目前有許多透過微流體晶片分選精子，但因受限於微流體晶片的尺度，因此很難進行大量分選(<10μl)，所以大部分微流體晶片都只能應用在ICSI的療程上。而目前傳統的分選方式有兩種；第一為上游法，此方法的缺點為無法收集到足夠的數量。第二種方法為離心法，而此缺點會破壞精子DNA的完整性，影響到後面的受精率。而根據文獻，具有活動力之精子具有逆流的特性，而超過90%的活動力精子具有靠表面游泳的生物特性。因此我們採取這兩種方法，逆流法是我們先前實驗室所開發的第一代晶片的核心技術，透果此法我們將有活動力之精子與死亡之精子分開；而具有活動力之精子有靠表面游泳行為，透過此特性我們設計了一個寬扁的流道，讓大部分的活動力之精子沿著流道上表面和下表面進行逆流，使分選的容積可以提升至100μl。另外透過此晶片能夠避免傳統方法會破壞DNA的問題，而不影響受精率。在本篇研究中，我們開發出扁平式流道來分選精子，應用於精子具逆流以及靠邊的特性，能在15分鐘內分選100ul的精漿容量，能達到90％的活動力。

Many sperm sorting microfluidic devices can’t accommodate semen samples above 10 μl because the micro-scale fluidic channel and separation method limits the capability of high volume semen sorting in the devices. Traditional sperm sorting methods include swim-up, and density gradient centrifugation. However, the swim-up method is only suitable for normal semen samples. And the density gradient centrifugation procedure decreases the sperm DNA integrity. In past studies, sperms tended to swim in the opposite direction of the surrounding fluid flow, so utilizing strong reverse flow filed could achieve the isolation of motile and dead sperms. Besides, for abnormal semen sample, this method avoids damage to sperm DNA integrity. Besides, developed sperms have been found that over 90% of sperms would like to swim to near rigid boundaries when swim against a flow field. In this research, we adopted this two concept and designed a channel (very flat rheotaxis shape) to increase the rigid boundary area that could increase the chance of sperms swimming to near the rigid boundaries and capable of handling semen samples of up to 100 μl at once. The result suggested sperm motility can reach about 90% in 15 mins, and sperm concentration is reduced from original 20.27 to 3.48 M/mL after sorting out dead sperms, and the collectable motile sperms are 174,000 cells in one time sorting process, which is suitable for ICSI and IVF

摘要.......................................................................II
ABSTRACT.............................................................III
致謝.......................................................................IV
目錄........................................................................V
圖目錄.....................................................................VII
表格........................................................................X
附件........................................................................XI
第1章 緒論...........................................................1
1.1 研究背景.....................................................1
1.1.1 微機電系統.................................................1
1.1.2 不孕症療程.................................................2
1.2 研究動機....................................................6
1.2.2 透過微流體晶片分選精子之缺點.................6
1.3 研究目的....................................................7
1.3.1 解決微流體晶片分選精子容積問題..............7
1.3.2 改善微流體晶片操作複雜度........................8
第2章 文獻回顧....................................................9
2.1 男性不孕症患者影響受精率之因素.............9
2.1.1 不孕症男性之精子......................................9
2.1.2 受精率因素探討........................................11
2.2 精子的趨向性...........................................14
2.2.1 精子的逆流性(upstream characteristic)...14
2.2.2 精子趨向於表面性質.................................16
2.3 應用微流體晶片分選精子..........................20
2.3.1 微流體晶片之流體特性.............................20
2.3.2 以穩定層流應用於精子篩選.......................20
2.3.3 利用移動距離來分選精子...........................27
2.3.4 利用精子逆流的特性分選具活動力的精子..30
第3章 晶片設計.................................................40
3.1 FUSS2ND .............................................40
3.1.1 FUSS2nd設計原理..................................40
3.1.2 流體力學理論 ........................................42
3.1.3 FUSS2nd流體模擬................................44
3.1.4 FUSS2nd操作步驟.................................45
3.2 透過扁平流道微流體裝置—HIT-FROSS.46
3.2.1 HiT-FROSS設計理論............................46
3.3.2 Hi-T FROSS流體模擬...........................48
3.3.3 Hi-T FROSS製程..................................50
3.3.4 Hi-T FROSS操作步驟...........................57
3.3 精蟲活力分析.......................................58
3.3.1 使用染劑分析.......................................58
3.3.2 透過CASA分析.....................................60
第4章 結果與討論..........................................62
4.1 FUSS2ND篩選結果..............................62
4.1.1 分選結果...............................................62
4.1.2 FUSS2nd設計缺陷..............................63
4.2 ＨI-T FROSS結果.................................65
4.2.1 晶片離流道內實際流速..........................65
4.2.2 Ｈi-T FROSS活動力精子逆流情形..........67
4.2.3 驗證精子具有靠邊行為在逆流期間........68
4.2.4 針對收集問題.......................................71
4.2.5 分選後結果...........................................73
4.3 ＨI-T FROSS與其他分選方式比較.........78
第5章 結論......................................................80
第6章 未來工作...............................................81
第7章 文獻參考...............................................82

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