Cell-Free Fetal DNA Increases Prior to Labor at Term and in a Subset of Preterm Births

Abstract

Cell-free fetal DNA in the maternal circulation has been associated with the onset of labor at term. Moreover, clinical studies have suggested that cell-free fetal DNA has value to predict pregnancy complications such as spontaneous preterm labor leading to preterm birth. However, a mechanistic link between cell-free fetal DNA and preterm labor and birth has not been established. Herein, using an allogeneic mouse model in which a paternal green fluorescent protein (GFP) can be tracked in the fetuses, we established that cell-free fetal DNA (Egfp) concentrations were higher in late gestation compared to mid-pregnancy and were maintained at increased levels during the onset of labor at term, followed by a rapid decrease after birth. A positive correlation between cell-free fetal DNA concentrations and the number of GFP-positive pups was also observed. The increase in cell-free fetal DNA concentrations prior to labor at term was not linked to a surge in any specific cytokine/chemokine; yet, specific chemokines (i.e., CCL2, CCL7, and CXCL2) increased as gestation progressed and maintained elevated levels in the postpartum period. In addition, cell-free fetal DNA concentrations increased prior to systemic inflammation-induced preterm birth, which was associated with a strong cytokine response in the maternal circulation. However, cell-free fetal DNA concentrations were not increased prior to intra-amniotic inflammation-induced preterm birth, but in this model, a mild inflammatory response was observed in the maternal circulation. Collectively, these findings suggest that an elevation in cell-free fetal DNA concentrations in the maternal circulation precedes the physiological process of labor at term and the pathological process of preterm labor linked with systemic inflammation, but not that associated with intra-amniotic inflammation.

Electronic supplementary material

The online version of this article (10.1007/s43032-019-00023-6) contains supplementary material, which is available to authorized users.

Introduction

Labor is a state of physiological inflammation [1–6] that is characterized by an increase in cellular and soluble inflammatory mediators in the cervix [1, 7–19], myometrium [8, 9, 11, 12, 20–24], chorioamniotic membranes [9, 11, 25–32], and decidua (e.g., maternal-fetal interface) [5, 9, 11, 25, 26, 29, 33–38]. This concept is supported by transcriptomic studies showing that gestational tissues from women who underwent spontaneous labor at term display a gene expression signature consistent with acute inflammation [28, 39–50]. In addition to this local inflammation, labor is accompanied by a maternal systemic inflammatory response [12, 51–57]. Such an immune process is thought to be initiated by cell-free fetal DNA (cffDNA) [58–62], since its concentrations increase from mid to late pregnancy in humans [60, 63–73] and animals [74–77] and are even greater during the onset of term parturition [60, 66].

Human studies have shown that there is a positive association between maternal circulating cffDNA and adverse pregnancy outcomes such as early- and late-onset preeclampsia [78–81], intrauterine growth restriction [81, 82], and spontaneous preterm labor [83–88]. However, the predictive value of cffDNA in the maternal circulation is controversial [89–93]. Animal studies have also mechanistically shown that the administration of CpG (oligodeoxynucleotide) to pregnant Il10^−/−or NOD mice induces an inflammatory response leading to fetal resorption and preterm birth in a TLR9-dependent manner [94, 95]. It was further established that such a TLR9-mediated inflammatory process was triggered by fetal, but not adult, cell-free DNA [86], suggesting that fetal cell-free nucleic acids are capable of inducing pregnancy complications. However, such mechanistic studies have relied on the administration of exogenous DNA. Herein, using an animal model of allogenic pregnancy, we investigated whether the presence of the gene for the green fluorescent protein can be detected in the cell-free DNA fraction of the maternal circulation from mid to late gestation, before and during term labor, and prior to preterm labor and birth. In addition, maternal inflammatory responses were evaluated by measuring plasma cytokine concentrations.

Methods

Mice

C57BL/6 Actb-Egfp (GFP-expressing) males and DBA/2 virgin females were purchased from The Jackson Laboratory in Bar Harbor, ME, USA, and bred in the animal care facility at C.S. Mott Center for Human Growth and Development at Wayne State University, Detroit, MI. All mice were kept under a circadian cycle (light:dark = 12:12 h). Females, 8–12 weeks old, were bred with males of proven fertility. Female mice were checked daily between 8:00 and 9:00 a.m. for the appearance of a vaginal plug, which indicated 0.5 days post coitum (dpc). Females were then housed separately from the males, their weights were monitored, and a gain of 2 or more grams by 12.5 dpc confirmed pregnancy. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University (Protocol No. A 07-03-15).

Plasma Collection During Mid and Late Pregnancy and Postpartum

Ten groups of pregnant DBA/2 mice were used for experiments at a designated time of gestation: 12.5 dpc, 13.5 dpc, 14.5 dpc, 15.5 dpc, 16.5 dpc, 17.5 dpc, 18.5 dpc, 19.5 dpc, in labor, and postpartum (n = 5–11 per group). The in labor group was defined as dams on 20.5 dpc that were actively delivering as indicated by the presence of the first pup in the cage bedding or a pup in the birth canal. The postpartum group was defined as dams that delivered at 20.5 dpc, from which samples were collected 12–36 h after delivery. On the designated day of gestation or postpartum, dams were anesthetized by the intraperitoneal injection of 250 mg/kg of Avertin [tribromoethanol (Sigma-Aldrich, St. Louis, MO, USA) dissolved in amylene hydrate (Sigma-Aldrich) and distilled water]. Whole blood (0.5–1.2 mL) was collected by cardiac puncture using a 22-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA) and immediately dispensed into a K₂ EDTA MiniCollect® tube (Greiner Bio-one GmbH, Kremsmünster, Austria). Anticoagulated blood was centrifuged at 800 x g for 10 min at 4 °C. The plasma was collected and stored in a DNA LoBind Eppendorf tube (Eppendorf, Hamburg, Germany) at −20 °C until analysis.

Epifluorescence Imaging of the Green Fluorescent Protein-Positive Pups

Once the blood was collected, the whole uterus with the cervix and ovaries still intact was removed and placed in a Petri dish with PBS. The number of fetuses expressing green fluorescent protein (GFP) from each dam was determined by ex vivo imaging with the IVIS Spectrum in vivo imaging system (Perkin Elmer, Waltham, MA, USA) and recorded. An excitation filter of 465 nm and an emission filter of 520 nm were used to determine GFP expression.

Intra-amniotic Administration of Lipopolysaccharide

Pregnant DBA/2 mice were anesthetized on 16.5 dpc by inhalation of 2–3% isoflurane (Aerrane, Baxter Healthcare Corporation, Deerfield, IL, USA) and 1–2 L/min of oxygen in an induction chamber. Anesthesia was maintained with a mixture of 1.75–2% isoflurane and 2.0 L/min of oxygen. Dams were positioned on a heating pad and stabilized with adhesive tape. Fur removal from the abdomen was accomplished by applying Nair depilatory cream (Church & Dwight Co., Inc., Ewing, NJ, USA). Body temperature was maintained at 37 ± 2 °C and detected using a rectal probe (VisualSonics, Toronto, Ontario, Canada). Respiratory and heart rates were monitored by electrodes embedded in the heating pad. An ultrasound probe (VisualSonics) was fixed and mobilized with a mechanical holder, and the transducer was slowly moved toward the abdomen. Ultrasound-guided intra-amniotic injection of lipopolysaccharide (LPS; Escherichia coli O111:B4; Sigma-Aldrich) at a concentration of 5 μg/100 μL of sterile 1X phosphate-buffered saline (PBS) (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac (3-10  pups per dam) using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson) (n = 15). Controls were injected with 100 μL of PBS into each amniotic sac (n = 13). The syringe was stabilized with a mechanical holder (VisualSonics Inc.). Following the ultrasound, dams were placed under a heat lamp until they regained full motor function, which typically occurred 5–10 min after heating.

Intraperitoneal Administration of Lipopolysaccharide

Pregnant DBA/2 mice were intraperitoneally injected on 16.5 dpc with 20 μg of LPS (Escherichia coli O55:B5; Sigma-Aldrich) in 200 μL PBS using a 26-gauge needle (BD PrecisionGlide Needle, Becton Dickinson) (n = 10). Controls were injected with 200 μL of PBS (n = 9).

Plasma Collection Prior to Inflammation-Induced Preterm Birth

Dams were anesthetized 21–22 h after intra-amniotic injection of LPS or 10–11 h after intraperitoneal injection of LPS. Whole blood (0.5–1.2 mL) was collected by cardiac puncture using a 22-gauge needle (BD PrecisionGlide Needle, Becton Dickinson) and immediately dispensed into a K₂ EDTA MiniCollect® tube (Greiner Bio-one GmbH). Anticoagulated blood was centrifuged at 800 x g for 10 min at 4 °C. The plasma was collected and stored in a DNA LoBind Eppendorf tube (Eppendorf) at −20 °C until analysis.

Cell-Free DNA Extraction and Real-Time PCR

Cell-free DNA was extracted from 200 μL of the previously collected maternal plasma using the Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit (Norgen Biotek Corp, Thorold, ON, Canada), following the manufacturer’s protocol (steps 1 through 9). Three positive controls for Egfp, obtained using DNA extracted from the semen of transgenic GFP-expressing male mice, were serially diluted tenfold through six concentrations (10 ng/μL–0.1 pg/mL) and used to generate standard curves. Samples and controls were preamplified with the Taqman™ PreAmp Master Mix (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions for reaction volumes, to increase the amount of DNA. The final concentration of each primer was 0.18 μM, and the final concentration of each probe was 0.05 μM. Fourteen cycles of the preamplification PCR were run on the Applied Biosystems QuantStudio 12K Flex Real-Time PCR instrument (Life Technologies).

A total of 1 μL of preamplified DNA was used for quantification of the amount of Egfp DNA present in the extracted cell-free DNA fraction. Kapa Probe Fast ABI Prism 2x qPCR Master Mix was used as recommended in the manufacturer’s protocol. A 75 base pair region of Egfp was detected with primers and probed as previously described [75, 96] (forward primer, 5’-ACTACAACAGCCACAACGTCTATATCA-3’; reverse primer, 5’-GGCGGATCTTGAAGTTCACC-3’; and Taqman probe, 5’-FAM-CCGACAAGCAGAAGAACGGCATCA-TAMRA-3’, where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine). Apolipoprotein B (Apob) was used as a housekeeping gene (forward primer, 5’-CGTGGGCTCCAGCATTCTA-3’; reverse primer, 5’-TCACCAGTCATTTCTGCCTTTG-3’; and Taqman probe, 5’-FAM-CCTTGAGCAGTGCCCGACCATTC-TAMRA-3’). Egfp primers had a final concentration of 900 nM each and a final probe concentration of 200 nM. Apob primers had a final concentration of 300 nM each and a final probe concentration of 200 nM. The samples were amplified using the Applied Biosystems QuantStudio 12K Flex Real-Time PCR instrument (Life Technologies). All samples were run in triplicate. Results were presented as relative concentrations of cell-free fetal DNA (Egfp, ng/μL) which were calculated by extrapolation from the standard curve established above.

Cytokine/Chemokine Plasma Concentrations

The ProcartaPlex Mouse Cytokine & Chemokine Panel 1A 36-plex (Invitrogen by Thermo Fisher Scientific, Vienna, Austria) was used to measure the concentrations of IFNγ, IL-12p70, IL-1β, TNFα, GM-CSF, IL-18, IL-17A, IL-22, IL-23, IL-27, IL-9, IL-15/IL-15R, IL-13, IL-4, IL-5, IL-6, IL-10, Eotaxin (CCL11), IFNα, IL-28, IL-2, IL-3, LIF, IL-1α, IL-31, GRO-α (CXCL1), MIP-1α (CCL3), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MIP-1β (CCL4), MIP-2 (CXCL2), RANTES (CCL5), G-CSF, M-CSF, and ENA-78 (CXCL5) in the plasma samples, according to the manufacturer’s instructions. Plates were read using the Luminex 100 SystemFill (Luminex, Austin, TX, USA), and analyte concentrations were calculated with ProcartaPlex Analyst 1.0 software from Affymetrix, San Diego, CA, USA. The sensitivities of the assays were as noted in Supplementary Table 1. Inter-assay and intra-assay coefficients of variation were less than 10%.

Statistical Analysis

The following data analysis was completed using SPSS version 19.0 software (IBM Corp, Armonk, NY, USA). Maternal plasma concentrations of cell-free fetal DNA (Egfp) throughout normal pregnancy were analyzed by the Kruskal-Wallis test followed by correction for multiple comparisons. The p values shown in Fig. 1 represent the comparisons between the 12.5 dpc group and the following groups: 17.5 dpc, 18.5 dpc, 19.5 dpc, and in labor. The Spearman rank correlation was used to analyze the relationship between the maternal plasma concentrations of cell-free fetal DNA (Egfp) and the number of GFP-positive pups per litter (Fig. 2). Cytokine and chemokine concentrations throughout normal pregnancy were analyzed using the Kruskal-Wallis test; the p values shown in Fig. 3B, 3C, 3D, and 3E represent the overall difference among groups. The Spearman rank correlation was used to analyze the relationship between the cytokine and chemokine concentrations and gestational age (Fig. 3F, 3G, 3H, and 3I). The Spearman rank correlation was also used to analyze the relationship between the cytokine and chemokine concentrations and number of GFP-positive pups or cell-free fetal DNA concentrations (Supplementary Figs. 2 and 3 ). Maternal plasma cell-free fetal DNA (Egfp) and cytokine/chemokine concentrations prior to preterm birth were compared using the Mann-Whitney U test. A p value ≤ 0.05 was regarded as statistically significant.

Fig. 1.

Cell-free fetal DNA concentrations from mid to late pregnancy, in labor, and postpartum. A Animal model of a paternal antigen (Egfp) expressed by some of the concepti, and timeline for the collection of maternal plasma for the quantification of cell-free fetal DNA (Egfp). B Concentrations of cell-free fetal DNA (Egfp) in the maternal plasma at 12.5–19.5 days post coitum (dpc), in labor, and 12–36 h postpartum. Each dot represents one maternal plasma sample. Midlines = medians, boxes = interquartile range, whiskers = minimum and maximum range. *p = 0.04, **p = 0.02. N = 5–11 dams per group

Fig. 2.

Correlation between maternal concentrations of cell-free fetal DNA (Egfp) and the number of GFP-positive pups. A Timeline for the collection of the whole uterus with pups for epifluorescence imaging. B Representative epifluorescence images of the whole uterus including GFP-positive pups at (from top to bottom) 12.5–19.5 days post coitum (dpc) and in labor. Implantation sites and GFP-positive pups are also shown 12–36 h postpartum. Upper left panel shows the epifluorescence color scale (red = lowest expression, yellow = highest expression). Noncolored pups are GFP-negative. C Correlation between maternal plasma concentrations of cell-free fetal DNA (Egfp) and the number of GFP-positive pups per litter. Each dot represents one maternal plasma sample. Spearman correlation coefficient ρ = 0.399, correlation significance p < 0.001. N = 88 dams

Fig. 3.

Maternal cytokine/chemokine plasma concentrations from mid to late pregnancy, in labor, and postpartum. A Timeline for the collection of maternal plasma for the quantification of cytokines/chemokines. Plots showing the maternal plasma concentrations of B CCL2, C CCL7, D GM-CSF, and E IL-5 at 12.5–19.5 days post coitum (dpc), in labor, and 12–36 h postpartum. Plots showing the positive correlation between gestational age and plasma concentrations of F CCL2, G CCL7, H CXCL2, and I IL-12p70 at 12.5–19.5 days post coitum (dpc), in labor, and 12–36 h postpartum. Each dot represents one maternal plasma sample. Midlines = medians, boxes = interquartile range, whiskers = minimum and maximum range. Red curves indicate trends over time. P values reflect overall difference among the groups (B–E) or correlation significance (F–I). Spearman correlation coefficients are also displayed. N = 5–11 dams per group

Results

Cell-Free Fetal DNA Concentrations During Mid and Late Pregnancy, in Labor, and Postpartum

A previous study indicated that cell-free fetal DNA levels are higher in allogeneic pregnancies compared to those which are congenic [75]. Therefore, we used an allogeneic murine model of pregnancy to evaluate whether cell-free fetal DNA concentrations increase prior to normal parturition. The mating strategy and sample collection time points are described in Fig. 1A.

The concentrations of cell-free fetal DNA (Egfp) in the maternal plasma were significantly elevated on 17.5 dpc compared to 12.5 dpc and were maintained until the onset of labor (Fig. 1B). Yet, cell-free fetal DNA (Egfp) concentrations dropped rapidly during the postpartum period (Fig. 1B).

Next, we evaluated whether the number of GFP-positive pups per litter would correlate with the cell-free fetal DNA (Egfp) concentration in the maternal circulation. To investigate this research question, we utilized an epifluorescence imaging system that allowed us to track the number of GFP-positive pups during mid and late pregnancy, in labor, and during the postpartum period (Fig. 2A). In Fig. 2B, representative images of the uterine horns including GFP-positive pups, placentas, and implantation sites (red-yellow color) are shown. The epifluorescence scale used to detect the green fluorescent protein is also shown in Fig. 2B. There was a positive and significant correlation between the number of GFP-positive pups per litter and the concentrations of cell-free fetal DNA (Egfp) (Spearman correlation coefficient ρ = 0.3989, p < 0.001; Fig. 2C).

Cytokine/Chemokine Plasma Concentrations During Mid and Late Pregnancy, in Labor, and Postpartum

Given that the cell-free fetal DNA concentrations are associated with a maternal systemic inflammatory response [60, 61, 86, 94, 95], we investigated the plasma cytokine/chemokine concentrations in the maternal circulation during mid and late pregnancy, in labor, and postpartum (Fig. 3A). Out of the 36 cytokines/chemokines determined in the maternal plasma, only four of them changed significantly during pregnancy, in labor, and the postpartum period after adjustment for multiple comparisons (data not shown). Specifically, CCL2 plasma concentrations were maintained at low levels throughout mid and late pregnancy and in labor but peaked during the postpartum period (p = 0.007; Fig. 3B). CCL7 plasma concentrations oscillated during mid and late pregnancy and were maintained at high levels during the postpartum period (p = 0.02; Fig. 3C). In addition, GM-CSF plasma concentrations increased from mid to late pregnancy, peaking on 19.5 dpc, and were maintained until the postpartum period (p = 0.001; Fig. 3D). On the other hand, IL-5 plasma concentrations were decreased in late gestation (14.5–18.5 dpc) compared to mid-pregnancy (12.5 dpc), resurged during labor, and were maintained in the postpartum period (p = 0.002; Fig. 3E). Interestingly, IL-10 plasma concentrations increased prior to labor and dropped soon after; yet, these changes were not statistically significant (Supplementary Fig. 1).

We also determined whether maternal cytokine/chemokine plasma concentrations were correlated with increasing gestational age. Maternal systemic concentrations of CCL2 (Spearman correlation coefficient ρ = 0.289, p = 0.02; Fig. 3F), CCL7 (Spearman correlation coefficient ρ = 0.458, p < 0.001; Fig. 3G), CXCL2 (Spearman correlation coefficient ρ = 0.306, p = 0.01; Fig. 3H), and IL-12p70 (Spearman correlation coefficient ρ = 0.248, p = 0.05; Fig. 3I) were positively correlated with increasing gestational age. All of these mediators were maintained elevated in the postpartum period. Furthermore, no significant correlations between plasma concentrations of cytokines/chemokines and the number of GFP-positive pups or cell-free fetal DNA concentrations were observed throughout gestation (data not shown).

Cell-Free Fetal DNA Concentrations Prior to Inflammation-Induced Preterm Birth

Given that cell-free fetal DNA concentrations were increased prior to term parturition (Fig. 1B) and cell-free fetal DNA levels are associated with preterm labor and birth [83–88], we investigated whether the levels of cell-free Egfp were increased prior to inflammation-induced preterm birth. To address this research question, we used two previously established models of endotoxin-induced preterm birth: a systemic model of inflammation (i.e., intraperitoneal administration of LPS) [97–106] and an intra-amniotic model of inflammation (i.e., intra-amniotic administration of LPS) [107, 108]. The animal models of inflammation-induced preterm birth are displayed in Fig. 4A. Maternal plasma samples were collected prior to preterm parturition (Fig. 4A). The rate of preterm birth induced by systemic inflammation was 72.7% (8/11 mice), similar to what has been reported [107, 108]. Concentrations of cell-free fetal DNA (Egfp) were increased prior to systemic inflammation-induced preterm birth (Fig. 4B). The rate of preterm birth induced by intra-amniotic inflammation was 87.5% (7/8 mice), as previously reported [107, 108]. Yet, concentrations of cell-free fetal DNA (Egfp) were not increased prior to intra-amniotic inflammation-induced preterm birth (Fig. 4C). It is worth mentioning that five dams intra-amniotically injected with LPS presented cell-free fetal DNA concentrations similar to those dams intraperitoneally injected with LPS (Fig. 4C vs. 4B). However, the median cell-free fetal DNA (Egfp) concentration of dams intraperitoneally injected with LPS was approximately eightfold higher than those that were intra-amniotically injected with LPS (Fig. 4B vs. 4C).

Fig. 4.

Cell-free fetal DNA (Egfp) concentrations prior to inflammation-induced preterm birth. A Timeline for the systemic (intraperitoneal) or intra-amniotic administration of lipopolysaccharide (LPS) to pregnant dams with GFP-positive pups on 16.5 days post coitum (dpc). Maternal plasma was collected at 17.0–17.5 dpc for the quantification of cell-free fetal DNA (Egfp). Concentrations of cell-free fetal DNA (Egfp) in the maternal plasma prior to B systemic (intraperitoneal) or C intra-amniotic inflammation-induced preterm birth. Each dot represents one maternal plasma sample. Red lines indicate medians. N = 9–15 dams per group

Cytokine/Chemokine Plasma Concentrations Prior to Inflammation-Induced Preterm Birth

To conclude our study, we determined the cytokine/chemokine concentrations in the maternal circulation prior to inflammation-induced preterm birth. As previously shown [108–110], the systemic concentrations of multiple cytokines/chemokines were increased prior to systemic inflammation-induced preterm birth (Fig. 5). The systemic concentrations of some cytokines/chemokines were also increased prior to intra-amniotic inflammation-induced preterm birth (Fig. 5). Yet, the systemic inflammatory response prior to intra-amniotic inflammation-induced preterm birth was milder than that of the systemic inflammation model (Fig. 5). Specifically, the plasma concentrations of GM-CSF, IL-1β, IL-6, IL-4, IL-12p70, IL-17A, CCL2, CCL3, CCL4, CCL5, CCL7, CXCL1, CXCL2, CXCL10, and G-CSF were higher prior to systemic inflammation-induced preterm birth than prior to intra-amniotic inflammation-induced preterm birth (Fig. 5).

Fig. 5.

Maternal cytokine/chemokine plasma concentrations prior to inflammation-induced preterm birth. Plots showing the maternal plasma concentrations of (left to right, top to bottom) GM-CSF, IL-1β, IL-6, IL-4, IL-12p70, IL-17A, CCL2, CCL3, CCL4, CCL5, CCL7, CXCL1, CXCL2, CXCL10, and G-CSF prior to systemic (intraperitoneal) or intra-amniotic inflammation-induced preterm birth. Each dot represents one maternal plasma sample. Red lines indicate medians. N = 9–15 dams per group

Lastly, we determined the correlations between the plasma concentrations of cytokines/chemokines and the number of GFP-positive pups or cell-free fetal DNA (Egfp) levels in our systemic and intra-amniotic inflammation-induced preterm birth models. Plasma concentrations of IL-6 and IL-4 were negatively correlated with the number of GFP-positive pups prior to systemic inflammation-induced preterm birth (Supplementary Fig. 2). Moreover, plasma concentrations of IL-4 were negatively correlated with cell-free fetal DNA (Egfp) levels prior to systemic inflammation-induced preterm birth (Supplementary Fig. 3). No significant correlations were observed between the plasma concentrations of cytokines/chemokines and the number of GFP-positive pups or cell-free fetal DNA (Egfp) levels prior to intra-amniotic inflammation-induced preterm birth (data not shown).

Discussion

Principal Findings

The principal findings of our study are as follows: 1) cell-free fetal DNA (Egfp) concentrations were higher in late gestation compared to mid-pregnancy and were maintained increased during the onset of labor at term, followed by a rapid decrease after birth; 2) there was a positive and significant correlation between cell-free fetal DNA (Egfp) concentrations and the number of GFP-positive pups; 3) cytokine/chemokine plasma concentrations followed different patterns from mid to late pregnancy, and most of them were increased in the postpartum period; 4) plasma concentrations of specific chemokines (i.e., CCL2, CCL7, and CXCL2) and IL-12p70 increased as gestation progressed and maintained elevated levels in the postpartum period; 5) cell-free fetal DNA (Egfp) concentrations increased prior to systemic inflammation-induced preterm birth, but not before intra-amniotic inflammation-induced preterm birth; and 6) cytokine/chemokine plasma concentrations increased prior to systemic inflammation- or intra-amniotic inflammation-induced preterm birth; however, the cytokine storm was greater in dams injected intraperitoneally.

Cell-Free Fetal DNA Concentrations Are Increased in Late Gestation and During Labor at Term

Consistent with Khosrotehrani et al. [75], whose animal model was used herein, cell-free fetal DNA concentrations in allogeneic pregnancies were higher in late gestation than in mid-pregnancy. The difference in cell-free fetal DNA concentrations between allogeneic and syngeneic pregnancies has been attributed to the apoptotic deletion of maternal fetal-reactive cells [75]. The activation of such cell death pathways may explain why there is a need for more immunoregulation (i.e., regulatory T cells) in allogenic than in syngeneic pregnancies [111]. While the abovementioned study included the first, second, and third weeks of murine pregnancy [75], in the current study, we determined the cell-free fetal DNA concentrations from mid to late pregnancy, since detection of circulating cffDNA during early/mid gestation is used to diagnose common fetal genetic abnormalities [112–124]. We further examined cell-free fetal DNA concentrations during the active process of labor and in the postpartum period. Consistent with human [60, 66] and animal [74–77] studies, cell-free fetal DNA concentrations were increased before and during term labor and decreased rapidly after birth. These data are consistent with the hypothesis suggesting that cell-free fetal DNA is a trigger for term parturition [58, 59, 61].

An important observation made in the current study is that the number of pups containing the paternally derived gene for green fluorescent protein (GFP) was positively correlated with the maternal concentrations of cell-free fetal DNA. One possible explanation is that greater numbers of pups containing the gene will result in a larger amount of gestational tissue that releases microparticles (e.g., syncytiotrophoblast microvilli) into the maternal circulation that can serve as a source of cell-free DNA [125–128]. Another possibility is that circulating cell-free fetal DNA is partially derived from fetal nucleated erythroid cells that enter into the maternal circulation as early as 12 weeks of gestation [129–134]. The frequency of such fetal cells increases as a function of gestation, peaking during late gestation and diminishing rapidly after birth [135], a pattern that is mirrored by cell-free fetal DNA concentrations. Yet, it is worth mentioning that the absolute quantity of fetal DNA in maternal circulation is greater in the cell-free fraction than in the cellular component [66]. Notably, maternal obesity has been shown to reduce the concentrations of circulating cell-free fetal DNA both in the clinical setting [136, 137] and in an animal model [138], possibly as a result of the differential gene methylation [139] or altered cell death pathways [140] observed in the placentas of obese women.

Telomere shortening [62, 141–143], cellular senescence [6, 141, 142, 144–148], and apoptosis [149] are thought to lead to cell-free DNA release, which in turn induces the onset of labor at term through the involvement of the TLR9 signaling pathway [58, 62]. Recent studies in humans have found a positive association between systemic cell-free fetal DNA and IL-6 concentrations [60]. In addition, DNA derived from mouse placental tissues and fetuses induced the release of IL-6 by macrophages in an in vitro model [62, 149, 150]. This pro-inflammatory effect was observed upon incubation with telomere-depleted mouse DNA and was inhibited by chloroquine, a TLR9 inhibitor [62, 150]. In the current study, the rise in cell-free fetal DNA in the maternal circulation was not linked to a surge in the systemic levels of any specific cytokine or chemokine; however, the maternal plasma levels might not accurately reflect the localized proinflammatory events occurring at the maternal-fetal interface and/or the placenta and fetal membranes. These studies did, however, demonstrate an increase in plasma concentrations of CCL7, GM-CSF, and IL-5 preceded or occurred during labor at term. In addition, we found that plasma concentrations of CCL2, CCL7, CXCL2, and IL-12p70 increased as gestation progressed. Together, these data suggest a role for these cytokines/chemokines in the physiological process of labor at term. This concept is supported by the following evidence: 1) CCL2 expressed by myometrial tissues regulates mechanical and endocrine signals implicated in the process of labor [21]; 2) GM-CSF amniotic fluid concentrations are higher in women with intra-amniotic infection [151, 152], and the blockade of this cytokine prevents preterm birth in mice [153]; 3) plasma concentrations of IL-12 are greater in mid gestation than in early pregnancy [154], and elevated levels of this cytokine in serum are associated with spontaneous preterm birth [155]; and 4) maternal plasma concentrations of IL-5 are increased in women who delivered at term with clinical chorioamnionitis [156]. Nonetheless, we found that the systemic concentrations of IL-5 and CCL7 (both anti-inflammatory mediators [157, 158]) peaked during postpartum, indicating that these cytokines, and possibly IL-10, may be important in preventing a systemic cytokine storm during normal parturition and in the healing processes taking place during the postpartum period.

Cell-Free Fetal DNA Concentrations Increased Prior to Systemic Inflammation-Induced, but Not Intra-amniotic Inflammation-Induced, Preterm Birth

We found that maternal plasma cell-free fetal DNA (Egfp) concentrations were increased prior to systemic (intraperitoneal) inflammation-induced preterm labor, but not prior to preterm labor resulting from intra-amniotic inflammation. These data indicate that not all cases of preterm labor are characterized by an increased cell-free fetal DNA concentration in the maternal circulation, which could explain why there is still controversy regarding the value of cell-free fetal DNA for predicting preterm labor and birth [83–93, 159].

Previous studies have consistently shown that Il10^−/−and NOD mice injected with CpG ODN delivered preterm [94, 95, 160]. However, wild-type mice injected with CpG ODN delivered at term [94, 95, 160], indicating that the mechanisms whereby cell-free DNA induces preterm parturition involve the IL-10 pathway. It is therefore not surprising that the adoptive transfer of regulatory T cells prevents CpG-induced preterm birth [160]. Contrary to the previous reports [94, 95, 160], another study showed that injection of CpG and fetal DNA, but not adult DNA, in wild-type mice induces fetal loss [86]. Such a process is mediated via the TLR9 pathway, since Tlr9^−/−mice injected with fetal DNA did not result in fetal loss [86]. This controversy has been recently reviewed [159]. Herein, we provide evidence that, in some cases of preterm labor driven by a systemic inflammatory response, a rise in cell-free fetal DNA in the maternal circulation precedes preterm birth.

Mechanistic studies have shown that intrauterine inoculation of TLR ligands (e.g., peptidoglycan and Poly:IC) caused preterm birth by inducing apoptosis [161]. This is consistent with research demonstrating that inflammatory stimuli induce apoptosis in the placental tissues [162–167]. The engagement of TLRs in the intrauterine space can also upregulate the expression of inflammasome components [161, 168–170]. In humans, the inflammasome is implicated in the mechanisms leading to spontaneous labor at term [169, 171–175] or preterm labor [171, 176–179]. Herein, we provide evidence that the systemic administration of endotoxin, the TLR4 ligand [180, 181], can lead to elevated concentrations of cell-free fetal DNA in the maternal circulation, a process that is most likely due to the activation of several apoptotic and non-apoptotic pathways.

In the current study, we also used a recently described animal model of intra-amniotic inflammation-induced preterm birth, which resembles the human syndrome of preterm labor since it occurs in the absence of a body temperature change (a readout of fever in humans) and a strong cytokine storm, both of which are observed in the intraperitoneal model [107]. Yet, the intra-amniotic administration of endotoxin induces a mild inflammatory response in the mother and the fetus [108]. Herein, we showed that intra-amniotic inflammation induced by an endotoxin leads to preterm birth in the absence of elevated cell-free fetal DNA concentrations in the maternal circulation. However, in some cases, intra-amniotic administration of endotoxin modestly increased the concentration of cell-free fetal DNA in the circulation, but these levels were not comparable to dams injected intraperitoneally with an endotoxin. These data suggest that intra-amniotic inflammation triggers inflammatory pathways (apoptotic and non-apoptotic mechanisms) leading to a minimal release of cell-free fetal DNA into the maternal circulation, which is insufficient to induce preterm labor and birth. In line with the concept that cell-free fetal DNA can activate non-apoptotic pathways, we have recently shown that intra-amniotic inflammation, either induced by microbial products or alarmins, induces pyroptosis by activating the NLRP3 inflammasome prior to preterm birth [182, 183]. Yet, other potential inflammatory pathways implicated in the mechanisms whereby cell-free fetal DNA leads to preterm birth should also be investigated.

Conclusion

In the current study, we provide evidence that cell-free fetal DNA concentrations are increased in the maternal circulation during late pregnancy and the onset of the labor and reduced rapidly in the postpartum period. In addition, cell-free fetal DNA concentrations were positively correlated with the number of pups carrying the paternally derived green fluorescent protein gene. The increase in cell-free fetal DNA concentrations was not associated with a surge in a specific cytokine/chemokine in the maternal circulation; however, these studies did not rule out the possibility of localized proinflammatory events within the maternal-fetal interface and/or placental tissues. We also found that cell-free fetal DNA concentrations were increased prior to systemic inflammation-induced preterm birth, which was associated with a strong cytokine storm in the maternal circulation. Yet, cell-free fetal DNA concentrations were not increased prior to intra-amniotic inflammation-induced preterm birth; however, in this model, a mild inflammatory response was observed in the maternal circulation. Collectively, these findings suggest that an elevation in cell-free fetal DNA concentrations in the maternal circulation precedes the process of labor at term. Furthermore, an elevation in cell-free fetal DNA concentrations in the maternal circulation is associated with a subset of preterm births that are induced by systemic inflammation.

Funding Information

This research was supported, in part, by the Perinatology Research Branch (PRB), Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services (NICHD/NIH/DHHS), and, in part, with federal funds from the NICHD/NIH/DHHS under Contract No. HHSN275201300006C. Dr. Roberto Romero has contributed to this work as part of his official duties as an employee of the United States Federal Government. This research was also supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal, and Child Health.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict(s) of interest.