Ovarian rejuvenation by Autologous stem cell ovarian transplant (ASCOT). Sonia Herraiz, PhD IVI, Spain Sonia.Herraiz@ivirma.com Paris, November 2, 2018 Disclosure information: Nothing to declare
Depletion of the ovarian reserve: follicles and AMH AMH Impairment of ovarian function Ovarian Aging Diminished ovarian reserve or poor ovarian response (PR) by non-determined causes Therapeutic alternatives Remaining pool of dormant follicles Acute premature ovarian insufficiency (POI) due to chemotherapy (ChT) Visser et al., Nature Reviews Endocrinology 2012. P a g e
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) Page 4
OUR EXPERIENCE DO IT AGAIN RCT
Modification of the original IVA technique Avoid IVA drugs. Kawamura et al.,2013 Minimize cryodamage/ischemia. Look for the best vascularized site. Allow for natural pregnancy 6
Ovarian fragmentation for follicular activation (OFFA) PI: César Díaz-García IVA OFFA Drug free Unique surgery No tissue cryopreservation On site transplant
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). Page 9
Fertility recover after bone marrow transplant Sterility after Chemoand radiotherapy Fertility restoration after BMT 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.
Bone marrow derived stem cells (BMDSC) BMDSC represent a heterogeneous group of mononuclear cells, including several hematopoietic and stromal stem/progenitor cells. Hematopoietic stem cell Stromal stem cell Astori et al. Am J Transl Res. 2010.
Ovarian rejuvenation by bone marrow derived stem cells Objectives and End points questions 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
14d 7d Experimental design 1 Cell labeling with Molday Ion Rhodamine B (MIRB) POI model Standard regimen: 120 mg/kg Cy 12 mg/kg Bu PR model Reduced regimen: 12 mg/kg Cy 1.2 mg/kg Bu Cy: Cyclophosphamide Bu: Busulphan MIRB nanoparticles MIRB labeled-human cells 18 NOD/SCID mice 18 NOD/SCID mice Control Group PBMNC Group BMDSC Group 100 μl of saline 1x10 6 human PBMNC 1x10 6 human BMDSC Ovarian hyperstimulation and mating Short-term effects Consecutive matings for 3 months Estrous cycle, ovaries, oocytes/embryos Long-term effects
POI model PR-model No-ChT Estrous cycle 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 Proestrous (P) Estrous (E) Metestrous (M) Diestrous (D) ChT Cell infusion P E M D Control PBMNC X X BMDSC Control X PBMNC X BMDSC Herraiz et al. Fertil Steril 2018;109:908 18
Litter size (n) % Pre-ovulatory follicles MII ovulated oocytes (n) 2-cell embryos Follicles, oocytes, embryos and offspring 2.5 2 1.5 1 0.5 0 PR * POI * 40 30 20 10 0 PR * POI 12 10 8 6 4 2 0 PR POI * Control PBMNC BMDSC BMDSCs infusion increased the number of preovulatory follicles, MII-ovulated oocytes and embryos. 14 POI-BMDSC PR-BMDSC 12 10 8 PR-BMDSC 6 4 POI-BMDSC PR-PBMNC First offspring Second offspring 2 PR-Control 0 1st offspring 2nd offspring 3rd offspring POI-Control POI-PBMNC Restored long term fertility Herraiz et al. Fertil Steril 2018;109:908 18
Ovarian stroma regeneration BMDSC infusion: Increase ovarian vascularization Improve cell proliferation Reduce apoptosis in stroma and follicles even in the high chemotherapy dose. Herraiz et al. Fertil Steril 2018;109:908 18
End points- Questions 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?
Experimental design 2: human ovarian tissue Xenograft Cell infusion Tissue sampling and analysis human ovarian cortex (OC) from Poor Responder (PR) women Control 100 μl of saline OC grafts Cell engraftment Follicular density and growth Vascularization and proliferation of ovarian stroma 7D BMDSC 1x10 6 human BMDSC Blood Estradiol secretion Ovariectomized NOD-SCID mice BMDSC Positive selection of their CD133+ cell fraction CD133+ 3x10 5 human CD133 Other organs Cell tracking D-7 D0 D+1 / D+7 / D+14
Prussian Blue BMDSC: human ovarian tissue Control BMDSC CD133+ Injected cells localized near blood vessels and close to follicles, in contact with GC. follicular density (follicles/mm3) 10 9 8 7 6 5 4 3 2 1 0 Secondary Primary Primordial CONTROL BMDSC CD133 CONTROL BMDSC CD133 CONTROL BMDSC CD133 Stem cell infusion promoted growth of human follicles D1 D7 D14 Secondary follicles were only observed in OC grafts receiving cell injection Herraiz et al. Fertil Steril 2018;109:908 18
Serum E2 (pg/ml) BMDSC: human ovarian tissue Control BMDSC CD133+ In human OC BMDSC Improved ovarian vascularization Increased cell proliferation 30 CONTROL BMDSC CD133 Blood samples Serum ELISA 17b estradiol 20 10 0 * Promoted estradiol secretion Herraiz et al. Fertil Steril 2018;109:908 18 Page 20
Are we recovering the ovarian function? 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. BMDSC-based therapies could be a suitable alternative to increase the reproductive potential of patients with impaired ovarian function
Next step: 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?
Study design 3: PR women 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
Autologous Stem Cell Ovarian Transplantation (ASCOT) 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). Clinical Trial Nº: NCT02240342 Herraiz et al. Fertil Steril 2018;110(3):496-505 Page 24
Study design: patient follow-up and IVF PRIMARY OBJECTIVE 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. SECONDARY OBJECTIVES ASCOT IVF PGT-A Pick-up & ICSI Previously reported for 12 PR (ESHRE, SRI 2016-2017) Page 25
AMH (pmol/l) FGF-2 (pg/ml) THSP-1 Results: Serum AMH and AFC PREVIOUSLY REPORTED OBSERVATIONS THAT WERE CONFIRMED WHEN ALL 20 PATIENTS WERE INCLUDED ** 50 7000 High variability among patients AMH response: Two peaks Day at after 15-20 ASCOT days and AMH(pM) 100-120 days Total AFC 40 6000 * Total AFC 10 significantly increased after 8 to 22 5000 Baseline 1.9±0.6(2.0) 4.0±1.3(4.0) 30 days 4000 D+2 PR12.2±1.7(2.0) 4.6±1.3(4.0) (on day 15 p=0.004). 9 PR2 20 3000 D+4 2.1±1.8(2.0) 4.9±1.7(5.0) PR3 8 A positive response was observed in 81% of patients considering: D+6 ASCOT 1.7±1.4(1.4) success 10 2000 3.0±1.4(3.0) PR4 7 1000 D+8 1.7±1.5(0.9) 5.2±2.7(5.0) PR5 Increase in AFC 3foll 0 No 6 0 D+11 PR61.8±1.4(1.6) 4.9±2.1(5.0) AFC W/o AFC response AMH increase W/o AMH PR7 Increase 5in AMH (2 SD) in increase 2 consecutive increase increase D+13 1.9±1.5(1.9) determinations 19% 4.7±2.4(4.5) PR8 D+15 2.1±2.1(1.7) 4.9±2.2(5.0) * PR9 High FGF-2 and 4 THROMBOSPONDIN-1 (THSP-1) values in aphaeresis were D+22 associated with improvement in AFC PR10 2.4±2.6(1.4) 6.0±2.7(6.5) and serum AMH 3 Positive D+29 PR11.9±1.4(1.6) 3.9±1.8(4.5) PR13 2 D+36 2.0±1.6(1.6) response 4.3±1.3(4.5) No difference was detected between the control and infused ovary PR14 1 D+43 1.5±1.1(1.2) 81% 4.0±2.8(3.0) PR15 D+71 PR16 1.0±0.7(0.8) 3.4±1.7(4.0) 0 PR17 0 20 40 60 80 100 120 140 D+92 160 180 2.5±2.5(1.6) 4.4±2.4(4.0) Days after ASCOT 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) Herraiz et al. Fertil Steril 2018;110(3):496-505
IVF study COS PROTOCOL AND OOCYTE PICK-UP ICSI AND PGT-A PGT-A ASCOT Pick-up & ICSI 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
Results: Cycle comparison and retrieved oocytes Parameter -Control ovary 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 Punctured follicles -Control ovary -Infused ovary MII oocytes -Control ovary -Infused ovary Embryos -Infused ovary 3[0-5] 2[2-3] 2[1-3] 3[1-6] 0[0-1] 0[0-1] 2[0-3] 0[0-1.2] 0[0-0.2] 1[0-2] 0[0-0.5] 0[0-0] 5[4-6] 3[2-3] 2[1-3] 3[1-4] 2[0-2] 1[0-2] 2[1-3] 1[0-1] 1[0-2] 1[1-2] 0[0-1] 1[0-1] 8[7-9] 5[4-5] 3[2-3] 3[2-4] 2[2-2] 1[0-2] 2.5[1-4] 1[1-2] 0.5[0-1] 1[0-4] 0.5[0-2] 0.5[0-1] <0.001 NS 0.02 Cancellation 7/24(29.2%) 4/28 (16.6%) 0/4(0%) <0.001 0.035 NS NS NS NS 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 Herraiz et al. Fertil Steril 2018;110(3):496-505 Page 28
Results: Individual outcomes 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 P a g e
OVERALL OUTCOMES 100 80 60 40 20 0 IVF outcomes (n=28 cycles/15pr) 14.3 17.9 85.7 82.1 32.2 67.8 Oocyte pick-up MII Embryo Positive Negative PREVIOUS CYCLES *Blastocysts screened acgh ** + 3 spontaneous pregnancies 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** Herraiz et al. Fertil Steril 2018;110(3):496-505 Page 30
TOTAL PREGNANCY RATE Patien t Age Years of infertility IVF Cycle before ASCOT IVF Pregnancies Spontaneous pregnancies Miscarriages 4 38 3 2 1 1 1 + 1 0 5 39 4 4 0 1 0 1 Live Births 9 39 5 2 1 1 0 1 + 1 5 pregnancies in 15 patients (33,3%) Last PR included have its embryos vitrified because they are still undergoing IVF attempts
Conclusions 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. 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 Xenograft mouse model: Ovarian cortex (OC) from PR women ASCOT (Autologous Stem Cell Ovarian Transplantation): Poor Responder patients OC from PR patients NCT02240342 POI PR
Acknowledgements Prof. Antonio Pellicer Dr. César Díaz-García Prof. Carlos Simón Anna Buigues Dr. Mónica Romeu Dr. Susana Martínez Dr. Nuria Pellicer Dr. Loida Pamplona Dr. Inés Gómez Dr. Pilar Solves Dr.José Martínez Dr. Lourdes Cordón