Oncofertility, and Cryopreservation:An update

Angelos Vilos

REI Division PGY7

Department of Obstetrics and Gynecology

Objectives & Outline

Definitions

Technical Updates

Goals in our department

What is Oncofertility?

“Oncofertility is a subfield that bridges oncology and reproductive research to explore and expand options for the reproductive future of cancer survivors.”

(Dr. Teresa K. Woodruff, Oncofertility Consortium, 2006)

Option Gap

Information Gap

Data Gap

Scientist Oncologist/ Clinician

Endocrinologist/ Fertility Specialist

ONCOFERTILITY

Oncofertility Specialist

Institutional Infrastructure / expertise

Reasonable chance for survival

Reasonable chance for fertility

Hope Choice

Oncofertility

1. Need?

2. What is Available?

3. What could be offered?

Need?

2011 US Stats: – 774 370 women/year in US are diagnosed with

some form of invasive cancer.– 8% (62 000) < 40 years of age.

• 33 000 – breast• 27 000- Leukemia/melanoma/NHL/leukemia/uterine• 4 000 – cervical (50% of all cases < 40 years)

(American Cancer Society: Cancer facts and figures, 2011)

Canadian Cancer Society n Canadian Cancer Statistics 2013

5 year survival rates:• Breast: 90%• Melanoma: 91%• Cervical: 71%• NHL: 69%• Leukemia: 55%

Canadian Cancer Society; Canadian Cancer Statistics 2013

Better detection, Better treatment, More Survivors!

Treatments: chemotherapy, radiation, BMT, SCT

– Cure rates for some types > 90%.• Pregnancy Rates < 1% post treatment

(Salooja N, et al. Pregnancy outcomes after peripheral blood or bone Marrow transplantation: retrospective survey. Lancet. 2001;358: 271-76)

ChemotherapyPremature Ovarian Failure: Infertility

• Alkylating agents:– Busulfan– Carboplatin– Chlorambucil– Cyclophosphamide– Dacarbazine– Ifosfamide– thiotepa

(American Cancer Society: Cancer facts and figures, 2011)

(Meirow D. Epidemiology and infertility in cancer patients. In: Preservation of fertility Gosden R, Tulandi T, editors. London: Taylor and Francis, 2004:21-38.

AA RR for POF

> 40 years = 80% Amenorrhea < 30 years = 20% Amenorrhea

RadiationPremature Ovarian Failure: Infertility

• Ionizing radiation

• LD50 of irradiation to oocyte causing POF is 2 Gy

• 5-10 Gy of radiation to ovary: POF 97% women• Doses between 14-30 Gy: Uterine dysfunction

*Cervical Cancer: Often receive 4 Gy.

CT abdo/pelvis: 8-30 mGy

Therapy often begins within 2-4 weeks of diagnosis.

*4000 children annually in US undergo sterilizing radiation treatment.

(American Cancer Society: Cancer facts and figures, 2011)

(Wallace WH. et al.. Int J Rad Oncol Biol Phys 2005;62:738-44)

What can we offer today, May 21, 2014?Fertility Sparing Techniques

• Oophoropexy: – Moving ovary out of intended radiation field.

• Radiation spray?• Stay where they are supposed to?• Post-op ischemia?

• GnRH agonist or OCPs during chemotherapy.– Convincing evidence? (Blumenfeld Z. et al. Fertil Steril. 2008;89:166-

73)

• Pre-treatment Ovarian stimulation (1-2 weeks)– Embryo banking (Partner? Frozen sperm?)

American Society of Reproductive Medicine (ASRM, 2006)

• Cryopreservation: “Standard Procedures”– Sperm– Embryo

(Requisite to embryo cryopreservation is male gamete source?)

Statement:• Creation and disposition of human embryos produced solely for future use

is dependent on:– Survival risk of patient– Ethical concerns– Legal concerns– Religious concerns

Alternative Options

ASRM (2012): Drops “Experimental” label

1. Cryopreservation of unfertilized Eggs– +/- In vitro maturation

2. Ovarian Tissue cryopreservation:– Subsequent autotransplantation

Cryopreservation

OOCYTE CRYOPRESERVATION

1. Background

2. Cryopreservation for dummies

3. Global Experience

Oocyte Cryopreservation

First pregnancy from oocyte cryopreservation:

- 25 years ago. (Chen C. Pregnancy after human oocyte cryopreservation. Lancet, 1986)

Worldwide (2009):

-Approximately 1000 births (Noyes N, et al. Over 900 oocyte cryopreservation babies born with no

apparent increase in congenital anomalies. Repro Biomed Online, 2009)

Oocyte Cryopreservation:Expansion of Techniques

Source of eggs:

1) Standard OHS: Retrieve mature oocytes • (Metaphase II)

2) Harvest Immature oocytes: • Primordial/Primary follicles

• Post oophorectomy aspiration• U/S guided aspiration

• Followed by In vitro Maturation (IVM)

Oocyte Cryopreservation:Expansion of Techniques

Freezing Technique:

1) Slow Freeze-Rapid Thaw technique

2) Vitrification- Rapid Freeze-Rapid Thaw

Oocyte Freezing: Challenges

Metaphase II oocyte:

Large size/High water content/Aligned chromosomes:

• Easily damaged spindle apparatus by ice crystals• Hardening zona pellucida adversely affects fertilization

(Shaw JM et al. Theriogenology 2000;53:59-72,

Baka SG et al. Hum Reprod 1995;10:1816-20,

Matson PL et al. Hum Reprod 1997;12:1550-3)

Cryopreservation Media

Nutrient and Dehydrating Components:• Human Serum Albumin• Propanediol• Sucrose

Cryoprotectant Components:• Dimethylsulphoxide (DMSO)• Ethylene Glycol (Anti-freeze)

Slow Cool-Rapid Thaw Process:

Slow Cool• Cooling process:

– 2oC/min to -7oC– 0.3oC/min to -40oC– 10oC/min to -120oC– Plunged in liquid

nitrogen to -196oC

Rapid Thaw• Rapid Warming

– 100oC/min (35oC bath)– Avoid re-crystallization

of large/cell damaging ice crystals

– Washed to minimize osmotic damage

-2oC

-5oC

< -10oC

Slow Cool

Rapid Cool

Very Rapid Cool

Slow Cool: Exosmosis

(Mazur P. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J Gen Physiol 1963.)

Drawbacks to Slow Freeze• Osmotic shock to cells

• Intracellular ice formation:– Estimated Oocyte survival rate 23-89% (n = 50%)

(Huang J. et al. Fertility preservation for female. J Rep Contr, 2006; 17: 109-28)

(Donnez J., Dolmans MM. Cryopreservation and Transplantation of Ovarian Tissue. Clinical Obs and Gynec. 2010: 53:4; 787-96)

Vitrification: An emerging technique for the new millennium

• Uses super high concentration of DMSO and ethylene glycol (antifreeze)

• Embryos/Eggs placed in successively higher concentrations of cryoprotectant, and sucrose.

• Rate of temperature drop: 23 000oC/min– 70 000x faster than slow cool method(0.3oC/min)– Ice crystals cannot form

Vitrification: Thawing Technique

• Instantaneously warmed• Immediately taken out of high concentration

Advantages:– Faster– Cheaper– Survival Rates 89-100%

(Katayama KP, et al. High survival rate of vitrified human oocytes

results in clinical pregnancy. Fertil Steril 2003)

Systematic Review and Meta-analysis:Comparing Vitrification vs SFRT Embryos

Analyzed 4 studies (Human blastocysts/cleavage stage embryos)– Included 3 RCT– 7482 vitrified– 1342 slow-frozen

• Day 3 embryo survival rate:– Vitrification greater (OR = 15.57)

• Day 5 blastocyst survival rate:– Vitrification greater (OR = 2.20)

(Loutradi KE, et al. Fertil Steril. 2008 Jul;90(1):186-93)

Status of oocyte cryopreservation in the United States

Percent (%)

ART Centers Responded 282/442 (64%)

Offer Oocyte Cryopreservation (OC) 143/282 (51%)

Offer OC only for cancer survivors, alternative to embryo freezing post IVF

36%

Offer OC electively for Advanced Mat. Age 64%

Elective for AMA 35-37 yr38-40 yr > 40 yr

87%49%26%

Method of freeze Slow Freeze (16%)Vitrification (56%)Both (28%)

Rudick B, et al. Fertility and Sterility, Dec. 2010

Success RatesOutome Percent (%)

No. of cryopreservation cycles 1847

No. of thawed cycles 857

Fertilization Rates 67%

Clinical Pregnancy Rates 33%

No. of live births (Live birth Rate) 337/857 (39%)*Multiples included

Rudick B, et al. Fertility and Sterility, Dec. 2010

Oocyte cryopreservation: Fears?

1) COH to retrieve oocytes prior to cancer therapy:

• Delay cancer therapy?– Currently Established technique for embryo freezing in cancer

patients.

• Fears regarding effect of gonadotropins on potentially malignant cells?– No Evidence of any adverse effects of fertility drugs on cancer

outcomes.– Breast: Aromatase Inhibitors

2) Cannot be done in prepubescent girls.

In Vitro Maturation

In Vitro maturation (IVM)• The isolation of primordial follicles from either fresh or

cryopreserved ovarian tissues.• Primordial follicles:

– Mechanically/Enzymatically isolated from cortical tissue – grown in serial cultures mimicking in vivo environment.

– Cortex isolates (containing PF) can be xenografted to host animals for growth to Pre-antral stage or grown on 3D gels.

SOUNDS EASY- RIGHT?

(Dolmans MM, et al. Reproduction 2007, 134: 253-62)

Copyright ©2010 Society for Reproduction and Fertility

Reproduction. 2008 Dec;136(6):703-715

Figure 1 Diagrammatic representation of the regulators and developmental time frames of mammalian follicle and oocyte development in vivo

~ 300 live births

Copyright ©2010 Society for Reproduction and Fertility

Reproduction. 2008 Dec;136(6):703-715

Multi-step strategy for the complete in vitro growth and maturation of follicles from large

animals and humans

•Support induction of steroidogenic function by GCs.

•Facilitate acquisition of oocyte cytoplasmic competence.

•Fully grown

•Cumulus enclosed oocyte

•Complete MII

3D culture media-support growing follicle

Copyright ©2010 Society for Reproduction and Fertility

Reproduction. 2008 Dec;136(6):703-715

Summary of the critical determinants of the success of in vitro growth and maturation systems for mammalian

oocytes

Ovarian Tissue Cryopreservation

1. History

2. Global Experience

3. Techniques

Ovarian Tissue Cryopreservation:A brief history

First Fresh ovarian transplantation reported 1906!!!!

(Morris RT. A case of heteroplastic ovarian grafting followed by pregnancy, and the delivery of a living child. Med Rec 1906;69:697-8)

Dr. RT Morris

• Medical training at Columbia Univ. 1880-1885.

• Performed 15 ovarian transplants.

• Tested various sites for implantation:– Broad ligament– Rectus muscle

Fresh Ovarian Transplant• Greatest triumph:

– 21 y/o recipient had ovaries removed for PCOS.– Donor 33 y/o, operated on for uterine prolapse,

removed 1 ovary.– Transplanted donor ovary into recipient in Peritoneal slits

on broad ligament.– Menstruation after 4 months.– Delivered live birth (followed by 2 full term pregnancies)

(Gosden RG. Robert T. Morris, MD- appreciation of an enlightened surgeon and pioneer of ovarian transplantation. Fertil Steril, Nov 2010)

Cryopreservation of whole ovarian tissue

Requires:

1. Surgical removal of part or whole ovary.

2. Create thin 1mm, (1x1 cm) cortex slices.

3. Freeze-Thaw process:– Slow-freeze*– Vitrification

4. Surgically re-implant:Orthotopic: Ovarian remnant or broad ligament.

Heterotopic: Anywhere else in body.

Advantages of whole tissue cryopreservation

• Performed without any ovarian stimulation.• Does not delay cancer therapy.• Can be banked indefinitely.• Can be used in prepubescent population.• Can be used anytime a surgeon must

remove an ovary, or ovarian reserve is in jeopardy.

Indications for cryopreservation in Nonmalignant disease

A) Uni/Bilateral oophorectomy

1. Benign ovarian tumors

2. Severe/Recurrent endometriosis

3. BRCA1/2 mutation carriers

B) Risk of Premature Menopause

1. Turner syndrome

2. Family history

3. Benign disease requiring chemotherapy:• Autoimmune disorders

C) Bone Marrow transplantation

1. Sickle cell anemia

2. Thalassemia major

3. Aplastic anemia

4. Autoimmune disorders refractory to immunosuppressive therapy.

(Donnez J., Dolmans MM. Cryopreservation and Transplantation of Ovarian Tissue. Clinical Obs andGynec. 2010: 53:4; 787-96)

Ovarian Tissue Cryopreservation: Evolution of the technique

1994:

Trials in sheep had demonstrated that orthotopic transplantation techniques could restore ovarian endocrine function, fertility, and yield viable offspring.

(Gosden RG, et al. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum Reprod 1994;9:597-603)

Ovarian Tissue CryopreservationHumans:Successful ovarian hyper-stimulation:

1) Ovarian function after transplantation of frozen, banked autologous ovarian tissue.

(Oktay K, Karlikaya G. N Engl J Med 2000;342:1919)

2) A technique for laparoscopic transplantation of frozen-banked ovarian tissue.

(Oktay K, Aydin BA, Karlikaya G Fertil Steril 2001;75:1212-6)

Spontaneous follicular development and estrogen production:

3) Orthotopic reimplantation of cryopreserved ovarian cortical strips after high

dose chemotherapy for Hodgkin’s lymphoma. (Radford JA, et al. Lancet

2001:357:1172-5)

Ovarian Tissue Cryopreservation

Human Live Birth:

The first human livebirth after orthotopic transplantation of cryopreserved ovarian tissue.

(Donnez J, et al. Lancet. Sept. 2004; 364:1405-10)

World wide:

25 subsequent live births by orthotopic transplantation:

- 50% of which registered in the last 3 years

- Both spontaneous and through COH

(Chung K et al, Conceptions, 2013;99:1534-42)

Ovarian Tissue Cryopreservation: Outcomes

• Average time to first menses: 4.7 months

• Duration of graft function: 9-86 months

• Interval from graft to first pregnancy: 6-11 months

(Chung K et al, Conceptions, 2013;99:1534-42)

Ovarian Tissue Cryopreservation

• Heterotopic autograft:Ovarian tissue grafted into the subcutaneous space

above the brachioradialis fascia of the forearm.

– Ovarian function restored in 2 patients for at least 2 years following heterotopic autograft.• 1 patient, oocytes aspirated percutaneously,

morphologically normal embryo transferred following IVF-OHS without pregnancy.

(Oktay K et al. Lancet 2004;363:837-40)

Ovarian Tissue Cryopreservation• Heterotopic autograft:

– 4 young cancer survivors (3 cx, 1 breast)• 29-38 yrs old

– Cryopreserved ovarian tissue thawed and transplanted between rectus muscle and fascia (2002-2007)

(Kim SS et al, Long term ovarian function and fertility after heterotopic autotransplantation f cryobanked human ovarian tissue : 8-year experience in cancer patients. Fertil Steril, June 2009)

Ovarian function MoniteringHormonal levels (FSH, LH, Estradiol, Progesterone)

Postmenopausal levels prior to transplantation.

(ie: FSH 80-100 mIU/mL, Undetectable E2 x 4 patients)

Transplant #1: • Restored ovarian function by 12-20 weeks. (FSH <20)• Effect lasted 3-5 months.

Transplant #2: • ¾ patients required 2nd transplant (4th relapsed disease)• Restored ovarian function in 3 patients 15-40 months (FSH <15)• 2/3: evidence of spontaneous ovulation (LH surge, elevated P)• 1/3: COH and IVF (no pregnancy)

Autografted ovarian tissue failure:Orthotopic superior to Heterotopic

• Key components in decreased success of autografted ovarian tissue:– Ischemia post transplantation

• Host-doner angiogenesis > Day 5 – Endocrine Function

• Folliculogenesis 4-6 months – Oxidative Stress – Significant loss of follicles during process (50-65%)

(Van Eyck AS, et al. Fertil Steril. 2010;93:1676-85)

(Donnez J., Dolmans MM. Clinical Obs and Gynec. 2010: 53:4; 787-96)

(Nottola SA, et al. Fertil Steril 2008;90;23-32)

Ovarian tissue cryopreservation and Re-implantation

I can read your thoughts!

Risks?

Re-Seeding tumor cells?Is there a potential risk of re-seeding tumor cells in transplanted ovarian tissue?

High Risk:

Leukemias - systemic by nature.

- AML/ALL require urgent chemotherapy, but not ovarian toxic.

Moderate Risk:

Neuroblastomas

Breast

Low Risk:

Wilms, lymphomas, osteosarcomas, squamous cell cervical, rhabdomyosarcomas

(Donnez J., Dolmans MM. Clinical Obs and Gynec. 2010: 53:4; 787-96)

Pathologic Evaluation

Prior to ovarian tissue autografting:

• Histological evaluation• Chromosomal analysis• Tumor markers (RT-PCR)

(Oktay K. Ovarian tissue cryopreservation and transplantation: preliminary findings and implications for cancer patients. Hum Reprod Update, 2001)

Potential indications for Ovarian autotransplantation based on the Analysis of 5571 autopsy findings of

females under the age of 40 in Japan

Leukemia – 2027

Gastric Carcinoma – 1095

Lymphoma – 736

Breast Cancer – 648

Uterine Cancer – 428

Pulmonary Carcinoma – 381

Colon Carcinoma – 256

(Kyono K, et al. Fert and Ster. Vol 93, No 7: May 2010)

Analysis of 5571 autopsy findings of females under the age of 40 in Japan

% Ovarian Mets

ConclusionComponents of a Successful Oncofertility Program:

1. Knowledgeable Oncologist/Primary Surgeon-Referral

2. Collaboration of Ethics support team

3. Fertility Specialist/ART skills-Infrastructure– Counseling– Social support network

4. Established Basic Science/Lab Teams– Cryobiology (Tissue Grafting, Oocyte Vitrification)– In vitro maturation

Experimental Vs Established

Oocyte Cryopreservation:

1. At least 25 year old history.

2. Is performed by at least 50% of ART clinics in US, and Europe.

3. Has Achieved over 1000 live births to date, 90% in the last 5 yrs.

4. Uses preexisting, well established OHS protocols.

5. Has evolved from the less successful slow-freeze /rapid-thaw, to vitrification method.

-Delivery Rate ~ conservatively at 10-15 % per oocyte thawed

Potential Concerns

1) Gonadotropin stimulation can potentiate malignant growth?

• Inconclusive evidence from either in vivo or in vitro studies.• Minimal stimulation protocols.• Aromatase Inhibitors to decrease circulating E2.

2) OHS can delay cancer therapy?

• Possible to use long or short protocols, luteal or proliferative start times limiting delay by no more than 2 weeks from referral to chemotherapy.

Experimental Vs Established

In Vitro Maturation of immature oocytes:1. Requires significant knowledge and replication of ovarian

physiology.

2. No delay in cancer therapy

3. Requires new training of technicians in:– cortex tissue sampling, – serial growth media, – precision timing of substrates.

4. Primary follicle – Mature oocyte: – Has not resulted in a live birth to date.– 300 live births from Antral to mature oocyte only.

Experimental Vs Established

Ovarian Tissue Grafting:

1. Has produced 26 live births world-wide, by orthotopic procedure.

2. Advantage of banking tissue indefinitely to allow technology to catch up with demand.

3. Does not require pre-treatment COH.

4. Only technology suitable for pediatric population.

5. Subject to surgical skill, requires 2x surgery.

6. Laboratory skills similar to embryo cryopreservation (slow freeze).

7. Dependent on a pathway between: Operating theatre - Pathology lab - Cryopreservation lab.

8. Tissue implant success:– Ischemic damage = Loss of PF = Poor Responders

Potential Concerns

1) Re-implanted tissue carries risk of malignant potential?

• Can be controlled for by careful:– Histologic analysis– Pathologic analysis– Stringent patient selection

The Fertility Clinic at LHSC

• Established Oncofertility Program:– Sperm cryopreservation for male patients– Embryo cryopreservation– Counseling on POF following treatment– HRT following treatment

• Future Goals:– Vitrification program: Embryo and Oocytes– Ovarian Tissue Banking– Ovarian Tissue Autografting

Further Readings

1) Nisker J., Baylis F., McLeod C. Choice in Fertility Preservation in Girls and Adolescent Women With Cancer. CANCER Supplement. 2006;vol 107: No 7.

2) Feyles V., Gianetto-Berruti A. Fertility preservation strategies in Female Cancer patients. MINERVA PSICHATR. 2008;49:71-80.

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