Drug-free IVA (in vitro activation) as an

infertility treatment for aging poor

responders

Professor, Department of OB/GYN

Director of Advanced Reproductive Medicine Research Center

International University Health and Welfare School of Medicine

Kazuhiro Kawamura

<1,000 follicles

birth Menarche menopause 40 yrs

Folli

cle

nu

mb

er

No follicle growth

Changes in follicle number during women’s life

Periodic follicle growth/follicle atresia

Young ovary Aging ovary

Aging

Problems

・Donor egg, adoption

・Regenerate oocytes

from iPS/stem cells

・MT injection/NT

・antioxidants, GH, etc

Solutions

Decreasing

oocyte/embryo quality

・Donor egg, adoption

・Drug-free IVA

・Regenerate oocytes

from iPS/stem cells Decreasing

residual follicle number

Aging infertility

Contents

1. Background of POI

1. Basic and translational studies for in vitro activation

of dormant follicles (IVA)

1. Clinical application of IVA

1. Drug-free IVA

Primary ovarian insufficiency (POI)

Diagnosis

1. Amenorrhea before 40 years of age

2. Hypergonadotropic hypogonadism

Symptoms

1. Infertility

2. Estrogen deficiency-hot flashes, mood disturbances, sexual

dysfunction etc.

Etiology

POI affects approx. 1% of women

Less than 1,000 follicles in POI patients before 40 yrs.

<1,000 follicles

Birth Menarche 40 yrs

Folli

cle

nu

mb

er

Normal women

POI patients

No follicle growth

・Egg donation is the most successful treatment

option, but…

・Resistant to traditional gonadotropin treatments

Treatments

・Lack of follicle growth and ovulation

・Exhaustion of ovarian follicles and

few residual follicles: <1,000 follicles

(undetectable AMH levels)

Specific features

Is it possible to activate residual

dormant follicles in POI patients?

Contents

1. Background of POI

1. Basic and translational studies for in vitro activation

of dormant follicles (IVA)

1. Clinical application of IVA

1. Drug-free IVA

Primordial

follicle

Primary

follicle

Growth factors (kit ligand, neurotrophins, BMPs, VEGF, LIF, etc.)

Among dormant follicles, ~0.1% follicles are selected to

activate. Because POI has < 1,000 residual follicles, these

growth factors can activate only 1 follicle.

We focused on intracellular signaling system

Involving in the activation.

Reddy et al. Science, 2008 Castrillon et al. Science, 2003

At early stage after birth, PTEN or FOXO3 deletion led to the activation of dormant

primordial follicles and resulted in depletion of follicles within 16-18 weeks.

PTEN null mice Foxo3 null mice

PTEN PIP3 PIP2 PDK1

SGK Akt

GSK

3

BAD

p27

Apoptosis,

Cell-cycle

arrest

TSC2 TSC1

RHEB 4EBP

EIF4E p70S6K

Protein

synthesis SGK

Akt

FOXO3 FOXO4

FOXO1

PI3K

Apoptosis, cell-cycle arrest

Nucleus

TOR

RTK

Growth

factors

CRK GRB2

Ras

Phosphatidylinositol 3-kinase (PI3K) signaling pathway

The PI3K signaling pathway begins PI3K activation by receptor tyrosine kinases (RTKs) after

binding growth factors. PI3K activates AKT, which inhibits the activities of FOXO3, resulting in

cell proliferation and survival. PTEN negatively regulates PI3K signaling.

In primordial follicles, local factors activate dormant follicles through PI3K-Akt-Foxo3

signaling pathway, whereas PTEN acts to block the signaling.

Is it possible to activate residual dormant

follicles in POI patients artificially by

transient PTEN suppression and/or PI3K

activation using drugs?

PI3K activator

PTEN inhibitor

A vanadyl complexed to hydroxypicolinic acid is a highly potent and

specific inhibitor at nano-molar concentrations.

RTK

SH2

Derossi et al. BBRC, 1998

A cell-permeable phospho-peptide

(740Y-P) binds to the SH2 domain of

p85 regulatory subunit of PI3K and

activates enzyme activity.

Ovaries

PTEN Inhibitor &PI3K activator

Control

transplanted into kidney capsule

FSH treatment

hCG treatment Oocyte retrieval

Mature oocytes

Adult ovariectomized

Method--mouse D3 mice

Epigenetic

analyses

IVF-ET

Pups

Histological

analyses

Culture 2 days

24h 48h

740YP

bpV (Hopic)

% t

ota

l fo

llic

les

primordial primary preantral antral large

antral

100

80

60

40

20

0

20

15

10

5

0

Follicular dynamics at day 14 after transplantaion of activated ovaries.

PTEN inhibitor+PI3K activator

*

*

*

*

*

primordial primary preantral antral large

antral

In vitro activation (IVA) - in vivo transplantation

-- ovarian histology

48h

96h

0

20

40

60

80

100

1 2

normal control

bpV(pic)+740Y-P

2-cel/mature oocytes

**

% e

mb

ryo

nic

develo

pm

en

t

blastocyst/2-cell

0h

In vitro activation (IVA) - in vivo transplantation - IVF-ET

24h

20

40

60

80

100

0

normal control

PTEN inhibitor+PI3K activator

Human ovarian cortical fragments PTEN

Inhibitor Control

Xeno-transplanted into kidney capsule

FSH treatment for 6 months

hCG treatment Oocyte retrieval Mature

oocytes

Ovariectomized SCID mice

Xeno-transplantation of human ovarian fragments to activate

dormant follicles: IVA, in vitro activation

Ovarian cortical fragments were obtained from patients with benign ovarian tumor with informed consent

from the patient and approval from local ethical human subject committee.

Culture 2 days

Morphology of human ovarian fragments after 6 months

of xeno-transplantation

control

PTEN

inhibitor

control

PTEN

inhibitor

Contents

1. Background of POI

1. Basic and translational studies for in vitro activation

of dormant follicles (IVA)

1. Clinical application of IVA

1. Drug-free IVA

Clinical application of IVA for POI patients

Ovariectomy

Cryo- Preservation

Culture of ovarian cubes with PI3K activators

Auto-transplantation Retrieve

mature eggs

In vitro fertilization

Embryo cryo-preservation

Preparation of ovarian cortical strips for freezing

Fragmentation of ovarian strips to

cubes

Embryo transfer

Histological analyses Conventional IVA

Fresh IVA

IRB approval: Human Subject committee of St. Marianna University

and Japan Society of Obstetrics and Gynecology

Localization of early follicles in ovarian cortex

medulla

cortex 1-2 mm

・Dissect ovarian cortices containing

residual follicles by removing medulla.

・Cut into small strips (1 x 1 cm2,

1-2 mm thickness, where residual

follicles are located).

・(Option: Cryo-preservation by vitrification method.)

・6-8 pieces of ovarian stripes could be

obtained from one POI ovary.

・Using 10% of volume of each

ovarian stripe, detect residual follicles.

histological analyses

Before dissection of medulla

After dissection of medulla

Small ovarian stripes ready

for use

・Fragment 2-3 ovarian pieces into 1-2 mm2 of cubes

・IVA drugs treatment (PTEN inhibitor and PI3K activator)

for 2 days to activate dormant follicles

Culture of ovarian cubes

In Vitro Activation

Fragmentation of ovarian

strips to cubes

・Before auto-transplantation,

wash cultured ovarian cubes by

warmed culture media alone to

avoid to introduce reagents inside

of body.

・Transplant beneath the serosa of

Fallopian tubes (20-40 cubes per site).

Beneath serosa of Fallopian tubes

— high vascularization,

convenience for trans-vaginal ultrasound monitoring

ease for oocyte retrieval

Culture of ovarian cubes

Auto- transplantation of activated ovarian

cubes

In Vitro Activation

Auto-transplantation

Cutting the serosa and making a pouch

between serosa (arrows) and Fallopian

tube (arrowhead).

Grafting multiple ovarian cubes (arrows)

beneath the serosa of Fallopian tubes.

Wound was covered by an oxidized

regeneration cellulose to avoid cube

loss from the graft site.

Is this site the best for grafting？

Remaining ovary is also good site, especially in the cases of early stage of POI.

・Monitor follicle growth weekly to biweekly: serum

estrogen and gonadotropin levels + transvaginal

ultrasound.

・Specific protocols for ovarian stimulation in POI patients

(Zhai, Kawamura, et al. JCEM 2016).

・Conventional IVF-ET

Retrieve mature eggs

In vitro fertilization

Patients’ follow up protocols

Tips for ovarian stimulation after IVA

In POI patients, due to absence of antral follicles before and

immediately after IVA, the first sign of follicle growth is elevation of

estrogen (E2) levels.

Early luteinization

Arrest of follicle growth

Oocyte degeneration

Without ovarian stimulation, follicles can grow spontaneously

followed by decline in FSH and LH levels based on negative feed

back of E2. However, in most of cases, the decrease in LH levels

is inadequate….

Zhai, Kawamura et al

JCEM 2016

Success rate of oocyte retrieval after ＩＶＡ in different protocols

・without LH level suppression+spontaneous growth: 27% (3/11) ・LH level suppression: 100% (2/2) ・without LH level suppression+HMG stimulation: 0% (0/2)

Orisaka Endocrinology 2013

cAMP production after FSH stimulation

Follicle growth after FSH stimulation Preculture with LH

FSH stimulation

Effects of hyper-LH on oocyte-granulosa-theca cell interactions

High chronic LH stimulation

GDF9↓

FSHR↓

CYP17↑

Preantral follicles exposed to high LH express low levels

GDF-9 in oocyte and FSHR in granulosa cells, resulting

in decreases in sensitivity of FSH stimulation and

suppression of follicle growth.

How can we stimulate ovaries after IVA?

1. Normalize LH levels by supplementation of estrogen and estrogen + progesterone with induction of withdrawal bleeding.

2. After confirmation of normal LH levels (<10 mIU/ml), maintain its low levels using GnRHa. (Daily GnRH AN injection is too expensive)

3. Similar to short protocol, treat patients with rFSH or pure HMG

(low LH content) for >2 weeks.

Results

Ovary grafting was performed in 46 patients

Follicle growth was found in 28 out of 46 patients containing

residual follicles based on the histological analyses.

After IVF, embryos were cryopreserved at day 2.

83 of POI patients (37.4 ± 4.9 years of age)

Duration of amenorrhea: 5.7 ± 3.5 years

Enrolled patients

Results

3 out of 8 patients became pregnant after

embryo transfer.

One miscarriage

Two successful deliveries

ーa male baby, 3254

ーa female baby, 2970g

Thawing embryo transfer was performed

in 8 patients. Others were accumulating

cryopreserved embryos.

Second babies by re-transplantation of frozen-

thawed cryopreserved ovaries after activation

1st patient: Dec. 2012-first baby

Sep. 2018-successful pregnancy

2nd patient: May. 2014-first baby

April. 2018-Second baby (female, 3091g)

After the delivery, we performed IVA again using cryopreserved

ovarian tissue.

Current clinical outcome of IVA

・Residual follicles based on histology:

Positive: n=96

Negative: n=56

・Ovariectomy: n=152

・IVA grafting with residual follicles with follow up

> 6 months: n=70

Total

N=152

Positive N=96

Negative N=56

・IVA grafting without residual follicles with follow up

> 6 months: n=16

0

10

20

30

40

50

60

70

80

90

100

64%

45/70

6%

1/16

% o

f fo

llicle

gro

wth

Our histological analyses were

effective to predict IVA outcome

Histological

analyses

Residual follicle (+) Residual follicle (-)

・Recent follicle growth after diagnosis of POI

・Positive AMH

・Short duration of amenorrhea

・POI/DOR patients with residual follicles.

How can we predict presence of residual

follicles?

Indication for IVA treatment

Duration of amenorrhea is a good predictive

factor for presence of residual follicles

0

2

4

6

8

10

Du

rati

on

of

am

en

orr

hea (

Year)

*

Residual follicle

(-) (+) 0

1

2

3

4

0-3 F

oll

icle

nu

mb

er

per

1x1x5m

m t

issu

e

3-6 6-9 >9

Duration of amenorrhea (Year)

Clinical application of IVA for POI patients

Ovariectomy

Cryo- Preservation

Culture of ovarian cubes with PI3K activators

Auto-transplantation Retrieve

mature eggs

In vitro fertilization

Embryo cryo-preservation

Preparation of ovarian cortical strips for freezing

Fragmentation of ovarian strips to

cubes

Embryo transfer

Histological analyses Conventional IVA

For long term

amenorrhea

Fresh IVA For short

term amenorrhea

IRB approval: Human Subject committee of St. Marianna University

and Japan Society of Obstetrics and Gynecology

・Success of oocyte retrieval : 42/46 patients (91%)

139/212 follicles (66%)

[1-16 cycles]

0

10

20

30

40

50

60

70

80

90

100

MII

oocyte

%

fertilization cleavage *high quality

D2-3 embryo *Veek: G1-3

116/139 85/116

76/85

32/76

Results of IVF after IVA

・Embryo transfer (cleavage stage D2 or 3): n=38

・Pregnancy: 15/38 (39%)

・Miscarriage: 5/15 (33%)

・7 babies, 3 ongoing pregnancies

Over all outcome after embryo transfer

・Mean age: 38.9 years of age

IVA grafting with residual follicles with follow up

> 6 months: n=124

IVA pregnancy/delivery

N=4 N=4 N=2

IVA in Poland: December 2016 IVA press conference in China: May 2015

Results

Reproducibility of IVA was already confirmed by China, Spain, Poland, and Mexico groups under our guidance. Kawamura et al Hum Reprod 2015

Zhai et al JCEM 2016

N=1

International patent：

Out-licensing to Ovascience. Inc

Kawamura et al. PNAS 2013 Hsueh, Kawamura et al. Endocrine Rev 2014 Suzuki, Kawamura et al. Hum Reprod 2015 Yuan, Kawamura et al FASEB J 2015 Kawamura et al. Hum Reprod 2016 Kawamura and Hsueh Curr Opin Obstet Gynecol 2016 Zhai, Kawamura et al. JCEM, 2016 Kawamura et al. Reproduction, 2017 Haino, Kawamura et al. JAYAO 2017 Sato, Kawamura et al. J Gynecol Women’s Health 2017 Kawamura et al. Syst Biol Reprod Med 2017

STIMULATION OF OVARIAN FOLLICLE DEVELOPMENT

AND OOCYTEMATURATION

PCT/US2013/059800

Follicle growth from primordial

to preovulatory stage takes more than

4-6 months.

In contrast to our expectation,

we found follicle growth before

4-6 months after grafting.

This result suggested that our IVA

method also stimulated growth of

secondary follicles in grafted ovaries.

Derived from

secondary follicles

Derived from

primordial follicles

Temporal follicle growth in transplanted ovaries

1.70

2.52

1.88

2.43

1.56

2.13

1.62

1.91

1.44

2.37

Ovary weight (mg)

Fragmentation of mouse ovaries followed

by allo-transplantation facilitated ovarian growth

Paired ovaries from

D10 mice with or

without

fragmentation were

grafted into kidney

capsules of adult

ovariectomized

mice. At 5 days after

grafting, ovaries

were dissected and

fixed before

measurement of

their weights.

Ovarian fragmentation followed by grafting promoted

growth of secondary follicles in mice

Follicle dynamics before and after grafting of intact and fragmented

(three pieces) ovaries from day 10 mice were evaluated at 5 days

after grafting.

What is the molecular

mechanisms underlying follicle

growth after fragmentation?

Hippo signaling pathway restricts cell proliferation

Hippo pathway regulates cell

proliferation. In normal tissue with

an intact cytoskeleton (purple),

hippos signaling inactivates a core

transcriptional factor (Yki or YAP)

and prevent it from activating

proliferation genes in the nucleus.

When cytoskeleton conformation is

changed, Yki/YAP is free to enter

the nucleus and produce CCN

growth factors leading to cell

proliferation.

GAPDH

YAP

pYAP

Wh

ole

Pie

ce

s

Wh

ole

Pie

ce

s

Ovarian fragmentation decreased phospho-YAP

(unactivated form) in mice

Paired ovaries from day

10 mice with or without

cutting into 3 pieces were

incubated for 1 h,

followed by

immunoblotting.

GAPDH

CCN2 W

ho

le

Pie

ce

s

Wh

ole

Pie

ce

s

Wh

ole

Pie

ce

s

3h 4h 5h

CC

N2

/

Paired ovaries from day 10 mice with or without cutting were incubated for 3–5 h

before immunoblotting.

Ovarian fragmentation increased CCN2 proteins in mice

0

1

2

3

4

* *

*

*

0

1

2

3

4

0

1

2

3

4 *

*

CCN2/CTGF CCN3/NOV

CCN5/WISP2 CCN6/WISP3

*

*

0

1

2

3

4

0 1 3 0 1 3

0 1 3 0 1 3 (h)

(h)

(h)

(h)

Thawed human cortical

strips were cut into pieces

or left intact before

incubation for different

intervals followed by real-

time RT-PCR analyses.

Increases in CCN transcripts after fragmentation of

human ovarian tissues.

Roles of CTGF (CCN2) in ovary

・CTGF treatment of ovaries induces the

expression of many genes related to cell-

cycle progression and cell differentiation,

thereby enhancing the growth of immature

follicles (Schindler R, Nilsson E, Skinner

MK 2010).

・Ctgf cKO mice exhibit severe subfertility

and multiple reproductive defects including

reduced numbers of preantral and antral

follicles, and a trend toward increased

numbers of atretic preantral and atretic

antral follicles (Nagashima et al. 2011).

Regulation of Hippo pathway by F-actin

Under conditions of low F-actin, Hippo pathway activity is high, leading to inhibition of Yki

/YAP activity and inhibition of tissue growth. When F-actin levels are high, Hippo pathway

activity is inhibited leading to nuclear import of Yki/YAP, and up0regulation of downstream

targets to promote tissue growth. Physiologically, F-actin accumulation may be regulated

by external tension cues, thereby controlling tissue growth via the Hippo pathway.

Paired ovaries from day 10 mice were

cut into three pieces or kept intact

before immunoblotting analyses of F-

and G-actin levels.

Ovarian fragmentation stimulated actin polymerization

followed by increased F-actin levels

Hippo pathway

genes

YA

P

YA

P

P

YA

P

CCN Growth factors

Secondary follicle growth

G-Actin F-Actin

14-3-3

Degradation

CCN

Ovarian fragmentation led to

changes in intercellular tension and

facilitated the conversion of G-actin

to F-actin.

Subsequent disruption of Hippo

signaling decreased pYAP to total

YAP ratios, leading to increased

expression of downstream CCN

growth factors.

Secretion of CCN growth factors

stimulated follicle growth.

Ovarian fragmentation suppresses Hippo

signaling, leading to follicle growth

Anti-apoptotic factors

Contents

1. Background of POI

1. Basic and translational studies for in vitro activation

of dormant follicles (IVA)

1. Clinical application of IVA

1. Drug-free IVA

Primordial

follicle

Primary

follicle

Secondary

follicle

Antral

follicle

Preovulatory

follicle

PI3K signal stimulation

Hippo signal disruption

Two-step follicle stimulation in IVA

New IVA approach targeting immature

secondary follicles

In early POI/DOR (poor responder) patients, is it

possible to stimulate residual secondary follicles to

increase number of antral follicles by disruption of

Hippo signaling only?

Ovarian cubes culture

1. Original IVA (PI3K stimulation and Hippo disruption)

Oophrectomy

Fragmentation and PI3K stimulators treatment

(2 days)

POI patients Two surgeries

Cryo- preservation

DOR/early

POI patients

Fragmentation and immediate return

Partial cortex removal

One surgery

2. Drug-free IVA (Hippo disruption only)

Drug-free IVA and Direct Activation

Drug-free IVA Direct

Activation

No positive evidence up to now A clinical study in progress:

Successful pregnancy in 7 out of 68 aging ovarian dysfunction patients (43-

48 yrs)

Limitation:

Drug-free IVA and orthologous grafting

Only applicable to DOR/early POI patients who likely have secondary follicles

Advantages:

IVI group, Spain (Bilbao workshop 2017) 3 spontaneous pregnancies/14 patients

・ Minimal damages to ovarian blood supply

・ No need to use Akt-stimulating drugs

・ Avoid potential follicle loss during culture

・ One laparoscopic surgery

・ Spontaneous pregnancy possible

Collaborators

Bunpei Ishizuka, Nao Suzuki,

Yorino Sato, Naoki Okamoto, Ikko

Kawashima, Midori Tamura

Seido Takae, Yodo Sugishita,

Nobuhito Yoshioka, Yuta Kawagoe,

Mariko Hoshina, Noriyuki Takahashi

Aaron JW Hsueh

Yuan Cheng

Jing Li

St. Marianna University

School of Medicine

Stanford University

School of Medicine