Poor ovarian response (POR) is a big challenge for in vitro fertilization. The traditional Chinese medicine, Cai’s Prescription of Tonifying Kidney and Strengthening Vitals (Cai’s Prescription) has yielded satisfactory results for POR treatment clinically, but systematic scientific research of Cai’s Prescription is not well reported. This study aimed to investigate the clinical effect of Cai’s Prescription on poor ovarian responders and its biological mechanism. Serum was collected from poor ovarian responders, and IL-1β, INFγ, FSH, E2 and AMH levels were analyzed by ELISA. Ovarian antral follicles were identified and counted using transvaginal ultrasound. The embryo quality grading were done on day 3 after retrieval. We used high-throughput sequencing of granulosa cells to investigate the gene transcription patterns of ovarian granulosa cells in poor ovarian responders after Cai’s Prescription pretreatment. The expression level of ARHGAP4 was analyzed by quantitative real-time PCR and western blot. The effects of ARHGAP4 for granulosa cells were analyzed by CCK-8 assay, annexin-V and PI staining, ELISA and western blot. The effects of Cai’s Prescription on the expression of PI3K-Akt pathway and apoptosis were analyzed by western blot. In this study, we found that Cai’s Prescription pretreatment had the tendency to improve the ovarian reserve function and could increase the number of high quality embryos for poor ovarian responders. Through high-throughput sequencing of mRNA in granulosa cells, we discovered ARHGAP4, which is a member of GTPase-activating proteins (GAPs) may be a candidate target for POR treatment. ARHGAP4 was significantly increased in poor ovarian responders and can be recovered after Cai’s Prescription pretreatment. Mechanically, combining the cell line model and clinical tissue samples, we found that ARHGAP4 can accelerate cell apoptosis and inflammation response in granulosa cells via PI3K-Akt signaling pathway. In addition, Cai’s Prescription pretreatment for three months significantly reduced the high level of ARHGAP4 in poor ovarian responders. This study shows that the traditional Chinese medicine, Cai’s Prescription yielded satisfactory results for poor ovarian responders clinically and ARHGAP4 may be a candidate target for POR treatment.

中文翻译：

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蔡氏方通过 ARHGAP4 抑制卵巢反应不良者的颗粒细胞凋亡

卵巢反应不良（POR）是体外受精的一大挑战。中药“蔡氏补肾固命方”(蔡氏方)在临床上治疗POR取得了满意的效果，但对蔡氏方的系统科学研究报道较少。本研究旨在探讨蔡氏方对卵巢反应不良者的临床疗效及其生物学机制。从卵巢反应不良者中收集血清，并通过 ELISA 分析 IL-1β、INFγ、FSH、E2 和 AMH 水平。使用经阴道超声对卵巢窦卵泡进行识别和计数。胚胎质量分级在取出后第3天进行。我们利用颗粒细胞的高通量测序来研究蔡氏方预处理后卵巢反应不良者卵巢颗粒细胞的基因转录模式。通过实时定量PCR和蛋白质印迹分析ARHGAP4的表达水平。通过CCK-8测定、膜联蛋白-V和PI染色、ELISA和蛋白质印迹分析ARHGAP4对颗粒细胞的影响。 Western blot分析蔡氏方对PI3K-Akt通路表达及细胞凋亡的影响。在这项研究中，我们发现蔡氏方预处理有改善卵巢储备功能的趋势，并且可以增加卵巢反应不良者的优质胚胎数量。通过对颗粒细胞中 mRNA 的高通量测序，我们发现 ARHGAP4（GTP 酶激活蛋白（GAP）的成员）可能是 POR 治疗的候选靶点。 ARHGAP4在卵巢反应不良者中显着升高，经蔡氏方预处理后可恢复。机械方面，结合细胞系模型和临床组织样本，我们发现ARHGAP4可以通过PI3K-Akt信号通路加速颗粒细胞的细胞凋亡和炎症反应。此外，蔡氏方预处理三个月显着降低了卵巢反应不良者的高水平ARHGAP4。本研究表明中药蔡氏方在临床上对卵巢反应不良者取得了满意的效果，ARHGAP4可能成为POR治疗的候选靶点。