1. McGill University Health Centre
Hananel Holzer, MD
Medical Director, MUHC Reproductive Center
McGill University Health Center
Director, REI Division, Dept. of Obstetrics & Gynecology
McGill University

2. A comprehensive approach for fertility preservation

3. Fertility preservation
713,220 new cases of female cancer were diagnosed in 2009 in the U.S. (Jemal 2009)
Childhood cancers 1%. 122 new cases per million children (Skinner 2006)
By 2010, one in 250 people will be a cancer survivor (Blatt 1999)
80% of children and adolescents diagnosed with cancer become long-term survivors
One of long-term side-effects of cancer therapy is premature gonadal failure – infertility

4. Ovarian reserve
Females born with a finite pool of oocytes ( Zukerman 1951)
Challenged by Johnson, Tilly et al; 2004, 2005; Germ line stem cells

5. Ovarian reserve
W. Hamish B. Wallace 2010

6. Ovarian reserve
W. Hamish B. Wallace 2010

7. Ovarian reserve
W. Hamish B. Wallace 2010

8. Chemotherapy Destruction of growing follicles
loss of primordial follicles
ovarian atrophy

9. Primordial Follicle Count ⇩⇩⇩
Ovarian Reserve ⇩⇩⇩
Early Menopause and Infertility

10. The effect of chemotherapy on the ovary is not “all or none”
No. of surviving primordial follicles following exposure correlates inversely to dose of chemotherapy
500
1000
1500
2000
2500
mg/kg
Dose of Cyclophosphamide
Number of PMF’s
(p = )
(Meirow et.al. Hum Reprod 1999)

11. (Meirow et al. Hum Reprod 2007)
Cortical fibrosis
Focal ovarian cortical fibrosis
No follicles in zone of fibrosis
“Wedge shaped”- corresponds to the blood supply
(Meirow et al. Hum Reprod 2007)

12. (Meirow et al. Hum Reprod 2007)
Chemotherapy: Direct and indirect damage
Vascular
injury
Cortical
fibrosis
Apoptosis
medulla
cortex
(Meirow et al. Hum Reprod 2007)

13. Loss of regulatory mechanisms
Loss of local regulatory factors (AMH?)
Accelerated recruitment of primordial follicles.
Decreasing ovarian reserve

14. Chemotherapy Alkylating agents:
Cyclophosphamide,ifosfamide, nitrosoureas, chlorambucil, melphalan, and Busulphan
not cell-cycle specific, highest risk of ovarian failure.
Antimetabolites
methotrexate, bleomycin, 5-fluorouracil, actinomycin-D, mercaptopurine, vincristine)
impact the cells of the metabolically active ovarian follicles , are considered to be low risk for gonadal dysfunction
Platin - intermediate risk

15. chemotherapy
Women >40 years have a 90% chance of amenorrhea subsequent to multi-agent chemotherapy
younger patients 20% - 90%.
All patients exposed to chemotherapy might have a diminished ovarian reserve
significant predisposition for developing premature ovarian failure. Infertility > 35 years.
The estimated probability of early menopause was at least 25% at age 30 years (Letourneau , Cancer 2011)

16. Radiotherapy Direct damage to ovaries
Damage to hypothalamic-pituitary axis
Dose dependent; total dose and fractionation schedule
Age dependent; size of primordial follicle pool
Radiation field dependent
Could also affect uterine function (M/C, IUGR, Premature deliveries)

17. Radiotherapy
for a given dose of radiation, the younger patients are, the later the onset of premature menopause.
LD50 <2 Gy.
the effective sterilising dose falls with increasing age
effective sterilising doses are 20·3 Gy at birth, 18·4 Gy at 10 years, 16·5 Gy at 20 years, and 14·3 Gy at 30 years.
model to reliably predict the age of ovarian failure after treatment with a known dose of radiotherapy (W Hamish B Wallace, Lancet 2005, International Journal of Radiation Oncology*Biology*Physics 2005)

18. Reproductive outcome
In animal studies, the risks of M/C and of malformed fetuses after chemotherapy are high.
In humans, no increases in fetal malformation or M/C after anticancer drug treatment.
No higher risk of genetic or chromosomal abnormalities in offspring from parents that had been cancer survivors (Winther JF 2004)
if conception occurs more than a year after cessation of the treatment
cancer treatment has mutagenic effects on the oocyte that are present in growing follicles and the risk of mutagenesis is related to the stage of oocyte development during exposure and the drug regime used. (Meirow 2005)

19. Fertility preservation

20. Fertility preservation
Indications:
Cancer patients before gonadotoxic treatment
Other diseases before gonadotoxic treatment
Young patients with Turner syndrome, Fragile X premutation (FMR 1), Galactosemia
Women in mid-thirties without partner ?

21. Options for fertility preservation
Should be tailored according to:
Patient’s age
Type of disease
Spread of the disease
Planned treatment
Time available
Whether she has a partner
(Holzer Tan 2005)

22. Options for fertility preservation
Ovarian protection:
Ovarian shielding
Ovarian transposition prior to local radiotherapy:
reduces dose to 5-15%
patients <40
Laparoscopy
Location depends on the planned radiation.
Does not protect against chemotherapy.
(Tulandi 2004)

23. Options for fertility preservation
GnRH agonist acts to protect gonads during chemotherapy by preferentially steering cells into less active cell cycle stage with decline in response to chemotherapeutic agents
Simulating pre-puberty.
Direct effect?
Decreased perfusion?
Young patients.
(Blumenfeld 2007 ,Huser 2008)

24. Options for fertility preservation
3 meta-analyses were published:
GnRHa are effective in preserving ovarian function and in reducing amenorrhoea (Clowse 2009, Ben Aharon 2010)
only prospective randomized studies:
odds ratio of 3.5 favouring the use of GnRHa.
Higher rates of resumption of menses and ovulation
No improvement of pregnancy rates (Bedaiwy 2010)

25. Options for fertility preservation
Some evidence that GnRHa has a protective effect.
the final conformation is still awaited.
GnRH antagnist – faster desensitization.
Flare up effect; combined treatment? (Mardesik 2004 von Wolff 2011)
The GnRHa could be used to induce ovulation in a stimulated cycle.
Reduced tumour cell sensitivity to chemo in E+ cases ?

26. Options for fertility preservation
Apoptosis inhibition:
Ceramide – 2nd messenger signals apoptosis
Sphingosine 1 phosphate (S1P) ceramid antagonist
Interesting and promising; still far from clinical implementation
(Morita, Paris, Perez, Tilly et al 1999,2000, 2002)

27. Cryopreservation for fertility preservation
Transplantation of whole intact ovary by vascular anastomosis
Wang et al Nature 2002;
Imhof et al 2006; Bedaiwy et al 2007
Huge technical challenge, perfusion of the cryoprotectant
In theory, risk of cancer cell transmission

28. Options for fertility preservation: Cryopreservation of ovarian tissue
Available for pre- and post-puberty patients
Hundreds to thousands of primordial follicles may be preserved.
No medical delay.
No ovarian stimulation.
Does not require a male partner
At least 2 surgical procedures (+ IVF)
Anesthesia- risks
Theoretic risk of neoplastic cells in transplanted tissue – recurrence?

29. Risk of presence of neoplastic cells in the transplanted tissue
Subclinical involvement of Hodgkin’s Lymphoma has not been identified in ovarian tissue (Seshadri Gook et al 2006)
detection of Hodgkin lymphoma within ovarian tissue taken at the time of harvest for cryopreservation. (Bittinger 2011)
58 patients with hematological malignancies underwent ovarian tissue cryopreservation
after thawing, markers to detect minimal residual disease used
real-time RT-PCR positive in one patient with CML.
this alarming result avoided tissue transplantation (Meirow et al 2008)
Positive markers of CML and AML in cryopresrved harvested ovarian tissue (Dolmans 2010)

30. Risk of presence of neoplastic cells in the transplanted tissue
Other organ transplants: donor derived malignancy ( Kauffman 2002, Ison 2011 AM J Transplant)
Extreme caution is warranted before we assume that we understand tumour biology well enough to estimate the risk of transmission of malignant cells in autotransplanted ovarian cortex.
BRCA 1&2 carriers - potential of developing ovarian cancer (Colgan 2001)
Prophylactic BSO, transplant ovarian fragments?

31. Options for fertility preservation: Cryopreservation of ovarian tissue
Loss of follicles during transplantation and initial ischemia. Absence of inhibitory mechanism? Longevity of the graft?
14 pregnancies and live births reported :
Donnez, Meirow*
Rosendahl* (6 pregnancies,3 live births, 2 women)/12 cases.
Demeestere (OTx2)
*IVF
Bias due to selective reporting?

32. Options for fertility preservation: Cryopreservation of ovarian tissue
Culture and IVM of primordial follicles: 2 step culture system: culture of tissue followed by isolation of follicles and culture. Or using 3D supportive matrix. Culturing to MII is the next chalange (Abir et al. Histol. Histopatho 2006 Picton et al. Reproduction 2008, Tefler et al. Hum Rep. 2008, Woodruff 2009)
Suspension of isolated primordial follicles (Dolmans et al 2008)
Xenotransplantation: human ovarian tissue to SCID mice. Aberrant microtubule and chromatin patern. Transmission of pathogens, short life span ethical issues, and … ( Lucifero 2002, Kim 2001)

33. Embryo cryopreservation
Integral part of IVF programs >25 years
Success rates 30-50% per embryo transfer, depending on the age at the time of oocytes retrieval.
Only well-established option of fertility preservation
Post pubertal patients.
Partner required
Donor sperm?

34. No male partner: Oocyte cryopreservation
1st live birth 1986 (Chen et al)
But:
oocytes vulnerable to freezing process
intracellular ice formation
membrane rupture, abnormal cortical granule reaction, zona hardening, meiotic spindle and cytoskeleton damage
: 5 live births

35. No male partner: Oocyte cryopreservation
Using PROH/sucrose slow freezing protocol and introduction of ICSI improved pregnancy rates (Porcu et al 1997)
Reported survival rates for mature oocytes (collected from stimulated ovaries) are 50-70%

36. Oocyte vitrification
Cryoprotectants in high concentration used to induce glasslike state, cell then rapidly frozen avoiding formation of intracellular ice
Kuleshova et al, 1999: survival rate 65%, PR/ET 21%
Yoon et al, 2003: survival rate 69%, implantation rate 6.4%, PR/ET 21.4%
Katayama et al, 2003: survival rate 94%, PR/ET 33%

37. Oocyte vitrification
165 live births (Chian et al ASRM 2007: McGill Reproductive Center in Canada, Instituto Mexicano de Infertilidad in Mexico and CECOLFES in Colombia)
A few thousand babies
Vitrification is emerging to be a better technique.

38. Embryo or oocyte cryopreservation after ovarian stimulation
Pregnancies reported are result of fertilization of frozen/thawed oocytes collected after ovarian stimulation
However:
time interval needed for ovarian stimulation 2-6 weeks.
Starting during menstruation
Time may not be available for cancer patients
Recent studies: stimulation luteal phase. (Von Wolff 2009, Sonmezer 2011)
ovarian stimulation associated with high estrogen levels which may not be safe in cases of hormone sensitive tumors such as breast cancer
Estrogens may have an indirect mitogenic effect on receptor negative cancers. (Gupta PB 2006)

39. Embryo or oocyte cryopreservation after ovarian stimulation
Aromatase inhibitor + FSH:
letrozole started 2 days before FSH administration, then given concomitantly, no increased risk of relapse (Oktay 2005, 2007, Azim 2008)
Lower estradiol levels
does not totally avoid stimulation
Early follicular start of treatment.
More studies are needed

40. Collection of immature oocytes from unstimulated ovaries
Pincus, J Exp Med 1935; Edwards, Nature, 1965,1969.
Cha et al,1991: Immature oocyte laparotomy for oocyte donation
Trounson et al, 1994: IVM: vaginal collection of immature oocytes, for PCO related infertility

43. Mature oocytes at oocyte collection in IVM cycles
8mm
12mm
14mm

44. Vitrification of IVM oocytes?
Could oocytes collected from unstimulated ovaries, matured in vitro, then vitrified survive thawing and be fertilized?
Could transfer of these embryos result in a viable pregnancy ?

45. Vitrification of IVM oocytes
No. of patients 20
Mean age
30.7 ±3.7
No. of mature oocytes retrieved 7
No. of immature oocytes retrieved
295
Mean oocyte maturation rate (%)
No. of oocytes vitrified and thawed
215
No. of oocytes survived (mean % + SEM )
148 ( )
No. of oocytes fertilized (mean % + SEM)
96 ( )
No. of embryos transferred (median; range)
64 (3.2; range 1 - 6)
No. of implantations (mean % + SEM)
5 ( )
No. of clinical pregnancies (%)
4 (20.0)
No. of live births (%)
4 (20)
Holzer et al ESHRE 2007

46. Conclusions
Vitrification of in-vitro matured oocytes collected from unstimulated ovaries followed by later thawing and fertilization can result in successful pregnancies and live births

47. Conclusions Preliminary results 20 patients, all with PCO
20% pregnancy rate (vs. 35% in “Fresh IVM” )
Lower implantation rate (10.8% vs. 14.4%)
Learning curve?

48. Conclusions
Collection of immature oocytes from unstimulated ovaries followed by IVM and vitrification of mature oocytes could be offered to patients with hormone-sensitive disease and/or when there is not enough time to stimulate ovaries
no risk of aggravating disease
no theoretic risk of re-instituting metastatic malignant disease
does not require same amount of time as that needed for ovarian stimulation, no need to wait for next cycle

49. When ovarian tissue is being harvested
Additional strategy of fertility preservation combines ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue, followed by in vitro maturation (IVM) and vitrification (Huang, Tulandi, Holzer, Tan, Chian Fertil Steril 2007)
When surgery performed to remove ovarian tumor or for other therapeutic indications

50. Fertility preservation in prepubertal children
Childhood and adolescence period of emotional and psychological instability; issues of sexuality, including fertility, are of particular importance
Depletion of primordial oocytes after gonadotoxic treatment proportional to size of oocyte pool
Younger patients have more oocytes; thus gonadal damage could seem to be less severe than in older patients

51. Fertility preservation in prepubertal children
Global incidence of acute ovarian failure in childhood cancer survivors ranges from 6.3 to 12% (Bakker 2004)
In Childhood Cancer Survivor Study ;the relative risk for survivors of ever being pregnant was 0.81 (Green DM 2009)
Very few treatments benefit younger patients at risk of infertility after treatment

52. Fertility preservation in prepubertal children; Ovarian tissue cryobanking
Primary method of fertility preservation for prepubertal girls
One ovary removed from 47 patients aged y (median: 5)
Ovarian tissue fragments frozen
strong inverse correlation found between age and follicular density
none of the cases had visible ovarian tumour components (Poirot 2007)
58 patients years, underwent ovarian
tissue cryopreservation.
1 case of ovarian lymphoma infiltration (Jadoul 2010)

53. Fertility preservation in prepubertal children: Combined approach
Unilateral oophorectomy: 19 patients aged 5-20 years (median 15 yrs.)
Antral follicles aspirated, average no. of oocytes: 9 (0-37)
GV oocytes – IVM
mature oocytes – frozen
Ovarian tissue – cryopreserved in fragments
(Revel 2008)

54. Fertility preservation in prepubertal children: Ovarian tissue cryobanking
Reimplantation of ovaries cryopreserved before puberty not yet performed in either nonpubertal or pubertal recipients
Animal models: puberty and fertility restored (Sauvat 2008)
Removal of entire ovary, although strongly advocated by some, may not be recommended for pediatric patients in whom fertility outcome is often uncertain.
How much? Based on the risk of the planned treatment and the ovarian volume.

55. Fertility preservation in pre-pubertal children
Fertility preservation should now be considered in children facing cancer treatment with high risk of gonadal toxicity, including high-dose chemotherapy and bilateral irradiation of gonads at toxic doses
Multidisciplinary team

56. McGill Fertility Preservation Center: Catchment Area
Greater Montreal
Quebec
Other Canadian provinces
USA
Trance Atlantic
Physicians: Oncology, Haematology, Radiation Oncology, Surgery, Paediatrics.
Nurses: nursing coordinators.
Non- medical professionals, self referrals

57. Accessibility Available and accessible:” next day appointment” policy.
Reciprocal: referring physician should be available, take part in planning.
Counsellor onsite or available via phone, Skype, webcam
On-going collaboration, individual cases
By-pass, current “road blocks” of the medical system.
Medico legal issues

58. Issues discussed with treating physician
Treatment: drugs, dose.
Time frame- coordinate treatments and tests.
Survival?
Risks of:
Stimulation
Collection
Anaesthesia.

59. Appointment at the fertility preservation center
Long term follow up
US (AFC)
FSH AMH
Consultation
Counselling
2nd collection
Luteal collection
Additional cycle
Treatment
Oocyte CP
Embryo CP
Ovarian tissue CP
GnRH-a
Follow up
Nursing consultation
Discuss and teach planned treatment
Draw blood tests
Feedback to physician
Consultation review of all options, tailored options
GnRH analog
Do nothing
3rd party
Ovarian tissue
Oocyte (IVF/IVM)
Embryo (IVM/IVM)
Appointment at the fertility preservation center
History taking (Fellow)
Ultrasound (AFC, cycle stage)
Initial contact secretary
Information
Schedule
TREAT I NG P H Y S C A N
COUNSELLING

60. McGill Fertility Preservation Center
396 women with various malignancies underwent oocyte retrieval
184 underwent oocyte retrieval without ovarian stimulation followed by IVM :
113 IVM cycles with vitrification of oocytes
71 IVM cycles, matured oocytes fertilized, resulting embryos vitrified

61. McGill Fertility Preservation Center
212 women underwent ovarian stimulation:
156 stimulation cycles, mature oocytes vitrified
56 stimulation cycles, mature oocytes fertilized and resulting embryos vitrified

62. Disease Categories for those who Froze Oocytes/embryos
Type of Cancer
N=183
Breast cancer 75
Hematological malignancies 47
Brain malignancies 15
Gynecological cancers 14
Sarcoma 11
Desmoid Tumour 5
Autoimmune disease 9
Melanoma 1
Colorectal cancer
GIT Malignancy
mean age = 29.7, SD =±5.6

63. Conclusions
Current methods of fertility preservation should be considered as investigational
Patients and their families should be advised about experimental nature of process
Cancer treatments should NOT be compromised

64. Conclusions
Fertility preservation is of utmost importance to patients and families of patients undergoing potentially gonadotoxic treatment
We provide option to try to preserve fertility potential
This hope alone may help in struggle to overcome the disease

65. Proper counseling and shared decision making
Fertility preservation center

66. C.K.N: Cancer Knowledge Network. http://multimed.current-oncology.com
ICRF: Israel Cancer Resreach Fund