Vitrification preserves SSCs, Wyns 2013.pdf

ORIGINAL ARTICLE Andrology

Vitrication preserves proliferationcapacity in human spermatogoniaJonathan Poels1,2, Anne Van Langendonckt1,2, Marie-Christine Many3,Francois-Xavier Wese4, and Christine Wyns1,2,*1Gynecology Unit, Medical School, Institut de Recherche Experimentale et Clinique, Universite Catholique de Louvain, Avenue Mounier, 52,1200 Brussels, Belgium 2Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Avenue Hippocrate, 10, 1200 Brussels,Belgium 3Experimental Morphology Unit, Medical School, Institut de Recherche Experimentale et Clinique, Universite Catholique de Louvain,1200 Brussels, Belgium 4Urology 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: [email protected]

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 stemcells (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 monthswere compared.

materials, setting, methods: Upon graft removal, histological and immunohistochemical analyses were performed to evaluatespermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3b-HSD). The entire piece of graftedtissue was assessed in each case.

main results and the role of chance: Seminiferous tubules showed good integrity after cryopreservation and xenograftingfor 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SGwere able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4+3.8, 4.1+7.3and 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 mousexenotransplantation model needs to be rened to study human spermatogenesis.

wider implications of the findings: The ndings of the present study have potential implications for cryobanking of ITT andfertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to beoptimized. There are so far no convincing data justifying modication of current clinical practice for ITT storage with slow-freezing, but thisstudy 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 N8 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: [email protected]

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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IntroductionWith increasing effectiveness of childhood cancer treatments, survivalrates are on the rise, and it is estimated that .80% of children survivetheir disease (Magnani et al., 2006; Arndt et al., 2007; Gatta et al.,2009). Unfortunately, improvements in treatment efcacy go hand inhand with increased toxicity, especially gonadotoxicity. Cryopreserva-tion of immature testicular tissue (ITT) containing spermatogonialstem cells (SSCs) is so far the only approach that can be proposedto 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 torestore 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 wereachieved in mice. Indeed, autotransplantation of ITT, cryopreservedby 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 6months of orthotopic xenografting of slow-frozen human ITT tonude mice (Wyns et al., 2008). However, rapid loss of SG wasrecorded, with recovery rates of 14.5% after 3 weeks (Wyns et al.,2007) and 3.7% after 6 months (Wyns et al., 2008), and differentiationappeared to be limited to the pachytene stage.

Vitrication is an innovative strategy preventing ice crystal formationby the use of high concentrations of cryoprotectant and ultrafastcooling speeds, which could minimize cellular damage (Amorimet al., 2011). In mice, promising results were obtained after ITT vitri-cation followed by 3 days of organotypic culture, since no differencein seminiferous tubule cellular density and integrity, cell viability, pro-liferation or apoptosis was observed between vitried and slow-frozentissue (Curaba et al., 2011a). Normal spermatogenesis and roundspermatids were also obtained after xenotransplantation of vitriedpig 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 cryopreservationmethod for human ITT with a view to fertility preservation inhumans.

(ii) To compare SG survival and differentiation after xenotransplant-ation of fresh, frozen-thawed (according to the protocol currentlyapplied in clinics) and vitried-warmed human ITT.

For this purpose, testicular tissue was orthotopically xenografted usingour mouse model previously developed for functional assessment ofcryopreserved tissue (Wyns et al., 2008).

Materials and Methods

Study designSmall pieces of ITT were obtained from 10 prepubertal boys. A smallsample was taken from each and xed in Bouins solution to serve asfresh non-grafted controls. The biopsy was then divided into three equalpieces allocated to the three grafting groups. One piece was immediatelygrafted 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, warmedand grafted to a third mouse (vitried grafts). After 6 months, the graftswere recovered and directly xed in Bouins solution, embedded in paraf-n and cut into serial sections.

Histological analysis was performed on hematoxylineosin (HE)-stainedsections to assess seminiferous tubule integrity and the germ celldifferentiation stage. SG were evidenced by immunostaining withmelanoma-associated antigen 4 (MAGE-A4; mouse anti-human monoclo-nal antibody puried from hybridoma 57B, kindly provided by GiulioSpagnoli, MD, University of Basel, Switzerland) and LCs were evaluatedafter immunostaining with 3b-hydroxysteroid dehydrogenase (3b-HSD),a key enzyme of steroidogenesis. Intratubular cell proliferation wasassessed after Ki67 immunostaining, and double immunostaining withMAGE-A4 and Ki67 was applied to identify proliferating SG.

AnimalsThirty 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 temperaturebetween 22 and 248C with a day/night cycle of 12 h. All housing materialand food were autoclaved before use. The mice were fed ad libitum onlaboratory chow (complete food for rats and mice; Pavan Carl,Oud-Turnhout, Belgium) and acidied water. All experiments in thisstudy were approved by the Ethics Review Board and the Committeeon Animal Research of the Catholic University of Louvain.

Donor testicular tissueITT 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 theparents and the childs ascent (where applicable). Patients were referredby pediatric oncologists or hematologists to the reproductive specialistin fertility preservation, when they considered that the risk of infertilitydue to treatment was high and/or the parents specically requested fertil-ity preservation techniques. All donors were scheduled for testicularbiopsy prior to gonadotoxic treatment. Disease and gonadotoxic treat-ment are shown in Table I. Unilateral testicular sampling of ,5% of thetotal testicular volume (based on theoretical size by age from 0 to12-year-old: 0.75 to 2.0 cm3; Beres et al., 1989) was performed by a pedi-atric urologist through scrotal incision. The majority of the collected tissuewas used for the boys fertility preservation. Individual testicular sampling isreported 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 mm3) xedin paraformaldehyde or Bouins solution (sent to the laboratory of anato-mopathology) served as non-frozen control for light microscopy (LM) andimmunohistochemical analysis. One piece of ITT (+1 1 3 mm) fromeach boy was used for our experiment and divided into three pieces(+1 mm3) allocated to the three grafting groups.

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Ethics approval and consent procedureAll experiments in this study were approved by the Ethics Review Board ofthe Catholic University of Louvain. The ethics committee agreed to tes-ticular biopsy for research purposes only when testicular surgery wasrequired for the childs fertility preservation and after obtaining informedconsent. Parents (or legal guardians) and the child had a consultationwith a specialist in reproductive medicine. Potential fertility restorationapproaches from stored samples were explained to each individual child(when applicable, usually from the age of 5 with adapted language) and

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Table II Testicular sampling.

No. Age(years)

Totalamount ofremovedtissue(mm3)

Totalamount ofremovedtissue forresearch(mm3)

Proportiondestined forresearch (%)

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 riskof gonadotoxicityof plannedtreatment aftertesticular biopsya

1 12 Homozygous sickle celldisease

High

2 9 Neuroectodermaltumor Intermediate

3 4 Acute lymphoblasticleukemia

Low

4 8 Acute lymphoblasticleukemia

Low

5 11 Osteosarcoma High

6 12 Ewings sarcoma High

7 11 Hodgkins lymphoma Intermediate

8 10 Embryonalrhabdomyosarcoma

Intermediate

9 2 Anaplasticmedulloblastoma grade 4

Intermediate

10 2 Abdominalneuroblastoma stage IV

High

aClassication refers to previously published data (Wyns et al., 2010).

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TableIIICon

tent

ofcollected

testicular

tissue

accordingto

Clerm

ontsclassication

(Clerm

ont,1963).

No.

Age

(years)

Anatomop

atho

logicalanalysis

Num

berof

SG(M

AGE-A-4-positivecells)pe

rseminiferous

tubu

leSpe

rmatogon

ialrecovery(%)

Ungrafted

tissue

Fresh

graft

Slow-frozengraft

Vitried

graft

Fresh

graft

Slow-frozengraft

Vitried

graft

112

SC,S

G,few

spcandLC

8.39

0.74

00.13

90.00

58.82

1.65

0.05

29

SC,S

GandLC

2.27

00.08

60.01

60

3.77

0.72

34

NC

ND

0.01

90.01

90.00

2ND

ND

ND

48

SC,S

GandLC

1.42

0.54

30.03

80.09

838

.32

2.66

6.94

511

SC,S

GandLC

2.12

0.12

60.09

80.01

65.91

4.59

0.73

612

SC,S

G,Spc,few

spzandLC

20.77

0.01

20

0.06

90.06

00.33

711

SC,S

G,Spc,few

spzandLC

13.33

0.35

70.08

60.58

82.68

0.65

4.41

810

NC

ND

0.02

70

0.00

6ND

ND

ND

92

SC,S

GandLC

2.82

0.26

20.66

60.39

09.29

23.65

13.83

102

SC,S

GandLC

2.58

ND

1.63

33.69

3NC

63.25

143.05

SC,S

ertolicell;SG

,spe

rmatogon

ia;S

pc,spe

rmatoc

yte;

Spz,spermatozoa

;LC,L

eydigcell;NC,n

oco

ntrib

utivesample;

ND,n

otde

term

ined

.

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his parents, making sure they understood that with stored ITT, there is noguarantee of success as yet. In all cases, parents or legal guardians gavetheir signed informed consent for cryobanking, as well as the youngboys themselves, if they were mature enough to understand the implica-tions of the procedure.

Cryopreservation protocolsThe 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) andsucrose 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 minbefore seeding manually at 288C. After holding for a further 15 min at288C, a cooling rate of 20.58C/min was used from 288C to 2408Cbefore 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. Seminiferoustubule 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, scalebar 100 mm (magnication 200) (ITT, immature testicular tissue).


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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 thenwashed 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 inLeibovitz L-15 (L-15, Sigma Aldrich), supplemented with 25 mg/ml humanserum albumin (HSA 20%, Cealb 2 g/10 ml, Brussels, Belgium) for 10 minat 48C. It was then transferred to the vitrication solution (5 ml) consistingof 15% EG, 15% DMSO and 0.5 M sucrose in L-15 medium, supplementedwith 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 CBS0.5 ml, Cryo Bio System, Aigle-France, France), and plunged into sterileliquid 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 andthe straws were quickly immersed in a 358C warming solution containingsucrose (1 mol/l) in L-15 medium, supplemented with 25 mg/ml HSA.The testicular tissue pieces were then serially transferred to three bathsof warming solutions with decreasing sucrose concentrations (0.5, 0.25and 0 mol/l) for 5 min each.

Liquid nitrogen sterilizationSterilization of liquid nitrogen (LN2) 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/cm2) was used to expose LN2 to UVC radiation. Thedewar with LN2 was placed 10 cm from the UVC lamp for 15 min basedon the UV dose required to eliminate the most UV-resistant micro-organism (330 000 UV dose for Aspergilus niger; Srikanth, 1995) usingthe calculation UV dose UV intensity (I ) resistance time (T ). Afterformula transformation, the following result was obtained: T 330 000/490 (at 10 cm) 673.5 s or 11.22 min.

XenograftingThe 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 ofthe same surgery, +1 mm3 pieces of fresh, slow-frozen or vitried-warmed donor testicular tissue were grafted without vascular anastomosisinto the scrotum, according to a previously described procedure (Wynset 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 theday of surgery and the following day.

Graft recoveryAfter 6 months, the mice were anesthetized by intraperitoneal injection ofketamine, euthanized by intracardiac blood puncture and the grafts wererecovered and directly xed in Bouins solution. The totality of thegrafted tissue was used for analysis.

Histological evaluation of grafted testiculartissueAfter 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 Plusslides and used for immunohistochemistry. Digital images were capturedwith a Mirax Midi digital camera (Zeiss Mirax Midi, Zeiss, Germany).

Seminiferous tubule integrity was evaluated on HE-stained sectionsunder a light microscope at 400 magnication. Tubules were consideredintact when good adhesion of cells to the basement membrane, good cellcohesion and no sclerosis were observed.

Immunohistochemical analysesMAGE-A4, Ki67 and 3b-HSD immunostainingMAGE-A4 mouse anti-human monoclonal antibody was used to evidenceSG. This antibody, puried from hybridoma 57B, was kindly provided byGiulio Spagnoli, MD (Yakirevich et al., 2003).

Figure 2 Spermatogonia differentiation to the pachytene stage inslow-frozen (A) and vitried (B) grafts from a 9-year-old donorand vitried (C) graft from a 2-year-old donor. Scale bar 50 mm(magnication 400).

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Ki67 mouse anti-human monoclonal antibody (DAKO M7240, Hever-lee, Belgium) was used to evaluate intratubular proliferation. Ki67 is anuclear antigen associated with cell proliferation and is present throughoutthe active cell cycle (late G1, S, G2 and M phases), but absent in restingcells (G0) (Scholzen and Gerdes, 2000).

Proliferating SG were counted after double immunostaining withanti-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 Plusslides were deparafnized and rehydrated. Endogenous peroxidaseactivity was blocked by incubating the sections with 0.3% H2O2

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. Alltubules 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 ingrafted 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 bar100 mm (magnication 200).


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(for MAGE-A4 and Ki67) or 3% H2O2 (for 3b-HSD) for 30 min atRT.

After washing under deionized water for 5 min, sections were placed incitrate buffer for 75 min at 988C (for MAGE-A4 and Ki67), followed bywashing 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 (forMAGE-A4 and Ki67) or 45 min (for 3b-HSD).

The primary antibody (diluted to 1/500 for MAGE-A4, 1/150 for Ki67and 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) wasadded and incubated for 60 min at RT, followed by washing in TBS0.05 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 Mayershematoxylin after washing under tap water for 3 min. Finally, the sectionswere dehydrated and mounted.

For double Ki67-MAGE-A4 immunostaining, sections immunostainedwith anti-Ki67 as described above were washed under acidied water(HCl 0.1 M) for 60 min, followed by distilled water for 5 min andthen TBS 0.05 M and 20% Triton X-100 three times for 2 min each.Non-specic antibody binding was blocked by incubation of samplesin 10% NGS and 1% BSA for 30 min at RT. MAGE-A4 antibody wasadded to the samples and incubated at 48C overnight in a humidiedchamber.

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) wasadded and incubated for 60 min at RT, followed by washing in TBS0.05 M and 20% Triton X-100 three times for 2 min each.

Sections were incubated with 3-amino-9-ethylcarbazole (AEC; DAKOK3464) as a chromogen for 10 min at RT and nuclei were counterstainedwith HE after washing under tap water for 3 min. Finally, the SuperfrostPlus slides were mounted.

Assessment of spermatogonial cell number, intratubular proliferationand interstitial LCsTo evaluate the number of SG in non-grafted control tissue and in fresh,frozen and vitried tissue grafts, one section every 50 mm was stainedwith MAGE-A4 antibody. The number of seminiferous tubules andMAGE-A4-positive cells were counted in the totality of the graft. Resultswere expressed as the mean number of MAGE-A4-positive cells pertubule. Recovery rates of SG were also calculated (number of SG pertubule in grafted tissue/number of SG per tubule in non-grafted controltissue 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 wereexpressed as the proportion of MAGE-A4-positive cells showing Ki67immunostaining. Three sections per graft were used for staining with3b-HSD for qualitative evaluation of LC function.

Statistical analysisAnalyses were performed using the JMP 7 program (Cary, NC, USA)based on SAS. Data are presented as mean+ SD or medians (P25P75). Statistical signicance between variables was evaluated using theMannWhitney U-test. A P-value of 0.05 was considered statistically sig-nicant. Comparisons were made between the groups (control versuseach grafting group and between grafting groups).

Results

Graft recoveryThe graft recovery rate after 6 months xenotransplantation was 96%(29/30). The only graft not recovered was from a 2-year-old boy.

Histological evaluationAn 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 vitriedgrafted tissue, respectively. Individual content of control testiculartissue is shown in Table III. Seminiferous tubule integrity was well pre-served after grafting in all groups, as indicated by a similar proportionof seminiferous tubules showing good cell cohesion, good adhesion ofcells to the basement membrane and no sclerosis. Indeed, 99.27%(88.26100), 98.34% (88.91100) and 100% (95.38100) intactseminiferous tubules were observed in fresh, slow-frozen and vitriedgrafted tissue, respectively, compared with 100% in fresh non-graftedtissue (Fig. 1). No statistical difference was observed between grafts(P 0.05).

Germ cell differentiation up to the pachytene stage was observed ingrafts 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 freshgrafts.

ImmunohistochemistrySpermatogonial cellsAn average of 301+ 88, 2100+3775, 986+ 1336 and 1425+ 2940seminiferous 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 andcontrol tissue. Columns show the mean and standard deviation.

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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 evidencedby MAGE-A4-positive cells (Fig. 3). There was a marked decrease inthe number of SG per tubule in all grafted tissue groups comparedwith 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 and7.3+ 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 (Band 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 tonude 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).


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Intratubular proliferative activityAn average of 227+112, 2013+3775, 946+1335 and 1425+3099seminiferous tubules were analyzed in non-grafted control, fresh grafted,slow-frozen grafted and vitried grafted tissue, respectively. Proliferativeactivity was similar (P 0.05) between the different groups, with amedian number and range of proliferating cells per seminiferoustubule of 0.03 (0.020.27), 0.32 (0.020.48), 0.13 (0.030.21) and0.14 (0.080.33) in non-grafted control, fresh grafted, slow-frozengrafted and vitried grafted tissue, respectively (Fig. 5).

Proliferative activity of SG cellsDouble immunostaining with MAGE-A4 (SG) and Ki67 (proliferation)revealed 4% (013.89), 5.5% (2.216.5) and 4.1% (016.4) of SGshowing proliferative activity in fresh, slow-frozen and vitriedgrafted tissue, respectively. No difference was observed betweengrafts (Fig. 6).

Leydig cellsThe presence of functional LCs, evidenced by 3b-HSD immunostain-ing in fresh and frozen-thawed-grafted tissue, is shown in Fig. 7.

DiscussionVitrication has been proposed as a potentially effective technique forlong-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-freezingand vitrication of human ITT was limited to reporting in vitro sperm-atogonial survival, and no functional evaluation of human SSCs aftervitrication was performed (Curaba et al., 2011b). The current invivo study yields encouraging results, showing that vitrication maywell 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-frozenand vitried grafted tissue, similar to non-grafted control tissue. SGand intratubular proliferating cell numbers, as well as differentiationcapacity, were similar in vitried-warmed and frozen-thawed ITTgrafts. Unfortunately, differentiation beyond the pachytene stage wasnot observed. A marked reduction in SG numbers was noted in slow-frozen (as previously reported) and vitried tissue grafts, as well as infresh grafts, compared with non-grafted tissue. This unexpectednding 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 bar100 mm (magnication 200).

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method, but possibly also the xenotransplantation model, may beimplicated.

Controlled slow-freezing is the procedure currently applied for ITTcryobanking in clinical practice (Wyns et al., 2011), based on studiesdemonstrating survival of human SG (Wyns et al., 2008; Van Saenet al., 2011) and attainment of offspring in mice after short-term(Shinohara et al., 2002) and long-term (Wu et al., 2012) storage offrozen tissue. Considering that both cryopreservation methods yieldsimilar 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 overcontrolled slow-freezing, namely there is no specic equipmentrequired and the method is potentially less harmful to the SG stemcell niche because of a lower risk of cell damage in the absence ofice crystal formation (Amorim et al., 2011). Although both cryopreser-vation protocols appear to maintain tubular cell integrity with good cellcohesion, good adhesion of cells to the basement membrane and nosclerosis, subtle cryodamage to the SG niche cannot be excluded.

Unexpected SG loss was encountered in the non-cryopreservedgroup, suggesting that the avascular transplantation procedure maybe implicated in tissue impairment. Indeed, a successful outcome forxenografts depends on a quick connection to the circulatory systemof recipient mice, providing supply of oxygen, nutrients and hormones.

A number of hypotheses may be put forward to explain SG loss andimpaired maturation.

First, ischemic stress experienced by testicular tissue transplantsbefore their revascularization may induce tissue necrosis or apoptosispathway activation in grafts, as reported for ovarian tissue (Israelyet al., 2006). Cell apoptosis was not analyzed in this study since this

phenomenon is an early event after transplantation, as observed in ourprevious transplantation experiment, where apoptotic markers werenot observed after 6 months (Wyns et al., 2008). However, using thesame xenotransplantation model, apoptosis was evidenced at earlierstages 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 SSCniche, consisting of Sertoli cells, LCs, peritubularmyoid 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. Aninsufcient 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 stem cell niche damage as well as en-suring faster graft reperfusion are therefore essential.

To promote revascularization of testicular tissue transplants, bothtesticular tissue vessels and host vessels may be targeted, as reperfu-sion is initiated by outgrowing vessels from the grafted tissue, whichwill connect to larger subcutaneous vessels formed by the host (VanEyck et al., 2010; Schlatt et al., 2010a). The use of endothelial cellapoptotic inhibitors or activators is an option, as they optimize thecontribution of human vessels to graft revascularization (Chavakisand Dimmeler, 2002).

Addition of vascular endothelial growth factor (VEGF) at the time oftransplantation may also stimulate neoangiogenesis (Nomi et al., 2002;Cao et al., 2005; Schmidt et al., 2006; Caires et al., 2009). Indeed, asingle treatment with VEGF at the time of grafting showed a higherpercentage of seminiferous tubules containing elongating spermatids(Schmidt et al., 2006).

Limiting oxidative stress responsible for germ cell apoptosis underhypoxic 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).


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such as N-acetylcysteine (NAC) at the time of transplantation couldreduce oxidative stress by enhancing intracellular generation of gluta-thione (GSH) in cells. This strategy has proven efcient in experimentsto prevent histopathological damage after testicular torsion/distortion,by reducing cell membrane lipid peroxidation (Cay et al., 2006; Aktaset al., 2010; Turkmen et al., 2012).

The second hypothesis concerns the inadequacy of the host envir-onment. Indeed, Schlatt et al. (2010b) recently demonstrated thatcontrol of endocrine function of grafted testicular tissue is extrinsicallymodulated by the hypothalamicpituitarygonadal axis of therecipient mouse. Species differences in SSC niche functioning andhormone interactions must therefore be considered. This is supportedby 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 inmice 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 SSCmaturation in transplants.

In conclusion, our study demonstrated that SG, while able tosurvive 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 forfuture fertility restoration purposes. Further studies are therefore es-sential to identify ways of limiting loss of SG and improving their abilityto differentiate after cryopreservation and transplantation.

AcknowledgementsThe authors are grateful to Mira Hryniuk, BA, for reviewing the Englishlanguage of the manuscript. The authors thank the laboratory ofmorphology 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 particularBernard Vanabelle and Sylvie Gantois, for their technical assistance.

Authors rolesJ.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 thediscussion.

FundingThis study was supported by a grant from the Fonds National de laRecherche Scientique de Belgique (grant Televie N87. 4.572.09.F).

Conict of interestThe authors declare that there is no conict of interest.

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