【作者】 肖彭莹；

【导师】 尤昭玲；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 目的：探讨中药护卵汤对GnRHa超促排卵大鼠子宫内膜容受性影响的作用机制,主要观察护卵汤对GnRHa超促排卵大鼠子宫内膜组织细胞形态、子宫内膜容受性相关标记物表达的影响。方法：第一部分研究背景应用计算机网络技术,进行大量相关文献检索,并对文献进行详细、系统地整理、分析,研究体外受精-胚胎移植(IVF-ET).控制性促排卵(COH)、子宫内膜容受性影响因素、中西药对子宫内膜容受性的影响及当前该领域的最新进展。大量研究表明中药在辅助治疗IVF-ET过程中具有疗效确切、安全、无耐药性的优势特点,值得进一步深入探索、研究。第二部分护卵汤对GnRHa超促排卵大鼠子宫内膜组织形态学的影响实验采用SPF级SD大鼠,将大鼠随机分为3组：分别为模型组、中药护卵汤组,同时设正常组做对照。观察阴道脱落细胞涂片,于妊娠第2、5天分别剖腹取子宫、称量并制作标本,进行光、电镜下内膜形态学指标的观察,主要对不同时段各组大鼠的子宫指数、内膜厚度、内膜成熟度、糖原、胞饮突等指标进行观察、检测。第三部分护卵汤对GnRHa超促排卵大鼠子宫内膜容受性相关因素表达的影响实验采用SPF级SD大鼠,将大鼠随机分为3组：分别为模型组、中药护卵汤组,同时设正常组做对照。于妊娠第2、5天分别取颈静脉血液、断颈后剖腹取子宫,制作标本,应用放射免疫方法、抗体芯片检测方法等对不同时段各组大鼠子宫内膜容受性相关影响因素E2、P、P/E2、IFN-γ、TNF-α、IL-4、 IL-6、TGF-β1、E-selectin、L-selectin、Leptin、OPN、TIMP-3、ICAM-1等进行检测,观察中药护卵汤对子宫内膜容受性相关因素的影响。结果：第一部分研究背景通过对网络数据库检索与大量文献整理、分析发现,常规IVF-ET的重要环节是COH,长方案在临床COH方案中应用最为普及；但COH药物的应用带来了不可避免的问题,直接、间接地影响了子宫内膜容受性,使妊娠率得不到提高。传统中医认为子宫内膜容受性与肾、气血、冲任密切相关,肾精充盈、气血充足,脏腑、经络、气血相互协调平衡则子宫能容物；现代医学认为子宫内膜容受性受多种激素、因子、酶类等相关因素的调控。导师根据多年临床治疗经验自拟护卵汤,此方以补肾为重、兼顾先后天之本、协调多个脏腑功能、调理冲任、理气调血并用。其药物亦有调控激素、因子等作用,能有效改善子宫内膜容受性,故将护卵汤作为被试因素。第二部分护卵汤对GnRHa超促排卵大鼠子宫内膜组织形态的影响1.一般情况动物实验过程中,大鼠精神状态可,活动自如,饮食、二便正常,毛发光泽。用药期间无不良反应。2.动情周期模型组于注射丙氨瑞林5d后阴道脱落细胞涂片示动情周期改变消失,注射HMG后周期改变恢复,中药组、正常组未见动情周期消失,呈现各期细胞表现,但中药组周期变化速度减缓,周期延长。3.大鼠子宫指数孕2d、孕5d模型组与中药组、正常组两两比较,子宫指数均有显著统计学意义(P<0.01)；孕2d、孕5d同组间比较,子宫指数亦有显著统计学意义(P<0.01)。4.大鼠子宫内膜厚度及子宫内膜成熟度孕2d及孕5d模型组与中药组、正常组两两比较,大鼠子宫内膜厚度均无统计学意义(P>0.05)；模型组、中药组、正常组同组比较,内膜厚度均有显著统计学意义(P<0.01)。孕2d模型组与中药组、正常组两两相比,大鼠子宫内膜成熟度无统计学意义(P>0.05)；孕5d模型组与中药组比,子宫内膜成熟度有统计学意义(P<0.05),与正常组比,子宫内膜成熟度有显著统计学意义(P<0.01),中药组与正常组相比,无统计学意义(P>0.05)。5.光镜下大鼠子宫内膜形态学观察孕2d模型组内膜腺体发育不良,形态不规则,数目不多,腺腔轻度扩张弯曲,间质疏松水肿,血管少；中药组腺体发育,部分形态不规则,数目不多,腺腔稍增大,分泌物稍增多,间质疏松水肿不明显,间质内可见少量血管增生充盈；正常组腺体发育,数目增多不明显,腺腔稍增大,间质疏密相间、细胞多呈梭形,间质内血管少。孕5d模型组腺体数目少,腺上皮细胞增生,腺腔狭窄,弯曲不明显,间质致密,多呈梭形细胞,血管较少；中药组腺体发育明显,数目增多,腺腔明显增大,分泌物明显增多,间质中基质细胞明显增生,间质疏松、水肿,间质细胞多呈圆形,少部分呈梭形,,间质内血管增生；正常组腺体数目多,腺体发育良好,腺腔大,并充盈着丰富的分泌物,间质中基质细胞明显增生,间质疏松、间质细胞多呈圆形,间质内血管明显增生。6.光镜下大鼠子宫内膜上皮细胞糖原观察孕2d、孕5d模型组与中药组、正常组比,大鼠子宫内膜糖原个数有统计学意义(P<0.01),而中药组与正常组比无统计学意义(P>0.05)。两时段同组间比较,均有统计学意义(P<0.01)。孕2d模型组糖原多于中药组及正常组,而孕5d明显少于中药组及正常组。7.电镜下大鼠子宫内膜胞饮突观察孕2d模型组大鼠子宫内膜表面形态呈不均一性表现,在内膜局部有少量胞饮突表达,形态大小不一,发育不同步,大部分内膜表面被浓密的微绒毛覆盖,无膜状突起,细胞间分界不清；中药组内膜表面形态规整,未见胞饮突表达,上皮细胞分布尚均匀,形态大小相对一致,发育基本同步,其顶端膜被长的微绒毛覆盖,细胞间分界欠清楚,微绒毛相互融合而构成小突起；正常组内膜表面形态较规整,未呈现胞饮突表达,上皮细胞分布均匀,形态大小基本一致,发育同步,其顶端膜被相对稍短的微绒毛覆盖,细胞间分界欠清楚,微绒毛相互融合而构成小突起。孕5d模型组内膜表面形态平整,但不均匀,胞饮突表达完全消失,上皮细胞形态大小不一,分布不均匀,其顶端膜被长短不一的微绒毛覆盖,细胞间界限不清楚,子宫内膜呈分泌晚期变化；中药组内膜表面有大量的膜状突起,胞饮突表达明显,其表面不甚光滑,分布相对均匀,形态大小相对一致,细胞间分界基本清楚,发育基本同步,膜状突起表面覆盖有较短而细的微绒毛；正常组内膜表面有大量胞饮突表达,顶端膜覆盖的微绒毛消失,表面光滑,突起明显,分布均匀,形态大小较一致,发育同步,彼此分界清楚,表现为完全发育的胞饮突。第三部分护卵汤对GnRHa超促排卵大鼠子宫内膜容受性相关因素表达的影响1.大鼠E2、P、P/E2值的比较孕2d模型组、中药组E2均高于正常组,有显著统计学意义(P<0.01),模型组与中药组相比,无统计学意义(P>0.05)；孕5d模型组、中药组E2也高于正常组,有统计学意义(P<0.05、P<0.01),模型组与中药组相比,无统计学意义(P>0.05)。孕2d及孕5d同组间比较,无统计学意义孕2d模型组、中药组P值均高于正常组,有统计学意义(P<0.01),模型组高于中药组,有统计学意义(P<0.01)；孕5d模型组、中药组均高于正常组,有统计学意义(P<0.05),模型组低于中药组,有统计学意义(P<0.05)。孕2d及孕5d同组间比较,模型组无统计学意义(P>0.05),中药组、正常组有统计学意义(P<0.01)。孕2d模型组、中药组P/E2较正常组低,有统计学意义(P<0.01),模型组较中药组稍高,但无统计学意义(P>0.05)。孕5d中药组、正常组P/E2高于模型组,有统计学意义(P<0.05),中药组于正常组无统计学意义(P>0.05)。2.大鼠子宫内膜Th1、Th2型细胞因子的蛋白表达孕2d、孕5d模型组IFN-γ、TNF-α均高于正常组,且表达上调均大于1.5倍,而中药组IFN-γ、TNF-α均低于模型组,且表达下调均大于1.5倍；孕2d中药组IL-4、IL-6均高于模型组及正常组,但上调幅度小,模型组有所下调,但下调亦不明显；孕5d模型组IL-4、IL-6均低于中药组及正常组,且表达下调均大于1.5倍,而中药组较模型组表达上调大于1.5倍。模型组Th1/Th2升高,而中药组Th1/Th2降低。3.大鼠子宫内膜容受性相关因素的表达孕2d与正常组比较,模型组TGF-β1、E-selectin、L-selectin及Leptin表达下调,且下调大于1.5倍,OPN、TIMP-3、ICAM-1微下调。与模型组比,中药组TGF-β1、TGF-β3、E-selectin、L-selectin、Leptin、TIMP-3表达均上调大于1.5倍,OPN及ICAM-1微上调；孕5d与正常组比,模型组TGF-β1、 TGF-β3、E-selectin、L-seleetin、Leptin、TIMP-3、ICAM-1表达下调,且下调大于1.5倍,OPN微下调。与模型组比,中药组TGF-β1、TGF-β3、E-selectin、 L-selectin、Leptin、TIMP-3表达均上调大于1.5倍,OPN及ICAM-1微上调。结论：1.通过对中医历代文献及现代医学研究进展的整理、分析、总结发现,子宫内膜容受性差是导致IVF-ET失败、妊娠率低的主要原因。中药可以改善子宫内膜的容受性,提高妊娠率。传统中医认为肾虚、肾精不足是子宫内膜容受性差的主要病机,气虚血弱、冲任不调是协同病机。导师从肾、肝、心、脾论治,时限与脏腑双重定位,注重气血冲任调节,主次分明论治,自拟护卵汤以改善子宫内膜容受性,提高妊娠率。2.GnRHa超促排卵大鼠着床窗口期提前开放和关闭,子宫指数及内膜成熟度低；内膜总体发育不良,腺体与间质发育不同步；内膜糖原含量少；胞饮突提前出现,表达呈现不均一性；E2、P明显增加、P提前升高,P/E2比值失衡；IFN-γ、TNF-α表达上调,IL-4、IL-6表达下调,Th1/Th2比值失衡,子宫内膜容受性标记物TGF-β1、TGF-β3、E-selectin、L-selectin.Leptin. TIMP-3、ICAM-1表达下调,说明COH药物直接和间接地影响了以上因素,降低了子宫内膜容受性,导致了着床的失败,妊娠率降低。3.护卵汤可以延迟GnRHa超促排卵大鼠子宫内膜着床窗的开放与关闭,提高子宫指数,改善内膜成熟度；促使内膜腺体与间质发育同步,增加内膜糖原含量；改变胞饮突表达的时限,使其呈现均一性；协调E2、P及P/E2比值；下调IFN-γ、TNF-α的表达水平,增强IL-4、IL-6的表达水平,使Th1/Th2细胞因子比值趋于平衡,从整体上调节免疫失衡状态；增强TGF-β1、TGF-β3、 E-selectin、L-selectin.Leptin.TIMP-3的表达,最终达到改善子宫内膜容受性的目的,促进胚胎着床,提高妊娠率4.本研究的创新点在于(1)应用多种实验方法从多方面多层次探讨了中药护卵汤对GnRHa超促排卵大鼠子宫内膜容受性的影响,为临床辅治IVF-ET提供科学依据,开辟新思路。(2)探讨了护卵汤改善子宫内膜容受性的作用机制,为进一步研制开发中药新药提供实验依据。(3)为中药辅助治疗IVF-ET,更深层次研究中药对子宫内膜容受性的作用机制及环节、适时启动着床的辅治效应与机制、进一步安全、有效、最大限度的提高IVF-ET妊娠率提供了初步思路。5.今后研究思路：完善研究设计,在现有研究基础上通过进一步研究相关细胞因子的信号通路及子宫内膜基因的表达,以期能更深层次地探讨护卵汤的作用机制和作用环节。更多还原

【Abstract】 ObjectiveResearch the effect of Huluantang on the endometrial receptivity of controlled ovarian hyperstimulation rats assisted by GnRHa. Main observation conclude the effects of Huluantang on rat uterus endometrium cell morphology, the related markers expression of endometrial receptivityMethodsThe first part Research backgroundApplication of computer network technology, searching for a large number of related literatures, doing thorough and systematic collation and analysis, research on IVF-ET, COH, endometrial receptivity, Chinese and Western medicine on the influence factors of endometrial receptivity and the latest developments in this field. A large number of studies have shown that Chinese medicine in the treatment process of IVF-ET with characteristics of good efficacy, safety, no advantage of drug resistance, worthy of further exploration and study.The second part Effects of Huluantang on endometrial tissue morphology of GnRHa hyperstimulation ratsExperiments using SPF level SD rats, divided rats randomly into3groups:model group, Huluantang group, respectively, while setting normal group. Observation on vaginal exfoliated cells in smear, removed uterus, weighed and produced specimens in pregnant2,5days, observed endometrial morphology under light and electron microscopic, mainly observed and tested on uterine maturity index, endometrial thickness, endometrial degree of maturation, glycogen, pinopodes indicators in different groups of pregnant rats.The third part Effects of Huluantang on endometrial receptivity related factors expression of GnRHa hyperstimulation rats Experiments using SPF level SD rats, divided rats randomly into3groups:model group, Huluantang group, respectively, while setting normal group. Taking jugular venous blood, removed uterus, produced specimens inpregnant2,5days, applied Radioimmunoassay method, protein chip detectionmethod for testing endometrial receptivity related factors in different groups of pregnant rats, such as E2、P、P/E2、 IFN-γ、TNF-α、IL-4、IL-6、TGF-β1、E-selectin、L-selectin、Leptin、OPN、 TIMP-3、ICAM-1, and observed effects of Huluantang on endometrial receptivity related factors expression.ResultsThe first part Research backgroundBy on network database retrieved and large literatures finishing and analysis, we found, important links of general IVF-ET is COH, the application of long programme is most universal; however application of COH drugs brings inevitable problems, effects the endometrial receptivity directly and indirectly, reduces pregnancy rate. Theory of TCM think that endometrial receptivity is closely related to kidney, Qixue, and chongren, kidney essence filling, and Qixue adequate, Zang-Fu, Meridian, and Qixue keep balance then uterine can capacity real; Modern medical think that endometrial receptivity is controlled bymultiple hormones, cytokines, and enzymes. Mentor based on many years of clinical experience, Self-made Huluantang, it respects kidney role, coordinating multiple viscera function, conditioning ChongRen, adjusting Qixue, herbs in Huluantang can control hormones, cytokines, and improve the endometrial receptivity effectively, so,we makes Huluantang as participants.The second part Effects of Huluantang on endometrial tissue morphology of GnRHa hyperstimulation rats1. General conditions: During the animal experiments, rats can be in good spirit; activities freely; bite and sup, defecation and urination will be normal, shiny hair. No adverse reaction during medication.2. Estrous cycle: Vaginal exfoliated cells smears shows that estrous cycle of model group is disappearing after injecting Alarelin5d, estrous cycle recovery after injecting HMG, estrous cycle of Huluantang group and normal group are not disappearing, showing the cells performance, but the cycle variation rate of Huluantang group slows down, cyclical extension.3. The uterus index of rats: Pregnant2d,5d model group, Huluantang group, normal group compared with each other, uterus index were statistically significant (P<0.01); compared with each same group, the uterus index also were statistically significant (P<0.01).4. Rats endometrial thickness and endometrial degree of maturity:Pregnant2d,5d model group, Huluantang group, normal group compared with each other, endometrial thickness were not statistical significant (P>0.05); Compared with each same group, were statistically significant (P<0.01). Pregnant2d model group, Huluantang group, normal group compared with each other, endometrial degree of maturity were not statistical significant (P>0.05), Pregnant5d model group compared with Huluantang group, were statistical significant (P<0.05), compared with normal group, were statistical significant (P<0.01); Huluantang group compared with normal group, was no statistical significant (P>0.05).5. Observation on the morphology of the rats’endometrium under light microscopic: Pregnant2d model group endometrial glands maldevelopment, form does not rules, not more, glands cavity mild expand and bent, mesenchyme loose edema, vascular less; Huluantang group glands have good development, part form does not rules, not more, glands cavity slightly increases, secretion real slightly increased, mesenchyme loose and edema does not obvious, less vascular hyperplasia, filling; normal group glands have very good development, increased does not obvious, glands cavity slightly increases, mesenchyme loose and density, majority cells are shuttle shaped, vascular less. Pregnant5d model group less glands, glands epithelial cell hyperplasia, glands cavity narrow, bent is not obvious, mesenchyme density, majority are shuttle shaped cell, contains large matrix cell gathered, and has giant peritoneal macrophages cell infiltration, vascular less; Huluantang group glands development obvious, more, glands cavity obvious increases, secretion real obvious increased, the matrix cell in mesenchyme obvious hyperplasia, mesenchyme loose and edema, more round, less shuttle shaped, vascular hyperplasia; normal group glands more, development good, cavity large and filled with rich glandular secretions, apparent proliferation of mesenchymal stromal cells, loose, cells more round, hyperplasia of vascular clearly.6. Observation on glycogen of ratsendometrium under light microscope: Pregnant2d,5d model group compared with Huluantang group, normal group were statistical significant in the number of rats endometrial glycogen (P<0.01), and Huluantang group compared with normal group was not statistical significant (P>0.05). Compared with each same group, were statistically significant (P<0.01). Pregnant2d model group’s glycogen was more than Huluantang group and normal group, Pregnant5d model group’s glycogen was significantly less than that Huluantang group and normal group.7. Observation on the rat endometrium pinopodes under the electron microscope: Pregnant2d model group endometrial surface form is hot uniform, has less pinopodes expression, form size varies, development does not synchronization, most surface is covered by thick micro-fluff, no shaped bumps, cell boundaries is clear; Huluantang group surface form structured, no pinopodes expression, epithelial cell distribution is uniform, form size is relative consistent, development basic synchronization, its top is covered by long micro-fluff, cell boundaries is clearly, micro-fluff mutual fusion and constitute small bumps; Normal group surface form structured, no pinopodes expression, epithelial cell distribution, size and form is consistent basically, development synchronization, the top is covered by relatively short micro-fluff, cell boundaries is lack of clarity, micro-fluff mutual fusion and constitute small bumps. Pregnant5d model group surface form flat, but not uniform, pinopodes are full disappeared, epithelial cell form size varies, distribution does not uniform, its top is covered by length varies of micro-fluff, cell boundaries is not clearly, endometrium is advanced secretion; Huluantang group surface has large of shaped bumps, pinopodes expression are clear, its surface is not very smooth, distribution relative uniform, form size relative consistent, cell boundaries is basic clearly, development is basic synchronization, bumps surface covers more short and fine micro-fluff; Normal group has a large number of pinopodes, micro-fluff disappeared, surface is smooth, bumps are significantly, distribution, form and size are consistent, synchronized development, mutual boundaries is clear, for the full development of pinopodes.The third part Effects of Huluantang on endometrial receptivity related factors expression of GnRHa hyperstimulation rats1. Comparison of E2、P、P/E2of pregnant rats: Pregnancy2d E2of model group, Huluantang group are higher than normal group, were statistically significant (P<0.01), model group compared with Huluantang group, was not statistically significant (P>0.05); pregnant5d model groups, Huluantang group are also higher than normal group, were statistically significant (P<0.05, P<0.01), model group compared with Huluantang group, was not statistically significant (P>0.05). Pregnant2d,5d compared between same groups, statistically significant in model group and Huluantang group, normal group was not statistically significant.Pregnant2d P of model group, Huluantang group is higher than normal group, was statistically significant (P<0.01), model group is higher than Huluantang group, was statistical significant (P<0.01); pregnant5d model group, Huluantang group is higher than normal group, was statistically significant (P<0.05), model group is lower than Huluantang group, was statistically significant (P<0.05). Pregnant2d,5d compared between same groups, model group has no statistical significant (P>0.05), Huluantang group and normal group were statistically significant (P<0.01).Pregnancy2d model group. Huluantang group P/E2are lower than normal group, were statistically significant (P<0.01), model group is slightly higher than Huluantang group, but was not statistically significant (P>0.05). Pregnant5d Huluantang group and normal group P/E2are higher than model group, were statistically significant (P<0.05). Huluantang group compared with normal group, was not statistically significant (P>0.05).2. Endometrial Thl,Th2cytokine expression of pregnant rats: Pregnant2d,5d IFN-γ, and TNF-a of model group are higher than normal group, and expression is raised than1.5times, Huluantang group are lower than model group, and expression is declined than1.5times; pregnant2d Huluantang group IL-4, IL-6are higher than model group and normal group, but raised less, model group is declined, but not clear; pregnant5d model group IL-4, IL-6are lower than Huluantang group and normal group, and expression is declined than1.5times, Huluantang group compared with model group expression is raised than1.5times. Model groupThl/Th2is raised; Huluantang group Th1/Th2is declined.3. Endometrial receptivity related factors expression of pregnant rats: Pregnant2d, compared with normal group, model group TGF-β1, E-selectin, L-selectin and Leptin expression is reduced more than1.5times, OPN, TIMP-3, ICAM-lis declined slightly. Compared with model group, Huluantang group TGF-β1, TGF-β3, E-selectin, L-selectin, Leptin and TIMP-3is raised more than1.5times, OPN and ICAM-1are raised slightly; pregnant5d, compared with normal group, model group TGF-β1, TGF-(33, E-selectin, L-selectin, Leptin TIMP-3and ICAM-1expression is declined more than1.5times, OPN also declined slightly. Compared with model group, Huluantang group TGF-(31, TGF-β3, E-selectin, L-selectin, Leptin, TIMP-3expression is raised more than1.5times, OPN and ICAM-1are also raised slightly.Conclusions:1. By on the collation, analysis, summary of ancient literatures of traditional Chinese medicine and modern medical advances, we found that bad endometrial receptivity is the main reason for the failure of IVF-ET, which caused low pregnancy rate.In recent years, modern research shows traditional Chinese medicine may improve endometrial receptivity, and increase pregnancy rate. TCM believes that deficiency of the kidney, deficiency of the kidney essence is of poor endometrial receptivity main pathogenesis, Qixue weak, ChongRen disharmony is synergistic pathogenesis. Mentor respects coordination of the kidney, liver, heart, spleen, dual position on time frames and Zang-Fu, pay attention to the coordination of the Qixue and ChongRen, Primary and secondary treatment, self-made Huluantang to improve endometrial receptivity, and increase pregnancy rate.2. GnRHa hyperstimulation model rats implantation window period ahead of open and close, uterine index and endometrial degree of maturation are lower; endometrial bad developed, development of glands and mesenchyme does not synchronize; less glycogen; pinopodes appears ahead, expression does not uniform; E2, P increased obviously, and P rised ahead; IFN-y and TNF-alpha are raised, IL-4, and IL-6are declined, Th1/Th2is imbalances, important molecular markers of embryo implantation:TGF-β1, TGF-β3, E-selectin, L-selectin, Leptin, TIMP-3, ICAM-1are reduced, it shows that COH drugs has direct and indirect impact on the above factors, reduced endometrial receptivity, led to the failure of implantation, declined the pregnancy rate.3. Huluantang can delay the open and close of implantation window, increase uterine index and endometrial degree of maturity; led glands and mesenchyme development synchronized, increased glycogen; change expression time range of pinopodes, makes it uniform; coordinated E2, P and P/E2ratio; reduced IFN-γ, TNF-α expression, increased IL-4, IL-6expression, makes Th1/Th2cytokines ratio be balanced, overall regulation of immune imbalance; increased TGF-β1, TGF-β3, E-selectin, L-selectin, Leptin, TIMP-3expression, and ultimately achieve the goal of improve endometrial receptivity, embryo implantation, and improve pregnancy rate.4. In this study, the innovation lies in:(1) Using various experimental methods to discuss the effects of Huluantang on endometrial receptivity of GnRHa hyperstimulation rats on multi-level protection. Provide the scientific basis for the clinical treatment of IVF-ET.(2) Discussion on the mechanism of improving endometrial receptivity by Huluantang then provides experimental basis for further research and development of new traditional Chinese medicine.(3) Provided a preliminary idea for treatment of IVF-ET by traditional Chinese medicine, deeper research of mechanism of traditional Chinese medicine on endometrial receptivity, timely start implantation of auxiliary therapeutic effect and mechanism, further improvement of IVF-ET pregnancy rates safely and effectively.5. Ideas for future research:improving research design, on the basis of existing research, through further research of cytokine signaling pathway and endometrial gene expression, in order to discuss on the mechanism and links of Huluantang in deep level.更多还原