Session 10 Storing ovarian tissue Debate- Slow freezing vs vitrification Vitrification

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The 5 th World Congress of the ISFP Vienna, AUSTRIA 217.11.18 Session 1 Storing ovarian tissue Debate- Slow freezing vs vitrification Vitrification Nao Suzuki, M.D, Ph.D Dept.

1 The 5 th World Congress of the ISFP Vienna, AUSTRIA Session 1 Storing ovarian tissue Debate- Slow freezing vs vitrification Vitrification Nao Suzuki, M.D, Ph.D Dept. of OB & GY, School of Medicine, St. Marianna University COI disclosure Nao Suzuki, MD, PhD Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa, Japan In conjunction with subject announcement, there are not the companies in the COI relations that I should disclose. 1

2 Ovarian tissue cryopreservation Kuwayama et al: Reproductive Bio Medicine Online 25 Ovarian tissue cryopreservation Vitrification Slow freezing Infiltration of CA Slow-freezing by program freezer Transfer to cryotube LN 2 Store at LN2 tank ovarian tissue Infiltration of CA Freezing from out-side and ice crystal formation Cell damage following grow ice crystal of out-side 2

Vitrification Ovarian tissue cryopreservation Infiltration of cryoprotective agent (CA) Setting piece of

solution (VS) LN 2 ovarian tissue Slow freezing Infiltration of CA Infiltration of CA Infiltration of VS

Store at LN2 tank ovarian tissue Infiltration of CA Freezing from out-side and ice crystal formation

3 Vitrification Ovarian tissue cryopreservation Infiltration of cryoprotective agent (CA) Setting piece of ovarian tissue to vitrification device rapid-freezing by LN2 Store at LN2 tank Infiltration of vitrify solution (VS) LN 2 ovarian tissue Slow freezing Infiltration of CA Infiltration of CA Infiltration of VS Rapidly freezing and ice crystal formation Slow-freezing by program freezer Transfer to cryotube LN 2 Store at LN2 tank ovarian tissue Infiltration of CA Freezing from out-side and ice crystal formation Cell damage following grow ice crystal of out-side Slow freezing vs vitrification Reprod BioMed Online, 211 3

ü Toxicity ü Feasibility Penetration of the entire tissue ü

4 Slow freezing vs vitrification Reprod BioMed Online, 211 Suitable Vitirification solutions? ü Toxicity ü Feasibility Penetration of the entire tissue ü Requires good training in the beginning ü Thinner is the best ü Risk in technical expertise Human Reproduction 29 4

Ovarian tissue vitrification Human Reproduction

1min Vitirification EG + DMSO + PrOH + Sucrose

preserved after vitrification than after slow

5 Ovarian tissue vitrification Human Reproduction 29 Vitrification Control 5min Slow freezing 1min Vitirification EG + DMSO + PrOH + Sucrose ü The ovarian stroma was significantly better preserved after vitrification than after slow freezing. ü On the other hand, the follicles were similarly preservaed after all freezing methods. RBM Online, 29 Ovarian tissue vitrification Vit LN2 5

6 Human Reproduction 213 VEG VEG VEG: 25% EG+ 25% Glycerol +.2% PVP +.2% PVA +.4% PG VED: 25% EG+ 25% DMSO +.2% PVP +.2% PVA +.4% PG JARG, 215 Slow freezing vs vitrification 1% DMSO+26% EG+2.5%+PVP +1M sucrose Methods of OTC Fresh ovarian tissue Slow freezing Vitrification vs Grafting sites A healed bed Freshly decorted bed vs 6

7 Slow freezing vs vitrification JARG, 215 Follicle stage (%) Proliferation rate (%) vitrified tissue grafted to a freshly decorticated vascular bed vitrified tissue grafted to a freshly decorticated vascular bed Slow freezing vs vitrification JARG, 215 Follicle stage (%) Proliferation rate (%) The best combination of treatments, cryopreser vation, and graft site vitrified tissue grafted to a vitrified tissue grafted to a preparation freshly was decorticated vitrification vascular and grafting to freshly the decorticated freshly decorted vascular bed. bed bed 7

5M sucrose VS2: 1% EG+1% DMSO+1% PrOH+1%PVP Slow freezing vs vitrification Fertility and Sterility, 216 Histologic evaluation and follicular

8 Fertility and Sterility, 216 Slow freezing vs vitrification SF SF: ethyl vinyl acetate bag vs Vit VT1: VS1 and metaric grids VT2: VS2 and metaric grids VT3: VS1 and ethyl vinyl acetate bag VT4: VS2 and ethyl vinyl acetate bag VS1: 2% EG+2% DMSO+.5M sucrose VS2: 1% EG+1% DMSO+1% PrOH+1%PVP Slow freezing vs vitrification Fertility and Sterility, 216 Histologic evaluation and follicular population In vitro study control SF: ethyl vinyl acetate bag VT1: VS1 and metaric grids VT2: VS2 and metaric grids VT3: VS1 and ethyl vinyl acetate bag VT4: VS2 and ethyl vinyl acetate bag 8

5M sucrose Slow freezing vs vitrification Fertility and Sterility, 216 Tissue viability Histologic evaluation and

9 Slow freezing vs vitrification Fertility and Sterility, 216 Cell death Histologic evaluation and follicular population In vitro study VT1: VS1 and metaric grids VS1: 2% EG+2% DMSO+.5M sucrose Slow freezing vs vitrification Fertility and Sterility, 216 Tissue viability Histologic evaluation and follicular population In vitro study VT2: VS2 and metalic grids The combination of a metallic grid with the VT1 solution obtained significantly better results than the other experimental groups. 9

Slow freezing vs vitrification Fertility and Sterility, 216 PF density In

DMSO, sucrose, and SSS does not affect the normal morphology of oocytes,

Ovarian tissue vitrifiation offers greater efficiency than slow freezing.

10 Slow freezing vs vitrification Fertility and Sterility, 216 PF density In vivo study fresh SF VT1 Fibotic area SF Human ovarian tissue using EG, DMSO, sucrose, and SSS does not affect the normal morphology of oocytes, follicles, and blood vessel distribution after transplantation. Ovarian tissue vitrifiation offers greater efficiency than slow freezing. VT1 Ovarian tissue vitrification Pre-clinical study: since M (35% v/v) EG + 5% PVP +.5 M sucrose 5min Human Reproduction 212 Collaborative research with Dr. Shu Hashimoto and Dr. Yoshiharu Morimoto (IVF Namba Clinic, Osaka, Japan) 1

Ovarian tissue vitrification & TP: 21.1-217.

others 4 2 Pts TP POI 193 153 Total 28 156 Pts.

for the POI patient Patient 1 2 3 4 5 6 7 Onset of POI AMH Age when IVA was done Pregnancy outcome 23y 33y 38y 29y 31y 28y 38y 4.9 pmol.4-.

11 Ovarian tissue vitrification & TP: Disease Pts OTC Pts OTC Breast Cancer (BC) 64 3 SLE 4 Uterine cervical cancer (SCC) Ewing sarcoma 1 87 Leukemia 5 Malignant Lymphoma 4 MDS 3 others 4 2 Pts TP POI Total Pts.: Number of patients, OTC: ovarian tissue cryopreservation, TP: transplantation Ovarian tissue vitrification: application to the new treatment for the POI patient Patient Onset of POI AMH Age when IVA was done Pregnancy outcome 23y 33y 38y 29y 31y 28y 38y 4.9 pmol ng/ml <.1 ng/ml <.1 ng/ml <.16 ng/ml <.1 ng/ml <.1 ng/ml 25y 33y 4y 31y 36y 3y 4y Live birth Live birth Chemical abortion Abortion Abortion Live birth Abortion Three live-births were achieved using vitrified-thawed ovarian tissue. Kawamura K, Suzuki N et al: PNAS 213 Suzuki N, et al. Human Reproduction

12 Research Grant Program on the survey for children and child care promotion on the Ministry of Health, Labor and Welfare in Japan Research about the effectiveness of Oncofertility treatments for the CAYA cancer patients By Dr. Seido Takae(St. Marianna University) and Nao Suzuki (PI) The number of OTC procedures in Japan 21 in total as of Research Grant Program on the survey for children and child care promotion on the Ministry of Health, Labor and Welfare in Japan Research about the effectiveness of Oncofertility treatments for the CAYA cancer patients By Dr. Seido Takae(St. Marianna University) and Nao Suzuki (PI) The number of OTC procedures according to the disease in Japan 1. Breast Cancer: Luekemia: Malignant Lymphoma: Soft tissue tumor(sarcoma): 8 5. Brain tumor: 7 12

Patients with breast cancer who underwent ovarian tissue transplantaion Breast Cancer(Luminal type): 39yo (at the time of a diagnosis) GP

1~: NAC(EC DTX) cpr 21X+1.3 Mastectomy + armpit lymph node dissection yct2n1m;ycstageⅡb, Luminal type 21X+1.

8 miu/ml, E2: <25 pg/ml, AMH: <.1ng/ml pst-surgery Menstrual cycle improvement (pre:85.±19.7days intervals post:41.6±17.4days intervals), AMH:.

13 Patients with breast cancer who underwent ovarian tissue transplantaion Breast Cancer(Luminal type): 39yo (at the time of a diagnosis) GP unmarried <FH> n.p. 21X.7: Diagnosis of breast cancer ct2n1m cstageⅡb Luminal type 21X.9: Marriage ovayectomy and OTC, AMH: 28.4pMol/L 21X.1~: NAC(EC DTX) cpr 21X+1.3 Mastectomy + armpit lymph node dissection yct2n1m;ycstageⅡb, Luminal type 21X+1.4~ Hormonal therapy (TAM+GnRHa:TAM: until 21X+5.2) 21X+5.9:44yo, Laproscopic ovarian tissue transplantation pre-surgery FSH: 59.8 miu/ml, E2: <25 pg/ml, AMH: <.1ng/ml pst-surgery Menstrual cycle improvement (pre:85.±19.7days intervals post:41.6±17.4days intervals), AMH:.2ng/ml, egg collection: two times but no eggs Ovarian Tissue Vitirification: New closed-type device: Cryosheet open-type closed-type 1 closed-type 2 Cooling Speed: -19,5 /min closed-type 3 New closed-type device: Cryosheet 13

Ovarian Tissue Vitirification: New closed-type device: Cryosheet

device (closed-type 2) The distance between ovarian tissue and liquid

LN 2 Ova tissue Improved device (closedtype 3: Cryosheet) Freezing

14 Ovarian Tissue Vitirification: New closed-type device: Cryosheet Improving the cooling speed Problem and improvement Conventional device (closed-type 2) The distance between ovarian tissue and liquid nitrogen is too great, therefore freezing is slow (<1, C/minute) Air LN 2 Ova tissue Improved device (closedtype 3: Cryosheet) Freezing could be speeded up by making the distance between ovarian tissue and liquid nitrogen shorter. LN 2 Ova tissue Ovarian Tissue Vitirification: New closed-type device: Cryosheet The sealed pouch Cryosheet in the bag Vitrification! 14

(Cryosheet):42, /min Ovarian Tissue Vitirification: New closed-type device: Cryosheet Donor ovary Vitrification After 2-3 weeks orthotopic

15 Ovarian Tissue Vitirification: New closed-type device: Cryosheet Cooling Speed Cryo Device TypeM:-19,5 /min Closed-type 2:-13, /min Closed-type 3 (Cryosheet):- 12, /min Temperature[ ] Time[s] 2 3 Heating Speed Cryo Device TypeM: 42, /min Closed-type 3 (Cryosheet):42, /min Ovarian Tissue Vitirification: New closed-type device: Cryosheet Donor ovary Vitrification After 2-3 weeks orthotopic implantation (2 piece / mouse) Green mouse C57BL/6J -Tg(CAG-GFP) Half cut Wild type mouse Control (Fresh implantation) C57BL/6J In Vivo fertility assay 15

Ovarian Tissue Vitirification: New closed-type device: Cryosheet Results : In

8 6 4 2 control (n=7) ns vitrification (n=1) Bearing / Mating (%) 8 6 4 2

birth ns control vitrification (n=5) (n=6) Ovarian Tissue Vitirification: New

4 1 uterus Area 1,4: Mesosalpinx 2,3: Omentum Type M-II Device ü 5μm Titanium

16 Ovarian Tissue Vitirification: New closed-type device: Cryosheet Results : In Vivo fertility assay Average of days The mean days until estrous cycle recovery control (n=7) ns vitrification (n=1) Bearing / Mating (%) Birthrate 5/6 6/1 control vitrification Average of live birth Live birth ns control vitrification (n=5) (n=6) Ovarian Tissue Vitirification: New closed-type device: Cryosheet cynomolgus monkeys: n=6 2 Omentum 3 Ovarian Tissue 4 1 uterus Area 1,4: Mesosalpinx 2,3: Omentum Type M-II Device ü 5μm Titanium device ü 8μm Titanium device Size and Shape ü Small Pieces 1,2: Right side: ü Sheet 2,3: Left side: 16

PrototypeⅡ (Titanium) 5μm device 3days 35 3 25 2 15 1 5 No.

Estradiol 5μm device 6days 35 12 3 1 25 8 2 6 15 4 1 2 5 No.

Cryo-Thawing Inplantation 5μm device 12days 35 Progesteron 3 No.

18 14days Hyperstimulation IVF Progesteron Estradiol 12 1 8 6 4 2 Cryo-Thawing Inplantation

17 PrototypeⅡ (Titanium) 5μm device 3days No.17 Ovarian Tissue Vitirification: New closed-type device: Cryosheet Progesteron Hyperstimulation IVF Estradiol 5μm device 6days No.14 Hyperstimulation IVF Estradiol Progesteron Cryo-Thawing Inplantation Cryo-Thawing Inplantation 5μm device 12days 35 Progesteron 3 No.15 Estradiol 25 Hyperstimulation IVF μm device No.18 14days Hyperstimulation IVF Progesteron Estradiol Cryo-Thawing Inplantation Cryo-Thawing Inplantation Ovarian Tissue Vitirification: New closed-type device: Cryosheet Retrieved Oocytes Mesosalpinx :Oocyte Number=11 Omentum :Oocyte Number=5 Sheet ovarian tissue :Oocyte Number=8 Pieces ovarian tissue :Oocyte Number=8 17

18 Ovarian Tissue Vitirification: New closed-type device: Cryosheet No18 Omentum :Gill s Haematoxylin stained Transplanted Ovarian tissue Oocytes Omentum Ovarian Tissue Vitirification: New closed-type device: Cryosheet No21 Omentum : Transmission-electron microscopy No Ice formation Control (non-frozen) Vitrified Thawed Tissue 18

New closed-type device for ovarian tissue

cryopreservation: Sf vs Vit Donor case1:39years Donor

Hart Attack BrooklynHospital Center White/ Unknown

bleeding NYC Health Hospitals Jacobi Sf Slow freezing

sulfoxide+human Serum Albumin Vitrification Vit

19 New closed-type device for ovarian tissue vitrification: Cryosheet Human ovarian tissue cryopreservation: Sf vs Vit Donor case1:39years Donor case2:23years Donor case3:31years African American/ Hart Attack BrooklynHospital Center White/ Unknown Brookhaven Memorial Hospital White/ intracranial bleeding NYC Health Hospitals Jacobi Sf Slow freezing Slow Freezing Solution MEMα+Sucrose+Dimethyl sulfoxide+human Serum Albumin Vitrification Vit Vitrification Solution H199 +Sucrose+Ethylene Glycol +Polyvinylpyrrolidone+Synthetic Serum Substitute 19

20 Human ovarian tissue cryopreservation: Sf vs Vit Sf Summary of Study Design Human Ovaries Vit Thawing Culture Freezing Embedding Sectioning & Staining Follicle Counting Human ovarian tissue cryopreservation: OVf vs CVit Vit vs 2

Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primordial Follicle Density, DNA DSBs and

Control Slow Freezing Vitrification Control vs. SF Control vs. VF SF vs. VF Primordial follicle Density (/mm3) 134.

5±2.3 2.1±2.7 12.8±3.1.3.12.6 Non-apoptotic Primordial follicle density (/mm3) 112.6±27.5 52.5±17.2 54.

66 Apoptosis 18 16 14 Primordial follicle Density (/mm3) * * 16 14 Non-apoptotic Primordial Density (/mm3) * γh2ax+

21 Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primordial Follicle Density, DNA DSBs and Apoptosis γh2ax positive : DNA double-strand-breaks AC3 positive : Apoptotic follicles Positive Negative H2AX H2AX AC3 AC3 Control Slow Freezing Vitrification Control vs. SF Control vs. VF SF vs. VF Primordial follicle Density (/mm3) 134.6±25 64±17 62± γh2ax+ Primordial follicle (%) 27.3± ± ± AC3+ Primordial follicle (%) 17.5± ± ± Non-apoptotic Primordial follicle density (/mm3) 112.6± ± ± Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primordial Follicle Density, DNA DSBs and Apoptosis Primordial follicle Density (/mm3) * * Non-apoptotic Primordial Density (/mm3) * γh2ax+ Primordial follicle (%) NS AC3+ Primordial follicle (%) NS NS 1 NS * : Significant NS: Non Significant 21

Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primary Follicle Density, DNA DSBs and

AC3 Control Slow Freezing Vitrification Control vs. SF Control vs. VF SF vs. VF Primary follicle Density (/mm3) 14.6±4.

16 Apoptosis 2 18 Primary follicle Density (/mm3) 25 Non-apoptotic Primary Density (/mm3) 5 γh2ax+ Primary follicle (%)

22 Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primary Follicle Density, DNA DSBs and Apoptosis γh2ax positive : DNA double-strand-breaks AC3 positive : Apoptotic follicles Positive Negative H2AX H2AX AC3 AC3 Control Slow Freezing Vitrification Control vs. SF Control vs. VF SF vs. VF Primary follicle Density (/mm3) 14.6± ±2 6.6± γh2ax+ Primary follicle (%) 11.6± ±12.8 3± AC3+ Primary follicle (%) 5.8± ±3.7 26± Non-apoptotic Primary follicle density (/mm3) 14.1±5 6.2± ± Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primary Follicle Density, DNA DSBs and Apoptosis 2 18 Primary follicle Density (/mm3) 25 Non-apoptotic Primary Density (/mm3) 5 γh2ax+ Primary follicle (%) NS 45 AC3+ Primary follicle (%) NS NS NS * * : Significant NS: Non Significant 22

Human ovarian tissue cryopreservation: Sf vs Vit Summary and Conclusion Impact of Vitrification vs SF on Follicle Density, DNA DSBs and Apoptosis ü Similar follicle density Sf ü Similar DNA DSB

Human ovarian tissue cryopreservation: Sf vs Vit A Collaborative research with New York Medical Collage and Yale University to improve the ovarian tissue cryopreservation method Human Ovar ies

23 Human ovarian tissue cryopreservation: Sf vs Vit Summary and Conclusion Impact of Vitrification vs SF on Follicle Density, DNA DSBs and Apoptosis ü Similar follicle density Sf ü Similar DNA DSB Breaks ü Similar apoptotic activation Vit Based on histological and immunohistochemical evaluation, Sf and Vit methods appear to be comparable. Human ovarian tissue cryopreservation: Sf vs Vit A Collaborative research with New York Medical Collage and Yale University to improve the ovarian tissue cryopreservation method Human Ovar ies Thawing Culture Embedding Sectioning & Staining γh2ax and AC3+ Follicle Counting Freezing Now on going Data analysis RNA-sequence (NextSeq 55 illumine) cdna amplification (Nextra XT Library prep kit) RNA extraction Reversetranscription Lazer Captured Microdicection 23

24 Slow freezing vs vitrification Scientific Reports, 217 Sf vs Vit Slow freezing vs vitrification Scientific Reports, % CI:.97 [ ] Sf Vit 24

25 Slow freezing vs vitrification Scientific Reports, % CI:.71 [.62-.8] Vit Sf Slow freezing vs vitrification Scientific Reports, % CI: 1.69 [ ] Sf Vit Sf 25

Slow freezing vs vitrification Scientific Reports, 217 95% CI: 3.44 [-5.9-11.

26 Slow freezing vs vitrification Scientific Reports, % CI: 3.44 [ ] Sf Vit Slow freezing vs vitrification Scientific Reports, 217 Vit LN2 less primordial follicular DNA strand breaks and better preservation of stromal cells. 26

Device Future: Ovarian tissue vitrification Vitrification? PrOH Glycerol EG PVA DMSO Trehalose PVP Sucrose Solution LN2 Vitrificaion of human ovarian tissue?? PROS 1.

Excellent survival in tissue culture CONS 1. No live births reported so far 2. Requires good training in the beginning 3.

27 Device Future: Ovarian tissue vitrification Vitrification? PrOH Glycerol EG PVA DMSO Trehalose PVP Sucrose Solution LN2 Vitrificaion of human ovarian tissue?? PROS 1. Excellent survival of ovarian stroma and blood vessels 2. A fast method 3. No particular apparatuses needed 4. Also feasible clinically Ovarian tissue cryopreservation 5. Excellent survival in tissue culture CONS 1. No live births reported so far 2. Requires good training in the beginning 3. Concentrations and osmolarities and the speed of the procedure are of extremely high importance 4.Toxic influence on the tissue is possible if less trained personnel Dr. Hovatta, World Congress on Fertility Preservation 29 in Belgium 27

28 Vitrificaion of human ovarian tissue?? PROS 1. Excellent survival of ovarian stroma and blood vessels 2. A fast method 3. No particular apparatuses needed 4. Also feasible clinically Ovarian tissue cryopreservation 5. Excellent survival in tissue culture CONS 1. No live births reported so far 2. Requires good training in the beginning 3. Concentrations and osmolarities and the speed of the procedure are of extremely high importance 4.Toxic influence on the tissue is possible if less trained personnel Dr. Hovatta, World Congress on Fertility Preservation 29 in Belgium Vitrificaion of human ovarian tissue?? PROS 1. Excellent survival of ovarian stroma and blood vessels 2. A fast method 3. No particular apparatuses needed 4. Also feasible clinically Ovarian tissue cryopreservation 5. Excellent survival in tissue culture CONS 1. No live births reported so far(at that time) 2. Requires good training in the beginning 3. Concentrations and osmolarities and the speed of the procedure are of extremely high importance 4.Toxic influence on the tissue is possible if less trained personnel Dr. Hovatta, World Congress on Fertility Preservation 29 in Belgium 28

29 216- ASFP (Asian Society for Fertility Preservation): countries 215 ESHRE The 1 st Congress of ASFP; Ho Chi Minh City, Vietnam 29

30 The 1 st Workshop and Symposium of ASFP; Shanghai, China Hands on seminar: Ovarian tissue vitrification At St. Marianna University, Japan: w/member of Seoul Natl Univ In India: Fertility Preservation Society (India) New Deli, India At St. Marianna University, Japan: w/ Dr. Kemar, Dr. Mila And Dr. Kock In Indonesia: Reproductive Medicine Society Medan, Indonesia Hands on seminar and Ovariectomy and ovarian tissue vitrification for 2 young canc er patients. Almaty, Republic of Kazakhstan At St. Marianna University, Japan: w/ Dr. Novero s team and Dr. Lee

Hands on seminar: Ovarian tissue vitrification In Philippines: OVARIAN TISSUE CRYOPRESERVATION POST GRADUATE

Marianna University, Japan: w/ Member of Dr. Sirayapiwat and Ms. Numchaisrika 216.3.

7 PROS of ovarian tissue vitrification are a fast method and no particular apparatuses needed.

Lee Ovariectomy and ovarian tissue vitrification for 2 POI patients. Shanghai, China. 216.11.14 At St.

27 Take home message u Based on several data, Sf and Vit methods appear to be comparable.

31 Hands on seminar: Ovarian tissue vitrification In Philippines: OVARIAN TISSUE CRYOPRESERVATION POST GRADUATE COURSE AND WORKSHOP. w/ Member of Dr. Novero s team Manila, Philippines At St. Marianna University, Japan: w/ Member of Dr. Sirayapiwat and Ms. Numchaisrika ASPIRE216, Jakarta, Indonesia PROS of ovarian tissue vitrification are a fast method and no particular apparatuses needed. It is easily handled for anyone at anywhere in anytime at 第二军医大学 Pr. Lee Ovariectomy and ovarian tissue vitrification for 2 POI patients. Shanghai, China At St. Marianna University, Japan: w/ Professor Lee s team from China Take home message u Based on several data, Sf and Vit methods appear to be comparable. u Ovarian tissue vitrification may be a promising new fertility preservation method for young cancer patients. ü Excellent survival of ovarian stroma and blood vessels ü A fast method ü No particular apparatuses needed ü Also feasible clinically ü Excellent survival in tissue culture ü It is easily handled for anyone at anywhere in anytime!! 31

Nao Suzuki, M.D, Ph.D Dept. of OB & GY, School of Medicine, St. Marianna University

The 5 th World Congress of the ISFP Vienna, AUSTRIA 2017.11.18 Session 10 Storing ovarian tissue Debate- Slow freezing vs vitrification Vitrification Nao Suzuki, M.D, Ph.D Dept. of OB & GY, School of Medicine,