Utility of Serum Ferritin for Predicting Myalgic Encephalomyelitis/Chronic Fatigue Syndrome in Patients with Long COVID

Objective: The most common symptom of post-acute coronavirus disease 2019 (COVID-19) is fatigue, and it potentially leads to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS); however, a specific prognosticator is lacking. We aimed to elucidate the clinical characteristics of patients who developed ME/CFS after COVID-19. Methods: In this retrospective observational study, patients who visited Okayama University Hospital for long COVID between February 2021 and March 2022 were investigated. Results: Of the 234 patients, 139 (59.4%) had fatigue symptoms. Fifty patients with fatigue symptoms (21.4%) met the criteria for ME/CFS (ME/CFS group), while the other 89 patients did not (non-ME/CFS group); 95 patients had no fatigue complaints (no-fatigue group). Although the patients’ backgrounds were not significantly different between the three groups, the ME/CFS group presented the highest scores on the self-rating symptom scales, including the Fatigue Assessment Scale (FAS), EuroQol, and the Self-Rating Depression Scale (SDS). Furthermore, serum ferritin levels, which were correlated with FAS and SDS scores, were significantly higher in the ME/CFS group (193.0 μg/L, interquartile range (IQR): 58.8–353.8) than in the non-ME/CFS group (98.2 μg/L, 40.4–251.5) and no-fatigue group (86.7 μg/L, 37.5–209.0), and a high serum ferritin level was prominent in female patients. Endocrine workup further showed that the ME/CFS group had higher thyrotropin levels but lower growth hormone levels in serum and that insulin-like growth factor-I levels were inversely correlated with ferritin levels (R = −0.328, p < 0.05). Conclusions: Serum ferritin level is a possible predictor of the development of ME/CFS related to long COVID, especially in female patients.


Introduction
Approximately two and a half years have passed since the coronavirus disease 2019 (COVID-19) pandemic broke out. COVID-19 involves both acute and chronic phases. Chronic prolonged symptoms are called post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, long COVID, or post-COVID-19 condition [1][2][3]. Patients with COVID-19 present with a variety of symptoms which make it difficult for patients to continue their previous social lives for months to years, and some patients have become unable to reintegrate into society after COVID-19 [3]. To respond to society's needs, we established a COVID-19 aftercare clinic (CAC) to treat patients with long COVID and to comprehend their pathophysiology [4]. The most common symptom in

Ethical Approval
Patients who wished to opt out were given the opportunity to do so after being informed about the study on our hospital's wall and website. This study was approved by the Okayama University Hospital Ethics Committee (no. 2105-030) and adhered to the Declaration of Helsinki. Informed consent from the patients was not necessary owing to the anonymization of the data. All authors had access to the study data and reviewed and approved the final manuscript.

Results
Of the 312 patients who visited the CAC during the study period, 78 patients did not meet the inclusion criteria. These 78 patients included 2 patients who visited the CAC less than 4 weeks after the onset of COVID-19, 75 patients whose disease onset was before 30 November 2021, and 1 patient who had hyperferritinemia prior to suffering from COVID-19. Data from the remaining 234 patients were analyzed in this study. All of these patients were followed up for at least 6 months from the onset of the disease. While 95 patients (40.6%) did not complain of general fatigue, the remaining 139 patients (59.4%) complained of general fatigue. Of the patients with fatigue symptoms, 50 patients (40.0%) met all of the three sets of diagnostic criteria for ME/CFS (Fukuda criteria, CCC, and IOM criteria), and these patients were included in the ME/CFS group, whereas the remaining 89 patients with fatigue were included in the non-ME/CFS group.

Patients' Backgrounds
The backgrounds of the patients, including age, BMI, number of days from the onset of COVID-19 to the first visit to the CAC, acute-phase treatments for COVID-19, and vital signs on admission, are summarized in Table 1. The ME/CFS group included 24 males (48.0%) and 26 females (52.0%) with a median age of 42 years (interquartile range (IQR): 30.3-51.8). Sex, age, and BMI were not significantly different between the three groups. The median duration from the onset of COVID-19 to the first visit to the CAC was 128 days (IQR: 72. 3-205.5) in the ME/CFS group, and it was significantly longer than that in the non-ME/CFS group (median: 73 days, IQR: 55.0-114.0) and that in the no-fatigue group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups. Table 1. Background information of patients with long COVID with or without symptoms of ME/CFS. 22.7 (19.8-27.0) 0.326 (b) Days from the onset to the first visit, median (IQR) 128 (72. 3-205.5) group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups. BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. ☨ : Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups. group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute ments, including place of care and oxygen or steroid administration, were not different between the three groups. Systolic blood pressure was slightly, bu cantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than two groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS me 5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had more severe scores for all self-rating scales than those in the other two group group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups. BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. ☨ : Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups.
: Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups. Self-rating scales in ME/CFS, non-ME/CFS, and no-fatigue groups of patients with long COVID. Self-rating symptom scales of each group, including fatigue assessment scale (FAS) of physical and mental evaluation, self-rating depression scale (SDS), EuroQol 5 Dimensions (EQ-5D) and 5 Levels (EQ-5D-5L), and EQ-5D visual analog scale (EQ-5D-VAS), were compared. The median is represented by the horizontal bar within the box, and the upper and lower horizontal lines of the box represent the 75th and 25th percentiles, respectively. The upper and lower horizontal bars outside the box represent the maximum and minimum values within 1.5 times the interquartile range, respectively, and the circles outside indicates outliers. The Kruskal-Wallis test was performed for statistical analysis, and * p < 0.05 and ** p < 0.01 indicate statistically significant differences between Figure 1. Self-rating scales in ME/CFS, non-ME/CFS, and no-fatigue groups of patients with long COVID. Self-rating symptom scales of each group, including fatigue assessment scale (FAS) of physical and mental evaluation, self-rating depression scale (SDS), EuroQol 5 Dimensions (EQ-5D) and 5 Levels (EQ-5D-5L), and EQ-5D visual analog scale (EQ-5D-VAS), were compared. The median is represented by the horizontal bar within the box, and the upper and lower horizontal lines of the box represent the 75th and 25th percentiles, respectively. The upper and lower horizontal bars outside the box represent the maximum and minimum values within 1.5 times the interquartile range, respectively, and the circles outside indicates outliers. The Kruskal-Wallis test was performed for statistical analysis, and * p < 0.05 and ** p < 0.01 indicate statistically significant differences between the indicated groups.

Laboratory Data and Correlations with the Self-Rating Scales
Blood cell counts and biochemical data were comprehensively analyzed in the three groups ( Table 2)  Interrelationships between the levels of serum ferritin and self-rating scales in patients with long COVID. Scatter diagrams between serum ferritin and self-rating symptom scales, including FAS, SDS, EQ-5D-5L, EQ-5D-VAS, and frequency scale for symptoms of gastroesophageal reflux disease (FSSG), are shown (see Figure 1 legend). The interrelationships between serum ferritin levels and these scores were analyzed using Pearson's correlation coefficient, and * p < 0.05 and ** p < 0.01 indicate statistically significant correlations.

Figure 2.
Interrelationships between the levels of serum ferritin and self-rating scales in patients with long COVID. Scatter diagrams between serum ferritin and self-rating symptom scales, including FAS, SDS, EQ-5D-5L, EQ-5D-VAS, and frequency scale for symptoms of gastroesophageal reflux disease (FSSG), are shown (see Figure 1 legend). The interrelationships between serum ferritin levels and these scores were analyzed using Pearson's correlation coefficient, and * p < 0.05 and ** p < 0.01 indicate statistically significant correlations. Table 2. Laboratory data of ME/CFS, non-ME/CFS, and no-fatigue groups.

ME/CFS (n = 48)
Non-ME/CFS (n = 87)  The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups. BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. ☨ : Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups. T ments, including place of care and oxygen or steroid adm different between the three groups. Systolic blood pres cantly, higher in the ME/CFS group (median: 129 mmH two groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, F 5L, EQ-5D VAS, and SDS, are shown in Figure 1. The more severe scores for all self-rating scales than those in Alb, albumin; ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BS, blood sugar; CK, creatine kinase; Cr, creatinine; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; Hb, hemoglobin; IgG, immunoglobulin G; LDL-C, low-density lipoprotein cholesterol; Plt, platelets; RBC, red blood cells; T-Bil, total bilirubin; TP, total protein; UN, urea nitrogen; WBC, white blood cells. The data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05.
J. Clin. Med. 2023, 12, x FOR PEER REVIEW 4 of 13 group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups.   BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. ☨ : Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups.    Table 3 shows the hormonal parameters for the three groups. Serum cortisol levels, plasma ACTH levels, and ACTH/cortisol ratios were not significantly different between the three groups. Serum TSH levels were significantly higher in the ME/CFS group (1.37 µIU/mL, IQR: 0.99-2.01) than in the non-ME/CFS group (1.06 µIU/mL, IQR: 0.80-1.56; * p < 0.05). Serum FT4 levels did not differ among the three groups. The FT4/TSH ratio was significantly lower in the ME/CFS group (0.87, IQR: 0.64-1.22) than in the non-ME/CFS group (1.15, IQR: 0.83-1.69; * p < 0.05). Serum GH levels in the ME/CFS group (0.22 ng/mL, IQR: 0.05-0.67) were significantly lower than in the no-fatigue group (0.37 ng/mL, IQR: 0.17-1.11; ** p < 0.01). Table 3. Endocrine data of ME/CFS, non-ME/CFS, and no-fatigue groups.

ME/CFS (n = 48)
Non-ME/CFS (n = 87) group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups. BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. ☨ : Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups.  REVIEW 4 group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase ments, including place of care and oxygen or steroid administration, were not signific different between the three groups. Systolic blood pressure was slightly, but not s cantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the two groups. BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-f groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Difference considered statistically significant at p < 0.05. ☨ : Statistically significant between the ind groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ 5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had signific more severe scores for all self-rating scales than those in the other two groups. group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups. BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. ☨ : Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS 5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME more severe scores for all self-rating scales than those in the group (median: 106 days, IQR: 73.0-152.0; ** p < 0.01). The variables of acute-phase treatments, including place of care and oxygen or steroid administration, were not significantly different between the three groups. Systolic blood pressure was slightly, but not significantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the other two groups.  BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-fatigue groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Differences were considered statistically significant at p < 0.05. ☨ : Statistically significant between the indicated groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ-5D-5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had significantly more severe scores for all self-rating scales than those in the other two groups. The variables of acute-phase ments, including place of care and oxygen or steroid administration, were not signific different between the three groups. Systolic blood pressure was slightly, but not s cantly, higher in the ME/CFS group (median: 129 mmHg, IQR: 112-140) than in the two groups.  BMI, body mass index; IQR, interquartile range. Data in the ME/CFS, non-ME/CFS, and no-f groups were analyzed using (a) Fisher's exact test or (b) the Kruskal-Wallis test. Difference considered statistically significant at p < 0.05. ☨ : Statistically significant between the ind groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including FAS, FAS physical, FAS mental, EQ 5L, EQ-5D VAS, and SDS, are shown in Figure 1. The ME/CFS group had signific more severe scores for all self-rating scales than those in the other two groups.

Severity of Fatigue-Related Symptoms
The scores of the self-rating scales, including F 5L, EQ-5D VAS, and SDS, are shown in Figure 1. more severe scores for all self-rating scales than tho : Statistically significant difference between the indicated groups.
We also analyzed the relationships between hormonal parameters and serum ferritin levels. Serum IGF-I levels were significantly and inversely correlated with serum ferritin levels in the ME/CFS group (R = −0.328, * p < 0.05) (Figure 4). Furthermore, the correlation between serum IGF-I and ferritin levels was stronger in the female patients in the ME/CFS group (R = −0.497, ** p < 0.01).  Table 3 shows the hormonal parameters for the three groups. Serum cortisol levels, plasma ACTH levels, and ACTH/cortisol ratios were not significantly different between the three groups. Serum TSH levels were significantly higher in the ME/CFS group (1.37 µIU/mL, IQR: 0.99-2.01) than in the non-ME/CFS group (1.06 µIU/mL, IQR: 0.80-1.56; * p < 0.05). Serum FT4 levels did not differ among the three groups. The FT4/TSH ratio was significantly lower in the ME/CFS group (0.87, IQR: 0.64-1.22) than in the non-ME/CFS group (1.15, IQR: 0.83-1.69; * p < 0.05). Serum GH levels in the ME/CFS group (0.22 ng/mL, IQR: 0.05-0.67) were significantly lower than in the no-fatigue group (0.37 ng/mL, IQR: 0.17-1.11; ** p < 0.01).

Endocrine Characteristics and Correlations with Serum Ferritin Levels
We also analyzed the relationships between hormonal parameters and serum ferritin levels. Serum IGF-I levels were significantly and inversely correlated with serum ferritin levels in the ME/CFS group (R = −0.328, * p < 0.05) (Figure 4). Furthermore, the correlation between serum IGF-I and ferritin levels was stronger in the female patients in the ME/CFS group (R = −0.497, ** p < 0.01).  cortisol, adrenocorticotropin (ACTH), free thyroxine (FT4), growth hormone (GH), insulin-like growth factor (IGF)-I, and thyrotropin (TSH), in the ME/CFS group. These interrelationships were analyzed using Pearson's correlation coefficient, and * p < 0.05 indicates statistically significant correlations.

Discussion
Our study revealed that serum ferritin levels were about two times higher in patients with long COVID (particularly in women) who thereafter developed ME/CFS than in patients with long COVID who did not develop ME/CFS. Ferritin synthesis is regulated by oxidation and anti-oxidation and is essential for in vivo inflammation and iron metabolism [28]. Hyperferritinemia is caused by hemochromatosis, inflammatory diseases, infectious diseases, hematological and malignant diseases, and liver and renal dysfunctions. According to an epidemiological study, the most common cause was infectious disease, followed by solid tumors and liver dysfunction [28][29][30]. ME/CFS has been hypothesized to develop under the condition of abnormal immune responses, viral infection, enhancement of oxidative and nitrosative stress pathways, or dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis [31][32][33]. Possible pathological mechanisms of long COVID include immune system abnormalities, direct viral tissue damage, and endocrine disability [34][35][36][37]. With regard to immune abnormalities, studies have focused on several cytokines. The hypothesis of immune abnormalities as ME/CFS pathomechanisms has previously led to research on immune abnormalities in ME/CFS. Although some biomarkers, including antibodies against β-adrenergic receptors and muscarinic acetylcholine receptors, oxidative stress and anti-oxidative activity, and inflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α, and nuclear factor-kappa B, have been studied, none of them has sufficient reliability for clinical use in the diagnosis or severity evaluation of ME/CFS [14,38,39]. The levels of serum ferritin have been reported to be elevated in patients with ME/CFS, as in our study, but the evidence is controversial [40]. Since iron deficiency can cause fatigue symptoms and mimic ME/CFS, it is recommended to exclude hypoferritinemia when observing patients with ME/CFS in primary care settings [41,42]. Female patients are more likely to suffer from iron deficiency and show lower serum ferritin levels than male patients [30]. Therefore, our results showing that serum ferritin levels were considerably higher in female patients with long COVID in the ME/CFS group, who would normally have lower ferritin levels, are clinically important.
In addition to the physiological elevation of serum ferritin levels associated with infection, the acute phase of COVID-19 causes localized inflammation of the lungs, leading to a systemic and extrapulmonary hyperinflammation syndrome and the production of excessive serum ferritin [43]. Hence, serum ferritin levels have been reported to reflect the inflammatory response and are useful markers for diagnosing and predicting the severity of the disease [44,45]. In contrast, in the chronic phase of COVID-19, hyperferritinemia has also been suggested to be a clinical feature of long COVID, as shown in our study, though the number of studies has been limited [46][47][48]. However, we cannot simply explain the phenomenon of hyperferritinemia as prolonged inflammation induced by the acute phase of COVID-19. This is because serum ferritin levels were only elevated in patients with ME/CFS without any other inflammatory markers, despite the longer time since the onset of COVID-19. In addition, no specific relationship between severity of the acute phase and development of ME/CFS was found.
In the chronic phase of COVID-19, hyperferritinemia may be caused by iron dyshomeostasis [49]. As an example of the possible mechanism in ME/CFS patients, hepcidin-like effects might be involved in this mechanism if SARS-CoV-2 specifically elevates ferritin levels in the context of direct and indirect metabolic involvement by viral infection [50]. In addition, hyperferritinemia in long COVID may be related to accompanying hypertension, sleep disturbances, or depression, all of which are consistent with the results of our studies showing that blood pressure and SDS were higher in patients with ME/CFS [46,47,51].
In the present study, we also uncovered a relationship between hyperferritinemia and endocrine profiles among patients with ME/CFS after COVID-19. Endocrine dysfunction, including hypothyroidism and impaired HPA axis, has been reported in patients with ME/CFS [32,52]. However, patients in the ME/CFS group in our study only had a tendency for occult hypothyroidism, and there was no significant difference in HPA axis function between the ME/CFS group and the other two groups. These findings may reflect the underlying mechanism specific to long COVID, and further investigation of the relationship between ferritin levels and hormonal characteristics showed a negative correlation between serum ferritin and serum IGF-I levels. IGF-I, which plays an important role in childhood growth and anabolism, also regulates ferritin synthesis at the messenger ribonucleic acid level [53,54]. Furthermore, IGF-I is known to be transiently downregulated in response to various stresses, malnutrition, and depression [55,56]. Taken together, serum IGF-I levels may be reduced by various factors associated with COVID-19, which may result in secondary dysregulation of serum ferritin levels in patients with long COVID. According to our study, this tendency was more prominent in female patients, suggesting that the presence of hyperferritinemia should be considered for ME/CFS, especially in female patients with post-COVID-19.
Our study had several limitations. First, all of the patients were referred from other hospitals; therefore, this study included only patients with more severe conditions. Second, we did not clearly distinguish patients who already had ME/CFS at the time of the visit to the CAC from those who developed ME/CFS over the time course. Both sets of patients were evaluated, and their blood samples were collected at the same time as the first visit. Third, since this study was a cross-sectional observational study without any follow-up, there may have been causal reversals, and the trends in serum ferritin levels could not be observed. Further prospective studies are warranted to validate the results. Fourth, monovariate analysis was repeated without adjustment, so a type I error could not be completely avoided. Fifth, our study was performed at a single center only with long COVID patients. A multicenter study with non-long COVID patients as controls is desired.
In conclusion, we revealed the clinical characteristics of patients who developed ME/CFS after COVID-19 and identified serum ferritin as a possible predictor of ME/CFS related to long-term COVID. Hyperferritinemia was objectively correlated with the severity of long-term COVID symptoms as well as endocrine hormone secretion. However, the mechanism remains hypothetical, and further in vivo and in vitro investigations are necessary to understand the characteristics of ferritin metabolism in patients with ME/CFS and long COVID.

Institutional Review Board Statement:
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Okayama University Hospital (no. 2105-030).
Informed Consent Statement: Informed consent from the patients was not necessary due to the anonymization of data. Information regarding the present study was provided on our hospital wall and on the website of our hospital, and patients who wished to opt out were offered that opportunity.

Data Availability Statement:
The datasets generated and analyzed in the present study are available from the corresponding authors on reasonable request.