Collection processing and storage of ovarian tissue clinical indications and best practice...
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OVARIAN TISSUE CRYOPRESERVATION OZGUR OKTEM MD AMERICAN HOSPITAL WOMEN`S HEALTH CENTER ISTANBUL TURKIYE
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Collection processing and storage of ovarian tissue-Clinical indications and best practice (by Ozgur Oktem_2010)
Transcript of 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
