IMPACT OF NEAR-CONTINUOUS LOW DOSE-RATE NEUTRON IRRADIATION ON PREGNANCY OUTCOMES IN MICE

The effects of galactic cosmic radiation on reproductive physiology remain largely unknown. We determined the impact of near-continuous low-dose-rate Californium-252 neutron irradiation (1 mGy/day) as a space-relevant analog on litter size and number of resorptions at embryonic day (E) 12.5 (n=19 radiated dams, n=20 controls) and litter size, number of resorptions, fetal growth, and placental signaling and transcriptome (RNA sequencing) at E18.5 (n=21 radiated dams, n=20 controls) in pregnant mice. A significantly increased early resorption rate and decreased placental weight was observed in irradiated mice. There were no statistically significant differences in litter size, fetal weight, length, or malformation rate between the groups. Near-continuous radiation had no significant effects on mechanistic target of rapamycin (mTOR), endoplasmic reticulum stress or inflammatory signaling, rate of double-stranded DNA breaks, and had minimal effects on gene expression in the placenta. These data suggest that near-continuous, low-level galactic cosmic radiation has a limited impact on pregnancy outcomes. Overall design: RNA sequencing was performed on one randomly selected placenta from each of 15 irradiated E18.5 litters and 15 control litters. The RNA from each of the 30 individual placentas was isolated using a Qiagen RNeasy mini kit following the manufacturer's instructions. Next, a reverse transcriptase reaction was performed using the Qiagen Quantitect RT kit according to manufacturer's instructions (Qiagen, Hilden, Germany). The resulting cDNA was then subjected to quantitative PCR. RNA purity, quantity and integrity was determined with NanoDrop (ThermoFisher Scientific, Waltham, MA) and TapeStation 4200 (Agilent, Santa Clara, CA) analysis prior to RNA-seq library preparation. The Universal Plus mRNA-Seq library preparation kit with NuQuant was used (Tecan, Männedorf, Switzerland) with an input of 200ng of total RNA to generate RNA-Seq libraries. Paired-end sequencing reads of 150bp were generated on NovaSeq 6000 (Illumina, San Diego, CA) sequencer and de-multiplexed using bcl2fastq. Illumina adapters and the first 12 base pairs of each read were trimmed using BBDuk (BBMAP; www.sourceforge.net/projects/bbmap) and reads <50bp post-trimming were discarded. Reads were aligned and quantified using STAR (2.6.0a)54 against the Ensembl mouse transcriptome (mg38.p6 genome release 96). Ensembl IDs were mapped to gene names and counts of genes with multiple IDs were aggregated. Low expression genes were removed if mean raw count <1 or mean counts per million (CPM) <1 for the dataset. Reads were normalized to CPM using the edgeR R package (Bioconductor; Boston, MA)55. Differential expression was calculated using the voom function in the limma R package with RNA integrity numbers as a covariate56. Gene set enrichment analysis (GSEA) was performed using the full ranked list of genes by fold change for the indicated comparison and the fgsea R package57 using Hallmark, Gene Ontology Biological Processes, and KEGG gene sets from the Molecular Signatures Database58-59. A heatmap was generated with the ComplexHeatmap R package58 following z-score transformation. Additional plots were generated using the ggplot2 R package61.