Citation: Zhai, R.; Shan, G.; Dai, C.; Hao, M.; Zhu, J.; Ru, C.; Sun, Y. Automated Denudation of Oocytes. Micromachines 2022, 13, 1301. https://doi.org/10.3390/ mi13081301 Academic Editor: Nam-Trung Nguyen Received: 20 July 2022 Accepted: 10 August 2022 Published: 12 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). micromachines Article Automated Denudation of Oocytes Rongan Zhai 1 , Guanqiao Shan 2 , Changsheng Dai 2 , Miao Hao 3 , Junhui Zhu 1 , Changhai Ru 4 and Yu Sun 1,2, * 1 School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China 2 Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ON M5S 3G8, Canada 3 School of Mechanical and Electrical Engineering, Research Center of Robotics and Micro Systems, Soochow University, Suzhou 215021, China 4 School of Electronic and Information Engineering, Suzhou University of Science and Technology, Suzhou 215009, China * Correspondence: sun@mie.utoronto.ca Abstract: Denudation is a technique for removal of the cumulus cell mass from oocytes in clinical intracytoplasmic sperm injection (ICSI). Manual oocyte denudation requires long training hours and stringent skills, but still suffers from low yield rate and denudation efficiency due to human fatigue and skill variations across operators. To address these limitations, this paper reports a robotic system for automated oocyte denudation. In this system, several key techniques are proposed, including a vision-based contact detection method for measuring the relative z position between the micropipette tip and the dish substrate, recognition of oocytes and the surrounding cumulus cells, automated calibration algorithm for eliminating the misalignment angle, and automated control of the flow rate based on the model of oocyte dynamics during micropipette aspiration and deposition. Experiments on mouse oocytes demonstrated that the robotic denudation system achieved a high yield rate of 97.0 ± 2.8% and denudation efficiency of 95.0 ± 0.8%. Additionally, oocytes denuded by the robotic system showed comparable fertilization rate and developmental competence compared with manual denudation. Our robotic denudation system represents one step towards the automation and standardization of ICSI procedures. Keywords: ICSI; oocyte denudation; robotic system 1. Introduction Human infertility has remained a highly prevalent global condition. Infertility is estimated to affect between 8% and 12% of reproductive-aged couples worldwide [1]. Intracytoplasmic sperm injection (ICSI) is a powerful therapeutic tool for the treatment of couples with severe infertility, and thousands of infertile couples have benefited from its use [2–4]. In ICSI procedures, the cumulus–oocyte complexes (COCs) are obtained through follicular aspiration, and the cumulus cells surrounding the oocyte must be removed to facilitate the injection of sperm [5] and the evaluation of oocyte morphology, in particular, the nuclear maturation stage for the extrusion of the first polar [6–8]. The removal of the cumulus cells from the oocyte is called oocyte denudation [9]. At present, oocyte denudation is done manually in ICSI clinics globally. An operator looks through the microscope eyepieces and manipulates oocytes using a mouth-controlled or hand-controlled pipette. Oocyte denudation is performed in two steps [10–12]. First, COCs are enzymolyzed in hyaluronidase to weaken the bonds between cumulus cells [13]. Due to the potential toxicity of the hyaluronidase on the oocytes, the enzymatic time (~40 s) is critical, which, however, is difficult to strictly control in manual operation. Then, cumu- lus cells are removed from oocytes by the pipette aspiration and deposition in a series of mediums without an enzyme. During pipette aspiration and deposition, it is difficult to recognize and manipulate the oocytes and the surrounding cumulus cells, especially when Micromachines 2022, 13, 1301. https://doi.org/10.3390/mi13081301 https://www.mdpi.com/journal/micromachines