You are on page 1of 21

Research

ajog.org

OBSTETRICS

Women with preterm birth have a


distinct cervicovaginal metabolome
Jeny Ghartey, DO, MS; Jamie A. Bastek, MD, MSCE; Amy G. Brown, PhD;
Laura Anglim, BA; Michal A. Elovitz, MD
OBJECTIVE: Metabolomics has the potential to reveal novel pathways

involved in the pathogenesis of preterm birth (PTB). The objective of


this study was to investigate whether the cervicovaginal (CV )
metab- olome was different in asymptomatic women destined to
have a PTB compared with term birth.
STUDY DESIGN: A nested case-control study was performed using CV

fluid collected from a larger prospective cohort. The CV fluid was


collected between 20-24 weeks (V1) and 24-28 weeks (V2).
The metabolome was compared between women with a spontaneous
PTB (n 10) to women who delivered at term (n 10). Samples
were extracted and prepared for analysis using a standard extraction
solvent method. Global biochemical profiles were determined
using gas chromatography/mass
spectrometry and ultraperformance liquid chromatography/tandem mass spectrometry. An
ANOVA was used to detect differences in biochemical compounds
between the groups. A

false discovery rate was estimated to account for multiple


comparisons.
RESULTS: A total of 313 biochemicals were identified in CV fluid.

Eighty-two biochemicals were different in the CV fluid at V1 in


those destined to have a PTB compared with term birth, whereas
48 were different at V2. Amino acid, carbohydrate, and peptide
metabolites were distinct between women with and without PTB.
CONCLUSION: These data suggest that the CV space is metabolically

active during pregnancy. Changes in the CV metabolome may be


observed weeks, if not months, prior to any clinical symptoms. Understanding the CV metabolome may hold promise for unraveling the
pathogenesis of PTB and may provide novel biomarkers to identify
women most at risk.
Key words: cervicovaginal metabolome, pregnancy, preterm birth

Cite this article as: Ghartey J, Bastek JA, Brown AG, et al. Women with preterm birth have a distinct cervicovaginal metabolome. Am J Obstet Gynecol
2015;212:776.e1-12.

reterm birth (PTB) is among the


leading cause of neonatal morbidity
and mortality, accounting for 11.2% of
live births in the United States as of

From the Maternal-Child Health Research


Program, Department of Obstetrics and
Gynecology, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA.
Received Feb. 12, 2015; revised March
16, 2015; accepted March 26, 2015.
This study was supported by the Maternal and
Child Health Research Fund, University of
Pennsylvania Perelman School of Medicine
grant R01NR014784.
The authors report no conict of interest.
Presented in oral format at the 35th annual
meeting of the Society for Maternal-Fetal
Medicine, San Diego, CA, Feb. 2-7, 2015. The
racing ag logo above indicates that this article
was rushed to press for the benet of the
scientic community.
Corresponding author: Michal A. Elovitz,
MD. melovitz@obgyn.upenn.edu
0002-9378/$36.00
2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ajog.2015.03.052

776.e1

1
2012. Studies have shown that women bolites less than 1 kDa necessary for
with a short cervix are at an increased
2-4
risk for PTB, yet most cases of PTB
are not preceded by a sonographic
short cervix. The sonographic short
cervix could be considered a phenotype
of premature cervical modeling, but in
fact, the immunological, biochemical,
mo- lecular, and cellular processes
involved
in
premature
cervical
remodeling have not been fully
5-9
elucidated.
Although there is some emerging
evidence regarding the cervicovaginal
10,11
(CV) microbiota and PTB,
how the
CV microbiota might alter cervical remodeling has not been studied. Whereas clinical data support the role of
infection and/or inammation in the
12
pathogenesis of preterm birth
and
understanding that cervical remodeling
must occur at any gestational age for
parturition to occur, the interaction of
these biological and molecular events to
induce spontaneous preterm birth have
not been elucidated.
Metabolomics, the study of meta-

American Journal of Obstetrics & Gynecology JUNE 2015

structure and function of an organism at


the cellular level, is able to provide information on multiple biological path13
ways acting in concert. It is a powerful
tool of fundamental discovery that relays information from the host, the
microbiota, and the environment to give
a comprehensive view of a biological
system in both healthy and diseased
states. Metabolomics has the potential to
uncover novel mechanistic pathways
involved in premature cervical remodeling and PTB.
The importance of the metabolome
may be best conceptualized by recent
advances in understanding the role of
gut microbiota in the development of
colorectal cancer in patients with in14
ammatory bowel disease. Although
it is well known that patients with inammatory bowel disease are at an
15
increased risk for colorectal cancer, it
seems that the bacterial metabolites,
more than the bacterial composition
itself, may protect from or predispose
toward cancer within the gastrointest16
inal tract. Metabolomic analysis has

JUNE 2015 American Journal of Obstetrics & Gynecology

776.e2

Research

revealed the actions of the microbiota


may result in the production of tumorpromoting metabolites, such as acetylaldehyde and nitrosamine, and may
mediate tumor suppressive effects
17,18
through inactivation of carcinogens.
Further- more, metabolomics have led
to the dis- covery of novel targets for
therapeutics
in
preventing
cell
13
proliferation in cancer.
Examination of the metabolome has the
potential to reveal novel mechanistic
pathways
involved
in
the
pathophysiology of disease and provide
therapeutic targets to prevent disease.
Metabolomics in pregnancy is in its
infancy. Existing studies have demonstrated the ability to test the
metabolome in maternal plasma,
amniotic uid, placenta, cord blood,
and urine. These studies have explored
the metabolome among women with
preeclampsia, gest- ational diabetes,
fetal growth restriction, and fetal
malformations and have shown key
differences in metabolites between
cases and controls.19-23
Despite these studies, the CV uid
metabolome and how it might be
altered in women with preterm birth
has not been studied. Investigating the
metab- olome in CV uid may provide
insight into the cellular metabolism as
it relates to the host and/or existing
microbiota involved with premature
cervical remo- deling. Therefore, the
objective of this study was to
investigate whether the CV uid
metabolome
was
different
in
asymptomatic women destined to have a
PTB compared with term birth.

M ATE R IALS

AND

ajog.org

Obstetrics

M ETHODS

Study population
A nested case-control study was performed from a prospective cohort. The
UK Prognostic Study of the Interferon
Gamma
Release
Assay
for
Tuberculosis (PREDICT) study was
performed at a single, urban tertiary
care institution between August 2011
and November
24,25
2012.
This study was approved by
the Institutional Review Board at the
University
of
Pennsylvania.
Women with a singleton pregnancy
who were at high risk for preterm birth

based on a prior history of preterm


birth or second-trimester loss,
previous cer- vical surgery without a
subsequent full

term birth, and/or uterine anomaly were


approached for recruitment into the
study. Women who were receiving systemic steroids or immunosuppressive
therapy, had human immunodeciency
virus, lupus, pregestational diabetes
mellitus, rheumatoid arthritis, Crohns
disease, ulcerative colitis, cancer or an
organ transplant were excluded.
Furthermore, women with activity in the
vagina in the 24 hours prior to specimen
collection (including sexual intercourse,
transvaginal ultrasound, digital cervical
examination, or vaginal lubricants), had
active vaginal bleeding, had evidence of a
rupture of membranes, and/or had a
cervical dilation of 3 cm or greater at the
time of enrollment were excluded.
Pertinent demographic, clinical, and
obstetric data were abstracted from the
24
medical record as previously described.
Women with spontaneous preterm birth
at less than 37 weeks of gestation were
identied (n 10). Controls were selected
at random from women having deliveries
at longer than 38 weeks of gestation
without evidence of chorioamnionitis.
Cervicovaginal biospecimens were collected at 2 time points: visit 1 (V1)
occurred at 20 weeks to 23 weeks 6 days
of gestation and visit 2 (V2) occurred at
24 weeks to 27 weeks 6 days of gestation.
All cases of preterm birth were reviewed
and conrmed
that
they
were
spontaneous preterm births.

Biospecimen collection
CV uid was collected following insertion of a sterile speculum using a sterile
cotton-tipped swab. Samples were
collected in 0.5 mL phosphate-buffered
saline, immediately placed in liquid nitrogen, and stored at e80 C for future
use.
Metabolomics analysis
Metabolomics analysis was performed
by Metabolon, Inc (Research Triangle
26,27
Park, NC) as previously described.
Samples were accessioned into the
Metabolon Laboratory Information
Management System (LIMS) and assigned a unique identier. The samples
(and all derived aliquots) were bar coded
and tracked by the LIMS. All samples
were maintained at e80 C until processed.
The samples were prepared using the

ajog.org
MicroLab
STAR system from Hamilton
Co. (Reno, NV).
To remove the protein fraction
while maximizing the recovery of
small molecules, the samples were
prepared using a proprietary series
of organic and aqueous extractions.
The resulting extract was divided
into 2 fractions: one for analysis by
ultraperformance
liquid
chromatography (UPLC)/tan- dem
mass spectrometry and one for
analysis by gas chromatography/mass
spectrometry (GC/MS). Samples were
placed briey on TurboVap (Zymark,
Hopkinton, MA) to remove the
organic solvent. Each sample was then
frozen and underwent vacuum desiccation. Samples were then prepared for
either liquid chromatography/mass
spectrometry or GC/MS.
For quality assurance/quality control
purposes, a selection of quality control
compounds was added to every sample,
including those that were being examined. These compounds were carefully
chosen so as not to interfere with the
measurement of the endogenous compounds. The quality control samples
are primarily used to evaluate the
control process for each study as well as
aiding in data curation.
Liquid chromatography/mass
spectrometry
The liquid chromatography/mass spectrometry portion of the platform was
based on a Waters ACQUITY UPLC
and Thermo Finnigan (San Jose, CA)
linear trap quadropole mass
spectrometer, which consisted of an
electrospray ioni- zation source and
linear ion trap mass analyzer. The
sample extract was split into 2
aliquots, dried, and then reconstituted in acidic or basic liquid
chromatography-compatible solvents,
each of which contained 11 or more injection standards at xed
concentrations. One aliquot was
analyzed using acidic positive ion
optimized conditions and the other
using basic negative ion optimized
conditions in 2 inde- pendent
injections, using separate
dedicated columns. Extracts reconstituted in acidic conditions were gradient
eluted using water and methanol, both
containing 0.1% formic acid, whereas

Obstetrics

Research

the basic extracts, which also used water/methanol, contained 6.5 mM


ammonium bicarbonate.
The mass spectrometry (MS)
analysis alternated between MS and
2
data- dependent MS scans using
dynamic exclusion, and the scan range
was from
80 to 1000 mass to charge ratio. Raw
data les were archived and extracted
as described below.

Gas chromatography/mass
spectrometry
The samples destined for GC/MS
analysis were redried under vacuum
desiccation for a minimum of 24 hours
prior to be- ing derivatized under dried
nitrogen us- ing bistrimethyl-silyltriouroacetamide.
The
gas
chromatography column was
5% phenyl and the temperature ramp
was from 40 C to 300 C in a 16 minute period. The samples were analyzed
on a Thermo-Finnigan Trace DSQ
fast-scanning, single-quadrupole mass
spectrometer using electron impact
ionization. The instrument was tuned
and calibrated for mass resolution and
mass accuracy on a daily basis. The information output from the raw data
les was automatically extracted as
discussed below.
Bioinformatics
The informatics system consisted of 4
major components, the LIMS, the data
extraction and peak-identication software, data processing tools for quality
control and compound identication,
and a collection of information interpretation and visualization tools for use
by data analysts. The hardware and
software foundations for these informatics components were the LAN
backbone, and a database server
running Oracle
10.2.0.1
EnterpriseEdition (Redwood
City, CA).
Data extraction and compound
identification
Raw data were extracted, peak identied,
and quality control processed using
28
Metabolons hardware and software.
Compounds were identied by comparison with library entries of puried

standards or recurrent unknown entities. Metabolon


maintains
a
library

based on authenticated standards that


contains the retention time/index, mass
to charge ratio, and chromatographic
data (including tandem MS [MS/MS]
spectral data) on all molecules present in
the library. Furthermore, biochemical
identications are based on 3 criteria:
retention index within a narrow window
of the proposed identication, nominal
mass match to the library
0.4 amu,
and the MS/MS forward and reverse
scores between the experimental data
and authentic standards.
The MS/MS scores are based on a
comparison of the ions present in the
experimental spectrum with the ions
present in the library spectrum. Although there may be similarities between
these molecules based on one of these
factors, the use of all 3 data points can
be utilized to distinguish and differentiate biochemicals. More than 3500
commercially available puried standard
compounds have been acquired and
registered into LIMS for distribution to
both the liquid chromatography-MS and
GC-MS platforms for the determination
of their analytical characteristics.

Statistical analyses
An analysis of variance (ANOVA) was
used to identify biochemicals that differed
signicantly between the groups
following log transformation and imputation of missing values, if any, with the
minimum observed value for each compound. The data were analyzed both
normalized to protein and nonnormalized, and results were similar between
the groups. However, because of the signicant differences in the protein hydrolysis between cases and controls, the data
presented here are those results that were
not normalized to the protein content.
Two-way ANOVA with repeated measures was used to identify biochemicals
exhibiting signicant interaction and
main effects for the experimental parameters of status and time. The data
were analyzed in 2 ways to account for
changes between the
groups and
changes that occurred over time within
the same sub- ject. ANOVA was used
when comparing metabolites between
groups across a sin- gle time point (in
which each subject was unique), whereas
a 2-way ANOVA with

repeated measures was used to take


into account metabolites that changed
over time within each subject
(accounting
for
within-subject
characteristics).
Only
metabolites with a value of P < .05
were considered signicant.
Additionally, because this
was a study of discovery, metabolites
that approached signicance (ie, had an
alpha level between 0.05 and
0.1)
were
explored.
An estimate of the false discovery
rate (q-value) was calculated to
account for multiple comparisons
29
that occur in metabolomics studies.
When analyzing numerous compounds,
statistical signi- cance may be found
because of random chance. The qvalue describes the false discovery rate;
a low q-value (q .10) is indicative of
high condence in a result. Random
forest (RF) is a supervised
classication technique reporting on the
consensus of a large number of decision
trees. In this study, the term and preterm
birth cohorts at both time points were
classied to achieve the following: (1)
assess the capacity to distinguish birth
cohorts on the basis of global metabolic
proles and (2) identify biochemicals
important to the classication. An accuracy of 25% is expected by random
chance when comparing 4 groups, so
the RF classication accuracy of 32%
is better than
random
chance.
Addition- ally, according to the
confusion matrix, when the RF model
misclassied sam- ples, it tended to
pick the wrong time point (ie, V1 vs
V2) but the correct birth cohort (ie,
term vs PTB), so the model may be
even more accurate than the 32%
accuracy belies.

Statistical analysis for clinical data


Categorical variables were compared
2
between groups by a c or Fisher exact
test, where appropriate. Continuous
variables were compared by the Student
t test or the Mann-Whitney U test,
depending on the distribution of the
data.
Spearmans
correlation
coefcients were
calculated
to
determine
correla- tions between
metabolites and cervical length less
than 2.9 cm (median). A value of P < .
05
was
considered
statistically
signicant. Clinical data were analyzed

using STATA
(version 11.0;
StataCorp, Inc, College Station, TX).

R ESULTS
Demographics
Clinical and obstetric data are listed in
Table 1. Women with a preterm
delivery were similar in age, race,
gestational age at V1 and V2, and
cervical length at V1 compared with
women with a term de- livery. Not
surprisingly, women who ul- timately
delivered preterm had a
signicantly higher number of prior
PTB, a signicantly lower cervical length
at V2 and gestational age at delivery as
compared with term birth (Table 1).
Cervicovaginal metabolome
A total of 313 biochemicals were identied in the CV uid. Signicant differences were noted in the CV uid
metabolome between women destined
to have a PTB compared with term
birth. Overall, women who had a term
delivery
had signicant changes in carbohydrate
and lipid metabolism between the second and third trimesters. In women
with a preterm delivery, signicant
changes in
amino acid metabolism were noted between the second and third trimesters.
Of note, the signicant change in carbohydrate metabolism observed among
women with a term delivery from V1 to
V2 does not occur in women with
PTB. At visit 1, women with a preterm
delivery had signicant changes in
amino acid and peptide metabolism
as compared with women with a term
delivery. Changes in multiple metabolic
pathways were noted at V2; women
with a preterm delivery had signicant
changes in amino acid, peptide,
carbohydrate, and lipid metabolism as
compared with women with a term
delivery (Table 2). A random forest plot
depicting classes of metabo- lites that
were important in distinguish- ing
between preterm and term delivery can
be found in the Figure.
A more in-depth look into the
metabolome among women with preterm and term delivery reveals
distinct differences in metabolites. In
women delivering at term, carbohydrate
and lipid metabolism appear to be
down-regulated between the second and
third
trimesters. Specically for

TABLE 1

Clinical and demographic characteristics of women with PTB and term


birth
Characteristic
b

Age, y
Race

PTB

Term

23.5 (17e38)

31.5 (26e35)

.22
.80

White

8 (20)

7 (70)

Black

1 (10)

2 (20)

1 (10)

1 (10)

8 (80)

2 (20)

Asian
Prior PTB

P value

Gestational age at V1, wks

20 4/7 (20e23)

Gestational age at V2, wks

26 3/7 (24 1/7e27 5/7) 26 (24 6/7e27 4/7)

20 3/7 (19 6/7e23 3/7)

.02
.70
1.00

Cervical length at V1, cm


b
Cervical length at V2, cm

3.3 (2.1e3.5)
2.9 (1.6e3.6)

Gestational age at delivery,


wksb

33 4/7 (26e36)

3.7 (2e4.8)
3.8 (2.4e4.7)
40 (39e41)

.07
.03
< .001

PTB, preterm birth; V1, visit 1; V2, visit 2.


a

P < .05 considered statistically significant; b Median (range); c n (percentage).

Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.

carbohydrate metabolism, glucose,


glucose-6-phosphate,
xylulose,
maltohexose, N-acetylglucosamine,
and
N-acetylgalactosamine
were
signicantly

decreased by the third trimester (0.2-,


0.14-, 0.59-, 0.35-, 0.45-, and 0.32-fold
change, respectively; Table 3) in women
who had a term delivery. Additionally, an
increase in methyl-4-hydroxybenzoate,
an antimicrobial agent, was noted among
women with term delivery between
the second and third trimesters (8.8-fold,
P < .1).
Similarly, important contributors of
lipid metabolism were signicantly
decreased in the third trimester among
women who delivered at term (myoinositol, 1-palmitoylglycerol [1-monopa
lmitin], dehydroisoandrosterone sulfate;
Table 3). In contrast, components of lipid
and carbohydrate metabolism were
signicantly up-regulated at V2 among
women with preterm delivery as
compared with term delivery (palmitoleate, 1.4-fold; 10-heptadecenoate, 1.6fold; 1-palmitylglycerol, 2.24-fold; glu
cose, 3.7-fold; glucose-6-phosphate, 3fold; Table 3).
At V1, a signicant down-regulation of
dipeptides was observed among women
with preterm delivery as compared with
term delivery. More than half of the 100
dipeptides detected in CV uid were

signicantly reduced among women who


had a preterm delivery (Table 3). At V2, a

signicant
increase
in
nacetylneuraminate (4.9-fold) was observed
among women with
preterm
delivery
as
compared with term delivery (Table
3). Finally, none of the metabolites
with sig- nicant fold change between
preterm and term delivery correlated
with cervical length less than 2.9 cm
(data
not
shown)
(Appendix;
Supplementary Tables).

C OM
MENT

Novel to this study, we demonstrate


that the CV space is metabolically
active dur- ing pregnancy and that the
CV metab- olome is different in
women destined to have a preterm
birth. Important ndings from this
study include the following: (1)
normal, term delivery is associated
with metabolic changes in the CV
space be- tween the second and third
trimesters; (2) these metabolic
changes (specically down-regulation
in the carbohydrate pathway) does
not appear to occur in women who
are destined to have a pre- term birth;
and (3) women destined to have a
PTB can be identied by differences in several metabolic processes
compared with women who deliver
at term.
Of 3500 standardized metabolites
that have been identied in other
biological

TABLE 2

Metabolic pathways that differ among women with preterm and term
a,b
birth
Metabolic pathway
2

Term V
Term V1

PTB V
2
PTB V1

PTB V1
Term V1

PTB V
2
Term V2

Amino acid

Peptide

22

Carbohydrate

Energy

Lipid

Nucleotide

Cofactors and vitamins

Xenobiotics

16

40

15

Total
a

Significantly different pathways between comparison groups with P < .05; Total number of pathways that are significantly
different (either increased or decreased) between comparison groups.

Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.

spaces, 313 were identied in the CV


space. There have been no other highthroughput metabolomic studies of the
CV space, and therefore, it is difcult
to compare our ndings with the
nonpregnant population. Of note, many
different classes of metabolites have
been identied in the gut metabolome,
which may represent the different
bacteria that reside in these respective
spaces. A similar number of metabolites
have been found in other studies
involving the gut; however, there is a
28
wide range.
Three investigators have examined
the metabolome and its association
with preterm birth, two in amniotic
30,31
32
uid,
and one in CV uid.
Both
studies of amniotic uid
demonstrated
alterations
in
xenobiotic metabolism among women
30,31
with PTB.
Only one other study has
examined the metabolome in CV
32
uid. Although these authors found
that in the setting of a short cervix, the
CV metabolome distinguished women
with term and preterm birth, they
did not report the distinct metabolic
pathways that differed between the 2
32
groups.
Our study is unique in that, using a
metabolomics approach, it provides the
rst comprehensive, detailed description

of metabolites that are present in the


CV space that were distinct in
women who ultimately had a PTB.
Although we would not expect the
metabolome in amniotic uid to
mirror the CV space, it

destined to have a term delivery may


represent a quiescent state in the CV
space that is necessary for the cervix
to maintain integrity; the lack of the
down-regulation of these carbohydrate
pathways in women destined to
deliver
preterm may indicate that this
quiescent state is never reached, and
hence, there is active energy utilization
that
may
be synonymous with
premature cervical remodeling.
Interestingly, n-acetylneuraminate or
sialic acid was signicantly increased
in the third trimester among women
with preterm birth as compared with
term birth. Sialic acid is a ubiquitous
cell-signaling molecule with important
37
roles in immunity. Specically, sialic
acid is thought to play a role in viral
38
entry into the cell.
Its notable
increase among women with PTB may
represent
is notable that these studies found a
metabolomic prole that was associated
with inammation. Therefore, these
studies provide validity in a metabolomic approach, which may serve to
advance our understanding of PTB. The
study herein further demonstrates the
strength of a metabolomic approach.
The signicant down-regulation of
carbohydrate metabolism observed in
women who had a term delivery is
notably absent among women with a
preterm
delivery. These metabolic
changes could represent contributions
from the host or the microbiota.
Epithelial cells store and metabolize
glycogen, which is deposited in large
amounts during highly estrogenic states
and is metabolized to lactic acid, hence
contributing to the acidic pH found in a
33
healthy genital tract.
Microbiota use glycogen as a substrate
and are thought to support lactobacilli
34,35
colonization,
which has been associated with a reduction in adverse preg36
nancy outcomes.
This signicant
down-regulation in women who had a
term delivery, together with the increase
in the antimicrobial agent, methyl-4hydroxybenzoate, may represent a stable vaginal microbiome that is not
observed in women destined for preterm
birth.

Alternatively, the down-regulation of


carbohydrate metabolism in women

an enhanced afnity for infection at


the cellular level.
More than 100 dipeptides were
detected in the CV uid and nearly
half were decreased among women
with PTB in the second trimester as
compared with term. Of note, the
average total protein among the PTB
and term group
differed more at V1 (605 mg/mL vs
745
mg/mL, respectively) than V2 (504
mg/
mL vs 543 mg/mL, respectively),
suggesting that the decreased dipeptides
might reect a reduced level of
protein hydrolysis among the women
with PTB. Changes in the expression
of proteases and protease inhibitors
have been detected
days before
39,40
spontaneous la- bor.
Therefore, it
is possible that the decrease in
dipeptides represents alter- ations in
proteases and their inhibitors among
women destined to have a PTB.
Whereas these data
demonstrate promise for
the study of the CV metab- olome
and understanding the pathogen- esis
of preterm birth, our sample size was
small. Although we did not nd a
corre- lation between metabolites and
a short- ened cervical length, our
study
might
have
been
underpowered
to
reveal an
association
between
metabolic
processes and sonographic short
cervix. Alterna- tively, the lack of
correlation may suggest that the
cellular activity of the CV space is more
biologically relevant
in
understanding and predicting PTB than a
short

FIGURE

Plot depicting classes of metabolites important in distinguishing between preterm and term births

Random Forest plot depicting classes of metabolites (along the y-axis) that were important in distinguishing between preterm and term births.
Mon- oacylglycerols (lipid metabolism), protein hydrolysis markers (dipeptides), and amino acid sugars (carbohydrate metabolism) were among the
metabolites that were most important in distinguishing the 2 groups.
Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol
2015.

cervix. However, larger studies are


needed before such conclusions can be
made.
Another limitation of our study is
that the cases had signicantly more
prior PTB and that the metabolic
changes observed may be unique to
these women and not mechanistically
involved in having a PTB in this

pregnancy. These results will need to


be validated in a larger

sample size in different populations.


Finally, the cohorts were not matched to
factors such as age, race, or parity, which
may have biased the results.
Noting these limitations, the results of
this study provide a new paradigm in
which to conceptualize the pathogenesis of
PTB. With our discovery-based approach,
this study provides an unbiased assessment

of the CV metabolic proles in


asymp- tomatic women destined to have
a PTB.
These ndings demonstrate the biological importance of the CV metabolome to pregnancy. Notably, these
ndings suggest unique metabolic proles in women who ultimately have a
term or PTB. As such, these data
challenge the current paradigm that
preterm labor

TABLE 3

Fold change of metabolites that differ in CV fluid among women with preterm and term birtha,b
Metabolic pathway

Subpathway

Biochemical name

Amino acid

Lysine metabolism
Phenylalanine and
tyrosine metabolism

Term V
Term V1

PTB
V2
PTB V1

Lysine

PTB V1
Term V1
b

0.48

Phenylacetylglutamine

0.19

3-(4-Hydroxyphenyl)lactate
Tryptophan metabolism

0.36

p-cresol sulfate

0.34b

Tryptamine

1.66

0.24b
0.12b

3-Indoxyl sulfate
Cystine

Urea cycle; arginine and


proline metabolism

Urea

0.25

0.55b
0.07b

Ornithine

Peptide

Creatine metabolism

Creatinine

0.65b

Guanidino and acetamido


metabolism

4-Guanidinobutanoate

0.66

Glutathione metabolism

5-Oxoproline

0.44b

Dipeptide

Dimethylarginine (SDMA ADMA)

0.37

Alanylphenylalanine

0.4

Arginylproline

0.33

Glycylisoleucine

0.42

Glycylleucine

0.4

Isoleucylalanine

0.47

Isoleucylglutamate

0.41

Isoleucylglycine

0.4

Isoleucylserine

0.37

Isoleucylthreonine

0.31

Leucylglutamate

0.44

Leucylleucine

0.49

Phenylalanylisoleucine

0.52

Phenylalanylleucine

0.45

0.64

Phenylalanylphenylalanine

0.66

0.6b

Threonylproline
Tyrosylglutamine

0.37

Tyrosylleucine

0.41

Valylalanine

0.37

Valylaspartate

0.34

Valylglycine

0.34

Valylleucine

0.33

Valylphenylalanine

0.36

Valylthreonine

Indolelactate
Methionine, cysteine, SAM,
and taurine metabolism

PTB V2
Term V2

0.48

0.41

0.59

0.49b

Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.

(continued)

TABLE 3

Fold change of metabolites that differ in CV fluid among women with preterm and term birtha,b (continued)
Metabolic pathway

Subpathway

Biochemical name

Carbohydrate

Glycolysis, gluconeogenesis,
and pyruvate metabolism

Term V
Term V1

PTB
V2
PTB V1

Glucose

0.2

Glucose-6-phosphate

0.14b

Glycogen metabolism

Aminosugar metabolism

3.73

0.59

Xylose

0.57b

Maltohexaose

0.35b

Maltopentaose

0.31b

Maltose

0.1b

N-acetylglucosamine

0.45

N-acetylgalactosamine

0.32b

0.43

0.62b

N-acetylneuraminate
Oxidative phosphorylation

Phosphate

Lipid

Long-chain fatty acid

Palmitoleate (16:1n7)
Myoinositol

Cofactors and vitamins

0.47

0.45b
b

0.44

Steroid

Dehydroisoandrosterone sulfate

0.67b

Purine metabolism,
(hypo)xanthine/inosine
containing

Xanthine

5.88c

Pyrimidine metabolism,
cytidine containing

Cytosine

0.25b

Nicotinate and
nicotinamide metabolism

Nicotinamide

0.43

0.64

1-Palmitoylglycerol (1-monopalmitin)

3-Hydroxyhippurate

0.36

Erythritol

Chemical

2-Pyrrolidinone

2.24

2.12
2.22

0.32b

Catechol sulfate
Food component/plant

0.42 Lysolipid

Monoacylglycerol

Benzoate metabolism

1.4
b

Nicotinamide adenine dinucleotide


reduced
Xenobiotics

1.56c
b

1-Stearoylglycerophosphoserine

Nucleotide

4.9
0.93b

10-Heptadecenoate (17:1n7)
Inositol metabolism

3.04c
0.4

Xylulose

Energy

PTB V2
Term V2

Term V1

Isobar: fructose 1,6-diphosphate,


glucose 1,6-diphosphate, myoinositol
1,4, or 1,3-diphosphate
Pentose metabolism

PTB V1

0.84

Glycolate (hydroxyacetate)

0.85

1.21c

1.21c
0.66

ADMA, asymmetrical dimethylarginine; CV, cervicovginal; SAM, S-adenosyl methionine; SDMA, symmetric dimethylarginine.
a

Significantly different pathways between comparison groups with P < .05; b A decrease in fold change between groups; c An increase in fold change.

Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.

is simply labor occurring earlier in


41
gesta- tion.
The identication of
distinct me- tabolites in the CV uid
metabolome among
women who

ultimately have a PTB compared likely involved in the pathogenesis of


with term birth unveiled important spontaneous PTB. Undoubtedly, future
metabolic pathways that are
work will need to reveal the contribution
of the host and microbiota to these

metabolic changes and will need to


elucidate the physiological changes that

are resulting in the metabolic


differences in women who ultimately
have a PTB. Investigating the CV
metabolome holds great promise for
unraveling mechan- isms involved in
term and preterm parturition.
-

REFERENCES
1. PeriStats. Available at: http://www.march
ofdimes.org/peristats/Peristats.aspx. Accessed
May 30, 2014.
2. Iams JD, Goldenberg RL, Meis PJ, et al. The
length of the cervix and the risk of spontaneous
premature delivery. National Institute of Child
Health and Human Development Maternal Fetal
Medicine Unit Network. N Engl J Med
1996;334:
567-72.
3. Fonseca EB, Celik E, Parra M, Singh M,
Nicolaides KH. Progesterone and the risk of
preterm birth among women with a short cervix.
N Engl J Med 2007;357:462-9.
4. Hassan SS, Romero R, Vidyadhari D, et al.
Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short
cervix: a multicenter, randomized, double-blind,
placebo-controlled trial. Ultrasound Obstet
Gynecol 2011;38:18-31.
5. Read CP, Word RA, Ruscheinsky MA,
Timmons BC, Mahendroo MS. Cervical remodeling during pregnancy and parturition: molecular characterization of the softening phase in
mice. Reproduction (Cambridge, England)
2007;134:327-40.
6. Timmons BC, Mitchell SM, Gilpin C,
Mahendroo MS. Dynamic changes in the cervical epithelial tight junction complex and differentiation occur during cervical ripening and
parturition. Endocrinology 2007;148:1278-87.
7. Nold C, Anton L, Brown A, Elovitz M. Inammation promotes a cytokine response and disrupts the cervical epithelial barrier: a possible
mechanism of premature cervical remodeling
and preterm birth. Am J Obstet Gynecol
2012;206:208.e1-7.
8. Timmons BC, Fairhurst AM, Mahendroo MS.
Temporal changes in myeloid cells in the cervix
during pregnancy and parturition. J Immunol
(Baltimore, MD: 1950) 2009;182:2700-7.
9. Xu H, Gonzalez JM, Ofori E, Elovitz MA.
Preventing cervical ripening: the primary mechanism by which progestational agents prevent
preterm birth? Am J Obstet Gynecol 2008;198:
314.e1-8.
10. Romero R, Hassan SS, Gajer P, et al. The
vaginal microbiota of pregnant women who
subsequently have spontaneous preterm labor
and delivery and those with a normal delivery at
term. Microbiome 2014;2:18.
11. Hyman RW, Fukushima M, Jiang H, et al.
Diversity of the vaginal microbiome correlates
with preterm birth. Reprod Sci ( Thousand Oaks,
CA) 2014;21:32-40.
12. Goldenberg RL, Culhane JF, Iams JD,
Romero R. Epidemiology and causes of
preterm birth. Lancet 2008;371:75-84.
13. Sevin DC, Kuehne A, Zamboni N, Sauer U.
Biological insights through nontargeted metabolomics. Curr Opin Biotechnol 2014;34C:1-8.
14. Louis P, Hold GL, Flint HJ. The gut microbiota, bacterial metabolites and colorectal cancer. Nat Rev Microbiol 2014;12:661-72.

15. Jess T, Gamborg


M, Matzen P,
Munkholm P, Sorensen TI. Increased risk of intestinal cancer in Crohns disease: a metaanalysis of population-based cohort studies.
Am J Gastroenterol 2005;100:2724-9.
16. Schwabe RF, Jobin C. The microbiome and
cancer. Nat Rev Cancer 2013;13:800-12.
17. Vanhaecke L, Knize MG, Noppe H, De
Brabander H, Verstraete W, Van de Wiele T. Intestinal bacteria metabolize the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]
pyridine following consumption of a single
cooked chicken meal in humans. Food Chem
Toxicol 2008;46:140-8.
18. Kassie F, Rabot S, Kundi M, Chabicovsky
M, Qin HM, Knasmuller S. Intestinal microora
plays a crucial role in the genotoxicity of the
cooked
food
mutagen
2-amino-3methylimidazo [4,5-f] quinoline. Carcinogenesis
2001;22:1721-5.
19. Bahado-Singh RO, Akolekar R, Mandal R,
et al. Metabolomics and rst-trimester prediction
of early-onset preeclampsia. J Matern Fetal
Neonatal Med ic 2012;25:1840-7.
20. Graca G, Goodfellow BJ, Barros AS, et al.
UPLC-MS metabolic proling of second
trimester amniotic uid and maternal urine and
comparison with NMR spectral proling for the
identication of pregnancy disorder biomarkers.
Mol Biosyst 2012;8:1243-54.
21. Dunn WB, Brown M, Worton SA, et al.
Changes in the metabolic footprint of placental
explant-conditioned culture medium identies
metabolic disturbances related to hypoxia and
pre-eclampsia. Placenta 2009;30:974-80.
22. Ivorra C, Garcia-Vicent C, Chaves FJ,
Monleon D, Morales JM, Lurbe E. Metabolomic
proling in blood from umbilical cords of low
birth weight newborns. J Transl Med
2012;10:142.
23. Fanos V, Atzori L, Makarenko K, Melis GB,
Ferrazzi E. Metabolomics application in
maternal- fetal medicine. Biomed Res Int
2013;2013:
720514.
24. Bastek JA, Hirshberg A, Chandrasekaran
S, et al. Biomarkers and cervical length to
predict spontaneous
preterm
birth in
asymptomatic high- risk women. Obstet
Gynecol 2013;122:283-9.
25. Elovitz MA, Brown AG, Anton L, Gilstrop
M,
Heiser L, Bastek J. Distinct cervical microRNA
proles are present in women destined to have a
preterm birth. Am J Obstet Gynecol 2014;210:
221.e1-11.
26. Iams JD; Clinical practice. Prevention of
pre- term parturition. N Engl J Med
2014;370:254-61.
27. Evans AM, DeHaven CD, Barrett T,
Mitchell M, Milgram E. Integrated, nontargeted
ultrahigh performance liquid chromatography/
electrospray ionization tandem mass spectrometry platform for the identication and relative quantication of the small-molecule
complement of biological systems. Anal Chem
2009;81:6656-67.
28. Russell WR, Hoyles L, Flint HJ, Dumas ME.
Colonic bacterial metabolites and human health.

Curr Opin Microbiol 2013;16:246-54.

29. Nieman DC, Shanely RA, Gillitt ND,


Pappan KL, Lila MA. Serum metabolic signatures induced by a three-day intensied exercise
period persist after 14 h of recovery in runners.
J Proteome Res 2013;12:4577-84.
30. Menon R, Jones J, Gunst PR, et al.
Amniotic
uid metabolomic analysis in spontaneous preterm birth. Reprod Sci ( Thousand Oaks, Calif
)
2014;21:791-803.
31. Romero R, Mazaki-Tovi S, Vaisbuch E, et al.
Metabolomics in premature labor: a novel
approach to identify patients at risk for preterm
delivery. J Matern Fetal Neonatal Med 2010;23:
1344-59.
32. Auray-Blais CRE, Gagnon R, Berthiaume M,
Pasquier JC. Metabolomics and preterm birth:
what biomarkers in cervicovaginal secretions are
predictive of high-risk pregnant women? Int J
Mass Spectrom 2011;307:33-8.
33. Boskey ER, Cone RA, Whaley KJ,
Moench TR. Origins of vaginal acidity: high D/L
lactate ratio is consistent with bacteria being the
primary source. Hum Reprod (Oxford, Englan d)
2001;16:1809-13.
34. Mirmonsef P, Hotton AL, Gilbert D, et al.
Free glycogen in vaginal uids is associated
with Lactobacillus colonization and low vaginal
pH. PloS One 2014;9:e102467.
35. Spear GT, French AL, Gilbert D, et al.
Human alpha-amylase present in lower-genitaltract mucosal uid processes glycogen to
support vaginal colonization by Lactobacillus.
J Infect Dis 2014;210:1019-28.
36. Verstraelen H, Verhelst R, Claeys G, De
Ba cker E, Temmerma n M, Vaneechoutte M.
Longitudinal analysi s of the vagina l microora
in pregnancy sugges ts that L crispatus promot es the stability of the normal vaginal
microora and that L gasseri and/or L iners
are mor e conducive to the occurrence of
abnorma l vagina l microora. BMC Microbiol
2009;9:116.
37. Varki A, Gagneux P. Multifarious roles of
sialic acids in immunity. Ann N Y Acad Sci
2012;1253:16-36.
38. Lewis WG, Robinson LS, Gilbert NM,
Perry JC, Lewis AL. Degradation, foraging, and
depletion of mucus sialoglycans by the vaginaadapted Actinobacterium Gardnerella vaginalis.
J Biol Chem 2013;288:12067-79.
39. Heng YJ, Di Quinzio MK, Liong S,
Permezel M, Rice GE, Georgiou HM. Temporal
investigation of matrix metalloproteinases and
their inhibitors in human cervicovaginal uid in
late pregnancy and labor. Reprod Sci ( Thousand Oaks, Calif ) 2012;19:55-63.
40. Heng YJ, Di Quinzio MK, Permezel M,
Rice GE, Georgiou HM. Cystatin A protease inhibitor and cysteine proteases in human cervicovaginal uid in term pregnancy and labor. Am
J Obstet Gynecol 2011;204:254.e1-7.
41. Romero R, Dey SK, Fisher SJ. Preterm labor: one syndrome, many causes. Science (New
York, NY) 2014;345:760-5.

A PP ENDI X

SUPPLEMENTAL TABLE 1

Comparison of metabolome in CV fluid among women destined to have a preterm or term birth
Term V 2
Term V1

Variable
Total biochemical (P

.05)a

Biochemicals ([Y)

0 j 16

Total biochemicals 0.05 less than P < .10


Biochemicals ([Y)
Total biochemical (P

16
21
2 j 19

.05)

Total biochemicals 0.05 less than P < .10

7
2j5
4
0j4

PTB V
1
Term V1

PTB V 2
Term V2

40

15

2 j 38
42
1 j 41

Status main effect

Time main effect

49

13

36

20

14

ANOVA, analysis of variance.


a

PTB V
2
PTB V1

Comparisons made using a 2-way ANOVA; b Comparisons made using ANOVA with repeated measures.

Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.

8j7
33
9 j 24

Status: time interaction

SUPPLEMENTAL TABLE 2

Metabolites that differ among women with preterm and term birth, grouped according to metabolic classa
Metabolic pathway
Amino acid

Peptide
Carbohydrate

Subpathway
Glycine, serine, and threonine metabolism

1b

Phenylalanine and tyrosine metabolism

Tryptophan metabolism

1j1

Leucine, isoleucine, and valine metabolism

Methionine, cysteine, SAM and


taurine metabolism

1j1

Urea cycle; arginine and proline metabolism

1j3
b

b
b

Creatine metabolism

Guanidino and acetamido metabolism

Glutathione metabolism

Dipeptide

22j25

Polypeptide

1b

Glycolysis, gluconeogenesis, and pyruvate


metabolism

2j1b

Pentose metabolism

2j1

3j2

2j1

4j16

2j1

Oxidative phosphorylation

Long-chain fatty acid

2j1

Polyunsaturated fatty acid (n3 and n6)

1
b

Inositol metabolism

Lysolipid
Glycerolipid metabolism

1j1b

1b

Monoacylglycerol

1j1

Steroid

1j1

Purine metabolism, (hypo)xanthine/inosine


containing

Purine metabolism, adenine containing

Pyrimidine metabolism, cytidine containing

Nicotinate and nicotinamide metabolism

1j2

Pantohenate and CoA metabolism

1b

Ascorbate and aldrate metabolism


Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.

Fatty acid, dihydroxy

0
b

TCA cycle

Carnitine metabolism

Cofactors and vitamins

Term V1
b

PTB V2
Term V2

Aminosugar metabolism

Nucleotide

PTB V1

Lysine metabolism

Fructose, mannose, and galactose metabolism

Lipid

PTB V
2
PTB V1

Gluamate metabolism

Glycogen metabolism

Energy

Term V2
Term V1

0
(continued)

SUPPLEMENTAL TABLE 2

Metabolites that differ among women with preterm and term birth, grouped according to metabolic classa (continued)
Metabolic pathway

Subpathway

Term V2

Xenobiotics

Benzoate metabolism

1j1

Xanthine metabolism

1b

Food component/plant

Term V1

PTB V
2
PTB V1

Term V1
b

PTB V2
Term V2
b

Drug

Chemical

1j2

Results provided in an alternative way by describing the pathways (not specific metabolites) that were differentially expressed by case or control status.
CoA, coenzyme A; TCA, trichloroacetic acid.
a

PTB V1

Significantly different pathways between comparison groups with P < .05; b 0.05, P < .10 or greater.

Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.