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Human oocyte cryopreservation: comparison between slow and ultrarapid methods
RBMOnline - Vol 19 No 2. 2009 171-180 Reproductive BioMedicine Online; www.rbmonline.com/Article/3843 on web 21 May 2009
Article Human oocyte cryopreservation: comparison between slow and ultrarapid methods Dr Fadini obtained his speciality in Gynaecology and Obstetrics and in Human Reproduction from the University of Milan, Italy. From 1985 to 1999 he directed the Unit of Reproductive Medicine and Endocrinology at San Gerardo University Hospital in Monza, University of Milan. He is Assistant Professor in the School of Specialization in Gynaecology and Obstetrics and at the School of Medical Biotechnologies, Faculty of Medicine, University of Milan-Bicocca. He has been Director of the Unit of Gynaecology and Obstetrics and Chief of the BIOGENESI Reproductive Medicine Centre at the Istituti Clinici Zucchi, in Monza since 2000.
Dr R Fadini R Fadini1, F Brambillasca1, M Mignini Renzini1, M Merola1, R Comi1, E De Ponti2, MB Dal Canto1,3 1 BIOGENESI Reproductive Medicine Centre, Istituti Clinici Zucchi, V. Zucchi, 24-Monza, Italy; 2Department of Medical Physics San Gerardo Hospital, V. Pergolesi 33, Monza, Italy 3 Correspondence: e-mail: [email protected]
Abstract The success of reproductive technologies is facilitated by the cryopreservation of embryos and gametes. In Italy, where legislation prohibits zygote and embryo cryopreservation, clinics have extensively introduced oocyte cryopreservation. Two different strategies of oocyte cryopreservation are available: slow freezing or ultrarapid cooling (vitrification). Although the results are very encouraging with both methods, there is still controversy regarding both the procedure itself and the most suitable method to use. This study reports the routine application of the two different oocyte cryopreservation methods in programmes running in two consecutive periods. The study centre carried out 286 thawing cycles for a total of 1348 thawed oocytes cryopreserved by the slow-freezing method and 59 warming cycles for a total of 285 warmed oocytes cryopreserved by vitrification. Comparison of the outcomes obtained with the slow-freezing method versus vitrification in women who underwent IVF for infertility showed survival, fertilization, pregnancy and implantation rates of 57.9% versus 78.9% (P < 0.0001), 64.6% versus 72.8% (P = 0.027), 7.6% versus 18.2% (P = 0.021) and 4.3% versus 9.3% (P = 0.043) respectively. These results suggest that oocyte vitrification is associated with a better outcome than the slow-freezing method. Keywords: cryoleaf, human oocyte cryopreservation, slow freezing, survival rate, vitrification
Introduction The wide use of ovarian stimulation protocols in assisted reproduction treatments has allowed the collection of a large number of oocytes; as a consequence, many embryos have been produced. Hopefully, all these efforts have significantly improved the success of IVF, and undoubtedly they have induced researchers to find methods of cryopreserving such numerous oocytes and embryos. At present embryo freezing is a highly reliable procedure and certainly reproductive techniques have been facilitated through the cryopreservation of surplus embryos which can improve treatment success and reduce multi-fetal pregnancies (Ashwood-Smith, 1986; Mandelbaum et al., 1987; Osmanagaoglu et al., 2004; Stehlik et al., 2005; Andersen et al., 2008). Oocyte cryopreservation is a more recent procedure that provides an alternative to embryo freezing without ethical and
religious problems and that can also be used to preserve fertility in patients at risk of ovarian failure. So far two methods have been proposed to freeze human oocytes, slow freezing and ultrarapid cooling. The slow-freezing method was the first to be introduced and the first pregnancy with a cryopreserved oocyte was obtained in 1986 (Chen, 1986). Only a few pregnancies were reported during the subsequent years because the method needed to be standardized. It took 10 years before suitable protocols were proposed to increase the efficiency of the slow-freezing method (Porcu et al., 1997, 1999, 2000; Fabbri et al., 2001). In recent years, the protocol has been better standardized by many Italian researchers; more favourable success rates using slow freezing have been reported and the number of pregnancies recorded has increased (Borini et al., 2004, 2006, 2007a,b; La Sala et al.,
© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
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Article - Slow and ultrarapid cryopreservation of human oocytes - R Fadini et al.
2006; Levi Setti et al., 2006; Bianchi et al., 2007; De Santis et al., 2007; Parmegiani et al., 2008). Another strategy to cryopreserve oocytes and embryos is the ultrarapid cooling or vitrification that allows glass-like solidification of a solution without ice-crystal formation. This technology has been widely used in animals but only recently has been studied in humans (Feichtinger et al., 1991; Mukaida et al., 1998; Vajta et al., 1998; Mauri et al., 1999; Chen et al., 2001; Vanderzwalmen et al., 2002; Son et al., 2003; Chian et al., 2004; Huang et al., 2007). The vitrification of embryos or blastocysts and more recently zygotes with good results has been extensively applied and accepted in the past few years (Mukaida et al., 2003; Vanderzwalmen et al., 2003; Huang et al., 2005; Kuwayama et al., 2005a; Koutlaki-Kourti et al., 2006; Liebermann and Tucker, 2006; Al-Hasani et al., 2007; Balaban et al., 2008; Youssry et al., 2008). The first birth after human oocyte vitrification was recorded in 1999 with a standardized protocol used on animals (Kuleshova et al., 1999). In the last decade, although few papers on the clinical outcome of oocyte vitrification have been published, very good results have been reported (Hong et al., 1999; Yoon et al., 2000, 2003; Katayama et al., 2003; Wright et al., 2004; Chian et al., 2005; Kuwayama et al., 2005b; Okimura et al., 2005; Lucena et al., 2006; Selman et al., 2006; Antinori et al., 2007). The study centre has been freezing oocytes since 2004, using the slow-freezing method, after many years of experience with slow embryo freezing. In 2007, vitrification was introduced to cryopreserve surplus oocytes produced during assisted reproduction procedures. The results obtained with the routine application of human oocyte cryopreservation performed with both freezing methods, applied in two consecutive periods, have been retrospectively analysed in order to discuss the viability and reliability of the two techniques.
Materials and methods This retrospective study included 254 women undergoing the IVF programme at Biogenesi Reproductive Medicine Centre, in whom a total of 345 oocyte thawing cycles were performed from April 2004 to December 2007. In this time no changes were made to the laboratory culture system.
Slow-freezing protocol From April 2004 to December 2006, 255 women undergoing IVF treatment agreed to freeze their surplus eggs using the slow-freezing protocol after signing a consent form. To date, out of 2291 oocytes cryopreserved by the slow-freezing protocol, 1348 oocytes from 208 patients have been thawed in 286 cycles.
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The cryopreservation protocol consisted of the slowfreezing and rapid-thawing method described by Fabbri et al. (2001). Freezing and thawing solutions were supplied by MediCult, containing 1,2-propanediol (PROH) and sucrose as cryoprotectants (nos. 10485010 and 10494010, respectively, MediCult, Denmark).
The oocytes were washed in Vial 1 solution at room temperature. Afterwards the oocytes were equilibrated for 10 min in Vial 2 solution and then transferred into Vial 3 solution for a maximum of 1 min. From one up to a maximum of three oocytes were loaded into a plastic straw (Paillettes Cristal 133 mm; Cryo Bio System, France) and transferred into an automatic biological vertical freezer (Kryo 360–1.7, Planer, UK). The cooling process was initiated reducing chamber temperature from 20oC to –7oC at a speed of freezing of 2oC/ min. Manual seeding was induced at –7oC. After a hold time of 10 min at –7oC, the straws were cooled slowly up to –30oC at a rate of freezing of 0.3oC/min and then rapidly to –150oC at a rate of 50oC/min. Then the straws were transferred into a canister for prolonged storage in liquid nitrogen. The thawing procedure started with removal of the straws from the liquid nitrogen, maintaining them in the air for 30 s, then the straws were plunged into a water bath at 30oC for 40 s. The oocytes were washed according to a four-step dilution procedure at room temperature to remove cryoprotectants: Vial 1 solution for 5 min; Vial 2 solution for 5 min; Vial 3 solution for 10 min; Vial 4 solution for 20 min (10 min at room temperature and 10 min at 37oC). The oocytes were finally cultured at 37oC and 5% CO2 for 2 h before intracytoplasmic sperm injection (ICSI).
Vitrification protocol From January 2007 to December 2007, 97 women undergoing IVF treatment agreed to freeze their surplus eggs, for a total of 765 metaphase II oocytes, using the vitrification procedure. Out of 765 vitrified oocytes, 285 oocytes from 46 patients were warmed in 59 cycles. Ethylene glycol, PROH and sucrose were used as cryoprotectants and cryoleaf as the carrier (no. 40771401A, MediCult). The cooling protocol consisted of four steps performed at room temperature: (i) denuded oocytes were incubated in a drop of equilibration medium (no. 12184001, MediCult) + flushing medium (no. 10840125A, MediCult) (25 Ml + 25 Ml 1:1) for 3 min; (ii) 25 Ml of equilibration medium were added to the first drop with oocytes and left for 3 min; (iii) oocytes were moved into a new drop of pure equilibration medium (25 Ml). The exposure time in the equilibration medium drop was up to a maximum of 9 min, according to the time needed for re-expansion of the cell to its original volume; (iv) oocytes were moved onto the surface of vitrification medium (no. 12184001, MediCult) at room temperature and washed for 60 s. Oocytes were then loaded on the cryoleaf tip using an extremely small volume (<2 Ml) of vitrification medium and plunged directly into clean liquid nitrogen. No more than two oocytes were placed on each cryoleaf. Then the cap was fixed on the cryoleaf and it was put into a canister for prolonged storage in liquid nitrogen in a dedicated storage tank. The warming protocol was performed using a vitrification warming kit (no. 12195002A, MediCult) by stepwise dilution of cryoprotectants. The cryoleaf was quickly immersed in a box previously filled with liquid nitrogen where the protective cap was removed while it was still submerged in RBMOnline®
Article - Slow and ultrarapid cryopreservation of human oocytes - R Fadini et al.
liquid nitrogen. Then the cryoleaf was directly immersed in 37oC pre-warmed warming medium for 1 min at room temperature. The oocytes were washed in dilution medium 1 for 3 min and in dilution medium 2 for a further 3 min. The oocytes were then washed twice in washing medium for 5 min at room temperature followed by 5 min on the warmer plate (37oC).
Ovarian stimulation The study was carried out over a long period so it included different ovarian stimulation protocols. To induce hypophysis down-regulation, long-protocol gonadotrophin-releasing hormone agonist was used (Decapeptyl 3.75 mg or 0.1 mg, Ipsen, Italy). Ovarian stimulation was carried out with recombinant-FSH (Puregon, Organon, Italy). Final oocyte maturation and ovulation was triggered with 5000 or 10,000 IU human chorionic gonadotrophin (HCG; Gonasi, AMSA, Italy, or Pregnyl, Organon). Oocyte retrieval was performed 34–36 h after HCG administration.
Selection of oocytes for cryopreservation The oocytes at metaphase II stage (MII) were carefully selected to choose the oocytes to cryopreserve according to the following morphological features: cytoplasmatic characteristics (homogeneous or with granularity, presence of vacuoles or smooth endoplasmic reticulum, presence of dark zone); polar body morphology (regular or fragmented/ degenerated). Only oocytes with regular shape and homogeneous cytoplasm were frozen.
Insemination and embryo culture After thawing, oocytes were cultured in ISM1 medium (no. 1050060A, MediCult) in an incubator at 37oC and 5% CO2 for 2 h before evaluation and ICSI. According to the Italian law on assisted reproduction, ICSI was been performed on only three mature oocytes per women. Fertilization was assessed 16–18 h after injection by the presence of two pronuclei. The resulting zygotes were individually cultured in a four-well Petri dish (no. 176740, NUNC, Denmark) in 0.5 ml of ISM1. The embryos were cultured for 2 days. The embryo quality was evaluated and graded by observing the percentage of fragmentation with respect to the overall cellular volume and blastomere symmetry according to the follow classification: Grade 1: embryos without any fragmentation and equal blastomeres, grade 2: embryos with less than 10% of fragmentation and equal size blastomeres, grade 3: 11–20% fragmentation with equal size blastomeres, grade 4: 21–40% of fragmentation with asymmetric blastomeres, grade 5: more than 40% of fragmentation with asymmetric blastomeres. As determined by Italian law, all the resulting embryos were transferred without any selection using a soft catheter under ultrasound guidance (Gynetics no. 2000, Semtrac Set 5, Belgium).
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Endometrial preparation and pregnancy assessment To time the embryo transfer, all women were examined by ultrasound scan at regular intervals. In order to prepare the endometrium, they received oestradiol hemihydrate supplementation 4 mg a day (17-B-oestradiol, Novo-Nordisk, Denmark), starting from the second day of the cycle up to an endometrial thickness of 8 mm, then 6 mg a day plus 600 mg progesterone (Progesterone, Rottafarm, Italy) to perform the embryo transfers after 4 or 5 days of progesterone supplementation. Both steroid supplementations were continued, after confirmation of clinical pregnancy, until week 12 of gestation. The presence of pregnancy was assessed 12 days after the transfer by quantitative definition of serum HCG. Clinical pregnancies were defined by the presence of a gestational sac with or without a fetal heart beat at the transvaginal ultrasound examination.
Statistical analysis Differences between slow-freezing and vitrification groups were analysed using the Student’s t-test and Fisher’s exact test depending on the distribution of data. Stata Software 9.0 (Stata Corporation, Texas, USA) was used for performing the statistical analysis. A level of P < 0.05 was adopted for significance.
Results Slow-freezing results A total of 2291 MII oocytes were cryopreserved by slow freezing from April 2004 to December 2006 in the centre, and 1348 oocytes from 208 patients (average age 34.1 p 3.5 years, range 24–39) were thawed in 286 cycles with an average number of 4.7 p 1.4 oocytes per cycle (Table 1). Out of 1348 thawed oocytes, only 780 oocytes survived the freezing–thawing procedure with a survival rate of 57.9% and an average number of oocytes per thawing cycle of 2.7 p 1.1. A total of 744 thawed oocytes were injected with an average of 2.6 ± 0.6 oocytes per cycle. Evidence of fertilization (two pronuclei; 2PN) was observed in 481 oocytes with a fertilization rate of 64.7%. Anomalous fertilization with 3PN (2.8%) was observed in 21 oocytes and 87 oocytes degenerated after insemination (11.7%). 155 oocytes (20.8%) did not show any evidence of fertilization. After 48 h of culture, 86 zygotes did not divide and 395 divided to a 2–4-cell embryo with a cleavage rate of 82.1%. Of the cleaved embryos 22.0% were Grade 1, 32.9% were Grade 2, 21.8% were Grade 3, 11.4% were Grade 4 and 11.9% were Grade 5 (Table 2). All embryos obtained were transferred, according to Italian law, without any embryo selection. The centre performed 224 embryo transfers in 161 patients with an average of 1.4 p 0.9 embryos per transfer. Of these, 27.7% of embryo transfers were with only one embryo, 46.9% with two embryos and only 25.4% of the embryo transfers
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Table 1. Comparison of cycle data for oocytes cryopreserved by slow-freezing or vitrification protocols.
No. of patients No. of thawing/warming cycles No. of thawed/warmed oocytes No. of survived oocytes (%) No. of injected oocytes No. of fertilized (2PN) oocytes (%) No. of cleaved embryos (%) No. of embryo transfers Mean no. of embryos per transfer ± SD a
Slow-freezing protocol
Vitrification protocol
P-value
208 286 1348 780 (57.9) 744 481 (64.6) 395 (82.1) 224 1.4 ± 0.9
46 59 285 225 (78.9) 162 118 (72.8) 108 (91.5) 55 1.9 ± 0.8
– – – <0.0001a – 0.027a 0.007a 0.003b
Fisher’s exact test; bStudent’s t-test.
Table 2. Comparison of morphological characteristics of embryos obtained from oocytes cryopreserved by slow-freezing or vitrification protocols. Embryo quality
Slow-freezing protocol
Vitrification protocol
P-valuea
Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
87/395 (22) 130/395 (32.9) 86/395 (21.8) 45/395 (11.4) 47/395 (11.9)
32/108 (29.6) 43/108 (39.8) 19/108 (17.6) 11/108 (10.2) 3/108 (2.8)
0.015
Values are number (percentage). a Fisher’s exact test – embryos obtained from vitrified oocytes showed a statistically significantly better grading distribution than those derived from oocytes cryopreserved by slow freezing.
were performed with three embryos (Table 3). Positive B-HCG was measured 14 days after embryo transfer, and 17 clinical pregnancies were observed, with a clinical pregnancy rate of 7.6% per embryo transfer and 10.6% per patient. The miscarriage rate was 29.4%. The implantation rate per embryo transferred was 4.3%, the implantation rate per injected oocyte was 2.4% and the implantation rate per fertilized oocyte was 3.5% (Table 4). For transfers of one, two or three embryos, the clinical pregnancy rates were 9.7%, 4.8% and 10.5% respectively. Five pregnancies ended in miscarriage and 12 healthy babies have been born.
Out of 162 inseminated oocytes, 118 oocytes showed evidence of fertilization (2PN) with a fertilization rate of 72.8% and 22 (13.6%) did not show any evidence of fertilization; four showed anomalous fertilization with 3PN (2.5%) and 18 degenerated after insemination (11.1%). After 48 h of culture, 10 zygotes did not divide and 108 divided to 2–4-cell embryos with a cleavage rate of 91.5%. According to the morphological characteristics of the embryos, 29.6% were Grade 1, 39.8% were Grade 2, 17.6% were Grade 3, 10.2% were Grade 4 and 2.8% were Grade 5 (Table 2). All embryos obtained were transferred, according to Italian law, without any embryo selection.
Vitrification results
The centre performed 55 embryo transfers in 43 patients with an average of 1.9 p 0.8 embryos per transfer. Of these, 34.5% of embryo transfers were with only one embryo, 34.5% with two embryos and 30.9% with three embryos (Table 3). Positive B-HCG was measured 14 days after embryo transfer, and 10 clinical pregnancies were observed, with a clinical pregnancy rate of 18.2% per embryo transfer and 23.3% per patient. The miscarriage rate was 20%. The implantation rate per embryo transferred was 9.3%, the implantation rate per injected oocyte was 6.2% and the implantation rate per fertilized oocyte was 8.5% (Table 4). For transfers of one, two or three embryos the clinical pregnancy rates were 10.5%, 15.8% and 29.4% respectively. At the time of writing, five pregnancies were ongoing, two ended in miscarriage and three healthy babies have been born.
A total of 765 MII oocytes were vitrified from January 2007 to December 2007 in the centre. Of these, 285 oocytes from 46 patients (average age 34.6 p 3.2 years, range 28–39) were warmed in 59 cycles with an average number of 4.8 p 1.2 oocytes each cycle (Table 1).
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Out of 285 warmed oocytes, only 225 oocytes survived the cooling–warming process with a survival rate of 78.9%. In total, 162 oocytes were inseminated, with an average of 2.7 p 0.5 oocytes per cycle and no more than 3 oocytes per cycle, in accordance with the Italian law.
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Article - Slow and ultrarapid cryopreservation of human oocytes - R Fadini et al.
Table 3. Comparison of the number of embryos transferred according to the oocyte cryopreservation protocol used. No. of embryos transferred
Slow-freezing Vitrification protocol protocol
P-value
1 2 3
62 (27.7) 105 (46.9) 57 (25.4)
NS
19 (34.5) 19 (34.5) 17 (31.0)
Values are number (percentage); NS = not significant (Fisher’s exact test).
Table 4. Comparison of clinical data between slow-freezing protocol and vitrification protocol.
No. of embryo transfers Patients at embryo transfer Positive B-HCG test Clinical pregnancies per embryo transfer Clinical pregnancies per patient Miscarriages Implantation rate per transferred embryo Implantation rate per thawed oocyte Implantation rate per injected oocyte Implantation rate per fertilized oocyte
Slow-freezing protocol
Vitrification protocol
P-valuea
224 161 21 17/224 (7.6) 17/161 (10.6) 5/17 (29.4) 17/395 (4.3) 17/1348 (1.3) 17/694 (2.4) 17/481 (3.5)
55 43 13 10/55 (18.2) 10/43 (23.3) 2/10 (20.0) 10/108 (9.3) 10/285 (3.5) 10/162 (6.2) 10/118 (8.5)
– – – 0.021 0.046 NS 0.043 0.012 0.019 0.025
Values in brackets are percentages. HCG = human chorionic gonadotrophin; NS = not significant. a Fisher’s exact test. According to Italian law, three oocytes/cycle were injected and no embryo selection was performed.
Discussion The reasons for choosing to freeze oocytes rather than embryos are not discussed here, even though it is still a debatable matter, considering that the results reported so far on cryopreserving human oocytes have not shown the same success rate as that obtained with embryo freezing (Oktay et al., 2006). In Italy, where embryo cryopreservation is forbidden by law, oocyte cryopreservation has become a necessary procedure in order to save oocytes and to ensure a good cumulative success rate (Boldt et al., 2003, 2006) avoiding reiterated ovulation induction. In this regard the Italian National Register on assisted reproductive technology has recently reported that more than 100,000 oocytes, 48.3% of all the oocytes retrieved in Italy in 2006, remained unused and were destroyed (Turco, 2008). Although many studies investigating the feasibility and efficiency of oocyte cryopreservation have reported good biological and clinical results supporting the viability of this technology, oocyte freezing is still considered an experimental procedure currently in a research phase and the efficiency of the method is debatable. The main problems concerning oocyte cryopreservation are due to the dimension of the cell, the high cytoplasmic water content and the low membrane permeability. In fact, during the freezing procedure, the water retained in the cytoplasm may produce ice crystals which could irreparably damage the ultrastructure RBMOnline®
of the oocyte. To protect the cell against chilling injuries, due to inner ice formation during freezing and thawing phases, the oocytes must be exposed to permeable and non-permeable cryoprotectants that induce oocyte dehydration by removing most of water from cytoplasm. Moreover the shrinkage and swelling of the oocytes, occurring during the dehydration phase, can also injure the morphology and functionality of oocytes (Rienzi et al., 2004; Ghetler et al., 2006; Coticchio et al., 2007; Nottola et al., 2007). The slow-freezing method implies the exposure of the oocytes to low cryoprotectant concentrations for a long time in order to induce a slow dehydration of the cells and a very slow decrease in temperature. The slow rate of cooling must be controlled by an appropriate freezer machine. A suitable sucrose concentration to be used in slow-freezing protocols is still under assessment. In fact the Italian studies, the most important published regarding this subject, suggest three different concentrations of sucrose: 0.1 mol/l (Borini et al., 2004; De Santis et al., 2007), 0.2 mol/l (Bianchi et al., 2007) and 0.3 mol/l (Borini et al., 2006; Levi Setti et al., 2006; De Santis et al., 2007; Parmegiani et al., 2008). The only randomized study published, which compared these three different sucrose concentrations, demonstrated that the highest concentration may ensure the best results (Fabbri et al., 2001). Although a paper reported a remarkable success rate also with 0.2 mol/l (Bianchi et al., 2007), the 0.3 mol/l protocol is, up to now, the most accepted, even if forthcoming studies may still keep the matter open. In 2004, the slow-freezing method
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was introduced, choosing 0.3 mol/l sucrose concentration and PROH, according to the previous experiences that had ensured better results (Fabbri et al., 2001). Vitrification allows an ultrarapid transition of the intracellular water in a glass-like phase, avoiding chilling injuries. Using this strategy to induce rapid dehydration, a high dose of cryoprotectants is used for a very short time, followed by an ultrarapid decrease in temperature (20,000oC/min). In order to speed up the cooling rate, the oocytes are directly plunged into liquid nitrogen. For this purpose, different protocols have been published and various devices have been proposed (Liebermann and Tucker, 2002; Liebermann et al., 2002a,b,c; Katayama et al., 2003; Cai et al., 2005; Chian et al., 2005; Isachenko et al., 2005; Kuwayama et al., 2005a,b; Sheehan et al., 2006). Cryoleaf was used as the carrier (Chian et al., 2005). This open carrier is simple to use and has a coloured security covering which mechanically protects the leaf where oocytes are placed. This makes handling the carrier in the liquid nitrogen easier and safer, although it is likely that every embryologist will become proficient in using their own device. This report is based entirely on experience with vitrification using PROH, ethylene glycol and sucrose as cryoprotectants. In order to better standardize the procedure, ‘homemade’ culture media were avoided. The commercially available kit from MediCult was found to be extremely convenient. This retrospective study consists of a comparison of the results achieved from both techniques. The two different cryopreservation procedures were applied sequentially in consecutive periods. During the whole period, the ovarian stimulation protocols used presented only minor differences; furthermore, no changes were made to the laboratory culture system or to the endometrial preparation. So, it can be confidently stated that results weren’t affected in any way by differences in the stimulation protocols used. The survival rate after thawing is one of the most critical measures in any oocyte cryopreservation programme. In the literature, a variable survival percentage has been reported with the slow-freezing method and with the ultrarapid cooling method (Tables 5 and 6). This study observed a significantly higher survival rate using the vitrification method. A survival rate of 57.9% was achieved in a 3-year period using the slowfreezing protocol for 286 thawing cycles with a total of 1348 oocytes thawed. This was considered sufficient experience to standardize the protocol and pass through the learning curve. Whilst in a few months, warming only 285 vitrified oocytes in 59 cycles, a higher percentage survival rate (78.9%, P < 0.0001) was achieved. It can be concluded that the difference could be due to the different methods.
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According to Italian assisted reproduction law, the same number of oocytes per cycle (not more than three per cycle) were inseminated in both groups but a higher fertilization rate was obtained after vitrification (72.8% P = 0.027), whilst the percentage of anomalous fertilization (1PN or 3PN) was comparable between the two methods. The better fertilization rate obtained using vitrified oocytes could be due to better health of the oocytes. The consequence of this would be that even though the same number of oocytes were inseminated, a
higher number of embryos were obtained for transfer (Table 1). The different mean number of embryos transferred is related to the technique used and it could have contributed to improve the outcome of the procedure. Regarding embryo quality, no cleavage delay in embryo development was observed with both methods but a different distribution among the five grades was recorded. Embryos obtained from vitrified oocytes showed a statistically significantly better grading distribution than those derived from slow-freezing oocytes (P = 0.015; Table 2). All this resulted in a higher number of good quality embryos in women where vitrification was performed and these biological elements might favourably affect the pregnancy rate. As far as the clinical pregnancy rate per transfer and the implantation rate are concerned, the literature reports a great variability with both methods (Table 5 and Table 6). Significant differences were observed when comparing the two methods. Vitrification resulted in a higher number of embryos to transfer, higher clinical pregnancy and implantation rates than the slow-freezing method. Very soon, a change was made from slow freezing to vitrification, increasing the success rate. So after 3 years of experience, the centre eventually decided to discontinue the slow-freezing method in favour of vitrification, because the latter method had shown a significant favourable impact on the whole outcome. As regards the vitrification success, it is quite difficult to compare these results, in terms of pregnancy rate, with those published by other groups (Table 5 and Table 6) because, according to the strict Italian law, the centre cannot produce more than three embryos nor perform a morphological embryo selection before embryo transfer, while strict selection is carried out in other countries. The centre transferred an average of 1.9 embryos per transfer, which means 34.5% of the embryo transfers were performed with only one unselected embryo (Table 3). This high percentage of unselected single embryo transfers is directly linked to the compulsory use of only three oocytes per cycle. So, it is difficult to compare the potential results that could have been obtained by fertilizing more than three oocytes. This might have reduced the pregnancy and implantation rates when compared with other published papers. Moreover the Italian results before and after the law are not comparable either. Nevertheless, the results of oocyte cryopreservation in this study appear worse when compared with other Italian studies previously published using slow-freezing oocyte cryopreservation (Borini et al., 2004, 2006, 2007a; Bianchi et al., 2007; De Santis et al., 2007; Parmegiani et al., 2008) as well as vitrification (Antinori et al., 2007). The difference could be explained by considering the selection of the women. In fact, the study centre has always carried out mild ovarian stimulation in IVF and has extensively applied in-vitro maturation of oocytes both in polycystic ovary syndrome/polycystic ovary patients and in young women with normal ovaries without any ovarian stimulation. This strategy definitely reduces the number of available oocytes, mainly in those women with a very good ovarian response who are actually the most favourable and whose oocytes can be frozen. So a sort of negative patient RBMOnline®
Article - Slow and ultrarapid cryopreservation of human oocytes - R Fadini et al.
Table 5. Studies reporting survival, fertilization, implantation and pregnancy rates of frozen–thawed oocyte cycles with slowfreezing method. Reference
Thawed Thawed cycles oocytes (n) (n)
Survival Fertilization Implantation rate rate (%) rate per (%) transfer (%)
Pregnancy Pregnancies Babies rate per (n) born transfer (%) (n)
Porcu et al., 1999 Winslow et al., 2001 Boldt et al., 2003 Borini et al., 2004 Li et al., 2005 Boldt et al., 2006 La Sala et al., 2006 Levi Setti et al., 2006 Borini et al., 2007a De Santis et al., 2007 Bianchi et al., 2007 Parmegiani et al., 2008
112 45 16 86 15 53 518 159 660 133 90 93
54.1 68.5 74.4 37.0 90.1 60.4 72.8 69.9 68.1 47.5 75.9 75.1
14.2 26.2 36.4 25.4 44.0 32.5 3.8 12.4 14.9 13.1 21.3 19.2
1502 324 90 737 81 361 1647 1087 3238 902 403 437
57.7 80.8 59.0 45.4 82.0 62.0 74.7 67.5 76.1 67.0 76.2 86.0
– 13.5 15.1 16.4 14.8 13.3 6.3 5.7 8.1 6.7 13.5 9.6
16 11 4 15 7 12 19 18 88 12 17 16
11 16 3 13 6 11 7 13 60 3 4 10
Table 6. Studies reporting survival, fertilization, implantation and pregnancy rates of cooled-warmed oocyte cycles with vitrification method. Reference
Thawed Thawed cycles oocytes (n) (n)
Survival Fertilization Implantation Pregnancy Pregnancies Babies rate rate (%) rate per rate transfer (n) born (%) transfer (%) (n)
Yoon et al., 2000 Yoon et al., 2003 Kuwayama et al., 2005b Chian et al., 2005 Lucena et al., 2006a Selman et al., 2006 Antinori et al., 2007 Cobo et al., 2008a
7 34 29 15 33 6 120 30
84.9 68.6 91.0 93.9 89.2 75.0 99.4 96.9
90 474 64 180 159 24 796 231
65.0 71.7 89.6 74.6 87.2 77.7 93.0 76.3
9.4 6.4 18.7 2.4 13.4 21.4 13.2 40.8
42.0 21.4 41.3 46.7 56.5 33.3 32.5 65.2
3 6 12 11 13 2 28 11
2 7 7 – – – 3 –
a
Egg donation programme.
selection has been introduced that could bias the outcome of the cycles. The Italian National Register (Turco, 2008) has recently published the results on oocyte cryopreservation carried out in 2006. The Italian Register has recorded data on 2977 thawing cycles which represent 7.3% of the whole IVF Italian cycles. A pregnancy rate of 10% per thawed and a pregnancy rate per transfer of 12.6% were reported. It is expected that this high number of oocyte cryopreservation cycles could possibly increase in the coming years, so that oocyte cryopreservation procedures will have a great impact on routine practice in the major Italian laboratories. Comparing the two available cryopreservation methods shows that vitrification has some advantages compared with slow freezing. Although the cost of materials for each patient is equal, the vitrification method does not need an expensive freezing machine. Regarding the time consumption for every single freezing procedure, slow freezing takes almost
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3 h to complete whilst the ultrarapid procedure can be completed in about 20 min. However, it should be remembered that, when there are many women undergoing a cryopreservation procedure, vitrification also takes a long time. Although the time taken is hardly a remarkable point, the appropriate timing of the freezing procedure is actually a critical element. In fact in a busy laboratory, if many samples must be simultaneously frozen with the slow procedure, the beginning of the procedure is, every so often, delayed so as to freeze the eggs retrieved from different women at once. Thereby, the delay in oocyte cryopreservation may age the oocytes. As reported, the ageing of oocytes may impair their developmental capability (Yanagida et al., 1998; Dozortsev et al., 2004; Parmegiani et al., 2008) and jeopardize the procedure. On the other hand, vitrification can be promptly performed after each oocyte retrieval without any delay.
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Another advantage of vitrification is the possibility to ‘individualise’ the process with respect to each oocyte. Using vitrification, the embryologist can check each oocyte during the different phases of shrinkage and swelling. In the study centre, each oocyte has been observed to have its own membrane permeability and shows a different response during exposure to equilibration and vitrification solutions. The importance of observing the changes in oocyte volume in an equilibration solution must be stressed, to decide when to move the cell into the vitrification solution and then into liquid nitrogen. In the authors’ experience, the best time is when the shrinkage/ swelling process is completed. So efforts must be made to individualize the incubation time as much as possible. Moreover, according to the individual response, a decision can be made at the dehydration and hydration phase whether to continue or suspend the cooling process. The slow method, however, cannot be individualized owing to the programmable freezer machine. The oocytes can only be observed in the equilibration solution for quite a short time before loading the freezing machine while the dehydration process in the first part of the cooling rate is still continuing. It is the authors’ view that vitrification easily fits all the laboratory demands, hence the different outcome of oocyte cryopreservation observed could be explained by its ability to be a safe and adaptable procedure to freeze oocytes. Even if oocytes were vitrified using an open system, clean factoryderived nitrogen and disposable materials can be used, changing the liquid nitrogen for each patient. The risk of potentially cross-contamination cannot be excluded, but as far as is known, there is no publication about vitrification that has reported the transmission of infectious diseases after cryotransfer. In any case, the use of sterile liquid nitrogen should be considered (Bielanski et al., 2000; Vajta and Nagy, 2006). Concerning the potential genetic and metabolic damage of oocyte cryopreservation, this report does not contribute any definite results. From all of the studies already published, more than 300 pregnancies have been obtained and 160 births have so far been reported. No disquieting reports have been published as yet. Interesting papers have recently recorded the outcome in infants conceived from slow-freezing/vitrification protocols. These studies confirm the safety of oocyte cryopreservation (Borini et al., 2007b; Gook and Edgar, 2007; Tur-Kaspa et al., 2007; Chian et al., 2008; Manipalviratn et al., 2008). In conclusion, at present in this clinical practice, oocytes are frozen as a routine part of the IVF procedure due to lack of another alternative, but it is considered to be a reliable method. Although there is a choice between two available methods to freeze human oocytes, according to this centre’s experience, the only reason for using slow freezing instead of ultrarapid cooling is habit in those centres which obtain good results with the slow method. This report supports introducing vitrification as new method because it can assure a more successful outcome. Oocyte cryopreservation procedures still need to be improved and although results are far from excellent, the results can be considered to be fairly good.
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vitrification: a new way to reduce cryo-injuries of bovine ova and embryos. Molecular Reproduction and Development 51, 53–58. Vanderzwalmen P, Bertin G, Debauche C 2003 Vitrification of human blastocyst with the hemi-straw carrier: application of assisted hatching after thawing. Human Reproduction 18, 1504–1511. Vanderzwalmen P, Bertin G, Debauche C 2002 Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Human Reproduction 17, 744–751. Winslow KL, Yang D, Blohm PL et al. 2001 Oocyte cryopreservation/a three year follow-up of sixteen births. Fertility and Sterility 76 (Suppl. 1), abstract S120. Wright DL, Eroglu A, Toner M, Toth TL 2004 Use of sugars in cryopreserving human oocytes. Reproductive BioMedicine Online 9, 179–186. Yanagida K, Yazawa H, Katayose H 1998 Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection. Human Reproduction 13, 2223–2226.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 30 July 2008; refereed 29 August 2008; accepted 11 February 2009.
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Article Human oocyte cryopreservation: comparison between slow and ultrarapid methods Dr Fadini obtained his speciality in Gynaecology and Obstetrics and in Human Reproduction from the University of Milan, Italy. From 1985 to 1999 he directed the Unit of Reproductive Medicine and Endocrinology at San Gerardo University Hospital in Monza, University of Milan. He is Assistant Professor in the School of Specialization in Gynaecology and Obstetrics and at the School of Medical Biotechnologies, Faculty of Medicine, University of Milan-Bicocca. He has been Director of the Unit of Gynaecology and Obstetrics and Chief of the BIOGENESI Reproductive Medicine Centre at the Istituti Clinici Zucchi, in Monza since 2000.
Dr R Fadini R Fadini1, F Brambillasca1, M Mignini Renzini1, M Merola1, R Comi1, E De Ponti2, MB Dal Canto1,3 1 BIOGENESI Reproductive Medicine Centre, Istituti Clinici Zucchi, V. Zucchi, 24-Monza, Italy; 2Department of Medical Physics San Gerardo Hospital, V. Pergolesi 33, Monza, Italy 3 Correspondence: e-mail: [email protected]
Abstract The success of reproductive technologies is facilitated by the cryopreservation of embryos and gametes. In Italy, where legislation prohibits zygote and embryo cryopreservation, clinics have extensively introduced oocyte cryopreservation. Two different strategies of oocyte cryopreservation are available: slow freezing or ultrarapid cooling (vitrification). Although the results are very encouraging with both methods, there is still controversy regarding both the procedure itself and the most suitable method to use. This study reports the routine application of the two different oocyte cryopreservation methods in programmes running in two consecutive periods. The study centre carried out 286 thawing cycles for a total of 1348 thawed oocytes cryopreserved by the slow-freezing method and 59 warming cycles for a total of 285 warmed oocytes cryopreserved by vitrification. Comparison of the outcomes obtained with the slow-freezing method versus vitrification in women who underwent IVF for infertility showed survival, fertilization, pregnancy and implantation rates of 57.9% versus 78.9% (P < 0.0001), 64.6% versus 72.8% (P = 0.027), 7.6% versus 18.2% (P = 0.021) and 4.3% versus 9.3% (P = 0.043) respectively. These results suggest that oocyte vitrification is associated with a better outcome than the slow-freezing method. Keywords: cryoleaf, human oocyte cryopreservation, slow freezing, survival rate, vitrification
Introduction The wide use of ovarian stimulation protocols in assisted reproduction treatments has allowed the collection of a large number of oocytes; as a consequence, many embryos have been produced. Hopefully, all these efforts have significantly improved the success of IVF, and undoubtedly they have induced researchers to find methods of cryopreserving such numerous oocytes and embryos. At present embryo freezing is a highly reliable procedure and certainly reproductive techniques have been facilitated through the cryopreservation of surplus embryos which can improve treatment success and reduce multi-fetal pregnancies (Ashwood-Smith, 1986; Mandelbaum et al., 1987; Osmanagaoglu et al., 2004; Stehlik et al., 2005; Andersen et al., 2008). Oocyte cryopreservation is a more recent procedure that provides an alternative to embryo freezing without ethical and
religious problems and that can also be used to preserve fertility in patients at risk of ovarian failure. So far two methods have been proposed to freeze human oocytes, slow freezing and ultrarapid cooling. The slow-freezing method was the first to be introduced and the first pregnancy with a cryopreserved oocyte was obtained in 1986 (Chen, 1986). Only a few pregnancies were reported during the subsequent years because the method needed to be standardized. It took 10 years before suitable protocols were proposed to increase the efficiency of the slow-freezing method (Porcu et al., 1997, 1999, 2000; Fabbri et al., 2001). In recent years, the protocol has been better standardized by many Italian researchers; more favourable success rates using slow freezing have been reported and the number of pregnancies recorded has increased (Borini et al., 2004, 2006, 2007a,b; La Sala et al.,
© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
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2006; Levi Setti et al., 2006; Bianchi et al., 2007; De Santis et al., 2007; Parmegiani et al., 2008). Another strategy to cryopreserve oocytes and embryos is the ultrarapid cooling or vitrification that allows glass-like solidification of a solution without ice-crystal formation. This technology has been widely used in animals but only recently has been studied in humans (Feichtinger et al., 1991; Mukaida et al., 1998; Vajta et al., 1998; Mauri et al., 1999; Chen et al., 2001; Vanderzwalmen et al., 2002; Son et al., 2003; Chian et al., 2004; Huang et al., 2007). The vitrification of embryos or blastocysts and more recently zygotes with good results has been extensively applied and accepted in the past few years (Mukaida et al., 2003; Vanderzwalmen et al., 2003; Huang et al., 2005; Kuwayama et al., 2005a; Koutlaki-Kourti et al., 2006; Liebermann and Tucker, 2006; Al-Hasani et al., 2007; Balaban et al., 2008; Youssry et al., 2008). The first birth after human oocyte vitrification was recorded in 1999 with a standardized protocol used on animals (Kuleshova et al., 1999). In the last decade, although few papers on the clinical outcome of oocyte vitrification have been published, very good results have been reported (Hong et al., 1999; Yoon et al., 2000, 2003; Katayama et al., 2003; Wright et al., 2004; Chian et al., 2005; Kuwayama et al., 2005b; Okimura et al., 2005; Lucena et al., 2006; Selman et al., 2006; Antinori et al., 2007). The study centre has been freezing oocytes since 2004, using the slow-freezing method, after many years of experience with slow embryo freezing. In 2007, vitrification was introduced to cryopreserve surplus oocytes produced during assisted reproduction procedures. The results obtained with the routine application of human oocyte cryopreservation performed with both freezing methods, applied in two consecutive periods, have been retrospectively analysed in order to discuss the viability and reliability of the two techniques.
Materials and methods This retrospective study included 254 women undergoing the IVF programme at Biogenesi Reproductive Medicine Centre, in whom a total of 345 oocyte thawing cycles were performed from April 2004 to December 2007. In this time no changes were made to the laboratory culture system.
Slow-freezing protocol From April 2004 to December 2006, 255 women undergoing IVF treatment agreed to freeze their surplus eggs using the slow-freezing protocol after signing a consent form. To date, out of 2291 oocytes cryopreserved by the slow-freezing protocol, 1348 oocytes from 208 patients have been thawed in 286 cycles.
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The cryopreservation protocol consisted of the slowfreezing and rapid-thawing method described by Fabbri et al. (2001). Freezing and thawing solutions were supplied by MediCult, containing 1,2-propanediol (PROH) and sucrose as cryoprotectants (nos. 10485010 and 10494010, respectively, MediCult, Denmark).
The oocytes were washed in Vial 1 solution at room temperature. Afterwards the oocytes were equilibrated for 10 min in Vial 2 solution and then transferred into Vial 3 solution for a maximum of 1 min. From one up to a maximum of three oocytes were loaded into a plastic straw (Paillettes Cristal 133 mm; Cryo Bio System, France) and transferred into an automatic biological vertical freezer (Kryo 360–1.7, Planer, UK). The cooling process was initiated reducing chamber temperature from 20oC to –7oC at a speed of freezing of 2oC/ min. Manual seeding was induced at –7oC. After a hold time of 10 min at –7oC, the straws were cooled slowly up to –30oC at a rate of freezing of 0.3oC/min and then rapidly to –150oC at a rate of 50oC/min. Then the straws were transferred into a canister for prolonged storage in liquid nitrogen. The thawing procedure started with removal of the straws from the liquid nitrogen, maintaining them in the air for 30 s, then the straws were plunged into a water bath at 30oC for 40 s. The oocytes were washed according to a four-step dilution procedure at room temperature to remove cryoprotectants: Vial 1 solution for 5 min; Vial 2 solution for 5 min; Vial 3 solution for 10 min; Vial 4 solution for 20 min (10 min at room temperature and 10 min at 37oC). The oocytes were finally cultured at 37oC and 5% CO2 for 2 h before intracytoplasmic sperm injection (ICSI).
Vitrification protocol From January 2007 to December 2007, 97 women undergoing IVF treatment agreed to freeze their surplus eggs, for a total of 765 metaphase II oocytes, using the vitrification procedure. Out of 765 vitrified oocytes, 285 oocytes from 46 patients were warmed in 59 cycles. Ethylene glycol, PROH and sucrose were used as cryoprotectants and cryoleaf as the carrier (no. 40771401A, MediCult). The cooling protocol consisted of four steps performed at room temperature: (i) denuded oocytes were incubated in a drop of equilibration medium (no. 12184001, MediCult) + flushing medium (no. 10840125A, MediCult) (25 Ml + 25 Ml 1:1) for 3 min; (ii) 25 Ml of equilibration medium were added to the first drop with oocytes and left for 3 min; (iii) oocytes were moved into a new drop of pure equilibration medium (25 Ml). The exposure time in the equilibration medium drop was up to a maximum of 9 min, according to the time needed for re-expansion of the cell to its original volume; (iv) oocytes were moved onto the surface of vitrification medium (no. 12184001, MediCult) at room temperature and washed for 60 s. Oocytes were then loaded on the cryoleaf tip using an extremely small volume (<2 Ml) of vitrification medium and plunged directly into clean liquid nitrogen. No more than two oocytes were placed on each cryoleaf. Then the cap was fixed on the cryoleaf and it was put into a canister for prolonged storage in liquid nitrogen in a dedicated storage tank. The warming protocol was performed using a vitrification warming kit (no. 12195002A, MediCult) by stepwise dilution of cryoprotectants. The cryoleaf was quickly immersed in a box previously filled with liquid nitrogen where the protective cap was removed while it was still submerged in RBMOnline®
Article - Slow and ultrarapid cryopreservation of human oocytes - R Fadini et al.
liquid nitrogen. Then the cryoleaf was directly immersed in 37oC pre-warmed warming medium for 1 min at room temperature. The oocytes were washed in dilution medium 1 for 3 min and in dilution medium 2 for a further 3 min. The oocytes were then washed twice in washing medium for 5 min at room temperature followed by 5 min on the warmer plate (37oC).
Ovarian stimulation The study was carried out over a long period so it included different ovarian stimulation protocols. To induce hypophysis down-regulation, long-protocol gonadotrophin-releasing hormone agonist was used (Decapeptyl 3.75 mg or 0.1 mg, Ipsen, Italy). Ovarian stimulation was carried out with recombinant-FSH (Puregon, Organon, Italy). Final oocyte maturation and ovulation was triggered with 5000 or 10,000 IU human chorionic gonadotrophin (HCG; Gonasi, AMSA, Italy, or Pregnyl, Organon). Oocyte retrieval was performed 34–36 h after HCG administration.
Selection of oocytes for cryopreservation The oocytes at metaphase II stage (MII) were carefully selected to choose the oocytes to cryopreserve according to the following morphological features: cytoplasmatic characteristics (homogeneous or with granularity, presence of vacuoles or smooth endoplasmic reticulum, presence of dark zone); polar body morphology (regular or fragmented/ degenerated). Only oocytes with regular shape and homogeneous cytoplasm were frozen.
Insemination and embryo culture After thawing, oocytes were cultured in ISM1 medium (no. 1050060A, MediCult) in an incubator at 37oC and 5% CO2 for 2 h before evaluation and ICSI. According to the Italian law on assisted reproduction, ICSI was been performed on only three mature oocytes per women. Fertilization was assessed 16–18 h after injection by the presence of two pronuclei. The resulting zygotes were individually cultured in a four-well Petri dish (no. 176740, NUNC, Denmark) in 0.5 ml of ISM1. The embryos were cultured for 2 days. The embryo quality was evaluated and graded by observing the percentage of fragmentation with respect to the overall cellular volume and blastomere symmetry according to the follow classification: Grade 1: embryos without any fragmentation and equal blastomeres, grade 2: embryos with less than 10% of fragmentation and equal size blastomeres, grade 3: 11–20% fragmentation with equal size blastomeres, grade 4: 21–40% of fragmentation with asymmetric blastomeres, grade 5: more than 40% of fragmentation with asymmetric blastomeres. As determined by Italian law, all the resulting embryos were transferred without any selection using a soft catheter under ultrasound guidance (Gynetics no. 2000, Semtrac Set 5, Belgium).
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Endometrial preparation and pregnancy assessment To time the embryo transfer, all women were examined by ultrasound scan at regular intervals. In order to prepare the endometrium, they received oestradiol hemihydrate supplementation 4 mg a day (17-B-oestradiol, Novo-Nordisk, Denmark), starting from the second day of the cycle up to an endometrial thickness of 8 mm, then 6 mg a day plus 600 mg progesterone (Progesterone, Rottafarm, Italy) to perform the embryo transfers after 4 or 5 days of progesterone supplementation. Both steroid supplementations were continued, after confirmation of clinical pregnancy, until week 12 of gestation. The presence of pregnancy was assessed 12 days after the transfer by quantitative definition of serum HCG. Clinical pregnancies were defined by the presence of a gestational sac with or without a fetal heart beat at the transvaginal ultrasound examination.
Statistical analysis Differences between slow-freezing and vitrification groups were analysed using the Student’s t-test and Fisher’s exact test depending on the distribution of data. Stata Software 9.0 (Stata Corporation, Texas, USA) was used for performing the statistical analysis. A level of P < 0.05 was adopted for significance.
Results Slow-freezing results A total of 2291 MII oocytes were cryopreserved by slow freezing from April 2004 to December 2006 in the centre, and 1348 oocytes from 208 patients (average age 34.1 p 3.5 years, range 24–39) were thawed in 286 cycles with an average number of 4.7 p 1.4 oocytes per cycle (Table 1). Out of 1348 thawed oocytes, only 780 oocytes survived the freezing–thawing procedure with a survival rate of 57.9% and an average number of oocytes per thawing cycle of 2.7 p 1.1. A total of 744 thawed oocytes were injected with an average of 2.6 ± 0.6 oocytes per cycle. Evidence of fertilization (two pronuclei; 2PN) was observed in 481 oocytes with a fertilization rate of 64.7%. Anomalous fertilization with 3PN (2.8%) was observed in 21 oocytes and 87 oocytes degenerated after insemination (11.7%). 155 oocytes (20.8%) did not show any evidence of fertilization. After 48 h of culture, 86 zygotes did not divide and 395 divided to a 2–4-cell embryo with a cleavage rate of 82.1%. Of the cleaved embryos 22.0% were Grade 1, 32.9% were Grade 2, 21.8% were Grade 3, 11.4% were Grade 4 and 11.9% were Grade 5 (Table 2). All embryos obtained were transferred, according to Italian law, without any embryo selection. The centre performed 224 embryo transfers in 161 patients with an average of 1.4 p 0.9 embryos per transfer. Of these, 27.7% of embryo transfers were with only one embryo, 46.9% with two embryos and only 25.4% of the embryo transfers
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Table 1. Comparison of cycle data for oocytes cryopreserved by slow-freezing or vitrification protocols.
No. of patients No. of thawing/warming cycles No. of thawed/warmed oocytes No. of survived oocytes (%) No. of injected oocytes No. of fertilized (2PN) oocytes (%) No. of cleaved embryos (%) No. of embryo transfers Mean no. of embryos per transfer ± SD a
Slow-freezing protocol
Vitrification protocol
P-value
208 286 1348 780 (57.9) 744 481 (64.6) 395 (82.1) 224 1.4 ± 0.9
46 59 285 225 (78.9) 162 118 (72.8) 108 (91.5) 55 1.9 ± 0.8
– – – <0.0001a – 0.027a 0.007a 0.003b
Fisher’s exact test; bStudent’s t-test.
Table 2. Comparison of morphological characteristics of embryos obtained from oocytes cryopreserved by slow-freezing or vitrification protocols. Embryo quality
Slow-freezing protocol
Vitrification protocol
P-valuea
Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
87/395 (22) 130/395 (32.9) 86/395 (21.8) 45/395 (11.4) 47/395 (11.9)
32/108 (29.6) 43/108 (39.8) 19/108 (17.6) 11/108 (10.2) 3/108 (2.8)
0.015
Values are number (percentage). a Fisher’s exact test – embryos obtained from vitrified oocytes showed a statistically significantly better grading distribution than those derived from oocytes cryopreserved by slow freezing.
were performed with three embryos (Table 3). Positive B-HCG was measured 14 days after embryo transfer, and 17 clinical pregnancies were observed, with a clinical pregnancy rate of 7.6% per embryo transfer and 10.6% per patient. The miscarriage rate was 29.4%. The implantation rate per embryo transferred was 4.3%, the implantation rate per injected oocyte was 2.4% and the implantation rate per fertilized oocyte was 3.5% (Table 4). For transfers of one, two or three embryos, the clinical pregnancy rates were 9.7%, 4.8% and 10.5% respectively. Five pregnancies ended in miscarriage and 12 healthy babies have been born.
Out of 162 inseminated oocytes, 118 oocytes showed evidence of fertilization (2PN) with a fertilization rate of 72.8% and 22 (13.6%) did not show any evidence of fertilization; four showed anomalous fertilization with 3PN (2.5%) and 18 degenerated after insemination (11.1%). After 48 h of culture, 10 zygotes did not divide and 108 divided to 2–4-cell embryos with a cleavage rate of 91.5%. According to the morphological characteristics of the embryos, 29.6% were Grade 1, 39.8% were Grade 2, 17.6% were Grade 3, 10.2% were Grade 4 and 2.8% were Grade 5 (Table 2). All embryos obtained were transferred, according to Italian law, without any embryo selection.
Vitrification results
The centre performed 55 embryo transfers in 43 patients with an average of 1.9 p 0.8 embryos per transfer. Of these, 34.5% of embryo transfers were with only one embryo, 34.5% with two embryos and 30.9% with three embryos (Table 3). Positive B-HCG was measured 14 days after embryo transfer, and 10 clinical pregnancies were observed, with a clinical pregnancy rate of 18.2% per embryo transfer and 23.3% per patient. The miscarriage rate was 20%. The implantation rate per embryo transferred was 9.3%, the implantation rate per injected oocyte was 6.2% and the implantation rate per fertilized oocyte was 8.5% (Table 4). For transfers of one, two or three embryos the clinical pregnancy rates were 10.5%, 15.8% and 29.4% respectively. At the time of writing, five pregnancies were ongoing, two ended in miscarriage and three healthy babies have been born.
A total of 765 MII oocytes were vitrified from January 2007 to December 2007 in the centre. Of these, 285 oocytes from 46 patients (average age 34.6 p 3.2 years, range 28–39) were warmed in 59 cycles with an average number of 4.8 p 1.2 oocytes each cycle (Table 1).
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Out of 285 warmed oocytes, only 225 oocytes survived the cooling–warming process with a survival rate of 78.9%. In total, 162 oocytes were inseminated, with an average of 2.7 p 0.5 oocytes per cycle and no more than 3 oocytes per cycle, in accordance with the Italian law.
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Article - Slow and ultrarapid cryopreservation of human oocytes - R Fadini et al.
Table 3. Comparison of the number of embryos transferred according to the oocyte cryopreservation protocol used. No. of embryos transferred
Slow-freezing Vitrification protocol protocol
P-value
1 2 3
62 (27.7) 105 (46.9) 57 (25.4)
NS
19 (34.5) 19 (34.5) 17 (31.0)
Values are number (percentage); NS = not significant (Fisher’s exact test).
Table 4. Comparison of clinical data between slow-freezing protocol and vitrification protocol.
No. of embryo transfers Patients at embryo transfer Positive B-HCG test Clinical pregnancies per embryo transfer Clinical pregnancies per patient Miscarriages Implantation rate per transferred embryo Implantation rate per thawed oocyte Implantation rate per injected oocyte Implantation rate per fertilized oocyte
Slow-freezing protocol
Vitrification protocol
P-valuea
224 161 21 17/224 (7.6) 17/161 (10.6) 5/17 (29.4) 17/395 (4.3) 17/1348 (1.3) 17/694 (2.4) 17/481 (3.5)
55 43 13 10/55 (18.2) 10/43 (23.3) 2/10 (20.0) 10/108 (9.3) 10/285 (3.5) 10/162 (6.2) 10/118 (8.5)
– – – 0.021 0.046 NS 0.043 0.012 0.019 0.025
Values in brackets are percentages. HCG = human chorionic gonadotrophin; NS = not significant. a Fisher’s exact test. According to Italian law, three oocytes/cycle were injected and no embryo selection was performed.
Discussion The reasons for choosing to freeze oocytes rather than embryos are not discussed here, even though it is still a debatable matter, considering that the results reported so far on cryopreserving human oocytes have not shown the same success rate as that obtained with embryo freezing (Oktay et al., 2006). In Italy, where embryo cryopreservation is forbidden by law, oocyte cryopreservation has become a necessary procedure in order to save oocytes and to ensure a good cumulative success rate (Boldt et al., 2003, 2006) avoiding reiterated ovulation induction. In this regard the Italian National Register on assisted reproductive technology has recently reported that more than 100,000 oocytes, 48.3% of all the oocytes retrieved in Italy in 2006, remained unused and were destroyed (Turco, 2008). Although many studies investigating the feasibility and efficiency of oocyte cryopreservation have reported good biological and clinical results supporting the viability of this technology, oocyte freezing is still considered an experimental procedure currently in a research phase and the efficiency of the method is debatable. The main problems concerning oocyte cryopreservation are due to the dimension of the cell, the high cytoplasmic water content and the low membrane permeability. In fact, during the freezing procedure, the water retained in the cytoplasm may produce ice crystals which could irreparably damage the ultrastructure RBMOnline®
of the oocyte. To protect the cell against chilling injuries, due to inner ice formation during freezing and thawing phases, the oocytes must be exposed to permeable and non-permeable cryoprotectants that induce oocyte dehydration by removing most of water from cytoplasm. Moreover the shrinkage and swelling of the oocytes, occurring during the dehydration phase, can also injure the morphology and functionality of oocytes (Rienzi et al., 2004; Ghetler et al., 2006; Coticchio et al., 2007; Nottola et al., 2007). The slow-freezing method implies the exposure of the oocytes to low cryoprotectant concentrations for a long time in order to induce a slow dehydration of the cells and a very slow decrease in temperature. The slow rate of cooling must be controlled by an appropriate freezer machine. A suitable sucrose concentration to be used in slow-freezing protocols is still under assessment. In fact the Italian studies, the most important published regarding this subject, suggest three different concentrations of sucrose: 0.1 mol/l (Borini et al., 2004; De Santis et al., 2007), 0.2 mol/l (Bianchi et al., 2007) and 0.3 mol/l (Borini et al., 2006; Levi Setti et al., 2006; De Santis et al., 2007; Parmegiani et al., 2008). The only randomized study published, which compared these three different sucrose concentrations, demonstrated that the highest concentration may ensure the best results (Fabbri et al., 2001). Although a paper reported a remarkable success rate also with 0.2 mol/l (Bianchi et al., 2007), the 0.3 mol/l protocol is, up to now, the most accepted, even if forthcoming studies may still keep the matter open. In 2004, the slow-freezing method
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was introduced, choosing 0.3 mol/l sucrose concentration and PROH, according to the previous experiences that had ensured better results (Fabbri et al., 2001). Vitrification allows an ultrarapid transition of the intracellular water in a glass-like phase, avoiding chilling injuries. Using this strategy to induce rapid dehydration, a high dose of cryoprotectants is used for a very short time, followed by an ultrarapid decrease in temperature (20,000oC/min). In order to speed up the cooling rate, the oocytes are directly plunged into liquid nitrogen. For this purpose, different protocols have been published and various devices have been proposed (Liebermann and Tucker, 2002; Liebermann et al., 2002a,b,c; Katayama et al., 2003; Cai et al., 2005; Chian et al., 2005; Isachenko et al., 2005; Kuwayama et al., 2005a,b; Sheehan et al., 2006). Cryoleaf was used as the carrier (Chian et al., 2005). This open carrier is simple to use and has a coloured security covering which mechanically protects the leaf where oocytes are placed. This makes handling the carrier in the liquid nitrogen easier and safer, although it is likely that every embryologist will become proficient in using their own device. This report is based entirely on experience with vitrification using PROH, ethylene glycol and sucrose as cryoprotectants. In order to better standardize the procedure, ‘homemade’ culture media were avoided. The commercially available kit from MediCult was found to be extremely convenient. This retrospective study consists of a comparison of the results achieved from both techniques. The two different cryopreservation procedures were applied sequentially in consecutive periods. During the whole period, the ovarian stimulation protocols used presented only minor differences; furthermore, no changes were made to the laboratory culture system or to the endometrial preparation. So, it can be confidently stated that results weren’t affected in any way by differences in the stimulation protocols used. The survival rate after thawing is one of the most critical measures in any oocyte cryopreservation programme. In the literature, a variable survival percentage has been reported with the slow-freezing method and with the ultrarapid cooling method (Tables 5 and 6). This study observed a significantly higher survival rate using the vitrification method. A survival rate of 57.9% was achieved in a 3-year period using the slowfreezing protocol for 286 thawing cycles with a total of 1348 oocytes thawed. This was considered sufficient experience to standardize the protocol and pass through the learning curve. Whilst in a few months, warming only 285 vitrified oocytes in 59 cycles, a higher percentage survival rate (78.9%, P < 0.0001) was achieved. It can be concluded that the difference could be due to the different methods.
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According to Italian assisted reproduction law, the same number of oocytes per cycle (not more than three per cycle) were inseminated in both groups but a higher fertilization rate was obtained after vitrification (72.8% P = 0.027), whilst the percentage of anomalous fertilization (1PN or 3PN) was comparable between the two methods. The better fertilization rate obtained using vitrified oocytes could be due to better health of the oocytes. The consequence of this would be that even though the same number of oocytes were inseminated, a
higher number of embryos were obtained for transfer (Table 1). The different mean number of embryos transferred is related to the technique used and it could have contributed to improve the outcome of the procedure. Regarding embryo quality, no cleavage delay in embryo development was observed with both methods but a different distribution among the five grades was recorded. Embryos obtained from vitrified oocytes showed a statistically significantly better grading distribution than those derived from slow-freezing oocytes (P = 0.015; Table 2). All this resulted in a higher number of good quality embryos in women where vitrification was performed and these biological elements might favourably affect the pregnancy rate. As far as the clinical pregnancy rate per transfer and the implantation rate are concerned, the literature reports a great variability with both methods (Table 5 and Table 6). Significant differences were observed when comparing the two methods. Vitrification resulted in a higher number of embryos to transfer, higher clinical pregnancy and implantation rates than the slow-freezing method. Very soon, a change was made from slow freezing to vitrification, increasing the success rate. So after 3 years of experience, the centre eventually decided to discontinue the slow-freezing method in favour of vitrification, because the latter method had shown a significant favourable impact on the whole outcome. As regards the vitrification success, it is quite difficult to compare these results, in terms of pregnancy rate, with those published by other groups (Table 5 and Table 6) because, according to the strict Italian law, the centre cannot produce more than three embryos nor perform a morphological embryo selection before embryo transfer, while strict selection is carried out in other countries. The centre transferred an average of 1.9 embryos per transfer, which means 34.5% of the embryo transfers were performed with only one unselected embryo (Table 3). This high percentage of unselected single embryo transfers is directly linked to the compulsory use of only three oocytes per cycle. So, it is difficult to compare the potential results that could have been obtained by fertilizing more than three oocytes. This might have reduced the pregnancy and implantation rates when compared with other published papers. Moreover the Italian results before and after the law are not comparable either. Nevertheless, the results of oocyte cryopreservation in this study appear worse when compared with other Italian studies previously published using slow-freezing oocyte cryopreservation (Borini et al., 2004, 2006, 2007a; Bianchi et al., 2007; De Santis et al., 2007; Parmegiani et al., 2008) as well as vitrification (Antinori et al., 2007). The difference could be explained by considering the selection of the women. In fact, the study centre has always carried out mild ovarian stimulation in IVF and has extensively applied in-vitro maturation of oocytes both in polycystic ovary syndrome/polycystic ovary patients and in young women with normal ovaries without any ovarian stimulation. This strategy definitely reduces the number of available oocytes, mainly in those women with a very good ovarian response who are actually the most favourable and whose oocytes can be frozen. So a sort of negative patient RBMOnline®
Article - Slow and ultrarapid cryopreservation of human oocytes - R Fadini et al.
Table 5. Studies reporting survival, fertilization, implantation and pregnancy rates of frozen–thawed oocyte cycles with slowfreezing method. Reference
Thawed Thawed cycles oocytes (n) (n)
Survival Fertilization Implantation rate rate (%) rate per (%) transfer (%)
Pregnancy Pregnancies Babies rate per (n) born transfer (%) (n)
Porcu et al., 1999 Winslow et al., 2001 Boldt et al., 2003 Borini et al., 2004 Li et al., 2005 Boldt et al., 2006 La Sala et al., 2006 Levi Setti et al., 2006 Borini et al., 2007a De Santis et al., 2007 Bianchi et al., 2007 Parmegiani et al., 2008
112 45 16 86 15 53 518 159 660 133 90 93
54.1 68.5 74.4 37.0 90.1 60.4 72.8 69.9 68.1 47.5 75.9 75.1
14.2 26.2 36.4 25.4 44.0 32.5 3.8 12.4 14.9 13.1 21.3 19.2
1502 324 90 737 81 361 1647 1087 3238 902 403 437
57.7 80.8 59.0 45.4 82.0 62.0 74.7 67.5 76.1 67.0 76.2 86.0
– 13.5 15.1 16.4 14.8 13.3 6.3 5.7 8.1 6.7 13.5 9.6
16 11 4 15 7 12 19 18 88 12 17 16
11 16 3 13 6 11 7 13 60 3 4 10
Table 6. Studies reporting survival, fertilization, implantation and pregnancy rates of cooled-warmed oocyte cycles with vitrification method. Reference
Thawed Thawed cycles oocytes (n) (n)
Survival Fertilization Implantation Pregnancy Pregnancies Babies rate rate (%) rate per rate transfer (n) born (%) transfer (%) (n)
Yoon et al., 2000 Yoon et al., 2003 Kuwayama et al., 2005b Chian et al., 2005 Lucena et al., 2006a Selman et al., 2006 Antinori et al., 2007 Cobo et al., 2008a
7 34 29 15 33 6 120 30
84.9 68.6 91.0 93.9 89.2 75.0 99.4 96.9
90 474 64 180 159 24 796 231
65.0 71.7 89.6 74.6 87.2 77.7 93.0 76.3
9.4 6.4 18.7 2.4 13.4 21.4 13.2 40.8
42.0 21.4 41.3 46.7 56.5 33.3 32.5 65.2
3 6 12 11 13 2 28 11
2 7 7 – – – 3 –
a
Egg donation programme.
selection has been introduced that could bias the outcome of the cycles. The Italian National Register (Turco, 2008) has recently published the results on oocyte cryopreservation carried out in 2006. The Italian Register has recorded data on 2977 thawing cycles which represent 7.3% of the whole IVF Italian cycles. A pregnancy rate of 10% per thawed and a pregnancy rate per transfer of 12.6% were reported. It is expected that this high number of oocyte cryopreservation cycles could possibly increase in the coming years, so that oocyte cryopreservation procedures will have a great impact on routine practice in the major Italian laboratories. Comparing the two available cryopreservation methods shows that vitrification has some advantages compared with slow freezing. Although the cost of materials for each patient is equal, the vitrification method does not need an expensive freezing machine. Regarding the time consumption for every single freezing procedure, slow freezing takes almost
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3 h to complete whilst the ultrarapid procedure can be completed in about 20 min. However, it should be remembered that, when there are many women undergoing a cryopreservation procedure, vitrification also takes a long time. Although the time taken is hardly a remarkable point, the appropriate timing of the freezing procedure is actually a critical element. In fact in a busy laboratory, if many samples must be simultaneously frozen with the slow procedure, the beginning of the procedure is, every so often, delayed so as to freeze the eggs retrieved from different women at once. Thereby, the delay in oocyte cryopreservation may age the oocytes. As reported, the ageing of oocytes may impair their developmental capability (Yanagida et al., 1998; Dozortsev et al., 2004; Parmegiani et al., 2008) and jeopardize the procedure. On the other hand, vitrification can be promptly performed after each oocyte retrieval without any delay.
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Another advantage of vitrification is the possibility to ‘individualise’ the process with respect to each oocyte. Using vitrification, the embryologist can check each oocyte during the different phases of shrinkage and swelling. In the study centre, each oocyte has been observed to have its own membrane permeability and shows a different response during exposure to equilibration and vitrification solutions. The importance of observing the changes in oocyte volume in an equilibration solution must be stressed, to decide when to move the cell into the vitrification solution and then into liquid nitrogen. In the authors’ experience, the best time is when the shrinkage/ swelling process is completed. So efforts must be made to individualize the incubation time as much as possible. Moreover, according to the individual response, a decision can be made at the dehydration and hydration phase whether to continue or suspend the cooling process. The slow method, however, cannot be individualized owing to the programmable freezer machine. The oocytes can only be observed in the equilibration solution for quite a short time before loading the freezing machine while the dehydration process in the first part of the cooling rate is still continuing. It is the authors’ view that vitrification easily fits all the laboratory demands, hence the different outcome of oocyte cryopreservation observed could be explained by its ability to be a safe and adaptable procedure to freeze oocytes. Even if oocytes were vitrified using an open system, clean factoryderived nitrogen and disposable materials can be used, changing the liquid nitrogen for each patient. The risk of potentially cross-contamination cannot be excluded, but as far as is known, there is no publication about vitrification that has reported the transmission of infectious diseases after cryotransfer. In any case, the use of sterile liquid nitrogen should be considered (Bielanski et al., 2000; Vajta and Nagy, 2006). Concerning the potential genetic and metabolic damage of oocyte cryopreservation, this report does not contribute any definite results. From all of the studies already published, more than 300 pregnancies have been obtained and 160 births have so far been reported. No disquieting reports have been published as yet. Interesting papers have recently recorded the outcome in infants conceived from slow-freezing/vitrification protocols. These studies confirm the safety of oocyte cryopreservation (Borini et al., 2007b; Gook and Edgar, 2007; Tur-Kaspa et al., 2007; Chian et al., 2008; Manipalviratn et al., 2008). In conclusion, at present in this clinical practice, oocytes are frozen as a routine part of the IVF procedure due to lack of another alternative, but it is considered to be a reliable method. Although there is a choice between two available methods to freeze human oocytes, according to this centre’s experience, the only reason for using slow freezing instead of ultrarapid cooling is habit in those centres which obtain good results with the slow method. This report supports introducing vitrification as new method because it can assure a more successful outcome. Oocyte cryopreservation procedures still need to be improved and although results are far from excellent, the results can be considered to be fairly good.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 30 July 2008; refereed 29 August 2008; accepted 11 February 2009.
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