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. Overall design: 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.