Discussion
Although a bunch of evidence is available regarding the association between NK cell cytotoxicity and female fertility (18), distinct opinions were formulated regarding the relationship between the cytotoxic activity of pNK and RM (19-21). In this study, the cytotoxic-related parameters, such as the percentage, immunophenotyping, and cytotoxicity of pNK cells in RM patients were evaluated. Interestingly, our results showed that NK cytotoxicity in the RM group was significantly lower than that in the control group. Moreover, the RM group showed a lower MFI of granzyme B and higher MFI of inhibitory receptors of pNK compared to that in the control group. These opposite results questioned the role of the cytotoxicity-related function of pNK cells in the occurrence of RM.
During implantation, the first step is the attachment of the embryo to the surface of the endometrium, followed by its migration and invasion until it is immersed in the depth of the uterine tissue (22). Uterine NK cells are a key factor in regulating the invasion of trophoblasts by secreting proteolytic enzymes such as matrix metalloproteinase-2 (MMP2) and MMP9 (23), and reducing extravillous trophoblast cell apoptosis (24). Nevertheless, it is still not clear whether the recruited pNK cells can regulate the pregnancy outcome or reflect what is happening at the maternal-fetal interface (19, 25, 26). Some scientists reported that patients with RM are accompanied with high pNK cells ratio (3, 20) or cytotoxicity (4). However, other scientists reported contradictory results indicating the same NK cell ratio (27) or lower pNK cytotoxicity (28) in RM patients compared with the fertile control, and our results are actually consistent with these results. A large cohort study suggested that the relationship between the subsequent pregnancy outcome and pNK cytotoxicity is not linear, and both patients with low and high pNK cytotoxicity tend to have high miscarriage rate (6). Although pNK cells contribute in creating an immune tolerant environment for placentation, they should still be equipped with a certain cytotoxic potential to fight infections during normal conditions (29). Therefore, both RM patients with higher pNK cytotoxicity and that with lower pNK cytotoxicity compared to fertile control should be taken into serious consideration.
NK cytotoxicity is controlled by the complex interaction between inhibitory and activating receptors (11, 30). Indeed, NK cells exert their cytotoxicity by secreting cytotoxic granules when the activating receptor signals are predominant (31). In order to explore the potential mechanism for low pNK cytotoxicity in RM patients, the expressions of receptors and cytotoxic granules in the pNK cells were also evaluated. However, our results showed no significant difference about their percentage between the RM and control groups, while the significant differences were observed in the MFI of granzyme B, CD158a, and CD158b in the pNK cells between the RM and control groups, which was consistent with the tendency for the cytotoxicity of pNK in the RM group. Thus, the predominant inhibitory signal might result in a low cytotoxicity in the RM group. It is also worth noting that some other pathways can actually be used by NK cells to kill infected or transformed cells. For instance, they can kill cells by the activation of the death receptor pathway, such as FasL-Fas signaling pathway and TNF-related apoptosis-inducing ligand (TRAIL) (32, 33). However, this was not the aim of our present work, and further studies should consider these different pathways used by the pNK cells to kill the target cells.
Our present work contains the following limitations that should not be underestimated. Firstly, this is a retrospective study. Secondly, several factors that affect the level and function of peripheral blood NK cells were not considered, including mental stress (34, 35), exercise (36, 37) and age. Although the patients with diseases that can influence peripheral blood NK cells were excluded, it is possible that some undetected autoimmune diseases or infections affected the results.