Alterations in the balance of leucocyte populations in uterine decidua may lead to the generation of an unfavourable cytokine environment that is associated with unsuccessful pregnancy. Single and double immunohistochemical labelling was used to examine leucocyte populations in decidua from normal third trimester, foetal growth-restricted and pre-eclamptic pregnancies. Placental bed biopsies from 12 women undergoing elective Caesarean section with no hypertension or foetal growth restriction (FGR), 8 women with FGR without maternal hypertension and 12 women with pre-eclampsia (PE) were used to quantify decidual CD56+ uterine NK cells, CD14+ macrophages, CD3+T-lymphocytes and CD8+ lymphocytes. CD3+CD56+, CD8+CD56+ and CD161+CD3+ double-labelled cells in decidua were compared in PE and control decidua. Decidual CD3+T-lymphocytes (P<0.01), CD8+ cytotoxic T-lymphocytes (P<0.05), CD14+ macrophages (P<0.0001) and CD56+ uterine natural killer (uNK) cells (P=0.01) were decreased in placental bed biopsies from women with PE compared with control third trimester decidua. By contrast, only CD56+ uNK cells were decreased in FGR decidua (P<0.05). Double-positive CD8+CD56+ cells were also decreased in PE compared with control third trimester decidua (P<0.05). The reduction in specific leucocyte subset numbers in PE and uNK cells in FGR suggests that altered local cytokine balance may be important in defective trophoblast invasion and spiral artery transformation in these pathological pregnancies.
Paula J Williams, Judith N Bulmer, Roger F Searle, Barbara A Innes and Stephen C Robson
H A Otun, B A Innes, G E Lash, B Schiessl, E Ball, R F Searle, S C Robson and J N Bulmer
Uterine spiral arteries undergo remodelling in normal pregnancy, with replacement of the musculoelastic arterial media by fibrinoid containing extravillous trophoblast cells. Deficient spiral artery remodelling is associated with several adverse pregnancy outcomes. Although there are distinct components of spiral artery remodelling, assessment is subjective and often based on an overall impression of morphology. We aimed to develop a quantitative approach for assessment of uterine spiral artery remodelling. Placental bed biopsies were immunostained using smooth muscle markers, digital images of spiral arteries were captured and Adobe Photoshop was used to analyse positive immunostaining. The method was then used to investigate variation in the same vessel at different levels within a paraffin block, and the effect of parity, pre-eclampsia or miscarriage on vascular smooth muscle cell content. Results were also compared with a more subjective morphology-based assessment system. There was good intra- and interobserver agreement and the method correlated well with the more subjective assessment system. There was an overall reduction in vascular smooth muscle, as detected by caldesmon 1 (h-caldesmon) immunopositivity, with increasing gestational age from 8 weeks to term. A previous pregnancy did not affect the amount of spiral artery smooth muscle. Comparison of pre-eclampsia and late miscarriage samples with controls of the appropriate gestational age demonstrated increased medial smooth muscle in pathological samples. This technique provides a simple, rapid, reproducible and inexpensive approach to quantitative assessment of spiral artery remodelling in normal and pathological human pregnancy, a process which although fundamental for successful pregnancy, is still incompletely understood.
J A Tamblyn, L E Jeffery, R Susarla, D M Lissauer, S L Coort, A Muñoz Garcia, K Knoblich, A L Fletcher, J N Bulmer, M D Kilby and M Hewison
Vitamin D deficiency is prevalent in pregnant women and is associated with adverse pregnancy outcomes, in particular disorders of malplacentation. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent regulator of innate and adaptive immunity, but its immune effects during pregnancy remain poorly understood. During early gestation, the predominant immune cells in maternal decidua are uterine natural killer cells (uNK), but the responsivity of these cells to 1,25(OH)2D3 is unknown despite high levels of 1,25(OH)2D3 in decidua. Transcriptomic responses to 1,25(OH)2D3 were characterised in paired donor uNK and peripheral natural killer cells (pNK) following cytokine (CK) stimulation. RNA-seq analyses indicated 911 genes were differentially expressed in CK-stimulated uNK versus CK-stimulated pNK in the absence of 1,25(OH)2D3, with predominant differentially expressed pathways being associated with glycolysis and transforming growth factor β (TGFβ). RNA-seq also showed that the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor were differentially expressed in CK-stimulated uNK vs CK-stimulated pNK. Further analyses confirmed increased expression of VDR mRNA and protein, as well as VDR-RXR target in CK-stimulated uNK. RNA-seq analysis showed that in CK-stimulated pNK, 1,25(OH)2D3 induced 38 and suppressed 33 transcripts, whilst in CK-stimulated uNK 1,25(OH)2D3 induced 46 and suppressed 19 genes. However, multiple comparison analysis of transcriptomic data indicated that 1,25(OH)2D3 had no significant overall effect on gene expression in either CK-stimulated pNK or uNK. These data indicate that CK-stimulated uNK are transcriptionally distinct from pNK and, despite expressing abundant VDR, neither pNK nor uNK are sensitive targets for vitamin D.