Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation.
Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation.
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DOI：
10.1093/humrep/deab123
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发表时间：
2021-07-19
期刊：
影响因子：
--
通讯作者：
Viganò P
中科院分区：
文献类型：
--
作者：
Giacomini E;Scotti GM;Vanni VS;Lazarevic D;Makieva S;Privitera L;Signorelli S;Cantone L;Bollati V;Murdica V;Tonon G;Papaleo E;Candiani M;Viganò P
子宫液衍生的细胞外囊泡（UF-EV）是用于实时监测子宫内膜状态的生物标志物的“液体活检”储库吗？ UF-EV 的转录组货物反映了子宫内膜组织的 RNA 谱以及非接受期和接受期之间的变化，可能支持其用于新型子宫内膜接受性测试。 EV 之前已从子宫液中分离出来，它们可能在植入过程中促进胚胎与子宫内膜的串扰。根据对子宫内膜组织植入相关基因和人类外泌体数据库研究的荟萃分析，被认为是接受性标记的 57 个转录本中有 28 个涉及人类外泌体中存在的蛋白质。然而，接受期 UF-EV 的具体转录组内容尚未确定。设立了两个实验系列。首先，我们同时对来自配对 UF-EV 的 RNA 物种和从生理周期女性收集的子宫内膜组织样本进行了测序。其次，我们分析了在已证实具有生育能力的女性的非接受期 (LH + 2) 和接受期 (LH + 7) 阶段以及从接受 ART 和整倍体囊胚移植的女性群体的接受期 (LH + 7) 阶段收集的 UF-EV 的 RNA 种类。对于配对超滤子宫内膜组织取样，在通过子宫内膜腔灌洗进行超滤收集后立即使用 Pipelle 获得子宫内膜组织活检。总体而言，收集了 n = 87 UF 样品并进行新鲜处理，用于 EV 分离和总 RNA 提取，同时使用蛋白质印迹来确认分离的囊泡的 EV 蛋白标记物的表达。 UF-EV 的物理表征通过纳米粒子跟踪分析进行。为了定义 UF-EV 样品的转录组货物，从 n = 83 UF-EV 样品成功制备了 RNA-seq 文库，并通过 RNA-seq 分析进行了分析。差异基因表达（DGE）分析用于比较不同组样本之间的RNA-seq结果。使用 g:Profiler 通过基因集富集分析进行功能富集分析。使用 WebGestalt 进行预排序基因集富集分析 (GSEA)，将 RNA-seq 结果与市售子宫内膜容受性阵列中评估的基因集进行比较。在子宫内膜活检和成对 UF-EV 样本的转录谱之间发现了高度显着的相关性（Pearson’s r = 0.70 P < 0.0001；Spearman’s ρ = 0.65 P < 0.0001）。在来自可育对照的 UF-EV 中，与 LH + 2 非接受期相比，LH + 7 接受期的 942 个基因转录本更加丰富，1305 个基因转录本更加丰富。 GSEA 评估了市售子宫内膜容受性阵列中包含的 n = 238 个基因与 LH + 7 与 LH + 2 UF-EV 比较之间转录谱的一致性，结果表明存在极其显着且一致的富集，标准化富集评分 (NES)=9.38 (P < 0.001)。商业阵列中的 LH + 7 并在 LH + 7 UF-EV 中富集，商业阵列中 LH + 7 中的转录物下调并在 LH + 7 UF-EV 中耗尽，NES = -5.40 (P < 0.001)。当分析下一个周期的一个整倍体囊胚移植后植入成功与失败的患者的 LH + 7 UF-EV 时，我们发现在实现妊娠的女性组中，有 97 个基因的转录水平增加，64 个基因的转录水平下降。 GSEA 旨在评估市售子宫内膜容受性阵列基因之间转录谱的一致性，并比较植入成功与失败的女性的 LH + 7 UF-EV，结果表明，商业阵列中接受期转录本显着富集，NES = 2.14（P = 0.001），并且在植入失败的女性的 UF-EV 中富集。构想的，并且对于在商业阵列中下调的转录物和在 UF-EV 中耗尽的转录物，NES = -1.18（P = 0.3）不显着。就身体特征而言，UF-EV 在分析的不同组中显示出同质性，除了 EV 大小略有但显着的差异外，与整倍体囊胚移植后未能受孕的患者相比，成功植入的女性的 EV 较小（平均直径±SD 分别为 205.5±22.97nm 和 221.5±20.57nm， P = 0.014）。转录组数据存放在 NCBI 基因表达综合库 (GEO) 中，并且可以使用 GEO 系列登录号：GSE158958 进行检索。与 EV 膜相关的 RNA 种类的分离可能不完全，膜结合的 RNA 种类（而不是 EV 的内部 RNA 含量）可能对我们的 RNA 测序结果有贡献。此外，我们无法明确区分外泌体、微泡和凋亡小体对我们的研究结果的相对贡献。当考虑接受 ART 的患者时，我们没有在整倍体胚胎移植的同一周期中收集 UF，而是在前一个周期中收集。我们认为这种方法最适合我们研究的新颖性、探索性。根据我们的结果，可以假设在进行胚胎移植的同一周期中验证 UF-EV RNA-seq 分析。这项研究利用 RNA-seq 分析的人类 EV 的最大样本量，建立了一个基因特征，用于微创子宫内膜容受性测试。该报告确实首次表明 UF-EV 的转录组与子宫内膜组织转录组相关，UF-EV 中的 RNA 特征随子宫内膜状态而变化，并且 UF-EV 可以作为潜在的侵入性较小的接受性标记物收集的储存库。因此，这篇文章代表了实时监测子宫内膜状态的微创方法的设计向前迈出了一步，这对于推进生殖医学领域是必要的。该研究由欧洲人类生殖和胚胎学学会竞争性资助（ESHRE 研究资助 2016-1）资助。作者没有需要披露的财务或非财务竞争利益。不适用。
Are uterine fluid-derived extracellular vesicles (UF-EVs) a ‘liquid biopsy’ reservoir of biomarkers for real-time monitoring of endometrial status? The transcriptomic cargo of UF-EVs reflects the RNA profile of the endometrial tissue as well as changes between the non-receptive and the receptive phase, possibly supporting its use for a novel endometrial receptivity test. EVs have been previously isolated from uterine fluid, where they likely contribute to the embryo-endometrium crosstalk during implantation. Based on a meta-analysis of studies on endometrial tissue implantation-associated genes and the human exosomes database, 28 of the 57 transcripts considered as receptivity markers refer to proteins present in human exosomes. However, the specific transcriptomic content of receptive phase UF-EVs has yet to be defined. Two experimental series were set up. First, we simultaneously sequenced RNA species derived from paired UF-EVs and endometrial tissue samples collected from physiologically cycling women. Second, we analyzed RNA species of UF-EVs collected during the non-receptive (LH + 2) and receptive (LH + 7) phase of proven fertile women and from the receptive (LH + 7) phase of a population of women undergoing ART and transfer of euploid blastocysts. For paired UF—endometrial tissue sampling, endometrial tissue biopsies were obtained with the use of a Pipelle immediately after UF collection performed by lavage of the endometrial cavity. Overall, n = 87 UF samples were collected and fresh-processed for EV isolation and total RNA extraction, while western blotting was used to confirm the expression of EV protein markers of the isolated vesicles. Physical characterization of UF-EVs was performed by Nanoparticle Tracking Analysis. To define the transcriptomic cargo of UF-EV samples, RNA-seq libraries were successfully prepared from n = 83 UF-EVs samples and analyzed by RNA-seq analysis. Differential gene expression (DGE) analysis was used to compare RNA-seq results between different groups of samples. Functional enrichment analysis was performed by gene set enrichment analysis with g:Profiler. Pre-ranked gene set enrichment analysis (GSEA) with WebGestalt was used to compare RNA-seq results with the gene-set evaluated in a commercially available endometrial receptivity array. A highly significant correlation was found between transcriptional profiles of endometrial biopsies and pairwise UF-EV samples (Pearson’s r = 0.70 P < 0.0001; Spearman’s ρ = 0.65 P < 0.0001). In UF-EVs from fertile controls, 942 gene transcripts were more abundant and 1305 transcripts less abundant in the LH + 7 receptive versus the LH + 2 non-receptive phase. GSEA performed to evaluate concordance in transcriptional profile between the n = 238 genes included in the commercially available endometrial receptivity array and the LH + 7 versus LH + 2 UF-EV comparison demonstrated an extremely significant and consistent enrichment, with a normalized enrichment score (NES)=9.38 (P < 0.001) for transcripts up-regulated in LH + 7 in the commercial array and enriched in LH + 7 UF-EVs, and a NES = −5.40 (P < 0.001) for transcripts down-regulated in LH + 7 in the commercial array and depleted in LH + 7 UF-EVs. When analyzing LH + 7 UF-EVs of patients with successful versus failed implantation after transfer of one euploid blastocyst in the following cycle, we found 97 genes whose transcript levels were increased and 64 genes whose transcript levels were decreased in the group of women who achieved a pregnancy. GSEA performed to evaluate concordance in transcriptional profile between the commercially available endometrial receptivity array genes and the comparison of LH + 7 UF-EVs of women with successful versus failed implantation, demonstrated a significant enrichment with a NES = 2.14 (P = 0.001) for transcripts up-regulated in the commercial array in the receptive phase and enriched in UF-EVs of women who conceived, and a not significant NES = −1.18 (P = 0.3) for transcripts down-regulated in the commercial array and depleted in UF-EVs. In terms of physical features, UF-EVs showed a homogeneity among the different groups analyzed except for a slight but significant difference in EV size, being smaller in women with a successful implantation compared to patients who failed to conceive after euploid blastocyst transfer (mean diameter ± SD 205.5± 22.97 nm vs 221.5 ± 20.57 nm, respectively, P = 0.014). Transcriptomic data were deposited in NCBI Gene Expression Omnibus (GEO) and can be retrieved using GEO series accession number: GSE158958. Separation of RNA species associated with EV membranes might have been incomplete, and membrane-bound RNA species—rather than the internal RNA content of EVs—might have contributed to our RNA-seq results. Also, we cannot definitely distinguish the relative contribution of exosomes, microvesicles and apoptotic bodies to our findings. When considering patients undergoing ART, we did not collect UFs in the same cycle of the euploid embryo transfer but in the one immediately preceding. We considered this approach as the most appropriate in relation to the novel, explorative nature of our study. Based on our results, a validation of UF-EV RNA-seq analyses in the same cycle in which embryo transfer is performed could be hypothesized. On the largest sample size of human EVs ever analyzed with RNA-seq, this study establishes a gene signature to use for less-invasive endometrial receptivity tests. This report is indeed the first to show that the transcriptome of UF-EVs correlates with the endometrial tissue transcriptome, that RNA signatures in UF-EVs change with endometrial status, and that UF-EVs could serve as a reservoir for potential less-invasive collection of receptivity markers. This article thus represents a step forward in the design of less-invasive approaches for real-time monitoring of endometrial status, necessary for advancing the field of reproductive medicine. The study was funded by a competitive grant from European Society of Human Reproduction and Embryology (ESHRE Research Grant 2016-1). The authors have no financial or non-financial competing interests to disclose. NA.
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影响因子：
16
作者：
Lötvall J;Hill AF;Hochberg F;Buzás EI;Di Vizio D;Gardiner C;Gho YS;Kurochkin IV;Mathivanan S;Quesenberry P;Sahoo S;Tahara H;Wauben MH;Witwer KW;Théry C
通讯作者：
Théry C
影响因子：
8.3
作者：
Franasiak, Jason M.;Forman, Eric J.;Scott, Richard T., Jr.
通讯作者：
Scott, Richard T., Jr.
影响因子：
3.4
作者：
Chaouat, G;Zourbas, S;Martal, J
通讯作者：
Martal, J
影响因子：
7.4
作者：
Campoy I;Lanau L;Altadill T;Sequeiros T;Cabrera S;Cubo-Abert M;Pérez-Benavente A;Garcia A;Borrós S;Santamaria A;Ponce J;Matias-Guiu X;Reventós J;Gil-Moreno A;Rigau M;Colas E
通讯作者：
Colas E
影响因子：
3.7
作者：
Ng YH;Rome S;Jalabert A;Forterre A;Singh H;Hincks CL;Salamonsen LA
通讯作者：
Salamonsen LA