Hum. Reprod. Advance Access originally published online on May 3, 2007
Human Reproduction 2007 22(6):1603-1611; doi:10.1093/humrep/dem062
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Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice
1 Gynecology Research Unit, Université Catholique de Louvain, 1200 Brussels, Belgium 2 Department of Urology, Université Catholique de Louvain, 1200 Brussels, Belgium
3 Correspondence address. Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium. Tel: +32-2-764-95-01; Fax: +32-2-764-95-07; E-mail: donnez{at}gyne.ucl.ac.be
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Abstract |
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BACKGROUND: Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days.
METHODS AND RESULTS: Single pieces of tissue from cryptorchid testes (2–9 mm3) of young boys (2–12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting.
CONCLUSIONS: Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.
Key words: cryopreservation/spermatogonia/testicular tissue/xenografting
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Introduction |
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Thanks to recent advances in oncological treatments, cure rates of childhood cancers are as high as 80% (Aslam et al., 2000
). Unfortunately, these treatments may prove toxic to the gonads. As it is estimated that, by 2010, one in 250 young adults (aged 20–29 years) will be a long-term survivor of childhood cancer (Bleyer, 1990), and the preservation of male germline cells in prepubertal boys is becoming an increasingly pressing clinical need.
The gonadotoxic effects of various oncological treatment regimens and the mechanisms leading to stem-cell depletion have already been reviewed (Meistrich, 1993; Meirow and Schenker, 1995; Howell and Shalet, 2005). Stem cells have the ability to self-renew and differentiate to regenerate adult tissue (Meistrich and van Beek, 1993; Weissman et al., 2001) in case of injury.
After a gonadotoxic insult, the restoration of spermatogenesis depends on the availability and integrity of the stem cells, as well as the existence of functional stem-cell niches. Loss of fertility will inevitably have a major impact on the future quality of life of young cancer patients and therefore needs due consideration. While the freezing of spermatozoa is a routine clinical practice for adults and adolescents, this option cannot be applied to prepubertal boys. Indeed, in immature testicular tissue, the seminiferous epithelium contains only Sertoli cells and different types of spermatogonia, among which the stem cells are found.
Because of the absence of mature gametes, cryopreservation of immature tissue, either in the form of a cell suspension or whole pieces of tissue, is the only way of preserving fertility in young boys. Several studies have investigated the freezing of isolated testicular cell suspensions in animals (Avarbock et al., 1996; Brinster and Nagano, 1998; Izadyar et al., 2002), as well as humans (Brook et al., 2001), and progeny from frozen rodent-derived stem cells (Kanatsu-Shinohara et al., 2003) have also been documented. Although this technique has proved successful for fertility restoration, even in larger animals (Honaramooz et al., 2003) when isolated non-frozen cells are transplanted, only 5–12% of these cells are able to recolonize the seminiferous tubule after autologous germ cell transplantation (Dobrinski et al., 1999; Nagano, 2003; Ogawa et al., 2003).
In addition, successful stem-cell transplantation requires functional recipient stem-cell niches (Ogawa et al., 2005). Because of the poor recolonization yield obtained with isolated cell transplantation, and since we cannot guarantee a remaining functional stem-cell niche in all cases of previous gonadotoxic insult, whole tissue cryopreservation, which preserves the microenvironment of the seminiferous tubule and interactions between germ and somatic cells throughout spermatogenesis, could be a better option. Moreover, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. In humans, there are only two publications on the cryopreservation of whole testicular tissue pieces. Both report a protocol that preserves good tissue integrity after freezing (Keros et al., 2005; Kvist et al., 2005), but no quantification or evaluation of functional capacity was carried out.
The present study was, therefore, designed to assess the survival and proliferation of spermatogonia and Sertoli cells after whole tissue cryopreservation and xenografting to nude mice, in order to evaluate the feasibility of immature testicular tissue cryobanking for later clinical application. There has been little research to date on the grafting of human tissue (Geens et al., 2006; Schlatt et al., 2006) and, to the best of our knowledge, this is the first report on the transplantation of cryopreserved immature human testicular tissue.
Our end points include histological evaluation of seminiferous tubule integrity, fibrosis and sclerosis of the graft, and immunohistological quantification and proliferative activity assessment of spermatogonia and Sertoli cells.
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Material and Methods |
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Design of the study
This study was designed to investigate the preservation and proliferation capacity of residual spermatogonia and Sertoli cells after cryopreservation and grafting. For this purpose, testicular tissue from 11 prepubertal boys was cryopreserved and xenografted for 21 days to the scrotum of immunodeficient nude mice. A sample of fresh tissue from each patient was used for identification of spermatogonia in the cryptorchid tissue and for controlled comparison of the number of spermatogonia and Sertoli cells before and after the freezing/thawing/grafting procedure. In fresh tissue and corresponding cryopreserved and grafted tissue, MAGE A4 and vimentin (a Sertoli cell marker) were used to clearly identify spermatogonia and Sertoli cells, respectively, and Ki67 to mark proliferative cells. Double-immunostaining for Ki67 and vimentin was applied to distinguish the proliferative activity of Sertoli cells from spermatogonia.
Sources of human testicular tissue
Immature testicular tissue was obtained from 11 boys aged between 2 and 12 years. All of them were undergoing unilateral orchidopexy for cryptorchidism. In all cases, the testes were located inguinally.
As standard procedure with this operation, a testicular biopsy was taken to evaluate the testicular histology (number of spermatogonia, absence of malignant cells) (Cortes et al., 2001). Part of the biopsy was used for this study after obtaining informed consent from the parents. Cryptorchid tissue was only considered for cryopreservation and grafting if spermatogonia were detected in the fresh fragment. Identification of spermatogonia was done according to Clermont and Leblond (1959) and by immunostaining with the MAGE A4 mouse anti-human monoclonal antibody purified from hybridoma 57B, kindly provided by Giulio Spagnoli MD (University of Basel, Switzerland) (Yakirevich et al., 2003).
The fragments, measuring 2–9 mm3, were immediately transferred to Falcon tubes containing Hanks balanced saline solution (HBSS) at 4°C and placed on ice.
Each biopsy of immature tissue was divided into two pieces in a Petri dish containing HBSS and placed on ice. The smallest piece was immediately fixed in 5% buffered formol saline for reference histology and immunohistochemistry. The remaining piece was cryopreserved within 10 min of recovery.
All experiments in this study were approved by the Ethical Review Board of the Catholic University of Louvain.
Freezing media and protocol
Testicular tissue samples were frozen using a modified protocol developed for spermatogonial preservation of adult human testicular tissue (Keros et al., 2005).
Dimethylsulfoxide (DMSO) 0.7 mol/l was supplemented with sucrose 0.1 mol/l and human serum albumin (HSA) 10 mg/ml as cryoprotectants. Tissue pieces were placed in 1 ml freezing media at 4°C in a 2 ml cryovial.
Using a programmable freezer (Minicool 40 PC Air Liquide, Marne-la-Vallee, France), the vials were maintained at 0°C for 9 min, cooled at a rate of 0.5°C/min to –8°C and then held for 5 min before seeding manually at –8°C. After holding for a further 15 min at –8°C, a cooling rate of 0.5°C/min was used from –8 to –40°C before final dehydration for 10 min at –40°C. After cooling at 7°C/min to –80°C, the vials were transferred to liquid nitrogen (–196°C).
Thawing
After retrieval from storage (6–51 days), the cryopreserved tissue was kept for 2 min at room temperature, thawed in a water bath at 37°C for 2 min, and then washed three times in a reversed sucrose concentration gradient solution (0.1, 0.05 and 0 M sucrose) for 5 min per bath, using HBSS medium on ice.
Xenografting
Recipient animals and surgical procedure
Five- to eight-week-old male nude mice (NMRI nu/nu, Janvier Laboratories, Le Genest-St-Isle, France) with a deficient T lymphocyte system were used for this study. The guidelines for animal welfare were approved by the Committee on Animal Research of the UCL.
One mouse was housed per cage under filtered hoods in rooms maintained at 28°C with a 12-h light:dark cycle. All housing material, food and water were autoclaved before use. The mice were fed ad libitum on laboratory chow and acidified water. During the experimental period, no hormonal therapy was administered to the mice.
The mice were anesthetized by intraperitoneal injection of ketamine (75 mg/kg; Anesketin®, Eurovet, Heusden-Zolder, Belgium) and medetomidine (1 mg/kg; Domitor®, Pfizer, Cambridge, USA) dissolved in phosphate-buffered saline. The recipients were first castrated. During the same surgery, one piece of donor testicular tissue (1–6 mm3) was grafted into the scrotum without fixation. Because of the poor results obtained with mature human testicular tissue grafts to the back of mice in terms of both graft survival (Schlatt et al., 2006) and graft sclerosis (Geens et al., 2006), the scrotum was chosen as the graft site. In addition, because of the known influence of temperature on spermatogenesis, as suggested by Wistuba et al. (2006) on differentiation, we felt that this site would provide the best model for future long-term grafting.
We previously addressed this issue in a preliminary experiment, in which mature human testicular tissue transplants under the skin of the axillary area were compared to scrotal grafts (Fig. 1). For the axillary grafts, a small skin incision was made and the testicular tissue was placed without suture close to the emergence of the lateral thoracic artery. Due to inferior preservation of seminiferous tubule integrity in this site [51 ± 0.03% versus 89 ± 0.03%; n = 2; P = 0.002 according to the scoring system of Keros (2005)], it was subsequently abandoned.
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The incisions were closed with 7/0 Prolene sutures, and anesthesia was reversed by injection of atipamezole (1 mg/kg; Antisedan®, Pfizer). Analgesia was provided by buprenorphine (0.1 mg/kg, Temgesic®, Schering Plough, Kenilworth, NJ, USA) on the day of surgery and the following day.
Recovery and analysis of donor grafts
Recipient mice were euthanized three weeks after grafting by cervical dislocation. This time interval was chosen to analyse the graft after the initial period of ischemia resulting from the inevitable delay before revascularization is established. Indeed, one of our previous studies on endometriotic implants in a nude mouse model demonstrated gradual human vessel regression and replacement by mouse neovessels up to 21 days after transplantation (Eggermont et al., 2005), and quantitative assessment of revascularization rates in bone grafts showed an increase up to 3 weeks before stabilization (Kirkeby, 1991). The grafts were dissected and fixed in buffered formol saline. After fixation, the grafts were routinely embedded in paraffin and cut into 5 µm thick sections. One section was stained with Mayer's hemalum and eosin for evaluation by light microscopy, and digital images were captured with a digital camera (Leica DFC 320, Zeiss, Germany). In each graft, all seminiferous tubules present in the widest cross-section were examined.
The morphology of the tissue and integrity of the seminiferous tubules were semi-quantitatively evaluated using a scoring method already described elsewhere (Keros et al., 2005). According to this scoring system, tubules with a normal morphology or slight damage were considered good and expressed as a percentage of the total sections examined. Adhesion of cells to the basement membrane, cell cohesion and pyknosis of cell nuclei were observed.
The degree of fibrosis in grafts was quantitatively evaluated by morphometry using computerized image capture. The degree of sclerosis was also measured by morphometry. Lamina propria thickness of 
10 µm in immature tissue (Santoro and Romeo, 2001) was considered normal.
All data were examined and compared to the reference histology (before freezing and grafting) by two blinded and trained observers.
Representative cross-sections of grafts were processed for immunohistochemistry.
The number of spermatogonia and Sertoli cells were evaluated after immunodetection by the MAGE A4 mouse anti-human monoclonal antibody purified from hybridoma 57B, kindly provided by Giulio Spagnoli MD (University of Basel, Switzerland) (Yakirevich et al., 2003), and the vimentin mouse anti-human monoclonal antibody clone V9 (Dako ref M0725), respectively. Their proliferative activity was further assessed by detection of the rabbit anti-human polyclonal antibody Ki67 (ImmunoSource ref K1745–25), directed against the nuclear Ki67 antigen of cells not in G0 of the cell cycle. In order to distinguish spermatogonial proliferation from Sertoli cell proliferation, double-immunodetection for vimentin and Ki67 was undertaken on the same sections using two different systems [alkaline phosphatase (AP) and horseradish peroxidase (HRP)].
Cross-sections were deparaffinized and rehydrated. Endogenous peroxidase activity was then blocked by incubating the samples in 0.3% H2O2 for 30 min at room temperature. Sections were placed in citrate buffer at 98°C for 75 min after washing under deionized water for 5 min, followed by washing in tris-buffered saline (TBS) 0.05 M and 20% Triton X-100. Non-specific antibody binding was subsequently blocked by incubation of samples in 10% non-immune goat serum and 1% bovine serum albumin (BSA) for 30 min at room temperature. The primary antibody (diluted to 1:500 for MAGE and 1:100 for Ki67) was added to the samples and incubated at 4°C overnight in a humidified chamber.
The following day, the samples were washed in TBS 0.05 M and 20% Triton X-100 three times for 2 min each. Secondary anti-mouse (for MAGE) or anti-rabbit (for Ki67) antibody (EnVision + System-Labelled Polymer-HRP; DAKO K4001 and K4003 for MAGE and Ki67, respectively) was added and incubated at room temperature for 1 h, followed by washing in TBS 0.05 M and 20% Triton X-100 three times for 2 min each. Diaminobenzidine (DAKO K 3468) was used as a chromogen (samples were incubated for 15 min at room temperature).
Nuclei were counterstained with hematoxylin after washing under tap water for 3 min.
For the double-immunostaining, Ki67 sections were used just before staining with hematoxylin and washed under tap water for 3 min followed by washing in TBS 0.05 M and 20% Triton X-100 three times for 2 min each. Non-specific antibody binding was blocked by incubation of samples in 10% non-immune goat serum and 1% BSA for 30 min at room temperature. The vimentin antibody, diluted to 1:50, was added to the samples and incubated at 4°C overnight in a humidified chamber.
The following day, the samples were washed in TBS 0.05 M and 20% Triton X-100 three times for 2 min each. Secondary antibody (EnVision + System/AP Rabbit/Mouse; DAKO K4018) was added and incubated at room temperature for 45 min, followed by washing in TBS 0.05 M and 20% Triton X-100 three times for 2 min each. Fast Red TR/Naphthol (Fast Red TR/Naphthol AS-MX tablet sets; SIGMA F4523) was used as the substrate for AP-conjugated antibody (samples were incubated in the dark for 30 min at room temperature). Nuclei were counterstained with hematoxylin after washing under tap water for 3 min. The stained sections were evaluated using light microscopy, and digital images were captured to count the number of marked spermatogonia, Sertoli cells and proliferative cells. The results were presented as the number of cells per cross-sectioned tubule. Mean tubule diameter (µm) was 42.37 ± 6.66 in fresh tissue and 80.17 ± 9.51 after the freezing/thawing/grafting procedure (P = 0.001).
Statistical analysis
Data were tested for normality using Littefors
Analyses were performed using the SPSS 11.5 program. A comparison was made between fresh controls and frozen-grafted tissue.
All data are presented as means ± SD. Statistical differences were analysed using Student's t-test. P 0.05 was considered statistically significant.
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Results |
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Histology
Almost all (91%) testicular xenografts were recovered from the host mice. The histology of the donor tissue at the time of freezing and after the freezing/thawing/grafting procedure is shown in Figure 2.
In fresh tissue, as well as cryopreserved and grafted samples, the immature tissue was composed of primitive seminiferous cords with immature Sertoli cells, spermatogonial cells of different types depending on donor age and interstitial tissue containing immature Leydig cells.
Histological analysis of the recovered immature grafts at 21 days (Fig. 2B) revealed well-preserved integrity of the tubules, with 82.19 ± 16.46% of sections of frozen tissue showing good morphology after grafting, similar to the 93.38 ± 6.00% observed in fresh control tissue samples (P = 0.083).
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Fibrosis was not induced after grafting of frozen-thawed tissue. Indeed, the degree of fibrosis was similar in both fresh controls (12.31 ± 0.08%) and cryopreserved grafts (11.10 ± 0.07%), as demonstrated by morphometric analysis (P = 0.692).
Transplantation of cryopreserved pieces of immature testicular tissue did not induce sclerosis. Indeed, after morphometric analysis, the thickness of the lamina propria was not significantly different between fresh controls (2.04 ± 0.15 µm) and frozen-grafted tissue (2.68 ± 0.23 µm) (P = 0.085).
Immunohistochemistry
Spermatogonial cell immunostaining
As indicated by the persistence of MAGE A4-positive cells in frozen-grafted tissue (Fig. 3B and C), spermatogonial cells were able to survive after cryopreservation and xenografting for three weeks. However, our data reveal a significant loss of spermatogonia after freezing and grafting. Their number decreased from 0.55 ± 0.52 per tubule in fresh tissue to 0.08 ± 0.13 per tubule in frozen-grafted tissue (P = 0.006).
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Sertoli cell immunostaining
As indicated by vimentin (a Sertoli cell marker) immunostaining, Sertoli cells were able to survive after cryopreservation and xenografting for three weeks. The number of Sertoli cells identified was similar, ranging from 41.8 ± 2.61 per tubule in fresh tissue to 47.1 ± 1.20 per tubule in frozen-grafted tissue (P = 0.300).
Cell proliferative activity
After grafting of cryopreserved tissue, we observed an increase in cell proliferation. The number of Ki67-positive cells significantly increased (Fig. 3E and F) from 0.07 ± 0.06 per tubule in fresh tissue to 2.27 ± 2.11 per tubule in frozen-grafted tissue (P = 0.008).
Double-immunostaining of frozen-grafted tissue with vimentin and Ki67 revealed that most proliferating cells were Sertoli cells.
Only 1.13% of proliferative cells were spermatogonia (Fig. 3G). However, 32% of spermatogonia continued to proliferate after freezing and grafting, compared to 17.8% (P = 0.382) in fresh tissue. For Sertoli cells, no proliferative activity was detected in fresh tissue, but Ki67 expression was observed in 5.1% of these cells after freezing and grafting.
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Discussion |
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Cryobanking of immature testicular tissue, either as a cell suspension or as tissue pieces, is the only available option for fertility preservation in young boys at risk of losing their testicular stem cells after radiotherapeutic or chemotherapeutic treatments. Indeed, as these patients do not yet produce spermatozoa for freezing, only immature tissue containing spermatogonia and spermatocytes is available for storage.
Survival rates of 70% were obtained after freezing and thawing of pure populations of type A spermatogonia (Izadyar et al., 2002).
Cryopreservation of testicular tissue is more challenging, however, because freezing of tissues generally requires greater permeation of cryoprotectants than cell suspensions (Leibo and Mazur, 1971). In addition, different cell types have different requirements, so early attempts to freeze testicular tissue pieces met with limited success (Nogueira et al., 1999).
Nevertheless, in animals, the technique has proved successful with the reported birth of offspring following transplantation of cryopreserved immature testicular tissue, and in vitro microinsemination in mice (Shinohara et al., 2002).
Very recently, successful cold storage and cryopreservation of immature primate testicular tissue prior to xenografting were reported (Jahnukainen et al., 2006). The authors showed that spermatogonial stem cells retained their capacity to reinitiate spermatogenesis, which has important implications for future clinical use.
As far as we know, ours is the first study to demonstrate that human spermatogonia are able to survive and grow in vivo after cryopreservation and xenografting of testicular tissue pieces obtained from young boys. A slow cooling protocol using 0.7 M DMSO supplemented with 0.1 M sucrose and HSA was applied. DMSO was used at low concentrations because better spermatogonial survival was demonstrated in adult tissue with this particular cryoprotectant by Keros et al. (2005). In their study, the assessment of cryoinjury was based on histological data, ultrastructural data and testosterone production in in vitro culture of adult human tissue (Keros et al., 2005).
In case of immature neonatal human tissue, only one reported study on cryptorchid testes has investigated whole tissue cryopreservation (Kvist et al., 2005). The authors observed spermatogonial survival after thawing and in vitro culture, as well as the ability of the tissue to produce testosterone in in vitro culture, but no quantification of the spermatogonia or evaluation of their functional capacity after cryopreservation was carried out.
To evaluate whether cryopreserved tissue retains its capacity for spermatogonial proliferation, we used a nude mouse xenografting model. Because this model will subsequently be used for the evaluation of differentiation during long-term grafting of normal immature tissue after this initial evaluation of the early postgrafting period, the scrotum emerged as the most promising localization. Indeed, it was hypothesized that hyperthermia was responsible for the spermatogenic arrest observed when ectopic autologous transplantation of immature testicular tissue was performed in marmosets (Wistuba et al., 2006). No comparisons have ever been made between different grafting sites for immature testicular tissue in primates. However, neonatal testes from rats have been transplanted into the anterior chamber of the eye, into the muscle, under the skin of the thoracic region, into the scrotum (Turner, 1938) and into the tips of the outer ears (Chan et al., 1969). Graft recovery rates obtained were respectively 90, 88, 55, 38 and 50%, but complete spermatogenesis was only observed in the two latter sites, again suggesting a negative influence of higher temperatures on grafts. This hypothesis may also explain the poor results observed in our preliminary experiment with axillary grafts. Our aim in this study was not to investigate germ cell differentiation, because it could be compromised by the fact that we used cryptorchid tissue, but to set up a xenografting model, which could be used for studies on normal immature human testicular tissue for longer grafting periods. We therefore chose a site where better differentiation could be obtained, rather than a higher graft recovery rate.
The potential of adult human testicular tissue to survive in fresh ectopic xenografts to mice has already been studied. (Geens et al., 2006; Schlatt et al., 2006). In the former study, poor recovery and survival rates of grafts were reported, but spermatogonial survival was nevertheless observed. In the latter study, some isolated spermatogonia were observed in 21.6–23.1% of grafts, and severe sclerosis was present in most of the grafts (69.2 and 57.4% for SCID-NOD and Swiss nude mice respectively), increasing with time after grafting. In both studies, tissue was grafted under the skin of the back and no differences were observed between nude and SCID mice. Apart from graft localization and the potentially deleterious effect of hyperthermia with grafts to the back, it should be stressed that this work on fresh human tissue utilized only mature tissue, while our study used immature human tissue grafted to the scrotum of nude mice.
As normal immature testicular tissue is difficult to obtain, cryptorchid tissue was used. We demonstrated that the overall morphology of the frozen-thawed tissue did not show any major freeze injuries in terms of tubule integrity, fibrosis or sclerosis after a 3-week grafting period. Moreover, the structural and functional characteristics of spermatogonial and Sertoli cells were preserved, as demonstrated by their proliferative capacity within seminiferous tubules.
Besides the favorable scrotal localization of the grafts, the apparently better preservation of tissue after freezing and grafting in our study may also be related to the immaturity of our tissue. Indeed, in rodents, it was reported that the establishment of spermatogenesis was better with immature donors (Schlatt et al., 2002), and in various animal donor species, differentiation of cryopreserved immature grafts (Honaramooz et al., 2002; Schlatt et al., 2002; Shinohara et al., 2002; Ohta and Wakayama, 2005) and fresh immature grafts (Honaramooz et al., 2002, 2004; Schlatt et al., 2002, 2003; Shinohara et al., 2002; Snedaker et al., 2004; Geens et al., 2006; Rathi et al., 2006; Yu et al., 2006; Zeng et al., 2006) in a mouse host resulted in complete spermatogenesis. This is further substantiated by the fact that we found a significant increase in tissue fibrosis (89.3%) and sclerosis (mean thickness of lamina propria: 19.93 ± 5.02 µm) after cryopreservation and grafting of adult human testicular tissue, compared to fresh tissue in a preliminary experiment using the same model (data not shown). Moreover, it has already been suggested that immature tissue could be better equipped to survive periods of ischemia or have greater capacity for angiogenesis in the host than adult tissue (Schlatt et al., 2002).
Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting.
Our results are presented as the number of cells per tubular section. We observed an increase in the mean tubular diameter after freezing and grafting, compared to fresh tissue. Whether this difference was due to the freezing or grafting procedure is unclear, but preliminary data have not revealed a modification in tubular size after cryopreservation (49.8 ± 11.58 µm in fresh tissue and 47.07 ± 3.81 µm in frozen-thawed tissue; n = 2; P = 0.736). It does not appear to result from an increase in the number of cells in the seminiferous tubules, as there was no difference in the number of Sertoli cells (P = 0.300), while spermatogonia numbers declined (P = 0.006). Nevertheless, we observed vacuolization in the tubules after freezing and grafting, which could account for this phenomenon, but the exact origin should be assessed by ultrastructural studies.
Interestingly, we observed increased proliferative activity in Sertoli cells after recovery of the grafts at 21 days. To establish whether this can be attributed to the freezing or grafting procedure, further studies are required. However, preliminary data on frozen prepubertal cryptorchid tissue appear to indicate that there is no increase in the number of Sertoli cells just after cryopreservation (31.95 ± 2.61 per tubule in fresh tissue and 42.95 ± 4.31 per tubule in frozen-thawed tissue; n = 2; P = 0.267). On the other hand, an increase in Sertoli cell number was previously reported after transplantation of fresh (non-frozen) prenatal and neonatal rat testes to a mouse host (Johnson et al., 1996). Considering that the number of Sertoli cells produced before puberty will determine the number of germ cells that can be supported through spermatogenesis, and hence the extent of sperm production in adulthood (Orth et al., 1988), understanding the factors involved in this increased Sertoli cell proliferation could prove vital.
Animal studies have conclusively demonstrated the crucial role of FSH in determining the mitotic proliferation of Sertoli cells (Meachem et al., 1996; Allan et al., 2004), and hemicastration of neonatal rats has been shown to induce hypertrophy in the contralateral testis (Cunningham et al., 1978), suggesting an increase in Sertoli cell number coincident with elevated levels of serum FSH. Thus, it is likely that the rapid multiplication of Sertoli cells after grafting is associated with raised FSH levels after mouse castration. The removal of some inhibitory mechanisms which normally operate in quiescent immature testes and/or other paracrine factors may also play a role in Sertoli cell multiplication.
Our observations also raise the question of whether induction of this proliferative activity in the graft is similar to the physiological proliferation waves observed in the seminiferous tubules during fetal, neonatal and peripubertal life (Sharpe et al., 2003) with, consequently, the same impact on the germ cell population. If so, we should observe an increase in spermatogonial proliferation. On the contrary, our results show a decrease in the number of spermatogonial cells after recovery of the grafts at 21 days and no significant difference in proliferative activity after cryopreservation and grafting (32.2%) compared to fresh tissue (17.8%). Again, whether this loss is related to an effect of cryopreservation or to the grafting procedure needs to be investigated. However, preliminary data on frozen prepubertal cryptorchid tissue indicate that freezing does not appear to be responsible for any spermatogonial loss (0.45 ± 0.35 per tubule in fresh tissue and 0.71 ± 0.89 per tubule in frozen-thawed tissue; n = 2; P = 0.267).
In a study on immature bull testicular tissue ectopically grafted to mouse hosts, the number of germ cells decreased significantly in grafts recovered between 1 and 2 months' post-transplantation (Rathi et al., 2005). The authors did not find any difference in germ cell apoptosis during this period compared to in situ controls. Beyond two months, however, the grafts showed an increase in the number of germ cells, but it remained significantly lower than in corresponding in situ controls. The initial lack of a blood supply was evoked as a reason, but the germ cells never appeared to recover from this deficit.
In rodent studies, apoptotic germ cell death was induced by decreasing both serum and intratesticular testosterone concentrations (Brinkworth et al., 1995; Henriksen et al., 1995). These studies are consistent with an in vitro study on adult human testicular tissue (Erkkila et al., 1997). Indeed, testosterone appears to be a critical and dose-dependent germ cell survival factor. The absence of intratesticular secretion of testosterone in our immature tissue at the time of grafting and the decreasing serum testosterone levels in the castrated mice could be factors involved in the observed spermatogonial loss. Ischemia induced by the freezing, thawing and grafting procedures and/or other unknown modulators may also be involved. Ki67 positivity, expressed in very few spermatogonial nuclei, simply implies that they are renewing but not differentiating. To further evaluate spermatogonial proliferation and differentiation potential, the grafting period should be extended.
Before clinical implementation of the technique, a number of questions relating to the life span of the grafts, the optimal size of pregrafted fragments, the influence of local testosterone and the need for gonadotropin supplementation must also be resolved.
The model used in this study could be a useful tool to address some of these pertinent questions, but should be extended to investigate grafting of normal immature human testicular tissue for longer periods. The use of cryptorchid tissue limits the conclusions that can be drawn from this study. Indeed, as an initially abnormal fetal environment is involved in the pathophysiology of cryptorchidism, and the functioning of germ cells is impaired by their potential for malignant transformation and incapacity to differentiate, data on spermatogonial cell functionality must be obtained from normal grafted immature testicular tissue. The short observation period in our study may have constituted a potential limitation if normal immature human tissue had been used, but such tissue was not readily available. We therefore focused our attention only on spermatogonial and Sertoli cell survival and proliferation following the initial period of ischemia and subsequent revascularization in the early post-grafting period (Kirkeby 1991; Eggermont et al., 2005).
In conclusion, through our in vivo orthotopic xenografting model, we have demonstrated the survival and proliferative capacity of both spermatogonial and Sertoli cells after cryopreservation of immature human testicular pieces. Further studies must, however, be conducted on normal immature human tissue to investigate its capacity to complete spermatogenesis after freezing and xenografting. Ethical and safety considerations must also be taken into account before cryopreserved immature testicular tissue grafting can be clinically implemented. One important issue that needs to be addressed is the potential risk of neoplastic cell contamination of cryopreserved tissue, which would contraindicate autotransplantation. In such cases, whole tissue pieces frozen according to our cryopreservation protocol could still be used for enzymatic digestion and purification of spermatogonial cells, eliminating neoplastic cells through magnetic cell sorting, as demonstrated in animal studies (Fujita et al., 2005) before transplantation of isolated cell suspensions. In addition, well designed and properly conducted studies on germ cell transplantation and testicular tissue grafting are required to evaluate the advantages and disadvantages of both potential clinical applications and establish their respective indications. For safety reasons, mainly unknown zoonosis risks, we stress the fact that cryopreserved tissue should not be used in clinical applications to produce human gametes through xenografting. At the moment, only autotransplantation of a patient's own tissue can be considered after oncological remission, and only if further studies demonstrate its capacity to reinitiate spermatogenesis.
Tissue banking may nevertheless be considered a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments.
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Acknowledgments |
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This study was supported by grants from the FNRS (Fonds National de la Recherche Scientifique), Televie: grant No. 7.4619.05, Fondation St. Luc, Belgian Federation Against Cancer, Baron Albert Frére and Comte Philippe de Spoelberch.
The authors are grateful to Dr Giulio Spagnoli for supplying the MAGE A4 antibody.
The authors thank Mira Hryniuk, B.A., for reviewing the English language of the manuscript. We also thank the Department of Anatomopathology of the UCL for their advice on the setup of double immunostaining and for specimen embedding.
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