drug-free iva (in vitro activation) as an infertility treatment for...
TRANSCRIPT
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