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

The study addresses the pressing need to understand how the hostile space environment—characterized by ionizing radiation and microgravity—affects female reproductive health, particularly in the context of renewed deep-space missions (e.g., Artemis, eventual Mars missions). Historically, spaceflight health research has been male-biased, with limited knowledge about female-specific risks and reproductive outcomes. Spaceflight induces oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic and gene regulatory changes, telomere dynamics, and microbiome shifts; some may persist after long-duration missions. A key clinical question is whether prolonged exposure to space stressors elevates risks for adverse pregnancy outcomes post-flight, especially small-for-gestational-age (SGA) infants (birthweight <10th percentile). SGA affects ~16% of infants globally and is linked to long-term cardiometabolic and other health risks. The authors hypothesize that maternal exposure to space stressors increases SGA risk and that circulating microRNAs (miRNAs), known to be conserved across species and involved in placental function and pregnancy complications, can serve as biosignatures for risk assessment and targets for countermeasures. The study aims to identify an miRNA signature shared by SGA and spaceflight and to leverage machine learning to propose FDA-approved drugs that could modulate these miRNAs as preventive countermeasures for women exposed to space conditions prior to future pregnancies.

Prior work has established core biological disruptions from spaceflight, including oxidative stress, DNA damage, mitochondrial and epigenetic alterations, telomere changes, and microbiome shifts, with varying persistence depending on mission duration. Female-specific data remain sparse; the National Academies’ 2023–2032 decadal survey highlights gaps regarding female reproductive risks. Physiological responses to spaceflight may precipitate early menopause, reduced fertility, endometriosis, and altered estrogen/insulin levels affecting reproductive function. For SGA, multiple maternal, placental, fetal, paternal, and environmental factors are implicated (e.g., smoking, preeclampsia, inadequate gestational weight gain, twins, paternal height/SGA status, pollution). Circulating miRNAs are minimally invasive biomarkers that regulate proliferation, differentiation, inflammation, mitochondrial function, and apoptosis, and have been linked to complicated pregnancies, maternal smoking, and fetal growth restriction. Placental miRNA patterns in SGA share features with cancer biology (invasion, rapid growth). Literature also links mitochondrial dysfunction in spaceflight with downstream metabolic and immune changes; estrogen signaling is known to modulate mitochondrial function and oxidative stress, potentially relevant to sex differences post-spaceflight.

Study design integrated human clinical miRNA data on SGA with murine and in vitro simulated spaceflight miRNA datasets, plus astronaut datasets, followed by systems biology and machine learning for drug repurposing.

• Human SGA dataset (ImmPort SDY1871): N=29 women (N=16 normal, N=13 SGA), maternal plasma at gestational time points A (12+0–14+6 weeks), B (15+0–17+6), and C (18+0–12+6 weeks). Nanostring nCounter (~800 miRNAs). Differential expression via DESeq2 (adj. p<0.05), PCA/heatmap visualizations.
• Simulated spaceflight miRNA datasets (NASA OSDR/GeneLab):
  • OSD-336 (mouse plasma, female C57BL/6): hindlimb unloading (HU) for microgravity simulation; irradiation with 0.5 Gy simplified GCR (7-ion mix), 1 Gy simulated SPE (protons 50–150 MeV), or 5 Gy gamma; sham controls. miRNA-seq pipeline (ACGT101-miR, mapping to miRBase v22). Significance via multiple tests (p<0.01/0.05) with FDR control; PCA and clustering analyses.
  • OSD-55 (human peripheral blood lymphocytes in vitro): modeled microgravity (rotating wall vessel bioreactor) vs 1 g; miRNA microarray (GEO GSE57400), FDR<0.05.
  • Hemolysis considerations: identical handling across conditions; variable miRNAs filtered during preprocessing.
• Pathway analyses: miRNA gene set analysis using RBiomirGS on MSigDB Hallmark and MitoPathways (MitoCarta-based), significance FDR<0.25; lollipop plots.
• Identification of common miRNAs: Overlap of significantly regulated miRNAs (adj. p<0.05) between SGA (SDY1871) and spaceflight datasets (OSD-55, OSD-336), retaining those with consistent direction; resulted in 13 shared miRNAs. Conservation analysis using miRBase v22.1, BLASTN to assess human-mouse pre-miRNA and mature miRNA homology.
• Disease, function, and gene target prediction: miRNet for disease/function enrichment (FDR<0.05). Gene targets compiled from six databases (miRmap, miRWalk, miRNet, miRDB, miRTarBase, mirDIP); kept targets shared by ≥3 databases; focused on genes targeted by ≥10 of the 13 miRNAs, yielding 45 hub genes. Networks via Cytoscape ClueGO/CluePedia and GeneMANIA; IPA for canonical pathways and upstream regulators.
• Additional transcriptomic analyses:
  • Long-term effects after GCR in mice: OSD-719 (50 cGy GCR, male/female C57BL/6J), blood RNA-seq at day 14 post-irradiation. DESeq2 analysis; fGSEA on Hallmark, Reactome, and MitoPathway (FDR<0.05).
  • Meta-analysis of 817 mouse samples (27 GeneLab datasets, 10 tissues): processed via MTD pipeline, batch correction with ComBat-seq, DESeq2 with covariate adjustment; assessed expression/target impacts of the 13 miRNAs and top 45 targets across ISS-flown tissues.
  • NASA Twins Study (human miRNA-seq): assessed expression of 13-miRNA signature across cell types during 340-day flight vs ground twin.
  • Inspiration4 (i4) astronaut snRNA-seq (OSD-570): 2 male, 2 female; PBMC subpopulations pre-flight and R+1, R+45, R+82; DE using Wilcoxon signed-rank (adj. p<0.05); sex-stratified pathway fGSEA (FDR<0.001). Cumulative distribution analyses of mRNA log2FC for miRNA seed matches using TargetScan v8.0 and Kolmogorov–Smirnov tests.
• Drug prediction: sChemNET deep learning trained on SM2miR associations; screening unlabeled small molecules from the Drug Repurposing Hub; predictions retained at ≥98th percentile score (20 repeats averaged). Enrichment for modes of action and indications with Fisher’s exact test (BH-adjusted). Selected FDA-approved, commercially available drugs targeting multiple miRNAs, prioritizing those covering ≥5 and especially all 13 signature miRNAs. Basal miRNA expression examined using the miRNA Tissue Atlas.

• Shared miRNA signature: Identified 13 miRNAs consistently dysregulated in both SGA maternal plasma and simulated spaceflight conditions (mouse plasma and human PBLs). Twelve were upregulated; miR-146b-5p was downregulated. Two shared the same seed sequence (AGCACCA; miR-29b-3p, miR-29c-3p). A significant positive correlation (p<0.05) in log2 fold-changes was observed when comparing SGA vs control with spaceflight vs terrestrial conditions for these 13 miRNAs.
• Conservation: The 13 miRNAs showed high cross-species conservation; pre-miRNAs had ≥88% homology, and all mature miRNAs were 100% identical between human and mouse, supporting translational relevance.
• Pathways and systems-level effects:
  • Hallmark overlaps: Both SGA and spaceflight conditions exhibited upregulation of DNA repair, downregulation of cell cycle, and suppression of immune response-related factors. Spaceflight-specific effects included upregulated coagulation and downregulated pancreas beta-cell signatures; SGA-specific effects included downregulated adipogenesis.
  • Mitochondria: Shared increases in OXPHOS complex I, lipid metabolism, mitochondrial ribosome/translation functions. Spaceflight samples showed stronger miRNA-associated dysregulation (e.g., upregulation of complex V, coenzyme Q metabolism, mtDNA maintenance). SGA samples showed comparatively less mitochondrial pathway damage and unique upregulation of vitamin B1 metabolism and TIM22 carrier pathways.
• Disease/function associations of the 13 miRNAs: Enrichment implicated conditions spanning pregnancy/placental disorders (preeclampsia, bladder outlet obstruction), bone (osteoarthritis), cardiovascular (atherosclerosis, heart failure), diabetes, liver injury, pulmonary diseases, CNS disorders, infections, and numerous cancers (acknowledging literature bias toward oncologic annotations). Two miRNAs (miR-29b-3p, miR-29c-3p) are upregulated in SGA children and may relate to catch-up growth.
• Gene targets and hubs: Across the 13 miRNAs, 9,387 gene targets were compiled; 45 hub genes were targeted by ≥10 miRNAs. NSD1 was targeted by all 13 miRNAs (linked to Sotos syndrome). Pathway analyses of these 45 genes highlighted development, immune regulation, RNA metabolism, PI3K-AKT, and estrogen receptor (ESR)-mediated signaling (ESR signaling ranked third in IPA canonical pathways; p=6.78E-05). Predicted suppression included androgen signaling, critical for female reproductive maintenance, and multiple innate/adaptive immune pathways.
• Long-term and sex-specific signals:
  • Mouse blood RNA-seq at 14 days post 50 cGy GCR showed widespread dysregulation of pathways associated with the 13 miRNAs; immune and mitochondrial pathways were suppressed long-term exclusively in females.
  • Inspiration4 astronauts (snRNA-seq): At R+1, stronger inhibition (via target gene suppression) in female T cell subsets (CD4, CD8, other T) and CD16 monocytes vs males; in females, T-cell target suppression persisted through R+82 in cumulative distribution analyses, whereas males showed minimal or transient effects. RNA metabolism pathways were suppressed across cell types. These patterns align with reports linking reduced fetal regulatory T cells to SGA risk.
  • NASA Twins Study: 7/13 miRNAs were overexpressed in at least one cell type during spaceflight; several remained elevated post-flight, suggesting potential persistence.
• Tissue context in flown mice: Across ISS datasets, liver showed the highest upregulation of signature miRNAs among surveyed tissues, followed by heart; soleus had the least. Target inhibition patterns suggested kidney, spleen, thymus, and liver as most affected by the 45-gene set.
• Basal expression: In healthy tissues (miRNA Tissue Atlas), signature miRNAs generally showed low expression in bladder, bowel, uterus, and arteries, contrasting with their upregulation in SGA and spaceflight conditions.
• Drug predictions and countermeasures:
  • sChemNET predicted 128 small molecules across development/approval stages targeting components of the 13-miRNA signature; modes of action enriched for estrogen/progesterone receptor agonists, vitamin D receptor agonists, glucocorticoid receptor agonists, and DNA polymerase inhibitors.
  • Two FDA-approved, commercially available agents—triamcinolone (corticosteroid) and perfluorodecalin (oxygen-carrying perfluorocarbon)—were predicted to target all 13 miRNAs. Literature suggests triamcinolone can modulate ROS/mitochondrial function; perfluorodecalin can deliver oxygen and may influence mitochondrial ROS. Predictions are hypothesis-generating and require functional validation; use would be pre-pregnancy as astronauts are not pregnant during flight.
• Overall implication: Spaceflight may induce a conserved, female-relevant miRNA signature that suppresses immune regulation (notably T cells), perturbs mitochondrial and hormonal signaling (including estrogen), and could elevate SGA risk if dysregulation persists into conception/post-flight periods.

The study integrates human SGA plasma miRNA profiles with simulated spaceflight miRNA data, cross-species conservation evidence, and astronaut omics to infer that spaceflight-associated stressors can elicit a miRNA signature overlapping with that seen in SGA. This signature converges on key biological axes implicated in SGA pathophysiology—mitochondrial dysfunction, DNA repair/cell cycle imbalance, immune suppression (notably T-cell regulation), lipid metabolism, and hormone signaling (estrogen/androgen/PI3K-AKT). Female-specific susceptibility is supported by murine long-term post-irradiation results (female-only suppression of immune and mitochondrial pathways) and Inspiration4 sex-stratified findings (sustained inhibition of target genes in female T cells through R+82). The persistence of some miRNAs post-flight in the NASA Twins Study suggests that flight-induced miRNA elevations could endure, potentially overlapping with peri-conception windows after mission completion. While causality between the 13-miRNA signature and SGA is not proven, the convergence of pathways, disease associations, and sex-dependent immune effects offers a mechanistic framework linking spaceflight exposures to elevated SGA risk. The drug repurposing analysis provides a preliminary set of pharmacologic strategies—particularly agents modulating glucocorticoid, estrogen/progesterone, and vitamin D pathways, and oxygen delivery/ROS modulation—that could be tested as pre-pregnancy countermeasures for women with prior space exposure. These findings argue for proactive monitoring of miRNA panels in female astronauts and targeted interventional studies to mitigate potential reproductive risks upon return to Earth.

This work identifies a 13-miRNA signature conserved between humans and mice that is shared by SGA and spaceflight exposures, implicating mitochondrial, immune, cell cycle/DNA repair, metabolic, and sex-hormone signaling pathways. Hub analysis revealed 45 common gene targets (NSD1 targeted by all), and astronaut datasets (Inspiration4, NASA Twins) provided indirect human evidence of target suppression and miRNA overexpression during and after flight, with notable female-specific T-cell effects. A machine learning framework (sChemNET) predicted 128 candidate small molecules; two FDA-approved agents (triamcinolone and perfluorodecalin) were identified as covering all 13 miRNAs, suggesting feasible pharmacologic avenues for pre-pregnancy risk mitigation post-spaceflight. Future directions include: prospective, sex-stratified astronaut studies measuring circulating miRNA panels longitudinally pre-/post-flight; targeted validation of the 13-miRNA signature in female reproductive tissues and placental models under simulated space conditions; mechanistic studies to establish causality between the signature and SGA phenotypes; and preclinical/clinical testing of predicted countermeasures (including vitamin D and hormone receptor modulators) for their capacity to normalize the miRNA signature and restore downstream pathways.

• Causality not established: The 13-miRNA signature is associated with both SGA and spaceflight but has not been proven to cause SGA or birth defects.
• Data heterogeneity and surrogates: Spaceflight datasets include simulated microgravity and radiation models, murine plasma/tissues, in vitro human PBLs, and limited astronaut cohorts; direct data from female reproductive tissues post-spaceflight are lacking.
• Sample size and sex balance: Astronaut datasets (N=4 in Inspiration4; male-only Twins Study) limit generalizability and power to detect sex-specific effects.
• Temporal persistence: While some miRNA elevations/target suppressions persist post-flight, the duration and clinical relevance relative to conception timing are unknown.
• Confounding and preprocessing: Potential hemolysis in plasma miRNA studies and platform differences were addressed but cannot be fully excluded; FDR thresholds differed across analyses (e.g., pathway FDR<0.25 for miRNA GSA).
• Drug prediction scope: sChemNET predictions do not specify directionality (inhibition vs promotion) on miRNAs; functional validation in appropriate models is required. Some agents (e.g., perfluorodecalin as a PFAS) carry exposure considerations despite being deemed safe in specific formulations.
• Generalizability to astronauts: Most space data are from mice; translation to human female astronauts requires further confirmation.