# Transcriptomic Signatures of the Foetal Liver and Late Prenatal Development in Vitrified Rabbit Embryos

## TL;DR

This study shows that embryo vitrification affects fetal liver development and gene expression, potentially leading to long-term health risks.

## Contribution

The study identifies transcriptomic changes in fetal livers of vitrified embryos, linking ART procedures to potential adult health risks.

## Key findings

- Vitrified embryos had lower fetal and liver weights and shorter body lengths at 24 days of gestation.
- RNA-Seq analysis revealed 43 differentially expressed genes in the fetal liver, many related to lipid metabolism and immune response.
- Offspring from vitrified embryos showed compensatory growth in the final week of gestation.

## Abstract

Assisted reproduction technologies (ARTs) are usually safe; however, recent evidence suggests we need to look at potential risks in adulthood for better safety. ART techniques, like embryo vitrification, differ from natural conditions, which can potentially impact foetal development and life after birth. This study examined whether hepatic changes previously described after birth are already present in foetal livers at the end of gestation. We performed a comparison of phenotype and hepatic genome-wide mRNA expression via RNA sequencing between fresh and vitrified transferred rabbit embryos. As a result, we found phenotypic differences at 24 days of gestation, with vitrified embryos having lower foetal and liver weights and shorter body lengths. Moreover, offspring derived from vitrified embryos tended to be heavier, indicating a growth spurt in the last week of gestation. Additionally, only a total of 12 differentially expressed genes (DEGs) were detected among foetus groups, some of which are known for their role in lipid metabolism and the stress and immune response. Therefore, our results suggest that vitrification and embryo transfer manipulation induce an adaptive response in embryos and foetuses, which is apparent in the hepatic tissue at the end of the gestation period.

Assisted reproduction technologies (ARTs) are generally considered safe; however, emerging evidence highlights the need to evaluate potential risks in adulthood to improve safety further. ART procedures like rederivation of embryos by vitrification differ from natural conditions, causing significant disparities between in vitro and in vivo embryos, affecting foetal physiology and postnatal life. This study aims to investigate whether hepatic transcriptome and metabolome changes observed postnatally are already present in foetal livers at the end of gestation. This study compared fresh and vitrified rabbit embryos, finding differences between foetuses obtained by the transfer of fresh and vitrified embryos at 24 days of gestation. Rederived embryos had reduced foetal and liver weights and crown-rump length. However, the offspring of vitrified embryos tended to be born with higher weight, showing compensatory growth in the final week of gestation (59.2 vs. 49.8 g). RNA-Seq analysis revealed 43 differentially expressed genes (DEGs) in the foetal liver of vitrified embryos compared to the fresh group. Notably, downregulated genes included BRAT1, CYP4A7, CYP2B4, RPL23, RPL22L1, PPILAL1, A1BG, IFGGC1, LRRC57, DIPP2, UGT2B14, IRGM1, NUTF2, MPST, and PPP1R1B, while upregulated genes included ACOT8, ERICH3, UBXN2A, METTL9, ALDH3A2, DERPC-like, NR5A2-like, AP-1, COG8, INHBE, and PLA2G4C. Overall, a functional annotation of these DEGs indicated an involvement in lipid metabolism and the stress and inflammatory process or immune response. Thus, our results suggest that vitrification and embryo transfer manipulation induce an adaptive response that can be observed in the liver during the last week of gestation.