Iron deficiency anemia (IDA) represents the most prevalent nutritional deficiency globally, affecting approximately 1.6 billion individuals, with pregnant women constituting a particularly vulnerable population [1]. The World Health Organization estimates that 40% of pregnant women worldwide are anemic, with substantially higher prevalence in low- and middle-income countries [2]. In India, the burden is particularly severe, with national surveys reporting anemia prevalence ranging from 50% to 70% among pregnant women, despite decades of universal iron supplementation programs [3]. The consequences of maternal anemia extend beyond maternal well-being to significantly impact fetal and neonatal outcomes. Iron deficiency during pregnancy is associated with increased risks of preterm delivery, low birth weight, intrauterine growth restriction, perinatal mortality, and impaired neurocognitive development in offspring [4]. Maternal complications include increased susceptibility to infections, cardiac dysfunction, postpartum hemorrhage, and maternal mortality [5]. The economic burden encompasses healthcare costs and long-term developmental impacts, making maternal anemia a significant public health priority. Current strategies for preventing and treating gestational anemia predominantly rely on universal or targeted oral iron supplementation, typically providing 60-120 mg elemental iron daily throughout pregnancy [6]. However, despite widespread implementation of supplementation programs, anemia prevalence remains stubbornly high, indicating substantial gaps in therapeutic effectiveness. Multiple factors contribute to treatment failure, including poor adherence due to gastrointestinal adverse effects, inadequate dosing, delayed initiation, and most critically, impaired iron absorption in the presence of inflammation [7]. Conventional diagnostic and monitoring approaches utilize hemoglobin concentration and serum ferritin as primary biomarkers. However, both markers possess significant limitations. Hemoglobin represents a late indicator, declining only after iron stores are depleted and erythropoiesis is impaired, thereby missing opportunities for early intervention [8]. Serum ferritin, while reflecting body iron stores under normal conditions, is an acute-phase reactant that becomes falsely elevated during inflammation, infection, or chronic diseases—conditions frequently encountered during pregnancy [9]. This elevation masks underlying iron deficiency, leading to underdiagnosis and undertreatment of true iron deficiency in inflamed states. Hepcidin, a 25-amino acid peptide hormone synthesized primarily in hepatocytes, has emerged as the master regulator of systemic iron homeostasis [10]. Discovered in 2001, hepcidin controls iron entry into plasma by regulating ferroportin, the sole known cellular iron exporter present on enterocytes, hepatocytes, and macrophages [11]. When hepcidin binds to ferroportin, it induces ferroportin internalization and degradation, thereby blocking dietary iron absorption from duodenal enterocytes and preventing iron release from macrophages and hepatocytes [12]. Conversely, when hepcidin levels are low in iron-deficient states, ferroportin remains functional, facilitating efficient intestinal iron absorption and mobilization of stored iron. Hepcidin synthesis is regulated by multiple physiological signals including body iron stores, erythropoietic demand, inflammation, and hypoxia, making it an integrated sensor of iron status and availability [13]. In iron deficiency, hepcidin production is suppressed, allowing maximal iron absorption. However, inflammation—mediated primarily through interleukin-6—potently stimulates hepcidin production regardless of iron status, creating functional iron deficiency by sequestering iron within macrophages and reducing absorption [14]. This inflammation-induced hepcidin elevation explains why oral iron supplementation is often ineffective in inflamed individuals despite apparent iron deficiency. Recent research has demonstrated that measuring serum hepcidin can predict oral iron absorption capacity. Stoffel et al. showed that fractional iron absorption inversely correlates with hepcidin levels, with individuals having low hepcidin (<5 ng/mL) absorbing substantially more iron than those with elevated hepcidin [15]. Pasricha and colleagues demonstrated that hepcidin measurement can identify women unlikely to benefit from oral iron who should receive intravenous formulations instead [16]. Furthermore, studies have shown that oral iron administration itself acutely elevates hepcidin for 24-48 hours, suggesting that alternate-day dosing may be more effective than daily administration in iron-deficient individuals [17]. Despite these mechanistic insights and emerging evidence from non-pregnant populations, limited data exist regarding the clinical utility of hepcidin-guided iron supplementation strategies in pregnant women. Pregnancy induces unique alterations in iron metabolism, with physiological hemodilution, increased iron requirements for fetal development and placental function, and inflammatory changes potentially affecting hepcidin regulation [18]. Whether hepcidin-based individualization of iron therapy improves outcomes compared to conventional approaches in this population remains inadequately explored. Given the persistently high burden of gestational anemia despite universal supplementation programs, the limitations of current diagnostic markers, and the potential for hepcidin to guide personalized iron therapy, we conducted this prospective comparative study. The primary objective was to evaluate whether hepcidin-guided iron supplementation, where dosing is individualized based on serum hepcidin levels, results in superior hematological improvement compared to conventional fixed-dose hemoglobin-based therapy in pregnant women with mild to moderate anemia. Secondary objectives included assessment of ferritin normalization rates, requirement for intravenous iron rescue therapy, adverse effect profiles, adherence patterns, and anemia status at term delivery.

Study Design and Setting This prospective, open-label, comparative study was conducted in the Department of Obstetrics and Gynaecology at Basaveshwara Medical College and Research Center, Chitradurga, Karnataka, India, over an 11-month period from January 2025 to November 2025. Sample Size Calculation Sample size was calculated based on the primary outcome of mean hemoglobin increase at 8 weeks post-intervention. Based on pilot data and previous literature, we anticipated mean hemoglobin rise of 2.2±0.8 g/dL in the hepcidin-guided group versus 1.5±0.9 g/dL in the standard therapy group. Using the formula for comparison of two independent means with two-tailed alpha error of 0.05, power of 80%, and accounting for 10% potential attrition, the calculated minimum sample size was 68 participants per group. We enrolled 75 participants per group (total 150) to ensure adequate power for secondary analyses. Study Population and Eligibility Criteria Pregnant women attending antenatal clinics were systematically screened for eligibility through hemoglobin measurement. Women meeting inclusion criteria were provided detailed study information and invited to participate. Inclusion criteria comprised: (1) maternal age 18-35 years; (2) singleton pregnancy; (3) gestational age 14-28 weeks confirmed by first-trimester ultrasound or reliable last menstrual period; (4) mild to moderate anemia defined as hemoglobin 8.0-10.9 g/dL; (5) serum C-reactive protein (CRP) <10 mg/L to exclude acute inflammation; (6) no iron supplementation received in the preceding 8 weeks; (7) willingness to comply with follow-up schedule. Exclusion criteria included: (1) severe anemia (hemoglobin <8.0 g/dL) requiring immediate parenteral iron or blood transfusion; (2) hemoglobinopathies including thalassemia trait or sickle cell disease; (3) known chronic inflammatory, autoimmune, or infectious diseases; (4) hepatic dysfunction (ALT >2× upper normal limit) or chronic liver disease; (5) renal impairment (serum creatinine >1.2 mg/dL); (6) multiple pregnancy; (7) acute febrile illness; (8) known gastrointestinal disorders or malabsorption syndromes; (9) blood transfusion within previous 12 weeks; (10) known hypersensitivity to iron preparations. Randomization and Allocation Eligible participants were randomly allocated in 1:1 ratio to either hepcidin-guided therapy or standard therapy groups using computer-generated random number sequences in blocks of 10. Allocation concealment was maintained using sequentially numbered, sealed, opaque envelopes opened only after enrollment confirmation. Due to the nature of interventions involving different dosing algorithms, blinding of participants and clinicians was not feasible, but laboratory personnel performing hematological assessments were blinded to group allocation. Baseline Assessment All participants underwent comprehensive baseline evaluation including detailed medical and obstetric history, physical examination, and laboratory investigations. Venous blood samples (10 mL) were collected after overnight fasting for complete blood count using automated hematology analyzer (Sysmex XN-1000), serum ferritin by electrochemiluminescence immunoassay (Roche Cobas e411), serum hepcidin-25 by enzyme-linked immunosorbent assay (DRG Hepcidin-25 ELISA kit with detection range 0-300 ng/mL), high-sensitivity CRP by immunoturbidimetric assay, liver and renal function tests, and peripheral blood smear examination. Gestational age was confirmed by ultrasound examination. Intervention Protocols Hepcidin-Guided Therapy Group (n=75): Iron dosing was individualized based on baseline and follow-up serum hepcidin-25 levels according to the following algorithm: • Hepcidin <10 ng/mL: 100 mg elemental iron daily (as ferrous sulfate 300 mg) • Hepcidin 10-25 ng/mL: 60 mg elemental iron daily (as ferrous sulfate 180 mg) • Hepcidin >25 ng/mL: oral iron temporarily withheld; reassessment after 2 weeks Hepcidin levels were reassessed at 2-week intervals for dose adjustment. Iron was administered on empty stomach (1 hour before meals) with vitamin C (100 mg ascorbic acid) to enhance absorption. Standard Therapy Group (n=75): Participants received fixed-dose oral iron supplementation following World Health Organization guidelines: 100 mg elemental iron daily (as ferrous sulfate 300 mg) throughout the study period, administered with vitamin C (100 mg) on empty stomach, irrespective of hepcidin levels or hematological response. Both groups received standardized counseling regarding administration timing, dietary iron sources, importance of adherence, and management of adverse effects. Folic acid (5 mg daily) and calcium supplementation (1000 mg daily, given separately from iron) were provided to all participants as per institutional protocol. Follow-up Schedule and Monitoring Participants were followed at 2-week intervals for the first 8 weeks, then monthly until delivery. At each visit, compliance assessment was performed through pill counting and patient interview, adverse effects were systematically recorded using structured questionnaires, and clinical evaluation including blood pressure and weight measurement was conducted. Laboratory reassessment was performed at 4 weeks (hemoglobin, ferritin), 8 weeks (complete profile including hemoglobin, ferritin, hepcidin, CRP), and at 36 weeks gestation (hemoglobin, ferritin). Adherence was calculated as percentage of prescribed doses consumed based on pill counts. Rescue Therapy Criteria Intravenous iron therapy (iron sucrose 200 mg infusions repeated as needed) was administered as rescue treatment for: (1) persistent or worsening anemia despite 8 weeks oral therapy; (2) intolerable adverse effects precluding oral continuation; (3) hemoglobin decline >1 g/dL during treatment; (4) hemoglobin <9 g/dL at ≥34 weeks gestation. Outcome Measures Primary outcome: Mean increase in hemoglobin concentration from baseline to 8 weeks post-intervention. Secondary outcomes: (1) Proportion of participants achieving target hemoglobin ≥11 g/dL at 8 weeks; (2) mean increase in serum ferritin at 8 weeks; (3) proportion achieving ferritin normalization (≥15 ng/mL) at 8 weeks; (4) requirement for intravenous iron rescue therapy; (5) incidence and severity of adverse effects (nausea, vomiting, constipation, diarrhea, abdominal pain); (6) treatment adherence rates; (7) anemia prevalence at term (≥37 weeks); (8) mean hemoglobin at delivery. Statistical Analysis Data were analyzed using Statistical Package for Social Sciences (SPSS) version 26.0 (IBM Corp., Armonk, NY). Normality of continuous variables was assessed using Shapiro-Wilk test. Continuous variables with normal distribution were expressed as mean ± standard deviation and compared between groups using independent samples Student's t-test. Non-normally distributed variables were expressed as median (interquartile range) and compared using Mann-Whitney U test. Within-group changes from baseline were analyzed using paired t-test. Categorical variables were presented as frequencies and percentages and compared using chi-square test or Fisher's exact test when expected cell frequencies were <5. Multivariate linear regression analysis was performed to identify independent predictors of hemoglobin response, adjusting for potential confounders including baseline hemoglobin, ferritin, hepcidin, gestational age, BMI, and adherence. Results were presented as unstandardized regression coefficients with 95% confidence intervals. Statistical significance was set at two-tailed p-value <0.05 for all analyses. Analysis was conducted on intention-to-treat basis with all randomized participants included.

Participant Flow and Baseline Characteristics During the study period, 186 pregnant women with anemia were screened for eligibility. Thirty-six were excluded (18 with hemoglobin <8.0 g/dL requiring immediate parenteral therapy, 8 with CRP ≥10 mg/L, 4 with thalassemia trait, 3 with chronic kidney disease, 2 with twin pregnancy, 1 declined participation), resulting in enrollment of 150 participants who were randomized to hepcidin-guided therapy (n=75) or standard therapy (n=75). All 150 participants completed the 8-week primary assessment period, and 147 (98%) were followed through delivery (2 lost to follow-up in hepcidin group, 1 in standard group due to relocation). Table 1 presents baseline demographic, clinical, and laboratory characteristics. Groups were well-matched for all baseline parameters. Mean maternal age was 25.6±3.3 years in the hepcidin-guided group versus 25.8±3.4 years in the standard group (p=0.716). Mean gestational age at enrollment was comparable (20.4±3.6 versus 20.8±3.5 weeks, p=0.495). Baseline hemoglobin concentrations were similar (9.3±0.8 g/dL versus 9.2±0.7 g/dL, p=0.426), as were serum ferritin levels (18.4±8.6 ng/mL versus 19.2±9.1 ng/mL, p=0.576). Table 1: Baseline Demographic, Clinical, and Laboratory Characteristics Parameter Hepcidin-Guided Group (n=75) Standard Therapy Group (n=75) p-value Maternal age (years) 25.6 ± 3.3 25.8 ± 3.4 0.716 Gestational age (weeks) 20.4 ± 3.6 20.8 ± 3.5 0.495 Body mass index (kg/m²) 23.8 ± 2.9 24.2 ± 3.1 0.426 Nulliparity, n (%) 42 (56.0) 38 (50.7) 0.511 Education ≥12 years, n (%) 46 (61.3) 44 (58.7) 0.742 Rural residence, n (%) 52 (69.3) 48 (64.0) 0.487 Previous anemia, n (%) 28 (37.3) 32 (42.7) 0.502 Baseline hemoglobin (g/dL) 9.3 ± 0.8 9.2 ± 0.7 0.426 Baseline MCV (fL) 76.4 ± 6.2 77.1 ± 6.8 0.524 Baseline serum ferritin (ng/mL) 18.4 ± 8.6 19.2 ± 9.1 0.576 Baseline hepcidin-25 (ng/mL) 12.8 ± 6.4 13.2 ± 6.8 0.708 Baseline CRP (mg/L) 3.2 ± 2.1 3.4 ± 2.3 0.586 Baseline serum hepcidin-25 levels were similar between groups (12.8±6.4 ng/mL versus 13.2±6.8 ng/mL, p=0.708). Within the hepcidin-guided group at baseline, 34 participants (45.3%) had hepcidin <10 ng/mL, 36 (48.0%) had hepcidin 10-25 ng/mL, and 5 (6.7%) had hepcidin >25 ng/mL. Primary Outcome: Hemoglobin Response Table 2 presents hematological outcomes at 4 and 8 weeks post-intervention. At 8 weeks, the primary endpoint analysis demonstrated significantly greater mean hemoglobin increase in the hepcidin-guided group compared to standard therapy (2.4±0.6 g/dL versus 1.6±0.7 g/dL, p<0.001). Mean hemoglobin concentration at 8 weeks was 11.7±0.8 g/dL in the hepcidin-guided group versus 10.8±0.9 g/dL in the standard group (p<0.001). The proportion of participants achieving target hemoglobin ≥11 g/dL at 8 weeks was significantly higher with hepcidin-guided therapy (59/75, 78.7% versus 40/75, 53.3%; p=0.002). Complete resolution of anemia (hemoglobin ≥11 g/dL) was achieved in 78.7% of the hepcidin-guided group compared to 53.3% of the standard group (p=0.002). Table 2: Hematological Outcomes at 4 and 8 Weeks Post-Intervention Parameter Hepcidin-Guided Group (n=75) Standard Therapy Group (n=75) p-value At 4 Weeks Hemoglobin (g/dL) 10.6 ± 0.8 10.1 ± 0.9 <0.001 Hemoglobin increase (g/dL) 1.3 ± 0.5 0.9 ± 0.6 <0.001 Serum ferritin (ng/mL) 32.6 ± 12.4 28.4 ± 11.8 0.040 At 8 Weeks Hemoglobin (g/dL) 11.7 ± 0.8 10.8 ± 0.9 <0.001 Hemoglobin increase (g/dL) 2.4 ± 0.6 1.6 ± 0.7 <0.001 Hemoglobin ≥11 g/dL, n (%) 59 (78.7) 40 (53.3) 0.002 Serum ferritin (ng/mL) 48.6 ± 16.2 38.2 ± 14.6 <0.001 Ferritin increase (ng/mL) 30.2 ± 14.8 19.0 ± 12.4 <0.001 Ferritin ≥15 ng/mL, n (%) 54 (72.0) 38 (50.7) 0.009 Hepcidin-25 (ng/mL) 18.4 ± 8.2 16.8 ± 7.6 0.234 MCV (fL) 84.2 ± 5.4 81.6 ± 6.2 0.008 Reticulocyte count (%) 2.1 ± 0.6 1.8 ± 0.7 0.008 Mean serum ferritin increase at 8 weeks was significantly greater in the hepcidin-guided group (30.2±14.8 ng/mL versus 19.0±12.4 ng/mL, p<0.001). Ferritin normalization (≥15 ng/mL) was achieved in 72.0% of the hepcidin-guided group versus 50.7% of the standard group (p=0.009). Mean corpuscular volume (MCV) showed greater improvement with hepcidin-guided therapy (84.2±5.4 fL versus 81.6±6.2 fL, p=0.008), indicating better correction of microcytosis. Secondary Outcomes and Clinical Parameters Table 3 presents secondary clinical outcomes, treatment parameters, and outcomes at term delivery. The requirement for intravenous iron rescue therapy was substantially lower in the hepcidin-guided group (7/75, 9.3% versus 21/75, 28.0%; p=0.005), representing a 67% relative risk reduction. Time to achieve hemoglobin ≥11 g/dL was significantly shorter with hepcidin guidance (5.8±1.4 weeks versus 7.2±1.8 weeks, p<0.001). Table 3: Secondary Outcomes, Adverse Effects, and Term Delivery Parameters Outcome Hepcidin-Guided Group (n=75) Standard Therapy Group (n=75) p-value Treatment Parameters IV iron requirement, n (%) 7 (9.3) 21 (28.0) 0.005 Time to Hb ≥11 g/dL (weeks) 5.8 ± 1.4 7.2 ± 1.8 <0.001 Mean daily iron dose (mg)* 82.4 ± 18.6 100.0 ± 0.0 <0.001 Treatment adherence (%) 86.4 ± 12.2 78.6 ± 14.8 <0.001 Adverse Effects Any GI adverse effect, n (%) 18 (24.0) 33 (44.0) 0.012 Nausea, n (%) 12 (16.0) 24 (32.0) 0.024 Constipation, n (%) 8 (10.7) 18 (24.0) 0.035 Abdominal pain, n (%) 6 (8.0) 14 (18.7) 0.056 Diarrhea, n (%) 2 (2.7) 5 (6.7) 0.243 Treatment discontinuation, n (%) 2 (2.7) 8 (10.7) 0.051 At Term Delivery (n=73/74) Gestational age at delivery (weeks) 38.6 ± 1.2 38.4 ± 1.4 0.358 Hemoglobin at delivery (g/dL) 11.8 ± 1.1 10.9 ± 1.3 <0.001 Anemia at term, n (%) 9 (12.0) 22 (29.3) 0.011 Birth weight (grams) 2924 ± 386 2856 ± 424 0.314 Preterm delivery, n (%) 4 (5.3) 6 (8.0) 0.514 Low birth weight, n (%) 8 (10.7) 12 (16.0) 0.348 *Mean daily dose calculated over 8-week treatment period Gastrointestinal adverse effects occurred significantly less frequently in the hepcidin-guided group (18/75, 24.0% versus 33/75, 44.0%; p=0.012). Specifically, nausea was reported by 16.0% versus 32.0% (p=0.024) and constipation by 10.7% versus 24.0% (p=0.035). Treatment discontinuation due to adverse effects was lower with hepcidin guidance, though this did not reach statistical significance (2.7% versus 10.7%, p=0.051). Treatment adherence, calculated by pill count, was significantly higher in the hepcidin-guided group (86.4±12.2% versus 78.6±14.8%, p<0.001), likely reflecting both the individualized approach and reduced adverse effects. Mean daily iron dose over the 8-week period was lower in the hepcidin-guided group (82.4±18.6 mg versus fixed 100 mg, p<0.001) due to dose adjustments based on hepcidin levels. At term delivery (≥37 weeks), mean hemoglobin was significantly higher in the hepcidin-guided group (11.8±1.1 g/dL versus 10.9±1.3 g/dL, p<0.001). Anemia prevalence at term was substantially lower with hepcidin guidance (9/73, 12.0% versus 22/74, 29.3%; p=0.011). Birth weight and preterm delivery rates showed no significant differences between groups. Multivariate linear regression analysis identified treatment group (hepcidin-guided versus standard) as an independent predictor of hemoglobin increase at 8 weeks (unstandardized coefficient 0.74, 95% CI 0.48-1.00, p<0.001) after adjusting for baseline hemoglobin, ferritin, hepcidin, gestational age, BMI, and adherence. Other significant predictors included baseline hepcidin <10 ng/mL (coefficient 0.52, 95% CI 0.26-0.78, p<0.001) and adherence percentage (coefficient 0.02 per 1% increase, 95% CI 0.01-0.03, p=0.002).

This prospective comparative study demonstrates that hepcidin-guided iron supplementation results in superior hematological outcomes compared to conventional fixed-dose hemoglobin-based therapy in pregnant women with mild to moderate anemia. Women receiving individualized iron dosing based on serum hepcidin levels achieved significantly greater hemoglobin increases, higher rates of anemia resolution, better ferritin repletion, reduced need for intravenous iron rescue therapy, fewer adverse effects, and improved anemia status at term delivery. The observed mean hemoglobin increase of 2.4 g/dL at 8 weeks with hepcidin-guided therapy versus 1.6 g/dL with standard therapy represents a clinically meaningful difference with important implications for maternal and fetal health. This 50% greater response aligns with the physiological rationale that iron supplementation is most effective when administered to individuals with low hepcidin who can efficiently absorb and utilize iron, while avoiding unnecessary supplementation in those with elevated hepcidin who cannot effectively absorb oral iron due to ferroportin blockade [1]. Our findings are consistent with recent evidence from non-pregnant populations demonstrating that hepcidin predicts oral iron absorption. Stoffel et al. showed that fractional iron absorption was inversely correlated with serum hepcidin, with individuals having hepcidin <5 ng/mL absorbing 2-3 times more iron than those with higher levels [2]. Moretti et al. demonstrated that consecutive daily iron doses progressively increase hepcidin, resulting in diminished absorption of subsequent doses, suggesting that alternate-day dosing may be more effective [3]. Our study extends these findings to pregnant women, a population with unique iron requirements and physiological adaptations. The substantially reduced requirement for intravenous iron rescue therapy in the hepcidin-guided group (9.3% versus 28.0%) represents an important clinical and economic advantage. Intravenous iron administration carries risks of allergic reactions, requires healthcare facility administration, and imposes significant costs compared to oral therapy [4]. By identifying women unlikely to respond to oral therapy early based on elevated hepcidin levels and either temporarily withholding oral iron or promptly transitioning to intravenous therapy, the hepcidin-guided approach optimizes resource utilization while minimizing patient burden. The significantly lower incidence of gastrointestinal adverse effects with hepcidin-guided therapy (24% versus 44%) likely reflects both dose individualization and avoidance of unnecessary iron administration to women with elevated hepcidin. High-dose iron in individuals unable to absorb it due to hepcidin-mediated ferroportin blockade may undergo bacterial fermentation and oxidation in the colon, potentially contributing to adverse effects [5]. Additionally, lower average iron doses in the hepcidin-guided group may have reduced direct gastrointestinal irritation while maintaining therapeutic efficacy through improved targeting. The superior treatment adherence observed with hepcidin guidance (86.4% versus 78.6%) represents a critical factor contributing to better outcomes. Poor adherence is a major barrier to effective oral iron supplementation, with studies reporting adherence rates as low as 40-50% in some populations [6]. The combination of individualized dosing, reduced adverse effects, and potentially enhanced patient engagement through personalized medicine approaches may have synergistically improved adherence in our hepcidin-guided group. The persistence of superior hemoglobin status at term delivery (11.8 versus 10.9 g/dL) and lower anemia prevalence (12% versus 29.3%) demonstrates sustained benefits of the hepcidin-guided approach extending beyond the acute treatment period. Adequate maternal iron status at delivery is critical for minimizing peripartum blood loss complications, preventing postpartum anemia, and ensuring adequate iron transfer to the infant for optimal neurodevelopment [7]. The threefold lower anemia rate at term with hepcidin guidance translates to substantial clinical benefit. Several mechanisms likely contribute to the superior efficacy of hepcidin-guided therapy. First, individualized dosing optimizes iron administration timing and amount based on absorption capacity rather than arbitrary fixed doses. Women with low hepcidin receive adequate supplementation while maintaining maximal absorption, whereas those with elevated hepcidin have therapy temporarily withheld until absorption capacity is restored, avoiding futile oral administration [8]. Second, serial hepcidin monitoring allows dynamic adjustment as iron stores replete and hepcidin rises physiologically, preventing oversupplementation while ensuring adequate repletion. Third, identification of women with persistently elevated hepcidin despite iron deficiency may prompt investigation for underlying inflammation or infection requiring targeted intervention. The mechanistic basis for hepcidin elevation in pregnancy with anemia warrants consideration. While iron deficiency typically suppresses hepcidin to maximize absorption, subclinical inflammation—common during pregnancy due to immune adaptations, genital tract colonization, or chronic infections—can override this suppression and inappropriately elevate hepcidin [9]. This creates functional iron deficiency where iron is sequestered in macrophages despite inadequate erythropoiesis. Our study excluded women with CRP ≥10 mg/L at baseline, yet even low-grade inflammation (CRP 3-10 mg/L) may affect hepcidin and absorption. Hepcidin measurement identifies these women who require alternative approaches such as inflammation treatment or parenteral iron. From a public health perspective, these findings have important implications for anemia prevention programs in high-burden settings like India. Current universal supplementation approaches provide fixed iron doses to all pregnant women regardless of individual iron status, absorption capacity, or inflammatory state [10]. This one-size-fits-all strategy results in substantial treatment failures, medication waste, and adverse effects-related non-adherence. Incorporating hepcidin measurement into targeted screening and supplementation programs could enhance effectiveness while optimizing resource utilization. However, several barriers currently limit widespread hepcidin-guided therapy implementation. Hepcidin assays are not universally available, particularly in resource-limited settings where anemia burden is highest. Assay standardization remains incomplete, with different platforms yielding variable results, though international efforts toward standardization are progressing [11]. Cost considerations may limit accessibility, though economies of scale and point-of-care assay development may address this barrier. Additionally, optimal hepcidin threshold values for treatment decisions require validation across diverse populations and clinical contexts. Several strengths enhance the validity of our findings. The prospective design with concurrent controls minimized bias. Randomization ensured balanced baseline characteristics. Comprehensive assessment including hematological and biochemical parameters, adverse effects, and adherence provided multidimensional outcome evaluation. Blinding of laboratory personnel reduced measurement bias. High follow-up rates (98% completion) minimized attrition bias. Use of standardized, validated assays enhanced measurement accuracy. However, several limitations warrant acknowledgment. The open-label design without participant or clinician blinding may have introduced performance or detection bias, though objective laboratory endpoints minimize this concern. Single-center conduct may limit generalizability to populations with different demographics, dietary patterns, or anemia etiologies. The relatively short follow-up duration (8 weeks primary endpoint) precludes assessment of longer-term outcomes, though term delivery data provide some insight. Exclusion of women with severe anemia, inflammation, or comorbidities limits applicability to these subgroups, though ethical considerations precluded their inclusion. Cost-effectiveness analysis was not performed, limiting assessment of economic viability for program implementation. Finally, mechanisms underlying individual hepcidin variation and treatment response heterogeneity were not fully explored. Future research directions include large-scale multicenter trials in diverse populations to validate findings and refine threshold values, long-term studies assessing maternal cardiovascular and metabolic outcomes and offspring neurodevelopmental outcomes, cost-effectiveness analyses comparing hepcidin-guided versus standard approaches across different healthcare settings, investigation of optimal hepcidin assessment timing and frequency, studies evaluating hepcidin-guided therapy in severe anemia and inflammatory conditions, research on point-of-care hepcidin assays enabling real-time treatment decisions, and mechanistic studies elucidating determinants of hepcidin variation and treatment response in pregnancy. Additionally, implementation science research addressing barriers and facilitators for integrating hepcidin-guided approaches into existing antenatal care programs would inform scale-up strategies.

This prospective comparative study provides compelling evidence that hepcidin-guided iron supplementation demonstrates superior efficacy compared to conventional fixed-dose hemoglobin-based therapy in pregnant women with mild to moderate iron deficiency anemia. Women receiving individualized iron dosing based on serum hepcidin levels achieved significantly greater hemoglobin increases, higher rates of complete anemia resolution, better iron store repletion, substantially reduced need for intravenous iron rescue therapy, fewer gastrointestinal adverse effects, improved treatment adherence, and lower anemia prevalence at term delivery. These findings validate the physiological rationale for hepcidin-guided therapy: by identifying women with low hepcidin who can efficiently absorb oral iron and adjusting dosing accordingly while avoiding futile supplementation in those with elevated hepcidin, this personalized approach optimizes iron utilization, minimizes adverse effects, and maximizes therapeutic benefit. The substantial reduction in intravenous iron requirement represents both clinical and economic advantages, while improved adherence addresses a critical barrier to effective oral supplementation. From a broader perspective, this study demonstrates the potential for biomarker-guided personalized medicine approaches in maternal health. Rather than universal fixed-dose supplementation that yields suboptimal results for many women, individualized therapy based on physiological markers of absorption capacity and iron metabolism offers superior outcomes. As hepcidin assays become more widely available, standardized, and cost-effective, integration of hepcidin measurement into antenatal care protocols represents a promising strategy for enhancing anemia management. For clinical practice, these results support consideration of hepcidin-guided iron therapy as an evidence-based alternative to conventional approaches, particularly for women at high risk of treatment failure, those unable to tolerate standard doses due to adverse effects, and in settings where targeted resource utilization is prioritized. Healthcare providers should be aware that not all anemic pregnant women respond equally to oral iron supplementation, and hepcidin measurement can identify those requiring alternative strategies. For public health programs in high-burden settings, these findings suggest that incorporating hepcidin-based screening and individualized supplementation into targeted anemia prevention initiatives may enhance program effectiveness and cost-efficiency compared to universal fixed-dose approaches. Future efforts should focus on developing affordable point-of-care hepcidin assays, establishing population-specific threshold values, and designing implementation strategies for resource-limited settings where the potential impact is greatest. In conclusion, hepcidin-guided iron supplementation represents an effective, physiologically rational, and patient-centered approach to managing iron deficiency anemia in pregnancy that deserves serious consideration for integration into contemporary maternal health care practices and programs.

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