Department of Development and Regeneration, KU Leuven, Leuven, Belgium.
Leuven Endometriosis Center of Excellence, University Hospital Leuven, Leuven, Belgium.
Hum Reprod. 2019 Sep 29;34(9):1650-1660. doi: 10.1093/humrep/dez116.
Can plasma miRNAs be used for the non-invasive diagnosis of endometriosis in infertile women?
miRNA-based diagnostic models for endometriosis failed the test of independent validation.
Circulating miRNAs have been described to be differentially expressed in patients with endometriosis compared with women without endometriosis, suggesting that they could be used for the non-invasive diagnosis of endometriosis. However, these studies have shown limited consistency or conflicting results, and no miRNA-based diagnostic test has been validated in an independent patient cohort.
STUDY DESIGN, SIZE, DURATION: We performed genome-wide miRNA expression profiling by small RNA sequencing to identify a set of plasma miRNAs with discriminative potential between patients with and without endometriosis. Expression of this set of miRNAs was confirmed by RT-qPCR. Diagnostic models were built using multivariate logistic regression with stepwise feature selection. In a final step, the models were tested for validation in an independent patient cohort.
PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Plasma of all patients was available in the biobank of the Leuven Endometriosis Centre of Excellence. Biomarker discovery and model development were performed in a discovery cohort of 120 patients (controls = 38, endometriosis = 82), and models were tested for validation in an independent cohort of 90 patients (controls = 30, endometriosis = 60). RNA was extracted with the miRNeasy Plasma Kit. Genome-wide miRNA expression analysis was done by small RNA sequencing using the NEBNext small RNA library prep kit and the NextSeq 500 System. cDNA synthesis and qPCR were performed using the Qiagen miScript technology.
We identified a set of 42 miRNAs with discriminative power between patients with and without endometriosis based on genome-wide miRNA expression profiling. Expression of 41 miRNAs was confirmed by RT-qPCR, and 3 diagnostic models were built. Only the model for minimal-mild endometriosis (Model 2: hsa-miR-125b-5p, hsa-miR-28-5p and hsa-miR-29a-3p) had diagnostic power above chance performance in the independent validation (AUC = 60%) with an acceptable sensitivity (78%) but poor specificity (37%).
LIMITATIONS, REASONS FOR CAUTION: The diagnostic models were built and tested for validation in two patient cohorts from a single tertiary endometriosis centre. Further validation tests in large cohorts with patients from multiple endometriosis centres are needed.
Our study supports a possible biological link between certain miRNAs and endometriosis, but the potential of these miRNAs as clinically useful biomarkers is questionable in women with infertility. Large studies in well-described patient cohorts, with rigorous methodology for miRNA expression analysis, sufficient statistical power and an independent validation step, are necessary to answer the question of whether miRNAs can be used as diagnostics markers for endometriosis.
STUDY FUNDING/COMPETING INTEREST(S): The project was funded by a grant from the Research Foundation - Flanders (FWO). A.V., D.F.O. and D.P. are PhD fellows from the FWO. T.D. is vice president and Head of Global Medical Affairs Fertility, Research and Development, Merck KGaA, Darmstadt, Germany. He is also a professor in Reproductive Medicine and Biology at the Department of Development and Regeneration, Group Biomedical Sciences, KU Leuven (University of Leuven), Belgium and an adjunct professor at the Department of Obstetrics and Gynecology in the University of Yale, New Haven, USA. Neither his corporate role nor his academic roles represent a conflict of interest with respect to the work done by him for this study. The other co-authors have no conflict of interest.
Not applicable.
血浆 miRNA 可否用于诊断不孕女性的子宫内膜异位症？
基于 miRNA 的子宫内膜异位症诊断模型在独立验证中未通过测试。
与没有子宫内膜异位症的女性相比，患有子宫内膜异位症的患者的循环 miRNA 表现出差异表达，表明它们可用于子宫内膜异位症的无创诊断。然而，这些研究显示出有限的一致性或相互矛盾的结果，并且没有基于 miRNA 的诊断测试在独立的患者队列中得到验证。
研究设计、规模、持续时间：我们通过小 RNA 测序进行全基因组 miRNA 表达谱分析，以确定一组具有区分患有和不患有子宫内膜异位症患者潜力的血浆 miRNA。通过 RT-qPCR 确认该 miRNA 集的表达。使用多元逻辑回归进行建模，采用逐步特征选择。最后，在独立的患者队列中测试模型进行验证。
参与者/材料、设置、方法：所有患者的血浆均在鲁汶子宫内膜异位症卓越中心的生物库中可用。在 120 名患者（对照组=38，子宫内膜异位症=82）的发现队列中进行了生物标志物发现和模型开发，在 90 名患者（对照组=30，子宫内膜异位症=60）的独立队列中测试了模型的验证。使用 miRNeasy Plasma Kit 提取 RNA。使用 NEBNext small RNA library prep kit 和 NextSeq 500 System 进行全基因组 miRNA 表达分析。使用 Qiagen miScript 技术进行 cDNA 合成和 qPCR。
我们根据全基因组 miRNA 表达谱分析确定了一组具有区分子宫内膜异位症患者和非患者能力的 42 个 miRNA。通过 RT-qPCR 验证了 41 个 miRNA 的表达，构建了 3 个诊断模型。仅最小轻度子宫内膜异位症的模型（模型 2：hsa-miR-125b-5p、hsa-miR-28-5p 和 hsa-miR-29a-3p）在独立验证（AUC=60%）中具有高于机会性能的诊断能力，具有可接受的灵敏度（78%）但特异性差（37%）。
局限性、谨慎的原因：在来自单个三级子宫内膜异位症中心的两个患者队列中构建和测试了诊断模型。需要在具有来自多个子宫内膜异位症中心的患者的大型队列中进行进一步的验证测试。
我们的研究支持某些 miRNA 与子宫内膜异位症之间可能存在生物学联系，但这些 miRNA 作为具有临床意义的生物标志物在不孕女性中的应用价值值得怀疑。需要在具有明确 miRNA 表达分析方法、足够统计能力和独立验证步骤的描述良好的患者队列中进行大型研究，以回答 miRNA 是否可作为子宫内膜异位症的诊断标志物的问题。
研究资金/利益冲突：该项目由研究基金会-佛兰德斯（FWO）资助。A.V.、D.F.O. 和 D.P. 是 FWO 的博士研究员。T.D. 是 Merck KGaA、达姆施塔特德国生殖医学、研究与发展部的全球医学事务副总裁和负责人，也是比利时鲁汶大学（天主教鲁汶大学）生殖医学与生物学系教授，美国耶鲁大学妇产科兼职教授。他的公司角色和学术角色都不代表他为这项研究所做工作的利益冲突。其他合著者没有利益冲突。
不适用。
