以羊水幹細胞治療大鼠多囊性卵巢症候群之潛力

Abstract

多囊性卵巢症候群（Polycystic ovary syndrome, PCOS），已在2026年確認將更名為多內分泌代謝卵巢症候群（Polyendocrine metabolic ovarian syndrome, PMOS），鑑於新名稱設有三年之過渡期並預計整合至2028年國際指南中，故本研究仍沿用PCOS作為疾病名稱。PCOS為育齡女性最常見的不孕症原因之一，其主要臨床症狀包括：高雄性素血症（hyperandrogenism, HA）、排卵功能障礙（ovulatory dysfunction, OD）和多囊性卵巢型態（polycystic ovarian morphology, PCOM）。PCOS的致病因素複雜且尚未完全釐清，目前認為係由遺傳、環境、生活型態誘發HA、代謝異常與慢性低度發炎阻礙卵母細胞成熟造成OD，進而發展為PCOS。鑑於全身慢性發炎為PCOS的重要發病機制，而羊水幹細胞具備優異之抗發炎的特性，且目前已有研究證實羊水幹細胞能夠幫助治療自然老化的卵巢與化療引起的卵巢早衰等難以恢復之疾病，然而對於羊水幹細胞治療PCOS之效果仍需更多的釐清。故本研究將先從懷孕大鼠中分離出羊水幹細胞（rat amniotic fluid stem cells, rAFSCs）後，再以脫氫異雄固酮（dehydroepiandrosterone, DHEA）誘導大鼠PCOS動物模型，隨後經由尾靜脈注射rAFSCs治療PCOS大鼠，以確認rAFSCs治療PCOS之可行性。 試驗一為rAFSCs之分離，結果發現自懷孕大鼠之羊水中分離rAFSCs後，將其擴增培養至第6代時其外觀型態呈現類纖維母細胞的紡錘狀型態；並經由流式細胞術分析後發現其表面抗原CD29、CD90以及MHC I呈陽性，而CD11b、CD45以及MHC II呈現陰性，證實其保有自我更新且免疫原性低之特性；最後從三系分化試驗中證實其可在體外誘導分化成爲脂肪、硬骨與軟骨細胞，具有多分化潛能，確定成功建立rAFSCs細胞系。試驗二為PCOS大鼠模型之建立，以皮下注射DHEA連續30天，結果表明經過DHEA處理後之大鼠表現出動情週期紊亂之情形；同時經DHEA處理後的大鼠卵巢中囊腫濾泡數量顯著增加，且黃體及腔濾泡數量顯著減少，上述結果表示大鼠符合PCOS患者之OD與PCOM症狀，說明此動物模型可作為後續評估rAFSCs治療PCOS可行性之依據。試驗三為rAFSCs治療大鼠PCOS的潛力，結果顯示經過rAFSCs治療的PCOS大鼠，於治療兩週後開始恢復較規律的動情週期，而DPBS處理後的PCOS大鼠則表現動情週期紊亂之情形直到試驗結束；再來是卵巢組織切片結果發現，相比於DPBS處理後的PCOS大鼠透過rAFSCs治療PCOS大鼠卵巢中囊腫濾泡的數量顯著減少；最後是配種試驗結果說明對照組之配種率達75％而DPBS與rAFSCs組皆達100％，且各組別間的胚胎數則無顯著差異。 綜合上述，以rAFSCs治療後能夠縮短PCOS大鼠恢復規律動情週期之時間，並且能減少PCOS大鼠卵巢中囊腫濾泡之數量，以達到緩解PCOS患者OD與PCOM病徵之功效。以上結果可提供人類臨床治療PCOS之參考。

Polycystic ovary syndrome (PCOS), which has been officially confirmed in 2026 to be renamed Polyendocrine Metabolic Ovarian Syndrome (PMOS), is referred to as PCOS in this study given the three-year transition period and its planned integration into the 2028 international guidelines. PCOS is one of the most common causes of infertility in women of reproductive age, characterized primarily by hyperandrogenism (HA), ovulatory dysfunction (OD), and polycystic ovarian morphology (PCOM). The pathogenesis of PCOS is complex and remains incompletely understood; however, it is currently believed that genetic, environmental, and lifestyle factors induce HA, metabolic abnormalities, and chronic low-grade inflammation, which hinder oocyte maturation and lead to OD, ultimately culminating in PCOS. Given that systemic chronic inflammation is a key pathological mechanism of PCOS, and amniotic fluid stem cells possess superior anti-inflammatory properties—with current studies demonstrating their efficacy in treating irreversible conditions such as naturally aging ovaries and chemotherapy-induced premature ovarian failure—the therapeutic potential of stem cells for PCOS requires further elucidation. Therefore, this study aimed to isolate rat amniotic fluid stem cells (rAFSCs) from pregnant rats, establish a PCOS rat model induced by dehydroepiandrosterone (DHEA), and subsequently administer rAFSCs via tail vein injection to evaluate the therapeutic feasibility of rAFSCs for PCOS. Experiment 1: Isolation and Characterization of rAFSCs. Following isolation from the amniotic fluid of pregnant rats, rAFSCs expanded to passage 6 exhibited a spindle-shaped, fibroblast-like morphology. Flow cytometry analysis revealed positive expression for surface antigens CD29, CD90, and MHC I, and negative expression for CD11b, CD45, and MHC II, confirming their self-renewal capacity and low immunogenicity. Furthermore, tri-lineage differentiation assays demonstrated their in vitro multipotency by successfully differentiating into adipocytes, osteoblasts, and chondrocytes, thereby confirming the successful establishment of the rAFSC line. Experiment 2: Establishment of the PCOS Rat Model. Rats were subcutaneously injected with DHEA for 30 consecutive days. Results demonstrated that DHEA treatment induced estrous cycle disruption in rats. Histological analysis revealed a significant increase in the number of cystic follicles, accompanied by a significant reduction in corpora lutea and antral follicles in DHEA-treated rat ovaries. These findings confirm that the rats exhibited symptoms mimicking OD and PCOM in PCOS patients, validating this animal model as a reliable foundation for evaluating the therapeutic feasibility of rAFSCs. Experiment 3: Therapeutic Potential of rAFSCs in PCOS Rats. The results showed that PCOS rats treated with rAFSCs began to regain more regular estrous cycles two week post-treatment, whereas DPBS-treated PCOS rats exhibited persistent estrous cycle disruption throughout the remainder of the study. Furthermore, histological analysis of ovarian sections revealed that rAFSC treatment significantly reduced the number of cystic follicles in PCOS rats compared to DPBS treatment. Finally, outcomes from the mating trial demonstrated that the pregnancy rate reached 75% in the Control group and 100% in both the DPBS and rAFSC groups, with no significant differences in the number of embryos observed among all groups. In summary, rAFSC treatment shortened the time required for PCOS rats to restore regular estrous cycles and reduced the number of cystic follicles in PCOS ovaries, thereby effectively alleviating the pathological features of OD and PCOM. These findings provide a valuable reference for future clinical applications in human PCOS therapy.