NETosis is a spectacular and violent form of cell death. When a neutrophil is activated, it decondenses its own nucleus, mixes its DNA with cytoplasmic and granule proteins, and casts the whole tangle outward as a web-like structure that can snare and neutralize pathogens. The process is a cornerstone of innate immunity, but it has a dark side. In rheumatoid arthritis, proteins externalized during NETosis can become citrullinated, sparking the generation of anti-citrullinated protein antibodies, or ACPA, the hallmark autoantibodies of the disease. NETs correlate with disease activity in RA patients, and in systemic lupus erythematosus, a relative inability to degrade NETs has been linked to a higher incidence of lupus nephritis, one of the most serious complications of the disease.

What makes the new findings especially provocative is the demographic backdrop. Autoimmune diseases in which NETs are implicated strike women far more often than men, and the skew is most extreme during the premenopausal years. Rheumatoid arthritis shows a female-to-male ratio of roughly 5:1 in premenopausal women, and lupus reaches an astonishing 10:1. Neutrophils from premenopausal women display a more mature and responsive phenotype than male neutrophils, with a correspondingly greater propensity for NET formation, and estrogen itself can drive this pro-NETotic behavior. That observation placed oral contraceptives squarely in the crosshairs of the Colorado team, led by Daniele Marcy and M. Kristen Demoruelle, because the pill commonly induces anovulation and thereby lowers total systemic estrogen exposure.

The researchers drew on the Studies of the Etiology of Rheumatoid Arthritis, or SERA, an observational project running since 2005 that follows first-degree relatives of people with established RA alongside healthy community controls. From stored plasma samples, the team assembled three groups of premenopausal women: a derivation cohort of 77 healthy women, a validation cohort of 87 healthy women, and a cohort of 104 women at elevated risk for RA because a first-degree relative had the disease. All participants completed detailed questionnaires on contraceptive, pregnancy, and smoking histories, and blood was drawn in EDTA tubes, processed within 30 minutes, and stored at minus 80 degrees Celsius until batch testing.

To gauge NETosis, the team measured plasma calprotectin, a heterodimer of the alarmin proteins S100A8 and S100A9 that are highly abundant in the neutrophil cytoplasm and released during NET formation. Calprotectin is a well-established marker of neutrophil activation and correlates with inflammation in NET-associated diseases, and because it can be quantified with a widely available commercial ELISA kit, the assay offers low inter-assay variability and a plausible path into routine clinical care. Importantly, the researchers excluded anti-CCP-positive individuals, since ACPA can independently induce NETosis and would muddy the interpretation. Laboratory technicians were blinded to each sample’s contraceptive status.

The results were strikingly consistent. Among healthy women in the derivation cohort, current pill users had a mean calprotectin level of 0.99 micrograms per milliliter compared with 1.38 in never or prior users, a difference that reached statistical significance at p equals 0.016. The validation cohort reproduced the pattern, with 0.9 versus 1.4 micrograms per milliliter and a p value of 0.001. In the at-risk relatives, current users again measured lower, at 1.19 versus 1.74 micrograms per milliliter, p equals 0.017. In multivariable linear regression models adjusting for age, ever smoking, and ever pregnancy—variables previously linked to NET levels—the association held, with borderline significance in the derivation cohort and a highly significant result in the validation cohort, where pill users averaged 0.56 micrograms per milliliter less calprotectin. Among the at-risk women, the adjusted difference was 0.73 micrograms per milliliter.

Notably, the effect did not appear to accumulate over time. There was no correlation between calprotectin levels and the duration of pill use, nor between calprotectin and the years elapsed since discontinuation among prior users. The authors argue this fits a model in which OCP-induced anovulation suppresses the high peri-ovulatory estradiol peaks that push neutrophils toward a NET-prone phenotype, an effect that begins shortly after initiation and reverses quickly upon stopping. When the team could confirm formulations through medical chart review, all 36 women documented were taking low-dose estradiol preparations of 0.035 milligrams or less, and estradiol dosage itself showed no correlation with calprotectin levels.

The numbers also sit sensibly within known biology. Mean calprotectin among healthy never or prior pill users, around 1.4 micrograms per milliliter, falls toward the upper end of the typical published range of 0.1 to 1.5, while the at-risk cohort’s never users averaged a modestly elevated 1.74. By contrast, patients with active rheumatoid arthritis commonly show calprotectin levels in the range of 10 to 50 micrograms per milliliter, underscoring that the study captured differences in a healthy or very early at-risk state rather than florid inflammation. The choice of EDTA-preserved plasma rather than serum also matters, since clotting during serum generation can activate neutrophils ex vivo and artificially inflate measured calprotectin.

The authors are careful about what the study can and cannot claim. As a cross-sectional analysis, it demonstrates association, not causation, and calprotectin release is not exclusive to NETosis—neutrophil necrosis and degranulation can also contribute. The team did not directly measure estradiol levels or confirm ovulatory status with luteinizing hormone surges, and no participants developed clinical RA during follow-up, precluding direct assessment of whether reduced NETosis actually mediates the lower RA risk previously reported in pill users. Still, multiple epidemiologic studies, including the Swedish EIRA study, have linked oral contraceptive use to reduced RA risk and to lower positivity for rheumatoid factor, and the new data offer a candidate mechanism: dampening NET formation and the citrullinated antigens it generates during a critical window of immune priming.

Future work, the researchers say, should extend these observations to women with established RA and lupus, examine neutrophil function before and after pill initiation in vivo, and test whether other hormonal contraceptives differing in estrogen dose, formulation, and route of administration produce similar effects. The study was funded by the National Institutes of Health and the University of Colorado Anschutz Ludeman Family Center for Women’s Health Research, and it appears in Frontiers in Lupus. If confirmed longitudinally, the findings could reframe a daily pill taken by millions as an unexpected modulator of innate immunity—one that may quietly influence which women’s neutrophils never fire their traps at all.

Subject of Research: Immunology

Article Title: Oral contraceptive pill Use is associated with lower levels of plasma calprotectin in premenopausal women

Article References: Marcy, D., Maretz, C., Sturm, K., Lugo, C., Deane, K. D., Holers, V. M., Norris, J. M., Zell, J., & Demoruelle, M. K. (2026). Oral contraceptive pill Use is associated with lower levels of plasma calprotectin in premenopausal women. Frontiers in Lupus, 4, Article 1819508. https://doi.org/10.3389/flupu.2026.1819508

Image Credits: AI Generated

DOI: 10.3389/flupu.2026.1819508

Keywords: oral contraceptives, calprotectin, NETosis, neutrophil extracellular traps, rheumatoid arthritis, lupus, autoimmune disease, estrogen, premenopausal women, immunology, women's health, biomarkers