Recent developments- Ovarian tissue transplantation versus in-vitro maturation of immature oocytes _Telfer_Evelyn_2010

1. In vitro growth of immature
follicles/oocytes
Evelyn E Telfer
Institute of Cell Biology
and
Centre for Integrative Physiology
University of Edinburgh

2. Options to use cortical tissue to
restore fertility
Currently limited to transplantation
46 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.

3. Ovarian Tissue Transplantation
Advantages
Restoration of endocrine and fertility function
Disadvantages
Uncontrolled loss of primordial follicles (freezing
method re-vascularisation)
Longevity of graft unpredictable
Risk of re-introducing cancer cells

4. In vitro growth of immature
oocytes
Frozen-thawed cortical strips Cortical strips contain
mainly primordial
follicles.
The challenge now is to
develop oocytes in vitro
from primordial stages
to maturation and
fertilisation.
Where are we now?
Rhabdomyosarcoma Pt 15yrs:thawed from slow freeze

5. Mice from Primordial Follicles...
Eggbert: First mouse born
from an in vitro grown
primordial follicle: 2 step
system total of 22 days in
vitro before IVM and IVF
Eppig & O’Brien 1996
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

6. Oocyte Development
In vivo
Oocyte
Growth 19-30µm 40-80µm 80-90µm 90-100µm 100-110µm
primordial primary preantral Early Mid antral Pre-ovulatory
antral
Growth/ Meiotic Arrest
Acquisition of Meiotic Competence
Acquisition of Developmental Competence
Transcription/Transcriptional Repression
Genomic imprinting

7. Developing a multistep
culture system for human IVG
• 1) Optimising growth from primordial stages
• 2) Supporting growth of isolated preantral
follicles to antral stages
• 3) Supporting final stages of oocyte
development out with large follicle
• 4) Testing function (IVM &IVF) and normality
(methylation profile etc)

8. Step one: Activation of quiescent
primordial follicles
Maintainers
Suppressors
Activation
of growth
Primordial
Pool
Activators

9. Micro-Cortex Culture
Cortical biopsy Micro-cortex
cut into strips
Underlying
Stroma and
larger follicles
removed
Tissue Architecture.
Surface area and
density of stromal
cells important feature
Free Floating cultures: basic
Telfer et al. 2008 conditions serum free medium

10. Growth within micro-cortex
Proportion of follicles
0.8
0.6 Primordial
Transitory
0.4 *
Primary
** Secondary
0.2
0
Day 0 Day 6
Days in Culture
Telfer,et al. (2008) Human
Reproduction

11. Improving rate of activation and early follicle
Figure 1
growth in vitro
Telfer, McLaughlin, Anderson, Hovatta and Liu
(2010)

12. Effect of inhibiting PTEN using 1 µM
bisperoxovanadium [bpV]
100 100
Non-Growing
Percentage of healthy
80 Growing Primary
Percentage of Follicles
80
Growing Secondary
oocytes
60 60
40 40
20 20
0 0
Uncultured Control 1uM uncultured control 1 um
d1 d3 d6

13. 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.

14. Step 2: Preantral – Antral stages.
Isolated Follicle Culture
Cultured Follicles before Isolated Follicle
micro-cortex isolation
Human Follicle development in vitro
(6 days)

15. Growth of isolated human preantral
follicles in vitro
200
n = 26
n = 33
Mean follicle diameter (microns)
150
* *
n = 24
n = 32
n = 36 n = 38 Control
100
Activin
50
0
0 2 4
Days in culture (step 2)
Telfer et al., 2008 Human
Reproduction

16. 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).
50
percentage of follicles
40
*
30
Control
Activin
20 **
10
0
Type 1 Type 2 Type 3 Type 4
Health/degeneration scale
Telfer et al., 2008 Human Reproduction

17. A B
C D

18. Applications of follicle/oocyte
culture systems (IVG)
Current
• Basic research tool
• Tissue viability assessment
Potential
• Fertility preservation (frozen tissue)

19. Follicles recovered after IVG from several
tissue sources (fresh and cryopreserved)
No. Biopsies Source Status Isolated Follicles
35 C-Section Fresh 480
(Edinburgh & Stockholm)
10 C-Section Vitrified 135
(Stockholm)
4 Gynae (35-52)(Edinburgh) Fresh 27
3 Fertility preservation Fresh 30
(Edinburgh) (12-21)
1 Fertility preservation Slow-Frozen 14 (2 fragments)
(Edinburgh) (15)
3 Fertility Preservation (26-30) Slow-Frozen 80
(Manchester) (Chemo
treated)

20. Cryo tissue before/after step 1 of
culture

21. 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

22. Step 3: Further oocyte growth
Almost fully grown oocytes (95microns) obtained within
alginate encapsulated follicles grown for 24 days
Xu et al. (2009) In vitro grown human ovarian follicles from cancer
patients support oocyte growth. Hum Reprod. 24: 2531-40

23. Multi-step Culture system to support
human oocyte development
Combining growth
techniques: Telfer,
Activation Woodruff, Hovatta,
Picton groups
Adapted from Telfer & McLaughlin 2007

24. Closing the gap between
IVG and IVM.

25. Bovine Follicles cultured for 8 days from primordial
(step 1) then 12 days from the preantral stage (step 2)
30μm
Fig 3.a
50μm McLaughlin & Telfer 2010 Reproduction

26. Bovine IVG oocytes 12 days step 2 Mean oocyte
diameter
P ercen tag e o f O o cytes
100 ≤ 50
80
50-80
60
80-100
40
20
0
Fix Day 6 Control rhActA rhAct+FSH
Further growth of complexes within alginate before IVM
Oocytes of up to 108 microns. MII and PB

27. Accelerated growth?
Or
Growth without brakes?….
How long does complete oocyte
development take?

28. Growth Rate of Follicles
in vivo
Preantral ‘Mature’ Time
Size
100-200μm 500-600μm 10-12 days
Mouse
150-300μm 1.5-3mm 40-50 days
Pig
100-150μm 3.8->8.5mm 40-50 days
Cow
4.00-6.00mm 70-100
Woman 120-300μm

29. Does a human oocyte really
need 70 days to develop or
is this time frame a
consequence of inhibition
regulating follicle
development?

30. ■
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31. Competing functions within the follicle
OOCYTE
DEVELOPMENT
Most important
feature of cumulus
lineage is physical
interaction with the
oocyte
ENDOCRINE
FUNCTION

32. In Vitro Growth of Oocytes
Optimising conditions to support
oocyte development through co-
ordinating oocyte-somatic cell
interactions
OOCYTE
DEVELOPMENT
Endocrine Function

33. Imaging of oocyte-somatic cell
interface
Focus on optimising oocyte-somatic cell communication during
IVG
McLaughlin, Bromfield, Albertini, Telfer (2010)
Molecular Human Reproduction

34. NEXT STEP…………….
Final Stages of Development
IVM +IVF
Complexes Isolated Tests for
from IVG antral “normality”
follicles Methylation etc
For further growth
Encouraging progress
But much to do before clinical applications may
be realised
Collaborative effort required to make significant
progress

35. 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]

36. Culture techniques at the centre of
Fertility Preservation Strategies
Ovarian tissue Test tissue viability by
cryopreserved IVG prior to
transplantation (density
testing etc)
Cultured in multi
step system
Production of
developmentally competent
oocytes

37. Acknowledgements
• Marie McLaughlin • Joo Thong (Edinburgh)
• David Albertini (Kansas)
• John Binnie • Joshua Johnson (Yale)
• Joyce Leyden • Dror Meirow (Tel Aviv)
• Christina Ding • Daniel Brison (Manchester)
• Hamish Wallace (Edinburgh)
• Outi Hovatta • Richard Anderson (Edinburgh)
(Karolinska) • Norah Spears (Edinburgh)
• Kui Liu (Umea)
• BBSRC (Cow work)
• NHS Endowment fund (Human work)
• Bio-incubator Fund
• MRC (Human work)