The role of macrophages in early pregnancy success.

Abstract

Macrophages are abundant in the ovary and uterus, with their densities and distribution changing throughout the estrous cycle in response to the sex steroid hormones, estrogen (E₂) and progesterone (P₄ ). In the uterus, macrophages are present in the endometrium, where they are located in close spatial proximity with uterine epithelial cells, and the myometrium. In the endometrium, macrophages are thought to have key roles in tissue remodelling in preparation for embryo implantation and trophoblast invasion. In the ovary, macrophages are present in the thecal cell layer where they surround developing follicles, and are also present in functional corpora lutea. They are thought to have a role in ovulation and in the extensive remodelling that occurs during formation of the corpus luteum. Other studies have suggested that macrophages enhance the output of P₄ from luteal cells. Their accumulation in the corpus luteum during luteolysis, as well as in atretic follicles, is suggestive of a role in tissue remodelling and removal of cellular debris. The specific role of macrophages during the pre- and peri-implantation period remains to be fully elucidated. Previous studies in mice lacking important macrophage-supporting cytokines suggest the role of these cells in endometrial receptivity and ovarian function may not be essential. However, interpretation of the importance of macrophages using these models is made difficult due to changes in macrophage phenotype, or incomplete macrophage depletion. The studies conducted herein make use of the Cd11b-Dtr transgenic mouse. The expression of the simian diphtheria toxin (DT) receptor (R) on CD11B-expressing macrophages enables transient and systemic depletion of these cells. The murine form of the DTR binds DT poorly and as such, injection of DT to wild-type mice has no effect. Immunohistochemistry and flow cytometry showed that using this model an 85% depletion of uterine macrophages and a 90% depletion of ovarian macrophages is achieved 24 h following DT injection to Cd11b-Dtr mice at estrus. Macrophage depletion in Cd11b-Dtr mice during the pre- and peri-implantation period caused complete pregnancy failure with loss during the peri-implantation phase. A direct adverse impact of DT on developing embryos was excluded as a contributing factor. Macrophage depletion did not affect uterine epithelial cell proliferation that occurs in response to E₂. Stromal cell proliferation that occurs in response to E₂ and P₄ in early pregnancy, prior to differentiation into decidual cells, also occurs normally following DT-elicited macrophage depletion. Macrophage depletion caused decreased expression of glycosylated structures involved in embryo attachment to uterine epithelial cells. There was a 25% reduction in the intensity of the lectin UEA-1 immunostaining (detects fucosylated structures on the uterine epithelium), and a 67% reduction in LewisX immunostaining. However, the process of artificially induced decidualisation was not impacted by macrophage depletion. Investigation into the impact of macrophage depletion on ovarian function showed that corpus luteum function was compromised, as evidenced by a 77% reduction in circulating plasma P₄ levels on day 4.5 post coitum (pc). Importantly, pregnancy could be rescued by the exogenous injection of P₄ and could be sustained into late gestation when P₄ support was maintained. The impact of macrophage depletion of the expression of mRNAs encoding enzymes responsible for the synthesis of P₄ was investigated using qRT-PCR. There was no change in mRNA expression of Star or Cyp11a1 following DT-elicited macrophage depletion, but there was a significant 29% decrease in Hsd3b1 mRNA expression. Hsd3b1 is the gene that encodes the protein HSD3B1, responsible for the conversion of pregnenolone to P₄. Following macrophage depletion, ovaries from Cd11b-Dtr mice were hemorrhagic at autopsy, and histology of ovarian tissue revealed evidence of impaired structural integrity of the corpora lutea. Staining for endothelial cell specific markers showed that endothelial cells were also depleted from some corpora lutea; the extent of endothelial cell depletion varied between corpora lutea within the same ovary. The lymphatic vasculature surrounding corpora lutea appeared not to be affected by macrophage depletion. In early pregnancy (day 1.5 and 2.5 pc), during the formation of the corpus luteum, the impact of macrophage depletion on genes involved in endothelial cell angiogenesis and survival was investigated. There was more than a 3-fold increase in the mRNA expression of Vegfa, but a significant decrease in the mRNA expression of Vegfc, Vegfd, Flt-1 and Kdr at both of these time-points following macrophage depletion. This study shows that macrophages are essential for the establishment of early pregnancy. In the endometrium, macrophages appear to influence uterine epithelial cell remodelling to increase the expression of markers of receptivity, and may facilitate the process of implantation. In the ovary, macrophages act to regulate the structure and function of the corpus luteum, potentially through regulating the expression of VEGFs and their receptors. These studies reveal new functions for macrophages in reproductive events and indicate that macrophage involvement in ovarian angiogenesis and support of steroidogenesis warrants further investigation.