【作者】 徐大宝；

【导师】 叶啟发；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：建立大鼠胚胎卵巢原位移植模型,探讨大鼠胚胎卵巢原位移植后的生殖内分泌功能恢复情况,为进一步研究其生育和子代健康安全性提供实验基础。方法：将90只日龄50-60天的雌性SD大鼠随机分为移植受体组(研究组)50只、正常对照组(阳性对照组)20只、去势对照组(阴性对照组)20只。研究组行孕17-19天胎龄胚胎卵巢原位移植(同时切除自身卵巢)、阳性对照组仅行开关腹手术、阴性对照组行自身卵巢切除而不行胚胎卵巢移植。于术后45天随机取研究组、阳性和阴性对照组大鼠分别10只,共30只(其余60只雌性大鼠继续饲养供第二章实验用),采用电发光方法检测各组大鼠血清雌、孕激素水平,并同时剖腹探查卵巢发育情况、切除卵巢行病理切片HE染色观察卵泡发育情况。结果：(1)移植术后雌孕激素恢复情况：移植后45天,研究组、阳性和阴性对照组(每组各10只雌性大鼠)的雌激素均数±标准差分别为114.65±15.59 pmol／L.121.71±14.92 pmol／L和56.51±7.82 pmol、L,孕激素分别为12.79±4.40 ng、ml.13.00±1.18 ng/ml和3.70±0.68 ng/ml。研究组和阳性对照组间雌孕激素水平比较无显著性差异(p>0.05),而研究组和阴性对照组间雌孕激素水平比较有显著性差异。(p<0.05)。(2)移植后卵巢发育情况：移植后45天,研究组、阳性和阴性对照组的卵巢容积均数±标准差分别为0.22±0.07 ml、0.24±0.09ml和0ml,研究组、阳性对照组间卵巢容积比较无显著性差异(p>0.05)。光镜下研究组和阳性对照组卵巢内均可见不同阶段发育的卵泡和黄体形成。结论：大鼠胚胎卵巢原位移植术后的卵巢可以存活,并可生长发育而具有排卵功能,其生殖内分泌功能可以恢复。目的：探讨大鼠胚胎卵巢原位移植后生育功能的恢复情况。方法：将第一章研究中没有处死的大鼠胚胎卵巢原位移植受体组(研究组)40只、正常对照组(阳性对照组)10只、去势组(阴性对照组)10只雌性大鼠纳入本实验中。将三组动物于术后45天后分笼分别与日龄45-65天雄性大鼠同笼饲养(雌雄比例为2:1),观察40天,比较各组雌鼠妊娠和生育情况(观察到母鼠妊娠后即和雄性大鼠分开饲养)。观察40天后解剖研究组未孕雌性大鼠探查卵巢发育及其周围粘连并与正常对照组盆腔解剖情况比较,并观察卵泡发育情况,以判断没有生育的雌性大鼠的不孕原因。结果：(1)妊娠情况：研究组、阳性对照组、阴性对照组妊娠率分别为47.5%(19/40)、100%(10/10)、0.0%(0/10)。研究组雌性大鼠观察40天内的妊娠低于阳性对照组的,两者比较有显著性差异(p<0.05)；研究组和阴性对照组的妊娠率比较有显著性差异(p<0.05)。(2)子代个数：研究组、阳性对照组、阴性对照组平均每次妊娠子代数目分别为7.0±2.2、10.5±2.3、0,研究组和阳性对照组每次妊娠生育的子代个数比较有显著性差异(p<0.05)。(3)不孕原因分析：研究组21只不孕大鼠卵巢内可见成熟卵泡发育及黄体；但其卵巢及输卵管周围均存在严重的粘连,而正常对照组无明显的盆腔粘连,这些可能是导致其不孕的主要原因。结论：雌性大鼠胚胎卵巢原位移植后具有自然生育功能；但由于术后盆腔局部粘连,它们术后妊娠率较正常对照组低,每次妊娠生育子代个数较正常对照组少。目的：初步探讨大鼠胚胎卵巢原位移植术后自然生育的子代的健康安全性问题。方法：(1)分别观察第二章研究中行胚胎卵巢原位移植术后确定妊娠的19只雌性大鼠和第二章研究中阳性对照组全部妊娠的10只雌性大鼠,妊娠后有无流产症象、有无死产、有无幼鼠死亡(观察至出生后3日),并比较观察结果；(2)随机抽取第二章研究中进行了胚胎卵巢原位移植术后自然生育的大鼠子代(研究组)及阳性对照组生育的大鼠子代(正常对照组)各40只,共80只子代大鼠；比较研究组(40只)和正常对照组(40只)35日龄时的体重；(3)应用常规G显带技术对两组子代大鼠进行核型分析,观察大鼠的染色体数目和结构的变异情况。以初步了解大鼠胚胎卵巢原位移植术后生育的子代的健康安全性。结果：(1)第二章研究中行胚胎卵巢原位移植术后确定妊娠的19只雌性大鼠和第二章研究中阳性对照组全部妊娠的10只雌性大鼠,妊娠后均无流产症象、无死产、无幼鼠生后3天内死亡；(2)子代生长发育比较：研究组和正常对照组35日龄的体重(x±s)分别为93.80±4.93g和94.13±4.53g。两组比较无显著性差异(P>0.05)。(3)胚胎卵巢原位移植大鼠子代(研究组)和正常对照组的核型均为42,XX或42,XY,两组均未发现染色体数目和结构异常。结论：大鼠胚胎卵巢原位移植后自然妊娠的孕鼠的流产、死产、幼鼠死亡率以及自然生育的子代的染色体数目、显带情况、子代的体重等与正常对照组比较无明显差异,初步提示大鼠胚胎卵巢原位移植后生育的子代在健康方面是安全的。更多还原

【Abstract】 Objective:To establish the animal models of orthotopic fetal ovarian allotransplantation in rats, and explore the restoration of reproductive endocrine function in the models, furthermore, to provide an experimental animal models for the study on its natural fertility restoration and the health safety of its offsprings.Methods:90 female Sprague Dawley rats aged 50-60 days were divided randomly into 3 groups. One was Study Group (Graft Recipient Group) which enrolled 50 rats, the other was Normal Control Group (Positive Control Group) which enrolled 20 rats, and the third was Negative Control Group (Spayed Group) which enrolled 20 rats too. Fetal ovaries aged 17-19 gestational days were allotransplanted to the rats in Study Group orthotopically (bilateral oophorectomy were carried out in one setting in all rats from the Study Group), while "Open and Close Only" the abdominal wall were assigned to Normal Control Group (all rats in this group remained their normal ovaries unremoved), and bilateral oophorectomy were carried out in all rats in Spayed Group (no grafts were transplanted after the oophorectomy). From each of the three groups, 10 rats were selected randomly and serum estradiol and progesterone level of each rat were measured by electroluminescence test on day 45 after the transplantation, and at the same time the pelvic cavity were explored, bilateral ovaries were identified and removed completely for ovarian volume measurement and follicle development evaluation by ovarian histomorphology.Results:(1) On day 45 after the operations, the estradiol level (x±s) of Study Group, Normal Control Group and Negative Control Group were 114.65±15.59 pmol/L、121.71±14.92 pmol/L and 56.51±7.82 pmol/L, respectively; and progesterone level (x±s) were 12.79±4.40 ng/ml、13.00±1.18 ng/ml and 3.70±0.68 ng/ml, respectively. There is no statistical difference between Study Group and Normal Control Group regarding the estradiol level and progesterone level (P>0.05). However, regarding either the estradiol level or progesterone level between the Study Group and Spayed Group, there was statistical difference (P<0.05). (2) On day 45 after the operations, the ovarian volume of each ovary (x±s) were 0.22±0.07 ml、0.24±0.09 ml and 0 ml in Study Group, Normal Control Group and Spayed Group, respectively. There was no statistical difference between the Study Group and the Normal Control Group regarding the ovarian volume of each group (P>0.05). At light microscope, all stages of follicles development, including the mature follicles and corpus luteum were observed in ovaries of both Study Group and Normal Control Group.Conclusion:The fetal ovary can survive, grow and develop to a status possessing the function of ovulation, and can restore its reproductive endocrine function after being allotransplanted orthotopically in adult female rats.Objective:To investigate the restoration of natural fertility after orthotopic fetal ovarian allotransplantion in rats.Methods:40 female rats from the Study Group (fetal ovarian recipient group),10 from the Normal Control Group and 10 from the Spayed Group which were left in Part I study were selected in the present study. All the 60 female rats were feeded, respectively, with male rats aged from 45 to 65 days in the same cage (the ratio of female and male rats was 2:1) 45 days after the allotransplantation, the spayed operation or the "open and close" operation for 40 days. The pregnancy rate (once the female rate was pregnant, it would be seperated from the male rat during the obseration period) and the number of baby rats given by each birth was recorded and compared between each of the three groups (Study Group, Normal Control Group and the Spayed Group). After the 40-day observation period, the infertile female rats in the Study Group together with female rats in the Normal Control Group were investigated regarding the anatomic changes around the adnexa, and the follicle development status in both groups were detected to help figure out the causes to the infertility of the rats from the Study Group.Results:(1) The pregnancy rate of Study Group, Normal Control Group and the Spayed Group were 47.5%(19/40)、100%(10/10)、0.0% (0/10), respectively. Compared with the Normal Control Group, the pregancy rate in Study Group was significantly lower (P<0.05), while compared with Spayed Group, the pregnancy rate in Study Group was significantly higher (P<0.05). (2) The mean baby rats number (x±s) of each labor in the Study Group and Normal Control Group was 7.0±2.2 and 10.5±2.3, respectively. Compared to the Normal Control Group, the mean baby rats number of each labor in the Study Group was significantly lower (P<0.05). (3) Analysis of the causes to the infertility of the rats in the Study Group:according to the results of histomorphology, mature follicles developed and corpus luteum could be seen in the ovaries of the 21 infertile rats from the Study Group, which indicated that the allotransplanted fetal ovaries had survived and developed to a normal status. However, severe peri-adnexal adhesion was witnessed in the Study Group and no obvious peri-adnexal adhesion was observed in the Normal Control Group, which indicated that the severe pelvic adhesion might contribute to the infertility in the Study Group.Conclusion:Restoration of natural fertility might occur after orthotopic fetal ovarian allotransplantation in female rats; however, the pregancy rate, as well as the infant number of each labor was significantly lower compared to the Normal Control Group. It was possibly because of the severe pelvic adhesion postoperatively.Objective:To investigate preliminarily the health safety of the offspring delivered following naturally getting pregnant after the orthotopic fetal ovarian allotransplantation in rats. Methods:(1) Observation of any symptoms of sportaneous abortion during pregnancy period and of any possible still birth and death of infant rats within 3 days after the delivery was carried out in 19 pregnant rats from the previous Study Group and in 10 pregnant rats from the previous Normal Control Group from the Part II.The observed results were compared between the two groups. (2) 40 offspring rats each from the previous Study Group and Normal Control Group were selected randomly and marked as Study Group and Normal Control Group in the present study, respectively. The mean weight at day 35 after birth of both groups was measured and compared. (3) By means of routine G-banding technique, the karyotype analysis and the observation of chromosomal number and the structure were carried out. This might help understand preliminarily the health safety of offsprings delivered after the orthotopic fetal ovarian allotransplantation in rats.Results:(1) In both of the previous Study Group and Normal Control Group in Part II, neither sportaneous abortions nor still births and deaths of the infants within 3 days after the birth was observed. (2) Comparison of physical growth and development between the two offspring groups:The mean body weight (x±s) on age day 35 in the Study Group and Normal Control Group was 93.80±4.93g and 94.13±4.53g, respectively. There was no significant difference between the two groups regarding the mean body weight on day 35 after the birth of the offsprings (P>0.05). (3) The karyotype analysis indicated that the karyotype from the offspring delivered by both orthotopic fetal ovarian allotransplantation female recipients and the female rats in previously Normal Control Group in PartⅡwere 42, XX or 42, XY. No chromosome abnormality (abnormal chromosomal number or abnormal chromosomal structure) was observed in both groups.Conclusion:After the female recipient rat of orthotopic fetal ovarian allotransplantation getting pregnant, the spontaneous abortion rate, the still birth and death rate, as well as the chromosomal number and structure and the body weight of its offsprings were normal, which implied preliminarily that the health status of such an offspring was safe.更多还原