ORIGINAL ARTICLE Andrology

Vitrication preserves proliferation

capacity in human spermatogonia
Jonathan Poels
1,2
, Anne Van Langendonckt
1,2
, Marie-Christine Many
3
,
Francois-Xavier Wese
4
, and Christine Wyns
1,2,
*
1
Gynecology Unit, Medical School, Institut de Recherche Experimentale et Clinique, Universite Catholique de Louvain, Avenue Mounier, 52,
1200 Brussels, Belgium
2
Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Avenue Hippocrate, 10, 1200 Brussels,
Belgium
3
Experimental Morphology Unit, Medical School, Institut de Recherche Experimentale et Clinique, Universite Catholique de Louvain,
1200 Brussels, Belgium
4
Urology Unit, Medical School, Institut de Recherche Experimentale et Clinique, Universite Catholique de Louvain,
1200 Brussels, Belgium
*Correspondence address. Tel: +32-2-764-95-01; Fax: +32-2-764-95-07; E-mail: christine.wyns@uclouvain.be
Submitted on October 25, 2012; resubmitted on November 28, 2012; accepted on December 10, 2012
study question: Does vitrication of human immature testicular tissue (ITT) have potential benets for future fertility preservation?
Does vitrication of human ITT have potential benets in an in vivo murine xenotransplantation model?
summary answer: Vitrication is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting.
what is known already: Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice.
Vitrication has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem
cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrication of ITT, in vitro survival of SSCs was demon-
strated, but their functionality was not evaluated.
study design, size, duration: Ten ITT pieces issuing from 10 patients aged 212 years were used. Fragments of fresh tissue
(serving as controls) and fresh, frozen-thawed and vitried-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months
were compared.
materials, setting, methods: Upon graft removal, histological and immunohistochemical analyses were performed to evaluate
spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3b-HSD). The entire piece of grafted
tissue was assessed in each case.
main results and the role of chance: Seminiferous tubules showed good integrity after cryopreservation and xenografting
for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG
were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 +3.8, 4.1 +7.3
and 7.3 +6.3%, respectively, for fresh, slow-frozen and vitried-warmed tissue after 6 months of xenografting.
limitations, reasons for caution: The study is limited by the low availability of ITT samples of human origin. The mouse
xenotransplantation model needs to be rened to study human spermatogenesis.
wider implications of the ndings: The ndings of the present study have potential implications for cryobanking of ITT and
fertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to be
optimized. There are so far no convincing data justifying modication of current clinical practice for ITT storage with slow-freezing, but this
study demonstrates that it is worth pursuing optimization of ITT vitrication as an alternative for preservation of SSCs.
study funding/competing interest(s): The present study was supported by a grant from the Fonds National de la Re-
cherche Scientique de Belgique (grant Televie N
8
7. 4.572.09.F). The authors declare that there is no conict of interest.
Key words: vitrication / cryopreservation / spermatogonia / testicular tissue / xenografting
& 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: journals.permissions@oup.com
Human Reproduction, Vol.28, No.3 pp. 578589, 2013
Advanced Access publication on January 12, 2013 doi:10.1093/humrep/des455

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Introduction
With increasing effectiveness of childhood cancer treatments, survival
rates are on the rise, and it is estimated that .80% of children survive
their disease (Magnani et al., 2006; Arndt et al., 2007; Gatta et al.,
2009). Unfortunately, improvements in treatment efcacy go hand in
hand with increased toxicity, especially gonadotoxicity. Cryopreserva-
tion of immature testicular tissue (ITT) containing spermatogonial
stem cells (SSCs) is so far the only approach that can be proposed
to preserve fertility in young boys, since spermatozoa are not pro-
duced before puberty. Slow-freezing of ITT is currently offered to pre-
pubertal boys whose fertility is threatened by gonadotoxic treatments
(Wyns et al., 2010). Two experimental options can be considered to
restore fertility from cryostored tissue: autotransplantation of testicu-
lar cells, cellular aggregates or tissue and in vitro maturation of SSCs
(Tournaye et al., 2004; Wyns et al., 2010). Encouraging results were
achieved in mice. Indeed, autotransplantation of ITT, cryopreserved
by slow-freezing, resulted in the birth of pups (Shinohara et al.,
2002; Wu et al., 2012).
In humans, a mouse xenotransplantation model was used to evalu-
ate cryopreservation protocols. In vivo survival, proliferation and initi-
ation of differentiation of spermatogonia (SG) were achieved after 6
months of orthotopic xenografting of slow-frozen human ITT to
nude mice (Wyns et al., 2008). However, rapid loss of SG was
recorded, with recovery rates of 14.5% after 3 weeks (Wyns et al.,
2007) and 3.7% after 6 months (Wyns et al., 2008), and differentiation
appeared to be limited to the pachytene stage.
Vitrication is an innovative strategy preventing ice crystal formation
by the use of high concentrations of cryoprotectant and ultrafast
cooling speeds, which could minimize cellular damage (Amorim
et al., 2011). In mice, promising results were obtained after ITT vitri-
cation followed by 3 days of organotypic culture, since no difference
in seminiferous tubule cellular density and integrity, cell viability, pro-
liferation or apoptosis was observed between vitried and slow-frozen
tissue (Curaba et al., 2011a). Normal spermatogenesis and round
spermatids were also obtained after xenotransplantation of vitried
pig ITT (Abrishami et al., 2009; Zeng et al., 2009). More recently, vit-
rication of non-human primate ITT showed preservation of tissue in-
tegrity, maintenance of proliferating SG and functional Leydig cells
(LCs) after xenotransplantation (Poels et al., 2012).
In humans, while the potential of vitrication to maintain proliferat-
ing SG after short-term organotypic culture has been reported, vitri-
cation of ITT has never been evaluated in vivo (Curaba et al., 2011b).
The objective of this study was:
(i) To evaluate vitrication as a potentially efcient cryopreservation
method for human ITT with a view to fertility preservation in
humans.
(ii) To compare SG survival and differentiation after xenotransplant-
ation of fresh, frozen-thawed (according to the protocol currently
applied in clinics) and vitried-warmed human ITT.
For this purpose, testicular tissue was orthotopically xenografted using
our mouse model previously developed for functional assessment of
cryopreserved tissue (Wyns et al., 2008).
Materials and Methods
Study design
Small pieces of ITT were obtained from 10 prepubertal boys. A small
sample was taken from each and xed in Bouins solution to serve as
fresh non-grafted controls. The biopsy was then divided into three equal
pieces allocated to the three grafting groups. One piece was immediately
grafted into the scrotum of nude mice, serving as fresh grafted controls.
One piece was frozen, stored for 24 h, thawed and grafted similarly
(frozen grafts). The third piece was vitried, stored for 24 h, warmed
and grafted to a third mouse (vitried grafts). After 6 months, the grafts
were recovered and directly xed in Bouins solution, embedded in paraf-
n and cut into serial sections.
Histological analysis was performed on hematoxylineosin (HE)-stained
sections to assess seminiferous tubule integrity and the germ cell
differentiation stage. SG were evidenced by immunostaining with
melanoma-associated antigen 4 (MAGE-A4; mouse anti-human monoclo-
nal antibody puried from hybridoma 57B, kindly provided by Giulio
Spagnoli, MD, University of Basel, Switzerland) and LCs were evaluated
after immunostaining with 3b-hydroxysteroid dehydrogenase (3b-HSD),
a key enzyme of steroidogenesis. Intratubular cell proliferation was
assessed after Ki67 immunostaining, and double immunostaining with
MAGE-A4 and Ki67 was applied to identify proliferating SG.
Animals
Thirty NMRI nu/nu mice (Janvier Laboratories, Le Genest-St-Isle, France)
aged between 4 and 8 weeks were used as recipients for the xenografts.
They were housed in cages under ltered hoods (MicroIsolator, Uno,
Brussels, Belgium) in rooms maintained at an ambient temperature
between 22 and 248C with a day/night cycle of 12 h. All housing material
and food were autoclaved before use. The mice were fed ad libitum on
laboratory chow (complete food for rats and mice; Pavan Carl,
Oud-Turnhout, Belgium) and acidied water. All experiments in this
study were approved by the Ethics Review Board and the Committee
on Animal Research of the Catholic University of Louvain.
Donor testicular tissue
ITT was retrieved from 10 boys aged between 2 and 12 years (2, 2, 4, 8, 9,
10, 11, 11, 12 and 12 years) after obtaining informed consent from the
parents and the childs ascent (where applicable). Patients were referred
by pediatric oncologists or hematologists to the reproductive specialist
in fertility preservation, when they considered that the risk of infertility
due to treatment was high and/or the parents specically requested fertil-
ity preservation techniques. All donors were scheduled for testicular
biopsy prior to gonadotoxic treatment. Disease and gonadotoxic treat-
ment are shown in Table I. Unilateral testicular sampling of ,5% of the
total testicular volume (based on theoretical size by age from 0 to
12-year-old: 0.75 to 2.0 cm
3
; Beres et al., 1989) was performed by a pedi-
atric urologist through scrotal incision. The majority of the collected tissue
was used for the boys fertility preservation. Individual testicular sampling is
reported in Table II.
Testicular tissue was transferred in Hanks buffered salt solution (HBSS,
Gibco, Merelbeke, Belgium) on ice to the laboratory. It was manually dis-
sected and cut into pieces. For each donor, a small piece (+1 mm
3
) xed
in paraformaldehyde or Bouins solution (sent to the laboratory of anato-
mopathology) served as non-frozen control for light microscopy (LM) and
immunohistochemical analysis. One piece of ITT (+1 1 3 mm) from
each boy was used for our experiment and divided into three pieces
(+1 mm
3
) allocated to the three grafting groups.
Vitrication preserves proliferation capacity in human SG 579

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Ethics approval and consent procedure
All experiments in this study were approved by the Ethics Review Board of
the Catholic University of Louvain. The ethics committee agreed to tes-
ticular biopsy for research purposes only when testicular surgery was
required for the childs fertility preservation and after obtaining informed
consent. Parents (or legal guardians) and the child had a consultation
with a specialist in reproductive medicine. Potential fertility restoration
approaches from stored samples were explained to each individual child
(when applicable, usually from the age of 5 with adapted language) and
........................................................................................
Table II Testicular sampling.
No. Age
(years)
Total
amount of
removed
tissue
(mm
3
)
Total
amount of
removed
tissue for
research
(mm
3
)
Proportion
destined for
research (%)
1 12 53.5 4 7.5
2 9 34 4 11.8
3 4 26 4 15.4
4 8 24 4 16.7
5 11 32 4 12.5
6 12 69 4 5.8
7 11 58 4 6.9
8 10 61 4 6.6
9 2 34 4 11.8
10 2 31 4 12.9
........................................................................................
Table I Patient background.
No. Age
(years)
Pathology Best estimated risk
of gonadotoxicity
of planned
treatment after
testicular biopsy
a
1 12 Homozygous sickle cell
disease
High
2 9 Neuroectodermaltumor Intermediate
3 4 Acute lymphoblastic
leukemia
Low
4 8 Acute lymphoblastic
leukemia
Low
5 11 Osteosarcoma High
6 12 Ewings sarcoma High
7 11 Hodgkins lymphoma Intermediate
8 10 Embryonal
rhabdomyosarcoma
Intermediate
9 2 Anaplastic
medulloblastoma grade 4
Intermediate
10 2 Abdominal
neuroblastoma stage IV
High
a
Classication refers to previously published data (Wyns et al., 2010).
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his parents, making sure they understood that with stored ITT, there is no
guarantee of success as yet. In all cases, parents or legal guardians gave
their signed informed consent for cryobanking, as well as the young
boys themselves, if they were mature enough to understand the implica-
tions of the procedure.
Cryopreservation protocols
The slow-freezing protocol was previously described by Wyns et al.
(2007). Briey, tissue pieces were placed in 1 ml freezing medium with
dimethyl sulfoxide 0.7 M (DMSO, Sigma Aldrich, Bornem, Belgium) and
sucrose 0.1 M (Sigma Aldrich) at 48C in a 2 ml cryovial (Nunc,
Denmark). Using a controlled freezer (Minicool 40 PC Air Liquide,
Marne-la-Vallee, France), the vials were maintained at 08C for 9 min,
cooled at a rate of 20.58C/min to 288C and then held for 5 min
before seeding manually at 288C. After holding for a further 15 min at
288C, a cooling rate of 20.58C/min was used from 288C to 2408C
before nal dehydration for 10 min at 2408C. After cooling at 278C/
min to 2808C, the vials were transferred to liquid nitrogen (21968C).
Figure 1 Histological appearance of non-grafted control tissue (A and A

: 12 years; A

: 2 years), and fresh (B and B

: 8 years; B

: 11 years),
slow-frozen (C and C

: 2 years; C

: 4 years) and vitried (D and D

: 9 years; D

: 2 years) ITT grafted for 6 months to nude mice. Seminiferous

tubule integrity was well preserved in all groups. A, B, C and D, scale bar 200 mm (magnication 100). A

, A

, B

, B

, C

, C

and D

, D

, scale
bar 100 mm (magnication 200) (ITT, immature testicular tissue).
Vitrication preserves proliferation capacity in human SG 581

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For thawing, the cryopreserved tissue was kept for 2 min at room tem-
perature (RT), thawed in a water bath at 378C for 2 min, and then
washed three times in a reversed sucrose concentration gradient solution
(0.1, 0.05 and 0 M sucrose) for 5 min per bath, using HBSS medium on ice.
For vitrication, the protocol of Abrishami et al. (2009) was applied,
slightly modied (Poels et al., 2012). Briey, testicular tissue was pre-
treated with an equilibration solution (5 ml) consisting of 7.5% (v/v) ethyl-
ene glycol (EG, Sigma Aldrich), 7.5% (v/v) DMSO and 0.25 M sucrose in
Leibovitz L-15 (L-15, Sigma Aldrich), supplemented with 25 mg/ml human
serum albumin (HSA 20%, Cealb 2 g/10 ml, Brussels, Belgium) for 10 min
at 48C. It was then transferred to the vitrication solution (5 ml) consisting
of 15% EG, 15% DMSO and 0.5 M sucrose in L-15 medium, supplemented
with 25 mg/ml HSA for 5 min at 48C.
The tissue was then placed on a piece of gauze to remove the surround-
ing vitrication medium, transferred to open cryostraws (Paillette CBS
0.5 ml, Cryo Bio System, Aigle-France, France), and plunged into sterile
liquid nitrogen according to Parmegiani et al. (2009). The straws were
inserted into precooled cryotubes (Nunc, Cryotube Vials, 1.8 ml,
Denmark), sealed and stored for 24 h in liquid nitrogen.
For warming, the cryotubes were removed from the liquid nitrogen and
the straws were quickly immersed in a 358C warming solution containing
sucrose (1 mol/l) in L-15 medium, supplemented with 25 mg/ml HSA.
The testicular tissue pieces were then serially transferred to three baths
of warming solutions with decreasing sucrose concentrations (0.5, 0.25
and 0 mol/l) for 5 min each.
Liquid nitrogen sterilization
Sterilization of liquid nitrogen (LN
2
) was performed according to Parme-
gianis protocol (Parmegiani et al., 2009) adapted to our materials.
Briey, an ultraviolet C (UVC) lamp (Osram 15W HNS, 253.7 nm, UV in-
tensity 1 m: 49 mW/cm
2
) was used to expose LN
2
to UVC radiation. The
dewar with LN
2
was placed 10 cm from the UVC lamp for 15 min based
on the UV dose required to eliminate the most UV-resistant micro-
organism (330 000 UV dose for Aspergilus niger; Srikanth, 1995) using
the calculation UV dose UV intensity (I ) resistance time (T ). After
formula transformation, the following result was obtained: T 330 000/
490 (at 10 cm) 673.5 s or 11.22 min.
Xenografting
The mice were anesthetized by intraperitoneal injection of ketamine
(75 mg/kg; Anesketin, Eurovet, Heusden-Zolder, Belgium) and medetomi-
dine (1 mg/kg; Domitor, Pzer, Cambridge, USA) dissolved in phosphate-
buffered saline. They underwent bilateral castration and, in the course of
the same surgery, +1 mm
3
pieces of fresh, slow-frozen or vitried-
warmed donor testicular tissue were grafted without vascular anastomosis
into the scrotum, according to a previously described procedure (Wyns
et al., 2007). After surgery, anesthesia was reversed by injection of atipa-
mezole (1 mg/kg; Antisedan, Pzer). Analgesia was provided by buprenor-
phine (0.1 mg/kg, Temgesic, Schering Plough, Kenilworth, NJ, USA) on the
day of surgery and the following day.
Graft recovery
After 6 months, the mice were anesthetized by intraperitoneal injection of
ketamine, euthanized by intracardiac blood puncture and the grafts were
recovered and directly xed in Bouins solution. The totality of the
grafted tissue was used for analysis.
Histological evaluation of grafted testicular
tissue
After xation in Bouins solution, tissue samples were embedded in paraf-
n and cut into 5 mm-thick serial sections.
One section every 50 mm was stained with HE for histological evalu-
ation by LM. Subsequent sections were mounted on Superfrost Plus
slides and used for immunohistochemistry. Digital images were captured
with a Mirax Midi digital camera (Zeiss Mirax Midi, Zeiss, Germany).
Seminiferous tubule integrity was evaluated on HE-stained sections
under a light microscope at 400 magnication. Tubules were considered
intact when good adhesion of cells to the basement membrane, good cell
cohesion and no sclerosis were observed.
Immunohistochemical analyses
MAGE-A4, Ki67 and 3b-HSD immunostaining
MAGE-A4 mouse anti-human monoclonal antibody was used to evidence
SG. This antibody, puried from hybridoma 57B, was kindly provided by
Giulio Spagnoli, MD (Yakirevich et al., 2003).
Figure 2 Spermatogonia differentiation to the pachytene stage in
slow-frozen (A) and vitried (B) grafts from a 9-year-old donor
and vitried (C) graft from a 2-year-old donor. Scale bar 50 mm
(magnication 400).
582 Poels et al.

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Ki67 mouse anti-human monoclonal antibody (DAKO M7240, Hever-
lee, Belgium) was used to evaluate intratubular proliferation. Ki67 is a
nuclear antigen associated with cell proliferation and is present throughout
the active cell cycle (late G1, S, G2 and M phases), but absent in resting
cells (G0) (Scholzen and Gerdes, 2000).
Proliferating SG were counted after double immunostaining with
anti-MAGE-A4 and anti-Ki67 antibodies.
LCs were evaluated after immunostaining with 3b-HSD (rabbit anti-
human polyclonal antibody; SantaCruz sc-28206, Heidelberg, Germany),
a key enzyme of steroidogenesis and marker of functionally active LCs
(Dupont et al., 1991; Gaskell et al., 2004).
For simple immunostaining, sections mounted on Superfrost Plus
slides were deparafnized and rehydrated. Endogenous peroxidase
activity was blocked by incubating the sections with 0.3% H
2
O
2
Figure 3 SG immunostaining with MAGE-A4 antibody. Non-grafted control tissue (A and A

: 12 years; A

: 2 years), and fresh (B and B

: 8 years,
B

: 11 years), slow-frozen (C and C

: 2 years, C

: 4 years) and vitried (D and D

: 9 years; D

: 2 years) tissue grafted for 6 months to nude mice. All

tubules were positive for MAGE-A4 in non-grafted control tissue (A, A

and A

), while only a few seminiferous tubules were positive for MAGE-A4 in

grafted tissue (B, C, D, B

, C

, D

, B

, C

and D

). A, B, C and D, scale bar 200 mm (magnication 100). A

, A

, B

, B

, C

, C

and D

, D

, scale bar
100 mm (magnication 200).
Vitrication preserves proliferation capacity in human SG 583

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(for MAGE-A4 and Ki67) or 3% H
2
O
2
(for 3b-HSD) for 30 min at
RT.
After washing under deionized water for 5 min, sections were placed in
citrate buffer for 75 min at 988C (for MAGE-A4 and Ki67), followed by
washing in tris-buffered saline (TBS) 0.05 M and 20% Triton X-100
(Sigma Aldrich) before incubation at RT with 10% normal goat serum
(NGS, Invitrogen, Merelbeke, Belgium) and 1% bovine serum albumin
(BSA, Invitrogen) to block non-specic binding sites for 30 min (for
MAGE-A4 and Ki67) or 45 min (for 3b-HSD).
The primary antibody (diluted to 1/500 for MAGE-A4, 1/150 for Ki67
and 1/100 for 3b-HSD) was added to the sections and incubated over-
night at 48C in a humidied chamber.
The following day, the slides were washed in TBS 0.05 M and 20%
Triton X-100 three times for 2 min each and secondary anti-mouse anti-
body (EnVision + System Labeled Polymer-HRP; DAKO K4001) was
added and incubated for 60 min at RT, followed by washing in TBS
0.05 M and 20% Triton X-100 three times for 2 min each. Diaminobenzi-
dine (DAKO K3468) was used as a chromogen, and sections were incu-
bated for 10 min at RT. Nuclei were counterstained with Mayers
hematoxylin after washing under tap water for 3 min. Finally, the sections
were dehydrated and mounted.
For double Ki67-MAGE-A4 immunostaining, sections immunostained
with anti-Ki67 as described above were washed under acidied water
(HCl 0.1 M) for 60 min, followed by distilled water for 5 min and
then TBS 0.05 M and 20% Triton X-100 three times for 2 min each.
Non-specic antibody binding was blocked by incubation of samples
in 10% NGS and 1% BSA for 30 min at RT. MAGE-A4 antibody was
added to the samples and incubated at 48C overnight in a humidied
chamber.
The following day, the slides were washed in TBS 0.05 M and 20%
Triton X-100 three times for 2 min each and secondary anti-mouse anti-
body (EnVision + System-Labeled Polymer-HRP; DAKO K4001) was
added and incubated for 60 min at RT, followed by washing in TBS
0.05 M and 20% Triton X-100 three times for 2 min each.
Sections were incubated with 3-amino-9-ethylcarbazole (AEC; DAKO
K3464) as a chromogen for 10 min at RT and nuclei were counterstained
with HE after washing under tap water for 3 min. Finally, the Superfrost
Plus slides were mounted.
Assessment of spermatogonial cell number, intratubular proliferation
and interstitial LCs
To evaluate the number of SG in non-grafted control tissue and in fresh,
frozen and vitried tissue grafts, one section every 50 mm was stained
with MAGE-A4 antibody. The number of seminiferous tubules and
MAGE-A4-positive cells were counted in the totality of the graft. Results
were expressed as the mean number of MAGE-A4-positive cells per
tubule. Recovery rates of SG were also calculated (number of SG per
tubule in grafted tissue/number of SG per tubule in non-grafted control
tissue 100).
Subsequent serial sections were used to analyze intratubular prolifer-
ation after Ki67 immunostaining. All sections were assessed and all intra-
tubular Ki67-positive cells as well as all seminiferous tubules were counted.
To evaluate the proportion of proliferating SG, sections were immunos-
tained with anti-Ki67 and anti-MAGE-A4 antibodies. Results were
expressed as the proportion of MAGE-A4-positive cells showing Ki67
immunostaining. Three sections per graft were used for staining with
3b-HSD for qualitative evaluation of LC function.
Statistical analysis
Analyses were performed using the JMP 7 program (Cary, NC, USA)
based on SAS. Data are presented as mean +SD or medians (P25
P75). Statistical signicance between variables was evaluated using the
MannWhitney U-test. A P-value of 0.05 was considered statistically sig-
nicant. Comparisons were made between the groups (control versus
each grafting group and between grafting groups).
Results
Graft recovery
The graft recovery rate after 6 months xenotransplantation was 96%
(29/30). The only graft not recovered was from a 2-year-old boy.
Histological evaluation
An average of 656 +237, 2420+4339, 1114+1309 and 1590+
3263 seminiferous tubules were examined on HE sections in non-
grafted control, fresh grafted, slow-frozen grafted and vitried
grafted tissue, respectively. Individual content of control testicular
tissue is shown in Table III. Seminiferous tubule integrity was well pre-
served after grafting in all groups, as indicated by a similar proportion
of seminiferous tubules showing good cell cohesion, good adhesion of
cells to the basement membrane and no sclerosis. Indeed, 99.27%
(88.26100), 98.34% (88.91100) and 100% (95.38100) intact
seminiferous tubules were observed in fresh, slow-frozen and vitried
grafted tissue, respectively, compared with 100% in fresh non-grafted
tissue (Fig. 1). No statistical difference was observed between grafts
(P 0.05).
Germ cell differentiation up to the pachytene stage was observed in
grafts from two donors (2 and 9 years of age) for slow-frozen and vit-
ried tissue (Fig. 2). No germ cell differentiation was found in fresh
grafts.
Immunohistochemistry
Spermatogonial cells
An average of 301+88, 2100+3775, 986 +1336 and 1425+2940
seminiferous tubules were analyzed in non-grafted control, fresh
Figure 4 Mean number of MAGE-A4-positive cells per seminifer-
ous tubule. N.B. The scale of the Y-axis is different for grafts and
control tissue. Columns show the mean and standard deviation.
584 Poels et al.

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grafted, slow-frozen grafted and vitried grafted tissue, respectively.
SG were identied in all groups, but not in all grafts, as evidenced
by MAGE-A4-positive cells (Fig. 3). There was a marked decrease in
the number of SG per tubule in all grafted tissue groups compared
with non-grafted controls (P 0.05) (Fig. 4). The mean number of
SG per tubule was similar in fresh, frozen and vitried grafts (P
0.05) (Fig. 4). The SG recovery rate was 3.4 +3.8, 4.1 +7.3 and
7.3+6.3% from fresh, slow-frozen and vitried grafted tissue, re-
spectively. Individual SG numbers per seminiferous tubule and recov-
ery rates are shown in Table III.
Figure 5 Intratubular proliferation evidenced by Ki67 immunostaining. Non-grafted control tissue (A and A

: 12 years; A

: 2 years), and fresh (B

and B

: 8 years; B

: 11 years), slow-frozen (C and C

: 2 years; C

: 4 years) and vitried (D and D

: 9 years; D

: 2 years) tissue grafted for 6 months to

nude mice. Few seminiferous tubules showed Ki-67-positive cells in control and grafted tissue. A, B, C and D, scale bar 200 mm (magnication 100).
A

, A

, B

, B

, C

, C

, D

and D

, scale bar 100 mm (magnication 200).

Vitrication preserves proliferation capacity in human SG 585

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Intratubular proliferative activity
An average of 227+112, 2013+3775, 946+1335 and 1425+3099
seminiferous tubules were analyzed in non-grafted control, fresh grafted,
slow-frozen grafted and vitried grafted tissue, respectively. Proliferative
activity was similar (P 0.05) between the different groups, with a
median number and range of proliferating cells per seminiferous
tubule of 0.03 (0.020.27), 0.32 (0.020.48), 0.13 (0.030.21) and
0.14 (0.080.33) in non-grafted control, fresh grafted, slow-frozen
grafted and vitried grafted tissue, respectively (Fig. 5).
Proliferative activity of SG cells
Double immunostaining with MAGE-A4 (SG) and Ki67 (proliferation)
revealed 4% (013.89), 5.5% (2.216.5) and 4.1% (016.4) of SG
showing proliferative activity in fresh, slow-frozen and vitried
grafted tissue, respectively. No difference was observed between
grafts (Fig. 6).
Leydig cells
The presence of functional LCs, evidenced by 3b-HSD immunostain-
ing in fresh and frozen-thawed-grafted tissue, is shown in Fig. 7.
Discussion
Vitrication has been proposed as a potentially effective technique for
long-term storage of ITT, with a view to preserving SSCs for future fer-
tility restoration in young boys with cancer (Curaba et al., 2011a,b;
Poels et al., 2012). However, comparison between slow-freezing
and vitrication of human ITT was limited to reporting in vitro sperm-
atogonial survival, and no functional evaluation of human SSCs after
vitrication was performed (Curaba et al., 2011b). The current in
vivo study yields encouraging results, showing that vitrication may
well be an alternative to slow-freezing for cryopreservation of ITT.
After 6 months of xenografting of human ITT, we observed good pres-
ervation of the integrity of seminiferous tubules in fresh, slow-frozen
and vitried grafted tissue, similar to non-grafted control tissue. SG
and intratubular proliferating cell numbers, as well as differentiation
capacity, were similar in vitried-warmed and frozen-thawed ITT
grafts. Unfortunately, differentiation beyond the pachytene stage was
not observed. A marked reduction in SG numbers was noted in slow-
frozen (as previously reported) and vitried tissue grafts, as well as in
fresh grafts, compared with non-grafted tissue. This unexpected
nding appears to indicate that not only the cryopreservation
Figure 6 Proliferating SG. Double immunostaining with MAGE-A4 and Ki67 in fresh (A and A

), slow-frozen (B and B

) and vitried (C and C

)
grafted tissue; black arrows show proliferating (brown staining of nucleus) spermatogonia (pink staining of cytoplasm), and red arrows show non-
proliferating spermatogonia (pink staining of nucleus and cytoplasm). A, B and C, scale bar 200 mm (magnication 100). A

, B

and C

, scale bar
100 mm (magnication 200).
586 Poels et al.

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method, but possibly also the xenotransplantation model, may be
implicated.
Controlled slow-freezing is the procedure currently applied for ITT
cryobanking in clinical practice (Wyns et al., 2011), based on studies
demonstrating survival of human SG (Wyns et al., 2008; Van Saen
et al., 2011) and attainment of offspring in mice after short-term
(Shinohara et al., 2002) and long-term (Wu et al., 2012) storage of
frozen tissue. Considering that both cryopreservation methods yield
similar outcomes, there are so far no convincing data to warrant modi-
cation of current clinical practice (Wyns et al., 2011).
However, research on vitrication of human ITT is worth pursuing.
Indeed, this approach presents several theoretical advantages over
controlled slow-freezing, namely there is no specic equipment
required and the method is potentially less harmful to the SG stem
cell niche because of a lower risk of cell damage in the absence of
ice crystal formation (Amorim et al., 2011). Although both cryopreser-
vation protocols appear to maintain tubular cell integrity with good cell
cohesion, good adhesion of cells to the basement membrane and no
sclerosis, subtle cryodamage to the SG niche cannot be excluded.
Unexpected SG loss was encountered in the non-cryopreserved
group, suggesting that the avascular transplantation procedure may
be implicated in tissue impairment. Indeed, a successful outcome for
xenografts depends on a quick connection to the circulatory system
of recipient mice, providing supply of oxygen, nutrients and hormones.
A number of hypotheses may be put forward to explain SG loss and
impaired maturation.
First, ischemic stress experienced by testicular tissue transplants
before their revascularization may induce tissue necrosis or apoptosis
pathway activation in grafts, as reported for ovarian tissue (Israely
et al., 2006). Cell apoptosis was not analyzed in this study since this
phenomenon is an early event after transplantation, as observed in our
previous transplantation experiment, where apoptotic markers were
not observed after 6 months (Wyns et al., 2008). However, using the
same xenotransplantation model, apoptosis was evidenced at earlier
stages and was high at 3 days (Wyns et al., 2008, PhD Thesis, unpub-
lished), but very low at 3 weeks post-transplantation (Wyns et al.,
2007). Ischemiareperfusion may thus induce damage to the SSC
niche, consisting of Sertoli cells, LCs, peritubular myoid cells and the inter-
stitial vascular network (Shetty and Meistrich, 2007; Caires et al., 2010),
essential for the maintenance of functional SSCs and tissue integrity. An
insufcient nutrient and oxygen supply also appeared to preclude semin-
iferous tubule maturation in some areas of grafted tissue (Rathi et al.,
2008). Limiting apoptotic tissue and stemcell niche damage as well as en-
suring faster graft reperfusion are therefore essential.
To promote revascularization of testicular tissue transplants, both
testicular tissue vessels and host vessels may be targeted, as reperfu-
sion is initiated by outgrowing vessels from the grafted tissue, which
will connect to larger subcutaneous vessels formed by the host (Van
Eyck et al., 2010; Schlatt et al., 2010a). The use of endothelial cell
apoptotic inhibitors or activators is an option, as they optimize the
contribution of human vessels to graft revascularization (Chavakis
and Dimmeler, 2002).
Addition of vascular endothelial growth factor (VEGF) at the time of
transplantation may also stimulate neoangiogenesis (Nomi et al., 2002;
Cao et al., 2005; Schmidt et al., 2006; Caires et al., 2009). Indeed, a
single treatment with VEGF at the time of grafting showed a higher
percentage of seminiferous tubules containing elongating spermatids
(Schmidt et al., 2006).
Limiting oxidative stress responsible for germ cell apoptosis under
hypoxic conditions may also be considered. Adding antioxidants
Figure 7 Immunostaining of LCs with 3b-HSD antibody. Non-grafted control tissue (A: 12 years) and fresh grafted (B: 8 years), slow-frozen grafted
(C: 2 years) and vitried grafted (D: 9 years) ITT. A, B, C and D, scale bar 50 mm (magnication 400) (3b-HSD, 3b-hydroxysteroid).
Vitrication preserves proliferation capacity in human SG 587

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such as N-acetylcysteine (NAC) at the time of transplantation could
reduce oxidative stress by enhancing intracellular generation of gluta-
thione (GSH) in cells. This strategy has proven efcient in experiments
to prevent histopathological damage after testicular torsion/distortion,
by reducing cell membrane lipid peroxidation (Cay et al., 2006; Aktas
et al., 2010; Turkmen et al., 2012).
The second hypothesis concerns the inadequacy of the host envir-
onment. Indeed, Schlatt et al. (2010b) recently demonstrated that
control of endocrine function of grafted testicular tissue is extrinsically
modulated by the hypothalamicpituitarygonadal axis of the
recipient mouse. Species differences in SSC niche functioning and
hormone interactions must therefore be considered. This is supported
by observations in pigs and monkeys, in whom administration of ex-
ogenous gonadotropins showed improved maturation and differenti-
ation of testicular tissue xenografted to mice (Zeng et al., 2006;
Rathi et al., 2008). By contrast, autologous transplantation in marmo-
sets (Wistuba et al., 2006) and marmoset or horse ITT xenografts in
mice receiving gonadotropin supplementation (Wistuba et al., 2004;
Rathi et al., 2006) showed inhibition of germ cell differentiation,
suggesting the involvement of non-hormonal factors affecting SSC
maturation in transplants.
In conclusion, our study demonstrated that SG, while able to
survive and proliferate, only partially initiate differentiation after vitri-
cation and orthotopic xenografting to nude mice, showing similar ef-
ciency to slow-freezing. Besides the cryopreservation method itself,
the transplantation procedure appears to be critical to ensure preser-
vation of spermatogonial cells and their differentiation capacity for
future fertility restoration purposes. Further studies are therefore es-
sential to identify ways of limiting loss of SG and improving their ability
to differentiate after cryopreservation and transplantation.
Acknowledgements
The authors are grateful to Mira Hryniuk, BA, for reviewing the English
language of the manuscript. The authors thank the laboratory of
morphology of the Institute of Experimental Research (IREC), in par-
ticular Prof. Marie-Christine Many, for access to laboratory facilities
(premises, morphology materials). The authors also thank the labora-
tory of andrology of the Cliniques Universitaires Saint-Luc, in particular
Bernard Vanabelle and Sylvie Gantois, for their technical assistance.
Authors roles
J.P. performed the experiments and wrote the manuscript. A.V.L.
revised the manuscript. M.-C.M. provided advice during the experi-
mental phase, and the premises. F.-X.W. performed surgical biopsies.
C.W. was responsible for the critical review of the manuscript and the
discussion.
Funding
This study was supported by a grant from the Fonds National de la
Recherche Scientique de Belgique (grant Televie N87. 4.572.09.F).
Conict of interest
The authors declare that there is no conict of interest.
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