The use of multiplex staining to measure the density and clustering of four endometrial immune cells around the implantation period in women with recurrent miscarriage: comparison with fertile controls.

Serval studies showed an increased uterine natural killer cell density in women with recurrent miscarriage. However, no study has previously investigated the density and clustering of major immune cells simultaneously in precisely timed endometrial specimen section of this group of women. This study aimed to investigate the profile of endometrial immune cells populations and clustering level simultaneously in women with recurrent miscarriage and compare the results to fertile controls. A total of 30 women with unexplained recurrent miscarriage and 30 fertile controls were included in this study. Endometrial biopsy was performed precisely 7 days after LH surge. The cells density was expressed as percentage of positive immune cell/total stromal cells and the clustering of different endometrial cells was measured by R language toolbox 'spatstat'. Multiplex immunohistochemical method was employed to stain a panel of human endometrium samples simultaneously with antibodies against CD3 for T cells, CD20 for B cells, CD68 for macrophages and CD56 for uterine natural killer cells. The median CD3, CD68 and CD56 cell density in the miscarriage group were significantly higher than those of the fertile controls. In addition, the clustering between CD56 uterine natural killer cells and CD68 macrophages in the miscarriage group was significantly increased compared with fertile controls. In conclusion, the significant change in numbers of three out of four endometrial immune cell density and a significant increase in clustering between CD68 and CD56 cells suggest that several immune cells and their interactions may be important in the function of the endometrium; abnormal interactions may predispose to recurrent miscarriage.