大鼠冻存卵巢组织自体异位移植后长期功能评估

Long-term ovarian function in rats after heterotopic autologous transplantation of cryopreserved ovarian tissues

目的评估成年大鼠冻存卵巢组织自体异位移植后的长期生殖、内分泌功能。方法78只Wistar成年大鼠,随机分为假手术组（1组,15只）、新鲜组织移植组（2组,24只）、冻存组织移植组（3组,24只）、去势组（4组,15只）。将去势后成年大鼠的新鲜卵巢组织及冻存卵巢组织自体异位移植于肾被膜下,分别于移植后5、8、10个月处死各处理组1/3数量动物,取卵巢及子宫组织作组织学检查。通过阴道脱落细胞学检查、动情期雌、孕激素水平监测评估移植后卵巢组织的生殖内分泌功能。结果所有动物模型中,新鲜及冻存卵巢组织移植后都可观察到存活组织块。2、3组动情期雌、孕激素水平与假手术组无显著性差异（P〉0.05）,且均明显高于去势组（P〈0.01）。冷冻复苏后组织与新鲜组织各级卵泡构成比无显著性差异（P〉0.05）,各组不同时间取得的卵巢组织各级卵泡构成比相比较均无显著性差异（P〉0.05）。移植后5个月,2、3组原始卵泡明显减少,分别为假手术组的59.1%和54.5%。结论小块卵巢组织可耐受冻融过程;冻存卵巢组织自体肾被膜下移植后,卵巢组织形态无明显改变,原始卵泡总数虽然明显减少,但有成熟卵泡发育并排卵,并能长期维持生殖内分泌功能。

Objective To assess the long-term efficacy of cryopreserved ovarian tissues after heterotopic autologous transplanted in rats. Methods Seventy-eight adult Wistar rats were divided into four groups randomly as follows： sham-operated group （group 1, n = 15）, fresh-transplanted group （group 2, n ： 24）, frozen-thawed-transplanted group （group 3, n = 24） and ovariectomized group （group 4, n = 15）. Fresh and frozen-thawed ovarian tissues of adult rats were autologous transplanted under renal capsule. One third animals of each group were slaughtered and their ovaries and uteruses were removed for morphometric analysis at 5 months after transplantation; one third additional animals were slaughtered at 8 months; and the remaining animals were slaughtered at 10 months. The measurement of serum estradiol （E2） and progesterone （P） concentrations during the estrus stage and vaginal cytology were performed to assess the secretary function of implanted ovarian tissues. Results Both fresh and frozen ovarian grafts survived in all the animal models. In groups 2 and 3, the serum E： and progesterone concentrations during estrus remained comparable to the sham-operated rats＇ hormone levels （groupl）, and significantly higher than the concentrations in group 4 at 5, 8, 10 months respectively （ P 〈 0.01 ）. Morphologically, no significant differences were observed in the proportion of each stage of follicles between frozen-thawed tissues and fresh ovarian tissues （ P 〉 0. 05）. And the proportion of each stage follicles in both types of grafts was comparable to that in group 1 （ P 〉 0.05 ）. The number of primordial follicles in fresh grafts and frozen grafts were significantly less than that in group 1. Grafts in group 2 contained 59.1% of the primordial follicles in sham-operated controls while frozen grafts contained 54.5% at 5 months after implantation. Conclusion Cryopreservation of small pieces of ovarian tissues is feasible for keeping cell viability. After autologous implanted under the renal capsule, the cryopreserved ovarian tissues have no significant morphological changes. The follicles survive and develop well, though the number of primordial follicles decreased. The secretary function of ovarian tissues could be preserved in a long term.