Obesity alters the endometrial transcriptome in a cell context-dependent manner. Obesity alters the endometrial transcriptome in a cell context-dependent manner

Obesity significantly impacts fertility, is positively correlated with endometrial cancer occurrence, and negatively correlated with endometriosis incidence. The endometrial epithelia often harbor disease driver-mutations, while the endometrial stroma are highly regulative of the neighboring endometrial epithelia. Two cohorts of CD-1 mice were fed high-fat diet or control diet ad libitum for 18 weeks, beginning at 8 weeks of age. Weight measurements were taken weekly and serum was collected bi-weekly. After 17 weeks, a glucose tolerance test was performed on all mice. After 18 weeks, uteri were collected. We profiled the transcriptomes of positively-enriched endometrial epithelia (cohorts 1 and 2) and macrophages (cohort 2), and epithelia- and macrophage-depleted stroma populations (cohort 2). Pieces of uterine tissue were saved for histological analysis. Purity of cell populations were confirmed by flow cytometry. RNA was isolated from each cell type and libraries were synthesized for RNA-seq. Mice fed a high-fat diet displayed significantly increased body mass compared to control diet mice after three weeks. High-fat diet mice developed glucose intolerance, hyperinsulinemia, and fatty liver. Obese mice displayed differential gene expression in the endometrial epithelia for 105 genes (FDR < 0.05), including factors related to innate immunity and leukocyte chemotaxis, such as calprotectin. The endometrial stroma of obese mice differentially expressed 141 genes (FDR < 0.05) including several factors related to circadian rhythm. Expression of many of these genes was significantly correlated with glucose tolerance or body mass (p < 0.05). We observed correlations between F4/80 macrophage marker, Cleaved Caspase 3 (CC3) apoptosis marker in the luminal epithelia and glucose intolerance. Among obese mice, we observed a subgroup with high CC3 staining in the luminal epithelium by immunohistochemistry. This subgroup displayed differential gene expression among all cell types, with pathways related to immune escape observed in epithelial and macrophage compartments, and pathways related to epithelial regulation observed in the stromal compartments. The endometrial macrophages of obese mice compared to control mice displayed differential gene expression of only one gene, although the abundance of F4/80+ macrophages was shown to be increased in the obese mouse uterus. There may be species-specific differences between humans and mice which limit interpretations. The results suggest an important role for differential response of both the epithelia and stroma in their response to obese phenotypes, while macrophages are dysregulated in the context of apoptotic epithelium. The obesity-related gene expression programs in cells found within the uterine microenvironment may influence the ability of the endometrium to function during pregnancy and contribute to endometrial cancer pathogenesis. Overall design: EPCAM+ epithelia, F4/80+ macrophages and VIM+ stroma were isolated from the uteri of mice following 18 weeks on high-fat diet or control diet. RNA was collected from cels and used for RNA-seq analysis.

肥胖对生育功能具有显著负面影响，与子宫内膜癌的发生呈正相关，与子宫内膜异位症的发病率呈负相关。子宫内膜上皮常携带疾病驱动突变，而子宫内膜间质对邻近的子宫内膜上皮具有高度调控作用。本研究以8周龄的CD-1小鼠为实验对象，设置两批队列，分别自由摄取高脂饲料或对照饲料，持续干预18周。实验期间每周记录小鼠体重，每两周采集血清样本。干预17周后，对所有小鼠实施葡萄糖耐量试验（glucose tolerance test）；干预18周后，采集子宫组织。我们对经阳性富集的子宫内膜上皮细胞（队列1与队列2）以及巨噬细胞（队列2）进行了转录组分析，同时对队列2中去除上皮细胞与巨噬细胞后的间质细胞群体开展转录组检测。留存部分子宫组织用于组织学分析，并通过流式细胞术（flow cytometry）验证了各细胞群体的纯度。从各细胞类型中提取总RNA并构建测序文库，用于RNA测序（RNA-seq）分析。

喂食高脂饲料的小鼠在干预3周后，体重显著高于对照组小鼠；高脂饮食小鼠还出现了葡萄糖耐量异常、高胰岛素血症（hyperinsulinemia）及脂肪肝（fatty liver）表型。肥胖小鼠的子宫内膜上皮细胞中共有105个基因存在差异表达（错误发现率False Discovery Rate, FDR < 0.05），其中包括与先天免疫及白细胞趋化相关的因子，如钙卫蛋白（calprotectin）。肥胖小鼠的子宫内膜间质细胞中共有141个基因存在差异表达（FDR < 0.05），包含多个与昼夜节律相关的调控因子。上述多数差异基因的表达水平与葡萄糖耐量或体重呈显著相关性（p < 0.05）。

我们观察到，F4/80巨噬细胞标志物、腔上皮细胞中裂解型半胱氨酸蛋白酶3（Cleaved Caspase 3, CC3）凋亡标志物的表达水平与葡萄糖耐量异常存在关联。在肥胖小鼠群体中，存在一个亚群其腔上皮细胞经免疫组化（immunohistochemistry）检测显示高CC3染色；该亚群的所有细胞类型均存在差异基因表达，上皮细胞与巨噬细胞组分中富集到与免疫逃逸相关的通路，间质细胞组分中则富集到与上皮调控相关的通路。与对照组小鼠相比，肥胖小鼠的子宫内膜巨噬细胞仅存在1个差异表达基因，但已有数据表明肥胖小鼠子宫内F4/80+巨噬细胞的丰度显著升高。

由于人与小鼠可能存在物种特异性差异，本研究结果的临床解读存在一定局限性。研究结果提示，上皮细胞与间质细胞对肥胖表型的差异响应均发挥关键作用，而巨噬细胞在凋亡上皮细胞所处的微环境中存在调控异常。子宫微环境内各细胞类型的肥胖相关基因表达程序，可能会影响子宫内膜在妊娠期间的正常功能，并参与子宫内膜癌的发病过程。

总体实验设计：分别在高脂饲料或对照饲料喂食18周后，从小鼠子宫中分离得到EPCAM+上皮细胞、F4/80+巨噬细胞及VIM+间质细胞，从各细胞类型中提取RNA并进行RNA-seq分析。