1 IVF Center, Human Reproductive Medicine Unit, Institute of Obstetrics and Gynecology, and 2 Department of Biology, University of Bologna, 40138 Bologna, Italy
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Abstract |
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Key words: cryoprotectants/exposure time/human oocyte cryopreservation/oocyte survival/sucrose concentration
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Introduction |
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The human metaphase II oocyte appears to be particularly susceptible to freezethaw damage, and it has been suggested that several forms of cryo-injury are responsible for the relative lack of success in preserving human oocytes. These include damage to the meiotic spindle and to unstably bound chromosomes (Magistrini and Szollosi, 1980; Sathananthan, 1988a, b; Pickering et al., 1990
; Van der Elst et al., 1992
; Gook et al., 1993
), to the microfilaments essential for polar body extrusion, pronuclear migration and cytokinesis (Vincent et al., 1990
), to the zona pellucida such as breaches and hardening (Johnson et al., 1988
; Johnson, 1989
; Todorow et al., 1989
), and to the cortical granules causing a premature cortical reaction (Schalkoff et al., 1989
; Vincent et al., 1990
; Gook et al., 1993
; Al-Hasani and Diedrich, 1995
).
It is important to mention that, today, an important problem limiting oocyte cryopreservation procedure is the survival rate after thawing. Studies using metaphase II oocytes showed that the oocyte survival rate after cryopreservation could be affected by morphological and biophysical factors.
Among the morphological factors, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. It was postulated that the cumulus cells may offer protection against the adverse effect of the cryoprotectant and/or cooling in a way not yet explained (Imoedemhe and Sigue, 1992). In the literature, few investigators have focused their attention on the effect of denuding the oocytes of their cumulus cells before cryostorage. However, the results reported in the few studies involving a low number of oocytes are controversial (Mandelbaum et al., 1988
; Imoedemhe and Sigue, 1992
; Gook et al., 1993
).
The main biophysical factor affecting the human oocyte survival is the intracellular ice formation that generally pierces the membrane causing cell lysis. Because the human oocyte is a large cell containing a large quantity of water, it requires a long time to reach adequate dehydration (osmotically balanced by the cryoprotectant solution) before lowering the temperature and thus it is more difficult to avoid ice crystal formation. Intracellular ice formation can be affected by the presence of the cryoprotectants in the freezing solutions, and by the freezing and thawing rate (Shaw, 1993).
The cryoprotectants generally used in oocyte freezing protocols are 1,2-propanediol (PROH, membrane-permeating cryoprotectant) and sucrose (membrane-non-permeating cryoprotectant). Their protective action is very complex and attributable to a number of properties (Shaw, 1993), the most important of which is the beginning of the dehydration process. In particular, sucrose does not enter the cell, but exerts its beneficial effects by causing cellular dehydration through changes in osmotic pressure (Friedler et al., 1988
): the increase of the extracellular solute concentration generates an osmotic gradient across the cell membrane, which draws water out of the cell, causing the cell to dehydrate before the freezing procedure.
Furthermore, it is extremely important to establish what the optimal exposure time of the oocyte to cryoprotectant solutions is. It has to be long enough to permit sufficient dehydration of the cell, but not so long as to damage the cell since it alters the intracellular pH as well as the developmental potential as seen in mouse zygotes (Damien et al., 1990). It was suggested that an exposure time of 10 min could be suitable for improving the survival rate of human oocytes (Al-Hasani and Diedrich, 1995
).
The aim of this study was: (i) to investigate the effect of the presence of cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to the cryoprotectant solutions on oocyte survival after thawing and (ii) to evaluate the efficiency of a human oocyte slow-freezing-rapid-thawing protocol using 1,2-propanediol and sucrose as cryoprotectants in a clinical trial in which the fertilization rate of thawed oocytes and the subsequent embryo development rate were assessed.
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Materials and methods |
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The oocyte-cumulus complexes were separated from their follicular fluid and transferred to 1 ml flushing medium (Medi-cult, CGA/Diasint, Florence, Italy) and incubated at 37°C in an atmosphere of 5% CO2 in air.
Cryoprotectant solutions
All cryoprotectant solutions were prepared using Dulbecco's phosphate-buffered solution (PBS) (Gibco, Life Technologies Ltd, Paisley, UK), 1,2-propanediol (PROH) (Fluka Chemika, Sigma Aldrich SrL; Milan, Italy) and a serum protein supplement (SPS; Pacific Andrology, CGA/Diasint, Florence, Italy). The `freezing solutions' were prepared: (i) 1.5 mol/l PROH + 30% SPS (15 mg/ml of plasma proteins) in PBS (equilibration solution) and (ii) 1.5 mol/l PROH + sucrose (0.1 or 0.2 or 0.3 mol/l) + 30% SPS in PBS (loading solution). For thawing procedure, the oocytes dehydrated in 0.1 or 0.2 mol/l sucrose were thawed in solution containing 0.2 mol/l sucrose and only the oocytes dehydrated in 0.3 mol/l sucrose were thawed in solutions with 0.3 mol/l sucrose.
Oocyte freezing/thawing programme
The cryopreservation protocol consisted of a slow freezingrapid thawing method.
After no more than 23 h of incubation, all the oocytes were transferred to Petri dishes containing PBS supplemented with 30% SPS at room temperature. One or two oocytes were placed in 0.5 ml of equilibration solution and maintained for 10 min at room temperature before transfer to 0.5 ml of the loading solution.
The oocytes were loaded in plastic straws (Paillettes Cristal 133 mm; Cryo Bio System, France) and transferred into an automated Kryo 10 series III biological vertical freezer (Planer Kryo 10/1.7 GB).
The initial chamber temperature was 20°C. Then the temperature was slowly reduced to 7°C at a rate of 2°C/min. Ice nucleation was induced manually at 7°C. After a hold time of 10 min at 7°C, the straws were cooled slowly to 30°C at a rate of 0.3°C/min and then rapidly to 150°C at a rate of 50°C/min. After 1012 min of stabilization temperature, the straws were transferred into liquid nitrogen tanks and stored until thawing.
To thaw, the straws were air-warmed for 30 s and then immersed in a 30°C water bath for 40 s until all traces of ice had disappeared. The cryoprotectant was removed at room temperature by stepwise dilution of PROH in the thawing solutions. The contents of the melted straws were expelled in 1.0 mol/l PROH + sucrose (0.2 or 0.3 mol/l) solution + 30% SPS and the oocytes were equilibrated for 5 min. Then the oocytes were transferred to 0.5 mol/l PROH + sucrose (0.2 or 0.3 mol/l) solution + 30% SPS for an additional 5 min and then in a sucrose (0.2 or 0.3 mol/l) solution + 30% SPS for 10 min before final dilution in PBS solution + 30% SPS for 20 min (10 min at room temperature and 10 min at 37°C). Finally the oocytes were cultured in IVF medium (Universal IVF medium Medicult) at 37°C in an atmosphere of 5% CO2 in air.
After 1 h, the oocytes were checked for survival. The oocytes were classified as `survived' if the zona pellucida and plasma membrane were intact, the perivitelline space was clear and normal in size and if there was no evidence of cytoplasmic leakage or oocyte shrinkage and there was virtually no space between the zona pellucida and the cytoplasm.
Experimental design
A. Oocyte survival rate: influence of cumulus and sucrose concentration
A total of 308 oocytes, collected from 18 counselling patients, were randomly divided into two groups: in the first, 184 oocytes had the cumulus oophorus partially removed mechanically using syringe needles; in the second, 124 oocytes were completely enzymatically denuded by a brief exposure to 40 IU/ml hyaluronidase (Type VIII; Sigma, Aldrich Srl, Milano, Italy) and by pipetting through a narrow bore glass pipette. The oocytes of both groups were cryopreserved using 1.5 mol/l PROH + 30% SPS (equilibration solution) and 1.5 mol/l PROH + 0.1 mol/l sucrose + 30% SPS (loading solution).
A total of 718 oocytes collected from 30 counselled patients were randomly divided into two groups: in the first, 354 oocytes had the cumulus oophorus partially removed mechanically; in the second, 364 oocytes were completely enzymatically denuded. The oocytes of both groups were cryopreserved using 1.5 mol/l PROH + 30% SPS (equilibration solution) and 1.5 mol/l PROH + 0.2 mol/l sucrose + 30% SPS (loading solution). The cryoprotectant removal was performed in the presence of 0.2 mol/l sucrose concentration in all the `thawing solutions' for the oocytes cryopreserved with 0.1 and 0.2 mol/l sucrose concentrations.
A total of 224 oocytes collected from 48 counselled patients were randomly divided into two groups: in the first one, 124 oocytes had the cumulus oophorus partially removed mechanically; in the second one, 100 oocytes were completely enzymatically denuded. The oocytes of both groups were cryopreserved using 1.5 mol/l PROH + 30% SPS (equilibration solution) and 1.5 mol/l PROH + 0.3 mol/l sucrose + 30% SPS (loading solution) and thawed using the `thawing solutions' containing 0.3 mol/l sucrose.
A. Oocyte survival rate: influence of exposure time to cryoprotectants (retrospective analyses)
In a group of 1130 thawed oocytes (86 patients), the effective oocyte exposure time to 0.2 mol/l sucrose solution was retrospectively calculated from the time in which the oocytes were loaded into the straw to the time the freezing programme started. This calculation was performed considering that a time of 30 s was necessary between the loading of one straw and the next; this is the time required to load the oocytes into the straw and to put them into the Planer Kryo 10 before starting the cooling programme. On the basis of this calculation, it was considered that when there were several patients with a large number of oocytes to be frozen simultaneously, by allocating two oocytes per straw, the oocytes were exposed to the sucrose solution for a time that could range from 30 s (i.e. if there were only two oocytes to cryopreserve) to 15 min (i.e. if there were
60 oocytes to cryopreserve). The oocytes were retrospectively divided into three groups depending on their exposure time: first group:
30 s and
5 min (0.55 min); second group: >5 min and
10 min (5.510 min); third group: >10 min and
15 min (10.515 min).
Clinical trial
Semen samples showed normospermic parameters according to World Health Organization (WHO, 1992) criteria. Sperm selection was done using the Percoll technique and the sperm suspension was kept in a 37°C incubator until intracytoplasmic sperm injection (ICSI) of the oocytes was performed.
Only the oocytes frozen and thawed in 0.2 mol/l sucrose solution, morphologically intact and with the polar body extruded were microinjected using ICSI (Van Steirteghem et al., 1993). The injected oocytes were then returned to the culture in IVF medium at 37°C in an atmosphere of 5% CO2 in air. The oocytes were examined between 16 and 20 h post microinjection to determine the presence of two pronuclei and the extrusion of the second polar body. Normally fertilized oocytes were transferred to the IVF medium.
The embryos were scored from best to worst as grade I, II, III, IV or V on day 2 according to development and morphological quality. The embryo grading was based on morphological appearance and the embryo development rate (Cummins et al., 1986). The morphological parameters were regularity of size, the shape of the blastomeres and the presence or absence of cytoplasmic vacuoles, granulations and extracellular fragments.
The results obtained in experiment A, expressed as percentages, were compared using 2-tests. P < 0.05 was considered significantly different using a two-tailed test. The data obtained in experiment B were statistically analysed using a comparison between the survival percentages obtained by the evaluation of the 95% confidence limits.
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Results |
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Furthermore the oocyte survival rate significantly increased in the presence of a tripled sucrose concentration. The oocytes cryopreserved in the presence of a 0.3 mol/l sucrose solution showed a survival rate of 82% (183/224) which was significantly higher than the 34 and 60% obtained in the group of oocytes cryopreserved in the presence of a 0.1 and 0.2 mol/l sucrose solution respectively (P < 0.001) (Table I).
B. Oocyte survival rate: influence of exposure time to cryoprotectants (retrospective analyses)
By a single observation performed under an inverted microscope to assess the oocyte morphological changes, it was found that at T0, when the oocyte had just been put into the loading solution (Figure 1a), the ooplasm showed regular dimensions and a regular perivitelline space. The same oocyte, after 15 min in 1.5 mol/l PROH supplemented with 0.2 mol/l sucrose (Figure 1b
), showed a shrunken ooplasm and plasmalemma with an increased perivitelline space as a consequence of contact with the high molarity of the solution. This reduction, reached after 15 min of exposure, has been estimated as ~20% of the diameter. The oocyte did not exhibit any other changes in its morphology after a 15 min exposure time.
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Discussion |
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Contrasting results are reported in the literature regarding the human oocyte survival rate after cryopreservation with or without the cumulus oophorus and using a solution consisting of 1.5 mol/l PROH to which 0.1 mol/l sucrose was added. It was observed (Mandelbaum et al., 1988) that the presence of the cumulus mass or the partial or the total removal of cumulus cells did not significantly modify the oocyte survival rate (36, 20 and 44% respectively). By contrast, it was found that the oocytes surrounded by a total cumulus and corona mass, as retrieved at ovum recovery, had a significantly reduced survival rate (48%) compared with those oocytes which had the mass removed prior to freezing (69%), suggesting that the presence of cumulus cells and the cumulus matrix causes a different rate and extent of dehydration during cryopreservation (Gook et al., 1993
). The cumulus-corona complex may also form a more rigid structure limiting the distortion of oocyte shape which occurs during ice formation in the cytoplasm (Ashwood-Smith et al., 1988
). The results of the current study agree with Mandelbaum's findings since the presence or absence of the cumulus oophorus does not influence the survival rate of the oocytes cryopreserved in the presence of 0.1 mol/l sucrose. Moreover, in the current study these data have also been confirmed by using 0.2 or 0.3 mol/l sucrose, showing no influence by cumulus oophorus on oocyte survival rate. These latter results are in contrast with those of others (Imoedemhe and Sigue, 1992
) who cryopreserved the oocytes in the presence of a 0.25 mol/l sucrose concentration. Imoedemhe and Sigue appear to be the only authors who used a sucrose concentration >0.1 mol/l, which was between the 0.2 and 0.3 mol/l concentrations experimented in the current study. They found that the oocyte survival was significantly better in oocytes with intact cumulus as compared with those without cumuli (54 and 27% respectively). The authors postulated that the presence of the cumulus mass may offer some protection against sudden osmotic changes and stresses which could be induced by rapid influx and/or efflux of the cryoprotectant during the procedures of equilibration and removal of the cryoprotectant in the pre-freeze and post-thaw periods respectively.
The variability in oocyte survival between this study and our previous results, in which a trend towards enhanced survival of the cumulus enclosed oocyte was observed with respect to cumulus denuded oocytes (Fabbri et al., 1998), may indicate such fluctuations in oocyte quality which may reflect some changes in water and cryoprotectant oocyte membrane permeability as was also previously reported (Gook et al., 1995
).
Furthermore, all these findings in the literature refer to experimental studies using a low number of oocytes, and, therefore, it is very difficult to make a definitive assessment regarding the role of the cumulus oophorus on oocyte survival after thawing. On the contrary, in the current study, the comparison was performed on a large number of oocytes (~900) allowing the suggestion that the cumulus mass has no effect on survival rate.
However, it is important to establish the exact role of the cumulus oophorus in the cryopreservation process in order to be confident as to the nuclear maturity and cytoplasmic health of the oocyte before cryopreservation. If the cumulus oophorus has no influence on oocyte survival, we suggest that it would be worthwhile to cryopreserve the oocytes completely denuded in order to make an exact evaluation of the quality and the maturation stage of the cryopreserved oocyte.
The presence of cryoprotectants (both permeating and non-permeating) in the freezing solution should minimize cell damage during the freezing and thawing process. For oocyte cryopreservation procedures, cryoprotectant concentrations are usually ~1.5 mol/l, many times higher than any other component in the medium. Thus the cryoprotectants enter the cell by osmosis. While the cryoprotectants readily cross the cell membranes, water usually crosses even more readily (Shaw, 1993).
The results presented here showed that a double, and, even more so, a tripled sucrose concentration significantly increased the oocyte survival rate (60 and 82% respectively; P < 0.001). Probably a 0.1 mol/l sucrose concentration was not sufficient to allow suitable oocyte dehydration before lowering the temperature as demonstrated by the overall low survival rate observed (34%). On the other hand, a 0.3 mol/l sucrose concentration, as shown by a significantly higher oocyte survival rate obtained using this sucrose concentration, probably causes a more adequate loss of intracellular water without excessive oocyte shrinkage which could lead to the collapse of the cellular membranes. This appears to be the first time that a 0.3 mol/l sucrose concentration was used in a human oocyte cryopreservation protocol.
The results obtained in this retrospective study suggest that poor results with the shorter times in 0.1 and 0.2 mol/l sucrose reflect insufficient oocyte dehydration and that the 15 min in 0.2 mol/l is approaching the extent of dehydration achieved with 0.3 mol/l sucrose for 30 s (time to load).
This appears to be the first time that a similar evaluation was performed, and it is suggested that satisfactory oocyte dehydration should be obtained before lowering of the temperature; this could further avoid the formation of intracytoplasmic ice crystals which are the main factor influencing the oocyte survival rate during cryopreservation procedures.
These encouraging results indicated that an increased sucrose concentration (0.3 mol/l) has enhanced the oocyte survival and suggests that insufficient dehydration results in poor survival rates. Further studies are required to verify if the combined action of a longer exposure to cryoprotectants with an increased sucrose concentration in freezing solutions could improve the oocyte survival rate after thawing.
In addition, based on the results of this study it appears possible not only to achieve a high survival rate of cryopreserved human oocytes but also to successfully fertilize these oocytes and obtain a high cleavage rate with satisfactory embryo development.
The results presented here indicate that it is possible to cryopreserve human oocytes and that ICSI could be an efficient method of achieving a satisfactory outcome in terms of fertilization. The overall survival rate obtained in the clinical trial after thawing was 54%, which is lower than that obtained in the experimental studies. This lower percentage could be explained considering that the clinical trial also includes those patients who have had their oocytes cryopreserved in the presence of 0.1 mol/l sucrose (with a low oocyte survival rate). The normal fertilization rate was 58%, which compares well with that obtained from routine IVF. Furthermore, the current data showed a lower abnormal fertilization rate (11%), with respect to Gook's findings (21%) (Gook et al., 1995). The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes.
The embryo development on day 2 shows a high rate of cleavage: 91% of the fertilized oocytes cleaved, in accordance with Gook's data (100% of cleavage in day 2) (Gook et al., 1995). Furthermore, embryo morphological quality does not seem to be compromised by cryopreservation inasmuch as 74% of the embryos were of good or fairly good quality.
In conclusion, it is suggested that the presence of the cumulus oophorus does not affect the oocyte survival rate; the presence of a higher sucrose concentration in the freezing solution, and a longer exposure time to the cryoprotectant (under particular conditions) positively affect the oocyte survival rate. These results should encourage further work to perfect the conditions for human oocyte cryopreservation.
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Notes |
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References |
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Submitted on August 4, 2000; accepted on November 14, 2000.