P-392 Peripheral blood naïve Treg (nTreg) cell subsets and implantation success in patients undergoing in vitro fertilization (IVF) treatment
Authors: Rakic K , Goropevsek A , Kovacic B , Reljic M 
Published: June 2022

Abstract

Study question
Which populations of circulating Treg cells, based on immunophenotyping with thirteen color flow cytometry and analysis with clustering algorithms play a role in implantation?

Summary answer
The phenotype of subpopulation within Treg cells that was most significantly increased in women who became pregnant was nTreg-like phenotype, which lacked CXCR5 expression.

What is known already
There are indications that Treg lymphocytes mediated immuno-regulatory mechanisms contribute to the occurrence of idiopathic infertility. Findings of studies indicate that higher nTreg cell subsets were associated with successful IVF treatment. These findings suggest that changes in the composition of the nTreg subset may allow increased tolerance induction during implantation. More detailed characterization of the Tregs in blood samples from women undergoing IVF/ICSI treatment has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 13-color flow cytometry Tregs cells can be divided into multiple complex subsets based on different markers.

Study design, size, duration
This prospective observational study included 36 patients undergoing IVF/ICSI treatment. Blood samples for 13-color flow cytometry, UMAP and FlowSOM cell clustering analyses were obtained from 19 patients undergoing controlled ovarian hyperstimulation and 17 patients undergoing vitrified-warmed blastocysts transfer (FET) in the natural cycle on the day of optimal blastocysts transfer, for data analysis GraphPad Prism was used. The study was performed between March 2020 and October 2021.

Participants/materials, setting, methods
In this study peripheral blood Treg cells composition was determined. We analyzed the phenotype of populations over or under-represented in samples from women who became pregnant compared to the women who did not within Treg cells. Treg cells were analyzed by both standard gating flow cytometry analysis and clustered by algorithms according to their level of expression of cell surface markers: CD25 (IL-2Rα), CD127 (IL-7Rα), CCR7, CXCR5, CD28, CD38, CD161, CD31, CD45RA, HLA-DR and CD15s.

Main results and the role of chance
Thirteen color flow cytometry and dimensionality reduction analyses were carried out, using the UMAP and FlowSOM cell clustering algorithms. Combining these tools, we were able to analyze the phenotype of populations in samples from women who became pregnant ( n = 23) compared to the women who did not (n = 13) within Treg cells. FlowSOM identified 15 phenotypically distinct populations within Treg cells. The most prevalent population that was increased in women who became pregnant was nTreg-like phenotype, which lacked CXCR5 expression. This related to our results by standard gating, where we observed that the percentage of CXCR5-nTreg cells and CD31+RTEnTreg cells within CD25+CD127low Treg cells was significantly higher in pregnant women (p = 0.003; p = 0.0162, respectively). Pregnant females had significantly higher percentage of CD45RA+nTreg cells within CD25hiCD127low Treg cells (p = 0.0162) which was in good correlation with the percentage of CD45RA+FOXP3low nTreg cells within CD25+FOXP3+ Treg cells (p &lt; 0.0001). Furthermore, the percentage of CD45RA+FOXP3+low nTreg cells within CD25+FOXP3+ Treg cells was significantly higher in pregnant women (p = 0.045). Meanwhile, the percentage of CD45RA-FOXP3hi effector (eTreg) Treg cells within CD25+FOXP3 Treg cells was lower in pregnant women (p = 0.0238). Finally, women who achieve a pregnancy had significantly lower percentage of CD45RA-FOXP3hiCD15s+eTreg cells within CD25+FOXP3+ Treg cells (p = 0.0233).

Limitations, reasons for caution
This study has some limitations. The sample size is too small. Standard gating in flow cytometry has the limitation of analysing only few markers at a time in a flow plot, the levels of expression of the markers are not considered, as an alternative, dimensionality reduction analyses were carried out.

Wider implications of the findings
Our findings imply that the composition of distinct populations within nTreg cells can influence the women’s chance of becoming pregnant, especially nTreg cells, lacking CXCR5 expression, which distingushes T-follicular regulatory cells. Findings help us to understand the role of nTreg subpopulations in process of implantation in women undergoing IVF/ICSI treatment.

Trial registration number
not applicable