Cryopreservation of human embryos using ethylene glycol in controlled slow freezing

Hee-Jun Chi1,3, Jung-Jin Koo1, Moon-Young Kim1, Jin-Young Joo1, Sang-Sik Chang1 and Kil-Saeng Chung2

1 IVF Center, Hanna Women's Clinic and 2 Department of Animal Sciences, Kon-Kuk University, Seoul, Korea


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian formula embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). This study was carried out to evaluate the permeation and toxicity of EG and to investigate the effects of its use in a slow-freezing protocol on post-thaw development of mouse embryos and on pregnancy outcome of frozen human embryos. METHODS: Spare human embryos after embryo transfer were cryopreserved using 1.5 mol/l EG or PROH using a slow-freezing protocol which had been tested previously in mouse experiments. RESULTS: The post-thaw survival rate of human embryos in the EG group (80.6%) was significantly higher than that in the PROH group (65.2%, P < 0.05). The implantation and clinical pregnancy rates of human embryos in the EG group (20.3 and 46.9%) were significantly higher than those in the PROH group (7.5 and 24.6%, P < 0.05). CONCLUSIONS: Ethylene glycol may be a good substitute for PROH to cryopreserve human embryos using a slow-freezing protocol.

Key words: ethylene glycol/permeation/pregnancy outcome/survival rate/toxicity


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
There are various potential causes of the cell damage that occurs during the cell freezing procedure. Cooling has been shown to exert a detrimental effect on the meiotic spindle of human oocytes (Pickering et al., 1990Go), and sodium ions have been shown to have a deleterious effect on the post-thaw viability of mouse oocytes (Stachecki et al., 1998Go). However, osmotic shock, intracellular ice formation and toxicity of the cryoprotectant are still regarded as the major causes of cell damage occurring during freezing and thawing procedures.

With slow freezing, due to the use of a low concentration of cryoprotectant, the effects of osmotic shock and toxicity of the cryoprotectant on cell damage are less severe than that of intracellular ice formation (Bryant, 1995Go). A vitrification method has been developed to circumvent the obstacle of slow freezing (Rall and Fahy, 1985Go). Although the use of high concentrations of cryoprotectant in vitrification dramatically reduces the risk of intracellular ice formation, the toxicity of the cryoprotectant is increased as a result of long-term exposure to high concentrations of cryoprotectant (Fahy et al., 1984Go; Valdez et al., 1992Go). Recently, rapid vitrification techniques have been developed in which only a brief exposure to cryoprotective agents is required (Kasai et al., 1990Go; Shaw et al., 1992Go), though by limiting the exposure to cryoprotectants to only one step, the degree of osmotic shock is increased. Thus, in the vitrification method, osmotically induced damage is the major reason for loss of viability (Shaw et al., 1991Go). Although debate persists with regard to superiority between the two protocols, recent studies have demonstrated better outcomes of embryos that were frozen using a slow-freezing protocol (Sommerfeld and Niemann, 1999Go; Uechi et al., 1999Go).

Ethylene glycol (EG) has been used widely with both the slow-freezing and vitrification methods for the cryopreservation of mammalian embryos such as mouse (Ali and Shelton, 1993Go; Zhu et al., 1993Go; Shaw et al., 1995Go; Emiliani et al., 2000Go), rat (Jiang et al., 1999Go), rabbit (Kasai et al., 1992Go), sheep (Cocero et al., 1996Go) and cow (Donnay et al., 1998Go; Sommerfeld and Niemann, 1999Go) due to its low molecular weight, high permeation ability (Oda et al., 1992Go; Gilmore et al., 1995Go; Zhu et al., 1996Go; Newton et al., 1998Go) and low toxicity (Sommerfeld and Niemann, 1999Go; Emiliani et al., 2000Go). Recently, EG was used for the vitrification of human oocytes (Kuleshova et al., 1999Go; Chung et al., 2000Go) and embryos (Vanderzwalmen et al., 1997Go; Mukaida et al., 1998Go), as well as for cryopreservation of human sperm (Gilmore et al., 1997Go). However, to date no data are available for the use of EG in cryopreserving human embryos in a slow-freezing protocol.

In preliminary experiments, mouse zygotes exposed to 1.5 mol/l EG exhibited a severe and rapid change in size compared with embryos exposed to 1.5 mol/l propylene glycol (PROH). Two-cell stage mouse embryos exposed to EG exhibited a higher percentage of embryos which developed to blastocyst (64.4%) than did embryos exposed to PROH (53.8%). These results indicated that EG has high permeation ability and low toxicity compared with PROH.

In the light of these preliminary studies, the possibility of using EG rather than PROH for the freezing of human embryos in a slow-freezing protocol was investigated, in order to minimize any resulting cell damage and improve clinical results. Hence, the present study was carried out to evaluate the feasibility of using EG in a slow-freezing protocol, with mouse embryos and human embryos, and to investigate the efficiency of EG in the cryopreservation of human embryos.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The study was approved by the authors' Institutional Review Board, and informed consent was obtained from all participating subjects.

Patients
In this prospective study, which was conducted between January 1999 and May 2000 (17 months), the spare embryos after embryo transfer of 62 patients were frozen using EG. Retrospective data from the frozen embryos of 60 patients (frozen using PROH between August 1997 and December 1998) were used as a control group. There was no difference in mean (±SD) patient age between the EG and PROH groups (32.7 ± 3.6 and 32.4 ± 3.8 years respectively). Patients with polycystic ovary syndrome, poor response to gonadotrophins, uterine factors or age >40 years were excluded from the study.

The women were down-regulated with GnRH agonist, buserelin acetate (Suprefact; Hoechst AG, Germany) and stimulated with FSH (Metrodin-HP; Serono, Geneva, Switzerland) and HMG (Humegon; Organon Phamaceuticals, The Netherlands). Oocytes were retrieved at 35–36 h after administration of HCG (Profasi; Serono, Geneva, Switzerland) using a vaginal ultrasound-guided procedure.

Preparation of human embryos
The aspirated oocytes were inseminated using either conventional IVF or by ICSI in modified P1 medium (glucose/phosphate-free medium) supplemented with 10% human follicular fluid (HFF). Embryos with normal fertilization were cultured in modified TCM-199 medium (Gibco, Grand Island, NY, USA) supplemented with 20% HFF (Chi et al., 1998Go). On day 2 or 3 after oocyte retrieval, the embryos were transferred and the spare embryos after embryo transfer were cryopreserved using either EG (Sigma) or PROH (Sigma) in a controlled slow-freezing protocol. Clinical pregnancy was determined ultrasonographically by confirming the presence of an intrauterine gestation sac.

Freezing and thawing solutions
Freezing and thawing solutions were prepared in Dulbecco's phosphate-buffered saline (PBS; Gibco-BRL, Grand Island, NY, USA) supplemented with 20% HFF, 0.2 mol/l sucrose (Sigma) and 1.5 mol/l EG or PROH. For the freezing procedure, four equilibration solutions were used with each cryoprotectant: 0.5 mol/l EG or PROH, 1.0 mol/l EG or PROH, 1.5 mol/l EG or PROH, and 1.5 mol/l EG + 0.2 mol/l sucrose or 1.5 mol/l PROH + 0.2 mol/l sucrose. Four equilibration solutions were also used for the thawing procedure: 1 mol/l EG + 0.2 mol/l sucrose or 1 mol/l PROH + 0.2 mol/l sucrose, 0.5 mol/l EG + 0.2 mol/l sucrose or 0.5 mol/l PROH + 0.2 mol/l sucrose, 0.2 mol/l sucrose alone, and PBS.

Slow freezing and thawing
Cryopreservation of human embryos was performed using a programmable controlled rate-freezing machine (Planer Series III Kryo 10, T.S. Scientific, Perkasie, PA, USA). The embryos were cryopreserved using PBS supplemented with 20% HFF, 0.2 mol/l sucrose and 1.5 mol/l EG or PROH. The embryos were exposed in a stepwise fashion to increasing concentrations of EG or PROH (0.5, 1.0, 1.5 mol/l EG or PROH, and 1.5 mol/l EG + 0.2 mol/l sucrose or 1.5 mol/l PROH + 0.2 mol/l sucrose, 5 min per step) in the freezing solution. The embryos were then loaded into a 0.25 ml sterile straw (Bicef, L'Aigle, France); this was loaded into the cryo-machine (which was precooled to 20°C) and kept at 20°C for 1 min for equilibration. The straw was subsequently cooled from 20°C to –7°C at a rate of 2°C/min, held at this temperature for 5 min, and then seeded manually. It was then cooled to –39°C at a rate of 0.3°C/min and plunged into liquid nitrogen. For thawing of the frozen human embryos, the straws were rewarmed by holding them in air for 15 s before plunging them into a water bath at 37°C. The cryoprotectant was then removed by reverse stepwise dilutions with thawing solutions (5 min each step). Prior to the use of EG for cryopreservation of human embryos in clinical settings, both EG and PROH were used to freeze sibling embryos obtained from the same patients over a few cycles to confirm the feasibility of EG as a cryoprotectant. The effect of equilibration temperature (room temperature and 37°C) for freezing and thawing procedures on post-thaw survival of human embryos frozen using EG was also investigated. Although freezing and thawing of human embryos using PROH was performed at room temperature, freezing and thawing using EG was carried out at 37°C in an IVF chamber.

Statistical analysis
Post-thaw survival rates and subsequent development of mouse embryos, and implantation and pregnancy rates of human embryos in the EG and PROH groups were compared by {chi}2 analysis. Patients' age and number of embryos transferred per cycle (presented as mean ± SD) were compared using Student's t-test. A P-value < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Mouse embryos at the 4-cell stage were exposed to the freezing solutions (EG or PROH) in a stepwise manner, and changes in morphology of the embryos were observed. Embryos exposed to EG or PROH did not show any recognizable morphological change prior to the exposure to the final step. However, when the embryos were exposed to the final step (1.5 mol/l EG + 0.2 mol/l sucrose or 1.5 mol/l PROH + 0.2 mol/l sucrose), the embryos in the EG and PROH groups exhibited a distinct shrinkage of cytoplasm. Such shrinkage of the embryos exposed to 1.5 mol/l PROH + 0.2 mol/l sucrose was more severe than that of the embryos exposed to 1.5 mol/l EG + 0.2 mol/l sucrose (Figure 1Go).



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Figure 1. Changes in morphology of mouse embryos exposed to 1.5 mol/l ethylene glycol (EG) + 0.2 mol/l sucrose or 1.5 mol/l propylene glycol (PROH) + 0.2 mol/l sucrose. The embryos were exposed to the freezing solutions in stepwise manner for 5 min each step; 0.5, 1.0, 1.5 mol/l (EG or PROH) + 0.2 mol/l sucrose. (A) Mouse embryos exposed to 1.5 mol/l EG + 0.2 mol/l sucrose; (B) mouse embryos exposed to 1.5 mol/l PROH + 0.2 mol/l sucrose. Original magnification, x400.

 
Mouse embryos at the 2-, 4- and 8-cell stages were frozen and thawed using either EG or PROH, after which the embryos were cultured in vitro. Post-thaw survival and subsequent development of embryos in the EG and PROH groups were observed in order to identify the efficiency of EG and the optimal stage of embryonic development for slow freezing using EG (Table IGo). Morphological survival rates of 2-cell stage embryos in the EG and PROH groups were 76.1 and 15% (P < 0.05) respectively—rates that were significantly higher than those of the 4- and 8-cell stage embryos in the EG (66.7 and 64.7% respectively) and PROH (3.7 and 8.7% respectively) groups. However, the proportion of 8-cell stage embryos that developed to expanded blastocysts in the EG and PROH groups (63.8 and 9% respectively, P < 0.05) was significantly higher than that of 4-cell (29.3 and 0% respectively) and 2-cell stage embryos (6.4 and 0% respectively).


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Table I. Post-thaw survival and development of mouse embryos according to the developmental stage
 
In order to confirm that EG could be used routinely for cryopreservation of human embryos in clinical settings, both EG and PROH were used to freeze sibling embryos obtained from the same patients over a few cycles (data not shown). As far as possible, the sibling embryos were allocated equally to EG and PROH groups according to their morphological grade. Although insufficient data were acquired for a comparison to be made between EG and PROH using sibling human embryos and to form any definitive conclusion, a better post-thaw survival rate (84.6%) was observed with embryos frozen using EG compared with those frozen using PROH (64.2%). There was no significant difference in the survival rates. In addition, two pregnancies were obtained from the four embryo transfer cycles in the EG group, but none from the PROH group.

The effect of equilibration temperature on post-thaw survival of human embryos frozen using EG in a few cycles was investigated (Table IIGo). Fourteen patients were allocated to two groups according to the equilibration temperature for the freezing and thawing procedure. There was no difference between the overall morphological qualities of the fresh embryos in the two groups. Post-thaw survival rates of the embryos equilibrated at room temperature and at 37°C were 81.8 and 80.5% respectively.


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Table II. The effect of equilibration temperature on post-thaw survival of the human embryos frozen using ethylene glycol
 
In comparing the use of EG and PROH in 122 patients, there were no statistical differences in the mean age of patients and the mean number of embryos transferred between the EG (32.7 ± 3.6 and 4.0 ± 1.6 respectively) and PROH (32.4 ± 3.8 and 4.4 ± 1.7 respectively) groups (Table IIIGo). The post-thaw survival rate of human embryos in the EG group (80.6%, P < 0.05) was significantly higher than embryos in the PROH group (65.2%). Implantation (55 implanted embryos from 270 transferred; 20.3%) and clinical pregnancy rates (31 pregnancy cycles from 66 frozen–thawed embryo transfer cycles; 46.9%, P < 0.05) of embryos in the EG group were significantly higher than implantation (22 implanted embryos from 291 transferred; 7.5%) and pregnancy rates (16 pregnancy cycles from 65 embryo transfer cycles; 24.6%) of embryos in the PROH group. The delivery rate of embryos in the EG group (33.3% per transfer) was higher than in the PROH group (16.9%), but there was no statistical difference between these rates.


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Table III. Pregnancy outcome of the human embryos frozen using ethylene glycol (EG) or propylene glycol (PROH)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In previous studies, EG exhibited a stable and high permeation into human ovarian tissue (Newton et al., 1998Go), spermatozoa (Gilmore et al., 1995Go) and mouse zygotes (Emiliani et al., 2000Go) compared with other cryoprotectants. In the present study, it was also observed that mouse zygotes exposed to 1.5 mol/l EG shrank rapidly to their minimum size, but were restored to close to their initial size within 2.5 min. In contrast, zygotes exposed to 1.5 mol/l PROH exhibited relatively slow shrinkage and recovery speed, as well as a smaller change in diameter. This result indicated that EG was more efficient at inducing dehydration than was PROH. Moreover, mouse embryos exposed to 1.5 mol/l PROH + 0.2 mol/l sucrose exhibited more severe morphological changes than those exposed to 1.5 mol/l EG + 0.2 mol/l sucrose (Figure 1Go). This result indicated that embryos exposed to 1.5 mol/l PROH + 0.2 mol/l sucrose underwent severe osmotic stress, unlike those embryos exposed to 1.5 mol/l EG + 0.2 mol/l sucrose. Ethylene glycol has a lower formula weight (62.07 g/mol) than either PROH (76.10 g/mol), dimethyl sulphoxide (78.13 g/mol) or glycerol (92.09 g/mol). Hence, it is suggested that EG enters and leaves embryos very rapidly due both to its low formula weight and high permeation ability, so that embryos do not undergo osmotic shock during the freezing and thawing procedure. Interestingly, unlike the mouse embryos, there was no distinct difference observed in morphological changes of human embryos between the EG and PROH groups, though this may reflect a difference between human and mouse embryos in terms of cell membrane permeability.

Although the 2-cell stage mouse embryos showed a higher survival rate than the 4- or 8-cell stage embryos, the subsequent development of 2-cell stage embryos was significantly lower than that of the 4- and 8-cell stage embryos (Table IGo). This suggests that the developmental potency of 2-cell stage mouse embryos is more sensitive to damage attributable to freezing and thawing procedures than either 4- or 8-cell stage embryos. In mouse embryos, the 2-cell stage is known to be a genomic activation stage. Embryonic genomic activation (EGA) in mice is sensitive to treatment with cycloheximide, indicating that protein synthesis plays an important role in mediating EGA (Wang and Latham, 1997Go, 2000Go). In addition, a deleterious effect of cryopreservation on DNA synthesis in the inner cell mass of bovine embryos has been observed (Takagi et al., 1996Go). Thus, the sudden shock of the freezing and thawing procedure at this important stage may critically damage the ability of the embryos to develop subsequently at the gene level.

In the present study, the high permeation, low toxicity and high efficiency of EG on cryopreservation of mouse embryos was confirmed. This result suggests that EG might be used successfully for the slow freezing of human embryos. This possibility is supported by results of other studies in which EG was used successfully for the vitrification of human oocytes (Kuleshova et al., 1999Go; Chung et al., 2000Go) and embryos (Vanderzwalmen et al., 1997Go; Mukaida et al., 1998Go).

It has been reported that transient cooling to room temperature could cause irreversible disruption of the meiotic spindle in human oocytes (Pickering et al., 1990Go). This suggests that the cooling to room temperature may also cause damage to the mitotic spindle of human embryos. However, no detrimental effect was observed due to cooling to room temperature on the post-thaw survival of human embryos in a few cycles (Table IIGo). Although freezing and thawing of human embryos in the EG group was performed at 37°C in order to minimize the potential damage incurred by cooling to room temperature, the potential damage on mitotic spindle of the embryos requires further investigation.

Implantation and pregnancy rates of embryos in the EG group were significantly higher than those of the PROH group (Table IIIGo). This finding indicates that, due to its low toxicity and high permeation, EG may be used to cryopreserve human embryos and thereby significantly improve post-thaw survival and subsequent embryo development, in turn improving pregnancy outcome. In addition, the proportion of day-3 embryos was higher in the EG group (45.3%) than in the PROH group (10.4%), which may also contribute to the improvement of the pregnancy outcome in the former group. This possibility is supported by previous data which showed the pregnancy rate of a day-3 embryo transfer group to be significantly higher than that of a day-2 embryo transfer group (Koo et al., 1999Go).

The post-thaw survival rate (80.6%) of human embryos that were frozen using EG in the present study was not different from that of human embryos that were vitrified using EG (80 or 78.8%) in previous studies (Vanderzwalmen et al., 1997Go; Mukaida et al., 1998Go). However, the pregnancy rate (46.9%) of the EG group in the present study was significantly higher than that of either vitrified EG group (16.6 or 5.5%). This suggests that the vitrification protocol is more damaging to the subsequent development of human embryos than the slow freezing protocol, though there was no difference in the morphological survival rate of embryos between the two protocols. The difference may be a result of osmotic shock effected by the high concentration of EG used in the vitrification protocol.

In conclusion, it is suggested that EG is a good substitute for PROH for the cryopreservation of human embryos, and provides better results. Although more studies are required to compare the advantages between the slow-freezing and vitrification protocols using EG, it is suggested that the slow-freezing protocol is physiologically less damaging to human embryos than the vitrification protocol. The efficiency and optimal concentration of EG for the cryopreservation of mouse and human oocytes and blastocysts in slow-freezing protocols, and the effects of cooling on the mitotic spindle of embryos, are currently under investigation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank S.J.Park and J.Y.Kim for technical assistance in animal care, and Y.S.Moon of the Gamete and Embryo Laboratory, Vancouver Hospital & Health Sciences Center, The University of British Columbia, Canada, for his valuable revision of the manuscript.


    Notes
 
3 To whom correspondence should be addressed at: Hanna Women's Clinic, 1499-5, Seocho-dong, Seocho-gu, Seoul, 137-070, Korea. E-mail: hanna129{at}hanmail.net Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 3, 2001; resubmitted on January 18, 2002; accepted on April 3, 2002.