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  • �� �������������� ���������������� �Table. 1��

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    Table. 2. The Percentage of Mice Which Reestablished Estrous Cyclicity,

    and the Mice in which Autotransplanted Ovaries were

    Detected

    Table. 1. The Percentage of Mice in Which Autotrans-

    planted Ovaries were Detected: Comparison of

    Autotransplanted Sites

    Although no significant di#erences were observed, the

    highest percentage was observed in subfascial dorsal

    muscle.

    Vitrification [*(P+,\W]^ 181

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    Fig. 2. �a� Days until reestablishment of estrous cycle, �b�uterine weight, and �c� serum estradiol �E2� levels:e#ects of vitrification

    The following ovarian tissues were auto-

    transplanted in subfascial of the dorsal muscle.

    Group D, fresh, minced ovaries; Group E,

    vitrified, minced ovaries; Group F, vitrified, whole

    ovaries. �P0.05�

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    Fig. 4. Imunohistochemistry for Ki67

    The ovary was obtained from mouse of Group E. �a� Green fluorescence showsKi67 immunoreactivity. �b� Phase contrast micrograph. �c� Blue fluorescence isnuclei with stained by DAPI. �d� The merged view of �a� and �c�.

    Fig. 5. E#ect of vitrification on ratio of follicles with Ki

    67-positive granulosa cells

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    Fig. 7. �a� The number of ovarian follicles at di#erent stages and �b� the ratio of follicleswith Ki67-positive granulosa cells

    Group G, non treatment mice �2-week-old�; Group H, mice in which ovaries wereremoved and vitrified at 2-week-old, auto-transplanted at 7-week-old, and ovarian

    morphology was examined at 12 -week-old; Group I, non treatment mice �12-week-old�� �P�0.05, ��P�0.01.

    Fig. 8. Ovarian morphology

    The ovarian section was obtained from Group

    H mouse and stained using methyl blue.

    Objective magnification, �20.

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    1� Wallace WH, Shalet SM, Crowne EC, Morris-Jones PH, Gattamaneni HR and Price DA.

    Gonadal Dysfunction Due to Cis-Platinum,

    Med Pediatr Oncol 1989; 17: 409�413.2� Donnez J, Martinez-Madrid B, Jadoul. P, Lan-gendonckt A, Demylle D and Dolmans M.

    Ovarian tissue cryopreservation and transplan-

    tation: a review, Hum Reprod Update 2006;

    12: 519�535.3� Wallace WH, Thomson AB and Kelsey TW.The radiosensitivity of the human oocyte, Hum

    Reprod 2003; 18: 117�121.4� Meirow D, Levron J, Elder-Geva T andHarden I. Pregnancy after transplantation of

    cryopreserved ovarian tissue in a patient with

    ovarian failure after chemotherapy, N. Engl J

    Med 2005; 353: 318�322.

    Fig. 9. �a� A straw containing the ovarian pieces, �b� injected ovarian pieces into thesubfascial dorsle muscle using the straw connected with a syringe, and �c� the ovarianpieces �arrow� 4 weeks after auto-transplantation

    Vitrification �����3�45 187

    21

  • 5� Demeestere I, Simon P, Buxant F, Robin V,Fernandez S, Centner J, Delbaere, A and

    Englert Y. Ovarian function and spontaneous

    pregnancy after combined heterotopic and or-

    thotopic cryopreserved ovarian tissue trans-

    plantation in a patient previously treated with

    bone marrow transplantation: Case report,

    Hum Reprod 2006; 21: 2010�2014.6� Israely T, Dafni H, Granot D, Nevo N,Tsafriri A, and Neeman M. Vascular Remodel-

    ing and Angiogenesis in Ectopic Ovarian Tran-

    plants: A Crucial Role of Pericytes and Vascu-

    lar Smooth Muscle Cells in Maintenance of

    Ovarian Grafts, Biol Reprod 2003; 68: 2055�2064.

    7� Jonathan L Tilly. Ovarian follicle counts-notas simple as 1, 2, 3, Reprod Biol Endocrinol

    2003; 1: 1�4.8� Liu J, Elst JV, Broecke RV and Dhont M,Early massive follicle loss and apoptosis in

    heterotopically grafted newborn mouse ova-

    ries, Hum Reprod, 2002; 17: 605�611.9� Aubard Y. Ovarian tissue xenografting, Eur JObstet Gynecol Reprod Biol 2003; 108: 14�18.

    10� Lee RK, Ho H, Yu S and Lu C, BlastocystDevelopment After Cryopreservation and Sub-

    cutaneous Transplantation of Mouse Ovarian

    Tissue, J Assist Reprod Genet 2005; 22: 95�101.11� Kagawa N, Kuwayama M, Nakata K, Vajta

    G, Silber S, Manabe N, Kato O, Production of

    the first o#spring from oocytes derived from

    fresh and cryopreserved pre-antral follicles of

    adult mice, Reprod Biomed Online 2007; 14:

    693�699.12� Migishima F, Suzuki R, Song S, KuramochiT, Azuma S, Nishijima M and Yokoyama M.

    Successful Cryopreservation of Mouse Ovaries

    By Virification, Biol Reprod 2003; 68: 881�887.13� Hasegawa A, Mochida N, Ogasawara T and

    Koyama K, Pup birth from mouse oocytes in

    preantral follicles derived from vitrified and

    warmed ovaries followed by in vitro growth, in

    vitro maturation, and in vitro fertilization, Fer-

    til Steril 2006; 86: 1182�1192.14� Salehaina M. Autogaraft of Vitrified MouseOvaries Using Ethylene Glycol as Cryoprotec-

    tant, Exp. Anim 2002; 51: 509�512.15� Gunasena K, Villines PM, Critser ES and Crit-ser JK, Live birth after autologous transplanta-

    tion of cryopreserved mouse ovaries, Hum Re-

    prod 1997; 12: 101�106.16� Mueller A, Maltaris T and Dimmler A. Devel-opment of sex cord stromal tumors after heter-

    otopic transplantation of cryopreserved ovar-

    ian tissue in rats, Anticancer Res 2005; 25:

    4107�4111.17� Maltaris T, Dimmler A, Ho#mann I, Beck-

    mann MW and Dittrich R. Comparison of two

    freezing protocols in an open freezing system

    for cryopreservation of rat ovarian tissue, J

    Obstet Gynaecol Res 2006; 32: 273�279.18� Gerdes J, Schwab U, Lemke H and Stein H,

    Production of a mouse monoclonal antibody

    reactive with a human nuclear antigen associ-

    ated with cell proliferation, Int. J. Cancer 1983;

    31: 13�20.19� Quirk SM, Cowan RG and Harman RM, Thesusceptibility of granulose cells to apoptosis is

    influenced by oestradiol and the cell cycle, J

    Endocrinol 2006; 189: 441�453.20� ����� ����� ����� ����������������� !"#$%� &'�()' 2004; 4: 523�529.

    *+,-. /01�2 3188

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  • Abstract

    Cryopreservation and Heterotopic Autotransplantation of

    Ovarian Tissue in Mouse

    Marie Ino1, Juichiro Saito1, Asako Taniuchi1, Noriyuki Takahashi1,

    Chiharu Tsuda1, Nao Suzuki1, Bunpei Ishizuka1, Masanori Itoh2,

    Yoshiharu Morimoto3, and Shu Hashimoto3

    High-dose chemotherapy and�or frequent irradiation for malignancy can cause sterility in young womenof reproductive age. Development of freezing�thawing techniques for ovarian tissues would thus be verysignificant for the restoration of ovarian function, and may help to maintain QOL via serum estradiol levels.

    Ovarian autografts of ICR mice have been performed in an attempt to preserve ovarian function and

    conception. Group A �Autotransplantation of fresh, minced ovary�: The ovaries were extirpated from micethen cut into small pieces, and autotransplanted to subfascial dorsal muscle �Group A-s�� mammary gland�Group A-m� and fibrous capsule of kidney �Group A-f�. Group B �Autotransplantation of vitrified, mincedovary�: The ovaries were cryopreserved by vitrification method after cutting ovaries into small pieces.Ovarian tissues were thawed after 1 week and autotransplanted to subfascial dorsal muscle. Group C

    �Autotransplantation of vitrified, whole ovary�: The same process was done without cutting ovaries intosmall pieces. Future, treatment of prepubescent girls who undergo chemotherapy or radiotherapy in mind,

    the ovaries of 2-week-old mice were cryopreserved after cutting into small pieces. Five weeks later, ovarian

    tissues were thawed and autotransplanted to subfascial dorsal muscle �Group D�� Vaginal smears weresubsequently observed, and the grafted ovary was extirpated at the time of diestrus approximately 4 weeks

    after transplantation.

    Although no significant di#erences in the percentage of mice in which autotransplanted ovaries were

    detected comparison of the autotransplanted site, the highest percentage was observed in Group A-s. The

    percentage of mice in which estrous cycle was resumed was 100� in Group A, 100� in the Group B, and77.8� in the Group C. The percentage was significantly lower in Group C than in Group B. The numberof primordial follicles were higher in Group A than in Group B. Also, total number of ovarian follicles was

    significantly larger in Group B than in Group C. Furthermore, the percentage of Ki67-positive ovarian

    follicles was significantly higher in Group B than in Group C. The percentage of Ki67-positive ovarian

    follicles in Group D was not di#erent as 12-week-old non treatment mice. These results indicate that the best

    site for autotransplantation of ovarian tissues in mice was the subfascial dorsal muscle. Minced ovary was

    less damaged than the whole ovary, so minced ovary was more suitable for vitrification than the whole

    ovary. The function of autotransplanted ovaries after freezing�thawing by vitrification was also maintainedin prepubatal mice.

    1 Department of Obstetrics and Gynecology, St. Marianna University School of Medicine2 Department of Anatomy� St. Marianna University School of Medicine3 IVF Nanba Clinlc

    Vitrification �������� 189

    23

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