The link between cell-free DNA, inflammation and the initiation of spontaneous labor at term

Abstract

Related article, page 583. Related article, page 583. In 1997, Denis Lo and colleagues first reported the presence of free, extracellular DNA derived from fetal sources circulating in the blood of pregnant women throughout gestation; they labeled it cell-free “fetal” DNA (cffDNA).1Lo Y.M. Corbetta N. Chamberlain P.F. et al.Presence of fetal DNA in maternal plasma and serum.Lancet. 1997; 350: 485-487Abstract Full Text Full Text PDF PubMed Scopus (2324) Google Scholar In a subsequent series of publications, these investigators reported that cffDNA makes up 3.4–6.2% of the total cell-free DNA in maternal plasma, that cffDNA is present in short fragments with only 20% >193 base pair and none >313 base pairs, that cffDNA circulating in maternal plasma has a half-life of only approximately 16 minutes, and that cffDNA levels in maternal plasma increase 11- to 12-fold from mid to late gestation.2Lo Y.M. Tein M.S. Lau T.K. et al.Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.Am J Hum Genet. 1998; 62: 768-775Abstract Full Text Full Text PDF PubMed Scopus (1388) Google Scholar, 3Chan K.C. Zhang J. Hui A.B. et al.Size distributions of maternal and fetal DNA in maternal plasma.Clin Chem. 2004; 50: 88-92Crossref PubMed Scopus (461) Google Scholar, 4Lo Y.M. Zhang J. Leung T.N. Lau T.K. Chang A.M. Hjelm N.M. Rapid clearance of fetal DNA from maternal plasma.Am J Hum Genet. 1999; 64: 218-224Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar Since that time, reports by multiple investigators have confirmed the observations by Lo et al, especially in regard to the marked increase in cffDNA levels at the end of gestation.5Ariga H. Ohto H. Busch M.P. et al.Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis.Transfusion. 2001; 41: 1524-1530Crossref PubMed Scopus (216) Google Scholar, 6Birch L. English C.A. O’Donoghue K. Barigye O. Fisk N.M. Keer J.T. Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation.Clin Chem. 2005; 51: 312-320Crossref PubMed Scopus (125) Google Scholar, 7Majer S. Bauer M. Magnet E. et al.Maternal urine for prenatal diagnosis: an analysis of cell-free fetal DNA in maternal urine and plasma in the third trimester.Prenat Diagn. 2007; 27: 1219-1223Crossref PubMed Scopus (36) Google Scholar, 8Wang E. Batey A. Struble C. Musci T. Song K. Oliphant A. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma.Prenat Diagn. 2013; 33: 662-666Crossref PubMed Scopus (288) Google Scholar Of note, the presence of cffDNA in maternal plasma and its increase at term does not appear to be restricted to human pregnancies; several published reports have described similar observations in other mammals, which include subhuman primates.9Khosrotehrani K. Wataganara T. Bianchi D.W. Johnson K.L. Fetal cell-free DNA circulates in the plasma of pregnant mice: relevance for animal models of fetomaternal trafficking.Hum Reprod. 2004; 19: 2460-2464Crossref PubMed Scopus (31) Google Scholar, 10Wang G. Cui Q. Cheng K. Zhang X. Xing G. Wu S. Prediction of fetal sex by amplification of fetal DNA present in cow plasma.J Reprod Dev. 2010; 56: 639-642Crossref PubMed Scopus (26) Google Scholar, 11De Leon P.M. Campos V.F. Dellagostin O.A. Deschamps J.C. Seixas F.K. Collares T. Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA).Theriogenology. 2012; 77: 694-698Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 12Kadivar A. Hassanpour H. Mirshokraei P. Azari M. Gholamhosseini K. Karami A. Detection and quantification of cell-free fetal DNA in ovine maternal plasma: use it to predict fetal sex.Theriogenology. 2013; 79: 995-1000Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 13Jimenez D.F. Tarantal A.F. Quantitative analysis of male fetal DNA in maternal serum of gravid rhesus monkeys (Macaca mulatta).Pediatr Res. 2003; 53: 18-23Crossref PubMed Scopus (37) Google Scholar, 14Mitsunaga F. Ueiwa M. Kamanaka Y. Morimoto M. Nakamura S. Fetal sex determination of macaque monkeys by a nested PCR using maternal plasma.Exp Anim. 2010; 59: 255-260Crossref PubMed Scopus (9) Google Scholar Although the clinical utility of cffDNA as a noninvasive tool to perform fetal genetic analyses that include gender determination, testing for Mendelian genetic disorders, and aneuploidy screening has become well-established,15Wright C.F. Burton H. The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis.Hum Reprod Update. 2009; 15: 139-151Crossref PubMed Scopus (191) Google Scholar the physiologic role for the highly regulated release of cffDNA by the placenta and fetal membranes is yet to be determined clearly.16Phillippe M. Cell-free fetal DNA: a trigger for parturition.N Engl J Med. 2014; 370: 2534-2536Crossref PubMed Scopus (54) Google Scholar In this regard, several published reports have provided data that confirm a significant relationship between elevated levels of cffDNA and the onset of parturition, both preterm and at term.7Majer S. Bauer M. Magnet E. et al.Maternal urine for prenatal diagnosis: an analysis of cell-free fetal DNA in maternal urine and plasma in the third trimester.Prenat Diagn. 2007; 27: 1219-1223Crossref PubMed Scopus (36) Google Scholar, 17Farina A. LeShane E.S. Romero R. et al.High levels of fetal cell-free DNA in maternal serum: a risk factor for spontaneous preterm delivery.Am J Obstet Gynecol. 2005; 193: 421-425Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Jakobsen T.R. Clausen F.B. Rode L. Dziegiel M.H. Tabor A. High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery.Prenat Diagn. 2012; 32: 840-845PubMed Google Scholar, 19Dugoff L. Barberio A. Whittaker P.G. Schwartz N. Sehdev H. Bastek J.A. Cell-free DNA fetal fraction and preterm birth.Am J Obstet Gynecol. 2016; 215: 231.e1-231.e7Abstract Full Text Full Text PDF Scopus (60) Google Scholar In addition, experimental studies have confirmed the ability of small DNA fragments (oligodeoxynucleotides) and DNA isolated from fetal tissue to stimulate a proinflammatory cascade within the pregnant mouse uterus that results in pregnancy loss (ie, preterm deliveries and/or fetal resorptions).20Thaxton J.E. Romero R. Sharma S. TLR9 activation coupled to IL-10 deficiency induces adverse pregnancy outcomes.J Immunol. 2009; 183: 1144-1154Crossref PubMed Scopus (132) Google Scholar, 21Sun Y. Qin X. Shan B. et al.Differential effects of the CpG-Toll-like receptor 9 axis on pregnancy outcome in nonobese diabetic mice and wild-type controls.Fertil Steril. 2013; 99: 1759-1767Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 22Scharfe-Nugent A. Corr S.C. Carpenter S.B. et al.TLR9 provokes inflammation in response to fetal DNA: mechanism for fetal loss in preterm birth and preeclampsia.J Immunol. 2012; 188: 5706-5712Crossref PubMed Scopus (137) Google Scholar The influx of inflammatory cells (especially macrophages and neutrophilic leukocytes) and the increase in proinflammatory cytokines in response to DNA that was observed during the experimental studies noted earlier are similar to the inflammatory events that were observed in the human uterus, cervix, and fetal membranes at or before the spontaneous onset of labor.23Thomson A.J. Telfer J.F. Young A. Campbell S. Stewart C.J. Cameron I.T. et al.Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process.Hum Reprod. 1999; 14: 229-236Crossref PubMed Scopus (443) Google Scholar, 24Osman I. Young A. Ledingham M.A. et al.Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term.Mol Hum Reprod. 2003; 9: 41-45Crossref PubMed Scopus (556) Google Scholar, 25Haddad R. Tromp G. Kuivaniemi H. et al.Human spontaneous labor without histologic chorioamnionitis is characterized by an acute inflammation gene expression signature.Am J Obstet Gynecol. 2006; 195: 394.e1-394.e24Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 26Unal E.R. Cierny J.T. Roedner C. Newman R. Goetzl L. Maternal inflammation in spontaneous term labor.Am J Obstet Gynecol. 2011; 204: 223.e1-223.e5Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar The DNA-mediated stimulation of a proinflammatory response appears to be produced, at least in part, by the Toll-like receptor-9 (TLR9), a pattern recognition receptor involved in innate immunity.20Thaxton J.E. Romero R. Sharma S. TLR9 activation coupled to IL-10 deficiency induces adverse pregnancy outcomes.J Immunol. 2009; 183: 1144-1154Crossref PubMed Scopus (132) Google Scholar, 21Sun Y. Qin X. Shan B. et al.Differential effects of the CpG-Toll-like receptor 9 axis on pregnancy outcome in nonobese diabetic mice and wild-type controls.Fertil Steril. 2013; 99: 1759-1767Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 22Scharfe-Nugent A. Corr S.C. Carpenter S.B. et al.TLR9 provokes inflammation in response to fetal DNA: mechanism for fetal loss in preterm birth and preeclampsia.J Immunol. 2012; 188: 5706-5712Crossref PubMed Scopus (137) Google Scholar, 27Goldfarb I. Adeli S. Berk T. Phillippe M. Fetal and placental DNA stimulation of TLR9: a mechanism possibly contributing to the proinflammatory events during parturition.Reprod Sc. 2017 Jan 1; (1933719117728798. https://doi.org/10.1177/1933719117728798. [Epub ahead of print])PubMed Google Scholar Of note, adult vertebrate DNA is normally a poor stimulator for TLR9 because of its increased levels of cytosine methylation and because adult DNA is poorly internalized into cells where it can interact with TLR9, which is present within intracellular endosomes.27Goldfarb I. Adeli S. Berk T. Phillippe M. Fetal and placental DNA stimulation of TLR9: a mechanism possibly contributing to the proinflammatory events during parturition.Reprod Sc. 2017 Jan 1; (1933719117728798. https://doi.org/10.1177/1933719117728798. [Epub ahead of print])PubMed Google Scholar, 28Phillippe M. Cell-free fetal DNA, telomeres, and the spontaneous onset of parturition.Reprod Sci. 2015; 22: 1186-1201Crossref PubMed Scopus (66) Google Scholar The exception to this situation appears to be DNA that is released by the placenta and fetal membranes (ie, cffDNA), which has been found to be hypomethylated and able to be internalized and results in TLR9 stimulation that leads to increased proinflammatory cytokine release.22Scharfe-Nugent A. Corr S.C. Carpenter S.B. et al.TLR9 provokes inflammation in response to fetal DNA: mechanism for fetal loss in preterm birth and preeclampsia.J Immunol. 2012; 188: 5706-5712Crossref PubMed Scopus (137) Google Scholar, 27Goldfarb I. Adeli S. Berk T. Phillippe M. Fetal and placental DNA stimulation of TLR9: a mechanism possibly contributing to the proinflammatory events during parturition.Reprod Sc. 2017 Jan 1; (1933719117728798. https://doi.org/10.1177/1933719117728798. [Epub ahead of print])PubMed Google Scholar The report in this issue of the American Journal of Obstetrics and Gynecology by Christina Herrera et al29Herrera C.A. Stoerker J. Carlquist J. et al.Cell-free DNA, inflammation, and the initiation of spontaneous term labor.Am J Obstet Gynecol. 2017; 217: 583.e1-583.e8Abstract Full Text Full Text PDF Scopus (41) Google Scholar sought to further test the hypothesis that cell-free DNA, especially when hypomethylated, is associated with increased proinflammatory cytokine levels and with spontaneous labor. Specifically, these investigators performed a prospective cohort study to assess the total cell-free DNA concentrations, the DNA methylation ratios, and interleukin-6 (IL6) levels at 26–30 and 34–36 weeks gestation and at the time of admission for delivery at term. Fifty-five women completed these studies, of which 20 women presented at term in active labor and the other 35 were not in labor on admission. The DNA in the cell-free portion of maternal plasma was extracted and measured by fluoroscopy; DNA methylation was assessed with the use of bisulfite conversion and comparative DNA sequencing, and the plasma IL6 concentrations were measured with the use of a commercial enzyme-linked immunosorbent assay. These investigators observed that the methylation ratio in total cell-free DNA that was isolated from maternal plasma was significantly higher for women who were admitted in spontaneous labor when compared with women who were not in active labor. They also observed that total cell-free DNA and IL6 levels increased significantly from 26 weeks gestation to term in plasma samples that were obtained from the pregnant women who went into spontaneous labor, compared with those who did not. The higher methylation ratio does not support the hypothesis that spontaneous labor is associated with decreased methylation of the cell-free DNA released by the placenta and fetal membranes; however, as previously noted, the cffDNA fraction represents only approximately 5% of the total cell-free DNA circulating in the maternal blood. Therefore, the hypomethylated cffDNA levels would be overshadowed by the 20-fold high amounts of normally methylated DNA in the maternal plasma. In contrast, the increasing levels of cell-free DNA and IL6 in the maternal blood during the third trimester that was observed by these investigators do provide support for the hypothesis that spontaneous labor is associated with factors that could lead to the proinflammatory events that result in the spontaneous onset of parturition. As mentioned previously, adult vertebrate DNA is a poor stimulant for a proinflammatory response; therefore, one could hypothesize that the high levels of methylated cell-free DNA that was found in the maternal systemic circulation by Herrera et al is a protective mechanism that prevents the potentially detrimental effects that would occur with a robust systemic inflammatory response if hypomethylated cffDNA was not overshadowed. In contrast, the proinflammatory events that potentially stimulate parturition occur within the pregnant uterus, where high levels of hypomethylated DNA that are released by the placenta and fetal membranes would be in position to stimulate local inflammatory and decidual cells directly. To better understand these relationships, future research by Herrera et al must assess the methylation status of the cell-free DNA fraction that is released by the gestational tissues (ie, the placenta and fetal membranes) and the effect of this hypomethylated DNA on the innate immune response that occurs locally within the pregnant uterus. Cell-free DNA, inflammation, and the initiation of spontaneous term laborAmerican Journal of Obstetrics & GynecologyVol. 217Issue 5PreviewHypomethylated cell-free DNA from senescent placental trophoblasts may be involved in the activation of the inflammatory cascade to initiate labor. Full-Text PDF