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S. Samuel KimReproductive Endocrinology & InfertilityUniversity of KansasKansas City, USA
Declared no potential conflict of interest.
Current status of heterotopicCurrent status of heterotopictransplantation of frozentransplantation of frozen--thawed ovarianthawed ovarian
tissuetissue
S. Samuel Kim, MD, FACOGS. Samuel Kim, MD, FACOGCenter for Advanced Reproductive Medicine,Center for Advanced Reproductive Medicine,
Reproductive Endocrinology & InfertilityReproductive Endocrinology & InfertilityUniversity of Kansas School of MedicineUniversity of Kansas School of Medicine
ISFP World Congress, 2009
Early Pioneers of Ovarian TransplantationEarly Pioneers of Ovarian Transplantation
•• 18951895 ROBERT MORRIS:ROBERT MORRIS:12 ovarian transplantation in human (by 1901)12 ovarian transplantation in human (by 1901)
•• 11906 ALEXIS CARREL: whole ovary transplant906 ALEXIS CARREL: whole ovary transplant
Parrott, D.M.V. (1960) The fertility of mice with orthotopic ovariangraft derived from frozen tissue. J. Reprod. Fertil., 1, 230–241.
Gosden, R.G., Baird, D.T., Wade, J.C. et al. (1994) Restoration offertility to oophorectomized sheep by ovarian autografts stored at–196°C. Hum. Reprod., 9, 597–603.
A. S. Parkes; Audrey U. Smith (Jan. 15, 1953)Regeneration of Rat Ovarian Tissue Grafted after Exposureto Low TemperaturesProceedings of the Royal Society of London. Series B, Biological
Sciences, Vol. 140, No. 901. pp. 455-470.
J. Donnez, M. Dolmans, D. Demylle, P. et al. (2004)Livebirth after orthotopic transplantation of cryopreserved ovariantissue. The Lancet, Volume 364, Issue 9443, 1405-1410
What is Ovarian Tissue Banking for?What is Ovarian Tissue Banking for?
For
•• PreservationPreservation ofof fertilityfertility•• RestorationRestoration ofof ovarian functionovarian function•• Prevention of POFPrevention of POF•• Conservation ofConservation of endangered animalsendangered animals•• Investigational tool for follicularInvestigational tool for follicular
biology and ovarian physiologybiology and ovarian physiology
At present,the main purpose ofovarian cryobanking
isfertility preservationfertility preservation
in cancer patients
How to restore fertility withHow to restore fertility withcryobanked ovarian tissue?cryobanked ovarian tissue?
Most growing and mature follicles do notMost growing and mature follicles do notsurvive after freezing and thawing ofsurvive after freezing and thawing ofovarian tissue.ovarian tissue.
Thus, primordial and primary follicles areThus, primordial and primary follicles arethe main components of frozenthe main components of frozen--thawedthawedovarian tissueovarian tissue
How to develop follicles in storedHow to develop follicles in storedovarian tissue to maturity?ovarian tissue to maturity?
Kim SS, Fertil Steril 2001
In vitroIn vitro cultureculture
heterotopic
Current DevelopmentCurrent Development in Heterotopicin HeterotopicOvarian Tissue TransplantationOvarian Tissue Transplantation
Heterotopic autotransplantation ofovarian cortical strips to the forearm
Oktay et al., JAMA. 2001;286
35 y female with Cx. CAfresh ovarian tissuetransplantation beforepelvic irradiation.Follicular growth 10 weeksafter transplantation
30 years old, stage II Breast Cancer• Restoration of ovarian function: 85 days• Eight consecutive percutaneous oocyte retrieval
(six after ovarian stimulation)• Of 20 oocytes retrieved, 8 were suitable for IVF
(5 IVM)• Two fertilized (one abnormal, one 4 cell stage
embryo)
Oktay et al., 2004, Lancet, 363
Rosendahl et al., Hum Reprod, 21, 2006
ET
hCG 22
28 y female with HD stage IIB,Follicle growth fromsub peritoneal transplantunder the abdominal wall
follicle aspiration x4,embryo transfer x2,1 biochemical pregnancy
T AA
AA
ET
KU MED
Heterotopic Autotransplantation ofHeterotopic Autotransplantation ofCryobanked Human Ovarian TissueCryobanked Human Ovarian Tissue
•• JH : 38 y old, adenocarcinoma of cervix;JH : 38 y old, adenocarcinoma of cervix; 20022002•• AS : 37 y old, carcinoma of cervix;AS : 37 y old, carcinoma of cervix; 2002 & 20032002 & 2003•• SH : 29 y old, carcinoma of cervix;SH : 29 y old, carcinoma of cervix; 2003 & 20042003 & 2004•• MS : 29 y old,MS : 29 y old, Breast cancer;Breast cancer; 2005 & 20062005 & 2006
Kim SS et al., 2009, F&S, 91
Restoration of ovarian function:100%Restoration of ovarian function:100%Duration: 3 months to 5 yearsDuration: 3 months to 5 yearsRestoration of fertility: ?Restoration of fertility: ?
Cryopreservation
Preparation of ovarian tissue:
Freezing solution (FS): 1.5 M DMSO +1% human serum albumin + 0.1 Msucrose (in Leibovitz L-15 medium)
Thawing solution (TS):TS1- 1.0M DMSO + 0.1M sucroseTS2- 0.5M DMSO + 0.1M sucroseTS3- 0.1M sucrose
Slow freezing of ovarian tissue
1) Start at 0°C and cool at 2°C/min to -7°C2) Soak for 5 min before manual seeding3) Seed at -7°C and hold for 5 min4) Continue to cool at 0.3°C/min to -40°C5) Cool at faster rate of 10°C/min to -1206) Store at -196°C
Transplantation
Autotransplantation of frozen-thawed human ovarian tissue
to the heterotopic sites
Abdomen: rectus muscle & rectus sheath
Endocrine Function
Restoration of Ovarian Function:• 3-5 months after transplantation, the
return of ovarian function was evidenced(FSH <20 IU/L, E2 >20 pg/ml), but lastedonly for 3-5 months.
• Three patients underwent secondtransplantation. The return of ovarianfunction was faster (2-4 months) aftersecond transplantation.
• Long term ovarian function lasting for 12-60 months has been established aftersecond transplantation in all 3 patients.
Estradiol
0
50
100
150
200
250
0 1 2 3 4 5 6 7 8 9 10 11 12
months after transplant
pg/m
l a
b
c
FSH
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10 11 12
months after transplant
IU/L
a
b
c
first transplantation
FSH
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10 11 12
months after transplant
IU/L
A
B
C
Estradiol
0
50
100
150
200
250
0 1 2 3 4 5 6 7 8 9 10 11 12
months after transplant
pg/m
l A
B
C
Re-Transplantation
Weekly serum FSH/LH/E2Weekly serum FSH/LH/E2 concentrations for 24 wk fromconcentrations for 24 wk from MayMay2004 (8 months post2004 (8 months post--transplantation) through November 2004transplantation) through November 2004
01 02 03 04 05 06 07 08 09 0
1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3weeks
mIU
/ml
FS HLH
E 2
02 04 06 08 0
1 0 01 2 01 4 0
1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3w e e k s
pg/m
l
E 2
Fertility
1
2
2 growing follicles in the ovarian graft2 growing follicles in the ovarian graft(subcutaneous, pre(subcutaneous, pre--rectus)rectus)
Ovarian stimulation wasinitiated when growingfollicles were identified byUTZ or palpation:300 IU rFSH and ganirelix0.25mg for 3 – 7 days.
HumanHuman oocytes retrievedoocytes retrieved from heterotopic graftsfrom heterotopic graftswhen the follicle size reached 14when the follicle size reached 14--20 mm20 mm..
MI
June 2005
MII
First oocyte retrieval: 9 months after transplantationFirst oocyte retrieval: 9 months after transplantation (2 MI oocytes)(2 MI oocytes)
( 6 cell & 3 cellembryos after3 day culture)
ICSIICSI
(MII oocytesafter IVMfor 24h)
( 2 cell & PNembryos after2 day culture)
Second oocyte retrieval: 14 months after transplantationSecond oocyte retrieval: 14 months after transplantation(1 MII oocytes, 2 MI oocytes)(1 MII oocytes, 2 MI oocytes)
Is there any future forheterotopic transplantation
of cryobanked ovarian tissue?
Pros Cons• Convenient for
repeated multipletransplantations
• Non invasiveprocedure
• Easily accessible foroccyte retrievalwithout anesthesia
• Feasible for patientswith severe pelvicadhesion
• IVF procedurerequired
• Efficacy not proven(No live birth yet)
• Suboptimal (unknown)environmental effectson follicle growth andmaturation
• Possible poor qualityoocytes
Issues with heterotopic ovariantransplantation
• Cryoinjury• Ischemic injury• Optimal heterotopic sites• Environmental factors/ Follicle
growth pattern• Egg retrieval technique• Efficacy
Issue 1: Cryoinjury
01020304050607080
fresh control slow freezing vitrification
before graftingafter grafting
Apoptotic rates of primordial folliclesApoptotic rates of primordial folliclesKim et al. (2004) Fertil Steril 82:679Kim et al. (2004) Fertil Steril 82:679--685685
Fresh Slow freezing Vitrification
2D gel electroporesis images (the protein expression patterns)
0
5
10
15
20
25
Fresh Slow Frz Vitri
beforeaf ter
protein expression changes
a b c
Vitrification, at least in theory, shouldeleminate ice formation and minimizecryoinjury.
Vitrification may replace slowfreezing in the future as a method ofovarian tissue cryopreservation (??)
Oxy
gen
cons
ump
tion
rate
(mM
/mg/
min
)
Incubation period (hour)
Room Temp (n=6)Ice (n=6)
0
.025
.05
.075
.1
0 3 24 48
**
Issue 2: Ischemic Injury
Significant ischemic damage can beseen about 24 h after transplantation,
but angiogenesis takes more than 48 h.
No treatment Ascorbic acid
3h
24h
e f
24h
c d
a b
We need to develop newstrategies that can protect
ovarian tissue from ischemia orfacilitate angiogenesis to makeovarian tissue transplantationclinically reliable and robust
technology.
• Rich in vasculature• Easily accessible• Stable in temperature• Enough space to accommodate
large ovarian graft and tosupport full follicular growth
Issue 3: Optimal heterotopic site
• Tested sites: subcutaneous (forearm,hip, abdominal wall), rectus muscle,uterus, breast tissue, superficialfascia of pectoralis muscle,subperitoneal tissue beneath theabdominal fascia between theumbilicus and the pubic bone
Issue 4: Environmental factors/Oocytes quality
• Temperature• Local pressure• Paracrine factors• Cytokines• Blood supplies
Issue 5: Egg retrieval
• Ovarian stimulation protocol• Aspiration needles (size, length)• Aspiration pressure• When to retrieve• IVM
Issue 6: Efficacy
• Follicle development: <50%• Egg retrieval: 3/5 (7/8)• Fertilization: 4/7 (2/20)• Embryo development: 3/4 (1/2)• Pregnancy rate: 0 (0)
Conclusions• Ovarian tissue cryobanking is a
promising strategy to preserve fertility incancer patients.
• Stored ovarian tissue can be graftedeither orthotopically or heterotopically.
• To date, there is no pregnancy withheterotopic transplantation of frozen-thawed human ovarian tissue.
Conclusions• Clinical practicability of heterotopic
transplantation is questionable at presentas the environment of heterotopic sitesmay not be as favorable for normalfollicular development.
• Nevertheless, there are many advantagesof heterotopic transplantation, especiallywhen the hostile conditions of pelviccavity preclude orthotopic transplantation.
AcknowledgmentAcknowledgment•• University of Kansas, KS,University of Kansas, KS,
USAUSA•• David Albertini, PhDDavid Albertini, PhD•• DJ Kim, MSDJ Kim, MS•• Rebekha Lang, MARebekha Lang, MA•• Kim Pomeroy, PhDKim Pomeroy, PhD
•• Cornell University, NY,Cornell University, NY,USAUSA
•• Roger Gosden, PhDRoger Gosden, PhD•• Hang Yin, PhDHang Yin, PhD
•• CHA Fertility Center,CHA Fertility Center,Seoul, KoreaSeoul, Korea
• WS Lee, MD• MK Chung, PhD
•• EulJi Life Institute, KoreaEulJi Life Institute, Korea•• HC Lee, MScHC Lee, MSc•• HH Lee, MScHH Lee, MSc
•• Seoul National Univesity,Seoul National Univesity,KoreaKorea
•• CS Suh, MDCS Suh, MD•• SH Kim, MDSH Kim, MD
•• ART Reproductive Center, CAART Reproductive Center, CAUSAUSA
•• David Hill. PhDDavid Hill. PhD
•• University of Washington,University of Washington,Seattle. USASeattle. USA
Kansas City
Thank you !Thank you !
Stimulation dose (FSH/HMG)and duration*
Peak oestradiol(pmol/L)†
Follicle size(mm)† Oocyte
1 None 888 10·9 GV
3 3675/2175, 13 days 980 14·2 FZ
10·7 Degenerated
8·9 GV
4 None 1093 9·1 FZ
5 3225/1725, 11 days 2257 14·5 FZ
13·1 M-I
9·5 FZ
6·4 GV
6 2025/2475, 10 days 1868 12·8 Mature
10·8 FZ
8·4 FZ
7 2100/2400, 10 days 921 11·8 Mature
10 GV
6·7 Degenerated
8 2025/1500, 10 days 987 11 Degenerated
9·9 Mature, GV, GV
Oktay et al., 2004, Lancet, 363
Ovarian function after autografting ofhuman ovarian tissue
Callejo et al., 2001, J Clin Endocrino Metab, 86
Serpinb-mice (8 X)laminin receptorhomolog – bovine (2 X)smooth musclegamma–actin-mammalian (7 X)
RAB4B(5 X)Serpinb(8 X)
Serpinb-mice (4 X)laminin receptor homolog (4 X)Lectin (Chain A)--bovine (4 X)Glutathione S-transferase (8 X)gamma-actin (4 X)actin-beta-mammalian (8 X)
After
RAB4BSerpinblaminin receptorhomolog- bovinesmooth musclegamma-actin-mammalian
RAB4BSerpinb
RAB4B-miceSerpinb-micelaminin receptor homologLectin (Chain A)---bovineGlutathione S-transferasegamma-actinactin-beta-mammalianCMP-NeuAc:GM3Sialyltransferase
Before
VitrificationSlowfreezing
Fresh
Identified proteins of significance byIdentified proteins of significance by MALDIMALDI--TOFTOF
Protein FunctionsProtein Functions
RAB4B ProteinA family of GTP-binding protein.Regulate vesicular trafficking
SERPINBs ProteinSerine proteinase inhibitor B family.Related with inflammation and tumor cells
LectinRelated cell structure (glycoprotein) and growth
Laminin receptorA family of extracellular matrix proteins.Multiple biological activities (growthdifferentiation, angiogenesis etc.)
ActinProtein related to microfilaments and fibers
Glutathione S-transferase
CMP-NeuAc:GM3sialytransferase
A family of catalytic enzyme.Antioxidant
Inhibition of EGF-mediated receptor activity andcell proliferation by HK1-ceramide
Stages of folliculogenesis in the adult human ovaryStages of folliculogenesis in the adult human ovary (Gougeon)(Gougeon)
Autotransplantation ofAutotransplantation of human ovarian tissuehuman ovarian tissue••OrthotopicOrthotopicOktay K, 2000 (frozenOktay K, 2000 (frozen--thawed)thawed)Radford J, 2001 (frozenRadford J, 2001 (frozen--thawed)thawed)Donnez J, 2004 (frozen-thawed)Meirow D, 2005 (frozen-thawed)Silber S, 2005 (fresh, frozenSilber S, 2005 (fresh, frozen--thawed)thawed)Anderson, 2007 , 2008 (frozenAnderson, 2007 , 2008 (frozen--thawed)thawed)Demeestere I, 2007 (frozenDemeestere I, 2007 (frozen--thawed)thawed)Piver P, 2009 (frozenPiver P, 2009 (frozen--thawed)thawed)
••HeterotopicHeterotopicOktay K, 2001 (fresh)Oktay K, 2001 (fresh)Callejo J, 2001 (fresh)Callejo J, 2001 (fresh)Kim SS, 2003 (frozenKim SS, 2003 (frozen--thawed)thawed)Oktay K, 2004 (frozenOktay K, 2004 (frozen--thawed)thawed)Rosendahl M, 2006 (frozenRosendahl M, 2006 (frozen--thawed)thawed)Kim SS, 2009 (frozenKim SS, 2009 (frozen--thawed)thawed)
Immunofluorescent localization ofImmunofluorescent localization ofmicrotubules and chromatinsmicrotubules and chromatins
Kim et al., Hum Reprod, 2005