超促排卵周期卵泡发育特点及卵母细胞发育潜能的相关机制研究
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摘要
研究背景
卵巢卵泡发育是一个涉及下丘脑—垂体—卵巢轴和局部旁分泌/自分泌机制(包括卵母细胞和周围颗粒细胞、膜细胞、间质细胞)的复杂生理过程,与女性的许多内分泌疾病有着密切的关系。许多研究致力于探索卵泡发育的正常生理精确过程,以更好的了解人类配子发育过程及生殖奥秘,更好的解决一些临床疾病;在辅助生育技术(Assistant reproductive technology,ART)中提高临床妊娠率,建立完善的体内外卵子成熟的体系,这是人类生殖研究中一个重要课题。然而对于卵泡发育的调节机制仍有许多未知的问题,特别是ART中不可回避的控制性超促排卵(Controlled ovarian stimulation,COH)下的卵泡发育和卵母细胞成熟的特点亟待进一步明确。
超促排卵是ART中几乎必不可少的一个部分,超促排卵过程中非生理剂量的促性腺激素运用,超生理的雌激素水平,引发了人们的担忧,而许多文献为这种担忧提供了依据:超促排卵对卵母细胞的正常减数分裂、表观遗传修饰和发育能力均有不良的影响,还能引发较高程度的颗粒细胞调亡。但是,超促排卵对正常卵泡发育和卵母细胞成熟的影响及其机制还缺乏一个系统的、整体水平的研究。目前新兴的蛋白质组学方法可为其在蛋白水平的改变提供技术上的支持。蛋白质组学(proteomics)是指研究一种细胞、组织或完整生物体所拥有的全套蛋白质的特征,包括蛋白质表达、翻译后修饰、蛋白与蛋白间相互作用等。卵泡液是卵母细胞的发育和成熟重要的微环境,研究超促排卵周期卵泡液的蛋白质组学改变能为探索超促排卵周期卵泡发育的特点提供一个整体的、蛋白质水平的确切依据,而就我们所知,目前国内外均无类似文献报道。
临床上ART中超促排卵周期中较多的卵泡被募集并发育至优势卵泡,然而超排周期中各个卵母细胞存在发育不同步的问题,hCG的运用则可能使一些没有完全成熟的卵母细胞提前发生减数分裂;而超促排卵周期卵母细胞自身胞浆、胞核成熟也存在不同步的情况。超促排卵周期卵母细胞发育不同步、胞浆成熟难以评价是全世界ART一个共同的难题,然而准确预测卵母细胞的成熟度和发育潜能,挑选首先移植的胚胎对提高ART妊娠结局有重要意义。卵泡液是卵母细胞的发育和成熟重要的微环境,通过检测卵泡液评价卵母细胞质量是一个精确、简便、无创、具有实用性的方法,具有极大的科学价值和临床应用价值。学者不断的探索卵泡液中的生物标志是否具有预测卵母细胞成熟的价值,许多文献报道了卵泡液中甾体激素、垂体激素、细胞因子和生长因子的水平与卵子的发育、受精和胚胎发育能力之间的联系,但争议较多,仍没有一个得到广泛认可的指标。而近年发现的卵母细胞来源的生长因子,已被证明在卵泡发育过程中占重要作用,其中骨形成蛋白-15(Bone morphogenetic protein-15,BMP-15)因其对卵泡发育具有的重要作用,已成为卵母细胞因子中最受关注的蛋白之一,其是否能反映卵母细胞的成熟状态,目前国内外均无类似报道。
超排卵过程卵泡发育的多寡决定了体外受精—胚胎移植(In VitroFertilization-Embryo Transfer,IVF-ET)的累计妊娠率,而不同的个体卵巢对外源性促性腺激素有着不同的反应。卵巢低反应是IVF工作中的一个难题和挑战,至今无很好的解决方法,既往的研究和我们的经验发现加大促性腺激素的用量往往无法提高低反应患者的卵泡发育数目,而其发生机制尚不明确。卵巢储备功能下降被认为是低反应发生的重要原因,然而相当一部分年轻、激素水平正常的妇女也会发生低反应。近年的研究发现卵巢局部的旁/自分泌机制可能会影响卵巢对外源性促性腺激素刺激的反应,而卵母细胞本身与周围卵泡细胞存在对话机制,其中BMP-15与颗粒细胞来源的干细胞因子(Stem cell facror,SCF)之间已被证明存在着反馈机制,共同调节卵泡发育。还有报道BMP-15能抑制颗粒细胞卵泡刺激素受体(Follicle stimulating hormone receptor,FSHR)的表达,而我们既往的研究发现低反应患者颗粒细胞FSHR较正常反应者下降,由此推测卵母细胞是否与卵巢低反应的发生有关?而国内外尚无该方面的报道。
综上所述,超排卵周期卵泡发育有其特异性,本研究根据当前卵泡发育和卵子成熟研究范畴内的趋势和国内的研究基础,以超排促排卵对卵泡发育的干预和影响、超排卵周期卵母细胞发育潜能预测以及卵巢低反应发生机制为切入点,探讨超促排卵周期卵泡发育、卵母细胞成熟的特点及其相关机制,为进一步明确卵泡发育调控机理和提高ART的安全性和成功率提供依据。
第一部分超促排卵周期卵泡液蛋白质组学研究
研究目的本研究的目的是通过研究超促排卵周期与自然周期卵泡卵泡液蛋白质组表达谱的改变,以助于阐明在超排周期中卵泡发育的特点,以及进一步探索超促排卵对卵泡发育的影响。
材料和方法采集自然周期与超促排卵周期卵泡液各6例,用二维凝胶电泳分离蛋白质组成分,银染染色后用PD-Quest软件分析数字化的凝胶图像,找出超促排卵周期差异表达的蛋白质点,采用胶内酶解消化方法得到差异蛋白斑点多肽混合物,用基质辅助激光解吸-串联质谱进行蛋白质鉴定。
结果1.通过分析二维凝胶电泳图谱,13个蛋白斑点在超促排卵周期卵泡液中表达量较自然周期组增加或减少2倍以上,并经Student's t-test检验,表达量两组有显著差异(P＜0.05)。其中11个点在超排周期中表达水平上调,另2个点在超排周期中表达水平下调。2.通过胶内酶解消化、基质辅助-激光解离/离子-飞行时间质谱或串联质谱分析得到差异表达的蛋白斑点的肽质指纹图谱,通过MASCOT搜索引擎在NCBI蛋白质数据库中检索,其中8个蛋白斑点成功得到鉴定。这些鉴定蛋白质包括触珠蛋白、富含亮氨酸α2糖蛋白、锌-α2-糖蛋白、转铁蛋白,α1-抗胰蛋白酶,补体C3等。这些蛋白参与排卵、细胞应激反应、类脂/类固醇代谢等过程。3.分别用速率散射比浊法和western印迹验证其中4个蛋白质的表达趋势:触珠蛋白、补体C3、转铁蛋白和α1-抗胰蛋白酶,其表达趋势与二维电泳结果一致。
结论通过蛋白质组学研究技术发现在超促排卵卵泡发育与卵母细胞成熟存在差异,其中与排卵、免疫反应、类脂/类固醇代谢相关的蛋白质表达发生异常变化,为进一步探索超促排卵对卵母细胞发育和成熟的影响提供了重要的线索。
第二部分人卵泡液骨形成蛋白-15水平与超排卵周期卵母细胞发育
研究目的研究卵母细胞来源因子骨形成蛋白-15(Bone morphogeneticprotein-15,BMP-15)在人卵泡液中的水平及其与卵母细胞质量、发育能力和后续胚胎质量的关系。
材料和方法对79例因男性因素不孕的进行卵胞浆内精子注射(ICSI)的患者,采用常规促超排卵方案,采集每位患者1.8～2.0cm卵泡卵泡液一份,共采集79例行ICSI治疗的患者的79个卵泡中的卵母细胞和卵泡液,卵母细胞单独培养,评价其受精和胚胎发育情况。western blot法检测卵泡液中的BMP-15水平;放免法检测卵泡液雌二醇(Estradiol,E2)、孕酮(Progesterone,P4)和促性腺激素(Follicle stimulating hormone,FSH)水平。
结果卵母细胞按其后续受精、卵裂和胚胎发育情况分别进行分组,受精组和卵裂组卵母细胞卵泡液BMP-15水平显著高于未受精和未卵裂的卵母细胞的卵泡液(P＜0.05)。优质胚胎组卵母细胞卵泡液BMP-15水平显著高于二级和三级胚胎(P＜0.01)。卵泡液BMP-15和E2水平呈显著性正相关(P＜0.01),与卵泡液FSH水平呈负相关(P＜0.01)。
结论卵泡液BMP-15水平可能是一个预测卵母细胞质量和后续胚胎发育能力的一个有价值的指标。
第三部分人卵泡液中BMP-15水平与卵巢低反应
研究目的:研究超排卵周期卵巢低反应患者卵泡液中BMP-15、SCF的水平及其与妊娠结局之间的关系。
材料与方法:对90例因管性因素不孕而行进行体外受精-胚胎移植(In VitroFertilization-Embryo Transfer,IVF-ET)的患者,采用本常规促超排卵方案,用Western印迹方法测定取卯时各组卵泡液中BMP-15的水平,用ELISA法检测卵泡液SCF水平。按取卵时卵泡发育数目不同,将卵巢对超排卵药物的反应分为低反应型(卵泡数≤3)、正常反应型(卵泡数4-13个)。将低反应组和正常反应组患者分别按卵泡液平均BMP-15或SCF水平将其分为高水平组和低水平组,比较其IVF-ET的结局。
结果:低反应组卵泡液中BMP-15(1.11±0.12)显著高于正常反应组(0.96±0.11,P＜0.01),低反应组平均SCF水平为164.83±23.97 pg/ml,也显著高于正常反应组139.88±11.24pg/ml,P＜0.01。低反应组高BMP-15患者妊娠率显著高于低BMP-15组(P＜0.05),与正常反应组妊娠率相当。
结论:高卵泡液BMP-15和SCF水平与卵巢低反应的发生有关。低反应患者高卵泡液BMP-15水平预示较好的IVF妊娠结局。
Introduction
Ovarian follicular growth is a process involving a complex exchange of hormonal signals between the hypothalamus-pituitary-ovary axis and by a localized para/autocrine mechanism within the ovary involving the oocyte and its adjacent somatic cells. Plenty of studies have been made towards unraveling the complex intraovarian control mechanisms that act in concert with systemic signals to solve some clinical problems, improve the outcome of assisted reproductive technology (ART) and establish the in vitro maturation (IVM) system. Exciting progress has been obtained such as the discovery of oocyte-derived factors; however, there are still many undiscovered mysteries in this field.
COH increase the numbers of oocytes ovulated and embryos produced in a variety of mammals. COH has been widely used in ART all over the world. However, artificial induction of ovulation with high doses of gonadotrophins has been demonstrated to decrease the viability of embryos and induce oocyte aneuploidy. Increasing evidences suggest that systemic studies should be conducted to reveal the impact of COH on follicle development and oocyte maturation. Proteomics is the large-scale study of proteins, modifications, complexes and interactions from a given cell line or organism. Proteomics is technically possible to offer detailed information on proteins with differences in protein expression and modification.
COH is suspected to influence the maturation of oocytes. Researchers have made great efforts to ameliorate IVF protocols to improve the outcome, such as trying to predicting the maturation and developmental competence of oocytes. The follicular fluid (FF) is the environment of the oocyte during its development and maturation. The composition in FF may influence and/or implicate oocyte quality, and some human follicular fluid proteins have been correlated with oocyte maturation during follicular development. However, no consistent biomarker has been found.
The number of acquired oocytes greatly influences the outcome of ART. Poor response to gonadotropin stimulation could not provide enough oocytes for treatment and significantly reduce the likelihood of conceiving in ART. There is accumulating evidence that oocyte plays an important role in regulating the function of neighboring somatic cells during mammalian folliculogenesis. The oocyte was demonstrated to be able to regulate its own maturation and affect the functions of neighboring somatic cells and the ovulation rate. Whether oocyte is associated with the poor response to COH is unknown.
In a word, upon the research background mentioned above, we investigate the characteristics of follicle development and oocyte maturation of COH cycle by exploring the proteomics analysis of COH FF, potential markers for oocyte maturation, the mechanism of poor response and to further uncover the intraovarian regulatory mechanisms.
Part One: Proteomic Analysis on the Alteration of Follicular Fluid Protein
Expression of Controlled Ovarian Hyperstimulation
Objective: To study the differences in protein expression of FF of COH cycle andnatural cycle, this is essential to understand the influence of COH on follicledevelopment.
Methods: To identify proteins with different expression profiles related to COH, weapplied a proteomic approach and performed two-dimensional gel electrophoresis(2-DE) on six COH FF samples and six FF samples from natural cycle women, followed by comparison of the silver-stained 2-DE profiles.
Results: The 2-DE patterns of COH FF and natural cycle FF with good quality havebeen obtained. When compared COH FF and natrual control with PDQuest software,it showed that 2 proteins were down-regulated and 11 proteins were up-regulatedsignificantly (P<0.05) in COH group as determined by spot volume. Among them, 1down-regulated and 7 up-regulated spots were identified by MALDI-TOF MS.Anomalies of several proteins such as, leucine-rich alpha-2-glycoprotein 1,Zn-a2-glycoprotein, transferrin,α1-antitrypsin, human complement component C3had been identified. Changes of haptoglobin, transferrin, human complement
component C3 andα1-antitrypsin expressions were further validated in by ratenephelometry analysis and western blot analysis respectively.
Conclusion: This study has indicated 8 differential proteins that are associated withovulation, immune response, lipid metabolism in COH FF, leading to a new insightinto the influence of COH on follicle development and oocyte maturation.
Part Two: High BMP-15 level in follicular fluid is associated with high qualityoocyte and subsequent embryonic development
Objective: Bone morphogenetic protein-15 (BMP-15) has displayed influences on oocyte maturation and quality. However, no dependence relation has been established between BMP-15 and oocyte quality/embryonic development in the human. The aim of this study is to investigate the BMP-15 levels in human follicular fluid (FF) and its role in assessing oocyte quality and developmental potential.
Methods: A total of seventy-nine occytes and their corresponding FF from 79 women undergoing intracytoplasmic sperm injection (ICSI) were examined. Each recruited oocyte was individually inseminated and thereafter assessed based on their fertilization, cleavage and preimplantation development. BMP-15, FSH, estradiol and progesterone levels of FF were also analysed via the techniques of western blot or radioimmunoassay.
Results: Higher FF BMP-15 levels were observed in group fertilization and groupcleavage (P < 0.05). The best embryo morphology had higher BMP-15 levels thanothers (P < 0.01). A significantly positive correlation was found between BMP-15 andestradiol levels in the same follicle.
Conclusion: The present study demonstrates that the BMP-15 in FF appears to be apotential factor in predicting oocyte quality and subsequent embryo development, andis regulated by estradiol, which may additionally be a valuable predictor of oocytefertilization.
Part Three: Increased BMP-15 and SCF levels in follicular fluid in poorresponders
Objectives: The aim of this study was to investigate the role of oocyte-derived factor bone morphogenetic protein-15 (BMP-15), stem cell factor (SCF) in follicular fluid in the ovarian response to gonadotropin stimulation.
Methods: Ninety infertile women undergoing ovarian stimulation with recombinant FSH were recruited. These women were divided into two groups: poor (n=33) and normal responders (n=57). Poor responder was defined as women with
卵巢卵泡发育是一个涉及下丘脑—垂体—卵巢轴和局部旁分泌/自分泌机制(包括卵母细胞和周围颗粒细胞、膜细胞、间质细胞)的复杂生理过程,与女性的许多内分泌疾病有着密切的关系。许多研究致力于探索卵泡发育的正常生理精确过程,以更好的了解人类配子发育过程及生殖奥秘,更好的解决一些临床疾病;在辅助生育技术(Assistant reproductive technology,ART)中提高临床妊娠率,建立完善的体内外卵子成熟的体系,这是人类生殖研究中一个重要课题。然而对于卵泡发育的调节机制仍有许多未知的问题,特别是ART中不可回避的控制性超促排卵(Controlled ovarian stimulation,COH)下的卵泡发育和卵母细胞成熟的特点亟待进一步明确。
超促排卵是ART中几乎必不可少的一个部分,超促排卵过程中非生理剂量的促性腺激素运用,超生理的雌激素水平,引发了人们的担忧,而许多文献为这种担忧提供了依据:超促排卵对卵母细胞的正常减数分裂、表观遗传修饰和发育能力均有不良的影响,还能引发较高程度的颗粒细胞调亡。但是,超促排卵对正常卵泡发育和卵母细胞成熟的影响及其机制还缺乏一个系统的、整体水平的研究。目前新兴的蛋白质组学方法可为其在蛋白水平的改变提供技术上的支持。蛋白质组学(proteomics)是指研究一种细胞、组织或完整生物体所拥有的全套蛋白质的特征,包括蛋白质表达、翻译后修饰、蛋白与蛋白间相互作用等。卵泡液是卵母细胞的发育和成熟重要的微环境,研究超促排卵周期卵泡液的蛋白质组学改变能为探索超促排卵周期卵泡发育的特点提供一个整体的、蛋白质水平的确切依据,而就我们所知,目前国内外均无类似文献报道。
临床上ART中超促排卵周期中较多的卵泡被募集并发育至优势卵泡,然而超排周期中各个卵母细胞存在发育不同步的问题,hCG的运用则可能使一些没有完全成熟的卵母细胞提前发生减数分裂;而超促排卵周期卵母细胞自身胞浆、胞核成熟也存在不同步的情况。超促排卵周期卵母细胞发育不同步、胞浆成熟难以评价是全世界ART一个共同的难题,然而准确预测卵母细胞的成熟度和发育潜能,挑选首先移植的胚胎对提高ART妊娠结局有重要意义。卵泡液是卵母细胞的发育和成熟重要的微环境,通过检测卵泡液评价卵母细胞质量是一个精确、简便、无创、具有实用性的方法,具有极大的科学价值和临床应用价值。学者不断的探索卵泡液中的生物标志是否具有预测卵母细胞成熟的价值,许多文献报道了卵泡液中甾体激素、垂体激素、细胞因子和生长因子的水平与卵子的发育、受精和胚胎发育能力之间的联系,但争议较多,仍没有一个得到广泛认可的指标。而近年发现的卵母细胞来源的生长因子,已被证明在卵泡发育过程中占重要作用,其中骨形成蛋白-15(Bone morphogenetic protein-15,BMP-15)因其对卵泡发育具有的重要作用,已成为卵母细胞因子中最受关注的蛋白之一,其是否能反映卵母细胞的成熟状态,目前国内外均无类似报道。
超排卵过程卵泡发育的多寡决定了体外受精—胚胎移植(In VitroFertilization-Embryo Transfer,IVF-ET)的累计妊娠率,而不同的个体卵巢对外源性促性腺激素有着不同的反应。卵巢低反应是IVF工作中的一个难题和挑战,至今无很好的解决方法,既往的研究和我们的经验发现加大促性腺激素的用量往往无法提高低反应患者的卵泡发育数目,而其发生机制尚不明确。卵巢储备功能下降被认为是低反应发生的重要原因,然而相当一部分年轻、激素水平正常的妇女也会发生低反应。近年的研究发现卵巢局部的旁/自分泌机制可能会影响卵巢对外源性促性腺激素刺激的反应,而卵母细胞本身与周围卵泡细胞存在对话机制,其中BMP-15与颗粒细胞来源的干细胞因子(Stem cell facror,SCF)之间已被证明存在着反馈机制,共同调节卵泡发育。还有报道BMP-15能抑制颗粒细胞卵泡刺激素受体(Follicle stimulating hormone receptor,FSHR)的表达,而我们既往的研究发现低反应患者颗粒细胞FSHR较正常反应者下降,由此推测卵母细胞是否与卵巢低反应的发生有关?而国内外尚无该方面的报道。
综上所述,超排卵周期卵泡发育有其特异性,本研究根据当前卵泡发育和卵子成熟研究范畴内的趋势和国内的研究基础,以超排促排卵对卵泡发育的干预和影响、超排卵周期卵母细胞发育潜能预测以及卵巢低反应发生机制为切入点,探讨超促排卵周期卵泡发育、卵母细胞成熟的特点及其相关机制,为进一步明确卵泡发育调控机理和提高ART的安全性和成功率提供依据。
第一部分超促排卵周期卵泡液蛋白质组学研究
研究目的本研究的目的是通过研究超促排卵周期与自然周期卵泡卵泡液蛋白质组表达谱的改变,以助于阐明在超排周期中卵泡发育的特点,以及进一步探索超促排卵对卵泡发育的影响。
材料和方法采集自然周期与超促排卵周期卵泡液各6例,用二维凝胶电泳分离蛋白质组成分,银染染色后用PD-Quest软件分析数字化的凝胶图像,找出超促排卵周期差异表达的蛋白质点,采用胶内酶解消化方法得到差异蛋白斑点多肽混合物,用基质辅助激光解吸-串联质谱进行蛋白质鉴定。
结果1.通过分析二维凝胶电泳图谱,13个蛋白斑点在超促排卵周期卵泡液中表达量较自然周期组增加或减少2倍以上,并经Student's t-test检验,表达量两组有显著差异(P＜0.05)。其中11个点在超排周期中表达水平上调,另2个点在超排周期中表达水平下调。2.通过胶内酶解消化、基质辅助-激光解离/离子-飞行时间质谱或串联质谱分析得到差异表达的蛋白斑点的肽质指纹图谱,通过MASCOT搜索引擎在NCBI蛋白质数据库中检索,其中8个蛋白斑点成功得到鉴定。这些鉴定蛋白质包括触珠蛋白、富含亮氨酸α2糖蛋白、锌-α2-糖蛋白、转铁蛋白,α1-抗胰蛋白酶,补体C3等。这些蛋白参与排卵、细胞应激反应、类脂/类固醇代谢等过程。3.分别用速率散射比浊法和western印迹验证其中4个蛋白质的表达趋势:触珠蛋白、补体C3、转铁蛋白和α1-抗胰蛋白酶,其表达趋势与二维电泳结果一致。
结论通过蛋白质组学研究技术发现在超促排卵卵泡发育与卵母细胞成熟存在差异,其中与排卵、免疫反应、类脂/类固醇代谢相关的蛋白质表达发生异常变化,为进一步探索超促排卵对卵母细胞发育和成熟的影响提供了重要的线索。
第二部分人卵泡液骨形成蛋白-15水平与超排卵周期卵母细胞发育
研究目的研究卵母细胞来源因子骨形成蛋白-15(Bone morphogeneticprotein-15,BMP-15)在人卵泡液中的水平及其与卵母细胞质量、发育能力和后续胚胎质量的关系。
材料和方法对79例因男性因素不孕的进行卵胞浆内精子注射(ICSI)的患者,采用常规促超排卵方案,采集每位患者1.8～2.0cm卵泡卵泡液一份,共采集79例行ICSI治疗的患者的79个卵泡中的卵母细胞和卵泡液,卵母细胞单独培养,评价其受精和胚胎发育情况。western blot法检测卵泡液中的BMP-15水平;放免法检测卵泡液雌二醇(Estradiol,E2)、孕酮(Progesterone,P4)和促性腺激素(Follicle stimulating hormone,FSH)水平。
结果卵母细胞按其后续受精、卵裂和胚胎发育情况分别进行分组,受精组和卵裂组卵母细胞卵泡液BMP-15水平显著高于未受精和未卵裂的卵母细胞的卵泡液(P＜0.05)。优质胚胎组卵母细胞卵泡液BMP-15水平显著高于二级和三级胚胎(P＜0.01)。卵泡液BMP-15和E2水平呈显著性正相关(P＜0.01),与卵泡液FSH水平呈负相关(P＜0.01)。
结论卵泡液BMP-15水平可能是一个预测卵母细胞质量和后续胚胎发育能力的一个有价值的指标。
第三部分人卵泡液中BMP-15水平与卵巢低反应
研究目的:研究超排卵周期卵巢低反应患者卵泡液中BMP-15、SCF的水平及其与妊娠结局之间的关系。
材料与方法:对90例因管性因素不孕而行进行体外受精-胚胎移植(In VitroFertilization-Embryo Transfer,IVF-ET)的患者,采用本常规促超排卵方案,用Western印迹方法测定取卯时各组卵泡液中BMP-15的水平,用ELISA法检测卵泡液SCF水平。按取卵时卵泡发育数目不同,将卵巢对超排卵药物的反应分为低反应型(卵泡数≤3)、正常反应型(卵泡数4-13个)。将低反应组和正常反应组患者分别按卵泡液平均BMP-15或SCF水平将其分为高水平组和低水平组,比较其IVF-ET的结局。
结果:低反应组卵泡液中BMP-15(1.11±0.12)显著高于正常反应组(0.96±0.11,P＜0.01),低反应组平均SCF水平为164.83±23.97 pg/ml,也显著高于正常反应组139.88±11.24pg/ml,P＜0.01。低反应组高BMP-15患者妊娠率显著高于低BMP-15组(P＜0.05),与正常反应组妊娠率相当。
结论:高卵泡液BMP-15和SCF水平与卵巢低反应的发生有关。低反应患者高卵泡液BMP-15水平预示较好的IVF妊娠结局。
Introduction
Ovarian follicular growth is a process involving a complex exchange of hormonal signals between the hypothalamus-pituitary-ovary axis and by a localized para/autocrine mechanism within the ovary involving the oocyte and its adjacent somatic cells. Plenty of studies have been made towards unraveling the complex intraovarian control mechanisms that act in concert with systemic signals to solve some clinical problems, improve the outcome of assisted reproductive technology (ART) and establish the in vitro maturation (IVM) system. Exciting progress has been obtained such as the discovery of oocyte-derived factors; however, there are still many undiscovered mysteries in this field.
COH increase the numbers of oocytes ovulated and embryos produced in a variety of mammals. COH has been widely used in ART all over the world. However, artificial induction of ovulation with high doses of gonadotrophins has been demonstrated to decrease the viability of embryos and induce oocyte aneuploidy. Increasing evidences suggest that systemic studies should be conducted to reveal the impact of COH on follicle development and oocyte maturation. Proteomics is the large-scale study of proteins, modifications, complexes and interactions from a given cell line or organism. Proteomics is technically possible to offer detailed information on proteins with differences in protein expression and modification.
COH is suspected to influence the maturation of oocytes. Researchers have made great efforts to ameliorate IVF protocols to improve the outcome, such as trying to predicting the maturation and developmental competence of oocytes. The follicular fluid (FF) is the environment of the oocyte during its development and maturation. The composition in FF may influence and/or implicate oocyte quality, and some human follicular fluid proteins have been correlated with oocyte maturation during follicular development. However, no consistent biomarker has been found.
The number of acquired oocytes greatly influences the outcome of ART. Poor response to gonadotropin stimulation could not provide enough oocytes for treatment and significantly reduce the likelihood of conceiving in ART. There is accumulating evidence that oocyte plays an important role in regulating the function of neighboring somatic cells during mammalian folliculogenesis. The oocyte was demonstrated to be able to regulate its own maturation and affect the functions of neighboring somatic cells and the ovulation rate. Whether oocyte is associated with the poor response to COH is unknown.
In a word, upon the research background mentioned above, we investigate the characteristics of follicle development and oocyte maturation of COH cycle by exploring the proteomics analysis of COH FF, potential markers for oocyte maturation, the mechanism of poor response and to further uncover the intraovarian regulatory mechanisms.
Part One: Proteomic Analysis on the Alteration of Follicular Fluid Protein
Expression of Controlled Ovarian Hyperstimulation
Objective: To study the differences in protein expression of FF of COH cycle andnatural cycle, this is essential to understand the influence of COH on follicledevelopment.
Methods: To identify proteins with different expression profiles related to COH, weapplied a proteomic approach and performed two-dimensional gel electrophoresis(2-DE) on six COH FF samples and six FF samples from natural cycle women, followed by comparison of the silver-stained 2-DE profiles.
Results: The 2-DE patterns of COH FF and natural cycle FF with good quality havebeen obtained. When compared COH FF and natrual control with PDQuest software,it showed that 2 proteins were down-regulated and 11 proteins were up-regulatedsignificantly (P<0.05) in COH group as determined by spot volume. Among them, 1down-regulated and 7 up-regulated spots were identified by MALDI-TOF MS.Anomalies of several proteins such as, leucine-rich alpha-2-glycoprotein 1,Zn-a2-glycoprotein, transferrin,α1-antitrypsin, human complement component C3had been identified. Changes of haptoglobin, transferrin, human complement
component C3 andα1-antitrypsin expressions were further validated in by ratenephelometry analysis and western blot analysis respectively.
Conclusion: This study has indicated 8 differential proteins that are associated withovulation, immune response, lipid metabolism in COH FF, leading to a new insightinto the influence of COH on follicle development and oocyte maturation.
Part Two: High BMP-15 level in follicular fluid is associated with high qualityoocyte and subsequent embryonic development
Objective: Bone morphogenetic protein-15 (BMP-15) has displayed influences on oocyte maturation and quality. However, no dependence relation has been established between BMP-15 and oocyte quality/embryonic development in the human. The aim of this study is to investigate the BMP-15 levels in human follicular fluid (FF) and its role in assessing oocyte quality and developmental potential.
Methods: A total of seventy-nine occytes and their corresponding FF from 79 women undergoing intracytoplasmic sperm injection (ICSI) were examined. Each recruited oocyte was individually inseminated and thereafter assessed based on their fertilization, cleavage and preimplantation development. BMP-15, FSH, estradiol and progesterone levels of FF were also analysed via the techniques of western blot or radioimmunoassay.
Results: Higher FF BMP-15 levels were observed in group fertilization and groupcleavage (P < 0.05). The best embryo morphology had higher BMP-15 levels thanothers (P < 0.01). A significantly positive correlation was found between BMP-15 andestradiol levels in the same follicle.
Conclusion: The present study demonstrates that the BMP-15 in FF appears to be apotential factor in predicting oocyte quality and subsequent embryo development, andis regulated by estradiol, which may additionally be a valuable predictor of oocytefertilization.
Part Three: Increased BMP-15 and SCF levels in follicular fluid in poorresponders
Objectives: The aim of this study was to investigate the role of oocyte-derived factor bone morphogenetic protein-15 (BMP-15), stem cell factor (SCF) in follicular fluid in the ovarian response to gonadotropin stimulation.
Methods: Ninety infertile women undergoing ovarian stimulation with recombinant FSH were recruited. These women were divided into two groups: poor (n=33) and normal responders (n=57). Poor responder was defined as women with
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