Simulated Physiological Oocyte

Maturation (SPOM) and Related

IVM Systems School of Women’s & Children’s Health

Robert Gilchrist

University of New South Wales Sydney,

Australia

In Vitro Maturation (IVM) (No or minimal ovarian stimulation)

IVF or ICSI

24 to 40 hours in vitro

Small antral

Conventional IVF Ovarian hyperstimulation (FSH) IVF or ICSI

hCG

Small antral Preovulatory

Thompson JG & Gilchrist RB (2013) In: Biology and Pathology of the Oocyte (Cambridge University Press)

IVF offspring

Human ~7 million

Cattle few

IVM offspring

Human ~6000

Cattle >400,000/year Walls M et al (2015) Hum Reprod

%

IV M IV F

0

2 0

4 0

6 0

8 0

Live Birth Rate/OPU

OPU

IVF or

ICSI

Menses Day 10

FSH

• Oocytes are matured in vitro

o from the GV-stage (intact cumulus-oocyte complexes)

o patient FSH-priming is compatible

Edwards RG (1962) Nature

Edwards RG (1965) Nature

hCG-priming ??

The standard definition of IVM

Bringing IVM to the ART clinic:

core biological challenges

1) Oocytes from small antral follicles are not fully

developmentally competent

2) Maturation in vitro can occur spontaneously without

appropriate somatic/maternal control

In Vitro Maturation (IVM) (No or minimal ovarian stimulation)

IVF or ICSI

24 to 40 hours in vitro

Small antral

COCs in small antral follicles are still developing

(oocyte capacitation)

BMP15/GDF9 BMP15/GDF9

FSH/cAMP

Developmental

competence

Developmental

competence

Small Antral Follicle

(EGF-p unresponsive)

Large Antral Follicle

(EGF-p responsive)

EGFR

signalling

EGF-p

Energy

Metabolite

supply

EGFR

signalling

Oocytes acquire developmental competence from

coordinated endocrine and paracrine signals

Sugimura S (2015) Dev. Biol. 403:139–149

Richani and Gilchrist (2018) Hum. Reprod. Update (Epub)

Bringing IVM to the ART clinic:

core biological challenges

1) Oocytes from small antral follicles are not fully

developmentally competent

2) Maturation in vitro can occur spontaneously without

appropriate somatic/maternal control

In Vitro Maturation (IVM) (No or minimal ovarian stimulation)

IVF or ICSI

24 to 40 hours in vitro

Small antral

Standard Spontaneous IVM IVF/ICSI

• complex medium, protein, FSH, EGFp

• LH, E2, cysteamine

Oocyte in vitro maturation (IVM)

FSH-priming

Basic Principals: oocyte “capacitation” in vitro -

Extended culture of meiotically competent COC

isolation

from

follicle

GVBD GV GV

prolonged gap-junction

communication

Meiotic inhibitors

• cAMP analogues, PDEi

• cGMP, CNP Nutrients & metabolic regulators

• Metabolites, IGFs, etc

CC regulators

• FSH, EGFp, E2, GDF9, BMP15, cumulin

cAMP

ATP

AC

5’AMP

ATP

AC

5’AMP

cAMP

GTP

Oocyte maturation: meiotic arrest

Gilchrist RB et al. (2016)

Reprod. 152:143-157

Oocyte maturation: meiotic resumption

Richani and Gilchrist (2018) Hum. Reprod. Update (Epub)

cAMP: in vivo and in various IVM systems

Gilchrist RB et al. (2016) Reprod. 152:143-157

Induced IVM (moderate cAMP)

end of maturation

CO

C c

AM

P

0 hours

Induced IVM (high cAMP, eg SPOM)

Biphasic IVM

Standard IVM

Oocyte in vivo maturation

+ GV GVB MII

± FSH/EGFp

GV GVB MII

+PDE inhibitor

+FSH/EGFp ++

GV GVB MII

+PDE inhibitor

± FSH/EGFp ++

± FSH/EGFp

GV GVB MII

+cAMP

± FSH/EGFp +++ ± PDE inhibitor

cAMP management during IVM improves outcomes

Gilchrist RB et al. (2016) Reprod. 152:143-157

22%

IVF/ET

8% IVF/ET

26% IVF/ET IVM Pre-IVM

IVM

Ovarian Hyperstimulation

Fetal Yield

Induced IVM (SPOM)

Standard IVM

Conventional IVF

Albuz FK et al. (2010) Hum. Reprod. 25:2999-3011

cAMP management during IVM improves outcomes

Thomas RE et al. (2004) Biol. Reprod. 71:1142-1149

Zeng HT et al. (2013) Hum. Reprod. 28:1536-1545

Richani D et al. (2014) Mol. Reprod. Dev. 81:422-35

Zeng HT et al. (2014) Biol. Reprod. 91:47, 1-11

Li HJ et al. (2016) Hum. Reprod. 31:810-821

Gilchrist RB et al. (2016)

Reprod. 152:143-157

Li HJ et al. (2016) Hum.

Reprod. 31:810-821 IVM (h)

cAMP pre-IVM preserves the benefits of CC-oocyte GJC

cAMP pre-IVM leads to decreased intra-oocyte ATP

Richani D et al. (unpublished)

cAMP pre-IVM leads to decreased intra-oocyte ATP

Richani D et al. (unpublished)

cAMP pre-IVM upregulates in intra-oocyte AMPK activity

2h of IVM

pAMPK 62 kDa

Actin 42 kDa

Actin 42 kDa

AMPK 63 kDa

pAMPK 62 kDa

Actin 42 kDa

AMPK 63 kDa

Actin 42 kDa

16h of IVM

Ph

osp

ho

ryla

ted

AM

PK

To

tal A

MP

K

Richani D et al. (unpublished)

Effect of cAMP pre-IVM on COC ATP and AMPK activity

pAMPK 62 kDa

β-actin 42 kDa

pAMPK 62 kDa

β-actin 42 kDa

2h of IVM 16h of IVM

Phosphorylated AMPK

Richani D et al. (unpublished)

Changes in energy metabolism associated with increased oocyte

developmental competence

Oocyte Cumulus

Increased energy demand ( ATP ADP ATP:ADP)

AMPK

Restore energy homeostasis

Increased ATP

AMPK

FSK/IBMX pre-IVM

Increased oocyte competence

Richani D et al. (unpublished)

Challenges in translating scientific advances to

the human IVM clinic

• Human oocytes are scarce and precious

• We have poor measures of oocyte quality

need to produce human embryos for research ..!!

• IVF status quo works – resistance from the ART sector

• Biological challenges (topic of presentation)

VUB Brussels: Oocyte donors and experimental

design

• Patient work-up: OCP, 3x days hMG (days 5-7), no hCG-priming, OPU 42h after last hMG

• Follicle size: typically ~5 mm,

Effect of pro-cumulin in human IVM (retrospective)

IVM: FSH + pro-cumulin Pre-IVM:

FSK+IBMX

ICSI

Control: n = 275 COC n = 19 patients and volunteers

Treatment: n = 246 COC n = 21 patients and volunteers

IVM: FSH Pre-IVM:

FSK+IBMX

ICSI

MII

(%

)

C o n tr o l T r e a tm e n t

0

2 0

4 0

6 0

8 0

1 0 0

Fe

rti

lis

ati

on

ra

te (

%)

C o n tr o l T r e a tm e n t

0

2 0

4 0

6 0

8 0

1 0 0

Da

y 3

GQ

E

mb

ry

o /

MII

(%

)

C o n tr o l T r e a tm e n t

0

2 0

4 0

6 0

8 0

Bla

sto

cysts

/ M

II (%

)

Control Treatment0

10

20

30

40*P

GV GVB MII

24 hours

+AREG+FSH +CNP +E2 +FSH

Human oocyte “capacitation” in vitro using CNP

(VUB Brussels)

ICSI

CNP

NPR2

cGMP

PDE3A cAMP 5’AMP

GVBD

30 hours

Sanchez F et al. (2017) Hum. Reprod. 32:2056-68

How to make a good quality oocyte for ART?

FSH + hCG

cAMP or CNP

GDF9/BMP15/cumulin

EGF-peptides or FSH

Jeremy Thompson

Lesley Ritter

Satoshi Sugimura

Navy Li

Xaioqian Wang

Firas Albuz

Maxime Sasseville

Acknowledgements

Robinson Research

Institute, University of

Adelaide, Australia

Funding

- COOK Medical

- NHMRC Australia: Fellowships, Project Grants, Development Grants, Program Grants

Vrije Universiteit Brussel

(VUB), Belgium

Laval University, Canada

Francois Richard

Johan Smitz

Heidi Van Ranst

Francesca Lolicato

Sergio Romero

Flor Sanchez

Wim Coucke

Ellen Anckaert

University of New

South Wales (UNSW),

Sydney, Australia

Dulama Richani

Michael Bertoldo

Angelique Riepsamen

Yiqing Zhao

Kirsty Walters

David Agapiou

David Robertson

Bill Ledger

Michel De Vos

Biljana Popovic-Todorovic

Samuel Dos Santos Ribeiro

Karlien De Bondt

Elsie Nulens

Ingrid Segers

Greta Verheyen

Herman Tournaye

University of Milano, Italy

Alberto Luciano

Sun Yat-sen University,

China

Haitao Zeng

Xiao-Yan Liang