Vitrification of Oocytes: Biological Lessons Learned From Mice, Applied to Women

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1 ESHRE Cryobiology Mtg Athens, Greece 9/26/09 Vitrification of Oocytes: Biological Lessons Learned From Mice, Applied to Women Gary D. Smith Ph.D., HCLD Associate Professor Director of Reproductive Sciences Program Departments of OB/GYN, Physiology, and Urology

2 Utility of Oocyte Cryopreservation 1) Preserving fertility in cancer patients and/or women undergoing oophorectomy. 2) Ethical / moral / social concerns with embryo cryopreservation. 3) Salvaging interrupted IVF cycles. 4) Oocyte banking in anticipation of reproduction at an advanced maternal age. - Slow-rate freezing - Vitrification

3 Cryo-Damage and Subsequent Oocyte Function Germinal vesicleintact (immature) Mechanical Thermal Metaphase II (mature) Chemical Veeck, 1999 BIG CELLS Lot of responsibilities 1) Spindle Formation / Function 2) Microfilament Function 3) Zona Pellucida

4 Recovery / Survival of Vitrified Mouse MII Oocytes (%) Oocytes / Straw Vitrification (n=40) 10 Oocytes / Straw Vitrification (n=1040) re Vitrification Morphology Percent Recovery Post-Warming Survival Post-Warming Befor After Vitrification

5 Influence of Vitrification and Time on Metaphase II Spindle Morphology 24/24 (100% a ) 8/39 (21% b ) 49/49 (100%) 24/24 (100%) a,b P<0.01 Gomes et al., Fertil & Steril, 2008

6 Polscope View of Spindle Assembly After Vitrification / Warming Gomes et al., Fertil & Steril, 2008

Does Vitrification Affect Spindle Morphology (Length)? Strict Criteria: 21.54 µm No measurement 30.

7 Does Vitrification Affect Spindle Morphology (Length)? Strict Criteria: µm No measurement µm Spindle Length (microns) NS n=30 NS n=17 NS n=32 Measurement 0 Control Solution Treatment Vitrified

spindle Normal polar body Abnormal * polar body Abnormal Chromatin Alignment Control (no

8 Does Vitrification Cause Aberrant Spindle Chromatin Alignment? Vitrified Warmed MII Oocytes Fix Oocytes Stain Chromatin Solution Exposure MII spindle MII spindle Normal polar body Abnormal * polar body Abnormal Chromatin Alignment Control (no vitrification) 1/37 = 2.7% a Solution Exposure Only 1/47 = 2.1% a Vitrification 3/49 = 6.1% a a P=0.63 Gomes et al., Fertil & Steril, 2008

9 Spindles and Cryopreservation The Bottom Line: 1) All three forces are detrimental - Mechanical (Ice crystals) - Thermal (Microtubule depolymerization) - Chemical (DMSO & PROP) 2) Debate on which meiotic stage most susceptible to damage 3) Remember microtubules are in dynamic flux - polymerization / depolymerization Today: greatest success is cryopreservation of MII oocytes

10 Oocyte Vitrification and Fertilization By Standard Insemination 24h Post-inse emination Cleavage e (%) a n=233 b n=115 b n=88 ICM (blue) Control 0 Standard Insem Oakes et al., Fertil & Steril, submitted Vitr/Warmed Standard Insem Vitr/Warmed No Insem (Parthenotes) Vitrified

11 Why Do Vitrified Oocytes Not Fertilize? MII Oocytes Vitrified Solution Exposure N=55 Vitrified a Warmed N=55 N=55 Vitrification Solution Exposure b Pronase Digestion Control (No Exposure / No Vitrification) b Zona Pellucida Solubility (sec) Oakes et al., Fertil & Steril, submitted a,b P< Control Normalized Zona Pellucida Dissolution (seconds)

12 Does Vitrification Induce Premature Cortical Granule Release?

13 Can Fetuin in Cryo-Media Influence Zona Pellucida Modifications? Zona Pellucida Dissolution (second ds) a a n=45 n=41 Control Oakes et al., Fertil & Steril, submitted Solution Exposure b n=42 Vitr/W (- Fetuin) a n=42 Vitr/W (+ Fetuin) a,b P<0.05

14 Mouse Oocyte Vitrification, ICSI, Fertilization, and Live-Births Oocytes Fertilized (% of injected) a N=89 Oakes et al., Fertil & Steril, submitted a N=75 Contr Vitr Contr Vitr a N=83 a N= Fetuses Born (% of transferred) a,b P<0.05

15 Oocyte Cryopreservation 1) Slow-rate freezing can work - 50 to 76% survival ~13 to 35% pregnancy rate/transfer (Porcu et al., 2000, Mol & Cell Endo) (Boldt et al., 2006, Reprod Biomed Online) (Bianchi et al., 2006, Reprod Biomed Online) 2) Vitrification current area of focus - two primary factors to consider for success a - vitrification means (container and coolant) b - solutions

16 Vitrification Containers What makes a good vitrification container? 1) Low volume of cryo-solution - rapid heat transfer - fastest cryopreservation / warming - no ice crystal formation 2) User friendly 3) Security EM Grids - Martino et al., 1996, Biol Reprod, Vol 54 Open Pulled Straws (OPS) - Vajta et al., 1998, Mol Reprod & Dev, Vol 51 Cryoloops - Lane et al., 1999, Fertil & Steril, Vol 72 Cryotop - Katayama et al., 2003, Fertil & Steril, Vol 80 CryoTip - Kuwayama and Smith

17 Closed Pulled Straw System 2 nd nd mark 1 st mark 40X - low volume ~ 0.4 µl (inner diam. = 200 µm) - secure and protected (heat-sealed at both ends)

18 Vitrification / Warming Solutions Vitrification Solutions 7.5% ethylene glycol 7.5% DMSO 20% Protein in M199-H 15% ethylene glycol 15% DMSO 0.5 M sucrose 20% Protein in M199-H Equilibration Solution Vitrification Solution Warming Solutions Initial Warming Solution (IWS) - M199-H M Sucrose - 20% Protein Dilution Solution (DS) - M199-H M Sucrose - 20% Protein Wash Solution (WS) - M199-H - 20% Protein

Objective Compare human metaphase II oocyte cryopreservation by slow-rate freezing and vitrification in a prospective, randomized, controlled clinical study.

19 Objective Compare human metaphase II oocyte cryopreservation by slow-rate freezing and vitrification in a prospective, randomized, controlled clinical study. Joyce Fioravanti, BSc 2004 Technical Training: 1) Oocyte slow-rate freezing - Dr. Porcu, M.D. - Bologna, Italy 2) Oocyte vitrification - University of Michigan

20 Prospective Randomized Controlled Study IRB approval and written informed consent Offered to women > 9 oocytes collected in fresh cycles Randomized to slow-rate freezing (Porcu) vs. vitrification Began January, 2005 As of Dec 2008: 230 cases cryopreserved, 78 thaws/warmings Four hours incubation before intracytoplasmic sperm injection Sol. Exp. Vitr. / Warm Initial Warm Solution Vitrification 4-Hrs Post- Wash T0hr Sol. Exp. T0hr Vitr. / Warm Slow-Rate Freezing T4hr T2hr

21 Pre-Cryo Survival and Fertilization 4-H Hour Survival Viable Non-Viable CSI Fertilization Post-I Normal Abnormal Age Thaws/ Survival Normal Warming Fertilization Cleavage Slow-Rate 31± % a 67% c 70% a Freezing Vitrification 32± % b 77% d 91% b Smith et al., 2009, submitted Fert & Stert a,b P<0.01; c,d P<0.05

22 Day 3 Embryo Development Following Oocyte Cryopreservation Grade (AU) a Day 3 Embryo Cell Number Day 3 Embryo Grade b Grade 1 Embryo Cell (#) and c d Grade 2 Grade 3 Grade 4 0 Vitrification/Warming SR Freeze/Thaw a,b&c,d P<0.05

23 Prospective Randomized Controlled Study: Pregnancy Rates Biochemical Pregnan ncy Rate (%) Slow-Rate Freeze/Thawing (N=30) Vitrification/Warming (N=48) a Embryos Transferred X: 3.2 SE: 0.3 b Embryos Transferred X: 3.1 SE: 0.2 c 34 oocy / 1 preg Per Thaw/Warming d a,b P<0.01; c,d P< oocy / 1 preg Clinical Pregnanc cy Rate

24 Oocyte Cryopreservation Meta- Analysis Vitrification Slow-Rate Freezing Age Fertilization Rate 74% 77% 65% 67% Clinical PR Per Oocytes Thawed/Warmed 4.5% 5.2% 2.3% 1.7% Clinical PR Per Thawing/Warming? 38%? 13% Clinical PR/ Transfer 46% 38% 21% 21% Oktay et al., Fertil & Steril, 2006

25 Conclusions 1) Oocyte cryopreservation by slow-rate freezing or vitrification are experimental procedures to be performed under IRB and informed consent. 2) Long-term follow-up of offspring obtained through oocyte cryopreservation is wanting (human and model systems). 3) Vitrification is not difficult. 4) Vitrification requires practice, can carry a technical signature, and can be unforgiving to technical mistakes.

26 Acknowledgements Program Support Timothy Johnson, MD Max Wicha, MD Physician Support Gregory Christman, MD Senait Fisseha, MD Eduardo LA Motta, MD, PhD Dana Ohl, MD John Randolph, MD Paulo Serafini, MD, PhD Yolanda Smith, MD Research Support Cristine Silva E Silva Joyce Fioravanti, BS Irvine Scientific Beto Alegretti, BS Pericles Hassun, PhD Keiko Hata, MS Satoshi Kishigami, PhD Masa Kuwayama, PhD Marcia Leonard, RN, PNP Thom Saunders, PhD Jason Swain, PhD National Institutes of Health - NICHD and NCI

27 Thank You

Cryopreservation of human oocytes with slow freezing techniques

ESHRE Campus Symposium Cryobiology and cryopreservation of human gametes and embryos Athens, Greece 25-26 September 2009 Cryopreservation of human oocytes with slow freezing techniques Giovanni Coticchio