Ovarian cryopreservation and transplantation program: the ... - eshre

Ovarian cryopreservation and transplantation
program: the Danish network
Claus Yding Andersen, Professor, MSc, DMSc
Laboratory of Reproductive Biology
University it Hospital Copenhagen,
University of Copenhagen
Copenhagen, Denmark
E-mail: yding@rh.dk
ESHRE Campus “Fertility preservation in cancer”
Bologna, Italy – 25-26 November 2010

Outline
The Danish network for ovarian
cryopreservation
Experience with transplantation:
transport and total in Denmark
Induction of puberty
Strategies for retrieving tissue an
for transplantation
ESHRE Campus “Fertility preservation in cancer”
Bologna, Italy – 25-26 November 2010

The concept of
Transport Ovarian Cryopreservation
The woman receives gonadotoxic treatment
at the local hospital
Ovarian tissue is removed at the local hospital
and transported to a central laboratory where
cryopreservation and storage is performed
Cryostored ovarian tissue is transported to
the local hospital for transplantation

Rationale:
Transport ovarian cryopreservation
Establish a possibility for women who may
otherwise be deprived the chance of
ovarian cryopreservation
Centralize the service of this relative
seldom performed procedure
The ischemic period following transplantation
reduces the follicle pool most dramatically

Transport of ovaries for cryopreservation
within Denmark

PROCEDURE:
Operation at 08 00 am
Partly isolation of the cortex
Pick-up at t09 00 am
Domestic flight/transport to Copenhagen
Grond transport from airport to Rigshospital
Arrival around 1 pm.
Transport of partly isolated ovarian cortex 4-5 h on ice

Cryopreservation of ovarian tissue from three
Danish centers

Preparation of human ovarian tissue
for cryopreservation

CRYOPRESERVATION PROTOCOL
(KLT Schmidt et al., 2003)
Cryoprotectant: t t 15M 1.5 Ethyleneglycol
l l
0.1 M Sucrose
10 mg/ml Human Serum Albumin
Temperature profile:
1. Equilibration –rotation (2 0 C in 30 min)
2. – 2 0 C/min until – 9 0 C
3. Manuel seeding
4. – 03 0,3 0 C/min until – 40 0 C
5. –10 0 C/min until – 140 0 C
6. Liquid nitrogen (– 196 0 C)

Transport of ovarian tissue – 6 year old girl
20 hours on ice prior to cryopreservation
Prior to transplantation
H i ti t l t d d th ki
Human ovarian tissue transplanted under the skin
of ovariectomised SCID mice for 4 weeks

Thawing of frozen ovarian tissue
I II III
0 ,75 M Ethyleneglycol 0,1 M sucrose
PBS
0,2 M sucrose in PBS
in PBS 10 min
10min
10 min

Transplantation of frozen/thawed
tissue in Denmark
In Denmark: 17 women/girls have been
transplanted with frozen/thawed ovarian
tissue a total of 23 times
Transport ovarian cryopreservation:
12 women/girls have been transplanted a
total of 15 times
No relapse

Diagnosis for transplantation of frozen/thawed
ovarian tissue in Denmark
Diagnosis
Number
Hogdkin’s lymphoma 4
Breast cancer 4
Non‐Hodgkin’s lymphoma 2
Ewing’s sarcoma 2
Aplastic anaemia 1
Cervical cancer 1
Autoimmune disease 1
Paraxymal nocturnal haemogloburi 1
Haemolytic urianary syndrome 1

Orthotopic transplantation of ovarian tissue

Heterotropic op transplantation a tat to a subperitoneal ea pocket

Longevity of transplanted frozen/thawed ovarian tissue
Age at
cryo
First Transplant
Second transplant
Remaining
Pieces Month Still work Pieces Month Still work Pieces
32 (1) 6 56 No 12 25 No 15
28 (2) 12 54 No 8 24 Yes 7
25 (3) 12 26 No 6 45 No 0
26 (4) 10 15 Yes 12 54 Yes 10
31 (7) 8 25 Yes 8 11 Yes 4
36 (9) 10 8 No 9 0 No 0
33 (6) 3 8 No 0
27 (8) 6 59 Yes 7

Longevity of transplanted frozen/thawed ovarian tissue
Age at
cryo
First Transplant
Second transplant
Remaining
Pieces Month Still work Pieces Month Still work Pieces
19 (10) 12 32 Yes 24
25 (11) 7 26 Yes 10
38 (14) 12 17 Yes 12
23 (15) 8 15 Yes 11
26 (12) 12 27 Yes 14
31(16) 10 8 Yes 25
25 (5) 8 20 Yes 8

Efficacy of transplanting cryopreserved
ovarian tissue in Denmark
17
14
13
10
Alleviating of menopausal symptoms: 3
Transplanted less that five months ago: 1
Divorced partner: 3

Results of transplanting frozen/thawed
ovarian tissue in Denmark
Transport:
• Three children born (two women)
• One legal abortion (natural)
• One clinical pregnancy (natural)
Immediately:
• One clinical pregnancy (IVF)
• Two biochemical pregnancies
(heterotropic transplants)

Considerations for transplantation
How much tissue should be collected?
How much tissue should be transplanted?
When should the oocytes be collected
after transplantation?
Could we envision stimulation with a
PTEN inhibitor prior to transplantation?
t ti

CONCLUSIONS
Ovarian cryopreservation, including transportation, is now
a clinical option
Transplanted frozen/thawed tissue restores ovarian function
with high efficacy and maintain function for prolonged
periods of time
Transplanted tissue restore fertility but the efficacy is
probably not high and refinements are needed
Results are encouraging for a continued research effort

HREGION
Laboratory of Reproductive Biology
The Copenhagen Workshop on
Cryopreservation of Ovarian Tissue
Learning the technique, hands on experience
T
The Copenhagen Workshop on Cryopreservation of Ovarian
Tissue is a three‐day laboratory and lecture course for those
interested in learning the technique behind the Danish
Cryopreservation Programme.
For more information
go to
www.cryoovary.dk
y
Course dates:
March 15‐17, 2011
or contact the course
coordinator:
stine.gry.kristensen@
rh.regionh.dk
CryoOvary.dk

Collaborators
University Hospital of Copenhagen: Laboratory of Reproductive Biology:
Anders Nyboe Andersen Kirsten Tryde Schmidt
Anne Loft Mikkel Rosendahl
The Fertility Clinic Stine Gry Kristensen
Christian Ottesen Inga Husum
Department of Gynaecology Tiny Roed
Mario Westphal
University Hospital of Aarhus: Tine Greve
EikE Erik Ernst Annette Rasmussen
Margit Dueholm Anne Grete Byskov
Ditte Trolle Claus Yding Andersen
Sara Markholt
The Fertility Clinic
Department of Gynaecology
The Danish Medical Research
Council
Odense University Hospital
Per Emil Rasmussen
The Fertility Clinic
Department of Gynaecology
The Danish Cancer Foundation

Laboratory of Reproductive Biology – Spring 2010