Urologist and male fertility doctor for vasectomy and vasectomy reversal, sperm retrieval, testicular mapping, varicocele repair and ejaculatory duct repair, Dr. Paul Turek, speaks about Fertility Restoration after Cancer: Current and Future Therapies. Dr. Turek is director of The Turek Clinic. Located in San Francisco, California, The Turek Clinic provides world-class patient care with an essential holistic approach. (WARNING: Images in slides not appropriate for all audiences due to subject matter.)

1. Fertility Restoration after Cancer: Current and Future Therapies Paul J. Turek MD, FACS, FRSM Director, The Turek Clinic San Francisco

2. Preservation Sperm banking Sperm harvest; electroejaculation Finding sperm during treatment Finding sperm after treatment Hormonal suppression Restoration Germ cell transplants Germ cell maturation in vitro Stem cell to germ cell maturation Techniques to Preserve or Restore Fertility

5. Sperm Banking with Cancer: Patient and Provider Attitudes PROVIDERS Survey of 718 oncology staff Two tertiary cancer centers 91% believed banking should be offered 52% actually offered it to patients Reasons: high cost, convenience PATIENTS 904 men with cancer Two tertiary cancer centers 51% wanted future fertility 60% recall being informed 24% banked sperm Reasons: Lack of information Serious room for improvement: Practice standards? Team medicine? Schover et al. J Clin Oncol. 20:1880, 2002 Schover et al. J Clin Oncol. 20:1890, 2002

7. Preservation Sperm banking Sperm harvest; electroejaculation Finding sperm during treatment Finding sperm after treatment Hormonal suppression Restoration Germ cell transplants Germ cell maturation in vitro Stem cell to germ cell maturation Techniques to Preserve or Restore Fertility

8. The Seager Electroejaculator

9. (n=44 couples @UCSF) Issues: Currently a limited/limiting resource Couples pregnant 64% Vib Stim only 14% Couples with ICSI 59% Preg. without tech. 10%

11. Preservation Sperm banking Sperm harvest; electroejaculation Finding sperm during treatment Finding sperm after treatment Hormonal suppression Restoration Germ cell transplants Germ cell maturation in vitro Stem cell to germ cell maturation Techniques to Preserve or Restore Fertility

12. Banking Sperm Before Radical Orchiectomy Banking Sperm During Radical Orchiectomy Not Doing Radical Orchiectomy…. Other Ways to “Bank….”

13. “ Smarter” Radical Surgery -Partial Orchiectomy

14. Finding Sperm after Cancer Treatment Microdissection FNA Mapping

15. How is FNA Mapping Different from Microdissection? Office FNA Map Office FNA Map Sperm Found? Yes Yes No No IVF/ICSI "Directed" TESE IVF/ICSI "Directed" TESE Donor Sperm Adoption Donor Sperm Adoption FNA Mapping Microdissection TESE in OR Sperm Found? IVF/ICSI Yes No Donor Sperm Adoption +/- Pregn Microdissection

16. Where is FNA Mapping Today? FNA Sites/Testicle Sperm Detection Rate Beliveau and Turek. Asian J Androl 13; 225, 2011

17. Damani et al. JCO 15: 930, 2002 Chan et al. Cancer 92: 1632, 2001 Chemotherapy and Fertility: Finding Sperm after Treatment 17 23 #Pts % With Sperm 9/20 (45%) 15/23 (65%) No birth defects…yet Zorn et al. Int J Androl 29: 521, 2006 13 58% Hsiao et al. JCO 29: 1607, 2011 73 27/73 (37%)

18. The Issue of Sperm Genotoxicity Chromosomal Damage DNA Damage T G A C G C T A A T

19. -100 0 100 200 300 0 50 100 150 200 Hyperhaploid sperm Days NOVP CHEMO Genotoxicity: Sperm Aneuploidy with Chemotherapy WA Robbins et al. Nat Genet. 16: 74-78, 1997 Hodgkins Disease FISH on chromos: X, Y, 8

20. 0 20 30 60 70 0 5% 10% 15% 20% Comet Tail Length (um) 25% 10 40 50 80 90 Frequency (%) Genotoxicity: Sperm DNA Strand Breaks with Chemotherapy Chatergee et al. Hum Reprod. 15: 762-66, 2000 CLL patient, fludarabine chemo, Comet assay of individual sperm 8 weeks chemo 11months post-chemo Baseline

21. Chemotherapy and Fertility: Finding Sperm after Treatment Special cases: Bone Marrow Transplant Paternity after hematologic malignancies that involve high dose chemotherapy , total body irradiation and bone marrow transplantation Ignatov et al. BMT 45; 1474, 2010

22. Special cases: Pubertal patients 13 y.o. Tanner III-IV male with L knee synovial sarcoma. Chemotherapy planned. During Broviac line placement, testis tissue extraction performed for cryopreservation. Mature sperm found.

23. Preservation Sperm banking Finding sperm during treatment Finding sperm after treatment Hormonal suppression Restoration Germ cell transplants Germ cell maturation in vitro Stem cell to germ cell maturation Techniques to Preserve or Restore Fertility

24. GnRH Flut Cytoxan Both 0 20 40 60 80 100 Percent Repopulation of Rat Testis Meistrich et al. J Androl. 16: 334, 1995 Chemotherapy and Fertility: Spermatogenic Shutdown Pre-chemotherapy treatment with total androgen blockade

25. Chemotherapy and Fertility: Spermatogenic Shutdown Issues: Promising research but feasibility unclear (lead time needed). Giving androgen blockade to rats AFTER chemotherapy improved spermatogenesis. Meistrich ML et al. Cancer Res. 59: 3557, 1999 Udagawa et al. Int J Urol. 8: 615-622, 2001 Androgen blockade in humans AFTER chemotherapy has not improved spermatogenesis (i.e. testis biopsy and semen quality). Thomson AB et al. Hum Reprod. 17: 1715, 2002

26. Preservation Sperm banking Finding sperm during treatment Finding sperm after treatment Hormonal suppression Restoration Germ cell transplants Germ cell maturation in vitro Stem cell to germ cell maturation Techniques to Preserve or Restore Fertility

27. Germ Cell Transplantation: Stem cells (LacZ) Brinster RL.Science. 296: 2174, 2002 1:5000 w-/w- Busulfan

29. Spermatogonial In Vitro Maturation 1N 4N SCF Induced 1 week Normal Mouse Testis Spermatogonial Cell line SCF Induced 3 weeks

30. Spermatogonial In Vitro Maturation L-X Feng et al. Science. 297: 392, 2002 Synaptonemal complexes Crossovers Spermatids! (Acr3-EGFP)

31. Embryonic stem cells Germ line stem cells Stem Cells, Germ Cells and Sperm Endoderm Mesoderm Ectoderm Kossack et al. Stem Cells 27:138, 2009 IPS

32. Germ Line Cells

33. Embryonic stem cells Germ line stem cells Stem Cells, Germ Cells and Sperm Endoderm Mesoderm Ectoderm Kossack et al. Stem Cells 27:138, 2009 IPS

34. How Far Can You Go in a Dish? Current Fertility

35. An Artificial Human Testicle

36. Sertoli Cells

37. Myoid cells/Leydig cells/Stroma

38. What Does an Artificial Human Testicle Look Like? Sertoli cells Fibronectin Stromal cells Leydig cells “ Germ cells” (hESCs, iPSCs)

40. Thanks to : Uche Ezeh M.D. Mark Fox Ph.D Shai Shefi M.D. Amander Clark Ph.D Connie John Ph.D Renee Reijo Pera Ph.D Lance Armstrong Foundation California Urology Foundation National Institutes of Health California Institute of Regenerative Medicine