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OBSTETRICS
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.
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-
776.e2
Research
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
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
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
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
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
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
20 4/7 (20e23)
.02
.70
1.00
3.3 (2.1e3.5)
2.9 (1.6e3.6)
33 4/7 (26e36)
3.7 (2e4.8)
3.8 (2.4e4.7)
40 (39e41)
.07
.03
< .001
Ghartey. Women with preterm birth have a cervicovaginal metabolome. Am J Obstet Gynecol 2015.
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
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
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.
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.
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
0.25
0.55b
0.07b
Ornithine
Peptide
Creatine metabolism
Creatinine
0.65b
4-Guanidinobutanoate
0.66
Glutathione metabolism
5-Oxoproline
0.44b
Dipeptide
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
Palmitoleate (16:1n7)
Myoinositol
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
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
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.
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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
16
21
2 j 19
.05)
7
2j5
4
0j4
PTB V
1
Term V1
PTB V 2
Term V2
40
15
2 j 38
42
1 j 41
49
13
36
20
14
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
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
Tryptophan metabolism
1j1
1j1
1j3
b
b
b
Creatine metabolism
Glutathione metabolism
Dipeptide
22j25
Polypeptide
1b
2j1b
Pentose metabolism
2j1
3j2
2j1
4j16
2j1
Oxidative phosphorylation
2j1
1
b
Inositol metabolism
Lysolipid
Glycerolipid metabolism
1j1b
1b
Monoacylglycerol
1j1
Steroid
1j1
1j2
1b
0
b
TCA cycle
Carnitine metabolism
Term V1
b
PTB V2
Term V2
Aminosugar metabolism
Nucleotide
PTB V1
Lysine 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.