Hum. Reprod. Advance Access originally published online on September 25, 2006
Human Reproduction 2006 21(12):3258-3269; doi:10.1093/humrep/del227
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cryopreservation of intact human ovary with its vascular pedicle
1 Department of Obstetrics and Gynecology, Minimally Invasive Surgery Center, The Cleveland Clinic Foundation, Cleveland, OH, USA 2 Department of Obstetrics and Gynecology 3 Department of Pathology, Assiut University Hospitals and School of Medicine, Assiut, Egypt and 4 Anatomic Pathology Department, Minimally Invasive Surgery Center, The Cleveland Clinic Foundation, Cleveland, OH, USA
5 To 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: falcont{at}ccf.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: The aim of this study was to assess the immediate post-thawing injury to the human ovary that was cryopreserved 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 and laparoscopic hysterectomy, respectively. Both women agreed to donate their ovaries for experimental research. In both patients, one of the harvested ovaries was sectioned and cryopreserved (by slow freezing) as ovarian cortical strips of 1.0 x 1.0 x 5.0 mm3 each. The other ovary was cryopreserved intact with its vascular pedicle. After thawing 7 days later, follicular viability, histology, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay (to detect apoptosis) and immunoperoxidase staining (to define Bcl-2 and p53 protein expression profiles) of the ovarian tissue were performed. Tissues from non-cryopreserved ovaries served as control specimens (two cases). RESULTS: The overall viability of the primordial follicles was 75 and 78% in intact cryopreserved–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 and weak Bcl-2 and p53 protein expressions were observed both in the intact C–T ovary and in the C–T ovarian cortical strips. CONCLUSIONS: Cryoperfusion and cryopreservation of entire human ovary can be achieved with the maintenance of excellent viability of the superficial and the deeper tissues using a slow-freezing protocol. Cryopreservation injury is associated neither with significant alteration in the expression pattern of Bcl-2 and p53 proteins in the ovarian tissues nor with significant follicular damage.
Key words: apoptosis/Bcl-2/cryopreservation/follicular viability/intact human ovary
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Although many reproductive-age patients can survive their cancers and lead normal lives, they are at increased risk of impaired reproductive functions. Consequently, fertility preservation is an important quality-of-life issue for them. Fertility preservation strategies were introduced to protect and/or regain reproductive function in patients exposed to cancer chemotherapy and/or radiotherapy. Advances in assisted reproductive technologies (ART), such as ovarian tissue cryopreservation and transplantation, oocyte cryopreservation and novel ovulation induction regimens, have renewed interest in fertility preservation in women scheduled to receive gonadotoxic chemotherapy and/or radiotherapy (Falcone et al., 2004
). Of these technologies, ovarian tissue cryopreservation may be a viable option for women who cannot delay treatment to undergo ovarian stimulation to create embryos or obtain oocytes for freezing. In this entirely experimental process, thawed tissue can be implanted after cancer treatment as an autograft to an orthotopic or a heterotopic site. Immature oocytes derived from thawed tissue can be matured in vitro if appropriate protocols are developed in the future. Also, the maturation process of the immature oocytes could be achieved by xenografting in immunodeficient mice (Gook et al., 2003).
To date, ovulation and creation of a human embryo from heterotopic ovarian transplant have been reported (Oktay et al., 2004). Moreover, two live births from orthotopic ovarian transplants following spontaneous (Donnez et al., 2004) and IVF (Meirow et al., 2005) cycles were reported, following modified cryopreservation and surgical protocols pioneered, in sheep, by Gosden and associates (Gosden et al., 1994; Baird et al., 1999). Despite these advances, ischaemic damage to the tissue and revascularization injury and the theoretical possibility of reintroducing malignant tumour cells remain as the main limitations for this option (Bedaiwy and Falcone, 2004).
The ischaemic damage to ovarian tissues can induce a high rate of follicular loss (Baird et al., 1999; Demirci et al., 2002). Therefore, eliminating this ischaemic damage could maintain both the viability and the functional integrity of the transplant. Ideally, this could be achieved by transplantation of an intact ovary with vascular anastomosis. Transplantation of an intact ovary with its vascular pedicle using microvascular anastomosis was achieved both in murine species (Yin et al., 2003) and in mammals (Jeremias et al., 2002; Bedaiwy et al., 2003). Partial restoration of hormonal functions in sheep (Bedaiwy et al., 2003) and pregnancy (Wang et al., 2002) after transplantation of intact cryopreserved–thawed (C–T) ovaries in rats were also reported. Recently, cryopreservation of intact human ovary with its vascular pedicle resulting in high post-thaw survival rates of follicles, small vessels and stromal cells as well as a normal histological structure in all the ovarian components was reported using a slow-freezing protocol. Slow freezing was achieved using a cryofreezing one-degree container (Martinez-Madrid et al., 2004).
The structural homeostasis of tissues is regulated by a delicate balance between cell survival and apoptotic cell death. In tissues, several molecules are involved in the survival (survival molecules such as Bcl-2) or cell death (apoptotic molecules such as p53) processes. Bcl-2 is a membrane-associated protein that resides in the nuclear envelope and mitochondria. It exerts its survival functions by modulating the mitochondrial release of cytochrome c and antagonizing the effects of Bax gene (Bcl-2-associated X). Bcl-2 is expressed in granulosa cells of both fetal and adult ovaries. p53 gene encodes a 53-kDa oncosuppressive nuclear protein that functions to antagonize Bcl-2 effects. In the ovary, p53 protein is expressed in the apoptotic granulosa cells of atretic follicles. In several organs such as the heart, ischaemia is associated with the induction of Bcl-2 and p53 protein expressions. Similarly, the induction of these molecules following ovarian ischaemia may have far-reaching effects on the outcome of the subsequent ovarian transplantation (Hussein, 2005).
Although previous studies examined the long-term effects of cryopreservation–thawing procedure injury on the ovarian cortical strips (Gosden et al., 1994), our understanding of the injury caused to ovarian tissue in the period immediately after thawing is still incomplete. The aims of this study were to: (i) describe a cryopreservation protocol using the programmable freezer allowing freezing and thawing of an intact human ovary with its vascular pedicle; (ii) assess the survival (viability) of the ovarian elements in the C–T tissues using Trypan Blue method; (iii) examine the frozen–thawed ovarian tissues for features of follicular health and atresia using the histological methods; (iv) evaluate the apoptotic changes in the ovarian tissues using combined histological methods and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay, immunoperoxidase method (p53 and Bcl-2 protein alterations, i.e. apoptotic and survival proteins) and (v) examine the status of the blood vessels using both histochemical [periodic acid Schiff (PAS) and Masson’s trichrome] and immunohistochemical (CD34, marker of vascular endothelium) staining methods.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The study group included both control (non-cryopreserved) and cryopreserved human ovaries. Also tissue sections from lymph nodes, squamous cell carcinoma, leukaemia cell lines, normal skin and liver were used as tissue-specific positive controls for Bcl-2, p53, apoptotic cells, PAS and Masson’s trichrome staining.
Patients in the cryopreservation group
The Institutional Review Board of The Cleveland Clinic Foundation approved this study. The first patient, aged 46 years, presented with menorrhagia and symptomatic fibroid uterus, who underwent total vaginal hysterectomy with bilateral salpingo-oophorectomy, McCall culdoplasty and cystoscopy. Intraoperative findings included an 8–10 week-sized bulky uterus and normal-appearing ovaries. The second patient, aged 44 years, presented with severe premenstrual dysphoric disorders, who underwent laparoscopic bilateral salpingo-oophorectomy. Normal-appearing uterus, tubes and ovaries were detected. Both ovaries and their pedicles, from both patients, were processed immediately as detailed below.
Patients in the control group
In the control group, formalin-fixed paraffin-embedded ovarian tissue was obtained from the Archives of the Department of Pathology, Assiut University Hospitals. Four ovarian tissue specimens were obtained from two women, 44 and 47 years old. The first was diagnosed with irregular uterine bleeding due to uterine fibroids and was treated by total abdominal hysterectomy with bilateral salpingo-oophorectomy. The second underwent total vaginal hysterectomy with bilateral salpingo-oophorectomy due to pelvic organ prolapse. The ovaries obtained from both patients appeared normal with a smooth white convoluted surface and firm consistency.
Cryopreservation
Cryopreservation of the intact ovary
We adopted our previous protocol described in 2003 (Bedaiwy et al., 2003). Briefly, immediately after oophorectomy, one ovary from each patient was perfused with heparinized Ringer’s solution followed by perfusion and immersion in a bath containing the cryoprotective mixture which was composed of Leibovitz L-15 medium (Irvine Scientific, Santa Ana, CA, USA), 10% fetal calf serum (FCS) (Irvine Scientific) and 1.5 M dimethylsulphoxide (DMSO) (Sigma, St. Louis, MO, USA). Both the ovarian vessels and excess hilar tissue were dissected, and ovarian ligaments were trimmed. The ovaries were perfused via the ovarian artery with the cryoprotective mixture using Horizon Modular Infusion System (McGaw, Irvine, CA, USA) to maintain a flow rate at 1.3 ml/min with continuous replenishment of the reservoir. After perfusion, the ovary was bisected and transferred to two cryovials 12.7 x 92 mm (Corning Coaster Corporation, Cambridge, MA, USA) containing the cryoprotective mixture for controlled freezing using Planer cryochamber (freezer; Planer, Middlesex, UK). Cooling began at +4°C and continued at 2°C per minute until ice nucleation was induced at –7°C. The temperature was then reduced at 2°C per 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 cortical strips of 1.0 x 1.0 x 5.0 mm3 each. The ovarian cortical strips were prepared as described by Gosden et al. (1994) and cryopreserved using the previously mentioned protocol but without perfusion.
Thawing
Thawing of the intact ovary
One week later, the vials were removed from the dewar and held for 1 min at room temperature before plunging in a bath of water at 37°C with gentle shaking. The contents of the vials were quickly emptied into a Petri dish containing Leibovitz L-15 medium supplemented with 10% FCS. The ovaries were washed and immediately perfused with Leibovitz L-15 medium supplemented with 10% FCS using a flow rate of 1.3 ml/min for 20 min. The cryoprotectant was gradually eliminated by pumping Leibovitz L-15 supplemented with 10% FCS into the reservoir (Bedaiwy et al., 2003).
Thawing of the ovarian cortical strips
Ovarian cortical strips were thawed using the same procedure as for intact ovary. Then, the strips were washed and held in Leibovitz L-15 containing 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 thawed ovarian tissues were evaluated for follicular viability in terms of plasma membrane function and structural integrity by the Trypan Blue exclusion test. Ovarian fragments were thinly sectioned in Leibovitz L-15 medium supplemented with 1 mg/ml (200 IU/ml) type 1 collagenase (Sigma), incubated at 37°C for 2 h and pipetted every 30 min. Collagenase activity was inhibited by 50% FCS. The suspension was filtered through a 70-µm nylon filter (Becton Dickinson Labware, Frankline Lakes, NJ, USA) and centrifuged at 400 g for 5 min. The precipitate was diluted with 50 µl of Leibovitz L-15 medium and kept in a water bath at 37°C. 20 l of 0.4% Trypan Blue (Sigma) was added to the suspension containing the follicles, deposited on a glass slide and examined under inverted microscope (400). For each fragment, 100 small intact follicles were examined while the partially or completely denuded oocytes were excluded. Both the number of stained cells and the total number of cells were counted. The percentage of viable follicles was determined by calculating the percentage of unstained cells.
Histological evaluation of the ovarian tissues for the features of follicular health and atresia in the C–T and control ovarian tissues
The maturing follicle is composed of an oocyte with granulosa layer and lacks reticulum. The hallmarks of the healthy follicles include intact oocyte, intact membrana granulosa, lack of necrosis and the presence of few pyknotic nuclei (<5% pyknotic nuclei) in this layer. The atretic follicles are characterized by the presence of attenuated membrana granulosa, loosely attached granulosa cells and increased number of pyknotic nuclei (>5% pyknotic nuclei) (Wood et al., 1997). Initially, the ovarian follicles were sorted into two groups, healthy and atretic, following Wood et al. (1997).
Histological evaluation of apoptotic changes in the C–T and control ovarian tissues
Evaluation of apoptosis was performed using combined histological and TUNEL assays. The results were expressed as mean and SEM. The histological criteria of apoptosis included condensed nuclear fragments, nuclei with marginated chromatin, multiple nuclear fragments, a single condensed nucleus, membrane-bound structures containing variable amounts of chromatin and eosinophilic 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 morphology with the TUNEL assay, using the commercially available QIA33TDT-FragELTM kit (Oncogen Research Products, Boston, MA, USA) following other groups (Hussein et al., 2006). Positive controls consisted of HL60 promyelocytic leukaemia cells and HL60 cells incubated with 0.5 µg/ml actinomycin D for 19 h to induce apoptosis. Some ovarian specimens were used as negative controls by substituting 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 x400 magnification. Ten different areas of each follicular wall were examined, and apoptotic index was determined as mean number of positively stained cells (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 of the blood vessels, we used immunoperoxidase staining methods and monoclonal antibodies targeting p53, Bcl-2 and CD34 proteins. Briefly, ovary sections mounted on glass slides were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.6% H202. Sections were then immersed in the retrieval solution (10 mM sodium citrate buffer, pH 6.0) and subjected to heat-induced antigen retrieval for 20 min (microwave at 
750 W). Non-specific protein binding was blocked with 10 min exposure to 10% normal goat serum. Sections were then incubated with primary antibodies for 1 h at room temperature. The following primary mouse monoclonal antibodies 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 brief rinsing in phosphate-buffered saline (PBS), a catalyzed signal amplification system (K1500, DAKO) was used according to the manufacturer’s instructions. Two observers independently evaluated the stained slides.
Positive controls
Sections from reactive lymphoid hyperplasia (lymph node), p53-positive squamous cell carcinoma and haemangioma were used as positive controls for Bcl-2, p53 and CD34 staining, respectively.
Negative controls
Additional sections of the tissues were stained in parallel but with omission of the primary antibody.
PAS and Masson’s trichrome stains
PAS stains carbohydrate moieties and basement membrane reticulin and therefore delineates the wall of the blood vessels. Masson’s trichrome stains myofibres, indicating skeletal or smooth muscle differentiation (versus non-muscle fibres). Thus, it stains the smooth muscle of the blood vessels.
Evaluation of Bcl-2 and p53 immunostaining
For the evaluation of Bcl-2 and p53 immunoreactivities, several rules were followed: (i) corresponding sections stained by haematoxylin and eosin were examined; (ii) sections were examined to detect the sites of the antibody positivity; (iii) a higher power magnification was used to evaluate the immunostaining; (iv) sections were examined independently by two observers and (v) Bcl-2 and p53 positivity was identified as diffuse brown cytoplasmic and nuclear staining, respectively. The numbers of follicles with positive and negative reactivity were counted in the ovarian tissues (Hussein et al., 2006).
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 the detection 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 (x40 and x100) to select the areas that showed the most intense vascularization (hot spots). Individual microvessels were counted in three fields at x200 magnification (x20 objective lens and x10 ocular lens; 0.7386 mm2/field). The final MVD was the mean value obtained from the counts of the three fields. SEM was expressed as mean (SD) (vessel/mm2). Any immunostained endothelial cells or endothelial cell clusters that were clearly separated from the adjacent microvessels were considered as a single and countable microvessel. Vessel lumens were not a prerequisite for a structure to be defined as a microvessel, and red blood 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 statistical significance of P < 0.05 were performed. Calculations were done with the Statistical Package for the Social Sciences for Windows, version 10.0.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Follicular viability in the C–T ovarian tissue
In the 46-year-old patient, the primordial follicular viability was 75 and 81% in C–T intact ovary and C–T ovarian cortical strips, respectively. In the 44-year-old patient, these values were 78 and 83%, respectively. The average value for both intact ovaries (76.5%) was comparable with that for cortical strips (82%). A summary of these data is presented in Table I.
|
Histological changes in the control and C–T ovarian tissues
In the control ovarian tissues, evaluation of the cortex and medulla revealed the absence of the histological features of necrosis such as: (i) swelling and clumping of the chromatin, (ii) pyknosis (condensation of the chromatin and shrinkage of the nucleus), (iii) karyorrhexis (fragmentation of the nucleus), (iv) karyolysis (dissolution of the nucleus) and (v) cytoplasmic opacification or eosinophilia (Figure 1). Similarly, in the cryopreserved ovaries, evaluation of the cortex and medulla of C–T intact ovaries and cortical strips revealed the absence of these histological features (Figure 2). Moreover, the blood vessels were not only viable and patent but also had intact muscle coat and endothelium (Figure 2).
|
|
In every sample, the primordial follicles were counted in 10 different fields and mean values were reported for the cryopreservation and control groups. Histological evaluation of the count of the primordial follicles revealed a trend towards higher values in the intact C–T ovary as compared with the cortical strips (4.5/high-power field versus 3/high-power field, respectively) (Figure 2). However, differences among these values did not reach the level of statistical significance, probably due to small sample size (two patients). Histological features of apoptosis were seen in the granulosa cells of atretic follicles either within the central region of the membrana granulosa layer or loosely attached to the membrana granulosa near its antral surface. 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 control and cryopreserved ovarian tissues, rare weak Bcl-2 expression was seen both in the healthy follicles and in the stromal cells (Figures 3 and 4). p53 expression (nuclear) was virtually absent in the healthy primordial, primary and secondary follicles, germinal epithelium and stromal cells subjected to warm ischaemia (Figures 3 and 4). Bcl-2 immunoreactivity was observed more frequently in the secondary follicles, followed 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 developmental 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 technique showed any immunoreactivity, whereas signal-positive TUNEL staining was observed in the positive controls (Figure 5). In the control ovarian tissues, granulosa cells with the histologic features of apoptosis were observed in the atretic follicles, either within the central region of membrana granulosa layer or loosely attached with membrana granulosa near its antral surface 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 fragmented nuclei of granulosa cells. The positively stained nuclei were observed either in the central layers of the membrana granulosa, at the antral surface or floating in the follicular atrium. DNA fragmentation was absent both in the interstitial cells and in the theca cells of the atretic follicles. Evaluation of apoptosis in the cryopreserved ovaries revealed similar findings: (i) the healthy (primordial, primary and secondary) follicles were TUNEL negative; (ii) TUNEL-positive signals were detected in the atretic follicles as indicated by the punctuated brown staining of the fragmented nuclei of granulosa cells (Figure 5) and (iii) the mean values of apoptotic cells in the intact cryopreserved ovarian tissues were comparable with those in the ovarian cortical strips and control ovarian tissues (1.8 ± 0.1 versus 1.7 ± 0.1 versus 1.8 ± 0.1, respectively). The results 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 evaluation of the basement membrane (PAS) and muscle coat (Masson’s trichrome) of ovarian blood vessels demonstrates persistent vascularity in both cryopreserved and control ovarian tissues. Microvessels are heterogeneously distributed within the ovarian 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 these results is presented in Table I and Figures 6–
8.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
In this investigation, the immediate post-thawing injury to the human ovary (cryopreserved both as a whole with its vascular pedicle and as ovarian cortical strips) was assessed using both histological and immunohistological methods. To achieve our goals, we established a study group formed of four patients. It included both control (non-cryopreserved) and cryopreserved human ovaries. Tissue sections from lymph nodes, squamous cell carcinoma, leukaemia cell lines, normal skin and liver were used as tissue-specific positive controls for the histochemical (PAS and Masson’s trichrome staining) and immunohistochemical (Bcl-2, p53 and apoptotic cells) parameters. Our investigation demonstrated the following findings: (i) the mean values of MVD, follicle counts and Bcl-2 protein expression were comparable between the cryopreserved and the control groups and (ii) no differences were seen between the two groups for the following parameters: healthy and atretic follicle counts, apoptotic index and Bcl-2 protein expression in the atretic follicles
Ovarian tissue banking is more popular as a fertility preservation option for patients without a partner or those who cannot delay gonadotoxic chemotherapy for the sake of ovarian stimulation to produce oocytes for freezing or embryos for cryopreservation. With the limited long-term survival upon reimplantation with ovarian tissue strips, ovarian transplantation with microvascular anastomosis could help avoid accelerated follicular loss and improve the longevity of the ovarian grafts. This helps to guarantee an immediate revascularization and establishment of the graft function. Although successful in rat and sheep animal models (Wang et al., 2002; Bedaiwy et al., 2003), microvascular anastomosis of intact frozen–thawed ovaries remains technically challenging. Moreover, cryopreservation of an entire organ with its vascular pedicle is much more complicated than ovarian cortical strips or suspended cell (oocyte) or group of cells (embryo).
In our previous study, we reported a 30% long-term patency rate of the ovarian vessels upon reimplantation of an intact frozen–thawed ovary in merino sheep (Bedaiwy et al., 2003). However, human ovaries are larger and may not be amenable to the same protocol. The technical challenges of the microvascular anastomosis could be circumvented adequately by appropriate training. On the contrary, the main determinant of success would be to develop an appropriate cryopreservation protocol of an entire ovary with its vascular pedicle. Adequate delivery of the cryoprotective agent (CPA) to almost every cell of the ovary is mandatory to guarantee an adequate post-thaw survival. Aiming at adequate CPA permeation to virtually every cellular component of the ovary, we used the Horizon Modular Infusion pump to ensure adequate CPA distribution throughout the ovarian substance. Infusing the CPA slowly through the ovarian artery and allowing it to come out of the ovarian vein ensures adequate use of the ovarian vascular channel as a delivery vehicle. To avoid intracellular ice formation, we used a slow-freezing protocol using the Planer freezer. The use of constant infusion pressure during the freezing and thawing phases ensured adequate distribution and clearance of the CPA.
The selection of DMSO as a CPA of choice was based on the initial observation by Newton et al. (1998) that exposure for 30 min to 1.5 M solutions of DMSO at 4°C achieved a mean tissue concentration that was almost 80%. In our study, 1.5 M DMSO was tolerated comparably by both an intact ovary and ovarian cortical strips. Interestingly, in a recent report where ovarian tissue was exposed to 10% DMSO for 15 min at 4°C, no significant differences in follicular viability before (99.4%) or after cryoprotectant exposure (98.1%) were documented. This could probably mean that ovarian tissue could tolerate adequately varying concentrations of DMSO with comparable post-thaw survival (Martinez-Madrid et al., 2004).
After thawing, viability assessment of the primordial follicles was comparable between intact ovaries and ovarian cortical strips. This demonstrates that delivering the CPA using an infusion pump was as efficient as the permeation of the ovarian cortical strips with the CPA avoiding cryoinjury to the follicles as well as the intraovarian vessels. The viable primordial follicular count in the intact ovaries in both of our patients (75 and 78%) was comparable with the 75.1% follicular viability reported by Martinez-Madrid et al. (2004), who tried a different freezing protocol for intact human ovary.
In this investigation, the presence of apoptotic activity in the ovarian follicles may be attributed to the presence of apoptosis-triggering signals and/or degradation of apoptosis-related proteins (caspase-3, caspase-8, Bcl-2 and actin) (Zhu et al., 1999; Schmidt-Mende et al., 2000). At the molecular level, cryopreservation and thawing had no effects on the values of apoptotic cell death and Bcl-2 and p53 protein expressions in both ovarian cortical strips and intact ovaries. A hypothesis to be tested is that cryopreservation and thawing can induce some effects on these values (apoptotic cell death and Bcl-2 and p53 protein expressions) over longer time intervals. This hypothesis stems from the fact that apoptosis is a dynamic process that requires active metabolism and usually takes several hours or even days to be executed (Hussein et al., 2006). These questions are open for future investigations. Apoptosis in the atretic follicles can effectively be present before the cryopreservation, but it does not seem to be increased by the cryopreservation process. Moreover, the presence of apoptotic changes in the atretic follicles supports the link between apoptosis and ovarian follicular atresia.
It is also possible that substances which stimulate apoptosis may have atretogenic effects on the ovarian follicles. The presence of Bcl-2 protein expression in the healthy ovarian follicles concurs with other reports (Tilly, 1996a,b; Felici et al., 1999) and suggests its possible physiologic role in the maintenance of follicular integrity. In support, mice lacking functional Bcl-2 protein possess reduced numbers of primordial follicles relative to their wild-type sisters (Ratts et al., 1995). The lack of p53 protein expression in the healthy follicles versus its presence of atretic ones concurs with previous studies and suggests lack of underlying DNA damage and therefore implies integrity of the genome in the healthy follicles (Hussein, 2005).
During organ transplantation, tissues are subjected to variable duration of ischaemic injury. In the heart, retina, liver and brain, ischaemic injury is associated with apoptosis as well as Bcl-2 and p53 alterations. Namely, the induction of Bcl-2 and p53 can occur as early as 0.5 and 1 h, respectively, following ischaemia (Peralta et al., 2002; Wang et al., 2003). Apoptosis requires active cell metabolism that could not be complete within 30 min and could therefore not alter the status of the follicles within an ovary. A hypothesis to be tested is that the differences observed in this investigation are the result of outright lysis (ischaemia could make cells more prone to lysis). It would be interesting for future investigations to examine this hypothesis.
Our investigation revealed the presence of persistent vascularity and comparable MVD in both control and cryopreserved groups. These findings indicate that our freeze–thawing protocol has no detrimental effects on the integrity of the vascular network of the ovarian tissue. Of note, the status of the ovarian vascular supply is central to dynamic changes occurring during the normal ovarian cycle. Follicular growth and the development of the corpus luteum are dependent on intact vasculature. The selection of a dominant follicle in monovular species is associated with angiogenesis. Moreover, selected follicles possess a more elaborate microvascular network than other follicles. The vasculature also plays a key role in the delivery of cholesterol to luteal cells for progesterone biosynthesis (Carr et al., 1981; Goede et al., 1998).
Many studies indicate that the ovaries of older women are smaller than those of young women. They are composed mainly of stromal cells that have receptors for, and respond to, gonadotrophins and secrete testosterone and lesser amounts of other androgens and estrogens. These ovaries remain gonadotrophin-driven, androgen-producing glands. Also, they are characterized by the presence of prominent cystic follicles, thick-walled vessels, granulomas, hyaline scars and low number of follicles. However, these follicles are not necessarily more sensitive to the freezing–thawing process (Couzinet et al., 2001; Longcope, 2001). In this investigation, the study group was formed of older patients knowing in advance the histopathological and follicular implications of ageing on the ovarian structure. Whether the aforementioned age-related changes have an impact on our results awaits further confirmation in younger age group upon ethical approval.
In this study, we assessed injury of the ovarian tissue in the period immediately after thawing. Our investigation provides further evidence that intact human ovary could be cryopreserved using a slow-freezing protocol. We have demonstrated comparable survival rates of follicles and limited molecular alterations between C–T intact ovaries and C–T ovarian cortical strips. The information herein reported is provided for the period immediately after thawing. The changes associated with long-term injury mandates further investigations.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The financial support for this study was a research grant from the Minimally Invasive Surgery Center, The Cleveland Clinic Foundation, Cleveland, OH, USA. This work was presented at ESHRE 20th Annual meeting, June 27–July 1, Berlin, Germany 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. (1999) Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at –196°C. Endocrinology 140:462–471.
Bedaiwy MA and Falcone T. (2004) Ovarian tissue banking for cancer patients: reduction of post-transplantation ischaemic injury: intact ovary freezing and transplantation. Hum Reprod 19:1242–1244.
Bedaiwy MA, Jeremias E, Gurunluoglu R, Hussein MR, Siemianow M, Biscotti C, Falcone T. (2003) Restoration of ovarian function after autotransplantation of intact frozen–thawed sheep ovaries with microvascular anastomosis. Fertil Steril 79:594–602.[CrossRef][ISI][Medline]
Carr BR, Sadler RK, Rochelle DB, Stalmach MA, MacDonald PC, Simpson ER. (1981) Plasma lipoprotein regulation of progesterone biosynthesis by human corpus luteum tissue in organ culture. J Clin Endocrinol Metab 52:875–881.[Abstract]
Couzinet B, Meduri G, Lecce MG, Young J, Brailly S, Loosfelt H, Milgrom E, Schaison G. (2001) The postmenopausal ovary is not a major androgen-producing gland. J Clin Endocrinol Metab 86:5060–5066.
Demirci B, Salle B, Frappart L, Franck M, Guerin JF, Lornage J. (2002) Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing–thawing of ovarian cortex in sheep. Fertil Steril 77:595–600.[CrossRef][ISI][Medline]
Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, van Langendonckt A. (2004) Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 364:1405–1410.[CrossRef][ISI][Medline]
Falcone T, Attaran M, Bedaiwy MA, Goldberg JM. (2004) Ovarian function preservation in the cancer patient. Fertil Steril 81:243–257.[CrossRef][ISI][Medline]
Felici MD, Carlo AD, Pesce M, Iona S, Farrace MG, Piacentini M. (1999) Bcl-2 and Bax regulation of apoptosis in germ cells during prenatal oogenesis in the mouse embryo. Cell Death Differ 6:908–915.[CrossRef][ISI][Medline]
Gavrieli Y, Sherman Y, Ben-Sasson SA. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501.
Goede V, Schmidt T, Kimmina S, Kozian D, Augustin HG. (1998) Analysis of blood vessel maturation processes during cyclic ovarian angiogenesis. Lab Invest 78:1385–1394.[ISI][Medline]
Gook DA, Edgar DH, Borg J, Archer J, Lutjen PJ, McBain JC. (2003) Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting. Hum Reprod 18:1772–1781.
Gosden RG, Baird DT, Wade JC, Webb R. (1994) Restoration of fertility to oophorectomized sheep by ovarian autografts stored at –196 degrees C. Hum Reprod 9:597–603.
Hussein MR. (2005) Apoptosis in the ovary: molecular mechanisms. Hum Reprod Update 11:162–177.
Hussein M, Bedaiwy M, Falcone T. (2006) Analysis of apoptotic cell death, Bcl-2, and p53 protein expression in freshly fixed and cryopreserved ovarian tissue after exposure to warm ischemia. Fertil Steril 85:1082–92.[Medline]
Jeremias E, Bedaiwy MA, Gurunluoglu R, Biscotti CV, Siemionow M, Falcone T. (2002) Heterotopic autotransplantation of the ovary with microvascular anastomosis: a novel surgical technique. Fertil Steril 77:1278–1282.[CrossRef][ISI][Medline]
Kerr JF, Wyllie AH, Currie AR. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257.[ISI][Medline]
Liu TJ, el-Naggar AK, McDonnell TJ, Steck KD, Wang M, Taylor DL, Clayman GL. (1995) Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck. Cancer Res 55:3117–3122.
Longcope C. (2001) Endocrine function of the postmenopausal ovary. J Soc Gynecol Invest 8:S67–S68.[CrossRef][ISI][Medline]
Martinez-Madrid B, Dolmans MM, Van Langendonckt A, Defrere S, Donnez J. (2004) Freeze–thawing intact human ovary with its vascular pedicle with a passive cooling device. Fertil Steril 82:1390–1394.[CrossRef][ISI][Medline]
Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, Schiff E, Dor J. (2005) Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 353:318–321.
Newton H, Fisher J, Arnold JR, Pegg DE, Faddy MJ, Gosden RG. (1998) Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod 13:376–380.
Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, Opsahl M, Rosenwaks Z. (2004) Embryo development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet 363:837–840.[CrossRef][ISI][Medline]
Peralta C, Bulbena O, Xaus C, Prats N, Cutrin JC, Poli G, Gelpi E, Rosello-Catafau J. (2002) Ischemic preconditioning: a defense mechanism against the reactive oxygen species generated after hepatic ischemia reperfusion. Transplantation 73:1203–1211.[CrossRef][ISI][Medline]
Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL. (1995) Ablation of Bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 136:3665–3668.[Abstract]
Schmidt-Mende J, Hellstrom-Lindberg E, Joseph B, Zhivotovsky B. (2000) Freezing induces artificial cleavage of apoptosis-related proteins in human bone marrow cells. J Immunol Methods 245:91–94.[CrossRef][ISI][Medline]
Tilly JL. (1996a) Apoptosis and ovarian function. Rev Reprod 1:162–172.[Medline]
Tilly JL. (1996b) The molecular basis of ovarian cell death during germ cell attrition, follicular atresia, and luteolysis. Front Biosci 1:d1–d11.[Medline]
Wang X, Chen H, Yin H, Kim SS, Lin Tan S, Gosden RG. (2002) Fertility after intact ovary transplantation. Nature 415:385.[Medline]
Wang F, Hu YL, Li YH. (2003) The expression of Bcl-2 protein on acute myocardial ischemia/reperfusion and the significance in forensic pathology. Fa Yi Xue Za Zhi 19:207–208 211.[Medline]
Weidner N, Semple JP, Welch WR, Folkman J. (1991) Tumor angiogenesis and metastasis – correlation in invasive breast carcinoma. N Engl J Med 324:1–8.[Abstract]
Wood TC, Montali RJ, Wildt DE. (1997) Follicle-oocyte atresia and temporal taphonomy in cold-stored domestic cat ovaries. Mol Reprod Dev 46:190–200.[CrossRef][ISI][Medline]
Yin H, Wang X, Kim SS, Chen H, Tan SL, Gosden RG. (2003) Transplantation of intact rat gonads using vascular anastomosis: effects of cryopreservation, ischaemia and genotype. Hum Reprod 18:1165–1172.
Zhu L, Yu X, Akatsuka Y, Cooper JA, Anasetti C. (1999) Role of mitogen-activated protein kinases in activation-induced apoptosis of T cells. Immunology 97:26–35.[CrossRef][ISI][Medline]
Submitted on December 4, 2005; resubmitted on March 15, 2006; resubmitted on May 11, 2006; accepted on May 16, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. A. Bedaiwy, S. A. El-Nashar, A. M. El Saman, J. L.H. Evers, S. Sandadi, N. Desai, and T. Falcone Reproductive outcome after transplantation of ovarian tissue: a systematic review Hum. Reprod., August 9, 2008; (2008) den301v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V.J. Onions, M.R.P. Mitchell, B.K. Campbell, and R. Webb Ovarian tissue viability following whole ovine ovary cryopreservation: assessing the effects of sphingosine-1-phosphate inclusion Hum. Reprod., March 1, 2008; 23(3): 606 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.P. Milazzo, L. Vaudreuil, B. Cauliez, E. Gruel, L. Masse, N. Mousset-Simeon, B. Mace, and N. Rives Comparison of conditions for cryopreservation of testicular tissue from immature mice Hum. Reprod., January 1, 2008; 23(1): 17 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Martinez-Madrid and J. Donnez Cryopreservation of intact human ovary with its vascular pedicle--or cryopreservation of hemiovaries? Hum. Reprod., June 1, 2007; 22(6): 1795 - 1796. [Full Text] [PDF]
|
|
|
|
|
|
M. A. Bedaiwy and T. Falcone Reply: Cryopreservation of intact human ovary with its vascular pedicle--or cryopreservation of hemi ovaries Hum. Reprod., June 1, 2007; 22(6): 1796 - 1797. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||