1 Unidad de Reproduccion, Servicio de Ginecologia y Obstetricia, Hospital General Universitario de Alicante, Alicante, Spain, 2 Department of Medical Genetics, Faculty of Medicine, University of Porto, 3 Centre for Reproductive Genetics Alberto Barros, Porto and 4 Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg Prof Abel Salazar 2, 4099-003 Porto, Portugal
5 To whom correspondence should be addressed at: Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg Prof Abel Salazar 2, 4099-003 Porto, Portugal e-mail: msousa{at}icbas.up.pt
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Abstract |
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Key words: blastocyst/human/morula/open pulled straws/vitrification
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Introduction |
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The open pulled straw method (OPS) has been used for vitrifying mammalian oocytes and embryos (Vajta et al., 1997, 1998
, 1999
), giving better success rates than those obtained with normal straws (Silvestre et al., 2003
). This method has also been applied to human oocytes and early embryos (Liebermann et al., 2002a
), but not to compacted morulae and early blastocysts. In the OPS technique, the low vessel inner diameter (0.8 mm versus 1.7 mm in French straws) and holding volume (12 µl versus up to 250 µl in French straws) increase the cooling rate and allow lower cryoprotectant concentrations to be used, thereby reducing toxic injury; direct contact with LN2 can be avoided by having extra air and cryoprotectant interfaces on either side of the bead of cryoprotectant solution containing the embryos (Chen et al., 2001
; López-Béjar and López-Gatius, 2002
). The cooling and warming rates can be further modified by using glass-pulled micropipettes, due to the higher heat conductivity of glass, reduced capillary size (0.33 mm inner diameter) and reduced loading volume (12 µl) (Kong et al., 2000
).
Two studies have recently investigated whether commercial fine pipette tips could improve vitrification and facilitate handling of bovine oocytes and human zygotes, when the tips were directly immersed in LN2. In the case of the in vitro-matured bovine oocytes, the vessel was a plastic gel-loading tip (Quality Scientific Plastics, USA) that was loaded with 3 µl of solution (Asada et al., 2002). In the case of human zygotes, the Cook-IVF-Flexipet denuding glass pipette (170 µm) was used with 2 µl of solution (Liebermann et al., 2002b
). In the present report, we assessed the survival rate of excess human compacted morulae and early blastocysts that were experimentally vitrified by the OPS method using a plastic micropipette tip with a reduced inner diameter (0.36 mm) and loading volume (0.5 µl).
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Materials and methods |
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Vitrification and warming
Embryos were incubated at 37°C in sperm preparation medium as holding medium (SPM, Medicult: HEPES-buffered IVF medium, with 10% synthetic serum substitute and 7% human synthetic albumin) for 1 min, then in SPM + 7.5% ethylene glycol (EG) + 7.5% dimethylsulphoxide (DMSO) for 3 min, and finally in SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose (Sigma, Spain; Sterile, Cell culture tested) for 25 s. They were then aspirated (one or two embryos) with an automatic 0.110 µl micropipette (Gilson) using a plastic micropipette tip with a long and soft extremity (0.36 mm inner diameter; Sorenson BioScience, Inc., USA; MiniFlex Round Tips, RNase/DNase-free, Sterile, 0.110 µl, Ref: 15110) (Figure 1). The tip was then exposed to LN2 vapour for 2 min (nearly in contact with LN2), first almost horizontal and then vertical, before being removed from the automatic micropipette, and then closed inside a pre-cooled 3.6 ml cryotube (Nunc, Denmark) and plunged into LN2. Embryos were thawed 1 month later: the tip was held with thumb and middle finger for 3 s and then immersed in SPM + 0.33 mol/l sucrose (37°C), at a 3045° angle (from horizontal), taking care that all the vitrified liquid column was immersed. As the solution softened, the outer medium started to enter the tip. At this moment, the open end of the tip was closed with the index finger and the solution flew out from the tip as the result of the increased pressure of the warming air inside the tip. After 1 min, embryos were transferred to SPM + 0.2 mol/l sucrose for 5 min, then to SPM for 2x5 min, and finally individually cultured for 24 h in 50 µl drops of Blast Assist System Medium-2.
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Results |
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Discussion |
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Embryo degeneration was found in 14/63 (22.2%) of the cases, 8/30 (26.7%) for morulae and 6/33 (18.2%) for early blastocysts. When this occurred, it was characterized by absence of expansion followed by rapid darkening of the cytoplasm and cell lysis, with decompaction having only been found in late stages of degeneration. This contrasts with signs of degeneration described for mouse blastocysts (Kasai et al., 2002). Although chemical toxicity was theoretically reduced by using a mixture of cryoprotectants and sucrose and by decreasing the time of exposure due to the very low diameter of the tip and volume of the solution (Arav et al., 2002
; Liebermann et al., 2002a
), in comparison with the study of mouse blastocysts (Kasai et al., 2002
) the most likely mechanism for the observed degeneration of compacted morulae and early blastocysts seems to be the chemical toxicity of the cryoprotectant. In this study, embryos were equilibrated at 37°C before and after vitrification. Equilibration at room temperature or 4°C would decrease evaporation and cryoprotectant toxicity, but also cryoprotectant diffusion. On the other hand, equilibration and dilution at a higher temperature can increase cell permeability and thus protect against osmotic swelling and osmotic shrinkage (Kasai et al., 2002
). To prevent excess swelling occurring when the cryoprotectant is removed after warming, the embryos are usually placed in a solution with a high concentration of sugar (thought of as non-permeating). It is not clear which dilution strategy is best for human embryos, as some data suggest that six-step sucrose dilution is better (Choi et al., 2000
; Cho et al., 2002
; Son et al., 2003
), while other groups report acceptable survival rates with two (Yokota et al., 2000
, 2001
; Cho et al., 2002
) or three-step dilution (Mukaida et al., 2001
, 2003
; Reed et al., 2002
; Vanderzwalmen et al., 2002
). The dilution strategy evaluated in this study, with all temperatures at 37°C and a three-step sucrose dilution, is based on the original warming OPS procedure (Vajta et al., 1997
, 1998
, 1999
), and gives results that are similar to those reported in earlier studies on vitrified (Table I) and slow-cooled (Kaufman et al., 1995
; Gardner et al., 2003
) human blastocysts.
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Acknowledgements |
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References |
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Submitted on June 12, 2003; resubmitted on August 12, 2003; accepted on September 18, 2003.