Hananel Holzer, MD Medical Director, MUHC Reproductive Center McGill University Health Center

Hananel Holzer, MDMedical Director, MUHC Reproductive Center

McGill University Health CenterDirector, REI Division, Dept. of Obstetrics & Gynecology

McGill University

McGill University

McGill University Health Centre

McGill University Health Centre

A comprehensive approachfor fertility preservation

Fertility preservation

• 713,220 new cases of female cancer were 713,220 new cases of female cancer were diagnosed in 2009 in the U.S. diagnosed in 2009 in the U.S. (Jemal 2009)(Jemal 2009)

• Childhood cancers 1%. 122 new cases per Childhood cancers 1%. 122 new cases per million children million children (Skinner 2006)(Skinner 2006)

• By 2010, one in 250 people will be a cancer By 2010, one in 250 people will be a cancer survivor survivor (Blatt 1999)(Blatt 1999)

• 80% of children and adolescents diagnosed 80% of children and adolescents diagnosed with cancer become long-term survivorswith cancer become long-term survivors

• One of long-term side-effects of cancer therapy One of long-term side-effects of cancer therapy is premature gonadal failure – infertilityis premature gonadal failure – infertility

Ovarian reserveOvarian reserve

• Females born with a finite pool of Females born with a finite pool of oocytes ( Zukerman 1951)oocytes ( Zukerman 1951)

• Challenged by Johnson, Tilly et al; 2004, 2005; Germ line stem cells


W. Hamish B. Wallace 2010





• Destruction of growing follicles• loss of primordial follicles• ovarian atrophy

ChemotherapyChemotherapy

Primordial Follicle Count ⇩⇩⇩

Ovarian Reserve ⇩⇩⇩

Early Menopause and Infertility

• 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

0

500

1000

1500

2000

2500

0 20 50 75 100mg/kg Dose of Cyclophosphamide

Nu

mb

er

of

PM

F’s

(p = 0.0001)

(Meirow et.al. Hum Reprod 1999)

• Focal ovarian cortical fibrosis

• No follicles in zone of fibrosis

• “Wedge shaped”- corresponds to the blood supply

(Meirow et al. Hum Reprod 2007)

Cortical fibrosis

Apoptosis Vascular

injury

Cortical fibrosis

Chemotherapy: Direct and indirect damage

cortex

medulla

(Meirow et al. Hum Reprod 2007)

Loss of regulatory mechanismsLoss of regulatory mechanisms

• Loss of local regulatory factors (AMH?)Loss of local regulatory factors (AMH?)

• Accelerated recruitment of primordial Accelerated recruitment of primordial follicles.follicles.

• Decreasing ovarian reserveDecreasing ovarian reserve

ChemotherapyChemotherapy

• Alkylating agents:Alkylating agents:

• Cyclophosphamide,ifosfamide, nitrosoureas, Cyclophosphamide,ifosfamide, nitrosoureas, chlorambucil, melphalan, and Busulphanchlorambucil, melphalan, and Busulphan

• not cell-cycle specific, highest risk of ovarian failure.not cell-cycle specific, highest risk of ovarian failure.

• AntimetabolitesAntimetabolites

• methotrexate, bleomycin, 5-fluorouracil, actinomycin-methotrexate, bleomycin, 5-fluorouracil, actinomycin-D, mercaptopurine, vincristine)D, mercaptopurine, vincristine)

• impact the cells of the metabolically active ovarian impact the cells of the metabolically active ovarian follicles , are considered to be low risk for gonadal follicles , are considered to be low risk for gonadal dysfunctiondysfunction

• Platin - intermediate riskPlatin - intermediate risk

chemotherapychemotherapy

• Women >40 years have a 90% chance of Women >40 years have a 90% chance of amenorrhea amenorrhea subsequent to multi-agent chemotherapysubsequent to multi-agent chemotherapy

• younger patients 20% - 90%.younger patients 20% - 90%.

• All patients exposed to chemotherapy might have a All patients exposed to chemotherapy might have a diminished ovarian reservediminished ovarian reserve

• significant predisposition for developing premature significant predisposition for developing premature ovarian failure. Infertility > 40% @ 35 years.ovarian failure. Infertility > 40% @ 35 years.

• The estimated probability of early menopause was at The estimated probability of early menopause was at least 25% at age 30 years least 25% at age 30 years (Letourneau , Cancer 2011) (Letourneau , Cancer 2011)

RadiotherapyRadiotherapy

• 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)

RadiotherapyRadiotherapy

• 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)

Reproductive outcomeReproductive outcome

• In animal studies, the risks of M/C and of malformed fetuses In animal studies, the risks of M/C and of malformed fetuses after chemotherapy are high.after chemotherapy are high.

• In humans, no increases in fetal malformation or M/C after In humans, no increases in fetal malformation or M/C after anticancer drug treatment.anticancer drug treatment.

• No higher risk of genetic or chromosomal abnormalities in No higher risk of genetic or chromosomal abnormalities in offspring from parents that had been cancer survivors offspring from parents that had been cancer survivors (Winther JF (Winther JF 2004)2004)

• if conception occurs more than a year after cessation of the if conception occurs more than a year after cessation of the treatmenttreatment

• cancer treatment has mutagenic effects on the oocyte that are cancer treatment has mutagenic effects on the oocyte that are present in growing follicles and the risk of mutagenesis is present in growing follicles and the risk of mutagenesis is related to the stage of oocyte development during exposure and related to the stage of oocyte development during exposure and the drug regime used. the drug regime used. (Meirow 2005)(Meirow 2005)

Fertility preservationFertility 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 ?

Options for fertility preservation Options for fertility preservation

Should be tailored according to:Should be tailored according to:

• Patient’s age Patient’s age

• Type of disease Type of disease

• Spread of the diseaseSpread of the disease

• Planned treatmentPlanned treatment

• Time available Time available

• Whether she has a partnerWhether she has a partner(Holzer Tan 2005)

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)


• 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)


• 3 meta-analyses were published:3 meta-analyses were published:• GnRHa are effective in preserving ovarian GnRHa are effective in preserving ovarian

function and in reducing amenorrhoea function and in reducing amenorrhoea (Clowse 2009, (Clowse 2009, Ben Aharon 2010)Ben Aharon 2010)

• only prospective randomized studies:only prospective randomized studies:

• odds ratio of 3.5 favouring the use of GnRHaodds ratio of 3.5 favouring the use of GnRHa..

• Higher rates of resumption of menses and Higher rates of resumption of menses and ovulationovulation

• No improvement of pregnancy rates No improvement of pregnancy rates (Bedaiwy 2010)(Bedaiwy 2010)


• Some evidence that GnRHa has a protective Some evidence that GnRHa has a protective effect. effect.

• the final conformation is still awaited.the final conformation is still awaited.

• GnRH antagnist – faster desensitization.GnRH antagnist – faster desensitization.• Flare up effect; combined treatment? Flare up effect; combined treatment? (Mardesik (Mardesik

2004 von Wolff 2011)2004 von Wolff 2011)

• The GnRHa could be used to induce ovulation The GnRHa could be used to induce ovulation in a stimulated cycle. in a stimulated cycle.

• Reduced tumour cell sensitivity to chemo in E+ Reduced tumour cell sensitivity to chemo in E+ cases ?cases ?


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)

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

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?

Risk of presence of neoplastic cells in the transplanted tissue

• Subclinical involvement of Hodgkin’s Lymphoma has not Subclinical involvement of Hodgkin’s Lymphoma has not been identified in ovarian tissuebeen identified in ovarian tissue (Seshadri Gook et al 2006)(Seshadri Gook et al 2006)

• detection of Hodgkin lymphoma within ovarian tissue detection of Hodgkin lymphoma within ovarian tissue taken at the time of harvest for cryopreservationtaken at the time of harvest for cryopreservation. . (Bittinger (Bittinger 2011)2011)

• 58 patients with hematological malignancies underwent 58 patients with hematological malignancies underwent ovarian tissue cryopreservationovarian tissue cryopreservation− after thawing, markers to detect minimal residual after thawing, markers to detect minimal residual

disease useddisease used− real-time RT-PCR positive in one patient with CML.real-time RT-PCR positive in one patient with CML.

− this alarming result avoided tissue transplantationthis alarming result avoided tissue transplantation (Meirow et al 2008)(Meirow et al 2008)

• Positive markers of CML and AML in cryopresrved Positive markers of CML and AML in cryopresrved harvested ovarian tissue harvested ovarian tissue (Dolmans 2010)(Dolmans 2010)

Risk of presence of neoplastic cells in the transplanted tissue

• Other organ transplants: donor derived Other organ transplants: donor derived malignancy malignancy ( Kauffman 2002, Ison 2011 AM J ( Kauffman 2002, Ison 2011 AM J Transplant)Transplant)

• Extreme caution is warranted before we assume Extreme caution is warranted before we assume that we understand tumour biology well enough that we understand tumour biology well enough to estimate the risk of transmission of to estimate the risk of transmission of malignant cells in autotransplanted ovarian malignant cells in autotransplanted ovarian cortex.cortex.

• BRCA 1&2 carriers - potential of developing BRCA 1&2 carriers - potential of developing ovarian cancer ovarian cancer (Colgan 2001)(Colgan 2001)

• Prophylactic BSO, transplant ovarian Prophylactic BSO, transplant ovarian fragments?fragments?


• 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?


• 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)

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?

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

• 1986-1997: 5 live births

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%

No male partner: Oocyte cryopreservation

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%

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.

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)

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

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

Mature oocytes at oocyte collection in IVM cycles

8mm 12mm

14mm

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 ?

No. of patients20

Mean age30.7 ±3.7

No. of mature oocytes retrieved7

No. of immature oocytes retrieved295

Mean oocyte maturation rate (%)67.9+4.1

No. of oocytes vitrified and thawed215

No. of oocytes survived (mean % + SEM )148 (67.5 + 5.8)

No. of oocytes fertilized (mean % + SEM)96 (64.2 + 4.5)

No. of embryos transferred (median; range)64 (3.2; range 1 - 6)

No. of implantations (mean % + SEM)5 (10.3+5.7)

No. of clinical pregnancies (%)4 (20.0)

No. of live births (%)4 (20)

Holzer et al ESHRE 2007

Vitrification of IVM oocytes

ConclusionsConclusions

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


• 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?


• 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

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

Fertility preservation in Fertility preservation in prepubertal childrenprepubertal 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

Fertility preservation in Fertility preservation in prepubertal childrenprepubertal 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

Fertility preservation in prepubertal Fertility preservation in prepubertal children; Ovarian tissue cryobankingchildren; Ovarian tissue cryobanking

• Primary method of fertility preservation for prepubertal girls

• One ovary removed from 47 patients aged 0.1-14 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 0.8-15.8 years, underwent ovarian

tissue cryopreservation.

• 1 case of ovarian lymphoma infiltration (Jadoul 2010)

Fertility preservation in prepubertal Fertility preservation in prepubertal children: Combined approachchildren: Combined approach

• Unilateral oophorectomy: 19 patients Unilateral oophorectomy: 19 patients aged 5-20 years (median 15 yrs.)aged 5-20 years (median 15 yrs.)

• Antral follicles aspirated, average no. of Antral follicles aspirated, average no. of oocytes: 9 (0-37)oocytes: 9 (0-37)

− GV oocytes – IVMGV oocytes – IVM

− mature oocytes – frozenmature oocytes – frozen

• Ovarian tissue – cryopreserved in Ovarian tissue – cryopreserved in fragmentsfragments

(Revel 2008)(Revel 2008)

Fertility preservation in prepubertal Fertility preservation in prepubertal children: Ovarian tissue cryobankingchildren: 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.

Fertility preservation in pre-Fertility preservation in pre-pubertal childrenpubertal 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

McGill Fertility Preservation Center: Catchment Area

• Greater Montreal Greater Montreal

• QuebecQuebec

• Other Canadian provincesOther Canadian provinces

• USAUSA

• Trance AtlanticTrance Atlantic

• Physicians: Oncology, Haematology, Radiation Physicians: Oncology, Haematology, Radiation Oncology, Surgery, Paediatrics.Oncology, Surgery, Paediatrics.

• Nurses: nursing coordinators.Nurses: nursing coordinators.

• Non- medical professionals, self referralsNon- medical professionals, self referrals

• Available and accessible:” next day Available and accessible:” next day appointment” policy.appointment” policy.

• Reciprocal: referring physician should be Reciprocal: referring physician should be available, take part in planning.available, take part in planning.

• Counsellor onsite or available via phone, Skype, Counsellor onsite or available via phone, Skype, webcamwebcam

• On-going collaboration, individual casesOn-going collaboration, individual cases

• By-pass, current “road blocks” of the medical By-pass, current “road blocks” of the medical system.system.

• Medico legal issuesMedico legal issues

AccessibilityAccessibility

Issues discussed with treating Issues discussed with treating physicianphysician

• Treatment: drugs, dose.Treatment: drugs, dose.

• Time frame- coordinate treatments and Time frame- coordinate treatments and tests.tests.

• Survival?Survival?

• Risks of:Risks of:

• Stimulation Stimulation

• CollectionCollection

• Anaesthesia.Anaesthesia.

TREATING

PHYSCIAN

COUNSELLING

McGill Fertility Preservation CenterMcGill 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

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

Disease Categories for those who Froze Oocytes/embryos

Type of Cancer N=183

Breast cancer75

Hematological malignancies47

Brain malignancies15

Gynecological cancers14

Sarcoma11

Desmoid Tumour5

Autoimmune disease9

Melanoma1

Colorectal cancer5

GIT Malignancy1

mean age = 29.7, SD =±5.6


• 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 Cancer treatments should NOT be compromisedcompromised


• 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

Proper counseling andProper counseling andshared decision makingshared decision making


center

C.K.N: Cancer Knowledge Network. C.K.N: Cancer Knowledge Network. http://multimed.current-oncology.comhttp://multimed.current-oncology.com

ICRF: Israel Cancer Resreach Fund ICRF: Israel Cancer Resreach Fund http://www.icrfmontreal.orghttp://www.icrfmontreal.org

Hananel Holzer, MD Medical Director, MUHC Reproductive Center McGill University Health Center

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