Identifying Fetal Macrophages from Sex Expression

Macrophages from male fetuses were identified on the basis of sum log Y chromosome expression above a threshold (0 in mouse and 0.4 in human). For mouse data, differentially expressed genes were defined between male fetal macrophages and all other cells, and any significant genes were used for hierarchical clustering of all cells into two groups. The group with an overrepresentation of male fetal macrophages was defined as fetal. To measure fetal correlation as a confirmation of identity of non‐Y chromosome‐expressing macrophages assigned to the fetal cluster, expression of each cell was compared with the average expression of the same set of differentially expressed genes across all confirmed male fetal macrophages. This analysis was repeated independently on all macrophages included in analysis (from Sham‐ KO‐ and WT‐infected conditions) at each time point to identify a fetal expression profile unique to that time point. Because analysis at each time point included cells processed from either 6 or 8 placentas, each fetal analysis included some cells from a male fetus to provide a ground truth at that time point. For human data, clusters were defined by hierarchical clustering on informative genes, defined as highly expressed and variable genes (fano‐factor and mean expression) and the group with an overrepresentation of male fetal macrophages was defined as fetal.