Oocyte cryopreservation: slow freezing vs. vitrification Ana Cobo IVI-Valencia, Spain ana.cobo@ivi.es www.ivi.es

Contents Cryopreservation Techniques Clinical outcome 3. Conclusions Slow freezing Vitrification Cryo-injury Clinical outcome Slow Freezing Slow Freezing vs. Vitrification Vitrification vs. Fresh oocytes 3. Conclusions Conclusions

Cryopreservation Techniques Slow freezing Vitrification Cryo-injury

Cryopreservation techniques Equilibrium Cryopreservation Procedures: When the osmotic equilibrium between the intra and extra cellular media is reached by applying very slow cooling rates. Slow Freezing: The cooling rate must be slow enough to allow a huge dehydration of the cell, thus avoiding the chance of intracellular ice formation.

Cryopreservation techniques Non Equilibrium Cryopreservation Procedures: It is not necessary to establish the osmotic balance between the intra and extracellular media along the period of cooling of the cells. Vitrificacation: The super-cooled intracellular water does not crystallize but turns into a very viscous solid of similar consistency to the glass.

Cryopreservation options Ice (Freezing) No Ice (Vitrification) Freeze Sol. Vitri

Benefits and disadvantages Slow freezing Vitrification Ice crystal ++++ / ++ - Chilling ++++ - / + Toxic ++ ++++ Osmotic ++ ++++ Mechanical +++ + Equipment ++++ - / ++ Skills ++ ++ Speed ++ ++++ Contamination + + / ++++ (???) Vajta Oocyte vitrification in ART

Injuries during cryopreservation Biological Factors (size, meiotic spindle) Technical Factors (cooling/warming rates, CPAs) Species, stage of development and origin of the samples Oocyte Vitrification in ART

Chilling: lipids of the cell membrane, meiotic spindle. Injuries during cryopreservation 2. Mechanisms of injury Chilling: lipids of the cell membrane, meiotic spindle. Ice Formation: from-5 and-15ºC to-80ºC. Intracellular ice formation (IIF) Osmotic damage Osmotic stress

Strategies to minimize injuries Slow-rate freezing: An attempt to maintain a balance between various sources of injury by using low CPA concentration and controlled ice formation Vitrification: Radical elimination of ice formation, and to reduce toxic and osmotic damage caused by high CPA concetrations Oocyte Vitrification in ART

Practical ways to achieve vitrification in embryology Cryoprotectant. concentration Practical ways to achieve vitrification in embryology Cooling-warming rates Oocyte vitrification in ART

Increasing the cooling rates How to perform? Use minimum volume in the loading solution allows to achieve extremely high cooling rates (Arav et al 1992) Establish direct contact Open Systems Oocyte vitrification in ART

Loading of Oocyte in minimum volume VS on Cryotop Cryotop Sheet X not minimum VS 〇　Minimum VS Oocyte in VS Cryotop Oocyte in VS Cryotop

Method of choice Cooling rate °C/min 20,000 2,000 Straw Cryotip Grid&loop OPS SOPS Cryotop Oocyte vitrification in ART

Clinical outcome Slow Freezing Slow Freezing vs. Vitrification Vitrification vs. Fresh oocytes Vitrification

Slow Freezing 1st generation protocols 2nd generation protocols EQ: 1.5M PROH / 0.3 M suc Dilution: PROH (1.0; 0.5; 0.2 M) /0.3 Msuc. Fabbri et al, 2001 Preg Impl EQ: 1.5M PROH / 0.1 M suc Dilution: PROH (1.0; 0.5; 0.2 M) /0.2Msuc Lasalle et al, 1985 Ref. Al-Hasani et al., 1987 Gook et al., 1994 Tucker et al., 1998 Porcu et. al, 2000 Borini et.al, 2004 Surv. (%) 33.9 41.0 24.0 46.8 37* Nº ovo 159 134 311 1640 737 1st generation protocols Pregnancy 25.4% Implantation 16.4%

Slow Freezing 2nd Generation Protocols 3rd Generation Protocols Replacement of Na+ for Choline EQ: 1.5M PROH HTF+ o PBS+/0.3M suc (26,3) (37,5) 2nd Generation Protocols 3rd Generation Protocols EQ: 1.5M PROH / 0.3 M suc Dilution: PROH (1,0; 0.5; 0,2 M) /0.2 M suc. Fabbri et al, 2001 % Bianchi, et.al., 2007

Eight centers Cycles involving MII cryopreservation between January 2006 and April 2008 “Under the conditions tested, the clinical outcome of oocyte slow-cooling cryopreservation is reduced compared with fresh cycles”.

Slow Freezing vs. Vitrification Human oocyte cryopreservation:comparison between slow and ultrarapid methods. Paffoni et. Al. RBM online 2008 Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and Vitrification. Cao et. al .Fertil Steril. 2009. Slow freezing or vitrification of oocytes: Their effects on survival and meiotic spindles, and the time schedule for clinical practice. Chen et.al. Taiwan J Obstet Gynecol 2009. Human oocyte cryopreservation: comparison between slow and ultrarapid methods. Fadini et. al. RBM on line 2009. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Smith et.al. Fertil. Steril. 2010. Oocyte vitrification is associated with a better outcome than the slow-freezing method.

Vitrified vs. Fresh oocytes Delivery rate using cryopreserved oocytes is comparable to conventional in vitro fertilization using fresh oocytes: potential fertility preservation for female cancer patients Griffo et al. 2009 Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Cobo et.al. Fertil. Steril. 2008. Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking Nagy et.al. Fertil. Steril. 2008. Embryo development of fresh ‘versus’ vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Rienzi et.al. Human Reprod. 2009. Embryo development and gestation using fresh and vitrified oocytes Almodin et.al. Human Reprod. 2010. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical. Cobo et.al. Human Reprod. 2010. Oocyte vitrification does not impair the outcome as compared with fresh oocytes

To identify randomized controlled trials that assessed the efficiency of oocyte vitrification compared to SF cryopreservation methods and fresh cycles (PRISMA statement principles). Oocyte vitrification in ART

Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials 2,799 records 16 assessed for eligibility 5 included Uni-centric studies (4282 vitrified oocytes, 3524 fresh oocytes and 361 slow-frozen oocytes). 2005 and 2009. Only MII oocytes. Three vitrification protocols using different cryo-protectant combinations and devices were analyzed: Cryotip® (349 oocytes), Cryotop® (2645 oocytes) and Cryoleaf® Cobo, Díaz, Fertil. Steril. 2011

Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials Oocyte vitrification in ART

Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials There were no differences regarding the fertilization rate when vitrified oocytes were compared to fresh ones (OR 1.02, 95% CI [0.91, 1.13]; heterogeneity: p=0.17, I2=44%, fixed effects model) but higher fertilization rates were observed when vitrification was compared to SF (OR 1.50, 95% CI [1.07, 2.11]; heterogeneity: p=0.56, I2=0%, fixed effects model) (Figure 3). Coclusions This review supports the hypothesis that the potential of fertilization, embryogenesis and pregnancy from oocytes derived from vitrification/warming cycles is not significantly different to that of fresh oocytes. It also suggests that oocytes coming from vitrification/warming cycles could result in better survival and fertilization rates than those coming from SF/thawing cycles. Vitrification also resulted in a higher rate top-quality embryo (22.4% vs. 8.0%, OR 3.32, 95% CI [1.37-8.02], p<0.01) and embryo cleavage rate (day 2: 64.6% vs. 47.7%, OR 2.00, 95% CI [1.33-3.00], p<0.01; day 3: 53.0% vs. 33.3%, OR 2.25, 95% CI [1.32-3.85], p<0.05) as compared to SF. In conclusion, based on the evidence provided by the randomized studies available, vitrification appears to be an efficient method to cryopreserve oocytes. However, more large-scale controlled trials, aimed at evaluating clinical outcomes after oocyte vitrification, will be required to further strengthen this conclusion. Oocyte vitrification in ART

To compare the outcome of vitrified-banked oocytes to the gold standard procedure employing fresh oocytes. Primary end point. Ongoing pregnancy rate (OPR) per intention to treat (ITT) population. Secondary outcomes. clinical pregnancy rate implantation rate fertilization rate embryo quality

Results Cobo et al Hum Reprod, 2010

Results 0.921 (0.667-1.274) Superiority of the OPR of the fresh group was not established Non-inferiority of vitrified group was shown with a margin of 0.667 (above the non-inferiority limit of 0.660) Cobo et al Hum Reprod, 2010

Donors base line characteristics Recipients base line characteristics 4 years-experience of an ovum donation program using cryo-banked oocytes Donors base line characteristics Recipients base line characteristics 95% CI Number of donors or recipients 1187 NA 1602 Nº donation cycles 1856 Mean donations/donor or recipient 1.6 1.4-1.8 1.2 1.0-1.4 Age (years) 27.1 26.9-27.3 41.1 40.9-41.3 BMI (Kg/m2) 22.5 22.3-22.7 23.7 23.5-23.9 Days of stimulation/endometrial preparation 12.2 11.3-13.2 15.4 13.6-17.1 rFSH dose (IU) 1761 1638.7-1883.4 E2 on day of hCG (pg/ml) 2726.6 2483.6-2969.6 329.5 301.3-357.8 P4 (ng/ml) 0.9 0.8-1.0 0.4 0.3-0.5 MII oocytes retrieved or received/donation cycle (mean) 22741 (12.2) 12.0-12.4 11.4-13.0 Survival rate (%) 20669 (90.1) 89.7-90.5 Mean oocyte survival /donation cycle 20669 (11.1) 10.9-11.3 Cobo et al ESHRE 2011. 4 years-experience of an ovum donation program using cryo-banked oocytes.

Results IVF outcome 95%CI Mean MII oocytes injected/donation cycle 20669 (11.1 ) 10.3-11.9 Fertilization rate 15163(73.4) 72.8-74.0 Cleavage day-2 14346 (94.6) 94.2-95.0 Cleavage day-3 12028 (83.8) 83.2-84.4 Top quality day- 2 /cleaved embryo 5998 (41.8) 41.0-42.6 Top quality day- 2 /injected oocyte 5998 (29.0) 28.4-29.6 Top quality day- 3/cleaved embryo 6323 (52.6) 51.7-53.5 Top quality day- 3/injected oocyte 6323 (30.5) 29.9-31.1 Blastocyst rate/embryo subjected to extended culture 4779 (58.9) 57-8-60.0 Good quality blastocyst 1773 (37.8) 36.4-39.2 Top quality blastocyst 1747 (36.5) 31.1-37.9 Cobo et al ESHRE 2011. 4 years-experience of an ovum donation program using cryo-banked oocytes.

Results Clinical outcome 95% CI Number of embryo transfers/donation 1607 (86.6) 85.1-88.2 Number of day-3 transfers 766 (47.7) 45.3-50.1 Number of blastocyst transfers 841 (52.3) 49.9-54.7 Number of embryos replaced (Mean ± SD) 3037 (1.6 ± 0.7) 1.2-2.0 Number of embryo cryopreservation cycles/donation cycle 1181 (63.6) 61.4-65.8 Mean number of re-vitrified embryos (Mean ± SD) 3491 (1.9 ± 2.0) 1.7-2.0 Implantation rate 1274 (41.9) 40.2-43.7 Implantation rate/day-3 embryo transfer 545/1447 (37.7) 35.2-40.2 Implantation rate/blastocyst embryo transfer 729/1590 (45.8) 43.4-48.3 Ongoing pregnancy /cycle 767 (41.3) 39.1-43.5 Ongoing pregnancy /embryo transfer 767 (47.7) Ongoing pregnancy / day-3 embryo transfer 348 (45.4) 41.9-48.9 Ongoing pregnancy /blastocyst embryo transfer 419 (49.8) 46.4-53.2 Number of Cryo-transfers 423/1181 (35.8) 33.1-38.5 Mean number of embryos replaced 2.2 ± 0.7 2.1-2.3 Ongoing pregnancy rate 178/423 (42.1) 37.4-46.8 Number of babies born (deliveries) “fresh ET” 604 (465) Number of babies born (deliveries) “Cryo-transfers” 97(75) 1274 (41.9) P<0.05 767 (41.3) 767 (47.7) NS 178/423 (42.1) Cobo et al ESHRE 2011. 4 years-experience of an ovum donation program using cryo-banked oocytes.

Results COPR according to the number of oocytes consumed. Kaplan-Meier plotting Oocytes consumed b. Day-3 and Blastocyst transfer Day-3 Blastocyst Day-3 cesnsored Blastocyst censored Oocytes consumed a. Overall data Survival fuction Censored 35 oocytes= COPR 97.6% * *P<0.05 (Long rank Breslow and Tarone-Ware tests) 20 oocytes= COPR 76.1% 13 oocytes= COPR 69.9% 16 oocytes= COPR 70.7% 15 oocytes= COPR 58.8% 13 oocytes= COPR 50% Cobo et al ESHRE 2011. 4 years-experience of an ovum donation program using cryo-banked oocytes.

“The excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg-cryobank and makes it likely that this approach becomes the future standard of care”.

Oocyte vitrification in infertile patients N (mean SD) Rank Nº patients 308 Age 35.1 ± 4.3 26-44 Nº aspirated oocytes 3534 (14.8 ± 7.9) 1-46 Nº vitrified oocytes 3104 (10.7 ± 5.0) 1-25 Nº warmed oocytes 2986 1-18 Survival N(%) 85.8 Per transfer Per cycle +BHGC CPR OPR IR Oocyte vitrification in ART

Oocyte vitrification in LR patients. Accumulation of oocytes COPR depending on the number of oocytes used, including fresh and warmed (accumulated) Kaplan Meier survival Curves Overall outcome <39 years old ≥39 years old Cobo et al., submitted Oocyte vitrification in ART

P=0,006 Oocyte vitrification in ART

Absolute number Mean (95%CI) % (95%CI)* High, consistent and predictable pregnancy rates after oocyte vitrification: a prospective longitudinal multicentric cohort study Rienzi L. 1, Cobo A. 2, Paffoni A. 3 et al., 1GENERA Centre for Reproductive Medicine, Rome, Italy; 2IVI Valencia, Spain; 3Fondazione Ca' Granda Ospedale Maggiore Policlinico, Infertility Unit, Milan, Italy  Characteristics Absolute number Mean (95%CI) % (95%CI)* Cycles 486 Warmed oocytes 2721 5.62 (5.32 - 5.93) Survived oocytes 2304 4.78 (4.54-5.03) 84.7 (83.3-86.0) Inseminated oocytes 2182 4.53 (4.28-4.77) 94.7 (93.7-95.5) Fertilized oocytes 1642 3.41 (3.19-3.62) 75.2 (73.4-77.0) Top quality embryos 796 1.89 (1.57-2.26) 48.1 (45.69-50.51) Embryos transferred 929 1.91 (1.83-1.99) Embryos cryopreserved 184 0.38 (0.29-0.48) Delivery rate per cycle per transfer 128/488 128/450 26.3(22.6-30.4) 29.4 (25.3-33.8) Newborn 147 15.8 (13.62-18.31) Rienzi et al; ESHRE 2011

Decisional making model based on recursive partitioning analysis (per patient basis) % N 0.000 71.6 322 1.000 28.4 128 Total 100.0 450 0 = delivery not obtained 1 = delivery obtained Number of vitrified MII ≤ 8 MII > 8 MII By recursive partitioning analysis it can be estimated that more than 8 oocytes vitrified are required to improve the outcome (22.6 vs 46.4% DR, respectively). % n 0.000 77.4 263 1.000 22.6 77 Total 75.6 340 % n 0.000 53.6 59 1.000 46.4 51 Total 24.4 110 Rienzi et al; ESHRE 2011 Reproductive clock ≤ 38 years > 38 years ≤ 3 days > 3 days % n 0.000 72.5 168 1.000 27.5 63 Total 50.9 229 % n 0.000 87.4 97 1.000 12.6 14 Total 24.7 111 % n 0.000 59.3 48 1.000 40.7 33 Total 18.0 81 % n 0.000 37.9 11 1.000 62.1 18 Total 6.4 29 Fig.1 Decisional making model based on recursive partitioning analysis (per patient basis)

Decisional making model based on recursive partitioning analysis (per patient basis) ≤ 8 MII > 8 MII Female age Day of transfer ≤ 38 years > 38 years ≤ 3 days Blastocyst % n 0.000 72.5 168 1.000 27.5 63 Total 50.9 229 % n 0.000 87.4 97 1.000 12.6 14 Total 24.7 111 % n 0.000 59.3 48 1.000 40.7 33 Total 18.0 81 % n 0.000 37.9 11 1.000 62.1 18 Total 6.4 29 Rienzi et al; ESHRE 2011

Perinatal outcome of child born after oocyte vitrification Perinatal outcome of child born after oocyte vitrification. Paired cohorts study. Perinatal outcome Vitrified Fresh Nº Deliveries 160 262 Nº New born 212 315 Perinatal mortality 3 (1.4) Mean maternal age at delivering 39.2 ± 4.9 39.6 ± 5.3 Mean gestational age (weeks) 37.4 ± 2.5 38.0 ± 2.3 No. of deliveries at <37 weeks (%) 37 (23.1) 47 (17.9) No. of deliveries at <34 weeks (%) 9 (5.6) 12 (4.6) Birth weight (mean ± SD) 2,718 ± 0.668 2,896 ± 0.658 No. of LBW (%) <2500gr. 65 (17.4) 75 (23.8) No. of VLBW (%) <1500 gr. 14 (1.9) 13 (4.1) Median Apgar score at 1 min. 8.9 ± 0.6 8.9 ± 0.8 Median Apgar score at 5 min. 9.3 ± 0.6 9.6 ± 0.3 Sex of new born. Female. 113 (53.7) 172 (54.6) Sex of new born. Male. 99 (46.3.) 143 (45.4) Incidence of major congenital anomalies 4(1.8) 2 (0.6) This slide shows the perinatal outcome of 147 new born after OV NS Cobo, Serna. Hum Reprod 2011

Conclusions Vitrified oocytes are currently offering excellent clinical outcomes either for ovum donation programs or for infertile patients. The safety of the strategy is being confirmed by the obstetric and perinatal outcome assessment.

Making possible the establishment and well functioning of this program