This study utilised high-throughput transcriptomic sequencing of placental tissue and peripheral blood flow cytometry to explore the effects of exercise intervention on pregnancy outcomes in patients with GDM. Compared with the non-intervention group, the exercise intervention group exhibited significantly lower neonatal birth weight, suggesting that exercise may modulate fetal growth toward a more normative trajectory. Moreover, transcriptomic analysis of the placenta revealed significant differential expression of key genes associated with immune regulation, oxidative stress, and regulation of fetal birth weight. In contrast, peripheral blood flow cytometric analysis confirmed mitochondrial membrane potential collapse and elevated oxidative stress in the placenta. Based on these findings, we hypothesise that exercise may improve immune inflammation and oxidative stress in GDM patients by modulating the expression of critical genes such as CHRDL1, RORB, CCL21, and MTCO1P40, and the regulation of fetal birth weight by the regulatory gene (IGFBP1).
Mechanisms of exercise intervention in immune regulation
This study identified the potential role of exercise in mitigating GDM-related inflammation and reducing pathological complications by flow cytometry. We discovered that exercise intervention significantly downregulated the expression of CCL21. These results highlight the therapeutic potential of exercise in restoring oxidative homeostasis, and its incorporation into GDM management strategies may attenuate oxidative stress-related complications. CCL21 is a pivotal pro-inflammatory cytokine that plays a role in T-cell homing and immune activation. The observed reduction implies that exercise might enhance immune dysregulation in GDM by inhibiting inflammatory signalling pathways, such as the NF-\(\kappa\)B pathway. This result aligns with those reported by Liu et al.29. Existing studies have shown that physical activity, including exercise by parents, can modulate inflammatory states in placental tissues. Several studies have shown that in pregnant women with GDM, placental tissues exhibit elevated expression of pro-inflammatory cytokines such as interleukin-6 (IL-6), TNF-\(\alpha\), and chemokines including CCL2(C-C motif chemokine ligand 21) and TLR4, which are associated with increased macrophage infiltration and systemic immune dysregulation30. Interventions that reduce systemic inflammation may inhibit these inflammatory pathways. For instance, pharmacological treatments like metformin can alleviate excessive glucose intake-induced placental inflammation in GDM patients, which reduces pro-inflammatory cytokines and enhances placental defence mechanisms31. Exercise may exert similar effects. In a study involving pregnant women, maternal exercise has been shown to improve offspring metabolic health by increasing the expression of anti-inflammatory enzymes such as superoxide dismutase-3 (SOD3), thereby reducing oxidative pressure and inflammation in the placenta and promoting placental and fetal health32. These findings suggest that exercise exerts anti-inflammatory effects by modulating inflammatory pathways in the placenta and suppressing immune cell recruitment, which could be relevant for inflammatory regulation in GDM patients.
Mechanisms of exercise intervention in alleviating oxidative stress
Additionally, exercise intervention notably upregulated the expression of antioxidant-related genes, including GPX3 (glutathione peroxidase 3) and MTCO1P40. These gene expression alterations contribute to the restoration of redox balance in the placenta of GDM patients, thereby decreasing oxidative stress levels and ultimately providing protective effects on the health of both the mother and fetus. GPX3, a critical antioxidant enzyme, is essential for scavenging ROS and alleviating oxidative stress. A human cohort study reported that GPX3 expression is often downregulated in placental tissues from GDM patients29, further corroborating our findings that exercise can effectively ameliorate oxidative stress in GDM pregnancies. Upregulation of genes such as MTCO1P40 and GPX3 reduces ROS in the placenta, thereby lowering oxidative stress, preventing oxidative damage, and maintaining cellular homeostasis33. Exercise promotes the expression of antioxidant enzymes, such as glutathione peroxidase (GPX) and superoxide dismutase (SOD), to restore placental redox balance34. Adaptive changes induced by exercise enhance mitochondrial antioxidant activity, which is critical for regulating oxidative stress in placental tissue. By improving redox homeostasis, exercise reduces placental oxidative damage and improves fetal health outcomes35. Although GDM placental tissues exhibit higher levels of oxidative stress markers, such as lipid peroxidation, exercise-induced antioxidant mechanisms can suppress these markers by enhancing mitochondrial adaptability and antioxidant enzyme expression36. This protective effect may be achieved by regulating mitochondrial dynamics, such as fusion and fission, to improve mitochondrial function and reduce oxidative stress markers like lipid peroxidation37. Moreover, exercise intervention was observed to suppress the expression of pro-oxidant genes such as CHRDL1, thus reducing the production of reactive oxygen species (ROS) and preserving normal placental function. In our study, flow cytometry results revealed that lymphocytes from women with GDM significantly reduced mitochondrial membrane potential, with 32.8% of cells in the Q3 quadrant and elevated ROS levels. These findings indicate the presence of substantial oxidative stress and mitochondrial dysfunction under GDM conditions. These results are consistent with previous studies. Francisco et al. reported that the chronic inflammatory state in GDM patients can impair mitochondrial biogenesis, affecting fatty acid \(\beta\)-oxidation and further exacerbating oxidative stress9. Similarly, Zhou et al. also observed a significant decrease in mitochondrial DNA copy number in peripheral blood mononuclear cells from GDM patients, suggesting that mitochondrial damage may be a key mechanism underlying GDM-related metabolic disturbances38. Our study further reveals that exercise intervention can significantly reverse this mitochondrial dysfunction. Importantly, our study demonstrates that exercise intervention can significantly reverse this mitochondrial dysfunction. In the GDME group, the proportion of cells in the Q3 quadrant decreased to 13.6%, approaching the level observed in the NC group (13.4%). These findings suggest that exercise has therapeutic potential in restoring mitochondrial homeostasis in GDM. These findings suggest that exercise has potential therapeutic value in restoring mitochondrial homeostasis. This result is consistent with the findings of Dempsey et al., who showed in a cohort of non-pregnant individuals that regular exercise training can enhance mitochondrial biogenesis, improve mitochondrial function, and reduce oxidative stress levels39.
Interaction between immune regulation and oxidative stress
It is noteworthy that a positive feedback loop between inflammation and oxidative stress commonly exists in patients with GDM, which may exacerbate the progression of the disease40. Exercise intervention has effectively disrupted this vicious cycle, modulating the body’s immune and oxidative stress status. This study employed high-throughput RNA sequencing to systematically analyse gene expression profiles in placental tissues. GO functional enrichment analysis revealed that DEGs were predominantly enriched in biological processes such as immune regulation, redox reactions, and cellular metabolism. Furthermore, KEGG pathway analysis highlighted the significant involvement of the AGE-RAGE signalling, energy metabolism-related pathways, and the MAPK signalling pathway in the response to exercise intervention27,28. These findings suggest that exercise may improve placental function and optimise fetal and maternal health outcomes by modulating these key signalling pathways. Pro-inflammatory markers such as IL-6 and TNF-\(\alpha\) are elevated in GDM patients and are known to increase ROS production. Conversely, ROS generated during oxidative stress can activate transcription factors such as NF-\(\kappa\)B, further promoting the production of pro-inflammatory cytokines and establishing a self-sustaining cycle41. Acute exercise can temporarily increase oxidative stress caused by muscle metabolism, leading to ROS production. Elevated IL-6 and TNF-\(\alpha\) levels post-exercise confirm this response, which may exacerbate oxidative stress, particularly in metabolic disorders like GDM42. Although antioxidant defence mechanisms, such as glutathione peroxidase and SOD, are activated during exercise to counteract oxidative stress, their efficiency may be compromised in the pro-oxidative environment of GDM patients, potentially allowing inflammation and oxidative damage to persist43. Acute exercise may transiently exacerbate oxidative stress and inflammation in GDM patients due to temporary increases in ROS and inflammatory cytokines. This highlights the importance of adjusting exercise intensity and incorporating recovery strategies44. However, long-term, moderate-intensity exercise has been shown to enhance mitochondrial efficiency and increase antioxidant enzyme expression, thereby reducing oxidative stress and inflammation associated with GDM. Over time, such interventions can disrupt the positive feedback loop between inflammation and oxidative stress45. Therefore, in GDM patients, the interaction between inflammation and oxidative stress is particularly significant, especially post-exercise, when ROS and pro-inflammatory cytokines reinforce each other. While acute exercise may temporarily worsen this cycle, chronic, appropriately tailored exercise can mitigate these effects by enhancing antioxidant defences and improving mitochondrial function.
Exercise affects fetal birth weight through placental function
Macrosomia and neonatal hypoglycemia are adverse pregnancy outcomes associated with GDM. In our study, neonatal birth weight in the GDME group was significantly lower than that in the GDM group (\(3050.0 \pm 312.2\) g vs. \(3855.5 \pm 336.0\) g), indicating that exercise intervention effectively controlled fetal weight gain and reduced the risk of macrosomia associated with GDM. Exercise is an effective methodology for preventing and managing GDM and has been shown to reduce the risk of these complications by modulating placental function46,47. Through placental transcriptomic analysis, several growth factors showed significant expression changes between the case and control groups, suggesting their potential involvement in regulating fetal weight by exercise. CHRDL1, which modulates the activity of bone morphogenetic proteins (BMPs), plays a key role in placental vascular development and fetal growth regulation48. Prolactin (PRL), a hormone with multiple functions in placental development and maternal metabolism49, was downregulated in the GDME group. Although PRL is associated with insulin resistance during pregnancy, its reduction in the exercise group may reflect improved metabolic balance and decreased inflammatory burden. Additionally, IGFBP1, a gene involved in fetal growth regulation, was downregulated, highlighting the role of exercise in fine-tuning growth factor signalling pathways50. IGFBP-1 has been identified as a crucial factor influenced by exercise. IGFBP-1 is integral to glucose metabolism and fetal development, and its downregulation through physical activity may explain how exercise mitigates GDM severity. Prior research has demonstrated that aerobic and resistance exercise in non-pregnant individuals enhances the PI3K/Akt signalling pathway, leading to increased levels of IGF-1 and IGF-1 receptor46. Moreover, prolonged endurance exercise has elevated circulating IGFBP-1 concentrations in both human and rodent models, suggesting a conserved mechanism across species51. Notably, IGFBP-1 infusion can neutralise the insulin-like effects of IGF-1, thereby raising fasting blood glucose levels52. This indicates that IGFBP-1 may be critical in regulating blood glucose during and after exercise53. Based on our findings, it can be inferred that exercise may modulate the expression of fetal growth-related genes, such as IGFBP1 and PRL, thereby influencing fetal birth weight and maternal adaptation to pregnancy, particularly under conditions of metabolic stress. The altered maternal environment observed in our study may impact key growth factor signalling pathways that directly regulate placental nutrient transport and fetal development, ultimately optimising the intrauterine growth environment and improving neonatal birth outcomes. These molecular alterations align with the observed clinical improvements in neonatal outcomes and maternal immune and metabolic profiles. This mechanism suggests a potential molecular pathway through which exercise benefits fetal birth weight by modulating placental function. Collectively, these findings provide a robust theoretical basis and molecular evidence supporting the implementation of exercise as an effective, non-pharmacological intervention strategy for pregnant women with GDM.
Impact of gestational age at intervention initiation
In our study, the diagnosis of GDM was performed at 24–28 weeks of gestation, consistent with current clinical screening recommendations54. Consequently, our intervention began shortly after diagnosis, typically in the late second trimester. The timing of intervention initiation is important because both maternal metabolic adaptation and placental development are already well underway by this stage, which may influence the magnitude of metabolic and molecular responses to exercise. Previous exercise interventions in GDM vary in start time, though most studies initiate protocols within a similar gestational window (24–28 weeks) following standard diagnostic procedures54. A systematic review by Davenport et al. found that most randomised controlled trials on exercise in GDM commenced interventions between 24 and 30 weeks of gestation55. Huang et al. reported that moderate-intensity aerobic and resistance training in pregnant women with GDM, initiated at 25–26 weeks, improved maternal glycemic control and reduced insulin requirements, while earlier-start interventions have shown additional benefits on birth weight and maternal weight gain55. Given that placental development and immune regulation are dynamic processes, the timing of intervention may significantly influence the molecular changes observed in the placenta transcriptome54. In our study, the observed upregulation of antioxidant genes and downregulation of pro-inflammatory markers may be partly attributed to the timely initiation of exercise during a critical window of placental development.
Limitations and future directions
We ensured that all participants adhered to the intervention protocol, with over 90% attendance for the required sessions, confirming the reliability of participant engagement in the study. This helped to mitigate potential confounding factors related to participant non-compliance. We also focused on the reliability of the experimental assays, particularly RNA-Seq. To ensure high-quality results, we implemented rigorous quality control measures during RNA sequencing, including obtaining over 42 million clean reads with Q30 scores above 95%. We also included detailed checks on sequencing depth and gene mapping rates, which validated the technical reliability of our data. Despite the meaningful results obtained, this study has several limitations. While transcriptomics and flow cytometry provided insights into the effects of exercise on placental immune and oxidative stress regulation, the specific functions and pathways of the identified genes require further investigation. Combining proteomics and single-cell transcriptomics in future studies could provide a more detailed understanding of the maternal-fetal interface. A limitation of this pilot study is the lack of baseline physical activity data, which could influence individual responses to the intervention. The small sample size and potential imbalance between groups may limit the generalizability of the findings. Future studies should involve larger cohorts and multi-centre collaborations to enhance external validity. This study focused on specific types and intensities of exercise but did not evaluate the differential effects of various exercise modalities, frequencies, and intensities. Future research should assess a broader range of exercise protocols to optimise intervention strategies for GDM patients. Recent studies have highlighted the influence of labour duration and oxytocin use on placental function and oxidative stress56. While our current study did not account for these factors, future research should consider their potential impact to refine the interpretation of molecular changes in placental tissues following maternal exercise interventions. Although this study indicated that the neonatal birth weight in the exercise intervention group was significantly lower than in the non-intervention group, changes in birth weight alone are insufficient to conclude an improvement in overall neonatal health outcomes. Clinically meaningful changes are typically defined based on deviations from the expected growth percentiles for gestational age (e.g., above the 90th percentile) or by extreme absolute values (e.g., below 2500 grams or above 4000 grams). Therefore, while the observed reduction in birth weight suggests that exercise may modulate fetal growth, further analysis is required to determine whether this change is beneficial. Clinically significant and to evaluate its actual impact on neonatal health outcomes. The current study primarily examined short-term effects during pregnancy. Longitudinal studies are needed to assess the long-term health benefits of exercise interventions for both mothers and offspring, providing more comprehensive data for clinical application.