Transcript Oncofertility, and Cryopreservation: An update

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)
Information Gap
Oncologist/ Clinician
Scientist
ONCOFERTILITY
Option Gap
Data Gap
Endocrinologist/
Fertility Specialist
Reasonable
chance for
survival
Institutional
Infrastructure /
expertise
Oncofertility
Specialist
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)
Chemotherapy
Premature Ovarian Failure: Infertility
• Alkylating agents:
–
–
–
–
–
–
–
Busulfan
Carboplatin
Chlorambucil
Cyclophosphamide
Dacarbazine
Ifosfamide
thiotepa
AA RR for POF
 > 40 years = 80% Amenorrhea
 < 30 years = 20% Amenorrhea
(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.
Radiation
Premature 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
Rapid Thaw
• Cooling process:
– 2oC/min to -7oC
– 0.3oC/min to -40oC
– 10oC/min to -120oC
– Plunged in liquid
nitrogen to -196oC
• Rapid Warming
– 100oC/min (35oC bath)
– Avoid re-crystallization
of large/cell damaging
ice crystals
– Washed to minimize
osmotic damage
< -10oC
-5oC
Slow Cool
-2oC
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 yr
38-40 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 Rates
Outome
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)
Figure 1 Diagrammatic representation of the regulators and developmental time frames of
mammalian follicle and oocyte development in vivo
~ 300 live births
Reproduction. 2008 Dec;136(6):703-715
Copyright ©2010 Society for Reproduction and Fertility
Multi-step strategy for the complete in vitro growth and
maturation of follicles from large animals and humans
•Fully grown
•Cumulus enclosed
oocyte
•Complete MII
Reproduction. 2008 Dec;136(6):703-715
3D culture media
-support growing follicle
Copyright ©2010 Society for Reproduction and Fertility
•Support induction of
steroidogenic function by GCs.
•Facilitate acquisition of oocyte
cytoplasmic competence.
Summary of the critical determinants of the success of in vitro growth
and maturation systems for mammalian oocytes
Reproduction. 2008 Dec;136(6):703-715
Copyright ©2010 Society for Reproduction and Fertility
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
•
•
•
•
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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 Cryopreservation
Humans:
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 Monitering
Hormonal 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
%
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Conclusion
Components 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.
2.
3.
4.
5.
At least 25 year old history.
Is performed by at least 50% of ART clinics in US, and Europe.
Has Achieved over 1000 live births to date, 90% in the last 5 yrs.
Uses preexisting, well established OHS protocols.
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.
2.
3.
4.
5.
6.
7.
8.
Has produced 26 live births world-wide, by orthotopic procedure.
Advantage of banking tissue indefinitely to allow technology to catch up
with demand.
Does not require pre-treatment COH.
Only technology suitable for pediatric population.
Subject to surgical skill, requires 2x surgery.
Laboratory skills similar to embryo cryopreservation (slow freeze).
Dependent on a pathway between:
Operating theatre - Pathology lab - Cryopreservation lab.
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.
THANK YOU