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. 2016 Oct;36(10):942-952.
doi: 10.1002/pd.4917. Epub 2016 Sep 25.

Connexin 43 is overexpressed in human fetal membrane defects after fetoscopic surgery

David W Barrett  1 , Anna L David  2 , Christopher Thrasivoulou  3 , Alvaro Mata  1 , David L Becker  4 , Alex C Engels  5 , Jan A Deprest  5 , Tina T Chowdhury  6
Affiliations

Connexin 43 is overexpressed in human fetal membrane defects after fetoscopic surgery

David W Barrett et al. Prenat Diagn. 2016 Oct.

Abstract

Objective: We examined whether surgically induced membrane defects elevate connexin 43 (Cx43) expression in the wound edge of the amniotic membrane (AM) and drives structural changes in collagen that affects healing after fetoscopic surgery.

Method: Cell morphology and collagen microstructure was investigated by scanning electron microscopy and second harmonic generation in fetal membranes taken from women who underwent fetal surgery. Immunofluoresence and real-time quantitative polymerase chain reaction was used to examine Cx43 expression in control and wound edge AM.

Results: Scanning electron microscopy showed dense, helical patterns of collagen fibrils in the wound edge of the fetal membrane. This arrangement changed in the fibroblast layer with evidence of collagen fibrils that were highly polarised along the wound edge but not in control membranes. Cx43 was increased by 112.9% in wound edge AM compared with controls (p < 0.001), with preferential distribution in the fibroblast layer compared with the epithelial layer (p < 0.01). In wound edge AM, mesenchymal cells had a flattened morphology, and there was evidence of poor epithelial migration across the defect. Cx43 and COX-2 expression was significantly increased in wound edge AM compared with controls (p < 0.001).

Conclusion: Overexpression of Cx43 in the AM after fetal surgery induces morphological and structural changes in the collagenous matrix that may interfere with normal healing mechanisms. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

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Figures

Figure 1
Figure 1
Scanning electron microscopy of fetal membrane defect. Image shows membrane surface and structural details within the amniotic membrane (top panel) and chorionic membrane (bottom panel) after fetoscopic surgery. The fetal membrane was taken from a 28‐year‐old patient who underwent fetoscopic surgery for treatment of stage I twin‐to‐twin transfusion syndrome at 19 weeks + 3 days and who was delivered at 29 weeks + 4 days by caesarean section. Scale bar indicated by white lines, with white boxes showing region of higher magnification
Figure 2
Figure 2
Collagen organisation and Cx43 protein expression in the amniotic membrane. The fetal membrane was taken from a 34‐year‐old patient who underwent fetoscopic laser coagulation in a monochorionic twin pregnancy complicated by twin reversed arterial perfusion. Gestational age at surgical intervention was 15 weeks + 0 days and gestational age at delivery by caesarean section was 35 weeks + 3 days. Merged images shows Cx43 expression in the fibroblast layer with dense regions of collagen fibres orientated along the length of the fibroblast layer in the wound edge of the amniotic membrane. Nuclei in blue were stained with DAPI and collagen fibres in red indicated by white lines detected by second harmonic generation confocal imaging. Inset shows higher magnification of the collagen fibres close to the wound edge. Merged image on left side shows control amniotic membrane taken from the same patient away from the wound. Scale bar = 100 µm
Figure 3
Figure 3
Collagen orientation in the amniotic membrane after fetoscopic surgery. Representative second harmonic generation confocal images show orientation of collagen fibres in four regions of the wound edge and compared to control amniotic membranes taken from the same patient away from the wound (top panel). A distribution of orientation values for each image is shown in the bottom panel with regions of highly polarised fibres in all regions of the wound edge compared with a disorganised arrangement in controls. The fetal membrane was taken from a 29‐year‐old patient who underwent fetoscopic surgery for treatment of stage IV twin‐to‐twin transfusion syndrome at 19 weeks + 5 days and who was delivered at 29 weeks + 5 days by caesarean section
Figure 4
Figure 4
Collagen organisation and Cx43 protein expression in the epithelial and fibroblast layer. Merged images show characteristic spotted staining of Cx43 protein expression in the fibroblast layer of the amniotic membrane with dense regions of collagen fibres aligned in parallel along the wound edge of the amniotic membrane. Blue signal is DAPI staining of nuclei and collagen fibres in red indicated by white lines detected by second harmonic generation confocal imaging. The dotted white lines show the border along the length of the wound edge in the amniotic membrane. Merged image on left side shows control amniotic membrane taken from the same patient away from the wound. Scale bar = 100 µm
Figure 5
Figure 5
Cx43 distribution and plaque formation in the tissue layers of the amniotic membrane after fetoscopic surgery. The distribution of Cx43 was analysed per unit tissue area for comparisons between epithelial and fibroblast layer (A) and per cell nuclei (B). Integrated density was quantified for cell fluorescence and compared between control and wound edge specimens. For quantification of Cx43 plaque density, each spike represents a Cx43 plaque (C). In all cases, error bars represent the mean and SEM values for n = 8 to 10 replicates representing amniotic membranes taken from three patients, where *p < 0.05, **p < 0.01 and ***p < 0.001
Figure 6
Figure 6
Gene expression levels in the amniotic membrane after fetoscopic surgery. Gene expression of Cx43, COX‐2 and Type I collagen was presented as ratio values and normalised to control amniotic membranes taken from the same patient away from the wound (B). In all cases, error bars represent the mean and SEM values for n = 12 to 18 replicates, representing amniotic membranes taken from three patients, where *p < 0.05, **p < 0.01 and ***p < 0.001
Figure 7
Figure 7
Cell migration and collagen organisation in the fetal membrane defect. Immunofluoresence confocal microscopy shows bulbous regions of epithelial cells (indicated by white dashed arrows, A, B) and increased Cx43 protein expression (D, E) and collagen alignment at the interface between the fibroblast layer and wound edge of the amniotic membrane (indicated by white arrows). Scanning electron microscopy shows flattened morphology of mesenchymal cells close to the wound edge with characteristic lamellipodium projections (C). Higher magnification shows typical punctate plaques of Cx43 at the cell to cell contacts (E) and densely aligned collagen fibres at the wound edge (F). Inset in D shows negligible Cx43 expression in fibroblast layer in patient matched control amniotic membrane. Wound edge in B and D is indicated by white dashed line. The thickness of the amniotic membrane is approximately 117 µm
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