1 Reproductive Biology Unit, Royal Women's Hospital, Carlton and 2 Melbourne IVF, East Melbourne, and 3 Department of Obstetrics and Gynecology, University of Melbourne, Victoria, Australia
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Abstract |
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Key words: cryopreservation/follicle/human/oocyte/ovary
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Introduction |
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Earlier studies on rodents suggested that restoration of fertility with subsequent births, following transplantation of ovarian tissue cryopreserved in glycerol, was possible (Parrott, 1960) and more recent studies demonstrated in-vitro maturation, fertilization, embryo development and live birth following cryopreservation of individual mouse follicles in dimethyl sulphoxide (DMSO) (Carroll et al., 1990
; Carroll and Gosden, 1993
; Cortvrindt et al., 1996
; Gunasena et al., 1997
) have confirmed the feasibility of such an approach. Further evidence of resumption of function following cryopreservation in DMSO has been reported for ovarian tissue obtained from sheep (Gosden et al., 1994
) and marmoset (Candy et al., 1995
), although it appears that the effects of cryopreservation and transplantation contribute to a relatively poor success rate compared to that obtained with fresh tissue (Candy et al., 1997
; Gunasena et al., 1997
).
Cryopreservation of ovarian tissue for potential clinical use is already being undertaken by a number of groups (Bahadur and Steele, 1996) despite a relative paucity of information on the likely survival of the tissue post-thawing. Histological evaluation of cryopreserved human ovarian tissue, as reported after orthotropic transplantation (Newton et al., 1996
) and following in-vitro culture (Hovatta et al., 1997
; Oktay et al., 1997
) suggests that some preservation of the follicles is possible. One study (Newton et al., 1996
) shows that variable results are obtained using a range of cryoprotectants in conjunction with a slow freeze/rapid thaw protocol, possibly reflecting the variation in infiltration rates of the cryoprotectants (Newton et al., 1998
). The inference from this is that an optimal regimen must be established for each cryoprotectant. As yet, no attempt has been made to optimize cryopreservation regimens for a given cryoprotectant with human tissue. The assumption that dehydration conditions and the rate of cooling which are used for embryos will be suitable for the cells of the ovarian cortex, the follicular cells and the oocyte, may be untenable, given the variation in size and the likely variation in water content in each of the cells within the follicle.
The present study was designed to investigate the effects of varying dehydration conditions and cooling rates on the histological appearance of thin slices of human ovarian cortex following cryopreservation in propanediol (PROH). The choice of cryoprotectant was based on its use in clinical protocols which give rise to the birth of healthy babies following cryopreservation of both embryos and mature oocytes. We have also adopted an approach designed to overcome an inherent difficulty in assessing human ovarian tissue. Previous studies, which have expressed survival as follicle numbers in frozen/thawed slices relative to numbers in non-frozen slices, do not account for the wide intra- and inter-patient variation in follicle numbers per slice (Osborn et al., 1997). We have, therefore, expressed our findings as the proportions of follicles, pre-granulosa cells and oocytes which remain intact and compared these to values for matched non-frozen tissue.
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Materials and methods |
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Cryopreservation
Rate of cooling
Slices were rinsed briefly in phosphate buffered saline (PBS; Trace, Clayton, Vic, Australia) and then dehydrated in 1.5 mol/l 1,2 propanediol (PROH; BDH, Kilsyth, Victoria, Australia) for 10 min followed by 1.5 mol/l PROH plus 0.1 mol/l sucrose for 30 min. All freezing and thawing solutions contained 10 mg/ml HSA in PBS. The slices were then transferred to vials containing the second solution and frozen at one of the following rates: (i) slow: 2°C/min to 8°C, at which temperature ice crystal nucleation was induced manually, followed by 0.3°C/min to 30°C, 50°C/min to 150°C then stored in liquid nitrogen; (ii) intermediate: achieved by suspending the vial in the liquid nitrogen vapour for >12 h followed by submerging in liquid nitrogen; (iii) rapid: achieved by plunging directly into liquid nitrogen.
Dehydration
Slices were prepared prior to cryopreservation using two dehydration regimens: (i) two-step method: 1.5 mol/l PROH for 10 min followed by 1.5 mol/l PROH plus 0.1 mol/l sucrose for 15, 30 or 60 min; (ii) one-step method: 1.5 mol/l PROH plus 0.1 mol/l sucrose for 30, 60 or 90 min.
Following dehydration slices were loaded into vials containing 1 ml of the final solution and frozen using the slow rate of cooling.
Slices frozen in all conditions were thawed using the rapid thaw method previously described (Gook et al., 1993) with one modification. To compensate for the use of vials, the air warming step was omitted and the water bath temperature was increased to 37°C. At this temperature the vials required between 23 min to thaw. After rehydration, slices were transferred to HEPESHTF plus HSA at 37°C for approximately 30 min and then fixed in glutaraldehyde in 0.1 mol/l cacodylate buffer (as above).
Histological evaluation
Following fixation, slices were treated with 1% osmium tetroxide (ProSciTech) in 0.1 mol/l cacodylate buffer and dehydrated stepwise through alcohol and finally acetone. Tissue was then embedded in Spurr's resin (Polysciences Inc., Warrington, PA, USA) and cured at 60°C for 24 h. Serial 1 µm sections were cut using a glass knife, mounted on slides and stained with 1% methylene blue (Schmid GmbH&Co., Stuttgart, Germany) in 1% sodium tetraborate (BDH). Sections were examined under an Olympus light microscope at x100 magnification.
Where possible, each condition in either the rate of cooling experiment or the dehydration experiment was compared to non-frozen slices from the same patient. Unfortunately, in some slices very few or no follicles were observed, which necessitated inclusion of slices from other patients to obtain adequate numbers for analysis.
The proportion of cortex that contained cellular material and matrix following treatment was estimated visually over three sections (1x2 mm), averaged, and expressed as the percentage of area intact. For each treatment, the numbers of intact follicles, pre-granulosa cells and oocytes (as described below) were divided by the total numbers examined for that treatment. Intact follicles had a complete basal lamina surrounding the pre-granulosa cell layer and showed no sign of cellular leakage into the cortex. Within each follicle the number of pre-granulosa cells which had an intact membrane, contained cytoplasm and were regular in shape was divided by the number of pre-granulosa cell nuclei present, to estimate the proportion of intact pre-granulosa cells. The circular shape of the oocyte, the presence of cytoplasm throughout the oocyte, and a membrane demonstrated that the oocyte was intact following the treatment. In contrast, oocytes which had not survived had, generally, no membrane, a small irregular area of cytoplasm which had retracted to around the germinal vesicle, and an empty area between this and the pre-granulosa cell layer. The normality in appearance and distribution of the organelles within the oocyte following treatment was compared to oocytes in non-frozen slices. Unless stated, all observations were made at the light microscopic level (x100). Electron microscopy was performed only where abnormalities were observed. Statistical analysis was performed using the 2 test, initially for the overall table and then for individual pairs. Where
2 was inappropriate, an exact test was used.
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Results |
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Rate of cooling
The effects of cooling rate on the ovarian cortex, pre-granulosa cells, and the oocyte are shown in Table I. The ovarian cortex in the non-frozen tissue consisted of compact matrix of stroma cells and collagen bundles (Figure 1A
) surrounding the follicles. In this tissue, it was estimated that 92% of the cortex was cellular or contained collagen, whereas in the frozen tissue the proportion was significantly reduced. Electron microscopic examination of the frozen tissue showed disruption of the collagen bundles and lysis of the stroma cells (Figure 1B
). The basal lamina enclosing the follicle remained complete in the majority of follicles following all rates of freezing (slow 100%, intermediate 75%, rapid 82%) compared to non-frozen follicles (100%). In contrast, the cellular components of the follicle, the pre-granulosa cells and the oocyte, showed a marked reduction in survival that was related to the rate of cooling. Although only 55% of the pre-granulosa cells survived the slow rate of cooling, this was higher than the proportion observed with the faster rates. Similarly, more intact oocytes were observed using the slow rate of cooling compared to the other rates.
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The effect of increasing the dehydration time on pre-granulosa cells and oocytes using the two-step protocol are shown in Table II. The proportion of intact pre-granulosa cells was reduced compared to the non-frozen tissue following dehydration for 15 or 30 min and further reduced with 60 min dehydration. However, although the proportion of intact oocytes was reduced compared to non-frozen controls, similar proportions were observed with all dehydration times. Within the oocyte cytoplasm, two effects of cryopreservation were apparent under the light microscope: the increased cytoplasmic area containing vacuoles or empty areas and the lack of dark staining organelles around the germinal vesicle. In the non-frozen oocytes, these dark organelles were identified, under the electron microscope, as round or oval mitochondria with concentric cristae and were observed in abundance close to the germinal vesicle (Figure 1E
). In some of the frozen oocytes examined under the electron microscope, the mitochondria were absent and the germinal vesicle was surrounded by an electron dense cytoplasm with many small, relatively empty vesicles (Figure 1F
). The presence of mitochondria in the oocyte cytoplasm (assessed at the light microscopy level) was included in the assessment of cryopreserved tissue. (The lack of mitochondria was also observed in oocytes following intermediate and fast rates of cooling but not included in the results due to the severity of the damage observed in these oocytes.) In some of the oocytes in the dehydration experiment, either lack of organelles or elevated vacuolation was observed, whereas in others both were observed together. The proportion of oocytes with an abundance of organelles similar to the non-frozen oocytes was significantly lower in the frozen tissue (Table II
, P < 0.001) and the lowest proportion was observed with the longest dehydration time (60 min compared to 15 and 30 min; P < 0.05). A similar trend was observed for the proportion of oocytes with
10% vacuolation. The relative proportion of `normal oocytes' (oocytes which were intact, had normal organelles and
10% vacuolation) was low for all dehydration times with between one-third to one-half of oocytes appearing morphologically normal. The proportion of intact pre-granulosa cells and oocytes with good preservation of cytoplasm was significantly lower after 60 min dehydration compared to the other times and may be due to the depth of the follicles within the tissue.
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Under the freezing conditions examined the highest proportion of intact and morphologically preserved primordial follicles was achieved with a single step dehydration for 90 min followed by slow freezing and rapid thawing.
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Discussion |
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In this study, evaluation of the effect of cooling rate on human ovarian tissue has indicated that both the pre-granulosa cells and oocyte achieve better survival with a slow rate. Although the rate of cooling has not been optimized further, it indicates that, at the concentration of cryoprotectant used, there is an overlap in the optimal cooling rate of each cell type in the follicle. In contrast, the optimal cooling rate for the stromal cells is closer to the intermediate rate. Damage to the stroma has not been reported in any of the human studies which have used a similar PROH method (Hovatta et al., 1996, 1997
) or comparable conditions with a slow rate of freezing and a similar concentration of other cryoprotectants (Newton et al., 1996
; Oktay et al., 1997
). Histological examination may not have detected this damage in the above studies because the tissue was embedded in paraffin and sections were 25 times the thickness evaluated in our study and the aim was to assess long term survival of follicles and not the fine structural integrity following cryopreservation. It was observed that the majority of stromal cells surrounding follicles, which had been frozen, thawed and subsequently isolated using enzymes, were dead (Oktay et al., 1997
). Whether cell death was a consequence of enzyme digestion or cryopreservation is unknown. Rates of cooling have not previously been compared for human or murine ovarian tissue. The better results obtained with the slow rate agree with the theoretical estimation (Mazur, 1970
) of <1°C/min for organ or tissue cryopreservation. Cell damage during the fast rate of freezing is likely to be due to intra- and/or extracellular ice formation, suggesting that a higher concentration of cryoprotectant may be beneficial at this rate. Although traditional ultrarapid rates of cooling are used in conjunction with high concentrations of cryoprotectants (2.58 mol/l; Gordts et al., 1990
; Nowshari et al., 1995
) to reduce ice formation, the balance between effective dehydration with minimal toxicity and osmotic injury is difficult to achieve for tissue. Ultrarapid regimens may, however, prove successful for human ovarian tissue following the success in preserving human fetal ovarian tissue (Zhang et al., 1995
) in high concentrations of DMSO plus sucrose.
Manipulation of the dehydration regimen prior to freezing has been shown, in our study, to affect cellular survival and morphological integrity. It is, therefore, difficult to compare results for different cryoprotectants without optimization for each cryoprotectant. This is supported by a study (Newton et al., 1998) which showed that penetration of various cryoprotectants into human ovarian tissue varies and is a function of the dehydration temperature. Unfortunately, the penetration rates were not examined at room temperature, the temperature at which dehydration was performed in our study. Newton's study would suggest that a minimum of 30 min dehydration in any of the commonly used cryoprotectants [DSMO, PROH, ethylene glycol (EG), glycerol] would have penetrated at least 70% of the tissue at 4°C. In contrast, similar rates of morphological survival of follicles was achieved in murine ovarian tissue dehydrated in different cryoprotectants for 5 min or longer at ~20°C (Candy et al., 1997
). A major difference between human and murine ovarian tissue is the degree of compaction of cells and it is likely that these results reflect the effect of the tissue density on the rate of penetration of cryoprotectants.
Multiple dehydration steps have been used mainly for embryo cryopreservation and are used generally to reduce osmotic stress. This approach does not appear to be beneficial for ovarian tissue and may be less successful than the single step approach due to greater variability, slightly lower preservation of pre-granulosa cells and no clear relationship with duration of dehydration. The variability in pre-granulosa cell survival between follicles in all conditions except the 90 min step may relate to the position of the follicle and its effect on dehydration.
Previous studies reporting morphological assessment of cryopreserved human ovarian tissue have expressed their results for follicle survival as the proportion of follicles present in the thawed relative to the number present in the non-frozen tissue (Hovatta et al., 1996; Newton et al., 1996
; Oktay et al., 1997
). An inherent assumption is that the number of follicles is approximately equivalent in each slice of tissue prior to treatment. This may not be a valid assumption for human ovarian tissue, due to wide variation in the distribution of follicles between slices within patients. By assessing the survival of individual follicles and cells within the follicle for each treatment and comparing this to non-frozen tissue, we have attempted to obtain a more accurate estimate of survival.
The main limitation of a morphological study, however, is that it gives no indication of functional integrity. This was shown clearly in a comparison of freezing rates for one-cell embryos in which the highest morphological survival rate was associated with lower cleavage and pregnancy rates (Van den Abbeel et al., 1997). At present, viability staining (Oktay et al., 1997
), maintenance of follicular structure in tissue cultured (Hovatta et al., 1997
) and transplanted (Newton et al., 1996
) indicates that some follicles from human ovarian tissue are viable following cryopreservation. Although functional integrity following cryopreservation has yet to be established for human ovarian tissue, live births have resulted from transplantation of cryopreserved tissue in the mouse (Parrott, 1960
; Gunasena et al., 1997
) and sheep (Gosden et al., 1994
). In addition, the demonstration of large antral follicles following transplantation of cryopreserved ovarian tissue from two species; marmoset (Candy et al., 1995
) and sheep (Salle et al., 1998
), and of DNA synthesis in preantral follicles following culture of thawed tissue from the domestic cat (Jewgenow et al., 1998
), show that folliculogenesis is possible. The relatively low rate of resumption of successful function, however, may indicate that few follicles have survived cryopreservation with the minimum number of cells required for follicular development and function. Given that the human primordial follicle contains only ~10 pre-granulosa cells, it may be that even minimal cell loss is incompatible with the resumption of function.
In conclusion, our study indicates that one-step dehydration for 90 min in PROH and sucrose prior to slow freezing followed by rapid thawing achieves more totally intact follicles than the other conditions and more oocytes with morphological characteristics similar to non-frozen oocytes than the other regimens tested. The study emphasizes the importance of establishing optimal conditions for the use of each cryoprotectant in complex multicellular systems.
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Acknowledgments |
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Notes |
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References |
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Submitted on December 29, 1998; accepted on April 28, 1999.