Extrauterine support of preterm lambs achieves similar transcriptomic profiling to late pre-term lamb brains

Our group has developed an extra-uterine environment for newborn development (EXTEND) using an ovine model, that aims to mimic the womb to improve short and long-term health outcomes associated with prematurity. This study’s objective was to determine the histologic and transcriptomic consequences of EXTEND on the brain. Histology and RNA-sequencing was conducted on brain tissue from three cohorts of lambs: control pre-term (106-107 days), control late pre-term (127 days), and experimental lambs who were born pre-term and supported on EXTEND until late pre-term age (125-128 days). Bioinformatic analysis determined differential gene expression among the three cohorts and across four different brain tissue sections: basal ganglia, cerebellum, hippocampus, and motor cortex. There were no clinically relevant histological differences between the control late pre-term and EXTEND ovine brain tissues. RNA-sequencing demonstrated that there was greater differential gene expression between the control pre-term lambs and EXTEND lambs than between the control late pre-term lambs and EXTEND lambs. Our study demonstrates that the use of EXTEND to support pre-term lambs until they reach late pre-term gestational age results in brain tissue gene expression that more closely resembles that of the lambs who reached late pre-term gestation within their maternal sheep’s womb than that of the lambs who were born prematurely. Our study investigated four brain sections per lamb from nine individual lambs. Two lambs were delivered pre-term and underwent immediate necropsy (Pre-term control); two lambs were delivered at 127 days and underwent immediate necropsy (Late Pre-term control); five lambs were delivered pre-term, underwent immediate cannulation and were supported by the EXTEND model (Experimental) and underwent necropsy after reaching a gestational age close to 127 days (gestational age similar to the Late Pre-term control cohort). The four brain sections investigated through both histology and RNA-sequencing were: hippocampus, motor cortex, basal ganglia, and cerebellum. Flash frozen tissue was procured from ovine brain at the time of necropsy and sent to GeneWiz for RNA extraction and RNA-sequencing. Total RNA was extracted from frozen cell pellet and fresh frozen tissue samples using Qiagen RNAeasy Plus Universal mini kit following Manufacturer’s instructions (Qiagen, Hilden, Germany). GeneWiz conducted RNA-seq library preparation as follows: A) mRNA enrichment, mRNA fragmentation, and random priming; B) first and second strand cDNA synthesis; C) end repair, 5’ phosphorylation, and dA-Tailing; D) adaptor ligation, PCR enrichment, and sequencing. Paired end sequences were assessed for quality using SeqKit. Adapter and poly G/X tails were removed using fastp. Post processing quality assessment is then repeated on the paired-end reads using SeqKit. Sequences were aligned to the ovis aries genome, version 107, using STAR. Reference files are available from Ensembl (https://ftp.ensembl.org/pub/release-107/fasta/ovis_aries/). Gene expression was quantified against the gene transfer format (GTF), v107 annotation file using STAR’s ‘--quantMode GeneCounts’ parameter. The reference GTF annotation file is also available from Ensembl (https://ftp.ensembl.org/pub/release-107/gtf/ovis_aries/). Sample-level quantifications from the resulting 'ReadsPerGene.out.tab’ file, column 2 for inward unstranded (IU) reads, were combined across all samples to generate the gene count matrix.

本课题组利用绵羊模型开发了新生动物宫外发育环境（Extra-uterine environment for newborn development, 简称EXTEND）系统，旨在模拟子宫内环境，以改善早产相关的短期与长期健康结局。本研究的目标是明确EXTEND对大脑产生的组织学与转录组学影响。
研究人员对三个羔羊队列的脑组织开展了组织学检测与RNA测序（RNA-sequencing）：早产对照组（孕106-107天）、晚期早产对照组（孕127天），以及实验组羔羊——早产出生后通过EXTEND系统支持至晚期早产胎龄（125-128天）的个体。生物信息学分析鉴定了三个队列间以及四种不同脑组织区域（基底神经节、小脑、海马体、运动皮层）的差异基因表达。
晚期早产对照组与EXTEND处理的绵羊脑组织之间未观察到临床相关的组织学差异。RNA测序结果显示，早产对照组羔羊与EXTEND处理组羔羊间的差异基因表达量，高于晚期早产对照组与EXTEND处理组羔羊间的差异水平。本研究证实，通过EXTEND系统支持早产羔羊至晚期早产胎龄，其脑组织的基因表达谱更接近在母羊子宫内发育至晚期早产胎龄的羔羊，而非早产出生未获支持的个体。
本研究共纳入9只个体羔羊，每只羔羊取4个脑组织区域进行分析：2只早产娩出后立即实施剖检（早产对照组）；2只于孕127天娩出后立即剖检（晚期早产对照组）；5只早产娩出后立即进行插管，并通过EXTEND系统支持，直至胎龄接近127天时实施剖检（实验组，胎龄与晚期早产对照组队列一致）。本次研究通过组织学与RNA测序分析的4个脑组织区域为：海马体、运动皮层、基底神经节与小脑。
剖检时采集绵羊（Ovis aries）脑组织并快速冷冻，送至GeneWiz进行RNA提取与RNA测序。按照制造商操作指南，使用Qiagen RNAeasy Plus Universal迷你试剂盒（Qiagen，德国希尔德）从冷冻细胞沉淀与新鲜冷冻组织样本中提取总RNA。
GeneWiz的RNA测序文库构建流程如下：A）mRNA富集、mRNA片段化与随机引物反转录；B）第一链与第二链cDNA合成；C）末端修复、5’端磷酸化与dA尾加尾；D）接头连接、PCR富集与测序。
使用SeqKit对双端测序序列进行质量评估；通过fastp去除接头与poly G/X尾序列；随后再次使用SeqKit对处理后的双端读取序列进行质量评估。
使用STAR将测序序列比对至绵羊（Ovis aries）参考基因组版本107；参考文件可从Ensembl获取（https://ftp.ensembl.org/pub/release-107/fasta/ovis_aries/）。
使用STAR的"--quantMode GeneCounts"参数，基于基因转移格式（GTF）v107注释文件对基因表达量进行定量。该GTF参考注释文件同样可从Ensembl获取（https://ftp.ensembl.org/pub/release-107/gtf/ovis_aries/）。
从生成的"ReadsPerGene.out.tab"文件中提取第2列对应正向非链（inward unstranded, IU）读取的样本水平定量结果，合并所有样本以构建基因计数矩阵。