卵巢新型玻璃化冷冻保存与移植重建化疗后大鼠卵巢功能的实验研究_《中国修复重建外科杂志》

作者：

李青 ¹ , 陈元诗 ² , 杨丽 ² , 孙愚 ³ , 汪燕 ⁴ , 朱江 ²

1四川大学华西临床医学院（成都，610041）;;
四川大学华西医院2胸部肿瘤科，3头颈肿瘤科;;
4四川大学华西第二医院生殖医学中心;

关键词：

化疗性卵巢损伤卵巢组织冷冻保存玻璃化冷冻法原位移植大鼠

DOI：

10.7507/1002-1892.20130244

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的探讨卵巢新型玻璃化冷冻法——细针穿刺浸入式玻璃化冷冻法（needle immersed vitrification，NIV）保存与移植重建化疗性卵巢损伤大鼠模型的卵巢功能效果。方法8～9周龄SPF级封闭群雌性Wistar大鼠52 只，体重250～300 g；选择至少有2次正常动情周期的50只大鼠纳入实验研究。随机取10只作为供体，采用NIV法冷冻保存卵巢组织并复苏。剩余40只大鼠根据处理方法不同随机分为3组：环磷酰胺组（C组，n=14）及环磷酰胺/移植组（C/T组，n=12）大鼠腹腔注射环磷酰胺，共21 d，建立化疗性卵巢功能损伤模型，C/T组将复苏的卵巢组织移植于大鼠左侧卵巢囊内；对照组（NS组，n=14）大鼠每日腹腔注射生理盐水，连续21 d。比较各组大鼠动情周期恢复情况、卵巢组织重量以及卵巢组织形态学改变、各级卵泡数目。结果C/T组1只大鼠于移植术后2 d死亡，其余大鼠均存活至实验完成。各组大鼠在注射结束后4周体重趋于一致，差异无统计学意义（P gt; 0.05）。NS组大鼠实验期间均具有规律、正常的动情周期，C组和C/T组大鼠在注射药物期间均出现动情周期紊乱。注射结束后，C组动情周期中位恢复时间为9 d（95%CI：7.9～10.1 d），C/T组为6 d（95%CI：4.9～7.1 d），两组比较差异有统计学意义（χ2=6.571，P=0.010）。C组在注射结束后4周卵巢组织重量较前减轻；C/T组大鼠移植卵巢组织均存活生长良好，其重量呈逐渐增加趋势。组织学观察示，C组与NS组、C/T组相比始基卵泡及初级卵泡数目均显著减少（P lt; 0.05），C/T组与NS组比较差异无统计学意义（P gt; 0.05）；各组次级卵泡和窦状卵泡数目比较，差异均无统计学意义（P gt; 0.05）。结论将NIV法冷冻保存的正常卵巢组织移植于化疗性卵巢损伤大鼠体内后，能显著缩短动情周期恢复时间，移植卵巢组织生长良好，各级卵泡计数接近正常水平，初步提示具有化疗后重建卵巢内分泌和生育能力的作用。

引用本文： 李青,陈元诗,杨丽,孙愚,汪燕,朱江. 卵巢新型玻璃化冷冻保存与移植重建化疗后大鼠卵巢功能的实验研究. 中国修复重建外科杂志, 2013, 27(9): 1116-1121. doi: 10.7507/1002-1892.20130244 复制

1.	Yan W, Xiao Z, Lei L, et al. Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation. Human Reproduction, 2008, 23(10): 2256-2265.
2.	Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod, 1986, 1(2): 81-87.
3.	Walshe JM, Denduluri N, Swain SM. Amenorrhea in premenopausal women after adjuvant chemotherapy for breast cancer. J Clin Oncol, 2006, 24(36): 5769-5577.
4.	De Bruin ML, Huisbrink J, Hauptmann M, et al. Treatment-related risk factors for premature menopause following Hodgkin’s lymphoma. Blood, 2007, 111(1): 101-108.
5.	Cvancarova M, Samuelsen SO, Magelssen H, et al. Reproduction rates after cancer treatment: experience from the Norwegian radium hospital. J Clin Oncol, 2009, 27(3): 334-343.
6.	Maltaris T, Seufert R, Fischi F, et al. The effect of cancer treatment on female fertility and strategies for preserving fertility. Eur J Obstet Gynecol Reprod Biol, 2007, 130(2): 148-155.
7.	Marhhom E, Cohen I. Fertility preservation options for women with malignancies. Obstet Gynecol Surv, 2007, 62(1): 58-72.
8.	Madalinska JB, van Beurden M, Bleiker EM, et al. The impact of hormone replacement therapy on menopausal symptoms in younger high-risk women after prophylactic salpingo-oophorectomy. J Clin Oncol, 2006, 24(22): 3576-3582.
9.	Mah PM, Webster J, Jönsson P, et al. Estrogen replacement in women of fertile years with hypopituitarism. J Clin Endocrinol Metab, 2005, 90(11): 5964-5969.
10.	Dahl SL, Chen Z, Solan AK, et al. Feasibility of vitrification as a storage method for tissue-engineered blood vessels. Tissue Eng, 2006, 12(2): 291-300.
11.	Keros V, Xella S, Hultenby K, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod, 2009, 24(7): 1670-1683.
12.	Xiao Z, Wang Y, Li L, et al. Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation. Fertil Steril, 2010, 94(6): 2323-2328.
13.	Meirow D. Ovarian injury and modern options to preserve fertility in female cancer patients treated with high dose radio-chemotherapy for hemato-oncological neoplasias and other cancers. Leuk Lymphoma, 1999, 33(1-2): 65-76.
14.	Ataya KM, McKanna JA, Weintraub AM, et al. A luteinizing hormoe-releasing hormone agonist for the prevention of chemotherapy-induced ovarian follicular loss in rats. Cancer Res, 1985, 45(8): 3651-3656.
15.	Letterie GS. Anovulation in the prevention of cytotoxic-induced follicular attrition and ovarian failure. Hum Reprod, 2004, 19(4): 831-837.
16.	Meirow D, Assad G, Dor J, et al. The GnRH antagonist cetrorelix reduces cyclophosphamide-induced ovarian follicular destruction in mice. Hum Reprod, 2004, 19(6): 1294-1299.
17.	Russel WR, Walpoe AL, Labhsetwar AP. Cyclophosphamide: induction of superovulation in rats. Nature, 1973, 241: 129-130.
18.	Meirow D, Dor J, Kaufman B, et al. Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. Potential mechanisms of ovarian injury. Hum Reprod, 2007, 22(6): 1626-1633.
19.	罗璐, 杨冬梓, 王箴, 等. 环磷酰胺对成年大鼠卵巢的损伤以及GnRHR 在卵巢的表达. 南方医科大学学报, 2007, 27(11): 1714-1717.
20.	罗满林, 顾为望. 实验动物学. 北京: 中国农业出版社, 2002: 145-149.
21.	Ghosh S, Misro M, Das UB, et al. Effect of human chorionic gonadotrophin coadministration on ovarian steroidogenic and folliculogenic activities in cyclophosphamide treated albino rats. Reprod Toxicol, 2001, 15(2): 221-225.

1. Yan W, Xiao Z, Lei L, et al. Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation. Human Reproduction, 2008, 23(10): 2256-2265.
2. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod, 1986, 1(2): 81-87.
3. Walshe JM, Denduluri N, Swain SM. Amenorrhea in premenopausal women after adjuvant chemotherapy for breast cancer. J Clin Oncol, 2006, 24(36): 5769-5577.
4. De Bruin ML, Huisbrink J, Hauptmann M, et al. Treatment-related risk factors for premature menopause following Hodgkin’s lymphoma. Blood, 2007, 111(1): 101-108.
5. Cvancarova M, Samuelsen SO, Magelssen H, et al. Reproduction rates after cancer treatment: experience from the Norwegian radium hospital. J Clin Oncol, 2009, 27(3): 334-343.
6. Maltaris T, Seufert R, Fischi F, et al. The effect of cancer treatment on female fertility and strategies for preserving fertility. Eur J Obstet Gynecol Reprod Biol, 2007, 130(2): 148-155.
7. Marhhom E, Cohen I. Fertility preservation options for women with malignancies. Obstet Gynecol Surv, 2007, 62(1): 58-72.
8. Madalinska JB, van Beurden M, Bleiker EM, et al. The impact of hormone replacement therapy on menopausal symptoms in younger high-risk women after prophylactic salpingo-oophorectomy. J Clin Oncol, 2006, 24(22): 3576-3582.
9. Mah PM, Webster J, Jönsson P, et al. Estrogen replacement in women of fertile years with hypopituitarism. J Clin Endocrinol Metab, 2005, 90(11): 5964-5969.
10. Dahl SL, Chen Z, Solan AK, et al. Feasibility of vitrification as a storage method for tissue-engineered blood vessels. Tissue Eng, 2006, 12(2): 291-300.
11. Keros V, Xella S, Hultenby K, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod, 2009, 24(7): 1670-1683.
12. Xiao Z, Wang Y, Li L, et al. Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation. Fertil Steril, 2010, 94(6): 2323-2328.
13. Meirow D. Ovarian injury and modern options to preserve fertility in female cancer patients treated with high dose radio-chemotherapy for hemato-oncological neoplasias and other cancers. Leuk Lymphoma, 1999, 33(1-2): 65-76.
14. Ataya KM, McKanna JA, Weintraub AM, et al. A luteinizing hormoe-releasing hormone agonist for the prevention of chemotherapy-induced ovarian follicular loss in rats. Cancer Res, 1985, 45(8): 3651-3656.
15. Letterie GS. Anovulation in the prevention of cytotoxic-induced follicular attrition and ovarian failure. Hum Reprod, 2004, 19(4): 831-837.
16. Meirow D, Assad G, Dor J, et al. The GnRH antagonist cetrorelix reduces cyclophosphamide-induced ovarian follicular destruction in mice. Hum Reprod, 2004, 19(6): 1294-1299.
17. Russel WR, Walpoe AL, Labhsetwar AP. Cyclophosphamide: induction of superovulation in rats. Nature, 1973, 241: 129-130.
18. Meirow D, Dor J, Kaufman B, et al. Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. Potential mechanisms of ovarian injury. Hum Reprod, 2007, 22(6): 1626-1633.
19. 罗璐, 杨冬梓, 王箴, 等. 环磷酰胺对成年大鼠卵巢的损伤以及GnRHR 在卵巢的表达. 南方医科大学学报, 2007, 27(11): 1714-1717.
20. 罗满林, 顾为望. 实验动物学. 北京: 中国农业出版社, 2002: 145-149.
21. Ghosh S, Misro M, Das UB, et al. Effect of human chorionic gonadotrophin coadministration on ovarian steroidogenic and folliculogenic activities in cyclophosphamide treated albino rats. Reprod Toxicol, 2001, 15(2): 221-225.

《中国修复重建外科杂志》

卵巢新型玻璃化冷冻保存与移植重建化疗后大鼠卵巢功能的实验研究

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《中国修复重建外科杂志》

卵巢新型玻璃化冷冻保存与移植重建化疗后大鼠卵巢功能的实验研究

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