Abstract
Introduction:
Neutrophil extracellular traps (NETs) are increasingly recognized as having important roles in autoimmune disease pathogenesis. In many autoimmune diseases, e.g., rheumatoid arthritis (RA) and lupus (SLE), premenopausal women have increased incidence. Understanding immunologic effects of exposures common in premenopausal women, such as oral contraceptive pill (OCP) use, is important to understanding autoimmune disease risk in women. We investigated the relationship between OCP use and circulating calprotectin, a NETosis marker relevant to autoimmune disease pathogenesis.
Methods:
164 samples from healthy premenopausal women (65 current OCP users; 99 never/prior users) and 124 samples from premenopausal women at-risk for RA by family history (57 current OCP users; 67 never/prior users) were tested for plasma calprotectin levels by ELISA. Levels were compared between current and never/prior users via Welch's T-test.
Results:
Healthy women currently taking OCPs had lower plasma calprotectin levels than never/prior OCP users in both the derivation (0.99 µg/mL vs. 1.38 µg/mL, p = 0.016) and validation (0.9 µg/mL vs. 1.4 µg/mL, p = 0.001) cohorts. Similarly, in women at-risk for RA, current OCP users had lower plasma calprotectin level compared to never/prior users (1.19 µg/mL vs. 1.74, p = 0.017).
Discussion:
Current OCP use is associated with significantly lower plasma calprotectin in healthy premenopausal women and premenopausal women at-risk for RA. Given calprotectin is considered a NETosis marker and NETosis is associated with pathogenesis in several autoimmune diseases, our findings suggest a reduction in NETosis via OCP use could be a potential mechanism influencing autoimmune disease in premenopausal women.
Introduction
Neutrophils are implicated in the pathogenesis of multiple autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) (). A particular feature of neutrophils that has been found to drive inflammation, tissue damage, and autoantibody generation in these autoimmune diseases is the formation of neutrophil extracellular traps (NETs). NET formation (NETosis) is a process by which activated neutrophils decondense their nuclei and release nuclear and cytoplasmic proteins (e.g., calprotectin) into the extracellular space in web-like structures that can trap and neutralize pathogens. NETosis is increasingly implicated in the initiation and amplification of autoimmunity (), including in RA, where citrullinated proteins externalized during NETosis can initiate and amplify the generation of anti-citrullinated protein antibodies (ACPA) (). Additionally, NETs in patients with RA have been shown to correlate with disease activity (), and a relative inability to degrade NETs has been associated with a higher incidence of lupus nephritis in patients with SLE (). As such, understanding the factors that regulate NET formation and the measurement of NET activity may provide important insights into the pathogenesis of several autoimmune diseases.
Autoimmune diseases in which NETs are implicated, including RA and SLE, occur more frequently in women than in men, with the greatest female predominance observed during the premenopausal years. For example, RA has a 5:1 female-to-male ratio in premenopausal females (), and SLE has a 10:1 ratio in premenopausal females compared to age-matched males (). Notably, neutrophils from premenopausal women have been shown to display a more mature and responsive phenotype compared to male neutrophils, with a corresponding increased propensity for NET formation (). This pro-NETotic phenotype in neutrophils of premenopausal women can be driven by estrogen (–), emphasizing the influence of sex-specific factors on NET formation.
One female-specific factor that can influence estrogen levels in premenopausal women is the use of oral contraceptive pills (OCPs). OCPs commonly induce anovulation, leading to lower total systemic estrogen levels. Previous studies have investigated the relationship between OCP use and autoimmune disease risk in RA and SLE. While the relationship between OCPs and SLE risk remains unclear, multiple studies have reported a reduced risk of developing RA in women who reported OCP use (, ). It is also reported that OCP use is associated with less positivity for the RA-associated autoantibody rheumatoid factor (RF) (). Yet, the biological mechanisms underlying the protective effect of OCP use and RA risk are not well defined. OCPs are the most commonly prescribed form of hormonal contraception in the United States, with 25% of women between ages 15–44 using OCPs (). Given the widespread use of OCPs, understanding their effects on immune function is critical for assessing potential influences on autoimmune disease risk, particularly in RA.
With this background, we aimed to examine the relationship between OCP use and NETosis, as measured by differences in circulating calprotectin, with potential implications for understanding the effects of OCPs on NET-mediated diseases. Of note, because ACPA can independently induce NETosis (, ), for this cross-sectional study, we focused on the relationship between OCP use and calprotectin levels among. ACPA-negative individuals. Moreover, several assays can measure NET activity. For our study, we selected plasma calprotectin because it has been associated with disease activity and inflammation in NET-associated diseases, including RA (–). It is also a widely available commercial assay that enables low inter-assay variability and potential translation into routine clinical care.
Materials & methods
Study cohorts
This cross-sectional study included two independent healthy population cohorts: a derivation cohort and a validation cohort. It also included a cohort of women with an increased risk of developing RA based on family history. Participants in all cohorts were identified from existing biospecimens previously obtained from the Studies of the Etiology of Rheumatoid Arthritis (SERA) cohort (). SERA is an observational study designed to understand the natural history of RA development. Briefly, the SERA project, which originated in 2005, identified probands with established RA and invited their first-degree relatives (FDRs) to be prospectively followed to explore factors associated with future development of RA. Inclusion criteria for SERA FDRs were having an FDR with established RA, and exclusion criteria were having inflammatory arthritis at or prior to the baseline study visit. In addition, SERA recruited healthy controls as a comparator group through local email and flyer advertisements throughout the community. Exclusion criteria for healthy controls were having a personal history of systemic autoimmune disease or inflammatory arthritis, having an FDR with RA, or having serum anti-CCP positivity (all healthy controls were confirmed o be anti-CCP negative at their study visit).
All participants included in the study reported being premenopausal. All participants had a detailed contraceptive exposure history, pregnancy history, and demographics collected by self-reported questionnaire. OCP status at the initial study visit was determined based on the following questions: “Have you ever used birth control pills?”, “Age when you first used this?”, “How many years in total did you use this?”, and “Are you using this now?”. Blood samples were obtained at the same study visit when questionnaires were completed.
Healthy population derivation cohort
A derivation cohort of healthy premenopausal women was used for the primary analysis Of the 336 stored samples from SERA healthy controls, 77 were premenopausal women who were current OCP users or not current OCP users (i.e., never or prior OCP users), and who also reported no prior use of other forms of hormonal contraception, including intrauterine devices (IUDs), implants, injectable contraceptives, vaginal rings, or transdermal patches. These 77 healthy controls were included in the derivation cohort.
Healthy population validation cohort
A validation cohort was used to independently assess the reproducibility of the primary findings. Of the 259 remaining stored samples from SERA healthy controls, 87 were premenopausal women who were current or never/prior OCP users, and had prior exposure to other hormonal contraceptive methods, but at the time of study visit/blood collection, were using OCPs (in the current OCP use group) or were not using any other forms of hormonal contraception (if in the never/prior OCP use group). These 87 healthy controls were included as the validation cohort.
First-degree relatives (FDRs) of RA patients
Because OCPs have been associated with reduced RA risk, we included a cohort of women with an increased risk for developing RA. Specifically, from the SERA cohort, we included premenopausal women without a history of RA or systemic autoimmune disease, and who had an FDR with established RA, which is associated with an increased risk of developing RA in the future (). Of the 1,775 stored samples from SERA FDRs, 171 were either current or never/prior OCP users, and who also reported no prior use of other forms of hormonal contraception. Only 17 of the 171 were serum anti-CCP positive. With this limited number of anti-CCP positive FDRs and because ACPA can independently induce NETosis (, ), we focused this study on anti-CCP negative FDRs who represent a very early at-risk state based on family history and not a more advanced at-risk phase of preclinical systematic RA-associated autoimmunity.
Blood collection
Blood was collected from each participant in EDTA-containing tubes. There was no requirement for fasting, and samples could be collected at any time of the day. Samples were not timed for phase of menstrual cycle. Processing of blood occurred within 30 min of collection and included centrifugation at 3,000 rpm for 10 min at room temperature. Plasma was then aliquoted into 2 mL cryovials and stored at −80°C until batch testing for this study.
NET remnant assays
Stored plasma samples were batch tested for quantification of calprotectin using commercially available ELISA kits from Werfen (QUANTA Lite® Calprotectin ELISA, catalog no. 704860; Barcelona, Spain) and following manufacturer's instructions. Technicians were blinded to the OCP status of the sample. Plasma calprotectin levels are reported as ug/mL. Calprotectin is a heterodimer of alarmins S100A8 and S100A9, which are highly abundant in neutrophil cytoplasm and released during NET formation. Plasma calprotectin has been widely used as a marker of neutrophil activation in previous studies and for its association with NET-associated inflammation in autoimmune diseases (–).
Statistical analysis
Demographic variables and calprotectin levels were compared between groups (current vs. never/prior OCP users) using Welch's t-test or chi-square testing, as appropriate. A multivariable linear regression model was used to account for potential confounding variables, which included age, ever smoking, and ever pregnancy based on prior studies reporting these variables associated with NET levels (, , ). Spearman's correlation coefficient was used to compare calprotectin levels and duration of OCP use or years since OCP discontinuation. Data were analyzed in IBM SPSS Statistics Package, version 31.
Results
Healthy population cohorts
Demographics
Participant characteristics for healthy population cohorts are included in Table 1. In both the derivation (N = 77) and validation (N = 87) groups, current OCP users were younger than never/prior users. In the derivation cohort, current OCP users were less likely to have had a history of pregnancy or breastfeeding. History of smoking was not different between current and never/prior OCP users.
Table 1
| Variable/Characteristic | Derivation cohort | Validation cohort | ||||
|---|---|---|---|---|---|---|
| Current OCP Users | Never/Prior OCP Users | P-value | Current OCP Users | Never/Prior OCP Users | P-value | |
| N = 31 | N = 46 | N = 34 | N = 53 | |||
| Age | 27.7 (6.1) | 34.2 (7.3) | <0.001 | 32.8 (5.4) | 35.3 (7.5) | 0.087 |
| Ever Smoker | 0 (0) | 2 (4.3) | 0.513 | 8 (24.2) | 15 (27.3) | 0.754 |
| Ever Pregnant | 3 (9.7) | 25 (54.3) | <0.001 | 21 (61.8) | 38 (69.1) | 0.489 |
| Ever Breastfeed | 0 (0) | 20 (43.5) | <0.001 | 18 (52.9) | 35 (63.6) | 0.329 |
| OCP Duration, years | 7.8 (5.1) | 8.6 (6.1)a | 0.605 | 11.7 (5.9) | 4.93 (5.0)* | <0.001 |
| Calprotectin, µg/mL | 0.99 (0.65) | 1.38 (0.72) | 0.016 | 0.9 (0.5) | 1.4 (0.8) | 0.001 |
Group characteristics for healthy population cohorts.
All values are either mean (standard deviation) or N (%).
For prior OCP users only.
OCP, oral contraceptive pill.
Plasma calprotectin levels based on OCP status
The mean calprotectin level amongst all healthy premenopausal women was 1.25 µg/mL. In the derivation cohort, current OCP users had significantly lower mean calprotectin levels (0.99 µg/mL) compared to never/prior users (1.38 µg/mL, p = 0.016, Table 1, Figure 1A). Importantly, in the validation cohort, plasma calprotectin was confirmed to be lower in current OCP users compared to never/prior users (p = 0.001, Figure 1B).
Figure 1
In 36 of 65 healthy women currently using OCPs, the specific OCP formulation could be confirmed by medical chart review. All women were taking low-dose estradiol formulations (i.e., ≤0.035 mg of estradiol). There was no correlation between calprotectin level and estradiol dosage (r = 0.13, p = 0.46).
In a multivariable model adjusted for age, ever smoking, and ever pregnancy, lower levels of calprotectin remained associated with current OCP use, with borderline association in the derivation cohort (p = 0.058) and a significant association in the validation cohort (p < 0.001).
In the derivation cohort, on average, those using OCPs had a 0.38 µg/mL lower calprotectin level than never/prior users. In the validation cohort, those using OCPs had a 0.56 µg/mL lower calprotectin level than never/prior users.
OCP duration
For current OCP users, there was no significant correlation between plasma calprotectin levels and OCP duration in years (r = 0.10, p = 0.59). In prior OCP users, there was no correlation between calprotectin levels and time since last use of OCPs (r = 0.01, p = 0.97).
FDR cohort at-risk for RA
Demographics
We included 104 FDRs who were at-risk for RA based on family history. Similar to the healthy population cohorts, FDR current OCP users were younger than never/prior OCP users (Table 2).
Table 2
| Variable/Characteristic | Current OCP Users | Never/Prior OCP Users | P-value |
|---|---|---|---|
| N = 57 | N = 67 | ||
| Age | 32.5 (8.8) | 35.7 (11.3) | 0.039 |
| Ever Smoker | 4 (22.2) | 13 (37.1) | 0.130 |
| Ever Pregnant | 8 (44.4) | 15 (42.9) | 0.457 |
| Ever Breastfeed | 15 (26.3) | 14 (21.2) | 0.256 |
| OCP Duration, years | 12.1 (13.7) | 3.3 (5.0)a | <0.001 |
| Calprotectin, µg/mL | 1.19 (0.75) | 1.74 (1.65) | 0.017 |
Group characteristics for at-risk cohort.
All values are either mean (standard deviation) or N (%).
For prior OCP users only.
OCP, oral contraceptive pill.
Plasma calprotectin levels based on OCP status
Plasma calprotectin levels were significantly lower in FDRs who were current OCP users (1.19 µg/mL) compared to never/prior OCP users (1.74 µg/mL, p = 0.017, Figure 1C). In a multivariable model adjusted for age, ever smoking, and ever pregnancy, lower levels of calprotectin remained significantly associated with current OCP use in FDRs (p = 0.019), with those using OCPs having a 0.73 µg/mL lower calprotectin level on average than never/prior users.
OCP duration
There was no significant correlation between plasma calprotectin levels and OCP duration in current OCP users in the FDR cohort (r = −0.07, p = 0.37) or between calprotectin levels and time since last use of OCPs in prior users (r = 0.01, p = 0.97).
Discussion
In this study, we demonstrate that current OCP use is associated with significantly lower circulating calprotectin levels in premenopausal women. Calprotectin is a well-established marker of neutrophil activation and NETosis and has been implicated in disease pathogenesis in NET-driven diseases such as RA and SLE. While the cross-sectional nature of our study limits causal inference, our findings may suggest that OCP use exerts immunomodulatory effects on neutrophils that have potential downstream consequences for inflammatory pathways relevant to autoimmune disease during the premenopausal period. Given that many systemic autoimmune diseases disproportionately affect women, defining how female-specific exposures, including hormonal contraception, shape immune function is essential, as such exposures may meaningfully influence autoimmune disease risk and evolution.
When interpreted in the context of prior reports (, ), calprotectin levels in our healthy cohorts were generally consistent with published ranges, in which most individuals exhibits a plasma calprotectin level between 0.1 and 1.5 ug/mL. Notably, mean levels among our healthy never/prior OCP users (1.4 ug/mL) were toward the upper end of this range, and in our at-risk cohort, mean calprotectin levels among never/prior OCP users (1.74 ug/mL) were modestly higher than those typicaly reported in healthy populations (–). In contrast, and as expected, calprotectin levels in both our healthy and at-risk groups were substantially lower than levels observed in patients with active RA (, ), which are commonly reported in the range of 10–50 ug/mL. It is also important to note that calprotectin in our study was measured in EDTA-preserved plasma rather than serum. Serum generation can be affected by clotting and induce neutrophil activation ex vivo, potentially leading to overstimulation of in vivo calprotectin levels ().
While we observed a consistent association between current OCP use and lower calprotectin levels across three independent cohorts, the mechanisms by which OCPs might reduce NETosis remain to be fully elucidated. Prior work has shown that estradiol can directly modulate neutrophil transcriptional programs and promote a more NET-prone phenotype, particularly at concentrations observed during peri-ovulatory estrogen surges in ovulating premenopausal women. Because OCPs suppress ovulation, they may reduce exposure to these high estradiol peaks, thereby shifting neutrophil phenotypes toward profiles more similar to those observed in men or postmenopausal women, which are less prone to NET formation. The absence of an association between calprotectin levels and duration of OCP use is consistent with this model, as OCP-induced anovulation occurs shortly after initiation and reverses quickly upon discontinuation. Alternatively, OCPs may indirectly influence NETosis through effects on the microbiome or systemic cytokine production (, ). Further mechanistic studies are needed to delineate the relative contribution of these pathways.
In RA, a disease in which OCP use has been associated with a reduced risk of disease development, our findings raise the possibility that suppression of NETosis represents a potential biological mechanism linking OCP use to disease risk. Many epidemiologic studies examining OCP exposure and RA risk do not distinguish between current and prior use, and RA onset may occur years after OCP discontinuation or following menopause. Nevertheless, attenuation of NET formation and the associated generation of citrullinated antigens during a critical window of immune priming could plausibly dampen downstream inflammatory amplification and autoantibody propagation. In the present study, we excluded ACPA-positive FDRs, because ACPA itself can promote NETosis and sustain inflammatory feedback loops. Larger longitudinal studies are needed to determine whether OCP-associated modulation of NETosis influences ACPA development and subsequent RA risk.
Several limitations warrant consideration. Our sample size was modest, and calprotectin represents only one measure of NET remnants. Additional NET-associated biomarkers and direct assessments of ex vivo NET formation could provide complementary insight. Moreover, calprotectin release is not exclusive to NETosis, and elevated levels may also reflect the effects of neutrophil necrosis or degranulation. Nonetheless, given its strong association with autoimmune disease activity and pathogenesis, our findings remain biologically relevant. We also did not directly measure estradiol levels or assess ovulatory status with luteinizing hormone surges, which would be required to definitively link lower levels of calprotectin with OCP induced anovulation. Finally, although we included an at-risk FDR cohort, no participants developed clinical RA during follow-up, precluding direct assessment of whether OCP-associated reductions in NETosis mediate reduced RA risk. Larger longitudinal studies will be necessary to address this question.
Important directions for future research include extending these observations to women with established autoimmune diseases, including RA and SLE, to determine whether OCP use is similarly associated with reduced calprotectin and altered neutrophil activity in disease states. In vivo studies examining neutrophil function before and after OCP initiation will be critical to confirm causal effects and to clarify the role of ovulation suppression and estradiol levels. Additionally, it will be important to determine whether similar effects are observed with other forms of hormonal contraception that differ in estrogen dose, formulation, and route of administration.
In summary, we show that premenopausal women who are currently using OCPs have lower circulating levels of plasma calprotectin, consistent with reduced NETosis. These findings provide insight into how hormonal contraception may shape innate immune function and suggest a potential biological pathway through which female-specific hormonal exposures could influence autoimmune disease risk and immune dysregulation.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation upon request.
Ethics statement
The studies involving humans were approved by Colorado Multiple Institution Review Board (COMIRB). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.
Author contributions
DM: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing. CM: Data curation, Formal analysis, Writing – original draft, Writing – review & editing. KS: Data curation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. CL: Data curation, Methodology, Writing – original draft, Writing – review & editing. KD: Writing – original draft, Writing – review & editing. VH: Writing – original draft, Writing – review & editing. JN: Writing – original draft, Writing – review & editing. JZ: Writing – original draft, Writing – review & editing. MD: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by funding from the National Institutes of Health [grant number, R01 AR076450 (MKD)] and the University of Colorado Anschutz Ludeman Family Center for Women's Health Research.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author VH declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Summary
Keywords
autoimmune disease, neutrophil extracellular traps (NETs), oral contraceptive pills (OCPs), rheumatoid arthritis (RA), women's health
Citation
Marcy D, Maretz C, Sturm K, Lugo C, Deane KD, Holers VM, Norris JM, Zell J and Demoruelle MK (2026) Oral contraceptive pill Use is associated with lower levels of plasma calprotectin in premenopausal women. Front. Lupus 4:1819508. doi: 10.3389/flupu.2026.1819508
Received
27 February 2026
Revised
12 April 2026
Accepted
18 May 2026
Published
16 June 2026
Volume
4 - 2026
Edited by
Antonia Mazzucato-Puchner, Medical University of Vienna, Austria
Reviewed by
Gabriela Vasquez Martinez, National Institute of Technology of Mexico, Mexico
Yajnaseni Chakraborti, University of Pennsylvania, United States
Updates
Copyright
© 2026 Marcy, Maretz, Sturm, Lugo, Deane, Holers, Norris, Zell and Demoruelle.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Daniele Marcy daniele.marcy@cuanschutz.edu
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.