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Home , Cryopreservation, Ovarian cortex, Vaginal transplantation

ANTICANCER RESEARCH 26: 4171-4176 (2006)

Xenotransplantation of Human Ovarian Pieces in -stimulated SCID Mice: The Effect of Ovariectomy

THEODOROS MALTARIS1, HEINZ KOELBL1, FRANZ FISCHL1, RUDOLF SEUFERT1, MARKUS SCHMIDT1, JOACHIM KOHL2, MATTHIAS W. BECKMANN3, HELGE BINDER3, INGE HOFFMANN3, ANDREAS MUELLER3 and RALF DITTRICH3

1Department of Obstetrics and Gynecology, Johannes Gutenberg University, D-55116 Mainz; 2Department of Obstetrics and Gynecology, Johann Wolfgang Goethe University, D-60590 Frankfurt; 3Department of Obstetrics and Gynecology, Erlangen University Hospital, University of Erlangen-Nuremberg, D-91054 Erlangen, Germany

Abstract. The number of follicles were compared in different Progress in the treatment of oncological diseases has developmental stages after the cryopreservation of human resulted in the last years to a great improvement of the ovarian tissue by open system followed by survival prognosis, especially in children and juvenile cancer into severe combined immunodeficient patients (1). In the USA alone, more than 20,000 children (SCID)-mice under stimulation, with and without ovariectomy. and young people of reproductive age are exposed every Ovarian tissue, cryopreserved for was year to known mutagens in the form of chemo- and/or partly examined by LIVE/DEAD viability staining or was radiotherapy for cancer. In the developed or Westernised transplanted in the neck muscle of 32 SCID-mice. The countries, women utilise better methods of contraception development of follicles, estradiol production, vaginal cytology and delay childbearing for social or financial reasons; as a and weight was assessed after 15 weeks under result an increasing number of women are anxious to gonadotropin stimulation, with or without ovariectomy. Viable preserve their fertility, when their early-stage cancers are follicles were detected in all frozen/thawed specimens using the discovered. A rising number of patients with non-malignant LIVE/DEAD assay. Ovariectomy caused a significant auto-immune diseases, such as rheumatoid arthritis or improvement of survival of follicles in the preantral and antral systemic lupus and haematological diseases are treated stages in the gonadotropin-stimulated animals (p<0.001), successfully with chemotherapy or radiation (2, 3). whereas there was no significant effect on the primordial and One of the serious side-effects of cytotoxic therapies primary follicle counts. In the non-ovariectomised group, only (chemo and/or radio therapy) is the threatening partial or isolated primordial and primary follicles could be detected. The complete destruction of the gonadal function (3). Quality-of- total follicle amount was significantly higher in the life is increasingly important to long-term survivors of cancer ovariectomised group (n=17, 9.2±7.8, mean±SD) than in the and one of the major quality-of-life issues is the ability to non-ovariectomized group (n=15, 0.3±1.0). This study produce and raise normal children. In a recent survey, 72% demonstrates that ovariectomy of stimulated recipient SCID- of women with a breast cancer diagnosis have been reported mice is essential for the development of follicles after to discuss fertility concerns with their doctors; 51% felt their xenotransplantation of cryopreserved human ovarian grafts. concerns were addressed adequately (4). One of the most promising methods for fertility preservation is the cryopreservation of ovarian tissue before oncological treatments because of the large number of Correspondence to: Theodoros Maltaris, MD, Department of follicles that survive the frozen/thawing procedure (5, 6). Obstetrics and Gynecology, Mainz University Hospital, The problem that arises after the cryopreservation is how to Langenbeckstrasse 1, 55124 Mainz, Germany. Tel: +49 6131 use this frozen material in order to achieve a pregnancy. 177995, Fax: +49 6131 174321, e-mail: [email protected] The vast majority of follicles that survive cryopreservation Key Words: Cryopreservation, gonadotropin stimulation, ovarian are primordial (7-10). tissue, primordial follicle, xenotransplantation, cancer, fertility There are three ways to achieve the development of these preservation. follicles to maturity. The first method is autografting, either

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Table I. Follicular development (mean±SD) of SCID-mice (ovarectomised or not) after transplantation of cryopreserved human ovarian tissue from 6 patients under stimulation.

Stgr: Indication (age) nSCID Ovariec. hMG Primordial Primary Pre-antral Antral

1 Hodgkin (24) 4 x x 4±4.8 4.2±3.9 4.3±2 4.3±2.5 1 CML (10) 5 x x 0.2±0.4 0.8±0.8 6±3.8 4.4±1.8 1 Hodgkin (34) 3 x x 0 0 3.7±2 4.3±4.9 1 Ewing sarc. (16) 5 x x 0 0.4±0.5 0.6±0.9 1±1 2 Hodgkin (17) 9 - x 0.1±0.3 0.3±1.4 0 0 2 Ovar. Ca. (29) 6 - x 0 0 0 0

Stgr = study groups; ovariec = ovariectomized; hMG = human menopausal gonadotropin.

orthotopic, which recently yielded the first human approval of the local university ethical committee. All patients pregnancy after cryopreservation of ovarian tissue (11, 12), suffered from malignant diseases (Table I) and wanted to preserve or heterotopic, e.g. subcutaneously, in the forearm (13). The ovarian tissue for a future pregnancy. A maximum of 5% of frozen tissue from each patient was used for our experiments. Prior to latter method necessitates the use of in vitro fertilization cryopreservation, a histological examination of the ovarian cortex (IVF) in order to achieve pregnancy. The second option is was performed in order to secure a sufficient amount of primordial the in vitro follicular maturation and IVF, a method which follicles. All patients had age-related normal follicular distribution. has already yielded pregnancies in animal experiments (14). This method is not applicable to the human species because Cryopreservation protocol. The ovarian cortex was obtained through of the long time period necessary for the primordial follicle an operative laparoscopy by dissecting an area of about 20x10x3 mm to reach the maturation stage (15). The third method is the ovarian tissue antimesenterically. The biopsies were cut into small pieces (approximately 1x1x1 mm) and were equilibrated in xenografting of human ovarian tissue in immunodeficient ascending aequimolar concentrations of animals [severe compined immonodeficient (SCID)-mice] (DMSO)/ propandiol up to a concentration of 1.5 M in stages of and their stimulation to full follicular maturation (16-20). 0.25 M. The tissue pieces remained in each concentration at 37ÆC Although only two pregnancies have been achieved to date, for 7 min and at the last concentration of 1.5 M for 30 min. The the cryopreservation of ovarian tissue is offered by many groups tissue was then placed in special cryovials (CTE, Erlangen, due to its future therapeutic potential. The aim of our study was Germany) and was loaded into an open freezing system which to examine the effect of ovariectomy in stimulated SCID-mice provides self seeding (CTE). The freezing protocol was as follows: a) cool at –5ÆC/min to –3.8ÆC; b) cool at –1ÆC to –5.3ÆC; c) cool at on the development of follicles after xenotransplantation of –0.2ÆC to –6ÆC; d) unchanged for 20 min; e) cool at –0.3ÆC to cryopreserved human ovarian grafts. The survival of the tissue –30ÆC; f) cool at –0.1ÆC to –35ÆC; g) cool at –0.3ÆC to –80ÆC; h) after thawing was examined by the fluorescent LIVE/DEAD cool at –10ÆC to –110ÆC; and i) immersion in liquid . After and follicular development was examined after storage in for at least 1 month, the probes were transplantation into SCID-mice. thawed at room . Removal of the was done in reverse order of the freezing equilibration procedure. The Materials and Methods tissue blocks were then cultured in an antibiotic supplemented Medicult IVF-Medium (Gück, Berlin, Germany). Animals. Thirty-two female SCID mice (C.B-17/IcrHanHsd scid, 6 weeks of age) were obtained from Harlan-Winkelmann (Borchen, Transplantation procedure. Surgery was performed under narcosis Germany). The animals were housed in a high efficiency with ketamin (80 mg/kg bodyweight, Ketavet, Pharmacia & Upjohn, particulate air-filtered positive pressure room. Cages (Techniplast, Erlangen, Germany) and xylazin (10 mg/kg bodyweight, Rompun, Milano, Italy) were filter topped and animals had free access to Bayer, Frankfurt, Germany), irrespective of the stage of the oestrus (Altromin 1314, Altromin, Lage, Germany) and water, under cycle. During surgery, mice were kept on a warming plate, the 12-h light and 12-h darkness conditions. Groups of 5-9 mice were incision site was disinfected with pure alcohol and covered with a housed in one cage. Upon arrival from the breeding company, the one way sterile towel. Both were removed by a small body mice were allowed to get acclimated for one week. All procedures, wall incision which was sutured with absorbable thread. Xenografting tests and injections were performed under a laminar flow hood in of ovarian cortex was performed within 2 h after removal from the a positive pressure room. Approval for the study was obtained from patient to minimize ischemic damage. The ovarian tissue pieces were the local ethics committee on animal experiments. The animals placed in an intramuscular pocket of the neck muscle. were maintained in accordance with Animal Care and Use Committee regulations. Gonadotropin stimulation. Mice received daily i.p. injections of human menopausal gonadotropin (hMG, Menogon, Ferring, Kiel; Patients. Six patients between 10 and 34 (median 20.5) years of age 1 IU FSH / 1 IU LH per animal/day) or saline, starting from day 14 were included in this study, following informed consent and after transplantation for 15 weeks.

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Table II. Pre-freeze follicular count (per mm2 of ovarian cortex) and survival of follicles assessed with the LIVE/DEAD viability assay after thawing.

Patient diagnosis Age Follicular count Survival count (%) mean number/mm2 (percentage of primordial) Complete Oocyte + Oocyte + Dead follicle >50% granulosa <50% granulosa

CML 10 36 (100%) 31 (41%) 35 (47%) 8 (11%) 1 (1.3%) Ewing sarcoma 16 24 (97%) 15 (39%) 16 (42%) 7 (19%) 0 Hodgkin 17 29 (100% ) 28 (45%) 27 (44 %) 7 (11%) 0 Hodgkin 24 21 (95.7%) 17 (42.5%) 15 (37.5%) 8 (20%) 0 Ovarian-CA 29 10 (95%) 11 (44%) 11 (44%) 2 (8%) 0 Hodgkin 34 9 (88%) 10 (45%) 9 (41%) 2 (9%) 1 (5%)

Table III. Follicle development (mean±SD) in the different study groups.

Stgr. nSCID Ovariec. hMG Primordial Primary Pre-antral Antral

1 17 x x 1.0±2.6 1.3±2.3 3.5±3.0* 3.3±2.6* 2 15 - x 0.1±3.1 0.4±1.2 0* 0*

*Significantly different; stgr = study group; ovariec = ovariectomized; hMG = human menopausal gonadotropin.

Oestrus cycle stage determination. Vaginal smears were taken once a week from all mice starting at day 10 after transplantation using sterile pipettes, in order to examine if follicular tissue survived the transplantation and could produce enough oestrogens to cornify the . Vaginal cells were left to dry after being smeared on a microscopic slide and were then stained with methylene blue. The epithelium cells were classified into one of the four following categories: a) pro-estrous, b) estrous, c) metestrous, d) diestrous (21).

LIVE/DEAD assay. One ovarian tissue piece from each patient was examined for vitality estimation. A fluorescence stain was used to estimate the number of follicles that had survived the freezing/thawing procedure in vitro. The LIVE/DEAD viability/cytotoxicity assay kit (L-3224, Molecular Probes®, Leiden, The Netherlands) provides a two-color fluorescence viability assay that is based on simultaneous determination of alive and dead cells. The method has been described elsewhere (17). In brief, the tissue was digested in PBS-medium supplemented with 1 mg/ml collagenase (Collagenase Type IV, Sigma-Aldrich®, Steinheim, Germany) and incubated at 37ÆC for 2 h. The samples were stained according to the assay instructions and were subsequently Figure 1. Histological staining of a primary follicle (x 250) after examined under a Zeiss fluorescence microscope (IM 35®, Zeiss, cryopreservation and grafting of human ovarian cortex in the SCID mouse. Oberkochen, Germany). The follicles were classified into 4 categories depending on the percentage of dead granulosa cells (Table II), and were considered dead, only when both the oocyte follicles that had survived the transplantation procedure (intact and and all the granulosa cells were dead. with ooplasm) were examined. The diameter of the nucleolus of primordial follicles was estimated to be ≈ 2 Ìm (17). Using a section Microscopic evaluation of the number of follicles. The grafts were thickness of 3 Ìm reduces the risk of overcounting, without totally recovered by a skin and neck muscle incision and were fixed in eliminating it. The follicles were classified as follows: primordial formalin. After routine paraffin embedding, the samples were follicles with one layer of flattened granulosa cells surrounding the serially sectioned (3 Ìm) and every tenth section was stained with oocyte; primary follicles with one layer of cuboid granulosa cells; haematoxylin and eosin and examined microscopically as a reference preantral follicles with two or more layers of granulosa cells, but no section. The numbers of primordial, primary, pre-antral and antral antrum; and antral follicles with an antral cavity.

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Statistical evaluation. SPSS was used for data evaluation. Nominal analogs (23) before the planned , ovarian data were expressed as mean±SD and compared using the transposition before radiation and the cryopreservation of Student’s t-test. A p-value of 0.05 was considered statistically cells or ovarian tissue. significant. In April 2004, the first pregnancy after of cryopreserved ovarian tissue was Results announced by a team of Belgian scientists (11). Many groups are cryopreserving ovarian tissue for future clinical The distribution of the ovarian tissue from the 6 patients in use (1). the two study groups, the follicular development and uterus The aim of the study was to examine the effect of weight (mean±SD) are presented in Table I. The pre-freeze ovarectomy on the number of follicles in different histological follicle count and the survival of follicles with developmental stages of cryopreserved human ovarian grafts the LIVE/DEAD assay after thawing are provided in Table transplanted to gonadotropin-stimulated SCID-mice. II. The follicular development and the uterus weights in the A slow freezing protocol was used with cryoprotective study groups, as well as the statistical analysis of the results agents and an open freezing system was already successfully are presented in Table III. applied for the cleavage stage (24). Many study groups have been using slow freezing equilibrium protocols Analysis of vaginal smears. Both study groups showed for the ovarian tissue cryobanking (summarized in 25). changes in vaginal cytology characteristics of oestrus cycles The pre-freezing histological follicular count showed at days 10-14 after surgery. These changes were evident normal age-related follicular distribution in the and through the presence of cornified epithelial cells. was in accordance with findings of other studies (26, 27). After thawing, we performed a LIVE/DEAD assay, Pre-freeze follicular histological count and follicular viability xenotransplanted the tissue in SCID-mice and tested the after thawing assessed with the LIVE/DEAD assay. All viability of the transplanted ovarian tissue by vaginal patients showed age-related normal follicular counts (Table cytology, uterus weight and follicle count. II), and were within 2 orders of standard deviation except The cornifying of the epithelial cells of the vaginal the pre-pubertal patient. The primordial follicle was the mucosa demonstrated that both groups produced ovarian most predominant follicle type. steroid hormones. The results of the fluorescence staining are also given in The results of the staining with the LIVE/DEAD Table II. Dead follicles were very rare. florescent assay confirm that a high percentage of oocytes, as well as granulosa cells survived the cryopreservation and Follicular survival and development. The results of the thawing procedure. Our results are in accordance with follicular count per patient are shown in Table I. Table III those of other publications (27, 28). It is known from other shows the results of the follicular count in the various studies that the main reason for the follicular loss after study groups. Ovariectomy caused significant development cryopreservation and xenografting is the ischaemic effect of follicles in the pre-antral and antral stages in the after transplantation rather than the cryopreservation (29). gonadotropin-stimulated animals (p<0.001), whereas The following factors influenced the follicular distribution there was no significant effect on the primordial and in transplantation studies: the inhomogeneous distribution primary follicle counts (Figure 1). In the non- of follicles in the ovarian cortex (intra-patient variation) ovariectomised group, only isolated primordial and (26), the age-related decline of follicles, the inter-patient primary follicles could be detected. The total follicle variation and the size of the grafts (7). amount was significantly higher in the ovariectomised Most researchers use human to maximise group (n=17, 9.2±7.8, mean±SD) than in the non- follicular development in the xenografted ovarian tissue (30, ovariectomised group (n=15, 0.3±1.0). 31). We also stimulated the SCID mice with exogenous gonadotropins in order to promote the development of Discussion primordial follicles. Our results indicate that ovariectomy improved the It is estimated that by 2010, one in every 250 women of survival of follicles in all developmental stages in the reproductive age worldwide will be a cancer survivor (22). xenotransplanted grafts in the gonadotropin-stimulated However, the lifesaving treatments administered can SCID mice. Since the stimulation was initiated two weeks provoke early menopause and subsequent , due to after the ovarian transplantation, we assumed that high the destruction of a significant proportion of ovarian follicles gonadotropin levels are necessary in the neovascularisation by chemo- and radiotherapy. Methods to preserve fertility in period directly after grafting, which is in accordance with the these young patients are chemoprotection with GnRH- work of Dissen et al. (32).

4174 Maltaris et al: Cancer and Fertility Preservation

Since the serum gonadotropins regulate the female 8 Candy CJ, Wood MJ and Whittingham DG: Effect of reproductive system and are essential for the cyclic growth on the survival of follicles in frozen mouse of follicles after the onset of antrum formation (33), it has ovaries. J Reprod Fertil 110: 11-19, 1997. 9 Gook DA, Edgar DH and Stern C: Effect of cooling rate and been assumed that one effect mediated by these hormones dehydration regimen on the histological appearance of human could be the local activation of angiogenic factors in ovarian cortex following cryopreservation in 1, 2-propanediol. developing follicles and corpora lutea (34, 35). Bex and Hum Reprod 14: 2061-2068, 1999. Goldman (36) were able to show that in bilaterally 10 Newton H, Aubard Y, Rutherford A, Sharma V and Gosden R: ovariectomised hamsters, both serum LH and FSH Low temperature storage and grafting of human ovarian tissue. concentrations were significantly elevated when compared Hum Reprod 11: 1487-1491, 1996. with unmanipulated animals. 11 Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Laschke et al. demonstrated by direct in vivo observation Squifflet J, Martinez-Madrid B and van Langendonckt A: Livebirth after orthotopic transplantation of cryopreserved that in ovarectomised hamsters, ovarian (although not ovarian tissue. Lancet 364: 1405-1410, 2004. cryopreserved) vascularisation was not only accelerated but 12 Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel also markedly enhanced (37). Y, Schiff E and Dor J: Pregnancy after transplantation of In conclusion, the xenotransplantation of small pieces of cryopreserved ovarian tissue in a patient with ovarian failure frozen/thawed human ovarian tissue in SCID mice provides after chemotherapy. N Engl J Med 353: 318-321, 2005. a practical method to assess the development potential of 13 Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, stored ovarian tissue. The observation of even a few growing Opsahl M and Rosenwaks Z: development after heterotopic transplantation of cryopreserved ovarian tissue. follicles is sufficient for this purpose, since almost only Lancet 363: 837-840, 2004. primordial follicles survive the cryopreservation. 14 Eppig JJ and O'Brien MJ: Development in vitro of mouse Ovariectomy of the mice improves the success of the oocytes from primordial follicles. Biol Reprod 54: 197-207, xenotransplantation procedure as a diagnostic method. 1996. 15 Gougeon A: Dynamics of follicular growth in the human: a Acknowledgements model from preliminary results. Hum Reprod 1: 81-87, 1986. 16 Maltaris T, Dimmler A, Muller A, Binder H, Hoffmann I, Kohl This work was partly supported by the Johannes and Frieda J, Siebzehnrubl E, Beckmann MW and Dittrich R: The use of an Marohn Foundation, Erlangen, Germany. open-freezing system with self-seeding for cryopreservation of mouse ovarian tissue. Reprod Domest Anim 40: 250-254, 2005. References 17 Maltaris T, Kaya H, Hoffmann I, Mueller A, Beckmann MW and Dittrich R: Comparison of xenografting in SCID mice and 1 Maltaris T, Boehm D, Dittrich R, Seufert R and Koelbl H: LIVE/DEAD assay as a predictor of the developmental potential Reproduction beyond cancer: a message of hope for young of cryopreserved ovarian tissue. In Vivo 20: 11-16, 2006. women. Gynecol Oncol, 2006 [Epub ahead of print]. 18 Maltaris T, Beckmann MW, Mueller A, Hoffmann I, Kohl J 2 Maltaris T, Seufert R, Fischl F, Schaffrath M, Pollow K, Koelbl and Dittrich R: Significant loss of primordial follicles after H and Dittrich R: The effect of cancer treatment on female prolonged gonadotropin stimulation in xenografts of fertility and strategies for preserving fertility. Eur J Obstet cryopreserved human ovarian tissue in severe combined Gynecol Reprod Biol, 2006 [Epub ahead of print]. immunodeficient mice. Fertil Steril, 2006 [in press]. 3 Maltaris T, Koelbl H, Seufert R, Kiesewetter F, Beckmann 19 Maltaris T, Dragonas C, Hoffmann I, Mueller A, Beckmann MW, Mueller A and Dittrich R: Gonadal damage and options MW and Dittrich R: Simple prediction of the survival of for fertility preservation in female and male cancer survivors. follicles in cryopreserved human ovarian tissue. J Reprod Dev Asian J Androl 8: 515-533, 2006. 52: 577-582, 2006. 4 Partridge AH, Gelber S, Peppercorn J, Sampson E, Knudsen K, 20 Mueller A, Maltaris T, Dimmler A, Hoffmann I, Beckmann Laufer M, Rosenberg R, Przypyszny M, Rein A and Winer EP: MW and Dittrich R: Development of sex cord stromal tumors Web-based survey of fertility issues in young women with breast after heterotopic transplantation of cryopreserved ovarian tissue cancer. J Clin Oncol 22: 4174-4183, 2004. in rats. Anticancer Res 25: 4107-4112, 2005. 5 Aubard Y, Poirot C, Piver P, Galinat S and Teissier MP: Are 21 Gunasena KT, Villines PM, Critser ES and Critser JK: Live there indications for ovarian tissue cryopreservation? Fertil births after autologous transplant of cryopreserved mouse Steril 76: 414–415, 2001. ovaries. Hum Reprod 12: 101-106, 1997. 6 Liu HC, He Z and Rosenwaks Z: Mouse ovarian tissue 22 Blatt J: Pregnancy outcome in long-term survivors of childhood cryopreservation has only a minor effect on in vitro follicular cancer. Med Pediatr Oncol 33: 29-33, 1999. maturation and gene expression. J Assist Reprod Genet 20: 23 Blumenfeld Z: Gynaecologic concerns for young women 421-431, 2003. exposed to gonadotoxic chemotherapy. Curr Opin Obstet 7 Broecke Van den R, Liu J, Handyside A, Van der Elst JC, Gynecol 15: 359-370, 2003. Krausz T, Dhont M, Winston RM and Hovatta O: Follicular 24 Siebzehnruebl ER, Todorow S, van Uem J, Koch R, Wildt L growth in fresh and cryopreserved human ovarian cortical grafts and Lang N: Cryopreservation of human and rabbit oocytes and transplanted to immunodeficient mice. Eur J Obstet Gynecol one-cell embryos: a comparison of DMSO and propandiol. Reprod Biol 97: 193-201, 2001. Hum Reprod 4: 312-317, 1989.

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25 Picton HM, Gosden RG and Leibo SP: Cryopreservation of 32 Dissen GA, Lara HE, Fahrenbach WH, Costa ME and Ojeda oocytes and ovarian tissue. In: Current Practise and SR: Immature rat ovaries become revascularized rapidly after Controversies in Assisted Reproduction. Vayena E, Rowe P, autotransplantation and show a gonadotropin-dependent Griffin PD (eds.). Geneva, World Health Organization, pp. 142- increase in angiogenic factor gene expression. Endocrinology 151, 2002. 134: 1146-1154, 1994. 26 Schmidt KL, Byskov AG, Nyboe Andersen A, Muller J and 33 Kumar TR, Wang Y, Lu N and Matzuk MM: Follicle Yding Andersen C: Density and distribution of primordial stimulating hormone is required for maturation follicles in single pieces of cortex from 21 patients and in but not male fertility. Nat Genet 15: 201-204, 1997. individual pieces of cortex from three entire human ovaries. 34 Makris A, Ryan KJ, Yasumizu T, Hill CL and Zetter BR: The Hum Reprod 18: 1158-1164, 2003. nonluteal porcine ovary as a source of angiogenic activity. 27 Siebzehnrubl E, Kohl J, Dittrich R and and Wildt L: Freezing Endocrinology 115: 1672-1677, 1984. of human ovarian tissue-not the oocytes but the granulosa is the 35 Gospodarowicz D, Cheng J, Lui GM, Baird A, Esch F and problem. Mol Cell Endocrinol 169: 109-111, 2000. Bohlen P: angiogenic factor is related to 28 Martinez-Madrid B, Dolmans MM, Van Langendonckt A, fibroblast growth factor. Endocrinology 117: 2383-2391, 1985. Defrere S and Donnez J: Freeze-thawing intact human ovary 36 Bex FJ and Goldman BD: Serum gonadotropins and follicular with its vascular pedicle with a passive cooling device. Fertil development in the Syrian hamster. Endocrinology 96: 928- Steril 82: 1390-1394, 2004. 933, 1975. 29 Liu J, Van der Elst J, Van den Broecke R and Dhont M: Early 37 Laschke MW, Menger MD and Vollmar B: Ovariectomy massive follicle loss and apoptosis in heterotopically grafted improves neovascularization and microcirculation of freely newborn mouse ovaries. Hum Reprod 17: 605-611, 2002. transplanted ovarian follicles. J Endocrinol 172: 535-544, 2002. 30 Gook DA, McCully BA, Edgar DH and McBain JC: Development of antral follicles in human cryopreserved ovarian tissue following xenografting. Hum Reprod 16: 417-422, 2001. 31 Oktay K, Newton H, Mullan J and Gosden RG: Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone. Hum Reprod 13: Received September 28, 2006 1133-1138, 1998. Accepted October 18, 2006

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