mouse ovarian follicle cryopreservation using vitriﬁcation ...€¦ · ovarian follicles may...

Mouse ovarian follicle cryopreservation using vitrificationor slow programmed cooling: Assessment of in vitrodevelopment, maturation, ultra-structure and meioticspindle organization

Nina Desai1, Faten AbdelHafez1,2, Mansour Y. Ali2, Ezzat H. Sayed2,Ahmed M. Abu-Alhassan2, Tomasso Falcone1 and James Goldfarb1

1Department of OB-GYN, Cleveland Clinic Foundation, Cleveland, Ohio, USA; and 2Department of OB-GYN, Women’sHealth Centre, Assiut University, Assiut, Egypt

Abstract

Aim: To compare different outcomes of vitrification and slow freezing of isolated pre-antral follicles and toevaluate different cryo-devices for vitrification of isolated follicles.Methods: Pre-antral follicles were isolated from mouse ovaries and cryopreserved using vitrification and slowfreezing. A preliminary experiment was carried out to select the optimal cryo-device for vitrification of isolatedfollicles. A total of 414 follicles were randomly distributed among four groups: control (CT) fresh (n = 100),nylon mesh (n = 96), electron microscopy grid (n = 102), and micro-capillary tips (n = 116). Subsequently, a totalof 979 follicles were randomly assigned to three different groups: CT fresh (n = 256), vitrification (n = 399) andslow freezing (n = 324). CT and cryopreserved/thawed follicles were cultured in vitro and examined daily fordevelopment. Final maturation was triggered with human chorionic gonadotrophin and rates of oocytematuration were calculated. The ultra-structure of cryopreserved/thawed follicles was studied using electronmicroscopy. Meiotic spindle presence and organization in mature oocytes were examined using the Oosightimaging system.Results: Micro-capillary tips resulted in poor immediate post-warming survival but no differences wereobserved in the subsequent in vitro development characteristics between different cryo-devices. Nylon meshproved to be the easiest carrier, particularly when large numbers of follicles were to be vitrified. Compared tovitrification, slow freezing resulted in a significantly lower number of intact follicles at the end of the cultureperiod (P < 0.0001). However all other outcome measures were comparable between both techniques.Conclusions: Isolated follicles were more vulnerable to cryodamage after slow freezing as compared tovitrification.Key words: in vitro maturation, meiotic spindle retardance, pre-antral follicle, ultra-structure, vitrification.

Received: June 16 2009.Accepted: February 5 2010.Reprint request to: Dr Nina Desai, The Cleveland Clinic Fertility Center, 26900 Cedar Road, Beachwood, OH 44122, USA.Email: [email protected] support: None.CapsuleIn vitro cultured vitrified/warmed isolated pre-antral follicles have significantly higher survival rates but comparable maturation rates,meiotic spindle assembly and chromosomal alignment compared to slowly frozen/thawed isolated pre-antral follicles.

doi:10.1111/j.1447-0756.2010.01215.x J. Obstet. Gynaecol. Res. Vol. 37, No. 1: 1–12, January 2011

© 2010 The Authors 1Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology

Introduction

Ovarian follicle/tissue cryopreservation has been pro-posed as an alternative fertility preservation option.1–6

Follicles can be cryopreserved in intact ovarian tissuepieces3,7–9 or after isolation of individual follicles fromthe fresh ovarian tissue using enzymatic or mechanicaltechniques.10–16 In both situations, follicles wouldrequire further maturation either in vivo or in vitro forthem to be useful in restoring fertility.

With ovarian tissue cryopreservation, follicle matura-tion upon thawing could be achieved in vivo with tissuetransplantation or alternatively in vitro by enzymaticdigestion of the cryopreserved/thawed ovarian tissue,followed by in vitro maturation (IVM). As an alternativestrategy, cryopreservation of isolated ovarian follicleshas many potential advantages over ovarian tissue cryo-preservation. First, the cryoprotectant agent (CPA) per-meation through follicular suspensions is expected tobe more effective compared to ovarian tissue pieces.10 Inaddition, ovarian tissue has more complex structurewith different cell types making the choice of a suitablecryopreservation protocol even more challenging.Moreover, the post-thaw in vitro assessment of the fol-licular pool will be easier with isolated follicles com-pared to the whole ovarian tissue pieces.17 Moreimportantly, cryopreserved/thawed follicles can be sus-pended in a plasma clot10,18 or collagen gel19 permittingauto-transplantation via a minimally invasive approach.Auto-transplantation of cryopreserved/thawed fol-licles could result in better survival compared to ovariancortical strips, as angiogenesis and revascularizationcould be faster.20 Added to these benefits is the fact thatthe basal lamina of isolated primordial follicles excludescapillaries, white blood cells and nerve cells, serving asa barrier to prevent cancer cell infiltration into the fol-licle.21 This could negate the risk of cancer recurrencethat has always been a concern when considering auto-transplantation of cryopreserved/thawed ovariantissue in cured cancer patients.

In light of this concern, cryopreservation of isolatedovarian follicles may provide an attractive fertilitypreservation alternative. Isolated ovarian follicles canbe cryopreserved by vitrification12–15 as well as bytraditional programmed slow freezing methodol-ogy.10,11,16,19,22 Prevention of intra-cellular ice formationis tantamount for the success with either methodology.In addition, the cryopreservation technique must mini-mize disruption of the oocyte–granulosa cell (GC) con-nections within the follicular unit to allow furthermaturation in vitro.

Slow freezing protocols require expensive instru-ments for controlled-rate freezing of cells/tissues. Thetime required for cryopreservation and thawing withslow cooling techniques is also substantially longer. Incontrast, vitrification is simpler, requires little special-ized freezing equipment, and less procedural time.However, vitrification protocols do involve the use ofCPA at high concentrations and specialized cryo-devices to achieve very high cooling rates.23 The highconcentrations of CPA used during vitrification may betoxic to cells. Combining several CPA and/or exposingcells to CPA at room temperature may help to decreasethe risk of toxicity.24 Although the vitrification tech-nique appears to be simple, its outcomes are affectedby numerous factors such as technical skills, the type ofCPA and the cryo-device used for vitrification.25

Selection of the optimal cryopreservation technologymay affect the survival of intact follicles and theirability to undergo IVM. To this end, we performed thecurrent study to evaluate the outcomes after vitrifica-tion or slow freezing of isolated mouse pre-antral fol-licles. The efficacy of different cryo-devices for isolatedfollicle vitrification was tested. We assessed not onlythe post-thaw survival of cryopreserved follicles butalso some functional parameters such as continued invitro growth (IVG), oocyte germinal vesicle breakdown(GVBD), and maturation to the metaphase II (MII)stage. Meiotic spindle organization in MII oocytes wasfurther scrutinized using the Oosight imaging system.The ultra-structure of oocytes from pre-antral folliclescryopreserved by both methodologies was also exam-ined. To our knowledge this is the first comprehensivestudy comparing the application of vitrification andslow freezing techniques to the cryopreservation of iso-lated ovarian follicles. Understanding the limitationsand optimizing the current freezing methodology is anecessary stepping stone to ultimately being able tocryopreserve human ovarian follicles.

MethodsAnimals and ovarian follicle isolation

The animal protocol was approved by the ClevelandClinic Foundation Institutional Animal Care and UseCommittee. Ovaries were harvested from B6D2F1 pups(14–18 days old) and placed in Leibovitz media (L15)supplemented with 10% synthetic serum substitute(SSS). Ovarian tissue was enzymatically digested using1 mg/mL collagenase Type I (132 U/mg) at 37°C. At30-min intervals, ovaries were transferred to a new dishcontaining L15/SSS and repeatedly pipetted using

N. Desai et al.

2 © 2010 The AuthorsJournal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology

Eppendorf pipette tips of decreasing bore sizes tofacilitate the release of pre-antral follicles. Follicleswere collected, rinsed free of enzyme and examinedunder a stereomicroscope. Intact pre-antral follicleswith two or more layers of GC surrounding the oocytewere collected for the study. Primary follicles and fol-licles with antral cavities were excluded. Care wastaken to select follicles of a similar size range. Follicleswere first pooled before random distribution into treat-ment groups. Homogeneity of follicles was further con-firmed by assessment at magnification ¥300 using aninverted microscope with Hoffman Modulation opticsand the Microsuite Basic Imaging software. Folliclesselected for these experiments measured between 120and 160 mm. Follicles exhibiting overt damage from theenzymatic digestion and/or an incomplete collar ofGC surrounding the oocyte were excluded from thestudy.

Follicle vitrification

Enzymatically isolated pre-antral follicles were vitrifiedusing an ethylene glycol (EG)-raffinose-based proto-col.12 The basal media for the preparation of equilibra-tion and vitrification solutions was L15 + 20%SSS.Follicles were equilibrated for 5 min in 2M EG followedby a 30–60-s incubation in vitrification solution contain-ing 6M EG + 0.3M raffinose. All equilibration and vitri-fication steps were performed at room temperature. Inthe first experiment, we compared the outcomes withthree different vitrification cryo-devices: nylon mesh(4 mm pores, Cat# 146502), electron microscopy grid(EM grid; Cat# G200T-Cu, Type: 200 mesh coppersquare), and gel-loading micro-capillary tips (0.5–1 mLin volume, 0.6 mm in diameter). The nylon mesh wascut into 10 ¥ 10 mm sections that could be easilyhandled and inserted into a cryovial.

Twenty pre-antral follicles in a fluid microdrop ofless than 3 mL were loaded on to either the nylon meshor EM grids and were immediately plunged into a1.5-mL cryovial pre-filled with liquid nitrogen (LN2).With micro-capillary tips, follicles were loaded withthe aid of a mouth pipette. After loading, the tip washeld for 2 min in the vapor phase, just above the surfaceof the LN2, before immersion into a 3.5-mL cryovialpre-filled with LN2.26 In the second series of experi-ments we elected to use a single carrier, the nylonmesh, to compare follicle cryopreservation by slow pro-grammed cooling versus vitrification.

Vitrified follicles were warmed by immersion inbasal medium containing 1M sucrose. Follicles wereleft in the warming solution for 10 min at room tem-

perature and then washed at 37°C. After 5 min, follicleswere pipetted into fresh basal medium at 37°C foranother 5–10 min before placing in culture.

Slow freezing of isolated follicles

A dimethyl sulfoxide (DMSO)-based slow freezingprotocol was used.11 The basal medium for the prepa-ration of freezing solutions was L15 + 20% SSS. Thefreezing medium consisted of 1.5M DMSO. Twentyfollicles were pipetted and transferred into a cryovialcontaining 150 mL freezing media at 4°C and equili-brated for 15 min. Vials were cooled in a PlanerKryo-10 programmable freezer (UK) at a rate of-2°C/minute until -7°C and manually seeded. Follow-ing seeding, samples were further cooled at -0.3°C/min to -50°C and then at a rate of -50°C/min until-110°C. Cryovials were plunged into LN2 to completethe freezing process.

For thawing, cryovials were held at room tempera-ture for 30 s before plunging into a water bath at 37°C,with agitation, until ice melted. CPA was removed byserial dilution in basal medium with decreasing con-centrations of DMSO from 1.5 M to 1 M to 0.5 M to 0Mfor 15 min intervals. The first two steps were carriedout at room temperature. The last step in basal mediawas performed at 37°C.

Evaluation of immediatepost-warming/thawing survival

Survival of cryopreserved/thawed follicles wasassessed microscopically based on morphology of thefollicle as viewed under the stereoscope and thereafterat magnification ¥300 using an inverted microscopewith Hoffman modulation contrast. A follicle was con-sidered to be intact if it possessed an oocyte sur-rounded by a complete tight collar of GC. Follicles withpartially or completely naked oocytes or large spaceswithin the granulosa-oocyte complex were graded asdamaged. Any dark atretic-looking follicles were alsograded as damaged. Only undamaged, intact pre-antralfollicles were selected for further in vitro culture (IVC).

In vitro culture of follicles and oocyte maturation

Fresh and cryopreserved/thawed pre-antral follicleswere cultured in Minimum Essential Medium a(a-MEM) supplemented with 1% Nu-serum, 100 mIU/mL follicle-stimulating hormone (FSH), 10 mg/mLinsulin, 5.5 mg/mL transferrin and 0.67 mg/mL sele-nium (ITS). Pre-antral follicles were group-cultured on

Vitrification of isolated ovarian follicles


Transwell inserts (24 mm in diameter and 0.4 mm poresize) in six-well plates. All cultures were performed at37°C with 5.5% CO2 in air.

Serum concentration was reduced after overnightculture to 0.1% Nu-Serum and medium was halfchanged every other day. Follicles were cultured invitro for up to 10 days. The number of attached folliclescontaining an oocyte was determined at the end of thisculture interval. Follicles were then placed in matura-tion medium to induce oocyte maturation. The matu-ration media consisted of a-MEM supplemented with5% Nu-serum, 100 mIU/mL FSH, ITS (10 mg/mLinsulin, 5.5 mg/mL transferrin, 0.67 mg/mL selenium),1 ng/mL epidermal growth factor (EGF) and1.5 IU/mL human chorionic gonadotrophin. After16–18 h, cultures were treated with 40 U/mL hyalu-ronidase to facilitate the removal of GC surroundingthe oocyte. Oocytes were examined under an invertedmicroscope (¥300) and graded for nuclear maturity.

Meiotic spindle evaluation

Meiotic spindle organization was evaluated using bothpolarized light retardance and immunochemical stain-ing. For meiotic spindle imaging in living oocytes, weapplied computer-assisted polarized microscopy (Pol-scope) using the Oosight imaging system. MII oocyteswere placed in micro-droplets of pre-warmed mediumand covered with mineral oil on a glass-bottomed dish.Care was taken to maintain temperature at 37°Cthroughout the examination to prevent transient depo-lymerization of spindle microtubules through oocytecooling. Oocytes were imaged at magnification ¥400. Incases where the spindle was not seen, the oocyte wasrotated two to three times using a holder and an intra-cytoplasmic sperm injection pipette, to make sure thatthe spindle was not overlooked. Once the meioticspindle was visualized, an image was captured. Thespindle retardance was determined using the imagingsoftware. The retardance gave a quantitative measure-ment of the degree of order and density of the micro-tubules within the spindle.

For immunostaining of the meiotic spindle, MIIoocytes were first fixed and permeabilized at 37°C with2% paraformaldehyde + 0.1% Triton X in phosphatebuffered saline (PBS) for 30 min.27 After rinsing inPBS + 1% human serum albumin (HSA), oocytes wereincubated in 10% mouse serum + 1% HSA in PBS for1 h. Oocytes were rinsed and then incubated in fluo-rescein isothiocyanate-conjugated anti-mouse alphaand beta tubulin in a 1:1 ratio for 2 h. Followinganother rinse, oocytes were incubated in 10 mg/ml pro-

pidium iodide in PBS for 45 min before being mountedon glass slides. All steps were carried out at 37°C.Meiotic spindles were visualized at magnification¥1000 using an oil immersion lens and an Olympus BX51 microscope fitted for reflected fluorescence. Anormal meiotic spindle system was defined as havingMII chromosomes aligned along the centre of a barrel-shaped spindle. For evaluation of normality of thespindle and chromatin, we used very strict criteria,within the limits of our imaging capabilities. Variationsin spindle morphology such as displaced spindlefibers, stray chromosomes, or chromosomal misalign-ments, all resulted in classification of the meioticspindle as abnormal.

Preparation of follicles for light and transmissionelectron microscopy

All the transmission electron microscopy (TEM)reagents were purchased from Electron MicroscopySciences. Cryopreserved pre-antral follicles were fixedimmediately upon thawing. Freshly isolated pre-antralfollicles were prepared in parallel as control (CT).Fixation of follicles was performed using 2.5%glutaraldehyde/4% paraformaldehyde in 0.2 Mcacodylate buffer overnight at 4°C. After being rinsedin cacodylate buffer, follicles were post fixed inosmium tetroxide. The samples were then dehydratedin increasing concentrations of ethanol, embedded inEpon and sectioned. Semi-thin sections were stainedwith 1% toluidine blue and examined with the lightmicroscope (LM). Ultra-thin sections were stained withuranyl acetate to be examined and photographed usingthe TEM.

Statistical analysis

Each experiment was performed in two to four repli-cates. The data were pooled for subsequent analysis.The initial outcome measures analyzed were the imme-diate post-thaw survival, morphology and ultra-structure. Further functional assessment includedsustained growth over the IVC period, antral cavityformation and oocyte maturation as evidenced byGVBD, progression to the MII stage and meioticspindle organization. Meiotic spindle analysis was con-ducted on all mature oocytes in a separate experimentusing either immunochemical staining or else theOosight imaging system. Pooled data was comparedand analyzed using c2 and anova tests as appropriate.A P-value of <0.05 was considered significant. TheStatsDirect program was used for statistical analysis.

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ResultsVitrification and cryo-device

A total of 414 pre-antral follicles were utilized in thisexperiment. The CT group consisted of fresh non-vitrified follicles (n = 100). The remaining follicles werevitrified using one of three cryo-devices: nylon mesh(n = 96), EM grid (n = 102) and micro-capillary tips(n = 116).

The nylon mesh proved to be the easiest of the testedcryo-devices for loading as many as 20 follicles at atime. The mesh could be easily cut into a variety ofsizes, which facilitated its handling and manipulation.In this experiment, we cut the nylon mesh into squares(10 ¥ 10 mm). At the time of loading, the edge of thenylon mesh was held above the dish surface with ahemostat and the follicles were easily pipetted on to thesurface in a miniscule volume of fluid. The EM grid wasloaded similarly but proved more tedious to handledue to its extremely small size (3 mm diameter disc).The micro-capillary tip also proved to be more difficultto control. It was technically challenging to quickly load20 follicles into the tip. Moreover inadvertent damageto the tip was possible during immersion into the vialcontaining LN2.

On warming, the micro-capillary tip resulted in thelowest percentage of intact follicles compared to thenylon mesh and EM grid (P < 0.0001 and P = 0.001,respectively). We plated and monitored the subsequentgrowth of all intact vitrified/warmed follicles. Non-vitrified follicles were cultured as a CT group. Platedfollicles from all three vitrification cryo-devices devel-oped similarly. There was no statistically significant dif-

ference in the subsequent development and maturationof all surviving pre-antral follicles among the groups(Table 1). Follicle survival at the end of the IVC periodwas 55–62%. Antral cavity formation ranged from 45%to 66%. No differences were observed between the treat-ment groups. GVBD occurred in 79% of oocytes fromCT follicles and in 79–91% of vitrified/warmed follicles.The rate of maturation to the MII stage was also notsignificantly different from that observed in the CTgroup (Table 1). Based on these data and the ease ofhandling experienced with the nylon mesh, the cryo-device was selected for all subsequent experiments.

Vitrification and slow freezing

A total of 979 pre-antral follicles were utilized in thisexperiment. The CT group consisted of fresh follicles(n = 256). The remaining follicles were either vitrifiedusing nylon mesh (n = 399), or frozen using slow pro-grammed cooling (n = 324).

Survival, in vitro development and maturation of isolatedpre-antral follicles

Immediate post-thaw survival (Fig. 1) did not differwith vitrification versus slow cooling (Table 2).However with prolonged IVC, the CT and vitrificationtreatment groups had a significantly higher proportionof follicles that survived to the end of the culture inter-val as compared to the slow freeze group (71%, 65%and 48%, respectively, P < 0.0001). Other outcome mea-sures such as antral cavity formation, GVBD and MIIformation were similar with both cryopreservationtechniques and in the CT group (Table 2).

Table 1 Post-warming survival and development of isolated pre-antral folliclesvitrified using different cryo-devices

CT Nylonmesh

EM grid Tips

Total follicles cryopreserved NA 96 102 116Post-warming survival rate (%)† NA 95% 91% 72%‡Number of follicles cultured 100 88 86 77Intact follicles at end of culture (%)§ 73% 55% 62% 61%Antral cavity formation (%)¶ 56% 52% 45% 66%GVBD (%)¶ 79% 90% 79% 91%MII maturation (%)¶ 52% 58% 51% 60%

†Post-warming survival was defined as follicles that were intact on warming. Only intactpre-antral follicles were plated for further culture. Survival rate is expressed as a percentageof the number of follicles initially cryopreserved. ‡Survival rate was significantly lower withthe microcapillary tips (P < 0.001). §The rate is expressed as a percentage of the number ofcultured follicles. ¶The rates of antral cavity formation, GVBD and MII maturation areexpressed as a percentage of the number of intact follicles at the end of the culture interval.CT, non-frozen control group; EM grid, electron microscopy grid; GVBD, germinal vesiclebreakdown; Tips, micro-capillary tips; NA, not applicable.



Meiotic spindle assessment

In a separate experiment, meiotic spindles were ana-lyzed using either immunochemical techniques orpolarized light imaging. All mature oocytes were pre-pared for microscopy (n = 194). Using immunochemi-cal staining techniques we could not distinguish anydifference in morphology of meiotic spindles afterslow freezing as compared to vitrification. The majorityof oocytes derived from IVC/IVM follicles in all threegroups displayed barrel-shaped meiotic spindles withcentrally arranged chromosomes typical of normalspindles (Fig. 2a). Figure 2b depicts an example of anoocyte with spindle abnormalities and disorganizedchromosomal alignment. The percentage of normal

spindles observed was similar in all three groups(Table 3).

A total of 110 MII oocytes were evaluated usingpolarized light microscopy and the Oosight imagingsoftware (Fig. 3). This system allowed us to not onlymonitor the visual presence of the spindle but also tomake morphometric measurements of the spindleassembly. Retardance values of meiotic spindles in CTand oocytes derived from the two cryopreservationtechniques were similar (Table 3).

By using the two different techniques for meioticspindle analysis, we had both morphological and mor-phometric data (retardance values) to support the ‘nor-malcy’ of the spindles. The fresh in vitro maturedfollicles served as a CT. These data suggest that the

Figure 1 Isolated pre-antral follicles photographed immediately after warming/thawing (¥150). (a) Vitrified/warmed fol-licles. (b) Frozen/thawed follicles.

Table 2 Summary of results for vitrified and slowly frozen follicles

Control Vitrification SlowFreeze

Total follicles cryopreserved NA 399 324Post-warming/thawing survival rate (%)† NA 95% 92%Number of follicles cultured 256 366 280Intact follicles at end of culture (%)‡ 71% 65% 48%§Antral cavity formation (%)¶ 57% 64% 61%GVBD (%)¶ 77% 75% 78%MII maturation (%)¶ 40% 41% 40%

†Post-warming/thawing survival was defined as follicles that were intact on thaw. Onlyintact pre-antral follicles were plated for further culture. Survival rate is expressed as apercentage of the number of follicles initially cryopreserved. ‡The rate is expressed as apercentage of the number of cultured follicles. §Significantly lower than control and vitrifiedtreatment groups (P < 0.0001). ¶The rates of antral cavity formation, GVBD and MII matu-ration are expressed as a percentage of the number of intact follicles at the end of the cultureinterval. NA, not applicable.

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cryopreservation technique did not affect the meioticspindle assembly.

Light microscopic (LM) and TEM evaluation

The LM examination of the semi-thin sections of CTand cryopreserved/thawed follicles showed intact fol-licles with a centrally located oocyte and intact base-ment membrane surrounded by a flattened thecal celllayer(s) (Fig. 4).

TEM examination of vitrified and slowly frozen fol-licles was similar to that of their fresh CT counterparts.Cytoplasmic organelles in the oocyte and GC werewell preserved. Also, finger-like processes (microvilli)were seen projecting into the zona pellucida. In caseswhere the ultra-thin sections were taken through thenucleus, the germinal vesicle and its nucleolusappeared normal (Fig. 5).

Discussion

We have shown that enzymatically isolated ovarianpre-antral follicles can be quite successfully vitrified

using at least two of the tested cryo-devices, the nylonmesh and the EM grid. Pre-antral follicle cryopreserva-tion by vitrification as compared to slow programmedcooling may better preserve the integrity of the follicu-lar unit allowing continued in vitro development andpreventing premature ovulation of the enclosedoocyte.

The vitrification process can be affected by the type ofthe cryo-device. Cryo-devices that permit the loading ofa very minimal amount of fluid expedite the rate oftemperature drop, which is particularly importantduring vitrification. Examples of such carriers includethe cryoloop,28 cryotop,7 open pulled straw29and EMgrid.30 To date, conventional straws,12,14 EM grids13 andsolid surface vitrification (SSV)15 have been attemptedtowards vitrification of isolated ovarian follicles, withvariable results. Although the cryotop was successfullyapplied for ovarian tissue vitrification,7,9 it has not beenused for isolated ovarian follicles. In the current study,the type of cryo-device did have an impact on immedi-ate post-warming survival. However, continued devel-opment in vitro, antral cavity development and oocyte

Figure 2 (a) Immunocytochemicalstaining of the meiotic spindleshowing a normal barrel-shapedspindle with the chromosomeswell aligned along the center. (b)Example of an MII oocyte withan abnormal spindle and dis-rupted chromosomal alignment.Magnification: ¥630; scale bar:20 mm.

(a) (b)

Table 3 Evaluation of meiotic spindle system in MII oocytes derived from cryopreserved/thawed in vitro matured pre-antral follicles using immunocytochemistry and polarized light imaging system

Technique Immunochemical Polarized light imagingNumber ofMII oocytes

Normal spindles& chromosomes

Number ofMII oocytes

Spindle retardance(Mean � SD nm)

No spindlefound

Control 28 19/28 43 2.54 � 0.52 6/43(67.9%) (7%)

Vitrification 31 18/31 44 2.55 � 0.65 3/44(58.1%) (7%)

Slow freeze 25 16/25 23 2.32 � 0.64 3/23(64.0%) (13%)

P-value – 0.73 – 0.22 0.5

SD, standard deviation.



maturation were comparable between differentcryo-devices.

Another consideration in evaluating the differentcryo-devices is whether they are ‘closed’ or ‘open’systems. Open systems allow direct contact between thesample and the LN2. To date, only two closed-systemvitrification vessels are commercially available: cry-otips31,32 and high-security vitrification (HSV) straws.33

Neither permits easy loading of large numbers of cells

(a)

(b)

(c)

Figure 3 The meiotic spindle birefringence shown byOosight spindle view (¥400). (a) Control group (b) vit-rification group, and (c) slow freezing group.

(a)

(b)

(c)

Figure 4 Thin sections of embedded pre-antral folliclesstained with Toluidine blue. (a) Control fresh (b)vitrified/warmed, and (c) slowly frozen/thawed. O,Oocyte; GC, granulosa cells; TC, theca cells.

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(unpublished data), which is an important practicalconsideration for follicle preservation. Dela Pena et al.(2002)12 used a 0.25-mL sealed plastic straw for vitrifi-cation. All other reports on vitrification of isolatedovarian follicles have involved open carrier systems.The potential for cross contamination during storageand theoretical safety risks have been debated.34,35 NewFood and Drug Administration and European TissueDirective regulations may require the exclusive use ofclosed systems for any type of cryopreservation as a

safety precaution. Modification and/or developmentof ‘closed’ cryo-devices specifically for isolated folliclesmay ultimately be necessary for long-term storage.

The EG-raffinose vitrification protocol selected foruse in this study resulted in excellent post-warmingsurvival. About 95% of follicles were morphologicallyintact immediately post warming and 65% survived tothe end of the IVC interval period, with 41% of oocytesmaturing to the MII stage. Our results are comparableto those reported by others.12,14 In vitro survival of

(a) 8,000x (b) 17,000x

(c) 3,000x (d) 17,000x

(e) 3,000x (f) 17,000x

Figure 5 Electron micrographs of(a,b) control (c,d) vitrified/warmed and (e,f) frozen/thawedpre-antral follicles. (a,c,e)Showing the relationshipbetween oocyte (O) and sur-rounding granulosa cells (GC).Note finger-like process(microvilli, arrow) projectinginto the zona pellucida (ZP).When the follicle was sectionedat the level of the germinalvesicle, the oocyte nucleus (N)and nucleolus (NU) were alsoseen. Note the areas of contactbetween the oocyte and GC (*).(b,d,f) Showing the relationshipbetween adjacent GC, GCnucleus (GCN) and GC cyto-plasm. Note the abundant mito-chondria (M) populating thecytoplasm. The contacts betweenadjacent GC were also preserved(arrow head).



plated follicles was 71% and 50% of IVM oocytes thatreached the MII stage.12,14 Lower in vitro developmentrates were obtained by Choi et al. (2007) using EM grids(40%).13 No information was presented on the finaloocyte maturation. More recently, Lin et al. (2008)15 suc-cessfully vitrified isolated pre-antral follicles on a metalsurface (SSV). Post-warming survival was 91–96%.After IVC and IVM, a 66% MII rate was reported.

It should however be noted that all of the above-mentioned studies used the mechanical isolationmethod for recovering individual pre-antral folliclesfrom the ovary. In the current work we used an enzy-matic technique for follicle isolation. The enzymatictechnique is less labor-intensive, allowing the harvestof a large number of follicles. However, enzymaticallyisolated follicles may be more vulnerable during freez-ing. The oocyte–GC connections within the follicularunit may be weakened by the enzymatic treatment.Nagano et al. (2007)14 reported lower post-warmingsurvival rates (49–57%) with this method of follicleisolation.

Enzymatically isolated ovarian follicles cryopre-served by slow cooling also have less developmentalcapacity.22 Despite high post-thaw survival, only 58%remained intact during culture with only 7.7% resumingmeiosis and only 3% developing to the MII stage.22 Thishas not been the case with slow freezing of mechanicallyisolated follicles. Xu et al. (2009)16 achieved quite excel-lent results with frozen/thawed secondary follicles cul-tured in alginate beads. After 12 days of culture, 74% offollicles remained intact and the maturation rate was64%. Cortvindt et al. (1996)11 also found that mechani-cally isolated follicles responded well to slow freezingand at least 80% were able to grow as intact units to theend of the culture interval, with 45% of IVM oocytesextruding a polar body.

Follicle segregation by enzymatic isolation mayhowever prove necessary when dealing with humanand large mammalian ovaries that have a dense ovariancortex.36 Establishing cryopreservation techniques thatgive good outcomes with enzymatically isolated fol-licles would therefore be an essential stepping stone inhuman fertility preservation. The current investigationcertainly indicates that enzymatically isolated folliclescan be successfully vitrified and in vitro matured. Therapid cooling rates associated with vitrification may beessential in preventing any further weakening or dis-ruption of the oocyte–GC complex.

We applied TEM to further evaluate the efficacy of thetwo cryopreservation techniques at the ultra-structurallevel.37 To our knowledge, this is the first report on

ultra-structure of vitrified isolated pre-antral follicles.Ultra-structural differences specific to the freezing tech-nique were not in evidence in the present study. TEM ofisolated pre-antral follicles after slow cooling11 alsoindicated little ultra-structural changes when comparedto fresh controls. Other researchers comparing ultra-structure of vitrified and slowly frozen ovarian tissuefrom mouse38 and human39 reported similar findings.

To complete this comparison on pre-antral folliclecryopreservation by vitrification versus programmedslow freezing, we examined the meiotic spindle mor-phology in the resultant IVM oocytes. The formation ofa normal meiotic spindle with well-aligned chromo-somes is vital for the normal functioning of the oocyteand the subsequent embryo.40,41 Immunochemicalstaining of fixed oocytes allows morphological assess-ment of meiotic spindle assemblies and evaluation ofnormalcy.27 Several studies have suggested that spindleassembly and chromosome alignment is better aftervitrification as compared to slow freezing of human42

as well as mouse oocytes.43,44 This difference was notobserved by Cobo et al. (2008)32 or by our group in thepresent work.

The availability of computer-assisted polarized lightmicroscopy has opened up a new avenue for studyingmeiotic spindles. Real-time non-invasive spindleimaging technology allows evaluation of spindle posi-tion and organization in living oocytes.45 Quantitativemeasurement of spindle birefringence or retardancemay be valuable in comparing different cryopreserva-tion protocols and their ability to preserve the orderand density of microtubules within the spindle. Withmature human oocytes, investigators using polarizedlight microscopy were able to demonstrate fasterrecovery of the meiotic spindle after vitrification ascompared to traditional slow freezing.46,47 To ourknowledge, the current study is the first to analyzemicrotubule birefringence in isolated pre-antral fol-licles after vitrification and IVC/IVM. The molecularintegrity and density of spindles in oocytes from cryo-preserved pre-antral follicles appeared to be unaf-fected by the cryopreservation technique.

One of the limitations of the present work may havebeen the optimization of IVC conditions and the timingfor IVM. The number of days selected for IVC beforefinal maturation has varied amongst investigatorsranging from 8 to 14 days.48–50 Increasing the cultureperiod to 16 days may lead to better results.8 We arecurrently exploring this possibility. Further groupingof follicles into small and large categories duringculture could also be beneficial in improving the

N. Desai et al.


maturation rates. In the present work, the size of fol-licles ranged from 120 to 160 mm in all groups.

The sum of the data presented indicate that althoughfollicle structure may not be optimally preserved byslow cryopreservation, resulting in lower yields ofmature oocytes after IVM, oocyte quality based onmeiotic spindle morphology is not different to that ofvitrification. The current data add to the limited pool ofinformation documenting outcomes with isolated pre-antral follicle cryopreservation. Further work in thisarea and functional analysis of the developmentalcapacity of embryos arising from both cryopreservationmethodologies are needed to establish the superiorityof one or the other technique. Efforts to maximize thedevelopmental competence of oocytes also need tofocus on the culture aspects of the maturation processand conditions for in vitro fertilization.

Follicle cryopreservation may serve as a ‘bridge’ tech-nology until such time that we are more successful withovarian transplantation or more capable of in vitromaturing primordial follicles from the human ovary.Indeed much more work is needed in this areaand proof of concept through ovarian follicle trans-plantation in animal models is necessary. Auto-transplantation of cryopreserved/thawed folliclesembedded in collagen matrices or plasma clots mayprove to be more effective than transplantation ofcryopreserved/thawed ovarian cortical strips, permit-ting faster neo-vascularization and slowing downatresia. Progress in this area is necessary if follicle cryo-preservation is ever to be considered a clinically viabletreatment option. Given the current limitation ofovarian tissue cryopreservation and transplantation, vit-rification of isolated ovarian follicles could be an invalu-able alternative approach for fertility preservation forpatients at risk of premature ovarian failure.

Acknowledgments

The authors would like to thank Dr Judith A DrazbaPhD and Mrs Mei Yin at the Imaging Core facility ofCleveland Clinic Foundation for their expert technicalassistance with the transmission electron microscopy.

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