Human Reproduction, Vol. 15, No. 8, 1787-1790,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids
1 Maria Infertility Medical Institute, 2 Department of Animal Sciences, Kon-Kuk University and 3 Maria Infertility Clinic, Seoul, Korea
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Abstract |
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Developmental capacity of human multipronuclear (PN) zygotes cryopreserved using an ultra-rapid freezing method and electron microscope (EM) grids was studied. Multipronuclear zygotes obtained from a human IVF programme were used as an alternative to normal 2PN zygotes; they were divided into 3PN or
4PN zygotes and their in-vitro development and cryo-injury were compared according to PN number. EFS30, which consisted of 30% ethylene glycol, 18% Ficoll, 0.5 mol/l sucrose and 10% fetal bovine serum with added modified Dulbecco's phosphate buffered saline was used as the freezing solution. After ultra-rapid freezing and thawing 85.5% of multipronuclear zygotes survived. A comparison of cleavage rates between the control and cryopreserved groups showed no significant difference (3PN; 81.3 and 85.4% and 4PN; 90.0 and 95.7% respectively). Comparing the in-vitro development after thawing up to blastocyst formation on day 5 after IVF, the outcome of the frozen 3PN group (22.0%) was not different from that of control 3PN group (38.5%), while the outcome of the frozen 4PN group (4.5%) was significantly lower than that of control 4PN group (44.4%) (P < 0.05).
Key words: developmental capacity/EM grid/human/multipronuclear zygotes/ultra-rapid freezing
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cryopreservation has now become an essential assisted reproduction technique in human IVF programmes so that patients have the opportunity of achieving pregnancy from more than one embryo transfer without having to be subjected to ovarian stimulation and oocyte retrieval each time (Baker, 1997
; Van den Abbeel et al., 1997). Human embryos have been cryopreserved at the unicellular pronuclear stage, at multicellular cleavage stages or at the blastocyst stage using different freezing protocols with either dimethylsulphoxide (DMSO), 1,2-propanediol (PROH) or glycerol as cryoprotective agents (Trounson and Mohr, 1983; Cohen et al., 1988; Feichtinger et al., 1991; Veek et al., 1993; Kaufmann et al., 1995). For these developmental stages, the freezing method has mainly employed slow controlled-rate cooling (Friedler et al., 1988). These methods require expensive equipment and are time-consuming. To overcome these disadvantages, simpler and faster freezing methods have been developed such as vitrification (Rall and Fahy, 1985) and ultra-rapid freezing (Trounson et al., 1987). Several pregnancies after ultra-rapid freezing of human embryos were reported (Barg and Feichtinger., 1990; Gordts et al., 1990; Trounson, 1990; Feichtinger et al., 1991). On the other hand, the efficacy of an ultra-rapid freezing method using electron microscope (EM) grids has already been reported (Martino et al., 1996; Kim et al., 1998a,b; Park et al., 1999) on bovine oocytes and blastocysts but there has still been no report of this protocol being tested on human embryos. Human embryos derived from multipronuclear zygotes as used for this study may serve as a model to evaluate a possible influence from the cryopreservation procedure, although such embryos obtained after abnormal fertilization may not have the same cryobiological properties and developmental potential as embryos obtained after normal fertilization (Noto et al., 1991; Joris et al., 1999). Our objective was to test the developmental capacity of human multipronuclear zygotes after ultra-rapid freezing using EM grids. |
Materials and methods |
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Chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA) and media from GIBCO (Grand Island, NY, USA) unless otherwise stated.
Recovery of human multipronuclear zygotes
Abnormally fertilized human oocytes were obtained from patients undergoing IVF. Briefly, the patients, after pituitary-gonadal suppression with a gonadotrophin-releasing hormone agonist (GnRHa) (Suprefact®, buserelin; Hoechst, Frankfurt am Main, Germany), were stimulated with gonadotrophin [follicle stimulating hormone (FSH) (Fostimon®; IBSA Institut Biochemique SA, Lugano, Switzerland)/human menopausal gonadotrophin (HMG) (Menogon®, Ferring, Kiel, Germany) or HMG alone] followed by the administration of human chorionic gonadotrophin (HCG; Pregnyl®; Organon, Oss, The Netherlands) for the final stage of follicular maturation. Pre-ovulatory oocytes were retrieved at 34–36 h after HCG injection, cultured and inseminated with a final concentration of 1x106 spermatozoa/ml. Zygotes were examined 16–18 h after insemination for the presence and number of PN, and polyspermic zygotes exhibiting more than 2 PN (multipronuclear zygotes) were separated from normally fertilized oocytes to be utilized for this study. Also, the multipronuclear zygotes were divided into the 3PN zygotes or more than 3PN zygotes (4PN).
Ultra-rapid freezing using EM grid
The procedure used to freeze the multipronuclear zygotes was the same as described in a previous study (Kim et al., 1998a,b). Equilibriation prior to freezing procedures was carried out at 25°C. Briefly, in this method, 400 mesh, 3.0 mm OD, copper EM grids (IGC 400; Pelco International, Redding, CA, USA) were used as a physical support for the zygotes in order to maximize cooling rates when they were plunged directly into liquid N2. EFS30, containing 30% ethylene glycol, 18% Ficoll, 0.5 mol/l sucrose and 10% fetal bovine serum with added modified Dulbecco's phosphate buffered saline, supplemented with sodium pyruvate (0.33 mmol/l), glucose (5.6 mmol/l), penicillin-G (0.0375 g/l) and streptomycin (0.025 g/l), was used as the freezing solution. The mean number of multipronuclear zygotes loaded onto one grid was about five. Freezing took place at 22–24 h after insemination. The total time that elapsed from the immersion of multipronuclear zygotes in cryoprotectant to the plunge of EM grids loaded with multipronuclear zygotes into liquid N2 was about 30 s.
Thawing and in-vitro culture
For thawing, cryoprotectants were removed by a three-step procedure at 37°C. The grids were transferred as quickly as possible into 0.5 mol/l sucrose diluted in phosphate buffered saline (PBS). They were then transferred into 0.25 mol sucrose PBS and 0.125 mol sucrose PBS. Each of the three steps needed about 1 min. After 3 min, the recovered multipronuclear zygotes were washed and co-cultured in cumulus cell monolayer drops (20 µl) of added CR1 medium (Rosencrans et al., 1993) supplemented with 10% FBS. CR1 medium is a simple serum-free medium containing 114.7 mmol/l sodium chloride, 3.1 mmol/l potassium chloride, 26.2 mmol/l sodium bicarbonate, 5 mmol/l hemi-calcium lactate, 0.4 mmol/l sodium pyruvate, 1 mmol/l L-glutamine, minimal essential medium non-essential amino acid (1% v/v), and basal medium Eagle essential amino acid (2% v/v).
Evaluation of frozen–thawed multipronuclear zygote
To assess the cryo-injury sustained by freezing and thawing of multipronuclear zygotes, survival, cleavage (24 h after thawing) and blastocyst formation (120 h after thawing) were examined (Figure 1). Embryo survival was defined as the percentage of recovered embryos that were morphologically intact after thawing and subsequent dilution of the cryoprotectant. In particular, passage through syngamy to the first cleavage division was used as an indicator of developmental potential of frozen-thawed multipronuclear zygotes. Final assessment of developmental capacity in this study was made at blastocyst formation on day 5 after thawing.
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Statistical analysis
Differences between treatment groups in each experiment were compared with
2 test using the Statistical Analysis System (SAS Institute, Cary, NC, USA) software package. |
Results |
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The survival of the human multipronuclear zygotes and the corresponding development rates after ultra-rapid freezing using EM grids are shown in Table I
. When the multipronuclear zygotes were ultra-rapidly frozen and thawed, the survival rates were high in both groups. Cleavage rates on day 2 between control and freezing groups were not significantly different for either the 3PN or 4PN groups. In addition, when the in-vitro development rates after thawing were examined on day 5 post-insemination, overall in-vitro development potential of multipronuclear zygotes after ultra-rapid freezing (15.9%) was significantly lower than that of control multipronuclear zygotes (40.9%) which had not been frozen (P < 0.05). However, the development result after freezing and thawing in the 3PN group did not differ from that of the corresponding control, although in the 4PN group it was significantly lower than in the control (P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Since the first report of a pregnancy from a frozen–thawed human embryo (Trounson and Mohr, 1983
) and the first recorded birth after such a transfer (Zeilmaker et al., 1984), many IVF groups have developed their protocols for a cryopreservation programme. However, the results obtained are clearly worse than those obtained with fresh embryos or in comparison with other mammalian species. In addition, there is no doubt that there is a marked advantage to ultra-rapid freezing when compared with conventional slow freezing methods in terms of cost and time. Human embryos have been frozen ultra-rapidly in media containing 2.0–3.0 mol/l DMSO and 0.24–0.5 mol/l sucrose with high rates of survival and development in vitro (Trounson et al., 1988; Trounson and Sjöblom, 1988). More recently pregnancies obtained by ultra-rapid freezing have been reported (Barg and Feichtinger, 1990; Gordts et al., 1990; Trounson, 1990; Feichtinger et al., 1991; Lai et al., 1996). Also, the mitochondrial distribution pattern in human multipronuclear zygotes and cleaved embryos after ultra-rapid freezing and thawing has been shown to be similar to that of control (Noto et al., 1993). These results demonstrate that the ultra-rapid freezing method should increasingly become the method of choice for human embryo freezing in IVF programmes. However, in human zygotes, ultra-rapid freezing was not proved to be an efficient method when compared with slow freezing (Van den Abbeel et al., 1997), while ultra-rapidly frozen pronucleate mouse ova showed high survival, cleavage and development rates, as effective as slow freezing (Van der Auwera et al., 1990). In our previous study using a new ultra-rapid freezing method (Kim et al., 1998a,b), we confirmed that the developmental capacity of bovine oocytes was maintained after thawing. In mature oocytes in particular, the blastocyst formation rate was shown to be similar to that in the control. Also, it is known that bovine protocols can be directly transferred to the human system (Ménézo and Joly, 1997).
In this study, we examined the new ultra-rapid freezing method for human PN stage embryos and, for experimental purposes, used multipronuclear zygotes produced by abnormal fertilization in human IVF as an alternative to normal 2PN embryos. The multipronuclear zygotes were grouped into 3PN or more than 3PN (4PN) zygotes to compare the in-vitro development and cryo-injury according to PN number. We confirmed the utility of the new ultra-rapid freezing method using EM grids when it was applied to human multipronuclear zygotes. After ultra-rapid freezing and thawing, a high survival rate (85.5%) and cleavage rate (88.7%) were obtained, although total blastocyst formation (15.9%) was significantly lower than that of control (40.9%) (Table I
). No difference was noted in the results according to PN number. Although the specimens used in this experiment were abnormally fertilized embryos, this study demonstrated that the new ultra-rapid freezing method using EM grids and EFS30 can be applied as a method of human pronuclear-stage zygote cryopreservation, the developmental capacity of human multipronuclear zygotes being maintained.
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Notes |
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4 To whom correspondence should be addressed at: Maria Infertility Medical Institute, 103–11 Sinseol-dong Dongdaemun-Gu, Seoul 130–110, Korea. E-mail: mimi60{at}unitel.co.kr
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References |
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Submitted on December 16, 1999; accepted on May 16, 2000.
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