EARLY EMBRYO LOSS IS ASSOCIATED WITH LOCAL PRODUCTION OF NITRIC-OXIDE BY DECIDUAL MONONUCLEAR-CELLS

In early embryo loss, the fetus may be considered to be an allograft and, therefore, may be rejected by maternal immunocytes. However, the cytotoxic mechanisms involved are still poorly understood. We have previously shown the involvement of natural killer (NK) cells and mononuclear cells expressing Mac-1 (CD11b) and F4/80 in resorbing compared to nonresorbing embryos. In this study, the role of nitric oxide (NO) in the mechanism of early embryo loss was studied. Pregnant CBA/J females mated with DBA/2 males (20-30% early embryo loss) and CD1 females mated with CD1 males (5-10% early embryo loss) were studied on days 8, 10, and 12 of gestation. Cells from the implantation sites of individual embryos were tested for the production of nitrite and nitrate with or without in vitro challenge with lipopolysaccharide (LPS) to determine whether decidual macrophages were primed in situ. On day 12 of gestation, when resorption was clearly visible, resorbing embryos showed more than a fivefold increase in both basal- and LPS-induced nitrite and nitrate production compared to nonresorbing embryos in both mouse strains tested, indicating that the decidual mononuclear cells were primed. Furthermore, more than 20% of CBA/J embryos showed a significant nitrate release on days 8 and 10 of gestation before any signs of embryo cytopathology. This percentage col-responded to the spontaneous resorption rate seen in CBA/J female X DBA/2 male matings. Similarly, 4% of the embryos from pregnant CD1 mice on days 8 and 12 of gestation produced a significant amount of nitrate, which again correlated with the low incidence of resorption observed in these mice. Using immunohistochemistry, the presence of inducible nitric oxide synthase (iNOS) was detected at implantation sites. Furthermore, decidual cells positive for both iNOS and the macrophage marker Mac-1 were demonstrated in implantation sites by double immunostaining. This strongly suggests that decidual macrophages could be the cellular source of NO production. Aminoguanidine, a selective inhibitor of the iNOS, inhibited the in vitro production of nitric oxide by cells isolated from individual implantation sites, and more strikingly, significantly reduced early embryo losses in CBA/J females mated by DBA/2 males when given orally or parenterally to the gravid females starting on day 6 of gestation. In addition, aminoguanidine-treated pregnant mice showed a significant increase in average litter size when the pregnancies were allowed to proceed to term. The results presented strongly gested a role for NO as an effector molecule in mediating early embryo loss and showed the in situ activation of decidual macrophages was an early event preceding spontaneous abortion.