卵巢新型玻璃化冷凍保存與移植重建化療后大鼠卵巢功能的實驗研究_《中國修復重建外科雜志》

作者：

李青 ¹ , 陳元詩 ² , 楊麗 ² , 孫愚 ³ , 汪燕 ⁴ , 朱江 ²

1四川大學華西臨床醫學院（成都，610041）;;
四川大學華西醫院2胸部腫瘤科，3頭頸腫瘤科;;
4四川大學華西第二醫院生殖醫學中心;

關鍵詞：

化療性卵巢損傷卵巢組織冷凍保存玻璃化冷凍法原位移植大鼠

DOI：

10.7507/1002-1892.20130244

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目的探討卵巢新型玻璃化冷凍法——細針穿刺浸入式玻璃化冷凍法（needle immersed vitrification，NIV）保存與移植重建化療性卵巢損傷大鼠模型的卵巢功能效果。方法8～9周齡SPF級封閉群雌性Wistar大鼠52 只，體重250～300 g；選擇至少有2次正常動情周期的50只大鼠納入實驗研究。隨機取10只作為供體，采用NIV法冷凍保存卵巢組織并復蘇。剩余40只大鼠根據處理方法不同隨機分為3組：環磷酰胺組（C組，n=14）及環磷酰胺/移植組（C/T組，n=12）大鼠腹腔注射環磷酰胺，共21 d，建立化療性卵巢功能損傷模型，C/T組將復蘇的卵巢組織移植于大鼠左側卵巢囊內；對照組（NS組，n=14）大鼠每日腹腔注射生理鹽水，連續21 d。比較各組大鼠動情周期恢復情況、卵巢組織重量以及卵巢組織形態學改變、各級卵泡數目。結果C/T組1只大鼠于移植術后2 d死亡，其余大鼠均存活至實驗完成。各組大鼠在注射結束后4周體重趨于一致，差異無統計學意義（P gt; 0.05）。NS組大鼠實驗期間均具有規律、正常的動情周期，C組和C/T組大鼠在注射藥物期間均出現動情周期紊亂。注射結束后，C組動情周期中位恢復時間為9 d（95%CI：7.9～10.1 d），C/T組為6 d（95%CI：4.9～7.1 d），兩組比較差異有統計學意義（χ2=6.571，P=0.010）。C組在注射結束后4周卵巢組織重量較前減輕；C/T組大鼠移植卵巢組織均存活生長良好，其重量呈逐漸增加趨勢。組織學觀察示，C組與NS組、C/T組相比始基卵泡及初級卵泡數目均顯著減少（P lt; 0.05），C/T組與NS組比較差異無統計學意義（P gt; 0.05）；各組次級卵泡和竇狀卵泡數目比較，差異均無統計學意義（P gt; 0.05）。結論將NIV法冷凍保存的正常卵巢組織移植于化療性卵巢損傷大鼠體內后，能顯著縮短動情周期恢復時間，移植卵巢組織生長良好，各級卵泡計數接近正常水平，初步提示具有化療后重建卵巢內分泌和生育能力的作用。

引用本文： 李青,陳元詩,楊麗,孫愚,汪燕,朱江. 卵巢新型玻璃化冷凍保存與移植重建化療后大鼠卵巢功能的實驗研究. 中國修復重建外科雜志, 2013, 27(9): 1116-1121. doi: 10.7507/1002-1892.20130244 復制

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.	羅璐, 楊冬梓, 王箴, 等. 環磷酰胺對成年大鼠卵巢的損傷以及GnRHR 在卵巢的表達. 南方醫科大學學報, 2007, 27(11): 1714-1717.
20.	羅滿林, 顧為望. 實驗動物學. 北京: 中國農業出版社, 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. 羅璐, 楊冬梓, 王箴, 等. 環磷酰胺對成年大鼠卵巢的損傷以及GnRHR 在卵巢的表達. 南方醫科大學學報, 2007, 27(11): 1714-1717.
20. 羅滿林, 顧為望. 實驗動物學. 北京: 中國農業出版社, 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.

《中國修復重建外科雜志》

卵巢新型玻璃化冷凍保存與移植重建化療后大鼠卵巢功能的實驗研究

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《中國修復重建外科雜志》

卵巢新型玻璃化冷凍保存與移植重建化療后大鼠卵巢功能的實驗研究

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