Isolation and characterization of uterine leukocytes collected using a uterine swab technique

Leukocytes from the maternal-fetal interface are a valuable tool to study local changes in immune function during pregnancy; however, sampling can be challenging due to inadequate tissue availability and the invasive nature of placental bed biopsy. Here, we aim to purify and characterize leukocytes from paired peripheral and uterine blood samples to assess whether a less invasive method of uterine blood collection could yield a population of enriched uterine leukocytes suitable for ex vivo and in vitro analyses.

Human peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) expressed from surgical gauze post C-section were isolated, and immunophenotypic information was acquired by multi-parameter flow cytometry. PBMC and UBMC were stained for markers used to define T and B lymphocytes, macrophages, regulatory T (T_Reg ) cells, and natural killer (NK) cells. Prime flow was performed to check expression and analysis of CD16^- CD56⁺⁺ and CD16^- CD56++ NK transcripts in PBMC and UBMC samples.

Immunophenotyping revealed that over 95% of both live PBMC and UBMC consisted of CD45⁺ leukocytes. Higher percentages of CD16^- CD56⁺⁺ , characterized as uterine NK (uNK) cells, were observed in UBMC samples as compared to PBMC samples (18.41% of CD45⁺ CD3^- vs. 2.73%, respectively), suggesting that CD16^- CD56⁺⁺ cells were enriched in these samples. In UBMC, 49.64% of CD3-negative cells were of peripheral NK phenotype (CD16⁺ CD56⁺⁺ ), suggesting infiltration of maternal peripheral NK (pNK) cell in the uterine interface.

Intrauterine leukocytes, especially CD16^- CD56⁺⁺ NK cells, can be collected in sufficient numbers with increased purity by sampling the uterine cavity postdelivery with surgical gauze. Our results suggest that this non-invasive protocol is a useful sampling technique for isolating CD16^- CD56⁺⁺ cells, however, due to peripheral blood contamination, the NK cell yield could be lower compared to actual decidual or endometrial samples post-partum which is more invasive.