Supplementary Material for: Comparative Transcriptomic Analysis of Embryo Implantation in Mice and Rats

<b><i>Background/Aims:</i></b> Embryo implantation is an essential process for eutherian pregnancy, but this process varies across eutherians. The genomic mechanisms that led to the emergence and diversification of embryo implantation are largely unknown. <b><i>Methods:</i></b> In this study, we analyzed transcriptomic changes during embryo implantation in mice and rats by using RNA-seq. Bioinformatics and evolutionary analyses were performed to characterize implantation-associated genes in these two species. <b><i>Results:</i></b> We identified a total of 518 differentially expressed genes in mouse uterus during implantation, of which 253 genes were up-regulated and 265 genes were down-regulated at the implantation sites compared with the inter-implantation sites. In rat uterus, there were 374 differentially expressed genes, of which 284 genes were up-regulated and 90 genes were down-regulated. A cross-species comparison revealed that 92 up-regulated genes and 20 down-regulated genes were shared. The differences and similarities between mice and rats were investigated further at the gene ontology, pathway, network, and causal transcription factor levels. Additionally, we found that embryo implantation might have evolved through the recruitment of ancient genes into uterine expression. The evolutionary rates of the differentially expressed genes in mouse and rat uterus were significantly lower than those of the non-changed genes, indicating that implantation-related genes are evolutionary conserved due to high selection pressure. <b><i>Conclusion:</i></b> Our study provides insights into the molecular mechanisms involved in the evolution of embryo implantation.

**背景与目的：** 胚胎着床是真兽类妊娠的必需过程，但该过程在不同真兽类物种间存在显著差异。目前，驱动胚胎着床起源与多样化的基因组机制尚未完全明确。
**方法：** 本研究通过RNA测序（RNA-seq）分析了小鼠与大鼠胚胎着床阶段的转录组变化，并采用生物信息学与演化分析方法，对两个物种的着床相关基因进行特征解析。
**结果：** 本研究共在小鼠子宫着床过程中鉴定得到518个差异表达基因，其中着床位点相较于非着床位点，有253个基因上调、265个基因下调；在大鼠子宫中则鉴定得到374个差异表达基因，包括284个上调基因与90个下调基因。跨物种比较显示，二者共享92个上调基因与20个下调基因。随后本研究进一步从基因本体（Gene Ontology，GO）、信号通路、调控网络以及因果转录因子层面，深入探讨了小鼠与大鼠在胚胎着床过程中的异同。此外，本研究发现胚胎着床的演化可能通过招募古老基因参与子宫表达体系而实现。小鼠与大鼠子宫中差异表达基因的演化速率显著低于未发生表达变化的基因，提示着床相关基因因受到较强的选择压力而在演化上较为保守。
**结论：** 本研究为解析胚胎着床演化过程中的分子机制提供了重要见解。