Ecole Nationale Vétérinaire de Lyon, Marcy l Etoile, and Hôpital Edouard Herriot, Lyon, France

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1 REPRODUCTIVE BIOLOGY FERTILITY AND STERILITY VOL. 75, NO. 4, APRIL 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols Banu Demirci, D.V.M., a Jacqueline Lornage, M.D., Ph.D., b Bruno Salle, M.D., Ph.D., b Lucien Frappart, M.D., Ph.D., c Michel Franck, D.V.M., a and Jean François Guerin, M.D., Ph.D. b Ecole Nationale Vétérinaire de Lyon, Marcy l Etoile, and Hôpital Edouard Herriot, Lyon, France Received May 23, 2000; revised and accepted October 2, Reprint requests: Banu Demirci, D.V.M., Laboratoire de Zootechnie, Ecole Nationale Vétérinaire de Lyon, Marcy l Etoile, France (FAX: ; b.demirci@vet-lyon.fr). a Laboratoire de Zootechnie, Ecole Nationale Vétérinaire de Lyon. b Laboratoire de Biologie de la Reproduction and CECOS, Hôpital Edouard Herriot. c Laboratoire d Anatomie Pathologique, Hôpital Edouard Herriot /01/$20.00 PII S (00) Objective: To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. Design: Small follicles ( 60 m in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. Setting: Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France. Animal(s): Lambs 5 to 6 months of age. Intervention(s): Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. Main Outcome Measure(s): Follicular mortality and histologic structure. Result(s): For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 C/min) without seeding and the standard very slow cooling protocol (0.3 C/min) were similar. Conclusion(s): Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2MofDMSO. (Fertil Steril 2001;75: by American Society for Reproductive Medicine.) Key Words: Cryopreservation, freezing protocols, ovarian tissue, sheep The long-term survival rate in young persons with malignant disease is constantly increasing. Ovarian or testicular failure is frequently reported after chemotherapy or radiotherapy (1), and it is especially difficult to protect the ovaries of young girls. Ovarian cryopreservation is an attractive concept, even though no human pregnancy after this technique has been reported. Animal studies have demonstrated that frozen-thawed ovarian tissue can restore cyclic secretion of ovarian steroids after autograft (2, 3) or transplantation (4 6). Pregnancy after frozen ovarian autograft has been reported in rats, mice, and ewes (7 10). Preservation of the pool of primordial follicles offers a means of conserving large numbers of female gametes. This can be realized by cryopreservation of primordial follicles after isolation (11) or by cryopreservation of the ovarian cortex. Sheep ovaries resemble human ovaries in terms of size and tissue composition. Primordial follicles in thin strips of sheep ovarian cortex remain viable after freezing to 196 C (3, 6, 8). Fertility can then be restored by ovarian autograft or, hypothetically, by isolating the primordial follicles of thawed ovarian tissue and performing in vitro maturation, fol- 754

2 lowed by IVF-ET. The latter procedure has been carried out successfully on fresh ovarian tissue in mice (12, 13). The cryopreservation procedure still could be improved in terms of the concentration of cryoprotectants and freezing and thawing techniques. Most investigators use the same protocol used for cryopreserving embryos manual seeding with very slow cooling (0.3 C/minute). This technique was used first in mouse primordial follicles in 1990 (11) and in sheep ovarian tissue in 1994 (8). We sought to assess the viability of small follicles after different freezing and thawing protocols. First, we compared the toxicity of two cryoprotectants dimethyl sulfoxide (DMSO) and 1,2-propylene glycol (PROH), on primordial follicles. We then compared the protective effect of the two cryoprotectants at different concentrations (1 M, 1.5 M, and 2 M) during the freezing procedure, using a slow cooling rate (2 C/minute) with and without semiautomatic seeding. Results were compared with those obtained by using the standard freezing protocol (cooling rate of 0.3 C/minute and manual seeding with 1.5 M DMSO). MATERIALS AND METHODS We collected 63 ovaries from 5- to 6-month-old lambs (breed unknown) at the slaughterhouse. The ovaries were placed in Minimum Essential Medium (MEM) (Sigma, St. Louis, MO) and were transported to the laboratory at 4 C in order to minimize ischemia. They were divided into two equal pieces at room temperature within hours of collection. The ovarian medulla was removed to obtain a layer of hemiovarian cortex 2 mm in thickness. Each fragment measured approximately 1 cm 2, and the mean weight was mg. Cryoprotectant Toxicity Ovarian fragments were incubated at room temperature for 10 minutes in BM1 medium (Ellios Bio-Media, Paris, France) with 10% fetal calf serum (FCS) (Sigma) supplemented with different concentrations of cryoprotectant. We used DMSO (Sigma) and PROH (Sigma) at concentrations of 10 M (80%), 9 M (70%), 6.5 M (50%), 4 M (30%), and 2 M (15%). Five ovarian cortex fragments were incubated with each concentration of cryoprotectant. Ten control ovarian fragments were incubated in BM1 medium (Ellios Bio-Media) without cryoprotectant. After incubation, fragments were washed in 1 ml of Leibovitz L-15 medium (Sigma) for 5 minutes to eliminate the cryoprotectant. One piece of tissue was fixed in Bouin liquid (Chimie-Plus, Denicé, France) for histologic examination; the rest of the tissue fragments were used to determine the rate of follicle viability. Ovarian Tissue Freezing Two freezing procedures were used: [1] freezing without seeding, in which the seeding temperature was determined by noting the spontaneous increase in temperature (T 2 ) that accompanied ice crystal nucleation, and [2] freezing with seeding, in which semiautomatic seeding was performed by release of negative calories at T 2. The freezing process was performed by using 1 M, 1.5 M, and2mofdmsoandproh. Five hemiovarian cortex fragments without seeding and five with seeding were frozen. Ten hemiovarian cortex fragments were frozen without cryoprotectant as controls for each procedure. Ovarian fragments were placed in cryogenic vials (Nunc Brand Products, Roskilde, Denmark) containing 1 ml of BM1 freezing medium supplemented with 10% FCS and the cryoprotectant. The fragments were incubated at room temperature for 10 minutes before freezing. After incubation, the cryogenic vials were placed in a programmable freezer (Minicool 40PC; Air Liquide Santé, Paris, France). All six cryogenic vials (five experimental and one control) were frozen at the same time. The freezing without seeding protocol consisted of a slow descent in temperature R 1 from 10 C (T 1 )to 40 C (T 3 )at a speed of 2 C/minute, then a descent R 2 from 40 C to 140 C (T 4 ) at a rate of 25 C/minute. The first cooling phase (R 1 ) was made much longer than the R 1 of the procedure with seeding to detect temperature changes in the cooling medium. During this phase, the point at which the temperature increased (T 2 ) was noted. For freezing with seeding, the tubes were cooled at the same rates: R 1 (2 C/minute) to 35 C, then R 2 (25 C/ minute) to 140 C. During the first descent, before reaching T 2, the latent heat generated was compensated by release of negative calories within the freezing chamber (semiautomatic seeding). The cooling curves were recorded throughout the procedure by computer. Temperature graphs were obtained for each freezing process. The cryogenic vials were transferred to liquid nitrogen and stored for 24 hours. We also tested the standard freezing protocol with 1.5 M of DMSO described by Gosden (8) in six hemiovarian cortex fragments. The vials were cooled at 2 C/minute to 7 C, then held at 7 C for 10 minutes for manual seeding. The temperature was then decreased by 0.3 C/minute to 40 C and thereafter by 10 C/minute to 140 C. Ovarian Tissue Thawing Ovarian tissues were thawed rapidly at 37 C and washed in Leibovitz L-15 medium for 5 minutes. Vials were shaken gently to promote efflux of cryoprotectant from the tissue. One tissue sample was fixed in Bouin liquid for histologic examination; the rest were used to determine the rate of follicle viability. Isolation of Small Follicles and Viability Testing Ovarian fragments were thinly sectioned in Leibovitz L-15 medium supplemented with 1 mg/ml (200 IU/mL) type 1 collagenase (Sigma). Fragments were incubated at FERTILITY & STERILITY 755

3 FIGURE 1 The morphology of primordial follicles (arrows) after incubation in 2Mofdimethyl sulfoxide (original magnification, 400). 37 C for 2 hours and pipetted every 30 minutes. Collagenase activity was inhibited by addition of 50% FCS. The suspension was filtered through a 60- m nylon filter (Bioblock Scientific, Illkirch, France) and centrifuged at 400 g for 5 minutes. The precipitate was diluted with 50 ml of Leibovitz L-15 medium and conserved in a water bath at 37 C. The suspension containing follicles (20 L) was deposited on a glass slide and examined by using an inversion microscope (original magnification, 400). Follicle staining was done at room temperature with trypan blue, 0.4% (Sigma); dead follicles stained blue, and live ones were not stained. For each fragment, we examined 100 small follicles ( 60 m in diameter). Only intact follicles were examined; partially or completely denuded oocytes were excluded and the decision based on oocyte viability. Histologic Examination The fragments were cut into sections that were 5 m in thickness after fixation in Bouin liquid. Staining with hematoxylin and eosin was done to examine follicular morphology. Statistical Analysis Coefficient correlations were obtained by using Pearson and nonparametric tests (Wilcoxon, Kruskal Wallis, and Mann Whitney). Differences were considered statistically significant at P Unistat software (Unistat, London, United Kingdom) was used for statistical analysis. RESULTS Because the number of primordial follicules varied among fragments, we chose to examine 100 follicles per fragment. Cryoprotectant Toxicity Microscopic examination of ovarian tissue showed that cryoprotectant toxicity resulted in cytoplasmic vacuoles and pycnotic nuclei, and the cells appeared shrunken. Follicle morphology improved at DMSO concentrations 6.5 M. At 2 M DMSO, follicle morphology was similar to that in control fragments (Fig. 1). At concentrations of PROH below 4 M, follicular morphology was similar to that in control fragments. In some control fragments, granulosa cells disrupted from thecal cells, and cytoplasmic vacuolation and nucleic condensation occurred. These anomalies may be histologic artifacts or the result of ischemia. Most follicles were dead at 10 M of DMSO ( %) (Fig. 2), but at 10 M of PROH, the mean mortality rate was only % (P 0.001). Primordial follicle mortality decreased as cryoprotectant concentrations decreased (r DMSO 0.99; r PROH 0.98). The mortality rate among primordial follicles was significantly lower at DMSO concentrations 4 M (8.0% 2.2%) and PROH concentrations 9 M (9.0% 2.6%) (Fig. 3). Results of viability tests and morphologic studies were 756 Demirci et al. Cryopreservation of sheep ovarian tissue Vol. 75, No. 4, April 2001

4 FIGURE 2 Results of trypan blue staining. (A), Dead follicles; (B), live follicles. Arrows 50 m. FIGURE 3 Follicular mortality after exposure of the ovarian tissue to dimethyl sulfoxide (shaded bars) or propylene glycol (white bars). * Significantly different from the dimethyl sulfoxide control group (r DMSO 0.99). ** Significantly different from the propylene glycol control group (r PROH 0.98). FERTILITY & STERILITY 757

5 discordant. Tissue morphology was similar with 10 M of DMSO and 10 M of PROH; however, the mortality rate among isolated follicles differed. In control fragments, cell morphology did not differ significantly at 4 M of PROH and 2MofDMSO. Determination of Seeding Temperature and Optimal Freezing Conditions for Ovarian Tissue For the freezing procedure, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity testing. During the freezing without seeding protocol, seeding temperature (T 2 ) was determined for each freezing medium. As the cryoprotectant concentration increased, T 2 decreased: 7 C, 8 C, and 11 C with DMSO and 6 C, 8 C, and 11 C with PROH at concentrations of 1 M, 1.5 M, and 2 M, respectively. After a slight increase in temperature, the temperature of the medium decreased, then increased to reference levels and followed the 2 C/minute descent curve once again (Fig. 4A). During the seeding with freezing protocol, negative calories were released at the ice nucleation temperature (T 2 ), which was previously determined for each freezing medium. With seeding, the temperature variations in the medium were constantly controlled (Fig. 4B). The increase in temperature never exceeded 5 C. Ovarian fragments that were frozen without cryoprotectant were completely structurally disorganized: The stroma was lysed, primordial follicles and the connective tissue were cleaved, primordial follicles had irregular contours, oocytes were lysed, and the distribution of chromatin was irregular. We could not isolate a sufficient number of small follicles for viability testing. Morphologic structure improved when a lower concentration of DMSO (1 M) was used, but the tissue was not well preserved. Primordial follicles had cytoplasmic microvacuolation and retracted cytoplasm, numerous nuclei were fragmented, and mature oocytes had lost their structure. The best primordial follicle morphology was obtained by using freezing with seeding and 2MofDMSO(Fig. 5). Tissue morphology was well preserved at cryoprotectant concentrations 1.5 M (DMSO or PROH), even though mature follicles still showed morphologic anomalies. The mortality rate was lowest for 1MofDMSO(19.0% 2.9% for freezing without seeding and 11.2% 1.3% for freezing with seeding). In contrast, at 1MofPROH, the mortality rate among small follicles was high (44.2% 5.5% and 24.0% 4.2% without and with seeding, respectively) (Fig. 6). As cryoprotectant concentrations were increased within nontoxic limits (up to 2 M), the mortality rate among isolated follicles decreased, as was expected from histologic observations. At 1.5 M of DMSO, the difference in follicle mortality rates between the standard very slow (0.3 C/minute) freezing protocol (17.6% 1.59%) and our protocol (2 C/ minute) without seeding (16.0% 1.0%) did not differ significantly (P 0.12). We observed a significant difference between the standard very slow protocol and our protocol with seeding (17.6% 1.59% vs. 12.0% 2.12%) (P 0.01), irrespective of the weight of the ovarian fragments. At most cryoprotectant concentrations; follicle morphology and isolated follicle vitality was slightly better when freezing included seeding, but not significantly so (Table 1). In fact, cytoplasmic vacuolation decreased when seeding was performed. In the freezing with seeding protocol, the difference among three concentrations of cryoprotectant was significant (Fig. 6). The weight of frozen ovarian fragments did not influence the mortality rate of isolated primordial follicles (P.2). Irrespective of weight, the thickness of ovarian tissue fragments was always 2 mm. DISCUSSION Mastery of cryoprotectant and freezing-thawing procedures can be of great importance in the prevention of infertility in young women receiving chemotherapy or total-body irradiation. The likelihood of permanent sterility is high after an aggressive cancer therapy (14). Ovarian tissue banking offers new possibilities of reinstalling fertility after treatment. The ovary contains a follicular reserve that is determined before birth and cannot be replenished. This reserve contains more than enough follicles for all menstrual cycles between puberty and menopause. Primordial follicles are the most important type of reproductive cells in ovarian tissue. The main goal of ovarian fragment cryopreservation is to preserve primordial follicles, rather than growing follicles, in order to reinstitute fertility. Since primordial follicles are small and less metabolically active, they may be more tolerant to the freeze-thaw process (8). Cryopreservation of primordial follicles requires not only the survival of oocyte and granulosa cells but also maintenance of the gap junctions and the metabolic cooperation essential for oocyte growth and development (15, 16). Primordial follicles have been successfully isolated (17 19). In our study, small follicles were isolated and characterized after incubation for 2 hours with collagenase type I by using a protocol tested in porcine (18) and human (19) ovarian tissue. Cryoprotectants are necessary for successful cryopreservation. We investigated the toxicity of two cryoprotectants in the first part of this study. After 10 minutes of incubation at room temperature, DMSO is more toxic than PROH at high concentrations. The viability of primordial follicles was sat- 758 Demirci et al. Cryopreservation of sheep ovarian tissue Vol. 75, No. 4, April 2001

6 FIGURE 4 Results of cooling without (A) and with (B) semiautomatic seeding. The temperature of tubes containing ovarian tissue is plotted as a function of time..... reference; medium; ---- N 2 tank. isfactory with 2 M of either DMSO or PROH. Recently, Jewgenow et al. (20) showed that the exposure of cat ovaries to cryoprotectants (1.5 M of DMSO and 1.5 M of PROH at 0 C for 15 minutes) did not influence the percentage of intact follicles. Incubation time is as important as the concentration of cryoprotectant and depends on the type of cryoprotectant utilized and the incubation temperature. Dimethyl sulfoxide penetrates human ovarian tissue more rapidly than PROH at 4 C. At 37 C, cell penetration is similar for the two cryoprotectants; however, they are more cytotoxic at this temperature (21). Our results are similar to those previously FERTILITY & STERILITY 759

7 FIGURE 5 Primordial follicle morphology after freezing-thawing. Semiautomatic seeding protocol with 2 M dimethyl sulfoxide (original magnification, 400). The long arrow indicates intact oocytes; the short arrow indicates an oocyte with pycnotic nucleus. obtained: DMSO is more toxic at room temperature, even though cell penetration appears to be similar on histologic examination. The standard incubation time for mouse ovarian tissue is 5 minutes at room temperature (2, 10). Cox et al. (9) increased incubation time to 15 minutes when the incubation temperature was decreased to 0 C in mouse fetal ovarian tissue. A high survival rate was observed among primordial follicles from mouse ovaries that were exposed to 1.5 M of DMSO and PROH for only 5 minutes at room temperature and then frozen (22). Incubation time is increased in human and sheep ovarian tissue because they are more fibrous and cryoprotectant penetration is reduced (5, 6, 8, 23, 24). Gook et al. (25) suggested that incubation time with PROH and sucrose can be as high as 90 minutes for human ovarian tissue; however, these investigators did not give the incubation temperature. In the second part of this experiment, we studied different freezing protocols: a slow freezing protocol with a more rapid rate of cooling (2 C/minute) vs. standard freezing protocol with very slow cooling rate (0.3 C/minute). Our protocol is original in that seeding was performed semiautomatically by releasing negative calories in the freezing chamber, without manual manipulation of the vials. Standard freezing protocols are based on embryo freezing techniques (11). Embryos are composed of few cells that have large volume. We propose a technique that is more currently used for freezing tissue samples composed of smaller cells, such as parathyroid tissue (26). This latter freezing technique, in which a more rapid rate of cooling (2 C/minute) was used, was as effective as standard freezing protocols. Most current protocols use DMSO at 1.5 M. We evaluated the follicular survival rate after freezing and thawing with different concentrations of DMSO or PROH. The morphologic structure of ovarian fragments appeared to be improved when cryoprotectants were added at low concentrations: At 1 M of DMSO, the cytoplasm of primordial follicles was retracted and cytoplasmic microvacuolation occurred, but the percentage of dead primordial follicles was low. All primordial follicles were highly disorganized, and the primordial follicle mortality rate was higher with 1Mof PROH. The best histologic characteristics and rates of follicle viability were obtained after freezing with seeding at 2M of DMSO or PROH. The difference between PROH and DMSO was no longer significant for concentrations greater than 1.5 M. Other investigators (5, 22) have obtained similar results. The architecture of ovarian fragments was well preserved 760 Demirci et al. Cryopreservation of sheep ovarian tissue Vol. 75, No. 4, April 2001

8 FIGURE 6 Follicular mortality after freezing-thawing of the ovarian tissue by using freezing without semiautomatic seeding (shaded bars) or freezing with semiautomatic seeding (white bars). *P 0.01 compared with propylene glycol freezing protocols with seeding. **P 0.02 compared with dimethyl sulfoxide freezing protocols with seeding. ***P 0.01 compared with propylene glycol freezing protocols without seeding. and the percentage of follicular mortality was very low in tissues cooled with 2 M of DMSO. Intracytoplasmic microvacuolization of oocytes has been described in marmoset ovaries (4). In our study, microvacuolization decreased when the freezing protocol included semiautomatic seeding. Sudden changes in temperature were avoided by release of negative calories. A cooling rate of 2 C/minute does not alter follicular survival rates. The effect of the cryoprotectant (nature and concentration) appears to be more important than the cooling rate. We show that a large fragment of ovarian cortex can be frozen TABLE 1 Mortality rate among isolated follicles according to freezing procedure and the type and concentration of cryoprotectant. PROH DMSO Freezing procedure 1 M 1.5 M 2 M 1 M 1.5 M 2 M Without seeding With seeding P value Note: Values are medians, and P values were calculated by using the Wilcoxon test. DMSO dimethyl sulfoxide; PROH propylene glycol. FERTILITY & STERILITY 761

9 properly as long as the thickness of the tissue does not exceed 2 mm, thus allowing adequate penetration of cryoprotectant. Many investigators (3, 6, 8, 24) have restored cyclic secretion of ovarian steroids after ovarian autograft by using freezing-thawing procedures. All reported large reductions in primordial follicle density after a long postgraft period. The grafting procedure itself may cause most of the follicular loss. As far back as 1956, it was observed that only 59% of follicles found in newly collected ovaries were present in grafts 2 to 30 days later (27). More recently, it was noted that grafting procedures are more deleterious than the freezing procedure to follicular survival (24). Revascularization of grafts takes 3 days; this period, in which the greatest proportion of follicular loss occurs, appears to be the most critical. Cryopreservation protocols need to be improved. Results are sufficiently encouraging for ovarian autoconservation to be considered as a method of fertility preservation, even though the future of ovarian fragments remains hypothetical. Autografts can restore ovarian function for a long period of time. At present, only one pregnancy has been reported in large mammals (8), and ovulation after transplantation of frozen banked ovarian tissue has been reported recently in humans (28). In vitro maturation of frozen-thawed primordial follicles may be an interesting alternative. Human primordial and primary follicles can be grown and maintained in ovarian tissue cultures in the long term. Frozen-thawed human follicles also appear to be viable and capable of further development in tissue culture (29). In conclusion, the toxicity of cryoprotectant is low at low concentrations: a large percentage of primordial follicles survive at concentrations of2mofproh and DMSO. Slow freezing of ovarian tissue with 2MofDMSOandsemiau- tomatic seeding results in minimal loss of primordial follicles. Acknowledgments: The authors thank Mehdi Benchaib, M.D., Ph.D., (Laboratoire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France) for statistical analysis and Marie T. Poirel (Laboratoire de Zootechnie, Ecole Nationale Vétérinaire de Lyon, Marcy l Etoile, France) for technical assistance. References 1. Byrne J, Fears TR, Gail MH. Early menopause in long terms survivors of cancer during adolescence. Am J Obstet Gynecol 1992;166: Harp R, Leibach J, Black J, Keldahl C, Karow A. Cryopreservation of murine ovarian tissue. Cryobiology 1994;31: Salle B, Lornage J, Demirci B, Vaudoyer F, Poirel MT, Franck M, et al. Restoration of progesterone secretion and histologic assessement after freezing, thawing and autograft of hemi-ovary in sheep. Fertil Steril 1999;72: Candy CJ, Wood MJ, Whittingham DG. 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Ovarian function after transplantation of frozen, banked autologous ovarian tissue [letter]. N Engl J Med 2000;342: Hovatta O, Silye R, Abir R, Krausz T, Winston RML. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long term culture. Hum Reprod 1997;12: Demirci et al. Cryopreservation of sheep ovarian tissue Vol. 75, No. 4, April 2001