The

n e w e ng l a n d j o u r na l

of

m e dic i n e

clinical implications of basic research

Elizabeth G. Phimister, Ph.D., Editor

Cell-free Fetal DNA A Trigger for Parturition

Mark Phillippe, M.D.
For decades, researchers have unsuccessfully
sought to understand the mechanisms underlying the onset of parturition. The apparent variation in mechanisms between other mammals and
humans has further confounded these efforts.
And yet, it does not make sense that an event so
essential for successful reproduction and the
propagation of evolutionarily related species
would have radically diverged in signaling mechanisms. Multiple lines of evidence now suggest
that an inflammatory process is involved in triggering parturition, a process that is modulated
and influenced by maternal, fetal, and placental
endocrine events. However, another important
factor may be cell-free fetal DNA.
In 1998, Lo et al.1 observed cell-free fetal
DNA in maternal plasma at concentrations of
3.4 to 6.2% of the total cell-free DNA in plasma,
along with a dramatic increase in levels of cellfree fetal DNA between early and late gestation.
Subsequent studies have shown that cell-free
fetal DNA is derived from placental trophoblasts
as they undergo cellular apoptosis and necrosis.
Others studies have documented an increase by a
factor of 12 in cell-free fetal DNA levels throughout gestation, with levels peaking at the end of
pregnancy,2 and an inverse correlation between
cell-free fetal DNA levels and the interval between blood collection and birth.3
Confirming the relationship between increased
levels of cell-free fetal DNA and parturition is the
observation that pregnant women who deliver
prematurely were found to have twice the levels
of cell-free fetal DNA as compared with women
in a control group or those with threatened preterm labor.4 Other studies have shown that
levels of cell-free fetal DNA above the 95th percentile are associated with increased rates of
preterm delivery. Notably, levels of cell-free
fetal DNA have been reported to be 30% higher
in twin gestations; such increases could be responsible for the shortened gestational length
in multifetal pregnancies.
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The presence of cell-free fetal DNA derived

from fetal or placental sources is not unique to
human pregnancies, since cell-free fetal DNA
has also been detected in the plasma of pregnant mice, cows, horses, sheep, and primates.
In mice and macaques, levels of cell-free fetal
DNA have been reported to increase markedly
during late gestation and then drop to undetectable levels after birth.5,6
The association between elevated levels of cellfree fetal DNA and parturition is interesting,
but how might these observations be mechanistically linked? The pattern-recognition receptor
TLR9, which is activated by free-circulating
hypomethylated DNA, is an excellent candidate
linker. TLR9 agonists have induced preterm deliveries when injected into pregnant mice. Fetal
DNA injected into pregnant mice produces gestational losses (resorptions), which do not occur
when adult DNA is used or when TLR9-deficient
mice are injected with fetal DNA.7
Multiple previous studies have provided compelling support for the premise that spontaneous parturition is mediated by the activation of
inflammation-related signaling pathways, leading to the increased secretion of proinflammatory cytokines and chemokines, the influx of
neutrophils and macrophages into the pregnant
uterus, and increased production of uterine-activation proteins, including the oxytocin receptor,
prostaglandin F receptor, and connexin 43. These
events also stimulate the activity of matrix metalloproteinases and the release of uterotonins,
leading to cervical ripening, membrane rupture,
and phasic myometrial contractions.
The missing link has been the fetal or placental signal that triggers these proinflammatory events in the absence of microbial invasion
and intrauterine infection. In aggregate, several
studies provide solid support for the hypothesis
that increased levels of cell-free fetal DNA, which
are released during placental maturation or
senescence and peak at the end of gestation, ac-

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The New England Journal of Medicine

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clinical implications of basic research

Term gestation

Placental maturation and senescence of

placental trophoblast cells

Mature placenta

Umbilical arteries
and vein

Increased cell-free fetal DNA in

maternal plasma

Chorion

Leukocyte

TLR9
activation

Pooled
maternal
blood

Macrophage

Innate immune response by the

chorion, decidua, myometrium,
and cervix

Decidua

Maternal vein
and artery

Increased proinflammatory
cytokines and chemokines
Myometrium

Increased uterine-activation protein

expression by the chorion, decidua,
myometrium, and cervix

Cervical
ripening

Membrane
rupture

Phasic uterine
contractions

Parturition
Figure 1. Proposed Signaling Pathway Leading from Placental Production of Cell-free Fetal DNA to Parturition.
COLOR
Several studies have suggested that levels of cell-free fetal DNA increase during placental maturation and peak at the end
ofFIGURE
gestation,
activating the innate immune system through stimulation of TLR9 (or another DNA-sensing pattern-recognition receptor).
This activity
Draft 5
6/23/14
Author rupture
triggers an innate immune response and the production of uterine-activation proteins, resulting in cervical ripening,
Phillippe of the fetal
1
Fig #
membranes, and phasic myometrial contractions and leading to parturition.
Title

Cell-free fetal DNA: A trigger

for parturition

ME

n engl j med 370;26

nejm.org

june 26, 2014

DE
Artist

Phimister
N Koscal

2535

AUTHOR PLEASE NOTE:

The New England Journal of Medicine
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clinical implications of basic research

tivate the innate immune system through stimulation of TLR9 (or another DNA-sensing patternrecognition receptor), leading to parturition
(Fig. 1). If the hypothesis is true, this mechanism
is potentially responsible for triggering the onset
of parturition in all mammals.
Disclosure forms provided by the author are available with the
full text of this article at NEJM.org.
From the Vincent Center for Reproductive Biology, Department
of Obstetrics and Gynecology, Massachusetts General Hospital,
Boston.
This article was updated on June 26, 2014, at NEJM.org.
1. Lo YM, Tein MS, Lau TK, et al. Quantitative analysis of fetal

DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768-75.
2. Birch L, English CA, ODonoghue K, Barigye O, Fisk NM,
Keer JT. Accurate and robust quantification of circulating fetal
and total DNA in maternal plasma from 5 to 41 weeks of gestation. Clin Chem 2005;51:312-20.

3. Majer S, Bauer M, Magnet E, et al. Maternal urine for prena-

tal diagnosis an analysis of cell-free fetal DNA in maternal

urine and plasma in the third trimester. Prenat Diagn 2007;27:
1219-23.
4. Leung TN, Zhang J, Lau TK, Hjelm NM, Lo YM. Maternal
plasma fetal DNA as a marker for preterm labour. Lancet 1998;
352:1904-5. [Erratum, Lancet 1999;354:780.]
5. Khosrotehrani K, Wataganara T, Bianchi DW, Johnson KL.
Fetal cell-free DNA circulates in the plasma of pregnant mice:
relevance for animal models of fetomaternal trafficking. Hum
Reprod 2004;19:2460-4.
6. Mitsunaga F, Ueiwa M, Kamanaka Y, Morimoto M, Naka
mura S. Fetal sex determination of macaque monkeys by a nested PCR using maternal plasma. Exp Anim 2010;59:255-60.
7. Scharfe-Nugent A, Corr SC, Carpenter SB, et al. TLR9 provokes inflammation in response to fetal DNA: mechanism for
fetal loss in preterm birth and preeclampsia. J Immunol 2012;
188:5706-12.
DOI: 10.1056/NEJMcibr1404324
Copyright 2014 Massachusetts Medical Society.

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The New England Journal of Medicine

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Copyright 2014 Massachusetts Medical Society. All rights reserved.