The goal is to demonstrate the relationship of the circulating pool of T-regulatory lymphocytes in the mother's peripheral blood with populations in the placentas and to compare with controls, what is the difference in the expression of individual regulatory molecules of T-regulatory lymphocytes according to new paradigms. The proportional and functional characteristics of T-regulatory lymphocytes will be correlated with the composition of the intestinal and vaginal microbiota.

OBJECTIVES

The primary objective of this project is to determine the differences between the circulating pool of regulatory Treg subpopulation proportions and the placental populations in patients with preterm birth and preeclampsia. Pilot study was conducted for the primary objective. Another objective is to evaluate the effect of microbiota on the regulation of Treg cells, especially in relation to mechanisms of preterm birth and preeclampsia. With growing knowledge of the role of microbiota, it is considered that the maternal microbiota could significantly influence setting of immunoregulatory responses even prenatally. Since Treg play a key role in regulating the immunological balance and are indispensable for induction and maintenance of peripheral tolerance, the investigators hypothesise that abnormalities in function of this population are important factors in increasing the risk of gestational pathologies (preterm birth and preeclampsia). Such dysregulation is most likely accompanied by disruption of the physiological Treg epigenetic profile which is in turn influenced by aberrant microbiota composition. Better understanding of these processes and identification of positively or negatively participating bacterial species could enable more reliable prediction of risk-pregnancy and allow for modulation of microbiota to be applied preventively.

Premature birth is defined as pregnancy termination before the end of the gestational week 37. It plays a role in 10% of all pregnancies including spontaneous or medically induced labours. The efforts to postpone premature labour are continuously developed to minimise the consequences such as low birth weight and other risk events. Preeclampsia is a serious blood pressure condition that develops during pregnancy. People with preeclampsia often have high blood pressure (hypertension) and high levels of protein in their urine (proteinuria). It plays role in 5% of all pregnancies. Preeclampsia typically develops after the 20th week of pregnancy. It can also affect other organs in the body and be dangerous for both the mother and her unborn child. Therefore, a great number of scientific studies are now focused on the early prediction of premature labour and preeclampsia and on finding a specific parameter to estimate female patients at premature labour and preeclampsia risk.

HYPOTHESIS AND AIMS

Hypothesis 1: Disbalance between tolerogenic T cell subsets (Treg, Th2, Th9 and Th22) and proinflammatory subsets (Th1 and Th17) is a sign of premature birth or preeclampsia.

Specific aim 1: Determine the differences between the circulating and the placental T cell subpopulations in patients with preterm birth and preeclampsia. Previously published analyses of Trees and their phenotypic properties give inconclusive or even contradictory results. For this reason, the investigators propose a more in depth approach combining deep immunophenotyping capable of identifying effector T cell, Treg and NK cell subsets and their qualitative phenotype together with assessment of functional capabilities of Tregs.

Hypothesis 2: Certain bacterial species of microbiota can be identified to modulate epigenetic maintenance of Treg phenotype.

Specific aim 2: Determine the influence of microbiota on the Treg regulation. Exposure to microbiota is one of the most important environmental factors introduced in the perinatal period. The capability of certain probiotic bacterial species to promote immunological tolerance, restrain inflammation and unwanted reactivity is being established in the context of intestinal health. Both bacteria and their metabolic products are known to shape the immune system. By influencing the epigenetic programming they modulate phenotype and function of a variety of immune cells including Tregs. The investigators hypothesise that beneficial bacterial species can be identified to promote appropriate epigenetic maintenance of Treg phenotype, function and stability, and thus support Treg tolerogenic role. On the contrary, certain bacterial species will contribute to Treg epigenetic dysregulation and introduce a more pro-inflammatory environment with functionally deficient Treg cells. To test this hypothesis, the investigators will characterise microbial composition of material collected from the mother by oral, rectal and vaginal swabs during pregnancy and at the time of birth.

CHARACTERISATION OF EXPECTED RESULTS

The main outcome of the project will be to improve the understanding of the regulatory processes taking part in the immune balance in preterm birth and preeclampsia. Special attention will be given to the dynamics and functional characteristics of Treg and their subpopulations, considering the role of maternal microbiota. The obtained results will be presented on both national and international congresses and symposia and also published in international impacted journals.

Inclusion Criteria:

• singletone pregnancy
• gestational age 9+0 - 12+0
• history of preeclampsia (PE)
• history of spontaneous preterm birth (PTL = preterm labour)
• history of pPROM (preterm premature rupture of membranes).

Exclusion Criteria:

• uterine malformations
• gestational age out of 9+0 - 12+0
• age out of 19-40

Pregnant women