/22 (18%) 1/7 (14%) 8/35 (23%) 9/38 (24%)

18. Testis Sperm Retrieval: Evidence-Based Guidelines Donoso, Tournaye, Devroey. Hum Reprod Upd. 2007, 13: 539-549 Obstructive vs. Nonobstructive Azoospermia Meta-analysis of 1103 cycles, non-randomized studies Obstructive vs Nonobstructive RR (CI) 2PN fertilization 1.18 (1.13-1.23) Clinical pregnancy rate 1.36 (1.1-1.69) Ongoing preg. rate 1.19 (0.87-1.61) Implantation rate 1.01 (.87-1.61) Miscarriage rate 0.84 (.48-1.48) Fixed model Nicopoullos et al. Fert Steril. 2004, 82: 691-701

21. History Physical Exam Hormones Semen Analysis Obstructive Non-obstructive The Problem of Azoospermia Repair ICSI ICSI 5% of infertile men

22. Vasal (MVSA; PVSA) Epididymal (MESA, PESA) Testicular (TESA, TESE, MicrodissectionTESE) Obstructive Azoospermia Everything works !!

23. Obstructive Azoospermia Guideline: Least invasive, least damaging, best yield. Turek et al. Ass Reprod Rev. 1999, 9: 60-64,

25. 1% 5% 40% 1% 5% 40% MESA PESA Why MESA and not PESA? 40% 8%

27. Testicular (TESA, TESE, MicrodissectionTESE) Nonobstructive Azoospermia Surgeon beware !!

28. Non Obstructive Azoospermia Finding Testicular Sperm Histologic Pattern Cases Recovery Rate (%) Normal 173 100% Hypoplasia 16 100% Maturation arrest 76 51% Sertoli cell-only 112 50% Tubular sclerosis 18 39% Tournaye et al. Hum. Reprod. 12: 80, 1997 52% in

29. Cryptorchidism 52-74% Variocele 63% Epididymitis 67% Mumps 67% Torsion >50% Post-chemotherapy 55-75% Genetic AZF a, b 0% Genetic AZF c 75% Idiopathic 50-60% Chance of Finding Sperm by NOA Diagnosis Shefi and Turek, submitted Raman and Schlegel. J Urol.170:1287, 2003 Hopps et al. Hum Reprod. 180:1660, 2003 Damani et al. JCO. 15: 930, 2002

30. Can we predict if sperm are present in NOA? History of ejaculated sperm FSH, inhibin level Testis biopsy histology Testicular size . Tournaye et al. Hum. Reprod. 12: 80, 1997. Seo JT and Ko W-J. Int J. Androl. 24: 306, 2001 Raman and Schlegel. J Urol.170:1287, 2003

31. Testis Biopsy: Results Normal Hypo Maturation Arrest Sertoli cell Only Only inform us of the biopsied area. How about elsewhere?

32. Why is sperm prediction important? 1. Can minimize emotional and financial cost of IVF cycles. 2. Can minimize trauma/damage to testis during sperm harvesting.

33. 0 20% 40% 60% 80% 100% Sensitivity and Sampling Technique in NOA Testes Likelihood of finding sperm 1 biopsy 3 FNA MAP Weiss et al. Harefuah 132: 614, 1997 Kim et al. J. Urol, 157: 144, 1997 30% 36% 50%

34. Approaches to the NOA Patient a) Who has sperm? b) Where is it? 1. Simultaneous diagnostic and therapeutic biopsies with &quot;Up front&quot; cryopreservation of sperm. 2. Realtime Multi-biopsy or deep (Micro) biopsy TESE with/without diagnostic biopsy. 3. Diagnostic Systematic FNA &quot;mapping&quot; of testes with subsequent &quot;directed&quot; TESE. Van Peperstraten A. et al. Cochrane Database Syst Rev 2006; 3:CD002807.

35. FNA Mapping, Directed TESE How Can We Find Sperm? And Microdissection TESE

36. How Can We Find Sperm? Microdissection TESE in OR Sperm Found? IVF/ICSI Yes No Donor Sperm Adoption +/- Pregn Microdissection Office FNA Map Sperm Found? Yes No IVF/ICSI &quot;Directed&quot; TESE Donor Sperm Adoption FNA Mapping

37. The Microdissection TESE Concept: Testis seminiferous tubules containing sperm are “thicker” than those that don’t. Schlegel PN. Hum Reprod 14: 131, 1999 Points: Systematic examination of testis lobules under 25x microscopy, general anesthesia

38. The Microdissection TESE Results: Retrospective comparison of multibiopsy to microdissection approaches Schlegel PN. Hum Reprod 14: 131, 1999 Multibiopsy 10/22 (45%) 720 mg Microdissection 17/27 (63%) 9.4 mg Approach Success (%) Tissue

39. The Microdissection TESE Amer M, et al. Hum Reprod 15: 653, 2000 Approach Success (%) Tissue TESE biopsy 30/100 (30%) 54 mg Microdissection 45/100 (45%) 4.6mg Concept: 100 men with “identical” bilateral histology. One side TESE, the other microdissection

40. Microdissection TESE Amer et al 2000 100 45% 30% Okada et al 2002 98 45% 17% Okubu et al 2002 17 48% 24% Tsujimura et al 2002 93 43% 35% Ramon et al 2003 321 62% 58% Study #Pts %MicroTESE %TESE Success Success 49% 33%

41. The Microdissection TESE Concerns: 1) Operating microscope needed 2) General anesthesia 3) Expertise with experience 4) Long term effects (especially if bilateral) 5) A repeatable procedure?

42. Prostate Biopsies 1 2 3 4 5 6 X X X X X X X X X Testis The Idea Testis FNA Mapping

43. Office FNA Map Sperm Found? Yes No IVF/ICSI &quot;Directed&quot; TESE Donor Sperm Adoption Testis FNA Mapping

44. Percutaneous Fine Needle Aspiration (FNA) The Set up The syringe/holder

45. 1. Local anesthesia 2. Testis “wrap” 3. Mark out sites (template) 4. Aspirate and smear 5. Apply pressure Percutaneous Fine Needle Aspiration (FNA)

46. Fine Needle Aspiration (FNA) Mapping Template 1 2 3 6 5 4 9 8 7 11 10 12 13 14 17 16 15 20 19 18 22 21 R L

47. FNA and “Directed TESE”

48. FNA Mapping-TESE Turek et al 2000 96 45% 95% (8) Lewin et al 1999 85 59% 59% (15) Hekimgil et al 2002 60 60% 60% (6) Cohen et al 2003 35 49% ----- Study #Pts % Sperm %TESE Success Success 53% Turek et al. J. Urol. 163: 1709, 2000 Hekimgil et al. BJU Inter. 87: 834, 2001 Cohen et al. Fertil Steril. 80: P344, 2003 Lewin et al. Hum Reprod. 14: 1785, 1999

49. FNA Mapping-Directed TESE Concerns: 1) No operating microscope needed 2) Local anesthesia 3) FNA expertise 4) Long term effects 5) A repeatable procedure?

50. What about the toughest (needle-haystack) cases? Replace “Directed” TESE with “Directed Micro-TESE”

51. Turek et al. ASRM 2004 Testis Biopsy (n=22) FNA Map (n=152) Sperm Present (n=174) TESE (n=29) MicroTESE (n=16) Proceed to IVF-ICSI (n=69 patients, 83 procedures) <2 sites sperm/map >2 sites sperm/map or hypo bx Success 13/16 (81%) Success 26/29 (90%) TESA (n=38) All FNA sites/map or hypo bx Success 37/38 (98%) Results

52. Testis sperm aspiration (TESA) Testis sperm extraction (TESE) Testis microdissection (MicroTESE) Not all sperm retrieval procedures are the same 20% of NOA men Least Invasive Most invasive

53. Laboratory Effort in Sperm Retrieval Cases Procedure MESA TESA TESE MicroTESE Mans-hrs 1 1-2 2-4 4-6 needed

54. Does “Mapping” Help in Redo NOA cases? TESA/TESE Attempts (n=51) (n=12) (n=3) % Patients Successful 90% 91% 100%

55. 1. Where are sperm generally found? 2. Are there any &quot;hot spots&quot; or &quot;sperm rich&quot; areas? 3. Is there any side to side correlation? 4. Are certain histologies more likely to have sperm? What have we learned? Testis FNA Mapping

56. 1 2 3 Sperm Frequency Maps Where are sperm found?

57. How “Patchy” or “Focal” is Sperm Production? Bx-FNA Discrepancy 27% FNA + Bx - Intratestis Variability - + 25% of testes Intertestis Variability - + 19% of testes Turek et al. J. Urol. 163: 1709, 2000

58. The FNA Map: An Archival Tool 1. What germ cells are present in the testis? 2. What other pathology is present? 3. Important for patient phenotyping. Meng MV et al. Hum Reprod. 16: 529, 2001 Nudell DM et al. Hum Reprod. 15, 1289, 2000 Fox M et al. Mol Reprod Dev. In press 4. Can it replace the standard biopsy? Meng et al. Am. J. Surg. Path. 25: 71, 2001

59. What’s Really New? MRI MRS Metabolic imaging of the testes with NMR

60. NMR Spectroscopy of the Testis Ex Vivo study of Testis Biopsies from Normal and Infertile Men Aronson, Kurhanewicz, Turek

61. MR Spectroscopy: Determining the Metabolic Fingerprint of the Normal and Abnormal Testis A non-invasive way to determine presence and location of spermatogenesis? Aaronson et al, 2007

63. A tough nut to crack No standard approach exists Be kind to your embryologists Be kind to your patients X X X X X X X X X How to Manage Truly and Virtually Azoospermic Men for IVF-ICSI?