Subcutaneous implants recapitulate the placental microenvironment for prenatal immunosurveillance

Introduction: Nearly half of all cases of prenatal complication, miscarriage, preeclampsia, gestational diabetes, and preterm labor, have an undefined immune basis. This immune cascade inhibits placenta development, yet the clinic focuses on later disease stages. No prenatal assay exists that can predict complications due to immune rejection of the fetus. Because of this, a minimally-invasive assay to predict prenatal complications could transform the field of obstetrics. In this work, we employ microporous scaffolds as a synthetic immunological niche to capture the longitudinal immune domain of healthy and preeclamptic-like, miscarriage-prone rodent pregnancies without needing to disrupt the womb and with greater specificity than blood.

Materials and

Methods: Microporous poly(ε-caprolactone) (PCL) scaffolds were implanted subcutaneously in C57BL/6 female mice prior to cohabitation with BALB/c (allogeneic) or C57BL/6 (syngeneic) males. Scaffolds were serially removed prior to cohabitation and on embryonic days (E) 5.5, 10.5, and 15.5 (early, mid, and late stage) of murine pregnancy. Scaffolds were also removed from age-matched non-breeders. Blood, spleen, uterus, placentae, and implanted scaffold cells were processed for flow cytometry and T cell activation assays, to characterize prenatal immune dynamics. Scaffolds during gestation were sequenced for gene expression (bulk RNA sequencing). For preeclamptic-like pregnancy, CBA females were bred with DBA/2 males. Scaffolds were serially biopsied at early, mid, and late gestation for similar analyses of immunological microenvironment through T cell proliferation assays and trophoblast migration. Immune and fetal cells in the scaffold were be analyzed via RNA sequencing to pathway enrichment and differential gene expression during healthy and preeclamptic-like pregnancies.

Results, Conclusions, and Discussions: In both healthy and preeclamptic-like pregnancies, the scaffold implant recapitulates tissue resident immune cell phenotypes (including NK cell and macrophage phenotypes) and the immune microenvironment of the uterus and placentae with greater tissue specificity than can be found in circulation. In addition to the innate immune cells present in the scaffold exhibiting tissue resident phenotypes and maturity similar to the placentae and uterus, gene expression in the scaffold can be used to distinguish between gestation at different stages and at different immunological states. Through a linear Elastic Net regression, we identified 8 genes that differentiate between allogeneic-bred and syngeneic-bred mice at embryonic day 5.5 (E5.5), prior to fetal or placental organogenesis, to construct a predictive biomarker panel for the risk of preeclampsia. Lastly, scaffolds capture a population of fetal cells identified in the placentae at E15.5.
We have developed an implant to remotely monitor immunological markers of prenatal progression. We determined that scaffolds accumulate both innate and adaptive immune cells, and scaffolds recapitulate the uterine microenvironment. Scaffold gene expression can differentiate prenatal immune state, and scaffolds capture a population of fetal cells from circulation. These scaffolds act as a minimally invasive prenatal monitoring tool. Future work will involve assessing scaffold capture of these cells at earlier gestational stages as well as characterizing these cells for aneuploidy and other conditions.

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