Vitrificationigakukai.marianna-u.ac.jp › idaishi › www › 354 › 03-35-4Marie Ino.pdf9:;? 2@...

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臩腺腽腪膁腵膇膐臸膗膽臂 Vol. 35, pp. 177189, 2007 Vitrification 腓腄腔腀腁腇腅腆腕腎腃腐腊腒腏腂腌腉腈腍腃腋腑 腝腣膕 1 腋腄 腕腅 腍腞 腄腒 腣腅 1 腐腗 腅腒 腃腋 1 腐腈 腙腌 腘腡 腟腉 1 腙腢 1 腎腏 腖腇 1 腄腌 腔腈 腚腤 腛腄 1 腕腅 腜腋 腘腡 2 腝腡 腝腕 腠腌 腙腢 3 腙腌 腝腕 腌腞腅 3 : 19 8 20 腫腅膰膝膻臆臌臮臆腙腡腣至膞腔腘臰腙膜腣腑腻臤腆膆臄腕腇腘腂臎臤膙臍腙臷腌至腧臵膯膅臟腍腤腠腡腆膱腃腢腥腔腂腤膶膔膒腛腺腫腱腙腄腂腔 Vitrification 腂腐至腚臵腙腓腂腔膫腌腐腠腡ICR 7 腺腫腱腧腂腔膅臟腚臞膪腧膖腍腤腐腞膅臟臝臖腗腦膍腧膫腌腐臅腙至臰臶腧腏腚腝腝膧腍腤腅至腧膺臬腌腐膯膧腍腤 腗腑腢腆膅臟膯腙至膞腐腥腔腂腤腅腧膫腌腐腝腐臚膋腖腌腔臕臼腆腌腔腂腘腂臗臃腆膰膝膻臆腧臐腉腘腈腔腛腂腉腘腂臝膴腧臲腌腔2 腚腺腫腱腚至腧臵腌5 臓膚膯臈膏膅臟腧膲腒腐膧膑膍腙膅臟腍腤腖至腆臨臻腍腤腅膎臤腆膸腟膳腂腊腖腆臇膷腋腥腐膺臬腌 至腧膧腌腐膤腆至腧腏腚腝腝膧腌腐膤腙腱腢Ki67 腛膳腅腒腐P0.05腝腐2 至腧膧腌腔 5 臓膚膯腙臈膏膅臟腌腐膤腕腛 12 至腙腌腔 腱腢Ki67 腌腈腐腥腔腂腐膱膼膍腛腺腫腱腙腖腒腔腡腂膅臟臝臖腕腁腣至腛膺臬腍腤腊腖腙腡腣膧膊腆 腌腠腍腈膧腙腡腤腲腼腀腰腆膍腍腤腁腤腂腛膺臬腍腤腊腖腙腡腣膅臟膯腙臨臻腌 腠腍腂腖膱腃腢腥腐腝腐 2 至腧膧腌臈膏膅臟腌腐膤腕腟至膞腐腥腔腂 腤腖臁腦腥腐ῐῌ῏ Vitrification 腑腻臤膌臵 膨膊膂臤臏臋膙膂臤腽膁腷臏膰膝膻臆臌臮臆腂腢腥腤腊腖腆腁腤 膙臍腆臎臗臤腚臝膴腛臘腰腱至膞腔腘臰腚腐 腘腑臛腖腘腤膎臤腆腁腤至膞腔腘臰腧膟腊 腌腠腍腂膰膝膻腖腌腔腡腈臺腢腥腔腂腤腚腆腯腮膀 腸腭腱腸腩腻腴腠腹腱腾腸腬膁腕腁腣腊腥腢腧腂腐臆腕腛 70至膞腧臱臉腍腤腖膵腋 腥腔腂腤 1腯腮膀腸腭腱腸腩腻腴腛40 膹膃臜腕 5g40 腨腧腕腛920 g 腕膘臰腘膩膦腙腘腤 腖膮腦腥腤 2臌臮臆腙膛腌腔腟腌腭臮臆腙腡腒腔 2 Gy 膃膍腚臮腕膬膿腱腢50膃膍腙膭臙腌腒腹腙腅腅腦腢腎 520 Gy 腚臮1 臩腺腽腪膁腵膇膐臸膗 臢膐膗膡臊 2 臩腺腽腪膁腵膇膐臸膗 膗膡臊 3 IVF 腘腨腜腮腽腶腳腮 177 11

Transcript of Vitrificationigakukai.marianna-u.ac.jp › idaishi › www › 354 › 03-35-4Marie Ino.pdf9:;? 2@...

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

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    %�100��� ! Vitirification $�� 35%& 35%�100��� Whole Vitrification $�� 27%& 21%�77.8������ ������� ! Vitrification$�'� Whole Vitrification $(�)��*�

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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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  • �� ������� Vitrification ���Whole Vitrification ������������������� Whole Vitrification �������� ����� stage ��������� !"#� �Fig. 3�� $%��&'()*���+�,-./� Vitrification/����������01���

    Fig. 4� Ki67�234+5��� Ki67���67�89:9/��;����?@A���B� Ki67 �CD� !"#�� Ki67 EF���GH+ Fig. 5 �5��� �����

    �� 444IJ 271I�61��� � Vitrification�� 239IJ 179I �74.8��� Whole Vitrification

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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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    16

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    ��� 2� ���������� Vitrific-ation ��������R8,S��GH��,S�,TU�VW� DEF��%GH�� VitrificationX%Y��� IJ!"��Z[�/2Q�11�� ��� DEF,��%GH\]� Vitirification X�56S��^_�!"�Z[�/2Q�`a�A012��13��bcd�;efg,h�� !"��,��ij_k� lmn+� op E2 q%GH Vitrification r�Whole Vitrification r$�TU��� GH Vitrific-ation r�;Whole Vitrification r�TU��efg,h��s2Q�789������ � �� GH Vitrification r�;tu�v9� Ki67we�v,x�T,y��z{1Q�� ���� op E2 q; Whole Vitrification r,|�GHIJ!"r56}~�?q�A6� ��GH Vitrificationr56?�%��� ,t�������2P 0�� Whole Vitrificationr�;!"�����Z0�� M����^_��������2 E2 �op�1Q�e�P /0� ����;op E2 q;�v��,:%���2Q��0� ���� !"����,;]�(op E2 q�1Q���P

    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

    �6   2184

    18

  • ����� �� E2 �������������������������������� !"#�$�� Ki67 % G0 �&'�"(�)���*+,-"#���.��/#012343�#18�� $�� 01�����)�,-"#����� ��,5�2343����/��/#19�� �6789%� ): Vitrification ;*?������ @��ABC�):���D8�����AE��D8��>?���,5�F���G�/HIJ!K�

    /����LMK��� ���):���ABC����N�O�/�� PQR� �S�TU�V"W� XY�V"/HI*�#�����#� $�� �ABC�):"#��Z*[D\�TU"#�� D8*>#)�]�^_3`�ab�����#� �c� Ki67%���)�*d-"#efgh�?� ���)����"#ij��>?�,5�����k*,-�� lm��%���)���,n�#�o Ki67%,-��/� lm���01�pq��/��%�Ars�2343��%�tE�#�/uv#�w�/�o� xR� y��Ars�2343�zu{|}����#���#��):��R*D8"#����~��lm��%������,-� U�� ���)���

  • ����� 3������� ��������������� 2���� �� �!��"����#$%�&'��� �!��( E2 )!*+,-./�'� ���0�!�!��(��1��234$�5678� E2 !9:,-�;�-� 1�!?@ABC�D!�(EF"GH��� 2�I VitirificationJ�(2K1L,-D�M�1LN� 2�IOPQRSTJ"UVWX�5F� �� Y1Z�[\]�!^1!_`,-ab�c!1�Ad_�5DY1Z�,-1L�M�e���/"�f8�

    � �� 2�I Vitrification J! Ki67g�1L!9:h( 12�IOPQRSTJ"UVWX�5F� ��i5ab�c!1�Ad_��

    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.

    jk�-l mno2p �186

    20

  • ��������������������������� ������ � !"��#��$%$&'�()*+,�-./0� ��12�3�45��67� +,89:�;'�?����@ABC��������

    � �

    DEF����GH���6$�� IJ%��KL���MNO�����P� QP� 3�R����ST������ � 3����UVWX�YO�� C�$Z��:�>[�\]�% � ��^A�4P�������P� _`aR�bc��B3��-d����:� ��ef������g�����hidP�

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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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