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Cryopreservation of human embryos at the morula stage and outcomes after transfer
FERTILITY AND STERILITY威 VOL. 82, NO. 1, JULY 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Cryopreservation of human embryos at the morula stage and outcomes after transfer Jun Tao, Ph.D., Randall H. Craig, M.D., Mark Johnson, M.D., Brenda Williams, B.Sc., Wendy Lewis, B.Sc., Jennifer White, B.Sc., and Nicole Buehler, B.Sc. Fertility Treatment Center, Chandler, Arizona
Objective: To evaluate the survival rate of human morula embryo freezing and the morphological alterations during freezing, during and after thawing, and their applications in embryo selection. Design: Retrospective observational study. Setting: Private infertility clinic. Patient(s): Consecutive patients under age 39 undergoing frozen morula embryo transfers from December 1999 to May 2003. Intervention(s): Embryo freezing was performed at the morula stage. Embryo thaw and post-thaw ETs were conducted on the same day, which is equivalent to a day 4 ET. Main Outcome Measure(s): Morphological alterations during freezing and thawing and after thawing. Post-thaw embryo survival rates, transferable rates, pregnancy rates, and implantation rates. Result(s): Morula embryos showed reversed morphological alterations during the freezing process; these alterations were recovered during thawing or shortly after the thawing. Post-thaw survival rates showed no significant difference between any of the morula substages. However, embryos scored as grade 3, which represented good quality, had significantly higher post-thaw survival and transferable rates than grade 2 and 1 embryos. Patients who received at least one grade 3 embryo had significantly higher pregnancy rates, implantation rates, and ongoing/live birth rates than other groups. Conclusion(s): An acceptable survival rate can be achieved after cryopreservation of human morula embryos, and morphological alterations that occur during and shortly after an embryo thaw can be a feasible index for determining viable embryos. (Fertil Steril威 2004;82:108 –18. ©2004 by American Society for Reproductive Medicine.) Key Words: Embryo cryopreservation, frozen embryo transfer, morphological alterations, morula
Received August 20, 2003; revised and accepted December 1, 2003. Reprint requests: Jun Tao, Ph.D., Fertility Treatment Center, Chandler AZ 85224 (FAX: 480-897-1283; Email: jtao@fertilitytreatment center.com). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 12.024
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With controlled ovarian stimulation, more eggs are produced than in natural cycles, which usually yields more embryos than can be transferred in a stimulated cycle. It has been about two decades since a surplus of embryos was first cryopreserved for subsequent pregnancy attempts. Transferring cryopreserved embryos is both less expensive and less invasive than a repeat stimulation and fresh ET. However, the general implantation rate and pregnancy rate after thawed ETs are still lower than after fresh ETs. Since 1983, when the first successful human pregnancy was achieved after transferring frozen embryos (1), numerous articles have been published about freezing human embryos at the pronucleate (2– 4), early cleavage (2– 8 cell) (3, 5, 6), and blastocyst stage (7, 8). Each embry-
onic stage has qualities that make it the right stage for freezing; however, each stage also has its disadvantages, which limit its success rate. Pronucleate freezing gives patients the best opportunity to have excess embryos in storage for subsequent ET attempts, and high post-thaw survival rates are usually achievable (3, 4, 9). Unfortunately, the further growth potential of post-thaw pronucleates is inconclusive. Cleavage-stage freezing improves embryo selection somewhat, but post-thaw survival rates are not always promising (3). When some blastomeres undergo lysis during the thawing process, which is not unusual, the potential for embryo growth is even less predicable (10 –12). Blastocyst freezing provides the best embryo selection, but the survival rates are generally lower than early embryonic-stage freez-
ing (13, 14). Recent studies demonstrated improved survival rates of blastocyst freezing, but there is a limited number of patients who have excess blastocysts for freezing (15, 16). Morula-stage embryo freezing has been successfully used in some farm animals (17, 18) but has never been considered as an option in human IVF practice. Not until recently were successful pregnancies and normal births reported after transferring embryos that were frozen at the morula stage (19). In addition to the uncertain growth potential of earlystage embryo freezing, embryos may also suffer some cryopreservation-related injuries. These injuries may be undetectable morphologically under a light microscope, which causes post-thaw embryos to appear intact, but may arrest further development (20). Therefore, requiring intact embryos or blastomeres after thawing cannot be considered a rigorous criterion for the evaluation of viability. The major problem in current frozen ET is that there is no feasible method to assess post-thaw embryo viability. Recent studies in mouse morula embryo freezing demonstrated morphological alterations during the freezing and thawing process (21). The morphological changes in thawing seem to help in judging post-thaw embryo viability. The ability to know which embryos are viable is a great benefit in determining the number of embryos that should be thawed to ensure that superior-quality embryos are available for transfer. This article describes the cryopreservation of human embryos at the morula stage and its implications for the pregnancy rate after transfer.
MATERIALS AND METHODS All patients under the age of 39 who underwent morula embryo freezing and thawing in our program from December 1999 through May 2003 were included in this study. Oocyte retrievals that took place during the same period and the occurrences of fresh ETs and embryo cryopreservation at this time were also analyzed. Institutional Review Board approval was not obtained since the freezing and thawing methods applied had been reported (19) and this study was a retrospective data analysis.
Patient Preparation in Stimulated Cycles and Embryo Management Patient stimulation, oocyte retrieval, insemination or intracytoplasmic sperm injection, and embryo culture have been described elsewhere (22). Embryo development was evaluated daily, and the grading criteria for day 2 and day 3 embryos were based on cell number, evenness of blastomeres, and the amount of fragmentation (22). Fresh ETs were performed on day 4. Day 4 embryo grading criteria were described in detail elsewhere (22) but simplified in this study. Briefly, grading on day 4 was based on [1] the proportion of blastomeres undergoing the compaction process; [2] the morphology of the compacted multicellular FERTILITY & STERILITY威
mass; [3] the embryo quality on days 2 and 3; and [4] the amount of fragmentation. Embryo quality was graded from 1 to 3, in which the grade of 3 represented good quality, grade 2 intermediate, and grade 1 poor. Grade 3 embryos (Fig. 1A–C) made up more than 75% of blastomeres undergoing compaction and the embryos looked sphere-shaped with a smooth profile. These embryos had about 4 and 8 cells on day 2 and 3, respectively, with even-sized blastomeres or a slight blastomere size difference and less than 25% fragmentation. Grade 2 embryos (Fig. 1D) were those which had 50%–75% of their blastomeres that underwent compaction or a morula which had a higher percentage of blastomeres compacted but with a slightly irregular-shaped profile. Grade 2 embryos might have also had blastomeres with a moderate size difference on day 2 and 3, or fragmentation between 25% and 50%. Grade 1 embryos had less than 50% of their blastomeres undergoing compaction, or had a higher percentage of blastomeres compacted but they were severely irregular shaped (Fig. 1E). On day 2 and 3 these embryos might have had low cell number, or severely uneven sized blastomeres and multinuclei might be observed; fragmentation was usually over 50% in these embryos. On day 4, two to three embryos with the best quality were selected for fresh transfer, and the remaining embryos were frozen only if they underwent compaction or showed signs of possible compaction. Otherwise, embryos were evaluated the next morning for possible freezing using the same criteria. The preference was to freeze grade 2 and 3 embryos. However, in practice, grade 1 embryos were frequently frozen for the sake of saving any possible conception opportunity for patients, especially when patients did not have any good embryos available or sometimes just because the patient requested it. To study the freezing/thawing survival rate at different compaction stages, morula embryos were classified into three substages: early, compact, and late morula. In the early morula (Fig. 1A), blastomeres are arranged tightly and some if not all of them start to “merge” together, but individual cells are still easily distinguishable. A compact morula (Fig. 1B) is defined as having blastomeres that appear completely “merged” together, in which the individual cell boundaries cannot be easily identified but in which nuclei are visible and intercellular space disappears. If the embryo score is grade 3, the embryo profile is smooth, which makes it look as if it were one big cell but with spread nuclei. Embryos that had passed the compact morula stage were considered to be in the late morula stage; morphologically they were considered to have postcompaction status (Fig. 1C), in which embryos again demonstrate individual cell membranes but the number of blastomeres is usually increased, about 16 cells in grade 3 embryos. Some blastomeres at the edge might become spindle shaped, and a small blastocele may start to form. Embryos having a clear blastocele were also classified as late 109
FIGURE 1 Photomicrographs of morulae at different substages with different quality. (A) Grade 3 early morula showing blastomeres starting to “merge” together, but individual cell profile still distinguishable. (B) Grade 3 compact morula embryo characterized by invisible individual cell, but nuclei spread and distinct. Embryo profile is smooth, which makes the embryo appear as one big cell. (C) Grade 3 late morula in which cell boundaries are distinguishable again and cell number increases compared with early morula. Blastomeres at the periphery become spindle shaped and blastulation starts. (D) Grade 2 compact morula embryo showing about 60%– 65% blastomeres undergoing compaction with ⬍20% fragments. (E) Grade 1 compact morula embryo exhibiting an irregular profile, although all blastomeres are undergoing compaction.
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morula embryos in this study. The morphological alterations of each embryo during freezing and during and after thawing were recorded for comparison studies.
Patient Preparation for Frozen Embryo Transfer (FET) One of the two protocols was employed: the first protocol was for patients with regular menstrual cycles who were administered E2 2 mg twice a day from cycle day 1. Progesterone administration, 50 mg IM in oil, was started on the evening of cycle day 12, and vaginal P gel (Crinone 8%; Wyeth-Ayerst Laboratories, Philadelphia) or vaginal P cream (10%) was administered from the evening of day 12 and then twice a day thereafter, in the morning and evening. Prednisone 10 mg was used from day 15 to day 19. Embryo thaw and transfer was performed on day 17, and a pregnancy test was conducted on cycle day 28. Patients with irregular menstrual cycles or who had failed treatment with the above protocol were then put on protocol 110
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2. Leuprolide acetate (Lupron; TAP Pharmaceuticals, Deerfield, IL) 1 mg daily was administrated from the midluteal phase, followed by 0.5 mg from cycle day 1 to day 9. E2 1 mg was administered twice a day, from day 1 to day 5, then 2 mg twice a day for 4 days followed by 2 mg three times a day for 5 days. After that, E2 was back to 2 mg twice a day until pregnancy test. Progesterone injection and P vaginal cream or crinone administrations were the same as in the first protocol but started from cycle day 15. Embryo thaw and transfer were performed on cycle day 20 and followed by the pregnancy test 11 days later. If a positive pregnancy test was achieved, E2 was continued to week 14, P injection to week 10, and P vaginal cream to week 12. The embryo thawing strategy was to achieve a goal of having two grade 3 post-thaw embryos. If embryo(s) did not survive or did not show morphological recovery changes after the thawing process, more embryo(s) would be thawed. Occasionally, the number of grade 3 embryos to be transferred might be more than two in patients that had repeated Vol. 82, No. 1, July 2004
failed implantation or repeated miscarriage due to embryo genetic abnormalities or per patient request. For patients without frozen grade 3 embryos, three to four or even more embryos of inferior quality were possibly thawed. Embryo transfers were usually performed 1–2 hours after the thawing procedure. Mechanical assisted hatching was applied to all embryos regardless of embryo quality (22), except embryos that had a ruptured zona or were missing part of the zona after retrieval from a cryovial during the thawing process.
Embryo Freezing The embryo freezing procedure was based on Testart’s method (23) with some modifications to minimize osmotic shock. In brief, embryos were first transferred into a Hepesbuffered human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% serum substitute supplement (Irvine Scientific) or serum protein substitute (SAGE BioPharma, Bedminster, NJ) at room temperature for 5–7 minutes. Embryos were then moved to a 0.75 M 1,2-propanediol/0.05 M sucrose (Sigma, St. Louis) Hepesbuffered HTF solution for 5–7 minutes, followed by a 1.0 M 1,2-propanediol/0.075 M sucrose solution for another 5–7 minutes. Finally, the embryos were transferred to a solution with 1.5 M 1,2-propanediol/0.1 M sucrose. Embryos were then loaded into cryogenic vials (Corning, NY) with 0.5 mL of cryomedium, which contained the 1.5 M 1,2-propanediol/0.1 M sucrose solution. The total time in this solution was about 5 minutes. The cryovials were moved to a programmable freezer (Planer, TS Scientific, Perkasie, PA) at a start temperature of 22°C. Initial cooling was carried out at the rate of 2°C/minute⫺1 to ⫺7.5°C and then remained at the same temperature for 10 minutes during which seeding was performed manually. After seeding, a cooling rate of 0.3°C/minute⫺1 was employed to ⫺30°C. After that, the temperature dropped at the rate of 50°C/ minute⫺1 to ⫺120°C. Cryovials were finally plunged into liquid nitrogen.
tific) or cleavage medium (SAGE) or global medium (IVFonline.com, Guilford, CT) until transfer. Embryos with more than 50% cells remaining intact were considered to have survived. Morphological alterations in each embryo during the thawing process were recorded and compared with their developmental status before freezing. Embryos showing morphological recovery changes were determined suitable for transfer unless no additional embryos were available. Embryos that failed to show recovery changes or that were of inferior quality were left in culture medium and then either discarded the next day or refrozen based on quality or patient choice.
Outcome Analysis Post-thaw embryo survival rates were compared within the different substages, and the morphological alterations during freezing and during and 1–1.5 hours after thawing were tracked for investigating their significance in embryo selection. Embryo survival rates and transferable rates were also analyzed based on embryo quality. The outcomes of FET were compared within the following groups in which patients had only grade 1 embryos transferred (group A), at least one grade 2 embryo but no grade 3 embryos transferred (group B), and at least one grade 3 embryo transferred (group C). Eleven days after ET, pregnancy tests were conducted. An hCG level over 10 mIU/mL was considered positive. Clinical pregnancy was defined as an intrauterine sac(s) identified by ultrasound examination. The implantation rate was defined as the number of gestational sacs per number of embryos transferred. Monozygotic twins were considered one implantation. Data are presented as mean ⫾ SD or percentage. Where appropriate, data were analyzed with unpaired Student’s t-test or 2-test. P⬍.05 was considered statistically significant.
Embryo Thawing The thawing procedure was performed about 3 hours before the transfer, during which cryovials containing embryos were quickly removed from liquid nitrogen and set on the bench top for 75 seconds. The cryovial was then immersed into a water bath at 37°C for 2.5 minutes followed by leaving the cryovial on the bench top for 1 minute before retrieving the embryos. Cryoprotectant was gradually removed by placing the embryos in Hepes-buffered HTF solutions with gradient decreasing concentrations of 1,2-propanediol and sucrose. Embryos were first transferred to a solution of 1.0 M 1,2-propanediol/0.2 M sucrose followed by a 0.5 M 1,2-propanediol/0.2 M sucrose solution. Then the embryos went through solutions with 0.15 M sucrose and 0.075 M sucrose, respectively. Embryos were in each solution for 6 –7 minutes. After rinsing in Hepes-buffered HTF medium, embryos were cultured in either P1 (Irvine ScienFERTILITY & STERILITY威
RESULTS Characteristics and outcomes of oocyte retrieval cycles and FET cycles are presented in Table 1. Thirty-five oocyte retrieval cycles had embryo freezing, but the fresh ETs were cancelled because of ovarian hyperstimulation syndrome (34 cycles) and prospective surgery (one cycle). Eighteen cycles completely failed, which resulted in fresh ETs cancelled and no embryos frozen because [1] no oocytes were retrieved (three cycles), [2] one or two mature oocytes were retrieved but no embryos were produced (eight cycles), or [3] all embryos were of poor quality and not worth transferring or freezing (seven cycles). In total, 1,762 embryos were frozen; 1,414 (80.2%) and 348 (19.8%) were frozen on day 4 in the afternoon and day 5 in the morning, respectively. No FET cycle was canceled for poor embryo survival. 111
TABLE 1 Characteristics and outcomes of oocyte retrievals and frozen ET cycles. Characteristics
Outcomes
No. of oocyte retrievals Age at oocyte retrieval (y) No. of oocytes retrieved Percent of cycles with fresh ET Percent of cycles with embryo freezing Percent of cycles with freezing but cancelled fresh transfer Percent of cycles that completely failed (no fresh transfer/no freezing) No. of embryos frozen per cycle No. of FET cycles (yield from 120 retrieval cycles) Age at FET (y) No. of embryos thawed per FET cycle No. of embryos transferred per FET cycle Positive pregnancy test per FET cycle (%) Clinical pregnancy per FET cycle (%) Ongoing/live birth per FET cycle (%) Implantation rate per transferred post-thaw embryo (%) Singleton per FET cyclea (%) Twins per FET cycleb (%) Triplets per FET cycleb (%)
415 33.1 ⫾ 3.4 16.9 ⫾ 10 87.2 (362/415) 63.4 (263/415) 8.4 (35/415) 4.3 (18/415) 6.7 ⫾ 4.6 137 33.1 ⫾ 3.1 3.2 ⫾ 1.3 (444/137) 2.5 ⫾ 0.7 (340/137) 72.3 (99/137) 57.7 (79/137) 49.6 (68/137) 36.2 (123/340) 60.3 (41/68) 33.8 (23/68) 5.9 (4/68)
a
Including one case that had fetal demise of monozygotic twins and delivered one healthy girl. b Including one set of monozygotic twins delivered. Note: FET ⫽ frozen ET. Tao. Morula embryo freezing and transfer. Fertil Steril 2004.
Morphological Alterations During Freezing Process Reversed morphological alterations were observed in all three subgroups (Figs. 2 and 3, and Table 2). Blastomeres of
early morula stage usually started to separate as soon as they were moved to the first freezing medium, a 0.75 M PROH/ 0.05 M sucrose solution. As the concentrations of cryoprotectants increased, cell separation became more obvious. When reaching the final step, in which the cryoprotectants were 0.15 M PROH/0.1 M sucrose, blastomeres of all early morula embryos (100%) were separated, which made them appear as cleavage-like embryos of day 3. Some embryos at the compact morula stage showed different degrees of cell separation and others remained at full compaction status during the freezing process. At the time of loading into a cryovial, 18.0% of embryos reverted back to cleavage-like embryos, which were characterized by either partially separated or completely separated blastomeres, and the rest (82.0%) remained at full compaction status. In late morula embryos, blastomere separation was not observed during the freezing process, but most embryos reversed to full compaction status as the freezing procedure continued. Spindle-shaped blastomeres disappeared, and distinct cell boundaries became obscure. Blastoceles that formed in some embryos decreased in size and usually disappeared completely before loading into cryovials (Fig. 3). At the time of loading, 91.3% late morula embryos morphologically reversed to full compaction status.
Morphological Alterations During Thawing Process Because of high concentrations of sucrose in the first two thawing solutions, blastomeres decreased in size when transferred from a cryovial to thawing media. As the thawing process proceeded, the size of the blastomeres increased proportionally to the reciprocal of the solution’s osmolality. As opposed to morphological reversal alterations in freezing, recovery alterations were demonstrated during the thawing process (Figs. 4 and 5 and Table 2). Blastomeres of early
FIGURE 2 Reversal morphological alterations during the freezing procedure. (A) Compact morula embryo before freezing. (B) The middle of freezing process. (C) Before being loaded into a cryovial showing visible individual cells.
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FIGURE 3 Reversal morphological alterations during freezing procedure. (A) Late morula embryo before freezing showing a small blastocele (arrow). (B) The middle of freezing process, showing a shrunken blastocele (arrow). (C) Before being loaded into a cryovial showing the embryo morphologically revert to full compaction status. The blastocele has completely disappeared.
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morula embryos usually remained separated at the beginning of thawing. The cells, although they appeared separated, were still clustered together. As thawing continued, some early morulae started to recompact and reverted to the original status that they had before freezing. More embryos at the compact morula stage before freezing had blastomere separation (81.5%) when released from a cryovial, compared with only 18.0% that had cell separation at the time of loading into a cryovial. The number of blastomeres seemed higher in most embryos at around 8 –16
cells. Different degrees of blastomere recompaction were frequently observed during the thawing process. Of the embryos that were frozen at the late morula stage, 46.2% had blastomere separation when retrieved from a cryovial, compared with no blastomere separation at the time of loading. Usually more cells, around 16 or more, could be identified. Only 45.3% of embryos remained at the full compaction status because of blastomere separation, compared with 91.3% of embryos that were at the full compaction status at the time of loading. At the end of the thawing process, the
TABLE 2 Post-thaw survival rates and morphological alterations during freezing/thawing process and after thawing in three substages of morula embryos. Embryonic substages before freezing
Early morula
Compact morula
Late morula
No. of embryos Survival ratea (%) Intact embryo after thaw (%) At the time of loading into cryovials (%) Blastomere separation Full compaction Postcompaction At the time of recovering from cryovials (%) Blastomere separation Full compaction Postcompaction 1–1.5 hours after thawing (%) Blastomere separation Full compaction Postcompaction
51 90.2 (46/51) 70.6 (36/51)
278 87.8 (244/278) 80.9 (225/278)
115 92.2 (106/115) 85.2 (98/115)b
100 (51/51) 0 (0/51) 0 (0/51)
18.0 (50/278) 82.0 (228/278) 0 (0/278)
0 (0/115) 91.3 (105/115) 8.7 (10/115)
100 (46/46) 0 (0/46) 0 (0/46)
81.5 (198/243) 18.5 (45/243) 0 (0/243)
46.2 (49/106) 45.3 (48/106) 8.5 (9/106)
67.4 (31/46) 32.6 (15/46) 0 (0/46)
26.7 (65/243) 67.5 (164/243) 5.8 (14/243)
10.4 (11/106) 41.5 (44/106) 48.1 (51/106)
a b
No significant difference between any two substages (P ⬎ .05). Significant difference compared with early morula group (P ⬍ .05).
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FIGURE 4 Recovery alterations of an embryo, which was at the compact morula stage before freezing, during and after a thawing procedure. (A) After being removed from a cryovial, the embryo is characterized by separated blastomeres as early morula but with a high number of cells. (B) At the end of the thawing process, blastomeres started to “merge” again. (C) One hour after thawing, the embryo recovered its prefreezing status.
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majority of these embryos in this group demonstrated different degrees of recovery alterations, either recompacting or even starting blastulation.
Morphological Alterations After Thawing Embryos continued to demonstrate morphological alterations after the thawing procedure (Figs. 4 and 5 and Table 2). About 1–1.5 hours after thawing, 32.6% of the early morula stage embryos became fully compacted. In the compact morula group, the number of embryos that had blastomere separation decreased from 81.6% at the time of
recovery from a cryovial to 26.7%, while the number of full compaction embryos increased from 18.5% to 67.5%. In addition to that, 5.8% of these embryos even advanced to postcompaction status. In the late morula group, the number of embryos with separated blastomeres dropped from 46.2% to 10.4%. Meanwhile, the number of embryos that went to postcompaction status increased from 8.5% to 48.1%. Embryos that were not selected for transfer because of no recovery alterations or inferior quality were cultured overnight, and only three embryos were recompacted and refrozen.
FIGURE 5 Recovery alterations of an embryo, which was at late morula stage before freezing, during and after a thawing procedure. (A) After being removed from a cryovial, the embryo appeared substantially at full compaction status. (B) At the end of the thawing process, the embryo advanced to postcompaction status. (C) One and a half hours after the thawing procedure the embryo developed to late compact status even with a blastocele and the potential formation of an inner-cell mass (arrow).
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TABLE 3 Relationship between embryo quality and post-thaw survival rate and transferable rate. Embryo quality Post-thaw survival rate No. of transfers per thaw
Grade 1
Grade 2
Grade 3
Average
84.9 (90/106) 69.8 (74/106)
84.4 (108/128) 68.8 (88/128)
94.3 (198/210)a,b 84.8 (178/210)b,c
89.2 (396/444) 76.6 (340/444)
Significant difference compared with grade 1 embryo (P ⬍ .05). Significant difference compared with grade 2 embryo (P ⬍ .01). c Significant difference compared with grade 1 embryo (P ⬍ .01). a
b
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Post-Thaw Survival Rates Post-thaw survival rates of embryos at three substages showed no significant difference between any two substages (Table 2). Embryos with less than 2–3 lysed blastomeres seemed to have no apparent effect on recompaction or blastulation, especially in the late morula group, because of the higher cell number. It was observed that 37 embryos had ruptured zona pellucida or were attached to an air bubble before being retrieved from a cryovial, which seemed to have some correlation with some embryo damage. Table 3 represents post-thaw survival rates and ET rates with different embryo quality. The results indicate that grade 3 embryos have a significantly higher post-thaw survival rate than grade 1 and grade 2 embryos. The percentage of embryos that were determined suitable for transfer after thawing was also significantly higher in grade 3 embryos.
Frozen Embryo Transfer Outcomes The results of a pregnancy test 11 days after transfer, clinical pregnancy rate, implantation rate, and ongoing/live birth rate are presented in Table 1. The outcomes of the two protocols applied to an FET patient preparation were combined because no significant differences between the two strategies were observed. No pregnancy had more than triplets.
Table 4 indicates the correlation between transferred postthaw embryo quality and outcomes. Patients who were transferred with at least one grade 3 embryo (group C) had significantly higher incidences of having positive pregnancy tests, clinical pregnancies, and ongoing/live births (P⬍.05). The implantation rate in group C is also significantly higher compared with groups A and B (P⬍.01).
DISCUSSION The current relatively poor outcomes from FETs may be more often caused by inferior embryo viability rather than poor endometrium receptivity, since the endometrial environment should be more optimal for implantation in frozen cycles than in gonadotropin-stimulated cycles. Several factors may be attributed to inferior outcomes of post-thaw ETs. The first is embryo selection during freezing. Traditionally, embryo freezing is performed at the early embryonic stages, such as the pronucleate (day 1) or cleavage stages of 4-cell (day 2) to 8-cell (day 3) blastomeres (1– 4). It is known that the number of good-quality embryos decreases daily, so the earlier the embryonic stage, the more uncertain the embryo viability. That is why thawed pronucleates are routinely cultured for several days before being transferred (24 –26). Since embryos at freezing are day(s)
TABLE 4 Correlations between transferred post-thaw embryo qualities and outcomes. Variables No. of cases No. of embryos thawed No. of embryos transferred Positive pregnancy test Clinical pregnancy Implantation rate Ongoing/live birth
Group A
Group B
Group C
Average
13 3.5 ⫾ 1.3 2.5 ⫾ 0.9 46.2 (6/13) 15.4 (2/13) 9.4 (3/32) 15.4 (2/13)
31 3.5 ⫾ 1.4 2.6 ⫾ 0.8 64.5 (20/31) 45.2 (14/31) 22.2 (18/81) 29.0 (9/31)
93 3.1 ⫾ 1.1 2.4 ⫾ 0.7 78.5 (73/93)a 67.7 (63/93)b,c 44.9 (102/227)b,d 61.3 (57/93)b,d
3.2 ⫾ 1.3 2.5 ⫾ 0.7 72.3 (99/137) 57.7 (79/137) 36.2 (123/340) 49.6 (68/137)
Significant difference compared with group A (P ⬍ .05). Significant difference compared with group A (P ⬍ .01). c Significant difference compared with group B (P ⬍ .05). d Significant difference compared with group B (P ⬍ .01). a
b
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before being at the stage desired for transfer, when post-thaw pronucleate develops poorly, which is not unusual, patients can end up with an insufficient number of good embryos for transfer and therefore a poor outcome can be expected. Freezing embryos at the 4- or 8-cell stage improves embryo selection, but predicting their growth potential is still difficult. Reported implantation rates of cleavage-stage embryos are only 10%–15% (25, 27, 28), compared with a 40%– 45% reported implantation rate for blastocyst transfers (25). Poor embryo selections at the pronucleate and cleavage stage make freezing embryos at these stages inconclusive with regard to viability. To ensure a sufficient number of viable post-thaw embryos available for transfer usually requires a higher number of early-stage embryos that are thawed and cultured for day(s) to select a good-prognosis embryo (26). In practice, the uncertainty of embryo development still makes it hard to get the desired number and quality of embryos for transfer that have good chance of resulting in a pregnancy. The second reason why the outcomes of post-thaw embryos are inferior is because of the inadequate post-thaw survival rates. The post-thaw pronucleate survival rate has been reported to be between 65% and 90% (4 – 6, 9, 20 –30). Survival rates of early cleavage embryos are usually lower than those of pronucleate-stage embryos (5, 30), but freezing at the cleavage stage is also popular because of a better embryo selection compared with the pronucleate freezing (31, 32). However, it should be noticed that reported survived cleavage-stage embryos include those with some blastomeres lysed after thaw, which is common (10 –12). The accepted definition of a survived cleavage embryo is one in which more than 50% of blastomeres remained intact after the thawing procedure (10, 33–35). The viability of these partially survived embryos is very likely impaired (36, 37). Recent studies have shown that the implantation rate of transferring embryos with some blastomere loss was significantly reduced (12). When the number of embryos for a transfer is kept low to avoid multiple gestations, the incidence of pregnancy after transferring these partially survived cleavage embryos cannot be expected to be high. The third reason why the outcomes of post-thaw embryos in early embryonic stages are inferior is the difficulty in determining viability and whether a determination can guide the embryo thaw strategy. Unlike embryo survival, some cryopreservation-caused injuries are not detectable by morphological assessment with a light microscope even though embryos or blastomeres appear intact after thaw (20). These injuries may be related to the low potential for further development. Taking this into consideration, plus poor embryo selection in early embryonic stages and possible blastomere lysis during thawing, post-thaw embryo viability prediction is very difficult. Although the difficulty can be overcome by culturing embryos overnight or longer to verify further embryo development, it would be too late to thaw 116
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additional embryos when necessary because of the concern of the synchronization between the endometrium and embryos. In practice, an insufficient number of good-quality embryos for transfer is not only the main reason for the poor FET outcomes, it even causes about a 10% cancellation rate in FET cycles (4 – 6). In summary, transferring post-thaw embryos with a good chance of achieving a pregnancy requires [1] freezing embryos at a late embryonic stage for a better embryo selection; [2] achieving a higher post-thaw survival rate with a low incidence of blastomere damage; and [3] determining the viability of post-thaw embryos at the time of thaw, rather than day(s) later. To achieve a higher success rate after FET, the above three conditions have to be met. Apparently, pronucleate- and cleavage-stage embryo freezing are not the optimal stages for achieving high clinical success rates. Other stages possible for freezing are the morula (mostly on day 4) and blastocyst (day 5 or 6) stages. Expanded blastocyst is the last embryonic stage before implantation and provides the best embryo selection because of a well-defined inner-cell mass. There have been many studies concerning the freezing of blastocysts, although many of them have been conflicting. However, recent reports seem promising (38, 39). Nevertheless, only a limited number of patients have excess blastocysts available for freezing (3, 15, 16). Morula-stage ET and freezing have not been used in human IVF programs. A recent study showed that there was a ⬃45% implantation rate for day 4 embryos after fresh morula ET (22), which is compatible with the reported blastocyst transfer, although no direct comparison can be drawn. The data presented in this study seem to support morula-stage freezing as another option in IVF practice because of the acceptable post-thaw survival rate, high implantation rate, and feasibility of viability determination. The 57.7% clinical pregnancy rate and 36.2% implantation rate after transferring of post-thawed embryos show a potential application. In addition, multiple pregnancies, a largely undesirable outcome, can be reduced by transferring a lower number of morula embryos. It was noticed in this study that blastomere separation and recompaction are highly correlated with environmental osmolality. After embryos are retrieved from cryovials during thawing, more embryos had blastomere separation compared with the time of loading. This indicates that blastomere separation continues after loading since the cryomedium osmolality still increases after seeding. In contrast, recompaction and blastulation occurred in the thawing process as the medium osmolality decreased. Embryos that show morphological recovery in thawing or shortly after thawing seem to have a better growth potential than those that failed to demonstrate recovery changes because the latter usually arrested or compacted poorly in overnight culture. Vol. 82, No. 1, July 2004
Within the three subgroups of morula embryos, the preference appears to be to freeze embryos at the compact morula or late morula stages although there is no significant difference in post-thaw survival rates from the early morula stage. This is because after reaching full compaction embryos provide information on the proportion of blastomeres undergoing compaction and the morphology of compacted embryos. In embryos at the early morula stage, it is uncertain what percentage of blastomeres will eventually compact and how well blastomeres will compact since it is not unusual that a large proportion of blastomeres undergo compaction but form irregularly shaped profiles. Moreover, compact morula and late morula embryos are more likely to recompact or even blastulate during the thawing process, which can immediately provide information regarding whether a good post-thaw embryo has been achieved or whether more embryos need to be thawed. Recompaction or blastulation is especially beneficial for embryo selection in patients who do not have good-quality embryos but who do have inferior-quality ones. In this population, embryos may have low cell number, unevenness of blastomeres, more than 25% fragments, or atypical compaction. The capability of recompaction and reblastulation seems to provide another opportunity for embryo grading, which helps in selecting relatively superior embryos from inferior ones. Freezing excess embryos before a fresh ET procedure is popular in current IVF practices. Routinely, the best-quality embryos are transferred in stimulated cycles to give patients the best opportunity for achieving pregnancy and avoid the possibility of losing good-quality embryos in the freezing and thawing process. Two dilemmas are frequently encountered. The first is how many embryos should be reserved for fresh ET since ET is conducted day(s) later and the embryo development is uncertain. Without reserving a sufficient number of early-stage embryos in the incubators, it is possible to end up with an insufficient number of good embryos on the day of the fresh ET. For day 5 ET, it is usually recommended that 4 –5 good day 3 embryos be reserved, even though only two blastocysts are desired to be transferred (40 – 42). It is more difficult to decide the number of embryos to be reserved if embryo freezing is performed at the pronucleate or 4-cell stages. The second dilemma occurs when more good-quality embryos than are needed are produced on the transfer day, when the majority of reserved embryos grow well, or when too many embryos are reserved. When this happens, a second embryo freezing has to take place and embryos are then frozen at a different embryonic stage, which may be not a preferred stage for that clinic. Cryopreservation of embryos at the morula stage seems to eliminate these dilemmas since embryo freezing can be conducted after a fresh ET, which ensures superior embryo selection for fresh ETs and there is FERTILITY & STERILITY威
no concern about either too many or an insufficient number of good embryos available for fresh ET. In conclusion, morula embryo freezing and thawing can be successfully applied in human assisted reproductive practice. When freezing embryos at the morula stage, not only can an acceptable FET outcome be achieved, but it can also significantly simplify some clinical decisions regarding the number of embryos that should be reserved for a fresh transfer and the number of embryos that should be thawed in FET cycles. References 1. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983;305:707–9. 2. Quinn P. Success of oocyte and embryo freezing and its effect on outcome with in vitro fertilization. Semin Reprod Endocrinol 1990;8: 272–80. 3. Demoulin A, Jouan C, Gerday C, Dubois M. Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum Reprod 1991; 6:799 –804. 4. 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 Steril 1993;59:1202–7. 5. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rates after early human embryo freezing. Fertil Steril 1986;46:268 –72. 6. Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3):161–74. 7. Cohen J, Simons RS, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fertil Embr Trans 1985;2:59 –64. 8. Quinn P, Kerin J. Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques. J In Vitro Fertil Embr Trans 1986;3:40 –5. 9. Miller KF, Goldberg JM. In vitro development and implantation rates of fresh and cryopreserved sibling zygotes. Obstet Gynecol 1995;85: 999 –1002. 10. 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;2:709 –15. 11. Edgar DH, Bourne H, Spiers AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000;15:175–9. 12. El-Toukhy T, Khalf Y, Al-Darazi K, Andritsos V, Taylor A, Braude P. Effect of blastomere loss on the outcome of frozen embryo replacement cycles. Fertil Steril 2003;79:1106 –11. 13. Troup SA, Matson PL, Critchlow JD, Morroll DR, Lieberman BA, Burslem RW. Cryopreservation of human embryos at the pronucleate, early cleavage, or expanded blastocyst stages. Eur J Obst Gyn Reprod Biol 1990;38:133–9. 14. Pantos K, Stefanidis K, Pappas K, Kokkinopoulos P, Petroutsou K, Kokkali G, et al. Cryopreservation of embryos, blastocysts and pregnancy rates of blastocysts derived from frozen-thawed embryos and frozen-thawed blastocysts. 2001;18:579 – 82. 15. Alper MM, Brinsden P, Fischer R, Wikland M. To blastocyst or not to blastocyst? That is the question. Hum Reprod 2001;16:617–9. 16. Blake D, Proctor M, Johnson N, Olive D. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Sys Rev 2002;2:CD002118. 17. Pugh PA, Ankersmit AE, McGowan LT, Tervit HR. Cryopreservation of in vitro–produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on postthaw survival. Theriogenology 1998;50:495–506. 18. Massip A. Cryopreservation of embryos of farm animals. Reprod Dom Anim 2001;36:49 –55. 19. Tao J, Tamis R, Fink K. Pregnancies achieved after transferring frozen morula/compact stage embryos. Fertil Steril 2001;75:629 –31. 20. Cocero MJ, Lopez-Sebastian A, Barragan ML, Picazo RA. Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol. Cryobiology 1996;33:502–7. 21. Tao J, Tamis R, Fink K. Cryopreservation of mouse embryos at morula/ compact stage. J Assist Reprod Genet 2001;18:235–43.
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22. Tao J, Tamis R, Fink K. The neglected morula/compact stage embryo transfer. Hum Reprod 2002;17:1513–8. 23. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil Steril 1985;44:645–51. 24. Nikolettos N, Al-Hasani S. Frozen pronuclear oocytes: advantages for the patient. Mol Cell Endocrinol 2000;27:169:55– 62. 25. Gorrill MJ, Kaplan PF, Patton PE, Burry KA. Initial experience with extended culture and blastocyst transfer of cryopreserved embryos. Am J Obstet Gynecol 1999;180:1472–4. 26. Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P, Van Steirteghem AC. Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 1997;12:1513–21. 27. Selick CE, Hofmann GE, Albano C, Horowitz GM, Copperman AB, Garrisi GJ, et al. Embryo quality and pregnancy potential of fresh compared with frozen embryos—is freezing detrimental to high quality embryos? Hum Reprod 1995;10:392–5. 28. Senn A, Vozzi C, Chanson A, De Grandi P, Germond M. Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage. Fertil Steril 2000;74:946 –52. 29. Virutamasen P, Boonkasemsanti W, Suwajanakorn S, Kosalanant V, Chaiyaput R, Parksamoot W. Pregnancy following transfer of frozenthawed embryo: a preliminary report. J Med Assoc Thai 1992;75:153–6. 30. Fugger EF, Bustillo M, Dorfmann AD, Schulman JD. Human preimplantation embryo cryopreservation: selected aspects. Hum Reprod 1991;6:131–5. 31. Freemann L, Trounson A, Kirby C. Cryopreservation of human embryos: progress on the clinical use of the technique in human in vitro fertilization. J In Vitro Fertil Embr Trans 1986;3:53–61. 32. Kattera S, Shrivastav P, Craft I. Comparison of pregnancy outcome of pronuclear- and multicellular-stage frozen-thawed embryo transfers. J Assist Reprod Genet 1999;16:358 –62.
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33. Trounson A. Preservation of human eggs and embryos. Fertil Steril 1986;46:1–12. 34. Freemann L, Trounson A, Kirby C. Cryopreservation of human embryos: progress on the clinical use of the technique in human in vitro fertilization. J In Vitro Fertil Embr Trans 1986;3:53–61. 35. Van Steirteghem AC, Van Den Abbeel E, Camus M, Van Waesberghe L, Braechman SP, Khan I, et al. Cryopreservation of human embryos obtained after gamete intra-fallopian transfer and/or in vitro fertilization. Hum Reprod 1987;2:593–8. 36. Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P, Van Steirteghem AC. Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 1997;12:1513–21. 37. Lornage J, Boulieu D, Mathieu C, Guerin JF, Pinatel MC, James R, et al. Transfers of frozen-thawed human embryos in cycles stimulated by HMG. Hum Reprod 1990;5:60 –5. 38. Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Reprod Biomed Online 2003;6:221–5. 39. Gaardner DK, Lane M, Stevens J, Schoolcraft WB. Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertil Steril 2003;79:407–10. 40. Rijnders PM, Jansen CAM. The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod 1998;13:2869 –73. 41. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R. Blastocyst quality affects the success of blastocyst-stage embryo transfer. Fertil Steril 2000;74:282–7. 42. Graham MC, Hoeger KM, Phipps WR. Initial IVF-ET experience with assisted hatching performed 3 days after retrieval followed by day 5 embryo transfer. Fertil Steril 2000;74:668 –71.
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Cryopreservation of human embryos at the morula stage and outcomes after transfer Jun Tao, Ph.D., Randall H. Craig, M.D., Mark Johnson, M.D., Brenda Williams, B.Sc., Wendy Lewis, B.Sc., Jennifer White, B.Sc., and Nicole Buehler, B.Sc. Fertility Treatment Center, Chandler, Arizona
Objective: To evaluate the survival rate of human morula embryo freezing and the morphological alterations during freezing, during and after thawing, and their applications in embryo selection. Design: Retrospective observational study. Setting: Private infertility clinic. Patient(s): Consecutive patients under age 39 undergoing frozen morula embryo transfers from December 1999 to May 2003. Intervention(s): Embryo freezing was performed at the morula stage. Embryo thaw and post-thaw ETs were conducted on the same day, which is equivalent to a day 4 ET. Main Outcome Measure(s): Morphological alterations during freezing and thawing and after thawing. Post-thaw embryo survival rates, transferable rates, pregnancy rates, and implantation rates. Result(s): Morula embryos showed reversed morphological alterations during the freezing process; these alterations were recovered during thawing or shortly after the thawing. Post-thaw survival rates showed no significant difference between any of the morula substages. However, embryos scored as grade 3, which represented good quality, had significantly higher post-thaw survival and transferable rates than grade 2 and 1 embryos. Patients who received at least one grade 3 embryo had significantly higher pregnancy rates, implantation rates, and ongoing/live birth rates than other groups. Conclusion(s): An acceptable survival rate can be achieved after cryopreservation of human morula embryos, and morphological alterations that occur during and shortly after an embryo thaw can be a feasible index for determining viable embryos. (Fertil Steril威 2004;82:108 –18. ©2004 by American Society for Reproductive Medicine.) Key Words: Embryo cryopreservation, frozen embryo transfer, morphological alterations, morula
Received August 20, 2003; revised and accepted December 1, 2003. Reprint requests: Jun Tao, Ph.D., Fertility Treatment Center, Chandler AZ 85224 (FAX: 480-897-1283; Email: jtao@fertilitytreatment center.com). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 12.024
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With controlled ovarian stimulation, more eggs are produced than in natural cycles, which usually yields more embryos than can be transferred in a stimulated cycle. It has been about two decades since a surplus of embryos was first cryopreserved for subsequent pregnancy attempts. Transferring cryopreserved embryos is both less expensive and less invasive than a repeat stimulation and fresh ET. However, the general implantation rate and pregnancy rate after thawed ETs are still lower than after fresh ETs. Since 1983, when the first successful human pregnancy was achieved after transferring frozen embryos (1), numerous articles have been published about freezing human embryos at the pronucleate (2– 4), early cleavage (2– 8 cell) (3, 5, 6), and blastocyst stage (7, 8). Each embry-
onic stage has qualities that make it the right stage for freezing; however, each stage also has its disadvantages, which limit its success rate. Pronucleate freezing gives patients the best opportunity to have excess embryos in storage for subsequent ET attempts, and high post-thaw survival rates are usually achievable (3, 4, 9). Unfortunately, the further growth potential of post-thaw pronucleates is inconclusive. Cleavage-stage freezing improves embryo selection somewhat, but post-thaw survival rates are not always promising (3). When some blastomeres undergo lysis during the thawing process, which is not unusual, the potential for embryo growth is even less predicable (10 –12). Blastocyst freezing provides the best embryo selection, but the survival rates are generally lower than early embryonic-stage freez-
ing (13, 14). Recent studies demonstrated improved survival rates of blastocyst freezing, but there is a limited number of patients who have excess blastocysts for freezing (15, 16). Morula-stage embryo freezing has been successfully used in some farm animals (17, 18) but has never been considered as an option in human IVF practice. Not until recently were successful pregnancies and normal births reported after transferring embryos that were frozen at the morula stage (19). In addition to the uncertain growth potential of earlystage embryo freezing, embryos may also suffer some cryopreservation-related injuries. These injuries may be undetectable morphologically under a light microscope, which causes post-thaw embryos to appear intact, but may arrest further development (20). Therefore, requiring intact embryos or blastomeres after thawing cannot be considered a rigorous criterion for the evaluation of viability. The major problem in current frozen ET is that there is no feasible method to assess post-thaw embryo viability. Recent studies in mouse morula embryo freezing demonstrated morphological alterations during the freezing and thawing process (21). The morphological changes in thawing seem to help in judging post-thaw embryo viability. The ability to know which embryos are viable is a great benefit in determining the number of embryos that should be thawed to ensure that superior-quality embryos are available for transfer. This article describes the cryopreservation of human embryos at the morula stage and its implications for the pregnancy rate after transfer.
MATERIALS AND METHODS All patients under the age of 39 who underwent morula embryo freezing and thawing in our program from December 1999 through May 2003 were included in this study. Oocyte retrievals that took place during the same period and the occurrences of fresh ETs and embryo cryopreservation at this time were also analyzed. Institutional Review Board approval was not obtained since the freezing and thawing methods applied had been reported (19) and this study was a retrospective data analysis.
Patient Preparation in Stimulated Cycles and Embryo Management Patient stimulation, oocyte retrieval, insemination or intracytoplasmic sperm injection, and embryo culture have been described elsewhere (22). Embryo development was evaluated daily, and the grading criteria for day 2 and day 3 embryos were based on cell number, evenness of blastomeres, and the amount of fragmentation (22). Fresh ETs were performed on day 4. Day 4 embryo grading criteria were described in detail elsewhere (22) but simplified in this study. Briefly, grading on day 4 was based on [1] the proportion of blastomeres undergoing the compaction process; [2] the morphology of the compacted multicellular FERTILITY & STERILITY威
mass; [3] the embryo quality on days 2 and 3; and [4] the amount of fragmentation. Embryo quality was graded from 1 to 3, in which the grade of 3 represented good quality, grade 2 intermediate, and grade 1 poor. Grade 3 embryos (Fig. 1A–C) made up more than 75% of blastomeres undergoing compaction and the embryos looked sphere-shaped with a smooth profile. These embryos had about 4 and 8 cells on day 2 and 3, respectively, with even-sized blastomeres or a slight blastomere size difference and less than 25% fragmentation. Grade 2 embryos (Fig. 1D) were those which had 50%–75% of their blastomeres that underwent compaction or a morula which had a higher percentage of blastomeres compacted but with a slightly irregular-shaped profile. Grade 2 embryos might have also had blastomeres with a moderate size difference on day 2 and 3, or fragmentation between 25% and 50%. Grade 1 embryos had less than 50% of their blastomeres undergoing compaction, or had a higher percentage of blastomeres compacted but they were severely irregular shaped (Fig. 1E). On day 2 and 3 these embryos might have had low cell number, or severely uneven sized blastomeres and multinuclei might be observed; fragmentation was usually over 50% in these embryos. On day 4, two to three embryos with the best quality were selected for fresh transfer, and the remaining embryos were frozen only if they underwent compaction or showed signs of possible compaction. Otherwise, embryos were evaluated the next morning for possible freezing using the same criteria. The preference was to freeze grade 2 and 3 embryos. However, in practice, grade 1 embryos were frequently frozen for the sake of saving any possible conception opportunity for patients, especially when patients did not have any good embryos available or sometimes just because the patient requested it. To study the freezing/thawing survival rate at different compaction stages, morula embryos were classified into three substages: early, compact, and late morula. In the early morula (Fig. 1A), blastomeres are arranged tightly and some if not all of them start to “merge” together, but individual cells are still easily distinguishable. A compact morula (Fig. 1B) is defined as having blastomeres that appear completely “merged” together, in which the individual cell boundaries cannot be easily identified but in which nuclei are visible and intercellular space disappears. If the embryo score is grade 3, the embryo profile is smooth, which makes it look as if it were one big cell but with spread nuclei. Embryos that had passed the compact morula stage were considered to be in the late morula stage; morphologically they were considered to have postcompaction status (Fig. 1C), in which embryos again demonstrate individual cell membranes but the number of blastomeres is usually increased, about 16 cells in grade 3 embryos. Some blastomeres at the edge might become spindle shaped, and a small blastocele may start to form. Embryos having a clear blastocele were also classified as late 109
FIGURE 1 Photomicrographs of morulae at different substages with different quality. (A) Grade 3 early morula showing blastomeres starting to “merge” together, but individual cell profile still distinguishable. (B) Grade 3 compact morula embryo characterized by invisible individual cell, but nuclei spread and distinct. Embryo profile is smooth, which makes the embryo appear as one big cell. (C) Grade 3 late morula in which cell boundaries are distinguishable again and cell number increases compared with early morula. Blastomeres at the periphery become spindle shaped and blastulation starts. (D) Grade 2 compact morula embryo showing about 60%– 65% blastomeres undergoing compaction with ⬍20% fragments. (E) Grade 1 compact morula embryo exhibiting an irregular profile, although all blastomeres are undergoing compaction.
Tao. Morula embryo freezing and transfer. Fertil Steril 2004.
morula embryos in this study. The morphological alterations of each embryo during freezing and during and after thawing were recorded for comparison studies.
Patient Preparation for Frozen Embryo Transfer (FET) One of the two protocols was employed: the first protocol was for patients with regular menstrual cycles who were administered E2 2 mg twice a day from cycle day 1. Progesterone administration, 50 mg IM in oil, was started on the evening of cycle day 12, and vaginal P gel (Crinone 8%; Wyeth-Ayerst Laboratories, Philadelphia) or vaginal P cream (10%) was administered from the evening of day 12 and then twice a day thereafter, in the morning and evening. Prednisone 10 mg was used from day 15 to day 19. Embryo thaw and transfer was performed on day 17, and a pregnancy test was conducted on cycle day 28. Patients with irregular menstrual cycles or who had failed treatment with the above protocol were then put on protocol 110
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2. Leuprolide acetate (Lupron; TAP Pharmaceuticals, Deerfield, IL) 1 mg daily was administrated from the midluteal phase, followed by 0.5 mg from cycle day 1 to day 9. E2 1 mg was administered twice a day, from day 1 to day 5, then 2 mg twice a day for 4 days followed by 2 mg three times a day for 5 days. After that, E2 was back to 2 mg twice a day until pregnancy test. Progesterone injection and P vaginal cream or crinone administrations were the same as in the first protocol but started from cycle day 15. Embryo thaw and transfer were performed on cycle day 20 and followed by the pregnancy test 11 days later. If a positive pregnancy test was achieved, E2 was continued to week 14, P injection to week 10, and P vaginal cream to week 12. The embryo thawing strategy was to achieve a goal of having two grade 3 post-thaw embryos. If embryo(s) did not survive or did not show morphological recovery changes after the thawing process, more embryo(s) would be thawed. Occasionally, the number of grade 3 embryos to be transferred might be more than two in patients that had repeated Vol. 82, No. 1, July 2004
failed implantation or repeated miscarriage due to embryo genetic abnormalities or per patient request. For patients without frozen grade 3 embryos, three to four or even more embryos of inferior quality were possibly thawed. Embryo transfers were usually performed 1–2 hours after the thawing procedure. Mechanical assisted hatching was applied to all embryos regardless of embryo quality (22), except embryos that had a ruptured zona or were missing part of the zona after retrieval from a cryovial during the thawing process.
Embryo Freezing The embryo freezing procedure was based on Testart’s method (23) with some modifications to minimize osmotic shock. In brief, embryos were first transferred into a Hepesbuffered human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% serum substitute supplement (Irvine Scientific) or serum protein substitute (SAGE BioPharma, Bedminster, NJ) at room temperature for 5–7 minutes. Embryos were then moved to a 0.75 M 1,2-propanediol/0.05 M sucrose (Sigma, St. Louis) Hepesbuffered HTF solution for 5–7 minutes, followed by a 1.0 M 1,2-propanediol/0.075 M sucrose solution for another 5–7 minutes. Finally, the embryos were transferred to a solution with 1.5 M 1,2-propanediol/0.1 M sucrose. Embryos were then loaded into cryogenic vials (Corning, NY) with 0.5 mL of cryomedium, which contained the 1.5 M 1,2-propanediol/0.1 M sucrose solution. The total time in this solution was about 5 minutes. The cryovials were moved to a programmable freezer (Planer, TS Scientific, Perkasie, PA) at a start temperature of 22°C. Initial cooling was carried out at the rate of 2°C/minute⫺1 to ⫺7.5°C and then remained at the same temperature for 10 minutes during which seeding was performed manually. After seeding, a cooling rate of 0.3°C/minute⫺1 was employed to ⫺30°C. After that, the temperature dropped at the rate of 50°C/ minute⫺1 to ⫺120°C. Cryovials were finally plunged into liquid nitrogen.
tific) or cleavage medium (SAGE) or global medium (IVFonline.com, Guilford, CT) until transfer. Embryos with more than 50% cells remaining intact were considered to have survived. Morphological alterations in each embryo during the thawing process were recorded and compared with their developmental status before freezing. Embryos showing morphological recovery changes were determined suitable for transfer unless no additional embryos were available. Embryos that failed to show recovery changes or that were of inferior quality were left in culture medium and then either discarded the next day or refrozen based on quality or patient choice.
Outcome Analysis Post-thaw embryo survival rates were compared within the different substages, and the morphological alterations during freezing and during and 1–1.5 hours after thawing were tracked for investigating their significance in embryo selection. Embryo survival rates and transferable rates were also analyzed based on embryo quality. The outcomes of FET were compared within the following groups in which patients had only grade 1 embryos transferred (group A), at least one grade 2 embryo but no grade 3 embryos transferred (group B), and at least one grade 3 embryo transferred (group C). Eleven days after ET, pregnancy tests were conducted. An hCG level over 10 mIU/mL was considered positive. Clinical pregnancy was defined as an intrauterine sac(s) identified by ultrasound examination. The implantation rate was defined as the number of gestational sacs per number of embryos transferred. Monozygotic twins were considered one implantation. Data are presented as mean ⫾ SD or percentage. Where appropriate, data were analyzed with unpaired Student’s t-test or 2-test. P⬍.05 was considered statistically significant.
Embryo Thawing The thawing procedure was performed about 3 hours before the transfer, during which cryovials containing embryos were quickly removed from liquid nitrogen and set on the bench top for 75 seconds. The cryovial was then immersed into a water bath at 37°C for 2.5 minutes followed by leaving the cryovial on the bench top for 1 minute before retrieving the embryos. Cryoprotectant was gradually removed by placing the embryos in Hepes-buffered HTF solutions with gradient decreasing concentrations of 1,2-propanediol and sucrose. Embryos were first transferred to a solution of 1.0 M 1,2-propanediol/0.2 M sucrose followed by a 0.5 M 1,2-propanediol/0.2 M sucrose solution. Then the embryos went through solutions with 0.15 M sucrose and 0.075 M sucrose, respectively. Embryos were in each solution for 6 –7 minutes. After rinsing in Hepes-buffered HTF medium, embryos were cultured in either P1 (Irvine ScienFERTILITY & STERILITY威
RESULTS Characteristics and outcomes of oocyte retrieval cycles and FET cycles are presented in Table 1. Thirty-five oocyte retrieval cycles had embryo freezing, but the fresh ETs were cancelled because of ovarian hyperstimulation syndrome (34 cycles) and prospective surgery (one cycle). Eighteen cycles completely failed, which resulted in fresh ETs cancelled and no embryos frozen because [1] no oocytes were retrieved (three cycles), [2] one or two mature oocytes were retrieved but no embryos were produced (eight cycles), or [3] all embryos were of poor quality and not worth transferring or freezing (seven cycles). In total, 1,762 embryos were frozen; 1,414 (80.2%) and 348 (19.8%) were frozen on day 4 in the afternoon and day 5 in the morning, respectively. No FET cycle was canceled for poor embryo survival. 111
TABLE 1 Characteristics and outcomes of oocyte retrievals and frozen ET cycles. Characteristics
Outcomes
No. of oocyte retrievals Age at oocyte retrieval (y) No. of oocytes retrieved Percent of cycles with fresh ET Percent of cycles with embryo freezing Percent of cycles with freezing but cancelled fresh transfer Percent of cycles that completely failed (no fresh transfer/no freezing) No. of embryos frozen per cycle No. of FET cycles (yield from 120 retrieval cycles) Age at FET (y) No. of embryos thawed per FET cycle No. of embryos transferred per FET cycle Positive pregnancy test per FET cycle (%) Clinical pregnancy per FET cycle (%) Ongoing/live birth per FET cycle (%) Implantation rate per transferred post-thaw embryo (%) Singleton per FET cyclea (%) Twins per FET cycleb (%) Triplets per FET cycleb (%)
415 33.1 ⫾ 3.4 16.9 ⫾ 10 87.2 (362/415) 63.4 (263/415) 8.4 (35/415) 4.3 (18/415) 6.7 ⫾ 4.6 137 33.1 ⫾ 3.1 3.2 ⫾ 1.3 (444/137) 2.5 ⫾ 0.7 (340/137) 72.3 (99/137) 57.7 (79/137) 49.6 (68/137) 36.2 (123/340) 60.3 (41/68) 33.8 (23/68) 5.9 (4/68)
a
Including one case that had fetal demise of monozygotic twins and delivered one healthy girl. b Including one set of monozygotic twins delivered. Note: FET ⫽ frozen ET. Tao. Morula embryo freezing and transfer. Fertil Steril 2004.
Morphological Alterations During Freezing Process Reversed morphological alterations were observed in all three subgroups (Figs. 2 and 3, and Table 2). Blastomeres of
early morula stage usually started to separate as soon as they were moved to the first freezing medium, a 0.75 M PROH/ 0.05 M sucrose solution. As the concentrations of cryoprotectants increased, cell separation became more obvious. When reaching the final step, in which the cryoprotectants were 0.15 M PROH/0.1 M sucrose, blastomeres of all early morula embryos (100%) were separated, which made them appear as cleavage-like embryos of day 3. Some embryos at the compact morula stage showed different degrees of cell separation and others remained at full compaction status during the freezing process. At the time of loading into a cryovial, 18.0% of embryos reverted back to cleavage-like embryos, which were characterized by either partially separated or completely separated blastomeres, and the rest (82.0%) remained at full compaction status. In late morula embryos, blastomere separation was not observed during the freezing process, but most embryos reversed to full compaction status as the freezing procedure continued. Spindle-shaped blastomeres disappeared, and distinct cell boundaries became obscure. Blastoceles that formed in some embryos decreased in size and usually disappeared completely before loading into cryovials (Fig. 3). At the time of loading, 91.3% late morula embryos morphologically reversed to full compaction status.
Morphological Alterations During Thawing Process Because of high concentrations of sucrose in the first two thawing solutions, blastomeres decreased in size when transferred from a cryovial to thawing media. As the thawing process proceeded, the size of the blastomeres increased proportionally to the reciprocal of the solution’s osmolality. As opposed to morphological reversal alterations in freezing, recovery alterations were demonstrated during the thawing process (Figs. 4 and 5 and Table 2). Blastomeres of early
FIGURE 2 Reversal morphological alterations during the freezing procedure. (A) Compact morula embryo before freezing. (B) The middle of freezing process. (C) Before being loaded into a cryovial showing visible individual cells.
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FIGURE 3 Reversal morphological alterations during freezing procedure. (A) Late morula embryo before freezing showing a small blastocele (arrow). (B) The middle of freezing process, showing a shrunken blastocele (arrow). (C) Before being loaded into a cryovial showing the embryo morphologically revert to full compaction status. The blastocele has completely disappeared.
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morula embryos usually remained separated at the beginning of thawing. The cells, although they appeared separated, were still clustered together. As thawing continued, some early morulae started to recompact and reverted to the original status that they had before freezing. More embryos at the compact morula stage before freezing had blastomere separation (81.5%) when released from a cryovial, compared with only 18.0% that had cell separation at the time of loading into a cryovial. The number of blastomeres seemed higher in most embryos at around 8 –16
cells. Different degrees of blastomere recompaction were frequently observed during the thawing process. Of the embryos that were frozen at the late morula stage, 46.2% had blastomere separation when retrieved from a cryovial, compared with no blastomere separation at the time of loading. Usually more cells, around 16 or more, could be identified. Only 45.3% of embryos remained at the full compaction status because of blastomere separation, compared with 91.3% of embryos that were at the full compaction status at the time of loading. At the end of the thawing process, the
TABLE 2 Post-thaw survival rates and morphological alterations during freezing/thawing process and after thawing in three substages of morula embryos. Embryonic substages before freezing
Early morula
Compact morula
Late morula
No. of embryos Survival ratea (%) Intact embryo after thaw (%) At the time of loading into cryovials (%) Blastomere separation Full compaction Postcompaction At the time of recovering from cryovials (%) Blastomere separation Full compaction Postcompaction 1–1.5 hours after thawing (%) Blastomere separation Full compaction Postcompaction
51 90.2 (46/51) 70.6 (36/51)
278 87.8 (244/278) 80.9 (225/278)
115 92.2 (106/115) 85.2 (98/115)b
100 (51/51) 0 (0/51) 0 (0/51)
18.0 (50/278) 82.0 (228/278) 0 (0/278)
0 (0/115) 91.3 (105/115) 8.7 (10/115)
100 (46/46) 0 (0/46) 0 (0/46)
81.5 (198/243) 18.5 (45/243) 0 (0/243)
46.2 (49/106) 45.3 (48/106) 8.5 (9/106)
67.4 (31/46) 32.6 (15/46) 0 (0/46)
26.7 (65/243) 67.5 (164/243) 5.8 (14/243)
10.4 (11/106) 41.5 (44/106) 48.1 (51/106)
a b
No significant difference between any two substages (P ⬎ .05). Significant difference compared with early morula group (P ⬍ .05).
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FIGURE 4 Recovery alterations of an embryo, which was at the compact morula stage before freezing, during and after a thawing procedure. (A) After being removed from a cryovial, the embryo is characterized by separated blastomeres as early morula but with a high number of cells. (B) At the end of the thawing process, blastomeres started to “merge” again. (C) One hour after thawing, the embryo recovered its prefreezing status.
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majority of these embryos in this group demonstrated different degrees of recovery alterations, either recompacting or even starting blastulation.
Morphological Alterations After Thawing Embryos continued to demonstrate morphological alterations after the thawing procedure (Figs. 4 and 5 and Table 2). About 1–1.5 hours after thawing, 32.6% of the early morula stage embryos became fully compacted. In the compact morula group, the number of embryos that had blastomere separation decreased from 81.6% at the time of
recovery from a cryovial to 26.7%, while the number of full compaction embryos increased from 18.5% to 67.5%. In addition to that, 5.8% of these embryos even advanced to postcompaction status. In the late morula group, the number of embryos with separated blastomeres dropped from 46.2% to 10.4%. Meanwhile, the number of embryos that went to postcompaction status increased from 8.5% to 48.1%. Embryos that were not selected for transfer because of no recovery alterations or inferior quality were cultured overnight, and only three embryos were recompacted and refrozen.
FIGURE 5 Recovery alterations of an embryo, which was at late morula stage before freezing, during and after a thawing procedure. (A) After being removed from a cryovial, the embryo appeared substantially at full compaction status. (B) At the end of the thawing process, the embryo advanced to postcompaction status. (C) One and a half hours after the thawing procedure the embryo developed to late compact status even with a blastocele and the potential formation of an inner-cell mass (arrow).
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TABLE 3 Relationship between embryo quality and post-thaw survival rate and transferable rate. Embryo quality Post-thaw survival rate No. of transfers per thaw
Grade 1
Grade 2
Grade 3
Average
84.9 (90/106) 69.8 (74/106)
84.4 (108/128) 68.8 (88/128)
94.3 (198/210)a,b 84.8 (178/210)b,c
89.2 (396/444) 76.6 (340/444)
Significant difference compared with grade 1 embryo (P ⬍ .05). Significant difference compared with grade 2 embryo (P ⬍ .01). c Significant difference compared with grade 1 embryo (P ⬍ .01). a
b
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Post-Thaw Survival Rates Post-thaw survival rates of embryos at three substages showed no significant difference between any two substages (Table 2). Embryos with less than 2–3 lysed blastomeres seemed to have no apparent effect on recompaction or blastulation, especially in the late morula group, because of the higher cell number. It was observed that 37 embryos had ruptured zona pellucida or were attached to an air bubble before being retrieved from a cryovial, which seemed to have some correlation with some embryo damage. Table 3 represents post-thaw survival rates and ET rates with different embryo quality. The results indicate that grade 3 embryos have a significantly higher post-thaw survival rate than grade 1 and grade 2 embryos. The percentage of embryos that were determined suitable for transfer after thawing was also significantly higher in grade 3 embryos.
Frozen Embryo Transfer Outcomes The results of a pregnancy test 11 days after transfer, clinical pregnancy rate, implantation rate, and ongoing/live birth rate are presented in Table 1. The outcomes of the two protocols applied to an FET patient preparation were combined because no significant differences between the two strategies were observed. No pregnancy had more than triplets.
Table 4 indicates the correlation between transferred postthaw embryo quality and outcomes. Patients who were transferred with at least one grade 3 embryo (group C) had significantly higher incidences of having positive pregnancy tests, clinical pregnancies, and ongoing/live births (P⬍.05). The implantation rate in group C is also significantly higher compared with groups A and B (P⬍.01).
DISCUSSION The current relatively poor outcomes from FETs may be more often caused by inferior embryo viability rather than poor endometrium receptivity, since the endometrial environment should be more optimal for implantation in frozen cycles than in gonadotropin-stimulated cycles. Several factors may be attributed to inferior outcomes of post-thaw ETs. The first is embryo selection during freezing. Traditionally, embryo freezing is performed at the early embryonic stages, such as the pronucleate (day 1) or cleavage stages of 4-cell (day 2) to 8-cell (day 3) blastomeres (1– 4). It is known that the number of good-quality embryos decreases daily, so the earlier the embryonic stage, the more uncertain the embryo viability. That is why thawed pronucleates are routinely cultured for several days before being transferred (24 –26). Since embryos at freezing are day(s)
TABLE 4 Correlations between transferred post-thaw embryo qualities and outcomes. Variables No. of cases No. of embryos thawed No. of embryos transferred Positive pregnancy test Clinical pregnancy Implantation rate Ongoing/live birth
Group A
Group B
Group C
Average
13 3.5 ⫾ 1.3 2.5 ⫾ 0.9 46.2 (6/13) 15.4 (2/13) 9.4 (3/32) 15.4 (2/13)
31 3.5 ⫾ 1.4 2.6 ⫾ 0.8 64.5 (20/31) 45.2 (14/31) 22.2 (18/81) 29.0 (9/31)
93 3.1 ⫾ 1.1 2.4 ⫾ 0.7 78.5 (73/93)a 67.7 (63/93)b,c 44.9 (102/227)b,d 61.3 (57/93)b,d
3.2 ⫾ 1.3 2.5 ⫾ 0.7 72.3 (99/137) 57.7 (79/137) 36.2 (123/340) 49.6 (68/137)
Significant difference compared with group A (P ⬍ .05). Significant difference compared with group A (P ⬍ .01). c Significant difference compared with group B (P ⬍ .05). d Significant difference compared with group B (P ⬍ .01). a
b
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before being at the stage desired for transfer, when post-thaw pronucleate develops poorly, which is not unusual, patients can end up with an insufficient number of good embryos for transfer and therefore a poor outcome can be expected. Freezing embryos at the 4- or 8-cell stage improves embryo selection, but predicting their growth potential is still difficult. Reported implantation rates of cleavage-stage embryos are only 10%–15% (25, 27, 28), compared with a 40%– 45% reported implantation rate for blastocyst transfers (25). Poor embryo selections at the pronucleate and cleavage stage make freezing embryos at these stages inconclusive with regard to viability. To ensure a sufficient number of viable post-thaw embryos available for transfer usually requires a higher number of early-stage embryos that are thawed and cultured for day(s) to select a good-prognosis embryo (26). In practice, the uncertainty of embryo development still makes it hard to get the desired number and quality of embryos for transfer that have good chance of resulting in a pregnancy. The second reason why the outcomes of post-thaw embryos are inferior is because of the inadequate post-thaw survival rates. The post-thaw pronucleate survival rate has been reported to be between 65% and 90% (4 – 6, 9, 20 –30). Survival rates of early cleavage embryos are usually lower than those of pronucleate-stage embryos (5, 30), but freezing at the cleavage stage is also popular because of a better embryo selection compared with the pronucleate freezing (31, 32). However, it should be noticed that reported survived cleavage-stage embryos include those with some blastomeres lysed after thaw, which is common (10 –12). The accepted definition of a survived cleavage embryo is one in which more than 50% of blastomeres remained intact after the thawing procedure (10, 33–35). The viability of these partially survived embryos is very likely impaired (36, 37). Recent studies have shown that the implantation rate of transferring embryos with some blastomere loss was significantly reduced (12). When the number of embryos for a transfer is kept low to avoid multiple gestations, the incidence of pregnancy after transferring these partially survived cleavage embryos cannot be expected to be high. The third reason why the outcomes of post-thaw embryos in early embryonic stages are inferior is the difficulty in determining viability and whether a determination can guide the embryo thaw strategy. Unlike embryo survival, some cryopreservation-caused injuries are not detectable by morphological assessment with a light microscope even though embryos or blastomeres appear intact after thaw (20). These injuries may be related to the low potential for further development. Taking this into consideration, plus poor embryo selection in early embryonic stages and possible blastomere lysis during thawing, post-thaw embryo viability prediction is very difficult. Although the difficulty can be overcome by culturing embryos overnight or longer to verify further embryo development, it would be too late to thaw 116
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additional embryos when necessary because of the concern of the synchronization between the endometrium and embryos. In practice, an insufficient number of good-quality embryos for transfer is not only the main reason for the poor FET outcomes, it even causes about a 10% cancellation rate in FET cycles (4 – 6). In summary, transferring post-thaw embryos with a good chance of achieving a pregnancy requires [1] freezing embryos at a late embryonic stage for a better embryo selection; [2] achieving a higher post-thaw survival rate with a low incidence of blastomere damage; and [3] determining the viability of post-thaw embryos at the time of thaw, rather than day(s) later. To achieve a higher success rate after FET, the above three conditions have to be met. Apparently, pronucleate- and cleavage-stage embryo freezing are not the optimal stages for achieving high clinical success rates. Other stages possible for freezing are the morula (mostly on day 4) and blastocyst (day 5 or 6) stages. Expanded blastocyst is the last embryonic stage before implantation and provides the best embryo selection because of a well-defined inner-cell mass. There have been many studies concerning the freezing of blastocysts, although many of them have been conflicting. However, recent reports seem promising (38, 39). Nevertheless, only a limited number of patients have excess blastocysts available for freezing (3, 15, 16). Morula-stage ET and freezing have not been used in human IVF programs. A recent study showed that there was a ⬃45% implantation rate for day 4 embryos after fresh morula ET (22), which is compatible with the reported blastocyst transfer, although no direct comparison can be drawn. The data presented in this study seem to support morula-stage freezing as another option in IVF practice because of the acceptable post-thaw survival rate, high implantation rate, and feasibility of viability determination. The 57.7% clinical pregnancy rate and 36.2% implantation rate after transferring of post-thawed embryos show a potential application. In addition, multiple pregnancies, a largely undesirable outcome, can be reduced by transferring a lower number of morula embryos. It was noticed in this study that blastomere separation and recompaction are highly correlated with environmental osmolality. After embryos are retrieved from cryovials during thawing, more embryos had blastomere separation compared with the time of loading. This indicates that blastomere separation continues after loading since the cryomedium osmolality still increases after seeding. In contrast, recompaction and blastulation occurred in the thawing process as the medium osmolality decreased. Embryos that show morphological recovery in thawing or shortly after thawing seem to have a better growth potential than those that failed to demonstrate recovery changes because the latter usually arrested or compacted poorly in overnight culture. Vol. 82, No. 1, July 2004
Within the three subgroups of morula embryos, the preference appears to be to freeze embryos at the compact morula or late morula stages although there is no significant difference in post-thaw survival rates from the early morula stage. This is because after reaching full compaction embryos provide information on the proportion of blastomeres undergoing compaction and the morphology of compacted embryos. In embryos at the early morula stage, it is uncertain what percentage of blastomeres will eventually compact and how well blastomeres will compact since it is not unusual that a large proportion of blastomeres undergo compaction but form irregularly shaped profiles. Moreover, compact morula and late morula embryos are more likely to recompact or even blastulate during the thawing process, which can immediately provide information regarding whether a good post-thaw embryo has been achieved or whether more embryos need to be thawed. Recompaction or blastulation is especially beneficial for embryo selection in patients who do not have good-quality embryos but who do have inferior-quality ones. In this population, embryos may have low cell number, unevenness of blastomeres, more than 25% fragments, or atypical compaction. The capability of recompaction and reblastulation seems to provide another opportunity for embryo grading, which helps in selecting relatively superior embryos from inferior ones. Freezing excess embryos before a fresh ET procedure is popular in current IVF practices. Routinely, the best-quality embryos are transferred in stimulated cycles to give patients the best opportunity for achieving pregnancy and avoid the possibility of losing good-quality embryos in the freezing and thawing process. Two dilemmas are frequently encountered. The first is how many embryos should be reserved for fresh ET since ET is conducted day(s) later and the embryo development is uncertain. Without reserving a sufficient number of early-stage embryos in the incubators, it is possible to end up with an insufficient number of good embryos on the day of the fresh ET. For day 5 ET, it is usually recommended that 4 –5 good day 3 embryos be reserved, even though only two blastocysts are desired to be transferred (40 – 42). It is more difficult to decide the number of embryos to be reserved if embryo freezing is performed at the pronucleate or 4-cell stages. The second dilemma occurs when more good-quality embryos than are needed are produced on the transfer day, when the majority of reserved embryos grow well, or when too many embryos are reserved. When this happens, a second embryo freezing has to take place and embryos are then frozen at a different embryonic stage, which may be not a preferred stage for that clinic. Cryopreservation of embryos at the morula stage seems to eliminate these dilemmas since embryo freezing can be conducted after a fresh ET, which ensures superior embryo selection for fresh ETs and there is FERTILITY & STERILITY威
no concern about either too many or an insufficient number of good embryos available for fresh ET. In conclusion, morula embryo freezing and thawing can be successfully applied in human assisted reproductive practice. When freezing embryos at the morula stage, not only can an acceptable FET outcome be achieved, but it can also significantly simplify some clinical decisions regarding the number of embryos that should be reserved for a fresh transfer and the number of embryos that should be thawed in FET cycles. References 1. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983;305:707–9. 2. Quinn P. Success of oocyte and embryo freezing and its effect on outcome with in vitro fertilization. Semin Reprod Endocrinol 1990;8: 272–80. 3. Demoulin A, Jouan C, Gerday C, Dubois M. Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum Reprod 1991; 6:799 –804. 4. 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 Steril 1993;59:1202–7. 5. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rates after early human embryo freezing. Fertil Steril 1986;46:268 –72. 6. Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3):161–74. 7. Cohen J, Simons RS, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fertil Embr Trans 1985;2:59 –64. 8. Quinn P, Kerin J. Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques. J In Vitro Fertil Embr Trans 1986;3:40 –5. 9. Miller KF, Goldberg JM. In vitro development and implantation rates of fresh and cryopreserved sibling zygotes. Obstet Gynecol 1995;85: 999 –1002. 10. 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;2:709 –15. 11. Edgar DH, Bourne H, Spiers AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000;15:175–9. 12. El-Toukhy T, Khalf Y, Al-Darazi K, Andritsos V, Taylor A, Braude P. Effect of blastomere loss on the outcome of frozen embryo replacement cycles. Fertil Steril 2003;79:1106 –11. 13. Troup SA, Matson PL, Critchlow JD, Morroll DR, Lieberman BA, Burslem RW. Cryopreservation of human embryos at the pronucleate, early cleavage, or expanded blastocyst stages. Eur J Obst Gyn Reprod Biol 1990;38:133–9. 14. Pantos K, Stefanidis K, Pappas K, Kokkinopoulos P, Petroutsou K, Kokkali G, et al. Cryopreservation of embryos, blastocysts and pregnancy rates of blastocysts derived from frozen-thawed embryos and frozen-thawed blastocysts. 2001;18:579 – 82. 15. Alper MM, Brinsden P, Fischer R, Wikland M. To blastocyst or not to blastocyst? That is the question. Hum Reprod 2001;16:617–9. 16. Blake D, Proctor M, Johnson N, Olive D. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Sys Rev 2002;2:CD002118. 17. Pugh PA, Ankersmit AE, McGowan LT, Tervit HR. Cryopreservation of in vitro–produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on postthaw survival. Theriogenology 1998;50:495–506. 18. Massip A. Cryopreservation of embryos of farm animals. Reprod Dom Anim 2001;36:49 –55. 19. Tao J, Tamis R, Fink K. Pregnancies achieved after transferring frozen morula/compact stage embryos. Fertil Steril 2001;75:629 –31. 20. Cocero MJ, Lopez-Sebastian A, Barragan ML, Picazo RA. Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol. Cryobiology 1996;33:502–7. 21. Tao J, Tamis R, Fink K. Cryopreservation of mouse embryos at morula/ compact stage. J Assist Reprod Genet 2001;18:235–43.
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22. Tao J, Tamis R, Fink K. The neglected morula/compact stage embryo transfer. Hum Reprod 2002;17:1513–8. 23. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil Steril 1985;44:645–51. 24. Nikolettos N, Al-Hasani S. Frozen pronuclear oocytes: advantages for the patient. Mol Cell Endocrinol 2000;27:169:55– 62. 25. Gorrill MJ, Kaplan PF, Patton PE, Burry KA. Initial experience with extended culture and blastocyst transfer of cryopreserved embryos. Am J Obstet Gynecol 1999;180:1472–4. 26. Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P, Van Steirteghem AC. Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 1997;12:1513–21. 27. Selick CE, Hofmann GE, Albano C, Horowitz GM, Copperman AB, Garrisi GJ, et al. Embryo quality and pregnancy potential of fresh compared with frozen embryos—is freezing detrimental to high quality embryos? Hum Reprod 1995;10:392–5. 28. Senn A, Vozzi C, Chanson A, De Grandi P, Germond M. Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage. Fertil Steril 2000;74:946 –52. 29. Virutamasen P, Boonkasemsanti W, Suwajanakorn S, Kosalanant V, Chaiyaput R, Parksamoot W. Pregnancy following transfer of frozenthawed embryo: a preliminary report. J Med Assoc Thai 1992;75:153–6. 30. Fugger EF, Bustillo M, Dorfmann AD, Schulman JD. Human preimplantation embryo cryopreservation: selected aspects. Hum Reprod 1991;6:131–5. 31. Freemann L, Trounson A, Kirby C. Cryopreservation of human embryos: progress on the clinical use of the technique in human in vitro fertilization. J In Vitro Fertil Embr Trans 1986;3:53–61. 32. Kattera S, Shrivastav P, Craft I. Comparison of pregnancy outcome of pronuclear- and multicellular-stage frozen-thawed embryo transfers. J Assist Reprod Genet 1999;16:358 –62.
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