1 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China
2 To whom correspondence should be addressed. Email: sunnnyjenny{at}yahoo.com.cn
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Abstract |
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Key words: biopsied embryo/cryopreservation/vitrification
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Introduction |
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Materials and methods |
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Embryo biopsy
Partial zona dissection (PZD) was carried out by creating an opening in the zona pellucida mechanically. Compacted embryos were incubated for 5 min in Ca2 + - and Mg2 + -free medium (SAGE, BioPharma, USA) before PZD. One blastomere was removed from the opening of each embryo, and the fragments were also removed as much as possible. After biopsy, the embryos were incubated for 68 h before freezing with the four methods described below. Frozen embryos were stored in liquid nitrogen for 17 days before thawing. All the freezing and thawing fluids were based on phosphate-buffered saline (PBS; Sigma St Louis, MO) supplemented with 20% (v/v) inactivated fetal bovine serum (FBS, Si Ji Qing Company, China).
Embryo cryopreservation
Standard freezing method. Embryos were equilibrated in 1.5 mol/l PROH (1, 2-propanediol; Sigma) for 10 min at room temperature. Then they were transferred to 1.5 mol/l PROH with 0.1 mol/l sucrose (Sigma) and loaded into plastic straws. Cooling was conducted in a programmable freezer (Kryo 10 Series, Planar Products, Sunbury Thames, UK) at a rate of 2 °C/min from room temperature to 7 °C, at which point seeding was carried out manually. Cooling was then continued at rates of 0.3 °C/min to 30 °C and 50 °C/min to 150 °C before plunging and storing in liquid nitrogen. For the thawing process, the straw was removed from storage, held in the air for 30 s and immersed in a water bath at 30 °C for 3040 s. In the dilution procedure, the embryos were serially transferred into 1.0 mol/l PROH with 0.2 mol/l sucrose, 0.5 mol/l PROH with 0.2 mol/l sucrose and 0.2 mol/l sucrose for 5 min each, followed by sucrose-free medium at room temperature for 10 min.
Modified freezing method. The modified freezing method of Jericho et al. (2003) was used: briefly the concentration of PROH in the freezing solutions remained at 1.5 mol/l, but the sucrose concentration was doubled to 0.2 mol/l. The concentration of sucrose was also increased up to 0.3 mol/l during the first thawing steps. Thawed embryos were rehydrated by sequential transfer to 0.75 mol/l PROH + 0.3 mol/l sucrose (5 min), 0.3 mol/l sucrose (5 min) and 0.2 mol/l sucrose (10 min). Rehydration was completed by transfer to sucrose-free HEPES-buffered HTF medium (10 min).
Modified thawing method. Based on the standard freezing method, modification was made only in the thawing procedure as follows: the sucrose concentration of the warming media was increased to 1.0 mol/l, and the solution was kept heated (37 °C) during the warming procedure. The straw was held vertically, just above the surface of the warming medium after being taken out from the liquid nitrogen, and the two ends were cut off. The ice content of the straw would slip in seconds into the 37 °C medium with 1.0 mol/l sucrose. The embryos were kept in the warm media for 30 s altogether and were serially transferred into 0.5, 0.4, 0.3, 0.2, 0.1 and 0 mol/l sucrose at room temperature (5 min each).
Vitrification. Biopsied embryos were pre-equilibrated for 3 min in 10% ethylene glycol (EG; Sigma) at room temperature. Embryos were then placed for the final equilibration in 30% EG with 0.5 mol/l sucrose for 30 s. In the meantime, one to three embryos were mounted on the tip of a Cryotop (Kitazato Ltd, Tokyo, Japan) and excessive medium was sucked away using a fine pipette. The Cryotops containing embryos were immediately plunged into liquid nitrogen, and a Cryocap was used to cover the tip of the Cryotop. For the thawing process, after removing the Cryocap, the Cryotop was quickly immersed in 1 mol/l sucrose at 37 °C for 1020 s. Then the embryos were transferred to 1.0 mol/l sucrose for 1 min at room temperature and subsequently to 0.5, 0.4, 0.3, 0.2, 0.1 and 0 mol/l of sucrose at intervals of 5 min at room temperature.
Assessment of the viability of frozenthawed embryos
After thawing, embryos were placed in 10% SSS HTF-cleavage medium (SAGE, BioPharma, USA) under mineral oil at 37 °C in a humidified atmosphere of 5% CO2 in air. After 2 h incubation, embryos were assessed to determine their integrity and the number of surviving blastomeres. Embryos with half or more blastomeres intact after thawing were defined as surviving embryos and those with all blastomeres fully intact were defined as intact embryos. Therefore, the survival rate was the percentage of embryos that have 50% intact blastomeres after thawing. Surviving embryos were incubated further for 3 days to assess the blastulation potential.
Statistical analysis
The 2 test was used to compare results for embryo survival, total blastomere survival and blastulation among five groups. Statistical significance was defined as a P-value <0.05.
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Results |
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Discussion |
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In this study, we also confirmed further that the modified freezing method of Jericho et al. (2003) with an increased sucrose concentration to 0.2 mol/l significantly enhances the survival of biopsied embryos compared with standard freezing methods. The survival rate of biopsied embryos with modified freezing methods (75%) was very similar to the survival rate of non-biopsied embryos with standard methods (85%). Similarly, a modified thawing method with 1.0 mol/l sucrose added to the warming media also significantly increased the survival rate of biopsied embryos (76%). The latter result suggests that the thawing procedures are also important for recovery of frozen biopsied embryos.
It is obvious that standard cryopreservation with slow programmed procedures is very different from vitrification. Vitrification could be obtained by combining the high concentration cryoprotectant with high cooling and warming rates. During vitrification, there will not have been cell damage caused by ice. EG is a common cryoprotectant used for vitrification of mammalian oocytes and embryos because of its low toxicity and fast permeation of the cell compared with other cryoprotectants (Ali, 1993; Newton, 1998
).
Increasing sucrose concentrations in the modified method of Jericho et al. (2003) appear to be beneficial to survival of frozenthawed biopsied embryos and the further development of blastocysts of surviving embryos. Others have also reported that increasing the concentration of sucrose to 0.2 mol/l improves survival of frozenthawed human oocytes (Fabbri et al., 2001
; Chen et al., 2004
; Stachecki and Cohen, 2004
). For slow freezing programmes for embryos, the cryoprotectant generally used is PROH (membrane-permeating cryoprotectant) and sucrose (non-membrane-permeating cryoprotectant). Sucrose does not enter the cell and it mainly induces cellular dehydration through changes in osmotic pressure. Therefore, varying the sucrose concentration can change the rate of cellular dehydration to draw water out of the cells sufficiently during freezing and change the speed of cellular rehydration during warming.
Biopsied embryos have a hole on the zona pellucida, and cryoprotectant and frozen medium can freely enter the previtelline space. It is suggested that the increased susceptibility of biopsied embryos could be a consequence of both zona drilling and blastomere removal (Joris et al., 1999). However, others found that micromanipulation does not appear to be a major factor for reducing the cryosurvival of human embryos since embryos derived from ICSI survive cryopreservation at similar rates to those derived from conventional IVF (Ludwig et al., 1999
). The hole on the zona pellucida after ICSI is obviously much smaller than that after biopsy and the small hole tends to close. We noted that blastomeres near to the hole of the zona pellucida were more likely to lyse after the warming of standard slow cryopreservation. It is possibly because water velocity is different among the blastomeres located close to and further away from the hole in the zona pellucida during the warming procedure. It is proposed that the zona pellucida acts as a partial water barrier to prevent blastomeres from rupturing by excessive high speed rehydration during the warming procedure. To verify the hypothesis, the cryopreservation protocols were revised accordingly. The common component of these successful modified protocols is that the concentration of sucrose of the warming media was increased to slow down the rehydration velocity. Also the cells shrink more after freezing with higher osmolality sucrose media. These factors help the cells to remain intact during the warming process.
There are limited normal spare human embryos available for methodological research to test cryopreservation methods for biopsied embryos, therefore using non-transferable embryos generated from clinical IVF/ICSI is a good alternative. Although mouse embryos have been widely used for evaluation of optimal conditions of culture medium and for frozen oocyte and embryo methods, species difference is still a major concern of validation of the methods for human embryos. Non-transferable (poor or abnormal) embryos derived from clinical IVF/ICSI still survived well with the modified protocols after biopsy.
In summary, the preliminary results of this study suggest that vitrification may be a better method for cryopreservation of biopsied human embryos. Also the modified method is better than the standard method for biopsied embryos. This preliminary finding is worthy of further investigation using normal human embryos.
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Acknowledgements |
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References |
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Submitted on November 9, 2004; resubmitted on January 6, 2005; accepted on January 19, 2005.
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