Age-Related Transcriptomic Profiling of Human Granulosa Cells Reveals mRNA-microRNA Regulatory Network Associated with Key Ovulation Dynamics [RNA-Seq]

Advanced maternal age (AMA) patients experience decreased success from assisted reproductive technologies (ART), attributed to quantity and quality of oocytes, which is significantly influenced by the health of surrounding granulosa cells (GCs). In this study, we compared mRNA and microRNA (miRNA) transcriptomes between young (<32 y.o.) and AMA (>38 y.o.) patients' GCs to identify ovarian aging molecular signatures. Granulosa cells were isolated from 18 patients’ follicular fluid, and RNA was isolated for sequencing and subsequent bioinformatics analysis. We identified 293 and 21 differentially expressed genes (DEGs) and miRNAs (DE miRNA), respectively, between young and AMA GCs (P value < 0.05, FDR < 0.25, Fold Change > 1.5). Highly expressed mitochondrial-encoded genes, MT-ND3, MT-ND6, and MT-CYB are upregulated in young GCs. Pathway analysis indicates DEGs play a role in inflammation, cytokine signaling, extracellular matrix (ECM) remodeling, and angiogenesis. Some key DEGs include CXCL8, IL1B, NLRP3, SIGIRR, ANGPT2, ADAM8, and ADAMTS14. Additionally, target gene prediction and pathway analysis of DE miRNA indicates both up- and downregulated miRNA in young GCs are likely targeting genes associated with cell signaling, mitochondrial function, oxidative stress, apoptosis, and senescence pathways in addition to cytokine signaling, angiogenesis, and ECM remodeling. To further investigate regulatory mechanisms, we looked at the convergence of DEGs with predicted target genes of DE miRNA and identified miR-483-3p, miR-1268a, miR-4497, miR-7704, miR-135a-5p, miR-1261, and miR-4791 as key regulators of pathways involved with inflammation, ECM, and angiogenesis. The gene expression data suggests aged GCs have an impaired ability to elicit the same pro-inflammatory response combined with a dysregulation of angiogenesis and ECM remodeling when compared to young GCs, and miRNA may be playing a key role in the regulation of these key ovulatory events. Human granulosa cells were isolated from patients' follicular fluid collected during hormone-stimulated oocyte retrieval cycles. Patients were split into two groups: young and aged. The young group consisted of patients under 32 years old, and the aged group consisted of patients over 38 years old. Patients with a BMI greater than 25, endometriosis, or polycystic ovarian syndrome were excluded from the study. RNA was isolated from a minimum of 1 million cells from each patient. Patient RNA samples were pooled into groups of three to limit individual variation. Each group contained nine patients pooled into three biological replicates.

高龄产妇（Advanced Maternal Age, AMA）患者接受辅助生殖技术（Assisted Reproductive Technologies, ART）的成功率显著下降，该现象与卵母细胞的数量及质量密切相关，而卵母细胞的状态又受到其周围颗粒细胞（Granulosa Cells, GCs）健康状况的显著影响。本研究对比了年轻（<32岁）与高龄（>38岁）患者颗粒细胞的信使RNA（mRNA）与微小RNA（microRNA, miRNA）转录组，旨在挖掘卵巢衰老的分子特征。

研究人员从18名患者的卵泡液中分离颗粒细胞，提取RNA并开展测序及后续生物信息学分析。结果显示，年轻与高龄颗粒细胞间共鉴定出293个差异表达基因（Differentially Expressed Genes, DEGs）与21个差异表达微小RNA（Differentially Expressed miRNAs, DE miRNAs）（P值<0.05，错误发现率（False Discovery Rate, FDR）<0.25，倍数变化（Fold Change）>1.5）。年轻颗粒细胞中，线粒体编码基因MT-ND3、MT-ND6及MT-CYB呈高表达上调趋势。

通路分析结果表明，差异表达基因主要参与炎症反应、细胞因子信号通路、细胞外基质（Extracellular Matrix, ECM）重塑及血管生成过程。关键差异表达基因包括CXCL8、IL1B、NLRP3、SIGIRR、ANGPT2、ADAM8及ADAMTS14。

此外，针对差异表达微小RNA的靶基因预测与通路分析显示，年轻颗粒细胞中上调及下调的微小RNA，其靶基因不仅涉及细胞信号通路、线粒体功能、氧化应激、细胞凋亡与细胞衰老通路，还涵盖细胞因子信号、血管生成及细胞外基质重塑过程。

为进一步探究调控机制，本研究分析了差异表达基因与差异表达微小RNA预测靶基因的交集，鉴定出miR-483-3p、miR-1268a、miR-4497、miR-7704、miR-135a-5p、miR-1261及miR-4791作为炎症、细胞外基质及血管生成相关通路的关键调控因子。

基因表达数据提示，相较于年轻颗粒细胞，衰老颗粒细胞触发促炎症反应的能力受损，同时伴随血管生成与细胞外基质重塑的失调，而微小RNA可能在这些关键排卵事件的调控中发挥核心作用。

本研究的人类颗粒细胞均分离自激素刺激取卵周期中收集的患者卵泡液。研究将患者分为两组：年轻组与高龄组，其中年轻组为32岁以下患者，高龄组为38岁以上患者。本研究排除了体质量指数（Body Mass Index, BMI）>25、子宫内膜异位症或多囊卵巢综合征的患者。从每名患者的至少100万个细胞中提取RNA，将患者RNA样本按每3例一组进行混合以减少个体差异，最终每组纳入9名患者，分为3个生物学重复样本。