Theriogenology 83 (2015) 285–293
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Effect of preoperative simvastatin treatment on transplantation of cryopreserved-warmed mouse ovarian tissue quality Jaewang Lee a, b, Jung Ryeol Lee a, b, Hye Won Youm a, Chang Suk Suh a, b, *, Seok Hyun Kim b a b
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, Republic of Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2014 Received in revised form 18 September 2014 Accepted 20 September 2014
After the ovarian tissue (OT) transplantation, the ischemia-reperfusion injury causes depletion and apoptosis of follicle. Recent reports stated that simvastatin reduces ischemic damage. Therefore, we used the mouse whole ovarian vitrification and autotransplantation models to investigate the effects of simvastatin. Five-week-old B6D2F1 mice were randomly divided into four groups. Three groups were given simvastatin orally (5 mg/kg) before ovariectomy, either 2 hours before (2H Tx) or once a day for 3 or 7 days. The control group was given saline 2 hours before ovariectomy. All ovaries were cryopreserved by vitrification, held in liquid nitrogen for 1 week before being warmed, and autotransplanted. The grafts were collected for analysis on 2, 7, or 21 days after transplantation. Ovarian follicle morphology and apoptosis were assessed by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Vessel integrity in ovary was evaluated by immunohistochemistry using anti-CD31 antibody. Serum FSH level was measured to estimate the transplanted ovarian reserve. The proportion of morphologically normal (G1) follicles at 7 and 21 days and the percentage of CD31 (þ) tissue at 21 days was significantly higher in the 2H Tx group than that in the control group. In addition, the 2H Tx group showed a significantly increased intact primordial follicle ratio at 2 and 21 days after OT transplantation. Administration of simvastatin 2 hours before ovariectomy could improve the quality after transplantation of cryopreserved mouse OT. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Fertilty preservation Simvastatin Ovary Vitrification Autotransplantation
1. Introduction Over the past few decades, the diagnosis and treatment of cancer have developed significantly in women, and the awareness of fertility preservation has increased [1–3]. There are several alternatives to preserve the fertility of women with a history of cancer, including oocyte, embryo, and ovarian tissue (OT) cryopreservation [3–6]. Recent reports on OT transplantation (OTT) have confirmed the resumption of * Corresponding author. Tel.: þ82 31 787 7251; fax: þ82 31 787 4054. E-mail address: [email protected] (C.S. Suh). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.09.027
the hypothalamus-pituitary-gonad axis, follicular development, and live births. However, ischemic injury is a major hurdle for fresh or frozen OT transplant as it results in the destruction of ovarian stromal cells and follicular depletion [7,8]. For this reason, swift establishment of a new blood supply is crucial to restore ovarian function. Many studies have investigated the strategies for reducing ischemic damage and improving neovascularization using treatments such as oxygen-free radical scavengers [9–12], gonadotropin [13–17], vascular endothelial growth factor (VEGF) [18], angiogenic factor [19], and antiapoptotic agents [20–24]. However, some of these studies are controversial and not all
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the agents studied showed positive effects on tissue survival. Therefore, further studies are needed to investigate these agents and to modify protocols. Simvastatin is a 3-hydroxymethylglutaryl coenzyme A reductase inhibitor, which reduces cholesterol synthesis, and has a protective effect on endothelial cell nitric oxide bioactivity and atherosclerotic plaque stabilization [25,26]. It also plays a role in cholesterolindependent immunomodulation and vasculoprotection [27,28]. These beneficial effects of simvastatin can protect the liver [29], heart [30–32], kidney [33], and other organs [34] against ischemia during storage and ischemia-reperfusion injury after grafting. However, no studies have investigated the effects of simvastatin treatment before ovariectomy or its ischemic-preventive effects after ovarian transplantation. The objective of this study was to evaluate whether simvastatin has a positive effect on mouse OT vitrificationwarming and transplantation. 2. Materials and methods 2.1. Experimental animals and simvastatin administration Five-week-old B6D2F1 female mice (Orient Co., Seongnam, South Korea) were housed under a 12-h light-dark cycle at 22 C and fed ad libitum. All experimental protocols and animal handling procedures were performed with the approval of the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (BA1208-110/ 061-01). Ovariectomy, vitrification, warming, and autotransplantation of whole ovaries were performed to assess the effects of peroral simvastatin administration on OT ischemic injury during the transplantation procedures. The mice were treated with 5 mg/kg/day of simvastatin (Sigma, St. Louis, MO, USA) 2 hours before ovariectomy, or once daily for 3 or 7 days before ovariectomy and vitrification (2H Tx, 3D Tx, and 7D Tx, n ¼ 10 mice per group, respectively). An equal volume of saline was administered perorally 2 hours before ovariectomy in the control group (n ¼ 10 mice per group). Dose of simvastatin was chosen from the study by Tuuminen et al. [33]. 2.2. Vitrification of whole mouse OTs The B6D2F1 female mice were randomly divided according to the treatment of simvastatin, and the mice were identified with a metal ear tag. Whole ovaries were removed and vitrification was performed in a two-step method as reported previously [35]. Briefly, the OTs were equilibrated in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% (v:v) fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 7.5% (v:v) ethylene glycol (Sigma), and 7.5% (v:v) DMSO (Sigma) for 10 minutes at room temperature (RT). The equilibrated OTs were then transferred into the vitrification solution, which was composed of D-PBS containing 20% FBS, 20% ethylene glycol, 20% DMSO, and 0.5 M sucrose (Sigma) for 5 minutes at RT. Each ovary was placed on an electron microscope copper grid (JEOL, Tokyo, Japan) to improve heat transfer, and the remaining vitrification solution eliminated using sterilized filter papers. The OTs on the top of the electron microscope grids were immediately plunged into liquid nitrogen and the vitrified ovaries placed into a 1.5-mL cryovial (Nunc, Roskilde, Denmark) filled with liquid nitrogen.
2.3. Warming and autotransplantation of cryopreserved ovaries One week after ovariectomy and vitrification, the cryopreserved OTs were exposed to RT for 10 to 20 seconds to warm and then immersed into 1.0 M sucrose solution for 5 minutes followed by stepwise incubation in 0.5, 0.25, and 0 M sucrose solution for 5 minutes, all at RT. Dulbecco’s PBS supplemented with 20% FBS was used as a basal medium. 2.4. Surgery The mice underwent surgery twice (ovariectomy and grafting) 1 week apart. They were anesthetized by intraperitoneal injection with a mixed solution containing ketamine (0.15 mg/g of body weight) and xylazine (0.016 mg/ g of body weight). The fur was shaved and the skin was swabbed with 70% (v:v) alcohol. A 1-cm incision was made on the dorsal skin and both kidneys were exteriorized through the incision site. A small hole was torn in the kidney capsule using fine forceps under sterile conditions. Following four serial steps of warming procedures, the vitrified-warmed ovaries were placed under each kidney capsule. After surgery, the incision site and skin were closed and sutured with 4-0 silk. 2.5. Recovery of ovarian grafts and blood collection The transplanted mice were euthanized by cervical dislocation at 2, 7, or 21 days after surgery, and the retrieved grafts were immediately fixed in 4% paraformaldehyde. Whole blood samples were obtained from cardiac puncture at 2, 7, or 21 days after grafting, and serum was separated by centrifugation at 13,000 rpm for 2 minutes. 2.6. Follicle classification and morphologic analysis After fixation with 4% paraformaldehyde, each group of ovaries was embedded in a paraffin block and cut into serial sections of 4 to 5 mm thickness. Representative sections were stained with hematoxylin and eosin solution (Merck, Darmstadt, Germany) to examine morphology under light microscopy (Nikon, Tokyo, Japan) at 400 magnification. Follicle grading was performed using the following criteria given by Lundy et al. [36]: (1) Primordial: single layer of flattened pregranulosa cells. (2) Primary: single layer of granulosa cells, with one or more cuboidal cells. (3) Secondary: two or more layers of cuboidal granulosa cells, without the antrum. (4) Antral: multiple layers of cuboidal granulosa cells, with the antrum. Follicular integrity was assessed using the following criteria given by Gandolfi et al. [37]: (1) Primordial and primary follicle: G1, spherical with an even distribution of the granulosa cells; G2, granulosa cells pulled away from the edge of the follicle but with
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the oocytes still spherical; G3, pyknotic nuclei, misshapen oocytes, or vacuolation. (2) Secondary and antral follicle: G1, intact spherical follicle with evenly distributed granulosa and theca cells, small space, and spherical oocytes; G2, intact theca cells, disrupted granulosa cells, and spherical oocytes; G3, disruption and loss of granulosa and theca cells, pyknotic nuclei, and missing oocyte. Atretic follicles were characterized by the presence of eosinophilia of the ooplasm, contraction, and clumping of chromatin material, and wrinkling of the nuclear membrane on the oocytes [38]. To avoid miscounting, only one slide was assessed for each ovary by two individual inspectors (J. Lee and H.W. Youm). We counted and evaluated the follicles when they contained oocytes ensuring that no preantral follicles were counted twice. 2.7. Analysis of apoptosis Paraffin sections of the OTs were used to assess apoptosis using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for the detection DNA fragmentation. In brief, after the removal of paraffin and rehydration of the samples, the sections were treated with 0.1% Triton X-100 (Amresco, Solon, OH, USA) in citrate buffer for 15 minutes at RT. This was then incubated with the TUNEL reaction mixture (enzyme-to-label solution, 1:9 dilution) for 1 hour at 37 C in a humidified chamber in the dark, and then rinsed with D-PBS. The slides were mounted in VECTASHIELD Mounting Medium with 40 ,6-diamidino-2phenylindole (Vector laboratories Inc., Burlingame, CA, USA), and examined under an inverted Zeiss AX10 microscope (Carl Zeiss AG, Oberkochen, Germany). The untreated TUNEL reaction mixture was used as a negative control, and a slide treated with 100 U/mL of DNase I was used as a positive control. Green fluorescence was visualized in cells that tested positive by TUNEL assay, using an excitation wavelength ranging from 450 to 500 nm and detection capability ranging from 515 to 565 nm. 40 ,6-Diamidino-2-phenylindole reached excitation at approximately 360 nm and emission at approximately 460 nm when bound to DNA, producing blue fluorescence in all the nuclei. When more than 30% of apoptotic cells were present in a follicle, the follicle was regarded as apoptotic [39]. 2.8. Immunohistochemical analysis for Cluster of differentiation Blood vessel density was investigated by immunohistochemistry using anti-CD31 antibody. Cluster of differentiation 31 (CD31) is expressed on the surface of a large portion of the endothelial cell intercellular junction. The paraffin slides were deparaffinized using three treatment steps with xylene for 8 minutes each, and rehydrated with several ethanol washes. Once rehydration of the slide was completed, microwave-mediated heat-induced epitope retrieval in a target retrieval solution (pH 9.0; Dako, Glostrup, Denmark) was performed. After this antigenic retrieval, the sections were treated with peroxidase-
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blocking solution (Dako, Glostrup, Denmark) for 10 minutes at RT, followed by incubation with anti-CD31 antibody (Abcam, Cambridge, UK) at a dilution of 1:200 for 1 hour at RT. After rinsing three times with Wash Buffer (Dako), the sections were treated with EnVision þ horseradish peroxidase (Dako) for 30 minutes at RT, then treated with liquid Diaminobenzidine þ substrate (Dako) for 10 minutes at RT. All sections were counterstained with hematoxylin (Dako) and dehydrated. Finally, the slides were mounted in Mounting Medium (Dako) and examined under an inverted Zeiss AX10 microscope (Carl Zeiss AG). The CD31-positive (þ) area of each ovary was estimated using i-solution image analysis software (IMT technology Inc., Seoul, Korea). 2.9. Measurement of FSH level Serum FSH concentrations were measured using an ELISA (Endocrine Technologies, Newark, NJ, USA) for the quantitative analysis of FSH levels in serum. In accordance with the manufacturer’s instructions, plates were measured at 450 nm using an optical density plate reader, and concentrations were calculated against serial standard dilutions. The intra- and interassay coefficients of variation were 6.35% and 5.88%, respectively, with a sensitivity of 0.5 ng/mL. 2.10. Statistical analyses The proportions of the follicle stages and normality within each of the groups were calculated. Data were analyzed by the chi-square test or ANOVA as appropriate. Statistical Package for the Social Sciences (SPSS) version 12.0 software (SPSS Inc., Chicago, IL, USA) and Graph pad Prism 5.0 (Graph pad software, San Diego, CA, USA) were used for the analysis. Values were considered significant when P < 0.05. 3. Results 3.1. Morphologic assessment of vitrified-warmed mouse OT Figure 1 (A, E, I) shows the representative images of the OT morphology as the transplantation duration increased. Two days after OTT, many ovarian follicles and stromal cells were damaged in all four groups after autotransplantation of cryopreserved-warmed OTs, and there were few developed follicles in all groups (Fig. 1A–D). On the contrary, stromal cell and follicle morphology was normal in 7 days after transplantation. Well-developed ovarian follicles can be seen in Figure 1 (E–H). After 21 days, most of the follicles and stromal cells were well established in the tissue grafts and ovarian follicle development increased in all four groups without pyknotic nuclei or edema (Fig. 1 I–L). Depending on the transplantation duration, these restoration tendencies increased. A total of 1874 ovarian follicles morphologically evaluated to determine the effects of simvastatin on follicle integrity. Table 1 represents the number and proportion of morphologically intact (grade I) follicles for the groups at 2, 7, and 21 days after OTT, respectively. On Day 2 after transplantation, the 2H Tx group showed the highest intact (grade I) follicle ratio; however, this difference did not reach statistical significance. Seven days after transplantation, the
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Fig. 1. A representative image of hematoxylin and eosin stains. X-axis shows types of different groups (A, E, I: control; B, F, J: 2H Tx; C, G, K: 3D Tx; and D, H, L: 7D Tx) and Y-axis represents period after ovarian tissue transplantation (A–D: 2 days; E–H: 7 days; and I–L: 21 days after transplantation). The magnification was 100 and scale bar indicates 500 mm. 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)
2H Tx group showed a significantly higher total grade I follicle ratio than the control group. A significantly higher total grade I follicle ratio was also seen 21 days after transplantation. In addition, the intact primordial follicle ratio in the 2H Tx group was also significantly higher than that in the control group (Fig. 2). 3.2. Evaluation of apoptotic follicles in OT grafts Figure 3 shows the representative images of the TUNEL reaction of transplanted OTs. A total of 1726 ovarian follicles from the four groups were evaluated for apoptosis using the TUNEL assay (Fig. 4). Two days after transplantation, all groups reported approximately 10% of the apoptotic follicle ratio. The apoptotic follicle ratio decreased not only in the 7-day groups, but also in all 21-day groups. However, there were no significant differences in the apoptotic follicle ratio among the four groups at 2, 7, and 21 days after OTT (Table 1). 3.3. Evaluation of blood vessel intensity Figure 5 shows CD31 (þ) cells localized in the transplanted OTs at 2, 7, and 21 days after OTT. The place where impaired stromal cells and ovarian follicles were visible, brown colored CD31 (þ) cells were evenly distributed in the ovaries 2 days after OTT (Fig. 5A–D). Figure 6 shows no significant difference in the proportion of the CD31 (þ) area (control group, 1.59 0.58; 2H Tx group, 2.22 0.64; 3D Tx group, 1.58 0.36; and 7D Tx group, 1.30 0.32). Around the ovarian follicles and stromal cells, CD31 (þ)
cells were expressed and blood capillaries were more detectable on Day 7 after the transplantation procedure (Fig. 5). However, there were no statistically significant differences in the proportion of CD31 (þ) among the four groups (Fig. 6: control group, 2.43 0.42; 2H Tx group, 1.62 0.40; 3D Tx group, 1.75 0.33; and the 7D Tx group, 2.88 0.57). Twenty-one days after transplantation, the ovarian follicles and the stromal cells were more recovered (Fig. 5 I–L). The blood capillary density, as evaluated by the CD31 (þ) area, was increased in all the groups compared with that of 2 and 7 days after transplantation (control group, 3.40 0.36; 2H Tx group, 5.99 0.76; 3D Tx group, 3.22 0.7; and 7D Tx group, 2.96 0.58), and the 2H Tx group showed a significantly higher CD31 (þ) area than the other groups. 3.4. Measurement of the serum FSH levels according to the duration of transplantation Serum FSH levels were increased 2 days after transplantation in all groups; however, the increased serum FSH levels were decreased at 7 and 21 days after transplantation (Fig. 7). There was no difference in the serum FSH levels between the groups at 2, 7, and 21 days after transplantation. 4. Discussion Ovarian tissue cryopreservation and transplantation are optimal for women who cannot delay chemotherapy or perform ovarian hyperstimulation to obtain oocytes, such as
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Table 1 The proportions of grade I and apoptotic follicle in accordance with a simvastatin treatment duration that is administered to preoperative ovariectomy on 2, 7 or 21 days after ovarian tissue transplantation.
2 days
7 days
21 days
Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles
Control
2H Tx
3D Tx
7D Tx
41/61 (67.2%)ab 10/23 (43.5%) 15/29 (51.7%) 0/0 (0%) 66/115 (57.4%) 20/146 (13.7%)
53/68 (77.9%)b 5/13 (38.5%) 23/38 (60.5%) 1/2 (50.0%) 82/121 (67.8%) 16/145 (11.0%)
66/89 (74.2%)ab 15/27 (55.6%) 20/35 (57.1%) 0/0 (0%) 101/151 (66.9%) 17/144 (11.8 %)
58/95 (61.1%)a 9/16 (56.3%) 21/36 (58.3%) 1/3 (33.3%) 89/150 (59.3%) 12/137 (8.8%)
27/51 (52.9%) 11/17 (64.7%)a 6/17 (35.3%)a 4/11 (36.4%)ab 48/96 (50.0%)a 6/117 (5.1%)
43/62 (69.4%) 24/41 (58.5%)a 14/18 (77.8%)b 5/7 (71.4%)a 86/128 (67.2%)b 5/132 (3.8%)
31/52 (59.6%) 22/36 (61.1%)a 9/16 (56.3%)ab 5/12 (41.7%)b 67/116 (57.8%)ab 9/144 (7.8%)
39/65 (60.0%) 7/22 (31.8%)b 10/14 (71.4%)ab 2/11 (18.2%)b 58/112 (51.8%)a 6/111 (5.4%)
87/173 (50.3%)a 40/85 (47.1%) 25/44 (56.8%)a 16/24 (66.7%) 168/326 (51.5%)a 12/214 (5.6%)
91/132 (68.9%)b 30/60 (50.0%) 12/22 (54.5%)a 12/21 (57.1%) 145/235 (61.7%)b 7/177 (4.0%)
63/105 (60.0%)ab 19/35 (54.3%) 15/26 (57.7%)a 14/23 (60.9%) 111/189 (58.7%)ab 11/155 (7.1%)
37/71 (52.1%)a 18/34 (52.9%) 15/37 (88.2%)b 11/13 (84.6%) 81/135 (60.0%)ab 4/104 (3.8%)
Different superscript letters indicate statistically significant differences (P < 0.05) and the superscripts were used for each group separately. No superscript means no significant differences among four different groups. 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy.
prepubertal cancer patients. Nevertheless, fertility preservation using OT banking and transplantation is still in the experimental stages. Despite numerous reports regarding the improvement of ovarian morphology, survival, and function in accordance with the different types of treatment, this methodology has not yet been established. Therefore, this study aimed to investigate the improvement of ovarian quality after cryopreservation and transplantation of the ovaries using a mouse model. Simvastatin is a hypolipidemic drug used with exercise, diet, and weight loss to control increased cholesterol level, or hypercholesterolemia. Many studies have investigated the protective effects of simvastatin to reduce ischemic damage on various organ transplants such as liver [29], heart [31,32], kidney [33], and endometriosis. Moreover, simvastatin may augment the efficacy of therapeutic angiogenesis induced by bone marrow–derived mesenchymal stem cells, as suggested by a murine hindlimb ischemia model [40,41], and
Fig. 2. Grade I follicle ratio according to four different groups.
improve vessel structure and function [27]. For this reason, we attempted to reduce the risk of ischemic injury and enhance angiogenesis during OTT via the preoperative administration of simvastatin. To our knowledge, the present study is the first to report the effects of simvastatin on cryopreserved-warmed mouse whole OTT. Because revascularization of blood vessels occurs within 48 hours in rodent models, any ischemic-induced tissue damage develops during that period. Our results also reveal that stromal cells and ovarian follicles were damaged on Day 2 after the transplantation procedure, and there were a few antral follicles that may have been affected by cryoinjury or ischemic injury. Hoeben et al. [42] reported that primordial follicles do not have their own vascular supply and are supported by vessels in the surrounding stroma. Relatively wellpreserved primordial follicles were found in the 2H Tx group, possibly because of the protective effect of simvastatin on stromal cells. Russo et al. [43] reported that treatment of simvastatin could improve endothelial cell dysfunction and reduce oxidative stress. On the basis of these results, we believe that the preoperative administration of simvastatin has a preventive or protective role on OT. Our results reveal that preoperative simvastatin administration 2 hours before ovariectomy has beneficial effects on grade I primordial follicles, grade I total follicle preservation, and the proportion of CD31 (þ) area. On the contrary, the apoptotic follicle ratio has no significant difference between the groups. On the basis of our results, we assumed that the protective mechanisms of simvastatin appeared through the improvement of angiogenesis and not the prevention of apoptosis of follicles. Tuuminen et al. [32] postulated that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia or reperfusion injury, and chronic rejection. Chen et al. also reported that simvastatin reduced hypoxia-induced endothelial leakage in
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Fig. 3. A representative image of terminal deoxynucleotidyl transferase dUTP nick end labeling reaction. X-axis shows types of different groups (A, E, I: control; B, F, J: 2H Tx; C, G, K: 3D Tx; and D, H, L: 7D Tx) and Y-axis represents period after ovarian tissue transplantation (A–D: 2 days; E–H: 7 days; and I–L: 21 days after transplantation). The magnification was 100 and scale bar indicates 500 mm. 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)
human umbilical vein endothelial cells in a dose-dependent manner in vitro, and low-dose simvastatin (0.2 mg/kg) induced the upregulation of endothelial nitric oxide synthase skewed vessels to a pericyte-coated and stable pattern, whereas high-dose simvastatin (10 mg/kg) decreased reactive oxygen species–induced hypoxia-inducible factor 1a and VEGF expressions, attenuating VEGF-derived tumor vessel hyperpermeability [27]. In the present study, mice in the three experimental groups were treated with 5 mg/kg of simvastatin 2 hours before surgery, or once a day for 3 or 7 days before ovariectomy and vitrification (2H Tx, 3D Tx, and 7D Tx groups, respectively). Another study reported that donor (5 mg/kg),
Fig. 4. Apoptotic follicle ratio in accordance with four different simvastatin treatment conditions.
but not recipient (2 mg/kg), simvastatin treatment prevented ischemia-reperfusion injury–induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury [32,33]. Their study also suggested that donor statin treatment targets the transplanted organ and the proximal events that may lead to early allograft injury and predispose the organ to the development of delayed graft function [32]. We also administered 5 mg/kg of simvastatin to the recipient after OTT and evaluated the effects of recipient simvastatin treatment in accordance with the study by Tuuminen et al. [33]. However, recipient simvastatin treatment did not show any significant effect on grade I total follicle ratio, primordial follicle preservation, apoptosis, and serum FSH (data not shown). In the present study, the beneficial effect of simvastatin administration was shown only in the 2H Tx group, and the relatively long-term administration of simvastatin was less beneficial in the other groups than in the 2H Tx group. Chen et al. [27] postulated that simvastatin has a pleiotropic and dose-dependent effect on tumor vascular stabilization. Moreover, Weis et al. reported that 3-hydroxy-methylglutaryl coenzyme A reductase inhibition has a biphasic dose-dependent effect on angiogenesis that is lipid independent and associated with alterations in endothelial cell apoptosis and VEGF signaling. They also reported that statins have proangiogenic effects at low therapeutic concentrations and angiostatic effects at high concentrations [44]. On the basis of these findings, we assume that accumulated simvastatin in the donor could reduce the beneficial effect on ovarian quality.
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Fig. 5. A representative image of immunohistochemistry for CD31. X-axis shows types of different groups (A, E, I: control; B, F, J: 2H Tx; C, G, K: 3D Tx; and D, H, L: 7D Tx) and Y-axis represents period after ovarian tissue transplantation (A–D: 2 days; E-H: 7 days; and I–L: 21 days after transplantation). 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy. The magnification was x100 and scale bar indicates 400 um. White arrows indicate the CD31-positive blood vessels in the transplanted ovarian tissue. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)
levels in accordance with the estrous cycles may cause these limitations. Hence, there is a possibility that some of the beneficial effects of simvastatin observed in morphology and angiogenesis were reflected in serum FSH levels. Further study is therefore required using more sensitive and direct markers of ovarian reserve and function, such as anti-Mullerian hormone, and these could reveal the effects on transplanted ovarian reserve and function more accurately.
Two days after transplantation, the FSH levels were increased in all groups; however, this was restored after 7 days. These results support the belief that the revascularization process begins within 48 hours of surgery and finishes at around 7 days, as reported in the murine model of a previous study [45]. In this study, some beneficial effects on morphologic evaluation and angiogenesis appeared in the 2H Tx group; however, there was no significant difference in the FSH levels among the four groups. Serum FSH is used as a ovarian reserve marker in experimental animals and in humans [46,47]; however, Lambalk et al. [48] reported that the roles of FSH as an ovarian reserve marker have limitations and relatively low sensitivity, and differences in FSH
The preadministration of simvastatin could improve the quality of ovarian follicles and enhance angiogenesis after
Fig. 6. The proportion of CD31 (þ) area in transplants after grafting in four different simvastatin treatment conditions.
Fig. 7. Measurement of serum FSH levels in all groups at different periods.
4.1. Conclusions
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mouse ovarian transplantation. However, further studies to identify the exact protective mechanism of simvastatin and research on other species such as the bovine and human are necessary. Acknowledgments This study was supported by a grant from the Korea Healthcare Technology R&D Project (A120080), Ministry of Health and Welfare, Republic of Korea (HI12C0055). The first two authors contributed equally to this work. References [1] Kim SS. Ovarian tissue banking for cancer patients. To do or not to do? Hum Reprod 2003;18:1759–61. [2] Newton H. [Freezing of ovarian tissue and outcome]. Contracept Fertil Sex 1992;1998:584–8 [in French]. [3] Oktay K, Sonmezer M. Ovarian tissue banking for cancer patients: fertility preservation, not just ovarian cryopreservation. Hum Reprod 2004;19:477–80. [4] Dath C, Van Eyck AS, Dolmans MM, Romeu L, Delle Vigne L, Donnez J, et al. Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites. Hum Reprod 2010;25:1734–43. [5] Hovatta O. Methods for cryopreservation of human ovarian tissue. Reprod Biomed Online 2005;10:729–34. [6] Demeestere I, Simon P, Buxant F, Robin V, Fernandez SA, Centner J, et al. Ovarian function and spontaneous pregnancy after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: case report. Hum Reprod 2006;21:2010–4. [7] Cox SL, Shaw J, Jenkin G. Transplantation of cryopreserved fetal ovarian tissue to adult recipients in mice. J Reprod Fertil 1996;107: 315–22. [8] Tryde Schmidt KL, Yding Andersen C, Starup J, Loft A, Byskov AG, Nyboe Andersen A. Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancerdfollicular growth, steroid production and oocyte retrieval. Reprod Biomed Online 2004;8: 448–53. [9] Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil 1998;114:341–6. [10] Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS, et al. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Steril 2004;82:679–85. [11] Hemadi M, Abolhassani F, Akbari M, Sobhani A, Pasbakhsh P, AhrlundRichter L, et al. Melatonin promotes the cumulus-oocyte complexes quality of vitrified-thawed murine ovaries; with increased mean number of follicles survival and ovary size following heterotopic transplantation. Eur J Pharmacol 2009;618:84–90. [12] Hemadi M, Saki G, Shokri S, Ghasemi FM. Follicular dynamics in neonate vitrified ovarian grafts after host treatment with melatonin. Folia Morphol 2011;70:18–23. [13] Wang Y, Chang Q, Sun J, Dang L, Ma W, Hei C, et al. Effects of HMG on revascularization and follicular survival in heterotopic autotransplants of mouse ovarian tissue. Reprod Biomed Online 2012; 24:646–53. [14] David A, Van Langendonckt A, Gilliaux S, Dolmans MM, Donnez J, Amorim CA. Effect of cryopreservation and transplantation on the expression of kit ligand and anti-Mullerian hormone in human ovarian tissue. Hum Reprod 2012;27:1088–95. [15] Yu X, Deng XH, Chao L, Yu HL, Liu WJ. [Response of cryopreserved ovarian tissue after autologous implantation in mouse stimulated with gonadotrophin]. Zhonghua Fu Chan Ke Za Zhi 2008;43:213–7 [in Chinese]. [16] Kakuta H, Tanaka M, Chambon P, Watanabe H, Iguchi T, Sato T. Involvement of gonadotropins in the induction of hypertrophyhyperplasia in the interstitial tissues of ovaries in neonatally diethylstilbestrol-treated mice. Reprod Toxicol 2012;33:35–44. [17] Abir R, Fisch B, Jessel S, Felz C, Ben-Haroush A, Orvieto R. Improving posttransplantation survival of human ovarian tissue by treating the host and graft. Fertil Steril 2011;95:1205–10.
[18] Schnorr J, Oehninger S, Toner J, Hsiu J, Lanzendorf S, Williams R, et al. Functional studies of subcutaneous ovarian transplants in non-human primates: steroidogenesis, endometrial development, ovulation, menstrual patterns and gamete morphology. Hum Reprod 2002;17: 612–9. [19] Labied S, Delforge Y, Munaut C, Blacher S, Colige A, Delcombel R, et al. Isoform 111 of vascular endothelial growth factor (VEGF111) improves angiogenesis of ovarian tissue xenotransplantation. Transplantation 2013;95:426–33. [20] Soleimani R, Heytens E, Oktay K. Enhancement of neoangiogenesis and follicle survival by sphingosine-1-phosphate in human ovarian tissue xenotransplants. PLoS One 2011;6:e19475. [21] Jee BC, Lee JR, Youm H, Suh CS, Kim SH, Moon SY. Effect of sphingosine-1-phosphate supplementation on follicular integrity of vitrified-warmed mouse ovarian grafts. Eur J Obstet Gynecol Reprod Biol 2010;152:176–80. [22] Friedman O, Orvieto R, Fisch B, Felz C, Freud E, Ben-Haroush A, et al. Possible improvements in human ovarian grafting by various host and graft treatments. Hum Reprod 2012;27:474–82. [23] Skaznik-Wikiel ME, Sharma RK, Selesniemi K, Lee HJ, Tilly JL, Falcone T. Granulocyte colony-stimulating factor in conjunction with vascular endothelial growth factor maintains primordial follicle numbers in transplanted mouse ovaries. Fertil Steril 2011;95:1405–9. [24] Wang L, Ying YF, Ouyang YL, Wang JF, Xu J. VEGF and bFGF increase survival of xenografted human ovarian tissue in an experimental rabbit model. J Assist Reprod Genet 2013;30:1301–11. [25] Farnier M, Guyton JR, Jensen E, Polis AB, Johnson-Levonas AO, Brudi P. Effects of ezetimibe, simvastatin and ezetimibe/simvastatin on correlations between apolipoprotein B, LDL cholesterol and nonHDL cholesterol in patients with primary hypercholesterolemia. Atherosclerosis 2013;229:415–22. [26] Steinberg H, Anderson MS, Musliner T, Hanson ME, Engel SS. Management of dyslipidemia and hyperglycemia with a fixed-dose combination of sitagliptin and simvastatin. Vasc Health Risk Manag 2013;9:273–82. [27] Chen Y, Zhang S, Peng G, Yu J, Liu T, Meng R, et al. Endothelial NO synthase and reactive oxygen species mediated effect of simvastatin on vessel structure and function: pleiotropic and dose-dependent effect on tumor vascular stabilization. Int J Oncol 2013;42:1325–36. [28] Li Y, Zhang D, Zhang Y, He G, Zhang F. Augmentation of neovascularization in murine hindlimb ischemia by combined therapy with simvastatin and bone marrow-derived mesenchymal stem cells transplantation. J Biomed Sci 2010;17:75. [29] Gracia-Sancho J, Garcia-Caldero H, Hide D, Marrone G, GuixeMuntet S, Peralta C, et al. Simvastatin maintains function and viability of steatotic rat livers procured for transplantation. J Hepatol 2013;58:1140–6. [30] Nykanen AI, Tuuminen R, Lemstrom KB. Donor simvastatin treatment and cardiac allograft ischemia/reperfusion injury. Trends Cardiovasc Med 2013;23:85–90. [31] Tuuminen R, Syrjala S, Krebs R, Arnaudova R, Rouvinen E, Nykanen AI, et al. Combined donor simvastatin and methylprednisolone treatment prevents ischemia-reperfusion injury in rat cardiac allografts through vasculoprotection and immunomodulation. Transplantation 2013;95:1084–91. [32] Tuuminen R, Syrjala S, Krebs R, Keranen MA, Koli K, AboRamadan U, et al. Donor simvastatin treatment abolishes rat cardiac allograft ischemia/reperfusion injury and chronic rejection through microvascular protection. Circulation 2011;124:1138–50. [33] Tuuminen R, Nykanen AI, Saharinen P, Gautam P, Keranen MA, Arnaudova R, et al. Donor simvastatin treatment prevents ischemiareperfusion and acute kidney injury by preserving microvascular barrier function. Am J Transplant 2013;13:2019–34. [34] Takata K, Sheng H, Borel CO, Laskowitz DT, Warner DS, Lombard FW. Simvastatin treatment duration and cognitive preservation in experimental subarachnoid hemorrhage. J Neurosurg Anesthesiol 2009;21: 326–33. [35] Jee BC, Youm HW, Lee JH, Kim JH, Suh CS, Kim SH. Impact of ketorolac administration around ovarian stimulation on in vivo and in vitro fertilization and subsequent embryo development. Gynecol Endocrinol 2013;29:436–9. [36] Lundy T, Smith P, O’Connell A, Hudson NL, McNatty KP. Populations of granulosa cells in small follicles of the sheep ovary. J Reprod Fertil 1999;115:251–62. [37] Gandolfi F, Paffoni A, Papasso Brambilla E, Bonetti S, Brevini TA, Ragni G. Efficiency of equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue: comparative analysis between human and animal models. Fertil Steril 2006; 85(Suppl. 1):1150–6.
J. Lee et al. / Theriogenology 83 (2015) 285–293 [38] Lass A, Silye R, Abrams DC, Krausz T, Hovatta O, Margara R, et al. Follicular density in ovarian biopsy of infertile women: a novel method to assess ovarian reserve. Hum Reprod 1997;12:1028–31. [39] Youm HW, Lee JR, Lee J, Jee BC, Suh CS, Kim SH. Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival. Hum Reprod 2014;29:720–30. [40] Zhang Y, Zhang R, Li Y, He G, Zhang D, Zhang F. Simvastatin augments the efficacy of therapeutic angiogenesis induced by bone marrow-derived mesenchymal stem cells in a murine model of hindlimb ischemia. Mol Biol Rep 2012;39:285–93. [41] Bruner-Tran KL, Osteen KG, Duleba AJ. Simvastatin protects against the development of endometriosis in a nude mouse model. J Clin Endocrinol Metab 2009;94:2489–94. [42] Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 2004;56:549–80. [43] Russo L, Gracia-Sancho J, Garcia-Caldero H, Marrone G, GarciaPagan JC, Garcia-Cardena G, et al. Addition of simvastatin to cold
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Effect of preoperative simvastatin treatment on transplantation of cryopreserved-warmed mouse ovarian tissue quality Jaewang Lee a, b, Jung Ryeol Lee a, b, Hye Won Youm a, Chang Suk Suh a, b, *, Seok Hyun Kim b a b
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, Republic of Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2014 Received in revised form 18 September 2014 Accepted 20 September 2014
After the ovarian tissue (OT) transplantation, the ischemia-reperfusion injury causes depletion and apoptosis of follicle. Recent reports stated that simvastatin reduces ischemic damage. Therefore, we used the mouse whole ovarian vitrification and autotransplantation models to investigate the effects of simvastatin. Five-week-old B6D2F1 mice were randomly divided into four groups. Three groups were given simvastatin orally (5 mg/kg) before ovariectomy, either 2 hours before (2H Tx) or once a day for 3 or 7 days. The control group was given saline 2 hours before ovariectomy. All ovaries were cryopreserved by vitrification, held in liquid nitrogen for 1 week before being warmed, and autotransplanted. The grafts were collected for analysis on 2, 7, or 21 days after transplantation. Ovarian follicle morphology and apoptosis were assessed by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Vessel integrity in ovary was evaluated by immunohistochemistry using anti-CD31 antibody. Serum FSH level was measured to estimate the transplanted ovarian reserve. The proportion of morphologically normal (G1) follicles at 7 and 21 days and the percentage of CD31 (þ) tissue at 21 days was significantly higher in the 2H Tx group than that in the control group. In addition, the 2H Tx group showed a significantly increased intact primordial follicle ratio at 2 and 21 days after OT transplantation. Administration of simvastatin 2 hours before ovariectomy could improve the quality after transplantation of cryopreserved mouse OT. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Fertilty preservation Simvastatin Ovary Vitrification Autotransplantation
1. Introduction Over the past few decades, the diagnosis and treatment of cancer have developed significantly in women, and the awareness of fertility preservation has increased [1–3]. There are several alternatives to preserve the fertility of women with a history of cancer, including oocyte, embryo, and ovarian tissue (OT) cryopreservation [3–6]. Recent reports on OT transplantation (OTT) have confirmed the resumption of * Corresponding author. Tel.: þ82 31 787 7251; fax: þ82 31 787 4054. E-mail address: [email protected] (C.S. Suh). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.09.027
the hypothalamus-pituitary-gonad axis, follicular development, and live births. However, ischemic injury is a major hurdle for fresh or frozen OT transplant as it results in the destruction of ovarian stromal cells and follicular depletion [7,8]. For this reason, swift establishment of a new blood supply is crucial to restore ovarian function. Many studies have investigated the strategies for reducing ischemic damage and improving neovascularization using treatments such as oxygen-free radical scavengers [9–12], gonadotropin [13–17], vascular endothelial growth factor (VEGF) [18], angiogenic factor [19], and antiapoptotic agents [20–24]. However, some of these studies are controversial and not all
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the agents studied showed positive effects on tissue survival. Therefore, further studies are needed to investigate these agents and to modify protocols. Simvastatin is a 3-hydroxymethylglutaryl coenzyme A reductase inhibitor, which reduces cholesterol synthesis, and has a protective effect on endothelial cell nitric oxide bioactivity and atherosclerotic plaque stabilization [25,26]. It also plays a role in cholesterolindependent immunomodulation and vasculoprotection [27,28]. These beneficial effects of simvastatin can protect the liver [29], heart [30–32], kidney [33], and other organs [34] against ischemia during storage and ischemia-reperfusion injury after grafting. However, no studies have investigated the effects of simvastatin treatment before ovariectomy or its ischemic-preventive effects after ovarian transplantation. The objective of this study was to evaluate whether simvastatin has a positive effect on mouse OT vitrificationwarming and transplantation. 2. Materials and methods 2.1. Experimental animals and simvastatin administration Five-week-old B6D2F1 female mice (Orient Co., Seongnam, South Korea) were housed under a 12-h light-dark cycle at 22 C and fed ad libitum. All experimental protocols and animal handling procedures were performed with the approval of the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (BA1208-110/ 061-01). Ovariectomy, vitrification, warming, and autotransplantation of whole ovaries were performed to assess the effects of peroral simvastatin administration on OT ischemic injury during the transplantation procedures. The mice were treated with 5 mg/kg/day of simvastatin (Sigma, St. Louis, MO, USA) 2 hours before ovariectomy, or once daily for 3 or 7 days before ovariectomy and vitrification (2H Tx, 3D Tx, and 7D Tx, n ¼ 10 mice per group, respectively). An equal volume of saline was administered perorally 2 hours before ovariectomy in the control group (n ¼ 10 mice per group). Dose of simvastatin was chosen from the study by Tuuminen et al. [33]. 2.2. Vitrification of whole mouse OTs The B6D2F1 female mice were randomly divided according to the treatment of simvastatin, and the mice were identified with a metal ear tag. Whole ovaries were removed and vitrification was performed in a two-step method as reported previously [35]. Briefly, the OTs were equilibrated in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% (v:v) fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 7.5% (v:v) ethylene glycol (Sigma), and 7.5% (v:v) DMSO (Sigma) for 10 minutes at room temperature (RT). The equilibrated OTs were then transferred into the vitrification solution, which was composed of D-PBS containing 20% FBS, 20% ethylene glycol, 20% DMSO, and 0.5 M sucrose (Sigma) for 5 minutes at RT. Each ovary was placed on an electron microscope copper grid (JEOL, Tokyo, Japan) to improve heat transfer, and the remaining vitrification solution eliminated using sterilized filter papers. The OTs on the top of the electron microscope grids were immediately plunged into liquid nitrogen and the vitrified ovaries placed into a 1.5-mL cryovial (Nunc, Roskilde, Denmark) filled with liquid nitrogen.
2.3. Warming and autotransplantation of cryopreserved ovaries One week after ovariectomy and vitrification, the cryopreserved OTs were exposed to RT for 10 to 20 seconds to warm and then immersed into 1.0 M sucrose solution for 5 minutes followed by stepwise incubation in 0.5, 0.25, and 0 M sucrose solution for 5 minutes, all at RT. Dulbecco’s PBS supplemented with 20% FBS was used as a basal medium. 2.4. Surgery The mice underwent surgery twice (ovariectomy and grafting) 1 week apart. They were anesthetized by intraperitoneal injection with a mixed solution containing ketamine (0.15 mg/g of body weight) and xylazine (0.016 mg/ g of body weight). The fur was shaved and the skin was swabbed with 70% (v:v) alcohol. A 1-cm incision was made on the dorsal skin and both kidneys were exteriorized through the incision site. A small hole was torn in the kidney capsule using fine forceps under sterile conditions. Following four serial steps of warming procedures, the vitrified-warmed ovaries were placed under each kidney capsule. After surgery, the incision site and skin were closed and sutured with 4-0 silk. 2.5. Recovery of ovarian grafts and blood collection The transplanted mice were euthanized by cervical dislocation at 2, 7, or 21 days after surgery, and the retrieved grafts were immediately fixed in 4% paraformaldehyde. Whole blood samples were obtained from cardiac puncture at 2, 7, or 21 days after grafting, and serum was separated by centrifugation at 13,000 rpm for 2 minutes. 2.6. Follicle classification and morphologic analysis After fixation with 4% paraformaldehyde, each group of ovaries was embedded in a paraffin block and cut into serial sections of 4 to 5 mm thickness. Representative sections were stained with hematoxylin and eosin solution (Merck, Darmstadt, Germany) to examine morphology under light microscopy (Nikon, Tokyo, Japan) at 400 magnification. Follicle grading was performed using the following criteria given by Lundy et al. [36]: (1) Primordial: single layer of flattened pregranulosa cells. (2) Primary: single layer of granulosa cells, with one or more cuboidal cells. (3) Secondary: two or more layers of cuboidal granulosa cells, without the antrum. (4) Antral: multiple layers of cuboidal granulosa cells, with the antrum. Follicular integrity was assessed using the following criteria given by Gandolfi et al. [37]: (1) Primordial and primary follicle: G1, spherical with an even distribution of the granulosa cells; G2, granulosa cells pulled away from the edge of the follicle but with
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the oocytes still spherical; G3, pyknotic nuclei, misshapen oocytes, or vacuolation. (2) Secondary and antral follicle: G1, intact spherical follicle with evenly distributed granulosa and theca cells, small space, and spherical oocytes; G2, intact theca cells, disrupted granulosa cells, and spherical oocytes; G3, disruption and loss of granulosa and theca cells, pyknotic nuclei, and missing oocyte. Atretic follicles were characterized by the presence of eosinophilia of the ooplasm, contraction, and clumping of chromatin material, and wrinkling of the nuclear membrane on the oocytes [38]. To avoid miscounting, only one slide was assessed for each ovary by two individual inspectors (J. Lee and H.W. Youm). We counted and evaluated the follicles when they contained oocytes ensuring that no preantral follicles were counted twice. 2.7. Analysis of apoptosis Paraffin sections of the OTs were used to assess apoptosis using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for the detection DNA fragmentation. In brief, after the removal of paraffin and rehydration of the samples, the sections were treated with 0.1% Triton X-100 (Amresco, Solon, OH, USA) in citrate buffer for 15 minutes at RT. This was then incubated with the TUNEL reaction mixture (enzyme-to-label solution, 1:9 dilution) for 1 hour at 37 C in a humidified chamber in the dark, and then rinsed with D-PBS. The slides were mounted in VECTASHIELD Mounting Medium with 40 ,6-diamidino-2phenylindole (Vector laboratories Inc., Burlingame, CA, USA), and examined under an inverted Zeiss AX10 microscope (Carl Zeiss AG, Oberkochen, Germany). The untreated TUNEL reaction mixture was used as a negative control, and a slide treated with 100 U/mL of DNase I was used as a positive control. Green fluorescence was visualized in cells that tested positive by TUNEL assay, using an excitation wavelength ranging from 450 to 500 nm and detection capability ranging from 515 to 565 nm. 40 ,6-Diamidino-2-phenylindole reached excitation at approximately 360 nm and emission at approximately 460 nm when bound to DNA, producing blue fluorescence in all the nuclei. When more than 30% of apoptotic cells were present in a follicle, the follicle was regarded as apoptotic [39]. 2.8. Immunohistochemical analysis for Cluster of differentiation Blood vessel density was investigated by immunohistochemistry using anti-CD31 antibody. Cluster of differentiation 31 (CD31) is expressed on the surface of a large portion of the endothelial cell intercellular junction. The paraffin slides were deparaffinized using three treatment steps with xylene for 8 minutes each, and rehydrated with several ethanol washes. Once rehydration of the slide was completed, microwave-mediated heat-induced epitope retrieval in a target retrieval solution (pH 9.0; Dako, Glostrup, Denmark) was performed. After this antigenic retrieval, the sections were treated with peroxidase-
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blocking solution (Dako, Glostrup, Denmark) for 10 minutes at RT, followed by incubation with anti-CD31 antibody (Abcam, Cambridge, UK) at a dilution of 1:200 for 1 hour at RT. After rinsing three times with Wash Buffer (Dako), the sections were treated with EnVision þ horseradish peroxidase (Dako) for 30 minutes at RT, then treated with liquid Diaminobenzidine þ substrate (Dako) for 10 minutes at RT. All sections were counterstained with hematoxylin (Dako) and dehydrated. Finally, the slides were mounted in Mounting Medium (Dako) and examined under an inverted Zeiss AX10 microscope (Carl Zeiss AG). The CD31-positive (þ) area of each ovary was estimated using i-solution image analysis software (IMT technology Inc., Seoul, Korea). 2.9. Measurement of FSH level Serum FSH concentrations were measured using an ELISA (Endocrine Technologies, Newark, NJ, USA) for the quantitative analysis of FSH levels in serum. In accordance with the manufacturer’s instructions, plates were measured at 450 nm using an optical density plate reader, and concentrations were calculated against serial standard dilutions. The intra- and interassay coefficients of variation were 6.35% and 5.88%, respectively, with a sensitivity of 0.5 ng/mL. 2.10. Statistical analyses The proportions of the follicle stages and normality within each of the groups were calculated. Data were analyzed by the chi-square test or ANOVA as appropriate. Statistical Package for the Social Sciences (SPSS) version 12.0 software (SPSS Inc., Chicago, IL, USA) and Graph pad Prism 5.0 (Graph pad software, San Diego, CA, USA) were used for the analysis. Values were considered significant when P < 0.05. 3. Results 3.1. Morphologic assessment of vitrified-warmed mouse OT Figure 1 (A, E, I) shows the representative images of the OT morphology as the transplantation duration increased. Two days after OTT, many ovarian follicles and stromal cells were damaged in all four groups after autotransplantation of cryopreserved-warmed OTs, and there were few developed follicles in all groups (Fig. 1A–D). On the contrary, stromal cell and follicle morphology was normal in 7 days after transplantation. Well-developed ovarian follicles can be seen in Figure 1 (E–H). After 21 days, most of the follicles and stromal cells were well established in the tissue grafts and ovarian follicle development increased in all four groups without pyknotic nuclei or edema (Fig. 1 I–L). Depending on the transplantation duration, these restoration tendencies increased. A total of 1874 ovarian follicles morphologically evaluated to determine the effects of simvastatin on follicle integrity. Table 1 represents the number and proportion of morphologically intact (grade I) follicles for the groups at 2, 7, and 21 days after OTT, respectively. On Day 2 after transplantation, the 2H Tx group showed the highest intact (grade I) follicle ratio; however, this difference did not reach statistical significance. Seven days after transplantation, the
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Fig. 1. A representative image of hematoxylin and eosin stains. X-axis shows types of different groups (A, E, I: control; B, F, J: 2H Tx; C, G, K: 3D Tx; and D, H, L: 7D Tx) and Y-axis represents period after ovarian tissue transplantation (A–D: 2 days; E–H: 7 days; and I–L: 21 days after transplantation). The magnification was 100 and scale bar indicates 500 mm. 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)
2H Tx group showed a significantly higher total grade I follicle ratio than the control group. A significantly higher total grade I follicle ratio was also seen 21 days after transplantation. In addition, the intact primordial follicle ratio in the 2H Tx group was also significantly higher than that in the control group (Fig. 2). 3.2. Evaluation of apoptotic follicles in OT grafts Figure 3 shows the representative images of the TUNEL reaction of transplanted OTs. A total of 1726 ovarian follicles from the four groups were evaluated for apoptosis using the TUNEL assay (Fig. 4). Two days after transplantation, all groups reported approximately 10% of the apoptotic follicle ratio. The apoptotic follicle ratio decreased not only in the 7-day groups, but also in all 21-day groups. However, there were no significant differences in the apoptotic follicle ratio among the four groups at 2, 7, and 21 days after OTT (Table 1). 3.3. Evaluation of blood vessel intensity Figure 5 shows CD31 (þ) cells localized in the transplanted OTs at 2, 7, and 21 days after OTT. The place where impaired stromal cells and ovarian follicles were visible, brown colored CD31 (þ) cells were evenly distributed in the ovaries 2 days after OTT (Fig. 5A–D). Figure 6 shows no significant difference in the proportion of the CD31 (þ) area (control group, 1.59 0.58; 2H Tx group, 2.22 0.64; 3D Tx group, 1.58 0.36; and 7D Tx group, 1.30 0.32). Around the ovarian follicles and stromal cells, CD31 (þ)
cells were expressed and blood capillaries were more detectable on Day 7 after the transplantation procedure (Fig. 5). However, there were no statistically significant differences in the proportion of CD31 (þ) among the four groups (Fig. 6: control group, 2.43 0.42; 2H Tx group, 1.62 0.40; 3D Tx group, 1.75 0.33; and the 7D Tx group, 2.88 0.57). Twenty-one days after transplantation, the ovarian follicles and the stromal cells were more recovered (Fig. 5 I–L). The blood capillary density, as evaluated by the CD31 (þ) area, was increased in all the groups compared with that of 2 and 7 days after transplantation (control group, 3.40 0.36; 2H Tx group, 5.99 0.76; 3D Tx group, 3.22 0.7; and 7D Tx group, 2.96 0.58), and the 2H Tx group showed a significantly higher CD31 (þ) area than the other groups. 3.4. Measurement of the serum FSH levels according to the duration of transplantation Serum FSH levels were increased 2 days after transplantation in all groups; however, the increased serum FSH levels were decreased at 7 and 21 days after transplantation (Fig. 7). There was no difference in the serum FSH levels between the groups at 2, 7, and 21 days after transplantation. 4. Discussion Ovarian tissue cryopreservation and transplantation are optimal for women who cannot delay chemotherapy or perform ovarian hyperstimulation to obtain oocytes, such as
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Table 1 The proportions of grade I and apoptotic follicle in accordance with a simvastatin treatment duration that is administered to preoperative ovariectomy on 2, 7 or 21 days after ovarian tissue transplantation.
2 days
7 days
21 days
Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles
Control
2H Tx
3D Tx
7D Tx
41/61 (67.2%)ab 10/23 (43.5%) 15/29 (51.7%) 0/0 (0%) 66/115 (57.4%) 20/146 (13.7%)
53/68 (77.9%)b 5/13 (38.5%) 23/38 (60.5%) 1/2 (50.0%) 82/121 (67.8%) 16/145 (11.0%)
66/89 (74.2%)ab 15/27 (55.6%) 20/35 (57.1%) 0/0 (0%) 101/151 (66.9%) 17/144 (11.8 %)
58/95 (61.1%)a 9/16 (56.3%) 21/36 (58.3%) 1/3 (33.3%) 89/150 (59.3%) 12/137 (8.8%)
27/51 (52.9%) 11/17 (64.7%)a 6/17 (35.3%)a 4/11 (36.4%)ab 48/96 (50.0%)a 6/117 (5.1%)
43/62 (69.4%) 24/41 (58.5%)a 14/18 (77.8%)b 5/7 (71.4%)a 86/128 (67.2%)b 5/132 (3.8%)
31/52 (59.6%) 22/36 (61.1%)a 9/16 (56.3%)ab 5/12 (41.7%)b 67/116 (57.8%)ab 9/144 (7.8%)
39/65 (60.0%) 7/22 (31.8%)b 10/14 (71.4%)ab 2/11 (18.2%)b 58/112 (51.8%)a 6/111 (5.4%)
87/173 (50.3%)a 40/85 (47.1%) 25/44 (56.8%)a 16/24 (66.7%) 168/326 (51.5%)a 12/214 (5.6%)
91/132 (68.9%)b 30/60 (50.0%) 12/22 (54.5%)a 12/21 (57.1%) 145/235 (61.7%)b 7/177 (4.0%)
63/105 (60.0%)ab 19/35 (54.3%) 15/26 (57.7%)a 14/23 (60.9%) 111/189 (58.7%)ab 11/155 (7.1%)
37/71 (52.1%)a 18/34 (52.9%) 15/37 (88.2%)b 11/13 (84.6%) 81/135 (60.0%)ab 4/104 (3.8%)
Different superscript letters indicate statistically significant differences (P < 0.05) and the superscripts were used for each group separately. No superscript means no significant differences among four different groups. 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy.
prepubertal cancer patients. Nevertheless, fertility preservation using OT banking and transplantation is still in the experimental stages. Despite numerous reports regarding the improvement of ovarian morphology, survival, and function in accordance with the different types of treatment, this methodology has not yet been established. Therefore, this study aimed to investigate the improvement of ovarian quality after cryopreservation and transplantation of the ovaries using a mouse model. Simvastatin is a hypolipidemic drug used with exercise, diet, and weight loss to control increased cholesterol level, or hypercholesterolemia. Many studies have investigated the protective effects of simvastatin to reduce ischemic damage on various organ transplants such as liver [29], heart [31,32], kidney [33], and endometriosis. Moreover, simvastatin may augment the efficacy of therapeutic angiogenesis induced by bone marrow–derived mesenchymal stem cells, as suggested by a murine hindlimb ischemia model [40,41], and
Fig. 2. Grade I follicle ratio according to four different groups.
improve vessel structure and function [27]. For this reason, we attempted to reduce the risk of ischemic injury and enhance angiogenesis during OTT via the preoperative administration of simvastatin. To our knowledge, the present study is the first to report the effects of simvastatin on cryopreserved-warmed mouse whole OTT. Because revascularization of blood vessels occurs within 48 hours in rodent models, any ischemic-induced tissue damage develops during that period. Our results also reveal that stromal cells and ovarian follicles were damaged on Day 2 after the transplantation procedure, and there were a few antral follicles that may have been affected by cryoinjury or ischemic injury. Hoeben et al. [42] reported that primordial follicles do not have their own vascular supply and are supported by vessels in the surrounding stroma. Relatively wellpreserved primordial follicles were found in the 2H Tx group, possibly because of the protective effect of simvastatin on stromal cells. Russo et al. [43] reported that treatment of simvastatin could improve endothelial cell dysfunction and reduce oxidative stress. On the basis of these results, we believe that the preoperative administration of simvastatin has a preventive or protective role on OT. Our results reveal that preoperative simvastatin administration 2 hours before ovariectomy has beneficial effects on grade I primordial follicles, grade I total follicle preservation, and the proportion of CD31 (þ) area. On the contrary, the apoptotic follicle ratio has no significant difference between the groups. On the basis of our results, we assumed that the protective mechanisms of simvastatin appeared through the improvement of angiogenesis and not the prevention of apoptosis of follicles. Tuuminen et al. [32] postulated that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia or reperfusion injury, and chronic rejection. Chen et al. also reported that simvastatin reduced hypoxia-induced endothelial leakage in
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Fig. 3. A representative image of terminal deoxynucleotidyl transferase dUTP nick end labeling reaction. X-axis shows types of different groups (A, E, I: control; B, F, J: 2H Tx; C, G, K: 3D Tx; and D, H, L: 7D Tx) and Y-axis represents period after ovarian tissue transplantation (A–D: 2 days; E–H: 7 days; and I–L: 21 days after transplantation). The magnification was 100 and scale bar indicates 500 mm. 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)
human umbilical vein endothelial cells in a dose-dependent manner in vitro, and low-dose simvastatin (0.2 mg/kg) induced the upregulation of endothelial nitric oxide synthase skewed vessels to a pericyte-coated and stable pattern, whereas high-dose simvastatin (10 mg/kg) decreased reactive oxygen species–induced hypoxia-inducible factor 1a and VEGF expressions, attenuating VEGF-derived tumor vessel hyperpermeability [27]. In the present study, mice in the three experimental groups were treated with 5 mg/kg of simvastatin 2 hours before surgery, or once a day for 3 or 7 days before ovariectomy and vitrification (2H Tx, 3D Tx, and 7D Tx groups, respectively). Another study reported that donor (5 mg/kg),
Fig. 4. Apoptotic follicle ratio in accordance with four different simvastatin treatment conditions.
but not recipient (2 mg/kg), simvastatin treatment prevented ischemia-reperfusion injury–induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury [32,33]. Their study also suggested that donor statin treatment targets the transplanted organ and the proximal events that may lead to early allograft injury and predispose the organ to the development of delayed graft function [32]. We also administered 5 mg/kg of simvastatin to the recipient after OTT and evaluated the effects of recipient simvastatin treatment in accordance with the study by Tuuminen et al. [33]. However, recipient simvastatin treatment did not show any significant effect on grade I total follicle ratio, primordial follicle preservation, apoptosis, and serum FSH (data not shown). In the present study, the beneficial effect of simvastatin administration was shown only in the 2H Tx group, and the relatively long-term administration of simvastatin was less beneficial in the other groups than in the 2H Tx group. Chen et al. [27] postulated that simvastatin has a pleiotropic and dose-dependent effect on tumor vascular stabilization. Moreover, Weis et al. reported that 3-hydroxy-methylglutaryl coenzyme A reductase inhibition has a biphasic dose-dependent effect on angiogenesis that is lipid independent and associated with alterations in endothelial cell apoptosis and VEGF signaling. They also reported that statins have proangiogenic effects at low therapeutic concentrations and angiostatic effects at high concentrations [44]. On the basis of these findings, we assume that accumulated simvastatin in the donor could reduce the beneficial effect on ovarian quality.
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Fig. 5. A representative image of immunohistochemistry for CD31. X-axis shows types of different groups (A, E, I: control; B, F, J: 2H Tx; C, G, K: 3D Tx; and D, H, L: 7D Tx) and Y-axis represents period after ovarian tissue transplantation (A–D: 2 days; E-H: 7 days; and I–L: 21 days after transplantation). 2H Tx, simvastatin treatment at 2 hours before ovariectomy; 3D Tx, simvastatin treatment once daily for 3 days before ovariectomy; 7D Tx, simvastatin treatment once daily for 7 days before ovariectomy. The magnification was x100 and scale bar indicates 400 um. White arrows indicate the CD31-positive blood vessels in the transplanted ovarian tissue. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)
levels in accordance with the estrous cycles may cause these limitations. Hence, there is a possibility that some of the beneficial effects of simvastatin observed in morphology and angiogenesis were reflected in serum FSH levels. Further study is therefore required using more sensitive and direct markers of ovarian reserve and function, such as anti-Mullerian hormone, and these could reveal the effects on transplanted ovarian reserve and function more accurately.
Two days after transplantation, the FSH levels were increased in all groups; however, this was restored after 7 days. These results support the belief that the revascularization process begins within 48 hours of surgery and finishes at around 7 days, as reported in the murine model of a previous study [45]. In this study, some beneficial effects on morphologic evaluation and angiogenesis appeared in the 2H Tx group; however, there was no significant difference in the FSH levels among the four groups. Serum FSH is used as a ovarian reserve marker in experimental animals and in humans [46,47]; however, Lambalk et al. [48] reported that the roles of FSH as an ovarian reserve marker have limitations and relatively low sensitivity, and differences in FSH
The preadministration of simvastatin could improve the quality of ovarian follicles and enhance angiogenesis after
Fig. 6. The proportion of CD31 (þ) area in transplants after grafting in four different simvastatin treatment conditions.
Fig. 7. Measurement of serum FSH levels in all groups at different periods.
4.1. Conclusions
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