Human Reproduction, Vol. 14, No. 9, 2362-2366,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Clinical experience with ultrarapid cryopreservation of human embryos resulting from intracytoplasmic sperm injection: Brief communication
1 Centre for Human Reproduction, Sinhá Junqueira Maternity Foundation, Rua D.Alberto Goncialves, 1500–14085–100 and 2 Department of Obstetrics and Gynecology, University of Ribeirão Preto (UNAERP), Ribeirão Preto, SP, Brazil
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Abstract |
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A total of 41 patients requested thawing of supernumerary embryos in an intracytoplasmic sperm injection (ICSI) programme. Mean patient age was 30.8 ± 3.8 years. Embryo freezing by the ultrarapid method was performed at room temperature in 3 mol/l DMSO and 0.25 mol/l sucrose. Total freezing time was 2.5 min including filling of the straw. In the thawing process, the embryos were removed from liquid N2, left at room temperature for 30 s, immersed for 40 s at 30°C, and then successively transferred at room temperature for 10 min to each of three sucrose solutions of decreasing concentration. The embryos were kept in culture and only those that presented cleavage after 24 h were transferred. Embryos from 42 cycles were thawed and a total of 24 transfers was performed. The mean number of thawed embryos was 5.0 ± 3.2 per cycle and the mean number of transferred embryos was 2.83 ± 1.3. The clinical pregnancy rate per cycle obtained after the thawing process was 16.6%. The clinical pregnancy rate per transfer was 29.2% and the implantation rate was 13.2%. The abortion rate was 14.3%. Six deliveries have been performed, with the birth of seven infants.
Key words: cryopreservation/human embryo/ICSI/ultrarapid method
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Introduction |
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Human embryo cryopreservation has greatly progressed since the first success (Trounson and Mohr,1983
) when 3- to 8-cell embryos were frozen with dimethylsulphoxide (DMSO). However, most cryopreservation techniques based on slow freezing require expensive equipment and are time-consuming.
The use of an ultrarapid method for mouse embryo freezing which only requires a rapid exposure of the embryos (2–2.5 min) to high DMSO concentrations (3–4 mol/l) and to 0.25 mol/l sucrose, followed by immersion in liquid nitrogen and rapid thawing in a 0.25 mol/l sucrose solution was reported (Trounson et al., 1987).
The development of intracytoplasmic sperm injection (ICSI) has allowed many couples to achieve fertilization through in-vitro fertilization (IVF) when they would have experienced little or no fertilization using standard IVF insemination methods (Palermo et al., 1993). The efficacy of the transfer of supernumerary multicellular human embryos obtained after ICSI was studied (Van Steirteghem et al., 1994). The embryos were cryopreserved on day 2 or 3 by a slow freezing protocol with DMSO. They reported a 53% survival rate and an overall pregnancy rate [positive human chorionic gonadotrophin (HCG) measurements] of 21.8% per transfer. The clinical pregnancy rate was 12.9% and the delivery rate 5.9%. The preclinical abortion rate was 40.9%.
Another group, using a slow freezing method with propanediol (Al-Hasani et al., 1996), concluded that pronuclear stage oocytes resulting from ICSI can be successfully frozen/thawed and the pregnancy rates (17%) achieved are comparable to those for zygotes obtained after IVF (20%). Using a slow freezing process, it was observed (Hoover et al., 1997) that pronuclear embryos resulting from ICSI could be cryopreserved successfully and thawed, with a clinical pregnancy rate (14%) comparable to conventional IVF (17.4%). It was later reported (Macas et al., 1998) that the zygotes arising from ICSI cycles survived cryopreservation at a rate similar to IVF zygotes, but their ability to implant and develop further was probably affected by the cryopreservation procedure. It was also observed (Simon et al., 1998) that embryos originating from ICSI were not vulnerable to cryopreservation and, when implanted, resulted in a comparable abortion rate to thawed embryos of conventional IVF. Furthermore, it was observed that blastocycsts obtained after ICSI and Vero cell co-culture can retain developmental competence after cryopreservation and thawing (Servy et al., 1998).
The objective of the present study was to obtain and report laboratory and clinical results of an ultrarapid method (URM) for cryopreservation of supernumerary human embryos obtained after ICSI.
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Materials and methods |
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A total of 41 patients had their supernumerary embryos cryopreserved by the ultrarapid method (see below) after patient participation in the ICSI programme of the Centre for Human Reproduction, Sinhá Junqueira Maternity Foundation, Ribeirão Preto, Brazil. During the same period, there were 215 other ICSI cycles carried out, of which 97 had supernumerary embryos frozen by the slow method. The mean age of the patients (215 cycles) was 33.4 ± 4.6 years.
In the ovarian stimulation cycle preceding ICSI we used blockade of the second phase with leuprolide acetate at the dose of 0.5 mg a day (Lupron®; Abbott, Saõ Paulo, Brazil). Fourteen days after the use of the analogue, with blockade established, we used pure follicle-stimulating hormone (FSH) (Metrodin HP®; Serono) at a fixed dose of 150 or 225 IU for a period of 7 days. Follicular development was monitored only by vaginal ultrasonography 8 days after ovarian stimulation began, with the doses of FSH being adapted according to the ovarian response. When a minimum of three follicles measuring 
17 mm in diameter was observed, human chorionic gonadotrophin (HCG) was administered at the dose of 10 000 IU. Oocyte retrieval was carried out by transvaginal ultrasound-guided puncture of follicles 34–36 h after HCG. After identification in the follicular fluid, the oocytes were classified for maturity. The cumulus–corona complex was removed by exposure to a solution of type IV hyaluronidase (H-4272; Sigma Chemical Co., St Louis, MO, USA) at a concentration of 80 IU/ml. The denuded oocytes were incubated in IVF-50 medium (Scandinavian IVF Science AB, Sweden) until the time for ICSI. Spermatozoa were separated from seminal fluid using discontinuous Sperm-Prep-100TM gradients (Scandinavian IVF Science AB) prepared with 40% and 90% fractions.
ICSI was performed by an established method (Svalander et al., (1995). The embryos were routinely transferred after 48 h in culture and supernumerary embryos were cryopreserved at end of the second day.
Ultrarapid method
Embryo freezing
In the ultrarapid freezing method we used 3 mol/l DMSO (Sigma, D-2650) and 0.25 mol/l sucrose (Sigma, S-1888) (Trounson et al., 1987) but we modified the thawing process.
Two solutions were first prepared: (i) culture solution consisting of IVF-50 medium plus 20% inactivated patient serum, and (ii) a DMSO solution consisting of 3.935 ml culture solution and 14.079 mol/l DMSO in a volume of 1.065 ml. A freezing solution was then prepared, in which 0.4278 g sucrose was added to the final 5.0 ml volume of the DMSO solution. The freezing solution containing 3 mol/l DMSO and 0.25 mol/l sucrose was filtered through a 0.22 µm membrane, exposed to an atmosphere of 90% N2, 5% CO2 and 5% O2 and kept at 4°C for a minimum of 12 h and a maximum of 72 h. The embryos were exposed to the freezing solution for exactly 2.5 min in the freezing straw at room temperature and the straw was then plunged directly into liquid N2.
Embryo thawing
A thawing solution (TS) is prepared with 0.4278 g sucrose and diluted with culture solution to a final volume of 5.0 ml on the thawing day. The TS (0.25 mol/l sucrose) is diluted at three different concentrations with culture solution: TS-1 consisting of TS at the final proportion of 0.17 mol/l sucrose solution; TS-2 consisting of TS at the final proportion of 0.13 mol/l sucrose solution, and TS-3 consisting of TS at the final proportion of 0.08 mol/l sucrose solution. The solutions were filtered through a 0.22 µm membrane, exposed to an atmosphere of 90% N2, 5% CO2 and 5% O2, placed on Nunc (Copenhagen, Denmark) culture plates and kept at room temperature.
The straws containing the frozen embryos were then removed from liquid nitrogen, left to stand at room temperature for 30 s and immersed in a water bath at 30°C for 40 s. The embryos were then transferred consecutively to the three sucrose dilutions (TS-1, TS-2 and TS-3) for 10 min each time at room temperature. The embryos were placed in culture solution at room temperature for 5 min and then in culture solution microdrops covered with oil at a temperature of 37°C and maintained in a 5% CO2 atmosphere for 24 h.
Evaluation of embryo survival and cleavage after 24 h
Survival was defined by the presence of only one intact blastomere after the thawing process (Lai et al., 1996). However, the embryos are inspected separately in terms of embryo development and only those that present a continuation of the embryo cleavage process are transferred to the patient (Van der Elst et al., 1997). Embryo cleavage after 24 h was considered to be present when the division of at least one of the blastomeres was observed (Lai et al., 1996).
Natural and substitutive transfer cycles
Two hormonal schemes were used for the transfer of thawed embryos, i.e. a natural cycle and a substitutive cycle. In the natural cycle, follicular development was monitored by serial vaginal ultrasonography starting on the 10th day of the cycle. A 5000 IU dose of HCG was administered when the follicle presented a diameter 18 mm, and thawing was routinely performed on the fourth day after HCG (day of HCG injection = day 1) and embryo transfer on the fifth day after HCG.
In the substitutive cycle, oestradiol valerate (Postoval®; Wyeth, São Paulo, Brazil) was administered from the first to the 13th day of the cycle at a daily dose of 4–6 mg. Progesterone was introduced on the 14th day at a dose of 50 mg/day i.m. as long as endometrial thickness was >6 mm (Oliveira et al., 1997). Thawing was performed on the fourth day of progesterone treatment and transfer on the fifth day of progesterone treatment.
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Results |
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A total of 41 female patients of mean (±SD) age 30.8 ± 3.8 years was enrolled on an ICSI programme and 222 of their supernumerary embryos from 42 cycles were cryopreserved using a modified URM regardless of their morphological quality. The embryos were frozen during the 2- to 6-cell stage, with the mean number of frozen embryos being 5.3 ± 3.2 per cycle. A total of 48% of the cryopreserved embryos showed >20% anucleate fragments (Table I
). Frozen embryos from a total of 42 patient cycles were thawed. The mean number per cycle of thawed embryos was 5.0 ± 3.2. In addition, 41% of the surviving embryos showed fragmentation >20%. In the patients who became pregnant, 83% of the embryos were in the 5- to 8-cell stage and 79% of the transferred embryos presented 
20% fragmentation (Table II).
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The mean number of embryos transferred after thawing was 2.8 ± 1.3 (Table III
). A total of 57% of the cycles presented at least one cleaved embryo after 24 h of culture (24 transfers). The frequency of embryo cleavage after 24 h in culture was similar (P = 0.77) for the patients who became pregnant (55.8 ± 23%) and for those who did not (56.8 ± 22%). The rate of embryo implantation was 13.2% (9/68), with a 10.3% rate (7/68) of ongoing implantation per embryo transferred, a 4.3% rate (9/211) of implantation per embryo thawed and a 3.3% rate (7/211) of ongoing implantation per embryo thawed. The clinical pregnancy rate per thawing cycle was 16.6% (7/42). The clinical pregnancy rate per embryo transfer was 29.2% (7/24). The abortion rate was 14.3% (1/7). Six deliveries have been performed, with the birth of seven infants (five singletons and one set of twins). One of these singleton pregnancies started as a twin gestation but spontaneous regression of one embryo occurred during the seventh week (Table III). For comparison, in the 215 contemporary ICSI cycles not involved in this study (see above), the implantation rate for fresh embryos was 18.2% and the clinical pregnancy rate per transfer was 33.8% (66/195).
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Out of the 42 cycles, 32 were natural (12.5% clinical pregnancy) and 10 were substitutive (30% clinical pregnancy). Four pregnancies were obtained in the natural cycle, three of them single (one of them ended with abortion) and one of them involving twins, although one of the twins regressed spontaneously. In the substitutive cycle there were three pregnancies, two of them single and one involving twins.
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Discussion |
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Several experimental studies on animals have investigated the applicability and safety of ultrarapid embryo cryopreservation. Chromosome analyses were performed (Bongso et al., 1988
) of 2-cell mouse embryos cryopreserved by a slow method and by an ultrarapid method using two different types of cryoprotective agents, propanediol (PROH) and DMSO. Three methods were used: slow with PROH, ultrarapid with DMSO and ultrarapid with PROH. The chromosome analysis performed on 1- and 2-cell embryos showed the same incidence of aneuploidy and polyploidy in embryos frozen–thawed by the three methods, which did not differ significantly from the control consisting of non-frozen embryos. However, better results were reported (Liu et al., 1993) with mouse embryo freezing using slow cryopreservation methods with PROH or DMSO as cryoprotectors compared with the ultrarapid method using DMSO. The distribution of the mitochondrial pattern was reported to be unchanged (Noto et al., 1993) reported that after ultrarapid cryopreservation of 2- to 4-cell human embryos. Isolated 2-cell mouse blastomeres, when cryopreserved by the ultrarapid method (3 mol/l DMSO)(Kang et al., 1994) and then thawed presented a level of development identical to that of non-frozen embryos. It was reported that 3- to 8-cell human embryos survived the freezing and thawing process (with 50% of the blastomeres intact) (Trounson et al., 1988; Trounson and Sjöblom, 1988) with an ultrarapid method that used a brief exposure to 3.5 mol/l DMSO and 0.25 mol/l sucrose. However, no pregnancy was obtained.
The results of ultrarapid cryopreservation of human embryos in the pronuclear stage (Gordts et al., 1990), with 94% normal morphological characteristics and 79% cleavage after the thawing process. A total of 34 thawed and cleaved embryos was transferred to 20 patients during spontaneous cycles and four pregnancies were obtained (20% pregnancy rate). Experience with an ultrarapid freezing method was reported (Feichtinger et al., 1991). A total of 181 embryos was frozen and thawed, with a 61% survival rate (110 embryos) with 50–100% intact blastomeres. The pregnancy rate was 9.8%, with the occurrence of one abortion and one ectopic pregnancy. These investigators stated that their results were similar to those obtained in their unit with a slow freezing method using PROH. Experience with an ultrarapid freezing method applied to human embryos was reported (Lai et al., 1996). A total of 63 embryos was thawed, 83% of which (52 embryos) survived with at least one intact blastomere. Nineteen transfers were performed with a mean number of transferred embryos of 2.7 and two term pregnancies were obtained (10% pregnancy rate). Although few units are currently using the ultrarapid method to freeze supernumerary human embryos, our centre was interested in working with this method because of its low operational cost and the rapidity of the cryopreservation process.
Thus, some modifications were introduced in this technique (three-step thawing) in order to improve its performance according to suggestions (Wilson and Quinn, 1989), where using an ultrarapid method for mouse embryos (3.5 mol/l DMSO), observed a protective effect on embryo development when the cryoprotectant was removed in different stages. In addition, no significant differences in rates of implantation were observed and fetal formation (Vasuthevan et al., 1992) when the cryoprotective agent was removed from mouse embryos in a single step as compared to removal in two steps. However, blastocysts formed after the one-step removal presented a low count of inner cells compared with those obtained in the two-step procedure.
Despite these modifications, our data demonstrate that only 24 thawing cycles (57%) were followed by transfer (at least one cleaved embryo). In the 18 cycles with no transfer (43%) there was embryo survival in five and absence of embryo cleavage after 24 h culture in 13. These values were lower than those obtained during the same period in our laboratory with the use of the slow method, in which embryo transfer did not occur in only 7% of the thawing cycles. On the other hand, using the parameter of embryo cleavage 24 h after thawing, 43% of the selected embryos were transferred to the patients who became pregnant (Table II
).
Recently, it was demonstrated (Van der Elst et al., 1998) that ultrarapid freezing of mouse oocytes with 3.5 mol/l DMSO can thus lead to day 5 in-vitro cultured blastocyst with significantly decreased inner cell mass numbers. It was agreed that, in view of the differences between blastomeres of early cleavage stages in mouse and human embryos (Van den Abbeel et al., 1997), modifications of the technique may be warranted to optimize survival and implantation of ultrarapidly frozen embryos. However, before rapid cooling is used routinely in clinical IVF programmes, its safety and reproducibility must be convincingly demonstrated.
Results of cryopreservation of supernumerary multicellular human embryos after ICSI were reported (Van Steirteghem et al., 1994). Only type A and B embryos were preserved (type A embryos had regular or irregular blastomeres and showed no anucleate fragments and type B embryos had 20% of their surface filled with anucleate fragments) while embryos with >20% anucleate fragments were not included in the analysis of the results.
In Brazil, supernumerary embryos are obligatorily cryopreserved. Since almost 50% of the cryopreserved embryos present 20% fragmentation, there is a low quality embryo population before the cryopreservation process takes place.
Despite these differences, the clinical results reported here (29.2% clinical pregnancy rate per transfer) are in agreement with those reported by other groups (Van Steirteghem et al., 1994; Macas et al., 1998) using supernumerary multicell embryos (21.8% clinical pregnancy rate per transfer) and pronuclear-stage embryos (a 20.4% clinical pregnancy rate per transfer) respectively.
The present results indicate that the ultrarapid method can be used for the cryopreservation of supernumerary embryos in an ICSI programme, although comparative studies with the slow method should be conducted.
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Acknowledgments |
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The authors wish to thank Mrs Elettra Greene for revising the English text.
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Notes |
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3 To whom correspondence should be addressed at: Centre for Human Reproduction, Sinhá Junqueira Maternity Foundation, Rua D.Alberto Concialves, 1500–14085–100, Brazil
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References |
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Bongso, A., Chye, N.S., Sathananthan, H. et al. (1988) Chromosome analysis of two-cell mouse embryos frozen by slow and ultrarapid methods using two different cryoprotectants. Fertil. Steril., 49, 908–912.[Web of Science][Medline]
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Submitted on December 8, 1998; accepted on June 10, 1999.
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