微小核糖核酸(miRNA)在預測植入窗期長度和時間扮演的角色

研究目的

以miRNA作為基礎的子宮內膜容受性測試 (MIRA) 和以mRNA作為基礎的子宮內膜容受性測試(ERA)的檢測結果有什麼區別？

What are the differences between results from a miRNA-based endometrial receptivity test (MIRA) and a mRNA-based one (ERA)?

研究結論

MIRA 和 ERA 的結果之間有 74.1% 的一致性。代表著MIRA中選擇的 miRNA 對於預測植入窗期可以做為選擇的指標。

There was a 74.1% concordance between MIRA’s and ERA’s results with additional implications from the select miRNAs that were indicators for window of implantation length.

前言

隨著科技的普及，不同類型的生物標誌物已被用於分析子宮內膜容受性，例如 microRNAs (miRNAs)及mRNAs，而這些現有技術的發展局限在於其預測植入窗期的能力。 過去發現透過mRNA分析而預測出屬於後容受期(post-receptive stage)的患者具有不同的長度的植入窗期。而目前的解決方案是對患者進行二次採檢，再次確認植入窗期，針對後續療程胚胎移植時間提供建議。

As more technologies become available, different types of biomarkers have been used to analyze endometrial receptivity such as microRNAs instead of mRNAs. A limitation to these existing technologies is in their ability to determine the length of window of implantation. Patients that are analyzed to be in the post-receptive stage have been seen to have varied lengths of window of implantation. The current solution is to obtain multiple biopsies from an IVF patient in order to confirm the length of their window in order to provide a suggestion on how to alter their embryo transfer time.

研究設計

本研究選用58 個子宮內膜組織活檢樣本進行MIRA分析，將這些結果與 ERA 進行比較以計算一致性。其中11位透過ERA判斷為後容受期(post-receptive stage)且胚胎移植成功的患者，這些患者皆進行二次採檢，活檢結果為“容受期”(receptive stage)”定義為植入“平均”窗期，“前容受期(pre-receptive stage)”定義為植入“縮短”窗期。針對比較植入樣本的縮短窗口和平均窗口之間的單個 miRNA 生物標誌物來進行額外的分析。

58 endometrial tissue biopsy samples were analyzed with MIRA. These results were compared to ERA to calculate concordance. Additional analysis was performed by comparing individual miRNA biomarkers between shortened and average window of implantation samples.11 post-receptive samples with a successful embryo transfer were chosen. Second biopsies for these samples with a “receptive” result indicated “average” window of implantation, while a “ pre-receptive” result from the second biopsy indicated a “shortened” window of implantation.

研究方法

本研究所使用之子宮內膜組織活檢樣本皆為之前進行過 ERA 測試的殘留臨床樣本中收集而來的。 透過 NextAmp™ 的多重定量 qPCR 平台提取和分析殘留組織樣本的 RNA，分析 MIRA 測試面板上 96 種 miRNA 生物標誌物的表達譜（expression profiles）。 隨後透過專有算法進行分析，以確定患者的子宮內膜階段（前容受期、容受期或後容受期）。

Endometrial tissue biopsy samples were collected from residual clinical samples that had previously undergone ERA testing. The residual tissue samples’ RNA was extracted and analyzed via a multiplex, quantitative qPCR-based platform called NextAmp™ to profile the expression profiles of 96 miRNA biomarkers on MIRA’s testing panel. The subsequent raw expression profiles were analyzed through a proprietary algorithm in order to determine the endometrial stage of the patient (pre-receptive, receptive, or post-receptive).

研究結果

根據比較結果，MIRA與ERA兩項測試結果的一致性為 74.1%。針對MIRA檢測中的個別miRNAs進一步分析，確認共有 9個差異表達 miRNA，可能作為縮短植入窗期的指標。子宮內膜分泌期的結束通常以如基質降解（MMP催化）、白細胞浸潤基質、腺體活動停止和整個組織的細胞凋亡等現象為特徵。透過 KEGG 通路分析結果，確定了這9個miRNA涉及這些特徵的通路，例如 ECM-受體相互作用、細胞衰老、壞死性凋亡、粘附連接和間隙連接。有了這些發現，我們發現所鑑定的 9 種 miRNA 是子宮內膜分泌期末期發生的形態變化的潛在調節因子，透過監測它們的表達量可以作為分泌期提前結束的早期指標，也意味著植入窗口縮短。

Concordance between the two tests was 74.1%. Further analysis of the individual miRNAs on MIRA’s panel identified 2 down-regulated and 7 up-regulated differentially expressed miRNAs that had potential acting as indicators for shortened window of implantation. The identified differentially expressed miRNAs were based on fold change (log2) = > 0.585 or < -0.585 and p-value = < 0.05 (t-test). Of the 9 identified differentially expressed miRNAs, KEGG analysis was performed to explore their implications in more depth. The end of the secretory phase of the endometrium is usually characterized by phenomena such as degradation of the stromal network (MMP catalysis), infiltration of the stroma by leukocytes, cessation of glandular activity, and apoptosis throughout the tissue. Through KEGG pathway analysis results, pathways that implicate these characterizations were identified such as ECM-receptor interaction, cellular senescence, necroptosis, adherens junctions, and gap junction. With these findings, we see that the 9 miRNAs identified are potential regulators of the morphological changes that occur at the end of the secretory phase in the endometrium, and by monitoring their expression levels can be early indicators of an advanced end to the secretory phase, implying a shortened window of implantation.

研究限制

本研究目前的研究樣本量有限，未來應進行更大規模的研究以確認這些發現。 此外，本研究目前先行對後容受期患者進行miRNA 生物標誌物分析；在未來會針對前容受期、容受期患者進行相同之分析。

Larger studies should be performed in order to confirm these findings as the current study sample size is limited. Additionally, this only takes into account post-receptive patients; therefore, the same analysis should also be done on patients that fall in the pre-receptive and receptive stages when undergoing endometrial receptivity testing.

研究結果的更廣泛意義

在這項初步研究中，我們發現miRNAs可以作為潛在的未來生物標誌，提供子宮內膜容受性檢測辨別患者縮短的植入窗期，對於辨識患者植入窗期時就無需多次採集活檢。

In this preliminary study, we see that miRNAs could act as potential future biomarkers for identifying shortened window of implantations for patients through endometrial receptivity testing, cutting out the need for multiple biopsies when identifying patients’ windows of implantation.

原文內容

E.P Yang, Y.J Lee, E.H Cheng, P.Y Lin, W.M Chen, W.C Hsu, P.C Chang, J.H Yang, Y.S Huang, C.Y Fan, A Hsu, Y.C Kao, T Wang, M.S Lee, P-385 miRNAs and their roles in predicting length and timing of window of implantation, Human Reproduction, Volume 37, Issue Supplement_1, July 2022, deac107.362, https://doi.org/10.1093/humrep/deac107.362