Effects of different freezing methods on the follicular viability of large pieces of human ovarian tissues

doi:10.3969/j.issn.0529-1356.2013.03.027

Abstract

Abstract:

Objective To study effects of cryopreservation of large pieces of human ovarian tissues and assess the follicular
viability after vitrification and two-step freezing. Methods Ovarian tissues from 15 patients were collected. The size of each
tissue block was about 15 mm×15 mm×2mm. The tissue block was cut into three pieces and randomly grouped as fresh(A), vitrification
(B), and two-step freezing (C) groups. All the pieces were cultured in vitro after thawing. The viability and proliferative capacity
of tissues were evaluated by in vitro production of estrogen and progestogen, histological analysis and immunohistochemical
staining of nuclear proliferation-associated antigens, Ki67 and B cell lymphoma/lewkmia-2（Bcl-2）. Results The ratios of normal
follicles were the highest in group A(91.9%), median in group C (82.9%) and the lowest in group B(71.3%) (P<0.05). The ratios of
morphologically normal primordial and primary follicles in group C were 86.8% and 54.4% which were higher than 73.8% and 44.4% in
group B(P<0.05), respectively. Difference of hormonal activity was not found among three groups（P>0.05). Positive expression of
Ki67 of group C（73%）was higher than group B（50%）and group A（55%）(P<0.05), where as that of group A was lower than group B
(P<0.05).There was no difference of positive expression of Bcl-2 staining between group A（57%）and group C（61%）(P>0.05), but
the ratio of positive Bcl-2 expression of group B（42%）was lower than groups A and C (P<0.05). Conclusions The frozen/thawed
large pieces of human ovarian tissues can reserve their vitality and the tissue with two-step freezing is better preserved than
the tissues with vitrification.

Key words: Ovarian tissue, Ki67, Cryopreservation, Vitrification, Two-step freezing, Human

CLC Number:

R711.6

ZHAO Shuo ZHAO Shu-qin SHI Qing QIN Xiao-min HAN Yi-long CHAO Lan DENG Xiao-hui*. Effects of different freezing methods on the follicular viability of large pieces of human ovarian tissues[J]. AAS, 2013, 44(3 ): 427-432.

References

［1］Suzuki N, Yoshioka N, Sugishita Y, et al. The possibility of the fertility preservation in young cancer patients based on practice of oncofertility treatment［J］. Gan To Kagaku Ryoho, 2012, 39(2):151-157. 
［2］Aubard Y, Piver P, Cognie Y, et al. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep［J］. Hum Reprod, 1999, 14(8):2149-2154.
［3］Poirot C, Vacher-Lavenu MC, Helardot P, et al. Human ovarian tissue cryopreservation: indications and feasibility［J］. Hum Reprod, 2002, 17(6):1447-1152. 
［4］Fahy GM, MacFarlane DR, Angell CA, et al. Vitrification as an approach to cryopreservation［J］. Cryobiology, 1984, 21(4):407-426.
［5］Moniruzzaman M, Bao RM, Taketsuru H, et al. Development of vitrified porcine primordial follicles in xenografts［J］. Theriogenology, 2009, 72(2):280-288.
［6］Sugimoto M, Maeda S, Manabe N, et al. Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification［J］. Theriogenology, 2000, 53(5):1093-1103.［7］Wang X, Catt S, Pangestu M, et al. Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice［J］. Reproduction, 2009, 138(3):527-535.
［8］Liu Y, Xu X, Ma X, et al. Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions［J］. Biotechnol Prog, 2010, 26(6):1635-1643.
［9］Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results［J］. Hum Reprod, 1986, 1(2):81-87. 
［10］Wang LH, Chen ZJ, Gao X. The morphological study of cryopreserved human ovarian cortical tissue［J］. Progress in Obstetrics and Gynecology, 2005, 14(1):16-19.(in Chinese)
王利红, 陈子江, 高选. 人卵巢组织冷冻保存的实验研究［J］. 现代妇产科进展, 2005, 14(1):16-19. 
［11］Isachenko E, Isachenko V, Rahimi G, et al. Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen［J］. Eur J Obstet Gynecol Reprod Biol, 2003, 108(2):186-193.
［12］Yue FZh, Gao BR. Experimental study on the viability of cryopreserved allograft valved aortic and pulmonary arteries［J］. Journal of Lanzhou Medical College, 2001, 27(2):12-15. (in Chinese)
岳凤珍, 高秉仁. 液氮超低温保存同种带瓣动脉活性实验研究［J］. 兰州医学院学报，2001, 27(2):12-15. 
［13］Mazur P. Freezing of living cells: mechanisms and implications［J］. Am J Physiol, 1984, 247(3 Pt 1): C125-142. 
［14］Gougeon A, Busso D. Morphologic and functional determinants of primordial and primary follicles in the monkey ovary［J］. Mol Cell Endocrinol, 2000, 163(1-2):33-42. 
［15］Hussein MR, Bedaiwy MA, Falcone T. Analysis of apoptotic cell death, Bcl-2, and p53 protein expression in freshly fixed and cryopreserved ovarian tissue after exposure to warm ischemia［J］. Fertil Steril, 2006, 85 (Suppl 1):1082-1092. 

[1]	LIU Xiao-li XU Lin WU Ai-xue WEN Cai-yun LI Ru-hua WU An-ting CHEN Dai-qian CHEN Cheng-chun. Analysis of cerebral gray matter structure in multiple sclerosis and neuromyelitis optica [J]. Acta Anatomica Sinica, 2024, 55(1): 17-24.
[2]	BAI He TENG Ye FENG Lei MENG Hai-wei1 TANG Yu-chun LIU Shu-wei. 3D hippocampal segmentation based on spatial and frequency domain features adaptive fusion and inter-class boundary region enhancement [J]. Acta Anatomica Sinica, 2024, 55(1): 73-81.
[3]	YIN Shi-qin YANG Si-yi WANG Rui-han YOU Gui-xuan YANG Ying-qiu ZHANG Lei. Distal tibiofibular syndesmosis fibular notch typing and its clinical significance based on CT [J]. Acta Anatomica Sinica, 2024, 55(1): 82-87.
[4]	YANG Yang SHI Jun LI Kun MA Yuan ZHANG Shao-jie HOU Er-fei WANG Chao-qun CHEN Jie WANG Xing LI Zhi-jun. Finite element analysis of cervical intervertebral discs after removing different ranges of uncinate processes [J]. Acta Anatomica Sinica, 2024, 55(1): 88-97.
[5]	SUN Lei WANG Xing-yu XIE Shui-hua. Risk analysis of re-fracture after percutaneous kyphoplasty in elderly patients with osteoporotic thoracolumbar compression fractures and construction of acolumnar graph prediction model#br# [J]. Acta Anatomica Sinica, 2024, 55(1): 98-104.
[6]	LIU Li-ping ZHU Meng-ni XU Hui . Effect of interleukin-6 on nucleated erythrocytes in lipopolysaccharide induced preeclampsia rats [J]. Acta Anatomica Sinica, 2023, 54(6): 722-729.
[7]	ZHAO Wei-ming LI Xiao YU Guo-ying WANG Gai-ping CHANG Cui-fang . Effect of serine protease inhibitor Kazaltype 1 on the proliferation of hepatocellular carcinoma cell and its mechanism [J]. Acta Anatomica Sinica, 2023, 54(6): 695-702.
[8]	HAN Yi CAI Li FENG Xiao . Risk analysis of bone cement leakage after percutaneous puncture vertebroplasty for osteoporotic spinal compression fractures and construction of a predictive model with column line drawings [J]. Acta Anatomica Sinica, 2023, 54(6): 710-715.
[9]	DU Da-peng SUN Yu ZHANG Min WANG Han-qin . Shear stress regulateing the phosphorylation of endothelial nitric oxide synthase Ser633 and Ser1177 in human umbilical vein endothelial cells through Pim1/Akt pathway [J]. Acta Anatomica Sinica, 2023, 54(5): 546-552.
[10]	ZHOU Chun-hui XIE Sheng-qiang WANG Jiang-ting LAN Xiao-juan ZHANG Jian-ning ZHAO Hu-lin . Application of multimodal electromagnetic navigation in endoscopic endonasal skull base anatomical measurement of fresh cadavers [J]. Acta Anatomica Sinica, 2023, 54(5): 560-566.
[11]	LIU Yan-jing SHI Yong-hong . Automatic extraction of point cloud on cartilage surface of intraoperative knee using FPFH-PointNet [J]. Acta Anatomica Sinica, 2023, 54(5): 553-559.
[12]	MI Si-rong LIU Guang-xing ZHANG Zhen-wu YU Peng-hui JU Xiao-jun RAO Li-bing LI Li . Accuracy of 3D printing skull under different CT layer thickness [J]. Acta Anatomica Sinica, 2023, 54(5): 575-581.
[13]	ZHU Qiu-liang YU Xiang-ping MA Jun CHEN Yun-yun LIN Fang RUAN Wen-bin . Adjustment of X-ray angle intraoperation based on the anatomic shape of femoral neck section [J]. Acta Anatomica Sinica, 2023, 54(5): 586-592.
[14]	LI Xiao-jun ZHANG Ya-min. Screening ferroptosis related genes influencing prognosis of colon cancer through bioinformatics analysis [J]. Acta Anatomica Sinica, 2023, 54(4): 445-452.
[15]	LIU Hai-lan MEI Yi-xuan ZHANG Yi LIU Cheng LIU Shu-wei. Classification and clinical significance of the branching patterns of the right pulmonary arteries based on the intravital CT data [J]. Acta Anatomica Sinica, 2023, 54(4): 453-459.