1 Department of Obstetrics and Gynaecology, Wales College of Medicine, Cardiff University, Cardiff CF14 4XN, UK, 2 Tecnobios Procreazione, Via Dante 15, 4 University of Bologna, 40125 Bologna and 3 Department of Obstetrics and Gynaecology, Vita-Salute University, H S. Raffaele, 20132 Milan, Italy
5 To whom correspondence should be addressed. Email: paynter{at}cardiff.ac.uk
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Abstract |
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Key words: cryopreservation/cryoprotectant/oocyte/permeability/propane-1,2-diol
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Introduction |
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Despite the live births attained, success using this cryopreservation technique is low (Paynter, 2000), the number of births, expressed as a percentage of the oocytes thawed, ranging from 1 to 10% and in general not exceeding 2%. In order to try to improve this, the freezing protocol has been modified. The first stage of the cryopreservation protocol involves addition of cryoprotectants; oocytes were exposed to 1.5 mol/l PrOH at room temperature and were then loaded into freezing straws and cryopreserved in 1.5 mol/l PrOH plus 0.1 mol/l sucrose. Addition of cryoprotectant can, if performed inappropriately, create potentially damaging fluctuations in cell volume. Exposure to cryoprotectant ideally needs to be performed in a manner that minimizes osmotic perturbations, as well as the duration of exposure to these potentially toxic chemicals, whilst allowing sufficient permeation to achieve protection from freezing injury. To this end, various modifications of the cryoprotectant addition strategy have been applied. These include increasing the temperature at which exposure to the cryoprotectant was performed so as to achieve faster permeation of the cryoprotectant thereby reducing the necessary exposure time (Yang et al., 1998
). The duration of exposure to 1.5 mol/l PrOH at room temperature has been reduced from 15 to 10 min, with improved survival (Fabbri et al., 2000
). Stepwise addition of the permeating cryoprotectant (PrOH) has also been adopted in an attempt to reduce osmotic perturbations (Quintans et al., 2002
). However, a comparison of stepwise and one-step addition protocols was not performed in the study of Quintans et al. (2002)
and the method included numerous other alterations to the freezing protocol such as the use of a sodium-depleted freezing medium. Sodium-depleted medium in combination with a reduced seeding temperature (6 versus 7 °C) and sucrose dilution rather than stepwise dilution of cryoprotectant has yielded good survival and live births (Boldt et al., 2003
). The most notable modification of the slow coolrapid thaw PrOH protocol has been to increase the concentration of the sucrose in an attempt to dehydrate the oocyte prior to freezing. Live births have been achieved by freezing in the presence of 1.5 mol/l PrOH plus 0.2 mol/l sucrose (Porcu et al., 1997
, 2000
) and 0.3 mol/l sucrose (Fosas et al., 2003
). Significantly better survival post-thaw has been reported when human oocytes were cryopreserved in the presence of 0.2 mol/l sucrose rather than 0.1 mol/l (Chen et al., 2004
), and the presence of 0.3 mol/l sucrose resulted in greater post-thaw survival compared with 0.1 or 0.2 mol/l sucrose (Fabbri et al., 2001
).
Changes to the cryoprotectant addition steps of cryopreservation protocols have thus been implemented successfully. However, no measurement of the actual response of the cells to these changes was performed in these studies. In some cryopreservation studies, the time of exposure to cryoprotectant prior to freezing has not been stated (Gook et al., 1993; Young et al., 1998
). In other studies, the time of exposure to the cryoprotectant varied greatly between experimental runs (Tucker et al., 1996
, 1998a
,b
; Fabbri et al., 2001
), in some cases on an arbitrary basis and in others being dependent upon the number of oocytes being frozen at one time. Such differences can be expected to result in extreme differences in the degree of hydration of the cell on freezing and hence to impact greatly on the fate of that cell following freezing and thawing.
This study measured the osmotic response of oocytes during a two-step addition of the permeating cryoprotectant PrOH. The response of mature human oocytes on exposure to 0.75 mol/l PrOH was monitored over a 10 min period. In a separate series of oocytes, the response on exposure to 1.5 mol/l PrOH for 10 min, following equilibration with 0.75 mol/l PrOH, was measured. In a further set of experiments, the osmotic response of oocytes on exposure to 1.5 mol/l PrOH plus 0.2 or 0.3 mol/l sucrose was measured following equilibration with 1.5 mol/l PrOH. The osmotic response of oocytes in the presence of 1.5 mol/l PrOH plus sucrose was compared with computer simulations generated using permeability data generated in the presence of PrOH or PrOH plus sucrose. This is the first report of such measured data and allows determination of the time required to achieve equilibration with permeating cryoprotectants, the relative merit of single-step versus two-step addition of PrOH and the quantification of the dehydrating effect of the presence of 0.2 or 0.3 mol/l sucrose in the cryoprotectant solution.
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Materials and methods |
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Microperfusion
The method for microperfusion of oocytes has been described previously (Paynter et al., 2001). Briefly, a single oocyte was placed in a 5 µl droplet of Dulbecco's phosphate-buffered solution (PBS) (Gibco, Life Technologies, Paisley, UK) supplemented with 20% (final concentration, 10 mg/ml) plasma protein solution (PPS) (Baxter AG, Vienna, Austria). The dish containing the oocyte was placed on the stage of a Nikon Diaphot 200 inverted microscope. A holding micropipette (Cook IVF, Brisbane, Australia) with a 1.5 µm diameter tip opening was used to hold the oocyte in the correct position during perfusion. The micropipette was filled with PBS and positioned adjacent to the oocyte using a Narishige micromanipulator. The pipette was then used to hold the oocyte by negative pressure generated by a Narishige IM-5A injector applied to the outer zona pellucida, care being taken not to deform the inner oolemma (measurement of the magnitude of negative pressure was not conducted in these experiments). The oocyte was then perfused by carefully adding 1 ml of perfusate by means of an air displacement pipette. The time taken between placing the oocyte in the 5 µl droplet and flushing it with the perfusate was minimized to reduce evaporation from the droplet.
Four sets of experiments were performed. (i) Oocytes were placed in a 5 µl droplet of PBS + PPS and perfused with 0.75 mol/l PrOH (Fluka, Milan, Italy) in PBS + PPS. (ii) Oocytes were placed in 0.4 ml of 0.75 mol/l PrOH in PBS + PPS for 7.5 min prior to being transferred to a 5 µl droplet of the same solution and perfused with 1.5 mol/l PrOH in PBS + PPS. For experiments (iii) and (iv), oocytes were placed in 0.4 ml of 1.5 mol/l PrOH in PBS + PPS for 10 min and were then transferred to a 5 µl droplet of the same solution and perfused with 1.5 mol/l PrOH plus (iii) 0.2 mol/l sucrose or (iv) 0.3 mol/l sucrose (Fluka, Milan, Italy) in PBS+PPS. The experiments were performed at room temperature (25±1 °C).
Data collection and analysis
Each oocyte was observed before, during and after perfusion using the inverted microscope and the images were recorded by a video camera. Using computer software designed by N.Gallaghan of Wales College of Medicine, Cardiff University, three approximately equidistant points were marked on the oocyte membrane and the radius of the circle formed by joining these points was calculated. This was repeated at a set of three different positions on the oocyte membrane and the mean radius was taken as the radius of the oocyte at a given time point. The volume of the oocyte at each time point was calculated and was normalized to the volume of the oocyte immediately prior to perfusion.
Simulated osmotic responses of oocytes in the presence of 1.5 mol/l PrOH plus 0.2 or 0.3 mol/l sucrose were generated using the mean values generated for the permeability of the cell to water (hydraulic conductivity), to the cryoprotectant PrOH and the reflection coefficient. Computer software (McGrath et al., 1992) was provided with values determined from oocytes exposed to 1.5 mol/l PrOH at 24 °C (Paynter et al., 2001
) (0.53 µm/min/atm, 0.28 µm/s and 0.94, respectively) or values generated in the presence of 1.5 mol/l PrOH plus 0.2 mol/l sucrose (0.43 µm/min/atm, 0.11 µm/s and 0.61, respectively) and instructed to model for an oocyte placed in a constant concentration of 1.5 mol/l PrOH and a step rise in non-permeating solute from 0.257 osm to either 0.457 or 0.557 osm (the changes contributed by the sucrose).
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Results |
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Discussion |
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A two-step addition of PrOH involving, for example, equilibration in 0.75 mol/l PrOH followed by exposure to 1.5 mol/l PrOH would take longer to achieve the same intracellular concentration of cryoprotectant than would a one-step addition of 1.5 mol/l PrOH, but would result in reduced osmotic stress. Increasing the temperature at which exposure to cryoprotectant is performed would reduce the time needed to achieve equilibration and, indeed, would further reduce the extent of cell volume excursions (Paynter et al., 2001). A further advantage of increasing the temperature of exposure would be to reduce temperature-related disruption of the cytoskeleton (Pickering et al., 1990
). However, increasing the temperature could increase any cytotoxic effects of PrOH, although short exposure (5 min) at temperatures between 37 and 32 °C has been shown to be tolerated by human oocytes (Yang et al., 1998
).
Exposure of oocytes, previously equilibrated in 1.5 mol/l PrOH, to 1.5 mol/l PrOH in the presence of sucrose causes human oocytes to shrink drastically with no recovery of volume because sucrose does not permeate into the cells. After 3 min exposure to the solution containing 0.3 mol/l, oocytes exceeded the 30% threshold of cell volume excursion whereas in the same situation, but with the sucrose concentration reduced to 0.2 mol/l, the 30% level of volume change was not reached until after 6 min of exposure. In a retrospective analysis, variation in the time of exposure of oocytes to 1.5 mol/l PrOH plus 0.2 mol/l sucrose was found to impact on oocyte survival, exposure for 10.515 min being better than either 5.510 min or 30 s5 min (Fabbri et al., 2001
). The significant difference in survival in the study of Fabbri et al. (2001)
corresponds, in this study, to only slight differences in oocyte volume (29% shrinkage versus 33%). It has been postulated that sucrose is beneficial during cryopreservation because it has a stabilizing effect on lipid membranes (Crowe et al., 1998
); it is possible that the longer exposure time better facilitates this effect. Microinjection of the sugar trehalose has been shown recently to be tolerated by murine oocytes up to an intracellular concentration of 0.15 mol/l (Eroglu et al., 2003
). The presence of a sugar both inside and outside the cell may increase its beneficial effect during cryopreservation. On the other hand, exposure to sucrose has been shown to have detrimental effects on oocytes. Exposure of cat oocytes to 0.5 mol/l sucrose for 5 min has been shown to significantly reduce development compared with exposure for just 1 min (Murakami et al., 2004
).
The dehydration caused by the presence of sucrose and resultant shrinkage to 30% volume immediately prior to the onset of cooling would appear not to be detrimental to the survival of the oocyte. In the presence of 0.3 mol/l sucrose plus 1.5 mol/l PrOH, oocytes shrink to 30% of their original volume by 3 min and to 40% of their original volume by 5.5 min exposure. In the two studies where 0.3 mol/l sucrose has been incorporated into the freezing solution (Fabbri et al., 2000
; Fosas et al., 2003
), the duration of exposure of human oocytes to this solution has not been defined. It has been shown that human oocytes retain integrity of the cell membrane following shrinkage to
40% of initial volume in the presence of saccharides (McWilliams et al., 1995
). However deformation of the spindle has been reported following exposure of human oocytes to hypo and hyperosmotic conditions (Mullen et al., 2004
). If, as was the case with Fabbri et al. (2001)
, the length of exposure to the freezing solution is dependent upon the number of oocytes to be frozen and 40% shrinkage prior to cooling is detrimental, then the use of a 0.2 mol/l sucrose solution would allow a longer window of opportunity for loading oocytes into freezing straws and placing them in a freezing machine, as well as allowing greater duration of exposure to the potentially beneficial effects of sucrose, than would the 0.3 mol/l sucrose solution.
The prediction of the osmotic response of human oocytes on exposure to PrOH plus 0.2 or 0.3 mol/l sucrose has been performed using permeability coefficients generated in the presence of PrOH alone or in the presence of PrOH plus sucrose. The presence of cryoprotectant has been shown to reduce the coefficient for water permeability (Adams et al., 2003). In this study, the presence of sucrose and PrOH resulted in reduced water and cryoprotectant permeability coefficients compared with those under similar conditions in the presence of PROH alone (Paynter et al., 2001
). The osmotic response of oocytes in the presence of 1.5 mol/l PrOH plus 0.1 mol/l sucrose has been accurately predicted using coefficients generated in the presence of PrOH (Paynter et al., 2001
) but, as the concentration of sucrose was increased, the predicition of osmotic response using these coefficients became less reliable.
The data generated in this paper have been based on relatively small numbers of oocytes, since the availability of human oocytes for use in research is understandably low. However, the variability within groups of data was sufficiently small to suggest that the data produced are representative of human oocytes in general. A small proportion of the oocytes perfused were not used for data generation, either because they were non-spherical, either before or during perfusion, making accurate volume calculation difficult, or because volume was shown to increase on initial exposure to the sucrose-containing solution.
Probably the most important factor in determining success in an oocyte cryopreservation programme is the quality of the oocytes prior to treatment (Paynter, 2000). The steps of a cryopreservation protocol are numerous and the effects are inter-related. In order to optimize and standardize survival post-cryopreservation, even with the best quality oocytes, it is essential to optimize survival at each stage and, having done so, to be rigorous in applying those steps in clinical practice. As demonstrated here, a difference of just a minute or two in the duration of exposure to a cryoprotectant can result in extreme differences in the volume of the cell and is likely to have drastic consequences during the subsequent freezing and thawing. The concept of storing cells in a frozen state is deceptively simple, but in reality requires an appreciation of a variety of biophysical events and a vigilance in applying the various steps required during a cryopreservation protocol (Paynter and Fuller, 2004
). This is particularly the case with the procedure of vitrification which involves higher concentrations of cryoprotectant (68 mol/l) and where the difference of just a few minutes in the duration of exposure to cryoprotectant can significantly alter survival (Wood et al., 1993
). Recently, vitrification has been applied to human oocyte storage (Kuleshova et al., 1999
; Katayama et al., 2003
; Yoon et al., 2003
) and, although the cryoprotectant used, namely ethylene glycol, appears to be well tolerated by oocytes, there is evidence that oocytes have greater sensitivity to osmotic stress following vitrification (Murakami et al., 2004
), thus care should be taken, particularly during dilution of the cryoprotectants. Greater attention to detail in applying vitrification clinically will be required because of not only the high cryoprotectant concentrations required but also the fast cooling and warming rates that are essential to the success of this technique.
By measuring the osmotic response of oocytes during the various steps involved in addition of cryoprotectant, protocols can be designed which keep cell volume within tolerated limits or achieve set levels of dehydration prior to freezing. In this way, time periods for each step can be defined which, if adhered to, should reduce the variability in survival currently seen in human oocytes post-cryopreservation.
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Submitted on March 8, 2004; resubmitted on December 7, 2004; accepted on December 13, 2004.