Helicobacter pylori (H. pylori), a microaerophilic, gram-negative bacterial organism, establishes a chronic infection within the gastric mucosal environment of approximately 50% of the global population, representing one of the most prevalent bacterial infections worldwide ^1,2. Since its discovery by Warren and Marshall in 1984 ³ H. pylori has been implicated in numerous gastrointestinal and extra-gastrointestinal manifestations ^4,5, with hematological disorders emerging as a significant area of clinical concern ⁶. The bacterium's association with iron deficiency anemia (IDA) ^7,8 Immune thrombocytopenic purpura ⁹, and vitamin B12 deficiency ^10,11 has been well documented. However, critical questions remain regarding the mechanisms underlying these associations and potential variations across demographic subgroups.

The relationship between H. pylori infection and hematological parameters extends beyond simple iron deficiency. Multiple pathophysiological mechanisms have been proposed, including chronic gastrointestinal blood loss from gastritis or peptic ulceration, impaired iron absorption due to hypochlorhydria, increased iron uptake by the bacterium itself, and systemic inflammatory responses that affect erythropoiesis ^12–16. Recent systematic reviews and meta-analyses have confirmed a strong association between H. pylori infection and iron deficiency anemia. Previous meta-analyses have consistently demonstrated increased odds of IDA in H. pylori infection, with pooled ORs ranging from 1.72–2.8) ^8,17. However, these aggregate figures may mask underlying heterogeneity in disease pathways and clinical outcomes.

A critical limitation in the current literature is the scarcity of sex-stratified analyses examining the hematological effects of H. pylori. Most published studies report only aggregate results without formal interaction testing to determine whether H. pylori effects differ between males and females ^12,18–20. This represents a significant gap in our understanding, especially considering well-established sex differences in iron metabolism ^21,22, baseline hematological parameters ²³, and the prevalence of H. pylori infection itself, which shows male predominance, even across diverse global populations (46.3% vs 42.7% in males vs females; 95% CI: 42.1–50.5 vs 39–46.5) ².

Sex differences in hematological parameters are multifaceted and influenced by numerous biological factors. Women of reproductive age experience regular menstrual blood loss, leading to higher iron requirements and increased susceptibility to iron deficiency ²². Conversely, males generally have higher baseline hemoglobin and hematocrit levels because of androgenic effects on erythropoiesis ²⁴. These baseline differences may modulate the hematological response to H. pylori infection; however, this hypothesis has been inadequately explored, particularly in contemporary cohorts.

The importance of sex-stratified research extends beyond academic interest to clinical practice implications. Current clinical guidelines for H. pylori screening and treatment do not incorporate sex-specific considerations ²⁵. Even though evidence suggests sex differences in diagnostic approaches ²⁶. If H. pylori affects males and females through different mechanisms, sex-specific diagnostic thresholds, screening protocols, and treatment strategies may be warranted. Moreover, understanding sex-specific effects could provide insights into the underlying pathophysiology and identify novel therapeutic targets ²⁷.

Recent advances in sex and gender medicine have highlighted the critical importance of disaggregated analyses in biomedical research ²⁸. NIH and major journals now mandate sex-specific reporting, recognizing that aggregated data may obscure significant biological differences and perpetuate health disparities. The Sex and Gender Equity in Research (SAGER) guidelines provide a framework for incorporating sex and gender considerations throughout the research process, from study design to data interpretation ^29,30. Despite these initiatives, implementation remains incomplete, particularly in the areas of infectious disease ^29,31,32.

The present study addresses this critical knowledge gap. We conducted a comprehensive sex-stratified retrospective cohort analysis of complete blood count (CBC) parameters to explore potential sex-specific effects of H. pylori infection on hematological parameters. Our objectives were to characterize the overall hematological profile of H. pylori-infected patients compared to H. pylori-negative participants, to test for sex × H. pylori interactions across nine CBC parameters, and to delineate sex-specific patterns of hematological abnormalities

This retrospective cohort study analyzed data from King Khalid University Hospital (KKUH), a tertiary care center in Riyadh, Saudi Arabia. Participants were identified and grouped based on their confirmed H. pylori infection status to compare hematological outcomes between H. pylori-positive and H. pylori-negative groups. The study protocol was approved by the Institutional Review Board at King Saud University, Riyadh, Saudi Arabia (Research Project No. E-24-8617), and the requirement for informed consent was waived due to the study's retrospective nature. All methods were performed in accordance with the relevant guidelines and regulations, including the Declaration of Helsinki and institutional guidelines.

Electronic medical records were systematically reviewed for the period January 2015 to December 2024 at KKUH. The following variables were collected: demographic characteristics (age, sex, nationality), anthropometric measurements (weight, body mass index), clinical diagnoses (H. pylori status, comorbidities), and laboratory parameters. Complete blood count parameters included red blood cell count (RBC), hemoglobin (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), white blood cell count (WBC), and platelet count (PLT). BMI and weight data were unavailable for some H. pylori-negative participants due to incomplete medical records.

From hospital electronic medical records, we identified 1,895 patients diagnosed with enteric infections between 2015 and 2024. Of these, 653 patients had confirmed H. pylori infection. After applying inclusion and exclusion criteria, 165 H. pylori-positive patients were included. H. pylori-negative participants were obtained from the family medicine clinic medical records within KKUH during 2023–2024. We reviewed records of individuals who had attended routine check-ups or treatment of acute, minor conditions (e.g., seasonal allergies, upper respiratory infections). H. pylori-negative status was defined as the absence of documented H. pylori infection in medical records. H. pylori-negative participants were frequency-matched to H. pylori-positive patients by age (± 5 years) and sex. After applying the same temporal criteria for CBC testing, 163 H. pylori-negative participants were included.

[Figure 1 here]

The inclusion criteria for both groups were age ≥ 18 years and the availability of complete CBC data. For H. pylori-positive patients, CBC parameters were assessed from tests performed within 10 days before or 2 days after H. pylori diagnosis to ensure temporal proximity to confirmed infection status. The final analysis included 328 participants (165 H. pylori-positive, 163 H. pylori-negative), providing adequate power for sex-stratified analyses.

A total of 328 participants were included, comprising 165 H. pylori-positive patients and 163 H. pylori-negative participants. The groups were well-matched for age (45.5 ± 16.6 vs. 45.9 ± 16.1 years, p = 0.828) and sex distribution (37.6% vs. 37.4% male, p = 1.000). No significant differences were observed in BMI between H. pylori-positive and H. pylori-negative participants (28.5 ± 6.8 vs. 28.8 ± 5.9 kg/m², p = 0.682). Table 1 summarizes the demographic and clinical characteristics of the study participants.

[Table 1 here]

Analysis of nine CBC parameters revealed four statistically significant differences between the H. pylori-positive and H. pylori-negative groups Table 2. Compared to H. pylori-negative participants, H. pylori-positive participants exhibited significantly lower hemoglobin (12.84 ± 1.87 vs. 13.39 ± 1.88 g/dL, p = 0.008), hematocrit (39.59 ± 5.27 vs. 41.45 ± 5.19%, p = 0.001), MCV (84.31 ± 7.54 vs. 86.58 ± 7.29 fL, p = 0.006), and higher RDW (14.41 ± 2.42 vs. 13.69 ± 1.63%, p = 0.002). This hematologic profile is consistent with IDA, commonly associated with H. pylori infection. No significant differences were observed in MCH, MCHC, RBC count, WBC, or platelet count.

[Table 2 here]

Clinical categorization analysis showed notable hematological abnormalities among H. pylori-positive patients compared to H. pylori-negative participants (Table 3). The prevalence of anemia was higher among infected individuals (27.3% vs. 19.0%), with corresponding increases observed in low hematocrit (21.2% vs. 12.3%) and red blood cell count deficiencies (16.4% vs. 14.1%). Microcytosis (MCV < 80 fL), hypochromia (MCH < 27 pg), and elevated RDW were also more prevalent in patients with H. pylori-positive status.

[Table 3 here]

Sex-stratified analyses revealed significant sex × H. pylori interactions for hematocrit (p = 0.0146) and RBC count (p = 0.0122), and a near-significant interaction for hemoglobin (p = 0.0511), as shown in Table 4 (Figure S1).

[Table 4 here]

Males with H. pylori infection demonstrated significant reductions in hemoglobin (d=-0.512, p = 0.005), hematocrit (d=-0.619, p < 0.001), and RBC count (d=-0.407, p = 0.026), indicating substantial anemia. Females primarily exhibited alterations in red cell indices, including decreased MCV (d=-0.392, p = 0.006), MCH (d=-0.295, p = 0.036), and increased RDW (d = 0.337, p = 0.017), without significant changes in hemoglobin or hematocrit. Figure S2 presents forest plots of sex-stratified effect sizes for all CBC parameters.

The sex-specific percentage changes shown in Fig. 2 underscore these differences, with males experiencing marked decreases in hemoglobin (-7.0%), hematocrit (-7.9%), and RBC (-6.5%), and females exhibiting reductions in MCV (-3.3%), MCH (-3.2%), and an increase in RDW (+ 5.3%).

[Figure 2 here]

Application of the WHO sex-specific anemia criteria revealed sex-dependent differences (Table S1). Among males, significantly more H. pylori-infected individuals met anemia criteria compared to H. pylori-negative participants (32.3% vs 11.5%, p = 0.010). In contrast, females showed a smaller, non-significant difference (31.1% vs 24.5%, p = 0.372), reinforcing distinct sex-specific patterns in H. pylori-associated anemia.

This retrospective cohort study demonstrates that H. pylori infection affects males and females through fundamentally different hematological mechanisms. The significant sex × H. pylori interactions for hematocrit (p = 0.0146) and red blood cell count (p = 0.0122), with a near-significant interaction for hemoglobin (p = 0.0511), provide statistical evidence for distinct biological responses. Males with H. pylori infection demonstrated substantial reductions in hemoglobin (d=-0.512, medium effect), hematocrit (d=-0.619, medium-large effect), and RBC count (d=-0.407, small-medium effect). At the same time, females exhibited predominant changes in red cell indices without significant alterations in hemoglobin or hematocrit. These sex-specific patterns suggest different underlying mechanisms and challenge current uniform approaches to H. pylori-associated hematological disorders.

The divergent hematological patterns observed reflect sex-specific pathophysiological mechanisms underlying H. pylori infection. Males demonstrate enhanced inflammatory responses, as evidenced by higher cyclooxygenase-2 (COX-2) expression in response to H. pylori infection, whereas females showed no COX-2 response ³⁴. Conversely, baseline TFF1 levels are significantly higher in women than in men, and H. pylori infection significantly reduces TFF1 in women ³⁴, suggesting sex-specific alterations in protective factors. Furthermore, H. pylori exhibits selective utilization of steroid hormones, which may contribute to sex-specific colonization patterns. The bacterium can absorb estrogens (which have 3-OH structures) but cannot metabolize them, while being unable to absorb testosterone or progesterone due to their 3 = O molecular structure, which prevents membrane interaction ³⁵.

Building on these differential inflammatory responses, hormonal mechanisms provide a crucial framework for understanding the observed sex differences. The protective effect of estrogen emerges as a key factor, with estrogen treatment eliminating H. pylori-induced gastric cancer in male mice and significantly decreasing neutrophil infiltration ³⁶. The molecular mechanism involves the estrogen-related receptor gamma (ESRRG), which suppresses H. pylori infection by directly binding to and upregulating the tumor suppressor TFF1 ³⁷. However, H. pylori-induced inflammation activates NF-κB signaling, which inhibits ESRRG-mediated TFF1 activation, creating a pathogenic feedback loop where infection suppresses its own protective mechanisms ³⁷. This hormonal regulation extends to iron metabolism, where clinical evidence demonstrates estrogen's potent effects: during IVF treatment, marked estrogen stimulation decreased median hepcidin levels from 4.85 to 1.43 ng/mL, demonstrating estrogen's regulatory role in iron homeostasis ³⁸. This hormonal regulation likely contributes to females' ability to buffer against full anemia, even with microcytic red cell changes; hemoglobin remains stable, explaining why females in our study maintained relatively stable hemoglobin levels despite developing microcytic changes characteristic of early iron deficiency.

Our findings both confirm and extend the growing literature on sex-specific effects of H. pylori. A comprehensive meta-analysis of 244 studies found males had greater odds of H. pylori infection than females did, supporting our observation of sex-specific vulnerability ³⁹. However, their analysis did not examine hematological outcomes or test for formal interactions as we have done. Recent studies from diverse populations have reported variable associations between H. pylori and anemia that vary by sex. Wang et al. (2024) similarly reported that H. pylori infection was associated with IDA specifically in women (p = 0.031) but not men, though their analysis was limited to iron markers without examining the full spectrum of CBC parameters ⁴⁰. A 2022 sub-Saharan study found complex relationships between H. pylori infection and anemia, with males showing higher susceptibility to certain hematological changes ⁴¹. Our findings provide a mechanistic explanation for these observations by demonstrating that males and females develop different types of hematological abnormalities. Males experience reductions in hemoglobin, hematocrit, and RBC count, indicative of overt anemia, while females show changes in red cell indices, suggesting early-stage iron deficiency without frank anemia.

These mechanistic insights from our cohort study have immediate clinical relevance for current practice guidelines. Current guidelines recommend H. pylori testing in patients with unexplained iron deficiency anemia, without sex-specific considerations ^25,26. Our findings suggest that the hematological presentation of H. pylori infection differs substantially by sex, indicating the need for tailored diagnostic approaches. For male patients, the presence of reduced hemoglobin, hematocrit, or RBC count should prompt evaluation for H. pylori, even in the absence of microcytic indices. The large effect sizes observed for hematocrit (d=-0.619) and hemoglobin (d=-0.512) indicate substantial clinical relevance for H. pylori screening in males with these abnormalities. Conversely, in females, the combination of low MCV, low MCH, and elevated RDW, even with normal hemoglobin, may indicate H. pylori-associated iron deficiency. Treatment considerations should account for potential sex-specific factors, with post-menopausal women who lose estrogen's protective effects requiring enhanced surveillance, given the potential loss of estrogen-mediated protection against H. pylori-associated complications.

This retrospective cohort study has several limitations. First, the retrospective design using electronic medical records limits our ability to establish temporal relationships between H. pylori infection and hematological changes. Second, our participant selection from patients with enteric infections ensures confirmed H. pylori status with appropriate clinical context, though this may not capture asymptomatic H. pylori carriers. Third, we lacked comprehensive data on factors that could influence hematological parameters, including iron supplementation, menstrual history, and dietary patterns.

Finally, our single-center study in Saudi Arabia may limit generalizability to other populations, as H. pylori strains vary geographically in virulence factors, and host genetic factors influencing iron metabolism may differ across ethnic groups.

H. pylori infection affects males and females through distinct hematological mechanisms. Males typically develop anemia characterized by reduced hemoglobin, hematocrit, and RBC count, whereas females exhibit changes in red cell indices. These sex-specific patterns from our retrospective cohort study support the need for tailored diagnostic approaches and treatment strategies in H. pylori-associated hematological disorders.