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)