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Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles*
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Aisilt"d reproductive fecl,no.logy FERTILITY AND STERILITY Copyright
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Vol. 64, No.6, December 1995
1995 American Society for Reproductive Medicine
Printed on acid-free paper in U. S. A.
Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles*
Robert A. Kaufmann, M.D.t:J: Yves Menezo, Ph.D.t§ Andre Hazout, M.D. II
Bernard Nicollet, M.D.§ Martine DuMont, Ph.D.11 Edouard J. Servy, M.D.t
Augusta Reproductive Biology Associates, Augusta, Georgia; Institut Rhonalpin Fondation Merieux, Lyon; and Clinique Pierre Cherest, Paris, France
Objective: To present our experience using cocultured cryopreserved and transferred blastocysts. Design: Retrospective study of patients undergoing transfer of cryopreserved blastocysts. Setting: Three different IVF centers. Patients: Four hundred sixty-seven thawed cycles from January 1991 to June 1994. Main Outcome Measure: Pregnancy rate per cycle after transfer of pre-embryos developed from thawed blastocysts. Results: One thousand two hundred thirty-nine blastocysts were thawed. Of these, 1,033 (83%) survived thawing and were transferred. Five hundred sixty-three thawed cycles resulted in 516 (92%) receiving intrauterine transfer. One hundred twelve clinical pregnancies were established, resulting in a 21.7% pregnancy per transfer with a 19% ongoing rate. The implantation rate of 13.4% results from 138 implanted pre-embryos. There was a higher PR in the programmed cycle (79/302; 26.2%) compared with the natural cycle (6/47;13%). Conclusions: Freezing at the blastocyst stage is a proven and reliable method in IVF technology. Although there may be fewer pre-embryos, their ability to implant appears to approach the potential of a fresh transfer. Fertil Steril 1995;64:1125-9 Key Words: Cryopreservation, coculture, blastocyst, freezing, thawing
Despite a number of improvements over the years, there have been few changes that have increased the overall live birth rate after IVF-ET. Recent evidence suggests that transfer of pre-embryos after coculture and at the blastocyst stage may improve success of IVF (1). In addition, cryopreservation of cocultured human blastocysts has been shown to produce excellent pregnancy rates (PRs) (2). Some of these benefits may be attributed to the usage of blastocysts Received January 12, 1995; revised and accepted June 9, 1995. * Presented in part at the 50th Annual Meeting of The American Fertility Society, San Antonio, Texas, November 5 to 10, 1994. t Augusta Reproductive Biology Associates. :j: Reprint requests: Robert Kaufmann, M.D., Augusta Reproductive Biology Associates, 812 Chafee Avenue, Augusta, Georgia 30904 (FAX: 706-722-2387). § IRH Fondation Merieux. II Clinique Pierre Cherest. Vol. 64, No.6, December 1995
(1, 2) and coculture systems (3, 4) (Kaufmann RA, Menezo YR, Nicollet B, Servy EJ, abstract). The first pregnancy after replacement of cryopreserved and thawed pre-embryos was in 1982 (5). The first births were reported in 1984 when early cleavage stage pre-embryos were cryopreserved in dimethylsulphoxide, thawed, and then transferred (6). Since that time many centers have been successful with cryopreservation and transfer of human preembryos at various stages of development. The first pregnancy after replacement of a frozen-thawed human blastocyst occurred in 1985 (7). To date, there has been more information accumulated regarding the use of blastocysts in cryopreservation (2, 8-13) (Menezo Y, Servy EJ, Kaufmann RA, Nicollet B, Hazout A, abstract). In our three different centers we have accumulated the largest series to date evaluating the use Kaufmann et a1.
Cocultured blastocyst cryopreservation
1125
.
of cryopreserved blastocysts for infertility patients undergoing IVF-ET. A retrospective study was performed to evaluate the use of cryopreserved blastocysts over a 3-year period using a very large patient population. MATERIALS AND METHODS Patients
In vitro fertilization cycles with blastocyst cryopreservation between January 1991 and April 1995 were studied (n = 563). The patients underwent ovarian stimulation with or without GnRH agonist (GnRH-a) treatment. The patients receiving GnRH-a were given either SC buserelin acetate (Hoechst Laboratories, Tour Roussel Hoechst, Puteaux, France) or leuprolide acetate (Lupron; TAP Pharmaceuticals, Chicago, 1L). Ovarian stimulation was performed with FSH (Metrodin; Serono Laboratories, Randolph, MA) and/or hMG (Pergonal; Serono Laboratories). Follicle growth was followed by sonography and E2 blood levels. Human chorionic gonadotropin (hCG; Organon, St. Denis, France or Profasi; Serono Laboratories) was given when the stimulation met the criterion for administration followed by oocyte retrieval 36 hours after hCG injection. In Vitro Fertilization and Culture Conditions
The 1VF procedures have been published previously (14). All 1VF procedures were performed in B2 medium (CCD, Paris, France; Fertility Technology, Nantik, MA; Pharmascience, Montreal, Quebec, Canada) without serum under 5% CO 2 in air for incubation (15). Sperm were prepared by various methods including Percoll (Sigma Chemical Company, St. Louis, MO) gradient centrifugation or standard swim-up methods. Patients had their pre-embryos evaluated for pronuclei status 14 to 20 hours postinsemination. The pre-embryos were transferred on either day 2 or 3 after insemination. Pre-embryos were cocultured on Vero cells according to protocols described previously (1, 14). Supernumerary pre-embryos were frozen on day 5, 6, or 7 after insemination at the expanded blastocyst stage. Glycerol (Sigma Chemical Co) was used as the cryoprotectant.
Table 1 Cryopreservation at the Blastocyst Stage Mter Coculture on Vero Cells No. of cycles thawed No. of transfer cycles* No. of thawed blastocysts No. of transferred blastocysts* No. of implanting embryos Implantation rate* No. of clinical pregnancies Pregnancies per transfer (%) Ongoing pregnancies per transfer (%)
563 516 (92) 1,239 1,033 (83) 138 138/1,033 (13.4) 112 21.7 19
* Values in parentheses are percentages.
curve was set -1°C/min from 22°C to -6°C using a programmed biologic freezer (Planer Kryo; T.S. Scientific, Perkasie, PA). Manual seeding then was performed after a 30-second delay. After the seeding, the pre-embryo was cooled slowly from -6°C to -37°C at a rate of -0.3°C/min. It then was plunged into liquid nitrogen. The blastocysts were kept at -196°C under liquid nitrogen until thawed. Thawing and Transfer Protocol
The protocol for thawing has been described previously (2). Briefly, the blastocysts were thawed quickly in air at room temperature. The cryoprotectant then was removed in seven steps, with decreasing concentrations of glycerol. After thawing, the pre-embryos were allowed to recover 3 to 4 hours in the culture medium before transfer. Only the morphologically normal pre-embryos that re-expanded were replaced in appropriately prepared patients. Patients were prepared for transfer either in natural cycles or by hormonal replacement. Natural cycles (n = 47) were evaluated and transfer was timed using serum LH, P, E 2, and ultrasound. Luteal phase P was administered to these patients. There were three types of hormonally supplemented cycles. Some patients (n = 107) received ovarian induction with gonadotropins. Others (n = 60) received GnRH-a and ovulation stimulation. Lastly, pre-embryos (n = 302) were transferred after administration of steroids in the form of transdermal E2 patches or oral E2 followed by P supplementation either intravaginally or through 1M injections (16). Generally, thawing and replacement was performed on day 20. Statistical Methods
Cryopreservation Protocol
Cryopreservation was performed in two steps. The pre-embryo initially was immersed in a 5% glycerol solution in B2 medium ± 10% serum for 10 minutes. Next the pre-embryo was placed in a solution of 9% glycerol in B2 + 10% serum containing 0.2 M sucrose for 10 minutes. The programmed freezing 1126
Kaufmann et al.
Cocultured blastocyst cryopreservation
Groups were compared using the X 2 test. A probability level of <0.05 was considered significant. RESULTS
The results are summarized in Table 1. The mean age of the patients was 32.4 years. Of 516 transfer Fertility and Sterility
Table 2 The Effect of Uterine Preparation on the Results of Cryopreservation and Thawing Spontaneous Replacement HMGAny cycle cycle FSH GnRH-a
47
No. of cycles with transfer No. of clinical pregnancies Pregnancy rate/transfer
6
12
302 79 26.2*
107 17 16
60
10 16
* Significantly different compared with spontaneous cycle, P < 0.005.
cycles, 112 pregnancies resulted. There were eight miscarriages with an ongoing PR per transfer of 19%. Overall 138 pre-embryos implanted out of 1,033 pre-embryos, resulting in an implantation rate of 13.4%. There were no significant differences in these results between the three institutions. The pregnancies in the differently prepared recipients were compared in Table 2. There was a higher implantation rate in the cycles that were prepared with exogenous steroids compared with spontaneous cycles. Figure 1 (pre-embryo not on coculture) and Figure 2 (pre-embryo on coculture) demonstrate blastocysts that expanded well and showed good morphological criteria after thawing and subsequently were transferred. Figure 3, on the other hand, shows a preembryo that is of fair quality after thaw (this preembryo is expanded, however, it has small vacuoles). There is a direct correlation between the quality of the pre-embryos and their morphology. The better the quality of the pre-embryo the greater the pregnancy potential. DISCUSSION
There appears to be some inherent advantages to using blastocysts for cryopreservation. Damage to
Figure 1
Figure 2
the trophectoderm of the blastocyst probably would have little effect on outcome if it occurs during cryopreservation (17). However, if this damage occurred to a pronuclear ovum, then the cell would be destroyed completely. One-cell damage to a two-cell embryo would compromise 50% of the pre-embryo. If the pre-embryos are cocultured before freezing then, because of an increase in pre-embryo cell number compared with classical culture conditions (1), they may be more suitable for cryopreservation. It also may be an advantage to increase the selection of normally developing pre-embryos, therefore reducing the number of pre-embryos to cryopreserve and therefore decreasing the cryoprservation and transfer cycle costs because the selection process has taken place before cryopreservation. Poorer quality pre-embryos may be cocultured on feeder cells and allowed to progress to the blastocyst stage frozen and then used in an alternate cycle. This ultimately
Figure 3
Expanded blastocyst.
Vol. 64, No.6, December 1995
Expanded blastocyst on co-culture.
Kaufmann et al.
Expanded blastocyst.
Cocultured blastocyst cryopreservation
1127
may increase the number of quality pre-embryos available for transfer and thus also increase PRs. The overall number of pre-embryos that reach the blastocyst stage and are able to be cryopreserved is 56%. The selection process, therefore, is before cryopreservation, whereas in pronuclear freezing it is after thawing. Our implantation rate of 13% is similar to the implantation rate of 11.2% recently published from a large series of thawed and transferred cryopreserved pronuclear pre-embryos (18). However, because of the numerous advantages mentioned previously, we believe there is still a strong argument in favor of the use of cryopreserved blastocysts. This investigation is the largest experience to date on cryopreservation of cocultured blastocysts. The implantation rate of 13% is approximately half of what is expected from fresh transfer of blastocysts but still is very similar to the results reported for transfer of early stage pre-embryos (19). The miscarriage rate of 13% was less then reported by Hartshorne and co-workers (9) (32%) for transfer of frozen blastocysts. Of note, the blastocysts in our study were exposed to coculture before cryopreservation, whereas, Hartshorne et al.'s (9) pre-embryos were grown in medium alone. Similar trends in miscarriages for pre-embryos cocultured on fetal bovine uterine fibroblasts (13%) compared with those cultured on conventional medium (29%) were found by Wiemer and co-workers (3). The co culture system may be promoting a better quality pre-embryo with better potential for implantation and survival. In fact most parameters were lower in Hartshorne et al.'s (9) study compared with ours, including number of transfer cycles (65% versus 92%), post-thaw viability (51 % versus 83%), implantation rate per blastocyst (10.3% versus 13.4%), and pregnancies per transfer (14% versus 21.7%). Our findings are in agreement with several other studies demonstrating the benefits of coculture on embryonic development (20, 21). Zetova and coworkers (20) found an implantation rate of 12.5% in the early coculture group compared with 8.4% in the medium alone group, which was highly significant. Embryonic morphology was improved markedly when early stage pre-embryos were cultured on bovine oviductal monolayers (21). The mode of action by which coculture systems exert their beneficial affects has been hypothesized. There may be embryotrophic factors released by the feeder cells, including glycoproteins. These glycoproteins may have growth-promoting properties to enhance embryonic growth. These cells also could be acting by removing toxic compounds, such as heavy metals or divalent cations. Massip et al. have shown in the mouse that in 1128
Kaufmann et al. Cocultured blastocyst cryopreservation
vitro culture and freezing will decrease the implantation rate (22). Prolonged in vitro culture, cryopreservation, and thawing will decrease the yield per pre-embryo. In our study, although there was a reduction in the implantation rate after thawing and transfer, the implantation rate of 13% still is similar to the rates expected for transfer of early stage preembryos. We found a significant improvement in PRs in the patients who had their endometrium prepared with exogenous steroid preparation. The reason for this may be a more physiologic preparation of the endometrium to receive the pre-embryo. Our results are similar to the findings of Schmidt et al. (23), who found a higher PR for thawed pre-embryo transfer in hormone replaced endometrium than during a natural cycle. In fact, it has been suggested that the endometrium in a cryopreservation-thawed preembryo transfer cycle may be more receptive to implantation than during a transfer of fresh pre-embryos in an IVF cycle (24). This is in contrast to the results of Queenan and co-workers (18) who did not demonstrate similar pregnancy results when transferring thawed early stage pre-embryos into natural or controlled cycles. These data suggest that freezing at the blastocyst stage is a reliable method in IVF technology. Although there may be fewer pre-embryos to cryopreserve, the ones remaining seem to be of superior quality. REFERENCES 1. Menezo Y, Hazout A, Dumont M, Herbaut N, Nicollet B. Coculture of embryos on Vero cells and transfer of blastocysts in humans. Hum Reprod 1992;7:101-6. 2. Menezo Y, Nicollet B, Herbaut N, Andre D. Freezing cocultured human blastocysts. Fertil Steril 1992;58:977-80. 3. Wiemer KE, Cohen J, Amborski GF, Wiker S, Wright G, Munyakazi L, et al. In-vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells. Hum Reprod 1989;4:595-600. 4. Wiemer KE, Hoffman DI, Maxon WS, Eager S, Muhlberger B, Flore I, et al. Embryonic morphology and rate of implant ation of human embryos following coculture on bovine oviductal epithelial cells. Hum Reprod 1993;8:97-101. 5. Trounson A, Mohr L. Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo. Nature 1983;303:707 -9. 6. Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CMPM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 1984;42:293-6. 7. Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts 1985;2:59-64. 8. Bolton VN, Wren ME, Parsons JH. Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil Steril 1991;55:830-2. 9. Hartshorne GM, Elder K, Crow J, Dyson H, Edwards RG. The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Hum Reprod 1991;6:136-41.
Fertility and Sterility i
10. 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 Obstet Gynecol Reprod BioI 1990;38:133-9. 11. Cohen J, Simons RS, Fehilly CB, Edwards RG. Factors affecting survival and implantation of cryopreserved human embryos. J Vitro Fert Embryo Transf 1986;3:46-52. 12. Fehilly CB, Cohen J, Simons RF, Fishel SB, Edwards RG. Cryopreservation of cleaving embryos and expanded blastocyst in the human: a comparative study. Fertil Steril 1985;44:638-44. 13. Hartshorne GM, Wick K, Elder K, Dyson H. Effect of cell number at freezing upon survival and viability of cleaving embryos generated from stimulated IVF cycles. Hum Reprod 1990;5:857-61. 14. Menezo Y, Guerin JF, Czyba JC. Improvement of human early embryo development in vitro by coculture on monolayers ofVero cells. BioI Reprod 1990;42:301-6. 15. Menezo Y, Testart J, Perrone D. Serum is not necessary in human in vitro fertilization, early embryo culture and transfer. Fertil Steril 1984;42:750-5. 16. Muasher SJ, Kruithoff C, Simonetti S, Oehninger S, Acosta AA, Jones GS. Controlled preparation of the endometrium with exogenous steroids for the transfer offrozen-thawed preembryos in patients with anovulatory or irregular cycles. Hum Reprod 1991;6:443-5. 17. Heyman Y, Chesne P. Development of cattle embryos after splitting of trophoblast biopsy and freezing. XIth International Congress on animal reproduction and artificial insemination. Communication number 167, Dublin: Elsevier, 1988.
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18. Queenan JT, Jr, Veeck LL, Seltman HJ, Muasher SJ. Transfer of cryopreserved thawed pre-embryos in a natural cycle or a programmed cycle with exogenous hormonal replacement yields similar pregnancy results. Fertil Steril 1994;62:54550. 19. Menezo YR, Janny L, Khatchadourian. Embryo quality and co-culture. In: Mastroianni L, Bennk C, Siuziki S, Vemer H, editors. Gamete and embryo quality. London: Parthenon Publishing Group, 1993:145-55. 20. Zetova L, Mardesic T, Mikova M, Muller P, Stroufova A. Improved development of human embryos cultured on a Vero cell monolayer. J Assist Reprod Genet 1993; 10:234-6. 21. Wiemer KE, Hoffman DI, Maxon WS, Eager S, Muhlberger B, Flore I, et al. Embryonic morphology and rate of implant ation of human embryos following coculture on bovine oviductal epithelial cells. Hum Reprod 1993;8:97-101. 22. Massip A, Zwalmen V, Puissant F, Camus M, Leroy P. Effects of in-vitro fertilization, culture, freezing and transfer on the ability of mouse embryos to implant and survive. J Reprod Fert 1984;71:199-204. 23. Schmidt CL, deZiegler D, Gagliardi CL, Mellon RW, Taney FH, Kuhar MJ, et al. Transfer of cryopreserved-thawed embryos; the natural cycle versus controlled preparation of the endometrium with gonadotropin-releasing hormone agonist and exogenous estradiol and progesterone (GEEP). Fertil Steril 1989; 52:609-16. 24. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril1986;46:268-72.
Kaufmann et aI. Cocultured blastocyst cryopreservation
1129
Aisilt"d reproductive fecl,no.logy FERTILITY AND STERILITY Copyright
~
Vol. 64, No.6, December 1995
1995 American Society for Reproductive Medicine
Printed on acid-free paper in U. S. A.
Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles*
Robert A. Kaufmann, M.D.t:J: Yves Menezo, Ph.D.t§ Andre Hazout, M.D. II
Bernard Nicollet, M.D.§ Martine DuMont, Ph.D.11 Edouard J. Servy, M.D.t
Augusta Reproductive Biology Associates, Augusta, Georgia; Institut Rhonalpin Fondation Merieux, Lyon; and Clinique Pierre Cherest, Paris, France
Objective: To present our experience using cocultured cryopreserved and transferred blastocysts. Design: Retrospective study of patients undergoing transfer of cryopreserved blastocysts. Setting: Three different IVF centers. Patients: Four hundred sixty-seven thawed cycles from January 1991 to June 1994. Main Outcome Measure: Pregnancy rate per cycle after transfer of pre-embryos developed from thawed blastocysts. Results: One thousand two hundred thirty-nine blastocysts were thawed. Of these, 1,033 (83%) survived thawing and were transferred. Five hundred sixty-three thawed cycles resulted in 516 (92%) receiving intrauterine transfer. One hundred twelve clinical pregnancies were established, resulting in a 21.7% pregnancy per transfer with a 19% ongoing rate. The implantation rate of 13.4% results from 138 implanted pre-embryos. There was a higher PR in the programmed cycle (79/302; 26.2%) compared with the natural cycle (6/47;13%). Conclusions: Freezing at the blastocyst stage is a proven and reliable method in IVF technology. Although there may be fewer pre-embryos, their ability to implant appears to approach the potential of a fresh transfer. Fertil Steril 1995;64:1125-9 Key Words: Cryopreservation, coculture, blastocyst, freezing, thawing
Despite a number of improvements over the years, there have been few changes that have increased the overall live birth rate after IVF-ET. Recent evidence suggests that transfer of pre-embryos after coculture and at the blastocyst stage may improve success of IVF (1). In addition, cryopreservation of cocultured human blastocysts has been shown to produce excellent pregnancy rates (PRs) (2). Some of these benefits may be attributed to the usage of blastocysts Received January 12, 1995; revised and accepted June 9, 1995. * Presented in part at the 50th Annual Meeting of The American Fertility Society, San Antonio, Texas, November 5 to 10, 1994. t Augusta Reproductive Biology Associates. :j: Reprint requests: Robert Kaufmann, M.D., Augusta Reproductive Biology Associates, 812 Chafee Avenue, Augusta, Georgia 30904 (FAX: 706-722-2387). § IRH Fondation Merieux. II Clinique Pierre Cherest. Vol. 64, No.6, December 1995
(1, 2) and coculture systems (3, 4) (Kaufmann RA, Menezo YR, Nicollet B, Servy EJ, abstract). The first pregnancy after replacement of cryopreserved and thawed pre-embryos was in 1982 (5). The first births were reported in 1984 when early cleavage stage pre-embryos were cryopreserved in dimethylsulphoxide, thawed, and then transferred (6). Since that time many centers have been successful with cryopreservation and transfer of human preembryos at various stages of development. The first pregnancy after replacement of a frozen-thawed human blastocyst occurred in 1985 (7). To date, there has been more information accumulated regarding the use of blastocysts in cryopreservation (2, 8-13) (Menezo Y, Servy EJ, Kaufmann RA, Nicollet B, Hazout A, abstract). In our three different centers we have accumulated the largest series to date evaluating the use Kaufmann et a1.
Cocultured blastocyst cryopreservation
1125
.
of cryopreserved blastocysts for infertility patients undergoing IVF-ET. A retrospective study was performed to evaluate the use of cryopreserved blastocysts over a 3-year period using a very large patient population. MATERIALS AND METHODS Patients
In vitro fertilization cycles with blastocyst cryopreservation between January 1991 and April 1995 were studied (n = 563). The patients underwent ovarian stimulation with or without GnRH agonist (GnRH-a) treatment. The patients receiving GnRH-a were given either SC buserelin acetate (Hoechst Laboratories, Tour Roussel Hoechst, Puteaux, France) or leuprolide acetate (Lupron; TAP Pharmaceuticals, Chicago, 1L). Ovarian stimulation was performed with FSH (Metrodin; Serono Laboratories, Randolph, MA) and/or hMG (Pergonal; Serono Laboratories). Follicle growth was followed by sonography and E2 blood levels. Human chorionic gonadotropin (hCG; Organon, St. Denis, France or Profasi; Serono Laboratories) was given when the stimulation met the criterion for administration followed by oocyte retrieval 36 hours after hCG injection. In Vitro Fertilization and Culture Conditions
The 1VF procedures have been published previously (14). All 1VF procedures were performed in B2 medium (CCD, Paris, France; Fertility Technology, Nantik, MA; Pharmascience, Montreal, Quebec, Canada) without serum under 5% CO 2 in air for incubation (15). Sperm were prepared by various methods including Percoll (Sigma Chemical Company, St. Louis, MO) gradient centrifugation or standard swim-up methods. Patients had their pre-embryos evaluated for pronuclei status 14 to 20 hours postinsemination. The pre-embryos were transferred on either day 2 or 3 after insemination. Pre-embryos were cocultured on Vero cells according to protocols described previously (1, 14). Supernumerary pre-embryos were frozen on day 5, 6, or 7 after insemination at the expanded blastocyst stage. Glycerol (Sigma Chemical Co) was used as the cryoprotectant.
Table 1 Cryopreservation at the Blastocyst Stage Mter Coculture on Vero Cells No. of cycles thawed No. of transfer cycles* No. of thawed blastocysts No. of transferred blastocysts* No. of implanting embryos Implantation rate* No. of clinical pregnancies Pregnancies per transfer (%) Ongoing pregnancies per transfer (%)
563 516 (92) 1,239 1,033 (83) 138 138/1,033 (13.4) 112 21.7 19
* Values in parentheses are percentages.
curve was set -1°C/min from 22°C to -6°C using a programmed biologic freezer (Planer Kryo; T.S. Scientific, Perkasie, PA). Manual seeding then was performed after a 30-second delay. After the seeding, the pre-embryo was cooled slowly from -6°C to -37°C at a rate of -0.3°C/min. It then was plunged into liquid nitrogen. The blastocysts were kept at -196°C under liquid nitrogen until thawed. Thawing and Transfer Protocol
The protocol for thawing has been described previously (2). Briefly, the blastocysts were thawed quickly in air at room temperature. The cryoprotectant then was removed in seven steps, with decreasing concentrations of glycerol. After thawing, the pre-embryos were allowed to recover 3 to 4 hours in the culture medium before transfer. Only the morphologically normal pre-embryos that re-expanded were replaced in appropriately prepared patients. Patients were prepared for transfer either in natural cycles or by hormonal replacement. Natural cycles (n = 47) were evaluated and transfer was timed using serum LH, P, E 2, and ultrasound. Luteal phase P was administered to these patients. There were three types of hormonally supplemented cycles. Some patients (n = 107) received ovarian induction with gonadotropins. Others (n = 60) received GnRH-a and ovulation stimulation. Lastly, pre-embryos (n = 302) were transferred after administration of steroids in the form of transdermal E2 patches or oral E2 followed by P supplementation either intravaginally or through 1M injections (16). Generally, thawing and replacement was performed on day 20. Statistical Methods
Cryopreservation Protocol
Cryopreservation was performed in two steps. The pre-embryo initially was immersed in a 5% glycerol solution in B2 medium ± 10% serum for 10 minutes. Next the pre-embryo was placed in a solution of 9% glycerol in B2 + 10% serum containing 0.2 M sucrose for 10 minutes. The programmed freezing 1126
Kaufmann et al.
Cocultured blastocyst cryopreservation
Groups were compared using the X 2 test. A probability level of <0.05 was considered significant. RESULTS
The results are summarized in Table 1. The mean age of the patients was 32.4 years. Of 516 transfer Fertility and Sterility
Table 2 The Effect of Uterine Preparation on the Results of Cryopreservation and Thawing Spontaneous Replacement HMGAny cycle cycle FSH GnRH-a
47
No. of cycles with transfer No. of clinical pregnancies Pregnancy rate/transfer
6
12
302 79 26.2*
107 17 16
60
10 16
* Significantly different compared with spontaneous cycle, P < 0.005.
cycles, 112 pregnancies resulted. There were eight miscarriages with an ongoing PR per transfer of 19%. Overall 138 pre-embryos implanted out of 1,033 pre-embryos, resulting in an implantation rate of 13.4%. There were no significant differences in these results between the three institutions. The pregnancies in the differently prepared recipients were compared in Table 2. There was a higher implantation rate in the cycles that were prepared with exogenous steroids compared with spontaneous cycles. Figure 1 (pre-embryo not on coculture) and Figure 2 (pre-embryo on coculture) demonstrate blastocysts that expanded well and showed good morphological criteria after thawing and subsequently were transferred. Figure 3, on the other hand, shows a preembryo that is of fair quality after thaw (this preembryo is expanded, however, it has small vacuoles). There is a direct correlation between the quality of the pre-embryos and their morphology. The better the quality of the pre-embryo the greater the pregnancy potential. DISCUSSION
There appears to be some inherent advantages to using blastocysts for cryopreservation. Damage to
Figure 1
Figure 2
the trophectoderm of the blastocyst probably would have little effect on outcome if it occurs during cryopreservation (17). However, if this damage occurred to a pronuclear ovum, then the cell would be destroyed completely. One-cell damage to a two-cell embryo would compromise 50% of the pre-embryo. If the pre-embryos are cocultured before freezing then, because of an increase in pre-embryo cell number compared with classical culture conditions (1), they may be more suitable for cryopreservation. It also may be an advantage to increase the selection of normally developing pre-embryos, therefore reducing the number of pre-embryos to cryopreserve and therefore decreasing the cryoprservation and transfer cycle costs because the selection process has taken place before cryopreservation. Poorer quality pre-embryos may be cocultured on feeder cells and allowed to progress to the blastocyst stage frozen and then used in an alternate cycle. This ultimately
Figure 3
Expanded blastocyst.
Vol. 64, No.6, December 1995
Expanded blastocyst on co-culture.
Kaufmann et al.
Expanded blastocyst.
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may increase the number of quality pre-embryos available for transfer and thus also increase PRs. The overall number of pre-embryos that reach the blastocyst stage and are able to be cryopreserved is 56%. The selection process, therefore, is before cryopreservation, whereas in pronuclear freezing it is after thawing. Our implantation rate of 13% is similar to the implantation rate of 11.2% recently published from a large series of thawed and transferred cryopreserved pronuclear pre-embryos (18). However, because of the numerous advantages mentioned previously, we believe there is still a strong argument in favor of the use of cryopreserved blastocysts. This investigation is the largest experience to date on cryopreservation of cocultured blastocysts. The implantation rate of 13% is approximately half of what is expected from fresh transfer of blastocysts but still is very similar to the results reported for transfer of early stage pre-embryos (19). The miscarriage rate of 13% was less then reported by Hartshorne and co-workers (9) (32%) for transfer of frozen blastocysts. Of note, the blastocysts in our study were exposed to coculture before cryopreservation, whereas, Hartshorne et al.'s (9) pre-embryos were grown in medium alone. Similar trends in miscarriages for pre-embryos cocultured on fetal bovine uterine fibroblasts (13%) compared with those cultured on conventional medium (29%) were found by Wiemer and co-workers (3). The co culture system may be promoting a better quality pre-embryo with better potential for implantation and survival. In fact most parameters were lower in Hartshorne et al.'s (9) study compared with ours, including number of transfer cycles (65% versus 92%), post-thaw viability (51 % versus 83%), implantation rate per blastocyst (10.3% versus 13.4%), and pregnancies per transfer (14% versus 21.7%). Our findings are in agreement with several other studies demonstrating the benefits of coculture on embryonic development (20, 21). Zetova and coworkers (20) found an implantation rate of 12.5% in the early coculture group compared with 8.4% in the medium alone group, which was highly significant. Embryonic morphology was improved markedly when early stage pre-embryos were cultured on bovine oviductal monolayers (21). The mode of action by which coculture systems exert their beneficial affects has been hypothesized. There may be embryotrophic factors released by the feeder cells, including glycoproteins. These glycoproteins may have growth-promoting properties to enhance embryonic growth. These cells also could be acting by removing toxic compounds, such as heavy metals or divalent cations. Massip et al. have shown in the mouse that in 1128
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vitro culture and freezing will decrease the implantation rate (22). Prolonged in vitro culture, cryopreservation, and thawing will decrease the yield per pre-embryo. In our study, although there was a reduction in the implantation rate after thawing and transfer, the implantation rate of 13% still is similar to the rates expected for transfer of early stage preembryos. We found a significant improvement in PRs in the patients who had their endometrium prepared with exogenous steroid preparation. The reason for this may be a more physiologic preparation of the endometrium to receive the pre-embryo. Our results are similar to the findings of Schmidt et al. (23), who found a higher PR for thawed pre-embryo transfer in hormone replaced endometrium than during a natural cycle. In fact, it has been suggested that the endometrium in a cryopreservation-thawed preembryo transfer cycle may be more receptive to implantation than during a transfer of fresh pre-embryos in an IVF cycle (24). This is in contrast to the results of Queenan and co-workers (18) who did not demonstrate similar pregnancy results when transferring thawed early stage pre-embryos into natural or controlled cycles. These data suggest that freezing at the blastocyst stage is a reliable method in IVF technology. Although there may be fewer pre-embryos to cryopreserve, the ones remaining seem to be of superior quality. REFERENCES 1. Menezo Y, Hazout A, Dumont M, Herbaut N, Nicollet B. Coculture of embryos on Vero cells and transfer of blastocysts in humans. Hum Reprod 1992;7:101-6. 2. Menezo Y, Nicollet B, Herbaut N, Andre D. Freezing cocultured human blastocysts. Fertil Steril 1992;58:977-80. 3. Wiemer KE, Cohen J, Amborski GF, Wiker S, Wright G, Munyakazi L, et al. In-vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells. Hum Reprod 1989;4:595-600. 4. Wiemer KE, Hoffman DI, Maxon WS, Eager S, Muhlberger B, Flore I, et al. Embryonic morphology and rate of implant ation of human embryos following coculture on bovine oviductal epithelial cells. Hum Reprod 1993;8:97-101. 5. Trounson A, Mohr L. Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo. Nature 1983;303:707 -9. 6. Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CMPM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 1984;42:293-6. 7. Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts 1985;2:59-64. 8. Bolton VN, Wren ME, Parsons JH. Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil Steril 1991;55:830-2. 9. Hartshorne GM, Elder K, Crow J, Dyson H, Edwards RG. The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Hum Reprod 1991;6:136-41.
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10. 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 Obstet Gynecol Reprod BioI 1990;38:133-9. 11. Cohen J, Simons RS, Fehilly CB, Edwards RG. Factors affecting survival and implantation of cryopreserved human embryos. J Vitro Fert Embryo Transf 1986;3:46-52. 12. Fehilly CB, Cohen J, Simons RF, Fishel SB, Edwards RG. Cryopreservation of cleaving embryos and expanded blastocyst in the human: a comparative study. Fertil Steril 1985;44:638-44. 13. Hartshorne GM, Wick K, Elder K, Dyson H. Effect of cell number at freezing upon survival and viability of cleaving embryos generated from stimulated IVF cycles. Hum Reprod 1990;5:857-61. 14. Menezo Y, Guerin JF, Czyba JC. Improvement of human early embryo development in vitro by coculture on monolayers ofVero cells. BioI Reprod 1990;42:301-6. 15. Menezo Y, Testart J, Perrone D. Serum is not necessary in human in vitro fertilization, early embryo culture and transfer. Fertil Steril 1984;42:750-5. 16. Muasher SJ, Kruithoff C, Simonetti S, Oehninger S, Acosta AA, Jones GS. Controlled preparation of the endometrium with exogenous steroids for the transfer offrozen-thawed preembryos in patients with anovulatory or irregular cycles. Hum Reprod 1991;6:443-5. 17. Heyman Y, Chesne P. Development of cattle embryos after splitting of trophoblast biopsy and freezing. XIth International Congress on animal reproduction and artificial insemination. Communication number 167, Dublin: Elsevier, 1988.
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18. Queenan JT, Jr, Veeck LL, Seltman HJ, Muasher SJ. Transfer of cryopreserved thawed pre-embryos in a natural cycle or a programmed cycle with exogenous hormonal replacement yields similar pregnancy results. Fertil Steril 1994;62:54550. 19. Menezo YR, Janny L, Khatchadourian. Embryo quality and co-culture. In: Mastroianni L, Bennk C, Siuziki S, Vemer H, editors. Gamete and embryo quality. London: Parthenon Publishing Group, 1993:145-55. 20. Zetova L, Mardesic T, Mikova M, Muller P, Stroufova A. Improved development of human embryos cultured on a Vero cell monolayer. J Assist Reprod Genet 1993; 10:234-6. 21. Wiemer KE, Hoffman DI, Maxon WS, Eager S, Muhlberger B, Flore I, et al. Embryonic morphology and rate of implant ation of human embryos following coculture on bovine oviductal epithelial cells. Hum Reprod 1993;8:97-101. 22. Massip A, Zwalmen V, Puissant F, Camus M, Leroy P. Effects of in-vitro fertilization, culture, freezing and transfer on the ability of mouse embryos to implant and survive. J Reprod Fert 1984;71:199-204. 23. Schmidt CL, deZiegler D, Gagliardi CL, Mellon RW, Taney FH, Kuhar MJ, et al. Transfer of cryopreserved-thawed embryos; the natural cycle versus controlled preparation of the endometrium with gonadotropin-releasing hormone agonist and exogenous estradiol and progesterone (GEEP). Fertil Steril 1989; 52:609-16. 24. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril1986;46:268-72.
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