1 / 39

Blastocyst Vitrification

Blastocyst Vitrification. BASAK BALABAN BSc. American Hospital of Istanbul Assisted Reproduction Unit. AMER IC AN HOSPITAL. Vitrification.

lotus
Télécharger la présentation

Blastocyst Vitrification

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Blastocyst Vitrification BASAK BALABAN BSc. American Hospital of Istanbul Assisted Reproduction Unit AMERICAN HOSPITAL

  2. Vitrification • Process that produces a glasslike solidification of living cells that completely avoids ice crystal formation during cooling. It completely avoids ice crystal formation in cryopreserved cells during warming to recover the cells for biological applications

  3. Vitrification Techniques • Traditional Vitrification (1998- early 2000s) • Ultrarapid vitrification (2000-...today)

  4. 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

  5. Base medium + Cryoprotectant 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 From Kasai et al. RBM Online 2004

  6. Slow-freezing low levels of cryoprotectants slow controlled rates of cooling (0.3oC/min) slow dehydration to minimize ice-crystal formation takes hours 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 Differences of slow freezing and vitrification

  7. Vitrification & Slow-cooling Kuleshova et al. F&S 2002

  8. 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)

  9. 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

  10. 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

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

  12. Cryoloop 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

  13. 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

  14. 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

  15. 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

  16. 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

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

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

  19. Superior results with Vitrification Stehlik et al.,RBM Online 2005 **Retrospective, carrier: Cryotop

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

  21. Vitrification versus slow freezing method for blastocyst cryopreservation Liebermann et al., F&S 2006 **Retrospective, carrier: cryotop

  22. Vitrification versus slow freezing method for blastocyst cryopreservation Liebermann et al., F&S 2006

  23. Slow freezing & vitrification method for human blastocysts Kuwayama et al., RBM Online 2005

  24. Higher survival rates with blastocyst vitrification *p<0.001 Vitrification: 13 patients, CPR: 53.8%, IR: 23.3% Huang et al., HR 2005

  25. 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

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

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

  28. 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

  29. Artifical shrinkage of the blastocoel cavity prior vitrification Mukaida et al.,HR 2006

  30. Better cryosurvival rates with vitrification of blastocysts after artifical zona opening Zech et al., RBM Online 2005 Galan et al., ESHRE 2003 reported 73% cryosurvival with blastocyst vitrification

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

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

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

  34. 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 thawing procedures are needed to develop more efficient and optimal vitrification methods

  35. 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

  36. Vitrification of human blastocysts with the Cryoloop 223 cycles, 725 blastocysts!! Mukaida et al.,HR 2003

  37. Successful Vitrification Liebermann et al., Biol. Reprod 2002

  38. 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) Zheng et al.,HR 2005

More Related