Introduction. Christiani A. Amorim, Anne Van Langendonckt, Anu David, Marie-Madeleine Dolmans, and Jacques Donnez 1. ORIGINAL ARTICLE Embryology

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1 Human Reproduction, Vol.24, No.1 pp , 2009 Advanced Access publication on September 23, 2008 doi: /humrep/den343 ORIGINAL ARTICLE Embryology Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix Christiani A. Amorim, Anne Van Langendonckt, Anu David, Marie-Madeleine Dolmans, and Jacques Donnez 1 Department of Gynecology, Université Catholique de Louvain, Avenue Emmanuel Mounier 52, bte 5247, 1200 Brussels, Belgium 1 Correspondence address. Tel: þ ; Fax: þ ; jacques.donnez@uclouvain.be table of contents... Introduction Materials and Methods Results Discussion Funding Authors roles Acknowledgement References background: Ovarian tissue cryopreservation is a promising technique to safeguard fertility in cancer patients. However, in some types of cancer, there is a risk of transmitting malignant cells present in the cryopreserved tissue. To avoid such a risk, pre-antral follicles could be isolated from ovarian tissue and grown in vitro. On the basis of this assumption, the aim of our study was to investigate in vitro survival and growth of pre-antral follicles after cryopreservation of ovarian tissue and follicular isolation, followed by encapsulation in alginate beads. methods: Ovarian biopsies from four patients were frozen and thawed. Pre-antral follicles were then isolated and embedded in an alginate matrix before in vitro culture for 7 days. results: Small pre-antral follicles ( mm) from frozen thawed tissue can survive and develop after enzymatic isolation and in vitro culture. A total of 159 follicles were incubated in a three-dimensional system (alginate hydrogel) and, after 7 days, all of them showed an increase in size (final size mm). The survival rate of the follicles was 90% (oocyte and all granulosa cells viable). conclusion: Our preliminary results indicate that alginate hydrogels may be a suitable system for in vitro culture of isolated human preantral follicles. However, more studies are required to establish whether follicular morphology and functionality can be maintained using this matrix. Key words: alginate / cryopreservation / follicle / in vitro culture / ovary Introduction In recent years, cryopreservation of pre-antral follicles in humans has emerged as a promising alternative to safeguard fertility in women at risk of premature ovarian failure, notably cancer patients having to undergo chemo/radiotherapy (Nisolle et al., 2000; Kim et al., 2001; Oktay, 2001; Gosden et al., 2002; Falcone et al., 2004; Martinez- Madrid et al., 2004a, 2007; Donnez et al., 2005, 2006a; Meirow et al., 2007a). Restoration of ovarian function with hormone production and follicular growth has been observed after transplantation of frozen thawed ovarian tissue (Oktay et al., 2000, 2004; Radford et al., 2001; Tryde Schmidt et al., 2004; Schmidt et al., 2005; & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 In vitro culture of isolated human pre-antral follicles 93 Rosendahl et al., 2006; Donnez et al., 2008), and a number of pregnancies (Donnez et al., 2004; Meirow et al., 2005, 2007b; Demeestere et al., 2006, 2007; Andersen et al., 2008) have been reported to date. Despite efforts to develop grafting procedures, a large percentage of grafted follicles are lost as a result of post-grafting ischemia and reperfusion-induced damage (Newton et al., 1996; Aubard et al., 1999; Donnez et al., 2006b). In addition, some studies have demonstrated a risk of transmitting malignant cells present in cryopreserved tissue (Shaw et al., 1996; Meirow et al., 1998, 2008). In order to avoid this risk, isolated follicles could be grown in vitro. This procedure would also allow the assessment of follicular quality after cryopreservation and direct monitoring of follicles during the culture period (Abir et al., 1999), which could also help to elucidate early folliculogenesis in humans and other mammal species. However, in vitro culture of isolated small pre-antral follicles from human ovaries has not yet yielded satisfactory results and only a few studies have been performed (Roy and Treacy, 1993; Oktay et al., 1997; Abir et al., 1999, 2001). Small pre-antral follicles (30 50 mm) from fresh and frozen thawed ovarian tissue have been shown to survive in vitro for up to 24 h (Abir et al., 1999, 2001) in collagen gel. The authors reported that isolated follicles could only grow in, but not on, a supporting matrix. This may possibly have been due to the preservation of intercellular interactions between granulosa cells (GCs) and the oocyte in this 3D system, which also provides optimal support for these fragile isolated follicles, similar to the ovary itself. Many aspects of oocyte growth and development are regulated by such interactions with adjacent GCs (Murray et al., 1998; Reynaud et al., 2000) and they can occur only within a 3D structure. This is probably why most studies are performed with pre-antral follicles enclosed in ovarian tissue (Hovatta et al., 1997, 1999; Carlsson et al., 2006). Recently, another 3D system using alginate hydrogels has been successfully applied to in vitro culture of isolated mouse (Pangas et al., 2003; Kreeger et al., 2005, 2006; Xu et al., 2006a,b; West et al., 2007) and rat (Heise et al., 2005) follicles. West et al. (2007) showed that this matrix is suitable for in vitro culture of isolated follicles due to its gentle gelling properties and biochemical characteristics. Its structure allows diffusion of hormones and other proteins that are essential for follicular development. Indeed, alginate is one of the most widely used biomaterials for microencapsulation because of its biocompatibility, high affinity to water and ability to form hydrogels under very mild conditions (Smidsrød and Skjåk-Braek, 1990; Draget et al., 1997; Amsden and Turner, 1999). The aim of this study was therefore to investigate in vitro survival of pre-antral follicles after cryopreservation of human ovarian tissue and follicular isolation, followed by encapsulation in alginate hydrogel in a basic culture system. Ovarian tissue freezing and thawing Freezing of the ovarian tissue strips was performed according to the method described by Gosden et al. (1994) with some modifications. The tissue was first suspended in 800 ml of minimal essential medium (MEM)þGlutamax TM (MEM, Gibco, Carlsbad, CA, USA; Glutamax is a more stable form of L-glutamine) in a cryovial. This medium was then replaced with the same amount of cryoprotective solution containing 10% DMSO (Sigma, St Louis, MO, USA) and 2% human serum albumin (Sanquin, Amsterdam, the Netherlands) in Leibowitz-15 (Gibco) at 08C. The cryovials were cooled in a programmable freezer with the following program: (i) cooled from 08C to 288C at 228C/min; (ii) seeded manually by touching the cryovials with forceps pre-chilled in liquid nitrogen; (iii) cooled to 2408C at 20.38C/min and transferred to liquid nitrogen (21968C) for storage. For thawing and cryoprotectant removal, the cryovials were exposed to room temperature for 2 min and immersed in a water bath at 378C until the ice completely melted. To remove the cryoprotective solution, the ovarian tissue was transferred from the cryovials to Petri dishes containing MEM-Glutamax, where it was washed three times (5 min per bath) before isolation and/or in vitro culture. After cryoprotectant removal, one fragment was cut from each biopsy and fixed in Bouin s solution for control after cryopreservation (Control 2). Ovarian follicle isolation The protocol previously described by Dolmans et al. (2006) was used to isolate small pre-antral follicles. Briefly, the cortical portion of the ovary was placed in a tissue chopper, adjusted to 0.5 mm. Ovarian fragments obtained were transferred to 50 ml conical flasks containing 10 ml of Dulbecco s phosphate-buffered saline (D-PBS; BioWhittaker, Verviers, Belgium) supplemented with 1 mg/ml collagenase type IA (Sigma), and incubated in a water bath at 378C for 60 min with gentle agitation. The ovarian digest was periodically (every 15 min) shaken with a pipette to mechanically disrupt the digested tissue. Digestion was completed by the addition of an equal volume of D-PBS at 48C supplemented with 10% fetal bovine serum (FBS; Gibco). Thereafter, the resulting suspension was centrifuged at 50g for 10 min at 48C. The supernatant was discarded and the pellet was transferred to Petri dishes and investigated for follicles under a stereomicroscope (Leica, Van Hopplynus Instruments, Brussels, Belgium). The follicles were picked up using a 135-mm-diameter stripper tip (Mid-Atlantic Diagnostics, Inc., Mount Laurel, NJ, USA) and washed three times in D-PBS supplemented with 10% FBS at 48C in order to avoid introduction of stromal cells into the alginate matrix. Follicle diameter was measured from the basement membrane using an ocular micrometer scale. Two to five follicles from each patient were subsequently processed for live/dead assays in order to evaluate follicular viability after isolation (Control 3). The remaining follicles were embedded in an alginate matrix (4 8 follicles/group). Materials and Methods Collection of ovarian tissue The use of human tissue for this study was approved by the Institutional Review Board of the Université Catholique de Louvain. After obtaining written informed consent, ovarian biopsies were taken from four women between 25 and 35 years of age. A small fragment from each biopsy was fixed in Bouin s solution for fresh controls (Control 1). The remaining ovarian tissue was cut into strips for cryopreservation. Calcium alginate embedding A 1% (w/v) solution of sodium alginate (Sigma) was prepared (Heise et al., 2005) and autoclaved. The isolated follicles were transferred with the stripper tips to droplets (20 ml) of alginate solution. To form the beads, the droplets were slowly released into a small beaker containing a solution of CaCl 2 (0.1 M). The beads containing the follicles were removed from the beaker and then washed three times in MEM-Glutamax. One bead from each patient was processed for live/dead assays in order to evaluate follicular viability after embedding in an alginate matrix (Control 4), and the rest of the beads were assigned for in vitro culture.

3 94 Amorim et al. In vitro culture of isolated follicles For every patient, three alginate beads (4 8 follicles/bead) were cultured per well. Since this is the first study to investigate how an alginate matrix affects isolated human pre-antral follicles, we chose a basic culture system. The in vitro culture procedure used is a modification of that described by Carlsson et al. (2006) for ovarian tissue culture: follicle incubation was performed in a four-well multidish (Nunc, Roskilde, Denmark) fitted with Millicell-CM inserts (12 mm in diameter, 0.4 ml pore size; Millipore, Bedford, MA, USA). The inserts were precoated with 100 ml of growth factor-reduced extracellular matrix (GFR Matrigel TM, BD, San Jose, CA, USA), pre-diluted 1:3 with MEM-Glutamax. Culture medium consisted of MEM-Glutamax supplemented with 10% FBS, 0.47 mm pyruvic acid (Sigma), 50 IU/ml penicillin (Sigma) and 50 mg/ml streptomycin sulfate (Sigma). Insulin, transferrin and selenium (ITS-G; Gibco) were also added to the medium at concentrations similar to those reported previously: 10 mg insulin/ml, 5.5 mg transferrin/ml and 6.7 ng sodium selenite/ml (Wright et al., 1999; Carlsson et al., 2006). It is important to point out that selenium is largely used as an antioxidant in culture media (Rose et al., 1999; Haidari et al., 2006). Culture medium was added to each well: 150 ml was pipetted into the inserts and 400 ml into the well outside the inserts. The isolated follicles in the alginate beads were cultured for 7 days at 378C in a 95% air and 5% CO 2 humidified environment. Every second day, 150 ml of culture medium was removed and replaced with fresh medium in the inserts. After 7 days of in vitro culture, the diameter of all the follicles was measured from the basement membrane using an ocular micrometer scale and all the beads were processed for live/dead assays in order to evaluate follicular viability. For a better understanding of the study, Fig. 1 shows the entire experimental design. Follicle viability Uncultured control groups included follicles immediately after isolation (Control 3) and isolated follicles immediately after embedding in the alginate matrix (Control 4). Follicles were incubated in 100 ml of D-PBS containing 2 mmol/l calcein-am and 5 mmol/l ethidium homodimer-i (Molecular Probes, Leyden, the Netherlands) for 45 min at 378C in the dark (Cortvrindt and Smitz, 2001). Non-fluorescent cell-permeant calcein-am enters cells and is cleaved by esterases in live cells, producing calcein, which is well retained within live cells, generating intense and uniform green fluorescence. Ethidium homodimer-i enters cells with damaged membranes and then binds to DNA with high affinity, resulting in a 40-fold enhancement of fluorescence, producing bright red fluorescence in dead cells. After exposure to fluorescent dyes, follicles were washed in D-PBS and observed under an inverted fluorescence microscope (Leica). Green fluorescence was visualized in live cells (ex/em 495/515 nm) and red fluorescence in dead cells (ex/em 495/635 nm), using two different filters (Maltaris et al., 2007; Santos et al., 2007; Aerts et al., 2008). The follicles were classified into four categories depending on the percentage of dead GCs live follicles: follicles with the oocyte and all GCs viable; minimally damaged follicles: follicles with,10% dead GCs; moderately damaged follicles: follicles with 10 50% dead GCs; dead follicles: follicles with both the oocyte and all GCs dead (Martinez-Madrid et al., 2004b). Histological analysis of ovarian tissue To evaluate follicular morphology in ovarian tissue (Controls 1 and 2), two small fragments were removed from each biopsy before (Control 1) and after (Control 2) cryopreservation. The fragments were fixed in Bouin s solution for 6 h to allow histological analysis of the follicles. The fragments were then dehydrated stepwise in increasing concentrations of ethanol, embedded in paraffin wax, cut into 5-mm-thick sections and stained with hematoxylin eosin. Evaluation of follicular quality was based on the integrity of the basement membrane, cellular density, presence or absence of pyknotic bodies and integrity of the oocyte. On the basis of these criteria, pre-antral follicles were classified as morphologically normal or degenerated (Amorim et al., 2000). Figure 1 Experimental design for the study of in vitro culture (IVC) of isolated human pre-antral follicles.

4 In vitro culture of isolated human pre-antral follicles 95 Statistical analysis The mean diameter of the pre-antral follicles was compared before and after 7 days of culture, and with the controls (1 4), using the Mann Whitney test. Comparisons between percentages of viable follicles found after in vitro culture and in control samples were analyzed by the x 2 test. P, 0.05 was considered statistically significant. Results Before in vitro culture, pre-antral follicles from biopsies were subjected to various procedures: cryopreservation, follicle isolation and embedding in alginate beads. All these steps could potentially damage the follicles (Amorim et al., 2003, 2006; Dolmans et al., 2006). We therefore added controls to each step to assess follicular quality. In a comparison of all controls, we did not observe any alterations in the morphology (Controls 1 and 2) or viability (Controls 3 and 4) of follicles (Fig. 2). After cryopreservation (Control 2), small pre-antral follicles had a similar morphology to follicles from fresh ovarian tissue (Control 1): GCs were well organized, without any signs of pyknotic bodies, and the oocyte was slightly stained, without any signs of degeneration or retraction. Follicular viability was not affected by follicle isolation (Control 3) or alginate embedding (Control 4): all 34 follicles from Controls 3 and 4 were found to be alive, with both the oocyte and all the GCs viable. A total of 159 follicles from four patients were cultured and all of them were found to have increased in size after 7 days, when culture was discontinued. The initial mean diameter was mm but, by the end of in vitro culture, follicular size had significantly increased to mm. Table I shows the number and size of follicles before and after in vitro culture, as well as that of follicles from all controls. No significant difference was observed in the initial size of follicles between the controls and those which were then cultured. Follicular viability, analyzed by vital fluorescent staining (calcein-am and ethidium homodimer-i), was assessed in all follicles recovered from each patient. Follicles were classified into four categories depending on the percentage of dead GCs found, according to the classification previously described. In Controls 3 and 4, all follicles were totally viable, whereas after 7 days of in vitro culture, 90% of isolated follicles were totally viable, 7% were minimally damaged and 3% were moderately damaged. No oocytes or not more than 50% of GCs were ever found to be dead. No difference in viability was observed Figure 2 Small pre-antral follicles in control groups. Scale bar: 10 mm. (A) Follicle in fresh tissue (Control 1) (35 mm). (B) Follicle in frozen thawed tissue (Control 2) (45 mm). (C) Isolated follicle stained with calcein-am (Control 3) (40 mm). (D) Isolated follicle after embedding in alginate hydrogel (Control 4) and staining (35 mm).

5 96 Amorim et al. Table I Pre-antral follicles (mean + SD) from controls and before and after in vitro culture Control 1 Control 2 Control 3 Control 4 Isolated follicles for in vitro culture... Number of follicles Initial size (mm) Final size (mm) * *Significantly higher than the initial size (P, 0.01). Control 1, fresh ovarian tissue; Control 2, frozen thawed ovarian tissue; Control 3, isolated pre-antral follicles; Control 4, isolated pre-antral follicles after embedding in an alginate matrix. Figure 3 Isolated pre-antral follicles before (upper line 45 mm) and after (lower line 60 mm) 7 days of in vitro culture. Scale bar: 15 mm. The second and third columns show follicular viability assessment by calcein-am and ethidium homodimer-i (Leica inverted fluorescence microscope). between Controls 2 and 3. Figure 3 shows follicular viability assessment by calcein-am and ethidium homodimer-i in isolated pre-antral follicles before and after 7 days of in vitro culture. Discussion To our knowledge, this is the first study to show that small human pre-antral follicles from frozen thawed ovarian tissue can survive after isolation and in vitro culture for 7 days. A total of 159 follicles were cultured and, after 1 week, all of them had increased in size (final size mm) and 90% remained alive (with both the oocyte and all GCs viable), proving that an alginate matrix can be an appropriate support for in vitro culture of isolated human pre-antral follicles. It should be pointed out that, although these follicles were mostly viable according to the fluorescent staining, they were not processed and examined for histology owing to technical difficulties. All studies that describe the method to culture isolated human preantral follicles in vitro (Roy and Treacy, 1993; Abir et al., 1999, 2001) use a 3D system, which mimics ovarian conditions more effectively, because many cellular processes in organogenesis occur exclusively in 3D (Xu et al., 2006a). Ovarian folliculogenesis is a complex developmental process that is regulated by various endocrine, paracrine and autocrine factors (Thomas et al., 2003; Demeestere et al., 2005), as well as intraovarian cell cell and cell matrix connections (Rodgers et al., 2003; Irving-Rodgers and Rodgers, 2005). Follicles need to be cultured under conditions that prevent dissociation of GCs from the oocyte, as contact between the two cell types is necessary for oocyte growth and maturation (Murray and Spears, 2000). In order to attain in vitro growth of small pre-antral follicles until later antral stages, it is furthermore important to prevent breakdown of the metabolic link between GCs and the oocyte, since this could lead to uncoordinated growth and differentiation of somatic and germ cells (Picton and Gosden, 2000). In this respect, alginate has proved to be a suitable matrix and, because it is firmer than collagen gel (Abir et al., 2001), it may well be a better support for the growth of isolated small human pre-antral follicles. An alginate matrix maintains integral

6 In vitro culture of isolated human pre-antral follicles 97 connections between the oocyte and somatic cells, as shown by Heise et al. (2005), with connexin 43 expression at similar levels in both encapsulated and in vivo rat pre-antral follicles. It also allows diffusion of proteins, such as FSH, necessary for follicular development (Heise et al., 2005). Moreover, it permits follicular growth and development, maintaining follicular morphology and function (Pangas et al., 2003; Kreeger et al., 2005, 2006; Xu et al., 2006a,b; West et al., 2007). Preservation of essential interactions between GCs and the oocyte may also be affected by the enzymatic procedure applied to isolate pre-antral follicles. In addition, membrane breakdown, as well as destruction of other intrafollicular components during enzymatic isolation from the stroma, represent major problems for further development of pre-antral follicles in vitro (Gosden et al., 2002). This erosion causes GCs to detach from the oocyte, which is an irreversible process that produces oocytes incapable of further development, though they may remain viable for several days (Bachvarova et al., 1980). Previous studies (Abir et al., 1999, 2001) on in vitro culture of small pre-antral follicles have demonstrated that fully isolated follicles are able to survive and grow for up to 24 h. The authors hypothesized that follicular death after such a short period of time may have been due to disruption of the basement membrane caused by the enzymatic treatment used to isolate the follicles (Abir et al., 2001). In one of the studies, Abir et al. (2001) compared fully and partially isolated follicles in order to assess whether the maintenance of interactions between follicles and stromal cells could improve in vitro culture results. The authors found, however, that only fully isolated follicles were able to grow in culture. Most of the partially isolated follicles detached from the collagen gels and died. Hovatta et al. (1999) also reported that partial isolation of pre-antral follicles is not beneficial in in vitro culture. After 2 weeks of culture, oocytes were shown to extrude from partially isolated follicles. Regarding the increase in follicular size observed after in vitro culture in this study, we cannot categorically state that this was due to follicular development, as more studies are needed to confirm the growth of preantral follicles. However, we cannot entirely exclude this possibility either, since in 37 follicles we could clearly see more than one layer of GCs around the oocyte after 7 days of in vitro culture (Fig. 3). Another explanation for the increase in follicular size could be swelling of the follicles due to fluid absorption during in vitro culture. Abir et al. (1999, 2001) encountered rapid follicular growth over 24 h, with a growth rate of 31% in their first study (Abir et al., 1999) and 90% in the second (Abir et al., 2001). Although Roy and Treacy (1993) reported growth of isolated pre-antral follicles ( mm) after 5 days of in vitro culture, they did not mention the growth rate. Due to the lack of information available on in vitro growth of isolated small human preantral follicles, we cannot know what kind of growth rate to expect from human follicles cultured in vitro. In conclusion, our preliminary results on alginate hydrogels indicate that they may be a suitable substance for in vitro culture of isolated human pre-antral follicles for a period of 7 days. However, further studies are required to establish whether the morphology and functionality of isolated human follicles can be maintained using this matrix. Funding This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant no ), the Fondation St Luc, the Foundation Against Cancer, and donations from Baron Albert Frère and Viscount Philippe de Spoelberch. Authors roles Study concept and design, statistical and histological analyses, manuscript preparation: C.A.A.; data collection: C.A.A. and A.D.; manuscript revision: A.V.L., M.-M.D. and J.D.; supervision of the laboratory components of the study: A.V.L.; biopsy collection: M.-M. D. and J.D.; final approval of the version to be published: J.D. Acknowledgement The authors thank Mira Hryniuk for reviewing the manuscript. References Abir R, Roizman P, Fisch B, Nitke S, Okon E, Orvieto R, Ben Rafael Z. Pilot study of isolated early human follicles cultured in collagen gels for 24 hours. Hum Reprod 1999;14: Abir R, Fisch B, Nitke S, Okon E, Raz A, Ben Rafael Z. Morphological study of fully and partially isolated early human follicles. Fertil Steril 2001;75: Aerts JM, Martinez-Madrid B, Flothmann K, De Clecq JB, Van Alest S, Bols PE. Quantification and viability assessment of isolated bovine primordial and primary ovarian follicles retrieved through a standardized biopsy pick-up procedure. Reprod Domest Anim 2008;43: Amorim CA, Lucci CM, Rodrigues AP, Carvalho FC, Figueiredo JR, Rondina D, Cecchi R, Giorgetti A, Martini A, Gonçalves PB. Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries. Theriogenology 2000;53: Amorim CA, Gonçalves PBD, Figueiredo JR. Cryopreservation of oocytes from pre-antral follicles. Hum Reprod Update 2003;9: Amorim CA, Rondina D, Lucci CM, Gonçalves PBD, Figueiredo JR, Giorgetti A. Permeability of ovine primordial follicles to different cryoprotectants. Fertil Steril 2006;85: Amsden B, Turner N. Diffusion characteristics of calcium alginate gels. Biotechnol Bioeng 1999;65: Andersen CY, Rosendahl M, Byskov AG, Loft A, Ottosen C, Dueholm M, Schmidt KL, Nyboe Andersen A, Ernst E. Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue. Hum Reprod 2008, in press. Aubard Y, Piver P, Cogni Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen thawed ovarian cortex in sheep. Hum Reprod 1999;14: Bachvarova R, Baran MM, Tejblum A. Development of naked growing mouse oocytes in vitro. J Exp Zool 1980;211: Carlsson IB, Scott JE, Visser JA, Ritvos O, Themmen AP, Hovatta O. Anti-Müllerian hormone inhibits initiation of growth of human primordial ovarian follicles in vitro. Hum Reprod 2006;21: Cortvrindt RG, Smitz JE. Fluorescent probes allow rapid and precise recording of follicle density and staging in human ovarian cortical biopsy samples. Fertil Steril 2001;75: Demeestere I, Centner J, Gervy C, Englert Y, Dalbaere A. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction 2005;130: Demeestere I, Simon P, Buxant F, Robin V, Fernandez SA, Centner J, Delbaere A, Englert Y. Ovarian function and spontaneous pregnancy

7 98 Amorim et al. after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: case report. Hum Reprod 2006;21: Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin s disease. Oncologist 2007;12: Dolmans MM, Michaux N, Camboni A, Martinez-Madrid B, Van Langendonckt A, Nottola SA, Donnez J. Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. Hum Reprod 2006;21: Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, Van Langendonckt A. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364: Donnez J, Dolmans MM, Martinez-Madrid B, Demylle D, Van Langendonckt A. The role of cryopreservation for women prior to treatment of malignancy. Curr Opin Obstet Gynecol 2005;17: Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, Van Langendonckt A. Restoration of ovarian function after orthotopic (intraovarian and periovarian) transplantation of cryopreserved ovarian tissue in a woman treated by bone marrow transplantation for sickle cell anaemia: case report. Hum Reprod 2006a;21: Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D, Dolmans MM. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update 2006b;12: Donnez J, Squifflet J, Van Eyck AS, Demylle D, Jadoul P, Van Langendonckt A, Dolmans MM. Restoration of ovarian function in orthotopically transplanted cryopreserved ovarian tissue: a pilot experience. Reprod Biomed Online 2008;16: Draget KI, Skjåk-Braek G, Smidsrød O. Alginate based new materials. Int J Biol Macromol 1997;21: Falcone T, Attaran M, Bedaiwy MA, Goldberg JM. Ovarian function preservation in the cancer patient. Fertil Steril 2004;81: Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at 2196 degrees C. Hum Reprod 1994;9: Gosden RG, Mullan J, Picton HM, Yin H, Tan SL. Current perspective on primordial follicle cryopreservation and culture for reproductive medicine. Hum Reprod Update 2002;8: Haidari K, Salehnia M, Valoujerdi MR. The effects of different concentrations of leukemia inhibitory factor on the development if isolated preantral follicles from fresh and vitrified mouse ovaries. Iran Biomed J 2006;10: Heise M, Koepsel R, Russell AJ, McGee FA. Calcium alginate microencapsulation of ovarian follicles impacts FSH delivery and follicle morphology. Reprod Biol Endocrinol 2005;3:47. Hovatta O, Silye R, Abir R, Krausz T, Winston RML. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture. Hum Reprod 1997;12: Hovatta O, Wright C, Krausz T, Hardy K, Winston RM. Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum Reprod 1999;14: Irving-Rodgers HF, Rodgers RJ. Extracellular matrix in ovarian follicular development and disease. Cell Tissue Res 2005;322: Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 2001;75: Kreeger PK, Fernandes NN, Woodruff TK, Shea LD. Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose. Biol Reprod 2005;73: Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials 2006;27: Maltaris T, Beckmann MW, Binder H, Mueller A, Hoffmann I, Koelbl H, Dittrich R. The effect of a GnRH agonist on cryopreserved human ovarian grafts in severe combined immunodeficient mice. Reproduction 2007;133: Martinez-Madrid B, Dolmans MM, Van Langendonckt A, Defrère S, Donnez J. Freeze-thawing intact human ovary with its vascular pedicle with a passive cooling device. Fertil Steril 2004a;82: Martinez-Madrid B, Dolmans MM, Langendonckt AV, Defrère S, Van Eyck AS, Donnez J. Ficoll density gradient method for recovery of isolated human ovarian primordial follicles. Fertil Steril 2004b;82: Martinez-Madrid B, Camboni A, Dolmans MM, Nottola S, Van Langendonckt A, Donnez J. Apoptosis and ultrastructural assessment after cryopreservation of whole human ovaries with their vascular pedicle. Fertil Steril 2007;87: Meirow D, Ben Yehuda D, Prus D, Poliack A, Schenker JG, Rachmilewitz EA, Lewin A. Ovarian tissue banking in patients with Hodgkin s disease: is it safe? Fertil Steril 1998;69: Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, Schiff E, Dor J. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005; 353: Meirow D, Baum M, Yaron R, Levron J, Hardan I, Schiff E, Nagler A, Yehuda DB, Raanani H, Hourvitz A et al. Ovarian tissue cryopreservation in hematologic malignancy: ten years experience. Leuk Lymphoma 2007a;48: Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Yemini Z, Dor J. Monitoring the ovaries after autotransplantation of cryopreserved ovarian tissue: endocrine studies, in vitro fertilization cycles, and live birth. Fertil Steril 2007b;87:418.e7 418.e15. Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra anani H, Slyusarevsky E, Amariglio N, Schiff E, Rechavi G et al. Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients. Hum Reprod 2008;23: Murray AA, Gosden RG, Allison V, Spears N. Effect of androgens on the development of mouse follicles growing in vitro. J Reprod Fertil 1998;113: Murray A, Spears N. Follicular development in vitro. Semin Reprod Med 2000;18: Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996;11: Nisolle M, Casanas-Roux F, Qu J, Motta P, Donnez J. Histologic and ultrastructural evaluation of fresh and frozen thawed human ovarian xenografts in nude mice. Fertil Steril 2000;74: Oktay K. Ovarian tissue cryopreservation and transplantation: preliminary findings and implications for cancer patients. Hum Reprod 2001;7: Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden RG. Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 1997;67: Oktay K, Newton H, Gosden RG. Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice. Fertil Steril 2000;73: Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, Opsahl M, Rosenwaks Z. Embryo development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;363:

8 In vitro culture of isolated human pre-antral follicles 99 Pangas SA, Saudye H, Shea LD, Woodruff TK. Novel approach for the three-dimensional culture of granulosa cell oocyte complexes. Tissue Eng 2003;9: Picton HM, Gosden RG. In vitro growth of human primordial follicles from frozen-banked ovarian tissue. Mol Cell Endocrinol 2000;166: Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, Watson AJ, Clayton JA, Harris M, Gosden RG et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin s lymphoma. Lancet 2001; 357: Reynaud K, Cortvrindt R, Smitz J, Driancourt MA. Effects of kit ligand and anti-kit antibody on growth of cultured mouse preantral follicles. Mol Reprod Dev 2000;56: Rodgers RJ, Irving-Rodgers HF, Russell DL. Extracellular matrix of the developing ovarian follicle. Reproduction 2003;126: Rose UM, Hanssen RG, Kloosterboer HJ. Development and characterization of an in vitro ovulation model using mouse ovarian follicles. Biol Reprod 1999;61: Rosendahl M, Loft A, Byskov AG, Ziebe S, Schmidt KT, Andersen AN, Ottosen C, Andersen CY. Biochemical pregnancy after fertilization of an oocyte aspired from a heterotopic autotransplant of cryopreserved ovarian tissue: case report. Hum Reprod 2006;21: Roy SK, Treacy BJ. Isolation and long-term culture of human preantral follicles. Fertil Steril 1993;59: Santos RR, Tharasanit T, Van Haeften T, Figueiredo JR, Silva JR, Van den Hurk R. Vitrification of goat preantal follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification method. Cell Tissue Res 2007;327: Schmidt KL, Andersen CY, Loft A, Byskov AG, Ernst E, Andersen AN. Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantation. Hum Reprod 2005;20: Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit cancer to graft recipients. Hum Reprod 1996;11: Smidsrød O, Skjåk-Braek G. Alginate as immobilization matrix for cells. Trends Biotechnol 1990;8: Thomas FH, Walters KA, Telfer EE. How to make a good oocyte: an update on in-vitro models to study follicle regulation. Hum Reprod Update 2003;9: Tryde Schmidt KL, Yding Andersen C, Starup J, Loft A, Byskov AG, Nyboe Andersen A. Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancer follicular growth, steroid production and oocyte retrieval. Reprod Biomed Online 2004;8: West ER, Xu M, Woodruff TK, Shea LD. Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials 2007;28: Wright CS, Hovatta O, Margara R, Trew G, Winston RM, Franks S, Hardy K. Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles. Hum Reprod 1999;14: Xu M, Kreeger PK, Shea LD, Woodruff TK. Tissue-engineered follicles produce live, fertile offspring. Tissue Eng 2006a;12: Xu M, West E, Shea LD, Woodruff TK. Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development. Biol Reprod 2006b;75: Submitted on March 3, 2008; resubmitted on August 20, 2008; accepted on August 26, 2008