【作者】 杜娟；

【导师】 陆华；

【作者基本信息】 成都中医药大学 ， 中医妇科学， 2022， 博士

【摘要】 目的:1.观察阳虚质对IVF-ET妊娠、早期流产的影响。2.探索IVF-ET 阳虚质卵泡微环境的代谢差异及病理性代谢标志物。3.初步探索IVF-ET 阳虚质卵泡微环境对细胞膜流动性和促性腺激素受体可及性的影响。方法:研究分为三个部分:1.第一部分采用双向性队列的观察性研究法。以阳虚体质为暴露因素,在IVF-ET患者中建立队列,根据体质调查结果将IVF-ET患者分为:阳虚质组和非阳虚质组(非阳虚质组分为平和质亚组、非阳虚质非平和质亚组)。分析各组基础资料,前瞻性观察或回顾性分析受试者首次取卵周期的临床、实验室资料以及首次胚胎移植周期的妊娠情况及早期流产情况,探索阳虚质对IVF-ET妊娠和早期流产的影响。2.第二部分研究从第一部分队列中筛选纳入IVF-ET典型阳虚质组和IVF-ET典型平和质组的患者各8例,分别收集血清、卵泡液样本各8个,提取卵泡外泌体样本8个。采用超高效液相色谱串联四级杆飞行时间质谱法对两组受试者的样本进行代谢组学分析,获悉两组血清、卵泡液和卵泡外泌体样本中的差异代谢成分及差异代谢通路。3.第三部分研究使用第二部分两组患者的卵泡液和卵泡外泌体样本,将经PKH67荧光标记的卵泡外泌体与KGN细胞(人卵巢颗粒细胞)共培养,观察颗粒细胞对卵泡外泌体的摄取,探索卵泡外泌体对细胞膜流动性的影响。分别用特异表达LH/hCG受体的细胞和特异表达hFSH受体的细胞,检测卵泡液中游离的促性腺激素成分与LH/hCG受体和FSH受体的结合功能活性,探索其对LH/hCG及FSH受体的可及性。结果:1.队列研究结果显示:(1)共纳入受试者369例,其中偏颇质约占71.54%,平和质约占28.46%;受试者中阳虚质76例,约占20.60%;偏颇质中主体质排名前四依次为阳虚质、湿热质、气郁质和血瘀质;偏颇质中兼夹体质者占58.33%,阳虚质最常见兼夹湿热质;(2)阳虚质组BMI低于平和质亚组,初潮年龄晚于非阳虚非平和质亚组,月经先期发生率高于非阳虚质组及其平和质亚组,不孕年限长于非阳虚质组,子宫内膜异位症率高于平和质亚组,输卵管因素率低于平和质亚组;(3)阳虚质组获成熟卵数低于非阳虚质组及其平和质亚组,优质胚胎数低于平和质亚组;(4)阳虚质组移植周期妊娠率和临床妊娠率均低于平和质亚组,妊娠周期生化妊娠+临床妊娠早期流产率高于平和质亚组。2.对IVF-ET典型阳虚质组及IVF-ET典型平和质组受试者的血清、卵泡液及卵泡外泌体样本的代谢组学分析发现:与IVF-ET典型平和质组相比,(1)IVF-ET典型阳虚质组血清存在9个差异代谢物,其中6个下调,3个上调,这些差异代谢物与1条代谢通路相关,即类固醇激素生物合成通路;(2)IVF-ET典型阳虚质组卵泡液存在5个差异代谢物,其中1个下调,4个上调,这些差异代谢物与4条代谢通路相关,分别为甘油磷脂代谢通路、磷脂酰肌醇信号通路、甘油脂类代谢以及精氨酸和脯氨酸代谢通路;(3)IVF-ET典型阳虚质组卵泡外泌体存在32个差异代谢物,其中17个下调,15个上调,这些差异代谢物与7条代谢通路相关,分别是亚油酸代谢、α-亚麻酸代谢、鞘脂代谢、花生四烯酸代谢、甘油磷脂代谢、类固醇生物合成和嘌呤代谢通路。3.卵泡液微环境中对颗粒细胞膜流动性和对促性腺激素受体可及性的影响研究结果显示:(1)IVF-ET典型阳虚质组和IVF-ET典型平和质组卵泡外泌体能被颗粒细胞摄取,但阳虚质组摄取的外泌体量显著低于平和质组,且其荧光信号以分布在细胞膜上为主,而平和质组荧光信号遍及细胞膜和细胞质内;(2)IVF-ET典型阳虚质组卵泡液中残留有高于平和质的游离LH/hCG水平,表明阳虚质者卵泡膜细胞对LH/hCG的利用下降,即LH/hCG对自身受体的可及性下降;(3)IVF-ET典型阳虚质组和IVF-ET典型平和质组的卵泡液中未检出FSH。结论:1.阳虚质是IVF-ET患者最常见的偏颇体质,阳虚兼夹湿热是最常见的兼夹体质;IVF-ET阳虚质组受试者BMI较平和质组低,可能存在代谢差异;IVF-ET阳虚质组获成熟卵数和优质胚胎数减少,妊娠率和临床妊娠率降低,孕早期流产率增加,体现了阳虚质对受孕和妊娠维持的影响;2.卵泡外泌体是研究卵泡微环境代谢特征更敏感的样本来源;IVF-ET 阳虚质的卵泡外泌体与血清存在相似的代谢通路,即类固醇/类固醇激素生物合成通路,卵泡外泌体与卵泡液存在相同的代谢通路,即甘油磷脂代谢通路,阳虚质血清、卵泡液、卵泡外泌体代谢组学特征主要集中于脂质代谢通路;卵泡外泌体和血清异中有同的差异代谢通路可能是“精血同源”的物质基础之一;3.IVF-ET典型阳虚质组卵泡外泌体磷脂代谢异常可能影响细胞膜流动性,影响LH/hCG受体的可及性;LH/hCG受体可及性可用于评估卵泡微环境中LH的活性及功能,阳虚质卵泡液对LH/hCG受体可及性弱于平和质,是引发黄体功能不足的可能机制之一。更多还原

【Abstract】 Objective 1.The influences of Yang-deficiency constitution on pregnancy and early miscarriage of IVF-ET patients were observed.2.The differential pathological metabolites and relative metabolic pathways of IVF-ET patients with Yang-deficiency constitution were explored.3.The effects of follicular microenvironment on cell membrane fluidity and hormone receptor accessibility in typical Yang-deficiency group of IVF-ET was explored preliminarily.MethodThere were 3 sections in this research.1.In Section 1,an observational study in the form of a bidirectional cohort was used.Taking Yang-deficiency constitution as the exposure factor,a bidirectional cohort was established among IVF-ET patients.The patients were divided into 2 groups as Yang-deficiency group and non-Yang-deficiency group,which had 2 subgroups as Balanced subgroup and non-Yang-deficiency and non-Balanced subgroup.The basic data of each group were analyzed,then the clinical and laboratory data related to assisted reproduction in the first oocyte retrieval cycle were prospectively observed or retrospectively analyzed,and the pregnancy rate and early miscarriage of the first embryo transfer cycle were compared.2.In Section 2,8 patients in typical Yang-deficiency constitution group and 8 in typical Balanced constitution group were included.8 samples of serum,8 samples of follicular fluid or 8 samples of follicular exosomes were collected or extracted.Metabolomics analysis by ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-Q-TOF-MS)was used on these samples to search for the different metabolic components in serum,follicular fluid and follicular exosomes samples of these two groups.Then,differential components were analyzed qualitatively and the relative metabolic pathways were concentrated.3.In Section 3,16 follicular fluid or follicular exosomes samples of the patients in section 2 were used in this section.The follicular exosomes labeled by PKH67 fluoresce were co-cultured with KGN cells(human ovarian granulosa cells)to observe the follicular exosomes englobement by granulosa cells and explore the effects on cell membrane fluidity.The LH/hCG Receptor Cell and FSH Receptor Cell were used in order to measure the binding accessibility with LH/hCG Receptor and FSH Receptor,and explore the effect of follicular microenvironment of Yang-deficiency constitution on the accessibility of LH/hCG and FSH receptors.Result 1.The results of this cohort showed as follows.(1)A total of 369 patients were in this cohort,and unbalanced constitutions accounted for about 71.54%,while balanced constitution about 28.46%.There were 76 patients of Yang-deficiency constitution(about 20.60%),The most common unbalanced constitutions were listed as Yang-deficiency constitution,Damp-heat constitution,Qi-stagnation constitution and Blood-stasis constitution.In unbalanced constitutions,the patients with 2 or more constitutions accounted for 58.33%.Damp-heat was the most common constitution accompanied with Yang-deficiency.(2)The mean BMI of Yang-deficiency constitution group was lower than Balanced constitution subgroup.Compared with non-Yang-deficiency group or Balanced subgroup or non-Yang-deficiency and non-Balanced subgroup,the menarche was later and epimenorrhea was more common in Yang-deficiency group.The duration of infertility was longer in Yang-deficiency constitution.And Yang-deficiency patients had more morbidity of endometriosis and less morbidity of salpingemphraxis.(3)The number of mature oocytes and high-quality embryos were less in Yang-deficiency group.(4)Compared with Balanced subgroup,the Yang-deficiency group had lower pregnancy rate and clinical pregnancy rate,and higher early miscarriage rate during transplantation cycle.2.The metabolites and metabolic pathways in serum,follicular fluid and follicular exosomes of the typical Yang-deficiency constitution group and typical Balanced constitution group showed as follows.(1)Comparing with typical Balanced constitution group,there were 9 different metabolites in serum of typical Yang-deficiency constitution,of which 6 down-regulated and 3 up-regulated.The relative metabolic pathway was Steroid hormone biosynthesis.(2)There were 5 different metabolites in follicular fluid of typical Yang-deficiency constitution,of which 1 down-regulated and 4 up-regulated.The relative metabolic pathways were Glycerophospholipid metabolism,Phosphatidylinositol signaling system,Glycerolipid metabolism,and Arginine and proline metabolism.(3)There were 32 different metabolites in follicular exosomes of typical Yang-deficiency constitution,of which 17 down-regulated and 15 up-regulated.The relative metabolic pathways were Linoleic acid metabolism,alpha-Linolenic acid metabolism,Sphingolipid metabolism,Arachidonic acid metabolism,Glycerophospholipid metabolism,Steroid biosynthesis,and Purine metabolism.3.The result of Gonadotropin receptor accessibility in follicular microenvironment showed as follows.(1)The follicular exosomes of typical Yang-deficiency and typical Balanced constitution were absorbed by granulosa cells,and the absorption of follicular exosomes in Yang-deficiency constitution were decreased.The fluorescence signal was mainly distributed on the cell membrane in Yang-deficiency group,while the fluorescence signal was throughout the cell membrane and cytoplasm in Balanced group;(2)The residual LH/hCG in the follicular fluid of the typical Yang-deficiency group was higher than that of the Balanced group,and it indicated that the utilization of LH/hCG by theca cells in the follicular fluid of the Yang-deficiency group was decreased,and the accessibility of LH/hCG to its own receptors in the follicular fluid of the Yang-deficiency group was decreased.(3)Neither Yang-deficiency group nor Balanced group had FSH in follicular microenvironment.Conclusion 1.Yang-deficiency constitution was the most common unbalanced constitution in IVF-ET patients,and Damp-heat was the most common concurrent constitution accompanied with Yang-deficiency.The mean BMI of Yang-deficiency group was lower,and might because of metabolic differences.Yang-deficiency group had less mature oocytes,less high-quality embryos,lower pregnancy and clinical pregnancy rates,and higher early miscariage rate,which reflected the influence of Yang-deficiency on pregnancy and pregnancy maintenance.2.More differential metabolites were found in follicular exosomes than in serum or follicular fluid,and it showed that follicular exosomes were more sensitive for metabonomics to evaluate the microenvironment in follicles.The same metabolic pathway of follicular exosomes and serum was Steroid biosynthesis/Steroid hormone biosynthesis,while the similar metabolic pathway of follicular exosomes and follicular fluid was Glycerophospholipid metabolism.The metabolomics of serum,follicular fluid and follicular exosomes focused on lipid metabolism pathway.The TCM theory of Homogeny ofEssence and Blood was proved by the similar metabolic pathway in both follicular exosomes and serum.3.The abnormal metabolism of exosome phospholipids in follicular microenvironment of IVF-ET patients affected the fluidity of cell membrane and the accessibility of LH/hCG receptor.The receptor accessibility of LH/hCG in follicular microenvironment was an important indicator for activity and functionality of LH.It was one of the possible mechanisms of luteal insufficiency that the LH/hCG receptor accessibility in follicle fluid of Yang-deficiency constitution was weaker than that of Balanced constitution.更多还原