Theriogenology 83 (2015) 854–861
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Transplantation of mouse ovarian tissue: Comparison of the transplantation sites Hye Won Youm a, Jung Ryeol Lee a, b, Jaewang Lee a, b, Byung Chul Jee a, b, Chang Suk Suh a, b, *, Seok Hyun Kim b a b
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, South Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2014 Received in revised form 23 October 2014 Accepted 20 November 2014
Many studies have shown that ischemic injuries during the transplantation process were more detrimental than cryoinjuries for follicle survival and death, and it has been reported that transplantation sites can affect the outcomes of grafted ovarian tissue (OT). The purpose of this study was to assess the impact of different OT transplantation sites on follicular integrity and function of OT grafts. B6D2F1 mice were randomly assigned to control (sham) and four experimental groups according to transplantation sites (back muscle [BM], fat pad [FP], kidney capsule [KC], and subcutaneous [SC]). The ovaries from four groups were autotransplanted to each site. The OT recovery ratios on Days 2, 7, and 21 were significantly decreased in the FP group. The mean numbers of follicles were significantly lower in all the grafting groups compared with the sham group, except in the KC group on Days 7 and 21 and the BM group on Day 21. On Day 2, all the experimental groups showed low intact (G1) follicle ratio when compared with the sham group; however, the BM, KC, and FP groups recovered their morphologic integrity on Day 7, and only the SC group presented a significant decrease in G1 follicle ratios. On Day 21, the G1 follicle ratios of the FP and KC groups were greater than the sham control group. The proportion of apoptotic follicles of the four OT graft groups was higher than in the sham group on Day 2, followed by a significant decrease in the KC group and an increase in the SC group on Day 7. The serum follicle-stimulating hormone levels were significantly increased in all grafting groups on Day 2. On Day 7, only the SC group showed the high follicle-stimulating hormone level compared with the other groups. The mean numbers of oocytes from OT grafts were the highest in the KC group, except in the control group, and the lowest in the SC group. The ratios of mature oocytes were also significantly greater in the sham and KC groups. However, the ratios of normal spindle did not differ among the five groups. In conclusion, the KC was the optimal site for OT transplantation in this murine model, whereas the SC site was unfavorable for this procedure. In this study, we confirmed that the different grafting sites influenced the outcomes of transplantation. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Ovarian tissue Transplantation Fertility preservation Kidney capsule Spindle
1. Introduction Recently, there has been a remarkable increase in the numbers of the patients with cancer and cancer survivors;
* 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.2014.11.026
however, cancer treatment such as high-dose chemotherapy and radiotherapy can negatively affect the ovarian follicular reserve, resulting in infertility [1]. In clinical practice, ovarian tissue transplantation (OTT) and cryopreservation have been used to restore the fertility of women with infertility caused by chemo/radiotherapy or ovarian loss and is used in many reproductive centers. Many reports on transplantation of cryopreserved–thawed ovarian tissues (OTs) have been
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published, and as a result of such advances, almost 30 live births have been achieved by transplanted human OTs [1,2]. Outside the body, follicles have to withstand physical restrictions such as ischemia, oxygen tension, unfavorable temperature, and nutrient depletion. Many follicles can be damaged and lost by cryoinjuries during the cryopreservation process. Nevertheless, many recent studies have shown that ischemic injuries during the transplantation process were more detrimental than cryoinjuries for follicle survival and death [3,4]. However, the optimal transplantation conditions of OT for follicular survival and graft continuity are not well documented yet. According to several studies, neovascularization after transplantation occurred within 48 hours in rats [5], 1 week in sheep [6,7], and 5 days in human xenotransplantation [8]. While neovascularization occurs, the OT grafts are vulnerable to ischemia and hypoxic environments. Therefore, the reduction of the ischemic period after transplantation is essential for the successful recovery of OT graft functions. Many studies have reported that OT could survive and achieve folliculogenesis after xenotransplantation into different species and sexes. This has become a practical tool for the study of in vivo follicular development and evaluation of malignancy recurrence in cancer patients. Additionally, because of the limited availability of humans and primates for transplantation research, the murine model has been widely used for OT xenotransplantation in many studies [8,9]. Before OTT, it should be considered whether the different grafting sites lead to different results; if so, it is necessary to determine which site is the most suitable for OT survival and recovery of function. In previous reports, several heterotopic sites for OTT have been investigated, including the back muscle (BM) [10,11], fat pad (FP) [12,13], kidney capsule (KC) [14,15], and subcutaneous (SC) tissue [16,17]. Soleimani et al. [18] recently found that the BM had some advantages as a mouse grafting site because of its angiogenic conditions. Dath et al. [9] stated that the intramuscular site was good for grafting because of the preservation of the stroma and reduction of fibrosis. The BM site is convenient and less stressful for surgery. However, muscle activity can cause movement of the OT grafts followed by escape from the grafting site. The FP site is usually used when cancer cells are injected into mice because of the good blood supply and the small changes in temperature and pressure. Lee et al. [12] reported that transplantation of vitrified-warmed mouse OT to the FP site was a useful procedure for fertility preservation. However, OTs transplanted into the FP site can be less functional and have increased risk of hemorrhage [13]. The KC site is popular as a platform for OT because of rapid revascularization [14]. Wang et al. [19] reported that successful production of healthy offspring was achieved using oocytes taken from OT grafted in KC. Yang et al. [20] showed that the KC and ovarian bursal cavity yielded more grafts and oocytes than the SC site owing to the relatively good blood supply. The SC as a grafting site has been used in several centers. This site is located close to the body surface, and it is very easy to apply OT grafting surgery and observe the follicle growth [21]. Schubert et al [17] reported that the grafts of fresh and cryopreserved ovarian cortex into the SC site of SCID mice
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were able to sustain OT function. However, several other articles reported that the environment is sensitive to the change of external temperature and pressure so that could disrupt ovarian processes; furthermore, the grafts can be moving inside the SC pocket because of the spacious environment [7,9]. However, few studies have compared the graft sites directly, so the optimal site for OTT has not been well determined yet [9,22]. The present study was performed to compare and assess the efficacy and influence of the four different transplantation sites (BM, FP, KC, and SC) by evaluating the graft recovery ratios, follicular density and integrity, the folliclestimulating hormone (FSH) concentration, follicle apoptosis, and the quantity and quality of oocytes obtained from OT grafts. 2. Materials and methods 2.1. Experimental animals This study was carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital. Four-week-old B6D2F1 female mice (Orient bio, Seongnam, Korea) were used for the OT autotransplantation model. The mice were housed in groups of five per cage, maintained at 22 C under controlled sterile conditions, with a 12-hour light/dark cycle and free access to an autoclaved pellet diet and water. 2.2. Autotransplantation of OTs The mice (n ¼ 175) were randomly assigned to five groups by OTT sites (sham, BM, FP, KC, and SC). At first, the mice were anesthetized by intraperitoneal injection of 30 mg/kg of zolazepam þ tiletamine (Zoletil, Virbac, France) and 10 mg/kg of xylazine (Rompun, Bayer, Germany). After analgesia, bilateral dorsal incision was made, and both ovaries were extracted. The excised ovaries were placed in Dulbecco’s phosphate-buffered saline (D-PBS: Gibco, Paisley, UK) and transplanted into four different transplantation sites, immediately. The procedure for OTT is shown in Figure 1. 2.2.1. Sham The dorsal–horizontal skin was incised bilaterally and closed immediately by suture. 2.2.2. Back muscle Both ovaries were grafted into the BM after making muscle pockets using a fine forceps, and then, the skin incisions were sutured. 2.2.3. Fat pad The inguinal mammary FP located 1 to 2 cm below the ovariectomy site was taken out. After making 1- to 2-mmlength incisions on FP, the ovaries were inserted into the FP, and the FP and skin incisions were sutured. 2.2.4. Kidney capsule The kidney was exteriorized through a dorsal–horizontal incision on the ovariectomy site. The ovaries were
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Fig. 1. Transplantation of mouse ovarian tissues into the four different sites (BM, FP, KC, and SC). Ovarian tissue and their 1-week-old grafts are shown in the green circles. BM, back muscle; FP, fat pad; KC, kidney capsule; OTT, ovarian tissue transplantation; SC, subcutaneous. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
2.2.5. Subcutaneous The both sides of dorsal skin were incised bilaterally. The ovaries were grafted into SC after making small pockets under the skin using a fine forceps; the skin incisions were sutured.
of ovaries was embedded in a paraffin block and cut into 4w5 mm sections serially. These sections were stained with Mayer’s hematoxylin–eosin solution (Merck, Darmstadt, Germany) for histologic examination. The other sections were used for immunohistochemistry and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL; Roche, Mannheim, Germany) assay.
2.3. Recovery of graft and histologic stain
2.4. Follicle classification and morphologic analysis
The OTs recovered on Days 2, 7, and 21 after transplantation were fixed in 4% paraformaldehyde. Each group
Follicle classification and morphologic analysis were performed using a light microscope (Nikon, Tokyo, Japan)
inserted beneath the KC through a small hole made by a fine forceps, and the skin incisions were sutured.
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at 400 magnification. Each follicle was classified into four types according to the categories of Lundy et al. [23]; primordial (single layer of flattened pregranulosa cells), primary (single-layer granulosa cells [GC] including cuboidal forms), secondary (at least two layers of cuboidal GC), and antral (multiple layers of cuboidal GC with antrum). The integrity of each follicle was evaluated using the following criteria by Youm et al. [24]; G1 (good: intact spherical follicle and oocyte), G2 (fair: GC pulled away from edge of follicles, but with intact oocyte), and G3 (poor: disruption and loss of granulosa and theca cells, pyknotic nuclei, and missing oocyte). To avoid miscounting, the follicles were analyzed in only one section per OT when they contained oocytes. 2.5. Analysis of apoptosis Apoptosis of OT follicles was evaluated by TUNEL assay. In brief, after treatment with 0.1% Triton X-100 in citrate buffer for 15 minutes at room temperature (RT), the slides were incubated with TUNEL reaction mixture for 1 hour at 37 C in a humidified dark chamber. After washing, the slides were mounted with VECTASHIELD mounting medium with 40 , 6-diamidino-2-phenylindole (DAPI; Vector laboratories Inc., Burlingame, USA). The labeled slides were examined under the inverted Carl Zeiss AX10 microscope (Carl Zeiss AG, Oberkochen, Germany). Green fluorescence was visualized in the TUNEL-positive cells at excitation wavelength ranging from 450 to 500 nm and detection capability ranging from 515 to 565 nm. 40 , 6-Diamidino-2phenylindole excited at approximately 360 nm and emitted blue fluorescence at approximately 460 nm when bound to DNA. When the portion of apoptotic cells in a follicle was greater than 30%, we considered it as an apoptotic follicle. 2.6. Measurement of FSH level The mouse blood sera were obtained, and the FSH concentrations were measured using ELISA assay kit (Endocrine technologies, Newark, USA) according to the manufacturer instructions. The intra-assay and inter-assay coefficients of variation were 6.35% and 5.88%, respectively, with a sensitivity of 0.5 ng/mL. 2.7. Oocyte collection and IVM Thirty-two mice (n ¼ 32) were divided into control (sham) and four OTT groups for oocyte collection. On Day 21 after OTT, the mice were treated with 7.5 IU pregnant mare’s serum gonadotrophin (Sigma Chemical, St Louise, USA) followed by 7.5 IU hCG (Sigma Chemical, St Louise, USA) 48 hours later. Then, the sham and grafted ovaries were collected 10 hours after hCG injection, and the cumulus–oocyte complexes (COCs) were collected mechanically in Medium-199 HEPES modified medium (Welgene, Daegu, Korea) supplemented with 20% fetal bovine serum (Gibco, Paisley, UK). The collected COCs were cultured in vitro in a maturation medium (Medium-199 supplemented with 10 mIU FSH, 10 mIU hCG, and 20% fetal bovine serum) for 4 hours, then the cumulus cells were
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removed by 85 IU/mL hyaluronidase (Cook, Brisbane, Australia). The retrieved oocytes were evaluated according to their maturity. Only mature oocytes were used for spindle and chromosome analysis. 2.8. Meiotic spindle and chromosome evaluation The mature oocytes were fixed in 4% paraformaldehyde for 1 hour, washed in D-PBS supplemented with 0.1% BSA (Sigma–Aldrich, Steinheim, Germany), and then transferred into 0.25% Triton X-100 for 10 minutes. After washing, the oocytes were moved into a 3% BSA/D-PBS blocking medium for 1 hour at RT and incubated with a-tubulin polyclonal antibody (1:100, Cell Signaling Technology, Boston, USA) for 2 hours. Then, the oocytes were treated with Alexa Fluor 488 (Abcam, MA, USA) and goat antirabbit IgG (H þ L) antibodies (1:200, Invitrogen, Grand Island, USA) for 1 hour in the dark at RT. Finally, the slides were mounted with VECTASHIELD mounting medium with DAPI. The localization of tubulin and chromatin was observed under the Carl Zeiss fluorescence laser confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany) at 400 magnification. We used optical filters specific for the wavelengths of 450 to 590 nm and 330 to 380 nm for the green (Alexa Fluor 488) and blue (DAPI) signals, respectively. Typical barrel-shaped microtubules traversing between both poles and centrally aligned chromosomes were considered as normal. Any other configurations were considered as abnormal. 2.9. Statistical analysis The proportion and normality in each sample were analyzed by the chi-square test or ANOVA using the SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA) and Graphpad Prism 5.0 (Graphpad software, California, USA). The values were considered significant when P < 0.05. 3. Results 3.1. Recovery ratios of OT grafts The images of the four OT grafting sites are shown in Figure 1. The OT graft recovery ratios on Days 2, 7, and 21 after transplantation were 98.5% (67 of 68) for the sham group, 94.6% (70 of 74) for the BM group, 80.3% (53 of 66) for the FP group, 93.1% (67 of 72) for the KC group, and 91.4% (64 of 70) for the SC group. In the FP group, the ovarian recovery ratios were significantly decreased. Separating the OT grafts from the transplantation site was easy on Day 2; however, it became progressively more difficult as the days of OTT increased because the grafts were perfused into the graft sites thoroughly on Day 21. 3.2. Mean follicle numbers and developmental stages of follicles Table 1 presents the follicular density and developmental stage of the five groups by histologic analysis. The mean numbers of follicles were significantly lower in all the grafting groups compared with the sham group, except in
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Table 1 The morphologic evaluation of follicular density and developmental stage of five groups according to the grafting sites and durations after transplantation. Duration after grafting Day 2 Sham BM FP KC SC Day 7 Sham BM FP KC SC Day 21 Sham BM FP KC SC
Number of transplanted ovaries
Number of ovarian grafts
Mean numbers of follicles (SE)
Primordial follicle ratios (%)
Antral follicle ratios (%)
22 20 24 24 22
22 20 21 23 21
22.9 10.8 12.6 9.9 12.1
2.4a 0.8b 1.2b 0.9b 1.3b
36.5ac 29.3ab 38.5c 26.9b 33.9abc
16.2a 0.9bc 0b 2.2c 0b
22 22 20 24 24
22 20 13 20 22
17.0 10.1 8.9 14.7 9.5
1.3a 1.7b 2.4b 1.3ab 1.5b
34.8a 36.0ab 27.6a 43.0b 36.5ab
16.3a 6.4b 6.0b 5.1b 1.4c
24 32 22 24 24
23 30 19 24 21
18.8 13.2 11.9 15.9 8.5
1.0a 1.5ab 1.5bc 1.6ac 1.6b
25.2a 26.8ab 31.7b 31.8b 27.4ab
17.1a 16.2a 17.6a 13.9a 7.8b
Different superscript letters indicate statistically significant differences (P < 0.05) and the superscripts were used for each duration group separately. Abbreviations: BM, back muscle; FP, fat pad; KC, kidney capsule; SC, subcutaneous; SE, standard error of the mean.
the KC group on Days 7 and 21 and the BM group on Day 21. The primordial follicle ratios were higher in the FP group (38.5%) on Day 2, KC group (43.0%) on Day 7, and FP and KC groups (31.7% and 31.8%) on Day 21. Antral follicles began to appear in the BM (0.9%) and KC (2.2%) groups initially on Day 2. However, the ratios of antral follicles increased in all the groups, except in the SC group, and there was no significant difference with the sham control group on Day 21.
3.3. Morphologic analysis of the grafts Figure 2A shows the proportion of the morphologically intact (G1) follicles among the groups. On Day 2, there was no significant difference in G1 follicle ratios between the four grafting groups, whereas all showed low G1 follicle ratios when compared with the sham group (sham: 61.3%, BM: 35.3%, FP: 37.7%, KC: 32.2%, and SC: 31.5%). The BM, KC, and FP
Fig. 2. Comparisons of the normality of ovarian tissues and their functions between the sham control and four different transplantation sites according to the durations after transplantation: (A) the intact (G1) follicle ratios, (B) the apoptotic follicle ratios, and (C) the serum FSH levels. Columns with different superscripts differ significantly (P < 0.05).
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groups recovered their morphologic integrity on Day 7, and only the SC group presented a significant decrease in G1 follicle ratios (sham: 64.5%, BM: 63.4%, FP: 62.1%, KC: 63.1%, and SC: 47.6%). On Day 21, the G1 follicle ratios of the FP and KC groups were higher than the sham control group (sham: 63.0%, BM: 59.5%, FP: 74.0%, KC: 71.9%, and SC: 70.9%).
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Fig. 3B). However, the ratios of normal spindle did not differ among the five groups (sham: 71.1%, BM: 56.0%, FP: 53.8%, KC: 64.1%, and SC: 57.1%; Fig. 3C). Figure 4 shows the immunofluorescence staining of a-tubulin in the meiotic spindle and the chromosomes in the nucleus of mature oocytes obtained from the sham OT and four OT grafts.
3.4. Apoptosis 4. Discussion Apoptosis was evaluated by TUNEL assay in a total of 961 follicles for Day 2, 874 follicles for Day 7, and 609 follicles for Day 21 from the five groups (Fig. 2B). The proportion of apoptotic follicles of the four OT graft groups was higher than in the sham group on Day 2 (sham: 5.9%, BM: 16.4%, FP: 17.8%, KC: 14.6%, and SC: 17.3%), followed by a significant decrease in the KC group (7.6%) and an increase in the SC group (23.8%) on Day 7 (sham: 4.4%, BM: 12.5%, and FP 15.1%). However, no differences among the five groups were observed on Day 21 (sham: 9.2%, BM: 8.4%, FP: 7.8%, KC: 8.3%, and SC: 7.2%). 3.5. FSH level We measured serum FSH levels to verify the recovery of ovarian function. As shown in Figure 2C, the serum FSH levels were significantly increased in all grafting groups on Day 2. On Day 7, only the SC group showed the high FSH level (5.5 ng/mL) compared with the other groups (1.6– 2.5 ng/mL) followed by no sign of significant difference among the groups on Day 21. The overall FSH levels were decreased as the OTT durations increased, except in the SC group on Day 7. 3.6. Evaluation of oocytes The OTs of five groups were collected on Day 21 after grafting. The numbers of ovarian grafts used for oocyte collection were 10, 12, 15, 13, and 12 from Sham, BM, FP, KC, and SC groups, respectively. The mean numbers of oocytes from OTs after ovulation stimulation were the highest in the KC group (10.4 2.3), except in the sham control group (15.8 4.8), and the lowest in the SC group (2.1 0.9; BM: 4.6 1.6 and FP: 3.5 1.3; Fig. 3A). The ratios of mature oocytes were also significantly higher in the sham (75.3%) and KC (60.7%) groups (BM: 45.5%, FP: 25.0%, and SC: 28.0%;
Xenotransplantation of human OT to mice is a very useful method for the assessment of malignancy recurrence, evaluation of ovarian function recovery, and elucidation of revascularization mechanisms after OTT with or without cryopreservation [17,25–28]. Furthermore, this murine transplantation model can provide the opportunities to improve grafting conditions for future reproductive medicine application. To date, several murine models and techniques for transplantation procedures have been developed. However, the most effective site for OT grafting has not been well established. Therefore, we evaluated and compared the outcomes of OTT in four grafting sites (BM, FP, KC, and SC). The OT grafts suffered from hypoxia and ischemic damage should undergo large amount of follicle loss during transplantation process, so it is required to shorten the ischemic duration to maintain the ovarian pool. In our study, the mean follicle numbers were comparable with those of the sham control group in KC group on Day 7 and BM and KC groups on Day 21, and these two sites were known to have good angiogenic conditions for OTT [9,18]. Therefore, it is suggested that the quick revascularization helped the KC and BM sites to comparatively well preserve their follicles. Whereas, the SC group showed constantly low mean numbers of follicles on Days 7 and Day 21 among the groups, which may be because of the relatively poor vascularization, stressful environment close to outside, and spacious SC pocket, which enable the OT to move [7,9]. Primordial follicles are resistant to the hypoxic environments. However, antral follicles are easily degenerated after grafting. Several studies reported that the absence of antral and secondary follicles in OT grafts could activate the primordial follicles and accelerate their growth because of the lack of inhibitory factors released by the antral follicles. Thus, rapid emergence of antral follicles appears to be
Fig. 3. Evaluation of oocytes retrieved from the ovarian tissues of the sham control and four grafts groups: (A) mean number of oocyte, (B) mature oocyte ratios, and (C) normal spindle ratios. Columns with different superscripts differ significantly (P < 0.05). BM, back muscle; FP, fat pad; KC, kidney capsule; SC, subcutaneous.
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Fig. 4. Immunofluorescence staining of a-tubulin showing morphology of meiotic spindle organization (green) and chromosome alignment (blue) in mature oocytes obtained from ovarian tissue grafts (400). Typical barrel-shaped microtubules traversing between both poles and centrally aligned chromosomes were considered as normal (A, B), and any other configurations were considered as abnormal (C, D). (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
important to maintain quiescent follicles [29]. In this study, the antral follicles were seen in the BM and KC groups on Day 2 firstly, but no antral follicle was observed in the FP and SC groups at that time. Then, the proportion of antral follicles increased as the grafting duration increased in all OTT groups; however, the SC group showed the lowest antral follicle ratio in all the durations among them. These conditions seem to affect their FSH levels. On Day 2, the serum FSH levels of all four OTT groups were significantly increased when compared with the control group followed by being decreased on Day 7 including the FP group but except the SC group. On Day 21, the four OTT groups including the SC group showed decreased FSH levels similar to that of sham control group. It can mean that OTs of all grafting were well transplanted and adapted to their grafting sites on Day 21, and so, the ovarian functions were recovered. We found that once the OTs were perfused into the recipient body, the FSH levels became decreased to the normal range similar to the control group, although there were significant differences in follicle integrity and/or oocyte maturity. Therefore, it is suggested that FSH measurement is necessary to confirm ovarian function recovery; however, more exact evaluations should be performed additionally to verify the results. Several studies reported that increased FSH levels were usually
observed in the OT grafting models during reestablishment of follicle development, which is possibly because of a deficiency of inhibin A produced by the growing follicles [30,31]. In the other words, when the OTs were perfused into the grafting site and develop the antral follicles after recovering their function, the FSH levels could be decreased to the normal range as shown in our outcome. The SC group also showed the lowest G1 follicle ratio and the highest apoptotic follicle ratio among the four grafting groups on Day 7. Therefore, it is suggested that the SC group needs more time to settle into the site, and the retardation of OT perfusion can induce some defects of OTs in SC site. The purpose of OTT is to obtain many intact oocytes; therefore, the assessment and comparison of oocyte quantity and quality are essential. We collected oocytes from the sham control ovaries and four OT graft groups 10 hours after hCG injection, followed by in vitro maturation for 4 hours. Usually, the oocytes were collected from ovaries 12 to 14 hours after hCG injection. However, the OTs in this study were transplanted into the four heterotopic sites which did not have oviduct. Therefore, we have collected the oocytes from OT grafts 10 hours after hCG injection before the ovulation time by mechanical dissection and performed IVM of oocytes for 4 hours. We have
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compared the oocyte collection time (10, 12, and 14 hours after hCG) and confirmed that the highest number of oocytes were obtained from the OT grafts 10 hours after hCG injection (unpublished data). The best outcome for the retrieved oocyte numbers and oocyte maturation ratios was achieved in the KC group among the four OTT groups, and the SC group together with the FP group have produced comparatively few oocytes and mature oocytes, which corresponded with the result of the evaluation of OT grafts. In conclusion, our study showed that the KC was the optimal site for OT grafting in the murine model, whereas the SC site was unfavorable for this procedure. And, we confirmed that the different OTT sites caused the different outcomes. Considering the results of this study, we recommend the KC site to optimize the experimental conditions and perform OT xenotransplantation for various purposes. Acknowledgments This study was supported by a grant (No. HI12C0055) from the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea. The first two authors contributed equally to this work. Competing Interests The authors have no conflict of interest to declare. References [1] Meirow D, Baum M, Yaron R, Levron J, Hardan I, Schiff E, et al. Ovarian tissue cryopreservation in hematologic malignancy: ten years’ experience. Leuk Lymphoma 2007;48:1569–76. [2] Von Wolff M, Donnez J, Hovatta O, Keros V, Maltaris T, Montag M, et al. Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapyda technique in its infancy but already successful in fertility preservation. Eur J Cancer 2009;45:1547–53. [3] Liu J, Van der Elst J, Van den Broecke R, Dhont M. Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod 2002;17:605–11. [4] Wang X, Huifang CH, Hang Y. Cryopreservation: fertility after intact ovary transplantation. Nature 2002;415:385. [5] Dissen GA, Lara HE, Fahrenbach WH. Immature rat ovaries revascularized rapidly after autotransplantation and show a gonadotrop independent increase in angiogenic factor gene expression. Endocrinology 1994;134:1146–54. [6] Gosden RG, Boulton MI, Grant K, Webb R. Follicular development from ovarian xenografts in SCID mice. J Reprod Fertil 1994;101:619–23. [7] Torrents E, Boiso I, Barri PN, Veiga A. Applications of ovarian tissue transplantation in experimental biology and medicine. Hum Reprod Update 2003;9:471–81. [8] Eyck ASV, Bouzin C, Feron O, Romeu L, Langendonckt AV, Donnez J, et al. Both host and graft vessels contribute to revascularization of xenografted human ovarian tissue in a murine model. Fertil Steril 2010;93:1676–85. [9] Dath C, Eyck ASV, Dolmans MM, Romeu L, Vigne LD, Donnez J, et al. Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites. Hum Reprod 2010;25:1734–43. [10] Maltaris T, Beckmann MW, Binder H, Mueller A, Hoffmann I, Koelbl H, et al. The effect of a GnRH agonist on cryopreserved human ovarian grafts in severe combined immunodeficient mice. Reproduction 2007;133:503–9.
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[11] Li F, Tao Y, Zhang Y, Li Y, Fang F, Liu Y, et al. Follicle growth and oocyte development after ovary transplantation into back muscle of immune-intact adult castrated male mice. Reproduction 2010;140: 465–76. [12] Lee DH, Kim JO, Yoo YK, Lee KJ, Kim JS, Ahn CJ. Restoration of reproductive potential after autotransplantation of frozen ovaries in mice pretreated with cyclophosphamide. J Breast Cancer 2008;11: 10–7. [13] Sundberg JP, Boggess D. Systematic approach to evaluation of mouse mutation. CRC Press; 2010. p. 10. [14] Oktay K, Newton H, Mullan J, Gosden RG. Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone. Hum Reprod Update 1998;13: 1133–8. [15] Hernandez FH, Bosch P, Sirisathien S, Wininger JD, Massey JB, Brackett BG. Effect of site of transplantation on follicular development of human ovarian tissue transplanted into intact or castrated immunodeficient mice. Fertil Steril 2004;81:888–92. [16] Kim SS, Kang HG, Kim NH, Lee HC, Lee HH. Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation. Hum Reprod Update 2005;20: 2502–8. [17] Schubert B, Canis M, Darcha C. Follicular growth and estradiol follow-up after subcutaneous xenografting of fresh and cryopreserved human ovarian tissue. Fertil Steril 2008;89:1787–94. [18] Soleimani R, Elst J, Heytens E, Broecke R, Gerris F, Dhont M, et al. Back muscle as a promising site for ovarian tissue transplantation, an animal model. Hum Reprod 2008;23:619–26. [19] Wang X, Catt S, Pangestu M, Temple-Smith P. Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice. Reproduction 2009;138:527–35. [20] Yang HY, Cox SL, Jenkin G, Findlay J, Trounson A, Shaw J. Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts. Reproduction 2006; 131:851–9. [21] Kim SS, Lee WS, Chung MK, Lee HC, Lee HH, Hill D. Long-term ovarian function and fertility after heterotopic autotransplantation of cryobanked human ovarian tissue: 8-year experience in cancer patients. Fertil Steril 2009;91:2349–54. [22] Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, et al. Embryo development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet Oncol 2004;363:837–40. [23] Lundy T, Smith P, Connell AO, Hudson NL, McNatty KP. Populations of granulosa cells in small follicles of the sheep ovary. J Reprod Fertil 1999;115:251–62. [24] 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. [25] Gook DA, Edgar DH, Borg J, Archer J, McBain JC. Diagnostic assessment of the development potential of human cryopreserved ovarian tissue from multiple patients using xenografting. Hum Reprod 2005;20:72–8. [26] Kim SS, Radford J, Harris M, Varley J, Rutherford AJ, Lieberman B, et al. Ovarian tissue harvested from lymphoma patients to preserve fertility may be safe for autotransplantation. Hum Reprod 2001;16: 2056–60. [27] Nisolle M, Casanas-Roux F, Qu J, Motta P, Donnez J. Histologic and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice. Fertil Steril 2000;74:122–9. [28] Oktem O, Oktay K. A novel ovarian xenografting model to characterize the impact of chemotherapy agents on human primordial follicle reserve. Cancer Res 2007;67:10159–62. [29] Baird DT, Campbell B, De Souza C, Telfer E. Long-term ovarian function in sheep after ovariectomy and autotransplantation of cryopreserved cortical strips. Eur J Obstet Gynecol Reprod Biol 2004;113:S55–9. [30] Callejo J, Vilaseca S, Medina M, Salvador C, Valls C, Lailla J. Inhibin and follicular development in heterotopical ovary transplants without vascular pedicle in syngeneic Lewis rats. Fertil Steril 2003; 79:743–8. [31] Campbell B, Telfer E, Webb R, Baird D. Ovarian autografts in sheep as a model for studying folliculogenesis. Mol Cell Endocrinol 2000;163: 131–9.
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Theriogenology journal homepage: www.theriojournal.com
Transplantation of mouse ovarian tissue: Comparison of the transplantation sites Hye Won Youm a, Jung Ryeol Lee a, b, Jaewang Lee a, b, Byung Chul Jee a, b, Chang Suk Suh a, b, *, Seok Hyun Kim b a b
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, South Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2014 Received in revised form 23 October 2014 Accepted 20 November 2014
Many studies have shown that ischemic injuries during the transplantation process were more detrimental than cryoinjuries for follicle survival and death, and it has been reported that transplantation sites can affect the outcomes of grafted ovarian tissue (OT). The purpose of this study was to assess the impact of different OT transplantation sites on follicular integrity and function of OT grafts. B6D2F1 mice were randomly assigned to control (sham) and four experimental groups according to transplantation sites (back muscle [BM], fat pad [FP], kidney capsule [KC], and subcutaneous [SC]). The ovaries from four groups were autotransplanted to each site. The OT recovery ratios on Days 2, 7, and 21 were significantly decreased in the FP group. The mean numbers of follicles were significantly lower in all the grafting groups compared with the sham group, except in the KC group on Days 7 and 21 and the BM group on Day 21. On Day 2, all the experimental groups showed low intact (G1) follicle ratio when compared with the sham group; however, the BM, KC, and FP groups recovered their morphologic integrity on Day 7, and only the SC group presented a significant decrease in G1 follicle ratios. On Day 21, the G1 follicle ratios of the FP and KC groups were greater than the sham control group. The proportion of apoptotic follicles of the four OT graft groups was higher than in the sham group on Day 2, followed by a significant decrease in the KC group and an increase in the SC group on Day 7. The serum follicle-stimulating hormone levels were significantly increased in all grafting groups on Day 2. On Day 7, only the SC group showed the high follicle-stimulating hormone level compared with the other groups. The mean numbers of oocytes from OT grafts were the highest in the KC group, except in the control group, and the lowest in the SC group. The ratios of mature oocytes were also significantly greater in the sham and KC groups. However, the ratios of normal spindle did not differ among the five groups. In conclusion, the KC was the optimal site for OT transplantation in this murine model, whereas the SC site was unfavorable for this procedure. In this study, we confirmed that the different grafting sites influenced the outcomes of transplantation. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Ovarian tissue Transplantation Fertility preservation Kidney capsule Spindle
1. Introduction Recently, there has been a remarkable increase in the numbers of the patients with cancer and cancer survivors;
* 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.2014.11.026
however, cancer treatment such as high-dose chemotherapy and radiotherapy can negatively affect the ovarian follicular reserve, resulting in infertility [1]. In clinical practice, ovarian tissue transplantation (OTT) and cryopreservation have been used to restore the fertility of women with infertility caused by chemo/radiotherapy or ovarian loss and is used in many reproductive centers. Many reports on transplantation of cryopreserved–thawed ovarian tissues (OTs) have been
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published, and as a result of such advances, almost 30 live births have been achieved by transplanted human OTs [1,2]. Outside the body, follicles have to withstand physical restrictions such as ischemia, oxygen tension, unfavorable temperature, and nutrient depletion. Many follicles can be damaged and lost by cryoinjuries during the cryopreservation process. Nevertheless, many recent studies have shown that ischemic injuries during the transplantation process were more detrimental than cryoinjuries for follicle survival and death [3,4]. However, the optimal transplantation conditions of OT for follicular survival and graft continuity are not well documented yet. According to several studies, neovascularization after transplantation occurred within 48 hours in rats [5], 1 week in sheep [6,7], and 5 days in human xenotransplantation [8]. While neovascularization occurs, the OT grafts are vulnerable to ischemia and hypoxic environments. Therefore, the reduction of the ischemic period after transplantation is essential for the successful recovery of OT graft functions. Many studies have reported that OT could survive and achieve folliculogenesis after xenotransplantation into different species and sexes. This has become a practical tool for the study of in vivo follicular development and evaluation of malignancy recurrence in cancer patients. Additionally, because of the limited availability of humans and primates for transplantation research, the murine model has been widely used for OT xenotransplantation in many studies [8,9]. Before OTT, it should be considered whether the different grafting sites lead to different results; if so, it is necessary to determine which site is the most suitable for OT survival and recovery of function. In previous reports, several heterotopic sites for OTT have been investigated, including the back muscle (BM) [10,11], fat pad (FP) [12,13], kidney capsule (KC) [14,15], and subcutaneous (SC) tissue [16,17]. Soleimani et al. [18] recently found that the BM had some advantages as a mouse grafting site because of its angiogenic conditions. Dath et al. [9] stated that the intramuscular site was good for grafting because of the preservation of the stroma and reduction of fibrosis. The BM site is convenient and less stressful for surgery. However, muscle activity can cause movement of the OT grafts followed by escape from the grafting site. The FP site is usually used when cancer cells are injected into mice because of the good blood supply and the small changes in temperature and pressure. Lee et al. [12] reported that transplantation of vitrified-warmed mouse OT to the FP site was a useful procedure for fertility preservation. However, OTs transplanted into the FP site can be less functional and have increased risk of hemorrhage [13]. The KC site is popular as a platform for OT because of rapid revascularization [14]. Wang et al. [19] reported that successful production of healthy offspring was achieved using oocytes taken from OT grafted in KC. Yang et al. [20] showed that the KC and ovarian bursal cavity yielded more grafts and oocytes than the SC site owing to the relatively good blood supply. The SC as a grafting site has been used in several centers. This site is located close to the body surface, and it is very easy to apply OT grafting surgery and observe the follicle growth [21]. Schubert et al [17] reported that the grafts of fresh and cryopreserved ovarian cortex into the SC site of SCID mice
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were able to sustain OT function. However, several other articles reported that the environment is sensitive to the change of external temperature and pressure so that could disrupt ovarian processes; furthermore, the grafts can be moving inside the SC pocket because of the spacious environment [7,9]. However, few studies have compared the graft sites directly, so the optimal site for OTT has not been well determined yet [9,22]. The present study was performed to compare and assess the efficacy and influence of the four different transplantation sites (BM, FP, KC, and SC) by evaluating the graft recovery ratios, follicular density and integrity, the folliclestimulating hormone (FSH) concentration, follicle apoptosis, and the quantity and quality of oocytes obtained from OT grafts. 2. Materials and methods 2.1. Experimental animals This study was carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital. Four-week-old B6D2F1 female mice (Orient bio, Seongnam, Korea) were used for the OT autotransplantation model. The mice were housed in groups of five per cage, maintained at 22 C under controlled sterile conditions, with a 12-hour light/dark cycle and free access to an autoclaved pellet diet and water. 2.2. Autotransplantation of OTs The mice (n ¼ 175) were randomly assigned to five groups by OTT sites (sham, BM, FP, KC, and SC). At first, the mice were anesthetized by intraperitoneal injection of 30 mg/kg of zolazepam þ tiletamine (Zoletil, Virbac, France) and 10 mg/kg of xylazine (Rompun, Bayer, Germany). After analgesia, bilateral dorsal incision was made, and both ovaries were extracted. The excised ovaries were placed in Dulbecco’s phosphate-buffered saline (D-PBS: Gibco, Paisley, UK) and transplanted into four different transplantation sites, immediately. The procedure for OTT is shown in Figure 1. 2.2.1. Sham The dorsal–horizontal skin was incised bilaterally and closed immediately by suture. 2.2.2. Back muscle Both ovaries were grafted into the BM after making muscle pockets using a fine forceps, and then, the skin incisions were sutured. 2.2.3. Fat pad The inguinal mammary FP located 1 to 2 cm below the ovariectomy site was taken out. After making 1- to 2-mmlength incisions on FP, the ovaries were inserted into the FP, and the FP and skin incisions were sutured. 2.2.4. Kidney capsule The kidney was exteriorized through a dorsal–horizontal incision on the ovariectomy site. The ovaries were
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Fig. 1. Transplantation of mouse ovarian tissues into the four different sites (BM, FP, KC, and SC). Ovarian tissue and their 1-week-old grafts are shown in the green circles. BM, back muscle; FP, fat pad; KC, kidney capsule; OTT, ovarian tissue transplantation; SC, subcutaneous. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
2.2.5. Subcutaneous The both sides of dorsal skin were incised bilaterally. The ovaries were grafted into SC after making small pockets under the skin using a fine forceps; the skin incisions were sutured.
of ovaries was embedded in a paraffin block and cut into 4w5 mm sections serially. These sections were stained with Mayer’s hematoxylin–eosin solution (Merck, Darmstadt, Germany) for histologic examination. The other sections were used for immunohistochemistry and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL; Roche, Mannheim, Germany) assay.
2.3. Recovery of graft and histologic stain
2.4. Follicle classification and morphologic analysis
The OTs recovered on Days 2, 7, and 21 after transplantation were fixed in 4% paraformaldehyde. Each group
Follicle classification and morphologic analysis were performed using a light microscope (Nikon, Tokyo, Japan)
inserted beneath the KC through a small hole made by a fine forceps, and the skin incisions were sutured.
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at 400 magnification. Each follicle was classified into four types according to the categories of Lundy et al. [23]; primordial (single layer of flattened pregranulosa cells), primary (single-layer granulosa cells [GC] including cuboidal forms), secondary (at least two layers of cuboidal GC), and antral (multiple layers of cuboidal GC with antrum). The integrity of each follicle was evaluated using the following criteria by Youm et al. [24]; G1 (good: intact spherical follicle and oocyte), G2 (fair: GC pulled away from edge of follicles, but with intact oocyte), and G3 (poor: disruption and loss of granulosa and theca cells, pyknotic nuclei, and missing oocyte). To avoid miscounting, the follicles were analyzed in only one section per OT when they contained oocytes. 2.5. Analysis of apoptosis Apoptosis of OT follicles was evaluated by TUNEL assay. In brief, after treatment with 0.1% Triton X-100 in citrate buffer for 15 minutes at room temperature (RT), the slides were incubated with TUNEL reaction mixture for 1 hour at 37 C in a humidified dark chamber. After washing, the slides were mounted with VECTASHIELD mounting medium with 40 , 6-diamidino-2-phenylindole (DAPI; Vector laboratories Inc., Burlingame, USA). The labeled slides were examined under the inverted Carl Zeiss AX10 microscope (Carl Zeiss AG, Oberkochen, Germany). Green fluorescence was visualized in the TUNEL-positive cells at excitation wavelength ranging from 450 to 500 nm and detection capability ranging from 515 to 565 nm. 40 , 6-Diamidino-2phenylindole excited at approximately 360 nm and emitted blue fluorescence at approximately 460 nm when bound to DNA. When the portion of apoptotic cells in a follicle was greater than 30%, we considered it as an apoptotic follicle. 2.6. Measurement of FSH level The mouse blood sera were obtained, and the FSH concentrations were measured using ELISA assay kit (Endocrine technologies, Newark, USA) according to the manufacturer instructions. The intra-assay and inter-assay coefficients of variation were 6.35% and 5.88%, respectively, with a sensitivity of 0.5 ng/mL. 2.7. Oocyte collection and IVM Thirty-two mice (n ¼ 32) were divided into control (sham) and four OTT groups for oocyte collection. On Day 21 after OTT, the mice were treated with 7.5 IU pregnant mare’s serum gonadotrophin (Sigma Chemical, St Louise, USA) followed by 7.5 IU hCG (Sigma Chemical, St Louise, USA) 48 hours later. Then, the sham and grafted ovaries were collected 10 hours after hCG injection, and the cumulus–oocyte complexes (COCs) were collected mechanically in Medium-199 HEPES modified medium (Welgene, Daegu, Korea) supplemented with 20% fetal bovine serum (Gibco, Paisley, UK). The collected COCs were cultured in vitro in a maturation medium (Medium-199 supplemented with 10 mIU FSH, 10 mIU hCG, and 20% fetal bovine serum) for 4 hours, then the cumulus cells were
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removed by 85 IU/mL hyaluronidase (Cook, Brisbane, Australia). The retrieved oocytes were evaluated according to their maturity. Only mature oocytes were used for spindle and chromosome analysis. 2.8. Meiotic spindle and chromosome evaluation The mature oocytes were fixed in 4% paraformaldehyde for 1 hour, washed in D-PBS supplemented with 0.1% BSA (Sigma–Aldrich, Steinheim, Germany), and then transferred into 0.25% Triton X-100 for 10 minutes. After washing, the oocytes were moved into a 3% BSA/D-PBS blocking medium for 1 hour at RT and incubated with a-tubulin polyclonal antibody (1:100, Cell Signaling Technology, Boston, USA) for 2 hours. Then, the oocytes were treated with Alexa Fluor 488 (Abcam, MA, USA) and goat antirabbit IgG (H þ L) antibodies (1:200, Invitrogen, Grand Island, USA) for 1 hour in the dark at RT. Finally, the slides were mounted with VECTASHIELD mounting medium with DAPI. The localization of tubulin and chromatin was observed under the Carl Zeiss fluorescence laser confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany) at 400 magnification. We used optical filters specific for the wavelengths of 450 to 590 nm and 330 to 380 nm for the green (Alexa Fluor 488) and blue (DAPI) signals, respectively. Typical barrel-shaped microtubules traversing between both poles and centrally aligned chromosomes were considered as normal. Any other configurations were considered as abnormal. 2.9. Statistical analysis The proportion and normality in each sample were analyzed by the chi-square test or ANOVA using the SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA) and Graphpad Prism 5.0 (Graphpad software, California, USA). The values were considered significant when P < 0.05. 3. Results 3.1. Recovery ratios of OT grafts The images of the four OT grafting sites are shown in Figure 1. The OT graft recovery ratios on Days 2, 7, and 21 after transplantation were 98.5% (67 of 68) for the sham group, 94.6% (70 of 74) for the BM group, 80.3% (53 of 66) for the FP group, 93.1% (67 of 72) for the KC group, and 91.4% (64 of 70) for the SC group. In the FP group, the ovarian recovery ratios were significantly decreased. Separating the OT grafts from the transplantation site was easy on Day 2; however, it became progressively more difficult as the days of OTT increased because the grafts were perfused into the graft sites thoroughly on Day 21. 3.2. Mean follicle numbers and developmental stages of follicles Table 1 presents the follicular density and developmental stage of the five groups by histologic analysis. The mean numbers of follicles were significantly lower in all the grafting groups compared with the sham group, except in
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Table 1 The morphologic evaluation of follicular density and developmental stage of five groups according to the grafting sites and durations after transplantation. Duration after grafting Day 2 Sham BM FP KC SC Day 7 Sham BM FP KC SC Day 21 Sham BM FP KC SC
Number of transplanted ovaries
Number of ovarian grafts
Mean numbers of follicles (SE)
Primordial follicle ratios (%)
Antral follicle ratios (%)
22 20 24 24 22
22 20 21 23 21
22.9 10.8 12.6 9.9 12.1
2.4a 0.8b 1.2b 0.9b 1.3b
36.5ac 29.3ab 38.5c 26.9b 33.9abc
16.2a 0.9bc 0b 2.2c 0b
22 22 20 24 24
22 20 13 20 22
17.0 10.1 8.9 14.7 9.5
1.3a 1.7b 2.4b 1.3ab 1.5b
34.8a 36.0ab 27.6a 43.0b 36.5ab
16.3a 6.4b 6.0b 5.1b 1.4c
24 32 22 24 24
23 30 19 24 21
18.8 13.2 11.9 15.9 8.5
1.0a 1.5ab 1.5bc 1.6ac 1.6b
25.2a 26.8ab 31.7b 31.8b 27.4ab
17.1a 16.2a 17.6a 13.9a 7.8b
Different superscript letters indicate statistically significant differences (P < 0.05) and the superscripts were used for each duration group separately. Abbreviations: BM, back muscle; FP, fat pad; KC, kidney capsule; SC, subcutaneous; SE, standard error of the mean.
the KC group on Days 7 and 21 and the BM group on Day 21. The primordial follicle ratios were higher in the FP group (38.5%) on Day 2, KC group (43.0%) on Day 7, and FP and KC groups (31.7% and 31.8%) on Day 21. Antral follicles began to appear in the BM (0.9%) and KC (2.2%) groups initially on Day 2. However, the ratios of antral follicles increased in all the groups, except in the SC group, and there was no significant difference with the sham control group on Day 21.
3.3. Morphologic analysis of the grafts Figure 2A shows the proportion of the morphologically intact (G1) follicles among the groups. On Day 2, there was no significant difference in G1 follicle ratios between the four grafting groups, whereas all showed low G1 follicle ratios when compared with the sham group (sham: 61.3%, BM: 35.3%, FP: 37.7%, KC: 32.2%, and SC: 31.5%). The BM, KC, and FP
Fig. 2. Comparisons of the normality of ovarian tissues and their functions between the sham control and four different transplantation sites according to the durations after transplantation: (A) the intact (G1) follicle ratios, (B) the apoptotic follicle ratios, and (C) the serum FSH levels. Columns with different superscripts differ significantly (P < 0.05).
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groups recovered their morphologic integrity on Day 7, and only the SC group presented a significant decrease in G1 follicle ratios (sham: 64.5%, BM: 63.4%, FP: 62.1%, KC: 63.1%, and SC: 47.6%). On Day 21, the G1 follicle ratios of the FP and KC groups were higher than the sham control group (sham: 63.0%, BM: 59.5%, FP: 74.0%, KC: 71.9%, and SC: 70.9%).
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Fig. 3B). However, the ratios of normal spindle did not differ among the five groups (sham: 71.1%, BM: 56.0%, FP: 53.8%, KC: 64.1%, and SC: 57.1%; Fig. 3C). Figure 4 shows the immunofluorescence staining of a-tubulin in the meiotic spindle and the chromosomes in the nucleus of mature oocytes obtained from the sham OT and four OT grafts.
3.4. Apoptosis 4. Discussion Apoptosis was evaluated by TUNEL assay in a total of 961 follicles for Day 2, 874 follicles for Day 7, and 609 follicles for Day 21 from the five groups (Fig. 2B). The proportion of apoptotic follicles of the four OT graft groups was higher than in the sham group on Day 2 (sham: 5.9%, BM: 16.4%, FP: 17.8%, KC: 14.6%, and SC: 17.3%), followed by a significant decrease in the KC group (7.6%) and an increase in the SC group (23.8%) on Day 7 (sham: 4.4%, BM: 12.5%, and FP 15.1%). However, no differences among the five groups were observed on Day 21 (sham: 9.2%, BM: 8.4%, FP: 7.8%, KC: 8.3%, and SC: 7.2%). 3.5. FSH level We measured serum FSH levels to verify the recovery of ovarian function. As shown in Figure 2C, the serum FSH levels were significantly increased in all grafting groups on Day 2. On Day 7, only the SC group showed the high FSH level (5.5 ng/mL) compared with the other groups (1.6– 2.5 ng/mL) followed by no sign of significant difference among the groups on Day 21. The overall FSH levels were decreased as the OTT durations increased, except in the SC group on Day 7. 3.6. Evaluation of oocytes The OTs of five groups were collected on Day 21 after grafting. The numbers of ovarian grafts used for oocyte collection were 10, 12, 15, 13, and 12 from Sham, BM, FP, KC, and SC groups, respectively. The mean numbers of oocytes from OTs after ovulation stimulation were the highest in the KC group (10.4 2.3), except in the sham control group (15.8 4.8), and the lowest in the SC group (2.1 0.9; BM: 4.6 1.6 and FP: 3.5 1.3; Fig. 3A). The ratios of mature oocytes were also significantly higher in the sham (75.3%) and KC (60.7%) groups (BM: 45.5%, FP: 25.0%, and SC: 28.0%;
Xenotransplantation of human OT to mice is a very useful method for the assessment of malignancy recurrence, evaluation of ovarian function recovery, and elucidation of revascularization mechanisms after OTT with or without cryopreservation [17,25–28]. Furthermore, this murine transplantation model can provide the opportunities to improve grafting conditions for future reproductive medicine application. To date, several murine models and techniques for transplantation procedures have been developed. However, the most effective site for OT grafting has not been well established. Therefore, we evaluated and compared the outcomes of OTT in four grafting sites (BM, FP, KC, and SC). The OT grafts suffered from hypoxia and ischemic damage should undergo large amount of follicle loss during transplantation process, so it is required to shorten the ischemic duration to maintain the ovarian pool. In our study, the mean follicle numbers were comparable with those of the sham control group in KC group on Day 7 and BM and KC groups on Day 21, and these two sites were known to have good angiogenic conditions for OTT [9,18]. Therefore, it is suggested that the quick revascularization helped the KC and BM sites to comparatively well preserve their follicles. Whereas, the SC group showed constantly low mean numbers of follicles on Days 7 and Day 21 among the groups, which may be because of the relatively poor vascularization, stressful environment close to outside, and spacious SC pocket, which enable the OT to move [7,9]. Primordial follicles are resistant to the hypoxic environments. However, antral follicles are easily degenerated after grafting. Several studies reported that the absence of antral and secondary follicles in OT grafts could activate the primordial follicles and accelerate their growth because of the lack of inhibitory factors released by the antral follicles. Thus, rapid emergence of antral follicles appears to be
Fig. 3. Evaluation of oocytes retrieved from the ovarian tissues of the sham control and four grafts groups: (A) mean number of oocyte, (B) mature oocyte ratios, and (C) normal spindle ratios. Columns with different superscripts differ significantly (P < 0.05). BM, back muscle; FP, fat pad; KC, kidney capsule; SC, subcutaneous.
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Fig. 4. Immunofluorescence staining of a-tubulin showing morphology of meiotic spindle organization (green) and chromosome alignment (blue) in mature oocytes obtained from ovarian tissue grafts (400). Typical barrel-shaped microtubules traversing between both poles and centrally aligned chromosomes were considered as normal (A, B), and any other configurations were considered as abnormal (C, D). (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
important to maintain quiescent follicles [29]. In this study, the antral follicles were seen in the BM and KC groups on Day 2 firstly, but no antral follicle was observed in the FP and SC groups at that time. Then, the proportion of antral follicles increased as the grafting duration increased in all OTT groups; however, the SC group showed the lowest antral follicle ratio in all the durations among them. These conditions seem to affect their FSH levels. On Day 2, the serum FSH levels of all four OTT groups were significantly increased when compared with the control group followed by being decreased on Day 7 including the FP group but except the SC group. On Day 21, the four OTT groups including the SC group showed decreased FSH levels similar to that of sham control group. It can mean that OTs of all grafting were well transplanted and adapted to their grafting sites on Day 21, and so, the ovarian functions were recovered. We found that once the OTs were perfused into the recipient body, the FSH levels became decreased to the normal range similar to the control group, although there were significant differences in follicle integrity and/or oocyte maturity. Therefore, it is suggested that FSH measurement is necessary to confirm ovarian function recovery; however, more exact evaluations should be performed additionally to verify the results. Several studies reported that increased FSH levels were usually
observed in the OT grafting models during reestablishment of follicle development, which is possibly because of a deficiency of inhibin A produced by the growing follicles [30,31]. In the other words, when the OTs were perfused into the grafting site and develop the antral follicles after recovering their function, the FSH levels could be decreased to the normal range as shown in our outcome. The SC group also showed the lowest G1 follicle ratio and the highest apoptotic follicle ratio among the four grafting groups on Day 7. Therefore, it is suggested that the SC group needs more time to settle into the site, and the retardation of OT perfusion can induce some defects of OTs in SC site. The purpose of OTT is to obtain many intact oocytes; therefore, the assessment and comparison of oocyte quantity and quality are essential. We collected oocytes from the sham control ovaries and four OT graft groups 10 hours after hCG injection, followed by in vitro maturation for 4 hours. Usually, the oocytes were collected from ovaries 12 to 14 hours after hCG injection. However, the OTs in this study were transplanted into the four heterotopic sites which did not have oviduct. Therefore, we have collected the oocytes from OT grafts 10 hours after hCG injection before the ovulation time by mechanical dissection and performed IVM of oocytes for 4 hours. We have
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compared the oocyte collection time (10, 12, and 14 hours after hCG) and confirmed that the highest number of oocytes were obtained from the OT grafts 10 hours after hCG injection (unpublished data). The best outcome for the retrieved oocyte numbers and oocyte maturation ratios was achieved in the KC group among the four OTT groups, and the SC group together with the FP group have produced comparatively few oocytes and mature oocytes, which corresponded with the result of the evaluation of OT grafts. In conclusion, our study showed that the KC was the optimal site for OT grafting in the murine model, whereas the SC site was unfavorable for this procedure. And, we confirmed that the different OTT sites caused the different outcomes. Considering the results of this study, we recommend the KC site to optimize the experimental conditions and perform OT xenotransplantation for various purposes. Acknowledgments This study was supported by a grant (No. HI12C0055) from the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea. The first two authors contributed equally to this work. Competing Interests The authors have no conflict of interest to declare. References [1] Meirow D, Baum M, Yaron R, Levron J, Hardan I, Schiff E, et al. Ovarian tissue cryopreservation in hematologic malignancy: ten years’ experience. Leuk Lymphoma 2007;48:1569–76. [2] Von Wolff M, Donnez J, Hovatta O, Keros V, Maltaris T, Montag M, et al. Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapyda technique in its infancy but already successful in fertility preservation. Eur J Cancer 2009;45:1547–53. [3] Liu J, Van der Elst J, Van den Broecke R, Dhont M. Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod 2002;17:605–11. [4] Wang X, Huifang CH, Hang Y. Cryopreservation: fertility after intact ovary transplantation. Nature 2002;415:385. [5] Dissen GA, Lara HE, Fahrenbach WH. Immature rat ovaries revascularized rapidly after autotransplantation and show a gonadotrop independent increase in angiogenic factor gene expression. Endocrinology 1994;134:1146–54. [6] Gosden RG, Boulton MI, Grant K, Webb R. Follicular development from ovarian xenografts in SCID mice. J Reprod Fertil 1994;101:619–23. [7] Torrents E, Boiso I, Barri PN, Veiga A. Applications of ovarian tissue transplantation in experimental biology and medicine. Hum Reprod Update 2003;9:471–81. [8] Eyck ASV, Bouzin C, Feron O, Romeu L, Langendonckt AV, Donnez J, et al. Both host and graft vessels contribute to revascularization of xenografted human ovarian tissue in a murine model. Fertil Steril 2010;93:1676–85. [9] Dath C, Eyck ASV, Dolmans MM, Romeu L, Vigne LD, Donnez J, et al. Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites. Hum Reprod 2010;25:1734–43. [10] Maltaris T, Beckmann MW, Binder H, Mueller A, Hoffmann I, Koelbl H, et al. The effect of a GnRH agonist on cryopreserved human ovarian grafts in severe combined immunodeficient mice. Reproduction 2007;133:503–9.
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