Detailed endometrial immune assessment of both normal and adverse reproductive outcome populations

Abstract

Purpose: Using a comprehensive flow cytometric panel, do endometrial immune profiles in adverse reproductive outcomes such as repeat implantation failure (RIF) and repeat pregnancy loss (RPL) differ from each other and male-factor controls?

Methods: Six-hundred and twelve patients had an endometrial biopsy to assess the immunophenotype. History on presentation was used to subdivide the population into recurrent implantation failure (RIF) [n = 178], recurrent pregnancy loss (RPL) [n = 155], primary infertility [n = 130] and secondary infertility [n = 114]. A control group was utilised for comparative purposes [n = 35] and lymphocyte subpopulations were described.

Results: Distinct lymphocyte percentage differences were noted across the populations. Relative to controls and RPL, patients with a history of RIF had significantly raised uterine NKs (53.2 vs 45.2 & 42.9%, p < 0.0001). All sub-fertile populations had increased percentage peripheral type NKs (p = 0.001), and exhibited increased CD69+ activation (p = 0.005), higher levels of B cells (p < 0.001), elevated CD4:CD8 ratio (p < 0.0001), lower T-regs (p = 0.034) and a higher proportion of Th1+ CD4s (p = 0.001). Patient aetiology confers some distinct findings, RPL; pNK, Bcells and CD4 elevated; RIF; uNK and CD56 raised while CD-8 and NK-T lowered.

Conclusions: Flow cytometric endometrial evaluation has the ability to provide a rapid and objective analysis of lymphocyte subpopulations. The findings show significant variations in cellular proportions of immune cells across the patient categories relative to control tissue. The cell types involved suggest that a potential differential pro-inflammatory bias may exist in patients with a history of adverse reproductive outcomes. Immunological assessment in appropriate populations may provide insight into the underlying aetiology of some cases of reproductive failure.

Keywords: ART; Endometrium; Immunophenotype; Lymphocytes; Natural killer cells.

Fig. 1

Flow cytometry image illustrating the selection of the lymphocyte gate using side scatter and antibodies to CD45. Lower panel (B) illustrates the total percentage expression of CD56 relative to all cells present in the biopsy

Fig. 2

Flow cytometry illustration indicating the uterine natural killer cells as the dominant lymphocyte (A) and expression of other markers which allow discrimination of NK subpopulations; CD16+ CD56^dim (pNK). NK-T are not illustrated here directly as CD3 or CD5 are not utilised on these channels, but can be seen below pNK and to the left of uNK. Panel (B) in the same patient illustrates CD57 expression and the localisation of this marker to CD56^dimcells alone

Fig. 3

Top panel illustrating all lymphocytes with specific isolation of CD4+ and CD8+ cells. The lower panel shows further differentiation of CD4+ cells into various bioactive subtypes, Th1, Th2, and T reg specifically. Positive CD3 staining is used to effectively set the CD4 and CD8 gates as it eliminates all the other non-T cell lymphocytes from the view. In the example below lymph gating, showing all available lymphocytes, rather than CD3+ gating is utilized

Fig. 4

Box and whisker plots illustrating the relative percentage spread of the two main patient population investigated, RIF and RPL versus that of the controls a pNK, b uNK, c CD56 total, d B-cells, e CD4 to 8 ratio and f Th1 to Th2 ratio

Fig. 5

Median percentage expression of immune cell subtypes between the first and repeat biopsy in 41 individuals. Mann Whitney U t-test shows no significant differences between paired samples