| 摘要: |
| 目的 构建非免疫因素致不孕症和免疫因素致不孕症子宫内膜类器官培养体系，并比较两者免疫细胞因子的差异。方法 标本采自海军军医大学（第二军医大学）第一附属医院生殖医学中心行宫腔镜检查的不孕症患者，获取非免疫因素致不孕症患者（n＝3）与反复着床失败（RIF）患者（n＝5）子宫内膜组织，通过洗涤、消化、重悬及接板，将组织嵌入基质胶中进行3D培养；在倒置显微镜下观察两组子宫内膜类器官生长情况；利用免疫荧光染色技术检测子宫内膜特异性标志物雌激素受体（ER)、角蛋白和上皮钙黏蛋白表达情况；利用ELISA法测定两组细胞因子水平，并比较两组细胞因子的差异。结果 在子宫内膜类器官体外培养过程中，类器官体积逐渐增大、细胞数量逐渐增多，培养7～10 d的类器官体积达到稳定状态且形状逐渐趋于圆形。在相同时间内，非免疫因素致不孕症子宫内膜类器官数量较RIF子宫内膜类器官多。免疫荧光染色显示类器官中子宫内膜相关标志蛋白ER、角蛋白和上皮钙黏蛋白均表达，提示子宫内膜类器官构建成功。ELISA检测结果显示，两组间干扰素γ、IL-10、TNF-α、IL-4水平和TNF-α/IL-4比值的差异均有统计学意义（P＜0.05或P＜0.01），TGF-β1、IL-17水平及IL-17/TGF-β1、干扰素γ/IL-10、TNF-α/IL-10比值差异均无统计学意义（均P＞0.05）。结论 在体外成功培养获得了具有增殖能力的非免疫因素致不孕症与RIF子宫内膜类器官，为免疫性不孕症的基础研究提供了新模型。 |
| 关键词: 子宫内膜类器官 不孕症 反复着床失败 免疫细胞因子 |
| DOI：10.16781/j.CN31-2187/R.20230597 |
| 投稿时间：2023-10-31修订日期：2024-04-24 |
| 基金项目:国家自然科学基金青年科学基金（82304619），中关村精准医学基金会（医健公益性-药学科研专项）. |
|
| Establishment and validation of endometrial organoids in infertility |
| CHEN Meiqi,FAN Shupan,CHEN Yuanjing,YANG Yunyun,WANG Zhuo* |
(Department of Pharmacy, The First Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200433, China
*Corresponding author) |
| Abstract: |
| Objective To construct a culture system of endometrial organoids for infertility induced by non-immune or immune factors, and to compare the immune cytokines between them. Methods The samples were collected from infertility patients undergoing hysteroscopy in Department of Reproductive Medicine Center of The First Affiliated Hospital of Naval Medical University (Second Military Medical University). Endometrial tissues were obtained from patients with infertility caused by non-immune factors (n＝3) and patients with repeated implantation failure (RIF) (n＝5). The tissues were embedded in matrix glue for 3D culture after washing, digestion, re-suspension and plate attachment. The growth of endometrial organoids of the 2 groups was observed under inverted microscope; the expression of estrogen receptor, keratin and E-cadherin, which were specific endometrial markers, was detected by immunofluorescence staining; and the cytokines of the 2 groups were determined by enzyme-linked immunosorbent assay (ELISA) and the differences of cytokines between the 2 groups were observed. Results During the process of in vitro culture of endometrial organoids, the volume of organoids and the number of cells gradually increased. After 7-10 d of culture, the volume of organoids reached a stable state, and the shape gradually became a perfect circle. At the same time, the number of organoids from the infertility patients caused by non-immune factors was more than that from the infertility patients caused by RIF. Immunofluorescence staining showed the expression of endometrial related marker proteins estrogen receptor, keratin and E-cadherin, indicating the successful construction of endometrial organoids. ELISA results showed that the levels of interferon (IFN)-γ, interleukin (IL)-10, tumor necrosis factor (TNF)-α, IL-4 and TNF-α/IL-4 ratio between the 2 groups were significantly different (P＜0.05 or P＜0.01). There were no significant differences in the levels of TGF-β1 or IL-17, the ratios of IL-17/TGF-β1, IFN-γ/IL-10 or TNF-α/IL-10 (all P＞0.05). Conclusion Endometrial organoids with proliferative ability from patients with non-immune infertility and RIF have been successfully cultured in vitro, which provides a new model for the basic research of immune infertility. |
| Key words: endometrial organoids infertility repeated implantation failure immune cytokines |
