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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Key words: developmental capacity/EM grid/human/multipronuclear zygotes/ultra-rapid freezing
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Introduction |
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Materials and methods |
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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 3436 h after HCG injection, cultured and inseminated with a final concentration of 1x106 spermatozoa/ml. Zygotes were examined 1618 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 2224 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 frozenthawed 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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Results |
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Discussion |
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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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References |
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Submitted on December 16, 1999; accepted on May 16, 2000.