Fetal microchimeric cells in blood of women with an autoimmune thyroid disease

Abstract

Context: Hashimoto's thyroiditis (HT) and Graves' disease (GD), two autoimmune thyroid diseases (AITD), occur more frequently in women than in men and show an increased incidence in the years following parturition. Persisting fetal cells could play a role in the development of these diseases.

Objective: Aim of this study was to detect and characterize fetal cells in blood of postpartum women with and without an AITD.

Participants: Eleven patients with an AITD and ten healthy volunteers, all given birth to a son maximum 5 years before analysis, and three women who never had been pregnant, were included. None of them had any other disease of the thyroid which could interfere with the results obtained.

Methods: Fluorescence in situ hybridization (FISH) and repeated FISH were used to count the number of male fetal cells. Furthermore, the fetal cells were further characterized.

Results: In patients with HT, 7 to 11 fetal cells per 1.000.000 maternal cells were detected, compared to 14 to 29 fetal cells in patients with GD (p=0.0061). In patients with HT, mainly fetal CD8(+) T cells were found, while in patients with GD, fetal B and CD4(+) T cells were detected. In healthy volunteers with son, 0 to 5 fetal cells were observed, which was significantly less than the number observed in patients (p<0,05). In women who never had been pregnant, no male cells were detected.

Conclusion: This study shows a clear association between fetal microchimeric cells and autoimmune thyroid diseases.

Figure 1. FISH and Repeated FISH.

A. FISH of female cells, showing two SpectrumOrange X FISH spots and of a presumed male cell indicated by an arrow, showing one SpectrumOrange X FISH and one SpectrumGreen Y FISH spot; B. Repeated FISH of the female cells and the presumed male cell, showing no SpectrumAqua Y FISH spots in the female cells. In contrary, the male cell shows one SpectrumAqua Y FISH signal on the exact same location as the SpectrumGreen Y FISH spot in image A (indicated by an arrow), indicating this is a true male cell. C. FISH of female cells and of one presumed male cell, indicated by an arrow. D. Repeated FISH of a male cell (C) shows no SpectrumAqua Y FISH spot. The SpectrumGreen Y FISH spot was probably caused by cellular debris or dust particles. The SpectrumOrange X FISH spot of that cell is larger than the other SpectrumOrange spots which may indicate two SpectrumOrange X FISH spots lying very closely to each other.

Figure 2. AxioVision Commander script for the automatic detection of male fetal cells.

A. Original image, split up in B. DAPI image and C. SpectrumGreen image; D. Threshold interactive on the DAPI image: segmentation based on the definition of a brightness range. Pixels within the defined gray level range are set to the maximum gray value 1 (pseudocolor blue); whilst pixels outside it are set to the minimum gray value 0 (black), resulting in a binary image; E. Scrap of image D: removing all artefacts too small to be possibly originating from cell nuclei; F. Close: filling in gaps in the contours of the nuclei; G. Dynamic Threshold of the SpectrumGreen image, resulting in a binary image showing the Y chromosome FISH spots; H. Scrap of image G: all regions smaller than 10 pixels and larger than 65 pixels are removed; I. Masking of the binary images F and H: retaining only the SpectrumGreen FISH signals lying in a nucleus. In the last step, to be included as a true FISH signal, the detected regions had to fulfil four measurement parameter conditions with regard to area of the region, lowest and highest pixel density and standard deviation of the pixel density (not shown).

Figure 3. Boxplot: fetal cells in patients with GD or HT, and healthy volunteers with son.

Minimum and maximum numbers of detected fetal cells are shown, as well as first quartile, median and third quartile. The number of fetal cells was significantly different between the three groups (p <0,05). Moreover, a significant difference between patients with GD and patients with HT was observed (MW, p = 0,0061).