The effects of laser-assisted zona pellucida manipulations on embryo competency and the mechanisms underlying embryo hatching in a mouse model

Mammalian embryo hatching from the zona pellucida (ZP) is a prerequisite to undergo subsequent implantation, but little is known about the involved molecules and regulatory mechanisms underlying the hatching process. Hatching failure can lead to implantation failure. Clinical laboratories have developed the non-contact infra-red diode laser to manipulate ZP for various purpose, such as assist embryo hatching and facilitate embryo biopsy procedure. Previous studies attempted to investigate the safety and efficacy of laser-assisted ZP manipulations; however, there is a significant paucity of safety evidence at the molecular level and the efficacy remains controversial. This thesis aimed to investigate the effect of laser-assisted ZP manipulations on embryo competency and transcriptome, and to uncover the regulatory mechanisms underlying the hatching process. In chapter 3, I analysed 181 questionnaires to characterise the clinical application pattern of ZP manipulation and found that laser-assisted ZP manipulation is extensively used in clinics, especially prior to embryo biopsy (89.5%). However, a huge disparity existed with regards to the current clinical practice, and no standardisation is evident at present. In chapter 4, mouse embryos were used to investigate the safety of the three most commonly used laser-assisted ZP manipulations, including ZP drilling (n=349), ZP partial drilling (n=319), and ZP thinning (n=297). No overt deleterious effects were identified with regards to the blastocyst development, cell apoptosis, heat shock stress (HSP70), and the expression of key developmental genes. However, the results demonstrated that laser-assisted ZP drilling significantly advance the hatching rate (81.1% (210) vs 40.1% (111), P<0.0001) and altered the pattern of embryo hatching at E4.5 (P<0.0001). In chapter 5, I further applied an Ishikawa cell in vitro implantation model to test the embryo attachment capability. The results demonstrated that laser-assisted drilling significantly increase the embryo entrapment rate (41.3% (19) vs 13.9% (28), P<0.0001) in a manner that may relate to the size of opening in ZP created by drilling. However, a potential benefit of laser-assisted drilling may occur for blastulation delayed embryos, shown by an improved attachment rate (100% (11) vs 50% (8), P=0.006). In chapter 6, I used the RNA-sequencing to compare the transcriptome of hatching (n=8) and pre-hatching mouse blastocysts (n=6), and 275 differentially expressed genes (DEGs) were identified (adjusted P<0.05). Furthermore, for the first time, the downstream analysis revealed that the in vitro blastocysts hatching is an ATP-dependent process; the significant activation of protein biosynthesis and organisation of cytoskeleton were also observed according to the transcriptomic alteration. In addition, the potential signaling pathway (such as RhoA signaling, Z-score=2.64, P<0.05) and upstream regulators (e.g., AGT, FGF2, and PRL) involved in the embryo hatching process were also revealed by the ingenuity pathway analysis. Finally, I further explored the effect of laser-assisted ZP drilling on embryo transcriptome using RNA-sequencing and found 48 DEGs (adjusted P<0.05). The downstream pathway analysis suggested the potential effect of laser-assisted drilling on embryo metabolic activity. In summary, this thesis provided safety evidence regarding the laser-assisted ZP manipulations, thus emphasising the importance of optimisation and refinement of clinical applications. Furthermore, this research also provides molecular profiling of hatching embryos, offering a useful asset for further exploring the mechanism underlying the embryo hatching process.

Keywords: cell apoptosis
zona pellucida
upstream regulator
assisted hatching
embryo attachment
mouse embryo
RNA sequencing
regulatory mechanism
laser-assited ZP manipulation
Infrared diode laser
pathway
Ishikawa cell
embryo hatching
embryo development
in vitro implantation model
assisted reproductive technology
Advisor: Coward, K
Granne, I