Estrogen hormones and their metabolites are implicated in the maintenance of healthy pregnancy and adequate fetal development. Abnormal levels were related to increased risk of pregnancy complications, particularly preeclampsia. Our aims were (1) to develop a methodological platform for the comprehensive assessment of estrogen metabolome in pregnancy; (2) to collect healthy reference data for relevant elements of estrogen metabolome in each trimester; (3) to assess unconjugated fractions of the estrogen metabolome, (4) to assess the dominant metabolic pathways of estrogen compounds. We enrolled healthy pregnant mothers between gestational week 5-15 (on the confirmation of pregnancy; 79 samples), gestational weeks 19-27 (70 samples), and gestational week 34-39 (54 samples). A method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to assess estrone, 17-beta-estradiol, estriol levels, and their metabolites as conjugated and unconjugated forms. Descriptive statistics were used to characterize the level of compounds in each trimester. Estrone, 17-beta-estradiol and estriol levels are dramatically increasing with the advancement of pregnancy. Measured levels were in a very wide range. 17-beta-estradiol is neither glucuronated nor sulphated. To the contrary, estriol and estrone are significantly conjugated; unconjugated fraction is <15% of total hormone levels in any trimester. Regarding metabolism, 4-methoxy-estradiol and 17-epiestriol were not detected. We concluded that (1) the levels of estrogen compounds and metabolites increase with advancing gestational age; (2) the wide ranges of levels challenge the establishment of a healthy reference range for clinical purposes; (3) 17-beta-estradiol is not conjugated significantly; (4) 4-methylation and 17-epimerization pathways of estrogens are negligible with our LC-MS/MS method.

Background: Biological therapy is frequently used for the treatment of inflammatory bowel disease (IBD); however, the long-term efficacy of anti-tumor necrosis factor-alpha (TNF−α) therapies, such as infliximab (IFX) and adalimumab (ADA), is often compromised by the development of antidrug antibodies (AIFX and AADA, respectively). While several individual factors are known to contribute to immunogenicity, the complex, interactive effects of various clinical variables have not been fully elucidated in a real-world setting. Methods: We conducted a hierarchical logistic regression analysis on a retrospective cohort of 153 pediatric and adult IBD patients receiving IFX or ADA therapy to identify clinical factors and their interactions associated with AIFX/AADA positivity. The analysis progressively incorporated demographic, disease-related, and treatment-related variables, culminating in a model that included two- and three-way interaction terms. Results: Our final model demonstrated modest predictive power, with a Nagelkerke R² of 0.287, explaining less than 30% of the variance in antibody positivity using readily available clinical data (AUC of 0.806, 71.0% sensitivity and 77.6% specificity). Key predictors included the type of biological therapy (IFX vs. ADA) and the duration of treatment, with IFX therapy being a significant independent predictor (OR = 6.940, p = 0.004) for antibody positivity. Importantly, we identified novel three-way interactions, revealing that the combined effect of age at disease onset, IBD subtype, and biological therapy type significantly influences antibody formation (p = 0.042), particularly in childhood-onset ulcerative colitis patients treated with IFX. A similar interaction was found for treatment duration, IBD subtype, and therapy type (p = 0.042), where the risk of antibody positivity with IFX increased significantly with treatment length, particularly in UC patients. Conclusions: This study highlights that the combination of routine clinical variables in IBD offers a data-driven, mechanistically insightful framework, supporting the prediction of AIFX/AADA positivity to a modest extent. This framework requires prospective and external validation before clinical implementation.

Background: Biological therapy is frequently used for the treatment of inflammatory bowel disease (IBD). The efficacy of the most commonly administered infliximab (IFX) and adalimumab (ADA) is impaired in the presence of anti-IFX (AIFX) and anti-ADA (AADA) antibodies. Methods: We analyze data from 153 patients to identify factors associated with AIFX/AADA positivity. We applied hierarchical logistic regression analysis that incorporates relevant clinical variables. Results: The results indicate that gender, age, localization, and IBD type alone do not influence AIFX/AADA positivity. However, the inclusion of treatment-related factors (e.g., type of biological therapy and treatment duration) improved model performance, with the final model achieving an explanatory power of 28.7%. One of the key predictors was the type of biological therapy: IFX treatment substantially increased the prevalence of AIFX positivity compared to ADA. Longer treatment correlated with a higher prevalence of positivity. Additionally, several interactions between variables were significant, particularly the combined effects of age at onset, IBD type, and biological therapy. Conclusion: In summary, the study highlights that the combination of routine clinical variables in IBD support the prediction of AIFX/AADA positivity to a significant, but minor extent.