本論文提出一整合型微流體晶片系統，已成功的透過微流道晶片所呈現的層流特性，以及符合不需離心即可篩選的原則，直接取代傳統篩選方式與人工挑選之過程，同時避免可能對精蟲造成的傷害，並結合馬克精蟲計數盤與流式細胞儀分析實驗，發展出能夠有效篩選精蟲的生醫晶片。為證實該系統的精蟲篩選能力，進而針對原始濃度及活動力不同的人精及豬精樣本進行一次性的篩選。
本研究晶片採用微機電製程與軟微影技術以聚二甲基矽氧烷 (Polydimethylsiloxanes, PDMS) 作為實驗之基材，其具備生物相容、低成本、可拋棄式、高精密度及製程簡易等特性，並可降低實驗樣本的使用量，相當符合微流體生醫晶片的需求。
以近期生殖醫學技術而言，可藉體外受精技術(In Vitro Fertilization, IVF)進行精蟲樣本之前處理，使部分男性為主因的生育障礙獲得改善，故精蟲的活動力、總數量、濃度等因素將直接影響受精結果。因此，可透過微流道生醫晶片於樣本前處理階段篩選出最適合進行IVF的精蟲。
本實驗研究共分為二階段，第一階段完成一微尺度層流篩選精蟲之晶片系統，主要針對精蟲的活動力進行分析以及探討篩選前後精蟲的死活比，其中包含晶片設計、流場模擬、馬克精蟲計數盤以及流式細胞螢光檢測的分析結果等。第二階段則延續第一階段的研究進一步改良成多重流道之晶片，以探討精蟲篩選率在質與量上的提升。最後本論文於第五章提出部分未來可望加以思考的研究方向。

The thesis presents an integrated microfluidic system which consists of laminar stream-based microchannels, makler counting chamber and flow cytometric analysis to enhance the sperm motility sorting efficiency. The working concept lies in the design of multiple channel systems that successfully replace traditional methods, which could be achieved motile sperms without centrifugation steps. Simultaneously, the damage to the sperms could be avoided. The fabrication process of our bio-chip include SU8 thick-film photolithography and soft-lithography, which has the advantages of biocompatibility, low-cost, high-precision, disposable, and easy to produce etc., so that it is suitable for biomedical chip. With the advances in the technologies, reproductive medicine has become an important role in the field of medical science. The main reasons of infertility from male are the abnormality of sperms and the lack of sperms. IVF (in vitro fertilization) technology is a natural in vitro fertilization process that comes after a series of sample pre-treatment. The semen property, including motility, total number, and the concentration of sperms will directly affect the outcome. Therefore, the optimized microfluidic system provides a good opportunity to use and an inexpensive requirement to select the most appropriate sperms before IVF process. The study is divided into two parts. In the first part, the goal is to construct a microfluidic based sperm sorting system, which focus on the sorting and the classification based on sperm motility and viability. The details including chip design, flow field simulation, makler counting, flow cytometric analysis, and the experiments of semen samples. In the second part, the microfluidic system with multiple channels enhances the sorting efficiency in the quality and quantity of sperm. At the end, some ideas of future works were proposed.

[1] Z. Zhan, C. Dafu, Y. Zhongyao, and W. Li, "Biochip for PCR amplification in silicon," in Microtechnologies in Medicine and Biology, 1st Annual International, Conference On. 2000, 2000, pp. 25-28.
[2] S. S. Howards, "Treatment of male infertility," N Engl J Med, vol. 332, pp. 312-7, Feb 2 1995.
[3] J. A. Januskauskas A, Rodriguez-Martinez H, "Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility," Theriogenology, vol. 60, pp. 743-58, 2003.
[4] E. RG, "Towards single births after assisted reproduction treatment," Reprod Biomed Online, vol. 7, pp. 506-8, 2003.
[5] D. RP, "The relative contribution of assisted reproductive technologies and ovulation induction to multiple births in the United States 5 years after the Society for Assisted Reproductive Technology/American Society for Reproductive Medicine recommendation to limit the number of embryos transferred," Fertil Steril, vol. 88, pp. 1554-61, 2007.
[6] W. R. Allen VM, Cheung A, "Pregnancy outcomes after assisted reproductive technology," J Obstet Gynaecol Can, vol. 28, pp. 220-50, 2006.
[7] C. M. Setti PE, Albani E, Morreale G, Novara PV, Cesana A, Parini V, "Outcome of assisted reproductive technologies after different embryo transfer strategies," Reprod Biomed Online, vol. 11, pp. 64-70, 2005.
[8] N. R. Katz P, Showstack J, "The economic impact of the assisted reproductive technologies," Nat Cell Biol, pp. s29-32, 2002.
[9] R. R. Henkel and W.-B. Schill, "Sperm preparation for ART," Reproductive Biology and Endocrinology, vol. 1, p. 22, 2003.
[10] C. R. Gordts S, Puttemans P, Brosens I, Valkenburg M, Norre J, Renier M, Coeman D, Gordts S, "Belgian legislation and the effect of elective single embryo transfer on IVF outcome," Reprod Biomed Online, vol. 10, pp. 436-41, 2005.
[11] B. S. Pandian Z, Ozturk O, Serour GI, Templeton A, "Number of embryos for transfer following in-vitro fertilisation or intra-cytoplasmic sperm injection," Cochrane Database Syst Rev, vol. 18, p. CD003416, 2004.
[12] Y.-A. Chen, Z.-W. Huang, F.-S. Tsai, C.-Y. Chen, C.-M. Lin, and A. Wo, "Analysis of sperm concentration and motility in a microfluidic device," Microfluidics and Nanofluidics, pp. 1-9, 2010.
[13] M. Yamada, K. Kano, Y. Tsuda, J. Kobayashi, M. Yamato, M. Seki, and T. Okano, "Microfluidic devices for size-dependent separation of liver cells " Biomedical Microdevices, vol. 9, pp. 637-645, 2007.
[14] C.-H. Lin, C.-Y. Lee, C.-H. Tsai, and L.-M. Fu, "Novel continuous particle sorting in microfluidic chip utilizing cascaded squeeze effect," Microfluidics and Nanofluidics, vol. 7, pp. 499-508, 2009.
[15] M. C. McCormack, S. McCallum, and B. Behr, "A novel microfluidic device for male subfertility screening," Journal of Urology, vol. 175, pp. 2223-2227, Jun 2006.
[16] D. J. Beebe, G. A. Mensing, and G. M. Walker, "Physics and applications of microfluidics in biology," Annu Rev Biomed Eng, vol. 4, pp. 261-286, 2002.
[17] R. Suh, S. Takayama, and G. D. Smith, "Microfluidic Applications for Andrology," Journal of andrology, vol. 26, pp. 664 - 670, 2005.
[18] B. S. Cho, T. G. Schuster, X. Zhu, D. Chang, G. D. Smith, and S. Takayama, "A microfluidic device for separating motile sperm from nonmotilesperm via inter-streamline crossings," in 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine & Biology, 2002, pp. 156-159.
[19] B. S. Cho, T. G. Schuster, X. Zhu, D. Chang, G. D. Smith, and S. Takayama, "Passively driven integrated microfluidic system for separation of motile sperm," Analytical Chemistry, vol. Vol. 75, No. 7, pp. 1671-1675, 2003.
[20] K. M. Horsman, S. L. R. Barker, J. P. Ferrance, K. A. Forrest, K. A. Koen, and J. P. Landers, "Separation of sperm and epithelial cells in a microfabricated device-Potential application to forensic analysis of sexual assault evidence," Analytical Chemistry, vol. 77, pp. 742-749 2005.
[21] S. Koyama, D. Amarie, H. A. Soini, M. V. Novotny, and S. C. Jacobson, "Chemotaxis assays of mouse sperm on microfluidic devices," Analytical Chemistry, vol. 78, pp. 3354 -3359, 2006.
[22] H. Mao, P. S. Cremer, and M. D. Manson, "A sensitive, versatile microfluidic assay for bacterial chemotaxis," Proceedings of the National Academy of Sciences, vol. 100, pp. 5449-5454, 2003.
[23] D. Huh, J. H. Bahng, Y. Ling, H.-H. Wei, O. D. Kripfgans, J. B. Fowlkes, J. B. Grotberg, and S. Takayama, "Gravity-Driven Microfluidic Particle Sorting Device with Hydrodynamic Separation Amplification," Analytical Chemistry, vol. 79, pp. 1369-1376, 2007.
[24] D. b. Seo, Y. Agca, Z. C. Feng, and J. K. Critser, "Development of sorting, aligning, and orienting motile sperm using microfluidic device operated by hydrostatic pressure," Microfluid Nanofluid, vol. 3, pp. 561-570, 2007.
[25] J. Wu, Y. Chung, K. Belford, G. Smith, S. Takayama, and J. Lahann, "A surface-modified sperm sorting device with long-term stability," Biomedical Microdevices, vol. 8, pp. 99-107, 2006.
[26] D. A. Chang-Yen, R. K. Eich, and B. K. Gale, "A monolithic PDMS waveguide system fabricated using soft-lithography techniques," Lightwave Technology, Journal of, vol. 23, pp. 2088-2093, 2005.
[27] W. T. Chu SC, Li CC, Kao RH, Li DK, Su YC, Wells DA, Loken MR, "Flow cytometric scoring system as a diagnostic and prognostic tool in myelodysplastic syndromes," Leuk Res, vol. 35, pp. 868-73, 2011.
[28] H. Kanno, K. Saito, T. Ogawa, M. Takeda, A. Iwasaki, and Y. Kinoshita, "Viability and function of human sperm in electrolyte-free cold preservation," Fertility and sterility, vol. 69, pp. 127-131, 1998.
[29] D. P. Froman and A. J. Feltmann, "Fowl (Gallus domesticus) sperm motility depends upon mitochondrial calcium cycling driven by extracellular sodium," Biol Reprod, vol. 72, pp. 97-101, Jan 2005.
[30] R. Felix, "Molecular physiology and pathology of Ca2+-conducting channels in the plasma membrane of mammalian sperm," Reproduction, vol. 129, pp. 251-62, Mar 2005.
[31] D. Zhang and M. Gopalakrishnan, "Sperm ion channels: molecular targets for the next generation of contraceptive medicines?," J Androl, vol. 26, pp. 643-53, Nov-Dec 2005.
[32] M. G. ZAMFIR TRUTA , STELA LERINTIU, ROMEO MICU, "A new method for human semen glucose concentration evaluation " Romanian Biotechnological Letters vol. 15, p. 6, 2010.
[33] K. Sato, K. Sato, M. Ozawa, K. Kikuchi, T. Nagai, and T. Kitamori, "Development of a micro breeder system for in vitro production of blastocysts," microTAS2005, 2005.
[34] M. Eisenbach, "Towards Understanding the Molecular Mechanism of Sperm Chemotaxis," The Journal of General Physiology, vol. 124, pp. 105 - 108, 2004.
[35] F. R. Salilew-Wondim Dessie, Michael Hölker, Markus Gilles, Danyel Jennen, Ernst Tholen, Vitezslav Havlicek, Urban Besenfelder, Vladimir L Sukhorukov, Ulrich Zimmermann, Joerg M Endter, Marc-André Sirard, Karl Schellander and Dawit Tesfaye, "Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern," Society for Reproduction and Fertility, vol. 133, pp. 931-946 2007.
[36] G. D. Smith and S. Takayama, "Gamete and embryo isolation and culture with microfluidics," Theriogenology, vol. 68, pp. S190-S195, 2007.
[37] Z. H. C. Glasgow.I.K., Beebe.D.J., Seong-Jun Choi, Lyman.J.T., Chan.N.G., Wheeler.M.B. , "Handling individual mammalian embryos using microfluidics," vol. 48, pp. 570 - 578, 2001
[38] M. T. J. John Paul Urbanski, David D. Craig, David L. Potter, David K. Gardner, Todd Thorsen, "Noninvasive Metabolic Profiling Using Microfluidics for Analysis of Single Preimplantation Embryos," Analytical Chemistry, vol. 80, pp. 6500-6507 2008.
[39] N. H. Kimura H, Akai T, Yamamoto T, Hattori H, Sakai Y, Fujii T., "On-Chip Single Embryo Coculture With Microporous-Membrane-Supported Endometrial Cells," IEEE Trans Nanobioscience, vol. 8, pp. 318-24, 2009.
[40] H. C. Zeringue, D. J. Beebe, and M. B. Wheeler, "Removal of Cumulus from Mammalian Zygotes using Microfluidic Techniques," Biomedical Microdevices, vol. 3, pp. 219-224, 2001.