Periodic Reporting for period 2 - Micro-GIFT (Microrobotic Gamete/Zygote IntraFallopian Transfer)
Reporting period: 2021-10-01 to 2023-03-31
Infertility is a worldwide problem affecting ~11% of the reproductive-age population. Severe cases are currently treated by in vitro fertilization (IVF) and intracytoplasmic injection techniques (ICSI) with high fertilization rates (~95%). However, embryo transfer is still the critical stage with only 32% of the cases resulting in clinical pregnancies. Moreover, the implantation rates per embryo remain very low (~17%) and often the procedure needs to be repeated several times with no success implying a high economic and social cost. Among the different methods used to overcome this issue, gamete or zygote intrafallopian transfer (GIFT or ZIFT) seems more promising offering appropriate physiological environment for zygote/embryo development at an optimal synchronization between embryonic and endometrial preparation. However, these methods are invasive and involve surgical procedures and anaesthesia to introduce macroscopic imaging and manipulation tools into the female body, increasing the risk of injury and ectopic pregnancies. The goal of Micro-GIFT is to seek for novel approaches to non-invasively transport and release high-quality gametes/zygotes in the fallopian tube in vivo (mice model). For that multifunctional untethered microrobots (~100 µm size) will be developed making use of smart materials and advanced microtechnologies. However, there are major challenges that need to be overcome to bring this technology close to the clinic, such as the in vivo imaging and control of such microrobots, and their removal after use. The project will also provide deeper insights on the contribution of the fallopian tube on the natural embryo development and implantation, being crucial to create more natural procedures with high success rates. The PI has contributed significantly to the field of sperm-based microbots for assisted fertilization and targeted drug delivery as well as developed a variety of novel microbiosensors for molecular and cellular analysis.
In the first reporting period, progress on all the planned Work-packages have been made despite delays caused by the current pandemic situation and a complicated pregnancy and parental leave of the PI. For example, in WP1, which is focused on the development of non-destructive devices for the assessment of gametes and embryos, we have fabricated different biosensing platforms for model analyte/sample detection by employing concepts of strain engineering and roll-up origami. We have developed tubular scaffolds for single cell analysis integrating impedimetric sensors for either performing impedance spectroscopy or tomography, allowing the simultaneous detection of single cells and the surrounding media conductivity. Moreover, we have reported the integration of actuators in such tubular-origami microdevices, to allow the gentle capture of oocytes, by demonstrating their functionality with model microobjects of different stiffness. This will then allow the assessment of the mechanical and electrical properties of the zygote membrane as means for detecting factors such as oxidative stress, morphology, zona pellucida thickness, cell metabolism, among other factors. In WP2, there hasve been some progress on stablishing proper cell culture conditions, employing bovine ex-vivo epithelial and ciliary cells, which are then cultured in three dimensional porous scaffolds with dimensions similar as the ones of a real oviduct. Different fabrication technologies are being explored such as 2-photon lithography, Gray lithography combined with plasma etching, and strain engineering to find out the optimal scaffold to create a functional in vitro-mimicking oviduct for the goal of embryo implantation analysis prior and after microrobotic transfer. WP3 deals with the development of untethered microcarriers to transport gamete/zygotes, and here one of the approaches was successfully concluded and published, which consist in a spiral-like micromotor actuated by external rotating magnetic fields which can capture and transport safely a fertilized oocyte from a microenvironment to another, while preserving its viability. The microcarrier was tested in different complex media and confined microfluidic channels, showing promising results. Currently there are two works which are in the phase of Manuscript preparation, one consisting on 3D printed biodegradable microgrippers for the same task, and alginate-based capsule-like micromotors with multiple functionalities. Finally, for WP4 and WP5, a feedback control system guided by ultrasound or optoacoustic is being developed. On one side we have reported on the open loop control of moving magnetically-driven microobjects of sizes similar as the zygote size, in real time and deep tissue employing US and Photoaocustic imaging. We succeeded in tracking such objects in vitro, in ex vivo tissues and in small mice models. In the past months we have been focused on implementing feedback control algorithms based on Machine learning for the precise localization of magnetically-driven microcarriers, making important steps towards their operation in complex and more realistic environments independently on the microcarrier size, velocity, locomotion principle and surrounding environmental factors.
The project is expected to promote the application of remotely controlled magnetic micro-carriers for in vivo assisted reproduction technology. Specifically, to transport zygotes or gametes under more natural conditions. This is expected to result in higher pregnancy rates, less invasive procedures and more natural conditions for the proper embryo implantation and development. Our approach will bring advances in the field of reproductive science. These magnetic carriers, together with appropriate imaging technologies, will serve as tools for diagnostics of infertility and other medical issues of the reproductive tract. The in vivo application of these technologies will provide deeper insights into zygote transport and embryo development through the reproductive tract and will reveal causes of infertility and low implantation rates. The proposed micro-carriers can also be equipped for drugs or gene delivery for treating zygote genetic problems or to avoid ectopic pregnancies. Furthermore, endangered animal species will benefit from this technology, as zygotes of selected gender can be delivered into the animal fallopian tube. Thereby, the proposed medical microrobots will have a tremendous application potential not only for in vivo assisted fertilization but also for diverse biomedical applications. In the future, many patients with repeated implantation failure and other infertility problems will also benefit from this technology. These carriers can also be used to deliver drugs to treat diseases like endometriosis, pelvic inflammatory diseases or other gynaecological diseases in the future. They can also be engineered to carry genes, mRNA, or imaging contrast agents, individually or in swarm to perform cooperative work to solve complex tasks. With this project, I expect not only to conceptionally change assisted reproduction technologies by making use of novel micro-and nanotechnologies, but also instigate progress in many other areas of high relevance for other scientific communities, such as imaging, microrobotics, medical technology, as well as fundamental cell biology.