Autologous Ectopic Grafting of Cryopreserved Testicular Tissue Preserves the Fertility of Prepubescent Monkeys That Receive Sterilizing Cytotoxic Therapy

Prepubertal boys facing a sterilizing cancer therapy cannot benefit from sperm banking. A potential alternative strategy for preserving their fertility involves cryopreservation of immature testicular tissue (1–3). Studies with nonhuman primates have revealed that fresh prepubertal testicular tissue survives well when xenografted into nude mice, and is able to differentiate to full spermatogenesis (4, 5). The testicular tissue can be removed from the grafting site, and sperm can be retrieved from the tissue. Xenogenic rhesus sperm generated in host mice have shown to be fertilization competent, allowing in vitro embryo development (4). This is contrary to adult testicular tissue that undergoes almost complete degeneration after grafting (6, 7).

Successful long-term survival and proliferation of human spermatogonia has been reported after xenografting of cryopreserved immature testicular tissue (8–10). However, these studies have failed to confirm the preservation of the functionality of spermatogonial stem cells in the cryopreserved tissues. Spermatogenic arrest at the pachytene spermatocyte stage occurred in cryopreserved testicular tissue for both humans and rhesus monkeys after xenotransplantation into an immunodeficient murine host (1, 9). Maximal preservation of spermatogonial stem cells in functional testicular tissue and an adequate transplantation or xenografting procedure are key factors to guarantee success of future fertility preservation approaches using testicular tissue transplantation and xenografting. In the terms of fertility preservation, all previous grafting strategies may be considered unsuccessful.

The reasons why the outcome after grafting of cryopreserved human and monkey xenografts has been poor, so far, remain unknown. It is possible that the cryopreservation protocols were not optimal and that the xenografting is leading to incomplete germ cell maturation due to differences between host and donor gonadotropic hormones (11), local testosterone levels within xenografts, or incompatibilities between the primate germ cells and the murine-supporting Sertoli cells (4, 12). Many, thus far, unexplored factors may affect success of germ differentiation in xenografted primate testis tissues. The effects of cold and warm ischemia before the reestablishment of blood supply to the grafts may also have relevant impact and to be explored would have required short-term studies. Warm ischemia may well depend on the site of grafting due to different degrees of vascularization at various grafting sites.

To evaluate if autologous grafting might serve as a fertility preservation method after testicular irradation and (hemi) castration, ectopic autologous grafting of cryopreserved testicular tissues from prepubertal and pubertal rhesus monkeys was used. Spermatogenic differentiation in autologous grafts was compared with that in xenografts in immunodeficient mouse host.

Five male rhesus monkeys aged 30 to 49 months (Macaca mulatta) from the University of Pittsburgh Plum Boro primate facility (Pittsburgh, PA) were used for the study. All animal experiments were approved by and carried out under the guidance of the Institutional Animal Care and Use Committee of the University of Pittsburgh School of Medicine. This study is a continuation of an irradiation study reported earlier (3).

One of the testes in each animal was biopsied under general anesthesia using isoflurane. In one monkey (#3199), unilateral orchidectomy was carried out. At the time of tissue collection, the testicular tissue from donor monkeys #3199, #3082, #3213, and #3117 consisted of seminiferous cords with preleptotene spermatocytes as the most advanced germ cell type and donor monkey #3212 showed full spermatogenesis as described earlier (3). The collected testicular tissue was dissected into small fragments (∼1 mm³). Altogether, 140 tissue fragments from monkey #3199; 10 fragments from monkeys #3082, #3212, and #3213; and 5 fragments from monkey #3117 were cryopreserved for tissue grafting experiment. Tissue fragments were kept in Dulbecco's Modified Eagle's Medium high glucose (4 g/L) containing 10% fetal calf serum, nonessential amino acids, and antibiotics on ice. Dimethylsulfoxide (DMSO) was added dropwise to the suspension to a final concentration of 1.4 mol/L. The tissue fragments were then transferred into cryovials and submitted to a slow-freezing protocol (1).

Six weeks later, in March 2006, testicular irradiation was carried out at the University of Pittsburgh Medical Center Presbyterian Hospital. The animals were castrated in March and April 2008, except monkey #3117, which was castrated in October 2008.

In June 2008, the testicular grafts were thawed in 37°C water bath for 2 minutes and were washed in fresh ice-cold medium. By using cancer implant G13 needle (Popper Precision Instruments), 5 testicular fragments were placed subcutaneously into the left side of the scrotum, and 5 under the skin on the left shoulder for monkeys #3082, #3212, and #3213. In monkey #3199, 30 testicular fragments were placed into the left side of the scrotum and 30 into the right side. Also 30 grafts each were placed subcutaneously on the shoulder and left arm. In monkey #3117, 5 grafts were placed subcutaneously on the left shoulder only. Five months later, in October 2008, monkeys were fully anesthetized again, and the grafts were dissected from under the skin and fixed in Bouins solution overnight.

Three adult (10–15 weeks old) immunodeficient male nude mice (Crl:Nu/Nu-nuBR; Charles River Laboratories) were used as recipients. Mice were castrated and 6 testicular fragments from monkey #3199 were placed in each mouse under the dorsal skin on either side of the dorsal midline by using cancer implant G13 needle (Popper Precision Instruments). Five months later, the mice were anesthetized and killed by exsanguination. The back skin was removed and grafts were dissected from the skin and fixed in Bouins solution.

The grafts were fixed for 18 to 24 hours and, washed and stored in 70% ethanol, and embedded in paraffin for serial sectioning at 4 μm throughout the entire graft. Tissue sections were stained with periodic acid-Schiff's reagent and Gill's hematoxylin and examined under the light microscope. Graft survival was defined by light microscopic observation of seminiferous tubules with Sertoli cells in the retrieved graft. The serial section showing best tissue survival was selected for analysis. The tubular diameter, fertility index (FI; percentage proportion of tubules containing spermatogonia), spermatogenic repopulation index (percentage proportion of tubules containing B-spermatogonia ore more advanced germ cell types), most advanced germ cell type, and percentage of tubules with the most advanced germ cell type were recorded in 50 tubular cross sections selected for analysis by random systematic sampling. All cross sections of seminiferous tubules were analyzed from smaller grafts, which did not contain 50 tubular cross sections. The identification of type A dark (Ad) and A pale (Ap) spermatogonia followed the scheme of Clermont and Leblond (13). The light microscopic determinations were conducted by one observer (K. Jahnukainen).

Five months after autologous grafting, 5% (8/155) of the cryopreserved grafts were recovered. No difference in graft survival was detected between different grafting sites. Altogether, 7% grafts (5/75) from scrotal, 4% (2/50) from shoulder, and 3% (1/30) from arm contained active testicular tissue. Survival of xenografts in the back skin of immunodeficient mice was 6% (1/18) for the same time period.

Five months after autologous grafting in the scrotum, 2 of 5 recovered grafts showed fully matured spermatogenesis (Fig. 1A and B). The 3 other scrotal autologous grafts had round spermatids and pachytene spermatocytes as the most advanced germ cell stages. Each graft that was recovered from other locations than from scrotal skin showed some germ cell maturation. However, maturation in these grafts did not reach beyond pachytene spermatocyte stage. The surviving xenograft that was recovered from immunodeficient mouse showed Sertoli cell-only (SCO) pattern and no germ cells. Table 1 summarizes the basic morphometric data of the recovered grafts.

In all autologous grafts, a morphologically normal interstitium with blood vessels, macrophages, peritubular, and Leydig cells was detected (Fig. 1A). The recovered xenograft showed interstitial infiltration with macrophages (Fig. 1C).

The present results provide first unequivocal evidence that cryopreservation of prepubertal primate testicular tissue maintains the functional capacity of spermatogonia and testicular tissue to produce sperm after grafting. Spermatogenesis up to level of spermatozoa was detected in the cryopreserved grafts autologously grafted into scrotal skin of the rhesus monkey. Testicular tissue that produced sperm was cryopreserved at the time when the donor has still been prepubertal (3). Grafting was carried out 2 years later when the donor was adult, but sterile, because of the effects of testicular irradiation and bilateral castration. Transferring our experimental scenario into a clinical setting, 2 subcutaneous testicular grafts containing seminiferous tubules with spermatozoa would have easily enabled assisted reproductive technology specialists to obtain sperm by testicular sperm extraction and to conduct intracellular sperm injection. Therefore, our study reveals a proof-of-principle for fertility preservation via grafting of testicular tissue in a sterile patient.

A major factor that guaranteed successful outcome by autologous grafting was the large number of cryopreserved grafts available in monkey #3199. Survival of the grafted tissue was low (3%–7%) independent of the site where the grafts were placed. Only the removal of one entire testis by hemicastration provided sufficient material to collect sperm. Altogether, 110 testicular fragments were cryopreserved from this single prepubertal testis. From the other 5 donors, small biopsies (20–40 mg of tissue) were collected and only 5 to 10 tissue fragments were cryopreserved. One of these donors had surviving testicular grafts but these did not contain fully matured sperm. No grafts were recovered from the donor that had shown intiation of puberty and spermarche before the cryopreservation of grafts (3). Present observations are in accordance with a previous autologous grafting experiment with marmoset using a low number of autologous grafts (14). None of the 7 cryopreserved grafts retransplanted in each marmoset were recovered after a 10-month period. A large amount of testicular tissue is therefore a critical factor to guarantee success of future fertility preservation with testicular tissue transplantation.

In this study, spermatogenic maturation beyond meiosis was seen only when ectopic autologous grafting was carried out into the scrotum. This was observed in all 3 grafts with good germ cell survival (FI, 43%–50%). Two other scrotal grafts with lower spermatogonial survival (FI, 2%–6%) had germ cell development up to the pachytene spermatocyte stage. All subcutaneous grafts from other locations showed some degree of spermatogenic recovery but did not develop beyond meiosis. This was despite good survival of spermatogonia (FI, 26%–44%). Our observations are in accordance with the previous autologous grafting study where grafting under the back skin was shown to contribute to spermatogenetic arrest but grafting into the scrotum allowed maturation (14). As previously hypothesized hyperthermia in back skin may be one of the factors responsible for the detected spermatogenic arrest. In this study, no autologous graft with Sertoli cell-only pattern was recovered and in most of the grafts, the spermatogenetic repopulation index was high (20%–70%). Thus, a significant part of surviving grafts showed potential to support spermatogenesis and more SCO tubules would most probably be repopulated with increased grafting time. Our results reveal that cryopreserved autografts survive with an all or nothing pattern. The loss of grafts seems to be the only limiting factor for fertility preservation procedures.

Further studies are needed to explore the reasons for graft disappearance that could be related to the cryopreservation protocol, the ectopic grafting site, graft rejection, or inflammation-like responses eventually induced by ischemia-induced death of cells in the grafts. Cryopreservation of testicular tissue requires adequate penetration of cryoprotectants through the seminiferous tubular membranes and barriers. A slow-cooling protocol using DMSO supplemented with sucrose and human serum albumin enabled survival of human xenografts in a short-term study (15). Improved survival of primate autografts will require further research on optimal protein support and concentrations of cryoprotectants in freezing solution as well as methods for adequate thawing, comparable to studies with ovarian tissue (16, 17). New grafting techniques that promote a fast and efficient graft revascularization will also be required. Orthotopic transplantation into an intratesticular location offers potentially intensive circulation and hormonal support to the testicular grafts (18). Also, other well-vascularized grafting locations such as muscle or kidney capsule could be selected to decrease posttransplantation anoxia as shown in the studies with ovarian grafts (19). It is also possible that host or gonadal grafts will be treated with hormones, growth factors, or biologic substances (such as vascular endothelial growth factor) to enhance graft vascularization (20).

Cryopreserved testicular grafts from rhesus monkey (#3199) showed very poor survival as xenograft. Only Sertoli cell-only tubules were detected in 1 surviving graft out of 18. The observed graft survival rate of 6% in immunocompromised mice is low compared with 73% previously reported using similar testicular material, freezing program, cryopreservation media, and follow-up time (1). Interstitial infiltration of macrophages was detected suggesting that the host immunoreaction may have affected graft survival and caused death of germ cells. Macrophage activation in the nude mice must be regarded as a factor that potentially affects the probability of spermatogenesis in primate testicular grafts. Macrophage activation can potentially be avoided by using more immunocompromised severe combined immunodeficient mice in future studies. In the present experimental setting, xenografting presented inferior potential for fertility preservation for this particular sterilized donor.

In conclusion, we were able to provide the proof-of-principle that cryopreserved primate spermatogonial stem cells and prepubertal testicular tissue have full functional capacity to mature to sperm after grafting. Optimal transplantation conditions were shown to be critical for maturation capacity. In this study, this was provided by autologous grafting of cryopreserved tissue into the scrotum. The observations provide valuable evidence that spermatogenic arrest reported previously in human testicular tissue (8–10) might be more associated to the xenografting method used than to the lack of spermatogenetic potential in the cryopreserved human tissue. We expect that an improvement of grafting strategies will open scenarios for clinical use of testicular tissue transplantation (1, 21, 22). The most promising approach will most probably involve autologous grafting of immature testicular tissue, stored before gonadotoxic therapy for nonmalignant pathologies or cautiously nonhematologic and nonmetastasizing cancers (1, 21, 22). For the other patients, testicular samples should be cryobanked after careful counseling.

No potential conflicts of interest were disclosed.

Conception and design: K. Jahnukainen, S. Schlatt

Development of methodology: K. Jahnukainen, J. Ehmcke, S. Schlatt

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Jahnukainen, J. Ehmcke, S. Schlatt

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Jahnukainen, J. Ehmcke, M. Nurmio, S. Schlatt

Writing, review, and/or revision of the manuscript: K. Jahnukainen, J. Ehmcke, S. Schlatt

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Jahnukainen, S. Schlatt

Study supervision: S. Schlatt

The nonhuman primate work was supported by Core B of the Center for Research in Reproductive Physiology at the University of Pittsburgh, including the skilful animal handling of Mike Cicco and Rachel Roslund. The authors thank Taija Leinonen and Jutta Salzig for skilful technical assistance.

This study was supported by NIH grants (1RO1 01050617-01; 2U54HD008610, project 1), the German Federal Ministry of Education and Research (Grant no. 01GN 0809), the Swedish Barncancerfonden, the Finnish Cancer Society, the Helsingin Sanomat Centennial Foundation, the Finnish Pediatric Research Foundation, Paulo Foundation, the Nona and Kullervo Väre Foundation, the Lance Armstrong Foundation, and Academy of Finland.

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