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

PROBLEM: 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. METHOD OF STUDY: 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 (TReg ) 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. RESULTS: 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. CONCLUSION: 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.

Bisphenol S and Epidermal Growth Factor Receptor Signaling in Human Placental Cytotrophoblasts.

BACKGROUND: Bisphenol S (BPS) is an endocrine-disrupting chemical and the second most abundant bisphenol detected in humans. In vivo BPS exposure leads to reduced binucleate cell number in the ovine placenta. Binucleate cells form by cellular fusion, similar to the human placental syncytiotrophoblast layer. Given that human placental syncytialization can be stimulated through epidermal growth factor (EGF), we hypothesized that BPS would reduce human cytotrophoblast syncytialization through disruption of EGF receptor (EGFR) signaling. OBJECTIVE: We tested whether BPS interferes EGFR signaling and disrupts human cytotrophoblast syncytialization. METHODS: We first tested BPS competition for EGFR using an EGF/EGFR AlphaLISA assay. Using human primary term cytotrophoblast cells (hCTBs) and MDA-MD-231 cells, a breast cancer cell line with high EGFR expression, we evaluated EGFR downstream signaling and tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional end points of EGFR signaling, including EGF endocytosis, cell proliferation, and syncytialization. RESULTS: BPS blocked EGF binding in a dose-dependent manner and reduced EGF-mediated phosphorylated EGFR in both cell types. We further confirmed that BPS acted as an EGFR antagonist as shown by a reduction in EGF internalization in both hCTBs and MDA-MD-231 cells. Finally, we demonstrated that BPS interfered with EGF-mediated cell processes, such as cell proliferation in MDA-MD-231 cells and syncytialization in hCTBs. EGF-mediated, but not spontaneous, hCTB syncytialization was fully blocked by BPS (200 ng/mL), a dose within urinary BPS concentrations detected in humans. CONCLUSIONS: Given the role of EGFR in trophoblast proliferation and differentiation during placental development, this study suggests that exposures to BPS at environmentally relevant concentrations may result in placenta dysfunction, affecting fetal growth and development. https://doi.org/10.1289/EHP7297.