微流體晶片精子泳動力分級及篩選研究

Abstract

人工協助生殖科技 (Assisted Reproductive Technology, ART) 如體外受精技術 (In Vitro Fertilization, IVF) 以及單一精蟲顯微注射技術(intracytoplasmic sperm injection, ICSI) 強調了高品質精子細胞篩選的重要性，精子活性分類為相關的技術提供不同活性的精子，此外，精子品質的檢測通常以精子的濃度，活性，形狀做為判斷男性不孕症的指標，使用統一的檢測方式來判斷男性的不孕症也是一個重要的議題。本研究藉由微流體生物晶片來進行活性精子特殊移動方式的觀察與分析，並提出可能之假設，有別於其他細胞，精子本身具有泳動能力，佐以實驗證實活性精子頭部會朝著流場的反方向進行定位而朝上游逆游前進。藉著此一特殊的現象以及有理論根據的流道設計，本生物晶片可以從未經處理的精液樣本中將高泳動性之精子從包括白血球、脂肪、無泳動力之精子…等其他雜質中篩選出來，更進一步的，依照精子本身泳動力強弱的不同，本晶片可以同時達到分級篩檢儲存和揀選最高泳動力精子之目的，此一簡單、不昂貴的微小晶片可以把活性精子的觀察、篩選、分級儲存這三項目標整合在一起，快速且持續的同時進行，並且不需要任何外接的電路或是其他類似幫浦的大型儀器。 靜水壓為本研究之穩定驅動壓力源，僅包含三個可以盛裝溶液之容器分別是未經處理精液之入口，緩衝液之入口，以及收集處之出口，而精液入口流道的流速精準的控制成三組不同的流速(最大流速為10μm/s, 30μm/s, and 50μm/s)，由於每個精子細胞的活性皆不同抵抗背景流速的能力亦不同，精子將被分級。由此機制篩選後的活性精子比率接近百分之百，並且實驗結果證明不同活性的精子明顯的被區分出來，並且可以依其活性的不同而進行儲存。 規格統一的生物晶片使精子泳動力分級的機制更有一致性，由不同樣本所得到之精子泳動力分布資料更可以進一步的運用於臨床的診斷不孕症狀況或甚至身體狀況，這可以減少人為判斷病人狀況的錯誤率以及節省人力資源的浪費。這個微小、可隨身攜帶、用完即丟的晶片可以協助達成分級制度之精子銀行、單一精蟲微影注射(ICSI)、體外受精技術(IVF)、或其他相關的人工生殖技術(ART)之議題，滿足臨床上的需求並提供一個一致性的精子泳動力分級平台。

Assisted reproductive technology (ART) like intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) reflect the importance of functional spermatozoa pre-selecting. Motility-based sperm preservation to provide competent sperm for clinic use is required. In addition, sperm quality examination such as sperm concentration, motility, and morphology are the index of the male infertility. The uniform standard for diagnosing male infertility is another significant issue. This thesis presents of sperm motion analysis to classify sperm motility via a microfluidic device in order to achieve assisted concepts. Based on the self- movement and against-flow phenomenon of motile sperm, the device can also isolate motile sperm from any other cellular debris. The device, an inexpensive microchip, can integrate observation, isolation, and classification of motile sperm in one chip without the need for electronics or external syringe pump. Hydrostatic pressure was used as the steady driving source in the three reservoirs: semen sample inlet, buffer inlet, and motile sperm outlet, where the flow field can be controlled specifically in three groups (maximum velocities are 10μm/s, 30μm/s, and 50μm/s), and motile sperm will be selected by the motility of each individual sperm. Results show successful separation via sperm motility. Comparatively, the motile sperm ratio was high (near 100%) after sorting, and the motility-based sperm classification show the positive relationship during the experiment. The uniform standard of sperm classification make it possible to diagnose the state of infertility, or even the health of the individual, it reduce the artificial mistake of determining sperm motility in whole semen sample and save a quantity of time and labor from diagnosis. The device provided a small, portable, and disposable platform on which to deal with issues, such as classification based sperm bank, ICSI, IVF etc, and should be useful to address clinical need of uniform standard for sperm classification.