Viability and function of the cryopreserved whole rat ovary: comparison between slow-freezing and vitrification

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1 ORIGINAL ARTICLES: REPRODUCTIVE BIOLOGY Viability and function of the cryopreserved whole rat ovary: comparison between slow-freezing and vitrification Milan Milenkovic, M.D., Ph.D., a,b Cesar Diaz-Garcia, M.D., a,c Ann Wallin, B.Sc., a and Mats Br annstr om, M.D., Ph.D. a a Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; b Division of Obstetrics and Gynecology, Telemark Hospital, Skien, Norway; and c Department of Obstetrics and Gynecology, La Fe University Hospital, University of Valencia, Valencia, Spain Objective: To investigate four different protocols for cryopreservation of the whole rat ovary with intact vasculature to evaluate whether differences exist in post-thawing viability of the ovary after either vitrification or slow freezing. Design: Experimental study. Setting: Obstetrics and gynecology department. Animal(s): Immature Sprague-Dawley female rats. Intervention(s): Ovaries were isolated with the vascular tree intact up to the bifurcation of the abdominal aorta and were subsequently cannulated. The ovaries were flushed with increasing concentrations of the cryoprotectant dimethyl sulfoxide (DMSO) to either 1.5 or 7 M. The ovaries underwent cryopreservation by vitrification or passive slow freezing. Main Outcome Measure(s): After thawing, the ovaries were subjected to neutral red viability staining to assess the density of viable small follicles and for long-term (48 hours) incubation evaluation of steroid secretion, histology, and apoptosis assay. Result(s): The follicular viability was decreased in both vitrification groups and in the slow-freezing group with the high concentration of DMSO, as compared with fresh controls. Estradiol levels in the incubation medium followed the same pattern. Light microscopy revealed well-preserved morphology in all groups after 48 hours incubation. Apoptosis was increased in both vitrified and cryopreserved ovaries. Conclusion(s): We have developed a new method that can be used in basic studies to improve cryopreservation protocols. Our initial findings suggest that a moderate concentration of the cryoprotectant DMSO is superior to a high DMSO concentration for both vitrification and slow freezing. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, ovary, rat, slow-freezing, vitrification Ovarian transplantation has been used for many years in the rat model for studies of ovarian endocrine function (1), and later the rat model was adapted for studies of ovarian function after cryopreservation (2). Ovarian cryopreservation research is performed for the purpose of strain rescue (3) and to optimize the procedure for use in programs for human female fertility preservation (4). Fertility preservation in the human female aims at preserving/restoring fertility in girls and young adult women who are planned to undergo potentially gonadotoxic cancer treatment (5, 6). However, in the light of that only a very limited number of live births have been reported and that most likely a large number of transplantation attempts have been performed, the procedure does most likely need further development to increase its effectiveness. Factors Received November 9, 2011; revised January 6, 2012; accepted January 25, 2012; published online February 16, M.M. has nothing to disclose. C.D.-G. has nothing to disclose. A.W. has nothing to disclose. M.B. has nothing to disclose. Reprint requests: Cesar Diaz-Garcia, M.D., Department of Obstetrics and Gynecology, La Fe University Hospital, Valencia, Spain ( diaz_cesgar@gva.es). Fertility and Sterility Vol. 97, No. 5, May /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert that should be improved are cryopreservation protocols (7, 8) and surgical transplantation procedures. Most of the follicular loss in cryopreserved tissue does not happen during the cryopreservation/thawing process itself but during the warm ischemic time after retransplantation (9, 10). Interventions such as transplantation to granulation tissue (11) or pretransplantation tissue incubation with growth factors (12), vitamin E (13, 14), or other antioxidants (15, 16) have shown moderate or no effect to increase follicular survival. Whole ovary transplantation has been suggested as an approach to overcome the deleterious effect of the prolonged ischemic time after the reintroduction of the tissue (17 21). Thus, research is 1176 VOL. 97 NO. 5 / MAY 2012

2 Fertility and Sterility ongoing to develop techniques for whole ovary cryopreservation and transplantation with vascular anastomosis (20 24). Indeed, live births have been demonstrated after whole ovary cryopreservation and vascular retransplantation both in the sheep (22) and in the rat (23), although the effectiveness of the entire procedure was very low with low live-birth rates. Our study developed a technique for isolation of the ovary and the vasculature pedicle in the rat, which we have used for an initial comparison of viability after cryopreservation by vitrification or slow freezing using fresh ovaries as controls. MATERIALS AND METHODS Animals Female immature Sprague-Dawley rats (Harlan Laboratories B.V.), aged 28 to 30 days and with a weight between 50 and 60 g, were used. This is an age/weight around 1 week before natural puberty. The animals were kept in group cages under controlled conditions (23 C) under a 12-hour light/dark cycle. They were fed pelleted food and tap water ad libitum. This experiment was approved by the Animal Ethics Committee of Gothenburg. Experimental Groups The experiment was designed to compare the viability of ovaries after different cryopreservation treatments. Each group consisted of five ovaries randomly allocated to the following groups: fresh control ovaries (F), or ovaries cryopreserved with low (L) or high (H) concentration of cryoprotectant by either vitrification (VIT) or slow-freezing (SLF). The four cryopreserved groups were thus referred as L-VIT or H-VIT after the vitrification procedure and as L-SLF or H-SLF after the slow-freezing procedure. Surgical Procedure The surgery was performed under aseptic conditions with the aid of a stereo operating microscope (6 to40 magnification; Leica Microsystems). The surgical procedure to isolate the ovary on a vascular pedicle was developed and modified in 20 animals before this study. Initial efforts (n ¼ 3) were made to place a smaller cannula directly into the ovarian artery on both sides to retrieve both ovaries from each animal. The arteries were too small to allow for successful cannulation. A second preliminary study (n ¼ 5) was performed to test an approach aiming to cannulate the aorta both from a cranial and from a caudal direction and then to ligate the aorta between the branching of the left and right renal vein to get separate cannulated specimens. However, this approach was not successful because the ligatures on the aorta would be too close to the branching of renal/ovarian arteries and would thereby disturb the flow. Finally, we retrieved the left ovary and a big vascular patch in 12 animals by the following methodology. The animals were anesthetized with isoflurane (4% induction and 1% maintenance). Bipolar diathermy (2-8W, Coa Comp Biocoagulator; Instrumenta AB) or coated sutures (Vicryl 6-0; Ethicon, Johnson & Johnson) were used before FIGURE 1 Schematic drawing of the abdominal vascular anatomy of the rat with the right ovary to be isolated. The rectangles represent points where vessels were ligated. The closed circle on the aorta indicates the insertion point of the cannula, and the open circle indicates the point where the cava was opened by a small slit. severance of any vessel (Fig. 1). A midline incision from the xiphoid process to the pubic bone was performed. The rectum was then transected to better expose the aorta and vena cava. The small bowel was taken out from the operatory field after severance of the mesenteric vessels. Afterward, isolation and severance of the lateral vessels of the aorta/cava was performed systematically with the bilateral iliolumbar vessels, the left common renal vascular trunk, and the left suprarenal and ovarian vessels being severed. After these steps, the left kidney with its adrenal gland was removed to get a good exposure of the cranial part of the surgical field. The ovarian artery and vein on the right side usually branch directly from the vena cava/aorta but in close proximity to the branching of the renal vessels and also with attachment to the lower pool of the left kidney (see Fig. 1). To not disturb the ovarian vessels at the dissection procedure, we gently incised the renal capsule on that side and pulled it over the kidney. A ligature was then placed over the renal vessels at a position in close proximity to the kidney, which was followed by nephrectomy. Two titanium clips (Hemoclip; Weck Closure System) were placed over the distal part of the right uterine horn, and the uterine branch of the ovarian artery was cut between the clips. The ovary with its vascular tree was at this point completely isolated but with intact arterial supply and venous drainage. Then, the dorsal lumbar arteries were identified, ligated, and severed. The distal ligation was tightened and secured with knots just before cannulation of the aorta. Cannulation of the aorta was performed at a site just caudal to the branching of the iliolumbar vessels and in a retrograde direction by the use of a 26G Teflon over-the-needle catheter (Terumo Sweden AB). After catheter fixation, the blood flow to the ovary was stopped by the cranial main ligature, which was placed around the aorta between the renal arteries and the VOL. 97 NO. 5 / MAY

3 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY celiac trunk. The whole vascular tree, including the ovary and its intact vasculature, could then be removed. At this step, a slit was made on the vena cava to allow for drainage of blood/fluid at flushing. The animals were killed at this stage. The ovarian specimen was then gently flushed by a handheld syringe with Ringer s acetate solution supplemented with 50 IU/mL of heparin sodium (Leo Pharma AB) and 0.2 mg/ml of xylocaine (Astra Zeneca) until only clear fluid was flushed out from the vena cava slit. Cryopreservation The ovaries were perfused with dimethyl sulfoxide (DMSO; Merck AG) diluted in appropriate concentrations in Leibovitz L-15 medium (GIBCO) at 4 C, according to the experimental protocol. The cannulated ovaries were placed in a tissue bath, and the cannula was connected to a pressure-infusion device (Rudolf Riester GmBH). The perfusion pressure remained constant at 100 mm Hg. The cryoprotectant was administered to the ovaries at increasing concentrations by M, 0.75 M, and 1.5 M for 5 minutes each for those with final concentrations of 1.5 M DMSO (L). Ovaries with a final concentration of 7 M DMSO (H) were perfused with increasing concentrations of DMSO (5 minutes each) of M, 1.75 M, 3.5 M, and 7 M. The ovaries subjected to slow freezing (L-SLF and H-SLF) were positioned into a 60 ml autoclavable, straight-sided, wide-mouth polypropylene jar (Nalgene Nunc) with 2 ml of either 1.5 M DMSO or 7 M DMSO; this cryovial container was then placed inside a precooled (4 C) container (Cryo Freezing Container; Nalgene Nunc) with isopropranolol. The larger container was placed inside a 80 C freezer for 24 hours to allow freezing at a rate of approximately 1 C/hours (24) and then was placed into liquid nitrogen and stored until thawing. The ovaries of the vitrification groups (L-VIT and H-VIT) were placed individually inside 60 ml autoclavable, straightsided, wide-mouth polypropylene jars (Nalgene Nunc) with 2 ml of either 1.5 M DMSO or 7 M DMSO. The container, with the cannulated ovary, was then plunged into liquid nitrogen and positioned there for 2 minutes before transfer to the storage tank. Thawing The cryovials were removed from the cryotank and positioned into a water bath at 37 C for 2 to 3 minutes to allow for complete thawing. The ovary was then dissected out from the bursa and subsequently rinsed in Leibovitz L-15 medium supplemented with decreasing concentrations of sucrose (0.5, 0.25, 0.1 M) for approximately 5 minutes in each washing step. After these steps to wash out the cryoprotectant, the ovary was divided into two hemiovaries and was processed for further viability tests. Density of Viable Primordial/Primary Follicles The ovarian cortex of one hemiovary was divided by scissors into small pieces (approximately mm), and these pieces were rinsed in phosphate-buffered saline (PBS) at room temperature. The pieces were transferred into a 35 mm culture dish (Nunclon; Nunc AS) containing 3mL of McCoy s 5a culture medium containing sodium bicarbonate and supplemented with 25 mm HEPES (Invitrogen-GIBCO), 0.1% bovine serum albumin (Roche Diagnostic), and 2 mm glutamate (Invitrogen-GIBCO). This culture medium was then supplemented with 50 mg/ml of neutral red solution (2-amino-3 methyl-7-dimethyl-aminophenazoniumchloride; Sigma-Aldrich), and the pieces were incubated at 37 C for 2 to 3 hours. Neutral red is a cationic vital dye, which at slightly acid ph gives a deep red color. It has been demonstrated that neutral red readily passes the cell membrane and that it concentrates in lysosomes of viable cells (25) so that viable and nonviable cells can be distinguished (26 29). After the incubation period, the medium was supplemented with 60 ml of a mixture of liberase (Liberase Blendzyme 3; Roche Diagnostics) and collagenase IV (Sigma-Aldrich) to final concentrations of 0.2 mg/ml of liberase and 0.04 mg/ml of collagenase IV to dissolve the tissue further and make follicles easily counted. The pieces were incubated in this mixture for 30 minutes, and the digestion was interrupted by adding 0.5 ml of fetal calf serum (FCS; Invitrogen-GIBCO). A small volume of the partially digested ovarian tissue was then transferred by a pipette onto a glass slide. Counting of number of viable small follicles was performed in an inverted microscope using a calibrated squared grid. Primordial follicles were oocyte surrounded by one layer of flattened granulose cells; primary follicles were oocyte surrounded by a single layer of cuboidal granulosa cells. Ovarian Culture and Estradiol Levels The other hemiovary was cut by scissors into small (approximately 1 1 mm) pieces and transferred into separate wells of a 24-well plate (Nunc AS) with 500 ml of McCoy s culture medium supplemented with 25 mm HEPES (Invitrogen- GIBCO), 0.1% bovine serum albumin (Roche Diagnostic), and 2 mm glutamate in each well. The ovarian tissue were subjected to a preincubation period of 24 hours; afterward, the medium was changed to a similar medium with an addition of 5 ng/ml of recombinant human follicle-stimulating hormone (FSH; Schering-Plough) for another 24 hours. The conditioned medium was then used for analysis of estradiol concentrations in duplicates by a DELFIA kit (Perkin-Elmer). Histology and Caspase-3 Immunohistochemistry Ovarian tissues that had been incubated for a 24-hour preincubation period and for 24 hours in the presence of FSH (5 ng/ml) were fixed in formaldehyde and then embedded in paraffin. Sections (approximately 4 mm thick) were stained using a rabbit antihuman polyclonal antibody against caspase-3 (AB4051; Abcam) to detect apoptotic cells according to the methodology previously described by Martinez- Madrid (18) with slight modifications (30). Sections were counterstained with hematoxylin and viewed on a Nikon EFD-3 (Nikon) microscope under bright field optics by two independent observers who were blinded to the experimental data. Caspase-3 positive follicles and stromal cells were 1178 VOL. 97 NO. 5 / MAY 2012

4 Fertility and Sterility quantified in terms of density, as previously reported elsewhere (30). FIGURE 3 Statistics Data are expressed as medians, percentages, quartiles, and ranges. Statistical comparisons were performed with the nonparametric Kruskal-Wallis test for multiple comparisons followed by Tukey s post-hoc test. P<.05 was considered statistically significant. Statistical calculations were made with the software PASW 18.0 (IBM). RESULTS Viability of Small Follicles after Cryopreservation The neutral red stain was used to assess the viability of primordial and primary follicles of both fresh and cryopreserved ovaries. The highest density of viable small follicles was seen in fresh ovaries, although the median of this group was not statistically significantly higher than that of the L-SLF group (Fig. 2). Follicular viability was very low in vitrified ovaries and also in the H-SLF group. Estradiol Production during Incubation The estradiol concentrations (Fig. 3) in FSH-conditioned medium after 24-hour incubation were statistically significantly higher in the cultures of fresh ovarian tissue (F) than in cultures of vitrified ovarian tissue (L-VIT and H-VIT) and slow-frozen tissue with a high concentration of cryoprotectant (H-SLF). Although the median level of estradiol in the conditioned medium of L-SLF was only about half of that of F, there was no statistically significant difference between the two groups. FIGURE 2 The number (median and ranges) of primordial and primary follicles within a cortex area of 0.06 mm 2 in ovaries (n ¼ 5 in each group) that were fresh (F) or cryopreserved either by vitrification in 1.5 M DMSO (L-VIT) or 7 M DMSO (H-VIT), or cryopreserved by slow freezing in 1.5 M DMSO (L-SLF) or 7 M DMSO (H-SLF). Statistical differences are indicated by brackets. Levels of estradiol (median and ranges) in medium after incubation of ovarian tissue with 5 ng/ml human recombinant follicle-stimulating hormone (FSH) for 24 hours (n ¼ 5 per group). The ovarian tissues were either fresh (F) or cryopreserved by vitrification in 1.5 M DMSO (L-VIT) or 7 M DMSO (H-VIT), or by slow freezing in 1.5 M DMSO (L-SLF) or 7 M DMSO (H-SLF). Statistical differences are indicated by brackets. Histology and Caspase-3 Immunohistochemistry The ovarian tissue that had been incubated for 48 hours appeared well preserved in all cryopreservation groups, with normal morphology of both small and secondary/preantral follicles (see Fig. 3). Blood vessels devoid of blood cells were a sign of efficient flushing of the specimen. Edema was not found in the tissue. The stromal tissue surrounding follicles was morphologically normal in all groups. Positive caspase-3 cells were found mostly in the stromal compartment surrounding both primordial and primary follicles (Fig. 4) in both vitrification and slow-freezing groups. There was no apparent difference in the density of these apoptotic cells between the groups. DISCUSSION Ovarian tissue cryopreservation has reached the clinical arena as a fertility preservation procedure in girls and women undergoing potentially gonadotoxic chemotherapy or radiotherapy. The effectiveness of this method is still fairly low; only around 15 live births have been reported (6) since the introduction of the method at an experimental stage more than 10 years ago (31), and the first healthy baby was reported in 2004 (32). The major hurdles to overcome in ovarian cryopreservation seem to be how to avoid damage to the cells induced by ice crystals (33) and how to optimize the transplantation procedure so that an ischemia-induced follicle loss is avoided. Our study evaluated an animal model for this research. Several animal models could be used to optimize cryopreservation protocols for human use. The animal model that has been used most extensively in ovarian cryopreservation research is the sheep (34, 35) because the ovine ovary is of VOL. 97 NO. 5 / MAY

5 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY FIGURE 4 Histologic section of areas with small follicles of ovaries that had been cryopreserved in 1.5 M dimethyl sulfoxide (DMSO) by either (A) vitrification (L-VIT) or (B) slow freezing (L-SLF). Immunohistochemical staining against caspase is indicated by red stain (original magnification: 200). a reasonable size and the animal is mono/diovulatory. However, this experimental model is rather expensive and cumbersome to work with. The rat has many advantages as an experimental model for research given that it is a small and inexpensive animal with high reproductive efficiency. Moreover, the knowledge of ovarian function in the rat, especially folliculogenesis, is vast (36). A disadvantage with the rat for ovarian cryopreservation research is, of course, that the size of the rat ovary is much smaller than the human ovary and that the distribution of the primordial follicle pool within the ovary is somewhat different as compared with humans (36). The reasons to cannulate the vessels before cryopreservation were to accomplish effective flushing of the ovarian tissue to remove blood cells before an effective distribution of the cryoprotectant. Moreover, the cannulated state of the ovary would enable post-thawing tests of functionality of the ovary later, which could involve in vitro perfusion (37) or even whole ovary retransplantation by vascular anastomosis (23). Indeed, functionality in terms of endocrine function and cyclicity with fertility would be the natural end points to evaluate the results after cryopreservation. There exist several different cryoprotectants used in research and in clinics. For ovarian tissue cryopreservation, the cryoprotectant DMSO has been the most widely used, but other cryoprotectants such as ethylene glycol have shown effectiveness in clinical ovarian fertility preservation procedures. We tested a low and a high concentration of DMSO to acquire fundamental information that can be used for further tests, including of other cryoprotectants or combinations of these. The concentration of 1.5 M DMSO was chosen because it is the most widely used DMSO concentration in ovarian tissue cryopreservation. It is based on the method described more than 15 years ago in the sheep (38). The high DMSO concentration used in our study was intended to test the toxicity of a high DMSO concentration within the tissue. In a recent study, the hemiovarian cortex of sheep was incubated at room temperature for 10 minutes in BM1 solution containing 10% FCS with either DMSO or propylene glycol in concentrations of 2 M, 4 M, 6.5 M, 9 M, or 10 M (39). Follicular survival was highest with the use of the 2 M concentration, regardless of the cryoprotectant. In our study, we perfused the ovary and distributed DMSO through the vascular tree. A stepwise increase of the concentration of DMSO was used to reduce osmotic stress. We also tested a high DMSO concentration (7 M) to confirm whether it had a toxic effect. We did not use higher concentrations of DMSO in this experiment because we found that ovarian perfusion with DMSO solutions over 7 M were unsuccessful owing to the marked viscosity of the solution (preliminary experiment, data not shown). Indeed, both follicular viability and estradiol production were decreased in the 7 M DMSO group as compared with the 1.5 M group. However, it remains to titrate out the optimal dose of DMSO for cryopreservation, and we believe these tests ought to examine in vitro viability and also retransplantation to test the essential in vivo function. In such a research, our currently developed rat method could be used for the larger dose-finding studies, the results of which could then be applied to larger and more costly animal models such as the sheep or a nonhuman primate species. We used ovaries of immature rats to work only with smaller follicles. Presence of large antral follicles, with a great volume of follicular fluid, would disturb the cryopreservation process. Because the follicles of the ovary depend on the surrounding stroma, we chose to not use culture of isolated follicles (40) but rather of small pieces of ovarian tissue. The small primordial and primary follicles do not secrete estradiol, but the secondary follicles do contain aromatase to form estradiol (36). Thus, the estradiol that was detected in the conditioned media in our study does not represent the viability of the large primordial/primary follicle pool but rather the pool of secondary and tertiary follicles. It is interesting to note that there was substantial estradiol production from ovaries that had undergone slow-freezing cryopreservation, indicating that the survival of y secondary and tertiary follicles is rather good. The histologic analysis of the ovaries demonstrated presence of follicles up to the preantral stage. In general, slow freezing has been the method most commonly used in ovarian cryopreservation. The rat has been 1180 VOL. 97 NO. 5 / MAY 2012

6 Fertility and Sterility proven to be a useful model for whole ovary vitrification (2). Sugimoto et al. (2) found that eight out of nine animals that had received transplanted vitrified ovary reestablished their cyclicity; histology revealed healthy antral follicles 3 months after transplantation. In other experiments on vitrification of whole ovaries in the sheep (41) and cow (42), some success have been reported. However, with human ovarian strips, the efficacy of slow-freezing versus vitrification is controversial (43, 44). It is also known that the toxicity of DMSO can be reduced by addition of nonpermeable cryoprotectants (45). Nevertheless, in this initial study of the whole rat ovary, we sought to test both a low and high concentration of DMSO for vitrification, although we expected that the outcome after vitrification would be poor. Our rationale for this was that we can use our data as a platform to build on in further studies examining addition of different cryoprotectants to find an optimal combination for vitrification. Of course, vascular transplantation to assess in vivo function should be added as a last step in such an investigation. In addition, we evaluated the presence of apoptosis using caspase-3 antibodies. We found apoptotic cells in the stroma around the primordial/primary follicles. It is not clear whether these cells are true stroma cells or represent cells that will later be derived into theca cells of the secondary follicles. In a study of cryopreserved whole human ovaries (18), apoptotic cells were detected in antral follicles and corpora lutea. It should be noted that some apoptosis occurs naturally in the ovary as there is a continuous depletion of follicles of all stages through a process generally referred to as atresia. 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