改良完整卵巢冷冻移植对去势大鼠内分泌功能的影响

Effect of optimized whole ovarian cryopreservation and transplantation on ovarian function in ovariectomized rats

目的探讨改良灌注后完整卵巢冷冻移植对卵巢去势大鼠内分泌功能的影响。方法选择8~10周龄的健康、雌性清洁级Lewis大鼠30只。10只成熟雌性Lewis大鼠作为供体,获取带蒂完整卵巢进行改良连续导入的精准灌注后冷冻。将20只Lewis大鼠随机分为正常对照组和去势移植组,去势移植组于卵巢去势后1周进行完整卵巢异体原位移植,并于移植前、移植后2周、移植后4周观察大鼠的一般情况、动情周期、血清激素水平改变,于移植后4周检测卵巢组织形态学改变,评估大鼠卵巢内分泌功能。结果去势移植组在大鼠完整卵巢异体原位移植术后3 d体重显著低于正常对照组[(197.1±0.8)g比(201.4±0.6)g,P<0.05];移植第4天后,体重增长显著高于正常对照组[(1.56±0.44)g/d比(1.51±0.52)g/d,P>0.05]。去势移植组大鼠完整卵巢冷冻移植术后(11±2)d恢复正常动情周期。移植前,去势移植组大鼠的卵泡刺激素(follicle-stimulating hormone,FSH)水平显著高于正常对照组,黄体生成素(luteinizing hormone,LH)、雌二醇、抗米勒管激素(anti-Müllerian hormone,AMH)水平则显著低于正常对照组(P值均<0.01)。移植2周后,去势移植组大鼠的FSH水平较移植前显著降低,但显著高于正常对照组(P<0.05);LH、AMH水平较移植前显著升高,但显著低于正常对照组(P值均<0.05);雌二醇水平较移植前显著升高(P<0.05),与正常对照组的差异无统计学意义(P>0.05)。移植4周后,去势移植组大鼠FSH水平较移植前显著降低(P<0.05),雌二醇、LH和AMH水平均显著高于移植前(P值均<0.05),FSH、LH和雌二醇与正常对照组的差异无统计学意义(P值均>0.05),但AMH仍显著低于正常对照组(P<0.05)。去势移植组大鼠的卵巢始基卵泡、初级卵泡和总卵泡数均显著少于正常对照组(P值均<0.05)。次级卵泡、窦卵泡数量与正常对照组的差异均无统计学意义(P值均>0.05)。结论改良完整卵巢冷冻移植至卵巢去势大鼠,动情周期和激素水平恢复正常,提示完整卵巢在连续导入的精准灌注后冷冻移植可有效重建卵巢内分泌功能。

Objective To explore the effect of optimized whole ovarian cryopreservation and transplantation on ovarian function in ovariectomized rats.Methods Thirty healthy,female clean-grade Lewis rats of 8 to 10 weeks of age were selected.Ten female adult Lewis rats were used as donors.Improved perfusion was applied after whole ovary harvesting.Twenty Lewis rats were randomly divided into the normal control group and the transplantation group.Whole ovarian cryopreservation and allotransplantation were performed after one week of ovariectomy in the transplantation group.The general state,estrous cyclicity,and gonadal hormones were observed before transplantation,2 weeks and 4 weeks after transplantation,respectively.The ovarian pathological changes were assessed after 4 weeks of transplantation.Results The body weight in the transplantation group was significantly lower than that in the control group after 3 d of transplantation of intact ovaries([197.1±0.8]g vs.[201.4±0.6]g,P<0.05).After 4 d of transplantation,the weight gain was similar with that in the control group([1.56±0.44]g/d vs.[1.51±0.52]g/d,P>0.05).In the transplantation group,the complete ovaries returned to normal estrous cycle after(11±2)d after transplantation.Before transplantation,the level of follicle-stimulating hormone(FSH)in the transplantation group was significantly higher than that in the control group,while the levels of luteinizing hormone(LH),estradiol and anti-Müllerian hormone(AMH)in the transplantation group were significantly lower than those in the control group(P<0.01).After two weeks of transplantation,the level of FSH in the transplantation group was significantly lower than that before transplantation,but still significantly higher than that in the control group(P<0.05).The levels of LH and AMH were significantly higher than those before transplantation,but still significantly lower than those in the control group(P<0.05).The level of estradiol was significantly higher than that before transplantation(P<0.05),and there was no significant difference between the two groups(P>0.05).After four weeks of transplantation,the level of FSH in the transplantation group was significantly lower than that before transplantation(P<0.05),and the levels of estradiol,LH and AMH were significantly higher than that before transplantation(P<0.05).There was no significant difference in the levels of FSH,LH and estradiol compared with the control group(P>0.05),but AMH was still significantly lower than that in the control group(P<0.05).Compared with the control group,the number of primordial follicles and primary follicles were significantly decreased in the transplantation group(P<0.05).There were no significant differences in the number of secondary follicles or antral follicles between two groups(P>0.05).Conclusion The estrous cycle and hormone level can be restored and ovarian function can be effectively reconstructed after whole ovarian cryopreservation and transplantation in ovariectomized rats by utilizing optimized perfusion.