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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: J Matern Fetal Neonatal Med. 2019 Dec 29;34(24):4009–4024. doi: 10.1080/14767058.2019.1702953

Preterm labor is characterized by a high abundance of amniotic fluid prostaglandins in patients with intra-amniotic infection or sterile intra-amniotic inflammation

Hassendrini N Peiris 1, Roberto Romero 2,3,4,5,6,7,*, Kanchan Vaswani 1, Sarah Reed 8, Nardhy Gomez-Lopez 2,9,10, Adi L Tarca 2,9,11, Dereje W Gudicha 2,9, Offer Erez 2,9,12, Eli Maymon 2,9,12, Murray D Mitchell 1,*
PMCID: PMC8314747  NIHMSID: NIHMS1722757  PMID: 31885290

Abstract

Spontaneous preterm labor is one of the “great obstetrical syndromes” which may lead to preterm delivery, a leading cause for neonatal and infant death. Prostaglandins are considered universal mediators for the onset of spontaneous labor at term. This concept is largely based on the observations that amniotic fluid concentrations of prostaglandins are elevated prior to and during the onset of labor; however, these studies have largely been performed using immunoassays. Distinguishing prostaglandins from similarly structured molecules (i.e. prostamides) is difficult given the cross-reactivity of available antibodies and the chemical similarity of this family of compounds. Herein, this limitation was overcome by utilizing mass spectrometry to determine prostaglandin and prostamide concentrations in the amniotic fluid of women who had an episode of preterm labor with intact membranes. Patients were classified into the following groups: 1 subsequent delivery at term (n=23); 2) preterm delivery in the absence of intra-amniotic inflammation (n=51); 3) preterm delivery with sterile intra-amniotic inflammation (amniotic fluid interleukin (IL)-6 >2.6 ng/mL without detectable microorganisms) (n=35); and 4) preterm delivery with intra-amniotic infection [amniotic fluid IL-6 > 2.6 ng/mL with detectable microorganisms] (n=16). Amniotic fluid samples were analyzed by liquid chromatography-tandem mass spectrometry. We found that 1) both prostaglandins and prostamides were detectable and distinguishable in the amniotic fluid of women with preterm labor; 2) amniotic fluid concentrations of PGE2, PGF2α, and PGFM were higher in patients with intra-amniotic infection than in those without intra-amniotic inflammation; 3) amniotic fluid concentrations of PGE2 and PGF2α were also greater in patients with intra-amniotic infection than in those with sterile intra-amniotic inflammation; 4) patients with sterile intra-amniotic inflammation had higher amniotic fluid concentrations of PGE2 and PGFM than those without intra-amniotic inflammation who delivered at term; 5) amniotic fluid concentrations of PGFM were also greater in women with sterile intra-amniotic inflammation than in those without intra-amniotic inflammation who delivered preterm; 5) amniotic fluid concentrations of prostamides (PGE2-EA and PGF2α-EA) were not different among patients with preterm labor; 6) amniotic fluid concentrations of prostaglandins, but no prostamides, were higher in cases with intra-amniotic inflammation (interleukin-6 >2.6 ng/mL); and 7) the PGE2:PGE2-EA and PGF2α:PGF2α-EA ratios were higher in patients with intra-amniotic infection compared to those without inflammation. Mass spectrometric analysis of amniotic fluid indicated that amniotic fluid concentrations of PGE2, PGF2α, and PGFM, but no prostamides, were significantly higher in women with preterm labor and intra-amniotic infection than in other patients with an episode of preterm labor. Yet, women with intra-amniotic infection had greater amniotic fluid concentrations of PGE2, and PGF2α than those with sterile intra-amniotic inflammation, suggesting that these two clinical conditions may be differentiated by using mass spectrometric analysis of amniotic fluid.

Keywords: chorioamnionitis, eicosanoids, mass spectrometry, parturition, prostamides

Introduction

Preterm birth (delivery before 37 weeks of gestation) and its complications are responsible for 35% of the 3.1 million neonatal deaths per year [1,2]. Each year, about 15 million preterm neonates are born worldwide [3]. The risk factors for preterm birth include prior preterm birth, maternal nutritional status, a very low maternal body mass index or maternal obesity, ethnicity, socioeconomic status, smoking during pregnancy, maternal age, parity, use of assisted reproductive technologies, a short cervix, and multi-fetal gestations [4–21]. Preterm birth is associated with severe short- and long-term complications, including bronchopulmonary dysplasia, cerebral palsy, blindness, and deafness [22–26].

Preterm labor is a syndrome caused by multiple pathologic processes [27,28]. Intra-amniotic infection/inflammation affects one in four preterm births and is largely subclinical in nature [29–35]. However, there is compelling evidence that the relationship between intra-amniotic infection/inflammation and spontaneous preterm labor is causal [31,35–48]. Intra-amniotic inflammation can result from microbial invasion of the amniotic cavity, referred to as intra-amniotic infection (IAI), or it can occur in the absence of detectable microorganisms using both culture and molecular microbiological techniques (i.e. “sterile intra-amniotic inflammation” or SIAI) [49–54]. The latter is an inflammatory process thought to be induced by danger signals or alarmins (endogenous molecules derived from cellular stress or necrosis [55–57]) found in the amniotic fluid [58–65]. Sterile intra-amniotic inflammation is more commonly observed than intra-amniotic infection in women with preterm labor and intact membranes [49,50]; however, to date, there are no biomarkers that can assist in the differential diagnosis of these two clinical conditions.

Prostaglandins are central mediators in the process of preterm and term parturition [66–78]. This family of molecules can induce uterine contractility and cervical ripening and participate in the mechanisms of extracellular matrix remodeling in the chorioamniotic membranes [27,71,79]. Both term and preterm labor require a combination of endocrine and mechanical stimuli from both mother and infant [27,79–85], including products of prostaglandin endoperoxide synthase-2 (PGHS, also known as PTGS-2 or cyclooxygenase-2 [COX-2]) [86,87]. Spontaneous parturition is preceded by an increased concentration of prostaglandins in the amniotic fluid, which is a cause—and not a result—of labor at term [88], given that it occurs before onset [89]. Therefore, the determination of prostaglandin concentrations could be of potential clinical value.

The accurate identification of endocannabinoids and eicosanoids by immunoassay has been a major challenge in parturition research. The lack of specific antibodies and the similarity of the molecular structures of these substances have led to difficulty in distinguishing among these moieties [90]. In recent years, the use of high-resolution mass spectrometry has enabled the more specific identification of prostaglandins and prostamides in biological fluids and placental explant culture media [74,76,77,91,92]. Therefore, herein, we used mass spectrometry to determine the prostaglandin and prostamide concentrations of the amniotic fluid of women presenting with an episode of preterm labor with intact membranes. Since prostaglandins are inflammatory mediators, we also determined whether the concentrations varied between patients with intra-amniotic infection and those with sterile intra-amniotic inflammation.

Methods

Sample collection

A retrospective cross-sectional study was conducted by searching the clinical database and Bank of Biologic Samples of the Detroit Medical Center, Wayne State University, and the Perinatology Research Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U. S. Department of Health and Human Services (NICHD/NIH/DHHS; Bethesda, MD, and Detroit, MI, USA). The inclusion criteria required 1) a singleton gestation; 2) a transabdominal amniocentesis performed between 20 and 36 weeks of gestation, before the rupture of the chorioamniotic membranes; and 3) sufficient amniotic fluid available for mass spectrometry studies.

All patients provided written informed consent before the collection of amniotic fluid samples. The collection and utilization of the samples were approved by the Institutional Review Boards of the participating institutions where the patients received care. Many of these samples had been used in previous studies of the biology of cytokines and inflammatory mediators in intra-amniotic infection/inflammation [49,50,54,64,76,93–96].

Clinical definitions

Preterm labor was diagnosed by the presence of at least two regular uterine contractions every 10 min associated with cervical changes in patients with a gestational age between 20 and 36/37 weeks. Microbial invasion of the amniotic cavity was diagnosed based on the results of amniotic fluid cultures for microorganisms [36,97–100] and broad-range polymerase chain reaction (PCR) assays, coupled with electrospray ionization mass spectrometry (PCR/ESI-MS; Ibis Technology; Athogen, Carlsbad, CA, USA) [49–51,53,54,64,77,101–108]. Intra-amniotic inflammation was defined as an amniotic fluid interleukin (IL)-6 concentration ≥ 2.6 ng/mL [32,62,93–96,109–115]. Intra-amniotic infection was defined as the presence of microbial invasion of the amniotic cavity with intra-amniotic inflammation [49–51,53,96,102–106,116–131]. Sterile intra-amniotic inflammation was defined as intra-amniotic inflammation without microorganisms detected by culture or PCR/ESI-MS [49–51,53,54].

Women who had an episode of preterm labor (PTL) were divided into four groups: 1) subsequent delivery at term (PTL-TD); 2) preterm delivery without intra-amniotic inflammation (PTL-PTD NI); 3) preterm delivery with sterile intra-amniotic inflammation (PTL-PTD SIAI); and 4) preterm delivery with intra-amniotic infection (PTL-PTD IAI).

Sample collection

Amniotic fluid samples were obtained by transabdominal amniocentesis performed for evaluation of the microbial and inflammatory status (i.e. Gram stain [132], white blood cell count [133], and amniotic fluid glucose concentration [134]) of the amniotic cavity in patients diagnosed with an episode of preterm labor. The results of microbiology and the inflammatory status of the amniotic fluid were used for clinical management. Samples of amniotic fluid were transported to the laboratory in a sterile, capped syringe and cultured for aerobic/anaerobic bacteria and genital mycoplasmas. Amniotic fluid IL-6 concentrations were used only for research purposes.

Detection of microorganisms with molecular methods

In addition to standard cultivation techniques, which determined aerobic and anaerobic bacteria as well as genital mycoplasmas, the amniotic fluid was analyzed by using broad-range real-time PCR/ESI-MS (Ibis Technology; Athogen) [49]. In brief, DNA was extracted from 300 μl of amniotic fluid by using a method that combines bead-beating cell lysis with a magnetic bead-based extraction method [135,136]. The extracted DNA was amplified by the previously described broad bacteria and Candida (BAC) detection assay, according to the manufacturer’s instructions. PCR/ESI-MS can identify 3400 bacteria and 40 Candida spp., which are represented in the platform’s signature database [137,138]. After PCR amplification, 30 μl aliquots of each PCR product were desalted and analyzed via ESI-MS. The presence of microorganisms was determined by signal processing and triangulation analysis of all base composition signatures obtained from each sample and compared to a database. The sensitivity (limit of detection) of the assay for the detection of bacteria in blood, on average, is 100 CFU/ml (95% CI, 6–600 CFU/ml). A comparison of detection limits between the blood and amniotic fluid showed that the assays have comparable detection limits (100 CFU/ml).

Determination of IL-6 in AF

The concentrations of IL-6 in amniotic fluid were determined by a sensitive and specific enzyme immunoassay obtained from R&D Systems (Minneapolis, MN, USA). The initial assay validation was performed in our laboratory before this study was conducted. The immunoassay uses the quantitative sandwich enzyme immunoassay technique, and the concentrations were determined by interpolation from the standard curves. The inter- and intra-assay coefficients of variation for IL-6 were 8.7 and 4.6%, respectively. The sensitivity of the assay for IL-6 was 0.09 pg/ml.

Mass spectrometry sample preparation

Standards and samples were subjected to an extraction protocol in organic solution. Extraction solution was prepared containing internal standards (250fmol each). Internal standards include the deuterated standards, namely, for prostaglandin E2-d4 (PGE2-d4), prostaglandin F2α-d4 (PGF2α-d4), 13,14-dihydro-15-keto-PGF(2alpha) -d4 (PGFM-d4), prostaglandin E2-ethanolamide-d4 (PGE2-EA-d4), and prostaglandin F2α-ethanolamide-d4 (PGF2α-EA-d4) in an extraction solution of methanol/formic acid (99:1). The extraction solution (250 μL) was added to a 96-plate well and vortexed (900 rpm, 5 min) followed by the addition of 850μL of chilled water, placed on a Teflon mat, and vortexed (900 rpm, 5 min). Using a vacuum manifold, the solid phase extraction (SPE) plate was pre-equilibrated consecutively using 500μL of methanol and 2 × 500μL of water. The lipid/eicosanoid extract was loaded onto the plate on low flow (< 5 mm Hg or dropwise and the gauge was slowly closed until <5mmHg was reached) onto the SPE plate and left to bind for 1 min. The plate was then washed twice with chilled water. The clean and bound lipid extract preparation was eluted and dried. The dried lipids were then reconstituted in an appropriate solvent (20% Methanol in water) and prepared in triplicate (sample replicates).

Mass Spectrometry Analyses

Ultra-performance liquid chromatography (UPLC; Shimadzu Ltd) was used for separation of the sample analytes coupled with a Kinetek C8 column (Phenomenex Australia Pty Ltd) attached to a guard column (Phenomenex Australia Pty Ltd). Oven temperature was set to 60°C and a 15-min gradient was set up using aqueous and organic mobile phase solvent preparations. A scheduled multiple reaction monitoring (sMRM) method was carried out for the analyses and quantitation of 5 analytes (PGE2, PGF2α, PGFM, PGE2-EA, and PGF2α-EA) and their respective deuterated versions (PGE2-d4, PGF2α-d4, PGFM-d4, PGE2-EA-d4, and PGF2α-EA-d4) by using both negative and positive modes. Each sample was injected in triplicate (technical replicates). A list of mass spectrometry conditions for both negative and positive modes is provided in Supplementary Material 1 (including limits of detection).

MultiQuant Analyses

Individual peaks for each analyte were selected using the retention time window of +/− 0.5 min. A signal-to–noise ratio of below 10 was excluded from the list. The multiquant data were used to generate area and peak area ratio (PAR) information for both standards and samples. The standard curve was plotted with individual concentrations against the PARs. The equation of the line was used to extrapolate the concentrations (pmol/L) for the samples.

Statistical analyses

The linear mixed model method was applied to analyze differences in amniotic fluid concentration among the study groups. Linear mixed modeling offers flexibility to account for dependency between observations from the same individual. Additionally, the linear mixed model method allows each sample to contribute to the variance estimation, suitable for analyzing repeated measurements with a missing value, contrary to the traditional analysis of variance approach. The amniotic fluid concentration for each analyte was log-transformed to better fit the normal distribution of the data. The Fisher’s exact test for categorical variables and the Kruskal-Wallis test for continuous variables were applied to compare maternal characteristics among the groups, with the P values for multiple testing corrected for the false discovery rate in post-hoc analysis. All the analyses were conducted using R language and environment for statistical computing (www.r-project.org).

Results

Demographic and clinical characteristics

The demographic and clinical characteristics of patients in the four study groups are presented in Table 1. A total of 125 patients, comprising the PTL-TD NI (n=23), PTL-PTD NI (n=51), PTL-PTD SIAI (n=35), and PTL-PTD IAI (n=16) groups, was involved in the study. Gestational ages at amniocentesis/delivery and birthweights were significantly different among the groups (Table 1). Lower gestational age at delivery and lower birthweight were observed in women with PTL-PTD IAI and PTL-PTD SIAI compared to those delivering at term (PTL-TD NI) and delivering preterm (PTL-PTD NI) without infection. No significant differences were identified in maternal age or body mass index among the study groups.

Table 1.

Maternal and clinical characteristics compared among the groups.

Clinical characteristics PTL-TD NI
(n=23)		 PTL-PTD NI
(n=51)		 PTL-PTD SIAI
(n=35)		 PTL-PTD IAI
(n=16)		 p-value
Age (years; median [IQR]) 25 (21–29) 23 (20–26) 23 (20–26.5) 25 (20.75–30.25) 0.47
Height (cm; median [IQR]) 160 (154–165) 162 (157–166) 162 (157–167) 166 (160–170) 0.31
Weight (kg; median [IQR]) 60 (54–76) 61 (54–74) 72 (52–86) 70 (63–77) 0.18
Body mass index (kg/m2; median [IQR]) 23.9 (21–30.8) 23.6 (21.1–28.9) 26.2 (22.1–32.4) 27 (24.9–33.5) 0.27
Parity (parous; n[%]) 17 (73.9) 33 (64.7) 20 (57.1) 7 (43.8) 0.25
Race (African-American; n[%]) 20 (87) 45 (88.2) 31 (88.6) 12 (75) 0.55
Smoking status (smoker; n[%]) 5 (21.7) 13 (25.5) 8 (22.9) 3 (18.8) 0.98
Gestational age at amniocentesis (weeks; median [IQR]) 31.9 (31–32.2) 31.4 (28.9–32.7) 24.35 (23.3–28.1) 26.5 (24.8–32.1) <.001
Gestational age at delivery (weeks; median [IQR]) 39 (37.5–39.9) 34.9 (33.2–36) 26.4 (24.2–30.5) 26.7 (25.6–32.6) <.001
Birthweight (grams; median [IQR]) 3080 (2772–3340) 2312 (1990–2588) 890 (558–1415) 1048 (747–1970) <.001
Sex (male; n[%]) 9 (39.1) 24 (48) 15 (42.9) 8 (50) 0.86
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IQR = interquartile range.

Mass spectrometric detection of prostaglandins in the amniotic fluid of women with preterm labor and intact membranes

Figure 1 shows the linear calibration range of prostaglandins and prostamides. Each molecule was positively identified by UPLC retention time with an authentic standard and unique MRM transition.

Figure 1. Linear calibration range of prostaglandins and prostamides.

Figure 1.

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LC-MS/MS multiple reaction monitoring (MRM) transitions for PGE2, PGF2a, PGFM, PGE2-EA, and PGF2a-EA. Each detectable molecule was positively identified by UPLC retention time with an authentic standard and unique MRM transition.

The amniotic fluid concentrations of prostaglandins and prostamides among the four study groups are described in Figure 2, and statistics are provided in Table 2 and Table 3. Patients with intra-amniotic infection had significantly higher median concentrations of PGE2 (p<0.001), PGF2α (p<0.001), and PGFM (p<0.001) than those who delivered either at term or preterm without infection or inflammation (Figure 2A–C). In addition, patients with intra-amniotic infection had greater median concentrations of PGE2 (p<0.001) and PGF2α (p<0.001) than those with sterile intra-amniotic inflammation (Figure 2A&B).

Figure 2. Amniotic fluid concentrations of prostaglandins and prostamides in patients with preterm labor.

Figure 2.

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Amniotic fluid concentrations of PGE2 (A), PGF2a (B), PGFM (C), PGE2-EA (D), and PGF2a-EA (E) were determined using LC-MS/MS. Women who had an episode of preterm labor (PTL) were divided into four groups: (1) term delivery (PTL-TD); (2) preterm delivery without intra-amniotic inflammation (PTL-PTD NI); (3) preterm delivery with sterile intra-amniotic inflammation (PTL-PTD SIAI); and (4) preterm delivery with intra-amniotic infection (PTL-PTD IAI).

Table 2.

Amniotic fluid analyte concentration differences among study groups.

Analyte Log2 fold change (SD) with PTL-TD NI as reference group p-value
PTL-PTD NI PTL-PTD SIAI PTL-PTD IAI
PGE2 0.318 (0.412) 1.142 (0.445) 4.166 (0.541) <.001
PGF2α 0.102 (0.261) 0.28 (0.278) 2.202 (0.344) <.001
PGFM 0.618 (0.323) 2.913 (0.345) 3.616 (0.427) <.001
PGE2.EA 0.13 (0.199) −0.083 (0.206) 0.267 (0.258) 0.378
PGF2α.EA 0.029 (0.266) −0.007 (0.279) −0.219 (0.361) 0.893
PGE2:PGE2.EA 0.071 (0.457) 0.858 (0.5) 3.793 (0.599) <.001
PGF2α:PGF2α.EA 0.088 (0.361) 0.156 (0.378) 1.798 (0.494) 0.001
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SD = standard deviation.

Table 3.

Post-hoc analysis of amniotic fluid concentration differences among study groups per analyte.

Analyte Group pair Fold change Log2 fold change Standard error Adjusted
p-value
PGE2 PTL–TD NI vs PTL–PTD NI 1.247 0.318 0.412 0.64
PTL–TD NI vs PTL–PTD SIAI 2.207 1.142 0.445 0.03
PTL–TD NI vs PTL–PTD IAI 17.951 4.166 0.541 <.001
PTL–PTD NI vs PTL–PTD SIAI 1.77 0.824 0.37 0.065
PTL–PTD NI vs PTL–PTD IAI 14.41 3.849 0.481 <.001
PTL–PTD SIAI vs PTL–PTD IAI 8.134 3.024 0.51 <.001
PGF2α PTL–TD NI vs PTL–PTD NI 1.073 0.102 0.261 0.813
PTL–TD NI vs PTL–PTD SIAI 1.214 0.28 0.278 0.512
PTL–TD NI vs PTL–PTD IAI 4.601 2.202 0.344 <.001
PTL–PTD NI vs PTL–PTD SIAI 1.131 0.178 0.228 0.64
PTL–PTD NI vs PTL–PTD IAI 4.287 2.1 0.305 <.001
PTL–PTD SIAI vs PTL–PTD IAI 3.789 1.922 0.32 <.001
PGFM PTL–TD NI vs PTL–PTD NI 1.535 0.618 0.323 0.122
PTL–TD NI vs PTL–PTD SIAI 7.532 2.913 0.345 <.001
PTL–TD NI vs PTL–PTD IAI 12.261 3.616 0.427 <.001
PTL–PTD NI vs PTL–PTD SIAI 4.908 2.295 0.282 <.001
PTL–PTD NI vs PTL–PTD IAI 7.994 2.999 0.378 <.001
PTL–PTD SIAI vs PTL–PTD IAI 1.629 0.704 0.397 0.158
PGE2.EA PTL–TD NI vs PTL–PTD NI 1.094 0.13 0.199 0.692
PTL–TD NI vs PTL–PTD SIAI 0.944 −0.083 0.206 0.813
PTL–TD NI vs PTL–PTD IAI 1.203 0.267 0.258 0.507
PTL–PTD NI vs PTL–PTD SIAI 0.863 −0.213 0.16 0.324
PTL–PTD NI vs PTL–PTD IAI 1.1 0.138 0.223 0.692
PTL–PTD SIAI vs PTL–PTD IAI 1.275 0.351 0.229 0.234
PGF2α.EA PTL–TD NI vs PTL–PTD NI 1.02 0.029 0.266 0.934
PTL–TD NI vs PTL–PTD SIAI 0.995 −0.007 0.279 0.981
PTL–TD NI vs PTL–PTD IAI 0.859 −0.219 0.361 0.692
PTL–PTD NI vs PTL–PTD SIAI 0.975 −0.036 0.223 0.921
PTL–PTD NI vs PTL–PTD IAI 0.842 −0.248 0.319 0.64
PTL–PTD SIAI vs PTL–PTD IAI 0.863 −0.212 0.331 0.692
PGE2:PGE2.EA PTL–TD NI vs PTL–PTD NI 1.05 0.071 0.457 0.921
PTL–TD NI vs PTL–PTD SIAI 1.813 0.858 0.5 0.17
PTL–TD NI vs PTL–PTD IAI 13.861 3.793 0.599 <.001
PTL–PTD NI vs PTL–PTD SIAI 1.725 0.787 0.397 0.111
PTL–PTD NI vs PTL–PTD IAI 13.196 3.722 0.517 <.001
PTL–PTD SIAI vs PTL–PTD IAI 7.642 2.934 0.556 <.001
PGF2α:PGF2α.EA PTL–TD NI vs PTL–PTD NI 1.063 0.088 0.361 0.909
PTL–TD NI vs PTL–PTD SIAI 1.114 0.156 0.378 0.813
PTL–TD NI vs PTL–PTD IAI 3.477 1.798 0.494 <.001
PTL–PTD NI vs PTL–PTD SIAI 1.048 0.068 0.301 0.909
PTL–PTD NI vs PTL–PTD IAI 3.272 1.71 0.438 <.001
PTL–PTD SIAI vs PTL–PTD IAI 3.119 1.641 0.453 <.001
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Patients with sterile intra-amniotic inflammation had higher median concentrations of PGE2 (p=0.03) and PGFM (p<0.0001) than those who delivered at term without intra-amniotic inflammation (Figure 2A&C). These patients also had a higher median concentration of PGFM than those with preterm labor who delivered preterm without intra-amniotic inflammation (p<0.001; Figure 2C).

No significant differences were observed in the concentrations of PGE2-EA and PGF2α-EA among the study groups (Figure 2D&E).

Amniotic fluid concentrations of PGE2 (p<0.001), PGF2α (p<0.001), and PGFM (p<0.001) were significantly higher in cases with intra-amniotic inflammation (IL-6 >2.6 n/ml) (Figure 3A–C). However, amniotic fluid concentrations of prostamides were not significantly higher in cases with intra-amniotic inflammation (IL-6 >2.6 n/ml) (Figure 3D&E).

Figure 3. Correlations between amniotic fluid concentrations of prostaglandins-prostamides and intra-amniotic inflammation in patients with preterm labor.

Figure 3.

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Correlations between amniotic fluid concentrations of PGE2 (A), PGF2a (B), PGFM (C), PGE2-EA (D), and PGF2a-EA (E) and elevated concentrations of IL-6 (2.6 ng/mL).

When the ratio of prostaglandin-to-prostamide was compared (Figure 4), significant differences were observed between patients with intra-amniotic infection or those with sterile intra-amniotic inflammation compared to the other study groups. Patients with either intra-amniotic infection or sterile intra-amniotic inflammation had higher ratios of PGE2: PGE2-EA (p<0.001) and PGF2α: PGF2α-EA (p<0.001) compared to patients without intra-amniotic inflammation (Figure 4A&B).

Figure 4. Amniotic fluid prostaglandin and prostamides ratios in patients with preterm labor.

Figure 4.

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Ratio of PGE2 to PGE2-EA and PGF2a to PGF2a-EA in the study groups. Women who had an episode of preterm labor (PTL) were divided into four groups: (1) term delivery (PTL-TD); (2) preterm delivery without intra-amniotic inflammation (PTL-PTD NI); (3) preterm delivery with sterile intra-amniotic inflammation (PTL-PTD SIAI); and (4) preterm delivery with intra-amniotic infection (PTL-PTD IAI).

Discussion

Principal findings of the study:

1) The amniotic fluid concentrations of PGE2, PGF2α, and PGFM were significantly higher in patients with intra-amniotic infection than in those without inflammation who delivered preterm or at term; 2) patients with intra-amniotic infection had significantly higher median amniotic fluid concentrations of PGE2 and PGF2α than those with sterile intra-amniotic inflammation; 3) the amniotic fluid concentrations of PGE2 and PGFM were higher in women with sterile intra-amniotic inflammation than in those without inflammation; and 4) the amniotic fluid concentrations of prostamides did not differ according to the presence or absence of sterile intra-amniotic inflammation or intra-amniotic infection in patients with preterm labor.

Prostaglandins in term labor

Eicosanoids, the essential lipid molecules, play a major role in inflammation [139] as well as in several key reproductive processes (e.g. ovulation [140,141], implantation [142], maintenance of pregnancy [143], and parturition [66–78]). Eicosanoids are synthesized via the metabolism of arachidonic acid through the action of several enzymes, giving rise to three major groups of molecules: prostanoids (derived from arachidonic acid), prostamides (derived from anandamide), and prostaglandin glycerol esters (derived from 2-acyl glycerol) [139]. During late pregnancy, the amniotic fluid contains factors that trigger the production of PGF2α by the fetal membranes [144–146], and concentrations of amniotic fluid PGF2α and PGE2 are elevated during the course of labor [70,73,74]. Thus, the evidence indicating that eicosanoids are involved in the physiological process of term parturition is solid.

Inflammation and infection in preterm labor

Preterm labor is a syndrome caused by multiple pathologic processes [28]. Intra-amniotic infection is an important cause of preterm labor [27,28]. Most cases of intra-amniotic infection follow an ascending pathway from the lower genital tract [147]. Microbial invasion of the amniotic cavity has been identified in 20% of preterm births [98] and 40–50% of preterm births with premature rupture of the membranes [148,149]. Indeed, with the application of molecular microbiologic techniques (detection of microbial DNA), the rate of intra-amniotic infection has become higher than with the use of cultivation techniques only [49,50,53,150–155]. Microorganisms elicit a response via the activation of the immune system of the mother and fetus [48,108,117,118,120,156–158], resulting in the release of inflammatory cytokines [156,158] as well as the stimulation of prostaglandin production [159–161].

Sterile intra-amniotic inflammation also contributes to spontaneous preterm parturition [49,50]. This condition is associated with elevated concentrations of endogenous danger signals or alarmins in the amniotic fluid [58–62,64,162]. The mechanisms whereby alarmins induce sterile intra-amniotic inflammation and preterm birth involve the activation of the inflammasome [54,65,163–166], a multi-protein complex responsible for the processing of mature IL-1β [167,168].

Intra-amniotic infection is the most important identifiable cause of preterm labor [169]. Infection may induce the secretion of cross-reacting prostamides as seen by the anandamide release that has been observed in response to hemorrhagic shock [170] and a challenge of bacterial endotoxin in human peripheral lymphocytes [171]. Moreover, COX-2, which can be induced by several inflammatory stimuli such as IL-1β and bacterial endotoxin [87,172–176], may be present at the site of inflammation or infection, and the increases in both anandamide and COX-2 may synergistically induce secretion of PGE2-ethanolamide.

Prostaglandins in preterm labor

Previous studies have reported an association between changes in the eicosanoid concentration and preterm parturition [68,76,78,91,177–179]. Using radioimmunoassays, higher amniotic fluid concentrations of PGE2 and PGF2α have been reported in patients with premature rupture of the membranes (PROM) who delivered preterm and had an identifiable infection compared to those without infection [178]. Moreover, women with preterm labor and intact membranes with intra-amniotic infection had increased amniotic fluid concentrations of PGE2, PGFM, and PGF2α [68,78,177]. Our findings are in agreement with the latter report, given the identification that amniotic fluid concentrations of PGE2, PGF2α, and PGFM are increased in pregnancies complicated by preterm labor with intra-amniotic infection compared to those without inflammation.

Using gas chromatography, investigators have measured concentrations of PGE2, PGD2, and PGF2α, among others, in the amniotic fluid of patients who delivered at preterm or term [91]. Higher concentrations of PGE2 and PGD2 were found in the amniotic fluid of term compared to preterm deliveries [91]. However, PGF2α concentrations were higher in preterm births compared to term deliveries [91]. The authors also reported that the presence of microorganisms in the amniotic cavity was not associated with increased concentrations of PGE2, PGD2, and PGF2α [91]. This finding contrasts with our previous report using radioimmunoassays, in which the amniotic fluid concentrations of PGE2, PGF2α, PGFM, 14-dihydro-15-keto-prostaglandin F2α, and 11-deoxy-13 were significantly higher in the context of intra-amniotic infection [68]. In our study, PGE2 and PGFM were higher in preterm pregnancies with intra-amniotic infection compared to those without inflammation. Moreover, in comparison to sterile intra-amniotic inflammation, significantly higher concentrations of PGE2 and PGF2α were observed in the amniotic fluid of women with intra-amniotic infection. This finding is consistent with previous reports showing that intra-amniotic inflammation, whether sterile or microbial-associated, resulted in higher amniotic fluid concentrations of PGE2 compared to those without inflammation [180]. Moreover, PGF2α concentrations were higher in the amniotic fluid of pregnancies with preterm premature rupture of the membranes and intra-amniotic inflammation, compared to those without this clinical condition (as determined by MMP-8 >23ng/mL and negative culture) [179]. Higher concentrations of PGF2α, regardless of their inflammatory status (and adjusted for gestational age), correlated with earlier delivery; therefore, it was proposed that PGF2α could be a predictor of early delivery [179]. Our observation was that higher concentrations of PGE2 and PGFM (but no significant difference in PGF2α) were found in the amniotic fluid of women with sterile intra-amniotic inflammation compared to those without inflammation delivering preterm or at term. Therefore, amniotic fluid PGF2α may serve as a predictor of early delivery in women with preterm premature rupture of the membranes and intra-amniotic inflammation but not in those with preterm labor and intact membranes.

It is worth mentioning that patients with an episode of preterm labor who delivered preterm without intra-amniotic inflammation did not have increased concentrations of prostaglandins compared to those who delivered at term. These data suggest that the pathophysiology of idiopathic preterm labor and birth (without intra-amniotic inflammation) involves mechanisms different from those implicated in intra-amniotic inflammation-associated preterm labor and birth. It would be relevant to investigate whether the concentrations of prostaglandins or other inflammatory/labor mediators are different between episodes of preterm labor; yet, serial amniotic fluid sampling would be required to conduct such a study.

The use of mass spectrometry for the detection of intra-amniotic inflammation and/or infection

The use of mass spectrometry enabled us to determine that the ratio of PGE2-EA and PGF2α-EA in pregnancies complicated with preterm labor and preterm birth with intra-amniotic infection was higher than that of the other study groups. The use of mass spectrometry helped to overcome one of the major obstacles of accurate identification of endocannabinoids and eicosanoids by immunoassays, resulting from the structural similarities of these compounds. Our findings suggest that a targeted mass spectrometric approach (as used herein) could prove to be more reliable in a clinical setting [90]. Indeed, other lipid mediators related to eicosanoid pathways have also been recently studied in amniotic fluids using mass spectrometry. In a recent study, it was found that leukotriene B4 is elevated in cases of microbial invasion of the amniotic cavity [76], while several eicosanoids of the epoxygenase pathway have also been measured in women undergoing spontaneous labor at term [74] and in patients with clinical chorioamnionitis at term [77].

Conclusions

In this study, we successfully employed a high-resolution mass spectrometric approach to characterize the relative changes in the amniotic fluid concentrations of five eicosanoids in pregnancies complicated with preterm labor. The profile of eicosanoids differs among the study groups. The highest concentrations of PGE2, PGF2α, and PGFM were found in patients with intra-amniotic infection. Further development of mass spectrometry for the separation of these products, thus reducing the non-specific identification of these compounds, may have potential utility in the clinical context.

Supplementary Material

Supplementary File

Acknowledgements

This research was supported, in part, by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U. S. Department of Health and Human Services (NICHD/NIH/DHHS); and, in part, with federal funds from NICHD/NIH/DHHS under Contract No. HHSN275201300006C. Dr. Romero has contributed to this work as part of his official duties as an employee of the United States Federal Government. NG-L is supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. HNP is funded by The Lalor Foundation, Boston, MA, USA.

Abbreviations:

AF

Amniotic fluid

PGHS or PTGS-2

prostaglandin endoperoxide synthase-2

COX-2

Cyclooxygenase-2

PGE2

prostaglandin E2

PGF2α

prostaglandin F2α

PGFM

13,14-dihydro-15-keto-PGF(2alpha)

PGE2-EA

prostaglandin E2-ethanolamide

PGF2α-EA

prostaglandin F2α-ethanolamide

IL-6

interleukin 6

IL-1β

interleukin 1β

LPS

Lipopolysaccharides

MRM

scheduled multiple reaction monitoring

PAR

peak area ratio

Footnotes

Disclosure: The authors report no conflicts of interest.

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