ovarian rejuvenation by autologous stem cell ovarian transplant...

Innovation Foundation

Foundation

Paris, November 2, 2018

Ovarian rejuvenation by Autologous stem cell ovarian transplant (ASCOT).

Sonia Herraiz, PhD

IVI Foundation, Spain

[email protected]

Disclosure information: Nothing to declare


Remaining pool of dormant

follicles

AMH

Depletion of the ovarian reserve: follicles and AMH

P

a

g

e

2

Visser et al., Nature Reviews Endocrinology 2012.

Ovarian Aging

Diminished ovarian reserve or poor ovarian response (PR) by non-determined causes

Impairment of ovarian function

Acute premature ovarian insufficiency (POI) due to chemotherapy (ChT)

Therapeutic alternatives


In vitro activation (IVA) of dormant follicles

Follicular rescue and live birth after IVA and ovarian fragmentation in POI patients.

Residual follicles are able to growth and be ovulated in an adequate ovarian environment.

Residual follicles


New ways of ovarian rejuvenation

Follicular rescue of remaining follicles by:

Ovarian fragmentation for follicle activation (OFFA).

Autologous Stem cell ovarian transplantation (ASCOT)


RCT DO IT AGAIN

OUR EXPERIENCE


Modification of the original IVA technique

6

Kawamura et al.,2013

Avoid IVA drugs.

Minimize cryodamage/ischemia.

Look for the best vascularized site.

Allow for natural pregnancy


Ovarian fragmentation for follicular activation (OFFA)

IVA OFFA Drug free

Unique surgery No tissue cryopreservation

On site transplant

PI: César Díaz-García


OFFA in POI

PATIENTS 14

OOCYTES 21

PREGNANCIES 5

LIVE BIRTHS 3


New ways of ovarian rejuvenation

Follicular rescue of remaining follicles by:

Ovarian fragmentation for follicle activation (OFFA).

Autologous Stem cell ovarian transplantation (ASCOT).


Sterility after

Chemo-

and radiotherapy

Fertility

restoration

after BMT

Bone

marrow

transplant

Fertility recover after bone marrow transplant

Veitia RA, Gluckman E, Fellous M, Soulier J. Recovery of female fertility after chemotherapy, irradiation, and bone marrow allograft: further evidence against massive oocyte regeneration by bone marrow-derived germline stem cells. Stem Cells. 2007.

Hershlag A, and Schuster MW. Return of fertility after autologous stem cell transplantation. Fertil Steril. 2002.

Salooja N, et al. Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey. Lancet. 2001.

Sanders JE, et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation. Blood. 1996.

Salooja N, at al.,. Successful pregnancies in women following single autotransplant for acute myeloid leukemia with a chemotherapy ablation protocol. Bone marrow transplantation 1994.


BMDSC represent a heterogeneous group of mononuclear cells, including several hematopoietic and stromal

stem/progenitor cells.

Hematopoietic stem cell

Stromal stem cell

Bone marrow derived stem cells (BMDSC)

Astori et al. Am J Transl Res. 2010.


Ovarian rejuvenation by bone marrow derived stem cells

1. Do Bone Marrow Derived Stem Cells (BMDSC) arrive into the ovarian tissue?

2. To assess if BMDSC could induce ovarian rescue in mice ovaries mimicking Primary Ovarian Insufficiency (POI) and Diminished Ovarian Reserve (DOR) conditions.

3. To analyze BMDSC in human cortex from Poor Responder (PR) patients xenografted into SCID mice.

4. To optimize ovarian reserve in PR patients by autologous stem cell ovarian transplant (ASCOT).

Page 2

Objectives and End points questions


Experimental design 1

Cy: Cyclophosphamide Bu: Busulphan POI model PR model

Standard regimen: 120 mg/kg Cy 12 mg/kg Bu

Reduced regimen: 12 mg/kg Cy 1.2 mg/kg Bu

18 NOD/SCID mice 18 NOD/SCID mice

Control Group PBMNC Group BMDSC Group 1x106

human

BMDSC

1x106

human

PBMNC

100 μl of

saline

7d

MIRB labeled-human cells

Cell labeling with Molday Ion Rhodamine B (MIRB)

MIRB

nanoparticles

Ovarian hyperstimulation and mating

14

d

Consecutive matings for 3 months

Long-term effects

Short-term effects

Estrous cycle, ovaries, oocytes/embryos


D-7 D-6 D-5 D-4 D-3 D-2 D-1 D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14

P E M D

Control

Control

ChT Cell infusion

BMDSC

PBMNC

PBMNC

BMDSC

Estrous (E) Proestrous (P) Metestrous (M) Diestrous (D)

PR

-mo

de

l P

OI

mo

de

l

X

✓

✓

X

X

X

No

-Ch

T

Estrous cycle

Herraiz et al. Fertil Steril 2018;109:908–18


Follicles, oocytes, embryos and offspring

BMDSCs infusion increased the number of preovulatory follicles, MII-ovulated oocytes and embryos.

0

2

4

6

8

10

12

14

1st offspring 2nd offspring 3rd offspring

Litte

r siz

e (

n)

PR-BMDSC

POI-BMDSC

PR-PBMNC

PR-Control POI-Control

POI-PBMNC Restored long term fertility

First offspring

Second offspring

PO

I-B

MD

SC

PR

-BM

DSC


0

2

4

6

8

10

12

PR POI

2-c

ell e

mb

ryo

s

Control

PBMNC

BMDSC

*

0

10

20

30

40

PR POI

MII

ovu

late

d o

ocy

tes

(n)

*

0

0.5

1

1.5

2

2.5

PR POI

% P

re-o

vula

tory

fo

llicl

es

* *


Ovarian stroma regeneration

BMDSC infusion:

Increase ovarian vascularization

Improve cell proliferation

Reduce apoptosis in stroma and follicles even in the high chemotherapy dose.



Do BMDSC arrive into the human ovaries?

Do these cells stimulate follicular growth in human ovarian tissue?

Is whole BMDSC active or only isolated CD133+ cells?

End points- Questions


Experimental design 2: human ovarian tissue

Ovariectomized NOD-SCID mice

human ovarian cortex (OC) from Poor Responder (PR) women

Control

100 μl of saline

BMDSC

1x106 human BMDSC

CD133+

3x105 human CD133

Cell infusion Xenograft

7D

D-7 D0 D+1 / D+7 / D+14

BMDSC

Positive selection of their

CD133+ cell fraction

Blood

Tissue sampling and analysis

OC grafts

Cell engraftment Follicular density and growth

Vascularization and proliferation of ovarian stroma

Other organs

Cell tracking

Estradiol secretion


BMDSC: human ovarian tissue

Injected cells localized near blood vessels and close to follicles, in contact with GC.

Secondary follicles were only observed in OC grafts receiving cell injection

Pru

ss

ian

Blu

e

Control BMDSC CD133+

0

1

2

3

4

5

6

7

8

9

10

CONTROL BMDSC CD133 CONTROL BMDSC CD133 CONTROL BMDSC CD133

D1 D7 D14

folli

cula

r d

en

sity

(fo

llicl

es/m

m3

) Secondary

Primary

Primordial


Stem cell infusion promoted growth of human follicles


BMDSC: human ovarian tissue

In human OC BMDSC

Improved ovarian vascularization

Increased cell proliferation

Promoted estradiol secretion


Control BMDSC CD133+

Blood samples

17b estradiol

Serum

ELISA

0

10

20

30Se

rum

E2

(p

g/m

l) CONTROL BMDSC CD133

*


Human BMDSCs rescued the already existing follicles to growth and to produce healthy offspring.

Follicular rescue could be promoted by the regeneration of the ovarian niche as suggested by the increased vascularization and proliferation, and suppressed apoptosis.

Are we recovering the ovarian function?

BMDSC-based therapies could be a suitable alternative to increase the reproductive potential of patients with impaired ovarian function


Provided that BMDSC transfer works in an animal model of human ovarian grafting:

Do we increase follicular recruitment in PR patients?

If so, how does it work?

Do we improve oocyte quantity and quality in PR patients?

Next step:


Pilot study with 20 PR patients based on the ESHRE criteria.

Patients are considered as their own control as only one ovary received intervention.

Autologous Stem Cell Ovarian Transplantation (ASCOT).

STUDY GROUP

• Maternal age ≤40 years.

• Two episodes of PR after COS (≤3 oocytes obtained).

• AFC> 2 follicles with at least 1 follicle in the ovary that will be perfused.

• AMH > 0,5 - ≤3pmol/L.

• Serum FSH levels ≤20IU/l.

• Regular menses.

Clinical Trial Nº: NCT02240342

Study design 3: PR women


Autologous Stem Cell Ovarian Transplantation (ASCOT)

Clinical Trial Nº: NCT02240342

1. Mobilization of BM derived stem cells by a 5-day treatment with Granulocyte colony stimulating factor (G-CSF, 10 ug/kg/ day).

2. Isolation of BM derived stem cells from peripheral blood by apheresis.

3. Infusion of stem cells (volume: 40.4± 12.3 ml) into the ovarian artery by catheterism (contralateral ovary served as control).

Herraiz et al. Fertil Steril 2018;110(3):496-505


Study design: patient follow-up and IVF

Patient follow-up

(6 months)

Serum AMH • Every 2 days during 15 days.

• Weekly up to 2 months.

• Monthly up to 6 months

Antral follicle count (AFC) • Weekly up to 2 months

• Monthly up to 6 month.

Previously reported for 12 PR (ESHRE, SRI 2016-2017)

Pick-up & ICSI

PGT-A

IVF

ASCOT

SECONDARY OBJECTIVES PRIMARY OBJECTIVE


High variability among patients AMH response: Two peaks at 15-20 days and 100-120 days

Total AFC significantly increased after 8 to 22 days

(on day 15 p=0.004).

A positive response was observed in 81% of patients considering:

Increase in AFC ≥3foll

Increase in AMH (2 SD) in 2 consecutive determinations

High FGF-2 and THROMBOSPONDIN-1 (THSP-1) values in aphaeresis were associated with improvement in AFC and serum AMH

No difference was detected between the control and infused ovary

PREVIOUSLY REPORTED OBSERVATIONS THAT WERE CONFIRMED WHEN ALL 20 PATIENTS WERE INCLUDED

0

1

2

3

4

5

6

7

8

9

10

0 20 40 60 80 100 120 140 160 180

AM

H (

pm

ol/

L)

Days after ASCOT

PR1

PR2

PR3

PR4

PR5

PR6

PR7

PR8

PR9

PR10

PR11

PR13

PR14

PR15

PR16

PR17

Day after ASCOT AMH(pM) Total AFC

Baseline 1.9±0.6(2.0) 4.0±1.3(4.0)

D+2 2.2±1.7(2.0) 4.6±1.3(4.0)

D+4 2.1±1.8(2.0) 4.9±1.7(5.0)

D+6 1.7±1.4(1.4) 3.0±1.4(3.0)

D+8 1.7±1.5(0.9) 5.2±2.7(5.0)

D+11 1.8±1.4(1.6) 4.9±2.1(5.0)

D+13 1.9±1.5(1.9) 4.7±2.4(4.5)

D+15 2.1±2.1(1.7) 4.9±2.2(5.0) * D+22 2.4±2.6(1.4) 6.0±2.7(6.5)

D+29 1.9±1.4(1.6) 3.9±1.8(4.5)

D+36 2.0±1.6(1.6) 4.3±1.3(4.5)

D+43 1.5±1.1(1.2) 4.0±2.8(3.0)

D+71 1.0±0.7(0.8) 3.4±1.7(4.0)

D+92 2.5±2.5(1.6) 4.4±2.4(4.0)

D+134 2.2±2.6(1.3) 3.3±2.3(2.5)

D+162 2.4±1.7(1.8) 4.9±1.9(5.0)

Positive response

81%

No response

19%

ASCOT success

0

10

20

30

40

50

AFCincrease

W/o AFCincrease

FGF-

2 (

pg/

ml)

**

0

1000

2000

3000

4000

5000

6000

7000

AMH increase W/o AMHincrease

THSP

-1

*

Results: Serum AMH and AFC



Pick-up & ICSI

PGT-A

COS PROTOCOL AND OOCYTE PICK-UP ICSI AND PGT-A

In these firsts attempts:

15 PR completed the IVF cycles, 2 were excluded and 3 are still ongoing.

COS protocol after ASCOT was the same than the used in the previous cycle

ASCOT

IVF study


Results: Cycle comparison and retrieved oocytes

Parameter Previous (n=24)

Post-ASCOT

(All, n=28)

Post-ASCOT optimum

(n=4)

p-value

Patient age (yr) 36 [34-37] 38 [36-38] 34 [33-37] <0.001

Partner age (yr) 36 [35-40] 37 [36-44] 35 [34-38] <0.001

Years of infertility 3[2-4] 4 [3-5] 3[2-3] <0.001

COS cycles 2 [1-2] 2 [2-3] 2[1-2] NS

BMI (%) 24[23-25] 24[21-25] 24[18-25] NS

FSH(IU/ml) 10.1[9.3-14.5] 12.3[9.7-21.5] NA NS

AMH (pM) 1.0 [0.9-3.0] 2.8 [0.7-3.7] 2.9[2.3-4.1] <0.001

Days of stimulation 10[9-12] 9[7-11] 9[9-12] NS

Gonadotrophin dose 3937[2475-4950] 4050[3150-4612] 4100[4050-4950] NS

AFC total - Control ovary - Infused ovary

3[0-5]

2[2-3]

2[1-3]

5[4-6]

3[2-3]

2[1-3]

8[7-9]

5[4-5]

3[2-3]

<0.001 NS

0.02

Punctured follicles -Control ovary -Infused ovary

3[1-6] 0[0-1] 0[0-1]

3[1-4]

2[0-2]

1[0-2]

3[2-4] 2[2-2] 1[0-2]

0.035

MII oocytes -Control ovary -Infused ovary

2[0-3]

0[0-1.2]

0[0-0.2]

2[1-3]

1[0-1]

1[0-2]

2.5[1-4]

1[1-2]

0.5[0-1]

NS

Embryos -Control ovary -Infused ovary

1[0-2]

0[0-0.5]

0[0-0]

1[1-2]

0[0-1]

1[0-1]

1[0-4]

0.5[0-2]

0.5[0-1]

NS

NS

NS

Cancellation 7/24(29.2%) 4/28 (16.6%) 0/4(0%) <0.001

• Optimum COS were considered when started

15 days after ASCOT or following an AFC

increase.

• Values are shown as Median [Inter quartile

range]

• Bonferroni adjustment was applied for multiple

test correction



Results: Individual outcomes

P

a

g

e

2

9

PR Age Cycles MII MII iO MII cO Embryos (E) Euploid E

1 38 4 3 0 3 2 0

3 38 1 3 2 1 3 0

4 38 3 6 2 4 4 1

5 39 2 3 3 0 2 0

6 40 1 1 0 1 0 0

7 39 1 0 0 0 0 0

8 37 3 6 2 4 2 0

9 35 2 8 3 5 6 1

10 38 1 2 2 0 1 0

11 34 2 1 0 1 0 0

13 33 2 3 1 2 3 0

14 38 2 4 2 2 2 0

15 33 1 5 2 3 5 3

16 37 1 1 1 0 1 0

17 36 2 5 4 1 5 0


OVERALL OUTCOMES

*Blastocysts screened aCGH

** + 3 spontaneous pregnancies

IVF outcomes

(n=28 cycles/15PR)

85.7 82.1 67.8

14.3 17.9 32.2

0

20

40

60

80

100

Oocyte pick-up MII EmbryoPositive Negative

PREVIOUS CYCLES ASCOT CYCLES

IVF Cycles 24 28

MII oocytes 35 51

Fertilization rate 69% 75%

Day 3 embryos 24 36 (31*)

Euploid embryos -- 5 (16 %)

Pregnancies 0 2 **

Live birth 0 1**



5 pregnancies in 15 patients (33,3%)

Last PR included have its embryos vitrified because they are still undergoing IVF attempts

Patient

Age Years of infertility

IVF Cycle before ASCOT

IVF Pregnancies

Spontaneous pregnancies

Miscarriages Live Births

4 38 3 2 1 1 1 + 1 0

5 39 4 4 0 1 0 1

9 39 5 2 1 1 0 1 + 1

TOTAL PREGNANCY RATE


ASCOT enhances ovarian reserve in 81% PR patients

ASCOT increases AFC 15 days after procedure. 1st COS attempt should be 2 weeks after ASCOT.

IVF cycle after ASCOT resulted in more follicles developed

The IVF cycle was not specially successful after ASCOT: 16% euploidy, 1 LB

But, a total of 5 pregnancies and 3 live births were achieved after ASCOT, 2 women having 2 pregnancies each.

Conclusions

FUTURE DIRECTIONS: Redefine inclusion criteria: Age<37, target population POI?

Improve ASCOT by using less invasive approaches.


SUMMARY: Ovarian niche rejuvenation by bone marrow derived stem cells

Chemotherapy- induced POI and

PR mouse models

POI PR

OC from PR

patients

Xenograft mouse model: Ovarian cortex

(OC) from PR women

NCT02240342

ASCOT (Autologous Stem Cell Ovarian

Transplantation): Poor Responder patients

Acknowledgements

Dr. Mónica Romeu Dr. Susana Martínez

Dr. Nuria Pellicer Dr. Loida Pamplona

Dr. Inés Gómez Dr. Pilar Solves

Dr .José Martínez Dr. Lourdes Cordón

Prof. Antonio Pellicer Dr. César Díaz-García

Prof. Carlos Simón

Anna Buigues

ovarian rejuvenation by autologous stem cell ovarian transplant...

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