Successful freezing and thawing of blastocysts cultured in sequential media using a modified method

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1 FERTILITY AND STERILITY VOL. 79, NO. 6, JUNE 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Successful freezing and thawing of blastocysts cultured in sequential media using a modified method Irma Virant-Klun, Ph.D., Tomaž Tomaževič, M.D., Ph.D., Lili Bačer-Kermavner, B.Sc., Jožica Mivšek, B.Sc., Brigita Valentinčič-Gruden, B.Sc., and Helena Meden-Vrtovec, M.D., Ph.D. Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia Objective: To evaluate the clinical role of blastocyst freezing and thawing after prolonged culturing in sequential media. Design: Retrospective analysis of 293 blastocyst freeze thawing cycles. Setting: University hospital infertility unit. Patient(s): Nonselected couples undergoing IVF. Intervention(s): Blastocysts were frozen and thawed by a modified method. Main Outcome Measure(s): Blastocyst recovery after freeze thawing and pregnancy rates after the transfer. Evaluation of the effect of the number of transferred blastocysts, the method of IVF, and of the woman s age on the results achieved by frozen thawed blastocysts. Result(s): Frozen thawed blastocysts provided a 29.5% clinical pregnancy rate per transfer. After the transfer of three blastocysts the pregnancy rate was 42.0%, and after the transfer of one or two blastocysts it was approximately the same (25.0% and 28.0%, respectively). The method of IVF did not affect pregnancy rates, but the increasing age of the woman did. Pregnancies were characterized by a low abortion rate (8.0%) regardless of the age of the woman. Conclusion(s): A modified method for blastocyst freeze thawing provides good clinical results. It offers the possibility for a single-thawed blastocyst transfer and represents a good alternative for older women because of its lower risk of spontaneous abortion. (Fertil Steril 2003;79: by American Society for Reproductive Medicine.) Key Words: Blastocysts, freezing and thawing, ICSI, IVF, pregnancies Received July 17, 2002; revised and accepted February 24, Supported by the Ministry of Education, Science and Sport, Slovenia, grant J Reprints requests: Irma Virant-Klun, Ph.D., Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Šlajmerjeva 3, SI-1000 Ljubljana, Slovenia (FAX: ; irma.virant@kclj.si) /03/$30.00 doi: /s (03) Blastocyst transfer is of great interest to many assisted reproductive technology (ART) centers. Selection of the best embryos to transfer to increase pregnancy rates and to replace a lower number of embryos to reduce the likelihood of an unwanted multiple pregnancy are being realized in more and more groups. Freezing of supernumerary blastocysts is still a matter of discussion, as adequate results are still awaited (1, 2). The first pregnancies after replacement of frozen thawed human blastocysts occurred in 1985 (3, 4). Blastocysts were cultured in a simple salt solution. The results showed that a significantly higher number of expanded blastocysts than of cleaving embryos survived cryopreservation, and relatively higher pregnancy rates were achieved by the replacement of thawed blastocysts than by the replacement of thawed cleaving embryos (3 6). On the other hand, some groups achieved no pregnancies with frozen thawed blastocysts (7). Different ART centers struggled with inconsistent results obtained by blastocyst freezing and thawing protocols, and searched for variants to improve on consistency. Cryopreservation of co-cultured human blastocysts has shown to produce excellent pregnancy rates (8 10). Revisiting the original two-step cryopreservation protocol (8), Menezo and Veiga (11) modified the protocol to become extremely convenient, and at least as 1428

2 successful as the earlier protocol. Some of these benefits may be attributed to the usage of co-culture systems. However, with increasing confidence in growth stage sequenced culture media, blastocyst culture for fresh transfer has become increasingly common (12 14). There are little data on clinical results achieved by frozen thawed blastocysts cultured in new sequential media without cell co-cultures by a slow cooling program (15 17). We have modified the two-step protocol for blastocyst freezing and thawing by Menezo and Veiga (11). The aim of this study was to evaluate the clinical role of blastocyst freezing and thawing in nonselected couples with supernumerary blastocysts after prolonged culturing in sequential media with a modified two-step freezing and thawing protocol. MATERIALS AND METHODS From January 2001 to February 2002 the embryos of all women (1,409 cycles) undergoing our program were cultured in sequential media (Blast Assist System; Medi-Cult, Jyllinge, Denmark) for 5 days to reach the blastocyst stage. Two blastocysts were transferred on day 5, whereas supernumerary blastocysts were cryopreserved using a modified two-step freezing protocol. Blastocysts were frozen in 410 (29%) cycles. If the woman did not conceive after fresh blastocyst transfer, frozen blastocysts were thawed and at most three survived blastocysts were transferred. Retrospectively, we analyzed 293 blastocyst freezing and thawing cycles in 292 nonselected couples to evaluate the recovery of frozen thawed blastocysts and clinical results (pregnancy, spontaneous abortion, and twin rates). The effects of the number of transferred blastocysts (1, 2, or 3), of the method of in vitro fertilization, classical IVF or intracytoplasmic sperm injection (ICSI), and of the age ( 30, 30 34, 35 39, 39 years) of the woman on clinical results achieved by frozen thawed blastocysts were evaluated. IVF, Blastocyst Culturing, and Transfer To start, the women were stimulated using a desensitizing protocol of GnRH-agonist (SC buserelin acetate; Suprefact: Hoechst AG, Frankfurt, Germany), started on day 22 of the menstrual cycle. After 14 days, if serum E 2 concentrations were 40 pg/ml, stimulation was started with hmg (Pergonal: Serono, Geneva, Switzerland) or FSH (Metrodin; HP 75 IU or Gonal F: Serono). Human chorionic gonadotrophin (Pregnyl: Organon, Oss, The Netherlands) was administered when the leading follicle measured 18 mm in diameter. Transvaginal ultrasound-guided aspiration of ovarian follicles was performed 36 hours after hcg administration. Classic IVF or ICSI was performed by the conventional methods, ICSI without using polyvinilpyrrolidone. All embryos of one patient were cultured as a group. To reach the stage of blastocyst, embryos were cultured in the sequential M1 and M2 media (Blast Assist System, Medi- Cult). On day 1, fertilized oocytes were transferred into 0.5 ml of M1 medium preincubated for at least 30 minutes in a double-well dish (Falcon, Becton Dickinson, Meylan Cedex, France). On day 3 they were transferred into the fresh preincubated M2 medium, the procedure was repeated daily until day 5. On day 5 at most two blastocysts were transferred (T.D.T. set; Prodimed, Neuilly-en-Thelle, France), and the supernumerary blastocysts were cryopreserved. Freezing and Thawing of Supernumerary Blastocysts To freeze the blastocysts cultured in sequential media, we modified the two-step method by Menezo et al. (8). Our modification of the method is as follows: our freezing solutions are based on Universal IVF Medium (Medi-Cult), because blastocysts have been cultured in Medi-Cult sequential media since the introduction of blastocyst culturing in our laboratory; blastocysts are incubated in freezing solutions at 37 C inaco 2 incubator to increase glycerol permeability; our slow cooling program stops at 150 C to prevent temperature shock when the blastocysts are plunged into the liquid nitrogen ( 196 C); blastocysts are thawed slowly. It has already been demonstrated that the termination of slow cooling is at about 80 C or at a lower temperature. We use this criteria in our method, with a subsequent plunge into liquid nitrogen that requires slow thawing. Embryos survived best when rewarmed slowly (10 C/min) (18). On the other hand, fast thawing (approximately 300 C/min) is needed when slow cooling is stopped at about 40 C with a subsequent plunge into liquid nitrogen. Cells that have been cooled to only 40 C will have more intracellular water than those cooled to 80 C or more. This water will freeze into relatively small ice crystals during this process. Then, if thawed too slowly the water will recrystallize into larger structures, which in turn can cause cell damage (19). Before freezing we did not make a special blastocyst selection. Compacted, expanded, and hatching blastocysts were frozen on day 5 or 6 according to Nakayama et al. (20) who reported no differences in survival and implantation rates between blastocysts frozen 5 or 6 days after insemination. All freezing and thawing solutions were preincubated for at least 30 minutes in a CO 2 incubator. Blastocysts were transferred into the first freezing solution (5% glycerol in Universal IVF Medium, Medi-Cult) for 10 minutes, and then into the second freezing solution (9% glycerol in Universal IVF Medium containing 0.2 M sucrose) for 10 minutes. During exposure to freezing solutions, blastocysts were incubated at 37 CinaCO 2 incubator. Then the blastocysts were placed into a plastic straw (Air Liquide, Bussy-Saint-George, France). One straw was filled with at most three blastocysts. The straws with blastocysts were cooled in liquid nitrogen vapor in a freezing machine (Minicool 40, Air Liquide) by a slow-cooling program: from 22 C FERTILITY & STERILITY 1429

3 to 6 C (manual seeding) at 2 C/min, from 6 Cto 40 C at 0.3 C/min, and from 40 C to 150 C at35 C/min. Then the straws were transferred into liquid nitrogen ( 196 C) and stored until use. For thawing, we have chosen the two-step thawing protocol with thawing solutions free of cryoprotectant. On the day of transfer, blastocysts were thawed at room temperature. They were transferred to two thawing solutions free of cryoprotectant glycerol: thawing solution 1 composed of 0.5 M sucrose in the Universal IVF Medium (Medi-Cult) and thawing solution 2 was composed of 0.2 M sucrose in the Universal IVF Medium. Blastocysts were exposed to each solution for 10 minutes at room temperature under a regulated 5% CO 2 stream (glass hood; K-Systems, Birkerød, Denmark). Then they were washed and transferred into the preincubated fresh Universal IVF Medium. Blastocysts were considered survived when more than 50% of inner mass and trophectoderm cells were undamaged and at most three blastocysts were transferred into the uterus about 1 hour after thawing. Coordination Between the Endometrium and Thawed Blastocysts In patients with a regular menstrual pattern, frozen thawed blastocysts were transferred into the uterus 4 days after the disappearance of the dominant follicle in the natural cycle. If the follicle did not disappear, utrogestan was administered three times daily for luteal phase supplementation. In these cases the transfer of thawed blastocysts was performed on day 4 of utrogestan therapy. Follicular growth monitoring was performed by determination of serum E 2 levels, vaginal ultrasound measurement of follicles and endometrium, and by quick urinary LH determination according to the established protocol for IVFembryo transfer in the natural cycle (21). Daily E 2 determinations started on day 9 of the menstrual cycle. Daily vaginal ulrasound monitoring and daily urinary LH determinations started when the E 2 level was 0.39 nmol/l (104 pg/ml). After a positive urinary LH and a decrease in E 2 level, the disappearance of the dominant follicle was observed on ultrasound. The transfer of frozen thawed blastocysts was performed 4 days later. In women with irregular menstrual cycles a minimal stimulation with 75 IU of FSH daily was started on day 7 of the menstrual cycle. Follicular growth monitoring was performed according to our protocol for follicle growth monitoring in the natural cycle (21). When the criteria for follicular maturity were achieved, and if the urinary LH was still negative, ovulation was induced with 5,000 IU of hcg, and the transfer of frozen thawed blastocysts was performed 6 days after hcg administration. If the urinary LH was positive, the transfer was planned for after the disappearance of the dominant follicle. TABLE 1 Clinical results achieved in 293 blastocyst freeze thawing cycles. Variables Thawing cycles 293 Couples 292 Female age (years) Thawed blastocysts 682 Survived blastocysts (%) 555 (81) Survival rate of blastocysts per couple 85.0% 23.7% Transferred blastocysts (%) 555 (100) Transferred blastocysts per cycle Blastocyst transfers (%) 288 (98) Clinical pregnancies (fetal heart beat) 85 Clinical pregnancy rate per blastocyst 29.5% transfer Clinical pregnancy rate per thawing 29% cycle Ectopic pregnancy (%) 1 (1) Twin pregnancies (%) 14 (16) Implantation rate (%) 99/555 (18) Spontaneous abortions (%) 7 (8) Statistical Analysis The SPSS program (SPSS Inc., Chicago, IL) was used for statistical analysis. We evaluated the differences among the groups by Mann-Whitney U test according to the number of transferred embryos, the IVF method, and the woman s age. Statistical significance was set at P.05. RESULTS Of the 682 thawed blastocysts, 555 (81%) survived the freezing and thawing procedure (Table 1). After thawing, 472 (85%) of the survived blastocysts were completely intact expanded or with no damaged cells. They were the same as before freezing (Fig. 1). Eighty-three (15%) survived blastocysts had less than 50% of damaged inner mass and trophectoderm cells, or were completely contracted with no signs of degeneration (Fig. 2). Contracted blastocysts recovered slowly. All survived blastocysts were transferred into the uterus. After the transfer of thawed blastocysts, 85 pregnancies were achieved; the pregnancy rate was 29.5% per embryo transfer and 29.0% per thawing cycle. The implantation rate per transferred blastocyst was 18%. No triplet pregnancy resulted, and there were 14 (16%) twin pregnancies. Eight percent of pregnancies ended in a spontaneous abortion (Table 1); the remaining 92% are either ongoing or ended in a delivery. Number of Frozen Thawed Blastocysts per Transfer After freezing and thawing, three blastocysts were transferred in 47 cycles, two blastocysts in 173 cycles, and one No Virant-Klun et al. Freezing thawing of human blastocysts Vol. 79, No. 6, June 2003

4 FIGURE 1 Frozen thawed expanded blastocyst (pregnant woman). FIGURE 2 Frozen thawed blastocyst that was completely contracted after freezing thawing. It did not recover before the transfer (nonpregnant woman). blastocyst in 68 cycles. The highest pregnancy rate (42%) was achieved after the transfer of three frozen thawed blastocysts, whereas pregnancy rates after the transfer of two or one thawed blastocysts were approximately the same: 28% and 25%, respectively. After the transfer of three thawed blastocysts, the twin pregnancy rate was significantly higher than after the transfer of two thawed blastocysts (35% vs. 14%; P.05), whereas after the transfer of one thawed blastocyst there was no twin pregnancy. The implantation rates achieved after the transfer of three, two, or one blastocyst did not differ (19%, 16%, and 25%, respectively). TABLE 2 Clinical results obtained by frozen thawed blastocysts according to the method of in vitro fertilization. Method of IVF IVF ICSI All Female age (years) Thawing cycles Blastocyst transfers (%) 234 (98) 54 (98) 288 (98) Thawed blastocysts Survived blastocysts (%) 453 (80) 102 (87) 555 (81) Transferred blastocysts Clinical pregnancies (fetal heart beat) Clinical pregnancy rate per blastocyst transfer 29 a 31 a 29.5% Ectopic pregnancy (%) 1 (2) 0 (0) 1 (1) Spontaneous abortions (%) 6 (11) 1 (6) 7 (8) Twin pregnancies (%) 11 (16) 3 (18) 14 (16) Implantation rate (%) 79/453 (17) 20/102 (20) 99/555 (18) a Statistically nonsignificant difference as detected by Mann-Whitney U test (P.05). FERTILITY & STERILITY 1431

5 Effects of the IVF Method and the Woman s Age In 238 freezing and thawing cycles IVF-derived blastocysts and in 55 cycles ICSI-derived blastocysts were transferred. The IVF method, whether classical IVF or ICSI, did not affect the blastocyst survival rate or pregnancy rate after the transfer of frozen thawed blastocysts (Table 2). In addition, there was no difference in twin pregnancy, implantation, and abortion rates (Table 2). The average age of the women who received frozen thawed blastocysts was years (range years). The survival rates of frozen thawed blastocysts did not differ among the age groups: 30 years (38 cycles), years (124 cycles), years (102 cycles), 39 years (29 cycles): 82%, 82%, 80%, and 83%, respectively. In women aged 30 years the pregnancy rate was 43%, whereas in the other age groups the pregnancy rates were approximately the same (29%, 27%, and 24%, respectively). In women 34 years the blastocyst implantation rate was lower than in younger women (14% vs. 23%; P.05) and there were no twin pregnancies (0% vs. 27%; P.05). The abortion rate was approximately the same in all age groups (6%, 8%, 7%, 0%, respectively). DISCUSSION We have found that by using a modified two-step freezing and thawing method there was a possibility for successful freezing and thawing of blastocysts cultured in sequential media. The method provided promising clinical results in nonselected patients. After the transfer of thawed blastocysts we achieved 85 pregnancies, that is, a 29.5% clinical pregnancy rate per transfer and a 29% pregnancy rate per thawing cycle; only 8% of the achieved pregnancies ended in a spontaneous abortion. Similarly, Langley et al. (16) and Behr et al. (17) obtained good clinical results after freezing and thawing of blastocysts cultured in sequential media; their results were comparable to ours. On the other hand, Pantos et al. (15) found a 56% survival rate and a 5.3% implantation rate after the transfer of frozen thawed blastocysts cultured in sequential media. In our study 81% of blastocysts survived the freezing and thawing procedure; 85% of the survived blastocysts were undamaged, indicating that due to good biological quality these blastocysts are appropriate for freezing and thawing. Blastocyst incubation in freezing solutions with glycerol at 37 C enables survival of blastocysts due to glycerol s permeability, efficient dehydration, and protection during cooling. Because cryoprotectants (glycerol included) are generally toxic, their concentrations, equilibration times, and equilibration temperatures are usually kept to a minimum room temperature or even lower (19). Using cryoprotectants at reduced temperatures may decrease toxicity but it contributes to osmotic problems (22). As in other substances, decreased temperature causes an increase in glycerol viscosity (22). Higher glycerol viscosity is related to worse penetration into embryonic cells during exposure to freezing solutions. Cocero et al. (23) reported a low reproductive yield after cryopreservation of ovine morulae and blastocysts using glycerol. Embryos cryopreserved with glycerol showed unequal degrees of conservation, even among blastomeres within a single embryo. Inner blastomeres were completely damaged, whereas external ones appeared to be intact. They found that a limited permeability of glycerol would explain the observed ultrastructural differences in blastomere integrity, which depends on the location of the blastomere. One of the most important features for successful cryopreservation is the biological quality of fresh blastocysts. The sequential media have proved to be efficient in the development of human embryos to the blastocyst stage, and also for ease of handling. After IVF, 60% of embryos reach the stage of blastocyst (16). In our program, the embryos of each couple are cultured in a group. There are other reports stating that the quality of human embryo growth is improved when embryos are cultured in groups rather than separately (24). It is known that embryos secrete various growth-enhancing factors as they develop (25). Culturing embryos together may counteract any dilution of these factors normally seen with singly cultured embryos. Gardner et al. (26) reported on improved development of embryos to the blastocyst stage when cultured in a group. On the other hand, Spyropoulou et al. (27) did not find any difference between the group cultured embryos and the separately cultured embryos. From our experience with sequential media we know that it is important to culture embryos in a sufficient volume of medium (0.5 ml), and to transfer embryos into a fresh medium each day. Thus, we avoid the accumulation of toxic compounds, such as heavy metal divalent cations (28 30) or other products of embryonic metabolism, such as ammonium (26, 31) and nitrate (32), and very likely compensate for the role of feeder cells. However, in a medium containing all amino acids, replacing the culture medium every 48 hours to alleviate ammonium toxicity significantly decreased the number of arrested embryos and significantly increased blastocyst cell number and the number of embryos developing to the blastocyst stage (26). Twin pregnancies represent a higher risk to the mother and neonates compared to a singleton pregnancy. In the present study approximately the same pregnancy and implantation rates were achieved after the transfer of one or two frozen thawed blastocysts. The transfer of one frozen thawed embryo that has self-selected to the blastocyst stage before freezing is supposed to result in a reduced risk of twin pregnancy. Blastocyst culture, freezing, and thawing are important techniques in the quest for the single conceptus transfer and the avoidance of multiple pregnancies with ART Virant-Klun et al. Freezing thawing of human blastocysts Vol. 79, No. 6, June 2003

6 The IVF method, whether classical IVF or ICSI, did not affect the pregnancy rate obtained by frozen thawed blastocysts, in spite of some reports that after ICSI a lower proportion of embryos reaches the blastocyst stage and that their biological quality is lower compared to that after IVF (33, 34). In humans, the age-related decline in a woman s fertility can be explained by a reduction in quality, either of the older uterus or of the embryos arising from aging oocytes. In the present study in women younger than 30 years the pregnancy rate after the transfer of thawed blastocysts was 43%, but the clinical pregnancy rate (24%) was relatively high also in women older than 39 years. Also, in seven pregnancies of women 39 years there was no spontaneous abortion. This might be the consequence of a more appropriate embryo selection for cryopreservation. Similarly, Janny and Menezo (35) observed a high delivery rate per oocyte retrieval (25.8%) in women 40 years after fresh blastocyst transfer. The modified method of freezing and thawing of blastocysts cultured in sequential media provides good pregnancy rates in nonselected patients. This might solve some dilemmas about blastocyst culturing. In addition to the good general clinical results this method offers the possibility for a single thawed blastocyst transfer and represents an alternative in older women because of a more appropriate embryo selection for freezing and thawing. Acknowledgments: The authors thank all the members of the IVF team: gynecologists Martina Ribič-Pucelj, M.D., Ph.D., Andrej Vogler, M.D., Ph.D., Eda Vrtačnik-Bokal, M.D., Ph.D., Bojana Pinter, M.D., Ph.D.; andrologists Branko Zorn, M.D., M.Sc., and Sašo Drobnič, M.D., Reproductive Unit; and biologist geneticist Alenka Veble, B.Sc., Genetic Unit, Dept. of Obstetrics and Gynaecology, University Medical Centre Ljubljana, Ljubljana, Slovenia, for their assistance during this study. The authors are also grateful to Mojca Pirc, B.Sc., Research Unit, University Medical Centre Ljubljana, for revising this manuscript, and biologist Bibi Fissbechus, Ph.D., from Medi-Cult, Jyllinge, Denmark, for all her support. References 1. Garcia-Velasco JA, Simon A. Blastocyst transfer: does it really affect the outcome? Curr Opin Obstet Gynecol 2001;13: Alper MM, Brinsden P, Fischer R, Wikland M. To blastocyst or not to blastocyst? That is the question. 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