clinical implications of basic research

The new engl and journal o f medicine
Elizabeth G. Phimister, Ph.D., Editor
n engl j med 370;26 nejm.org june 26, 2014
2534
Cell-free Fetal DNA — A Trigger for Parturition
Mark Phillippe, M.D.
For decades, researchers have unsuccessfully
sought to understand the mechanisms underly-
ing the onset of parturition. The apparent varia-
tion 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 mech-
anisms. Multiple lines of evidence now suggest
that an inflammatory process is involved in trig-
gering 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 cell-
free 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 through-
out gestation, with levels peaking at the end of
pregnancy,
2
and an inverse correlation between
cell-free fetal DNA levels and the interval be-
tween 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 pre-
term labor.
4
Other studies have shown that
levels of cell-free fetal DNA above the 95th per-
centile 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 re-
sponsible for the shortened gestational length
in multifetal pregnancies.
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 preg-
nant 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 undetect-
able levels after birth.
5,6
The association between elevated levels of cell-
free fetal DNA and parturition is interesting,
but how might these observations be mechanis-
tically linked? The pattern-recognition receptor
TLR9, which is activated by free-circulating
hypomethylated DNA, is an excellent candidate
linker. TLR9 agonists have induced preterm de-
liveries when injected into pregnant mice. Fetal
DNA injected into pregnant mice produces ges-
tational 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 com-
pelling support for the premise that spontane-
ous parturition is mediated by the activation of
inflammation-related signaling pathways, lead-
ing to the increased secretion of proinflamma-
tory cytokines and chemokines, the influx of
neutrophils and macrophages into the pregnant
uterus, and increased production of uterine-acti-
vation proteins, including the oxytocin receptor,
prostaglandin F receptor, and connexin 43. These
events also stimulate the activity of matrix metal-
loproteinases and the release of uterotonins,
leading to cervical ripening, membrane rupture,
and phasic myometrial contractions.
The missing link has been the fetal or pla-
cental signal that triggers these proinflamma-
tory 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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n engl j med 370;26 nejm.org june 26, 2014
2535
clinical implications of basic research
Mature placenta
Myometrium
Decidua
Pooled
maternal
blood
Umbilical arteries
and vein
Chorion
Maternal vein
and artery
Placental maturation and senescence of
placental trophoblast cells
Increased cell-free fetal DNA in
maternal plasma
TLR9
activation
Leukocyte
Macrophage
Cervical
ripening
Membrane
rupture
Phasic uterine
contractions
Parturition
Term gestation
Increased proinflammatory
cytokines and chemokines
Innate immune response by the
chorion, decidua, myometrium,
and cervix
Increased uterine-activation protein
expression by the chorion, decidua,
myometrium, and cervix
1
Phimister
6/23/14
AUTHOR PLEASE NOTE:
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Phillippe
N Koscal
6/26/14
Cell-free fetal DNA: A trigger
for parturition
Figure 1. Proposed Signaling Pathway Leading from Placental Production of Cell-free Fetal DNA to Parturition.
Several studies have suggested that levels of cell-free fetal DNA increase during placental maturation and peak at the end of gestation,
activating the innate immune system through stimulation of TLR9 (or another DNA-sensing pattern-recognition receptor). This activity
triggers an innate immune response and the production of uterine-activation proteins, resulting in cervical ripening, rupture of the fetal
membranes, and phasic myometrial contractions and leading to parturition.
The New England Journal of Medicine
Downloaded from nejm.org by LUIS ENRIQUE MOSCOSO ORTIZ on July 18, 2014. For personal use only. No other uses without permission.
Copyright © 2014 Massachusetts Medical Society. All rights reserved.
n engl j med 370;26 nejm.org june 26, 2014
2536
clinical implications of basic research
tivate the innate immune system through stimu-
lation of TLR9 (or another DNA-sensing pattern-
recognition 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 noninva-
sive prenatal diagnosis. Am J Hum Genet 1998;62:768-75.
2. Birch L, English CA, O’Donoghue 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 gesta-
tion. 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
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5. Khosrotehrani K, Wataganara T, Bianchi DW, Johnson KL.
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mura S. Fetal sex determination of macaque monkeys by a nest-
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7. Scharfe-Nugent A, Corr SC, Carpenter SB, et al. TLR9 pro-
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DOI: 10.1056/NEJMcibr1404324
Copyright © 2014 Massachusetts Medical Society.

The New England Journal of Medicine
Downloaded from nejm.org by LUIS ENRIQUE MOSCOSO ORTIZ on July 18, 2014. For personal use only. No other uses without permission.
Copyright © 2014 Massachusetts Medical Society. All rights reserved.