多囊性卵巢症候群之臨床表徵與致病機轉

Abstract

多囊性卵巢症候群 (Polycystic ovarian syndrome, 簡稱PCOS) 是育齡婦女最常見的內分泌疾病，其臨床表徵十分多元且個體變異性極高，在青少年時期，病患可能出現無月經或者月經不規則、多毛症、嚴重痤瘡、肥胖、雄性禿等症狀；到了生育年齡則可能面臨不孕，並在懷孕期間出現較高機率的流產、妊娠糖尿病、子癲前症、早產等風險；在新陳代謝方面，胰島素阻抗或第二型糖尿病、高血壓、高血脂等代謝症候群的發生率顯著上升，子宮內膜增生或者子宮內膜癌的機率也增加，因此對婦女健康的影響極為廣泛而深遠。而此複雜疾病無論是從診斷標準、致病機轉、治療方針甚至於長期預後，都尚有許多未解之謎題仍待進一步研究釐清，因此博士班期間將針對此疾病進行一系列臨床與基礎研究。 在第一個研究中，為了進一步釐清此複雜症候群之臨床表徵分群模式，並針對其新陳代謝風險找出預後危險因子，我們使用了強大且創新的統計學圖表分析法--廣義相關圖（generalized association plots，GAP），來分析多囊性卵巢症候群病患的症狀模式和特徵分群。我們首先運用關聯性分析，找出與多囊性卵巢症候群之新陳代謝異常最為相關的四個內分泌指標 (FSH, LH, free androgen index, DHEA-S)，接著依照這四個內分泌指標表現值相近度，將總共460名多囊性卵巢症候群患者分為四個次族群，每個次族群中的個體即這四個內分泌指標表現最相近的一群病患。結果可發現，所分出的四個次族群，不僅各自具有獨特的內分泌表徵，也同時具有顯著不同的新陳代謝異常風險。當病患出現高free androgen index數值且低LH濃度時，將會是罹患新陳代謝症候群的高風險群，這些患者可更密集地接受新陳代謝相關追蹤檢查，倘若病患出現低free androgen index數值且高LH濃度時，則可告知病患日後得到新陳代謝異常風險與同齡女性無異，應可降低不必要的追蹤檢測和健保花費，也可降低病患之焦慮不安。我們的研究成功的將患者進行了良好分組，也找出了與新陳代謝風險相關的內分泌特徵。 在第二個研究中我們針對多囊性卵巢症候群的致病機轉進行研究，由於多囊性卵巢症候群最重要的臨床症狀之一是濾泡成長異常和排卵失調，而卵巢濾泡液的生成也是濾泡成長過程重要的一環，故我們首先分析了卵巢濾泡液是如何調控相鄰內皮細胞之通透性，並針對其成分進行分析。我們先以單層人類臍靜脈內皮細胞裝置分析11 位多囊性卵巢症候群和9位對照組受試者卵巢濾泡液對於內皮細胞通透性的調節，發現多囊性卵巢症候群患者的濾泡液會使得相鄰內皮細胞通透性顯著下降，由於細胞通透性與血管生成因子有關，故進一步以晶片分析13位多囊性卵巢症候群和11位對照組受試者卵巢濾泡液中的血管生成因子濃度，晶片所偵測的55種血管生成因子中，我們發現多囊性卵巢症候群病患的濾泡液表現較高濃度的Platelet factor 4(PF4)，且PF4會與IL-8進行拮抗性結合，抑制細胞間隙的生成並降低相鄰內皮細胞的通透性，我們的發現不僅提供了多囊性卵巢症候群致病機轉的另一種新解釋，而且也讓我們對於PF4與卵巢功能相關之細胞生理機轉有更進一步的了解。 多囊性卵巢症候群具有高度遺傳性，但在過去眾多基因關聯性研究中卻找不出顯著致病基因，故被認為可能是一個多基因遺傳疾病。然而針對其高度遺傳性的另一個可能解釋是超基因調控 (epigenetics)，過去相關文獻甚少，故在第三個致病機轉相關研究中，我們將針對超基因調控其中一種重要機轉 – DNA甲基化進行研究，針對卵巢顆粒細胞運用晶片分析全基因體DNA甲基化表現，試圖釐清多囊性卵巢症候群和對照組之間是否有顯著DNA甲基化差異。在這個研究中我們分析了兩種卵巢顆粒細胞，一種是成人卵巢顆粒細胞，代表成熟的疾病細胞，另一種是從誘導性多功能幹細胞 (induced pluripotent stem cells, iPSCs) 再次分化衍生出的卵巢顆粒細胞(分化第十二天)，代表處於剛分化階段的初始細胞。DNA甲基化晶片的分析結果顯示多囊性卵巢症候群患者的iPSC衍生顆粒細胞和成人顆粒細胞均出現CREB訊息傳導路徑過度活化的情形，後續的西方墨點法亦驗證了多囊性卵巢症候群組有較高的CREB蛋白和CBP蛋白表現量，此發現可為多囊性卵巢症候群的致病機轉和臨床表徵提供嶄新而合理的解釋。 由於微小核醣核酸 (miRNA) 調節也是超基因調控重要的機轉之一，且血液游離微小核醣核酸的表現在許多疾病研究中已經被證實有良好的臨床診斷及預測預後功效，故在第四個研究中，我們欲分析血液游離微小核醣核酸是否可以作為多囊性卵巢症候群的臨床診斷和療效預測之生物指標。我們從文獻中選擇了14種可能與多囊性卵巢症候群致病機轉相關的標的微小核醣核酸，在75個多囊性卵巢症候群病患和20個對照組受試者的周邊血液進行逆轉錄聚合酶鏈式反應 (rtPCR) 測量。在第一個階段的標的微小核醣核酸測量中，我們發現多囊性卵巢症候群患者與對照組受試者的血液游離微小核醣核酸表現模式有顯著不同，多囊性卵巢症候群患者的血液miR-93, miR-132, miR-221, miR-223, miR-27a, miR-212表現值相較於對照組顯著較高，而miR-222和miR-320a表現值顯著較低。此外，metformin藥物在臨床上可改善50%患者的排卵功能和月經週期，然而目前並無方法可預測其療效，故我們分析了metformin治療有效和無效兩組之間的微小核醣核酸表現，亦發現有顯著差異。第二個階段為模型建立，我們以miR-93、miR-132、miR-222、miR-27a、miR-125b、miR-212這六個最具顯著差異的微小核醣核酸表現值，建立了多囊性卵巢症候群的診斷模型，其接收者操作特徵 (receiver operating characteristic, ROC) 曲線下面積 (area under curve, AUC) 高達0.96，對於受試者是否符合多囊性卵巢症候群臨床診斷有極佳的預測性。此外我們針對metformin是否具有改善排卵的臨床療效，也以miR-132, miR-27a, miR-222, miR-93這四個微小核醣核酸表現值建立了預測模型，並成功在另一個獨立的多囊性卵巢症候群病患族群中驗證其預測效果，藉由此預測模型，可以幫忙預測患者是否可以在接受metformin治療後，出現排卵功能及月經週期上的改善，對於臨床治療的療效預測和藥物選擇有所助益。 總結以上四個研究所獲得的成果，從病患卵巢濾泡液、卵巢顆粒細胞、血液檢體、到病患專一性iPSC細胞株的建立，透過各種分析方法，在臨床症狀、疾病診斷、疾病預後、致病機轉以及臨床治療上都有許多新的發現，包括：臨床症狀的分組解析和新陳代謝症候群危險因子的探尋、濾泡液中血管生成因子與其潛在影響內皮細胞通透性之機轉解析、病患專一性及多囊性卵巢症候群特異性iPSC之建立與再分化研究、全基因體DNA甲基化分析所發現之CREB訊息調控路徑異常對於致病機轉之可能影響、以血液游離微小核醣核酸建立之多囊性卵巢症候群診斷模型和metformin療效預測模型，無論是研究過程所積累的經驗，或者是所獲得的種種研究成果，都為日後多囊性卵巢症候群相關基礎或臨床研究提供了大量的工具和材料，在臨床醫療的診斷和治療也有諸多貢獻。

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women with reproductive age. There is a broad spectrum of clinical presentations and the heterogeneity of symptoms is extremely high. In adolescence, the common presentations include amenorrhea, irregular menstruation, hirsutism, severe acne, obesity and alopecia. The patients of childbearing age have higher risks of infertility and pregnancy complications, such as abortion, gestational diabetes, preeclampsia and preterm delivery. And they also have higher risk of metabolic aberrations such as insulin resistance, type II diabetes, hypertension and dyslipidemia. Therefore its impact on women health is huge and lifelong. Until now, there are still many unanswered questions regarding to its diagnostic criteria, pathophysiology, treatment and screen strategies, and long-term prognosis. Therefore the aim of the proposal is to conduct series of basic and clinical researches on PCOS. In the first study, we applyed a matrix visualization and clustering approach, generalized association plots (GAP), to divide 460 PCOS patients into four distinct clusters according to the correlation of four endocrine parameters. Each cluster exhibited specific endocrine characteristics and the prevalence of metabolic syndrome (MS) was significantly different among each cluster (P < 0.0001). A common endocrine characteristic of the metabolically unhealthy clusters was relatively lower LH level. While high FAI level did correlate with more severe metabolic aberrations, high LH level (>15 mIU/ml) during early follicular phase showed better predictive value than low FAI level to become a metabolically healthy cluster. Our results could stratify women with PCOS into meaningful subtypes and provide a better understanding of related risk factors. This could potentially enable the design and delivery of more effective screening and treatment intervention. The second study was focusing on the pathogenesis of PCOS. Abnormal folliculogenesis is one of the cardinal presentations of PCOS and the formation of follicular fluid (FF) have been proposed to be involved in the normal follicular growth. However, whether or not there is a change in intrafollicular angiogenetic/angiostatic factors and in endothelial permeability underlies PCOS is unknown. Therefore we collected the FF from 13 PCOS and 11 ovulatory control subjects. The influence of FF on endothelial cell permeability was evaluated using a human umbilical vein endothelial cell monolayer permeability assay. The FF from PCOS patients caused significantly poorer endothelial cell permeability comparing with the effect of FF from the control group. The intrafollicular expression profiles of angiogenesis-related proteins were analyzed using a Human Angiogenesis Protein Array Kit. Among the 55 angiogenesis-related proteins tested, there was a significantly higher level of intrafollicular platelet factor 4 (PF4) and PF4/IL-8 complex in the PCOS group (p = 0.004). The anti-permeability effect of PF4 was related to the decrease in the intercellular gaps and antagonistic binding with IL-8. Our study provided the first evidence of the pathophysiologic contribution of the well-known angiostatic protein, PF4, on human reproductive biology. The increase of the intrafollicular PF4 and its anti-permeability effect might affect the formation of FF and folliculogenesis in PCOS. In the third study, we wanted to elucidate whether and how the pathogenesis of PCOS is related to epigenetic aberrations. We established patient-specific induced pluripotent stem cells (iPSCs) from skin fibroblasts through the application of nonviral episomal reprogramming and were differentiated into ovarian granulosa cells (GCs) with the use of a cocktail of growth factors. This could serve as a valuable model to evaluate the presymptomatic pathogenic events in early cellular differentiation and developmental status. The combination of DNA methylomic analysis in the adult GCs and iPSC-derived GCs revealed several differentially methylated genes and differentially expressed pathways between the PCOS and control groups, and a preserved persistent hyperactivation of the CREB signaling pathway might be involved in the pathogenesis of PCOS. CREB and its coactivators are known to be critical sensors for both hormonal and metabolic signals, and it is possible that the aberration of the CREB signaling pathway could be related to complex hormonal and metabolic disorders such as PCOS. These results could have implications on the early developmental origin, familial nature, and environmental interaction effects of this disease, providing possible therapeutic targets in the future. The aim of the fourth study is to investigate the role of microRNAs (miRNAs) in the pathogenesis of PCOS, and to evaluate the feasibility of miRNA expression as clinical diagnostic biomarkers for PCOS. We selected 14 targeted miRNAs which had been reported to be related with glucose metabolism or diabetes, ovarian or pituitary function in the literatures. Totally 75 patients with PCOS and 20 control subjects had their plasma sent for rtPCR analysis. The results showed that the expression levels of miR-93, miR-132, mR-221, miR-223, miR-27a and miR-212 were significantly higher in PCOS and the levels of miR-222 and miR-320a were significantly lower in PCOS group. The prediction model for the diagnosis of PCOS was further established according to six discriminative miRNAs and the area under curve (AUC) was as high as 0.96. The second part of the study is to evaluate the feasibility of circulatory miRNAs as the predictive biomarkers for the therapeutic effect of metformin. Clinically about 50% of PCOS patients showed improvements in ovulation and menstrual cyclicity when taking metformin. Therefore we compared the expression level of circulatory miRNAs between metformin-effective and metformin-effective patients. And then we established a predictive model for the therapeutic effects of metformin according to the expression level of four discriminative miRNAs (miR-93、miR-222、miR-223、miR-429). The AUC was 0.72 and could be validated with another separate PCOS cohort. Our study showed that the expression pattern of circulatory miRNAs could be effective prediction models for the diagnosis of PCOS and the therapeutic effect of metformin. The pathophysiological contribution of the differentially expressed miRNAs to PCOS needs further researches to elucidate. In summary, the GAP analysis stratified women with PCOS into meaningful subtypes and provide a better understanding of related risk factors. There was an increase in the level of intrafollicular PF4 protein and its anti-permeability effect might affect the formation of FF and folliculogenesis in PCOS. The aberrant epigenetic regulation was shown to be involved in the pathogenesis of PCOS, including differentially expressed DNA methylation involving CREB signaling pathway. The differential levels of circulatory miRNAs between PCOS and control group could be an excellent diagnostic tool for PCOS and the regulatory roles of these miRNAs on PCOS deserved further studies. The above results provide considerable contributions for both the basic researches and clinical practices in PCOS.