超排卵中孕激素抑制卵泡自发排卵的机制研究

Premature ovulation is a challenge for controlled ovarian hyperstimulation so GnRH analogue is used to prevent LH surge and premature ovulation, with the disadvantages of long injection, complex protocols and high risk of OHSS. Our recent studies reported that progesterone was able to be used to prevent premature ovulation during the controlled ovarian stimulation in combination with the freeze-all techniques. The embryological and clinical pregnancy outcomes were approved by clinical data but the inherent mechanism was still unknown. Previous researches have shown that progesterone can block LH surge by feedback function on the hypothalamus level. However, the downstream mechanism of controlling spontaneous ovulation in ovary is still largely unknown. .We presumed that the progesterone receptor (PR) and related downstream proteases (ADAMTS1, EDN2 and PPARγ) were directly and indirectly inhibited in PPOS, which resulted into the delay or block of spontaneous ovulation. Moreover, the extent of inhibiting ovulation could be possible to be fine- tuned if the dose-dependent and time-dependent changes of progesterone were approved. Our research would firstly explore the molecular mechanism of progesterone and PR pathway on inhibiting spontaneous ovulation using the rat progesterone-primed superovulated model. By adopting the 3 dimensional (3D) in vitro follicle growth model, the whole process of follicle growth dynamic and ovulation was witnessed in the progesterone-primed milieu. The expression of PR and their downstream proteases (ADAMTS1, EDN2 and PPARγ) were examined in the control of progesterone and PR antagonist using Real-time PCR, Western blotting. This study will fill out the blank of the research of the follicle growth and ovulation mechanism under ovarian stimulation background and explore the relationship of ovulation rupture and oocyte developmental potential in progesterone-primed condition from indirect and direct perspective. At the same time, the study will be helpful to explain the association between extent of progesterone priming and inhibiting ovulation as well as the mechanism of high progesterone influencing oocyte developmental competence, also, to find the possible novel site in PR pathway for drug manufacture and provide a new perspective for safer, more efficient and optimal strategy for controlled ovarian hyperstimulation.

如何控制提早排卵是超促排卵技术中的核心问题，目前常规方法是使用GnRHa类似物，不足之处是用药复杂、注射时间长、并发症多。在全胚冷冻的基础上，本中心提出了以利用孕激素抑制自发排卵为理念的超排卵新方法（progestin-primed ovarian stimulation, PPOS), 目前尚缺少相关的机理研究。我们假设PPOS 在卵巢层面上的机制之一，是直接或间接的下调孕激素受体（PR）及其下游调节蛋白(ADAMTS1、EDN2和PPARγ)，造成卵泡破裂延迟或阻滞。本项目拟通过大鼠超排卵模型及体外3D卵泡培养模型，研究孕激素对卵泡发育、成熟和排卵的调节，以及孕激素化状态下颗粒细胞PR及其下游调节蛋白的表达，并通过细胞及在体动物两个水平转染PR，验证PR表达与排卵抑制的关联性。研究将拓展生殖内分泌机制，优化PPOS治疗新策略。

高孕激素状态下超排卵（Progestin-primed ovarian stimulation, PPOS）周期呈现卵泡“不易破裂”的特点，自发LH峰发生率很低（0-3%），说明PPOS周期有良好的卵泡控制性。本研究针对孕激素可抑制卵泡自发排卵这一临床现象，从卵巢及下丘脑两个不同层面研究了孕激素对卵泡发育及抑制排卵的作用机制。我们通过：1）建立高孕激素促排卵小鼠模型，研究孕激素化作用下卵泡发育特征、甾体激素合成酶功能以及卵泡发育相关信号通路（Akt-PI3K-mTOR）信号通路的变化，我们发现小鼠PPOS模型中动情周期延长，窦卵泡及黄体数量减少，囊状卵泡增多、甾体激素合成酶等表达未见差异。PPOS组卵巢颗粒细胞p-Akt及p-mTOR显著高于MPA组，但是p-PI3K, p-Akt及p-mTOR表达低于hMG组，说明促性腺激素(FSH)的刺激使得颗粒细胞 PI3K-Akt-mTOR磷酸化增强，而MPA的添加抑制了这一过程；2）应用去卵巢的小鼠模型研究孕激素抑制提早排卵的机制，首先给予雌孕激素处理连续监测到LH峰被抑制的现象，随后在AVPV 脑区灌注dynorphin、GABA-A 和 GABA-B 受体拮抗剂，观察被孕激素抑制的 LH 峰是否可被挽救，结果说明在灌注dynorphin、GABA-A抑制剂的小鼠出现了LH峰，而GABA-B抑制剂未见LH峰被挽救，说明孕激素通过下调dynorphin 和GABA-A神经递质抑制下丘脑AVPV 脑区kisspeptin神经元出现自发LH峰，为孕激素的临床应用提供了明确了神经生物学机制。3）通过体外小鼠次级卵泡培养模型中添加孕激素，发现在体外培养的卵泡中孕激素抑制了次级卵泡生长，FSH可克服孕激素的抑制作用，孕激素对卵子成熟和颗粒细胞甾体激素合成功能没有显著的不良影响。以上研究结果从不同的实验角度论证了孕激素抑制自发排卵的发生机理，为PPOS应用和推广提供了科学的理论依据和支撑。