Collection processing and storage of ovarian tissue-Clinical indications and best practice (by Ozgur Oktem_2010)

1. OVARIAN TISSUE
CRYOPRESERVATION
OZGUR OKTEM MD
AMERICAN HOSPITAL
WOMEN`S HEALTH CENTER
ISTANBUL TURKIYE

2. QUALITY OF LIFE ISSUES
IN CANCER SURVIVORS
FERTILITY PRESERVATION
CHEMOTHERAPY RADIOTHERAPY

3. FERTILITY PRESERVATION
Preservation of reproductive function
became an important quality of life issue
in cancer patients
Life expectancy is increasing

4. 5 year survival rates have increased in
cancer patients
5 YEAR SURVIVAL
CHILDHOOD CANCERS
1975-1977 1996-2004
58% 80%
ADULT CANCERS
50% 66%
Jemal et al. CA Cancer J Clin 2009;58:71–96

5. Adult survivors of childhood
cancers a new population!
Oktem et al Ann N Y Acad Sci. 2008;1135:237-43
Oktem et al Pediatr Blood Cancer 2009 Aug;53(2):267-73

6. A Age:22
Dx: Hodgkin’s lymphoma Age:27 Age:28
HSCT Cure Amenorrhea
Chemotherapy
Infertility
Jan 2002 Apr 2007 Jan 2008
diagnosed Menstrual Amenorrhea Return of
with irregularity menses
cancer
FSH:42mIU/mL
Premature ovarian
failure!
A MATTER OF LIFE AND DEATH QUALITY OF LIFE ISSUE

7. OVARIAN TISSUE BANKING
Ovarian tissue freezing is the only fertility
preservation options for
Pediatric and adolescent cancer patients
Adults who have
No time for embryo freezing or
Contraindication for embryo freezing
No husband or partner for embryo freezing
•Oktem et al. Cancer 2007
•Oktem and Oktay Fertil Steril 2008

9. Jemal et al. CA Cancer J Clin 2009;58:71–96

11. Chemotherapy and cell
death

12. DAMAGE TO DNA.
as neutrons and particles
Indirect actions due to
formation of free radicals
and DNA damage. This
mechanism is particularly
true for sparsely ionizing
radiation such as x-rays.
HSCT
The higher the dose of radiation
The higher the dose of radiation TBI
The higher the risk of premature ovarian failure !! - 20-30 Gy⇒37/38
The higher the risk of premature ovarian failure
Ovarian failure
Single dose is more toxic than fractionated dose.
Single dose is more toxic than fractionated dose. TBI + Cyc
- OR:~1 (1 yıl)
The LD50 of the human oocytes may be 1.99 Gy∗;less
The LD50 of the human oocytes may be 1.99 Gy∗;less - 135/144 patients have
than the previously thought (4 Gy)∗∗
than the previously thought (4 Gy)∗∗ POF
100cGy=1Gy=100 Rad

13. Other indications for fertility
preservation requiring
chemotherapy and/or stem cell
transplantation
Systemic lupus erythematosus
Myelodysplasia
Aplastic anemia
Wegener’s vasculitis
Auto-immune hemolytic anemia
Sickle-cell disease
Thalassemia

14. GONADOTOXICITY
1 Patient’s age
Younger the patient higher the follicle counts
2 Cytotoxic potential of therapy
Alkylating agents more toxic
3 Dose and duration of therapy
Longer duration and higher doses more toxic

15. GONADOTOXICITY
1 Patient’s age
Younger the patient higher the follicle counts
More likely to retain some ovarian function after
therapy
Oktem and Oktay Am J Hem Oncol 2008;7;1-7

16. Growing phase 10%
Resting phase 90%
Primordial follicles determines
ovarian reserve. Drugs mainly
targeting PF have more
impact on ovarian reserve.
SHORTER REPRODUCTIVE
LIFE SPAN
HIGHER RISK FOR
PREMATURE OVARIAN

17. How to assess the damage in the human ovary
Hormonal and USG markers
Currently there is not a hormonal marker of
primordial follicle counts.
FSH, AFC , and AMH levels are commonly used
reserve markers.
Reh et al. Fertil Steril 2007
Oktem et al. Fertil Steril 2007

18. FSH action
Oktem ANYAS 2008

19. GONADOTOXICITY
1 Patient’s age
Younger the patient higher the follicle counts
2 Cytotoxic potential of therapy
Alkylating agents more toxic
3 Dose and duration of therapy
Longer duration and higher doses more toxic

20. CHEMOTHERAPY AGENTS

21. GONADOTOXIC
CHEMOTHERAPEUTICS
CHEMOTHERAPY
Different toxicity potential
Alkylating agents most toxic
Cyclophosphamide
Platinum group
Chlorambucil
Taxanes Melphalan
Antracyclines Busulfan
+
Oktay et al. Hum Reprod. 2004 Mar;19(3):477-80
++
Oktem and Oktay Fertil Steril 2006;86:S312 P-725
Oktem et al. Cancer:2007 110(10):2222-9

22. ALKYLATING AGENTS
Nitrogen mustards Platinum (alkylating-like) Aziridines
Chlorambucil Carboplatin
Chlormethine Carboquone
Cisplatin
Cyclophosphamide Nedaplatin ThioTEPA
Ifosfamide Oxaliplatin Triaziquone,
Melphalan Triplatin tetranitrate Triethylenemelamine
Bendamustine Satraplatin
Trofosfamide
Uramustine Alkyl sulfonates
Busulfan
Nitrosoureas Mannosulfan
Carmustine Treosulfan
Fotemustine
Lomustine Hydrazines
Nimustine Procarbazine
Prednimustine Triazenes
Ranimustine Dacarbazine
Semustine Temozolomide
Streptozocin

23. A Age:22
16.6 ± 3.5 PF
Dx: Hodgkin’s lymphoma
Ovarian freezing
diagnosed with cancer
March 2004 April 2004
6.17 ± 0.7 PF
B Age:22
Dx: Non-Hodgkin lymphoma
Chemotherapy 1XCHOP
Oktem et al Cancer 2007

24. Oktem et al. Cancer 2007
Oktem et al. Am J Hem Oncol 2008

25. 16.6 ± 3.5 PF 6.17 ± 0.7 PF
%63 loss
Chemotherapy
1XCHOP
AGE 22 AGE 30
The cost of one course of CHOP in the ovary
8 YEARS AGING
Oktem et al. Cancer 2007

26. A Age:33 5.66 ±0.9
Dx: Breast cancer
Ovarian freezing
diagnosed with cancer
May 2003
1.5 ±0.6
B Age:33
Dx: Non-Hodgkin lymphoma
Chemotherapy 7XCHOP
GnRH analog
FSH: 20.8 mIU/mL

27. A Age:18 16.6 ±1.6
Dx: Hodgkin’s lymphoma
Ovarian freezing
diagnosed with cancer
May 2003
14.4 ±1.6
B Age:18
Dx: AML
Chemotherapy 2XADE-GMTZ

28. Control VACA + RT
7.6 ±1.7 AGE 24 4.52 ±0.9 AGE 24
Oktem et al Cancer 2007

29. TWO IMPORTANT QUESTIONS
TO BE ANSWERED...
How to Measure THE DAMAGE?
How to assess the toxicity of NEW DRUGS?

30. Ovarian
Xenografting
Severe Combined Immune Deficient (SCID) Mice
T cell B cell
Cellular immunity
Cellular immunity Humoral immunity
Humoral immunity
NO GRAFT REJECTION

31. GRAFT VASCULARIZATION
Gross
300um

32. Cy-induced damage in human ovary as
assessed by tunnel assay
Oktem et al Cancer Res 2007; 67: 10159-62

33. Follicle loss after single dose Cy
Oktem et al Cancer Res 2007; 67: 10159-62

34. OVARIAN TISSUE FREEZING

35. Ovarian Transplantation
Techniques
Orthotopic
Orthotopic Heterotopic
Heterotopic
(Pelvic)
(Pelvic) (Subcutaneous)
(Subcutaneous)
Transplant
Transplant Transplant
Transplant
Resumption
Resumption
of Ovarian
of Ovarian IVF
IVF
Functions
Functions
Spontaneous
Spontaneous Embryo
Embryo
Conception
Conception Transfer
Transfer

36. Ovarian Transplantation
Techniques
Orthotopic
Orthotopic Heterotopic
Heterotopic
(Pelvic)
(Pelvic) (Forearm)
(Forearm)
Transplant
Transplant Transplant
Transplant
Resumption
Resumption
of Ovarian
of Ovarian IVF
IVF
Functions
Functions
Spontaneous
Spontaneous Embryo
Embryo
Conception
Conception Transfer
Transfer

37. Patient A
Patient B

38. Patient A
Patient B
Oktay et al, JAMA, 2001

39. Estradiol Output From
Heterotopic Transplant
RH Estradiol
250
200
pg/mL
150
100
50
0
1 5 13 15 20 22 28 32 33 34 36 39 40 RH
Cycle Day (arbitrary)
RCV
RCV Estradiol
6000
5000
4000
pg/mL
3000
2000
1000
0
1 5 13 15 20 22 28 32 33 34 36 39 40
Cycle Day (arbitrary)

41. Percutaneous Oocyte
Retrieval

42. Percutaneous Oocyte
Retrieval

43. First Embryo After Ovarian
Transplant
24 Hours
18 Hours
24 Hours

44. Pelvic Ovarian
Transplantation
Oktay et al, NEJM 2000

45. Meirow et al, NEJM 2005
Comparison of Two
Orthotopic Transplant
Techniques
Ovarian Function & No Ovarian Function
Pregnancy via IVF

46. Oktem, Sonmezer, Oktay
Textbook of Assisted Reproductive Technologies, 2005

47. ISCHEMIA AFTER
TRANSPLANTATION
HYPOXIA INDUCIBLE FACTOR-1 ALPHA (HIF-
1α)
BEFORE TRANSPLANTATION AFTER TRANSPLANTATION

49. Author Year Transplantat Cryo indication IVF / Age at Age at tx. Outcome
ion site spontaneous ovarian
cryo.
Oktay 2004 Heterotopic Breast cancer IVF 30 36 Embryo
development
Donnez 2004 Orthotopic Hodgkin’s Spontaneous 25 31 Healthy live
disease birth
Meirow 2004 Orthotopic Hodgkin’s IVF 26 28 Healthy live
disease birth
Demeestere 2006 Orthotopic/het Hodgkin’s Spontaneous 24 29 One
erotopic disease miscarriage at
7 weeks, one
healthy live
birth
Oktay 2006 Heterotopic Hodgkin’s Spontaneous 28 32 Healthy live
disease birth
Rosendahl 2006 Orthotopic/het Hodgkin’s IVF from 28 30 Biochemical
erotopic disease heterotopic pregnancy
site
Silber* 2008 Orthotopic Idiopathic Spontaneous 14 28 Ongoing
premature pregnancy
ovarian failure
Anderson 2008 Orthotophic Non Hodgkin’s IVF 32 34 Ebryo dev.
lympohoma
Anderson 2008 Orthotopic/het Hodgkin’s IVF 25 27 Clinical
erotopic disease pregnancy
Anderson 2008 Orthotopic Hodgkin’s diseas IVF 26 28 Healthy live
birth
Anderson 2008 Orthotopic Ewings sarkomu IVF 27 30 Healthy live
birth
Sonmezer&Oktay, 2008

50. Oktay and Oktem 2008 Fertil Steril
ALL PATIENTS

51. Oktay and Oktem 2008 Fertil Steril
ALL PATIENTS

52. Ovarian Freezing in Childhood Cancers
Oktem et al Ann N Y Acad Sci. 2008;1135:237-43
Oktem et al Pediatr Blood Cancer in press

53. INDICATIONS FOR FERTILITY
PRESERVATION PEDIATRIC
POPULATION
Oktem et al Ann N Y Acad Sci. 2008;1135:237-43
Oktem et al Pediatr Blood Cancer in press

54. Slow freezing vs. Vitrification
Controlled rate (slow) freezing is the most
commonly used cryopreservation method
for human ovarian tissue* .
Ultrarapid freezing (vitrification) is being
widely used in embryo and oocyte
freezing.
Data on its applicability on ovarian tissue
freezing is very limited.
*:Oktem Fertil Steril 2008

55. The structure of primordial follicles are
preserved better in slow frozen samples
Slow freezing Vitrification
Oktem Balaban and Urman ASRM 2009 USA
WFPC 2009 Belgium

56. Growing follicles are preserved
better in slow frozen samples
Fresh Slow freezing Vitrification

57. RESULTS
Slow frozen ovaries contain significantly
higher number of primordial follicles than
vitrified ones.
2
2,5
Primordial follicle/mm
a,b
2
1,5 a,c
1.97 b,c
1
1.27 0.97
0,5
0
Control SF VF
a:p>0.05
b:p<0.0001
c:p<0.001

58. RESULTS
Antimullerian hormone production from slow
frozen ovaries is significantly higher than vitrified
ones.
0,8
a,b
0,7
AMH (ng/mL)
0,6
0,5
0,4 a,c
0,3
0.47
0,2 b,c
0,1 0.21
0 0.07
Control SF VF
a:p>0.05
b:p<0.05 Oktem Balaban and Urman ASRM 2009 USA
c:p<0.05 WFPC 2009 Belgium

59. ANTI-MULLERIAN HORMONE
Oktem et al. Ann N Y Acad Sci 2008;1127:1-9

60. Literature
Isachenko et al Cyro letters 2008
Vitrification (2.62 M dimethylsulphoxide + 2.6
M acetamide + 1.31 M propylene glycol +
0.0075M polyethylene glycol) no comparison
with slow freezing.
Vitrification preserves ovarian follicles and
stroma better than slow freezing
SF PrOH Sucrose and EG
VF PrOH EG PVP DMSO
(Hovatta et al Hum reprod 2009)

61. UNKNOWNS…
Following questions are waiting to be
answered
Which method ?
SF vs. VF
Which cryoprotectant or combination of
different cry0protectants?
DMSO, EG, PrOH etc..
Incubation, seeding times, exposures?

62. CONCLUSION
Fertility preservation has recently
emerged.
The right option should be offered to
carefully selected patients.
Success rates of ovarian freezing is stilll
low due to
Underutilization (%94.9 -56 of 59 have
not used their tissues yet)
54% personal-social
38% still under therapy
8% death

63. THANK YOU

64. Bulent Urman, MD ACKNOWLEDGEMENT
Basak Balaban MSC
Aycan Isiklar MSc
Ebru Alper MD
Cengiz Alatas MD
Ramazan Mercan MD
Alper Mumcu MD
Cem Ayhan MD
Kayhan Yakın MD
Erhan Palaoglu MD
Kamil Peker MD