Embryo Cryopreservation Slow freezing vs. Vitrification BASAK BALABAN BSc. American Hospital of Istanbul Assisted Reproduction Unit AMERICAN HOSPITAL

Cryopreservation Slow freezing of human oocytes Vitrification of bovine oocytes İn small drops Slow freezing of human embryos Slow freezing of domestic animal embryos Vitrification of mouse oocytes OPS Vitrification of bovive oocytes and embryos Vitrification of bovine blastocysts Slow freezing of mouse embryos Vitrification of mouse embryos 1972 1973/74 1983 1985 1986 1989 1993 1996 1997/98

Cryopreservation Techniques Slow conventional freezing Traditional Vitrification Ultrarapid vitrification

From Kasai et al. RBM Online 2004 Vitrification solutions DMSO+Acetamide+ propylene glycol Ethylene glycol+ Ficoll+Sucrose Ethylene glycol+ DMSO Ethylene glycol+ glycerol Slow Freezing solutions DMSO /1-2 PROH + Sucrose Glycerol+ Sucrose Base medium + Cryoprotectant From Kasai et al. RBM Online 2004

Differences of slow freezing and vitrification high levels of cryoprotectants very fast cooling rates (~20,000oC/min) fast cooling rates result in solidification of solution into glass-like structure (no crystallization) takes seconds Slow-freezing low levels of cryoprotectants slow controlled rates of cooling (0.3oC/min) slow dehydration to minimize ice-crystal formation takes hours

Vitrification & Slow-cooling Control of solute penetration Yes No Control of dehydration rate Duration out of the incubator 10min. 3 hrs. Prolonged temperature shock Fracture of ZP Possible Capture by growing ice crystals Equipment and running costs Inexpensive Expensive Kuleshova et al. F&S 2002

Vitrification of Human Embryos Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M. Hum Reprod 1998 13:2874-9 Implantation rate of 17% for best embryos Implantation rate of 9.8% for all embryos

Problems Associated with Traditional Vitrification Procedures High levels of cryoprotectants are toxic to embryos (4-10 M compared to 0.5-1.0M) Procedure must be performed at 4oC Technically demanding Advantages of Ultra-Rapid Vitrification Increases in cooling rates alleviates toxicity of high levels of cryoprotectants Can be performed at room temperature or 37oC

Variables in Vitrification Cooling &warming rates:Ideal vitrification protocol must pass rapidly through the critical temperature zone of 15 to – 5ºC to decrease chilling injuries. High warming rates by directly plunging cells into the warming solution is suggested (-196 to 37ºC)

Variables in Vitrification Concentration of the cryoprotectant: To achieve high cooling rates requires the use of high concentrations of the cryoprotectant solution which depresses ice crystal formation, so a critical concentration is required but in some cryoprotectants, this minimal concentration (Cv) can lead to either osmotic or chemical toxicity

Variables in Vitrification Sample size and carrier systems Sample size should be minimized to reduce the duration of vapour coat and to increase the cooling rate, minimizing the volume of the vitrification solution as much as possible is necessary to facilitate vitrification by higher cooling rates To minimize the volume of the vitrification solution special carriers are used for vitrification process ** Open pulled straws ** Flexipet- denuding pipette ** Microdrops ** Electron-microscopic copper grids ** Hemistraw system ** small nylon coils or nylon mash ** Cryotop,cryotip ** Cryoloop

Carriers for vitrification Cryotop Cryotip Cryotip Kuwayama et al.,RBM Online 2005

Cryoloop Hampton Research, Laguna Niguel, CA, USA Nylon loop (20µm wide; 0.5-0.7 mm in diameter) Thin film of cryoprotectant solution by surface tension Embryos are placed by pipette Hampton Research, Laguna Niguel, CA, USA

Advantages of Cryoloop Vitrification Lack of thermoinsulating layer maximizes heat transfer (>20,000oC/min) Easy manipulations Constant visualization of embryo Cryoloop stored within cryovial Procedure is performed at 37oC

Concerns with Regards to Sterility of Liquid N2 storage Tedder et al., 1995 Hepatitis B transmission Bielanski et al., 2000 Viral contamination Bielanski et al., 2003 Microbial contamination, no viral cross contamination Kyuma et al., 2003 No microbial or viral cross contamination

Clinical Results on Ultra-Rapid Vitrification of Human Cleavage Stage Embryos Author Carrier Cryosurvival(%) CPR% IR% Mukaida 1998 Conventional straws 71.8 15.5 4.9 Saito 2000 16.0 Danasouri 2001 OPS 49.3 30.5 10.4 Kuwayama 2005 CRYOTIP 98.0 27 Raju 2005 Locally prepared cryovial 95.3 35.0 14.9 Desai 2007 CRYOLOOP 85.0 44.0 20.0 Balaban 2008 92.2 29.7

Cryopreservation of human embryos by vitrification or slow freezing: A systematic review and meta-analysis Pubmed search: 873, only 4 included!!, Primary outcome: Postthaw survival rate, Sec.Outcome: Cleavage&Blastocyst dev.& hatching, CPR Loutradi et al., F&S 2008

Heterogenity of the studies included Loutradi et al., F&S 2008

Pooled data on cleavage, blastocyst development &hatching, CPR, IR, and LBR were NOT feasible **Valojerdi et al.,JARG 2009 retrospective study: higher CPR/IR, Kolibianakis et al.,Curr.Op.Obst.Gyn. 2009 review no dif. İn CPR

Cryopreservation Results with Slow Freezing Urman&Balaban et al. F&S 2007

CBS High security straw Heat sealed Heat sealed Filling noozle part Connection part with aspiration system Identification plug Hydrophobic Safety plug e Seeding point Conventional straw e Seeding point Cotton plug connected to the aspiration system Plastic plug Air bubble Medium

More Successful Results in Slow Freezing Cycles with HSS Balaban et al., F&S 2007

First Trials of ultra-rapid vitrification on human day 3 cleavage stage embryos GIII/G5 series (Vitrolife, Sweden) sequential culture system was used for fresh embryo culture Day 3 embryos of good quality that were donated for research with concent were used Ethylene glycol and 1,2-propanediol was used as the cryoprotectant. All procedures were performed at 37ºC (Lane M and Gardner DK (2001) Vitrification of mouse oocytes using a nylon loop. Mol. Reprod. Devel. 58: 342-347). Cryoloop was used as the carrier system American Hospital 2006-2008

Cryosurvival rates of vitrified day 3 cleavage stage embryos No.of day 3 embs. Vitrified (Total) 165 No.of 8c vitrified (%) 92(55.7) No.of warmed embs. No.of lost embs.during warmed (%) 3(1.8) (3-8c) Total Cryosurvival (%) 152 (93.8) (152/162) Cryosurvival of 8c top quality embs. (%) 89 (100) (89/89) Embs. with 100% intact blastomeres after warming 104 (68.4) 8c with all blast.intact (%) 68 (76.4) (68/89) American Hospital Data 2006

Blastocyst formation rates of vitrified day 3 cleavage stage embryos Parameter Total Day 5 Day 6 Blastocysts from 100% survived embs.(%) 66(63.4) Blastocysts (%)(Total) 84(55.2) 76(50.0) 8(5.2) Good quality blasts.(≤3AA)(%) 39(46.4) 38(50.0) 1(12.5) Blast from 8c (%) 55(59.7) - Hatching blastocysts(%)(Total) 26(30.9) 20(26.3) 2(3.5) H.Blasts. from 8C embs.(%) 18(32.7) 17(30.9) 1(1.8) American Hospital Data 2006

A randomized controlled study on human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation To compare the blastocyst development between embryos that were cryopreserved by either slow freezing or vitrification on day 3 To determine whether slow freezing and vitrification have different effects on human cleavage stage embryo metabolism Human Reproduction 2008 Balaban B¹, Urman B¹, Ata B, Isiklar A¹, Larman MG², Hamilton B² and Gardner DK²

Table-1- Embryo development Slow Freezing Vitrification 95%CI of difference P value Cryosurvival (%) 206/232 (88.7) 222/234 (94.8) +1 – 11% 0.02 Embs. with 100% blastomere survival (%) 106/206 (51.4) 173/222 (77.9) +18 - %35 0.00 Blastocyst formation (%) 102/206 (49.5) 134/222 (60.3) +1 – 20% Blasts.≥ 3AA (%) 43/102 (42.1) 70/134 (52.2) - 2.7 – 22.8% 0.12 Hatching blastocysts (%) 22/102 (21.5) 42/134 (31.3) - 1.4 – 20.9% 0.09 Balaban & Gardner et al., HR 2008

Analysis of Embryo Metabolism Non-invasive fluorometric assays based on the reduced pyridine nucleotides NADH or NADPH Fluorescence Pyruvate (mM) Pyruvate + NADH + H+ Lactate + NAD+ Balaban & Gardner et al., HR 2008

Table 2- Metabolic Analysis 5 10 15 20 25 Slow Freezing Vitrification * Pyruvate uptake (pmol/embryo/h) Balaban & Gardner et al., HR 2008

Table-3- Clinical Outcome with Vitrification No.of patient’s warmed 73 No.of embryos vitrified(Mean) 314 (4.2) No.of embryos warmed 241(3.3) No.of warmed embs. with 8C 85(35.2) No.of warmed embs. with >8C 49(20.3) No.of warmed embs with <8C 107(44.3) Cryosurvival (%) 222 (92.1) No.of embs. with 100%survival 160(72.1) No.of morula on day 4 (day of ET) 138(62.1) Balaban & Gardner et al., HR 2008

Table-3- Clinical Outcome with Vitrification No.of ET (Mean) 168(2.3) No.of morula ET (%) 146(86.9) CPR(%) 36(49.3) IR(%) 50(29.7) OPR(%) 33(45.2) MPR(%) 13(36.1)(1-triplet, 12 twins) Abortion(%) 3(8.3) Deliveries(%) 8(24.2:8/33)(2-twins, 6 singleton) Balaban & Gardner et al., HR 2008

Conclusion Human embryo metabolism is significantly depressed following cryopreservation by slow freezing In contrast, embryos that were vitrified had a metabolism activity almost twice that of the frozen embryos Subsequent embryo development appeared to be elevated following vitrification These data therefore support the hypothesis that vitrification is associated with less cellular trauma when slow freezing and should be considered as the primary method of human embryo cryopreservation Balaban & Gardner et al., HR 2008

Clinical Outcome with Embryo Vitrification No.of patient’s warmed 339 No.of embryos vitrified (Mean) 1458 (4.3) No.of embryos warmed (Mean) 1051 (3.1) No.of warmed embs. with 8C(%) 354 (33.6) No.of thawed embs. with >8C(%) 210 (19.9) No.of thawed embs with <8C (%) 487 (46.3) Cryosurvival (%) 955 (90.8) No.of embs. with 100%survival(%) 640 (67.0) No.of morula on day 4 (day of ET)(%) 546 (57.1) American Hospital of Istanbul Data: 01.11.09

Clinical Outcome with Embryo Vitrification No.of ET (Mean) 780 (2.3) No.of morula ET (%) 553 (70.8) CPR(%) 147 (43.3) IR(%) 203 (26.0) OPR(%) 133 (39.2) MPR(%) 48 (32.6) (4-triplet, 44-twins) Abortion(%) 14 (9.5) Deliveries(%) 52 (39.0) (19-twins, 33-singleton) American Hospital of Istanbul Data: 01.11.09

First trials with the Closed System-RAPID I CARRIER USED RAPID-I CRYOLOOP No.of embryos vitrified 64 66 No.of embryos warmed No.of warmed embs. with 8C 24 (37.5) 24 (34.8) No.of warmed embs. with >8C 11 (17.1) 12 (18.1) No.of warmed embs with <8C 29 (45.3) 30 (45.4) Cryosurvival (%)* 52 (81.2) 60 (90.9) No.of embs. with 100%survival 28 (53.8) 39 (65.0) No. of blastocysts (day 5)* 23 (44.2) 31 (51.6) Good quality blasts. (≤3AA)* 10 (43.4) 13 (42.0) Hatching blasts (day 5)* 7 (30.4) 9 (29.0) *NS, Fisher’s exact test Larman & Balaban Oral presentation ASRM 2009

Neonatal Outcome of Vitrified Cleavage Stage Embryos Rama Raju et al. F&S 2009 No.sig. dif. for neonatal parameters: Mean gestational age, birth weights for singleton & MPR, PR induced complications, Incidence of birth defects ( major & minor malformations)

Necessity of blastocyst vitrification ? Increasing application of BT especially for some selected cases results with supernumerary blastocysts for freezing to increase cumulative pregnancy rates per oocyte retrieval A reliable procedure for the cryopreservation of blastocysts is needed, because after fresh ET, only small number of supernumerary blastocysts are likely to be available for cryopreservation Based on the published cochrane data (2008), vitrification appears to result in significantly higher survival and pregnancy rates

Blastocyst vitrification First pregnancy after human blastocyst vitrification was achieved by Yokota et al., HR 2000 EG- based vitrification solutions are widely used as it has a low toxicity with rapid diffusion into the cell through ZP and cellular membrane 1st. Vit.sol. EG+DMSO 2nd. EG+DMSO+Ficoll+ Sucrose, Warming: Decreasing concentrations of Sucrose sol. are preferred Concentration of cryoprotectants are decreased to 7.5% from 25% over the years of experience

>10.000 blasts. vitrified Youssry et al.,RBM Online 2008

Blastocyst vitrification Is it the most effective and successful method to cryopreserve embryos at blastocyst stage???

Faster re-expansion after thawing with vitrification method Stehlik et al.,RBM Online 2005

Pooled data on cleavage, blastocyst development &hatching, CPR, IR, and LBR were NOT feasible

Artifical shrinkage by microneedle Artifical shrinkage by laser Large blatocoele of more developed blastocysts may disturb the efficacy of vitrification due to inappropriate Dehydration and permeation of cryoprotectant, which may cause ice crystal formation in the rapid cooling and warming steps of vitrification. Ice crystal formation can be a voided by reducing fluid content of the blastocoele of more developed blastocysts Mukaida et al., HR 2006 1st.clin.appl- Vanderzwalmen et al., HR 2002,3,4

Takahashi et al.,F&S 2005 Liebermann et al., F&S 2006 also reported no adverse effect

Vitrification results with higher cryosurvival rates for biopsied human embryos Poor cryosurvival rates (approx. 30%) and clinical outcome reported after conventional slow freezing of biopsied cleavage stage embryos ( Joris et al., HR 1999, Magli et al., HR 1999) Escriba et al., F&S 2008 had shown similar cumulative OPR/OPU for PDG and non-PGD group with blastocyst vitrification Zheng et al.,HR 2005

RESULTS Vitrification as a cryopreservation method has many primary advantages and benefits based on the published data Vitrification protocols are now starting to enter the mainstream of human ART The reports of successfully completed pregnancies following vitrification are encouraging for further research More studies on vitrification and warming procedures are needed to develop more efficient and optimal vitrification methods

Concerns regarding Vitrification LN2 still remains to be a potential source of contamination since the technique is based on direct contact between the vitrification solution containing cryoprotectant agents and LN2. So from a clinical point of view: Is there a need to sterilize LN2? How is it possible to maintain its sterility Cross contamination with viruses?? ( No publication since 1985, about 450 publications) Closed systems should be used in clinical human IVF in the future to avoid this concern.(Like CBS HS vitrification straws, Cryotip……) New clinical trials with safer closed systems should be applied Low toxicity vitrification solutions must be designed in the future Genetical structure of the vitrified cell?? Chromosal abnormalities, gene expressions ...... More studies are needed to prove the safety of the technique

MAIN TOPICS I. Basic Cryobiology and Cryopreservation Overview 1. Principles of cryobiology 2. The evolution of cryopreservation techniques: Slow-freeze, vitrification, freeze drying and beyond…. 3. Cryopreservation for wildlife conservation: Lessons for human IVF 4. What can we learn from the livestock industry? 5. The future of cryopreservation in assisted reproduction II. Oocyte Cryopreservation 1. What, when, and how to cryopreserve oocytes? Lessons from oocyte physiology 2. Cryopreservation of oocytes for social or medical reasons: Is there a difference? 3. The rise of the new banking system? Cryopreservation of donor oocytes 4. Recovery of high quality oocytes for cryopreservation: Optimising clinical protocols 5. To vitrify or not to vitrify? Molecular markers to assess cryopreservation techniques 6. Safety of oocyte cryopreservation: Outcomes and long-term follow-up III. Sperm Cryopreservation 1. Principles of sperm cryopreservation: Cryopreservation methods and effectiveness 2. Frozen versus fresh sperm: Are the outcomes really any different? 3. Cryopreservation of testicular and epididymal sperm: Outcomes, safety and long term follow-up 4. Cryopreservation of sperm for oncology patients: Pre- and post-treatment quality, utilization, success and safety 5. Molecular assessment of frozen-thawed sperm: Cytogenetics, DNA fragmentation, methylation and more… 6. Safety of sperm cryopreservation: Outcomes and long-term follow-up? IV. Embryo Cryopreservation 1. Assessing frozen embryo survival after thawing: Revisiting the 50% rule 2. Embryological criteria for successful cryopreservation: What stage and grade of embryos should be cryopreserved? 3. Slow-freezing versus vitrification: Are molecular markers or success rates driving cryopreservation techniques? 4. Optimizing frozen-thaw embryo cycles: Clinical aspects 5. A critical assessment of assisted hatching and lysed cell removal on frozen-thawed embryos 6. Safety of embryo cryopreservation: Outcomes and long-term follow-up V. Cryopreservation of Ovarian Tissue 1. Cryopreservation of whole ovaries or ovarian tissue: Clinical indications and outcomes 2. Collection, processing and storage of ovarian tissue: A review of best practice 3. Ovarian tissue transplantation: What constitutes success? 4. In Vitro Maturation from cryopreserved ovarian tissue: As good as fresh? VI. Regulation, Law and Ethics of Cryopreservation 1. Regulatory aspects of cryopreservation around the world: Global village or dystopia? 2. Posthumous collection and storage of gametes: Do the living have any rights? 3. Whose embryo is it anyway? Storage terms, future dispositions, effective consent, joint ownership and other dilemmas 4. An embryologist’s dream: The perfect consent form VII. Training and Quality Management Aspects of Cryopreservation 1. Optimizing cryopreservation techniques – What’s critical, what’s not? 2. Keeping a lid on cryopreservation: Key performance indicators in the IVF lab 3. Open forum for delegates to share their experiences (with panel discussion)

Material & Methods Clinical protocols Hipothalamopituitary axis downregulation with GnRH analogues started on midluteal phase and discontinued after 10 days of estradiol treatment Endometrium is prepared using incremental doses of Estradiol valerate (2- 8 mg) Progesteron vaginal gel (%8) is started for luteal supplementation Balaban HR 2006, Urman 2007, Balaban 2007

No difference in Neonatal parameters for fresh&vitrified embryos Rama Raju et al., F&S 2009

No difference in Neonatal parameters for fresh&vitrified embryos Rama Raju et al., F&S 2009

Slow Freezing & Vitrification of Cleavage stage embryos No sig. differences between 2 carriers: Cryotop, Cryotip, V.Sol: EG+DMSO+ Sucrose-Vit.kit(Ir.S) Kuwayama et al.,RBM Online 2005

Similar OPR after ET of biopsied vitrified blastocysts Escriba et al., F&S 2008 Retrospective study, Carrier: 0.25ml. French straws

Vitrification of blastocysts after PGD yields similar cumulative OPR Escriba et al., F&S 2008