Frozen Dreams Vitrification, as becoming the key procedure in IVF Lab: Is 100% survival a reality? Laura Rienzi Senior Clinical Embryologist GENERA Centres for Reproductive Medicine Rome, Marostica, Umbertide, Napoli, Italy

Agenda 1. Review of different approaches utilized in the IVF Lab. for oocyte and embryo cryopreservation 2. The Revolution of Vitrification Approach - New possibilities in in the Lab - New clinical indications for cryopreservation 3. What do we expect for the future? © Merck KGaA Darmstadt/Germany

Cryopreservation is essential in ART Cryopreservation of gametes and embryos is an essential aspect of Assisted Reproductive Technologies (ART). Its widespread application has opened new clinical perspectives, allowing to increase efficacy and safety of IVF treatments. The proportion of cryopreserved embryo transfers compared with fresh cycles is growing in Europe. It has been estimated that overall cryopreservation contributed to 32% of the transfers in 2011 (compared to 28% in 2010). (Kupka et al., 2016). The observed differences are mainly due to specific legal situations that in some nations impose restricted embryo transfer policies aimed at reducing the incidence of multiple pregnancies (Coetsier and Dhont, 1998). Fondation to build a beautiful house we need solid fondation © Merck KGaA Darmstadt/Germany

Review of different approaches for oocyte and embryo cryopreservation

History of cryopreservation in ART Slow freezing of domestic animal embryos Slow freezing of human oocytes Slow freezing of human embryos Ultrarapid Vitrification with EM grids OPS Ultrarapid Vitrification Vitrification of mouse oocytes Slow freezing of mouse embryos Vitrification of mouse embryos CryoLoop CryoTip Ultrarapid Vitrification Vitrification of bovine blastocysts 1935 1948 1972 1973/1974 1983 1985 1989 1993 1996 1997 1999 “It is more difficult to destroy a prejudice than an atom” Albert Einstein

PRONUCLEAR, CLEAVAGE STAGE and BLASTOCYST SLOW FREEZING HISTORY DMSO Slow-freezing 9% Glycerol + 0.2M sucrose Slow freezing 1.5M PrOH + 0.1M sucrose Slow freezing 1.5M PrOH + 0.2M/0.3M sucrose 1983 Trounson and Mohr Cleavage-stage First delivery 1984 Zeilmaker et al. 1985 Cohen et al. Blastocyst 1995 Kaufman et al. Blastocyst 83% 2002 Behr et al. Blastocyst 98% 2003 Gardner et al. Blastocyst 69% 2009 El-Toukhy et al. Biopsied blastocyst 88% 1985 Lasalle et al. 1986 Testart et al. Cleavage stage 87.5% 1990 Hartshorne et al. Cleavage stage 80% 1991 Demoulin et al. Pronuclear stage 54% Cleavage stage 47% 1993 Veek et al 2.000 pronuclear stage 68%, 1996 Al Hasani et al Pronuclear stage 77% 1998 Mandelbaum et al Cleavage stage 73% 1999 Damario et al., Pronuclear stage 90% Joris et al. Biopsied cleavage stage 2001 Tiitinen et al. Cleavage stage 76.6% SET 2002 Rienzi et al. Cleavage stage Blastomere removal 2007 Edgar et al. Cleavage stage SET 2003 Jericho et al Biopsied cleavage-stage 2009 Edgar et al. Cleavage stage 92.6% 2011 Wood et al. Cleavage stage 90%

The Vitrification Approach Vitrification is a pseudo second order phase transition (IUPAC Compendium of Chemical Terminology, 1997) converting a material into a glassy amorphous solid that is free from crystalline structure. Cooling/warming rates × Viscosity Probability of vitrification ̴ Volume of the sample Types of cells

Biophysical Aspects: Binary Phase Diagram 10-4 poise CPs Liquid phase T° Equilibrium 0°C freezing Thermal hysteresis Heterogenous curve (Tm) nucleation area curve (Th) Homogenous nucleation - 40°C Cooling rate Ice phase -100°C Glass phase No crystal 1013 poise curve (Tg) transition -120°C Glass Concentration of solute

PRONUCLEAR, CLEAVAGE STAGE and BLASTOCYST VITRIFICATION HISTORY EG Vitrification DMSO Vitrification 15% EG, 15% DMSO + 0.75-0.5M sucrose Vitrification 16% PROH, 16% EG + 0.65M sucrose 1998 Mukaida et al. Cleavage stage 79% 2001 El-Danasouri and Selman Cleavage stage 49.3% 2002 Cho et al. Blastocyst 2005 Rama Raju et al. Cleavage stage 95.3% 1999 Hsieh et al. Cleavage stage 62.5% 2000 Yokota et al. Blastocyst 2003 Venderzwallen et al. 2005 Kuwayama et al. Cleavage stage 98% 2006 Mukaiada at al. Artificial shrinkage of blastocyst 2007 Desai et al. Cleavage stage 85% 2009 Ebner et al. Closed system 2010 Kahraman and Candan Day 4 embryos 85.9% Biopsied day 4 89.8% 2015 Decrock et al. Cleavage stage vitrification RCT 2008 Balaban et al. RCT Cleavage stage 94.8%

Minimum Volume – Open System Approach Concentration of cryoprotectant in VS 30% (EG and DMSO) Volume of 0.1 µL Cooling rate of approximately 23,000ºC/min Osmolarity of VS ≈8.000 to 4.000 mOsm/L Kuwayama et al., 2005, 2007 Ana Cobo, 2008

Open vs closed system Efficiency and safety To avoid possible contamination sterile nitrogen can be used (Vajta et al., 1998, Parmegiani et al., 2009). In alternative, specific devices able to avoid direct contact of the samples with the nitrogen during vitrification (closed system) and/or storage are available (Kuleshova and Shaw 2000, Isachenko et al., 2006, Abdelhafez et al., 2011, Vanderzwalmen et al., 2009, Parmegiani 2011).

The revolution of vitrification approach – in the lab

Vitrification has changed the structure of the lab - Incredible improvements in survival rate (also for oocytes and biopsied embryos) - New timings of cryopreservation (according to cell developmental stage) - New flexibility of the lab. © Merck KGaA Darmstadt/Germany

Slow-freezing vs vitrification: clinical pregnancy and survival rates RCTs Cohort studies A typical laboratory could improve a ≈60% embryo survival rate using slow-freezing to somewhere between 74% and 99% by using vitrification  Rienzi et al., Hum Reprod update, 2016

Vitrification is successful for biopsied embryos Joris et al., Hum Reprod. 1999 Reduced survival after human embryo biopsy and subsequent cryopreservation. Jericho et al, Hum Reprod. 2003 A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos. Escribá et al., Fertil Steril. 2008 Vitrification of preimplantation genetically diagnosed human blastocysts and its contribution to the cumulative ongoing pregnancy rate per cycle by using a closed device. Zhang et al., Reprod Biomed Online. 2009 Vitrification of biopsied embryos at cleavage, morula and blastocyst stage. Chang et al., Hum Reprod. 2013 Blastocyst biopsy and vitrification are effective for preimplantation genetic diagnosis of monogenic diseases. Schoolcraft et al., Fertil Steril. 2011 Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based comprehensive chromosome screening in infertile patients.

Vitrification is successful for all stages of development: including oocytes

It has been difficult to overcome the intrinsic sensitivity of the cell zona pellucida hardening Cytoplasmic and cytoskeleton damage Oocyte ageing membrane permeability Meiotic spindle depolymerization Polar body degeneration/fusion Impact on oocyte physiology

Oocyte vitrification validation 2008: Efficiency in donation program not compromised with vitrification (Cobo et al., 2008; Nagy et al., 2008) 2008: The clinical pregnancy rate double with the introduction of vitrification (Tulandi, 2008; Cao et al., 2009; Smith et al., 2010 (RCT)) 2010: Prospective randomized study with own sibling oocytes demonstrates the lab efficiency of the technique (RCT) (Rienzi et al., 2010) 2010: Cumulative ongoing pregnancy rate with oocyte vitrification in a standard infertility program (Ubaldi et al., 2010) 2010: Prospective randomized study with donor oocytes demonstrates clinical efficiency of the technique (RCT) (Cobo et al., 2011) Prospective randomized study comparing slow freezing cs vitrification (RCT) (Smith et al., 2010) 2011: Efficiency of oocytes vitrification in the infertile population (RCT) (Parmegiani et al., 2011) 2012: Multicentric longitudinal cohort study to confirm reproducibility (Rienzi et al., 2012) Meta-analysis of randomized controlled studies (Oktay et al. 2006, Cobo et al., 2011; Cil 2013, Glujovky et al., 2014, Potdar et al 2014)

Pregnancy rate: Vitrification vs slow freezing

Oocyte survival: Vitrification vs slow freezing Rienzi et al., Hum Reprod update, 2016

RCT: cryo-banked oocytes Vitrification has almost allowed overpassing the gap in lives birth between fresh and cryopreserved oocytes in good prognosis patients.

Oocyte cryopreservation applications in ART Oocyte cryopreservation is an emerging discipline that has already a key place for different applications: Fertility preservation for medical reasons Fertility preservation for social reasons Use of cryo-banked oocytes for egg donation Avoids the production of supernumerary embryos in IVF Accumulation of excess oocytes in IUI cycles Accumulation of oocytes in case of poor response Fondation to build a beautiful house we need solid fondation © Merck KGaA Darmstadt/Germany

Medical indications In ITALY: 61.000 women between 10-30 years at risk of premature ovarian failure (ISTAT 2012) 75.000 women suffering endometriosis (Benaglia et al, 2010) 2.500 women <40 years with diagnosis of breast cancer Oktay et al., 2010

Clinical live births in cancer patients

Personal considerations The issue of persevering oocytes instead of embryos is of considerable importance. Oocyte-cryopreservation gives women a reproductive autonomy. Instead, the use of male-partner/donor sperm to create embryos introduces several ethical, moral and legal concerns. For this reason, we emphasize that there are scientific, moral and ethical reasons to promote oocyte (and NOT embryo) cryopreservation as gold standard in female onco-fertility (women and post-puberal girls, when possible before oncological treatment). Rienzi and Ubaldi, 2015 

Cryopreservation can be personalized for each embryonic stage Vitrification can be applied for all stages of development: at the right moment before oocyte aging before singamy when viability is assessed according to the expansion EGA Cryopreservation can be personalized for each embryonic stage

The revolution of vitrification approach – clinical implications

© Merck KGaA Darmstadt/Germany Efficient cryopreservation program for oocytes and embryos Enhances cumulative live birth rate per oocyte retrieval cycle Allows systematic application of elective single embryo transfer policy Provides the opportunity to perform cycle segmentation Extends time for embryo evaluation Permits fertility preservation for medical and non-medical indications Enables egg banking for donation and/or for oocyte accumulation © Merck KGaA Darmstadt/Germany

Elective SET is equally effective (cumulatively) but significanlty safer than double ET meta-analysis of individual patient data from 8 RCTs CUMULATIVE LIVE BIRTH RATE RISK OF MULTIPLE BIRTH eSET (n = 683) Double ET (n = 684) Now what is the right number of embryos to transfer. Today we know!! This number is ONE single embryo per transfer: Why? Because we have cumulatively the same livebirth rate… BUT it is more safe!!!! Less risk multiple pregnancy.. Essential to be guarranted to the patients. Essential if we want healphy child Adapted from McLernon, et al. BMJ., 2010 © Merck KGaA Darmstadt/Germany

Contribution of cryo-cycle to LBR It has been estimated that in Europe in 2011, cryopreservation contributed to the overall live birth rate by 4% (raising from 19.7% with only fresh cycles to 24.0% including cryo-cycles) (Kupka et al., 2016). In countries where cryopreservation is systematically applied, the rise is even more significant: Finland +13.4%, Switzerland +10.2% and Australia and New Zealand +13,5% (Kupka et al., 2016, Macaldowie et al., 2012). These data reflect the improvements obtained in embryo culture, cryopreservation and/or the more conservative embryo transfer policy adopted.

Cycle segmentation SEGMENT A SEGMENT B GnRH AGONIST TRIGGERING Lower extrauterine pregnancies (Shapiro et al., 2012; Li et al., 2015) Improved LBR (Roque et al., 2013) GnRH AGONIST TRIGGERING in a GnRH ANTAGONIST CYCLE Almost no ovarian hyperstimulation sindrome (Youssef et al., 2014 ) SEGMENT A SEGMENT B What can we infer from these evidences? The best stimulation should be performed to fully exploit ovarian reserve WITHOUT exposing the patient to hyperstimulation risks.. This can be done by agonist triggering and cryopreservation of all oocytes/embryos/blastocyst. Today possible with vitrification approach Transfer in a receptive endometrium.. To be improved by new approaches of endometrial receptivity assessment, lower risks of extrauterine pregnancies has been reported Embryo transfer in a receptive endometrium Devroey, et al. Human Reproduction, 2011. © Merck KGaA Darmstadt/Germany

Cycle segmentation for PGD-A in AMA Elective single embryo transfer policy combined with enhanced embryo selection and vitrification is a realistic option also in poor prognosis patients of advanced maternal age. First evidence – RCT – too low numbers but a beginning! To be continued!! © Merck KGaA Darmstadt/Germany

Oocyte accumulation for poor-responder patients Fondation to build a beautiful house we need solid fondation © Merck KGaA Darmstadt/Germany

The future is already here

Standardization of the technique Unlike slow freezing, vitrification does not need a freezing machine to provide specific cooling parameters. The technique is totally performed manually and is thus operator-dependent. Vitrification effectiveness may thus be highly variable and depending on the laboratory protocols and experience. The heterogeneity of methods applied cause also serious difficulties with transportation of vitrified samples between laboratories using different cryoprotectants mixture and/or vitrification carriers. A radical process of standardization is necessary.

Semi-automated vitrification is already available

Conclusions Cryopreservation is essential in ART and should be optimized in each single IVF laboratory. According to the updated evidences, vitrification is the best strategy for all stages of development and allows: Increased cumulative live birth, reduced multiple pregnancy and ovarian hyperstimulation syndrome rates; Female fertility preservation and cryo-egg donation programs; New perspectives for embryo evaluation are imaginable; If standardized and/or automated its consistency and efficiency could be assured. Validation is necessary to asses the efficiency and the power of the new technologies © Merck KGaA Darmstadt/Germany