Towards Automation of In vitro Fertilisation (IVF)

Abstract

One in six couples globally suffer from infertility. The most commonly accepted treatment for infertility is in vitro fertilisation (IVF). The suite of manual procedures used in an IVF laboratory are technically challenging. Thus, it is not surprising that IVF success is influenced by the expertise and experience of the embryologist. In this thesis I design and manufacture a novel device and investigate its capability to simplify several IVF procedures. The device consists of two components, the Pod and Garage. The Pod houses an individual oocyte or embryo, while the Garage accommodates numerous Pods. With this, multiple oocytes or embryos can be docked in an array within a Garage. First, I investigated the toxicity of the polymer used to fabricate the device. When comparing mouse embryos cultured within the device compared to standard culture there were no differences in development or DNA damage. Towards utilising the device for intracytoplasmic sperm injection (Katalinic et al.), I microinjected fluorescent microspheres into presumptive zygotes (PZs) within our device. My results showed that injected zygotes were able to develop to the blastocyst stage. Furthermore, I quantified the time it takes to perform standard oocyte microinjection and compared this with microinjection within the device. My results showed that my device simplified the microinjection procedure by removing the need for a holding pipette. Furthermore, our device improved traceability of injected vs non-injected oocytes and allowed high-throughput microinjection. Importantly, microinjection time was significantly decreased using our device compared to standard microinjection. Next, I investigated the cryopreservation of mouse oocytes and blastocyst-stage embryos within our device. The results of this study demonstrated no structural impact on the device when it was directly and repeatedly exposed to liquid nitrogen (LN2). Furthermore, compared to standard cryopreservation practice, vitrification and warming within our device showed no significant differences in viability for both oocytes and embryos. Additionally, vitrification and warming within our device occurred within ~3 nL, an approximate 300-fold reduction in exposure to the cytotoxic cryoprotectants compared to standard vitrification. Moreover, our device minimised direct handling and improved traceability of oocytes and embryos within cryoprotectants, thereby simplifying the procedure. Finally, I assessed embryo development under a single-step vs a two-step culture system. Using commercial human IVF media, my results demonstrated no statistically significant difference in developmental competency in resultant blastocyst-stage embryos cultured under the two culture systems. However, there was a negative impact of changeover of culture medium at Day 3 in the two-step culture system when transferred from the first medium to a new first medium of the two-step culture system. To obviate the need for intervention at Day 3, I designed and manufactured polydimethylsiloxane (PDMS) microfluidic manifold to accommodate docking of three Garages housing a total of nine Pods and investigated its use for seamless media delivery and exchange during embryo culture. Collectively, this thesis is the first to consider and examine devices fabricated using two-photon polymerisation technology and their application in and simplification of IVF procedures.