collection processing and storage of ovarian tissue clinical indications and best practice...

OVARIAN TISSUE CRYOPRESERVATION

OZGUR OKTEM MDAMERICAN HOSPITALWOMEN`S HEALTH CENTERISTANBUL TURKIYE

FERTILITY PRESERVATION

QUALITY OF LIFE ISSUES IN CANCER SURVIVORS

CHEMOTHERAPY RADIOTHERAPY

FERTILITY PRESERVATION

Preservation of reproductive function became an important quality of life issue in cancer patients

Life expectancy is increasing

Jemal et al. CA Cancer J Clin 2009;58:71–96

1975-1977 1996-2004

58% 80%

50% 66%

CHILDHOOD CANCERS

ADULT CANCERS

5 year survival rates have increased in cancer patients

5 YEAR SURVIVAL

Adult survivors of childhood cancers a new population!

Oktem et al Ann N Y Acad Sci. 2008;1135:237-43Oktem et al Pediatr Blood Cancer 2009 Aug;53(2):267-73

Jan 2002

A Age:22Dx: Hodgkin’s lymphoma

Apr 2007

HSCT

Age:27

CureChemotherapy

Jan 2008

Age:28

diagnosed with cancer

Menstrual irregularity

Amenorrhea Return of menses

Amenorrhea

Infertility

FSH:42mIU/mL

Premature ovarian failure!

A MATTER OF LIFE AND DEATH QUALITY OF LIFE ISSUE

OVARIAN TISSUE BANKING

Ovarian tissue freezing is the only fertility preservation options for

Pediatric and adolescent cancer patientsAdults who have

No time for embryo freezing or Contraindication for embryo freezing No husband or partner for embryo freezing

•Oktem et al. Cancer 2007•Oktem and Oktay Fertil Steril 2008

Jemal et al. CA Cancer J Clin 2009;58:71–96

Chemotherapy and cell death

DAMAGE TO DNA. as neutrons and particles

Indirect actions due toformation of free radicals and DNA damage. This mechanism is particularly true for sparsely ionizing radiation such as x-rays.

The higher the dose of radiation

The higher the risk of premature ovarian failure !

Single dose is more toxic than fractionated dose.

The LD50 of the human oocytes may be 1.99 Gy∗;less than the previously thought (4 Gy)∗∗

100cGy=1Gy=100 Rad

The higher the dose of radiation

The higher the risk of premature ovarian failure !

Single dose is more toxic than fractionated dose.

The LD50 of the human oocytes may be 1.99 Gy∗;less than the previously thought (4 Gy)∗∗

100cGy=1Gy=100 Rad

TBITBI-- 2020--30 Gy30 Gy⇒⇒37/38 37/38

Ovarian failureOvarian failureTBI + CycTBI + Cyc

-- OR:OR:~~1 (1 y1 (1 yııl)l)-- 135/144 patients have 135/144 patients have

POFPOF

HSCT

Other indications for fertility preservation requiringchemotherapy and/or stem cell transplantation

Systemic lupus erythematosusMyelodysplasiaAplastic anemiaWegener’s vasculitisAuto-immune hemolytic anemiaSickle-cell diseaseThalassemia

GONADOTOXICITY

Patient’s ageYounger the patient higher the follicle counts

Cytotoxic potential of therapyAlkylating agents more toxic

Dose and duration of therapyLonger duration and higher doses more toxic

1

2

3

GONADOTOXICITY

Patient’s ageYounger the patient higher the follicle countsMore likely to retain some ovarian function after therapy

1

Oktem and Oktay Am J Hem Oncol 2008;7;1-7

Resting phase 90%10%Growing phase

Primordial follicles determines ovarian reserve. Drugs mainly targeting PF have more impact on ovarian reserve.SHORTER REPRODUCTIVE LIFE SPANHIGHER RISK FOR PREMATURE OVARIAN FAILURE

How to assess the damage in the human ovary

Hormonal and USG markersCurrently there is not a hormonal marker of primordial follicle counts.

FSH, AFC , and AMH levels are commonly used reserve markers.

Reh et al. Fertil Steril 2007Oktem et al. Fertil Steril 2007

FSH action

Oktem ANYAS 2008

GONADOTOXICITY

Patient’s ageYounger the patient higher the follicle counts

Cytotoxic potential of therapyAlkylating agents more toxic

Dose and duration of therapyLonger duration and higher doses more toxic

1

2

3

CHEMOTHERAPY AGENTS

GONADOTOXIC CHEMOTHERAPEUTICSCHEMOTHERAPY

Different toxicity potentialAlkylating agents most toxicPlatinum groupTaxanesAntracyclines

Cyclophosphamide

Busulfan

Chlorambucil

Melphalan

+ Oktay et al. Hum Reprod. 2004 Mar;19(3):477-80+ +Oktem and Oktay Fertil Steril 2006;86:S312 P-725

Oktem et al. Cancer:2007 110(10):2222-9

ALKYLATING AGENTS

Nitrogen mustardsChlorambucilChlormethineCyclophosphamideIfosfamideMelphalanBendamustineTrofosfamideUramustine

NitrosoureasCarmustineFotemustineLomustineNimustinePrednimustineRanimustineSemustineStreptozocin

Platinum (alkylating-like)CarboplatinCisplatinNedaplatinOxaliplatinTriplatin tetranitrateSatraplatin

Alkyl sulfonatesBusulfanMannosulfanTreosulfan

HydrazinesProcarbazine

TriazenesDacarbazineTemozolomide

AziridinesCarboquoneThioTEPATriaziquone, Triethylenemelamine

March 2004

diagnosed with cancer

A

B

Ovarian freezing

Chemotherapy

Age:22Dx: Hodgkin’s lymphoma

Age:22Dx: Non-Hodgkin lymphoma

April 2004

1XCHOP

16.6 ± 3.5 PF

6.17 ± 0.7 PF

Oktem et al Cancer 2007

Oktem et al. Cancer 2007 Oktem et al. Am J Hem Oncol 2008

Chemotherapy

1XCHOP

16.6 ± 3.5 PF 6.17 ± 0.7 PF

AGE 22

%63 loss

AGE 30

The cost of one course of CHOP in the ovary8 YEARS AGING

Oktem et al. Cancer 2007

May 2003

diagnosed with cancer

A

B

Ovarian freezing

Chemotherapy

Age:33Dx: Breast cancer

Age:33Dx: Non-Hodgkin lymphoma

7XCHOPGnRH analog

5.66 ±0.9

1.5 ±0.6

FSH: 20.8 mIU/mL

May 2003

diagnosed with cancer

A

B

Ovarian freezing

Chemotherapy

Age:18Dx: Hodgkin’s lymphoma

Age:18Dx: AML

2XADE-GMTZ

16.6 ±1.6

14.4 ±1.6

Control VACA + RT7.6 ±1.7 AGE 24 4.52 ±0.9 AGE 24

Oktem et al Cancer 2007

TWO IMPORTANT QUESTIONS TO BE ANSWERED...

How to Measure THE DAMAGE?

How to assess the toxicity of NEW DRUGS?

Severe Combined Immune Deficient (SCID) Mice

T cell B cell

Cellular immunityCellular immunity Humoral immunityHumoral immunity

NO GRAFT REJECTION

Ovarian Xenografting

GRAFT VASCULARIZATIONGross

300um

Cy-induced damage in human ovary as assessed by tunnel assay

Oktem et al Cancer Res 2007; 67: 10159-62

Follicle loss after single dose Cy

Oktem et al Cancer Res 2007; 67: 10159-62

OVARIAN TISSUE FREEZING

Orthotopic(Pelvic)

Transplant

Orthotopic(Pelvic)

Transplant

Heterotopic(Subcutaneous)

Transplant

Heterotopic(Subcutaneous)

Transplant

Resumptionof Ovarian Functions

Resumptionof Ovarian Functions

SpontaneousConception

SpontaneousConception

IVFIVF

Embryo Transfer Embryo Transfer

Ovarian Transplantation Techniques

Orthotopic(Pelvic)

Transplant

Orthotopic(Pelvic)

Transplant

Heterotopic(Forearm)Transplant

Heterotopic(Forearm)Transplant

Resumptionof Ovarian Functions

Resumptionof Ovarian Functions

SpontaneousConception

SpontaneousConception

IVFIVF

Embryo Transfer Embryo Transfer

Ovarian Transplantation Techniques

Patient A

Patient B

Patient A

Patient B

Oktay et al, JAMA, 2001

Estradiol Output From Estradiol Output From Heterotopic TransplantHeterotopic Transplant

RCV Estradiol

01000

20003000400050006000

1 5 13 15 20 22 28 32 33 34 36 39 40

Cycle 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 40

Cycle Day (arbitrary)

pg/m

L

RH

RCV

Percutaneous Oocyte Percutaneous Oocyte RetrievalRetrieval

Percutaneous Oocyte Percutaneous Oocyte RetrievalRetrieval

24 Hours24 Hours

18 Hours18 Hours24 Hours24 Hours

First Embryo After Ovarian First Embryo After Ovarian TransplantTransplant

Pelvic Ovarian Transplantation

Oktay et al, NEJM 2000

Comparison of Two Orthotopic Transplant Techniques

Ovarian Function &Pregnancy via IVF

No Ovarian Function

Meirow et al, NEJM 2005

Oktem, Sonmezer, Oktay Oktem, Sonmezer, Oktay Textbook of Assisted Reproductive Technologies, 2005 Textbook of Assisted Reproductive Technologies, 2005

ISCHEMIA AFTER TRANSPLANTATIONHYPOXIA INDUCIBLE FACTOR-1 ALPHA (HIF-1α)

BEFORE TRANSPLANTATION AFTER TRANSPLANTATION

AuthorAuthor Year Year TransplantatTransplantation siteion site

CryoCryo indicationindication IVF / IVF / spontanspontaneeousous

Age at Age at ovarian ovarian cryo. cryo.

Age at tAge at tx. x. OutcomeOutcome

Oktay 2004 Heterotopic Breast cancer IVF 30 36 Embryodevelopment

Donnez 2004 Orthotopic Hodgkin’s disease

Spontaneous 25 31 Healthy live birth

Meirow 2004 Orthotopic Hodgkin’s disease

IVF 26 28 Healthy live birth

Demeestere 2006 Orthotopic/heterotopic

Hodgkin’s disease

Spontaneous 24 29 One miscarriage at 7 weeks, one healthy live birth

Oktay 2006 Heterotopic Hodgkin’s disease

Spontaneous 28 32 Healthy live birth

Rosendahl 2006 Orthotopic/heterotopic

Hodgkin’s disease

IVF from heterotopic site

28 30 Biochemical pregnancy

Silber* 2008 Orthotopic Idiopathic premature ovarian failure

Spontaneous 14 28 Ongoing pregnancy

Anderson 2008 Orthotophic Non Hodgkin’s lympohoma

IVF 32 34 Ebryo dev.

Anderson 2008 Orthotopic/heterotopic

Hodgkin’s disease

IVF 25 27 Clinical pregnancy

Anderson 2008 Orthotopic Hodgkin’s diseas IVF 26 28 Healthy live birth

Anderson 2008 Orthotopic Ewings sarkomu IVF 27 30 Healthy live birth

Sonmezer&Oktay, 2008Sonmezer&Oktay, 2008

Oktay and Oktem 2008 Fertil Steril

ALL PATIENTS

Oktay and Oktem 2008 Fertil Steril

ALL PATIENTS

Ovarian Freezing in Childhood Cancers

Oktem et al Ann N Y Acad Sci. 2008;1135:237-43Oktem et al Pediatr Blood Cancer in press

INDICATIONS FOR FERTILITY PRESERVATION PEDIATRICPOPULATION

Oktem et al Ann N Y Acad Sci. 2008;1135:237-43Oktem et al Pediatr Blood Cancer in press

Slow freezing vs. Vitrification

Controlled rate (slow) freezing is the most commonly used cryopreservation method for human ovarian tissue* .Ultrarapid freezing (vitrification) is being widely used in embryo and oocyte freezing.Data on its applicability on ovarian tissue freezing is very limited.

*:Oktem Fertil Steril 2008

Slow freezing Vitrification

The structure of primordial follicles are preserved better in slow frozen samples

Oktem Balaban and Urman ASRM 2009 USAWFPC 2009 Belgium

Growing follicles are preserved better in slow frozen samples

Slow freezing VitrificationFresh

RESULTSSlow frozen ovaries contain significantly higher number of primordial follicles than vitrified ones.

0

0,5

1

1,5

2

2,5

Control SF VF

Prim

ordi

al fo

llicl

e/m

m2

1.97

1.27 0.97

a,b

a,cb,c

a:p>0.05b:p<0.0001c:p<0.001

00,10,20,30,40,50,60,70,8

Control SF VF

AM

H (n

g/m

L)

RESULTSAntimullerian hormone production from slow frozen ovaries is significantly higher than vitrified ones.

0.47

0.210.07

a,b

a,c

b,c

a:p>0.05b:p<0.05c:p<0.05

Oktem Balaban and Urman ASRM 2009 USAWFPC 2009 Belgium

ANTI-MULLERIAN HORMONE

Oktem et al. Ann N Y Acad Sci 2008;1127:1-9

LiteratureIsachenko et al Cyro letters 2008

Vitrification (2.62 M dimethylsulphoxide + 2.6 M acetamide + 1.31 M propylene glycol + 0.0075M polyethylene glycol) no comparison with slow freezing.

Vitrification preserves ovarian follicles and stroma better than slow freezing

SF PrOH Sucrose and EGVF PrOH EG PVP DMSO (Hovatta et al Hum reprod 2009)

UNKNOWNS…

Following questions are waiting to be answered

Which method ?SF vs. VF

Which cryoprotectant or combination of different cry0protectants?

DMSO, EG, PrOH etc..Incubation, seeding times, exposures?

CONCLUSIONFertility preservation has recently emerged.The right option should be offered to carefully selected patients.Success rates of ovarian freezing is stilll low due to

Underutilization (%94.9 -56 of 59 have not used their tissues yet)

54% personal-social38% still under therapy8% death

THANK YOU

ACKNOWLEDGEMENTBulent Urman, MD

Basak Balaban MSC

Aycan Isiklar MSc

Ebru Alper MD

Cengiz Alatas MD

Ramazan Mercan MD

Alper Mumcu MD

Cem Ayhan MD

Kayhan Yakın MD

Erhan Palaoglu MD

Kamil Peker MD