Human immature oocytes can be

grown to maturity in vitro: next

steps towards clinical application

Evelyn E Telfer

Institute of Cell Biology

and

Genes and Development Group CDBS

University of Edinburgh

Ovarian Club Paris

November 2018

Disclosure

• The University of Edinburgh has filed a

patent on aspects of this work. Patent

application number: GB 1416858.7

• I have no other financial or commercial

interests to declare in relation to the work

presented here.

• This work has been funded by the UK

Medical Research Council

Developing systems to grow human oocytes in vitro

>100 Live Births

Tissue freezing in Edinburgh since 1996. First EdinburghTissue

reimplanted 2015

Preantral

Antral

Decreasing numbers

with development

Primordial

Increasing levels

of apoptosis Late

Preantral

Store of non-growing (NGFs) (primordial)

follicles utilised throughout reproductive

life

Primordial Follicles are present in the ovarian cortex at all

ages. Can we utilise the large numbers that are lost in vivo?

Why develop oocytes in vitro?

• Define the fundamental mechanisms of oocyte development (basic science)

• Clinic: Fertility Preservation

• Clinic: Next generation IVF (IVG)

• Animal Production

• Endangered species

• Toxicity testing

Mice from Primordial Follicles

(and now PGCs and iPSCs)

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, O’Brien et al., 2003

More recently: Complete in vitro generation of fertile oocytes from primordial germ cells

Morohaku et al., 2016 and iPSCs Hikabe et al., 2016

Developing human primordial

follicles from cortical strips in vitro

Human ovarian cortical

biopsies taken for

fertility preservation

contain mainly

primordial/unilaminar

follicles.

The challenge is to

develop oocytes in

vitro from primordial

stages to maturation

and fertilisation.

Frozen-thawed cortical strips

Rhabdomyosarcoma Pt 15yrs:thawed from slow freeze

Oocyte

Growth

In vivo

19-30µm

40-80µm 80-90µm 90-100µm 100-110µm

Pre-ovulatory primordial primary preantral Early

antral

Mid antral

Making an Egg is Complicated

Growth/ Meiotic Arrest

Acquisition of Meiotic Competence

Acquisition of Developmental Competence

Transcription/Transcriptional Repression

Genomic imprinting

Developing a multistep culture

system for human oocytes

• 1) Optimising growth from primordial stages

• 2) Supporting development of isolated growing follicles

• 3) Final stages of oocyte development

• 4) Testing function (meiotic and fertilisation potential) and normality

Source of Human OvarianTissue

For Research

• Small strip of ovarian cortex donated after informed consent:

• Caesarean section (Healthy women)

• Fertility Preservation (various cancers and Turners syndrome) Some tissue obtained after chemo treatment.

• Tissue from 15 months-45 years (fresh and cryopreserved)

• Transgender patients (whole ovaries at time of gender reassignment surgery)

Step 1: Micro-Cortex Culture

Micro-cortical fragments

Free floating cultures: basic

conditions serum free medium

Underlying

stroma reduced

larger follicles

removed

Cortical biopsy cut into

strips

Tissue Architecture.

Surface area and

density of stromal

cells important feature

Telfer et al., 2008 Human Reproduction 23:

1151-8

Step one: Activation and Growth of

Quiescent Follicles

• Optimal time & size to remove growing follicles from micro-cortex environment

• 6-8 days; ≥ 100µM mean diameter

• Prolonging Step 1 results in increased death and poor quality follicles/oocytes

Telfer et al., 2008 Human Reproduction 23: 1151-8

Step 2: Isolating Growing Follicles

Cultured

micro-cortex

Follicles

before

isolation

Isolated Follicles

Manual dissection using needles and fine scalpel

Activin A supplementation of medium

Additional 6-8 days in vitro

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 three: Isolating Oocyte-Granulosa

Cell Complexes

In vitro Grown Follicles

(after 2 steps)

Remove oocyte and

surrounding cells

Step 3: Culture

on membranes

McLaughlin, Albertini, Wallace, Anderson & Telfer 2018 Mol Hum Reprod 24: 135-142

0

20

40

60

80

100

120

140

0h 2days 4days

Time on membranes

Oo

cyte

dia

met

er (m

icro

ns)

Mean Oocyte Diameter at end of Multi-step Culture

Step 3

After

IVM

McLaughlin, Albertini, Wallace, Anderson & Telfer 2018 Mol Hum Reprod 24: 135-142

Metaphase II oocytes from human IVG follicles

McLaughlin, Albertini, Wallace, Anderson & Telfer 2018 Mol Hum Reprod 24: 135-142

McLaughlin, Albertini, Wallace, Anderson & Telfer 2018 Mol Hum Reprod 24: 135-142

Oocytes that matured showed large polar bodies

McLaughlin, Albertini, Wallace, Anderson & Telfer 2018 Mol Hum Reprod 24: 135-

142

Oocyte Yield

Approx 30% of oocytes that complete culture

process can reach Metaphase II: Epigenetic

Status and Fertilisation potential?

Multi-step Culture system to support human

oocyte development

McLaughlin, Albertini, Wallace, Anderson & Telfer 2018 Mol Hum Reprod 24:

135-142

In Vitro Growth of Oocytes from

Prebubertal Girls

Oocytes developed in vitro from pre-pubertal mice:

What about oocytes from young girls?

Patients and samples

Samples from Edinburgh Fertility preservation service (3-16)

(all laparoscopic biopsies)

Diagnosis Age (years) Biopsy Menarche

Rhabdomyosarcoma 3.0 Fresh Premenarche

Ependymoma 8.2 Fresh Premenarche

Rhabdomyosarcoma 7.9 Cryopreserved Premenarche

Rhabdomyosarcoma 10.6 Fresh Premenarche

Ewing’s Sarcoma 12.2 Fresh Premenarche

Ewing’s Sarcoma 12.0 Fresh Post menarche

Sacral Sarcoma 12.3 Fresh Premenarche (early puberty)

Sickle Cell Anaemia 14.6 Fresh Premenarche (early puberty)

Acute Myeloid

Leukaemia 14.4 Fresh Premenarche (early puberty)

Hodgkin’s Disease 14.1 Fresh Post menarche

Hodgkin’s Disease 15.3 Fresh Post menarche

Rhabdomyosarcoma 16.0 Cryopreserved Post menarche

0

5

10

15

20

25

30

35

0 5 10 15 20

% G

V A

bs

en

t F

ollic

les

Age (yrs)

0

10

20

30

40

50

60

70

Prepubertal Pubertal Adult

Me

an

Fo

llic

le D

iam

ete

r (m

icro

ns

)

* *

A B

C D

25µm

50µm

Morphologically abnormal oocytes (Big Fat

Oocytes) found in young ovaries. 8 yrs 14 yrs

Follicles activate growth (step 1) in young

tissue and growing follicles can be

isolated for step 2

Tissue cultured from young girls (3-10 and 12-15)

Effect of age on In Vitro Growth

(IVG) (Step 2)

60

90

120

150

180

210

240

270

Day 6 Day 8 Day 10 Day 12

Me

an

Fo

llic

le D

iam

ete

r (m

icro

ns

)

3-10yrs

12-15yrs

25-38yrs

Growth rate during step 2 of culture

Anderson RA et al., 2014 Human Reproduction 29: 97-106

Conclusions

• Follicles can initiate growth at all ages, to

secondary stage

• Follicles from younger girls grow slowly, and

show little oocyte growth

• Follicles from adolescent girls grow more slowly

than those from adult women but show

significant oocyte growth compared to younger

girls

• Culture System needs to be adapted according

to age and tissue origin

Range of ovarian tissue developed in vitro

Tissue Source Endpoint achieved in vitro

(multi-step system)

Healthy women (end of

pregnancy)

Metaphase II oocytes

Prepubertal girls (FP

source)

Multilaminar stages

Turner’s Patients Multilaminar/early antral

Chemo treated Variable depends on age and

treatment

Gender Reassignment Metaphase II oocytes

IVG: System has to be adapted for tissue type

Summary

• Multi step culture system supports

human oocyte growth and

development

• Optimisation of each step required

• Further testing required (epigenetic

status)

• Fertilisation potential?

• A model system for human oocyte

development

Next Steps

• Improving Culture Media

(additives/timings)

• Compare Culture systems (FastGrow)

versus SlowGrow (Picton system)

• Physical Conditions to improve polar body

formation

Previous conditions always

resulted in large polar

bodies. New conditions are

promising and spindles

currently being analysed

Next Steps Towards Clinical

Application • Determining health and developmental

competence of IVG oocytes (sequencing,

epigenome, metabolome)

• Fertilisation of IVG human oocytes: HFEA

approval

• Embryo Testing

• Parallel studies on a large animal model

(sheep and cow) embryo testing and

transfer. Life Young (Collaboration with

Prof Helen Picton, Leeds)

Acknowledgements

The patients who

kindly donate their

ovarian tissue and the

clinical teams that

support this

Dr Marie McLaughlin

Dr Yvonne Clarkson

Dr Cheryl Dunlop

Dr Kelsey Grieve

Dr Christina Ding

Mr John Binnie

Ms Joan Creiger

Ms Anne Saunderson

Ms Norma Forson

Prof Hamish Wallace

Prof Richard Anderson

Dr Joo Thong

Prof David Albertini

(Kansas/NYC)

Dr Neale Watson

Funding: