Autoimmune thyroid disease (AITD) is the most common endocrine disorder in women of reproductive age, including during pregnancy. It encompasses two clinical conditions: autoimmune hyperthyroidism and hypothyroidism. Pregnancy significantly affects thyroid function regulation, while thyroid dysfunction can influence fertility, pregnancy progression, and the postpartum health. Although the role of acquired immunity in AITD is well understood, recent research increasingly emphasizes innate immunity. Key cells involved in innate immunity include neutrophils, monocytes, and NKT cells. However, limited data exist on their role during pregnancy, especially in women with AITD.

This study aims to investigate the dynamic changes in neutrophils, monocytes and NKT cells during pregnancy and compare findings between women with normal pregnancies and those with AITD.

The study will analyze the frequency and activation status of these innate immune cells in peripheral blood samples collected during the first, second, and third trimesters. In addition, concentrations of thyrotropin, thyroid hormones, thyroglobulin, and thyroid autoantibodies, will be measured. A thyroid ultrasound will also be performed.

Autoimmune thyroid disease (AITD) affects approximately 2-17% of women of reproductive age, and represents the most common endocrine disorder in pregnancy. It includes autoimmune hyperthyroidism and hypothyroidism. Pregnancy has a significant impact on the regulation of thyroid function, and thyroid dysfunction may contribute to adverse pregnancy outcomes and neonatal complications.

Early detection of thyroid dysfunction during pregnancy is critical for optimal maternal-fetal health. While acquired immunity is a recognized contributor to the development of AITD, increasing attention has been given to the role of innate immunity. Innate immune mechanisms are essential for early immune response initiation and maintaining maternal tolerance toward the fetus, which represents a semi-allogeneic entity. This tolerogenic state is regulated both systemically and locally at the maternal-fetal interface.

The most relevant innate immune cells include neutrophils, monocytes/macrophages, and natural killer T (NKT) cells. Neutrophils participate in immune defense through cytotoxicity, phagocytosis, and cytokine secretion. Their numbers increase during pregnancy, and they play a role in implantation and placental development. Monocytes and decidual macrophages contribute to immune tolerance and placental remodeling. NKT cells, which bridge innate and adaptive immunity, perform immunoregulatory functions and have been observed in increased proportions in the decidua compared to peripheral blood. However, limited data are available regarding their role in pregnancy with thyroid autoimmunity.

The hypothesis of this study is that the numbers of neutrophils, monocytes and NKT cells and activation state of monocytes and NKT cells change during normal pregnancy and in pregnancy affected by AITD. The primary objective is to analyze differences in the frequency and activation marker expression of these immune cells between the two groups.

Peripheral blood mononuclear cells will be isolated, labeled, and analyzed using flow cytometry.

Expression of HLA-DR on monocytes and CD69 and CD25 on NKT cells will be quantified. In parallel, thyroid function parameters will be measured, including thyrotropin (TSH), free thyroxine (FT4), free triiodothyronine (FT3), thyroglobulin (Tg), and thyroid autoantibodies (TPOAb, TgAb, antiTSHR).

Pregnant women will be enrolled prospectively and will undergo blood sampling in each trimester. Ultrasound evaluation of the thyroid gland will be conducted to aid in diagnosis and classification. Participants will be categorized into groups based on thyroid hormone status and autoantibody titers: (1) normal pregnancy and (2) pregnancy with AITD.

The expected enrollment is 80 pregnant women (40 per group). All analyses will be conducted according to standardized laboratory and immunophenotyping protocols.

Statistical analysis will include group comparisons using t-tests or ANOVA for parametric data and Mann-Whitney or Kruskal-Wallis tests for non-parametric data. Correlation analyses will be conducted using Pearson or Spearman coefficients. A p-value < 0.05 will be considered statistically significant.

Inclusion Criteria: adult healthy pregnant women and pregnant women with autoimmune thyroid disease

• with a single pregnancy
• older than 18 years
• who have signed the informed consent form

Exclusion Criteria:

• pregnant women who refuse to give informed consent
• pregnant women under the age of 18
• pregnant women with malignant diseases
• pregnant women with thyrotoxicosis or hypothyroidism of a non-autoimmune nature and previous thyroidectomy or ablative therapy with iodine-131
• pregnant women with an acute or chronic disease that is not an autoimmune thyroid disease

Only adult pregnant women are eligible for the study

The planned prospective observational study will include pregnant women referred by primary care clinics in Rijeka and Primorsko-goranska County to the Clinical Department of Nuclear Medicine and the Department of Endocrinology, Diabetes and Metabolic Diseases of the Clinic of Internal Medicine of the Clinical Hospital Center Rijeka for thyroid examination and who have signed an informed consent form.

All IPD that underlie results in a publication will be available upon request from Tatjana Bogović Crnčić

Beginning 3 months and ending 3 years after the publication of results

The principal investigator and collaborators involved in the research will have access to the IPD and supporting information.

Working versions of the data will be stored during the project on the principal investigator's computer, the project collaborator's laptop, and an external drive to which both the collaborators and the principal investigator will have access.

The final version of the data will be made available by the principal investigator through the repository of the Faculty of Medicine in Rijeka, established in the national Dabar system, where publications and other project documents will be stored.