Increase incidence of cancer during the reproductive age. Survival and cure rates of cancer are improving. Resulting in Increasing demand for fertility preserving interventions.

1. Fertility Preservation
in Cancer Patient
Marwan Al-Halabi MD. PhD
Professor in Faculty of Medicine
Damascus - University
And
Medical Director
Orient Hospital
assisted Reproduction center
Damascus – Syria

2. Introduction
Increase incidence of cancer during the reproductive age.
Survival and cure rates of cancer are improving.
One in 1000 adults is a survivor of childhood cancer.
Better attention has been paid to prevention of
reproductive failure.
Increasing demand for fertility preserving interventions.

3. Childhood CA Survival Rate

4. Factors to Consider
Desire of patient to retain fertility potential
Age, obstetrical history, family history, history of
infertility
Extent of the patient’s cancer
Optimal cancer therapy should always supersede
fertility preservation

5. Six distinct issues should be considered
The risk of sterility with the proposed treatment program
The overall prognosis for the patient
The potential risks of delaying chemotherapy
The impact of any future pregnancy upon the risk of tumor
recurrence
The impact of any required hormonal manipulation on tumor
itself
The possibility of tumor contamination of the harvested tissue

6. The Forgotten Female?
Studies suggest that only
about half of oncologists
discuss fertility preservation
with their patients

7. ASRM Guidelines
The ASRM report therefore offers guidelines
to cancer and fertility specialists to assist
them in preserving fertility in cancer
patients and in facilitating reproduction
after treatment.

8. Cancer specialists should inform patients about
these options
refer them to fertility specialists.
counseling by a qualified geneticist
ASRM Guidelines

9. should not deny cancer patients
assistance with reproduction??.
An Expected Shortened Life

10. Cancer Treatment
&
Fertility

11. Chemo – Therapy
Fallicular destruaction .
Ovarian Fibrosis .
Premature Ovarian Failure
Reduced E2 and P4 Levels

13. 13
Temporal intervals in
folliculogenesis

14. Ovulation

15. Differential sensitivity of different cellular
components of the ovary
Impaire follicular maturation.
Deplete primordial follicles.

16. Effects of cancer treatment on
male fertility
Agents Effect on sperm
•Radiation to the testes, Chlorambucil,
Cyclophosphamide, Procarbazine, Melphalan,
Cisplatin
Prolonged azoospermia
•BCNU, CCNU Azoospermia in adulthood after treatment
before puberty
•Busulfan, Ifosfamide, BCNU, Nitrogen Mustard,
Actinomycin D
Azoospermia likely, but always given with other
highly sterilizing agents
•Carboplatin Prolonged azoospermia not often observed at
indicated dose
•Doxorubicin, Thiotepe, Cytosine arabinoside,
Vinblastine, Vincristine
Can be additive with above agents in causing
prolonged azoospermia, but cause only
temporary reductions in counts when used
alone
•Amacrine, Bleomycine, Dacarbazine, Daunorubicin,
Epirubicin, Etoposide, Fludarabine, 5-Fluorouracil, 6-
Mercaptopurine, Methotrexate, Mitoxantrone,
Thioguanine
Only temporary reductions in counts at doses
used in conventional regimens, but additive
effects are possible
•Newer agents ?
Lee et al. JCO, 2006

17. Degree of Risk Treatment
High risk
(>80%)
•Hematopoietic stem cell transplantation with cyclophosphamide/total body irradiation or
cyclophosphmide/busulfan
•External beam radiation to a field that includes the ovaries
•CMF, CEF, CAF x 6 cycles in women age 40 and older
Intermediate risk •CMF, CEF, CAF x 6 cycles in women age 30-39
•AC x 4 in women age 40 and older
Lower risk
(<20%)
•CHOP X 4-6 cycles
•CVP
•AML therapy (anthracycline/cytarabine)
•ALL therapy (multi-agent)
•CMF, CEF, CAF x 6 cycles in women age less than 30
Very low or
no risk
•Vincristine
•Methotrexate
•5-fluorouracil
?
•Taxanes
•Oxaliplatin
•Irinotecan
•Monoclonal antibodies
•Tyrosine kinase inhibitors
Risks of Permanent POF after
Chemotherapy & Radiotherapy
Lee et al. JCO, 2006

18. Factors affecting the extent of
radiotherapy induced gonadotoxicity
1. Patient’s age.
2. Dose of radiation (Breaking point 300cGy).
3. Extent.
4. Type of radiation (abdominal, pelvic external beam,
brachytherapy).
5. Fractionation of the total dose.

19. Effect of radiation dose and age on ovarian
function
Ovarian dose (cGy) Risk of ovarian failure
60 No deleterious effect
150 No deleterious effect in young
women ; some risk for sterilization in
women older than 40
250-500 In women aged 15-40, 60%
permanently sterilized; remainder
may suffer temporary amenorrhea. In
women older than 40, 100%
permanently sterilized
500-800 In women aged 15-40, 60%-70%
permanently sterilized; remainder
may experience temporary
amenorrhea. No data available for
women over 40 .
>800 100% permanently sterilized

20. Break point for radiation is
around 300cGy
11-13% had POF <300cGy.
60-63% had POF >300cGy.
>6Gy irreversible ovarian failure.
< 2Gy 50% of the oocyte population is
destroyed.

21. Factors affecting the extent of chemotherapy
induced gonadotoxicity.
Type, duration, dose.
Gonatotoxicity induced by chemotherapy is almost
irreversible.
(• decreased number of follicles to absent follicles)
(• fibrosis )
Amenorrhea ranges 0-100 %
– younger age group 21 -71%
– older age group 49 - 100%
The risk of gonadal damage increases with age (lower
number of oocytes).
Temporary amenorrhea or permanent.

22. Effect of age on risk of premature
ovarian failure

23. indicator of ovarian damage
The best biochemical indicator of ovarian damage and failure
is :
FSH
E2
Inhibin – B
AMH

24. Fertility
Preservation

25. Options for preserving fertility
Fertility – sparing surgical procedures
Pharmacological protection ( GnRHa )
Embryo cryopreservation
Oocyte cryopreservation
Ovarian transposition
Ovarian tissue cryopreservation
In vitro oocyte maturation
Stem Cells Therapy

26. Ovarian Cancer
3-17% of ovarian cancer patients are < 40
7-8% of all stage I epithelial ovarian cancers occur
in women < 35
Possible candidates for fertility-sparing treatment
– Malignant germ cell tumors
– Sex cord – stromal tumors
– Borderline tumors
– Stage IA invasive epithelial ovarian cancer
Duska, L et al. Epithelial ovarian cancer in the reproductive age group. Cancer 1999, 85: 2623-2629.

27. Fertility-Sparing Surgical Procedures
Ovarian cystectomy
Unilateral salpingo-oophorectomy
Bilateral salpingo-oophorectomy
All should be performed with comprehensive
surgical staging

28. Pharmacological protection
GnRH – Agonist
GnRH – Antagonist : under investigation , no result
to date .
Oral Contraceptives : not work .
Progesterones : not works .
Apoptosis inhibitors : Mice only .
Sphingosinc -1- Phosphate

29. GnRH agonist
The concept is to
induce
hypogonadism
before starting
chemotherapy,

30. GnRH agonists
– Premenarchal gonads appear to be least
sensitive to cytotoxic drugs.
– By suppressing gonadotrophin.
– No protection effect of radiation therapy.
– No protetive effect on male gonads.

31. GnRHa for protection of ovary and preservation of
fertility during C/T
-a preliminary report(I)
pereyra, Gynecologic Oncology 81, 371-7
Method: the patients were divided into three groups:
– Group A: premenarchal Pt, age 3~7.5(n=5), GnRHa(-).
– Group B: postmenarchal Pt,age: 14~20(n=12),GnRH(+)
– Group C: postmenarchal Pt,age: 15~20(n=4),GnRH(-)
– All Pt, received PCT regimens for Tx lymphoma, leukemia
or thymoma. In group B, leuprolide acetate inhibition was
obtained with a deport injection administered each months
before and during treatment.
–

32. GnRHa for protection of ovary and preservation
of fertility during C/T -a preliminary report(II)
Result:
– Group A:spontaneous menarche between
12~17.9y/o. followed by normal menstruction
and ovulatory cycles.
– Group B: After withdrawal GnRHa, continue
normal ovulatory cycles. 2 Pt became preg.
– Group C: hypergonadotrophic hypoestreogenic
amenorrhea

33. GnRHa for protection of ovary and preservation
of fertility during C/T
-a preliminary report(III)
Conclusion:
– Polychemotherapy(PCT) administered at early
age, when ovarian follicles have not reached
maturation, produces less damage.
– GnRHa provide a powerful protection of
ovarian follicle during C/T.
– We suggested GnRHa in all adolescents with
maligancies prior and during C/T.

34. Embryo Cryopreservation

35. Embryo Cryopreservation
15 – 40 % success rates.
Survival rates of embryos between 35 and
90%.
8 – 30% implantation rates.
Not acceptable to prepubertal, adolescent and
women without a partner.
Need IVF.

36. Ovarian stimulation protocols in estrogen–
sensitive cancers.
Short flare – up protocol.
Natural cycle IVF.
Tamoxifen ( Anti–estrogen)
Letrozole suppresses plasma ostradiol,
estrone
and estrone sulphate levels.

37. Oocyte Cryopreservation

38. Oocyte Cryopreservation.
for single women, ethically accepted.
Oocytes are more sensitive to freezing–thawing
procedures than embryos.
Results are still very low.
Alternative strategy is to freeze immature oocytes
( primordial follicle).
Other alternative is vitrification .

39. Pregnancy Rate 2 – 11 %
Oocyte Freezing
68% Survival
48% Fertilization
21% Pregnancy Rate
Oocyte Cryopreservation.
Oocyte Vitrification

40. In vitro Oocyte Maturation
(IVM)
Harvesting immature follicles (they may
become atretic).
More oocytes became available for clinical
treatment.
No large doses of gonadotropic hormones
for stimulation.
Eliminates risks of ovarian stimulation

41. Ovarian
Transposition

42. Ovarian Transposition
Ovaries(one) surgically moved away
from the radiation field to minimize
exposure and damage.
available before or after puberty as an
outpatient surgical procedure.

43. Technique
Techniques for ovarian transposition using a
laparoscopic approach vary according to the
radiation field shape, size, and location
Ovarian Transposition

44. Ovarian Transposition
Pregnancy rates are approximately 50 – 75 %
Spontoneouselly.
11% Conceived with IVF
Benefit :
Prevention of premature menopause .
Preservation of fertility ?? !

45. Complications of Ovarian
Transposition
Fallopian tube infarction.
Chronic ovarian pain.
Ovarian cyst formation.
Migration of ovaries back to their original
position.
Ovarian metastasis (No increased risk).

50. Ovarian Tissue Freezing
and transplantation

52. Ovarian cortical freezing

53. 24 year-old patient with Hodgkin’s disease, pre-SCT

54. Case report
Belgian day-old
baby Tamara
Bouanati nestles
in the arms of her
mother Ouarda
Touirat, 32. Touirat
beat cancer and
gave birth after an
ovarian tissue
transplant

55. It is still a Hope
Stem cells Therapy

56. Conclusion.
GnRH analogues are the only available
medical protection for chemotherapy.
Laparoscopic ovarian transposition is a good
option if radiotherapy is to be used.
Oocyte cryopreservation is gaining
popularity.
Embryo cryopreservation is the most
successful
fertility preservation.

57. Slow-freeze protocol for
ovarian tissue preservation
1) Equilibrate cortical slices in
cryoprotectant for 30 min at 0o C
2) Cool at 2o C/min to -9o C
3) Soak for 10 min and seed
4) Continue at -0.3o C/min to -40o C
5) 10o C to -140o C
6) Transfer to liquid nitrogen (-196o C)

58. Risk of ovarian involvement in
cryopreservation candidates
Low risk Moderate risk High risk
Wilms’ tumor
Stage IV
breast cancer
Leukemia
Lymphomas
Stage I–III lobular
breast cancer Neuroblastoma
Stage I-III
breast cancer
(infiltrating ductal)
Adenocancer of the
cervix
Stage IV lobular breast
cancer
Non-genital-
rhabdonyosarcoma
Colorectal cancer
Osteogenic sarcoma
Squamous cell cancer of
the cervix
Ewing’s sarcoma

59. Screening of ovarian tissue
Best screening is light microscopy
Molecular markers can be used in rare cancers
Test tissue before freezing and after thawing

60. Transplantation

61. High rate of follicle loss
after transplantation
5% of primordial
follicles lost during
freezing and thawing
65% of primordial
follicles lost during
revascularization
(xenografting)

62. Whole ovary cryopreservation as
an attempt to improve follicle
survival
Successful in mice
Partial success in sheep
– 3/11 patent after 8–10 days1
– Long-term function with new
freezing technology2

63. Whole ovary freezing by
directional freezing in sheep
8 sheep ovaries frozen with MTG technology
5/8 grafts survived but only 2/8 had
long-term cyclical function (24–36 months)
Oocyte retrieval in 2
Parthenogenic activation

64. Whole ovary freezing is
challenging in humans
Human ovary is larger
– 4 x 2 x 0.8 cm (20–35 gm)
vs 2.5 x 1.5 x 0.5 (3–8 gm)
Need to optimize the protocol for germ cells and
somatic cells
Whole ovary cryopreservation not yet
technically possible in humans

65. Orthotopic
(pelvic)
transplant
Heterotopic
(subcutaneous)
transplant
Resumption
of ovarian
functions
Spontaneous
conception
IVF
Embryo
transfer
Ovarian transplantation
techniques

66. Orthotopic
(pelvic)
transplant
Heterotopic
(forearm)
transplant
Resumption
of ovarian
functions
Spontaneous
conception
IVF
Embryo
transfer
Ovarian transplantation
techniques

67. “Successful” cases of orthotopic
ovarian transplantation
Author Outcome
Oktay & Karlikaya
N Engl J Med 2000;342:1919
Ovulation, endocrine function for
9 months
Radford et al.
Lancet 2001;357:1172–5
Follicle development and endocrine
function up to 9 months
Donnez et al.
Lancet 2004;364:1405–10
Spontaneous pregnancy and live
birth
Meirow et al.
N Engl J Med 2005;353:318–21
Live birth after IVF

68. Pelvic ovarian transplantation

69. 0
20
40
60
80
100
0 5 10 15 20 25 30
Days after follicle seen
Estradiol(pg/mL)
Progesterone(ng/mL)
hCG injections
First orthotopic ovarian transplant with frozen ovarian tissue

70. First pregnancy after
ovarian transplant?
Ovarian biopsies frozen in 1997 at age 25
Received MOPP for Hodgkin’s disease
Birth control pills after chemotherapy
Transplant in 2003
Spontaneous conception & delivery in 2004

71. Questions with Donnez et al. report
Risk of ovarian failure relatively low
– 20% at ≤ 25 years of age
– 50% chance of pregnancy1
Both ovaries remain
Three ovulations from the right (intact) ovary
No hormonal or ultrasound monitoring of
ovulation from the transplant

72. Live birth following transplantation
of cryopreserved ovarian tissue
after chemotherapy-induced
ovarian failure

73. Comparison of two orthotopic
transplant techniques
Ovarian function &
pregnancy via IVF
No ovarian function

74. Orthotopic
(pelvic)
transplant
Heterotopic
(subcutaneous)
transplant
Resumption
of ovarian
functions
Spontaneous
conception
IVF
Embryo
transfer
Ovarian transplantation
techniques

75. Advantages of
heterotopic transplant
Non-invasive
Repeated procedures feasible
Can inject agents directly in the graft
Easy monitoring (risk of recurrence)
Easy removal
Suitable after pelvic radiation

76. “Successful” cases of heterotopic
ovarian transplantation
Author Outcome
Oktay et al.
JAMA 2001;286:1490–3
Follicle development and endocrine
function for 3 years
Oktay et al.
JAMA 2001;286:1490–3
Ovulation, endocrine function for
3 years
Oktay et al.
Lancet 2004
Folliculogenesis, endocrine function
for > 2½ years; embryo
development
Oktay
Hum Reprod 2006;21:1345–8
Live birth?

78. Patient A
Patient B

79. Patient A
Patient B

80. Estradiol output from
heterotopic transplant
RCV estradiol
0
1000
2000
3000
4000
5000
6000
1 5 13 15 20 22 28 32 33 34 36 39 40
Cycle day (arbitrary)
pg/mL
RH estradiol
0
50
100
150
200
250
1 5 13 15 20 22 28 32 33 34 36 39 40
Cycle day (arbitrary)
pg/mL
RH
RCV

81. Ovarian transplant in a
patient with breast cancer
36-year-old patient
Ovary cryopreserved before chemotherapy
High-dose chemotherapy before bone marrow
transplant
In menopause for 6 years

84. Follicle development in a
frozen-thawed transplant

85. Percutaneous oocyte retrieval

86. First embryo after ovarian
transplant
24 hours
18 hours24 hours

87. Embryo #3: 1 PN after ICSI
16h post-ICSI 24h post-ICSI
6/03/04z
PGD: Female pronucleus, partially decondensed sperm DNA

88. “Normal” embryo yield
22 oocytes
9 suitable for IVF (8 ICSI)
2 abnormal embryos
1 grade 1/2, 4-cell embryo
Yield: 4.5%

89. Follicle maturity is achieved at
a smaller follicle diameter
Follicle stage GV M-I Mature
Mean follicle size
(range)
8.7 mm
(6.4–10.9)
10.4 mm
(7.6–13.1)
11.5 mm
(9.9–12.8)
Average number of stimulation days similar to controls
(10.8 days, range 8–13)

90. Is Pregnancy Possible
after heterotopic
transplantation?

91. Brenda: first primate pregnancy
after ovarian transplant

92. Latest case of heterotopic
ovarian transplant
Hodgkin’s disease at age 28: receives
ABVD + RT to chest & spleen
Ovarian tissue cryopreserved at age 29 prior
to SCT due to relapse
Amenorrhea and elevated FSH for 2.5 years
(45–95 IU/L) after receiving preconditioning
chemotherapy
Subcutaneous ovarian transplant performed
on 12 August 2004

93. Subcutaneous transplantation in a
menopausal woman
post-stem cell transplantation
15 pieces thawed
5 x 5 x 1 – 15 x 5 x
2 mm
Suture pull-through technique

94. Ovarian function after
heterotopic ovarian transplant
Felt follicle growth 7 weeks post-transplant
10 weeks post-transplant
– E2: 250 pg/mL
– P4: 14 ng/mL
Transvaginal ultrasound performed 11 weeks
post-transplant

95. Pregnancy after
heterotopic transplant

96. Corpus luteum in
menopausal ovary?

97. Spontaneous recurrent pregnancies
after heterotopic ovarian transplant
D&C performed as no FH detected
Cytogenetics of POC revealed trisomy 16
Patient felt follicle growth 3 weeks after D&C
and menstruated a week later
Had intercourse on the day of ovulation
and conceived again!

98. Long-term follow-up
Resumed follicular activity in graft 3 months
postpartum
Ovulation in graft? and “menopausal” ovary a
week ago
Attempted pregnancy
B-hCG pending

99. Spontaneous pregnancy after
heterotopic ovarian transplant
What is the true source of pregnancies after
ovarian transplants?
How did the two conceptions occur in a
menopausal women contemporaneously
with follicle growth in the graft?

100. Evidence for germline stem cells in
adult human female bone marrow
Analysis of human female bone
marrow (BM) reveals expression of
germline marker genes
1 BM collected from donors between 24–36 years of age
2 Ut1 and Ut2: adult human uterus used as a negative
control

101. Ovariectomy causes a near-complete elimination of Mvh expression in BM.
Replacement of estrogen, norgesterone, or both does not alter BM Mvh levels in
ovariectomized females
Ovariectomy abolishes Mvh
expression in the bone marrow

102. Impact of chemotherapy
on stromal function

103. Ovarian graft
Germ cell
Signal for induction of germ stem cell
production in bone marrow or other sites
Bone
marrow
Migration to the menopausal ovary
Ovulation
Fertilization

104. Summary
Ovarian transplantation is an emerging
experimental technique that can be offered for
fertility preservation if studied under
IRB-approved protocols
Germ stem cell story intriguing
– needs confirmation

105. Cryopreservation and
transplantation of ovarian tissue.
Still experimental procedure.
Limited studies.
Primordial follicles should have better survival rates.
In vitro – growth of primordial follicles.
(after immune deficient animal host).
trans–species viral infections.
Transplanted back into patient,
(Cancer nidus).
after cryopreservation.

106. Ovarian Cancer and
Infertility
Ovulation is associated with an increased
risk of epithelial ovarian cancer. (epithelia
proliferation, inclusion cyst formation).
Oncogenes HER-2/meu
K-ras
c-myc
mutations P53 tumor-
suppressor gene.

107. S. AL SAMAWI MD. Gyn. Obs.
A. TAHA MD. Gyn. Obs.
M. ABDUL WAHED MD. Gyn. Obs.
J. SHARIF Senior Biologist
N. ABO HASSAN Androlgist
F. RAHMEH Androlgist
S. MATOUK Executive Secretary
F. HAMAD Administration Manager
A. ALKHATEB M.D Micro Biologist
R. ALKHATEB MD. Gyn. Obs. Ph. D.
Acknowledgement

108. Thank You