近年來除了先天性的原因，現代人生活的不良習慣越來越普遍與複雜，使得不孕症發生的機率有越來越高的趨勢。根據統計，不孕症的發生率約15%，大約7對夫婦就有一對有不孕症的問題，而治療不孕症的方法就是通過體外受精，也就是試管嬰兒，相關的議題越來越所被關注，特別是體外人工受精需要大量且複雜的人工操作，而根據統計台灣的試管嬰兒的存活率只有27.4%，但是提高胚胎品質的控制因素有限，且需要繁複的人工操作，因此如何才能提升胚胎的品質就成為了未來主要的研究方向。
本研究的目標主要通用微流體的技術，整合自動定位、動態灌流，建造體外輸卵管的培養環境等之多功能輸卵管晶片。利用流阻抓取的設計成功地抓取胚胎在晶片的微結構上，並且針對性地給予胚胎滾動之物理刺激，此滾動物理刺激類似於輸卵管之環境。同時我們利用晶片流道上設計標記以評估胚胎運動速度。配合上注射幫浦的流速控制使胚胎在晶片上貼附滾動，從而達到控制胚胎在晶片裡的速度為500μm/min。此外搭配上動態灌流功能適時地帶走代謝廢棄物，用以提高胚胎培養品質。此晶片完成了整合自動化的設計，大大地減少不必要的人為操作對胚胎造成之影響。

In recent years, in addition to congenital reasons, the bad habits of modern life become more common and complex. The incidence of infertility comes to about 15%, which means that every 1 out of 7 couples has the problem of infertility. In vitro fertilization (IVF), also named test tube baby, is the most common way of treating infertility. Unfortunately, in vitro artificial insemination requires complicated manual operation. The statistic results indicate that the survival rate of test tube baby is only 27.4%. Improving the quality of the embryo becomes a major research topic now.
In this research, many microfluidics technologies, including automatic positioning, dynamic perfusion, in vitro bionic uterine culture environment, are integrated into this uterine chip. First, the embryos were captured via the design of the specific microfluidic structure. Then the physical stimulation and the rotation induced by the shear force were applied to mimic the environment of the fallopian tube. The rotation of embryos on chip were achieved with the translational speed of 500μm/min under the pump velocity control. The dynamic perfusion was also applied to flush the metabolic waste away and provide the required nutrition for cell culture. Our chip has the potential feature targeting for greatly reducing the impact of unnecessary human manipulation during IVF.

ABSTRACT I
摘要 II
致謝 II
目錄 IVV
圖目錄 VII
表目錄 VIII
第一章 緒論 1
1.1 研究背景 1
1.1.1 不孕因素 1
1.1.2 輔助生殖技術(ART) 3
1.1.3 生物微機電與實驗室晶片 4
1.1.4 胚胎體內發展 6
1.1.5 自體子宮內膜細胞與受精卵共培養 6
1.2 文獻回顧 7
1.2.1 傳統體外培養方式 7
1.2.2 傳統培養材料 8
1.2.3 體外微流體胚胎培養裝置 8
1.2.4 追蹤單一胚胎發展 13
1.2.5 體外微流體胚胎滾動裝置 15
1.3 動機與目的 17

第二章 晶片設計與原理 19
2.1 設計基礎與理論 19
2.1.1微流體流阻分析 19
2.1.2微流體流速分析 20
2.1.2微流體剪應力分析 20
2.2 設計概念 21
2.2.1 胚胎抓取定位之微結構設計 22
2.2.2 晶片操作 23
2.2.3 動態流阻抓取系統 24
第三章 晶片製程 26
3.1 晶片製作流程 26
3.1.1 微流道母模製程 26
3.1.2 微流道晶片製程 27
3.2製程結果 28
3.2.1 微流道結構 29
第四章 材料準備與實驗架設 30
4.1 材料準備 30
4.1.1 聚苯乙烯乳膠微粒 30
4.1.2 冷凍小鼠胚胎 31
4.1.3 培養基 33
4.2 實驗架設 33
4.2.1 儀器架設 33
第五章 實驗結果 34
5.1 胚胎在晶片上抓取結果 34
5.2 傳統培養方式結果 34
5.3 晶片上標籤制作結果 35
5.4 胚胎在晶片上滾動結果與討論 35
5.5 膠原蛋白對胚胎滾動之影響結果 37
5.6 膠原蛋白在不同濃度下胚胎行為之結果 38
5.7 胚胎控制在不同流速下與不同膠原蛋白濃度之下之滾動結果與討論 40
第六章 總結 42
參考文獻 43

[1] C. Gnoth, E. Godehardt, P. Frank-Herrmann, K. Friol, J. Tigges, and G. Freundl, “Definition and prevalence of subfertility and infertility,” Hum. Reprod., vol. 20, no. 5, pp. 1144–1147, 2005.
[2] S. Gurunath, Z. Pandian, R. A. Anderson, and S. Bhattacharya, “Defining infertility-a systematic review of prevalence studies,” Human Reproduction Update, vol. 17, no. 5. pp. 575–588, 2011.
[3] K. A. O’Connor, D. J. Holman, and J. W. Wood, “Declining fecundity and ovarian ageing in natural fertility populations,” Maturitas, vol. 30, no. 2, pp. 127–136, 1998.
[4] D. B. Dunson, B. Colombo, and D. D. Baird, “Changes with age in the level and duration of fertility in the menstrual cycle,” Hum. Reprod., vol. 17, no. 5, pp. 1399–1403, 2002.
[5] E. H. Yu, W. S. Yeung, E. Yee Lau, W. W. So, and P. C. Ho, “High serum oestradiol concentrations in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent frozen-thawed embryo transfer cycles,” Hum. Reprod., vol. 15, no. 2, p. 250, 2000.
[6] F. Talib, Z. Rahman, and M. Azam, “Best practices of total quality management implementation in health care settings,” Health Mark. Q., vol. 28, no. 3, pp. 232–52, 2011.
[7] A. B. Jose-Miller, J. W. Boyden, and K. A. Frey, “Infertility,” American Family Physician, vol. 75, no. 6. 2007.
[8] Mae Tao Clinic, “Mae Tao Clinic Annual Report 2013,” 2013.
[9] U. Wahrburg, “Taschenatlas der Ernhrung,” Pharm. Ztg., vol. 144, no. 1, p. 86, 1999.
[10] L. I. Barmat, H.C. Liu, S.D. Spandorfer, A. Kowalik., “Autologous endometrial co-culture in patients with repeated failures of implantation after in vitro fertilization- embryo transfer,” J. Assist. Reprod. Genet., vol. 16, no. 3, pp. 121–127, 1999.
[11] C. Deachapunya and S. M. O’Grady, “Epidermal growth factor regulates the transition from basal sodium absorption to anion secretion in cultured endometrial epithelial cells,” J. Cell. Physiol., vol. 186, no. 2, pp. 243–250, 2001.
[12] F. Dominguez, B. Gadea, A. Mercader, F. J. Esteban, A. Pellicer, and C. Simón, “Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system,” Fertil. Steril., vol. 93, no. 3, 2010.
[13] M. Okabe, “The cell biology of mammalian fertilization,” Development, vol. 140, no. 22, pp. 4471–4479, 2013.
[14] H. Kimura, T. Yamamoto, H. Sakai, Y. Sakai, and T. Fujii, “An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models,” Lab Chip, vol. 8, no. 5, pp. 741–6, 2008.
[15] A. P. Sommer, M. K. Haddad, and H. J. Fecht, “It is Time for a Change: Petri Dishes Weaken Cells,” J. Bionic Eng., vol. 9, no. 3, pp. 353–357, 2012.
[16] J. E. Swain and G. D. Smith, “Advances in embryo culture platforms: Novel approaches to improve preimplantation embryo development through modifications of the microenvironment,” Hum. Reprod. Update, vol. 17, no. 4, pp. 541–557, 2011.
[17] K. S. Kolahi, A. Donjacour, X. Liu, W. Lin, R. K. Simbulan, “Effect of substrate stiffness on early mouse embryo development,” PLoS One, vol. 7, no. 7, 2012.
[18] D. A. Rappolee, C. Basilico, Y. Patel, and Z. Werb, “Expression and function of FGF-4 in peri-implantation development in mouse embryos,” Development, vol. 120, no. 8, pp. 2259–2269, 1994.
[19] S. Raty, E. M. Walters, J. Davis, H. Zeringue, D. J. Beebe, “Embryonic development in the mouse is enhanced via microchannel culture,” Lab Chip, vol. 4, pp. 186–190, 2004.
[20] R. L. Krisher and M. B. Wheeler, “Towards the use of microfluidics for individual embryo culture,” Reproduction, Fertility and Development, vol. 22, no. 1. pp. 32–39, 2010.
[21] D. L. Hickman, D. J. Beebe, S. L. Rodriguez-Zas, and M. B. Wheeler, “Comparison of static and dynamic medium environments for culturing of pre-implantation mouse embryos,” Comp. Med., vol. 52, no. 2, pp. 122–6, 2002.
[22] Y. Xie, F. Wang, W. Zhong, E. Puscheck, H. Shen, and D. a Rappolee, “Shear stress induces preimplantation embryo death that is delayed by the zona pellucida and associated with stress-activated protein kinase-mediated apoptosis,” Biol. Reprod., vol. 75, no. 1, pp. 45–55, 2006.
[23] K. Matsuura, N. Hayashi, Y. Kuroda, C. Takiue, “Improved development of mouse and human embryos using a tilting embryo culture system,” Reprod. Biomed. Online, vol. 20, no. 3, pp. 358–364, 2010.
[24] Y. S. Heo, L. M. Cabrera, C. L. Bormann, C. T. Shah, S. Takayama, and G. D. Smith, “Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates,” Hum. Reprod., vol. 25, no. 3, pp. 613–622, 2010.
[25] Y. S. Hur, J. H. Park, E. K. Ryu, S. J. Park, J. H. Lee, “Effect of micro-vibration culture system on embryo development,” J. Assist. Reprod. Genet., vol. 30, no. 6, pp. 835–841, 2013.
[26] J. R. Alegretti, A. M. Rocha, B. C. Barros, P. Serafini, “Microfluidic dynamic embryo culture increases the production of top quality human embryos through reduction in embryo fragmentation,” Fertility and Sterility, vol. 96 Suppl, no. 3. pp. S58-S59 O – 196, 2011.
[27] M. S. Kim, C. Y. Bae, G. Wee, Y. M. Han, and J. K. Park, “A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos,” Electrophoresis, vol. 30, no. 18, pp. 3276–3282, 2009.
[28] C. Y. Bae, M. S. Kim, and J. K. Park, “Mechanical stimulation of bovine embryos in a microfluidic culture platform,” Biochip J., vol. 5, no. 2, pp. 106–113, 2011.
[29] V. Isachenko, R. Maettner, K. Sterzik, E. Strehler, “In-vitro culture of human embryos with mechanical micro-vibration increases implantation rates,” Reproductive BioMedicine Online, vol. 22, no. 6. pp. 536–544, 2011.
[30] E. Isachenko, R. Maettner, V. Isachenko, S. Roth, R. Kreienberg, and K. Sterzik, “Mechanical agitation during the in vitro culture of human pre-implantation embryos drastically increases the pregnancy rate,” Clin. Lab., vol. 56, no. 11–12, pp. 569–576, 2010.
[31] Y. Xie, F. Wang, E. E. Puscheck, and D. A. Rappolee, “Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos,” Mol. Reprod. Dev., vol. 74, no. 10, pp. 1287–1294, 2007.
[32] C. Simón, A. Mercader, J. Garcia-Velasco, “Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure,” J. Clin. Endocrinol. Metab., vol. 84, no. 8, pp. 2638–46, 1999.
[33] V. Eyheremendy, F. G. E. Raffo, M. Papayannis, J. Barnes, C. Granados, and J. Blaquier, “Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure,” Fertil. Steril., vol. 93, no. 3, pp. 769–773, 2010.
[34] H. Kimura, H. Nakamura, T. Akai, “On-chip single embryo coculture with microporous-membrane-supported endometrial cells,” IEEE Trans. Nanobioscience, vol. 8, no. 4, pp. 318–324, 2009.
[35] M. Cruz, N. Garrido, J. Herrero, I. Pérez-Cano, M. Muñoz, and M. Meseguer, “Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality,” Reprod. Biomed. Online, vol. 25, no. 4, pp. 371–381, 2012.
[36] W. X. Li, G.T. Liang, W. Yan, Q. Zhang, W. Wang, X.M. Zhou, “Artificial uterus on a microfluidic chip,” Fenxi Huaxue/ Chinese J. Anal. Chem., vol. 41, no. 4, pp. 467–472, 2013.
[37] H. Y. Huang, H.H. Shen, C.H. Tien, C.J. Li, S.K. Fan, C.H. Liu, “Digital microfluidic dynamic culture of mammalian embryos on an Electrowetting on Dielectric (EWOD) chip,” PLoS One, vol. 10, no. 5, 2015.
[38] G. Vajta, T.T. Peura, P. Holm, A. Paldi, “New method for culture of zona-included or zona-free embryos: The Well of the Well (WOW) system,” Mol. Reprod. Dev., vol. 55, no. 3, pp. 256–264, 2000.
[39] S. Sugimura, T. Akai, T. Somfai, M. Hirayama, “Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos,” Biol. Reprod., vol. 83, no. August, pp. 970–978, 2010.
[40] R. Ma, L. Xie, C. Han, K. Su, T. Qiu, L. Wang, “In vitro fertilization on a single-oocyte positioning system integrated with motile sperm selection and early embryo development,” Anal. Chem., vol. 83, no. 8, pp. 2964–2970, 2011.
[41] W.-H. Tan and S. Takeuchi, “A trap-and-release integrated microfluidic system for dynamic microarray applications,” Proc. Natl. Acad. Sci. U.S.A., vol. 104, no. 4, pp. 1146–51, 2007.
[42] D. Di Carlo and L. P. Lee, “Dynamic Single-Cell Analysis for Quantitative Biology,” Anal. Chem., vol. 78, no. 23, pp. 7918–7925, 2006.
[43] A. N. Terävä, M. Gissler, E. Hemminki, and R. Luoto, “Infertility and the use of infertility treatments in Finland: Prevalence and socio-demographic determinants 1992-2004,” Eur. J. Obstet. Gynecol. Reprod. Biol., vol. 136, no. 1, pp. 61–66, 2008.
[44] C. Leung, Z. Lu, X. P. Zhang, and Y. Sun, “Three-dimensional rotation of mouse embryos,” IEEE Trans. Biomed. Eng., vol. 59, no. 4, pp. 1049–1056, 2012.
[45] J. C. Li, M. Koji, K. Yuka, F. Hiroaki, and N. Keiji, "Application of mechanical stimuli using a microfluidic air actuating system to cultured mammalian embryos," Micro-NanoMechatronics and Human Science (MHS), 2010 International Symposium on. IEEE, 2010.
[46] J. Chung, Y. J. Kim, and E. Yoon, “Highly-efficient single-cell capture in microfluidic array chips using differential hydrodynamic guiding structures,” Appl. Phys. Lett., vol. 98, no. 12, 2011.
[47] N. Saiz and B. Plusa, “Early cell fate decisions in the mouse embryo,” Reproduction, vol. 145, pp. R65-80, 2013.