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Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation
FERTILITY AND STERILITY威 VOL. 75, NO. 2, FEBRUARY 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation Philip F. Brook, Ph.D.,a John A. Radford, M.D.,b Steven M. Shalet, M.D.,c Adrian D. Joyce, M.D.,d and Roger G. Gosden, D.Sc.a Leeds General Infirmary, University of Leeds, Leeds, and Christie Hospital, University of Manchester, Manchester, United Kingdom.
Received May 17, 2000; revised and accepted August 25, 2000. Supported by the Kay Kendall Leukaemia Research Fund (UK). Reprint requests: Roger G. Gosden, D.Sc., Department of Obstetrics and Gynecology, Women’s Pavilion (F3.38), Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada (FAX: 514-843-1662; Email: roger.gosden@muhc. mcgill.ca). a Centre for Reproduction, Growth, and Development, Leeds General Infirmary. b Department of Medical Oncology, Christie Hospital. c Department of Endocrinology, Christie Hospital. d Department of Urology, Leeds General Infirmary. 0015-0282/01/$20.00 PII S0015-0282(00)01721-0
Objective: To develop a new protocol for conserving fertile potential in men undergoing sterilizing chemotherapy by low temperature banking of germ cells which can be returned to the patient’s testes after thawing. Design: Isolation of human and murine germ cells for comparing cellular viability after cooling to liquid nitrogen temperatures by the use of different cryoprotective agents and for infusion into the testis. Setting: Laboratory research environment. Patient(s): Men undergoing routine surgery in a urology department. Intervention(s): Testicular biopsy. Main Outcome Measure(s): Cellular viability and infusion of seminiferous tubules. Result(s): After isolation using a two-step enzymatic disaggregation protocol, 66% to 87% of germ cells from human and murine specimens, respectively, were still viable. Cell survival was similar in four commonly used cryoprotective agents after cooling to liquid nitrogen temperatures. Seminiferous tubules infused by back flow with dye solution via the rete testis were filled with an efficiency of 55%. Conclusion(s): Judging from the high viability of unfractionated germ cells, it is feasible to isolate germ cells from testicular biopsies for low temperature banking with the aim of attempting to restore fertility after iatrogenic sterilization. (Fertil Steril威 2001;75:269 –74. ©2001 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, spermatogonia, sterilization, testes, transplantation
Chemotherapy for treating cancer is frequently gonadotoxic and can render patients of either sex and any age temporarily infertile or even permanently sterile. Like the malignant cells that are the intended targets of alkylating agents, nitrosoureas, and platinum compounds used in chemotherapy, germ cells are highly susceptible. Once the populations of spermatogonial stem cells in the testes or primordial follicles in the ovary have been destroyed, fertile potential is lost irreversibly (1–3). The development of increasingly effective conventional dose regimens and high-dose chemotherapy with stem cell rescue has led to steady improvements in remission and longterm survival in cancer patients. This progress has been most impressive for Hodgkin’s lymphoma and testicular cancer, which commonly affect young people. In consequence, the num-
bers of long-term survivors of cancer are rising. It has been estimated that, by the year 2010, one in 250 of the adult population will be a long-term survivor of a malignant disease contracted in childhood (4). Cryostorage of semen prior to commencing cancer therapy is offered routinely to preserve reproductive options of patients at risk of iatrogenic sterilization. For men who fail to store semen or produce poor quality specimens, the chances of spermatogenesis recovery vary, and they may require assisted conception technology to father a genetic child (5). Some patients do not store semen because they had a good fertility prognosis when their disease was first diagnosed but, after relapsing, they may be unable to produce a useful specimen as a result of oligospermia/azoospermia induced by cytotoxic therapy (3). 269
Attempts have been made to protect germ cells from cytotoxic treatment in animal studies by inhibiting spermatogenesis using GnRH analogues (6). This is an attractive idea because of its simplicity and relative safety, but we have yet to see a convincing demonstration of clinical benefit (7–9). Although there are no epidemiological data indicating any higher risk of abnormality for the children of former cancer patients, these men have more aneuploid sperm in their semen (10) and there is more pregnancy wastage from male rats that have recovered spermatogenesis after treatment with cyclophosphamide (11, 12). We should not be complacent, therefore, about fertility options in men undergoing sterilizing treatment, and prepubertal patients currently have none. A radical alternative to semen banking and hormonal protection has emerged from evidence that spermatogenesis and fertility can be restored by micro-injecting a crude suspension of germ cells into mice rendered sterile with busulphan or by a mutation (13–15). Subsequently, these encouraging results were extended to germ cells that had been cryopreserved before transplantation (16). These technical feats indicate the possibility of harvesting and cryopreserving spermatogonial stem cells from cancer patients before high-dose chemotherapy so that when the individuals are in full remission from disease the cells might be reimplanted in their testes. A fully successful method might reinstate both spermatogenesis and natural fertility, as in the animal model. To be successful, stem cells have to undergo a succession of chemical and thermal insults, including enzymatic isolation from tissue, cooling and long-term storage, thawing, and transfer, before they can migrate to the adluminal compartment of the tubules to resume spermatogenesis. Although animal data are encouraging, there is, to date, no published information for human germ cells. We have therefore developed a protocol for harvesting human germ cells for low temperature banking and, in addition, have tested a simple method for transferring them into testes in a manner that would be clinically feasible.
MATERIALS AND METHODS Sources of Tissue Preliminary studies were performed using testes obtained from F1 hybrid mice (C57BL/6 ⫻ CBA/Ca) from the animal colony in the Department of Biology, University of Leeds, United Kingdom. Eleven patients (age range 22 to 35 years) allowed their human testicular tissue to be used for research, and attended the urology service at the Leeds General Infirmary. The specimens were used within 3 hours of biopsy and confirmed as histologically normal by routine histology. In addition, eight whole testes were obtained from patients undergoing orchidectomy for prostate cancer at the Christie Hospital NHS Trust, Manchester. These organs were refrig270
Brook et al.
Isolation of male germ cells
erated for up to 24 hours before they were transported by express courier to Leeds for experimental study. Tissues were collected under protocols approved by the respective hospital research ethics committees (the equivalent of institutional review boards).
Tissue Disaggregation After we killed the mice, their testes were decapsulated and disaggregated using a two-stage procedure (n ⫽ 20 replicates). Each pair of organs was transferred to 4 mL of Leibovitz L-15 medium (Gibco BRL, Paisley, Scotland) containing 1 mg/mL collagenase Type 1a (Sigma Chemical Company, Poole, Dorset) and agitated gently for 8 to 10 minutes on a Spiramix at 37°C. Separation of the seminiferous tubules from adherent interstitial tissue was assisted by repeated aspiration with a wide-bore pipette. The products were filtered through a 120-m nylon mesh stretched across a wide-mouthed vessel (Lockertex, Locker Wire Weavers Ltd, Warrington, Cheshire). The retained tissue was rinsed with 2 to 3 mL of Leibovitz medium to wash away traces of collagenase and any free-floating cells. The mesh was inverted over a second conical vessel and the tubules were transferred into a Universal tube containing fresh medium for the second stage of disaggregation to prepare a cell suspension. Leibovitz medium was replaced with Dulbecco’s PBS containing 6 g mL⫺1 bovine pancreatic trypsin, 2 mM EDTA, 16 g mL⫺1 ovine hyaluronidase, 0.4 g mL⫺1 DNase I, and 0.2 mM sodium pyruvate (Sigma). The vessel was agitated and incubated for 10 minutes at 37°C, and the remaining lumps of tissue were disaggregated by pipetting. Enzymatic digestion was halted by adding a concentrated stock of soybean trypsin inhibitor (Sigma) adjusted to produce a final concentration of 500 g mL⫺1. The supernatant fluid was removed after gentle centrifugation at 500g for 10 minutes at 4°C. The cell pellet was resuspended in 1 mL of Leibovitz medium containing trypsin inhibitor at half the previous concentration. It was subsequently washed by centrifugation, as before, and resuspended in 1 mL of medium. A small fraction was transferred to 0.1% trypan blue (w/v) in 0.15M NaCl and 100 cells per preparation were scored at ⫻400 to estimate the percent unstained (i.e., viable). In another series of tests, the results with the trypan blue dye test were found to correspond less than 95% consistent with the results of propidium iodide (1 ng mL⫺1) staining for dead cells. Human testicular biopsies were disaggregated using a similar protocol with few modifications. The tissue was cut into cubes measuring approximately 3 ⫻ 3 ⫻ 3 mm and incubated in collagenase medium for 12 to 20 minutes followed by 12 to 15 minutes in trypsin-EDTA medium, according to the fibrosity of the tissue, to produce cell suspensions comparable to those from the mice. Vol. 75, No. 2, February 2001
Cryopreservation of Disaggregated Tissue Cell suspensions from five human testicular biopsies were each divided into four aliquots and transferred to Nunc cryotubes (Life Technologies Ltd, Glasgow, Scotland). In the total volume of 1 mL there was 4% fetal calf serum plus 1.5M of one of the following cryoprotective agents: dimethyl sulphoxide, 1,2-propanediol, ethylene glycol, glycerol (Sigma). The cells were gently agitated and allowed to equilibrate at 20°C for 15 minutes. A small fraction was reserved for testing with trypan blue. The tubes were cooled on a Planar Kryo 10 series II automated freezer (Planar Products, Sudbury-on-Thames, Middlesex, UK) using the following basic protocol after experimental optimization: for the first ramp, the rate of cooling was 2°C min⫺1 between 20° to 4°C; the second ramp was at 0.3°C min⫺1 down to ⫺30°C; and the third ramp was 50°C min⫺1 to ⫺180°C. Seeding was carried out manually at ⫺7°C. Finally, the tubes were plunged into liquid nitrogen and transferred to a storage dewar for up to 8 days. The tubes were rapidly thawed by holding them in air for 1 minute and then plunging them in a bowl of water at room temperature for 2 minutes and stirring vigorously. The cryoprotective agents were removed by washing the tissues in progressively more dilute solutions (2 minutes in 1.0M, followed by 0.5M and then fresh Leibovitz medium). In experiments that were restricted to the more readily available murine tissue, comparisons were made between the cellular viability after different rates of cooling between 0.1° to 1°C min⫺1 in the second ramp using glycerol as the cryoprotective agent (CPA). The loss of viability due to the cooling rate was obtained by subtracting the initial percentage of dead cells. Cell suspensions from six pairs of testes were tested using 1.5M glycerol and 4% fetal calf serum as cryoprotective medium. Samples were retested for viability after thawing. A further set of five experiments with human cells was performed using the cooling rate that was found to be optimal (0.3 min⫺1).
Cytological Analysis To verify that cell suspensions contained spermatogonial cells, the cell surface marker, c-kit protein, was investigated immunocytochemically (17). A rabbit anti-human antibody (PC34) was chosen for specificity to the amino acid residues 962 to 976 in the c-kit tyrosine kinase receptor, which are homologous in these species. The staining procedure involved blocking endogenous peroxidase with 0.6% H2O2 and nonspecific blocking with 20% normal serum. Apart from negative controls, the sections were covered with primary antibody diluted 1:200 in a humidified box for 2 hours at room temperature. After washing, anti-rabbit IGG preconjugated with peroxidase was applied to the sections at 1:500 (antibodies from Sigma Chemical). After 40 minutes, this antibody was also removed and 0.02% diaminobenzidine was applied as a substrate before counterstaining with Light Green. FERTILITY & STERILITY威
Cell suspensions from disaggregated murine and human testes, both fresh and cryopreserved specimens (n ⫽ 5 and n ⫽ 8, respectively), were stained as air-dried specimens on slides by Cytospin centrifugation (300g). In some specimens, the first antibody was omitted as a negative control and specificity was confirmed in others by immunolabeling 4-m sections of mouse testes fixed in paraformaldehyde and embedded in paraffin wax. A single layer of stained cells was observed in the basal compartment of the tubules corresponding to the expected location of spermatogonia.
Infusion into Seminiferous Tubules Attempts were made to infuse eight whole human testes with trypan dye solution/red blood cells. The method was modified from one tested previously in primates (18) and involved infusing 200 L of dye via the rete testes at either a single site or at four points along their length. The fluid was introduced by drip feed under gravity with a column height of 75 cm. The extent of fluid loss, if any, was slight, and each infusion was chased into the tubules by an equal volume of PBS. Immediately afterward, the testicular capsules were incised for examining dye distribution and estimating the proportion of stained seminiferous tubules under a dissecting microscope (⫻20). In another experiment, a suspension of erythrocytes in heparinized PBS was injected into the tubules using the same procedure. The tubules were dissected, fixed in 4% formaldehyde, sectioned in wax, and stained with Harris’s hematoxylin and eosin.
Statistics Statistical analyses were carried out using ANOVA.
RESULTS Tissue Disaggregation The two-stage procedure produced almost completely disaggregated tissue from murine and humans testes, although the latter required a longer incubation and sometimes there was a small residue of human tissue that was resistant to treatment and was discarded (⬍5%). The viability index, indicated by trypan blue exclusion, consistently showed higher rates of cell survival with mouse testes than with human testes, averaging 87% and 66%, respectively (Table 1). When the cells were observed by phase contrast microscopy, the percentage of unstained cells appeared to be similar for all morphologic types, although specific cell types were not identifiable.
Cryopreservation Before immersion in the CPA and freeze-thawing, human testicular cell viability fell by 10% from approximately 60% (Table 2). Mean cellular viability varied from 52% to 58% for the four CPAs tested, but these differences were not significant in five replicates for each group (P ⬎.05). After correcting for variations in initial viability, the rate of cooling was found to be a significant factor affecting the viability after freezing and thawing (Table 3). Because cell death was 271
TABLE 1
FIGURE 1
Comparison of cellular viability after enzymatic disaggregation of murine and human testes.
(A) A human testicle that had been injected with trypan blue dye in four places along the length of the rete testis. (B) An organ that received only a single injection of the same volume.
Cell viability (%)a Species
Number of testes
Mean
Range
Mouse Human
20 11
87 66
79–91 62–69
P ⬎ .05.
a
Brook. Isolation of male germ cells. Fertil Steril 2001.
increased by rates of cooling ⱖ0.4°C min⫺1, 0.3°C min⫺1 was adopted for the standard protocol. A tiny proportion (Ⰶ1%) of the cells in human and murine preparations was stained for c-kit, and in both fresh and frozen-thawed preparations. It was not possible, of course, to verify whether these had been viable before processing. Brook. Isolation of male germ cells. Fertil Steril 2001.
TABLE 2
Infusion into Seminiferous Tubules
Viability of isolated cells from human testes after cryopreservation: comparison of four cryoprotective agents. Cryoprotective agents (1.5M)
Mean % viability (range)a
Glycerol DMSO 1,2-propanediol Ethylene glycol
54 (52–57) 54 (51–57) 58 (55–59) 52 (51–53)
P ⬎ .05.
a
Brook. Isolation of male germ cells. Fertil Steril 2001.
TABLE 3 Viability of murine testicular cells after disaggregation and cryopreservation: effect of rate of cooling. Rate of cooling (°C min⫺1) from 4°C to ⫺30°C 0.1 0.2 0.3 0.4 0.6 1.0
Viability (%)
Postdigestion
Post-thawing (range)
⌬a
85 87 84 88 92 90
77 (75–78) 77 (76–77) 76 (72–79) 75 (73–76) 71 (68–73) 65 (59–68)
8 10 8 13 21 25
P ⬍ .01.
a
Brook. Isolation of male germ cells. Fertil Steril 2001.
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Gravity feed into human rete testes was successful provided that the flow was kept open by holding the needle tip securely in the lumen. Judging by the dispersal of dye into the tubules, injection was more effective when multiple sites were used rather than a single one, but the overall percentage of stained tubules was 55.3 ⫾ 3.2% (SEM) for the eight organs studied (Fig. 1). Staining was more concentrated in the tubules than in the interstitium, and deep blue tubules sometimes lay next to unstained ones, indicating that most of the dye had arrived by direct infusion rather than passive diffusion from the puncture site(s). Likewise, erythrocytes injected via the rete testes were identifiable by histology in the lumens of seminiferous tubules, though never found in uninjected controls.
DISCUSSION This study indicates that crude suspensions of germ cells, including spermatogonia, can be isolated from human testes for cryopreservation. The methods could form a basis for storing stem cells to restore human fertility, paralleling bone marrow transplantation technology. The protocol is straightforward enough to be performed in any assisted reproduction center with minor modifications, such as the substitution of the patient’s serum for fetal calf serum and careful screening of enzyme sources. The two-step disaggregation lengthens the procedure, but carries the advantage of delaying exposure to the aggressive trypsin-EDTA treatment until absolutely necessary. Additionally, this preferred method enables Vol. 75, No. 2, February 2001
the interstitial cells to be removed in the washing step and opens the possibility of storing the tubules after the first stage. Cryopreservation of intact tubules is also practicable (19) and provides options after thawing for either 1) performing the second step for cell infusion, or 2) transplanting the tubules without further treatment to a heterotopic site for spermatogenesis to be initiated in the hope of producing sperm for an intracytoplasmic sperm injection (ICSI) procedure (20). The lower viability of isolated human cells compared to murine cells probably reflects the greater fibrosity of the tissue and the correspondingly longer incubation in enzymes, though this time might be reduced for prepubertal testes. Cryopreservation was responsible for the loss of fewer cells (about 10%) than from disaggregation. More surprisingly, the choice of CPA did not affect the viability when cells were cooled with the use of the optimized protocol. Glycerol has been adopted for clinical cases because, although it permeates cells more slowly than the other CPAs, it is a physiological compound (though used at nonphysiological concentrations) and widely used for cryopreserving spermatozoa. Vitrification has not been attempted yet, and the cells were cooled slowly using the so-called “equilibrium” method to minimize osmotic and thermal gradients. The full procedure from the receipt of the specimen in the laboratory to depositing the cells in a storage dewar was less than 4 hours. In the original studies by Brinster et al. (14), a crude suspension of murine gonocytes restored spermatogenesis by progressive expansion of clones and differentiation over the course of several months (21). The stem cells are a subpopulation of c-kit positive cells that survived all the procedures and includes the spectrum of spermatogonia from type Ao to B. No absolutely specific marker for stem cells has yet been identified, nor is the number of cells required to restore fertility known. Research needs to address both of these issues if a fertile outcome of infusing testes is to be reproducible. In pilot experiments attempting to enrich the total spermatogonial population, both density gradient centrifugation and centrifugal elutriation, have been tested, and although the latter gave a superior harvest of c-kit positive cells and cell viability (22), more work is needed to refine the methodology. Another strategy for cell enrichment that could be tested on human germ cells is to bind cells to immunomagnetic beads coated with antibodies to c-kit, which has been successful in animal studies (23). In mice, direct microinjection of gonocytes into the seminiferous tubules was effective, but this route is not practicable with the more fibrous tubules in the primate testis. Preliminary evidence from monkeys indicated that the rete testis is a more satisfactory route because the wall is less fibrous and cells can be flushed backward into the seminiferous tubules (18). This method has now been demonstrated with the use of excised human gonads in which over half of the organs were infused, and this result will undoubtedly be FERTILITY & STERILITY威
improved in future. In clinical practice, it would be feasible to locate the rete with the use of ultrasound so that a cell suspension can be injected directly into its lumen through the scrotal skin. Storage and autotransplantation of germ cells could be of clinical utility for prepubertal patients for whom no option for fertility conservation exists. Some adult men might elect for this strategy as a backup to semen banking or for the chance of restoring natural fertility. There would be great concern, of course, if there was a risk of returning the old disease to the body, but this should be avoidable by careful selection of patients. In Hodgkin’s lymphoma, which is probably the most relevant disease affecting young males, testicular involvement or relapse is virtually unknown. Nevertheless, many questions need to be answered before these methods should be offered routinely. Is Sertoli cell function impaired by chemotherapy? How many stem cells are required for transfer? Does the age of the patient affect the chances of success? How soon should transfer be attempted after chemotherapy and how long is the delay before sperm reappear in the ejaculate? Finding answers to these questions may be slow because research material is scarce and the nature of the work is long term, but if these efforts help improve the reproductive health of people who have survived cancer they will be worthwhile.
Acknowledgments The authors thank Noel Clarke, consultant urologist at the Christie Hospital, Manchester, for his cooperation.
References 1. Apperley JF, Reddy N. Mechanisms and management of treatmentrelated gonadal failure in chemoradiotherapy. Blood Rev 1995;9:93– 116. 2. Meirow D, Schenker JG. Cancer and male infertility. Hum Reprod 1995;10:2017–22. 3. Schlatt S, von Scho¨nfeldt V, Nieschlag E. Germ cell transplantation in the male: animal studies with a human perspective. Hum Fertil 1999; 2:143– 8. 4. Blatt J. Pregnancy outcome in long-term survivors of childhood cancer. Med Pediatr Oncol 1999;33:29 –33. 5. Naysmith TE, Blake DA, Harvey VJ, Johnson NP. Do men undergoing sterilizing cancer treatments have a fertile future? Hum Reprod 1998; 13:3250 –5. 6. Ward JA, Robinson J, Furr BJA, Shalet S, Morris ID. Protection of spermatogenesis in rats from the cytotoxic procarbazine by the depot formulation of Zoladex, a gonadotrophin-releasing hormone agonist. Cancer Res 1990;50:568 –74. 7. Johnson DH, Linde R, Hainsworth JD, Vale W, Rivier J, Stein R, et al. Effect of luteinizing hormone releasing hormone agonist given during combination chemotherapy on posttherapy fertility in male patients with lymphoma: preliminary observations. Blood 1985;65:832– 6. 8. Waxman JH, Ahmed R, Smith D, Wrigley PF, Gregory W, Shalet S. Failure to preserve fertility in patients with Hodgkin’s disease. Cancer Chemother Pharmacol 1987;19:159 – 62. 9. Kreuser ED, Hetzel WD, Hautmann R, Pfeiffer EF. Reproductive toxicity with and without LHRHA administration during adjuvant chemotherapy in patients with germ cell tumors. Horm Metab Res 1990; 22:494 – 8. 10. Martin RH, Hildebrand K, Yamamoto J, Rademaker A, Barnes, M, Douglas G, et al. An increased frequency of human sperm chromosomal abnormalities after radiotherapy. Mutat Res 1986;174:219 –25. 11. Hales BF, Crosman K, Robaire B. Increased post-implantation loss and
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malformations among the F2 progeny of male rats chronically treated with cyclophosphamide. Teratology 1992;45:671– 8. Trasler JM, Hales BF, Robaire B. Paternal cyclophosphamide treatment causes fetal loss and malformations without affecting male fertility. Nature 1985;316:144 – 6. Brinster RL, Zimmerman JW. Spermatogenesis following male germ cell transplantation. Proc Natl Acad Sci USA 1994;91:11298 –302. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transfer. Proc Natl Acad Sci USA 1994;91: 11303–7. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat Med 2000;6:29 –34. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nature Med 1996;2: 693– 6. Dym M, Jia MC, Dirami G, Price JM, Rabin SJ, Mochetti I, et al. Expression of the c-kit receptor and its autophosphorylation in immature rat type A-spermatogonia. Biol Reprod 1995;52:8 –19.
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18. Schlatt S, Rosiepen G, Weinbauer GF, Rolf C, Brook PF, Nieschlag E. Germ cell transfer into rat, bovine, monkey and human testes. Hum Reprod 1999;14:144 –50. 19. Allan JA, Cotman AS. A new method for freezing testicular biopsy sperm: three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule. Fertil Steril 1997;68:741– 4. 20. Gosden RG, Matthews SJ, Kim SS, Schlatt S. Potential fertility in mice after isografting cryopreserved testicular tissue. Biol Reprod 2000; 62(suppl 1):190. 21. Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999;60:1429 –36. 22. Brook PF, Radford JA, Shalet SM, Joyce A, Gosden RG. Frozen banking of isolated germ cells for protection against chemotherapy induced sterility of male cancer patients. J Reprod Fertil Abstr Ser 1997;19:27. 23. von Scho¨nfeldt V, Krishnamurthy H, Foppiani L, Schlatt S. Magnetic cell sorting as a fast and efficient method of enriching viable spermatogonia from rodent and primate testes. Biol Reprod 1999;61:582–9.
Vol. 75, No. 2, February 2001
Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation Philip F. Brook, Ph.D.,a John A. Radford, M.D.,b Steven M. Shalet, M.D.,c Adrian D. Joyce, M.D.,d and Roger G. Gosden, D.Sc.a Leeds General Infirmary, University of Leeds, Leeds, and Christie Hospital, University of Manchester, Manchester, United Kingdom.
Received May 17, 2000; revised and accepted August 25, 2000. Supported by the Kay Kendall Leukaemia Research Fund (UK). Reprint requests: Roger G. Gosden, D.Sc., Department of Obstetrics and Gynecology, Women’s Pavilion (F3.38), Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada (FAX: 514-843-1662; Email: roger.gosden@muhc. mcgill.ca). a Centre for Reproduction, Growth, and Development, Leeds General Infirmary. b Department of Medical Oncology, Christie Hospital. c Department of Endocrinology, Christie Hospital. d Department of Urology, Leeds General Infirmary. 0015-0282/01/$20.00 PII S0015-0282(00)01721-0
Objective: To develop a new protocol for conserving fertile potential in men undergoing sterilizing chemotherapy by low temperature banking of germ cells which can be returned to the patient’s testes after thawing. Design: Isolation of human and murine germ cells for comparing cellular viability after cooling to liquid nitrogen temperatures by the use of different cryoprotective agents and for infusion into the testis. Setting: Laboratory research environment. Patient(s): Men undergoing routine surgery in a urology department. Intervention(s): Testicular biopsy. Main Outcome Measure(s): Cellular viability and infusion of seminiferous tubules. Result(s): After isolation using a two-step enzymatic disaggregation protocol, 66% to 87% of germ cells from human and murine specimens, respectively, were still viable. Cell survival was similar in four commonly used cryoprotective agents after cooling to liquid nitrogen temperatures. Seminiferous tubules infused by back flow with dye solution via the rete testis were filled with an efficiency of 55%. Conclusion(s): Judging from the high viability of unfractionated germ cells, it is feasible to isolate germ cells from testicular biopsies for low temperature banking with the aim of attempting to restore fertility after iatrogenic sterilization. (Fertil Steril威 2001;75:269 –74. ©2001 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, spermatogonia, sterilization, testes, transplantation
Chemotherapy for treating cancer is frequently gonadotoxic and can render patients of either sex and any age temporarily infertile or even permanently sterile. Like the malignant cells that are the intended targets of alkylating agents, nitrosoureas, and platinum compounds used in chemotherapy, germ cells are highly susceptible. Once the populations of spermatogonial stem cells in the testes or primordial follicles in the ovary have been destroyed, fertile potential is lost irreversibly (1–3). The development of increasingly effective conventional dose regimens and high-dose chemotherapy with stem cell rescue has led to steady improvements in remission and longterm survival in cancer patients. This progress has been most impressive for Hodgkin’s lymphoma and testicular cancer, which commonly affect young people. In consequence, the num-
bers of long-term survivors of cancer are rising. It has been estimated that, by the year 2010, one in 250 of the adult population will be a long-term survivor of a malignant disease contracted in childhood (4). Cryostorage of semen prior to commencing cancer therapy is offered routinely to preserve reproductive options of patients at risk of iatrogenic sterilization. For men who fail to store semen or produce poor quality specimens, the chances of spermatogenesis recovery vary, and they may require assisted conception technology to father a genetic child (5). Some patients do not store semen because they had a good fertility prognosis when their disease was first diagnosed but, after relapsing, they may be unable to produce a useful specimen as a result of oligospermia/azoospermia induced by cytotoxic therapy (3). 269
Attempts have been made to protect germ cells from cytotoxic treatment in animal studies by inhibiting spermatogenesis using GnRH analogues (6). This is an attractive idea because of its simplicity and relative safety, but we have yet to see a convincing demonstration of clinical benefit (7–9). Although there are no epidemiological data indicating any higher risk of abnormality for the children of former cancer patients, these men have more aneuploid sperm in their semen (10) and there is more pregnancy wastage from male rats that have recovered spermatogenesis after treatment with cyclophosphamide (11, 12). We should not be complacent, therefore, about fertility options in men undergoing sterilizing treatment, and prepubertal patients currently have none. A radical alternative to semen banking and hormonal protection has emerged from evidence that spermatogenesis and fertility can be restored by micro-injecting a crude suspension of germ cells into mice rendered sterile with busulphan or by a mutation (13–15). Subsequently, these encouraging results were extended to germ cells that had been cryopreserved before transplantation (16). These technical feats indicate the possibility of harvesting and cryopreserving spermatogonial stem cells from cancer patients before high-dose chemotherapy so that when the individuals are in full remission from disease the cells might be reimplanted in their testes. A fully successful method might reinstate both spermatogenesis and natural fertility, as in the animal model. To be successful, stem cells have to undergo a succession of chemical and thermal insults, including enzymatic isolation from tissue, cooling and long-term storage, thawing, and transfer, before they can migrate to the adluminal compartment of the tubules to resume spermatogenesis. Although animal data are encouraging, there is, to date, no published information for human germ cells. We have therefore developed a protocol for harvesting human germ cells for low temperature banking and, in addition, have tested a simple method for transferring them into testes in a manner that would be clinically feasible.
MATERIALS AND METHODS Sources of Tissue Preliminary studies were performed using testes obtained from F1 hybrid mice (C57BL/6 ⫻ CBA/Ca) from the animal colony in the Department of Biology, University of Leeds, United Kingdom. Eleven patients (age range 22 to 35 years) allowed their human testicular tissue to be used for research, and attended the urology service at the Leeds General Infirmary. The specimens were used within 3 hours of biopsy and confirmed as histologically normal by routine histology. In addition, eight whole testes were obtained from patients undergoing orchidectomy for prostate cancer at the Christie Hospital NHS Trust, Manchester. These organs were refrig270
Brook et al.
Isolation of male germ cells
erated for up to 24 hours before they were transported by express courier to Leeds for experimental study. Tissues were collected under protocols approved by the respective hospital research ethics committees (the equivalent of institutional review boards).
Tissue Disaggregation After we killed the mice, their testes were decapsulated and disaggregated using a two-stage procedure (n ⫽ 20 replicates). Each pair of organs was transferred to 4 mL of Leibovitz L-15 medium (Gibco BRL, Paisley, Scotland) containing 1 mg/mL collagenase Type 1a (Sigma Chemical Company, Poole, Dorset) and agitated gently for 8 to 10 minutes on a Spiramix at 37°C. Separation of the seminiferous tubules from adherent interstitial tissue was assisted by repeated aspiration with a wide-bore pipette. The products were filtered through a 120-m nylon mesh stretched across a wide-mouthed vessel (Lockertex, Locker Wire Weavers Ltd, Warrington, Cheshire). The retained tissue was rinsed with 2 to 3 mL of Leibovitz medium to wash away traces of collagenase and any free-floating cells. The mesh was inverted over a second conical vessel and the tubules were transferred into a Universal tube containing fresh medium for the second stage of disaggregation to prepare a cell suspension. Leibovitz medium was replaced with Dulbecco’s PBS containing 6 g mL⫺1 bovine pancreatic trypsin, 2 mM EDTA, 16 g mL⫺1 ovine hyaluronidase, 0.4 g mL⫺1 DNase I, and 0.2 mM sodium pyruvate (Sigma). The vessel was agitated and incubated for 10 minutes at 37°C, and the remaining lumps of tissue were disaggregated by pipetting. Enzymatic digestion was halted by adding a concentrated stock of soybean trypsin inhibitor (Sigma) adjusted to produce a final concentration of 500 g mL⫺1. The supernatant fluid was removed after gentle centrifugation at 500g for 10 minutes at 4°C. The cell pellet was resuspended in 1 mL of Leibovitz medium containing trypsin inhibitor at half the previous concentration. It was subsequently washed by centrifugation, as before, and resuspended in 1 mL of medium. A small fraction was transferred to 0.1% trypan blue (w/v) in 0.15M NaCl and 100 cells per preparation were scored at ⫻400 to estimate the percent unstained (i.e., viable). In another series of tests, the results with the trypan blue dye test were found to correspond less than 95% consistent with the results of propidium iodide (1 ng mL⫺1) staining for dead cells. Human testicular biopsies were disaggregated using a similar protocol with few modifications. The tissue was cut into cubes measuring approximately 3 ⫻ 3 ⫻ 3 mm and incubated in collagenase medium for 12 to 20 minutes followed by 12 to 15 minutes in trypsin-EDTA medium, according to the fibrosity of the tissue, to produce cell suspensions comparable to those from the mice. Vol. 75, No. 2, February 2001
Cryopreservation of Disaggregated Tissue Cell suspensions from five human testicular biopsies were each divided into four aliquots and transferred to Nunc cryotubes (Life Technologies Ltd, Glasgow, Scotland). In the total volume of 1 mL there was 4% fetal calf serum plus 1.5M of one of the following cryoprotective agents: dimethyl sulphoxide, 1,2-propanediol, ethylene glycol, glycerol (Sigma). The cells were gently agitated and allowed to equilibrate at 20°C for 15 minutes. A small fraction was reserved for testing with trypan blue. The tubes were cooled on a Planar Kryo 10 series II automated freezer (Planar Products, Sudbury-on-Thames, Middlesex, UK) using the following basic protocol after experimental optimization: for the first ramp, the rate of cooling was 2°C min⫺1 between 20° to 4°C; the second ramp was at 0.3°C min⫺1 down to ⫺30°C; and the third ramp was 50°C min⫺1 to ⫺180°C. Seeding was carried out manually at ⫺7°C. Finally, the tubes were plunged into liquid nitrogen and transferred to a storage dewar for up to 8 days. The tubes were rapidly thawed by holding them in air for 1 minute and then plunging them in a bowl of water at room temperature for 2 minutes and stirring vigorously. The cryoprotective agents were removed by washing the tissues in progressively more dilute solutions (2 minutes in 1.0M, followed by 0.5M and then fresh Leibovitz medium). In experiments that were restricted to the more readily available murine tissue, comparisons were made between the cellular viability after different rates of cooling between 0.1° to 1°C min⫺1 in the second ramp using glycerol as the cryoprotective agent (CPA). The loss of viability due to the cooling rate was obtained by subtracting the initial percentage of dead cells. Cell suspensions from six pairs of testes were tested using 1.5M glycerol and 4% fetal calf serum as cryoprotective medium. Samples were retested for viability after thawing. A further set of five experiments with human cells was performed using the cooling rate that was found to be optimal (0.3 min⫺1).
Cytological Analysis To verify that cell suspensions contained spermatogonial cells, the cell surface marker, c-kit protein, was investigated immunocytochemically (17). A rabbit anti-human antibody (PC34) was chosen for specificity to the amino acid residues 962 to 976 in the c-kit tyrosine kinase receptor, which are homologous in these species. The staining procedure involved blocking endogenous peroxidase with 0.6% H2O2 and nonspecific blocking with 20% normal serum. Apart from negative controls, the sections were covered with primary antibody diluted 1:200 in a humidified box for 2 hours at room temperature. After washing, anti-rabbit IGG preconjugated with peroxidase was applied to the sections at 1:500 (antibodies from Sigma Chemical). After 40 minutes, this antibody was also removed and 0.02% diaminobenzidine was applied as a substrate before counterstaining with Light Green. FERTILITY & STERILITY威
Cell suspensions from disaggregated murine and human testes, both fresh and cryopreserved specimens (n ⫽ 5 and n ⫽ 8, respectively), were stained as air-dried specimens on slides by Cytospin centrifugation (300g). In some specimens, the first antibody was omitted as a negative control and specificity was confirmed in others by immunolabeling 4-m sections of mouse testes fixed in paraformaldehyde and embedded in paraffin wax. A single layer of stained cells was observed in the basal compartment of the tubules corresponding to the expected location of spermatogonia.
Infusion into Seminiferous Tubules Attempts were made to infuse eight whole human testes with trypan dye solution/red blood cells. The method was modified from one tested previously in primates (18) and involved infusing 200 L of dye via the rete testes at either a single site or at four points along their length. The fluid was introduced by drip feed under gravity with a column height of 75 cm. The extent of fluid loss, if any, was slight, and each infusion was chased into the tubules by an equal volume of PBS. Immediately afterward, the testicular capsules were incised for examining dye distribution and estimating the proportion of stained seminiferous tubules under a dissecting microscope (⫻20). In another experiment, a suspension of erythrocytes in heparinized PBS was injected into the tubules using the same procedure. The tubules were dissected, fixed in 4% formaldehyde, sectioned in wax, and stained with Harris’s hematoxylin and eosin.
Statistics Statistical analyses were carried out using ANOVA.
RESULTS Tissue Disaggregation The two-stage procedure produced almost completely disaggregated tissue from murine and humans testes, although the latter required a longer incubation and sometimes there was a small residue of human tissue that was resistant to treatment and was discarded (⬍5%). The viability index, indicated by trypan blue exclusion, consistently showed higher rates of cell survival with mouse testes than with human testes, averaging 87% and 66%, respectively (Table 1). When the cells were observed by phase contrast microscopy, the percentage of unstained cells appeared to be similar for all morphologic types, although specific cell types were not identifiable.
Cryopreservation Before immersion in the CPA and freeze-thawing, human testicular cell viability fell by 10% from approximately 60% (Table 2). Mean cellular viability varied from 52% to 58% for the four CPAs tested, but these differences were not significant in five replicates for each group (P ⬎.05). After correcting for variations in initial viability, the rate of cooling was found to be a significant factor affecting the viability after freezing and thawing (Table 3). Because cell death was 271
TABLE 1
FIGURE 1
Comparison of cellular viability after enzymatic disaggregation of murine and human testes.
(A) A human testicle that had been injected with trypan blue dye in four places along the length of the rete testis. (B) An organ that received only a single injection of the same volume.
Cell viability (%)a Species
Number of testes
Mean
Range
Mouse Human
20 11
87 66
79–91 62–69
P ⬎ .05.
a
Brook. Isolation of male germ cells. Fertil Steril 2001.
increased by rates of cooling ⱖ0.4°C min⫺1, 0.3°C min⫺1 was adopted for the standard protocol. A tiny proportion (Ⰶ1%) of the cells in human and murine preparations was stained for c-kit, and in both fresh and frozen-thawed preparations. It was not possible, of course, to verify whether these had been viable before processing. Brook. Isolation of male germ cells. Fertil Steril 2001.
TABLE 2
Infusion into Seminiferous Tubules
Viability of isolated cells from human testes after cryopreservation: comparison of four cryoprotective agents. Cryoprotective agents (1.5M)
Mean % viability (range)a
Glycerol DMSO 1,2-propanediol Ethylene glycol
54 (52–57) 54 (51–57) 58 (55–59) 52 (51–53)
P ⬎ .05.
a
Brook. Isolation of male germ cells. Fertil Steril 2001.
TABLE 3 Viability of murine testicular cells after disaggregation and cryopreservation: effect of rate of cooling. Rate of cooling (°C min⫺1) from 4°C to ⫺30°C 0.1 0.2 0.3 0.4 0.6 1.0
Viability (%)
Postdigestion
Post-thawing (range)
⌬a
85 87 84 88 92 90
77 (75–78) 77 (76–77) 76 (72–79) 75 (73–76) 71 (68–73) 65 (59–68)
8 10 8 13 21 25
P ⬍ .01.
a
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Gravity feed into human rete testes was successful provided that the flow was kept open by holding the needle tip securely in the lumen. Judging by the dispersal of dye into the tubules, injection was more effective when multiple sites were used rather than a single one, but the overall percentage of stained tubules was 55.3 ⫾ 3.2% (SEM) for the eight organs studied (Fig. 1). Staining was more concentrated in the tubules than in the interstitium, and deep blue tubules sometimes lay next to unstained ones, indicating that most of the dye had arrived by direct infusion rather than passive diffusion from the puncture site(s). Likewise, erythrocytes injected via the rete testes were identifiable by histology in the lumens of seminiferous tubules, though never found in uninjected controls.
DISCUSSION This study indicates that crude suspensions of germ cells, including spermatogonia, can be isolated from human testes for cryopreservation. The methods could form a basis for storing stem cells to restore human fertility, paralleling bone marrow transplantation technology. The protocol is straightforward enough to be performed in any assisted reproduction center with minor modifications, such as the substitution of the patient’s serum for fetal calf serum and careful screening of enzyme sources. The two-step disaggregation lengthens the procedure, but carries the advantage of delaying exposure to the aggressive trypsin-EDTA treatment until absolutely necessary. Additionally, this preferred method enables Vol. 75, No. 2, February 2001
the interstitial cells to be removed in the washing step and opens the possibility of storing the tubules after the first stage. Cryopreservation of intact tubules is also practicable (19) and provides options after thawing for either 1) performing the second step for cell infusion, or 2) transplanting the tubules without further treatment to a heterotopic site for spermatogenesis to be initiated in the hope of producing sperm for an intracytoplasmic sperm injection (ICSI) procedure (20). The lower viability of isolated human cells compared to murine cells probably reflects the greater fibrosity of the tissue and the correspondingly longer incubation in enzymes, though this time might be reduced for prepubertal testes. Cryopreservation was responsible for the loss of fewer cells (about 10%) than from disaggregation. More surprisingly, the choice of CPA did not affect the viability when cells were cooled with the use of the optimized protocol. Glycerol has been adopted for clinical cases because, although it permeates cells more slowly than the other CPAs, it is a physiological compound (though used at nonphysiological concentrations) and widely used for cryopreserving spermatozoa. Vitrification has not been attempted yet, and the cells were cooled slowly using the so-called “equilibrium” method to minimize osmotic and thermal gradients. The full procedure from the receipt of the specimen in the laboratory to depositing the cells in a storage dewar was less than 4 hours. In the original studies by Brinster et al. (14), a crude suspension of murine gonocytes restored spermatogenesis by progressive expansion of clones and differentiation over the course of several months (21). The stem cells are a subpopulation of c-kit positive cells that survived all the procedures and includes the spectrum of spermatogonia from type Ao to B. No absolutely specific marker for stem cells has yet been identified, nor is the number of cells required to restore fertility known. Research needs to address both of these issues if a fertile outcome of infusing testes is to be reproducible. In pilot experiments attempting to enrich the total spermatogonial population, both density gradient centrifugation and centrifugal elutriation, have been tested, and although the latter gave a superior harvest of c-kit positive cells and cell viability (22), more work is needed to refine the methodology. Another strategy for cell enrichment that could be tested on human germ cells is to bind cells to immunomagnetic beads coated with antibodies to c-kit, which has been successful in animal studies (23). In mice, direct microinjection of gonocytes into the seminiferous tubules was effective, but this route is not practicable with the more fibrous tubules in the primate testis. Preliminary evidence from monkeys indicated that the rete testis is a more satisfactory route because the wall is less fibrous and cells can be flushed backward into the seminiferous tubules (18). This method has now been demonstrated with the use of excised human gonads in which over half of the organs were infused, and this result will undoubtedly be FERTILITY & STERILITY威
improved in future. In clinical practice, it would be feasible to locate the rete with the use of ultrasound so that a cell suspension can be injected directly into its lumen through the scrotal skin. Storage and autotransplantation of germ cells could be of clinical utility for prepubertal patients for whom no option for fertility conservation exists. Some adult men might elect for this strategy as a backup to semen banking or for the chance of restoring natural fertility. There would be great concern, of course, if there was a risk of returning the old disease to the body, but this should be avoidable by careful selection of patients. In Hodgkin’s lymphoma, which is probably the most relevant disease affecting young males, testicular involvement or relapse is virtually unknown. Nevertheless, many questions need to be answered before these methods should be offered routinely. Is Sertoli cell function impaired by chemotherapy? How many stem cells are required for transfer? Does the age of the patient affect the chances of success? How soon should transfer be attempted after chemotherapy and how long is the delay before sperm reappear in the ejaculate? Finding answers to these questions may be slow because research material is scarce and the nature of the work is long term, but if these efforts help improve the reproductive health of people who have survived cancer they will be worthwhile.
Acknowledgments The authors thank Noel Clarke, consultant urologist at the Christie Hospital, Manchester, for his cooperation.
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18. Schlatt S, Rosiepen G, Weinbauer GF, Rolf C, Brook PF, Nieschlag E. Germ cell transfer into rat, bovine, monkey and human testes. Hum Reprod 1999;14:144 –50. 19. Allan JA, Cotman AS. A new method for freezing testicular biopsy sperm: three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule. Fertil Steril 1997;68:741– 4. 20. Gosden RG, Matthews SJ, Kim SS, Schlatt S. Potential fertility in mice after isografting cryopreserved testicular tissue. Biol Reprod 2000; 62(suppl 1):190. 21. Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999;60:1429 –36. 22. Brook PF, Radford JA, Shalet SM, Joyce A, Gosden RG. Frozen banking of isolated germ cells for protection against chemotherapy induced sterility of male cancer patients. J Reprod Fertil Abstr Ser 1997;19:27. 23. von Scho¨nfeldt V, Krishnamurthy H, Foppiani L, Schlatt S. Magnetic cell sorting as a fast and efficient method of enriching viable spermatogonia from rodent and primate testes. Biol Reprod 1999;61:582–9.
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