Micromotors – untethered, motile, microscopic devices – are implemented in this dissertation for two applications in the field of assisted reproductive technology. First, as synthetic motor units for individual sperm cells, representing a novel approach to counteract sperm immotility (asthenozoospermia), which is one of the most prevalent causes of male infertility. Second, as synthetic carriers of fertilized oocytes (zygotes) towards the realization of non-invasive intrafallopian transfer, representing a novel alternative to the current keyhole surgery (laparoscopy) approach to achieve early embryo transfer after in vitro fertilization. In both applications, magnetically actuated micromotors are utilized to capture, transport, and deliver individual cells in a reproducible, controllable manner. In comparison with established in vitro fertilization routines, the crucial advantage of employing micromotors for the manipulation of gametes, i.e. sperm and (fertilized) oocytes, lies in the potential transfer of decisive steps of the fertilization process back to its natural environment – the fallopian tube of the female patient – taking advantage of the untethered, non-invasive motion and manipulation capabilities of magnetic micromotors. When sperm motility can be restored with magnetic micromotors, sperm can travel to the oocyte under external actuation and control, and the oocyte does not need to be explanted for in vitro fertilization. However, if in vitro fertilization was necessary, fertilized oocytes can be transferred back to the fallopian tube by micromotors in a non-invasive manner, to undergo early embryo development in the natural environment. These novel concepts of micromotor-assisted reproduction are presented and investigated in this thesis, and their potential is analyzed on the basis of proof-of-concept experiments.:1 Introduction 6 1.1 Background and Motivation 6 1.2 Objectives and Structure of this Dissertation 9 2 Fundamentals 11 2.1 Micromotors Definition and Concept 11 2.2 Micromotors for Biomedical Applications 13 2.3 Magnetic Micropropellers 15 2.3.1 Theory 15 2.3.2 Implementation 20 2.4 Microfabrication: Direct Laser Writing 21 2.5 Assisted Reproductive Technology 23 2.5.1 In vitro Fertilization and Intracytoplasmic Sperm Injection 24 2.5.2 Embryo Transfer and Zygote Intrafallopian Transfer 25 2.5.3 The Sperm Cell and the Oocyte 26 2.6 Towards Micromotor-Assisted Reproduction 28 3 Materials and Methods 30 3.1 Fabrication of Microfluidic Channel Platforms 30 3.1.1 Tailored Parafilm Channels 30 3.1.2 Polymer Channels Cast from Micromolds 31 3.1.3 Tubular Channels to Mimic In vivo Ducts 32 3.2 Fabrication of Magnetic Micropropellers 32 3.2.1 Direct Laser Writing of Polymeric Resin 33 3.2.1.1 Design and Programming 33 3.2.1.2 Exposure and Development 35 3.2.1.3 In Situ Direct Laser Writing 35 3.2.2 Critical Point Drying 35 3.2.3 Magnetic Metal Coatings 36 3.2.4 Surface Functionalization 37 3.3 Sample Characterization 38 3.3.1 Optical Microscopy 38 3.3.2 Scanning Electron Microscopy 38 3.4 Cell Culture and Analysis 39 3.4.1 Sperm Cells 39 3.4.2 Oocytes 39 3.4.3 In vitro Fertilization 41 3.4.4 Hypoosmotic Swelling Test 44 3.4.5 Cell Viability Assays 44 3.5 Magnetic Actuation 45 3.5.1 Modified Helmholtz Coil Setup 46 3.5.2 MiniMag Setup 47 3.5.3 Experimental Procedure 48 3.5.3.1 Micromotor Performance Evaluation 48 3.5.3.2 Cell Transport Experiments 49 3.5.3.3 Cell Transfer Experiments 50 4 Micromotor-assisted Sperm Delivery 51 4.1 Micromotor Design and Fabrication 51 4.2 Actuation and Propulsion Performance 53 4.3 Capture, Transport, and Release of Sperm 56 4.4 Delivery to the Oocyte 59 4.5 Sperm Viability and the Ability to Fertilize 61 5 Micromotor-assisted Zygote Transfer 68 5.1 Micromotor Design and Fabrication 68 5.2 Actuation and Propulsion Performance 70 5.3 Capture, Transport, and Release of Zygotes 76 5.4 Transfer between Separate Environments 80 5.5 Zygote Viability and Further Development 82 6 Conclusions and Prospects 85 Appendix 87 Bibliography 93 List of Figures and Tables 108 List of Abbreviations and Terms 109 Theses 111 Selbstständigkeitserklärung 112 Acknowledgments 113 List of Publications 115 Curriculum Vitae 116

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