fertility preservation in cancer patients

Fertility Preservationin Cancer Patient

Marwan Al-Halabi MD. PhDProfessor in Faculty of Medicine Damascus - University

And

Medical DirectorOrient Hospital assisted Reproduction center Damascus – Syria

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.

Childhood CA Survival Rate

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

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

The Forgotten Female?

Studies suggest that only

about half of oncologists

discuss fertility preservation

with their patients

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.

Cancer specialists should inform patients about

these options

refer them to fertility specialists.

counseling by a qualified geneticist

ASRM Guidelines

should not deny cancer patients assistance with reproduction??.

An Expected Shortened Life

Cancer Treatment&

Fertility

Chemo – Therapy

Fallicular destruaction .

Ovarian Fibrosis .

Premature Ovarian Failure

Reduced E2 and P4 Levels

13

Temporal intervals in folliculogenesis

Ovulation

Differential sensitivity of different cellular components of the ovary

Impaire follicular maturation.

Deplete primordial follicles.

Effects of cancer treatment onmale 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

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

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.

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; remaindermay experience temporary amenorrhea. No data available for women over 40 .

>800 100% permanently sterilized

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.

Factors affecting the extent of chemotherapyinduced 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.

Effect of age on risk of premature ovarian failure

indicator of ovarian damage

The best biochemical indicator of ovarian damage and failure

is :

FSH

E2

Inhibin – B

AMH

Fertility

Preservation

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

Ovarian Cancer

3-17% of ovarian cancer patients are < 407-8% of all stage I epithelial ovarian cancers occur in women < 35Possible 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.

Fertility-Sparing Surgical Procedures

Ovarian cystectomy

Unilateral salpingo-oophorectomy

Bilateral salpingo-oophorectomy

All should be performed with comprehensive

surgical staging

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

GnRH agonist

The concept is to

induce

hypogonadism

before starting

chemotherapy,

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.

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.

–

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

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.

Embryo Cryopreservation

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.

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.

Oocyte Cryopreservation

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 .

Pregnancy Rate 2 – 11 %

Oocyte Freezing

68% Survival

48% Fertilization

21% Pregnancy Rate

Oocyte Cryopreservation.

Oocyte Vitrification

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

Ovarian

Transposition

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.

TechniqueTechniques for ovarian transposition using a laparoscopic approach vary according to the radiation field shape, size, and location

Ovarian Transposition

Ovarian Transposition

Pregnancy rates are approximately 50 – 75 %

Spontoneouselly.

11% Conceived with IVF

Benefit :

Prevention of premature menopause .

Preservation of fertility ?? !

Complications of Ovarian Transposition

Fallopian tube infarction.Chronic ovarian pain.Ovarian cyst formation.Migration of ovaries back to their originalposition.Ovarian metastasis (No increased risk).

Ovarian Tissue Freezing and transplantation

Ovarian cortical freezing

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

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

It is still a Hope

Stem cells Therapy

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.

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)

Risk of ovarian involvement in cryopreservation candidates

Low risk Moderate risk High risk

Wilms’ tumor Stage IV breast cancer Leukemia

LymphomasStage 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

Screening of ovarian tissue

Best screening is light microscopy

Molecular markers can be used in rare cancers

Test tissue before freezing and after thawing

Transplantation

High rate of follicle loss after transplantation

5% of primordial

follicles lost during

freezing and thawing

65% of primordial

follicles lost during

revascularization

(xenografting)

Whole ovary cryopreservation as an attempt to improve follicle

survival

Successful in micePartial success in sheep– 3/11 patent after 8–10 days1

– Long-term function with new freezing technology2

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 2Parthenogenic activation

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 cellsWhole ovary cryopreservation not yet technically possible in humans

Orthotopic(pelvic)

transplant

Heterotopic(subcutaneous)

transplant

Resumptionof ovarian functions

Spontaneousconception

IVF

Embryo transfer

Ovarian transplantation techniques

Orthotopic(pelvic)

transplant

Heterotopic(forearm)transplant

Resumptionof ovarian functions

Spontaneousconception

IVF

Embryo transfer

Ovarian transplantation techniques

“Successful” cases of orthotopic ovarian transplantation

Author Outcome

Oktay & KarlikayaN 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

Pelvic ovarian transplantation

0

20

40

60

80

100

0 5 10 15 20 25 30Days after follicle seen

Estra

diol

(pg/

mL)

Prog

este

rone

(ng/

mL) hCG injections

First orthotopic ovarian transplant with frozen ovarian tissue

First pregnancy after ovarian transplant?

Ovarian biopsies frozen in 1997 at age 25Received MOPP for Hodgkin’s diseaseBirth control pills after chemotherapyTransplant in 2003Spontaneous conception & delivery in 2004

Questions with Donnez et al. report

Risk of ovarian failure relatively low– 20% at ≤ 25 years of age

– 50% chance of pregnancy1

Both ovaries remainThree ovulations from the right (intact) ovaryNo hormonal or ultrasound monitoring of ovulation from the transplant

Live birth following transplantation of cryopreserved ovarian tissue

after chemotherapy-induced ovarian failure

Comparison of two orthotopic transplant techniques

Ovarian function &pregnancy via IVF

No ovarian function

Orthotopic(pelvic)

transplant

Heterotopic(subcutaneous)

transplant

Resumptionof ovarian functions

Spontaneousconception

IVF

Embryo transfer

Ovarian transplantation techniques

Advantages of heterotopic transplant

Non-invasive

Repeated procedures feasible

Can inject agents directly in the graft

Easy monitoring (risk of recurrence)

Easy removalSuitable after pelvic radiation

“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?

Patient A

Patient B

Patient A

Patient B

Estradiol output from heterotopic transplant

RCV estradiol

0100020003000400050006000

1 5 13 15 20 22 28 32 33 34 36 39 40Cycle day (arbitrary)

pg/m

L

RH estradiol

0

50

100

150

200

250

1 5 13 15 20 22 28 32 33 34 36 39 40Cycle day (arbitrary)

pg/m

L

RH

RCV

Ovarian transplant in a patient with breast cancer

36-year-old patientOvary cryopreserved before chemotherapyHigh-dose chemotherapy before bone marrow transplantIn menopause for 6 years

Follicle development in a frozen-thawed transplant

Percutaneous oocyte retrieval

First embryo after ovarian transplant

24 hours

18 hours24 hours

Embryo #3: 1 PN after ICSI

16h post-ICSI 24h post-ICSI

6/03/04z

PGD: Female pronucleus, partially decondensed sperm DNA

“Normal” embryo yield

22 oocytes

9 suitable for IVF (8 ICSI)

2 abnormal embryos

1 grade 1/2, 4-cell embryo

Yield: 4.5%

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)

Is Pregnancy Possibleafter heterotopic transplantation?

Brenda: first primate pregnancyafter ovarian transplant

Latest case of heterotopic ovarian transplant

Hodgkin’s disease at age 28: receives ABVD + RT to chest & spleenOvarian tissue cryopreserved at age 29 prior to SCT due to relapseAmenorrhea and elevated FSH for 2.5 years (45–95 IU/L) after receiving preconditioning chemotherapySubcutaneous ovarian transplant performed on 12 August 2004

Subcutaneous transplantation in a menopausal woman

post-stem cell transplantation

15 pieces thawed5 x 5 x 1 – 15 x 5 x 2 mm

Suture pull-through technique

Ovarian function after heterotopic ovarian transplant

Felt follicle growth 7 weeks post-transplant10 weeks post-transplant– E2: 250 pg/mL– P4: 14 ng/mL

Transvaginal ultrasound performed 11 weeks post-transplant

Pregnancy after heterotopic transplant

Corpus luteum in menopausal ovary?

Spontaneous recurrent pregnancies after heterotopic ovarian transplant

D&C performed as no FH detectedCytogenetics of POC revealed trisomy 16Patient felt follicle growth 3 weeks after D&C and menstruated a week laterHad intercourse on the day of ovulationand conceived again!

Long-term follow-up

Resumed follicular activity in graft 3 months postpartumOvulation in graft? and “menopausal” ovary a week agoAttempted pregnancyB-hCG pending

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 contemporaneouslywith follicle growth in the graft?

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 age2 Ut1 and Ut2: adult human uterus used as a negative

control

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 Mvhexpression in the bone marrow

Impact of chemotherapy on stromal function

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

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

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.

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-rasc-mycmutations P53 tumor-suppressor gene.

S. AL SAMAWI MD. Gyn. Obs.A. TAHA MD. Gyn. Obs.M. ABDUL WAHED MD. Gyn. Obs.J. SHARIF Senior BiologistN. ABO HASSAN AndrolgistF. RAHMEH AndrolgistS. MATOUK Executive SecretaryF. HAMAD Administration ManagerA. ALKHATEB M.D Micro BiologistR. ALKHATEB MD. Gyn. Obs. Ph. D.

Acknowledgement

Thank You