Consensus meeting on fertility preservation Barcelona, June 6 th -7 th 2011 Oocyte cryopreservation: slow freezing and vitrification Laura Rienzi, Rome, Italy Senior Clinical Embryologist
Cryopreservation in ART Cryopreservation of gametes, embryos and blastocysts is an essential component of modern ART. Successful cryopreservation program: 1. allows to reduce the number of embryos transferred, thereby reducing multiple pregnancies and maximizing cumulative pregnancy rates per oocyte retrieval. 2. allows delayed embryo transfer during a natural menstrual cycle. 3. allows to preserve male and female fertility.
OUTLINES: oocyte cryopreservation Need Methods Efficiency Concerns
Oocyte cryopreservation Medical reason Malignant diseases Surgical ovary removal Polycystic ovary Hyperstimulation sydrome Premature ovarian failure
Oocyte cryopreservation Logistic reasons Sperm collection problem Legal reasons Restrictions in embryo cryopreservation Fate of embryos of separated couples Social reasons Wish to delay motherhood Moral reasons
Oocyte cryopreservation Oocyte donation Oocyte banks may result in - widespread availability - shortened, eliminated waiting list - safety (quarantine) - choice
OUTLINES Need Methods Efficiency Concerns
Vitrification is not a new technique it is more difficult to destroy a prejudice than an atom Albert Einstein Slow freezing of domestic animal embryos Slow freezing of human embryos Slow freezing of human oocytes Ultrarapid Vitrification with EM grids Slow freezing of mouse embryos Vitrification of mouse embryos Vitrification of mouse oocytes Vitrification of bovine blastocysts OPS Ultrarapid Vitrification Cryoloop Cryotip Ultrarapid Vitrification 1935 1948 1972 1973/1974 1983 1985 1989 1993 1996 1997 1999 Living matter can survive freezing, but only if the molecules are not ordered, but solidified where they are.. in disordered or uncrystallized form. Luyet 1935
Vitrification: biophysical aspects 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 Probability of vitrification = Cooling/warming rates * Viscosity Volume of the sample Types of cells
Biophysical aspects: binary phase diagram 20 C T 0 C Thermal hysteresis ~-40 C Heterogenous Ice phase 10-4 poise Liquid phase CPs Cooling rate -100 C -120 C Glass phase No cristal 10 13 poise Concentration of solute
Minimum volume-direct contact approach Volume of 0.1 µl Cooling rate can be increased to - 23.000ºC/min Required CPA concentration in VS 50% (v/v) to 30% (v/v) Osmolarity of VS ~8.000 to 4.000 mosm/l Ana Cobo, personal comunication
OUTLINES Need Methods Efficiency Concerns
Ooocyte sensitivity Cytoplasmic and Cytoskeletron damage Meiotic spindle depolymerization Oocyte ageing zona pellucida hardening membrane permeability Impact on oocyte physiology Polar body degeneration/fusion
Slow freezing Different protocols have been proposed: A) 1.5M PROH + 0.2/0.2 Sucrose: Gook et al., 1993; Porcu et al., 2000; Winslow et al., 2001; Yang et al.,2002 B) 1.5M PROH + 0.3/0.3 Sucrose: Fabbri et al.,2001; Fosas et al., 2003; Chen et al., 2005; Levi Setti et al., 2006; Borini et al.,2006; La sala et al., 2006; De Santis et al., 2007; Parmegiani et al., 2009 C) 1.5M PROH + 0.2/0.3 Sucrose: Bianchi et al., 2007; Borini et al., 2007 D) Sodium deplemented protocol: Quintas et al., 2002; Boldt et al., 2006; Petracco et al., 2006
Slow freezing vs vitrification Slow Freezing literature 1996-2005 Vitrification literature 2003-2005 Age, mean 33.7 32.3 Fertilization rate 64.9 (2,478/3,818) 74.2 (637/859) Clinical pregnancies per thawed oocyte Clinical Pregnancies per injected oocytes Clinical Pregnancies per transfer 2.3 x10-2 (153/6720) 4.5 x10-2 (61/1354) 4.0 x10-2 (153/3818) 7.2 x10-2 (61/859) 20.6 (153/742) 45.5 (61/134) Implantation rate 10.1 (185/1828) 17.2 (81/473) Oktay et al., 2006
Oocyte vitrification: what is the evidence? The clinical pregnancy rate has doubled with the introduction of vitrification (Tulandi, 2008) Efficiency in donation program not compromised with vitrification (RCT) (Cobo et al., 2007; 2010; Nagy et al., 2007) Prospective randomized study with own sibling oocytes demonstrates the Lab efficiency of the technique (RCT) (Rienzi et al., 2010) Cumulative ongoing pregnancy rate with oocyte vitrification without embryo selection in a standard infertility program is comparable to what obtained with embryo cryopreservation (Ubaldi et al., 2010) Schoolcraft et al., 2009; Chian et al., 2008; Kim et al., 2010
Clinical application: infertile population
Study design In order to validate the effectiveness of a vitrification approach for oocyte cryopreservation a prospective comparison was designed in our population of infertile patients (september 08 - march 09). This study was set-up as a non-inferiority trial with a prospective target of 240 sibling metaphase II oocytes obtained from an estimated 40 ICSI patients Oocyte fertilization rates after ICSI (per warmed oocyte and per injected oocyte) were evaluated as primary outcomes. Secondary outcomes were pronuclear morphology and embryo development Rienzi et al., Human Reproduction 2010
Material & Methods The general idea of the study was to minimize extra stress on oocytes often related with cryopreservation procedures, namely: 1. Long exposure to Hepes buffered media, with uncertain temperature control, for oocyte denudation and selection under the inverted microscope 2. Prolonged oocyte in vitro culture without the protection of cumulus and corona cells 3. Oocyte ageing In this way, by using randomized sibling oocytes the only difference between the fresh and the vitrified group was the vitrification procedure itself followed by 2 hours of in vitro culture. Rienzi et al., Human Reproduction 2010
Patient population Rienzi et al., Human Reproduction 2010
Laboratory outcomes 17.2% ongoing implantation rate per transferred embryo 12.9% ongoing implantation rate per warmed oocyte Rienzi et al., Human Reproduction 2010
Cumulative pregnancy rates www.generaroma.it
Study design o The study was design as a prospective longitudinal cohort study. o The baseline characteristics, embryological data, clinical and ongoing pregnancy rate were analyzed on a per cycle basis. o The cumulative pregnancy rate obtained with fresh and vitrified oocytes from the same stimulation cycle was analyzed on a per patient basis. Ubaldi et al., Human Reproduction 2010
Material & Methods o All consecutives patients undergoing ICSI treatment in the Centre for Reproductive Medicine GENERA between September 2nd 2008 and May 15th 2009 were considered for this study o Only patients with supernumerary oocytes available for cryopreservation were included. A single fresh attempt was included for each patient. Ubaldi et al., Human Reproduction 2010
Laboratory results 44.6% of our patients, 39.9% of cycles Ubaldi et al., Human Reproduction 2010
Clinical results Ubaldi et al., Human Reproduction 2010
Clinical results P=0,006 647 vitrified oocytes are still available Ubaldi et al., Human Reproduction 2010
Oocyte vitrification: clinical application in donor program
Oocyte vitrification: clinical application in donor program
Oocyte vitrification: efficacy o Embryo development in the laboratory is not affected by the vitrification procedure o High ongoing pregnancy rates are achieved in standard infertility program and egg donation program with transfers of embryos derived from vitrified oocytes o Among various infertility factors, only female age influenced significantly the outcome o The overall efficiency justifies the application of this strategy in routine infertility work
Multicentric longitudinal cohort study All consecutive oocyte warming cycles performed in standard infertile population between september 2008 and May 2010 in 3 different centres: -GENERA Rome -IVI Valencia -Ospedale Mangiagalli Milan Vitrification procedure was performed according to Cobo et al., 2007; 2010; Rienzi et al., 2010. AIM of the study is to evaluate the effect of patients and cycles characteristics (female age, infertily factor, stimulation protocol, sperm quality, number of oocytes retrieved, number of oocytes vitrified, oocyte incubation time, day of transfer) on outcomes. PRIMARY OUTCOME MEASURE: Delivery
Patients and cycles characteristics Number % Infertility factor male 165/488 33.8 female tubal 66/488 13.5 endometriosis 31/488 6.4 ovulatory 73/488 15.0 idiopathic 84/488 17.2 combined 26/488 5.3 other 43/488 8.8 Stimulation Protocol (fresh cycle) agonist 278/488 58.0 antagonist 210/488 42.0 Sperm origin >1000,00/ml (ejaculated) 469/488 96.1 <1000,00/ml (ejaculated) 4/488 0.8 Surgically extracted 15/488 3.1
Fresh and warming cycles characteristics N (mean) SD Fresh cycles Female age 35.99 3.93 CCOCS retrieved 4949 (10.9) 6.34 MII 3899 (8.63) 4.71 Vitrified MII 2965 (6.56) 4.01 Warming cycles Warmed MII 2740 (5.61) 3.37 Survived MII 2321 (4.76) 0.85 Inseminated oocytes 2183 (4.47) 0.94 Fertilized oocytes 1655 (3.39) 2.41 Excellent and good quality embryos 915 (1.88) 1.64 Embryo transferred 929 (1.90) 0.92 Embryos crypreserved 187 (0.38) 1.05
Warming cycles clinical outcomes N % Embryo transfers 436/488 89.3 Clinical Pregnancy rate per cycle 166/488 34.0 Clinical Pregnancy rate per transfer 166/436 38.1 Implantation rate 186/929 20.0 Abortion rate 37/166 22.3 Delivery rate per cycle 129/488 26.4 Delivery rate per transfer 129/436 29.6 Babies born 147/929 15.8
Factors influencing the outcome: delivery rate Multivariate Logistic Regression Analysis B P OR 95% CI Female age -0.05 0.03 0.94 0.88-0.98 Number of vitrified oocytes 0.06 0.01 1.07 1.01-1.13 Day of transfer 0.33 0.02 1.39 1.04-1.88
0 = not obtaind 1 = obtained DELIVERY RATE PER CYCLE % n 0.000 73.6 359 1.000 26.4 129 total 100.0 488 Number of vitrified MII Improvement = 0.015 8 MII % n 0.000 78.8 279 1.000 21.2 75 total 72.5 354 > 8 MII % n 0.000 59.7 80 1.000 40.3 54 total 100.0 134 Female age Improvement = 0.006 Day of transfer Improvement = 0.015 38 years > 38 years 3 days > 3 days % n 0.000 74.4 183 % n 0.000 88.9 96 % n 0.000 69.0 69 % n 0.000 32.4 11 1.000 25.6 63 1.000 11.1 12 1.000 31.0 31 1.000 67.6 23 total 50.4 246 total 22.1 108 total 20.5 100 total 7.0 34
OUTLINES Need Methods Efficiency Concerns
Obstetric outcomes Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Castellón LA, García Amador MI, Montoya Sarmiento JE. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed online 2008 Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed online 2009
Toxicity and Sterility Concerns The most widely emphasized concerns are toxicity and danger of contamination. Unfortunately, available vitrification methods still struggle with these problems to date Son and Tan, 2009
Toxicity and Sterility OPEN systems are required for success (lower cryoprotectants concentration higher cooling warming rate) CLOSED systems are required for safety?? (same for slow freezing) Sterile LN2 for vitrification would be the solution. Unfortunately, sterilization in large volume is very difficult (impossible?)... but... Sterilization in small volume is possible: FILTRATION (Vajta et al., 1998) UV (Arav et al., 1998, Parmegiani et al., 2009) The problem of storage can be solved: in Vapor phase LN2 tanks or closed containers
Sterile Nitrogen: UV irradiation
Vitrification in ART Excellent survival and development ability; Consistent and reproducible results; Optimal timing of cryopreservation; EGA Survival 85 90% 90 95% 85 95% 70 95% Implantation 15%- 18% 15 20% 20-30% 25-40% To him who devotes his life to science, nothing can give more happiness than increasing the number of discoveries, but his cup of joy is full when the results of his studies immediately find practical applications Louis Pasteur
Conclusions - Vitrification procedure maintain oocyte competence to develop in vitro (also in the population of infertile patients). - Oocyte vitrification is effective to improve clinical results in ART (~60 ongoing cumulative clinical pregnancy rate in young patients) and can be applied for fertility preservation. - In a more general sense, oocytes cryopreservation may be regarded as a step towards decreasing differences between genders regarding choice in reproduction.
www.generaroma.it CLINICA VALLE GIULIA, Roma SALUS ASI MEDICAL, Marostica GENERA UMBERTIDE, Perugia Gynecology: Filippo Ubaldi Elena Baroni Silvia Colamaria Maddalena Giuliani Fabio Sapienza Embryology: Laura Rienzi Stefania Romano Laura Albricci Antonio Capalbo Roberta Maggiulli Benedetta Iussig Nicoletta Barnocchi