Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes

Abstract

Inflammation is central to the mechanisms of parturition, but the lack of understanding of how it is controlled in normal parturition hampers our ability to understand how it may diverge resulting in preterm birth. Cell-free fetal DNA is found in the amniotic fluid, and it is thought to be able to activate inflammation as a danger-associated molecular pattern. Although its levels increases with gestational age, its effect has not been studied on the human fetal membranes. Thus, the aim of this study was to determine if the fetal DNA can trigger inflammation in the human fetal membranes and, thus, potentially contribute to the inflammatory load. Isolated human amniotic epithelial cells and fetal membrane explants were treated apically with fetal DNA causing the translocation of NF-KB into the nucleus of cells and throughout the cells of the explant layers with time. Fetal membrane explants were treated apically with either small or larger fragments of fetal DNA. IL-6, TNFα, and GM-CSF secretion was measured by ELISA, and pro-MMP2 and pro-MMP9 activity was measured by zymography from apical and basal media. Increased apical IL-6 secretion and basal pro-MMP2 activity was seen with small fragments of fetal DNA. When the data were disaggregated based on fetal sex, males had significant increases in IL-6 secretion and basal increased activity in pro-MMP2 and 9, whereas females had significantly increased basal secretion of TNFα. This was caused by the smaller fragments of fetal DNA, whereas the larger fragments did not cause any significant increases. Male fetal DNA had significantly lower percentages of methylation than females. Thus, when the cytokine and pro-MMP activity data were correlated with methylation percentage, IL-6 secretion significantly correlated negatively, whereas GM-CSF secretion positively correlated. These data support the role of fetal DNA as an inflammatory stimulus in the FM, as measured by increased NF-κB translocation, cytokine secretion, and increased pro-MMP activity. However, the data also suggested that the responses are different from FM tissues of male and female fetuses, and both the fragment size and methylation status of the fetal DNA can influence the magnitude and type of molecule secreted.

Keywords: cytokine; fetal DNA; fetal membranes; fetal sex; inflammation; methylation.

FIGURE 1

Characterization of fetal DNA fragments isolated from AECs. (A) Ethidium bromide stained gel of DNA either; whole—the cffDNA upon immediate isolation from primary human amnion epithelial cells or, sonicated—cffDNA isolated from primary human amnion epithelial cells but sonicated for 4 min. Two different patient samples of fetal DNA are shown as examples. (B) Quantification of methylation percentage of DNA from the individual patients’ collected FM samples. Each dot indicates separate patients’ DNA n = 16.

FIGURE 2

Fetal DNA causes the translocation of the NF-κB subunit p65. (A) Immunocytochemistry of the nuclear translocation of p65 (a) not treated with cffDNA and (b) treated with 100 ng/ml of 4 m.s. cffDNA. Green—p65. (B) Quantitation of the percentage of p65 translocation in AECs treated with 1, 10, 100, and 1,000 ng/ml 4 m.s. cffDNA or LPS 1000 ng/ml for 30 min n = 3 AEC from different patients’ isolated cells. (C) Fetal membrane explants treated only apically with 100 ng/ml 4 m.s. cffDNA for 30 mins, 1, and 2 h. The dotted white line represents untreated control, AEC—amnion epithelial cells. AMC—amnion mesenchymal cells. The data displayed as fold change from no treatment control. (n = 3 different patients’ fetal membrane explants). *p = < 0.05.

FIGURE 3

Cytokine secretion from fetal membrane explants after treatment with w.cffDNA or 4 m.s cffDNA. Fold change secretion, compared to no treatment control, of cytokines into the apical (top) well or basal (bottom) well of the transwell fetal membrane explant after treatment with (red) w.cffDNA or (blue) 4 m.s. cffDNA at 100 ng/m. (A) IL-6, (B) IL-6 after treatment with LPS (100 ng/ml), (C) TNF-ɑ, and (D) GM-CSF. n = 7–10 patients. *p = < 0.05.

FIGURE 4

Matrix metalloproteinase activity from fetal membrane explants after treatment with w.cffDNA or 4 m.s. cffDNA. The fold change activity, compared to no treatment control, of MMPs from the apical (top) well or basal (bottom) well of the transwell fetal membrane explant after treatment with (red) w.cffDNA or (blue) 4 m.s cffDNA at 100 ng/ml. (A) Example zymography gel. T = apical, B = basal, (B) Pro-MMP9, and (C) pro-MMP2. n = 10 patients. *p = < 0.05.

FIGURE 5

Sex-specific response of FM explants to 4 m.s cffDNA. (A) Example of PCR product gel shows that in lanes 2–4 the expression of SRY indicates a female fetus, whereas the expression of both SRY and ALT1 in lanes 5–7 indicated a male fetus. The fold change secretion, compared to no treatment control, of cytokines into the apical (top) well or basal (bottom) well of the transwell fetal membrane explant after treatment with 4 m.s cffDNA at 100 ng/ml. (B) IL-6, (C) TNF-alpha, and (D) GM-CSF (E) pro-MMP9 (F) pro-MMP2. Black—untreated control. Blue- male fetus, and pink—female fetus. n = 7–10 patients. *p = < 0.05 **p = < 0.001.

FIGURE 6

Sex-specific response of FM explants to w.cffDNA. The fold change secretion, compared to no treatment control, of cytokines into the apical (top) well or basal (bottom) well of the transwell fetal membrane explant after treatment with w.cffDNA at 100 ng/ml. (A) IL-6, (B) TNF-alpha, and (C) GM-CSF (D) pro-MMP9 (E) pro-MMP2. Black—untreated control. Blue- male fetus, and pink—female fetus. n = 7–10 patients. *p = < 0.05 **p = < 0.001.

FIGURE 7

Fetal membrane response to fetal DNA relative to methylation percentage. (A) Percentage methylation of male and female fetal DNA. The correlation of cytokine secretion from FM correlated with percentage methylation of fetal DNA used for apical treatment. (B) Apical secretion of IL-6 regardless of fetal DNA fragment size. (C) Table illustrating the correlation between the percentage of methylation and the apical secretion of IL-6 with w.cffDNA; the apical secretion of IL-6 from the male explants with 4 m.s cffDNA. (D) Basal GM-CSF secretion regardless of fetal DNA size is used for treatment. (E) Table illustrating the correlation between the percentage of methylation and the basal secretion of GM-CSF of FM treated with w. cffDNA; the basal GM-CSF secretion of FM treated with 4 m.s. cffDNA; the basal secretion of GM-CSF from the female explants treated with w.cffDNA.

FIGURE 8

Summary of the effect of fetal DNA on fetal membrane explants from male and female fetus pregnancies. (A) Table of treatment conditions, secretion locations with DNA species causing a response. (B) Transwell system with fetal membrane explant. The secretion into the apical media compartment of IL-6, and pro-MMP-2, into the basal media compartment of IL-6, TNF-α, pro-MMP2, and pro-MMP9. The dotted purple arrow indicates secretion regardless of fetal sex. The dotted blue arrows indicated secretion by male fetus FM. The dotted pink arrows indicated secretion by female fetus FM. n = 7–10 patients. *p = < 0.05 **p = < 0.001. (C) Diagram of fetal membranes (modified from Richardson et al., 2020) with attached decidua, illustrating the wave of NF-κB p65 subunit translocation to the nucleus over time (amnion→chorion→decidua) in response to fetal DNA treatment (apical).