© 2008 Universitair Ziekenhuis Gent1 QC/QA in cryopreservation laboratories within assisted reproduction units: future recommendations Prof Dr Etienne Van den Abbeel Department of Reproductive Medicine, University Hospital Gent, Belgium Istanbul 8 June 2013

22 © 2008 Universitair Ziekenhuis Gent Introduction Cryopreservation of human oocytes: why? Legal Ethical Donor-acceptor programmes Fertility preservation Cancer Non-medical reasons Cryopreservation of human embryos: Why? Increase efficiency of ART Tool to reduce multiple pregnancies Transfer in natural cycle Fertility preservation Efficient and safe cryopreservation procedures

33 © 2008 Universitair Ziekenhuis Gent Introduction Cryopreservation of reproductive cells Stopping biological time -196°C Lethal intra-cellular ice formation Fate of cellular water Equilibrium (quasi-equilibrium) cooling Non-equilibrium cooling ( Freezing ) ( Vitrification)

44 © 2008 Universitair Ziekenhuis Gent Dilemma: vitrification or freezing? There are two circumstances in which vitrification methods should definitively be considered: where it is clear that extra-cellular ice is responsible for significant damage, and where the results of classical freezing methods are unsatisfactory (Pegg, 2005) Classical freezing of oocytes is suboptimal Classical freezing of blastocysts is suboptimal

55 © 2008 Universitair Ziekenhuis Gent Introduction Cryopreservation within assisted reproduction units: future recommendations Vitrify …. But take care

66 © 2008 Universitair Ziekenhuis Gent Vitrification Rationale for vitrification Very simple procedure? Reduces the time of the cryopreservation procedure? Flexibility No ice crystallization? Eliminates the cost of expensive programmable freezing equipment? More efficient?

77 © 2008 Universitair Ziekenhuis Gent Vitrification current status: No standard procedure is available Several roads can lead to Rome The current plethora of protocols has to become canalized to a few of proven efficiency, which will greatly facilitate the comparison of data between centers as well as the troubleshooting of disappointing results within a center (Gosden, 2011)

88 © 2008 Universitair Ziekenhuis Gent Recommendations concerning vitrification now and in the future: Closed vitrification techniques should be the gold standard Closed vitrification methods should be robust, standardized and biologically safe QC/QA in vitrification laboratories Technical issues

99 © 2008 Universitair Ziekenhuis Gent T° Concentration of solute Th Tg Equilibrium Freezing Curve Liquid phase Glass phase Molecular organization as in a crystal structure Phase diagram Glass transition curve Ice phase molecular structure of a viscous liquid and is not crystalline

10 © 2008 Universitair Ziekenhuis Gent 1. Equilibrium Vitrification of reproductive cells (Rall et al 1985) Probability of vitrification: Cooling rates x [CPA] 2. “Minimal Volume open pulled straw Vitrification”(1997 Vajta et al) Probability of vitrification: Cooling rates x [CPA] Sample Volume

11 © 2008 Universitair Ziekenhuis Gent 3. The effect of warming rates Mouse model Seki, Mazur ( 2009, 2010, 2012) In minimal volume vitrification, the warming rate is dominant over the cooling rate Cooling rate/warming rate x [CPA] Sample volume

12 © 2008 Universitair Ziekenhuis Gent 4. The device: open versus closed vitrification Human model Cobo et al (2011) open devices better for minimal volume vitrification than closed devices because of very high cooling rates in open devices Cobo et al (2013) The warming rate is perhaps the best determinant factor for succes in minimal volume cooling…. This can be best achieved with open system?! Open vitrification: cross contamination issues (Bielanksi et al, 2009) Vapour storage, sterile liquid nitrogen De Munck et al (2013) Using a CBS HS closed VIT device a 90% survival rate can be achieved when human oocytes are immediately warmed in a large volume at 37°C

13 © 2008 Universitair Ziekenhuis Gent The device and the warming rate: open versus closed vitrification Vitrification Warming Storage Cryo TOP open open open Cryo Loop open open open Flexipet open open open Cryologic open open open …… Cryo TIP closed closed closed Cryopette closed closed closed …… Rapid i closed open closed CBS HS VIT closed open closed Vitrisafe closed open closed

14 © 2008 Universitair Ziekenhuis Gent Other variables of vitrification CPA toxicity Type and concentration of CPA PG, EG, DMSO, Glyc …. Temperature of exposure Permeability of cells to water and CPA Glyc<EG<DMSO<PG Temperqture of exposure Variability amongst oocytes and embryos Oocytes<zygotes<embryos<blastocysts

15 © 2008 Universitair Ziekenhuis Gent Take home message on technical issues Minimal volume vitrification is non-equilibrium vitrification. It is a Vitrification method that not always leads to vitrification) Succes of the vitrification method depends on a correct interplay between a “sufficient” high cooling rate, “sufficient” permeation of a sufficient high concentration of penetrating cryoprotectant, “sufficient” dehydration by a non-penetrating cryoprotectant, and a “sufficient” high warming Standardization will be a challenge

16 © 2008 Universitair Ziekenhuis Gent Recommendations concerning vitrification now and in the future: Closed vitrification techniques should be the gold standard Closed vitrification methods should be robust, standardized and biologically safe QC/QA in vitrification laboratories Clinical issues Can it work? Does it work? Is it efficient?

17 © 2008 Universitair Ziekenhuis Gent Vitrification: Evidence for practice Slow freezing versus vitrification Open versus closed vitrification Closed versus closed vitrification

18 © 2008 Universitair Ziekenhuis Gent Results from literature: some caution! Different devices and different media formulations used Oocyte collection cycle characteristics Patient selection Cryo policy (selection of oocytes/embryos before cryo) Warming and transfer policy (selection oocytes/embryos after warming) No uniform reporting of data and (or) study endpoints Commercial bias?

19 © 2008 Universitair Ziekenhuis Gent

20 © 2008 Universitair Ziekenhuis Gent Oktay et al., 2006/2008 (abstract) VariableSlow Freezing (2006)Vitrification (2006) Age, mean Fertilization rate64.9 (2,478/3,818)74.2 (637/859) Clinical pregnancies per thawed oocyte (153/6720)0.045 (61/1354) SLOW FREEZING VERSUS VITRIFICATION - oocytes Slow freezing/Vitrification (2008) Clinical pregnancies per thawed oocyte (314/14215)/0.058 (212/3672)

21 © 2008 Universitair Ziekenhuis Gent Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. (Cobo et al, 2011) Grade A level of evidence? OBJECTIVE: To perform a systematic review of the literature to identify randomized controlled trials assessing the efficacy of oocyte vitrification in terms of oocyte survival, fertilization, embryo development, and pregnancy rates. DESIGN: Systematic review and meta-analysis of randomized controlled trials (>2500 papers and abstracts). Five eligible studies were finally included. They involved 4,282 vitrified oocytes, 3,524 fresh oocytes, and 361 slow- frozen oocytes between 2005 and 2009.

22 © 2008 Universitair Ziekenhuis Gent

23 © 2008 Universitair Ziekenhuis Gent Discussion and critical points of vitrification of human oocytes More prospective studies should be done! Evident clinical heterogeneity was present Statistical heterogeneity between studies especially for morphological survival The authors state that to obtain good results “open” vitrification should be used The efficiency (implantation per oocyte warmed) of open vitrification of human oocytes from young donors is 10% Unsufficient data for “older” patients

24 © 2008 Universitair Ziekenhuis Gent Pelin Ci et (2013) Age-specific probability of live birth with oocyte cryopreservation: an individual patient data meta-analysis. Fertil Steril (Article in press) Non-donor egg cycles Original data from 10 studies including cycles from patients showed that live birth success rates declined with age regardless of the freezing technique (slow freezing vs vitrification). Women whose SF eggs were preserved before age 30 had a greater than 8.9% likelihood of implantation per embryo which declined to 4.3% for embryos from eggs frozen after 40. For vitrification cycles, implantation success declined from 13.2% for embryos from eggs frozen at 30 to 8.6% for embryos from eggs frozen at 40. So, estimated probabilities of live birth for vitrified oocytes were higher than those for slowly frozen

25 © 2008 Universitair Ziekenhuis Gent Open versus closed vitrification Stoop et al (2012) Cryo TOP vs CBS HS VIT RCT, two devices, same vitrification and warming technology 144 oocytes, survival 83% versus 92% (NS) Paffoni et al (2011) Cryo TOP vs Cryo TIP Retrospective trial, two devices, different vitrification and warming technology 529 oocytes, survival 82.8 % versus 57.9 (p< 0.001) Papatheodorou et al (2013) Open vitrisafe vs closed vitrisafe Prospective randomised study, open vs closed same device, different Vitrification technology (CPA concentration) 1206 oocytes, survival 82.9% vs 91.0% (p< 0.05), no differences in pregnancy and implantation rates

26 © 2008 Universitair Ziekenhuis Gent Closed versus closed vitrification Closed (CBS HSS) vitrification of oocytes: Stoop et al (2012) Number of oocytes warmed: 123 Number of oocytes survived (%): 111 (90.2) Implantation rate per oocyte warmed: 13/123 (10.6%) Closed (Vitrisafe) vitrification of oocytes: Van der Zwalmen et al (2010) Number of oocytes warmed: 146 Number of oocytes survived (%): 137 (94%) Implantation rate per oocyte warmed: 6% Closed (cryo-TIP) vitrification of oocytes: Smith et al (2009) Number of oocytes warmed: 349 Number of oocytes survived (%): 260 (75%) Implantation rate per oocyte warmed: 5.2% Closed (cryo-TIP) vitrification of oocytes: Paffoni et al (2011) Number of oocytes warmed: 261 Number of oocytes survived (%): 151 (57.9%) Implantation rate per oocyte warmed: 2.6%

27 © 2008 Universitair Ziekenhuis Gent VITRIFICATION Day 3 embryos

28 © 2008 Universitair Ziekenhuis Gent Cryopreservation of human embryos: freezing vs vitrification(review paper) Kolibianakis et al (Current opinion in OB/GYN 21, , 2009) Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Review to evaluate whether the published literature offers data to allow the clinician to choose the best between two cryopreservation methods Vitrification as compared with slow freezing, appears to be better in terms of post- thawing survival rates for cleavage-stage embryos (odds ratio (OR): 6.35,95% CI: ) Postthawing blastocyst development of embryos cryopreserved in the cleavage stage is significantly higher with vitrification as compared with slow freezing (OR: 1.56, 95% CI: ) No significant difference in clinical pregnancy rates per transfer could be detected between the two cryo methods (OR: 1.66, 95% CI: ).

29 © 2008 Universitair Ziekenhuis Gent Cryopreservation of embryos: freezing versus vitrification AbdelHafez et al, 2010 RBM-online Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis Locate randomized controlled trials comparing embryo cryopreservation methods Conclusions: Vitrification is superior to slow freezing which in turn is superior to ultra- rapid freezing. However, more well designed and powered studies are needed to further corroborate these findings

30 © 2008 Universitair Ziekenhuis Gent Results from literature: embryos (open vitrification) El-Danasouri (2001) Kuwayama (2005) Mukaida (2007) Desai (2007) Balaban (2008) Raju (2009) Valojerdi (2009) Clin P / ET Impl /E Transferred Impl / E Warmed 596/ / /4242 (30.8%) (16.5%) (13.6%)

31 © 2008 Universitair Ziekenhuis Gent Results from literature: embryos (closed vitrification) Van Landuyt et al, 2010 (CBS HS VIT) Clin P / ET Impl /E Transferred Impl / E Warmed 55/257 64/423 64/515 (21.4%) (15.1%) (12.4%)

32 © 2008 Universitair Ziekenhuis Gent VITRIFICATION BLASTOCYST

33 © 2008 Universitair Ziekenhuis Gent Cryopreservation of human embryos: freezing vs vitrification(review paper) Kolibianakis et al (Current opinion in OB/GYN 21, , 2009) Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Review to evaluate whether the published literature offers data to allow the clinician to choose the best between two cryopreservation methods Vitrification as compared with slow freezing, appears to be better in terms of post-thawing survival rates for blastocysts (OR:4.09, 95% CI: ) No significant difference in clinical pregnancy rates per transfer could be detected between the two cryo methods (OR: 1.66, 95% CI: ).

34 © 2008 Universitair Ziekenhuis Gent Results from literature: blastocysts (open vitrification) Choi (2000), Yokota (2001), Cho (2002), Reed (2002), Hiraoka (2004), Hu (2004), Stehlik (2005), Huang (2005), Kuwayama (2005), Mukaida (2007), Liebermann (2007), Son (2007), Van der Zwalmen (2007), Hiraoka (2008), Ebner (2009) Liebermann (2009), Rama Raju (2009) Clin P / ET Impl /E Transferred Impl / E Warmed 4974/ / /13629 (48.8%) (28.1%) (22.9%)

35 © 2008 Universitair Ziekenhuis Gent Results from literature: blastocysts (closed vitrification) Stachecki (2008), Van der Zwalmen (2009, 2013), Liebermann (2009), Van Landuyt (2011), De Croo (2013) Clin P / ET Impl /E Transferred Impl / E Warmed 229/ / /1004 (52.6%) (30.8%) (26.2%)

36 © 2008 Universitair Ziekenhuis Gent Take home messages on clinical issues Good survival, fertilization, embryo development and pregnancy rates can be obtained with closed and open vitrification of oocytes Oocyte morphological survival rates are higher with vitrification as compared with classical freezing The external validity of oocyte vitrification maybe limited to good responders or donors and there are insufficient data for other patient categories. Age might be a considerable factor Vitrification provided a significant clinical breakthrough for the preservation of blastocysts There is some debate as to the real benefit of VIT over slow freezing for D3 embryos The long term safety of the technique remains to be confirmed

37 © 2008 Universitair Ziekenhuis Gent Cryopreservation Human oocyte, embryo, blastocysts vitrification: no standard procedure available Future recommendations? Standardization through automation True vitrification