7 Cryopreservation of human embryos ANDRI~ VAN STEIRTEGHEM ETIENNE VAN DEN ABBEEL MICHEL CAMUS PAUL DEVROEY
EMBRYOS FOR FREEZING
Infertility treatment by in vitro fertilization (IVF) and subsequent embryo transfer or gamete or zygote intrafallopian transfer (GIFT or ZIFT) always includes ovarian hyperstimulation. Different protocols can be used to induce multiple follicular maturation: clomiphene citrate alone or in combination with human menopausal gonadotropins (HMG), purified folliclestimulating hormone (FSH) or a combination of gonadotropin-releasing hormone (GnRH) agonists with HMG. In these different stimulation regimens, ovulation will be induced by administration of human chorionic gonadotropins (HCG). Several oocytes may be retrieved and after in vitro insemination it is not uncommon for several embryos to develop. The chances of a successful pregnancy are increased if two, three or more embryos can be replaced. This implies, however, that multiple pregnancies can occur after multiple embryo transfers in IVF and a similar problem exists in GIFT and ZIFT when multiple oocytes or zygotes are replaced. It is a generally accepted practice to limit the number of embryos transferred to two or three in order to limit the well-documented risk of multiple fetuses, since high-rank multiple pregnancies in particular can cause perinatal and postnatal morbidity and mortality. Any supernumerary embryos can be cryopreserved for some time before a subsequent second attempt at replacement, should the first attempt have failed. Embryos from an oocyte and embryo donation programme can also be cryopreserved. Freezing these embryos may circumvent the requirement of synchrony between ovarian cycles of donor and recipient when fresh embryos are used. If a donated oocyte or embryo is available for a recipient it may be replaced as a fresh embryo if the ovarian cycles of donor and recipient are in synchrony or it may be frozen as an embryo for later use, being thawed and transferred at an appropriate moment in the recipient's cycle. If, for medical reasons, fresh embryos cannot be replaced during the Bailli~re's Clinical Obstetrics and Gynaecology-313 Vol. 6, No. 2, June 1992 Copyright © 1992, by Baillitre Tindall ISBN 0-7020-1633-0 All rights of reproduction in any form reserved
314
A. V A N S T E I R T E G H E M ET A L
treatment cycle, they can be cryopreserved and transferred during a subsequent cycle. It seems appropriate that couples should be counselled about cryopreservation before the start of any treatment of assisted procreation. If infertile couples choose to cryopreserve their excess embryos, they may be asked to sign an appropriate consent form stating that (1) they both agree, (2) cryopreserved embryos will only be transferred if they are of adequate morphological quality after thawing, (3) the duration of storage of these embryos will be limited in time. Human embryo freezing implies a number of additional requirements to normal IVF practice. A controlled biological freezer system is needed to allow accurate cooling to subzero temperatures at rates of 0.1-0. 5°C/min. Liquid nitrogen facilities are needed, including the liquid nitrogen storage tanks that must be maintained either by close supervision or by automatic liquid nitrogen filling. The embryologists who do embryo freezing must have extensive experience in the freezing and thawing of experimental embryos such as mouse embryos. A group should not embark on human embryo freezing before the experimental system works well. GENERAL PRINCIPLES AND METHODS OF CRYOPRESERVATION OF EMBRYOS
Spermatozoa were the first human cells to be cryopreserved successfully (Polge et al, 1949). Major advances have since been achieved in the cryopreservation of many cells and even tissues and organs. The general cryobiological principles have been extensively reviewed in recent articles (Ashwood-Smith, 1986; Trounson, 1986; Friedler et al, 1988; Mandelbaum, 1990). The two critical periods for cell damage during cryopreservation are the initial cooling to a low temperature and the later return to physiological temperature. Survival will not be impaired by storage at the temperature of liquid nitrogen ( - 196°C). It is generally accepted that most of the damage in cryopreservation is caused by the formation of intracellular ice crystals. The cryopreservation process is done in various steps: (1) exposure and equilibration of the embryo to a permeating cryoprotectant in a one-step or multiple-step procedure in the presence or absence of a non-permeating cryoprotectant such as sucrose, (2) cooling to subzero temperature, (3) induction of extracellular ice formation, the so-called seeding process, to prevent excessive supercooling of the embryo, (4) storage at -196°C in liquid nitrogen, (5) thawing to room temperature, (6) removal of the permeating cryoprotectant by stepwise dilution in the presence or absence of the non-permeating cryoprotectant sucrose, and (7) the transfer of the frozen-thawed embryo to a physiological environment that may allow resumption of development. Cryoprotectants are used to protect against damage caused by freezing and thawing. The exact mechanism by which each of the various cryoprotectants exerts its protective action remains to be determined. Permeating (such as glycerol, dimethyl sulphoxide and propanediol) and non-permeating cryo-
C R Y O P R E S E R V A T I O N OF H U M A N EMBRYOS
315
protectant agents (such as sucrose) are used for the freezing and thawing of embryos. So far, most human embryo freezing has been done by slow freezing protocols: (1) DMSO (1.5 M) may be used for day 2 or day 3 embryos (Trounson and Mohr, 1983), (2) glycerol (8-10%) is appropriate for expanding blastocysts (Cohen et al, 1985a) and (3) propanediol (PROH) for day 1 or day 2 embryos (Lassalle et al, 1985). These procedures have many common features. After equilibration with the cryoprotectant, the embryos are slowly cooled (l°C/min) to - 6 or -7°C; after seeding, the embryos are slowly cooled (0.3°C/min) to -30 or -80°C and then immersed into liquid nitrogen. The thawing procedure is done rapidly (300°C/min) for the PROH and glycerol procedure or slowly (8°C/min) for the DMSO protocol. More recently Trounson et al (•988) have reported excellent survival and developmental potential of mouse and human embryos after ultrarapid freezing. The embryos are exposed for a few minutes to high concentrations of DMSO (3.5 M) and sucrose (0.25 M) and then immediately immersed into liquid nitrogen. Most embryos survived after thawing rapidly in a warm water bath. Ultrarapid freezing is quick and inexpensive since it does not require a programmable biological freezer. FACTORS AFFECTING H U M A N E M B R Y O C R Y O P R E S E R V A T I O N Data from surveys
International and national surveys are useful indicators of the performance of the practice of human embryo cryopreservation. Two world surveys were done on the occasions of two World Congresses of IVF (Norfolk, USA, 1987; Jerusalem, Israel, 1989). These surveys reported data about the practice of, respectively, 21 and 132 centres throughout the world (Van Steirteghem and Van den Abbeel, 1988, 1990). Supernumerary embryos were frozen after the transfer of fresh oocytes, zygotes and embryos. More than half of the centres reported replacement of four or more concepti. Up to 31 December 1988, 30850 embryos had been frozen by three slowfreezing procedures (DMSO, PROH and glycerol); 18322 embryos had been thawed and examined in anticipation of transfer. Slightly more than half of these embryos (56.6%) were found suitable for transfer after thawing. After 6441 embryo transfers, 738 clinical pregnancies were established. The pregnancy rate per embryo transfer procedure was 11.5 %, and per thawed embryo 4.1%. Spontaneous abortions occurred in 29.3 % of the pregnancies, usually early in pregnancy. Four centres mentioned the occurrence of malformations. In countries such as Australia, France and the United States of America the results of medically assisted procreation are registered at a national level. Data about cryopreservation are included in the yearly reports. Fugger (1989) reported on the clinical status of human embryo cryopreservation in the USA. The mean number of embryos transferred per pregnancy was 15.0 (1767 embryos transferred in 118 cycles) and the
316
A. VAN S T E I R T E G H E M ET A L
pregnancy rate per transfer was 13.4% (118 pregnancies out of 884 transfer cycles). The results of individual centres are quite similar to the international and national surveys. Camus et al (1991) reported 631 consecutive transfers of cryopreserved supernumerary embryos at the Brussels Free University's Centre for Reproductive Medicine between December 1985 and March 1990. Excess embryos after IVF, GIFT and ZIFT were frozen at the 2- to 8-ceU stage (n = 4048) using the slow DMSO protocol and at the pronuclear stage (n = 1139) using the PROH procedure. After thawing 2883 embryos during this period, 973 embryos (33.7%) were actually transferred. The pregnancy, delivery and spontaneous abortion rates per transfer were respectively 15% (95/631), 10.5% (66/631) and 29.5%. Mandelbaum (1990) reported that 4.2% of the frozen-thawed embryos will finally result in the birth of a child (122/2909) which corresponds to 6.7% of the embryos frozen and thawed and transferred (122/1813). This is similar to their results from the transfer of fresh embryos in 1987 and 1988:431 children born from the transfer of 6163 embryos (7%).
Cryopreservation procedure The three slow freezing protocols for human embryos have been successful in establishing pregnancies and births but so far no controlled comparative studies have been done between the three protocols. Literature data were used by Testart et al (1990) to compare PROH and DMSO; the transfer and implantation rates of frozen and thawed embryos were significantly higher for PROH + sucrose (60% and 6.8%) than for DMSO (43% and 3.8%). Camus et al (1991), however, reported recently that the percentage of frozen-thawed embryos able to he transferred was 32.8% (732/2230) when DMSO was used for cleaving embryos and 35.5% (232/653) when PROH was used for the cryopreservation of pronucleate stage embryos. The addition of 0.1M sucrose to the 1.5M PROH plays a part in (1) dehydrating the embryo before cooling, (2) diluting the PROH more rapidly during thawing and (3) promoting membrane stability. Embryo survival is significantly reduced if the PROH protocol is done without adding sucrose (Mandelbaum et al, 1988). Two French teams reported that the quality of seeding has a significant effect on embryo survival after thawing (Gu6rin et al, 1989; Mandelbaum, 1990). The clinical application of ultrarapid freezing was reported on by Gordts et al (1990) and Feichtinger et al (1991). After ultrarapid freezing and thawing, 68% (160/235) of the embryos had at least half of their initial number of blastomeres and 112 replacement procedures resulted in 13 pregnancies (11.6%) of which 5 ended prematurely.
Stage of embryonic development Pregnancies have been reported after transfer of embryos cryopreserved at the different stages of preimplantation development. But there are no conclusive data indicating that a particular stage of embryonic development is
CRYOPRESERVATION OF H U M A N EMBRYOS
317
more appropriate for freezing. The cryopreservation of excess day 2 embryos has the practical advantage of allowing selection of embryos for fresh transfer and of providing supernumerary embryos of sufficient morphological quality for cryopreservation. If pronucleate embryos are frozen in cases where couples are being treated by IVF-ET, the situation may arise in which the embryos left in culture have not cleaved further and the fresh embryo transfer procedure has to be cancelled (Mandelbaum, 1990). The freezing of pronucleate embryos can be applied without reservation with regard to patients treated by GIFT or ZIFT (Van Steirteghem et al, 1987a). Several other publications report the usefulness of the cryopreservation of pronucleate stage embryos (Cohen et al, 1988a; Fugger, 1989; Testart et al, 1990; Camus et al, 1991; Demoulin et al, 1991). It is theoretically feasible to freeze embryos on day 3 of embryo culture; this may have the practical advantage of avoiding the necessity to freeze on non-working days. As reported by the Bourn Hall group, it is also possible to have a successful freezing programme of human blastocysts (Fehilly et al, 1985; Cohen et al, 1986). Twice as many cleaving embryos were frozen as expanding blastocysts because of the low developmental potential of human embryos in vitro but significantly more expanded blastocysts survived cryopreservation than did cleaving embryos and relatively more pregnancies were established by the replacement of thawed blastocysts than by the replacement of thawedcleaving embryos. The French Clamart group (Lassalle et al, 1985) reported that 2-, 4- and 8-cell embryos survived thawing after freezing with PROH better (67 %) than did intermediate cleavage stage embryos (22%). This difference was not confirmed by Mandelbaum et al (1987) who used the same cryopreservation procedure or by Camus et al (1989) who used the DMSO protocol for freezing 2- to 8-cell embryos. The latter did not detect any difference between the freezing of 4-cell (day 2) and 8-cell (day 3) embryos.
Morphological appearance of the cryopreserved embryo The morphology of cleaving embryos can be assessed under the inverted microscope (magnification x 200) and the embryos can then be classified (Staessen et al, 1989). It is obvious from many reports in the literature that the survival of frozen-thawed embryos is influenced by the morphological quality of the embryos at the time of freezing (Cohen et al, 1986; Freeman et al, 1986; Mandelbaum et al, 1987; Testart et al, 1987; Fugger et al, 1988). Our group's results indicate that for both the DMSO and the PROH protocols the survival and potential for implantation are strongly correlated with the prefreezing embryonic quality; if embryos contain ~oo many anucleate fragments they will not survive the stress of cryopreservation and cannot implant into the uterus (Van Steirteghem et al, 1987a; Van den Abbeel et al, 1988; Camus et al, 1989). At least one-third of frozen embryos is fully intact after thawing (Van Steirteghem et al, 1987a; Mandelbaum et al, 1988; Testart et al, 1990). The viability of a frozen-thawed embryo is not affected if at least half of the initial number of blastomeres is still present after thawing (Freeman et al, 1986; Testart et al, 1987). The Monash University group recommended as
318
A. V A N S T E I R T E G H E M ET A L
cut-off point for the transfer of a frozen-thawed embryo that half of the blastomeres should remain intact after thawing. This recommendation was based on observations that 8-cell embryos which had lost more than four blastomeres during cryopreservation did not develop further in vitro. However, each blastomere of mammalian embryos up to the 8-cell stage has totipotency and may develop normally to term (Willadsen, 1981). Some teams transfer cryopreserved embryos which have less than half of their blastomeres intact after thawing; certain groups will systematically replace all frozen-thawed embryos if at least one blastomere has survived. At least six births have been reported after the replacement of frozen-thawed embryos with less than half of the initial number of blastomeres (Mohr et al, 1985; Veiga et al, 1987; Testart et al, 1990; Mandelbaum, 1990). The survival of individual blastomeres was clearly related by Hartshorne et al (1990) to the stage at which the cleaving embryo is frozen. The highest rates of cell survival were found among the pronuclear embryos compared with the cleavage stage embryos. The number of embryos with all cells surviving was found to correlate inversely with cell number and this result may be related to the theoretical surface area of embryos at the different cleavage stages. Our experience of the influence of blastomere loss on the implantation of frozen-thawed embryos after transfer was reported on by Van den Abbeel et al (1990). In 213 replacements, 275 fully intact embryos were replaced, compared with 146 transfers of 207 embryos which had lost some blastomeres during freezing and thawing. The pregnancy rate per transfer was 15.9% for intact embryos and 10.9% for embryos with some blastomere loss (not significant); the implantation rate per embryo transferred was, respectively, 13.8 and 7.7% (P
EMBRYOS FOR FREEZING
Infertility treatment by in vitro fertilization (IVF) and subsequent embryo transfer or gamete or zygote intrafallopian transfer (GIFT or ZIFT) always includes ovarian hyperstimulation. Different protocols can be used to induce multiple follicular maturation: clomiphene citrate alone or in combination with human menopausal gonadotropins (HMG), purified folliclestimulating hormone (FSH) or a combination of gonadotropin-releasing hormone (GnRH) agonists with HMG. In these different stimulation regimens, ovulation will be induced by administration of human chorionic gonadotropins (HCG). Several oocytes may be retrieved and after in vitro insemination it is not uncommon for several embryos to develop. The chances of a successful pregnancy are increased if two, three or more embryos can be replaced. This implies, however, that multiple pregnancies can occur after multiple embryo transfers in IVF and a similar problem exists in GIFT and ZIFT when multiple oocytes or zygotes are replaced. It is a generally accepted practice to limit the number of embryos transferred to two or three in order to limit the well-documented risk of multiple fetuses, since high-rank multiple pregnancies in particular can cause perinatal and postnatal morbidity and mortality. Any supernumerary embryos can be cryopreserved for some time before a subsequent second attempt at replacement, should the first attempt have failed. Embryos from an oocyte and embryo donation programme can also be cryopreserved. Freezing these embryos may circumvent the requirement of synchrony between ovarian cycles of donor and recipient when fresh embryos are used. If a donated oocyte or embryo is available for a recipient it may be replaced as a fresh embryo if the ovarian cycles of donor and recipient are in synchrony or it may be frozen as an embryo for later use, being thawed and transferred at an appropriate moment in the recipient's cycle. If, for medical reasons, fresh embryos cannot be replaced during the Bailli~re's Clinical Obstetrics and Gynaecology-313 Vol. 6, No. 2, June 1992 Copyright © 1992, by Baillitre Tindall ISBN 0-7020-1633-0 All rights of reproduction in any form reserved
314
A. V A N S T E I R T E G H E M ET A L
treatment cycle, they can be cryopreserved and transferred during a subsequent cycle. It seems appropriate that couples should be counselled about cryopreservation before the start of any treatment of assisted procreation. If infertile couples choose to cryopreserve their excess embryos, they may be asked to sign an appropriate consent form stating that (1) they both agree, (2) cryopreserved embryos will only be transferred if they are of adequate morphological quality after thawing, (3) the duration of storage of these embryos will be limited in time. Human embryo freezing implies a number of additional requirements to normal IVF practice. A controlled biological freezer system is needed to allow accurate cooling to subzero temperatures at rates of 0.1-0. 5°C/min. Liquid nitrogen facilities are needed, including the liquid nitrogen storage tanks that must be maintained either by close supervision or by automatic liquid nitrogen filling. The embryologists who do embryo freezing must have extensive experience in the freezing and thawing of experimental embryos such as mouse embryos. A group should not embark on human embryo freezing before the experimental system works well. GENERAL PRINCIPLES AND METHODS OF CRYOPRESERVATION OF EMBRYOS
Spermatozoa were the first human cells to be cryopreserved successfully (Polge et al, 1949). Major advances have since been achieved in the cryopreservation of many cells and even tissues and organs. The general cryobiological principles have been extensively reviewed in recent articles (Ashwood-Smith, 1986; Trounson, 1986; Friedler et al, 1988; Mandelbaum, 1990). The two critical periods for cell damage during cryopreservation are the initial cooling to a low temperature and the later return to physiological temperature. Survival will not be impaired by storage at the temperature of liquid nitrogen ( - 196°C). It is generally accepted that most of the damage in cryopreservation is caused by the formation of intracellular ice crystals. The cryopreservation process is done in various steps: (1) exposure and equilibration of the embryo to a permeating cryoprotectant in a one-step or multiple-step procedure in the presence or absence of a non-permeating cryoprotectant such as sucrose, (2) cooling to subzero temperature, (3) induction of extracellular ice formation, the so-called seeding process, to prevent excessive supercooling of the embryo, (4) storage at -196°C in liquid nitrogen, (5) thawing to room temperature, (6) removal of the permeating cryoprotectant by stepwise dilution in the presence or absence of the non-permeating cryoprotectant sucrose, and (7) the transfer of the frozen-thawed embryo to a physiological environment that may allow resumption of development. Cryoprotectants are used to protect against damage caused by freezing and thawing. The exact mechanism by which each of the various cryoprotectants exerts its protective action remains to be determined. Permeating (such as glycerol, dimethyl sulphoxide and propanediol) and non-permeating cryo-
C R Y O P R E S E R V A T I O N OF H U M A N EMBRYOS
315
protectant agents (such as sucrose) are used for the freezing and thawing of embryos. So far, most human embryo freezing has been done by slow freezing protocols: (1) DMSO (1.5 M) may be used for day 2 or day 3 embryos (Trounson and Mohr, 1983), (2) glycerol (8-10%) is appropriate for expanding blastocysts (Cohen et al, 1985a) and (3) propanediol (PROH) for day 1 or day 2 embryos (Lassalle et al, 1985). These procedures have many common features. After equilibration with the cryoprotectant, the embryos are slowly cooled (l°C/min) to - 6 or -7°C; after seeding, the embryos are slowly cooled (0.3°C/min) to -30 or -80°C and then immersed into liquid nitrogen. The thawing procedure is done rapidly (300°C/min) for the PROH and glycerol procedure or slowly (8°C/min) for the DMSO protocol. More recently Trounson et al (•988) have reported excellent survival and developmental potential of mouse and human embryos after ultrarapid freezing. The embryos are exposed for a few minutes to high concentrations of DMSO (3.5 M) and sucrose (0.25 M) and then immediately immersed into liquid nitrogen. Most embryos survived after thawing rapidly in a warm water bath. Ultrarapid freezing is quick and inexpensive since it does not require a programmable biological freezer. FACTORS AFFECTING H U M A N E M B R Y O C R Y O P R E S E R V A T I O N Data from surveys
International and national surveys are useful indicators of the performance of the practice of human embryo cryopreservation. Two world surveys were done on the occasions of two World Congresses of IVF (Norfolk, USA, 1987; Jerusalem, Israel, 1989). These surveys reported data about the practice of, respectively, 21 and 132 centres throughout the world (Van Steirteghem and Van den Abbeel, 1988, 1990). Supernumerary embryos were frozen after the transfer of fresh oocytes, zygotes and embryos. More than half of the centres reported replacement of four or more concepti. Up to 31 December 1988, 30850 embryos had been frozen by three slowfreezing procedures (DMSO, PROH and glycerol); 18322 embryos had been thawed and examined in anticipation of transfer. Slightly more than half of these embryos (56.6%) were found suitable for transfer after thawing. After 6441 embryo transfers, 738 clinical pregnancies were established. The pregnancy rate per embryo transfer procedure was 11.5 %, and per thawed embryo 4.1%. Spontaneous abortions occurred in 29.3 % of the pregnancies, usually early in pregnancy. Four centres mentioned the occurrence of malformations. In countries such as Australia, France and the United States of America the results of medically assisted procreation are registered at a national level. Data about cryopreservation are included in the yearly reports. Fugger (1989) reported on the clinical status of human embryo cryopreservation in the USA. The mean number of embryos transferred per pregnancy was 15.0 (1767 embryos transferred in 118 cycles) and the
316
A. VAN S T E I R T E G H E M ET A L
pregnancy rate per transfer was 13.4% (118 pregnancies out of 884 transfer cycles). The results of individual centres are quite similar to the international and national surveys. Camus et al (1991) reported 631 consecutive transfers of cryopreserved supernumerary embryos at the Brussels Free University's Centre for Reproductive Medicine between December 1985 and March 1990. Excess embryos after IVF, GIFT and ZIFT were frozen at the 2- to 8-ceU stage (n = 4048) using the slow DMSO protocol and at the pronuclear stage (n = 1139) using the PROH procedure. After thawing 2883 embryos during this period, 973 embryos (33.7%) were actually transferred. The pregnancy, delivery and spontaneous abortion rates per transfer were respectively 15% (95/631), 10.5% (66/631) and 29.5%. Mandelbaum (1990) reported that 4.2% of the frozen-thawed embryos will finally result in the birth of a child (122/2909) which corresponds to 6.7% of the embryos frozen and thawed and transferred (122/1813). This is similar to their results from the transfer of fresh embryos in 1987 and 1988:431 children born from the transfer of 6163 embryos (7%).
Cryopreservation procedure The three slow freezing protocols for human embryos have been successful in establishing pregnancies and births but so far no controlled comparative studies have been done between the three protocols. Literature data were used by Testart et al (1990) to compare PROH and DMSO; the transfer and implantation rates of frozen and thawed embryos were significantly higher for PROH + sucrose (60% and 6.8%) than for DMSO (43% and 3.8%). Camus et al (1991), however, reported recently that the percentage of frozen-thawed embryos able to he transferred was 32.8% (732/2230) when DMSO was used for cleaving embryos and 35.5% (232/653) when PROH was used for the cryopreservation of pronucleate stage embryos. The addition of 0.1M sucrose to the 1.5M PROH plays a part in (1) dehydrating the embryo before cooling, (2) diluting the PROH more rapidly during thawing and (3) promoting membrane stability. Embryo survival is significantly reduced if the PROH protocol is done without adding sucrose (Mandelbaum et al, 1988). Two French teams reported that the quality of seeding has a significant effect on embryo survival after thawing (Gu6rin et al, 1989; Mandelbaum, 1990). The clinical application of ultrarapid freezing was reported on by Gordts et al (1990) and Feichtinger et al (1991). After ultrarapid freezing and thawing, 68% (160/235) of the embryos had at least half of their initial number of blastomeres and 112 replacement procedures resulted in 13 pregnancies (11.6%) of which 5 ended prematurely.
Stage of embryonic development Pregnancies have been reported after transfer of embryos cryopreserved at the different stages of preimplantation development. But there are no conclusive data indicating that a particular stage of embryonic development is
CRYOPRESERVATION OF H U M A N EMBRYOS
317
more appropriate for freezing. The cryopreservation of excess day 2 embryos has the practical advantage of allowing selection of embryos for fresh transfer and of providing supernumerary embryos of sufficient morphological quality for cryopreservation. If pronucleate embryos are frozen in cases where couples are being treated by IVF-ET, the situation may arise in which the embryos left in culture have not cleaved further and the fresh embryo transfer procedure has to be cancelled (Mandelbaum, 1990). The freezing of pronucleate embryos can be applied without reservation with regard to patients treated by GIFT or ZIFT (Van Steirteghem et al, 1987a). Several other publications report the usefulness of the cryopreservation of pronucleate stage embryos (Cohen et al, 1988a; Fugger, 1989; Testart et al, 1990; Camus et al, 1991; Demoulin et al, 1991). It is theoretically feasible to freeze embryos on day 3 of embryo culture; this may have the practical advantage of avoiding the necessity to freeze on non-working days. As reported by the Bourn Hall group, it is also possible to have a successful freezing programme of human blastocysts (Fehilly et al, 1985; Cohen et al, 1986). Twice as many cleaving embryos were frozen as expanding blastocysts because of the low developmental potential of human embryos in vitro but significantly more expanded blastocysts survived cryopreservation than did cleaving embryos and relatively more pregnancies were established by the replacement of thawed blastocysts than by the replacement of thawedcleaving embryos. The French Clamart group (Lassalle et al, 1985) reported that 2-, 4- and 8-cell embryos survived thawing after freezing with PROH better (67 %) than did intermediate cleavage stage embryos (22%). This difference was not confirmed by Mandelbaum et al (1987) who used the same cryopreservation procedure or by Camus et al (1989) who used the DMSO protocol for freezing 2- to 8-cell embryos. The latter did not detect any difference between the freezing of 4-cell (day 2) and 8-cell (day 3) embryos.
Morphological appearance of the cryopreserved embryo The morphology of cleaving embryos can be assessed under the inverted microscope (magnification x 200) and the embryos can then be classified (Staessen et al, 1989). It is obvious from many reports in the literature that the survival of frozen-thawed embryos is influenced by the morphological quality of the embryos at the time of freezing (Cohen et al, 1986; Freeman et al, 1986; Mandelbaum et al, 1987; Testart et al, 1987; Fugger et al, 1988). Our group's results indicate that for both the DMSO and the PROH protocols the survival and potential for implantation are strongly correlated with the prefreezing embryonic quality; if embryos contain ~oo many anucleate fragments they will not survive the stress of cryopreservation and cannot implant into the uterus (Van Steirteghem et al, 1987a; Van den Abbeel et al, 1988; Camus et al, 1989). At least one-third of frozen embryos is fully intact after thawing (Van Steirteghem et al, 1987a; Mandelbaum et al, 1988; Testart et al, 1990). The viability of a frozen-thawed embryo is not affected if at least half of the initial number of blastomeres is still present after thawing (Freeman et al, 1986; Testart et al, 1987). The Monash University group recommended as
318
A. V A N S T E I R T E G H E M ET A L
cut-off point for the transfer of a frozen-thawed embryo that half of the blastomeres should remain intact after thawing. This recommendation was based on observations that 8-cell embryos which had lost more than four blastomeres during cryopreservation did not develop further in vitro. However, each blastomere of mammalian embryos up to the 8-cell stage has totipotency and may develop normally to term (Willadsen, 1981). Some teams transfer cryopreserved embryos which have less than half of their blastomeres intact after thawing; certain groups will systematically replace all frozen-thawed embryos if at least one blastomere has survived. At least six births have been reported after the replacement of frozen-thawed embryos with less than half of the initial number of blastomeres (Mohr et al, 1985; Veiga et al, 1987; Testart et al, 1990; Mandelbaum, 1990). The survival of individual blastomeres was clearly related by Hartshorne et al (1990) to the stage at which the cleaving embryo is frozen. The highest rates of cell survival were found among the pronuclear embryos compared with the cleavage stage embryos. The number of embryos with all cells surviving was found to correlate inversely with cell number and this result may be related to the theoretical surface area of embryos at the different cleavage stages. Our experience of the influence of blastomere loss on the implantation of frozen-thawed embryos after transfer was reported on by Van den Abbeel et al (1990). In 213 replacements, 275 fully intact embryos were replaced, compared with 146 transfers of 207 embryos which had lost some blastomeres during freezing and thawing. The pregnancy rate per transfer was 15.9% for intact embryos and 10.9% for embryos with some blastomere loss (not significant); the implantation rate per embryo transferred was, respectively, 13.8 and 7.7% (P
