What is the best cryopreservation protocol for human ......electron microscopic evaluations. The...

ORIGINAL ARTICLE Infertility

What is the best cryopreservationprotocol for human testiculartissue banking?Y. Baert1,*, D. Van Saen1, P. Haentjens2, P. In’t Veld3,H. Tournaye1,4, and E. Goossens1

1Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel, Laarbeeklaan103, Brussels 1090, Belgium 2Centre for Outcomes Research, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, Brussels 1090, Belgium3Department of Pathology, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels 1090, Belgium 4Centre for Reproductive Medicine,Universitair Ziekenhuis Brussel, Laarbeeklaan 101, Brussels 1090, Belgium

*Correspondence address. Tel: +32-2-477-46-44; Fax: +32-2-477-46-32; E-mail: [email protected]

Submitted on December 12, 2012; resubmitted on February 22, 2013; accepted on March 1, 2013

study question: Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking?

summary answer: Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotec-tants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue.

what is known already: Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment arecommonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied inseveral animal models but need further exploration with human tissue.

study design, size, duration: Fragments (n ¼ 160) of testicular tissue from 14 patients undergoing vasectomy reversal wereassigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M inthe presence (+S) or absence of sucrose (2S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV)or direct cover vitrification (DCV).

materials, setting, methods: Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability,spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultra-structural alterations were studied by scoring cryodamage to four relevant testicular cell types.

main results and the role of chance: The USF 1.5 M DMSO + S protocol proved not solely to prevent cell death and topreserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicu-lar cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4+5.6 in control tissue to 4.9+ 2.1, 8.2+ 5.4,11.6+5.1, 8.8+3.9, 12.6+4.4 and 11.7+5.7 was observed after cryopreservation combined with at least one other form of cryoinjurywhen using CSF 0.7 M DMSO 2S, CSF 0.7 M DMSO + S, CSF 1.5 M DMSO + S, USF 0.7 M DMSO + S, SSV and direct cover vitrification(DCV), respectively (P , 0.001).

limitations, reasons for caution: Supplementary research is required to investigate the effect on tissue functionality and toconfirm this study’s findings using prepubertal tissue.

wider implications of the findings: An optimal cryopreservation protocol enhances the chances for successful fertility res-toration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenevertissue is to be procured from a diseased child at a site distant from the banking facility.

study funding/competing interest(s): This study is supported by a PhD grant from the Agency for Innovation by Scienceand Technology and research grants from the Flemish League against Cancer-Public Utility Foundation, the Scientific Research FoundationFlanders, Methusalem grant and the Vrije Universiteit Brussel. E.G. is a postdoctoral fellow of the Scientific Research Foundation Flanders.The authors declare that no competing interests exist.

Key words: cryopreservation / electron microscopy / germ cells / immunohistochemistry / male infertility

& 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.7 pp. 1816–1826, 2013

Advanced Access publication on April 7, 2013 doi:10.1093/humrep/det100

IntroductionThe number of men surviving cancer at a young age has increased dra-matically in the past 40 years thanks to early detection and improvedcancer treatments. Nowadays, the overall cure rates for paediatricmalignancies approach 80% and are still improving (Ginsberg, 2011).Unfortunately, as most of these patients receive polychemotherapyand/or irradiation, a major concern is impairment or loss of their fer-tility due to spermatogonia (SG) depletion (Howell and Shalet, 2005).Cancer treatment during childhood is assumed to impair fertility inabout one-third of male childhood cancer survivors (Rendtorff et al.,2010). Not surprisingly, fertility preservation is an important issue inregard to the long-term quality of life. Cryopreservation of small frag-ments of testicular tissue is an experimental strategy for fertility pres-ervation in cases where cryopreservation of sperm is not applicable(see Geens et al., 2008; Schlatt et al., 2009 for review).

Post-pubertal and adult cancer patients could also benefit from theupcoming fertility preservation strategy, since some of them mayalready be azoospermic at the moment of sperm banking as a resultof a spermatogenic arrest caused by pretreatment or factors relatedto their malignancy (Sabanegh and Ragheb, 2009; Dohle, 2010).Other potential indications include systemic or haematological non-cancerous diseases which require gonadotoxic treatments (Geenset al., 2008), gonadectomy (Jahnukainen et al., 2006), Klinefelter’s syn-drome (Paduch et al., 2009), Yq deletions (Krausz and Forti, 2006) andcryptorchidism (Kvist et al., 2006).

At present, controlled slow freezing (CSF) using dimethylsulphoxide(DMSO) as a cryoprotectant is the method used to cryopreserve imma-ture testicular tissue in animal models (Schlatt et al., 2002; Shinoharaet al., 2002; Luetjens et al., 2008; Milazzo et al., 2010). In rodents, micro-insemination using sperm retrieved from grafted cryopreserved pre-pubertal testicular tissue has even led to the birth of healthy offspring(Shinohara et al., 2002). Because these findings are supportive for a clin-ical application, several teams started freezing human testicular tissueusing a programmable freezer. Kvist et al. (2006) reported a CSFmethod, initially developed for cryopreserving human ovarian tissue,to be feasible for the cryopreservation of human testicular tissue too.More recently, Keros et al. (2007) reported a programmed freezingprotocol with induced ice nucleation using 0.7 M DMSO for prepubertaltesticular tissue, which was originally developed for adult testicular tissue(Keros et al., 2005), yielding good structural integrity. Wyns et al. (2008)adapted this protocol by adding 0.1 M sucrose and observed survival ofprepubertal human SG able to proliferate and initiate differentiation infrozen–thawed xenografts. It is, therefore, the first-choice techniqueto cryopreserve immature testicular tissue in their reproductiveprogram (Wyns et al., 2011). Nevertheless, since CSF requires expen-sive computerized equipment and the process is time-consuming, amore convenient alternative would be preferable.

Several publications reported uncontrolled slow freezing (USF) tobe a successful cryopreservation strategy for testicular tissue of differ-ent animal species (Honaramooz et al., 2002; Ohta and Wakayama,2005; Jahnukainen et al., 2007; Baert et al., 2012). However, USFhas not yet been considered for human testicular tissue, despite thefact that this method is inexpensive, convenient and time-saving, andthus, possibly a good substitute for CSF.

Recent studies also propose vitrification, which is an ultrarapidice-free cryopreservation technique, as a potential alternative

method for prepubertal testicular tissue cryopreservation. Thegroup of Wyns reported preservation of immature mouse (Curabaet al., 2011), immature non-human primate (Poels et al., 2012) andimmature human testicular tissue (Poels et al., 2013) by vitrificationusing open straws. In the present study, we developed a new open vit-rification technique, termed DCV, based on a promising study onovarian tissue cryopreservation (Zhou et al., 2010). Despite manyadvantages, concerns have been raised regarding the biologicalsafety of open vitrification systems, as liquid nitrogen may mediatethe transfer of pathogenic agents (Bielanski and Vajta, 2009). Theclosed solid-surface vitrification (SSV) system would be a saferoption for clinical application. SSV was reported to yield good preser-vation of prepubertal mouse (Baert et al., 2012) and piglet testiculartissue (Abrishami et al., 2010). So far, no study determined whetherhuman testicular tissue can be successfully preserved by DCV or SSV.

The present study was designed to explore USF, SSV and DCV forcryopreservation of human testicular tissue and to compare these newmethods with conventional CSF. To evaluate the efficiency of freezingand vitrification, we systematically examined human testicular tissue bylight microscopy and transmission electron microscopy.

Materials and Methods

Donor testicular tissueA testicular tissue biopsy measuring �50 mm3 was obtained from 14 adultpatients undergoing a vasectomy reversal after written informed consent.All men had normal spermatogenesis, as proven by histology. The biopsiedspecimens were immediately transported to the laboratory on ice, washedto remove any residual blood and cut into 6–8-mm3-sized fragments forthe freezing procedures or 3–4 mm3 in case vitrification was performed.The fragments were allocated to different groups until each group con-tained 20 fragments. Fresh tissue pieces were immediately fixed and sub-sequently used as non-cryopreserved controls for light microscopic orelectron microscopic evaluations. The remaining pieces were preparedfor cryopreservation (Fig. 1). This study was approved by the internalreview board of the UZ Brussel (99/131D).

Controlled slow freezingTesticular tissue was cryopreserved using three CSF protocols, two USFprotocols and two vitrification methods.

CSF was performed by placing two tissue pieces in each cryovial filledwith sterile Hank’s balanced saline solution (HBSS; 14175-129; Life Tech-nologies, Merelbeke, Belgium), containing 0.7 M DMSO (D2650;Sigma-Aldrich, Bornem, Belgium) without sucrose (2S) and 5 mg/mlhuman serum albumin (HSA; 10046; Vitrolife, Goteborg, Sweden) orwith 0.1 M sucrose (+S; 10274-5c; VWR, Leuven, Belgium) and 10 mg/ml HSA as described by Keros et al. (2007) and Wyns et al. (2008), re-spectively. As a third CSF procedure, we modified the latter protocolby using 1.5 M instead of 0.7 M DMSO to test whether an increasedfinal DMSO concentration would enhance its protective effect on tissueor cells. Equilibration was performed at +48C for 30 min. Using a pro-grammable freezer (Planer Kryo 10 series, Planer Products, Middlesex,UK), the vials were cooled at 18C/min with holding at 08C for 5 min, fol-lowed by cooling at 0.58C/min until 288C. At this temperature, theprogram was put on hold for 10 min to allow manual seeding. Theprogram continued at a rate of 0.58C/min until 2408C, held for10 min, and continued to 2708C at 78C/min, with subsequent plungingto liquid nitrogen. The samples were thawed according to Keros et al.

Comparing cryoprotocols for human testis tissue 1817

(2007) or Wyns et al. (2008), in a water bath at 378C until the ice melted(2 min) and then washed twice for 5 min in sterile HBSS on ice or, whenapplicable, three times in a reversed sucrose gradient solution (0.1, 0.05and 0 M sucrose). Afterwards, the fragments were immediately fixed forlight or electron microscopy.

Uncontrolled slow freezingThe USF 1.5 M DMSO + S protocol described earlier by our team formouse tissue contained a slightly higher sucrose concentration (Baertet al., 2012). In each cryovial, two tissue pieces were equilibrated in cryo-protective medium for 15 min on ice water. The cryomedium for USF con-sisted of Dulbecco’s minimal essential medium/F12 (DMEM/F12;31330-095; Life Technologies) supplemented with 1.5 M DMSO, 0.15 Msucrose and 10% HSA. An USF 0.7 M DMSO + S protocol was addedin order to study possible DMSO toxicity at higher concentration. Thecryovials were placed in an isopropyl alcohol container (479–3200; MrFrosty Freezing Container; VWR) and put in a 2808C freezer. Sampleswere cooled with an uncontrolled rate of �18C/min. When 2808Cwas reached, cryovials were transferred to liquid nitrogen for storage.The samples were thawed at 378C in a water bath for 2 min as describedin Baert et al. (2012), washed twice in DMEM/F12 supplemented with10% HSA to dilute the cryoprotectant and fixed for further assessment.

VitrificationTesticular tissue samples were vitrified and warmed as previouslydescribed for prepubertal mouse testicular tissue (Baert et al., 2012).The testicular fragments were exposed to a DMEM/F12-based vitrificationsolution containing 1.05 M DMSO and 1.35 M ethylene glycol (EG;E-9129; Sigma-Aldrich) for 10 and 2.1 M DMSO and 2.7 M EG for5 min. At the second step, the vitrification solution was supplementedwith 20% HSA. The following step differed between the two vitrificationmethods. For SSV, the tissue pieces were transferred to an aluminiumfloater partially immersed in liquid nitrogen to allow vitrification. After-wards, two vitrified samples were transferred to a liquid nitrogen-cooledcryovial before the storage in a liquid nitrogen tank. For DCV, the frag-ments were immediately put in a cryovial and directly exposed to liquidnitrogen. Samples were warmed according to Baert et al. (2012) byadding a pre-warmed solution (378C, DMEM/F12 + 0.5 M sucrose +20% HSA) to the cryovials and by keeping the samples at 378C for

2 min. Samples were washed in DMEM/F12 with 20% HSA for 2 min at378C and finally immersed in a fixative for light or electron microscopy.

Light microscopySamples for light microscopy were fixed in a hydrosafe fixative (R10S7-16-60; Labonord, Rekkem, Belgium). This is an alcohol-based formol-free fixative. After fixation, the samples were embedded in paraffin andcut into 5 mm-thick serial sections for immunohistochemistry. Three sec-tions separated by 40 mm were evaluated for each marker. For thepurpose of quantifying cells, three random fields were imaged per section.

Inhibina (M3609; Dako, Heverlee, Belgium) was used as a marker for activeSertoli cells (SCs). This marker also allowed assessing the effect of cryopreser-vation on the seminiferous epithelial integrity by determining the percentage ofintact tubules. Normal seminiferous epithelium consists of SCs which extendfrom the basal membrane to the lumen of the tubule and show a good adhe-sion between each other, with germ cells and with the basement membrane(Mruk and Cheng, 2004). A disrupted epithelium is characterized by rupturedcell–cell and cell–matrix connections (Keros et al., 2005). Immunohistochem-ical stainings for steroidogenic acute regulatory protein (StAR; sc-25806;Tebu-bio, Boechout, Belgium), a Leydig cell (LC)-specific marker, wereused to study the integrity of the interstitial tissue after cryopreservation bycounting the number of intact LC clusters per tubule (Gotoh et al., 1984).Such intact clusters (ICs) are seen as a small or larger group of LCs in closecontact with the surrounding connective tissue and in the vicinity or aroundblood vessels (Davidoff et al., 2009). Staining procedures for inhibin a andStAR were performed as previously described (Van Saen et al., 2011).

Terminal deoxynucleotidyl transferase-mediated nick and end-labeling(TUNEL), with in situ end-labeling of fragmented DNA, was used to evalu-ate apoptosis. The percentage of TUNEL-stained tubular and interstitialnuclei to all nuclei in the tubules or interstitium was recorded. ATUNEL assay was performed with the DeadEnd Colorimetric TUNELSystem (Promega, Leiden, The Netherlands). The sections were deparaf-finized and rehydrated, followed by fixation in 4% paraformaldehyde for15 min. They were incubated with 20 mg/ml of proteinase K for 10 minat room temperature, and then the sections were rinsed with phosphatebuffered saline and kept in the equilibration buffer for 10 min at room tem-perature. Thereafter, they were incubated with 100 ml of a working ter-minal deoxynucleotidyltransferase (TdT) reaction mix [containingrecombinant TdT (rTdT) enzyme] in a humid atmosphere for 1 h at378C. The reaction was terminated by washing the slides for 15 min.The sections were then immersed in 0.3% hydrogen peroxide for 5 min

Figure 1 Experimental scheme of the study design. DMSO, dimethylsulphoxide; 2S, without sucrose; +S, with sucrose.

1818 Baert et al.

at room temperature to inactivate endogenous peroxidase and subse-quently incubated with 100 ml of streptavidin for 30 min at room tempera-ture. Finally, the sections were stained with diaminobenzidine (DAB).Positive controls consisted of incubating sections with DNase I. A negativecontrol was performed by incubating the section with a TdT reaction mixwithout the rTdT enzyme.

Cell proliferation ability was assessed by detection of proliferating cellnuclear antigens (PCNAs) in the basal germ cells of the cryopreservedfragments. PCNA is a central component of the DNA replication machin-ery and is also involved in many cell processes, such as DNA recombin-ation and repair, chromosome assembly and cell cycle progression viaits protein–protein interactions. Therefore, it has been promoted as agood predictive marker for cryoinjury (Milazzo et al., 2010). The PCNAindex was estimated as the percentage of PCNA-positive germ cellslocated at the basement membrane. Germ cells in the basal region ofthe seminiferous tubule were recognizable by their large, roundednuclei, while SCs have an ovoid or triangular-shaped nucleus (Mruk andCheng, 2004). The average number of ubiquitin carboxy-terminal hydro-lase L1 (UCHL1)-positive SG per tubule was determined to study andcompare the actual spermatogonial survival after cryopreservation.UCHL1 has been employed in various animal models and the human toidentify SG (von Kopylow et al., 2010). The cross-sections were deparaf-finized and rehydrated in descending series of alcohol. Peroxide activitywas quenched using 0.3% H2O2 (for PCNA stainings) or 3% H2O2 (forUCHL1 stainings) in methanol for 30 min. Sections were placed incitrate buffer (0.01 M, pH 6.0) for 75 min in a 958C water bath (forPCNA stainings) or for 4 min in a microwave at 500 W (for UCHL1 stain-ings) and cooled for 30 min at room temperature. Non-specific bindingwas blocked in 10% normal goat serum for 30 min at room temperature.Sections stained for PCNA underwent an additional permeabilization stepwith 0.1% Triton X-100 for 30 min at room temperature. The primaryantibody of PCNA (1/400, NA03; VWR) or UCHL1 (1/2500,7863-0504; Bioconnect, Huissen, The Netherlands) was added to thesamples and incubated at 48C overnight or for 2 h, respectively. A horse-radish peroxidase-labelled secondary anti-mouse antibody (K5007; DakoReal Envision Detection System, Dako) was added and incubated atroom temperature for 1 h. DAB was used as a chromogen.

All sections were counterstained with haematoxylin, rehydrated andmounted. Examinations were performed on an inverted light microscope(Olympus IX81). Digital images were captured with a digital camera(CC12 Soft Imaging System, Olympus) at a final magnification of ×200for Inhibin a and StAR stained slides or ×400 for PCNA, UCHL1 andTUNEL-stained slides.

Transmission electron microscopySamples for electron microscopy were fixed in 2.5% glutaraldehyde in coca-dylate buffer (0.1 M, pH 7.4) at 48C. Fresh and cryopreserved samples of+1 mm3 were post-fixed in 1% osmium-tetroxide and 2% uranyl acetate.Next, the samples were dehydrated through an ascending series ofethanol, immersed in propylene oxide for solvent substitution and embed-ded in a Poly/bed 812 Araldite resin (21960; Polysciences Inc., Eppelheim,Germany). Ultrathin sections (50–100 nm) were cut with an ultracut ultra-microtome (Reichert-Jung, Depew, NY, USA), mounted on a copper gridand contrasted with uranyl acetate and lead citrate. Ultrastructural examina-tions were performed using a Tecnai 10 Philips electron microscope. Digitalimages were taken at magnifications between ×1850 and ×12 500 using amega view G2 CCD camera (SIS-company, Munster, Germany). Imageswere saved in TIFF format with a resolution of 1376 × 1032 and viewedwith the Olympus Soft Imaging Viewer (version 5.1, Olympus Soft ImagingSolutions) software. A semi-quantitative analysis of the cell integrity was per-formed by studying the ultrastructural morphology of 20 SG, SCs, LCs and

myoid cells (MCs) in each fresh and cryopreserved sample. As defined byTrump et al. (1974), subcellular alterations characteristic of acute cryo-preservation injury range from reversible changes (dilated endoplasmatic re-ticulum; dense mitochondria with enlarged cristae) to those which areirreversible (high-amplitude swollen mitochondria containing flocculentdensities; distorted membrane systems). When there were no signs ofcryoinjury (intact cell) or when reversible morphological alterations wereobserved (influenced cell), testicular cells were categorized as undamaged.If irreversible cryoinjury was noted, testicular cells were considered asdamaged. The proportions of undamaged (intact and influenced) anddamaged testicular cells were calculated in each sample.

Statistical analysesData are presented as mean values+ standard deviation, and statistical ana-lyses were conducted using one-way analysis of variance with post hoc Bonfer-roni adjustments (IBM SPSS Statistics Version 19, IBM Corporation, Somers,NY, USA). A P-value of ,0.05 was considered statistically significant.

Results

Apoptosis, germ cell proliferation abilityand SG survivalSections stained by TUNEL were analysed to study apoptosis aftercryopreservation. The frequency of apoptotic cells in the tubular com-partment was not different from the control for all tested protocols.The mean value of apoptotic cells in the interstitium slightly increasedafter cryopreservation, though it did not reach statistical significance(Fig. 2).

Cell proliferation ability was assessed by estimating the PCNA index(PCNA-positive basal germ cells to all basal germ cells) in the differentexperimental groups (Fig. 3A, B and E). In addition, the averagenumber of UCHL1-positive SG per tubule was determined to studyspermatogonial survival (Fig. 3C–E). When compared with the freshcontrol (PCNA index of 36.8+9.7% and 21.4+5.6 SG pertubule), PCNA indices and the number of SG decreased significantlyto 18.9+9.0% (P , 0.001) and 4.9+ 2.1 (P , 0.001) after CSF0.7 M DMSO 2 S and to 23.9+5.0% (P , 0.02) and 11.6+5.1(P , 0.001) after CSF 1.5 M DMSO + S, respectively. Higher PCNAindices were obtained with CSF 0.7 M DMSO + S (25.4+6.7%),USF 0.7 M DMSO + S (28.9+10.0%), SSV (41.4+ 14.0%) andDCV (36.2+ 17.3%). Compared with the control, our data neverthe-less reveal a significant loss (P , 0.001) of SG per tubule after CSF0.7 M DMSO + S (8.2+5.4), USF 0.7 M DMSO + S (8.8+3.9),SSV (12.6+ 4.4) and DCV (11.7+5.7). In the fragments cryopre-served with USF 1.5 M DMSO + S, the PCNA index (32.4+13.1%) and the number of SG per tubule (16.9+ 8.7) were notaffected compared with the fresh control (Fig. 3E).

Seminiferous epithelium coherence andinterstitial compartment integrityThe average percentage of intact and distorted tubules was deter-mined on inhibin a immunostained sections. On average in the freshtissue fragments, 89.7+ 5.8% of the tubules was considered tohave a structurally normal epithelium. The tissue samples subjectedto CSF 0.7 M DMSO + S (74.5+8.0%, P , 0.05), USF 0.7 MDMSO + S (72.4+ 11.7, P , 0.005), SSV (71.0+ 21.6%, P ,


0.002) and DCV (59.6+20.9%, P , 0.001) displayed severe deform-ation of the seminiferous epithelium compared with the control, char-acterized by a rupture of cell–cell and cell–matrix connections. CSFand USF could preserve the epithelial morphology when using 1.5 MDMSO and sucrose (Fig. 4A–C). Surprisingly, also CSF using 0.7 MDMSO without the addition of sucrose could preserve the tubules.

Immunohistochemistry for StAR allowed visualization of the LCclusters to study the impact of the cryoprotocols on the integrity ofthe interstitium. In the control tissue fragments, 1.9+0.4 LC clustersper tubule showed IC morphology and normal connectivity to sur-rounding interstitial components. This number was not statistically dif-ferent after cryopreservation (Fig. 4D–F).

Protection of testicular cell ultrastructureThe impact of the different cryopreservation procedures on testicularcell morphology was studied semi-quantitatively at high resolutionusing the transmission electron microscopy.

The majority of testicular cells in fresh fixed tissues was undamaged;up to 66.7+ 29.3% of LCs, 90.0+17.3% of MCs, 100.0+0.0% ofSG and 93.3+11.5% of SCs. Surviving testicular cells in CSF 0.7 MDMSO 2 S, CSF 1.5 M DMSO + S, both USF and both vitrificationgroups that have an undamaged ultrastructure were observed infrequencies not different from the control. CSF 0.7 M DMSO +S-cryopreserved fragments contained a percentage of cells showing

Figure 2 Protection from apoptosis. Apoptotic cells in a fresh (A) and SSV-treated fragment (B) detected by TUNEL. Negative control incubatedwithout terminal transferase (C). Positive control treated with DNase I (D). No significant differences in apoptosis were observed between fresh andcryopreserved fragments (E). A red arrow in A and B indicates a negative cell. A black arrow in A and B indicates a positive cell. Results in (E) areexpressed as means+ SD and were compared with the values obtained from a fresh control (Fr). In all groups, n ¼ 10 fragments. CSF, controlled slowfreezing; DCV, direct cover vitrification; DMSO, dimethylsulphoxide; S, sucrose; SSV, solid-surface vitrification; USF, uncontrolled slow freezing;Vi, vitrification.

1820 Baert et al.

an undamaged morphology not different from the control for all butone cell type, i.e. SG. Using the latter protocol, the SG that survivedcryopreservation exhibited distinct ultrastructural signs of cryoinjury,thereby reducing the percentage of undamaged SG significantly to33.3+2.9% (Fig. 5A–E).

DiscussionIn the present study, we explored USF, SSV and DCV as new cryo-preservation methods for human testicular tissue and comparedthem with the conventional CSF. Our data demonstrate that only

USF 1.5 M DMSO + S proved to efficiently preserve human testiculartissue.

Cryopreservation of human testicular tissue by CSF, being themethod of choice in current fertility preservation programmes, isbased on a lengthy slow-rate cryopreservation procedure requiringan expensive controlled rate freezer and liquid nitrogen supply. More-over, different cycles have to be performed in series for this type offreezing. On the contrary, USF only takes up about half the time ofthe CSF procedure and requires a rather inexpensive small 2808Cfreezer, which might be portable, and isopropyl alcohol containersto place cryovials in it. The vitrification technique needs just +

Figure 3 Impact on cell proliferation ability and spermatogonial survival. Immunostaining done for PCNA on a fresh sample (A) and a CSF 0.7 MDMSO 2 S-treated sample (B). Immunostaining showing UCHL1-positive cells in a control sample (C) and a CSF 1.5 M DMSO + S-cryopreservedsample (D). The lowest PCNA indices were found when using CSF 0.7 M DMSO 2 S and CSF 1.5 M DMSO + S. Other cryogroups had comparablePCNA indices to the control. However, importantly, only cryopreservation with USF 1.5 M DMSO + S could retain the absolute number of SG (E).Insets in (A–D) represent negative control. A red arrow in A and B indicates a negative cell. A black arrow in A and B indicates a positive cell. Resultsin (E) are expressed as means+ SD and were compared with the values obtained from a fresh control (Fr). In all groups, n ¼ 20 fragments for PCNAand UCHL1 staining. *,#P , 0.001; **P , 0.02. CSF, controlled slow freezing; DCV, direct cover vitrification; DMSO, dimethylsulphoxide; PCNA, pro-liferating cell nuclear antigen; S, sucrose; SG, spermatogonia; SSV, solid-surface vitrification; USF, uncontrolled slow freezing; Vi, vitrification.


Figure 4 Preservation of the seminiferous epithelial coherence and interstitial compartment integrity. Tubules of a specimen cryopreserved withUSF 0.7 M DMSO + S showing an intact (IT) or distorted (DT) epithelium that were stained for inhibin a at low (A) and high (B) magnification.Tissue treated with CSF 0.7 M DMSO + S, USF 0.7 M DMSO + S and both vitrification protocols showed a poor outcome, as indicated by significantlydecreased percentages of intact tubules (C). Cryoinjury was not prominent when the capacity to preserve the interstitial compartment was evaluatedby StAR immunostaining (D). High magnification image of an IC and distorted cluster of LCs in an USF 0.7 M DMSO + S-cryopreserved fragment (E).Every tested cryoprotocol resulted in a comparable number of LC clusters per tubule showing a normal structure (F). Insets in (A) and (D) representnegative controls. Results in (C) and (F) are shown as means+ SD and were compared with values obtained from fresh controls (Fr). In all groups,n ¼ 20 fragments. *P , 0.05; **P , 0.005; ***P , 0.002; ****P , 0.001. CSF, controlled slow freezing; DCV, direct cover vitrification; DMSO, dimethyl-sulphoxide; LC, Leydig cell; S, sucrose; SSV, solid-surface vitrification; USF, uncontrolled slow freezing; Vi, vitrification.

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30 min of preparation and allows an immediate transfer to liquid nitro-gen, thereby, completely eliminating the need for cryopreservationequipment. USF and vitrification are, therefore, potentially useful tech-niques not only when the procurement site is distant from the bankingsite, but also for laboratories in developing countries (Vajta and Nagy,2006).

So far, data comparing CSF, USF and vitrification for testicular tissueare limited. In mice, Milazzo et al. (2008, 2010) tested distinct CSF andUSF protocols to determine the optimal parameters for immaturemouse testicular tissue cryopreservation. They showed the superiorityof CSF 1.5 M DMSO + S in preserving seminiferous cord morphology,cell proliferation ability and functional integrity among tested condi-tions. In our previous work, we have performed comparative investi-gations of USF 1.5 M DMSO + S and SSV for prepubertal mousetesticular tissue. We showed that SSV resulted in success rates com-parable with the USF protocol in maintaining testicular cell

ultrastructure, tubular morphology and tissue function (Baert et al.,2012). In piglets, Zeng et al. (2009) compared cell viability and invivo developmental potential after CSF 1.3 M DMSO 2 S, USF 1.3 MDMSO 2 S and a closed vitrification system. No significant differenceswere observed between these three methods. In agreement, resultsfrom comparative investigations by Abrishami et al. (2010) demon-strated SSV to be as efficient as CSF 1.3 M DMSO + S in cryopreserv-ing piglet testicular tissue. The group of Wyns showed theirvitrification system using open cryostraws to be as efficient as CSF0.7 M DMSO + S in preserving the integrity of prepubertal mouse(Curaba et al., 2011) and prepubertal human testicular tissue (Poelset al., 2013).

In this study, we used two well-established CSF protocols that weredeveloped by the group of Keros et al. (2005) and Wyns et al. (2008)for freezing human testicular tissue. Like Keros et al. (2005), we wereable to preserve the tubular epithelial structure and interstitial cells in

Figure 5 Effect of the tested cryoprotocols on the testicular cell ultrastructure. Transmission electron micrographs of a fresh sample demonstratingan overview of the testicular cells of interest (A), a cell considered as intact (B), an influenced cell with reversible ultrastructural changes (C) and a cellscored as being irreversibly damaged (D). Percentages of undamaged (intact and influenced) or damaged testicular cells (E). Testicular cells still presentafter cryopreservation show similar testicular cell integrity in CSF 0.7 M DMSO 2 S, CSF 1.5 M DMSO + S, both USF and both vitrification groups.CSF 0.7 M DMSO + S resulted in a pronounced altered ultrastructure of surviving SG. Results in (E) are expressed as mean values and were comparedwith the values obtained from fresh controls (Fr). In all groups, n ¼ 3 fragments and n ¼ 20 cells of each cell type. *P , 0.05. CM, cell membrane;CSF, controlled slow freezing; DCV, direct cover vitrification; DMSO, dimethylsulphoxide; ER, endoplasmatic reticulum; LC, Leydig cell;M, mitochondrion; MC, myoid cell; N, nucleus; NM, nuclear membrane; S, sucrose; SC, Sertoli cell; SG, spermatogia; SSV, solid-surface vitrification;USF, uncontrolled slow freezing; Vi, vitrification; arrowhead indicates mitochondrial flocculent densities.


human testicular tissue with CSF using 0.7 M DMSO 2 S. However,we were unable to preserve the SG. TUNEL staining did not revealmore apoptotic cells in the tubules, but analyses targeting the SGshowed a reduced cell proliferation ability and spermatogonial losswhen using CSF 0.7 M DMSO 2 S. This discrepancy in results canbe due to the use of MAGE-A4 as a SG marker by Keros et al.Given that primary spermatocytes also express this protein makes itplausible that Keros et al. have included these cells in their analysis(Aubry et al., 2001). The SG that did survive and all studied somaticcell types displayed a comparable ultrastructure to control cells. Mul-tiple factors may have contributed to the less favourable results of CSF0.7 M DMSO 2 S. First, it was reported that the induced ice-nucleation step included in this CSF protocol may not be necessaryand may even be harmful (Milazzo et al., 2008). Second, theabsence of sucrose in the cryopreservation medium of this CSFmethod could have resulted in insufficient cell dehydration. Macromo-lecules, such as sugars, can act as a non-permeable cryoprotectant, in-creasing water withdrawal from cells and, thus, counteractingintracellular ice crystal formation. It was reported that the additionof sucrose as a cryoprotectant improved the cryopreservationoutcome of embryos (Van den Abbeel et al., 1994), ovarian tissue(Amorim et al., 2010) and spermatogonial stem cells (Izadyar et al.,2002). However, in this study, CSF 0.7 M DMSO + S-frozen tissuestill exhibited cryoinjury: a reduction in tubules with a coherent sem-iniferous epithelium, poor spermatogonial survival and the survivingSG showing clear ultrastructural changes compared with the control.A third factor concerns the low concentration of DMSO (0.7 M).This may cause insufficient dehydration and eventually the formationof harmful intracellular ice crystals. Elevating the concentration to1.5 M DMSO may improve protection against tissue cryoinjury(Milazzo et al., 2008; Milazzo et al., 2010). Surprisingly, we found noimprovement in the outcome when cryopreserving human testiculartissue by CSF 1.5 M DMSO + S.

The USF 1.5 M DMSO + S protocol was developed earlier by ourteam for testicular tissue preservation in a mouse model (Goossenset al., 2008; Baert et al., 2012). Apparently, this USF protocol canalso maintain human testicular tissue during cryopreservation, sincethe present results showed no major differences between USF1.5 M DMSO + S-frozen tissue and fresh control tissue. Reasonswhy USF 1.5 M DMSO + S gave better results than any CSF protocolcould be the freezing rate itself or the shorter equilibration time. Pro-longed exposure to DMSO before storage can be harmful (Flemingand Hubel, 2006). The testicular cells in both fresh and USF tissuepieces, which were categorized using the TEM as damaged, displayedstructural changes which could result from hypoxia and handling of thematerial before fixation.

Vitrification aims at removing a high proportion of cellular water andsolidification of the solution without crystal formation. To achieve this,it is necessary to use both high cooling rates and high concentrationsof cryoprotectants. The SSV technique was applied because it is aclosed system and thereby it minimizes the potential hazard of patho-genic contamination of samples. Furthermore, it provides enoughspace for tissue (Abrishami et al., 2010). In order to give a vitrifiableconcentration of total cryoprotectants while decreasing their individualspecific toxicity, DMSO and EG were combined. SSV could notprotect human testicular tissue; more seminiferous tubules werefound displaying a ruptured epithelium after cryopreservation and

SSV had a negative impact on the spermatogonial number. We hy-pothesize that this may be due to stress on the cells as a result ofmechanical forces generated by the extracellular ice. Incomplete vitri-fication, and thus, crystallization, may have occurred because in SSVhigh cooling rates are difficult to attain. High cooling rates are easierto obtain with DCV in which liquid nitrogen is directly applied tothe testicular tissue. Nevertheless, by using the latter procedure, theoutcome of vitrification did not improve. Vitrification is a promisingtechnology in testicular tissue cryopreservation, but still in an initialstage. Further optimization of this technique should be considered.Elevating concentrations of cryoprotectants and/or reducing tissuesize may help to achieve complete vitrification (Yavin and Arav, 2007).

Because normal prepubertal testicular tissue is difficult to obtain, wecryopreserved adult human testicular tissue. Protocols similar to thosedescribed here have been used successfully to preserve prepubertaltesticular tissue from various animals (Honaramooz et al., 2002; Jahnu-kainen et al., 2007; Goossens et al., 2008; Abrishami et al., 2010; Baertet al., 2012). Yet the newly introduced protocols for human testiculartissue cryopreservation should be tested for cryopreserving prepuber-tal human testicular tissue. A study in rats already highlighted the dif-ferential sensitivity of immature tissue to the selection of thecryoprotectants and their concentrations (Unni et al., 2011). Further-more, functional integrity evaluation of cryopreserved tissue is funda-mental to validate a freezing–thawing/cooling–warming procedure,since previous studies concluded that a high survival rate of morpho-logically normal SG does not necessarily imply that those SG are actu-ally functional (Frederickx et al., 2004; Jahnukainen et al., 2007; Zenget al. 2009). The opposite may also be true; our previous studyshowed that cryoinjured tissue retained its function and couldrecover after transplantation (Baert et al., 2012). It remains, thus,important to assess cell or tissue functionality by xenografting or byin vitro assays.

In conclusion, this study showed that USF 1.5 M DMSO + S is anefficient alternative to CSF for human testicular tissue. USF 1.5 MDMSO + S yielded the best results in preventing apoptosis and pro-tecting the tubular epithelium coherence, interstitial compartment in-tegrity, cell proliferation ability, spermatogonial number and testicularcell ultrastructure. However, further studies should be conducted toevaluate cell or tissue functionality and to confirm the present findingsusing prepubertal testicular tissue.

AcknowledgementsThe authors are grateful to Mrs Marleen Berghmans of the Depart-ment of Pathology for skilful preparation of the tissue for transmissionelectron microscopy and Mr Pierre Hilven and Mr Fabian Van Haelstfrom the Department of Embryology and Genetics for their helpwith data acquisition.

Authors’ rolesY.B.: conception and design of the study, acquisition of samples foranalysis, interpretation of data, drafting of the article and final approvalfor submission. D.V.S.: acquisition of samples for analysis and revisionof the article. P.H.: interpretation of data and revision of the article.P.I.V: transmission electron microscopy-sample acquisition, discussionof the results and revision of the article. H.T.: conception and design of

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the study and final approval for submission. E.G.: conception anddesign of the study, interpretation of data, discussion of the results, re-vision of the article and final approval for submission.

FundingThis study is supported by a PhD grant from the Agency for Innovationby Science and Technology and research grants from the FlemishLeague against Cancer-Public Utility Foundation, the Scientific Re-search Foundation Flanders, Methusalem grant and the Vrije Universi-teit Brussel. E.G. is a postdoctoral fellow of the Scientific ResearchFoundation Flanders.

Conflict of interestNone declared.

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