To boldly go where no microRNAs have gone before: spaceflight impact on risk for small-for-gestational-age infants

Abstract

In the era of renewed space exploration, comprehending the effects of the space environment on human health, particularly for deep space missions, is crucial. While extensive research exists on the impacts of spaceflight, there is a gap regarding female reproductive risks. We hypothesize that space stressors could have enduring effects on female health, potentially increasing risks for future pregnancies upon return to Earth, particularly related to small-for-gestational-age (SGA) fetuses. To address this, we identify a shared microRNA (miRNA) signature between SGA and the space environment, conserved across humans and mice. These miRNAs target genes and pathways relevant to diseases and development. Employing a machine learning approach, we identify potential FDA-approved drugs to mitigate these risks, including estrogen and progesterone receptor antagonists, vitamin D receptor antagonists, and DNA polymerase inhibitors. This study underscores potential pregnancy-related health risks for female astronauts and proposes pharmaceutical interventions to counteract the impact of space travel on female health.

Fig. 1. The global response of miRNA expression comparing SGA patients to controls and spaceflight samples to controls and the core pathways being influenced.

Principal Component Analysis (PCA) plots demonstrate the clustering of the samples for each group. a Comparative miRNA expression analysis between Small for Gestational Age (SGA) patients (triangles) and control (healthy) patients (circles) at three distinct time-points: A ($12^{+0}-14^{+6}$ weeks) (blue), B ($15^{+0}-17^{+6}$ weeks) (yellow), and C ($18^{+0}-12^{+6}$ weeks) (green). Individual timepoint PCA plots can be found in Supplementary Fig. 1. For all SGA analysis there were a total of N = 29 patients included in the study with N = 16 normal birth outcomes and N = 13 SGA births for each time point. b miRNA expression PCA plots comparing SGA and control patients, irrespective of time. c miRNA expression clustering of OSD-336 mouse plasma under various treatments: 0.5 Gy Galactic Cosmic Radiation (GCR, red), 1 Gy Solar Particle Event (SPE, yellow), 5 Gy Gamma radiation (blue), and Sham (black); in both microgravity (circles) and normal gravity (triangles). Individual radiation-type PCA plots can be found in Supplementary Fig. 2. d miRNA expression analysis of OSD-55 human peripheral blood leukocytes (PBLs) under normal Earth gravity (circles) and microgravity (triangles). e miRNA expression analysis comparing SGA patients and control patients at three different time-points: A, B, and C, focusing exclusively on significant genes (p < 0.05). f Time-independent analysis of significant miRNAs differences (p < 0.05) between SGA and control patients. g Significant miRNA expression analysis (p < 0.05) of OSD-336 mouse plasma under different simulated radiation and microgravity conditions. h Significant miRNA expression analysis (p < 0.05) of OSD-55 human PBLs under microgravity and normal gravity conditions. Gene set analysis of miRNAs on common (i) Hallmark pathways and (j) MitoPathways in SGA and simulated spaceflight pathways compared to control, using the Molecular Signatures Database (MSigDB) Hallmark pathways to assess miRNA expressions impact on these pathways and functions. SGA human miRNA regulation is compared to control both time-independently and at different timepoints (left). Space radiation miRNAs with and without simulated microgravity are compared to Sham (right). The x-axis represents a coefficient term indicating pathway inhibition (negative value) or activation (positive value). The point size indicates the degree of significance, denoted by False Discovery Rate (FDR). Only significant values (FDR < 0.25) are displayed. The full list of significantly regulated pathways can be found in Supplementary Figs. 3 and 4.

Fig. 2. Common miRNAs in SGA and space environment.

a Heatmap depicting miRNAs significantly regulated in SGA vs. controls (adj. p < 0.05) present in simulated spaceflight datasets. b Heatmap displaying miRNAs consistently regulated in the same direction between SGA vs controls and PBL samples or mouse plasma. Analysis focuses on 13 common miRNAs from the plasma comparison. For the heatmaps, log₂(Fold-Change) is color-coded, with red shades indicating upregulated genes and blue shades indicating downregulated genes. Significance is denoted by * adj. p-value < 0.05 and # p-value < 0.05. c Correlation plot of log₂(Fold-Change) values for the 13 miRNAs, indicating positive correlation in red shades and negative correlation in blue shades. d Radar plot illustrating homology of pre-miRNAs (red) and mature miRNAs (black) in humans and mice for the 13 common miRNAs in SGA and space.

Fig. 3. Diseases linked to common SGA and space miRNAs.

Predicted diseases associated with the 13 miRNAs identified through miRNet. Curated into main groups, only diseases with FDR < 0.05 are displayed on the x-axis.

Fig. 4. Biological functions linked to common SGA and space miRNAs.

Predicted KEGG, Reactome, and miRNA functions associated with the 13 miRNAs identified through miRNet. Curated into main groups, only pathways with FDR < 0.05 are displayed on the x-axis.

Fig. 5. Gene targets of the SGA-associated spaceflight miRNA signature.

a Upset plot depicting the 45 gene targets shared by 10 or more of the 13 miRNAs. b Network illustrating the connections between the 13 miRNAs (gray nodes) and the 45 gene targets (red highlighted nodes). The gene target interactions with the miRNAs were determined by CluePedia. c Network displaying the global pathways associated with the 45 genes, predicted by CluePedia and visualized in Cytoscape. d Top 50 pathways regulated by the 45 genes, determined by GeneMANIA, with black nodes representing genes and gray diamonds indicating pathways. The red diamond nodes indicate the pathways that are related to female-specific functions and disorders. The full list of gene targets for the miRNAs can be found in Supplementary Data 2.

Fig. 6. Sex difference comparison of gene expression changes in the top 45 gene targets of the 13 specific miRNAs from the Inspiration 4 (I4) astronaut data derived from scRNA-sequence analysis of whole blood.

Bar plots depict the ratio of increased (red) or decreased (cyan) gene expression values between female and male I4 astronauts. a The values obtained 1 day after return to Earth (R1) versus the average of the 4 pre-flight time points. b The average values for 45 days (R45) and 82 days (R82) after return to Earth compared to the pre-flight average across the 4 time points.

Fig. 7. Sex-specific cumulative plots illustrate the impact on the top 45 gene targets of 13 specific miRNAs in Inspiration 4 (I4) astronaut data, derived from scRNA-sequence analysis of whole blood.

These cumulative plots focus on T cells (i.e., CD4 and CD8 T cells and others) in the I4 astronaut scRNA-seq data, comparing 1 day after return to Earth (R1) (red line), 45 days after return to Earth (R45) (orange line), and 82 days after return to Earth (R82) (gold line) to pre-flight levels. The x-axis represents log₂(fold-change) values for the comparisons, while the “no-site” line serves as a baseline for genes without targets to the 13 miRNAs. Various shades of gray in the no-site lines correspond to specific comparisons, as indicated in the figure legend. a Cumulative plots specifically for the female astronauts. b Cumulative plots specifically for the male astronauts.

Fig. 8. Basal miRNA expression in healthy tissues and predicted small molecule drugs.

a Heatmap presenting miRNA expression levels as z-scores across various healthy tissues, sourced from the miRNA Tissue Atlas. b Heatmap illustrating the mode of action and enriched drug indicators for predicted small molecule drugs targeting the miRNAs, as determined by sChemNET. Color intensity represents −log10(adj. p-value). c Upset plot revealing specific predicted small molecule drugs that target the miRNA signature associated with Small-for-Gestational-Age (SGA) in spaceflight. The drugs are in common with 5 or more of the 13 miRNAs. Notably, two small molecule drugs, triamcinolone and perfluorodecalin, are shared among all the identified miRNAs. All predicted small molecule drugs can be found in Supplementary Fig. 11 and Supplementary Data 3.

Fig. 9. Overall summary of the findings from this study.

In panel (I) we define the experimental data utilized for this study with: (1) the mice under simulated space environment experiments, (2) the simulated microgravity experiments done in vitro, and (3) the plasma data obtained from females during pregnancy with and without SGA outcome. For panel II) we define the databases (i.e. ImmPort and GeneLab) that the data was obtained from and the 13 miRNAs identified to be key for potential SGA risk during spaceflight. In panel III) we defined the key endpoints of the study which are (A) disease association, (B) development, (C) mitochondrial dysfunction, (D) immune dysfunction, (E) female reproductive function, and (F) predicted FDA-approved drugs as potential countermeasures. This figure was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.