Sociodemographic characteristics and health-related conditions during pregnancy for the 530 women included in this study are shown in table 1. The median serum ferritin value was 15.2 μg/L (IQR = 8.7 - 25.05). Only one participant presented hyperferritinemia (serum ferritin > 200 pg/L) ³².

Table 1

Characteristics
Age (mean ± SD)		24.6 ± 5.3
Living in an urban area [n (%)]		428 (80.8)
Socioeconomic status		[n (%)]
	1	494 (93.2)
	2	28 (5.3)
	3	7 (1.3)
Blood count		(mean ± SD)
	Hemoglobin (g/dl)	11.0 ± 1.3
	Hematocrit	33.9 ± 3.7
	White blood cells	11,507.9 ± 3,429.4
	Granulocytes (%)	72.6 ± 10.0
	Monocytes (%)	5.2 ± 2.0
	Lymphocytes (%)	20.2 ± 7.5
Iron status		[n (%)]
	Gestational anemia	229 (43.2)
	Serum ferritin depletion	213 (40.2)
Delivery mode
	Cesarean section	243 (45.8)
Newborn
	Gestational age at birth (mean ± SD)	38.5 ± 1.9
	Birth weight (mean ± SD)	3,134.9 ± 494.4
	Birth size (mean ± SD)	50.3 ± 3.1

Under the hypothesis that high iron levels may promote inflammatory states, we explored the relationship between various immune markers and indicators of iron status in the participants. As observed in figure 1, we found significant and positive weak correlations between the absolute lymphocyte count and hemoglobin (r = 0.09, p = 0.02), hematocrit (r = 0.13, p = 0.003), red blood cells (r = 0.13, p = 0.004), and serum ferritin (r = 0.12, p < 0.001). Monocytes also correlated with serum ferritin (r = 0.09, p < 0.001).

Figure 1 SF: Serum ferritin; HB: Hemoglobin; HTC: Hematocrit; RBC: Red blood cells; WBC: White blood cells; LC: Lymphocyte count; MC: Monocyte count; GC: Granulocyte count; GA: Gestational age; BW: Birth weight; SB: Size at birth * Significant correlations (p < 0.05)

In separate multivariate linear regression models, we observed that lymphocyte and monocyte counts were associated with serum ferritin, considering maternal age, socioeconomic status, and gestational weeks as covariates (table 2). Interestingly, white blood cells inversely correlated with birth weight (r = -0.17, p < 0.001). When this relationship was evaluated by linear regression, the variables remained associated even after adjustment for maternal age, gestational age, and socioeconomic status.

Table 2

Variable		β	SE	p value
Lymphocyte counts
	Maternal age	0.031	0.069	0.478
	Socioeconomical status	-0.049	0.041	0.263
	Gestational age	-0.010	0.238	0.813
	Serum ferritin	0.123	0.017	0.005
Monocyte counts
	Maternal age	-0.189	0.097	<0.0001
	Socioeconomic status	0.025	0.058	0.565
	Gestational age	-0.054	0.337	0.206
	Serum ferritin	0.092	0.024	0.032

β: Beta coefficient; SE: Standard error

Understanding iron balance during pregnancy remains a complex issue. While much nutritional advice during pregnancy focuses on preventing anemia to ensure fetal health ³³, previous findings, as in other underdeveloped countries, have indicated an inverse association between serum ferritin and hemoglobin levels with adverse fetal outcomes such as low birth weight ^11,18. Given the hypothesis that iron may contribute to inflammatory states, we investigated, for the first time in the Colombian population, the relationship between serum ferritin and various inflammatory markers, including white blood cell counts and serum proinflammatory cytokine levels.

The relationship between iron status, inflammatory markers, and pregnancy outcomes has been primarily investigated in preeclampsia, where higher neutrophil and lymphocyte counts and elevated serum iron and ferritin concentrations have been detected in pregnant women with this condition compared to those with healthy pregnancies ^34,35. This phenomenon may be attributed to endothelial dysfunction from oxidative stress secondary to increased maternal iron levels.

Although Lee et al. used a different study design than ours, they found that IL-6 concentrations at delivery were positively correlated with maternal serum ferritin (p < 0.01) ²⁴. However, the relationship between maternal serum ferritin and IL-6 is controversial, as other studies have found no association between them in healthy or high-risk pregnancies, which often include autoimmune diseases, obesity, diabetes mellitus, and preeclampsia ^25,36. Nevertheless, studies evaluating the correlation between serum ferritin and interleukins in pregnant patients have small sample sizes, limiting the extrapolation of the results.

Similar findings have been published regarding white blood cells and iron status. Tang et al. found a direct association between hemoglobin concentration and T-cell count in healthy third-trimester pregnant women ³⁷. Consistent with the aforementioned findings, some studies reported non pregnant individuals with anemia exhibiting lower counts of lymphocytes and natural killer cells ³⁸. However, the precise mechanism of how hemoglobin levels influence peripheral lymphocytes is not completely understood. Some white blood cell populations demonstrate functional alterations in anemic states, such as diminished bactericidal activity in macrophages ³⁹ or reduced enzymatic activity in neutrophils (in mice) ⁴⁰.

Iron participates in the immune response by inducing immune cell differentiation and regulating cellular response pathways and cytokine functions ⁴¹. High white blood cell counts may result from augmented cell proliferation facilitated by iron availability ⁴². Moreover, under such conditions, macrophage M1 polarization is induced by inflammatory cytokines such as IL-6 or TNF-α, along with increased expression of other iron metabolism proteins, such as ferroportin, transferrin receptor, and ferritin ^43,44. Similarly, high iron levels may enhance neutrophil recruitment and promote inflammatory states through the secretion of proinflammatory cytokines, as evidenced in patients with hemochromatosis ⁴⁵.

White blood cell values represent the activity of the immune response and have been identified as predictive factors for some chronic inflammatory conditions ^46-49. Although white blood cell values increase physiologically during late pregnancy ⁵⁰, its count may be used in predicting adverse perinatal events, such as preterm birth and preeclampsia severity ^51-54. Consistent with this, the white blood cell count was inversely related to birth weight and gestational age. This result complements previous findings in this cohort, where serum ferritin was inversely associated with birth weight ¹⁷. Similar findings have been reported in Israeli pregnant women, in whom the white blood cell count also showed an inverse association with birth weight ⁵⁵ and is related to adverse perinatal outcomes ^56-58. The previous information suggests a potential connection between maternal iron levels and inflammatory immune activity, which could have adverse effects on perinatal outcomes.

Our results suggest that maternal iron status before delivery could influence the systemic inflammatory response. Studies on the proinflammatory effects of serum ferritin have identified that stimulation of macrophages with the heavy subunit (FeH) of ferritin induced a significant increase in the mRNA expression of IL-Ιβ, IL-6, IL-12, and TNF-α, as functionally verified -by ELISA- with elevated levels of IL-1 β and IL- 12p70 secreted by macrophages ⁵⁹. These results could suggest a proinflammatory role of elevated iron levels during pregnancy, negatively impacting maternal-fetal outcomes.

IL-6 -secreted by monocytes, macrophages, and T cells in response to recognition of pathogen-associated molecular patterns- stimulates C-reactive protein and hepcidin hormone secretion by hepatocytes through the involvement of the transcription factor STAT3 and NF-ke ^60,61. C-reactive protein, in turn, induces the release of reactive oxygen species and cytokines by macrophages ⁶². Inflammatory states within placental tissue may trigger the production of IL-6 by circulating monocytes or macrophages, initiating a systemic-type response. This response includes the release of cytokines, such as IL-1, TNF-α, IFN-γ, or IL-6 in the serum. Consequently, these changes result in decreased serum iron levels (hypoferremia) ⁶³, leading to iron sequestration intracellularly or in circulating storage proteins, such as ferritin, or increased storage in macrophages of the reticuloendothelial system ⁶⁴.

Despite the results of our study, it is important to highlight that outcome such as low birth weight are usually not acute. Therefore, the findings of this study could represent the consequences of changes that occurred in the early stages of pregnancy or before ^{3,12,15,65,66}.

Our study has several limitations. Enrollment of participants in the peripartum implies higher cytokine levels observed due to their physiological increase during this period. Thus, the measurements do not represent the third trimester of pregnancy ^67,68. Nevertheless, we excluded participants from the ancillary cohort with comorbidities associated with systemic inflammation. Other causes, such as obesity, were not analyzed in this study, and may have resulted in a biased interpretation of the results.

We also recognize the limitations of the extreme group approach design in determining effect size, although it is informative for identifying a positive association between iron markers and proinflammatory conditions in late pregnancy ⁶⁹.

Additionally, the study design does not allow for causality, as we cannot ascertain whether iron overload is a stimulus to produce proinflammatory cytokines or, on the contrary, if inflammation induces the elevation of serum ferritin during pregnancy. The multiplex cytokine assay used was not sensitive enough to detect blood levels of IL-10, an anti-inflammatory cytokine relevant in fetal tolerance, associated with iron-deficiency anemia in the adult population ⁷⁰.

Further studies are required to functionally corroborate the influence of iron overload on maternal serum and tissue (placenta) inflammation. In addition, serum ferritin can be affected by the expansion of plasma volume, so other indicators of iron status, such as the concentration of the soluble transferrin receptor, could better represent the maternal iron status.

In summary, in a representative sample of pregnant women in Cartagena, Colombia, iron markers (serum ferritin, hemoglobin, and hematocrit) are correlated to lymphocyte count and associated with inflammation markers such as IL-6. Furthermore, we observed that women with elevated white blood cell counts and high serum ferritin levels tended to have infants with lower birth weights. This fact suggests a potential involvement of iron in inflammatory processes during pregnancy; conditions associated with inflammation in the final trimester may have adverse effects on perinatal outcomes.

However, further studies are required to establish causality between these events. Also, associations were generally of weak magnitude. Then, other aspects that may have an influential role in systemic inflammation must be identified. The direct effects of iron on the fetoplacental unit should be evaluated in tissue (placenta) for a closer approximation to the biological background of this finding.