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Human Reproduction of 12 doi:10.1093/humrep/del227

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Cryopreservation of intact human ovary with its vascular pedicle

Mohamed A.Bedaiwy1,2, Mahmoud R.Hussein3, Charles Biscotti4 and Tommaso Falcone1,5

1Department of Obstetrics and Gynecology, Minimally Invasive Surgery Center, The Cleveland Clinic Foundation, Cleveland, OH, USA, 2Department of Obstetrics and Gynecology, 3Department of Pathology, Assiut University Hospitals and School of Medicine, Assiut, Egypt and 4Anatomic Pathology Department, Minimally Invasive Surgery Center, The Cleveland Clinic Foundation, Cleveland, OH, USA5To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, A81, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail: [email protected]

BACKGROUND: The aim of this study was to assess the immediate post-thawing injury to the human ovary that wascryopreserved either as a whole with its vascular pedicle or as ovarian cortical strips. MATERIALS AND METHODS:Bilateral oophorectomy was performed in two women (46 and 44 years old) undergoing vaginal hysterectomy andlaparoscopic hysterectomy, respectively. Both women agreed to donate their ovaries for experimental research. Inboth patients, one of the harvested ovaries was sectioned and cryopreserved (by slow freezing) as ovarian corticalstrips of 1.0 ´ 1.0 ´ 5.0 mm3 each. The other ovary was cryopreserved intact with its vascular pedicle. After thawing 7days later, follicular viability, histology, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigeninnick-end labelling (TUNEL) assay (to detect apoptosis) and immunoperoxidase staining (to define Bcl-2 and p53 pro-tein expression profiles) of the ovarian tissue were performed. Tissues from non-cryopreserved ovaries served as con-trol specimens (two cases). RESULTS: The overall viability of the primordial follicles was 75 and 78% in intactcryopreserved–thawed (C–T) ovaries and 81 and 83% in ovarian cortical strips in the 46- and 44-year-old patients,respectively. Comparable primordial follicle counts, absence of features of necrosis, mean values of apoptosis andweak Bcl-2 and p53 protein expressions were observed both in the intact C–T ovary and in the C–T ovarian corticalstrips. CONCLUSIONS: Cryoperfusion and cryopreservation of entire human ovary can be achieved with the main-tenance of excellent viability of the superficial and the deeper tissues using a slow-freezing protocol. Cryopreserva-tion injury is associated neither with significant alteration in the expression pattern of Bcl-2 and p53 proteins in theovarian tissues nor with significant follicular damage.

Key words: apoptosis/Bcl-2/cryopreservation/follicular viability/intact human ovary

Introduction

Although many reproductive-age patients can survive theircancers and lead normal lives, they are at increased risk ofimpaired reproductive functions. Consequently, fertility pres-ervation is an important quality-of-life issue for them. Fertilitypreservation strategies were introduced to protect and/or regainreproductive function in patients exposed to cancer chemother-apy and/or radiotherapy. Advances in assisted reproductivetechnologies (ART), such as ovarian tissue cryopreservationand transplantation, oocyte cryopreservation and novel ovula-tion induction regimens, have renewed interest in fertility pres-ervation in women scheduled to receive gonadotoxicchemotherapy and/or radiotherapy (Falcone et al., 2004). Ofthese technologies, ovarian tissue cryopreservation may be aviable option for women who cannot delay treatment to undergoovarian stimulation to create embryos or obtain oocytes forfreezing. In this entirely experimental process, thawed tissue

can be implanted after cancer treatment as an autograft to anorthotopic or a heterotopic site. Immature oocytes derived fromthawed tissue can be matured in vitro if appropriate protocolsare developed in the future. Also, the maturation process of theimmature oocytes could be achieved by xenografting in immu-nodeficient mice (Gook et al., 2003).

To date, ovulation and creation of a human embryo fromheterotopic ovarian transplant have been reported (Oktay et al.,2004). Moreover, two live births from orthotopic ovarian trans-plants following spontaneous (Donnez et al., 2004) and IVF(Meirow et al., 2005) cycles were reported, following modifiedcryopreservation and surgical protocols pioneered, in sheep, byGosden and associates (Gosden et al., 1994; Baird et al.,1999). Despite these advances, ischaemic damage to the tissueand revascularization injury and the theoretical possibility ofreintroducing malignant tumour cells remain as the mainlimitations for this option (Bedaiwy and Falcone, 2004).

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The ischaemic damage to ovarian tissues can induce a highrate of follicular loss (Baird et al., 1999; Demirci et al., 2002).Therefore, eliminating this ischaemic damage could maintainboth the viability and the functional integrity of the transplant.Ideally, this could be achieved by transplantation of an intactovary with vascular anastomosis. Transplantation of an intactovary with its vascular pedicle using microvascular anastomo-sis was achieved both in murine species (Yin et al., 2003) andin mammals (Jeremias et al., 2002; Bedaiwy et al., 2003).Partial restoration of hormonal functions (Bedaiwy et al.,2003) and pregnancy (Wang et al., 2002) after transplantationof intact cryopreserved–thawed (C–T) ovaries were alsoreported. Recently, cryopreservation of intact human ovarywith its vascular pedicle resulting in high post-thaw survival ratesof follicles, small vessels and stromal cells as well as a normalhistological structure in all the ovarian components wasreported using a slow-freezing protocol. Slow freezing wasachieved using a cryofreezing one-degree container (Martinez-Madrid et al., 2004).

The structural homeostasis of tissues is regulated by a delicatebalance between cell survival and apoptotic cell death. In tis-sues, several molecules are involved in the survival (survivalmolecules such as Bcl-2) or cell death (apoptotic moleculessuch as p53) processes. Bcl-2 is a membrane-associated proteinthat resides in the nuclear envelope and mitochondria. It exertsits survival functions by modulating the mitochondrial releaseof cytochrome c and antagonizing the effects of Bax gene (Bcl-2-associated X). Bcl-2 is expressed in granulosa cells of bothfetal and adult ovaries. p53 gene encodes a 53-kDa oncosup-pressive nuclear protein that functions to antagonize Bcl-2effects. In the ovary, p53 protein is expressed in the apoptoticgranulosa cells of atretic follicles. In several organs such as theheart, ischaemia is associated with the induction of Bcl-2 andp53 protein expressions. Similarly, the induction of these mol-ecules following ovarian ischaemia may have far-reachingeffects on the outcome of the subsequent ovarian transplantation(Hussein, 2005).

Although previous studies examined the long-term effectsof cryopreservation–thawing procedure injury on the ovar-ian cortical strips (Gosden et al., 1994), our understandingof the injury caused to ovarian tissue in the period immedi-ately after thawing is still incomplete. The aims of thisstudy were to: (i) describe a cryopreservation protocol usingthe programmable freezer allowing freezing and thawing ofan intact human ovary with its vascular pedicle; (ii) assessthe survival (viability) of the ovarian elements in the C–Ttissues using Trypan Blue method; (iii) examine the frozen–thawed ovarian tissues for features of follicular health andatresia using the histological methods; (iv) evaluate theapoptotic changes in the ovarian tissues using combinedhistological methods and terminal deoxynucleotidyl trans-ferase (TdT)-mediated dUTP-digoxigenin nick-end labelling(TUNEL) assay, immunoperoxidase method (p53 and Bcl-2protein alterations, i.e. apoptotic and survival proteins) and(v) examine the status of the blood vessels using both histo-chemical [periodic acid Schiff (PAS) and Masson’s tri-chrome] and immunohistochemical (CD34, marker of vascularendothelium) staining methods.

Materials and methodsThe study group included both control (non-cryopreserved) and cryo-preserved human ovaries. Also tissue sections from lymph nodes,squamous cell carcinoma, leukaemia cell lines, normal skin and liverwere used as tissue-specific positive controls for Bcl-2, p53, apoptoticcells, PAS and Masson’s trichrome staining.

Patients in the cryopreservation group

The Institutional Review Board of The Cleveland Clinic Foundationapproved this study. The first patient, aged 46 years, presented withmenorrhagia and symptomatic fibroid uterus, who underwent totalvaginal hysterectomy with bilateral salpingo-oophorectomy, McCallculdoplasty and cystoscopy. Intraoperative findings included an 8–10week-sized uterus and normal-appearing ovaries. The second patient,aged 44 years, presented with severe premenstrual dysphoric disor-ders, who underwent laparoscopic bilateral salpingo-oophorectomy.Normal-appearing uterus, tubes and ovaries were detected. Both ova-ries and their pedicles, from both patients, were processed immedi-ately as detailed below.

Patients in the control group

In the control group, formalin-fixed paraffin-embedded ovariantissue was obtained from the Archives of the Department ofPathology, Assiut University Hospitals. Four ovarian tissue speci-mens were obtained from two women, 44 and 47 years old. The firstwas diagnosed with irregular uterine bleeding due to uterinefibroids and was treated by total abdominal hysterectomy withbilateral salpingo-oophorectomy. The second underwent total vag-inal hysterectomy with bilateral salpingo-oophorectomy due topelvic organ prolapse. The ovaries obtained from both patientsappeared normal with a smooth white convoluted surface and firmconsistency.

Cryopreservation

Cryopreservation of the intact ovary

We adopted our previous protocol described in 2003 (Bedaiwyet al., 2003). Briefly, immediately after oophorectomy, one ovary(from each patient) was perfused with heparinized Ringer’s solu-tion followed by perfusion and immersion in a bath containing thecryoprotective mixture which was composed of Leibovitz L-15medium (Irvine Scientific, Santa Ana, CA, USA), 10% fetal calfserum (FCS) (Irvine Scientific) and 1.5 M dimethylsulphoxide(DMSO) (Sigma, St. Louis, MO, USA). Both the ovarian vesselsand excess hilar tissue were dissected, and ovarian ligaments weretrimmed. The ovaries were perfused via the ovarian artery with thecryoprotective mixture using Horizon Modular Infusion System(MCGaw, Irvine, CA, USA) to maintain a flow rate at 1.3 ml/minwith continuous replenishment of the reservoir. After perfusion,the ovary was bisected and transferred to two cryovials 12.7 × 92mm (Corning Coaster Corporation, Cambridge, MA, USA) con-taining the cryoprotective mixture for controlled freezing usingPlaner cryochamber (Planer Freezer, Middlesex, UK). Coolingbegan at +4°C and continued at 2°C per minute until ice nucleationwas induced at –7°C. The temperature was then reduced at 2°Cper minute until –35°C and subsequently at 25°C per minute until–140°C before cryovials were plunged into liquid nitrogen.

Cryopreservation of the ovarian cortical strips

The other ovary, for each patient, was divided into ovarian corticalstrips of 1.0 × 1.0 × 5.0 mm3 each. The ovarian cortical strips wereprepared as described by Gosden et al. (1994) and cryopreservedusing the previously mentioned protocol but without perfusion.

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Thawing

Thawing of the intact ovary

One week later, the vials were removed from the dewar and held for 1min at room temperature before plunging in a bath of water at 37°Cwith gentle shaking. The contents of the vials were quickly emptiedinto a Petri dish containing Leibovitz L-15 medium supplementedwith 10% FCS. The ovaries were washed and immediately perfusedwith Leibovitz L-15 medium supplemented with 10% FCS using aflow rate of 1.3 ml/min for 20 min. The cryoprotectant was graduallyeliminated by pumping Leibovitz L-15 supplemented with 10% FCSinto the reservoir (Bedaiwy et al., 2003).

Thawing of the ovarian cortical strips

Ovarian cortical strips were thawed using the same procedure as forintact ovary. Then, the strips were washed and held in Leibovitz L-15containing 10% FCS for 20 min.

In vitro assessments of the C–T and control ovarian tissues

Evaluation of the ovarian follicular viability in the C–T ovarian tissue

The cryopreserved ovarian tissues were evaluated for follicular viabil-ity in terms of plasma membrane function and structural integrity bythe Trypan Blue exclusion test. Ovarian fragments were thinly sec-tioned in Leibovitz L-15 medium supplemented with 1 mg/ml (200IU/ml) type 1 collagenase (Sigma), incubated at 37°C for 2 h andpipetted every 30 min. Collagenase activity was inhibited by 50%FCS. The suspension was filtered through a 70-μm nylon filter (BectonDickinson Labware, Frankline Lakes, NJ, USA) and centrifuged at400 g for 5 min. The precipitate was diluted with 50 ml of LeibovitzL-15 medium and kept in a water bath at 37°C. Trypan Blue (0.4%,Sigma) was added to the suspension containing the follicles (20 μl),deposited on a glass slide and examined under inverted microscope(×400). For each fragment, 100 small intact follicles were examinedwhile the partially or completely denuded oocytes were excluded.Both the number of stained cells and the total number of cells werecounted. The percentage of viable cells was determined by calculatingthe percentage of unstained cells.

Histological evaluation of the ovarian tissues for the featuresof follicular health and atresia in the C–T and control ovariantissues. The maturing follicle is composed of an oocyte with granu-losa layer and lacks reticulum. The hallmarks of the healthy folliclesinclude intact oocyte, intact membrana granulosa, lack of necrosis andthe presence of few pyknotic nuclei (<5% pyknotic nuclei) in thislayer. The atretic follicles are characterized by the presence of attenu-ated membrana granulosa, loosely attached granulosa cells andincreased number of pyknotic nuclei (>5% pyknotic nuclei) (Woodet al., 1997). Initially, the ovarian follicles were sorted into twogroups, healthy and atretic, following Wood et al. (1997).

Histological evaluation of apoptotic changes in the C–T andcontrol ovarian tissues. Evaluation of apoptosis was performed usingcombined histological and TUNEL assays. The results were expressedas mean and SEM. The histological criteria of apoptosis includedcondensed nuclear fragments, nuclei with marginated chromatin,multiple nuclear fragments, a single condensed nucleus, membrane-bound structures containing variable amounts of chromatin and eosi-nophilic cytoplasm (Kerr et al., 1972).

DNA fragmentation assay for the detection of apoptotic cells in the C–T and control ovarian tissues

To evaluate the apoptotic changes, we examined cellular morphol-ogy with the TUNEL assay, using the commercially available

QIA33TDT-FragEL™ kit (Oncogen Research Products, Boston, MA,USA) following other groups (Hussein et al., 2006). Positive controlsconsisted of HL60 promyelocytic leukaemia cells and HL60 cellsincubated with 0.5 μg/ml actinomycin D for 19 h to induce apoptosis.Some ovarian specimens were used as negative controls by substitut-ing distilled water for deoxynucleotidyl transferase (Gavrieli et al.,1992; Liu et al., 1995).

Results of the TUNEL assay were evaluated following other groups(Hussein et al., 2006). All slides were examined at ×400 magnifica-tion. Ten different areas of each follicular wall were examined, andapoptotic index was determined as mean number of positively stainedcells (Hussein et al., 2006).

Immunohistochemical evaluation of p53, Bcl-2 and CD34 protein expressions in the C–T and control ovarian tissues

To examine the prosurvival, proapoptotic proteins and the status ofthe blood vessels, we used immunoperoxidase staining methods andmonoclonal antibodies targeting p53, Bcl-2 and CD34 proteins.Briefly, ovary sections mounted on glass slides were deparaffinizedand rehydrated. Endogenous peroxidase activity was blocked with0.6% H202. Sections were then immersed in the retrieval solution (10mM sodium citrate buffer, pH 6.0) and subjected to heat-induced anti-gen retrieval for 20 min (microwave at ∼750 W). Non-specific proteinbinding was blocked with 10 min exposure to 10% normal goatserum. Sections were then incubated with primary antibodies for 1 hat room temperature. The following primary mouse monoclonal anti-bodies were used: clone DO-7, Catalogue No. M7001 and clone 124,Catalogue No. M0889 for p53 and Bcl-2, respectively (DAKO,Carpinteria, CA, USA). Anti-CD34 monoclonal QBEnd/10 (Novocastro,Newcastle, UK) was used to examine the blood vessels. After briefrinsing in phosphate-buffered saline (PBS), a catalyzed signal amplifi-cation system (K1500, DAKO) was used according to the manufac-turer’s instructions. Two observers independently evaluated thestained slides.

Positive controls

Sections from reactive lymphoid hyperplasia (lymph node), p53-pos-itive squamous cell carcinoma and haemangioma were used as posit-ive controls for p53, Bcl-2 and CD34 staining, respectively.

Negative controls

Additional sections of the tissues were stained in parallel but withomission of the primary antibody.

PAS and Masson’s trichrome stains

PAS stains carbohydrate moieties and basement membrane reticulinand therefore delineates the wall of the blood vessels. Masson’s tri-chrome stains myofibres, indicating skeletal or smooth muscle differ-entiation (versus non-muscle fibres). Thus, it stains the smooth muscleof the blood vessels.

Evaluation of Bcl-2 and p53 immunostaining

For the evaluation of Bcl-2 and p53 immunoreactivities, severalrules were followed: (i) corresponding sections stained by haema-toxylin and eosin were examined; (ii) sections were examined todetect the sites of the antibody positivity; (iii) a higher power mag-nification was used to evaluate the immunostaining; (iv) sectionswere examined independently by two observers and (v) Bcl-2 andp53 positivity was identified as diffuse brown cytoplasmic andnuclear staining, respectively. The numbers of follicles with posit-ive and negative reactivity were counted in the ovarian tissues(Hussein et al., 2006).

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Evaluations of the status of the blood vessels in the C–T and control ovarian tissues

We chose CD34, as it is more sensitive than other markers for thedetection of endothelial cells in the ovary. Microvessel density(MVD) was assessed by light microscopy following Weidner et al.(1991). Ovarian tissue was scanned at low magnification (×40 and×100) to select the areas that showed the most intense vascularization(hot spots). Individual microvessels were counted in three fields at×200 magnification (×20 objective lens and ×10 ocular lens; 0.7386mm2/field). The final MVD was the mean value obtained fromthe counts of the three fields. MVD was expressed as mean (SD) (vessel/mm2). Any immunostained endothelial cells or endothelial cell clus-ters that were clearly separated from the adjacent microvessels wereconsidered as a single and countable microvessel. Vessel lumens werenot a prerequisite for a structure to be defined as a microvessel, and redblood cells were not used to define a vessel lumen (Weidner et al., 1991).

Statistical analysis

Analysis of variance (ANOVA) and Student’s t-tests with a statisticalsignificance of P < 0.05 were performed. Calculations were donewith the Statistical Package for the Social Sciences for Windows,version 10.0.

Results

Follicular viability in the C–T ovarian tissue

In the 46-year-old patient, the primordial follicular viabilitywas 75 and 81% in C–T intact ovary and C–T ovarian corti-cal strips, respectively. In the 44-year-old patient, these val-ues were 78 and 83%, respectively. The average value forboth intact ovaries (76.5%) was comparable with that forcortical strips (82%). A summary of these data is presentedin Table I.

Histological changes in the control and C–T ovarian tissues

In the control ovarian tissues, evaluation of the cortex andmedulla revealed the absence of the histological features ofnecrosis such as: (i) swelling and clumping of the chromatin,(ii) pyknosis (condensation of the chromatin and shrinkage ofthe nucleus), (iii) karyorrhexis (fragmentation of the nucleus),(iv) karyolysis (dissolution of the nucleus) and (v) cytoplasmicopacification or eosinophilia (Figure 1). Similarly, in the cryo-preserved ovaries, evaluation of the cortex and medulla of C–Tintact ovaries and cortical strips revealed the absence of thesehistological features (Figure 2). Moreover, the blood vesselswere not only viable and patent but also had intact muscle coatand endothelium (Figure 2).

In every sample, the primordial follicles were counted in 10different fields and mean values were reported for the cryop-reservation and control groups. Histological evaluation of thecount of the primordial follicles revealed a trend towards highervalues in the intact C–T ovary as compared with the corticalstrips (4.5/high-power field versus 3/high-power field, respec-tively) (Figure 2). However, differences among these values didnot reach the level of statistical significance, probably due tosmall sample size (two patients). Histological features of apop-tosis were seen in the granulosa cells of atretic follicles eitherwithin the central region of the membrana granulosa layer orloosely attached to the membrana granulosa near its antral sur-face. A summary of these data is presented in Table I.

Bcl-2 and p53 protein expressions in the control and C–T ovarian tissues

The positive and negative controls were positive and negative,respectively, indicating the validity of our results. In both

Table I. Morphological analysis of the non-cryopreserved (control), cryopreserved intact ovary and cortical ovarian tissue strips

The healthy follicles include primordial, primary and secondary follicles. Plasma membrane function and structural integrity were determined by the Trypan Blue exclusion test (viability assay), in which both the number of stained follicles and the total numbers of follicles (100 follicles) were counted. The percentage of viable cells was determined by calculating the percentage of unstained cells. Follicular health and atresia were determined using the histological methods and were reported in descriptive terms. Apoptotic index was evaluated using combined terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling and histological methods where 10 different areas of each follicular wall were examined. The apoptotic index was calculated as mean number of positively stained cells. For the evaluation of Bcl-2 and p53 immunoreactivities, the numbers of follicles with positive and negative reactivity were counted in the ovarian tissues. NA, not applicable; NS, not significant (P > 0.05).

Aspects Cryopreserved ovarian tissue Non-cryopreserved ovarian tissue P-value

Ovarian cortical strips

Intact ovary Cortical strips and intact ovaries

Control group

Follicular viability [unstained cells/total cells (%)]

82/100 (82) 76/100 (76.5) 79/100 (79) NA NA

Healthy follicles Present Present Present Present NSAtretic follicles Present Present Present Present NSApoptotic index 1.7 ± 0.1 1.8 ± 0.1 1.7 ± 0.1 1.8 ± 0.1 NSApoptotic changes

Healthy follicles Absent Absent Absent Absent NAAtretic follicles Present Present Present Present NS

Bcl-2 protein expressionHealthy follicles (mean ± SEM) 1.75 ± 0.3 1.70 ± 0.3 1.70 ± 0.3 1.76 ± 0.3 NSAtretic follicles (mean ± SEM) 0.06 ± 0.7 0.06 ± 0.7 0.06 ± 0.7 0.06 ± 0.7 NS

p53 protein expressionHealthy follicles Absent Absent Absent Absent NAAtretic follicles Absent Absent Absent Occasional NA

Status of the blood vessels Persistent vascularity Persistent vascularity Persistent vascularity Persistent vascularityMicrovessel density (mean ± SEM) 5.3 ± 0.8 5.3 ± 0.8 5.3 ± 0.8 5.6 ± 0.9 NSPeriodic acid Schiff stain Persistent vascularity Persistent vascularity Persistent vascularity Persistent vascularity NAMasson’s trichrome stain Persistent vascularity Persistent vascularity Persistent vascularity Persistent vascularity NA

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control and cryopreserved ovarian tissues, rare weak Bcl-2expression was seen both in the healthy follicles and in thestromal cells (Figures 3 and 4). p53 expression (nuclear) wasvirtually absent in the healthy primordial, primary and secondaryfollicles, germinal epithelium and stromal cells subjected towarm ischaemia (Figures 3 and 4). Bcl-2 immunoreactivitywas observed more frequently in the secondary follicles, fol-lowed by primary and primordial follicles. In these follicles,Bcl-2 immunoreactivity was restricted to granulosa cells. The

theca cells were never immunoreactive regardless of the devel-opmental stage of the follicles (Figures 3 and 4).

DNA fragmentation assay (TUNEL) in the control and C–T ovarian tissues

None of the negative controls stained with TUNEL techniqueshowed any immunoreactivity, whereas signal-positive TUNELstaining was observed in the positive controls (Figure 5). In the

Figure 1. Histological features of the control (non-cryopreserved) ovarian tissue. The figures show primordial follicle (A, ×200), primary (B andC, ×400), secondary (D, ×400), Graafian (E, ×200) and atretic follicles (F, ×400).

Figure 2. Upper panel: Histological features of both cryopreserved–thawed intact ovary and ovarian cortical strips. The figures show primordialfollicle (A, ×400) and intact intraovarian blood vessels (B, ×400). The primordial follicle is formed of primary oocyte, surrounded by a singlelayer of flattened mitotically inactive, granulosa cells resting on a thin basal lamina. Lower panel: Histological features of the human ovarian fol-licles in both cryopreserved intact ovary and ovarian cortical strips. The follicles include (A) primary, (B) Graafian and (C) atretic follicles. A(×200), the primary follicle is formed of enlarged oocyte, surrounded by a single layer of cuboidal to columnar mitotically inactive granulosacells. B (×400), the Graafian follicle is formed of an antrum, several layers of granulosa cells and theca layers. C (×200), the atretic follicle isformed of thin inner layer of small, exfoliating granulosa cells and an outer theca layer.

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control ovarian tissues, granulosa cells with the histologic fea-tures of apoptosis were observed in the atretic follicles, eitherwithin the central region of membrana granulosa layer orloosely attached with membrana granulosa near its antral sur-face or in the antral follicular fluid. The healthy (primordial,primary and secondary) follicles were TUNEL negative.TUNEL-positive signals were detected in the atretic follicles.This was evident as punctuated brown staining of the frag-mented nuclei of granulosa cells. The positively stained nucleiwere observed either in the central layers of the membranagranulosa, at the antral surface or floating in the follicularatrium. DNA fragmentation was absent both in the interstitialcells and in the theca cells of the atretic follicles. Evaluation ofapoptosis in the cryopreserved ovaries revealed similar find-ings: (i) the healthy (primordial, primary and secondary) folli-cles were TUNEL negative; (ii) TUNEL-positive signals weredetected in the atretic follicles as indicated by the punctuatedbrown staining of the fragmented nuclei of granulosa cells(Figure 5) and (iii) the mean values of apoptotic cells in theintact cryopreserved ovarian tissues were comparable withthose in the ovarian cortical strips and control ovarian tissues(1.8 ± 0.1 versus 1.7 ± 0.1 versus 1.8 ± 0.1, respectively). Theresults are summarized in Table I.

Evaluation of CD34 protein expression in the control and C–T ovarian tissues

Endothelial immunostaining with anti-CD34 as well as evaluationof the basement membrane (PAS) and muscle coat (Masson’strichrome) of ovarian blood vessels demonstrates persistentvascularity in both cryopreserved and control ovarian tissues.Microvessels are heterogeneously distributed within the ovar-ian tissue. The mean values of ovarian MVD were 5.6 ± 0.9,5.3 ± 0.8 and 5.3 ± 0.8 for control, cryopreserved intact and

cortical ovarian tissues, respectively. A summary of theseresults is presented in Table I and Figures 6–8.

Discussion

In this investigation, the immediate post-thawing injury to thehuman ovary (cryopreserved both as a whole with its vascularpedicle and as ovarian cortical strips) was assessed using bothhistological and immunohistological methods. To achieve ourgoals, we established a study group formed of four patients. Itincluded both control (non-cryopreserved) and cryopreservedhuman ovaries. Tissue sections from lymph nodes, squamouscell carcinoma, leukaemia cell lines, normal skin and liver wereused as tissue-specific positive controls for the histochemical(PAS and Masson’s trichrome staining) and immunohistochem-ical (Bcl-2, p53 and apoptotic cells) parameters. Our investiga-tion demonstrated the following findings: (i) the mean values ofMVD, follicle counts and Bcl-2 protein expression were com-parable between the cryopreserved and the control groups and(ii) no differences were seen between the two groups for the fol-lowing parameters: healthy and atretic follicle counts, apoptoticindex and Bcl-2 protein expression in the atretic follicles

Ovarian tissue banking is more popular as a fertility preser-vation option for patients without a partner or those who cannotdelay gonadotoxic chemotherapy for the sake of ovarian stimula-tion to produce oocytes for freezing or embryos for cryopreserva-tion. With the limited long-term survival upon reimplantation withovarian tissue strips, ovarian transplantation with microvascularanastomosis could help avoid accelerated follicular loss andimprove the longevity of the ovarian grafts. This helps to guar-antee an immediate revascularization and establishment of thegraft function. Although successful in rat and sheep animalmodels (Wang et al., 2002; Bedaiwy et al., 2003), microvascularanastomosis of intact frozen–thawed ovaries remains technically

Figure 3. Bcl-2 and p53 protein expressions in the control (non-cryopreserved) ovarian tissue. Bcl-2 protein expression in the primary (A, ×400),secondary (B, ×400) and atretic (C, ×400) follicles and the ovarian stroma (C). The expression appears as golden yellow cytoplasmic staining.Note the lack of p53 protein expression in the primary (D, ×400), Graafian (E, ×200) and atretic (F, ×400) follicles and the ovarian stroma (F)(arrowheads).

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challenging. Moreover, cryopreservation of an entire organwith its vascular pedicle is much more complicated than ovar-ian cortical strips or suspended cell (oocyte) or group of cells(embryo).

In our previous study, we reported a 30% long-term patencyrate of the ovarian vessels upon reimplantation of an intactfrozen–thawed ovary in merino sheep (Bedaiwy et al., 2003).However, human ovaries are larger and may not be amenableto the same protocol. The technical challenges of the microvas-cular anastomosis could be circumvented adequately by appro-priate training. On the contrary, the main determinant ofsuccess would be to develop an appropriate cryopreservation

protocol of an entire ovary with its vascular pedicle. Adequatedelivery of the cryoprotective agent (CPA) to almost every cellof the ovary is mandatory to guarantee an adequate post-thawsurvival. Aiming at adequate CPA permeation to virtuallyevery cellular component of the ovary, we used the HorizonModular Infusion pump to ensure adequate CPA distributionthroughout the ovarian substance. Infusing the CPA slowlythrough the ovarian artery and allowing it to come out of theovarian vein ensures adequate use of the ovarian vascularchannel as a delivery vehicle. To avoid intracellular ice forma-tion, we used a slow-freezing protocol using the Planer freezer.The use of constant infusion pressure during the freezing and

Figure 4. Upper panel: Bcl-2 and p53 protein expressions in both cryopreserved intact ovary and ovarian cortical strips. Bcl-2 protein expressionin the primordial (A, ×200), atretic (B, ×400) follicles and the ovarian stroma (C, ×200). The expression appears as golden yellow cytoplasmicstaining. Note the lack of p53 protein expression in the primordial (D, ×400), atretic follicles (E, ×400) and the ovarian stroma/Graafian follicle(F, ×400) (arrowheads). Lower panel: Bcl-2 and p53 protein expressions in positive controls. Bcl-2 protein expression in the lymph node withreactive hyperplasia (G, ×200) and p53 protein expression in the cells of squamous cell carcinoma (H, ×400) (arrowheads).

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thawing phases ensured adequate distribution and clearance ofthe CPA.

The selection of DMSO as a CPA of choice was based onthe initial observation by Newton et al. (1998) that exposurefor 30 min to 1.5 M solutions of DMSO at 4°C achieved amean tissue concentration that was almost 80%. In our study,1.5 M DMSO was tolerated comparably by both an intactovary and ovarian cortical strips. Interestingly, in a recentreport where ovarian tissue was exposed to 10% DMSO for 15min at 4°C, no significant differences in follicular viabilitybefore (99.4%) or after cryoprotectant exposure (98.1%) weredocumented. This could probably mean that ovarian tissuecould tolerate adequately varying concentrations of DMSOwith comparable post-thaw survival (Martinez-Madrid et al.,2004).

After thawing, viability assessment of the primordial folli-cles was comparable between intact ovaries and ovarian corti-cal strips. This demonstrates that delivering the CPA using aninfusion pump was as efficient as the permeation of the ovariancortical strips with the CPA avoiding cryoinjury to the folliclesas well as the intraovarian vessels. The viable primordial fol-licular count in the intact ovaries in both of our patients (75 and78%) was comparable with the 75.1% follicular viabilityreported by Martinez-Madrid et al. (2004), who tried a differ-ent freezing protocol for intact human ovary.

In this investigation, the presence of apoptotic activity in theovarian follicles may be attributed to the presence of apoptosis-triggering signals and/or degradation of apoptosis-related pro-teins (caspase-3, caspase-8, Bcl-2 and actin) (Zhu et al., 1999;Schmidt-Mende et al., 2000). At the molecular level, cryop-reservation and thawing had no effects on the values of apop-totic cell death and Bcl-2 and p53 protein expressions in bothovarian cortical strips and intact ovaries. A hypothesis to betested is that cryopreservation and thawing can induce someeffects on these values (apoptotic cell death and Bcl-2 and p53protein expressions) over longer time intervals. This hypothesisstems from the fact that apoptosis is a dynamic process thatrequires active metabolism and usually takes several hours oreven days to be executed (Hussein et al., 2006). These questions

are open for future investigations. Apoptosis in the atretic folli-cles can effectively be present before the cryopreservation, butit does not seem to be increased by the cryopreservation pro-cess. Moreover, the presence of apoptotic changes in the atreticfollicles supports the link between apoptosis and ovarian follic-ular atresia.

It is also possible that substances which stimulate apoptosismay have atretogenic effects on the ovarian follicles. The pres-ence of Bcl-2 protein expression in the healthy ovarian folliclesconcurs with other reports (Tilly, 1996a,b; Felici et al., 1999)and suggests its possible physiologic role in the maintenance offollicular integrity. In support, mice lacking functional Bcl-2protein possess reduced numbers of primordial follicles rela-tive to their wild-type sisters (Ratts et al., 1995). The lack ofp53 protein expression in the healthy follicles versus its pres-ence of atretic ones concurs with previous studies and suggestslack of underlying DNA damage and therefore implies integ-rity of the genome in the healthy follicles (Hussein, 2005).

During organ transplantation, tissues are subjected to varia-ble duration of ischaemic injury. In the heart, retina, liver andbrain, ischaemic injury is associated with apoptosis as well asBcl-2 and p53 alterations. Namely, the induction of Bcl-2 andp53 can occur as early as 0.5 and 1 h, respectively, followingischaemia (Peralta et al., 2002; Wang et al., 2003). Apoptosisrequires active cell metabolism that could not be completewithin 30 min and could therefore not alter the status of the fol-licles within an ovary. A hypothesis to be tested is that the dif-ferences observed in this investigation are the result of outrightlysis (ischaemia could make cells more prone to lysis). Itwould be interesting for future investigations to examine thishypothesis.

Our investigation revealed the presence of persistent vascu-larity and comparable MVD in both control and cryopreservedgroups. These findings indicate that our freeze–thawing proto-col has no detrimental effects on the integrity of the vascularnetwork of the ovarian tissue. Of note, the status of the ovarianvascular supply is central to dynamic changes occurring duringthe normal ovarian cycle. Follicular growth and the develop-ment of the corpus luteum are dependent on intact vasculature.

Figure 5. Apoptotic changes in the HL60 promyelocytic leukaemia cells, cryopreserved intact ovary and ovarian cortical strips. A (×200) Apop-tosis in the HL60 promyelocytic leukaemia cells (control), with positive staining (inset, arrowhead). B (×400) Apoptosis in atretic ovarian folli-cles, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling-positive cells (arrowheads).

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The selection of a dominant follicle in monovular species isassociated with angiogenesis. Moreover, selected folliclespossess a more elaborate microvascular network than other fol-licles. The vasculature also plays a key role in the delivery of

cholesterol to luteal cells for progesterone biosynthesis (Carret al., 1981; Goede et al., 1998).

Many studies indicate that the ovaries of older women aresmaller than those of young women. They are composed

Figure 6. Upper panel: Evaluation of the status of the blood vessels in the control (non-cryopreserved) ovarian tissue. CD34 protein expressionin the ovarian stroma. The ovarian blood vessels show persistent vascularity and moderate density of microvessels. The endothelial cells of themicrovessels are stained brown by anti-CD34 antibodies. The inset represents CD34 staining in the control (haemangioma) (arrowheads) (×400).Lower panel: Evaluation of the status of the blood vessels in the control (non-cryopreserved) ovarian tissue. Periodic acid Schiff (PAS) (A–C)and Masson’s trichrome (D–F) stain within the ovarian stroma. The ovarian blood vessels show persistent vascularity and moderate density ofmicrovessels. The muscle coat (Masson’s trichrome) and basement membrane (PAS stain) outline the blood vessels (arrowheads) (A, C and D:×200; B, E and F: ×400).

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Figure 7. Upper panel: Evaluation of the status of the blood vessels both in cryopreserved intact ovary and in ovarian cortical strips. CD34 proteinexpression within the ovarian stroma. The ovarian blood vessels show persistent vascularity and moderate density of microvessels. The endothe-lial cells of the microvessels are stained brown by anti-CD34 antibodies (arrowheads) (×400). Lower panel: Evaluation of the status of the bloodvessels both in cryopreserved intact ovary and in ovarian cortical strips. Periodic acid Schiff (PAS) and Masson’s trichrome stain within the ovar-ian stroma. The ovarian blood vessels show persistent vascularity and moderate density of microvessels. The muscle coat (Masson’s trichrome)and basement membrane (PAS stain) outline the blood vessels (arrowheads). (All pictures are on scale of ×200 except the lower right one, ×400.)



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mainly of stromal cells that have receptors for, and respond to,gonadotrophins and secrete testosterone and lesser amounts ofother androgens and estrogens. These ovaries remain gonado-trophin-driven, androgen-producing glands. Also, they arecharacterized by the presence of prominent cystic follicles,thick-walled vessels, granulomas, hyaline scars and lownumber of follicles. However, these follicles are not necessar-ily more sensitive to the freezing–thawing process (Couzinetet al., 2001; Longcope, 2001). In this investigation, the studygroup was formed of older patients knowing in advance thehistopathological and follicular implications of ageing on theovarian structure. Whether the aforementioned age-relatedchanges have an impact on our results awaits further confirma-tion in younger age group upon ethical approval.

In this study, we assessed injury of the ovarian tissue in theperiod immediately after thawing. Our investigation providesfurther evidence that intact human ovary could be cryopre-served using a slow-freezing protocol. We have demonstratedcomparable survival rates of follicles and limited molecularalterations between C–T intact ovaries and C–T ovarian corti-cal strips. The information herein reported is provided for theperiod immediately after thawing. The changes associated withlong-term injury mandates further investigations.

AcknowledgementsThe financial support for this study was a research grant from the Min-imally Invasive Surgery Center, The Cleveland Clinic Foundation,Cleveland, OH, USA. This work was presented at ESHRE 20thAnnual meeting, June 27–July 1, Berlin, Germany 2004.

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Submitted on December 4, 2005; resubmitted on March 15, 2006, May 11,2006; accepted on May 16, 2006


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