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compact stage embryos
FERTILITY AND STERILITY威 VOL. 75, NO. 3, MARCH 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Pregnancies achieved after transferring frozen morula/compact stage embryos Jun Tao, Ph.D., Robert Tamis, M.D., and Katharine Fink, A.A. Arizona Institute of Reproductive Medicine, Phoenix, Arizona
Objective: To report two cases of frozen embryo transfers in which embryos were frozen at the morula/ compact stage and pregnancies were achieved after transfer. Design: Case report. Setting: Private assisted reproductive program. Patient(s): Two women had transfer of embryos that were frozen at the morula/compact stage. Intervention(s): Human morula/compact embryos were cryopreserved and transferred after subsequent thaw. Main Outcome Measure(s): Embryo survival after thawing and subsequent pregnancy outcome. Result(s): Three and five compact embryos were frozen on day 4 for two patients, respectively. In the first case, all three embryos survived after thawing, and all were transferred. In the second patient, three of five embryos survived after thawing, and those three surviving embryos were transferred. Pregnancies were achieved in both patients. The first woman became pregnant with twins and delivered two girls weighing 2,270 g and 2,071 g, respectively. The second patient became pregnant with a singleton and delivered a boy weighing 2,837 g. Conclusion(s): Human embryos can be frozen and thawed in the morula/compact stage and achieve normal pregnancy. Advantages of embryo freezing/thawing at the morula/compact stage include the following: [1] compared with earlier embryonic stage freezing, morula/compact-stage embryos provide better embryo selection and [2] it is easier and safer to perform assisted hatching on compact-stage embryos when compared with those from other preimplantation stages. (Fertil Steril威 2001;75:629 –31. ©2001 by American Society for Reproductive Medicine.) Key Words: Morula/compact embryo, freezing and thawing, frozen embryo transfer.
Embryo freezing and thawing has been applied to human embryos for many years. However, embryo survival rates after thawing and pregnancy rates after transfer are still suboptimal. The key issues include selecting embryos with good potential before freezing and successfully freezing and thawing the embryos.
Received May 16, 2000; revised and accepted August 30, 2000. Reprint requests: Jun Tao, Ph.D., 5333 West Mercury Place, Chandler, Arizona 85226 (FAX: 480-897-1283; E-mail: [email protected]). 0015-0282/01/$20.00 PII S0015-0282(00)01751-9
Embryonic cleavage from the pronuclear to the eight-cell stage does not provide an accurate prognosis for future development, which may be partially due to the embryonic genome not being activated. Blastocyst stage embryos do provide better selection; however, achieving good results after freezing and thawing currently is still a challenge. In addition, assisted hatching, which is a widely applied procedure before embryo transfer and may also be of benefit for “poor” blastocysts, is not easy and safe to perform on the blastocyst embryos because of the reduced or absent perivitelline space.
This article reports two cases in which embryos were frozen at the morula/compact stage; in both cases, assisted hatching was performed before transfer.
CASE REPORT The first patient was a 32-year-old woman with 12 years of infertility and a diagnosis of premature menopause. Therefore, donor oocytes were used for this patient and two fresh and one frozen embryo transfer occurred before this cycle. Both fresh transfers achieved clinical pregnancy, but one had no fetal heartbeat, and the other miscarried 4 weeks after transfer. The second woman was a 37-year-old who had received a diagnosis of mild endometriosis. Her partner had a failed vasectomy reversal. This patient delivered twin boys after therapeutic donor insemination (TDI) 5 years ago. However, since then, she had had five failed TDI cycles. 629
In the egg donor for the first patient and for the second patient, ovarian stimulation was started with ovarian suppression. Leuprolide acetate (Lupron, 0.5 mg/day; TAP Pharmaceuticals, Deerfield, IL) was administered in the midluteal phase. After confirmation of adequate ovarian suppression by transvaginal ultrasound (no ovarian cysts ⬎10 mm) and adequate serum E2 level (⬍50 pg/mL), the leuprolide acetate dose was discontinued in the donor and was decreased to 0.25 mg in the patient and continued until the hCG trigger. Highly purified FSH (Fertinex; Serono Laboratories, Norwell, MA) was used for gonadotropin therapy at a dosage of 6 ampules per day for the patient and 4 ampules per day for the egg donor. Monitoring of follicular development and serum E2 levels was initiated on the fourth day of gonadotropin therapy. Ovulation was induced by 5,000 IU of hCG (Profasi; Serono), and transvaginal oocyte retrieval was performed 36 hours later. After oocyte retrieval, all mature oocytes were inseminated by ICSI. Embryo culture was performed using P1 medium (Irvine, CA) supplemented with 10% Serum Substitute Supplement (SSS; Irvine, CA) under mineral oil (Sigma, St. Louis, MO) and in conditions of 37°C and 5% CO2. To achieve better embryo selection, embryo transfers were performed on day 4 after embryos reached the morula/ compact stage. The grading of morula/compact embryo was based on the following criteria: grade 4 was the best-quality embryo, in which all blastomeres underwent the compacting process and formed a sphere-shaped, compacted cell mass. Grade 3 embryo was one in which embryos had about two thirds to three quarters of their blastomeres undergo the compact process. Grade 3 embryos could have ⬍25% fragments. Grade 2 embryos were those in which only about half of the blastomeres underwent the compacting process and formed a small, compacted cell mass that filled half of the zona; they could contain 25%–50% fragments. Grade 1 embryos were the lowest quality, in which only a less than one-third–sized compacted cell mass was formed and there might be ⬎50% fragments. Embryos graded as 4 and 3 were considered good embryos, and those graded as 2 and 1 were considered poor embryos. In both cases, three grade 3 embryos were selected for fresh embryo transfer. Mechanical assisted hatching was applied about 30 minutes before the transfer. Briefly, the embryo was secured onto a holding pipette at the 9-o’clock position, and the zona pellucida was pierced by a microneedle at the 1- or 5-o’clock position. The microneedle kept moving forward and passed through the other side of the zona pellucida. Then the embryo was released from the holding pipette, and the microneedle holding a portion of the zona pellucida was rubbed against the holding pipette until the zona pellucida was opened. Pregnancy was achieved in the first patient, but spontaneous abortion occurred at 4 weeks after transfer. No pregnancy was achieved in the second patient. 630
Tao et al.
Morula/compact embryo freeze and transfer
There were three (all graded as 3) and five (one graded as 3 and four graded as 2) compacted embryos remaining for patients 1 and 2, respectively. Because of the concerns regarding the capability of the remaining embryos to reach the blastocyst stage, we chose to freeze these embryos right after the fresh embryo transfer. Because these patients were not part of a clinical trial, institutional review board approval was not attempted. On the basis of the embryo qualities, we believed that freezing the remaining embryos after the fresh transfers provided the best benefit for these patients. The freezing process was modified from the method of Lasalle et al. (1). Embryos were first transferred to a HEPESbuffered human tubal fluid (HTF) medium (HTF/HEPES) supplemented with 10% SSS at room temperature for 5 minutes. Then embryos were moved to an HTF/HEPES medium containing 0.5 M 1,2-propanediol (Sigma)/0.05 M sucrose (Sigma) for 5 minutes, then transferred to a medium with 1.0 M 1,2-propandiol/0.075 M sucrose for another 5 minutes. Finally, the embryos were transferred to an HTF/ HEPES medium with 1.5 M 1,2-propandiol/0.1 M sucrose and were loaded into 0.25-mL straws (TS Scientific, Perkasie, PA). A freezing process was performed in a Cryogenesis freezer (model CL-863, Biogenics, Napa, CA). A standard procedure was applied; in other words, °C/min⫺1 from 18°C to ⫺7.5°C, followed by seeding and holding for 10 minutes. Then the temperature was lowered to ⫺30°C at the rate of 0.3°C/min⫺1. From that point, the cooling rate was 10°C/min⫺1 to ⫺140°C. The embryos were then transferred to liquid nitrogen. Four and 2 months, respectively, after failed fresh-embryo transfer, both patients went through our frozen-embryo transfer program. Briefly, leuprolide acetate (1 mg) was administered in the midluteal phase until the following cycle day 14. On cycle day 1, Estrace (1 mg/b.i.d.) was started and given until day 8 and then replaced by Estradiol Valerate (5 mg i.m. twice weekly, SCHEIN, Phoenix, AZ). In the evening of cycle day 15, 25 mg of progesterone in oil was given i.m.; on cycle day 16, 50 mg was given i.m., and thereafter, that dosage was given b.i.d. Estradiol Valerate was increased to 10 mg i.m. on cycle day 18. Embryo transfers were performed on cycle day 19. For the first patient, there was no leuprolide acetate administration, and Estradiol Valerate (5 mg i.m.) was started on cycle day 1. Thawing was conducted the morning of transfer. The straws were first taken out of the liquid nitrogen and set at room temperature for 30 – 40 seconds in a horizontal position. Then, the straws were transferred to a 30°C water bath for about 15–20 seconds. After release from the straw, embryos were first moved to an HTF/HEPES medium containing 1.0 M 1,2-propandiol/0.2 M sucrose for 5 minutes, followed by a medium with 0.5 M 1,2-propandiol/0.15 M sucrose for 5 minutes. Finally, the embryos were transferred to a medium with 0.1 M sucrose for another 5 minutes. After rinsing two to three times in plain HTF/HEPES medium with Vol. 75, No. 3, March 2001
10% SSS, embryos were cultured in P1 medium. For the first patient, all three embryos survived and were all still graded as 3. In the second patient, three out of five embryos survived; they were graded as 2, 2, and 3. About 30 minutes before embryo transfer, embryos were moved to HTF/HEPES medium with 10% SSS. Mechanical zona opening, as described above, was performed on all embryos to be transferred. Embryos were then moved back to P1 medium until transfer. Both patients achieved pregnancy. A positive level of -hCG (147 IU/L) was observed in the first woman 10 days after transfer and increased 2 days later (367 IU/L). Transvaginal ultrasound showed two gestational sacs and two fetal heartbeats 4 weeks after transfer. Two girls, one weighing 2,270 g and the other weighing 2,071 g, were delivered by cesarean section at 34 weeks of gestation. The second patient had a positive -hCG (84 IU/L) 10 days after transfer that increased 2 days later (130 IU/L). One gestational sac with a heartbeat was observed under transvaginal ultrasound 4 weeks after transfer. At 35 weeks of gestation, a boy weighing 2,837 g was delivered vaginally.
DISCUSSION Embryos are routinely frozen at the pronuclear stage (day 1), at the two-cell to eight-cell stages (day 2 and day 3), and at the expanded blastocyst stage (day 5 or day 6). The morula, or compact-stage, embryo (day 4) seems not to be considered to be at an optimal stage for freezing. After an extensive literature search using the MEDLINE database, we failed to identify any other cases in which human embryos at the morula/compact stage were frozen and transferred. To our best knowledge, this is the first report of freezing human embryos at the morula/compact stage and of pregnancies achieved after transfer. For these two patients, remaining embryos were frozen the same day after the fresh embryo transfer, that gave the lab the opportunity to choose the best embryos for the transfer. There are several reasons why blastocyst freezing was not considered: [1] the capability of further development to the expanded blastocysts was a concern because usually only a limited number of embryos reach that stage and [2] postthaw blastocyst survival was a concern, though good pregnancy rates had been reported when blastocyst survived cryopreservation. Before these two cases, we had three similar cases. One was a patient aged 43 years and had four frozen embryos, one graded as 3 and the others as 2. The second patient was aged 48 years and had five frozen embryos, one graded as 3, two as 2, and two as 1. In these two cases, all embryos survived the freezing and thawing process, but no pregnancy was achieved after transfer. Poor prefreezing embryo quality might have accounted for the failed pregnancy. The third patient was 38 years of age and had five frozen embryos, of which two were graded as 3, two as 2, and one as 1. Four of the five embryos survived the FERTILITY & STERILITY威
freezing procedure, but again, no pregnancy was achieved after transfer. Our early animal experiments showed that mouse fully compacted and early blastocyst stage embryos had very high postthaw survival rates and that the majority of them reached the expanded blastocyst stage. After embryo transfer, normal offspring were delivered (unpublished observations). Compared with earlier embryonic stages, from pronuclear to eight-cell stage, morula/compact-stage embryos should allow better embryo selection. Though blastocyst stage embryos can provide even better embryo selection compared with morula/compactstage embryos, it was not our intention to transfer embryos at blastocyst stage. The reasons for choosing the morula/compact stage rather than the blastocyst stage are that [1] we prefer to perform assisted hatching on all embryos transferred; [2] at the blastocyst stage, embryos may be more vulnerable to injury during the transfer process; and [3] extended culture requires more labor and incubator space. Many investigators have reported that assisted hatching improves implantation rates (2– 4). The value of assisted hatching is debatable for blastocyst transfer. For healthy, fully expanded blastocyst embryos, assisted hatching may not be necessary for a natural hatching process. However, the reality is that there are always some patients in the population who have only relatively low-quality blastocysts. These poor blastocysts may expand slightly but be unable to complete a normal hatching process. Those blastocysts are usually trapped and finally collapse inside the zona pellucida. Assisted hatching may benefit these embryos. However, the risk of performing assisted hatching on blastocyst-stage embryos is of concern because of the reduced perivitelline space. Performing assisted hatching at this stage is very risky, especially if an acid Tyrode’s solution is used. In contrast, the morula/compact stage may be the safest stage at which to perform the assisted-hatching procedure. This is not only because morula/compact-stage embryos have established extensive cell junctions, which excludes the probability of losing an individual blastomere, but it is also because this is the stage in which the largest perivitelline space can be observed during the entire preimplantation process. Further experience will be necessary to define whether transfer of morulae after assisted hatching may be advantageous for an optimized and efficient cryopreservation program. References 1. 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. 2. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairment of the hatching process following in vitro fertilization in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5:7–13. 3. Check JH, Hoover L, Nazari A, O’Shaughnessy A, Summers D. The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil Steril 1996;65:254 –7. 4. Tao J, Tamis R. Application of assisted hatching for 2-day-old, frozenthawed embryo transfer in a poor-prognosis population. J Assist Reprod Genet 1997;14:128 –30.
631
Pregnancies achieved after transferring frozen morula/compact stage embryos Jun Tao, Ph.D., Robert Tamis, M.D., and Katharine Fink, A.A. Arizona Institute of Reproductive Medicine, Phoenix, Arizona
Objective: To report two cases of frozen embryo transfers in which embryos were frozen at the morula/ compact stage and pregnancies were achieved after transfer. Design: Case report. Setting: Private assisted reproductive program. Patient(s): Two women had transfer of embryos that were frozen at the morula/compact stage. Intervention(s): Human morula/compact embryos were cryopreserved and transferred after subsequent thaw. Main Outcome Measure(s): Embryo survival after thawing and subsequent pregnancy outcome. Result(s): Three and five compact embryos were frozen on day 4 for two patients, respectively. In the first case, all three embryos survived after thawing, and all were transferred. In the second patient, three of five embryos survived after thawing, and those three surviving embryos were transferred. Pregnancies were achieved in both patients. The first woman became pregnant with twins and delivered two girls weighing 2,270 g and 2,071 g, respectively. The second patient became pregnant with a singleton and delivered a boy weighing 2,837 g. Conclusion(s): Human embryos can be frozen and thawed in the morula/compact stage and achieve normal pregnancy. Advantages of embryo freezing/thawing at the morula/compact stage include the following: [1] compared with earlier embryonic stage freezing, morula/compact-stage embryos provide better embryo selection and [2] it is easier and safer to perform assisted hatching on compact-stage embryos when compared with those from other preimplantation stages. (Fertil Steril威 2001;75:629 –31. ©2001 by American Society for Reproductive Medicine.) Key Words: Morula/compact embryo, freezing and thawing, frozen embryo transfer.
Embryo freezing and thawing has been applied to human embryos for many years. However, embryo survival rates after thawing and pregnancy rates after transfer are still suboptimal. The key issues include selecting embryos with good potential before freezing and successfully freezing and thawing the embryos.
Received May 16, 2000; revised and accepted August 30, 2000. Reprint requests: Jun Tao, Ph.D., 5333 West Mercury Place, Chandler, Arizona 85226 (FAX: 480-897-1283; E-mail: [email protected]). 0015-0282/01/$20.00 PII S0015-0282(00)01751-9
Embryonic cleavage from the pronuclear to the eight-cell stage does not provide an accurate prognosis for future development, which may be partially due to the embryonic genome not being activated. Blastocyst stage embryos do provide better selection; however, achieving good results after freezing and thawing currently is still a challenge. In addition, assisted hatching, which is a widely applied procedure before embryo transfer and may also be of benefit for “poor” blastocysts, is not easy and safe to perform on the blastocyst embryos because of the reduced or absent perivitelline space.
This article reports two cases in which embryos were frozen at the morula/compact stage; in both cases, assisted hatching was performed before transfer.
CASE REPORT The first patient was a 32-year-old woman with 12 years of infertility and a diagnosis of premature menopause. Therefore, donor oocytes were used for this patient and two fresh and one frozen embryo transfer occurred before this cycle. Both fresh transfers achieved clinical pregnancy, but one had no fetal heartbeat, and the other miscarried 4 weeks after transfer. The second woman was a 37-year-old who had received a diagnosis of mild endometriosis. Her partner had a failed vasectomy reversal. This patient delivered twin boys after therapeutic donor insemination (TDI) 5 years ago. However, since then, she had had five failed TDI cycles. 629
In the egg donor for the first patient and for the second patient, ovarian stimulation was started with ovarian suppression. Leuprolide acetate (Lupron, 0.5 mg/day; TAP Pharmaceuticals, Deerfield, IL) was administered in the midluteal phase. After confirmation of adequate ovarian suppression by transvaginal ultrasound (no ovarian cysts ⬎10 mm) and adequate serum E2 level (⬍50 pg/mL), the leuprolide acetate dose was discontinued in the donor and was decreased to 0.25 mg in the patient and continued until the hCG trigger. Highly purified FSH (Fertinex; Serono Laboratories, Norwell, MA) was used for gonadotropin therapy at a dosage of 6 ampules per day for the patient and 4 ampules per day for the egg donor. Monitoring of follicular development and serum E2 levels was initiated on the fourth day of gonadotropin therapy. Ovulation was induced by 5,000 IU of hCG (Profasi; Serono), and transvaginal oocyte retrieval was performed 36 hours later. After oocyte retrieval, all mature oocytes were inseminated by ICSI. Embryo culture was performed using P1 medium (Irvine, CA) supplemented with 10% Serum Substitute Supplement (SSS; Irvine, CA) under mineral oil (Sigma, St. Louis, MO) and in conditions of 37°C and 5% CO2. To achieve better embryo selection, embryo transfers were performed on day 4 after embryos reached the morula/ compact stage. The grading of morula/compact embryo was based on the following criteria: grade 4 was the best-quality embryo, in which all blastomeres underwent the compacting process and formed a sphere-shaped, compacted cell mass. Grade 3 embryo was one in which embryos had about two thirds to three quarters of their blastomeres undergo the compact process. Grade 3 embryos could have ⬍25% fragments. Grade 2 embryos were those in which only about half of the blastomeres underwent the compacting process and formed a small, compacted cell mass that filled half of the zona; they could contain 25%–50% fragments. Grade 1 embryos were the lowest quality, in which only a less than one-third–sized compacted cell mass was formed and there might be ⬎50% fragments. Embryos graded as 4 and 3 were considered good embryos, and those graded as 2 and 1 were considered poor embryos. In both cases, three grade 3 embryos were selected for fresh embryo transfer. Mechanical assisted hatching was applied about 30 minutes before the transfer. Briefly, the embryo was secured onto a holding pipette at the 9-o’clock position, and the zona pellucida was pierced by a microneedle at the 1- or 5-o’clock position. The microneedle kept moving forward and passed through the other side of the zona pellucida. Then the embryo was released from the holding pipette, and the microneedle holding a portion of the zona pellucida was rubbed against the holding pipette until the zona pellucida was opened. Pregnancy was achieved in the first patient, but spontaneous abortion occurred at 4 weeks after transfer. No pregnancy was achieved in the second patient. 630
Tao et al.
Morula/compact embryo freeze and transfer
There were three (all graded as 3) and five (one graded as 3 and four graded as 2) compacted embryos remaining for patients 1 and 2, respectively. Because of the concerns regarding the capability of the remaining embryos to reach the blastocyst stage, we chose to freeze these embryos right after the fresh embryo transfer. Because these patients were not part of a clinical trial, institutional review board approval was not attempted. On the basis of the embryo qualities, we believed that freezing the remaining embryos after the fresh transfers provided the best benefit for these patients. The freezing process was modified from the method of Lasalle et al. (1). Embryos were first transferred to a HEPESbuffered human tubal fluid (HTF) medium (HTF/HEPES) supplemented with 10% SSS at room temperature for 5 minutes. Then embryos were moved to an HTF/HEPES medium containing 0.5 M 1,2-propanediol (Sigma)/0.05 M sucrose (Sigma) for 5 minutes, then transferred to a medium with 1.0 M 1,2-propandiol/0.075 M sucrose for another 5 minutes. Finally, the embryos were transferred to an HTF/ HEPES medium with 1.5 M 1,2-propandiol/0.1 M sucrose and were loaded into 0.25-mL straws (TS Scientific, Perkasie, PA). A freezing process was performed in a Cryogenesis freezer (model CL-863, Biogenics, Napa, CA). A standard procedure was applied; in other words, °C/min⫺1 from 18°C to ⫺7.5°C, followed by seeding and holding for 10 minutes. Then the temperature was lowered to ⫺30°C at the rate of 0.3°C/min⫺1. From that point, the cooling rate was 10°C/min⫺1 to ⫺140°C. The embryos were then transferred to liquid nitrogen. Four and 2 months, respectively, after failed fresh-embryo transfer, both patients went through our frozen-embryo transfer program. Briefly, leuprolide acetate (1 mg) was administered in the midluteal phase until the following cycle day 14. On cycle day 1, Estrace (1 mg/b.i.d.) was started and given until day 8 and then replaced by Estradiol Valerate (5 mg i.m. twice weekly, SCHEIN, Phoenix, AZ). In the evening of cycle day 15, 25 mg of progesterone in oil was given i.m.; on cycle day 16, 50 mg was given i.m., and thereafter, that dosage was given b.i.d. Estradiol Valerate was increased to 10 mg i.m. on cycle day 18. Embryo transfers were performed on cycle day 19. For the first patient, there was no leuprolide acetate administration, and Estradiol Valerate (5 mg i.m.) was started on cycle day 1. Thawing was conducted the morning of transfer. The straws were first taken out of the liquid nitrogen and set at room temperature for 30 – 40 seconds in a horizontal position. Then, the straws were transferred to a 30°C water bath for about 15–20 seconds. After release from the straw, embryos were first moved to an HTF/HEPES medium containing 1.0 M 1,2-propandiol/0.2 M sucrose for 5 minutes, followed by a medium with 0.5 M 1,2-propandiol/0.15 M sucrose for 5 minutes. Finally, the embryos were transferred to a medium with 0.1 M sucrose for another 5 minutes. After rinsing two to three times in plain HTF/HEPES medium with Vol. 75, No. 3, March 2001
10% SSS, embryos were cultured in P1 medium. For the first patient, all three embryos survived and were all still graded as 3. In the second patient, three out of five embryos survived; they were graded as 2, 2, and 3. About 30 minutes before embryo transfer, embryos were moved to HTF/HEPES medium with 10% SSS. Mechanical zona opening, as described above, was performed on all embryos to be transferred. Embryos were then moved back to P1 medium until transfer. Both patients achieved pregnancy. A positive level of -hCG (147 IU/L) was observed in the first woman 10 days after transfer and increased 2 days later (367 IU/L). Transvaginal ultrasound showed two gestational sacs and two fetal heartbeats 4 weeks after transfer. Two girls, one weighing 2,270 g and the other weighing 2,071 g, were delivered by cesarean section at 34 weeks of gestation. The second patient had a positive -hCG (84 IU/L) 10 days after transfer that increased 2 days later (130 IU/L). One gestational sac with a heartbeat was observed under transvaginal ultrasound 4 weeks after transfer. At 35 weeks of gestation, a boy weighing 2,837 g was delivered vaginally.
DISCUSSION Embryos are routinely frozen at the pronuclear stage (day 1), at the two-cell to eight-cell stages (day 2 and day 3), and at the expanded blastocyst stage (day 5 or day 6). The morula, or compact-stage, embryo (day 4) seems not to be considered to be at an optimal stage for freezing. After an extensive literature search using the MEDLINE database, we failed to identify any other cases in which human embryos at the morula/compact stage were frozen and transferred. To our best knowledge, this is the first report of freezing human embryos at the morula/compact stage and of pregnancies achieved after transfer. For these two patients, remaining embryos were frozen the same day after the fresh embryo transfer, that gave the lab the opportunity to choose the best embryos for the transfer. There are several reasons why blastocyst freezing was not considered: [1] the capability of further development to the expanded blastocysts was a concern because usually only a limited number of embryos reach that stage and [2] postthaw blastocyst survival was a concern, though good pregnancy rates had been reported when blastocyst survived cryopreservation. Before these two cases, we had three similar cases. One was a patient aged 43 years and had four frozen embryos, one graded as 3 and the others as 2. The second patient was aged 48 years and had five frozen embryos, one graded as 3, two as 2, and two as 1. In these two cases, all embryos survived the freezing and thawing process, but no pregnancy was achieved after transfer. Poor prefreezing embryo quality might have accounted for the failed pregnancy. The third patient was 38 years of age and had five frozen embryos, of which two were graded as 3, two as 2, and one as 1. Four of the five embryos survived the FERTILITY & STERILITY威
freezing procedure, but again, no pregnancy was achieved after transfer. Our early animal experiments showed that mouse fully compacted and early blastocyst stage embryos had very high postthaw survival rates and that the majority of them reached the expanded blastocyst stage. After embryo transfer, normal offspring were delivered (unpublished observations). Compared with earlier embryonic stages, from pronuclear to eight-cell stage, morula/compact-stage embryos should allow better embryo selection. Though blastocyst stage embryos can provide even better embryo selection compared with morula/compactstage embryos, it was not our intention to transfer embryos at blastocyst stage. The reasons for choosing the morula/compact stage rather than the blastocyst stage are that [1] we prefer to perform assisted hatching on all embryos transferred; [2] at the blastocyst stage, embryos may be more vulnerable to injury during the transfer process; and [3] extended culture requires more labor and incubator space. Many investigators have reported that assisted hatching improves implantation rates (2– 4). The value of assisted hatching is debatable for blastocyst transfer. For healthy, fully expanded blastocyst embryos, assisted hatching may not be necessary for a natural hatching process. However, the reality is that there are always some patients in the population who have only relatively low-quality blastocysts. These poor blastocysts may expand slightly but be unable to complete a normal hatching process. Those blastocysts are usually trapped and finally collapse inside the zona pellucida. Assisted hatching may benefit these embryos. However, the risk of performing assisted hatching on blastocyst-stage embryos is of concern because of the reduced perivitelline space. Performing assisted hatching at this stage is very risky, especially if an acid Tyrode’s solution is used. In contrast, the morula/compact stage may be the safest stage at which to perform the assisted-hatching procedure. This is not only because morula/compact-stage embryos have established extensive cell junctions, which excludes the probability of losing an individual blastomere, but it is also because this is the stage in which the largest perivitelline space can be observed during the entire preimplantation process. Further experience will be necessary to define whether transfer of morulae after assisted hatching may be advantageous for an optimized and efficient cryopreservation program. References 1. 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. 2. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairment of the hatching process following in vitro fertilization in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5:7–13. 3. Check JH, Hoover L, Nazari A, O’Shaughnessy A, Summers D. The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil Steril 1996;65:254 –7. 4. Tao J, Tamis R. Application of assisted hatching for 2-day-old, frozenthawed embryo transfer in a poor-prognosis population. J Assist Reprod Genet 1997;14:128 –30.
631