Assisted reproductive technology

Assisted reproductive technology FERTILITY AND STERILITY Copyright 1994 The American Fertility Society Vol. 62, No.4, October 1994 Printed on acid-free paper in U. S. A. Cryopreservation of supernumerary multicellular human embryos obtained after intracytoplasmic sperm injection*t Andre C. Van Steirteghem, M.D., Ph.D.:j: Josiane Vander Elst, Ph.D. Etienne Van den Abbeel, B.Sc. Hubert Joris, M.T. Michel Camus, M.D. Paul Devroey, 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 pregnancy rate of frozen-thawed human supernumerary multicellular embryos that were cryopreserved after intracytoplasmic sperm injection or IVF. Design: A clinical study. Setting: Consenting patients in an academic research environment. Patients: Couples with severe male infertility, indicated by failed or sporadic fertilization after IVF or subzonal insemination or by <500,000 progressively motile spermatozoa in the entire ejaculate, and couples for IVF during the same period. Interventions: After microinjection or IVF, the three best-quality embryos were transferred, and 1,171 embryos from intracytoplasmic sperm injection compared with 2,495 embryos from IVF were frozen with dimethyl sulphoxide. Of these, 413 and 969 embryos were thawed, respectively. Main Outcome Measure: The survival rate, the total and clinical pregnancy rates, the delivery rate, and the preclinical abortion rate were calculated. Results: Fifty-three percent of the thawed intracytoplasmic sperm injection embryos survived. Twenty-two pregnancies have been established in 101 transfers, corresponding to a total pregnancy rate of 21.8% per transfer. The clinical pregnancy rate was 12.9% per transfer and the delivery rate was 5.9% per transfer. Of the IVF embryos, 51% survived and 37 pregnancies have been established in 253 transfers. The total and clinical pregnancy rates and the delivery rate were 14.6%, 10.7%, and 7.1 %, respectively. The preclinical abortion rate was 40.9% for cryopreserved intracytoplasmic sperm injection embryos and 27.0% for IVF embryos. Conclusions: The high incidence of preclinical abortions after transfer of human embryos cryopreserved after intracytoplasmic sperm injection requires extension of the series. Fertil Steril 1994;62:775-80 Key Words: Intracytoplasmic sperm injection, cryopreservation, pregnancy Received December 22, 1993; revised and accepted May 27, 1994. * Supported by the Fund for Medical Scientific Research (grant 3.0018.92), Brussels, Belgium. t Presented at the VIIIth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Kyoto, Japan, September 12 to 15, 1993. :j: Reprint requests: Andre Van Steirteghem, M.D., 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: 32-2-477-5060). Senior Research Assistant of the National Fund for Scientific Research, Brussels, Belgium. Intracytoplasmatic sperm injection (1-4) recently has been introduced as the most successful technique to obtain live births in cases of severe male infertility in couples who could not be treated by traditional IVF or even by any previously described technique of assisted fertilization, such as subzonal insemination (SUZI). The fertilization and implantation rates after intracytoplasmic sperm injection are higher than those after standard IVF or after SUZI for malefactor infertility in our center. Of approximately 90% successfully injected oocytes, 65% develop two Vol. 62, No.4, October 1994 Van Steirteghem et al. Human embryo freezing after intracytoplasmic sperm injection 775

pronuclei and hence show normal fertilization. After 24 hours of further in vitro culture, >70% of these two-pronucleated oocytes will have progressed through one, two, or three mitotic divisions. Furthermore, these cleaved embryos are of good morphological quality and show <20% of their surface filled with anucleate fragments. After transfers of these good-quality embryos, a clinical pregnancy rate of 39% per ET has been reported (4). Furthermore, 47% of the started cycles provide multicellular embryos of good quality for cryopreservation. The present study reports on the outcome of thawing of 413 supernumerary multicellular embryos obtained after intracytoplasmic sperm injection compared with 969 embryos obtained after IVF during the same period. Patients MATERIALS AND METHODS From November 1991 until July 1993, a series of 329 treatment cycles of assisted fertilization by intracytoplasmic sperm injection with cryopreservation of 1,171 supernumerary embryos was carried out at the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University. Patients were selected for intracytoplasmic sperm injection when they fulfilled the following inclusion criteria: [1] total absence or <5% of normal fertilization after standard IVF, [2] <500,000 progressively motile spermatozoa in the whole ejaculate, or [3] failed or sporadic fertilization after SUZI. The patients were counseled on the novelty of the intracytoplasmic sperm injection procedure. The patients who agreed to cryopreserve supernumerary embryos signed a consent form that included prenatal diagnosis by chorionic villus sampling or amniocentesis as well as a prospective follow-up of the children born after intracytoplasmic sperm injection and cryopreservation. The protocols were approved by the ethical committee of the Medical Campus of the Dutch-speaking Brussels Free University. The outcome of transfer of cryopreserved embryos in patients undergoing intracytoplasmic sperm injection was compared with the outcome in patients undergoing standard IVF during the same period. Ovarian Stimulation and Standard IVF Ovarian stimulation was carried out by a desensitizing protocol of intranasally administered GnRH agonist (GnRH-a) buserelin acetate (Suprefact; Hoechst, Brussels, Belgium) in association with hmg (Humegon; Organon, Oss, The Netherlands or Pergonal; Serono, Brussels, Belgium) and hcg (Pregnyl; Organon, or Profasi; Serono). The details of this stimulation protocol have been described previously (5). The procedure for conventional IVF in our center has been extensively described previously (6, 7). Semen Evaluation and Preparation for Microinjection Semen evaluation includes the assessment of conventional semen characteristics. The evaluation of semen density and motility is carried out according to the recommendations of the World Health Organization (8). The morphology was assessed by strict Kruger criteria (9). For semen preparation before intracytoplasmic sperm injection, the seminal fluid is removed from the semen by washing in Earle's medium containing 3% bovine serum albumin (fraction V, A 9647; Sigma Chemical Co., StLouis, MO) and centrifuging at 1,800 X g for 5 minutes. Subsequent sperm selection is done through two or three layers of Percoli depending on the number of motile spermatozoa that are present in the semen. Three layers of Percoll are used if ~5 X 10 6 spermatozoajml with a motility of 40% are present, whereas two layers are used for the other semen samples. The sperm suspension is put on top of the lowest density gradient. After centrifugation at 300 X g for 20 minutes, the bottom layer is aspirated and transferred into approximately 5 ml of Earle's medium in a Falcon tube. After centrifugation at 1,800 X g for 5 minutes, the supernatant is removed and the pellet is gently resuspended in 0.2 ml Earle's medium. The sperm suspension is kept in a 37 C incubator (5% 0 2,5% C0 2, 90% N 2 ) until the moment for intracytoplasmic sperm injection of the oocytes. Oocyte Preparation for Microinjection Oocyte retrieval was done by vaginal ultrasoundguided pick-up. The cumulus and corona radiata were removed by incubation for <1 minute in HEPES-buffered Earle's medium containing 80 IU/mL hyaluronidase (type VIII, Sigma). The removal of the cumulus and the corona was continued by aspirating the complexes in and out of handdrawn glass pipettes. The oocytes were observed under the inverted microscope at 200X or 400X magnification to assess integrity and maturational 776 Van Steirteghem et al. Human embryo freezing after intracytoplasmic sperm injection Fertility and Sterility

stage by observation of the presence or absence of the germinal vesicle or the first polar body. This procedure of oocyte preparation has been described in detail recently (4). Intracytoplasmic Sperm Injection Procedure The details of the preparation of the microtools and of the protocol for intracytoplasmic sperm injection have been described extensively in previous reports (1-4). Briefly, holding and injection pipettes were made by drawing glass capillary tubes with a pipette puller and by further processing on a microgrinder and a micro forge. An almost immotile spermatozoon was aspirated tail first into the tip of the injection pipette. The oocyte was held with the polar body at 12 or 6 o'clock by the holding pipette and the injection pipette was introduced through the zona pellucida into the ooplasm at 3 o'clock. After injection of a single spermatozoon, the injection pipette was gently withdrawn and the oocyte was released from the holding pipette. Assessment of Fertilization and Embryo Cleavage After the microinjection procedure, the further handling of oocytes and the evaluation of fertilization and cleavage was done as in our standard IVF procedure (6, 7). Fertilization was assessed 16 to 18 hours after the injection and was considered to be normal where two pronuclei were present. The state of embryo cleavage was assessed another 24 hours later. Embryo quality was scored according to the presence of anucleate fragments and the extent to which they filled the embryo's surface. Briefly, three types of embryos were considered: type A embryos had regular or irregular blastomeres and showed no anucleate fragments, type B embryos had ~20% of their surface filled with anucleate fragments, and type C embryos had > 20% and up to 50% of their surface filled with anucleate fragments. Cleaved embryos of types A, B, and C were eligible for transfer. Embryo Freezing If type A or B embryos in excess of the two or three best selected for fresh transfer were available, they were cryopreserved on day two or three by a slow freezing protocol with dimethyl sulphoxide (10). The developmental stage of these embryos varied from the 2- to the 16-cell stage. Evaluation of Survival After Thawing The morphology of the embryo after thawing was evaluated under the dissecting microscope at X40 and under the inverted microscope at X100. The criterion for survival of a multicellular embryo was that at least half of the initial number of blastomeres was intact after thawing. Surviving embryos were put in culture for a further 4 to 6 hours before transfer. Transfer of Frozen-Thawed Embryos Frozen-thawed embryos were transferred during natural unstimulated ovarian cycles or during cycles that were stimulated with clomiphene citrate. Up to three embryos were loaded in a few microliters of Earle's medium and into a Frydman catheter (LG 4.5; Prodimed, Neuilly-en-Thelle, France) and transferred into the uterine cavity. Biochemical pregnancy was defined as a significant increase in serum hcg concentrations on at least two occasions ;::::10 days after 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. Prenatal diagnosis was carried out by chorionic villous sampling at 9 to 10 weeks of gestation or by amniocentesis at 16 weeks of gestation. Genetic counseling was done in view of the prenatal diagnosis and for the planned prospective follow-up study of the children born after intracytoplasmic sperm injection and cryopreservation. RESULTS Developmental Stage and Quality of Supernumerary Multicellular Embryos Frozen After Intracytoplasmic Sperm Injection From November 1991 until July 1993, 1,171 supernumerary embryos were frozen in 329 cycles of assisted fertilization by intracytoplasmic sperm injection. Of these embryos, 413 were thawed to correspond to 120 cycles that were planned for the replacement of frozen-thawed embryos. Table 1 gives detailed information on the developmental stage and the morphological quality ofthe cohort of embryos that were frozen. The majority of embryos (64.8%; 759/1,171) was at the 3- to 4-cell stage as expected when cryopreservation is done at day 2 after insemination. The second highest frequency class was formed by 5- to 8-cell embryos (25.7%; 301/1,171). Approximately 8% of embryos were at the 2-cell stage (95/1,171). Few embryos were be- Vol. 62, No.4, October 1994 Van Steirteghem et al. Human embryo freezing after intracytoplasmic sperm injection 777

Table 1 Developmental Stage and Embryonic Quality of 1,171 Multicellular Human Embryos Cryopreserved during 329 Cycles of Intracytoplasmic Sperm Injection Stage Type A* Type Bt Total 2-cell embryos 14 81 95 3- and 4-cell embryos 74 685 759 5- to 8-cell embryos 22 279 301 9- to 16-cell embryos 2 14 16 Total 112 1,059 1,171 * Type A, embryos showing no anucleate fragments. t Type B, embryos with ~20% oftheir surface filled with anucleate fragments. tween the 9- and 16-cell stage (1.4%; 16/1,171). Embryos whose quality was judged insufficient for freezing at day 2 or 3 (>20% anucleate fragments in their surface) were left in culture and those that developed to the blastocyst stage were frozen as such. A total of 35 blastocysts was frozen in this study. When classifying the frozen 2- to 16-cellstage embryos according to morphological quality, 1,059 embryos out of 1,171 (90.4%) were of type B, whereas the remaining 9.6% of embryos (112/ 1,171) were of type A. Developmental Stage and Quality of Supernumerary Multicellular Embryos Frozen After IVF From November 1991 until July 1993, 2,495 supernumerary embryos were frozen in 1,055 cycles of IVF. Of these embryos, 969 were thawed to correspond to 316 cycles that were planned for the replacement of frozen-thawed embryos. Table 2 gives detailed information on the developmental stage and the morphological quality of the cohort of embryos that were frozen. Two major groups of embryos were distinguished: embryos at the 3- to 4-cell stage (41%; 1,023/2,495) and embryos at the 5- to Table 2 Developmental Stage and Embryonic quality of 2,495 Multicellular Human Embryos Cryopreserved during 1,055 Cycles of IVF Stage Type A* Type Bt Total 2-cell embryos 92 288 380 3- and 4-cell embryos 172 851 1,023 5- to 8-cell embryos 151 903 1,054 9- to 16-cell embryos 8 30 38 Total 423 2,072 2,495 * Type A, embryos showing no anucleate fragments. t Type B, embryos with ~20% oftheir surface filled with anucleate fragments. Table 3 Survival, Pregnancy, and Implantation Rates after Transfer of Frozen-Thawed Embryos Obtained from Intracytoplasmic Sperm Injection or IVF Intracytoplasmic sperm injection No. of cycles with freezing 329 No. of embryos frozen 1,171 No. of cycles with thawing 120 No. of embryos thawed 413 No. of embryos transferred 218 No. of embryo transfers 101 No. of positive hcg measurements 22 No. of preclinical abortions 9 No. of clinical pregnancies 13 Singletons 10 Twins 2 Triplet 1 No. of clinical implantations 17 Evolution of clinical pregnancies No. of first-trimester miscarriages 6* No. of extrauterine pregnancies 0 No. of second-trimester interruptions 1 No. of deliveries 6 Singletons 4 Twins 2 No. of children born 8 * Including 5 singletons and one vanishing twin. t Including 6 singletons. :j: Including two vanishing twins. Including one vanishing triplet. IVF 1,055 2,495 316 969 493 253 37 10 27 25 2 0 29 6t 3 0 18 18:j: 0 18 8-cell stage (42.3%; 1,054/2,512). Approximately 15% of embryos were at the 2-cell stage (380/2,495). Few embryos were between the 9- and 16-cell stage (1.5%; 38/2,495). A total of 17 blastocysts was frozen. This distribution is significantly different from the distribution of types of embryos frozen after intracytoplasmic sperm injection (x 2 = 183.3, P = 0.0001, df = 3). When classifying the frozen 2- to 16-cell stage embryos according to morphological quality, 423 embryos out of 2,495 (16.9%) were of type A, whereas a majority of83.1% of the embryos (2,072/2,495) were of type B. This distribution of embryos of type A and B is significantly different from the distribution of embryos frozen after intracytoplasmic sperm injection (x 2 = 34.9, P = 0.0001). Outcome of Thawing and Transfer in Supernumerary Multicellular Embryos Cryopreserved After Intracytoplasmic Sperm Injection The outcome of the 413 embryos thawed in 120 planned thawing cycles is presented in Table 3. The overall survival rate was 53%, meaning that 218 of the 413 thawed embryos were suitable for transfer. This resulted in 101 transfer procedures, which corresponds to 84% of the planned cycles. Twentytwo pregnancies were established on the basis of positive hcg measurements corresponding to a to- 778 Van Steirteghem et al. Human embryo freezing after intracytoplasmic sperm injection Fertility and Sterility

tal pregnancy rate of 21.8% per transfer (22/101) and of 18.3% per thawing cycle (22/120). Nine pregnancies of the 22 were biochemical and were recorded as preclinical abortions (40.9% preclinical abortion rate). The remaining 13 clinical pregnancies included 10 singletons, two sets of twins, and one set of triplets. This corresponds to a clinical pregnancy rate of 12.9% per transfer (13/101) and of 10.8% per thawing cycle (13/120). The clinical implantation rate was 7.8% per embryo transferred (17/218) and 4.1% per embryo thawed (17/413). The evolution of the clinical pregnancies was as follows: 6 clinical pregnancies ended in a first-trimester miscarriage (5 singletons and 1 vanishing twin), 1 second-trimester ongoing pregnancy was interrupted, and 6 pregnancies ended in a delivery ( 4 singletons, 1 original set of twins, and 1 set of twins deriving from the triplet). This corresponds to a delivery rate of 5.9% per transfer (6/101) and of 5% per thawing cycle (6/120). The first-trimester abortion rate was 27.3% (6/22). The clinical ongoing implantation rate was 3.7% per embryo transferred (8/218) and 1.9% per embryo thawed (8/413). It should be mentioned that this series of 22 pregnancies included one patient with two recurrent preclinical abortions and one first-trimester miscarriage. Outcome of Thawing and Transfer of Supernumerary Multicellular Embryos Cryopreserved After IVF The outcome of the 969 embryos thawed in 316 planned thawing cycles is also presented in Table 3. The overall survival rate was 51%, meaning that 493 of the 969 thawed embryos were suitable for transfer. This resulted in 253 transfer procedures, which corresponds to 80% of the planned cycles. Thirty-seven pregnancies were established on the basis of positive hcg measurements, corresponding to a total pregnancy rate of 14.6% per transfer (37/253) and of 11.7% per thawing cycle (37/316). Ten pregnancies of the 37 were biochemical and were recorded as preclinical abortions (27% preclinical abortion rate). The remaining 27 clinical pregnancies were all singletons, among which there were two vanishing twins. This corresponds to a clinical pregnancy rate of 10.7% per transfer (27 I 253) and of 8.5% per thawing cycle (27 /316). The clinical implantation rate was 5.9% per embryo transferred (29/493) and 3% per embryo thawed (29/969). The evolution of the clinical pregnancies was as follows: 6 clinical pregnancies ended in a first-trimester miscarriage, 3 pregnancies were extrauterine, and 18 pregnancies lead to the delivery of 18 singletons. This corresponds to a delivery rate of 7.1% per transfer (18/253) and of 5.7% per thawing cycle (18/316). The first-trimester abortion rate was 16.2% (6/37). The clinical ongoing implantation rate was 3.7% per embryo transferred (18/493) and 1.9% per embryo thawed (18/969). DISCUSSION The present study is the first report on pregnancies established after transfer of frozen-thawed human embryos obtained after intracytoplasmic sperm injection. We describe the outcome of 329 cycles of cryopreservation in which 1,171 supernumerary multicellular embryos were frozen. In 120 of these cycles, 413 embryos were thawed and about half of these embryos (53%) were found suitable for transfer in 101 procedures. The total pregnancy rate was as high as 21.8% per transfer (22/101), but there was a high incidence of preclinical abortions and of first-trimester miscarriages. Of the 22 pregnancies established on the basis of hcg measurements, 9 ended as a preclinical abortion; the clinical pregnancy rate (12.9%) became approximately half of the initial total pregnancy rate. Of the 13 pregnancies that continued to a clinical pregnancy, 6 ended in a first-trimester miscarriage; the delivery rate fell to 5.9%, which was only 46% of the initial clinical pregnancy rate. When comparing the results of cryopreservation after IVF, we found a similar embryo survival rate and delivery rate, but the initial total pregnancy rate, as well as the incidence of preclinical abortions and first-trimester miscarriages, was higher when the cryopreserved embryos resulted from intracytoplasmic sperm injection. Indeed, the initial total pregnancy rate of 21.8% for embryos cryopreserved after intracytoplasmic sperm injection was higher than the rate of 14.6% observed after transfer of cryopreserved embryos after IVF. This was also the case when compared with total pregnancy rates observed in previous studies by our team, that is, 15% (10) and 12.5% (Joostens M, Camus M, Van den Abbeel E, Maes R, Devroey P, Van Steirteghem AC, abstract). In particular, however, the discrepancy between the total and clinical pregnancy rates is greater for embryos obtained from intracytoplasmic sperm injection than for embryos obtained from IVF. More pregnancies are established, but more pregnancies are lost. In the series of embryos resulting from IVF, a preclinical abortion rate of 27.0% and a first-tri- Vol. 62, No.4, October 1994 Van Steirteghem et al. Human embryo freezing after intracytoplasmic sperm injection 779

mester abortion rate of 16.2% were observed. In the group of embryos resulting from intracytoplasmic sperm injection, the preclinical abortion rate reached 40.9% and the first-trimester abortion rate was 27.3%. In another recent study by our center (Joostens M, Camus M, Van den Abbeel E, Maes R, Devroey P, Van Steirteghem AC, abstract) on a large series of 1,434 transfers of frozen-thawed embryos, a 17.8% preclinical abortion rate and a 13% first-trimester abortion rate were mentioned, which are also lower than the 40.9% preclinical and the 27.3% first-trimester abortion rates reported here. An explanation for the high rate of preclinical abortions and first-trimester miscarriages after transfer of cryopreserved embryos obtained from intracytoplasmic sperm injection is not obvious. The effect does not seem to be related to the procedure of intracytoplasmic sperm injection itself because the total and clinical pregnancy rates in the fresh intracytoplasmic sperm injection cycle were reported to be 49.6% and 39.2% per transfer, respectively (4). It is well known that prefreezing embryo quality is a crucial parameter in determining postthaw survival rate and implantation rate. In the study by Camus et al. (10), an implantation rate of 21% per embryo transferred was found for excellent embryos with regular blastomeres without any fragments, an implantation rate of 14.5% was found for embryos with up to 20% fragments, and none ofthe embryos with >20% fragments resulted in a pregnancy in single transfers. Embryos obtained after intracytoplasmic sperm injection are of good quality so that fresh ET is possible in 90% of the cycles and even triple ET in 75% ofthe cycles (4). Cryopreservation of supernumerary embryos is performed in 4 7% of the cycles, and as was evident in this study, only embryos with ~20% of fragments in their surface were frozen. However, in the cohort of embryos frozen after IVF, significantly more embryos of excellent quality were present, so that the availability of excellentquality embryos for transfer may be different between the two groups. Also, male and female patient-related factors may be involved, such as patient age, endometrial receptiveness, or sperm defects. We will have to investigate on a larger series of transfers what is the impact of embryo-or patient-related factors on the outcome of transfer of cryopreserved embryos obtained after intracytoplasmic sperm injection compared with IVF. The findings of the present report demonstrate that pregnancies and live births after transfer of cryopreserved embryos obtained from intracytoplasmic sperm injection are possible. The only currently puzzling aspect related to this procedure is the high percentage of preclinical abortions and first-trimester miscarriages. Neither the procedure itself nor embryo quality seem to be plausible causative factors. Further evaluations on a larger series will have to show whether this effect is consistent. Acknowledgments. We are indebted to many colleagues: Jiaen Liu, M.D., Zsolt Nagy, M.D., Geertrui Bocken, M.T., Bart Desmet, M.T., An Vankelecom, M.T., and Heidi Van Ranst, M.T., of the microinjection team and Ms. Ria Maes of the cryobiology team. Frank Winter, M.A., of the Language Education Centre of our University edited. REFERENCES L Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17-8. 2. Palermo G, Joris H, Derde M-P, Camus M, Devroey P, Van Steirteghem A. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993;59:826-35. 3. Van Steirteghem AC, Liu J, Joris H, Zsolt N, Janssenswillen C, Tournaye H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993;8:1055-60. 4. 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;8:1061-6. 5. 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 superovulution in IVF or GIFT. Hum Reprod 1988;3:585-90. 6. Staessen C, Camus M, Khan I, Smitz J, Van Waesberghe L, Wisanto A, et al. 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