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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.
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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.
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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
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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
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