增膜助孕方对COH小鼠种植窗期子宫内膜容受性的的影响
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摘要
目的：观察增膜助孕方对控制性超排卵小鼠种植窗期子宫内膜组织形态学、性激素及其受体、局部因子和着床数的影响,分析其疗效,从分子和基因水平探讨其作用机制,为增膜助孕方辅助IVF-ET增加妊娠率提供实验依据。
方法：本研究以清洁级健康昆明种雌性未孕小鼠为研究对象,通过建立控制性超排卵小鼠模型,采用增膜助孕方进行实验研究。
SPF级健康昆明种雌性未孕小鼠100只,饲养一周后,随机分为5组,每组各20只,即空白组(生理盐水组)、模型组(GnRHa+HMG+HCG组,即GnRHa长周期促排卵方案)、阳性对照组(GnRHa+HMG+HCG+阿司匹林组)、中药低剂量组(GnRHa+HMG+HCG+增膜助孕方低剂量组)和中药高剂量组(GnRHa+HMG+HCG+增膜助孕方高剂量组)。模型组、阳性对照组、中药低剂量组和中药高剂量组小鼠建立控制性超排卵小鼠模型：于动情周期第3天开始,每日上午9时定时腹腔内注射丙氨瑞林40μg/100g体重,连续9d,至d9同时注射HMG0.01IU/g体重促排卵,48h后注射HCG0.1IU/g体重。
1模型组、阳性对照组、中药低剂量组和中药高剂量组小鼠在注射HCG同时,空白组小鼠在动情期,每组随机取10只小鼠,按雌：雄=1：1比例分别合笼饲养,次日上午9：00检查阴栓,见阴栓者定为孕1d,余类推。合笼后第8天(孕8d)上午9：00脱颈处死小鼠,开腹取出子宫,剖开两侧子宫计算着床数。
2模型组、阳性对照组、中药低剂量组和中药高剂量组小鼠,在注射HCG后48h,空白组自排卵后(阴道涂片呈排卵征象)48h,将每组其余10只小鼠在种植窗期脱颈处死小鼠,进行取材及指标测定。用HE染色法对照观察子宫内膜组织形态学变化,用ELISA法检测子宫内膜中E2和P的含量,用免疫组化法检测子宫内膜中ER、PR、整合素β3、血管内皮生长因子(VEGF)及同源异型盒因子(HOXA10)的表达水平,用逆转录聚合酶链反应法检测子宫内膜中白血病抑制因子(LIFmRNA)的表达水平。
结果：
1与空白组相比,模型组子宫内膜腺体发育不良、腺体小而直、数目减少、腺腔轻度扩张弯曲、腺体和间质发育不同步,子宫内膜中E2、P、E2/P、ER、PR、整合素β3、VEGF、HOXA10及LIFmRNA的表达显著减少(P<0.01),着床数无统计学意义(P>0.05)。
2与模型组相比,阳性对照组子宫内膜组织形态学变化略有改善,子宫内膜中E2、P、ER、PR、整合素β3、VEGF及HOXA10的表达有显著差异(P<0.01),着床数明显增加(P<0.01),E2/P平衡失调未得到纠正(P>0.05),LIFmRNA的表达差异无统计学意义(P>0.05)。中药低剂量组子宫内膜形态学变化得到了明显改善,子宫内膜中E2、P、E2/P、ER、PR、LIFmRNA、整合素β3、VEGF及HOXA10的表达有显著差异(P<0.01),着床数明显增加(P<0.01)。中药高剂量组子宫内膜形态学变化得到了明显改善,子宫内膜中E2、P、E2/P、ER、PR、整合素β3、VEGF、HOXA10及LIFmRNA的表达有显著差异(P<0.01),着床数明显增加(P<0.01)。
3与阳性对照组相比,中药低剂量组子宫内膜形态学变化得到了明显改善,与空白组相似,子宫内膜腺体发育形态规则、数目增加,腺腔扩张弯曲明显,腺体和间质同步发育；子宫内膜中E2/P、ER、PR、LIFmRNA、整合素P3、VEGF及HOXA10的表达差异有显著统计学意义(P<0.01),E2的表达差异有统计学意义(P<0.05),着床数明显增加(P<0.01),P的表达差异无统计学意义(P>0.05)中药高剂量组子宫内膜组织形态学变化得到了明显改善,子宫内膜中E2、P、E2/P、ER、PR、整合素(33、VEGF、HOXA10及LIFmRNA的表达有显著差异(P<0.01),着床数明显增加(P<0.01)。
4与中药低剂量组相比,中药高剂量组子宫内膜组织形态学变化无明显差异,与空白组相似；子宫内膜中E2、P、PR、整合素p3及HOXA10的表达有显著差异(P<0.01),VEGF的表达差异有统计学意义(P<0.05),着床数明显增加(P<0.05),E2/P、ER及LIFmRNA差异无统计学意义(P>0.05)。
结论：
1增膜助孕方能有效的预防或纠正控制性超排对小鼠子宫内膜组织形态学造成的不良影响。
2增膜助孕方能显著提高GnRHa降调节引起的黄体功能不足而导致的E2和P低水平,改善E2/P的平衡失调,且能明显增强控制性超排卵小鼠子宫内膜中ER及PR的表达。
3增膜助孕方能显著提高控制性超排卵小鼠子宫内膜中LIFmRNA、整合素β3、VEGF、HOXA10的表达水平。
4增膜助孕方能显著提高控制性超排小鼠的子宫内膜容受性,进而增加着床数,效果优于阿司匹林,且安全可靠。
Objective:To observe the influence of Increasing Endometrium Zhu Yun Recipe on tissue morphology、sex hormone and its receptor and local factors of the COH mice endometrium in the implantation window and the number of implantation, analyze its effects, and to explore its mechanism in molecular and gene level, and to provide experimental basis for Increasing Endometrium Zhu Yun Recipe assisting IVF-ET to increase pregnancy rate.
Methods:SPF clean healthy female Kunming non-pregnant mices were the research objects in the study, through the establishment of controlled ovarian hyperstimulation mouse model, Increasing Endometrium Zhu Yun Recipe was used in experimental study.
Healthy Kunming female non-pregnant100mices, a week after breeding, were randomly divided into5groups, with20rats in each group, namely blank group (physiological saline group)、 model group (GnRHa+HMG+HCG group、GnRHa long period inducing ovulation scheme)、positive control group (GnRHa+HMG+HCG+aspirin group)、 low dose group (GnRHa+HMG+HCC+Increasing Endometrium Zhu Yun Recipe low dose group) and high dose group (GnRHa+HMG+HCG+Increasing Endometrium Zhu Yun Recipe high dose group). Model group、positive control group、low dose group and high dose group of mices were established the COH mice model:in the third day of estrous cycle, the timing intraperitoneal injection of Alarelin40μg/100g weight was at nine every morning, continuous nine days, on the ninth day injecting HMG0.01IU/g weight was used to induce ovulation at the same time, and after48h injecting HCG0.1IU/g body weight was used to the further maturation of oocytes.
1When mices of model group, positive control group, low dose group and high dose group in Chinese medicine were injected of HCG and the blank group rats in estrus,10mices in each group were randomly taken out, according to female:male=1:1proportion, were respectively closed cage. On the next morning9:00vaginal suppository was checked, vaginal plug for the first day of pregnancy, and so on. Mices were killed at9a.m. on the eighth day after closing cage. The uterus was opened out and the both sides of the uterus were opened and the number of implantation was counted.
2The rest10mices of model group, positive control group, low dose group and high dose group after injection of HCG, in48h, blank group after ovulation (vaginal smear showed signs of ovulation)48h, were taken off the neck to death in the implantation window and materials were collected and indexes were determined. Morphological changes of endometrium were observed by HE staining, and the content of endometrial E2and P were detected by ELISA, and the expressions of endometrial ER、PR、integrinβ3、vascular endothelial growth factor (VEGF) and homeobox factor (HOXA10) were detected by immunohist-ochemistry. Leukemia expression level was detected by reverse transcrip-tion polymerase chain reaction.
Results:
1Compared with the blank group, in the model group, endometrial glandular dysplasia was small and straight, and the number of glands decreased, and glandular cavity was mild expanding and bendind. Glands and stroma growth was not synchronous. The expressions of endometrial E2、P、E2/P、ER、PR、integrin β3、 VEGF. HOXA10and LIFmRNA significantly reduced (P<0.01), but the number of implantation had no statistical significance (P>0.05).
2Compared with the model group, in the positive control group, the endometrial morphological changes slightly improved, and the expr- essions of endometrial E2、P、ER、PR、integrinβ3、 VEGF and HOXA10were significantly different (P<0.01), and the number of implantation significantly increased (P<0.01), but the imbalance of E2/P was not corrected (P>0.05) and LIFmRNA expression was of no significant difference (P>0.05). Changes of endometrial morphology had been significantly improved in the low dose group, and the expressions of E2、P、E2/P、ER、PR、leukemia inhibitory factor LIFmRNA、integrinβ3、 VEGF and HOXA10in the endometrium were significantly different (P<0.01), and the number of implantation significantly increased (P<0.01). Changes of endometrial morphology in high dose group had been significantly improved, the expressions of endometrial E2、P、E2/P、ER、 PR、integrinβ3、VEGF、HOXA10and LIFmRNA had significant difference (P<0.01), and the number of implantation significantly incre-ased (P<0.01).
3Compared with the positive control group, in the low dose group changes of endometrial morphology had been significantly improved, similar to the blank group, and the number of endometrial gland morphogenesis of rules increased, and glandular cavity expansion bend significantly. Glands and stroma synchronously developed. There were significant difference in the expressions of endometrial E2/P、ER、PR、 LIFmRNA、integrinβ3、VEGF and HOXA10(P<0.01), and there was difference in the expression of E2(P<0.05), and the number of implantation significantly increased (P<0.01); the expression of end-othelial P was of no difference (P>0.05). Morphological changes of endometrial tissue in high dose group were significantly improved, and the expressions of endothelial E2、P、E2/P、ER、PR、integrinβ3、VEGF、 HOXA10and LIFmRNA had significant difference (P<0.01), and the number of implantation significantly increased (P<0.01).
4Compared with the low dose group, in high dose group there was no significant difference in morphological changes of endometrial tissue, similar to the blank group; and the expressions of endometrial E2、P、PR、 integrinβ3and HOXA10had significant difference (P<0.01), and the expressions of VEGF had difference (P<0.05), and the number of implantation significantly increased (P<0.05), but in the expressions of E2/P and ER and LIFmRNA there were no significant differences (P>0.05).
Conclusions:
1Increasing Endometrium Zhu Yun Recipe can effectively prevent or correct adverse effects on the mice endometrial morphology from controlled superovulation.
2Increasing Endometrium Zhu Yun Recipe can significantly improve the low levels of E2and P caused by inadequate luteal function from GnRHa down regulation, improve the balance of E2/P, and increase the expressions of the COH mice endometria ER and PR.
3Increasing Endometrium Zhu Yun Recipe can significantly increase the expression level of the COH mice endometrial LIFmRNA, integrinβ3, VEGF and HOXA10.
4Increasing Endometrium Zhu Yun Recipe can significantly impr-ove the endometrial receptivity of COH mice and increase the number of implantation, the effect is better than that of aspirin, safe and reliable.
方法：本研究以清洁级健康昆明种雌性未孕小鼠为研究对象,通过建立控制性超排卵小鼠模型,采用增膜助孕方进行实验研究。
SPF级健康昆明种雌性未孕小鼠100只,饲养一周后,随机分为5组,每组各20只,即空白组(生理盐水组)、模型组(GnRHa+HMG+HCG组,即GnRHa长周期促排卵方案)、阳性对照组(GnRHa+HMG+HCG+阿司匹林组)、中药低剂量组(GnRHa+HMG+HCG+增膜助孕方低剂量组)和中药高剂量组(GnRHa+HMG+HCG+增膜助孕方高剂量组)。模型组、阳性对照组、中药低剂量组和中药高剂量组小鼠建立控制性超排卵小鼠模型：于动情周期第3天开始,每日上午9时定时腹腔内注射丙氨瑞林40μg/100g体重,连续9d,至d9同时注射HMG0.01IU/g体重促排卵,48h后注射HCG0.1IU/g体重。
1模型组、阳性对照组、中药低剂量组和中药高剂量组小鼠在注射HCG同时,空白组小鼠在动情期,每组随机取10只小鼠,按雌：雄=1：1比例分别合笼饲养,次日上午9：00检查阴栓,见阴栓者定为孕1d,余类推。合笼后第8天(孕8d)上午9：00脱颈处死小鼠,开腹取出子宫,剖开两侧子宫计算着床数。
2模型组、阳性对照组、中药低剂量组和中药高剂量组小鼠,在注射HCG后48h,空白组自排卵后(阴道涂片呈排卵征象)48h,将每组其余10只小鼠在种植窗期脱颈处死小鼠,进行取材及指标测定。用HE染色法对照观察子宫内膜组织形态学变化,用ELISA法检测子宫内膜中E2和P的含量,用免疫组化法检测子宫内膜中ER、PR、整合素β3、血管内皮生长因子(VEGF)及同源异型盒因子(HOXA10)的表达水平,用逆转录聚合酶链反应法检测子宫内膜中白血病抑制因子(LIFmRNA)的表达水平。
结果：
1与空白组相比,模型组子宫内膜腺体发育不良、腺体小而直、数目减少、腺腔轻度扩张弯曲、腺体和间质发育不同步,子宫内膜中E2、P、E2/P、ER、PR、整合素β3、VEGF、HOXA10及LIFmRNA的表达显著减少(P<0.01),着床数无统计学意义(P>0.05)。
2与模型组相比,阳性对照组子宫内膜组织形态学变化略有改善,子宫内膜中E2、P、ER、PR、整合素β3、VEGF及HOXA10的表达有显著差异(P<0.01),着床数明显增加(P<0.01),E2/P平衡失调未得到纠正(P>0.05),LIFmRNA的表达差异无统计学意义(P>0.05)。中药低剂量组子宫内膜形态学变化得到了明显改善,子宫内膜中E2、P、E2/P、ER、PR、LIFmRNA、整合素β3、VEGF及HOXA10的表达有显著差异(P<0.01),着床数明显增加(P<0.01)。中药高剂量组子宫内膜形态学变化得到了明显改善,子宫内膜中E2、P、E2/P、ER、PR、整合素β3、VEGF、HOXA10及LIFmRNA的表达有显著差异(P<0.01),着床数明显增加(P<0.01)。
3与阳性对照组相比,中药低剂量组子宫内膜形态学变化得到了明显改善,与空白组相似,子宫内膜腺体发育形态规则、数目增加,腺腔扩张弯曲明显,腺体和间质同步发育；子宫内膜中E2/P、ER、PR、LIFmRNA、整合素P3、VEGF及HOXA10的表达差异有显著统计学意义(P<0.01),E2的表达差异有统计学意义(P<0.05),着床数明显增加(P<0.01),P的表达差异无统计学意义(P>0.05)中药高剂量组子宫内膜组织形态学变化得到了明显改善,子宫内膜中E2、P、E2/P、ER、PR、整合素(33、VEGF、HOXA10及LIFmRNA的表达有显著差异(P<0.01),着床数明显增加(P<0.01)。
4与中药低剂量组相比,中药高剂量组子宫内膜组织形态学变化无明显差异,与空白组相似；子宫内膜中E2、P、PR、整合素p3及HOXA10的表达有显著差异(P<0.01),VEGF的表达差异有统计学意义(P<0.05),着床数明显增加(P<0.05),E2/P、ER及LIFmRNA差异无统计学意义(P>0.05)。
结论：
1增膜助孕方能有效的预防或纠正控制性超排对小鼠子宫内膜组织形态学造成的不良影响。
2增膜助孕方能显著提高GnRHa降调节引起的黄体功能不足而导致的E2和P低水平,改善E2/P的平衡失调,且能明显增强控制性超排卵小鼠子宫内膜中ER及PR的表达。
3增膜助孕方能显著提高控制性超排卵小鼠子宫内膜中LIFmRNA、整合素β3、VEGF、HOXA10的表达水平。
4增膜助孕方能显著提高控制性超排小鼠的子宫内膜容受性,进而增加着床数,效果优于阿司匹林,且安全可靠。
Objective:To observe the influence of Increasing Endometrium Zhu Yun Recipe on tissue morphology、sex hormone and its receptor and local factors of the COH mice endometrium in the implantation window and the number of implantation, analyze its effects, and to explore its mechanism in molecular and gene level, and to provide experimental basis for Increasing Endometrium Zhu Yun Recipe assisting IVF-ET to increase pregnancy rate.
Methods:SPF clean healthy female Kunming non-pregnant mices were the research objects in the study, through the establishment of controlled ovarian hyperstimulation mouse model, Increasing Endometrium Zhu Yun Recipe was used in experimental study.
Healthy Kunming female non-pregnant100mices, a week after breeding, were randomly divided into5groups, with20rats in each group, namely blank group (physiological saline group)、 model group (GnRHa+HMG+HCG group、GnRHa long period inducing ovulation scheme)、positive control group (GnRHa+HMG+HCG+aspirin group)、 low dose group (GnRHa+HMG+HCC+Increasing Endometrium Zhu Yun Recipe low dose group) and high dose group (GnRHa+HMG+HCG+Increasing Endometrium Zhu Yun Recipe high dose group). Model group、positive control group、low dose group and high dose group of mices were established the COH mice model:in the third day of estrous cycle, the timing intraperitoneal injection of Alarelin40μg/100g weight was at nine every morning, continuous nine days, on the ninth day injecting HMG0.01IU/g weight was used to induce ovulation at the same time, and after48h injecting HCG0.1IU/g body weight was used to the further maturation of oocytes.
1When mices of model group, positive control group, low dose group and high dose group in Chinese medicine were injected of HCG and the blank group rats in estrus,10mices in each group were randomly taken out, according to female:male=1:1proportion, were respectively closed cage. On the next morning9:00vaginal suppository was checked, vaginal plug for the first day of pregnancy, and so on. Mices were killed at9a.m. on the eighth day after closing cage. The uterus was opened out and the both sides of the uterus were opened and the number of implantation was counted.
2The rest10mices of model group, positive control group, low dose group and high dose group after injection of HCG, in48h, blank group after ovulation (vaginal smear showed signs of ovulation)48h, were taken off the neck to death in the implantation window and materials were collected and indexes were determined. Morphological changes of endometrium were observed by HE staining, and the content of endometrial E2and P were detected by ELISA, and the expressions of endometrial ER、PR、integrinβ3、vascular endothelial growth factor (VEGF) and homeobox factor (HOXA10) were detected by immunohist-ochemistry. Leukemia expression level was detected by reverse transcrip-tion polymerase chain reaction.
Results:
1Compared with the blank group, in the model group, endometrial glandular dysplasia was small and straight, and the number of glands decreased, and glandular cavity was mild expanding and bendind. Glands and stroma growth was not synchronous. The expressions of endometrial E2、P、E2/P、ER、PR、integrin β3、 VEGF. HOXA10and LIFmRNA significantly reduced (P<0.01), but the number of implantation had no statistical significance (P>0.05).
2Compared with the model group, in the positive control group, the endometrial morphological changes slightly improved, and the expr- essions of endometrial E2、P、ER、PR、integrinβ3、 VEGF and HOXA10were significantly different (P<0.01), and the number of implantation significantly increased (P<0.01), but the imbalance of E2/P was not corrected (P>0.05) and LIFmRNA expression was of no significant difference (P>0.05). Changes of endometrial morphology had been significantly improved in the low dose group, and the expressions of E2、P、E2/P、ER、PR、leukemia inhibitory factor LIFmRNA、integrinβ3、 VEGF and HOXA10in the endometrium were significantly different (P<0.01), and the number of implantation significantly increased (P<0.01). Changes of endometrial morphology in high dose group had been significantly improved, the expressions of endometrial E2、P、E2/P、ER、 PR、integrinβ3、VEGF、HOXA10and LIFmRNA had significant difference (P<0.01), and the number of implantation significantly incre-ased (P<0.01).
3Compared with the positive control group, in the low dose group changes of endometrial morphology had been significantly improved, similar to the blank group, and the number of endometrial gland morphogenesis of rules increased, and glandular cavity expansion bend significantly. Glands and stroma synchronously developed. There were significant difference in the expressions of endometrial E2/P、ER、PR、 LIFmRNA、integrinβ3、VEGF and HOXA10(P<0.01), and there was difference in the expression of E2(P<0.05), and the number of implantation significantly increased (P<0.01); the expression of end-othelial P was of no difference (P>0.05). Morphological changes of endometrial tissue in high dose group were significantly improved, and the expressions of endothelial E2、P、E2/P、ER、PR、integrinβ3、VEGF、 HOXA10and LIFmRNA had significant difference (P<0.01), and the number of implantation significantly increased (P<0.01).
4Compared with the low dose group, in high dose group there was no significant difference in morphological changes of endometrial tissue, similar to the blank group; and the expressions of endometrial E2、P、PR、 integrinβ3and HOXA10had significant difference (P<0.01), and the expressions of VEGF had difference (P<0.05), and the number of implantation significantly increased (P<0.05), but in the expressions of E2/P and ER and LIFmRNA there were no significant differences (P>0.05).
Conclusions:
1Increasing Endometrium Zhu Yun Recipe can effectively prevent or correct adverse effects on the mice endometrial morphology from controlled superovulation.
2Increasing Endometrium Zhu Yun Recipe can significantly improve the low levels of E2and P caused by inadequate luteal function from GnRHa down regulation, improve the balance of E2/P, and increase the expressions of the COH mice endometria ER and PR.
3Increasing Endometrium Zhu Yun Recipe can significantly increase the expression level of the COH mice endometrial LIFmRNA, integrinβ3, VEGF and HOXA10.
4Increasing Endometrium Zhu Yun Recipe can significantly impr-ove the endometrial receptivity of COH mice and increase the number of implantation, the effect is better than that of aspirin, safe and reliable.
引文
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