Regulatory T (Treg) cells play a critical role in orchestrating maternal immune tolerance, allowing pregnancy success. Deficiency in Treg cell abundance and/or function is implicated in reproductive and pregnancy complications, including recurrent pregnancy loss (RPL). No definitive diagnostic exists to identify and characterise immune dysfunction in RPL patients, consequently treatment interventions including immunotherapies cannot be matched to patient subtypes.
We are developing a flow cytometry-based diagnostic test to identify T cell-based immune dysregulation in RPL patients using peripheral blood immune cells. A key knowledge gap is to quantify the temporal within-individual variation in abundance and phenotype of peripheral blood T cells in women. Hence, this pilot study aims to determine within-individual variation in T cell populations collected from healthy reproductive-aged women (n=15) during the mid-luteal phase of the menstrual cycle.
Medical and reproductive history were recorded, and blood samples collected over three menstrual cycles within a 3-6 month period. Leukocytes were isolated by density gradient centrifugation and analysed by 15-colour multi-parameter flow cytometry to identify T cells subsets and characterise Treg cell phenotype. The mean coefficient of variation (CV) was calculated for each T cell parameter to determine the degree of within-individual variability.
Treg cell abundance and expression of key phenotypic markers, including CTLA4 (CV=2.4%), Helios (CV=1.3%) and FOXP3 MFI (CV=10.1%) varied little within an individual over time. Within memory Treg cell subsets, there was little variation in central memory Treg cells (CV=11.8%) and naïve Treg cells (CV=8.9%). However, RORγt and Tbet expression varied moderately (CV=31.3% and 21.6% respectively).
These results indicate low within-individual variation in Treg cell abundance and phenotype over time, raising the prospect that a single blood draw will be broadly informative for T cell parameters relevant to RPL risk. The findings of this study will govern the development of protocols for immune-phenotyping diagnostics in RPL.