recent developments ovarian tissue transplantation versus in-vitro maturation of immature oocytes ...
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Recent developments- Ovarian tissue transplantation versus in-vitro maturation of immature oocytes _Telfer_Evelyn_2010TRANSCRIPT
In vitro growth of immature follicles/oocytes
Evelyn E TelferInstitute of Cell Biology
andCentre for Integrative Physiology
University of Edinburgh
Options to use cortical tissue to restore fertility
Currently limited to transplantation46 cases from 25 studies (fresh and frozen)9 pregnancies in 8 women
Bedaiwy, M.A., et al., Reproductive outcome after transplantation of ovarian tissue: a systematic review. Hum Reprod, 2008. 23(12): p. 2709-17.
Ovarian Tissue Transplantation
AdvantagesRestoration of endocrine and fertility function
DisadvantagesUncontrolled loss of primordial follicles (freezing
method re-vascularisation)
Longevity of graft unpredictable
Risk of re-introducing cancer cells
In vitro growth of immature oocytes
Cortical strips contain mainly primordial follicles.
The challenge now is to develop oocytes in vitrofrom primordial stages to maturation and fertilisation.
Where are we now?
Frozen-thawed cortical strips
Rhabdomyosarcoma Pt 15yrs:thawed from slow freeze
Mice from Primordial Follicles...
Eggbert set back primordial follicle culture research: feared abnormal. Subsequent improvements in mouse method led to birth of “normal” pups O’Brien et al 2001
Eggbert: First mouse born from an in vitro grown primordial follicle: 2 step system total of 22 days in vitro before IVM and IVFEppig & O’Brien 1996
OocyteGrowth
In vivo
19-30µm 40-80µm 80-90µm 90-100µm 100-110µm
Pre-ovulatoryprimordial primary preantral Early antral
Mid antral
OocyteOocyte DevelopmentDevelopment
Growth/ Meiotic Arrest
Acquisition of Meiotic Competence
Acquisition of Developmental Competence
Transcription/Transcriptional Repression
Genomic imprinting
Developing a multistep culture system for human IVG• 1) Optimising growth from primordial stages
• 2) Supporting growth of isolated preantralfollicles to antral stages
• 3) Supporting final stages of oocytedevelopment out with large follicle
• 4) Testing function (IVM &IVF) and normality (methylation profile etc)
Suppressors
Maintainers
Activators
Step one: Activation of quiescent primordial follicles
Primordial Pool
Activation of growth
Micro-Cortex Culture
Underlying Stroma and larger follicles removed
Micro-cortex
Tissue Architecture.
Surface area and density of stromalcells important feature
Telfer et al. 2008
Cortical biopsy cut into strips
Free Floating cultures: basic conditions serum free medium
Days in Culture
0
0.2
0.4
0.6
0.8
Day 0 Day 6
PrimordialTransitoryPrimarySecondary
Pro
porti
on o
f fol
licle
s
*
**
Telfer,et al. (2008) Human Reproduction
Growth within micro-cortex
Figure 1Improving rate of activation and early follicle growth in vitro
Telfer, McLaughlin, Anderson, Hovatta and Liu (2010)
Effect of inhibiting PTEN using 1 µM bisperoxovanadium [bpV]
0
20
40
60
80
100
Uncultured Control 1uM
Non-GrowingGrowing PrimaryGrowing Secondary
Per
cent
age
of h
ealth
y oo
cyte
s
0
20
40
60
80
100
uncultured control 1 um
Per
cent
age
of F
ollic
les
d1 d3 d6
Growth within micro-cortex
• Optimal time and size to remove growing follicles from micro-cortex environment.
• In our hands: 6-8 days (depending on size)
• Leaving growing follicles longer in step 1 results in increased death and poor quality follicles/oocytes.
Human Follicle development in vitro (6 days)
Step 2: Preantral – Antral stages. Isolated Follicle Culture
Cultured micro-cortex
Follicles before isolation
Isolated Follicle
Growth of isolated human preantralfollicles in vitro
0
50
100
150
200
0 2 4
Days in culture (step 2)
Mea
n fo
llicl
e di
amet
er (m
icro
ns)
Control Activin
n = 36 n = 38n = 32
n = 33
n = 24
n = 26
* *
Telfer et al., 2008 Human Reproduction
0
10
20
30
40
50
Type 1 Type 2 Type 3 Type 4
ControlActivin
*
**
perc
enta
ge o
f fol
licle
s
Health of in vitro grown human follicles after 6 days in cortical strip culture (step 1) followed
by 4 days in isolated culture (step 2).
Health/degeneration scale
Telfer et al., 2008 Human Reproduction
A
C
B
D
Applications of follicle/oocyteculture systems (IVG)
Current • Basic research tool• Tissue viability assessment
Potential • Fertility preservation (frozen tissue)
No. Biopsies Source Status Isolated Follicles
35 C-Section(Edinburgh & Stockholm)
Fresh 480
10 C-Section(Stockholm)
Vitrified 135
4 Gynae (35-52)(Edinburgh) Fresh 27
3 Fertility preservation (Edinburgh) (12-21)
Fresh 30
1 Fertility preservation (Edinburgh) (15)
Slow-Frozen 14 (2 fragments)
3 Fertility Preservation (26-30) (Manchester) (Chemo treated)
Slow-Frozen 80
Follicles recovered after IVG from several tissue sources (fresh and cryopreserved)
Cryo tissue before/after step 1 of culture
Antral development from in vitro grown human primordial follicles within 10 days
Telfer et al., 2008: A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin. Human Reproduction 23: 1151-1158
Step 3: Further oocyte growth
Xu et al. (2009) In vitro grown human ovarian follicles from cancer patients support oocyte growth. Hum Reprod. 24: 2531-40
Almost fully grown oocytes (95microns) obtained within alginate encapsulated follicles grown for 24 days
Multi-step Culture system to support human oocyte development
Activation
Combining growth techniques: Telfer, Woodruff, Hovatta, Picton groups
Adapted from Telfer & McLaughlin 2007
Closing the gap between IVG and IVM.
Bovine Follicles cultured for 8 days from primordial (step 1) then 12 days from the preantral stage (step 2)
30μm
50μm
Fig 3.a
McLaughlin & Telfer 2010 Reproduction
020406080
100
Fix Day 6 Control rhActA rhAct+FSHPerc
enta
ge o
f Ooc
ytes
≤ 50
50-80
80-100
Mean oocytediameter
Bovine IVG oocytes 12 days step 2
Further growth of complexes within alginate before IVM
Oocytes of up to 108 microns. MII and PB
Accelerated growth?Or
Growth without brakes?….
How long does complete oocytedevelopment take?
Growth Rate of Folliclesin vivo
Preantral ‘Mature’Size
Time
100-200μm 500-600μm 10-12 days
150-300μm 1.5-3mm 40-50 days
100-150μm 3.8->8.5mm 40-50 days
120-300μm 4.00-6.00mm 70-100
Mouse
Pig
Cow
Woman
Does a human oocyte really need 70 days to develop or
is this time frame a consequence of inhibition
regulating follicle development?
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Competing functions within the follicle
OOCYTE DEVELOPMENT
ENDOCRINE FUNCTION
Most important feature of cumulus lineage is physical interaction with the
oocyte
Optimising conditions to support oocyte development through co-ordinating oocyte-somatic cell interactions
In Vitro Growth of Oocytes
OOCYTE DEVELOPMENT
Endocrine Function
Imaging of oocyte-somatic cell interface
Focus on optimising oocyte-somatic cell communication during IVG
McLaughlin, Bromfield, Albertini, Telfer (2010) Molecular Human Reproduction
Final Stages of Development
Complexes Isolated from IVG antralfollicles
For further growth
IVM +IVF
Tests for “normality”Methylation etc
NEXT STEP…………….
Encouraging progress
But much to do before clinical applications may be realised
Collaborative effort required to make significant progress
EURO CULTURE CLUB:ESHRE INITIATIVE TO ADVANCE
FOLLICLE CULTURE TECHNIQUES
• Smitz J, Dolmans MM, Donnez J, Fortune JE, Hovatta O, Jewgenow K, Picton HM, Plancha C, Shea LD, Stouffer RL, Telfer EE, Woodruff TK, Zelinski MB.
• Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation. Hum Reprod Update. 2010 Feb 1. [Epub ahead of print]
Culture techniques at the centre ofFertility Preservation Strategies
Ovarian tissue cryopreserved
Test tissue viability by IVG prior to transplantation (density testing etc)
Cultured in multi step system
Production of developmentally competent oocytes
Acknowledgements• Marie McLaughlin• John Binnie• Joyce Leyden• Christina Ding
• Outi Hovatta(Karolinska)
• Kui Liu (Umea)
• Joo Thong (Edinburgh)• David Albertini (Kansas)• Joshua Johnson (Yale)• Dror Meirow (Tel Aviv)• Daniel Brison (Manchester)
• Hamish Wallace (Edinburgh)• Richard Anderson (Edinburgh)• Norah Spears (Edinburgh)
• BBSRC (Cow work)• NHS Endowment fund (Human work)• Bio-incubator Fund • MRC (Human work)