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Cryopreservation of Bovine Ovarian Tissue: Structural Normality of Follicles after Thawing and Culturein Vitro
Cryobiology 38, 301–309 (1999) Article ID cryo.1999.2170, available online at http://www.idealibrary.com on
Cryopreservation of Bovine Ovarian Tissue: Structural Normality of Follicles after Thawing and Culture in Vitro S. J. Paynter, 1 A. Cooper, B. J. Fuller, and R. W. Shaw Department of Obstetrics & Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom The recovery of viable follicles from cryopreserved ovarian tissue would be of benefit in many areas of assisted reproduction. Structural integrity needs to be maintained following cryopreservation of ovarian tissue in order to retrieve healthy follicles which can then be cultured in vitro to produce viable oocytes. We have assessed the effect of in vitro culture of bovine tissue for 0, 1, 4, 24, or 48 h after exposure to, or cryopreservation in, dimethylsulphoxide. Immediately after freezing, normality of primary and preantral follicles within the tissue was significantly lower than for tissue exposed to the cryoprotectant without freezing or for control tissue. After 4 h in culture, cryopreserved tissue appeared to have recovered from damage caused by freezing, although the percentage of tissue with normal morphology declined after 24 and 48 h of culture. There was no significant difference between percentage normality in control tissue and tissue exposed to the cryoprotectant without freezing for any of the culture times studied. These data indicate that it is possible to freeze/thaw bovine ovarian tissue while retaining a reasonable yield of morphologically intact follicles and that a short period of post-thaw culture may enhance follicle recovery. © 1999 Academic Press Key Words: cryopreservation; ovary; bovine; follicles.
There is a specific need for therapies to protect fertility in young female patients receiving radiotherapy or chemotherapy for various cancers for which, in some cases, long-term curative rates are high (2). For male patients, sperm cryopreservation may be appropriate in such circumstances, but for female patients, the option of gamete cryopreservation is not, as yet, offered routinely, largely due to the many unanswered questions about reliability and efficacy of oocyte cryopreservation (4). It is increasingly being suggested that cryopreservation of ovarian tissue should be offered to young female cancer patients as a way of enhancing future fertility (9). Replantation of frozen– thawed ovarian grafts resulted in successful pregnancies in mice as long ago as 1960 (21). However, the technology does not yet exist for either autotransplanting the ovarian tissue in humans or for retrieving immature oocytes from the thawed tissue and maturing them in vitro for subsequent in vitro fertilization. Recent excellent reReceived January 20, 1999; accepted March 29, 1999. 1 To whom correspondence should be addressed. Fax: 01222 743722. E-mail: [email protected]. 301
sults in mice (13) and success in sheep, a good model for the fibrous human ovary (11), in which live births have been achieved following autoimplantation of cryopreserved ovarian tissue, do provide grounds for optimism. Nevertheless, there are many questions to be answered surrounding ovarian tissue cryopreservation, particularly concerning isolation of immature oocytes for subsequent maturation in vitro. It is now becoming established that to achieve in vitro maturation, it is very important to maintain normal structure and cellular organization, not only of the maturing oocyte, but also of the surrounding cell layers (theca and granulosa) which are essential for several signaling processes. The present study was undertaken to assess the effects of cryopreservation on the interrelated structures of ovarian tissue and to address the question of whether a period of recuperative tissue culture post-thawing would allow repair of damage induced during cryopreservation. Bovine ovaries were used because the ovary is closer in size and fibrous tissue content to the human than those of small animals such as mice. Also, cows are similar to humans in that singleton births are the norm. The relative contributions of freezing dam0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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age and damage due to addition/removal of cryoprotectant without freezing were also assessed. MATERIALS AND METHODS
Source of Tissue and Tissue Preparation Bovine ovaries were obtained from the local abattoir from cattle over 30 weeks old with approval from the Ministry of Agriculture Fisheries and Food. The ovaries were collected within 2–4 h of slaughter and transported to the laboratory in phosphate-buffered saline (PBS; Gibco BRL) at room temperature. The tissue was prepared for the experiments within 2 h of collection; therefore, all processing was undertaken within 4 – 6 h of slaughter. At least six ovaries were required for each experimental run. The ovaries were cleaned of adhering tissue and washed in sterile PBS prior to removal of the outer ovarian cortex with a sterile scalpel. The outer cortex was removed in strips 1–2 mm thick which were placed into a phosphate-buffered medium (PB1; Gibco BRL Cat. No. 21300-17 plus 0.06 g/L benzylpenicillin, 0.036 g/L sodium pyruvate, 1 g/L glucose, and 0.012 g/L phenol red) supplemented with heat-inactivated foetal calf serum (FCS; 10%; Gibco BRL.). The strips of ovarian cortex were pooled in this medium prior to selection, without intentional bias, into one of three groups. The strips of ovarian tissue were further cut into cubes of 1–2 mm 3; at least 150 cubes were required for each experimental run. This tissue was held in PB1–FCS prior to either being frozen in 1.5 M dimethylsulphoxide (Me 2SO) or exposed to 1.5 M Me 2SO without freezing. Untreated tissue was immediately placed into in vitro culture. Exposure to Cryoprotectant and Cryopreservation of Ovarian Tissue At least 10 cubes of tissue were placed into each of 10 vials (Nunc cryotubes, 1.8 ml, Cat. No. 363401) containing 1 ml of 1.5 M Me 2SO (made up in PB1–FCS). These vials were held for 20 min on ice to allow equilibration with the cryoprotectant. To assess the effect of exposure to Me 2SO without freezing, the cryoprotectant was diluted in 5 vials by the addition of 1 ml PB1–
FCS at room temperature. This was followed with three washes, each involving placing the tissue plus 1 ml of solution into 9 ml PB1–FCS for 20 min, two washes being performed at room temperature and the final wash at 37°C. This tissue was then placed into in vitro culture. The five remaining vials were cryopreserved by placing them into a precooled (4°C) controlledrate freezer (Planer UK Ltd.) cooled at 2°C/min to 28°C and held at this temperature for 10 min. At this point the samples were manually seeded and then cooled at 0.3°C/min to 240°C. At 240°C the vials were cooled more rapidly (10°C/min) to 2150°C before being plunged into liquid nitrogen. The frozen vials were stored in liquid nitrogen for up to 2 weeks before being thawed. They were thawed rapidly by placing the vials in a waterbath at 37°C until the ice had melted. Dilution of the Me 2SO was performed as described above for tissue exposed to cryoprotectant without freezing. In Vitro Culture Protocol At least 10 cubes of tissue which had been frozen, exposed to cryoprotectant without freezing, or untreated as control tissue, were placed into each of five organ culture dishes (Falcon Cat. No. 3037) containing 1 ml of culture medium. The culture medium was TCM 199 (Gibco BRL) supplemented with 0.06 g/L benzylpenicillin, 0.05 g/L streptomycin, and 0.25 g/L fungizone (Sigma UK Ltd.) plus 10% FCS. The dishes were incubated at 39°C in a humidified atmosphere with 5% CO 2 in air. At time 0, 1, 4, 24, and 48 h after placement in culture, one dish was removed and the cubes of tissue were fixed in 10 ml of 4% paraformaldehyde. The fixed tissue pieces were embedded in paraffin wax and three serial sections were taken at 100-mm intervals. The sections were stained with hematoxylin– eosin and the follicular structure was scored for normality in all treatment groups. Each experiment was carried out in triplicate. Scoring of Follicular Normality and Statistical Analysis Normality of follicular structure was scored microscopically, in a blinded fashion, on each
EFFECT OF POST-THAW CULTURE ON OVARIAN TISSUE
set of serial sections for each treatment group. Follicles were scored according to the following scoring system: primary follicles (small follicles containing a germinal vesicle-stage oocyte surrounded by 1–2 layers of cuboidal granulosa cells) were graded 1–3 (Fig. 1), Grade 1 being spherical in shape with even distribution of granulosa cells, intact theca, and a spherical oocyte, Grade 2 having theca and granulosa cells pulled away from the edge of the follicle but the oocyte still spherical, and Grade 3 having theca and granulosa cells pulled away from the edge of the follicle with severe pyknotic nuclei in the granulosa cells and oocyte misshapen with/without vacuolation. Preantral follicles (larger follicles surrounded by several layers of granulosa cells and containing a germinal vesicle-stage oocyte) were also graded 1–3 (Fig. 2) as follows: Grade 1 is an intact spherical follicle with evenly distributed granulosa and theca cells, small spaces between cells being acceptable with a normal spherical oocyte; Grade 2 is an intact spherical follicle with intact theca cells but with disruption of the granulosa cells and apparent loss of cells and containing a normal spherical oocyte; and Grade 3 shows great disruption and loss of granulosa cells with theca cells pulled away from the follicle edge with many pyknotic nuclei visible and a misshapen vacuolated oocyte. One hundred primary and 25 preantral follicles (reflecting the frequency of abundance of each type in the processed tissues) were assessed for normality in each group on three occasions. The results were expressed as overall percentage normality for each treatment group. Statistical differences were assessed by the x 2 test (P , 0.05) with the Yates correction for multiple comparisons. RESULTS
The percentage of follicles in each of the three grades is shown in Figs. 3 and 4 for primary and preantral follicles, respectively. Between 5 and 10 tissue pieces were examined, depending upon the abundance of follicles. For
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the purposes of further data processing and comparison with other studies, grade 1 was classed as normal, with grade 2 and 3 being abnormal. The percentage normality of primary and preantral follicles after the various treatments are displayed in Tables 1 and 2. From the results displayed in Figs. 3 and 4, it can be seen that for both control primary and preantral follicles (Figs. 3A and 4A), there was a small drop in the percentage of high-grade (grade 1) follicles seen comparing freshly isolated tissues and tissues cultured for up to 4 h, with a concomitant small increase in the lowest grade (grade 3). During the later stages of culture (24 and 48 h) there was a continued shift in normalities of the control follicles, with further drops in grade 1 and increases in grade 3. Similar patterns were seen in both types of follicles after exposure to Me 2SO alone (Figs. 3B and 4B), with stable percentages of grade 1 follicles over 4 h in culture, while beyond that time there was a decrease in grade 1 and an increase in grade 3. Immediately after cryopreservation, the numbers of primary and preantral follicles that were judged as grade 1 (Figs. 3C and 4C) were lower than in the control tissue or tissue exposed to Me 2 SO, with grade 2 follicles (intermediate damage) being noticeably higher than in control tissues. Over the next 4 h, for both follicle types, an increase in grade 1 follicles and parallel changes in grades 2 and 3 were observed (Figs. 3C and 4C). Thereafter, during long-term culture the cryopreserved follicles exhibited the same changes in follicle structure as seen in control tissues after the same period, such that by 48 h, the distributions among the three grades were similar in all groups. When the results were expressed as percentage of normal follicles (grade 1) from the total numbers counted, the values in Table 1 show that immediately after freezing there was significantly lower percentage normality of primary follicles in the cryopreserved tissue than in the tissue exposed to cryoprotectant without freezing and control tissue. The situation was the same after 1 h of culture but the damage appeared to have been repaired by 4 h. At 24 and 48 h the percentage normality was reduced in all cases.
FIG. 1. Photomicrographs of bovine primary follicles within ovarian tissue (bar, 30 mm). (a) Normal (grade 1) follicle. (b) Abnormal (grade 2) follicle. (c) Abnormal (grade 3) follicle.
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FIG. 2. Photomicrographs of bovine preantral follicles within ovarian tissue (bar, 30 mm). (a) Normal (grade 1) follicle. (b) Abnormal (grade 2) follicle. (c) Abnormal (grade 3) follicle.
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in post-thaw culture appeared to take place a little faster (being completed within 1 h). For both primary and preantral follicles, there were
FIG. 3. Percentage of primary follicles of each grade at each post-thaw culture period. (A) Control untreated tissue. (B) Tissue exposed to cryoprotectant without freezing. (C) Cryopreserved tissue. Grade 1 s, Grade 2 u, Grade 3 o.
As shown in Table 2, the results for preantral follicular normality showed a pattern similar to that of primary follicles, although the recovery
FIG. 4. Percentage of preantral follicles of each grade at each post-thaw culture period. (A) Control untreated tissue. (B) Tissue exposed to cryoprotectant without freezing. (C) Cryopreserved tissue. Grade 1 s, Grade 2 u, Grade 3 o.
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TABLE 1 Median (Range) Percentage of Normal (Grade 1) Primary Follicles from 5–10 Tissue Pieces Time in culture (h)
Control
Exposed to CPA
Cryopreserved
0 1 4 24 48
57 (42–57) 39 (39–46) 36 (36–43) 26 (20–40) 12 (4–20)
46 (40–48) 40 (40–48) 35 (35–36) 32 (14–39) 12 (4–22)
*18 (11–25) *26 (18–35) 35 (27–40) 16 (12–34) 18 (8–26)
* Significantly different from values in same row.
no significant differences between percentage normality in control tissues and that in tissue exposed to cryoprotectant without freezing, after any of the culture times. DISCUSSION
During normal ovarian function, a natural maturation process occurs throughout consecutive hormonal cycles which permits development of a number of the primary follicles through secondary and tertiary follicular stages to large antral follicles containing a mature oocyte for ovulation, while other follicles within the cohort become atretic and degenerate (12). Thus, there is a background of natural follicular change within an ovary against which any effects of cryopreservation must be set. In addition, the methods of harvesting and manipulation of the ovarian tissue prior to cryopreservation may have an impact on the normality of follicle structure. From our current studies it would appear that approximately half of the primary or preantral follicles in the fresh, control tissues were of the highest morphological grade after initial tissue collection and dissection, and these would be the type of follicles selected for maturation in vitro. These percentages are of a similar order to those reported in studies on ovarian tissues from other species such as primates (6). Greater percentages of high-grade primary follicles (up to 90%) have been reported in ovaries of prepubertal mice (7) but this probably reflects the physiology of the ovary in such immature animals. In the current
study, the small decrease in percentage of normal follicles seen once the tissues were returned to 37°C probably reflects latent damage incurred in the tissues during surgical removal and transport and which is only fully expressed once the tissues were returned to 37°C (a form of hypoxia/reoxygenation damage (19)). The fact that the percentages of grade 2 follicles in each case stayed relatively constant might suggest that there was a progression of follicle degeneration with time in culture, i.e., grade 1 follicles degenerating into grade 2 scoring and, concomitantly, equivalent amounts of grade 2 follicles degenerating into grade 3 scoring. Although restoration of fertility following autografting of ovarian tissue has resulted in live births in sheep (11), the use of this course of treatment for cancer patients is questionable due to the risk of transmission of the disease following grafting of tissue (23). It has also been possible to achieve follicular development to the large antral stage under laboratory conditions by xenografting ovarian tissues from a donor species (e.g., marmoset) into a well-vascularized site in immune-incompetent mice (7), but for human infertility treatment such a technique raises several questions about the ethics and safety (from transmissible diseases) of using an intermediate living host. In the clinical setting, in vitro maturation of follicles to yield oocytes for fertility treatment would at present probably be the preferred option. The later stages of follicle growth (antral), while being the most conducive to maturation in vitro, are less frequently found in ovarian tissue biopsies TABLE 2 Median (Range) Percentage of Normal (Grade 1) Preantral Follicles from 5–10 Tissue Pieces Time in culture (h)
Control
Exposed to CPA
Cryopreserved
0 1 4 24 48
48 (44–56) 36 (32–36) 36 (36–40) 24 (16–40) 10 (8–12)
44 (24–48) 36 (36–48) 44 (32–44) 8 (4–28) 10 (10–28)
*12 (12–28) 32 (16–32) 32 (20–31) 20 (4–44) 12 (10–20)
* Significantly different from values in same row.
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and may also be more susceptible to freeze/thaw damage (14). Candy et al. (7) found in a murine model that while most primordial follicles survived cryopreservation, less than 49% of primary follicles survived when intact murine ovaries (;2 mm 3) were preserved. When primate (6) ovarian tissue was cryopreserved and implanted in a “pseudo” in vivo environment (i.e., in immune-incompetent mice), no large antral follicles could be found immediately after, or for a few days following, cryopreservation but some of the surviving preantral follicles did mature to form large antral stages after a few weeks of implantation. For follicles to be matured in vitro they would have to be isolated from the fibrous ovarian tissue which would most practically involve a combination of enzymatic digestion and mechanical disruption (22). To achieve any success, as many follicles as possible must be of high morphological grade in the tissue after cryopreservation and before the isolation procedures are started. Human primordial follicles have been shown to survive freezing (14) and grafting into immuno-deficient mice as tissue slices (17) and have been successfully isolated from human tissue after cryopreservation (20). As yet no methods exist for maturation of primordial follicles in vitro from large mammalian species, the technique having only been achieved in mice (10). Thus primary and preantral follicle survival may be of more significance at a practical level. Long-term (several weeks) in vitro culture of preantral and early antral human follicles has recently been demonstrated (1). From the current studies, it appears that the procedures for exposure and removal of cryoprotectant did not significantly affect the morphological grading of the follicles. These conditions were selected on the basis of previous work investigating cryoprotectant diffusion in ovarian tissue (18, 25) and are likely to result in the Me 2SO permeating the tissue, at least to a large extent. However, the procedures of cryopreservation in our study did cause a significant drop in the percentage of normal primary, and especially preantral, follicles when assessed immediately after thawing and cryoprotectant re-
moval. Similar effects have been noted when studying primate and murine ovarian tissues recovered from cryopreservation (6, 7). This may reflect structural damage following osmotic dehydration during slow cooling or ice nucleation and crystal growth during rewarming. When both fresh control and cryoprotectant-exposed tissues were placed into culture, there was a gradual decline in the high-grade morphology group of follicles over 4 h and a more significant fall over the next 24 and 48 h, suggesting that the culture conditions were not optimal for follicle survival within the tissue pieces. Long-term (5-day) culture of isolated murine follicles has certainly been possible in our laboratory (3) and in that of others (5, 8, 24); thus, it may be that diffusion of nutrients, oxygen, or autocrine factors are a limiting factor within three-dimensional tissue pieces. When the cryopreserved tissues were examined, an increase in the number of high-grade early follicles was seen over 4 h, particularly for the preantral follicles which more than doubled compared with those seen immediately after cryopreservation. In fact by 4 h of culture, the numbers of high-grade follicles of both primary and preantral stages were the same as those of the fresh tissue. This suggests that the process of rewarming the cryopreserved tissue in a nutrient-rich environment allowed the follicular cells to reestablish metabolic activity, normal cell volume control, and cell– cell contacts. The slower recovery seen in post-thaw culture of the primary follicles may reflect the lower metabolic rate in such immature follicles (16). Again, during the later (24- and 48-h) periods of culture, the normality of the follicles from the cryopreserved tissues declined in a fashion similar to that in fresh untreated tissues. In summary, these results suggest that there may be a place for post-cryopreservation culture of ovarian tissues to maximize yields of follicles for oocyte maturation. In addition, there is room for improving both the cryopreservation and the culture conditions for ovarian tissues, with strategies such as the recently reported use of extracellular matrix (15) being likely to assist in the latter. However, the long-term goal is to achieve
EFFECT OF POST-THAW CULTURE ON OVARIAN TISSUE
isolation of follicles capable of maturation from cryopreserved tissues, and studies are underway to investigate this aspect. REFERENCES 1. Abir, R., Franks, S., Mobberley, M. A., Moore, P. A., Margara, R. A., and Winston, R. M. L. Mechanical isolation and in vitro growth of preantral and small antral human follicles. Fertil. Steril. 68, 682– 688 (1997). 2. Apperley, J. F., and Reddy, N. Mechanisms and management of treatment-related gonad failure in recipients of high dose chemoradiotherapy. Blood Rev. 9, 93–116 (1995). 3. Ashley, P. J., Paynter, S. J., Cooper, A., Fuller, B. J., and Shaw, R. W. A comparison between host serum and foetal calf serum in an in vitro maturation system for pre-antral mouse ovarian follicles. Fertil. Steril. Abstract Series 19, 40 (1997). 4. Bernard, A., and Fuller, B. J. Cryopreservation of human oocytes: A review of current ideas and future perspectives. Hum. Reprod. Update 2, 193–207 (1996). 5. Boland, N. I., and Gosden, R. G. Effects of epidermal growth factor on the growth and differentiation of cultured mouse ovarian follicles. J. Reprod. Fertil. 101, 369 –374 (1994). 6. Candy, C. J., Wood, M. J., and Whittingham, D. G. Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Hum. Reprod. 10, 2334 –2338 (1995). 7. Candy, C. J., Wood, M. J., and Whittingham, D. G. Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries. J. Reprod. Fertil. 110, 11–19 (1997). 8. Cortvrindt, R., Smitz, J., and Van Steirteghem, A. In vitro maturation, fertilization and embryo development of immature oocytes from early pre-antral follicles from prepubertal mice in a simplified culture system. Hum. Reprod. 11, 2656 –2666 (1996). 9. Donnez, J., and Bassil, S. Indications for cryopreservation of ovarian tissue. Hum. Reprod. Update 4, 248 – 259 (1998). 10. Eppig, J. J., and O’Brien, M. J. Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54, 197–207 (1996). 11. Gosden, R. G., Baird, D. T., Wade, J. C., and Webb, R. Restoration of fertility to oophorectomised sheep by ovarian autografts stored at 2196°C. Hum. Reprod. 9, 597– 603 (1994). 12. Gougeon, A. Dynamics of human follicular growth. A morphologic perspective. In “The Ovary” (E. Y. Adashi and P. C. K. Leung, Eds.), pp.21–39. Raven Press, New York, 1993.
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13. Gunasena, K. T., Villines, P. M., Critser, E. S., and Critser, J. K. Live births after autologous transplant of autologous mouse ovaries. Hum. Reprod. 12, 101–106 (1997). 14. Hovatta, O., Silye, R., Krausz, T., Abir, R., Margara, R., Trew, G., Lass, A., and Winston, R. M. L. Cryopreservation of human ovarian tissue using dimethyl sulphoxide and propanediol-sucrose as cryoprotectants. Hum. Reprod. 11, 1268 –1272 (1996). 15. Hovatta, O., Silye, R., Abir, R., Krausz, T., and Winston, R. M. L. Extracellular matrix improves survival of both primordial and primary ovarian follicles in long term culture. Hum. Reprod. 12, 1032– 1036 (1997). 16. Krohn, P. L. Transplantation of the ovary. In “The Ovary” (S. Zukerman and B. J. Weir, Eds.), Vol. II, pp. 101–128. Academic Press, New York, 1977. 17. Newton, H., Aubard, Y., Rutherford, A., Sharma, V., and Gosden, R. Low temperature storage and grafting of human ovarian tissue. Hum. Reprod. 11, 1487–1491 (1996). 18. Newton, H., Fisher, J., Arnold, J. R. P., Pegg, D. E., Faddy, M. J., and Gosden, R. G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum. Reprod. 13, 376 –380 (1998). 19. Nugent, D., Newton, H., Gallivan, L., and Gosden, R. G. Protective effect of vitamin E on ischaemiareperfusion injury in ovarian grafts. J. Reprod. Fertil. 114, 341–346 (1998). 20. Oktay, K., Nugent, D., Newton, H., Salha, O., Chatterjee, P., and Gosden, R. G. Isolation and characterisation of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil. Steril. 67, 481– 486 (1997). 21. Parrott, D. M. V. The fertility of mice with orthotopic ovarian grafts derived from frozen tissue. J. Reprod. Fertil. 1, 230 –241 (1960). 22. Roy, S. K., and Treacy, B. J. Isolation and culture of human preantral follicles. Fertil. Steril. 59, 783–790 (1993). 23. Shaw, J. M., Bowles, J., Koopman, P., Wood, E. C., and Trounson, A. O. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit cancer to graft recipients. Hum. Reprod. 11, 1668 – 1673 (1996). 24. Spears, N., Boland, N. I., Murray, A. A., and Gosden, R. G. Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile. Human Reprod. 9, 527–532. 25. Thomas, N., Busza, A., Cooper, A., Paynter, S., Fuller, B., and Shaw, R. Measurement of permeating levels of cryoprotectant during ovarian tissue cryopreservation using 1H NMR spectroscopy in human and porcine ovaries. CryoLetters 78, 179 –184 (1997).
Cryopreservation of Bovine Ovarian Tissue: Structural Normality of Follicles after Thawing and Culture in Vitro S. J. Paynter, 1 A. Cooper, B. J. Fuller, and R. W. Shaw Department of Obstetrics & Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom The recovery of viable follicles from cryopreserved ovarian tissue would be of benefit in many areas of assisted reproduction. Structural integrity needs to be maintained following cryopreservation of ovarian tissue in order to retrieve healthy follicles which can then be cultured in vitro to produce viable oocytes. We have assessed the effect of in vitro culture of bovine tissue for 0, 1, 4, 24, or 48 h after exposure to, or cryopreservation in, dimethylsulphoxide. Immediately after freezing, normality of primary and preantral follicles within the tissue was significantly lower than for tissue exposed to the cryoprotectant without freezing or for control tissue. After 4 h in culture, cryopreserved tissue appeared to have recovered from damage caused by freezing, although the percentage of tissue with normal morphology declined after 24 and 48 h of culture. There was no significant difference between percentage normality in control tissue and tissue exposed to the cryoprotectant without freezing for any of the culture times studied. These data indicate that it is possible to freeze/thaw bovine ovarian tissue while retaining a reasonable yield of morphologically intact follicles and that a short period of post-thaw culture may enhance follicle recovery. © 1999 Academic Press Key Words: cryopreservation; ovary; bovine; follicles.
There is a specific need for therapies to protect fertility in young female patients receiving radiotherapy or chemotherapy for various cancers for which, in some cases, long-term curative rates are high (2). For male patients, sperm cryopreservation may be appropriate in such circumstances, but for female patients, the option of gamete cryopreservation is not, as yet, offered routinely, largely due to the many unanswered questions about reliability and efficacy of oocyte cryopreservation (4). It is increasingly being suggested that cryopreservation of ovarian tissue should be offered to young female cancer patients as a way of enhancing future fertility (9). Replantation of frozen– thawed ovarian grafts resulted in successful pregnancies in mice as long ago as 1960 (21). However, the technology does not yet exist for either autotransplanting the ovarian tissue in humans or for retrieving immature oocytes from the thawed tissue and maturing them in vitro for subsequent in vitro fertilization. Recent excellent reReceived January 20, 1999; accepted March 29, 1999. 1 To whom correspondence should be addressed. Fax: 01222 743722. E-mail: [email protected]. 301
sults in mice (13) and success in sheep, a good model for the fibrous human ovary (11), in which live births have been achieved following autoimplantation of cryopreserved ovarian tissue, do provide grounds for optimism. Nevertheless, there are many questions to be answered surrounding ovarian tissue cryopreservation, particularly concerning isolation of immature oocytes for subsequent maturation in vitro. It is now becoming established that to achieve in vitro maturation, it is very important to maintain normal structure and cellular organization, not only of the maturing oocyte, but also of the surrounding cell layers (theca and granulosa) which are essential for several signaling processes. The present study was undertaken to assess the effects of cryopreservation on the interrelated structures of ovarian tissue and to address the question of whether a period of recuperative tissue culture post-thawing would allow repair of damage induced during cryopreservation. Bovine ovaries were used because the ovary is closer in size and fibrous tissue content to the human than those of small animals such as mice. Also, cows are similar to humans in that singleton births are the norm. The relative contributions of freezing dam0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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age and damage due to addition/removal of cryoprotectant without freezing were also assessed. MATERIALS AND METHODS
Source of Tissue and Tissue Preparation Bovine ovaries were obtained from the local abattoir from cattle over 30 weeks old with approval from the Ministry of Agriculture Fisheries and Food. The ovaries were collected within 2–4 h of slaughter and transported to the laboratory in phosphate-buffered saline (PBS; Gibco BRL) at room temperature. The tissue was prepared for the experiments within 2 h of collection; therefore, all processing was undertaken within 4 – 6 h of slaughter. At least six ovaries were required for each experimental run. The ovaries were cleaned of adhering tissue and washed in sterile PBS prior to removal of the outer ovarian cortex with a sterile scalpel. The outer cortex was removed in strips 1–2 mm thick which were placed into a phosphate-buffered medium (PB1; Gibco BRL Cat. No. 21300-17 plus 0.06 g/L benzylpenicillin, 0.036 g/L sodium pyruvate, 1 g/L glucose, and 0.012 g/L phenol red) supplemented with heat-inactivated foetal calf serum (FCS; 10%; Gibco BRL.). The strips of ovarian cortex were pooled in this medium prior to selection, without intentional bias, into one of three groups. The strips of ovarian tissue were further cut into cubes of 1–2 mm 3; at least 150 cubes were required for each experimental run. This tissue was held in PB1–FCS prior to either being frozen in 1.5 M dimethylsulphoxide (Me 2SO) or exposed to 1.5 M Me 2SO without freezing. Untreated tissue was immediately placed into in vitro culture. Exposure to Cryoprotectant and Cryopreservation of Ovarian Tissue At least 10 cubes of tissue were placed into each of 10 vials (Nunc cryotubes, 1.8 ml, Cat. No. 363401) containing 1 ml of 1.5 M Me 2SO (made up in PB1–FCS). These vials were held for 20 min on ice to allow equilibration with the cryoprotectant. To assess the effect of exposure to Me 2SO without freezing, the cryoprotectant was diluted in 5 vials by the addition of 1 ml PB1–
FCS at room temperature. This was followed with three washes, each involving placing the tissue plus 1 ml of solution into 9 ml PB1–FCS for 20 min, two washes being performed at room temperature and the final wash at 37°C. This tissue was then placed into in vitro culture. The five remaining vials were cryopreserved by placing them into a precooled (4°C) controlledrate freezer (Planer UK Ltd.) cooled at 2°C/min to 28°C and held at this temperature for 10 min. At this point the samples were manually seeded and then cooled at 0.3°C/min to 240°C. At 240°C the vials were cooled more rapidly (10°C/min) to 2150°C before being plunged into liquid nitrogen. The frozen vials were stored in liquid nitrogen for up to 2 weeks before being thawed. They were thawed rapidly by placing the vials in a waterbath at 37°C until the ice had melted. Dilution of the Me 2SO was performed as described above for tissue exposed to cryoprotectant without freezing. In Vitro Culture Protocol At least 10 cubes of tissue which had been frozen, exposed to cryoprotectant without freezing, or untreated as control tissue, were placed into each of five organ culture dishes (Falcon Cat. No. 3037) containing 1 ml of culture medium. The culture medium was TCM 199 (Gibco BRL) supplemented with 0.06 g/L benzylpenicillin, 0.05 g/L streptomycin, and 0.25 g/L fungizone (Sigma UK Ltd.) plus 10% FCS. The dishes were incubated at 39°C in a humidified atmosphere with 5% CO 2 in air. At time 0, 1, 4, 24, and 48 h after placement in culture, one dish was removed and the cubes of tissue were fixed in 10 ml of 4% paraformaldehyde. The fixed tissue pieces were embedded in paraffin wax and three serial sections were taken at 100-mm intervals. The sections were stained with hematoxylin– eosin and the follicular structure was scored for normality in all treatment groups. Each experiment was carried out in triplicate. Scoring of Follicular Normality and Statistical Analysis Normality of follicular structure was scored microscopically, in a blinded fashion, on each
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set of serial sections for each treatment group. Follicles were scored according to the following scoring system: primary follicles (small follicles containing a germinal vesicle-stage oocyte surrounded by 1–2 layers of cuboidal granulosa cells) were graded 1–3 (Fig. 1), Grade 1 being spherical in shape with even distribution of granulosa cells, intact theca, and a spherical oocyte, Grade 2 having theca and granulosa cells pulled away from the edge of the follicle but the oocyte still spherical, and Grade 3 having theca and granulosa cells pulled away from the edge of the follicle with severe pyknotic nuclei in the granulosa cells and oocyte misshapen with/without vacuolation. Preantral follicles (larger follicles surrounded by several layers of granulosa cells and containing a germinal vesicle-stage oocyte) were also graded 1–3 (Fig. 2) as follows: Grade 1 is an intact spherical follicle with evenly distributed granulosa and theca cells, small spaces between cells being acceptable with a normal spherical oocyte; Grade 2 is an intact spherical follicle with intact theca cells but with disruption of the granulosa cells and apparent loss of cells and containing a normal spherical oocyte; and Grade 3 shows great disruption and loss of granulosa cells with theca cells pulled away from the follicle edge with many pyknotic nuclei visible and a misshapen vacuolated oocyte. One hundred primary and 25 preantral follicles (reflecting the frequency of abundance of each type in the processed tissues) were assessed for normality in each group on three occasions. The results were expressed as overall percentage normality for each treatment group. Statistical differences were assessed by the x 2 test (P , 0.05) with the Yates correction for multiple comparisons. RESULTS
The percentage of follicles in each of the three grades is shown in Figs. 3 and 4 for primary and preantral follicles, respectively. Between 5 and 10 tissue pieces were examined, depending upon the abundance of follicles. For
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the purposes of further data processing and comparison with other studies, grade 1 was classed as normal, with grade 2 and 3 being abnormal. The percentage normality of primary and preantral follicles after the various treatments are displayed in Tables 1 and 2. From the results displayed in Figs. 3 and 4, it can be seen that for both control primary and preantral follicles (Figs. 3A and 4A), there was a small drop in the percentage of high-grade (grade 1) follicles seen comparing freshly isolated tissues and tissues cultured for up to 4 h, with a concomitant small increase in the lowest grade (grade 3). During the later stages of culture (24 and 48 h) there was a continued shift in normalities of the control follicles, with further drops in grade 1 and increases in grade 3. Similar patterns were seen in both types of follicles after exposure to Me 2SO alone (Figs. 3B and 4B), with stable percentages of grade 1 follicles over 4 h in culture, while beyond that time there was a decrease in grade 1 and an increase in grade 3. Immediately after cryopreservation, the numbers of primary and preantral follicles that were judged as grade 1 (Figs. 3C and 4C) were lower than in the control tissue or tissue exposed to Me 2 SO, with grade 2 follicles (intermediate damage) being noticeably higher than in control tissues. Over the next 4 h, for both follicle types, an increase in grade 1 follicles and parallel changes in grades 2 and 3 were observed (Figs. 3C and 4C). Thereafter, during long-term culture the cryopreserved follicles exhibited the same changes in follicle structure as seen in control tissues after the same period, such that by 48 h, the distributions among the three grades were similar in all groups. When the results were expressed as percentage of normal follicles (grade 1) from the total numbers counted, the values in Table 1 show that immediately after freezing there was significantly lower percentage normality of primary follicles in the cryopreserved tissue than in the tissue exposed to cryoprotectant without freezing and control tissue. The situation was the same after 1 h of culture but the damage appeared to have been repaired by 4 h. At 24 and 48 h the percentage normality was reduced in all cases.
FIG. 1. Photomicrographs of bovine primary follicles within ovarian tissue (bar, 30 mm). (a) Normal (grade 1) follicle. (b) Abnormal (grade 2) follicle. (c) Abnormal (grade 3) follicle.
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FIG. 2. Photomicrographs of bovine preantral follicles within ovarian tissue (bar, 30 mm). (a) Normal (grade 1) follicle. (b) Abnormal (grade 2) follicle. (c) Abnormal (grade 3) follicle.
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in post-thaw culture appeared to take place a little faster (being completed within 1 h). For both primary and preantral follicles, there were
FIG. 3. Percentage of primary follicles of each grade at each post-thaw culture period. (A) Control untreated tissue. (B) Tissue exposed to cryoprotectant without freezing. (C) Cryopreserved tissue. Grade 1 s, Grade 2 u, Grade 3 o.
As shown in Table 2, the results for preantral follicular normality showed a pattern similar to that of primary follicles, although the recovery
FIG. 4. Percentage of preantral follicles of each grade at each post-thaw culture period. (A) Control untreated tissue. (B) Tissue exposed to cryoprotectant without freezing. (C) Cryopreserved tissue. Grade 1 s, Grade 2 u, Grade 3 o.
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EFFECT OF POST-THAW CULTURE ON OVARIAN TISSUE
TABLE 1 Median (Range) Percentage of Normal (Grade 1) Primary Follicles from 5–10 Tissue Pieces Time in culture (h)
Control
Exposed to CPA
Cryopreserved
0 1 4 24 48
57 (42–57) 39 (39–46) 36 (36–43) 26 (20–40) 12 (4–20)
46 (40–48) 40 (40–48) 35 (35–36) 32 (14–39) 12 (4–22)
*18 (11–25) *26 (18–35) 35 (27–40) 16 (12–34) 18 (8–26)
* Significantly different from values in same row.
no significant differences between percentage normality in control tissues and that in tissue exposed to cryoprotectant without freezing, after any of the culture times. DISCUSSION
During normal ovarian function, a natural maturation process occurs throughout consecutive hormonal cycles which permits development of a number of the primary follicles through secondary and tertiary follicular stages to large antral follicles containing a mature oocyte for ovulation, while other follicles within the cohort become atretic and degenerate (12). Thus, there is a background of natural follicular change within an ovary against which any effects of cryopreservation must be set. In addition, the methods of harvesting and manipulation of the ovarian tissue prior to cryopreservation may have an impact on the normality of follicle structure. From our current studies it would appear that approximately half of the primary or preantral follicles in the fresh, control tissues were of the highest morphological grade after initial tissue collection and dissection, and these would be the type of follicles selected for maturation in vitro. These percentages are of a similar order to those reported in studies on ovarian tissues from other species such as primates (6). Greater percentages of high-grade primary follicles (up to 90%) have been reported in ovaries of prepubertal mice (7) but this probably reflects the physiology of the ovary in such immature animals. In the current
study, the small decrease in percentage of normal follicles seen once the tissues were returned to 37°C probably reflects latent damage incurred in the tissues during surgical removal and transport and which is only fully expressed once the tissues were returned to 37°C (a form of hypoxia/reoxygenation damage (19)). The fact that the percentages of grade 2 follicles in each case stayed relatively constant might suggest that there was a progression of follicle degeneration with time in culture, i.e., grade 1 follicles degenerating into grade 2 scoring and, concomitantly, equivalent amounts of grade 2 follicles degenerating into grade 3 scoring. Although restoration of fertility following autografting of ovarian tissue has resulted in live births in sheep (11), the use of this course of treatment for cancer patients is questionable due to the risk of transmission of the disease following grafting of tissue (23). It has also been possible to achieve follicular development to the large antral stage under laboratory conditions by xenografting ovarian tissues from a donor species (e.g., marmoset) into a well-vascularized site in immune-incompetent mice (7), but for human infertility treatment such a technique raises several questions about the ethics and safety (from transmissible diseases) of using an intermediate living host. In the clinical setting, in vitro maturation of follicles to yield oocytes for fertility treatment would at present probably be the preferred option. The later stages of follicle growth (antral), while being the most conducive to maturation in vitro, are less frequently found in ovarian tissue biopsies TABLE 2 Median (Range) Percentage of Normal (Grade 1) Preantral Follicles from 5–10 Tissue Pieces Time in culture (h)
Control
Exposed to CPA
Cryopreserved
0 1 4 24 48
48 (44–56) 36 (32–36) 36 (36–40) 24 (16–40) 10 (8–12)
44 (24–48) 36 (36–48) 44 (32–44) 8 (4–28) 10 (10–28)
*12 (12–28) 32 (16–32) 32 (20–31) 20 (4–44) 12 (10–20)
* Significantly different from values in same row.
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and may also be more susceptible to freeze/thaw damage (14). Candy et al. (7) found in a murine model that while most primordial follicles survived cryopreservation, less than 49% of primary follicles survived when intact murine ovaries (;2 mm 3) were preserved. When primate (6) ovarian tissue was cryopreserved and implanted in a “pseudo” in vivo environment (i.e., in immune-incompetent mice), no large antral follicles could be found immediately after, or for a few days following, cryopreservation but some of the surviving preantral follicles did mature to form large antral stages after a few weeks of implantation. For follicles to be matured in vitro they would have to be isolated from the fibrous ovarian tissue which would most practically involve a combination of enzymatic digestion and mechanical disruption (22). To achieve any success, as many follicles as possible must be of high morphological grade in the tissue after cryopreservation and before the isolation procedures are started. Human primordial follicles have been shown to survive freezing (14) and grafting into immuno-deficient mice as tissue slices (17) and have been successfully isolated from human tissue after cryopreservation (20). As yet no methods exist for maturation of primordial follicles in vitro from large mammalian species, the technique having only been achieved in mice (10). Thus primary and preantral follicle survival may be of more significance at a practical level. Long-term (several weeks) in vitro culture of preantral and early antral human follicles has recently been demonstrated (1). From the current studies, it appears that the procedures for exposure and removal of cryoprotectant did not significantly affect the morphological grading of the follicles. These conditions were selected on the basis of previous work investigating cryoprotectant diffusion in ovarian tissue (18, 25) and are likely to result in the Me 2SO permeating the tissue, at least to a large extent. However, the procedures of cryopreservation in our study did cause a significant drop in the percentage of normal primary, and especially preantral, follicles when assessed immediately after thawing and cryoprotectant re-
moval. Similar effects have been noted when studying primate and murine ovarian tissues recovered from cryopreservation (6, 7). This may reflect structural damage following osmotic dehydration during slow cooling or ice nucleation and crystal growth during rewarming. When both fresh control and cryoprotectant-exposed tissues were placed into culture, there was a gradual decline in the high-grade morphology group of follicles over 4 h and a more significant fall over the next 24 and 48 h, suggesting that the culture conditions were not optimal for follicle survival within the tissue pieces. Long-term (5-day) culture of isolated murine follicles has certainly been possible in our laboratory (3) and in that of others (5, 8, 24); thus, it may be that diffusion of nutrients, oxygen, or autocrine factors are a limiting factor within three-dimensional tissue pieces. When the cryopreserved tissues were examined, an increase in the number of high-grade early follicles was seen over 4 h, particularly for the preantral follicles which more than doubled compared with those seen immediately after cryopreservation. In fact by 4 h of culture, the numbers of high-grade follicles of both primary and preantral stages were the same as those of the fresh tissue. This suggests that the process of rewarming the cryopreserved tissue in a nutrient-rich environment allowed the follicular cells to reestablish metabolic activity, normal cell volume control, and cell– cell contacts. The slower recovery seen in post-thaw culture of the primary follicles may reflect the lower metabolic rate in such immature follicles (16). Again, during the later (24- and 48-h) periods of culture, the normality of the follicles from the cryopreserved tissues declined in a fashion similar to that in fresh untreated tissues. In summary, these results suggest that there may be a place for post-cryopreservation culture of ovarian tissues to maximize yields of follicles for oocyte maturation. In addition, there is room for improving both the cryopreservation and the culture conditions for ovarian tissues, with strategies such as the recently reported use of extracellular matrix (15) being likely to assist in the latter. However, the long-term goal is to achieve
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isolation of follicles capable of maturation from cryopreserved tissues, and studies are underway to investigate this aspect. REFERENCES 1. Abir, R., Franks, S., Mobberley, M. A., Moore, P. A., Margara, R. A., and Winston, R. M. L. Mechanical isolation and in vitro growth of preantral and small antral human follicles. Fertil. Steril. 68, 682– 688 (1997). 2. Apperley, J. F., and Reddy, N. Mechanisms and management of treatment-related gonad failure in recipients of high dose chemoradiotherapy. Blood Rev. 9, 93–116 (1995). 3. Ashley, P. J., Paynter, S. J., Cooper, A., Fuller, B. J., and Shaw, R. W. A comparison between host serum and foetal calf serum in an in vitro maturation system for pre-antral mouse ovarian follicles. Fertil. Steril. Abstract Series 19, 40 (1997). 4. Bernard, A., and Fuller, B. J. Cryopreservation of human oocytes: A review of current ideas and future perspectives. Hum. Reprod. Update 2, 193–207 (1996). 5. Boland, N. I., and Gosden, R. G. Effects of epidermal growth factor on the growth and differentiation of cultured mouse ovarian follicles. J. Reprod. Fertil. 101, 369 –374 (1994). 6. Candy, C. J., Wood, M. J., and Whittingham, D. G. Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Hum. Reprod. 10, 2334 –2338 (1995). 7. Candy, C. J., Wood, M. J., and Whittingham, D. G. Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries. J. Reprod. Fertil. 110, 11–19 (1997). 8. Cortvrindt, R., Smitz, J., and Van Steirteghem, A. In vitro maturation, fertilization and embryo development of immature oocytes from early pre-antral follicles from prepubertal mice in a simplified culture system. Hum. Reprod. 11, 2656 –2666 (1996). 9. Donnez, J., and Bassil, S. Indications for cryopreservation of ovarian tissue. Hum. Reprod. Update 4, 248 – 259 (1998). 10. Eppig, J. J., and O’Brien, M. J. Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54, 197–207 (1996). 11. Gosden, R. G., Baird, D. T., Wade, J. C., and Webb, R. Restoration of fertility to oophorectomised sheep by ovarian autografts stored at 2196°C. Hum. Reprod. 9, 597– 603 (1994). 12. Gougeon, A. Dynamics of human follicular growth. A morphologic perspective. In “The Ovary” (E. Y. Adashi and P. C. K. Leung, Eds.), pp.21–39. Raven Press, New York, 1993.
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13. Gunasena, K. T., Villines, P. M., Critser, E. S., and Critser, J. K. Live births after autologous transplant of autologous mouse ovaries. Hum. Reprod. 12, 101–106 (1997). 14. Hovatta, O., Silye, R., Krausz, T., Abir, R., Margara, R., Trew, G., Lass, A., and Winston, R. M. L. Cryopreservation of human ovarian tissue using dimethyl sulphoxide and propanediol-sucrose as cryoprotectants. Hum. Reprod. 11, 1268 –1272 (1996). 15. Hovatta, O., Silye, R., Abir, R., Krausz, T., and Winston, R. M. L. Extracellular matrix improves survival of both primordial and primary ovarian follicles in long term culture. Hum. Reprod. 12, 1032– 1036 (1997). 16. Krohn, P. L. Transplantation of the ovary. In “The Ovary” (S. Zukerman and B. J. Weir, Eds.), Vol. II, pp. 101–128. Academic Press, New York, 1977. 17. Newton, H., Aubard, Y., Rutherford, A., Sharma, V., and Gosden, R. Low temperature storage and grafting of human ovarian tissue. Hum. Reprod. 11, 1487–1491 (1996). 18. Newton, H., Fisher, J., Arnold, J. R. P., Pegg, D. E., Faddy, M. J., and Gosden, R. G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum. Reprod. 13, 376 –380 (1998). 19. Nugent, D., Newton, H., Gallivan, L., and Gosden, R. G. Protective effect of vitamin E on ischaemiareperfusion injury in ovarian grafts. J. Reprod. Fertil. 114, 341–346 (1998). 20. Oktay, K., Nugent, D., Newton, H., Salha, O., Chatterjee, P., and Gosden, R. G. Isolation and characterisation of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil. Steril. 67, 481– 486 (1997). 21. Parrott, D. M. V. The fertility of mice with orthotopic ovarian grafts derived from frozen tissue. J. Reprod. Fertil. 1, 230 –241 (1960). 22. Roy, S. K., and Treacy, B. J. Isolation and culture of human preantral follicles. Fertil. Steril. 59, 783–790 (1993). 23. Shaw, J. M., Bowles, J., Koopman, P., Wood, E. C., and Trounson, A. O. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit cancer to graft recipients. Hum. Reprod. 11, 1668 – 1673 (1996). 24. Spears, N., Boland, N. I., Murray, A. A., and Gosden, R. G. Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile. Human Reprod. 9, 527–532. 25. Thomas, N., Busza, A., Cooper, A., Paynter, S., Fuller, B., and Shaw, R. Measurement of permeating levels of cryoprotectant during ovarian tissue cryopreservation using 1H NMR spectroscopy in human and porcine ovaries. CryoLetters 78, 179 –184 (1997).