Human Reproduction, Vol. 19, No. 2, 300-305,
February 2004
© 2004 European Society of Human Reproduction and Embryology
Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes
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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BACKGROUND: Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. METHODS: Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37°C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 µl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN2) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN2. Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. RESULTS: The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. CONCLUSIONS: The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.
Key words: blastocyst/human/morula/open pulled straws/vitrification
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Introduction |
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Vitrification is a cryopreservation method that has been successfully applied to human blastocysts using electron microscope grids (Choi et al., 2000
; Cho et al., 2002; Son et al., 2003), cryoloops (Mukaida et al., 2001, 2003; Reed et al., 2002), open plastic straws (Yokota et al., 2000, 2001) and closed plastic straws (Vanderzwalmen et al., 2002), as well as to compacted morulae using closed plastic straws (Vanderzwalmen et al., 2002). Grids and cryoloops have the highest cooling rate due to the extremely low volume of vitrification medium and immersion contact with liquid nitrogen (LN2). However, they require laborious embryo handling and carry the risk of LN2 contamination. Vitrification of expanded blastocysts may be further improved by reducing the volume of the blastocoelic cavity (Vanderzwalmen et al., 2002; Son et al., 2003), or by using a sucrose six-step dilution after warming (Cho et al., 2002).
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 (1–2 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 (1–2 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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Embryo culture and morphological assessment
Excess embryos from clinical IVF/ICSI treatment cycles (Sousa et al., 2002
) were donated by couples for whom embryo cryopreservation was declined. Cells were used after patients gave their informed consent according to Ethical Committee guidelines. In cycles with embryo transfer at day 5, injected oocytes were cultured (37°C with 5% CO2 in air) in IVF medium (Medicult, Denmark) up to the zygote stage, transferred to Blast Assist System Medium-1 (Medicult) until day 3 and then to Blast Assist System Medium-2 (Medicult). In cycles with embryo transfer at day 3, excess embryos were taken from IVF medium and cultured in Blast Assist System Medium-2 until day 5. Embryo quality was evaluated according to Staessen et al. (1995). Electron microscopy processing of embryos was as described in Sousa and Tesarik (1994).
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 0.1–10 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, 0.1–10 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 30–45° 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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For the first set of experiments, 36 excess embryos (19 compacted morulae and 17 early blastocysts) were made available for this research. These came from cycles with embryo transfer at day 5, after the embryos with the higher morphological and developmental scores had been removed for clinical transfer. These embryos were all processed for ultrastructural studies at different times after warming, 3 h after warming if degenerated or after 24 h of culture if they survived. For the second set of experiments, embryos came from cycles with embryo transfer at day 3, after the embryos with the higher morphological and developmental scores had been selected for clinical transfer. A total of 54 excess day 3 embryos were experimentally cultured for 2 further days. Of these, 27/54 (50%) developed with 11 (20.4%) reaching the compacted morulae stage and 16 (29.6%) the early blastocyst stage by day 5. These embryos were all processed for developmental studies after warming. Survival of cryopreserved embryos after warming was determined by development of morulae to blastocysts and of early blastocysts to expanded or hatching blastocysts.
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Results |
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A total of 63 embryos was vitrified. In the first experiment, the overall survival rate was 27/36 (75%), being 14/19 (73.7%) for compacted morulae and 13/17 (76.5%) for early blastocysts. In the next 24 h, all live morulae evolved to early blastocysts and all but one live early blastocyst evolved to late blastocysts, with 2/13 (15.4%) attaining the hatching stage. After warming, embryos shrunk but then re-expanded, except when degenerated. Degenerated embryos showed darkening of most of the cells, followed by the disappearance of the nuclei and cytoplasmic swelling and lysis. No zona fracture was noticed in this set of experiments. In the case of morula degeneration, blastomere darkening and lysis began at the periphery, with decompaction of the remaining blastomeres being observed only at a late stage. Focal blastomere degeneration did not compromise further evolution of morulae. In the case of blastocyst degeneration, total cell lysis was rare and in most of the cases the trophectoderm was more severely affected than the inner cell mass. When only a few cells in the trophectoderm and inner cell mass showed evidence of degeneration these did not compromise embryo development (Figure 2). In the second set of experiments, the overall survival rate was 22/27 (81.5%), being 8/11 (72.7%) for compacted morulae and 14/16 (87.5%) for early blastocysts (Figure 3). After 24 h culture, 22 embryos developed further into expanded blastocysts. One of the 16 (6.3%) early blastocysts showed a zona pellucida fracture, but did not degenerate. Two of the 14 (14.3%) early blastocysts that survived hatched. Thus, in total, 49/63 (77.7%) of the vitrified embryos survived, 22/30 (73.3%) from compacted morulae and 27/33 (81.8%) from early blastocysts, with 4/27 (14.8%) of the surviving blastocysts reaching the hatching stage.
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Discussion |
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During vitrification, water does not form ice crystals but solidifies into a glassy state (Rall and Fahy, 1985
). Vitrification depends on temperature conduction, the concentration of the cryoprotectant and the volume of the solution (Arav et al., 2002; Kasai et al., 2002; Liebermann et al., 2002a). We have assessed the survival rate of excess human compacted morulae and early blastocysts after vitrification in fine pipette tips. The method differs from those described by other studies on human (Choi et al., 2000; Yokota et al., 2000, 2001; Mukaida et al., 2001, 2003; Cho et al., 2002; Reed et al., 2002; Vanderzwalmen et al., 2002; Son et al., 2003) and animal (Vajta el al., 1999; Kong et al., 2000; Kasai et al., 2002) blastocysts in that: (i) the holding solution was adapted to human embryos using an IVF medium buffered with HEPES that contained 10% synthetic serum substitute and 7% human synthetic albumin, thus avoiding sera; (ii) the OPS plastic straw (0.8 mm inner diameter, 0.07 mm wall thickness) was replaced by a plastic micropipette tip with a reduced size (0.36 mm inner diameter, 0.077 mm wall thickness); (iii) embryos were vitrified in a smaller volume (0.5 µl versus 1–2 µl in OPS); and (iv) the tip was exposed to LN2 vapour before plunging it into LN2 inside a cryotube. This would avoid LN2 contamination and may have a further beneficial effect, in that the heat transfer would have been more uniform than if the tips had been immersed directly into LN2 (Arav et al., 2002). This, together with the low tip inner diameter and volume of the medium may also reduce the probability of zona fractures. To reduce the likelihood of fracture injury even further, the tips were warmed by holding them in the hand for 3 s (Vajta et al., 1997, 1998, 1999; Kasai et al., 2002), before immersing them into the warm dilution solution. Immediate contact with the dilution solution might cause unequal expansion of the tip and its contents (Arav et al., 2002) as well as causing the dilution solution to freeze around the tip. These factors may thus explain why this simple procedure gave an overall survival rate of 73% for compacted morulae and of 82% for early blastocysts, as well as a low incidence (1/63, 1.6%) of zona fractures.
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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The procedure reported here has several advantages over previously published protocols. The tips are sterile, commercially available, very small, simple to load with an exact, very small amount of cryoprotectant and easy to handle as they are attached to a Gilson pipette throughout most of the procedure. This is important, as complex embryo handling increases the time of exposure to the vitrification solution before cooling (Kasai et al., 2002
). Whether this technique will give clinical pregnancy rates as high as previously reported slow-cooled and vitrified human embryos now needs to be established. |
Acknowledgements |
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We acknowledge the gynecological collaboration of J.Beires and N.Montenegro (MD, PhD). This work was partially supported by the Ministry of Science and Higher Education (FCT: 36363/99, 43462/01, 35231/99, 42812/01, 48376/02, UMIB).
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Submitted on June 12, 2003; resubmitted on August 12, 2003; accepted on September 18, 2003.
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