Confocal laser scanning microscope study of cytokeratin immunofluorescence differences between villous and extravillous trophoblast: Cytokeratin downregulation in pre‐eclampsia

Abstract

Pre-eclampsia is a disease characterized by failures in interstitial implantation. One product of the implantation process is the basal plate; a structure whose complexity makes it hard to fully appreciate the pathological changes in significant diseases of pregnancy. This article describes our use of CLSM immunofluorescence to examine the cytokeratin composition of the cells of trophoblastic origin in the term placental basal plate. Large differences in the content of the structural polymeric protein were compared using analysis of digital images. We show that greater pancytokeratin immunofluorescence is observed in extravillous cytotrophoblast cells as compared with villous trophoblast. There is a >30-fold difference in the mean area percent of the most intensely immunofluorescent pixels in the tissue containing these cells. This is a very high, statistically significant difference as defined by the Wilcoxon Signed Ranks Test Asym. Sig. (two-tailed): P < 0.001. The most invasive population of cells of the trophoblast lineage (the extravillous trophoblast) exhibits a significant reduction in cytokeratin immunofluorescence when comparisons of healthy and pre-eclamptic pregnancies are made. This ratio was 2.4:1. It was tested using the Mann-Whitney U-test. From healthy to pre-eclamptic the reduction was from mean rank 83.42((healthy)) to 51.13((pre-eclamptic)). The difference was very highly statistically significant (n = 53 + 75 = 128; U = 984.500; Z = -4.852; P < 0.001). There was also less cytokeratin-related immunofluorescence in villous trophoblast when healthy villi were compared with pre-eclamptic villi. The observed alterations in trophoblastic cytoskeletal components are expected to damage the anchorage and motility of cells. The extravillous trophoblast is known to be necessary for implantation. This leads to a cellular hypothesis of the failure of implantation resulting in reduced depth of uterine invasion and failure to adapt the spiral arterioles for low-pressure perfusion of the intervillus space, two well-known features of pre-eclampsia. The reduction in cytokeratin-related immunofluorescence in the villus trophoblast seen on comparing healthy term placentae with those from pre-eclamptics implies that the trophoblast is a weaker epithelial layer in the hypertensive pregnancy. This could account for the rise in deported trophoblast associated with pre-eclampsia. Deported trophoblast has been invoked as the systemic messenger that leads to generalized maternal hypertension seen in this condition.

Immunofluorescence confocal laser scanning microscopy and immuno‐electron microscopic identification of keratins in human materno‐foetal interaction zone

“…Previous experiments using anti-pan-cytokeratin antibodies had shown an up-regulation of pankeratin in extravillous trophoblast as compared with villous trophoblast [5]. Here, we identify the specific molecular species of keratins that are involved (Table 6); (Figs 2 and 3).…”

“…Initial experiments used a mouse monoclonal anti-pan-cytokeratin-IgG 1 isotype as a trophoblast marker, which is a ‘cocktail’ of K4, 5, 6, 8, 10, 13 and 18 (Sigma-Aldrich, MO, USA, C2931) [5]. This was diluted in TBS-T containing 20% foetal calf serum, 1:800.…”

“…Its cells and syncytia perform several key roles in implantation, placentation, pregnancy maintenance and parturition [2,3]. It is also of significance in pregnancy pathologies [4,5]. A characteristic cytoskeletal marker of extra-embryonic membrane epithelia is the 10 nm diameter bundles of intermediate filaments comprised of keratins [6][7][8].…”

Healthy and pre‐eclamptic placental basal plate lining cells: Quantitative comparisons based on confocal laser scanning microscopy

“…Such "anchoring villi" display trophoblastic immunofluorescence characteristics in a solid core of cyto-trophoblast cells close to the basal plate. These exhibit the bright immunofluorescent anti-cytokeratin characteristics of the extravillous trophoblast and differ from the relatively less intense chorionic villus covering syncytiotrophoblast (Ockleford et al, 2004).…”

“…Any complete explanation of the cause of the disease will need to include a reason for the cytokeratin-based cytoskeletal aspects described recently (Ockleford et al, 2004). These observations are a useful step in understanding how alterations in the developmental programme of transcription (Beck et al, 1995;Knöfler et al, 2001) lead to the implantation deficits typical of pre-eclampsia.…”