Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide

S.J. Paynter1, A. Cooper, L. Gregory, B.J. Fuller and R.W. Shaw

Department of Obstetrics & Gynaecology, University of Wales College of Medicine, Cardiff CF14 4XN, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 µl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10°C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem–Katchalsky (K–K) passive coupled transport coefficients, namely Lp (hydraulic permeability), PDMSO (permeability to DMSO) and {sigma} (reflection coefficient) were derived. The resulting coefficients were Lp = 1.65 ± 0.15, 0.70 ± 0.06 and 0.28 ± 0.04 µm/min.atm; PDMSO = 0.79 ± 0.10, 0.25 ± 0.04 and 0.06 ± 0.01 µm/s and {sigma} = 0.97 ± 0.01, 0.94 ± 0.03 and 0.96 ± 0.01 at 30, 24 and 10°C respectively. The activation energy for Lp was 14.70 and for PDMSO was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects.

Key words: cryoprotectant/dimethylsulphoxide/freezing/oocyte/permeability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovulation induction techniques used in the treatment of infertility often result in the retrieval of numerous oocytes. At present, the policy of most clinics is to attempt to fertilize all of the oocytes retrieved and to place embryos, in excess of those required for the current treatment cycle, into low temperature storage. Whilst this protocol is successful, it creates legal and ethical concerns related to such issues as the length of time for which the embryos should be stored and the fate of the stored material should the parents separate or either partner die. Storage of oocytes would raise fewer ethical concerns than embryo storage and would also facilitate oocyte donation, which is currently limited due to the need to synchronize donor and recipient cycles. Perhaps the greatest advantage of oocyte storage would be to young female cancer patients, allowing them to store oocytes prior to their receiving fertility-threatening chemo/radiotherapy (Apperley and Reddy, 1995Go).

Only a small number of pregnancies and live births have been reported following human oocyte cryopreservation (Chen, 1986Go, 1988Go; van Uem et al., 1987Go). Oocytes are often rendered impenetrable by spermatozoa following freezing, although this can be overcome by using ICSI post-cryopreservation (Porcu et al., 1997Go; Polak et al., 1998Go, Young et al., 1998Go). The microtubular spindle may also be disrupted during exposure to reduced temperatures (Sathananthan et al., 1988Go; Pickering et al., 1990Go; Almeida and Bolton, 1995Go). The fact that oocytes are large cells and therefore have a low ratio of surface area to volume hinders survival post cryopreservation – oocytes are likely to retain intracellular water on freezing which will form ice, and intracellular ice formation is almost always lethal. In order to store cells at low temperatures, they must be pre-exposed to cryoprotectants which help minimize damage due to ice formation. Exposure to cryoprotectants, even without freezing, can result in extreme fluctuations in cell volume, causing cell damage or rendering the cell more susceptible to damage during subsequent cooling and ice formation. Thus exposure to cryoprotectant must be performed gradually in order to minimize cell volume fluctuations, whilst at the same time the length of exposure to cryoprotectants needs to be limited in order to keep toxicity to a minimum. The temperature of exposure to cryoprotectant is also important; lower temperatures will help reduce chemical toxicity, but as previously mentioned, low temperatures are in themselves damaging to oocytes. Exposure of human oocytes to the cryoprotectant dimethylsulphoxide (DMSO) at 37°C has been shown to impair subsequent fertilization, whereas exposure at 4°C did not (Pickering et al., 1991Go). The procedures used for the equilibration of oocytes with cryoprotectant have thus far been based on empirical techniques used for embryo freezing. The recent spate of pregnancies/live births following cryopreservation and ICSI of human oocytes have all used a freezing protocol involving exposure to 1.5 mol/l propane-1,2-diol + 0.1 mol/l sucrose with slow cooling and fast warming (Porcu et al., 1997Go; Polak et al., 1998Go; Young et al., 1998Go) without any evidence that this is indeed the best protocol. Clearly, now is the time to design cryopreservation protocols specifically for oocytes.

Oocyte freezing in animals has been considerably improved using empirically derived modifications to techniques (Bernard and Fuller, 1996Go). However, further improvements are necessary, since cryopreservation of immature murine oocytes led to loss of surrounding cumulus cells and poor development following fertilization (Cooper et al., 1998Go) and cryopreservation of mature murine oocytes resulted in lowered cell number in the inner cell mass of resultant blastocysts (Van der Elst et al., 1998Go). The design of cryopreservation protocols using theoretical models has been shown to be feasible for murine oocytes (Karlsson et al., 1996Go).

In order to optimize procedures for exposure to and removal of cryoprotectant, knowledge of several biophysical properties of cells is required (Mazur, 1990Go). The characteristics that determine the osmotic response of a cell include (i) the osmotically inactive volume (Vb), (ii) the hydraulic permeability (Lp), (iii) the solute permeability (Ps), (iv) the activation energy (Ea) of Lp and Ps and (v) cell surface area to volume ratio (Mazur, 1963Go, 1977Go, 1984Go). Such data would allow prediction of osmotic events during exposure to and dilution of cryoprotectant as well as during freezing. When cells are cooled, ice will form outside the cells initially which will draw water from within the cells (Mazur, 1963Go). The extent of cellular dehydration is determined by the permeability characteristics of the cell. In order for cells to survive freezing, they must be sufficiently dehydrated, or contain sufficient cryoprotectant, that intracellular ice formation does not occur.

Data have been accumulated concerning the permeability to water of human oocytes (Bernard et al., 1988Go; Hunter et al., 1992aGo,bGo) and the cryoprotectant permeability of murine oocytes (Jackowski et al., 1980Go; Fuller et al., 1992Go; Paynter et al., 1997Go; Agca et al., 1998Go). Whilst the murine data are useful, murine and human oocytes differ in size and hence their surface area to volume ratio. Unfortunately, few data are available on the permeability characteristics of human oocytes in the presence of cryoprotectant; Ps has been determined at 3°C in the presence of DMSO (McGrath et al., 1995Go) and at room temperature in the presence of propane-1,2-diol (Fuller et al., 1992Go). What is required are comprehensive data on permeability characteristics of human oocytes at a range of temperatures to allow determination of activation energy and hence prediction of osmotic response. Since the osmotic response will differ depending on the cryoprotectant used, such studies will need to be performed with a range of cryoprotectants. In this study, permeability data were calculated for fresh human oocytes by measuring cell volume changes following exposure to 1.5 mol/l DMSO. This is the most commonly used cryoprotectant, and the only cryoprotectant with which human live births have been reported from frozen oocytes without the use of ICSI to achieve fertilization. Best estimates of the permeability coefficients Lp, PDMSO (permeability to DMSO) and {sigma} (reflection coefficient, relative permeability of a cell to water and cryoprotectant) were generated using computer software. These measurements were determined at 30, 24 and 10°C and from these the temperature dependencies of Lp and PDMSO were then calculated using an Arrhenius relationship to determine the activation energy (Leibo, 1980Go).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Source of oocytes
Oocytes were obtained from patients, with informed consent, undergoing treatment for infertility at University Hospital Wales, Cardiff or Singleton Hospital, Swansea. Oocytes were retrieved from follicles following treatment with ovulatory drugs and were placed into modified (Whittingham, 1974Go) phosphate buffered saline (Gibco, Paisley, UK) supplemented with 4 mg/ml bovine serum albumin (Sigma, Poole, Dorset, UK, Cat. No. A3311 – PB1) at 37°C. The oocyte cumulus complexes were then placed into PB1 plus hyaluronidase (150 IU/ml; Sigma) for 4–5 min at 37°C followed by gentle pipetting to remove the cumulus cells. The oocytes were then washed three times in PB1 and were assessed for maturational stage. They were held in PB1 at 37°C until required, a maximum of 2 h.

Microperfusion
The method for microperfusion of oocytes has been described previously (Paynter et al., 1997Go). Briefly, a single oocyte was selected at random and placed in a 5 µl droplet of PB1 in the lid of a cell culture dish (3.5 mm; Falcon, Plymouth, UK). A micropipette was made, pulled from a borosilicate glass capillary (GC100–10, Clark Electromedical Instruments, Reading, UK), using a pipette puller (MPP11, Research Instruments, Penryn, UK) and a microforge (MF11, Research Instruments, UK) to have a tip opening of ~10 µm diameter. The dish containing the oocyte was placed on the stage of an inverted microscope (Axiovert 35M, Zeiss). The micropipette was filled with paraffin oil (BDH) and manoeuvred to a position adjacent to the oocyte using a micromanipulator (Zeiss, Welwyn Garden City, UK). The pipette was then used to hold the oocyte by negative pressure [generated by a threaded plunger syringe (Shardlow Micrometers Ltd, Sheffield, UK)]. The oocyte was held only by 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 adding 1 ml of DMSO (Sigma, Poole, UK), diluted in PB1 to give the required concentration (1.5 mol/l), to the 5 µl droplet by means of a Gilson pipette. The time taken between placing the oocyte in the 5 µl droplet and flushing it with the cryoprotectant was minimized to reduce evaporation from the droplet.

The experiments were performed at room temperature (24°C), 10 and 30°C. The temperature of the perfusate was controlled at 30°C by a heated stage and control unit (TRZ 3700, Zeiss) and at 10°C by a brass stage containing circulating cooled water (RTE-5DD, Neslab, Leighton Buzzard, UK). The temperature of the perfusate was monitored at frequent intervals throughout the perfusion using a copper–constantan thermocouple (Comark, Rustington, Sussex, UK). The maximum temperature variation of the perfusate during each experiment was ±2°C.

Data collection and analysis
Each oocyte was observed before, during and after the perfusion using an inverted microscope (Axiovert 35M, Zeiss) and the images were recorded by a video camera. Measurements of cell diameter across three axes were then taken, to the nearest millimetre, from freeze-frame images on the videoscreen (x740). The values measured in the presence of 1.5 mol/l DMSO were entered into the parameter estimation portion of the computer software (McGrath et al., 1992bGo) as a matrix of time and corresponding mean cell radius. The software includes a model for the coupled membrane transport in the classical Kedem–Katchalsky format (Kedem and Katchalsky, 1958Go) which comprises two coupled first-order non-linear ordinary differential equations describing the total transmembrane volume flux and the transmembrane permeable solute flux. In the present case, cells were exposed to a physiological solution with cryoprotectant present. Hence the solution was modelled as a ternary solution consisting of water, salt (non-permeating solute) and cryoprotectant (permeating solute). Details have been published elsewhere (Paynter et al., 1997Go).

The software was instructed to model for an extracellular step rise in cryoprotectant concentration from 0 to 1.5 mol/l with a corresponding decrease in non-permeating solute from 0.29 osm to 0.245 osm. Oocytes were assumed to be spherical and only oocytes that retained a circular cross-section during osmotic responses were analysed. The osmotically inactive volume was taken as 20% of initial oocyte volume. The software, which includes a model for the coupled membrane transport in the classical Kedem–Katchalsky format, derives best estimates of Lp, PDMSO and {sigma} based on the experimental data and mathematically predicted volumes using the Box–Kanemasu method of parameter estimation. At least six oocytes were analysed at each temperature.

Values for the activation energy (Ea) of the permeability coefficients in the presence of cryoprotectant were obtained by plotting Ln(Lp) or Ln(PDMSO) against 1000T–1 where T is temperature in degrees Kelvin. The activation energy is given by –{Delta}Ln(Lp or PDMSO versus 1000T–1)Rx1000 where R is the gas constant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean (±SEM) osmotic response of oocytes exposed to 1.5 mol/l DMSO at 30, 24 or 10°C is shown in Figure 1Go. Oocyte volume was plotted as normalized volume, that is the volume of an oocyte at a given time following addition of cryoprotectant solution divided by the volume of the same oocyte in isotonic medium (PB1) immediately prior to perfusion. Each oocyte shrank on initial exposure to cryoprotectant and then gradually re-expanded. As the temperature of exposure to cryoprotectant decreased, the extent of the initial shrinkage increased. The time taken to reach a minimum volume and to recover to initial volume was greater the lower the temperature.



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Figure 1. Mean normalized volume of human oocytes following exposure to 1.5 mol/l DMSO. Oocytes were exposed at 10°C ({blacksquare}), 24°C ({square}) and 30°C ({blacklozenge}). Results are expressed as mean ± SEM.

 
Figure 2Go shows the osmotic response of one oocyte from each treatment group together with the plot of the predicted response for each oocyte based on the statistical best parameter estimates for the K–K coefficients (Lp, PDMSO and {sigma}). The predicted response showed a good fit to the measured response in each case. The mean values of Lp, PDMSO and {sigma} generated for oocytes in each treatment group are given in Table IGo. The oocytes perfused at 24°C were a mixture of mature metaphase II and immature germinal vesicle breakdown (GVBD) oocytes and the combined results are presented in Table IGo. Lp was 0.73 ± 0.13 and 0.67 ± 0.04, whilst PDMSO was 0.245 ± 0.05 and 0.25 ± 0.07 for mature and immature oocytes respectively. Values for Lp and PDMSO increased with temperature, whereas {sigma} appeared to be temperature independent.



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Figure 2. Normalized volume of a typical human oocyte exposed to 1.5 mol/l DMSO. Oocytes were exposed at 10°C ({blacksquare}), 24°C ({square}) and 30°C ({blacklozenge}). The solid line for each case represents the predicted osmotic response based on the statistical best parameter estimate for the Kedem–Katchalsky permeability coefficients.

 

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Table I. Kedem–Katchalsky permeability coefficients of human oocytes exposed to 1.5 mol/l DMSO at a range of temperatures (mean ± SEM)
 
Figures 3 and 4GoGo show Arrhenius plots of the Lp and PDMSO data respectively. There is a linear relationship between Ln(Lp) or Ln(PDMSO) and 1000 T–1 over the temperature range studied (r2 = 0.97 in each case). The Ea values calculated from these data were 14.70 and 20.82 kcal/mol for Lp and PDMSO respectively.



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Figure 3. Arrenhius plot of Lp (hydraulic permeability) data in the presence of DMSO. Results are expressed as mean ± SEM.

 


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Figure 4. Arrenhius plot of PDMSO (permeability to DMSO) data (results are expressed as mean ± SEM).

 

    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
This is the first time that a value for the activation energy of Lp and PDMSO has been reported for human oocytes.

On exposure to 1.5 mol/l DMSO, the oocytes initially shrank due to water efflux from the cell, indicating that the oocytes were more permeable to water than to the cryoprotectant. As DMSO began to enter the oocytes, so their volumes increased. The movement of water and cryoprotectant across the cell membrane was slower the lower the temperature.

The value obtained for Lp in the presence of DMSO was higher than the value for Lp for human oocytes in the absence of cryoprotectant (0.40 ± 0.12) at 20°C and lower than that found at 10°C (0.40 ± 0.18) (Hunter et al., 1992aGo). Lp has been reported to be elevated for human oocytes in the presence of propane-1,2-diol (Fuller et al., 1992Go). However, these unusually high values generated for Lp may have been the result of inaccuracies in modelling the movement of molecules across the diffusion membrane. Lp has been reported to be elevated for murine oocytes in the presence of DMSO (McGrath et al., 1992aGo; Marlow et al., 1994Go). However, using the microperfusion system used in the present study, values for Lp of murine oocytes in the presence of cryoprotectant have been found to be similar to those in the absence of cryoprotectant (Paynter et al., 1997Go).

The value obtained for PDMSO at 24°C in the present study was similar to the solute permeability generated in the presence of propane-1,2-diol at a similar temperature (Fuller et al., 1992Go). The permeability characteristics of murine oocytes have been found to be almost identical in the presence of the cryoprotectants propane-1,2-diol and DMSO (Paynter et al., 1997Go).

Values for Lp and PDMSO were generally higher for human oocytes than for murine oocytes under similar conditions (Paynter et al., 1997Go), the values being significantly higher for Lp at 30°C and 24°C and for PDMSO at 30°C and 10°C (Mann–Whitney U, P < 0.05). The reflection coefficient ({sigma}) was found to be independent of temperature with values from human oocytes similar to those of murine oocytes in the presence of DMSO (Paynter et al., 1997Go). One major difference between the human and murine oocytes used in the two studies was the stage of development of the oocytes. Murine oocytes were collected after ovulation into the ampulla and were all at metaphase II, the first polar body having been extruded. Human oocytes were aspirated from follicles prior to ovulation and were, therefore, at a variety of maturational stages. The oocytes in the present study were used between 1 and 4 h after collection. Half of the oocytes perfused at 24°C were mature, i.e. had the first polar body present, whilst all oocytes perfused at 30°C and 10°C were mature. At 24°C, there was no significant difference between values for Lp and PDMSO for mature and immature (i.e. germinal vesicle breakdown – GVBD) oocytes. In the goat, immature oocytes have been shown to have a higher permeability to water and a lower permeability to propane-1,2-diol than mature oocytes (Le Gal et al., 1995Go). In the Rhesus monkey, germinal vesicle stage and metaphase I oocytes showed irregular shrinkage when subjected to hyperosmotic stress but shrinkage was regular in metaphase II oocytes (Younis et al., 1996Go). Similarly, we have found shrinkage to be non-spherical in germinal vesicle stage murine oocytes in the presence of cryoprotectant (unpublished observation). Bovine oocytes matured in vitro had a higher Lp than immature, in-vivo matured or in-vitro fertilized oocytes (Ruffing et al., 1993Go).

Previous studies on the permeability of human oocytes to water have reported large variability in the values obtained for individual oocytes (Hunter et al., 1992aGo,bGo), when perfusion took place at 5–6 h post-collection. In the present study, variability was not extensive, and was maximum at 30°C. However, only those oocytes that remained spherical on shrinkage were used for calculation of permeability coefficients, with several oocytes in each group being discarded [ten oocytes (eight mature and two GVBD) at 10°C; four oocytes (three mature and one GVBD) at 24°C and five mature oocytes at 30°C].

The value for the osmotically inactive volume (Vb) of oocytes used in the present study was 20%. Other workers have shown that Vb can vary quite widely for individual oocytes (Hunter et al., 1992aGo) and found the mean value for Vb to be 29%. When permeability coefficients were generated using the data reported and a Vb of 29%, the values were not significantly different (Mann–Whitney U (P > 0.05) from those reported (at 30°C Lp = 1.66 ± 0.17, PDMSO = 0.61 ± 0.08, {sigma} = 0.94 ± 0.01; at 24°C Lp = 0.75 ± 0.07, PDMSO = 0.18 ± 0.03, {sigma} = 0.87 ± 0.03; and at 10°C Lp = 0.33 ± 0.04, PDMSO = 0.05 ± 0.01, {sigma} = 0.94 ± 0.03).

This study is the first to report an activation energy for the values Lp and PDMSO for human oocytes. The values calculated were similar to those found with murine oocytes (11.65 kcal/mol for Lp and 23.52 kcal/mol for PDMSO). These data can be used to predict the osmotic response of oocytes in the presence of DMSO at a range of temperatures and will, therefore, facilitate the design of cryopreservation protocols which minimize osmotic stress and could lead to improved survival of oocytes post-cryopreservation. The permeability parameters for human oocytes are higher than those of murine oocytes, suggesting that the former require a shorter exposure period to DMSO. Toxic effects can thus be reduced without affecting the loading of intracellular cryoprotectant.


    Acknowledgments
 
We would like to thank the patients and staff of Cardiff Assisted Reproduction Unit, University Hospital of Wales and Singleton Hospital, Swansea without whose co-operation this work would not have been possible. Financial support for this work was provided by Wellbeing, the Health Research Charity for Women and Babies (Grant Ref. B1/93).


    Notes
 
1 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 23, 1998; accepted on May 13, 1999.