Single-Cell Transcriptomic Profiling of Fixed PBMCs using FLEX panel Reveals Trimester-Specific Immune Adaptations in Pregnancy

Single-cell RNA sequencing (scRNA-seq) has transformed molecular biology by enabling high-resolution mapping of cellular heterogeneity. However, conventional scRNA-seq technologies are primarily restricted to fresh or methanol-fixed samples, posing challenges for the analysis of formaldehyde-fixed or formalin-fixed, paraffin-embedded (FFPE) tissues. Recently, 10x Genomics introduced the Single Cell Gene Expression Flex (10x-FLEX) platform to facilitate scRNA-seq profiling of formaldehyde-fixed or FFPE samples. However, only a limited number of studies have applied this method. Here, we employed 10x-FLEX scRNA-seq on formaldehyde-fixed peripheral blood mononuclear cells (PBMCs) from first- and third-trimester pregnant women to investigate immune adaptations during pregnancy. Our findings validate that 10x-FLEX identifies the same immune cell types as conventional 10x 3′ chemistry, while providing enhanced sensitivity for sparsely populated immune subsets and subtle transcriptional variations. We observed functional enrichments in CD14⁺ monocytes, CD16⁺ monocytes, and NK cells across trimesters. Notably, peripheral NK cells showed enrichment of epithelial–mesenchymal transition (EMT) and tissue remodeling pathways, suggesting a potential role in developmental regulation during late gestation. Furthermore, we detected trimester-specific activation of immune signaling pathways, including SN, THBS, TNF, GAS, TWEAK, and NRGR signaling during early pregnancy, and TRAIL and complement pathways during late pregnancy. We also identified APP–CD74 ligand–receptor interactions upregulated in CD14⁺ monocytes in the third trimester, reflecting altered intercellular communication across gestation. Together, these results demonstrate the robustness of 10x-FLEX in capturing biologically meaningful transcriptional and signaling dynamics in fixed PBMCs, highlighting its promise for single-cell transcriptomic analysis of fixed clinical specimens in maternal–fetal immunology.