Expression of CD4+CD25+FOXP3+ regulatory T cells in women with idiopathic recurrent spontaneous abortion and implantation failure: 3-color flow cytometric analysis



Introduction:  A population of CD4+CD25+ regulatory T (Treg) cells is thought to regulate alloreactive T cells in many autoimmune diseases. Lack of Treg cells resulted in abortions in mice and transfer of them prevented miscarriage. FOXP3 is now considered the most specific marker for Treg cells. In this study, we investigated whether levels of peripheral blood Treg cells in women with recurrent spontaneous abortion (RSA) of unknown etiology or with repeated implantation failures (IF) are different from those of normal fertile women.

Materials and Methods:  Non-pregnant women with a history of idiopathic RSA or repeated IF were enrolled in the study group (n = 15) and non-pregnant fertile females served as controls (n = 7). A flow cytometry assay was used. Peripheral blood mononuclear cells (PBMCs) were isolated and stained with appropriate monoclonal antibodies that identify Treg cells: for surface markers, such as anti-CD4 and anti-CD25, and for intracellular marker, anti-FOXP3.

Results:  The proportions of CD4+CD25+FOXP3+ Treg cells were significantly different between the patients and controls, 1.5% versus 2.4%, respectively (P < 0.05). In addition, an alteration in the expression of surface CD25 was noted after permeabilization of PBMC which allows antibodies to enter the cells and bind to FOXP3. The mean percentage of CD25+ cells before permeabilization, 9.9 + 4.8%, was decreased to 5.3+3.5% after permeabilization (P < 0.01). The ratios of CD4+CD25+/CD4+ between the surface and the intracellular staining also decreased by 46% (P < 0.001).

Conclusion:  CD4+CD25+FOXP3+ cells were significantly lower in the patients with idiopathic RSA or multiple IF than in the controls. Permeabilization for intracellular staining induces a decrease in expression of surface markers.