Cleavage Stage Embryo Cryopreservation Slow Freezing Versus Vitrification Basak Balaban VKF American Hospital of Istanbul Assisted Reproduction Unit Head of IVF Laboratory Turkish Society of Clinical Embryologists (KED) Chair AMERICAN HOSPITAL XVI Annual Meeting of MSRM April 21-24 2016 Cesme, Izmir Turkey
Cryopreservation Slow conventional freezing Vitrification Techniques
From Kasai et al. RBM Online 2004 Slow Freezing solutions DMSO /1-2 PROH + Sucrose Glycerol+ Sucrose Vitrification solutions DMSO +Acetamide+ propylene glycol Ethylene glycol + Ficoll +Sucrose Ethylene glycol + DMSO Ethylene glycol + glycerol Base medium + Cryoprotectant
Embryo Cryopreservation
Overview of the Lecture Clinical outcome 1.Which method is more successful for embryo cryopreservation; Slow or vitrification? 2.Perinatal outcome, safety 3.Other advantages of vitrification 4.Concerns and future directions
Cryopreservation of human embryos by vitrification or slow freezing: A systematic review and meta-analysis 0.001 0.01 0.1 10 100 1000 Slow Vit. Pubmed search: 873, only 3 included!!, Primary outcome: Postthaw survival rate, Sec.Outcome: Cleavage&Blastocyst dev.& hatching, CPR Pooled data on cleavage, blastocyst development &hatching, CPR, IR, and LBR were NOT feasible Loutradi et al., F&S 2008
Cryopreservation of cleavage stage embryos by vitrification vs. slow freezing?? Which one is better? Survival Blastocyst development 0.002 0.1 Slow 10 500 Vit. **Biopsied embs. Li and Rama Raju found no stat sig. dif for CPR Efstratios et al.,cur.op.obs&gynec. 2009
Meta-analysis indicate that embryo vitrification is superior to slow freezing based on direct comparison of embryo survival and CPR, OPR, and IR Desai et al.,rbm Online 2010
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
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% 0.02 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
Table 2- Metabolic Analysis Pyruvate uptake (pmol/embryo/h) 25 20 15 10 5 * 0 Slow Freezing Vitrification Balaban & Gardner et al., HR 2008
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) 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) 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(%) Abortion(%) 3(8.3) 13(36.1)(1-triplet, 12 twins) Deliveries(%) 8(24.2:8/33)(2- twins, 6 singleton) Balaban & Gardner et al., HR 2008
Once the embryo succeeds further cleavage upon thawing IR did not differ for SF vs. Vit Van Landuyt HR 2013
Mainly SET, closed carriers, stat. >2500 embs. were needed for sig.in clinical outcome
Sig. more embryos were fully intact after vitrification ( 75.4% vs. 28.6%) Debrock HR 2015
Outcome of vitrified cleavage-stage embryos: 1872 cycles Chi et al. Zygote 2015 showed in a retrospective study that day 3 vit. is superior to day 2 in terms of CPR Cobo et al., F&S 2012
A critical appraisal of cryopreservation (Slow cooling vs. Vitrification) of human oocytes and embryos All prospective randomized trials from the avaliable evidence compares longstanding traditional method for slow cooling with vitrification even though modified method of slow cooling (with single-step dehydration in PROH with a higher(0.2m) concentration of sucrose) has been shown to be significantly inferior More prospective randomized trials comparing modified slow cooling method with vitrification for cleavage stage embryos are needed to conclude whether vitrification is a more succesful method at this stage Edgar & Gook HR Update 2012
Which stage is more successful with vitrification? Cleavage or blastocyst abstract All used slow freezing for embryos and blastocysts!!
Pros.rand. 120 patients with 1st.IVF/ICSI cycle, any female age Fernandez-Shaw 2015
Neonatal Outcome of Vitrified Cleavage Stage Embryos No.sig. dif. for neonatal parameters: Mean gestational age, birth weights for singleton & MPR, PR induced complications, Incidence of birth defects ( major & minor malformations) Rama Raju et al. F&S 2009
Perinatal &neonatal outcomes of 494 babies from 972 vitrified day 3 ET ** Shi et al., F&S 2012
Neonatal outcome and birth defects in 6623 singletons born following minimal ovarian stimulation and vitrified versus fresh SET Kato O et al., Eur J Obst.Gyn. Reprod Biol. 2012 Vitrification of embryos/blastocysts did not increase the incidence of adverse neonatal outcomes or birth defects following SET, no sig. diff. in LGA Obstetric and Neonatal outcomes after transfer of vitrified early cleavage stage embryos. Liu SY et al., HR 2013 No adverse effect on neonatal outcome. Birthweight was higher in the vitrified group versus slow or fresh ET implying an improved perinatal outcome
Age, parity, child sex, year of birth, birth order 1st. report 2008 from Australia by Shih, than Pelkonen 2010 and Sazonova 2012 reported LGA in FET singletons. ALL in slow-frozen embryos.. 3 studies for vitrified embryos- not included in the meta-analysis 1. Kato 2012 that showed no diff. for embryos and blastocysts 2. Shi 2012 showed in a small population that mean birthweight is higher for vit. day 3 emb. BUT no risk calculations for LGA 3. Liu 2013 showed sig. higher birthweight with vit. compared to slow f. &fresh How does slow freezing or vitrification affect early embryo and plasental development and intrauterine growth environment should be further explored?
NO SIG. DIFFERENCES IN NEONATAL PARAMETERS Kaartinen JARG 2016
Poor cryosurvival rates (~30%) and clinical outcome after conventional slow freezing of biopsied cleavage stage embryos ( Joris et al., HR 1999, Magli et al., HR 1999) Vitrification results with higher cryosurvival rates for biopsied human embryos Zheng et al.,hr 2005 Survival and IR of biopsied cleavage embs. and vit. at blastocyst stage is similar with non-biopsied counterparts. El-Toukhy HR 2009 Higher survival and preg.for biopsied and vit. embryos compared to biopsied and slowly frozen Keskintepe JARG 2009
Flexibility of re-cryopreserving cells by vitrification method It s presumed that refrozen & thawed embryos using conventional methods results with detrimental cryoinjury Chang C. RBM Online 2008 Two successful pregnancies obtained following oocyte vitrification and embryo re-vitrification. Kumasako et al., F&S 2009 The efficacy of the transfer of twice frozen thawed embryos with the vitrification method Peng et al.,rbm Online 2011 Live birth after transfer of a twice vitrified warmed blastocyst that had undergone trophoectoderm biopsy James et al., RBM Online 2012 Vitrification of human embryos previously cryostored by either slow freezing or vitrification results in high pregnancy rates Cobo et al., F&S 2013 Outcome of cryotransfer of embryos developed from vitrificed oocytes: double vitrification has no impact on delivery rates Greco et al., Springerplus. 2015 Successful implantation and live birth of a healthy boy after triple biopsy and double vitrification of oocyte-embryoblastocyst.
Conclusion Which method is more successful for cleavage embryo cryopreservation Based on current evidence vitrification results with better clinical outcome when compared with traditional slow freezing, however more randomized trials maybe needed with modified slow freezing Vitrification results with better clinical outcome for biopsied cleavage stage embryos Vitrification provides flexibility for recryopreservation of embryos with successful clinical outcome
Concerns for long term storage of vitrified cells Closed carriers prevent the direct contact of cells with LN2, reducing the risk of pathogens as well as cross contamination with reactive chemical compounds during storage As chemical reaction is avoided aseptic storage is of particular interest for long-term storage Increased possibility of toxic effect related with higher concentration of CP Increased susceptibility to molecular changes in the vitrified cell structures due to the glassy state Higher sensitivity to deviations in storage temperature Wirleitner HR 2013
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