Human Reproduction, Vol. 16, No. 3, 411-416,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Human oocyte cryopreservation: new perspectives regarding oocyte survival
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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The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Various attempts to cryopreserve human oocytes have been performed with contrasting results. Therefore the effect of some factors, such as the presence or absence of the cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to cryoprotectants, on human oocyte survival after thawing were investigated. The oocytes were cryopreserved in 1,2-propanediol added with sucrose, using a slow-freezing-rapid-thawing programme. After thawing, the oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and the outcomes of insemination and subsequent embryo development were also recorded. The post-thaw cryosurvival rate was not different for the oocytes cryopreserved with their cumuli partially removed mechanically (56%) when compared with those cryopreserved with their cumuli totally removed enzymatically (53%). On the contrary, a significantly higher survival rate was obtained when the oocytes were cryopreserved in the presence of a doubled sucrose concentration (0.2 mol/l) in the freezing solution and the survival rate was even higher when the sucrose concentration was tripled (0.3 mol/l) (60 versus 82% P < 0.001). Furthermore, a longer exposure time (from 10.5 to 15 min) to cryoprotectants, before lowering the temperature, significantly increased the oocyte survival rate (P < 0.005). Intracytoplasmic sperm injection produced a good fertilization rate (57%) of thawed oocytes and a high embryo cleavage rate (91%) and a satisfactory embryo morphology was observed (14 and 34% for grade I and grade II embryos respectively).
Key words: cryoprotectants/exposure time/human oocyte cryopreservation/oocyte survival/sucrose concentration
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Introduction |
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Human oocyte cryopreservation represents an attractive option to the range of infertility treatments available at present. Although good fertilization and cleavage rates (Al-Hasani et al., 1987
; Chen, 1988; Gook et al., 1994, 1995) as well as a few pregnancies (Chen, 1988; Van Uem et al., 1988; Serafini et al., 1995; Tucker et al., 1996; Porcu et al., 1997; Antinori et al., 1998; Borini et al., 1998; Polak de Fried et al., 1998; Yang et al., 1998; Young et al., 1998) have been reported using human oocytes that survived cryopreservation, results have been variable and not sufficiently successful for routine use.
The human metaphase II oocyte appears to be particularly susceptible to freeze–thaw 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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Ovarian stimulation, oocyte retrieval and culture protocol
Fresh human oocytes were obtained from 96 counselled patients undergoing an IVF programme. Ovarian stimulation was achieved as described previously (Porcu et al., 1994
), using a combination of a gonadotrophin-releasing hormone analogue (Triptoreline, Decapeptyl 3.75; Ipsen Biotec, Paris, France) and menotrophins (Metrodin HP, 75 IU; Serono, Milan, Italy). Follicular growth was monitored by serum oestradiol-17ß measurements and ovarian ultrasonography. Ovulation was induced with 10 000 IU of human chorionic gonadotrophin (Profasi; Serono, Milan, Italy) when at least two follicles of 
18 mm in diameter were observed, with oestradiol concentrations corresponding to the number of follicles. Transvaginal ultrasound-guided oocyte retrieval was performed 34 h later. 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 freezing–rapid thawing method.
After no more than 2–3 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 10–12 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 (0.5–5 min); second group: >5 min and 10 min (5.5–10 min); third group: >10 min and 15 min (10.5–15 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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Experimental design
A. Oocyte survival rate: influence of cumulus oophorus and sucrose concentration
For the relationship between oocyte survival rate and the presence of cumulus, no statistically significant difference was found between the oocytes cryopreserved with the cumulus partially or totally removed in 0.1 mol/l (31 versus 39% respectively), 0.2 mol/l (60 versus 58% respectively) or 0.3 mol/l (81 versus 83% respectively) sucrose concentration (Table I
).
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By comparing the overall survival rate of the 308 oocytes cryopreserved in the presence of 0.1 mol/l sucrose with that of the 718 oocytes cryopreserved in the presence of 0.2 mol/l sucrose (Table I
), a significantly higher oocyte survival rate was found in the second group with respect to the first group (34 versus 60%; P < 0.001) (Table I).
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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A gradual enhancement of the oocyte survival rate was observed in relation to the increase of the exposure time to 0.2 mol/l sucrose solution. A significantly higher survival rate was found when the exposure time to the sucrose solution was between 10.5 and 15 min as compared with an exposure time of 30 s to 5 min and one of 5.5 to 10 min (Table II
).
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Human oocytes cryopreservation clinical trial
Ninety-six patients (mean age 30.7 ± 3.5 years) were enrolled in the clinical trial and 1769 oocytes were collected and cryopreserved in 0.2 mol/l sucrose solution. A total of 1502 oocytes were thawed (112 thawing cycles). A total of 796 out of 1502 survived to cryopreservation showing a survival rate of 54% (Table III
).
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On day 2, the embryos showed a high cleavage rate (91%). In all, 74% of embryos showed a good or fairly good quality, considering good quality to be those embryos graded I (no fragmentation), II (<10% fragmentation), III (<30% fragmentation), in particular, 14% of them were grade I, 34% were grade II, and 26, 13 and 13% were grades III, IV and V respectively.
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Discussion |
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In this study, two factors influencing human oocyte survival after cryostorage were investigated, namely the effect of the presence or absence of the cumulus oophorus and the influence of different sucrose concentrations in the freezing solutions.
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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3 To whom correspondence should be addressed at: IVF Center, Human Reproductive Medicine Unit, Institute of Obstetrics and Gynecology, University of Bologna, 40138 Bologna, Italy. E-mail: rfabbri{at}orsola-malpighi.med.unibo.it
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Submitted on August 4, 2000; accepted on November 14, 2000.
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