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