Capacitation-IVM culture

of oocytes from small follicles

in PCOS patients

Johan Smitz & IVM Team

Follicle Biology Laboratory

Center for Reproductive Medicine

Vrije Universiteit Brussel (VUB)

Brussels , Belgium

Conflict of interest declaration

Speaker fees from :

BioMérieux,

Besins Female Healthcare,

Ferring Pharmaceuticals,

Merck

LEARNING OBJECTIVES

Acquire knowledge on :

- oocyte development in small human follicles

- oocyte Maturation in-vitro (IVM) :

recent developments

- improved method for clinical IVM :

CAPACITATION – IVM (“CAPA”)

IVM : what is it ?

DEFINITION

- is NOT a truncated IVF

- is NOT ‘rest’ material from IVF

DeVos et al. Human

Reproduction, 2016

SELECTION

PROCESS

Gougeon A. Human Reproduction 1986;2:81 - 87.

HUMAN FOLLICLE DEVELOPMENT

A. GOUGEON

IVF

Why IVM systems stay under-utilised

1- FOLLICLE HETEROGENEITY

the HCG injection on 6-12 mm follicles

causes asynchronous maturation of oocytes

2 - INAPPROPRIATE SIGNALLING is

induced “spontaneous” vs “induced” meiosis

re-initiation

Effect of hCG triggering in IVM

( Son et al., RBM Online 2008 )

compact cumulus

expanded

cumulus

GV

GV

MI

MII

PR 23,3%

PR 40%

Post HCG = In vivo + in vitro

maturation

Type of cumulus-oocytes obtained and OUTCOME

NOTE :

What the data show on “+hCG” - IVM

Generally low implantation rates

Increased early pregnancy loss

Need for multiple ET(2-4 Embryos)

Results: hesitant adoption by clinics

IVM and assessment of

oocyte/embryo quality

Property In vivo maturation In vitro maturation

Transcriptional

silencing Complete Leaky

Maternal RNA

processed Complete Partial

Metabolic loading Maximal Partial

Gap junctional comm. Maintained Retracted

Ca stores Maximal Partial

Pace of progression Slow Fast

Meiotic progression Complete Incomplete

D. Albertini, 2nd Biogenesi Conference, 2010

SMALL Follicle LARGE Follicle

How to IMPROVE

1- FOLLICLE HETEROGENEITY

the HCG injection on 6-12 mm follicles

causes asynchronous maturation of oocytes

2 - INAPPROPRIATE SIGNALLING is

induced “spontaneous” vs “induced” meiosis

re-initiation

Positive stimulus

HOW TO IMPROVE ?

Use a system inspired from animal production

Better exploit the small (2-8 mm) follicles ? Learn about

Gamete competences

Receptors expression

Signalling cascades

FIVE KEY STEPS TO IMPROVE IVM

ABANDON HCG INJECTION

GIVE FSH or HP-hMG PRIMING: 2-3 DAYS

USE DEFINED CULTURE MEDIA, NO SERUM

APPLY POSITIVE IN-VITRO MATURATION STIMULUS

PRE – MATURATION ( “CAPACITATION“ ) CULTURE

AVOID HCG to FOSTER CUMULUS- OOCYTE COMMUNICATION

POTENTIAL of CAPACITATION-IVM versus Standard of Care

Sanchez et al.,2017 HR

100

79

62

50

32

25

100

70

53

43

23

18

100

48

31

23

14

8

COCs MII 2PN GQED3 ED3 Q1-top quality

Blastocysts D5-D6

ICSI (PCOS, n=97)

CAPA (n=15)

Standard IVM (n=15)

TRANSFERABLE BLASTOCYSTS OBTAINED

PER 100 COC ( sibling oocytes )

‘stimulated ICSI’ CAPA-IVM“NEW” STANDARD IVM

OOCYTE PHYSIOLOGY : ‘flash’ REVIEW

1- In the small antral follicle (2-8 mm) the

oocyte is actively kept under meiotic arrest

what are the main mechanisms ?

2- By LH rise: meiosis reinitiation and

cumulus expansion ?

what are the prerequisite conditions ?

MEIOTIC ARREST

Regulation

cAMP

ATP

AC

5’AMP

ATP

AC

5’AMP

cAMP

LH ACTIVATES THE EGF- LIKE SYSTEM ESSENTIAL TO OOCYTE MATURATION

Theca

cells

Mural granulosa

cells BM

Cumulus

cells

Oocyte

LH PDE3A

cAMP

Gs

LHR GPR3 / GPR12

PDE4D

PDE4D

BM: basal membrane; hCG: human chorionic gonadotropin; LH: luteinising hormone; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; LHCGR: lutropin-

choriogonadotropic hormone receptor; PDE4E, Phosphodiesterase 4E; PDE3A, Phosphodiesterase 3A; AC, adenylyl cyclase.

Conti et al. Mol Endocrinol 2006;20:715–723

EGFR

LHCGR

AC3/9

Epi

AR

BTC

EGF-LIKE

FACTORS

OOCYTE SECRETED

FACTORS (OSF)

CUM GDF-9 FGF2 BMP15

GONADOTROPHINS

A. GOUGEON

Is Nuclear

Maturation

completed ?

FSH-R

POSITIVE

Stimulus

PRIMARY IMPRINTS

EGF-Like ?

EGF-R ?

PCO OVARY

PICK-UP of SMALL ( 2-8 mm) Follicles

from PCOS

When COC are detached from the follicle wall

1- essential receptor systems and growth factors are not yet fully functional

2- meiosis inhibition is missing in simple media

NON-hCG IVM : Characterising GV stage oocyte from 2 to 6 mm follicles ( = 70% of harvest )

96 98 100

102

104

106

108

110

112

114

116

118

120

122

124

0

4

8

12

16

20

24

28

Diameter (µm)

Nu

mb

er o

f o

oc

yte

s

ControlA Meiotically incompetent

94 96 98 100

102

104

106

108

110

112

114

116

118

120

0

10

20

30

40

50

60

70

Diameter (µm)

Nu

mb

er o

f o

oc

yte

s

N = 293

B

C D<105µm' ≥105'and'<110µm' ≥110µm'

4" 19" 84"

3,7%" 17,8%" 78,5%"

<105µm' ≥105'and'<110µm' ≥110µm'

32# 98# 163#

10,9%# 33,4%# 55,6%#

Dispersed(%)

Intermediate(%)

Condensed(%)

Controlsmall <105µm 3(75) 0 1(25)

medium ≥105<110µm 3(27.3) 3(27.3) 5(45.5)large ≥110µm 20(31.3) 10(15.6) 34(53.1)

Total(N=79) 26(32.9) 13(16.5) 40(50.6)

Meioicallyincompetent

small <105µm 17(65.4) 6(23.1) 3(11.5)medium ≥105<110µm 46(59.7) 21(27.3) 10(13.0)

large ≥110µm 49(38.0) 51(39.5) 29(22.5)

Total(N=232) 112(48.3) 78(33.6) 42(18.1)

Chromatinconfiguration

Mean (n=107) 112,6 ± 5,1 µm

Diameter of immature

oocytes before IVM

Chromatin configuration in

immature oocytes before

IVM

Dispersed Intermediate CondensedDispersed Intermediate CondensedDispersed Intermediate Condensed

96 98 100

102

104

106

108

110

112

114

116

118

120

122

124

0

4

8

12

16

20

24

28

Diameter (µm)

Nu

mb

er o

f o

oc

yte

sControlA Meiotically incompetent

94 96 98 100

102

104

106

108

110

112

114

116

118

120

0

10

20

30

40

50

60

70

Diameter (µm)

Nu

mb

er o

f o

oc

yte

s

N = 293

B

C D<105µm' ≥105'and'<110µm' ≥110µm'

4" 19" 84"

3,7%" 17,8%" 78,5%"

<105µm' ≥105'and'<110µm' ≥110µm'

32# 98# 163#

10,9%# 33,4%# 55,6%#SANCHEZ et al. Human

Reproduction, 2015 TRANSCRIPTIONAL

SILENCING Variable Stage

STANDARD IVM

CAPACITATION + IVM

•Works for COCs from follicle Ø

diameter ≥ 8 mm

•STANDARD IVM METHODS are

not focused on increasing

oocyte competence

•COC from 2-6 mm follicles

require capacitation before

IVM

•Tailor capacitation time to

follicle size (24–48 hours)

•HYPOTHESIS :

“CAPACITATION CULTURE”

o ENHANCES MATURATION

POTENTIAL

o INCREASES THE BLASTOCYST

YIELD

Ø : Average

CAPACITATION (CAPA) culture

Can IMPROVE IVM from smaller follicles

Improve maturation

by a 2-STEP CULTURE

STEP 1 : actively inhibit the immediate GVBD STEP 2 : administer the positive maturation trigger

By keeping up cAMP in COC :

persistent communication between cumulus -

oocyte leading to a slower GVBD : this results in

an increased oocyte developmental capacity.

TRANSLATIONAL RESEARCH

- Luciano (2002) - bovine

- Rebecca Thomas & Rob Gilchrist (2003)- bovine

- Kawashima (2008) - pig

- Daniela Nogueira (2004)- mouse – human

OPTIONS TO CONTROL MEIOTIC ARREST

CAPA SPOM

CNP

(Pre-IVM)

ZHANG M LUCIANO AM (2011)

Gilchrist RB and Thompson J, 20062

CNP

OSF

1. Gilchrist RB et al. Reproduction 2016;152:R143–R157; 2. Gilchrist RB and Thompson JG. Theriogen 2006; 67:6–15; 3.

2. Zhang M et al. Endo 2010;152:4377-4385

C-TYPE NATRIURETIC PEPTIDE PDE- INHIBITORS

CAPA- IVM TREATMENT SCHEME

shortened CLINICAL PART reduced monitoring and drugs cost

OPU

IN VITRO STUDY :

Capacitation and Maturation Culture

Meiosis

Stimulators

EGF-LIKE

Natural Meiotic inhibitors

•C-type Natriuretic Peptide

GV GV

CAPACITATION culture

24 or 48 hrs

AT RETRIEVAL AFTER PRE-MATURATION

Physiological level of essential components

growth factors – anti-oxydants – GSH …

PB

IVM

AFTER IVM

After EXPANSION

ICSI

2-8 mm Follicles

CAPACITATION-IVM compared to STANDARD IVM

LABORATORY PART

CAPA

system

OPU Pre-IVM IVM

Standard

IVM

IVM

30h

OPU

IVM Patients (sibling oocyte study):

women with PCOS; personalized

stimulation 3/4x225-150-75IU hMG,

OPU 42h after last hMG

At retrieval Post CAPA Post CAPA-IVM

ICSI

Embryo

culture till D3

or blastocyst

stage (D5 or

6)

Embryo culture

till blastocyst

stage (D5 or 6) Vitrification

Vitrification

and / or ET

NGS:

SAFETY DATA

Ploidy

methylation

30h (24-46h)

CONTROL :

Cooperation with Prof Gavin Kelsey

The Babraham Institute, Cambridge

12 conventional ICSI

20 CAPA-IVM / ICSI

• Day 5 or 6 Day 6

• Good quality blastocysts (BL3, BL4; AA, AB, BA, BB)

DNA methylation analysis by PBAT (post-bisulfite adaptor tagging) in single

blastocysts

Methylation data were compared to Okae et al, 2014 : (deeply

sequenced pool of 80 human blastocysts)

Whole genome methylation analysis in human blastocysts derived from CAPA-IVM

Global methylation variation in human

blastocysts was NOT affected by

• Type of ART: CAPA-IVM vs ICSI

• Culture medium

• Sex of the embryo

• Day 5 or Day 6 culture

• Blastocyst expansion grade (BL3/BL4)

CLINICAL DEVELOPMENT OF IVM

RESULTS CAPACITATION-IVM

by 2018

5 Pilot Studies

169 PCOS Patients

Brussels @ UZbrussel (Profs Devos M & Anckaert E ) : Safety DATA

Hoshiminh@ MyDuc ( Profs Lan Vuong & Manh Tuong ) : RCT

Osaka @ IVF Japan (Profs Morimoto & Fukuda ) : Application

Total Patients Patients COC MII% Total Available

N with ET Day 3 Embryon Transferred

Embryos

Implantations

(number of sacs)%

Ongoing

Pregnancy% Deliveries

Total

Babies

STD-IVM 40 33 653 48 141 67 19 28% * 12 9 11

2,0 ** 36%

Per patient data 16,3 7,8 3,5

CAPA-IVM 40 37 723 63 176 78 31 40% * 19 16 22

2,1 ** 51%

Per patient data 18,1 11,4 4,4

* Per Transferred Embryos (Total embryos transferred)

** Per Embryo Transfer (All patients with embryo transfer)

UPDATED TILL 07-09-18First ET cycle

CLINICAL OUTCOME

RCT CAPA-IVM vs. STANDARD IVM combined with

FREEZE ALL and ET D3

Only DATA of FIRST frozen DAY-3 EMBRYO TRANSFER

G. Griesinger 2015

Calculation of IMPLANTATION rate

CONFIDENTIAL PRELIMINARY DATA VUB and MyDuc

Unpublished data confidential

SUMMARY IMPLANTATION RATE from Fresh vs VITRIFIED DAY 3 EMBRYOS

RCT´s – Vietnam NO HCG

Lan et al 2018 + HCG

Cohen et al 2018 +HCG

TRANSFER TYPE

STD IVM Vitrif N=33

CAPA-IVM Vitrif N=37

FRESH N=823

Vitrified N=50

FRESH N=263

Vitrified N=51

IR (%) 28% 40% 25% 21% 14% 4%

Unpublished data confidential

Infertility treatment in PCOS :

conventional approach

CURRENT TREATMENT

• Lifestyle intervention

• First line therapy: - clomiphene or letrozole

• Second line therapy:

- gonadotrophins

- laparoscopic surgery (drilling)

• If additional infertility factors: IVF/ICSI

- GnRH antagonist / Agonist stimulus in

hyperandrogenic anovulatory PCOS

RESULTS / COMPLICATIONS

• Ovulation in 80%

• Pregnancy in 35 %

• Multiples : 10%

• Pregnancy in 20% per cycle

• Cancelled for Risk : 10-30%

• Multiples : 15-25%

• OHSS developed : 5-10%

• cum birth rate 58% ( 3 cy )

• OHSS : few (GnRHa trigger)

• Discomfort : high

• Multiples : 11-30 %

(Balen et al., HRU 2016 ; Molenaer et al , 2016)

NEW APPROACH PCOS

•Lifestyle intervention

•First line therapy: - clomiphene or letrozole

• Second line therapy:

CAPA – IVM : no HCG Single eFET

RESULTS versus

COMPLICATIONS

Ovulation in 80%

Pregnancy in 35 %

Multiples : 10%

Short treatment

Zero multiples

short time to pregnancy

Treatment of PCOS in 2018 : CAPA-IVM

CONCLUSIONS

• IVM is the only ART that totally excludes OHSS

• IVM reduces burden and cost for the patient

• GV oocytes from 2-6 mm follicles from PCOS patients can reach full

developmental capacity after capacitation culture (“CAPA”)

• CAPA-IVM increases the number of embryos per oocyte pick–up (4-5)

• CAPA- IVM embryos are chromosomally and epigenetically normal

• High implantation rates (40%) from vitrified/warmed Capa-IVM embryos

• Healthy children born (N=30) from CAPA-IVM in JAPAN and VIETNAM

ACKNOWLEDGEMENTS

Funding

- Fund For Research Flanders (FWO)

- Fund for Innovation in Technology (IWT)

- Free University Brussels (VUB)

Vrije Universiteit

Brussel (VUB)

Michel De Vos

Ellen Anckaert

Flor Sanchez

Sergio Romero

Heidi Van Ranst

Francesca Lolicato

Nurses, embryologists

&medical staff of CRG

at UZBRUSSEL

Jeremy Thompson

David Mottershead

SPRH, Robinson

Institute, University of

Adelaide

NSWU ( Sidney )

Rob Gilchrist

National University

Vietnam

MyDuc Hospital (

Hochiminh City )

Vuong Thi Ngoc Lan

Ho Manh Tuong

Le Hoang Anh

Ma Pham Que Mai

Nurses, embryologists

IVF JAPAN GROUP

IVF OSAKA

Yoshiharu Morimoto

Isaku Fukuda

Nurses, embryologists

And medical staff at IVF

Japan

3.9 2.2

RESULTS: EMBRYOLOGY

STD

CAPA

60%

51%

At oocyte Retrieval

Post CAPA 24h

Post IVM 32h

CAPA-IVM System

CNP – ARREST :

ADVANTAGES of CAPA for

the embryology laboratory

PHYSIOLOGICAL ARREST BY NATURAL PEPTIDE

• NO CHEMICAL SUBSTANCES NEEDED

AFTER CAPA CULTURE

POSITIVE MEIOTIC TRIGGER • Gives max MII at 30 hrs • Allows SINGLE SPERM

INJECTION TIME

COC CAPACITATION TIME WINDOW

• SHIFTS e-LAB ACTIVITIES

WITHIN NORMAL WORK HOURS