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1 RBMOnline - Vol 16 No Reproductive BioMedicine Online; on web 19 March 2008 Article Development of antral follicles after xenografting of isolated small human preantral follicles Marie-Madeleine Dolmans received her medical degree from the Université Catholique de Louvain (UCL) in Brussels in 2000 and her PhD degree on cryopreservation and transplantation of human ovarian tissue in 2006, with Professor Jacques Donnez and Anne Van Langendonckt acting as promoters. Her research work focuses on human ovarian follicle isolation and xenotransplantation. Currently, she is a resident in the department of gynaecology at the UCL. Dr Marie-Madeleine Dolmans Marie-Madeleine Dolmans 1,3, Wu Yuan Yuan 1,2,3, Alessandra Camboni 1, Antoine Torre 1, Anne Van Langendonckt 1, Belen Martinez-Madrid 1, Jacques Donnez 1,4 1 Gynecology Research Unit, Université Catholique de Louvain, 1200 Brussels, Belgium; 2 Reproductive Medicine Centre, The 1st Affiliated Hospital of Sun Yat-sen University, Guangzhou, , China 3 M-M Dolmans and W Y Yuan are joint first authors. 4 Correspondence: Tel: ; Fax: ; donnez@gyne.ucl.ac.be Abstract Small human pre-antral follicles can be enzymatically isolated from the surrounding stroma, and are able to survive after 7 days of xenografting. The aim of the present study was to assess the developmental capacity of enzymatically isolated human follicles after long-term xenografting to severe combined immunodeficient (SCID) mice. Ovarian biopsies were obtained from three women years of age. Human ovarian tissue was enzymatically dissociated using collagenase or a purified collagenase blend to obtain isolated follicles that were xenografted to SCID mice for 5 months. Recombinant FSH was given to the mice for the last 2 weeks. Five months after xenografting, follicular morphology was assessed by histology, and follicular proliferation by Ki-67 immunohistochemistry. Four grafts containing a total of 84 follicles were recovered. This follicular population was composed of 11 primordial follicles, 38 primary follicles, 31 secondary follicles and four antral follicles. Ki-67 was found to stain granulosa cells in antral follicles intensively. The results demonstrate, for the first time, that isolated human follicles are able to survive after long-term xenografting, and can develop into antral follicles after FSH stimulation. Keywords: collagenase, fertility preservation, isolated human follicles, xenografting Introduction With all the recent advances in cancer treatments, many young cancer patients find themselves facing the prospect of losing their fertility after aggressive chemotherapy, especially with alkylating agents, and/ or radiotherapy (for review, see Donnez et al., 2006). Cryopreservation of ovarian primordial follicles has emerged as a potential option to restore fertility in these young women (Gosden, 2001; Demeestere et al., 2003; Isachenko et al., 2006). It has been shown that these follicles are very resistant to cryoinjury because they have a relatively inactive metabolic rate, contain only a few granulosa cells around the oocyte, and are small in size (Hovatta, 2005). In addition, their oocyte does not have a zona pellucida, metaphase spindle or peripheral cortical granules (Shaw et al., 2000a,b). Another advantage of cryopreserving primordial follicles is that they represent the vast majority of the follicular reserve in the ovary (Gougeon, 1986). Successful grafting of fresh and cryopreserved isolated follicles was previously reported in mice, producing live offspring (Gosden, 1990; Carroll and Gosden, 1993). Isolation of human follicles from their surrounding tissue is difficult, however, because of the density of human ovarian stroma and the relatively low number of follicles in adult ovarian tissue (Hovatta et al., 1997). Oktay et al. (1997) reported the first successful isolation of primordial follicles from human ovaries using a gentle enzymatic technique with collagenase, combined with manual dissection. However, complete (Abir et al., 1999, 2001) or partial (Hovatta et al., 1999; Abir et al., 2001) follicle isolation severely impairs Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 706 follicular viability in culture. After isolation, primordial and primary follicles degenerate within the first 24 h of culture (Abir et al., 1999, 2001). Studies have revealed alterations in the basal lamina of follicles after collagenase digestion (Eppig, 1994), and Hovatta et al. (1999) reported more markedly premature oocyte extrusions from follicles that had been partially isolated using collagenase. An optimized protocol using a purified collagenase blend (PCB) for digestion was therefore developed (Dolmans et al., 2006), yielding good quality isolated human follicles, which is an absolute prerequisite for the further successful processing of these follicles, either for culture or transplantation. In a previous study, it was demonstrated that PCB-isolated follicles survive and show granulosa cell proliferation 1 week after xenotransplantation, and are able to form stroma-like tissue (Dolmans et al., 2007). The aim of the present study was to assess the preservation of the developmental capacity of enzymatically isolated human follicles 5 months after xenografting to severe combined immunodeficient (SCID) mice, and their ability to reach the antral stage after gonadotrophin stimulation. Materials and methods Follicle isolation The use of human tissue for this study was approved by the Institutional Review Board of the Université Catholique de Louvain. Ovarian biopsies were taken from three women (26 29 years of age) after obtaining written informed consent. They were all undergoing laparoscopic surgery for benign gynaecological disease. Small preantral follicles were isolated according to a previously described procedure (Dolmans et al., 2006). Briefly, each biopsy was cut into two equal parts: one for PCB digestion and the other for collagenase digestion. The cortical ovarian tissue was cut with a tissue sectioner (McIIwain Tissue Chopper; Mickle Laboratory, Guildford, UK), and uniform-size pieces of mm were obtained. The fragments were digested either with 1 mg/ml collagenase type IA (Sigma, St Louis, MO, USA) for 60 min, or 0.04 mg/ml PCB (Liberase Blendzyme 3; Roche, Indianapolis, USA) for 75 min. Only fully isolated follicles were recovered using a 130 μm micropipette (Flexipet; Cook, Ireland). They were then rinsed three times in phosphatebuffered saline (PBS) supplemented with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO, USA) to remove the stromal cells. The follicular population was not characterized before grafting, as evaluation of the size of each follicle would have prolonged the time to grafting, altering follicle viability. In previous experiments, however, the proportion of primordial and growing follicles was evaluated after isolation and showed 20% of total follicles to be μm in size, 61% to be μm in size, 15% to be μm in size and only 0.01% to be over 100 μm in size (data not shown). Clot preparation After isolation, the follicles were embedded in plasma clots, serving as vehicles to facilitate subsequent grafting (Carroll and Gosden, 1993). Clot preparation was carried out according to a previously described procedure (Dolmans et al., 2007), wherein follicles were placed in 20 μl of fresh human autologous venous plasma and clotting was induced by adding 20 μl of 25 mmol/l CaCl 2 (1:40 dilution), followed by incubation at 37 C for 30 min. Transplantation to SCID mice and gonadotrophin stimulation The guidelines for animal welfare were approved by the Committee on Animal Research of the Université Catholique de Louvain. Plasma clots containing human isolated follicles were transplanted to SCID mice as previously described (Dolmans et al., 2007). Briefly, intraperitoneal injection of ketamine (75 mg/kg, Anesketin; Eurovet, Heusden-Zolder, Belgium) and medetomidine (1 mg/kg, Domitor; Pfizer, Cambridge, USA) was administered for anaesthesia, and buprenorphine (0.1 mg/kg, Temgesic; Schering Plough, Kenilworth, NJ, USA) for analgesia. A small slit was made bilaterally in the ovarian bursal membrane of three 8-week-old female SCID mice to graft the clots with the follicles. Then, the abdominal wall and skin were closed with 7/0 suture (Prolene). After surgery, anaesthesia was reversed by injection of atipamezole (1 mg/kg, Antisedan; Pfizer). The mice were kept for 5 months in sterile conditions, and after this period, they were stimulated with 7.5 IU of recombinant FSH (Puregon; Organon, Kloosterstraat, BH Oss, Netherlands) every other day for 2 weeks, in order to stimulate growth of the FSH responsive follicular pool (Vegetti and Alagna, 2006). The mice were then sacrificed by cervical dislocation. The ovarian bursae containing the grafts were recovered, fixed in 4% formaldehyde for 24 h and embedded in paraffin. Histological evaluation and immunohistochemistry The ovarian bursae containing the grafts were serially sectioned (5 μm thick sections) and every third slide was stained with haematoxylin eosin (Merck, Darmstadt, Germany). Follicles were counted and classified according to Smitz and Cortvrindt (2002) into primordial, primary, secondary, early antral and small antral follicles as follows: primordial follicles with one layer of flattened granulosa cells around the oocyte; primary follicles with one layer of cuboidal granulosa cells; secondary follicles with two or more layers of cuboidal granulosa cells; early antral follicles with a diameter of 200 μm and patchy fluid-filled spaces within the granulosa cells; small antral follicles with an antral cavity formed as a result of coalescence of the fluid-filled spaces. Follicular proliferation was measured by evaluating the presence of follicles with Ki-67-positive granulosa cells. Ki-67 is a nuclear antigen associated with cell proliferation (Hall and Levison, 1990) and is present throughout the active cell cycle (late G1, S, G2 and M phases), but absent in resting cells (G0). Follicles with at least one Ki-67-stained granulosa cell were considered to have initiated growth (Gougeon and Busso, 2000; Oktay et al., 2000). In order to assess the origin of the stroma-like cells in the

3 grafts, immunostaining was carried out with anti-human vimentin-specific antibodies. Vimentin is a cytoskeletal protein considered to be a marker of cells of mesenchymal origin. It is strongly expressed in ovarian stromal cells and granulosa cells from growing follicles (Czernobilsky et al., 1985). Immunohistochemistry was performed according to a previously described procedure (Dolmans et al., 2007). Paraffin sections were deparaffinized and rehydrated and endogenous peroxidase activity was blocked by incubating the sections with 0.3% H 2 O 2 (Merck) for 30 min at room temperature. Antigen retrieval was carried out in citrate buffer for 75 min at 98 C. Non-specific binding sites were blocked with normal goat serum (NGS) for 30 min. The sections were incubated with primary antibodies overnight: rabbit antihuman Ki-67 immunoglobulin (IgG) (4 C, 1:250 dilution; A0047 Dako, Glostrup, Denmark) or mouse monoclonal anti-human vimentin IgG1 (room temperature, 1:50 dilution; Clone V9, Novocastra, Newcastle, UK). A second incubation was performed for 60 min at room temperature with goat anti-rabbit IgG (Dako, K4003) or goat anti-mouse IgG (1:2 dilution, Dako, K4001). Diaminobenzidine (Dako) was used as a chromogen and nuclei were counterstained with haematoxylin. Human endometrium of proliferative status and grafted human ovarian tissue were used as positive controls for Ki-67 immunolabelling. The specificity of the antibodies was shown on human and murine ovarian tissue as illustrated in Figure 1. Results Isolation of follicles After collagenase and PCB digestion respectively, 105 and 88 follicles were isolated from patient 1, 59 and 53 follicles from patient 2, and 92 and 90 follicles from patient 3. Mouse A was grafted with clots containing 105 and 88 follicles in the right and left ovarian bursae respectively, mouse B with clots of 59 and 53 follicles, and mouse C with clots of 92 and 90 follicles. Histological evaluation The right and left bursae (containing both mouse ovary and human grafts) were removed after 22 weeks of grafting, including 2 weeks of FSH stimulation. Four of the six grafts were recovered, as the clots with collagenase-isolated follicles in mouse A and mouse C could not be found. Serial histological sections were analysed to determine follicle number and stage. Follicles were classified according to developmental stage. Primordial, primary, secondary and antral follicles were all found on histological analysis (Figures 2 and 3). From the four grafts, 84 follicles were recovered in total: 11 primordial, 38 primary, 31 secondary and four antral. As 290 follicles were initially grafted, this gives a recovery rate of 28.97%. The two grafts that could not be found were not included as their loss was due to technical problems. In mouse A, grafted with 88 PCB-isolated follicles on the right side, one antral follicle was found 5 months after grafting. In mouse B, 31 follicles (six primordial, 11 primary, 12 secondary and two antral) were recovered from the left ovarian bursa grafted with 59 collagenase-isolated follicles, and 13 follicles (four primary and nine secondary) from the right ovarian bursa grafted with 53 PCB-isolated follicles. In mouse C, grafted with 90 PCB-isolated follicles on the right side, 39 follicles were recovered (five primordial, 23 primary, 10 secondary and one antral). Morphologically, follicles at all stages looked well preserved. Primordial (Figure 2A) and primary (Figure 2B) follicles appeared as rounded structures, about μm in diameter, containing an intact oocyte with a large nucleus, spherical or slightly ovoid in shape. The oocyte was surrounded by a single and complete layer of granulosa cells, flattened or cuboidal in shape, in primordial and primary follicles respectively. Healthy-looking secondary follicles also appeared as rounded structures, μm in diameter (Figure 2B, 3D). Four antral follicles were recovered: three early antral follicles measuring μm, two visible in Figure 2B, and one small antral follicle measuring 600 μm (Figure 2C). The fluid-filled antral cavity of this small antral follicle was encapsulated by numerous layers of granulosa cells and some theca cells. The oocyte with its nucleolus was clearly visible, surrounded by an intact zona pellucida (Figure 2C). Histological analysis also demonstrated the presence of stroma-like cells in the isolated follicle grafts (Figure 2A C), which showed a similar architecture to human ovarian cortical tissue after grafting. They were further analysed by immunohistochemistry. Immunohistochemistry As shown in Figure 3, intensive Ki-67 staining of granulosa cells was observed in antral follicles (Figure 3B, D), and staining of a few granulosa cells was observed in primordial, primary and secondary follicles, proving that these follicles were still growing and had not arrested their development. This also indicates the human origin of these cells, as Ki-67 staining is human-specific (Figure 1). In order to assess the origin of the stroma-like cells in the grafts, immunostaining was carried out with anti-human vimentin antibodies, specific for human tissue. Staining was found to be strong in ungrafted human ovarian tissue, but not detectable in mouse ovarian tissue (Figure 1A). In control human tissue, vimentin immunoreactivity was detected in the cytoplasm of human stromal cells (Figure 1B). In isolated follicle grafts, the stroma-like tissue surrounding follicles stained positive for human vimentin (Figure 3E, F), confirming the human origin of the stromal cells, as previously shown in isolated follicles grafted for 1 week (Dolmans et al., 2007). 707

4 Figure 1. Positive and negative controls of Ki-67 and vimentin immunohistochemical staining. (A) Negative control of vimentin staining; murine ovarian section not stained by anti-human vimentin antibody. Scale bar 50 μm. (B) Positive control of vimentin staining; human ovarian section stained by anti-human vimentin antibody. Staining is strong in ovarian stromal cells. Scale bar 100 μm. (C) Negative control of Ki-67 staining; murine ovarian section not stained by anti-human Ki-67 antibody. Scale bar 100 μm. (D) Positive control of Ki-67 staining; granulosa cells from human follicles stained by antihuman Ki-67 antibody in ovarian tissue grafted for 1 week. Scale bar 50 μm. 708 Figure 2. Histological sections of isolated human follicles xenografted for 5 months to severe combined immunodeficient (SCID) mice. (A) Haematoxylin eosin-stained histological section of a primordial follicle and two follicles undergoing transition from the primordial to primary stage, surrounded by well developed stroma-like cells. The morphology of these follicles looks normal. Scale bar 50 μm. (B) Haematoxylin eosin-stained histological section of one early antral and one primary follicle both with visible oocyte. One other antral follicle and three secondary follicles are also present. The morphology of these follicles looks normal. The surrounding stroma also appears normal, although less compact than native stroma. Scale bar 100 μm. (C) Haematoxylin eosin-stained histological section of a 600 μm antral follicle. H = human ovarian graft, M = mouse ovary. The oocyte with a visible nucleolus is surrounded by an intact zona pellucida. The antral cavity is encapsulated by multiple layers of apparently normal granulosa cells and a few theca cells. Scale bar 200 μm.

5 Figure 3. Immunohistochemical staining of the grafts. H = human ovarian graft, M = mouse ovary. (A) and (B) Anti-human Ki-67 immunostaining of a small antral follicle. Intensive brown staining of the granulosa cells indicates proliferation. Note that the mouse follicles on the right are not stained. Scale bar 1 mm for (A) and 200 μm for (B). (C) and (D) Anti-human Ki-67 immunostaining of two early antral follicles and a secondary follicle. Proliferating cells can be identified by Ki-67 immunolabelling of their cell nuclei. Intensive Ki-67 staining of granulosa cells was observed in antral follicles, and staining of a few granulosa cells in secondary follicles. Scale bar 200 μm for (C) and 100 μm for (D). (E) and (F) Anti-human vimentin immunostaining of an isolated follicle xenograft. Staining is strong in stroma-like cells surrounding grafted human follicles. Scale bar 200 μm for (E) and 50 μm for (F). Discussion As far as is known, this is the first study to show that isolated human preantral follicles can survive and grow to the antral stage 5 months after xenografting. Antral follicles were previously obtained after xenotransplantation of human ovarian cortical tissue, but this involved tissue fragments (Gook et al., 2001, 2005; Van den Broecke et al., 2001). Previous attempts to assess the viability of isolated human follicles by in-vitro culture approaches have yielded poor results. Enzymatically isolated human primordial follicles did not grow well in in-vitro culture (Abir et al., 1999, 2001; Hovatta, 2004), and rapid disruption of the basement membrane was observed (Abir et al., 1997). Abir et al. (1999) cultured enzymatically isolated human follicles on collagen gel but the follicles only survived for 24 h. The viability of isolated human follicles after xenotransplantation was studied, and it was proven that they can survive for 1 week after grafting (Dolmans et al., 2007). In the present study, development of some antral follicles was observed 5 months after transplantation of isolated small preantral follicles to SCID mice. This demonstrates that human primordial and primary follicles retain their developmental potential after enzymatic isolation. The follicles were obviously growing, as demonstrated by intensive Ki-67 staining of granulosa cells. However, it is of crucial importance that the quality of the oocyte be further assessed, since isolation and transplantation procedures may affect its developmental competence. As pointed out by Hutt and Albertini (2007), the oocyte plays a key role in the regulation of growth and differentiation of the follicle and is highly sensitive to assisted reproductive technologies. Morphologically normal primordial follicles were also found, indicating that some follicles are maintained at a resting stage after long-term transplantation, as observed for ovarian tissue transplantation (Nisolle et al., 2000; Oktay et al., 2000; Gook et al., 2001). A global graft recovery rate of 4/6 was obtained, which may have been due to technical problems, as suggested by Carroll and Gosden (1993). It was observed that for PCB-isolated follicles, 3/3 grafts were recovered, compared with only 1/3 grafts for collagenase-isolated follicles. As discussed elsewhere, it is believed that the purified endotoxin-free enzyme preparation, Liberase, is a promising alternative to impure collagenase preparations for the reproducible isolation of intact primordial and primary follicles from human ovarian tissue (Dolmans et al., 2006). The results here show, however, that both Liberase and collagenase enzymatic protocols can preserve follicular morphology and developmental potential, 709

6 710 since well-developed follicles were found in both groups 5 months after xenotransplantation. As it is known exactly how many isolated follicles were grafted, it was possible to calculate the recovery rate, which was 28.97% (84 recovered follicles/290 grafted). This is in the same range as the follicle recovery rate described in the literature for grafted human tissue fragments. For example, Baird et al. (1999) reported that about 35% of primordial follicles survived transplantation when fresh sheep tissue was transplanted to SCID mice. This was reduced by another 7% when frozen thawed tissue was grafted. In a study on autografting of sheep ovarian cortex, Aubard et al. (1999) estimated that no more than 5% of primordial follicles survived the freezing grafting procedure. It was shown that the isolated follicle recovery rate to be at least comparable with that of transplanted fragments, as long as 5 months after grafting. The formation of a new, well-vascularized, stroma-like structure is likely to be a key factor in sustaining follicular growth in the in-vivo model described here. In studies by Carroll and Gosden (1993), the ability of dispersed ovarian follicular and stromal cells to reorganize into a functioning organ in vivo led to the restoration of fertility in mice. In a previous study (Dolmans et al., 2007), formation of a new ovary-like structure was observed 7 days after grafting of human follicles, although follicles were separated from stromal cells after digestion of the ovarian cortex. This was done by picking up the follicles with a pipette and washing them three times with fresh medium to remove remaining stromal cells. This intriguing phenomenon was also observed here, 5 months after grafting. The stroma-like cells of human origin, as proved by positive vimentin staining, may have originated either from contaminating stromal cells grafted together with the isolated follicles or from the involvement of granulosa cells with endothelial-like characteristics into a stromal cell type (Antczak and Van Blerkom, 2000). Studies are currently under way to characterize further the species-specific origin of these cells using the fluorescence in-situ hybridization technique. The stroma-like structure, although less compact than native stroma, resembles cortical tissue after grafting. Previous studies have indeed shown the stroma to exhibit poor cellularity after autotransplantation (Camboni et al., 2007) or xenotransplantation (Nisolle et al., 2000) of cryopreserved human ovarian tissue. Unexpectedly, the stroma-like tissue observed after isolated follicle grafting was able to support the development of follicles, even up to the antral stage, as suggested by the presence of antral follicles and theca cells surrounding the 600 μm antral follicle. In-vivo xenografting of isolated ovarian follicles is thus a promising approach to study the interaction between follicles and stromal cells. Indeed, isolated primordial follicles do not grow beyond the secondary stage in culture (Hovatta et al., 1999), and organ culture renders follicle-stroma interaction study almost impossible (Cortvrindt and Smith, 2001). In conclusion, the present study demonstrates, for the first time, preservation of the developmental potential of enzymatically isolated small human preantral follicles 5 months after xenografting. This may provide a model to investigate the cross-reaction between stromal and follicular cells. It also opens up new perspectives for pathologies at risk of cancer cell transmission, as an alternative to ovarian cortical transplantation, once the safety of the procedure has been fully assessed. Acknowledgements The present 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, the Foundation Martine Midi and the CNGOF (to A Torre) and donations from Baron Albert Frère and the Vicomte Philippe de Spoelberch. The authors thank Mira Hryniuk, and Christiani Andrade Amorim for reviewing the manuscript. References Abir R, Fisch B, Nitke S et al Morphological study of fully and partially isolated early human follicles. Fertility and Sterility 75, Abir R, Roizman P, Fisch B et al Pilot study of isolated early human follicles cultured in collagen gels for 24 h. 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