P-279 The role of autophagy in the development of mouse preimplantation embryos and morphokinetics
Authors: Nguyen T.D , Nguyen T.N.Q , Ziller V 
Published: June 2022

Abstract

Study question
Which role does autophagy play in the development of mammalian preimplantation embryos?

Summary answer
This study suggests the critical role of autophagy in the fertilized oocytes and embryo development.

What is known already
Cellular nutrition plays an important role in improving reproductive efficiency in all animals. Homeostasis of nutrient metabolism is tightly controlled by a cellular pathway called autophagy, which can recycle nutrients and organelles to allow cells adapting to nutritional stress such as nutrient deficiency or excess. Increasing evidence shows that autophagy is involved in a wide range of cellular events within mammalian reproduction. Since it is known that autophagy has paradoxical functions in many cellular processes, it is still unclear which role autophagy plays in the development of mammalian preimplantation embryos.

Study design, size, duration
To determine the role of autophagy in the development of mammalian preimplantation embryos, a randomized parallel group study with hybride Bl6CBa/ca mouse embryos was designed.

Participants/materials, setting, methods
207 zygotes of Bl6CBAca mice were randomly distributed into two groups. The control embryos were cultured in 2ml GTL media (Vitrolife) supplemented with DMSO (vehicle), while the treatment embryos were cultured in 2ml GTL media supplemented with 100 nM autophagy inhibitor Bafilomycin (Baf). Embryos were non-invasively monitored with a Primovision time-lapse system (Vitrolife) for 5 days. Blastocyst, degeneration rate and morphokinetics were analyzed. Furthermore, confocal microscopy staining of 2PN embryos with autophagy marker LC3 was performed.

Main results and the role of chance
Our study showed a statistically significant difference in the expanded blastocyst as well as degeneration rate among groups (p &lt; 0.0001). 89,19% of embryos in the control group (non-treated) (N = 111) reached to expanded blastocysts, while most of the embryos in Baf-treated group (N = 96) stopped development at 4C-stage. No embryo in Baf-treated group reached expanded blastocysts (blastocyst rate = 0) and 100% embryos degenerated. However, there is no statistically significant difference in the development time from 2C to 4C stage between non-treated (1330 ± 17.35 min, N = 50) and Baf-treated group (1364 ± 22.20 min, N = 50).
Moreover, confocal microscopy staining of 2PN embryos with autophagy marker LC3 showed that non-treated embryos expressed weakly and dispersed LC3 signal in the whole cell. In contrast, LC3 signal in Baf-treated embryos were significantly accumulated and translocated in the cell membranes.
The accumulation of autophagy-marker LC3 after treatment with Bafilomycin, an autophagy inhibitor, indicated the high expression of autophagosomal activity during the embryo development. This may explain the toxicity in Baf-treated embryos, which prevent the embryos to develop further than 4C-stage. Altogether our study suggests the critical role of autophagy in the fertilized oocytes and embryo development

Limitations, reasons for caution
Murine embryos model is widely used for safety testing in assisted reproduction technologies, however human embryos might behave differently. Moreover, although treatment with Bafilomycin is the gold standard in autophagy research in vitro, it might not mimic exactly the autophagy dysfunction in human embryo development.

Wider implications of the findings
Emergent evidences show that dysfunction of autophagy, which maintains the cellular homeostasis, correlates with metabolic disorders including obesity. In women, obesity causes anovulation and menstrual irregularities. Therefore, we aim to investigate the potential role of autophagy in the obesity, which may help improving the pregnancy rate in the obesity-associated infertility.

Trial registration number
Not applicable