Ficoll density gradient method for recovery of isolated human ovarian primordial follicles

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1 FERTILITY AND STERILITY VOL. 82, NO. 6, DECEMBER 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Ficoll density gradient method for recovery of isolated human ovarian primordial follicles Belen Martinez-Madrid, V.M.D., Ph.D., Marie-Madeleine Dolmans, M.D., Anne Van Langendonckt, Ph.D., Sylvie Defrère, B.Sc., Anne-Sophie Van Eyck, M.D., and Jacques Donnez, M.D., Ph.D. Department of Gynecology, Université Catholique de Louvain, Brussels, Belgium Objective: To develop a simple and efficient technique to allow rapid recovery of a maximum number of good quality isolated follicles. Design: Prospective experimental study. Setting: Academic research unit of the department of gynecology in a university hospital. Patient(s): Biopsies were obtained from five women (between 26 and 31 years of age). Intervention(s): Biopsies were cut with a tissue sectioner. Enzymatic digestion was performed in a collagenase solution for 90 min at 37 C. The follicles were recovered using a discontinuous Ficoll density gradient method. Main Outcome Measure(s): The number of follicles present in the interface layers of Ficoll gradient was quantified. Follicular viability of these recovered follicles was assessed with live-dead stains, using calcein-am and ethidium homodimer-i. Result(s): Out of a total of 6,811 recovered follicles, we found 63% (n 4,201) at the medium 1.06 Ficoll interface and 36.9% (n 2,590) at the Ficoll interface, which represents 99.9% of total recovered follicles. Analysis by vital fluorescent staining showed that 95.8% of the follicles treated with Ficoll were totally viable. Conclusion(s): The Ficoll density gradient method allows us to maximize the recovery of isolated human ovarian follicles and minimize the manipulation time while maintaining high follicular viability. (Fertil Steril 2004;82: by American Society for Reproductive Medicine.) Key Words: Human, isolation, ovary, primordial follicle, viability stain, recovery, Ficoll density gradient Received November 5, 2003; revised and accepted May 3, Supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (FNRS) no and FNRS Televie no Reprint requests: Jacques Donnez, M.D., Ph.D., Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires St Luc, Avenue Hippocrate 10, Brussels 1200, Belgium (FAX: ; donnez@ gyne.ucl.ac.be) /04/$30.00 doi: /j.fertnstert The development of techniques for the isolation of ovarian follicles has contributed to the study of mammalian folliculogenesis in vitro. The primordial follicle is the follicular stage of choice for research and clinical studies because the majority of oocytes within the human ovarian cortex are located in the primordial follicle resting pool (1, 2), representing the main source of oocytes in the ovary. Studies on culture, transplantation, or cryopreservation can be conducted using follicles embedded in cortical tissue or isolated follicles. The maintenance of follicle quality during the isolation and recovery process is extremely important for their subsequent survival and, therefore, for the success of these studies. Culture of follicles as isolated units allows a fair comparative study of optimum culture conditions, because it is possible to compare groups with an equal number of follicles and at a particular developmental stage (3). However, in the case of ovarian cortical pieces, the number of follicles in culture is unpredictable owing to the variable density and uneven distribution of follicle clusters in adult women, depending on age and physiologic status (1, 4). Culture of isolated follicles also has the advantage of allowing their direct monitoring during the culture period (5), which is not possible in the culture of ovarian tissue because follicles within the cortex are not visible under stereomicroscopic examination (6). Moreover, the individual development of isolated folli- 1648

2 cles in culture can be assessed by morphologic, cellular, and molecular analysis (7 9). However, long-term culture of human isolated preantral follicles has not been very successful to date (10, 11). Nor has the alternative approach of culturing partially isolated follicles yielded better results than either small tissue slice culture (12) or complete isolated follicle culture (11). Indeed, an increase in follicular size (doubling the diameter) and granulosa cell number was observed only in fully isolated, but not partially isolated, primordial and primary follicles cultured in collagen gels for 24 h, in case of both fresh and frozen-thawed follicles (11). Transplantation of isolated follicle suspensions would allow us to introduce a high and known number of follicles into the host and to obtain faster angiogenesis, minimizing ischemic and reperfusion damage, which can induce follicular loss of more than 50% in case of cortical strip transplantation (13, 14). Furthermore, it would reduce the risk of transferring malignant cells through the transplant (15, 16) in case of autografting in cancer patients. Cryopreservation and transplantation of isolated primordial follicles has been successfully achieved in mice (17), yielding normal offspring. The small size of isolated follicles, compared to tissue slices, allows a shorter equilibration time during conventional cryopreservation (18) as well as the alternative of vitrification methods. In addition, primordial follicles are at a relatively undifferentiated stage with a low metabolic rate, which makes them more resistant to cryoinjury (7, 18, 19). The process of oogenesis and folliculogenesis requires a coordinated program of gene expression events to occur in developing follicles. According to Huntriss et al. (20), a better understanding of the regulation of early human oogenesis requires analysis of the genes expressed either specifically or predominantly during these stages of development. Isolation of follicles is a tool that will enable us to study stage-dependent gene expression, as has already been done for the expression of FSH receptors (21) and FIGLA (20) genes in human preantral follicles. However, the above-described procedures are dependent upon efficient methods to isolate and recover primordial follicles. Filtration of the digested cortical cell suspension, to remove stromal debris and harvest the follicles, is a method widely employed in different species (22 28). Nevertheless, when this method was used in our laboratory, extensive follicular loss was noted on the mesh filter surface, with follicles remaining stuck to the filter after rinsing (Fig. 1A). To overcome this problem, we developed a new method for the recovery of isolated preantral follicles, based on the Ficoll discontinuous density gradient separation technique used in pancreatic islet purification (29). The present study describes the modified Ficoll density gradient method for FIGURE 1 (A) Mesh filter surface after filtration of digested cortical cell suspension and rinsing, stained with calcein-am and ethidium homodimer-i. Arrows show follicles remaining stuck to the filter (Leica inverted fluorescence microscope; original magnification 100). (B E) Follicular viability assessment by calcein-am and ethidium homodimer-i (Leica inverted fluorescence microscope). (B) Live follicle (original magnification 400). (C) Moderately damaged follicle with 50% of dead granulosa cells (original magnification 400). (D) Dead follicle (original magnification 400). (E) Group of live follicles with a dead follicle (arrow) (original magnification 100). isolated human ovarian follicles, providing a suitable means of recovering greater numbers of viable primordial follicles. MATERIALS AND METHODS Collection of Ovarian Cortical Tissue and Isolation of Follicles The use of human tissue for this study was approved by the Institutional Review Board of the Université Catholique de Louvain. Ovarian biopsies were obtained from five women (between 26 and 31 years of age), three undergoing FERTILITY & STERILITY 1649

3 laparoscopic surgery for nonovarian disease, and two undergoing oophorectomy during a Wertheim-Meigs procedure. Tissue was immediately transported from the operating room to the research laboratory in HEPES-MEM medium (GIBCO, Scotland, UK) on ice. At 4 C, the rest of the medulla was removed from the biopsies using surgical scissors, and the cortical portions were put into a tissue sectioner (McIlwain Tissue Chopper, The Mickle Laboratory, Guildford, UK), adjusted to 0.5 mm. The cutting procedure was fast ( 5 minutes), and uniform-size pieces of mm were obtained. The fragments were then transferred to 50-mL conical tubes containing 10 ml of Dulbecco phosphate-buffered saline (PBS) medium (Biochrom AG, Berlin, Germany) supplemented with 1 mg/ml collagenase type IA (Sigma, St. Louis, MO) and incubated in a water bath at 37 C for 90 minutes with gentle agitation. The ovarian digest was periodically (approximately every 20 minutes) agitated by a micropipette to mechanically disrupt digested tissue. Digestion was terminated by the addition of an equal volume of cold PBS medium supplemented with 10% of fetal bovine serum (FBS) (Sigma). The resulting suspension was then centrifuged at 50 g for 5 min at 4 C, the supernatant was discarded, and the pellet was further processed as described below. Recovery of Isolated Follicles by the Ficoll Discontinuous Density Gradient Method For the preparation of the discontinuous Ficoll density gradient, the pellet containing the follicles was resuspended in 7.5 ml of Ficoll (Biocoll Separating Solution, Biochrom AG) solution (density 1.1 g/cm 3 ) at the bottom of a 50-mL conical tube, constituting the first Ficoll density layer (1.1 Ficoll layer). The successive density layers were subsequently added on top to complete the discontinuous gradient: 3.5 ml of 1.09-g/cm 3 Ficoll solution (1.09 Ficoll layer), and 2.5 ml of 1.06-g/cm 3 Ficoll solution (1.06 Ficoll layer) and 2.5 ml of PBS medium (PBS layer). The gradient tube was centrifuged at 50 g for 17 min at 4 C. Finally, the different interface layers were recovered, transferred to a Petri dish, and investigated for follicles under the stereomicroscope (Leica MZ 9.5, Heerbrugg, Switzerland). The Ficoll layers and the resting pellet were examined to check that all the follicles had migrated into the interfaces. The recovered follicles were examined by phase-contrast microscopy under an inverted microscope (Leica) with an ocular micrometer to measure their size and visualize their morphologic appearance. Viability Assessment Follicular viability was analyzed in order to test if Ficoll treatment has a toxic effect on follicles. The recovered follicles were picked up using a glass micropipette and transferred to a droplet of PBS containing 2 mol/l of calcein-am (Molecular Probes) and 5 mol/l of ethidium TABLE 1 Mean number of recovered follicles per mm 3 in different patients. Specimen Age No. of recovered preantral follicles Mean no. of recovered follicles/mm , homodimer-i (Molecular Probes, Leiden, The Netherlands). Follicles were incubated with the fluorescent dyes for 45 minutes at 37 C in the dark (6). Nonfluorescent cell-permeant calcein-am enters the cell and is cleaved by nonspecific esterase activity in living cells, producing calcein. The polyanionic dye, calcein, is well retained within live cells, giving an intense uniform green fluorescence. Ethidium homodimer-i enters permeable cells (cells with damaged membranes) and then binds to DNA with high affinity, undergoing a 40-fold enhancement of fluorescence, thereby producing a bright red fluorescence in dead cells. After exposure to fluorescent dyes, the follicles were washed in PBS, mounted between coverslips, and observed under an inverted fluorescence microscope (Leica DMIL). Green fluorescence could be visualized in live cells after exposing the cells to light with an excitation (ex) wavelength of 495 nm and observing the emitted (em) light at a wavelength of 515 nm, and red fluorescence was visible in dead cells with ex and em of 495 and 635 nm, respectively. The follicles were classified into four categories depending on the percentage of dead granulosa cells: [1] live follicles: follicles with the oocyte and all the granulosa cells (GC) viable; [2] minimally damaged follicles: follicles with 10% of dead GC; [3] moderately damaged follicles: follicles with 50% of dead GC; and [4] dead follicles: follicles with both the oocyte and all the GC dead. RESULTS Recovery of Isolated Follicles by the Ficoll Discontinuous Density Gradient Method The enzymatic digestion procedure combined with the Ficoll discontinuous density gradient method for the recovery of isolated follicles provided a total of 6,811 isolated preantral follicles, collected from a volume of 1,141.5 mm 3 of cortical tissue, which demonstrates the reasonable efficacy of the technique considering the age of the patients (between 26 and 31 years of age). Table 1 shows the mean number of 1650 Martinez-Madrid et al. Recovery of isolated human follicles Vol. 82, No. 6, December 2004

4 TABLE 2 Percentage of follicles recovered from each interface layer and from the pellet. Layers No. of recovered follicles Percentage of recovered follicles SEM Interface: 1.06 Ficoll PBS 4, Interface: 1.09 Ficoll 1.06 Ficoll 2, Interface: 1.1 Ficoll 1.09 Ficoll Layer 1.1 Ficoll pellet recovered follicles per mm 3 of ovarian cortex in each specimen. Large individual differences in the number of preantral follicles per mm 3 were found, varying from 0.4 to 21.6 follicles/mm 3. Ninety-five percent of recovered follicles were between 30 and 50 m in diameter, measured using inverted microscopy, which, according to published criteria (20), corresponds to primordial or early primary follicles. The rest of the recovered follicles (5%) ranged between 50 and 120 m, constituting follicles from the primary to preantral stage. The follicles exhibited a normal morphology with an intact basement membrane. After investigating for follicles in the different interface layers, a mean of 63.04% (n 4,201) of total recovered follicles was found at the interface between the 1.06 Ficoll layer and PBS layer, and a mean of 36.90% (n 2,590) at the interface between the 1.09 and 1.06 Ficoll layers, which represents 99.94% of total recovered follicles. At the interface between the 1.1 and 1.09 Ficoll layers, only 0.02% of follicles were recovered, and in the 1.1 Ficoll layer, which contained the resting pellet, 0.04% were found (Table 2). The other Ficoll layers were completely devoid of follicles. Effect of Ficoll Treatment on Follicular Viability Follicular viability, analyzed by vital fluorescent staining (calcein-am and ethidium homodimer-i), was assessed in more than 95% of follicles recovered from each patient, with the exception of patient 4, where only 8.5% of recovered follicles were analyzed owing to the high number of follicles (6,360) collected. The follicles were classified into four categories, depending on the percentage of dead granulosa cells found (Fig. 1B E), according to the classification described above. Out of a total of 977 follicles analyzed, 95.8% were totally viable (oocyte and granulosa cells) after exposure to Ficoll discontinuous density gradient and less than 1% of follicles were really damaged (Table 3). DISCUSSION The ovarian cortex contains mainly primordial follicles. In women at risk of undergoing premature ovarian failure due to cancer treatment or other pathologies, it is recommended that primordial follicles be cryopreserved, to maximize the chances of recovering fertility potential in the future. In humans, however, growing oocytes from the primordial follicular stage to mature and fertile oocytes, either in vivo (transplantation) or in vitro (culture), remains a challenge. There are two possible routes to be taken in the cryopreservation, culture, and grafting of primordial follicles: use of follicles embedded in cortical tissue or isolated follicles. Among other advantages, culture of isolated follicles permits direct monitoring during the culture period (5), not possible in the case of culture of cortical slices because follicles enclosed within the cortex cannot be observed (6). However, culture systems must be optimized to allow long-term culture of isolated primordial follicles. Transplantation of suspensions with a known number of isolated follicles avoids the risk of grafting empty cortical pieces from women with advanced depletion of the resting pool. Moreover, angiogenesis following grafting of isolated follicles is fast (30), reducing ischemic and reperfusion damage and, therefore, the huge follicular loss that such damage provokes. Furthermore, in the case of cancer patients, it would minimize the risk of transferring malignant cells through the transplant (15, 16). In healthy follicles, the follicular basal lamina excludes capillaries, white blood cells, and nerve processes from the granulosa compartment until ovulation (31). Therefore, cancerous cells would be unable to pass through the follicular basal lamina of primordial follicles. To ensure the growth of grafted isolated primordial follicles, it would be necessary to add stromal cells, which could be taken from the patient after cancer remission. If taken before the treatment, healthy stromal cells would need to be separated from possibly cancerous cells by purification techniques, such as fluorescence-activated cell sorting for specific cell surface markers. The main advantage of cryopreserving isolated follicles compared to tissue slices is that, owing to their small size, the equilibration time is shorter during conventional cryopreservation, while allowing the possibility of attempting vitrification methods. The goal of obtaining offspring originating from isolated primordial follicles has only been achieved in mice, by grafting isolated primordial follicles into the ovarian bursa (17). Despite this limited success, it is nevertheless a promising start in the hard task of imitating nature. The growth and development of oocytes requires a coordinated program of gene expression events to occur in developing follicles. The development of techniques for the FERTILITY & STERILITY 1651

5 TABLE 3 Follicular viability of isolated follicles after Ficoll treatment. Follicular viability categories No. of follicles in each category Percentage of follicles in each category SEM Live follicles Minimally damaged follicles Moderately damaged follicles Dead follicles isolation of ovarian follicles has contributed to the study of folliculogenesis, having already identified the stage-dependent expression of some genes in isolated human preantral follicles (20, 21). But progress in this area of basic research is hindered by the small size and limited availability of human ovarian follicles, and requires the use of sensitive molecular techniques (20). Ovarian biopsies are frequently donated by adult women, in whom depletion of the follicular pool is advanced, leaving a poor reserve for experimentation. In addition, follicles are enclosed in dense interstitial tissue, which makes their isolation technically difficult. Therefore, effective isolation and recovery techniques are of vital importance to obtain a large yield of competent follicles for further studies. It is also very important to avoid any damage to follicles during isolation and recovery processes, thus assuring their survival and, consequently, the success of these studies. For the recovery of isolated follicles, several possible methods have been described. Harvesting follicles from the suspension of digested cortical cells with a precalibrated pipette (7, 10) is time consuming because it entails picking them up, one by one, and transferring stromal cells at the same time. The centrifugal elutriation method has been used to separate follicles from somatic cells (25, 32), but large amounts of tissue were required and most of the oocytes were recovered bare. The filtration method does not require great amounts of tissue and purifies follicles from the resting cells, being widely employed in different species such as bovines (22), ovines (23), and wild animals (28). Nevertheless, we observed a loss of follicles that remained stuck to the filter surface after rinsing (Fig. 1A). The method presented in this study employs the Ficoll discontinuous density gradient separation technique for the recovery of isolated preantral follicles. It provides a suitable means of recovering large numbers of viable primordial follicles and separating them from the stromal cells. Nevertheless, large differences in follicular density were found according to the specimen, varying from 0.4 to 21.6 follicles/ mm 3. Surprisingly, the highest recovery rate was obtained from the oldest patient. These results confirm those of Lass et al. (1), who studied follicular density in ovarian biopsies obtained from 60 women and found a range from 0 to 160 follicles/mm 3, with a median of 8 follicles/mm 3. In a recent study (33), not only were differences in follicular density found from patient to patient (from 1.1 to follicles/mm 3 in 21 different women), but also between different fragments of the same ovary, ranging from 1.8 to 166 follicles/mm 3. Moreover, in a recent review of follicular distribution, Lass (34) concluded that the ovarian cortex indeed varies significantly in its follicular content and it is not clear whether a particular area is more predisposed to follicular nesting than another, or if it is due to purely random allocation. This variable density of follicles makes it difficult to compare the efficacy of this technique with other methods described in the literature. For example, Roy and Treacy (10) recovered a yield of small isolated follicles per quarter of human ovary, and Oktay et al. (7) retrieved an average of primordial follicles per block of mm of ovarian cortex, both harvesting follicles from the digested tissue with a pipette. In order to evaluate the efficacy of the technique, Oktay et al. (7) compared the numbers of follicles isolated and recovered from tissue with those counted in histologic sections from tissue blocks of the same size. This is a good way to calculate effectiveness if patients are young, with a homogeneous follicular distribution, or if a large amount of tissue is available for the study. If not, this type of comparison is subject to the uneven distribution of clusters between the different cortical pieces analyzed in the same woman. The maintenance of follicular quality during the isolation and recovery process is extremely important for the subsequent survival of follicles placed in culture or grafted. In our study, the majority of the follicles (99.94%) were recovered from only two interface layers. This means that collection of the follicles from this small volume did not take long, which is of vital importance in preserving their quality. Ficoll is a polymer of sucrose and behaves as an ideal neutral sphere, not permeable to biological membranes. Ficoll solutions have long been used in the formation of density gradients for the separation and isolation of eukaryotic cells, as a separation medium for the isolation of lymphocytes, and as a component in defined culture media. Recently, it has been shown that Ficoll is nontoxic to embryos (35), and it has been employed as a component of cryoprotectant solutions to preserve ovarian tissue pieces (36) and blastocysts (37). In our study, the viability of follicles exposed to Ficoll was assessed with calcein-am and ethidium homodimer-i, to test whether Ficoll treatment has a toxic effect on them. Analysis by vital fluorescent staining showed that 95.8% of follicles treated with Ficoll were totally viable Martinez-Madrid et al. Recovery of isolated human follicles Vol. 82, No. 6, December 2004

6 We have described, for the first time, a Ficoll density gradient method for the recovery of isolated human ovarian follicles. This technique allows us to maximize the recovery rate and minimize follicle manipulation time, while maintaining high follicular viability. Acknowledgments: B. Martinez-Madrid, V.M.D., Ph.D., and M.-M. Dolmans, M.D., contributed equally to this article. The authors thank D. Dufrane, M.D., for his technical assistance. References 1. Lass A, Silye R, Abrams DC, Krausz T, Hovatta O, Margara R, et al. Follicular density in ovarian biopsy of infertile women: a novel method to assess the ovarian reserve. Hum Reprod 1997;12: Gougeon A. Age-related changes of the population of human ovarian follicles: increase in the disappearance rate of nongrowing and earlygrowing follicles in ageing women. Biol Reprod 1994;50: Smitz JEJ, Cortvrindt RG. The earliest stages of folliculogenesis in vitro. Reproduction 2002;123: Qu J, Godin PA, Nisolle M, Donnez J. 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