RNA-Sequencing based analysis of bovine endometrium during the maternal recognition of pregnancy. RNA-Sequencing based analysis of bovine endometrium during the maternal recognition of pregnancy

Purpose: Perform RNA-seq study on bovine endometrial tissues during the peri-implantation period (Day16-17 of gestation) to reveal important genes and biological pathways required for conceptus growth and development Methods:RNA sequencing was done using Illumina NextSeq500. Single-end reads in the FASTQ format were explored using FastQC, low-quality reads were trimmed from both 3’ and 5’ ends until a base pair of Phred quality score of 30 (99.9% accurate) or greater was not found, reads having a mean quality score less than 30 and length below 30 nucleotides were filtered out. Cleaned reads were aligned against the bovine reference genome (Bos_taurus.ARS-UCD1.2) using HiSAT2. The resulting SAM files were sorted, converted to BAM files using SAMtools. Read counts mapped to bovine gene models were generated using htseq-count script from HTSeq package. Bioconductor DESeq2 was used to get the differentially expressed genes among pregnant vs. non-pregnant (P vs. NP), pregnant vs. cyclic (P vs. C), and non-pregnant vs. cyclic (NP vs. C) groups. Conclusions: The study demonstrated that the presence of conceptus at Day 15-17 of gestation affects the endometrial gene expression related to endometrial remodeling, immune response, nutrients and ion transporters, and relevant signaling pathways in the caruncular region of bovine endometrium during the maternal recognition of pregnancy. Overall design: There are 4 Cyclic, 4 Pregnant, 4 Non-Pregnant samples. Differential gene expression between each group (pregnant vs. non-pregnant, pregnant vs. cyclic, and non-pregnant vs. cyclic) are compared.

研究目的：针对妊娠植入窗口期（妊娠第16-17天）的牛子宫内膜组织开展RNA测序（RNA-seq）研究，以揭示孕体生长发育所需的关键基因与生物学通路。
实验方法：采用Illumina NextSeq500平台开展RNA测序。首先使用FastQC对FASTQ格式的单端测序读段进行质量评估，随后对3'端与5'端的低质量读段进行修剪，直至未检出Phred质量评分低于30（准确率99.9%）的碱基对；同时过滤掉平均质量评分小于30、序列长度不足30个核苷酸的读段。将质控后的清洁读段与牛参考基因组（Bos_taurus.ARS-UCD1.2）通过HiSAT2进行序列比对。将生成的SAM格式文件进行排序，并借助SAMtools工具转换为BAM格式文件。通过HTSeq软件包中的htseq-count脚本，计算比对至牛基因模型的读段计数。使用Bioconductor的DESeq2工具，分别比较妊娠组与非妊娠组（Pregnant vs Non-Pregnant，P vs NP）、妊娠组与发情周期组（Pregnant vs Cyclic，P vs C）以及非妊娠组与发情周期组（Non-Pregnant vs Cyclic，NP vs C）之间的差异表达基因（differentially expressed genes）。
研究结论：本研究证实，在妊娠第15-17天，孕体的存在会影响牛子宫内膜子宫阜区域在妊娠母体识别过程中，与子宫内膜重塑、免疫应答、营养转运及离子转运相关的基因表达，以及对应的信号通路。
实验设计：本研究共设置4个发情周期组样本、4个妊娠组样本与4个非妊娠组样本，对各组间（妊娠组vs非妊娠组、妊娠组vs发情周期组、非妊娠组vs发情周期组）的差异基因表达情况进行比较分析。