In vitro growth of immature
       follicles/oocytes
           Evelyn E Telfer
      Institute of Cell Biology
          ...
Options to use cortical tissue to
        restore fertility
Currently limited to transplantation
 46 cases from 25 studies...
Ovarian Tissue Transplantation
                   Advantages
Restoration of endocrine and fertility function
             ...
In vitro growth of immature
                    oocytes
Frozen-thawed cortical strips        Cortical strips contain
     ...
Mice from Primordial Follicles...

                         Eggbert: First mouse born
                         from an in ...
Oocyte Development

In vivo
Oocyte
Growth      19-30µm             40-80µm               80-90µm            90-100µm      ...
Developing a multistep
culture system for human IVG
• 1) Optimising growth from primordial stages

• 2) Supporting growth ...
Step one: Activation of quiescent
            primordial follicles
                Maintainers




Suppressors

          ...
Micro-Cortex Culture
    Cortical biopsy                             Micro-cortex
    cut into strips
                    ...
Growth within micro-cortex
Proportion of follicles




                          0.8
                          0.6        ...
Improving rate of activation and early follicle
    Figure 1

                        growth in vitro




Telfer, McLaughl...
Effect of inhibiting PTEN using 1 µM
                                     bisperoxovanadium [bpV]
                        ...
Growth within micro-cortex
• Optimal time and size to remove growing
  follicles from micro-cortex environment.
• In our h...
Step 2: Preantral – Antral stages.
    Isolated Follicle Culture




Cultured       Follicles before   Isolated Follicle
m...
Growth of isolated human preantral
          follicles in vitro
                                       200




           ...
Health of in vitro grown human follicles after 6
days in cortical strip culture (step 1) followed
    by 4 days in isolate...
A   B




C   D
Applications of follicle/oocyte
     culture systems (IVG)
                    Current
• Basic research tool
• Tissue viab...
Follicles recovered after IVG from several
     tissue sources (fresh and cryopreserved)
No. Biopsies   Source            ...
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 seru...
Step 3: Further oocyte growth




Almost fully grown oocytes (95microns) obtained within
alginate encapsulated follicles g...
Multi-step Culture system to support
        human oocyte development




                                        Combinin...
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)




         ...
Bovine IVG oocytes 12 days step 2 Mean oocyte
                                  diameter
P ercen tag e o f O o cytes




 ...
Accelerated growth?
           Or
Growth without brakes?….
 How long does complete oocyte
       development take?
Growth Rate of Follicles
          in vivo

        Preantral   ‘Mature’      Time
                    Size

        100-2...
Does a human oocyte really
need 70 days to develop or
    is this time frame a
 consequence of inhibition
     regulating ...
■
               ♦
        ▲
                   ♦
■   ■              ■
                   ▲
    ▲              ■

■




  ...
Competing functions within the follicle


                              OOCYTE
                            DEVELOPMENT
   ...
In Vitro Growth of Oocytes

  Optimising conditions to support
  oocyte development through co-
  ordinating oocyte-somati...
Imaging of oocyte-somatic cell
                         interface




        Focus on optimising oocyte-somatic cell comm...
NEXT STEP…………….
         Final Stages of Development
                                   IVM +IVF
  Complexes Isolated     ...
EURO CULTURE CLUB:
  ESHRE INITIATIVE TO ADVANCE
 FOLLICLE CULTURE TECHNIQUES
• Smitz J, Dolmans MM, Donnez J, Fortune JE,...
Culture techniques at the centre of
   Fertility Preservation Strategies
Ovarian tissue              Test tissue viability...
Acknowledgements
•   Marie McLaughlin   •   Joo Thong (Edinburgh)
                       •   David Albertini (Kansas)
•   ...
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Recent developments ovarian tissue transplantation versus in-vitro maturation of immature oocytes alpha-telfer_evelyn_2010

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Recent developments ovarian tissue transplantation versus in-vitro maturation of immature oocytes alpha-telfer_evelyn_2010

  1. 1. In vitro growth of immature follicles/oocytes Evelyn E Telfer Institute of Cell Biology and Centre for Integrative Physiology University of Edinburgh
  2. 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. 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. 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. 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. 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. 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. 8. Step one: Activation of quiescent primordial follicles Maintainers Suppressors Activation of growth Primordial Pool Activators
  9. 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. 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. 11. Improving rate of activation and early follicle Figure 1 growth in vitro Telfer, McLaughlin, Anderson, Hovatta and Liu (2010)
  12. 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. 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. 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. 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. 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. 17. A B C D
  18. 18. Applications of follicle/oocyte culture systems (IVG) Current • Basic research tool • Tissue viability assessment Potential • Fertility preservation (frozen tissue)
  19. 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. 20. Cryo tissue before/after step 1 of culture
  21. 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. 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. 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. 24. Closing the gap between IVG and IVM.
  25. 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. 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. 27. Accelerated growth? Or Growth without brakes?…. How long does complete oocyte development take?
  28. 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. 29. Does a human oocyte really need 70 days to develop or is this time frame a consequence of inhibition regulating follicle development?
  30. 30. ■ ♦ ▲ ♦ ■ ■ ■ ▲ ▲ ■ ■ ▬▬▬▬
  31. 31. Competing functions within the follicle OOCYTE DEVELOPMENT Most important feature of cumulus lineage is physical interaction with the oocyte ENDOCRINE FUNCTION
  32. 32. In Vitro Growth of Oocytes Optimising conditions to support oocyte development through co- ordinating oocyte-somatic cell interactions OOCYTE DEVELOPMENT Endocrine Function
  33. 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. 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. 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. 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. 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)

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