Human Reproduction, Vol.28, No.4 pp. 897–907, 2013
Advanced Access publication on February 20, 2013 doi:10.1093/humrep/det039
REVIEW Andrology
Spermatogonial stem cell preservation
and transplantation: from research
to clinic
E. Goossens 1,*, D. Van Saen 2, and H. Tournaye 1,2
1
Biology of the testis (BITE), Department for Embryology and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels 1090,
Belgium 2Centre for Reproductive Medicine, UZ Brussel, Laarbeeklaan 101, Brussels 1090, Belgium
*Correspondence address. Tel: +32-2-474-92-13; Fax: +32-2-477-46-32; E-mail: [email protected]
Submitted on December 13, 2012; resubmitted on January 17, 2013; accepted on January 28, 2013
study question: What issues remain to be solved before fertility preservation and transplantation can be offered to prepubertal
boys?
summary answer: The main issues that need further investigation are malignant cell decontamination, improvement of in vivo fertility
restoration and in vitro maturation.
what is known already: Prepubertal boys who need gonadotoxic treatment might render sterile for the rest of their life. As
these boys do not yet produce sperm cells, they cannot benefit from sperm banking. Spermatogonial stem cell (SSC) banking followed
by autologous transplantation has been proposed as a fertility preservation strategy. But before this technique can be applied in the
clinic, some important issues have to be resolved.
study design, size duration: Original articles as well as review articles published in English were included in a search of the
literature.
participants/materials, setting, methods: Relevant studies were selected by an extensive Medline search. Search
terms were fertility preservation, cryopreservation, prepubertal, SSC, testis tissue, transplantation, grafting and in vitro spermatogenesis.
The final number of studies selected for this review was 102.
main results and the role of chance: Cryopreservation protocols for testicular tissue have been developed and are
already being used in the clinic. Since the efficiency and safety of SSC transplantation have been reported in mice, transplantation
methods are now being adapted to the human testes. Very recently, a few publications reported on in vitro spermatogenesis in mice, but
this technique is still far from being applied in a clinical setting.
limitations, reasons for caution: Using tissue from cancer patients holds a potential risk for contamination of the collected
testicular tissue. Therefore, it is of immense importance to separate malignant cells from the cell suspension before transplantation. Because
biopsies obtained from young boys are small and contain only few SSCs, propagation of these cells in vitro will be necessary.
wider implications of the findings: The ultimate use of the banked tissue will depend on the patient’s disease. If the
patient was suffering from a non-malignant disease, tissue grafting might be offered. In cancer patients, decontaminated cell suspensions
will be injected in the testis. For patients with Klinefelter syndrome, the only option would be in vitro spermatogenesis. However, at
present, restoring fertility in cancer and Klinefelter patients is not yet possible.
study funding/competing interest(s): Research Foundation, Flanders (G.0385.08 to H.T.), the Institute for the Agency
for Innovation, Belgium (IWT/SB/111245 to E.G.), the Flemish League against Cancer (to E.G.), Kom op tegen kanker (G.0547.11 to H.T.)
and the Fund Willy Gepts (to HT). E.G. is a Postdoctoral Fellow of the FWO, Research Foundation, Flanders. There are no conflicts of
interest.
Key words: clinic / fertility / spermatogonial stem cell / transplantation / prepubertal boys
& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
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Goossens et al.
Introduction
Results
Throughout Europe, more and more institutions are now offering
fertility preservation to prepubertal boys facing the loss of spermatogonial stem cells (SSCs). At UZ Brussel, we started banking testicular
tissue in 2002 and since then, tissue from 47 boys has been stored
(Table I). One of these patients was a 4-year old boy, who was suffering from drepanocytosis. His parents came to our fertility department in 2002 to discuss the fertility preservation options for their
son. The boy had been treated with hydroxyurea for 6 months
and amoxicillin daily per os. When the boy was under anaesthesia
for placement of the portacath, a testicular biopsy was taken. A
small piece of the tissue was prepared for histological examination,
while the major part was stored for later use. The boy recovered
well and in 2011, the parents returned to discuss the possibility of
transplantation.
Although fertility preservation options have been discussed extensively in the literature, some important information is still lacking. Clinicians wanting to start testicular tissue banking in their centre often
turn to us if they have questions regarding patient inclusion, cryopreservation protocols or transplantation strategies. Therefore, our
aim in the present review was to compile these questions and find
answers or discuss potential solutions.
In the near future, patients will be referred back for transplanting
their banked SSCs, therefore there is an urgent need to resolve
unknown issues, so that we are able to counsel and treat our patients
and their parents appropriately.
Who should be offered preservation of SSC?
Methods
We conducted an extensive Medline search using following search terms:
fertility preservation, cryopreservation, prepubertal, SSC, testis tissue,
transplantation, grafting and in vitro spermatogenesis. Original articles as
well as review articles published in English were included. The final
number of studies selected for this review was 102.
Table I Overview of indications for testis tissue banking
at the UZ Brussel, Belgium.
Malignant diseases
n
Non-malignant diseases
n
........................................................................................
Leukaemia
8
Drepanocytosis
14
Testicular cancer
1
Klinefelter syndrome
10
Neuroblastoma
1
Thalassemia
3
Osteosarcoma
1
Granulomatous disease
2
B-cell lymphoma
1
Ideopathic medullar aplasia
1
Rhabdomyosarcoma
1
Medulloblastoma
1
Anaplastic ependymoma
1
Ewing sarcoma
1
Nasopharynxcarcinoma
Total
1
17
Total
n: samples collected and stored since 2002.
30
The first aim should always be to store sperm cells. In case the ejaculate does not contain mature sperm cells, testicular sperm extraction
(TESE) might be an option. However, when the testes are still immature, a testicular biopsy will be taken. Several groups of patients might
benefit from SSC banking.
Patients facing gonadotoxic treatments
Treatment regimens for cancer patients involve chemotherapy and
radiotherapy which target rapidly dividing cells. Although there is no
active production of spermatozoa in the prepubertal testis, it is not
a quiescent period of testicular development. The SSCs are constantly
dividing to populate the growing seminiferous tubules and are thus
targets for gonadotoxic treatments (Jahnukainen et al., 2011a).
Acute lymphoblastic leukaemia (ALL), being the most common
cancer type in children, involves treatment with low-risk regimens
(Jahnukainen et al., 2011a). Long-term follow-up of childhood ALL
survivors indicates that treatment not involving high-dose cyclophosphamide does not totally deplete SSCs and that spermatogenesis is
re-initiated from the surviving stem cell population (Nurmio et al.,
2009; Jahnukainen et al., 2011b). However, one-fifth of the children
with ALL experience relapse requiring intensive therapy including
alkylating agents, testicular irradiation and/or haematopoietic stem
cell transplantation, bringing them in the high risk population for
permanent sterility. When these patients want to bank testicular
tissue before a subsequent—and more severe—therapy, a substantial
reduction in spermatogonia may have occurred.
Spontaneous recovery of spermatogenesis is possible but this
depends on the survival of SSCs and their ability to differentiate.
Gonadal damage is related to the type of drugs, the total dose, the
fractionation schedule and the field of treatment. Irradiation doses
of 0.1 –1.2 Gy can cause impairment of spermatogenesis, while
doses of more than 4 Gy might cause permanent damage (Wallace
et al., 2005). Treatment regimens may thus be categorized into lowand high-risk treatments. High-risk treatments involve whole-body
irradiation, pelvic or testicular radiotherapy, chemotherapy conditioning for bone marrow transplantation and treatment with alkylating
drugs (Wallace et al., 2005). However, not all patients receiving alkylating agents are at high risk. The cumulative dose is the major risk
factor for permanent sterility (Table II). Patients receiving these treatments should be counselled for fertility preservation options.
Next to cancer patients, patients suffering from blood diseases are
often treated with chemo- and radiotherapy as a conditioning therapy
for bone marrow transplantation. These patients are at high risk for
lifelong sterility and should thus be offered testicular tissue banking.
Other drugs, such as hydroxyurea, which are administered to
patients with drepanocytosis, could also influence testicular function.
Long-term treatment with hydroxyurea can affect fertility (Berthaut
et al., 2008). Oligo-and azoospermia was reported in four patients
who received hydroxyurea starting from childhood to adulthood
(Lukusa and Vermylen, 2008). The impact of these treatments on
the ability to restore spermatogenesis in fertility preservation
methods is currently unknown.
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Translating transplantation strategies
Table II Alkylating agents with major sterilizing effects
in men.
Alkylating agent
Gonadotoxic
cumulative dose
........................................................................................
Cyclophosphamide
19 g/m2
Busulphan
600 mg/kg
Melphalan
140 mg/m2
Procarbazine
4 g/m2
Ifosfamide
42 g/m2
Chlorambucil
1.4 g/m2
At the moment, follow-up data on childhood cancer survivors are
scarce. In a follow-up study in 51 long-term survivors of childhood
ALL, testicular size and semen quality were not altered in patients
treated with ,10 g/m2 cyclophosphamide. However, decreased
serum-free testosterone levels were observed which are indicative
of impaired Leydig cell function. On the other hand, no or very few
spermatozoa were observed in patients who received higher doses
of cyclophosphamide or testicular irradiation (Jahnukainen et al.,
2011b). Sertoli cell impairment was observed 18 years after receiving
combination therapy in a 31-year-old azoospermic man (Bar-Shira
Maymon et al., 2004). More studies are definitely needed to estimate
the risk for infertility associated with different treatment conditions.
1999) or electroejaculation (Seager and Halstead 1993). However, if
sperm collection is not possible, testicular tissue recovery might be
performed. For patients receiving gonadotoxic treatment, the biopsy
can be taken under general anaesthesia at the time of placing the
central line. For Klinefelter patients, the testis should be biopsied as
soon as possible after diagnosis. We opt to take a large biopsy from
the lower pole of the largest testis (Gies et al., 2012). The technique
of a single large volume biopsy instead of the multiple biopsy sampling
method is preferred to avoid the risk of post-operative fibrosis and to
preserve maximal endocrine testicular function. We propose to
remove at least half of one testis, for two reasons: (i) testes of children
are small and contain only very few SSCs. To obtain enough SSCs to
recolonize an adult testis, small biopsies might be inadequate and (ii)
to have enough testicular tissue for several fertility restoration
methods/trials.
While the majority of the biopsy should be kept for later use, a
small testis fragment (6 mm3) should be fixed for histological analyses.
Performing analyses for tubular integrity and the presence of SSCs is
recommended.
There is a slight risk of bleeding or infection and the area may be
sore for 2–3 days after the biopsy. The scrotum may swell or
become discoloured and this should clear up within a few days of
the procedure. In general, the risks for acute and late-onset complications of testicular biopsy in children are considered low and hence
elective biopsies are frequently obtained in children, for example children with leukaemia (Chan et al., 1988) or even in children with
genetic disorders (Vogels et al., 2008).
Patients with Klinefelter syndrome
Apart from gonadotoxic treatments, sterility can also occur in patients
with Klinefelter syndrome (KS). KS is caused by the presence of one or
more extra X chromosomes (80% have 47,XXY, while the remaining
20% represent higher grade chromosome aneuploidies or mosaicisms). KS represents the most common genetic cause of azoospermia
and occurs in 1 in 600 newborn males. KS patients have a relatively
normal germ cell number in the testis at birth, but this gradually
declines during childhood (Wikström et al., 2004). In adult life the syndrome is associated with extensive fibrosis and hyalinization of the
seminiferous tubules, and Leydig cell hyperplasia. Patients experience
hypergonadotrophic hypogonadism and inhibin B levels are undetectable (Aksglaede et al., 2006). Most of the patients are diagnosed
because of their fertility problems. In 50% of the patients spermatozoa
can be collected using TESE which can be used in assisted reproduction techniques (ARTs) (Staessen et al., 1996; Tournaye et al., 1996;
Friedler et al., 2001; Vervaeve et al., 2004). Unfortunately, the remaining other half of the patients cannot father their own children. Cryopreservation of testicular tissue before SSC loss could be an option
for these patients. We started banking testicular tissue from KS
boys (age range: 13– 16 years) in 2009. So far, testicular tissue from
ten patients has been cryopreserved and spermatogonia were
observed in about half of them, an observation that is in agreement
with a Finnish study (Wikström et al., 2004, Van Saen et al., 2012).
Clinical testicular biopsy
How to biopsy testicular tissue?
The first aim should always be to retrieve spermatozoa. Spermatozoa
can be collected by masturbation, penile vibrostimulation (Brackett
Cryopreservation of SSCs
Should we store cell suspensions or tissue?
There are different options to cryopreserve SSCs: as a testicular cell
suspension, as an enriched cell suspension or as testicular tissue. Testicular cell suspensions contain several cell types differing in size, shape
and water content, requiring different freezing conditions and media
compositions for optimal preservation of viability and functionality.
The type of cryoprotectant, its concentration and the cooling rate
could all influence cell survival. For SSC transplantation (SSCT) it is
particularly important that the SSCs survive the freezing procedure
and maintain their functionality. Therefore, protocols have been established focusing on the preservation of spermatogonia (Avarbock et al.,
1996; Izadyar et al., 2002; Frederickx et al., 2004; Sá et al., 2012).
Frozen-thawed bovine spermatogonia were able to colonize the
mouse testis, although with lower efficiency compared with the
fresh population (Izadyar et al., 2002).
However, the cryopreservation of testicular tissue might be a better
option. Testicular tissue freezing preserves the cell –cell contacts, but
requires more permeable cryoprotectants. The best preservation of
morphology and hormonal activity was achieved when dimethylsulphoxide (DMSO) was used to freeze adult testicular tissue (Keros et al.,
2005). The feasibility of the selected protocol was later confirmed
using prepubertal testicular tissue (Keros et al., 2007). Most of the
centres perform tissue freezing, leaving the options open to perform
testicular tissue grafting, SSCT or in vitro maturation (IVM). It is not
clear whether enzymatic digestion of frozen-thawed tissue would
result in better viability compared with digestion before freezing. It
is possible that testicular tissue is better adapted to the hypoxic
900
conditions after freezing, since only the ‘strongest’ cells will survive the
freezing procedure.
Which protocol is optimal?
Most of the centres offering testicular tissue banking use a controlled
slow freezing protocol with DMSO as cryoprotectant (Keros et al.,
2007; Wyns et al., 2011). As this method is time consuming and
requires expensive equipment, a user-friendly and time-saving uncontrolled slow-freezing protocol was proposed. After having proved the effectiveness in a mouse model (Baert et al., submitted for publication),
the uncontrolled slow-freezing method using 1.5 M DMSO and 0.1 M
sucrose was validated using adult human testis tissue (Baert et al., submitted). This protocol did not only preserve the seminiferous epithelium
and the interstitial compartment at a (ultra)structural level, but the
spermatogonia also maintained the potential to divide. More research
is required to confirm these findings using human prepubertal tissue.
An alternative to conventional freezing is vitrification. Vitrification
involves the use of increasing concentrations of cryoprotectants and
ultrarapid cooling to avoid ice crystal formation. Recently, the efficiency of vitrification was explored using immature human testicular
tissue. The tubular integrity and the proliferation potential of spermatogonia could be maintained (Curaba et al., 2011).
Although difficult to assess, the effect on tissue functionality has to
be investigated by xenotransplantation or IVM. The effect of uncontrolled slow freezing of non-human primate testicular tissue has
been evaluated by xenografting. Different conditions were compared
and freezing with 1.4 M DMSO yielded better graft survival than
0.7 M DMSO and ethylene glycol (Jahnukainen et al., 2007).
However, xenotransplantation of primate or human tissue to the
mouse testis does not restore full spermatogenesis. In human xenografts, at least early germ cell differentiation can be observed up to
the spermatocyte level (Van Saen et al., 2012). Eventually, xenotransplantation to higher animal species, such as primates, can be considered. Recently, IVM of mouse testicular fragments has been
reported, but the efficiency was very low (Sato et al., 2011). At
present, this culture method is not yet reported for human tissue,
but it may be an option to assess functionality after cryopreservation
in the future.
As long as there is no efficient way to evaluate the functionality of
human testicular tissue after freezing, it is difficult to conclude which of
the tested protocols is optimal. It has been shown that cell viability
does not necessarily correspond to the functional capacity of the
SSCs (Frederickx et al., 2004).
Goossens et al.
could improve graft outcome. Cooled grafts were significantly
heavier, showed higher graft survival and more seminiferous tubules
containing spermatogenesis compared with samples grafted immediately (Jahnukainen et al., 2007). To optimize the conditions for transporting human prepubertal testis tissue, these experiments should be
repeated with human material.
Transplantation: different patients, different
strategies
Two transplantation strategies are currently being studied: the injection
of SSCs and the grafting of testicular tissue: the latter has the advantage
that SSCs can remain within their natural niche, thereby preserving
interactions between the germ cells and their supporting cells. Therefore, grafting may be regarded as a first-choice strategy. Only in cases
where the risk for malignancy recurrence is real, transplantation of
SSCs by injection might be proposed. An alternative approach would
be to mature SSCs in vitro in combination with ART. But as mentioned
earlier, this method has not been studied in men thus far (Fig. 1).
Grafting
Storage or transport of tissue before cryopreservation
Where to graft? Tissue can be grafted to an ectopic or homotopic
location. Initially, in mouse models, grafting was performed to
ectopic sites, such as in the peritoneal space, the ear or under the
skin on the back (Boyle et al., 1975; Schlatt et al., 2002). Full spermatogenesis was reported in xenografts from several species (Honaramooz et al., 2002; Schlatt et al., 2002, 2003, Honaramooz et al.,
2004; Oatley et al., 2004; Snedaker et al., 2004; Rathi et al., 2006,
Zeng et al., 2006; Abrishami et al., 2010), but never in marmoset
(Schlatt et al., 2002; Wistuba et al., 2004). The higher temperature
at these ectopic sites compared with the scrotum was suggested to
be the cause of either sclerosis of the graft or meiotic arrest. Consequently, tissue was grafted to the scrotum (Leutjens et al., 2008). A
recent study in which testicular fragments were autologeously
grafted to several locations in the irradiated primate body showed
that spermatogenesis could only be re-established when the graft
was placed in the scrotum (Jahnukainen et al., 2012). However, although many fragments were transplanted, a very low graft survival
was reported. Transplantation of the tissue under the tunica albuginea
of the testis (intratesticular grafting) might improve the efficiency
because, in mice, this technique proved highly efficient (Van Saen
et al., 2009). Compared with ectopic xenografting (Goossens et al.,
2008a), xenotransplantation of prepubertal human testis tissue into
the mouse testis also resulted in better graft survival and initiation
of germ cell differentiation (Van Saen et al., 2011).
In some cases immediate cryopreservation is not possible or wanted.
This may occur when, after procurement, the tissue has to be transported to a nearby hospital or laboratory. Therefore, short-term preservation of testis tissue may be required before cryopreservation. The
effect of storage medium, tissue sample size and hypothermia have
been investigated on porcine tissue (Yang and Honaramooz, 2010;
Yang et al., 2010). The results showed that testicular cells can be preserved for 6 days at 48C and maintain a viability of more than 80%.
The tissue sample size did not affect viability. In contrast, the
medium had a significant effect on viability, with Leibovitz
medium + 20% fetal bovine serum yielding the highest scores.
Cooling primate testicular tissue for 24 h at 48C before grafting
Are additional interventions necessary? In the mouse, a considerable loss
of SSCs after intratesticular grafting was reported (Van Saen et al.,
2011). This loss resulted from degeneration of tubules in the centre
of the graft, possibly due to hypoxia during the first days after grafting,
with the first 4 days after transplantation being critical (Van Saen
et al., in press). To reduce this SSC loss, testicular tissue fragments
should be kept as small as possible and/or early blood supply to the
grafted tissue should be stimulated. Vascular endothelial growth factor
(VEGF) is an important regulator of angiogenesis during development
as well as in adulthood (Holmes and Zachary, 2005). VEGF-treated
grafts showed increased graft weight and more tubules contained
Translating transplantation strategies
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Figure 1 Disease-specific strategies. According to the patient’s disease, the ultimate use of the banked tissue will be different. If the patient was
suffering from a non-malignant disease, testicular tissue will be biopsied and cryopreserved during treatment. Once cured, the patient might
return for intratesticular tissue grafting (green arrows). If, however, the patient has a risk of malignant-cell contamination in the testis (red dots),
the frozen-thawed biopsy has to be enzymatically digested to enable the removal of malignant cells by flow cytometry or differential plating. To
increase the chance of fertility restoration, spermatogonial stem cell (SSC) numbers need to be increased and/or SSCs will need to be enriched
in vitro. SSCs will be transplanted to the testis by the SSC transplantation technique (red arrows). For patients with Klinefelter syndrome, whose
testes might have atrophied by adulthood, the only option would be in vitro differentiation of SSCs to spermatozoa through organ or cell cultures,
and the spermatozoa produced could be used for ICSI (purple arrows).
elongating spermatids. These VEGF-treated grafts tended to have a
better vasculature compared with control grafts (Schmidt et al., 2006).
Next to its role in angiogenesis, VEGF also regulates SSC homeostasis
(Caires et al., 2012). Prior in vitro treatment with VEGF prevented
germ cell death and stimulated differentiation in bovine grafts (Caires
et al., 2009). Other factors which stimulate angiogenesis and germ cell
survival could also improve graft outcome. Treatment with
sphingosine-1-phosphate, for example, resulted in a higher blood
vessel density in ovarian transplants (Soleimani et al., 2011). However,
it is not clear which treatment strategy should be applied: in vitro stimulation of the tissue fragments with VEGF prior to grafting, injections with
VEGF at the graft site at the time of grafting or a combination of both.
Further research is needed to elucidate the role of these factors and
treatment strategies in reducing SSC loss after grafting.
When to graft? At present, it remains unknown whether testicular
grafts are better supported by an immature prepubertal environment
or by a mature one. The influence of the recipients’ maturation state
(pre-pubertal, pubertal or post-pubertal) on the efficiency of reestablishing spermatogenesis still has to be explored.
Initiation and conservation of spermatogenesis depends on the constant and controlled interaction of hormones. High FSH levels stimulate Sertoli cell proliferation and maturation, while LH induces
Leydig cell maturation and subsequent androgen production. The
endocrine environment at the time of grafting is important for
proper re-establishment of spermatogenesis. For ectopic grafting in
animal models, high FSH and LH levels were obtained by castration
(Schlatt et al., 2002). However, for obvious reasons, castration is
not considered in the human. Because high levels of FSH and LH
are observed just before puberty, this may be a good time for autologous grafting. However, at that time point, there may still be uncertainty about the health prognosis and the final impact of
gonadotoxic treatment on fertility. Indeed, patients’ fertility can
recover spontaneously, sometimes even after more than 10 years
(Lampe et al., 1997). Therefore, transplantation in adulthood would
be preferable. Adult patients will return to the fertility clinic when
902
natural conception does not occur. In contrast to other adult men,
these patients have undergone testicular biopsy or (hemi)orchiectomy and had gonadotoxic treatment. Hence, they have
already higher levels of FSH and LH, making both ectopic and homotopic grafting also feasible at adult age.
Increased FSH and LH levels might change the micro-environment
in the remaining testicle allowing proliferation and maturation of
Sertoli cells and Leydig cells. As a result, spontaneous fertility restoration might be advanced and grafting might not be necessary at all.
A good follow-up of the patients who have banked testis tissue is
thus extremely useful and indispensable for good counselling.
How long will grafts last? Evidence shows that spermatogenesis in
ectopic grafts may not be everlasting. Due to the lack of an excretory
system in the grafts, spermatozoa and fluid may accumulate causing
damage to the epithelium. At present, it is unclear whether macrophages will remove these excess sperm cells as they do after vasectomy. If not, spermatozoa will undergo ageing and lose their
fertilization capacity over time. Therefore, the ideal grafting period
should be determined allowing the collection of spermatozoa before
these processes begin. In mouse ectopic grafts, the increase in FSH
levels correlated with the damage of the seminiferous epithelium.
Changes in the serum FSH levels could thus be used to determine
the ideal time for sperm retrieval from the grafted tissue (Schlatt
et al., 2003). Ectopic grafting always has to be combined with ART.
Also for intratesticular grafts, it is still an open question whether
connections can be established between grafted and endogenous
tubules, ensuring a functional excretory system. As part of the endogenous tubules are removed—and thus pulled apart—before
placing the graft, this is theoretically possible. However, after grafting
in rodent models, the presence of donor-derived spermatozoa in
the epididymis has not been reported and, thus, the chance for
natural reproduction may be very small.
Fertilizing capacity after grafting. Little is known about the functionality
of the sperm generated in the grafts. Only a few groups have
addressed this question using mouse and rabbit donor tissue. ICSI
using sperm retrieved from ectopic and intratesticular mouse allografts
demonstrated that the spermatozoa were able to support full-term
development of progeny (Schlatt et al., 2003; Ohta and Wakayama,
2005). It was also possible to obtain offspring using rabbit sperm
that had developed in intratesticular xenografts (Shinohara et al.,
2002) and porcine sperm extracted from ectopic xenografts in mice
(Nakai et al., 2010). Normal blastocyst development in vitro was
obtained after performing ICSI with sperm from ectopic porcine and
monkey xenografts (Honaramooz et al., 2004, 2008). As it is not possible to perform such experiments using sperm obtained from human
xenografts, research on large mammals (e.g. non-human primates) will
be of great value.
SSC injection
In contrast to grafting strategies, natural conception might be possible
after the injection of SSCs into seminiferous tubules. This technique
was first described in the mouse almost 20 years ago (Brinster and
Zimmerman, 1994) and has since been used widely in both fundamental and translational research. Five years later, SSC injection was first
Goossens et al.
proposed as a fertility preservation strategy (Bahadur and Ralph,
1999).
How to transplant SSCs? In the mouse, a testicular cell suspension containing SSCs is introduced through the efferent duct in the rete testis.
This allows the filling of several seminiferous tubules with one single
puncture (Ogawa et al., 1997). Because of differences in anatomy
and consistency (texture) of the tissue, larger testes, for example
bovine, primate, human, are not easy to inject via the efferent
ducts. The rete testis, which can be visualized by ultrasound, proved
to be a better injection site (Schlatt et al., 1999). Compared with a
single injection, multiple injections of acellular solutions did not
improve the filling efficiency of human cadaver testes (Ning et al.,
2012). We experienced that testes from aging donors (aged 40–65
years) were more difficult to inject than the ones from aged donors
(aged 65–83 years), because the older testes have less tension.
Since testes of boys who were treated with testicular irradiation or
with high doses of alkylating agents do not contain differentiated
germ cells (Relander et al., 2000), their testes may have a consistency
similar to those from the aged donors. Because the inner testicular
pressure in testes in vivo might be higher, the infusion technique
might have to be adapted to be applicable in a clinical setting. As
these experiments are not possible in the human, experiments on primates or other large mammals are of high value. Very recently, rhesus
monkey SSCs have been used for autologous and allogeneic transplantation. SSC infusion was performed using ultrasound-guided rete
testis injections. Cells were injected under slow constant pressure
and chased with saline. After having reached maturity, mature
sperm cells could be found in the ejaculate in 60% of the
prepubertal recipients and sperm cells were able to fertilize oocytes
in ICSI. This demonstration of functional donor spermatogenesis
following SSCT in non-human primates is an important milestone in
the translation of SSC transplantation to a clinical setting (Hermann
et al., 2012).
Are additional interventions necessary? Only SSCs will relocate to the
basement membrane, while the somatic and more advanced germ
cells will be eliminated primarily through uptake and phagocytosis by
Sertoli cells (Parreira et al., 1998). The quantity of cells injected
(and thus the fraction of SSCs in the injected suspension) is the determining factor for the success of transplantation.
Several enrichment strategies have been proposed. The first
attempts made use of sedimentation velocity in combination with
differential plating (Morena et al., 1996; Dirami et al., 1999), but the
efficiency was improved when sorting techniques [using
magnetic-activated cell sorting (MACS) or a fluorescence-activated
cell sorter (FACS)] were employed. In mice, highly enriched SSC
populations were obtained using the following marker sets:
b1-integrin+/a6-integrin+/c-kit2/aV-integrin2 (Shinohara et al.,
2000) or MHC-I2/Thy-1+/c-kit2 (Kubota et al., 2003). During the
last few years many other markers for spermatogonia have been discovered (for review: Phillips et al., 2010). Inhibitor of DNA binding 4
(ID4) might be the marker that is most specific for SSCs (Oatly et al.,
2011). Human SSCs share several markers with mouse SSCs: a6-integrin, GFRa1 and THY1. However, other markers are not
shared: a1-integrin, TSPY1, CD133 and SSEA4 (Dym et al., 2009).
The search for (more) specific SSC markers is ongoing. But for clinical
903
Translating transplantation strategies
applications, a stringent selection to obtain only SSCs might not be
needed. In the injured testis, undifferentiated spermatogonia might
revert to SSCs and restore spermatogenesis (Nakagawa et al., 2007).
However, even if spermatogonia could be highly enriched, for clinical application it might still be unsatisfactory. Indeed, biopsies
obtained from young boys are small and contain only few SSCs. Therefore, the propagation of these cells in vitro will be necessary. In 2009,
such a culture system was described starting from adult testicular
tissue (Sadri-Ardekani et al., 2009). Two years later, the same group
validated their results on prepubertal testis tissue (Sadri-Ardekani
et al., 2011). They obtained a more than 18000-fold increase in SSC
number after 2 months of culture. As it has been estimated that a
1300-fold increase would be adequate to repopulate an adult
human testis, a 1-month period of culture should be sufficient.
Before this culture system can be applied in the clinic, more research
has to be carried out on the (epi)genetic stability of the cells in culture,
the competence of these cells to repopulate the basal membrane and
the fertilizing capacity of the spermatozoa generated from SSCs propagated in vitro. Also, animal-derived media components need to be
replaced by clinical grade components.
examining the methylation pattern in mouse spermatozoa did not
reveal any differences in three studied genes (Goossens et al.,
2009). The general methylation status and specific histone modifications were studied in post-transplantation germ cells in a stagedependent manner and were no different compared with control
spermatogenesis. However, a deficient expression of two histone
modifications was found after SSCT (Goossens et al., 2011). Although
H4K5ac and H4K8ac expression in early germ cells differed from controls, the acetylation of H4 was correct at the time when histones have
to be exchanged for protamines (in spermatids). The relationship
between epigenetic changes and spermatogenesis is still largely
unknown. It is still unclear why some modifications occur only in
certain stages and what role they play, for example H4 acetylation
in spermatogonia and spermatocytes. It is therefore interesting to
carry out fundamental research into the exact functions of these modifications and their impact on germ cell development or fertility.
It would also be worthwhile to perform efficiency and safety studies
after autologous transplantation in larger mammals.
When to transplant SSCs? In mice, it has been reported that colonization efficiency is higher when SSC transplantation is performed in
immature recipients compared with adult recipients who have been
sterilized by chemotherapy (Ogawa et al., 1999). However, because
in these experiments, immature recipients did not receive a sterilizing
treatment the validity of the results can be questioned. The study of
Hermann et al. (2012) showed that spermatozoa could be obtained
both after autologous transplantation of SSCs in either prepubertal
or adult recipients. This is an important finding for any future clinical
application, because it implies that SSC transplantation may be postponed for several years until it becomes clear whether the patient is
infertile.
In contrast to spermatogenesis established in testicular tissue grafts,
spermatogenesis might be retained lifelong after SSC injection. Indeed,
serial transplantation experiments in mice showed that SSCs were still
able to self-renew and to colonize recipient mouse seminiferous
tubules after at least nine successive transplantations (Ryu et al.,
2006). As SSCs in human have different characteristics than those in
rodents, similar experiments in animals more closely related to
humans should be conducted.
What if there is a risk that the biopsy contains malignant cells?
Fertilizing capacity after SSC transplantation. A number of studies evaluating the efficiency of fertilization after SSCT have been performed.
Compared with fertile controls, the fertilization capacity of spermatozoa obtained after SSCT was reduced both after in vivo conception and
IVF, but not after ICSI (Goossens et al., 2003). The lower fertilization
rate after IVF could be explained by a reduced motility of the spermatozoa as was shown by computer-assisted motility analysis (Goossens
et al., 2008b). Live born offspring obtained after SSCT were found to
be healthy. They showed normal weights and lengths and were fertile.
Numerical chromosomal aberrations could not be detected in
spermatozoa from transplanted males, or in their offspring (Goossens
et al., 2010).
Since epigenetic modifications are essential for several developmental events, it is important that epigenetic characteristics are established
correctly during spermatogenesis and in the offspring. A study
Other unanswered questions
Many paediatric malignancies are capable of metastasizing through the
blood, with a potential risk for contamination of the collected testicular tissue. The transplantation of as few as 20 leukaemic cells could
cause malignant recurrence in rats (Jahnukainen et al., 2001). In the
human, the threshold number of malignant cells able to cause malignant relapse when transplanted to the testis is unknown. Therefore,
it is of immense importance to detect even the slightest contamination
of the testicular tissue. In case of contamination, the separation of
SSCs from malignant cells before transplantation is necessary. For
this purpose, the use of MACS and/or FACS for depleting cancer
cells from mouse and human testicular cell suspensions has been
studied but was reported to be insufficient (Fujita et al., 2005 and
2006; Geens et al., 2007). Also, cell selection by selective matrix
adhesion was not sufficiently efficient (Geens et al., 2011). The poor
specificity of spermatogonial surface markers and the aggregation of
germ and leukaemic cells was the limiting factor in positive selection
of germ cells. Applying singlet discrimination can circumvent
miss-sorting resulting from cell clumping (Hermann et al., 2011).
Also, immunophenotypic variation among lymphoblastic leukaemia
cells can hamper the efficient removal of leukaemic cells. This immunophenotypic variation could be caused either by the enzymatic treatment or by the intratesticular environment. Therefore, a combination
of both positive and negative selection will be necessary to remove all
cancer cells and prevent leukaemia transmission (Hou et al., 2007a).
Since decontamination results are not conclusive, other decontaminating strategies should be tested. It is possible that the culture system
for propagating SSCs does not support the growth of malignant cells,
but this still needs to be proved.
Xenografting could offer an alternative. Germ cells produced in
ectopic xenografts could then be used in ART. However, up to now
only spermatogonial survival was achieved in ectopic human prepubertal xenografts (Goossens et al., 2008b). Xenografting also raises ethical
and safety concerns. An important issue for public health with regard
to xenotransplantation is the risk of unintended introduction of xenogeneic infections into the human population. Endogenous retroviruses
904
exist as proviral DNA in the germ lines of all mammals. These retroviruses remain as an inherited part of the genetic structure and do not
cause disease in the host species. However, they are able to infect
cells from other species (Chapman, 2009).
Xenotransplantation could, however, serve to detect contamination
in cryopreserved tissue (Kim et al., 2001; Hou et al., 2007b). While
classical histology failed to detect malignant cells in ovarian tissue
from leukaemic patients, grafting and quantitative real-time PCR
showed the presence of contaminated cells (Dolmans et al., 2010).
Quantitative real-time PCR could be a very sensitive technique to
detect residual minimal diseases using patient-specific primers encoding the rearranged B- or T-cell receptor of the malignant clone (Geens
et al., 2007). Still the question remains of how safe transplantation will
be. The absence of contaminating cells in the screened piece of tissue
cannot guarantee their absence in another part of the tissue. Therefore, for patients at risk for malignant contamination, autologous testicular tissue grafting will not be possible, so that the only option to
restore fertility will be by transplanting cell suspensions.
What in case of atrophied testes at the time of transplantation?
Apart from malignant diseases, sterility because of germ cell loss can
also occur in patients with KS (47,XXY). Fifty per cent of these
boys still have germ cells in their testes around puberty. Nevertheless,
these germ cells will disappear after puberty (Wikström et al., 2004).
Whether SSC transplantation or grafting may be an option in boys
with KS remains unknown because the majority of them will show
degenerated testes at adult age (Aksglaede et al., 2006). Alternatively,
SSCs may be cultured in vitro to produce sperm for ART, for example
ICSI. In the initial attempts to achieve in vitro spermatogenesis, spermatogonia were co-cultured with Sertoli cells. Haploid cells were
detected by flow cytometry and spermatid-specific markers were
expressed in rat and bull cultures (Lee et al., 2001; Iwanami et al.,
2006). However, their functionality could not be proved as no
viable offspring were produced after performing microinjection into
activated oocytes (Iwanami et al., 2006). The spatial arrangement of
testicular cells in their natural environment cannot be achieved by
the conventional culture methods. A three-dimensional
soft-agar-culture system was designed to mimic the in vivo features
of the testis. When mouse SSCs were cultured in a three-dimensional
soft agar culture system in the presence of somatic testicular cells, differentiation up to the level of post-meiotic cells was achieved (Stukenborg et al., 2008). Spermatozoa were observed when cell suspensions
were cultured in the presence of gonadotrophins (Stukenborg et al.,
2009). However, the functionality and safety of these in vitro produced
spermatozoa still needs to be proved. Recently, Sato et al. (2011)
reported the production of spermatozoa in organ culture in which
fresh and frozen-thawed testis tissue fragments of mouse pups were
cultured on agar half-soaked in medium. Round spermatids and
spermatozoa were collected from cultured tissue fragments and
used for round spermatid injection and ICSI, respectively. Live
progeny were born.
So far, in vitro generation of human sperm cells from SSCs has not
been reported. Late spermatids could be produced from late pachytene spermatocytes and secondary spermatocytes in co-cultures
obtained from azoospermic patients. The in vitro matured spermatids
were microinjected into oocytes, but showed low fertilization rates
and chromosomal abnormalities were found in all generated
Goossens et al.
embryos (Sousa et al., 2002). The initiation of meiosis in vitro
remains the critical event to achieve full spermatogenesis. More recently, haploid human cells were generated using isolated CD49f+
cells from an azoospermic patient (Riboldi et al., 2012). Future research is definitely needed to establish full human spermatogenesis
in vitro.
In vitro spermatogenesis could also be an alternative to circumvent
the transmission of malignant cells to patients at risk for malignant contamination in their testis, for example leukaemic boys. However, in
contrast to SSC transplantation, this strategy entails the need for ART.
Conclusion
Taking into account the progress that already has been made and
the barriers that still have to be overcome, the first patients who
will probably undergo SSC transplantation are those who suffered
from non-malignant diseases (for example sickle-cell anaemia
treated by bone marrow transplantation) as for these patients there
will be no risk of re-introducing malignant cells into the testis. Grafting
testicular tissue is a relatively easy technique but the question remaining is how much, and where, the tissue should be grafted. As long as
there is no solution for the early hypoxia seen in testicular tissue grafts,
necrosis will occur during the first few days after grafting (Van Saen
et al., in press). Because the testicular fragments are small and might
contain only few SSCs, the loss of even a few of these cells may
have a substantial impact on fertility restoration. Also, as shown in a
recent study on primates, multiple tissue fragments should be
grafted to have a realistic chance of retrieving spermatozoa (Jahnukainen et al., 2012). Therefore, we would recommend transplanting multiple tissue fragments to different sites on the body: under the skin, in
the scrotum and in the testis. It would also be useful to keep some of
the stored tissue for a—possible—second grafting or for other strategies (e.g. in vitro spermatogenesis, xenografting).
At this point in time, restoring fertility in patients who have been
treated for cancer is a bridge too far. The frozen-thawed biopsy has
to be enzymatically digested to enable the removal of malignant
cells using cell sorting strategies or differential cell culture. However,
at present, this is not possible. Once the tissue is digested, grafting
is no longer feasible, and the only remaining option is to inject the
cell suspension in the testis. As in the mouse, it has been estimated
that only 12% of the SSCs can colonize the recipient testis (Nagano,
2003) and, because biopsies taken from boys are small, SSCs should
be propagated in vitro prior to injection to increase the chance for fertility restoration. However, to date, the safety aspects of the in vitro
culture systems, as regards (epi)genetic stability, remain to be
examined.
For patients with KS, yet another strategy is required, because
47,XXY testes may become atrophied by adult age. Grafting and
cell injection are thus inappropriate approaches. The only option
would be in vitro differentiation followed by ICSI. As research on
in vitro spermatogenesis in mice is only starting, this method will probably not be applicable for humans in the years to come.
This review started with the presentation of a boy who had been
suffering from a non-malignant disease and who later returned to
the fertility department to ask about fertility restoration strategies.
His indication would have favoured the grafting of testicular tissue fragments. However, as the boy was still pubertal at the time of
Translating transplantation strategies
consultation, the impact of gonadotoxic treatment remained unclear.
Therefore, we advised the boy and his parents to wait a few more
years and return for transplantation only if normal fertility appeared
to be compromised.
Authors’ roles
E.G. designed the review concept, performed the research and
drafted the review, D.V.S. and H.T. performed the literature research
and revised the review.
Funding
Our research is supported by grants from the Research Foundation,
Flanders (G.0385.08 to HT), the Institute for the Agency for Innovation, Belgium (IWT/SB/111245 to EG), the Flemish League against
Cancer (to EG), Kom op tegen kanker (G.0547.11 to HT) and the
Fund Willy Gepts (to HT). E.G. is a Postdoctoral Fellow of the
FWO, Research Foundation, Flanders.
Conflict of interest
None declared.
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