探討骨盆底肌肉功能在女性骨盆機能障礙的角色與應用

Abstract

背景：婦女骨盆機能障礙最常見的為尿失禁（urinary incontinence）與骨盆脫垂（pelvic organ prolapse），不僅會導致患者生殖泌尿道感染、解尿障礙、尿液滯留、骨盆垂墜疼痛、排便困難、同房不適，還且會衝擊患者生活品質，致病主要原因牽涉到懷孕、分娩、停經等因素導致的神經損傷，以及泌尿生殖系統骨骼肌細胞（urogenital skeletal muscle cells）包括尿道括約肌（sphincters）與骨盆底肌肉（pelvic floor muscles）的功能失調，除了保守治療方法，目前最有效的尿失禁或骨盆脫垂治療方式仍為手術治療，婦女一生因尿失禁或骨盆脫垂而須接受手術的機率將近20%，相較於傳統手術，使用合成網膜能達到較好的手術效果，然而過去文獻中所報導某些嚴重的併發症如傷口癒合不良、疼痛等狀況，導致學者專家們對網膜使用於骨盆脫垂產生警告與疑慮。 幹細胞（stem cells）在再生醫學（regenerative medicine）的應用具有極大的潛能，其中人類誘導型多能幹細胞（human induced pluripotent stem cells, hiPSCs）不僅較不具倫理或宗教上的爭議，且具有與胚胎幹細胞（embryonic stem cells, ESCs）相當的分化潛能，將人類誘導型多能幹細胞應用於婦女泌尿生殖系統或婦女骨盆機能的治療具有極大的優勢，不僅可能藉由骨盆組織的再生而提高治療成功率，且有望能因避免植入人工合成物質而降低目前網膜手術的副作用，目前關於此方面的研究仍處於待開發的狀態。 本研究在臨床醫學研究部分的主要目的，在於利用臨床評估方式，先探討罹患骨盆機能障礙婦女的骨盆底肌肉功能與疾病表現、治療成效等的關係，在基礎醫學研究的部分，希望能先初步探討將胚胎幹細胞與人類誘導型多能幹細胞細胞株分化為骨骼肌先驅細胞的可行性，以期能在未來能將罹患尿失禁或骨盆脫垂婦女的周邊血液細胞重新編序成為人類誘導型多能幹細胞，並應用所得之人類誘導型多能幹細胞於婦女尿失禁與骨盆脫垂的個人化醫療。 材料及方法：本研究為一觀察性的研究，在臨床醫學研究的部分，所有的診療方法皆依照醫療應有的原則與常規來進行。診療內容依患者情況包含臨床問診、骨盆檢查（pelvic examination）、超音波檢查（ultrasound）、或尿動力學檢查（urodynamic studies），患者可依據本身意願與醫師溝通，決定接受或拒絕某些診療項目，完成臨床相關的檢查之後，將邀請因尿失禁、骨盆脫垂、或手術併發症就診之婦女參與研究，對於同意參加研究並簽署受試者同意書之受試者，均將相關資料前瞻性地輸入資料庫，包括基本資料、下段尿路症狀、骨盆檢查、超音波檢查、與尿動力學檢查；本研究需收案87位罹患尿失禁或骨盆脫垂的婦女，參與研究的受試者僅須依循一般醫療常規接受診療與回診；在基礎醫學研究的部分，利用非轉基因（non-transgenic）的分化方法，將胚胎幹細胞與人類誘導型多能幹細胞的細胞株分化為骨骼肌先驅細胞（skeletal progenitor cells），然後利用組織免疫染色（immunohistochemistry）與定量即時聚合酶鏈鎖反應（quantitative real time polymerase chain reaction）分析生產效能。 結果：在評估可靠性、一致性、以及有效性之後，證實我們用來評估骨盆肌肉功能的立體陰道口超音波（four-dimensional introital ultrasound）方法，可實用於評估膀胱脫垂女性手術前後的提肛肌完整性以及自主性和非自主性骨盆底肌肉功能。此外，我們有以下發現：在患有骨盆底症狀的女性當中，停經與較弱的靜止時骨盆底支持和較弱的不自主的骨盆底肌肉收縮對腹壓上升的反應能力有關，但與自主性骨盆底肌肉收縮無關；骨盆脫垂在患有下泌尿道症狀的婦女中很常見，骨盆脫垂的存在與較弱的靜止、非自主、和自主的骨盆肌肉功能均有關。在基礎醫學研究部份，我們發現以抑制BMP和激活Wnt的分化方式所產生的細胞，無論是表現PAX7的骨骼肌先驅細胞，或者是表現MYH3的骨骼肌細胞（skeletal myocytes），產生的效能都比只以激活Wnt的分化方式來得高。 討論：骨盆底肌肉功能對維持女性骨盆底功能具有重要的角色。識別骨盆底肌肉功能障礙可能有助於定制適合患者的治療方針並改善治療效果。來自人類多能幹細胞的骨骼肌先驅細胞很有潛力成為將來治療女性骨盆底功能障礙的細胞來源。 結論：骨盆底肌肉功能在女性骨盆障礙的致病機制、疾病嚴重程度、與治療成效，均佔有重要的影響力；骨骼肌先驅細胞可以通過非轉基因方法有效地獲得。

Background: Pelvic floor dysfunctions are common among women. Common female pelvic floor dysfunctions include urinary incontinence, pelvic organ prolapse, and lower urinary tract symptoms such as frequency and urgency. Pelvic floor dysfunctions frequently cause urogenital infection, voiding dysfunction, urinary retention, pelvic pain, constipation, and coital difficulty, as well as remarkable impact on the quality of life of women. Risk factors of female pelvic floor dysfunctions include pregnancy, vaginal delivery, forceps delivery, obesity, older ages, menopause, prior pelvic reconstructive surgeries, and chronic straining. With a crucial role playing in the pathophysiology of female pelvic floor dysfunctions, the urogenital skeletal muscular dysfunction cannot be fully corrected via the current treatment modalities. In order to overcome the current dilemma of management for female pelvic dysfunction, deeper understanding of the roles of pelvic floor muscles in female pelvic floor function may help to improve treatment outcomes and potentially to develop a better treatment method. Stem cells have great potential in the application of regenerative medicine. The human induced pluripotent stem cells represent (iPS cells; iPSCs) a prime candidate cell type for current research and future cell therapy because of their significant self-renewal, differentiation potential and the relative lack of ethical conflict. Skeletal muscle progenitor cells derived from human pluripotent stem cells are a promising cell source for regeneration in skeletal muscle-related diseases. Skeletal muscle progenitor cells can be obtained by non-transgenic approaches, i.e., by exposing human pluripotent stem cells to differentiation cues that enable the sequential recapitulation of key stages of skeletal myogenesis. The common approaches involve the activation of Wnt signaling in monolayer cells by treating with CHIR99021 with or without inhibition of bone morphogenetic protein (BMP) signaling pathways. The serial investigations of the study aim to use clinical evaluation methods such as four-dimensional introital ultrasound to explore the significance of pelvic floor muscle function among women with pelvic floor dysfunctions including urinary incontinence, pelvic organ prolapse, and lower urinary tract symptoms, the association of surgical outcomes with basic information, type of pelvic floor repair, types of meshes used in the pelvic repair, pelvic morphology, pelvic floor muscle function, and molecular cell biology of the vagina. Additionally, we aim to assess the skeletal muscular differentiation capacity of various human induced pluripotent stem cell (hiPSCs) lines and human embryonic stem cell (ESCs) lines and compare the following two protocols of skeletal myogenesis from human pluripotent stem cells: (1) Wnt signaling activation alone and (2) Wnt signaling activation and the inhibition of BMP. Materials and methods: We prospectively recruited women with urinary incontinence, pelvic organ prolapse, pelvic floor symptoms, or lower urinary symptoms and collect the subjects’ data including basic information, clinical data, methods of pelvic floor repairs, types of slings or meshes used in the pelvic repairs, pelvic morphology, pelvic floor function, four-dimensional introital ultrasound, and molecular cell biology of the vagina. The skeletal myogenesis from human pluripotent stem cells with two protocols was compared by the analysis of skeletal muscle progenitor-associated PAX7 and skeletal myocyte-associated MYH3. Results: Our method applying four-dimensional introital ultrasound and post-processing analyses are feasible to assess levator muscle integrity as well as voluntary and involuntary pelvic floor muscle function of women with cystocele before and after operations. The feasibility was approved after evaluations of reliability and agreement as well as validity, which were respectively determined by intraclass correlation coefficients with 95% confidence interval and Bland-Altman analysis as well as correlation of squeezing ultrasound measurements with modified Oxford scale. Furthermore, we disclosed the following findings: Menopause is associated with a weaker resting pelvic floor support and impaired responsiveness of involuntary pelvic floor muscle contractions to sudden intra-abdominal pressure rise but not with voluntary pelvic floor muscle contractions in women with pelvic floor symptoms. Pelvic organ prolapse is common in women with lower urinary tract symptoms and the presence of pelvic organ prolapse is associated with weaker resting, involuntary, and voluntary pelvic floor muscle functions. Both of the two protocols used in this study can recapitulate the myogenic developmental sequence. However, significantly greater expression levels of skeletal muscle progenitor -associated PAX7 and skeletal myocyte-associated MYH3 were found in cells obtained through the protocol with BMP inhibition and Wnt activation. Discussion: Pelvic floor muscle is important in maintaining female pelvic floor function. Identification of pelvic floor muscle dysfunction may help in tailored management and improving treatment outcomes. Skeletal muscle progenitor cells from human pluripotent stem cells may provide a potential source of treatment for female pelvic floor dysfunction. Conclusions: Pelvic floor muscle is important for female pelvic floor dysfunction. Skeletal muscle progenitors can be obtained by non-transgenic approaches from human pluripotent stem cells.