母兔卵巢异体异位移植的实验研究

目的:建立雌兔异体卵巢组织移植模型,探索非血管吻合卵巢组织移植的可行性。方法:以雌兔为实验动物,A组:阳性对照,不做任何处理;B组:阴性对照,切除双侧卵巢;C组:卵巢组织异体异位移植组,分离卵巢组织,切成约1mm×1mm×1mm大的组织块,移植于异体颈部皮下,术后观察阴道脱落细胞涂片变化,同时于移植后第18、25、35、45、55天检测血清E2及P水平,60天处死母兔,对移植物及子宫内膜进行组织学检查。结果:(1)C组3只雌兔阴道呈红色或粉红色、湿润、肿胀,进入发情期;(2)A、C组术后25天血清E2及P水平均高于B组,并逐渐下降,35天达到最低水平,随后又呈上升趋势,至55天已明显高于B组(P<0.05),B组则始终维持在较低水平,未出现升高趋势;(3)A、C组卵巢组织内可见不同发育阶段的卵泡。结论:兔卵巢组织异体异位移植后移植物可存活,并释放内源性激素,对靶器官产生作用。     

人卵巢组织异种移植模型研究进展

近年来,肿瘤治疗的进展明显提高了年轻女性患者的生存率。但抗肿瘤治疗的长期不良反应会使女性患者产生卵巢早衰和不育。对尚未生育的女性患者来说,保留生育功能至关重要。卵巢组织异种移植技术可以避免将肿瘤细胞重新带回体内的风险,也可以降低体内促排卵带来的危害,而近年来多种异种移植模型的建立为保留人类生殖功能提供了可能。从移植物的来源、培养温度及处理时间、移植部位、受体的选择与处理、促性腺激素的应用等方面对当前人卵巢组织异种移植的研究进展作一综述。     

自体冷冻卵巢组织移植重建生育功能

由于卵巢组织对烷化剂、放疗甚为敏感．随着女性恶性疾病中放化疗应用的增多，引起卵巢功能衰竭，导致内分泌失调和生育功能丧失者随之增多。报道1例应用化、放疗前获取自体卵巢组织，待其治疗后再行移植术，重建内分泌和生育功能的成功经验。     

冻融后移植的小鼠卵巢组织对促性腺激素的反应

目的 探讨冻融后移植的小鼠卵巢组织对促性腺激素的反应.方法 将36只性成熟雌性小白鼠随机分为新鲜移植组、冻融移植组和对照组,每组12只.新鲜移植组小鼠切除双侧卵巢,将卵巢切成小组织块,立即移植人双侧肾被膜下;冻融移植组小鼠切除双侧卵巢,将卵巢切成小组织块,采用玻璃化冷冻方法冷冻保存,2周后将冷冻卵巢组织复苏,移植入小鼠双侧肾被膜下.卵巢组织移植2周后,新鲜移植组和冻融移植组每组随机取6只小鼠应用7.5 IU人绝经期促性腺激素及10 IU绒毛膜促性腺激素,观察移植后的卵巢组织对促性腺激素的反应.同时应用免疫组化染色方法观察各组卵泡中卵泡刺激素受体的表达情况.结果 新鲜移植组、冻融移植组和对照组未应用促性腺激素小鼠卵巢组织内近成熟卵泡百分率分别为2.3%、2.3%和2.6%,应用促性腺激素小鼠卵巢组织内近成熟卵泡百分率分别为4.2%、4.0%和5.8%,各组内分别比较,差异均有统计学意义(P＜0.05);新鲜移植组和冻融移植组与对照组比较,差异均元统计学意义(P＞0.05).新鲜移植组、冻融移植组和对照组卵泡刺激素受体表达积分吸光度值在窦状卵泡中分别为9408±2777、9175±3093和8838±2064,在窦前卵泡中分别为4531±1903、4808±1386和5516±1136,各组间分别比较,差异均无统计学意义(P＞0.05).结论 卵巢组织冷冻保存、复苏及移植过程未影响卵巢卵泡刺激素受体的表达,冻融后移植的小鼠卵巢组织对外源性促性腺激素的反应未受冷冻、复苏及移植等过程影响.     

17β-雌二醇与N-甲基-N-硝基-N-亚硝基胍诱导ICR小鼠子宫内膜癌的研究

目的 :用 17β 雌二醇 (E2 )与N 甲基 N 硝基 N 亚硝基胍 (MNNG)诱导ICR小鼠子宫内膜癌 ,研究子宫内膜癌变的诱发因素。方法 :选择 6周龄ICR雌性小鼠 90只 ,分为 4组。Ⅰ组 (空白对照组 ) ,小鼠 12只 ,常规喂养 ;Ⅱ组 (MNNG +E2 组 ) ,小鼠 2 6只 ,经阴道宫腔给予MNNG ,每周 1次 ,共 3次 ,在给予MNNG的同时喂E2 ;Ⅲ组 (MNNG组 ) :小鼠2 6只 ,仅给予MNNG ;Ⅳ组 (E2 组 ) :小鼠 2 6只 ,仅给予E2 。于实验后 3个月、6个月及 9个月各组取若干只小鼠 ,测定血清雌激素 (E)及孕酮 (P) ,并作子宫内膜病理组织学检查。结果 :实验 3个月时 ,Ⅳ组子宫内膜发生复杂型 (3 5 )或不典型增生性改变 (2 5 ) ;实验 6个月时 ,Ⅱ组、Ⅲ组、Ⅳ组均有部分发生复杂型或不典型增生过长及癌变 ,但 3组间差异无显著性 (P >0 .0 5 ) ;实验 9个月时Ⅱ组复杂型增生、不典型增生、内膜癌分别为 35 .7% (5 14)、2 8.6% (4 14)、35 .7% (5 14) ,Ⅳ组以上病变分别为 14.3% (2 14)、4 2 .9% (6 14)、4 2 .9%(6 14) ,Ⅱ组、Ⅳ组间相比差异无显著性 (P >0 .0 5 )。实验 6个月与 9个月时 ,喂E2 组E P均明显高于喂水组 (P <0 .0 1或P <0 .0 5 )。结论 :单独给予E2 或MNNG后 ,即能较好地诱发子宫内膜癌。E2 与MNNG联合给药时 ,效果并未较?     

复发性子宫内膜异位症根治术时自体卵巢组织移植的价值

目的 探讨保守手术后复发的重症子宫内膜异位症(内异症)根治术时自体卵巢组织移植的应用价值。方法 1997年2月-2000年3月,对17例40岁以下,内异症保守手术后出现复发性疼痛和盆腔包块,药物治疗无效的Ⅳ期内异症患者,施行根治术时将自体卵巢组织片移植于大网膜之中。术后雌激素替代治疗3个月。停药后1个月后每月测定卵泡刺激素(FSH)、黄体生成素(LH)和雌激素(E2),直至卵巢功能恢复。E2水平上升后,测定基础体温,每周做阴道脱落细胞检查,计算成熟指数(MI)。每4次为1个周期。结果17例患者中,8例术后4个月血清E2水平上升,7例术后6个月E2水平上升,2例术后12个月E2维持在低水平,FSH、LH上升被列为卵巢组织失活。卵巢组织移植成活率为88.2％。卵巢功能恢复后,基础体温呈双相型,阴道脱落细胞涂片呈现周期性变化片型。随访17例患者均无复发性疼痛和盆腔包块。结论 对卵巢组织破坏严重的复发性重症内异症患者,根治术时实施卵巢组织移植是避免内异症复发,重建卵巢功能的有效方法。     

玻璃化冷冻保存羊卵巢组织异种移植的实验研究

目的:探讨羊卵巢组织玻璃化冷冻复苏后异种移植的卵泡生长发育情况。方法:取绵羊的卵巢,将卵巢皮质切块,应用玻璃化快速冷冻法保存。羊卵巢组织片复苏后在裸鼠的颈部皮下移植,每只裸鼠移植2块组织,于移植1个月后获取移植物,HE染色观察存活卵泡的情况。结果:共移植组织16块,取材时肉眼可见其中一块组织纤维化萎缩,镜下见此块组织纤维化,移植组织存活率87.5%(14/16)。HE染色观察每只移植小鼠的2块移植物中基本只有1块有可视卵泡。1只鼠未见可视卵泡;1只鼠见1个不典型原始卵泡;1只鼠有2个不典型原始卵泡;余下5只鼠的移植卵巢组织中均可见多个典型的存活卵泡。结论:羊卵巢组织经冷冻复苏后能够成功移植于裸鼠的颈部皮下,大部分卵泡处于良好的存活状态。     

冻存卵巢组织移植的影响因素

冻存卵巢组织移植作为一种保存年轻女性生殖内分泌功能的治疗方法,正日益受到重视,但其影响因素众多,技术程序有待进一步优化.检索综述中英文文献,探索冻存卵巢组织移植的影响因素发现,冻存卵巢组织移植的影响因素包括冷冻保存过程、移植部位、抗氧化损伤、生长因子、激素、机械因素、临床因素等.目前卵巢移植还存在许多亟待解决的问题,随着冷冻保存技术、血管吻合技术和辅助生殖技术的发展,冻存卵巢组织移植将成为解决卵巢早衰、保存年轻癌症患者生殖内分泌功能的有效途径.     

子宫内膜异位症根治术时自体卵巢组织移植的应用

目的　评价保守手术后复发的重症子宫内膜异位症 (内异症 )根治术时实施自体卵巢组织移植的价值。方法　常州市妇幼保健院 1997年 2月至 2 0 0 1年 12月对 2 5例子宫内膜异位症保守手术后出现复发的Ⅳ期内异症患者 ,施行根治术或剩余卵巢切除术 ,术时将自体卵巢组织片移植于大网膜之中。术后测定卵泡刺激素(FSH)、黄体生成激素 (LH)和雌二醇 (E2 )。E2 水平上升后 ,测定基础体温 ,做阴道脱落细胞学检查。结果　 2 5例患者中 ,2 0例术后 4～ 6个月E2 水平上升 ,卵巢组织移植成活率为 80 0 %。卵巢功能维持时间最长者已达 4年以上。仅 1例出现内异症复发 ,复发率为 5 0 %。结论　对卵巢组织破坏严重的复发性重症内异症患者 ,根治术时实施卵巢组织移植是保留卵巢功能 ,减少内异症复发的有效方法。     

显微血管吻合卵巢自体移植研究

治疗早期宫颈癌的传统手术为子宫广泛切除加盆腔淋巴组织清扫,该手术包括切除两侧卵巢。年轻患者人工去势,会过早出现类似更年期综合征的表现,发生骨质疏松症和心血管疾患。从心理学、生理学、内分泌学的角度出发,在确保肿瘤手术疗效的前提下,力求保留年轻宫颈癌患者的卵巢功能,提高其生存质量,已引起了学者们的重视［１－３］。在多年从事显微血管吻合、器官移植的基础上［４－７］,我们就卵巢移植的手术方式,移植部位,术后性激素变化进行了初步研究。１资料与方法１．１临床资料选择５例年轻宫颈癌 Ⅰｂ期患者,１９－３８岁,…     

Heterotopic ovarian transplantation without vascular pedicle in syngeneic Lewis rats: six-month control of estradiol and follicle-stimulating hormone concentrations after intraperitoneal and subcutaneous implants.

OBJECTIVE: To assess the recovery, maintenance, and quality of ovarian function by comparing the success of autotransplantation in intraperitoneal (IP) and SC locations over a 6-month period in syngeneic Lewis rats. DESIGN: Experimental animal study. SETTING: Unit of Experimental Research at the Barcelona University School of Medicine. ANIMAL(S): Female syngeneic Lewis rats. INTERVENTION(S): The animals were randomized to one of three groups: group A, control group with ovariectomy (n = 15); group B, ovariectomy and IP autologous heterotopic transplant of ovarian tissue without vascular pedicle (n = 14); and group C, ovariectomy and SC autologous heterotopic transplant (n = 15). MAIN OUTCOME MEASURE(S): Serum levels of FSH and 17beta-E2 and vaginal cytology. RESULT(S): In groups B and C, E2 serum concentrations from day 7 and day 10 onward remained comparable to basal levels, and significantly higher than in group A, throughout the 6-month period. In group B from day 7 after surgery onward, and in group C from day 10 after surgery onward, FSH concentrations remained low (comparable to basal levels) throughout the follow-up period. Vaginal cytology of groups B and C showed trophic maturation between days 4 and 10 after ovariectomy and insertion of the ovarian tissue implant, whereas the control group remained atrophic. There were no statistically significant differences between IP and SC implants. CONCLUSION(S): A heterotopic autotransplant of ovarian tissue without vascular pedicle in syngeneic Lewis rats is successful for > or =6 months.     

Heterotopic ovarian transplantation without vascular pedicle in syngeneic Lewis rats: long-term evaluation of effects on ovarian structure and function

OBJECTIVE: To assess the long-term efficacy of intraperitoneal (IP) and subcutaneous (SC) ovarian autotransplantation in rats. DESIGN: Experimental animal study. SETTING: Unit of Experimental Research, Barcelona University School of Medicine. ANIMAL(S): Female syngeneic Lewis rats aged 14 weeks. INTERVENTION(S): Group A, control group undergoing ovariectomy (n = 15); group B, undergoing ovariectomy and IP autologous heterotopic transplant (n = 15); and group C, ovariectomized with SC autologous heterotopic transplant (n = 15). In groups B and C, five animals were killed and their ovaries removed for morphometric analysis at 30 days after transplantation; five additional animals were killed at 180 days, and the remaining five animals were killed at 360 days. MAIN OUTCOME MEASURE(S): Ovarian morphometric analysis and serial measurement of E(2) and FSH serum levels. RESULT(S); The mean number of antral follicles in the control group A was significantly higher than that observed in the ovarian grafts collected and examined 30 days after grafting in rats from groups B and C, but the mean granulosa cell area was significantly higher in both transplantation groups than in controls because of ovarian follicular hyperplasia. Histological examination of ovaries removed at 6 and 12 months after grafting in groups B and C showed increasing degrees of fibrosis, loss of primordial follicles, and the presence of epithelial cysts. In groups B and C, from day 30 after surgery onward, serum E(2) was significantly higher and FSH significantly lower, respectively, than in group A. E(2) and FSH patterns in groups B and C were similar throughout the study period. CONCLUSION(S): Heterotopic ovarian transplantation without vascular pedicle in rats is characterized by follicular hyperplasia endocrinologically functional, followed by progressive loss of follicles in heterotopic ovarian autografts.     

Morphologic,endocrinologic and natural pregnancy assessment of allogeneic ovarian orthotopic transplantation without a vascular pedicle in rabbits

Objective

The objective was to assess the natural pregnancy of rabbits subjected to bilateral oophorectomy and orthotopic allogeneic intact and sliced ovarian transplantation without a vascular pedicle, and to determine the morphofunctional aspects of the transplanted ovaries.

Study design

Thirty-two female rabbits were studied. The ovaries were removed and orthotopically transplanted without vascular anastomoses between the two breeds of rabbits. In Group 1 (n = 8), only laparotomy and laparorrhaphy were performed, in Group 2A (n = 8) intact ovaries were transplanted on both sides, in Group 2B (n = 8) both ovaries were sliced and orthotopically transplanted, and in Group 2C (n = 8) an intact ovary was transplanted on one side and a sliced ovary on the other side. Three months later, the animals were paired with males for copulation. Estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone levels were assessed. A histologic study was carried out and the numbers of pregnancies and litters were also determined.

Results

Pregnancies occurred in seven rabbits in Group 1, 37.5% of rabbits in Group 2A, 50% in Group 2B, and 62.5% in Group 2C. Hormone levels and histology confirmed the vitality and function of all ovaries.

Conclusions

Intact or sliced orthotopic allogeneic ovarian transplantation without a vascular pedicle is viable in rabbits, and preserves their hormonal and fertile functions.     

荷人卵巢上皮癌裸鼠冻融卵巢组织移植的效果评价

目的:获取人卵巢上皮癌裸鼠原位移植瘤癌旁正常卵巢组织,经安全筛查并冻融后移植至去势裸鼠体内,探讨移植后效果,为临床应用提供依据。方法:将人卵巢上皮癌OVCAR3细胞种植于裸鼠皮下以获取瘤源,并进行卵巢原位移植,建立卵巢癌原位移植瘤模型,解剖裸鼠获取癌旁正常卵巢组织。癌旁组:取筛选后的癌旁卵巢组织进行玻璃化冷冻,复苏后分别进行皮下及原位移植,各20例;对照组:同龄正常裸鼠卵巢组织,皮下移植及原位移植各20例;去势裸鼠组:20只同龄去势裸鼠;正常卵巢组:20只同龄正常裸鼠。移植12周后,分析各组裸鼠卵巢组织内卵泡形态及激素分泌功能。结果:40只人卵巢上皮癌裸鼠原位移植瘤模型中共获取35份活检正常的癌旁卵巢组织,获取率87.5%(35/40)。玻璃化冷冻前后卵巢组织中卵泡形态及各级卵泡比例均无显著差异(P>0.05),癌旁冷冻组织和癌旁新鲜组织的异常卵泡比例差异无统计学意义(P>0.05),冷冻组织以初级卵泡及次级卵泡等窦前卵泡为主。癌旁组皮下移植组织存活率80%(16/20),对照组皮下移植组织存活率90%(18/20),癌旁组原位移植组织存活率90%(18/20),对照组原位移植组织存活率95%(19/20)。移植后组织内卵泡以次级卵泡及窦状卵泡为主,各级卵泡的形态及构成比和未移植的同龄正常裸鼠卵巢相似(P>0.05);癌旁组卵泡数明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植组织内的各级卵泡数差异无统计学意义(P>0.05)。癌旁组卵泡刺激素水平明显低于去势裸鼠组,而雌二醇水平明显高于去势裸鼠组(P<0.01);癌旁组卵泡刺激素水平明显高于对照组(P<0.05)及正常卵巢组(P<0.01),而雌二醇水平明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植的卵泡刺激素水平和雌二醇水平差异无统计学意义(P>0.05)。结论:癌旁卵巢组织冻融移植后有正常卵泡发育及激素分泌功能;皮下移植和原位移植均可取得较好的效果;癌旁卵巢组织冻融移植有望作为卵巢上皮癌患者治疗后恢复卵巢内分泌功能的有效手段。     

Impact of the cryopreservation technique and vascular bed on ovarian tissue transplantation in cynomolgus monkeys

Purpose

The aim of this study was to determine the best combination in terms of cryopreservation techniques and vascular bed preparation before grafting in order to obtain functional ovarian tissue after transplantation.

Methods

Five cynomolgus monkeys were used. Strips from 10 ovaries were cryopreserved, 5 by vitrification (V), and 5 by slow-freezing (SF). Pieces of fresh ovarian tissue were used for controls. After 1 month, the strips were autografted to two different vascular beds, healed (HB) or freshly decorticated (FDB), constituting four study groups: SF-HB, SF-FDB, V-HB, and V-FDB. These were compared to fresh tissue. After 6 months, the ovaries were removed and several parameters analyzed: follicle quality, stage, density, proliferation, apoptosis, functionality, vascularization, and fibrosis. Mixed effect linear regression models were built to assess the impact of cryopreservation and vascular bed preparation on ovarian tissue viability and functionality. p values were adjusted for multiple testing using the Benjamini-Hochberg method, and q values < 0.20 were considered significant in order to achieve a 20 % false discovery rate.

Results

Compared to fresh tissue, no difference was observed in the percentage of morphologically normal follicles, while a significant increase was noted in the follicle proliferation rate (41 %, q = 0.19), percentage of antral follicles (12 %, q = 0.14), and number of vessels per area (3.3 times, q = 0.07) in the V-FDB group.

Conclusions

Vitrification associated with FDB vascular bed preparation is the best combination to obtain functional autografted ovarian tissue. Further studies are nevertheless required, with confirmed pregnancies and live births before introducing the procedure into clinical practice.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-015-0542-y) contains supplementary material, which is available to authorized users.