Oocyte deformation during injection is a major cause of potential cell damage which can lead to failure in the Intracytoplasmic Sperm Injection (ICSI) operation used as an infertility treatment. Injection speed plays an important role in the deformation creation. In this paper the effect of different speeds on deformation of zebrafish embryos is studied using a specially designed experimental set-up. An analytical model is developed in order to link injection force, deformation, and injection speed. A finite element (FE) model is also developed to analyse the effect of injection speed, allowing the production of additional information that is difficult to obtain experimentally, e.g., deformation and stress fields on the oocyte. The numerical model is validated against experimental results. Experimental results indicate that by increasing the injection speed, the deformation decreases. However, higher speeds cause higher levels of injection force and force fluctuation, leading to a higher vibration during injection. For this reason, an optimum injection speed range is determined. Finally, the FE model was validated against experimental results. The FE model is able to predict the force-deformation variation during injection for different speeds. This proves to be useful for future studies investigating different injection conditions.
The central role of the oviduct, as the site of zona pellucida (ZP) maturation, fertilization and early embryogenesis, has been recognized. The objective of this study was to investigate whether ampullary and isthmic derived epithelial cells have different effects on in vitro ZP hardening, in vitro fertilization (IVF) and in vitro culture (IVC) of the resulting embryos. Cumulus oocyte complexes (COCs) were matured in a coculture system with ampullary/isthmic epithelial cells, TCM199 supplemented with insulin-like growth factor I (IGF-I) and epithelial derived growth factor (EGF) (GF treated group), conditioned media produced using ampullary (ACM), isthmic (ICM), COCs+ampullary, and COCs+isthmic epithelial cells, contactless culture system, oviductal fluid, GF+ACM/ICM, and drops of TCM199 (control), for 24h. The matured oocytes were randomly divided into two groups: Group I was subjected to ZP digestion; Group II underwent IVF. The duration of the ZP digestion, in a coculture system with ampullary epithelial cells (AE) was significantly increased (p<0.05), compared with other groups. Penetrated oocytes and monospermic fertilization were significantly increased (p<0.05) in the AE group. The mean number of spermatozoa per penetrated oocyte was reduced dramatically for the AE group (p<0.05). A significant increase (p<0.05) in the embryo development was observed in all treated groups, compared to the control. Results revealed that epithelial cells harvested from the ampullary segment of the oviduct had in vitro specialized role in ZP hardening and have subsequent IVF and IVC outcomes.
Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technology used in infertility treatment, where a single sperm is selected and immobilized using a glass injection pipette and is inserted directly into the cytoplasm of an oocyte under the microscope. Auto-alignment of the injection pipette is a prerequisite for any proposed automated oocyte injection procedure. In this paper, an autoalignment procedure has been proposed. This technique requires the positioning of the injection and holding pipettes in the three orthogonal axes under microscopy which is complex. The existing proposed methods are system-specific and require appropriate algorithms. In this paper, 12 commonly used focusing algorithms were evaluated to verify the optimal one for the ICSI application. These algorithms were assessed by measuring focusing accuracy, range, the number of false maxima, and the width of the curve. The focus level for each pipette is calculated by the algorithm using focus measure functions (FMA). The Fibonacci search algorithm is employed for controlling the z-axis of the motorized stage to obtain the focal plane of the injection and holding pipette. The experimental results verified that the Brenner gradient has demonstrated the highest overall performance for injection pipette focalization, while the energy of gradient has presented a highest overall performance for holding pipette focalization.INDEX TERMS Intracytoplasmic sperm injection (ICSI), autofocusing, computer vision, focus measurement algorithm, Fibonacci search.
A rapid prototyping technique is demonstrated which uses a red femtosecond laser to produce a metallic mould which is then directly used for the replica moulding of PDMS. The manufacturing process can be completed in less than 6 h making it a viable technique for testing new designs quickly. The technique is validated by creating a microfluidic device with channels of height and depth of 300 µm, with a ramp test structure where the height and width of the channels reduces to 100 µm to demonstrate the techniques 3D capabilities. The resulting PDMS device was easily removed from the metallic mould and closely replicated the shape aside the expected shrinkage during thermal curing. As the technique uses a single replica process, the surface roughness at the base of the channels corresponds to the un-ablated polished metal mould, resulting in a very low surface roughness of 0.361 nm. The ablated metallic mould surface corresponds to the top of the PDMS device, which is bonded to glass and does not affect the flow within the channels, reducing the need for optimisation of laser parameters. Finally, the device is validated by demonstrating laminar flow with the no-slip condition.
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