Human Reproduction Vol.21, No.10 pp. 2656–2660, 2006 doi:10.1093/humrep/del228 Advance Access publication June 12, 2006. Cryopreservation of biopsied embryos at the blastocyst stage M.C.Magli1, Luca Gianaroli1,3, N.Grieco1, E.Cefalù2, G.Ruvolo2 and A.P.Ferraretti1 1 S.I.S.ME.R, Reproductive Medicine Unit, Bologna and 2Biology of Reproduction Center, Reproductive Medicine Unit, Palermo, Italy 3 To whom correspondence should be addressed at: S.I.S.ME.R, Reproductive Medicine Unit, Via Mazzini, 12, 40138 Bologna, Italy. E-mail: luca.gianaroli@sismer.it Preliminary data were presented during the 21st Annual Meeting of ESHRE (Copenhagen, June 20–23, 2005). Key words: 1,2-propanediol/blastocysts/cumulative pregnancy rate/embryo biopsy/PGD Introduction Within the general framework of assisted reproductive techniques (ART) safety, the definition of criteria that predicates the selection of healthy, viable embryos has become increasingly important in reproductive medicine. Since the development of amniocentesis in the early 1970s, couples at risk of an affected pregnancy have been recommended to undergo prenatal diagnosis, having the possibility of giving birth only to children who are healthy for the investigated pathology. More recently, PGD has been proposed as an early form of prenatal diagnosis that is based on the analysis of a single cell: a blastomere biopsied from morphologically normal day 3 embryos or the first and second polar body retrieved from the oocyte (Handyside et al., 1990, 1992; Verlinsky et al., 1990). In both cases, the prevention of genetic and chromosomal disorders can be made before embryos are transferred to the patients’ uterus. Basically, PGD is recommended for the screening of singlegene disorders and the analysis of chromosomal abnormalities, both numerical and structural while, more recently, it has also been proposed to screen carriers of genetic predisposition to late-onset disorders and to screen couples having an affected child requiring bone marrow transplantation from an HLAidentical sibling (Fiorentino et al., 2004; Kuliev and Verlinsky, 2004; Van de Velde et al., 2004; Kuliev et al., 2005). PGD for aneuploidy is by far the most common technique as the selection of chromosomally normal embryos for transfer has a positive effect on the clinical outcome (Gianaroli et al., 1999a; Munné et al., 1999). This is probably due to the strong association between chromosomal abnormalities and embryo non-viability, mainly resulting in spontaneous abortions and implantation failure (Gianaroli et al., 2005; Munné et al., 2005; Verlinsky et al., 2005). In consideration of the complexity and labour intensity of PGD, as well as of the psychological condition to which patients are exposed, the availability of an efficient cryopreservation program is especially important in the case of biopsied embryos. In view of the above considerations, embryos diagnosed as transferable by PGD are of particular value and the possibility of storing them prevents the couple from having to repeat all the steps of the PGD cycle. Unfortunately, the cryopreservation of biopsied embryos has given disappointing results when performed at the cleavage stage following conventional protocols (Joris et al., 1999; Magli et al., 1999). In this study, biopsied embryos that were diagnosed as normal or healthy carriers after PGD (for aneuploidy, translocations or genetic disorders) were grown to the blastocyst stage and frozen. The outcome of the thawing cycles is reported here. 2656 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org Downloaded from http://humrep.oxfordjournals.org/ by guest on August 23, 2015 BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact. Cryopreservation of biopsied embryos Materials and methods Experimental design Before starting its clinical application, the reported protocol for blastocyst freezing was tested on 18 blastocysts. They were derived from consenting PGD patients and had been diagnosed as abnormal after genetic analysis and therefore not suitable for transfer. According to patients’ consent, they were frozen and thawed after at least 1 week in liquid nitrogen. After thawing and overnight culture, they were morphologically evaluated and then discarded. Patients—conventional cycles During the same period, 88 IVF/ICSI patients underwent 92 thawing cycles in which 150 blastocysts were thawed. These blastocysts had been cryopreserved in previous treatment cycles following the same protocol as for biopsied embryos. All the embryos generated by these patients were thawed in the 92 thawing cycles. The culture system was the same as described for PGD cycles. Freezing and thawing The cryopreservation solutions were based on the use of HEPESbuffered T6 medium supplemented with 10% human serum albumin (HSA; SAGE, Biopharma, Trumbull, CT, USA). The freezing solution included 1.5 M 1,2-propanediol (PROH) in 0.1 M sucrose in the classical slow-freezing protocol used for zygote or embryo cryopreservation (Lassalle et al., 1985; Ferraretti et al., 1999). Cooling was performed in a programmable freezer (Kryo 10 Series, Planer Products, Sunbury Thames, UK) at a rate of 2°C/min from 20 to 7°C. At this point, manual seeding was done, and freezing was continued at a rate of 0.3°C/min to –30°C, and 50°C/min to –150°C. Straws, containing one blastocyst each, were then plunged into liquid nitrogen and stored. Blastocyst thawing was performed in the late afternoon of the day before transfer. The cryoprotectant was removed through sequential steps in 1.0 M PROH in 0.2 M sucrose (5 min), 0.5 M PROH in 0.2 M sucrose (5 min) and finally 0.2 M sucrose (5 min). The thawed blastocysts were thoroughly washed, transferred to culture medium (CCM, Vitrolife) and cultured overnight. Results In the initial part of this study, the freezing protocol was applied to 18 blastocysts diagnosed as abnormal by PGD. After thawing, 11 survived (61%) and 8 hatched in vitro. These results supported the clinical application of the technique that is summarized in Table I. A total of 47 blastocysts were thawed and 25 fully survived the procedure (53%). Soon after thawing, the blastocele cavity of all 25 blastocysts was collapsed, but on the following morning, 15 blastocysts were fully expanded (Figure 1) and the remaining 10 had an expanding cavity. In seven of the expanded blastocysts, the hatching process had already started, irrespective of the thickness of the zona pellucida that in some cases had not thinned (Figure 2). Embryo transfer was possible for 18 patients (53% of the thawing cycles) yielding seven clinical pregnancies (39% per transferred cycle, 21% per thawing cycle) with an implantation rate of 32.0%. Two pregnancies miscarried, and five went regularly to term delivering six healthy infants. The transfer of fully expanded blastocysts was tightly related to implantation, as seven of the eight gestational sacs originated from blastocysts with a fully expanded blastocele at the time of transfer. In five patients who were scheduled for PGD of single-gene disorders or translocations, two blastomeres had been removed at embryo biopsy. After thawing, three blastocysts survived out of seven; a single embryo transfer was performed in the three cycles and two embryos implanted to term. Table II represents the results obtained by the thawing of blastocysts that had been frozen during conventional IVF/ICSI cycles. In all, 150 blastocysts from 88 IVF/ICSI cycles were thawed in 92 cycles, and 87 (58%) survived. Embryo transfer Table I. Outcome of thawing cycles in PGD patients Thawed cycles (n) Thawed blastocysts (n) Survived blastocysts [n (%)] Transferred cycles [n (%)] Clinical pregnancies (n) Percentage per transferred cycle Percentage per thawed cycle Implantation rate (%) Abortions (n) Infants born (n) 34 47 25 (53) 18 (53) 7 39 21 32 2 6 2657 Downloaded from http://humrep.oxfordjournals.org/ by guest on August 23, 2015 Patients—PGD cycles Between January 2001 and February 2004, a total of 49 PGD patients had 89 supernumerary embryos that were cryopreserved at the blastocyst stage. Following the treatment cycle, 34 of them returned for a frozen embryo transfer in which all their embryos were thawed. In the fresh cycle, oocytes were inseminated and cultured in IVF medium in microdroplets under pre-equilibrated mineral oil (Vitrolife, Göteborg, Sweden). Embryo biopsy was performed on day 3 embryos in standard HEPES-buffered medium (Gamete, Vitrolife) by chemical opening of the zona pellucida with removal of one nucleated blastomere (aneuploidy screening, n = 29) or two (PGD for monogenic disorders and translocations, n = 5) (Gianaroli et al., 2003). Biopsied embryos were transferred to fresh culture medium (CCM, Vitrolife) and cultured until the time of transfer or cryopreservation. Embryo transfer was performed at completion of the genetic analysis on day 4 or 5 (Gianaroli et al., 1999b). When more than two transferable embryos were available, the transfer was always postponed to day 5 with the aim of reducing the need to resort to embryo cryopreservation on the basis of natural selection. Only morphologically normal blastocysts (grades 3–4, A or B for inner cell mass and trophectoderm according to Gardner and Schoolcraft, 1999) diagnosed as normal at the genetic analysis were cryopreserved. Clinical outcome Hormonal replacement therapy was administered for the thawed cycles (Ferraretti et al., 1999). Briefly, oestradiol valerate (Progynova, Schering, Milan, Italy) was given 2 mg/day from day 1 to day 5 of the menstrual cycle; 4 mg/day from day 6 to day 10; 6 mg/day from day 11 to day 13 and 4 mg/day from day 14. From day 15, 50 mg/day progesterone was given, and from day 17, the dose was increased to 100 mg/day. Clinical pregnancies were defined by the presence of a gestational sac with fetal heartbeat at ultrasound scanning. The implantation rate represented the percentage of gestational sacs with fetal heartbeat divided by the total number of embryos transferred. Before embryo transfer, PGD patients were especially recommended to undergo prenatal diagnosis to confirm and complete the results from PGD. Data were analysed by Fisher’s exact test and chi-square analysis applying the Yates’ correction, 2 × 2 contingency tables. M.C.Magli et al. Table II. Outcome of thawing cycles in conventional IVF/ICSI patients Thawed cycles (n) Thawed blastocysts (n) Survived blastocysts [n (%)] Transferred cycles [n (%)] Clinical pregnancies (n) Percentage per transferred cycle Percentage per thawed cycle Implantation rate (%) Abortions (n) Infants born (n) 92 150 87 (58) 63 (68) 18 29 20 20.7 5 13 Table III. Cumulative data in PGD and conventional IVF/ICSI cycles IVF/ICSI cycles 34 35.9 ± 4.4* 416 (12.2 ± 2.9) 416 337 (81) 310 (92) 272 140 (51) 54 (17)** 47 (15)*** 29a 13 (45) 25.9 47 79 (1.8 ± 0.5) 88 33.8 ± 3.4* 899 (10.2 ± 3.3) 695 542 (78) 515 (95) – – 141 (27)**** 150 (29)***** 75b 23 (31) 19.9 138 228 (1.6 ± 0.5) 20 43 59 5 29.1 44 41 30 47 13 20.2 32 *P < 0.005. **, *** versus ****, *****P < 0.001. a Five cycles were not transferred in the fresh cycle being at risk for ovarian hyperstimulation syndrome (OHSS). b Thirteen cycles were not transferred in the fresh cycle being at risk for OHSS. Figure 2. Expanded blastocyst at the observation performed on the morning after thawing. The hatching process is advanced despite the thickness of the zona pellucida. was performed in 63 cycles (68% of thawed cycles) resulting in 18 clinical pregnancies (29% per transferred cycle and 20% per thawed cycle) and an implantation rate of 20.7%. Spontaneous abortion occurred in 5 pregnancies, while 13 went to term with 13 healthy infants born. 2658 The cumulative data of the patients included in the study are reported in Table III. Due to the prevalence of indications to PGD for aneuploidy (29 patients out of 34), the mean maternal age was significantly higher in the PGD group [35.9 ± 4.4 (SD) years] compared with the IVF/ICSI group (33.8 ± 3.4 years; P < 0.005). In addition, consequent to the genetic analysis, the proportion of embryos suitable for transfer and/or cryopreservation was greater in the IVF/ICSI group (141 and 150, respectively, representing 56% of the generated embryos) than in the PGD group (54 and 47, respectively, representing 33% of the generated embryos; P < 0.001). Despite a significantly higher maternal age in the IVF/ICSI group, the cumulative pregnancy rate per patient was similar in the two groups (59% in the PGD group and 47% in the IVF/ICSI group) as well as the take-home baby rate (44% in the PGD group and 32% in the IVF/ICSI group). Discussion Previously reported experiences have demonstrated that the standard freezing protocols yield unsatisfactory results when applied to biopsied embryos, when using both propanediol Downloaded from http://humrep.oxfordjournals.org/ by guest on August 23, 2015 Figure 1. Expanded blastocyst at the observation performed on the morning after thawing. The zona pellucida is thin and the hatching process has already started. Patients (n) Age in years (M ± SD) Oocytes [n (M ± SD)] Inseminated oocytes (n) Fertilized oocytes [n (%)] Embryos [n (%)] Biopsied embryos (n) PGD normal [n (%)] Transferred embryos [n (%)] Cryopreserved embryos [n (%)] Transferred cycles (n) Clinical pregnancies [n (%)] Implantation rate (%) Transferred cycles—cumulative (n) Transferred embryos—cumulative [n (M ± SD)] Clinical pregnancies—cumulative (n) Percentage per transferred cycle Percentage per patient Abortions—cumulative (n) Implantation rate (%)—cumulative Take-home baby rate per patient (%) PGD cycles Cryopreservation of biopsied embryos standard freezing protocol (80 versus 50%, respectively). Unfortunately, no clinical application was reported. A valid alternative could be represented by vitrification that avoids ice-crystal formation by combining high cryoprotectant concentration and high cooling and warming rates. Recently, encouraging results have been reported using non-transferable biopsied embryos with a survival index of 90% (Zheng et al., 2005). Neither in this case a clinical application was achieved, whereas in another preliminary study the vitrification of blastocysts developed from biopsied embryos yielded a survival index of 73% and a clinical pregnancy (Galan et al., 2003). Basically, two considerations supported the decision of cryopreserving biopsied embryos at the blastocyst stage, as it was done in the current study: (i) it permits the selection of the most viable embryos for cryopreservation by discarding those having developmental arrest. In this way, the number of frozen embryos is kept at a minimum. (ii) The thinning of zona pellucida in the expanding blastocyst possibly reduces its effect of water barrier, making the presence of a breach due to blastomere biopsy of less effect as far as water permeability and turbulence is concerned. In addition, the structure of the blastocyst itself, with ∼100 tightly connected cells, could possibly make it more resistant to high-speed rehydration. Accordingly, in a recent study, blastocysts were cryopreserved after undergoing trophectoderm biopsy; 27 of them were thawed, 24 had >50% cells survived and yielded an implantation rate of 26% (McArthur et al., 2005). The decision to use PROH for blastocyst freezing instead of glycerol was based on its characteristics of permeability and capacity of replacing H2O molecules to protect against thermal shock or dilution stress (Sakkas et al., 2001; Quintans et al., 2003). This was considered to be especially important for the presence of the breach in the zona pellucida and the consequent relevance of water flow at warming. As the preliminary data derived from the experimental part of this study were promising, the same protocol was also applied to blastocysts originated from non-biopsied embryos for which the traditional use of glycerol as cryoprotectant had not provided satisfactory results in terms of implantation and incidence of miscarriage. According to the data obtained with the use of PROH, the survival rate still remains the critical point corresponding to 53% of thawed blastocysts in the PGD group. This figure was not different from that obtained in non-biopsied embryos which were frozen with the same protocol (58%). However, compared with the data reported in the literature, the survival rate of the IVF/ICSI group is lower than expected by using the glycerol as cryoprotectant (Ménézo and Ben Khalifa, 1995; Gardner et al., 2003; Veek, 2003) suggesting that possibly different strategies could be optimal for the freezing of blastocysts generated from biopsied or intact embryos. In both study groups, surviving embryos could further develop and give rise to gestational sacs with fetal heartbeat in a proportion corresponding to 32.0% after PGD and 20.7% in IVF/ICSI cycles. Despite the significant difference between the proportion of transferable embryos in the fresh cycle of the two groups, that was superior in IVF/ICSI cycles, the high implantation rate in the PGD group compensated for the strong selection due to the genetic analysis (Table III). As a result, the 2659 Downloaded from http://humrep.oxfordjournals.org/ by guest on August 23, 2015 (Magli et al., 1999) and dimethyl sulphoxide (Joris et al., 1999). This negative performance is probably related to the breach created in the zona pellucida that might alter the diffusion of the cryoprotectant solution compared with zona-intact embryos. It can be postulated that the zona pellucida has an important role in preserving embryo integrity, especially through the steps of dehydration, shrinking and rehydration occurring during freezing and thawing. According to a recent report, blastomeres near to the breach in the zona are more prone to lysis after thawing (Zheng et al., 2005). This could be due to a different water velocity during thawing in the proximity of the hole and far from it, implying that, although water and cryoprotectants pass freely through the zona pellucida, the zona could act as a barrier to modulate speed rehydration during warming. In a recent report, an opening was performed in the zona pellucida on days 3–4 embryos; after vitrification of the resulting blastocysts and subsequent warming, 80% survived with an implantation rate of 26% (Zech et al., 2005). These findings suggest that the empty space consequent to the removal of blastomeres might have an additional effect on embryo viability after cryopreservation. The poor survival rate obtained after thawing biopsied embryos is of great concern for the general outcome of a PGD cycle, especially when considering the complexity, both technical and psychological, of the procedure. For this reason, the supernumerary embryos are considered to be particularly valuable having being screened for a genetic abnormality. More recently, the technique of comparative genomic hybridization (CGH) has been proposed for the analysis of all chromosomes in single cells (Voullaire et al., 2000; Wells and Delhanty, 2000). The time currently needed to complete the diagnosis is not compatible with a fresh embryo transfer and embryos must be cryopreserved after biopsy (Wilton et al., 2001). This makes the availability of an efficient cryopreservation protocol mandatory to minimize the damage related to embryo freezing and thawing. In the effort of investigating the cryopreservation process and designing a protocol more suitable for biopsied embryos, the role of sucrose, a protein source and freezing medium has been reconsidered. Jericho et al. (2003) have proposed an increased concentration of sucrose in the freezing solution that could enable a more complete dehydration of blastomeres, in combination with the use of human serum instead of albumin. Due to their chemical structure (polyhydroxy domains facilitating the interaction with water molecules), the content of globulins in human serum could substantially contribute to an adequate dehydration, improving the recovery of viable embryos after thawing (Pool and Martin, 1994). The use of this protocol for biopsied embryos significantly improved the results compared with a standard freezing protocol: the survival index was 67 versus 46%, respectively, with a pregnancy rate of 16.7% per transfer (6 clinical pregnancies yielded by 36 transfers and 41 thawing cycles) and an implantation rate of 12% (Jericho et al., 2003). Another study has proposed the use of choline-based, sodium-free medium to replace the conventional sodium-based medium (Stachecki et al., 2005). The proportion of survived blastomeres was significantly improved compared with a M.C.Magli et al. References Ferraretti AP, Gianaroli L, Magli MC, Fortini D, Selman HA and Feliciani E (1999) Elective cryopreservation of all pronucleate embryos in women at risk of ovarian hyperstimulation syndrome: efficiency and safety. Hum Reprod 14,1457–1460. Fiorentino F, Biricik A, Karadayi H, Berkil H, Karlikaya G, Sertyel S, Podini D, Baldi M, Magli MC, Gianaroli L et al. 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Hum Reprod 20,1615–1618. Submitted on March 7, 2006; resubmitted on April 12, 2006; accepted on May 8, 2006 Downloaded from http://humrep.oxfordjournals.org/ by guest on August 23, 2015 cumulative clinical pregnancy rate after PGD was not lower than that obtained in IVF/ICSI cycles, with a resulting takehome baby rate per patient of 44% after PGD and 32% in IVF/ ICSI cycles. Although these figures are not significantly different, the trend is clinically relevant. These results are especially valuable when considering the significantly higher mean maternal age in the PGD group. These data confirm that an efficient cryopreservation program permits to give a substantial contribution to the cumulative pregnancy rate. This is especially notable for biopsied embryos for which the clinical advantages associated with the identification of transferable embryos based on PGD results are maintained after thawing. Although the number of patients included in the study group was relatively too small to draw robust conclusions, the method described here demonstrates that the implantation rate of biopsied embryos that were cryopreserved at the blastocyst stage is comparable with that obtained with fresh cycles. The current results suggest that, if the embryo survives the cryopreservation/thawing process, its chances of implanting are not negatively affected. Research should now be concentrated on improving the survival rate. If this obstacle is overcome, the impact on the efficiency of PGD treatment would be of great relevance.