United States Marine Corps Officer Candidates School (OCS) is an intensive and demanding 10-week military training course wherein male and female candidates execute the same rigorous training events in pursuit of the same highly coveted goal: to be commissioned as United States Marine Corps officers. Barriers to successful completion of OCS include musculoskeletal injury (MSKI), which has been identified as one of the predominant threats to military readiness. The burden of MSKI is particularly high during initial military training wherein recruits undergo high volume, prolonged, intensive and often unaccustomed mechanical loading (O’Leary, Wardle, Rawcliffe, et al., 2020). Known risk factors that affect MSKI include military-specific and repetitive weight-bearing activities such as load carriage (Hughes et al., 2019), military drill (Rice et al., 2018), and female sex (O’Leary, Wardle, Rawcliffe, et al., 2020). Bone stress injuries, a specific type of MSKI, represent a critical and pervasive military health issue, afflicting upward of 7% of men and 20% of women (Lovalekar et al., 2023) during initial military training, rendering bone a critical target tissue for MSKI reduction. Additionally, musculoskeletal outcomes may relate to physical performance which is a priority for military populations (Bird et al., 2022). Therefore, there is a need to optimize MSK health to limit injury, improve performance, and reduce attrition, particularly in women and during early career training.
One avenue of inquiry for understanding and mitigating MSKI risk relates to micronutrient status, which represents a modifiable factor for optimizing military readiness. Iron and vitamin D, in particular, are reciprocally related (Azizi-Soleiman et al., 2016) and have been separately investigated for effects on MSK health (Hennigar et al., 2016). Vitamin D and iron deficiencies are highly prevalent globally and are commonly seen in military personnel, with greater deficits observed in women (Andersen et al., 2010). Prior longitudinal observational studies have consistently demonstrated diminished serum 25-hydroxy-vitamin D (25[OH]D), the major circulating form of vitamin D, resulting from military training (Andersen et al., 2010). However, the definitive mechanism contributing to this diminished status remains unclear.
Prior work has also shown that diminished iron status resulting from military training is independent of energy deficits (O’Leary, Wardle, & Greeves, 2020) and further, that increased dietary intake during training is insufficient in circumventing this decline in men and women alike (Yanovich et al., 2015). While supplemental iron during military training has been shown to be efficacious in women (McClung et al., 2009), little data exists in men. Likewise, vitamin D and calcium supplementation imparts increases in bone parameters, including bone compressive strength in both men and women (Gaffney-Stomberg et al., 2014) and lowers the incidence of stress fracture in women (Lappe et al., 2008) without exacerbating iron status decline (Hennigar et al., 2016). Furthermore, even marginal iron deficiency has been shown to impair physical performance in women (Brownlie et al., 2002); however, results remain limited in young adult men and previous work often only included limited assessments of iron status. However, despite prior observations of vitamin D and iron deficiencies occurring simultaneously, little data exists examining both deficiencies concurrently in men and women undergoing the same intense training.
Therefore, the primary purpose of this study was to characterize hematological status in male and female Marine OCS candidates and determine associations between micronutrient deficiency (i.e., iron status and 25-hydroxy-vitamin D) and operationally relevant outcomes (i.e., skeletal health, MSKI) at baseline and post-10 weeks of arduous military training. We hypothesized that candidates would suffer a combined decline in hematological status over the course of 10 weeks with more pronounced changes occurring in female candidates than in male. Furthermore, those candidates with compromised hematological status would also demonstrate attenuated bone quality and greater incidence of MSKI.
Materials and Methods
Experimental Approach to the Problem
The proposed study was an ancillary component to Institutional Review Board-approved protocol involving developing a Warfighter mobility signature and predictive algorithm for MSKI risk during Marine Corps Officer Candidate School, Grant Number: N00014-21-1-2725 (Bird et al., 2022; Koltun et al., 2022; Koltun, Bird, Forse, et al., 2023; Koltun, Bird, Lovalekar, et al., 2023; Lovalekar et al., 2023). This study was approved by the University of Pittsburgh Institutional Review Board and Office of Naval Research Human Research Protection Office, endorsed by the OCS Human Research Program, and performed in accordance with the Declaration of Helsinki.
Participants
Candidates were recruited for the OCS study via large, in-person information sessions and group briefings by study personnel. No OCS leadership was present during briefings to remove the potential threat of coercion. Study participants were healthy male and female United States Marine Corps officer candidates aged ≥18 years. All individuals entering the 10-week training course were eligible to participate. A study team member briefed the volunteers who provided written informed consent prior to participation. Candidates were not excluded on the basis of age, prior injuries, or health conditions due to being deemed “fit for duty” by Marine Corps standards. Baseline characteristics by sex can be found in Table 1.
Baseline Characteristics by Sex
| Baseline characteristics | Women (n = 50) | Men (n = 177) |
|---|---|---|
| Age (years) | 24 ± 3 | 23 ± 3 |
| Race/ethnicity (n, %) | ||
| White | 41 (82%) | 103 (58%) |
| Asian | 2 (4%) | 6 (3%) |
| Black or African American | 2 (4%) | 6 (3%) |
| Hispanic or Latino | 3 (6%) | 14 (8%) |
| Other | 1 (2%) | 5 (3%) |
| Not specified | 3 (6%) | 43 (24%) |
| Height (cm) | 165.00 ± 5.87a | 177.05 ± 6.74a |
| Body mass (kg) | 65.49 ± 6.27a | 80.76 ± 8.67a |
| Body mass index (kg/m2) | 23.9 ± 2.0a | 25.9 ± 2.2a |
| Season testing occurred (n, %)b | ||
| Fall | 5 (10%) | 27 (15%) |
| Winter | 14 (28%) | 82 (46%) |
| Summer | 31 (62%) | 68 (38%) |
| Lower-extremity overuse injury | 15 (30%) | 31 (17.5%) |
aSignificantly different by sex.
bSeason determined by arrival date.
Experimental Procedures
The proposed study was a prospective, longitudinal study in OCS candidates, which consisted of two assessment time points, baseline, and posttesting. A total of 12 ml of blood was collected during medical screening that occurred prior to the initiation of the 10-week program and again following completion of it. Officer Candidates were not fasted for blood draws. Serum blood tubes sat at room temperature for 30 min to clot and were then centrifuged at 1,500g for 10 min. Plasma tubes were centrifuged at 1,500g for 10 min at room temperature immediately after collection. Supernatant was aliquoted and stored on dry ice, then transferred overnight on dry ice to the University of Pittsburgh, where it was stored at −80 °C until further analysis. In addition to blood collection, candidates performed peripheral quantitative computed tomography (pQCT) scans.
Outcome Measures
Anthropometric Variables
Prior to the start of physical training, height and weight were recorded by a stadiometer and digital scale (Healthometer Professional 500KL) and used to calculate body mass index.
Musculoskeletal Variables
Parameters of volumetric bone density, bone geometry, and estimated bone strength were assessed via pQCT (XCT2000, Stratec) with a voxel size of 0.4 mm at both timepoints. Three tibial sites were imaged: the metaphysis (4%), mid-diaphysis (38%), and proximal diaphysis (66%). For analysis, sites were down selected to only the two proximal sites. The two proximal sites were chosen as we have demonstrated previously that predominantly cortical sites are associated with bone stress injury (BSI) during OCS (Koltun et al., 2022). Tibial scans were performed of the nondominant leg, except in instances of metal hardware implantation or history of previous fracture. A trained technician measured tibial length (tibial plateau to the midline of the medial malleolus, mm) at the baseline visit. Image analysis was performed in accordance with expert recommendations (Bone Diagnostic LLC; Rantalainen et al., 2011). Cortical volumetric bone mineral density and endosteal and periosteal circumference were acquired at the proximal diaphyseal regions (38, 66%). Polar strength strain index (SSI = moment of resistance = polar moment of inertia of the cortical bone area [mm × 4]/[max distance to the center] density weighted section modulus) is a measure of bone strength and quantifies bone’s resistance to bending and torsional loads.
Hematological Variables
Total serum iron (reference range: 28–170 μg/dl) was measured via 2,4,6-Tri-(2-pyridyl)-5-triazine (TPTZ) using Beckman Coulter AU680 and AY5800 analyzers. Serum ferritin (reference range: 10–282 ng/ml) was measured via the immunoenzymatic method using Beckman Coulter UniCel Dxl 800. Total iron-binding capacity (TIBC; reference range: 250–420 μg/dl) was measured via immunotubidimetric method using Beckman Coulter AU680 and AU5800 analyzers. TIBC is the sum total of serum iron and unsaturated iron-binding capacity and was calculated as = (Transferrin) × (1.4). Hepcidin was measured via biotechne ELISA (Krepuska et al., 2024). Transferrin saturation (reference range: 25%–50%) was calculated as (serum iron/TIBC) × 100 and reported as a percentage. 25-hydroxy vitamin D (reference range: 25–100 ng/ml) was measured by mass spectrometry on a Water’s XEVO Triple Quadrupole tandem liquid chromatography–mass spectrometry. Sample preparation was completed using a solid-phase extraction in a 96-well plate using Water’s Oasis plates. Iron deficient was defined as ferritin cutoff of ≤30 ng/ml. Iron replete was defined as ferritin of >30 ng/ml. While thresholds vary, iron overload is typically defined as ferritin of >200 ng/ml. Vitamin D deficient was defined as 25(OH)D <20 ng/ml. Vitamin D insufficient was defined as 25(OH)D 20–30 ng/ml and sufficient was defined as 25(OH)D >30 ng/ml.
Musculoskeletal Injury
Injury data was deidentified from internal OCS records and collected on candidates who presented to the Physical Therapy Department for MSKI treatment. All BSIs were diagnosed by trained medical staff based on radiographs or magnetic resonance imaging. Only lower extremity overuse MSKI occurring during OCS and not preexisting were included in the present analysis.
Statistical Methods
Statistical analysis was performed using SPSS Statistics (version 28.01.1). Paired samples t tests were performed to evaluate significant within-subject differences. For variables that did not meet assumptions, data transformation was attempted and, if unsuccessful, Wilcoxon signed-rank tests were used to determine significance. Significance was set a priori at .05 (two sided). Changes in vitamin D and iron classification from baseline to posttesting were assessed using McNemar’s test. Iron and vitamin D concentrations were assessed with 2 × 2 (Sex [male vs. female] × Time [pre vs. post OCS]) mixed-measures analyses of variance. Mean percentage change from pre to post was reported. To assess the effect of baseline micronutrient status on bone and muscle outcomes, 2 × 2 (Sex [male vs. female] × Group [Group 1: optimal (i.e., no deficiency) vs. Group 2: vitamin D insufficient + deficient + iron deficient]) analyses of variance were performed. The association between micronutrient concentrations and performance was measured using Pearson correlation coefficients. Binomial logistic regression analyses were performed to assess the effect of baseline micronutrients on development of lower extremity overuse MSKI during OCS training. Separate analyses were conducted for men and women.
Results
A total of 227 (177 men, 50 women) Marine OCS candidates who completed OCS training were included in this analysis. Men were taller (p < .001), heavier (p < .001) faster (p < .001), and had a greater body mass index (p < .001). Men and women did not significantly differ by age, race, or lower-extremity MSKI (all p > .053; Table 1).
Micronutrient Status at Baseline and Posttesting
Micronutrient classification data are presented in Table 2. At baseline, 75.0% of men were sufficient in 25(OH)D, as were 82.0% of women. At posttesting, the percentage of candidates with vitamin D insufficiency significantly increased in men (22.7–38.6%, p < .001), but not women (p = .267). There was no change in the percentage of men and women with vitamin D deficiency over time (p > .50). Conversely, at baseline, 90.3% of men were replete in iron status and just 54.0% of women. The percentage of candidates who were iron replete declined in men (p = .011) and women (p < .001), which resulted in a greater percentage of men (p = .036) and women (p < .001) with iron deficiency posttesting.
Nutritional Status Groupings by Sex
| Baseline | Posttesting | McNemar test (p) | ||
|---|---|---|---|---|
| Vitamin D groups | ||||
| Sufficient (25(OH)D > 30 ng/ml) | M | 132/176 = 75.0% | 104/176 = 59.1% | <.001 |
| W | 41/50 = 82.0% | 34/50 = 68.0% | .065 | |
| Insufficient (25(OH)D 20–30 ng/ml) | M | 40/176 = 22.7% | 68/176 = 38.6% | <.001 |
| W | 8/50 = 16.0% | 13/50 = 26% | .267 | |
| Deficient (25(OH)D < 20 ng/ml) | M | 4/174 = 2.3% | 4/174 = 2.3% | 1.00 |
| W | 1/50 = 2.0% | 3/50 = 6.0% | .500 | |
| Iron groups | ||||
| Replete (ferritin > 30 ng/ml) | M | 159/176 = 90.3% | 144/176 = 81.8% | .011 |
| W | 27/50 = 54.0% | 10/50 = 20.0% | <.001 | |
| Iron deficient (ferritin cutoff of ≤30 ng/ml) | M | 17/176 = 9.7% | 29/176 = 16.5% | .036 |
| W | 23/50 = 46.0% | 39/50 = 78.0% | <.001 | |
Note. n = 1 (1%) woman missing from post 10-week vitamin D; n = 4 (2%) men missing from post 10-week iron; and n = 1 (2%) woman missing from post 10-week iron.
p < .05 results are in bold.
Changes in concentrations of biomarkers are presented in Table 3. A main effect of time for 25(OH)D (41.65 ± 1.13 to 36.21 ± 0.81 ng/ml, p < .001, η2 = .174), total iron (107.13 ± 3.08 to 75.17 ± 2.40 μg/dl, p < .001, η2 = .327), ferritin (62.99 ± 4.39 to 42.31 ± 3.37 ng/ml, p < .001, η2 = .207), iron saturation (31.33 ± 1.08 to 21.72 ± 0.97%, p < .001, η2 = .284), and hepcidin (59,765.81 ± 3,201.91 to 40,871.28 ± 3,127.26 ng/ml, p < .001, η2 = .126) indicates that concentrations of biomarkers associated with candidates’ micronutrient status decreased during OCS. There was a significant Sex × Time interaction, such that TIBC concentration decreased over time in men (342.72 ± 47.88 to 330.12 ± 50.06 μg/dl, p < .001, η2 = .096), but not women (p = .150). For 25(OH)D, there was a main effect of sex, such that women demonstrated greater concentrations than men (p = .019, η2 = .019). There was no main effect of time on 25(OH)D. Men demonstrated greater total iron (p < .001, η2=.073), ferritin (p < .001, η2=.130), hepcidin (p < .001, η2 = .081) concentrations, and percent iron saturation (p < .001, η2 = .102) than women.
Change in Nutritional Status and Performance Metrics Following 10 Weeks of OCS Among Men and Women
| Baseline | Posttesting | Change (%) | Effect | ||||
|---|---|---|---|---|---|---|---|
| Sex | Time | Sex × Time | |||||
| Hematological measures | |||||||
| Total 25-OH Vitamin D (ng/ml) | M | 40.86 ± 13.82 | 34.85 ± 9.22 | −8.9 | .019 | <.001 | ns |
| W | 44.80 ± 15.88 | 38.98 ± 12.16 | −10.64 | ||||
| Total iron (μg/dl) | M | 115.14 ± 37.26 | 86.60 ± 31.00 | −20.49 | <.001 | <.001 | ns |
| W | 102.43 ± 32.68 | 63.33 ± 26.89 | −35.39 | ||||
| Ferritin (ng/ml) | M | 86.75 ± 60.29 | 63.56 ± 46.67 | −18.85 | <.001 | <.001 | ns |
| W | 41.33 ± 26.33 | 21.80 ± 13.93 | −37.12 | ||||
| TIBC (μg/dl) | M | 342.72 ± 47.88 | 330.12 ± 50.06 | −3.81 | <.001 | ns | <.001 |
| W | 366.95 ± 82.48 | 382.08 ± 76.24 | 5.71 | ||||
| Transferrin saturation (%) | M | 34.18 ± 11.95 | 26.28 ± 10.56 | −18.04 | <.001 | <.001 | ns |
| W | 28.43 ± 10.41 | 17.19 ± 10.53 | −37.78 | ||||
| Hepcidin (ng/ml) | M | 77.69 ± 46.27 | 60.59 ± 43.27 | −14.22 | <.001 | <.001 | ns |
| W | 49.59 ± 36.03 | 29.93 ± 42.78 | −26.72 | ||||
Note. TIBC = total iron binding capacity; ns = nonsignificant (p ≥ .05); OCS = Marine Officer Candidates School.
p < .05 results are in bold.
Micronutrient Status and pQCT Musculoskeletal Parameters
Differences between micronutrient status groups for pQCT-derived musculoskeletal outcomes at baseline and posttesting are presented in Table 4. At baseline, a significant Sex × Group interaction was present for endosteal circumference at the 66% proximal site (interaction effect: p = .023), such that in women (p = .037,
Baseline and Changes in Select Bone Parameters Based on Micronutrient Status
| Effect | ||||||
|---|---|---|---|---|---|---|
| Sex | Baseline group | N | Mean ± SD | Sex | Group | Sex × Group |
| 38% cortical density (mg/cm3) | ||||||
| Men | Optimal | 78 | 1,131.67 ± 30.79 | <.001 | .279 | .773 |
| Deficiency | 32 | 1,127.59 ± 30.84 | ||||
| Women | Optimal | 22 | 1,164.91 ± 22.25 | |||
| Deficiency | 26 | 1,157.87 ± 22.66 | ||||
| Δ 38% cortical density (mg/cm3) | ||||||
| Men | Optimal | 78 | 2.37 ± 17.41 | .210 | .149 | .437 |
| Deficiency | 32 | −5.62 ± 20.90 | ||||
| Women | Optimal | 22 | −4.93 ± 15.97 | |||
| Deficiency | 26 | −7.33 ± 20.15 | ||||
| 38% periosteal circumference (mm) | ||||||
| Men | Optimal | 78 | 77.91 ± 4.63 | <.001 | .963 | .150 |
| Deficiency | 32 | 76.72 ± 4.93 | ||||
| Women | Optimal | 22 | 68.04 ± 3.54 | |||
| Deficiency | 26 | 69.16 ± 3.99 | ||||
| Δ 38% periosteal circumference (mm) | ||||||
| Men | Optimal | 78 | 0.13 ± 0.76 | .475 | .333 | .060 |
| Deficiency | 32 | 0.38 ± 0.88 | ||||
| Women | Optimal | 22 | 0.83 ± 3.07 | |||
| Deficiency | 26 | 0.07 ± 0.78 | ||||
| 38% endosteal circumference (mm) | ||||||
| Men | Optimal | 78 | 34.06 ± 4.58 | .001 | .305 | .177 |
| Deficiency | 32 | 33.81 ± 4.24 | ||||
| Women | Optimal | 22 | 30.44 ± 2.81 | |||
| Deficiency | 26 | 32.28 ± 4.49 | ||||
| Δ 38% endosteal circumference (mm) | ||||||
| Men | Optimal | 78 | 0.030 ± 0.53 | .569 | .902 | .198 |
| Deficiency | 32 | 0.16 ± 0.67 | ||||
| Women | Optimal | 22 | 0.24 ± 0.87 | |||
| Deficiency | 26 | 0.08 ± 0.39 | ||||
| 38% SSI (mm3) | ||||||
| Men | Optimal | 76 | 2,218.41 ± 357.92 | <.001 | .993 | .116 |
| Deficiency | 31 | 1,543.12 ± 199.51 | ||||
| Women | Optimal | 22 | 2,117.04 ± 384.08 | |||
| Deficiency | 26 | 1,628.84 ± 281.27 | ||||
| Δ 38% SSI (mm3) | ||||||
| Men | Optimal | 76 | 25.09 ± 110.00 | .906 | .049 | .313 |
| Deficiency | 31 | 2.38 ± 92.62 | ||||
| Women | Optimal | 22 | 51.43 ± 210.36 | |||
| Deficiency | 26 | −18.44 ± 48.96 | ||||
| 66% cortical density (mg/cm3) | ||||||
| Men | Optimal | 71 | 1,085.42 ± 34.56 | <.001 | .075 | .699 |
| Deficiency | 30 | 1,072.10 ± 36.74 | ||||
| Women | Optimal | 22 | 1,124.36 ± 33.71 | |||
| Deficiency | 27 | 1,115.77 ± 28.45 | ||||
| Δ 66% cortical density (mg/cm3) | ||||||
| Men | Optimal | 71 | 0.54 ± 19.85 | .021 | .971 | .519 |
| Deficiency | 30 | 2.93 ± 17.48 | ||||
| Women | Optimal | 22 | −6.04 ± 20.14 | |||
| Deficiency | 27 | −8.69 ± 24.14 | ||||
| 66% periosteal circumference (mm) | ||||||
| Men | Optimal | 71 | 92.61 ± 5.91 | <.001 | .540 | .099 |
| Deficiency | 30 | 91.51 ± 6.48 | ||||
| Women | Optimal | 22 | 80.42 ± 4.78 | |||
| Deficiency | 27 | 82.81 ± 5.70 | ||||
| Δ 66% periosteal circumference (mm) | ||||||
| Men | Optimal | 71 | 0.22 ± 0.90 | .380 | .962 | .474 |
| Deficiency | 30 | 0.11 ± 0.72 | ||||
| Women | Optimal | 22 | −0.04 ± 0.91 | |||
| Deficiency | 27 | 0.08 ± 0.78 | ||||
| 66% endosteal circumference (mm) | ||||||
| Men | Optimal | 77 | 58.11 ± 6.55 | <.001 | .312 | .023 |
| Deficiency | 33 | 56.64 ± 6.20 | ||||
| Women | Optimal | 22 | 49.47 ± 4.77 | |||
| Deficiency | 27 | 53.26 ± 7.08 | ||||
| Δ 66% endosteal circumference (mm) | ||||||
| Men | Optimal | 77 | 0.16 ± 0.98 | .634 | .703 | .895 |
| Deficiency | 33 | 0.11 ± 0.66 | ||||
| Women | Optimal | 22 | 0.28 ± 1.48 | |||
| Deficiency | 27 | 0.18 ± 0.97 | ||||
| 66% SSI (mm3) | ||||||
| Men | Optimal | 71 | 3,325.93 ± 576.60 | <.001 | .852 | .191 |
| Deficiency | 30 | 3,218.86 ± 645.04 | ||||
| Women | Optimal | 22 | 2,306.12 ± 391.41 | |||
| Deficiency | 27 | 2,422.94 ± 412.48 | ||||
| Δ 66% SSI (mm3) | ||||||
| Men | Optimal | 71 | 35.84 ± 111.78 | .003 | .527 | .807 |
| Deficiency | 30 | 27.15 ± 109.54 | ||||
| Women | Optimal | 22 | −25.95 ± 77.32 | |||
| Deficiency | 27 | −45.54 ± 155.83 |
Note. 38, 66% SSI were LN transformed due to failure to meet assumptions; Δ = delta (posttesting minus baseline); SSI = polar strength strain index (SSI = moment of resistance = polar moment of inertia of the cortical bone area [mm × 4]/[max distance to the center]) density weighted section modulus); optimal group = sufficient vitamin D concentrations and replete ferritin concentrations; deficient group = insufficient or deficient vitamin D concentrations and/or deficient ferritin concentrations.
p < .05 results are in bold.
When examining changes in musculoskeletal parameters based on micronutrient status, only SSI at the 38% site was significant. Specifically, at the tibial mid-diaphysis (38% site), there was a main effect of group in absolute change (posttesting minus baseline) in SSI (p = .049, ), such that the mean of the optimal group was significantly greater (M = 38.26 mm3, SE = 15.59) than was the deficient group (M = −8.03 mm3, SE = 17.27). No other significant interaction effects or differences between micronutrient groups were observed.
Micronutrients and Operationally Relevant Outcomes
MSKI data are presented in Table 1. A binomial logistic regression model was performed to ascertain the effects of each baseline micronutrient on the likelihood that participants suffer a lower extremity overuse MSKI during OCS training. In men but not women, baseline hepcidin, χ2(1) = 3.915, p < .048 explained 3.6% of the variance in injury and correctly classified 82.5% of cases. Increased baseline hepcidin was associated with an increased likelihood of suffering a MSKI in men. No other micronutrient parameters were predictive of injury (all p > .07).
Discussion
Vitamin D status declined over the course of 10 weeks. However, due to men and women beginning training with robust Vitamin D concentrations, posttesting levels remained, on average, sufficient.
Men and women demonstrated evidence of degraded iron status following OCS with profoundly greater magnitude declines occurring in women.
In the present study, men and women demonstrated a decline in total iron, serum ferritin, iron saturation, and hepcidin, indicative of degraded iron status and more specifically, of iron deficiency. Though men demonstrated degraded status, due to starting with more than double the ferritin concentrations compared with women, substantially less men concluded training meeting the threshold of iron deficiency (≤30 ng/ml) compared with women. During the 10 weeks, TIBC significantly decreased in men, whereas in women it increased. This TIBC observation in women is likely due to the increases in transferrin levels with declining iron status (Cichoń et al., 2022). Moreover, transferrin saturation concentrations fell below the clinical threshold of 20% in women, indicative of iron deficiency. These findings are consistent with prior studies in military training including a marked decline in iron, transferrin saturation, and ferritin in male Israeli Defense Forces combatants undergoing 4 months of basic training (Moran et al., 2012). Similar declines in ferritin of −15% (Gofrit et al., 2023) and −32% (Martin et al., 2019) were observed in female combatants during military training of the same duration (4 months).
Those with optimal micronutrient status exhibited preferential bone morphology and improved adaptation in response to training compared with those who were micronutrient deficient.
In the present study, bone strength (SSI) over the course of training significantly increased in the optimal group (M = 38.26 mm3, SE = 15.59) compared with a decline in the deficient group (M = −8.03 mm3, SE = 17.27). Moreover, micronutrient deficient women exhibited greater endosteal circumference at the proximal tibia, despite similar periosteal circumference and density. In men, cortical mass is placed further from the neutral axis of the long bone, increasing cross-sectional area, and bestowing greater resistance to bending. Conversely, women typically construct bones that are narrower than are men’s due to greater periosteal apposition in the male skeleton during puberty. To compensate for this difference, women tend to maintain structural integrity via endosteal apposition and increased mineral density at proximal sites (Hart et al., 2017). However, in the present study, results suggest that women suffering from micronutrient deficiency exhibited greater endosteal circumference, without greater periosteal circumference or density to compensate for resultant thinner cortices, leaving them at mechanical disadvantage.
Increased serum hepcidin concentrations in men, not women, exclusively predicted lower extremity overuse injury sustained during OCS training.
Increased circulating hepcidin results in increased iron sequestering thereby limiting biologically available iron for physical performance (McClung, 2019). Increased hepcidin has been observed in female marathoners (Roecker et al., 2005) and in military personnel following arduous operational activity (McClung, 2019), while others found that hepcidin concentrations decreased in women undergoing exhaustive running training (Auersperger et al., 2013). In the present study, hepcidin declined in both men (−14.22%) and women (−26.72%) following training and, in men only, was the only micronutrient parameter to predict incidence of lower extremity overuse injury sustained during OCS training, such that men who became injured exhibited greater hepcidin concentrations at baseline. Prior work has consistently demonstrated that regulatory factors that exert a significant influence on hepcidin activity include baseline iron status and hepcidin levels preexercise (Fensham et al., 2023). The decline in hepcidin seen here may indicate a protective adaptation due to the candidates declining iron status whereby hepcidin is suppressed so that dietary iron absorption and replenishment of iron stores can increase and recover from a deficient state (Peeling et al., 2014). Interestingly, this occurred despite the majority of men beginning and ending training with replete status. Further study is warranted to discern the change in hepcidin in response to military training, specifically as it relates to declines in iron status and MSKI risk.
Limitations
The present study has some limitations. This study did not measure soluble transferrin receptor or hemoglobin, negating our ability to diagnose iron deficiency anemia based on clinical thresholds. However, according to the Centers for Disease Control, the sensitivity of serum ferritin is 89% for diagnosis of iron depletion compared with hemoglobin, which is only 26% (Pfeiffer et al., 2013). We did not measure parathyroid hormone, limiting our ability to fully elucidate the plausible disturbance to calcium homeostasis resulting from intense exercise. We did not control for menstrual cycle or status in female candidates, which may have helped to explain our results. Due to the limited access to candidates typical to military field environments, candidates were not exclusively fasted prior to their blood draw, and physical activity prior to testing was not controlled for, therefore diurnal variation and changes in response to exercise and nutrition may have influenced our results. As previously noted, supplementation data was not directly measured. Finally, pQCT is able to measure volumetric bone mineral density but lacks the necessary resolution to examine bone microarchitecture.
Practical Applications and Conclusions
Parameters of vitamin D and iron status changed over the course of Marine OCS training, suggesting a decline in micronutrient status. However, robust vitamin D concentrations in women and high iron stores in men prior to the onset of training appeared to attenuate declines. Optimal micronutrient status was associated with improved bone strength and a more mechanistically advantageous tibial shaft, suggesting optimal vitamin D and ferritin concentrations may exert beneficial consequences on bone fatigability and fracture reduction during military training. Moreover, hepcidin was predictive of lower-extremity injury risk suggesting the need for additional research to more carefully investigate this potential association. While additional research is needed to further elucidate this relationship, dietary and supplemental intake of vitamin D and iron should be prioritized both preceding and during military training to counteract detriments to bone observed under conditions of insufficiency.
Acknowledgments
Authorship, Declarations: We would like to acknowledge the OCS candidates that volunteered to participate in this study as well as the OCS command staff at Marine Corps Base Quantico, without whom this work would not have been possible. We would also like to acknowledge Varun Patel, Jack Holiat, Samantha Cooper, and Raj Patel for their technical assistance. We thank the Military Performance Division, United States Army Research Institute of Environmental Medicine, for their provision of the pQCT. The authors’ responsibilities were as follows— Project administration: Sekel, Koltun, Bird, Forse, and Martin. Supervision: Lovalekar and Nindl. Formal analysis: Sekel and Lovalekar. Writing-original draft: Sekel. Methodology: Lovalekar, Koltun, Bird, Forse, and Martin. Funding acquisition: Martin. Study conceptualization: Nindl. Writing-review and editing: All authors. Grant 1: Office of Naval Research N-00014-21-1-2725: Development of a Physical Readiness Decision Tool to Leverage Wearable Technologies for Monitoring Warfighters Mobility and Load Exposure.
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