Prospective randomized study on the cryopreservation of human embryos with dimethylsulfoxide or 1,2-propanediol protocols*t

Transcription

1 FERTILITY AND STERILITY Vol. 63, No. I, January 1995 Copyright 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Prospective randomized study on the cryopreservation of human embryos with dimethylsulfoxide or 1,2-propanediol protocols*t Josiane Vander Elst, Ph.D.:j: Michel Camus, M.D. Etienne Van den Abbeel, B.Sc. Ria Maes, M. T. Paul Devroey, M.D., Ph.D. Andre C. Van Steirteghem, M.D., Ph.D. Centre for Reproductive Medicine, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel) Medical School and University Hospital, Brussels, Belgium Objective: To investigate the optimal protocol for cryopreservation of human embryos obtained from IVF. Design: Prospective randomized study. Setting: Consenting patients in an academic research environment. Patients: Couples undergoing IVF. Interventions: A cohort of 2,220 supernumerary multicellular embryos were obtained from 488 patients who were randomized over slow freezing protocols with dimethylsulfoxide (DMSO, 819 embryos), 1,2-propanediol (699 embryos) or a mixture of DMSO and 1,2-propanediol (702 embryos). A total of 725 embryos have been thawed (DMSO, 232 embryos; 1,2-propanediol, 250 embryos and DMSO and 1,2-propanediol, 243 embryos) for transfer in natural ovarian cycles. Main Outcome Measures: Embryo survival rate, embryo implantation rate, clinical pregnancy rate (PR), delivery rate, live-birth rate. Results: The embryo survival rate was significantly higher with the DMSO protocol (52.6%) than with the 1,2-propanediol (32.0%) or the DMSO and 1,2-propanediol protocols (34.9%). The clinical PR per thawing cycle was significantly higher in the DMSO protocol (17.2%) than in the 1,2-propanediol protocol (3.9%). The clinical implantation rate per embryo thawed was significantly different between a DMSO-frozen embryo (4.7%) and a 1,2-propanediol-frozen embryo (1.2%). A DMSO and 1,2-propanediol-frozen embryo had a 3.7% chance of implantation. The delivery rate per thawing cycle was significantly higher in the DMSO protocol (12.5%) than in the 1,2-propanediol protocol (2.6% ). The live-birth rates per embryo thawed were 3.5%, 0.8%, and 2.9% in the DMSO, 1,2-propanediol, and DMSO and 1,2-propanediol groups, respectively. Conclusion: Supernumerary multicellular embryos as presented in daily clinical IVF practice have the highest chance of survival and of implantation after cryopreservation when DMSO has been used. Fertil Steril 1995;63: Key Words: Cryopreservation, human, embryos, survival, implantation, pregnancy, dimethylsulfoxide, 1,2-propanediol The advantage of a cryopreservation program to store the supernumerary embryos produced after ovarian hyperstimulation for IVF, GIFT, or zygote intrafallopian transfer (ZIFT) has already been well documented (1-12). This coupling of embryo cryopreservation and assisted reproduction will Received January 24, 1994; revised and accepted July 29, * Supported by the Fund for Medical Scientific Research (grant ), Brussels, Belgium. t Presented at the IXth Meeting of the European Society of Human Reproduction and Embryology, The Hague, The Netherlands, July 5 to 8, :j: Senior Research Assistant of the National Fund for Scientific Research, Brussels, Belgium. Reprint requests: Josiane Vander Elst, Ph.D., Centre for Reproductive Medicine, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel) Medical School and University Hospital, Laarbeeklaan 101, B-1090 Brussels, Belgium (FAX: ). 92 Vander Elst et al. Human embryo cryopreservation Fertility and Sterility

2 gain even more in importance in the future from the fact that the numbers of embryos available for cryopreservation are increasing steadily as a result of the introduction of new and more efficient techniques, such as intracytoplasmic sperm injection (13), and of the optimization of transfer procedures. The novel technique of intracytoplasmic sperm injection is characterized by high fertilization and embryo cleavage rates, such that 4 7% of the started cycles lead to cryopreservation of good-quality embryos. Moreover, the new transfer policy in our center of electively transferring two good quality embryos instead of three to avoid triplet pregnancies (14) also is leaving increasingly more embryos available for cryopreservation. The first successful pregnancy after transfer of a frozen-thawed human embryo was described by Trounson and Mohr (1) in 1983 using a slow freezing protocol with dimethylsulfoxide (DMSO) for four- and eight-cell embryos. Later on, freezing results have been reported using glycerol for the blastocyst stage (3) and 1,2-propanediol for one-cell and multicellular embryos (4, 5). In recent years, a general trend has developed to freeze more earlystage one- to four-cell embryos and from the literature it is obvious that the 1,2-propanediol protocol has become the most popular method (4, 5, 8, 10-12). In the same period in which 1,2-propanediol became a popular cryoprotectant, the use of GnRH agonists (GnRH-a) for ovarian stimulation also was introduced (15). This leads to a situation in which the freezing results described for DMSO mostly are associated with the use of clomiphene citrate-hmg stimulation, whereas the freezing results described for 1,2-propanediol mostly are associated with the use of GnRH-a and hmg for ovarian stimulation. Although the literature provides us with many data series on the freezing of different stages of embryonic development with different freezing protocols under different stimulation situations (1-12), comparison between published series almost is impossible as a result of patient-, stimulation-, or embryo-related selection biases. The absence of clear guidelines 10 years after the first report on successful freezing of human embryos accentuates the fact that human embryo freezing continues to be a challenging problem. The existing confusion led us to conduct a prospective randomized study to compare the outcome of different freezing protocols for clearly supernumerary multicellular human embryos obtained after the use of one and the same ovarian stimulation protocol. The final aim was to select an optimal protocol for the use of the cryobiologist who is confronted in daily clinical IVF practice with such a heterogeneous cohort of human embryos. We compared three slow-cooling protocols with DMSO, 1,2-propanediol, or a mixture of DMSO and 1,2- propanediol (16). We evaluated post-thaw survival rate, clinical pregnancy rate (PR), delivery rate, and live-birth rate. MATERIALS AND METHODS Patients and Embryos for Cryopreservation This study was conducted between September 1990 and July All patients in this trial were undergoing IVF and received ovarian stimulation by a combination of GnRH-analogs and hmg in the form of a long protocol (17). Ovarian suppression was obtained by daily administration of the GnRH-analog buserelin acetate (Suprefact; Hoechst, Brussels, Belgium) for at least 2 weeks starting on day 1 of the cycle. Ovarian superovulation was induced by hmg (Humegon; Organon, Oss, The Netherlands or Pergonal; Serono, Brussels, Belgium). When at least three follicles of 18 rom diameter were observed on ultrasound (US) and when serum 17fJ-E 2 was >1,000 pg/ml (conversion factor to SI units, 3.671), 10,000 IU hcg (Pregnyl; Organon or Profasi; Serono) was injected to trigger ovulation. Oocyte retrieval was performed by vaginal USguided puncture under local anesthesia. Oocytes were collected, cultured, and inseminated as described previously (18). Fertilization was assessed 16 to 18 hours after insemination by checking the presence of two distinct pronuclei. The fertilized oocytes were cultured further and, at 42 to 44 hours after insemination, embryonic development was assessed. Embryos were classified according to stage of development and morphological quality. The morphological score included four types of embryos of decreasing quality: [1] type A, embryos with equally or unequally sized blastomeres and no anucleate fragments; [2] type B, embryos with up to 20% of their volume filled with anucleate fragments; [3] type C, embryos with >20% and ::::;50% of their volume filled with anucleate fragments; and [4] type D, embryos with >50% of their volume filled with anucleate fragments. The three best embryos were selected for transfer in the collection cycle at day 2. Once a patient had more than three embryos presenting with <20% fragmentation (type A or B), the supernumerary embryos were al- Vol. 63, No.1, January 1995 Vander Elst et al. Human embryo cryopreservation 93

3 located to one of the three cryopreservation protocols described below: protocol A, DMSO; protocol B, 1,2-propanediol; and protocol C, DMS0-1,2- propanediol. Embryos were frozen at the two- to eight-cell stage with the majority of embryos at the four-cell stage. Allocation was done according to simple randomization tables. Protocol A: Slow Freezing With 1.5 M DMSO The slow-freezing protocol with 1.5 M DMSO was adapted from Trounson and Mohr (1) and from Camus et al. (9). The freezing medium consisted of HEPES-buffered Earle's medium with 2.25% (volj vol) of a 20% human serum albumin solution (HSA; Belgian Red Cross, Brussels, Belgium) and DMSO (British Drug House Ltd., Poole, Dorset, United Kingdom). Embryos were equilibrated in 0.75 M DMSO at room temperature for 10 minutes. They then were transferred to glass ampules (Wheaton 6201-B36; Polylab, Merksem, Belgium) containing 400,uL of a 1.5 M DMSO solution and these ampules were kept at 4 oc on crushed ice for another 10 minutes. After transfer to a biologic freezer preset at 4 C (Planer Kryo 10; VEL, Leuven, Belgium), embryos were cooled further at a rate of -2 C/min to -6 C. Seeding then was induced manually by touching the ampule with a liquid N 2 -cooled forceps. The temperature was held at -6 C for 10 minutes. Further freezing was performed at a cooling rate of -0.3 C/min down to -80 C, at which temperature the ampule was immersed in liquid N 2 Thawing was done by removing an ampule from the liquid N 2 and putting it immediately on crushed ice for 15 minutes. The ampule was emptied and the embryo was transferred to a solution containing 1 M DMSO at room temperature for 10 minutes. Gradual dilution was continued in solutions of M and M DMSO for 10 minutes each at room temperature. Thereafter, embryos were rinsed twice in HEPES-buffered Earle's medium and twice in embryo culture medium and finally were transferred to a 25-,uL droplet in a culture dish (18). Protocol B: Slow Freezing With 1.5 M 1,2-Propanediol and 0.1 M Sucrose The slow freezing protocol with 1.5 M 1,2-propanediol was adapted from Mandelbaum et al. (4). The freezing medium consisted of HE PES-buffered Earle's medium with 2.25% (voljvol) of a 20% HSA solution and 1,2-propanediol (P 1009; Sigma Chemical Company, St. Louis, MO). Embryos were equilibrated in 1.5 M 1,2-propanediol at room tempera- ture for 15 minutes and subsequently in 1.5 M 1,2-propanediol and 0.1 M sucrose (British Drug House Ltd.) for 5 minutes. Embryos then were transferred to glass ampules containing 400,uL of a 1.5 M 1,2-propanediol and 0.1 M sucrose solution. After transfer to a biologic freezer preset at room temperature, the embryo was cooled at a rate of -2 C/min to -6 C. Seeding was induced manually by touching the ampule with a liquid-n 2 -cooled forceps. Further freezing was performed at a cooling rate of -0.3 C/min down to -40 C, at which temperature the ampule was immersed into liquid N 2 Thawing was done by removing an ampule from the liquid N 2, transferring it immediately to a 37 C water bath and shaking it gently for approximately 30 seconds. The ampule was emptied and the embryo was transferred to a solution of 1 M 1,2-propanediol and 0.2 M sucrose for 5 minutes at room temperature. Gradual dilution was continued in solutions of 0.5 M 1,2-propanediol and 0.2 M sucrose and of 0.2 M sucrose alone for 5 minutes each at room temperature. Thereafter, embryos were rinsed twice in HEPES-buffered Earle's medium and twice in culture medium and finally were transferred to a 25-,uL droplet in a culture dish. Protocol C: Slow Freezing With 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M Sucrose The protocol for the combined use of M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose was adapted from Russell (16). The freezing medium consisted of HEPES-buffered Earle's medium with 2.25% (voljvol) of a 20% HSA solution and a mixture of M DMSO and M 1,2-propanediol. Embryos were equilibrated in this mixture at room temperature for 15 minutes and subsequently in 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose for 5 minutes. Embryos then were transferred to glass ampules containing 400,uL of the 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose solution. After transfer to a biologic freezer preset at room temperature, the embryo was cooled at a rate of -2 C/min to -6 C. Seeding was induced manually by touching the ampule with a liquid N 2 -cooled forceps. Further freezing was performed at a cooling rate of -0.3 C/min down to -40 C, at which temperature the ampule was immersed into liquid N 2 Thawing was done by removing an ampule from the liquid N 2, transferring it immediately to a 37 C water bath and shaking it gently for approximately 30 seconds. The ampule was emptied and the embryo was transferred 94 Vander Elst et al. Human embryo cryopreservation Fertility and Sterility

4 to a solution of 0.5 M DMSO, 0.5 M 1,2-propanediol, and 0.2 M sucrose and of 0.2 M sucrose alone for 5 minutes each at room temperature. Thereafter, embryos were rinsed twice in HEPES-buffered Earle's medium and twice in culture medium and were finally transferred to a 25-p,L droplet in a culture dish. Evaluation of Survival After Thawing The morphology of the embryos after thawing was assessed under a dissecting microscope at X40 magnification and under an inverted microscope at X100 magnification. Acceptable survival was defined where at least 50% of the initial number of blastomeres were intact. The surviving embryos were put into culture for another 4 to 6 hours before replacement. Transfer of Frozen-Thawed Embryos Frozen-thawed embryos were transferred during natural ovarian cycles. Transfer cycles were monitored in the periovulatory period by daily measurement of the serum concentration of 17{3-E 2, LH, and P by means of direct radioimmunoassay procedures from day 8 on. If the LH surge occurred and P levels were ~1.5 J.Lg/L (conversion factor to SI units, 3), multicellular embryos were thawed 3 to 4 days after the day of ovulation as judged by LH measurements. Two- to four-cell embryos were thawed on day 3 and five-to-eight-cell embryos on day 4 after the day of the LH surge. If P levels were <1.5 J.Lg/L, the thawing was postponed for 1 day. The transfer of the embryos occurred within 12 hours after the time of thawing, after confirming their viability. In case a mixed transfer of two-to-four- and five-toeight-cell embryos had to be done, the transfer was planned in synchrony with the four-cell embryos. From day 10 after the LH rise on, the concentration of hcg in the serum also was measured. Initial pregnancy was defined as a significant increase in serum hcg concentrations in at least two measurements within 10 days of embryo replacement. Clinical pregnancy implied the observation of a gestational sac by means of echographic screening at 7 weeks of pregnancy. Ongoing pregnancy was defined when endocrine and echographic parameters evolved normally beyond 20 weeks. Statistics Data for the three different freezing protocols were compared with contingency table analysis (x 2 analysis). A difference was considered significant at p s Patients RESULTS From September 1990 until July 1992, a total of 488 patients were enrolled in 499 cycles of the cryopreservation program and were allocated randomly over the cryopreservation protocols: 177 patients were allocated to protocol A (DMSO), 165 to protocol B (1,2-propanediol), and 146 to protocol C (DMS0-1,2-propanediol) as shown in Table 1. Patients with different indications for infertility were represented equally in the three groups and there was no significant difference in patients' mean age (32.7, 32.7, and 33.1 years, respectively). Oocyte and Embryo Yield of the Collection Cycle Table 2 presents the results of the collection cycles in terms of oocyte and embryo yield. There was no difference in the mean number of oocytes retrieved, of oocytes fertilized, or the number of embryos frozen. The number of embryos transferred in the collection cycle was approximately three in accordance with the design of the study. Embryos for Cryopreservation Table 3 presents information on the distribution of multicellular embryos over the three freezing protocols. A total of 2,220 embryos were frozen in 499 cycles: 819 using protocol A, 699 using protocol B, and 702 using protocol C. There was no difference between the three protocols as regards the stage of development of the frozen embryos or prefreeze embryonic quality. The stage of development varied from the two-cell to the eight-cell stage and the majority of embryos were in the expected fourcell stage at day 2 after insemination. Two-cell embryos formed the second highest frequency class. Few five- to eight-cell embryos that were undergoing or had completed their third mitotic division were found. The majority of the embryos (approximately 80%) were of quality type B. The remaining 20% of embryos were of excellent quality (type A). Outcome of Transfer and Pregnancy of the Collection Cycle Table 4 shows the results of the outcome of pregnancies ensuing from the transfer of fresh human Vol. 63, No. 1, January 1995 Vander Elst et al. Human embryo cryopreservation 95

5 Table 1 Indication of Infertility and Mean Age in 488 Patients Randomized Over Cryopreservation Protocols with DMSO, 1,2-Propanediol, or DMS0-1,2-propanediol* DMSO 1,2-Propanediol DMS0-1,2-propanediol No. of patients Percent with tubal indication Percent with idiopathic indication Percent with andrological indication Mean age (y) * DMSO: 1.5 M dimethylsulfoxide; 1,2-propanediol: 1.5 M propanediol and 0.1 M sucrose; DMS0-1,2-propanediol: 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose. embryos in 499 collection cycles for 488 patients who had cryopreservation of their supernumerary embryos: 517 embryos were replaced in 183 transfers in patients enrolled in freezing protocol A, 4 79 embryos in 166 transfers in protocol B, and 434 embryos in 150 transfers in protocol C. The percentage of positive hcg measurements per transfer was 47% in the DMSO group, 45.8% in the 1,2-propanediol group, and 41.3% in the DMS0-1,2-propanediol group. The preclinical abortion rate was 13.9% in the DMSO group, 18.4% in the 1,2-propanediol group, and 11.3% in the DMS0-1,2-propanediol group. The clinical PRs per transfer were 40.4%, 37.4%, and 36.7% in the DMSO, 1,2-propanediol, and DMS0-1,2-propanediol groups, respectively. The clinical implantation rate per embryo transferred was 21.7% in the DMSO group, 17.1% in the 1,2-propanediol group, and 17.7% in the DMS0-1,2-propanediol group. Of the clinical pregnancies, 82.4% (61 of 74) in the DMSO group, 79.8% (47 of 62) in the 1,2-propanediol group, and 81.8% (45 of 55) in the DMS0-1,2-propanediol group were ongoing. The delivery rate per transfer was 33.3% in the DMSO group, 28.3% in the 1,2-propanediol group, and 30.0% in the DMS0-1,2-propanediol group. This led finally to 86 children born after transfer of fresh embryos in the patients enrolled in the DMSO group, 60 children in the 1,2-propanediol group, and 61 children in the DMS0-1,2-propanediol group. This corresponds to a live-birth rate of 16.6% per embryo transferred in the DMSO group, 12.5% in the 1,2-propanediol group, and 14.1% in the DMS0-1,2-propanediol group. No significant difference was found between the three groups as regards clinical PR, embryo implantation rate, delivery rate, or live-birth rate. Outcome of Transfer and Pregnancy of the Thawing Cycle Table 5 shows the outcome of 725 embryos that were thawed: 56 patients had 232 embryos thawed in protocol A, 67 had 250 embryos thawed in protocol B, and 55 had 243 embryos thawed in protocol C. The survival rate of embryos significantly was higher in the DMSO group at 52.6% (122 of 232) than in the 1,2-propanediol group at 32.0% (80 of 250, P < ), and the DMS0-1,2-propanediol group at 34.9% (85 of 243, P < ). The mean number of embryos transferred was 2. 77, , and 2.02 per transfer, respectively. The initial PR per transfer based on positive hcg measurements was Table 2 Oocyte and Embryo Yield in 499 Collection Cycles for 488 Patients Who Had Cryopreservation of Their Supernumerary Multicellular Embryos According to a Cryopreservation Protocol With DMSO, 1,2-Propanediol or DMS0-1,2-Propanediol* DMSO 1,2-Propanediol DMS0-1,2-Propanediol No. of patients No. of cycles No. of oocytes retrieved Mean no. of retrieved oocytes per patient No. of fertilized oocytes Mean no. of fertilized oocytes per patient No. of embryos transferred Mean no. of embryos transferred per patient No. of embryos frozen Mean no. of embryos frozen per patient , , , , , , * DMSO: 1.5 M dimethylsulfoxide; 1,2-propanediol: 1.5 M propanediol and 0.1 M sucrose; DMS0-1,2-propanediol: 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose. 96 Vander Elst et al. Human embryo cryopreservation Fertility and Sterility

6 Table 3 Developmental Stage and Embryonic Quality of Multicellular Human Embryos Randomized over Cry~preservation Protocols with DMSO, 1,2-Propanediol, or DMS0-1,2-Propanediol* No. of embryos frozen Two-cell (%) Three-cell(%) Four-cell(%) Five-cell(%) Six-cell(%) Eight-cell(%) Type At(%) Type B:j: (%) DMSO DMSO 1,2-Propanediol 1,2-Propanediol * DMSO: 1.5 M dimethylsulfoxide; 1,2-propanediol: 1.5 M propanediol and 0.1 M sucrose; DMS0-1,2-propanediol: 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose. t Type A: embryos showing no anucleate fragments.. :j: Type B: embryos with,;;20% of their volume filled With anucleate fragments. 25.0% in the DMSO group (11 of 44), 8.9% in the 1,2-propanediol group (4 of 46), and 21.4% in the DMS0-1,2-propanediol group (9 of 42). This difference was not significant. There was a significant difference in the clinical PR per transfer between the DMSO group (25%; 11 of 44) and the 1,2-propanediol group (6.5%; 3 of 46). The clinical PR per transfer was 16.7% in the DMS0-1,2-propanediol group. There also was a significant difference in the clinical PR per thawing cycle between the DMSO group (17.2%; 11 of 64) and the 1,2-propanediol group (3.9%; 3 of 78). The clinical PR per thawing cycle was 10.9% in the DMS0-1,2-propanediol group. When the clinical implantation rate per thawed embryo was calculated, we observed that it was significantly higher for embryos frozen with DMSO (4.7%; 11 of232) than with 1,2-propanediol (1.2%; 3 of 250). The clinical implantation rate per embryo thawed in the DMS0-1,2-propanediol group was 3. 7% (9 of 243) and fell in between that of the DMSO and the 1,2-propanediol groups and was not significantly different from either. The delivery rate per thawing cycle was higher significantly in the DMSO group (12.5%; 8 of 64) than in the 1,2- propanediol group (2.6%; 2 of 64). The live-birth rate per embryo thawed was 3.5% in the DMSO group; 0.8% in the 1,2-propanediol group, and 2.9% in the DMS0-1,2-propanediol group. DISCUSSION It was the aim of the present study to find an answer to the question as to which existing slow- freezing protocol is the most appropriate for cryopreserving a heterogeneous cohort of multicellular supernumerary embryos obtained after IVF. We performed a randomized prospective comparison between three different slow freezing protocols where DMSO, 1,2-propanediol, or a mixture of DMSO and 1,2-propanediol were used as cryoprotectants. We demonstrate that a slow-freezing protocol with 1.5 M DMSO down to -80 C may be the optimal protocol for freezing two-to-eight-cell supernumerary embryos after transfer of the three morphologically best embryos in the collection cycle. The importance of this study to the field of human embryo cryopreservation is its prospective randomization. To our knowledge, this is the first report where patients in the different cryopreservation groups did not differ as to mean age, indication of infertility, stimulation protocol, treatment procedure, embryo stage, embryo quality, outcome of the collection cycle, or nature of the transfer cycle. Furthermore, it is the first randomized study in which patients enrolled in different cryopreservation groups were compared simultaneously. In previous studies by our own center (6, 7, 9) as well as by other authors (1-5, 8, 10, 11) one protocol generally was tested for selected stages of embryo development and the outcome was compared with data from the literature. Also, results often only were expressed in terms of PR per transfer, whereas in the present study emphasis is placed on other important parameters, such as individual embryo survival and implantation rates, in appreciating the success rate of a freezing procedure. The data of the present study confirm one of the first studies on human embryo cryopreservation (1) where the use ofdmso to -80 C for the freezing of four-cell and eight-cell human embryos was advised. We cannot confirm any advantage of a combination of DMSO and 1,2-propanediol over each cryoprotectant alone, as was claimed by Russell (16) for mouse eight-cell embryos. We also cannot recommend the use of 1,2-propanediol. The results of our study conflict with literature data that propose 1,2-propanediol as the preferential protocol for freezing embryos left in culture after IVF (4, 5, 10, 11, 19). The major difference in this respect is the selection of the embryos at the prefreezing stage. In our study, the embryos that are cryopreserved clearly are excess embryos after the three best embryos have been transferred in the collection cycle. This situation definitely is different Vol. 63, No. 1, January 1995 Vander Elst et al. Human embryo cryopreservation 97

7 Table 4 Outcome of Pregnancies Ensuing From the Transfer of Fresh Human Embryos in 499 Collection Cycles for 488 Patients Who Had Cryopreservation of Their Supernumerary Multicellular Embryos According to a Cryopreservation Protocol with DMSO, 1,2-Propanediol, or DMS0-1,2-Propanediol* DMSO 1,2-Propanediol DMS0-1,2-Propanediol No. of transfers No. of embryos transferred No. of positive hcg measurements Percent of transfers No. of preclinical abortions No. of clinical pregnancies Percent of transfers Singleton Twins Triplets No. of clinical embryo implantations Percent of embryos transferred No. of first-trimester miscarriages No. of extrauterine pregnancies No. of second-trimester miscarriages No. of lost for follow-up No. of induced abortions No. of deliveries Percent of transfers Singletons Vanishing twins Vanishing triplets Twins Vanishing triplets Reduced triplets Triplets No. of children born Percent of embryos transferred * DMSO: 1.5 M dimethylsulfoxide; 1,2-propanediol: 1.5 M propanediol and 0.1 M sucrose; DMS0-1,2-propanediol: 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose. from the situation where one-cell embryos are allocated randomly either to fresh transfer or to freezing procedure as well as from a situation in which the best embryos are selected for freezing. Indeed, in many groups (4, 8, 10, 11), all pronucleate embryos in excess to four or five are frozen with 1,2- propanediol on day 1 while the remainder are left in culture to be transferred in the collection cycle. High survival rates of 70% to 80% were obtained for pro nucleate embryos. Another approach by Testart et al. (5) was to freeze two- to four-cell embryos of very good quality with 1,2-propanediol on day 2 while fresh transfer was done with sibling embryos that were less suitable for survival after freezing and thawing. An important argument against both these procedures is that, while trying to improve the success rate of cryopreservation, one may lower the chances of pregnancy in the collection cycle. Although the opposite is claimed and it is stated that the PR in the collection and frozen cycle are high and equal (20), a randomized study will be needed to substantiate this hypothesis. In our experience, the PR per transfer of cryopreserved super- numerary embryos is lower than the PR per transfer of their sibling fresh embryos (6, 7, 9). The overall PR after freezing remains modest in our experience, mainly because of the lesser quality of real excess embryos. The work by Mandelbaum et al. (4) resembles our study more, in that all available embryos were left to develop to day 2 or 3 and that the crucial decision to transfer or cryopreserve them was made at that point. One major difference, however, is the strictness in selection of embryos in terms of morphological quality of blastomeres and the presence of anucleate fragments. Only 16% of patients starting an IVF cycle were reported to provide embryos for freezing (21), whereas in our center approximately 50% of patients have embryos cryopreserved (6, 7, 9, 13). Implantation rates reported in the present study with DMSO (10.7% per embryo transferred and 5.4% per embryo thawed) are similar to those reported by Mandelbaum et al. ( 4) using 1,2-propanediol for multicellular embryos (14% per embryo transferred, 8% per embryo thawed). The embryo selection process in this study might ex- 98 Vander Elst et al. Human embryo cryopreservation Fertility and Sterility

8 Table 5 Outcome of Pregnancies Ensuing From the Thawing of 725 Human Multicellular Embryos That Were Frozen With DMSO, 1,2-Propanediol or DMS0-1,2-Propanediol and Transferred in Natural Ovarian Cycles DMSO 1,2-Propanediol DMS0-1,2-Propanediol No. of patients with thawing cycles 56 No. of planned thawing cycles 64 No. of embryos thawed 232 No. of embryos suitable for transfer 122 Percent of embryos thawed 52.6t No. of transfers 44 No. of positive hcg measurements 11 Percent of transfers 25.0 No. of preclinical abortions 0 No. of clinical pregnancies 11 Percent of transfers 25.0:j: Percent of thawing cycles 17.2 Singleton 11 Twins 0 No. of clinical implantations 11 Percent of embryos transferred 9.0 Percent of embryos thawed No. of first-trimester miscarriages 2 No. of extrauterine pregnancies 1 No. of deliveries 8 Percent of transfers 18.2 Percent of thawing cycles 12.5~ Singletons 8 Twins 0 No. of children born 8 Percent of embryos transferred 6.6 Percent of embryos thawed 3.5 * DMSO: 1.5 M dimethylsulfoxide; 1,2-propanediol: 1.5 M propanediol and 0.1 M sucrose; DMS0-1,2-propanediol: 0.75 M DMSO, 0.75 M 1,2-propanediol, and 0.1 M sucrose. t Group DMSO is significantly different from groups 1,2-propanediol and DMS0-1,2-propanediol, respectively, P < , p < :j: Group DMSO is significantly different from group 1,2-propanediol, P < Group DMSO is significantly different from group 1,2-propanediol, P < II Group DMSO is significantly different from group 1,2-propanediol, P < ~ Group DMSO is significantly different from group 1,2-propanediol, P < plain the differences with 1,2-propanediol in our center. That 1,2-propanediol is less suitable for freezing a cohort of multicellular embryos may be a result of changed permeability characteristics and surfaceto-volume ratios of multicellular, as opposed to one-cell, embryos. 1,2-Propanediol is a very rapidly permeating cryoprotectant and may cause rehydration during prefreeze equilibration, hence, increasing the chance of intracellular freezing. Similarly, at the subzero plunging temperature of -40 C associated with the 1,2-propanediol protocol, dehydration may be less than at the -80 C plunging temperature that is used for DMSO. However, it cannot be concluded from our study that the lower embryo survival rate in the 1,2-propanediol group is due to the cryoprotectant itself. Any other variable of the cryopreservation protocol such as the equilibration schedule, the cooling schedule, the method of thawing, and the training skill may play a role in thawing outcome. The final effect is that, due to lower survival rates in the 1,2-propanediol group, less embryos are available for transfer in a thawing cycle with 1,2-propanediol than with DMSO or D MS0-1,2-propanediol. In a previous study in which we described an advantage of the use of DMSO over 1,2-propanediol for multicellular embryos (7), bias also was introduced by allocation of embryos from different treatment groups to either protocol. The majority of multicellular embryos were frozen with DMSO and originated from IVF whereas 20% of the total number of multicellular embryos were frozen with 1,2-propanediol and originated mostly from GIFT or ZIFT. We can conclude from this prospective randomized study that, to optimize the efficiency of cryopreservation of multicellular embryos obtained after IVF, a cryopreservation protocol with the cryoprotectant DMSO seems to have the best po- Vol. 63, No. 1, January 1995 Vander Elst et al. Human embryo cryopreservation 99

9 tential for cryopreserving multicellular embryos at different developmental stages and of different morphological qualities. Dimethylsulphoxide provides a significantly higher survival and implantation rate per embryo thawed than 1,2-propanediol in the way that both cryoprotectants were used here. This study was not conducted to find the optimal protocol for a specific stage of embryo development or a specific degree of embryo quality because this conclition rarely coincides with the daily reality of IVF. A definite conclusion will be possible only when all the cryopreserved embryos have been thawed and replaced, but this condition would, if pushed to the limit, render any study on embryo cryopreservation impossible. Acknowledgments. We acknowledge the clinical, scientific, technical, and nursing staff of the Centre for Reproductive Medicine. Special thanks go to Frank Winter, M.A., of the Language Education Centre who edited the manuscript and to Mr. Johan Schiettecatte, M.T., Chief Technician of the Endocrinology Department, who assisted in the data collection. Ms. Iris Fatoux is acknowledged for assistance in typing of the manuscript. REFERENCES 1. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. N a ture 1983;305: Mohr L, Trounson A, Freemann L. Deep freezing and transfer of human embryos. J In Vitro Fert Embryo Transf 1985;2: Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fert Embryo Transf 1985;2: Mandelbaum J, Junca AM, Plachot M, Alnot MO, Alvarez S, Debache C, et al. Human embryo cryopreservation, extrinsic and intrinsic parameters of success. Hum Reprod 1987;2: Testart J, Lassalle B, Forman R, Gazengel A, Belaisch-Allart J, Hazout A, et al. Factors influencing the success rate of human embryo freezing in an in-vitro fertilization and embryo transfer program. Fertil Steril1987;48: Van Steirteghem AC, Van den Abbeel E, Camus M, Van Waesberghe L, Braeckmap.s P, Khan I, et al. Cryopreservation of human embryos obtained after gamete intra-fallopian transfer and/or in-vitro fertilization. Hum Reprod 1987;2: Van den Abbeel E, Van der Elst J, Van W aesberghe L, Camus M, Devroey P, Khan I, et al. Hyperstimulation: the need for cryopreservation of embryos. Hum Reprod 1987;3 Suppl2: Fugger EF. Clinical status of human embryo cryopreservation in the United States of America. Fertil Steril 1989;52: Camus M, Van den Abbeel E, Van Waesberghe L, Wisanto A, Devroey P, Van Steirteghem AC. Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant. Fertil Steril 1989;51: Veeck LL, Amundson CH, Brothman LJ, DeScisciolo C, Maloney MK, Muasher SJ, et al. Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil Steril1993;59: Quinn P. Success of oocyte and embryo freezing and its effect on outcome with in-vitro fertilization. Semin Reprod Endocrinol 1990;8: Van Steirteghem AC, Van den Abbeel E, Camus M, Devroey P. Cryopreservation of human embryos. Bailliere's Clin Obstet Gynaecol 1992;6: Van Steirteghem AC, Zsolt N, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8: Staessen C, Janssenswillen C, Van den Abbeel E, Devroey P, Van Steirteghem AC. Avoidance of triplet pregnancies by elective transfer of two good-quality embryos. Hum Reprod 1993;8: Smitz J, Ron-El R, Tarlatzis BC. The use of gonadotrophin releasing hormone agonists for in vitro fertilization and other assisted procreation techniques: experience from three centres. Hum Reprod 1992;7: Russell JB. The combination of dimethylsulfoxide with 1,2- propanediol to cryopreserve embryos at different stages of development. Assist Reprod Rev 1991;1: Smitz J, Devroey P, Camus M, Deschacht J, Khan I, Staessen C, et al. The luteal phase and early pregnancy after combined GnRH-agonist/HMG treatment for superovulation in IVF or GIFT. Hum Reprod 1988;3: Staessen C, Camus M, Khan I, Smitz J, Van W aesberghe L, Wisanto A, et al. An 18-month survey of infertility treatment by in-vitro fertilization, gamete and zygote intra-fallopian transfer, and replacement offrozen-thawed embryos. J In Vitro Fert Embryo Transf 1989;6: Fugger EF, Bustillo M, Dorfmann AD, Schulman JD. Human preimplantation embryo cryopreservation: selected aspects. Hum Reprod 1991;6: Testart J, Lassalle B, Belaisch-Allart J, Forman R, Hazout A, Volante M, et al. Human embryo viability related to freezing and thawing procedures. Am J Obstet Gynecol 1987;157: Mandelbaum J, Junca AM, Plachot M, Alnot MO, Salat Baroux J, Alvarez S, et al. Cryopreservation of human embryos and oocytes. Hum Reprod 1988;3: Vander Elst et al. Human embryo cryopreservation Fertility and Sterility