CURRENT APPROACHES IN SEVERE MALE INFERTILITY

1. CURRENT APPROACHES IN SEVERE MALE INFERTILITY Prof. Semra Kahraman M.D. Bio.Çağrı Beyazyürek, Zafer Candan, Sevil Ünal, Semra Mılık, Assoc. Prof. Semih Özkan M.D. Istanbul Memorial Hospital, ART and Reproductive Genetics Center Istanbul, Turkey

2. SEVERE MALE INFERTILITY • Genetic factors • Preimplantation genetic diagnosis • Surgical sperm recovery techniques • Sperm DNA fragmentation • Derivation of gamete cells from embryonic stem cells • Development of artificial gametes

3. Male Infertility and Genetics • Structural Chromosomal Abnormalities Translocations (Robertsonian, Reciprocal, Cryptic) Duplication, inversion, insertion • Numerical Chromosomal Abnormalities Gain or loss of entire chromosomes • Micro or macrodeletions on Y chromosome • Gene defects

4. GENETIC FACTORS İMH ART and Reproductive Genetics Center • Karyotype analysis of 1935 infertile men with severe oligozoospermia or azoospermia • 1214 cases: Non-obstructive azoospermia (NOA) • 721 cases: Severe oligoasthenoteratozoospermia (OAT) (total sperm concentration in the whole ejaculate< below 5 million). • (1364 cases: Y-microdeletion analysis)

5. RESULT In cases with severe male factor infertility the incidence of having an abnormality in at least one test(karyotype analysis or Y-microdeletions) is 16,6% in our study.

6. Distribution of normal and abnormal karyotypes in infertile menİMH ART and Reproductive Genetics Center • Klinefelter Syndrome was the most frequently detected abnormality (57.3% of detected abnormalities)

7. Chromosomal Variants • Heterochromatin polymorphism is considered as a variant of a normal karyotype, but is more frequent in infertile men. • More attention must be directed to infertile men with heterochromatin polymorphism

8. Y chromosome microdeletion results in severe male infertility AZFc deletion 82%

10. Y-microdeletion rate in several studies CountryPatient Number% Our study13647.7 India839.6 Spain50 16.0 Japon63 15.8 USA, Australia50 20.0 USA108 7.0 France53 9.4 Finland201 9.0 China101 11.0 Slovenia226 4.4 Taiwan94 11.7 New Zealand657.7

11. Patients having both karyotype abnormality and Y-micro-deletion

12. Why Y-chromosome Micro-deletion analysis before TESE procedure? The deletion types of AZF a,b and c loci on Yq11, are the potential prognostic factors in patients planned to undergo TESE/mic-TESE procedures. cAZF c = Approximately 50% of the cases, mature spermatozoa cAZF b = Nearly impossible to find mature spermatozoa cAZF b+c and cAZF a+b+c = Total absence of testicular spermatozoa

13. Y-microdeletion and Mic-TESE

14. Sperm retreival rates of TESE patients with same micro-deletions

15. Conclusions • The high frequencies of cytogenetic abnormalities and Y micro deletions definitely suggest the need for genetic screening and counselling in severe male factor cases. • Karyotyping should be regarded as a mandatory part of the pre-treatment screening process for all men referred for ICSI. • Y-deletion analysis test is neccessary before deciding TESE procedure.

16. Preimplantation Genetic Diagnosis for Male Infertility(Aneuploidy)

17. Golden Standarts • Biopsy: Single Blastomere • Fixation: Hypotonic+Fixative method • Hybridization: FISH at least 9chromosomes: 13,15,16,17,18,21,22,X,Y • Analysis at least 2 rounds+recheck • Transfer on day 4

18. MF 13.2% (43.5%) RIF 3.8% 12% 10.2% 14% %9.2 1.1% 5.2% 5.8% 9.3% RPL 15.9% AMA Indications and combined factors in 1000 PGD cycles

19. RIF RPL AMA Male Factor and PGD (n=433) 29.6% n=129 27.2% 8.5% 11.6% 2.5% 20.6%

20. Sperm Source

21. Number of cycles 34% * 20.4%

22. PGD for Only Male Factor Infertility Istanbul Memorial Hospital ART and Genetics Center

24. Distributing of Chromosomal Abnormality

25. PGD RESULTS Ejaculated vs Testicular Sperm(Maternal ages below 38)

26. Conclusion • The results of our study shows that the rate of aneuploidy is as high as 60% in patients with severe male factor infertility • Aneuploidy rate increases with the presence of other combined contributing factors such as AMA, RIF and RSA • PR dramatically decreases as more indications are combined with male infertility.

27. Preimplantation Genetic Diagnosis (PGD) Translocations

28. Probes PGD-translocations n=104 • Locus Spesific (LSI) (200-500kb) • Centromeric (CEP) (alpha satellite p11-q11) • Telomeric (Tel) (60-170kb) • Whole Chromosome Painting Probes (WCP)

29. PGD for Male translocation Carriersn=104

30. Sperm FISH Aneuploidy screening for translocation cases. Is There Any Interchromosomal Effect? (n=5)

31. Conclusion • . PGD should be a viable alternative for translocation carriers to reduce miscarriages • Spermatozoa FISH testing may be used as an indicator of aneuploidy and segregation rate in gametes in translocation carriers and can give good approximation of success in a PGD cycle • Aneuploidy screening should be a part of genetic evaluation if female partner is >38 years

32. Micro-Dissection TESE Procedures in Azoospermic Patients

33. SPERM RECOVERY in NOA PATIENTS (n=1023) İMH Andrology Unit • Micro-TESE: (NOA) 729 • TESA 294 Sperm recovery rate in NOA Cases375 / 729 = % 51.4

34. Results of Mic-TESE • Patients with first Mic-TESE trials : • No of patients : 591 • Sperm recovery: 354 • No sperm : 237 Sperm recovery rate: 354 / 591 = (%60) • Patients with previously conventional TESE trial with no sperm recovery : • No of patients: 60 • Sperm recovery: 32 • No sperm: 28 Sperm recovery rate: 32 / 60 = %53.3

35. Secondary MicroTESE success rates in patients • Secondary MicroTESE n=242 Sperm recovered: 178 No Sperm : 64 Success rate :178/242=73.5%

36. TESE/mic-TESE • TESE 36% vs Mic-TESE (Schlegel) 68%

37. Klinefelter’s Syndrome • No of cases: 65 Micro-TESE cases Sperm found: 26 No sperm : 39 Success rate 26/65 = % 40

38. Surgical sperm recovery rate according to hystopathology • Sertoli cell only (Germ cell aplasia) No of cases: 56 Sperm found: 20 No sperm : 36 Success rate: 20/56 = % 35

39. Surgical sperm recovery rate according to hystopathology • Maturation arrest Total number of cases : 37 Sperm found : 19 No sperm found : 18 Success rate : 19 / 37 = 51%

40. Conclusion • Mic-TESE is one of the most recent and important advance in surgical sperm retrieval techniques. It’s success in sperm retrieval and less complication rates made this technique is the most preferable procedure in sperm retrieval.

41. Conclusion • Mic-TESE procedure has been you used in our clinic since June 2002. mic-TESE operation improved our sperm retrieval rates and fulfilled some of our patients hopes of having biologically their own offsprings.

42. Sperm DNA Fragmantation

43. Meta-analysis:SCSA, performed in semen, cannot predict the outcome of ICSI (Evenson D. 2006)

44. Sperm DNA fragmantation/TUNEL TEST • Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling assay. • To detect the DNA damage, accounting for apoptotic sperms • At least 500 sperm are count for evaluation • The clinical value of TUNEL in predicting of IVF/ICSI outcomes in terms of fertilization rate and clinical outcome is not clear yet; • < 20 % low degree of sperm DNA damage group • ≥ 20 % high degree of sperm DNA damage group

45. Representative Images of TUNEL Asssay Green stained ones are apoptotic sperms, high DNA fragmentation

46. Tunel TEST in RIF Cases with Low Sperm Motility

47. Tunel TEST in RIF Cases with Low Sperm Motility

48. 210 mouse oocyctes (ICSI): • 65 fertilization • 7 transgenic births Nayernia et al.,2006

49. This study shows the higher plasticity potential of adult stem cells, like hES cells • In April 2007, Nayernia declared that his team obtained the bone marrow stem cells, called mesenchymal stem cells, from four adult men who were about to undergo bone marrow transplants • BMS cells are able to differentiate to early germ cells, primordial germ cells (PGCs) and even spermatogonial stem cells (SSC) and spermatogonia in vitro and in vivo.

50. In-vitro generatedartificial gametes: ultimate solution Patients with absent gametes or gonads Somatic cell haploidization. Converting somatic cells from mitotic division to meiotic division directly. De-differentiating somatic cells into embryonic stem cell and re-differantiating ES cells into gametes. Extracting adult stem cell and re-differentiating them into gametes.