目的
子癲前症為嚴重的產科併發症之一，子癲前症特徵為妊娠20周後出現高血壓和蛋白尿症狀，並伴隨其他嚴重併發症，造成孕婦及胎兒有較高死亡率及致病率。子癲前症病因尚不清楚，部分研究顯示早期胎盤微環境的免疫狀態改變可能導致子癲前症。近年的研究指出自組織、器官釋放的外泌體會參與細胞內訊息傳遞並調控免疫發炎反應，外泌體中含有來源細胞訊息，如蛋白質及遺傳訊息，可作為產科相關併發症直接標誌物。先前研究亦顯示Th17及Treg細胞間平衡對預防子癲前症發生扮演重要角色。本研究旨在探討參與免疫發炎反應調節的Th17和Treg細胞所分泌之細胞激素失衡，另一方面分析血液中外泌體的數量與其中的細胞激素是否可能與子癲前症致病機制相關，並且進一步探討細胞激素含量和外泌體數量是否和疾病嚴重度相關。

方法
本研究分別評估孕婦周邊血液與外泌體中Th17及Treg細胞激素是否可作為子癲前症潛在生物標誌物，我們設計前瞻性世代研究(prospective cohort study)，孕婦懷孕初期即納入研究，並追蹤至生產或是疾病發生，共招募39名確診子癲前症孕婦及127名健康孕婦作為對照組，共採集懷孕期間三個時間點，分別從第一孕期至第三孕期，檢測血液與外泌體中的Th17、Treg類型的五種細胞激素(IL-10、IL-17、IL-21、IL-22及TGF-β)濃度，以及血液中總體外泌體(CD63)與胎盤外泌體(PLAP)數量。並於產婦生產後收集孕婦生產相關資訊、慢性病史及新生兒健康狀態。建立鑑別分析模型，利用接收者操作特徵曲線(ROC)與支援向量機等機器學習演算法，計算曲線下面積(AUC)評估預測性生物標誌物的疾病預測能力。
 
結果
結果顯示子癲前症孕婦，在第二孕期其所有細胞激素濃度和健康孕婦皆有顯著的差異，而細胞激素的平衡亦能觀察到Th17細胞激素在病人中佔主導地位，此外亦使用支援向量機評估細胞激素作為預測或診斷子癲前症的表現，單一細胞激素IL-22的預測表現最好，結合全部Th17細胞激素(IL-17、IL-21、IL-22)則有最高的曲線下面積(AUC)；導入機器學習演算法－支援向量機，用以評估並提升細胞激素預測的準確率，血液中單一細胞激素IL-22的交叉驗證準確率為75%，而TGF-β則為77%。此外結果亦指出子癲前症孕婦在第二孕期的總體外泌體與其中的IL-21、IL-22及TGF-β皆與健康孕婦有顯著差異，單一外泌體中的細胞激素TGF-β預測能力表現最好，其交叉驗證準確率為89%，而結合胎盤外泌體與外泌體中TGF-β濃度則有最高準確率，其交叉驗證準確率為90%。

結論
結果表明Th17代表的促發炎和Treg代表的抗發炎細胞激素於懷孕期間的變化在子癲前症病人中皆有明顯不同，而外泌體中含有的細胞激素亦有部分有顯著差異，分析Th17與Treg間細胞激素平衡亦和孕婦發炎反應相關，其潛在機制和孕婦免疫調節有相關並可能導致子癲前症，未來仍需要近一步研究探討外泌體與細胞激素在子癲前症的潛在機制。

Purpose
Preeclampsia is one of the most serious complications of pregnancy and characterized by new onset hypertension with coexisting proteinuria after 20 weeks of gestation. Some studies have been suggested that immunological alterations in the early placental microenvironment may participate in the induction of preeclampsia. Exosomes are involved in intracellular communication and contain molecular information of origin cell, such as proteins which could serve as direct markers of pregnancy-related complications. Previous studies were reported that the balance between Th17 and Treg cells is critical to prevent preeclampsia. We hypothesized that Th17 and Treg cytokines imbalance and maternal exosomes were involved in immunoregulation and as potential biomarkers for preeclampsia in early gestation. This study was aimed to evaluate the relationship of maternal Th17/Treg cytokines and exosome in preeclampsia. We further investigate whether the plasma and exosomal cytokines would be associated with severity of preeclampsia. In addition, we assess whether these biomarkers could be used as predictive biomarkers for preeclampsia.

Methods
This study recruited 39 pregnant women with preeclampsia and 127 healthy pregnant women as controls. We determined the both Th17 and Treg cytokines (IL-10, IL-17, IL-21, IL-22 and TGF-β) in plasma and exosome. In addition, the concentrations of total and placental exosomes were also determined. We further collected the maternal medical history and neonatal characteristics of each participant. We estimated the odds ratios of each biomarker through logistic regression which also illustrated the ROC curve and indicated the AUC. In addition, we implemented the support vector machine in order to evaluate the predictive performance of each biomarkers.

Results
We found the significant differences in plasma levels of all cytokines during the second trimester. The AUC of plasma cytokine IL-22 through logistic regression analysis demonstrated a good classification efficiency. The results manifested the best performance when combined all Th17 cytokines (IL-17, IL-21 and IL-22). We further conducted the support vector machine to evaluate the predictive accuracy of each biomarker. The results showed a good cross-validation accuracy of IL-22 and TGF-β. We also found the significant differences in exosomal level of IL-21, IL-22, TGF-β and total exosome concentration during the second trimesters. The results of ROC analysis revealed a good classification efficiency of TGF-β. The cross-validation accuracy of TGF-β through support vector machine manifested a best discriminant ability in single exosomal marker.

Conclusion
The results indicated that the significant different patterns of plasma and exosomal markers between preeclampsia patients and healthy controls across the gestation. Moreover, both plasma and exosomal markers were associated with severity of preeclampsia. These results suggest a potential mechanism of Th17 and Treg cytokines as well as exosomes that may contribute to maternal immune activation and result in preeclampsia. Further studies are needed to clarify the potential mechanisms of exosomes and cytokines in preeclampsia.

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