May analysis of Th1, Th2, Th17 and Treg cells in peripheral blood of women with early pregnancy (11-13 weeks) be predictive of risk of developing fetal intrauterine growth restriction?

Background: In pregnancy a balance of immune tolerance and suppression is needed to protect the fetus.T cells regulate immune responses and together with cytokines form a regulatory network maintaining homeostasis between the fetus and the maternal immune system.The cytokines drive the differentiation program of CD4+T cells toward Th1,Th2,Th17 and T regulatory (Tregs).CD4+CXCR3+ Th1,characterized by the transcription factor T-bet and the production of IL-2,IFN-γ,and TNF-α,are involved in the cellular immunity and rejection process.CD4+CCR4+ Th2,characterized by the transcription factor GATA-3 and the production of IL-4,IL-5,IL-13,play a role in the maintenance of pregnancy by inhibiting Th1 responses.CD4+CCR6+ Th17, characterized by the transcription factor RORc,induced by TGF-β and IL-6,IL-21 and IL-23,produce IL17-A,IL17-F and IL-22 and favour inflammation and autoimmunity.CD4+CD25+ Tregs,characterized by the transcription factor Foxp3,induced by TGF-β together with IL-2 and retinoic acid,suppress immune response and maintain peripheral tolerance producing IL-10 and TGF-β.In early phase of normal pregnancy a predominance in peripheral blood (PB) of Th2 and Treg cells and upregulation of their cytokines occurs,with a concurrent decrease of Th1 and Th17 cells and of their cytokines.In contrast alterations in the percentage and number of PB T cells were observed in patients with recurrent miscarriage and pre-eclampsia caused by imbalance of Th1/Th2 and Treg/Th17 ratios.The breakdown of peripheral immunotolerance could negatively affect the mechanisms of embryo implant and may therefore lead to defects in placental framework and vascular function,with consequent increase in blood flow resistance and decreased fetal nutritive intake. This pathogenic mechanism may underlie,at least in part,the intrauterine growth restriction (IUGR).Aim of this work is to verify whether in the early pregnancy (11-13 weeks) the Th1/Th2 and Treg/Th17 ratios in women’s PB may be predictive of IUGR. Materials and Methods: We will collect peripheral blood samples from women at early pregnancy (11-13 weeks). The mononuclear cell will be isolated by Ficoll-Hypaque density gradient centrifugation and taken at interphase, washed twice in PBS and counted. An aliquot of these cells will be immediately used to evaluate CD4+CXCR3+ (for Th1), CD4+CCR4+ (for Th2), CD4+ CCR6+ (for Th17) and CD4+CD25+ (for Tregs) membrane marker expression by indirect immunofluorescence. The remaining cells will be assayed for T-bet (for Th1), GATA-3 (for Th2), RORc (for Th17) and Foxp3 (for Tregs), and β-actin (as endogenous control) mRNA levels by real-time PCR. We will follow-up the recruited patients to assess if they will develop normal pregnancy outcome (as controls) or fetal IUGR (as pathology group). Finally, we will correlate these data with pregnancy outcome. Expected Results: In the pathology group we expect to find an increase of Th1 (CD4+CXCR3+) and Th17 (CD4+CCR6+) cells and of T-bet and RORc mRNA expression, with a concurrent decrease of Th2 (CD4+CCR4+) and Treg (CD4+CD25+) cells and of GATA-3 and Foxp3 mRNA expression. On the contrary, in controls we expect to find increase of Th2 and Treg cells and of GATA-3 and Foxp3 mRNA expression, with a concurrent decrease of Th1 and Th17 cells and of T-bet and RORc mRNA expression. These Th1/Th2 and Treg/Th17 imbalances could alter the physiologic process of trophoblastic invasion and causing vascular function defect that may underlie one of the causes of fetal IUGR. If these findings will be confirmed, the assay of Th1, Th2, Treg and Th17 in PB at 11-13 weeks of gestational age could be considered as an early non-invasive marker of risk of developing fetal IUGR.