Pregnancy is a significant physiological condition marked by substantial increases in metabolic and hematologic demands. To facilitate fetal growth and placental function, maternal blood volume increases by roughly 50%, necessitating a corresponding rise in red blood cell mass and, as a result, a considerable uptick in iron utilization [1,2,3]. This elevated requirement, estimated at 1000-1200 mg of elemental iron throughout gestation, makes pregnant women particularly susceptible to iron deficiency (ID) and its advancement to iron deficiency anemia (IDA). IDA during pregnancy poses a significant global health issue, linked to serious adverse effects such as maternal fatigue, heightened risk of preterm delivery, low birth weight, and hindered cognitive development in children [4,5].

The current clinical approach to evaluating iron status during pregnancy, especially at the vital first antenatal booking visit, relies heavily on traditional hematological indicators such as hemoglobin (Hb), serum ferritin, and transferrin saturation. Although essential, these markers exhibit considerable limitations in the fluctuating context of early pregnancy. Hemoglobin serves as a late indicator, decreasing only after iron reserves are depleted. While haemoglobin serves as an indicator of existing anaemia, ferritin detects early-stage iron deficiency (depleted iron stores), enabling timely intervention to avert severe anaemia and its associated complications during pregnancy [6,7]. Serum ferritin, the main marker of iron reserves, acts as an acute-phase reactant that may be elevated due to the low-grade inflammation typical in pregnancy, potentially obscuring genuine iron deficiency [6,7,8]. This diagnostic uncertainty can postpone timely intervention.

Iron homeostasis and erythropoiesis are controlled by two important hormones: hepcidin and erythropoietin (EPO). Hepcidin, recognized as the primary regulator of systemic iron availability, regulates dietary iron absorption and the recycling of iron by macrophages through the degradation of the cellular iron exporter ferroportin [9,10]. Inflammatory signals increase hepcidin levels, which sequester iron and contribute to the phenomenon known as “anemia of inflammation”, but iron deficiency and heightened erythropoietic demand generally lead to reduced hepcidin production [11,12]. Importantly, urinary hepcidin has been identified as a reliable, non-invasive indicator of systemic hepcidin activity [13]. Erythropoietin (EPO) is a primary stimulator of red blood cell production, secreted by the kidneys in response to tissue hypoxia. During the early stages of pregnancy, EPO levels increase to facilitate the necessary expansion of red cell mass, generating a legitimate signal for iron demand that, under normal physiological conditions, should be accompanied by a corresponding suppression of hepcidin to release iron [13,14].

Nevertheless, the exact relationship between the demand signal (EPO) and the regulatory signal (hepcidin) during the critical first trimester remains inadequately defined. It is postulated that early subclinical inflammation or other pregnancy-related adaptations may impair the appropriate hepcidin response to increasing EPO levels, resulting in functional iron deficiency even before any changes in standard parameters. Consequently, a comprehensive assessment that integrates the stimulus (EPO), the regulatory gatekeeper (urinary hepcidin), and the resulting iron status (ferritin, Hb, etc.) provides a robust, mechanistic framework for comprehending the earliest disruptions in iron metabolism during pregnancy.

To this point, only a few studies have simultaneously assessed serum EPO and urinary hepcidin in pregnant women [15,16], and none have specifically examined this triad of biomarkers at the crucial first antenatal booking. This indicates a notable gap in understanding, as this moment provides the earliest chance for predictive evaluation and targeted intervention. This cross-sectional study aims to evaluate alterations in iron metabolism and erythropoietic activity among pregnant women by assessing serum erythropoietin, hepcidin, and iron status indices, and to determine the influence of gestational age at antenatal registration and anaemia status on these parameters.

Figure 1 shows the flow of participants through the study from the beginning to the end. A total of 350 women, consisting of 200 pregnant women at various stages of pregnancy at first antenatal booking, and 150 non-pregnant women (controls), were investigated in this study. There was no significant difference in mean age between pregnant women and non-pregnant women. Weight and BMI were significantly higher (p < 0.001) among pregnant women than in controls. There was no statistical difference in the educational status of cases and controls. The anaemic status of the pregnant women according to the WHO anemia classification showed that 132 (66.0%) of the pregnant women were anaemic while 68 (34.0%) were non-anaemic, while in the non-pregnant women, 40 (26.7%) were anemic as against 110 (73.3%) which were non-anaemic but using Lawson Anemia classification, 120 (60.0%) of the pregnant women were anaemic while 80 (40.0%) were non-anaemic, similarly, in the non-pregnant women, 29 (19.3%) were anaemic as against 121 (80.7%) which were non-anaemic. Of the 200 pregnant women, 38 (19.0%) registered at ≤12 weeks of gestation, while the majority, 162 (81.0%), registered at >12 weeks of gestation, as shown in Table 1.

Table 2 shows the mean values of measured hematological parameters for pregnant and non-pregnant women. The mean red blood cell count, haemoglobin, haematocrit, platelet count, lymphocyte, and monocyte were significantly lower (p < 0.05) among pregnant women than non-pregnant women. Conversely, total white blood cell count, neutrophil, erythrocyte sedimentation rate, mean cell volume, and mean cell hemoglobin were significantly higher (p < 0.05) in pregnant women than in non-pregnant women.

Table 3 shows mean serum erythropoietin, urinary hepcidin and iron status for pregnant n and non-pregnant women. The mean serum erythropoietin (p < 0.001) and total iron binding capacity (p < 0.002) were significantly higher. In contrast, urinary hepcidin (p < 0.001), serum ferritin (p < 0.001), iron (p < 0.03), and percentage saturation transferrin (p < 0.004) were significantly lower among pregnant women than non-pregnant women.

The red blood cell count, haemoglobin (p > 0.001), and hematocrit were higher (p < 0.05) among pregnant women who registered at ≤12 weeks than those who registered at >12 weeks of pregnancy. Conversely, the mean MCHC, platelet, and erythrocyte sedimentation rate were significantly lower (p < 0.05) among pregnant women who registered ≤12 weeks than those who registered for antenatal care at >12 weeks of pregnancy. The mean concentrations of mean cell volume, mean cell haemoglobin, total white blood cell count, lymphocytes, monocytes, and granulocytes were not significantly different between the groups (Table 4).

The mean serum iron and ferritin in pregnant women who registered for antenatal care at ≤12 weeks of pregnancy were significantly higher (p < 0.05) than those who registered at >12 weeks of pregnancy. The mean urinary hepcidin, total iron binding capacity, and percentage saturation transferrin were significantly lower (p < 0.05) in pregnant women who registered for antenatal at ≤12 weeks of pregnancy than those who registered for antenatal at >12 weeks of pregnancy. Serum erythropoietin levels did not differ significantly between the groups, as shown in Table 5.

Table 6 shows the measured hematological parameters by anemic status of pregnant women according to Lawson’s criteria. The red blood cell count, hemoglobin, hematocrit, mean cell volume, and mean cell hemoglobin concentration in anemic pregnant women were significantly lower (p < 0.05) compared to non-anemic pregnant women. The platelet count and erythrocyte sedimentation rate in anemic pregnant women were, however, significantly higher compared to those of non-anemic pregnant women. But the mean cell haemoglobin, total white blood cell, lymphocyte, monocyte and granulocyte in anemic pregnant women were not significantly different from those in non-anaemic pregnant women.

Table 7 indicates the comparison of the levels of serum erythropoietin, urinary hepcidin and iron status parameters between anaemic and non-anaemic pregnant women based on Lawson criteria (Hb < 10 g/dL). The mean serum erythropoietin and urinary hepcidin of anaemic pregnant women were significantly higher (p < 0.05) than those of non-anaemic pregnant women. Still, serum ferritin, iron, total iron binding capacity, and percentage saturation transferrin in anaemic pregnant women were significantly lower (p < 0.05) compared to non-anaemic pregnant women.

Table 8 shows the cut-off points, sensitivities, and specificities of the different parameters as predictors of anemia in pregnancy. Haemoglobin cut-off point is 10.95 g/dL with 100% sensitivity and 95% specificity, haematocrit cut-off was 30.45% with 95% sensitivity and 87.0% specificity, serum erythropoietin cut-off for anemia is 5.05 mIU/mL with 92% sensitivity and 62% specificity. In contrast, the cut-off, sensitivity, and specificity of urinary hepcidin for anemia detection was 11.9 ng/mL, 95%, and 73%. Also, the serum iron cut-off was 30.5 µg/dL with 96.0% sensitivity and 100% specificity, while the serum ferritin cut-off was 18.5 ng/dL with 92% sensitivity and 100% specificity, respectively.

This research indicates that pregnancy is associated with marked changes in iron metabolism, marked by increased erythropoietic activity and reduced iron stores. The significantly elevated serum erythropoietin and total iron-binding capacity, and decreased ferritin, serum iron, hepcidin, and transferrin saturation, suggest a state of high iron demand and relative iron deficiency among pregnant women. Early antenatal registration (≤12 weeks) appears beneficial, as it is associated with better iron status and more favorable regulatory profiles compared to late registration.

The study finds that a substantial majority (81%) of women scheduled for their first antenatal visit after 12 weeks of gestation do so later than the World Health Organization’s recommended guidelines. This prevalent real-world trend of late-first bookings suggests that these adaptive and potentially deficient conditions may already be present before formal antenatal care commences. This finding aligns with previous research [21,22,23]. However, this figure exceeds the rates of 49.5% [24], 68.8% [25], and 56.98% [26] reported in Tanzania and Ethiopia. Late bookings result in pregnant women missing timely early pregnancy assessments that are crucial for favorable pregnancy outcomes. Authors have suggested that women who made late bookings were found to be twice as likely to experience anaemia compared to those who booked early. Additionally, late bookers were reported to be six times more likely to have low-birth-weight infants than their early booking counterparts [24,25,26].

The absence of significant differences in socioeconomic factors such as occupation and education between the cases and controls reinforces the conclusion that the observed changes in biomarkers are mainly influenced by the pregnancy itself, rather than by confounding demographic factors [27]. The haematological profile of women in the early stages of pregnancy is characterized by a unique pattern of dilutional anaemia accompanied by reactive erythropoietic characteristics, a physiological increase in leukocytes attributed to neutrophils, and mild thrombocytopenia. These alterations align with and support previously documented hormonal influences (increased EPO, suppressed hepcidin [28]). It is vital to recognize this pattern as a normal physiological response to prevent unnecessary investigations, while remaining alert to identify any values that may deviate significantly from this expected adaptive baseline.

The comparative examination of hematological parameters illustrated in Table 2 offers a detailed overview of the significant physiological adjustments the maternal hematopoietic system undergoes by the time of the first antenatal booking. The identified pattern does not suggest a pathological condition; instead, it reflects a coherent and complex response to the requirements of pregnancy.

The findings directly support the study’s emphasis on biomarkers related to iron demand and regulation. They indicate that by the first antenatal booking, the maternal organism has already transitioned into a state of “regulated iron deficiency,” characterized by a hepcidin-EPO axis that is actively mobilizing iron for erythropoiesis. The measurable iron reserves are being systematically depleted. This condition is notably more pronounced in women who book after 12 weeks, highlighting the dynamic aspect of this adaptation. The clinical implications are clear: reliance solely on conventional haematological indices such as haemoglobin is inadequate. Evaluating ferritin (a marker of iron stores) and understanding the altered regulatory environment (via hepcidin and EPO) are crucial for the early identification of genuine iron deficiency and the prompt initiation of treatment, potentially before overt anemia manifests. This biomarker profile offers a physiological basis for advocating early and proactive iron supplementation during pregnancy, ideally commencing in the first trimester.

The identified pattern, significantly higher serum erythropoietin (EPO) and total iron-binding capacity (TIBC) alongside significantly reduced urinary hepcidin, serum ferritin, serum iron, and transferrin saturation, provides a coherent representation of a system that prioritizes iron mobilization for erythropoiesis at the cost of iron reserves. This triad of findings is essential for understanding the physiology of iron demand during pregnancy.

The notable suppression of urinary hepcidin signifies a vital adaptive response. Hepcidin, which regulates the entry of iron into plasma, is downregulated to enhance dietary iron absorption and promote the release of recycled iron from macrophages. This suppression directly addresses the perceived need for iron.

Simultaneously, the substantial increase in serum EPO, which catalyzes heightened red blood cell production, is driven by both the relative anaemia resulting from plasma volume expansion and the emerging iron deficiency. The alterations in both regulatory hormones (hepcidin decreasing and EPO increasing) compellingly illustrate the body’s coordinated effort to satisfy the rising iron requirements of the fetus and the expanding maternal red cell mass [28,29].

Conventional parameters of iron status validate the implications of this regulatory shift. The significantly reduced levels of serum ferritin, serum iron, and transferrin saturation indicate a swift depletion of iron reserves and a reduction in the circulating iron available for hemoglobin synthesis. The increased TIBC further indicates the body’s ability to optimize iron transport capacity in a context of limited iron availability. This overall profile is indicative of developing iron-deficient erythropoiesis, which precedes and underpins the high incidence of anemia observed in this group [30,31].

Moreover, the comparison between women booking at ≤12 weeks and >12 weeks of gestation (Table 4) provides a significant temporal perspective on the evolution of these changes. The observation that haemoglobin, haematocrit, and RBC count were elevated in the earlier booking group implies a less pronounced dilutional anaemia during the initial trimester [32,33]. In contrast, the notably lower MCHC and elevated ESR observed in the later booking group suggest a progression towards more pronounced hemoglobin dilution and inflammation-related alterations as pregnancy progresses. The absence of a significant difference in MCV and MCH between these groups indicates that macrocytic compensation (potentially due to EPO stimulation or folate) is an early and persistent response. Most critically, this comparison of gestational age suggests that the essential regulatory changes (hepcidin suppression, EPO increase, and depletion of iron stores) are already initiated early in the first trimester and become more firmly established by the second trimester, underscoring a very limited window for preventive intervention. This aligns with previous reports indicating that elevated hepcidin levels in the first trimester are linked to a reduced risk of iron deficiency anaemia in late pregnancy [15].

The findings presented in Table 5 offer a crucial temporal perspective on the study of iron biomarkers, demonstrating that the physiological adaptation to the iron demands of pregnancy is a dynamic and rapidly changing process. The comparison between women booking at or before 12 weeks of gestation and those booking after 12 weeks reveals a significant transition in iron homeostasis during the first trimester, shifting from a state of relative iron repletion to one of active depletion.

The observation that serum iron and ferritin levels were significantly higher in the ≤12 weeks group is critical. It suggests that, on average, women in the very early stages of pregnancy still retain measurable iron reserves. This implies that the considerable demand for iron stores for fetal-placental development and maternal erythrocyte expansion has not yet completely surpassed the body’s reserves or absorptive capacity at this stage [34].

In contrast, the notably reduced urinary hepcidin levels in the ≤12 weeks group indicate that the regulatory response initiates almost immediately. The decrease in hepcidin represents an early, proactive adjustment aimed at improving iron absorption in anticipation of future needs, even before significant depletion of stores. This initial downregulation of hepcidin likely facilitates optimal iron acquisition during the crucial period of organogenesis [35].

Furthermore, the markedly lower Total Iron-Binding Capacity (TIBC) and transferrin saturation in the earlier booking group further clarify this staged adaptation. A reduced TIBC implies that the production of transferrin, the protein responsible for iron transport, has not yet reached its maximum upregulation. The diminished saturation percentage, despite elevated serum iron levels, suggests that the circulating transport system is not yet fully activated or saturated, which aligns with a less stressed condition [34].

The most compelling finding is the lack of a significant difference in serum erythropoietin (EPO) levels between the two gestational-age groups. This suggests that the stimulus for erythropoiesis, that is, primarily tissue hypoxia resulting from dilutional anemia, may not vary substantially between these early time points. Alternatively, it may imply that the EPO response is already at its peak very early in pregnancy due to the initial expansion of plasma volume, and therefore does not show significant differences between these subgroups. This separation between EPO (which remains similar) and hepcidin (which is more significantly suppressed early on) underscores that these two regulatory hormones, while frequently coordinated, can be affected by distinct physiological signals during early pregnancy [9,10].

The examination of hematological parameters in relation to anaemic status (Table 6) offers essential insights into the functional implications of altered iron regulation during early pregnancy, as delineated by Lawson’s criteria. The observed pattern strongly supports the conclusion that the predominant anaemia in this cohort is microcytic and hypochromic, characteristic features of iron-deficient erythropoiesis. This establishes a direct connection between the hematological profile and the biomarker evidence of iron depletion [36].

The notably elevated platelet count (thrombocytosis) in anaemic women is a well-documented reactive phenomenon associated with iron-deficiency anemia, even in the absence of pregnancy. Iron deficiency can stimulate platelet production, potentially as a compensatory mechanism or due to overlapping cytokine influences on megakaryopoiesis and erythropoiesis [37].

Upon reviewing Table 7, the findings extend the conversation beyond mere iron deficiency, emphasizing a possible dysregulation of iron metabolism in anemia during early pregnancy. Increased levels of hepcidin in anaemic women serve as a significant warning sign, suggesting that the anaemia may not solely stem from insufficient dietary intake but could also involve an inflammatory factor that hinders iron utilization [38]. Consequently, evaluating hepcidin and inflammatory markers in conjunction with standard iron assessments could facilitate a more tailored and effective approach to managing anaemia in early pregnancy.

The examination of cut-off values, sensitivity, and specificity for various parameters (as shown in Table 8) shifts the focus of the study from merely describing physiological adaptations to assessing the clinical diagnostic value of these biomarkers in identifying anaemia during early pregnancy. The findings indicate a hierarchy in diagnostic efficacy, with conventional hematological indices and iron storage markers demonstrating superior performance compared to regulatory hormones when evaluated as standalone tests, thereby highlighting the beneficial role of a multi-parameter approach.

The efficacy of haemoglobin (cut-off at 10.26 g/dL), exhibiting 100% sensitivity and 95% specificity, is predictably robust, as it serves as the primary measure for defining anaemia (according to Lawson’s criteria: Hb < 10 g/dL). The nearly perfect sensitivity confirms its effectiveness in accurately identifying individuals with anaemia. The 95% specificity suggests that a measurement below this threshold is a strong indicator of true anaemia, with a minimal false-positive rate.

The remarkable diagnostic efficacy of serum ferritin (cut-off 18.5 ng/mL) and serum iron (cut-off 30.5 µg/dL), both achieving 100% specificity alongside high sensitivity (92% and 96%, respectively), represents the most crucial finding from a clinical etiological standpoint.

A specificity of 100% indicates that no non-anemic woman within this cohort had a ferritin level below 18.5 ng/mL or an iron level below 30.5 µg/dL. This establishes low ferritin as an almost flawless confirmatory test for iron-deficiency anemia in this scenario. A ferritin level dropping below this threshold at the initial visit strongly indicate that depleted iron stores are likely responsible for the anaemia, warranting immediate and potentially aggressive iron therapy.

Conversely, the regulatory hormones demonstrate a different performance profile. Serum erythropoietin (EPO) shows high sensitivity (92%) but lower specificity (62%). This reflects its physiological function: it is elevated in most cases of anemia (high sensitivity). Still, it is also raised in many non-anemic pregnant women due to the physiological dilution and EPO stimulation associated with normal pregnancy, resulting in a higher number of false positives (lower specificity). Consequently, EPO serves as a sensitive marker of erythropoietic stress but cannot independently differentiate between physiological adaptation and pathological anemia.

Urinary hepcidin exhibits a balanced yet moderate profile, with a sensitivity of 95% and a specificity of 73%. The notably high sensitivity suggests that dysregulation of hepcidin is a prevalent characteristic. The specificity of 73% indicates that it is more specific than EPO, although less so than ferritin. This pattern reinforces its role as a pathophysiological marker of potential iron blockage, but it should not be regarded as a definitive diagnostic tool for iron deficiency.

The findings highlight significant changes in iron metabolism during pregnancy, marked by increased erythropoietic activity and diminished iron reserves. Elevated levels of serum erythropoietin and total iron-binding capacity, paired with reductions in ferritin, serum iron, hepcidin, and transferrin saturation, point to a state of heightened iron demand and relative deficiency in pregnant women. Early antenatal registration (within 12 weeks) is associated with improved iron status and more favorable regulatory profiles compared to late registration. Overall, the results underscore the importance of early antenatal care and timely monitoring of iron status to prevent and manage iron deficiency and anaemia during pregnancy.