In vitro development of human ovarian follicles. Evelyn E Telfer Institute of Cell Biology University of Edinburgh

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1 In vitro development of human ovarian follicles Evelyn E Telfer Institute of Cell Biology University of Edinburgh

2 Fertility preservation options Embryo Freezing Oocyte Freezing (hormonal stimulation) Immature oocyte freezing (IVM) Whole Ovary Freezing Ovarian Tissue Freezing (Cortical strips)

3 Developmental Regulation of oocyte number in vivo Primordial Preantral Decreasing Numbers with Development Late Preantral Increasing levels of apoptosis Antral Follicle Interactions Endocrine Regulation

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

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

6 FOLLICLE CULTURE Promises so much Slow to develop in humans and domestic species Where are we now?

7 Developing a multistep culture system 1) Optimising growth from primordial/primary stages 2) Supporting growth of isolated preantral follicles 3) Final stages of oocyte development 4) Testing function and normality

8 Proposed Multi-step Culture System to support development of bovine/human oocytes from primordial stages (IVG) Micro-cortex culture Removal of preantral follicles by micro-dissection Individually cultured and monitored for oocyte/follicle health markers Removal of OGC s to membrane culture system for final growth and development IVM of isolated oocyte-cumulus complexes (i) (ii) (ii) (iii) (iii) Developmentally Competent oocytes for Fertilisation Step one Step two Step three Embryos available for use in ART, stem cell derivation cloning etc

9 Step 1: Preparation of cortex Ovarian cortical biopsies Stroma and larger follicles removed Micro-cortex Tissue Architecture. Surface area and density of stromal cells important feature

10 Stages of follicle development present in micro-cortex at end of culture Proportion of follicles * ** Primordial Transitory Primary Secondary 0 Day 0 Day 6 Days in Culture Telfer,McLaughlin,Ding & Thong (2008, Human Reproduction)

11 Follicle growth within microcortex Cultured micro-cortex Histology follicles grown within micro-cortex Human Follicle development in vitro (6 days)

12 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 follicles longer results in increased death and poor quality follicles/oocytes.

13 Isolating Growing Follicles Cultured micro-cortex Follicles before isolation Isolated Follicle Human Follicle development in vitro (6 days)

14 Two Step Serum Free Culture System for Human oocytes Step one 6 days Cortical Biopsy (n=6) from women undergoing elective C section Strips cultured for 6 days in serum free medium Preantral follicles (66-132µm) Mean 100+/-3.4. Isolated by microdissection Step Two 4 days Telfer et al., 2008a Follicles cultured in control medium (McCoys) (n=36) or McCoys + 100ng/ml Activin (n=38)

15 Growth of isolated human preantral follicles in vitro 200 Mean follicle diam meter (microns) n = 36 n = 38 n = 32 n = 33 n = 24 n = 26 * * Control Activin 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 * ** Control Activin Type 1 Type 2 Type 3 Type 4 Health/degeneration scale Telfer et al., 2008 Human Reproduction

17 A B C D

18 Morphological Health of In Vitro Grow n Human Follicles: Activin versus Activin + FSH rtio n o f licles Pro p o F o l * Type 1 Type 2 Type 3 Type 4 rhacta rhacta&rfsh Healthy Morphological Follicle Health Category Health/degeneration scale Degenerate Telfer et al., 2008b

19 Activin and FSH have a positive effect on isolated growing follicles but what about on initiation of Primordial Follicles?

20 Effect of FSH on Primordial activation P ercen tag e o f F o llicles P ercen P entage of of Follicles a a a b b b b d b c,d b c c,d e e c e e c Day 0 Control 1ng/ml FSH c,d Day 0 Control 1ng/ml FSH Day 0 Control 1ng/ml FSH e e Quiescent Quiescent Primary Primary Secondary Secondary Quiescent Primary Secondary (ii) 6 days in vitro (iii)

21 Regulating the rate of depletion of the Primordial Pool Maintainers Suppressors Activators

22 Low dose FSH increases size of growing follicles 50 µm (microns) Mean Follicle Diameter Growth of Isolate d Human Se condary Follicle s Disse cte d from Culture d Cortical Fragme nts c 100 a Control Exposed Fragments FSH Exposed Fragments 6 8 Total Time In Vitro (days) 10

23 PI3K Figure signalling 1 regulating Activation of Primordial Follicles

24 Inhibiting mtor affects initiation of human primordial follicles in vitro 100 Percentage of Follicle es * ** Fresh Fixed Control Rap 0.01pM Rap 0.1nM Rap 100nM Low dose High dose Quiescent Primary Secondary follicles per treatment

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

26 Development of follicles within cortical strips from vitrified tissue (Step one) follicles of follicles Percentage follicles Primordial transitory primary secondary 0 day 0 day 6 days in culture Vitrified Tissue (n=10 biopsies) Telfer et al., 2008c

27 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). of follicles percentage Control Activin Type 1 Type 2 Type 3 Type 4 Healthy Degenerate Health/degeneration scale Vitrified tissue source Telfer et al., 2008c

28 + Activin A Vitrified cultured - Activin A 6 days strip culture + 4 days isolated culture Fresh Cultured

29 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:

30 Bovine Folicles cultured for 8 days from primordial (step 1) then 12 days from the preantral stage (step 2) 30µm Fig 3.a 50µm Oocytes of up to 108 microns produced in vitro (Met II & PB, IVM)

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

32 Growth Rate of Follicles in vivo Preantral Mature Size Time Mouse Pig Cow Woman µm µm days µm 1.5-3mm days µm 3.8->8.5mm days µm mm

33 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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35 Cultured Follicles transplanted under KC of SCID mouse Brakes back on 6 weeks in vivo 50µm Collaboration with Daniel Brison and Helen Picton

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

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

38 Confocal imaging of oocyte-somatic cell interface Optimising oocyte-somatic cell communication during IVG David Albertini KUMC

39 Multi-step Culture system to support human oocyte development Activation

40 NEXT STEP. Final Stages of Development IVM +IVF Complexes Isolated from IVG antral follicles For further growth Tests for normality Methylation etc

41 Acknowledgements Marie McLaughlin John Binnie Joyce Leyden Christina Ding Outi Hovatta (Karolinska) Kui Liu (Umea) Joo Thong (Edinburgh) Suresh Kini (Edinburgh) David Albertini (Kansas) John Bromfield (Kansas) Joshua Johnson (Yale) Hamish Wallace (Edinburgh) Richard Anderson (Edinburgh) Norah Spears (Edinburgh) BBSRC (Cow work) NHS Endowment fund (Human work)

42 John Eppig Hello from Bar Harbor!

43 Follicular Development Oocyte Growth Decreasing numbers and Increasing death