Exposure to real or simulated microgravity induces skeletal muscle wasting, osteoporosis, and anemia1–3. Alterations in iron metabolism could participate to these imbalances due to the central role of iron in energy metabolism, cell respiration, oxygen transport, and muscle function4. Previous researches have demonstrated that young males exposed to real or simulated microgravity rapidly exhibit iron redistribution, characterized by increased serum iron availability (i.e. serum iron and transferrin saturation levels) and spleen iron sequestration5,6. This redistribution is accompanied in human and rodents males by an increase of both hepcidin mRNA levels in the liver and peptide levels in plasma5,7–9. Hepcidin, which limits the membrane expression and activity of ferroportin – the sole known Fe2+ cell exporter, particularly from spleen macrophages and enterocytes10 – could potentially contribute to these early iron redistribution observed in male astronauts and bedridden patients.
Despite the increasing number of female astronauts and known sex-related differences in basal iron metabolism regulation11,12, all current data have been collected only from males. To address this gap, we recently participated to the AGBRESA clinical study involving 16 males and 7 females exposed to 60 days of head-down tilt bed rest13. In this sample of participants, our preliminary findings suggest that females appear to exhibit increased iron availability in plasma after 6 days of bed rest. However, unlike males, they did not exhibit a concomitant increase in serum hepcidin levels13. Furthermore, after 60 days, unlike males, females no longer showed increased transferrin saturation, suggesting sex-differences in iron metabolism regulation over the long term13. Such plasma iron excess could progressively lead to oxidative stress in some organs, and raise the possibility of exacerbating organ damage in astronauts, particularly osteoporosis, muscle atrophy, cancer, or liver injuries. In addition, a better understanding of the effects of microgravity on iron metabolism could also improve, on Earth, the follow-up of bedridden patients, who also experience extreme physical inactivity.
To further substantiate these short-term results, we investigated the regulation of iron metabolism in both sexes exposed to simulated microgravity. The first campaign included eighteen females (Vivaldi) and the second nineteen males (Vivaldi 2) exposed to 5 days of dry immersion, a ground-based model mimicking microgravity14. Both well controlled campaigns were conducted in MEDES (Toulouse) and similarly set up (i.e., diet, biological sampling, and management).
Serum ferritin, an iron-storage protein, is widely recognized as a marker of body iron stores in the absence of inflammation. Serum transferrin, the iron transporter, becomes increasingly saturated as circulating iron levels rise10. The transferrin saturation coefficient is thus calculated as the ratio of iron to transferrin and serves as a marker of plasma iron availability.
In basal condition, before exposure to dry immersion (i.e., BDC-1), serum iron and transferrin saturation levels, which characterize plasma iron availability, did not differ between both sexes (Figure 1A and 1B). However, females exhibit lower serum ferritin levels (p<0.001; Figure 1E) and higher serum transferrin levels compared to males (p<0.001; Figure 1B), confirming sex-specific differences, that are generally associated to higher iron storage in males12. Taken together, these findings suggest that in basal state females 1) as males, have same iron availability levels in plasma and 2) unlike to males, exhibit lower body iron stores. In absence of an iron active excretion mechanism in humans, this could be attributed to menstrual bleeding, which constitutes an important source of iron release in premenopausal females15.
At the 5th day of dry immersion, both sexes exhibit increased serum iron levels (+57% for males and +40% for females, Time: p<0.001, Figure 1A), and an increase of transferrin levels, which is more pronounced in males compared to females (+8.5% vs. 3.2%, compared to basal situation, Time: p<0.001, interaction: p=0.037, Figure 1B). Concomitantly, in males and females, the increase in transferrin saturation levels after dry immersion (+12.2 and +8.0%, Time: p<0.001, Figure 1C) clearly indicates enhanced plasma iron availability. These results characterize a similar modulation of iron metabolism in both sexes exposed to short-term simulated microgravity, confirming our data previously collected in males and more recently in a smaller group of females5,13. Spaceflight and simulated microgravity are well known to induce anemia, characterized by a decrease of hemoglobin mass in both sexes13,16–18. In the present study, we also found a substantial decrease in hemoglobin mass in females after 5 days of simulated microgravity (-8.0%, Table 1), whereas twice lesser decrease in males (-3.9%, Table 1). Given that 65-70% of total body iron being bound within hemoglobin of red blood cells, this earlier reduction of total hemoglobin mass in females, already observed during bed rest experiments13, suggests that males and females would not be necessary exposed to the same magnitude of iron metabolism alteration or misdistribution during the first days of simulated microgravity. Concomitantly with the increase of plasma iron availability, both sexes present an increase in serum ferritin levels (Time: p<0.001 compared to baseline, Figure 1E) that is greater in males compared to females after 5 days of dry immersion (+33.9±27.0 vs. +13.6±15.2 µg/l, Interaction: p=0.008, respectively). As an increase of serum ferritin levels also occurs during inflammation19, it must be underlined that serum hsCRP levels were not increased in females, whereas we observed a slight but significant increase in males, though remaining in normal ranges (Table 1). Moreover, serum hsCRP levels was weakly, but significantly correlated with serum ferritin levels in males (r=0.4 p=0.013, Figure 2A). These findings suggest that during the first days of exposure to simulated microgravity, the increase of serum ferritin levels in males could be partly related to mild subclinical inflammation in addition to body iron storage increase and/or cytolysis releasing ferritin in serum.
Hepcidin is mainly synthetized by the liver in response to elevated hepatic iron stores and inflammation10. Here, both sexes exhibit an increase in serum hepcidin levels (Time: p<0.001, Figure 1D), that is greater in males than females after 5 days of dry immersion (+5.9±3.5 vs. +2.0±3.1 nmol/L, interaction: p=0.001, Figure 1D). Importantly, this response was however heterogeneous in females with a “responder” group (i.e., increase of serum hepcidin levels) and a “non-responder” group (i.e., non-increase of serum hepcidin levels), unlike males who uniformly exhibit increased serum hepcidin levels. Surprisingly, an increase of serum hepcidin levels generally induces a decrease of iron transferrin saturation levels, which is not observed here both in males and females, questioning the source(s) of iron released into the plasma. As hepcidin, CRP is produced by the liver during inflammatory state throughout the IL-6/STAT3 pathway20. Although hsCRP only slightly increases in males under immersion, we cannot exclude regarding the strong correlation between hepcidin and hsCRP (r=0.67, p<0.001, Figure 2A), that the increase of inflammatory state in the liver could promote hepcidin mRNA transcription, leading to the serum hepcidin increase. Concomitantly, we also found a strong correlation between serum hepcidin and ferritin levels in both males and females (r=0.70, p<0.001, r=0.71, p<0.001, respectively, Figure 2A and 2B). Importantly, we observed that participants with baseline serum ferritin values lower than 30 µg/L (all females) exhibited no increase in serum hepcidin levels, whereas the magnitude of the increase becomes more significant when baseline serum ferritin values exceed 100 µg/L (Figure 2D). Despite the fact that hepcidin synthesis is reported to be downregulated by estrogen levels21, we did not report statistically significant relationship between serum estrogen levels and serum hepcidin levels in females (r=-0.01; p=0.53; Figure 2A). Menstrual losses in most premenopausal female are also an additional potential source of iron loss22. During the 5 days of dry immersion, no females experienced menstrual bleeding, and we did not identify statistically significant relation between menstrual cycle (detailed in Robin et al.23) and “responder” / “non-responder” groups. These results support the hypothesis that unidentified factors would be responsible for the sex differences in the amplitude of iron metabolism responses to simulated microgravity.
In conclusion, our data support that short-term exposure to simulated microgravity induces an increase of iron availability, serum ferritin and hepcidin levels in both sexes suggesting an alteration of iron metabolism. In females, we identified a non-responsive subgroup that did not exhibit elevated serum ferritin and hepcidin levels. These findings suggest sex-related differences likely related to basal iron status and metabolism. It becomes imperative to elucidate the origin of this iron misdistribution, as the accumulation of iron in tissues, combined with cosmic radiation exposure, could present significant long-term hazards to astronauts of both sexes. A comprehensive understanding of these mechanisms in males and females will facilitate the development of dietary strategies and menstrual management protocols aboard the ISS and Gateway, to mitigate excessive body iron accumulation.